Study On Quality Standard Of Pongamia Pinnata And Safety Evaluation Of Its Extract

Objective: Established quality standard of Pongamia pinnata by pha- emcognostic and determination,and selected the extraction and purification process of total flavonoids from Pongamia pinnata that its safety was evaluated.Method:⑴To study the Pharmcognosy of Pongamia pinnata systematically,and macroscopical identification ,microscopic identification,physicochemical identification,TLC identification and UV spectrometry methods were carried out.⑵The contents of karanjin and total flavonoids in Pongamia pinnata were determined by spectrophotometry and HPLC.⑶The single factor experiment and orthogonal test design were adopted to select the optimum extraction of total flavones in Pongamia pinnata root that purified with smacroporous absorbing resin.⑷The toxicity of PRF was studied through the acute toxicity test in mice with Bliss method:PRF were administered by gastric infusion to mice one time daily,and observed toxic reation, weight change and death for 14 days continuously, the mediam lethal dose(LD50) and the confidence limit of 95%.⑸The different doses of PRF preparation were administered to SD rats by gavage for 90 days.And rats were observed for 14 days after drug withdrawl.The rat,s weights,organ coefficients,the indexes of hematology and biochemisty were determined.Pathological examinations were carried out.Results:⑴The original plant morphology and microscopic characteristics were put forward of Pongamia pinnata. Results of physicochemical identification showed that there are flavonoids and triterpenes in Pongamia pinnata which contains karanjin form the result of TLC.There is no absorption of water extract in 200 nm～800 nm,and 60% alcohol eatract has absorption maximum at 260 nm and 303 nm; chloroform extract has absorption maximum at 244nm and 322nm.⑵The tatal flavonoid content is 1.06% in Pongamia pinnata root determined by UV spectrometry; and the karanjin content are 0.164% in Pongamia pinnata root by HPLC.⑶The optimum extraction process of total flavones in Pongamia pinnata root is as follows:the concentration of ethanol 60%,ratio of material to liquid 1:20,and refluxing for 3times,1.0h every time in water bath with 80℃.The result shows that the optimum conditions of D101 included that the concention of sample was 0.6 mg·mL-1 with pH value 3 of sample solution and feeding rate of 2 mL·min-1,first elute with purified water(molish reaction is negative),then elution eluted with 3BV 20% ethanol after removing impurity with 3BV 20% ethanol, the last, using 70% ethanol as eluent.⑷LD50 of PRF in mice was 3.3016g/kg(ig) and 95% confident limit was 2.8895g/kg～3.7953g/kg.The diet mices were dissected,In visual observation, there were not abnormal phenomena in the main organs of mice such as bleeding.the surviving mice from groups were observed daily for 14 days, Theirsappearance characteristic, hair color ,behavioral activity,diets,mental status,fecal charcterand returned to normal next day after administration, At the end of the experiments, mices' weight increase significantly.(5) The long-term toxicity showed that, the effects of PRF on the rat body weight and food intake were not observed,and the PRF had some influence on organ coefficient and haematological indexes,but returned to normal after completion of recovery period. Hyperemia,inflammatory cell infiltration were observed in liver,kidney,heart,uterus and prostate.Conclusion:(1) Identification methods of microstructure, TLC and UV to Pongamia pinnata were established. (2)The determination methods of total flavonoids and by UV and HPLC respectively were established, they are stable and good repeatability, can be ued to control quality of Pongamia pinnata. (3) Extraction and purification of total flavonoids from Pongamia pinnata root are selected, the content of total flavonoids that purified are more than 50%,can achieve goal of purification.(4) The LD50 for PRF in mice was 3.3016g/kg,there were no significant pathological changes and delayed toxicity in main organs.(5)The results of long term toxicity test for 3 months showd that repeated administration of PRF in rats with high dose(760mg/kg) may reduce RBC,Hb and HCV, and change some liver function index, and induce pathology of liver ,but all of the change above are recovered at the end of the recovery,the toxicity of PRF to rats are reversible.The safe does of PRF in rat is 190mg/kg.