Free Radical-Scavenging, Anti-Inflammatory, and ...

Research ArticleFree Radical-Scavenging Anti-Inflammatoryand Antibacterial Activities of Water and Ethanol ExtractsPrepared from Compressional-Puffing Pretreated Mango(Mangifera indica L) Peels

Chun-Yung Huang Chia-Hung Kuo Chien-Hui Wu Ai-Wei Kuan Hui-Ru GuoYu-Hua Lin and Po-Kai Wang

Department of Seafood Science National Kaohsiung University of Science and Technology Kaohsiung Taiwan

Correspondence should be addressed to Chun-Yung Huang cyhuangwebmailnkmuedutw

Received 20 September 2017 Revised 16 January 2018 Accepted 28 January 2018 Published 21 February 2018

Academic Editor Alberto Fiore

Copyright copy 2018 Chun-Yung Huang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

During the processing of mango a huge amount of peel is generated which is environmentally problematic In the present study acompressional-puffing process was adopted to pretreat the peels of various mango cultivars and then the bioactive compounds ofmango peels were extracted by water or ethanolThe phenolic compound compositions as well as the free radical-scavenging anti-inflammatory and antibacterial activities of water extract (WE) and ethanol extract (EE) from nonpuffed (NP) and compressional-puffed (CP) mango peels were further evaluated It was found that compressional-puffing could increase the yield of extractsobtained frommostmango varieties and could augment the polyphenol content of extracts from Jinhwang and Tainoung number 1(TN1) cultivars The WE and EE from TN1 exhibited the highest polyphenol content and the greatest free radical-scavengingactivities among themango cultivars tested Seven phenolic compounds (gallic acid pyrogallol chlorogenic acid p-hydroxybenzoicacid p-coumaric acid ECG and CG) were detected in CPWE (compressional-puffed water extract) and CPEE (compressional-puffed ethanol extract) from TN1 and antioxidant stability of both CPWE and CPEE was higher than that of vitamin C Furtherbiological experiments revealed that CPEE from TN1 possessed the strongest anti-inflammatory and antibacterial activities andthus it is recommended as a multibioactive agent which may have applications in the food cosmetic and nutraceutical industries

1 Introduction

Mango (Mangifera indica L) is recognized as one of the mosteconomically productive fruits in tropical and subtropicalareas throughout the globe Mango has excellent nutritionalvalue and health-promoting properties A variety of studieshave been performed showing high concentrations of antiox-idants including ascorbic acid carotenoids and phenoliccompounds in mango [1] Mango fruit is the main edible partand is usually processed into various products such as pureenectar jam leather pickles chutney frozen mango dehy-drated products and canned slices During the processing ofmango a huge amount of peel is generated which constitutesapproximately 15ndash20 of the mango fruit [2] Mango peel isa waste by-product and its disposal may have a substantial

impact on the environment Previous studies reported thatmango peel contains a variety of valuable compounds suchas polyphenols carotenoids enzymes and dietary fiber [2]Extracts from mango peel also exhibit antioxidant activity[3] anti-inflammatory activity [4] protection against mem-brane protein degradation and morphological changes inrat erythrocytes caused by hydrogen peroxide (H2O2) [5]antibacterial activity [6] and anticancer activity [7] Hencethe utilization of mango peels may be an economical meansof ameliorating the problem of waste disposal from mangoproduction factories as well as converting a by-product intomaterial for food cosmetic and pharmaceutical industrialusages

Free radicals including superoxide anion radical (O2∙minus)hydroperoxyl radical (HO2

∙) hydroxyl radical (HO∙)

HindawiJournal of Food QualityVolume 2018 Article ID 1025387 13 pageshttpsdoiorg10115520181025387

2 Journal of Food Quality

peroxyl radical (ROO∙) and alkoxyl radical (RO∙) aredefined as any molecules or atoms with one or moreunpaired electrons and are often involved in human diseases[8] Many studies have shown that free radicals in livingorganisms cause oxidative damage to different moleculessuch as lipids proteins and nucleic acids and these areinvolved in the interaction phases of many diseases suchas cancer atherosclerosis respiratory ailments and evenneuronal death [9] Antioxidants are substances that delayor prevent the oxidation of cellular oxidisable substratesThey exert their effect by scavenging reactive oxygen species(ROS) or preventing the generation of ROS [10] Syntheticantioxidant compounds such as butylated hydroxytoluene(BHT) and butylated hydroxyanisole (BHA) have potentantioxidant activity and are commonly used in processedfoods However they have been restricted because of theircarcinogenicity and other toxic properties [11 12] Thus inrecent years there has been considerable interest in naturalantioxidants derived from biological materials becauseof their presumed safety and potential nutritional andtherapeutic value

A large number of publications have suggested that fruitpolyphenols are related to immunomodulatory and anti-inflammatory properties via in vitro and animal studies [13]Inflammation is a complicated physiological phenomenonthat occurs when the immune system in the body isactivated to counter threats such as injury infection andstress Macrophages often play a unique role in the immunesystem because they not only elicit an innate immuneresponse but also act as effector cells in inflammation andinfectionWhenmacrophages encounter bacterial endotoxinlipopolysaccharide (LPS) they can be stimulated to generatea variety of inflammatory mediators such as nitric oxide(NO) tumor necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573) IL-6 prostaglandin E2 (PGE2) and adhesion moleculesto help eradicate the bacterial assault [14] Generally sub-stances with inhibitory effects on the expression and activityof enzymes (eg inducible NO synthase (iNOS)) involved inthe generation of inflammatory mediators such as NO in themouse macrophage-like cell line RAW 2647 are consideredto possess immunomodulatory activity [15] Since a varietyof polyphenols exist in mango peels further research on theuse of mango peel extracts as immunomodulatory or anti-inflammatory agents is warranted

Antibacterial agents are the synthetic or natural com-pounds that interfere with the growth and division ofbacteria A number of studies have shown that pathogenicmicroorganisms in humans and various animal species havedeveloped resistance to drugs This drug resistance is due tothe random or otherwise inappropriate usage of commercialantimicrobial agents As such there is an urgent need fornew antibacterial agents In addition synthetic antibioticshave been known to induce side effects such as the appear-ance of resistant bacteria skin irritation organ damageand immunohypersensitivity [16] Accordingly many studieshave attempted to develop new agents with high antibacterialactivity but with fewer or possibly even no side effects Thereis a particular demand for antibacterial compounds fromnat-ural resources [17] Plants produce a range of antimicrobial

compounds in various parts such as bark stalk leaves rootsflowers pods seeds stems hull latex and fruit rind [6] Fruitpeel is the outer covering of a fruit which functions as aphysical barrier It also serves as a chemical barrier by virtueof the presence of many antimicrobial constituents whichprotect the fruit from exposure to external pathogens or otherfactors that may tend to decrease the quality of the fruitTherefore fruit peels are good sources for obtaining naturalantibacterial agents

Bioactive compounds in mango peel are generallyextracted via the following methods extraction with 80ethanol by sonication for 3 days at room temperature [18]extraction performed three times with methanol for 3 h pertime [19] extraction with 95 ethanol three times 72 h pertime [20] extraction with acetone or ethyl acetate for upto 20 h [21ndash23] extraction by microwave-assisted method[24 25] or extraction with supercritical CO2 followed bypressurized ethanol [26] However these methods generallyinvolve the use of a large volume of solvents require a longextraction time consume a lot of energy are costly and some-times are not eco-friendly The present study builds uponon the research reported in our previous investigation [27]In brief we previously developed a compressional-puffingprocess that has been successfully implemented to increasethe extraction yield of fucoidan from brown seaweed [27 28]and augment the extraction yields of total phenolics andtotal flavonoids from pine needles [29 30] Compressional-puffing can be utilized as a pretreatment step to disruptthe cellular structure of samples thereby better enablingthe release of bioactive compounds by solvent extraction[27] In this study compressional-puffing was utilized forpretreatment of mango peels and water extract (WE) andethanol extract (EE) extracted from nonpuffed (NP) andcompressional-puffed (CP) mango peels were comparedThe phenolic compound composition and the free radical-scavenging anti-inflammatory and antibacterial activitiesof WE and EE from mango peels were also evaluated Tothe best of the authorsrsquo admittedly limited knowledge thisis the first study to elucidate the free radical-scavenginganti-inflammatory and antibacterial activities of WE andEE extracted from compressional-puffed mango peels TherecoveredWEandEE are expected to possessmultifunctionalactivities providing a wide range of benefits The utilizationof mango peel will also help to play a role in minimizing thegeneration of waste worldwide

