Study On Hypolipidemic Effect And Mechanism Of Allantoin Extract From Cistanche Salsa

Objective:To establish a HPLC method for content determination of allantoin extract from C.salsa;To study on optimum purification technology conditions of allantoin extract by macroporous resin;To investigate effects and hypolipidemic mechanism of allantoin extract;To evaluate the safety of allantoin extract,and to provide scientific support for the reasonable utilization and development of C.salsa.Methods:1.Detemination of allantoin was accessed by using an Hypersil BDS C₁₈ column?4.6mm×250 mm,5?m?and a mobile phase of methanol-water at a ratio of 0.4:99.6.The flow rate was 0.5 mL/min,temperature at 30?and detection wavelength at 224 nm.2.With absorption rate and eluent rate of allantoin as indexes,single factor tests were adopted to investigate adsorption and elution conditions of macroporous resin,such as the sample concentration,eluent concentration,eluent rate and so on.Further orthogonal test was used to investigate the optimum purification parameters.3.Eighty-four Wistar rats were randomly divided into normal control group,model group,positive control group,low-dose group of allantoin extact?0.05 g/kg?,middle-dose group of allantoin extact?0.2g/kg?,high-dose group of allantoin extact?0.8 g/kg?.After treatment for 6 weeks,rats were killed with anesthesia,and the serum levels of TG,TC,HDL-C,LDL-C,SOD,MDA,GSH-PX,ALT,AST,LPL,HL were measured.Rats'livers of each group reserved at a temperature of-80?.HMG-CoA reduetase,FAS,ACC,HSL relative mRNA and protein expression level in rats'livers of each dosage group of allantoin extract were determined by Real-time PCR and Western Blot technology.4.According to the national standard procedures and methods in food safety and toxicological evaluation,a series of toxicological studies on the ingestion of allantoin extract were conducted,which included acute toxicity,genetic toxicity and subacute toxicity tests.Result:1.A good linearity of allantoin was achieved in the range of 40²40?g/mL,r=0.9993;Average recovery was98.9%,RSD was 1.3%?n=6?.The average content of allantoin in C.salsa was 1.09%.2.AB-8 type macroporous resin had the best effect of separation and purification,its optimum purification technology conditions were:the sample concentration 10 mg/mL,eluent of 100%ethanol,eluent rate 1 mL/min and diameter to height ratio of resin 1:10.Under these conditions,purity of allantoin from C.salsa was up to 20.01%from 1.09%.After purification by AB-8 macroporous resin,the purity of allantoin from C.salsa was increased by 18 times.3.Compared with the normal control group,the outcomes of TC,TG,LDL-C,MDA in the model group were increased apparently?P<0.05?,and HDL-C,SOD,GSH-PX,ALT,AST,ALP,LPL,HL decreased apparently?P<0.05?.Compared with the model group,the outcomes of TC,TG,LDL-C,and MDA were deceased apparently?P<0.05?,and HDL-C,SOD,GSH-PX,ALT,AST,ALP,LPL,HL increased apparently?P<0.05?in high,middle-dose groups and positive control group.Compared with the normal control group,there was no significant differences in the indexes of high and middle-dose group of allantoin extract?P>0.05?.Compared with the normal control group,the HMG-CoA reduetase,FAS relative mRNA and protein expression level in rats'livers were deceased apparently in high and middle-dose group of allantoin extract?P<0.05?,there was no significant differences in the ACC,HSL relative mRNA and protein expression level in rats'livers of each dosage group?P>0.05?.4.For acute toxicity,the maximum tolerated dose?MTD?was higher than 20 g/kg.The results of Ames test,mouse bone marrow micronucleus test and mouse sperm abnormality test were negative.After thirty days of feeding,rats showed no significant difference in body growth,organ development or histological observation?P>0.05?.Conclusion:1.The method has advantages of good stability,high precision,good reproducibility,which can be used in content determination of allantoin from C.salsa.2.AB-8 macroporous resin was suitable for separation and purification of allantoin extract,optimized purification technology was stable and feasible,it could be extended for production applications.3.Allantoin extract exert certain lipid-lowing effect and hypolipidemic mechanism perhaps in connection with expression of HMG-CoA reduetase and FAS.Its hypolipidemic effect in that allantoin extract could inhibit synthesis of cholesterol by inhibiting the expression of HMG-CoA reduetase and FAS.The result could provide a basis that allantoin extract develop into hypolipidemic medicine.4.Under the conditions of this experiment,the allantoin extract has neither acute nor genetic toxicity.This results for the allantoin extract from C.salsa subsequent exploitation provide a reference.