Spices Flavor Chemistry and Antioxidant Properties

-

A C S S Y M P 0 S I U M S E R I E S 660

Spices

Flavor Chemistry and Antioxidant Properties

Sara J. Risch, EDITOR Science By Design

Chi-Tang Ho, EDITOR Rlltgers, 71le Stale University of New Jersey

De~e.l~ped from ~ symposiu m sponsor~:---~ the DIVIsIOn of Agn cultural and Food . .c!t~rp~~try; .,~

American Chemicol Society, WOshington, DC

Uhnry or Congr''!! Ca lalos;ng.!n. I'u bUu !lon Ila!a

Sl'tc,,: Il.wor che,niWy and antio~idant pmpcrtie .• I S,,.n I, Ri""h, rJilOI,

Chi ·T"n~ 110. wiU)",

p. cm.-{ACS .• ympo<ium series, ISSN 0097--61 ~6: 660)

··n.:",lopa l from a , ym posi um l ponsored by tile Divi~ion of Al'i~uh"I,,1 ~"J F("O~Il'lIc",i,"y al ti,e 2 111h National Muting oflhe Anoerio.:an Cloemic"1 S<lci.,ly. New Orlean~.looi~i.fta. Much 24_28, 1\I'}6.'"

ISHN O· M412_3495_7

.. SI'I<:<:.,·-Congre. .. e... 2. C'm, lime nt~-Cong,e.',es.

I. Ri«:h. Sara 1., 1958- n. HO,o.i·Tang. 19044- III. American C1Icmk.,1 SOCiely. Division of Agrie ultulal und F"od O'CII,iSlry. IV. AUlerican Otc:,"IC~1 SOCtcty. Meeting (2 1 Ith: 1996: New OrIeIlllS, La .. ) V. SC'.!eS.

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PRll\'TED];O; Tim UNIll;D STAll~ OF AMERICA

Advisory Board

ACS Symposium SerIes

Robert J. Alaimo l'roeler & Gamble PhJ'lI1l1<:euticals

Mark Arnold University of Iowa

David Baker Uoivenity ofTenne5$<'e

Arindam Bose I'filtr Cel1!r~1 Rcsearcl,

Robert F. Brady. Jr. N~v,11 Re.o;car(h t abomtory

Mary E. Castellion ChemEdil Company

Margaret A . Cavanaugh NallQn.1 Science Foundatioo

Arthur B. Ellis Univer,;ity of Wiscon!in at Madison

Gunda I. Georg Un;~cui ty or Kansas

Mnddcine M. Joullie Uni"cl"$ity o f l'cnnsylvania

Lawrence P. Klemann Nabj~o 1'00<15 GIOUp

Douglas R. Lloyd 'Ille University of Teus at Au~l in

Cynthia A. Maryanoff R . w, JOII05011 J'llarmaccut ;cal R~ • • dt Instit ute

Roger A. Minear University of 1I1 ioois

M Urbnna- Chalnp"ign

Omkaram Nalarnasu AT&T Ikll LaboralOri~

Vincent Pecoraro Uni~rsily of MichiG~n

George W. Roberts Non" Carolina StAte UniVeriiiity

John R. Shapley University of Illinois

I! Urba.na-013mpalgn

Douglas A . Smith Concurrent Teeh!lolvgies Corporation

L. Somasundaram DuPont

Midlad D. Taylor Par~e· DaYi, l'hilftnaCeutical Reseuch

William C. Walker DuPont

Peter Willett University or Sheffield (England)

Foreword

THE ACS SYMPOS IUM SERIES W3S first published in 197410 provide a mechanism for publishing symposia quickly in book form. The purpose of Ihis series is to publish comprehensive books developed from symposia, which are usually "snapshots in time" of the current research bei ng done on a topic. plus some review material on the lopic. For this reason, it is nccess:uy that Ihe papers be published as quickly as possible.

Before a symposium-based book is put under contract, the proposed tllble of contents is reviewed for appropriateness 10 the topic and for comprehensiveness of the collection. Some papers are excluded al this point, and others are added to round Qut the scope of the volume. In addi tion, a draft of each paper is peer-reviewed prior (0

fi nal acceptance or rejection. This anonymous review process is supervised by the organizer(s) of the symposium, who become the editor(s) of the book. TIlt": authors then revise their papers according to the recommendations of both the reviewers and the editors. prepare camera-ready copy, and submit the final papers to the editors, who check that all necessury revisions have been made.

As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previously published papers are not :!ccepled.

ACS BOOKS DEPARTMENT

Contents

Preface ............................................................................................................. ix

GENERAL OVERVIEW Al"'D M ETHODS

I . Sllices: Sources, Processing, and Chemistry..... ......................... ..... 2 Sara J. Risch

2. Methods of Dactel"ial Reduction in Spices ................... .................... 7 W. Leistr itz

FLAVOR CIl EMIS1"RY

3. The Principal Flal'or Components of Fenugreek ( l'rigo1lelfa foe1lum-graecum L) ............................................................................... 12

Imre Blank. Jianming Lin, Stephanie Dev3ud, Ren~ Fumeaux, and L:l.Urent n. Fay

4. Vanil\ll............................... ........................................................ .............. 29 Daph na Havkin-Frcnkel and Ruth Dorn

5. Onion Flavor Chemistry a nd Factors Influencing Flavur Intensity .................................................................................................. 41

William M . Randle

6. Cuntribu lion IIf Nonvolati le SuUur-Containing Flavor PI'ecursors of the Genus Allium to the Flavor of Thermally I'rocessed Allium Vegetables ................................ "............................ 53

Tung·"lsi Yu

ANALYTICAL 1):CHNIQUES

7. Characterization of Saffron Flavor by Aroma Extract I)ilution Analysis................................................................................... 66

Keith R. Cadwallader, Hyung !-Icc 8aek. and Min COli

,

H. Tile (]wracteri7.atinn of Volatil e :lntl Se mivulatile Cllm llIInen"~

in I'uwdered Turmeric hy Dired Therm:ll Extractiun ( ;a5 ChruIIllIlugr:lphy- M:lss Sllectrumetry ..................................... 1:10

Rich:lrd D. Iliscrodt, a li-Tang 1-10, and Robert T. Rosell

'J. !,unge nt Flavor I'rnfil es and Co mponents of Spices by Chrolll.,tugraphy and Chemiluminescen t Nitr ngen Ilc tectinn ...................................... " .............................. ..... ".................... 98

E. M. Fujina ri

10. Supercritical "' Iuid Extraction nf AlliulII Species ............................ 11 3 Eli;r.abcth M. C.,lvey and Erie Block

11. Determin ation of Glu cosinolates in Mu stard by High .Performance: Liquid Chromatography-Electrospray M:lss Spectrometry ....................................................... 125

Caro l L. Zrybko and Robert 1'. Rosen

12 . Reasons fur the Variation in Compos ition of Some COInnlercial Essential Oils ........................................... ..................... .. 138

Chi-Kuen Shu and Brian M. L1wrence

13. Cumpon ent AII ~llyses of Mixed Spices .............................................. 160 C. K. Che ng, C. C. Chen, W. Y. Sh u, L. L Shih , and H. H. Feng

ANll0XIDA."lT PROPERTIES

14. Antioxidative Activity of Spices a nd Spice Extracts ....................... 176 Helle Lindberg Madsen, Grete Bertelsen, and Leif H. Skibsted

15. Antioxidat ive Effect and Kinetics Study of Capsanthin on the Chlorophyll-Sens itized I)hotooxidation of Soybean Oil and Se lected Flavor Compounds ....................................................... 188

Chung-Wen Chen, Tung Ching Lee, and Chi-Tang Ho

16. Cureumin : An Ingredient that Red uces Plate let Aggregation and Hyperlipidemia, and Enhances Antioxidant and Inunune Fu nctions ................................................................................................ 199

Yaguang Li u

17. Antioxidant Activity of L:lvandin (L tlVam/lIia x illfermedill ) Cell Culture s in Relati lln to Their R"s marinic Acid Content._ ............ 206

T. L6pez-Arnaldos, J. M. Zapata, A A. Calder6n , and A. Ros Barccl6

18. Anti-innanunatory Antioxidants from Trupic:!1 Zin giberaceae Pla nts: Isolation and Synthes is of New Curcuminoids ................. 219

Toshiya Masuda

19. Curcumin: A Pulse Radiolys is In vestigll tinn of the Radical In Micellar Systems: A Model for 8eha\'ior 3S a Biological Antioxidant in Both Hydrnphobic a nd Hydrophilic EnvironJllents ......................................................................................... 234

A A Gorman, I. Hamblett, T. J. Hi ll . H. Jones, V. S. Sri nivasan, and P. O. Wood

INDEXES

Author Index .................................................................................................. 246

Affiliation I ndex ............................................................................................. 246

Subject Index .................................................................................................. 247

Preface

S PICES ARE WIDELY USED IN FOOD PRODUCTS to create the distinctive flavor and I:haruClcr that is representative or di fferent cuisines. The symposium on the flavor and antioxidant propenies o f spices was organized \0 look al new developments in the area o[ spicc chemistry.

111C nature of the volatile components in different spic..:s is important in understanding the fl avors they impart 10 food products. Research is being conducted \0 determine the significant volut ilc and nonvolatile compounds thnl create the distinct fla vor of various spices. Variations in spi ces C;l1l occur depending on the region in which Ihey arc grown and the climatic condition ... which can dramatically infl uence their composition. Th is information j-.;

imponant in formulating food products and maintaining their cons is t~ncy (I\,...:r time_ As spices vary in their flavor profile, adjustments may need to be made in the fonnulution of a product or spice blend to maintain the desired !lavOL Computer modcls are being developed to eVlllullte spices [lnd spice blends to determine thcir composition and other rekvllnt variables_

Another major arcu of interest today is the antioxidant properties of spices. Although spices have long been used to help preserve food, it has not been known what components give the preservative eITect. Research in this area has expanded beyond the use of spices as preservatives 10 the potcntiol hcalth benefits they confer as antioxidants in the body_ There is considerable evidence tha t specific components in spices may provide these benefi cia l cl1ccts. Research is being carried oul to determine the aclive components and \0 explain the mechanism of action. With increasing interest in the use of food products to help maintain health and prevent disease, spices may playa significtlnt role.

-Ille symposium on which this book is based was sponsort..-d by the Division of Agricu ltural and Food Chemistry at the 21lth National Meeting of the Americtlll Chemicill Socicty, which took place in New Orleans, Louisiana, from March 24- 28, 1996. ·n lis symposi um provided a forum for researchers from all over the world to prcsent information on possible specific roks .)J" spices ill disease prevcntion.

We thank all of the pan icipants in thc symposium as well as I h~· ad,litioll;11 authors who have contributed papers \0 this hook_ All or 'lIlT c"I1~·_ ' !-!Ul-" wi", gtlve of their time to Hssist in the review of the mnnuslTirl.~ an: r,-n':'I !i/l-.I , ; lIltl

Iheir work is ~inccrdy appreciatcd. Thc American Spice Trauc Associatiun was helpful in providing general information about spices and their usage.

SARA J. Ri sen Scicnce Oy Design 50S :-Jorth Lake Shore Drive, 113209 Chicaso. IL 606 11

C III·TANG 1-10 Dcp:_u1ment ofFoocl Science Cook College Rutgers, '111C Siale Un iversity orNcl\' Jersey New Brunswick. NJ 08903- 023 (

Novcmber 22, 1996

GENERAL OVERVIEW AND M ETHODS

Chapter 1

Spices Sources, ProceSSing, and Che mist rY

Sara J . Risch

Science By DeSig n, 505 North L'lke Shore Drive, #3209, Chicago, IL 60611

Spices are used throughout the world to season food products and create the unique characterist ic fl avors of different cuisines, Spices are grown primarily in tropical count ries although the United States has recently started growing a number of different spices to supply the domestic needs. The use of spices has increased significantly over the past few years, due in pan to the high level of interest in different types of foods that use a wide variety of spices. Interest is also developing in the ability of spices to act as antioxidants in addition to seasoning a product

Spices have long been important for food products. It was found that small amounts o r various plants could be used to enhance the flavor of a food and also served to help preserve that food . In somc cases spices wefe even used to mask spoilage or off-flavors in products. This use continued for centuries wi thout any real understanding of how the spices were being effective. People simply understood 1hat spices helped create a more desi rable taste in the foods that were being prepared. Different cuisines are noted for using s~cific types of spices to create their characterist ic fl a\·ors.

People also viewed spices as being imponant because in early history the entire ecollomy of many regions was based solely on spice trade. Spices were the major item of trade and the region tha t could contro l spice trade dominated as a world power. That situation has changed dramat ically with spices now accounting for less than 0 .1 percent of world trade. Other raw materials as well us processed foods account for a larger percentagc of world-wide trade than spices. There are still several countries that rely heavily on the trade of a specific spice. In Tan~ni a,

the production of clove accounts for a large percentage of that country's economy. Tanzania grows about 213 of tile world requirement for cloves. About half Granada's revenue is derived from the sales of nutmeg and mace. !fyou also consider vanilla,

(tI l991 American Chemicat Society

1. RISCH Sp i"fls: S(//m:u, l'rocb'l';ng, allll CJII'IIris(ry

which is noIteclmicaJly /I spice but is a Vf%'J 'Nidcly used flavoring material that comes from a plant grawn in tropical regions, it is the second largest prodllct in the Malagasy Republic.

There are 36 different herbs and spices that are generally recosnized and commonly used, White and black pepper account for the largest amount of spices used in terms of dollar value The ne.'l:t highest value spices consumed are cloves, nUlmeg, cardamom, cinnamon ginger, mace and allspice It should be noted that accurate figures for the value of spice imports and exports are difficult to track. , Countries typically do nOt repon spices separately from other food products

Most spices that are imported into the United States come in whole or unprocessed, There are requi rements that they meet minimum standards for purity and cleanliness. The Food and Drug Administration performs inspections on the incoming spices mainly to conlirm that they are free from undesirable filth and that they are safe for use in food . The American Spice Trade Association (I) and AOAC (2) have defined specific tests for spices. A complete list of typicallcsl~ for the quali ty of herbs and spices was developed a number of years ago by Heath (3) This provides a comprehensive listing o f tesls that can give infor mation on Ihe volatile oil content as well as other quality re lated factors in spices.

Spices and herbs come from a number of di fferent pans of plants. This cnn include the bark, seeds, berries Dnd leavcs of a plant. In the United States, spices have a legal definition that is spelled out in the Code o f Federal Regulations (4). This states that spices are "any aromm ie vegetable substance in whole, broken or ground fonn, except those SllbS1anc~s which have been traditiona lly regarded as foods, such as onions, garlic and celery; whose significant function in food is seasoning rather than nutritional; that is tAre to name; and from which no ponion of any vola:ile oil or other na\'oring principle has been removed". nle form that the spice is use<! in ollen depends on the application in the food product In some cases it is desirable to have large, visible pieces of spice while in others the spice may be finely ground to be more easily incorporated into the product. Some of the appl ications for whole spices include peppercorns in sausages, bay leaves in soups and various seeds thllt are used on or in breads and other baked products including caraway seeds, sesame seeds and poppy seeds.

For the vast majority of applicat ions, the spice is processed before using in a food product. General processing of spices usually involves some means of panicle size reduction. Processors use different techniquc~ to achieve the desired panicle size. The process of grinding spices breaks down some of the cell structure to make the volatile oils in the plant more readily released when used in a product than if the spice were left in the whole form. Care must be laken during the grinding process to insure that the desired volatile oil level is maintained in the spice. One process Ihal has been investigated to help preserve the qual ity of the spice is cryogenic grinding.

In add it ion to grinding or comminution of the plant part , a mcans or bacterial reduction is also needed. The different methods employed to reduce the bacterial load on spices. including irradiation, steam trcatment and ethylene oxi{!c, will be covered in a subsequent chapter in Ihis book. A comprehensive review ofindividllill

,

S I' II.:F.S: flA VOR CIII': MJSTIl V fi NO ANTlOXJUflNT l' IIOI'lmTJK"

sp i..:;cs intludinij thcir flavor characters and various applications is included in the Source Book of Flavors by Reineccius (5). This rererence also includes inrormation aboUI processing ofspice5 as well as the specific volatile oils and the procedures that arc lI sed to extract the oils from spices. The flavor of herbs and spices is derived from the volatile viis that arc present in the plant. The only exceplion to this is the cap~icum~ which give The flavor to peppen. The essential oil content of herbs is Ollcn less Ihan 1% . The conlenl of other spices ranges up to 18% essential oil in clove buds Essential oils arc recovered from herbs and spices by steam distillation. '111i5 recovers The vollllile oils but not the non-volat ile components which contribute to the pungency of some spices such as ginger and pepper. Oleoresins are oblained by solvent extraction which yields both volatile and non-volatile components.

While spices have long been used in food products, their usage continues to gro w al a significant rate. A recent estimate by Ihe American Spice Trade Association indicates that the usage in the U. S. alone could reach one billion pounds by the year 2000 (AST A personal communication, 1996). Interest in spices started growing aner World War II and saw a sharp rise beginning in the 1980's. This c~n likely be attributed to first, the exposure that many people had to other spices in the 1940's and. more recently. to increased interest in different ethnic cuisines. 11 should be noted that the consumption figures that are reported include dehydrated onion and garlic even though by legal definition they are not spices. These items may be used as spices in a food in the U. S. but must be listed separately on an ingredient statement. The average consumption today is about 50"10 greater than the average a decade AgO with the annual per capita consumption of spices in the United States ovcr three pounds (ASTA, personal communication, 1996). The spices that have shown the largest increase in use include sesame seeds., oregano and paprika. II is likely that the growth in usc of sesame seeds parallels the growth in fast food restaurants using sesame seeds on sandwich buns.

The other trends in spice consumption in thc U.S. follow thc growth of cenain food categories. One area is in hot foods where the AInerican public is becoming much more adventuresome in the foods that they will eat. Consumption of rcd peppcrwas up 105% from 1988 to 1992. Our bland foods arc becoming much spicier One company rc:ccnlly in troduced a fudge sauce for icc cream that contains blnck pepper, red pepper and cinnamon ill addition to dark chocolate. At the 1996 Institute of Food Tcdlnologists convenlion, one demonstration showed thc use of red pepper sauce in brownies. In contrast, another area of growth is in the mild herbs including basil and oregano. This growth follows the increase in consumplion or piua and a proliferation of commercially available spaghcui sauces. As the desire for ncw and unique foods continues, it is expected the use of spices will continue to grow.

Spices are commonly grown in tropiCI'll regions ofthc world. This can present challenges to companies that are trying to fmd a consistent source of spices both from a qlJal ity and supply perspective. Fanners in the U. S. have Slarted growing a number of di fferent spices to meet our growing demand. When dehydrated onion and garlic arc includcd in the figures, the U.S. in 199] was able to supply 38% of its needs

I. JUSCI! Spices: SOllrces, Pmce.I'.I'i/'I:. and Chemistry

(ASTA, personal communication, 1996). The other countries that are impoJ'lant sources of spices include India, Indonesia, Mexico, Guatemala, China and Canada. Canada has gained prominence: for its production of mustard, caraway and coriander seeds.

With the growth in the use of spices., there has been continued research into the active components of spices not only from a flavor standpoint but also from functional perspective 10 explore the antioxidant properties of spices. Differcnt spices are known to contain hundreds of acli .... e compounds. One area of interest is to identify Ihe specific compounds in a spice that make the most significant contribution to the flavor of that product. A technique that has been applied to flavor compounds to detennine thclr relative importance to Oavor of a product is gas chromatography coupled with effluent sniffing, referred to as GC-O (gas chromatography-olfactometry). Several variations on this te<:hnique, including aroma extract dilution analysis, can be used to determine the compounds that are most important to the aroma. This uses the human nose to determine characteristic and importance ofindividual compounds to compliment gas chromatography which can only give an indication of relative amounts of different compounds but cannot give any information on importance to the flavor profile. It is known that flavor compounds can have widcly varying sensory thresholds so having only the analylical dala does not teUthe entire story regarding importance of the individual compounds. The two specific spices that were evaluated and are presented in later chapters were salll'on (Cadwallader and Back) ~nd fenugreek (Blank et el). This data e~n be used in developing flavor systems that may be able to replace or supplcment spices in food products.

There is ongoing de\-dopmcnl of new analytical techniques to evaluate spices to dcvclop specific information about their composi tion. These techniques may nol be applicable only to spices but are ~erving to expand our knowledge of spices and their active components. One method is supercr it ical extraction which is used to more effectively removc Ihe volatile compounds from a spice and eliminate Ihe interference of olher plant malerials. Once the compounds are isolated, one detector that is being employed is specific to nilTogen and can aid in determining Ihe amounts of capsaicinoids which contribute the heat or pungency to different types of peppers.

Spice companies and food manufacturers arc also concerned about maintaining the consistency of the spices and spice blends that they li re using. It is know that a number offactors can influence the abundance of the aelive components in spices. The climatic conditioJlll can have a significant impact on spice quality from one year to the ne.'!:t . One chapter la ter in Ihis hook will address the influence or different growing conditions on the volatile compounds that are present (Randle). The same species of plant that is grown in different regions of the country can have a diffcrent volatile profile from that grown in another region. Understanding these dilTerences"is important for manufacturers to be able to maintain consistent flavor of a product when the spice being used may be sourced from different areas. A variety of plant species have been investigated to develop fingerprints of spices from different growing regions. This information can be used to determine authenticity or a spice

,

6

. ,

SPICES: H .AVOM CIlt-:l\IISTMY ANIl ANTlUXILIANT j' IWI'EMT1KIi

and determine optimum blends that should be used to maint!!.in the desired flavor profile. Statistical programs arc also bting developed \0 be used 10 detemune the most likely constituents in a spice blend and aid in replication oCa slJCCilic blend. This is presented in the chapter by Chen el al.

An area of considerable interest and the focus of many research projects is the antioxidant propenies of spices. As was mentioned earlier, one of the earliest uses of spices was to help preserve foods. Liule was understood III that time about the mode of aaion of the spices. It was simply known that they would help maintain the quality of the food to be stored, The area of antioxidant propenies of spices is imponant for preserving the quality of foods but may also provide beneficial health effects fo r people consuming the spices. This area will be covered in much more detail in an ollCl"View of the antioxidant properties of spices that is presented later ill this book in the chapter by Madsen et a!. One spice component in particular that has received atlemion is curcumin. Several chapters address the effectiveness of rurcumin and possible ways to synthesize new curruminoids. The investigators have looked at the various ways in which the compounds may be effective as an antioxidant in different systems. This is an area that will continue to receive attention in the research conununity as people cominue [ 0 look for Ihe specific health benefilS Ihal certain foods may offer. Spices will remain important for Ihe flavor lhat Ihey impart to foods and may also gain significance for other benefits they have to offer.

Literat ure C ited

I . Analytical Methods orthe American Spice Trade Association, )rd ed. ASTA.., Englewood Cliffs, NJ.,1978.

2. Official Methods of Analysis. 14th ed. AGAC, Washington, D.C. 1984. 3. lieath, t-I . B. Call. lnSI. Food 'f"e,·h. J. 1968, 1(1), pp. 9·36. 4. Code of Fcderal Regulations, Title 2 1, U.S. Government Printing Office.

1995. 5. Source Book oj Flavors; Reineccius, G. A., Ed., Second ed.; Chapman and

Hall, One Penn Plaza, New York, NY, 1994, pp234· 255.

Chapte r 2

Methods of Bacterial Reduction ill Spices

w. L..e is tr itz

Spie~Tec limited, 185 Alexllndra Way, Carul Stream, IL 60 188

There IIfC three major Incthods for b.lclerial reduction currently being us.:d in the spicc industry. These arc ethyknc oxide, irnlliiation and Sh::.m. Irradiation has received a gl"l!lll deal of pn:ss and although tnc popul;Hity uf this mcthod is increasing, there is still whether or nol Ilk: consurno::r will IIcccpt it. The induslry h.1S $t:icntific theory but the consulTler activists :lrc concerned about the unknown ruture consequences o f the k chnolo);.v. Another method that is not new bu has nOl been used in thc U.S. i;; stcam ste riliLation using supcrhealcd steam. This is a consumcr friendly method that may be the solution tlmt the world is lookin~ ror. The nlCth(l\l is efti!ctive and viewed liS being safc. A comparison of the three Ill<:toods will be presenll""d and the adv.lrltages and disndvuntages of cach method delllikd.

Spices are grown all o~r the world, in many cases it is in areas wh..'re the cle:'lnlincss is not closely controlled. There is 110 dian made to limit contamination of the spices wither during growing or harvesting. While there are some specifications and regulations concerning imponed spiccs, it is oncn necessary to process the spices to reduce the bacteriallond prior to use in food products. The oot:h:rialload varies with thc type or spice and total plate counts in excess of o ne mill ion colony formin~ units per g~ 11:1\·e been reponed in some spkt:s (I). Diflerenl types of pcpp..:r are known to ()fien h:'lVC very high bacterial counts.

The original need sterilized spices cnmc from a demand by the U.S. armed r(\Tecs. I'rior to World War 11 , they were seeking roods that coukllx: ItI,: ld for long pcri<..'lls (l rtill"ll: without spoiling. One source orlh.: bacterial contamination that rcduced the .~ho:1 r-lir ... or some products was the spice or seasoning being used. By reducing the l~ wl \J f IxJl·t~rii\ in the spice, products could be made "ith significant I)' longer slk:lf-l i\"cs. Till: nll . ."thud !;lr doing this rJCedcJ to dcstroy the bacteria without changing the Havor or color "I" th,· spicc. Sterili7.ation tcduliqucs \\ierc developed and were so successrul ror prndu<."ls n~l.k I;,r th.: I1rmcd rorces, that it soon became a gcnemlly accepted tedl!lilJllc Ii,r m',\ ' I'in·' 'lIkl

c:r t9!17 Amcrit.·~n ChCI1lK.":li S ... ;kly

St'U · t ·~ .. , .· t ~\ VOlt Cl IEM tS'fllY A:"I II ,\ NTIOXIIJANT 1 'lIo t'EKTn~ ..

"l·a". ,nm~s . All 'I'to.:e" ,m.1 .,",·a:«mings tk, lint ro:ttuin: h:to.: lel'i;11 reJudMlI1. hUI 111I.·r.: is a

l i,1.. I:,el"f wilh all ,'Ilhe n~"\lerials. '1 he "w,d in;1I111t:1llllrcr nC<'d~ In wcil:!h lito.: ri"l..s " f lI"m~: il1;1 ll·ri;.ls 111:,1 p"lentially 1~1n::;1 high ha( lerial loao wil h any !le~ .. tin· r-.;r(CI'1Mm tl1l' I,' 111;1)" he ,dth th,: pr'lI.:cs.sin!!- nfthe spi..:e.

Ill<' treal n .. "t nf spices h i r"'llll(e Ihe h."lI:kri;d k'ad is "nl'lI IdeneJ h' ....

\l': ll li/"tMIII. In !<Ome c .. ~, II ... · spke nk!) ;lCt'l:llIy be ~erili:lJ;d. IMwl'{.""er. H m,'re a<.:l"1 .rat .. h:n ll ~' h:I(I .. ,-i:11 ,-.. duetM UI. TIll: lirst mctlK.d 111;11 W;lS dc,·ck'po."d 1<1 retlu .. e the h:'dlT~11 l".,,~ <,n "I'icl's ill\'lIl\'cd lhe u <;c o f cthykne oxide ~as . nne (omllany th;lt is using a ,,·I'-.KIIl ,,(this met l ~>d to,by is (iriflith MicruSeicnee. The trealment nm~i~ts (If a lillll.'lI, I",," tnnpern t,u'e (yel<.: "r .... 1eulll11 gas.sing with Clhylcn( "xide 0' propyknc oxide. The

;:"IK'ral pr"l,ess now is de~rihcd below.

t . t'rodu..:t i~ p!;J.;eu in a scak:d cham l'l(."r about the width or" standurd l l.S. palkt (.j() x 42 in). The chamber may ... ary in k,ngth and height, hut it is critical thnt it

1\ot be too wide. 2. A \,<I": UUI11 is pulled within the chanlbcr. 3. "l'he..:h.1111hcr is heated to bring the temperature up to 110·120 I:. 4. I tumidit)' is introduced. ([t slllluld he IlOteJ thatlhe .. ombinatioll of lle,lt and humidi ty are essential to ac tivnte the microorgan i~ms within the sllice.) 5. I:.thylcllc oxide gas is int roduced into the ch.'lmber. 6. Th..: spice is held in Ihe dl'lmber under Ihesc conditions fu r a ~pcdlicd period uf time which is ,Iire .. tly re lah .. d to the bulk density of the spice lind the level u f ooc tcr~"\1 reduction thai is d~ircd.

1. The gas is c)(pclk.xI rrom Ih,: chambcr and it is Ilushed scveral limes with air prior to rcluming it to auno~pherie pressure. S. The product is then ren ... J\·ed to a '-Iuarantine area until it is pro ... en th.1t IMl

rc~itJtlal gas can be detected.

TIl;S i~:1 pro"cn method fo r spices .. particularly with wlMl\e sp;"; .. ~ and sectls. I ln"" .. \" .. r. the usc of gas for grourMl spices is eontro'-crsial today. Thi~ method is e'tp:lhlc of r..:dllc ing Ihc bacterial load by 31 least I x I 0'. nit' proecss may be repeated if the initial re~u l l docs IMlt llleCI customer requirements. Using ethylenc oxide gas doc~ rC\jui .. · a luner time period than irradialion (If steal11 .. wilh a lYl'ical process requiring any«hc re Ir01;1 12 to 18 hours. l'roduclivity is il,nucnced by the Si7,C and amount uf eham[:.,:rs :l\"3 ibblc. nle cost pcr pound of spice trellted is based upon volun .. trcated, bul l.. dens it ), ami iength ofli1ne Te(luirl'd to obtain the desired b.1cterial reduction.

Irwdiation h;ls become a popular method o f rcUueing b:leteria in spkes anJ s,~asoT1in~s since its introduction in the early 1980's, lIcc,lrding to the personnel at

SkriGeJ1ics ill Schaumberg, 1L. Throl.l~h the cont ro lled release of Cob:llt 60, ~piCC5 P:1SS

through a hetun or gamma energy that dcstroys b.1cter ia. The amount o f energy is clil led the radiatioll absorbed dose !IIKI is nleasureo in fads ( I rad = 100 ergs/g). Most ~pi .. es arc tre .. ted at a Icvcl of 1.0 • 1.5 ntds which is less than 50% of the rate allowed by Ihe Food and Drug Administ rat iun (FDA). TIM; gamma process requires (onlf()1 of only o11C 1).'lf'U1lCter and that is time of exposure. SleriGeni(s literature lK)tes thnl their process is klh,11lH spoi~lge mlol! pathogenic microorganisms in spices. The following Sleps describe the SteriGcnies method fo r usc o f irradiation to reduce bacterin in spkes.

2. U :tSTKIT"l. Mell!l)(l~ 11 Baelcri(l/ Helillel iOlI in Spitt{ 9

I. I'roduct is loaJnl 011tO a ( arrier and travels alo ng a .. onveyor through a series or doors and locks int" the cdl ll r .... containing eoixlh 60. 2. The product pa.<;scs IImWJJ and then tl .. ou~h t ..... o shelvcs nfisotopes where the greatest pcrcent .. ge () f OOelt.:r~1 1 reduction occurs. While in the cell. the product i:« hombarded with energy lit lower lc\'cI~ as .... 'CII. 3. The product rctUfl\.<; through Ihe ~ric:« o f doors ,mo locks o n a tin){'(.( schedule and pro~ralll to its origin. The entire rHlCes.~ takes between 5 and IS hours, depending upun the initial bacterial klad and the reduction that is required.

l11is is a cost clfoxtivc method IIk!tllffers 11 number of1:ll:ocfits to the consumcr. The main I>.:ndit is its overall effecti"encss. There is no nM)i~t l1re add .. d to the spice and thcre are no ad ... erse cfkets on the wlatilc oi ls o r rMlI1volatiJe components so the fla vor pmfile i~ !lOt negatively impat:ted. The irrnJ ilil ion wi ll completely penctra te packaging materials so that they can be processed withoulthe need o r opening a package.

The major concern with irradiatilln centcrs upon the fear ofeOllsumer a(ti ... isl groups, bot h domest ic and international. tha t nOI enough dnla hns been gathered to dl .. terrnine the long term effects o f eoting rood products which have been irradia ted or which contain ingr .. xlicnts tl101t have b<..~n imld~1ted. For this reason. the usc o f irradiation or spices and ~asl)t1i[lgs has been Nmn .. d in scveral fo reign countries. Some U.S. I{)od manufacturers ha ... e also made it a policy not to usc any ingredients which have been irradiated. SteriGenics, the leading company offering irradiated spices is currently addrcs.~ing these cono;crns both to go\,emmental ageneics and to consumer groups.

The usc or steam to reduce the b.1e terial count in spices has been gaining momentum due to the C"oneerns with the ~Ife t )' ofbolh gas and irradiat ion. Sleam is considered III bc a safe form orblle terial reduction throughout the workl . There arc I WO

melhods o f steam bacteria reduc tion being olli:red in the U.S. today. O ne o f tllcse is a wet method and the Oilier is a dry pwc .. ss.

'I'hcrc are scvcrnJ mClhods o f ..... et Sleam systems for bacterial reduction available in the U.S. These include lhe MicroMaster proccs..~ ufoCd by McCormick. Fuchs Micro c<)ntfoJ and the Kikkom.1n continuous steam process. These systems use eitne r closed chambers or continuous fced systemS. The key to all orthe:'\C systems is the ability to penetrate the whole spices or Sl.'Cds with wet steam at II temperature o f 100 C or great .. r for a ~pcei1kJ period o r time. The bulk density is another critical facto r v.hk h helps determine till: flow rate. moisture piek.up and e ... entually the cost per pound.

A l'(lnsistent cOllecn! with tho:: we t steam process is the anility to properly dry the ~I' iee during with OIM; I'as~ or treatmcnt after ~ter i1i7ation. It may bei;ome ncces.o;.ary to re.dry lhe product at a II>\\ cr temperalure 10 achieve the desired moisture le,·els. TIle overaU .. " sls for using wet s team 11\.11ch those nfusing gas.

The usc or dry steam (superheated) is the lotesl stl'<lmlechnology being used for ~ rin"s lind seasonings. The cquipmentlo produce spices by this method was recently installed by SpiceTec Ltd. in their plant in Carol S tream. II.. [t is the first usc of this me thod in thc United Slales. The process tlow for thi~ system is desc ribed below.

I. Continuous precle:med product is drupped through <In airlock into a closed chamber that contains a shaking bed.

10 S I' I( "K'i: ."IA Vtllt ClI EMIST RY AN I) ANTl()XIIlANT I'JI;O I'ERTI~:<;

2. The product is sterilized (Iuiddy "t a Icmpcrulurc between 108 and 125 C. DUring this process, the stearn is supcr+hcall:d and turns into saturated steam or vapor. A continuous airflow system bcltins the cooling Of111<: spi<.:cs immediately. 3. TIle product drops through im airlock inlo a St:cond closed chamber where the cooling prol:CSS is continued with ambient air withom Jhe dcvc10prncnl of condensation. Dust is fLhered out during the entire process.

' I bi.~ proc<''SS, which is viewed as being safe also offers a number ofbcncfits. COntinuous flow of the produd yields exccl!cnl productivi ty. AU different fo rms of spkcs can oc treated including whole and ground spices, herbs and ground seasoning blends. 'I'll<: efficiency of the process o ilers the potential for cost savings when compaflxl to both the gas and wet steam process.

All t hree mcthods of ~tcri lizal ion o r bacterial reduction offer both benefits and disadvantages. Irradiation i;; the most effective means of bacterial reduction, however, there are COtlSlUllCr oonccms about the safety of tile product. Ethylene oxide is a proven method that has been in usc fo r years but has recently come under the scrutiny of FDA and the Enviroruncntal Protect io n Agency (EPA). The weI steam process is not quite as e ffective as irradia tion and the COSI is slightly higher, howevcr, it is regarded as a SlIle

method. The dry steam method is highly effective and the newest method available. It i.~ more expensive than irradiation but k ss expensive a process than both cthylene oxide and weI steam. No one nlCthod is perfect. The selection of thc method to be used is up 10 the individually processor and must meet the needs o f the customer.

I. Reineccius, G., Ed., Source Book of Ftav(Jrs. Chapman and Hall , New York, 1994, p. 244.

FLAVOR CHEMlSTRY

Chapler 3

The Principal Flavor Components of Fenugreek (Trigonelia Joenum-graecum L.)

Imre Blank, J innming Lin, Su! phanie De\'aud, Ren~ J<"unleaux, and uwrent B. fay

Nestec Limited, Nestl ~ Research Center, Vers_chcz_les_Ulanc, P.O. Box 44, CJI- IOOO t.au sa nne 26, Swil7.erla nd

3- l-I ydroxy-4,5-dimethyl-2(5N)-furanone (sotolone) was established as the character impact flavor compound of fenugreek on the basis of gas chromatography-olfactomelry. Solo[one was found to occur predominantly in the (5.\) enantiomeric form (95%) and to have a olJCroo value of -19.7%0 About 2-25 ppm sotoJone were determined

in fenugreek of different origins using the isotope dilution assllY technique. Sotolone was generated in model systems by themlally induced oxidative deaminalion of 4-hydroxy-L-isoleucine (lUI.) using different carbonyl compounds. Up to 24 11101% yields were obtained by boiling HIL and melhylg[yoxal as react ive « -dicarbonyl at pH 5 for 10. h. Strecker degrada lion of HIL was found to be a competi tive react ion resulting in the formal ion of 3-hydroxy-2-melhy[bulana1. The I~clone of HJL, 3.arnino-4,S-dimelhyl-3.4-dihydro-2(5fl)-furanone, was found 10 be a better precursor of sotoione. It generated about 36 1001% SOlolone in the presence of methy[glyoxal

f enugreek is the dried seed of 7i-igoneflajoelllllrl-graeclim L. (Fabaceae). The plant is an annual herb widely cultivated in Medilerranean countries and Asia (I) The pods contain about 10-20 yellowish seeds with an appetil':ing pleasing aroma. Toasted and ground fenugreek seed is an essential ingredient of curry powders and is often mixed with breadstum. It is used as a seasoning in food products such as pickles, chutneys, vanilla extracts, artificial maple syrup, Hnd olhers.

Several volatile constituents have been reported in fenu greek (1), mainly terpenes and fatty acids. However, 110 systematic wOlk has yet been published on compounds that contribute to the characteristic aroma of fenugreek , 3.Hydroxy-4,Sdimcthyl-2(5H)-furanone (sololooe) was suggested as an important volatile constituent offenugreek due to ils seasoning-l ike flavor note (3, -I) .

I{) t997 American Chemical SocH:ty

J. IlI.ANK IT AL. Principal Havor CDmpQfI~nls of Ft nllgTUIc IJ

SOlo[one is a powerful flavor compound found in several food! and spi,,;es (.') It contributes to the burnt/sweet note oCaged sake (6), cane sugar (7), and conce (S).

10 Ihe spicy/curry no te of lovage (9) and condiments (J(J); M well as tn the nutty/sweet flavor ofbolrytized wines (/I) and fl or-sherry wines (/1). The lIavoring potenlial of soto[one is due 10 its low threshold values, that of 0.02 ngll air (8). 0.3 ~g11 water (detection/nasal, 10), and 0.036 ~gIl water (deteclionJl'etronasal. f Jl.

The structural similarity beTween soto[one and 4.hydroxy-L.isolcllcine (111 1.). Ihe mosl abundant free amino aeid in fenugreek seeds, was pointed oul (/4. fJ). I I was postulated thai Ihis unusual amino acid could be the pre<:ursor of sotolune in fenugreek (/5) (Figure I). This hypothesis has rec;ent[y been supponed by the f.1C1 that only the (5S) enantiomer of soto[one was found in fenugreek (16). This is in gond agreement with the stereochemistry of HIL isolated from fenugreek, i.e. (2S, 31l. '1.\) (/7). However, these authors failed to discuss possible formation pathways.

OH '1Hl ~OH , • 1 2 !!.

o (lS,3RJ.'l)-HJL (5S)·Sotolone

Figure I . STereochemistry of 4-hydroxy-L-isoleucinc (HIL) and sotolone found in the seeds of fenugreek (Trigo/ref/a jrx!IIum-gracclIlII L).

The purpose of the present sludy was to identify those vola tile compounds which significantly contribute to the seasoning.like note of fenugreek using the approach of sensory directed chemical analysis. Gas chromatogmphy in combination with olfactometry and mass speclrometry have been used as key sleps of this approach (/8, /9). The formation of flavor impact compound(s) was studied in model systems using the quantification technique Isotope Dilution Amy (10, 2/). The mechanist ic study was based on a hypothetical pathway proposed for The formation of sotolone via thermally induced oxidative deamination of HIL (10).

E:lperiOlcntal

Materials. Commercially available fenugreek seeds of different geographical origins and fenugreek oleoresin were used. Sotolone was from Aldrich and diethy[ 2-methy[-3-oxobutancdioate, L- isoleueine, Olethylglyo){al (40% in water), phenylglyoxal, 2,3-butanedione. and 2,3-pentanedione from Fluka, Solvents and other chemicals were of anal)1ical gradl'l'from Merck.

Synl hesis. 3 -Amino-4, 5-dimethyl-3, 4-dihydro-2(5H}-furanone hydrochloride (ADF) and 4-hydroxy-L-isoleucine (HIL) were obtained as diaslereomeric mixtures by photochemical chlorination ofL.-isoleucine (Figure 211), i,e. (3S,4R,5R13S,4R,5S) and (2S,3R,4R12S,3R,4S), respectively (17, 12, 13). (S,6_ 13C]-3 -Hydroxy-4,S-dimethyl

-- ,

14 SPICFS: HAVOM CIIEM ISTHY A."il) ANT.oxmANT 1·IWI·~;HTlr.s

2(5H)-furanone (fUC1]-sotolone) was prepared by condensat ion of diethyl 2-methyJ-3-oxobutanedioate and (I,2. IJQ-acetaldchyde followed by lactonization and subsequent decarboxylation under strongly acidic condit ions (Figure 2b) (1U) , The structures of the synthesized compounds were verified by elemental analysis. mass spectromtlry (MS), and nuclear magnetic resonance spectroscopy measurements CHNMR, IlC_NMR) (14).

Gas Ch romfilogra phy·Olfaclometry. GC-O was performed on a Carlo Erba (Mega 2) equipped Wilh a cold on-column injector, FID and II sniffing-pon. Fused sil ica capillary columns of medium (DB-OV 1701) and high polarity (DB-tFAP) were used as previously described (9), both 30 m x 0.32 mm with a film thickness of 0.25 I-Im. The temperature program was: 50"e (2 min), 6°C/min 10 180"C, IO"C/min to 240"C (10 min). Linear retention indices (RJ) were calculated according 10 van den 0001 and Kratz (25). The sensory significance of each odorant was evaluated and expressed as the flavour dilution (FO) factor (/8).

M:l$s Spectromr try

QUlllitative Analys is_ Elet.:tron impact (EI) and positive chemical ionisation (PCI, ammonia) mass spectra were obtained on a Finnigan MAT 8430 mass spectrometer MS-EI were gener-.ded at 70 eV and MS-CI at 150 eV with ammonia as the reagent gas Non-volatile samples were directly introduced into the ion source held at 200 ec. Vola tile component s were introduced via a Hewlett-I'ackard HP-S890 gas chromatograph (GC-MS) using a cold on-column injection. DB-FFAP fused silica capillary columns were used (30 m x 0.25 mm, film thickness 0 25 ~lm). The carrier gas was helium (90 kPa). The temperature program was: SOec (2 min), 4°C/min to 180"C, lOoCJmin to 240"C (10 min).

Quantitative Amllysis. Sotolone was quant ified by isotope dilution assay using [ 13C:zl-sotolone as internal standard (10, 2.f) . Quantification experiments were performed wi th a HP-597 I GC·MS using the following conditions: DB-Wax capillary column (30 m x 0.25 111m, film thickness 0.25 1-1111); carrier gas: helium (100 kPa); spli tless injection (250"C); temperature program: 20eC (0.5 min), 300C/min 10 lOO"C, 4"Cllllin to 145"C, 70°C/min to 220"C (10 min); El ionisation at 70 tV; selected ion monitoring (SIM) of sotolone (mh 128) and the labeled internal standard (mil 130). The concemration of sotolone was calculated frolll Ihe peak areas us ing II calibration factor of 1. 1 (Figure 3). A good linearity was found in the cOllcentration range 3- 150 )1g1ml. All samples were injected twice.

Fast Atom Bombul'dlll f nt (FA8-l\1S). This was applied to study the lactonization of IIiL 10 ADF. FAI3-MS was performed on a Finnigan MAT 8430 double focusing mass spectrometer. FAB ionisation was carried out with a saddlefield atom gun (Ion Tech, Teddington, UK) which was operated at 2 rnA and 7-8 kV with xenon. Glycerol was used as matrix. The positive io ns at mlz 130 (protonated molecular ion of AOF) and 148 (protonated molecular ion of HIL) were recorded.

lsotOlle Ra tio Mass Spectrometry (GC-IIU\'IS), This was performed with a Finnigan MAT delta S isolOpe MS coupled on-line with a Varian 3400 GC via a combustion interface. Isotope ratios were expressed as Ii-values [%oj versus the 1'01'

3. Il IANK ~71' 1\1_ Prilll:ipal Ha~/Jr COl1lptmtn'.f v/ Ftllll/:reek 15

A

Lel,hv --. 2. Hydrolysis ~OH

" B

n 0

EIO~O OE<

'CH;CHO •

EtOH

AOF HIL

J=( o

F!gure 2. Synthesis of 4-hydroxy. L-isoh:ucine (}-IIL) and 3-amino-4,5-dimcthyl _3 4-d~hydro-2(5H)-furanone hydrochloride (AoF) (A), and [S,6-13Cj-3-hydro .... y-4:5_ dlmethyl-2(5H)-furanonc ([ nC21-soIOJone, used as internal standard) (D)

ISOOOO I,.

" Ion mh 128 0 H3C OH • sototone ~ 100000 ):0( • 0

H3C o .. )~'":-O ~ « SOOOO

' .00 W.OO II (1) 11.00 lJ.OO 14.00

0 80000 " 0 Ion mh 130 ii H]C OH ~ GOOOO labeled sotolone

HJ'C~O • 0 ~ '0000 «

20000

9.00 1000 11.00 12.00 ~.-

JJ.OO 14.00

Retention time (min.)

:igure 3. Quantification of sotoJone by isotope di lution IIssay using lJCl-sotololle as mternal standftrd. The ions mlz 128 Mnd mlz ))0 wer, ',"o,d"d by GC '10 . • • ...... -IV .~ opcralmg III the selected Ion monitoring mode. (Adapted from ref. 24.)

S I' [CES: nA\'OK CI [ EMI~-rKY ANIl ANTIOX IIl" ." T I'I(OI '~;IITI K'i

siandani having II ["CJ/I"C) i~ololle mlio uf 0.011237 fur CO! yielded by combusliun of fos.~i l C;lCO) (reedee Belemnite) The GC wa~ elluillred with n 013-FFAI' fused silica capillary column (30 m x 0 25 mrn, film thickness 0.25 J.m) using hdium as carrier gas (100 kPa) and Ihe split injection mode (220°C). The tempcralUre pmgrnm was the same liS mentioned above.

S" lI lplc l'I"fl'3ra iioli

Qualit:.l \i l·r Almlys i.~ for GC-Olraflomrlry. The gr(lund fenugreek seeds {(OO 1;) wcre extracted wilh dicthyl elher (EtlO. 200 1111) by stirring the ~lIspensi()n for ~ h The S(livent was separated and the extraction W;lS c(lntinlled with Addition~1 ,;"Ivent (200 1111) overnight. The extracts were combined. filtered and cuncentrated (SO ml) llil ~ Yigrellx column. Non-volatile by-products were separated by vacuum

~lIblil1la tiUIi (YS). i.e. \Tapping volatiles under high vacuum conditions (2'10' ~ mbaT) illto 1raps cuoled with liquid N] (2/). The sample was introduced drop-by-drop into the vaCU1I1Il ~ystelll to increase the yields. Then. EIlO (30 ml) was added to the residlle and lite i501~tion procedure was repeated. The condensates of the traps were cOlllbilled and concentrated to I IllI on a Yigreux column.

Q ualitative Aualysis for GC- IRM S. Sotolone W;lS isolated from fenugreek oit:oresin, An aqueous solution of the oleoresin (35 .5 gllOO ml) was extracted wilh Et ~O containing 5 % etlmMI (5 x 100 011). The organic phase was concentrated to 100 mland extracted with NalCOI (0.5 mol/L. 3 x 100 ml). After ;lcidification of the aqueous phase to pH 2 (HC1. S moUL). sotolone was re-extracted with EtlO (4 x 100 ml). The sample WitS dried O\'er anhydrous Na!SO. and concentrated to 20 ml Sotololle was separated from nonvolatile by-products by sublimation in vacuum (10.1

mbar) (1/). The condensates of the traps were collected and concentrated 10 2 mi. Q ualil:Hh'e Analysis ror FAR-MS. HIL was dissolved in phosphate buITers

(0. 1 moUL) with different pH values (pH 3.0, 4.0. 5_0, 6.0, and 7.0). The soilit ions were boiled for I h in scaled glass tubes. The samples were rapidly cooled down and directly analysed by FAD-MS.

QUlI. llt ila l ive Analysis in Ftllugreek Serds (10). The material (5-10 g) was homogenized in wateT"ethanol (50 ml, 95:5). [ IJC2]-Sotolone (10-20 ~g) was added as internal standard and the suspension was stirred for 30 min. After centrifugation (30 min. 10000 rpm). the supernatant was extracted with EtlO. The acidic components were isolated with NalCOJ (0.5 moVL). The aqueous solution was acidified to pH J (5 mollL HCI) and re-extracted with EtlO Finall}'. the organic layer was washed with saturated NaCI solution. dried over Na2S04 and concentratcd 10 ilbOiIl 0 .2 ml using a Yigreux column and micro-distillation.

Q uanlilativt AII l_l ys is in Model Experilll ~ nls (14). I-IIL (2-10 IIIg) and

ADFHCI (2-10 mg) were each dissolved in a phosphate buffer (0.1 molll.... pH 5.0). ACter adding the carbonyl reactant. the solution was boiled for I h in a sealed glass lube. The molar ratio of precursor to carbonyl was 1:10. Water and the internal standard ([ 13Cl ]-sotolone) were addcd to the cooled reaction mixlure. The sample was saturated with NaCl and the pH adjusted to 4 (Hel, I moVL). Sotolone was c."m acted from the rcaclion mixture with Et20 for 8 n. The extract was dried (Na2S04) and concentrated to I 011. All experiments were performed in duplicate.

3. RlANK ET AI~ Principal H al'flr Cmnpnntnl.\· of Fenugreek 17

Resulls 311d Diu uuion

Aroma Compusi lion of Fr-nugruk. Liquid.liquid ex'raetion using diethyl ether resulted in an aroma exlract which represented the characteristic note of the origmal product. Ihal i.e. is seasoning-like, spicy, herbaceous, and fenugreek-like. The representativeness of the SIImples before and after purification was checked by sensory evaluation.

GC-Olf;lctometry (GC-O) was used 10 detect Ihe odor-aclive cornpound~

present in the aroma extract orrenugreek. It is II simple but effective mel hod to select those volatiles which contribute to Ihe overall flavor (18,19). On the basis of Ge-o. seventeen odorants were detected in the original aroma extract (Figure 4). An an.ma extract dilution analysis (AEDA) was applied to eiassify the aroma compo~itjlln into three groups having different sensory relevance. "high"' (no. 17), "medium" (nos. ,t, 6, 16). and "background" (nos. 1-3, S, 7-15). Hence. identification experiments were focused on the odorants belonging to the firs' two groups. Compound no. 17 was of particular in terest because of its high FD·factor lind ch~raclcrislie seasoning-like nole.

n FD-factor (2 ) ..

- - - - .. - -- - - - --- - -- - - - - - .. --- - -- - - -- - -- - - - -. - - - - - - - - - - -- 17 · -14

12

high sellst)ly relcI'flnce

10 - - -- -- - ----- --- --- .. - , . - •. -- .--- •• - - .- ... ------- --- . .. - -

• 6

",eJi,il1, S"'"S0ry rde.unce

- --- -- _. - ---- .. _- --_ ... -•

4 I.

4 - backgrOlmd nm", -- - ~- --- II" - ------------- · 15

"Ie TlT ,

2

L-~--T~--~-L~Lll~L-~.LLII _.--~L-~ -, BOO 1000 1200 1400 1600 1BOO 2000 2200

(Retention index, FFA P)

Figure 4. I'D-c~romatogram of an aroma extract obtained Crom fenugreek seeds.

The chemical struct ure~ (Figure 5) of the odorants were mainly elucidated by GC-MS (Table I). Compound no. 17 was identified as sotolone (Figure 6A). Sotolone is likely the character impact compound of fenugreek as indicated by the high FDfactor of 2". correspondingly low sensory threshold and characteristic aroma note.

,.

18 SPICES: ruvolt CIIEf, II ~'TMY ANI) ANTIOXIDANT I'ROi'M(Tlt~')

Sowlone was detected by GC-O even after mo re than IOOOO·fold dilution of the original aroma c){lraCI. Its FD-faclor was si~ nificantly higl1t~r than those of acet ic acid (FD'" 21

), (Z)-I ,5-ocladiene-3-one (FD- 2), and 3-amino-4,5-dimethyl-3,4-dihydro_ 2(5H)-furanonc (FO" 21) which belong to Ihe group wilh medium sensory relevance (Figure 4).

The FO-faclOrs of the remaining compounds were lower. They mosl likely cont ribute to the background of Ihe fenugreek [Iavor. These odorants art short chain falty acids (nos. 10, 11 , 14), lipid degradation products (nos. 3, 12), and alkylaled melhoxypyrazines (nos. 5, 7). All odorants listed in Table I, except nos. 15 and 17, were identified for the first time as constituents o f fenugreek aroma.

Table I. Odor-a ctive Compounds Detected in a n Aroma Extrac t or Fenugreek Seedli 011 the Basis of GC-O (II is the IHllllber of dilution steps)

No Compound Relention index Arol1la qlwliry FD-filClor FFAP OV-170f (Ge-O) r1")

I Diacety[' 990 '" BUllery 2 Unknown 1020 , d . Fruity. metallic 3 3 I ·Oclcne-3-one~ 1296 106> Mushroom-like • 4 (l)- I,S-Octadicne-3-oroc· 1369 1090 Metallic, tteran iu1\l .likc , , 3-lsopropyl-2· Inethoxypyr:l.7.,ueb 14211 1145 Roasl)', earthy 3 6 A~1ie aCid' 1445 '" Acidic, pUlIgent 7

7 3·lsobuty-2- methoxyp)'razin~· 1518 1235 Roasty. plprika·like 3 8 Linalool ' 1543 11 93 Flowery 4

9 U""""". 1554 n.d. Sulfury, roasty 3 to Butanoic acid' 1624 968 S ..... eaty, rancid 3 II Isovaleric acid' 1663 102S S ..... eaty, rancid 4

12 U""""". 1760 n.d. Fatty 3 13 UnknoWll 1823 n.d. Flowery, citrus-like 3 14 Caproic ac id' 1845 1! 63 Musty 3

" Eugenol' 2163 I SIlO Spiq· 4

16 3-Amil\O-'l,5-dimcthyl- 21 90 , d. ScaSOfling.like , 3, 4.dihydro-2(5J f)-furanone'

17 SOIolone' 2210 1350 Seasoning.like 14

Idcll1ifical ion by comparison wilh the refcrCllCe compound on ,I>< basis of retention indices, aron18 quality and GC-MS. Identification by comparison with the reference compound OIl "" basis of rdcntion indices and aroma quality. The amounts were too small for verification by GC·MS.

n.d. I~ detennincd

Terpenes and terpenoid compounds do not playa major rolc. Only linalool was detected. by GC·O (no. 8). Most of the Icrpenes ident ified by GC·MS wefe odorless at the concentration prescnt in the arOma extract, i.e. 0.- and p-pinene, sabinene, 3-carene, menthol, p-Ierpineol, cineol, anethol, p-terpinyl acetate, 1·1'·

3. BlANK I:,'T AI_ Principal Ha~or COl/lfHml'll1~' (if f£lIIlgruk 19

mcnth~n.8.yl acetate, carvone, and several sesqui terpenes. Further volatiles of low or no sensory relevance were I-pentano!, l . hexanoJ, 2-rnethyl-2.butcnc-l.ol, 2-methyl. 2-butenal, 2-pentylfuran, formic acid, propanoic acid, and fu rther longer chuin f;nty

acids, -y-butyrolactone and several 5-alkylated -y- Iactones, )-amino-4,5-dilllethyl_ 2(3}f)-fllnmone, and others.

0

)lOti

6

o

~OH

~OH

8

4: 17

0

~ ,

DOH II

"

.hIll

o 0

16

0

~

3

o ~OH

"

, 7

Figure 5. Sensory relevant compounds idemified in the arom~ cxt .... ct uf Ji.·m,!-',cc:' The numbers correspond to those in Table I.

20 SI'ICES: t"IAVOR Ult:M ISTJtY ANI) ANlIOXl llANT I'H.OI'Elnll·~'i

~ 100") A '" :. 80 ": .£ " c 50

1 ID c 29 ID 40 -.~ ro

20 Lj 0; 0:

72

~ 113

"--~ '-.. -~ - .

43

55 83

128

20 40 60 BO 100 120 140 mass I charge

100 70

" B

0

80 b"Jl' .£ " o 0 c 60 .. C

ID " 85 £ 56

-" 20 43 ID

,.1 .1.

129 0:

.,..,..,.,.,.,lrrt·'TTT~~ 20 40 60 BO 100 120 140

mass I charge

Figure 6. Mass spectra of3-hydroxy-4,5-dimelhyl-2(5H)-fur3none (sololone) (A) and of :; -ami no-4, 5-d ime! hyl-3, 4-dihydro-2(51f)-furanone (8).

Sterenisn meric C h:ml.cterisat ion of 50101001'. In fenugreek seeds, sotolonc occurs p~cdominantly in the (50) enantiorneric form (95%). This is in good agreement with the data reported by Sauvaire et aL (16)

The OI ·'C values of natural and 5ynthesi7.ed sotolone were determined by

isotope ratio mass spectrometry (IRMS) using the GC combustion technique (26), As shown in T~blc II, natural sololone was characterised by a OllCroa value of -19.7%0. On the contrary, a racemic mi:dure of synthesized 50tolone showed a significantly higher value (-23.3%0).

Sotolone isolated from a commercial product revealed a ,sl lCPOII value of

-22.3%0 indicating a mixture of natural and synthetic compounds. This was confirmed by chirospeciflc GC analysis resulting in a ratio of 65:35 for (5S):(SR). Hence, both

3. ntAN K ET AI . Principal Ffa~o,. Comp(mellis of Fenugreek 21

GC·IRMS and chirospeciflc GC suggest that about 213 of the sotolone found in rhe conunercially available liquid seasoning was contaminated with synthetic sOlolonc

Table II. slle Values (%e PDP) of Solo lone of Synthdic and N:lIural Origin

SQ/o/Olle

Synthetic

Natural (fenugreek oleoresin)

Natural (fenugreek seeds)

Liquid seasoning (commercial)

·23 .30 ± 0. 20

-19.69±O.20 -19.75 ± 0.20

-22 .28 ± 0.20

Quantitalion or SotolOllf in Fenugreek. The concentralion of sololone w~s determined by isotope dilution assay (10, 24) using labelled SOlolone as internal standard . As shown in Table !II, the typical concenlralion range of sotolone was about 3-12 mglkg fenugreek seeds. However, the amouots depend on the geographical origio. Fenugreek seeds from Egypt smelled more intensely and, in agreement with that, more sotolon was found in these samples. Some Trigoncl/Q species do not contain any 501010ne as reported by Sauvaire et a!. (11i) and, consequcl1tly, they lack the characteristic seasoning-like note.

The sensory relevance of 50tolone is due to ils low threshold value of 0.3 ~Iglkg water (10). In fenugreek seeds, the concentration of sotolone is usually at least 300{) times higher than its threshold, thus indicating the sensory impact of sololooe to the overall flavor of fenugreek and products cont~ining fenugrtck . As shown io Table III, high amounts of 501010ne were found in curry powder and some commercial liquid seasonings

Table HI. Co ncentration of 50tolone in Fenugreek and Products Conta ining '<enugreek

Sample

Fenugreek, seed (Egypt, 1985) Fenugreek, seed (Egypt, 1995) Fenugreek, seed (Australia, 1991) Fenugreek, seed (lOrance, 1996) Fenugreek, seed (India, 1996) Fenugreek, seed (Turkey, 1995) Curry powder (containing fenugreek) Liquid seasoning A Liquid seasoning B

S%lone /mglkgj

25.1 12.2 4.2 3.3 5.1 3.4

39.7 1.4

88 .9

I

! I

22 SPICf.:S: nAVOk CIIi':MI!o.'TJ.l.Y ANI) ANTlOXIUMH l 'IWf'Elnn:s

Form ation of So to lone from !' ,'tcu rsol'S

PretursorJ Present in Ihe Allueous EXlracls of Fenugreek Seeds. The presence of g!ycosidically bound sUlolone was examined by treat ing an aqueous extract of fenugreek wi th ex- and p-glucosidases. As shown in Table IV, this enzymatic treatment did 1101 si!,lni licantly enhance the amounts 'of 5010100e compared 10 the reference sample. On the other hand, boiling of the exlrHcl under acidic conditions (pH 2.4) for I h led to a more than 10 fold increase.

These trials indicate the presence of precursors in fenugreek that can be Iransfomled to sotolone using specific conditions, particularly an acidic medium and heat treatment. 4.1·!ydroxy-L.isoleucine, known to be a characteristic amino acid of fenugreek (14, J 5), may be Ofle of these precursors.

Ta ble IV. Formation of So to lone from Pre(ursors Present in the Aqueous [:ltra cts of Fenugreek Seells.