2 Materials and Methods

21 Materials Folin-Ciocalteursquos phenol reagent gallic acidprotocatechuic acid chlorogenic acid p-hydroxybenzoicacid pyrogallol caffeic acid mangiferin epicatechin p-coumaric acid ferulic acid epicatechin gallate (ECG) cate-chin gallate (CG) ellagic acid rutin quercetin kaempferolhomogentisic acid tannic acid vanillic acid 22-diphen-yl-1-picrylhydrazyl (DPPH) sodium nitrite LPS dimeth-yl sulfoxide (DMSO) and 221015840-azino-bis(3-ethylbenzothia-zoline-6-sulphonic acid) diammonium salt (ABTS) werepurchased from Sigma-Aldrich (St Louis MO USA) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide

Journal of Food Quality 3

(1) (2) (3) (4) (5) (6)

Figure 1 Appearance of various Taiwanese mango varieties (1) Haden (2) Tainoung number 1 (3) Tu (4) Jinhwang (5) Yuwen (6) Irwin

(MTT) was purchased from Calbiochem (San Diego CAUSA) Dulbeccorsquos modified Eaglersquos medium (DMEM)trypsinEDTA fetal bovine serum (FBS) penicillin andstreptomycin were purchased from Gibco Laboratories(Grand Island NY USA) Methanol acetic acid andpotassium persulfate were obtained from Nihon ShiyakuIndustries Ltd (Tokyo Japan) All other reagents if notdeclared were purchased from Sigma-Aldrich (St LouisMO USA) and were all of analytical grade

22Mango Fruits and Peels Sixmango cultivars produced inTainan City Taiwan were utilized in this studyThe varietiesnamely Jinhwang Tainoung number 1 (TN1) Irwin YuwenHaden and Tu (Figure 1) were collected from a local grocerymarket in Xinhua District Tainan City Taiwan The fruitswere used after they had completed ripening Samples of peelswere separated manually from six varieties of mango fruitsand were then oven-dried and stored in aluminum bags at4∘C until use

23 Compressional-Puffing Procedure A compressional-puffing method [27 28 31] with minor modification wasadopted to pretreat mango peels In brief the dried peelsamples were crumbled and sieved using a 20-mesh screenThe portion retained by the screen was collected and thencompressional-puffed using a continuous compressional-puffing machine with the temperature set at 220∘C Thecorresponding mechanical compression pressure and steampressure levels inside the chamber are listed in Table 1 Afterthe compressional-puffing the peel samples were groundinto fine particles and stored at 4∘C for further extractionexperiments

24 Extraction Procedure We followed the methods of Yanget al (2017) [28] Briefly the nonpuffed and compressional-puffed peel samples were pulverized and sieved using a20-mesh screen The portion passed through the screenwas collected and extracted by 95 ethanol (wv = 1 10)for 4 h at 25∘C with shaking The resultant solution wasthen centrifuged at 9170timesg for 10min and the supernatantwas collected NPEE (nonpuffed ethanol extract) and CPEE(compressional-puffed ethanol extract) were thus obtainedafter oven-drying the supernatant at 40∘C In additionthe precipitates after 95 ethanol extraction were further

extracted by double-distilled water (wv = 1 10) for 1 h at70∘C with shaking Then the mixture was centrifuged at9170timesg for 10min and the supernatant was collected NPWE(nonpuffed water extract) and CPWE (compressional-puffedwater extract) were obtained after oven-drying the super-natant at 50∘C All dried extracts were milled to fine par-ticles and stored at 4∘C for further analyses The combinedcompressional-puffing pretreatment and extraction processis depicted in detail in Figure 2 The extraction yield wascalculated using the following equation

extraction yield () = (119892A119892B) times 100 (1)

where 119892A represents the dry mass weight of the extract and119892B is the weight of the mango peel sample on a dry basis

25 Determination of Polyphenol Content Polyphenol con-tent was estimated by the Folin-Ciocalteu colorimetricmethod based on the procedure of Singleton and Rossi (1965)[32] and using gallic acid as the standard agent

26 High-Performance Liquid Chromatography (HPLC) Anal-ysis of Total Phenolic CompoundComposition Theseparationof total phenolic compounds was performed by the methodof Schieber et al (2000) [33] and using a Shimadzu HPLCsystem (Shimadzu Kyoto Japan) equipped with a UV-visdetector A reversed-phase Inspire C18 column (250mmtimes 46mm id 5120583m) purchased from Dikma Technologies(USA) was used for all chromatographic separations Thecolumn was operated at 25∘C The mobile phase consisted of2 (vv) acetic acid in water (eluent A) 05 acetic acid inwater and acetonitrile (50 50 vv eluent B) The gradientprogram was as follows 20ndash55 B (50min) 55ndash100 B(10min) and 100ndash20 B (5min) The injection volumeof all samples was 20120583l The spectra were monitored at280 nm and performed at a flow rate of 1mlmin Gallicacid pyrogallol protocatechuic acid chlorogenic acid p-hydroxybenzoic acid caffeic acid mangiferin epicatechinp-coumaric acid ferulic acid ECG CG ellagic acid rutinquercetin kaempferol homogentisic acid tannic acid andvanillic acid were used as standards for HPLC analyses

27 DPPHRadical-Scavenging Activity The scavenging activ-ity of the DPPH radical in the samples was determined


Table 1 Process variables for compressional-puffing and extraction and extraction yields for various Taiwanese mango peel extracts

Operational variables NPWE CPWE NPEE CPEE

Mechanical compression Pressure (kgcm2) 0 5 0 5Number of compression times 0 3 0 3

PuffingTemperature (∘C) 0 220 0 220Pressure (kgcm2) 0 11 0 11

Time (sec) 0 10 0 10

PretreatmentSolvent 95 EtOH 95 EtOH NAlowast NA

Temperature (∘C) 25 25 NA NATime (h) 4 4 NA NA

ExtractionSolvent ddH2O ddH2O 95 EtOH 95 EtOH

Temperature (∘C) 70 70 25 25Time (h) 1 1 4 4

Extraction yield of extract ()lowastlowast NPWE CPWE NPEE CPEEJinhwang cultivar 335 plusmn 04cBClowastlowastlowast 366 plusmn 23bC 234 plusmn 12bA 302 plusmn 10cBTainoung number 1 cultivar 295 plusmn 12bA 348 plusmn 10bB 292 plusmn 07dA 337 plusmn 09dBIrwin cultivar 309 plusmn 09bcB 400 plusmn 22bC 226 plusmn 03bA 296 plusmn 04cBYuwen cultivar 312 plusmn 14bcB 370 plusmn 18bC 263 plusmn 09cA 374 plusmn 10eCHaden cultivar 255 plusmn 15aB 286 plusmn 27aB 188 plusmn 08aA 204 plusmn 05aATu cultivar 259 plusmn 03aB 291 plusmn 01aD 229 plusmn 05bA 270 plusmn 01bClowastNA not applicable lowastlowastExtraction yield of extract () = (119892solid extract dry basis119892mango peel sample dry basis) times 100

lowastlowastlowastValues are mean plusmn SD (119899 = 3) values in thesame column with different letters (in a b c d and e) and in the same row with different letters (in A B C and D) are significantly different (119901 lt 005)

Various mango peels

Hot-air dried

Nonpuffed (NP)

Crumbled

Pulverized

Supernatant collected and oven- NPEE

CPEE

Supernatant collected and oven-

Compressional-puffed (CP)

NPWECPWE

Centrifuged at 9170 timesＡ for 10 mindried at 40∘C

Precipitate collected and ddH2O added

Centrifuged at 9170 timesＡ for 10 min

95 ethanol added and shaken for 4 h

dried at 50∘C

Shaken for 1h at 70∘C

Figure 2 Flowchart of the compressional-puffing process and extraction methods for NPEE CPEE NPWE and CPWE

using the method described previously [28 34] In brief50 120583l of mango peel extract (concentrations ranging from 0to 300 120583gml for Tainoung number 1 and Haden cultivars0ndash600 120583gml for Jinhwang and Tu cultivars and 0ndash900 120583gmlfor Irwin and Yuwen cultivars) was added to 200120583l 01mMDPPH solution (in methanol) The mixture was shakenvigorously for 1min and left to stand for 30min in the darkat room temperature After the reaction the absorbance of

all sample solutions was then measured at 517 nm usingan ELISA reader (PowerWave 340 BioTek InstrumentsWinooski VT USA) The radical-scavenging activity wascalculated as the percentage inhibition using the followingequation

DPPHradical-scavenging () = [1 minus 119860 sample

119860control ] times 100 (2)


where 119860 sample is the absorbance of the methanol solutionof DPPH with tested samples and 119860control represents theabsorbance of the methanol solution of DPPH without thesample

