FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2001; 16: 212214 DOI: 10.1002/ffj.

985

Chemical composition of the volatiles of Hemidesmus indicus R. Br.

S. Nagarajan, L. Jagan Mohan Rao and K. N. Gurudutt
Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute, Mysore 570 013, India

Received 21 July 2000 Revised 6 November 2000 Accepted 17 November 2000

ABSTRACT: The fragrant roots of Hemidesmus indicus (Indian sarsaparilla) is used in Indian native medicine and herbal tea preparations. The volatiles obtained by steam distillation (yield, 0.25%) contained 2-hydroxy-4methoxybenzaldehyde (91%) and ( ) ledol (4.5%), which are isolable in pure form, as the major constituents. The GCMS analysis of the residual oil showed the presence of over 40 minor constituents. Among them, nerolidol (1.2%), borneol (0.3%), linalyl acetate (0.2%), dihydrocarvyl acetate (0.1%), salicylaldehyde (0.1%), isocaryophyllene (0.1%), -terpinyl acetate (traces) and 1,8-cineol (traces) are important as aromatic and bio-active principles. Copyright 2001 John Wiley & Sons, Ltd. KEY WORDS: Hemidesmus indicus; Asclepiadaceae; volatile oil; GCMS analysis; 2-hydroxy-4-methoxybenzaldehyde; ledol

Introduction
Indian sarsaparilla (Hemidesmus indicus R.Br.), which belongs to the monotypic genus Hemidesmus of the family Asclepiadaceae, is a twining slender shrub found in India. Its root is woody with a diameter of 0.51.5 cm, brownish or purple in colour and of aromatic odour. The root-bark is easily separable from the inner core, which is the aromatic part. The dried roots are medicinal and well known in Indian pharmacopoeia as the drug Anantmul,1 which is a tonic and blood purier and is used for treating a variety of physiological disorders. Syrupy extract made from the roots is used as a avouring agent and in the preparation of sherbet, which is reported to have cooling properties.1 The methanol extract of the root is known to achieve neutralization of snake venom.2 While 2-hydroxy-4methoxybenzaldehyde is reported as the major constituent of its essential oil,3 no information is available on the minor constituents. Therefore, we undertook a systematic analysis of the volatile oil and report its composition here.

(Hemidesmus indicus R.Br./MGM/ LJM/ 8/ 2000) was deposited at the herbarium of the University.
Isolation of the Volatiles

The roots were cleaned with water and cut into small pieces, 23 cm long, for distillation by the following alternative methods:
Clevenger Distillation

The material (275 g) was placed in a round-bottomed ask containing 1 l distilled water, two drops of antifoam added, and subjected to Clevenger distillation for 8 h. The distillate was extracted with diethyl ether 100 ml 3 , the ethereal layers combined and dried over anhydrous Na2 SO4 . The extract was concentrated on a water bath to 10 ml and further evaporated to dryness by passing argon (yield, 0.11 g).
LikensNickerson Method

Experimental
H. indicus root

Fresh roots were collected from the herbal garden of University of Mysore, Mysore. A voucher specimen
Correspondence to: K. N. Gurudutt, Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute, Mysore 570 013, India. E-mail: cif@cscftri.ren.nic.in

A 3 l round-bottomed ask containing the material (250 g) and 1 l water was connected to the one arm of a LikensNickerson apparatus and a 250 ml roundbottomed ask containing a mixture of diethyl ether (100 ml) and petroleum ether (50 ml) to the other arm. Simultaneous distillation and extraction was carried out for 5 h. After cooling, the organic layer was separated, dried over anhydrous Na2 SO4 , concentrated in a rotary evaporator to 10 ml, and further evaporated to dryness by purging with argon (yield, 0.12 g).

Copyright 2001 John Wiley & Sons, Ltd.

