Essential oil from the fruits of Amomum cannicarpum (Wight) Bentham ex Baker (Zingiberaceae), was hydrodistilled and characterized by gas chromatography-mass spectrometry (GC-MS). The major constituents in the oil were betha-pinene (14.00%), elemol (10.45%) and alpha-cadinol (8.50%). Thirty-three (91.48%) out of forty-one constituents were identified by GC-MS and subsequent data analysis. The antimicrobial activity of the oil against Gram-positive and Gram-negative bacteria and some fungi were determined by the disc diffusion assay. The oil showed good antibacterial activity against Salmonella typhi, Pseudomonas aeruginosa and Proteus vulgaris and very good antifungal activity against Candida albicans and C. glabrata.Eterično ulje iz plodova biljke Amomum cannicarpum (Wight) Bentham ex Baker (Zingiberaceae) dobiveno je destilacijom vodenom parom, a zatim je pomoću plinske kromatografije i masene spektrometrije (GC-MS) određen njegov sastav. Najvažniji sastojci u ulju bili su -pinen (14.00%), elemol (10.45%) i -kadinol (8.50%). Ukupno je od 41 sastojka identificirano 33 sastojka (91.48%). Disk-difuzijskom metodom određeno je antimikrobno djelovanje ulja na Gram-pozitivne i Gram-negativne bakterije i neke gljivice. Dosta snažno antimikrobno djelovanje zapaženo je na bakterije Salmonella typhi, Pseudomonas aeruginosa i Proteus vulgaris ten a gljivice Candida albicans i C. glabrata

DAN MATHEW

GEORGE VARUGHESE

RENJU KRISHNA VALSAMMA

SABULAL BABY

SUKUMARAN PRADEEP NEDIYAMPARAMBU

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Zingiberaceae is one of the essential oil bearing plant families. The genus Amomum inthis family has over ninety species distributed in Africa, tropical Asia, Australia and thePacific Islands (1, 2). These plants are mostly terrestrial, rhizomatous herbs. Amomumseeds are used as spices and their plant parts are used in traditional medicine for curingtoothache, dysentery, diarrhoea, rheumatism, vomiting, dyspepsia and lung diseases (3,4). Amomum subulatum or 'large cardamom' distributed in the eastern Himalayas is themost investigated Amomum species. The chemical composition of essential oils from theseeds of A. subulatum have been studied by various groups and the major constituent,1,8-cineole, has been found to vary from 61 to 86% (5, 6). Also, oils from different partsof other Amomum species such as A. thyrsoideum (7), A. longiligulare (7), A. villosum (8), A.473Acta Pharm. 56 (2006) 473–480 Short communicationComposition and antimicrobial activity of essential oilfrom the fruits of Amomum cannicarpumBABY SABULAL1MATHEW DAN2NEDIYAMPARAMBU SUKUMARAN PRADEEP3RENJU KRISHNA VALSAMMA3VARUGHESE GEORGE1*1 Phytochemistry Division, TropicalBotanic Garden and Research InstitutePacha-Palode, Thiruvananthapuram--695 562, Kerala, India2 Horticulture and Garden DevelopmentDivision, Tropical Botanic Garden andResearch Institute, Pacha-PalodeThiruvananthapuram-695 562Kerala, India3 Microbiology Division, Tropical BotanicGarden and Research InstitutePacha-Palode, Thiruvananthapuram--695 562, Kerala, IndiaAccepted October 5, 2006Essential oil from the fruits of Amomum cannicarpum(Wight) Bentham ex Baker (Zingiberaceae) was hydrodis-tilled and characterized by gas chromatography-massspectrometry (GC-MS). Major constituents of the oilwere -pinene (14.00%), elemol (10.45%) and -cadinol(8.50%). Thirty-three (91.48%) out of forty-one constitu-ents were identified by GC-MS and subsequent dataanalysis. Antimicrobial activity