基于理化性质—药效—体内外吸收评价葛根素和天麻素配伍应用的合理性
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摘要
1研究目的
葛根和天麻近年来在中药内服治疗颈椎病中应用较多,且多配伍使用。二者的主要活性成分分别为葛根素(puerarin, Pur)和天麻素(gastrodin, Gas)。它们都具有改善微循环、扩张冠状动脉、降血压、抗氧化等药理活性。临床上二者单独使用常被用于高血压、突发性耳聋、椎基底动脉供血不足所致的颈椎病、眩晕等微循环障碍引起的疾病,两者的注射液亦常联合静注给药用于治疗上述疾病。基于葛根素和天麻素的临床应用,探讨其配伍应用的合理性及其相互影响十分必要。为此,本研究将从葛根素和天麻素配伍后理化性质、抗氧化改善微循环药效作用、大鼠体内药代动力学和Caco-2细胞中的吸收机制等多层面,探讨葛根素和天麻素配伍应用的合理性。
2研究内容与方法
围绕以上研究目的,本实验拟从葛根素和天麻素配伍后溶解度和油水分配系数等理化性质、清除自由基和改善微循环等药效作用、大鼠体内药代动力学及在Caco-2细胞中吸收机制方面,以二者配伍后溶解度和油水分配系数、清除二苯代苦味肼基自由基(DPPH自由基)和抗凝血抗血小板聚集率、大鼠体内药代动力学参数及Caco-2细胞模型表观渗透系数为指标,探讨葛根素和天麻素配伍应用合理性。
2.1.葛根素和天麻素配伍理化性质研究
2.1.1葛根素和天麻素配伍后表观溶解度
采用紫外分光光度法测定葛根素、天麻素单独及配伍使用后在水中的平衡溶解度。
2.1.2葛根素和天麻素配伍后油水分配系数
采用摇瓶-HPLC法测定葛根素、天麻素单独及配伍使用后在不同pH值正辛醇-磷酸盐缓冲体系中的油水分配系数。
2.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
采用二苯代苦味肼基自由基(DPPH自由基)清除率为指标,考察葛根素和天麻素单独及配伍使用后体外抗氧化能力;并测定体内外活化部分凝血酶原时间(APTT)和ADP诱导的血小板聚集抑制率,考察葛根素和天麻素单独及配伍使用后抗凝血作用及抗血小板聚集作用,研究两者合用后改善微循环的药效作用。
2.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
建立同时测定葛根素、天麻素、天麻苷元和内标对羟基苯乙醇(Tyr)4种成分在大鼠血浆中药物浓度的HPLC方法,并应用该法研究单独或合并静脉注射及灌胃给药葛根素、天麻素后,两者在大鼠体内药代动力学过程及相互作用。以Tyr为内标,血浆样品经甲醇沉淀蛋白后,上清液用N2吹干,残渣用乙腈-0.05%磷酸(20：80)复溶后,以乙腈-0.05%磷酸水为流动相梯度洗脱,用Agilent ZORBAX SB-Aq C18(4.6×150mm,5μm)色谱柱分离,在250nm波长处测定葛根素,221nm处测定天麻素和内标。单独或合并给药葛根素、天麻素后,检测大鼠血浆中二者浓度,用WinNonlin5.2.1药动学软件和SPSS17.0软件分别计算各给药组药动学参数并进行统计分析,比较单独或合并给药后葛根素、天麻素药动学过程。
2.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
采用HPLC(?)(?)测定葛根素、天麻素在Caco-2细胞模型中转运后的含量,计算其表观渗透系数,研究二者单独或合并给药后在Caco-2细胞中的跨膜转运特性和吸收机制,并考察浓度、转运蛋白抑制剂对葛根素和天麻素吸收的影响。
3研究结果
3.1葛根素和天麻素配伍理化性质研究
3.1.1葛根素和天麻素配伍后平衡溶解度
室温下,葛根素平衡溶解度为162g·L-1,天麻素在水中的平衡溶解度为303.81g·L-1；与不同浓度天麻素配伍后,葛根素溶解度有所变化,加入1.0、1.5g·L-1天麻素后,葛根素平衡溶解度与单独葛根素组无显著性差异,而其余浓度组均能显著提高葛根素平衡浓度,最高者可为原来的5.1倍,且葛根素溶解度的增加与天麻素浓度呈线性关系,表明一定浓度的天麻素可改善葛根素溶解度。
3.1.2葛根素和天麻素配伍后油水分配系数
(1)单用葛根素、天麻素时,测得的Log P均值分别为0.4803、-0.8573,与预测值接近(用Chem Draw Ultra6.0软件通过分子结构预测的Log P值分别为0.4826、-1.0595)。
(2)葛根素油水分配系数
①单用组及合用组中葛根素在不同pH磷酸盐缓冲液为水相的介质中log P值均<1,提示葛根素的亲水性大于亲脂性,且在肠道中不易被吸收；
②随着pH的升高,葛根素P值不断降低,在pH8.0磷酸盐缓冲溶液中P值最小,提示葛根素在胃液中脂溶性较大,可能在胃中的吸收要高于小肠；
③葛根素与天麻素合用后,油水分配系数P值较单用组降低,在pH1.2-5.8范围内尤为明显,说明此时葛根素亲水性大大提高,这与本课题前述溶解度研究结果相一致。
(3)天麻素油水分配系数
①单用组及合用组中天麻素在不同pH磷酸盐缓冲液为水相的介质中log P值均为负数,表明天麻素的亲水性很大,这与前述天麻素溶解度结果吻合。虽然它的油水分配系数比葛根素小,但因其分子量小,在肠道中吸收仍高于葛根素；
②pH对天麻素的油水分配系数并无影响,推测天麻素在胃肠道中的吸收无部位特异性；
⑧天麻素与葛根素合用后,油水分配系数较单用组并无差异,推测葛根素并不能改变天麻素在肠道中的脂水分配情况。
3.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
3.2.1葛根素和天麻素配伍后抗氧化作用
(1)以抗坏血酸(Vc)为阳性对照,其IC50(DPPH自由基清除率为50%时的浓度)为0.024g·L-1,葛根素的IC5o为17.56g·L-1,虽不及Vc,但显示有一定的抗氧化能力；天麻素在浓度为0.5-200g·L-1浓度范围内DPPH自由基清除率为0-1.86%,未超过50%,故无法计算IC5o,可推断其IC50值大于200g·L-1。
(2)不同浓度葛根素、天麻素单独或合用后,DPPH自由基清除率结果表明,葛根素清除率随浓度增加而增大,天麻素各浓度组间清除率均无显著性差异(P>0.05)。加入天麻素后,葛根素的清除率与单独使用葛根素组比较无统计学差异,即天麻素并无清除DPPH自由基能力,也不能增加葛根素的清除能力,推测各合用组的清除率基本系葛根素产生的作用。
3.2.2葛根素和天麻素配伍后改善微循环作用
(1)体外APTT结果
①单用葛根素低、中剂量组无抗凝血作用,高剂量组显示有抗凝血作用(P<0.01);中剂量葛根素与中剂量或高剂量天麻素合用后,均具有抗凝血作用(P<0.01)。
②单用天麻素低、中剂量组无抗凝血作用,高剂量组显示有抗凝血作用(P<0.01)；但天麻素低剂量组与高剂量葛根素合用后,有抗凝血作用(P<0.01);天麻素中剂量组与中剂量或高剂量葛根素合用后,均具有抗凝血作用(P<0.01)。
⑧合用组中,高剂量Pur+低剂量Gas组、中剂量Pur+中剂量Gas组、高剂量Pur+中剂量Gas组、中剂量Pur+高剂量Gas组、高剂量Pur+高剂量Gas组均具有抗凝血作用(P<0.01)。
④葛根素在20-40g／L、天麻素在150-300g／L剂量范围内,其APTT值均随剂量增加而延长,呈剂量依赖趋势。
(2)体内APTT结果
与空白组比较,除葛根素高剂量组外,其它给药组均有极显著性差异,说明Pur、 Gas均有抗凝血作用；且二者合用中剂量组的抗凝血作用强于各单用组,与体外APTT的该项结果一致。
(3)抗血小板聚集结果
单用葛根素低、中、高剂量组间或天麻素低、中、高剂量组间均无统计学差异；但直观分析可知,其抗血小板聚集能力(以抑制率计),均随给药剂量增加而增大。但与单用中剂量组比较,中剂量葛根素与中剂量天麻素配伍后,其抗血小板聚集抑制率均有提高(P<0.05)。
3.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
(1)建立HPLC同时测定葛根素、天麻素体内含量的方法,结果表明：葛根素、天麻素分别在0.05～5.98μg/mL禾口0.101～101μg/mL范围内线性关系良好(r>0.9960),其最低定量限分别为0.05、0.101μg/mL,检测限分别为0.0245、0.0486μg/mL,精密度、稳定性RSD均小于12%,样品提取回收率均可达80%以上,说明建立的方法准确可靠、灵敏度高,可较好地应用于葛根素、天麻素同时给药的药代动力学研究。
