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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. 生物活性追蹤分離與純化
8.5 L發(fā)酵肉湯經(jīng)4000rpm離心30分鐘以分離生物質(zhì)。用甲醇萃取該400克生物質(zhì)，過濾得到清晰溶劑層。（然后）進(jìn)行三次后續(xù)提取。將萃取液合并并濃縮，使其不含溶劑。將干燥的粗提物懸浮于去離子水中，并通過填充有有DIAION HP-20樹脂的玻璃柱，柱床體積為225毫升。用3升去離子水洗滌樹脂兩次，以除去未結(jié)合組分和介質(zhì)顆粒。用水和具有不同極性的甲醇梯度進(jìn)一步洗脫該樹脂柱。合并具有生物活性組分并用旋轉(zhuǎn)蒸發(fā)器蒸發(fā)直至干燥。先用石油醚再用二氯甲烷將甲醇提取物進(jìn)一步分級。將活性二氯甲烷萃取物（500毫克）進(jìn)行硅膠柱層析并用氯仿 - 甲醇梯度洗脫。檢測各組分抗假絲酵母的生物活性（瓊脂擴(kuò)散法）。合并活性組分，濃縮至干燥，得到半純樣品。通過制備型柱層析法對半純樣品（100毫克）在下述條件下進(jìn)行進(jìn)一步純化：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磷酸緩沖液（35:65）洗脫，洗脫流速為20ml /分鐘，并用220nm波長紫外檢測。將純的活性組分（11 - 13分鐘的洗脫液）進(jìn)行減壓蒸發(fā)，并用HP-20樹脂脫鹽，并用甲醇洗脫。洗脫的溶劑進(jìn)行濃縮，得到純的化合物（20毫克）。用1H NMR、MS和紅外光譜分析鑒定所純化的化合物。所有用于萃取的溶劑都是LR級，其中，HPLC等級的溶劑用于分析和制備型HPLC。分析HPLC純度用Water’s Empower 軟件LC進(jìn)行，采用Kromasil RP-18, 序列號: 39673 （150毫米×4.6 毫米），3.5微米柱。普通柱層析（CC）用硅膠（60 - 120＃），薄層色譜法在默克公司的硅膠60F 254（20×20厘米）鋁板上進(jìn)行。核磁共振光譜記錄在DMSO-d6上（Bruker 300 MHz）�；瘜W(xué)位移以ppm表示，并以四甲基硅烷作為內(nèi)標(biāo)。用Bruker Daltonics系統(tǒng)進(jìn)行質(zhì)譜分析。紅外光譜用Perkin Elmer PARAGON1000光譜儀記錄在加壓KBr壓片上。

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