Addition (0 the Reaclioll cOIlfiiliollS Solo/Qlle aqueous extrocl pH Trel I {mi'" {mglkg]

None 6.8 25 120 18.9 a-Glucosidase (200 U) 6.8 25 120 19.2 ~-Glucosidase (200 U) 6. ' 25 120 20.2 None 2.4 100 60 252.3

SOURCE: Adapted from ref 10.

Formation of Solololl t frolll 4-lIydro:ly-L-isoleudne (II IL). The formation of soto lone was studied in phosphate·buftered model systems (pH 5.0) by react ing HIL with different mono- and a-dicarbonyl compounds at 100"C for I h. Roth 2,3· butanedione and 2,3.pentanediont rormed only low amounts of sotolont (Table V) Higher yields were achieved with the u . \.:eloaldehydes methylglyoxal (7.4 mol%) and phenylglyoxal (2.5 mol"!.), producing about 70-200 times more sotolone than Ihe corresponding reaction with the u-dikefones. Monocarbonyl compounds, such as propionaldehyde and phenylacetaldehyde, generated less than 0 I molo/. 50tololle (U , 27).

Formation or SOlolone f .. olll 3-Amino-4,S-dim cthy!-l,4.dihydro-2(S/J)furanon e (ADF). The emciency of ADF, the lac tone of HIL. to generate sotolone was tested in the same modd system as described above. As shown in Table V, significantly higher amounts of sotolone were generated from ADF as compared to !ilL Using methylglyoxal, the yields were increased from 64 Ilg (7.4 mol%) to 274 ~Ig (35.9 mol%), thus indicating ADF to be a more efficient precursor than the free anlino acid (HIL).

3. HLANK t.T At- PrillCipai Flayor Comptmtlll.f of ,.',rwgn:t!k

T able V. Fornllllion of SOlolon c from the ,'recurJo rs 4. lI ydroxy. I,,..jsolrucine (H IL)~ and 3_Amino_4,S_dimt lhyl_3,4_d ihydro-2(SII)_fur:lIIone (A I)F)b I' re.~rlll

in Fenu greek Seeds.

(l.DiC(lf"bollyf ",ccllrxo,c StJIO/OIle<i 1"1.:'" JIlL AJ)F {jlg/mg filL] {mo/%j

2,J ·Outanedione , 0.34 ± 0.03 <0 I

2,3.Pentanedione , 0.30 ± 0.03 <0. 1

Methylglyoxale + 64.2 ± 0.3 74

Phenylglyoxal + 22.2 ± 0.3 2S

2,3·Pentanedione + 5.4 ± 0.3 0.7

Methylglyoxal + 274.4 :t 3.4 35 .9

'Control experiment (without a.carbonyl) yie lded less than 0.01 molo/. sotolonc. b Control experiment (without a-carbonyl) yielded 0.03 11101% sotolone. c The molar ra tio of precursor to a.dicarbonyl was I : 10. d Data are means of at least twO experiments, each injected twict. e Control experiment (without HIL) yielded 0.07 ~Ig sotolont.

Mechan ism of the FOl"Ill;lt ion of Sotolone (J<'igure 7, 1);lt hwny A). The data reported above confinn the hypothesis of the formation o~ so~olone by thermally induced oxidative deamination of HlL (10) Acid camlyzed cyciLzalion of HIL leads to the corresponding lactone (ADF) which rtacts with an a-dicarbonyl (e.g. methylglyoxal) to form a SchilT base (pathway A) Rearr~ngement and subsequent. hydrolysis gives rise to sotolone. The data show that a-<hcarbonyls are cap.ablt ot generating sotolone from both HIL and the lactone ADF. Iloweve~, the lat~e!' ,~ m~re emcient in producing sotolone. Furthermore, the relatively low Yields achieved With HlL indicate an alternative degrRdation pathway.

SO'ecker ()egndation of n i L as ;) Comlleti tive Reac~ion (Figure ?, Ilathway B). The lower yitlds obtained wilh Hi t might be expla~ned b~ a pamal Strecker degradation of the amino acid I ilL in the ~rest:nce of an aCII~e Q.dlcarbonyl, e,g. Illt:thylglyoxal. As shown in Figure 7, the anlln~-carbony~ reaction of 1·IlL III~d methylglyoxal results in a SchilT base which may tither cychze or decomp:Jse v~~ decarboxylation. 11le Strecker aldehyde of I·IIL .. J_~ydro.x.y_2.lI\elhylbul"n~l , IS

released by hydrolysis and this compound was tentatIVely Ident,hed by GC·M S ( . ./) as

a mixture of diastereomers. In the sample based on tilL and methylglyoxal, Ihe ra tio of Strecker aldehyde

to sotolont was about 1:2 at pH 5 (Table Vl). Consequently, the Slr(!(:ker degradation of liIL is a competit ive reaction to Ihe form at ion of SO\(l~()!IC. 1 II ~1\1l1I'asl.

I I o f Strecker aldehyde were detecled in Ihe sample conl;\IIuns Ihe la<:I" "I' on y races .' ... . . I . ADF · bout 50 times less than in the reactIon wllh HI!. I he lurlll;ltllUl "t ",In " lit , I.e. a from ADF is the favoured reaction. most likely due to the hluded 1:;lIb(l~yl )" ''''')'

S I'I O~'i: HAVIlK Cl l~:MJ .... wn{Y ANI> ANTlnXJllA NT l 'IUll'~:KTlES

Tahl!' VI. Jt!lfio or Solololl e 10 3- lIydrO:fYM 2-1II!'lhylbu l:lllal l'Or llled in tll otlrl HI';\f li on~ Based 011 Mr_tbylglyoul and Ih e Prrcursors 4- lI ydro:fy- l...- isolelidne

(III L) !llld 3w Amino-4,S-dimclhyl-3,4-dihydro-2(511)-rurallolle (A 1>,.' )

I,ff IIlL AJ)F

J I : 17 , 50 5 1.2 ' 50 b , 2 : ]0 7 I I.S : 40

_~H~. ____ • '\---(NII,

'\ ,)...j, H20 0 0

IlI L o

/y" 11 20 .. MG 0

"~ .-.. ) N"0 II, o:~

o 0 .. ./

11,-., N =CI/ - {; -CII

~X u~) , o u ,"

t-. CO, 1l N-CH=C -(;11 'r---I' I •

/-. ..... A, mJ o 0

It 20

~OH o 0

figure 7. Formation of 5Ololone (palhway A) and 3.hydroxy-2.methylbutanal (pathway 8 ) from 4.hydroxy.L-isoleucine (HIL) and 3-amino-4,S-dimclhyl'],4. dihydro·2(5/1).furanone (AOF) using mcthylg1yoxal (MG) as carbonyl reac:tant.

3. tHANK Kf A IM PrinciJl(JI HMor Compollents of f enugredc 25

Innuenee or Ihe pH . II is well known that Doth lactooiz.ation and the format ion of Ihe Schiff base strongly depend on Ihe pH of the reaction medium. As shown in Figure 8, the lactonlzation step (I-liL ~ ADF) was favored under acidic: oondi tions and the yields were about 50 .;. at pH ], but only 10 % at pH 5. On the other hand, Schiff bases are readily formed under neutral and sl ightly basic condi tions.

The reactivity of the carbonyl c:ompound is another crucial parameter for the formation of the Sc:hiff base. o.-Ketoaldehydes are much more effic:ient than adiketones (Table IV) and a-keto acids, whic:h generate only low amounts of sotolone

from HIT- (27).

7 6 5

~I

,

}-·igure 8_ Lac:loni7,..i1tion of 4.hydroxy-L·isoleudne (!-lIT-) as a function of pH.

To fllld the optimum pH, methylglyoxal was reacted with HlL and the lactone AD!', respectively. The reac:lion of methylglyo)(at and HIL was favoured at pH 5 whic:h apparenlly is the best c:ompromise belween the lactonizalion slCP and the reac:livily of the amino group to form the Schiff base (Figure 7). Once Ihe lactone (ADF) r.~

formed the amino-c:arbonyl reac:lion was favoured al pH 5·6. In general, Ihe yields obtained with the lactone were significantly higher compared to the amino acid, partic:ularly al pH 6 (40_2 molo/. ).

The reaction of HJL and methylglyoxal at pH 5 performed in waler yielded 2 .8 mol% sotolone c:ompared to 7.4 mol% when using the phosphate buffered system. This suggests a catalytic: effeC:1 of phosphate on the formation of sotolone from HIL

2. SI'IC.:S, FlAVOR CII t:MISTRY AND AN .... O)(IUMIIT l'IW"t:RTIES

!o:(ftcl of Reaclion Temperalufe ~nd Time. The sotolone yields from tilL and mcthylglyoxal were slrongly dependent on both reaction lime and temperature. Significant amounts were generated above 70°C Crable VJJ ). At II constant temperature of 100°C, the yield of sotorone continuously increased over a period of to h, wilh no significant increase thereafter (Table VUI). About 23 ~(g to 210 ~\S sotolone were gcneraled from 30 min to ]0 h which corre~ponds ' !O 2.7 mol% and 23 .8 rnol%, respectively.

Methylglyoxal reacted wilh 1-fTL al SO°C for 48 h resulted in 3.8 mol% 5010100c, thus indicating that II long reaction lime and mild reaction conditions arc also suitable for generating significant amounts of sotolone. Therefore, hot climatic conditions might favour the fomlal ion of so to lone from HIL

Table vn. fo rmation of Solo lone fro lll n iL as a FUllcl ion of lhe React ion TemllcrMure Using !\Iethylglyoxrli as Carbonyl Reacta nt ~

Temperature Sol()lolI'.b Yield

, b

• b

fOCI {~lgllllg HILI {mol%{

50 0.31 ± 0.01 0.03 60 1.15±0.03 0. 13 70 4.00 ± 0 05 0.46 80 12.5 ± 0.5 1.44 90 27.9 ± 1. 8 3.2

100 64.2 ± OA 7A

Reaction conditions: phosphate buffer (0.1 mol/I., pH 5.D), 50·1 OO°C, \ h. Data are means of at least two experiments, each injected twice.

Trible VIII. Fon n:Hion of Sotolone frolll H[L as Affected by the Reaction Time Using Melhylglyoxal lU C;lrbunyl lleacla nt :t

Time SOloloneb Yield {II] Ipglmg HlL] {mul%]

0.5 23.4 ± 0.8 2.7 1 64.2 ± 0.4 7A 2 102./ ± 4,5 11 .7 5 170,2±5,1 19.5

10 206.7 ± 10 23 .8 15 208.3 ± 6.4 24.0 24 229.9 ± 5.) 26.4

Reaction conditions: phosphate buffer (0. I moVL, pH 5.0), lOO"C, 0.5·24 h. Data are means of at least two experiments, each injected twice.

3. Bl ANK t>:1· AI . Principtli "lavQr Cvmponents lif f'tnu/;rtek

Concl usion

The role of (5~1,so lolone as a charac ter impact compound of fenugreek was corroborated and its formation from 4.hydroxy·L-isoleueinc (I·IIL) via thermally induced oxidative dcamination WilS SllbSl~n t iated, The lactone of !-IlL, 3-atnino·4.5-dimcthyl.3,4.dihydro.2(5fl).furanone (ADF), was found 10 be lI. better precursor 11ulI\

the ~mino acid. a -Ketoaldehydes wcre more effective in generating sotalone frum both I·IIL and ADF than a·diketoncs. The reactivity of the dicarbonyl and the lactonization step are important parameters, particularly for the formation of the Schill' base. The transformation yields from IUL into sototone greatly depend on the reaction conditions., such as temperature, time, pH and amount of dicarbonyl Iligh amounts ofsot olone were obtained by boiling methytgtyoxal with HJL for 10 h:lt pI-! 5 (24 mulOV.). Even better results (40 mol%) were achieved using ADF as precursor (I h, pH 6), most likely due to inhibition of the Strecker degradation by the blocked

carboxyl group,

Acknuwlcdgmeul s

We are gratt-ful to ~'t r V Krebs for expert technical assistance, Dr. H. Schierbeek for performing the GC-IRlvIS rne~5Uremen ts, and Dr. E. I' rior for cri tically reading the manuscript . We also thank Prof A. Mosandl, Universi ty of Frankfurt, Germany, for

the chirospecifle GC analysis.

Li terat ure C ited

I.

2

3.

4 .

5.

6. 7.

•• 9. 10.

Lew;s, V,S, (Ed.), SpiCf!~' tlml Herbs/ur Ihl! Pucxl 'l/dusfry; Food Trade Press. Orpington, England, 1984; pp. 141 - \42. Girardon, 1'.; Ilessiere, 1.M., Baccou, J C.; Sauvaire, Y. PIal/faMed., 1 98~ , 51.

533-534 Rijkens, F.; Doelens, H. In Proc. 1111. S)lmp. Aruma R".~carch; Maarse, r I. , Groenen, 1) .1 ., Eds.; Pudoc: Wageningen, The Netherlauds, 1915; pp. 203·220 Girardon, P.; Sauvaire, V.; Baccou, J -C,; Bessiere, J -M. I.eben.",". /V,.\ ..... Tedmol. 1986, 19, 44-46. Kobayashi, A. In FI<lI"Or 01,,",i.I·I1)'. Trelllil- and IJew:lopmellf.\·;. Teranishl .. R , BUllery, R.G., Shahidi, E, Eds.; ACS Symp. SeT. 3S~, Amencan Ch,..nllclil Society: Washington, DC, 19119; pp 49-59. Takahashi, K,; Tadenuma, M. ; SalO, S, Agr. /Jiol. ('lwlIl. 1'176 , ./0, 325·330 TokitunlO, Y.: Kobayashi, A. , V~manishi, T ,: MurHki. S. /'/"11('. Japall A('(/d

1980, 56/J, 451-462. Blank, I.; Sen, A.; Grosch W. Z. Leb"mm. U'Jlers. F()r.l'~·h 1992, 195,2 3')-245 Blank, I ; Schiebtrle, P. Plav. hagr. J. 1993, 8, 191·195. Blank, I., Schieberle, P .: Grosch W. III I'rogrtss ill Plltllf)Jlr a",1 Prenll'sur

Sflldie.f; Schreier, P.; Winterhalter, P., Eds.; Allur I'ubl.: WhealOn, USA. 19'.13.

pp. 103.109.

" SPICt:S, ... .AVOR I.: IIEM ISTIl\' ANI) ANTlO XILlA NT l'IW,' ,·;KTlE.-;

II Masuda, M , Qkawa, E., Nishimura, K.; Yunomc, H A.I!r. /J/(}f. Chem. 19114,

48,2707-271 0. 12. Martin, B.; Etievarll, P.x. ; Le Quere, J.L., Sehlieh, P. J. A;:ric. Food ('//(.",.

1992,40,475-478, lJ. Wild , H. Cirelli. Ind., 1988, 580-SS6. 14 . I'owden, L. ; Pratt, H.M.; Smith, A PhYfochemistry 1973,12, 170 1·17 11. J~. Sauvaire, Y.; Girardon, P.; BaceQu, l e .; Risteruoci, A.M. I'IIYffldll.'misllY

1984, }1, 479-486. If> Sauvaire, Y.; Srenae, P.; Guichard, E.; Fournier, N. In FOllr/h Imemllfifmal

Works/lOll all Seeds, VO/lllne 1; Co me. D.; Corbineau, F., Eds., ASFIS. Paris, France, 1993; PI'. 201-206.

17. Alcock, N.W., Crout, 0 H.; Gregorio. M.V.M., Lee, E.; Pike, G , Samuel, C J. Phyfochemistry 1989, 28, 1835· 184 J.

ll! , Grosch, W. Trends F()()(/ Sci. Teehl/ol. 1993, 4, 68-73, 19, Acree, T .E. III F/OI'(Ir Science . S/lll~'ib/e Pr;lIcip/e.~ alld reehlliqlll'.~ Acree,

T E.: Ter~ ni shi , R., Eds : American Chemical Society. Wa~hill!!IOn , DC, 1993 ; pp. 1-18 .

20. Schieberle, P. : Grosch, W. J. Agrie. Food Chem. 1987,35,252-257. 21. Sen, A., Laskawy, G ; Schieberle, P.: Grosch, W. J. Agrie. Food Chem. 199 1,

39, 757-759. 22. Faulstich, H., [}QlIing, J ; Michl, K.: Wieland T. Liebigl· AIIII. Chem. 1973,560-

'6' 23 . Hasan, M. In Nelli Trellds ill Natllral Products Chemi.flry. Rahman, A.: Le

QUesnc, P.W., F..ds.; Studies in Organic Chemistry, Vol. 16, Elsevier Sci r ubl. · Am.~tcrdam, The Netherl~nd 5, 1986; pp. 123-14 1.

2... Blank, I. ; Lin, J., Furncaux, R, Welti, D,H., Fay, LB. J. Agrle. Frw Chem. 1996,44, (in press).

25 . vlln def 0001, H.: Kratz, r . J . Chmll/alogr. 1963, I J, 463-471. 26. Bruche, G., Dietrich, A.; Mosandl, A. Z. Lebcmm. Ulller.I·. ,"'or.feh. 1995,201,

249-252. 27 Blank, I. ; Lin, J. ; fay, L.n .; FUllleaux, R. In fj/Ofl(/\'OlIr.~ 9.5. Analysis -

Precllrsor Siudies • fjiolec/molagy Etievant, X.; Schreier, r ., Eds.; Lcs CoHoques no. 75, lNRA, Paris, 1995; pp. 385-J88.

Chapter 4

Va nilla

Da ph na lIavkin-Frc nke l a Dd Ruth Do rn

David Michael & Company, Inc., l080 1 Decatu r Road, Philadelphia, PA 19154

Vanilla is the most widely used flavor in the food and confectionery induf>1ries. Worldwide annual consumption was 1900 tons in 1995, witb 1400 Ions imported to tbe USA aloue. . . .

Vanilla flavor is extracted from the cU(ed bean of Vamlla p/allijo/m, a member of the orchid family origina ted in Mexico. A small.mount of beans is produced from yet another species - Yani/Ja lahiteflsis. Beans (pod-like fruit) arc produced after 4-5 years of cultivation. Fruit maturation occurs after 10 months. The characteristic flavo r and aroma de-'elnps in the fruit after a process called ·curing~, lasting an addit ional 3-6 mODth.s. We ~ablisbe~ tissue culture fo r vanilla , identified major components w the blOS}lIthetlC pathway ofvanjUin and found tbat tissue culture extract contains major components of vanilla fla vor present in the bean. Hence, plant t issue culture may be an altemative method for vanilla fl avor production, for f>1 udying the vanillin biosyntbetic pathway and for micropropagal ion.

With a better unden"tanding and ability 10 manipulate the biosYl1 thetic pathway and to regula te cultivar quality and flowering, we lUay control production and consistent quality of the vanilla beans.

I. Agronomic Prod uction or Va nilla

1J1troduction, Vanilla is tbe world's IllOSl popular flavor. Its fruit y, flora l fragrance combined with a deep, aronatic body makes it unique and universally favored .

Vanilla is an epipbytic orchid native oftbe tropical region of Mexico. TIle flavoring material is obtained from dry cured pod-like fruits commercially called "beans". The generic name Vanilla is derived from the Spanish " vauillia", a diminutive of\.o;1Io, a pod. Its species name, plani/o/io, refers to the broad, fl at leaf

of the plant. VaniUa became kllOW'tl in Europe following Cortexs conquest of the Aztec

kingdom in 15 19. Many centuries earlier, vanilla was. source of{l .voring and used

(D 1997 American Chcmical Society

30 S I'ICES: HAVON; l: II EMISTKY ANI} ANTIUXLIlANT PROI'EIlTI I':';

by the Aztec emperors who used vanilla \0 flavor a cocoa drink, which in the present day is hot chocolate. The Indians called vanilla ~TIi;mchilf', or black flower (6). The Spanish took vanilla back to their homeland and, shortly after, began manufacturing

chocolate with vanilla flavoring. In England, Hugh Morgan recOllllllended that vanilla flavoring should be used with chocolate to serve Queen Elizabeth I, [",Uowing the example oftbe Aztecs.

For more than three centuries after Henlan Cortes lived, Mc.'dco was Ihe only vanilla producing country in the world. Many attempts were made to grow the plant in Oilier tTopical countries but these efforts failed since the plant or vine grew and flowered, but DO fruit s were produced. It was not UJllil 1836 thaI Charles Morren, a Belgian bOlanist, discovered why vanilla was not able 10 produce fruit oul of Mexico. TIle anatomy of the flower is such that self-pollination is impossible. Morren discovered that pollination is c3rried out by a tiny bee of the Mellipone family which lived in tbe vanilla growing region of Mexico. It is difficult for other insects to reillace the tiny bees (3).

To achieve pollination one needs to remove the rostellum, a flower structure that is a modification oflbe stigma lying between the male and female organs and prevent access oflhe pollen 10 the stigma. Pollinalion is done by removing tbe rostellum wilh a sharp objecl, so Ihat the pollen from the anther can be in contact mth the st igma. Because Ihe blossom lasts for a very short time (less than a day), pollination must take place as soon as tbe flower opens (6). Charles Morren was the first to propose hand-pollination and he was the first to produce vanilla heans outside of MeKico. This discovery laid the fOUJldalion for a new vanilla indU.1ry and broke Mexico's monopoly.

In 1841 Edmond Albium, a slave in the Frencb-owued island of ReUllion, discovered a rapid pollination method. With the use of a poimed tip of a small bamboo stick, he picked up the adhesive pollen and prying up the flap-like rostellum inside the flower, he pressed the male pollen mass onto the sticky female stigma. This method ofpollinatioD is still used commercially today. Using the technique described above, the French staRed vanilla cultivation on many ofthe islands in the Indian Ocean. VaniUa plantings were established in Reunion, Mauritius, Madagascar, Comoros, Thafila, Bra:.cil, Jamaica and other islands in the West Indies. Vanilla was first introduced to the island of Java in 1819 by a Dutch scientist, using vines from Madagascar. Conunercial productioll in central Java started in the early 20th century and is known as "Java Vanillc~ (3).

Today. the primary growing regions in the Indian Ocean are Madaga~ar, Comoros and ReUllion, Beans from this area are called Bourbon and represent half of the world:s vanilla production. Indonesia produces the other half of the world's supply with an increase in production and quality. Tahitian vanilla beans, which represent another variety, are grown mainly on the island of French Polynesia. Vanilla is also beginning to be grown on Tonga, Although vanilla started in Mexico, al present Mexico produces a small percent of the world's consumption (Fig. I). Many other countries with suitable climates grow vanilla, bnt at present remain minor production regions (Fig.2).

4. 11,\ VKIN·~·RENKEI. & DOltN

Indonesia 127,llon5

Madaga5c;or 650.3 tons

Vanilla

Costa Rica 4.6 tons

31

'-Figure I. U.S General Imports of Vanilla Beans (U.S. Department or Commcrce, 1995).

Tahiti

Madagascar

Figure 2. World Vanilla Production.

r--25"

25" f

r

SI'[CES: FlAVOR CII E~I ISTRV tl.N I> tl.NTIOXII)tl.NT I'ROI'I';ltTt£S

Cullh';lljon

Sl,ui~. Van.iIJa is a tropical climbing orchid. I' rom rnorc than 110 species that have been described, on.ly 3 are of commercial value. TIley arc Vawfla plallijolia Salisb Ames (VRnilla fragran s Andrews), Vanilla lahiltlleSiS, and Vanilla pompon(l. Qnly V. plal/ijolia and Vanilla fahifellesis are pennitled to be used in rood. V. pl(llli/alia. the most important COllllllcrcia lly, is cullivated in all lhc vani lla gTowing areas, except ror Tahiti and Hawaii. The heans arc 10 to 25 cm long and I to 1.5 elll wide.

V. fall/lellesis is indigenous to Tahiti and is different from V. pfallijofia by h~villg slender siems and narrower leaves. Th e pods are also sllOrter thall V. pkmijolia (6). Thc V. fahitellesis beans arc perfume}" and contain anisyl alcohol, anisyl aldehydc, anisic acid, and m~ybc heliou opine which Ire absent in V. pfallijolia bC~II s. The bcans of V. lahitellesis colllmand higher market price than V. p'cllli/oIw bcan ~.

Economically, thc l e~st imponant is Vanilla pompall(l, also known as van ilion. It produces an inferior quality bean and in the past was IIsed for pcrfume and tobacco fla vor. 1be plant produces less beans than V. plal/ijoli(l and has larger and I,ider leaves. However, V. pompaflfl has important traits, including growth under more adverse conditions, and resist3tlce to root rot disease that may be used ill cross Im.:cding.

Climate. Vanilla needs II warn} and moi~1 tropical climate wi th fr~IUCllt , hilt 1I0t excessive rain. Under excessive rainfall, vaoilla may be aUlcked by mildew and rool dise~se. Under drought eonditioos, Ihe vlnilla plant can suffcr considerable damagc, wh.ich will result in a smallllumber oflloweTS and low yicld. In gcneral , sloping land that bas soil with high organic 1IIltter, high w~ter holding capacity and access to irrigation in dry years will overcome thc problems caused by wcather (12).

Vanilla grows best ill 40-50% orllomlal sunlight intensity. In excessive sunlight , the apical buds teud to lose moisture lind the gro\'\-1h of the vines is stunted. Heavy shade \vill result in vines with thin stelllS, small beans, and reduced number of flowcrs and fruils. A slladed nea, rainfall be1ween 1000-3000 mm and an average temperature of 23° to 29°C year round are the preferred conditions.

Soil :Jnd Nutrient!. Vanilla is a shanow rooting plantlnd grows well iu welldrained humus-rich soil. A top layer of mulch helllS keep Ihe moimire in and the roots to spread. Soil plrameters, such as lexture and pH, appear 10 be more imponantthan uutrients. In. hcavy rainfalluea, good drainage is essellliat The best soil appears 10 be limestone with a pH of 6.0 to 7.0 and a deep layer of mulch, which provides the nu trients, over Rnd around Ihc rools. Although the level ofcalciullI, nitrogen, potassium, phosphorous and micronutrients in vanilla are essential for thc grO\~1h and production of beaus, much IOOre allention has heen given 10 the type of TlIulch, because mulch aJlpears to be more imponant than nutrienls (II).

4. IItl.VKIN·~·RENKEI. (. IJOR.N Va"illa .IJ

Support. '111e vllnilla pl3tlt is a climbing orcllid 3tld needs me~han.ical support. Support is l lso needed to provide convenient ICcess for pollwauon a~d harvesting. Fast growing trees with smllil branches are preferred and also provide shade. Howevcr, it is irnportantthat the support ing trce will not compete for water and nutrients. Trees native to the Iropics are commonly used, such as avocado, coffee, annatto, orange, etc. In some cases, il is possible to ~se ~pport trees wbich. themselves, can produce clsh crops. In MldagasclT. caSUlfUll pme trees Ife:lso widely used. In Dlost pianlations, the support trees Ire planted before the vlllUlla piants ( 12). In greenhouses, vanilla can be grown faUing dowli or hanging.

P,-opagalion. Vanilla is commonly propagated from eutlin~ usually with 8-12 nodes. Small cuttings will produce vines, bUI tbc larger thc cuttmg, the faster the plant will flower. Thc situplest way to plant is t~ lay thc cu~tiog 011 the ~~und and cover it \vilb thick laycrs of mulch. However, tillS propagatlo~ ~elhod lmuts .tbe amount of pia lit materiallhat ru.ay be used. Also, the many orlgms of the cutllugs represcnt genetic vlriabili ty. .

A vine becomes productive after 3-4 years, depending upon the weather and soil conditions, and the healtb of the vine. 111e vine [Jowers ooce a year f~r 5 or ~ successive years. After 8 10 10 years, productivity goes down. Drought, m~~clent mulch, overexposure to sun, and over-pollinatioo can eootribute to ~ redUClion 10 the productive period of the vine (4). WIlen planning a new plantation, one has to account for the life expectancy of the vines. An hectare of land cln hold about 4000 productive vines. The avcuge yield is 1.5 to 2 kg green beans per vine. A nJ.;lture good quality bean weighs betwcen 15 to 30 grams ( 12).

"loweriog, "'ruit Set, G rowtb li nd Maturation. II is well known tbat vanilla vines need to reach a certain maturity before starting to flov.'tr. Appareotly Ibis is to accumulale growth factors or nutrients needed for blossoming. The fa ctors affecting the timing and abundance of flowers are nOI fully ~derstood .. However, according to Childers et . al. (4) drought , le~perature, prumng and .the SI1.e of the . original cutting may In influence thc flowel1ng. Removal of 4 to 6 Inches o f the aplcRI bud 6 to 8 weeks bcfore blossoming prOUlOles flowerin g.

Vines usually blossom for I to 2 months. The flowers open early in Ih.e morning and last for one day. PoUinat ion must be done Ihe same day. Also. ill a raceme (the flowcr c1ustcr) only one f10wcr opeos per day and about I ~ to 20 flowers open in daily succession, although nOI aU of them \vill be fe rt ile. It is desirllble 10 pollioate 10 obtain between 5-10 beans because over-pollinltion wi ll result in small

beaos and a short production life. Because self_pollination is impossible to achieve (l'ig.3), hand-pollinalioll must

be used to ohtain a comolercial crop. Hand-pollinat ion is donc as originally deS(;ribed by Edmond Albium in 1841 in Reunion, whicb consists or. reruovnl~ Ihe rosteliuDl blck so that the pollen hearing anlher can be pressed agamst the Slig.ma. Once pollination takes place, Ille development of the pod ensuing with the enlargemelll of the OVlry is completed in about I and a half to 2 months (3).

SI'ICK"" nAVOR CII £.\II~~rRY ,\ NI) ANTIOXIIiANT 1'lI.tH'ERTIES

Afler the fruit hIS amined its full size, it takes an additional 5 to 6 months to mature and ripen. l>tlling the maturation period, the dry weight and the protein content illcreases. Manyother physiological changes occur, U yet orunknowD nature, Ihal resull in the productioll orflavor precursors,

. Curing. The green, mature van illa bean has no flavor or vauiJia aroma. If

the bean stays on the vine, the typical vanilla flavor will develop after a prolonged ripening period. 110wever, in commercial practice, the characterist ic fla vor and aroma of vanilla beans is due to chlnges lak.ing place during the curing process (2).

The curing process consists offour steps: I. Killing 2. Sv.-eating 3. Drying, aud 4. Conditioning.

Killing designates a process aimed at abolishing tissue aud cellular activity but retaining heal. Tolerant enzymatic activity is imponaru fo r the curing I,roeess. The ruost common processes are hot water killing, sun killing, and oven killing. Beans are placcd in a wire basket and dipped ill 65°C waler for a few minutes. Killing by heat can be done also in an oven or by exposure to the stlU for a llrolonged time. 1bere is a patent ro r using free7Jng as IUOIher killing method ( [). TIle killing proces.~ apparently entails the breaking or cell membranes and mixing ofprl!Viously compartmentalized substrates aud enzymes.

Killing is rollowed by "swcating~, which allows excess moisture to escape. This reduces bacterial and rungal spoilagc, and yct Icaves enough moisture for enzymatic activity. Tbis is the most impon ant step. Glucosides, like giucovaniliin, are broken doml to vanillin. 'Ibis is the step by which the t)Tl ical characteristics or the vanilla bean flavor, aroma and color are devcloped. The duralion orthe sweating process is between 7· [ 0 days.

Following the sweating step is "drying", which reduces the moisture content rrom 60· 70% to 20..30%. 11le rcduced moisture content is t)"llical ror a good quality cured bean and helps to eliminate any microbia l activity and to secure a long shelrtife. 'I1Le allloIDLt or moisture also affects appearauce, flexibility, and quality of the beans. In this step it is necessary IIOt to obtailltoo hot a temperature, because some of the flavo r and aroma components can evaporate or break dov.ll . Tbe drying lakes 7·10 days in the sun, 2· 3 hours a day. Oven drying can bc employed at 45°·50°C. Overdryiug of the bean will result in In inferior quality bean.

·I"he list stage is "conditionillg", or aging. 'Ibis elll last from a month to severa l months, Duriog this period, the vanilla bean obllins the typi~l ·rullflavo r· .

Chemical reaclions, such as ox.idalion and hydrolysis are the main evcnts (12).

11. Biotechnology Production

Recent developments in ]IISnll issue culture and the need to gain control 011 hean quality and yield turns our attention to this method as Rilother approach to solve SOUle oflhe problems associated with {he agronomic producl ion of vanilla (5). Plant tissue culture calL be used ror three different objectives: 1. micropropagat ion 2. production of secondary products and 3. study ofbiosyllIhetic pathways.

-

4. tLWKIN· t ·R£NKEI. & UOkN Vallilla 35

M icropropagation. In IUOst vanilla tbat is cultivated, conmlCrcial propagalion is done by cUll iug. Several repo rc s have shOWlI the Ilossibility of using tissue cuhure tecbniques to obtain clollal propagation and disease-rreo; vanilla plants (8,9). We used a different approacll using seeds as starting material. We were able 10 g~rlllinatc vanilla seeds ULLd ei' controlled stelile conditions in ft petri dish. 1bc seed l)rerCrs to gemlinatc on the beau flesh (Fig. 4). YOIUlg grecn be~n s., I to 2 months old afi.cr being pollinated, lVere surface stcrili7.ed with I S% bleach. The beans wcre cu~ LntO segments aud placed on Gamborg's, n·s basalllledium (7) rree ofhornl?lIe \\~th the addition or cefolaxin and vancomycin It concentr-~t ions of 100 Illg per hter. Ibe bean sections were subcultured to I clean plate every two weeks. Gennination of seeds embedded in the bean sections staned after 3·6 mouths. The emerging embryos lVere tl1Lnsferred to rifts (mesh size 25 microns, Sigllla Chenucai COUipany, St. Louis., MO), using the same m.:diuru without agar (Fig. 5), Each embryo was CUI illto a rew small pieces mt eacb subculturing. After a few mouths, plsllt lets \vere ronned and moved to the greenhouse for hardening, using orchid mix. In an establislu:d lab, the number ofplantlcts that ca ll be Ilroduced is unlimiled. Seeds rrom young beans germinat.: fhtcr than those from IIUltu re beans probably because tILey have not become dOflll\lut.

The t echniqUt: just described gives us the lools to produce a vast nUilLber IIr disease.free plallliets that lIUly be used to select for dcsi rable {raits, such as high yiL;1d. early flowcrin g aud superior navor aud arOOla orbeans.

Secondary )'roduct. In the last rew years, there has been an altempt by I:scat!t.:LLct.il:s Corporatiollto conune rcia!i7.c vani lla flavor by using plant cell culture (8). I\ \."..:onlmg 10 the published da ta, they oblained a yield of nearly 100 mg vani llin per li te l ~f . culture, or roughly 14 mg vanillin per one gram ofdl)' wcight ofeell m~ss .. I tl ~ ILkd~· that a several rold increase in vanillin yield would be necessary for pmd uctlOll to bo.: ecoDomical. Another but alttmative biotechnological approach for the prUllucti l)LL "I" vanilla is being investigated by Westcott et 81 (1 3). They IISC vanilla. roots to c~lIl\'~ rt rerolic acid to vanillin. 11Le limiting step here is where one can obla Ui LL atLL\'~LI lel'u ll..: acid fur the biocollversion. We used embryo cult\Jre, reasoning that dillercnti:lt..:d tissue win be \I1Ore conducive than cell suspension to produce secondary metabolites. We were able to grow tbe embryo in up to a 10 L biorcaCior with a m.arine impelh:r at 100 rpLIl, because the erubiYos are sensit ive to high sllear. The culture prouIKcd a larger number of the flavor COIllJJonenls thl t Ire in vlnilla bean (Fig. 6 (a) and (b». The profile of the flavor components in cuhured tissues is different t..han in the bcan . It is nOI expected that undifferentiated cultured tissues will produce the same ~ro~le and similar proportions ofnavor components that are produced in the bean ",lllch I.S differentiated aud a specialized organ. We, thererore, concentrated on the prodUC\lOll of vanillin, which has the highest ecouomie value. We found Ihat embryo tissue produced up to 0, 16% vanillin and that 8 vanilla beau produced betwe<::ll 1.3%. . vanillin ou a dry weigllt basis. At present, it appears that tissue culture IlroductlOIi IS appreci~bly lower and may not be economical.

, , .j

J6 SI'ICES: n .... VOR CII EMISTR¥ ANI) ANTIOXID .... NT PROl'ERTlES

Figure 3. Cluster o f Vllnilla Flowers.

Figure 4. Germinllting Vanilla Seeds on the Bean Flesh.

4. I IAVKIN.nu-;r.IKt:L <'II DORN Vanilla

The germinating embryos were held on noal ing fafts to maximize access to air and nutrients. By the end of 60 days, the resulting plantlets were transferred \0

a pot with orchid mix in the greenhouse for hardening.

Figure 5. Growth of Gcrminaling Embryos.

J7

Vanillin UiosynthcCic: r ath wlIY. Using the approach described previously, we grew the embryo culture 10 study the bio~~l l hct ic pathway of vanillin to flnd out the limiting step(s) in the production or the compound. After growing the cuhurc and analyzing most of the exlracllble material, we propose that vanillin biosynthesis Slarls from chain shortening of p.hydroxycoumaric acid, resulting in the production of p.hydroxybcllzoic Acid, p-hydroxyben zyl aldchyde and p-hydroxybenzyl alcohol. We observed an accumulation of p-hydroxybenzyl alcohol up to 10% of tbe dry weight, suggesting thai the enzyme Ihal catal)'7,.es the hydro."<ylllion of p-bydroxybenzyl alcohol 10 3, 4.dihydro."<ybenzyl alcohol is not active. In homogenized tissue, IDOst of Ihe Il-hydroxybenzyl alcohol is convened 10 3, 4-dibydroxybenzyl alcohol aner a fcw hours., suggesting that in intact tissues the enzyrne and the substrate are companmentalized. Feeding experimeuls to the embryos with J , 4-dihydroxybenzyl aldehyde, a by-product ofJ,4.dihydroxybenzyl alcohol, led to the accumulation of vanillin, indicating thaI methylat ion is not a limiting step.

GTven this ill fomlltion , understanding the limiting step call lay the foundation for future work. fly isolating and purifying the em:.yme Ihat bydroxylates p-bydroxybenzyillcohol and cloning the gene, we lilly obtaill a culture with I high yie ld Ofvluillin that call be economically produced.

Future Ollportunitie! For Hiotcchnolgy_ We bave shown Ibat microprop'gation of vIIl i11a is Ilossible with the use of tissue culture. This technology lUay be developed further to select for cultivars with sullcrior traits. Illlllortantiy, it may be used to propagate transgenic tissues tunsfected, fo r example, "ith genes to increase the yield of vanillin, induce early nowering or disease resistance.

SI' ICES: HAVON: CII B IISl'N:Y ANI) ANT!OXIIlANT I'JW I'ERTlf:~

HPLC chromatogram of embryo culture extract.

A

1 . 00

o.so

• i • , • • • ; • • ; • • •

• •

~ 0.16t or 0.".

;

; • 0 so "

• • .. , , 0 . 40

, 00 < . )( H)l M inu tes

Figure 6A & 613. HPLC Chromatogram. Isocratic separ-.ltion was used with the mobile phase, 85% acidified water and 15% addilk-d melhanol (1 .25% acetic acid), conlrollt:d by a Walers 600E system. A I'hcnomenox Selectosil CI8 column (250 x 4.6 111m) wilh 5 ~m panicle size was used. The peaks were detcclcd [II 280 nm using a Waters 994 photodiode array.;

4. IL\VKIN-FRENU:L & l)ORN Vanilla .19

ltPLC chromatogram of Bourbon vanilla beans extract (I fold). i •

" ,

• • l •

, 00 , •

• 1.)l ~I 1>." . . . , , .. 4 . 00 • 0 , g • • ,

::I 0 0

)< 1 0 1 M'"'' I . .,. •.

Figure 6. COlllimlf!1i

'" SI'ICK.<ii, FI..AVOH CIIE"'lI ~"RY ANII A .... TIOXIllANT I'ROl'EIHI K<;

/{rfcrcn CC$ I. Ansaldi , Gwcl1sdc; Gil, Gerard ; Le PClit, Jean, u.s. PalmI 11 4,956, 192,

Sell· 11 . 1990. 2. 3 4.

<,. 7. , 9. 10.

" 12.

D.

Arana, F.E. Circular No. 15, I!Imhillglon, D. C , USDA 1945 Ch~1I1crs, N. r:.; C~bes, H. R. Cirell/ar No. 28, Washing/OIl, D. C, USDA 1948 Childers, N. " .; Clbes, II. R. ; ~Icn'.nde:c-Medina E. In rhe orchids a Scil'I,lfific slin-ey; Withoer, C.L. Ed.; ROllald PrcS:S: New York, 1959 Colhn, I-I. A. ; Watts, M. In Halldbook O/pklllf celf cullllre • techlliques for I'mfJ(lgatlOlI alld breeding Vol I Evano D A . Shan, \" " . A . P _ . . . .., . " ,1,. " . ..... , . nmurato, . V.: Yamada, Y. Eds; Macmillan PUblishing Co. : New York, 1983 COiTcll, D. S. J &0,.. Bol. 1953, ', 29 1 Gamborg, O. ~.; MiII~r, R. A.; Ojima , K. Exp. Cell Re:;. 1968, 50,148- 151 Knuth, Mark E.; Sahal, Om P., U.S. P(I(ellf #5,057,424, OC(. 15, 199J and U.S. Pa/t'II/ 1I5. 068,J84, NOII.l6, 1991. KOll01l0\IIiCl., 1-1.; Janick. I. lIor/sciellce. 1984 19 5g Philip , V.J.; Naillar, S.A.Z. J. 1'1011/ Physiol. 1986: 122,2 11 PUlesegJove, J.W.; BrowI!, E.G.; Green, C.L.; Robbins, S.R.J Spices Vol 2 1981

~na~ive, ~S . In Spices, herh$(l1/d edible jilllg; ; CharalamOOus, G. Ed. ; ElscVler SCIence n. V.:AmstcrdalH. 1994 Westcott, R.I.; Cheetham, P.S.J.; Barraclough A.I. Phy /ochem. 1994 35(1) 135·138 " ,

Chapter 5

Onion Flavor Chemistry and Factors Influencing Flavor Intensity

William M. Randle

Vcparlment of liorticulture, 111 1 Plant Science Buildin g, Unh·ersity of Georgia, Athcns, GA 30602-7273

Although onions are an importam vegelable and halle nUlriliollal IIalue ill diets around the world, Ihey are primarily consumed for Ihcir distinctille flavor or their ability to enhance navors in other foods . Onion flavor is dominated by organosul fur compounds aris ing from the enzymatic decomposition of S·alk(en)yl-L-cysteine S-oxidc navor precursors following tissue disruption. Compounds arising frolll prccursor decomposition, such as the lachrymatory factor and various thiosulfin.ates gille onions their characteristic navors. Sul fate is absorbed by lhe planl and incorporaled Ihrough cysteine 10 glutathione. From glutalhione, sulfur can proceed Ihrough several peptide pathways and terminate in Ihe synthesis of one of three flavor pre<:ursors. Plallor intensity is governed by genetic factors within the onion and crwironmenlal conditions under which the onions grow. Onion cul tivars differ in the ability to absorb sulfate and in the efficiency of synthesi7jng fla vor precursors. Inc reased sulfate fertilit y, higher grow ing temperatures and dry growing conditions all contribute to increased navor intensity in onion.

Onions (AWl/lIZ CI!(IO t .) hallc world-wide imparlance, ranking second among all IIegetahles in economic imporlanee with an estimated value of $6 billion (I) . Although onions cont ribute significantly to the human diet and howe therapeutic properties. they arc primarily consumed for their unique nallor or for their ahility 10 enhance the n~vor of other foods. Onions arc an ancient IIcgelahle and can be traced back through archeological records and early wri tings. OniOllS were used as food, as medicines. in mummification. in art . and as spiritual objects (2) . Interestingly. onion~ hallc been domesticated for so long that they no longer have the

~ 1997 American ChcmlCilI Socicly

42 SI'ICK"' : flAVOR CII EMISTRY AND ANTIOXIO,\NT PROPERTlFS

ability to exist in [he wild and require human intervent ion for survival In contempor.try society, onions weave (heir way through OUf diet and are consumed daily by most people. United States Pl;: f capita consumption of oniom in 1995 was approximately 18 pounds compared to 9 pounds in 1975 (per. com., National Onion Association).

Onion Flavor Chemistry

While compounds such as the water·soluble carbohydrat.:s (sugars) and organic acids can contribUle [Q the sensory experience when consuming onions, onion flavor is dominated by a special class of biologically active orgaTlOsulfur compounds (3-4), Intact dry-bulb onions have lillie onion flavor or aroma, Flavor and aroma develops only when the onion is damaged or cut and flavor precursor compounds undergo enzymatic decomposition to form a variety of volatile sulfur compounds which give onions their ch~racteristic taste and aroma, The first sulfur compound associated with onion flavor was identified in the 1890's (5). Pioneering rese~rch in th is century among scientists such; as Chester Cavall ito, Authur Stoll, Ewald Sl:ebeck, nolx:l laureate Artturi Vinanen, Sigmund Schwimmer, Mendel Mazelis , George Freeman, Jane Lancaster , and Eric Block, has char~cterized many of the sulfur compounds contribut ing to fl~vor, the biosynthetic pathway for flavor precursor development, and the process by which flavor develops once the Onion is cui or cooked,

Onion flavor precursor formatiOn begins with the uptake of sulfate (SO/) by the onion, its reduction to sulfide, and suhscquent assim ilation to cysteine by lightdependent reactions in the leaves of the plant (6), From cysteine, the sulfur can be further metabolized to foml other sulfur·conta ining pl~nt compounds, Sulfur'S proposed entry into the !lavor pathway is via glutathione, Early studies using radioactive isotopes suggested tha t sulfur passed through glutathione and was incorporated into S-2·carboxy propy l cysteine or S-2-carboxy propyl glutathione, eventually te rminating into S·propenyl cysteine sulfox ide (7), Using radioactively laheled sulfate in pulse chase experiments, Lancaster and Shaw (8) demonstrated that sulfur was first incorporated into ),-glutamyl peptides as biosymhetic intemlediales prior to terminating in the S-alk(en)yl cysteine sul foxide precursors,

There are 3 S-alk(en)yl cysteine sul foxide !lavor precursors in oniom: S-(E)I ·propenyl cysteine sulfoxide is usually found in highest concentration and is responsible for tearing and pungency assoc iated with onions; S·methyl cysteine sulfoxide which nomlaJly occurs in lesser concentrations; and S·propyl cysteine sulfoxide which is gener,dly found is the lowest concentration (9), A founh precursor, S-2-propenyl cysteine sulfoxide, is the predominate precursor of garlic, and fou nd in other Alfiurns, but it is not detectable in onions, The pathways leading to the synthesis of each flavo r precursor are not fully understood, nor do we understand the regulation of sulfur through these various pathways. Sulfur is thought to be transformed through several peptide intemlediate pathways, unique to each of flavor precursors COmpounds (f0; Fig. I),

The precursors are synthesized and stored in the cytoplasm of the plants's

S. KANI)U; OniIJTI Fla~ur Chemistry

cells (l }) . Al1 iinase is compartlllentalized in Ihe ct:JJ's vacuole. When lhe membrane of the vacuole is broken, alliinasc is released and the precursors ,IrC

broken dowll producing a chain of events. Primary products include shon-Jived imemlcd iatc compounds, such as the I-propenyl cys!cim: sulfoxide dcrivcd lachrymatory faelOr (LF, propanethial S·oxide), and other sulfeni!; acids from the different precursor specics. Other primary products arc compounds pyruvate and ammonia which are marc stable. The LF, common to only I·propenyl cysteine sulfoxide a(;(;umulating AlliulIIs, produces the tearing, mouth burn, and pungency scnsations (12). A series of thiosu lfinmes arc then formed which chMaclerize the unique flavors and aromas of onion. Early reports of di- and polysulfidcs and th iosulfonates were shown to Ix: "artif,lcts" of hot injection port and gas chromatographic column l13). 111C different flavor precursors give risc to diffen:nt thiosulfinates which impart dist inet flavors to the sensory experience (14). ror example, the propenyl/propyl th iosulfinates have green raw fresh onion !lav{)r, and the methyl/methyl th iosulfinates have a cabbagc note (Table I) .

The thiosulfinates then serve as the progenitor species of vinually all Iht: snlfur compounds formed from the cut plillll. These compounds are un~tab l c amI undcrgo disassociation and rearrangcment to form primary headspace vol~tile~ (initial products formed from cut onions at room tcmperature such as the thiosulfinmes), S(."Condary vola tiles (secondary products produced from the thiosulflnatcs ,11 rUOIll lCmperature), and secondary solution components (products formed when thiosu lfinates stand in so\tllion at room temperatuare) (15), Cut onions sitting on a kitchen counter or on a salad bar, therefore, change flavor over time. For a thorough discussion of cut onion compounds. see Block, 1992 ,

F~ctors Innucncing FJaYOr Intensity

Onion tlavor hltensity is governed by plam genetics and the environmental condilions in which the onion grows, In the marketplace, onious vary widely in sulfur-based tlavor intensity, Culti var Factors and Flavor Intensity , Some onioll'l arc very pungent 3nu aromatic while others can be eaten raw hy the average person with relativi! case. Onion color has very little 10 do with how pungent it might be , Red and yellow skinned Onions can range from being mild \0 very pungent, White onions, howel'cr, are usually only pungent to very pungent. Some onions, such as the Grano and Granex cult ivars can be grown mild with lillie flavor. Other unions, su~h ;lS the Danvers and Southport cuhivars, are very pungent while some, such as the Sweet Spanish eultivars, falllx:tween mild and pungent. The gt:netic potential of a Cl!itiv:n to ahsorb sulfur and synthesize the flavor precursors great ly determines how flavorful an onion will be,

While the her itability of !lavor prceursor accumulation inl)nions has not been determined, it is most likely a quantitatively inheri ted trait. As descrihed e:lI'licr, the biosymhetic pathway is compit:x with many peptide intenlleliiates, [n ;ldditioll, 11 proposed enzymes regulate compound synthesis in the llavur pathw:,y (/7;. Further empirical evidencc to support qu~ntitative inhl!ritan~t: of the l1avo)"

SI'ICt:S: F1AVOIt CH EMISTRY ANIl ANTI OXIIlANT I'IWI'ERTIES

Sti ll ale~Cystelne ~ g ·Glutamylcysleine > Glutathione

glutam~ GIYCC /

S.2.carboxy propylglulalhione

g.g I "t~ myl. S · pro pyl

~GIYcine g.glll lamyl-S-2-c a rboxy S ·melhylg!u1 .. thion.

propyl cysteine

1 1 cystelno ...:;:-

g -glutnmyl ·S· ' . propcnylcys teinc

g. glutamyl·S -mllthy tcys leine

1 g. g l yt~myl· S · p r opy l cysteine S .oK1<I",

g . glutamyl·S " ·propcnyl cys tc ine S · oKide

01 -01 lut~ rnyt . S ·m ethyl cysllline S · oxldc

S. pro pylcys ' e ine S·oKide

S ·(E)- ' .P ropeny cysteine S·oxide S·methylcysleinl! S ,oKide

Figure I . The proposed biosynlhctic pathway for S-alk(en)yl cysteine sul folt ides in onion~.

Tallie I . Sensory experience of d ifferent onion thiosulfinates and lachrymatory factor

ComI22!!Jl!1 Methyl methane thiosulfinate Me thyl propyl thiosulfinate Propyl methyl thiosulfinale Dipropyl thiosuHinate Propenyl methylthiosulfinate Mctbyl propenyl thiosulfinate Propcnyl propylthi05ulfinatc Thiopropanal S-oxide

Flavor NQte cabbage cabbage/onion cabbage/ollion green onion, chive cabbage, onion, metallic cabbage raw frcsh onion pungent, heat, mouth bum

S. RANIlU: Ollion Ffa~f)r CllcmLI'f ry 45

precursors is the continuOtis variat ion found for the different navor precuf ..... JrS within onion and the faCl that the growing environment greatly influences precursur accumulation (18). Qualitatively inherited trai ts, which are governed by one hI several genes, usually fonn discrete classes and arc unaffected by the environment.

Cuhivar differences can begin 10 be explained in several ways. Some eultivars accumulate 2 to 3 times the amou nt of sul fu r as others, and sulfur accumulation can be a key element in detennining onion fl avor intensilY (/9). If we were to assume that cuhivars assimilate the avai lable sulfur inlo the fl avor precursor"" with the same efficicncy, differences among cultivars CQuid be explaincd simply on how much sulfate enters the plant. However, cultivars differed in their efficicncy at moving sulfur through the fl avor pathway and in synthesizing the vari(lu~

precursors (20). The correlation, however, between total bulb sulfur accumulatiun and bulb pungency was poor among onions of broad genetic background, ranging between r = -0. 05 and 0.36 (2/ -22). Some mild cult ivars accumulate large amounts of sulfate, but do not accumulate correspom)ing concentrations of flavor precursor~, while pungent cuhivars will assimilate more of Ihe available sulfur into fla vor precursors (23). As an example. Sav~nnah Sweet . a mild eultivar, had OAO% total bulb sulfur (dry weight basis) and 4, 05 mg/g fresh weight of total flavor p re!,:ursor~. On the other hand . Rio Grande, it pungent cult ivar. had similar tota l bulb sulfur accumulat ion (0.43%), but accumulated 5.73 Illg/g fre sh weight total flavo r precursors (a 42 % inerease in total precursor accumulation).

The rat io o f the various fl avor precursors also differs among cul tivars. Flavor precursor composit ion and concentration can significantly influence fl avor and navor intensity . Some cultivars synthcsi1.c and accumulated mostly I-propenyl cysteine sulfoxide, the lachrymator and pungency producing precursor, but little methyl or propyl cyste ine sul foxide (24). Other cult ivars accumulate significant amount o f methy l ~ and propylcysteine sul foxide relative to the I -propenYl precursor (25). The different precursor ra tios give rise to different tastes and aroma . For example, if [otal precursor concentrations were equal, a cultiva r with a rat io of 12:4:2 I-propenyl :methyl:propyl cysteine sulfoxide would be highly pungent and [ear producing, while another eulti\'ar with a 6:9:3 rat io would be less pungent, and have significant fresh onion flavor. Why eul tivars differ in partit ioning sulfur into [he various pathways leading to the 3 cysteine suI fox ides or how environmental signals regulate synthesis among the pathways has yet to be dctennined.

Onion cultivars differ in alliinasc concentrat ion which may affect fla vor development and intensity . Pukekohe Long Keeper. a pungent cuhivar, had a 4 fold increase in bulb a11iinasc content compared to the mild Granex 33 cullivar (per. com .• J.E. Lancaster, Crop and Food , NZ). In addition, onion al1iinase decomposes the three cysteine su lfoxide flavor precursors al different ra tes (26). The kineti!':S of I-Propenyl cyste ine sulfoxide decomposition is quickest, followed by the methyl and propyl cysteij!e sulfo~ id es. /II vivo. I -propenyl cyste ine sulfoxide decomposilion was almost instantaneou~ and complete, whereas methyl cysteine sulfoxide ~nd propyl cysteine sulfoxide were decomposed to a much lesser extent (27). Betwcen 60 and 80% of the methyl cysteine sulfoxide, and approximately 50 % of the propyl cysteine sulfoxide was left intact in onion macerate , even after 2 hours following maceration

46 SPICES, FlAVOR C1IEI'oI ISTRY AN[) ANT/OXIIlANT l'IHH'nfI'lES

3

--"' '" 2.5

'" ~ "' ~ 2 '" ~ '" -'" 1.5 .s ~ ~

1 0 ~ ~

" U

'" 0.5 ~

CL

0 0.10 0.48 0 .85 1.60 3.10

Sulfur Fertitity (meq/titer)

F igure 2. S-alk(en)yl cysteine sulfoxide flavor precursor accumulation from mature onion bulbs in response to increasing SU](Uf fe rtility from appl ied nutrient solutions. Solid bars are I -propenyl cysteine sulfoxide. Lined bars are methyl cysteine sulfoxide. Dotted bars are propyl cysteine sulfoxide.

S. RANIlI.~; Onitm 1'lal'or Chemi.l'lry 47

of bulb tissue. Cultivars ais() diffe red in the degree of decomposition of each precursor (28). -nlcrefore, onion cuhivars diffe ring in fl avor pretursor composi/ion will vary in flavo r intensity do 10 their precursor conccll1ralion and degrt:c to wh idl lhe precursors afC decomposed.

Flavor Distribution. Flavor is unequally dimibU!cd within the onion planl :md is dependent on onion ontogeny _ Within the bulb, Ihere is a flavor gradient. The highest concentrat ion of precursors occurs in the inlerior hase of the bulb, while the lowest concemration of precurSors aTC in Ihe top outside scales (29) . As the plant and bulb grows, flavor first increases in intensity , but then decreases as the bulb swells to maximum size and matures (30). Onion flavor and intensity also change during Storage. Some cuUivars increase in flavor inten~ ity during storage while others decrease (31). The ratio of lhe d iffcrem precursors can also change duri ng storage . In one onion cuhivar, the l -propenylthiosulfinates significantly increased in concentration during six months of storage while the methy l methane lhiosul rin~ tes

completely disappeared (per com. Norman Schmidt, Dep\. of Chem., Georgia SOuthern University).

Environmental Factors and Flavor (nlensity. The environment in which an onion grows and develops will greatly infl uence how mild or pungenl an individual cullivar will be . Yearly and locational flavor intensity differences among onion cultiv(ifs have been known and reported (32). When isolated individually, environmental factors such as sulfate availability , temperature, and water supply afree! flavor intensity.

Sulfate Fertility . Sulfate availahility greatly influences onion flav or inrcnsi ty (33) . When onions were grown with sulfur fenility levels ranging from deficient to luxuriant, the concentration and ratio of the three flavor precursors changed (34: fi g 2). At sulfur fert il ity levels which cau~ sulfur deficiency symptoms in onion plants, methyl cysteine su lfoxide was the dominant precursor while I -propenyl cy"wine sulfoxide was a minor precursor. As sulfate feniJity increased to ndequate Jt: vcls, I-propenyl cysteine sulfoxide increased in concentration and methyl Cy~ l ci ne

sulfoxide dt.-creased in concc:ntralion. At luxuriant sulfate fcrlility , i -propt:nyl cysteine sulfoxide was the dominant precursor while methyl cysteine sul l"o xidc became a minor precursor. Interestingly, total precursor concelliration was similar from those onions grown al defi cient sulfate fenility and those grown at luxuriant levels. I! appeared that when onions were stressed for sulfate, the methyl cystd nc sulfoxide biosynthetic pathway became a st rong sink for the available sul f"J[ and large amounts of methyl cysteine sulfoxide aeculllulmed, even at the cxpcnse o f plant growth and bulb developmem. The prupanethial S-oxide and !hiosulfina te concentrations follo wed the same pallem as the cysteine sulfoxide precursor concentrations when responding to increasing sulfate feni Jity (35). Scnsory evaluation from professional tastt: panels confirmed the chemical analysis. As available sulfate levels increased in the growing envirOlUnent, se nsory notes ~\l ch ;IS

S I'ICES; HAVOI{ CII I~M I ,!,"TI{Y ANI) hNTIOXI ,)A"'T I'ROI'EI{Tr ES

Tallie II. Aroma and navor notes from 'Granex 33' onions grown at increasing .\III"a le fertili ty 11'1'1'15. Higher numbers represent more intense nOll'S.

Auribute low S mod S high S exeess S

Green aroma 6A 6.0 5.' 6.6

Pungency aroma 7A 8.0 7.2 8.4

Total arom~ 8.3 8.' 7.7 9.5

l1iuer 3.3 5.0 4 .1 6.7

GrCCIi Flavor 6.8 6.' 6.2 7.3

Ileat 4.9 7.2 6.6 8.1

Pungent navor 5.7 8.1 7. 2 9A Sweet 7.6 6 .0 6.2 4.8

Total Flavor 8.2 9.5 9.2 ll.l

Total Sulfur Flavor 5.2 7.8 7.0 8.2 .' .

Uoilcd Onion Flavor 4A 3.1 3.7 3.

Cabbage Flavor 3A 3.1 3.1 3

Fn:sh Sulfur Hwor 7.0 7.5 6,7 7.5

Fruit Sulfur Flavor 4.7 4.6 4.6 4.7

Hydrogen Sulfide 1.4 1.8 1.5 22

Rubbel)' Sulfur 2.3 4.0 3.1 4.4

S. IthNI>I,E 011;011 n avor Chemistry

a5tota l aroma. biuerness, heat, pungency and total .~ulrur navor also increased (Table 11).

Grou-iug TemlK!ra ture. Tempentture is important for onion growth ill lll development. Minimum temperalUres for bulbing a~ around IO"C and reach a maximum around 35"C. In an early slUdy . although increasing tempcratun: increased volatile sulfur compounds in onions. the author was unable 10 specify if navor differences were due to temperalUre re lated plant growth (i.e., the faster tILe plant grew, the more pungent the plant became) or to the direct effect of temperature on n ilvor development (36). Recently , a study was completed where plalUs eX]lo~eu to 4 different growing tcmperatures we~: I) grown for a specific length of time and harvested when the plants were of different developmental age. or. 2) grown 10 maturilY which took increasing lengthli of lillie as the tempcracure dttrea.'ied. In both cases, increasing the growing lemperamre from IU'C to 31"C increaSCtl the pungency ( a~ measured by enzymatically fonn pyruvate) of the onions. and the increase was lincar in response to increasing temperature (Figure 3). The hotter the growing conditions. the more pungent the onions will be.

Water Supply . Growing onions under dry cond itions will also increase bulb pungency compa~d to onions grown under well irrigated conditions . When onions werc grown under na tural rainfall or supplemented with irrigation WOlle r, the nonirrigated onions produced a higher volatile sulfur content compare<! to irrigated onions (37). or produced increased flavor strength as measured hy volatile headspace analysis. enzymatically developed pyruvate, and .'\Cnsol)' evaluation (38). II.s bulb size was smaller in the non-irrigated plots. it was thought that increased flavor strength was due to a concentration of the flavor prc<:ursor compounds in smaller cells. The exact mechani~m for flavor increases in water-stressed plants. however, is yet to be detennined.

