四逆散中枳实—芍药、芍药—甘草药对配伍肠吸收特性研究
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摘要
复方配伍是中药临床用药的特色之一,药对是复方配伍中最小的单位,具有结构简单、取效明确、易于开展科学研究等优势,是研究复方配伍机理有利的切入点。长期以来,中医药学家对中药复方配伍机理进行了深入的研究和探索,但多侧重于复方(药对)配伍的临床、化学、药理方面的作用机理研究,较少关注复方(药对)配伍对药物体内过程的影响。现代医药学研究证明,口服后药物产生药理作用必须先经过胃肠道吸收,中药多为复方口服给药,因此,有必要研究中药复方有效成分和有效组分的吸收代谢动力学及复方配伍后的体内相互作用,从肠吸收代谢的角度阐述中药复方(药对)配伍的作用机理。
四逆散方始见于汉代张仲景《伤寒论》,是调和肝脾的名方,由芍药、枳实、柴胡、甘草四味药组成,包含了枳实-芍药、芍药-甘草、柴胡-芍药、柴胡-枳实四个药对,具有疏肝解郁作用,临床常以本方加减治疗肝脾气郁所致胁肋脘腹疼痛等症。本研究重点应用研究药物肠吸收代谢的经典模型:大鼠在体肠灌流模型对四逆散中枳实-芍药、芍药-甘草药对中主成分和提取物配伍的肠吸收特性进行了深入的研究,以期从肠吸收代谢的角度阐明四逆散药对的配伍机理。
本论文首先分别对中药复方配伍机理的研究进展、四逆散中药对研究进展及药物吸收代谢研究手段的研究进展进行了文献综述。实验部分主要完成了以下研究:
1.本论文首先从枳实、芍药、甘草药材提取物的制备入手,建立了枳实药材中黄酮类成分柚皮苷、橙皮苷、新橙皮苷的UPLC测定方法,比较了不同产地枳实药材中上述3种成分的含量,选择了含量比例适中的枳实药材供实验用。以四逆散临床使用汤剂为依据,制备了各药材水提取物,并对枳实中柚皮苷、橙皮苷和新橙皮苷,芍药中芍药苷,甘草中甘草酸、甘草苷的含量进行了测定。结果:枳实提取物中柚皮苷、橙皮苷、新橙皮苷的含量分别为52.65 mg·mL~(-1)、9.25 mg·mL~(-1)、66.25 mg·mL~(-1),芍药提取物中芍药苷的含量为22.41 mg·mL~(-1),甘草提取物中甘草酸、甘草苷的含量分别为16.35 mg·mL~(-1),22.38 mg·mL~(-1)。
2.考察了枳实-芍药、芍药-甘草药对中主成分的理化性质。
(1)参照《中国药典》方法考察了枳实-芍药、芍药-甘草药对中主成分柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷的近似溶解度;采用摇瓶法测定了各成分的油水分配系数(logP值),结果分别为柚皮苷(-0.44)、橙皮苷(-0.39)、新橙皮苷(-0.31)、芍药苷(-0.67)、甘草酸(0.78)和甘草苷(0.16),结合Marvin在线软件对各成分理化参数的进行预测,并根据预测口服药物生物利用度的“五规则”,推测各成分的体内吸收情况,结果:仅从理化性质上判断,上述各成分的体内吸收情况可能均较差。
(2)为保证大鼠在体肠灌流试验的准确可靠,对各成分的化学稳定性进行了考察。采用恒温法,以pH值为5.5～8.0的Hank's平衡盐溶液作为溶解介质,在37℃条件下考察各成分在24h内的稳定性。实验结果表明柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷在pH值为5.5～8.0的HBSS溶液中均比较稳定,可以进行在体实验。
3.建立了药对中各主成分大鼠在体肠灌流样品的分析方法。
建立了柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷在体肠灌流样品的UPLC测定方法,血浆和胆汁样品的HPLC测定方法。实验结果表明,各成分的线性良好,精密度、重复性、稳定性、回收率符合要求,所建方法可行。
4.应用大鼠在体肠灌流模型对枳实-芍药、芍药-甘草药对中主成分和各提取物的肠吸收代谢进行了研究,考察了20、50、100μmol·L~(-1)3个浓度各成分及提取物的肠吸收情况。结果表明,各成分及其在提取物中,在大鼠十二指肠、空肠、回肠、结肠的吸收都较差,有效渗透系数(P_(eff)~*值)均＜0.5,但提取物中成分的吸收与相应单体成分比较有不同程度的提高。经SPSS13.0统计软件分析,枳实提取物中柚皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠41.1%,空肠39.9%,回肠32.57%;其中橙皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠45.83%,空肠47.17%,回肠30.85%;其中新橙皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠53.83%,空肠47.61%,回肠32.05%,其它浓度枳实提取物中成分与相应浓度单体比较也均有显著性增加,表明枳实提取物中其他成分会促进柚皮苷、橙皮苷、新橙皮苷的肠内吸收。芍药提取物中芍药苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):空肠24.92%,回肠36.33%,其它浓度芍药提取物中芍药苷与相应浓度芍药苷比较也有显著性增加,表明芍药提取物中其他成分也会促进芍药苷的肠内吸收。甘草提取物中甘草苷(20μmol·L~(-1))与单体相比在回肠段吸收百分率显著增加20.22%(p＜0.05),其它浓度甘草提取物中甘草苷与相应浓度甘草苷比较也有显著性增加,而甘草酸则无显著性变化,表明甘草提取物中其它成分会促进甘草苷吸收,但对甘草酸无显著影响。
5.研究了枳实-芍药、芍药-甘草药对主成分配伍和提取物配伍的肠吸收特性,初步阐述枳实-芍药、芍药-甘草药对的配伍机理。
选择不同配伍比例研究药对配伍的肠吸收情况,单体成分配伍以浓度比例为1∶1,1∶5,5∶1进行试验,提取物配伍以生药量比例为1∶1,1∶5,5∶1进行试验。