(2)葛根素和天麻素联合给药后,大鼠体内主要药代动力学参数(AUC、Cmax、T1/2、 Tmax、MRT)与单独给药后有显著性差异(P<0.05)。无论是灌胃还是静脉注射,清除率(CL)降低,平均滞留时间(MRT)延长,相对生物利用度均有提高,尤以灌胃组显著,灌胃葛根素合用组为单用组的106倍,灌胃天麻素合用组为单用组的1.5倍。
3.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
(1)50μg·mL-1葛根素在Caco-2细胞的转运无明显的方向性,且转运速率基本恒定,表明此浓度范围内葛根素的转运为不需要耗能的被动转运过程；100、200μg·mL-1葛根素在Caco-2细胞的跨细胞转运呈现较强的方向性(Papp(BL→AP)/Papp(AP→BL)均大于1.5),且这种有向性可被维拉帕米和环孢素抑制,故认为葛根素的跨膜转运除被动扩散外,还存在着外排泵的作用。
(2)100μg·mL-1天麻素在Caco-2细胞的跨细胞转运无明显的方向性,且转运速率几乎恒定,提示天麻素的转运为不需要耗能的单纯被动转运过程。
(3)葛根素和天麻素合用后,葛根素吸收量增加(Papp(AP→BL)由1.285×10-6cm/s增加至1.425×10-6cm/s),外排量减少(Papp(BL→AP)由4.539×10-6cm/s减少至3.108×10-6cm/s,P<0.05),外排比率由3.531下降至2.181,减少了38.22%,推测天麻素可发挥类似于维拉帕米或环孢素的作用,即天麻素为P-gp或MRP2抑制剂,可促进葛根素的跨膜转运,其促进程度与维拉帕米接近。
4结论
4.1葛根素和天麻素配伍理化性质研究
(1)室温下,葛根素在水中的平衡溶解度为1.62g·L-1,天麻素为303.81g·L-1,按2010版药典规定,前者属于微溶范畴,后者属于易溶范畴。一定浓度的天麻素可改善葛根素溶解度,且葛根素溶解度的增加与天麻素浓度呈线性关系。
(2)葛根素、天麻素单独测定时Log P均值分别为0.4803、-0.8573,均小于1,表明二者的亲水性大于亲脂性。葛根素的P值随着pH的升高而降低,表明葛根素在胃液中脂溶性较大,可能在胃中的吸收要高于小肠,而pH对天麻素的P值并无影响,表明天麻素在胃肠道中的吸收无部位特异性。在一定pH范围内,天麻素可使葛根素的亲水性提高,但葛根素并不能改变天麻素在肠道中的脂水分配情况。
(3)以溶解度和油水分配系数为测定指标,从理化性质层面解释葛根素和天麻素配伍应用的合理性。
4.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
(1)葛根素具有DPPH自由基清除作用,而天麻素并无此作用,但这并不意味着天麻素无抗氧化作用,它有可能通过其它途径(如抑制脂质过氧化、增强抗氧化酶活性等)产生作用。
(2)以体内外APTT、ADP诱导的血小板聚集抑制率为指标,葛根素和天麻素一定剂量配伍后,其抗凝血、抗血小板聚集作用增加。
(3)从体内外抗凝血和抗血小板聚集层面,解释了葛根素和天麻素配伍应用的合理性。
4.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
(1)葛根素和天麻素合用后,可彼此影响在大鼠体内的药代动力学过程,增加吸收,降低清除率,延长平均滞留时间,提高生物利用度。
(2)从体内药动学角度,解释葛根素和天麻素配伍应用的合理性。
4.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
(1)低浓度葛根素在Caco-2细胞的转运无明显的方向性,为不需要耗能的被动转运过程；随着浓度升高,葛根素的跨膜转运呈现较强的方向性,且这种有向性可被维拉帕米和环孢素抑制,表明葛根素除被动扩散外,还存在着外排泵的作用,其可能为P-gp和MRP2的底物。天麻素在Caco-2细胞的跨膜转运无明显的方向性,为不需要耗能的单纯被动扩散。
(2)天麻素可使葛根素吸收量增加,外排量减少,推测天麻素可发挥类似于维拉帕米或环孢素的作用,即天麻素为P-gp或MRP2抑制剂,可促进葛根素的跨膜转运,其促进程度与维拉帕米接近,而葛根素对天麻素吸收促进作用不明显。
(3)天麻素(被动转运)这类吸收良好的药物,Caco-2细胞模型是一个研究药物吸收非常好的模型,而对于像葛根素这类吸收差的药物,Caco-2细胞模型只能作为体内吸收的一个定性而非定量指标。
(4)从药物在体外Caco-2细胞模型中吸收机制层面,解释葛根素和天麻素配伍应用的合理性。
5创新点
(1)基于葛根素和天麻素的临床应用,从两者配伍后理化性质、抗氧化改善微循环药效作用、大鼠体内药代动力学和在Caco-2细胞模型中的吸收机制等多层面探讨其配伍应用的合理性。为葛根素和天麻素在临床合用上的实践性提供了一个重要依据,对探索中药成分的配伍理论有一定实践意义。
(2)基于椎-基底动脉供血不足所致的颈椎病、眩晕中医发病机制为缺氧、缺血等微循环障碍,本研究以DPPH自由基清除、体内外活化部分凝血活酶时间、ADP诱导的血小板聚集抑制率为指标,考察药物抗氧化及改善微循环作用,以点带面,多指标验证了葛根素和天麻素配伍的合理性。
(3)首次对葛根素和天麻素在大鼠体内药动学特征进行较深入的探讨,并以Caco-2细胞模型为工具,研究葛根素和天麻素吸收转运机制及相互影响,从药代动力学角度和细胞层面阐明两者吸收机制及配伍应用的合理性；从肠吸收角度,研究两种有效成分的吸收及其相互作用,有助于对中药复方成分配伍规律科学性的理解。
1Objective
Radix Puerariae and Gastrodia elata Blume, the active components of which are puerarin (Pur) and gastrodin(Gas), are used frequently in cervical spondylosis, and they are always been used together recently. Both of them can improve microcirculation, expand coronary artery, lower blood pressure, anti-oxidation, ect. Each of them is used for treatment of hypertension, sudden deafness, cervical spondylosis caused by vertebro-basilar artery insufficiency, vertigo and other diseases caused by microcirculation disturbance in clinic. Their injections were used in combined way for treatment of some cardiocerebrovascular diseases, especially for vertigo due to vertebrobasilar ischemia. Based on their clinical application and theory of TCM on combination, it is necessary to investigate the rationality of combined applications and interaction of the two constituents. Our study focused on the active component, Pur and Gas, to investigate the rationality of their compatibility from the aspects of physico-chemical properties, anti-oxidation, improving microcirculation function, pharmacokinetics in rat and absorption mechanism on Caco-2monolayer cell.
