Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 5(7), 40-44, July (2015) Res. J. Chem. Sci. International Science Congress Association 40 Compositional analysis and Antimycobacterium Tuberculosis Activity of Essential Oil of Hyptis Suaveolens LamiceaeRunde M., Kubmarawa D. and Maina H. Department of Chemistry, School of Pure and Applied Science Modibbo Adama University of Technology P.M.B. 2076 Yola Adamawa State NIGERIAAvailable online at: www.isca.in, www.isca.me Received 30th June 2015, revised 4th July 2015, accepted 16th July 2015 AbstractFresh leaves of Hyptis suaveolens were collected and pretreated for essential oil analysis with the sole aim of linking the ethnomedicinal uses of this plant by the people of Adamawa State Nigeria to its essential oil composition. The results obtained from the analysis shows that 68 compounds were present in varying concentration out of which 16 compounds have appreciable concentration making 75.135 % of the total abundance. The major compound being Caryophyllene (20.643 %) followed by Sabinene (16.711 %) and Terpinolene (8.490 %). The essential oil of Hyptis suaveolens also showed anti mycobacterium activity when tested on strain 7H9/ADC with MIC of 3.13 %. This activity was compared with standard drug Rifampicin which also has MIC of 0.1 µg/ml. Keywords: Hyptis suaveolens, essential, antimycobacterium, tuberculosis, microbroth dilution. Introduction Hyptis suaveolens is a medium aromatic annual shrub distributed in the tropical and subtropical regions. It is usually applied in Asian food recipes as an appetizer because of its flavored essential oil. It was reported to be used for traditional medicine as an anticancer. The leaves of Hyptis suaveolens have been utilized as a sudorific, galactogogue and as a cure for parasitic cutaneous diseases. Crude leaves extract is also used as a relief to colic and stomachache. Leaves and twig are considered to be antispasmodic and used in anti-rheumatic and anti-suporific bath, as anti-inflammatory, fertility agent and also applied as an antiseptic in burns, wound and various skin complaints. The ethanolic extract from its leaves exhibited healing properties with a supportive role of antioxidant enzymes. The decoct ion of the roots is highly valued and is reported to contains urosolic acid, a natural HIV-Intergrase and inhibitorPresence of sterols, flavonoids, alkaloids, saponins, terpenoids tanins in various extracts was also confirmed by preliminary phytochemical investigation, TLC and HPTLC methods6,7 . Studies are ongoing throughout the world in search for compounds that are biologically active with a low profile of side effects. Essential oils from plants usually show antimicrobial activity against a wide range of microorganisms including resistant bacteria and fungi. In this study steam distillation was used to extract essential oils from Hyptis suaveolens with the aim at determining the anti-mycobacterium tuberculosis and antioxidant activities. Material and Methods Plant material: Fresh leaves of Hyptis suaveolens was collected in January 2015 at Sangere, Girei local government of Adamawa State North-east Nigeria. The plants was isolated and conserved for extraction. Essential oil extraction: The leaves of hyptis suaveolens (1 kg) was immediately subjected to extraction to avoid loss of some essential oils as a result of drying process, and using a modified type of steam distillation apparatus (in which the receiver end of the pass through another vessel containing ice) for 2.5 h essential oils of the plant which has a yield of 0.4 % was collected over water and later kept at 4 C until further required. Gas chromatography Mass spectroscopy (GC-MS): GC-MS analysis were performed on a J and W