枳实-芍药药对研究结果显示,柚皮苷与芍药苷1∶1配伍时,与单体成分比较,柚皮苷在十二指肠和空肠的吸收百分率分别增加5.94%和5.37%,芍药苷在十二指肠、空肠段的吸收百分率分别降低8.37%和8.92%,1∶5和5∶1比例配伍时柚皮苷和芍药苷的变化与1∶1配伍时趋势相同;橙皮苷与芍药苷1∶1配伍时,与单体成分比较,橙皮苷的吸收百分率分别增加十二指肠11.76%,空肠8.09%,回肠9.57%,结肠18.78%,芍药苷在十二指肠、结肠段的吸收百分率分别降低2.89%和6.89%,1∶5和5∶1比例配伍时橙皮苷的变化与1∶1配伍时趋势相同;新橙皮苷与芍药苷1∶1配伍时,与单体成分比较,新橙皮苷在回肠段的吸收百分率增加9.23%,1∶5和5∶1比例配伍时新橙皮苷的变化与1∶1配伍时趋势相同。枳实芍药提取物以5∶1配伍时,与单独枳实提取物比较,柚皮苷在十二指肠和空肠的吸收百分率分别提高6.93%和14.02%;枳实芍药提取物以1∶5配伍时,与单独芍药提取物比较,芍药苷在十二指肠、回肠和结肠段的吸收百分率分别提高3.79%、5.25%和12.94%,但经统计分析无显著差异,另两个比例则几乎无变化。
甘草-芍药药对研究结果显示,甘草酸与芍药苷1∶1配伍,与单体成分比较,甘草酸在大鼠肠段的吸收百分率分别降低十二指肠3.3%、空肠6.25%、回肠13.08%、结肠3.18%,芍药苷在回肠段的吸收百分率降低12.93%;甘草酸与芍药苷1∶5和5∶1配伍时,甘草酸在十二指肠段吸收百分率略有增加,分别为9.53%、4.59%,芍药苷在十二指肠段吸收百分率略有降低,分别为12.94%、11.32%。甘草苷与芍药苷1∶1配伍,与单体成分比较,芍药苷在回肠段的吸收百分率降低6.02%。芍药甘草提取物1∶1配伍,与单独芍药提取物比较,芍药苷在十二指肠的吸收百分率增加13.76%;芍药甘草提取物1∶5配伍,与单独甘草提取物比较,甘草酸的吸收百分率分别增加十二指肠11.34%、空肠9.39%、回肠10.71%、结肠6.63%,但经统计分析无显著差异。
6.初步考察枳实中柚皮苷苷元柚皮素和橙皮苷苷元橙皮素的肠吸收情况。
枳实-芍药、芍药-甘草药对中主成分均为苷类化合物,这些苷类化合物在人体中会被肠菌酶等水解成苷元后,进一步被吸收或代谢,因此本实验初步考察了枳实中柚皮苷苷元柚皮素和橙皮苷苷元柚橙皮素在大鼠肠道的吸收情况。结果表明,柚皮素和橙皮素在大鼠不同肠段的有效渗透系数(P_(eff)~*值)均较大,柚皮素的有效渗透系数(P_(eff)~*值)由高到低依次为结肠3.15±0.30、十二指肠2.77±0.43、空肠2.39±0.30、回肠1.90±0.53,橙皮素的P_(eff)~*值由高到低依次为为结肠3.05±0.28、十二指肠2.63±0.31、空肠2.50±0.26、回肠1.99±0.17。柚皮素和橙皮素均生成了2个代谢产物,但液相方法未能分离,代谢产物的极性增大,推测可能为同类型的二相代谢产物。
综上,枳实-芍药和芍药-甘草药对中主成分柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷的肠吸收均较差,而药材提取物中相应成分的吸收有显著增加,药对配伍后主成分和提取物的肠吸收有所变化。中药复方(药对)配伍机理复杂,从肠吸收代谢角度探讨药对的配伍规律,有待从多成分/组分(苷、苷元)、多角度(代谢酶、肠道菌、转运载体)着手,进一步深入研究。
The compatibility of prescription in traditional Chinese medieine(TCM) is the special feature of Chinese Materia Medica.Traditional Chinese medicine matchs is the smallest unit in the compatibility of prescription.It is composed simply,take a clearly effect,edge scientific research easily,etc.Hence,it is a favorable point to study the mechanism of the compatibility of prescription.The experts of chinese medical science have researched and explored the rule of the compatibility for a long time.However,the researches were more emphasis on compatibility of clinical application,chemistry,pharmacology,than the correlation with process of drug in vivo and therapeutic effect.Now the researchs of modern medicine demonstrated drugs of oral administration have to be absorbed by gastrointestinal tract firstly.The oral administration is the main form of Chinese herbs,so it is necessary to research absorption,metabolism and drug interaction in vivo,that active components and active priciples of the compatibility of prescription in traditional Chinese medicine.Then it would be expounded the mechanism of the compatibility of prescription.In this study,the main components of Zhishi-Shaoyao and Shaoyao-Gancao Sini contained were selected as a model medicine.Character of its absorption and metabolism were studied in a four-site perfused rat intestinal model.