28 ABTS Radical Cation-Scavenging Activity The ABTSradical cation-scavenging activity was performed accordingto the method described previously [28 34] The ABTS∙+solution was produced by mixing 5ml of 7mM ABTS solu-tion with 88120583l of 140mM potassium persulfate and allowingthe mixture to stand in the dark for 16 h at room temperaturebefore use The ABTS∙+ solution was diluted with 95ethanol so that its absorbance at 734 nm was adjusted to070 plusmn 005 To determine the scavenging activity 100 120583ldiluted ABTS∙+ solution was mixed with 100 120583l of mangopeel extract (concentrations ranging from 0 to 100 120583gmlfor Tainoung number 1 and Haden cultivars 0ndash300 120583gmlfor Irwin Yuwen and Tu cultivars and 0ndash500 120583gml forJinhwang cultivar) and the mixture was allowed to reactat room temperature for 6min After the reaction theabsorbance of all sample solutions was then measured at734 nm using an ELISA reader (PowerWave 340 BioTekInstruments Winooski VT USA) The blank was preparedin the same manner except that distilled water was usedinstead of the sample The scavenging activity of ABTS∙+ wascalculated using the following equation

ABTSradical cation-scavenging () = [1 minus 119860 sample


where 119860 sample is the absorbance of ABTS with tested samplesand 119860control represents the absorbance of ABTS without thesample

29 Cell Line and Culture Murine macrophage cell linesRAW 2647 were obtained from the Bioresource Collectionand Research Center the Food Industry Research and Devel-opment Institute (FIRDI Hsinchu Taiwan) The cells weregrown in DMEM supplemented with 10 FBS and 100Umlpenicillin-streptomycin solution at 37∘C in a humidifiedchamber with 5 CO2 The medium was changed every twodays

210 Measurement of Cell Viability TheMTT assay was usedto evaluate cell viability Briefly RAW 2647 cells (2 times 105mlin a 96-well plate) were plated with culture medium andincubated for 24 h at 37∘C with 5 CO2 in a humidifiedatmosphereThemedium was removed and fresh serum-freemedium containing different concentrations of mango peelextracts (concentrations ranging from0 to 25120583gml for CPEEof TN1 and CPWE of TN1) was added After 24 h of incu-bation at 37∘C with 5 CO2 the MTT reagent (01mgml)was added After incubating at 37∘C for 4 h the MTT reagentwas removed and DMSO (100 120583l) was added to each well andthoroughlymixed by pipetting to dissolve theMTT-formazancrystals The absorbance was then determined by an ELISAreader (PowerWave 340 BioTek Instruments Winooski VTUSA) at a wavelength of 570 nm The cell viability () wascalculated using the following equation

Cell viability () = (119879119862) times 100 (4)

where 119879 is the absorbance in the test and 119862 is the absorbancefor the control

211 Measurement of Nitrite Oxide in Culture Media RAW2647 cells (2times105 cellsml) were seeded in a 96-well flat bot-tom plate for 24 h at 37∘C with 5 CO2 The culture mediumwas removed and replaced with fresh medium containingtested samples at various concentrations prior to challengingwith 1 120583gml of LPS The nitrite concentration was measuredin the culture supernatant after 24 h of coincubation In brief50 120583l of the cultured supernatants was added in the 96-well plate and 100 120583l of Griess reagent was added to eachwell and allowed to stand for 10min at room temperatureThe absorbance at 540 nm was measured using an ELISAreader (PowerWave 340 BioTek Instruments Winooski VTUSA) and the quantification of nitrite was standardized withNaNO2 at 0ndash100 120583M concentrations [35]

212 Zone of Inhibition Five bacteria were tested for antibac-terial activity of mango peel extractsThese were three Gram-negative bacteria (Escherichia coli ATCC 11775 Salmonellatyphimurium ATCC 13311 and Vibrio parahaemolyticusATCC 17802) and two Gram-positive bacteria (Staphylococ-cus aureus ATCC 12600 and Bacillus cereus ATCC 14579)which were obtained from the Culture Collection andResearch Center of the Food Industry Research and Devel-opment Institute Hsinchu Taiwan Antibacterial activity wasmeasured using the standard method of diffusion disc plateson agar [36] In brief E coli S typhimurium S aureus and Bcereus were grown in tryptic soy broth (TSB) medium (DifcoLaboratories Detroit MI USA) andV parahaemolyticuswasgrown in TSB medium + 3 NaCl for 24 h at 37∘C and01ml of each culture of bacteria at proper cell density wasspread on tryptic soy agar (TSA Difco Laboratories DetroitMI USA) plate surfaces (3 NaCl was added to TSA forV parahaemolyticus) Paper disc (8mm in diameter) wasplaced on the agar medium to load 50120583l containing 2mgof mango peel extract (4 wv in 005M acetate bufferpH 60) Control paper discs were prepared by infusing with50 120583l Antibiotic-Antimycotic Solution (containing 10000unitsml penicillin 10mgml streptomycin and 25 120583gmlamphotericin) (Corning Corning NY USA) or 50 120583l 005Macetate buffer The plates were incubated at 37∘C for 24 hAfter 24 h antibacterial activity of the extracts against thetest bacteria was observed by growth-free zone of inhibitionnear the respective disc and the inhibition diameters weremeasured

213 Statistical Analysis Experiments were performed atleast three times Values represent themeansplusmn standard devi-ation (SD) Statistical analyses were done using the StatisticalPackage for the Social Sciences (SPSS) The results obtainedwere analyzed using one-way analysis of variance (ANOVA)followed by Duncanrsquos Multiple Range tests 119901 lt 005was considered statistically significant Correlation analyseswere performed using the square of Pearsonrsquos correlationcoefficient (1198772)


3 Results and Discussion

31 Effects of Mango Varieties Compressional-Puffing andExtraction Methods on Extraction Yields of Peel ExtractsSix varieties of mango fruits namely Jinhwang Tainoungnumber 1 (TN1) Irwin Yuwen Haden and Tu were col-lected from a local grocery market in Xinhua District TainanCity Taiwan Samples of peels were separated manually andthe peels were oven-dried till the moisture content reached4ndash7 (wet basis) The dried peel samples were crumbled andsieved using a 20-mesh screen and the portion retained bythe screen was collected and compressional-puffed accordingto the technique developed previously [27] Compressional-puffing applies a mechanical compression force of approx-imately 5 kgcm2 to the sample three times before puffingwhich can account for the difference between compressional-puffing and the conventional puffing gun processThe puffingtemperatures were set at 220∘C and the correspondingpressure level inside the chamber was found to be 11 kgcm2(Table 1) The NP and CP peel samples were ground andsieved using a 20-mesh screen The portion passing throughthe screen was collected and then the bioactive compoundswere extracted by either ethanol or hot water as shownin Figure 2 In the preliminary experiment we extractedpuffed peel sample directly using 70∘C hot water and foundthat the extract after being dried exhibited a stone-likehard structure which stuck tightly to the inner surfacesof the container and was difficult to dislodge Thus the70∘C hot water extraction condition was not adopted in thepresent study After extraction four peel extracts namelyNPWE (nonpuffed water extract) CPWE (compressional-puffed water extract) NPEE (nonpuffed ethanol extract) andCPEE (compressional-puffed ethanol extract) were obtainedaccording to their puffing pretreatments and extractionmethods for each mango cultivar (Figure 2) The yields ofthese extracts are indicated in Table 1 In the comparison ofextraction yields among different mango varieties for thesefour extracts it was found that the yields of extracts for thetestedmango cultivarswere similar except thatHaden andTucultivars had relatively lower extraction yieldsThus the peelsof Jinhwang TN1 Irwin and Yuwen cultivars with higheryields of extracts would have advantages for further commer-cial production It was reported that compressional-puffingcould primarily rupture the structure of the puffed samplesand then augment the extraction yield of crude fucoidan frombrown algae [27 28] and increase the extraction yields of totalphenolics and total flavonoids from pine needles [29 30] Inthe present study we also found that compressional-puffingcould rupture the structure of mango peel (data not shown)and increase the extraction yields in both CPWE and CPEEas compared to NPWE and NPEE respectively (Table 1)Therefore compressional-puffing can also be effectively usedin mango peels to facilitate the release of bioactive com-pounds by simple extraction operations A comparison ofthe extraction yields between water and ethanol extractionsrevealed that water extraction tended to have higher yieldsof extracts as compared to ethanol extraction A higher yieldof extract has the potential for commercialized productionIn addition previous reports revealed that the composition

of mango peel extract is complicated and it may containpolyphenols flavonoids carotenoids vitamin E vitamin Cpectin unsaturated fatty acids and other biologically activecomponents that positively influence health [25 37ndash39]Mango peel extract has also exhibited biological functionssuch as antioxidant properties [25 39] and inhibition of HeLahuman cervical carcinoma cell proliferation [38] Generallyphenolic compounds are the major bioactive componentsof mango peels [18] and these have exhibited antioxidantactivity and an antiproliferative effect on HeLa cells [2537ndash39] Thus the phenolic compound composition in ourmango peel extracts and their effects on biological functionswarrant further examination Taken together peel extractsfrom Jinhwang TN1 Irwin and Yuwen cultivars had higherextraction yields than those of Haden and Tu cultivarsCompressional-puffing pretreatment resulted in a worth-while incremental increase in the extraction yields of mangopeel extracts Water extraction tended to have higher yieldsof extracts as compared to ethanol extraction which wouldbe beneficial in commercialized production The phenoliccompound composition and biological functions of mangopeel extracts require further characterization