Scientific gas chromatography directly couple to the mass spectrometer system (model GC Agilent technologies 7890A, Agilent technologies Inert MSD 5975C) HP 5 ms, 5 % phenyl methyl silox: 469.56 509. Capillary column (30M x 250µm) was used under the following condition: ovum temperature 50C for 1 min, then 10C/min to 200C for 1min, and 20/min to 300 for 2 min. Injector temperature 230c carrier gas He, flow rate 1ml/min; the volume of the injected sample was 0.2µL of diluted oil in hexane, split less injection techniques, lionization energy 70ev, in the electron ionization (EI) mode, ion source temperature 230c scan mass range of M/Z 60-335; the constituents of the essential oils were identified base on comparison of the retention indices and mass spectra of most of the compound with data generated under identical experimental conditions by applying a two dimensional search algorithm considering the retention index as well as mass spectral similar with those of Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 40-44, July (2015) Res. J. Chem. Sci. International Science Congress Association 41 authentic compounds available in NBS75K and NIST08 Libraries. The retion indices (RI) are in relation to a homologous series of n-alkanes on the GC column under the same chromatographic condition components. Relative concentration will be obtained by peak area normalization as describe by Ramzi A. M. et al. Antituberculosis activity of pure oil sample using Microbroth dilution technique: Dilution of the oils One microliter of the oil was dissolved in 0.1 mI of 10% dimethylsulphoxide (DMSO) to give a concentration of 50% oil. Organism preparation: Five hundred microliter of test organism mycobacterium bovis (BCG) freshly thawed stock was inoculated into 50 mI of sterile Middlebrook 7H9/ADC broth medium and incubated at 30C for 5-7 days. The optical density was measured at 650 nm wavelength. This in our work the OD WAS 0.2 and this is equal to 10 cfu/ml. Sample screening for antituberculosis activity: Into each well of 96 microwell plate was transferred 50µl of sterile 7H9 broth starting from well 2 to 12. To each of the first well was added 100µl of 10% DMSO, 100 µl of 25 µg/ml solution of rifampicin (control drug) and 100 µl of the diluted oil sample. Using a multichannel pipette 50µl was carefully removed from well 1 to 2, mixed thoroughly and the process continued to well 11 from which 50µl was withdrawn and discarded. The well were inoculated with 50 µl of diluted BCG culture and incubated at 30C for 7 days. The results were confirmed by adding tetrazolium dye after the incubating period. The wells where there was no color change were regarded as activity of test samples indicating inhibition of test organism. The last well where there was no growth is regarded as the minimum inhibitory concentration (MIC) of the sample and the result shown in table-2.Results and Discussion Essential oil composition: Figure-1is the gas chromatography spectrum of essential oil of Hyptis suaveolens leaves extract. The mass spectrum reveals the presence of 68 compounds as presented in table-1; out of which 16 compounds were presented with appreciable percentage abundance in which Caryophyllene (20.643 %) has the highest concentration followed by Sabinene (16.711%) and Terpinolene (8.49 %). Other researchers have also reported the present of major components of essential oil of Hyptis suaveolens cultivated in Italy as follows: Sabinene (34 %), beta-Caryophyllene (11.2 %) and Terpinolene (10.7 %), whereas beta-Caryophyllene (34.65 %), Germacrene (10.32 %), alpha-Bergamotene (6.56 %), Rimuen (6.46 %) and alpha-Copaene were shown to be the major compounds in essential oil of Hyptis suaveolens from Indonesia10,11. In India, a