Sini was first found in the Treatise on Exogenous Febrile Diseaseine of Zhang Zhongjing in Han Dynasty.It is well-known to reconcile the liver and spleen,and is composed of Paeoniae Radix,Fructus Aurantii Immaturus,Bupleurum,Glycyrrhiza uralensis,including Zhishi-Shaoyao,Shaoyao-Gancao,Chaihu-Shaoyao,Chaihu-Zhishi, with the role of liver qi stagnation and in clinic it was uesd to treat the Rib epigastric abdominal pain caused by liver and spleen qiyu decoction.This study focused on the research of intestinal absorption that the main components of Zhishi-Shaoyao and Shaoyao-Gancao in Sini in perfused rat intestinal model,In order to clarify mechanism of the compatibility of Sinisan.
In this thesis,firstly reviewed the researched advance of the mechanism of the compatibility of Chinese herbals,Sinisan medicine and absorption and metabolism of drugs respectively.Completed the following studies in experimental part:
1.Extracts from Fructus Aurantii Immaturus,Paeoniae Radix and Glycyrrhiza uralensis were prepared.
Firstly determination of UPLC for naringin,hesperidin and neohesperidin of the extract of Fructus Aurantii Immaturus were established,and compared to contents of those components of Fructus Aurantii Immaturus from different areas.In this study,we selected the moderate content in Fructus Aurantii Immaturus herbs for the experiment.The extracts of medicinal herbs were prepared respectively,naringin,hesperidin and neohesPeridin in Fructus Aurantii Immaturus,paeoniflorin in Paeoniae Radix,glycyrrhizin acid,glycyrrhizin in Glycyrrhiza uralensis as indicators,then established determination method of UPLC to ensure the content of extracts.The result was Fructus Aurantii Immaturus extract containing naringin 52.65 mg,hesperidin 9.25 mg,neohesperidin 66.25 mg;Paeoniae Radix extract containing paeoniflorin 22.41 mg;Glycyrrhiza uralensis extracts containing glycyrrhizin acid 16.35 mg,glycyrrhizin 22.38 mg per mL.
2 Research on physical and chemical properties of the main components of Zhishi-Shaoyao and Shaoyao-Gancao.
(1) Research on physical and chemical properties of the main components that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin of Zhishi-Shaoyao and Shaoyao-Gancao.Reference to Chinese Pharmacopoeia investigated the approximate solubility of each component;Determined the components of the oil-water partition coefficient(logP values)by shake flask method,and the results were naringin (-0.44),hesperidin(-0.39),neohesperidin(-0.31),paeoniflorin(-0.67),glycyrrhizin acid(0.78) and glycyrrhizin(0.16),and combines with prediction that physical and chemical parameters of each component by Marvin online software,to speculate absorption of the components in vivo according to the Five Rules.Only judged from the physical and chemical properties of the above components,that they may have poor absorption in vivo.
(2)Study on the stability of model drugs was necessarily.Firstly chemical stability of compounds in model drugs was investigated in order to ensure there liability of results in Perfused rat intestinal model.Stability of tested drugs in hank,s balanced salt solution during temperature 37℃and PH differ from5.0 to 8.0 for 24 h was investigated.The results showed that compounds naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid, glycyrrhizin were stable in differ PH from 5.0 to 8.0 solutions.So all tested drugs were suitable for the vitro perfused test.
3 Methodology ofperfused sample analysis was established.
The UPLC determination method of naringin,hesperidin,neohesperidin,paeoniflorin, glycyrrhizin acid,glycyrrhizin contained in perfused rat intestinal model sample and the HPLC determination in plasma and bile samples was established.Results indicated that linearity,precision,repeatability,stability,recovery of these compounds fit the bill in this method.
