Toxicity/Efficacy Evaluation Of Coumarin Components Of Psoraleae Fructus And Quality Study Of Its Capsules

Psoraleae Fructus is a traditional Chinese medicine commonly used in clinical practice.It has the effects of warming the kidney and promoting yang,soothing qi and relieving asthma,warming the spleen and stopping diarrhea.It mainly treats the symptoms of spleen and kidney weakness,bone atrophy,and bone arthritis.Its effective components can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,which has become a research focus in the prevention of osteoporotic disease.Psoraleae Fructus mainly includes coumarins,flavonoids and monoterpene phenolic compounds,and still contains carbohydrate compounds such as Psoralea corylifolia polysaccharides(PPs),glucose,raffinose,volatile oils,fatty oils and resins Wait.In recent years,adverse reactions of Psoraleae Fructus and its compounds,such as Xianling Gubao and Zhuanggujianwan,had been common in clinical practice.However,due to the complex chemical composition of Psoraleae Fructus,the main toxic substances have not been fully elucidated in modern studies.In this subject,it is of great significance to discover the attenuated and effective components of Psoraleae Fructus and develop its preparation.In this paper,the chemical constituents,toxicity,anti-osteoporosis pharmacological effects of Psoraleae Fructus and zebrafish model were reviewed.The main purpose of this study was to evaluate the toxicity/efficacy of Psoraleae Fructus and to study the quality of Coumarin Components of Psoraleae Fructus capsules.The first part:zebrafish model was used to evaluate the toxicity/efficacy of the main components of Psoraleae Fructus.1.Zebrafish model was used to identify the toxicity difference of the main components of Psoraleae Fructus.The toxicity of coumarins,flavonoids and monoterpenoid phenols was compared by correlation between toxicity evaluation of zebrafish and composition analysis.The results showed that the toxicity of zebrafish was strongly correlated with flavonoids and bakuchiol(r=-0.789 6,-0.766 7,P<0.01),and weakly correlated with coumarins(r=-0.4332).It is suggested that the coumarin is relatively safe.2.Objective to investigate the effect of the changes of glucoside/coumarin composition of Coumarin Components of Psoraleae Fructus(CCPF)on the safety of zebrafish.CCPF is mainly composed of coumarin glycosides(such as psoralenoside and isopsoralenoside)and coumarins(such as psoralen and isopsoralen).Previous studies had found that the toxicity of glycosides and coumarins are quite different.In this study,a zebrafish model was used to evaluate the effect of changes in the internal composition ratio of Coumarin Components of Psoraleae Fructus(1:0;20:1;10:1;0:1)on toxicity.The results showed that the lowest toxic concentration of zebrafish organs were 500 μg/mL,300 μg/mL,200 μg/mL,10 μg/mL,and the LC50(6 dpf)were 400.7μg/mL,279.3 μg/mL,192.8 μg/mL,22.3 μg/mL,respectively,suggesting that the increasing proportion of(iso)Psoralen in CCPF will increase the toxicity to zebrafish.3.The effects of PPs on the metabolic-efficacy/toxic properties of CCPF was evaluated by zebrafish model.PPs were prepared by water extraction and alcohol precipitation,and PPs content was determined to be about 21.51%by phenol-sulfuric acid method.Secondly,zebrafish juveniles were used to evaluate the toxicity changes of PPs with CCPF.It was found that PPs(LC50:382.7μg/mL)and CCPF(LC50:210.1 μg/mL)were basically non-toxic substances,but the toxicity of PPs with CCPF increased rapidly,PPs:CCPF4:1(LC50:61.8μg/mL)and PPs:CCPF2:1(LC50:80.0 μg/mL)were a slightly toxic substance,speculating that the toxicity of zebrafish gradually increased with the increase of PPs content.Thirdly,Zebrafish model was used to evaluate the metabolic effects of PPs on CCPF.The results showed that PPs could accelerate the metabolism of psoralenoside and isopsoralenoside to psoralen and isopsoralen.Finally,the effect of PPs on CCPF anti-osteoporotic activity was evaluated by the bone loss model of zebrafish induced by prednisolone.The results showed that CCPF had obvious anti-osteoporosis activity at the concentration of 2.5 μg/mL,but the anti-osteoporosis activity of PPS was weak.After the compatibility of PPs with CCPF,the mineralized area and IOD value of zebrafish skull in 0.5μg/mL and 2.5μg/mL concentration groups were significantly increased compared with the model group(P<0.001),suggesting that after CCPF combined with PPS,the anti-osteoporosis activity was significantly increased.The second part:Study on the quality standard and stability of Coumarin Components of Psoraleae Fructus capsules.1.Quality research of Coumarin Components of Psoraleae Fructus capsules:Quality standard research including five aspects:character,identification,inspection,specific chromatogram and content determination.The content of capsules was determined as earthy yellow powder with bitter slightly taste.Psoralenoside,isopsoralenoside,psoralen and isopsoralen in the capsules were qualitatively identified by HPLC at 246 nm.The retention time of the chromatographic peak of the test substance was the same as that of the above four reference substances,and there was no interference from the negative control.According to the test under the general rule 0103 of the fouth parts of the Ch.P(2020),the water content was less than 9.0%,and the difference of the filling volume was less than±10%,which was in line with the regulations.According to the general rule 0931 of the fourth parts of the Ch.P(2020),the dissolution method was established and the method was verified.The HPLC specific chromatogram of Coumarin Components of Psoraleae Fructus capsules was established.Four common peaks were identified:peak 1(S)psoralenoside,peak 2 isopsoralenoside,peak 3 psoralen,and peak 4 isopsoralen.Peak 1(S)was the reference peak of fingerprint.HPLC method was established to determine contents of psoralenoside,isopsoralenoside,psoralen and isopsoralen in the capsules.The ultrasonic extraction method was determined.methodological investigations show that the precision of the determination method,stability,repeatability,the sample recovery rate are all good.The total content of psoralenoside,isopsoralenoside,psoralen and isopsoralen in each capsule shall not be less than 30.0 mg.2.Stability evaluation of Coumarin Components of Psoraleae Fructus capsules:the influencing factors,accelerated and long-term stability test were investigated.The test of influencing factors showed that except a small amount of agglomeration in the contents of the sample under high humidity conditions,the properties and content of the other test samples did not change significantly.It should be dry and sealed during storage and transportation.The accelerated test lasted for 6 months,and the long-term test lasted for 9 months.The results showed that there were no significant changes in the properties,moisture content,characteristic map,content and microbial limit of the three batches of samples in the pilot test,and they had good stability.Conclusion:the toxicity/efficacy evaluation of zebrafish showed that the safety of CCPF was better than flavonoids and bakuchiol;CCPF is mainly composed of glycosides and coumarins,and the increase of the content of coumarins can increase toxicity of zebrafish.PPs could accelerate the conversion of glycosides into coumarins in CCPF,and increase the toxicity and anti-osteoporosis activity.The established quality control methods of identification,inspection and content determination could effectively detect the main index components(iso)psoralenoside and(iso)psoralen,which could provide sufficient basis for the quality control of Coumarin Components of Psoraleae Fructus capsules.