Water usage and sulfate uptake by onions was also poorly correlated (r = O.09; Randle, unpublished data). Whcn plants were grown hydroponically to detemline sulfate uptake requiremcnts o\·er lhe course of the growing season. water usage was greatly affected by daily differences in rolar radiation while sulfate uptake was unaffected. TIle greater the solar radiation. the more water was transpired through thc leavcs.

Summary

The chemistI)' of onion navor from sulfur compounds is quite complex. The hiosynthetic pathway leading to the three foml.~ of S-alk(ell)yl-L-cystcinc S-o.lide navor pre<:ursor synthesis is eomplicatcd arKI still being developed and proven. Onion cultivars differ in thei r ability to synt hesize the navor precursors and differ in the ratio of,precursors synthesized Each precursor gives rise to sulfenie acids and thiosulfinates which define different fla vor experiences and navor intensity . The environment in which the onions grows is also important in detennining navor intensity and composition. Increasing sulfate fertility. increasing the growing temperature . and/or decreasing the water supply will increase onion flavor sirength.

-OJ '-' :J

u 0(3

5 -

ro 3 -o!d > :J ~

>"-

SI'IC":'" ~· I .\vOM CHEM ISTRY ANI) ANTIOXIIJANT l'IIOI'i<IO"IES

35 days of bulbing

To maturity

2+---------r-------~~------_4 10 17 24 31

Growing Temperature (Celcius)

~; ig ure .3. Oni~n pungency (umo! pyruvale per Illl of o nion juice) in response II) mCICaSltli; growlIlg temperalures. Solid line reprcsenls planls g rown allhe diffe rcnt Icmrer3111res for 35 days in a bulbing photoperiod . Dashed line represenlS planls grown 10 IllHlurilY at Ihe different lemperatures.

5. MAN!)I.!': 011;1)11 FIMor Chemi!;try 51

Li terature C ited (I) FAD, Prodllc/ion Yellfbook; Food and Agricuhure Organizalion , Rome . ... 7, 1994 . (2) 1·landl , P . Ill: Olliolls and Alliell Crups ; Brew~ler , J . L.; Rabinowitch, II .D .. Ed. CRC Press, Inc. l10ca Ralon, FL, 1990, Vall ; . (3) Block, E. Angew. Chem. 1m. Ed. 1!)92 , 31, 1135· 1178 . (4) Darbyshire , n.; Steer , U: r. In: Oniolls and Allied Crops; Brewsle r. J .L.; Rabioowi tch , 11 .0 ., Ed . CRC I're~s, Inc . Boca Raton, FL, 1990 , Vol 3; 1-16. (5) Semmler , F.W. Arcll. PJwrm. 1892 , 230,43 ... ·443. (6) Laocaster, J .E.; Boland , MJ . In: Onions allli Allied Crops; Brewster. 1. L. : Rabinowilch, I-I. D., &I . C RC PreSS, Inc. Boca R:lIon, F L, 1990, Vol 3 ; 33·72. (7) Granroth, B. AntI. Acad. Sci. Fel/n. Ser . 1970, A2 154, 1-71 (8) LancaSler, J .E .; Shaw, M.L. Phytochemistry. 1989 ,28,455-460. (9) Randle, W.M. ; L:mcaslcr, J .E .; Shaw, M.L.; SUllon, K.H. , Hay, R.L., Buss:ud ; M.L. 1. Amer. Soc. lIort. Sci. 1995 , 120, 1075· 1081. (10) Block, E. Alisew. ('hem . /111 . Ed. 1992 , 3{, 1135· 11 78. (1/) Lancasler, J . E.; Coll in, 1-I .A . Plant Sci . l..ell. 1981, 22, 169· 176 . (/2) Block, E.; Naganathan, S.; P1.Jlman, D.; Zhao, S. 1. Agrc. Food Chell!. 1992. 40, 2418-2430. (13) mock, E. Angt'w. Cllt'm. 1111 . Ed. 1992 , 31, 1135· 1178 . (14) Dlock, E.; NaganallulII, S.; P\,Jhn~o . D .; Zhao. S. 1. Agre. Food ('hem. 1992. 40,2418-2430. (15) Block, E. AlIgf'll-'. CJI t'm. 1m. Ed. 1992 , 31, 1135· 1178 . (16) Block, E. A"Kl'w. Cllt'lII . 1m. I:.i l . 1992 , JJ, 1135· 1178 . (/7) Whilaker , 1.R. Adl-'. F/JOlI Rn. 1976, 22, 73-133 . (/8) Randle , W.M.; LancaSler, J .E.; Shaw, M.L. ; Sullon, K.H., Hay. R.L; Bussard; M.L. J . Amer. Soc. Nort. Sci. 1995 , 120, 1075-1081. (l!1) Randle, W.M. Euphyrim. 1992 ,59, 15 1-156. (21) Randle , W.M. Ellphylim. 1992 ,59, 15 1· 156. (22) Randle , W.M.; Bussard , M.L. 1. Amer. Soc. lion . Sci. 1993, 118, 766·770. (21) Randle , W.M .; Laneasler, J .E.; Shaw , M.L.; Sulton, K. H .; Hay. R.l.. ; Bussard; M.L. 1. Allier. Sot'. Hon. Sci. 1995, no, 1075-108 1 (24) Thomas, O.J .; Parkin, K.L. 1. Asr. Food Chell! . 1994 , 42 . 1632-1638. (25) Randle, W.M.; uneasie r, J .E.; Shaw, M.L ; SUlion, K.II., 11 3Y. H. L. : Bussard ; M.L. 1. Amer. !We. lion . Sci. 1995,120, 1075-1081. (26) Schwimmer, S .; Mazcl is , M. Ar('/I. lJi(x:hl'm. and lJivphy . 1963. tOO, 63·73. (27) Lancaster, J .E .; Shaw , M.L. ; Randle, W.M. PrOf'. Natl. Oniun Res. Conj. 1995 , Madison, WI , 53·58 . (28) Lancaster, J .E .; Shaw, M.L ; Randle, W.M. /'roc. NlIIl. Olliull R(~.I" . COli/. 1995 , Madison, WI, 53·58. (29) Freeman, G.G, J. Sci. FOUlI AKrir. 1975,26,471 -481. (30) unCasta, J.E .; McCallion, B. Il . ; Sh:IW, M.L {)hysioll'lalll. 1986 . 6r,. :! lJ3· 297. (3{) Kopscll, D.E. ; aandle. W.M. l/ortSril'llCl! . 19')5 , 31.766.

-, ,. SI'I t:K>.;, F1AVOIt CHEM IST RY ,\N IJ ANl"JOX II)ANT l 'IU>l'~:ln l F,S

V':l l ~ IIK:~~lcr. J. P. .: Reay. 1'.1'. ; M~nn. J .D. : Bcnncn , W.O.: Scc.lcoic, J .R. New Z"o/(Illd J. £'.1'. ARrie. 1988, 16, 279-285 . cJ3} I:rccman, G,G,: Mossadeghi, N. J . Sci. Food Agrk. 1970,11, 610-61 5. (N) 1{ 'II1Ulc . W.M.; m ock . E.: UUlcjohn. M.; !'ulman. D.; Bussard. M .L. 1. ,·lgri<.', Fond (.1rt!lII. 1994, 42. 2085-2088 . US) Randle, W.M .: i...1rlCaSlcr. J .E. : Shaw, M.L. ; Suno n, K. II. ; Hay, R.L. ; lIu ~~anJ: M.L. J. Amer. Soc. /lor/. Sci. 1995 , 120, 1075·1081. U(j) Pl:llcn;u~, 11. 1 . Agrie. Nu. 1944.62,371 -379. (J7} I'lalenius, II . J. Agric. Rr:s. 19.t4. 62. 37 1-379. • (3S) Freeman, G.G.; Mossadcghi, N. 1. 11Qrt. Sci. 1973,48,365·378 .

Chapler 6

Contribution of Nonvolutile Sulfur·Containing Flavor Precursors of the Genus Allium to the Flavor of Thermally Processed

Allium Vegetables

Tun g- lls i YII

Department !If .'ood Engineering, Du-Yeh Ins titute of Technology 11 2, Shan-jea u Road, Da-Ts uen , Chan g- UwlI , Tniwun , Republic of Chiml

This article discusses the contributions of nonvolat ile sulfur-containing flavor precursors of the genus Allium to the flavor of thermally processed Allium vegetables through two approaches. In the first approach we have analyzed the volatile compounds generated from thermally processing blanched Allium \·egctalJles. In the second approach we have analyzed the volatile compounds produced during thermal degradaTion o r thermal interaction solut ions of S-alk(en)ylcysleine sulfo)(ides, the major nonvolatile sulfur-containing flavor precursors of Allium vege tables. A la rge number of volatile compounds can be generated from thermally processed blanched Allium vcgetables and from the thermal degradation or thermal interaction solutions o f these sulfoxides, and many of these volatile compounds can be fO\Jnd in thermally processed Allium vegetables. We have demonstra ted that the nonvolatile sulfur-co n(aining !Javor precursors of Allium vegctables can not only generate volat ile compounds through thermal degradation or thermal interactions but also t hrough the thermal degradation or interaction products of these sulfoxides 10 make important contributions to the flavor of thermally processed Allium vegetables.

Leaves or bulbs of Tile genus Allium, as represented by garlic (AI/illm .5Olil1ll11 L.), onion (Allilllil C"lXl L_), shallot (Al/iulII {l.sallulliclillI auct ), lind .... 'Cish onion (A llillm ji.WII/OJIIIII

1..), have been widely used aT home and in the food industry as vege tables or flavoring materi als because of their strong flavor properties. The characteristic aromas of the Allium species are mainly allribUied to the sulfilr.containing volatile compounds. Unlike the preformed volatile compounds, such as esters and terpene compounds in fruits and some spices which are biosynthesized as plants develop, thc volatile components of the genus Allium are released from their nonvolatile sulfur-containing flavor precursors, especially S.a!k(cn)ylcystcine sulfoxides, by lin enzymatic-mediated degradation that takes place when the plants are disrupted. Depending on the species. in the alk(en)yl

54 S I'IO:S: FlAVO R l:I IEM[STHY ANI ) ANTII) XLIIANT I' ROI' t:RTl ES

groups the sulfoxides are mainly a combination of allyl. methyl. propyl, and I·propenyl group (1-3).

Most of the research on the flavor of the Alliulll vegetables before 1991 focused mainly on the analysis of the volatile compounds of these vegttables, the formation of the volati le compou nds in the genus Al lium through the enzymic de,gradation and tht transformation of the nonvolatile sulfur-containing flavor precursors, such as the Salk(en)ylcysteine sulfoxides, and the stability of the thiosulfinates which are the enzymic reaction products of the S-alk(en)ylcysteine sulfoxides. There has been very li tt le research on how amino acid characteristics affect flavor precursors. Since Salk(en)ylcysteine sulfoxides are amino acids, they should undergo thermal degradalion or interactions with other food components., especiaUy reducing sugars. The focus of this research has been on lhe contributions of nonvolatite fl avor precursors of Ihe genus Allium to the characterist ic flavor ofth~mally processed Allium vegetables. especially gartic. shallot, and welsh onion We approach this research in two ways. First, we used blanched Allium vegetables to see if significant amou nts of volatile compounds could be generated from the nonvolatile sulfur-containing flavor precursors which wcre retained in Ihe tissues of All ium vegelables ancr blanchiug these vegetables during Ihennal treatment, especially baking and fry ing. In our second approach. we synthesized four important S-alk(en)y]cysteine sulfoxides: S.allyl-, melhyl-, propyl-, and 1-propenylcysteine sulfoxides. These sulfollides were Ihen subjecled to thermal degradation or interaclions wilh other food components in the Allium vegetables to determine what their conlributions were to the characteristic fla\'or of themlally processed Al lium vegetables. 111e following reviews some of our research.

Vo latile Compounds Genera led frolll Th ermally Tru ted Blanched Allium Vegel:lblu

To determine the potential contribution of the nonvola tile sulfur-containing fl avor precursors of the genus Allium to the navor of thermal ly processed Allium vegetables, we IIsed blanched Allium vegetables to which we applied Ihermal treatment. BlanChing Ireallllent can deactivate the Ilavor enzymes and retain most or Ihe flavor precursors in Ihe tissues of Allium vegetables. Since no signiftcanl amount of volatile compounds existed in the intact tissues of Allium vegetables, volati le compounds detected in thermally treated Allium vegetables could have been generated by thermally degrading Ihe nonvolali le precursors in the tissues or Ihrough the Ihermat inleractions of these precursors and olher components. especial ly sugars, in the tissues.

We were able to identi ry some impon ant volatile compounds in blanched and Ihermally trealed blanched garlic slices, shallot slices and Welch onions as found in Tables I, 11 and III , respectively, As you can see in these three tables, no significant amou nts of volat ile compounds were idenlified from blanched garlic (BG), blanched shallot (BS), blanched green leaf of welsh onion (BGL), and blanched white sheath o f welsh onion (BWS), These results prove thai blanching treatment of Allium vegelables can deactivate the Ilavor elllymes in these vegetables efficiently and inhibit the enzymic format ion of volatile compounds from the flavor precursors in Allium vegetables. Hllwever, you will note that in Tables I, 11, and III , baking or frying treatment of lhe

6. YU Jo1a'l'or of TI,ermally P~·tll All ium Vl'!:t1ablt.~

Table I , Some imponant volatile compounds identified in garlic samples Yield, ppm

Compound BBG· F13G · Compoullt!s Probably Genera ted from TIJerill al D" grlld :llion of

Nonvolat ile FlJn'or Precunmrs I-propene acetaldehyt!e melhyl allyl sulfide ally sulfide methyl allyl disulfide I ,2 -dithiacyclopenl-3-ene allyl disulfide methyl al1yl trisullide 3·vinyl-4 H- l ,2-dithiin 2-vinyl-41'1-1.3-di thiin allyllrisulfide

712 1.85 0.06 1.16 0,80 0.77 9 .14

153 OA9 0.31 0.52

10.55 25.24

1.31 8.99 1.26 2. IS

50.74 4 .34 1.44 0.72 0.66

liG'

2.20 0.70 nd' •

0.G9 0.04 006 2,40 0.17 0. 14

ed 006

1,2,3,4-tetrathiepane 221 S.98 nd dimethyltrithiepanes 2.42 7.63 003 Co mpou nds Probably Generated from Thenll allnltrllCl ioll$ of Sugars

a nd Nonvolatile "' avor Precursors 2,5 -dimet hylpyrazine 0.51 0.69

elhylmethylpyrazines 0.35 0 .72 3,S-diethyl.2-methylpyrazines 0,47 OA5 Comlloulltls I'robably Generated from T hermallnteractiolls of Upitls

and Nonvolatile f.'[:Jvor Precursors 4-heptenal 2-ethylpyridine 2-pentylfuran methylethylpyridine phenylacetaldehyde buytlbenzene pentylbenzene hexylbenzene bem:othiophene

0, 11 0. 12

ed 0.06 0. 10 0.06 0.25 043 0,70

0.68 0.20 0.22 0.19 OA4 0.19 0.34 0 .62 OAO

• liRG: Baked Blanched Garlic; FBG: Fried Blanched Garl ic; BG: Blanched Garlic. Data regenerated from ref. (-I ).

•• nd: not delected

nd ed ed

ed ,d ,d ,d ed , d ed

" ed

51> .sl ·H': ~~";: F/.II\'O K 1.;1I ~:M l :n·K\, ANIl ANT1(IXIIlANT l'IU'I ' UtTI1'~";

T~ble II . Some iJl1~nant volatile com~unds idenlified in shallOI sameles

Yield, 2Em

COll12ound BBS ' FilS' .S ' Compounds P.-obably Gen ~raltd from Thermall>egradatiOIl of

Non volatile lilavor l'rf<' lI r5ors

I-propene 0.43 0 .86 nd " melhanethiol 0.58 196 M dimethyl disulfide 0.46 1.91 "' dipropyl disulfide 1.66 2.00 "' l_propanethiol 0.29 0.30 0.03

3-mclhyhhiopheltC "' 0.78 "' methyl propyl disulfide 2.49 8.50 0.01 dimethylt hiophenes 4 .83 15.44 "d I-propeny] methyl disulfide s 0.75 5.46 008 dimethyl trisulfide 3. 52 7.28 "' I-propenyl propyl disulfide "' 1.42 "' methyl propyl trisolfidc 19.12 1644 "' dipropyl trisulfide 7.29 3.94 "' Compounds Probably Gentra ltd from Thf rmallntf rllclions o(Sugan

a nd Nonvola tile Flavor prtcunon

pyridine "' 0 .41

2-pentylfuran "' 5.80

methylpyr31.ine 1.14 5.48 dilliethylpyrazines Jl64 59.24

2,3 -di met hylpyridi ne 1.23 "' ethyl melhylpyrazines 6.11 15.38

Irimethylpyrazine 4.53 10.77 S-ethyl-2-methylpyridine "' 0.82 2, 6-diet hylpyrazine "' 338 ethyl dimethylpyrarines 33.85 29.38

methyl propylpyrazincs 443 331 dimethyl propylpyrazincs 2.70 12.83

2,3 -dimethyl-5-[ I-methylpropyl]pyrazine 1.51 "d j -eth>:l-2. 6-dimeth:r: IE>:ridi ne 0.94 "d

BBS: Baked Blanched Shallol: Fl1S: Fried Blanched Shallot;

ns: Blanched Shallot . Data regenerated from ref. (S) .

nd: not detected

"' "' "' "d "d

"' "' "' "' "' "' "' "' "'

,. YO Flal'or "f TJrermall)" l 'mcn,\·td Al lium Vegt'labll''\" 57

Table III . Some im~nllnl volatile eom~unds idenlified in welsh onion salll ~les. Yield, ppm

Compound BDGL' FflGL' DGL· BBWS' FBWS' BWS'

Co mpound Probllbly Gener:Hf ti from T herma l Drgradation of

t'IIonvolllli le Flavor Pfe<:urson

methanethiol 002 0.04 trace" 0.02 0.07 trace

I_propancthiol 0.08 nd '· · 0 .02 0 .08 "d 0.02

dimethyl disulfide 0. 10 0.58 trace 0.05 0 .67 ,,' 2_methylthiophene 0.02 00 1 "' 0 .01 0.01 "' methyl propyl disulfide 0.06 0.04 "' 0.05 0 .03 rnI

dimethylthiophenes 2.02 0.72 0 .16 U8 078 "d

I-propenyl methyl disulfides 0.63 2 .64 "' 054 2.25 "d

dipropyl disulfide 0.02 0.03 "' 0.02 0.05 "d

dimethyllrisulfide 0.63 0.96 "' 0.72 1.44 "d

I_propenyl propyl disulfides 0.14 0.15 "d 0 .11 1.01 "d

methyl propyl trisulfide 0 .23 0.39 "d 0.33 0.69 "d

I-propenyl methyltriSlllflde 0.08 0 .2 1 "' 0 .08 0.20 "d

dipropyl lrisulfide 0. 18 "' "' 0.25 "d M

I-propenyl propyl trisulfide 0.08 0 .21 trace 0.07 0.20 0 .01

Conlpou nds Probably Gtll trll lM f.-om Thtrmallnleraclions ofSugfln

lind Nonvolatile Flavor Precunon

2-penty! lUran "' 0.13 "d "' 0.09 " dihydro-2-met hyl-3 _[2 I-i J_furanon "' "d 0.28 "' "d 0.24

dimet hylpyrujnes 0.04 "' "' 0.05 "' "d

3-(methylthio) propanal "' 0.08 "d "' 0 .11 "' 2-ethyl-3 ,S_dimethylpyrazine 0.03 "' " 0 .03 ,,' "d

dimelhylpyridine 0.04 "d "' 0.01 "' M

2-acetylfuran 0 .26 0.40 " 0 .34 0.49 "' 2-acelylpyrrole 0.08 "' 0.04 0 .20 "' 0 .0 1

2_melhox:r:_4_vin>:l~henol 0.27 0.Q7 004 0.03 0 .02 0.05

, llllGL: Baked Blanched Grcen Leaf; FllGL: Fried Blanched Green Leaf;

,. ...

DGL: Blanched Grcen Leaf; BBWS: Baked Blanched White Sheath;

FBWS: Fried l11anehed White Sheath; BWS Blanched White Sheath.

Data regenerated From ref. (5 ).

Trace: < O.OOS ppm nd : not detected

58 SI'ICES; t'i.AVOl{ CII EMLSTR't' ANO ANTIO X([lANT !'IUll'mIT!I':"

blanched AlliUm vegetable slices generated significant amounts of volatile compounds. These volatile compounds arc proposed to be mainly generated from the thermal degradation of nonvolatile flavor precursors ill the Allium vegctabks and the interactions of these precursors and other components, especially sugars and lipids, in thcse vegetables.

The major volatile compounds listed in Tables I, II and III separated into two groups; Ihose that were probably generated from thermal degradation of nonvolalile flavol' precursors and those generated from thermal interactions of nonvolatile flavor precursors of garlic and sugars. The major volatile compounds generated from thermally degraded nonvolatile flavor precursors of garlic listed in Table I were allyl disulfide, 1-propene, acetaldehyde, allyl sulfide, methyl allyl trisulfide, and other polysulfurcontaining cyetic compounds. Thc major volat ile compounds generated from thermal interactions of nonvolatile flavor precursors of garlic and sugars were pyra7.ines, Formation ofpyra1.ines in these thermally treated blanched garlic sliccs are proposed through the Maillard type reactions of the amino-containing precursors and the reducing group-containing sugars existing in the garlic tissues. The major volatile compounds Ihat were identified in baked blanched and fried blanched garlic slices which were probably generated from thermal interactions of nonvolatile flavor precursors of garlic and lipids were pyridines and alkyl benzenes.

Table II lists the major volatile compounds from baked blanched and fried blanched shallot slices generated from thermal degradation of nonvolatile flavor precursors of shallot were methyl propyl trisulfide, dimethylthiophenes, mcthyl propyl disullide, and dipropyl trisulfide, The major volatile compounds that were probably generated from thernlal intemctions of nonvolatile flavor precursors of shallot and sugars were pyrazines, especially ethyl dimethyl pyral.ines, dimethyl-pyrazines, ethyl methyl pyrazines, and trimethylpyrazine.

The major volatile compounds listed in Table llf are from baked blanched and fried blanched welsh onion slices. The first group, generated from the thermal degradation of nonvolatile flavor precursors, were dimethylthiophenes, and alk(en)yl disullides and trisulfides. The a1k(en)yl group mentioned above could be methyl, propyl, and I-propenyl. Those major volatile compounds generated from thermal interactions of nonvolatile flavor precursors of welsh onion and sugars were pyrazincs, especially dimethylpyraz.ines, 2-methoxy-4-vinylphcnol, 2-acetylfuran, and 2-acetylpyrrole

Volatile Compounds Generaled from Thenn:11 Degradation or Thermal Intenlc(ions of of Alk(en)yl Cysteine Sulfo l: ides

To delennine the potential contributions of the nonvolatile flavor precursors of the genus Allium to the flavor of thermally processed Allium vegetables, in our second approach we analyzed the volati le compounds generated from the thermal degradation of the Salk(cnhoJcysteine sulfoxides and fi 'om the interactions of these sulfoxides with other food compllnelUs existing in Allium vegetables. Four S-alk(en)ylcysteine sulfoxidcs were syJl{h~si7,,",d by us and were subjected to thermal degradation or thermal interactions; the VOI:tlilc compounds generated WCfe analyzed to determine what contribution these $ull,'xities made tll the navor of (hermally processed Allium vegetables. The su!foxides

6. '" Hal'or oj Th~rmally ProCfHeu Allium Vegfllab/es

Table IV. Some important ~olati 1e compounds identified in the [henna! degradation or them!al intcmction solutions of S-all\'lcyslcinc sulfoxide (alliin)

Yield, rnllLrnolt: of ;'Illi;n Compound A' A'IG* I -propene 39.90 "do, acetaldehyde 1199.60 16.50 allyl alcohol 93 ,)0 340.70 ethyl acetate 23230 0' acetic acid 72.70 4.30 thiawlc 9.90 13.30 acetal 26.40 0' dimethyl disulfide Il.ID nd 2-methyllhiazole 7.50 0,70 1,2 ·dithiacyclopc:ntanc 1.70 0' methyl elhyl disulfide 61.QO ,,' methyl allyl disulfide 1.70 ,'" dimcthyltrisulfidc 11.50 ,,' formylthiophcnc5 3.50 29.90 2-ace\yllhia:role 359.50 140.30 methyl-l ,1 ,3-triuliacyclopentane 188,00 0' dimethyl tctrasulfidc 530 "d ethyl methyhctrathi~nc 0' I 10 methyl- I ,2,3,4 -tctrathianc 57.50 ,,' 1 ,2,3, 4-tdralhiepane 9.70 10.60 dimcthyltetrathianes 20.90 ,,' pyra1.ine "d 4.10 methrlpyrazine ,,' 43.80 dimclhylpyrazines ,,' 10.00 cthylpyrazine ",I 15.10 ethyl mctllylpyrazincs od 9.40 2-acetylthiophcnt "" 13.40 ethyl dimcthylpyrazincs ,d 2.20 mctllyl propyhhiazolcs ,,' 30.60 bcll1.othiophcne ,,' 20.80 phcnylacetaldchydc ,,' 1.90 acetyl methylthiophcnes 0' 6.00 2-blllylthiophene ,,' 0' 2-pcntyhhiophene 0' ,d 2-pentylpyridine 0' "d 2-hexylthiophcnc 0' ,,' 2-hcxanoyllhiophcne "d ud 2 -pentylbcnzaldchyde ,d ,,' 2 -form~' 1-2-~nt~·1thioQhenc ,d od

• A: Alliin was themlally degmdcd in a "H 5 aqueous sotut ion. Data legener31ed from ref (6).

A+G: Alliin was thermall)' inlera(;ted willl glucose in 3 pH 7.5 aqucous sotUlion, Data regenerated from ref. (7),

A+ D' 4.50

0' 2193.60

od "d

42, 50 nd

,,' 1.30

15 .90 od

,,' 0' 6.00

16 .20 4,00

,,' 24.90

"d 16.50

"d ,,' "d ,d

,,' 0' od od "d od

,,' ,,' 3 1.50 6.10

18.40 til [0 31 .50

39~ .10

110.20

A+D: Alliin was thennall~ interac1ed wil1l2,4-i"1ecadien31 in a pH 6 aqucous solution. Data regeneraled fro'" ref. (8)

•• nd: nOl dctocted

:;9

S I'ICI':S, FlAVOR CHU,IISTRY AN Il ANTIOXIOA:'I.T I'ROl'ERTtK"

Table V. Some imponant volatile compounds identified in the thennal

degradation or thermal interaction solutions of S-methy1cystcine

sulfoxide

mg{mole of MeC~SO

Comeound MeC~SO' MeC~SO+G ·

methanethiol 8.20 0.90

dimethyl disulfide 3320.80 2598,90

dimethyl trisulfide 21.60 59.80

lhiaz:ole 18.20 2,90

thioeyanie acid, methyl estcr 65.30 1.30

Illcthylthiol furan 157.50 86,60

pyrazine 69.80 114 ,60

methylpyrazine 8 90 264.00

ethylpyrazine \.70 99.90

di lllcthylpyrazincs 4.60 336.90

trimcthylpyrazinc n.d. u 40.5Q,

ethyl methylpyrazine 23.80 115.20

2,6.diethylpyrazine 19.30 6.30

ethenylpyrazinc 5.40 53 .20

ethyl dimethylpyrazines 6.50 74.90

ethenyl methylpyrazines 330 176.90

2-mcthylpyridine 4.30 3.10

2·acelylpyridinc n,d. 8,30

J -( mcthylthio )pyridine 17.40 5.70

t H Elrrole 11.00 40.20 • McCySO . S-methyl-L--cysteine sulfoxide

.. MeCySO+G S-methyl-L-cysteine sulfoxide + glucose

nd : not dctectcd

6_ YU Flavor of Tilf!rll1nify Pmcl's.~ed All ium Vegclnhll'.l'

Table VI Some important volatile compounds identified in the thermal

degradation or thenna! interaction solutions of S-propy1cysteine

sulfoxide

mglmole of PrCySQ

ComEound PrC~SO -+

I-propanethiol 88.80

1,1 '·thiobis propane 7.20

methyl propyl disulfide 8.60

dipropyl disulfide 6246.40

dipropyl trisulfide 7088.80

ethanethioic acid, S-propyl ester 44.00

pyrazine 450

methylpyrazine nd · ·

dimet hylpyrazines 3.60

ethylpyra1;inc ed 2-ethyl-3-methylpyrazine e' 2, 6-diet hylpyrazine od ethenylpyrazine 4.60

ethyl dimethylpyrazines 5.50

methyl propyJpyrazines 12.60

isopropenylpyrazine 0' dimethyl propylpyrazinc 0' mcthyl-{ l -propenyl)pyra7.inc 7.50

2-ethenyl-6-methy!pyrazine ed 3_(propylthio )pyridinc 10.30

methyl (propylthio)pyridinc 5.40

IH-E~rrole od • PrCySO . S-propyl-L-cysteine sulfoxide

•• PrCySO+G : S-propl-L-cystcine sulfoxide + glucose

nd: nbt detected

PrC~SO+G·

5.60

33 .40

38.70

8367.80

3853,00

2.70

60.90

126,80

11 9.40

70.90

1330

9.20

2 1 10

65.30

86.30

42.40

9 ,30

65 ,80

87.50

11.90

4 .80

18.40

',I

62 ~I>ICES: ~' I A"() lt CII EMISTln" ANIl Ai'I'T IOXJI)lI N'r 1'IHlI'EIHJES

Table VII . Some important volatile compounds identified in the thermal

degradation or themla! interactions of S-(+)-cis- ! -propenyJcysteine

sulfoxide

mglmoie of PrenCySO

Compound PrenCySO* PrenCySO+ G' (I-propenylth io) acetaldehyde 7.20

2-methyl-I,3-dithiane 15.80

methylthiophcncs 33 .60

2,4-dimethylthiophene 1.40

tetrahydrothiophene-3 -one nd *·

thiophenecarboxaldehydes 2.40

S-methyl -2( SH J-thiophene 0' formyl methylthiophenes 16,20

2-acet yl-S-methylthiophene 10.40

thiazole 3.20

4-methyl isothiazole 5.20

2,4-dimehtylthiazole 2.80

2-acetylthiazole 499,80

pyrazine 0' melhylpyrazine ' .00 2,6-dimethylpyrazine 4.60

ethyl methylpyrazines od

trimethylpyrazine od

3 -ethyl-2,S-dimethylpyrazine od

3-methylpyridine 10.60

3,S-diethylpyridine 7.6<)

3 butylpyridine od

PrenCySO. (+)-S-cis-l-propenyl-L-cysteine sulfoxide

PrenCySO+G · (+)-S-cis-l-propenyl-L-cysteine sulfoxide + glucose

*. nd: not detected

' .6() 3.00

253.00

16.00

4.40

58,00

9.80 84 ,00

g.20

29.40

5.80

2.00

158.40

52 .60

86.40

51.40

10.00

5.20

10.20

32.60

15.60

16.20

6. YU Flaw}r of Thermally I'rlJCl.'s,mi All ium Vegettlb/",\'

were S-;llIyl. , S-mclhyl-, S-propyl-, and (+)-S-d.\'-l -propcnylcysleinc slllfoxidcs. Some imponanl volatile compounds identified by thermal degradation or lhr nnni

interaction solutions ofS-allylcystcinc sulfoxide (alliin) afC shown in Table IV. Thermni dcgradalion of alliin mainly generated acclaldehyde, 2-acctyhhiawle, ethyl aCC1 <l le, mClhyl. l ,2,)-lrilhiacyc!opcntane, and acetic acid. Thermal interaction of alli in :lIld glucose mainly generated pyrazincs, especially melhylpyrazinc, dimcthylpyrazines, cthylpyrazinc, ethyl methylpyrazines, methylpropylthiazolc:s, 2-acctylthiophene, and benzothiophene. l1lemlal interactions of alliin and 2,4-decadienal represented the m~j o r

degr~dation products of veget~ble oil containing linolenic acid, and were mainly alkyhhiophenes, especially 2-formyl-S-pentyllhiophene, 2-hcxyhhiophenc, 2-bUlyllhiophene, and 2-hexanoylthiophene, 2-pentylpyridine, and 2-pcntyl-bcnzaldchydc,

In Table V we havc identified some imponant volatile compounds from the thermal degradation or thcrmal intcraction solutions of S-mcthylcystcinc sulfo >: idc. Thellllal degmdation of S-methylcysteine sulfoxide mainly generated dimethyl disulfide and methylthio furan. Thermal interactions of S-methyl-cysteine sulfoxide and glucose mainly generated pyrazines, especially dimethylpyrazines, methylpyrazinc, ~I hyl

mc!hyJpyrazine, cthenyl methyJ-pyra1.ines, pyrazinc, and ethylpyrazine. Table VI lists some important volatile compounds idcntilied from the thermal

degra(btion or thermal interaction solutions of .'I'-propylcysteine sulfoxide. T hermal degradation of S-propylcysteine sulfoxide mainly generated dipropyl Irisullidc and dipropyl disullide. Thermal interactions ofS-propylcysteine sulfoxide and glucose m:\i 'lly generated pyral.ines, especially methylpyral.ine. dimethylpyra7.ines, methyl propylpyrazines, ethyl dimethyl-pyrazines, and 2-ethenyl-6-mcthylpyrazine.

Some important volatile compounds identiflcd in the thermal degradati on or thermal interaction solutions of S-L'is-l-propenylcysteine sulfoxide arc shown in ~' ~. b ie

VII. As shown in Table VII, the rmal degradation of S-d~·-l-rropenylcysteine sull'oxide mainly generated 2-acetylthiazole and methyl-thiophenes. The addition of glucose to the aquoous solution of S-ci!i-I -propenylcysteine 'sulfoxide increased the yield of thiophenes but decreased the yie ld of 2-acctylthiozole, Thermal interactions of S-cis-lpropenylcystcine sulfoxide and glucose mainly generated pyrazines, especiil lly melhylp)'razine. pyrazine, 2,6-dimethyl pyrazine. elhyl mcthylpyrazine, and 3 -Clhyl - ~.5-

dimethyl-pyrazine, 3,S.diethylpyridine, and 3-bUlylpyridine. We have demonstrated that a large number of volatile compounds genemtcd

frolll the thermal degradation of S-alk(~n)yJcysteine sulfoxides or from thenn~l

intemctions of these sulfoxides with food componems can also be found in the thenn:\lly processed Allium vegetables (5,\1). proof that the nonvolatile sulfur-containing !lavor precursors of Allium vegetables can generate volatile compounds th rough thcrmal degradation or thermal in teractions but abo that the thermal degradation or thennal interaction products of these sulfoxides make an importam contribution to the fb vor of thenllally processed Allium vegetable.

Literatllre Cited

Block, E.; Naganathan, S.; Putman, D, Ziao, S.·H. J. Agric. Food 011:111. 1992 , 40. 24 IS.

s l'le)';''': FIAVOW: t: 11 ~;M tSTRY ,\NIl ,\NTIOX tllANT I'tIOJ'EIITtK"

2. Block, E.; I'ulman, D.; Zhao, S.-H. J_ Ab,,.ic, FQotlChem, 1992,40,2431 . 3. IJIock, E.; Naganathan S ; Putman D.; Zhao S.-H. l'u/'t & Appl. Clwm. 1993,65,

625 , 4 YI!, T.H.; Lin, L.Y.; 1-10, C.-T.J. Ag/'ic. Prxxl Chem. 1994,42,1342. 5. ellen, Y.N. Sludy 011 Ihe CO/llrihllliOIl 0/ jlm'OI' PI'<'cli/'sor.t I(J Ihe jlm'Or /orlllolioll

a/ lIreI'll/oily IN'OC/!!i$l!d jj,nllol alld ",eI,.·" onioll, 1996, M.S. dissertation of DI!. -Yeh Institute of Technology, Taiwan. ROC.

6. Yu, T ,B ; Wu, C.M.: Rosen, R.T ; Ual1man, T G. ; Bo, c.-T. J. Ag,.ic. FoocJChem. 1994,42, 146.

7 Y\!, T .B .; Wu, C.M .; 110, c.-T. .J. Agrk. FoodClU'III. ' 994, 42,1005. ::; VII. T .H.; Lee, M.H., Wu, C.M .; Ho, C.-T. In Upid!; ill Fo<xl Fla~'O,.s, Ho.c.r.,

Han man, T_G_ Ed.; ACS Symp_ Sec 558; American Chemical Society: Washington, D_C , 1994,61-76_

9. Yu, ·r.I·!. ; Wu, C,M,; 110, C.-T. 1 Agric. FoodChcm, 1993,800. A NALYfICAL T ECHNIQUES

Chapter 7

Characterization of Saffron by Aroma Extract Dilution

Flavor Analysis

Keith R. Cndwalloder, Hyung lIee Raek, ond Min Cai

Depurtment or .'nod Scie nce and Techn ology, Missin ippi Agric ulturlll lind .'n~es.'ry Experimen t Slation, r-,·lissinippi State Un ive rsity,

lIer.r:er BUlldmg, Stone Bu u leva rd, Mississippi Slll te, M S 39762- 9805

Volatile compounds were isolated from Spanish "Mancha Superior" s~fff<)n by simultaneous steam distillntion-sulvcnt e.~t rJc l ioll (S DE) nnJ dlrect solvent extraction ( DE). Extmcts wcre analyzed by gas chromatography (GC)-mass spectrometry, GC-ol fnctometry (OC-O), and aroma extract dilution analysis (AEDA). Total ion chro!JJ:llograms o f SDE.and DE extr::lcts were different with respect to levels andtypo:s of \'ohlllies prescnt; however, both e;(trncts had distinct saITro!l- like quniities. A tOlal of 25 uroma-active components were consistently de tected by OC-O und AF.DA, with 18 common to both SDE lind DE e~trne ts. One compound telllatively identi lied as 2-hydroxy-4,4,6-trlmethyl-2,5.cyelohcxadien-l.one (saffron, dried hay-like) had the hi.gllest 10g,(Oavor dilution fac tor) in hath extracts, followed by 2,6,6-t~!me thyl - I ,3-cyclohexadiene-l-carboxaldehyde (safranal) (saffron, tealike) alld an unknown compound having a saffron, drit.:d hay·like aroma.

Saffron, the dried dilrk-red stigmas of CmCIll" salil'w· L. flowers, is used hI impart both col.or a~d flavor to foods. Considered to be onc of the most expcnsive spices, saffron IS pTllnarily produced in Spain U.l). but is grown commercially in :;(\'eral ot~r countries. Production, chemistry, adulteration, und quality assurance aspeets of saffron ha\'e been reviewed (1. J). The imense yellow color of sam·on primarily is dUe! to .the presence o~ crocin, a mi~tu re of water solublc carotenoid glycosidcs (./-8). The p~ lmar)' taste-actIVe component of saffron is picrocrocin, a bitter glycoside of 2,6,6-tflmethyl.4-hydroxy-l-formyl-l -cyc!ohexene (./-8). Jlydrolysis of picrocrocin leads to thc formatio n of ~afnlllal , the lllnjor component of the essential oil of sam·on (9). Safralllil. having :I typical saffron-like aroma, Ims been generally considered 10 t>e the charac ter-impact component of saffron (10). Rlklel and Pclrlika (II), using gas ehronmtography.olfuctometf)' (GC-O), confirmed the importance of safranaito saffron :lroma: !lOwc,·er, they detected several unidemified aroma-aeti\'<' components.

@ 1997 Amerk-~ Il Chemical SIJdet)"

" GC.() is used for the di:l~~ l ion of llromu-m;livc cOinponcnls in a volatik \"\;r:11:1

(12). The n:lal iv~ aroma intensity of each compoll~l1 t (;[\Il be determined hy C1Wrll:l extract dilution armlysis (AEDAJ. which involves OC-O cV:lluntion of [\ serial d:itl \i"n series of a voblilc (!tImet (I J). rrolll these results. <I Ilavvr dilution (FO) t~\r l.l r. Ih<' highest dilution :II ..... I\i.;h [\ spetific aroma compound was last UClcch:d b)' GC-O, i ~ obtained for each aroma-acth'e COlllponent o f Ihe (xlraCI. ro factors arc then u'C"\! 10 arrange the aroma-aclive compounds according to their significance in \11\: ("lr.l("1. Ihu~ prudding a bc!ncr understanding of the rule each compound plays in the on'ra!l !Javor of the food . A critical c"uJuiltion 01' ,\ ED.I\ can be found d~cwh<!l"<: ( J.:. J 5) . AEDA hilS been cmployed for th~ itl~ntification of irnportml\ nroma cOlllrOnCl\l~ in wid..: vark ty uf foods, e.g. lobst("r (16), crab (17), beef (18), and brc:1d cno<! (/9).

Thc prcsenl study dt"aJs wi lh th..: identification of predominant arorn:I-:ICli\"e compounds ill saffron by AEDA. In ordcr to "ssur..: that [he resu lts of AED,\ \wrc representat ive of s,'1ffron aruma, ,·olatile components w..:re isol:.ih:d hy IWO cxtr:u:lion

tcchniques.

Mat eri:l ls & MethOds

i\.1aterials. Dried Sllffrull, type "Mancha Superior" , W:lS purchnseti from twO d,Jl1lc~tic spice suppliers. A total of thf\.'C I 01.. samples were ohtain..:d for this study. Ori£lnal packing date of Ihis material was 1995. Samples were stored in the dark .:11 room

temperature until analysis. St:lndard fl avor compounds were purchas.."'<i from Aldrith Chemical Co.

(Milwaukee, WI).

Simull ancous Sttalll Distillation-Soh·NII E:).:tradiun (S in:,. \Vh,)le saffron stigmlls (U.5 g), 50 IIlL of deodorized-distilled .... 'tIter. and I 0 ~d. of internal standard SI)hltion (3.07 mw mL 3-11cptanol in methanol) were continuously exlrael.;d \\ith \0 l1IL of r.:distilled dichlorometh,lI1c in a micro SCille SOE apflaratus (c:l talog 1\0 . 16415 J. C'hrompack, Raritan, NJ) for 3 h under atmosph..:ric condi tions. snE colJ tinger temperature was mainl3incd at _4°C. Elich extract was dried over. 2 g. ~f :1II1.1 ~uroli s sodiulll sulfate and COnCCnlmted to 0.5 IIIL under a gentle stream tol punll..:J nltrugen. Duplicate exlrm:tions wer~ prd"urmed fllr each sample.

Birc:ct Soh· ~ nt Extrat:lion ( BE). Who!.: salTron ~tignl:ls (2.) g) were ground for 3() s in a spice milt (Model K74)OC, Reg:!1 Wilfe, Inc., Kewaskum. WI) and then .~ie\ed Ihrough a no. 60 nylon mesh screen. Saffrnn powder (0.5 g), 10 ~IL of i11lcmnl stnnJard !IOlutiun {as atM,,·cJ, and 3 mt of redistilled t1ichloromclharle were \"i~ .... rou~ly sh:lken (or 5 min in a 16l' 125 mill serew capped testlut-.e scalt.'I.I with 11 I>TI L-lincd cap. The suspension wa~ allowed to stand ;n the dark for 2 h, shakt:n ugnin. <Ind th~ll t:en tril"llgctl al 3,OOOxg ror 30 min. Alkr recovering the supernatant, the rc~i d\l~· ,'us extractetltwu more times with 3 IllL of dichlo[mneth:me ~s d..:snih~d ahove I ·~ tr;lc\ was dried over 2 g of anhydrous sodium sulfate .. nd concentratcd to U.s Ill !. unt!.:r i\

gentle stream of purili ... "li nitrogen. Each saffron sample II'US extracted in lillpli..;atc'.

Gas C hromatography-Mass Spect rometry (GC-MS). <.;C-fl.·\S sy,,!em c"u'.i~k.1 " f an HI' 51190 Series II GCIIl I' 5972 mass sdccti\"c ,!ettxlor (fI.·'SI), Itcwlcll· I';,,·k;ud.

I

" SI'le..:" , YiAVOR CIIEMISTRY ANI} ANTIUXlDANT j'KOI'ERTl ES

Co. , Palo Alto. CA.) One ~IL of each extract was injected (spJilless mode; 30 s valve delay; 200"C injcctor temperature) into a capillary column (DB-wax or DO-5ms, 60 m Icnglh x 0.25 mm i.d. x 0.25 ).lm film thitkness (dr); J & W Scientific, Folson, CAl· Helium was used as carrier gas at a constant flow rate of 0.96 mU min. Oven IcmpcralUre was programmed from 400C 10 lowe 81 a rate of 3°Clmin with initial and finn! hold times of 5 and 60 min, respcrlively. MSn condilions were as foll ows: capillary direct interface temperature, 280"C; ionization energy, 70 cV; mass range. 33·350 a.m. II.; EM voltage, 1956 (Atune + 200V); scan rate, 2.2 scansls. Each SDE or DE eXlraCI was analyzed in duplicate.

Arom a EJl: trAct I>ilu lion AnAlysi.! (AEllA). GC-olfactometry (OC-O) system consistro o f a Varian 3300 GC (Varian Instrument Group, Walnut Creek, CAl cquippe<l with a flame ioni7,.8tion detcctor (FlO) and sniffing port. Column crouent was split 1:1 between FlO and sniffing port by using deactivated capillary columns (I m length x 0.25 mm Ld.). Serial di lutions (I :3) were prepared from SDE and DE extracts using dichloromethane as diluent. Each dilution (1 ].IL) was injected into an FSOT column (DU.wax or DIl-Sms, 30 m length x 0.32 mm i.d. x 0.25 ].1m dr. J & \V Scientific). GC conditions were same as GC-MS except that oven temperature was program med at a rate of 6°C/min and with initial and final hold times of Sand 30 min, respectively. FID and sniffing port .... 'Cre held at 250°C.

GClO wa.s performed by two trained panelists familiar wilh saffron aroma. Each panelist evaluated a dilution series for only one SOE or DE el(lraet for each saffron sample. Because of the similarity between GClO results for SDE elltraels from all three saffron samples, and likewise DE elliracts, final AEDA results were summarized by calculating average 10glFD factors) (n '" 6) for each type of extract.

Com pound hlentifica lion . nd Q ua ntila lion. Compound identifications wcre based on comparison o f GC retention indices (RI )(2O), mass spectra, and aroma properties of unknowns with those of authentic standard compounds analyzed under identical cxpc~imental conditions. Tentative identifications were based on matching mass spectra of unknolVns lVi th those in the Wiley 138K mass speclral database (John Wilcy and Sons, Inc., 1990) and literature or on matching Rl values and aroma properties of unkllOwos with those of au thentic standard compounds.

Relative concentrations of positivcly identified compounds .... 'Cfe dctcnnined using their MS rcsponse factors compared with the internal standard. Response factors wcre determined by analyzing standard compounds at three levels under identical GClMS conditions. ll1e relative concentration of each tentatively identified compound was estimated from its peak area relative to the internal standard.

Results & IJiscussion

Vola ti le co mponenU or saITron. Typical total ion chromatograms of SOE and DE volatile cxtracts of saffron arc shown in Figures I and 2. respectively. Quantitative results arc prescnted in Tablc I. A total of 46 core volatile componcnts wcre identificd. or the~, 30 compounds were found common to both SDE and DE extracts. Sarranal (2,6,6·trimethyl-1 ,3-cyclohexadicnc- l .carboxaldchyde, no. 31) was the most abundant volatilc component, followed by isophorone (3,5,5-t rimethyl-2-cyc!ohcxen- l.one, no. 26), and 2,6,6-trimethyl-2-cYclohclIenc-1 ,4-dione (no. 36). Thc

7 . CAJ)WAUAOEK lIT AI~ Analysis of Saffron f7a.,()r .9

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Time (min)

figure: 2. Total ion chromatogram of saffron volatiles isolated by dircct solvent extraction. Peal.: !lumbers correspond \0 those in Tables I and II and Figure I.

Table l. Volatile Co mpounds in Saffron

No. ' Compound JtJ& Cone. (u glL:)' DB-wax DB-Sms SDEd DE<

966 '" 6.O±J.9 2.0±O.2 975 <700 9.3±1.7 ,,' 2 5-Tcrt-butyl-I.3-cyclopt:ntadiener

3 2,3-ButanedioneJ '

5 3-Hydroxy-2-bUlanoncC· 1285 711 nd; 7}±30 6 j.Heptanol (I.S.') 1295 899 10 3,5,5-Trimclhyl-3-cyclohcxen-l-one f 14 12 104' 61=10 ,d 11 Acclic acid" 1446 <700 ,d 754±108 13 Megasligma-7,9. 13-tricnel'" 1452 1262 11±2 "" 14 2.Furancarboxaldehydcl" 1463 '" ,- 23:t9 "" 15 2 -Methy1enc-6.6-dimclhyl-3 -cyclohcxenc- l-carboxaldehyde' 149K 1112 120±22 34:::4 18 2,6.6-Trimclhyl-3-oxo-3-cyclohexcne-l-carboxaJdchyde' 1541 \ I!I 26±J 19 LinalooJ;' 1546 J 104 26±5 2.4:tO.3 20 2-McthyJpropanoic acid" 1569 , d 9.4±1.6 2 1 5-Memyl-2-furancarboxaldehyde" 157; 7.3±2.8 "" 22 2,3-Bulan..::.diol~' 1577 '" ,d 21:t2 23 Sulfinylbis methane" 1582 '44 ,d lS±2

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32 33 J5 36 37 J8 J9 41 44 45 46 47

48

Compound

Mcgastigma-4,6,8-trienc isomcrf' 3,5,5-T rimcthyl_ 2 -cyclohexen-l-one (isophorone)' Megastigma-4,6,8-trienc isomer!' Mcgastigma-4,6,8-tricne isomer" Oihydro-::!(J /1)-fur:monel" 2,6,6-Trimcthyl-I,J-cydohcxadiene-I-carboxaldehydc (safr:maJ)1 2 -Hydrox),-3,5.5-trimethYI_ 2-c)"d ohexcn_l _onef 3·Mcthylbuulnoic acid" 4-Hydroxy. 3,5 ,5-trimeth}'1-2 -cyc!ohc)(en-]-{)nei 2,6,6-T rimelhyl-2-cydohcxene_l. <I-dioncl 2-H ydroxy-4,4,6-trimcthyl-2.5-cycJohexadien_ l_one' 2,6,6· Trimethyl-2, 4-cYdoheptadien_ l_onc f'

2(SfI)-Furanom." 2.2,6-T rimethyl.j ,4-cyclohexilnedionef) l-PhenethvlaceullL" DihYdro-n-iononef" Hexanoic :lcid i '

(E)-Gcraniol" 6.10-Dlmelhyl-S,9-uooo:cadicn_2 -ono: isomer"

T ab le I Continued

No.' Compound

49 Benzcnemcthanol l '

51 Sulfonylbis methanc'" 52 2,4,6-Trimcthylbenzaldehyde f>

53 2-Phenylethanol' 54 2,4,6,6-T etramclhyl- ! -cyelohexenc-I-earboxaldchydei

55 /l-Jononel ' 56 2,6,6-T rimethyl-3 -o.~o-I ,4-eyclohexadicne-I-earboxaldchydei

58 2- Hydroxy-3,5,5-trimethyl-2-cyclohexcn-1 A-dioner; 60 2,6,6-T rimethyl-4-oxo-2 -eye lohexen-I-earboxaldehyde'" 61 2,6.6-Trimethyl-5-oxo-1 ,3-gelohexadiene-I-earboxaldchydei

62 4- Hydroxy-2,6,6-l rimethyl-2-eyclohexen-I -onef'

63 4- H ydroxy-2.6,6-1rimethyl· 3-oxo- l-cyelohexcne- I-carboxaldchydei

64 5-( BUla-1 ,3-dicnyl)-4, 4,6·trimethyl- 1 ,S-cyelohcxadien-l-ol'

B.! ' DB-wo.~ DB-51ll~

1586 1323 1602 1129 1620 1363 1628 1365 1633 1656 1210 1668 1152 1672 869 1683 1698 115 1 1734 1163 1740 1229 1761 912 1785 1179 1820 1258 18J9 1849

1849 1857

Bt DB-wax DB-5ms

1880 1043 1903

1904 1321 1917 1121 191 7 1313 1947 1484 1970 1312 2023 1240 2083 2127 1369 2141 1258 2152 1346 2179 1501

l:Qnc. (~,g IG)' SDE" DE'

D±S oct 1270±130 95-k::130 9. 7± 1.9 "d 80:!:18 7A±11 7.S:rJ A 5~12 525<>::390 3-180=300 207±39 97±12 od 90± I J 55±21 2J±6 1280:::170 921::1 20 258±211 84±8 21:::!± 18 128±15 od 400±S5 -07±135 554:t 70 4.5±0.7 4.4'±OA 7.·k!: 1.0 3.610.6 od 33±7

11±4nd 7.I±LO 3.9±0.8

-Continued on n(XI ~ge.

I);;Qnc. (~, gL i::l'

SOE' DE'

4.8±1.5 6.9±L2 od 7.S10.9 58±iO u 231±63 264129 N/A' NIA 8.6± 1.1 4.7± 1.1 70±1 2 42±7 281±130 37S±53 37±24

" 1 SiS 28±10 15±S 93137 I 29±75 74±18 40±1J 39±10

' Peak numbers I;orrespond to those in Figures I and 2 and Table II . ~RJ. retention index. <Avenge re lative concentration =: standard deviation (n= 12). ~SDE, simultaneous sleam distillation-soh'ent extral;tion. ' OE, di rect solvcnt e)(lraction. 'Compound tcntativcly identified by comparing its mass spt{:\rum 10 Wiley 138K mass spectral database. 'Compound nOI previously identified in saffron. 'Compound positively identified as des<:ribed in materials and mdhods. "tr. trace. 'Compound lent:ltively identified by I;omparing its mass spectrum with published literature ( II) . indo not dctt:cted. ~I .S .. internal stMd:ud. 'N/A, not available, peak I;ould not be resolved.

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cOllccmmtions of safnmal pre~nted in Tabl.: I were similar tu pub1i!;lwJ values (6). Likcw;s<:, the rci:uive (lnundllnees (with resl)<:ct to s3franal) for prominent volatiles (e.g., nos. 15,26.31,36.38,41,53. and 511) were in reasonnble agrt"Cm..:nt with previous studies (9,11).

Dcspite agrcc ll1ent with published literature, volmile profiles of SOE and DE t:xtracts dillcred frOIll eilch other with respect 10 levels of major volatiles liS well as in I) I'<s of compounds identified. DE allowed for the isolation of acidic COmponents (nus. 11, 20,34, 3nd 46), whil.: no aciJs were delttted in SDE extracts. On tht: other hand, SDE extracts cont:aincd higher levels of major saffron volat;l.:s (e.g .. nos. 26, 31 , (lnd 36). Iligher le\'els of thes<: components may hilv,;: been a resilit of thcrnmUy induced hydrolysis of their glucoside pr\.'(:ursors during SDE (9). FUrlhermlJfI: , il also is lil.el)' that other tht:rmal1y generated compounds were form..:d during SUE. Fllr example, the sugilr hreakdown products, 2-furancarhoxyaldehyd<.: (110. 14) and 5-llletllyl-2-fllnlncarboxaldchyde lno. 2 1), wcre oilly de tected in SDE extra~N. SOt:: e.~tr;lcts also contained four megastigmatriene isorrn.:rs (nos. 13, 24, 27, ami 28), which have been previously reported 115 \'olatile constituents of starfruit (]f). In (lJJition tn the above mentioned eOlllJXlUnds, IllUIlY (lthers wert' idenli tied for the firsttilllt: as saffron components (see compounds indicated by an asterisk ill Table I). Of [lo1rticular interest and importance were the following compounds detected hy both GC-MS and r.C·0: 2,3-hutanedinne (no. 3), acetic 3cid (no. II ), linalool (no. 19), and 3-mcthylbutunoic acid (no. 33). Le\'i.'ls of these compound were cOInJXlmt ivd) low with respect \0 previously ideillilicd enillponelllS.

Aroma·acliv~ com iloncnts of saffron_ The characteristic aroma of s.1Jfron has been previously defined in the literature as "sweet, spicy, l10ml odor wi th a fany, herhac..:ous ulldertone (I)". Despi le quantitative and qUlllitutivc differences h..: t w~'CIl

SDE and DE extracts, both had distinct saffron-li ke aromas, although their :lromas were not identic.11. DE extr:lCts had sweet, spicy, and l10ral nol1.:5 ami were representative of dried saffron ; whcreas, SDE extracts hac! nUlly, cooked ri<:c- and hay-like notes but st ill maintained a d..:finite saffron-like quality.

A \otal of 25 aroma·activc compounds were consistently detected by GC-O and AEOA in SDE and DE extrl1ets (Table II). More compounds (23) were detected in SDE extracts lhan DE eXTracts (22), with 18 common to both extf:l<:IS. Aromas deleclec! during GC-O could be grouped into Ihe following general catcgnries: Silfrron-likc (nos. 10, 30,3 1,34,37, nnd 57), falty, stale, biller (nos. 16,17,42,43. and 50), sweet, floral (nos. 19, 25, 53, and 59), sour (nos. I. II, and 33), nutty, cooked (nos, R, 9, and 12), eilrthy, plilstie (nos. -1 and 7), buttery (no. J) and gl'<.~n onion (no. 40). In general, compounds that were not COmmon 10 buth extracts had low 10gj(FO-factors) « I). For compounds detected in both extracts, most had higher loglFD-raclor.;) in SDE extracts. This was csp<.ocially true for nUlly, cooked aromas whieh may have been thermally generated during SDE exlraction.

Compounds having high 10gj(FD-factors) (>2) in ooth extmets were described as S;lffron, drit:d hay (unknown, nos. 34 ilnd 37), saffron, tea (s.1franal, no. 31) and cooked rice, baked bread (no. 9). COtllPOU!U! no. 37 had the highest log)(FD-factor) in both t:xtraets. Interestingly, this compound and no. 3.1 were detec ted at only high dilutions (> I :9), i.e., these compounds were not detected Juring GC/O of concentr:lled extwcts. This phenomenon was observed previously (1-1) ami may hal'c h..:en due to

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(27) and 0.2 Jig/L (30), r.:spt:\:lively. A compound willi a saffron, !Ioral, hay- like aroma \ViiS tenHllivdy identi fied by its mass s~clrum a~ J,5,S-trimcthyJ.).cyclohcxcnI-one (no. 10). Unidcntilied compounds within this group wcr~' described as stale, bill ... r (nos. 16 and 17), green union (no. 40), lmu flowl, rose, saffron (nil_ 57).

Compounds con~idcn:d to have only minor roles in s.a llron aroma 1I0£)(FO factors) < II werc ,]cscribcd as sour, dark chocolme (unknown, no . 1), bultery, crcam chc~'sc (2,3·bulanedionc, no. 3), plastic water boule (unknO\\11 , no. 4), vineg;!f, acidic (acetic acid, no. II), SInk, soapy (unknown, no. 42), fruity , stale (urWlown, no. 50) lllld COllon candy, strawberries [4.hydmxy·2,5-dimethyl-3{2!-l)· furrulOne, Ihl. 59[. With its low threshold compound no. 59 is <HI important component of scveral foods (18.3/) .

Conclusion

Through usc of AEDA and two extraction tcchniqucs it was po~~ibk to indicmc important arorna-:lctive components in saffron. Aromas contributed by 2- hydroxy-4,4,6.t rimelhyl-2,5-cyc1ohexadicn- l -one (telllatively identilied), safranal, and an unidC'ntificd compound (saffron, dried hay Moma) were predominant in Mffron. I [o\\'cver, it also was app:lrent that other componenlS contributc 10 saffron aroma, sumc of which are thermally gcncmted during cook ing of s..1.ffron. Many compounds were identified for Ihe firstlimc as constituents of saffron. Results uf this sludy may Ix: useful in devclopment of analytical strategies [or monitoring s..1.ffron Oav(>[ quality .

Acknowledgemcnts

Approved for publication by the Mississippi Agricultural and Forestry [xjJ\:riment Station as manuscript No. PS·&919. Support for this study was provided by the Missi~sippi Agricuhl!r:ll and Forestry EXjJ\:rim('!lt Station.

Literature C ited

I. Ft'II(1l"o/i's llondhook of MaYor Ingrcdil:nl.l". Burdock, G.A . Ed., CRC Press . Bt)Ca Raton, FL, 1995; Vol I, 3rd cd., pp 247-24K.

2. Sllmpathu, S.R., Shivashankar. S.; Lewis, Y.S. Cdt. Rev. Food Sci. NlIlr 19!i4, 20. 123·157.

3. Oberdk-ck , R. D/wh. Lebensm.-Rulldsch. 1991, 87. 246-252. 4. CastcllM, M.R., Montijano, H. ; Manjon, A; lborra, J.L. J. Chruma/ogr .. A 199J,

648. 187·190. 5. lborra, 1. L; Caslcllar, M.R., Canovas, M., Manjon, A. J. Food St.·i. 1992, 57. 714·

731. 6. Sujata, V.; Ravishankar, G.A.; Vc[[kataram;}n, LV. J. Chroma/ogr. 1992, 624,

497·502. 7. Tarantilis, P.A.; Polissiou, M.; MiUlfait, M. J. Chrolllli/ogr .. if 1994, 664,55-6 1. 8. Tarantilis, P.A.; Tsoupras, G., l'olissiou, M. J Chromu/ogr .. A 1995, 669, 107-

11K. 9. Zarghami, N.s.; lIeim:, D.E. Phylochem. 1971,1 0, 2755-2761. 10. Nanlsimhllm, S.; Chand, N.; Rajalakshmi , D. J Food QUill. 1992, 15. 303-3 14.

..

II Riidel, W.; P~Ir1:ika, 1'1'1. J fligh Res. Chmnw/ogr. 1991 , 1-1, 771-77·1. 12 . Auee, T. In Hm'or M"aSllrelllt'lJI; 1-10, ('.-T., [I.-lanley, (;.1-1 .• Ells., Dekker: :\"C\\·

York. 1993; Vol. I, Chapter 4. 13 . Grosch, W. Trends ill Foml Sci rrrllllol. 1993, 4.61::-73. 14. Abbott , N.; Etievant, 1'. , lss.:lIlchou, S; Langlois, D . .I. Agric. Fo"d ('hem 1')')3 .