4 In perfused rat intestinal model to study intestinal absorption of the 20、50、100μmol.L~(-1) three concentration compounds and extracts in the combination drug Zhishi-Shaoyao,Shaoyao-Gancao.The results showed that all the components and extracts were poorly absorbed in the duodenum,jejunum,ileum,colon of rat.Effective permeability coefficient of the compounds(P_(eff)~* values) were＜0.5.However,compared with the corresponding compounds,the absorption of extracts had increased.Through the statistical software SPSS13.0 analysis the percent absorption of naringin(20μmol.L~(-1)) in extracts of Fructus Aurantii Immaturus increased by 41.1%in duodenum,39.9%in jejunum,32.57%in ileum and the percent absorption of hesperidin(20μmol·L~(-1)) in the extracts significantly increased by 45.83%in duodenal,47.17%in jejunum,30.85%in ileum;neohesperidin(20μmol·L~(-1))in the extracts had a significant increase in percent absorption that 53.83%in duodenal,47.61%in jejunum,32.05%in ileum and intestinal abosoption of the other concentration extracts of Fructus Aurantii Immaturus also increased significantly compared with their corresponding monomers.It is indicated that other components in of extracts in Fructus Aurantii Immaturus would promote intestinal absorption of naringin,hesperidin, neohesperidin.The percent absorption of Paeoniflorin(20μmol·L~(-1)) in extracts of Paeoniae Radix increased by 24.92%in jejunum,36.33%in ileum compared with monomer and the others concentration extracts also increased more than compound.All of these indicated that other components of extracts in Paeoniae Radix will also promote intestinal absorption of paeoniflorin.The percent absorption of glycyrrhizin(20μmol·L~(-1)) in extracts of Glycyrrhiza uralensis increased by 20.22%(p＜0.05)in ileum and the others concentration of extract had a increase compared with the same concentration of compound,while glycyrrhizin had no significantly change.It is indicated that other components of extracts in Glycyrrhiza uralensis would promote absorption of glycyrrhizin,but had no effect on glycyrrhizic acid.
5 Research on Intestinal absorption of the main components and extracts of Zhishi-Shaoyao and Shaoyao-Gancao and explained their mechanism of the compatibility initially.
Select the different ratio of compatibility to study the intestinal absoption of math-pair, the ratio of compounds and extracts were both 1:1,1:5,5:1.
The results of compatibility in Zhishi-Shaoyao showed when the ratio was 1:1 naringin combined with paeoniflorin,paeoniflorin can promote absorption of naringin 5.94%in duodenum and 5.37%jejunum in rat while absorption of paeoniflorin decreased 8.37%in duodenum and 8.92%in jejunum.when ratio was 1:5,5:1,the absoption of naringin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 hesperidin combined with paeoniflorin,the percent absoption of hesperidin increased by 11.76%in duodenal,8.09% in jejunum,9.57%in ileum and 18.78%in colon while the percent absoption of paeoniflorin decreased by 2.89%in duodenum and 6.89%in colon,when ratio was 1:5,5:1,the absoption of hesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 neohesperidin combined with paeoniflorin,the percent absoption of neohesperidin increased by 9.23%in ileum.when the ratio was 1:5,5:1,the absoption of neohesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 5:1 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the absorption of naringin increased by 6.93%in duodenum 14.02%in jejunum.When the ratio was 1:5 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the percent absoption of paeoniflorin increased by 3.79%in duodenum 5.25%in ileum 12.94%in colon compared with the percent absoption of extracts of Paeoniae Radix.
The results of compatibility in Shaoyao-Gancao showed that,with the ratio of compatibility,of glycyrrhizin acid and paeoniflorin was 1:1,compared to compounds,the percent absorption of the glycyrrhizin acid in rat decreased by 3.3%in duodenal,6.25%in jejunum,13.08%in ileum,3.18%in colon respectively;with the ratio of compatibility of glycyrrhizin acid and paeoniflorin was 1:5 and 5:1,the percent absorption of the glycyrrhizin acid in rat increased a little that by 9.53%,4.59%in the ileum,the percent absorption of the paeoniflorin in rat decreased a little that by 12.94%,11.32%in the ileum; with the ratio of compatibility of glycyrrhizin and paeoniflorin was 1:1,compared to compounds,the percent absorption of paeoniflorin in rat decreased by 6.02%in ileum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:1,compared to single extracts of Paeoniae Radix,the percent absorption of paeoniflorin in rat increased by 6.02% in duodenum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:5, compared to single extracts of Glycyrrhiza uralensis,the percent absorption of glycyrrhizin acid in rat increased by 11.34%in duodenum,9.39%in jejunum,10.71%in ileum,6.63%in colon respectively,but there is no significant difference after statistical analysis.
6 Research on Intestinal absorption that naringenin(aglycone of naringin) and hesperidin(aglycone of hesperetin) preliminarily
The principal components of Zhishi-Shaoyao and Shaoyao-Gancao are glycosides,that would be into aglycones by hydrolysis of intestinal bacteria in the human body,then absorbed or metabolized,therefore a preliminary examination of this experiment studied on intestinal absorption of naringenin(aglycone of naringin) and hesperetin(aglycone of hesperidin) in Fructus Aurantii Immaturus in rat.The results showed that effective permeability coefficient(P_(eff)~* value) of.naringenin and hesperidin in rat were higher in different segments of intestine of rat.It was demonstrated that P_(eff)~* of naringenin decreased in order as flows:3.15±0.30 in colon,2.77±0.43 in duodenum,2.39±0.30 in jejunum, 1.90±0.53 in ileum;And P_(eff)~* of hesperidin decreased in order as flows:3.05±0.28 in colon, 2.63±0.31 in jejunum,2.50±0.26 in duodenum,1.99±0.17 in ileum.There were two metabolic products which naringenin and hesperidin produced,but UPLC can not separate effectively.The polarity of Metabolites increased,so they were probably the same type of two-phase metabolites.
In summary,the main components of Zhishi-Shaoyao and Shaoyao-Gancao that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin were poorly intestinal absorption.The compatibility of drugs had no significant effect on intestinal absorption of each component.It is a further study on the intestinal absorption in order to reveal the mechanism of the compatibility of match-pair,from the points of Multi-components/components(glycosides,aglycone),multi-angles(metabolic enzymes, intestinal bacteria,transporters).
