Study On The Difference Of Chemical Composition Of Jujube Seed From Different Producing Areas And Comparison Of Antioxidant Activity

Objective:Ziziphi Spinosae Semen(ZSS),the dried seeds of Ziziphus jujube Mill.var spinosa(Bunge)Hu ex H.F.Chou,a well-known traditional Chinese medicine for the treatment of insomnia and anxiety.In modern pharmacological and clinical studies,ZSS is widely used for its anxiolytic and sedative effects,and other activities including protection of the cardiovascular system,antihyperlipdemia and antioxidant actives,etc.With the accelerated pace of life,more people suffer from insomnia,anxiety,and cardiovascular diseases.As a medicinal material with both medicinal and edible value,the demand for ZSS is increasing Ziziphus jujube var.spinosa is widely distributed in the low mountain-hill regions of the north of China,and most of them are wild.The ecological environment between producing areas is quite different,and there are significant differences in the quality of ZSS among different producing areas.The sporadic cultivation plantations are lack of good agriculture practice.The eco-environments of different production areas are quite deviating,and the quality of ZSS from various regions have significant differences.However,there are few studies on the influence of origin factors on its internal chemical compositions,and mostly just contents determinations of a few major components by HPLC.But,as a complex chemical system,the quality differences of TCMs cannot be fully reflected by only a few index components.In addition,biological activity is also an important aspect of the quality evaluation of Chinese medicinal materials,and there are few studies on the differences in biological activity of ZSS between different origins.So the influence of origin factors on the quality of ZSS still needs to be systematically studied from multiple anglesBased on the above considerations,this study will be carried out from the following four aspects:(1)Ultra high-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS)was applied to comprehensively characterize the chemical constituents in ZSS.(2)UHPLC-LTQ-Orbitrap MS metabolomics followed by multivariate statistics was applied to discriminate ZSS samples from different origins according to their chemical constituents.(3)The concentrations of nine elements in ZSS samples from different regions were analyzed by inductively coupled plasma mass spectrometry(ICP-MS),and the statistical characteristics of elemental composition were used to characterize the characteristics of inorganic elements in ZSS according to different regions.(4)In vitro antioxidant activity evaluation method was used to evaluate the antioxidant activity of ZSS samples from different regionsMethods:1.UHPLC-LTQ-Orbitrap MS was used to comprehensively characterize the chemical constituents of ZSS.The data-dependent acquisition(DDA)-MS2 strategy was employed to obtain fragment ions information.The parent ion list(PIL)strategy was used to analyze the compounds that did not obtain fragment ions information.Through the analysis of mass spectrometry data and literature data to identify target components.Based on the Metwork technology and non-target components library that based on two C-flavonoid glycoside subtypes in ZSS,the non-target compounds are identified.2.To discriminate ZSS from ten different regions of China according to their chemical constituents based on comparative untargeted metabolomics by using UHPLC-LTQ-Orbitrap MS.UHPLC-LTQ-Orbitrap MS analysis was first performed on samples of ZSS from different origins.The pretreated data were further processed using principal component analysis(PCA)and the orthogonal projections to latent structures discriminant analysis(OPLS-DA)to ensure marker authenticity.3.The concentrations of nine elements(Na?Mg?Al?K?Ca?Mn?Cu?Zn)in ZSS samples from different regions were analyzed by ICP-MS.Samples were digested in a microwave system.Principal component analysis,correlation analysis,and cluster analysis(CA)were applied to the data to evaluate the analytical results.4.The antioxidant activity of ZSS extracts from different origins was evaluated through four different antioxidant assays:1,1-diphenyl-2-picrylhydrazyl(DPPH·),2-2'-azino-bis(3-ethylbenzo-thiazoline-6-sulphonate)diammonium salt(ABTS·+),Ferric Reducing Antioxidant Power(FRAP)assays and hydroxyl radical scavenging ability.Results:1.A novel UHPLC-LTQ-Orbitrap-MS method that combined with multiple data collection and mining strategies was established to rapidly separate and identify components from the extract of ZSS.58 compounds were identified,including 36 flavonoids,14 saponins,6 triterpenes,and 2 alkaloids.Among them,christinin D,jujubesaponin I,jujubasaponin VI,and ziziberanalic acid were detected from ZSS for the first time.Besides,9 new components were inferred through non-target compounds mining strategies.2.Based on UHPLC-LTQ-Orbitrap-MS metabolomics,a method for evaluating the chemical composition of ZSS from different regions was established.The PCA and OPLS-DA can discriminate ZSS samples from 10 different regions.In particular,the samples from Huabei(Shanxi,Hebei),Shaanxi,Henan,and Dongbei(Liaoning)have obvious differences,can be well distinguished.The OPLS-DA,t-test,and variable importance in projection(VIP)plot were used to ensure differential markers between 4 groups(Huabei,Shanxi,Henan,Dongbei).There were 34 significant differences between Shaanxi and Henan group,26 significant differences between Shaanxi and Huabei group,15 significant differences between Shaanxi and Dongbei group,33 significant differences between Dongbei and Huabei group,39 significant differences between Henan and Huabei group,and 33 significant differences between Henan and Dongbei group.3.The concentrations of 9 elements(Na?Mg?Al?K?Ca?Mn?Fe?Cu?Zn)in ZSS from 10 origins were determined by ICP-MS,and feature maps were established.The results showed that the contents of elements in ZSS from different regions were different,but there was consistency and characteristics in the element composition and distribution.The PCA showed that Mg,K,Ca,Fe,Cu,and Zn may be the characteristic components of ZSS.The CA showed that there existed regional differences of the elements distribution in ZSS.4.The antioxidant activity of ZSS extracts were determined by ABTS assay,FRAP assay,DPPH assay,and hydroxyl radical scavenging capacity.The results showed that the ZSS extracts had remarkable antioxidant activity.Antioxidant activities of ZSS extracts from different places were significantly different,and the antioxidant abilities of the same sample were different in various evaluation systems.In comprehensive evaluation,the antioxidant activity of the samples from Hebei was stronger,and samples from Henan were weaker in the four evaluation systems.This study had comprehensively characterized the quality diversity of ZSS according to the origins from the aspects of organic chemical composition,elements,and antioxidant activity.It provided research ideas and basic data for further study on the quality differences of ZSS from different origins.It can also provide the theoretical basis for fine breeding,and selection of suitable planting areas,and further development and utilization of resources.