Study On Separation, Purification And Hypoglycemic Effect Of Corosolic Acid From Eriobotrya Japonica Leaves

In recent years, it has become research focus extracting hypoglycemic active components from natural products diabetes since the prevalence of diabetes was increasing. Corosolic acid(CA), which belongs to triterpenoid acid, has many biological activities. This thesis took traditional Chinese medicinal plant Eriobotrya japonica Leaves as raw material, established a HPLC method for determination of CA, optimized extraction, separation and purification methods of CA and studied its hypoglycemic effect in small mice.1. A simple HPLC has been developed and validated for analysis of corosolic acid from Eriobotrya japonica leaves. The sample was accomplished on a C18 column(250 mm×4.6 mm, 5 μm) and recorded at 210 nm. The greatest resolution was achieved with methanol-1%acetic acid water(92:8,v/v) as mobile phase at a flow rate of 1.0 mL/min. The calibration curve was: y=3.78×106x-6227.14, and the correlation coefficient for the calibration curve plot(R2 = 0.9991) showed linearity good over the range tested(0.0125-0.4 mg/m L). The average recovery was 99.67%. This method is convenient, fast, accurate and reliable.2. The technique conditions for extracting CA from Eriobotrya japonica leaves by using ultrasonic and microwave assisted extraction were investigated. The results showed that by ultrasonic assisted extraction the yield of CA was 0.68% with the optimal extracting conditions: extraction temperature 50℃, time 30 min and 90% ethanol as sovent, 1:20(g/m L) as the ratio of material to liquid. By microwave assisted extraction, in condition of extraction time 4 min and microwave power 200 W, the yield of CA was 0.52%. Under the optimal extracting conditions, the yield of CA is 30.77% higher by ultrasonic than microwave assisted extraction, however, by microwave assisted extraction 87% of extraction time and 50% of extraction solvent can be saved. Advantage of extracting CA assisted by ultrasonic and microwave both are effective and convenient.3. The optimum technologies of separation and purification were established by macroporous resin asorption, silica gel colum chromatography and recrystallization. The results of macroporous resin separation static test implied that HZ802 was the best resin for the separation. The dynamic test indicated that the optimal process parameters were as follows: 3 BV of 8 mg/mL sample was loaded onto the column at flow rate of 2BV/h, then eluted with 2.5BV of 90% ethanol solution at flow rate of 2BV/h, obtaining a CA purity of 34.12%. The conditions of silica gel colum chromatography were as follows: Dry sample volume was CA: silica gel(1:50), petroleum ether: ethyl acetate(2:1,v/v) as eluent. The purity of CA was 53.28%. The purity of CA can be futher reached 96.43% by recrystallization.4. To study the hypoglycemic effect of CA from Eriobotrya japonica Leaves in diabetic mellitus mice(DM) induced by streptozotocin(STZ). DM were established by intraperitoneal administration of STZ at 160 mg/kg. 56 male ICR mice were selected as 7 groups: nomal and DM control groups, low, medium and high dose treatment groups which were administrated extracts with CA at 50, 150 and 300 mg/kg respectively, positive control group which was administrated with gliclazide at 50 mg/kg and CA standard sample group which was treated with 5 mg/kg. The concentraction of fasting blood glucose(FBG), oral glucose tolerance test(OGTT), insulin(INS), blood lipid index and ogan index were determined. The results showed that the administration groups of fasting blood glucose(FBG) decreased significantly(P < 0.01). INS and HDL-C were significantly improved in gliclazide, middle and high dose treatment groups(P <0.05). TC and TG were significantly reduced in gliclazide and middle dose treatment group(P <0.05). And there were no significant effects on LDL-C and organ index. The results indecated that CA can regulate FBD and lipid metabolism. It provides a certain reference for the development of new drugs with CA.