2Methods and Contents
According to the above objective, our experiment plan to study the following aspects of Pur and Gas after their combination, that were physico-chemical properties like solubility and oil-water partition coefficient, pharmacodynamic effects of free radical-scavenging and microcirculation improvment, pharmacokinetics in rats and absorption mechanism on Caco-2monolayer cell. The solubility, oil-water partition coefficient, DPPH free radical-scavenging rate, anti-platelet aggregation, pharmacokinetics parameter and apparent permeation coefficient of Caco-2monolayer cell were taken as the indexes to investigate the compatibility rationality of Pur and Gas.
2.1Study of physico-chemical properties after compatibility of Pur and Gas
2.1.1Apparent solubility
Determining the solubility of Pur and Gas used alone or together with ultraviolet spectrophotometry.
2.1.2Oil-water partition coefficient
Shake flask-HPLC method was used to determine oil-water partition coefficient of Pur and Gas used alone or together in n-octanol/phosphates buffer system of different pH value.
2.2Study of antioxidation and microcirculation improvment after compatibility of Pur and Gas
Taken DPPH free radical-scavenging rate as the index to investigate the antioxidant ability of Pur and Gas used alone or together; Taken APTT and ADP-induced anti-platelet aggregation rate in vitro vivo as indexes to evaluate anticoagulation and anti-platelet aggregation effects of Pur and Gas used alone or together, eventually investigating their effect of improving microcirculation.
2.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
A reliable HPLC method was developed for simultaneous determination of Pur, Gas, HBA and internal standard Tyr in rat plasma, which has been successfully applied to the pharmacokinetic study of the analytes in rats after i.g./i.v. administration of Gas and Pur alone or combined with each other. Chromatography was carried out on an Agilent ZORBAX SB-Aq C18column (4.6×250mm,5μm) equipped with an Agilent analytical guard column (4.6×12.5mm,5μm) using a gradient mobile phase consisted of ACN-H2O with0.05%phosphoric acid as a modifier at a flow rate of1.0mL/min. The UV detector wavelength was set at250nm for Pur, whilst221nm for Gas and IS. Comparisons of the pharmacokinetic data were performed using the software of WinNonlin5.2.1and SPSS statistical software package.
2.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
Concentration of Pur and Gas after transporting through Caco-2monolayer cell was determined by HPLC. Then calculate the apparent permeation coefficients to study the characteristics of transport and absorption mechanism. Meanwhile investigate the influences of concentration and transport protein inhibitor on the absorption of Pur and Gas.
3Results
3.1Physico-chemical properties after compatibility of Pur and Gas
3.1.1Apparent solubility
The solubility of Gas in water was303.81g·L-1and that of puerarin was1.62g·L-1, which could be increased by at most5.1times when adding more than1.5g·L-1Gas. The increasing solubility of Pur and the concentration of adding Gas were in a linear fashion.
3.1.2Oil-water partition coefficient
(1) The oil-water partition coefficients of Pur and Gas were0.4803and-0.8573, respectively, which were close to predictive value(We used software of Chem Draw Ultra6.0to calculate the Log P value, which were0.4826、-1.0595for Pur and Gas, respectively).
(2) Oil-water partition coefficients of Pur
①The Log P value of Pur in all groups were less than1in phosphates buffer system of different pH. It was indicated that puerarin was more hydrophilic and could be difficult to absorb in the intestine.
②The P value of Pur will decrease with the increase of pH. It was indicated that Pur would absorb more easily in stomach than in intestine.
③The P value of Pur in combined group was lower than that in single group, especially in the range of pH1.2to5.8. It showed that adding Gas could increase the hydrophilcity of puerarin, which was consistent with the preliminary solubility results.
(3) Oil-water partition coefficients of Gas
①The Log P value of Gas in all groups were negative in phosphates buffer system of different pH which inferred that Gas was high hydrophilic. Although the Log P value of Gas was less than that of Pur, absorption would be easier for Gas because of its small molecular weight.
②The change of pH have little influence on oil-water partition coefficients of Gas. It was indicated there was no site specificity in the absorption of Gas.
③There was no significant difference between the P value of Pur in single and combined groups, which indicated that Pur could not increase the oil-water partition coefficient of Gas.
3.2Antioxidation and microcirculation improvment after compatibility of Pur and Gas
3.2.1Antioxidation
(1) Pur has effect of antioxidation to certain extent, the IC50of which was17.56g·L-1, while that of the Vc was0.024g·L-1. Meanwhile, Gas has little effect of antioxidation because its IC50is higher than200g·L-
(2) The DPPH free radical-scavenging rate of Pur increased with its concentration, while that of Gas didn't change with its concentration (P>0.05). When added Gas, the DPPH free radical-scavenging rate of Pur didn't increase which indicated that it was just Pur contributed to the anticxidation effect.
3.2.2Improving microcirculation
(1) In vitro APTT
①Low or middle dose groups of Pur had no effect of anticoagulation while high dose group did. There was anticoagulation when middle dose of Pur used together with middle or high dose of Gas.
