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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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 C_(18),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3μL;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、667μg/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、0.16～10.00μg/mL(r>0.99),定量下限分别为0.22、0.34、1.99、0.15、0.16μg/mL,最低检测限分别为0.028、0.085、0.251、0.035、0.010μg/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、K_a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、K_a值均显著低于对照组,而A4的A、K_a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、K_a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K_a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、K_a值,空肠组、回肠组A5的A值以及空肠组A5的K_a值均显著低于十二指肠组;而空肠组、回肠组、结肠组A3的K_a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。
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 m L/min. The column temperature was 45 ℃,and sample size was 3 μ L. 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(K_a )as indexes,the effects of different doses of ethanol extract from B. striata(low-,medium-,high-dose were 166,333,667 μ g/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/m L(r>0.99). The limits of quantitation were 0.22,0.34,1.99,0.15,0.16 μg/m L,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 K_a 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 K_a values of A1 and A3 in non-ligation group were significantly lower than control group,while A and K_a values of A4 were significantly higher than control group(P<0.05 or P<0.01). A and K_a 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,K_a value of A1 in colon group,A and K_a values of A2 in colon group,A value of A3 in ileum group,A and K_a values of A4 in ileum group and colon group,A values of A5 in jejunum group and ileum group as well as K_a value of A5 in jejunum group were all significantly lower than duodenum group. K_a 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.
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 m L/min. The column temperature was 45 ℃,and sample size was 3 μ L. 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(K_a )as indexes,the effects of different doses of ethanol extract from B. striata(low-,medium-,high-dose were 166,333,667 μ g/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/m L(r>0.99). The limits of quantitation were 0.22,0.34,1.99,0.15,0.16 μg/m L,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 K_a 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 K_a values of A1 and A3 in non-ligation group were significantly lower than control group,while A and K_a values of A4 were significantly higher than control group(P<0.05 or P<0.01). A and K_a 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,K_a value of A1 in colon group,A and K_a values of A2 in colon group,A value of A3 in ileum group,A and K_a values of A4 in ileum group and colon group,A values of A5 in jejunum group and ileum group as well as K_a value of A5 in jejunum group were all significantly lower than duodenum group. K_a 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.
引文
[1]国家药典委员会.中华人民共和国药典:一部[S].2015年版.北京:中国医药科技出版社,2015:103.
[2]杨铠银.白及药理作用研究进展[J].科技展望,2016,26(24):312.
[3]HE X,WANG X,FANG J,et al.Bletilla striata:medicinal uses,phytochemistry and pharmacological activities[J].JEthnopharmacol,2017.DOI:10.1016/j.jep.2016.11.026.
[4]孙爱静,庞素秋,王国权.白及化学成分与抗肿瘤活性研究[J].中国药学杂志,2016,51(2):101-104.
[5]仰莲,彭成,李小红,等.白及的化学成分及生物活性研究进展[J].中药与临床,2014,5(6):59-64.
[6]李伟平,何良艳,丁志山,等.白及的应用及资源现状[J].中华中医药刊,2012,30(1):158-160.
[7]戴鸥,仰莲,周勤梅,等.白及化学成分分离鉴定[J].中国实验方剂学杂志,2018,24(14):43-47.
[8]马先杰,崔保松,韩少伟,等.中药白及的化学成分研究[J].中国中药杂志,2017,42(8):1578-1584.
[9]饶瑞瑛,任元雪,李欣瑜,等.白及提取物抗变异链球菌致龋效果的研究[J].中国微生态学杂志,2018,30(3):291-295、299.
[10]张兵,史亚,周芳美,等.白及提取物体外抗流感病毒药效及其机理研究[J].中药材,2017,40(12):2930-2935.
[11]刘珈羽,郭换,肖佳雯,等.不同粒径白及粉的粉体学性质及体外溶出度比较[J].中国实验方剂学杂志,2018,24(3):25-29.
[12]陈鹏程,侯靖宇,胡杰,等.杜仲提取物中4个主要成分的大鼠在体肠吸收[J].中国医药工业杂志,2015,46(7):730-735.
[13]伍萍,李梅,巩仔鹏,等.基于在体循环肠灌流模型分析羊耳菊提取物的肠吸收特性[J].中国实验方剂学杂志,2018,24(2):1-8.
[14]赵菲菲,杨馨,徐丹,等.白及非多糖组分的止血作用及其机制的初步研究[J].中国药理学通报,2016,32(8):1121-1126.
[15]国家药典委员会.中华人民共和国药典:四部[S].2015年版.北京:中国医药科技出版社,2015:363-368.
[16]杨武,侯佳,陆苑,等.头花蓼提取物的大鼠在体肠吸收研究[J].中国中药杂志,2015,40(21):4281-4287.
[17]刘跃.头花蓼的口服吸收机制及其药代动力学研究[D].贵阳:贵州医科大学药学院,2015.
