目的 應(yīng)用體外實(shí)驗(yàn)方法篩選淫羊藿次苷Ⅱ在肝微粒體中的尿苷二磷酸葡萄糖醛酸轉(zhuǎn)移酶（UGT）抑制劑，為改善淫羊藿次苷Ⅱ生物利用度提供新思路。方法 首先選擇槲皮素、山柰酚、桔皮素、柚皮素、水飛薊素、草質(zhì)素、胡椒堿、甘草查爾酮A以及異銀杏雙黃酮作為抑制劑篩選對(duì)象，對(duì)以上9種化合物在人肝微粒體、人腸微粒體、大鼠肝微粒體、猴肝微粒體及小型豬肝微粒體中對(duì)淫羊藿次苷Ⅱ葡萄糖醛酸化反應(yīng)的抑制作用進(jìn)行初步研究。抑制劑分別選擇低、中、高3個(gè)濃度（1、10、100 μmol/L），采用超快速高效液相色譜（UFLC）法測(cè)定代謝產(chǎn)物生成速率，以UGT代謝酶殘余活性評(píng)價(jià)抑制能力。從中篩選出抑制能力較強(qiáng)的化合物（半數(shù)抑制濃度IC₅₀≤10 μmol/L），對(duì)其在人肝微粒體中的抑制機(jī)制進(jìn)行系統(tǒng)研究并計(jì)算IC₅₀及抑制常數(shù)（K_i）值。IC₅₀值的測(cè)定采用單一底物濃度法，在不同濃度代謝酶抑制劑的孵育體系中，代謝產(chǎn)物的生成速率不同，應(yīng)用非線性回歸分析計(jì)算而得。K_i的測(cè)定需在孵育體系中設(shè)計(jì)3～4個(gè)底物濃度以及4～5個(gè)包括0點(diǎn)在內(nèi)的抑制劑濃度，抑制動(dòng)力學(xué)類型通過Dixon作圖法和Lineweaver-Burk作圖法確定，采用二次作圖法計(jì)算K_i。結(jié)果 山柰酚、槲皮素及甘草查爾酮A對(duì)淫羊藿次苷Ⅱ在不同種屬肝微粒體及人腸微粒體中的葡萄糖醛酸化反應(yīng)均具有較強(qiáng)的抑制作用；對(duì)在人肝微粒體中的葡萄糖醛酸化反應(yīng)抑制作用的IC₅₀值分別為（1.01±0.26）、（4.65±0.51）、（5.34±1.00） μmol/L；Dixon作圖法及Lineweaver-Burk作圖法表明，甘草查爾酮A能夠競(jìng)爭(zhēng)性抑制淫羊藿次苷Ⅱ在人肝微粒體中的葡萄糖醛酸化反應(yīng)，K_i值為0.18 μmol/L；槲皮素遵循混合型抑制動(dòng)力學(xué)模型，K_i值為0.23 μmol/L；山柰酚符合非競(jìng)爭(zhēng)型抑制動(dòng)力學(xué)模型，K_i值為0.36 μmol/L。結(jié)論 山柰酚、槲皮素及甘草查爾酮A能夠降低淫羊藿次苷Ⅱ在不同種屬肝微粒體中的葡萄糖醛酸化反應(yīng)速率，使代謝產(chǎn)物生成減少，清除減慢。

Objective To screen the UDP-glucuronosyltransferase (UGT) inhibitors of icariside Ⅱ in liver microsomes in vitro and provide a new idea to improve the bioavailability of icariside Ⅱ. Methods In this study, the inhibitory effects of quercetin, kaempferol, hesperetin, naringenin, silymarin, piperine, herbacetin, licochalcone A, and isoginkgetin against icariside Ⅱ glucuronidation were assessed firstly. Icariside Ⅱ was incubated in human, rat, monkey, minipig liver microsomes and human intestinal microsomes with varying inhibitors concentration (1, 10, and 100 μmol/L). An UFLC based method was used to quantify the glucuronide of icariside Ⅱ. The residual activity of UGT enzymes was used to evaluate the inhibitory capacity. The inhibitory mechanism and kinetic parameters of inhibitors with the IC₅₀ values less than or equal to 10 μmol/L were investigated in human liver microsomes. IC₅₀ values were determined by using a single substrate concentration with various concentration of inhibitors and were calculated by non-linear regression analysis. Inhibition kinetic parameters (K_i) were determined by using various concentration of icariside Ⅱ in the presence or absence of various concentration of inhibitors. The inhibition kinetic type was evaluated by determining the intersection point in Dixon and Lineweaver-Burk plots. The second plot of slopes from Lineweaver-Burk plot vs inhibitors concentration was utilized to calculate the corresponding inhibition parameter values. Results The catalytic activities of human, rat, and monkey minipig liver microsomes and human intestinal microsomes were strongly inhibited by quercetin, kaempferol, and licochalcone A in icariside Ⅱ glucuronidation. The calculated IC₅₀ values of quercetin, kaempferol, and licochalcone A in human liver microsomes were (1.01±0.26), (4.65±0.51), and (5.34±1.00) μmol/L, respectively. Both Lineweaver-Burk and Dixon plots demonstrated that licochalcone A was a competitive inhibitor for icariside Ⅱ glucuronidation in human liver microsoms with the K_i value of 0.18 μmol/L. Quercetin was a mixed inhibitor for human liver microsomes mediated icariside Ⅱ glucuronidation with the K_i value of 0.23 μmol/L. Kaempferol is a noncompetitive inhibitor for icariside Ⅱ glucuronidation in human liver microsomes with the K_i value of 0.36 μmol/L. Conclusion The glucuronidation of icariside Ⅱ in different species liver microsomes can be strongly inhibited by quercetin, kaempferol, and licochalcone A.

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃（973計(jì)劃）項(xiàng)目（2013CB531800）