4/. 1698-1703. 15 . Guichard, 11.; Guichard, E.; Langlois, D., bsandlOu, S.; Abho!!, N. 7..

/'cb..".ml. UIIICIT /<,,'ol"sch 1995, 201. 344-350. 16. Cadwallader. K.R.; T(lIl , Q.; Chen, F., 1\kycrs. S.I' J Agric F"od Ch,.lII. 1')95,

43. 2432·2437. 17. Chung, H.Y.; Cadwallad.::r, K.R. J. Agrh-. Food e"1'III. 1994, 42. 2867·2869. 18. Guth, II. ; Grosch, W Lebeo.wl.·WiJ.~. 1I.·r"elll/o!. 1993,26. 171-177. 19. Schieberie, P., Grosch, W Z I.ebell.l"m. Unlen. Fondl. 1987,1 85. 111·11 3. 20. van d.:: n 0001, II. ; Krat~., I'. D . .I. Chroll/olOgr. 1963, II, 463-471. 2 1. MacLcod, G.; "me~, 1.M. J'hywch"m. 1990, 29, 165-172 . 22. VasscrOt, Y; Arnaud, A.; Galzy, 1'. AclO 8io/edlllol. 1995,15.77-95. 23. Shu, C.-K.; LawrcncO', I1.M. J. A,;ric. Food e "e",. 199-1, 42. 1732- 1733. 24. Lipid Oxida/ioll in Food, 51. Angdo, t\.J." ACS Symposium Series No. 500 ..

Amcrkan Chemical Society: Washington D.C., 1992. 25 . Whillicld, F.13.; Freeman, D.J.; La~l, HI. AII.llr. J. Chelll. 191::2, J5, J73-.18:; . 26. Forss, D.t\., Dunston.:: , E.t\.; [hullshaw, E. II.; Slark, W. J. Food Sci. 1962, 2 7.

90-93 . 27. Bullery, RG .; Seifer, R.M., Ling, I..C . .I. Agric. Food Chem. 1988, )6. 100G·

1009. 28 . Schicberle. P. Z. l.ebl:lISIII. Unrcn. F()1"Sch 1990, IYi. 206-209. 29. Fmss, D.A. J. /)airy Rc.~. 1979, 46.691-706. 30. Glladagni, D., l3ut1cry , RG .; Tllrnbaugh, J.G. J. Sci. fi,(,,1 Agric. 1972, 23. 1 .. :;)-

1444 . 31. Meyerl, F, \{cgula, N.; Thomas, A. "111'/0<"111'111. 1981), 28. 631-633

"

'i I ., i I I

Ch apter 8

The Charactel"i7 .. at ion of Volatilc and SCl11 ivnlatile Components in Powdered

Tunncrk by Direct Thcrmal Extraction Gas Chromatography- Mass Spcct rom ctry

Ric hard II. lIiscrudt l•l, Ch i-Tan g li n', and I~HtlC l· t T. 1{ (I .~ell l

' I)CI':trtnlen t of fn~1d Scie nce and ICenter ti lr ,\dv:l nced F fllMI

Tt ... hnulu!U', Couk Culkge, Ru tger s, The State Uni" er si ty or New J e r sey New Ilrun~'~ic k, NJ 0890J - 02:l 1 '

Fivc commercial powdered turmeric samples werl' unalYled to identify vulallk and semi-volat ile Components. Thi~ analysis did not include tl~e lIoll-volatile eurcuminoids. Structural infomlution was obtainoo by direct tl.lennal extraction Kas Chromatography-mass spectrometry (GC. MS) uSing the electron ioniz.l1ion (EI) mode. Semi-qulIIttitativc values lire also rcportcd.

Turmeric tJClongs to the famil y Zillgiheruceae along with the other noteworthy memhers ginger, cardamom, and galallgal root. It belongs to the genus CIJrn/;',u

whic ~l cons i .~ts of hundr~ds of species of plants th"t grow from rhi:wml'~, undergf()ulld roolhkc stems. EconomIcally, the most importallt species is l/I)/l/1'.wicll. Tunneric is grown in \\~drm. rainy regions of the world such !IS China. Indonesia. India, JHllI"ie:l. antll'eru (/).

Tunn<:rie has been used since carly times 10 cure cverything frOIll leprnsy to th~ c<.>mmon cold but is probably best known for its properties as a carminat ive. It is used to provide aroma and taste to foods and as a dye for f.lbric . lIS propcrtie~ as a dye arc poor because tunneric is not light stable. Turmeric is also used in some cultures as a c('>~lItl·t i e, to lightell the skin.

. The prim:,ry II ses ofturmcrie today arc liS a component in curT}'. us a colnring 111 J \'ilrtety o f drted and frozen foods and as a po\\'dercd spice. Tht."" powdered spire is prcp;I: t.""d by harvcsling tbe fresh rhil..omes and boil ing them to gelatini7.e the stareh ,IUd dls~rse the color. 1he rhil..ome$ arc dried in the sun for 10 to 15 days and then grollnd IIItO a powder (I). The work prescnted in this report is on the analysis of volD.tilc and sem i-voll1tile components in powdered tu rmeric. This work does not include the non-volatile curcuminoids

. . The primary focus oftunneric research today is based on its properties as an antluxl.dant (1.3) and as an antieareinogen (4,5.6). The antioxidant propcr1ie~ of tunnerte <Ire based on the abi lity of eurcumin to form complexes with metals and to fOrln.3 resonance stabi lized free radical. CUTCumin, along with Ihe other eurcumilloids,

@ 1!197 Amelican Chemical Society

8. 1I1SEIWllT ~::r '\I ~ Voln'ik~ in I'tJlI'df'rf'l/ Turmeric

delllethQxycureumin ami bi.nlemclhoxycureu111in, arc nOllvolalile eompOllcnls of turmeric that impart its di~linelive orange-yellow color. Curcumin l,olllp1cxes" ilh metals, such as copper and nickel. thilt init iale or catalyze free mdie:ll ox idation. This property enables cureumin to act synergistically with other antioxidantS in inhibiting free radical oxidation (7).

The bond dissociatiOll energy for the phenolic hydroxyl groups in eurcumin is low and a phenoxide free radical is easily formed (Figure J). This free radical is resonance stahili1..ed. The odd electron can be delocali"led through the entire molecule hecausc this molccule is a completely cOlljugated system. Figure 2 shows curcumin acting as a free radical SCa\'enger, reacting wilh a pero.'(y free radical , fonning a hydropcroxide and the eurcurnin free radical. The other property ofeurcllrnin thai m:lkes it a good antioxidant is that once the free radic31 is fonncd it will not go on 10

react with unsaturated falty acids and initiate or propagate oxidation reactions. Tunneric has been shown to inhibit the initiation and progression of cancer.

Azuine and Flhide (4) demonstrated Ihal turmeric inhibited forestomach tumors induced by benzo[ajpyrene (01') and skin tUlll(lrS induced by 7.12- dimethylbenz[ajanthraeene (DMBA) in mice. These studies were conducted al the 2% and 5"10 levels. BenzolaJpyrene. which is a component in eigarellc smoke and barbecue foods, is a procareinogen that is o.'(idized during I'hase I oxidation in the liver to an ultimate carcinogen (Figure 3). The ultimale carcinogen forrned is 11 good aikylating agent and can react with DNA to fonn a mutat ion. Tunnerie h:ls been shown to inhibillllmors formed by this mechanism by inhibiting Ihe P450 monooxygenase system. Tunnerie also increases levels of gluta thione and glutllthione S-transferase :lelivity. GIUlilihioile is an emlogenous nuc1eophillie chemical that can react with ultimate carcil1\""Igens formed during l'hllSC I oxidation without forming tumors. Turmeric has als(I been shown to inhi bi t the progression of cancer by inhibiting orn ithine decarbo xylase. This <: nzyme dccarboxylates ornithine, an endogellous :Imino acid, fomling a polyamine. This polyamine s timulates cell and consequently tumor gro\\1h.

In another study. Mukundan, e/ (If (5) showed that as little as 0. 1 % IUrmnie in the diet significantly removed UP-DNA adduct.~ or inhibited the binding of DNA with 01' formed in rat liver. They also observed 0.03% eureumin to he more effective in inhibiting OP-DNA adduCIS. They did not propose:l mechanism for this obse .... .-ation.

n ac kgrnund

Preliminary work by these authors involved the analysis of components in lht."" methanol extracl of powdered tu rmeric using thennospray and particle beam liqu id chromatography-mass spectrometry (LC-MS). Thermospray is a soft ioni1..ation tcchniquc producing protonaled and dcprolonated molecular ions. providing mokcular weight infonnation about analytes. The therrnospray LC-MS chromatogram for the methanol extract of powdered tu rmeric is shown in Figure 4. The eureuminoids were identified based on their molecular weight. Also detected were three major late eluting components as wel1 as numerous minor components. The molecular weights ror these components were obtained from thc thermospray Illass spectra. Particle beam LC-MS was attempted 10 obtain structural infonnation from EJ-mass spectra, for the

S I' I C ~:S, ~'L\VOR C1Ui.MISTJ.tY AND ANTII'XI1)ANT "!lOI'ERTIES

01-1

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H o '0

01-1

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l - OO· + HO - C P,ro,v

Fr" R' lIlul Cu,curnln

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f, u Radle.1

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f . tty Acid Fret Radlc.1

"' igure 2. Reaction Mechanism for Curcumin ACling as" !'ree R~(Jirlil ScavenJ:e r

8. UISEIHlllT \,:'1' AI ~

bentO lil! pyrenc procarcinogen

ONA·Nt-I:.!

N_:_~_Po' H)o-""",~c~_~"'_P_~ ~ 1I~

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I , 5

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SI'ICt::S, HAVOM CIIEMISTRY AND ANTIOXIIJANT I'ROPEKTI ES

components detected by thennospray. The problem encountered with particle beam LC·MS was that none of the latc eluting peaks detec ted by thcrmospray were observed. This phenomenon was attributed 10 the volatility of the components that were lost during removal of the mobile phase: in the particle beam interface. Structural infom131ion for the volatile components in the methanol extract o flUnneric was then obtained by dinxt thennal extraction GC·MS.

Materia ls

Samples of powdered turmeric, consisting of 5 different eommerciallabcls, wcre purchased locally. Chromosorb WHI' (8011 00 m) was purchased from Supelco, Inc ., Bellefonte, PA, USA. Chromosorb WHP was used to dilute the powderec tunnerie and was conditioned at 300 GC for 2 hrs. prior to use. d,-Naphthalene (98+ atom %D) was purchased from Aldrich Chemical Company, St. Louis, MO, USA for use as an in ternal standard. The internal standard solution WIIS prepared by diluting 10.0 mg ofdl·naphthalene with 10.0 mL methanol. A 60 m x 0.32 mm (i .d.) dj= 0.25 ].1m, 00-1 capillary GC column was purchased from J&W Scienti fic, Folsom, CA, USA.

Experimenta l

There are many IIltematives to direct thelTnai extraction methodology. For instance compounds could be isolated using the Likens Nikerson system, which provides for steam distillation of the aqueous sample wi th ether extraction on a continuous basis in onc apparatus. The disadvantages of this sys tem are that the steam distillation utilizes high temperatures ( IOOGC) which may dccompose labile compounds or cause other thermal induced reaetiOI15 to occur in the product being llI'Ialyzcd thereby complicating Ihe data interpretation. Additiona1Jy, the ether extract from the Likens Nikerson system m:eds to be concentrated prior to GC·MS analysis IlI'Id there is always a risk of losing highly volatile components from the sample during the concentration step which is usually performed in a spinning band still or Kudema Danish concentrator.

Another alternative is to usc vacuum steam distillation combined with cryotrapping. In this technique the sample is steam distilled under reduced pressure so that lower temperature isolation is permissible. The steam distillate is then coooenst.'d in a series of four or more eryotraps cooled with liquid nit rogen and or dry ice acetone slurries. 11le major disadvantage of this tcchnique is that the first several cryotraps rapidly become filled wilh icc from the large volume of distillate. After thawing the traps, the water and traps must be extracted with organic solvent such as ether or mcthylene chloride to isolate the volatile organics. There are additional problems with cxtraction efficiency for polar organics that are not eas ily extracted from the water. This results in large volumes o r organic solvent that must be concentrated, once again risking thc loss or highly volatile compounds in the process. The extracting solvents used must be or ultra high purity. Frequently the extracting solvents must be distilled before use because concentrating large volumes of solvent will concenlrate solvent impurit ies as well. Dhmk rons of solvent, with no sample, are often needed to identify

8. IIISEROOT ET AI~ Volotifl'.$ in Powderrd Trmneric 85

artifacts and impurities arising from the solvent concentration step. Extraction experiments arc lime consuming and costly since elaborate glassware se tups, vacuum

systems. and concentration equipment are employed. . . Direct lhemlal extraction involves the extraction of volati le and scnlH'olall lc

components from solid samples using heat (8). Since volatile compo.ncnts are .. desorbed directly onto the head of a GC capillary column at subamblent conditions, samples should contain less than 5·10% water to prevent icc formation at the .h~ad of the column. Direct thermal extract ion is a single stcp process that involvcs mlmmal or no sample preparat ion making it advantageous over other extraction techniques. Additional benefits are no solvent disposallll'ld no anifact peaks in the sllI'Ilple chromatogram from the concentration of large volumes of extracting solvent.

The instrument used for this experiment was the Shon Path Thermal Desorption (SPTD) system ID·]. This is a commercial system developed join!!)' by the Center for Advanced Food Technology (CAFT) at Rutgers University and Scientific Instrument Services, lnc_ (SIS), Ringoes, NJ, USA. The TD-3 was connected to a Varian ]400 GC (Sugarland, TX, USA).

Powdered turmeric samples were prepared for quantitative analysis by thoroughly mixing 20.0 mg with 180.0 mg of Chromo sorb WH~ (80/100 ~). Fifteen (15.0) mg of this mixture was weighed into a 10.2 em x 4 mm (I.d) glass hne~ stainless steel thermal desorption tube (Stientific Instrument Services, Inc., Ringoes, NJ, USA). Si lani7.ed glass wool \\-"as added to both ends to conta in the sample. Both

the glass wool and thermal desorption tube were conditioned at . 300 "c for 2 hrs. prior to usc. Five (5.0) ~L ofdl-naphthaiene solullon (1.0 mg! mL CH,OJ·I) was spiked into each sample. This was equivalent to 3,333 ppm based on the weight of turmeric in the turmeric-Chromosorb mixture. A s~rcam of nit rogen (80 mUmin.) at room temperature was used to purge the desorptIon tube (30

min.) of methanol from the internal standard solution. The thermal desorption tube has a needle at one end and is threaded at the

other for connection to the SPTD system. When an injection is made, the thennal desorption tube ucedle pierces the GC injection port septum. Heating blocks close around the thcmlal desorption tube aud provide rapid heating. Helium passes through the desorption tube at I mUmin. and the volatile components are desorbed onto a 013·

I capillary column held at·20 °c with dry ice (see Figure S). In this analysis the volati le components were trapped onto the head of the

capillary column at suOOmbient temperatu~e (-20 "S) and .separated by temperature 0

programming the column from -20 to 150 Cat 20 CJ mm. fo!lo~.by ISO to 280 C at 5 °CJ min. Quantitative results were obtained using a flame 100l7.ation deteeto~ (FlO). Structural information was obtained using a Finnigan MAT 8230 magnellc sector mass spectrometer, operating in the EI·mode, and interfaced to a SS300 data system (San lose. CA, USA). TIle details of the instrument condit ions lire listed in

Table I.

86 S I'I C I'~": FI .... \ \'O I! Cll E,\ JIS'I" ln' '\/'1 11 ANTI CIXI IL\ NT ]' ll( ) I'FnTIFs

_ _ - C:",-i" Gil) flow

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ln strurn~nl : Varian 3 -l0U G C

Column Temp. prog. luj. h: l1lp. I'll) temp.

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10 11 source IClllP

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Figur;; (, shows ;l GC chromatOgrllll 1)11urmcri~ powd"r oblaincd by dir..,..:1 lhcrm::1

extraction. The major laic eluti ng pt:aks in Ihis I:hrornalognlill were corr..:I"h;u lI'i\h lhe ]all' eluting peaks JClcd<.'d by lhnnwspray LC-MS based on lll<>lccular wei ght ,llId n.:la livc intensity.

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~ellli - 4ut\lllil"lil'C n.:~lll1~ for the \·ulali k c,>mpvnellb in fivc hmlleric p(lWJ~I"S .

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f'igllrc 10_ KU(llI'u :mtll'roposed S'rurluns for Vnl.lli le ami SCllIi ' \'o!alilc Componcn ts in Turmeric

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96 SJ' ICES: HAVOK CHEMISTRY AND ANTlOXIDA..Yl" I'ROr ERTlF..s

PERCENT is the percent, expressed as a decimal, oftunneric in the tum lcric-Chromosorb mixture.

EI-mass spccu<t were obtained for each componenl. figures 7, 8, & 9 show the El-mass spectra for the three major components detected by di rect thermal extraction GC-MS. Table 1II contains a [isl of the eight most abundant ions for the minor conlponcnts in luoncric powder and their relative intensities. Figure 10 lists the structures for the components detected. Some ofthe!;e identifications are based on correlation with mass spectra in the NISTIEPAlNIH Mass Spei: tral Library, However, the mass spet:t ra for most of the components could not be found in the library and many of ,the structures in Figure 10 are proposed structures. For proposed structures, the Identlfications nre based on similarities with components for which there was a good librolry match and by evaluation of the fragmentation patterns.

Figure 7 shows the EJ-mass spectra for ar-tunnerone, the character impact compound for turmeric. The identification of this component was based on into,:rpre~ti ol\ of the ma.~s spectrum and correlation with previously reported data (9, /0). FIgure 8 shows the mass spectrum ofturmerone. Mass spectra l data for this c~mponcnt was also reported by Su, el 0/ (9). They reported a base peak at mil. '" 121 WI th a probe temp equal to 70°C. ll1ey also reported the appearance of an ion at ml7. -119 when the probe temperature was increased 150~C which they attributed to the aromalization of the eyclohexadienyl moiety at the high temperatures o f the GC· MS interface line. Figure 9 shows the mass spectrum of curl one. This agrees with the data reported by Kisco, et al (/2).

Addit ional research on the analysis of volatile components in Curcuma species can be found in the ]itcrdture (10, /), 14,15). , .. Conclus ion

Extraction of natural products yields complex mixtures of volatile, semi-volatile, and nonvolati le components. No one technique for obtaining mass spectral data for these components is generally applicable. Direct thennal extTllclion GC·MS proved to be a valuable tcchnique for the identification o f volati le and semi-volatile componcnts in powdered turmeric.

Acknowledgemen ts

We acknowledge the Center for Advanced Food Technology (CAFT) mass spectrollletry facility for providing instrumentation support. CAFT is an initiative of the New Jersey Commission on Science and Technology. Th is is New Jersey Agrieullural Experiment Stalion (NJA ES) publication #D·10570· 1·96.

.-

8. JIISEROOT ET ,\ L VtJiatiles in POM'd~nd Turmeric

L.ilenture C ited

I) Govindarajan, V. S. Crit. Rev. in FoodSci. andNldr. 1980, 12, pp. 199·]01 2) Masuda, T .; lsobe, J.; Jitoc, A.; and Nakatani , N. Phytochem. 1992,] 1,

pp. ]645·]647.

97

3) Toda, S.; Miyasc, T.; Arichi , II .; Tanizawa, H.; and Takino. Y. Chem. Pharm. Bull. 1985, 33, pp. 1725-1728.

4) Azuine, M. A. and Bhide, S. V. Nutr. Cancer 1 ~92 17, pp. 77·83. 5) Mukurnian, M. A.; Chacko, M. C.; Annapuma, V. V.; and Kirshaswamy, K.

Carcinogenesis 1993. 14 , pp. 493-496 6) Azuine, M. A.; Kayal, 1. J.; and Bhide, S. V. J. Cancer Res. Clin. Oneal. 1992,

118, pp. 447·452 7) Tonnesen, H. H. In Phenolic Compounds in Food and Their Effects Oil flea/lh

, . Analysis, Oceurance. ondChemiSIrY Editor, Ho, C.·T.; Lee, C. Y. ; and Huang, M-T. ACS Symposium Series 507, American Chemical Society: Washington, DC, 1992; pp. 144-153.

8) Hartman, T. G.; Overton, S.; Manum, l ; Baker, C. W. ; and Manos, J. N. food Teehnol. 1991 , 45 , pp. 104·1 05.

9) Su, H. c. F.; Horvat, R.; and Jilani , G. J Agrie. Food Chem. 1982 , ]0, pp. 290·292.

10) Rao, A. S.; Rajanikanth, B.; and Seshadri, R. J. Agrie. Food Chern. 1989,37, pp.740·74].

II ) Kingston. D. G. I. ; Bursey, J. T .; and Bursey, M. M. Chemical Reviews 1914,

74, pp. 2 15· . 12) Kiso. Y.; Suzuki, Y.; Oshima, Y.; and Hikino, 1-1. Phytochem. 1983,22,

pp.596·597. 13) Gtwlap, A. S. and Bandyopadhyay, C. J. Agric. FoodChem. 1984,32,

pp.57·59. 14) Dung, X. N.; Tuyd, N. T. B.; and Leclercq, P. A. J. £$senJ. Oil Res. 1995,7,

pp.261.264. 15) Ky, I>. T.; van de Ven, L. J. M.; Leclcrcq, P. A.; and Dung, N . X. J. Esse" t. Oil

Res. 1994,6, pp. 213·21 4. 16) Majiat , P.; Erdos, Z.; and Takacs, 1. J. Chromatogr. 1974 , 91, pp. 89·103. 17) Khurana, A., and Ho, C·T J. UquidChroma/ogr. 1988, II , pp. 2295·2304. 18) Hiscrodt, R.; Hartman, T.G.; Ho, C.·T.; and Rosen, R.T. J. Chroma/ogr. 1996 ,

740, pp. 51·64

Chapte r 9

Pungent Flnvor Profile s and Components of Spices by Chromatography and

Chemi lumin cscen t Nitrogen Detection

E. 1\1. Fujinari

An lek In struments, Inc., 300 Hammel Weslficld I{uad, Huu ston, 'rX 77090- 3508

The pungent characteri st ic of hot n avm.~ is ort en dUl: TO the presence of a class o f nitrogen (;onlainillg co mpo unds s u(;h as capsa ki no ids. These co mpo llent s ca n he an'l lyzcd by c hromatography wit h c he milumincsccn t n ilrogen detect io n (C I.ND). T hi s .~ens il i" e nilrogc rH;peci fie deled or l:an s impl ify com plex analyses by eliminating no n-nilrogenous compo ne nt s in the sn rnplc. Thi s allolVs the chromlilographer to easil y focus on the scp'Hali oH of the nitrogen ('umaining compo nenTS respons ibl e for Ihe "ho tness" o f spices.

Man y kinds o f nitroge n cont a inin g com pounds res po ns ihl e for pungent or "ho t" navors in s pices hn ve heen reported . Struct ures of hoi compo nents (I.y ) in horseradis h o il :Ire s ho wn in Fig ure I. The red ho t c hi li pe ppers ('onl:l ill n il rogcno u.c; com pounds know n as capsa i ci noid.~ which nrc quite si mi lar in struc tllre (Fig ure 2)_ Hyhrid peppe rs possess diffe re nt deg rees of ho tness, t~.g. j:l lapcno> Indian birds·eyc's> Mexi can habanero vnricti es. Piperin e ( IX ) is the hot component in hlack pe ppe r. Si nce Ihese nnal y tes con tai n nitrogen , l'l lro mnt og raphi c det ectio n usi ng the c hem ilumincscent n i l rog(~n de tect o r is inhe re ntl y s ui l:lble. S impli fied l: hro lll:ltog ra llls arc o btai ned s ince no n-n itrogeno us clJlllpo ll nd.~ in the samp le s nrc tTa nspnre nt to the detecto r.

Hi s to ril:ll lly, capsaic inoids in foOl.!!; have bee n anal yzed hy organ ol epti c e valuation CD. co lo rimelry (£). n nd llY spectrophOlo ll1c tric me thods Q, i). C hrom;ltog mphi (' methods have also been used, incl uding thin layer c hrummog mphy ('1'1,( ') (,5,. ~ and gas c hrumntography (Ge) (7·9). T ypically, GC metlu>\ls require n

"

III

IV

V

II"", It

" C=C -C-C~N

/ II II

BUI . ) Cllv"ihilc

II"", II C = C--C - S- C= N

If /I II I\lIyllhi O<:Y~Il"I~

AII)I i '(J lloi{Jey:Ul~lc

t1 ",," ~ 1 elhyl irorhiocpnate

Figure I. Stnlclme of hot compoll nds itt horSeradi sh oil.

'19

I

100 51' ICES: n~\vOR CIUJ',tI ~"RY ANI) ANTIOXIDANT 1' ltol'ER1U:S

eH,o):)N VIII I H

~I O #

° I

° IX CN-~-CU = CH- CH = CH~O)

Pipctille ~o

rigurc 2. Structure of c<lpsaicinoids and pi perine.

, .

9. H 1JINAIU Pungent Fla~o,. Profiles & ComfXJnelfiS of Spicn 101

derivatization step for these compounds prior to analysis in order to make them more volat ile. However. capsaicins have been analyzed without derivatization by high performance liquid chromatography (HPLC) with UV detectio n using norma l- and reversed-p hase techniques (10- 17). Better resolution of capsaicins (11) has been reported using reversed-phase (RP) rather than normal-phase (NP) chromatography. Reversed-phase HPLC separation of piperine followed by UV detection has been reported earl ier (H. lID· Using mass spectrometry (MS) as a means fo r GC detection provides a powerful tool for structura l charac teri zation of Oavors. e.g. ginger o il (W. Howeve r, quantitati on of ana[ytes by MS detection in chromatography may be difficult because of the response variation of the detector due to sample andlor solvent induced matrix; effects. O n the other hand, the CLND response is stable and not affected by co mplex sa mple matrices. Q uantit ati on of ca psaic in and dihydrocapl>aicin in red pepper by HPLC*C LND was previously reported aID. The linear response of the CLND is shown in Figure 3.

Corantnt lun

capsaicin blg) r-0.99952; m- O.00189; b--O.00202

Figure 3. HPLC*CLND calibration curve of capsaicin. Reprinted with permission from E. M. Fujinari, in "Spices, Herbs and EUible Fungi". G. Charalrunbous (Ed.), 1994 , pp 36n79. with kind permission from Elsevier Science * NL, Sara Burgerhartstraat 25 , 1055 KV Amsterdam, The Netherlands.

102 s riCES: )'IAVOR CIIEM ISTRY ANI} ANT IOXlnANT PIWI' EKTIES

Benn et. al. (ill demonstrated the use of GC-CLND for the detection of nitrogen containin g compounds in navors and essc nl ial oils. The advanlage of this technique is illustrated in Figures 4a-b using a CP-Si l 5 CB (Chrompack, 25m x O.53mm 10, 1.0 ~lm film thickness) column . Fi gure 43 is Ihe GC-FID profile of a green pepper flavor containing nitrogenous components from the horseradish oil and (he three pyrazine compounds. As Figure 4b shows, us ing GCCLN D. the Ili. ragen-containing compounds are eas il y deJected wilhout interference from the sampl e matrix . Peak identification was achieved using the horsemdish oil and the pyrazi nc siandards.

SU I>ercrilical fluids (s uch as supercritical CO2) possess simi lar viscosi ti es to those of gases, yet their diffusivities are much greater than liquids. These phys ical prope rties together provide hi gher separation effi ciencies for SFC. with sharper peak.s than fo r HPLC. Since hi gh molecular weight and thermall y labi le compounds can be anal yzed by this techniqu e, SFC also provides an added advantage over Gc. Taylor et. al. (2 2 ) reported a feas ibi lity s tudy fo r supercritical Ouid chromatography - chemil uminescent nitrogen detect ion (SFC-CLNO) with open tubular columns. SFC-CLND of hot mustard extract is show n in Fig ure 5. Hot components were identified as allyl isothiocya natc (peak. A) and butyl isothiocya nate (peak B) usi ng corresponding analytical standards.

This papcr will focus on nitrogen-speci fi c detection for liquid chromatography incl ud ing HPLC-CLNO profi les of chili powder. paprika oleores in. black pepper, and capsai ci ns in onion and garli c navors.

Experimental

Appa r-dtus. Hi gh performance liquid chrom atographi c separations were achieved on a binary gradi ent mi crobore HPLC system: primary pu mp (A) Model 305, secondary pump (B) Mode l 306, manometric module Model 805. and a dynamic mixer Modcl 81 IC from Gi lson Inc. (Middleton, WI). Sample injections were achie ved with a 20!-!L loop on a Model EQ-36 injection valve from Valco Instruments Co. Inc. (Houston, TX). A stainl ess steel V-splitter also from Valco was used in order to achi eve a post-column split of Ihe mobile phase now 10 Ihe CLNO. A Supelcosil LC- 18S analytical HPLC column was purchased from SUPELCO Inc. (Bellefonte, PA). The V-s plitter was all ached to the analytical col umn by a SLIPFREE connector, ava ilable from Keystone Scientific Inc. (Bellefonte, PA). Analyses of nor-dihydrocapsai ci n, capsaicin and dihydrocapsai ci n in spi ces as well

•

9. ~'U JIN,\RI Pungent flavor Profiles &: Compotl~nts of Spices

.)

'·b)

, • '" '" " 1·ln .. In,lnl

" •

1 ,I , , , , • " " " Tlmt (mini

f igure 4. GC profile of green pepper navor with horseradish oil and pyrazine mixtures. . a) FIJ): pCllks E :;:: 2_ lllel h y l _3_methoxypyr~zlne and

G = 2·methoxy -3-clhylpyrazme. b) CLND: peaks A :;:: bU I -3-enonit r i l~, B =:' allyl thi ocyan<ltc , C:;:: allyl isothiocyanate, D :;:: 2: buty llsotllLocyanate. E:;:: 2_mclhyl _3_ lllcthoxypyrazlllc, F :;:: 2-lllethyl.-5-melhox ypyrazi ne, G :;:: 2_lllclhoxy_3_ethylpyrazlIlc; <lnd H :;:: phenyl ethyl isothiocyanatc. Rcpn nted \v ltl~ .. . permission frolll S. M. Bcnn, K. Myung, a~l~ E .• M. h lJlnarl. in "Food Flavors, Ingredients and Composition, G. Charalambous (Ed.). 1993. pp65.-73., . with ki nd permission frolll ElseVier SC ience - NL, Sara Burgerhallstraat 25.1055 KV Amsterdam, The Netherlands.

103

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104 S I' ICES: FlAVOR Cl lEMISrRY AND ANTIOXlnAl'iT rROl' ~;RTles

, o

A

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Time (min)

20

, .•

Figure 5. Capi llary SFC-CLND profile of ho i mustard extract LO.I gram of mustard powder extracted in I mL water (30%) and met han~ 1 (7~%) solution I. Peaks A = allyl isothiocyanatc. B = 2.-b.utyl lsotluocyanate. and C = unknown nitrogen cOl1laltlll1g compound. ChrOlnatographic conditions: pressure program from 80 aIm (hold 5 min). ra mp 10 150 atm at 10 aIm/mi n, then to 200 atm al 15 atm/min ; Cyano (20 m x .I~ n~m 10, 0.25 mm film thickness) column; time split IIlJCCtlOI1 0.2 sec. Reprinted wi th pcrmiss ion from H. Shi , J. T. B. Strode III. E. M. Fujinari and L T. Taylor, J Chrornatog r. A, 734 ( 1996) 303. with kind permiss ion from Elsevier Science - NL Sara Burgcrhartstraat 25, 1055 KV Amsterdam, The Netherlands.

9. I'UJINAKl PUflgelll f1a~or Projifts & ComfKmenu of Spices lOS

as piperine ill black pepper were accomplis hed with the nitrogen speci fi c detector. model 7000 HPLC·CLND, from Anlek Instruments Inc. ( HolI~ ton , TX) and Della chromatography software from Digital Solutions (Margate,-Australia) run on an IBM 486 compatible computer.

Reagents and Standards. T he nalU ral capsaicin standard mixture was composed of 65% capsaicin and 35% dihydroca psaicin. Piperine (97%) and citric acid (99+%) were also purchased from Aldrich Chemical Co. (M il waukee, WI) . HPLC gmdc methanol (99+%) was obtained from Fisher Scientific Co. (Fair Lawn, NJ). Sodium free distilled wate r was obtained from Q7.arka Drinking Water Co. (Hou ston, TX). All standards and reagents were used without further purification. The HPLC mobile phase was filtered through a Millipore Co rp. (Bedford, MA ) !-IV filter with a 0.45 Ilm pore size. Paprika oleoresin , onion, garlic. black pepper. and chili powders wcre obtained from commercial sources.

Analytical method. The capsaici n/dihydroca psaicin and piperine refere nce standards were prepared as 13. 19 mg/mL and 4. 16 mglrnL solutions in methanol, respectively . Sampl es were prepared by separately weighing the following and bringing the volume to 25 mL with methanol: red chil i powder (2.5 136 g), black pepper powder (2.5154 g), onion powder ( 1.5 126 g). and garlic powder (1.5297 g). Each sam ple (5 mL) was concentrated to I mL final vol ume with a gentle stream of helium. Sam ples for the hot garli c and onion flavor profiles we re prepared usi ng a ( I + I v/v) mixture of the capsaici n reference standard and 100 IlL of the concentrated fl avOrs. HPLCCLND anal yses were accomplished using a 15 ilL partial fill ed injection to a 20 J.lL sample loop into a Supelcosil LC- ISS analytical col umn: 250mm x 4.6mm 10, 5 1lm particle size, 100 A pore size. An isocratic mobile phase, methanol/water wilh 0. 1 % citric acid at pH 3.0 (65:35 v/v). was utilized with a fl ow rate of 0.650 mU min. A Ysplitter was config ured post-column and used to deliver a flow of 100

~lUmin to the CLN D. The CLND condi ti ons werc: 11000 C pyrolysis temperature, PMT voltage 780. range x 10. and I volt detector output.

Results and Discussion

Thi s chemilumin csccnt nitrogen detector fo r HPLC was firs t desc ribed in (W. The detecti o n mec hanis m fo r nitrogen determination is show n below:

I

106 SPICES; Fl.4.VOR CHEM ISTRY AND ANTIOXIDANT PROPERTIES

-------~... 'NO + other oxides

Ni lrogen containi ng analytes (R-N) are oxidized in a furnace al high temperatures 10 nilric oxide (·NO). Chemiluminescence as show n in the second eq uation is detected by a photomultiplier lube (PMT). The photons detected are proport ional to th e amount of nitrogen in the analyte(s).

The HPLC-CLND profile of red chi li pepper is shown in Figure 6. The capsaicinoids lIordihydrocapsaicin CAl, capsa icin (8), and di hydrocapsaicin (C) are easily observed without interference from the red chi li powder matrix in this chromatogram. This separation was achieved via isocratic reversed- phase chromatography using a mobile phase consisting of MeOH/water with 0.1 % c itri c acid at pH 3.0 (65:35 v/v). A Supelcosil LC- ISS (250mm x 4.6mm ID) column with a mobile phase now rate of 0.65 mUmin was used. A post-column split was used to direct 100 l.d .Jmin to the CLND. The three capsaicinoid compounds (A, B. and C) are also very easily detected in onion (Figure 7) and garlic (Figure 8). Several pola r nitrogen contai ning component'; eluted at the solvent front for the onion, garlic and red chili powder samples .. Figure 9 shows the HPLC-CLND profile of black pepper. Paprika oleoresin was ex.t racted by the method reported by Coopcr (m and analyzed by HPLC -CLNO (Figu re 10) showi ng the presence of capsaicin (A) and dihydrocapsaici n (8).

Conclusion

Pungent (hot) components can be separated using gas chromatography, s upercriticat nuid chromatography, and high performance liquid chromatography and detected wilh the chemiluminescent nitrogen detectors (CLND). These nitrogen-specific detectors provide a means of analyzing nitrogen-contain ing compounds rree of matrix interfcrencc.

9. FUJINARI Put/gent Flavor Profile,f & Components of Spice.1i

, .. .. -"

.. ... "' .. B

c A

--Time (min)

Figure 6. HPLC-CLND profile of red (;hili powder. Peaks: A = nordihydrocapsaici n, B = capsaicin , and C= dihydrocapsaicin.

107

-•

108 S I' ICES: FlAVOR C1H;M ISTRY ANIl ANTiOXIDANT I·ROI·~:RTiE."

" . .... '" f-

" ........ f-

., ~~ ..

- -- •

B

A

-" Time (min)

c

Figure 7. I-I PLC-CLND profile of capsHicins with onion navor. Peaks: A = nordihydrocapsaicin, B =: capsaicin, and C= dihydrocapsaicin.

9. FUJINAIU Pungent F1a~o' Profiles & CrJmponenls of Spices 109

.... ... .,." -

B

........ " -c

... "' ...... f--

- -• ,;

Time (min)

Figure 8. HPLC-CLND profile of capsnicins wilh garlic navor. Peaks: A = nordihydrocapsaicin, B = capsaici n, and C= dihydrocapsaicin.

I 110 SI'ICK'l: H AVON. CHEMISTRY AN n ANTIOXII>ANT I'KO I'ERTI\';s

A -."--

.. .......

I

• _. ~'---'-J ~"-.N' '------.J

. - - - -- • • • • • , Time (min)

Figure 9. HPLC-CLND profile of piperine in black pepper. Peak: A = pi peri ne.

•

9. niJINARI PIIll~nl Ha~l)r ProjiJu & Components of Spices III

~

. ,

... ..... .. A B . , .. ....... A

-... ..... ..

I 0 0 , I ..... - .......... - .. ";..;!! l!.: l:iil'i~:;;:.-t::::~:;: _:c

Time (ruin)

Figu re 10. HPLC-CLND profi le of paprika oleores in . Peaks: A = capsaicin and B = di hydrocapsaici n .

Literature Cited

I. Gov indarajan. V.S.: Narasimhan, S.; Dhanara, SJ. J. Food Sci. Techno!' 1977 • .M. 28-34.

2. Van Fedor, K.; Unters, Z. Lebcnsm 193 1, hl. 94- 100. 3. DiCecco, J.J . J. Assoc. Qff. Anal. Chern. 1979,@..998- 1000. 4. Trejo. G.; Wila, A. J. Food Sci. 1973.lH. 342-344. 5. Spanjar. P.; Bla'l.ovich. M. Analyst 1%9,21. 1084-1089. 6. Andre, L.; Mi le , L. Acla Alimen. 1975,1. 113- 121 . 7 . HarIman, K.T. 1. food Sci. 1970. Ji. 543-547. 8. DiCecco. J. J. Assoc, Off. Anal. Chern. 1976.22. 1-3 . 9. Todd, P.H.; Bensinger, M.G.; Biflu. T. J. Food Sci. 1977.11.

660-668. 10. Woodbury. J .E. J. Assoc. Ofr. Anal. Chern .. 1980. Ql. 556-558 . 11. Saria, A; Lembeck, F.; Skofitsch, G. J. Ch romatogr. 1981.

208,41-46. 12. Hoffman, P.O.: Lego, M.e.; Oalello. W.O. J. Agric. Food

Chcm .. 1983.21 1326-1330. 13. L'lIv , M.W. J . Assoc. Ofr. Anal. Chern. 1983, M.. 1304- 1306. 14. Weaver, K.M.; Luker, R.O; Nealc, M. E. J. Chrornatogr. 1984.

301. 288-29L 15. Kawada , T .; Wfltanabc, T .; KUlsurn, K.; Takami, H.; Iwai, K· 1

Chromatogr. 1985, 329, 99- 1 05 .

.,

112

16.

17.

18.

19.

20.

21.

22.

23.

SI' ICF$: FlAVOR CII ~;"W;TRY ANn ANTlOXlDANT I'ROPERTIK.,

Weaver, K.M.; Awdc, 0 .13 . J. C hromalogr. 1986. 367. 438-442. Cooper. T.H.; Guzinski. 1.A.; Fisher. C. 1. Agrie. Food Chern. 1991. 39 2253-2256. Wood, A.B.: Barrow, M.L.; James. DJ. Flavour Fragr. J. 1988, J.55 -64. Yemin. G.; Parakanyi, C. In Spices, Herh.~, lind Edihle Fllngi; C haralamhous, G" Ed. ; Devel opments in Food Science Series 34; Elsevier Science Publishers, Amsterdam The Netherlands 1994. 34579-594. " ~uji nari, E.M. In Spice,~, Herh .... (llfd Edihle Fllllgi; Charail.llnbous, G., Ed.; Developments in Food Science Series 34; Elsev ier Science Publ ishers, Amsterdam, The Netherlands, 1994, 34 367-379. Berlll, S. M.; Myun g, K.; Ftrji nari , E. M. In Food Fll1vor,f, l/1gredie/lI.~ (llId COII/pMifioll; Charalam bo us. G., Ed.; Developmen ts in Food Sciellce Series 32; Elsevier Science Pu?lishcrs, Amsterdam, The Netherlands, 1993, lZ 65-73. Stu. H.; Slro(k, J.T.B. III ; Fujinan , E.M.: Taylor, L.T . J . Chromatogr.A, 734 ([996) 303-3 10. -Fuj inari E, M.: Cou rthaudon L. O. J. Chromatoer. 1992,592 209-214. - -'

Chapter 10

Supercritical Fluid Extraction of Allium Species

E1iUlbeth M. Calveyl and Eric Blockl

' Center for Jo'nod Safety Bnd Applied Nutrition, U.S. Food and Drug Administration, 200 C Street Southwest, Washington, DC 20204

lDepartment of Chemistry, State University of New York- Albaoy, Albany, NY 12222

Supcrcri tical fluid teclmologies are viable alternatives for the extraction and analysis of natural products because of the heightened awareness of the cost and safety hazards as~oc i ated with the use and disposal of conventional organic solvents. Supercritical C02 (SC-C01) is of particular interest to the food industry. Because of its low critical temperature, SC-C02 exlrllction can provide an accurate representation of the taste, color and odor of naturally occurring materials found in spices, fla vors and foods. The apptication of supercritical fluid extraction technology to the analysis and/or production of spice and flavor cltracts is discussed. The use of the technology in the extraction of organosulfur compounds found in Allium species is emphasized.

Applications of supercriticat fluid extraction (SFE) technologies are being investigated extensively by the food industry (1-9). The extensive patent activity related to food processing applications since the early 1970s is evidence of interest in SFE. Over 50 patents have been issued or applied for in the US or e lsewhere. Table I lists some of the patents related to the fl avor and spice industry (10-23). Supereritical C02 (SC· COl) with its low critical temperature (3 t .3 0c) is of particular in terest to the food industry because many of the components in food matrices react or degrade at elevated temperatures. The advantages of SFE for the food industry include potentially higher yields, better quality products and the use of a nonflammable, nontoxic solvent. Current Food and Drug Administration regulations [21 Code of Federal Regulat jons 184.l24O(c)] list COl as "generally recognized as safe"' when used as a di rect human food ingredient. For th is reason, CO2 can be used in food with virtually no limitations other than current good manufacturing rrac tiee. In contrast, the use of traditional organic sol vents requires the removal 0 residual solvent to permitted levels. This removal usually requires some distillation, which can cause off-flavors due to decomposition of components at the elevated temperatures used. The chance of offnavors resulting from residual solvents is eliminated when extracting wi th SC-C0 2 because of the ease of removing C02 from the food matrix. Other motives for the investigation of SFs include the potential for new product development and compliance with more stringent pollution control regulations that increase the cost of waste disposal of traditional solvents (24).

Although SC-COl can solubilize non-polar to moderately polar compoundS, the use of entrainers or modifiers (e.g .• ethanol) enhances the solvating properties of the

~ 1997 AmeriCiln ChemiCil I Sociely

I

"' S I'ICES, t' IAVOR CIIEM I!>-rR't' AN)) ANTIOXlIlANT " RO!' t;RTIES

TABLE I. Represc:ntalivc Palen!s Related to SFE of Aavol'S and Spices

Patent ##

us 3 477 856

us 4123559

DE3133032

us 4 400 398

us 4 470 927

us 4 474 994

US 4 490398

JP 84 232 064

EP AppJ. 206 738/ US Appl. 746 607

EP Appl. 154258

l P 63 87 977

JP Oll17761

JP02 135069

JP 02 235 997

us 5 120558

1111 If69

IOl3Ins

3/3/83

8123/83

9111184

100/8'

12125/84

12126/84

1113006 6119/85

12111/85

4119188

5110189

512""" 9118/90

6/9/92

Title Reference

Process for the Extr.lCtion of Flavors 10

Process for the Production of Spice 10, II Extracts

Apparatus and Methods for Extr.lCtion 12

Method for Obtaining Aromatics and 10 Dyestuffs from Bell Peppers

Method of Extracting the Flavoring 10,13 Substances from the Vanilla Capsule

Purification of Vanillin 10, 14

Process for the Preparation of Spice 10, 15 Ex",," Manufacture of Flavoring Substances 16

Process for the Production of Citrus 17 Flavor and Aroma Compositions

Flavoring Extracts 18

[solation of Spices, Peroxides and 19 Other Useful Components from Cruciferous Plants

Extraction of Flavor Components of 20 Alcoholic Beverages

ManufactufC of Spice Extracts 21

Extraction of Flavoring fro m 22 Seaweeds

Process fo r the Superc r itical 23 Extract ion and Fractionation of Spices

10. CALVEY &. BLOCK SCFE of Allium Species 115

fluid. Whereas a neat compound may be soluble in 5C-C02. it may not be extractable from its matrix without the addition of an entrainer. This phenomenon is demonSll1Itcd in the dc:caffeination of coffee: ( 10); neal caffeine is soluble in dry SC-C02. bUI moist 5C-C02 or moist coffee is necessary for the e.uraclion of caffeine from coffee beans. This same phenomcnon occurs with decaffeination by traditional organic solvents. Investigators have hypOlhcsiud thai water frc:es the: "chemically bound" caffeine in the

coffee matrix. An SFE system contains five basic components: pump(s), extraction vessel.

temperature controls. pressure controls and separator{s), For processing, a variety of recovery strategies is viable: a) change of temperature; b) change of pressure; andlor c) use of a suitable adsorbent material. The complexity of the proceSSing SFE system depends on the desired application and the mode of product recovery. Rizvi et al. (8) divided food-related SFE applications into three basic categories: a) total extraction, b) deodorization, and c) fT".lctionalion. Total extraction is the removal of a component or group of related compounds from an insoluble matrix. This type of process is exemplified by the extraction of vegetable oils with SC-C02. Deodori1.ation relates to operating the extraction system at less than the maximum density of the solvent. The extraction conditions are usually held constant while the components of interest, gencrally the more soluble ones, are preferentially removed from the matrix. This type of application is appropriate for the removal of objectionable aromatics or the extraction of desirable odor components such as from spices. Whereas SFI! in gencral fractionates a matrix, Rizvi et al. (8) use the term fractionat ion to describe the separati ~m of coextracted components from each other. They also usc the term to describe concentration of components either as the ext ractant or in the res idual material. In sample preparation, the SFE systcm can be directly attached to a chromatographic system and used as an on-line injection technique. If SFE is performed off-line, the resulting extractant is int roduced in to a chromatographic system via conventional injection techniques. Multiple examples of on-line and off-line analytical scale SFE re lated to food products can be found in the literature (9.25-44).

SFE of SpictS and Flavors

Although many compounds have been extracted by using SC-CO:!, the majority of the work rdated to food productS can be divided into three broad categories: flavors/spices, herbicides/pesticides and lipids. Table lI lists the extract ion conditions which use SCCO2 for a variety of spice and flavor applications, The loss of the volatile components desired in spice extracts is reduced because C02 is a gas at room temperature and the higher temperatu reS required in distillation are not needed. Hubert and Vitzthum (47) indicated that the best spice extracts have all the organoleptic factors of the whole spice even after dilution of the extract. They investigated black pepper, nutmeg and chilies and indicated tha t the products obtained from the SC-C02 extractions were organoleptically simitar to the commercially obtained extrac ts. In 1981 Caragay (48) reviewed the extraction conditions for cloves, cinnamon and vanilla pods. Stahl and Gerard (45) studied the solubility and fractionation of essential oils in SC-C02. They were able to obtain quantitative recovery of these volatile oils free of undesirable substances without fractionation of the essential oils themselves. Once the essential oil components wefC extracted, further frac tionation into ccrtain substance groups (i.e" monoterpene hydrocarbons, ~e squiterpene hydrocarbons, oxygen containing monoterpenes and oxygen containing scsqu iterpenes) was possible. Several laborato ries hav~ employcd off-line extraction techniques to fractionate the navor components of ginger, pimcnto berries, apple essence (29) and lemon peel (30). By using gas chromatography (GC) as the means of analysis. these laboratories were :Iblc to show some fractionation of the fl:lvor components as a function of thc extmclil'll densi ty. Other laboratories have investigated the use of SFE in flavor analysis hy din.'..:! coupling of the extrac tor to a chromatographic technique such as GC (25,26,3J,.IX ) and SFC (27.34).

..

• '" SI'ICFS: n A VOR Cm ;"m 'TKY AI''W ANTIOX1OANT I'KQI'IO.RTIES

TABLE II. Representative Applications of SFE Related to Flavors and Spices

C01l1l11odit:t Conditions Reference

Rosemary 45 °C; 300 atm; 10 min; cryolrnpping 25 -6S or ·10 "C

Eucalyptus leaves. lime peel, 45 °C; JOOalm; 10 min; cryogenic 26 Icmon peel, ba.~it trapping -SO to 30 "c Cold·pre.~scd grapefruit oil 70 "C; 0.1767-0.8579 glmL; 12 min; 27

cryotrapping -65 or _10 °C

Garlic and onion 35 "C: 240 aim; 30 min; cryOirapping 28 ().] "C

Ginger, pimemo berries SO GC; 1500-5000 psi 2' Lemon peel oil 30-58 "C; 90-250 kglcml 30 Mexican spices (OrigcU!um vulga~ and Pimpinella ulli.sum)

55 "C; 167 bars; separation vessel: 55 "C; 26.7 bar,

31

Chamomile essential oi l 40 "C: 90 bar: separation vessels in series: 0 GC, 90 bar; -5 "c, 30 har.

32

Clovcs 40-140 GC; 82-400 atm; 4.5 h 33 Tunncric 60 "C; 250-280 bar; 15-20% McOH 34 , . Shiitake mushrooms 40 "C; 3000 psi; fractionation

through $C\'eral trap.~ 35

Hops essential oil, biller principles 50 °C; 0.05-0. 1 glmL; 15-30 min 36 Lavender essential oil, waxes 48 GC; 90 bar; separation vessels in 37

series: - 10 "C, 80 bar; 0 GC, 25 bar.

Caraway fruits 50 "C; 9.7 MPa 38 Ginger 40 "C; 0.85 glmL; 3 min static, 30

min dynamic; I mUmin 39

Es..~ent ial oil components (limonene, carvone, anethole

40-120 "C; 20- I 20 bar 45

cugenol, caryophyUene, valcranone)

Orange juice {treatment used to 35-6O "C;7-34MPa; IS- 180min 4. deactivale ~tinestcrasel

10. CALV~Y &; BLOCK SCFE 0/ Allium SptCj~s 117

SFE of Allium Specle5

The natural flavors from garlic (Allium sot;vum), onion (A. ct!pa), and other Allium species, like those from many other common vegetables and fruits, are noc present as such in the intact planls but arc fonned by cntymatic processes when the plants arc chewed or cut (49). Addilionai flavors, also considered natural, are formed during cooking as a result of the thennal breakdown o f the initial enzymatically produced navoraots in either an aqueous on nonaqueous (e.g. , cooking oil) medium. If the breakdown products are unstable, other compounds can be formed, which can contribute to the aroma and taste of the food .

Supercritical fluid extraction of Allium s~cies with C02 provides an effective and environmentally friendly alternative to tradItional organic extractions. Miles and Quimby (SO) extracted garl ic products by using SC-C02 under mild conditions, They analyzed the extracts by GC with atomic emission detection. Sinha (SI) extracled onions with SC-C02 and analyzed the extracts by GC-mass spectrometry (MS). The use of traditional GC methodologics in the analyses of these SF extracts by the above research groups precluded the identification of those compounds primarily responsible for the charactcristic fla vorll of freshly cut members of the genus Allium (S2). We have previously shown that the chromatographic profiles of extracts of Alii14m species (garlic and onion) obtained with SC-C02 at thc low temperature of 3S GC were similar to the chromatographic profiles of corresponding organic solvent extracts (28), The flavor qualities of the SF extraclS were judged 10 be comparable to Ihose of fresh garlic and onion. Our experience with SFE of garlic contrasts with that of Wagner and Breu (S3). They reported the complete dccomposition of garlic compounds, such as allicin (S-2-propenyl 2-propenethiosuUinate, AlIS(O}SAII), after garlic juice was stored at room temperature for 3 h and then extracted wilh CO2, It is di fficu lt 10 evaluate their work because they did not report Ihe SFE condit ions employed. We observe very little change in the liquid chromatographic profile of SF extracts of aqueous homogenates of garlic that were extracted at 35°C following storage for 10 min or 2 h at room temperature (27.5°C) (Figure I). Our observations agree with those of Lawson (54), who has shown thai the half·li fe of allicin at room temperatun: is 4 days in water.

Supercritical fluid extraction of the major garlic flavorant, allicin, is 25% more efficient than the best procedure which uses organic solvents if the SFE is done at or below 35°C. Liquid chromatography (LC)-MS under thermospray conditions confirmed the identity of allicin from the garlic (28). Analyses of SF extracts of commercial garlic products which have been reconstituted by addition of water showed LC profiles similar to those seen with fresh garlic. Al licin and related Ihiosulfinales (RS(O)S R') have been identified in SF eXlracts of garlic (Figure 2A), freeze-dried ramp (A. tricoccum) and frozen ramp (Figure 28), The ratio of the thiosulfinates foond are signifi cantly dirferent in the garlic and ramp. The major constituent in the garlic extract is allicin, peak (pk) 4. The major constituents in the frozen ramp e;1.t ract are ally l methyl dtiosulfinates (AIIS(O)SMe, pks 213) with a significant contribution of the dimethylthiosulfinate (McS(O)SMe, pI:. I). The I-propenyl isomers o f allicin are also present in both the garlic and ramp extracts. The chromatographic profi le of the freeze-dried ramp extract more closely resembles lhe garlic extract than the extract from the frozen whole ramp. Ajoene (AllS(O)ClhCH=CHSSAlI), a major compone nt found in oil-macerated garHc products, has been found in small quantit ies in SF extracts of garlic and (tenta tively) in those from ramp. The identity of these compounds has been verified by LC-MS employing atmospheric pressure chemical ionitation (unpubl ished data).

A similar study employing onion juice showed SFE to be ca. 69% as efficient as conventional organic solvents for extracting the major organosul fur compounds (28). The diminished efficiency of SFE for onion compared with gart ic may be due to

1I8 SI'ICKS: .. LAVOR CIlt:1.f1~"TRY AND ANTIOXIDA NT I'}I:C)PERTI ES

3.0 A

• 2.0 0

~ ~ ~

-< 1.0

3.0 B

t! 2.0

J 1.0

0.0 L~~~~;::::::;:;;:~~=; 5 10 15 20 25 30

Time (min)

Figure I. LC chromatograms (UV, 254 nm) representing: (A) SF extract of a garlic homogenate stored at room temperature for 10 min and (8) SF exnact of a garlic homogenate stored al room temperature for 2 h.

10. CALVt,Y & 8LOCK sen: of Allium SpKieJ.' 119

0.8

4 A

0.6

~ of 0.4 0

" ~ -<

0.2 ,

2 , I

3 ,[ 0.0

, :1

·e , , I

0.8 2

B

0.6

• 0 < ~O.4

4 .. 3 <:

0.2

0.0 , 2 4 6 8 10 12 14

, Time (min)

Figure 2. LC ChromalOarams (UV. 254 om) of SF extracts: (A) garlic and (D) frozen fresh ramps. Pc identification: ( I) MeS(O)SMe; (2/3) AIIS(O)SMc; and (4) allicin.

':' , :

·1-, , ~.I , ,:! !\

,

120 SI'ICl';S: FlAVOR CIfEMI.!o'l'RY AND ANTIOXIDANT "IWPEKTIt.:S

2.5

A

2.0

• u :;iu

~ " ~ « 1.0

0.5

0.0

2.5

II

2.0

• g I.S • ~ 0 llLO <

0.5 6

1 00

5 10 15 20 2S 3D Timr (min)

Fi ~ure 3: LC .chromatograms (U V, 210 nm) o f garlic SF extracts: (A) 35 cC, 3 ~nm ~tallc. renod ~I 240 atm. (B) 50 "c, 28 min slatic period at 240 atm. Peak Idl;Ull fi eat lon: (5) aJoene(s); (6) I ,3-vinyldithiin; and (7) dially l trisulfide.

10. CAI.V,,-Y &: nWCK SCFE of Allium Species 121

the IOO-fold decrease in levels of flavorants in the former andlor our failure [0 detect non-volatile flavoranls present in the SF extracts of onions. The profile of the navorants in SF C1{tracts of onion, as detcnnincd using GC-MS, was very similar to that previously reponed for ether extracts of onion (55). Analysis of SF extracts of onion by LC showed many more componenlS than seen by GC-MS but peak overlap makes component identification difficult In brief. analysis o f an SF extract rapidly prepared from 10· IS g of fresh yellow onions and inuncdiately analyzed showed the presence of var ious thiosulfinales. propilnelhiai S-oxide (LF), bissulfine(s), zwiebelanes, signifieant quantities of a series o f eepaenes (RS(O)CHEtSSR') and a few related compounds (56,57). Because many of these compounds were nonvolati le, GC-MS may have significantly underestimated the efficiency of the SFE procedure. Moreover many of these I;ompounds had weak UV absorption so that monitoring LC analyses by UV detection at 254 nm was problematic. Instead, the ident ification of compounds was achieved by LClMS techniques that resolved overlapping conlpounds of different masses (58). This analysis employed synthetie standards that had been authenticated by OIlier spectroscopic: methods.

The Erred of SFE Conditions on the Distribution of Organos ulfu r C ompounds F ouud in Garlic

Yields of biologically active rearrangement products (ajoenes, dithiins and alkyl sulfides) derived from all icin vary with the type o f solvent used. lberl el a!. (59) reponed on the transformation products from crushed garlic that had been stored in ethanol for 9 days (216 h). Approximately 10% of the ini tial allicin was converted to the ajoenes and 8% of the allicin remained: the major product fonned from the initial thiosulfinates prescnt was the diallyl trisulfide (>70%). This pattern contrasted with lherl et aI's results for crushed garlic stored in oil. In this case, no allicin remained after 24 h, and the ajoenes, dithiins and diallyl disulfide const ituted ca. 25, 60 and 10% of the transformation products, respectively. At 48 and 216 h slight changes in these ratios of the transfonnation products were found in the crushed garlic ~tored in oil.

We have continued our SFE studies of Allium species by investigating the effects of di fferent SFE conditions (i .e., temperature, stat ic extraction time and modifiers) on the distribution of organosulfur compounds in the result ing ext racts . These extracts were analyzed by reversed-phase LC. Initially, we investigated the effect of temperature IlJId the duration of statIc extral;tion time on the distribution of the organosulfur compounds in garlic SF ext racts by comparing chromatographic profiles. An SF extract obtained at 35°C with a 5 min static extraction time was used as the reference. Our J)£Climinary resu lts showed that an SF extract obtained at 5O"C with a 30 min sta tic period produced the most dramatic changes in the chromatographic prori les. These c hanges were observed after IS min (Figure 3). By comparing LC retention times and on-line UV spectra with synthesized standards (Figure 4), two of the peaks in this region can be assigned as ajoenc(s) (pk 5) and diallyl trisulfide (pk 7). The most intense peak in this region can be tentatively identified as I,J-vinyldithiin (pk 6) by comparing Its UV spectra with that found in the literature (59). The other peaks in this region have UV spectra characteristic or al.kylsulfides.

Exp~ rimenta l

Extraction. Allium extracts were obtained by using a PrepMaster and Accutrap (SuJ)£Cx, Inc .. Pittsburgh, PAl. Whole cloves of garlic (2-4 g) or- ramp bulbs (2-5 g) were homogenized in 10 mUg of water at ambient temperature with a Tissumizer (Tekmar, Cincinnat i, OH). Freeze dried ramp (0.5 g) was homogenized with 20 mL of water. The solutions were allowed to stand at room temperature for 5-10 min to ensure complete enzymatic conversion to the th iosulfinates and related compounds. Hydro matrix (Varian, Palo Alto, CAl was mixed with 15 mL of homogenate and

122 SI' ICES : nAVOR CII £M ISTRY AN I) ANTIOXIDANT I' KO I'EllTI ~;S

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•

10. CALVEY & Il LOCK SCF£ of Allium Sp«;t.f 12.1

placed in a 50 mL extraction vesseL Unless olherwise indicated, .he homogenates were extracted at 35°C and 240 aIm at a flow rate of 2 mUmin unt il 60 g of C02 (SFE/SFC grade. Air Products, Allentown, PAl were consumed. A static eXlratliOn time of 5 m.in was used to ensure [hennal equilibrium. The effluent was trapped on glass beads at Joe and dcsorbcd with I mL of methanol, mcthanoUwater (50150). or ethanol. The ramp exlfaClS were analyzed directly, whereas the garlic extracts were diluled to 5 mL with water.