VOLATILE CONSTITUENTS OF HEMIDESMUS INDICUS

213

Steam Distillation

Results and Discussion

The volatiles from fresh Hemidesmus indicus roots were isolated by Clevenger distillation, simultaneous distillation and extraction and steam distillation methods, with 0.13%, 0.16% and 0.25% yields, respectively. The last method was not only the most efcient but also convenient for large-scale extraction. From a pentane solution of the distillate, a colourless crystalline material was isolated (91%) and characterized from its m.p. (4041 C; lit.3 4143 C) and 1 H NMR spectrum6 and conrmed by its mass spectral fragmentation [m/z (% abundance): 53(28), 63(25), 81(15), 95(30), 108(25), 134(3), 151(100) and 152(20)] as 2-hydroxy4-methoxybenzaldehyde. Incidentally, the volatile oil obtained from the roots of Decalpis hamiltonii, which also belongs to the family Asclepiadaceae, contains this compound to an extent of 96%.7 Total ion chromatogram (TIC) of the residual volatile oil, generated by GCMS on a polymethylsiloxane capillary column, indicated the presence of at least 40 constituents. The second most abundant constituent, which eluted closely after nerolidol, did not give a clear mass spectrum, indicating that it was probably a mixture. It was then isolated in pure from by SiO2 column chromatography of the residual oil, followed by crystallization (yield, 4.5%; purity by GC, 95%). Its mass spectral fragmentation pattern [m/z (% abundance): 43(100), 55(32), 69(30), 81(30), 93(20), 109(28), 122(25), 161(15), 189(10) and 204(8)] and 222(3)] indicated it to be a sesquiterpene alcohol. These data, together with the physical data [m.p. 104105 C, []D 5.6 (c,0.6 in CHCl3 ); lit.8 m.p. 103104 C, []D 5.8 (c,1.5 in CHCl3 )] and 1 H NMR spectrum9 led to its identication as ( ) ledol. Rest of the minor constituents were identied by a library match of their mass spectra and determination of their retention indices on the SPB-1 column, and in some cases, on the DB wax column (see Table 1). Among them, nerolidol, borneol, ciscaryophyllene, linalyl acetate, dihydrocarvyl acetate, salicylaldehyde, isocaryophyllene, caryophyllene, linalyl acetate, dihydrocarvyl acetate, salicylaldehyde, isocaryophyllene, -terpenyl acetate and 1,8-cineol are important as avour constituents.10 The presence of dodecanoic and hexadecanoic acids in the volatile oil was probably due to the vigorous distillation conditions.

Steam was passed steadily into a suspension of the material (400 g) in 4 l water for 4 h. The steam distillate (3 l) was cooled and extracted with dichloromethane (100 ml 3). The combined dichloromethane extracts were dried over anhydrous Na2 SO4 and evaporated to dryness (yield, 0.3 g).
Isolation of the Major Constituents

The sticky mass from steam distillation was dissolved in n-pentane (10 ml) and stored overnight in a refrigerator, when needle-like crystals were formed. They were separated by decanting off the mother liquor. The latter was reprocessed to obtain two more crops of crystals. The solids were combined and recrystallized from pentane (yield, 0.27 g). The mother liquor on evaporation gave a viscous oil (28 mg, 9.3%) which was loaded onto a column of silica gel (100200 mesh, 5 g) and eluted with hexaneethyl acetate (15%) mixtures to collect ve fractions. The needle-like crystals (13 mg) that separated out from the second fraction were ltered and recrystallized from hexane. 1 H NMR spectra of the crystalline solids were recorded on a Varian EM 390 NMR spectrometer (90 MHz) using CDCl3 as solvent and tetramethylsilane as the internal standard. Optical rotation was recorded on Perkin-Elmer 243 polarimeter.
GCMS Analysis of the Volatiles

