Indian Journal of Chemistry Vol. 42B, March 2003, pp. 641-647

Variations in the essential oil composition of Persea bombycina (King ex Hook. f.) Kost and its effect on mug a silkworm (Antheraea assama Ww) - A new report

S N Choudhury*

Plant Sciences and Ecology Division, Regional Research Laboratory , Jorhat -785006, Assam, India

and

Ingrid Vajczikova

Department of Chemistry, Institute of Chemical Technology , Prague, Czech Republic

Received 19 September 200 I .. accepted (revised) 20 November 2002

Persea bombycina, with the local name 'Som', is a primary food plant for the 'muga' silk worm (Antheraea assam a Ww). Hydrodistillation of the fresh green leaves of P. bombycina gives a clear and sweet scented oil in 0.02 to 0.054% (w/w) yield . The oil composition is investigated by GC and GC/MS. A total of 79 compounds have been identified . Substan­tial differences in the composition of the oils from 9 leaf samples are noticed. Aldehydes constitute a major portion of the oil (28 .0 to 53.2%) followed by acids (11.4 to 40.8%) and alcohols (7.4 to 14.8%). Dodecanal (3.1 to 25 .8%), decanal (2.4 to 25.8%), II-dodecenal (1.5 toI6.3%) and undecanal (1.3 to 9.5%) are present at extremely variable concentrations. Vari a­tions of (E)-nerolidol is quite conspicuous (0.5 % to 21.9%). Out of the two oxides, caryophyllene oxide is a major compo­nent (3.0 to 12.1 %). Experiments on rearing performances of muga silkworm on nine different collections of 'som', their most favoured host plant, reveal that growth of the worms are influenced unevenly by the different chemical constituents present in the essential oil. Correlation studies indicated that aldehyde per cent in the essential oil has positive influence on the growth of silkworm and cocoon quality , while increased ac id per cent in the oil is found to be detrimental for growth and cocoon quality.

Persea bombycina (King ex Hook. f.) Kost (Lau­raceae)1 formerly named MachiLus bombycina King. is a medium size non-decidous tree found in the As­sam valley in India up to an elevation of 500m (MSL)I.2. This tree, locally known as 'som', is perhaps a cultivated form of Machi/us gambLee or M. kurzii. Apart from Assam, its distribution extends to the Khasi and laintia Hills in India2 along the lower Hi­malaya, and as far as to the west of Nepal3. In Assam 'som' provides the primary food for 'muga' silk worms (Antheraea assama WW)4-6, which produce the golden silk very specific to the northeastern states of India and found nowhere else on the globe4.7-

11 . Lakhimpur district of Assam is one of the good

producers of 'muga' cocoons. Rearers use only 'som' plant as a main host for rearing of 'muga' worms. Dur­ing the course of floristic survey of this district we encountered this species abundantly in natural stand. Organoleptic and olfectory tests made on the leaves at sources, revealed that their essential oil composition may differ from plant to plant.

It has been reported that 'som' plant have many va­rieties4, different morphotypes 12 and found different taste of leaves 13 but extensive work has not been done

on the chemical constituents of essential oil on this plantI4-16.

The role of inhibitors in host plant selection indi­cates that many different chemicals may be expected to have an inhibitory effect on feeding habits of dif­ferent insects. Literature survey on essential oil and insect effect revealed that the terpenoids included u­pinene, B-pinene, limonene and eugenol contact of few insect was sufficient to evoke vitellogenesis and reproductive activi ty 17. Terpenoids present both in foliage and seeds of the cotton plant (Gossypium her­sutum) is of importance in insect utilization of cotton varieties I8.19. On the other hand essential oils contain acyclic monoterpenes, such as geraniol, citronellal , citral and limonene have been identified as alarm de­fense subsrances in numerous insects2o.21. Repellent properties of aromatic hydrocarbons on insects have also been described22

-24. Aldehydes and ketones are

repellent at high concentrations to Erioschia brassi­cae25

, while the larvae of B. mori are attracted by the odour of n-butyl aldehyde. Decanoic acid reduces feeding on elm twigs by ScoLytus muLtiotriatui 6

,

while the pentatomids, thyanta sp and euschistus sp are repelled by the odour of methacrylic acid23. This

642 INDIAN J. CHEM., SEC B, MARCH 2003

indicating that essential oil constituents have promi­nent role on insect feeding and host selection.

