Studies On The Chemical Components Of Clematis Species

Members of the genus Clematis L.,belonging to the family Ranunculaceae,are mostly perennial woody vines together with a small part of herbs,shrubs or subshrubs. There are more than 350 species(spp.) in all,with about 150 distributed throughout China,mainly in southwestern China.Until now,studies on the chemical components have only been carried out on twenty or so spp.from this genus.So,it is worth studying the chemical components of the other Clematis spp.Some spp.of the genus are used for dispelling wind and eliminating dampness, tendon relaxation and activation of blood circulation,and detumescence and relieving pain.Five Clematis spp.,namely C.chinensis,C.hexapetala,C.terniflora var. mandshurica,C.armandii and C.montana,are documented in the Chinese Pharmacopoeia,with the radix and rhizoma of the first three as the sources of the famous traditional Chinese medicine 'Radix et Rhizoma Clematidis' and that of the last two as the sources of 'Caulis Clematidis Armandii'.In addition,more than 40 spp. are used in traditional or folk remedies with radix and rhizome,and stem and vine, and misuse of these drugs often occures.As a result,an accurate and reliable analytical method is needed for the identification of the drugs from this genus.According to the above study background,the main target of this thesis is concentrated on studies on determination and characterization of the triterpenoid saponins in various Clematis taxa from different areas in China using HPLC-ELSD, HPLC-MS and HPLC/ESI-MSⁿ method,based on the studies on chemical components of two Clematis spp.(C.terniflora and C.puberula var.ganpiniana). And further,the phylogenetic relationship of this genus was discussed from the view of chemotaxonomy,which provided scientific information for the research and development of new medicinal resources from this genus.The main innovation and academic contributions of this thesis are presented as follows:1.Column chromatography techniques(including silica gel and Sephadex LH-20) were used to separate constituents from the ethanol extract of the whole plants of C. terniflora and the structures of seven compounds were elucidated by ESI-MS,¹H NMR,¹³C NMR and physicochemical properties.Four of them were isolated from the genus Clematis for the first time,i.e,tricosanol,heptacosanoic acid, 3,5,7,3'-tetrahydroxyflavone and 5,7,3',5'-tetrahydroxy-dihydroflavone.2.Column chromatography techniques(including silica gel and Sephadex LH-20) were used to separate constituents from the 70%ethanol extract of the whole plants of C.puberula var.ganpiniana.The structures of eight triterpenoid saponins and one lignan were elucidated by IR,ESI-MS,TOF-MS,¹H NMR,¹³C NMR,HMQC, ¹H-¹HCOSY,HMBC and physicochemical properties.All of them were isolated from this plant for the first time.And one new triterpenoid saponin was identified as 3β, 23α-dihydroxy 12-olean-3-O-β-D-ribopyranosy1-(1→3)-α-L-rhamnopyranosyl-(1→2) -β-D-xylopyranosy1-28-O-α-L-rhamnopyranosy1-(1→4)-β-D-glucopyranosy1-(1→6)-β-D-glucopyranosyl ester and named as clematiganoside A.3.An HPLC-ELSD method was established to determine the five triterpenoid saponins in the 70%ethanol extracts of 16 samples(nine spp.and five varieties) from the genus Clematis.The results of method validation showed that this method was simple,precise and repetitive.The overall recoveries for the five analytes were between 91.3 and 99.5%.The LOD and LOQ were 0.0507～0.152μg and 0.169～0.506μg,respectively.However,sensitivity of this method was not so satisfactory,for the contents of the saponins were less than LOD in many samples.As a result,it is applicable for the quantification of just the abundant triterpenoid saponins in the TCM from Clematis spp.and identification of its plant sources.4.An HPLC-MS method was established to determine the four triterpenoid saponins in the 70%ethanol extracts of 20 samples(ten spp.and four varieties) from the genus Clematis.The results of method validation showed that this method was rapid,sensitive,accurate and repetitive.The overall recoveries for the four analytes were between 95.1 and 103.1%.The LOD and LOQ were 0.0220～0.207 ng and 0.0733～0.656 ng,respectively,so the sensitivity of this method was several hundredfolds higher than that of the HPLC-ELSD method established in this thesis. As a result,it is applicable for the quantification of the triterpenoid saponins in the TCM from Clematis spp.and identification of its plant sources.5.An HPLC/ESI-MSⁿ method was established to characterize and identify the triterpenoid saponins in crude extracts of C.puberula var.ganpiniana and C chinesis. Firstly,MSⁿ experiments were carried out on the eight reference compounds to obtain the fragmentation patterns of the tfiterpenoid saponins according to the fragment ions. Based on the fragmentation patterns of the reference compounds,this method was applied to studies on the chemical substance in the 70%ethanol extracts of C. puberula var.ganpiniana and C chinesis,and 33 triterpenoid saponins were identified.6.The above HPLC-MSⁿ method was employed to study the distribution of the triterpenoid saponins in some Clematis taxa from China and 17 of them were used as markers for discussing the phylogenetic relationship between these taxa.The results showed that the phylogenetic relationship between them based on these saponins was not all in accordance with that of classic morphological taxonomy,and some even got different results.As a result,further study is needed for research and development of new medicinal resources from this genus and establishing a more reasonable taxonomic system of it.