四逆散方始见于汉代张仲景《伤寒论》,是调和肝脾的名方,由芍药、枳实、柴胡、甘草四味药组成,包含了枳实-芍药、芍药-甘草、柴胡-芍药、柴胡-枳实四个药对,具有疏肝解郁作用,临床常以本方加减治疗肝脾气郁所致胁肋脘腹疼痛等症。本研究重点应用研究药物肠吸收代谢的经典模型:大鼠在体肠灌流模型对四逆散中枳实-芍药、芍药-甘草药对中主成分和提取物配伍的肠吸收特性进行了深入的研究,以期从肠吸收代谢的角度阐明四逆散药对的配伍机理。
本论文首先分别对中药复方配伍机理的研究进展、四逆散中药对研究进展及药物吸收代谢研究手段的研究进展进行了文献综述。实验部分主要完成了以下研究:
1.本论文首先从枳实、芍药、甘草药材提取物的制备入手,建立了枳实药材中黄酮类成分柚皮苷、橙皮苷、新橙皮苷的UPLC测定方法,比较了不同产地枳实药材中上述3种成分的含量,选择了含量比例适中的枳实药材供实验用。以四逆散临床使用汤剂为依据,制备了各药材水提取物,并对枳实中柚皮苷、橙皮苷和新橙皮苷,芍药中芍药苷,甘草中甘草酸、甘草苷的含量进行了测定。结果:枳实提取物中柚皮苷、橙皮苷、新橙皮苷的含量分别为52.65 mg·mL~(-1)、9.25 mg·mL~(-1)、66.25 mg·mL~(-1),芍药提取物中芍药苷的含量为22.41 mg·mL~(-1),甘草提取物中甘草酸、甘草苷的含量分别为16.35 mg·mL~(-1),22.38 mg·mL~(-1)。
2.考察了枳实-芍药、芍药-甘草药对中主成分的理化性质。
(1)参照《中国药典》方法考察了枳实-芍药、芍药-甘草药对中主成分柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷的近似溶解度;采用摇瓶法测定了各成分的油水分配系数(logP值),结果分别为柚皮苷(-0.44)、橙皮苷(-0.39)、新橙皮苷(-0.31)、芍药苷(-0.67)、甘草酸(0.78)和甘草苷(0.16),结合Marvin在线软件对各成分理化参数的进行预测,并根据预测口服药物生物利用度的“五规则”,推测各成分的体内吸收情况,结果:仅从理化性质上判断,上述各成分的体内吸收情况可能均较差。
(2)为保证大鼠在体肠灌流试验的准确可靠,对各成分的化学稳定性进行了考察。采用恒温法,以pH值为5.5～8.0的Hank's平衡盐溶液作为溶解介质,在37℃条件下考察各成分在24h内的稳定性。实验结果表明柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷在pH值为5.5～8.0的HBSS溶液中均比较稳定,可以进行在体实验。
3.建立了药对中各主成分大鼠在体肠灌流样品的分析方法。
建立了柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷在体肠灌流样品的UPLC测定方法,血浆和胆汁样品的HPLC测定方法。实验结果表明,各成分的线性良好,精密度、重复性、稳定性、回收率符合要求,所建方法可行。
4.应用大鼠在体肠灌流模型对枳实-芍药、芍药-甘草药对中主成分和各提取物的肠吸收代谢进行了研究,考察了20、50、100μmol·L~(-1)3个浓度各成分及提取物的肠吸收情况。结果表明,各成分及其在提取物中,在大鼠十二指肠、空肠、回肠、结肠的吸收都较差,有效渗透系数(P_(eff)~*值)均＜0.5,但提取物中成分的吸收与相应单体成分比较有不同程度的提高。经SPSS13.0统计软件分析,枳实提取物中柚皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠41.1%,空肠39.9%,回肠32.57%;其中橙皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠45.83%,空肠47.17%,回肠30.85%;其中新橙皮苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):十二指肠53.83%,空肠47.61%,回肠32.05%,其它浓度枳实提取物中成分与相应浓度单体比较也均有显著性增加,表明枳实提取物中其他成分会促进柚皮苷、橙皮苷、新橙皮苷的肠内吸收。芍药提取物中芍药苷(20μmol·L~(-1))与单体相比吸收百分率显著增加(p＜0.05):空肠24.92%,回肠36.33%,其它浓度芍药提取物中芍药苷与相应浓度芍药苷比较也有显著性增加,表明芍药提取物中其他成分也会促进芍药苷的肠内吸收。甘草提取物中甘草苷(20μmol·L~(-1))与单体相比在回肠段吸收百分率显著增加20.22%(p＜0.05),其它浓度甘草提取物中甘草苷与相应浓度甘草苷比较也有显著性增加,而甘草酸则无显著性变化,表明甘草提取物中其它成分会促进甘草苷吸收,但对甘草酸无显著影响。
5.研究了枳实-芍药、芍药-甘草药对主成分配伍和提取物配伍的肠吸收特性,初步阐述枳实-芍药、芍药-甘草药对的配伍机理。
选择不同配伍比例研究药对配伍的肠吸收情况,单体成分配伍以浓度比例为1∶1,1∶5,5∶1进行试验,提取物配伍以生药量比例为1∶1,1∶5,5∶1进行试验。
枳实-芍药药对研究结果显示,柚皮苷与芍药苷1∶1配伍时,与单体成分比较,柚皮苷在十二指肠和空肠的吸收百分率分别增加5.94%和5.37%,芍药苷在十二指肠、空肠段的吸收百分率分别降低8.37%和8.92%,1∶5和5∶1比例配伍时柚皮苷和芍药苷的变化与1∶1配伍时趋势相同;橙皮苷与芍药苷1∶1配伍时,与单体成分比较,橙皮苷的吸收百分率分别增加十二指肠11.76%,空肠8.09%,回肠9.57%,结肠18.78%,芍药苷在十二指肠、结肠段的吸收百分率分别降低2.89%和6.89%,1∶5和5∶1比例配伍时橙皮苷的变化与1∶1配伍时趋势相同;新橙皮苷与芍药苷1∶1配伍时,与单体成分比较,新橙皮苷在回肠段的吸收百分率增加9.23%,1∶5和5∶1比例配伍时新橙皮苷的变化与1∶1配伍时趋势相同。枳实芍药提取物以5∶1配伍时,与单独枳实提取物比较,柚皮苷在十二指肠和空肠的吸收百分率分别提高6.93%和14.02%;枳实芍药提取物以1∶5配伍时,与单独芍药提取物比较,芍药苷在十二指肠、回肠和结肠段的吸收百分率分别提高3.79%、5.25%和12.94%,但经统计分析无显著差异,另两个比例则几乎无变化。
