An antibacterial constituent, has been isolated from Ficus deltoideus Jack leaves. Based on spectroscopic data (IR, 1H-NMR, 13 C NMR 1D and 2D and MS), the structure of this compound was identified as Olean-12en-3β-ol, (β-amyrin), C30H50O. This compound showed antibacterial activities against E. coli, B. subtilis and S.  aureus.  The minimum inhibition concentration (MIC) agains  E.  coli,  B. subtilisand  S. aureusare 230, 380 and 460 (µg/mL) respectively.Key words: Antibacterial activity,Ficus deltoideusJack, β-amyri

., Dachriyanus

., Suryati

Lajis, Md Nordin Hj

Nurdin, Hazli

INDONESIAN JOURNAL OF PHARMACY

English

An antibacterial constituent, has been isolated from Ficus deltoideus Jack leaves. Based on spectroscopic data (IR, 1H-NMR, 13 C NMR 1D and 2D and MS), the structure of this compound was identified as Olean-12en-3β-ol, (β-amyrin), C30H50O. This compound showed antibacterial activities against E. coli, B. subtilis and S.  aureus.  The minimum inhibition concentration (MIC) agains  E.  coli,  B. subtilisand  S. aureusare 230, 380 and 460 (µg/mL) respectively

Suryati

Hazli Nurdin

Dachriyanus

Md Nordin Hj Lajis

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Characterization antibacterial constituent from Ficus deltoideus Jack leaves

