Elicitation effect of Saccharomyces cerevisiae yeast extract on ...

N. Happyana et al. Proceeding <strong>of</strong> The International Seminar on Chemistry 2008 (pp. 257-261)Jatinangor, 30-31 October 2008Materials and MethodsGeneral experimental proceduresOptical rotations were determined using a Perkin-Elmer 141 polarimeter. UV spectra were performedon a Varian Corry 100 Conc spectrometer. FTIRspectra were carried out on a Perkin Elmer spectrumOne FTIR as a film on a KBr plate. NMR spectrawere recorded on JEOL JNM A 5000 at 400 MHz( 1 H) and 100 MHz ( 13 C). VLC and radialchromatography were carried out using Merck Si gel60 GF254 and, for TLC analysis, precoated Si gelplates (Merck Kieselgel 60 GF254, 0.25 mm) wereused.Plant and material1. M. macroura Miq. 509 hairy root culturesTransformation <strong>of</strong> the M. macroura Miq. nucleus byAgrobacterium rhizogenes 509 has assisted byBiotechnology Laboratory, Indonesian Institute <strong>of</strong>Sciences. The cultures <strong>of</strong> M. macroura Miq. 509were grown in 50 ml liquid medium <strong>of</strong> MurashigeSkoog (MS) with 0.5 ppm <strong>of</strong> indol-3-buteric acid(IBA) and shaken at 100 rpm at room temperarure.The subculture was carried out every 4 weeks. Theaddition <strong>of</strong> 0.5 ml <strong>Saccharomyces</strong> <strong>cerevisiae</strong> <strong>yeast</strong><strong>extract</strong> elicitor (2.5% w/v) was carried out when thehairy root cultures reach the age <strong>of</strong> 8 weeks, then theywere incubated for 7 days and harvested.2. Cultures <strong>of</strong> S. <strong>cerevisiae</strong>S. <strong>cerevisiae</strong> <strong>yeast</strong> was obtained from MicrobiologyLaboratory, School <strong>of</strong> Life Sciences, InstitutTeknologi Bandung. It was grown in 50 ml liquidmedium <strong>of</strong> GYE and shaken at 100 rpm at roomtemperature. The cultures <strong>of</strong> S. <strong>cerevisiae</strong> weresubcultivated every two days by transferring 10 ml <strong>of</strong>the culture into 40 ml <strong>of</strong> the fresh liquid medium. Thecultures were harvested at 16 hours old. Thesupernatants part was removed and the cells weredried and grinded to powders.Extraction and isolationThe dried powdered <strong>of</strong> hairy roots <strong>of</strong> M. macrouraMiq. 509 (35 gram) was macerated with MeOH. TheMeOH <strong>extract</strong> was fractionated by VLC (Si-gel,hexane-EtOAc, 1:1 to 2:8) into 7 fractions. Thesecond fraction (258 mg) was subjected to silica gelradial chromatography, eluted with hexane : EtOAc(6 : 4) to yield mulberr<strong>of</strong>uran P (I) (21 mg), while thefourth fraction (300 mg) was subjected to silica gelradial chromatography, eluted with hexane: EtOAc(4: 6) to afford chalcomoracin (II) (161 mg). The fifthfraction was successively purified by silica gel radialchromatography (CHCl 3 : MeOH, 9: 1) and flashsilica gel column chromatography (hexane : acetone,6:4) to yield a new compound (5 mg).HPLC analysisHPLC analysis was performed using a Shimadzu-VPsystem (Shimadzu, s’-Hortengenbosch, TheNetherlands) consisting <strong>of</strong> a LC-10AT pomp, aKontron 360 auto sampler, a SPD-M10A DADdetector, a FCV-10AL low pressure gradient mixer, aSCL-10A system controller, a FIAtron systems CH-30column heater, operated with CLASS-VP s<strong>of</strong>tware,version 6.12SP4. The column used was a Licrosphere5 RP-18 (250 x 4 mm; Chrompack, Middelburg, theNetherlands). The injection volume was 20 µl with aflow rate <strong>of</strong> 1 ml mim -1 using a time program <strong>of</strong> 45minutes. The mobile phase was consisted <strong>of</strong> Water(0.05% formic acid) (solvent A) and MeCN (0.05%formic acid) (solvent B) and used a linear gradientsystem with given time endpoints: 0.01 min: 5% B; 15min: 30% B; 30 min: 95% B; 35 min: 95% B; 40 min:5% B; and 45 min: 5% B.For HPLC analysis, the sample was prepared withdissolving 1 mg sample in 1 ml methanol, transferredinto a micro tube and closed immediately, andsubjected into a HPLC instrument. This procedure wasalso applied toward the control, the sample media, andthe control media.Cytotoxic activityThe human ovarian carcinoma cell line (OVCAR-3),at a density <strong>of</strong> 5 x 10 4 cell/ml, was seeded in 96 wellmicroplates and andalasin C (1), mulberr<strong>of</strong>uran P, andchalcomoracin were added to the cell cultures atconcentrations <strong>of</strong> (500–0.98 µg/ml). The cells werethen incubated for 48 h in 5% CO 2 incubator at 37 °Cand MTT solution was added 4 h before the end <strong>of</strong> theincubation time. Cell survival was evaluated with amultiwell scanning spectrophotometer (Asys Hitec,Expert96, Asys Co. Japan) at 540 nm. All data arepresented as the mean values <strong>of</strong> triplicates.Results and DiscussionThe MeOH <strong>extract</strong> <strong>of</strong> M. macroura hairy root wasfractionated with vacuum liquid chromatography intoseven fractions. Purification <strong>of</strong> the second fractionyielded mulberr<strong>of</strong>uran P (I) (Hano, 1986) while thefourth and the fifth fractions successively affordedchalcomoracin (II) (Takasugi, 1988) and a newcompound. The structures <strong>of</strong> these compounds weredetermined based on spectroscopic data including UV,IR, NMR, and MS spectra.In this research, 0.5 ml <strong>of</strong> S. <strong>cerevisiae</strong> <strong>yeast</strong> <strong>extract</strong>elicitor (2.5% w/v) was added into M. macroura Miq509 hairy root cultures which has achieved thestationary phase (8 weeks), and then it was incubated258