苦参异戊烯基黄酮与甘草黄酮联用体外抑菌活性评价及抗小鼠乳腺炎作用研究
Bacteriostasis of Prenylated Flavonoids from Sophora Flavescens<\i> and Flavonoids from Glycyrrhiza Uralensis<\i> Alone and Their Combination on Staphylococcus Aureus<\i> and Experimental Mastitis
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摘要: 目的 探讨苦参异戊烯基黄酮与甘草黄酮以及两者联用的体内、体外抗菌活性，为苦参和甘草的资源价值发现及其综合开发利用提供科学依据。方法 制备获得苦参异戊烯基黄酮部位（KSHT）及甘草黄酮部位（GCHT），采用UPLC-Q-TOF-MS技术对所制备的组分进行定性分析。采用金黄色葡萄球菌生长、细菌生物被膜生成及细菌黄素生成等指标来评价2种黄酮部位单用及联合用药的体外抑菌活性；进一步采用金黄色葡萄球菌致小鼠乳腺炎模型进行体内抗菌活性评价。结果 从KSHT中鉴定出20个黄酮类成分，其中18个为异戊烯基取代黄酮类化合物。从GCHT中共鉴定出22个黄酮类化合物，均为黄酮及二氢黄酮类化合物。KSHT对金黄色葡萄球菌生长抑制作用、生物被膜形成和黄素生成的抑制效果均优于GCHT，且二者联用优于各自单用，表现出协同抗菌作用。体内抗菌实验结果显示，二者高、低剂量单用及联用对小鼠乳腺炎各项指标均有一定改善作用，二者联用对乳腺炎小鼠脏器指数、组织载菌量的改善作用优于单用组，且KSHT优于GCHT；但GCHT对模型组小鼠乳腺组织病理学改善作用及对炎症因子IL-1β、IL-2、TNF-α的降低作用均优于KSHT，且与联用组相当。结论 苦参异戊烯基黄酮与甘草黄酮联用抗金黄色葡萄球菌作用优于单用，在畜牧养殖业中乳腺炎的防治中具有一定的应用前景。为苦参和甘草的资源价值发现及其综合开发利用提供科学依据，同时为制备防治奶牛乳腺炎及金黄色葡萄球菌感染的日化用品、中成药和中兽药提供参考，并为畜牧养殖业替代抗生素研究提供支撑。Abstract: OBJECTIVE To explore the antibacterial activity of prenylated flavonoids from Sophora flavecens and licorice flavonoids and their combination in vitro and in vivo， so as to provide scientific basis for the resource value discovery and comprehensive development of Sophora flavescens Ait. and Glycyrrhiza uralensis Fisch. METHODS The UPLC-Q-TOF-MS technology was applied to qualitatively analyze the prepared prenylated flavonoids from Sophora flavescens Ait. (KSHT) and licorice flavonoids (GCHT). The in vitro antibacterial activity was evaluated by investigating the effect of a single use and combined use of KSHT and GCHT on inhibiting the growth of Staphylococcus aureus and the generation of biofilm and flavin. The in vivo antibacterial activity was evaluated by building a mouse mastitis model induced by Staphylococcus aureus. RESULTS A total of 20 and 22 flavonoids were identified from KSHT and GCHT， respectively. KSHT outperformed GCHT in inhibiting the growth of Staphylococcus aureus and the generation of biofilm and flavin. Moreover， the combined use of KSHT and GCHT had better inhibitory effect than their independent use， suggesting that they have synergistic antibacterial action. Whether they were independently or jointly used in high or low doses， all the indexes of mastitis in mice were improved. Compared with the independent use of KSHT and GCHT， the combined use can better improve the viscera index and fungal burden of tissue in mice with mastitis. Regarding the improvement of indexes， KSHT was better than GCHT. In terms of the effect on improving the histopathology of mammary gland and reducing IL-1β， IL-2 and TNF-α in model group， GCHT was better than KSHT， but equivalent to the combination group. CONCLUSION The results of in vitro and in vivo tests indicated that the combined use of KSHT and GCHT has the best effect in resisting Staphylococcus aureus. It is expected that they can be jointly used in the prevention and treatment of mastitis in animal husbandry.
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