LC Ana lyses. LC analyses were perfonned by using a Waters 600 pump equipped with a Rheodyne Modd 7725i manual injector (50 ilL loop) and a Waters Model 991 photodiode array detection system. Chromatographic separation was achicved on a YMC-Pac!r:: PolymcrC I8 (YMC Inc, Wilmington, NC) column with a OPTI-SOL V mini fillcr (Optimize Technolo~ies Inc., Portland, OR). Thc linear grati ient was 60% H20 (sol ... ent A)J40% acetomtrile (solvent 8) which was held for 10 min, then increased by 8% acetonitri le/min to 20% H20/80% acetonitrlle, and held for 15 min . The fl ow rate was 0.8 mUmin,

Acknowled gme nts

We gratefully acknowledge support from the National Science Foundation (E.B.), the NRI Competi ti ... e Grants ProgramlU.S. Department of Agriculture (Award No. 92-37500.8060), and McCormick & Company, Inc. (E.B.). We thank R. Epply for walk ing the banks of the Potomac in search of wild ramp and P. Whangcr for pro ... iding the freeze dried ramp.

Literature Cited

l. Grimmell, C. Cliemistry and Industry, 1981, 359. 2. King, I.W .; lohnson, I .H.; Friedrich, J.P. 1. Agric. Food Chern. 1989,37, 95l. 3. Hoyer, G.c. ChemTech 1985,440. 4. Banlc, K.D.; Clifford, A.A. Spec. Publ . . R. Soc. Chern. 1994, 160, I. 5. King, J.W., INFOR M 1993,4, 1089. 6. Eckert, C.A.; Van Aislen, J.G.; Stoicos, T. En ... iroll . Sci. Technol. 1986,20,319. 7. Dzie:zak, J.D. Food Teelinology 1986, June, 66. 8. Ri:z ... i, 5.S.H.; Daniel, l A.; Benado, A.L.; Zollweg, I.A. Food Techllology 1986, 57 . 9. Hawthorne, S.8 . AI/al. Cllern. 1990,61, 633A. 10. McHugh, M.A.; Krukonis, V.J ., Eds. Supercritical Fluid Extroction Principles (IIu/ Practice, Butterworths, Doston, 1986. 11. Vitzthum, 0.; Huben, P. US Patent 4 123 559, 1978: Chern. Ahstr .. 1973, 79, 306780. . 12. Strauss, K. Ger. Offen. DE Patent 3 133032, 1983: Cllern. Abstr .. 1983 , 99, 7546k . 13. Schil tz, E. ; Vollbrecht, Il. -R. ; Sandner. K.; Sand, T.; Muhlnickel , P. US Patent 4 470 927, 1984; Chern . Abslr. 1983,99, 4363n. 14. Makin, E.C. US Patent 4 474 994, 1984; ClielTl . Abslr. 1984, 101, 2 10742y. 15. Behr, N.; Van der Mei, H.; Sirtl , W.; Schnege1berger, H.; Von Ettinghausen, O. US Patent 4 490 398, 1984; Chell! . AbSlr. 1985 , 101, 111886n. 16. Japan Oxygen Co., Ltd, Jpn. Kokai Tokkyo Koho JP Patent 59 232 064, 1984; Chem. Abslr. 1985, 101, 202893d. 17. Japikse, C.H.; Van Brockin, L.P.; Hembree, lA.; Kitts, R.R. ; Meece, D.R. Eur. Pat. App!. EP 206 738, 1986; Chern, Abslr. 1987, 107, 6048w. 18. Calame, J.P.; Steiner, R. Eur. Pat. App!. EP 154258,1985; Chern. Abstr. 1986 , 104, 33288u. 19. Kobayashi, T.; Taniguchi, M. lpn. Kohi Tokkyo Koho JP Pa.ten! 63 87 977, 1988; Chern. Abstr. 1988 ,109,2075275 .

• •

, ", ., I

.1

124 SI' ICt:. ... : FlAVO ll CIlEM. I ~"RY ANI) ANTlOXI1>ANT I' ROI't;RTIES

20. Sugyama, K.; TOlllohiro, Y. Jpn. Kohi Tokkyo Koho JP Patenl 01 J 17 761. 1989; Cluml. Abslr. 1989, 1/1, 76540g. 2 1. KaZllYuk..i , Y.; Kobayashi, M. l pn. Kokai Tokkyo Koho JP Patent 02 135069, 1990; CIr~m. Abstr. 1990, 1/3, 96385e. 22. Kobayasbi. M.; Shiraishi. S.; Matsukura, K.; Yamashi ta, K. Jpn. Kokai Tokkyo Koho JP Palent 02 235 997, 1990; Ch~m. Abstr. 1991 ,1/4, 60706s. 23. Nguyen, U.; Evans, O.A.; Berger, 0.1 .; Calderon, J.A. US patentS 120558, 1992. 24. Coenen, II .; Kriegel. E. Ger. Chem. £lIg. 1984,7, 335. 25. I-ia ..... lhorne , S.B.; Krieger, M.S.; Miller, 0 .1. Allaf. Chern. 1988, 60, 472. 26. liawtho rne, S.B.; Miller, 0.1.; Krieger, M.S. 1. Chroma/ogr. Sci. 1989, 27, 347. 27. Anderson. M.R.; Swanson, J.T. ; Porter, N.L. ; Richter, B.E. 1. ChromalOgr. Sci. 1989, 27,371. 28. Cal vey, E.M.; Matusik, J.E.; White, K.D.; Betl, J .M.; Block, E.; Li nlej ohn, M.H.; Naganathan, S. ; Putman, D.J . 1. Agric. Food Chem. 1994, 42, 1335. 29 . Krukonis, V .J. A CS Symp. Su. 1985.289, 154. 30 . SUf;iyama, K.; Saito, M. J. C/lromalOgr. 1988,442 , 121. 3 1. Ondarla. M.; Sanchez, A. CflrOll1alogruphia 1990, 30, 16. 32. Reverchon, E.; Senatore, F. 1. Agric. Food Chem. 1994,42. 154 . 33. Huston, C. K.; Ji, H. J. Agric. Food Chem. 1991 ,39, 1229. 34. Sanagi, M.M.; Ahmad, U.K.; Smith. R.M. J. Chromatogr. Sci. 1993, 3f, 20. 35. Charpentier, B.A. ; Sevenants, M.R.; Sanders, R.A. in The Shelf Lif~ of Foods (!l Id B~~'~rag~s, Charalambous, G. (Ed.), Elsevier. Amsterdam, 1986, 4 13 . 36. Verschuere, M. ; Sandra, P.; David, F. J. ChrOllla /ogr. Sci. 1992,30,388. 37. Reverchon. E.; Della Porta, G.; Senatore, F. J . Agric. FoO(/ Ch~nr. 1995 ,43, 1654. 38. Kal lio, H.; Kerrola, K.; Alhonmaki, P. J. Agric. Food Ch~m. 1994,42,2478. 39. Bart ley, J.P. J. Sci. Food Agric. )995,68, 21 5. 40. Taylor, S.L.; King, 1.W.; Snyder, 1.M . J. Microcofumn Sep. 1994, 6, 467·473. 4 1. Blanch, G.P.; Ibanez, E.; Herraiz, M.; Regle ro, G. Anal. ClI~nI. 1994, 66, ,~88· 892. 42. Nam. K.S.: King. J.W. HRC 1994, f 7, 577·582. 43. Lembke, P.: Bornet, J.; Englehardt, H. 1. Agric. Food Cllelll. 1995,43, 38·55. 44. Blanch, G.P.; Reglero, G.; Herraiz, M. J . Agric. Fomi Ch~m. 1995 ,43, 1251· 1258. 45. Siahl, E.; Gerard, D. P~if/4l,,~r &: FfOl"oris/. 1985, /0, 29. 46. Arreo la, A.G.; Balaban, M.O.; Marshall , M.R. ; Peplow, A.1.; Wei, C.I.; Cornell , J.A. l . Food Sci. 1991 , 56, 1030. 47. Hubert, P.: Vitzthum, O.G. Ang~w. Cllen!. In l. Ed. Engl. 1978,17,710. 48. Caf3gay, A.B. Perfum~r &: FfavoriSI. 198 1,6, 43. 49. Virtanen. A.I. PIrYlochem. 1965,4, 207. 50. Miles, W.S.; Quimby, B.D. Am. Lab. 1990, Jll fy. 28F. 51. Sinha, N.K.; Guyer, D.E.; Gage , O.A.; Lira , C.T. J. Agric. Food Chem. 199 2 , 40, 842. 52. Block, E. J. Agric. Food Chem. 1993, 4/,692. 53. Wagner, H.; Breu. W. DISch. Apolh. ZIg. 1989, 129, 2 1. 54 . Lawson, L.D. in Human Medicillal Aserlls f rom PfOIllS, Kinghorn , A.D.; Ba13ndrin. M.F. (Eds.) ACS Symposium Series No. 534, American Chemical Society, Washington, D.C. 1993 , 306·330. 55. Block, E.M.; Putman, D.; Zhao, S.·H. J. Agric. Food Chern. 1992,40,243 1. 56. Block, E. Angell'. Cllem. fil l. Ed. Engl. 1992.31. 1135· 1178. 57. Block, E. ; Calvey, E.M. in Sulfur Compounds in Foods, Mussinan, C.1.; Keelan, M.E. Eds. ACS Symposium Series 564, ACS, Washington, D.C. 1994,63·79. 58. Matusik, J.E.; White, K.D.; Block, E.; Calvey, E.M. "Supercritical Fluid Extrac tion and Atlllosphe ric Pressure: Chemical lonilation MSIMS of Cepaenes." presented al44th ASMS Annual Meeting. Port land OR. May 19% . 59. lberl . B.; Winkler. G.: Knobloch, K. Pfarl/a M~d. 1990, 56, 202.

Chapter 11

Determination of Glucosinolates in Mustard by High-Performance Liquid

Chromatography- Electrospray Mass Spectrometry

Carol L. Zrybko' and Robert T. Rosen!

INabisco, Inc., 200 D~Fores t AVC!nue, East Hanover , NJ 07936 !C~nter for Advanced Food Technology, Cook College, Rutge rs,

The Slate Univ~rsity of New J~rsey, New Brunswick, NJ 08903- 0231

A method was developed to detemline glucosinolates in mustard seeds by reverse phase HPLC using vola tile ion· pairing reagents followed by UV d~tection at 235 nm. Two external standards. phenethylglucosinolate and sinigrin wer~ used to quantify results. The identities of individual mustard glucosinolalcs were confi rmed by negative ion elcctrospray mass spectrometry as was LC peak puri ty. This LCIMS method may be used to identify species which are not commercially available as pure standards since mass spectrometry can be used to check for all known glucosinolate anions_ Three mustard types were chosen rrom the Brussica species. y~lIow

(Bross;ca hirfo), brown, and oriental (8rassica j lll/Cca). Eleven mustard samples representing harvest areas of southwestern Canada were analyzed in triplicate for glucosinolale content. Percent coefficient of variat ion l>ctween triplicate samples or the same batch was often less than 10"10.

In mustard and other members or the Bra~'ica species of the Cruciferea family of vegetables, the important nonvolatile precursors are the hydrophil ic glucosinolates. The structure of glucosinolates. as seen in Figmc I , imparts strong acidic properties to the compound duc to the sulfate group. Other important structural moieties include the glucose and cyano groups, G lucosino l a t e ~ vary due to differences in the R group. The side group can be alkyl, branched alkyl. indol~, aromatic. or unsatura ted_ The differences in the side chain impart different l1avors to rood , AJI glucosinolates are fo rmed in plants from L·amino acids and sugars through a common biosynth~t ic pathway.

<D 1991 American Chemica t Society

126 SPICES: HAVOR CII KPtIlSTRY AN n ANTIOXIDANT I'ROI'ERTIES

R

~CJ 12011 s-c/

~ Oli NOSO)'

0/-1

Il

Figurt I· The structure of glucosinolates.

Glucosinolates breakdown dut to the action of enzymes and heat to give many fla vor compounds. Myrosinases (thioglucoside glucohydrolases) are found compar1menlalized within the plant, and are released when lhe cell walls break through bruising, sample grinding, Of during normal growth as a result of damage and death of cells_ Olucosinolates are easily degradt:d by the myrosinase enzyme at both acidic and basic pH to give such products as isothiocyanates. nit riles, thiocyanates, m;:azolidine·2-thioncs. hydroxynitrilo:lS, and epithionitriles. all of which impan flavor to mustards (1).

Kalt. kohlrabi, broccoli, cabbage, mustard, ClIuliflower, rapeseed. turnip. and rutabaga. of the Brassi(""(1 group of cruciferous vegetables, are best known for their pungent flavor. In ancient limes, these crops were thought to have medicinal power over headaches. goul, diarrhea, deafness, and stomach disorders (1). Olucosinolate breakdown products, specifically )-indole carbinol, )·indole acetonitrile, and 3,)'diindolylmtthane, produced by myrosinase degradation of giucobrassicin. are considered Phase II anticarcinogens (2). These compounds have also been provtn to increase enzymatic activity of glutathione-S-transferase (OS H), ethoxycoul11arin O-detlhylase (ECD), aryl hydrocarbon hydroxylase (AHH), and epoxide hydratase (EI-/) , all of which in tum increase the polarity of lipophilic carcinogens so that Ihey art: more readily excreted in the urine. Olher benefits from the glucosinolates in Bro.\".~ka plal"llS include decreased growth of microorganisms. fungi, and Ihe ability to repel mOSI insects (3).

To date, most research into glucosinotale idenlificalion has been dOlle by isolating glucosinolate breakdown products and then determining the original precursor (-I). This method of back tracking was favored because il was inexpensive and fast. Analysis of glucosinolales has also retied heavily on Gc/MS techniques on either gJucosinolate degradation products such as the isothiocyanates or o n the desutfated and trimethylsilyated (TMS) derivit ized gtucosinotates (S) . Mosl often, in the case of MS, the isolhiocyanates and other volalile compontnts are identified rather than the g!ucosinolate itself(6}.

In this study, negative ion dect rospray mass spectrometry is used because il detects molecular anions and at!ows for direct identi fication ofgJucosinotates, most of which are unavailable as pure standards. We apply a novel method of reversed phase liPLC ulilizing volatile ion-pairing reagents followed by negative ion electfospray mass spectromelry to semi-quantify and identify the glueosinolates in mustard seeds. Formic acid and triethylamine (TEA) were used to produce the ionpairing agent , triethylammonium formate which interacted with the glucosinotates to increase retention and prevent their elution in the void volume.

•

1I . ZRYBKO" ROS~:N Glucosillolaln in Mustard l27

Materials & Method

Analytically pure sinigrin (98%) was purchased from Aldrich (51. Louis, MO). as were the ion-pairing reagents, formic acid and triethylamine. OluconaslUniin (phenethylglucosinolate) was obtained from LKT Laboratories (St Paul, Minn) Sinapic acid (98Y.) was supplied by Aldrich (St. Louis, MO)_ HPLC-grade methanol was purchased from Fisher Scientific (Pittsburgh, PA)_ HPLC-grade watcr was generated in the laboratory through the lise ofa Milli-Q UV Plus Ultrapure waler system by Millipore (Milford, MAl.

The externat standard. phenethylgtucosinolate, was c~refully weighed (10.27 mg) and dissolved in 10 mt m'LC'grade water 10 obtain a solution with a conctntratiotl of 1.027 mgfml. Sinigrin monohydrate (10.27 mg) was also dissolved in HPLCgrade water to obtain the same ooncentration as the phenethylglucosinolMe Sinapine bisulfate (98%) was purchased through Spectrum Cht:lI1ical (Gardenia. CA) and was used as an external standard to quantify sinapine found in all three mustard types. All standards were filtered using a 0.4 5 micron Acrodisc purchased from Gelman Scientitic (Philadelphia, PA). Standards were stored in ~l11bcr autoS.1mpler vials under refrigeration until needed_

Drown, oriental, alld yellow mustard seeds were obtained from various suppliers. Continental Grain (Albena, Canada), North Dakota Mustard and Spice, and McConnick & Company (Hunt Valley, MD). Samples were prepared by grinding -25 grams of mustard seed in a 250 1111 IKllypropylene co:lntrifuge lube, Thomas Scientific (Swedesboro. NJ), with 100 ml methanol. A Deckman Polytron was used for three minutes 10 insure a homogeneous grind. Tho:l sftmples were then centrifuged at 2700 rpm for 5 minutes. The supernatant was poured off into a 250 mt round bottom flask and then evaporated using a Rotovap set at 55°C and an rpm of 55 . Samples were evaporated to a volume of approximately 2 ml methanol. The samples were Ihen dissolved in 100 ml HPLC-grade water. All samples were filtered using a 0.45 micron nylon Acrodisc as used during 51andacd prcparalion

Analyses of mustard samples were performed by injecting a 20 ul aliquot of the sample into a Waters (Milford, MA) high ' perfonllallce liClUid chromatograph linked to a Waters 490 ultra-violet detector set at 235 nm. A Waters 600E System Controller and a 712 WISP Autoinj~l; t or were also used. The tillire f-IPLC system was controlled using the Walers Millenium data acquisition software program version 2.1. The column used was a Phenomenex (Torrence, CA) 5 pill 00S(20) column measuring 250 mm X 4.6mm with a 55 mm X 4.6 mm Walers C-18 guard I;olumn attached

The two mobile phase solvents, methanol and water. were prepared with O. I 5% triethylamine and 0.18% formic acid added as ion.pairing reagents_ Both solutions were filto:lrcd using a 0.45 11m filte r, sonicated for 30 minutes, and degassed with helium throughout Ihe chromatographic procedure at a sparge ralt: of 50 ml/minute. The initial mobile phase was 100% HPLC-grade waler. At ten minutes. the mobile phase was switched to a linear gradient of loew. water to 100%

• 128 SI'ICt;S: n .... VOR t: llliMISTRY ANIl ANTIOXIDANT !'IW,'EM.Tl K'i

methanol over 60 minutes After each run the illilia! mobile phase conditions were set and the system allowed 10 equilibrate. The now rllle was kepI conslant at I mUminute. The column temperature was held al 406 C

Confinuation of peak identity was accomplished through the use or a Fisons (Danvers. MA) VG Platform II mass spe<:\romeler connected 10 a YG Mass Lynx data system. The system was operated in the negative ion eledrospray mode. The ion source temperature was sct al 150·C while the cone voltage was -20V. The HI'LC conditions were identical to those obtained off-line except that a Varian 9012 HPLC (Fernando, CA) was used along with a Varian 9050 UV-Vis detector.

Quantilication of the nonvolatile flavor precursors, sinigrin, progoit rin, sinal bin. ~nd sin~pine was determined using external standards. A solulion conta ining seW. phel1ethylgl uco~inola!e and 50% sinigrin monohydrate was analy..:ed to determine the response f~ctor5 of both aromatic and alkenyl glucosinolates, respectively. The ratio of response factor was 1 19, indicating that the aromatic glucosinolate was more sensitive. The concentration of the aromatic compounds, sinalbin and sinllpinc, were calculated using this response factor since pure stand~rds were commercially un~vai l~ ble .

I~ cs lllt s li nd I)iscussion

Glucosinolat e Q lIlIntil1ClI tion. Ye!low, brown, and orielllal mustards were analYl.ed for their glucasinolate content. The major compounds identified by mass speclromelry can be seen in Table I. A chromatogram of ycllow mustard can be seen in Figure 2, whereas Figure 3 shows a chromatogram of brown mustllrd Many oflhe minor peaks seen on the HPLC chromatograms were nol visible by the mass spectrometry method utihed. This indicates that these compounds are um;harged and therefore are not gillcosinolates. Results for the qllant ificalio~ of compounds in yellow mustard (Brassica hirta) identified by mass spec. can be seen in Table 11 .

Ta ble I. Non\'olaciles J' ound in Yellow, Brown, a nd Orient ll ll\luSlards

RT I~eak "til: hlelltijictJtiQII 1# (obseM'CtJ)

10. 5 minutes 358 AJ lylglucosinolate (Sinigrin) II 2 388 2-hydroxy-3-

butenylglucosinolate(Progoitrin) 21 3 42. l2-hydroxybenzylgluCQsinolate (Sinalbin) 33 4 35. 3.5-dimethoxy-4-hydroxycinnamoyl

choline (Sinapine) 35 5 586 Sinalbin + glucose

II . ZRYBKO&ROSEN GlUC05inoltltl.f in MtJ/j'laNI

• 3

;; L ,1 , 0 -" 1

, , , , , 0 20 '0 60

Time (min)

Figure 2- [fPLC chromatogram of methanol extract from yellow mustard seeds (See Table 1 for idcmilications)

•

~ 1

0 l.wL

0 , • , , , . 0 20 '0 60

Time (min)

Figure 3- HPLC chromatogram of methanol extract of brown mustard seeds (See Table I for identifications)

Table II. Gluc(lsinolate Contellt of Veil ow i\"lust:ml Seeds

Il) /I Htln'!!st Ar!!" Progoitr;" SimI/bin Silltlpine Amou", (mg/g)

4-6 Calgary, Canada 0.51:,::0. 14 27.46:!: 1.20 27.68:!: 1.41 10-12 Manaloba, Canada 0.60:,:: 0.17 27.1!7.:t..!.09 32.09+ 0.69

16- 18 Alberta, Canada 0.56:,:: 0 10 24 .81± 1.92 30.60:'::2.21 19-21 Saskatchewan, Canada 0.45.:':: 0.04 23.00+ 2.85 28.05+ 4.1 5

•

12.

130 SI 'H': E."'; fL\ VOI( CIIK\IISTRY AN I) ANTlOXIUANT 1 ' lml '~: H:rU:S

I' roguilrin. 2.hydroxy-J-butcnylslucosillolah.:, (figure 4) is Ihe precursur I() g,l i]rin whie'h lms been shown 10 C~USc hypothyroidism in ani ln:!ls fed la rge ~Ill(llll\lS of cnlCi rerOIiS vegetables. The avenge Ieve! of progllilrin tillllld in yellow ll11btilrd was 0 53 mgtg! 0,06. The identification of progoiuin was (ktcrmiucd directly lioml hc I1l.:gali,'c iOIl C"lC<:l fOSllray mass spectnml The mass spccrnnn CIUI De seen in Figll"'~ 5. The molrcular aniun can be seen al 11111. J:S1! The mulo..'(:u lnr weight of I' rngoilrin is ltctualty 427 (a<ldi. iol1 o f potassium (lIIio n) The llIass spl-.:: trolHelcf sees o nly the lInion when in lhe negl1!ive iOIl mode,

Oil

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Figure 4. StrucTure ofprogoilrin ~niOIl

Sioi.lbill, p~ra·hydroxyb<:n l)' lgJllcosi noJaie (Figure 6), is found al hi~h levels in yellow mustard The coocentf',Hion of sinalbin was 25.79 mglg 1: 2 .30, which is consiSU!n1 wilh Fenwick's Crilical Review ofGlucosinol:ues and thei r llre:lkdow/l l' rulh l!,;lS (1982) and The USDA I'hytochcmiC;J1 datll b~sc available on the Int ernet ( I !TTP .lll'robe,Nalu~da , Gov : II)OO) Si nalbin is n01 found ill 1I11{\Jl C >I S a !lOtassium sail. but mlher has sillapinc as Ihe posi tive iOll The stn lcture and information lu r silmpinc is Illentioned below bt."ClIusc sinapine is ~I so present liS :I peak Oil the chrol11~lograms of brown, and OI'icnlalliluSlards.

Sinalbin was idemified by 1ll1lSS specn omclry The molecula r aniun is seen at nih 424 (Fi~ure 7).

Figure 6· StntCtUfC of sinalbin anion

The cOlllpound wit h a retenlion lime of 35 minutes has been hypot hesizc!! as sina!bin plus I:\IUC05C. The m/z of 586 shows 424 mass unit s from ~inalbin lim! 162 from gilleose Rcse~rchcrs have rCllOrled Ihe presence of glucosinn!ates wit h II second sugar in Illustard lind OI lIer vegetables liom the Hm .... ,·inl spccies (I) FUr1hcr res ... arch, illcludill!; fractionation and NMR, must lJe done on Ihis cnlllJ'lOuml in order 10 determine Ihe exact stl1lcture

..

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<'l 0 ~ '" ~ 0;

e _ -------'~'-~~" ... ''l.

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o

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1.32 SPICE.Ii: HAYOR Cm : M/STltV ANI) ANTlOXIJ)ANT 1'I(oI'~;RTIES

i I I

j

11. ZRvnKO & ROSl<;N Glucosillll/ale.f in Muslard 133

Brown and oriental mustard are from the same species, Brassica jlfllcca. and therefore. had very similar gluoosinolate content. The individual results for each mustard sample can be seen in Table III.

Tahle UI. G lucosinolate Content of Brown Mustard Seeds 1/) /1 1)"pc Hlln'Q/ Arca Sinigrin Sinnpinc

A mount (!lIgfJIJ.

1--3 Orienlal Calgary. Canada 9.59± 0.94 22 .05± J..'j0 7··9 Oriental Saskatawan. Canada 9. 10+0.19 24 .50± 1.09 22--24 Oriental Saskatchewan. Canada 9.3 1± 0.52 25.00± 1.97 25--27 Orienlal Alberta, Canada 8.16± 0.36 20.24± 1.77 13·-15 Brown Saskatchewan, Canada 8.29± 0.26 24.20;t 0.92 28--30 Brown Alberta, Canada 8.S0± 1.97 14.36± 2.70 31--33 Brown Saskatchewan, Canada 8.20! 0.95 12.98;+;; 1.78

The major compound in brown and oriental mustards is sinigrin. commonly known as allylglucosinolate. (Figure 8) with a retention time of approximately 10.5 minutes. Sinigrin degrades to allylisothiocyanate (AITC) by the myrosinase enzyme in the presence of water to give mustard its pungency. The concentra tion of sinigrin was found to be 9.04 mg/g ± 0.62 in oriental mustard and slightly lower than that for brown. The CV of the sinigrin results for all twenty one samples in the 8rassicn jill/ceo group was 1069'10. The identity of sinigrin was proven by negative ion c1ectrospray mass spectrometry when the MS showed a peak with a mass to charge ratio of)S8 as seen in Figure 9_ The negative ion is seen by the mass spcctrometry method used. whereas the positive ion. potass ium. is no t observed. The actual molecular weight of sinigrin is 397.

Figure 8- Structure of sinigrin anion_

Sinapine. a choline ester ofsinapic acid (Figure 10) was identilied in yellow, brown. and oriental mustard. This compound, 3,5-dimethoxy-4.hydroxy cinnamoyl chorine. is common in the 8rassica group. Researcher.; have shown that although sinapine is the positive ion to sillalbin, the glucosinolate and phenolic choline ester contents are not correlated with one another (7). For this reason sinapine is seen in both brown

134 SI'ICt;S: FlAVOR CHEM ISTRY AND ANTIOXIIlANT .'ROJ'EN-TIES

o

" . ., •• ." '0 E 2

I

•

11. ZltYDKO & ROSt:N Gluco:tinolaU5 ;11 Mustard 135

lind oriental mustards although sinalbin is not present. The neglllive ion eleclTospray mass spectrum of sinapine using a low cone voltage of 20 V, showed an ion al mJz 354 , The peaks seen above the mlz 354 peak in Figure 11 al'e 46 mllSS units higher, which is the compound fonning adducis wilh formic acid, the mobile phase modifier. Since very liule fragmentation occurs at such II low cone voltngc. 8 second mass spectrum was run at II higher cone vol tage, 120 volts, to give fragmentation . This data and II NMR of the fractionated peak showed Iha\ the compound was an adduct of sinapine and formate, 3 10 mass units coming froln the sinapine calion and 4S amll frOIll the format~ anion. The [M-Hr o f this comple ... occurs at nlfz 354. Interestingly. becKuse of the ion-pairing, the sinapine did not give a spectrum in the positive ion mode.

o II •

CIIJ~ CH - cu -CO-CI 12CJ I1 N(CI'!lh

HOT OCI~

Figure 10- Structure of sinapine.

Conclusion

To date, the major nonvolatiles in mustard seeds have been isolated. identifio:'d. and quantified by reversed phase HPLC using ion-pairing reagents and negative iOI1 etcctrospray mass spectrometry. Accuracy and I)recision o f the method was determined by bolh spiking studies and replicate analysis of individual smnples. respectively. The accuracy was detennined by spiking both brown and yellow mustard samples with sinigrin and calculating the percent recovery. Recovery was olien greater than 100-/ . . Precision was detennined by running all samples in tripl icate and calculating the standard d~viation and coefficient of variation. CVof most SIImples was less than 10"10. This data indicates that this method works well for identifYing intact glucosinolates and therefore eliminates the need for desulfating and derivitil.ing glucosinolatcs as seell in previous studi~s (5) , Use of mass spCl,;trometry allows identification withoul glllcosinolate startdards. most of which caullot be purchased.

Ackllowlrdglll tlits

The author gratefully acknowledges the work of Dr Elaine M . FukudH, CAFT, Rutgers Universi ty and Dr. Janet M. Deihl, Nabisco, Inc. ntis is New Jersey Agricultural Experiment Station Publication /I DI0570-96-2 .

•

136 SI'ICK'), FlAVOR CH):;MISTRY AND ANTIOXIDANT PROI'I' )l.Tl ES

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lL ZRYRKO & ROSEN Glm;os;lIoiate!l" in Mustnrd

Literature Cittd

Fenwick, G. R.; Heaney, R. K. . Mullin, W. J" e Re CriT. Rev. Food Nul., 1982,

18, 123·20 1. 2. Bradfield, C. A , Bjcldanes, L. F Vtl. Chern. Toxic. 1984,22,977-982. 3 Delaquis, PJ; Ma7..za G. Food Tech, 1995,11,73-84. 4. Oleson, 0 ., Sorensen, H .• 1. Allier. Oil Chem. SOC;. 1981 , 857-865 . 5. Helboe. l'. Olsen, 0 ., Sorensen, H., J. Chrolll_ . 1980, 197, 199-205.

131

6. Kjaer. A., Ohashi, M.: Wilson, J. M., Djerassi, C. Acta Chemica Scal1dillavica, 1963,17,2143-2 154

1.Bouchereau. A , Hamelin, 1; Lamour, T., Renard, M; LaTher, F; Phyux:hemis/I)'.

1991, 30 (6),1973- 188 1

Reasons for of Some

Chapter 12

the Variation in Composition Commercial Essential Oils

Chi·Kuen Sh u and Brian M. Lawrence

R. J. Reynolds Tobacco Company, 950 Reynolds Bou levard, Win ston.Salem, NC 27102

The reasons which cause the compositional changes for some commercial essential oils are due to (1) the effect of exlrinsic c~nditions, (2) the effect of interspecific and infraspecific dtffercl:ces, (3) the effeci of ontogeny, (4) the efft(;l of processmg parameters, and (5) the effect of adulteration Using examples, each effect is discussed_ .

Over the years it has long been known that commercially available essential oi ls vary from se~son to ~eason and from geographic source to geographic source_ However,. thIS questt?n of variation has not been carefully examined across a range of oils to detemune why such variations take place. Usually it is explained that chang~s. fro~ one se.ason to another result in compositional changes; however, thiS IS a little too Simple to explain some of the composi tional changes that are encountered.

As a result, we would lik~ to discuss some basic reasons why compositional ch~nges are encountered usmg examples of the magnitude of such changes The effects that will be discussed are: .

I . The effect of ~xtrinsic ~ondilio.ns (climate/geographical origin). 2. T~e ~ffect ~fmterspec'fic and mfraspecific differences (between and

wlthm species). 3. The effect of ontogeJ.ly (growth stage of plant material harvested). 4. T.he. eff~ct of processing parameters (dry 'Is. weI plant material on

dlSl1llal1on changes) S. The effect of adul teration (the addition of synthetic or other natural

components to "stretch" an oi l.

© t997 American Chemical Soci~ly

•

12. SH U & lAWRENCE Variations ill Compol·jtion oj Some Essential Oils

The EfTect ofClimlite and Geographic Origin on Oil Composition.

In 1989 Lawrencc et aI. (I) showed that peppermint oil, which is produced from clonally reproduced plams that are grown in different regions of the United States, could be readily differentiated based on Iheir area of cultivation and oil production. The areas of commercial production oflhese oils are the Midwest , Idaho, Yakima-Kennewick Valley in Washington, and the Madras and WiHamette valleys of Oregon. It was proved that geographical location and microenvironment were influential faclors which affect, albeit slightly, the composition of the oils produced in each region. Examination of the quantitative data of the most constituents revealed that it was extremely dimcult to differentiate the oils because of the similarity in the data. As a result, selected component ralios were used to prove differences between the oils. To simplify the component ratio data obtained from ca. SO samples of oil obtained from each growing region, Ihey were presented as polygonal representations. Such a presentation of data can be thought of as being pictorial pallern recognition of

the oils produced in each region.

139

The components ratios selected to differentiate between the oils can be

seen in Table I. Many of these ratios have multipliers that were used to ensure 1hat the polygons formed would lit the scale of the circular graph. What this means is that only oils produced in a specific region will have their component ratio data fit within the polygon. Oils whose component ratio dala does nOI fit within the polygon cannot be a pure oil produced in that specific region. The pallern recognition of the five different types of peppermint oil can be seen in

Figures 1-5 . To further usc this concept to differentiate between other oils produced

in the United States from different regions we attempted to differentiate between Native spearmint oils (ex. Mentba spicata 1..) grown in the Midwest and in the Farwest. Similarly, we also attempted to differentiate between Scotch spearmint oils (ex. Menlha iracilis sole), which are also produced in the Midwest and the rll.lWcst We could not use lighter regional differences between the oils because the oils are not sold according to the state or valley in which they are produced,

only by the broad regions of Midwest and Farwest. For both speannint oils the component ratios used can be seen in Table

n. Again, multipliers were used to ensure that the component ratio data for bOlh spearmint oils would fit on the same circular graph. The results of the component ratio data that were obtained for 20 samples of each oil type plus 10 samples of a second culling oil obtained from Farwest Native spearmint can be seen in Figures 6-10. The compounds chosen for component ratios were those whose raw analytical data varied so that a differentiation between Scotch and Native spearmint oils and 2"" cutting Native spearmint oils could be made. For example, Scotch speannint oil is slightly richer in limonene, 3-octanol, menthone and carvone as compared to Native spearmint oil, which in tum, is slightly richer in mycrene, 1-8-cineole, 3-octyl acetate. trans-sabinene hydrate, P-bourboncne and trans-carveol than Scolch spearmint oil. However, because it is difficult 10 look at raw analytical data and readily see slight differences, the use of

.i , '. ',' I 'I

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I

,"

'J ,

-140 SI' ICt;S, H A VOR CIIEMISTRY ANU ANTIOXIOANT l'IW Pt;RTlES

T able I. CO"' IIOnl'; lI l Ral lo) Selected for Peppermin t Oil OifTt rt nl ili l ion Accordin g 10 Origin I. 1,8·CineoleILimonene 2. 1,8·CincoldM:enthofu ran x 112 J. 1,8·Cineole/Menlhyl AcelBle 4. 1,8·CineoleIMenlhol x 50 5. 1,8 CineoleIMenthone x 25 6. MenlhoruranIMenthOllC x 100/6 7. Menthofuran/Mentho] x SO 8. Meru hofuranlMcnt hyl Acetate x 5 9. McnthofuranILimonene 10. MenthoneiMcnlhyl Acetate x 112 II . Mentho l/Menthane ! 2. Melltho l/Menthyl Acetate x 112

, 12

Midwest

FigUi'CS 1-5 I'attern Recognitio lls of th t Five Types of Peppermint Oil

• 12. S IIU & LAWRF",,"CE Vtl riations in Composition of Some E.~.umtioJ OiLf 141

7

4 12

2 Wilamette

Figure 2.

7

,

Madras

fo' jgure 3 .

.•

142 SJ'JCf;S: HAVOR CIIEM ISTRY AND ANTIOXIDANT !'U.O\'ERTIES

7

12

9 Kennewick

Figure 4.

4

12

Idaho

Figure S.

12. SIIU & Lo\WRENCE Varia/iolls in Composition of Some w 'rn/iot Oils

Table n. Componen l R:uios Ustd to Differentiale between !\'Iidweslllnd Farwesl Native Spearm int Oils !Iud Scotch Spellrln int Oils

•

I . LimoncneIMyrcentx In 2. Limoneneli ,8·Cincoie x 112 3. i.irnonenel) ·Qc\aool x 115

Limonenelp.Bourbonene x lIS MyrceneJj3.Bourbonene x 2 I ,8-Cineolc/Myrcene x 5 CaIVone/Myrcc!!c x 1110 CarvonelJ3-Bourbonene x 1/10 3.OctanollJ-0cIyl acetate x 112

4.

5. 6. 7 8 9. 10. II 12

I ,8-Cineo]eltrans.Sabinene hydrate x 112 cis·CarveoVlrans-Carveol x 1/ 10 Carvone/trans-Carveol x 11150

6 " "

2 " 3

..

9

' 12

Midwest Native Speannint

Figum 6-JO " I Uern Recognitions oflhe Five Types ofSpcllrmill1 Oil

143

144 srIC ES: HAVOM C IIEMJSTRY AND Al'/TIOXII)ANT I'WQ I'!:: H.T IF,S

Figure 7.

3

Figure 8.

Far West Native Spearmint

Midwest Scotch Spearmint

12. S II U & LA .... 'RENCE Jlariatiotu in Composition of Smne Esselltial Oils

Figure: 9.

3

I-igure 10.

Far West Scotch Spearmint

2nd Cutting Far West Native Spearmint

i

145

-146 5I'ICES: FlAVOR CIIEMIS1'RV AND ANTIOXIDANT PROPERTU:S

component rat ios magnifies these differences so that they can both be readily discernible and can be easily displayed pictorially.Examination of the limoneneimyrcene ratios for each different speanninl oil , we have 1.81-3_39 (Midwest Native), 0.88-2.68 (Farwest Native), 1.60-2.02 (2"" cutting FarweSI Native), and 6.99-9,27 (Midwest Scotch), 8. 12-1 1.48 (Farwest Scotch). From these data it can be seen that it is easy to different iate Native spcannint oil from Scotch speannint oil. Using the eleven other component ratios merely makes Ihis differentiat ion task easier. When Ihis data is presented as a polygonal representation. this differentiation is readily evident from these pattern recognition figures (Figure 6-10) that:

(a) Native and Scotch spearmint oils can be readily differentiated. (b) Farwest and Midwest Scotch and Native spearmint oil can be

readily differentiated. (c) A second cutting of oil ofFarwest Native spearmint can be

readily differentiated from all orthe other spearmint oils.

The Effect oflnlerspecific and Infraspecific Differences on Oil Composition.

In the previous above example, it was shown that two spearmint oils, which were produced fro m don.a Uy reproduced distinctly different species of bkmhA. produced oils whose compositions were similar but could be different iated; however, the detailed oil composit ional data were not presented. To address this point, the analysis of oils that are known commercially as cedarwood will be the next subject o f discussion.

Around the world a number of oils are produced that are described as being cedarwood oil. Each of these oils is produced from a different species as can be seen in Table m. The oils produced fro m Atlas cedar. t-limalayan cedar, Incense cedar, Lebanon cedar, East African cedar, Oregon cedar, Mulanje cedar, Alaskan cedar, Eastern white cedar and Western red cedar are known to possess different chemical compositions. The major components of these oils can be seen in Table IV. 10 contrast, the Texas, Virginia, Chinese, and Atlantic cedarwood oils, although produced from differen t species, all possess composi tions thaI are qui le similar (Table V) Using the hypothesis that it is easier to look II component rat ios rather than raw data, the components that show most variation between the four cedarwood oils were a-cedrene, thujopsene, j}-himachalene and cedro!. As a result, the component ratios were examined rather than the raw data (Table VI) , A comparison between these data reveals that Virginia cedarwood oil can be clearly differentiated by the thujopseneia-cedrene and j}-himachaleneicuparene ratios. It is more difficul t to differentiate Texas lind Chinese cedarwood; however, from the thujopsene/cedrol ratios it is possible. Finally Atlantic cedarwood oil possesses a unique j}-himachalene/cuparene ratio, thereby different iating it from the other oi ls.

To !Unher discuss this qUes1ion of interspecific differences in composition. lemongrass oil wi ll be used as II second example. There are two forms of lemongrass oil produced commercially, one produced in Asia from Cymbopo8QO 0txU0SU$ (Nees ex Steud.) Wats. and the other produced in the

1.2. SHU & IAWRENCJo: Vllriat;ons in Composition of Some ~ntial Oiu 147

Table In. Various "C,dan'" that have heeD 1I.'ed 19 produce ceda rwood oil I Virginia Cedar Juniperus yirg iniaoll L. 2. Texas or Mexican Cedar Juniperus ashej Buchholz 3. Western Red Cedar Thuja pljClIllI Donn ex. D. Don 4. Northern or Eastern White Cedar Wa occidentalis L, 5, Kenyan or East African Cedar .illni~ Hochst ex. Endl 6. Chinese Cedar Chamaecyparjs funebris (End!,) Franco

7.

8. 9. 10 .

II 12. IJ

" 15

Alaskan Cedar

Oregon Cedar

Mulanje Cedar Atlas Cedar Lebanon Cedar Himalayan Cedar Atlantic Cedar Incense Cedar

(syn. CU P feSSUS funebris End!.) Chamaecyparis nootkatensis (D. Don) Spach Chamaecvparis lawsoniana (Andr Murray) Widdringto nia liupressoides (L ) End!. Cedrus a!lal11ica G. Manett Cedrus libani A. Rich. Cedrus deodara (Roxb.) Loud. CbAtllilecypads tbYQjdes (L) B,S.P. l .jbQccdrus decurreos Torrey

Table IV. !\b jor Com l,Qnents in Various CcdltI"WQod Oils

3.

4.

5.

7.

8.

9.

methyl thujale 10. a-himachalene T-muurolol j}_himachalene tropolooes l" -himllchalene

occidentatol occidol

cedro!

l"-cadinene I ( IO)-cadinen-413-ol

o-cadinene a-pinene borneol

thujopsene cedrol B-himachalene

II .

12 .

14 .

a-torosol ~-Iorosol (E)_a llanton_6.0!

(Z}-a-atlantone (E)-o.-atlanlone

p. tnethoxythymol carvacrol tropolones

I !

I

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14. SI'ICES: HAVOI{ CII EMI!t-rRY ANt) ANTIOXIDANT I'ROI'I-;RTmS

T"blt'. V. J\lflin ComlloncnlS of Four Crd!!nvood Oils C(l nJpolJ ud V T C A «-cedrene 38_0 226 33.8 20.5 j3-cedrene 9.2 5.5 ' .1 5.0 thujopseoe 23.4 ". 360 31.7 cx.·h imachalenc 0 .' 07 j)-chamigrene 14 L5 (3-sclincne 0.' p.himachalenc 2. 1 II 0.7 0.2 a.-charnigrcne L5 1.6 ar-Curcumene trace 0.3 cuparene 0.' I., 22 J5 cedrol 12_3 122 85 lOA widdrol I.' 1.1 I I 14 a-bisabolol Irace trace Irace 0.) Ccdanvood Oils: V . Virginia C - Chinese

T - Texas A - Atlantic

Table VI. Component Rat ios Used to Differentiale Between Th ujoluene-rich Ced:uv.·ood Oils

Com pound Ratios V T C A tllujopseneJa-cedrene 2 - 3 • - 9 4 _ 5 6.5 - 7.5 thujopsene/cedrol 1.7-21 3.6 - 4.0 4.0 - 4.4 2.9 - 3. 1 13-himachalen!iCjll2ar~n; 2.2 - 2,1; Q ~ - Q 7 Q 2: - Q 4 001 - Q, I Cedarwood Oils: V · Virginia C · Chinese ,. T - Texas A " Atlantic

nhle VII. C hem ical Composition of t;, Indian Lcmongran Oi l Comtlound ludiau I Indi HlI II I1hytanese Il.-thujene trace 2.8 Il.-pinenl;: 0.4 5.4 camphene 0.5 1.0 10. 1 myrcenc Irace 10.3 limonene 2.7 3.8 ' .3 4-nonanone 0.5 0 .3 trace 6-mel hyl- 5 -hepten-2 -one 2.6 2.5 32 citronellal O. 0.' 08 linalool 14 I I 2.1 Il-caryophyllene 13 0' 0.4 neral 32.6 34. 1 18.5 geranial 40.8 44.3 '1.2 .7 geran)'1 acetate 3.' 2.2 3.4 cit ronellol 0.5 0.6 0.6 geraniol 4.2 )8 22

12. SHU &: LA''VIU:N<':E VariatioftJ in Compo.vitioR of ScIM Esuntial Oils

New World from CyroboPOiQO c jlratus (DC) Stapf Authentic oi ls obtained from each species were subjected 10 analysis and by GC/MS. A summary o r lhe differences betwun their compositions can be seen in Table VII and Vii t Although the differences between these oils appear to be major be<:ause of the high limonene content of the West Indian oils, il is easier and more prudent to use component ratios to differentiate between the oil as shown in Table IX. It is believed thai examination of the hydrocarbons and oxygenated compounds separately is II good way to examine Ihe authenticity of an oil.

The existence of morphologically identical plants which possess oils that have differing chemical compositions is not a new concept . In fact, the existence of infraspecific differences is widespread in the Labiatae and Compositae families, but it is IIOt limited to them. A few years ago Lawrence (2) showed that Qcimum basjljcum L. can contain oils thai possess a variety of compositions. It was also found that oils contained constituents that were biosynthesized either via the shikimic acid pathway, or the mevalonic acid pathway, or both (Figure I I ) . A summary of the data obtained during the analysis of more than 200 separate Q basilicym plants revealed that they possessed oils that, IIOt only contained components from single pathways or dual pathways, but also wi thin these groupings a wide quantitative variation of constituents was also observed. A summary of these dala can be seen in Tables X and Xl.

The Effect of Growth Stage of the lJar"l'uted Plant on the Composition of Its Oil.

Although the effect of growth stage of the plant on oil composition should be well known, oils produced commercially are not always produced year after year from plants that were harvested at their same growth stage. Two examples will be used to illustrate this point.

First, oils produced from Coriaodrum satjyym L differ chemically quite drastically (3) as can be seen from the dala presented in Table XII. The oils that are known as cilantro oil are generally those produced from plants at stages I and 2; however, the oi l yield is higher the later the plant is harvested. Nevertheless, using the ratio between decanal & (E)-2-decenat and linalool and (E)-dodecenal, a reproducible cilantro oil can be obtained (Table Xl1I) .

A second example of a change in chemical composition as the plant matures can be found with the oil ofTagetc:s mjnula L. (4). E:tamination of the main acyclic hydrocarbon and the acyclic ketones (Table XIV and Figure 12) will allow the user to select an oil that is most liked. From these data, component ratios can be used as a quality check for oil reproducibility. The reason why oils can fall outside the desired component ratio can be understood based on the data presented in Table XlV.

T he E rred of Processing Parameters on Oil Com position

In 1985, Schmaus and Kubeczka (5) examined the influence of oil isolation condit ions on the composition oflinalyl acetate-rich oils. They found that the increased water content in plants distilled when fresh, resulted in a corresponding

II

14'

ISO SI'ICES: n AVOR CIIEI\USTRY AND ANl'IQXIIlANT PROPERTIES

Table Y in. COlU lJ OU lid

Chemic!! 1 Composi lion ofW. lndian umongrll$$ Oil Gualtmllin I Guatemala n

limonene 4-nonene 6-methyl-5-heplen-2-one citronella! linaloo1 /3-caryophyllene !leral geranial gerany! acetate citronellol geraniol

12.3 15.9 1.8 2.4 3.0 44 0.' 0 .• 1.8 2.1 2.2 1.3

30.5 28.6 37.3 37.1

L1 1.1 04 0.2 46 26

Table IX. Componerll Ratios Used to Differentiate between E. Indian lind W . India n J.emongrass Oil

CllmpOntntlRfi tio E. Indi lln methyl heptenonel4-nonanone 5.0 - 9.5 geraniollgeranyl acetate 1.2 - 1.8 citronel1allcit ronel1ol \ . 1 - 1.7 neralllimonene 8 5 - 12 5

W, India n I 0 - 2.0 2 .2 - 3.0 2.0 - 3.2 ! 5 - 3.0

Table X. Major Components of Various OC;lIIum bm"ilicum Selections 'Siog l~ Rios!nthetk Pl!thwfI:\:s)

Compound I(a) Hbl 2{a) 2(b) 3(/1) 4(a)

1.8-cineole 3.2 4 .• 1.4 2.7 2.4 3.4 (Z)-p-ocimene 0 .1 0.3 0.2 0.1 tracc Irace (E)-p-ocimene 0 .• 5.4 1.0 2.2 0.4 Irace camphor 0.2 1. 8 OJ 4.3 0.' 1.8 linalool 0.5 4.8 83. 51.6 0 .• . .2 methyl chavico! 85.3 49.0 0.4 0.7 1.5 5.0 geraniol Irace trace 0 .3 22.8 Irace ,"" methyl eugenol I.. \2.2 trace trace 67.7 0) (E)-methyl cinnamate Irace trace trace trace 03 640 fi!g~DQI I[l!et Q,Z Q.l trace Z·O ,~" Legend :Chemolypes of OcimulII basilicllnI with a Single Biosynlhetic Pathway:

Type! (.) methyl chavicol-rich (b) methyl chavicol>lI1cthyl eugenol

Type 2_ (.) linalool-rich (b) tinalool>geraniol

Type 3_ (.) methyl eugenol-rich Type 4. (.) methyl cinnamate-rich Type 5. (.) eugenol-rich (not found)

12. SHU & lAWRENCE Variatioll\' in Compm'ition of Some El'senlial Oi/.f 151

T llble XI. Major Components of Various Odmum bllSifi(.uIII Sd cctions (Dual Biosyn lhelic PalhwllYs)

Compound He) IIC) ' zee) 1,8-cineole 5.7 3.8 4.8 (z)-I}-ocimene 0. 1 (E)-I)-octmene 2.0 camphor 1.5 linalool 22,9

lCc)' I.. 0 .• 1.0 0.2

51.0

2(dl 3.8

'4 2.5 4.'

30.2 methyl chavicol 52.1 2 1 0 2.1 geraniol trace 0.2 0.4 methyl eugenol 0 .1 0 I 10.2

Z(e)

2.8 0.2 1.0 LS

34.2 1.2 0.2 0. 1

Ji b) 7.7 0 .1 3. 1 0.8

21.2 0.7

Irace 38.4

(E)-methyl cinnamaletrace trace trace 27.8 trace eugenol trace lrace 37 139 148 42 5 I Legend : Chemotypes orOcinllIm basilicum with a Dual Biosynthetic Pathway:

Type I. (c) methyl chavicol>linalool (c)'melhyl chavicol>linalool>methyl cinnamate

Type 2. (c) linalool> mcthyl chavicol (c) 'Iinalool>methyl chavicol>eugenoJ (d) linaloot>eugenoJ>methy\ eugenol

.... (e) linalool>methyl cinnamate Type 3. (b) mcthyl eugenol>linaloo\

Ta ble XII. Conlpa rative chern ital co mposi tion of Cor;fIIltlru m snt;I'uIII 1_ at Various Sugts 2fMaturit!

Stages of Plant Maturity2

!::ol11 lH!!!nd I 2 3 4 5 6

Octaoal 1.20 1.20 0.85 0.66 0.44 0.35

Nonanal 0 .5\ 020 0.11 0.05 0.05 0 .08

Oecanal 30.0 18.09 11.91 6.30 6.24 1.61

Camphor 0.08 Irace 0_52 1.26 2,18 2.44

(E)-2-0ecenat 20.6 46.5 46.5 40.6 30.2 3.9

Oodeeana! 3.)0 ,.7 0 .26 064 0.52 0.41 (E)-2-Undeeenal 2.56 2 1.7 13' Tridecanal 3.07 J.87 2.02 0 .92 1.08 0 .46

(E)-2-Dodecanal 7.63 8.14 5.95 4.59 4 ,78 2.19

Telradecanal 0.68 0.30 0 ,12 0. 15 O.I! 0. 15

(E)-2.Tridecena! 0.42 0.21 0 .14 0.09 009 O. i3

(E)-2-Tetradecenal 4.45 2.57 1.73 1.53 15' 1. 73

Linalool 0.34 4.27 17.47 30.05 40.88 60.37

Geraniol 0.12 0. 1 I 0.35 0.7 1 0.93 1.42

Q~[ln:t1 !!!i=tlal~ ! 11 Q 1~ Q 16 0 ,69 Qli:2 Q 26 l Stages of maturity: 1 - noral initiation; 2 - nearly full nowering; 3 .. full flowering,

primary umbel young green fruit; 4 '"' past fu ll flowering 50010 nower, SOOIo fru it; 5 - full green fruit; 6" brown fruit on lower umbels, green fruit on upper umbels: -- - nOI detected

I

I.

.! " ·1 ., ,

,~

• . /

[52 SI' ICES, H.AVOf{ C I I .~IISTRY ANU ANTIOXII)ANT j'ROl'ERTIES

Table XU I. Component Rat io! of CillUll ro O il Consti luenlS as II I1I C11suremt ll 1 of oil gualily

Com ponen t Rll iio Siages of Matu rity , 3 • 5 • dccanaV(E)-2-dccanal 1.5 OA 0.3 0.2 0.2 OA lill1\loo!/(El·2-dodecenal O,Q Q,52 294 6.55 8,55 24,J

Table X I V. Chll.nge in per(cnlnge com posil ion of T(lgclc$ minI/til oil dur ing m, S;xs:I, of plnn t

Development Dihyd ro- (Z)- (E)- (Z)- (E)-

St~l:!.: {~}·O·os;imtne t:li:;etone Tnl:,etone T IIKetenoue vcgetativc 16.9 46 .4 22.4 3.2 L5 2.0 buds visiblc 11.8 51 .3 18.5 14 44 3.0 buds opening 21.9 36.0 24. 1 LJ 6.9 L5 !lowers opening 33.3 30.0 17.5 20 , .• 1.9 full flower 35 .3 30.5 16.9 22 ' .9 " immature fruit 41.3 24 .1 ' .2 03 18.2 2.0 ma!yr~ fruil 4~ 2 148 7 . ~ 04 20,4 J2

Table XV. Compllrllt ive Chem iclI l CO lli position of French a nd R ussian Clllry Sage Qib

Compound Frtnch Oils Russian Oil . , A {dried} n {fresh}

a -pinene 0 .20-0.21 0 .08 0.02

p-pinene 0 .34 - 0.J8 0.98 0.22 limoncne + 1,8.cineole 0 .19-0.20 0.29 0.41 (Z)-I}-oeimcne 0. 16 0.40 0 .09 (E)-I}..()Cimene 0.2 1-0.23 0. 72 0.06 linalool 888·8.98 28.48 14.43

a-terpineol 0 .20 - 0.21 2.64 2. 11 11<':1"01 0 .05·0.06 0.56 0.42 geraniol 0. [ I 1.54 0.98 linalyl acetate 72.41-14 . 18 50.86 63 . 18 lI<.:ryl acetate 0.13-0. 17 0.75 1.03 1;;eranyl acetate 0 .30 - 0.40 1.42 2.01

(3-caryophyllcne 1.85· 1.89 1.65 202 £Cl"1ll3Crene D 3.29 - 4.09 3.54 0.36 caryophyllene oxide 0.35·040 0.16 0. 16

~cI!ir!<2 1 121-1.27 0.91 1.812

12. SHU & lAWR~'1IICE Varia/imll' in Compru'itiorl of Some Esnmlial Oils

increase in distillation time and a decrease in pH causing partial hydrolysis of linaly! aCdate followed by partial acid catalyzed degradation orlinalool result ing in an increase of (E)- and (Z).p-otimene, limoncne, terpinolene, a-terpineol, geraniol. ncryl acetate, and geranyl aeclale. In 1985, Dore and Jaubert (6) presented an example ofthesc effects as it applies to clary sage oil (Table XV~. Similar results were presented by lawrence (7) (Table XVI) where the U.S. 011 was produced from fresh plam material, while the other two oils were produced from dried material. The effects described by Schmaus and Kubeczka (5) are very evident in these results. A processing parameter that needs to be mentioned is storage. If an oil is stored under poor conditions such as in a half·filled drum in which some dissolved water has deposited causing some minute rusting in the drum, oxidation of the oil can and does occur. 1\ self-explanatory example of the oxidation of coriander oil can be seen in Table XVII.

The EfTect of Ad ultera tion on the Composition or a n Oil

Two examples of oil adulteration will be discussed namely, lemongrass oil and peppermint oil. Examination of the data presented in Table XVIll shows the analysis of three commercial samples oflcmongrass oil. Sample Nos. 2 and 3 are grossly adulterated because they were found to contain ca. 20% of either diacetone alcohol or triacctin as an adulterant . In contrast, Sample No. I has to be examined from the standpoint of component ratios. A comparison between the component ratios ofE. Indian, W . Jndian lemongrass oi l and Sample No. I (Table XIX) reveals thai the oil is not a troe example of either lemongrass oil; therefore, it must be adulterated.

Over the past few years the production of Indian peppennint oil has increased. both in the Punjab area as well as in the Dareilly region of Uttar Pradesh. As this oil is substantially lower in price than North American peppermint oil, an economic temptation exists 10 mix Indian peppermint oil with one oftne U.S. peppermint oils and sell it as U.S. oil. I\s a result, it was decided to determine if this practice al the 10'10 adulteration level could be readi ly

detected.

[53

Initially, 18 oils from both regions were analyzed by Ge/MS 10 ensure that there were TIQ unusual constituents Ihat would interfere with further studies. The component ralios shown in Table I were calculated, and these were plotted on a circular graph to show the typical pattern of an Indian peppermint oil (Figure 13). From the individual Indian peppermint oil samples, a composite was made, and it was added ala \0% inclusion rale into a composite oil of the Midwest, Idaho, Kennewick, Madras, and Willamette peppermint oils that were analyzed earlier. These adulterated oils were then analyzed by GC and the important peak identities were confirmed by GClMS. The component ratios of each adulterated oil were calculated and .",ere plotted against the original pattern recognition polygonal representations of the oils from each region oil (Figures

14-18). I\s can be seen, the data plot for the U.S. oi l containing 10% of the

Indian composite oil fell outside of lhe polygons, thereby confinning that each oil was adulterated.

I

IS4 SI'ICES: H AVOR ClIEI'tIl:nRY AND ANTIOXIOA NT I·RO I·EM.TI~.:s

SHIKIMIC ACID

PATHWAY VIA

PHENYLALANINE

MEVALONIC ACIO PATHWAY

VIA GERANYL PYROPHOSPHATE

_ METHY~ CHAVICOL 1

l: I ----UNALOOL

METHYL EUGEN~L I I 7 GERANIOL

EUGENOL

METHY~ CINNAMATE

«'igure 11 OUIII Biosynthetic Pathway Fou nd in Ocimum JJilsificum

(Z)- (E)-

tllgetenones or J!-oclmenones

(Z)- (E)-

tagetones or 2.3_dlhYdro_p_odmenones

dlhydrotagetone

Figure 12 Tagetes Oil Ketones

12. Sil l! & LAWRENCE Variations ;/1 Composition of Some &sential Oils IS5

Table XVI. ComQllralive Chemiclll Composi tion of Commtrical Cl:u), Sage Oil Compound U,S. O il Frc lI ( h Oil Russia n Oil

a-pinene myrcene limonene (Z)·Il-ocimene (E).p-ocimene linalool

0.17-0.22 0.1-0.3 0.2-0.3 1.25-1.7 1 0.1-0.2 0.3-0.5 040-0.77 0.1-0.2 01-0.2

linalyl acetate p-caryophyllcne a . terpineol germacrene 0 nery! acetate geranyl acetate ocrol

0.42 - 0.70 0 .43 - 1.36

20.29 - 28.6] 44.9-S34 0.86 - 1.3 1 1.05 - ] .05 2.6] - 3.55 1.00 - 1.67 19]-].24 0.62 - LI S 1.67-326 geraniol

caryophyllene oxide sclacco\

0.2 1 - 0.27 017-044

"'''' 0. 1 - 0 .2 9.0 - 16.0

49.0 - 73.6 \.4 - 1.6 02 - 0.6 \.6-20 0.2 - 0.] 0.]-05

trace - 0 I 0. 1 - 0,] 0.3 - 0.5 0 1 - 0 2

trace - 0 .2 0. 1 -0.4

10.4 - 19.] 45.]-61.8

1.1 - 1.8 1.2 - 2.5 0.7 - 2.0 0.4 - 0 .6 0.8 - 1.2 0.] - 0.5 0.6 - 1.2

0.5 0. 1 - 0.2

Tahle xvn. The Effeci of Stnrfli!.e (Oticialion) on Coriander O il Compound p-cymene cis-linalool oxide (furan) trans-linalool uxide (furan) camphor linalool 4-methyl-5-hexen-4-olide cis-linalool oxide (pyran) trans-linalool oxide (pyran) p~ymen-8-Q!

Good Oil Oxidized Oil 1.7 -], 8 1.9 0.2 - 0 5 0 .2-04 2. 1 - 4 4

70.3 - 77.1

14 . 1 12.] 63

]8.1 3.1 1.5 14 06

T ll ble XVIl!. ChemiclI l Com llosi tion of ,\d uhtra ted Lt mongrass Oil Compound I 2 3 myrcene 1.8 limonene 4-nonanone 6-methyl-4-heptenone 3,4 diacctone alcohol triacetin citronella! 0.6 linalool 2.9 I}-caryophyl lene 0 .5 neral 36,2 geranial 45.5 geran)'l acetate 0.7 citronellol 0.3 geraniol 26

trace Irace

I.' 1.6 0 .6 0.4 1 9 1.6

21.3 25.8

0.5 0.5 J.l 1.5

1.0 0 .9 26.3 24. 1 32 .7 J 1.0

4. 1 3.8 0.3 0.3 6,0 56

~:

i j

I >

}. '. , t i i "

I

t I 1 J ., ., 1

i I

156 SPICl'~": FlAVOR CJl EMISTI{Y AND ANTIOXIDANT I'IWI'ERTIF.5

Table X[X.Componen t R:lIios Used to Differenliate Bel ween 1<:. ind ian and W. Ind ian Lelllollerass Oil lllnt an Adult rated Oil (Sample No. L)

Com pollent Rat io E. lndian W. lnd ia n Samllh: No.1 methyl heptenoncl4-nonanone geraniol/geranyl acetate citronellal/citronello1 n"rall1imOIlCIlC

6

5

1

2

5.0 - 9.5 1.2 - 1.8 L1 - I. 7

8.5 - 125

3

1.0 - 2.0 2.2 - 3.0 2.0 - 3.2 L5 - 3 0

9

10

12

11

India

3.7 2.0

Figure 13 Pattern Recogn ition or all Indian l'cpperl11in t Oi l

12. SHU & IAWI{~:NC£ Variations in Composition of Some Es.wntial Oils

Midwest 3

Figures 14-18 Pattern \{ecognit ions or the Five Adulterated Pellperminl

Oils

•

Willamette 3

Figure 15.