甘草-芍药药对研究结果显示,甘草酸与芍药苷1∶1配伍,与单体成分比较,甘草酸在大鼠肠段的吸收百分率分别降低十二指肠3.3%、空肠6.25%、回肠13.08%、结肠3.18%,芍药苷在回肠段的吸收百分率降低12.93%;甘草酸与芍药苷1∶5和5∶1配伍时,甘草酸在十二指肠段吸收百分率略有增加,分别为9.53%、4.59%,芍药苷在十二指肠段吸收百分率略有降低,分别为12.94%、11.32%。甘草苷与芍药苷1∶1配伍,与单体成分比较,芍药苷在回肠段的吸收百分率降低6.02%。芍药甘草提取物1∶1配伍,与单独芍药提取物比较,芍药苷在十二指肠的吸收百分率增加13.76%;芍药甘草提取物1∶5配伍,与单独甘草提取物比较,甘草酸的吸收百分率分别增加十二指肠11.34%、空肠9.39%、回肠10.71%、结肠6.63%,但经统计分析无显著差异。
6.初步考察枳实中柚皮苷苷元柚皮素和橙皮苷苷元橙皮素的肠吸收情况。
枳实-芍药、芍药-甘草药对中主成分均为苷类化合物,这些苷类化合物在人体中会被肠菌酶等水解成苷元后,进一步被吸收或代谢,因此本实验初步考察了枳实中柚皮苷苷元柚皮素和橙皮苷苷元柚橙皮素在大鼠肠道的吸收情况。结果表明,柚皮素和橙皮素在大鼠不同肠段的有效渗透系数(P_(eff)~*值)均较大,柚皮素的有效渗透系数(P_(eff)~*值)由高到低依次为结肠3.15±0.30、十二指肠2.77±0.43、空肠2.39±0.30、回肠1.90±0.53,橙皮素的P_(eff)~*值由高到低依次为为结肠3.05±0.28、十二指肠2.63±0.31、空肠2.50±0.26、回肠1.99±0.17。柚皮素和橙皮素均生成了2个代谢产物,但液相方法未能分离,代谢产物的极性增大,推测可能为同类型的二相代谢产物。
综上,枳实-芍药和芍药-甘草药对中主成分柚皮苷、橙皮苷、新橙皮苷、芍药苷、甘草酸、甘草苷的肠吸收均较差,而药材提取物中相应成分的吸收有显著增加,药对配伍后主成分和提取物的肠吸收有所变化。中药复方(药对)配伍机理复杂,从肠吸收代谢角度探讨药对的配伍规律,有待从多成分/组分(苷、苷元)、多角度(代谢酶、肠道菌、转运载体)着手,进一步深入研究。
The compatibility of prescription in traditional Chinese medieine(TCM) is the special feature of Chinese Materia Medica.Traditional Chinese medicine matchs is the smallest unit in the compatibility of prescription.It is composed simply,take a clearly effect,edge scientific research easily,etc.Hence,it is a favorable point to study the mechanism of the compatibility of prescription.The experts of chinese medical science have researched and explored the rule of the compatibility for a long time.However,the researches were more emphasis on compatibility of clinical application,chemistry,pharmacology,than the correlation with process of drug in vivo and therapeutic effect.Now the researchs of modern medicine demonstrated drugs of oral administration have to be absorbed by gastrointestinal tract firstly.The oral administration is the main form of Chinese herbs,so it is necessary to research absorption,metabolism and drug interaction in vivo,that active components and active priciples of the compatibility of prescription in traditional Chinese medicine.Then it would be expounded the mechanism of the compatibility of prescription.In this study,the main components of Zhishi-Shaoyao and Shaoyao-Gancao Sini contained were selected as a model medicine.Character of its absorption and metabolism were studied in a four-site perfused rat intestinal model.