The literature survey failed to uncover any pub­lished work on the existing chemotypic variations of the essential leaf oil in relation to rearing performance of 'muga' silk worms. Hence this paper is the first re­port on the searching out the chemical variations of the essential oil of leaves of P. bombycina and their effect on the growth and cocoon quality of muga silk worm.

Materials and Methods Nine 'som' plants were selected from Lakhimpur

district of Assam. These plants were demarcated as accession (A) number from 1 to 9. Air layering tech­nique was adopting for collecting the plant samples in the month of April/May 1996. Six to eight air layer­ings were prepared in each selected plant. Four good layerings were planted in prepared pits at a spacing of 3.5 x 3.5 m. Plants height was maintained at 4.0 m.

Rearing of 'muga' worms was carried out during October-November CKatya' brood) of 1999. Each plant was covered by nylone net (3.5 x 3.5 x 4.0 m) over a bamboo frame made according to the size of the net. Di sease free lay ings(dfls) were first brushed into a bamboo tray and 100 numbers of newly emerged 1st instar larvae were transferred to each plant and allowed to complete larva l period. Matured larvae were collected from each plant and transfened to bamboo trays (one tray for each plant) filled up with dry leaves, after recording their weight. Cocoon characters such as average weight of cocoons, shell weight, silk ratio, effective rate of rearing were de­termined after 10 days of collection of matured larvae. Sericin and fibroin per cent were estimated from each shell27

. All the experiments were carried out at Re­gional Research Laboratory, JOI'hat, Assam during 1996-200 l.

Plant material. Fresh leaves were collected from each matured tree from the experimental farm of RRL, Jorhat, Assam.

Isolation of essential oils. Fresh leaves (300g) plant samples were hydrodisti lled in a clevengers apparatus, yielding 0.02 to 0.054% (w/w) of a clear oil. Qualitative and quantitative analysis

Gc. A Parkin-Elmer 8500 gas chromatograph equipped with a FID detector and a HP-l fused silica colimn (24 m x 0.32 mm, 0.17 11m film thickness) was used. The samples were injected in the sp lit mode, using pressure controlled helium as carrier gas at a

linear velocity of 30 cmls (at 60°C) Injector and de­tector temperatures were maintained at 250°C. The column oven temperature was programmed from 60°C (after 2 min) to 250°C at 4°C/min. The final tempera­ture was held for 20 min. Peak areas and retention times were measured by electronic integration. The relative amounts of individual components are based on the peak areas obtained, without FlO response fac­tor correction. Temperature programmed (linear) re­tention indices of the compounds were determined relative to n-alkanes.

GCIMS. Analyses were carried Ol,;.t on a Shimadzu GC-17A1GCMS-QP5000 system. A 25mxO.20 mm fused silica HP-l column, with a fi lm thickness of 0.33 Jlm, was employed. The column oven tempera­ture was programmed from 60°C (after 3 min) to 300°C at 5°C/min. The injector and GC/MS interface temperatures were maintained at 280 and 300°C, re­spectively. Helium canier gas was pressure controlled to give a linear velocity of 44 cmls (at 60°C).Electron ionization mass spectra were acquired over the mass range 10-400 Da at a rate of 2/s.

Component identification. The constituents of the oils identified by matching their 70 cY mass spectra and linear temperature programmed retention indices with reference libraries28

.37

.

Results and Discussion Nine plants were selected at the source using or­

ganoleptic and smearing smell of leaf test and essen­tial oil compositions were further confirmed. Detailed GC and GCrMS analysis indicated that the oil was a complex mi xture consisting of more than 79 com­pounds. The volatile compounds indi ~ated in the oil are li sted in Table I.

The leaf oil was found to be very rich in aldehyde, constituted by twelve compounds. Total aldehyde percentage in the oil of different 'SO;11' plant varied significantly. The minimum (28.0%) was recorded in A9 whereas this percentage went up to 53.2% in case of AS. Detailed scrutiny of the Table I revealed that decanal itself contributed 25.8% out of 50.7% of total aldehyde per cent in A6. On the other hand this re­duced to only 2.4% in A4. Dodecanal was another dominating constituents in AS (25.8 %), A7 (25.6%), A3 (23.3%), A8 (18.6%), and A4 (17 .2%). Choud­hury and Leclercq reported higher percentage of do­decanal in the leaf of this plant from Jorhat district of Assaml 4. But the same constituent was found onl y 3. 1 % and 5.8% in A9 and A2 respectively. 11-dodecenal was found in varied percentages ( l.5 to 16.3).