②Low or middle dose groups of Gas had no effect of anticoagulation while high dose group did. There was anticoagulation when low or meddle dose of Gas used together with high dose of Pur, or meddle dose of Gas used together with middle dose of Pur.
③Groups of M_Pur+M_Gas, M_Pur+H_Gas, H_Pur+L_Gas, H_Pur+M_Gas, H_Pur+H_Gas had the effect of anticoagulation(L, M, H stands for low, middle, high dose).
④The APTT were dose-dependent within the concentration of20-40g/L and150-300g/L for Pur and Gas, respectively.
(2) Invivo APTT
The APTT of all groups had significant difference comparing with blank group, except high dose group of Pur. It indicated that both Pur and Gas had anticoagulation, especially when they were both used in middle dose.
(3) Anti-platelet aggregative activity
There was no significant difference among all groups of Pur or Gas. From the visual analysis results, it showed that the inhibit ratio increased with the dose. However, the anti-platelet aggregation of combined group was significant increased compared with each single group of Pur and Gas.
3.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
(1) A reliable HPLC method was developed for simultaneous determination of Pur and Gas in rat plasma with a linear range of0.05-5.98μg/mL for Pur and0.101-101μg/mL for Gas (r>0.9960). The LLOQ, LOD of Pur and Gas were determined to be0.0486,0.101μg/mL,0.0245and0.05μg/mL, respectively. The intra-day and inter-day precision were all less than12.0%, whilst the extract recovery were all above80%. The fully validated method was successfully applied to the pharmacokinetic study of the analytes in rats after intragastric/intravenous administration of Pur and Gas alone or combined with each other.
(2) The pharmacokinetic profiles of combined administration were found to be distinct from those of given alone, which could have higher bioavailability (F) and lower clearance rate (CL), as well as longer mean residence time (MRT) both through i.g. and i.v. routes, particularly notable via i.g. administration. The relative oral bioavailability of Pur in combined administration is10.7-time as much as that of single administration, while1.5-fold in Gas.
3.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
(1) It was passive transport for50μg·mL-1Pur while directional (Papp(BL→AP)/Papp(AP→BL)>5) for100μg·mL-1,200μg·mL-1Pur across Caco-2monolayer cell. The efflux rate will decrease when added verapamil and cyclosporine, which indicated that the transepithelial transporting process of Pur was partly actively carrier-mediate transport besides the passive diffusion.
(2) It was passive diffusion for100μg·mL-1Gas when transporting across Caco-2monolayer cell.
3) When100μg·mL-1Gas and Pur used together, the permeability coefficient of apical to basolateral was increased from1.285×10-6cm/s to1.425×10-6cm/s, and the permeability coefficient of basolateral to apical was decreased from4.539×10-6cm/s to3.108×10-6cm/s. The efflux rate had reduced from3.531to2.181.This suggests Gas has the similar effect of varapamil, which is the inhibitor of P-gp. Gas could promote the transport of Pur.
4Conclusions
4.1Physico-chemical properties
(1) The solubility of Gas in water was303.81g·L-1and that of puerarin was1.62g·L-1, which belong to slight soluble and easily soluble, respectively according to2010edition of pharmacopoeia. The increasing solubility of Pur and the concentration of adding Gas were in a linear fashion.
(2) The oil-water partition coefficients of Pur and Gas were0.4803and-0.8573, respectively, both of which were less than1. It was indicated that both of them was more hydrophilic. The increase of pH would decrease the P value of Pur while had little influence on the P value of Gas. It was indicated that Pur would absorb more easily in stomach than in intestine, while there was no site specificity in the absorption of Gas. It showed that adding Gas could increase the hydrophilcity of puerarin in a certain pH range, while Pur could not increase the oil-water partition coefficient of Gas.
(3) Taken solubility and oil-water partition coefficient as indexes, investigate the Compatibility Rationality of Pur and Gas from the aspect of physico-chemical properties.
4.2Antioxidation and microcirculation improvment after compatibility of Pur and Gas
(1) Pur has DPPH free radical-scavenging activity while Gas don't, but it doesn't mean Gas have no antioxidation through another ways (inhibit lipid peroxidation, enhanced activity of antioxidant enzymes, etc.).
(2) Taken APTT and antiplatelet aggregation induced by ADP as indexes, effects of anticoagulation and anti-platelet aggregation can be increased when Pur and Gas used together in a certain dose.
(3) Investigate the Compatibility Rationality of Pur and Gas from the aspect of anti coagulation and anti-platelet aggregation in vitro/vivo.
4.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
(1) Pur and Gas will affect each other on pharmacokinetic profiles, such as improving bioavailability (F), lowering clearance rate (CL) and prolong mean residence time (MRT) when they were co-administrated to rats.
(2) Investigate the Compatibility Rationality of Pur and Gas from the aspect of pharmacokinetics.
4.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
(1) It was passive transport for low concentration of Pur while directional with the increase of concentration when transporting across Caco-2monolayer cell. This directional transpot will be weakened when added verapamil and cyclosporine, which indicated that the transepithelial transporting process of Pur was partly actively carrier-mediate transport besides the passive diffusion. Pur may be the substrate of P-gp and MRP2. It was passive diffusion for Gas when transporting across Caco-2monolayer cell.
(2) Gas can promote the permeability coefficient from apical to basolateral and reduce the permeability coefficient from basolateral to apical of Pur, suggesting that Gas has the similar effect of varapamil (the inhibitor of P-gp), could promote the transport of Pur.
(3) Caco-2monolayers are an excellent model of the passive transcellular pathway (like Gas), while just be an index of qualitative rather than quantitative concerning the slowly and incompletely absorbed drugs (like Pur).
5Innovations
(1) Based on clinical application of Pur and Gas and theory of TCM on combination, investigate the rationality of their compatibility from the aspects of physico-chemical properties, improving microcirculation function, pharmacokinetics in rat and absorption mechanism on Caco-2monolayer cell. These results might lay a foundation for explaining the combination of traditional Chinese medicine in prescriptions containing Pur and Gas and provide an important basis in clinical practice with these two components.
(2) The pathogenesis of cervical spondylosis and dizziness caused by the vertebrobasilar insufficiency from perspective of traditional Chinese medicine are hypoxia and ischemia, the symptoms of microcirculation disturbance. The DPPH free radical-scavenging activity, APTT and ADP-induced anti-platelet aggregation rate were selected as indexes to investigate the antioxidation and microcirculation improvment of Pur and Gas.We used multi-index to prove the rationality of their compatibility, faning out from point to area.
(3) The pharmacokinetic characterizations of Pur and Gas in rat were discussed in-depth. The HPLC method for simultaneous determination of Pur, Gas, HBA and internal standard (Tyr) in rat plasma was developed and validated for the first time.
(4) Caco-2monolayer cell model was used to study the absorption mechanism and the interaction between Pur and Gas. Investigate the Compatibility Rationality of Pur and Gas from the cellular level.
