Food Sci. Technol. Res., 7 (4), 303–306, 2001 Note

Volatile Components of the Essential Oils in Galanga (Alpinia officinarum Hance) from Vietnam

Tram Ngoc LY,1 Ryo YAMAUCHI2 and Koji KATO 2,*

1The United Graduate School of Agricultural Science, Gifu University, 1-1, Yanagido, Gifu 501-1193, Japan 2Department of Food Science, Gifu University, 1-1, Yanagido, Gifu 501-1193, Japan

Received April 11, 2001; Accepted September 27, 2001

The essential oils from fresh and dried rhizomes of galanga (Alpinia officinarum Hance) were obtained by hydro- distillation, and fractionated to the hydrocarbon and oxygenated compound fractions by silica gel column chromatog- raphy. Twenty-eight hydrocarbons and 29 oxygenated compounds were identified by gas chromatographic (Kovat’s index) and mass spectrometric data. In the fresh rhizome, the main components (over 1.0% in content) were 1,8-cine- ole (50.0%), exo-2-hydroxy-1,8-cineole acetate (11.2%), -caryophyllene (6.4%), - and -pinenes (1.7 and 2.6 %), - bisabolene (2.6%), chavicol (2.0%), limonene (2.0%), 4-terpineol (1.6%), chavicol acetate (1.2%), and methyl eugenol (1.0%). On drying the rhizome, the monoterpene fraction (including hydrocarbon and oxygenated compounds) de- creased in content, and the sesquiterpene and aromatic compound fractions increased. Major components of the oil from dried rhizome (over 2.0% in content) were -bisabolene (9.6%), 1,8-cineole (8.2%), chavicol acetate (5.9%), chavicol (5.3%), eugenyl acetate (3.7%), -farnesene (3.3%), methyl eugenol (3.3%), -caryophyllene (2.9%), -bisab- olol (2.6%), spathulenol (2.5%), farnesyl acetate (2.4%), 4-hydroxycinnamyl acetate (2.3%).

Keywords: essential oil, 1,8-cineole, galanga, Alpinia officinarum Hance, Zingiberaceae

Galanga (also called galangal, galingale or galangale) is a pun- Materials and methods gent and aromatic rhizome, which is used as a flavoring agent as Materials Fresh rhizome of galanga (Alpinia officinarum well as a traditional medicine in southeastern Asia. It is said that Hance) was purchased at a local market in Hanoi, Vietnam and two different species of galanga are present: one is the so-called botanically authenticated at the National Center for Scientific smaller (or lesser) galanga (Alpinia officinarum Hance), a pere- Research Technology, then refrigerated until analysis. The rhi- nial herb native to China; the other is the so-called greater galan- zome (moisture ca. 70%) was also sliced, dried at 45ºC, ground ga (Alpinia galanga or Languas galanga), a stemless perennial to a powder and used as a dried rhizome (moisture ca. 10%). herb of southeastern Asia (Yang & Eilerman, 1999). Both plants Reagents Authentic eugenol, -pinene, linalool, and - are members of the ginger family (Zingiberaceae) and their rhi- farnesene were obtained from Wako Pure Chemicals (Osaka). - zomes resemble ginger in shape. Greater galanga is used in the Caryophyllene and 1,8-cineole were obtained from Tokyo preparation of meat dishes and curries in its natural form or for Chemical Industries (Tokyo). Authentic n-paraffins with the flavoring foods as dried powder, whereas smaller galanga is used number of carbon atom from 9 to 20 were purchased from Sig- mainly as a drug (Burkill, 1966; Do, 1995). In recent years, ma (St. Louis, MO), and were used as standard compounds for many biological activities of galanga have been reported: antitu- calculation of Kovat’s index. mor (Itokawa et al., 1987; Kondo et al., 1993), antiulcer (Mitsui Preparation of the volatile oil One kilogram of the fresh et al., 1976), antibacterial and antifungal (Janssen & Scheffer, rhizome which corresponds to 400 g of the dried rhizome was 1985; Ray & Majumdar, 1975, 1976) properties. The essential pulverized with distilled water and submitted to hydro-distilla- oil in the rhizome of A. galanga (greater galanga) has been re- tion in a modified Clevenger apparatus for 8 h. The dried rhi- ported by many researchers (Scheffer et al., 1981; De Pooter et zome was treated in the same way. The oil layer obtained was al., 1985; Charles et al., 1992; Mori et al., 1995), while the oil in separated and dried over anhydrous sodium sulfate. Fresh and the rhizome of A. officinarum (smaller galanga) has been studied dried oil yields, on a dry weight basis, were 4 mg/1000 mg and only by Lawrence et al. (1969). Since they could not effect com- 2.7 mg/1000 mg, respectively. ponent separation, constituents were analysed as a mixture; we Separation of hydrocarbon and oxygenated hydrocarbon therefore attempted to identify individual constituents. Since fractions in the oil The essential oil (100 l) thus obtained was both the fresh and the sliced and dried rhizomes are used in Viet- dissolved in a small amount of pentane and submitted to a silica nam, we also compared the oil from the fresh rhizome with that gel 60 micro column (40–60 m) to separate the hydrocarbon from the dried rhizome. and oxygenated compound fractions (Mastelic et al., 1998). The column was successively eluted with pentane (20 ml), a mixture *To whom correspondence should be addressed. of pentane and diethyl ether (1:1, v/v; 15 ml) and diethyl ether E-mail: [email protected] (15 ml). The eluate was collected in 4 ml fractions and each frac- 304 T.N. LY et al. tion was examined by thin layer chromatography (silica gel 60 chromatogram was tentatively identified by its Kovat’s index

