Screening And Evaluation Of Hypoglycemic Supramolecular System Based On Chinese Herbal Active Ingredients Gallic Acid

Diabetes Mellilus?DM?is a lifelong metabolic disease.The patient of DM needs take medicine over lifetime period once DM occurs,so drug safety is of vital importance.Therefore,scientific treatment and rational drugs combination are essential.In particular,the combination of traditional Chinese and western medicine is a common treatment for diabetes.Rhus chinensis Mill.?RCM?belongs to the Anacardiaceae family and the Rhus chinensis Mill.genera.It has the functions of lung constriction and decreasing internal heat,detoxification and hemostasis,astringent intestines and antidiarrhea,dampness constriction and so on.Moreover,RCM has been documented to treat thirst quenching?diabetes mellitus?.The chemical composition of Galla chinensis is complex,and the main active ingredients are tannin and gallic acid.Gallic Acid?GA?has a preventive effect on cardiovascular diseases,nervous system diseases,diabetes,liver fibrosis and tumors,which provides broad application prospects for disease treatment.GA is soluble in hot water,ethyl ether,ethanol,acetone and glycerol,but not in cold water.The dissolution behavior of drug has great influence on the bioavailability.The improvement on bioavailability can be achieved by increasing the solubility of drug.Repaglinide?RG?,which is a short-acting oral insulin-producing hypoglycemic agent,belongs to the BCS II drugs.It has the characters of low solubility?almost insoluble in water?and high permeability.As a result,RG shows only about 50%of oral bioavailability and short half-life.Long-term taking medication causes serious medication compliance problems in patients.Pharmaceutical cocrystal and coamorphous phases are supramolecular systems which are constructed using active pharmaceutical ingredients?API?and biocompatible small molecule?Cocrystal former,CCF;Coamorphous former,CAF?through non-covalent forces of hydrogen bonds and van der Waals forces.Cocrystal and coamorphous phases can keep the intact structure and pharmacological activities of API,while,produce advantages on solubility,dissolution rates and even bioavailability.Recently,Cocrystal and co-amorphous have received much attention on improving the physical andchemical properties of the API.Part I In vivo evaluation of RCM and its extractObjective:To evaluate the pharmacokinetic behaviors and the hypoglycemic efficiencies of RCM and its extract in rats.Methods:According to the determination method of RCM in Chinese Pharmacopoeia 2015,RCM extract was prepared by hydrochloric acid hydrolysis method.The in vivo pharmacokinetics of RCM and its extracts in rats were studied through determing the concentration of GA in rat plasma using LC-MS/MS method.The diabetic mouse models were established using streptozotocin?STZ?-inducing method.The hypoglycemic efficiencies of RCM and its extracts in rats were evaluated using diabetic mouse models.Results:The C_max of GA in RCM and its extract were 0.31 and 0.77 mg·L^-1,respectively.Compared with RCM,the pharmacokinetic parameters C_max,AUC_0-t and AUC_0-?of the extract were significantly improved?P<0.05?.Both RCM and its extract showed hypoglycemic effect on diabetic mouse,and the hypoglycemic efficiency of extract was better than than of RCM.Conclusion:Compared with traditional Chinese medicine RCM,the extract of active components showed higher bioavailability and hypoglycemic efficiency.The positive correlation between the improvement of GA in vivo pharmacokinetic parameters C_max and AUC and the hypoglycemic efficiency indicated that the main active component of gallnut extract with hypoglycemic effect was GA.Based on this,we carried out the project on screening and evaluation of GA-based supramolecular complexes in the following parts.Part II Screening and physicochemical properties evaluation of supramolecular cocrystal complexes based on GAObjective:To screen cocrystal supramolecular complexes using GA as the model API to improve the physicochemical properties involving solubility and dissolution rate of GA.Methods:Using GA as API and biocompatible small molecules such as organic acids,amino acids,amides and piperazines as CCFs,GA-based cocrystals were screened using solvent-assisted grinding method.The Powder x-ray diffraction?XRD?,Dfferential scanning calorimeter?DSC?,Termogravimetric analysis?TG?,Ifrared spectroscopy?IR?,Scanning electron microscope?SEM?were performed to characterize the screened complexes.Density functional theory?DFT?simulation was applied to investigate the hydrogen bondings in cocrystals.The equilibrium solubilities and dissolution rates of complex in vitro were determined by saturation solubility method and paddle method,respectively.Results:Seven cocrystals were identified by structural characterization,namely