The Research Of The Preparation Of Flavonoids In The Leaves Of Carya Cathayensis Sarg.and Their Effcts On Reducing The Level Of Uric Acid

ObjectiveWe determine and compare the content of primary flavonoid aglycones in the leaves of Carya cathayensis Sarg.from different areas in Zhejiang province in order to guide us find the leaves with higher content of flavonoid aglycones and purify total flavonoids and pinostrobin in the the leaves with high content flavonoids.Hyperuricemia(HUA),one of chronic metabolic disease,is closely related to hypertension,coronary heart disease,arteriosclerosis,stroke and its incidence is increasing considerably.HUA may be the independent factor on acting on kidney,liver and joint negatively.It has reports that the flavonoids could adjust the production and excretion of UA through many ways and have significant preventive and therapeutic effects.In this paper,we explored whether total flavonoids and pinostrobin could reduce the level of SUA in the hyperuricemia mice and rats and other indexes related to SUA in order to provide a basis for developing and utilizing the leaves of Carya Cathayensis Sarg.futher.Methods1.Comparative Study of Primary Flavonoid Aglycones Content in Leaves of Carya Cathayensis Sarg.from Different Areas in Zhejiang Province:Using the RP-HPLC method,the content of chrysin,cardanomin,pinostrobin chalcone and pinostrobin in leaves from different areas were determined and compared with the detection wavelengths of 262nm(chrysin),342nm(cardanomin,pinostrobin chalcone),284nm(pinostrobin)and the mobile phase consisting of methanol and 0.1%acetic acid aqueous solution(70:30).2.The method of preparating of total flavonoids:Using the RP-HPLC method,the content of pinostrobin chalcone in sample solutions were determined.The optimal macroporous resin SD200 was screened by static adsorption tests,and various technological parameters of pinostrobin chalcone enrichment were optimized by dynamic adsorption and desorption tests.3.The method of purifying pinostrobin:Signal factor test was investigated to optimize the manufacturing process of pinostrobin.Pinostrobin was obtained by alcohol-backflow extracting,sodium hydroxide degeneration,adsorption and desorption of polyamide resins,extract of petroleum ether,alcohol crystallization and determined with RP-HPLC method.4.Effect of total flavonoids and pinostrobin on nomal Kunming mice:Kunming mice were successively administrated for seven days with total flavonoids and pinostrobin.We drawn blood after the last administration for an hour,which were tested by biochemical analyzer including UA,BUN,Cr,AST,ALT and to observe the effect of total flavonoids and pinostrobin on the level of serum UA in normal mice.5.Effect of total flavonoids and pinostrobin on Hyperuricemia mice induced by intraperitoneal injection of uric acid:Kunming mice were successively administrated for seven days with total flavonoids and pinostrobin.We gave an intraperitoneal injection of uric acid suspension quickly after the last administration.An hour later,we drawn blood from the mice and used the biochemical analyzer to test the level of serum UA,BUN,Cr,AST,ALT and utilized the ELISA to test the activity of XOD in serum and liver in order to observe the effect of different dosage of total flavonoids and pinostrobin on lowering uric acid.6.Effect of total flavonoids and pinostrobin on Hyperuricemia mice induced by potassium oxonate with hypoxantine:We combined the potassium oxonate with hypoxantine to mice model every day,at the same time the mice were successively administrated for seven days with total flavonoids and pinostrobin.We drawn blood after the last administration for an hour,which were tested by biochemical analyzer including UA,BUN,Cr,AST,ALT.We used the ELISA to test the activity of XOD in serum and liver and utilized the RT-PCR to detect the expression of URAT1,GLUT9 and ABCG2 genes and used the WB to detect the protein expression of URAT1,GLUT9 and ABCG2 in kidney in order to observe the mechanism and the effect of different dosage of total flavonoids and pinostrobin on lowering uric acid.7.Effect of total flavonoids and pinostrobin on Hyperuricemia rat