of the oil against Gram--positive and Gram-negative bacteria and some fungi,was determined by the disc diffusion assay. The oilshowed good antibacterial activity against Salmonellatyphi, Pseudomonas aeruginosa and Proteus vulgaris andvery good antifungal activity against Candida albicansand C. glabrata.Keywords: Amomum cannicarpum (Zingiberaceae), essentialoil, GC-MS, -pinene, elemol, -cadinol, antibacterial ac-tivity, antifungal activity* Correspondence, e-mail: sabulal@gmail.commuricarpum (9), A. kwangsiense (10), A. schmidtti (11), A. xanthioides (12), A. tsao-ko (12), A.linguiforme (4, 13) and A. testaceum (14) were studied previously.We have recently reported the chemical composition and antimicrobial activities ofthe rhizome and leaf oils of Amomum cannicarpum (15, 16). We have also studied thechemical compositions of the leaf oil of A. muricatum from the Western Ghats region inSouth India (17). -pinene is the major constituent of these oils and this is in agreementwith previous reports on other Amomum oils (7, 8, 10–14).A. cannicarpum is a stout, gregarious herb growing up to 3.5 m. It is endemic toSouth India and is fairly common in its evergreen forests (1). This is the first report onthe chemical composition and antimicrobial activity of the essential oil from the fruits ofA. cannicarpum.EXPERIMENTALPlant collection and oil isolationMature fruits of A. cannicarpum were collected from Cheruthoni, Idukki district inKerala (India) in February 2005. A voucher specimen was deposited in the Herbarium ofTropical Botanic Garden and Research Institute (Pacha-Palode, Thiruvananthapuram,India). Hydrodistillation of the fresh fruits (959 g) using a Clevenger-type apparatus forthree hours yielded 0.8 mL of a colourless, pleasant smelling oil.Gas chromatography-mass spectrometry (GC-MS) analysisGC-MS analysis of the essential oil was carried out by splitless injection of 1.0 L ofthe oil on a Hewlett Packard 6890 gas chromatograph (Hewlett Packard, USA) fittedwith an HP-5 MS cross-linked 5% PH ME siloxane, 30 m  0.32 mm  0.25 m capillarycolumn, coupled with a model 5973 mass detector. GC-MS operation conditions: injectortemperature 220 °C, transfer line 240 °C, oven temperature programme 60–243 °C (3 °Cmin–1), carrier gas He 1.4 mL min–1, mass spectra: electron impact (EI+) mode 70 eV, ionsource temperature 240 °C. Individual components were identified by Wiley 275.L data-base matching and by comparison of mass spectra with published data (18). Relativepercentages of individual components in the oil (Table I) were calculated from theirpeak areas in the total ion chromatogram obtained from the GC-MS experiment. Rela-tive retention indices (RRI) of constituents in Table I were determined on the above PHME siloxane capillary column using C5-C30 straight chain alkanes (Aldrich, India) asstandards.Bacterial and fungal strainsGram-positive bacteria, Staphylococcus aureus (MTCC 96), Bacillus cereus (MTCC 430),B. subtilis (MTCC 441), Gram-negative bacteria, Serratia marcescens (MTCC 97), Pseudo-monas fluorescens (MTCC 103), P. aeruginosa (MTCC 741), Klebsiella pneumoniae (MTCC109), Proteus vulgaris (MTCC 426), Escherichia coli (MTCC 443), Salmonella typhi (MTCC474B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.733) and the fungi, Candida albicans (MTCC 227), C. albicans (MTCC 1637), C. albicans(MTCC 3017), C. glabrata (MTCC 3019) were obtained as the Microbial Type Culture Col-lection (MTCC) from the Institute of Microbial