Study On Biological Assay For Quality Evaluation Of Bupleuri Radix Associated With Its Relieving Actions On Exterior Symptoms And Fever

The quality of Chinese materia medica(CMM)is the fundamental guarantee of the clinical efficacy of traditional Chinese medicine(TCM),and the quality control(QC)of CMM has always been one of the key issues in its modernization and internationalization.The current QC standards for CMM have gradually changed from no or single indicative component to several constituents.However,the lack of correlation between indicative components and clinical efficacy as well as the real internal quality of CMM has always been the key bottleneck in CMM’s QC.Against this background,biological assay was introduced because of its advantages and characteristics in the strong correlation with drug efficacy.And biological evaluation has gradually become an important direction for quality standardization of CMM.However,the current biological evaluation research still has the problems:1)the chosen biomarkers for quality evaluation of some CMM are disconnected from clinical application;2)single bioactivity cannot represent the overall quality of CMM with multifunctional characteristics.Bupleuri Radix(BR),first recorded in Shen-nong’s Herbal Classic and described as"upper herbs",is a commonly used CMM in clinics whose effects is to release the exterior,reduce fever,soothe the liver,resolve depression,and upraise yang qi.In Chinese pharmacopoeia(2020 edition),two varieties[Bupleurum chinense DC.(Bei Chaihu,BC)and B.scorzonerifolium Willd.(Nan Chaihu,NC)]of BR were listed,and the total content of saikosaponin A and D(≥0.30%)was used as its quality control standard.This study focused on the clinical efficacy of BR in the treatment of exogenous fever.And four types of in vitro models related to this efficacy,including anti-macrophage activation,anti-mast cell activation,anti-oxidative stress and antipyretic effect,were used to find reliable and sensitive bioactive indicators of BR,for comprehensively evaluating the strength of its"release the exterior and reduce fever" actions.Through the selection of 12 bioactivity indicators,the biological quality control system of BR was established by four indicators:NO production induced by LPS in macrophages,total antioxidant capacity,TNF-αproduction induced by LPS in whole blood cells,and complement activation.Given that the releasing action on exterior symptoms of BR is mainly characterized by the antipyretic effect,we assigned a total weight coefficient of 0.7 to the two indicators directly associated with fever:TNF-α production in whole blood cells(0.5)and complement activation(0.2);and the weights of NO production induced by LPS in macrophages and total antioxidant capacity were 0.2 and 0.1,respectively.Subsequently,we used this evaluation method to appraise six extracts of BR(1#～6#)of different varieties or origin sources,and the comprehensive score from high to low is 6#>1#>3#>5#>4#>2#.Therefore,we chose 6#(BC from Shaanxi)with the highest score ranking as the research object for the antipyretic mechanism of BR.As we know,BR’s effects on treating "intermingled cold and heat,and pathogenic qi"was recorded as early as in Shen-nong’s Herbal Classic.If the main mechanism of BR in infectious fever can be clarified,it will be of great significance to improve the biological assay for its quality evaluation associated with the relieving actions on exterior symptoms and fever and to clear its effective material basis.We prepared the water extract of 6#(XBCE)in accordance with the clinical way,and then explored its antipyretic characteristics and peripheral mechanism.The obtained results showed that,in LPS(i.v.)induced febrile response rat model,XBCE(i.g.)exhibited a confirmed and mild antipyretic effect.We further investigated the peripheral mechanism of antipyretic,and the results showed that XBCE had a definite suppression effect on TNF-α of the three peripheral endogenous pyrogens——it could not only significantly inhibit the secretion of TNF-α in LPS-stimulated whole blood cells and THP-1 monocytes(in vitro),but also markedly decreased serum TNF-α in mouse and rat endotoxemia models(in vivo);XBCE could also strikingly restricted LPS-dependent complement activation(in vitro and in vivo),which might also be advantageous to its suppression on TNF-α.On the basis of the antipyretic peripheral mechanism of BR being clarified(suppressing peripheral TNF-α),studies of saponins-rich extract of BC(SRBC)related to antipyretic effect were further conducted.The results showed that SRBC could also significantly reduce the serum TNF-α level in endotoxemia mice,and alleviate the fever induced by LPS in rats,even showing a better antipyretic effect than that of XBCE.To clear the contribution of the two pharmacopoeia quality control substancessaikosaponin A and D in these effects,we mixed them in proportion to their concentrations in the extract to obtain a mixture of the two standard substances(MAD).Unexpectedly,MAD neither inhibited LPS-induced TNF-α production in THP-1 cells nor reduced serum TNF-α levels in endotoxemia mice,which suggested that SRBC may be more beneficial to enrich the antipyretic substances of BR,but saikosaponin A and D may not be the antipyretic constituents.Based on the peripheral antipyretic mechanism of BR and the fact that monocytes are the main contributors to TNF-α secretion in whole blood cells,we further optimized the previously established biological quality evaluation method associated with its relieving actions on exterior symptoms and fever:1)replacing whole blood cells with THP-1 cells to evaluate the effect of BR on endogenous pyrogen TNF-α;2)focusing in vitro complement activation indicators on alternative pathway.Subsequently,we evaluated 7 new samples of BR containing 4 BC(B-1#～B-4#)and 3 NC(N-5#～N-7#)with this optimization method,and the comprehensive score from high to low is B-1#>N-6#>N7#>N-5#>B-3#>B-4#>B-2#.Except for the top-ranked B-1#(which happens to be the 6#we chose before),the scores of the 3 NC were higher than that of the other 3 BC(in which the total content of saikosaponin A and D was higher than the limit of pharmacopoeia,and much higher than that in NC).There was no correlation between the total content of saikosaponin A and D and the score of biological quality control(P>0.05),which also suggested that saikosaponin A and D should not be the antipyretic substance of BR.Taken together,based on the pathophysiology of exterior symptoms and fever and the in vitro effects of BR,this study,for the first time,establishes a biological quality evaluation method using multiple bioactivity indexes,and uses it to evaluate the quality associated with the relieving actions on exterior symptoms and fever of 6 extracts of BR.Subsequently,the antipyretic characteristics and mechanism of the top-ranked 6#BC(from Shaanxi)were studied.After the antipyretic peripheral mechanism was clarified,the biological quality control system was optimized,and 7 extracts of BR were further evaluated.From the results that the total content of saikosaponin A and D was not correlated to the score of biological quality control,and MAD could not suppress TNF-αin vitro and in vivo,it is suggested that saikosaponin A and D may not be the material basis of the antipyretic effect of BR.This study carries out a beneficial exploration of the biological assay for quality evaluation of BR,and can provide a valuable reference for other CMM’s quality biological evaluation research.