Study On The Discovery Of Anti-Inflammatory And Antioxidant Bio-Active Groups In ZN Based On Spectrum-Effect Relationship Analysis And The Green Extraction Methods

The bio-active components of traditional Chinese medicines(TCMs)are complex,and usually their therapeutic effects achieved by internal interactions via multicomponent and multitargets,which brings great challenges to the systematical study of the material basis and mechanism in TCMs.Besides,the extraction of compounds in TCMs has an over-reliance on traditional organic solvents,which has been one of the key issues in the global environmental governances.Zanthoxylum nitidum(Roxb.)DC.(ZN)is one of the Guangxi "Gui-Shi-Wei" authentic medicinal materials,which has the effect of promoting blood circulation and removing blood stasis,regulating qi-flowing for relieving pain,Qufeng Tongluo prescription and removing toxicity for detumescence.In addition,it has been reported that ZN has varies the bioactivities,including anti-inflammatory,antioxidant activities,antimicrobial activity and anticancer activity,etc.At present,the material basis of anti-inflammatory and antioxidant activities in ZN is still unclear.Meanwhile,the conventional extraction methods are eco-unfriendly,which seriously restricts the popularization and application of ZN.Base on this,this research focused on the unclear bio-active compounds of ZN,and the eco-unfriendly conventional extraction methods.This research hopes to provide references for ZN’s mining of bio-active ingredients and the development of green extraction methods.Additionally,researches in the area of TCMs also can get inspired by our research.The main research contents are as follows:1.In view of the unclear anti-inflammatory and antioxidant active ingredients in ZN,the research discovered the anti-inflammatory and antioxidant bio-active groups in ZN based on spectrum-effect relationship analysis.Firstly,a structure diagnostic ion-oriented network was established based on the mass defect filteringbased structure classification(MDFSC)and diagnostic fragment-ion-based extension(DFIBE).Then,together with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC Q-TOF MS),a total of 48 chemical components in ZN were rapidly and comprehensively profiled,including 35 alkaloids,seven coumarins,three phenolic acids,two flavonoids,and one lignan.Secondly,the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography(HPLC)and 18 common characteristic peaks were chosen as research object.Combined with the results of cluster analysis and similarity evaluation,we found that ZNs from Guangxi province had good uniformity and similarity.Thirdly,20 batches of Zanthoxylum samples were tested for anti-inflammatory and antioxidant activities in vitro models.Then,the partial least squares regression(PLSR),gray relational analysis(GRA)models,and Spearman’s rank correlation coefficient(SRCC)were applied to discover the bioactive compounds in ZN.The results showed that peak 4(nitidine),12(oxynitidine)and 17(dihydronitidine)were closely related to the anti-inflammatory activity,whereas peak 3,5(chelerythrine),7(hesperidin),12 and 13 were the possible antioxidant components.2.It was found in the previous research that alkaloids and flavonoids are closely related to the anti-inflammatory and antioxidant activities of ZN.To establish a green and efficient extraction method for ZN,a urea-based deep eutectic solvent(DES)system was developed.Firstly,A total of 50 DESs were prepared and their extraction rate were screened,and the enzyme conditions affecting the rate were also evaluated.As a result,it was found that the Ur/Pa based cellulase system owned the best extraction efficiency to 5 main compounds in ZN(including magnoflorine,nitidine chloride,chelerythrine,diosmin,and hesperidin)with the rate of 34.56 ± 1.09 mg/g,which was higher than the methanol(25.06 ± 0.19 mg/g).Secondly,optimized by the one factor experiments and response surface methodology(RSM),the best condition was shown as follows: 27% water content,35 min extraction time,39℃extraction temperature,25:1 solid/liquid ratio,1.3 mg/m L enzyme concentration.Thirdly,the scanning electron microscopy(SEM)results showed that the Ur/Pa based cellulase system entirely destroys the cell wall of ZN,which brought excellent extraction rate.In all,this study provides some references for the discovery of bio-active components and green extraction of alkaloids and flavonoids from Chinese herbal medicine.