葛根和天麻近年来在中药内服治疗颈椎病中应用较多,且多配伍使用。二者的主要活性成分分别为葛根素(puerarin, Pur)和天麻素(gastrodin, Gas)。它们都具有改善微循环、扩张冠状动脉、降血压、抗氧化等药理活性。临床上二者单独使用常被用于高血压、突发性耳聋、椎基底动脉供血不足所致的颈椎病、眩晕等微循环障碍引起的疾病,两者的注射液亦常联合静注给药用于治疗上述疾病。基于葛根素和天麻素的临床应用,探讨其配伍应用的合理性及其相互影响十分必要。为此,本研究将从葛根素和天麻素配伍后理化性质、抗氧化改善微循环药效作用、大鼠体内药代动力学和Caco-2细胞中的吸收机制等多层面,探讨葛根素和天麻素配伍应用的合理性。
2研究内容与方法
围绕以上研究目的,本实验拟从葛根素和天麻素配伍后溶解度和油水分配系数等理化性质、清除自由基和改善微循环等药效作用、大鼠体内药代动力学及在Caco-2细胞中吸收机制方面,以二者配伍后溶解度和油水分配系数、清除二苯代苦味肼基自由基(DPPH自由基)和抗凝血抗血小板聚集率、大鼠体内药代动力学参数及Caco-2细胞模型表观渗透系数为指标,探讨葛根素和天麻素配伍应用合理性。
2.1.葛根素和天麻素配伍理化性质研究
2.1.1葛根素和天麻素配伍后表观溶解度
采用紫外分光光度法测定葛根素、天麻素单独及配伍使用后在水中的平衡溶解度。
2.1.2葛根素和天麻素配伍后油水分配系数
采用摇瓶-HPLC法测定葛根素、天麻素单独及配伍使用后在不同pH值正辛醇-磷酸盐缓冲体系中的油水分配系数。
2.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
采用二苯代苦味肼基自由基(DPPH自由基)清除率为指标,考察葛根素和天麻素单独及配伍使用后体外抗氧化能力;并测定体内外活化部分凝血酶原时间(APTT)和ADP诱导的血小板聚集抑制率,考察葛根素和天麻素单独及配伍使用后抗凝血作用及抗血小板聚集作用,研究两者合用后改善微循环的药效作用。
2.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
建立同时测定葛根素、天麻素、天麻苷元和内标对羟基苯乙醇(Tyr)4种成分在大鼠血浆中药物浓度的HPLC方法,并应用该法研究单独或合并静脉注射及灌胃给药葛根素、天麻素后,两者在大鼠体内药代动力学过程及相互作用。以Tyr为内标,血浆样品经甲醇沉淀蛋白后,上清液用N2吹干,残渣用乙腈-0.05%磷酸(20：80)复溶后,以乙腈-0.05%磷酸水为流动相梯度洗脱,用Agilent ZORBAX SB-Aq C18(4.6×150mm,5μm)色谱柱分离,在250nm波长处测定葛根素,221nm处测定天麻素和内标。单独或合并给药葛根素、天麻素后,检测大鼠血浆中二者浓度,用WinNonlin5.2.1药动学软件和SPSS17.0软件分别计算各给药组药动学参数并进行统计分析,比较单独或合并给药后葛根素、天麻素药动学过程。
2.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
采用HPLC(?)(?)测定葛根素、天麻素在Caco-2细胞模型中转运后的含量,计算其表观渗透系数,研究二者单独或合并给药后在Caco-2细胞中的跨膜转运特性和吸收机制,并考察浓度、转运蛋白抑制剂对葛根素和天麻素吸收的影响。
3研究结果
3.1葛根素和天麻素配伍理化性质研究
3.1.1葛根素和天麻素配伍后平衡溶解度
室温下,葛根素平衡溶解度为162g·L-1,天麻素在水中的平衡溶解度为303.81g·L-1；与不同浓度天麻素配伍后,葛根素溶解度有所变化,加入1.0、1.5g·L-1天麻素后,葛根素平衡溶解度与单独葛根素组无显著性差异,而其余浓度组均能显著提高葛根素平衡浓度,最高者可为原来的5.1倍,且葛根素溶解度的增加与天麻素浓度呈线性关系,表明一定浓度的天麻素可改善葛根素溶解度。
3.1.2葛根素和天麻素配伍后油水分配系数
(1)单用葛根素、天麻素时,测得的Log P均值分别为0.4803、-0.8573,与预测值接近(用Chem Draw Ultra6.0软件通过分子结构预测的Log P值分别为0.4826、-1.0595)。
(2)葛根素油水分配系数
①单用组及合用组中葛根素在不同pH磷酸盐缓冲液为水相的介质中log P值均<1,提示葛根素的亲水性大于亲脂性,且在肠道中不易被吸收；
②随着pH的升高,葛根素P值不断降低,在pH8.0磷酸盐缓冲溶液中P值最小,提示葛根素在胃液中脂溶性较大,可能在胃中的吸收要高于小肠；
③葛根素与天麻素合用后,油水分配系数P值较单用组降低,在pH1.2-5.8范围内尤为明显,说明此时葛根素亲水性大大提高,这与本课题前述溶解度研究结果相一致。
(3)天麻素油水分配系数
①单用组及合用组中天麻素在不同pH磷酸盐缓冲液为水相的介质中log P值均为负数,表明天麻素的亲水性很大,这与前述天麻素溶解度结果吻合。虽然它的油水分配系数比葛根素小,但因其分子量小,在肠道中吸收仍高于葛根素；
②pH对天麻素的油水分配系数并无影响,推测天麻素在胃肠道中的吸收无部位特异性；
⑧天麻素与葛根素合用后,油水分配系数较单用组并无差异,推测葛根素并不能改变天麻素在肠道中的脂水分配情况。
3.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
3.2.1葛根素和天麻素配伍后抗氧化作用
(1)以抗坏血酸(Vc)为阳性对照,其IC50(DPPH自由基清除率为50%时的浓度)为0.024g·L-1,葛根素的IC5o为17.56g·L-1,虽不及Vc,但显示有一定的抗氧化能力；天麻素在浓度为0.5-200g·L-1浓度范围内DPPH自由基清除率为0-1.86%,未超过50%,故无法计算IC5o,可推断其IC50值大于200g·L-1。
(2)不同浓度葛根素、天麻素单独或合用后,DPPH自由基清除率结果表明,葛根素清除率随浓度增加而增大,天麻素各浓度组间清除率均无显著性差异(P>0.05)。加入天麻素后,葛根素的清除率与单独使用葛根素组比较无统计学差异,即天麻素并无清除DPPH自由基能力,也不能增加葛根素的清除能力,推测各合用组的清除率基本系葛根素产生的作用。
3.2.2葛根素和天麻素配伍后改善微循环作用
(1)体外APTT结果
①单用葛根素低、中剂量组无抗凝血作用,高剂量组显示有抗凝血作用(P<0.01);中剂量葛根素与中剂量或高剂量天麻素合用后,均具有抗凝血作用(P<0.01)。
②单用天麻素低、中剂量组无抗凝血作用,高剂量组显示有抗凝血作用(P<0.01)；但天麻素低剂量组与高剂量葛根素合用后,有抗凝血作用(P<0.01);天麻素中剂量组与中剂量或高剂量葛根素合用后,均具有抗凝血作用(P<0.01)。
⑧合用组中,高剂量Pur+低剂量Gas组、中剂量Pur+中剂量Gas组、高剂量Pur+中剂量Gas组、中剂量Pur+高剂量Gas组、高剂量Pur+高剂量Gas组均具有抗凝血作用(P<0.01)。
④葛根素在20-40g／L、天麻素在150-300g／L剂量范围内,其APTT值均随剂量增加而延长,呈剂量依赖趋势。
(2)体内APTT结果
与空白组比较,除葛根素高剂量组外,其它给药组均有极显著性差异,说明Pur、 Gas均有抗凝血作用；且二者合用中剂量组的抗凝血作用强于各单用组,与体外APTT的该项结果一致。
(3)抗血小板聚集结果
单用葛根素低、中、高剂量组间或天麻素低、中、高剂量组间均无统计学差异；但直观分析可知,其抗血小板聚集能力(以抑制率计),均随给药剂量增加而增大。但与单用中剂量组比较,中剂量葛根素与中剂量天麻素配伍后,其抗血小板聚集抑制率均有提高(P<0.05)。
3.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
(1)建立HPLC同时测定葛根素、天麻素体内含量的方法,结果表明：葛根素、天麻素分别在0.05～5.98μg/mL禾口0.101～101μg/mL范围内线性关系良好(r>0.9960),其最低定量限分别为0.05、0.101μg/mL,检测限分别为0.0245、0.0486μg/mL,精密度、稳定性RSD均小于12%,样品提取回收率均可达80%以上,说明建立的方法准确可靠、灵敏度高,可较好地应用于葛根素、天麻素同时给药的药代动力学研究。