The GCMS analysis of the total volatiles was performed on a Shimadzu GC-17A coupled with a QP 5000 MS by injecting 2 l of its dichloromethane solution (100 mg in 2 ml) using a SPB-1 column (Supelco, USA, 30 m 0.32 mm, lm thickness 0.25 m) under the following conditions: oven temperature was programmed at at 50 C for 2 min, rising at 2 C/min to 250 C (5 min); injection port temperature, 250 C; detector temperature, 250 C; carrier gas, helium; ow rate 1 ml/min. GCMS analysis of the residue obtained on removal of the major crystalline constituent was performed on a DB wax column (J&W Scientic, USA; 30 m 0.25 mm, lm thickness 0.25 m) with the oven temperature programmed at 60 C for 5 min, rising at 2 C/min to 220 C, then held for 5 min; injection port temperature, 225 C; detector temperature, 250 C. Retention indices of all the constituents were determined by the Kovats method,4 using n-alkanes as standards. The volatile constituents were identied by a comparison of their Kovats indices with those reported in literature4,5 and their identication conrmed by computer matching of their mass spectral fragmentation patterns with those of compounds in the NISTMS Library.
Copyright 2001 John Wiley & Sons, Ltd.

Conclusion
For the extraction of aroma volatiles from H. indicus roots, steam distillation is the most suitable method. 2-Hydroxy-4-methoxybenzaldehyde, which can be isolated in pure crystalline form, accounts for ca. 91% of the extract. The remaining portion consists of over 40

Flavour Fragr. J. 2001; 16: 212214

214 K. N. GURUDUTT, L. JAGAN MOHAN RAO AND S. NAGARAJAN

Table 1. Chemical composition of the volatiles from the roots of Hemidesmus indicus SPB-1 No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

DB WAX KI
1636 1493

Compound
Salicylaldehyde 1,8-Cineol Camphor Pinocarveol -pinene oxide Pinocarvone Borneol 4-Terpenenol Bornyl acetate Myrtenal Methyl salicylate -Terpineol Verbenone Myrtenol Linalyl acetate Isobornyl acetate 2-Hydroxy-4-methoxybenzaldehyde Dihydrocarvyl Acetate -Terpinyl acetate -Elemene cis-Caryophyllene Isocaryophyllene -Selinene Nerolidol Ledol Dodecanoic acid Hexadecanoic acid

KI
1003 1011 1105 1109 1119 1124 1140 1152 1155 1158 1164 1167 1170 1194 1256 1296 1305 1325 1370 1427 1453 1457 1563 1629 1636 1957

MC
122 154 152 152 152 150 154 154 196 150 152 154 150 152 196 196 152 196 196 204 204 204 204 222 222 200 256

(%)
0.1 Traces 0.5 Traces Traces Traces 0.3 Traces Traces Traces Traces Traces Traces Traces 0.2 Traces 91.0 0.1 Traces Traces 0.2 0.1 0.1 1.2 4.5 0.2 0.2

1545 1735 1767 1552 2145

1589

2239 2502

Isolated by SiO2 column chromatography and characterized by physical and spectral methods.

constituents, among which ledol (4.5%) and nerolidol (1.2%), the sesquiterpene alcohols, are major. The minor but olfactorily and biologically signicant constituents are borneol, linalyl acetate, dihydrocarvyl acetate, salicylaldehyde, -terpinyl acetate and 1,8-cineol.

References
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4. Jennings W, Shibamoto T. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography. Academic Press: New York, 1980. 5. Adams RP. Identication of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press: San Diego, CA, 1989. 6. Pouchert CJ. The Aldrich Library of NMR spectra. vol. 2(2), 2nd edn. 1983; 115. 7. Nagarajan S, Jagan Mohan Rao L, Gurudutt KN. Flavour Fragr. J. 2001; 16: 27. 8. Gijsen HJM, Wijnberg JBPA, Stork GA, de Groot A. Tetrahedron 1992; 48(12): 2465. 9. Weyerstahl P, Marschall H, Thefeld WK, Surburg H. Flavour Fragr. J. 1998; 13: 295. 10. Pakrashi SC, Ghosh Dastidar PP. J. Org. Chem. 1980; 45: 4765. 11. Burdock GA. Fenarolis Handbook of Flavor Ingredients vol. 2, 3rd edn. CRC Press: Boca Raton, FL, 1995.

Copyright 2001 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2001; 16: 212214