Study On Synergistic Effect Of Berberine Hydrochloride And Berbamine Hydrochloride Based On Intestinal Absorption-liver Metabolism

Diabetic Retinopathy is one of the most serious microangiopathy of Diabetes Mellitus.And DR is the key reason of blindness in patients of DM.Therefore,the tasks of research and development of new drugs for the prevention and treatment of DR are very urgent.It has been shown that the blood glucose level and the expression of VEGF in DR’s models can be obviously lowered by Berberine hydrochloride,and obviously ameliorate the damage of Microvascular morphology.So Berberine hydrochloride will have great development prospects in the prevention and treatment of DR.Both Berberine and Berbamine are components of Tibetan medicine Berberis Cortex Extracts.Berberine hydrochloride is a substrate for P-glycoprotein（P-gp）.The efflux of P-gp leads to low bioavailability of Berberine hydrochloride.Berbamine hydrochloride is a calcium channel antagonist.The extracellular calcium influx after activation of the body-regulated calcium channel has obvious inhibitory effect,which can inhibit the efflux of P-gp,suggesting that Berberine hydrochloride can synergize with Berberine hydrochloride to increase the absorption and metabolism of Berberine hydrochloride and enhance its therapeutic effect.Therefore,the two are used as the first step in the in vitro study of antioxidant matrix,the first step of in vivo intestinal absorption of the drugs and the first hepatic metabolism test of intestinal liver metabolism to study on synergistic effect of Berberine hydrochloride and Berbamine hydrochloride.Objective:1.To determine the antioxidant activity of Berberine hydrochloride and Berbamine hydrochloride and the two different compatibility groups,and to find the synergy between the two.2.To determine the absorption parameters of intestinal absorption in different compatibility of Berberine hydrochloride and Berbamine hydrochloride,and to find the synergy between the Berberine hydrochloride and Berbamine hydrochloride in intestinal absorption.3.To determine the metabolic parameters of Berberine hydrochloride and berbamine hydrochloride in different liver metabolism of different species,and to find the synergy between the Berberine hydrochloride and Berbamine hydrochloride in liver particle metabolism.Method:1.The DPPH rate of Berberine hydrochloride and Berbamine hydrochloride and their compatibility group were determined by TLC and DPPH method.The IC50 of Berberine hydrochloride and Berbamine hydrochloride was obtained.2.The unilateral intestinal perfusion method was used to determine the absorption parameters of different compatibility groups Berberine hydrochloride and Berberine hydrochloride.3.The metabolic rate of rat and human livers microparticles were determined by hepatic particle metabolism in different compatibility groups of Berberine hydrochloride and Berbamine hydrochloride.Result:1.The IC50 of Berberine hydrochloride,Berbamine hydrochloride and VC were819.00,275.91,180.75μg·mL^-1,respectively.The IC50 was the same in the antioxidant activity of Berberine hydrochloride and Berbamine hydrochloride.The stronger the ability of antioxidants to scavenge free radicals,indicating that the antioxidant activity of Berbamine hydrochloride is greater than that of Berberine hydrochloride.J20:A60 has the strongest antioxidant activity in the combination of Berberine hydrochloride and Berbamine hydrochloride,suggesting that the antioxidant activity of Berbamine hydrochloride is greater than that of Berberine hydrochloride.2.In the unidirectional perfusion test of Berberine hydrochloride and Berbamine hydrochloride’s different compatibility groups,the perfusate’s pH 7.4 was selected.In each intestinal segmenthe,Berberine hydrochloride’s absorption was cut back according to the jejunum,duodenum and ileum.The decrease in Berberine hydrochloride and Berbamine hydrochloride was compared with the Berberine hydrochloride control group.It was found that the Berberine hydrochloride’s Ka,Papp and Q in the compatibility group was larger than the the control group,indicating that it was in the compatibility group.The absorption of Berberine hydrochloride in different intestinal segments increased to some extent,indicating that Berberine hydrochloride promoted the absorption of Berberine hydrochloride in the small intestine,which may be related to the inhibition of P-gp protein.The mass concentration ratio of the compatibility group was J40:A50 and J30:A20’s Ka were significantly different from other compatibility groups（P<0.05）,suggesting that the Berberine hydrochloride concentration ranged from 10 to 60μg·mL^-1.In the compatibility groups,the absorption of J40:A50 and J30:A20 are better.3.In the study of the metabolism of Berberine hydrochloride in rat and human liver microsomes,Berberine hydrochloride was incubated in rat and human liver microsomes’s the best condition is that temperature was 37℃,time was 60 min,liver microsomal protein concentration was 0.5 g·L^-1.When Berberine hydrochloride mass concentration 2.5²0μg·mL^-1,in rat liver the Km in the microsomes was 1.6157μg·mL^-1,which was greater than the Km value of human liver microsomes of 1.4775μg·mL^-1,suggesting that the enzyme in human liver microsomes may have an affinity for Berberine hydrochloride compared with that in rat.The maximum size of the microsomes and the maximum reaction rate Vmax in rat liver microparticles is12.8535μg·g^-1·min^-1,which is less than the Vmax value of human liver microsomes14.99701μg·g^-1·min^-1.Human liver microparticles have a stronger ability to metabolize berberine hydrochloride.The combination of Berberine hydrochloride and Berbamine hydrochloride showed a significant difference in the metabolic rate of J15:A5 in the compatibility group compared with J2.5:A2.5（P<0.05）.There wasn’t significant difference in other compatibility groups;in human liver microsome metabolism,compared with J2.5:A2.5 group,the metabolic rate of J15:A5,J10:A20in the compatibility group was significantly different（P<0.05）,other’s weren’t significant difference in the compatibility group,and the metabolic rate of J15:A5 was the highest in the liver microsome metabolism of rats and humans,indicating that J15:A5 was the best compatibility under the incubation conditions.Conclusion:When Berberine hydrochloride is combined with Berbamine hydrochloride,antioxidant is almost entirely determined by Berbamine hydrochlorid,and Berbamine hydrochloride can promote the absorption and metabolism of Berberine hydrochloride in intestine and liver,and both drugs have synergistic effect.Got the best compatibility groups of Berberine hydrochloride and Berbamine hydrochloride in the intestinal absorption and liver microparticles.It provides a scientific basis for the compatibility research and formulation development of Berberine hydrochloride.