(2)葛根素和天麻素联合给药后,大鼠体内主要药代动力学参数(AUC、Cmax、T1/2、 Tmax、MRT)与单独给药后有显著性差异(P<0.05)。无论是灌胃还是静脉注射,清除率(CL)降低,平均滞留时间(MRT)延长,相对生物利用度均有提高,尤以灌胃组显著,灌胃葛根素合用组为单用组的106倍,灌胃天麻素合用组为单用组的1.5倍。
3.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
(1)50μg·mL-1葛根素在Caco-2细胞的转运无明显的方向性,且转运速率基本恒定,表明此浓度范围内葛根素的转运为不需要耗能的被动转运过程；100、200μg·mL-1葛根素在Caco-2细胞的跨细胞转运呈现较强的方向性(Papp(BL→AP)/Papp(AP→BL)均大于1.5),且这种有向性可被维拉帕米和环孢素抑制,故认为葛根素的跨膜转运除被动扩散外,还存在着外排泵的作用。
(2)100μg·mL-1天麻素在Caco-2细胞的跨细胞转运无明显的方向性,且转运速率几乎恒定,提示天麻素的转运为不需要耗能的单纯被动转运过程。
(3)葛根素和天麻素合用后,葛根素吸收量增加(Papp(AP→BL)由1.285×10-6cm/s增加至1.425×10-6cm/s),外排量减少(Papp(BL→AP)由4.539×10-6cm/s减少至3.108×10-6cm/s,P<0.05),外排比率由3.531下降至2.181,减少了38.22%,推测天麻素可发挥类似于维拉帕米或环孢素的作用,即天麻素为P-gp或MRP2抑制剂,可促进葛根素的跨膜转运,其促进程度与维拉帕米接近。
4结论
4.1葛根素和天麻素配伍理化性质研究
(1)室温下,葛根素在水中的平衡溶解度为1.62g·L-1,天麻素为303.81g·L-1,按2010版药典规定,前者属于微溶范畴,后者属于易溶范畴。一定浓度的天麻素可改善葛根素溶解度,且葛根素溶解度的增加与天麻素浓度呈线性关系。
(2)葛根素、天麻素单独测定时Log P均值分别为0.4803、-0.8573,均小于1,表明二者的亲水性大于亲脂性。葛根素的P值随着pH的升高而降低,表明葛根素在胃液中脂溶性较大,可能在胃中的吸收要高于小肠,而pH对天麻素的P值并无影响,表明天麻素在胃肠道中的吸收无部位特异性。在一定pH范围内,天麻素可使葛根素的亲水性提高,但葛根素并不能改变天麻素在肠道中的脂水分配情况。
(3)以溶解度和油水分配系数为测定指标,从理化性质层面解释葛根素和天麻素配伍应用的合理性。
4.2葛根素和天麻素配伍抗氧化和改善微循环药效研究
(1)葛根素具有DPPH自由基清除作用,而天麻素并无此作用,但这并不意味着天麻素无抗氧化作用,它有可能通过其它途径(如抑制脂质过氧化、增强抗氧化酶活性等)产生作用。
(2)以体内外APTT、ADP诱导的血小板聚集抑制率为指标,葛根素和天麻素一定剂量配伍后,其抗凝血、抗血小板聚集作用增加。
(3)从体内外抗凝血和抗血小板聚集层面,解释了葛根素和天麻素配伍应用的合理性。
4.3葛根素和天麻素配伍在大鼠血浆中药代动力学研究
(1)葛根素和天麻素合用后,可彼此影响在大鼠体内的药代动力学过程,增加吸收,降低清除率,延长平均滞留时间,提高生物利用度。
(2)从体内药动学角度,解释葛根素和天麻素配伍应用的合理性。
4.4葛根素和天麻素配伍在Caco-2细胞中吸收机制研究
(1)低浓度葛根素在Caco-2细胞的转运无明显的方向性,为不需要耗能的被动转运过程；随着浓度升高,葛根素的跨膜转运呈现较强的方向性,且这种有向性可被维拉帕米和环孢素抑制,表明葛根素除被动扩散外,还存在着外排泵的作用,其可能为P-gp和MRP2的底物。天麻素在Caco-2细胞的跨膜转运无明显的方向性,为不需要耗能的单纯被动扩散。
(2)天麻素可使葛根素吸收量增加,外排量减少,推测天麻素可发挥类似于维拉帕米或环孢素的作用,即天麻素为P-gp或MRP2抑制剂,可促进葛根素的跨膜转运,其促进程度与维拉帕米接近,而葛根素对天麻素吸收促进作用不明显。
(3)天麻素(被动转运)这类吸收良好的药物,Caco-2细胞模型是一个研究药物吸收非常好的模型,而对于像葛根素这类吸收差的药物,Caco-2细胞模型只能作为体内吸收的一个定性而非定量指标。
(4)从药物在体外Caco-2细胞模型中吸收机制层面,解释葛根素和天麻素配伍应用的合理性。
5创新点
(1)基于葛根素和天麻素的临床应用,从两者配伍后理化性质、抗氧化改善微循环药效作用、大鼠体内药代动力学和在Caco-2细胞模型中的吸收机制等多层面探讨其配伍应用的合理性。为葛根素和天麻素在临床合用上的实践性提供了一个重要依据,对探索中药成分的配伍理论有一定实践意义。
(2)基于椎-基底动脉供血不足所致的颈椎病、眩晕中医发病机制为缺氧、缺血等微循环障碍,本研究以DPPH自由基清除、体内外活化部分凝血活酶时间、ADP诱导的血小板聚集抑制率为指标,考察药物抗氧化及改善微循环作用,以点带面,多指标验证了葛根素和天麻素配伍的合理性。
(3)首次对葛根素和天麻素在大鼠体内药动学特征进行较深入的探讨,并以Caco-2细胞模型为工具,研究葛根素和天麻素吸收转运机制及相互影响,从药代动力学角度和细胞层面阐明两者吸收机制及配伍应用的合理性；从肠吸收角度,研究两种有效成分的吸收及其相互作用,有助于对中药复方成分配伍规律科学性的理解。
1Objective
Radix Puerariae and Gastrodia elata Blume, the active components of which are puerarin (Pur) and gastrodin(Gas), are used frequently in cervical spondylosis, and they are always been used together recently. Both of them can improve microcirculation, expand coronary artery, lower blood pressure, anti-oxidation, ect. Each of them is used for treatment of hypertension, sudden deafness, cervical spondylosis caused by vertebro-basilar artery insufficiency, vertigo and other diseases caused by microcirculation disturbance in clinic. Their injections were used in combined way for treatment of some cardiocerebrovascular diseases, especially for vertigo due to vertebrobasilar ischemia. Based on their clinical application and theory of TCM on combination, it is necessary to investigate the rationality of combined applications and interaction of the two constituents. Our study focused on the active component, Pur and Gas, to investigate the rationality of their compatibility from the aspects of physico-chemical properties, anti-oxidation, improving microcirculation function, pharmacokinetics in rat and absorption mechanism on Caco-2monolayer cell.
