超声辅助酶法提取茶麸黄酮及其对Ⅱ型5α-还原酶抑制活性

Ultrasound-assisted enzymatic hydrolysis of flavonoids from camellia seed cake and the inhibitory activity on 5α-reductase type Ⅱ

通过正交试验优化超声辅助酶法提取茶麸黄酮的工艺条件,同时建立了Ⅱ型5α-还原酶体外酶促反应体系考察茶麸提取物对Ⅱ型5α-还原酶的抑制活性,并通过分子对接模型对茶麸中主要黄酮组分抑制Ⅱ型5α-还原酶的机理进行了模拟,为茶麸中黄酮类活性物开发和应用提供了基础。正交试验优化的提取工艺为:50℃下,50%乙醇,酶解p H=6,添加茶麸质量2.5%的纤维素酶酶解1 h,再以45 kHz,50℃超声提取30 min,此优化条件下总黄酮得率为(10.13±0.32)mg/g。通过超高效液相色谱质谱(UPLC Q-TOF MS)初步推断茶麸中主要含有山奈酚3-O-[2-O-β-D-半乳糖-6-O-α-L-鼠李糖]-β-D-葡萄糖苷和山奈酚3-O-[2-O-β-D-木糖-6-O-α-L-鼠李糖]-β-D-葡萄糖苷两种特征黄酮类化合物。茶麸总黄酮具有Ⅱ型5α-还原酶抑制活性,分子对接模拟结果显示山奈酚3-O-[2-O-β-D-木糖-6-O-α-L-鼠李糖]-β-D-葡萄糖苷与5α-还原酶的结合能力与非那雄胺相近,可能是茶麸总黄酮中抑制Ⅱ型5α-还原酶的主要活性分子。

The process conditions of ultrasonic-assisted enzymatic hydrolysis of flavonoids from camellia seed cake were optimized by orthogonal tests,and the enzymatic reaction system of 5α-reductase type Ⅱ in vitro was established to investigate the activity of camellia seed cake extract on5α-reductase type Ⅱ.Moreover,the molecular docking model was used to simulate and predict the inhibition of 5α-reductase type Ⅱ by flavonoids from camellia seed cake,which provided the basis for the development of camellia seed cake and other plants.The results show that the extraction process optimized by the orthogonal experiment is as follows:50℃,50%ethanol,adjusting pH to 6 for enzymatic hydrolysis,adding 2.5% of camellia seed cake for enzymatic hydrolysis for 1 h,and then ultrasonically extracting at 45 kHz,50 ℃ for 30 min.The yield of total flavonoids is(10.13±0.32) mg/g under this optimized condition.It is preliminarily deduced that camellia seed cake mainly contains kaempferol 3-O-[2-0-β-D-galactose-6-O-α-L-rhamnose]-β-D-glucoside and kaempferol 3-O-[2-O-β-D-xylose-6-O-α-L-rhamnose]-β-D-glucoside by chromatography coupled with mass spectrometry(UPLC Q-TOF MS).Camellia seed cake extract has a certain inhibitory activity of 5α-reductase type Ⅱ.Through molecular docking simulation,it is speculated that the binding energy of kaempferol 3-O-[2-O-β-D-xylose-6-O-α-L-rhamnose]-β-D-glucoside to 5α-reductase is close to that of finasteride,which is considered as the main active molecule of 5α-reductase inhibitor in the total flavonoid of camellia seed cake.