[18]刘亚丽,孙振,张文秀,等.白头翁皂苷B3的溶解度及油水分配系数与大鼠在体肠吸收研究[J].药物评价研究,2016,39(1):87-91.
[19]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[20]陈慧慧,姚银辉,鲁澄宇.在体肠灌流法用于中药有效成分吸收的研究进展[J].中国药师,2011,14(11):1677-1679.
[21]翟永松,杜守颖,徐冰,等.三七总皂苷油水分分配系数及大鼠在体肠吸收动力学研究[J].中国中药杂志,2010,35(8):984-988.
[22]苗燕飞,陈国广,任丽莉,等.盐酸齐拉西酮大鼠在体肠吸收动力学[J].南京工业大学学报(自然科学版),2017,39(1):133-136.
[23]徐术,刘忠良,胡晋红.药物肠吸收的实验研究方法概述[J].中国药房,2009,20(16):1266-1268.
[24]雷燕,张建华,张明,等.P-糖蛋白抑制剂研究进展[J].广州化工,2016,44(6):27-30.
[25]高秀蓉,蒋学华,杜青青,等.P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响[J].华西药学杂志,2013,28(2):135-139.
[2]杨铠银.白及药理作用研究进展[J].科技展望,2016,26(24):312.
[3]HE X,WANG X,FANG J,et al.Bletilla striata:medicinal uses,phytochemistry and pharmacological activities[J].JEthnopharmacol,2017.DOI:10.1016/j.jep.2016.11.026.
[4]孙爱静,庞素秋,王国权.白及化学成分与抗肿瘤活性研究[J].中国药学杂志,2016,51(2):101-104.
[5]仰莲,彭成,李小红,等.白及的化学成分及生物活性研究进展[J].中药与临床,2014,5(6):59-64.
[6]李伟平,何良艳,丁志山,等.白及的应用及资源现状[J].中华中医药刊,2012,30(1):158-160.
[7]戴鸥,仰莲,周勤梅,等.白及化学成分分离鉴定[J].中国实验方剂学杂志,2018,24(14):43-47.
[8]马先杰,崔保松,韩少伟,等.中药白及的化学成分研究[J].中国中药杂志,2017,42(8):1578-1584.
[9]饶瑞瑛,任元雪,李欣瑜,等.白及提取物抗变异链球菌致龋效果的研究[J].中国微生态学杂志,2018,30(3):291-295、299.
[10]张兵,史亚,周芳美,等.白及提取物体外抗流感病毒药效及其机理研究[J].中药材,2017,40(12):2930-2935.
[11]刘珈羽,郭换,肖佳雯,等.不同粒径白及粉的粉体学性质及体外溶出度比较[J].中国实验方剂学杂志,2018,24(3):25-29.
[12]陈鹏程,侯靖宇,胡杰,等.杜仲提取物中4个主要成分的大鼠在体肠吸收[J].中国医药工业杂志,2015,46(7):730-735.
[13]伍萍,李梅,巩仔鹏,等.基于在体循环肠灌流模型分析羊耳菊提取物的肠吸收特性[J].中国实验方剂学杂志,2018,24(2):1-8.
[14]赵菲菲,杨馨,徐丹,等.白及非多糖组分的止血作用及其机制的初步研究[J].中国药理学通报,2016,32(8):1121-1126.
[15]国家药典委员会.中华人民共和国药典:四部[S].2015年版.北京:中国医药科技出版社,2015:363-368.
[16]杨武,侯佳,陆苑,等.头花蓼提取物的大鼠在体肠吸收研究[J].中国中药杂志,2015,40(21):4281-4287.
[17]刘跃.头花蓼的口服吸收机制及其药代动力学研究[D].贵阳:贵州医科大学药学院,2015.
[18]刘亚丽,孙振,张文秀,等.白头翁皂苷B3的溶解度及油水分配系数与大鼠在体肠吸收研究[J].药物评价研究,2016,39(1):87-91.
[19]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[20]陈慧慧,姚银辉,鲁澄宇.在体肠灌流法用于中药有效成分吸收的研究进展[J].中国药师,2011,14(11):1677-1679.
[21]翟永松,杜守颖,徐冰,等.三七总皂苷油水分分配系数及大鼠在体肠吸收动力学研究[J].中国中药杂志,2010,35(8):984-988.
[22]苗燕飞,陈国广,任丽莉,等.盐酸齐拉西酮大鼠在体肠吸收动力学[J].南京工业大学学报(自然科学版),2017,39(1):133-136.
[23]徐术,刘忠良,胡晋红.药物肠吸收的实验研究方法概述[J].中国药房,2009,20(16):1266-1268.
[24]雷燕,张建华,张明,等.P-糖蛋白抑制剂研究进展[J].广州化工,2016,44(6):27-30.
[25]高秀蓉,蒋学华,杜青青,等.P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响[J].华西药学杂志,2013,28(2):135-139.