induced by lipid emulsion:We used the lipid emulsion to rats model every day,meanwhile the rats were successively administrated for eight weeks with total flavonoids and pinostrobin.We drawn blood after the last administration of fasting 12h and used the biochemical analyzer to test the level of serum UA,BUN,Cr,AST,ALT and utilized the ELISA to test the activity of XOD in serum and liver in order to observe the effect of total flavonoids and pinostrobin on lowering uric acid.Results1.Comparative Study of Primary Flavonoid Aglycones Content in Leaves of Carya Cathayensis Sarg.from Different Areas in Zhejiang Province:The calibration curves of chrysin,cardanomin,pinostrobin chalcone and pinostrobin were linear in the range of 0.0025?0.025,0.0025?0.025,0.025?0.600 and 0.0125-0.250mg/mL,respectively.The content of chrysin and pinostrobin chalcone in the leaves from Wangchuan village Weiping town Chun'an county were the highest,and the content of cardanomin and pinostrobin in the leaves from Zhejiang Chinese medical university were the highest.But the content of 4 flavonoid aglycones in the leaves from Liangxi village Tuankou town Lin'an county were the lowest.2.The method of purifying total flavonoids:The SD200 macroporous resin was used for the following tests because it possessed of higher adsorption/desorption capacity and desorption ratio for pinostrobin chalcone.The best process of pinostrobin chalcone enrichment with SD200 macroporous resin was as follow:the adsorption time was 150min,and 25? was selected as optimal temperature.The proper initial concentration of pinostrobin chalcone in sample solution was 3.58 mg/mL.The volume and flow rate of sample solution were 6.5 BV and 2.0 BV/h in the adsorption process,respectively,and the volume and flow rate of eluting solution were 10 BV ethanol-water solution(70:30,v/v)and 4.0 BV/h in the desorption process,respectively.Through treatment in above perfect conditions,the content of pinostrobin chalcone in product was increased by 4-fold from 7.02%to 26.06%,with a recovery yield of 82.81%.3.The best preparation process of pinostrobin was as follow:the powdered leaves were extracted twice with 95%aqueous ethanol by reflux for 30min at 100?.Sample solution rest over night after 1M of sodium hydroxide added in with the ratio of 400:7.The polyamide resins were added to a round bottomed flask and then mixed with alkaline sample solution with the solid-liquid ratio of 20:1.The flask was rotated under vacuum to remove ethanol solvent in a rotary evaporator at 55? until there was no ethanol solvent in it.The sample-laden polyamide resins were added to a glass column and first washed with 15 BV deionized water,second with 15 BV 20%ethanol-water solution and last with 15 BV 60%ethanol-water solution which was concentrated and dried.Crude from 60%ethanol-water solution was dissolved in methanol and extracted 10 times continuously with petroleum ether.The product from petroleum ether was dissolved and crystallized in ethanol-water solution and the pinostrobin with high purity was obtained.4.Effect of total flavonoids and pinostrobin on nomal Kunming mice:Compared with the control group,total flavonoids and pinostrobin had little effect on the Cr,BUN,AST,ALT levels in normal mice serum and maintained the level of serum UA in normal mice,while allopurinol and benzbromarone significantly(P<0.01)decreased the level of serum UA in normal mice.5.Effect of total flavonoids and pinostrobin on Hyperuricemia mice induced by intraperitoneal injection of uric acid:Compared with control group,the level of serum UA in model group was significantly(P<0.01)increased which meant acute Hyperuricemia model was well founded by intraperitoneal injection of uric acid suspension(300mg/kg).Compared with model group,high-dose(200mg/kg)total flavonoids,high-(200mg/kg)and middle dose(100mg/kg)pinostrobin significantly(P<0.01)decreased the level of serum UA in model mice,also low-,high-dose total flavonoids and low-,high-dose pinostrobin significantly(P<0.05)decreased the level of serum XOD in model mice.Total flavonoids and pinostrobin had no effect on the Cr,BUN,AST,ALT levels of serum and XOD level of liver in