| Objective herba epimedii is a good medicine for tonifying liver and kidney and strengthening muscles and bones,which has been used in traditional Chinese medicine for many years.However,with the wide application of Epimedium and its preparations in clinical practice,the adverse events of herba epimedii and its preparations are frequent.China Food and Drug Administration,Pharmacovigilance Journal and clinicians have all reported that herba epimedii and its preparations have the risk of liver injury.The safety issue of herba epimedii and its preparations has attracted extensive attention at home and abroad.Therefore,the key problems to be solved in the drug development and clinical application of herba epimedii are to deeply explain the toxicity mechanism of herba epimedii and the means of processing and reducing toxicity.Our previous studies have demonstrated that both herba epimedii and Icarinidin II can affect ASC aggregation and mitochondrial damage during the activation of NLRP3 inflammasomes in vivo,and promote the activation of NLRP3 inflammasomes induced by mycortin in vitro,thereby promoting the expression of caspase-1 protein and the secretion of IL-1β inflammatory cytokines.Thus,the activation of NLRP3 inflammasomes causes immunospecific liver damage,and direct administration has no obvious inherent toxicity.Based on this,the prophase research food safety related preparations containing herba epimedii(faeries bone remains capsule bones,joints,pills,etc.)of the adverse reactions reported finding,which used for herba epimedii,rather than the main herba epimedii,however,is generally clear writing is used in traditional tonic processed products as a medicine,such as: marked with suet main of herba epimedii herb medicine include:Hipaemon Kidney-Protective Pill and Sanshen Pill,which can strengthen the kidney and replenish qi and nourish Yin,and Tiger Bone Wine,which can promote blood circulation and dispel wind and strengthen the muscles and bones.It is noted that the use of cooked herba epimedii in medicine includes: "Guiling Ji"(tonifying the kidney and strengthening the Yang)and "Jiawei Jingang Pan"(strengthening the bones and strengthening the tendons).At the same time,the processing method of herba epimedii in Lei Gong Pao Zhi Lun,the earliest record of the processing method of herba epimedii,was processed by suet,which was also the most frequently used processing method in the past several thousand years and is still in use today.Therefore,it is possible to reduce the risk of abnormal hepatotoxicity caused by herba epimedii by using suede.It is worth further study.Studied in this paper based on previous herba epimedii diathesis liver toxicity mechanism research,screening of herba epimedii diathesis to the risk of liver toxicity composition,further explore suet main reduces the risk of herba epimedii to diathesis of liver toxicity composition change and biological mechanisms,for herba epimedii the suet processing,reduce the risk of diathesis to liver toxicity.At the same time,in order to solve the problem of the risk of heterogeneous hepatotoxicity caused by herba epimedii,this study proposed that "combined risk" should be included on the basis of the current "component search and content measurement",and the biological evaluation method of risk component index should be established to provide technical support for the processing and quality control of herba epimedii.Methods The research methods are as follows:1.The samples of herba epimedii in different batches were collected,processed into herba epimedii prepared with suede in batches according to the pharmacopoeia and literature conditions,and the alcohol extracts were prepared respectively;Standard mother liquor of 16 chemical components of herba epimedii was prepared and stored.2.Ultra-high performance liquid chromatography(UPLC)and liquid-mass spectrometry(UPLC-QTOF-MS)were used to detect the contents of various chemical components in different batches of herba epimedii and before and after processing,and to analyze the causes of component changes.3.The immune stress model of mice was used to verify the differences in ALT,AST,Caspase-1,IL-1β,MDA and other biochemical indexes before and after the preparation of Epimefuli.After HE staining,the differences in liver injury were observed.The differences of key proteins before and after processing were further determined by Western blot.4.Based on the previous studies on the mechanism of herba epimedii and icariside II causing heterotoxicity,the key target proteins of caspase-1 and IL-1 β were selected to screen the active components in herba epimedii with high risk of heterotoxicity by using the LPS-induced immune stress model in