Sini was first found in the Treatise on Exogenous Febrile Diseaseine of Zhang Zhongjing in Han Dynasty.It is well-known to reconcile the liver and spleen,and is composed of Paeoniae Radix,Fructus Aurantii Immaturus,Bupleurum,Glycyrrhiza uralensis,including Zhishi-Shaoyao,Shaoyao-Gancao,Chaihu-Shaoyao,Chaihu-Zhishi, with the role of liver qi stagnation and in clinic it was uesd to treat the Rib epigastric abdominal pain caused by liver and spleen qiyu decoction.This study focused on the research of intestinal absorption that the main components of Zhishi-Shaoyao and Shaoyao-Gancao in Sini in perfused rat intestinal model,In order to clarify mechanism of the compatibility of Sinisan.
In this thesis,firstly reviewed the researched advance of the mechanism of the compatibility of Chinese herbals,Sinisan medicine and absorption and metabolism of drugs respectively.Completed the following studies in experimental part:
1.Extracts from Fructus Aurantii Immaturus,Paeoniae Radix and Glycyrrhiza uralensis were prepared.
Firstly determination of UPLC for naringin,hesperidin and neohesperidin of the extract of Fructus Aurantii Immaturus were established,and compared to contents of those components of Fructus Aurantii Immaturus from different areas.In this study,we selected the moderate content in Fructus Aurantii Immaturus herbs for the experiment.The extracts of medicinal herbs were prepared respectively,naringin,hesperidin and neohesPeridin in Fructus Aurantii Immaturus,paeoniflorin in Paeoniae Radix,glycyrrhizin acid,glycyrrhizin in Glycyrrhiza uralensis as indicators,then established determination method of UPLC to ensure the content of extracts.The result was Fructus Aurantii Immaturus extract containing naringin 52.65 mg,hesperidin 9.25 mg,neohesperidin 66.25 mg;Paeoniae Radix extract containing paeoniflorin 22.41 mg;Glycyrrhiza uralensis extracts containing glycyrrhizin acid 16.35 mg,glycyrrhizin 22.38 mg per mL.
2 Research on physical and chemical properties of the main components of Zhishi-Shaoyao and Shaoyao-Gancao.
(1) Research on physical and chemical properties of the main components that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin of Zhishi-Shaoyao and Shaoyao-Gancao.Reference to Chinese Pharmacopoeia investigated the approximate solubility of each component;Determined the components of the oil-water partition coefficient(logP values)by shake flask method,and the results were naringin (-0.44),hesperidin(-0.39),neohesperidin(-0.31),paeoniflorin(-0.67),glycyrrhizin acid(0.78) and glycyrrhizin(0.16),and combines with prediction that physical and chemical parameters of each component by Marvin online software,to speculate absorption of the components in vivo according to the Five Rules.Only judged from the physical and chemical properties of the above components,that they may have poor absorption in vivo.
(2)Study on the stability of model drugs was necessarily.Firstly chemical stability of compounds in model drugs was investigated in order to ensure there liability of results in Perfused rat intestinal model.Stability of tested drugs in hank,s balanced salt solution during temperature 37℃and PH differ from5.0 to 8.0 for 24 h was investigated.The results showed that compounds naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid, glycyrrhizin were stable in differ PH from 5.0 to 8.0 solutions.So all tested drugs were suitable for the vitro perfused test.
3 Methodology ofperfused sample analysis was established.
The UPLC determination method of naringin,hesperidin,neohesperidin,paeoniflorin, glycyrrhizin acid,glycyrrhizin contained in perfused rat intestinal model sample and the HPLC determination in plasma and bile samples was established.Results indicated that linearity,precision,repeatability,stability,recovery of these compounds fit the bill in this method.
4 In perfused rat intestinal model to study intestinal absorption of the 20、50、100μmol.L~(-1) three concentration compounds and extracts in the combination drug Zhishi-Shaoyao,Shaoyao-Gancao.The results showed that all the components and extracts were poorly absorbed in the duodenum,jejunum,ileum,colon of rat.Effective permeability coefficient of the compounds(P_(eff)~* values) were＜0.5.However,compared with the corresponding compounds,the absorption of extracts had increased.Through the statistical software SPSS13.0 analysis the percent absorption of naringin(20μmol.L~(-1)) in extracts of Fructus Aurantii Immaturus increased by 41.1%in duodenum,39.9%in jejunum,32.57%in ileum and the percent absorption of hesperidin(20μmol·L~(-1)) in the extracts significantly increased by 45.83%in duodenal,47.17%in jejunum,30.85%in ileum;neohesperidin(20μmol·L~(-1))in the extracts had a significant increase in percent absorption that 53.83%in duodenal,47.61%in jejunum,32.05%in ileum and intestinal abosoption of the other concentration extracts of Fructus Aurantii Immaturus also increased significantly compared with their corresponding monomers.It is indicated that other components in of extracts in Fructus Aurantii Immaturus would promote intestinal absorption of naringin,hesperidin, neohesperidin.The percent absorption of Paeoniflorin(20μmol·L~(-1)) in extracts of Paeoniae Radix increased by 24.92%in jejunum,36.33%in ileum compared with monomer and the others concentration extracts also increased more than compound.All of these indicated that other components of extracts in Paeoniae Radix will also promote intestinal absorption of paeoniflorin.The percent absorption of glycyrrhizin(20μmol·L~(-1)) in extracts of Glycyrrhiza uralensis increased by 20.22%(p＜0.05)in ileum and the others concentration of extract had a increase compared with the same concentration of compound,while glycyrrhizin had no significantly change.It is indicated that other components of extracts in Glycyrrhiza uralensis would promote absorption of glycyrrhizin,but had no effect on glycyrrhizic acid.