work carried on essential oil of Hyptis suaveolens reveals that, 1,8-Cineole (44.4 %) followed by beta-Caryophyllene, beta-Pinene and Camphene12. In a similar work by Okonogi et al on the essential oil of the same plant obtained from Northen Thailand revealed beta-caryophyllene, 1,8-Cineole and Phellandrene are the major compounds of the oil, whereas Fun and Baerhein reported the major compound of the essential oil of the plant species as 1,8-Cineole (27-38 %), and Sabinene (12-18 %)13,14. Brazilian source of Hyptis suaveolens essential oil was presented with Sabinene, Limonene, Bicyclogermacrene, beta-Caryophyllene and 1,8-Cineole as the major compounds of the oil14In the same vein, analysis of essential oil obtained from Togo showed the predominace of beta-Caryophyllene (33.8 %), alpha-Bergamoten (11.3 %) and alpha-Caryophyllene (7.4 %)16. From the above discussions, Caryophyllene is common in all the samples obtained from various locations except in the sample obtained from Brazil which has Limone, Bicyclogermacrene and beta-Phellandrene. On the other hand Rimuene is another compound that has appeared only in oil sample from Indonesia. These results shows that differences in locations affect the composition of essential oil obtain from Hyptis suaveolens.However, some of the results are in line with our finding. Anti-mycobacterium tuberculosis activity of the essential oil: After the evaluation of the essential oil of Hyptis suaveolensfollowed the anti- mycobacterium tuberculosis screening using 7H9/ADC strain of mycobacterium bovi. The result obtained are shown intable-2 which shows that the essential oil of Hyptis suaveolens is active against the strain 7H9/ADC with MIC of 3.13 % although in comparison the control rifampicin drug was more active with MIC of 0.1 µg/ml. Dorman and Deans state that the oils antimicrobial properties is related to the composition of the plants volatile oils, the structural configuration of the oils constituent compounds and their functional groups and potential synergistic interactions among the compounds17. Therefore in our case the anti-mycobacterium activity observed in the essential oil of Hyptis suaveolens can be attributed to the presence of high percentage concentration of Caryophyllene, Sabinene, Terpinolene and to other minor compounds that may contribute to the antimicrobial activity. Conclusion The GC-MS analysis of the essential oil of leaves of Hyptis suaveolens source from Girei local government of Adamawa state, North-eastern Nigeria, indicate that the major compounds are caryophyllene (20.643 %), Sabinene (16.711 %) and Terpinolene (8.49 %). The essential oil of Hyptis suaveolenswas active against 7H9/ADC strain of mycobacterium bovi as utilized in this study with MIC of 3.13 %. Therefore the result of this studies support the use of this plant in ethnomedicine as an alternative remedy for symptoms of tuberculosis; as such the essential oil of this plant may be a potential candidate for further studies to isolating the active compounds effective against Mycobacterium tuberculosis. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 40-44, July (2015) Res. J. Chem. Sci. International Science Congress Association 42 Figure-1 Gas chromatography (GC) spectrum of hyptis spicigeraTable-1 GC-MS Analysis of Hyptis suaveolens S.No. Constituents RT(min) % conc. KI MW 1. Alpha. - phellandrene 5.131 0.575 902 136 2. Alpha. - pinene 5.251 2.644 905 136 3. Camphene 5.533 0.122 910 136 4. Benzene, propyl - 5.597 0.337 811 120 5. Benzene, 1 - ethyl - 2 - methyl - 5.734 0.671 814 120 6. Benzene,1 - ethyl - 2 - methyl - 5.784 0.600 815 120 7. Benzene, 1,2,3, - trimethyl - 5.896 0.344 817 120 8. Bicyclo [3.1.0] - hexene, 4 - methylene - 5.996 16.711 919 136 9. . - Pinene 6.052 5.490 921 136 10 . 