vitro.5.The results of the above content determination were combined with the biological activity of the selected high-risk components of herba epimedii causing heterotoxicity to liver injury,and the risk weight was assigned.The risk component index(RCI)of herba epimedii causing heterotoxicity was evaluated by multiple indexes.The representativeness of risk component index of herba epimedii heterotoxicity was verified by activity detection of different batches of herba epimedii samples.Results 1.A total of 8 batches of herba epimedii were collected and 8 batches of herba epimedii prepared with suede were prepared.The alcohol extracts were prepared and stored for later use.The mother liquor of 16 standard components of herba epimedii were prepared and stored.2.The fingerprint of herba epimedii was constructed by UPLC-HPLC,and the contents of 15 components were determined.It was found that the contents of 10 components in herba epimedii were significantly decreased after processing.Liquid mass spectrometry(UPLC-QTOF-MS)was used to detect the peak area of the chemical components of herba epimedii before and after processing.It was found that the increase of icariin was significant.The results showed that with the change of processing time and temperature,the glycoflavones in herba epimedii could be converted into flavonoid aglycones by deglycogroup.3.The results of immune stress model experiment in mice showed that ALT,AST,Caspase-1,IL-1β,MDA and other biochemical indexes were significantly decreased after the processing of herba epimedii.Meanwhile,the results of HE staining showed that the liver damage in the treated herba epimedii group was decreased compared with that in the treated herba epimedii group.The results of bone marrow macrophage assay showed that the activity of key target protein Caspase-1 and the secretion of inflammatory cytokine IL-1 β were significantly decreased after processing.Furthermore,the significant differences between key proteins Caspase-1 and IL-1β before and after processing were confirmed by Western Blot.In vivo and in vitro immune stress induced by lipopolysaccharide induced by herba epimedii,the results showed that the processing of herba epimedii can reduce the risk of idiopathic hepatotoxicity caused by herba epimedii.4.Based on the previous studies on the mechanism of herba epimedii and icariside II causing heterotoxicity,Caspase-1 and IL-1β,two key target proteins,were used to screen the high-risk components of herba epimedii causing heterotoxicity.The screening results showed that there were mainly four components contributing to the risk of heterotoxicity of herba epimedii.They were icariside II,baohuoside II,sagittatoside B,and icariside I.5.Put that herba epimedii diathesis hepatotoxicity high-risk components of biological activity and the content of the joint,adopt inverse normalization method on the risk of toxicity component efficacy of weight distribution,further use of multiple index comprehensive evaluation method to construct herba epimedii diathesis risk of toxicity component index,the biological activity of different batch samples of herba epimedii test to validate the results found that risk component index,Correlation analysis of 1/EC50 of herba epimedii heterotoxicity and risk component index showed that 1/EC50 was significantly positively correlated with the risk component index(R2=0.8316,**P <0.01),that is,the higher the risk component index was,the higher the 1/EC50 was,and the lower the EC50 was,the higher the risk of herba epimedii heterotoxicity was.These results indicated that risk component index could better characterize the risk of idiopathic hepatotoxicity in herba epimedii.Conclusion in this paper,based on previous herba epimedii and herba epimedii glycoside II diathesis liver toxicity mechanism research,herba epimedii was established by multiple chemical detection method,then study of changes in chemical composition before and after processing,by pharmacological experiments following the suet main reduces the risk of herba epimedii to diathesis of liver toxicity phenotype,Then based on the immune stress induced by LPS in vitro model of herba epimedii diathesis to active ingredients at high risk of developing liver toxicity screening,and joint before and after the processing of herba epimedii diathesis to high-risk chemical composition changes of liver and explain herba epimedii processing material base of reducing the risk of diathesis to liver toxicity and mechanism,to reduce the risk of herba epimedii to diathesis of liver toxicity to provide evidence to support,Finally,the risk component index is established for quality control. |
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