32 Polyphenol Contents and Free Radical-Scavenging Activi-ties of Peel Extracts from Various Mango Cultivars Phenoliccompounds are reported to be the major bioactive compo-nents that exist in mango peels [18] In the present study fourpeel extracts (NPWE CPWE NPEE and CPEE) from sixmango cultivars were utilized to determine their polyphenolcontents by the Folin-Ciocalteu colorimetric method Theresults presented in Table 2 suggest that peel extracts fromthe TN1 cultivar possessed the highest amount of totalphenolic compounds as compared to other peel extractsThus it is reasonable to postulate that peel extracts of theTN1 may exhibit high biological activities and thereforefurther investigation is warranted Moreover a comparisonof the polyphenol contents between NPWE and CPWE in allmango cultivars revealed that polyphenol content of CPWEwas higher than that of NPWE (Table 2) indicating thatcompressional-puffing could increase the polyphenol contentof water extracts in all mango cultivars However in thecase of ethanol extracts only CPEEs from Jinhwang andTN1 had higher polyphenol contents than those of NPEEsfrom Jinhwang and TN1 (Table 2) Moreover for all mangocultivars polyphenol contents of ethanol extracts were higherthan those of water extracts (Table 2) indicating that ethanolextraction was effective in the extraction of polyphenolsPolyphenols are well known to exhibit antioxidant activitydue to their ability to scavenge free radicals via hydrogendonation or electron donation and the reactivity of thephenol moiety [40] Accordingly the antioxidant capacitiesof NPWE CPWE NPEE and CPEE of six mango peels werecharacterized using DPPH and ABTS radical-scavengingassays DPPH is a stable free radical and is widely usedto evaluate the antioxidant activity in a relatively shorttime compared to other methods [41] The SC50 values(concentration of mango peel extract capable of scavenging50 of DPPH radical) of the peel extracts (NPWE CPWENPEE and CPEE) from six mango cultivars for DPPH


Table 2 Polyphenol content DPPH radical-scavenging activity and ABTS radical cation-scavenging activity of extracts from variousTaiwanese mango peels

Polyphenols ()lowast NPWE CPWE NPEE CPEEJinhwang cultivar 140 plusmn 011aAlowastlowastlowastlowast 211 plusmn 024aB 531 plusmn 025aC 913 plusmn 016cDTainoung number 1 cultivar 159 plusmn 09eA 166 plusmn 11eA 235 plusmn 04eB 285 plusmn 07eCIrwin cultivar 309 plusmn 018bA 292 plusmn 019aA 706 plusmn 029bC 507 plusmn 011aBYuwen cultivar 236 plusmn 025bA 463 plusmn 090bB 721 plusmn 005bD 626 plusmn 005bCHaden cultivar 641 plusmn 020dA 731 plusmn 019cB 189 plusmn 03dD 134 plusmn 03dCTu cultivar 525 plusmn 027cA 101 plusmn 16dB 147 plusmn 02cD 130 plusmn 05dCDPPH SC50 values (120583gml)lowastlowast NPWE CPWE NPEE CPEEJinhwang cultivar 499 plusmn 7fD 368 plusmn 13fC 197 plusmn 12dA 251 plusmn 0fBTainoung number 1 cultivar 570 plusmn 22aC 670 plusmn 22aD 460 plusmn 14aB 417 plusmn 13aAIrwin cultivar 368 plusmn 11eC 255 plusmn 2dB 195 plusmn 9dA 222 plusmn 8eAYuwen cultivar 324 plusmn 3dD 303 plusmn 5eC 165 plusmn 5cA 206 plusmn 4dBHaden cultivar 124 plusmn 3bD 101 plusmn 5bC 69 plusmn 5bA 86 plusmn 4bBTu cultivar 183 plusmn 2cD 158 plusmn 5cC 783 plusmn 49bA 967 plusmn 47cBVitamin C 113 plusmn 01ABTS SC50 values (120583gml)lowastlowastlowast NPWE CPWE NPEE CPEEJinhwang cultivar 186 plusmn 0eD 139 plusmn 0eC 700 plusmn 00fB 540 plusmn 33cATainoung number 1 cultivar 282 plusmn 38aC 233 plusmn 05aB 157 plusmn 09aA 130 plusmn 08aAIrwin cultivar 113 plusmn 7cC 101 plusmn 6dC 590 plusmn 08eA 763 plusmn 28eBYuwen cultivar 137 plusmn 2dC 102 plusmn 2dB 520 plusmn 09dA 620 plusmn 17dAHaden cultivar 553 plusmn 17bC 373 plusmn 24bB 273 plusmn 09cA 307 plusmn 17bATu cultivar 115 plusmn 5cD 773 plusmn 34cC 247 plusmn 13bA 340 plusmn 16bBVitamin C 358 plusmn 007lowastPolyphenols () = (119892119892solid extract dry basis) times 100

lowastlowastSC50 values (concentration of mango peel extract capable of scavenging 50 of DPPH radical) forDPPH radical-scavenging of different mango peel extracts lowastlowastlowastSC50 values (concentration of mango peel extract capable of scavenging 50 of ABTS cationradical) for ABTS radical cation-scavenging of different mango peel extracts lowastlowastlowastlowastValues are mean plusmn SD (119899 = 3) values in the same column with differentletters (in a b c d e and f) and in the same row with different letters (in A B C and D) are significantly different (119901 lt 005)

radical-scavenging activity are presented in Table 2 As shownin Table 2 all peel extracts from TN1 exhibited the mostDPPH radical-scavenging activity as compared to othermango cultivars and the most potent was CPEE of TN1 withan SC50 value of 417 plusmn 13 120583gml Kim et al (2010) reportedthat the SC50 value of the DPPH radical-scavenging activityof Irwin mango peel ethanol extract was about 40 120583gml[18] which was similar to the SC50 value of CPEE of TN1reported here A comparison of theDPPH radical-scavengingactivities of the CPWE group with those of the NPWEgroup revealed that compressional-puffing could increase theDPPH radical-scavenging activities of peel extracts (Table 2)Moreover DPPH radical-scavenging activity of all EE groups(including NPEE and CPEE) was greater than that of theWEgroups (NPWE and CPWE) which appeared to be positivelycorrelated with the higher polyphenol amount in the EEgroups as shown in Table 2 Regarding the scavenging activityof ABTS∙+ the relatively long-lived ABTS∙+ was decolorizedduring the reaction with hydrogen-donating antioxidant[42] The SC50 values (concentration of mango peel extractcapable of scavenging 50 of ABTS radical cation) of thepeel extracts (NPEE CPEE NPWE and CPWE) from sixmango cultivars for ABTS radical cation-scavenging activityare also presented in Table 2 The results show that amongthe extracts from six mango cultivars peel extracts fromthe TN1 exhibited the most ABTS radical cation-scavenging

activity and the SC50 value for the most potent CPEE ofTN1 was 130 plusmn 08 120583gml Kim et al (2010) reported that theSC50 value of the ABTS radical cation-scavenging activity forIrwin mango peel ethanol extract was about 200 120583gml [18]which was less effective in ABTS radical cation-scavengingcapacity as compared to our CPEE of TN1 Regarding NPWEand CPWE compressional-puffing could increase the ABTSradical cation-scavenging activity in CPWE of mango cul-tivars which was similar to the finding for DPPH radical-scavenging activity All EEs (including NPEE and CPEE) hadgreater ABTS radical cation-scavenging activity comparedto WEs (including NPWE and CPWE) (Table 2) To betterunderstand the relationship between polyphenol contentsand free radical-scavenging activities of peel extracts acorrelation plot was performed and the results are shownin Figure 3 A high correlation between the polyphenolcontents of peel extracts and their corresponding free radical-scavenging activities (DPPH and ABTS radical-scavengingactivities) was found in NPWE CPWE NPEE and CPEEwhich was also consistent with previously reported observa-tions [43] In summary peel extracts from the TN1 had thehighest amount of total phenolic compounds and possessedthe most DPPH and ABTS free radical-scavenging activitiesFor all water extracts compressional-puffing had a tendencyto increase the contents of total phenolic compounds inCPWEs and resulted in an incremental increase in free


DPPHABTS

05

10152025303540

(1S

C50)times10

00(l

g)

2 4 6 8 10 12 14 16 180Polyphenol content ()

R2 = 09599

R2 = 09892

(a)

0 2 4 6 8 10 12 14 16 1805

101520253035404550

(1S

C50)times10

00(l

g) R2 = 07667

R2 = 08297

DPPHABTS

Polyphenol content ()

(b)

DPPHABTS

0

10

20

30

40

50

60

70

(1S

C50)times10

00(l

g)

2 4 6 8 10 12 14 16 18 20 22 24 260Polyphenol content ()