Technology, Chandigarh (India).475B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.Table I. Chemical composition of the essential oil from the fruits of Amomumcannicarpum determined by gas chromatography-mass spectrometryConstituent RRIa Concentration (%)-pinene 972 14.00p-cymene 1022 3.88Limonene 1025 3.411,8-Cineole 1027 0.48trans-linalool oxide 1069 0.43cis-linalool oxide 1086 0.70Linalool 1102 3.60-pinene oxide 1105 0.67-campholenal 1123 0.70trans-pinocarveol 1135 3.04Pinocarvone 1158 1.19Cryptone 1184 3.07-terpeniol 1190 1.51Myrtenal 1193 2.07Verbenone 1206 0.63Cuminal 1235 1.01Ascaridole 1252 1.33-amorphene 1472 0.5610,11-Epoxy calamenene 1485 0.93-bisabolene 1504 1.22Elemol 1548 10.45E-nerolidol 1564 1.41Widdrol 1592 2.04Humulene epoxide II 1598 0.721,10-di-epi-cubenol 1605 0.791-epi-cubenol 1619 1.13-eudesmol 1625 3.98epi--muurolol 1637 4.74-eudesmol 1642 5.92-cadinol 1651 8.50(z)--trans-bergamotol 1714 3.20Oplopanone 1732 2.5014-Hydroxy--muurolene 1781 1.66Total 91.48a Relative retention indexAntibacterial and antifungal activityAntibacterial activity of essential oil from the fruits of A. cannicarpum was testedagainst the above-mentioned Gram-positive and Gram-negative bacteria by the disc agardiffusion method (19, 20). The bacteria were grown on Mueller-Hinton agar media (pH7.3). Agar media were poured into the plates to a uniform depth of 5 mm and allowed tosolidify. The microbial suspensions at 5  106 cfu mL–1 were streaked over the surface ofmedia using a sterile cotton swab to ensure confluent growth of the organism. The discsused were Whatman No. 1 papers, 6 mm in diameter. 10-L aliquots of the oil 33.3% (V/V)in DMSO, spotted on filter paper discs, which were then aseptically applied to the surfaceof agar plates at well-spaced intervals. The plates were incubated at 37 °C for 24 hoursand the observed growth inhibition zones, including disc diameters were measured. Con-trol discs impregnated with 10 L of the solvent DMSO and streptomycin (2 g per disc),reference for bacteria, were used alongside the test discs in each experiment (Table II).The above-listed fungi were obtained as MTCC, cultured in modified Sabouraud’sagar and suspensions at 5 x 106 cfu mL–1 were used. 10 µL of oil (33.3%, V/V) in DMSO,was impregnated on discs amplitude with 10 L of DMSO and fluconazole (2 g perdisc) used as controls (Table II).476B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.Table II. Antimicrobial and antifungal activity of the essential oil from the fruitsof Amomum cannicarpum determined by the disc diffusion assayBacteria/fungiDiameter of inhibition zone (mm)aOilb ControlcGram-positive bacteriaStaphylococcus aureus 11 25B. cereus 0 17Bacillus subtilis 0 11Gram-negative bacteriaSerratia marcescens 7 16Pseudomonas fluorescens 10.3 20P. aeruginosa 12 11Klebsiella pneumoniae 0 15Proteus vulgaris 11 15Escherichia coli 0 17Salmonella typhi 10 0FungiCandida albicans 11.7 10C. albicans 11 7C. albicans 11.7 25C. glabrata 11 9a Experiments were done in triplicate and the results are mean values.b 10 µL of oil (33.3%, V/V) in DMSO.c Control – 10 µL streptomycin per disc for bacteria, and 10 µL fluco-nazole (2 g per disc) for fungi.RESULTS AND DISCUSSIONThirty-three constituents out of forty-one, containing 91.48% of the essential oil fromthe fruits of A. cannicarpum, were identified by GC-MS (Table I). The gas chromatogramof the oil on a HP-5 MS capillary column is shown in Fig. 1. The percentage of oxygen-ated sesquiterpenes in the oil