CHOUDHURY et al.: ESSENTIAL COMPOSITION OF PERSEA BOMBYCINA 643

Table 1-Chemical composition of the leaf oils of Persea bombycina from Lakhimpur district of Assam, India

Compd Sesquiterpene hydrocarbons

a-copaene

~-caryophyllene

aromadendrene humulene

~-selinene

a-selinene

8-cadinene

Sesquiterpene alcohols

Elemol (E)-nerolidol caryophyllene alcohol globulol viridiflorol T-cadinol T-muurolol

~-eudesmol

selin-II-en-4 -01

a-cadinol

a-bisabolol (E,E)-farnesol

Aldehydes

heptanal octanal nonanal

a-campholenal 9-decenal decanal 10-undecenal undecanal II-dodecenal dodecanal 12-tridecenal tetradecanal

Esters

isobornyl acetate octyl decanoate nonyl decanoate decyl decanoate undecyl decanoate decyl II-dodecenoate decy l dodecanoate und.:cy I II-dodecenoate undecyl dodecanoate dodecy I I 1-dodecenoate dodecyl dodecanoate

R.I

1370

1411

1431 1444 1476 1486

1510

1530 1547 1555 1566 1574 1621 1623 1628

1629 1632

1664

1700

876 980 1082 1099

1174 1184 1275 1286 1378 1388 1480 1593

1265 1963 2061 2160 2259 2340 2358 2439 2450 2538 2550

Al

t

0.2 2.3

2.5

1.9

0.4 0.5

0.6 0.9

4.3

0.3 0.2 2.1

0.2 3.7 3.4 3.9

10.8 10.8 0.6 0.3

36.3

0.5

0.1

0.1 0.1

0.8

A2

0.9

t

0.2

1.1

21.9

1.0 0.5

0.1 1.4

0.5 25.4

0.1 0.1 5.3

12.0 0.4 1.6 4.0 5.8

0.2 29.5

0.8 t

0.1 t

0.1

0.1

0.1

1.2

A3

0.3

0.3

1.0

0.3 0.1

0.1 0.6

0.3 2.4

t

0.1 2.1

0.1 4.0 1.5 9.5 3.1

23.3

0.3 44.0

0.4

t

0.1 0.2 0. 1 0.1

t

0.4

t

1.3

A4

0.5

0.5

0.7 1.0

0.3 0.4

0.6 0.8

0.2 4.0

0.2 0.6

0.1 2.4 2.5 4.5

14.1 17.2 0.5 0.4

42.5

0.2

t

t

0.1 0.1

t 0.2 0.2

0.8

FID A5

0.3

1.1

0.9

2.3

1.5 1.6 1.4

0.7 0.3

1.0

0.3 6.8

0.1 0.5

0.1 3.8 2.2 4.3

16.3 25 .8

t

0.1 53.2

0.2

0.1

0.1 0.1

0.5

A6

0.6

0.1 t

0.4

0.1

1.2

0.2 5.3 0.1

25 .8 l.l 1.3 8.2 8.6 0.1

t

50.7

0.4

0.2 0.1

0.7

A7

1.1

1.1

2.0

0.9 0.1

0.7

3.7

1.7

3.3 0.4 5.9 4.2

25 .6 t

0.1 41.2

0.2

t

0.1 0.1

0.1 0.3

0.1 0.9

A8

0.1

0.3

0.4

0.4 l.l 3.9 0.6

5.9 1.4 4.8 2.4

18.6 0.6

39.7

0.2

0.2

A9

0.1

0.1

0.5 0.5 1.3

2.3

0.1 0.2

10.2

9.7 1.0 1.7 1.5 3.1 0.1 0.1

28.0

0.2 0.6 0.2 0.5

1.3 (-Contd)