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Majalah Farmasi Indonesia, 21(2), 134 – 138, 2010  Majalah Farmasi Indonesia, 21(2), 2010 134Characterization antibacterial constituent from   Ficus deltoideus Jack leaves  Karakterisasi konstituen antibakteri dari daun Ficus deltoideus Jack   Suryati1*), Hazli Nurdin2, Dachriyanus3 and Md Nordin Hj Lajis4 1. Politechnic, Andalas University, Padang, Indonesian 2. Faculty of Mathematics and Natural Science,Andalas University, Padang, Indonesian 3. Faculty Pharmacy,  Andalas University,Padang, Indonesian 4. Institute of BioScience, University Putra Malaysia, Malaysia  Abstract  An antibacterial constituent, has been isolated from Ficus deltoideus Jack leaves. Based on spectroscopic data (IR, 1H-NMR, 13C NMR 1D and 2D and MS), the structure of this compound was identified as Olean-12en-3β-ol, (β-amyrin), C30H50O. This compound showed antibacterial activities against E. coli, B. subtilis and S. aureus. The minimum inhibition concentration (MIC) agains E. coli, B. subtilis and  S. aureus are 230, 380 and 460 (µg/mL) respectively. Key words : Antibacterial activity, Ficus deltoideus Jack,  β-amyrin  Abstrak  Suatu konstituen antibakteri, telah diisolasi dari daun Ficus deltoideus Jack. Karakterisasi struktur menggunakan data spektroskopi IR, 1H-NMR, 13C-NMR, 1D dan 2D serta data MS, menunjukkan bahwa konstituen hasil isolasi adalah Olean-12en-3β-ol,  (β -amyrin), C30H50O. Uji aktivitas antibakteri terhadap E. coli,  B.subtilis dan S. aureus menunjukkan bahwa β-amyrin dapat menghambat pertumbuhan bakteri, dengan nilai MIC 230 µg/mL untuk E. coli, 380 µg/mL untuk B. subtilis, dan 460 µg/mL untuk bakteri S. aureus. Kata kunci: Aktivitas antibakteri , Ficus deltoideus Jack, β-amirin  Introduction Ficus deltoideus Jack is an epiphytic shrub which is native and widely distributed in several countries of the Southeast Asia (Musa and Lip, 2007). It is easily found in the coastal, but not in mangrove area. Different parts of the plant are used traditionally to  treat various kinds of ailments. The fruits are chewed to relieve headche, tootache and cold. Powdered root and leaves of the plant has been applied externally to wounds and sores and for relief of rheumatism (Musa and Lip, 2007). Decoction from the whole plants is well known as traditional herbal drink for women after childbirth to help strengthen the uterus (Sulaiman, et al., 2007). The plant sap was used to   detach   wart from  the skin (Burkill, 1966).    Moreover it improves blood circulation and pharmacologically  blood glucose (Aminudin, et al., 2007). On the other hand, there is no             report related to its chemical constituent           and bioactivity. In this report, the structur elucidation of the isolated compound from ethyl acetate fraction of F.deltoideus Jack leaves extracts  and its antibacterial activity againts E. coli, B.subtilis and  S. aureus are discussed.      Methodology  General Vaccum Liquid Chromatography (VLC), using silica gel PF254 Merck (7749), column chromatography, using silica gel 7734 (70-230 mesh) and silica gel 9385 (230-400 mesh) (Merck). IR spectrum was measured    with   FT-IR   Perkin   Elmer   1650.  Suryati  Majalah Farmasi Indonesia, 21(2), 2010 135Table I. The comparizon of 1H and 13C-NMR (1D,2D) data of  β-amyrin and  13C-NMR data of β-amyrin  β-amyrin isolated compound Literature8 No δC (ppm) DEPT δH(ppm), (ΣH multiplisity) HMBC COSY δC  β-amyrin (ppm) 1 38.9 CH2 1.67 (2H, t) 47.9 (C-9)  38.7 2 27.5 CH2 1.60 (2H, m)  3.21 (H-3) 27.3 3 79.2 CH 3.21 (1H, t)  1.60 (H-2) 79.0 4 39.7 C    38.8 5 55.4 CH 0.75 (1H, t)   55.3 6 18.5 CH2 1.52 (2H, m) 37.3 (C-10); 39.8 (C-8)  18.5 7 33.1 CH2 1.54 (2H, t) 17.0 (C-26)  32.8 8 39.8 C    38.8 9 47.9 CH 1.53 (1H, t) 23.5 (C-11) 1.90 (H-11) 47.7 10 37.3 C    37.6 11 23.5 CH2 1.90 (2H, dd) 124.6 (C-12); 139.8 (C-13); 47.9 (C-9) 1.53 (H-9); 5.12 (H-12) 23.6 12 124.6 CH 5.12 (1H, t) 42.3 (14); 47.9 (C-9) 1.90 (H-11) 121.8 13 139.8 C    145.1 14 42.3 C    41.8 15 26.8 CH2    26.2 16 28.2 CH2    27.0 17 33.9 C    32.5 18 47.8 CH    47.4 19 47.0 CH2    46.9 20 31.3 C    31.1 21 37.1 CH2    34.8 22 37.0 CH2    37.2 23 28.3 CH3 0.99 (3H, s) 55.4 (C-5); 79.2 (C-3)  28.2 24 15.8 CH3 0.78 (3H, s) 55.4 (C-5); 79.2 (C-3); 28,3 (C-23)  15.5 25 15.9 CH3 0.95 (3H, s) 55.4 (C-5); 38.9 (C-1); 47.9 (C-9)  15.6 26 17.0 CH3 1.00 (3H, s) 42.3 (C-14); 47.9 (C-9); 33.1 (C-7)  16.9 27 21.6 CH3 0.91 (3H, s) 39.8 (C-8)  26.0 28 28.9 CH3 0.79 (3H, s) 33.9 (C-17)  28.4 29 33.5 CH3 0.86 (3H, s) 47.0 (C-19); 23.4 (C-30); 31.3 (C-20)  33.3 30 23.4 CH3 1.06 (3H, s) 26.8 (C-21)  23.7   Characterization antibacterial constituent………………. Majalah Farmasi Indonesia, 21(2), 2010 1361H and 13C-NMR spectra were recorded with a JEOL JNM ECA-500, at 500 MHz (1H) and         125 MHz (13C). TLC analysis was performed           on   precoated   Si   Gel   plates (Kiesegel 60F254 ,Merck). MS spectra (EI-MS) was obtained on Finnigan LCQ-Deca,70 eV. The melting points were measured on Fisher John Melting point apparatus.    Plant Material Ficus deltoideus Jack leaves, were collected from Kambang, West Sumatera. The plant was identified at Herbarium of the  Biology Department, Andalas University (ANDA), and a voucher specimen (MM 001), is deposited at the Herbarium.   Extraction and Isolation The dried powder of  leaves (1 kg), of Ficus deltoideus Jack was macerated sequentially with hexane, ethyl acetate and methanol at room temperature. The combined extracts were concentrated in-vacuo, to give the hexane extract (47 g), ethyl acetate (16 g) and methanol (29 g). The ethyl acetate extract (16 g), was further fractionated by VLC with gradient   elution,   using  hexane-ethyl    acetate (10:0 – 0:10) afforded 5 fractions (F1-F5). Fraction F2 (2.8 g) was  rechromatographed    on   silica  gel  eluted  with hexane-ethyl acetate (10:0 – 0:10), to give 4 subfraction (F2.1-F2.4).  F2.3 (98 mg), was rechromatographed on silica gel eluted with hexane : ethyl acetate  9 : 1, the yellowish solid mass, was obtained and then washed with hexane to give white crystal (14 mg).   Bioassay Antibacterial activity test was carried out by measuring growth inhibition zone of E. coli, B. subtilis, and  S. Aureus, at various concentration, using disk diffusion susceptibility method (Rojas, et al., 2008 ; Zakaria, et al., 2006). The minimum inhibition concentration (MIC), was determined by  dilution method (Edberg, et al., 1986).  β-amyrin, C30H50O (Fig.1.): mp:198-199 oC ; IR (νmaks, cm-1) : 3362 (OH), 2927 and 2865 (C-H alifatik), 1139 (C-O), 1456 and 1377  (methyl and methylene) ; EI-MS (m/z) : 426 (M+), 411, 218 (base peak), 203, 189 and 175. The comparizon of 1H and 13C-NMR data of  β-amyrin and 13C-NMR β-amyrin literature8, see Table I, and  antibacterial activity, see Table II.   Figure  1.  Structure of β-amyrin.  Table II. Inhibition zone (cm) and MIC of β-amyrin againts E. coli, B. subtilis, and  S. Aureus bacteria   Compound Bacteria Concentration  % (b/v) in ethyl acetate MIC (µg/mL)  Control 0.25 0.50 1.00 2 β-Amyrin E. coli 0.0 0.9 1.0 1.2 1.3 230 B. subtilis 0.0 0.9 0.9 1.0 1.1 380 S. aureus 0.0 0.8 0.9 0.1 1.2 460  Suryati  Majalah Farmasi Indonesia, 21(2), 2010 137Result and Discussion The 1H and 13C-NMR spectra of this compound is identical to the triterpenoid. The 8 methyl protons signal (0-2 ppm) was  assigned (s,3H) at  δH(ppm) : 0.78 (H-24);           0.79  (H-28); 0.86 (H-29); 0.91 (H-27); 0.95 (H-25); 0.99 (H-23); 1.00 (H-26) and 1.06 (H-30), respectively. The 13C NMR showed 30            carbon atom signals, related to triterpenoids. DEPT analysis gave 8 CH3, 10 CH2, 5 CH          and 7 C quarternary carbon signals.               