TLC plate, 0.25 mm thickness; Merck, Darmstadt, Germany). (KI), which was calculated using of n-paraffins (C9–C20) as refer- Diethyl ether/hexane, (4:1, v/v) was used for the solvent and the ences (Majlat et al., 1974) and compared to the published index compounds were detected with vanillin in H2SO4. The hydrocar- (Breheret et al., 1997; Bylaite et al., 1998; Davies, 1990; De bon fraction (ca. 0.95 of Rf value) was eluted with pentane, and Pooter et al., 1985; Jantan et al., 1998; Jirovetz et al., 1998). the oxygenated compound fraction (~ca. 0.52 of Rf value) with Gas chromatography-mass spectrometry (GC-MS) Mass both solvents, a mixture of pentane/diethyl ether, and diethyl spectrometric analysis of the compounds was performed with a ether. Shimadzu GC-MS QP5000 gas chromatograph-mass spectrome- Gas chromatography (GC) Gas chromatographic analy- ter fitted with a column (0.25 mm i.d¥30 m, film thickness 0.25 ses of the hydrocarbon and oxygenated compound fractions were m) of methyl silicon DB-1. Column temperature was pro- carried out on a Shimadzu GC-18A gas chromatograph (Shimad- grammed from 80ºC to 250ºC at 5ºC/min, and the spectrum was zu Co., Kyoto) equipped with a flame ionization detector (FID). recorded at an ionizing potential of 70 eV. The spectrum was The column was methyl-silicon CBP-1 capillary (0.25 mm compared to confirm the compound with an authentic one from i.d¥25 m, film thickness 0.25 m), and programmed first at 50ºC the library of the National Institute of Standards and Technology for 2 min, then raised to 210ºC at a speed of 3ºC /min, with a gas (NIST) for the GC-MS. flow-rate of 1.0 ml/min of nitrogen (split ratio of 1/50). Peak areas were measured with a SIC Chromatocorder 21 integrator Results and Discussion (System Instruments Co., Tokyo). Each peak (compound) on the Volatile hydrocarbons from the fresh and dried rhizo- mes Gas chromatograms of hydrocarbon fractions from the fresh and dried rhizomes are shown in Fig. 1-A and -B. Twenty- one hydrocarbon compounds (Table 1) were identified from KI and MS data, and seven compounds from MS data. Peaks in chromatograms Fig. 1-A and -B could be divided into three groups: A (peaks 1–10), B (peaks 11–27), and C (peaks 28–31) corresponding to monoterpene, sesquiterpene and other (satur- ated and unsaturated) hydrocarbon groups, respectively. Of the