GA-paminobenzoic acid?GA-PABA,mole ratio is 1:1?,GA-succinimide?GA-SM,1:2?,GA-amino acetic acid?GA-AAA,1:1?,GA-glutaric acid?GA-GLA,1:1?,GA-L-proline?GA-LP,1:1?,GA-piperazine?GA-PZ,1:1?,GA-caffeine?GA-CA,1:1?cocrystals.The hydrogen bond in GA-PABA cocrystal was formed between C=O of GA and N-H of PABA.The GA-SM cocrystal was produced in 1:2 molar ratio,therefore,two hydrogen bonds were constructed by C=O and O-H in GA molecule and N-H and C=O in the two SM molecules,respectively.The hydrogen bonds of GA-AAA and GA-CA cocrystals were derived from O-H of GA and C=O of CCFs.The formation of hydrogen bond between GA and GLA involved in the respective-COOH in the two molecules,which produced double hydrogen bonds?C=O…H-O and O-H…C=O?.The hydrogen bonds of the GA-LP and GA-PZ were produced between C=O of GA and N-H of LP and PZ.The results of evaluation on physicochemical properties showed that the solubilities and dissolution rates of GA-PABA,GA-SM,GA-AAA,GA-GLA and GA-LP were significantly improved compared with GA?P<0.05?.Conclusion:Most cocrystal complexes improved the solubility and dissolution rate in vitro.Screening of supramolecular complexes has great potential in improving the physicochemical properties of GA.Part III:?-Glucosidase inhibition and molecular docking of GA and its cocrystalsObjective:To evaluate the inhibitory efficiency of GA and its cocrystals on?-glucosidase under physiological p H conditions,and explore the changes and mechanisms of inhibition efficiency of?-glucosidase of cocrystals compared with GA at the molecular level,which provide a reference for further study on action of GA-based cocrystals in vivo.Methods:GA,and GA-PABA,GA-SM,GA-AAA,GA-GLA and GA-LP cocrystals were selected for?-glucosidase inhibition evaluations.The p-nitrophenol-?-D-glucoside?PNPG?was used as a substrate to measure the inhibitory activity of GA and its cocrystals on?-glucosidase by spectrophotometry.Molecular Docking?MD?technology was used to simulate the binding pattern of GA?or its cocrystals?with?-glucosidase.The binding pattern of GA and its cocrystals to?-glucosidase was theoretically studied to provide theoretical guidance to the experimental results.Results:The results of the enzyme activities of GA and its cocrystals in vitro showed that the?-glucosidase inhibition efficiency of cocrystals were different from that of GA.The inhibitory activities on?-glucosidase of GA-PABA,GA-SM,GA-GLA and GA-LP cocrystals were higher than GA.Among those cocrystals,GA-PABA and GA-GLA cocrystals were particularly prominent in improving the inhibitory activities on?-glucosidase,while,the GA-SM cocrystal showed slight improvement of inhibitory activity than that of GA.However,the?-glucosidase inhibition of the GA-AAA was lower than that of GA.The GA-PABA,GA-SM and GA-AAA cocrystals were selected to perform MD simulations with?-glucosidase protein molecule.Combined the results of MD and inhibitory activity evaluation,it indicated that GA-PABA system entered the?-glucosidase active cavity in the form of GA-PABA supramolecular,which made the whole structure more stable and the binding pattern stronger.However,in the case of GA-SM?1:2?cocrytal system,if it entered the?-glucosidase active cavity in the GA-SM supramolecular form,one of the SM molecule did not really enter the active cavity due to the large size of the whole supramolecular.This binding pattern of GA-SM system with?-glucosidase resulted in no significant difference of the binding energy between the whole supramolecular system and a single GA molecule.Combined with the results of?-glucosidase active of GA and GA-SM?1:2?cocrytal,it was reasonable speculation that the single GA molecule,but not GA-SM supramolecular,entered the active cavity of?-glucosidase protein to perform binding procedure.After the formation of supramolecules between GA and AAA,the overall GA-AAA supramolecules could not penetrate into the active cavity of?-glucosidase protein,but only bound with?-glucosidase near the cavity mouth.As a result,the binding energy with?-glucosidase protein of GA-AAA system was lower than that of GA.The results further confirmed the experimental results of decreased enzyme activity of GA-AAA cocrystal.Conclusion:The binding mechanisms of GA and its cocrystals with?-glucosidase protein were investigated based on the results of MD simulation and inhibitory activity evaluations.Among the selected three cocrystal systems,API-CCF whole supramolecules entered the active cavity of?