157

15' SPICES, FlAVOR CHEMISTRY AN D ANTIOXIDANT ,'ROI'ERTIES

Madras 3

Figure 16.

12

Kennewick

Figurt 17.

•

12. SHU & I.AWRENCE Yar;aliollS in Compositiot1 of Sorm &selltial Oils

Idaho 3

•• <

Literature Cited

I. Lawrence, D, M,; Shu, C-K.; Hams, W. R. Per/11m. Flav. 1989, 14(6), pp.

21-30. 2. Lawrence, B. M. In Flavors alld Fmgrallcts: A World Perspective. Lawrence. B. M.; Mookerjee, B. D.; Willis, B. 1., Eds., Elsevier SeL Pub!., Amsterdam, 1988, pp. 161-169. 3. Lawrence, B. M. In New CropS; Janick, 1.; Simon, J. E., Eds .• 1. Wiley & Sons, New York, 1993, pp. 620-627. 4. Lawrence, B. M Progress ill Essential Oils. 1992. / 7(J). pp. 131-133. 5. Schmaus, G.; Kubeczka, K. H. In Essential Oils and Aromatic Plams. Baerheim Svendsen, A.; Scheffer, 1. 1. C., Eds., Maninus NijhoffJDr. W. Jank Publishers, Dordrecht, Netherlands, 1985 pp. 127-134. 6. DOTl~, D.; Jauben, J-N, Par/11m. Casmef. Ar6me.r. 1985,61, pp_ 79·85 . 7. Lawrence, B. M . In Advo/J(:t:s ill LAbiatae Science; Harley, R. M., Reynolds, T., Eds.; p, Royal Botanic Gardens Kew, 1992, pp. 399-436.

15,}

Chapter 13

Component Analyses of Mixed Spices

C. K. Ch engl, C. C. Che n l, W. Y. Shu l , L L. Shih l, a nd U. II . Feng l

Lrood Indu stry Reseatl,:h a nd Developme nt Institute, P.O. Box 246, Ihinchu 300, Ta iwa n, Republic of Chin a

l lnstitute of Sta tistics, National Tsing-Hua Uni\'crsity, lIs inch u 300, Tniwan, Rc public or Ch ina

A qualitative and quantitative technique for recogniling spices as

well as the mixing ratios in an unknown spice blend is established

via numerical analysis based on a database consisting of 355

difTerent spices. Formulating the spice recognition problem as a

constrained opt imitation problem is a key step. A similarity index

measuring the degree of similarity between two spices is proposed,

which is very useful in the process o f spice recognition. For

purposes of testing, an existing mixture (basi l, 47.9%; clove,

12.8%; mint, 9.4%; sage, 23 .8%; wintergreen. 6. 1%) and a trial

spice blend with known composition were analYled by the .< technique. The results are surprisingly accurate.

Humans have been consuming spices and spice blends for several thousand years.

Even to this day, spices and spice blends are highly valued commodities from all

over the world (J - 2).

From the flavor point of view, the major attributes of most spices are

contributed by essential oils. These can be extracted by steam distillation (or

similar process) of ground spi~. With the advent of gas chromatography (GC)

and the combined technique of GCI mass spectrometry (MS), analyses and

identifications of volatile components of essential oils extracted from different

spices have been very thoroughly and well documented (3-5). However, most

of the published reports were based on the analysis of an individual spice. When

it comes to food processing, usually it is a spice blend instead of thc individual

spice which has been added to the fonnulation.

Cl t997 Amcrican Chcmia] Socicly

13. CIJ FJ~G ET AI.. ComfHJmmt Analps of Mir£d Spices

Detection ofan individual spice in a spice blend can be achieved through

sensory analysis, instrumental analysis, or the combination of both. When

analyz.cd by a sensory method, which requires trained panelists, only quali tative

results can be obtained (6). flor semi-quantitative analysis, there have been two

types of approaches to invest igate the content of an individual spice in a spice

blend. One is based on calculation of major components in a mixture as

compared to published results . A good example has been dcmonstrated by

Lawrence and Shu (4). The more advanced approach is based on a computer

pattern recognition method. Chien (7) has shown a very genius mathematical

method to analyze the presence of an individual essential oil in a mixture. In that

report, fou r out of six essential oils with contents ranging from 5% (geranium,

cedar wood, patchouli) to 15% (bergamot) were correctly identi fi ed. The other

two oils, galbanum and Dourgeon de Cassis, were not picked up by the computer

because of their low level (0.25%).

This paper presents a qualitative and quantitative analysis of an individual

spice in a spice blend. The ratios of each spice were calculated by using a

computerized numerical analysis wi th a data matrix composed orJ55 spices and

accumulated total of922 volatile compounds. The principal part oflhis method

is the solving of a constrained optimil.3tion problem.

Th w ry

161

Thc problem of rC(;ogniling the individual spice as well as the mixing ratios in

a spice blend is first fonnulated as a constrained optimization problem. We then

solve the problem by numerical methods. The procedure is dcscribed below.

Vector r cprcsenta lion or spices. Each spice can be characterized by its

components and composing ratios. Mathematically. each spice can be treated as

a vector X=(x/.x1o".,xJ . where x, represents the ratio of the I-I h compound in

that specific spice. In the present study. 355 different kinds of spices were

collected and 922 volatile compounds (n=922) were counted among these 355

spices. Therefore. n is equal to 922 and x/+x2 + •.. +xm" l. The 355 spices were

represented by 355 vectors X~X) ...• XJl7 Using Anise # I collected in the present

study as an example, this is shown in Table t . In Table I, each code number represents a single volat ile compound

identified in the essential oil. e.g., code no. 236 represents estragole, code no.

162 S I>I C ~:S, n A VOH CIIJ.;;\USTRY AND ANTIOXIDANT " ROI't;RTIES

Table I . The volat ile compounds o r anise #1.

Code No. of Ratio el.)

compounds

"6 2.513 187 1,487

236 4.975 250 0,121 292 0.312 29J 0.221

'" 0010 454 2.382 511 0.784 5J2 0.030 540 86.683 666 0.22 1 667 o 76J

Som 100

13. CII"NG);'f A I~ COmpollt 1lt AI/(llyses of Mi:ad Spit'S 163

Equations 3-5 indicate thaI if spice blend X is compared with itself, then

JlIlier(J(X)= I , if component s in spice blend X can not be found in Yand \lice versa,

then '"dex(X.I')-o, otherwise, the index shall be ranged between I and 0 Therefore,

Jlldex(X, Y} can be used to measure the similarity between X and Y.

Similari ty index has ils rna lhematical meaning. Any spice vector can be

considered 15 a probability distribution on the sel {I.Z, .... ,922}. The well ·kllown I squared Hellinger distance between two probability distributions X and Y is defined I

" 9'2~ 2 911

'jj E: - 50) = 2 -2 I E:5o = 2[1-lndex(X, y)], (6) ,.,

It is very clear that the larger the bldex(X, t) is, the smaller the Hellinger distance

bctween X and Y wi!! be. Therefore Imlex(X.t) directly refl ects Their similarity. For

example. Anise#2 (9) and Anise #4(10) have many volatile compounds in common

(Table II) . The similarity index between these two spices is 0 .964 indicat ing high

degree of similarity. On the contrary. when volatile compounds of Anise #2 are

'I !

540 represents trans-anethole. A spice blend X ofindividual spices contained in compared wi th those of coriander # I (/I) • as shown in Table III , an index value of 0

our collection can be viewed as a complex combination of vectors X,.X}o .... Xm: is obtained, indicating no similarity between these two spices.

where tJ i is the mixing ratio of the ;-th spice in the spice blend _ For a given

unknown spice blend Y, identirying the spices contained in Y amounts to finding a

t;orwex combination X'" fj /X,+ fj :X}+ ... + fJ J.uXJJJ as "close" to Y as possible

Methods for measuring the ctoseness between spice blends X and Y will be

discussed next.

Similarity ind ex For any two spice blends X=(x,.xz, " '''9}}). and Y=(y,.yZ, .. . Y9U),

the similarity index. denoted '"de.x(X.Y). is defined as:

'" llldex( X ,Y) = I .r;;.;y;, Basically. Inde.x(X,Y) has the following propenies.

For any two spice blends X and Y. 1.0 :i lmlu:(X. y) ~ I

2. If X- Y, l llllex(X, 17- 1

3.U cO lll llo nt nls in X CAn not bf fo und in Ya nd vice Yfrs a, thf n

ltUlt! I (X, Y)=O.

(2)

(3)

(4)

(5)

Table 1[. Comparison of the volatile compositions of anise #2 and star ~nise

#4. The similarity index " 0.964 Codo: No. Rallo Code No. Ratio

Anise'2 SUir IIllise'~ Anise .2 Star anise u 20 000000 0.00081 436 000000 0.00081 40 0.00000 0.00092 437 0 .00010 0.00581

60 0.00000 0 .00 102 440 000000 0.00112

IS. 0,00567 0.04563 450 0.00000 0.00051 236 0.00304 0 ,00346 454 0.00000 0,00112

257 000000 0.00509 467 0.00010 0.00051 292 0 .00608 0 .02954 480 0.00365 0.00428

29J 0 .00263 0 .00428 499 0.00020 0.00346

lOJ 0.00000 0 .00092 51' 0.00010 0.00000

331 0.00000 0.0006 1 531 0,00000 0.01039

315 0.00000 0.00 122 512 0,00010 0 .00183

175 0.00010 0.0005 1 540 0.97376 0.87044

386 0.00020 0.00387 552 0 .00000 0.0014)

38' 0.00010 0.00000 666 0,00405 0.00000 425 0.00000 0.()()()41 745 0 .00010 0 .00000

164 SPICIiS: FlAVOR Cn EMISTRY AND ANT IOXIIJANT 1'R:Oi'ERl'll':S

Table 111 Comparison of the volat ile compositions of anise 112 and coriander

III . The similarit:r: index - O.

Code No. Ra1io Code No. Ratio

Anise 112 Coriander H\ Anise #2 Coriander ' I , 0.00000 0.465 19 211 0.00000 0.00505 7 0.00000 0,09284 231 000000 0.01 6 15 , 0 .00000 0. 10394 236 0,00304 000000

II 0.00000 0 .00303 267 000000 0.00303

IJ 0.00000 0.05853 292 0.00608 0.00000

14 0.00000 0.00706 293 0.00263 0.00000

J5 0.00000 0.0565 1 348 000000 0,00202

32 0.00000 0.00303 349 0.00000 0.00202

50 0.00000 0.04339 353 0.00000 0.00505

58 0.00000 0 .00202 357 0.00000 0.00101

61 000000 0.00]0) 375 0.00010 0.00000

62 0 .00000 0.00 101 386 0.00020 0 .00000

64 0 .00000 OJ)()202 388 0.00010 0 .00000

65 0.00000 O.c)0202 390 0.00000 0.00706

" 0.00000 0.0010 1 399 0.00000 0.00 101

67 0.00000 0.0 1413 402 0.00000 0.00505

71 0.00000 0.00404 437 0.000\0 0.00000

83 0.00000 0.00706 467 0.00010 0.00000

87 0.00000 0.00605 480 0.00365 0.00000

9 1 0.00000 0,00908 499 0.00020 0.00000

"5 0.00000 0.00101 51' 0,00010 0,00000

143 0.00000 0.01312 5J2 0,00010 0 .00000

144 0.00000 0.00807 540 0.97376 0.00000

14 5 0.00000 0.00 101 '" 0.00405 0.00000

156 0.00567 0.00000 745 0.00010 0.00000

210 0.00000 0.04440

tJ. CHENG lIT AI. Component AnalJ·sts of Mind Spica 165

Numerical Analysis. Given an unknown spice blend Y, we consider the spice

recognition problem a.s that of finding the best rat io in wltich one can mi){ the 355 spices X',xl •.. . ,xJ55 so as 10 achieve the maximal similarity to the target spice

blend Y. This problem is e){actly equivalent to the following constrained optimization

problem:

(maximize S<P)

P o (7) SubjeCI lo p , + p,+ .... +Pm = 1 , ,~,

where .fl... ~(/3 ,,/3 ]> ... , /3 JJJ) represents a set of mi ){ing ratios and the objective

function S is defined as:

(')

To determine the best ratio J!.... ' so as to achieve the ma){imal value of S(p), we

employ optimization theory and numerical methods (see Luenbcrger (8» an~ computer programs are designed accordingly. The nonzero components of .Ii... indicate the presence of the corresponding spices.

EX llerimenlal Seclion

Pupara tion of mixed spices for test ing. A testing mixture of spice blend was

prepared by mi){ing powdered spices purchased from local trading company. The

formulat ion is shown in Table IV .

Table IV. The formulation of mi){ed spices for testing.

Spices Wt(g)

Black pepper 27.00

Allspice 27.02

Celery seed 13.52

Clove 1.62

Garlic 27.01

Star anise 6 1.03

Sum 157.20

Isolat ion of volatile components from spices. One hundred grams of ground spice blend shown in Table IV were added to a three-neck bottle containing 300ml of disti lled water. This slurry was steam distilled and extracted with dichloromethane (Fisher Scientific) for 2 hours in an apparatus similar to that described by LikensNickerson(l2). The organic: layer was separated, dried over anhydrous sodium sulfate (E Merck). evaporated 10 minimal volume in a Vigreu){ column, and then concentrated to about 0.5 ml under a gentle stream of nitrogen.

166 SYICES: nAVOR CII J.:l.U~"TRY ANI) ANTIOXIDANT I'ROI'F.RTIES

Gc. GC analyses were carried out on a Varian 3400 chromatograph equipped

with a 30 m x 0.25 mm i,d. fused silica capillary column (DB-WAX, J&W

Scientific, USA). 1be linear flow rate of the carrier gas (H1) was 43 emfs. The

oven temperature was programmed from 40·C to 200· C at a rate ofloC/min

with initial holding at 40·C for 2 min. The injector and detector (FID)

temperatures were set aI200·e and 210 ·C, respectively.

GC/l\1S. GCIMS was carried out on a Finnigan Mal ITO. ac conditions were

the same as those mentioned aOOve. Identifica tion of volatile compounds was

based on comparison of retention index and mass spectra with those of authentic

compounds.

Sel up of the data malril. The raw data were collected from published

Jitcrarure such as those cited by TNO-ClVO (3), ESO (5), or those listed in the

present study. Take essential oil ofbJack pepper (I J) as an example, the original

data format is shown in Table V, the volatile compounds were then arrangcd into

coded mlmher as those of Anise #1 shown in Table I. So far, a 355 (spices) by

922 (volatile compounds) data matrix has been established. Of the 355 spices,

there exists repeated collection of data from the same spice from different

geological areas or differcnt publications. f or examples, 4 doves, 5 star anises,

9 anises, 6 corianders, 8 allspices, 2 black peppers lind 27 basils, are included in

the present data matrix.

Results a nd Discussion

Test nm of known spice bl ~nds from published li terature.. The spice blends

which contain basil, cinnamon leaf, peppermint, sage and wintergreen as

reponed by Lawrence and Shu (4) were used to test the effectiveness of the

present tOcory. The ratios of individual spice are shown in Table VI, the coded

I\llmbers of identified volatile compo1Jnds in the spice blends are shown in Table

Vll. Computer output of numerical analyses of compounds shown in Table vn are listed in Table VIII, the similarity index of the testing result is 0.959,

• indicating a high level of confidence. In Table VI II, there arc repeated

identifications of the same type of spice, e.g. , the appearance of basi Is #3, #19,

1124,1126 and #27; cloves III and II 4; and mentha # 1 and II 12. In fact, the

repeated appearance of the same spice indicates the closeness of these spices.

It was also confirmed that the similarity index within the same spice listed in

Table VIII usually ranged from 1.0 to 0.90 (data not shown). In ordcr to

13. CIl t:NG t;'f '\1_ Component Analysts of Miud Spict.f

Compounds %

Sabinene 22.50

alpha-Pinene 17.50

Limonene 17.00

del ta-3-Carene 13.50

beta-Caryophyllene 5.00

alpha-Humulene 1.00

Carvone 0 .10

Eugenol 0. 10

Linalool 0. 10

Myrist icin 0 .10

Nerolidol (unknown isonler) 0. 10

Piperonal 0 .10

cis-Sabinene hydrate 0 .10

trans-Sabinene hydrate 0.10

Safrole 0. 10

Terpinen-4-ol 0 .10

alpha-Terpineol 0. 10

TOIal 17 components 77.60

(Source: Adapted from ref . J J )

Table VI . Reported composition of testing mix ture of essential oi ls

Essential oils "I.

Exotic basi l

Cinnamon leaf

Peppermint

Sage

Wintergreen

42. 1

IS .8

15 .8

21.1

' .2

(Source: Adapted from ref . .J)

167

,j •

:~

;i ,

1611 8I'10 :S, HAV()U CHEM ISTRY AND ANTIQXIUANT ,'ROI'fo;RTIt:s

Table VII . Volatile comEounds identified ITom Table VI .

, Code No. Com~md %

4J7 alpha-Pinene 1.3

182 Camphene Il

467 beta-Pinene 0.7

m Myrco.::nc 0.5

292 Limonenc \I

.0 l.lI-Cineolc 4.' 444 alpha-Thujonc , .• 470 1x..1a·ThIUone I..

'" Camphor 4.7

297 Mcnthonc 3.0

762 Mcnthofur.an 0.4 .-293 Linalool 0.9

296 Menthol 4.' 23. Methyl ehavieol 34 .7

177 IJornool L7

m Methyl salieylale .., 197 Cinnamaldehyde 0. '

250 Eugenol 9A

25' Eusen~'1 aectate OA

(Source: Adapted from ref . -I)

13. CIIENG "1' AI_ Compom",t Ana/y.Wl' 0/ Mi.ud Spica

Table VI1l . Ratios of essential oils IS analyzed by numerical analyses,

Ihe similari ty index. - Q 252

Spice II

Basil 113

Basil #19

Basil #24

Basil #27

Basil #26

Ray leafNI

Cinnamon N4

Clove #1

Clove #4

Commint #8

Mentha #1

Mentha #12

Roscmary #3

Sage #2

WinteriTeen 1# I

% Assisnmem

2.7393

2.6\87

2.8070

12.8562

18.7463

2.7733

0,4658

0.5672

10.6274

7.4766

0.5556

2.6013

2,7350

19,4858

59446

Basil #26

Clove #4

Commint #8

Sage #2

WinterSTeen #1

169

simplify tbe result, thc ralios of those spius and herbs which appeared repeatedly

were summed up and assigned to the spice with the highest ratio, as shown in the

third column of Table vm. Table IX shows the summed up ratios of spices

from Table VllI, as compared with tbose proposed by Lawrence and Shu (4).

In Table IX. clove 114 and commint 118 were not reported in tbe previous study

(.f), instead, cinnamon leaf and pcppennint. respectively, were assigned. When

the similarity index between these two seemingly differenl pairs of spices (clove

#4 vs. cinnamon leaf; commint #8 vs. peppermint) were compared, an index

value of 0.95 was observed in tbe former, indicating a high degree of closcness

(similar in composition of essential oil), the latter pair showed a less satisfactory

index valuc ofO.83, slillthis was a good matcb wi th acceptable confidence It

should be considered that the results in Table IX were from data in reference 4

that was used in the data matrill of the present study. The ratios of spices

analyzed by the numerical mctbod were very accurate when compared to the

original data. This is a good demonstration that tbe approach used in the present

study can be used to examine the ratios of any spice blend, as long as the

individual spice is included in tbe data matrix (355 spices).

"0 SI'ICES: nAVOR CHEMISTRY AND ANTIOXIJ)ANT i'ROI'EHTII!:S

Tahle IX. Comparison of the numerical melhod with previous publications

Spices assigned % lI(cal) Spicesb Yo

Basil #26 47.9 144 Exolicbasil 42.1 Clove 114 c 12.7725 Cinnamon leaf 15.8 Comminl #8 d 9.3919 Peppermim 15.8

Sage #2 23.8251 Sage 21 I

Wintergreen #1 6.0962 Wintergreen 5.2

a·summed up values from Table VIII .: b: data from ref 4.: c:index (Clove

#4, Cinnamon Leaf 114 )- 0.95: d:lndex (Cornmint 118 , Pepper #8 )- 0.83

"

,,-L li\I1 fl ~~ , ,

0 '0 ' 0 . 0 _ 0 ' 0 , ,

'0 _ 0 .0 .0 ' 00 " 0 .. ... C( .... "u ,. )

Fig I Gas chromatogram of the vola tile components of the mixed spices from Table X.

13. CHENG ET AL Component Analysts of Mixed Spices 17l

Allll lysis ofte5t ing spice blend prepa rw in the la bontol")'. The formulation

of a real spice blend used in the present study is shown in Table IV. Volatile

compounds from this spice blend were isolated by simuillneous steam distillation

and solvent extraction. Identification of volatile compounds was acromplished by

comparing the retention index lind mass spectra with those of authentic samples

and published publications (/4 - IS) . Figure I shows the gas chromatogram of

isolated volatile compounds from the testing spice blend , Percent ratios orthe5e

volatile compounds identified are shown in Table X. Compounds in Table X were

then coded by number in the same manner as those of Table VII. The orig inal

resllits of numerical analyses are shown in Table XI, similar in fomlat to those of

Table VIU. Recognition of spices in this spice blend showed a very high similarity

index (0.981), indicating high level of confIdence. Those spices th At appeared

repeatedly with a high similarity index were assigned to the spice wi th highest ratio,

i.e. the ratios of anise and star anise were combined, as ment ioned earlier, that is

because both anise and star anise listed are similar in composition. The same holds

true for the combinations of black pepper and allspice. The only spice which is

determined by numerical analysis but not added into the testing mixture is

marjoram (ca. 0.43%).

The comparisons of ratios of spices as determined by numerical method am!

those calculated from the content of essent ial oil are shown in Table XII. Sensory

analysis of a 0.4% solution of the actual spice blend as compared wi lh that of a

predicted spice blend have shown lillie difference. The accuracy of Ihis test is

surprisingly good for star anise, black pepper and garlic. The mlios of allspice and

clove are quite good, although some deviations from the actual data exists. Celery

has been correctly identified but the ratio determined in the present test (only 0.03

%) is qui te low, probably caused by technical difficul ty of our MS database in

assigning the correct volatile compounds of celery. As to the appearance of

mrujoram, although the ratio assigned to this spice is smllil (0.43%), the similarity

index between mrujoram and other identified spices has not shown any high degree

of similarity. Probably this deviation is caused by the low value assigned to celery

(0.03% vs. \.78%).

Conclusion

The present study has shown that the ratios of individual spices in a spice hlend can

be accurately solved by a numerical method when compared with previous refKln5

(4. 7). Spices in a spice blend with an essential oil coment as low as 0.370/. can be " I • ,

,I , ) , I

Ii d ! ' i '. il 'I " I I i '/

l rj "

• ,

172 SI' I CF.s: nAVOH CIIP'}'U!>'TRY AN I) ANTIOX.IDANT I'ROI'ERTI I;:S

Table X. Identified volatile compounds from the s~ice blends from Table IV Peak N O a -6. Compollnds

0.29 alpha-pinene 2 0.67 beta-pinene ) 0.19 sabinene 4 1. 50 della-J-carene 5 0.16 alpha-pheJiandrene 6 0.21 myrccne 7 0.05 alpha-terpinene 8 2.28 limonene

• 0.10 1,8-cincole 10 0.12 gamma-terpinene II 0.18 para-cymene 12 0,04 altyl methyl disulfide IJ 0.07 lerpinolene 14 0.0 1 methyl trans-I.propenyl disulfide 15 0.0 1 dimethyl trisulfide 16 0.03 penlylbenzenc 17 002 4-isopropcnyt-l-melhylbenzcnc 18 0,02 lioalool oxide I. 0 .25 alpha-copaenc 20 0.34 linalool 21 0.02 trans-alpha ·famesene ,.. 22 3.37 bela-caryo phyllene 23 0.49 terpinen-4-o1 24 0.45 deha-cadinene 25 0 ,08 cSln.gole 26 0.25 alpha-terpineol 27 0.09 carvone

" 0.<15 cis-anethole

2' 0.90 diallyl tri sulfide ) 0 70,16 trans-anethole 1I 0.06 para-cymcn-8_01 32 0.1<1 3 -methyl-I-phenyl-I -butanonc JJ 0.39 beta-caryophyllene oxide l4 3.02 anisaldehydc 35 2. 17 methyleugellOl

~~ 11.46 CII8SOnQ! TOIa! JOO 00

a , Number refers 10 Fig I

13. CHENG t:r AL CQmponent Analyses of Mixed Spices 173

Table XI , Computer output of ralios of essential oils as

anal:tzed b:z: numerical anal:tses. The similarit:t index " 0.981

Sl!ice N Calculated % Assignment

Anise #1 3.92<15 Anise #2 17.8]<11 Anise 11<1 9.5 1<19 Anise #5 7.0813 Anise 116 9.6]66 Anise 117 0.7293

Star anise 1i2 9.92<11 Star anise 1i4 19.3589 Star anise 114

Celery #5 0.0305 Celery 115

Clove 11<1 <15803 Clove #<1

Garlic iii 0.<1170 garlic #J

Marjoram #2 0.4275 marjoram #2

Black pepper " 0.278<1 Black pepper #2 5.7291 Black pepper #2

Allspice #<1 0.3164 Allspice liS 1.9528 Allslli,,1I:8 J 2518 Alislli",1I:8

Table XII. Ratios of individual spice in the spice blends after adjustment

3CCQrding to closeness of simiiarit:t inde)!.

spices WI (g)1 essential oil % oil in the Yo oi l in the

contcnt (%)b mixture (cal)C mixture (exp)d

black pepper 27.00 1.76 6.<13 6.29 allspice 27.02 2.58 9.4<1 6.31

celery 13 .52 0.97 1.78 0.03

clove 1,62 7,67 1.68 4 .80

Slar anise 6 1,03 9 ,72 80.31 81.70

garlic 27.01 0.10 0.37 0 .<14

marjoram 0.<13

I 'Dala from Table IV.;b: Data obtained from this s\udy.;c:Nonnalized per cent

distribution; d :Ralios combined from Table XI.

174 SI'ICES: HAVOR cln:~lISTRY AND ANTIOXIIlANT I'IWI'ERTlK'i

accuflltely ,Ietermincd without significant deviation from the actual value. It is the

utmost hope of the authors that application of this technique can definitively

shorten Ih~ time and effort used to determine the ratio of individual spice in a spice

blends

Acknowle(lgrmenu

This work was supported by the Ministry of Economic Affai rs, Taipei, Taiwan,

Republic of China. We uc also gfale!ul lo our laboratory slaffs for technical assistance.

Liarllltur( C ited

I_Farre ll." T. In SpiCt!l'.COlldiIllI!IIIS,(llld Se(lsollillg~·. Van Nostrand Reinhold, New

York.U, gO.

2 Giese, f. Food Techllol .• 1994. 48(4),88.

J . Volmill Compol/llds ill Foods M(KIf~, H .• Vis5(;her, CA., Eds., TNO-CI va, Zeist, tile Netherlancs 1989, Vol 1-2.

4.Lawrel,ce, B., Shu, C. K. In Plavor Measurtmelll. Ho, C.T .; Manley, C.B , Eds ,

Marcel P ekker, New York, 1993, pp 267·328 ..

S. ESO: 'lhe COli/plett Database of wenfial OiLf. BACIS, the Netherlands, 1995.

6.Salzer,!JJ . CRC Crit. Rev. Food Sci. Nutr;., 1977,9,345 .

7 Chien PA. A/KII. Ciltm. 1985, 57,348. , , 8 Luenbt!:rger,D.G. Optimizatioll by Yeetor Space Methods. John Wiley & Sons,

New y p rk.1968.

9 Tabacctu, R., Gamero, 1 ; Buil, R. Rivista Iial., 1974, 56,683 .

IO.Cu, J Q In /Olh /I~. Congress Ess. Oils, Fragr.& Flav., Washington, 0 C .• 1986,

pp231 ,2 4 1

[I.Poller, TL., Fagerson, 1.5. J. Agric. Food Chem., 1990, 38, 2054.

12. Likens~ S. T ., Nickerson, G n ., Am. !XJ<:. Brewing ChemislS Proc. 1964, 5.

13.WrolstjJ'dt , RE., Richard, H.M.; Jennings, W.G. J. Food Sci., 1971, 36,584.

14. Jennin¢ls, W.; Shibamoto, T . In QII(I/itath'e AI/aly:l'is of Flavor alld Fragrallce

Vola/ii' s by Glass C(If/ilIary Gas ChromalOgrt/phy Academic Press, New York

1980. 15. MOlI()t~rpelles: II/fit/red, Mass, H·NMR, (md CI J NMR S/Hctra alld KOI'rIfS

II/dice.f. Swigar, A. <\ .; Silverstein, R. M. Eds.; Aldrich Chern. Co. Inc.,

Milwaufkee, WisconJin, 1981.

ANTIOXIDANT P ROPERTIES

"

" ,

;

) i

..l I

j "

I

I

I

, ,

Chapter 14

Antioxidative Activity of Spices and Spice Extracts

lIelle Undberg Madsen. Grete Bertelsen. and l..eif U. Skibsted

Department of Dairy a nd I<·ood Science. The Royal Veterinary and Agricultura l Univers ity. Rnlighednej 30,

DK- 1958 .' rederiksberg C, Denmark

The alllio,,;dative activity of spices and spice extracts can usually be traced back to their content ofpheDolic compounds. Plant phenols may scavenge free radicals involved in lipid peroxidation .s has beeD documented in several model systems, although other mechanisms should be considered especially in relation to the carty stages of oxidative deterio ra tion. Phenolic compOlwds isolated frOIll spices have been foun d to react with hydroxyl radicals wiOI nearly diffusion controlled reaction rates. An assay based on a combination of detennination ofpbenol equivalents and determination of radical scavenging capacity by the ESR spin trapping technique confiTU.1S the nearly diffusion controUed reaction rates and may prove useful for exploration of new plaut materials and for adjustment of e"'Taction procedllfes, including selection of solvent. This and otber assays based on oxygell deplet ion measurements are recommended prior to final testing in real foods. Co-extracted cbJorophyUs in spice extract present a problem as photosensitizers in food cxposed to light during storage IIld use.

Addi~ion of spices to food is ao established procedllfe in most cult llfes. The seasooing contl'lbutes a pleasant flavour, and at the same time a number of compounds possessing amioxidative activity are added. Scientific investigatioll of the antioxidative activity of spiccs has been initiated duriug the last few decades and an understanding of the mechanism of the antioxidative activities is emerging. Early work by Chipauh and coworkm (1-3) in the ftfties documented significant reduction in oxidation in lard and e.dible oi~s. The most pronounced cffects were found for rosemary aod sage, observa. lions which lIave been verified io most later investigations (4-6). However, a great va riety of other spices invest igated show similar antioxidative activity and notably their relative efficiencies are strongJy dependent on the actual food.

© 1997 Am~rican ChcmiCilt Society

14. MADSEN ET AL ,v,tioxidati,·, Acti~ily of Spias lind Spicl Extracts 177

OJidation and Antioxidants

During oxidation free radicals are generated cOnlinuously in the propagation phase. In this phase a lipid radical (R') reacts very quickly with (triplet) oxygen in a reaction controlled by diffusion. The formed peroxyl radical ( ROO') can abstract a hydrogen from an unsaturated lipid molecule (R' H). This reaction lead5 to generation of a new lipid radical (R'·) and continued activity in the chain reaction. The hydroperoxides (ROOHl, the products fonned in the chain reaction, are without smell and taste. Hydroperoxides are easily decomposed to yield alkoxyl radicals (RO·). These radicals can funher abstract a hydrogen from a lipid molecule in effect starting a new chain reaction contributing further to the propagation phase (7) (Figure I). Reaction products from the degradation of the initial fonned hydroperoxides, the secondary oxidation products, are carbonyl and carboxyl compounds which give rise to the unpleasant taste and smell o f oxidized food (8).

An efficient way to delay oxidation is scavenging by antioxidants of the free radicals generated in the propagation phase or during the break down of the hydroperoxides, i.e. scavenging of either the peroxyl radicals or the alkoxyl radicals. The critical level needed of such primary antioxidants to be effective in a given product corresponds to the concentration necessary to inhibit all chain reactions started by the ;nitiation process. M long as tlte concentration of the antioxidants is above this critical concentration, the total number of radicals is kept at a constant low level, a time period which is defined as the induction period. During the induction period the antioxidant is gradually depIcted and when the critical concentration is reached, radicals will escape from reaction with the antioxidant, and the concentration of hydroperoxides will increase_ The high level of hydro peroxides wi ll funher increase the concentration of radicals, and the rcmaining antioxidant will be used up completely (9). With all the antioxidants consumed, the oxidative processes will aceelerate, and the increase in the production of secondary oxidation p£oducts will lead to a progressing deterioration of the product.

Phenolic compounds act as primary antioxidants by donating a hydrogen atom to either the alkoxyl radicals (equat ion 1) or the peroxyl radicals (equation 2) in irreversible reactions. The reactions lead to generation of an antioxidant free radical (A·) with a lower energy compared to the energy ofRO· and ROO·, ;.~. the reaction is enthalpy driven.

AH + RO· J\H + ROO· -

A· + ROH A- + RooH

( I) (2)

The relatively high stability of A- reduces its ability to abstract a hydrogen from a lipid molecule in effect breaking the chain reaction. The efficiency of phenolic compounds as scavengers offree radicals is due to the high stability of the generated pheno)(y radical, whieh is caused by delocalisation of the unpaired electron in the aromatie ring.

However, phenols are not act ive as antioxidaots unless substitution at either the onho or para position has increased the elect roo density at the hydroxy group and lowered the oxygen-hydrogen bond energy, in effect increasing the reactivity towards the lipid free radicals. Substitution in phenolic compounds at the meta pOsition has a rather limited effect, and compounds like resorcinol with a hydroxyl group in the meta

178

In itiation

Propagation

Termination

SI'ICES, n.AVOR CII~~IlSTR\, ANI) ANtiOXIDANT I'ROrERTIF.s

ROOH

RH ROO·

RH

~X' 1"'----- XH

R·

ROO,

-yRO'

Non radical products

0 ,

J-igure I. Lipid Olcidation wilh an iuiliatioD pbase, propagation pbase and termination phase.

-.

14. l\lAIlSJ.:N ~"T AL An/ioxid(Jt;vt Adi!'i" of Spias and Spice Extracts 179

position are thus poor scavengers offree radicals compared to similar compounds with substitution al the 011110 or para position. The preSCllce of additional hydroxy groups, either 31 tile onho or para position, further iucreases the an tiolridative aClivily of the corupowld IS illtn -molecular hydrogen bouds stabilize Ihe phenoxyl radicals (9).

Antiolidative Compou nd, in Spices

Phenolic comlloUJlds constitute the IHgcSl proport ion of know nalUral antioxidants. It should, however, be noted that only H minor part of the compounds in spices which have antioxidative aetivity has been isolated and identified. Nakatani and coworkers (1 0./1) have reported the structure aud characterized the antioxidative prop,mies of §cveral phenolic diterpeno.'S isoLtted from ro§clllary (RosmarillllS officinalis L.). From the SlIme plillt , eompounds like eamosic acid, eamosol (12-/4), rosnuridiphenol and rosm3Tiquinone ( />./6) have been identified (Figure 2) as antioxidants. Moreover a number of the cOllvouuds fOlU1d UI rosemary have been found in the botanically closely related s.1ge (Sa/via officil/a/;s L.) (12. / 3) Hnd summer savory (Soll/reja harle/ISis L.) (/7./8).

In other spices, fiavolloid s alolle or together wilh other phenolic compounds have been found 10 contribute to the antioxidative activity. In oregano (Origanum vulgare L.) various autioxidative compounds have beeD isolated, and among the act ive components four flavonoids were identified (/9). From thynJe (Thymus vulgaris L.), which like oregano also is a ruelJlbcr of tbe LabialfU! family, antioxidBtive compounds have been isolated and identified as dimers oftllyruol and f1avonoids (20). The antioxidalive aClivilY of pepper (Piper lIigrum L.) can, at least partially, be ascribed to the presence of glycosides of the f1avolloid s kaempferol, rhamlletin and quercetin and at least fivc different phenolic amides (21.22).

A variety ofdilli::rent volatile compounds such as the terpeuoids thymol, caT\'ocrol, eugenol., carvone and thujone. which are character-impact compounds for imponalll spices (23.24), have 3ntioxidative activity hili the use of these compounds as amioxi· dants for different foods are limiled by the cbaracteristic flavour of the plnicular compound.

Evaluation of the Uadic:l1 Scavengin~ Activity

Screening of IIltioxidative activity in various mode! systems is imponan! prior 10 te!>ling or application of IIltioxidanls in foods. Such model systenl$ are more rapid compared to food storage experiments. and the model systcms might eveu be more illfonn:uive in relation 10 Intioxidant mechanisms.

Electron spin resonance (ESR) spectroscopy is curre1Llly being introduced to different areas of 311plicatioll ill food science. The lechniquc may provide valuable information conceming thc elemeutary processes in lipid o)(idalion and the nature of reaction iU!ennedi~tes. A number of ant ioxidants iocluding extracts or compounds isoLaled from spices have been investigated by differelll ESR technique. The amio)(ida· live activity of the fractiou containing tlle essential oils fro m oregano (OrigallulIl \"Ignre L.), summer savory (Sawreja horUltti$ I~ ) and thyme (Thymus vulgaris L.) has been documented in • system where oxidative stress and free radical reactions were induced either by UV irradiation or the supcmxide radical. Free radicals Cill be detected directly

, , i r

, 1,<

'J

.1 , .1

•

.J ;.

J , , j l

. i , .,

,

, 1

OH

Carnasel

OH

Epirosmanol

HO OH

•

Rosmaridiphenol

9H

, .... \

Carnosie acid

Too

HO

' OH

Isorosmanol

OH

o,~/

-,/

CHO 'CHO

Rosmadial

OH ,

"" '\ UH

Rosmanol

~OH Hoae 0 o OH

~O H

Rosmarinic acid

., ....

Rosmariquinone

F.gurt 2. Chemical structure for compounds isolaled from rosemary (Rosmarinus officinalis L.).

---- ---------

~

~

R ~ -. ): < 0 • 9 , • ~ < ~ 0

~ 0 • ~ z .;

• • 0 • ~ , , .. ~

;: ~ ~ ~ ~ ~

~ • s' ". ~ ~.

~ ,;.' ... .g> S' • it .g> ~.

r ~

-

•

182 SI'ICES: flAVOR CIn1l-mi'TKY AND ANTIOXIDANT PROI't:RTl ES

0.6

0.5

~

"" 0.4 • ~ ~ 0 0. 3

0

• • 0.2 " c

0 . 1

0 .0 ~ E • 0 0 .- ~ ~ ~ ,

~ c c • , c c 0 0 0 0 0 0 0 • E , " "" "" CD > > • c 0 0 • • • ~ , , , • • • 0 0 0 0 , ,

" ~ • • c ~ E 0 • c • ~ E " .- ,

0 ~ 0

"' U ~

Figure 3. The hydroxyl radical sc.vcngiug activity of nine different spices all belonging to the family Lobimae. The activity was measured by using electron spiu resonance technique, and Ihe indexes were calculated on the basis orlop height from oata of reference (28). A low value of the index indicates an efficient selvenger. Columns with different letters arc significantly dilTereDl at 5% level

-

14. MADSt:N F:r AI. Alltiandative ActMty of Spices tllld Spiu Extrar:l$ 183

by ESR spectroscopy, and have in the spice extract of oregano, thyme and summer savory been identified as free radicals derived from thymol and carvacrol. NOlably, thymol and carvacrol radicals were also present in untreated essential oils indicating thc high stability of the radicals and the potential o f the compounds as scavengers of oxygen-centred free radicals (15). Experiments using spin trapping technique are based on compet ition between the compound expected to have antioxidative propenies and a "spin trap" in reaction with free radicals. 1be it! vitro anlioxidant properties of rebam;· pide, a nove! antiulcer agent, have thus been documented by the spin trapping technique (26). and the scavenging activity of carnosine and related dipeptides have been fun her investigated (27) In an investigation with focus on the early stages of oxidat ion in food s, the ability of spice ext racts to inhibit the initiation of free radical process was evaluated by adapting the ESR spin trapping technique (18). The cxtracts or tile spices obta ined by ethanoVwater extraction were freeze dried and redissolved in waler. The water soluble compounds, panly phenols, were found to reduce the signal intensity in the ESR spectrum ofthe spin trap/free rad ical adduct when hydroxyl radicals were generated by the Fenton reaction, indicating a high scavenging potential of t he spice extracts against hydroxyl radicals. Ext racts of each of nine different spices investigated, all belonging to the Labiatae fami ly, were capable of scavenging hydroxyl radicals (Figure J ) Most efficient was winter savory. rosemary, sage and the two oregano species. Kinetic studies gave an estimate of the rate constant between hydroxyl radicals alld phellols in the extract of Turlcish oregano. The value of the second order rate constant was slightly above that for a diffusion controlled bimolecular reaction in water, and the high value indicated that compounds other than phenols were involved in the compeTition with the spin trap in the reaction for the hydroxyl radicals. Rate constants for reactions between compounds isolated from spices and different radicals have, however, o nly been determined for a few substances Ilnd funher studies should be encouraged. An estimate of the rate constant of 4.8·10!0 M ·! S·I between hydroxyl radicals Ilnd eugenol is, however, an example (29). TIris corresponds to a veT)' fast reaction with a rate constani similar 10 the rate constant found for the reaction between hydroxyl radicals and phenols in extracts of Turkish oregano (28). These results together document that hydroxyl radicals are very reactive, and model systems based on other radicals should be developed for a differentiation between different groups of frce rad ical scavengers. The second-order rate conslant detennined for lhe superoxide scavenging act ivity of eugenol (a volat ile present in clove, cinnamon and basil (29» has been used for comparison of phenolic antio,odants and the enzyme superoxide dismutase. Superoxide dismutase was most effective in deactivating superoxide, inte~dilles ratc constants were found for both eugenol and guaiacol, while the activity of phenol was rather poor (30) .

At later stages in the oxidation process the scavenging activity of antioxidan ts towards peroll.yl radicals is of relevance. Aruoma et al. ( 31) investigated the rate of reaction or a number of ant ioxidanu with the trichloromethyl peroxyl radical . Carnosic acid had a r1lteconstant of 2.7· 101 M·' 5·', about 10 times higher than the rate const Jnt of camosoI. The react ivity of carnosic acid with the peroxyl rad icals was comparable 10

propyl gallate, and it could be concluded that camosic acid is effective as a scavenger of trich1oromethyl peroxyl radicals, although the reactivi ty of carnosic acid was not as high as the reactivity of ascorbic acid and the waler soluble tocopherol analogue, trolOl( C. Determination of the rate constants for the reaclion between hydroxyl rad icals and

"

1

-I .;

'84 srICI:t.'i: HAYU\{ (; II I::MISTRY AND ANTIOXIDANT "IWI'ERl"I F:S

either camosic acid or camosol again showed ute constants whicb indicated that the reactions were essentially diffusion controlled (31).

AnlioI.idativt ICli"ity in model systems

While dcterolination of rate constants fo r scavenging of free radicals by antioxidants is highly relevant for establishment of structure/activity relationships. it is essential for pr;l~tical food application to constnlct betefogeooous model systems with phase transfer. Frankel et al. (31) evaluated rosmarinic acid, caruosic acid and camosol in such model systems by mel5Uring both primary and secondary oxidation products and showed that the antioxidat ive activity depends strongly upon the substrate. The compounds coluaining a carboxy group (rosmarinic acid and camosic a~id) were the IliOst effective aotioxidauts ill bulk COllI oil whereas the antioxidative activity of camosol WIS only limited in this oil. However, in an olw emulsion a bigb antioxidathre activity was seen fOJ" camosol and camosic acid while only slight autioxidative or even prooxidative activity was found fOl" rosmarinic acid. The observation tbat tbe more polar antioxidants aTe more active in pure lipids, and the nOli polar antioxidaniS most acthre in a polar substra te, and for wh.icb the tenll ')lOlar paradox" has been introduced, confimls previous findings for tocopherols .and ascorbic acid derivatives as an tioxidants (33). In the bulk oil the hydrOI)hi lic ant ioxidants are oriented in the oillair interface providing optimal protection of tbe lipids aglin!.t oxygen radicals while the hydrophobic antioxidants dissolves in the hOlllOgeneous lipid pbase. The opposite situation with hydrOI)hobic antioxidant conccntntion in the oil/water interface is encoulllered in the emulsion syst;om, where the hydrophobic antioxidants, like camoso!, are most efficient. These obsclVRtions may at least paniaUy explain tbe variation of antioxidative activity seen for different spices in different foods (3), and the need for investigations in such bet erogenous model SySlemS and. in the actual food products prior to prtlctical use is obvious.

Antiol.idative activity in food

Thl: results obtained by Frankel et a1. (32) using food models mig\Jt explain the (IUalilalive di.fl:erence observed between antioxidative activity of summer savory and rosemary in a meat storage eX]leriment and in a storagc experiment with dressing. In precooked pork meat balls no sig;nificant difference between the antioxidative activity of rosemary and SllllI.lt1er savory was seen, and both spices were able to retard the development of oxidative off·Oavour, measured by determination ofthiobarbituric acid reactive substances (Madscn et al/. TIle Royal Veterinary and Agriclilrural Unhrersity, Fredcriksberg, OK, unpUblished da1\a). l11csc results were, however, different from those fouud for a dressing with a high fat content (50%). In the dressing the addition of rosemary was found to be significantly more efiective in proteclioll against oxidation compared to summer savory (M:adscn et al. The Royal Veterinary and Agricultural Uni .... ersity, Frederiksberg, OK, wl:published data). Although the nature and quantity of the ~ctive compowlds prescnt ill the two spices were not determined, it may be speculat ed that differences in lhe hydrollhilic or bydrophobic properties of the anlioxidants lUay be imponall t. Ie is well-known, tbat rosmarinic acid is ao important

14. MADSEN t.T AL. AnJioridativt Activity 0/ Spices and Spia Extracts 185

antioxidative coqJooenl in summer savory (/8). The low efficiency of this substance in bulk oil might provide I rationale for the reduced antioxidative activity observed for summer savory in the dressing. Other factors such IS differences in pH in the two foods may also oontnoute to the different activities. Frankel el 01. (32) have also studied the a" tioxidative activity of camosa!, clmosK; acid and rosmarinic acid in emuJsions with diJrerent pH. At pH 4 and pH 5, where the carbo1C}'1 group is protonatcd to a significant extent the aotioxidative activity of camosol and camosic acid were much higher than the .ctivny at pH 7 where the carboxylate form dominates. This pH dependency might be explaioed by the cbange in polarity and oxidation potential or by a higher stability of the compounds at lower pH. An increased knowledge of tbe effects of pH will be beneficial for a more rational use of plant antioxidants.

Further perspectivu and d evelopment5

A thrce-step prOCedllfe as the one outlined encompas.~ing (i) determination of radical scavcnging activity of spice extract, (ii) te~ti.llg ill model systems, and (ill) fuJal application in foods may prove useful for future work on natural antioxidants. Different solvents should also be explored for extraction of spices. Different polarity of the solvents might be used for "tuning" of extracts to differcnt products of differen t hydropbobidbydrophilic balance. A higller antioxidative activity was found of a hexane extract of rosemary and sage compared to other solvents by using successive extraction of solvents of increasing I,olarity (34'). I-:I owever, most. investigations report a high antioxidative activity in extracts obtained from polar solvents (/.35,36). The use oflarge amounts of org:anic solveuts is in general unwanted for environmeotal resonance, so altemllive extraction methods should be considered. lne use of supercritical COl extraction, which has already shown positive results (37), might be getting more attention since the tecbnique is efficiellt and II.so protects the phenolic compounds against degradation during extraction.

The interaction between different antioxidants such as tocopherols. ascorbic acid and spice componenls and possible synergisms should also be explored. Fang & Wada (]8,39) already invCSl:igated the synergism between a-tocopherol and extract of rosemary in ethanol The results support tbe hypothesis that rosemary antioxidants regmCTIte oxidized a-tocopherol. Similarly Ternes & Schwarz (4'0) found an increased stability oftoeopherols wben rosemary extract was added to meat samples. To obtain more information about the antioxidathre compounds a determination of the reduction potential of key components (6,4' /) would be of interest for ranking of antioxidants io order to determiue \o\"IDch oftbe spice Illtioxidants can regenerate the tocopherols. 11le problctllS encountered with photosensiti7jng ehlorophylls co-extraction from the spices making the food produ~t sensitive to lig\Jt (Madsen el 01. Royal Veterinary and Agricultural University, Frederiksberg, DK, unpublished data) also needs further

investigation.

Acknowledgments

This research was part of the frame program "Natural antioxidants from plants" sponsored by the F0TEK-program t.hrough. National hod Agency and LMC-Centre for

Advanced Food Studies.

I 186 SPICES: FLAVOR CIiEM ISTItY AN D ANTIOXIDANT \,ROl't:RT1ES

Liara lUre Cited

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2. ChipIUIt , J.lL ; Mizuno, G.R. ; Lundberg, W.O. Food Res. 1955,10, pp. 443-448. 3. Chip.ull, l lL ; Mizuno, GR ; Lundberg, W.O. Food Techno!. 1956, 10, pp. (5)

209·2 11 . 4. P. litzsch, A.; Schulze, H.; Metzl, F. ; Baas, H. Fll!iscJnYirtsch. 1969, 49. PI'.

1349·1 353. 5. Gcrhardt , U.; Schr6ter, A. Gordimr 1983, 9, pp. 171· 176. 6. Gerhardt , U.; B6hm, T. Fleischwirtsch. 1980,60. pp. 1523·1526. 7. Kanner, 1.; German, J. B. ; Kinsella, lE. CRC Crit. Rev, Food Sci. NIl/f. 1981,15,

PI' . 3 17· 364. 8. Shahidi, F. III Upidoxidation illfood:. SI.Angelo, A. J. Ed. American Chemical

Society:.Washington DC, 1992, pp. 161-182. 9. "okomy, J. In AU(Qxidorofl ofullsotllrared lipids;. Chan, H.W., -5. Ed. Academic

Prcss:.London, 19M1, pp. 14 1·206. 10. Nakatani, N.; (!latani, R. Agric. Bioi. Chelll . 1981,45, pp . 238S·2186. I I. Nakatani, N.; Inatani, R. Agric. Bioi. Chem. 1984, 48, PI). 208 1·2085. 12. Brieskom, C.B. ; Fuchs, A.; Bredenberg, J.B.; McChesney, J.D,; Wenken , E, 1,

Org. Chl!m. 1964,19. pp. 2293·2298. 13. Brieskoru, C. H.; 06mling, H.·1. Z. ubmsm. · Unters. ·Forsch. 1970, U/. pp,

10·16. 14. Wu, J.W.; Lee. M.· H.; Chang, S. J. Am. Oil Chern. Soc. 1982, 59, pp. 339-34 S. 15 . Houlihan , C.M.; tlo, C.-T. ; Chaug, S.S. 1. Alii. Oil Clrem. Soc. 1984, 6/. pp.

1036- 1039. 16, Houlihan, C.M.; Ho, C.·T. ; Chang, S.s. J Am. Oil Clrem. Soc. 1985,62, pp.

96-98. 17. Reschke. A. Z. Ll!benslll. ,Ulllers. ·Forsch. 1983,176. pp. 116· 119. 18. Bectelsc:n, G.; Ouigopberseu, c.; Nielsen, PH ; Madsen, H.L. ; Stadel, P. 1. Agric.

FoodChelll. 1995,43, pp. 1272·127S. 19. Vekiari, SA; Oreopouiou, Y.; Tzia, C. ; Thomopouloll, C.D. J. Am. OJJ Chern.

Soc. 1993, 70. PI'. 483·487. 20. Nakallll~ N. 10 Plrenolic compounth illfood and their effl!cts 0/1 heallh 1/;. HUIlIg,

M.· T., Ho, C.' T., Lee, C.·Y. Ed. American Chemical Society:.Washlogtoo DC, 1992, pp. 72-86.

2 1. V6sgen,D. Hernnano, K. Z. Lebensm. -Ulllers. -Forseh. 1980, 170, pp. 204·207. 22. Nakatani, N.; Inalani, R. ; Ohts, H. ; Nishioka, A. EIIyiroll. Healtlr Pl!fspect. 1986,

6 7, pp. \3S-142. 23. Far.g, lL S.; Bade~ AZ.MA ; Hewedi, F.M.; EI·B.roty, G.S.A. JAm. 011 Clle",.

Soc. 1989,66, pp. 792-799. 24. Kramer, lLE. J. Am. Oi/Chem. Soc. 1985,62, pp. 111 · 113. 25. Deighton, N.; Glidewel~ S.M. ; Deaus, S.G.; Goodman, B.A. J & i. Food Agric.

1993,63. pp. 221-225. 26, Yoshik.lwa, T.; Naito, Y.; Tanigawa, T.; Kondo, M. An"tim. ·FlYsch. IDrug Res.

1993, 43. pp. 363·366.

14. MADSEN ~;r AI~ AlilioxidDtive Activity of Spit.'tS twd Spice Extracts 187

27. Chan, W.K.M.; Decker, E.A. ; Lee, J,I1 .; Butterfield, D.A. J. Agric. Food Chf'lII.

1994, 42. pp. 1407- 1410. 28. Madseu, I-lL ; Nielsen, JUt. ; Derlelsen, G.; Skibsted, L H. Food Chemistry. III

press. 29. Nagababu. E.; Lakshmaiah. N, Biocllem. Pharmacol. 1992,43, pp. 2393· 2400. )0. Tsujimolo, Y.; Hashizume, tl ; YamaZlk~ M. /111. J. Biochem. 1993, 15, pp .

491·494. 31. Arnoma, 0 .1. ; Halliwell , 8 .; Aeschblch, R. ; Loliger, J. Xe"ohiotica 1992, 22, pp.

257-268. 32. Frankel. E.N.; Huang, SAY.; Aeschbach, R. ; mor, E. J. Agric. Food Chem. 1996,

44, pp. 13 1-135. 33. Frauke~ EN.; Huang. S.-W.; Kawler, J.; Gennan, lB. J. AgriC. Focx1Chem. 1994,

42, pp. \054· 1059, 34. Svoboda, K.I'.; Deans, S.G. Flavour alld Fragrance Jor/rnal 1992, 7, pp. 8 1-87. 35. Chang, 5.5.; "Ostric-Matijuevic, B.; Hsieh, O.A.L. ; Huang, C.-L. J. Food Sci.

1977, 42, pp. \102-1106. 36. Palitzsch, A .; Schulze, N.; Lotte r, G.; Steichcle, A. Fleisc/nvirlsch. 1974,54, pp .

63-68. 37. Djarmat~ Z.; lankov, lLM.; Schwirtlich, E.; Djulinac, D.; Djordjevic, A. J. Am. Oil

Chem. Soc. 1991,68. pp. 73 1-734. 38. Wada, 5.; Fang, X. JOllrl/ol of Food ProcesSing alld Preservatioll 1992, / 6. pp.

263-274. 39. Fang, X.; Wada, S. Food Research /lllerl/o/iOlraI 1993, 16, pp. 405-411 . 40. Ternes., W.; Schwlrz, K. Z. Lebellsm. _Utllers. -Forsch. 1995,201 , PI'. 548· 550. 41. Palic, A.; KrizanC(;, D.; Oikanovio-Luean. Z. Fleischwirtsch. 1993, 73. pp.

670-672.

~ " " I:

" ., , , "

, , " '.

, ' I

Chapter 15

Antioxidative Effect and Kinetics Study of Capsanthin on the Chlorophyll-Sensitized Photooxidation of Soybean Oil and Selected

Flavor Compounds

Chung-Wen Chen, Tung Ching Lee, and Chi-Tang Ho

Depar tment of Food Science, Cook College, Rutgers, The Sta te Unive rs ity of New Je rsey, New Brun swick, NJ 08903-023 1

The antioxidat ive effect of capsanthin and lutein on the chlorophyllsensitized photooxidation of 2-ethylfuran, 2,4,5-trimethyloxazole, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) was studied using a spectrophotometric method. Both capsanthin and lutein exhibited the antiphotooxidative effect on these flavor compounds. The antiphotooxidative activity of capsanthin was higher than that of lu tein. The antioxidalive effect of capSlnthin, p-carotene, and lutein on the photooxidation of soybean oil containing chlorophyll was studied by the Rancimat method, in which the induction time and the anti-photooxidation index (API) were measured. The induction times of soybean oil which contained the carotenoids were longer than that oflhe control sample which contained no carotenoid. As the number of conjugated double bonds increased, the API of soybean oil increased. The capsanthin, which contains II conjugated double boods, a conjugated keto group and a cyc10pentane ring, had higher API than Il-carotene, which contains II conjugated double bonds but neither a conjugated keto group nor a cydopentane ring. The steady-state kinetics study of capsanthin on the chlorophyll-sensitized photooxidation of soybean oil was conducted by oxygen depletion method. The result showed that capsanthin quenched singlet oxygen only and its quenching rate constant was 5.746 x 109 M- I s -I in methylene chloride,

Some food constituents are sensitive to photooxidation, Oil and fats are the most sensitive food constituents for photooxidation becausc thcy contain a large number of double bonds in their structures and probably due to the greater solubili ty of molecular oxygen in the lipid phase as compared to the aqueous phase (1,1) . Flavor compounds especially five membered heterocyclic flavor compounds such as alkylfuran, alkylpyrrole and alkyloxazolc compounds easily undergo pholooxidation reaction (3).

Cl I997 American Chemical Sociely

I S. CIIEN f::T AI... Effoct o/OIp.fanthin 011 Plwtooxidotion 0/ Soybean Oil 189

The whole photosensitized oxidation reaction can be described as follows (4):

A sensitizer, SlIch as chlorophyll , can absorb light energy and becomes an excited singlet sensitizer ('Sen O), which is then rapidly converted to the excited triplet sensitizer eSen O

) by an intersystem crossing (JSC) mechanism, a process of electron rearrangement. The )Seno transfers its energy to triplet oxygen ('01) to generate the singlet oxygen (101) by a triplet-triplet annihilation reaction, a process which occurs via a collision of two excited )01 Singlet oxygen is a very reactive form of oxygen and easily attacks the substrates (A) which have double bonds to generate oxidized products (A01),

The process of photosensitio>:ed oxidation can be minimized by quenchers, which can quench singlet oxygen andlor excited sensitizers. The antiphotooxidative effect of nickel chelates on the singlet oxygen oxidation of soybean oil have been reported by Lee and Min (.5), Ascorbic acid and tocopherols are not only free radical scavengers but also good singlet oxygen quenchers (6.7). len-Butyl hydroquinone (fDHQ) as weI! as other phenolic compounds can also acl as singlet oxygen quenchers (8-/2). Rosemary oleoresin and rosemariquinone, a rosemary extract, can inhibit light-induced oxidation (13./4) . Among these quenchers, carotenoids appeared to be the most plausible candidates for quenching the singlet oxygen. The quenching mechanisms and kinetics of several carotenoids. including f3-apo-8'carotenal, Il-carotene, canthaxanthin, lutein, zeaxanthin, lycopene, isozeaxanthin, and astaxanthin have been well studied (/5-17) .

Capsanthin (Figure I) is the most abundant carotenoid in the paprika spice. The concentration of capsanthin is about 1590 mg per kg of dry mailer and 41 -54% of total carotenoids in paprika ( /8,/9). The antiphotooxidative effect and kintics study of capsanthin on soybean oil has not been well studied. In addition, the study of the antiphotooxidative effect of carotenoids mostly focused on lipid or fatty acids. The antioxidative elTect of carotenoids on the photooxidation of flavor compounds has not been well studied, either.

This paper reports the antioxidative effect of capsanthin on the chlorophyllphotosensitized oxidation of soybean oil and selected flavor compounds including 2-ethylfuran, 2,4, 5-trimethyloxazole, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), which are unstable when exposed to light in the presence of sensitizer (3,10). The quenching mechanism and kintics of capsanthin on the photosensitized oxidation of soybean ' oil are also reported. Il-carotene and lutein were used as cont rols for comparing the antiphotooxidative activity with capsanthin.

EXPERIMENTAL

Materials. Soybean oil was purchased from a local supermarket and purified in our laboratory according to the method of Lee and Min (/5). Chlorophyll was extracted and purified from spinach according to the method of Ornata and Murata (1/). 11-carotene was purchased from Sigma Chemical Co. (St. Louis, MO). Capsanthin,

190 S t>I CES; ~UVOR CIIF .. M ISTRY AND ANTIOX!I)A NT "KO t'ERTlE.."

Compound Structure

Capsanthln

B·Carotene

Lutein u

on

0 "

No. of conjugated double bond

11

11

10

Figu~ I. Structures of cap sa nIh in, J3-carotene, and lutein.

coolant ol.ltld +--

condenser

oxygen Inld .. coolant lold

reaction n ask

light 5Oun-e water light so~n-e

Figure 2. Apparatus for the photosensitized mtidation of flavor compounds.

I S. CII I£N ~.,. AL Effect of Capsollthitl 011 P//IJlO(Jxidation oj Soybean Oil 191

which was extracted from paprika spice, and lutein were obtained from KALSEC Co. (Kalamazoo, Ml). 2-Ethylfuran, 2,4,S-trimethyioxazoie, and DMHF werc purchased from Aldrich Chemical Co_ (Milwaukee. WI), Ab$Olute alcohol was obtained from Pharmco Products Co. (BrOOKfield, CT).