R2 = 09154

R2 = 09739

(c)

DPPHABTS

0102030405060708090

(1S

C50)times10

00(l

g)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 300Polyphenol content ()

R2 = 09875

R2 = 0962

(d)

Figure 3 Association between polyphenol content and DPPHABTS radical-scavenging activities of mango peel extracts (a) NPWE (b)CPWE (c) NPEE (d) CPEE SC50 concentration for scavenging 50 of DPPH or ABTS free radicals

radical-scavenging activities as compared to NPWEs For allethanol extracts only CPEE of TN1 had a higher contentof total phenolic compounds and possessed higher freeradical-scavenging activities as compared to NPEE of TN1Moreover for all extracts ethanol extracts generally hada higher amount of total phenolic compounds and causedgreater free radical-scavenging activities as compared towater extracts Therefore in summary both CPWE andCPEE of the TN1 cultivar warrant further analyses of thephenolic compound composition and storage stability of theirantioxidant capacity as well as their anti-inflammatory andantibacterial activities

33 Analysis of Phenolic Compound Composition StorageStability of Antioxidant Capacity Anti-Inflammatory ActivityandAntibacterial Activity inCPWEandCPEEof TN1CultivarPeel extracts of TN1 cultivar have the highest amount of totalphenolic compounds and the most free radical-scavengingactivities Moreover CPWE and CPEE from TN1 had higherextraction yields and greater polyphenol contents as com-pared toNPWE andNPEE fromTN1Therefore the phenoliccompound composition of CPWE and CPEE from TN1 wasanalyzed by RP-HPLC coupled with UV-vis detector Theresults are shown in Figure 4 and Table 3 In Figure 4 itcan be seen that seven phenolic compounds namely gallic

Table 3 Phenolic compound composition in the CPWE and CPEEof Tainoung number 1 cultivar

Compound Tainoung number 1 cultivarCPWE (mg100 g)lowast CPEE (mg100 g)

119901-Hydroxybenzoic acid 1863 plusmn 318 3313 plusmn 2Gallic acid 579 plusmn 72 1052 plusmn 1Pyrogallol 566 plusmn 55 930 plusmn 90Chlorogenic acid 125 plusmn 8 245 plusmn 7Catechin gallate (CG) 125 plusmn 43 189 plusmn 52p-Coumaric acid 689 plusmn 94 131 plusmn 0Epicatechin gallate (ECG) 320 plusmn 39 508 plusmn 70lowastThe concentration of phenolic compound is expressed as mg100 g peelweight dry basis

acid pyrogallol chlorogenic acid p-hydroxybenzoic acid p-coumaric acid ECG and CG were tentatively identified inCPWE and CPEE of TN1 by HPLC analysis Table 3 showsthe quantitative data of phenolic compound compositionin the CPWE and CPEE of TN1 It was found that bothCPWE and CPEE of TN1 contained large amounts of p-hydroxybenzoic acid gallic acid and pyrogallol and smalleramounts of chlorogenic acid CG p-coumaric acid and ECGA comparison of the phenolic compound composition in


(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)

Figure 4 (a) High-performance liquid chromatography of peel extracts (CPWE and CPEE) of Tainoung number 1 cultivar (b) high-performance liquid chromatography of polyphenol standards gallic acid (1) pyrogallol (2) chlorogenic acid (3) p-hydroxybenzoic acid(4) p-coumaric acid (5) ECG (6) and CG (7)

CPWE and CPEE revealed that CPEE of TN1 had greateramounts of p-hydroxybenzoic acid gallic acid pyrogallolchlorogenic acid CG p-coumaric acid and ECG than thoseof CPWE (Table 3) These results are consistent with the datashown in Table 2 which illustrates that CPEE of TN1 hashigher total phenolic compounds compared toCPWEofTN1We found that p-hydroxybenzoic acid was the predominantphenolic compound detected (up to 3313 plusmn 2mg100 g peelweight dry basis) in CPEE of TN1 and the results were alsosupported by other studies reporting that p-hydroxybenzoicacid could be detected in the extract of mango cultivar [44]The concentrations of gallic acid for CPWE and CPEE of TN1were recorded as 579 plusmn 72 and 1052 plusmn 1mg100 g peel weightdry basis respectivelyThese data are comparably higher thanthose reported previously for the ethanol extract of mangopeel with an average gallic acid concentration of 15220 plusmn014mg100 g mango peel dry weight [45] Previous studiessuggested that pyrogallol can be detected in the ethanolicextract of mango kernel (the mango tested was purchasedfrom an Egyptian local market) with a concentration of13379 plusmn 031mg100 g mango kernel dry weight but itwas absent in the ethanolic extract of mango peel [45]However we found that pyrogallol could be detected inCPWE and CPEE of TN1 with a concentration of 566 plusmn 55and 930plusmn90mg100 g peel weight dry basis respectively Wespeculate the reason may be due to differences between thetested mango varieties Structurally p-hydroxybenzoic acidgallic acid and pyrogallol are monophenolic compoundswhich exhibit antioxidant activity owing to their hydrogen-donating or electron-donating properties [46] Thereforethe high free radical-scavenging activities of CPWE andCPEE of TN1 may be attributed to the high contents ofp-hydroxybenzoic acid gallic acid and pyrogallol Besidesphenolic compounds previous studies reported that a syn-ergistic effect of combinations of phytochemicals may alsoresult in beneficial biological functions such as inhibitionof proliferation of human cancer cells [38 47] Thus thesynergistic effects of constituents in CPWE and CPEE of TN1with respect to their effects on biological functions warrantfurther investigation The storage stability of antioxidantagent is important with respect to its potential industrialapplicationHerewe evaluated the storage stability of vitaminC CPWE of TN1 and CPEE of TN1 by DPPH radical-scavenging assayThe test sample powderswere redissolved in

double-distilled water at various concentrations and the sam-ple solutionswere stored at room temperature for 1 2 4 and 8hours and then the correspondingDPPH radical-scavengingactivities were determined The data presented in Figure 5(a)suggest that the well-known natural antioxidant vitaminC would dramatically reduce its DPPH radical-scavengingactivity after 1ndash8 hoursrsquo storage However the DPPH radical-scavenging activities in either CPWE of TN1 or CPEE of TN1were not obviously changed after 1ndash8 hoursrsquo storage (Figures5(b) and 5(c)) These findings clearly indicate that the peelextracts of mango exhibited a high storage stability in termsof antioxidant activity Fruit polyphenols have been reportedto be related to immunomodulatory and anti-inflammatoryproperties via in vitro and animal studies [13] NO is aninflammatory mediator induced by inflammatory cytokinesor bacterial LPS in various cell types including macrophages[48] Samples with NO inhibitory activity thus have thepotential to possess anti-inflammatory activity CPEE andCPWE from TN1 were tested for their anti-inflammatoryactivities by investigating their effects on NO productionin LPS-induced RAW2647 macrophages Neither CPEE norCPWE obviously affected the viability of RAW2647 cellsat the 625ndash25 120583gml concentrations that were tested inthe presence of 1 120583gml LPS (Figure 6(a)) As shown inFigure 6(b) when RAW2647 cells were treated with 1 120583gmlLPS theNOproductionwas increased from 311plusmn025 120583Mto128 plusmn 01 120583M Moreover when RAW2647 cells were treatedwith 1 120583gml LPS in the presence of various concentrationsof CPEE it was found that NO production was significantlydecreased from 128 plusmn 01 120583M to 954 plusmn 008 120583M whereasin the presence of various concentrations of CPWE NOproduction was only slightly reduced These results indicatethat CPEE of TN1 had apparent anti-inflammatory activityand thus it may have potential as a natural and safe agent inthe protection of human health by modulating the immunesystem Previous studies demonstrated that extractswith highpolyphenol content exhibited high antibacterial activity [49]As such we evaluated the antibacterial activity of CPEE andCPWE of TN1 by the diffusion disc method Five bacteriathree Gram-negative bacteria (E coli S typhimurium and Vparahaemolyticus) and two Gram-positive bacteria (S aureusand B cereus) were adopted to assess the antibacterial prop-erties As can be seen in Figures 7(a)ndash7(f) both CPEE andCPWE of TN1 exhibited antibacterial activities against the


0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)

Figure 5 DPPH scavenging activities of vitamin C CPWE of TN1 and CPEE of TN1 under different storage times (a) Vitamin C (b) CPWEof TN1 (c) CPEE of TN1

0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)

Figure 6 (a) Effects of CPEE of TN1 CPWE of TN1 and LPS on cell viability of RAW 2647 cells (b) Effects of CPEE of TN1 CPWE of TN1and LPS on NO secretion in RAW 2647 cells The data are the means plusmn SD of triplicate samples Bars with different letters are significantlydifferent (119901 lt 005)


A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)

