Compatibility Rationality Study Of Puerarin And Gastrodin Based On Physico-chemical-Pharmacological Function-absorption Of Drugs

1ObjectiveRadix 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 ContentsAccording 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 Gas2.1.1Apparent solubility Determining the solubility of Pur and Gas used alone or together with ultraviolet spectrophotometry.2.1.2Oil-water partition coefficientShake 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 GasTaken 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 togetherA 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 cellConcentration 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. 3Results3.1Physico-chemical properties after compatibility of Pur and Gas3.1.1Apparent solubilityThe 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 Pur①The 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 Gas①The 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 Gas3.2.1Antioxidation(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 APTT①Low 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 APTTThe 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 activityThere 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.4Conclusions4.1Physico-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.