model mice.6.Effect of total flavonoids and pinostrobin on Hyperuricemia mice induced by potassium oxonate with hypoxantine:Compared with the control group,the blood UA level of model group increased significantly(P<0.01)which indicated that hyperuricemia model of mice could be build successfully with the combination of potassium oxonate and hypoxanthine.Compared with the model group,the blood UA level of total flavonoids middle group,total flavonoids low group and pinostrobin high group reduced significantly(P<0.01)and the effect of reducing UA level from total flavonoids group was stronger than from pinostrobin group at the same time the blood Cr and BUN level of total flavonoids and pinostrobin group reduced significantly(P<0.05).The blood and liver XOD levels of total flavonoids and pinostrobin group reduced significantly(P<0.05)and the effect of reducing XOD level from pinostrobin group was stronger than from total flavonoids group(P<0.01).Total flavonoids could significantly downregulate the expression of URAT1 mRNA(P<0.01).not GLUT9 and ABCG2 mRNA.Total flavonoids could significantly downregulate the expression of protein of GLUT9(P<0.01),URAT1(P<0.05).Pinostrobin could significantly downregulate the expression of protein of GLUT9(P<0.05).7.Effect of total flavonoids and pinostrobin on Hyperuricemia rat induced by lipid emulsion:Compared with the control group,the blood UA level of model group increased significantly(P<0.01)which indicated that hyperuricemia model of rats could be built successfully with lipid emulsion.Compared with the model group,the blood UA level of total flavonoids and pinostrobin groups reduced significantly(P<0.01)and the effect of reducing UA level from total flavonoids group was stronger than from pinostrobin group.The blood AST and ALT levels of pinostrobin group or not total flavonoids group were reduced significantly(P<0.05).The blood XOD levels of pinostrobin group or not total flavonoids group were reduced significantly(P<0.05).The urine UA level of total flavonoids and pinostrobin groups increased significantly(P<0.05)and the effect of increasing UA level from total flavonoids group was stronger than from pinostrobin group(P<0.01).Conclusion1.The content of 4 flavonoid aglycones in the leaves of Carya cathayensis Sarg.from different areas in Zhejiang province had large difference,thus choose a different origin leaves for development and utilization according to the need is significant.2.The method established was a promising basis for industry-scale preparation of pinostrobin chalcone from the leaves of Carya cathayensis Sarg.3.Sodium hydroxide degeneration,adsorption and desorption of polyamide resins and alcohol crystallization were the key steps to obtain high purity pinostrobin which could lay the foundation for pinostrobin preparation at large scale.4.Kunming mice were successively administrated for seven days with total flavonoids and pinostrobin and their blood UA level were no difference with who were not administrated with them.5.The acute hypenricemia mice model was established with intraperitoneal injection of uric acid suspension successfully.The blood UA level of total flavonoids and pinostrobin groups reduced significantly(P<0.01)and the effect of reducing UA level from pinostrobin group was stronger than from total flavonoids group.6.The hypenricemia mice model was established with the combination of potassium oxonate oxonate and hypoxnthine successfully.The blood UA level of total flavonoids and pinostrobin groups reduced significantly(P<0.01)and they could protect the kidneys of hypenricemia mice at the same time.Total flavonoids may reduce the XOD level in the blood and liver,downregulate the expression of URAT1mRNA and of GLUT9,URAT1,ABCG2 proteins against hypenricemia.Pinostrobin may reduce the XOD level in the blood and liver and downregulate the expression of GLUT9,ABCG2 proteins against hypenricemia.7.The hypenricemia rat model was established with lipid emulsion.The blood UA level of total flavonoids and pinostrobin groups reduced significantly(P<0.01).Total flavonoids focused on increasing excretion of UA,whereas pinostrobin on reducing the blood XOD level.