2Methods and Contents
According to the above objective, our experiment plan to study the following aspects of Pur and Gas after their combination, that were physico-chemical properties like solubility and oil-water partition coefficient, pharmacodynamic effects of free radical-scavenging and microcirculation improvment, pharmacokinetics in rats and absorption mechanism on Caco-2monolayer cell. The solubility, oil-water partition coefficient, DPPH free radical-scavenging rate, anti-platelet aggregation, pharmacokinetics parameter and apparent permeation coefficient of Caco-2monolayer cell were taken as the indexes to investigate the compatibility rationality of Pur and Gas.
2.1Study of physico-chemical properties after compatibility of Pur and Gas
2.1.1Apparent solubility
Determining the solubility of Pur and Gas used alone or together with ultraviolet spectrophotometry.
2.1.2Oil-water partition coefficient
Shake flask-HPLC method was used to determine oil-water partition coefficient of Pur and Gas used alone or together in n-octanol/phosphates buffer system of different pH value.
2.2Study of antioxidation and microcirculation improvment after compatibility of Pur and Gas
Taken DPPH free radical-scavenging rate as the index to investigate the antioxidant ability of Pur and Gas used alone or together; Taken APTT and ADP-induced anti-platelet aggregation rate in vitro vivo as indexes to evaluate anticoagulation and anti-platelet aggregation effects of Pur and Gas used alone or together, eventually investigating their effect of improving microcirculation.
2.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
A reliable HPLC method was developed for simultaneous determination of Pur, Gas, HBA and internal standard Tyr in rat plasma, which has been successfully applied to the pharmacokinetic study of the analytes in rats after i.g./i.v. administration of Gas and Pur alone or combined with each other. Chromatography was carried out on an Agilent ZORBAX SB-Aq C18column (4.6×250mm,5μm) equipped with an Agilent analytical guard column (4.6×12.5mm,5μm) using a gradient mobile phase consisted of ACN-H2O with0.05%phosphoric acid as a modifier at a flow rate of1.0mL/min. The UV detector wavelength was set at250nm for Pur, whilst221nm for Gas and IS. Comparisons of the pharmacokinetic data were performed using the software of WinNonlin5.2.1and SPSS statistical software package.
2.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
Concentration of Pur and Gas after transporting through Caco-2monolayer cell was determined by HPLC. Then calculate the apparent permeation coefficients to study the characteristics of transport and absorption mechanism. Meanwhile investigate the influences of concentration and transport protein inhibitor on the absorption of Pur and Gas.
3Results
3.1Physico-chemical properties after compatibility of Pur and Gas
3.1.1Apparent solubility
The solubility of Gas in water was303.81g·L-1and that of puerarin was1.62g·L-1, which could be increased by at most5.1times when adding more than1.5g·L-1Gas. The increasing solubility of Pur and the concentration of adding Gas were in a linear fashion.
3.1.2Oil-water partition coefficient
(1) The oil-water partition coefficients of Pur and Gas were0.4803and-0.8573, respectively, which were close to predictive value(We used software of Chem Draw Ultra6.0to calculate the Log P value, which were0.4826、-1.0595for Pur and Gas, respectively).
(2) Oil-water partition coefficients of Pur
①The Log P value of Pur in all groups were less than1in phosphates buffer system of different pH. It was indicated that puerarin was more hydrophilic and could be difficult to absorb in the intestine.
②The P value of Pur will decrease with the increase of pH. It was indicated that Pur would absorb more easily in stomach than in intestine.
③The P value of Pur in combined group was lower than that in single group, especially in the range of pH1.2to5.8. It showed that adding Gas could increase the hydrophilcity of puerarin, which was consistent with the preliminary solubility results.
(3) Oil-water partition coefficients of Gas
①The Log P value of Gas in all groups were negative in phosphates buffer system of different pH which inferred that Gas was high hydrophilic. Although the Log P value of Gas was less than that of Pur, absorption would be easier for Gas because of its small molecular weight.
②The change of pH have little influence on oil-water partition coefficients of Gas. It was indicated there was no site specificity in the absorption of Gas.
③There was no significant difference between the P value of Pur in single and combined groups, which indicated that Pur could not increase the oil-water partition coefficient of Gas.
3.2Antioxidation and microcirculation improvment after compatibility of Pur and Gas
3.2.1Antioxidation
(1) Pur has effect of antioxidation to certain extent, the IC50of which was17.56g·L-1, while that of the Vc was0.024g·L-1. Meanwhile, Gas has little effect of antioxidation because its IC50is higher than200g·L-
(2) The DPPH free radical-scavenging rate of Pur increased with its concentration, while that of Gas didn't change with its concentration (P>0.05). When added Gas, the DPPH free radical-scavenging rate of Pur didn't increase which indicated that it was just Pur contributed to the anticxidation effect.
3.2.2Improving microcirculation
(1) In vitro APTT
①Low or middle dose groups of Pur had no effect of anticoagulation while high dose group did. There was anticoagulation when middle dose of Pur used together with middle or high dose of Gas.
