论文部分内容阅读
目的 探讨10批不同发酵时间的纳豆芽孢杆菌纯种发酵淡豆豉提取物的高效液相色谱 (HPLC) 图谱中主要色谱峰与促大鼠成骨细胞增殖活性的相关性,为表征纯种发酵淡豆豉抗骨质疏松作用物质基础和优化单一菌种发酵淡豆豉工艺提供依据.方法 HPLC建立纯种发酵不同时间淡豆豉的乙醇提取物指纹图谱 (纳豆芽孢杆菌纯种发酵6、12、18 h、1～7 d:H6、H12、H18、D1～D7) ;噻唑蓝 (MTT) 法测定各提取物的促大鼠颅骨成骨细胞增殖活性,并通过高效液相色谱-质谱联用 (HPLCMS) 技术对色谱峰进行确认;灰色关联度分析法研究其谱效关系.结果 纯种发酵不同时间的淡豆豉乙醇提取物在质量浓度100μg·L-1时均具有较明显的促成骨细胞增殖作用,发酵4 d时活性最强,其促成骨细胞增殖率可达43.6％.谱效关系分析表明,与药效关联度较高的是黄豆黄素、染料木苷、黄豆黄苷和大豆苷.结论 关联度分析结果中黄豆黄素,染料木苷,大豆苷与课题组前期体外活性实验结论相互佐证,证实了本实验中谱效关系分析的科学性.与前期研究结果相比,纯种发酵淡豆豉与自然发酵淡豆豉的促成骨细胞增殖活性成分基本一致.“,”OBJECTIVE To establish the fingerprint of the extract from Semen Sojae Praeparatrm fermented by different preparation, to investigate the effects on the proliferation in rat osteoblasts, and to analyze the spectrum-effect relasionship. METHODS The fingerprint of alcohol extract from Semen Sojae Praeparatrm fermented by one functional bacteria for 6, 12, 18 h and 1-7 d ( H6, H12, H18 and D1 ～ D7) were established by HPLC, and the proliferation of each extract on rat osteoblasts was measured by MTT. The spectrum-effect relasionship was analyzed by grey relation analysis. Qualitative analysis was performed by HPLC-DAD-MS for analysis of major active components in alcoholic extract. RESULTS Alcoholic extract of Semen Sojae Praeparatrm fermented by different preparation was demonstrated to show similar proliferative activity at the concentration of 100 μg·L-1. Semen Sojae Praeparatrm fermented for 4 d led to the osteoblasts proliferation rate of 43. 6％. On the basis of UV and mass spectral analysis, sixteen flavonoids were tentatively characterized. The glycitein had the highest correlation with pharmacodynamics, genistin, glycitin and daidzin also show higher correlation than the other chromatography peaks. The proliferation were promoted significantly after the treatment with glycitein, genistin, and daidzin ( 10-9-10-6 mol·L (-1) ) by the verification experiment. CONCLUSION Fermentation time of 4 d is more appropriate. The results of grey relational analysis and the activity in vitro of glycitein, genistin and daidzin mutually support each other, which confirms the scientificness of the spectral-effect relationship analysis in this study. The substance basis of anti-osteoporosis effect in the pure bacterial fermented soybean and natural fermented soybean are basically the same.