Evaluat ion of the IIn lioxid.uj"t effect or carolenoids on the chlorophyllsensiliud phOlOOl id llion or 2-ethylrunn, 2.4,5-lrimelhylollzole, lind DMHF by 5pectrophotometric method. Mixtures of I x 1003 M 2-ethylfuran, 2,4,5· lrimethyloxazoie, or DMHF in]OO rol absolute alcohol containing 10 ppm chlorophyll and either 1.0 x iO-~ or 2.5 x iO-~ M carotenoids were prepared in 250 ml of flask. The antiphotooxidative effect of carotenoids including capsanthin and lutein on these flavor compounds was carried out under light exposure at 20"C while bubbling with oxygen It 260 ml/min according to the method of Chen lind Ho (3) as shown in Figure 2. The light intensity at the sample posit ion was 67,000 lux. The effect of carotenoids was detennined using I Hitachi U-31l0 spectrophotometer (Danbury, Cf) in which the percentage of substrate remaining was calculated by measuring the decrease of optimum wavelength absorbance of test compounds. The optimum wavelength absorbance of 2-ethylfuran, 2,4,S-trimethyloxa.tole, and DMHF was 215, 225, and 29Onm, respect ively.

Evaluation of th e antioxidative effect of carotenoids on the chlorOI)h), lI sensit ized photooJidation of purilied soybean oil by the Rancimat meth od. Samples of 100 or 200 ppm carotenoids in 2.5 g purified soybean oil containing 200 ppm chlorophyll were prepared in 2.0 cm i.d. x 15cm glass open cylinders. The cylinders were then placed in the light box, which was modified from the device as reported by Lee and Min (15). The light intensi ty at the sample level was 10,000 lux . The temperature of the light storage box was kepI al 25°C. After 4 hrs storage in the light box, the cylinders were placed in a Model 679 Rancimat instrument (Metrohm, Switzerland). The oxidative stabili ty of this soybean oil was measured by determining the induct ion time at ]()()OC. The ai r supply was maintained at 20 IIhr.

Quenching mecbanisol and kinetics study of capsanthin by olygen depletion method. The quenching mechanism and kinetics of capsanthin were studied using the steady Slate kinelic method of Foote and Denny (22) and Lee and Min (16). Samples of 0.03, 0.06, 0.10 or 0.16 M purified soybean oil in methylene chloride containing 4 ppm chlorophyll and 0, 0.25 x 10-$,0.5 x 10-$,0.75 x lO-s or I x 1O-~ M capsanthin were prepared. Fifteen mt of prepared sample was transferred inlo 30m! serum bottles and sealed air-tight with rubber septa. Duplicate samples were placed in the light box for 2hrs at 25°C and the percentage of heads pace oxygen was analyzed using a Hewlett-Packard 5890 II gas chromatograph (Ge) equipped with a thennal conductivity detector (TCD) and a Hewlett-Packard )396A integrator. The GC column was a two-layer concenlric column (Alltech CTRI, Avondale, PAl; the outer column was 6 ft x 1/4 in. o.d. packed with an activated molecular sieve; the inner column was 6 ft x JIB in. o.d. packed with a porous polymer mixture. The flow

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192 SPIC.;S: H AVOR Cm :i\W,TRY AN I) A.NTIOX IDANT I'ROPER1H:S

rate of helium carrier gas was at 20 ml/min. The temperature of thc injection pon, oven and detector wcre 50, 35 and 100°C, respectively.

RESULTS AND DICUSSION

Antipholooxidative effect of ca rolenoids on 2-elhylfuran, 2,4,5-trimethylolnole. and DMRF. The effect of 1.0 Ie iO-s or 2.5 x lO-s M capsanthin or lutein on the photooxidative stabili ty of2-ethylfuran, 2,4,S-trimethyloxuole, and DMHF in absolute alcohol containing chlorophyll was conducted under light exposure while bubbling with oxygen. The percentage of substrate remaining was determined using spectrophotometry and was used as an index of ant iphotooxidative activity of carotenoids. The gre<lter the percentage of substrate remaining, Ihe higher the anlipootooxidative activity exhibited.

The ant ipholooxidative effect of capsanthin and lutein on the 2-ethyJfuran, 2,4,5-trimethyloxazole, and DMHF is shown in Figure 3. Both capsanthin and lutein showed the antipholooxidative effect on these flavor compounds. The antiphotooxldative activity of carotcnoids increased when the concentration increased from 1.0 x 10-5 to 2.5 x lO-s M . Comparison of the same concentration of capsanthin and lutien, the antiphotooxidative activity of capsanthin was higher than lhal oflUlein.

AnliphotoOlidlitive elTed of carotenoids on the induction time ofsoybean oil a5

measured by the Rancimat method. The effect of 100 ppm or 200 ppm capsanthin, p-carotene, or lutein on the pholooxidative stability o f soybean oil containing 200 ppm chlorophyll was measured by the Rancimat method after 4 hours light cxposure at 25°C. Preliminary studies showed that Ihe induction time of purified soybean oil containing 100 Of 200 ppm carotenoids wi thout chlorophyll was slightly lowef than Ihat of the blank sample which contained no carotenoids (data nol shown). Therefore, there is no anti-autoxldative effect of carotenoids on the soybean oil. Table I shows that as the concentration or the carotenoids increased from 100 to 200 ppm, the induction time as well as the anti-photooxidation index (API) increased. The induction lime of soybean oil containing the carotenoids was longer than that of the control sample which containcd no carotenoid; however, it was still shoner than that of the blank sample which contained no carotenoid and no chlorophyll .

Table II shows that as the number of conjugated double bond increased from 10 to II, the API of soybean oil increased at the concentrations of 100 and 200 ppm carotenoids. It has been reponed that as the number of conjugated double bonds of carotenoids increascd, the peroxide values of chlorophyll-sensitized photooxidation of soybean oil decreased significantly (16.1 1). Therefore, the singlet oxygen quenching abi lity of carotenoids was dependent on the number of conjugated double bonds of the carolenoids as reported by Lee and Min (16), Jung and Min (17). and Hirayama et a!. (23).

Table II also shows that the soybean oi l containing capsanthin had a higher API than the oil containing j3-carotene ahhough both of the carotcnoids have the

L5. cm:N ET At... EJ/tcl o{ Cap.~nthin on Pho(ooxiriation o{ Soj'briJn Oil "3

~

II' 100 no chlorophyll and ~

~ carotenoid

: 80 ·c •• e '" capunthln ( 2.S J: 10-5 M) • • lutein (2.5 x 10.5 M) • 40 ~ • capsanthin (1.0/10.5 M) • ~ • 20 lutein (1.0 J: 10' M) .Q

chlorophyll only , '" 0

0 , 3 4 5

Time (min)

II' 100 no chlorophyll and ~ carotenoid ~

.5 : ••

80 capsanthln (2.5 J: 10.5 M)

e lutein (2oS J: 10.5 M) • • .0 capsanthin (1.0 I( 10.5 M)

~ • lutein (1.0" 10·J 1\1) • ~ 40 (B) .lI ehlorophyll only , '" 20

0 , 3 4 5

Time (min)

II' '00 ~

no chlorophyll

= and carotenoid ·c 80 .; e " capsanthin (2.S x 10.5 M) • • ~ .. lulein (2.S I( 10.5 M) 5 • capsanthln ( 1.0 J: 10' 1\1) • ~ " (C) lutein (I.0 I( 10.5 1\1) .Q , chlorophyll on ly

'" 0 0 " 12' "" Time (min)

Figure 3. Antipootooxldative effect of capsanthin and IUlein on (A) 2-ethylfuran, (8) 2,4,S-trimethyloxazole, and (C) 2,S-dimethyl. S-hydroxy-3(2H} furanone (OMHF) in absolute alcohol conlaining \0 ppm chlorophyll at 20 °C.

I

19' SI'IC ~;S: n A VOR CHEMISTRY AND ANTIOXIDANT " ROI't;RTl ES

Ta ble I. Effect of carotenoids on the induction time detected by the Rancimat method of soybean oil containing 200 ppm chlorophyll during light exposure for 4 hours.

Carotenoid (ppm)

Control) Capsanlhin (100) Capsanthin (200) Blank)

Controf p.Carotene (100) p·Carotene (200) Blank1

Controll

I.utein (100) Lutein (200) Blank}

Induct ion lime OT)I (hIs)

7.96 8.94 9.64

11.20

7.96 8.90 9.60

11.20

7.96 8.74 9.39

11 .20

Ant;-phOlooxidation index (API)I

0 .303 0.518

0.292 0 .505

0.241 0.440

1. API - (carotenoid IT· control IT) I (blank IT - control IT) Each value is the means of duplicates

2. soybean oil + 200ppm chlorophyll 3. soybean oil only

T a ble U. Effect orthe number of conjugated double bond or carotenoids on the anti -photooxidation index (API) of soybean oil during light exposure.

Carotenoid

(100 ppm) Lutein p·Carotene CapsBnthin

(200 ppm) Lutein !3·Carotene Capsanthin

Number of conjugated double boud

\0 I I II

\0 II II

API

0 .241 0 .292 0 .303

0.440 0 .505 O.S Ig

15. e llEN ET At. Effect o/Cnpllonlhin on I'Jlf)tooxidation of Soybean Oil 195

same number of conjugated double bonds. It has been reported that the singlet oxygen quenching ability of carotenoids was determined not only by the number of conjugated double bonds.. but by the functional groups of the carotenoid (23). As reported by Uirayama el II (13), conjugated keto groups and the presence of a cydopenlane ring in the carOienoids stimulate quenching efficiency. Comparison of the struclUre of capsanlhin wilh that of Il-carotene reveals that there is one conjugated keto group and one cydopentane ring in the SlNClure of capsanlhin but !}-carotene comains neilher of them (Figure I). Therefore, the anliphotoo)(;dative act ivity of c.apsanthin is higher than that of p-carotene, as shown in Table II This result confirms Hirayama's prediction.

The Rancimat method has been used to measure the antioxidant activity of synthetic and natural antioxidanu (U.}6) and has correlated well with oil stability measured by the Active Oxygen Method (17) and peroxide value measurement (28). Our study showed that using the Rancimat method to study the antiphotooxidative effect of carotenoids on the soybean oil was in agreement with the results using the headspace oxygen depletion method (16) and the peroxide value method (16,1 7) .

Quench ing mecha nism a nd kinet ics of caps!lnt hin. The following steady·state kinetic equation would be established if carotenoids reduced the chlorophyll· photosensitil:ed singlet oxygen oxidation by singlet oxygen quenching (4,22).

{.d[02Vdt}·t - {d[A0211dt}·\ - K'\ (1 + (kq[Q] + kox-Q[Q) + kd)l1:r{All

A02 : oxidized soybean oil K . rale of sing let oxygen formation kq : reaction rate constant o r physical singlet oxygen quenching by capsanthin Q . capsanthin kox-Q : reaction rate constant or chemical singlet oxygen quenching by carotenoid kd : decaying rate constant or singlet oxygen kr : reaction rate constant or soybean oil with singlet oxygen A . soybean oil.

The intercepts and slopes of the plots of (-d[02Vdt)-\ vs [AJ-l at various concentrations of quencher (Q) are K'\ and K-\ {(kd + kq[QJ + kOX-Q[Q}/krl, respectively. The plot of SQlSo (slopes in the presence and absence or quencher) vs [Ql is a straight line, and the slope or the straight line is (kq + kOX-Q)/kd (4,}9.30). The rate constants (kd) of singlet oxygen decay in different solvents have been repon ed (31). Therefore, the total singlet oxygen quenching rate constant (kq + kOX_Q) or quencher can be determined rrom the slope of the plot of SQlSo vs lQl

[4]. The plots or(-d[021/dt)- L vs [soybean oil)-\ at di fferent levels orcapsanthin

are shown in Figure 4 . The intercepts were the same at different levels or the capsanthin, but the slopes or the plots increased as the concentration or capsanthin increased from 0 to 1 x 10-5 M . This resul t showed that capsanthin as well as other

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5 1'l eKS: FlAVOR CHEMI~1'RY AND ANTIOXIDANT I' WOI'ERTn:s

• no c:ap llmlhin • 0.25 1l10·5M • 030 x IO·5M • 0.15 x IO·5M Q 1.00 x IO-5M

10 20 30

1/011 in methylene chloride (11M)

'0

Figure 4. Effect of capsanthin on the headspace oxygen depletion of soybean oil in methylene chloride containing 4 ppm chlorophyll when exposed to light for 2 hours at 25 0c.

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5

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0 0.0 0.2 0.' 0.' 0.8 1.0 1.2

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Figure S. Plot 0( 5«'30 vs {capsanthin}.

••

IS. c m:N ET AI~ Effect oj CapSll rllhin 011 Photooxidatiotl ojSo),bean Oil 197

carotenoids quenched the singlet oxygen but did not quench the excited triplet state of chlorophyll to reduce the photosensi tized oltidation of soybean oil (4,5, /5,/6).

To measure the 10lal singlel oxygen quenching rate constant (kq + kox-Q) o f capsanlhin, the regression line of setSo V5 (capsanthin] was plotted as shown in Figure 5. So and So are the slope! of the plot of (-d[02ydl)"1 vs {soybean oi l]- I in the presence and absence of capsanlhin, respcetively. As shown in Figure 5, the slope of the plOI ofSQfSo vs {capsanthin1 is 5.746 x ,lOS M-l . Since the slope of the plol ofSQfSo vs [quencher] is (kq + kOX-Q)lkd «(11.30) and the kd value of singlet oxygen in methylene chloride is 1.0 x 1()4 s-I (3/), Ihe total singlet oxygen quenching rate constant (kq + kox-Q) of capsanlhin is 5.746 x 109 M-Is-t in methylene

chloride.

CONCLUSION

II has been known that carotenoids are able to quench singlet oxygen to reduce the photosensitized oxidation reaction (/6./7) . Our study showed that capsanthin, a major carotenoid from paprika, as well as p-carotene and lutein exhibited the ability to quench singlet oxygen to reduce the photosensitized oxidation of both soybean oil and selected flavor compounds including 2-ethylfuran. 2,4.5-trimethyloxazole, and DMHF. These results suggest that capsanthin as well as other carotenoids may be applied to food system which contains food lipids or flavor compounds to minimize food photodeterioration.

REFERENCES

I.

2.

3. 4.

5. 6. 7. 8.

9. 10.

11 12 .

13.

Rosenthal, I. in Singlet O~ Vol IV Polymers alld Biorna/ecule.f. Frirner, A.A., Ed.; C RC Press: Florida, 1985, pp. 145-163. Samuel, D.; Steckel. F. in Molecular Oxygen ;11 Biology, Hayaishi, 0 ., Ed.,

Elseviu: New York, 1974, I. Chen, C-W.; Ho, C-T. J. Agric. FoodChem. in press. Foote, C .S. in Singlet Orygell, Wasserman, H.H. and Murray, R.W., Eds., Academic Press: New York, J979, Pl'. 139-171. Lee, S-.H ., Min, D.B. l . Agric. FoodChem. 1991 ,39,642-646. lung, MY.; Choe, E.; Min, D.M. l . Food Sci. 1991 ,56,807. Chou, P-.T.; Khan, A.U. Biochcm. Blophys. Res. Cornm. 1983, 1/5, 932-9]7. Sherwin, E.R. in Food Aciditil'eS. Branen, P.M.; Davidson and Salminen,S., Eds., Marcel Dekker, Inc; New York. 1990, p. 139. Clough, R.L.; Yee, B.G.; Foote, C.S. l. Amer. Chem. Soc. 1979,101,83 . Matsuura, T. ; Yoshimura, N.; Nishinaga, A.; Sai to, 1. Te/ra. Lett. 1969,2/, 1669-\671 Thomas, M.l .; Foote, C.S. Pno/ochclII. ,'n%hiol. 1978,27,683-693. Tournaire, C.; Croux, S.; Maurette, M-.T. 1. Photochem. Ph%biol. 1993, 19, 205·215. Hal111I, c., Cuppett, S. l . Amer. Oil Chem. Soc. 1993, 70,477.482.

I ,98 SI' ICF..S, FlAVOR CHEMI!o.'TRY AND ANTIOXmANT I' IW I'ERTIES

14. Hall III , C.; Cuppel!. S.; Wheeler, D., Fu, X. J. Amer. Oi/ Chem. Soc. 1994, 71, 533-535.

15. Lee, E.C.; Min, D.B. J. Food Sci. 1988,53, 1894·1895. 16. Lee. S-.W.; Min. O.B. 1. Agrie. FoodChem. 1990,38, 1630.1634. 17. Jung, M.Y.; Mjn, D.B. J. Amer. Oil Chem. Soc. 1991, 68, 653-658. 18. Mlnguu-Mosquera. M.I., Homero-Mindez, D. J. Agrie. FoodChem.J994 , 42,

1555·1560. 19. Biaes, P.A.; Daood, H.G.; Huszka, T.T.; Biaes, PK 1. Agrie. Food Chem.

1993, 41,1864-1867. 20. Chen, C.-. W.; Shu, C.-K.; Ho, C.-T. 1. Agrie. Food Chem. in press. 21. Ornata, T.; Murata, N. Pho/achem. Ph%biol. 1980, 31,18]-185. 22. Foote, C.S.; Denny, R.W. 1. Amer. Chem. Soc . 1968,90, 623 3.6235. 23 . Hirayama, 0 .; Nakamura,K. ; Hamada, S., Kobayasi, y , Lipids 1994, ]9,149-

ISO. 24. Chen, C .-W.; Ho, C.-T. 1. Food Lipids 1995, 2, 35-46. 25. Zhang., K.Q.; Bao, Y.; Wu, P., Rosen, R.T.; Ho, C.-T. 1. Agric. FoodChem.

1990,38, 11 94-1197. 26. Ho, C-.T .; Chen, Q .; Shi, H.; Zhang, K.Q., Rosen, R .T. Prel'ellliVl! Med. 1992,

21,520-525 , 27. Laubli, M.W.; Bruttel, P.A. 1. Amer. Oil Chem. Soc . 1986, 63, 792-795. 28. Gordon, M.H.; Mursi , E. 1. Amer. Oil Chern. Soc. 1994, 71, 649-65 1. 29. Foote, C.S.; Ching, Ta-Yen.; Geller, G.G. Pho/ochern. Ph% biol. 1974,20,

511-513 . )0. Yamauchi, R.; Matsushita, S. Agric. Bioi. Chern. 1977,41 , 1425-14) 0. ) I. Hurst, J.R. ; McDonald. J.D.; Schuster, G.B. 1. Arner. Chem. Soc. 1982, 104,

2065-2067.

•

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Chap'er 16

Curcumin: An Ingredient that Reduces Platelet Aggregation and Hyperlipidemia, and Enhances Antioxidant and Immune

Function s

Vaguaog U u

1.. Y. Resea rch Corporat ion, 67- 08 106A Street, Flushing, NY 11365

The effects of curcumin on blood platelet aggregation, hyperlipidemia, lipid peroxidation and immune fun ction wcre determined. Curcumin exhibi ted st rong inhibitio n of blood platelet aggregat ion in humans and lipid peroxidation. in~uced by adenosine diphosphate (ADP) of rau The marked nse til serum Chole5tcrol levels in rats following 4 weeks of cholesterol fceding was significantly reduced by curcumin. Addi tionally, curcumin showed that it increased immune function, including macrophage rate and Iymphoblastoid transformation in imlnunosuppression rats induced by cyclophosphane (CY). The above pharmaceutical effects o f curcumin with addition of flavone of Malricaria L. were stronger than curcumin alollt. It can be seen that c~rcumin effectively prevents cardiovascular disease, increases Immune function and exhibits anti-oxidant activity.

Curcumin is isolated from the rhizomes of the plant , CllrCl/flla lal/ga L. which is a medicinal plant widely used in China, India and Southeast Asia. The rhizomes of the plant has been used in China as a reduction in serum hyperlipidemia lind

stomachic drug (I) . . . . Curcumi" has demonstrated anti_inflammatory and anll-oxldant properties and

it is essentially non-toxic (1-6). Recent studies have illdicated that a group of four ingredients, which include cureumin, scoparone, ferulic acid and flavone . of Malr icaria L. has demonst rated marked hyperlipidemia and platelet-aggregatIOn

reducing propert ies (7). . . This paper describes the effects of curcumm and flavone of M. c~J(mlOml{fa L.

on platelet-aggregation, hyperlipidemia, anti-oxidant and immune funcllons.

C 1997 American Chemical Society

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200 SI'ICt:s, HAVOK CHEMI~"TRY AND ANTIOXIDANT "KOI' EKTI ES

Materinls

Curcumin was extracted from C. 10llgel L. according to the following steps. Rhizome of C IQ"ga L. o r C. aromaliea salish or C. zeJoaria RQ5CO was

driC<l and powered. One kilogram of the powder was soaked in 5 liters of 95% ethanol, fOf 12 hoors at room temperature_ The resulting ethanol extract was fiitered. The ethanol was then recovered under reduced pressure distillation. A rcsid~c was dissolved in 200 ml o f I N NaOH. The resul ting solution was adjusted to pH 7 with I N HCI and a light ycllow precipi tate was formed. The precipitate was dissolved in 300 ml o f 95% ethanol and the last procedure was repeated to form a light yellow precipitate again. The light yellow precipitate was washed with acetone and ether successively and then dried under vacuum and was found to have a melting point at 18) ° C.

Flavone was e:o: tracted fW1l1 M. chamQmilla L. according to the following steps.

Plants o f M. dutll/omillo L. were dried and powdered. One kg o f the powder was e:o:tracted in 2 liters of 95% ethanol for about 24 hours at room temperature. The solution was fi ltered afld the filtrate saved. Two liters of 950/. ethanol were add~d to the residue and renuxed in a water bath for 6 hours. The renuxed ethanol was cooled and filtered and the filtrate combined wi th the ext ract fiitrate. Ethanol w~s then recovered by reduced pressure distillation and the residue was saved. One thousand ml of acetic ether was added to the residue and rcnu:o:ed in a waler bath for 6 hours. The renuxing procedure was repeated. Acetic ether W3$ then concentrated under reduced pressure distillation. Crystals were formed which were then washed wilh waler. The final crystals were dried under vacuum and were found to have a melting point of about 25f1 C (7).

C. 10llga I •. aod M. chanlt)milla L. are recognized by Food and Drug administration (FDA) as safe for human consumption.

Methods

m ood Ilhutiet Aggregatioll . The blood platelet aggregat ion lest was performed according to the method of 130m et al. (8). Blood was collected from veins of humans using a needle attached to a plastic disposal syringe. The blood was immedia tely transferred to a siliconized glass tube containing 0. 1 volume of 3.130/. soohlm citrate. Platelet-rich plasma (PR!') was oblained by centrifugation of the whole blood at 1000 rpm for 10 min at room temperature_ Platelet-poor plasma (1'1'1') was prepared by centri fugation of the remaining blood at )000 rpm for 10 min. Platelet aggregation was performed at 37°e. Human platelet studies were carricd 0 11\ wilh a COnstant platelet number () x 10' Iml). With regard to detcflllillatior1 of plAtelet aggregation. maximum aggregation was induced by 0.2 I-\ M (ADI') . 0 ,4 Ill I PRP was added into each tube. Thirty six tubes were divided into three groups. Fifiy III of saline was added 10 each tube of control group, 50 1-\1 of curculllin (0.5 lIIg/ml) was added to each tube of the crucumin (t reatmE:nt I)

16. U U Curcumin: Platelet All1JT"f8atiQn d: flyperlipidemia 20'

group. and 50 ~I of curcumin (0.45 mg/ml) + 5 III flavone of M. chanwmilla I •. (0.05 mg/ml) were added to each tube of w rcumin plus flavone of M. chamomilla L. (treatment 2) group. After incubation for 3 minutes at )1' C., 50 I.d of2 M ADP was added 10 each tube. A five minute's aggregation GUrvc for eath lube was plotted.

Percent inhibition of aggregat ion by PHP was calculated by:

% 8"~l'Ilion in control· % IUn:gal'OQ ,..il ll tmtIUlC'" %inhibilionofagrrgalion - ---- ------------- .~ 100 %

% aggregation in control

Determination of Lipid Per"Oxidation in Mitochondria and Mkr"050mt!J. Rats were killed by decapitation afier fasting for 24 hours and their liver tissue was quickly removed. Microsomal and mitochondria fract ions were isolated from the liver ti ssue by the method ofOda (9). The liver tissues wefe cut into small slices in 0.25 M sucrosc containing 3 mM Tris-HCI and 0. 1 mM EDTA (pH 7.4) at 4°C, and then small slices of liver ti ssues were homogenized with nine limes (Ihe amount by weight) 0.25 M sucrose solulion conlaining 3 mM Tris-HCI and 0. 1 mM EDTA (pH 7.4) at 4° C. The homogenate solutions were adjusted to pH 7.4 by the addition or o. t N KCI, and then the homogenate was centrifuged at 50 x g for 10 minutes al 4D

C to remove the nuclear fractions and red blood cells. The supernatant phase was centri fuged al 700 :0: 8 for 10 minutes at 4°C, and then further centrifuged at 5,000 x g for 45 minutes at 40C to give mitochondrial fractions. The isolated mitochondrial fract ions were washed twice with Krebs-Ringer phosphate buffer (pH 7.4). The supernatant phase centrifuged at 5,000 :0: 8 for 45 minutes was further centrifuged at 24,000:0: g for 10 minutes 8t4° C. The precipitation was removed and the supernatant phase was again centrifuged at 54,000 x g for 60 minutes at 4' C to obtain microsomal fractions. The isolated microsomal fractions were washed twice with Krebs-Ringer phosphate buffer (pH 7.4). Mitochondria and microsomes were suspended in Krebs-Ringer phosphate bulTer (pH 7.4). The final protein concentrations of the mitochoodrial and microsomal suspensions were adjusted to to mg protein/ml.

A mi:o:lure of mitochondrial suspension (0.5 ml, \0 mg protein/ml), KerbsRinger phosphate bulTer conlaining 40 mM ADP, 12 mM ascorbic acid and the indicated amoullt of curcumin and flavone of M chamoll/illa L. were incubated at 31' C for 15 minutes in a final volume of I ml, In the treatment I group, buffer contained 5 :0: 10-4 M curcumin. In the treatment 2 group, buffer contained 0.45 x 10.4 M curcumin and 0.05:0: 10-4 M flavone , Lipid peroxides o f mitochondria were determined by the method ofOhkawa ct al. (10).

Reducillg Hypcrlipidemia Ted. Young male Wislar rats with an average weight of 130 g were fed 1I0rmallaboratory dict and were housed individually in cages. Room temperature was controlled at 25 ± l a C with 60"10 re lative humidity. Lighting was controlled with cycles of 12 hours light followed by 12 hours of dark.

202 SI'!CES: n,AVOR CIIEMISTRY ANIl ANTIOXIDANT PROl'ERTIFS

Fony male rats were separated into four groups. The normal gTOOp was given II regular laboratory diet . Cont rol and treatment groups 1 and 2 were given II regular diet supplemented with 1% cholesterol and 0.2% eholie acid. A restricted diet (8g) was prescribed every morning. Curcumin (dose 100 mglkglday) was dissolved in distilled water (2 ml/rat) and was orally administered to rats of Irealmenl 1 group. Curcumin (dose 90 mg/kg/day) plus flavone (dose 10 mglkglday) were dissolved in distilled water (2 O1ll(81) and was orally administered to rats of treatment 2 group. After 4 weeks of feeding. the ralS were starved J 8 hours, then sacrificed. Blood was collected from Ihe jugular vein. The blood and liver were removed immediately and portions of the tissue were examined to detcRTline cholesterol, triglyceride and total lipid.

Lipid Conte'lI l . Total lipid contents of the' liver and serum were determined by the methods of Sperry and Braoo (IJ). Total cholesterols were determined by the methods of Carr and Drekter (11), frcc cholesterol by the method of Sperry (13), and triglyceddes by the method of Van handel-Zilversmit (14) .

Immune Flilielioli. Male mice weighting 18-20 g were used in the experiments and were divided into treatment 1,2 and control groups. f or treatment I group, the dosage or curcumin was 5.5 mglkg injected intrapctitoneally. For treatment 2 group, the dosage o f curcumin was 5.0 mglkg and 0.5 mglkg flavone. The mice of control group were injected with the same volume o f normal saline. These injections were repeated daily for 7 days. On the last day, both trealed and control groups were injected intraperitoneally with CY. The dosage ofCY was 4.5 mg/kg. On the seventh day, the animals were killed by decapitation. The blood was removed immediately and was examined to determine immune fimelion.

Ly mphoblastoid Transrormlltion Test . Lymphoblastoid transformation test was performed using the method of Hogan (15). Cells (J x 10'/ml) were maintained in Eagles minimum essent ial medium containing 10% fetal bovine serum (FIlS) and 10 pglml polysaccahride-free purified phytohemagglutinin (PI·IA.P). The cells were incubated at J7oC/5% CO, 195¥o humidity and the medium changed every day.

I\-bcrophllge. The macrophage cells were cultured in PPM.I- I640 medium with 10% FBS. Aller overnight incubation at 31' C in an aunosphare of 50/. C~ aoo 95% air, non-adherent cells were washed away. The adherent cells were funher incubated with 4 rul of the medium for 24 hours at 31' C. Antigens were added to macrophage monolayers, After 6 hours incubation at 31' C, the monolayers were washed four times to remove antigens and immediately used for experiments. Macrophage cytotoxicity was measured as previously described (16). Results are expressed as a specific percentage of !lCr release (percentage cylotoxicity) as calculated by the following formula: percentage specific cylotoxieity - 100 x (experimental C.p.m. - spontaneous c.p.m.)I(tolal c.p.m. -spontaneous c.p.m.) .

16. U U Curcumin: Platelet Aggregation & Hyperlipidemia 203

Result! Alld Discussion

The significant inhibitory effect of curcumin on platelet-aggregation is summarized in Table I below. Curcumin with flavone of M. dUll/lOll/ilia L. appeared to be more effective than curcumin alone. Platelet aggregation and subsequent adhesion under the influence of local fl ow irregularities are primary and injury of the intimal tissue is secondary (17) . Organ damage and dysfunction may be a consequence of platelet awegation and embolization of platelet thrombi in micro-circulation Aggregation of platelets might cause damage result ing in obst ruction of vessels. Intermittent infusions of adenosine diphosphate into the coronary circulation of pigs caused circulatory collapse, electrocardiographic evidence of myocardial ischemia, ventricular dysrhylhmias and formation o f platelet aggregates in the microcirculation (18, 19).

Table I. Effects or C urCUlllin on Aggregation or Platelet

Rate of asgregation of platelet Control Treatment 1 Treatment 2

67.6 ± 5.0 26.S ± 0.8· 24,7 ± 0.7·

Each value represents the mean of 10 animals ± S.E. • P<O.OI, significantly different from the normal group. Treatment I: Curcumin only. Treatmelll 2: Curcumin plus flavone of Matricaria c/llUIlomilla I ..

As shown in Table II , the blood cholesterol level in rats fed I % cholesterol was elevated 21:14% and supplementation of curcumin and curcumin plus flavone of M. chamomifla L. to the cholesterol diet depressed this elevation significn!l!l)' Phospholipid levds showed no significant change. The addition of flavone or At! challlomil/a L , appeared to be more effective than the curcumin-only group.

TAble II . ElTect OfCurc:ulllin 011 Cholesterol Levels in Blood

Diet Cholesterol Phos~holi~id CholesteroVPhos~holi~id

(mgldl) (mgldl) (%)

Normal 125 ± 6 201 ± 5 62 Cholesterol 4S0 ± 40 230 ±9 209 Treatment I 2 10 ± 10· 20S ± 6 · 101 Treatmenl2 176tS' 195 ± 5' 90

As shown in Table III , total lipid levels, free and total cholesterol, and triglyceride levels of the liver decreased significantly when curcumin and navone of M, chamolllilla L. wefe added to the hypercholesterolemic diet but free cholesterol in the liver appeared no significant change.

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204 SI' IC,.:.s, FlAVOR C II EMJI'I"TRY AND ANTIOXIOANT l'ROI" ~RTIES

Tlll)lc lIJ. [ fTecl orCurcumin on Lipid uvd

Per 100 g li,"er li$SUe T~al lipid Total cholCSlcroi Flee cholesk:roI

III ( lng) (mg)

Norma! 7.!I.t I.O j I6 J:90 lJl.i21 Choleslerol 17.1:1:2.1 2472:1:151 327.t26 Tlc-llmenl I 9.0 :to.S' 1101 :1:92 ' 29 1 :i2l' Trc.,IIlICUll 8. 1 :t0.8 ' 921 :1:80' 280:1:24'

Each value represents the mean of 10 samples ± S.E.

Trigl)Wridc (mg)

397:1: 2S j20:t 27" " !II :tlS" "]].ill '

'I' < 0.05, significantly different from the normal group; .. P < 0.01

Reccnt clinical studies have shown that the plant C. lUI/go L . significantly reduces blood lipid (I). As shown in Table III , curcumin can also significantly reduce blood and liver cholesterol levels. Therefore, these studies suggest that curcuntin may be the active ingredient responsible for the lipid-lowering effects of the plant C. IVI/ga L

Table IV lists Ihe lipid pero:o.:idlltion in rat liver mitochondria. Treatment I and 2 significantly reduced the level of pero:o.:idation. Pero:o.:idation of lipids in the liver has been shown to result in many kinds of to:o.:icity such as of membrane function and damage to membrane bound enzymes (20).

Tallie IV, Effects or Cur<:um in on ADP plus Ascorbic Acid-induced Lipid PeroxidAl ion in Rllt Liver Mitochndria

Group Normal Treatment I

Inhibition

Treatment 2 69.1% ± 5.0 · Each value represents the mean of I 0 samples ± S.E. • P<O.OI, significantly different from the normal group.

CY is a very st rong immune inhibitor. It inhibited phagocytic rate to 77% and lymphoblastoid to ~8°/. in rats. As shown in Table V and VI, curcumin and curcumin with navone of M. chamvlI/if{a L. can significantly increase immune function which has been markedly decreased by CY.

Tllble V. Effects orCurcumin on MacrOllhllges

Group Control Cy CY + Treatment I CY + Treatment 2

Phagocytic rate ± SO (%)

35.10 ± 2.01 8.00 ± 0 .36

21 .00 1. 0.20· 23 .20 ± 0.20·

16. LlV CllTCUmi,.: Platelet Aggregation & I/)perlipitlf'mia

Table VI. EIT« ! or Cul"(: unlin 011 LY"lpholllasloili Trl'lliSrormRlion

Group

Control

Cy

CY + Treatment I

CY + Treatment 2

CPM ±SD

I3~O ±Sl

697 ± 38

905 ± 70·

985 ± 75·

Each value represents the mean of 10 samplcs ± S.E. ' P<O.OI. $igniflcanlly different rrom cydophosph.unide group.

,.,

On the basis of the above results, we conclude that curcumin may protect against cardiovascular disease, increase immune function and has anti-oxidant activity.

Litenture Cited

1. Xue C.S. Pharmocology and application of Chinese Medicine; Wang Y. Ed.; People Health Published House: Beijing, 1983 ; pp 849-850.

2. Shanna, C .P. Bioc. Phann. 1976,25,181 1-18 12. 3. TOOa, S., Miyase, T .; Anchi, H.; Tanizawa, H.; Takino, Y. Chern. Pharm. OulL

1985,33,lnS-I728. 4 . Srimal R. C.; and Dhawan, B.N. J. Pharo Pharmacol. 1973,25,447-452. 5. Rao, T. S.; Basu, N .; Siddiqui, H.H. Indian J. Med. Res. 1982,75, 574-578. 6. Mukhopadhyay, A.; Basu, N .; Gujral, P.K. Agents and Act ions 1982,12,508-

SIS. 7. Yaguang Liu. U.S. patent 1#4,842,859, 1989, pp 2-3. 8. Born, F.V.R; Cross, MJ . J. Physiiol. 1963, 158, 178. 9. Ohkawa, H; Crush;, N; vagi, K. Biochcm. 1979,95,35 1. 10 . Oda., T; Seki,S. J. Elec. Micro. 1965, 12,2 10 . II . Sperry, W.M .; Brand, F.C. J. Bioi Chern. 1955, 213,69. 12. Carr, J.J .; Drekter,I.J. Clin. Chern. 1956,2,353. l3 . Sperry, W.M. ; Webb, M. J. Bioi Chern. 1959, 187,97. 14. Vanhandel, E.; Zilversmit, 0 .8 . 1. Lab Clin. Med . 1967, 50,152. IS. Hogan, M.M.; Vogel, S.N. J. lmmuno. 1987, IJ 9, 3697-3703. 16. Schwartz, RH.L., Jackson 1..; Paul, W.E. J. Immuno!. 1975, 115, 1330. 17. Jorgensen, 1.. ; Haeren, lW.; Moe, N. Throm. Dial. Haem. 1973,29,470. 18. Jorgensen, L.; Rowosll, H.G.; Hovig, T. Lab Invest. 1967,17,616. 19. Moore, S.; Merserrcau, W.A. Art Pathol. 1968,85, 623 . 20. Rice-Evans, C., Hochstein, P. Biochem. Biophys. Res. Comm. 1981, 100.15)7.

• Chapter 17

Antioxidant Activity of Lavandin (Lavandula x intermedia) Cell Cu ltures in Relation to Their Rosmarinic Acid

Content

T. 16pcz-Arnaldos l , J. M. Zapata1, A. A. Calderon ', and A. Ros Barcelo!

lIkpartment of Plant Biology, Universi ty of Murcia, E- JO I OO M Ufcia Spoin

' D ' eparlment or Plant Biology, University IIf Alca la de H enn res, E- 2887 1 Alcahi de Henares, Spain

AllIioxidants are onc oflhe principal ingredients thaI protect food quali t ~Y prev~lting oxidative deterioration ofbiomole<:ules. There is a grOwin~ !nl,erest m. the appli,c8tion of antioxidants from plant material. herbs and SpiCes. SPICes provide one oflhe most promising sources of an Ii oxidant compounds. Extracts from the Lamiaceae and Boraginaceae families have be,en ~pon~ to be effectjv~ anlioxidams. The antioxidant activity is pnm,anly attnbu~ed to phenolic compounds that functio n as free radical tenrunators~, In some cases, also ~ m~tal chelators. The present study was u.ndenaken to assess the antiOXidant act ivity of extracts from lavand~ (~lIdli/a x illttrmedia) cell cultures. We have compared the supcroJOde arnon scavenger activity of rosmarinic acid (main constituent of lavandin eJttracts) wi.th that di s.played by other structurally-related compounds. Finally, the Iron-chelaung propenies ofrosmarinic acid are reponed.

The food industry routinely u~s s~nthetic anlioxidants such as DBA, DHT and TBHQ to p~otect. products from OXidative deteriorat ion. Although these antioxidants are effechVC, hi~y sta.ble. and inexpensive, there is concem about potential adverse effects fro~ ~ynthetl c antiOXidants (J). Toxicological data suppon the safety of the synthetic antlOludants wh.en u~ at no~na1 use levels. Public perception that sYnlhetic compounds are dangerous IS dnvmg the mdustry to search for "natural" antioxidants

Traditionall.y spices have been u~ to improve flavor and stabilit~ of food. One m~e o f preservation by plant extracts IS the antioxidant activity whlch protects foods against. off fl avor related to lipid oxidation. The antioxida tive activity found in many plants IS due to the presence of compounds of a phenolic nature (I).

C 1997 American Chemicat Stlciely

•

17. UWE'l .... ARNALDOS .... A I~ Antioxidant Activity of LavQlldin Cell CuJtlm!~ 207

Phenolic antioxidants function as free radical tenninators and in some cases as metal chelaton. Phenolic acids panicularly have been identified as potent antioxidants. Caffeic acid and its esters are good examples of phenolic antioxidants (2).

Rosmarinic acid (a-O-caffeoyl-J,4-dihydroxyphenyllactic acid) (Figure I) is an example of a caffei c acid ester occurring in plants. Rosmarin ic acid is mainly found in species of the Doraginaceae and Lamiaceae fami lies, but can also be detected in other families (i .e. Apiaceae), ferns and homwons (Table I) (3,4). This suggests that the ability 10 synthesize this caffeoyl ester may actually be widespread as evidenced by rosmarinic acid accuOlulation in a range of species (Tab!e I) (5).

Current research on rosmarin ic acid centers on its physiological and pharmacological act ivities, and it is regarded as a potential pharmaceutical plant product (5). So, rosmarinic acid has been shown to suppress the complement-dependent components of endotoxin shocks in rabbits and t he oxidized compound displays antithyrotropic activity on human thyroid membrane preparations (5). Funhermore, there is increasing concern about the suitability of rosmarinic acid as a food additive.

One approach to recovery of uscful plant metabolites such as rosmarinic acid is to extract plants as they are currently produced. Unfortunately the agronomic conditions for production of such plants are extremely variable. Unreliable cultivation, htuvesting, shipping techniques, infestat ion with pests and natural status of the plant can all impart thC:' ]ost of the product produCi!d by the plant (6). Changes in any of these condi tions clln adversely influence the production of compounds such as rosmarinic acid. The application of modern culturing of plant cells offers an alternative means of production. Condi tions of culturing can be optimized such that a cdl culture would produce rosmarinic acid on a continuous basis at high yield and in a foml where it can b..: eusily extracted (7).

Occurrence ofRos marinic Acid in Lavandi n Cell C ullurn

Cultures have been successfully established from a number o f plllnt speci..:s which accumulate rosmarinic acid in their tissues. However, only a few of these culture.< hnve demonstrated the ability to continuously produce the ester ill I'ilro (5). In 1994. 1_1' !'C7.Amaldos et al. (8) established cell cultures derived rrom leaves lind spikcs oflilviltltlin, a sterile hybrid between i..,m'01xh,l" mlf:!lslijolia and La1't:mdll/(l illU!rmedia . Microscupic analyses of the cells forming the callus showed the presence ofbille fluorescencc in th~ vacuoles when the preparations were observed under l lV light. This fluorescence is indicative of the presence ofrosmarinic acid in the vacuoles. This result is in agreement with those of Uiiusler et al. (9), who have recently demonstrat~d the accumulation of fOsmari nic acid in vacuoles of suspension-cul tured cells of COII:II$ by protoplast and vacuole isolation (9).

To corroborate the presence ofrosmarinic acid in lavandin cells cultured ill I'itro, SO % (plv) methanolic extracts were obtained from the ce lls. Tn Figure 2, the UV spectrum of non-purified methanolic extracts is prnctically superimposible on that of pure rosmarinic acid. This suggests that rosmarinic acid is the main phenolic present in the methanolic extracts of the lavandin cells. Evidence in suppon of this suggestion arises ITom the HPLC analyses of non-purified methanolic extracts obtained from !avandin cell cultures (Figure 3).

Preparative low pressure chromatography on Sephadex LH-20 (0 26 x 420 mm; methanol as eluent) ofmethanolic extracts of the cel ls and subsequent analysis of the

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208 SI'lCES, FlAVOR CHEMISTRY AND ANTIOXIIJAI'i'T I'ROPERTIES

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of non-purified

17. LOPEZ.ARNALOOS n AL. Antioxidant Activity 0/ Lavandin Cell Cultures 209

Table I. Occurence of rosmarinic acid in some plant families

Plant

Apiaceae

£ryngium campeSlre

Sanicula europaea

Uoraginaccac

Alle/IIISG officinalis

Boraga officinalis

Uthospermum ojJicinaie

Pulmonaria o/ficinalis

Lamiaceae

Calamintha sylvatica S.l.

l.avandula angustifolia s .l.

Lovondu{o .f bumatii

Lavandula lo/ijolio

Lycopus europaeus

Melissa officilWlis s.l.

Memha x piperilo

Melllha pu/egium

Memha spicala

Manurda didylllll

Nepela cataria

Ocimum basilicuIII

Origanum vulgare

Orthosiphon ariS/alILS

Prunella vil/garis

RosmarinllS officinalis

Sa/via lavandillifolia s.l.

Salvia ojficinalis

Salureja //lontalla s.l.

Thy//l1lS vII/garis

Zosteraceac

Zooslera marina

Organ

leaf

flower

leaf

flower

'eaf !lower

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2.7

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1.8

0.2

1.9

5.0

0.8

6.1

2.5

0.6

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1.2

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1.9

21 . SPICES: HAVOR CII EMISTRY .... NI> ANTIOXIDA..'tT " ROPERTIES

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17. I .UJ>~"Z·ARNAll)OS ET A I ~ Antioxidant Activity of f.Avandin Celt Cu/turf'.f 2 11

resultant fractions by TLC (silica gcl; acetic acid:mclhanol:dichloromclhane, 4:15 '35 v/v/v) and UV-Visible spectroscopy revealed that, under the assay conditions. no phenolic other Ihan rosmarinic acid could be detected aller examination of TLC plates under UV light (A"'36S run) and by spraying them with II solution of ferric chloride (2 %, p/v) (da til nol shown). Analyses oflhe plates showed Ihat only II few fractions were enriched in rosmarinic acid, as judging by comparison orlhe It, orlhe spots wilh Ihl\l of pure rosmarinic acid. Furthermore, the UV spectra of these fractions were identical to that showed by pure rosmarinic acid (data not shown).

The determination by HPLC ofrosn1alinic acid in methanolic extracts oflavandin cell cultures revealed a content of about 3 micromoles per graAi of fresh weight (10)_ This means a concentration or about 2-3 % in weight as regards the dry weight. Similar concentrations have been reported working wi th other plant materials (Table I) (3)

Although rosmarinic acid has been previously detected and qUllntitied in ti eldgrown plant material from the genus LawlI/dllla (3), LOpez-Amaldos e/ a/. (8) were the first to demonstrate production of rosmarinic acid in cell cultures derived from plants belonging to this genus. Funher studies are necessary in order to optimize production and evaluate the potential for large-scale production of rosmarinic acid in lavumlin cell cultures_

Su pero l id e allion-scavenging ac tivity of Cllrllcts obta ined from lavllndin cell cultures

The presence of rosmarinic acid in methallOlic extracts o f lavandin cell cultures suggcstS that these extracts may offer potential as antioxidants. Despite the fact that lavandin belongs to the Lamiaceae family, there is little information about its antioxidant properties (J I).

In order to determine ant ioxidative activity of compounds or extracts obtained from plants, several methods have been developed (/2). Comparison of the results obtained by ditfercnt tcchniques is complicated because antioxidative activi ty d('pcnds on several fadars including the substrate used in the evaluation. However, for screening purpo~es, a model system is much less time-consuming than traditional storage 51\1t1ies (I),

In our study, the anlioxidative activity of methllnol ic extracts from lavandin eell cultures was monitored by following the ability of these extracts to scavenge sup('roxide anions generated in a phenazine methosulphate (PMS)-NADH system. Figure 4 shows the absorbance at 270-280 nm (indicative o r lOtal phenolics) and at 325 nm (indicat ive of rosmarinic acid) as well as the inhibit ion or the superoxide dismutase-scnsitive nitroblue tetrazolium chloride (J\'DT) reduction in fractions obtained after chrom:lIography of lavandin methanolic extracts on Sephadex LH-20. From this figure, it can be observed that maximum superoxide anion scavenging ability is associated wi th those fractions containing rosmarinic acid. This fact suggests that rosmarinic acid is the main compound responsible for the superoxide anion-scavenging activity of methallOlic extract s obtained from lavamlin cell eultures_

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FiSlire 4. Total phel/olic cOlllent, expressed a.r absorbance at 270-280 flm (-.), rOSlIl{lI"inic acid COl1fent, expressed as absorbance at 325 nm (-), and slIperoxide dismutase-sensitive NET reduction (---) of fractions obtained after chromatographic separatioll all Sephadex LH-20 of methanolic extracts from laV(mdill cell Cll/lares.

17. J,Ol'cZ-ARNAL OOS lIT AI.- Antioxidant Adj~ity of Lamndin Cell Cultures 2 13

SlJpero}(id e 3nion-scavenging adivity of rosmariuic acid a nd otber related com pounds

To better understand how rosmarinic acid aclS as a superoxide anion scavenger, the ability of olher structurally-related phenolics to inhibit the NBT reduction in a PMSNADH system was determillcd for comparison. The compounds tested were: (A) Protocatechuic acid (a dihydroxybenroic acid), (8) Caffeic acid (a dihydroxycillllamic acid), (C) Chlorogenic acid (an ester of caffeic acid and quinic acid), and (D) Rosmarinic acid (which can De considered as a d imer of caffeic acid) (Figure 5). lC.IO values for the scavenging of superOldde anions by phenolic compounds wcre calculated from the inhibition's percentage- concentration regres.~ion

lines of the titrations represented in F igure 5. Table II contains the 1C.IO values obtained from data in Figure 5. As can be

seen in this Table, of all the phenolics tested, rosmarinic acid was the most efficient compound in deactivating the superoxide anion, inhibiting the supcroxide dismutasesensitive NBT reduction by 50 % with a concentrat ion of 33 p M.

The antioxidative potency of a compound is re lated to its structure, in particular to electron delocaliz.ation of the aromatic nucleus and is a lso influenced by the number of hydroxyl groups (13). In th is way, the higher antioxidative activity of rosmarinic acid with rcspect to that of the other phenolics tested could De related with the presence of two catechol groups on its molecule . However, since the lC.IO value for rosmarinic acid is less than half of that for caffe ic acid (Table II), other mechanisms for superoxide anion deactivation may be involved.

When othcr assays are used in order to compare antioxidant activity of rosmarinic acid with that of the other mentioned phenolics, contradictory results are found. Thus. in the methyl- linoleate test (14), the antioxidant efficiency increases in the following order: chlorogenic acid, protocatechuic acid , caffeic acid, rosmarinic acid. However. using the method involving the free radical 2,2-Diphenyl-lpicrylhydraz.yl (DPPH') (15) the increasing order of antioxidative efficiency is rosmarinic acid, protocatechuic acid, caffeic acid. These data illustrate the above mentioned difficulty of comparing rcsults obtained by application of different experimental methods.

Iron-chclatillg act ivity or rnsmarinic acid

Superox ide allion is a relat ively nonreactive species in aqueous solution, but in the presence of hydrogen peroxide and a transition metal such as iron, the extremely reactive hydroxyl radical may be generated through a superoxidc anion-driven, metal-cata)ysed Fenton reaction. The hydroxyl radical is able to initiate lipid peroxidation directly through abstraction of hydrogen from fatty acids leading to the formation of off-flavors and other undesirable compounds.

One p05sible strategy for minimizing pcrox idat ive damage consists in removing traces of heavy metals by the use of chelators. In this way, rosmarinic acid seems \0 be a good candidate for chelating iron and other metals since it bears two catechol and a carboxyl groups on its molecule.

When rosmarinic acid is incubated in the presence of iron ions. the formation of a r05marinic acid-iron complex takes place. The formation of this complex can

214 SI'ICt:S: fLAVOR CHEMISTRY AN U ANTIOXIL>ANT PROI' ERTIES

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Phenolic concentration Cu.M )

F~' 14 ft! 5. EffeCl of protocatechuic acid (A). caffeic acid (8), chlorogenic acid ( ,and rasmarinie acid (D) concelltrations on superoxide anions generated in a MS/NADII system and dttermined by rhi! superoxide dismllfau-sensiti ve NBT red/laiQ/!. Bars show standard errors.

17. I"OPEZ_A RNALDOS.:1' AI.. Antioxidant ACli~ity oj Lo.viJ/ldin Cell Cultures 2 15

Table n . Superoxide anion-scavenging efficieucy and str ucture of the phenolic acids tested

Compound Structure

Protocatechu ic acid 500

"

Caffeic acid 157

Chlorogenic acid 114 'O~'O ~-. COOH

' 0 O.

Rosmarinic acid 33 .,,!\.CH.C.=</ Q -~ ~_ OH

HO HOOC OH

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216 SP1Ct; .... : H .A VO R CIIY.MISTRY AND ANll OXIDANT 1'H.OPf.:lfrlt;S

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0 0

• • u u c c 0 0 -" .0

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Figure 6. f/PL C cliromarogrmllT o/the rosmarillic acid-iroll complex mOllitored at 572 nm (A) and at 333 nm (8) . Arrows indicate retention time/or rosmarinic acid (Bar ... 4 mil/Utes) .

17. , .oPEZ-AR NALDOS "', AI~ Antioxidant Adil'ity oj Lal'o.ndin C~1l Cu!tUrt., 217

he followed by VIS spectroscopy and it presents an absorp tion maximum at sn nm

(I"'. Titrat ion curves for rosmarinic acid in the presence of different concentra tiolls

of iron ions indicate a stoichiometry of 1 [0 1 for the complex. These dala were confirmed by elemental analysis and inductively coupled plasma emission spectrometry (10). This I to I complex seems to be lhe only absorbent species in the reaction media. Support o f this assertion come from graphical analysis of the consecutive spectra according to Coleman a 0.1. (16) and from HPLC analysis of the reaction media (Figure 6). From this figure . it can also be deduced thai , under the assay conditions, almost all rosmarinic acid is forming part of the complex, since absorbance at 333 nm for the expected retention lime of rosmarinic acid is very low.

Although chelating activity of compounds bearing catechol groups has been largcly known, there is no report on complexing properties of rosmarinic acid towards iron ions. In 1985 Banthorpe et al. , working with callus cultures of lAvandula angustifolia, demonstrated the secretion into the supporting agar of the (2, E)-2-(3, 4-d ihydroxyphcny1)cthenyl ester of 3-0, 4-dih ydrox yphenyl)-2-propenoic acid and of its (E,E)- isomer (17). These compounds, which are probably rclated biosynthetically with rosmarinie acid (18), are able to chelate FeH ions forming intensely blue pigments (IT} . Since )avandin cell cultures also display blue pigmentation in the supporting agar and rosmarinic acid has been found to be the main phenolic constituent present in extracts of the medium (19), a participation of rosmarinic acid in the pigmentation of the culture media through the formation of complexes with iron ions cannot be discarded (19).

Conclusions

Rosmarinic acid fu lfils the requirements for being considered as a potent antioxidant since it is not only capable of eHiciently scavenging superoxide anions, but is also able to chelate iron ions. Studies on ()(hcr possible mechanisms for rosmarinic acid acting as an antioxidant, such as hydrogen perox ide detox ification through peroxidase-catalyzed reactions (8) , are now in progress. In the search for sources of this comJ>Ound, lavandin cell cultures must be considered since they accumulate relatively high amounlS of easy-to-purify rosmarinic acid .

Acknowledgments

This work has bcen partially supported by grants fron the CICYT (Spain), Projects # ALI 93/573 and ALI 9511018.

Literature cited

I. Madsen. H. L.; Bertelsen, G. Trends Food Sci. Techno/. 1995,6,271. 2. Maruta, Y.; Kawabata, 1.; Nik i, R. 1. Agric. Food Chem. 1995,43,25<.)2. 3. Lamaison, 1. L. ; Petitjean-Freytet, C.; Carnal, A. Ann. PllOrm. Fr. 1990, ·{8.

103. 4. Petersen, M.; Hausler, E.; Meinhard , 1.; Karwatzki, S .; Gcrtlnw.\ki. (. I'hllJ/

Cell TiS5. Org. Cull . 1994,38, 171.

I 218 S},ICES: FL.\vOR CIIEMIS'J'RY AND ANTlOXJOANT " IWPF;JtTJES

S. De-Eknamkul , W .; Ellis, 8. E. In Biouchnology in AgricuilUft' alld Fortslry; Bajaj, Y. P. S., Ed.; Medicinal and Aromatic Plants I; Springer-Verlag : Berlin, 1988, Vol. 4; pp 310-329.

6. Whitaker, R. J.; Hashimoto, T.; Evaru;, D. A. Ann. NY Acad. Sci. 1984,435, 364.

7. De-Eknamkul. W.; Ellis, B. E. Pfama Med. 1984.51,346. 8. L6pcz-Arnaldos, T .; LOpez-Serrano, M.; Ros Barcel6, A.; Calder6n, A. A.;

Zapata. J. M. Biochem. Mof«. Bioi. 1m. 1994,34,809. 9. HlIusler, E.; Petersen, M.; Alfermann, W. Plant Cell Rtp, 1993,12,510. 10. L6pez-Arnaldos, T.; L6pez-Serrano, M.; Ros Barcel6, A.; Calder6n, A. A.;

Zapala, J. M. Frestnius J, Anal. Chtm. 1995, 351, 311. 11. Economou, K. D.; Oreopou\ou, V.; Thomopoulos, C. D. J, Am. Oil Chern. Soc.

1991 ,68, 109. 12. Frankel, E. N. Trends Food Sci. Technof. 1993,4,220. 13. Shahidi, F.; Wanasundara, P. K. J. P. D. Crit. Rev. Food Sci. Nutr. 1992, 32,

67. 14. Cuvelier, M. E.: Richard, H. ; Bersel , C. Biosci. Biotech. Biocl1em. 1992,56,

324. 15. Brand-Williams, W.: Cuvelier, M. E.; Bersel, C. Lebensm. WiJ"S. Technol.

1995, 28, 25. 16. Coleman. J. S.; Varga, L. P.; Maslin, S. H . lnorg. Chern. 1970,9,1015. 17 . Banlhorpe, D. Y.: Bilyard, H. 1.; WalSOn, D. G. Phytochemistry 1985, 24.

2677. 18. Bamhorpe , D. V.; Bilyard, H. J .; Brown, O. D. Phytochemistry 1989, 28, 2109. 19. L6pel.-Arnaldos, T. ; L6pez-5errano, M.; Ros Barcel6. A.; Calder6n, A. A.;

Zapata, 1. M. Nat. Prod. Len. 1995, 7, 169.

•

Chapter 18

Anti-inflammatory Antioxidants from Tropical Zingiberaceae Plan ts

Isolation and Synthesis of New C urcuminoids

Toshiyo Masuda

Faculty or Integrated Arts and Sciences, Unh·ersity of Toku shima, Minamijosanjima i- I , Tokushima 770, Japan

Antiinflammatory active lUlt iol(idants were isolated from tropical Zingiberaceae plant rhi zomes. Their chemical structures were determi ned to be new curclll11 illoids. In particu lar, Ihe compounds from Zil1giber CUSSWIWll(lr (cassumunins and cassumunarins) have complex cLircuminoid structures and bOlh groups have sHonger

., ant iol<.idant and antii nflammatory ac tivities than those of curCli lTli ll. The total syttthesis of one of thcse complex curcuminoids. cassumunin A, was accomplished in 10 steps starting from 0 -

vanillin. The regio-specilic effcct of the hydroxyl and methoxyl groups on the antioxidant ac tivi ty of curcuminoid was investigated using synthctic curcumin analogs.

Natural antioxidants are important materials for the oxidative deterioration of food, and some antioxidants also have potential prevention activity ill the initial stages of oxidatioo-rdated diseases. I'lams used fOf food. includillg spice, are well known to be rich in phenol s_ The phenols have been targeted as natuml antioxidants because most phenolic compounds have a radical trapping property which produces an antioxidant activity ( I). There are about fourteen hundred species of the Zingiberaceae plants in the world. Most of these grow or are cuhivaled in tropical and subtropical Asia (2). The Zingiberaceae .plants typically have large rhiwmes. Asian people have used these for f~ and. spIce, and also for traditional medicines to m~intain health (3). In conllectlon wllh our research 10 discover new natuml all1io~idants, which are beneficial for human health, we have been investi gating IropiClll Zingibcraceae plants as 11 source for new biologically activc antioxidants.

Antioxidant ACli,,] t)" of Tropicul Zingibcraccac Plant Rhizomes

Wc have examined Ihe alt,ioxidant activity of ninc cultivated species of Zingiberaccae rhizomes which were collccted in Indonesia and Okinawa. The ant ioxidant act i¥i ty was judged by the inhibition of linoleic acid oxidation in an

C) 1997 American Oemiell l Society

I 220 S PICES: HAVOR CIiEMISTRY '\'~ J) ANTIOXIDANT PROPERTJt;S

ethanol-buffer system. The results obtained using the thiocyanate: and TBA l11ethods (4) are shown in Figure I. The "ntioll idant activi ty of the acelone extract of the rhizomes incrcased in the order Curcuma heym!tlTUl < Phutomtda speciosa < Curcuma (It Tuginosa < AmOnlum kepu/aga < Curcuma rna/rgga < Zingi/)er CQS$umunar < Curcuma xanthorrhi<.J1 < Alpinia galanga < Curcuma domestira using the thiocyanate method, and in the order Curcuma hqneana < Plllleomeria SIH!ciosa < Curcuma aeruginosa "" Curcuma mangga < AnlOmum kepu/aga < Zillgiber CaSSl/lllllnOr < Curcuma .mfl/horrhiw < Alpinia ga/ang(l < Curcuma dUlllesrku usi ng the TBA method.

Curcumin is a typical constitue nt of tTopical Zingiberaceae plant (5) and has strong antioxidant activi ty (6). We allalYl.ed the quantity of curcumin and two known analogs in the acetone extract of the rhizomes. Although the extracts from Curctlma domestica, Curcuma xa/lthorrlril.(l, and Zingiber cassumunar were determined to contai n a re latively high amount of curcumin by HPLC analysis, thei r strong amioxidant ac tivity could nOI be explained only by the curcurninoid contents (7). The results indicated the presence of new additional antioxidants ill Ihe rhizomes.

New Antiinnam matory Anlioxidants from Tropical Zingiberareae Pla ll ts

Recently, furtumin has received much attelltion because of ils interesting biological activity. whicb may be related to ils antioxidant property (8). I\nliinnammatory activiTY is an important biological activiTy of curcomin (9). because innammation is one of the peroxidation-related events in living organisms and its inhibition suggests tha t eurcumi n works as an ant ioxidant even in living cells. Huang e/ al. reported antitUlllor promotion ac tivi ty of curcumi n and clarified tha t its ac tivity is linked with the suppression of arachidonic acid ruelabolislll ( 10). This metabolic pathway is one of the lipid peroxidation events in living organisms. InnalllmHlion is also well known to be closely re latcd to the metabolism of arachidonic acid. We have been invest igating antiinn~mmatory ~cTive ~ntioxidants in tropical Zi ngiber~ce~e plants and succeeded in i~lat i ng such ant ioxidants from three species of tropical Zingiberaccae plants.

