清肺排毒汤对二肽基肽酶-Ⅳ的抑制作用研究

王安琪 田莉娟 陈晓鹏 张静 邹立伟 杨凌

摘要 目的：以自主研发的二肽基肽酶-Ⅳ（DPP-Ⅳ）特异性双光子荧光探针甘氨酰-脯氨酰-N-丁基-4-氨基-1，8-萘二甲酰亚胺（GP-BAN）为底物，人血浆为DPP-Ⅳ酶原，评价方剂清肺排毒汤及处方中中药材对DPP-Ⅳ的抑制作用，并进一步評价具有DPP-Ⅳ抑制活性中药材的成分单体化合物。方法：以建立的DPP-Ⅳ抑制剂筛选体系，分别将清肺排毒汤汤剂的喷干粉水溶液、中药颗粒水溶液、中药单体化合物与DPP-Ⅳ特异性探针GP-BAN在37 ℃条件下共同孵育，通过测定探针底物代谢产物BAN的荧光值来判断上述物质对DPP-Ⅳ的抑制程度。结果：清肺排毒汤具有较弱的DPP-Ⅳ活性的抑制作用，而其组分中药水溶液浓度为1 000 μg/mL时，麻黄、枳实、广藿香颗粒水溶液对DPP-Ⅳ的活性明显的抑制作用，酶残余活性分别为40.00%、61.05%、62.04%。橙皮素、芦丁、芹菜素、槲皮素、山柰酚、去甲基麻黄碱、川陈皮素对DPP-Ⅳ有一定的抑制作用，其中最强的3种单体成分槲皮素、山柰酚及芹菜素对DPP-Ⅳ的IC50值分别为（75.39±6.17）μmol/L、（9.16±0.69）μmol/L和（7.91±1.09）μmol/L。结论：清肺排毒汤对DPP-Ⅳ的有一定的抑制作用，存在抑制DPP-Ⅳ的成分群和单体化合物，这些发现为“清肺排毒汤”对感染新型冠状病毒肺炎（COVID-19）患者，尤其是COVID-19合并T2DM的患者的治疗提供了参考。

关键词 二肽基肽酶-Ⅳ;抑制剂;2型糖尿病;清肺排毒汤;新型冠状病毒肺炎;芹菜素;槲皮素;山柰酚

Inhibitory Effect of Qingfei Paidu Decoction on DPP-Ⅳ

WANG Anqi1，TIAN Lijuan1，CHEN Xiaopeng2，ZHANG Jing1，ZOU Liwei1，YANG Ling1

（1 Institute of Interdisciplinary Integrative Medicine Research，Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China; 2 Department of General Surgery，Shangrao Municipal Hospital，Shangrao 334000，China）

Abstract Objective：With the self-developed two-photon fluorescent probe glycyl-prolyl-N-butyl-4-amino-1，8-naphthalimide（GP-BAN） for dipeptidyl peptidase-Ⅳ（DPP-Ⅳ） detection with high specificity and sensitivity as the substrate and human plasma as the source of DPP-Ⅳ，this study aims to evaluate the inhibitory effect of Qingfei Paidu Decoction and the medicinals in the prescription on DPP-Ⅳ，and to further explore the effective compounds in the medicinals.Methods：With the established DPP-Ⅳ inhibitor screening system，the solution of Qingfei Paidu Decoction powder，the aqueous solution of the Chinese medicinal granules，and the effective compounds in the medicinals were respectively incubated with GP-BAN at 37 ℃.The inhibition on DPP-Ⅳ was evaluated based on the fluorescence value of the probe metabolite BAN.Results：Qingfei Paidu Decoction showed mild inhibitory effect on DPP-Ⅳ.The aqueous solution of Herba Ephedrae，Fructus Aurantii Immaturus，and Herba Pogostemonis granules at 1 000 μg/mL had obvious inhibitory effect on DPP-Ⅳ，and the residual activity of the enzyme was 40.00%，61.05%，and 62.04%，separately.Hesperetin，rutin，apigenin，quercetin，kaempferol，norephedrine，and nobiletin significantly suppressed DPP-Ⅳ，particularly quercetin，kaempferol，and apigenin whose half maximal inhibitory concentration（IC50） on DPP-Ⅳ was（75.39±6.17） μmol/L，（9.16±0.69） μmol/L，and（8.29±0.93） μmol/L，respectively.Conclusion：Qingfei Paidu Decoction inhibits DPP-Ⅳ，and the medicinals in the prescription and the compounds in the medicinals also show inhibitory effect on DPP-Ⅳ.These findings can serve as a reference for the treatment of coronavirus disease 2019（COVID-19） with Qingfei Paidu Decoction，especially COVID-19 complicated with type 2 diabetes mellitus（T2DM）.C8E55514-531D-42F5-A73B-86BE2AFDB859

Keywords DPP-Ⅳ; Inhibitor; Type 2 diabetes mellitus; Qingfei Paidu Decoction; COVID-19; Apigenin; Quercetin; Kaempferol

中图分类号：R289.5;R284文献标识码：Adoi：10.3969/j.issn.1673-7202.2022.08.003

二肽基肽酶-Ⅳ（Dipeptidyl Peptidase-Ⅳ，DPP-Ⅳ）是哺乳动物体内分布的一种重要的丝氨酸水解酶[1]，其参与体内多种生物活性多肽如生长因子、趋化因子、神经肽等的水解进而导致其部分或完全失活。……