was 47.97%, followed by monoterpene hydrocarbons(21.29%) and oxygenated monoterpenes (20.43%). The percentage of sesquiterpene hy-drocarbons in the oil was relatively low (1.78%). The major constituents of the oil were-pinene (14.00%), elemol (10.45%) and -cadinol (8.50%). Similar studies on essentialoils from the rhizomes (15) and leaves (16) of A. cannicarpum and other Amomum species(7, 8, 10–14) showed that -pinene is one of the major constituents in Amomum oils.477B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.Fig. 1. Gas chromatogram of the essen-tial oil from the fruits of Amomumcannicarpum on a HP-5 MS cross-lin-ked 5% PH ME siloxane (30 m x 0.32mm x 0.25 m) capillary column.Antibacterial and antifungal activities of the essential oil were tested by the disc dif-fusion assay. The oil at a 33.3% (V/V) concentration in dimethyl sulfoxide showed goodactivity against the Gram-negative bacteria Salmonella typhi, Pseudomonas aeruginosa andProteus vulgaris in comparison with streptomycin at 2 g per disc and against the fungiCandida albicans and C. glabrata, in comparison with the antifungal control, fluconazoleat 2 g per disc (Table II). No activity was observed against Bacillus subtilis, B. cereus,Klebsiella pneumoniae and Escherichia coli.CONCLUSIONSChemical characterization and antimicrobial screening studies on plant-based es-sential oils could lead to a discovery of new natural antimicrobials. The present study isthe first report on chemical characterization of the essential oil from the fruits of A. can-nicarpum. The oil showed promising antimicrobial activity against, Salmonella typhi,Pseudomonas aeruginosa and Proteus vulgaris and the fungi Candida albicans and C. gla-brata.Acknowledgements. – We express our sincere thanks to the Director, TBGRI, for laboratory facili-ties and to the Director (Research), Textiles Committee, Kannur, Kerala, India for providing the GC--MS facility.REFERENCES1. S. K. Jain and V. Prakash, Zingiberaceae in India, Rheedea 5 (1995) 154–169.2. D. J. Mabberley, The Plant-Book: A Portable Dictionary of the Higher Plants, Cambridge UniversityPress, Cambridge 1990, p. 25.3. The Wealth of India, Raw Materials (Ed. B. L. Manjunath), Council of Scientific and Industrial Re-search, Delhi 1948, Vol. 1, p. 68.4. S. Dutta, R. Ahmed and M. G. Pathak, Essential oil composition of Amomum linguiforme Benth.from north-east India, Indian Perfum. 44 (2000) 11–13.5. N. Gurudutt, J. P. Naik and P. Srinivas, Volatile constituents of large cardamom (Amomum su-bulatum Roxb.), Flav. Fragr. J. 11 (1996) 7–9.6. P. K. Rout, D. Sahoo, K. S. Jena and Y. R. Rao, Analysis of the oil of large cadamom (Amomumsubulatum Roxb.) growing in Sikkim, J. Essent. Oil Res. 15 (2003) 265–266.7. T. T. Nguyen, V. T. Nguyen, X. Q. Cao, X. T. Nguyen and T. Q. Tran, Preliminary studies onAmomum species in Ninh Thuan Province, Tap Chi Duoc Hoc 11 (2001) 10–13; ref. Chem. Abstr.137 (2002) 388996r.8. F. Pu, J-Q. Cu and Z-J. Zhang, The essential oil of Amomum villosum Lour., J. Essent. Oil Res. 1(1989) 197–198.9. L-F. Zhu, Y-H. Li, B-L. Li, B-Y. Lu and N-H. Xia, Aromatic Plants and Essential Oil Constituents,Peace Book Co., Hong Kong 1993, p. 294.10. L-F. Zhu, Y-H. Li, B-L. Li, B-Y. Lu and W-L. Zhane, Aromatic Plants and Essential Oil Constituents,Supplement 1, Peace Book Co., Hong Kong 1995, p. p. 164–165.478B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.11. N. X. Dung, D. L. Phuong and P. A. Leclercq, Trans-p-(1-butenyl)-anisole: the main componentin the leaf, stem and root oils of Amomum schmidtti from Vietnam, J. Essent. Oil Res. 4 (1992)239–242.12. H. Okugawa, M. Moriyasu, K. Saiki, A. Kato, K. Matsumoto, A. Fujioka and Y. Hashimoto, Onflavor components in seeds of Amomum xanthioides, A. tsao-ko and Alpinia katsumadai, Shoyaku-gaku Zasshi 41 (1987) 108–115.13. A. K. Hazarika and S. C. Nath, Methyl chavicol – the major component of the rhizome oil ofAmomum linguiforme Benth., J. Essent. Oil Res. 7 (1995) 325–326.14. M. Sirat, L. F. Hong and S. H. Khaw, Chemical composition of the essential oil of the fruits ofAmomum testaceum Ridl., J. Essent. Oil Res. 13 (2001) 86–87.15. J. Mathew, B. Sabulal, M. Dan, S. Shiburaj and V. George, Chemical composition and antimi-crobial activity of essential oil from the rhizomes of Amomum cannicarpum (Wight) Bentham exBaker, Fitoterapia 77 (2006) 392–394.16. J. Mathew, B. Sabulal, M. Dan, S. Shiburaj and V. George, Chemical composition and antimi-crobial activity of the leaf oil of Amomum cannicarpum (Wight) Bentham ex Baker, J. Essent. OilRes. 18 (2006) 35–37.17. B. Sabulal, M. Dan and V. George, Volatile constituents from the leaf oil of Amomum muricatumBeddome, J. Essent. Oil Res. 18 (2006) 35–37.18. R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Quadrupole MassSpectroscopy, Allured Publishing Corporation, Carol Stream 2001.19. D. A. V. Berghe and A. J. Vlietinck, Screening Methods for Antibacterial and Antiviral Agents fromHigher Plants, in Methods in Plant Biochemistry (Eds. P. M. Dey and J. B. Harborne), Vol. 6, Aca-demic Press, London 1991, pp. 47–69.20. G. Cappuccino and N. Sherman, Microbiology: A Laboratory Manual, Benjamin Cumming SciencePublishing, California 1998, p. 254.S A @ E T A KSastav i antimikrobno djelovanje eteri~nog ulja iz plodovabiljke Amomum cannicarpumBABY SABULAL, MATHEW DAN, NEDIYAMPARAMBU SUKUMARAN PRADEEP,RENJU KRISHNA VALSAMMA i VARUGHESE GEORGEEteri~no ulje iz plodova biljke Amomum cannicarpum (Wight) Bentham ex Baker(Zingiberaceae) dobiveno je destilacijom vodenom parom, a zatim je pomo}u plinske kro-matografije i masene spektrometrije (GC-MS) odre|en njegov sastav. Najva`niji sastojciu ulju bili su -pinen (14,00%), elemol (10,45%) i -kadinol (8,50%). Ukupno su od 41sastojka identificirana 33 sastojka (91,5%). Disk-difuzijskom metodom odre|eno je anti-mikrobno djelovanje ulja na Gram-pozitivne i Gram-negativne bakterije i neke gljivice.Dosta sna`no antimikrobno djelovanje zapa`eno je na bakterije Salmonella typhi, Pseudo-monas aeruginosa i Proteus vulgaris te na gljivice Candida albicans i C. glabrata.Klju~ne rije~i: Amomum cannicarpum (Zingiberaceae), eteri~no ulje, GC-MS, -pinen, elemol, -kadi-nol, antibakterijsko djelovanje, antimikotsko djelovanje479B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.Phytochemistry Division, Tropical Botanic Garden and Research Institute, Pacha-PalodeThiruvananthapuram-695 562, Kerala, IndiaHorticulture and Garden Development Division, Tropical Botanic Garden and Research InstitutePacha-Palode, Thiruvananthapuram-695 562, Kerala, IndiaMicrobiology Division, Tropical Botanic Garden and Research Institute, Pacha-PalodeThiruvananthapuram-695 562, Kerala, India480B. Sabulal et al.: Composition and antimicrobial activity of essential oil from the fruits of Amomum cannicarpum, Acta Pharm. 56 (2006)473–480.

Sastav i antimikrobno djelovanje eteričnog ulja iz plodova biljke Amomum cannicarpum

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