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3.3. Isolation and Characterization of Bioactive Compound
The active compound was found to be intracellular, hence mycelial extract was subjected to bioactivity guided
isolation and purification as mentioned in 2.7. The active compound was able to adsorb on HP-20 resins. Further
fractionation of crude material using petroleum ether helped removing the impurities and subsequent dichloro-
methane fraction was found to be active. The pure compound was finally isolated by silica gel chromatography
followed by preparative HPLC using RP-18 resin. The characterization of isolated compound was carried out
based on data obtained from mass, IR and 1 H NMR spectra (Figures 3-5). In 1 H NMR spectra, the signals at δ
7.8 and 7.35 were assigned for protons attached at C-3 and C-5 respectively. Proton at unsaturated C-7 appeared
at δ 8.13 due to nitrogen and OH group being in proximity. The aromatic protons associated with other pyridine
ring were assigned in the range of δ 7.4 - 8.3. The O-methyl group showed a singlet for 3 H at δ 3.9. Structural
confirmation was done by comparing the spectral values with the published data [19]. Based on the data, the ac-
tive compound was characterized as Caerulomycin A. Figure 6 and its chemical properties are shown in Table
5. From the final yield of the isolated compound (20 mg from 8.5 L broth), the back calculated titer in the har-
vested broth was estimated as 2.3 mg/L. In order to simplify the extraction method for scale-up purification, the
whole broth extraction with equal quantity of ethyl acetate was attempted. Caerulomycin A was found to be
completely extractable in ethyl acetate hence this method could be used for large scale extraction batches.

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3.3. 生物活性化合物的分離與鑒定
生物活性化合物發(fā)現(xiàn)于細(xì)胞內(nèi)，因此，根據(jù)上述在2.7中所述的生物活性指導(dǎo)的方法對(duì)菌絲體提取物進(jìn)行分離和純化�；钚曰衔锬鼙籋P-20樹脂吸附。用石油醚對(duì)粗制品進(jìn)一步分餾以幫助除去雜質(zhì)，隨后的二氯甲烷組分發(fā)現(xiàn)具有生物活性。先用硅膠柱層析、再用RP-18樹脂進(jìn)行制備型HPLC，最終分離得到純的化合物。所分離化合物的鑒定是基于質(zhì)量、紅外線光譜和1H NMR光譜所得到的數(shù)據(jù)（圖3-5）。在1H-NMR光譜中，在δ7.8和7.35處的信號(hào)被定為分別連于C-3和C-5上的質(zhì)子。由于氮和OH基團(tuán)很近，所以位于不飽和C-7上的質(zhì)子出現(xiàn)在δ8.13。與其他吡啶環(huán)相連的芳香質(zhì)子位于δ7.4－8.3之間。 O－甲基在δ3.9處呈單峰。通過將光譜數(shù)據(jù)與已發(fā)表的數(shù)據(jù)[19]比較以進(jìn)行結(jié)構(gòu)確認(rèn)。根據(jù)該數(shù)據(jù)，將活性化合物被鑒定為青藍(lán)霉素。（結(jié)構(gòu)見）圖6和它的化學(xué)性質(zhì)見表5。根據(jù)所分離的化合物的最終產(chǎn)率（從8.5 L液體培養(yǎng)基中得到20毫克），往回推算出收獲培養(yǎng)基中的滴度約為2.3毫克/升。為了簡化提取方法以進(jìn)行規(guī)�；兓�，嘗試用等量乙酸乙酯對(duì)全培養(yǎng)基進(jìn)行提取。發(fā)現(xiàn)青藍(lán)霉素可以完全被乙酸乙酯萃取，因此可用該方法進(jìn)行大規(guī)模批次提取。

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