Curcuma domeslica . Half-processed rhizomes of Curcuma dOl/u!Stica are known as turmeric and are used as a food coloring agent. The rhizomes are also used as ItlIditional mediciues. New ~nt i oxidan ts (conlpounds 2 and 3) were isolaled from the dry rhizomes of the plant, the stmctures of which are shown in f-lgure 2. ,.\nlioxidant act ivity of the compounds are displayed in !--lgure 3 ( 11).

Curcuma XQ/llllO"hiza. Curcuma .mntl/{)rrlliza is a medicinal ginger in Indo nesia, however. thc rhilomeS are sometimes used for spice as a substitnte for CurCl/lllel dOlllc.l"licu. Wc isolated new ant ioxidants (compoullds 4 and 5) fro m the rhizomes. Their slructures and IIl1tio:>lid3nl activity are presented in !--lgures 2 and 4. respectively ( 12).

Zingibu cassumunar. Zingiber Ca$$umwrar has large yellow rhizomes similar to Curcullla dOlllcs/ica. The rhizomes are known to have anti innammatory activity (13) and are used as traditional medicine~ in Thai land and Indonesia. We have succeeded in isolating ne w antioxidants with an tiinnammatory activity (rom the rhizomes based on both the antio~idant and antiinnammatory assays (14. IS). Figure 2 shows the stroclllres of six new compounds Icassumunins A-C ( 16) (compoullds 6, 7, and 8. respectively) and cassumunarins A-C (17) (compounds 9. 10. aud II , respectivelY)1 isolated along with curC llrn in aud S'-rnethollycurcurniu. The antioxidant activity of the cassumunins and cassumunari ns is also shown in figures Sand 6. respectively.

18. MASUIJA Arlii-inflammatory AI,tiQxidanls from Zingiberoceat Plants

r---------r"

" ~u

I'" ...:, 01 u,

r-----------,'"

"I ~ 0\ ~ " . "i ~ ·8 • 2 ~ . . " <i , .. ~I V I u,

m

SI' ICK'i, FlAVOK CHEMISTRY ANI> ANTIOXIDANT I'ROI'liK:nES

%~: " 0 0

oc, -Figure 2. The Structures of Antioxidants from the Rhi zomes of Tropical Zingibcraceae Plants

•

18. MASUDA kl/i.injlamtrw.lory Antioxidanl$ from Zingibtrtlct!at Plants

E c o o ~ -• • " c • "' -o ~

"' -<

E c

o o ~ -• • " c • "' -o ~

"' -<

0 .5 ~ no addi t ive --- 1

0.4 ---- 2 ~ 3

0.3

0.2

0 .1

0 .01~~~~ o 2

R gure 3. Ant ioxidant Activity domestico.

0 .5

4

Time (days)

6

of the Isolated Compounds from CurCI/rna

~ no additive --- 1

0. 4 --- 4 --0- 5

0 .3

0 .2

0.1

O.O~~~~ o 2 4 6

T i me (days)

Figure 4. Antioxidant Activity of the isolated Compounds from Curcum(1 Xllnrhorrliil.ll .

i

'" SI'I(.:I\S, HAVOR CII E.'ttl,nRY AND ANT10XlOANT I·ROI'~;R1U~"

E = e Q on -• • 0 c

" "' • 0 • .0 <:

-= Q Q on

o

O.S -0- n o add i tive

• 1

0.4 • 6 -0- 7

• 8

0.3

0.2

0 . 1

0 .0 0 4 6 8 1 0

T im e (d ays)

Figure 5. Antioxidarlt AClivily of Cas sum un ins A (6 ), B (7), and C (8) ,

0.2

o·,f ~=====:J o. o~

o 2 4 6 8

T ime (d ays)

Figure 6. AntiOxidllnt Activity of Cassumullarins A (9 ), B (1 0), and C ( I I ) .

1 0

18. MASUDA Anti-inflammatory Anlioxifitlnt.\' jrom ZinKibtrllaar " lallU 225

Anli innam malory Acth 'ily or the 150111100 Antioxidant" Newly isolated compounds from the chree Zinl=iberaccac phill is previously rmnlioneti are SlrllclUrally related to curcumin. We eXAmined their anliinnammillory act ivi ty (18). Curcumi" (0.6 I.mol) showed inhibitory Rellvil), of edema formation, which is caused by an innammalion inducer. TPA (l2-0 -ltlradeunoylphorbol-l3 . aCel&le.2 IIg), on mouse car, The activity of Ihe isola ted compound$ IIsinji! the mouse ear method is summari7.ed in Table 1.

Synthe$is of Cassumunin A, a Complex Cun:uminold from ZitfgiiJer cossumutfar

We have begun the tOial synthesis of several new complu: curcuminoids and sllcceeded in the synthesis of caSSUlllUnin A. Ihe moSI potent antiinIlHmm~to~y an t io~idan t from Zingiber cU$Yllmllnar. The synlhesis of curcumin was achieved by many groups (19), howcver. thc most efficient mcthod was first reportcd by Pabon (20), which involvcs thc Knocvenagel condensation of thc boron complex of 2,4-pentadione wilh vanil1in. In Ihe synthesis strategy, we employed this method for the coupling of two differcnt Iypes of benzaldehyde wilh 2,4·pendadionc.

Metho~ymelhylated v-vanillin 12, which was prepared from o-y~ni l lin and metho~ymethylchloride, was condensed wi th 3"4'-dimetho~yacetophellone, giving the conjugated ketone t3 in 97% yield. One methyl group was introduccd at the ~-position of the carbonyl group in 13 by treatment of (CIIJhCuLi in 94% yield (compound 14). The methoxymethyl group of 14 was removed by acid treatmellt (I N HCI) in methanol to give 15 in 76% yield. The carbonyl group of 15 was reduced to a hydro~yJ group by Nal3ll~ in 98% yield (compound Hi~ After diacdylation of 16, the diacetatc 17 was heated at 160 OC in DMSO to introduce a double bond at the appropriate position in 90% overall yield (compound 18) from 15. Afte r hydrolysis of the acetyl group of 18 (compound 19), regiO$ekctiye introduction of aldehyde functio n at the 4-posi tion was achieved by treatment with chloroform under alkaline conditions. giving aldehyde 20 in 40% yield, The coupling reaction of 20 with diketone 21 under Pabon's condi tions (20) gave cassumunarin A in 41 % yield. The synthetic cassumunin A was identical with the nalural compound based on spectroscopic methods (i-Igure 7, Synthesis of New Cu~umin A nalog.~ and Their Antioxida nt a nd AnliiTinamma lory Aclil'ilies

Among planl consli tuents, there are many kinds of phenolil;: compounds, such as navonoids. coumarins. and phenol carboxylic acids. It is well knC'wn that they have various regioisomers in positions of the hydroxyl and melho~yl group substi tuents. The st ructure and antio~idanl efficiency rel ~tjo!1 ships for tocopherols (21) and navonoid.~ (22) have been well investigated. however. little is known about curcurninoids. We have been interested in the regio-effect of the hydroxyl and methoxyl groups in the benzene rings of cureumin on their ant iox idant and antiinflammatory activities.

We synthesi7.ed cleven curcumin 311alogs as shown in Figure 8, and eMmined their antioxidant activity using linoleic acid as a substrate and AlBN as ~n oxidation inducer. The data are shown in Figures 9-12 (23). Figure 9 shows that the hydroxyl group at 2 or 4-posilions is important for antioxidant activity. Figures 10-12 show that a methoxyl group at the ortllo or pam-I)()sitions in(fC;iscS the activity. The 4-hydroxy-3-methoxy compound 31 (curcumin), the 2-hydmAY 3-methoxy compound 26, and the 2 -hydroxy-5-melho~y coml)()und ~ sh"w Ih"

I iii

". Sr IC.:S, FlAVOR CHEMISTRY AND ANTIOXIDANT I'ROrERTl fS I'. MASUDA Allti·j"flamnwlory Alltio.rid(mls from ZingibuQcrtu Planls 227

Ir: · • r " " " r a r i 1 8 " a r : i " 8 r

fa

;ii 8 , I N 0

~. :£ Tl$ble I. Inhib itory Act ivity of the Isolated Compounds (0.6 " nlOl) " on TPA {2 l!~.Induced Mouse Ear Innammation C •

Tr~(J/m(!nt - " f \ {~~ 'j

Experimant Left Ear R igh I Ear "" Oh:t:SE(ms.! Inhibil;OII( '1bJ Significance a •• • ,i I. f , i TPA 10 16.MO.9

:l- 'l. I+TPA TPA 10 9.7:t1.1 58 p<O.OI r 9. 'l. is 2+TPA TPA 5 8.7:t. 1.4 52 p<D.Ol u u

a 'l. r r · , l+TPA TPA 5 5.310.4 32 p<O.OI " u u f , • r r 4+TPA T PA 6 r 1.5:t:t.3 69 p41.0 1 u u

~l a 'I

~l eli £ i u < • .1

2. 15 " , TPA • 17.hO.9 Q '\ • l +TPA TPA 5 8,'h{),9 51 p<O.OI " " " r r ,

6+TPA TPA 5 14.5% 1.0 83 p<O.Ol r , 8 8 a a p<.O.O I

, 8 " 7+TPA TPA 5 13.3:t:1.2 76 '" d · 8+TPA TPA 5 1l.1%1.3 75 pdWI a <

'j a a ., ~ r r # ~ 3. ~ f ~ , • TPA 6 16.5:tO.6 0 0 ~ '" a ;;

r " " I +TPA TPA 5 9.2%1.3 56 p<O.OI r 8 B 0 9+TPA TPA 5 12.h2. 1 75 p<Q.0I

~ • I O+TPA TPA 5 1O.h1.4 62 p<O.Ol II +TPA TPA 5 IO.hO.8 62 £01<0.01 • " ~

number of mice. bO: lnean of weight differnce betwee n right ear and left ear. 'l. 0 'l. :l- ~ lin: a a " r £ " r r " r u u u u u u

~l ~ I " ;1% f , , 0' . ~ '" e %

Table II. Inhibilory Atl ivity orCun:uminoids (0.6 I'fllol) ~ • " • r r 011 TPA (2 rS)-lndut'cd Mouse Ear Inn a mmation RJ 8 f 8 'l. 9. r r Trl.'almtfll u u

uft Ear Righi £Or '" Db~£ Inhibjlion(%) Significonu " TPA 4 !7.4iO.9 a a 0 > • 31(1)+TPA TPA 4 9.7:t 1.9 56 p<O.O I r a 28+TPA TPA 4 10.0%1.2 5' pd101 > ~ -it ~ 26+TPA TPA 4 4.4% 1.5 25 p<Q.OS • · all : nu mber of mice, bO: mean of weight diITernce between right ear and left ear. e ,

• ~

a R g 'l. 'l. :l- :l-r r u '-' u u

r

$ I'ICt;S: n~vow CIiEMI.!.7K't' AND ANTIOXIDANT l'ItOP£llTiFS

I) 1310 y' o 0

0 AA

i~yH R, 2) B(OBuh

• Y J) ,,·BuNH1

R.

MO~1a . TBDM$ia

\",-,J-H

U OR

R"~IOM. TBDM$i

R.

R,

0 0

'" R, R, &'

R,

220 11, .11 , lI, gH. 11,_ 11. II, _U. "". H 2J: 112"11. IIj=lI . R..o-OII. lI,all. ""~ II

U: 112"11. IIJ..otl. II.o-H. II".H. "". H 25.: II~. II j2H. a..- Il. II,..H. l!"aH U: 1I~1. II j-OM .. Jt..a H. II,. H. ~H 21: 11:=011. R,a H. R..o-oM~. IIJ" H. l!"a H lS: R,coOll. RJ211. a... It . II,o()M~. ",,_H :t9: 11,=011. R,=Il. R.~It.It,_H . ~< 30: R~M •. R,=H. R,,-oll . R,. tt R,,- It 3 1: 1I1a H. R, =OM •. Iyo.OU. Rla H, R,,: U Jl: II,_U, 11.1 0011. R,e>(}M<. II,.H, R •• II

o 0

OR RO

Figure 8. Struclures and Symhesis or Curcuminoids (2 3-3 2) .

'.2 , _ ______ _ ______ -,

, . E 0: 0.8

~

" ~ O.6

~ ,!O.4

• 0.2

o

o nIIlnMbllor

" "

2 , • , , 7 • , Time {hrJ

Fi gure 9 . Inhibitory AClivilY of CurCUlllinoids (22-25) against AIBN. initiated Uooidc Acid Oxidation (Liooleic Acid: 54 mM . AIBN: 81 mM. Curcumilloid: 170 1.M).

R,

R.

LS. MASUDA Anti.inflammatory Antioxidants from ZingiJnraCMt Plants

u ~---------------~

t:. U()'OIl)

E o 111 ().OU.t.OM.)

0," ~.

" ~O.6 c ~ " ~ 0.4

• 0.7

0 0 2 , . ,

'rime (hr)

FigurelO. Inhibitory Activity of CUfCuminoids initiated UlIOleic Acid Oxidation (LillOleic Acid: Curcuminoid: 170 JIM).

. , (2 5-29) 54 mM .

. , against AIBNAIBN: 81 mM.

u.-- ---- --- ---------,

0 .... !"~!hl l ...

" E 13("011)

C 0.' ~ 0 :Je("01l 100M.)

~

" • JI(4011 )-0.1. )

• 0.' ~ 2 " E 0.'

• 0.2

Time {hrJ

Figure ll. Inhibi tory ACli vity of Curcumiooids (23. 30. and 31) against AIBN·initiated Uno/eic Acid Oxidation (Linoleic Acid: 54 mM. AIBN: 81 111M, Curcuminoid: 170 t.M).

'"

230 SI' ICES: FUVOR CHEMISTRV AND ANTIOXIDANT l'ROI'ERTn ;s

..,,------------------, o .. Iol>llli ....

• J.I (.l.ONi

! ~

0.'

r' .B 0 .4 ~

0.'

Time (br)

Figure l2. InllibilOry Actiyity of Curcuminoids (24 and 32) againsl A ISNinitiated Linoleic Acid O xidation (Linoleic Acid: 54 mM, AIBN: 8 1 mM, Curcuminoid: .70 I'M).

~ -~ 3 2 •

FigureD. Stabiliza tion Effect of Phenolic Radical by Mtthoxyt O)(ygen and Olefin of Curcuminoids (2 6, 28, and J 1).

18. MASUDA Anti·inflammatory Antioxidants from Zingiberaceae Plants

o

o

-£ g I

o -£ g

o

o

o I

231

I !. I

I i ! 1

i: i !

"

!

I ! ,

SI' ICt:.,,: .. LA VOl{ CHEMISTRY ANI) ANTIOXIDANT I'ROI'ERTI ES

'" 0 ... Iwllli_ , - 0 .)J E,,"OHJ,s.o~I'1

• .)6CJ.OH 4,!-_'bj .......... '))

0.' - • J I (. OK ).()j.<<)

0.'

Time (hr)

Finllre 15. Inhibitory Activity of Curcuminoids (3 3 and 34) and Curcumin (3i) against AIBN-initiated Lino[eic Acid Oxidation (Unoleic Acid: 54 mM, AIBN : 81 mM , Cun:: umi lloid: 170mM).

s trongest activity ill th is assay sys.tem and Ihei r ?ffici ency is allTlO5t equi~al?rl,t. These data indicate that the follow lllg factors are Important for the curcum1l1old s alllioxidant activity; I) the p?~nolic radical can.be cO~ljugatcd to the ~?uble bond in the alkyl part at the I-posmon. 2) the phenoliC radical call be stabilized by the lone pair of the mcthoxyl oxygen, as shown in Figur~ 13. These pam.meters arc si mi lar to those in tocopherol's case (24). We syntheSized two curcumll1 analogs, the 4-hydroJly-2 3-dimethoxy analog 33 and the 2~hydroxy-3,4-methylencdioxy analog J4 ( Fig~re 14). In the former compound the phenolic radical can be stabi lized by the two methOJlyl oxygcns and the rad ical of the latter cOIll~~nd call be stabilized by one of the mr:thylcndioxyl oxygens, whose lone pair ~ s well overlapped wi th the phenolic mdical because the five membered nn.g rc:SIrIClS the lone ()3lr obi tal of the oxygen in a good direction f~r the delocahl'.allo,:, o~ the phenolic radical to the oxygen (25).T hey showed slight ly stronger antlOJl.ldanl activity than that of cun::umin (Figure 15).

The allt iinflammatory activity of the two analogs (c?lllpounds 26 a~d ~), which showed equivalent antioxid~~lt activi ty to cur~~mlll,. a~e sllmmafl7.ed .In Tllblc II . These analogs have antllllflammatory activ ity SIIIII1 3(. to.CUrcUlm n, indicating th~1 the regio-specificity of s llbstituents on the aromatic rlllg.s t? the 1I111iinflammatory ~ct iv ity may not be so important a~d only the antl.oxldant effici ency of the substance may important FUl1hcr studIes along these 11IIe$ are now in progress.

Acknowledgments

This work was supponed in part by gronts from the Ue.hara M.emorial Foundation, Takano Research Foundation, and Ministry of EducatIOn, SCience and CulllJre of Japnn. The author thanks Dr. A. Jitoe, Ms. J. Isobe, and Ms. A. Kida of Osaka Ci ty Uni versi ty ror their work in this project. The author also thanks Dr. Tom 1.

18. MASUDA Anli.injIammalllfY Antioxidants /I'llm ZinRiMrl1UrJe PlanL,

Mabry of the Univer.;ily of T CAaS a t Austin for Ollf I;lIl1aborat itln in Tu u, and Ik N. Nakatani of Osaka City Universily aod Dr. S. Yonemori uf the Univ. of Ryukyus (Of" the ir suppor1 of plant collection.

Refercoces

1. Osawa, T.; Ramaralham, S.; Kawakishi. S.: !'la miki. M., "htnfl/i(" COntf/(J/lnli.f in FQQd and Their 1:.1Jects on Hl!.alth II, ACS Symposium Series ~(Ji: American Chemical Society; WaShington. DC, 1992. Chapter [0, pp 122-134.

2. Hegnauer. R., Chem01axonQmie der PjlanUII, Birkhauser Verlag: Basel, 1963, Vol. 2. : Chapter 48, pp 451-471 .

3. Corner. E. 1. H.; Watallabe, K .• Illustrated Guidt to Tropical Pluws, lIi rokawa Publishing; Tokyo, 1969, 1'1'1069-1086.

4. O!;awa. T .; Namiki, M., Agric BioI. Chl!lIl., 1981 ,45,735-739. 5. Kuroyal1agi, M.; Natori, S., Yakuguku w sshi, 1970,90, [467 -1470. 6. TOOa, S.: Miyase, T.; Arichi, H., Tanizawa, H.; Takino, Y., Chem. Phl"flm.

8ull .. 1985, 33,1725-1728. 7. Jitoe, A.: Masuda, T.; Tellgah, [. G. P.; Suprapata, D. N.; Gara. I. W.;

Nakatani. N., J. Agric, Food Chem., 1992,40, 1337- 1340. 8. Ammon, H. P. T .; Wahl, M. A., Planla Ml!d., 1991, 57,1 -7. 9. Rao, T . S.; Basu, N.; Siddiqui , H. H., Indian J. Ml!d. Res., 1982, 75,574-578. 10. Huang, M.-T.; Sman , R. c.; Wong, C.-Q.; Conney, A. H., Cancer Res .. 1991 ,

51.813-819. I L Masuda, T.; litoe. A.; lsobe, 1.; Nakatani, N., PhYIOChtmislry , 1993, 32, [557.

1560. 12. Masuda. T., [robe, J.; J itoe. A.; Nakatani, N .. PhYlochl!mislry, 1992,31,3645-

3647. 13. Ponglux. D. ; Wongseripipatana, S.; I'tiadungcharoen, T .; Ruangrungsri, N.;

Likhiwitayawuid, K., Mtdicinal l'Ioms; VictOfY Poim; Bangkok, 1987. pp 275.

14. Masuda, T.: Ji toe, A., J. Agric. FoodChem., 1994, 42, 1850-1856. IS. Masuda, T.; Jitoe, A.; Mabry, T . J., 1. Am. Oil Chl!m. Soc. , 1995, 72, 1053-

1057. 16. Masuda, T .; Ji toe, A.: Nakatani, N., ChI!III;s/ry l.ttl., 1993, 189- 192. [7. Jitoe, A.: Masuda, T.; Mabry, T . J., TetrahedrQu Uti., 1994,35,981 -984. 18. Gshwendt, M.; Kittstein. K .. FUl"Stenberger, G.; Marks, F., Cancer utt., 1984,

25, 177-187. 19. Arrieta. A. E, Pharmaxie, 1993, 48, (f)(j-691 . 20. Pabon, H. 1. J ., Ru uil, 19\54. 83. 379-385. 21. Barclay. L. R. c.: Edwards, C. D.; Mukai, K.; Egawa. Y., Nishi, T ., J. Org.

Clu~m., 1995, 6{), 2139-2744. 22. Mora, A., Rios, P. 1. L: Alcara7., M. J.; 8iocltem. PIU/rnU/cal., 1990,40,79.\ ·

797. 23. Masuda, T; Kida, A., Proceedi ng of Annual Meet ing of Japancse Society "f

Nutri tion and Food Science, 1995, pp 103. 24. Hughes, L.; Burton, G. W.; Ingold, K. U.; Slaby, M.; Fosler, D.O., I'lu·n"Ii<·

Compounds iu Food and Their Effects on Heal/h II, ACS Symposium Snies 507; American Chemical Society; WaShington, DC, 1992. Chaplcr 14. I'P 184-199.

25. Burton, G. W.; Ingold, K. U., Ace. Chem. Res., 1986, 19, 194·201.

I

I

Curcumin: of the

Chapter 19

A Pulse Radiolysis Investigation Radical in Micellar Systems

A Model ror Behavior as a Biological Antioxidant in Both Hydrophobic and Hydroph ilic Environments

A. A. Gorman ' , J. Oamblen', T. J. Hill', H. Jooes' , V. S. Srlnivasa nJ, and P. D. Wood!

tDepartment or Chemistry, Univers ity or Manchester, Manchester M13 9PL, United Kingdom

ICenter ror I'hotochemleal Sciences, Bowling Green Slate Univers ity, Bowlin g Green, on 43403

Curcumin has been solubilised in aqueous anionic, cationic and neutral micellar systems and in aqueous buffer. The formation of its radical has been ooSl.:rved in all situations. The radical is effectively inert to oxygen and reacts with the natural antioxid~nt s vitamin C and vitamin E and Trolox 10 a degree dependent on localion. II is concluded that the anlicarcinogenic ac tivity of Cllreutllin may welt be II. consequence of its ability (0 access both hydrophobic nnd hydrophilic environmcrus, act as a mdical scavenger and be rcpaired by both vitamin C and vitomin E,

Curcumin ( I ), the major coostituem of the spice turmeric, is a molecule of considerable inte rest liS a consequence of ils known biological activity. This includes the lightinduced ollidative killing of thic leria (/.2 ) and anlicarcioogenesis re lated to inhibition of lipid peroKidation (3-6). As part of a program aimed at undemanding the anticarcillogcnic activi ty of this molecule (cj Ref. 7) we have examined the behavior of

curcumin in miccU<lr systems with cll1phasis on its location, the formation of the radica l and the reac tion of that radical with o~ygen and key 'tntioKidants. The SUrfll1;talllS employed were sodium dodecyl sulfate (S])S), cetyl IrimcthylammolliulII bromide (crAll) and Tri ton X- HlO (TX) with allionic. cationic and neutral head groups respectively. The cun:umin radica l was formed by the Slandard pul se radiolytic sequence (8.9) summarised in etllt:ltiolls 1·5 where curcumin (Cur-OIl) dOIl:I1C~ ,III electron 10 the azide radic:tl prior to Inss uf a I'f{)lnn.

~ 1997 American Chemical Sudely

19. COllMAN ~-:T At. Curtumin: A Pub."r Radiolysu' Inv£Higation

H 20 + e ~ H' • Oll' • e(solvated)

e(solvated) • N,o + H2O ~ N, • OB: • OH' OU' • N,. ~ 01-1 ' • N,·

N,· + Cur·OH ~ N," + Cur·OI'I" Cur·QH'-' Cur..o-

Experimental

235

(I)

(2)

(')

(4)

(I)

Pllise radiolysis CKperimcnts WeT!! essentially as prtviously described (10). Digitized data were Ir:lflsferred to the memory of a DAN 486 DX 33 PC for analysis. \Vmer was doubly.distilled from potassium pemlanganate. CUTCunlin was as described (7). Cetyl trimelhy!ammonium bromide (Fioons), sodium azide (BDH). sodium dodecyl sulf,ne (Aldrich), Triton X- I(X) (Aldrich). Trolox (Aldrich). vitamin C (Sigma) and vit:lmin E (Aldrich) were used as received_ Solutions were bubbled with either nitrous o:<ide (B ritish Oxygen Company) or nitrous oxide containing 10% oxygen (Air Products),

Rale const:lms delennim:d by lime-resolved lechniques are accurate to ± 5%.

Res ult s .

Curcumin was readily solubiliscd in aqueous surfactant systems in which the surfactant concentr:l tion was above the critieal micelJe conccrur:uion (cme). In each Clse the electronic :Ibsorption spectrum was independent of surfoctant concentration above the cllle.

Absoq )\ iull Spectr a . The electronic absorptioll spectr3 of cl1rcl1min solubilised ill

:Jqueous SDS and TX (Figure la) systcms were essentially idcnlical O.'!la~ - 417 nm) and ahsohllely typical of the corresponding spectra in hydrocarbon solvents such tiS cyc1ohexane. a clear indication that, in such systems, a eurcutllin molec ule on nvcrage occupies a hydrophobic region of the micellar system, i.e. is "within" a micelle. In conlr:lst, the spectrum of curcumin in the aqueous crAB system (Figure lb) was

typical of Ihe or"nge curcumin anion ()"mv; - 480 nm) and it would appear tha! , in this SYStCtll, c.:UfCUllIi ll is predominantly On Ihe outer surface o f the m icelles, anionic, and aSSOl.~ialcd with the teoiary ammonium head groups of the CrAB componentS, These con<:imion., arc sllpponed by pnlse radiolys is experiments (vide infra) .

I'U b l' ]{ a diul yl ic fo r mu ti on o f Ih e Rad icu l in Micellar Systems. Pulse radiolysis of N20-bubbled aqueous. pU 7 buffered, micellar solut ions of curcumin (2.0 x !U·S moll · t) comain ing sodium azide (2.0 x 10-2 moll-I) led 10 fonm.tion of a

species with )"mu - 490 nnl, 111is is ellemplified for the case of crAil in Figure 2 which shows the formation of the 490 nm species and the bleaching of the curcumin ground state as a function of time. These processes take place on the same microsecond li nH;~e~lc (cf. Figures 2a and 2b) and the slow decDy of the 490 nm species on milliscwnd timcscalcs (Figure 2c) was second-order, as cxpected for a mdical-radical combin:l tion process. There were only minor variations in the kinetics of mdic:ll growth and decay for the differem micellar systems for a given curcumin concemr:llion. Of kcy impon,lIIce was the linding thai use of N:!O containing 10% oxygen led 10 absolutely no change in the kinetics of decay of the rad ical absorption ;It 490 nm. This :llIuwS:III upper limi t of - 1.0 K 1()2 1 mol· t s· l tO be placed on the rate COlISlan t for r';:KtioliWith oxygen. It is clear that in these systems the eUTCumin r.ldic:1I is indeed beill>! pn"lun'd and is inert to oKygen on the timescales of its exiStence in our experiments. II III11SI I ... · recognised that in the real biologic:!l siatation the rJdical wuuld IIOt tli .. ""'''y vi" s~>\·"!III ·

236 SPICES: FlAVOR CII EM1STRY AND Ar-'TIOXII)ANT I'ROPERTlF..s

00

a

0.5

b

0.5

400 500 600 nm

Fieu r e I. Ekctron ic absorpt ion spc~:t r ;1 of cun:umin (2.0 :t 1O.~ mol 1. 1) SO!libi!iscJ in iln iUluCOUS solution of (a) TX (5.0:t J()-3 mol I-I) and (b) crAB (5.0 .... 10-3 mol I-I).

19, GORMAN to" AI- Curcumin: A Puls~ Rndiol)'$u Invtstigation 237

00

a 0.1

'~ ;/~~!1 0.0

i j •• 1 , ,

; 1 t,·, , , -0.1 n

i i" i ib r i i i c' , r I , -T' I , I r ,

300 400 500 nm

Fi gure 2. Transient absorptiOn spcctr:1 measured 6.4 jlS (Xl. 22 ~s (D). 58 )1 S (6) and 1261-15 (9) after pulse radiolysis of a solu tion of curcumin (2.0)( 10-5 mol \-1). crAB (5.0)( 10-5 mol J.l) and sodium azide (2.0 K 10-2 01011-1) in N20-saturated pH 7 bu ffer. Insets: lime dependence of (a) transient format ion 31 490 nm

with first-order fil, t' - 1.0)( lOS 5. 1, 4.6 % absorp tion/division, 10 )ls/division. (b) corresponding ground-stale bleac hing at 430 nm with first-order fi l, t' '" 0.71 x

105 s", 6.7 % absorptioll/div is ion,](J !-Is/division alK! (el lransient decay a1490 nm with second-order fil, 4.2 % absorplion/divisiol1, SO nls/division.

23' SI'IC"::S: Jo"I-",VOR CIIEM I!oo1"RY ANI) ANTIOXlDANT PROPERTIES

'±'! I U I I" :. I I -.1. 1, Ll ,1 IJI

~Pf~I--llfJ ++ 1, j i b,

-! -j ! I -t1~ i --- i~ 1

, I .. -I ,

! j

Figure J. (a) and (b): Repe;u of c.xp<:tltnents com:sponding to Figure 2 showing tntnsient dec:IY :1I 41)0 11m ill the present:e of (a) vi tamin C (2.0 x 10.5 mol 1'\) wi th

first-order fit , k' .. 2.U x 101 S· I, 4.9 % absorption/division, 300 !-,s/division and (b) Trolox (2.0 x 10.5 mol 1.1) with first -order fit, t'= 8.5 x 103 s·l, 4.6 %

absorption I division, 50 !-Is/division. (e) and (d): Repeat of experiments corresponding 10 t-igurc 2 using TX (5.0 x 10·) mol I- I) in the presence of vitamin E (2.0 x 10.5 mol 1. 1) showing (c) u-,lIIsiem dccay at 490 nm with fi~l-order fit, /t.'

-= 1.1 x 103 S-l, 1.2 % :Ibsorption/division. 250 lis/division and (b) corresponding growth of absorption at 430 nm wi th fi rs l ·ord~r fil. t': 1.2 x 103 s", 0.6 %

absorption/division, 250 !-,s/division.

19. GORMAN ET A l~ Currumin: A Pum RadiolY5is Inl'~tigation

order combination with itself, hut by a much slower pseudo first -order process involving ambient scavengers. Inefficient reaction with oxygen might become morc importpm in that Silu:lIion.

2

° O_I/'OH

4

The nC)(1 (IUeslion co be addressed al Ihis slUge was whether the location of radical fomlP\ion would influence ils ability 10 react wilh additive mole!;ules expected to be good radical scavengers. The potential scavengers chosen were vitamin E (2), vilamin C (3) and the waler-soluble model of Ihe fonne r, Trolox (4). In the evenl of efficient repair of the curcumin radical one would amicipll1e firstly a significam shonen ing of the lauer's lifet ime and a change to pseudo first-order decay. if the quencha concentration was significantly higher than th<l t of the radical. Thus. the above experiments were repea ted in the presencc of 2.0 x 10.5 mol l-I sc:lvenger. In all of Ihese experiments the scavenger will of cOurse compete with CllrCl.lmin for the azide radical. The cons<:quence of this is formation of both Ihe curcumin radical and thc potential scavcngcr radical on microsecond limesc:lles (cf. FiguTC 2a). The subsequent interaclions can be monitored as a consequence of the hct thai 1111: absorption m:l.~inlllrll of the cU!"Cllmin radical (490 nm) is signifkant ly shifted from those of vitamin C (- 3(iO nm) ;"tnd vitamin E and Trolm; (- 430 nm).

Sc::.ven gin g of Ihe Curcumin R::.dic::.1 P roduced in CTAU Micelles. In lhe presence of vitamin E the curcuntin radical decayed via clean second-order kinctics on a timescale identical 10 that of Figure 2c, i.e. in this situation vitamin E , which must be solubilised wi thin thc micelles, is unable to scavengc the curcumin radical. Since subsequent experiments demonstrate repai r of the laller by vi tamin E, these data clearly agree wi th the propos;!1 that Ihe curcumin in this p;lT\ieubr microhelcrogeneous environment occu pies lin cKtramicellar location as Ihe (;orresponding mono-3nion. Strong support for this conclusion C3me from the fillding 111:11 the w:lIer·soluble vitamin C and Trolox bolh quenched the cllTCumin r:ldic:ll cfficiellily as shown in FigllTCS 3a and 3b. The decay of the mdical absorption fo llo wed dc;m first-order kinetics, corr~sponding 10 rate COllStalll~ for reaCtion betwcen the l:urcumin radical and the scavenger of 1.0 x lOS J mol· t s· 1 for vitamin C and 4.3 x l(jll mol-I sol for Tro!lI.X. Although the latter is somewhat more efficient as a sC;lvenger, the c )( perimcm:d r:\to,: conStantS are too close to allow significant conclusions to be dr~wn.

Scave nging of thl' C Ufcunli n Il adkal in S US and TX \\·I icelll's. In SI:11k COntr,tSIIO the situation for crAB above , the cu!"Cumin radical produced in eitl !<.: r SUS· or TX-bascd micellar systems was efficiently tluenched by vitamin £. solubilised within the micelles as exemplified for TX in Figures 3c and 3d. These show the dec:ly of the

I , I , I

I : I

240 SI'ICF.S: FlAVOR CIIEMISTRY AND ANTIOXIDANT PROPERTI ES

OD

0.07

0.00

-0.07

400 500 nm 600

Fig\lr~ 4. Transient absorptiOn spectr;1 Illc;lsurcd 5.2)ls (X), 12 Ils (0 ), 2S)ls

(6) and 58 llS ('i7) afler pul~e radiolysis of a sol ution of cun.:umin (2.0 x 10.5 1ll~11 ' , d 00' 'd (20' 10.2 mol \_1) in N, O-saturuted pH 7 buffer. Insets: lUoe ' )all , \U111a 2;1 C . h • "_ Q4 dependence of (,I) Ir,lIlsient fornl;l\ion ,\[ 490 nm With fi rst-o:der fit, k - 7.2 x 1

s.l , 1.0 % absorption/diviSion, 10 !-Is/division :md (0) tranSient decay at 49Q nm

wilil sccond·()I"der fit. n.7 % absorplion/divisiOll. 25() ~ls/division

19. GOHMAN lIT AJ.. Curcllmin: A Puhe Radiolysi.f im'estigation 241

curcumi n radical al 490 nlll and the corresponding growth of the vitamin E radical at 430 nm on lhe snme limcscaleS. These dHta clearly indicate that the quenching of the curcumin radical by vit:lTlIin E does indeed involve repair and supports thc cadler conclusion that in the SDS and TX syStems the c li rcumin is solubilised within the hydrophobic region of the micelle_ When Ihese experimenls were repeated in the presence of vitamin C (20 x lO·S moll-t), the decay or the euremnin radical was only margi nally faster than the blank and still largely second-order. In the presence of the same concentration o f Trolox quenching occurred lO'an extent which made the decay predominantly first-ord er but the Tate constant was an order of magnitude lower than in the CTAB-based system (vide supra) . Clearly Trolox is more hydrocarbon-like than vitamin C and has a higher probability of accessing the hydrophobic regions_

Fo rmn(i oll of Ihe Curcll J11in Rad ical in Wa ler. It was possible to produce 2.0 x 10-5 mol I-I eUfemnin in water by dropping a solution in 0.1 M NaOH into a large excess of pH 7 buffer. Th:u this was a real homogeneuus solUlion, as opposed for instance to a microc rys ta lline disperSion, was clearly demonstrated by the speclrum which was essenti all y identicnl to thm in other hydroxylie solvents ~uch as ethanol and in panicular the kinetics of its fCact ion with the azide radical, with and without addition of vitamin C ~nd Trolox. Thu" pulse r'ldiolysis of such a solulion, N20-bubbled, containing sodiutll azide led to formation of fhe cllrcumin radical (Figure 4a) on timescales essentially idelllicalto those for the CrAB-based micellar sySTem for whieh eurcumin is thoughl to be in the ~nionic foml on the micellar surface, The spcctra! changes with time (Figure 4) were vinuully identical to those for the micellar ~ystems, panicularly the SOS and TX systems where the cUTCumin is in the neutral fornI , and the radical dec~y was second-order as expected (Figure 4b).

Scave nging of the C un:wni n Radical in Wuler _ Addition of either vitamin C or 1'rolox (2.0 .x 10.5 mol 1'\ in each case) led to efficient quenching of the curcum in radic!l!. Rutecollstallts for scavenging were 1.2 x 1()81 mol-I s·1 and 1.6 x 108j mol-t s·1 for vitamin C and Trolox respeCTively . The raw data are exemplified in fhe case of Trolo:>:; in FigufC 5. Insets a and b show the decay of the cUfCumin radieal at 490 nm and Ihe growth of the 1'rolox radical at 430 nm respectively. These occur on the same timescales. Inset b shows the initial 'immediate' fOIUlation of the Trolox radical as a consequence of competition for the ilzide radica l. The spectra in FIgure 5 clearly show the loss of the cmcumin radical at 490 Hill with a concomitant increase in the intensity of Ihe Trolox radical at -130 nm. An isosbestic poini close to 450 111 11 is apparent. Again these data confiml that the scavenging involves rep~ir of the cUTCllmin radical.

Co ttcl lls iutl ~

It is clea r from the e.xperiments desl"fibcd that the molecule cllTCumin can access both hydrophobic aud hydrophilic environments of a microhelerogeneous system. In addition, the corresponding r'ldical is stable with respect 10 oxygen , aT least on our experimental tit11cs(;ak~, but can be repaired by both the na turalaJltiuxidanfs vitamins C and E in hydroph il ic and hydrophobic environments respectively. It is currently our view th~llhe combi nation of ~mphiphilic charac ter ~nd radical stability/repair behavior referred to is the key to the importance of cure urn in as ~ln anticareinogenlc agent. One can imagine a scenario where mole(;ules of this type ~ct as an interphasic shunk for radical repair.

Ack n owle dgme nts

Experiments werc perfortlled at the Paterson lnst iwte for Cancer Research Free Radical Research Facility, the Christ ie Hospital NBS Trust, Manchester. TIle Fa(;ility is funded under the European COlllmissiOl1 TMR PROGRAMME - ACCESS TO LA RGE SCALE

242 SPICJ.:S: Fl.AVOR CHEMISTRV ANI) ANTIOXIDANT !·IWI·fRTIE. ...

00

0.01 ! . , b

, ,

~-./ ~ 0.0 '-~------:-=7"""------::=.:...J

450 nm 550 350

Fillure S. Tr:msient absorptio n spe!;lr;t me:lsured 111 IlS (X), 171 }.Is (0 ),327 ).Is

(6) and 576 ~lS (V) after pulse radiolysis o f;t soit llion of curcumin (2 .() x 10,5 mol \- 1). sodium ~zide (2.0 x 10-2111011,1) in N20-satur:lled pH 7 buffer conmining Trolax (2.0 x \0-5 mo l J. I), ]11 SCIS: 1in)( dependt':llCC: o f (a) transient decay at 490 nm

with rirsl-order fi l, k' = J.3 x 103 S· l , 0.4 % absorplion/division, 150 ).Is/division and (b) cOrTcsponding growth o f absorption :1I 430 mn with firs l-order 111,1:' '" 3.2

x loJ s· I, OA % absorption/division. 150 ).ls/division .

19. GORMAN":1' AI_ Curcumin: A Pulu Radiolysis Invutigation

f ACILITIES. Gnln! ERBFMGECT 950084· Access 10 Pi e R FRR Facility. This work was supported by NIH Grant I RJ5GM4435-0 JAI (VSS) and the EPSRC (U.K.).

Literature Cited .

I.

2.

3. 4. 5

6. 7.

8 . 9.

10.

Tonnesen, H. ; De Vries, I I. ; Karlson. J .; V3n t Jenegouwen, G. G . J . Pharm. Sci . 19K7, 76, pp. 371 -373. Dahl, T . A.; McGow;m, M. A.; Shand, M. A.; Srinivasan, V. S. Arch. Microbiol. 1989, /5/, pp. 183-185. $h:lf111:I, O. P. 810(."111:111 . Pharmacul. 1976,25, pp. 1811·1812. Nag.lhh ush:ul, M.; Bhide , S. V. Nurr. CO/lcer 1986,8, pp. 201-210. Harlfll:UI. P. E.; Sh;U1kel, D. M. Environ. Mol. MUloge/l. 1990, /5, pp. 145-182. Nagabhushan, M.; Bhide, S. V. J . Am. Coli. Nutr. 1992. II , pp. 192-1 98. Gom lan, A. A.; Hamblet! , I.; Srinivasan, V. S.; Wood, P. O. Photochem. Photoblo l. 1994 ,59, pp. 389-398. Hayon E.; Simic, M. J . Amer. Chem. Soc. 1970.92, pp. 7486·7487. Butl er, 1.; Land, E. 1.; Swallow, A. 1.; Prutz, W. Radial. Phys. Chern. 1984, 23, pp. 265-27(1. GOflll:lI1. A. A.; Hamblett, I. Chem. Phys. Lett. 1983,97, pp. 422-426. , ,

• , , I ,

I

! I

.I I

i INDEXES I , I , , I

01

Ii .l P Ii Ii ~ .. . , ~; ,I

N h' o. " ~;,'

[", I' I, ,. f, I ,

:',~

:' ~ -: ii J

r , , •

t"

I

Author Index

Dad:. Hyung Bee, 66 Barcel6, A. Ros, 206 Bene lscn. Grete, 176 Blank. Imre, 12 Block, Eric. 113 Cadwallader, Keith R., 66 Cai, Min, 66 Calderon, A. A., 206 Calvey, Elizabeth M., 113 Chen, C. C., 160 Chen, Chung-Wen. 188 Cheng, C. K., 160 Devaud, Stephanie, 12 Darn, Ruth, 29 Fay, Laurent B., 12 Feng. H. H., 160 Fujillori, E. M., 98 Fumeaux, Rene, 12 Gorman, A. A., 234 Hamblen, I .. 234 lIavldn-Frenkel, Daphna, 29 Hill. T.l., 234 Hiserodl, Richard D., 80

Ho, Chi-Tang, 80,188 Jones, H., 234 Lawrence, Brian M., 138 Lee, Tung Ching. 188 leistrilz, W", 7 Lin, Jianming. 12 Liu, Yaguang. 199 L6pez-Amaldos, T .• 206 Madsen, Helle Lindberg, 176 Masuda, Toshiya, 219 Randle, Will iam M., 4 1 Risch, Sara J., 2 Rosen, Robert T ., 80, 125 Shih, L. L., 160 Shu, Chi-Kuen, 138 Shu, W. Y., 160 SkibSled. LeifH., 176 Srinivasan. V. S .• 234 Wood, P. 0" 234 Yu, Tung-Hsi, 53 Zapata, J. M" 206 Zrybko, Carol L., 125

Affiliation Index

Antek Instruments, Inc" 98 Bowling Green Stale Univ~rsily, 234 Da-Yeh Institute o f Technology, 53 David Michael & Company, Inc., 29 Food Industry Research and Development

Institute, 160 L. Y. Research Corporation. 199 Mississippi State University. 66 Nabisco. Inc., 125 National Tsing-Hua University, 160 Nestec Limited, 12 R. J. Reynolds Tobacco Company, 138 RUlgers. The State University of New

Jersey, 80, I 25, 188

Science By Design, 2 SpiceTec Limited, 7 State University of New York

Albany. 113 The Royal Veterinary and Agricultural

Universi ty, 176 U.S. Food and Drug Administration. 113 UniverSity of AJcall'i de Henares, 206 University of Georgia. 41 University of ManchesICr, 234 University of Murcia, 206 University of Tokushima. 2 I 9

246

INDEX 247

Subject Index

A

Aduileration. role in commercial essential oil composition, 153,155- 159

Agronomic production of vanilla, 30-34,36/

S-A1k(en)y1-L-cysteine S-oxide flavor precursors. role in onion flavor. 41-50

Allium supercritical flui d extraction, 112- 123 volatile component fonnalion. 53-54

Allium (lsca/onicum aue!., Set Shallot Allium ecpu L., Set Onion Alliumjislu/osum L., See Welsh onion Allium sat;vum L. , Set Garlic S-Ally1cysteine sulfoxide, identification

of volatile compounds. 591,63 3-Alnino·4,5-dimethyl-3.4-dihydro-

2(5H)-furanone, formation of sotolone, 22-23

Anti-inflammatory antioxidants. anti -inflammatory ac tivity. 225,226r

Antioxidant ac tivity relationship to rosmarinic acid content

for iavandin cell cultures, 206-2 17 spices, 6 Zingiberaceae plant rhizomes,

219,221/ Antioxidant(s)

delay of oxidation, 177,179 in spices and extracts

activity. 184- 185 evaluation of radical scavenging.

179-184 identification. 179- 181/

natural, 219 synthetic. 206

Amiphotooxidation index, caps3l1thin, 188- 197

Aroma extract dilution analysis. saffron flavor characterization. 66-78

B

Bacterial reduction in spices dry steam procedure, 9-10 ethylene oxide gas procedure, 8 irradiation procedure, 8-9 methods, 3-4 wet steam procedures. 9

Benzoto:lpyrene. phase I oxidation. 81,83/ Biological antioxidant, curcumin radical

as model. 234-242 Biosynthctic pathways, production

of vanilla. 37 Biotechnological production of vanilla

biosynthetic pathways, 37 micropropagation, 35-37/ secondary products. 35,38-39/

Blood plate let aggregation. effect ofcurcumin, 199- 205

Bra55ieo, See Mustard

c

a-O-Caffeoyl-3 ,4-dihydroxyphen y11ac tic acid, See Rosmarinic acid

Capsaicin. analysis using chromatography and chemiluminescent nitrogen de tection, 98- 111

Capsaicinoids, analyt ical methods. 98.101 - 104

Capsanthin antioxidative effect, 189 concentration in paprika. 189.190/ effect on chlorophyll-sensitized

photooxidation of soybean oil and flavor compounds

2,5-di methy[-4-hydrox y-3(21f)-furanone, 192.193/

2.ethylfuran, 192,193/ 2.4.5-trimethyloxazole, 192.193/ experimental procedure, 189-192

I

..

24S SI'ICES: FL.,\VOR CIIEl'o llSTkY AND ANTIOX IDANT PROPERTI ES

Clpsanthin- COIrtilllled induction time, 192,194- 195 kinetics, 195 quenching mechanism, 195-197

p.Carolcne, antioxidative effect, 188- 197

Cassumunin A, synthesis. 225,227[ Cc lyllrimcthylammonium bromide

micelles, curcurnin radical formation,

238/.239 ChcmiluminesccnI nitrogen detection,

pungent flavor profiles and components ofspiccs,98- 111

Chlorophyl l·sensilized pholooxidalion (1f soybean oil and navor compounds, 188- 197

Cholesterol levels in blood, effect o/" curcumin. 203

Chromatography with CLNO to analyze pUllgenl navor profiles and components of spices, 98- 111

Ciim'l!c, role in commercial essential oil composition, 139- 146

Commercial essential oil composit ion variation faclors

ildultcrmion, 153,155- 159 cl imate, 139- 146 experi mental desc ripl ion. 138 geographic origin, 139- 146 growth ~tage o f hnrvcslcd plant,

149.1 51- 1 52/,1541 infraspecifie differences. 149- 151 I, 1541 inte r~pceific differences, 146-150 process ing parameter. 149, 152-153,155/

Component analyses o f nuxed spices accuracy, 171 experimental procedure,

161,165- 166,167/ methods, 160-161 num..:rical :malysis, 165 simi lar ity index, 162- 164 vector representat ion o f ~pices,

161-1 62 Crocus sflliv /Is L,. See Saffron Cullivar, role in flavor intensity

of onions, 43,45,47

Curcuma domes/ic(/. anti-inflammatory antioxidants, 220

C/lrcum(/ 10llga L., See Cureumin Curcuma :calllhorrhiz(/, anti-inflammatory

antioxidants, 220,2231 Cureumin

antioxidant properties, 6 applications, 199 biological activ ity, 234 cholestcrol 1eve1s in blood, 203 description, 80--8] experimental description, 200-202 lipid level. 203-204 lipid peroxidation, 204 Iymphoblastoid transformation,

204-205 macrophage, 204 piatelet aggregation, 203 properties, 199 radical formation, 234-242 reaction mechanism as free radical

scavenger, 81.821 resonance stabilization of frce radical,

8 1,82/ structure, 234

Curcumin analogues anti -inflammatory ac tivities,

, . 226t,232

antioxidant act ivities, 225,228-232 synthesis, 225,228/

Curcumin radical in miceihrr systems absorption spectra, 235.236/ experimental procedure, 235 pulse radiolytic formation of rad ical,

235,237/.239 radical formation in water, 240/.24 1 scavenging of radical

in cetyltrimethylammonium bromide micelles, 238/,239

in sodium dodecyl su lfate and Triton X-JOO micelles, 239,241

in water, 241,242/ Curcuminoids, isolation and synthesis,

2 19-232 Curing, steps, 34 Curlone, electron ionization- MS, 92/,96

INIlEX

D

Dihydrocapsaicin analysis using chromatography and

chemiluminescent nitrogen detection, 98- 111

structure, 98,100/ 2,5- Dirncthyl-4-hydroxy.3(2 /1)-furanone,

role of capsanthin on chlorophyllsensit i7.cd photoox idation, 192,1931

Direct solvcnt extraction, saffron flavor charactcrization, 66-78

Direct thennal extraction GC-MS to characterize components of lunncric, 80- 96

Dry stearn systcm, bacterial reduction in spices, 9-10

E

Electron spin resonance spectroscopy evaluat ion of an tioxidative activity

of spices and cxtracts, 176-185 evaluat ion of rad ical scavenging activity,

179,182-184 Electrospray-HPLC- MS. glucosinolate

determination in mustard, 125-136 Esse ntial oils

factors affccting composit ion variat ions, 138- 159

role in flavor, 160 See II /SO Commercial essential oil

composit ion variat ion factors Ethylene oxide gas procedure, bacterial

reduction in spices, 8 2.Elhylfuran, role of capsanthin on

chlorophyll -sensitized photooxidation, 192,193/

r

Fats , scnsitivity to photooxidation, 188 Fenugreek

aroma composi tion, 17- 20 experimental descript ion, 13-16 flavor components, 13-26

24.

Fenugreek-Con/ilru~d

formation of sotolone from preeUfSOI'S, 22-26

quantilation of sotolone , 21 stereoisomeric characteriution of

sotolone, 20-21 volati le const ituents, 12

Flavone, role in pharmacelllicai effect of curcumin, 199-205

Flavor compounds, role of capsanthin on chlorophyll-sensitized photooxidation, 188-197

Flavor distribution, role in flavor intensity of onions, 47

Flavor of thcrmally processed Allium vegetables, contribut ion of nonvolatile sulfur-containing flavor precursor of Allium, 53-63

G

Garlic contribution of nonvolatile sulfur

containing flavor precursors to flavor, 53-63

pungent flavor profiles and components us ing chromatography and ehcmiluminescent nitrogen detection, 106.108-109/

supercriticaJ fluid extraction, 112- 123 Gas chromatography-chcmiJuminescent

nitrogen detection, 102, I 03/ Gas chromatography-MS, saffron flavor

characterization, 66-78 Gas chromatography~]factomctry

description, 5,67 saffron flavor characterization, 66-78

Geographic origin, role in commercial essential oil composit ion, 139-146

Glucosinolate determination in mustard using HPLC-electrospray- MS

accuracy, 135 applications, 126 experimental procedure. 126-128 identificat ion methods, 126 nonvolatile compounds, 128

I

I 25. srlCI>S: .' LAVOR CII £MISTRY AND ANTIOXIDANT PNON! RTI F.5

Glucosinol ate dctcnnination in mustard using HPLC-eJeclrospray-MSContinued

plllgoilrin. 130,13 1/ sinalbin, 130, 132/ sinapine. 133, 135,136/ sinigrin. 133.1341

Ii

Il igh-performance liquid chromatographyc1eclfospray-MS. gtucosinolate determination in mustard, 125-136

Hybrid peppers, degrees o f hOlness, 98 Hydrophilic environment, curcumin

radical formation, 234-242 Hydrophobic environment, curcumin

rlldical formation, 234-242 ). H ydrox y4 .5-dimclhyJ -2( 5H)-fu ranone,

See SOlolooc 4-Ii ydrox y.L. isoleucinc

formation of sotolone, 22 struclUrc, 13

2-Hydroxy-4,4,6-lrimclhyl-2,S-cycJo· hexadicn- I-one, Set Saffron

Hyperlipidemia. curcumin effect. 199-205

Immune func tion, effects of cun:umin, J99- 205

Induction time, capsanthin, 188-197 Infraspeci fici ty , role in commercial

essential oil composi tion, 149-151/, 154/ hllcrspecifici ty, role in commercial

essential oil composition, 146-150 Iron-che/ming activity, rosmarinic acid,

2 13,2 16-217 IlTlldiation, bacterial reduction in spices, 8-9

L

Lavandin cell culture(s) an tiOJlOidant activity related 10 rosmarinic

acid content, 206-217 occurrence of rosmarinic acid,

207-208,210-21 1

Lavandin cell culture(s)--COlltillu~d superoxide anion scavcnging activity,

211-212 Lavandula x inurm~dja , See Lavandin

cell eulture(s) Lipid(s), generation of compounds, 54-56 Lipid level, effects of curcumin, 203-204 Lipid peroxidation, effects of curcumin,

199-205 Lute in, an tioxidative effect on

photooxidation of 2-ethylfuTlln, 188-197 Lymphoblastoid transformation, effccts

of curcumin, 204-205

M

Macrophage, curcumin effect, 204 S-Mcthylcysteine sul f01dde, id;:ntifica tion

of vo latile compounds, 6Ot,63 Micellar systems, curcumin radical

formation, 234--242 Micropropagation, production of vanilla,

35-37/ Miled spices, componenl analyses,

160--173 Mass spectrometry_H PLC-elccttOspray,

glucosinolate determination in mustard, 125- 136

Mustard, glucosinolate determination using HPLC---electrospray-MS, 125-136

N

Natural an tjolidants, phenols, 219 Nitrogcn-containing compouuds, source

of puugenl flavors in spices, 98 Nonvolatile sulfur-containing Oavor

precursor of AlliwlI , contribution to flavor of thermally processed Allium vegetables

volatile compounds generated from thermal degl"Jdation or thermal interw.:t ions or alk(en)yl cysteine sul foxidcs, 58-63

volati le compounds gcnerJtcd ftOm thermally treated blanched Allium vegetables_ 54- 58

INllEX

Nordihydrocapsaicin, analysis using chromatography and chemiluminescent nitrogen d;:tec\ion, 98-1 11

o

Onion contribution of nonvolatile sul fur

containing flavor precursors to flavor, 53--63

factors affecting flav or intensi ty cult ivar, 43,45.47 environmental factors, 46/- 50/ flavor distribution, 47

flavor chemistry, 42-43,44/,1 pungent flavor profiles and components

using chromatography and chemiluminescent ni trogen detection, 106,108- 109/

supercrit ical fluid extraction, 112- 123 Oxidation

delay using antioxidants, 177,179 frce radical generation, 177 ,178/

p

Paprika, pungent flavor profiles and components using chromatography and chemiluminescent nitrogen detect ion, 106,111/

Peppermint oils, composi tion variation factors , 138- 159

pH, role in sotolone formation, 2S Phenolic compounds in plants, related

to ant ioxidative activity, 176-179 Photooxidation sensit ivity of food constituents, 188 soybean oi l and flavor compounds,

capsanthin effect , 188- 197 Photosensitized oxidat ion reaction, 189 Piperine

pungent fl avor profiles and compouents using chromatography and chemiluminescent nitrogen detection, 106,110/

structure, 98,100/

-,- -

251

Platelet aggregat ion, effects of curcumin, 203

Proct:ssing parameter, role in commercial essential oil composition, 149, 152- 153,155t

Progoitrin, determination in mustard using HPLC-electroSpray- MS. 125-136

S-( +)-cis- I-Propenyleysteine sulfoxide, identification of volatile compounds, 621,63

S-Propylcysteine sulfoxide, identificat ion of volatile compounds, 61t,63

Pulse radio lysis, curcumin radical formation in micellar systems, 234-242

Pungent flavor profiles and components of spices anllly7.cd by chromatography and chcmiluminescent nitrogen dctection

advantages, 102, I 03/ detection mechanism for nitrogen

determination, 105-106 experimental procedure, 102,105 HPLC--chemilumincscent

nitrogen detection garlic, 106, 108-109/ onion, 106,108-109/ paprika oleoresin, 106,111/ piperine, 106, 110/ red chit i powder, 106,107/

linear response, 101

R

Radical seavenging activity, evalUlltion using ESR spectroscopy, 179,182- 184

Ramp, supercri tical fluid extraction, 112- 123

Rancimat method, antiphotooxi(i;ltive effect of carotenoids on induction time of soybean oi\. 191 -192,194- 195

Red chili powder, pungent fl'lvor proJ'jks and COmponents using c hwlllal",!!ral'hy and chemiJuminescellt nilf11gl'U ,1<;1",·t i" n. 106,107/

I

I ~ , r i' ! t I

I i ~ "

r , :1 :i I' I ~ , t ~ i I, " ) .. ,

252 SPICES: I'I.AVOR CII I':M ISTRY AND ANTIOX IIlANT I'ROI' I:: RTIF.5

Rosmarinic acid iron-chelnting activ ity. 2 13,2 16-2 17 occurrence, 207-2 ! 1 structure, 207,2081 supcroxidc Dnion scavenging activity.

213,214j,215f

s

Saffron, protIuclion, 66 Saffron flavor characterization using

aroma extract dilution analysis aroma-aclivecomponcnts, 74-78 detection of aroma-active component

using GC-olfactomctry, 67 experimental procedure, 67-68 volatile compone nts, 68-74

Sa[ranal, role in navor, 66-78 Semh'olatile components in powdertd

turmeric, characterization using direcl thermal extraction GC- MS. 80-96

ShailOl. contribut ion o f nonvolatile sulfur-containing flavor PfcClIrsors to navor, 53-63

Sillalbin, determination in mustard u~ing HPLC--clcctrospray- MS, 125-136

Sinapinc, determination in mustard using HPLC--clectrospray- MS, 125- 136

Sinigrin, determination in mustard using HPLC--clectrospray-MS, 125-136

Sodium dodccyl sulfate micelles, cureumin fad ical format ion, 239,241

Soto!onc, 12- 26 Soybean oil. role o f t;apsanthin on

chlorophyl l-sensitiud photoox idation, 188- 197

Spcurmint oi ls, composi tion variat ion f3cto/'S,138- 159

Spi.;cs ,mlioxidati \'c activity, 6,176-185,206 bacleri;11 reduction, 3-4,7- 10 component analyses, 160-173 facto rs affect ing consistency, 5-6 rcsearch into active components, 5 role o f e.~se ntial oils in favor, J 60

Sterili7.ation techniques. development for b;lcteria l reduction in spices, 7

Sugars, generation of compounds, 54-56

Sulfate fertility, role in flavor intensity of onions. 46/.47-49

Supcrcritical carbon dioxide, c xt rac tion of Allium species. 1[3-123

SupcrcrilicaJ fluid chromalographychemilumineseent nitrogen detcction, \02.104/

Supereriticai fluid extraction Allium species

conditions 'Is. organosulfur compound distribution, 120/, 121.122/

cxperimcntal procedure, 121, 123 extraction conditions. 115,1161 garlic. 117-119/ onion, 117,121 ramp. J 17,119/

applic~tions in food industry, 113 entrainer, I 13,115 patents. 11 3,1 141 syste m components, 11 5 use for analysis of spices, 5

Superoxide anion scavenging activity lavandin cell culture extracts,

211- 2 12 rosmarinic acid, 213,214/,2 151

Synthetic antioxidants. 206

T

Thermosprny. descriptio n, 8 1 TrigoTII!lla/~num-gratculI\ L , Ste

Fenugreek 2,6,6-Trimcthyl-I ,3-cyc1ohexadiene-l

(;arboxaldchyde, Ste Safranal 2,4,5-Trimethylollazole, role of

capsanthin on chlorophyll-sensitized photOQx idation, 192. 193/

Tropical Zingiberaceae plants posscssing ncw ant i-inflammatory ant ioxidants

ant i· inflammatory activity of isolated antioxidant, 225,2261

Curcuma domestica, 220.222-223/ Curcuma :rMI1!orrhiZfI, 220,223/ ZillgibercclSsumar.220,224/

INDEX

Turmeric components characterized by direct thcrmal extraction GC- MS

di rectthemlal extraction chromatogram, 87,88/

clectron ionization- MS for components, 90-93,96

experi mental procedure, 84-87 methods, 81,84 semiquantitative values fOl' volatile

and semi volatile components. 87,891,96

structures. 94-95/,96 thcrmospray LC- MS chromatogram.

8 1,83/ ar-Turmcronc, electron ionization- MS,

90- 91/.96 Triton X- I 00 micelles, cureumin radical

formation, 239,241

v

Vanilla agronomic production. 29- 34,36 biotechnology production, 34-39 secondary products, 35.38- 39/

Vanilla I'lani/ofia. Su Vilnillil

Volatile componenL~ in powdered tU9 characterization lIsing d irect thermal extraction GC- MS. 80-96

w

Welsh onion. contribution of nonvolatile ' sulfur-containing flavor precursors to fla vor, 53-63

Wei steam system, bacterial reduction in spices, 9

z

Zil1giber Ca,{SUmUllar anti-inflammatory an tioxidants,

220.224/ synthesis of cassumunin A, 225,227/

Zingiberaceae, members, 80 Zingiberaceae plant(s), source o f

antioxidants. 2 19 Zingiberaceae plan t rhizomes, antioxidant

activity, 2 19- 220,221/

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