Figure 7 Zone of inhibition of CPEE of TN1 and CPWE of TN1 at concentration of 4 wv in 005M acetate buffer pH 60 against (a)Escherichia coli (b) Salmonella typhimurium (c) Vibrio parahaemolyticus (d) Staphylococcus aureus and (e) Bacillus cereus In each dish AB C and D represent antibiotic acetate buffer CPEE of TN1 and CPWE of TN1 respectively (f)The bar graph summarizes the four separateantibacterial experiments and shows the zone of inhibition according to treatments Values are expressed as the mean plusmn SD (119899 = 4) Themeans that have at least one common letter do not differ significantly (119901 gt 005)

five bacteria tested The Gram-negative bacteria were moresensitive than Gram-positive ones to CPEE and CPWE ofTN1 (Figure 7(f)) In addition for these five bacteria exceptV parahaemolyticus CPEE exhibited higher antibacterialactivity compared to CPWE These results may be attributedto the higher polyphenol content detected in CPEE (Table 2)which is also consistent with previous findings [50] Inter-estingly for V parahaemolyticus CPEE had less antibacterialactivity compared to CPWE We speculate the reason maybe due to the presence of 3 NaCl in the medium of Vparahaemolyticus However further experimental studies areneeded to elucidate the mechanism of action In summarythe present study demonstrated that CPEE and CPWE fromTN1 had high amounts of phenolic compounds possessedgood and stable free radical-scavenging activities and exhib-ited anti-inflammatory and antibacterial activities CPEE ofTN1 exhibited the most antioxidant anti-inflammatory andantibacterial properties and thus has potential for use in thefood cosmetics and nutraceutical industries

4 Conclusion

In this study we employed a compressional-puffing pre-treatment process and two extraction methods to extractbioactive compounds from six Taiwanese mango peels The

compressional-puffing process increases the extraction yieldsand polyphenol contents of peel extracts Ethanol extractsof peels had higher amounts of total phenolic compoundsand greater free radical-scavenging activities as compared towater extracts of peels The polyphenol contents of extractspositively correlated to the free radical-scavenging activitiesof extracts Among these extracts CPEE of TN1 exhibitedthe most antioxidant anti-inflammatory and antibacterialproperties Thus it is suggested as a natural safe and stableantioxidant agent with anti-inflammatory and antibacterialproperties which may have a wide range of applications infood cosmetics and nutraceuticals Future studies on thepolyphenol composition and biological activities of mangopeel extracts after an in vitrodigestion aswell as investigationsof the in vivo biological activities of mango peel extracts arewarranted

Conflicts of Interest

The authors have no conflicts of interest to declare

Authorsrsquo Contributions

Chun-Yung Huang and Chia-Hung Kuo contributed equallyto this work


Acknowledgments

This work was supported by a grant provided by the Ministryof Science and Technology Taiwan awarded to Chun-YungHuang (no MOST 106-2320-B-022-001)

References

[1] SMR Ribeiro L CA Barbosa JHQueirozMKnodler andA Schieber ldquoPhenolic compounds and antioxidant capacity ofBrazilian mango (Mangifera indica L) varietiesrdquo Food Chem-istry vol 110 no 3 pp 620ndash626 2008

[2] CM Ajila S G Bhat andU J S P Rao ldquoValuable componentsof raw and ripe peels from two Indian mango varietiesrdquo FoodChemistry vol 102 no 4 pp 1006ndash1011 2007

[3] CM Ajila K A Naidu S G Bhat andU J S P Rao ldquoBioactivecompounds and antioxidant potential of mango peel extractrdquoFood Chemistry vol 105 no 3 pp 982ndash988 2007

[4] M Knodler J Conrad E M Wenzig et al ldquoAnti-inflammatory5-(111015840Z-heptadecenyl)- and 5-(81015840Z111015840Z-heptadecadienyl)-resorcinols from mango (Mangifera indica L) peelsrdquoPhytochemistry vol 69 no 4 pp 988ndash993 2008

[5] C M Ajila and U J S Prasada Rao ldquoProtection against hydro-gen peroxide induced oxidative damage in rat erythrocytes byMangifera indica L peel extractrdquo Food and Chemical Toxicologyvol 46 no 1 pp 303ndash309 2008

[6] A Prakash K Mathur A Vishwakarma S Vuppu and BMishra ldquoComparative assay of antioxidant and antibacterialproperties of Indian culinary seasonal fruit peel extractsobtained from Vellore Tamilnadurdquo International Journal ofPharmaceutical Sciences Review and Research vol 19 no 1 pp131ndash135 2013

[7] V L T Hoang J-T Pierson M C Curry et al ldquoPolyphenoliccontents and the effects of methanol extracts from mangovarieties on breast cancer cellsrdquo Food Science and Biotechnologyvol 24 no 1 pp 265ndash271 2015

[8] J Mimic-Oka D V Simic and T P Simic ldquoFree radicals in car-diovascular diseasesrdquo The scientific journal Facta UniversitatisSeries Medicine and Biology vol 6 no 1 pp 11ndash22 1999

[9] P Vijayabaskar N Vaseela and G Thirumaran ldquoPotentialantibacterial and antioxidant properties of a sulfated polysac-charide from the brown marine algae Sargassum swartziirdquoChinese Journal of Natural Medicines vol 10 no 6 pp 421ndash4282012

[10] B Halliwell ldquoAntioxidants in human health and diseaserdquoAnnual Review of Nutrition vol 16 pp 33ndash50 1996

[11] A L Branien ldquoToxicology and biochemistry of butylatedhydroxyanisole and butylated hydroxytoluenerdquo Journal of theAmerican Oil Chemistsrsquo Society vol 52 no 2 pp 59ndash63 1975

[12] N Ito S Fukushima A Hagiwara M Shibata and T OgisoldquoCarcinogenicity of butylated hydroxyanisole in F344 ratsrdquoJournal of the National Cancer Institute vol 70 no 2 pp 343ndash352 1983

[13] J Gonzalez-Gallego M V Garcıa-Mediavilla S Sanchez-Campos and M J Tuno ldquoFruit polyphenols immunity andinflammationrdquo British Journal of Nutrition vol 104 supplement3 pp S15ndashS27 2010

[14] K-J Yun D-J Koh S-H Kim et al ldquoAnti-inflammatoryeffects of sinapic acid through the suppression of induciblenitric oxide synthase cyclooxygase-2 and proinflammatory

cytokines expressions via nuclear factor-120581B inactivationrdquo Jour-nal of Agricultural and FoodChemistry vol 56 no 21 pp 10265ndash10272 2008

[15] Y-C Liang Y-T Huang S-H Tsai S-Y Lin-Shiau C-FChen and J-K Lin ldquoSuppression of inducible cyclooxygenaseand inducible nitric oxide synthase by apigenin and relatedflavonoids in mouse macrophagesrdquo Carcinogenesis vol 20 no10 pp 1945ndash1952 1999

[16] M Wawruch L Bozekova S Krcmery and M Kriska ldquoRisksof antibiotic treatmentrdquo Bratisl Lek Listy vol 103 no 7-8 pp270ndash275 2002

[17] A-S H Abdullah M E S Mirghani and P Jamal ldquoAntibac-terial activity of Malaysian mango kernelrdquo African Journal ofBiotechnology vol 10 no 81 pp 18739ndash18748 2011

[18] H Kim J Y Moon H Kim et al ldquoAntioxidant and antiprolif-erative activities of mango (Mangifera indica L) flesh and peelrdquoFood Chemistry vol 121 no 2 pp 429ndash436 2010

[19] J C Barreto M T S Trevisan W E Hull et al ldquoCharacter-ization and quantitation of polyphenolic compounds in barkkernel leaves and peel of mango (Mangifera indica L)rdquo Journalof Agricultural and Food Chemistry vol 56 no 14 pp 5599ndash5610 2008

[20] V S Falcao-Silva D A SilvaM D F V Souza and J P SiqueiraJr ldquoModulation of drug resistance in Staphylococcus aureus bya kaempferol glycoside from Herissantia tiubae (Malvaceae)rdquoPhytotherapy Research vol 23 no 10 pp 1367ndash1370 2009

[21] C H Sai Srinivas M kumar Mediboyina U J S PrasadaRaoand A Kothakota ldquoStudies on isolation and antioxidant prop-erties of bioactive phytochemicals from mango peel harvestedat different developmental stagesrdquo International Journal ofAgriculture vol 2 no 2 pp 136ndash148 2017

[22] A Kuganesan G Thiripuranathar A N Navaratne and PA Paranagama ldquoAntioxidant and anti-inflammatory activitiesof peels pulps and seed kernels of three common mango(Mangifera indical L) varieties in Sri Lankardquo InternationalJournal of Pharmaceutical Sciences and Research vol 8 no 1p 70 2017

[23] M N Safdar T Kausar and M Nadeem ldquoComparison ofultrasound and maceration techniques for the extraction ofpolyphenols from the mango peelrdquo Journal of Food Processingand Preservation vol 41 no 4 Article ID e13028 2017

