ABSTRACT Antibacterial Terpenoid from The Bark of Lansium domesticum Corr cv. Kokossan (Meliaceae) Tri Mayanti, 1 Siti Soidah 1, W. Drajat Natawigena, 2 Unang Supratman, 1 dan Roekmi-ati Tjokronegoro 1 1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor 45643, Sumedang 2 Department of Plant Diseases and Plant Control, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor 45643, Sumedang In the course of our continuing search of novel active compounds from Indonesian plants, the methanolic extract of bark of L. domesticum cv kokossan showed significant antibacterial activity against B. cereus and E. coli. The methanolic extract of the bark of L. domesticum cv kokossan was concentrated and extracted with ethyl acetate. The ethyl acetate extract exhibited an antibacterial activity with inhibit zone respectively 12.5 and 14 mm against B. cereus and E. coli. The ethyl acetate fraction was separated by combination of column chromatography on Kieselgel 60 to afford an antibacterial compound as white crystal with melting point 166-168 o C. Based on UV, IR, H 1 -NMR, C 13 -NMR and 2D-NMR spectroscopic evidences and comparison with previously data reported indicated that isolated compound as terpenoid group. An antibacterial compound showed antibacterial activity with inhibit zone 10 mm at concentration of 1000 ppm against E. coli.
Antibacterial Terpenoid from The Bark of Lansium domesticum Corr cv. Kokossan (Meliaceae) 1 Tri Mayanti, 2 Siti Soidah 2, W. Drajat Natawigena, 3 Unang Supratman, 2 dan Roekmi-ati Tjokronegoro 2 ABSTRACT In the course of our continuing search of novel active compounds from Indonesian plants, the methanolic extract of bark of L. domesticum cv kokossan showed significant antibacterial activity against B. cereus and E. coli. The methanolic extract of the bark of L. domesticum cv kokossan was concentrated and extracted with ethyl acetate. The ethyl acetate extract exhibited an antibacterial activity with inhibit zone respectively 12.5 and 14 mm against B. cereus and E. coli. The ethyl acetate fraction was separated by combination of column chromatography on Kieselgel 60 to afford an antibacterial compound as white crystal with melting point 166-168 o C. Based on UV, IR, H 1 -NMR, C 13 -NMR and 2D- NMR spectroscopic evidences and comparison with previously data reported indicated that isolated compound as terpenoid group. An antibacterial compound showed antibacterial activity with inhibit zone 10 mm at concentration of 1000 ppm against E. coli. Keywords: antibacterial, terpenoid, Lansium domesticum, kokosan, Meliaceae 1 Research artikel was presented at International Confererence On Medicine and Medicinal Plants, Surabaya, September 8 th -9 th, 2007. 2 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor 45643, Sumedang 3 Department of Pest Control and Plant Diseases, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor 45643, Sumedang
INTRODUCTION Lansium domesticum Corr (Meliaceae) is a popular fruit in southern Asia. The plant family Meliaceae is noted for the production of useful bitter principles which are generally used in folk medicine as a drug for diarrhea which caused by bacteria. The bark and leaf infusion of L. domesticum cv. duku showed antibacterial activity against Echerichia coli, Staphylococcus aureus, and Bacillus subtiles (Loekitowati and Hermansjah, 2000). Phytochemical investigation of the fruit peel of L. domesticum revealed the presence of lansic acid, 3β-hydroxyonocera-8(26),14-dien-21-one and 21αhydroxyonocera-8(26),14-dien-3-one (Tanaka et al., 2002). The seed contain tetranortriterpenoids named dukunolides A-F (Nishizawa et al., 1985, 1989). The bark contain antifeedant triterpenoid namely iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, onoceradienedione, lansiolic acid, lansiolic acid A and 3-keto lansiolic acid. In our continuing search on bioactive compounds from Indonesian plants, the methanolic extract of bark of L. domesticum cv kokossan showed significant antibacterial activity against B. cereus and E. coli. In this research, we report the isolation and characterization of antibacterial triterpenoid, 14-hydroxy-7-onoceradienedione (1). O O HO (1) EXPERIMENTAL General Experimental Procedure. UV and IR spectra were measured with System Perkin Elmer Spectrum One. 1 H and 13 C-NMR spectra were recorded with a JEOL JNM ECA- 500, operating at 500 and 400 MHz ( 1 H), 125 and 100 MHz ( 13 C), using residual and deutered solvent peaks as internal standards. Vacuum liquid chromatography was carried out using Kieselgel 60.