644 INDIAN 1. CHEM., SEC B, MARCH 2003

Table I - Chemical composilion of Ihe leaf oils of Persea bombycina from Lakhimpur district of Assam, India (-Contc£)

Compd R.I FlO Sesquiterpene hydrocarbons AI A2 A3 A4 A5 A6 A7 A8 A9

Hydrocarbons

oclane 800 I 0.3 0.1 I 1.5 0.1 0.4 nonane 900 0.3 1.2 0.3 0.1 0.1 2.6 0.2 0.3 0.8 I-decene 988 0.4 0.1 t 0.1 0.4 0.7 0.4 decane 1000 0.3 0.1 0.6 0 .3 0.2 0.5 0 .3 p-cymene 1010 0.5 0.1 t I t I-undecene 1089 1.2 0.2 0.3 0 .5 0.6 1.0 0.3 0 .2 0 .2 undecane 1100 1.4 0.5 1.8 0.8 1.1 1.3 1.4 0.5 0.2 I-undecyne 111 2 tridecane 1300 I drima-7,9 (II)-diene 1460 0.5 heplacosane 2700 0.1 t I 0. 1 0.1 0.1 nonacosane 2900 0.1 0.1 0.2 t 0.1 I

4.3 2.5 3.2 2.01 2.6 6.8 2.6 2.1 2.1

Alcohols

I-heplanol 953 0.4 Isoborneal 11 35 t 0.1 I-nonanol 1158 1.3 5.3 1.3 0.5 0.4 4.9 1.4 2.7 6.5 terpinen-4-o1 1169 1.1 0.2 9-decen-l-ol 1246 I I I

I-decanol 1256 I 0.1 0.2 0.2 0.1 10-undecen-I-ol 1347 4.8 0.4 0.9 2.1 1.5 0.7 0 .7 1.0 0.6 I-undecanol 1358 5.8 2.0 6.9 4.0 3.4 1.1 5.6 6.6 3.0 I-alkano l 1501 1.8 1.2 3.0 2.2 0.4 2.0 4.1 0 .2 Irans-phYlol 2097 I 0.1 0.3 0.2 0.4 I 0.3

13.7 7.9 10.8 10.n 9.1 7.4 10.0 14.8 10.3

Oxides

caryophyllcne oxide 1562 3.3 3.4 6.2 12.1 3.3 5.1 8.0 3.0 3.0 humulene 1,2-epox ide 1588 1.9 0.6 I 1.8 1.0

3.3 5.3 6.8 12.1 5.1 5.1 9.0 3.0 3.0

Acids

oClanoic (capry lic) acid 1165

nonanoic acid 1259 I 0.1 I I

dccanoic (capric) ac id 1362 0 .9 2.7 1.1 1.4 0.3 9.6 2.0 5.9 32.8

I O-undecenoic acid 1445 0.4 0.6 1.1 t 0 .2 I 0.2 0.8

undecanoic acid 1458 0.4 1.0 1.3 1.2 0.4 0.1 0. 1 1.4

II-dodecenoic ac id 1545 6.0 1.9 I 2.7 3.0 2.9 0.1 6.5 2.0

dodecanoic (lauric) acid 1550 7.1 4.7 14.5 9.3 7.1 2.9 11.7 12.1 5.0

12-lridecenoic acid 1645 0.3 0.3 0.4

tridecanoic acid 1650 0.1 0. 1

tctradecanoic (myristic) acid 1751 0.2 0.2 0.3 0.3

hexadecanoic (pa lmi tic) acid 1940 0.1 0.1 0.2 0.2 I 0.2

15.4 11.4 18.9 14.9' 11.6 15.5 13.9 26.1 40.8

Ketone

2-t ridecanone 1478 0.3 0.6

Terpene

a-pincne 928 0.4 0.2 0.1

CHOUDHURY el aL.: ESSENTIAL COMPOSITION OF PERSEA BOMBYCINA 645

Heptanal, octanal, a-campholenal, 9-decenal, 10-undecenal, 12-tridecenal and tetradecanal were ap­peared as minor constituents in the leaf oils.

