W.Z.W.M. Zain et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 520-523) ISBN 978-979-18962-0-7 Jatinangor, 30-31 October 2008 -(ε)-Viniferin an antifungal polyphenol from Vatica odorata Wan Zuraida Wan Mohd Zain 1*, Shaari Daud 1 , Neni Kartini Che Mohd Ramli 1, Liliwirianis Nawi 1, Norizan Ahmat 2 1Faculty Of Applied Sciences, UiTM Pahang, 26400 Jengka, Pahang, Malaysia, 2 Faculty Of Applied Sciences, UiTM Malaysia, 40450 Shah Alam, Selangor, Malaysia. Tel:09-4602341 Fax:09-4602455 *e-mail:[email protected] Abstract A new resveratrol dimer, -(ε)-viniferin has been isolated from the stem bark of Vatica odorata . (-)-ε-viniferin has showed antifungal activity towards these four genus of fungal i.e Rhizoctonia solani , Fusarium s p, Pythium sp . and Phytophthora sp ., the pathogens of vegetables. The in–vitro bioassay of this compound against fungal revealed that there were inhibition zone occurred for four genus of fungal tested. Keywords : antifungal, resveratrol, ε-viniferin, Vatica odorata, Dipterocarpaceae Introduction flavonoides, arylpropanoids and resveratrol oligomers. Many of the later class of compounds which form a Damping off disease refers to sudden plant death in major polyphenoic constitutens , show useful the seedling stage due to the attack of fungi, in which biological activities such as chemopreventive, most are soil burne. It caused by these four genus hepatoprotective, antiflammation, antibacterial and which has been used in this work, Rhizoctonia solani , cytotoxic (Hakim 2002). To date, there are neither Fusarium sp , Pythium sp . and Phytophtora . This medicinal nor commercial values recorded for this disease is widely distributed and is a problem on a particular species although Dipterocarpaceae species worldwide basis. It affects seeds and seedling of were known to produce high quality timber various crops. Application of the synthetic fungicides (Symington 1974). has been considered to be one of the cheapest and most common approaches for control of this disease. However, there are some reports that show the Materials and Methods resistance of this diseases to fungicide (FRAC, 2005). Furthermore, the chemical control of the disease has General Experimental Procedures several deleterious effects and hazardous to the environment. The use of natural products for control Melting points (uncorrected) were determined on a of fungal diseases in plants is considered interesting micro-melting point apparatus. Optical rotations alternative to synthetic fungicides due to their lower were recorded on a Perkin-Elmer 341 polarimeter. negatitive impacts on the environment (David and UV and IR spectra were measured with a Varian 100 Conc and FTIR Spectrum One Perkin-Elmer Barbara, 2006). Many plant extracts have been 1 13 confirmed to exhibit inhihibitory effects on some instruments, respectively. H and C NMR spectra were recorded with a JEOL ECP400 spectrometer pathogenic organisms and such have been used for the 1 13 control of pest problems (Awuah, 1994). It also have operating at 400 ( H) and 100 ( C) MHz, using been reported that plant extracts and plants essential residual and deuterated solvent peaks as reference oils are effective as antimicrobial agents against food standards. MS spectra were measured at 70eV. and grain storage fungi (Bishop and Thorton, 1997) HRFABMS were recorded on a JEOL JMS AX- and nematodes (Walker 1998). Vatica odorata 500. Vacuum liquid chromatography (VLC) and (Dipterocarpaceae) is a medium size trees ranges from radial chromatography were carried out using Indo-China to Malaysia, occurs in the lowland area at Merck Si gel 60 GF 254 and, for TLC analysis, 350-500 m elevation. The plant can be easily precoated Si gel plates (Merck Kieselgel 60 GF 254 , identified from other Dipterocarpaceae species due to 0.25 mm) were used. its hairy rough pink twigs and pale yellow flowers. This family of plant produces a wide variety of natural products, including among others terpenoides, 520 W.Z.W.M. Zain et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 520-523) Jatinangor, 30-31 October 2008 Plant Materials Results and Discussion Samples of the tree bark of V. odorata were collected from the Pulau Mata Kail, Belum Forest Reserve, Perak, Malaysia. The plant was identified by botanist, University Putra Malaysia, and a voucher