荭草中6种活性成分在正常和心肌缺血模型大鼠体内的肠吸收特征差异研究
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篇名: 荭草中6种活性成分在正常和心肌缺血模型大鼠体内的肠吸收特征差异研究
TITLE: Study on Intestinal Absorption Characteristic Differences of 6 Active Constituents of Polygonum orientale in Normal and Myocardial Ischemia Model Rats
摘要: 目的:研究荭草中6种活性成分(荭草素、异荭草素、牡荆素、原儿茶酸、山柰素-3-O-β-D-葡萄糖苷、槲皮苷)在正常和心肌缺血模型大鼠体内的肠吸收特征差异。方法:采用超高效液相色谱-串联质谱法测定大鼠肠循环灌流液中6种活性成分的含量。将80只雄性SD大鼠分为正常组和模型组,每组40只,模型组大鼠皮下注射盐酸异丙肾上腺素(50mg/kg)复制心肌缺血模型,正常组大鼠注射等量生理盐水,每日1次,连续注射2d。造模成功24h后,取正常组和模型组大鼠肠段进行在体肠循环灌流实验。分别考察荭草提取物不同质量浓度(5.0、10.0、20.0mg/mL)、不同肠段(十二指肠、空肠、回肠、结肠)、P-糖蛋白(P-gp)抑制剂(盐酸维拉帕米)和胆汁对各成分肠吸收的影响。结果:荭草素、异荭草素、牡荆素、原儿茶酸、山柰素-3-O-β-D-葡萄糖苷和槲皮苷的质量浓度线性范围分别为3.15~50.40、3.21~51.31、1.63~52.43、1.60~50.94、1.31~20.97、8.07~129.25µg/mL(r均大于0.999),定量限分别为7.86、8.45、6.52、4.00、3.28、16.14ng/mL,精密度、基质效应和稳定性试验的RSD均小于11%,准确度为85.64%~107.65%,符合生物样品定量分析要求。除模型组大鼠十二指肠中荭草素的吸收在不同质量浓度下无显著性差异外,其余5种成分在正常和模型大鼠十二指肠的吸收均随活性成分质量浓度的升高而增加,且模型组大鼠中质量浓度和/或高质量浓度活性成分(槲皮苷除外)的吸收显著少于正常组(P<0.05)。在正常组大鼠中,荭草素主要在空肠、回肠、结肠吸收较多,异荭草素主要在回肠吸收较多,牡荆素、原儿茶酸主要在空肠、回肠吸收较多,山柰素-3-O-β-D-葡萄糖苷主要在十二指肠、空肠、结肠吸收较多,槲皮苷主要在结肠吸收较多;在模型组大鼠中,荭草素主要在空肠、结肠吸收较多,异荭草素在4种肠段的吸收差别不大,牡荆素主要在回肠吸收较多,原儿茶酸、山柰素-3-O-β-D-葡萄糖苷主要在空肠吸收较多,槲皮苷主要在十二指肠、回肠吸收较多;且在同一肠段中,模型组各成分的吸收少于正常组。加入盐酸维拉帕米后,正常组大鼠中各成分的吸收均增加,但差异无统计学意义(P>0.05);模型组大鼠中,荭草素、异荭草素、牡荆素、原儿茶酸和山柰素-3-O-β-D-葡萄糖苷的吸收均显著增加(P<0.05),槲皮苷吸收也有所增加但差异无统计学意义(P>0.05)。胆汁流入十二指肠后,正常组大鼠中原儿茶酸的吸收显著增加(P<0.05);模型组大鼠中除异荭草素、槲皮苷外,其余各活性成分的吸收均显著增加(P<0.05)。结论:荭草中6种活性成分在正常和心肌缺血模型大鼠的全肠段均有吸收,且在病理状态下P-gp抑制剂和胆汁可更明显的促进上述成分的肠吸收。
ABSTRACT: OBJECTIVE:To s tudy the intestinal absorption differences of 6 kinds of active constituents of Polygonum orientale (kaempferol,isokaempferol,vitexin,protocatechuic acid ,kaempferol-3-O-β-D-glucoside and quercetin )in normal and myocardial ischemia(MI)model rats. METHODS :UPLC-MS/MS method was adopted to determine the contents of 6 active components in the intestinal circulatory perfusion fluid. Totally male SD 80 rats were divided into normal group and model group ,with 40 rats in each group. Model group was given isoproterenol hydrochloride (50 mg/kg) subcutaneously to induce MI model;normal group was given constant volume of normalsaline, once a day , for consecutive 2 days. 24 h after successful molding ,normal group and model group received in-situ intestinal circulatory perfusion experiment. The effects of different concentration s of P. orientale extract(5.0,10.0, 20.0 mg/mL),different intestinal segments (duodenum,jejunum,ileum,colon),P-glycoprotein(P-gp)inhibitors(verapamil) and bile on the intestinal absorption of each constituent were explored. RESULTS :The linear ranges of concentrations of kaempferol, isokaempferol, vitexin, protocatechuic acid , kaempferol-3-O-β-D-glucoside and quercetin were 3.15-50.40, 