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2.7. Bioactivity Guided Isolation and Purification The 8.5 L fermented broth was centrifuged at 4000 rpm for 30 min to separate the biomass. The 400 g of biomass was extracted with methanol and clear solvent layer was obtained by filtration. Three subsequent extractions were performed. The extracts were pooled and concentrated to make it solvent free. The dried crude extract was suspended in demineralized water and further passed through a glass column packed with diaion HP-20 resin having bed volume 225 ml. The resin was washed twice with 3 L of demineralized water to remove the unbounded components and media particles. The resin column was further eluted with water and methanol gradient with varying polarity. The bioactive fractions were pooled together and evaporated till dryness with a rotary evaporator. The methanolic extract was further fractionated with petroleum ether followed by dichloromethane. The active dichloromethane extract (500 mg) was subjected to silica gel column chromatography andeluted with gradient of chloroform-methanol. The fractions were tested for bioactivity against Candida sp. (discdiffusion assay). The active fractions were pooled together and concentrated till dryness to get semi pure sample.The semi-pure material (100 mg) was further purified by preparative chromatography using the following conditions: C-18 Eurospher column (20 mm × 250 mm, 10 μm) in isocratic mode and eluted with acetonitrile and0.01 M phosphate buffer (35:65) at a flow rate 20 ml/min with UV detection wave length at 220 nm. The pureactive fraction (eluted at 11 - 13 min) was evaporated under reduced pressure and desalted using HP-20 resinfollowed by elution with methanol. The eluted solvent was concentrated to get pure compound (20 mg). The purified compound was characterized by 1 H NMR, MS and IR spectroscopy. All the solvents used for extraction were of LR grade, where as HPLC grade solvents were used for analytical and preparative HPLC. Analytical HPLC purity was determined in Water’s Empower software LC using Kromasil RP-18, SN: 39673 (150 mm ×4.6 mm), 3.5 μm column. Normal column chromatography (CC) was performed on Silica gel (60 - 120 #) andTLC on Silica Gel 60F 254 (20 × 20 cm) aluminum sheets from Merck. NMR Spectra were recorded in DMSO-d6 on Bruker 300 MHz. Chemical shifts are expressed in ppm and tetramethylsilane was used as an internal standard. Mass spectra were taken on Bruker Daltonics system. IR spectra were recorded in pressed KBr discs with Perkin Elmer PARAGON1000 spectrometer. |
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2.7. 生物活性追踪分离与纯化 8.5 L发酵肉汤经4000rpm离心30分钟以分离生物质。用甲醇萃取该400克生物质，过滤得到清晰溶剂层。（然后）进行三次后续提取。将萃取液合并并浓缩，使其不含溶剂。将干燥的粗提物悬浮于去离子水中，并通过填充有有DIAION HP-20树脂的玻璃柱，柱床体积为225毫升。用3升去离子水洗涤树脂两次，以除去未结合组分和介质颗粒。用水和具有不同极性的甲醇梯度进一步洗脱该树脂柱。合并具有生物活性组分并用旋转蒸发器蒸发直至干燥。先用石油醚再用二氯甲烷将甲醇提取物进一步分级。将活性二氯甲烷萃取物（500毫克）进行硅胶柱层析并用氯仿 - 甲醇梯度洗脱。检测各组分抗假丝酵母的生物活性 （琼脂扩散法）。合并活性组分，浓缩至干燥，得到半纯样品。通过制备型柱层析法对半纯样品（100毫克）在下述条件下进行进一步纯化：C-18 Eurospher柱（20毫米×250毫米，10微米）在等梯度模式下用乙腈和0.01M磷酸缓冲液（35:65）洗脱，洗脱流速为20ml /分钟，并用220nm波长紫外检测。将纯的活性组分（11 - 13分钟的洗脱液）进行减压蒸发，并用HP-20树脂脱盐，并用甲醇洗脱。洗脱的溶剂进行浓缩，得到纯的化合物（20毫克）。用1H NMR、MS和红外光谱分析鉴定所纯化的化合物。所有用于萃取的溶剂都是LR级，其中，HPLC等级的溶剂用于分析和制备型HPLC。 分析HPLC纯度用Water’s Empower 软件LC进行，采用Kromasil RP-18, 序列号: 39673 （150毫米×4.6 毫米），3.5微米柱。普通柱层析（CC）用硅胶（60 - 120＃），薄层色谱法在默克公司的硅胶60F 254（20×20厘米）铝板上进行。核磁共振光谱记录在DMSO-d6上（Bruker 300 MHz）。化学位移以ppm表示，并以四甲基硅烷作为内标。 用Bruker Daltonics系统进行质谱分析。红外光谱用Perkin Elmer PARAGON1000光谱仪记录在加压KBr压片上。 |
2楼2014-10-26 11:23:35