Plant Material. Samples of the bark L. domesticum cv kokossan were collected in March 2006 from Cililin, Bandung, Indonesia. The plant was identified by the staff at Department of Biology, Padjadjaran University. Bioassay. A 7,2 g nutrient broth (2 g sodium chloride 0,85%; 1,2 g beef extracts and 4 g pepton) were suspenced in 400 ml hot aquadest which contain 12 g gel. This media was further sterilized for 15 minutes at 121 o C. The bacteria (E. coli and B. Cereus) were incubated at 37 o C for 18-24 hours in this media. Paper disk which contain the sample was put on the media. The sample was incubated at 37 o C for 18-24 hours and inhibit zone was further measured. Extraction and Isolation. The dried and milled bark of L. domesticum cv kokossan (3 kg) was extracted exhaustively by methanol at room temperature. The methanol extract (200 g) was partitioned between n-hexane and 10% aqueous methanol, and the aqueous phase was further extracted with ethyl acetate. The ethyl acetate-soluble fraction was subjected to Kieselgel 60 column eluted with 0-100% dichloro metane/n-hexane. The fraction eluted with dichloro metane/n-hexane (6:4) contained (1). The fraction was further separated by Kieselgel 60 column eluted with acetone/n-hexane (1:9) to yield (1) (10,7 mg). 14-hydroxy-7-onoceradienedione (1): colorless amorphous solid, IR (KBr) ν max 3444, 2962-2931, 1705, 1698, 1458, 1338, 1315, 1261-1076, 802 cm -1. 1 H-NMR (CDCl 3 ) δ H 7.3 (1H, s), 5.4 (1H, s), 2.75, 2.73, 2.70 (2H, dt), 2.6 (2H, m), 2.1 (3H, m), 1,9 ( 2H, m), 1.8 (2H, m), 1.7 (2H, m), 1.6-1.4 (6H, m), 1.3 (3H, s), 1.09 (3H, s), 1.08 (3H, s), 1.05 (3H, s), 1.01 (3H,s), 0.96 (3H, s), 0.92 (3H,s); 13 C-NMR (CDCl 3 ) δ C see Table 1. DISCUSSION Compound 1 was obtained as colorless amorphous solid. The IR spectrum of 1 showed a sharp absorption band at 3444 cm -1 and strong absorption band at 1705 cm -1 indicating the presence of hydroxyl, alkene and ketone groups. The 1 H and 13 C-NMR spectrum of 1 showed the presence of hydroxyl group on C-14 (δ C 74,0) (Table 1). Therefore, it may be concluded that 1 is 14-hydroxy-7-onoceradienedione.