[24] E Dorta M G Lobo and M Gonzalez ldquoImproving theefficiency of antioxidant extraction from mango peel by usingmicrowave-assisted extractionrdquo Plant Foods for Human Nutri-tion vol 68 no 2 pp 190ndash199 2013

[25] R Rojas J C Contreras-Esquivel M T Orozco-EsquivelC Munoz J A Aguirre-Joya and C N Aguilar ldquoMangopeel as source of antioxidants and pectin Microwave assistedextractionrdquo Waste and Biomass Valorization vol 6 no 6 pp1095ndash1102 2015

[26] M P Garcia-Mendoza J T Paula L C Paviani F A Cabraland H A Martinez-Correa ldquoExtracts from mango peel by-product obtained by supercritical CO2 and pressurized solventprocessesrdquo LWT- Food Science and Technology vol 62 no 1 pp131ndash137 2015

[27] C-Y Huang S-J Wu W-N Yang A-W Kuan and C-YChen ldquoAntioxidant activities of crude extracts of fucoidanextracted from Sargassum glaucescens by a compressional-puffing-hydrothermal extraction processrdquo Food Chemistry vol197 pp 1121ndash1129 2016


[28] W-N Yang P-W Chen and C-Y Huang ldquoCompositionalcharacteristics and in vitro evaluations of antioxidant and neu-roprotective properties of crude extracts of fucoidan preparedfrom compressional puffing-pretreated sargassum crassifoliumrdquoMarine Drugs vol 15 no 6 article no 183 2017

[29] P-S Chiang D-J Lee C G Whiteley and C-Y HuangldquoAntioxidant phenolic compounds from Pinus morrisconicolausing compressional-puffing pretreatment and waterndashethanolextraction Optimization of extraction parametersrdquo Journal ofthe Taiwan Institute of Chemical Engineers vol 70 pp 7ndash14 2017

[30] P-S Chiang D-J Lee C G Whiteley and C-Y HuangldquoExtracting antioxidant phenolic compounds from compres-sional-puffing pretreated Pinus morrisonicola Effects of opera-tional parameters kinetics and characterizationrdquo Journal of theTaiwan Institute of Chemical Engineers vol 75 pp 70ndash76 2017

[31] C-Y Huang C-H Kuo and P-W Chen ldquoCompressional-puffing pretreatment enhances neuroprotective effects offucoidans from the brown seaweed Sargassum hemiphyllumon 6-hydroxydopamine-induced apoptosis in SH-SY5Y cellsrdquoMolecules vol 23 no 1 p 78 2018

[32] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdicphosphotungstic acid reagentsrdquo Ameri-can Journal of Enology and Viticulture vol 16 pp 144ndash158 1965

[33] A Schieber W Ullrich and R Carle ldquoCharacterization ofpolyphenols in mango puree concentrate by HPLC with diodearray and mass spectrometric detectionrdquo Innovative Food Sci-ence and Emerging Technologies vol 1 no 2 pp 161ndash166 2000

[34] C-YWang T-CWu S-L Hsieh Y-H Tsai C-W Yeh andC-YHuang ldquoAntioxidant activity and growth inhibition of humancolon cancer cells by crude and purified fucoidan preparationsextracted from Sargassum cristaefoliumrdquo Journal of Food andDrug Analysis vol 23 no 4 pp 766ndash777 2015

[35] H Kobuchi M T Droy-Lefaix Y Christen and L PackerldquoGinkgo biloba extract (EGb 761) Inhibitory effect on nitricoxide duction in themacrophage cell line RAW2647rdquoBiochem-ical Pharmacology vol 53 no 6 pp 897ndash903 1997

[36] C-Y Huang C-H Kuo and C-H Lee ldquoAntibacterial andantioxidant capacities and attenuation of lipid accumulation in3T3-L1 adipocytes by low-molecular-weight fucoidans preparedfrom compressional-puffing-pretreated Sargassum crassifoliumSargassum crassifoliumrdquoMarine Drugs vol 16 no 1 p 24 2018

[37] M R Ali M J Yong R Gyawali A Mosaddik Y C Ryuand S K Cho ldquoMango (Mangifera indica L) peel extractsinhibit proliferation of HeLa human cervical carcinoma cell viainduction of apoptosisrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 55 no 3 pp 397ndash405 2012

[38] H Kim H Kim A Mosaddik R Gyawali K S Ahn and S KCho ldquoInduction of apoptosis by ethanolic extract ofmango peeland comparative analysis of the chemical constitutes of mangopeel and fleshrdquoFoodChemistry vol 133 no 2 pp 416ndash422 2012

[39] Y-G Liu X-M Zhang F-Y Ma and Q Fu ldquoThe antioxidantactivitives of mango peel among different cultivarsrdquo in IOPConference Series Earth and Environmental Science vol 61Thailand March 2017

[40] F Shahidi and P K Wanasundara ldquoPhenolic antioxidantsrdquoCritical Reviews in Food Science and Nutrition vol 32 no 1 pp67ndash103 1992

[41] J Wang Q Zhang Z Zhang H Song and P Li ldquoPotentialantioxidant and anticoagulant capacity of lowmolecular weightfucoidan fractions extracted from Laminaria japonicardquo Interna-tional Journal of Biological Macromolecules vol 46 no 1 pp 6ndash12 2010

[42] L P Leong and G Shui ldquoAn investigation of antioxidantcapacity of fruits in Singapore marketsrdquo Food Chemistry vol76 no 1 pp 69ndash75 2002

[43] S Tachakittirungrod S Okonogi and S ChowwanapoonpohnldquoStudy on antioxidant activity of certain plants in Thailandmechanism of antioxidant action of guava leaf extractrdquo FoodChemistry vol 103 no 2 pp 381ndash388 2007

[44] A Elzaawely and S Tawata ldquoPreliminary phytochemical inves-tigation on mango (Mangifera indica L) leavesrdquo World Journalof Agricultural Sciences vol 6 no 6 pp 735ndash739 2010

[45] U E SMostafa ldquoPhenolic compound and antioxidant potentialof mango peels and kernels (Mangifera indica L) on the fryingoil stability lipid profile and activity of some antioxidant serumenzymes in ratsrdquo Journal of American Science vol 9 no 11 pp371ndash378 2013

[46] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWT - FoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[47] N P Seeram L S Adams M L Hardy and D HeberldquoTotal cranberry extract versus its phytochemical constituentsantiproliferative and synergistic effects against human tumorcell linesrdquo Journal of Agricultural and Food Chemistry vol 52no 9 pp 2512ndash2517 2004

[48] E J Swindle and D D Metcalfe ldquoThe role of reactive oxygenspecies and nitric oxide in mast cell-dependent inflammatoryprocessesrdquo Immunological Reviews vol 217 no 1 pp 186ndash2052007

[49] K Fukai T Ishigami and Y Hara ldquoAntibacterial activity oftea polyphenols against phytopathogenic bacteriardquo Agriculturaland Biological Chemistry vol 55 no 7 pp 1895ndash1897 1991

[50] R Naz H Ayub S Nawaz et al ldquoAntimicrobial activity toxicityand anti-inflammatory potential of methanolic extracts of fourethnomedicinal plant species from Punjab Pakistanrdquo BMCComplementary and Alternative Medicine vol 17 no 1 articleno 302 2017

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


peroxyl radical (ROO∙) and alkoxyl radical (RO∙) aredefined as any molecules or atoms with one or moreunpaired electrons and are often involved in human diseases[8] Many studies have shown that free radicals in livingorganisms cause oxidative damage to different moleculessuch as lipids proteins and nucleic acids and these areinvolved in the interaction phases of many diseases suchas cancer atherosclerosis respiratory ailments and evenneuronal death [9] Antioxidants are substances that delayor prevent the oxidation of cellular oxidisable substratesThey exert their effect by scavenging reactive oxygen species(ROS) or preventing the generation of ROS [10] Syntheticantioxidant compounds such as butylated hydroxytoluene(BHT) and butylated hydroxyanisole (BHA) have potentantioxidant activity and are commonly used in processedfoods However they have been restricted because of theircarcinogenicity and other toxic properties [11 12] Thus inrecent years there has been considerable interest in naturalantioxidants derived from biological materials becauseof their presumed safety and potential nutritional andtherapeutic value

A large number of publications have suggested that fruitpolyphenols are related to immunomodulatory and anti-inflammatory properties via in vitro and animal studies [13]Inflammation is a complicated physiological phenomenonthat occurs when the immune system in the body isactivated to counter threats such as injury infection andstress Macrophages often play a unique role in the immunesystem because they not only elicit an innate immuneresponse but also act as effector cells in inflammation andinfectionWhenmacrophages encounter bacterial endotoxinlipopolysaccharide (LPS) they can be stimulated to generatea variety of inflammatory mediators such as nitric oxide(NO) tumor necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573) IL-6 prostaglandin E2 (PGE2) and adhesion moleculesto help eradicate the bacterial assault [14] Generally sub-stances with inhibitory effects on the expression and activityof enzymes (eg inducible NO synthase (iNOS)) involved inthe generation of inflammatory mediators such as NO in themouse macrophage-like cell line RAW 2647 are consideredto possess immunomodulatory activity [15] Since a varietyof polyphenols exist in mango peels further research on theuse of mango peel extracts as immunomodulatory or anti-inflammatory agents is warranted

