Toxicology And Efficacy Study Of Cihuangzengdan Powder

Objective:The overall goal of this study is to evaluate the safety and efficacy on reproduction to provide the required data for IND application of Cihuangzengdan powder as a new veterinary drug.Methods:1)The acute toxicity study was performed to determine the maximum tolerance dose(MTD).2)To perform the long-term toxicity study,the rats were randomly divided into negative control group and three experimental groups for high(36 g crude/kg),middle(24 g crude/kg)and low(12 g crude/kg)dose of Cihuangzengdan powder.20 rats per group(half male and half female)were administrated with the Cihuangzengdan powder for 4 consecutive weeks.Following the last administration,14 rats of each group were used for hematology,blood biochemical indexes,systematic autopsy and histopathological examination.The remaining 6 rats were observed for 2 more weeks and analyzed exactly same as that of the above 14 rats.3)To perform the efficacy study,Kunming female mice were divided into negative control group,positive control group(Jiangzhizengdan San,16 mg/10 g)and three experimental groups(160 mg/10 g,16 mg/10 g,1.6 mg/10 g of Cihuangzengdan powder).Cihuangzengdan powder was administered via gavaged on daily,five times total.On day 5,10 and 15 following the administration,the mice were weighed and the serum was collected to determine the serum level of FSH,LH and E2 by ELISA.The uterine ovarian tissues were collected to detect the relative mRNA expression of FSHR,LHR and ER? by qPCR.HE staining was performed to observe the morphology of ovarian tissues.Result.1)Acute toxicity study demonstrated that the maximum tolerated dose(480 g crude/kg)was 800 times higher than that will be used in chicken.2)Long-term toxicity study demonstrated that there were no significant differences in appearance,physical activity,body weight gain,hematology and blood biochemical indexes comparing with that of the negative control group(P>0.05).The histopathological changes of the main organs were not observed.Drug delayed toxicity was not observed after the drug withdrawal.3)The efficacy study demonstrated that(1)there was no significant effect on body weight gain,uterus and ovary index in female mice with three different doses on day 5,10,and 15 following the drug administration.However,on day 15,the uterus and ovary index in the middle dose group increased insignificantly.(2)On day 15,FSH in the middle dose group and low dose group was increased significantly than that in other groups(P<0.01).No significant difference was observed with the serum LH level within all of the groups on day 5,10 and 15 following the drug administration(P>0.05).On day 10,a significant difference was observed for the serum E2 level between the middle dose group and the other groups including negative control group(P<0.01).On day 15,compared with the negative control group,the E2 levels were significantly increased for all of the three dose experimental groups(P<0.01)with the middle dose group as the highest.(3)Comparing with the negative control group,on day 10,the expression of FSHR mRNA in the middle dose group and positive drug group were significantly higher(P<0.01).On day 15,the expression of FSHR mRNA in the high and middle dose groups were significantly higher(P<0.05).Comparing with the negative control group,on day 5,the expression of LHR mRNA in the middle dose group was significantly higher(P<0.05).On day 10,the expression of LHR mRNA in the low dose group was significantly higher(P<0.01).On day 15,the expression of LHR mRNA in the middle dose group was significantly higher(P<0.01).Comparing with the negative control group,on day 15,the expression of ERP mRNA in the middle dose group and positive drug group were significantly higher(P<0.05).(4)The HE staining showed that the follicles were well developed and the structure was intact.There was no significant difference in the number of follicles in each treatment group on day 5,10 and 15 following the administration.However,on day 15,the number of follicles was increased with the middle dose group.Taken together,the middle dose group was the best.Conclusion:1)No toxicity was observed for Cihuangzengdan powder to mice and rats.Therefore it is safe to use.2)Cihuangzengdan powder was able to increase the levels of reproductive hormones including FSH and E2 to regulate the mRNA expression of the reproduction hormone receptors of FSHR,LHR and ER? so as to improve the follicular development and reproductive function.