Table 1. Vo latile compounds identified in the hydrocarbon fraction. Fresh Dried Peak Kovat’s Published Compound rhizome rhizome No indices indices (%)a) (%)a) 1 -Thujene 922 924c) 0.1 n.d. 2 -Pinene 929 928c) 1.7 n.d. 3 Camphene 941 940c) 0.1 n.d. 4 -Pinene 969 967c) 2.6 n.d. 5 Myrcene 982 981c) 0.5 n.d. 6 -Terpinene 1006 1006c) 0.1 n.d. 7 p-Cymene 1009 1008c) 0.1 n.d. 8Limonene 1019 1020c) 2.0 n.d. 9 -Terpinene 1048 1047c) 0.4 n.d. 10 Terpinolene 1076 1077c) 0.1 n.d. 11 -Copaene 1369 1369d) n.d. 0.3 12 -Elemene 1382 1381d) n.d. 0.6 13 -Caryophyllene 1410 1403e) 6.4 2.9 14 -Santalene 1412 1412d) n.d. 0.2 15 -Bergamotene 1429 1436d) 1.3 4.7 16 Unknown 1443 — 0.3 0.3 17 -Farnesene 1446 1448d) 0.1 0.8 18 Unknown 1447 — 0.1 0.3 19 Patchouleneb) 1466 — n.d. 0.2 20 -Caryophylleneb) 1468 — 0.5 1.4 21 -Curcumene 1475 1475d) n.d. 0.4 22 Germacrene 1483 1482d) 0.2 1.5 23 Zingibreneb) 1489 — 0.2 1.3 24 -Bisabolene 1497 1496d) 2.6 9.6 25 Pentadecane 1501 1500e) 2.4 1.9 26 Unknown 1510 — 1.2 8.9 27 -Farnesene 1518 1501d) 0.8 3.3 28 3-Hexadecyneb) 1663 — 0.5 1.5 29 8-Heptadeceneb) 1672 — 0.6 1.2 30 3-Octadeceneb) 1684 — n.d. 0.4 31 Heptadecaneb) 1700 — 0.1 0.3 Total (%) 25.0 42.0 n.d: not detected. a)Relative percentage based on peak area on the gas chro- Fig. 1. Gas chromatograms of the hydrocarbon fractions obtained from matogram (FID), refers to the total essential oil. b)Tentative identification fresh (A) and dried (B) rhizomes. The GC conditions were described in based upon mass spectrum only. c)Bylaite et al., 1998; d)Davies, 1990; e)De Materials and Methods. Pooter et al., 1985. Essential Oils from the Rhizomes of Alpinia officinarum Hance 305 peaks (compounds) in sesquiterpene group B, nos. 16, 18 and 26 the scale of analysis. This is the first time to detect, from the could not be identified. The major hydrocarbons in the oil from smaller galanga A. officinarum (Lawrence et al., 1969) as well as the fresh rhizome shown with their relative percentage based on the greater galanga A. galanga (Scheffer et al., 1981; De Pooter GC peak area were -pinene (1.7%), -pinene (2.6%) and et al., 1985; Charles et al., 1992; Mori et al., 1995), the sesquiter- limonene (2.0%) as monoterpene, and -caryophylene (6.4%), penes patchoulene, germacrene, zingibrene and -farnesene and -bergamotene (1.3%), -bisabolene (2.6%) as sesquiterpene four other hydrocarbons (group C). and pentadecane (2.4%). Lawrence et al. (1969) also reported Volatile oxygenated compounds from the fresh and dried that the three compounds mentioned above were the major com- rhizomes Gas chromatograms of oxygenated compound hydro- ponents of the monoterpenes in the fresh rhizomes. It is remark- carbon fractions from the fresh and dried rhizomes are shown in able that these three compounds are also major components in Fig. 2-A and -B. The twenty-nine oxygenated compounds (Table the monoterpene fraction from the greater galanga Alpinia galan- 2) were identified in the same way as the hydrocarbon fraction. ga (Scheffer et al., 1981). The compounds in the monoterpene These compounds could not be grouped by KI as the hydrocar- fraction including the major compounds mentioned above were bon fraction, however, they were identifiable in groups of monot- found to disappear when the rhizome was dried. A similar com- erpenes and aromatic compounds (peaks 1–22), and of parison had been reported for the greater galanga (De Pooter et sesquiterpenes and aromatic compounds (peaks 23–37). Peaks al., 1985). The major compounds in the sesquiterpene fraction (compounds) 21, 22, 24, 30, 31, 34, 35, and 37 could not be were also major components in the oil from the dried sample. It identified even by MS data. But from their KI, compounds 21 is hard to explain why such a compound as -caryophyllene de- and 22 (peaks 21, 22) were presumed to be aromatic compounds, creased, while most compounds increased in their percentage of and compounds 24, 30, 31, 34, and 35 sesquiterpenes or aroma- content by drying. Five sesquiterpenes (peaks 11, 12, 14, 19 and 21) and another hydrocarbon (peak 30) were detected for the dried sample, but not for the fresh one. This could be ascribed to