-glucosidase protein during the binding procedure for GA-PABA and GA-AAA cocrystal systems,but single GA molecule entered the active cavity in the case of GA-SM system due to the dissociation of hydrogen bond between GA and SM.In the cocrystal supramolecular system,the combination of CCF and API would lead to the conformational change of API,which further affects the binding of API and receptor,as a result,produce difference in the enzyme activity.Different API-CCF cocrystal structures induce different changes,which may be synergistic or inhibitory.Part IV The evaluation on pharmacokinetic and hypoglycemic efficiency of GA and its cocrystalsObjective:To evaluate the pharmacokinetics and hypoglycemic efficiency of GA and its cocrystals in vivo,and to explore the differences of pharmacokinetics and hypoglycemic efficiencies between GA and its cocrystals.Methods:Using SD rats as animal models,the pharmacokinetics of GA,GA-PABA,GA-SM,GA-AAA and GA-GLA cocrystals were evaluated by detmining the concentration of GA in plasma of SD rats using LC-MS/MS method.A diabetic mouse model was established by STZ-inducing method.The hypoglycemic efficiency of GA and the GA-PABA,GA-SM and GA-AAA cocrystals were investigated to elucidate the difference between GA and cocrystal on hypoglycemic activity in vivo.Results:The pharmacokinetic parameters involving C_max,AUC_0-t and AUC_0-?)of cocrystals were improved compared with GA?P<0.05?.Especially GA-GLA showed the most significant improvement.The C_maxs of GA and GA-GLA cocrystal were 1.53 and 2.67 mg·L^-1,respectively;the AUC_0-ts were respectively.The hypoglycemic efficiencies of all cocrystals were higher than that of GA.Especially,the improvement on hypoglycemic efficiency of GA-PABA cocrystal was the most significant;the maximum hypoglycemic rates were 58.7%and 12.8%for GA-PABA cocrystal and single GA,respectively.The difference was statistically significant.Conclusion:Screening of GA-based supramolecule cocrystals had great potential in improving its pharmacokinetic parameters(C_max,AUC).With the increase of bioavailability and the enhancement of inhibitory activity on?-glucosidase,the hypoglycemic efficiency of GA-based cocrystal was improved significantly.Part V Construction and evaluation of GA-PABA-repaglinide ternary supramolecular hypoglycemic systemObjective:To construct a ternary supramolecular system using double API?GA and RG?as a model,and to evaluate its physicochemical properties in vitro,pharmacokinetic and hypoglycemic effect in vivo.To explore whether the combination of GA and repaglinide produce synergistic effects and increase hypoglycemic efficiency on the premise of lowering the dose of western drug repaglinide.Construction ternary supramolecular hypoglycemic system provide new ideas for the treatment of diabetes using integrated traditional Chinese active ingredient and western medicine.Methods:The ternary supramolecular system was screened by solvent evaporation.The screened supramolecular complex was characterized by PXRD,TMDSC and IR.The equilibrium solubility and dissolution rate of the complex were determined by saturated solubility and paddle methods.The pharmacokinetic of repaglinide and its ternary supramolecular complex were evaluated through determining the concentration of repaglinide in SD rat plasma using LC-MS/MS method.A diabetic mice model was established by STZ-inducing method and used to evaluate the hypoglycemic efficiency of repaglinide and its ternary supramolecular complex.Results:The GA-PABA-RG ternary coamorphous supramolecular system was successfully constructed.The formation of the new phase of ternary coamorphous was confirmed using PXRD and TMDSC characterization.The hydrogen bonds were formed between C=O of RG and N-H of PABA,and between O-H of GA and C=O of PABA;PABA as a bridge molecule in the ternary system.The results of physiochemical properties showed that the solubility of RG in coamorphous was 1.2 times higher than that of pure RG;the cumulative dissolution rate increased from 72.1%to80.3%?P<0.01?.The C_maxs of single RG and RG in the coamorphous were and 5.09 mg·h·L^-1,respectively.Compared with single RG,the coamorphous phase were increased by 1.9 times?P<0.05?,1.5 times?P>0.05?and 1.8 times?P>0.05?,respectively.The maximum hypoglycemic rates were62.30%and 39.42%for GA-PABA-RG ternary coamorphous and single RG,respectively.The coamorphous complex with GA improved the hypoglycemic efficiency compared with single RG.Conclusion:The ternary coamorphous supramolecular system was constructed,in which western medicine RG and Chinese medicine monomer GA were linked together by PABA as a bridge molecular.The formation of ternary coamorphous effectively improved the physicochemical properties of RG.The combination of RG and GA produced synergy on hypoglycemic efficiency.In the case of using the same dose of western medicine RG,the efficiency of hypoglycemic was improved nearly doubled with the introduction of low toxicity component GA.The construction of the western medicine component-bridge molecule-Chinese medicine monomer ternary supramolecular system provided a novel idea for the combination of Chinese medicine and western medicine in the treatment of diabetes.