5 Research on Intestinal absorption of the main components and extracts of Zhishi-Shaoyao and Shaoyao-Gancao and explained their mechanism of the compatibility initially.
Select the different ratio of compatibility to study the intestinal absoption of math-pair, the ratio of compounds and extracts were both 1:1,1:5,5:1.
The results of compatibility in Zhishi-Shaoyao showed when the ratio was 1:1 naringin combined with paeoniflorin,paeoniflorin can promote absorption of naringin 5.94%in duodenum and 5.37%jejunum in rat while absorption of paeoniflorin decreased 8.37%in duodenum and 8.92%in jejunum.when ratio was 1:5,5:1,the absoption of naringin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 hesperidin combined with paeoniflorin,the percent absoption of hesperidin increased by 11.76%in duodenal,8.09% in jejunum,9.57%in ileum and 18.78%in colon while the percent absoption of paeoniflorin decreased by 2.89%in duodenum and 6.89%in colon,when ratio was 1:5,5:1,the absoption of hesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 1:1 neohesperidin combined with paeoniflorin,the percent absoption of neohesperidin increased by 9.23%in ileum.when the ratio was 1:5,5:1,the absoption of neohesperidin and paeoniflorin were the same as ratio of 1:1.When the ratio was 5:1 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the absorption of naringin increased by 6.93%in duodenum 14.02%in jejunum.When the ratio was 1:5 extracts of Fructus Aurantii Immaturus combined with extracts of Paeoniae Radix,the percent absoption of paeoniflorin increased by 3.79%in duodenum 5.25%in ileum 12.94%in colon compared with the percent absoption of extracts of Paeoniae Radix.
The results of compatibility in Shaoyao-Gancao showed that,with the ratio of compatibility,of glycyrrhizin acid and paeoniflorin was 1:1,compared to compounds,the percent absorption of the glycyrrhizin acid in rat decreased by 3.3%in duodenal,6.25%in jejunum,13.08%in ileum,3.18%in colon respectively;with the ratio of compatibility of glycyrrhizin acid and paeoniflorin was 1:5 and 5:1,the percent absorption of the glycyrrhizin acid in rat increased a little that by 9.53%,4.59%in the ileum,the percent absorption of the paeoniflorin in rat decreased a little that by 12.94%,11.32%in the ileum; with the ratio of compatibility of glycyrrhizin and paeoniflorin was 1:1,compared to compounds,the percent absorption of paeoniflorin in rat decreased by 6.02%in ileum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:1,compared to single extracts of Paeoniae Radix,the percent absorption of paeoniflorin in rat increased by 6.02% in duodenum;with the ratio of compatibility of the extracts of Shaoyao-Gancao was 1:5, compared to single extracts of Glycyrrhiza uralensis,the percent absorption of glycyrrhizin acid in rat increased by 11.34%in duodenum,9.39%in jejunum,10.71%in ileum,6.63%in colon respectively,but there is no significant difference after statistical analysis.
6 Research on Intestinal absorption that naringenin(aglycone of naringin) and hesperidin(aglycone of hesperetin) preliminarily
The principal components of Zhishi-Shaoyao and Shaoyao-Gancao are glycosides,that would be into aglycones by hydrolysis of intestinal bacteria in the human body,then absorbed or metabolized,therefore a preliminary examination of this experiment studied on intestinal absorption of naringenin(aglycone of naringin) and hesperetin(aglycone of hesperidin) in Fructus Aurantii Immaturus in rat.The results showed that effective permeability coefficient(P_(eff)~* value) of.naringenin and hesperidin in rat were higher in different segments of intestine of rat.It was demonstrated that P_(eff)~* of naringenin decreased in order as flows:3.15±0.30 in colon,2.77±0.43 in duodenum,2.39±0.30 in jejunum, 1.90±0.53 in ileum;And P_(eff)~* of hesperidin decreased in order as flows:3.05±0.28 in colon, 2.63±0.31 in jejunum,2.50±0.26 in duodenum,1.99±0.17 in ileum.There were two metabolic products which naringenin and hesperidin produced,but UPLC can not separate effectively.The polarity of Metabolites increased,so they were probably the same type of two-phase metabolites.
In summary,the main components of Zhishi-Shaoyao and Shaoyao-Gancao that were naringin,hesperidin,neohesperidin,paeoniflorin,glycyrrhizin acid,glycyrrhizin were poorly intestinal absorption.The compatibility of drugs had no significant effect on intestinal absorption of each component.It is a further study on the intestinal absorption in order to reveal the mechanism of the compatibility of match-pair,from the points of Multi-components/components(glycosides,aglycone),multi-angles(metabolic enzymes, intestinal bacteria,transporters).
引文
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