| Depression is a common mental disorder.There are up to 350 million people worldwide suffering depression that leads to serious consequences.In the screening of antidepressant drugs from the Chinese Meteria Medica,it was found that Fructus Psoraleae(FP)and its main components have antidepressant effects.However,during the development of antidepressant products of FP,preclinical safety evaluation studies have found that FP could cause toxic reactions on some organs such as liver and reproductive system.In addition,the clinical adverse reaction events of FP are increasing.Therefore,its toxic and side effects have become the bottleneck of the clinical application and product development of FP,urgently needed to make a comprehensive and systematic study.The problem of potential safety hazard of FP has not been properly solved,and its toxic components and toxic mechanism have not been elucidated.Therefore,in this study,the systematic toxicity evaluation is firstly performed to determine the toxic dose,cycle,target organ and its toxic mechanism(toxic cause)of FP,and toxicity screening was conducted for each main component group to find out its main toxic components and elucidate its toxic mechanism;the toxicological study is conducted to determine the attenuation principle of the preparation of the antidepressant effective fraction of FP,i.e.,removing the main toxic components,maintaining the main component with non-toxic or low toxicity and antidepressant effect.After obtaining an antidepressant effective fraction,the pharmacological and safety evaluation of this effective part is carried out to ensure that this effective fraction is effective and safe.Three aspects of extract,principal component group and principal component from FP are applied for to explore the toxicity of FP and clarify its main toxic components and its toxic mechanism,providing theoretical support for the preparation method to reduce or remove the toxicity of FP and ensuring the safety of clinical medication,so as to serve as a scientific basis for its development as a safe and effective new Chinese medicine as soon as possible.1.Systematic evaluation on the toxicity of FPBased on the screening of different extraction methods of FP,it is found that the main components in FP could fully extracted by refluxing with 70%ethanol;Wistar Rats were administered with the EEFP at doses of 1.5,1.0 and 0.5 g/kg during 28 days orally.Histopathologic and clinicopathologic analyzes were performed,and hormone level of serum and the mRNA of enzymes produced by steroids in adrenal glands were detected.The area of each band of adrenal glands and the steroids level in the adrenal glands were also measured.During the treatment,both the histopathologic and clinicopathologic examination showed that EEFP contributed to liver,prostate,seminal vesicle and adrenal gland damage.Among the enzymes involved in regulation of adrenal steroid hormone secretion,NET,VMAT2,and CYP11B1 were upregulated,while CYP17A1 was downregulated.Among the adrenal steroid hormones,COR and NE were upregulated,while levels of DHT and serum ACRH and CRH decreased.Adrenal gland,prostate,and seminal vesicles could also be the target organs of FP toxicity.Abnormal enzyme and hormone metabolism related to the hypothalamic pituitary adrenal(HPA)axis caused by the EEFP may be the potential toxic mechanism for changes in the adrenal gland and secondary sex organs of male animals.The toxicity study of the extracts provided the dose and period reference for the subsequent toxicity study of the main components and main components of FP.2.Screening of major toxic components of FPFP is divided into three main groups by chemical separation analysis,including Furocoumarin group(mainly containing psoralen,isopsoralen and glycosides),Flavonoids group(mainly containing psoralen flavonoids such as psoralen flavonone and psoralidin)and Monoterpenoids group(mainly containing bakuchiol);according to the dose and cycle determined by the toxicity study of FP system,toxicological evaluation of the three main groups showed that after oral gavage administration of 80 mg/kg of furanocoumarin for 28 days in rats,abnormal increases in serum transaminases and pathological changes characterized by liver injury were observed;however,after oral gavage administration of 1.5 g/kg of flavonoids and 900 mg/kg of Flavonoids in rats for 28 days,there was no significant change.Therefore,it indicates that the main toxic component group in FP is furanocoumarin.3.Toxicity of psoralen and isopsoralen and its mechanism of liver injuryThe psoralen and isopsoralen in FP were prepared by petroleum ether extraction,polyamide column and silica gel column;The toxicology of psoralen and isopsoralen was studied to explore the toxic mechanism of liver injury.In the present study,psoralen and isopsoralen,the two main constituents of FP,were administered orally to rats(80 and 40 mg/kg,respectively)and mice(320 and 160 mg/kg,respectively)for 28 days,followed by biochemical and histopathological examinations to evaluate their hepatotoxicity.The results showed that psoralen and isopsoralen could induce the toxic reactions of liver and other organs