1 - octen - 3 - ol 6.107 1.021 907 127 11. .myrcene 6.255 0.536 925 136 12. Benzene, 1,2,4 - trimethyl - 6.306 1.614 826 120 13. 3 - octanol 6.398 0.189 939 130 14. Decane 6.442 0.246 928 136 15. 3 - Carene 6.556 0.804 931 136 16. (+) - 2 - Carene 6.704 0.505 93 4 120 17. Benzene, 1 - ethyl - 3 - methyl - 6.749 0.509 834 134 18. Benzene, 1 - methyl - 2 - (1 - methylethyl - ) 6.838 0.295 936 136 19. D - Limonene 6.936 8.832 938 134 20. Benzene, 1,4 - diethyl - 7.207 0.189 944 134 21. Benzene, 1 - methyl - 3 - propyl - 7.262 0.278 945 136 22.  - Terpimene 7.396 1.403 947 134 23. Benzene, 1 - methyl - 2 - propyl - 7.491 0.119 949 154 24. 3 - cyclohexen - 1 - ol, 4 - methyl - 1(1 - methylethyl) - 7.613 0.556 952 134 25. Benzene, 2 - ethyl - 1,4 - dimethyl - 7.664 0.167 953 134 26. Benzene, 1 - methyl - 4 - ( - methylethyl) - 7.7 08 0.241 954 136 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 40-44, July (2015) Res. J. Chem. Sci. International Science Congress Association 43 S.No. Constituents RT(min) % conc. KI MW 27. Terpinolene 7.867 8.496 957 152 28. Bicyclo [2.2.1] heptan - 2 - one, 1,3,3 - trimethyl - 7.912 0.547 958 154 29. 1,6, - Octradien - 3 - ol, 3,7 - dimethyl - 8.099 0.859 961 134 30. 1,3,8 - P - menthatriene 8.274 0.137 965 134 31. Benzene, 1,2,3,5 - te tramethyl - 8.313 0.216 966 154 32. Bicyclo [2.2.1] heptan - 2 - ol,1,3,3 - trimethyl - 8.397 0.928 967 154 33. 2-Cyclohexen-1-ol,1-methyl-4-(1-methylethyl)-, trans 8.481 0.090 969 134 34. 1,3,8 - P - menthatriene 8.614 0.156 972 132 35. 1H - indene,2,3 - dihydro - 4 - me thyl - 8.673 0.193 973 94 36. Bicyclo [3.2.0] hept - 6 - ene 8.51 0.194 840 94 37. Benzene, 2 - ethyl - 1,4 - dimethyl - 8.810 0.390 1063 121 38. Pyridine, 2 ethyl - 5 - methyl - 8.888 0.045 1055 148 39. 2 - ethyl - 2 - methyl - 1,3 - dithiolane 8.985 0.104 998 134 40. Benzene, 1 - methyl - 4 - (1 - methylethyl) - 9.072 0.095 981 154 41. Borneol 9.022 0.495 984 154 42. 4 - Terpineol 9.384 1.214 986 150 43. Thymol 9.454 0.057 989 150 44. Methylsalicylate 9.490 0.266 949 152 45. Benzene, 1,3 - dimethyl - 5 - (1 - methylethyl) - 9.538 0.061 990 134 46. Benzene, ethyl - 1,2,4 - trimethyl - 9.702 0.090 994 148 47. 6 - Methyl - 4 - indanol 10.745 0.222 1014 142 48. Naphthalene, 2 - methyl - 11.002 0.123 1020 142 49. Naphthalene, 1 - methyl - 11.214 0.084 1024 142 50. Santolina triene 11.361 0.800 1027 136 51. Camphere 11.504 0.178 1030 136 52. Copaen 12.100 0.211 1242 204 53. Cyclobuta [1,2.3,4] dicyclopentene, decahydro-3a-methyl-6-methylene-1 (1-methylethyl)-, [Is- (1.alpha., alpha., 3b. ., 6a. ., 6b alpha alpha.,)] - 12.209 1.182 1244 204 54.  - Elemene 12.290 0.274 1245 204 55. Isocaryophyllene 12.493 17.750 1249 204 56. Caryophyllene 12.750 4.459 1255 204 57. Bicycle [3.1.1] hept-2-ene,2,6-dimethyl-6-(4-methyl 3 - pentenyl) - 12.867 4.459 1257 204 58. 1H-cycloprop (e) azulene, decahydro-1,1,7- trimethyl - 4 - methylene - , [1aR] - 12.945 0.116 1258 204 59. 1,6,10-Dodecatriene, 7,11-dimiethyl-3-methylene-, (E) - 13.104 0.401 1262 204 60. Alpha. - caryophyllene 13.166 2.160 1263 204 61. Naphthalene, decahydro-4a-methyl-1-methylene-7- (1 - methylethylidene) - 13.377 0.157 204 62. 1H cyclopenta [1,3] cyclopropa [1,2] benzene, octahydro-7-methyl-3-methylene-4-(1-methylethyl)-, [3as - (3a. alpha. 3b. . 4.., 7 alpha., 7a s )] - 13.489 0.802 1269 204 63. 