Gut Microbiota Mediated Metabolism and CYP1A1 Inhibition Effects of Scutellaria Barbata D. Don
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摘要:
目的 研究半枝莲提取物经肠道菌转化前后主要化学成分含量的变化及其对CYP1A1酶抑制能力的影响。 方法 采用超高效液相色谱串联三重四级杆质谱(UPLC-Q-TRAP-MS)技术检测半枝莲提取液在肠道菌中温孵0、0.5、1、2、4、6、12、24 h后主要活性成分的经时变化过程；通过CYP1A1酶活性分析实验研究半枝莲提取液经肠道菌转化不同时间点后对CYP1A1酶抑制作用的变化，并进一步研究半枝莲中主要黄酮类成分，野黄芩素、木犀草素、汉黄芩素及其苷对CYP1A1酶的抑制作用；利用分子对接验证野黄芩素、木犀草素、汉黄芩素与CYP1A1之间的结合情况。 结果 半枝莲提取液经过肠道菌转化后，野黄芩素含量先增加后减少，木犀草素、汉黄芩素含量增加，黄酮苷类成分野黄芩苷、木犀草苷、汉黄芩苷含量随时间延长显著减少，而二萜类成分半枝莲碱A、半枝莲碱B、半枝莲碱X含量无明显变化；半枝莲提取液经肠道菌温孵2、4、6 h后对CYP1A1酶活性的抑制作用增强，而12 h后这种增强作用逐渐减弱；野黄芩素、木犀草素、汉黄芩素与CYP1A1蛋白对接后的结合自由能分别为-6.33、-6.61、-7.09 kcal/mol，配体与CYP1A1中Phe224形成的π-π堆积作用为主要相互作用。 结论 半枝莲中的黄酮类化合物是其发挥抑制CYP1A1酶活作用的主要活性物质，可能与半枝莲的清热解毒功效有关。 Abstract: OBJECTIVE To study gut microbiota mediated the changes in the concentration of main chemical components of Scutellaria barbata D.Don (S. barbata) and the vary of its inhibitory effect on CYP1A1 enzyme.METHODS Ultra-fast liquid chromatography coupled with quadrupole linear ion trap mass spectrometry (UPLC-Q-TRAP-MS) was established to quantify the constituents in co-incubation solution. CYP1A1 activity assay was used to detect the inhibitory effect of co-incubation extract, scutellarein, luteolin and wogonin on CYP1A1 enzyme. Molecular docking was used to study the binding of ligand to CYP1A1.RESULTS After the S. barbata extract was transformed by intestinal bacteria, the content of cutellarein increased at the earliest stage and then decreased, the content of luteolin and wogonin were increased, the content of scutellarin, luteoloside and wogonoside were decreased. Additionally, there was no significant change in the content of Scutebarbatine A, B, and X. The inhibitory effect of S. barbata extract on CYP1A1 enzyme activity was increased after incubating with intestinal bacteria for 2, 4, 6 h, and this enhancement decreased after 12 h. The binding energy between scutellarin, luteolin, wogonin with human CYP1A1 were -6.33, -6.61 and -7.09 kcal/mol, respectively. The main interaction was the formation of π-π stacking between the ligand and Phe224 of CYP1A1.CONCLUSION The flavonoids in S. barbata might be the main active substances that inhibit CYP1A1 enzyme activity.-
Key words:
- gut microbiome /
- Scutellaria barbata D.Don /
- CYP1A1 enzyme /
- molecular docking
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表 1 线性回归方程和相关系数
化合物 回归方程 R2 线性范围/ (μg·mL-1) 野黄芩素 Y=0.156 8X+0.054 6 0.993 4 0.401~8.02 野黄芩苷 Y=0.774 6X+0.177 8 0.997 2 0.041 5~8.300 木犀草素 Y=0.330 8X-0.005 2 0.999 0 0.062 4~1.560 木犀草苷 Y=1.140 3X+0.000 5 0.999 3 0.001 67~0.334 汉黄芩素 Y=8.201 7X+0.005 5 0.999 7 0.001 67~0.067 半枝莲碱A Y=1.310 6X+0.05 0.999 0 0.08 37~1.670 半枝莲碱B Y=7.831 7X+0.111 4 0.990 9 0.020 0~0.400 半枝莲碱X Y=1.841 6X-0.000 9 0.999 9 0.010 0~0.200 
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表 2 配体与人CYP1A1的结合能及相互作用
配体分子 结合自由能/(kcal·mol-1) 相互作用的氨基酸残基 野黄芩素 -6.33 SER122，ASN255，ASP313，SER116，PHE224，PHE123，PHE258，ALA317，GLY316，ILE115，ILE386，LEU312, LEU496，THR321，THR497 木犀草素 -6.61 SER122，ASN222，ASP313，ASP320，GLY316，PHE224，ALA317，PHE123，PHE319，PHE258，LUE254，LEU496，ILE386，THR321 汉黄芩素 -7.09 SER122，ASP313，GLY316，ALA317，PHE224，PHE123，PHE319，PHE258，ASN222，ILE115，ILE115，ILE386，LEU496，LEU254，THR321，THR497 注：1 kcal=4.18585 kJ。
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