in rats,while mice were not sensitive to these two compounds.Furthermore,the corresponding results indicated that administration of psoralen and isopsoralen repressed the expression of CYP7A1,BSEP,MRP2 and SULT2A1 and increased the expression of FXR and MRP3 in the rat liver.In summary,the toxic reactions of psoralen and isopsoralen are different in different species.In this study,multiple organ toxicity,such as cholestatic liver injury,occurs in rats,but not in mice.Psoralen and isopsoralen are the two main toxic constituents of FP.In addition,psoralen and isopsoralen cause liver injury,possibly through inhibiting bile acid excretion in the liver,leading to the accumulation of toxin in hepatocytes.According to the above toxicity studies,the preparation principle of the antidepressant effective part of FP was determined,that is,removing the main toxic components of furanocoumarins(psoralens and isopsoralens),and maintaining the non-toxic and effective groups of components(flavonoids,estrogenic endocoumarins,and monoterpenoids).Technologies such as Ethanol extraction,macroporous resin and polyamide resin separation were used to prepare the antidepressant effective part of FP,so as to establish a simple and scientific method for the preparation of antidepressant effective part of FP to ensure the pharmacological effect of its antidepressant and the safety of its application.4.Preparation of effective components group of FP(EGFP)and study on antidepressant pharmacologyBy removing the main toxic ingredients of psoralen and isopsoralen,an effective component group containing flavonoids,psoralidin and bakuchiol was obtained.The main components of this effective component group had significant antidepressant effect.Preliminary safety screening of this effective component group revealed that large doses of this effective component group could cause death and abnormal changes in liver index of rats,while the effective component group reducing the content of bakuchiol was not significantly abnormal.It is speculated that bakuchiol may be a potential toxic component of FP.Therefore,the preparation process of the effective fraction was optimized,and the method of petroleum ether extraction was added to control the content of bakuchiol in the effective part.To sum up,the effective part of FP prepared in this study removes psoralen and isopsoralen,the main toxic components of FP,retains the main antidepressant active components of flavonoids and psoralidin,and then controls the content of the potentially toxic component of bakuchiol.Pharmacological studies revealed that the effective part of FP(60 and 30 mg/kg)significantly reduced the immobility time of rats with behavioral limitation(swimming and tail suspension)and increased 5-HT content in serum and hippocampal tissues in a dose-dependent manner,indicating that this effective part has significant antidepressant effect.In order to ensure the safety of FP anti-depressant active ingredients,we conducted a systematic safety evaluation of FP(acute toxicity and subchronic toxicity tests).5.Safety evaluation of EGFPThe results of acute toxicity test showed that no significant acute toxicity reaction or animal death was observed in rats given a total of 27.38 g/kg of the preparation solution of antidepressant active part of FP by oral gavage twice a day,with LD50>27.38 g/kg;The results of subchronic toxicity test showed that the preparation solution of antidepressant active part of FP was orally administered to rats for 28 consecutive days,with doses of 1.2,0.6 and 0.3 g/kg,respectively.The results of clinical pathology and histopathological examination showed that none of the treated animals had significant drug-related abnormal changes.Based on above safety study,the effective part showed that it was safe and non-toxic.After this thesis made studies on systematic toxicological of FP,it is found that the toxicity target organs of FP are liver,adrenal gland,prostate and seminal vesicle.Psoralen and isopsoralen are the main toxic components of FP,the main toxic mechanism of which is the inhibition of bile acid excretion leading to liver injury,and the disorder of HPA axis-related enzymes and hormones production after liver injury is the main factor of adrenal gland and secondary sex organ injury in male rats.On the basis of toxicological studies,the principle of attenuation of the antidepressant effective part of FP was determined,that is,removing the main toxic components,controlling the potential toxic components,retaining the antidepressant active components,and obtaining the antidepressant effective part containing flavonoids,psoralidin and bakuchiol;On the basis of pharmacodynamics and safety evaluation,it was determined that the effective part of FP had antidepressant pharmacological effects,which was safe and non-toxic.On the one hand,the above research ensures the safe use of FP and provides a scientific basis for its development as a safe and effective new antidepressant drug as soon as possible;on the other hand,it serves as a new idea for the research and development of other toxic traditional Chinese medicines. |
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