Table 2. Vo latile compounds identified in the oxygenated compound frac- tion. Pub- Fresh Dried Peak Kovat’s Compound lished rhizome rhizome No indices indices (%)a) (%)a) 11,8-Cineole 1028 1025d) 50.0 8.2 2Linalool 1084 1086d) n.d. 0.2 3Verbenolb) 1105 — 0.2 0.4 42,8-Menthadien-1-ol 1113 1120d) 0.2 0.3 5Terpinen-1-olb) 1145 — 0.6 0.7 64-Terpineol 1160 1160d) 1.6 1.4 7 p-Cymen-8-ol 1167 1167d) 0.2 0.3 8 -Terpineol 1172 1178d) 1.2 0.9 9Piperitolb) 1179 — 0.3 0.4 10 Trans-Carveol 1196 1200d) 0.2 0.2 11 Chavicol 1237 1243e) 2.0 5.3 12 Bornyl acetate 1266 1264e) 0.1 0.3 13 4-Thujen-2-yl acetateb) 1277 — 0.3 n.d. 14 Chavicol acetate 1315 1309e) 1.2 5.9 15 Exo-2-Hydroxy-1,8-cineole 1323 — 11.2 0.4 acetateb) 16 Eugenolb) 1328 — 0.2 1.0 17 Carveol acetateb) 1340 — n.d. 0.2 18 Citronellyl acetate 1341 1335d) 0.1 n.d. 19 Geranyl acetate 1361 1363d) 0.4 1.2 20 Methyl eugenol 1371 1368e) 1.0 3.3 21 Unknown 1373 — 0.2 n.d. 22 Unknown 1410 — n.d. 0.3 23 Eugenyl acetate 1484 1483e) 0.3 3.7 24 Unknown 1511 — 1.1 4.4 25 trans-Nerolidol 1561 1553c) 0.1 0.4 26 Caryophyllene oxide 1582 1575d) 0.1 1.3 27 Spathulenolb) 1615 — 0.7 2.5 28 Guaiolb) 1632 — 0.2 1.2 29 -Cadinolb) 1640 — n.d. 0.4 30 Unknown 1649 — n.d. 0.4 31 Unknown 1656 — n.d. 0.2 32 4-Hydroxycinnamyl acetate 1666 — 0.1 2.3 33 -Bisabolol 1670 1666d) 0.3 2.6 34 Unknown 1681 — n.d. 0.3 35 Unknown 1794 — 0.6 2.9 36 Farnesyl acetateb) 1815 — 0.2 2.4 37 Unknown 1949 — 0.1 2.1 Total (%) 75.0 58.0 n.d: not detected. a)Relative percentage based on peak area on the gas chro- Fig. 2. Gas chromatograms of the oxygenated compound fractions ob- matogram (FID), refers to the total essential oil. b)Tentative identification tained from fresh (A) and dried (B) rhizomes. The GC conditions were de- based upon mass spectrum only. c)Bylaite et al., 1998; d)Davies, 1990; e)De scribed in Materials and Methods. Pooter et al., 1985. 306 T.N. LY et al.