Antibacterial agents are the synthetic or natural com-pounds that interfere with the growth and division ofbacteria A number of studies have shown that pathogenicmicroorganisms in humans and various animal species havedeveloped resistance to drugs This drug resistance is due tothe random or otherwise inappropriate usage of commercialantimicrobial agents As such there is an urgent need fornew antibacterial agents In addition synthetic antibioticshave been known to induce side effects such as the appear-ance of resistant bacteria skin irritation organ damageand immunohypersensitivity [16] Accordingly many studieshave attempted to develop new agents with high antibacterialactivity but with fewer or possibly even no side effects Thereis a particular demand for antibacterial compounds fromnat-ural resources [17] Plants produce a range of antimicrobial

compounds in various parts such as bark stalk leaves rootsflowers pods seeds stems hull latex and fruit rind [6] Fruitpeel is the outer covering of a fruit which functions as aphysical barrier It also serves as a chemical barrier by virtueof the presence of many antimicrobial constituents whichprotect the fruit from exposure to external pathogens or otherfactors that may tend to decrease the quality of the fruitTherefore fruit peels are good sources for obtaining naturalantibacterial agents

Bioactive compounds in mango peel are generallyextracted via the following methods extraction with 80ethanol by sonication for 3 days at room temperature [18]extraction performed three times with methanol for 3 h pertime [19] extraction with 95 ethanol three times 72 h pertime [20] extraction with acetone or ethyl acetate for upto 20 h [21ndash23] extraction by microwave-assisted method[24 25] or extraction with supercritical CO2 followed bypressurized ethanol [26] However these methods generallyinvolve the use of a large volume of solvents require a longextraction time consume a lot of energy are costly and some-times are not eco-friendly The present study builds uponon the research reported in our previous investigation [27]In brief we previously developed a compressional-puffingprocess that has been successfully implemented to increasethe extraction yield of fucoidan from brown seaweed [27 28]and augment the extraction yields of total phenolics andtotal flavonoids from pine needles [29 30] Compressional-puffing can be utilized as a pretreatment step to disruptthe cellular structure of samples thereby better enablingthe release of bioactive compounds by solvent extraction[27] In this study compressional-puffing was utilized forpretreatment of mango peels and water extract (WE) andethanol extract (EE) extracted from nonpuffed (NP) andcompressional-puffed (CP) mango peels were comparedThe phenolic compound composition and the free radical-scavenging anti-inflammatory and antibacterial activitiesof WE and EE from mango peels were also evaluated Tothe best of the authorsrsquo admittedly limited knowledge thisis the first study to elucidate the free radical-scavenginganti-inflammatory and antibacterial activities of WE andEE extracted from compressional-puffed mango peels TherecoveredWEandEE are expected to possessmultifunctionalactivities providing a wide range of benefits The utilizationof mango peel will also help to play a role in minimizing thegeneration of waste worldwide

2 Materials and Methods

21 Materials Folin-Ciocalteursquos phenol reagent gallic acidprotocatechuic acid chlorogenic acid p-hydroxybenzoicacid pyrogallol caffeic acid mangiferin epicatechin p-coumaric acid ferulic acid epicatechin gallate (ECG) cate-chin gallate (CG) ellagic acid rutin quercetin kaempferolhomogentisic acid tannic acid vanillic acid 22-diphen-yl-1-picrylhydrazyl (DPPH) sodium nitrite LPS dimeth-yl sulfoxide (DMSO) and 221015840-azino-bis(3-ethylbenzothia-zoline-6-sulphonic acid) diammonium salt (ABTS) werepurchased from Sigma-Aldrich (St Louis MO USA) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide


(1) (2) (3) (4) (5) (6)

















Time (sec) 0 10 0 10







Various mango peels

Hot-air dried

Nonpuffed (NP)

Crumbled

Pulverized


CPEE



NPWECPWE





dried at 50∘C
































DPPHABTS

05

10152025303540

(1S

C50)times10

00(l

g)


R2 = 09599

R2 = 09892

(a)

0 2 4 6 8 10 12 14 16 1805

101520253035404550

(1S

C50)times10

00(l

g) R2 = 07667

R2 = 08297

DPPHABTS


(b)

DPPHABTS

0

10

20

30

40

50

60

70

(1S

C50)times10

00(l

g)


R2 = 09154

R2 = 09739

(c)

DPPHABTS

0102030405060708090

(1S

C50)times10

00(l

g)


R2 = 09875

R2 = 0962

(d)









(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)





0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(1) (2) (3) (4) (5) (6)

















Time (sec) 0 10 0 10







Various mango peels

Hot-air dried

Nonpuffed (NP)

Crumbled

Pulverized


CPEE



NPWECPWE





dried at 50∘C
































DPPHABTS

05

10152025303540

(1S

C50)times10

00(l

g)


R2 = 09599

R2 = 09892

(a)

0 2 4 6 8 10 12 14 16 1805

101520253035404550

(1S

C50)times10

00(l

g) R2 = 07667

R2 = 08297

DPPHABTS


(b)

DPPHABTS

0

10

20

30

40

50

60

70

(1S

C50)times10

00(l

g)


R2 = 09154

R2 = 09739

(c)

DPPHABTS

0102030405060708090

(1S

C50)times10

00(l

g)


R2 = 09875

R2 = 0962

(d)









(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)





0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







Time (sec) 0 10 0 10







Various mango peels

Hot-air dried

Nonpuffed (NP)

Crumbled

Pulverized


CPEE



NPWECPWE





dried at 50∘C
































DPPHABTS

05

10152025303540

(1S

C50)times10

00(l

g)


R2 = 09599

R2 = 09892

(a)

0 2 4 6 8 10 12 14 16 1805

101520253035404550

(1S

C50)times10

00(l

g) R2 = 07667

R2 = 08297

DPPHABTS


(b)

DPPHABTS

0

10

20

30

40

50

60

70

(1S

C50)times10

00(l

g)


R2 = 09154

R2 = 09739

(c)

DPPHABTS

0102030405060708090

(1S

C50)times10

00(l

g)


R2 = 09875

R2 = 0962

(d)









(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)





0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



























DPPHABTS

05

10152025303540

(1S

C50)times10

00(l

g)


R2 = 09599

R2 = 09892

(a)

0 2 4 6 8 10 12 14 16 1805

101520253035404550

(1S

C50)times10

00(l

g) R2 = 07667

R2 = 08297

DPPHABTS


(b)

DPPHABTS

0

10

20

30

40

50

60

70

(1S

C50)times10

00(l

g)


R2 = 09154

R2 = 09739

(c)

DPPHABTS

0102030405060708090

(1S

C50)times10

00(l

g)


R2 = 09875

R2 = 0962

(d)









(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)





0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(mAU

)

600

400

200

CPEE of TN1

CPWE of TN1

(1)

(2)

(3)

(4)

(5) (6)(7)00 5 10 15 20 25 30 35 40

(minute)

(a)

200400600

0

(mAU

)

(1)(2) (3) (4) (5) (6)(7)

0 5 10 15 20 25 30 35 40(minute)

(b)





0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0102030405060708090

100

0 10 20 30 40 50 60 70

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(a)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

Concentration (gml)

8 hr4 hr

2 hr1 hr0 hr

(b)

Concentration (gml)

0102030405060708090

100

0 30 60 90 120 150 180 210

DPP

H sc

aven

ging

activ

ity (

)

8 hr4 hr

2 hr1 hr0 hr

(c)


0102030405060708090

100110

Cel

l via

bilit

y (

)

0 1 1 1 1 1 1 10

0000

000

0 0

A A A A A A AA

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(a)

02468

101214

Nitr

ite (

M)

A

DBC

B

ECD CDE

0 1 1 1 1 1 1 10

0000

000

0 0

LPS (gml)CPEE (gml)

CPWE (gml)625 125 25

625 125 25

(b)



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



A

B

D

C

(a)

A

B

D

C

(b)

A

B

D

C

(c)

A

B

D

C

(d)

A

B

D

C

(e)

Esch

erich

ia co

li

Salm

onell

aty

phim

uriu

m

Vibr

iopa

raha

emol

yticu

s

Stap

hylo

cocc

usau

reus

Bacil

lus c

ereu

s

CPEECPWE

CDAB

GEF DE F

BCA

BCA

02468

1012141618202224

Zone

of i

nhib

ition

(mm

)

(f)



4 Conclusion








Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Acknowledgments


References























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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