Study On Chemical Composition And Quality Control Methods Of Bupleurum Chinense

Objective:Bupleurum bicaule Helm（Chinese name:Zhuiye Chaihu）is from the genus Bupleurum（Umbelliferae）,which is mainly distributed in Inner Mongolia and Northeast of China.It is a standard variety of Chinese medicinal materials in Inner Mongolia,and has been used medicinally for a long history in the locality.At present,the circulation market of B.bicaule is large,so it has a good application prospect.The purpose of this paper is to review the research on plant resources,chemical components,pharmacological effects and quality control of genus Bupleurum in recent years.In addition,the chemical constituents and quality control methods of B.bicaule were studied in order to provide a basis for expanding the application of B.bicaule and expanding the medicinal source of Bupleurum.Methods:1.Study on chemical composition:The chemical components of different polar parts of B.bicaule were separated and purified by macroporous resin,silica gel column chromatography,Sephadex LH-20,reverse silica gel column chromatography and flash column chromatography.The structures of the compounds were identified by mass spectrometry,1D and 2D NMR spectroscopy.The NO inhibitory activity and hepatoprotective activity of 13 isolated compounds were evaluated.Where the essential oil of B.bicaule was extracted by hydrodistillation and its components were analyzed by gas chromatography-mass spectrometry（GC-MS）.The contact and repellent activities of the oil and four identified compounds against Lasioderma serricorne and Liposcelis bostrychophila adults were evaluated.2.Study on quality control methods:The plant was identified by primitives,characters,DNA,microtlc,TLC,specific chromatogram identification.The HPLC method for simultaneous determination of saikosaponin A,C,D and prosaikogenin F,G,I was established.Based on the above research,the draft of quality standard of B.bicaule was drawn up.Results:1.Results of chemical composition research:A total of 23 compounds were isolated from the roots of B.bicaule.They are:13,28-epoxy-11-oleanene-16-one（1）,chaihuterpene A（2）,13,28-epoxy-olean-11-en-16-ol-3-one（3）,saikogenin E（4）,sa ikogenin D（5）,a-spinasterol（6）,13,28-epoxy-2,23-dihydroxy-olean-11-en-16-one（7）,16a,23a,28β-trihydroxyoleana-11,13（18）-diene-3-one（8）,caffeic acid（9）,saikogenin A（10）,saikogenin F（11）,alaternin（12）,a-spinasterol-3-O-β-D-glucopyranoside（13）,3-[(6-deoxy-（β-D-galactopyranosyl）oxy]-13,28-epoxy-23-hydroxy-olean-11-en-16-one（14）,st igmasterol-3-O-β-D-glucopyranoside（15）,prosaikogenin G（16）,3β,16p,23,28-tetrahydro xy-11a-methoxy-olean-12-ene-3-O-β-D-fucopyranoside（17）,prosaikogenin F（18）,prosai kogenin D（19）,saikogenin B（20）,saikosaponin b2（21）,saikosaponin A（22）,saikosa ponin D（23）.Preliminary investigation of NO inhibitory activity and hepatoprotective activity of compounds 1-4,7-9,12-17 indicated that compounds 14,16 and 17 show ed significant NO inhibitory activity at 10⁵ M,All of the compounds had any signifi cant protective effect on HepG2 cell damage at 10 μM.26 components were determined in the essential oil and the main compounds are trans-2-isopropylbicyclo[4.3.0]non-3-en-8-one（25.9%）,4,5-dimethyl-1,2,3,6,7,8,8a,8b-oct ahydrobiphenylene（23.5%）and 1,4-dimethoxy-2-tert-butylbenzene（4.3%）.It was found that the contact toxicity and repellent activity of the essential oila gainst L.serricorn e were better than L.bostrychophila,the percent repellency against L.serricorne reac hed to 100%.Among the four selected compounds,（2E,4E）-2,4-nonadienal exhibited s trong contact toxicity against L.serricorne with LD₅0 value of 2.31 μg/adult,while β-bisabolene possessed higher repellency.2.Results of quality control research:The characteristics,microscopical,thin layer and DNA molecular identification methods of B.bicaule were established.In addition,the HPLC specific chromatogram of B.bicaule was established and 10 characteristic peaks were found.The contents of saikosaponin A,C,D and prosaikogenin F,G,I in 12 samples of B.bicaule were 0.11%-0.34%,0.05%-0.20%,0.35%-1.92%,0.13%-0.87%,0.18%-1.49%and 0.04%-0.37%,respectively.It was found that the content of saikosaponin D in B.bicaule is significantly higher than that in B.chinense and B.scorzonerifolium.Prosaikogenin G and I with high content in B.bicaule were not detected in two pharmacopoeia varieties,which could be used for the identification of B.bicaule and pharmacopoeia varieties.Conclusions:1.This paper investigated the chemical constituents of different polar parts of B.bicaule.Among the 23 isolated compounds,compounds 1 and 2 are new compounds,and compound 1 has a new skeleton.Compounds 3,7,12,14 were isolated from the genus Bupleurum for the first time,compounds 8,10,17,20 were isolated from B.bicaule for the first time,some saponins showed strong NO inhibitory activity.The main components in B.bicaule were terpenoids.In this paper,the bioactivities of B.bicaule essential oil and its main components against two stored-product insects were also been investigated,the essential oil was found to have the potential of development and utilization as natural pesticides.Essential oil is the main component of Bupleurum,so the safety of B.bicaule essential oil for human remains to be assessed.2.The systematic identification of B.bicaule was conducted through a variety of methods and the specific chromatogram was established.The HPLC method for simultaneous determination of six saponins in B.bicaule was established at the same time,the established method is simple and accurate,which could be served as a reference for the quality control of B.bicaule.