②Low or middle dose groups of Gas had no effect of anticoagulation while high dose group did. There was anticoagulation when low or meddle dose of Gas used together with high dose of Pur, or meddle dose of Gas used together with middle dose of Pur.
③Groups of M_Pur+M_Gas, M_Pur+H_Gas, H_Pur+L_Gas, H_Pur+M_Gas, H_Pur+H_Gas had the effect of anticoagulation(L, M, H stands for low, middle, high dose).
④The APTT were dose-dependent within the concentration of20-40g/L and150-300g/L for Pur and Gas, respectively.
(2) Invivo APTT
The APTT of all groups had significant difference comparing with blank group, except high dose group of Pur. It indicated that both Pur and Gas had anticoagulation, especially when they were both used in middle dose.
(3) Anti-platelet aggregative activity
There was no significant difference among all groups of Pur or Gas. From the visual analysis results, it showed that the inhibit ratio increased with the dose. However, the anti-platelet aggregation of combined group was significant increased compared with each single group of Pur and Gas.
3.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
(1) A reliable HPLC method was developed for simultaneous determination of Pur and Gas in rat plasma with a linear range of0.05-5.98μg/mL for Pur and0.101-101μg/mL for Gas (r>0.9960). The LLOQ, LOD of Pur and Gas were determined to be0.0486,0.101μg/mL,0.0245and0.05μg/mL, respectively. The intra-day and inter-day precision were all less than12.0%, whilst the extract recovery were all above80%. The fully validated method was successfully applied to the pharmacokinetic study of the analytes in rats after intragastric/intravenous administration of Pur and Gas alone or combined with each other.
(2) The pharmacokinetic profiles of combined administration were found to be distinct from those of given alone, which could have higher bioavailability (F) and lower clearance rate (CL), as well as longer mean residence time (MRT) both through i.g. and i.v. routes, particularly notable via i.g. administration. The relative oral bioavailability of Pur in combined administration is10.7-time as much as that of single administration, while1.5-fold in Gas.
3.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
(1) It was passive transport for50μg·mL-1Pur while directional (Papp(BL→AP)/Papp(AP→BL)>5) for100μg·mL-1,200μg·mL-1Pur across Caco-2monolayer cell. The efflux rate will decrease when added verapamil and cyclosporine, which indicated that the transepithelial transporting process of Pur was partly actively carrier-mediate transport besides the passive diffusion.
(2) It was passive diffusion for100μg·mL-1Gas when transporting across Caco-2monolayer cell.
3) When100μg·mL-1Gas and Pur used together, the permeability coefficient of apical to basolateral was increased from1.285×10-6cm/s to1.425×10-6cm/s, and the permeability coefficient of basolateral to apical was decreased from4.539×10-6cm/s to3.108×10-6cm/s. The efflux rate had reduced from3.531to2.181.This suggests Gas has the similar effect of varapamil, which is the inhibitor of P-gp. Gas could promote the transport of Pur.
4Conclusions
4.1Physico-chemical properties
(1) The solubility of Gas in water was303.81g·L-1and that of puerarin was1.62g·L-1, which belong to slight soluble and easily soluble, respectively according to2010edition of pharmacopoeia. The increasing solubility of Pur and the concentration of adding Gas were in a linear fashion.
(2) The oil-water partition coefficients of Pur and Gas were0.4803and-0.8573, respectively, both of which were less than1. It was indicated that both of them was more hydrophilic. The increase of pH would decrease the P value of Pur while had little influence on the P value of Gas. It was indicated that Pur would absorb more easily in stomach than in intestine, while there was no site specificity in the absorption of Gas. It showed that adding Gas could increase the hydrophilcity of puerarin in a certain pH range, while Pur could not increase the oil-water partition coefficient of Gas.
(3) Taken solubility and oil-water partition coefficient as indexes, investigate the Compatibility Rationality of Pur and Gas from the aspect of physico-chemical properties.
4.2Antioxidation and microcirculation improvment after compatibility of Pur and Gas
(1) Pur has DPPH free radical-scavenging activity while Gas don't, but it doesn't mean Gas have no antioxidation through another ways (inhibit lipid peroxidation, enhanced activity of antioxidant enzymes, etc.).
(2) Taken APTT and antiplatelet aggregation induced by ADP as indexes, effects of anticoagulation and anti-platelet aggregation can be increased when Pur and Gas used together in a certain dose.
(3) Investigate the Compatibility Rationality of Pur and Gas from the aspect of anti coagulation and anti-platelet aggregation in vitro/vivo.
4.3Pharmacokinetics in rat plasma after administration of Pur and Gas together
(1) Pur and Gas will affect each other on pharmacokinetic profiles, such as improving bioavailability (F), lowering clearance rate (CL) and prolong mean residence time (MRT) when they were co-administrated to rats.
(2) Investigate the Compatibility Rationality of Pur and Gas from the aspect of pharmacokinetics.
4.4Absorption mechanism of Pur and Gas in Caco-2monolayer cell
(1) It was passive transport for low concentration of Pur while directional with the increase of concentration when transporting across Caco-2monolayer cell. This directional transpot will be weakened when added verapamil and cyclosporine, which indicated that the transepithelial transporting process of Pur was partly actively carrier-mediate transport besides the passive diffusion. Pur may be the substrate of P-gp and MRP2. It was passive diffusion for Gas when transporting across Caco-2monolayer cell.
(2) Gas can promote the permeability coefficient from apical to basolateral and reduce the permeability coefficient from basolateral to apical of Pur, suggesting that Gas has the similar effect of varapamil (the inhibitor of P-gp), could promote the transport of Pur.
(3) Caco-2monolayers are an excellent model of the passive transcellular pathway (like Gas), while just be an index of qualitative rather than quantitative concerning the slowly and incompletely absorbed drugs (like Pur).
5Innovations
(1) Based on clinical application of Pur and Gas and theory of TCM on combination, investigate the rationality of their compatibility from the aspects of physico-chemical properties, improving microcirculation function, pharmacokinetics in rat and absorption mechanism on Caco-2monolayer cell. These results might lay a foundation for explaining the combination of traditional Chinese medicine in prescriptions containing Pur and Gas and provide an important basis in clinical practice with these two components.
(2) The pathogenesis of cervical spondylosis and dizziness caused by the vertebrobasilar insufficiency from perspective of traditional Chinese medicine are hypoxia and ischemia, the symptoms of microcirculation disturbance. The DPPH free radical-scavenging activity, APTT and ADP-induced anti-platelet aggregation rate were selected as indexes to investigate the antioxidation and microcirculation improvment of Pur and Gas.We used multi-index to prove the rationality of their compatibility, faning out from point to area.
(3) The pharmacokinetic characterizations of Pur and Gas in rat were discussed in-depth. The HPLC method for simultaneous determination of Pur, Gas, HBA and internal standard (Tyr) in rat plasma was developed and validated for the first time.
(4) Caco-2monolayer cell model was used to study the absorption mechanism and the interaction between Pur and Gas. Investigate the Compatibility Rationality of Pur and Gas from the cellular level.
引文
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