3.21-51.31,1.63-52.43,1.60-50.94,1.31-20.97,8.07-129.25 µg/mL(r>0.999). The lower limits of quantification were 7.86, 8.45,6.52,4.00,3.28,16.14 ng/mL,respectively. RSDs of precision ,matrix effect and stability tests were all lower than 11%; the accuracy were 85.64%-107.65%,which were in line with the requirements of biological sample quantification analysis. Except for there was no statistical significance in the absorption of kaempferol absorption in duodenum of model group at different concentrations,absorption of other five constituents in duodenum of normal and model rats increased with the increase of the concentration of active constituents ,and absorption of medium- and/or high- concentration active constituents (except quercetin )in model group was significantly lower than normal group (P<0.05). In normal group ,the absorption of kaempferol was more in jejunum,ileum and colon ,isokaempferol was more in ileum ,vitexin and protocatechuic acid were more in jejunum and ileum , kaempferin-3-O- β-D-glucoside was more in duodenum ,jejunum and colon ,quercetin was more in colon ;in the model group ,the absorption of Polygonum orientale in jejunum and colon was more ,the absorption of isokaempferol in 4 intestinal segments was little different ,vitexin was mainly absorbed in ileum ,protocatechuic acid and kaempferol- 3-O-β-D-glucoside was mainly absorbed in jejunum ,quercetin was mainly absorbed in duodenum and ileum ;in the same intestine ,the absorption of constituents in the model group was less than normal group. After adding verapamil ,absorption of all constituents in the normal group increased ,but the difference was not statistically significant (P>0.05);absorption of kaempferol ,isokaempferol,vitexin,protocatechuic acid and kaempferol- 3-O-β-D-glucoside were all increased significantly in model group (P<0.05),while there was no statistical significance in the increase of quercetin (P>0.05). After the bile flowed into the duodenum ,absorption of protocatechuic acid was increased significantly in normal group (P<0.05);absorption of other active constituents were increased significantly in model group,except for isokaempferol and quercetin (P<0.05). CONCLUSIONS :Six active constituents of P. orientale were absorbed in the whole intestine of normal and MI model rats ,and the absorption of above constituents may be enhanced more significantly by P-gp inhibitor and bile under pathological condition.
期刊: 2020年第31卷第13期
作者: 潘洁,杨淑婷,孙佳,刘春花,王永林,陆苑
AUTHORS: PAN Jie,YANG Shuting ,SUN Jia,LIU Chunhua ,WANG Yonglin ,LU Yuan
关键字: 心肌缺血;荭草;活性成分;肠吸收;肠灌流;大鼠
KEYWORDS: Myocardial ischemia ;Polygonum orientale ;Active constituent ;Intestinal absorption ;Intestinal perfusion ;Rat
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