白及醇提物中5种主要活性成分的在体肠吸收特征研究
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篇名: 白及醇提物中5种主要活性成分的在体肠吸收特征研究
TITLE:
摘要: 目的:考察白及醇提物中主要活性成分4-(葡萄糖氧基)-肉桂酸葡萄糖氧基苄酯(A1)、2-异丁基苹果酸(A2)、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯(A3)、二氢菲1(A4)、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯-2-(4-O-肉桂酰基-6-O-乙酰基)葡萄糖苷(A5)在大鼠肠道中的吸收动力学特征。方法:以葛根素为内标,采用超高效液相色谱-串联质谱法(UPLC-MS/MS)检测肠循环液中A1~A5的质量浓度。色谱柱为Acquity UPLC BEH C18,流动相为乙腈(含0.1%甲酸)-水(含0.1%甲酸)(梯度洗脱),流速为0.35 mL/min,柱温为45 ℃,进样量为3 μL;采用电喷雾离子源,以多反应监测模式进行正、负离子扫描,用于定量分析的离子对分别为m/z 593.2→431.1(A1)、m/z 189.0→129.0(A2)、m/z 725.3→457.2(A3)、m/z 347.1→332.1(A4)、m/z 1 059.3→793.1(A5)、m/z 417.0→267.0(内标)。采用大鼠在体肠循环灌流模型,以累计吸收转化率(A)和吸收转化速率常数(Ka)为指标,考察不同剂量白及醇提物(低、中、高剂量分别为166、333、667 μg/mL)、胆汁、P-糖蛋白(P-gp)抑制剂(维拉帕米)、不同肠段对上述5种成分肠吸收的影响。结果:A1、A2、A3、A4、A5检测质量浓度的线性范围分别为0.22~14.00、0.34~21.75、1.99~127.16、0.15~9.75、0.16~10.00 μg/mL(r>0.99),定量下限分别为0.22、0.34、1.99、0.15、0.16 μg/mL,最低检测限分别为0.028、0.085、0.251、0.035、0.010 μg/mL,日内、日间RSD均小于10%,方法回收率为83.60%~106.91%,基质效应不影响待测物的测定。白及醇提物低、中剂量组A1的A、Ka值均显著高于高剂量组,低剂量组A3的A值显著高于中、高剂量组(P<0.05或P<0.01)。不结扎组A1、A3的A、Ka值均显著低于对照组,而A4的A、Ka值均显著高于对照组(P<0.05或P<0.01);P-gp抑制剂组A1、A3的A、Ka值均显著低于对照组(P<0.05或P<0.01)。空肠组、回肠组、结肠组A1的A值以及结肠组A1的Ka值,结肠组A2的A、Ka值,回肠组A3的A值,回肠组、结肠组A4的A、Ka值,空肠组、回肠组A5的A值以及空肠组A5的Ka值均显著低于十二指肠组;而空肠组、回肠组、结肠组A3的Ka值均显著高于十二指肠组(P<0.05或P<0.01)。结论:本研究建立的UPLC-MS/MS法专属性强、灵敏度高、操作简便,可用于A1~A5的定量分析及药动学研究。白及醇提物中5种活性成分均为全肠道吸收,且吸收肠段各有不同;A1、A3在肠道中的吸收较多,可能已达饱和;胆汁可抑制A1、A2的肠吸收,但可促进A4的肠吸收;A1~A5可能均不是P-gp的底物。
ABSTRACT: OBJECTIVE: To investigate absorption kinetic characteristics of main active components as 4-(glucoseoxy)- glucoseoxybenzyl cinnamate (A1), 2-isobutyl malic acid (A2), 1,4-bis [4-(glucoxy) benzyl]-2-isobutyl malic acid ester (A3), dihydrophenanthrenes 1 (A4) and 1,4-bis [4-(glucosoxy) benzyl]-2-isobutyl malic acid ester-2-(4-O-cinnamoyl-6-O-acetyl) glucoside (A5) from ethanol extract of Bletilla striata in the intestines of rats. METHODS: Using puerarin as internal standard, UPLC-MS/MS was used to determined the concentration of A1-A5 in intestinal circulation fluid. The determination was performed on Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile (containing 0.1% formic acid)-water (containing 0.1% formic acid) (gradient elution) at the flow rate of 0.35 mL/min. The column temperature was 45 ℃, and sample size was 3 μL. The positive ion and negative ion scanning were carried out in the multiple reaction monitoring mode by electrospray ion source. The ion pairs for quantitative analysis were m/z 593.2→431.1 (A1), m/z 189.0→129.0 (A2), m/z 725.3→457.2 (A3), m/z 347.1→332.1 (A4), m/z 1 059.3→793.1 (A5), m/z 417.0→267.0 (internal standard). In the in vivo intestinal circulation perfusion model, using accumulative absorption transfer rate (A) and