Enhancement Of Hypoglycemic Activity Of Extract Of Ginkgo Biloba Leaf By Biotransformation

Ginkgo biloba,as one of the most popular herbal medicines,was planted in China widely,and the annual production of its leaf was approximately 70%in the world. Ginkgo biloba Leaf has attracted great attention as agents for decreasing the blood glucose level,balancing the blood lipid level,scavenging the free radicals,improving circulation,particularly cerebral circulation.However,due to the inefficiency of Ginkgo biloba Leaf directly ulitilized as the herb medicines,the problem how to elevate the efficiency of the herb seems to be more serious.Now increasing interests has attracted on the biotransformation of Ginkgo biloba Leaf by the medicinal mushrooms which might improve the bioactive components and their medical effects of the herbs.In current investigation,the comparisons of various indexes such as glucose, fructosamine,sugar,NADH,haemoglobin,glycosylated haemoglobin in blood,the systemic enzymes related to the glycolytic metabolism such as hexokinase,citratse synthase and pyruvate dehydrogenase in kidney and liver in the normal and alloxan-induced hyperglycemic rats were carried out.Results showed that the fructosamine level was significant higher in the model rats than that in the normal group, which indicated that the non-enzymatic glycation reaction was enhanced in the model rats.Significant declining of the content of total haemoglobin,increasing of the ratsio of glycosylated haemoglobin/total haemoglobin and NADH level,and decreasing of the related enzymes such as hexokinase,citratse synthase and pyruvate dehydrogenase activities of hexokinase,citratse synthase and pyruvate dehydrogenase showed that the Tricarboxylic acid cycle（TCA） and Embden-Meyerhof pathway（EMP） of glycolytic metabolism were inhibited and finally accelerates the blood glucose levels.According to above results,the hypothesis that non-enzymatic glycosylation reaction could increase the blood glucose of the diabetic rats was put forward.The reaction systems to detect the non-enzyme glycation reaction and Fenton reactive system were optimized,respectively,and based on the bioactivities scavenging the free radicals and inhibition of non-enzymatic glycation reaction andα-glucosidase activity,11 strains such as Hericium erinaceus,Coriolus versicolor,and yeast were chosen to biotransformate extract of Ginkgo biloba L.（EGB）.Results showed that comparing with other strains,all three indexes were enhanced after the biotransformation of EGB by Hericium erinaceus,and especially,the inhibition of non-enzymatic glycation reaction was most significantly improved.Based on the results of inhibition of non-enzymatic glycation reaction in vitro, optimization of the operating parameters such as pH,temperature,fermentation period and media composition were carded out by on-factor-at-a-time method,and the suitable culture conditions was set as:pH 6.0,temperature 27℃,fermentation period,and 0.5% EGB addition content.And four media factors were also confirmed as glucose,peptone, wheat bran,and corn powder.The orthogonal test design L₉（3⁴） demonstrated the optimal composition containing 2%glucose,0.4%peptone,2%wheat bran,1.5%corn powder 0.12%KH₂PO₄、0.06%MgSO₄ and CaCO₃ 1%.The temperature and initial pH for highest inhibitory ratio were 27℃and 6.0,respectively.In 20-L scale fermenter,the optimal operating factors were:8 L before 72 h,4.8 L after 72 h,and agitation rate was 120rpm.The hypoglycemic,inhibiting nonenzymatic glycosylation reaction,hypolipidemic, and antioxidant effects of various treatments,such as extract of Ginkgo biloba Leaf （EGB）,biotransformation products of EGB by H.erinaceus,submerged culture of H. erinaceus and the culture combined with EGB,were compared in alloxan-induced hyperglycemic rats.Results showed that in the group treated with biotransformation products of EGB by H.erinaceus:①The levels of blood sugar（14.59mmol/L） and fructosamine（3.10mmol/L） were significant lower that other groups,which indicated that the biotransformation products of EGB by H.erinaceus could remarkably inhibit the non-enzyme glycation reaction in the TypeⅠdiabetes rats;②the ability of glucose tolerance was enhanced;③the levels of blood sugar,TC and TG were decreased and that of HDLC were increased.The mechanism analysis of anti-hyperglycemic activities of biotransformation products of EGB by H.erinaceus was conducted,and the results revealed that haemoglobin in blood,hexokinase（58.16and 97.34umol/min/g protein）, citratse synthase（776.5 and 643.2umol/min/g protein） and pyruvate dehydrogenase （90.25 and 69.85umol/min/g protein） in kidney and liver of the group treated with biotransformation products of EGB by H.erinaceus was significantly higher than those in the control group and the mefformin treated group,synchronously,the content of glycosylated haemoglobin in blood was significantly lower than that of the control group and the metformin-treated group.According to these results,it could be concluded that the biotransformation products of EGB by H.erinaceus could generate significant anti-hyperglycermic and hypoglycemic effect on alloxan induced diabetic rats by inhibiting non-enzymatic glycosylated reaction,decreasing glycosylated haemoglobin level,improving total haemoglobin level and supply of oxygen,enhancing respiration of tissue,decreasing the levels of NADH,speeding up catabolism of glucose, and regulating the enzymes activities related to the glycolytic metabolism coupled with its elevating level of insulin of plasma.The hypoglycemic,inhibiting nonenzymatic glycosylation reaction,hypolipidemic, and antioxidant effects of various treatments,such as extract of Ginkgo biloba Leaf （EGB）,biotransformation products of EGB by H.erinaceus,submerged culture of H. erinaceus and the culture combined with EGB,were compared in streptozotocin-induced type 2 diabetic rats.Results prove that the biotransformation products of EGB by H.erinaceus could inhibit nonenzymatic glycosylation reaction of protein in vivo by reducing the content of fructosamine in the treated group,decrease the blood glucose level（5.92 mmol/L） and enhance SOD activity（182.3UN/mL） compared with control group and other treated-group.However,no significant influences were observed on body weight and lipid,and the blood insulin level was higher in transformation group than in model group,but distinctly lower than other treated group.The isolation effects of 9 types macro-porous resins were compared and results revealed that AB-8 resin was most suitable to extract the active compositions with inhibitory activity on non-enzymatic glycation reaction from the broth of H.erinaceus. And the optimum technological conditions for adsorption were as fellows:pH=2.0, flow ratio=4%volume of resin/min,40%ethanol was selected as the most suitable agent for resolving the main bioactive fractions,and 10-cycle repeating experiments were also carried out and the results showed that the eluting factors were stable enough for the future applicant.The crude extraction resolving from the AB-8 resin biotransformation products of EGB by H.erinaceus,had been testified to inhibit non-enzymatic glycation reaction andα-glucosidase,as well as to scavenge free radical in vitro.The corresponding IC₅₀ values were 1.223mg/mL,15.1 mg/mL and 6.3mg/mL, respectively.It was presume d that the crude extraction could combine with products of non-enzymatic glycation reaction,change their structure and finally attenuate their fluorescence intensive.