1H-cycloprop [e] azulene, decahydro-1,1,7-trimethyl-4-methylene-, [1aR-(1a. alpha., 4a.., 7. Alpha., 7a.. 7b alpha)] - 13.598 1.447 1271 204 64. Germacrene B 13.687 5.280 1273 204 65. Germacrene A 1 3.829 0.192 1276 204 66. Caryophyllene oxide 14.802 2.893 1296 220 67. Bicyclo [3.1.1] hept-2-ene, 2,6-dimethyl-6-(4- methyl - 3 - pentenyl) - 16.621 0.161 1332 204 68. Pyrido [3,4-d] pyridazine-4,5(3H.6H)-dione, 1-(2- furfuryl) - 7 - methyl - 20.137 1.377 1302 257 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 40-44, July (2015) Res. J. Chem. Sci. International Science Congress Association 44 Table-2 Antimycobacterium bovi (7H9/ADC) of essential oil of Hyptis spicigera% Conc. 25 12.5 6.25 3.125 0.45 0.28 0.14 0.07 0.035 MIC Hyptis suaveolens - - - - + + + + + 3.13% Rifampicin - - - - - - - + + 0.1µg/ml KEY: (-) No growth in the well, means there is anti-TB activity. (+) Growth in the wel,l means no anti-TB activity, Rifampicin (Control drug) References 1.Kingston D.G., Rao. M.M. and Zucker W.V., Plant Anticancer against IX Constituent of Hyptis suaveolens, J. of Nat. pro.,42(5 496-499 (1997)2.The wealth of India (Raw materials), CSIR New Delhi., 5, 159 (1964)3.Kirtikar K.R. and Basu B.D., Indian Medicinal Plants, Singh B and Singh M.P. Publishers India., 3, 2032 (1991) 4.Annie S., Rhadika S., Udupa A.L., Udupa S.L. and Somashekar S., Wound Healing Property of Ethanolic Extract of Leaves of Hyptis suaveolens with supportive role of Antioxidant Enzymes, Ind. J. of exp. Bio., 41(3),238-241 (2003)5.Chatterjee A. and Pakrashi S.C., The Treatise on Indian Medicinal Plants, PID, New Delhi: 5, 15 (1997)6.Chitra S., Patil M.B. and Ravi K., Wound Healing Activity of Hyptis suaveolens (L)Poit, Int. J. of Pharm. Tech. Res., 1(3), 737-744 (2009)7.Umedum N.L., Nwajagu U., Udeozo I.P., Anarado C.E. and Egwuatu C., The efficacy of Hyptis suaveolens: A review of its Nutritional and Medicinal Application, Eur. J. of Medi. Pl., 4(6) 661-674 (2014)8.Carson C.F. and Riley T.V., Antimicrobial Activity of a Major Component of the Essential oil of Melaleuca alternifolia. J. of App. Bacterio.,78(3), 264-269 (1995)9.Ramzi A.M., Mansour S.A., Mohammed A.A., Adnan J.A. and Jamal M.K., GC and GC/MS Analysis of Essential Oil Composition of the Endemic Soqotraen Leucas virgata Balf. f. and Its Antimicrobial and Antioxidant Activities. Int. J. Mol. Sci.,14(11), 23129-23139; doi:10.3390/ijms141123129 (2013)10.Giovanni B., Guido F., Angelo C., Illaria M., Pier L.C and Barbara C., Repellent of Hyptissuaveolens whole Essential oil and major Constituents against Adult of Granary weevil sitophilus granaries. Bullet. of insectol., 65(2), 177-183 (2012)11.Chatri M., Amri B. and Mansyurdin P.A., Chemical Components of Essential oils of the Leaves of Hyptis suaveolens (L) poit from Indonesia, Amer. J. of Res. Comm.,2(10), 30-38 (2014)12.Sharma N., Verma U.K. and Tripatti A., Bioactivity of Essential oil from Hyptissuaveolens against Storage mycoflora. Proc. Inc. cont. Controlled Atmosphere and Fumigation in Stored Product, Gold-cast Austrilia. 8-13thFTIC Ltd. Publishing, Israel., 99-116 (2007)13.Okonogi S., Chansakaow S., Vejabhikul S., Tharavichikul P., Herphokanount J, Nakano A. and Ikegami F., Antimicrobial Activity and Pharmaceutical Development of Essential oil from Hyptis suaveolens. Proc. WOCAMP.,3(4), 163 (2005)14.Fun C.E and Baerheim A.S., The Essential oil of Hyptis suaveolens poit Grown on Aruba. 10. 1002/ffj.2730080306, available on linelibrary.wiley.com visited., 14-6-2015 (2006) 15.Azevedo N.R., Campos I.F., Ferreira H.D., Portes T.A., Santos S.C. Seraphin J.C and Ferri P.H., Chemical Variability in the Essential oil of Hyptis suaveolens. U. S Nat. lib. of Med. Nat. Inst. of Health,57(5), 733-6 (2001)16.Koba K., Raymoud C., Millet J., Chaumout J.P. and Sanda K., Chemical Composition of Hyptis pectinataL.H. lanceolata poit H. Suaveolens L and H. spicigeraLam, Essential oil from Togo, J.of Essent.oil Bea. Pl.10(5), 357-364 (2007) 17.Dorman H.J.D. and Deans S.G., Antimicrobial Agent from Plant: Antibacterial Activity of Plant Volatile oils, J. of App. Microbiol.,88, 308-316 (2000)