Total alcohol of each oil sample contributed a major percentage of the bulk of the oi l but significant variation existed among the percentages. Maximum percentage was recorded in A8 (14.8%). This percentage was drastically reduced to 7.4% in A6. 1-Undecanol was found as a major indi vidual compound in A3 (6.9%), A8 (6.6%), Al (5.8%) and A7 (5.6%). The second dominating compound, I-nonanol was found 6.5% and 5.3% in A 9 and A2 respectively.

Sesquiterpene alcohol was found to be extremely variable in 'som' leaf oil. Total 25.4% of sesquiterpene alcohol was detected in A2, whereas this was only 0.4% in case of A8. Considering the individual com­ponent, (E)-nerolidol was also recorded highest in A2 (21.9%) same chemical was found only 0 .5% in A9. The rest of the compounds occured in amounts less than 1.0% except for caryophyllene alcohol (1.6%), a-cadinol (1.4%) and globulol (1.4%).

The bulk of the leaf oil of 9 samples comprised ~f 12 hydrocarbons. Maximum total percentage (6.8) was recorded in A6 and lowest was only 2.0% in A4. None of the individual compounds amounted to more than l.2% except octane and undecane.

Sesquiterpene hydrocarbon occupied compera­tively less percentage in the bulk of the oil. The varia­tions of total sesquiterpene hydrocarbon was 0.1 % (A9) to 2.5% (A 1).

Acid percentage in the oil was quite high in all the samples, although there was a marked fluctuation in percentage. A9 contained highest percentage of acid (40.8%) of the bulk of the oil followed by 26.1 % in A8. It reduced to 11.4% in A2.

Eleven ester compounds have been detected from 9 leaf samples. But its contribution in the bulk of the oil remained only 1.3%.

Oxide percentage showed remarkable variation from 3.0% (A9) to 12.1 % (A4) . Caryophyllene oxide was a major compound in A4 (12.1 %) followed by A7 (8.0%), A3 (6.2%) and A6 (5.1 %). 2-Tridecanone was found in a very small quantities in Al (0.3%) and A 7 (0.6%). a-Pinene was detected only in three leaf samples and the percentage was less than 0.5.

A considerable variation in weight of individual muga larvae was found to exist while fed on nine 'som' plant (leaf) collected from only one district of Asssam (Table II). The plants having high aldehyde per cent in their oils produced healthy worms (14.0 to 14.3g), in less larval duration, with greater cocoon weight (6.0 to 6.2g), higher shell weight (0.65 to 0.67 g) and maximum ERR% (30.9 to 3l.3). This sug­gested that the aldehyde per cent in the leaf oil is beneficial for the muga worm. Ishikawa and Hirao,196538

. reported that larvae of B. mori are at­tracted by aldehyde at low concentrations while being repelled at high concentration. On the contrary the affinity of A. assama towards aldehydic compounds was found to increase with the increased concentra­tion of the component. Acceptability and repellent character of different insectes on same chemicales at different concentrations have been well men­tioned24

.39.4o. Fibroin, the filament forming true silk was also enhanced at higher aldehyde per cent. Strong positive correlation effect between aldehyde per cet in oil and larval weight (r=0.939), cocoon weight (r=0.855), shell weight (r=0.976), ERR% (r=0.665) and fibroin per cent (r = 0.71) also support this view (Table III). In contrast sericin per cent of the silk was low at higher aldehyde in oil. A negative correlation was recorded between the aldehyde per cent and sericin (r = -0.619), indicating that higher aldehyde per cent might have detrimental effect for the produc­tion of sericin (Table III).

Table II -Larval and cocoon characters of 'muga' silkworm (A. assama) as affected by major essential oi l constitu-ents of 'Som' (P. bombycina) collected from one district of Assam

Accession number of P.bombyc ina Elants collected from LakhimEur district of Assam Characters I 2 3 4 5 6 7 8 9

Mean larval wt (g) 12.00 12.00 13.50 13.70 14.30 14.00 13.60 12.80 11.60 Rearing period(days) 30.00 29.00 29.00 28.00 27.00 27.00 28.00 29.00 32.00 Mean cocoon wt (g) 5.00 4.60 5.10 5.70 6.20 6.00 5.80 5.60 5.00 Shell wt (g) 0.52 0.55 0.60 0.63 0.67 0.65 0.63 0.57 0.47 Silk ratio (0/0 ) 10.40 11.90 11.70 11.00 10.80 10.80 10.80 10.20 9.40 ERR (0/0 ) 30.8 22.9 24.3 25.5 31.3 30.9 27.3 22.6 18.3 Sericin (0/0 ) 21.80 18.40 16.40 19.90 16.00 16.10 17.00 21.40 22.20 Fibroin (0/0 ) 78.20 81.60 83.60 80.10 84.00 83.90 83.00 78.60 77.80

