Research On Effect Of Salt-fried Process On The Tissue Distribution And Metabolism Of Active Components In Qing’e Wan

Qing’e wan is a classical traditional prescription.It was originally published in the prescription of Taiping Huimin Heji Bureau in Song Dynasty.The Qing’e wan contained in the 2015 edition of the Pharmacopoeia of the People’s Republic of China consists of four traditional Chinese medicines(salt-processed Eucommiae cortex,salt-processed Psoraleae fructus,Juglandis semen,and Allii sativi bulbus).It has the functions of nourishing liver and kidney,strengthening bones and muscles,blacking hair,and is used for the treatment of postmenopausal osteoporosis.Eucommiae cortex and Psoraleae fructus are all required to be salt-processed.The results of the our previous research showed that the effect of the salt-processed Qing’e Wan in the treatment of osteoporosis was significantly better than that of the raw group.In addition,salt processing can promote the absorption of active components in the prescription to play a role of enhancing the efficacy.In order to further explore the scientific connotation of salt-process enhancing the efficacy,this experiment studied the material basis by threeaspects:content determination of the index components of Qing’e Wan,distribution of active components in tissues and organs of rats,metabolic analysis of Qing’e Wan in vivo,and effect of active components of Qing’e Wan on the activity of osteoclast in vitro.Part one:On the basis of the determination of 15 major components in Qing’e Wan,we investigated the distribution of these 12 components(psoralenoside,isopsoralen,psoralen,geniposidic acid,bavachinin,neobavaisoflavone,bavachin,psoralidin,isobavachalcone,pinoresinol diglucoside,isobavachin,bavachalcone)in heart,liver,spleen,lung,kidney,uterus,ovary and testis of rats through tissue distribution experiment.The results showed that these 12 components in Qing’e Wan were promoted targeting into the "kidney meridian" after salt processing.Part two:Rats were taken raw and salt-processed Qing’e Wan orally.Then,The difference components of raw and salt-processed Qing’e Wan in rats were find out from the components in plasma,feces,urine,and bile of rats by PCA and OPLS-DA.Combined with the tissue distribution of 12 active components,it was found that the differential components in plasma contained these 12 active components.Besides,the metabolic pathways and metabolites of 12 different components in the blood were analyzed.We found that the quantity of metabolites from 12 components of phase Ⅰ and phase Ⅱ are equivalent.Moreover,the isomers have the same metabolic pathway,and its metabolites are also isomers.Furthermore,the components can be interconverted into each other.Qing’e Wan contains various flavonoids,coumarins,iridoids and etc.It reported that these components had phytoestrogen-like effect.Hence,it indicated that the anti-osteoporosis efficacy of Qing’e Wan is the result of synergistic effect of multi-component and multi-target.Part three:we optimized the traditional osteoclast culture method,and established an osteoclast culture method with rapid growth and accurate determination of enzyme activity to improve the screening efficiency of active components s in Qing’e Wan.Compared with the traditional methods,the osteoclasts obtained by the new culture method have the better effects in morphology,mRNA expression of marker gene and bone resorption function.The EC50(μmol/L)can be determined more accurately due to wider range of enzymatic activity and osteoclasts number.And the cost were greatly reduced due to the evidently shorter culture cycle of new culture method.As a result of screening the components by osteoclasts activity,it was found that ge niposidic acid,pinoresinol diglucoside,psoralenoside and isopsoralenoside had no significant inhibitory effect on osteoclasts.The inhibition intensity of components on oste oclasts ranged from large to small:Corylifol A(0.1926)>4’-O-methylbavachalcone(0.2316)>isopsoralen(1.85)>isobavachalcone(5.113)>bavachalcone(5.408)>psoralidin(8.4)>corylin(11.97)>bavachinin(12.56)>psoralen(13.64)>isobavachin(15.07)>bavachin(15.26)>bakuchiol(30.63)>neobavaisoflavone(35.47).We studied the effects of bavachinin on NF-kB signaling pathway,PI3k/AKT signaling pathway and MAPK signaling pathway about osteoclasts.The results showed that bavachinin could inhibited phosphorylation of p65 in NF-kB signaling pathway,phosphorylation of AKT in PI3k/AKT signaling pathway,phosphorylation of p38,JNK,ERK in MAPK signaling pathway to inhibit osteoclastogenesis,differentiation and function of osteoclasts,especially acting on PI3k/AKT signaling pathway and MAPK signaling pathway.In sum,the material basis of salt-process enhancing the efficacy of Qing’e Wan are mainly about geniposidic acid,pinoresinol diglucoside in Eucommiae cortex,psoralenoside,isopsoralen,psoralen,bavachinin,neobavaisoflavone,bavachin,psoralidin,isobavachalcone,isobavachin,bavachalcone in Psoraleae fructus.These 12 ingredients have not only high content in Qing’e Wan,but also can reach high drug concentration in various organs after entering the body,which target the "kidney meridian" to a certain extent.Furthermore,they also have a great inhibitory effect on osteoclasts.Geniposidic acid and pinoresinol diglucoside have the effect of promoting osteoblasts among them,while psoralenoside can be transformed into psoralen which inhibits osteoclastogenesis and promotes osteogenic activity to exert the anti-osteoporosis effect.