specimen H O OH was deposited in the herbarium. HO Extraction and Isolation H The dried powdered tree bark (0.45 kg) of V. odorata was macerated with acetone (3 x 4L) followed by methanol (3 x 4L), and each extract was evaporated HO under reduced pressure to give dark brown residues. The MeOH (75g) extract was also subjected to fractionation using VLC (silica gel, n-hexane-EtOAc OH = 10:0, 8:2, 6:4, 4:6, and 2:8) into five major fractions F-J. Fraction G (2.2g) was refractionated by flash column chromatography (silica gel, n-hexane-EtOAc = 10:0 to 8:2) followed by radial chromatography (silica gel, CHCl 3-MeOH = 10:0 to 8:2) afforded (-)- OH ε-viniferin (313mg ). (-)-εεε-viniferin Assays for Antifungal Activity MP: 187-189ºC. [α] : -42º( c 0.1, MeOH). Tested Extracts D Rf : 0.62 (CHCl -MeOH, 8:2). 3 IR (KBr): 3420, 2920, 1600, 1512, 834 cm -1. (-)-ε-viniferin was tested for antifungal activity. It was UV/Vis λ (MeOH) nm : 203, 226, 322. filtered sterile through 0.45 mm membrane filter max before testing. The methanol solvent was used as a test 1H NMR (400 MHz, CD COCD : 7.20 (2H, d, J = control. 3 3 8.4, H-2a/H-6a), 6.83 (2H, d, J = 8.6, H-3a/H-5a), 5.41 (1H, d, J = 5.5 Hz, H-7a), 4.46 (1H, d, J = 5.1 Microorganisms Hz, H-8a), 6.24 (3H, br s, H-10a/H-12a/H-14a), 7.17 (2H, d, J = 8.4 Hz, H-2b/H-6b), 6.73 (2H, d, J = 8.4 Four genuses of fungi were tested for anti-fungal Hz, H-3b/H-5b), 6.91 (1H, d, J = 16.5 Hz, H-7b), 6.70 activity i.e Rhizoctonia solani , Fusarium sp , Pythium (1H, t, J = 16.5 Hz, H-8b), 6.32 (1H, d, J = 2.2 Hz, H- sp. and Phytophthora sp ., the pathogens of vegetables. 12b), 6.72 (1H, d, J = 2.2 Hz, H-14b). The fungi was isolated from diseased vegetables. The fungi was isolated on Potato Dextrose agar (PDA) and 13 C NMR (100 MHz CD 3COCD 3): 133.6 (C-1a), was identified based on their morphological 127.9 (C-2a/C-6a), 116.1 (C-3a/C-5a), 158.2 (C-4a), characteristics. They were maintained on PDA plates 93.9 (C-7a), 57.1 (C-8a), 147.6 (C-9a), 106.9 (C- for further experiment. 10a/C-14a), 159.9 (C-11a/C-13a), 102.1 (C-12a), 129.9 (C-1b), 128.6 (C-2b/C-6b), 116.4 (C-3b/C-5b), Preparation of Inoculate 158.2 (C-4b), 130.1 (C-7b), 123.5 (C-8b), 136.3 (C- 9b), 119.8 (C-10b), 162.4 (C-11b), 96.8 (C-12b), The 5 mm mycelia plug of each fungi was taken from 159.7 (C-13b), 104.2 (C-14b). + the leading edge of 4 old day culture and placed at the HRMS-EI: m/z [M ] calcd for C 28 H22 O6: 454.1425; centre of the Potato Dextrose Agar (PDA) plate. The 5 found: 454.1416. mm paper disc was then soaked with crude extracts (10µL) and placed at 2 sides of the plates. The plates was then incubated at 28ºC and observed for inhibition zone everyday for 5 days incubation. The mycelial growth of the fungi was measured. 521 W.Z.W.M. Zain et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 520-523) Jatinangor, 30-31 October 2008 Table 1: Results (mean of radial growth inhibition of fungal pathogens) of antifungal screening of (-)-ε-viniferin Radial growth (cm)/mean Fungi Days 1 2 3 4 5 Rhizoctonia solani 0.5 0.9 1.4 1.4 1.6 Control 0.4 1.0 1.6 2.0 3.1 Fusarium sp 0.6 1.3 1.4 1.4 1.5 Control 0.6 1.2 1.8 2.0 3.5 Phytium sp 0.2 0.2 0.7 1.1 1.4 Control 0.3 1.1 0.8 1.0 2.4 Phytophtora sp 0.6 0.9 1.2 1.3 1.6 Control 0.4 1.0 1.4 1.5 3.0 Growth inhibition of Fusarium sp 4 3.5 A B 3 2.5 Viniferin 2 Control 1.5 1 Radial growth Radial (cm) growth 0.5 0 C D D/1 D/2 D/3 D/4 D/5 Day Growth inhibition of Pythium sp ε 3 Figure 2: Antifungal activity screening of (-)- - 2.5 viniferin against fungal pathogens. 2 A: Rhizoctonia solani B: Fusarium sp . Viniferin 1.5 C: Phytium sp. D: Phytophtora sp. Control 1 0.5 In our previous papers, we have studied Radial growth (cm0 0 antifungal activity on the acetone crude extract of D/1 D/2 D/3 D/4 D/5 Vatica odorata (Wan Zuraida et. al. 2006). We Day have conclude that the positive effects of antifungal activities of acetone crude extracts against tested Growth inhibition of Phytophthora sp fungi may be due to a few factors such as the 3.5 3 existence of antifungal substances in extracts 2.5 (phytoalexins) and the synergistic effects of the 2 Viniferin chemical compounds. 1.5 Control 1 For ongoing study on antifungal of the Vatica Radial growth (cm) growth Radial 0.5 odorata plant we have purified and isolate the 0 compound first before tested to the fungi.