Table 1. 13 C-NMR data of 14-hydroxy-7-onoceradienedione (1) on 500 Hz δ H (Σ H, Posisi C δ C multiplisitas, HMBC COSY J dalam Hz 1/19 38,54/ 38,37 2,08; 1,91 (2H,m) H-2/H-20 2 34,82 2,23; 2,41 (2H, dt, dq, J=15) H-1 3 216,96-4/22 47,62-5 51,57 1,57 (1H, m) C-9,C-10,C-24 H-6 6 28,98 1,12 (2H, m) H-5/H-17 7 121,70 5,41 (1H, m) C-5, C-9, C-25 H-26 8 135,27-9 55,47 1,59 (1H, m) C-26 10/18 36,58-11/12 21,54 1,61 (1H, m) C-9 13 61,80 1,12 (1H, m) C-17 14 74,03-15 44,23 1,46 (2H, m) C-14, C-17 H-16 16 31,41 1,51 (2H, m) C-1,C-4 H-15 17 55,23 1,42 (1H, m) C-18, C-22 20 34,13 2,26;2,73 C-19 H-19 (2H, dt; td, J=15) 21 217, 12-23 25,14 1,04 (3H, s) C-5, C-24 24 22,25 1,08 (3H, s) C-3, C-23 25 13,43 0,96 (3H, s) C-5, C-9, C-10 26 22,33 1,77 (3H, s) C-9 27 24,20 1,21 (3H, s) C-13,C-14,C-15 28 15,13 0,93 (3H, s) C-13,C-16,C-17 29 21,43 1,02 (3H, s) C-17,C-22,C-30 30 26,49 1,09 (3H, s) C-17,C-29 O 2 11 25 1 9 10 3 5 6 20 19 12 21 13 18 22 26 8 27 7 HO 17 14 28 16 15 O 30 29 24 23 (1) Antifungal activity of the methanol, ethyl acetate, and n-hexane extracts of the bark of L. domesticum kultivar cv. kokosan were tested against B. cereus and E. coli. The results are tabulated in Table 2, showing that ethyl acetate extracts have inhibit zone 12.5 mm against B. cereus and 14.0 mm against E. coli at 1% w/v. The compound (1) showed
antibacterial activity with inhibit zone 10 mm at concentration of 1000 ppm against E. coli (Table 3, Fig.1). Table 2. Antibacterial activity of the methanol, ethyl acetate, and n-hexane extracts of the bark of L. domesticum kultivar cv. kokosan with paper disk method. Inhibit Zone (mm) Methanol n-hexane Ethyl acetate Bacteria 0,1 0,5 1,0 0,1 0,5 1,0 0,1 0,5 1,0 B. cereus 10,5 9,5 12,0 - - - 11,0 10,5 12,5 E. coli - 13,5 10 - - - 9,0 10,5 14,0 Table 3. Antibacterial Activity of Isolate Bacteria Inhibit zone (mm) Acetone Isolate (500 ppm) Isolate (1000 ppm) B. cereus 8 8 8 E. coli 8 8 10 Fig 2. Antibacterial Test of Isolate against B. cereus (a) and E. coli (b).
CONCLUSION Based on UV, IR, H 1 -NMR, C 13 -NMR and 2D-NMR spectroscopic evidences and comparison with previously data reported indicated that antibacterial compound from the bark of L. domesticum cv. kokosan is 14-hydroxy-7-onoceradienedione. The compound (1) showed antibacterial activity with inhibit zone 10 mm at concentration of 1000 ppm against E. coli. REFERENCES Loekitowati, H.P. and Hermansjah. 2000. Studi pemanfaatan biji duku (Lansium domesticum) untuk obat diare secara in vitro. Jurnal Penelitian Sains. 7: 41-48. Tanaka, T., M. Ishibashi, H. Fujimoto, E. Okuyama, T. Koyano, T. Kowithayakorn, M. Hayashi, dan K. Komiyama. 2002. New onoceranoid triterpene constituents from Lansium domesticum. Journal Natural Product. 65: 1709-11. Nishizawa, M., Y. Nademoto., S. Sastrapradja., M. Shiro, dan Y. Hayashi. 1985. Structure of dukunolide, bitter principles of Lansium domesticum. Journal of Organic Chemistry, 50(26): 5487-90. Washington DC. Nishizawa, M., M. Emura., H. Yamada., M. Shiro., Chairul, Y. Hayashi dan H. Tozuda. 1989. Isolation of a new cycloartanoid triterpenes from leaves Lansium domesticum: novel skin-tumor promotion inhibitors. Tetrahedron Letter. 30 (41): 5615-18.