All of these signals are identical with β-amyrin carbon signals8 (Table I, Fig. 1). The              olefinic proton at δH 5.12 ppm (H-12;1H,t)  was coupled by  two neighboring methylene protons    at   δ    1.90   ppm     (H-11;2H,dd).     Correlation between H-11 and H-12 was supported by COSY analysis. So that, the double bond must be located at C-12  (δC 124.6) and C-13 (δC139.8) (Table I, Fig. 1). The signal at δH 3.21ppm (1H,t), belongs to methyneoxy proton C-3 (δC 79.2 ppm; νC-O = 1179 cm-1). This methyneoxy proton H-3 was coupled by methylene protons H-2 at 1.60 ppm (2H,m).  Correlation between H-3 and H-2    also established by COSY analysis. HMBC correlation between to methyl group protons (H-23 and H-24) with carbon C-5 and C-3, established a gem-dimethyl system at C-4.  The methyl groups position at C-10, C-8, C-17 and C-20, was also established in the same manner (Table I, Fig.1).     Figure 2. Mass fragmentation of β-amyrin. Characterization antibacterial constituent………………. Majalah Farmasi Indonesia, 21(2), 2010 138The mass spectrum of this compound showed the molecular ion (M+) at m/z: 426. The fragment ion at m/z : 218 (base peak) is characteristic to triterpenoid fragmentation. The other fragments at m/z: 203, 189           and 175 supported the proposed structure  (Fig. 2).  Based on the  antibacterial activity test, it is shown that this compound is more sensitive against E.coli, than B. subtilis and more sensitive than S. aureus. The minimum inhibition concentration (MIC) against  E. coli, B. subtilis and  S. aureus are 230, 380 and 460 µg/mL respectively. As describe early, there is no report the isolated compound from F. deltoidea and the related plants in the same genus. Althought β-amirin is a well known compound, but this is the first report of this compound from F.deltoidea Jack.  Conclusion  An antibacterial constituent, β-amyrin (C30H50O),  has been isolated from Ficus deltoideus Jack leaves. The minimum inhibition concentration (MIC) against  E. coli,  B. subtilis  and S. aureus are 230, 380 and 460 µg/mL respectively.   References  Aminudin N., Sin, C.Y., Chee, E.S., Nee, K.I., and Renxin, L. 2007., Blood glucose lowering effect of Ficus deltoidea aqueous extract. Malaysian Journal of Science 26: 73-78.   Burkill, I. H. A 1966., “Dictionary of the Economic Product of Malay peninsula”, Goverment of Malaysia and singapore by the ministery of Agriculture and Cooperatives, kuala lumpur,  Edberg, S. C. and Berger. 1986., Tes Kerentanan Antimikroba In Vitro, Penerbit Buku Kedokteran, Jakarta, 199 - 211. Musa. Y, and Mohd. Lip, J., 2007., Mas Cotek (Ficus  deltoideus), A. New potential medicinal plant in Malaysia Rojas. J, Velasco. J, Morales. A, Diaz. T, and Meccia, G. ,2008,  Evaluation of Antibacterial Activity on Different Solvent Extracts of Euphorbia caracasana B and Euphorbia cotinifolia L Collected in Venezuela. Bol. Latinoam. Caribe Plant med. Arimatic. 7(4), 198-201 Shashi. B. Mahato and Asish P. Kundu, 1994., 13C-NMR Spectra of pentacyclic       triterpenoids, A compilation and some salient featurs , Phytochemistry, 37(6), 1517-1575.  Sulaiman, M. R., M. K. Hussain, Z. A. zakaria, M. N. Somchit, S. Moin, A. S. Mohamad, and D. A. Israf,  2008, Evaluation of the antinociceptive activity of Ficus deltoidea aqueous extract, J. Fitoterapia, Zakaria, z.A, Jais, A. M. M, Henie, E.F.P, Zaiton, H. Somchit, M.N. Sulaiman, M.R, and faisal, F.O, 2006, The in-vitro Antibacterial Activity of Tinospora crispa extracts, biological science, 6(2), 398-401      * Correspondent : Suryati     Politechnic, Andalas University, Padang, Indonesian    Email : suryati_chemua@yahoo.co.id 

Characterization antibacterial constituent from Ficus deltoideusJack leaves

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Characterization antibacterial constituent from Ficus deltoideusJack leaves

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