Table 3. Percentage aroma compounds of essential oil from the rhizome References of Alpinia officinarum Hance. Breheret, S., Talou, T., Rapior. S. and Marie Bessiere, J. (1997). Compound fresh rhizome (%)a) dried rhizome (%)a) Monoterpenes in the aromas of fresh wild mushrooms (Basidio- Monoterpenes mycetes). J. Agric. Food Chem., 45, 831–836. Hydrocarbons 7.7 n.d. Burkill, I.H. (1966). In “Dictionary of the Economic Products of the Oxygenated compounds 65.9 13.5 Malay Peninsula,” Vol. 2, Ministry of Agriculture and Co-opera- Sesquiterpenes tives, Kuala Lumpur (Malaysia), pp 1327–1332. Hydrocarbons 12.1 27.2 Bylaite, E., Venskutonis, R.P. and Roozen, J.P. (1998). Influence of Oxygenated compounds 2.3 12.4 harvesting time on the composition of volatile component in differ- Others ent anatomical parts of Lovage (Levisticum officinale. Koch.). J. Hydrocarbons 3.6 5.3 Oxygenated compounds 4.8 21.5 Agric. Food Chem., 46, 3735–3740. Unknown Charles, D.J., Simon, J.E. and Singh, N.K. (1992). The essential oil of Hydrocarbons 1.6 9.5 Alpinia galanga Willd. J. Essent. Oil Res., 4, 81–82. Oxygenated compounds 2.0 10.6 Chen, S-H., Huang, T-C., Ho, C-T. and Tsai, P-J. (1998). Extraction, n.d.: not detected. a)Relative percentage based on peak areas on the gas analysis and study on the volatiles in Rosells tea. J. Agric. Food chromatogram (FID), refers to the total essential oil. Chem., 46, 1101–1105. Davies, N.W. (1990). Gas Chromatographic retention indices of mono- terpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J. Chromatogr., 503, 1–24. De Pooter, H.L., Nor Omar, M., Coolsaet, A.B. and Schamp, N.M. tic compounds. The major oxygenated compounds in the oil (1985). The essential oil of greater galangal (Alpinia galanga) from from the fresh (and dried) rhizome were 1,8-cineole [50.0 (and Malaysia. Phytochemistry, 24, 93–96. 8.2%)], which is also a major component of the oil from greater Do, T.L (1995). Medicine plants and drugs for digestive system. In “Medicine Plants of Vietnam,” Science and Technique, Hanoi (Viet- galanga (Scheffer et al., 1981; De Pooter et al., 1985; Mori et al., nam), pp 498. 1995). This is not unexpected as both plants belong to the same Itokawa, H., Morita, H., Sumitomo, T., Totsuka, N. and Takeya, K. Alpinia genus. Lawrence et al. (1969) also reported that 1,8-cine- (1987). Antitumour principles from Alpinia galanga. Planta Med- ole was a major component in the oxygenated compounds. Cine- ica, 53, 32–33. ole and the unusual exo-2-hydroxy-1,8-cineole acetate, one of Jantan, I., Ahmad, A.S., Bakar, S.A.A., Ahmad, A.R., Trockenbrodt, M. and Char, C.V. (1998). Constituents of the essential oil of the major components (cf. Table 2) showed a significant decrease Baeckea frutescens L. from Malaysia. Flavour Frag. J., 13, 245– in concentration during drying of the rhizome. Thirteen of the 29 247. compounds identified were detected in both galanga including Janssen, A.M. and Scheffer, J.J.C. (1985). Acetoxychavicol acetate, an greater one for the first time. These were verbenol, 2,8-methadi- antifungal component of Alpinia galanga. Planta Medica, 51, 507– en-1-ol, terpinen-1-ol, p-cymen-8-ol, piperitol, 4-thujen-2-yl 511. Jirovetz, L., Buchbauer, G., Puschmann, C., Shafi, M.P. and Geetha- acetate, carveol acetate, trans-nerolidol, spathulenol, guaiol, - Nambiar, M.K. (1998). Analysis of the essential oils of the leaves of cadinol, 4-hydroxycinnamyl acetate, and farnesyl acetate. the medicinal plants Vitex negundo var. negundo and Vitex negundo 1’-Acetoxychavicol acetate (ACA) was not found in the oil var. purpurscens from India. Acta. Pharm., 48, 179–186. from Alpinia officinarum Hance, although it is known to be one Kondo, A., Ohigashi, H., Murakami, A., Suratwadee, J. and Koshi- mizu, K. (1993). 