absorption and transformation rate constant (Ka) as indexes, the effects of different doses of ethanol extract from B. striata (low-, medium-, high-dose were 166, 333,667 μg/mL,respectively), bile, P-glycoprotein (P-gp) inhibitors (verapamil) and different intestinal segments on the absorption of above 5 components were investigated. RESULTS: The linear range of A1, A2, A3, A4 and A5 were 0.22-14.00, 0.34-21.75, 1.99-127.16, 0.15-9.75, 0.16-10.00 μg/mL(r>0.99). The limits of quantitation were 0.22, 0.34, 1.99, 0.15, 0.16 μg/mL, respectively. The lowest detection limits were 0.028, 0.085, 0.251, 0.035 and 0.010 μg/mL. RSDs of inter-day and intra-day were all lower than 10%. The recoveries ranged 83.60%-106.91%. Matrix effect did not affect the determination of the substance to be measured. A and Ka values of A1 in B. striata ethanol extract low-dose and medium-dose groups were significantly higher than high-dose group; A value of A3 in low-dose group was significantly higher than medium-dose and high-dose groups (P<0.05 or P<0.01). A and Ka values of A1 and A3 in non-ligation group were significantly lower than control group, while A and Ka values of A4 were significantly higher than control group (P<0.05 or P<0.01). A and Ka values of A1 and A3 in P-gp inhibitor group were significantly lower than control group (P<0.05 or P<0.01). A values of A1 in jejunum group, ileum group and colon group, Ka value of A1 in colon group, A and Ka values of A2 in colon group, A value of A3 in ileum group, A and Ka values of A4 in ileum group and colon group, A values of A5 in jejunum group and ileum group as well as Ka value of A5 in jejunum group were all significantly lower than duodenum group. Ka values of A3 in jejunum group, ileum group and colon group were significantly higher than duodenum group (P<0.05 or P<0.01). CONCLUSIONS: Established UPLC-MS/MS method is specific, sensitive and simple, and it can be used for quantitative analysis and pharmacokinetic study of A1-A5. The 5 active components in B. striata ethanol extract are absorbed by the whole intestine, and the intestinal segments are different. A1 and A3 are absorbed more in intestinal tract and may be saturated. Bile can inhibit intestinal absorption of A1 and A2, but promoted intestinal absorption of A4. A1-A5 may not be the substrate of P-gp.
期刊: 2019年第30卷第6期
作者: 孙慧园,陈浩,梅朝叶,郑林,巩仔鹏,李月婷,李勇军,黄勇
AUTHORS: SUN Huiyuan,CHEN Hao,MEI Chaoye,ZHENG Lin,GONG Zipeng,LI Yueting, LI Yongjun,HUANG Yong
关键字: 白及醇提物;活性化合物;在体肠循环灌流模型;超高效液相色谱-串联质谱法;肠吸收特征
KEYWORDS: Bletilla striata ethanol extract; Active compound; in vivo intestinal circulation perfusion model; UPLC-MS/MS; Intestinal absorption characteristics
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