| Berberine has been found to have a wide range of pharmacological activities in recent years,such as antioxidant,hypoglycemic,hypolipidemic,anti-cancer,and cardiovascular and cerebrovascular diseases.But its poor intestinal absorption,low bioavailability,long-term use can perhaps result in serious intestinal symptoms.(Huang-gui solid dispersion,HGSD)was successfully prepared by the combination of berberine and sodium caprate(SC),and the national patent(patent number)was obtained.: ZL2008 1 0050679.X),and the preliminary work of the research group shows that the anti-diabetic effect of the new form is significantly better than pure berberine.Because of the need to dissolve berberine in anhydrous ethanol during the preparation of solid residue,it is necessary to dissolve berberine in anhydrous ethanol,and there is a high cost of anhydrous ethanol in the process of large-scale industrial preparation and the high risk of preparation process.Therefore,in the first part,the process of Huang-gui solid dispersion was optimized.The specific experimental methods were as follows:①Huang-gui solid dispersion with different concentrations of ethanol solvent were analyzed by solvent evaporation method,X-ray diffraction analysis,electron spectroscopy and infrared spectroscopy were used to analyze the HGSD.The existence of the state of the material and the interaction between the initial materials were studied to study whether the chemical reaction between the material.②The optimum process of yellow decene solid dispersion was screened out by process cost and dissolution curve.③Study on the bioavailability of the Best Process Huang-gui solid dispersions.④Acute toxicity and subacute toxicity studies to ensure its safety.The experimental results show that①There is no significant difference in the content of the yellow decene solid dispersion under different preparation conditions.The infrared spectra of the solid dispersions prepared by different conditions are almost all of the superposition of berberine,sodium caprate and PEG6000,and there is no obvious difference,which can show that there is no chemical reaction in each material.X-RD indicates that the berberine crystal peak in the physically mixed sample still indicates that berberine is present as a crystalline form.In the solid dispersion,there are two characteristic absorption peaks of PEG6000,while the characteristic absorption peak of berberine is greatly weakened,which indicates that the majority of berberine in the solid dispersions of Huang-gui under various conditions are mainly in amorphous form.Electron scanning electron microscopy shows berberine pure product was smooth needle-like,part of the cohesion into a bundle.In the physical mixing state,berberine is present in the form of crystals,while PEG6000 is the attachment of berberine and capric acid in different shapes and sizes.The Huang-gui solid dispersion is very different,and the crystallinity of the crystal is obviously decreased,only a very small number of berberine crystals exist,the crystal attachment disappeared shows that the vast majority of berberine and sodium caprate dispersed in PEG6000.②We found that the Huang-gui solid dispersion prepared by the ethanol solvent under 70% concentration was more gradually,indicating that the dispersion of the yellow decene solid dispersion prepared under these conditions was poor.The dissolution rate of HGSD-1,HGSD-2,HGSD-3 were higher than that of the original research and berberine hydrochloride.So the final choice is 80% ethanol solution.③Compared with commercially available berberine,the Cmax,Tmax and AUC0-24 h of HGSD-3 were significantly increased,and Cmax increased to about 5 times,Tmax was advanced from 60 min to 40 min,and the bioavailability was significantly increased.In the acute toxicity,the activity and appetite of mice were not significantly changed with the increase of berberine dosage,and the dosage was 1 g / kg,2 g / kg,4 IX G / Kg,6 g / kg,the mice were alive,at 6 g / kg,and the mice had a significant decrease in activity and appetite.When the concentration reached 8 g / kg,mice had died,but given the greater concentration,the Huang-gui solid dispersion were no longer soluble in the solvent,so we did not further study the survival rate of the mice with greater dosage.Subacute toxicity,200-220 g wistar rats were divided into Control,100 mg / kg,200 mg / kg,400 mg / kg,800 mg / kg dose group,each group of 10,respectively,after 14 days,Organs,blood tests and whole blood biochemical,coagulation factors and other indicators.The rest of the mice to stop the drug,withdrawal 7 days after the relevant indicators.The mean red blood cell count,erythrocyte count,hemoglobin,erythrocyte volume,mean erythrocyte volume,mean erythrocyte hemoglobin,mean erythrocyte hemoglobin concentration,white blood cell count,platelet count,prothrombin time,alanine