1’-Acetoxychavicol acetate as a potent inhibitor of of the major components of the oil from Alpinia galanga (Mori tumor promoter-induced Epstein-Barr virus activation from Languas et al., 1995). Since both oils were obtained in a similar way, the galanga, a traditional Thai condiment. Biosci. Biotechnol. Biochem., difference could be attributed to a difference in the species, A. 57, 1344–1345. galanga and A. officinarum. This may therefore, be an effective Lawrence, B.M., Hogg, J.W. and Terhune, S.J. (1969). Essential oils way to distinguish lesser galanga from greater galanga. and their constituents. Part 2. The oil of Alpinia officinarum Hance. Perfum. Essent. Oil Rec., 60, 88–96. Quantitative change of the oil component during the drying Majlat, P., Erdos, Z. and Takacs, J. (1974). Calculation of application process of the rhizome After identification of the compounds, of retention indices in programmed temperature gas chromatogra- their percentages in the essential oil were calculated from the phy. J. Chromatogr., 91, 89–103. peak areas of the gas chromatogram and are summarized in Mastelic, J., Milos, M., Kustrak, D. and Radonic, A. (1998). The Table 3. The percentage of hydrocarbon fraction in the oil from essential oil and glucosidically bound volatile compounds of Cala- mintha nepeta (L.) Savi. Croatica Chemica Acta., 71, 147–154. the fresh rhizome was 25.0% [=7.7% (monoterpene)+12.1% Mitsui, S., Kobayashi, S., Nagahori, H. and Ogiso, A. (1976). Constit- (sesquiterpene)+3.6% (others)+1.6% (unknown)]. This value for uents from seeds of Alpinia galanga Willd. and their anti-ulcer the dried sample was similarly calculated at 42.0%. For the oxy- activities. Chem. Pharm. Bull., 24, 2377–2382. genated compound fraction, these values were 75.0% (fresh rhi- Mori, H., Kubota, K. and Kobayashi, A. (1995). Potent aroma compo- nents of rhizomes from Alpinia galanga Willd. L. Nippon Shokuhin zome) and 58.0% (dried). It is interesting that the ratio of hy- Kagaku Kogaku Kaishi, 42, 989–995 (in Japanese). drocarbons to oxygenated constituents (0.72) for the dried sam- Ray, P.G. and Majumdar, S.K. (1975). New antifungal substance from ple is almost double that for the fresh rhizome (0.33) in spite of Alpinia officinarum Hance. Ind. J. Exp. Biol., 13, 409. the disappearance of monoterpene hydrocarbons (7.7%, group Ray, P.G. and Majumdar, S.K. (1976). Antifungal flavonoid from A) from the dried rhizome. This could be of aid in evaluating Alpinia officinarum Hance. Ind. J. Exp. Biol., 14, 712–714. Scheffer, J.J.C., Gani, A. and Baerheim-Svendven, A. (1981). Mono- which rhizome can be used as an aromatic material, fresh or terpenes in the essential rhizome oil of Alpinia galanga (L.) Willd. dried. Sci. Pharm., 49, 337–346. Yang, X. and Eilerman, R.G. (1999). Pungent principal of Alpinia Acknowledgement The authors are grateful to Dr. Shingo Kawai of Gifu galangal (L.) Swartz and its applications. J. Agric. Food Chem., 47, University for the GC-MS analysis of the samples. 1657–1662.