646 INDIAN 1. CHEM., SEC B, MARC H 2003

Table III - Showing the corre lation co-efficient values (r) be­tween larva and cocoon qualities of muga silkworm (A. assama)

and essenti a l o il compos iti ons of P. bombycina

C harac ters r va lues

Aldehyde% and larva l WI. 0.939*

Aldehyde% and cocoon wI. 0 .855*

Aldehyde% and she ll wI. 0.976*

Aldehyde% and ER R% 0.665

Aldehyde% and seric in% -0.6 19

Aldehyde% and fibroin % 0 .7 10

Acid% and la rva l wI. -0.607

Acid% and cocoon wI. -0.270

Acid% and shell wI. -0.684

Acid% and ERR % -0.948*

Acid% and sericin % 0.62 1

Acid% and fibroin % -0.62 1

Acid content in the essential oil found to have negative effect on the yield and cocoon qua lity char­acters of 'muga' silk (Table II). Detrimental effect of acid on insect feeding was mentioned by earlier workers23

.26

. Significant negative correlation between

acid per cent in oil and larval weight (r = - 0.607),

shell weight (r= - 0 .684), ERR% (r=-0.948) and fibroin per cent (r =-0.621) confirm this findings (Table III).

Acknowledgement

The authors are grateful to North Eastern Council, Shillong, for providing financial help in conducting the investigation. We also thank Director, RRL, Jor­hat fo r providing us the facilities to conduct the ex­periment. The authors are also grateful to (Late) Dr P A Leclercq, the then Scientist of Laboratory of In­strumental Analysis, Department of Chemical Engi­neering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands for quantitative analys is of oi l samples.

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CHOUDHURY er al.: ESSENTIAL COMPOSITION OF PERSEA BOMBYCINA 647

35 Henncbcrg 0 , Weimann B & loppek , Mass spectrometry li­brary search system Masslab, version 7.4 (for Ultrix), Max­Planck-Institute fur Kohlenforschung, Mulheim/Ruh, Ger­many, 1990.

Using MassLib the following data bases were searched:

a) McLafferty F W & Stauffer, Th e Wiley/NBS Registry of Mass Spectral Dara, 4th Edn, (Wiley-Interscience, New York), 1988. b) Henneberg 0 , Weimann B & loppek W, MPI Library of Mass Spectral Dara, Max- Planck Instiut fur Kohlenfor­schung, Mulheim/Ruhr, Germany, 1994. c) Brauw M C ten Noever de, Bouwman J, Tas A C & Vos G F L, COll/pilarion of mass spectra of volarile compounds in food, TNO-HVV -CSIA, Zeist, The Netherlands, 1988. d) Posthumus M A & Teuni s C J, WAU Library of Mass Soecrral of Narural Products, Wageningen Agricultural Uni-

versity, Wageningen, The Netherlands, 1977.

e) Leclercq P A & Snijders H M, EUT Library of EL Mass Spectra, Edindhoven University of Technology, Eindhoven, The Netherland, 1998.

36 National Institute of Standards and Technology, PCV Version of the NIST/NIH mass spectral darabase, version 4.5 , U.S. Department of Commerce, Gaitherburg, MD, USA, 1994.

37 Mclafferty F W & Stauffer 0 B, Mass Specrromerry LibrGlY Search Sysrem Bench Top/PBM, version 3.0, Pali sad Co. , Newfield, 1993, Using Bench Top/PBM the followin g data­base was searched: McLafferty F W & Stauffer 0 B, The Wiley/Mbs Regi stry of Mass Spectral Data, 5th edition, Wiley and Sones, New York, 1991.

38 Ishikawa S & Hirao T, Bull Seric. Exp. Stn , 20, 1965, 21. 39 Long W H, Fm. Chern, 125, 1962, 22.

40 Nayer J K & Fraenkel, J Insect Phy, 8, 1962, 505.