aminotransferase,There were no significant difference in the concentrations of urea nitrogen,creatinine,total protein,albumin,blood glucose,total cholesterol,potassium,nitrogen and sodium.Alanine aminotransferase,aspartate aminotransferase increased to varying degrees,but 100 mg / kg,200 mg / kg,400 mg / kg withdrawal 7 days after the return to normal,800 mg / kg is still a small increase.It was demonstrated that sub-acute toxicity,the solid dispersion was safe at doses of 100 mg / kg,200 mg / kg,400 mg / kg.Second,the preliminary work of this group has proved that berberine and berberine as the main drug Huang-gui solid dispersion on type 2 diabetes with hypoglycemic effect,and diabetes-induced myocardial injury has a protective effect,but caused by type 1 diabetes Whether the myocardial injury has a protective effect and its mechanism is not clear.It is known that the Huang-gui solid dispersion prepared under the new technological process is not significantly different from the solid dispersion in the previous study.Therefore,based on the preliminary basis,we successfully established the model of type 1 diabetes mellitus to explore the new process on the protective effect of type 1 diabetes mellitus on myocardial injury.We explored the protective effect of the solid dispersion of Huang-gui on the apoptosis of cardiomyocytes induced by diabetes mellitus from the whole level.The specific experimental method was used to replicate Wistar type 1 diabetes mellitus(T1DM)model by intraperitoneal injection of streptozotocin(STZ).The mice were randomly divided into three groups: 15 rats in each group: blank group(Con),model group,T1 DM rats were given the same amount of normal saline.The rats in the control group were treated with HGSD-3(200 mg / Kg,bw);administration time is 4 weeks.Echocardiography showed changes in cardiac function in each group.HE staining was used to detect heart disease in each group.TUNEL staining was used to detect the apoptosis of cardiomyocytes in each group.The expression of apoptosis-related protein,autophagy-related protein,P-AMPK / AMPK and P-m TOR / m TOR were detected by Western blotting.The results showed that①The body weight of the Model group was significantly lower than that of the control group(Con),and the body weight was significantly increased after 4 weeks of administration of HGSD-3.Compared with the control group(Con),fasting blood glucose was significantly increased in Model group,and fasting blood glucose decreased after 4 weeks of administration of HGSD-3,but it was not obvious.②Compared with Con group,the ventricular septum(IVS)was significantly increased in T1 DM group,the level of cardiac ejection fraction(EF%)decreased,the level of cardiac function(E / A)decreased,and the HGSD-3 group improved the cardiac function Incomplete phenomenon.The results of HE staining showed that the myocardium of Con group was neat and the nuclear staining was clear.The myocardium of the Model group was disordered,the nucleus was deeply stained,the hypertrophy of myocardium was deformed,the infiltration of inflammatory cells and the interstitial fibrosis were obvious.Chaos,nuclear staining is basically clear,the cell morphology is generally complete.TUNEL staining showed that the number of TUNEL positive cells was increased in the Model group compared with the Con group,and the number of positive cells was decreased in the administration group compared with the DM group.③The expression of Bcl-2 in myocardium of model rats was significantly decreased,and the expression level of apoptosis-related protein Cleaved Caspase-3 was significantly increased.The expression of autophagy-related proteins P-ULK1 / ULK1,Beclin-1,LC3(P <0.05,P <0.01).The expression of P-AMPK / AMPK was significantly decreased and the expression of P-AMPK / AMPK was significantly decreased(P <0.05,P <0.01).Compared with Model group,the expression of Bcl-2 protein in HGSD-3 group was significantly higher than that in model group(P <0.05).The expression level of apoptosis-related protein Cleaved Caspase-3 was significantly decreased in HGSD-3 group,and the expression of apoptosis-related protein P-ULK1 / ULK1(P <0.05,P <0.01).The expression of P-AMPK / AMPK was significantly increased,and the expression of P-AMPK / AMPK was significantly increased(P <0.05,P <0.01).In summary,the preparation scheme of 80% solvent is the best production,and the safety is high.At the same time,the experiment also proved that the Huang-gui solid dispersion can protect the myocardial injury caused by type 1 diabetes.The mechanism may be through the inhibition of myocardial cell apoptosis to protect the cardiomyocytes,and AMPK may be its key target,the specific mechanism may be through the activation of AMPK / m TOR pathway,thereby activating autophagy,and then inhibit cardiomyocyte apoptosis. |
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