Studies On Quality Control Of Small Leaves Ku-Ding-Cha And Preliminary Pharmacophylogeny On Boraginaceae Ku-Ding-Cha

"Ku-Ding-Cha" is a traditional Chinese tea used mainly in south of China for almost 2,000 years.Although different species used in different places of China are all called "Ku-Ding-Cha".Large leaves Ku-Ding-Cha which belongs to family Aquifoliaceae and small leaves Ku-Ding-Cha which belongs to family Oleaceae are two main species used in the markets of all the country.Several plants of family Guttiferae and Boraginaceae are also used as Ku-Ding-Cha in sparing places of China.In the present study,the chemical constituents in the Ligustrum robustum;quality control method of small leaves Ku-Ding-Cha;chemical constituents and preliminary pharmacophylogenetic study of Boraginaceae Ku-Ding-Cha were studied.Sixteen compounds were isolated and identified in L.robustum,they were ligustaloside B(1),ligustaloside B di-Me acetal(2),ligustaloside A(3),acteoside(4), ligupurpuroside A(5),isoacteoside(6),ligupurpuroside B(7),ligupurpuroside D(8), osmanthuside B₆(9),ligupurpuroside C(10),3,4-dihydroxyphenyl(11),lipedoside B-â…¢a(12),lipedoside B-â…¢b(13),glucose esters of(E)-2,6-dimethyl-6-hy droxyocta-2,7-dienoic acid(14),luteolin-7-O-β-D-glucopyranoside(15),rutin(16).Eight of them were first isolated from this species;two of them were first isolated from this genus.84 samples including raw materials and the commercial products were collected from the tea markets or from their habitats around China and the quality control methods were developed.The HPLC fingerprint used for the quality control of small leaves Ku-Ding-Cha was established.After investigating chromatography and extracting condition,two mutual patterns were established from 35 old leaves original plants and 18 buds of products, respectively.The data were used for Hierarchical Cluster analysis and similarity evaluation.It was indicated that 35 old leaves samples were identified into three types, the identified plant L.robustum were all in the typeâ… and typeâ…¡.Through the mutual pattern established the alternative species can be classified out of the main species of L. robustum.The investigation of the mutual pattern of the bud products in the market indicated although the same sample of the buds maybe belong to different species,the global chemical characters of all the bud products are very similar.The quality control can be evaluated through the common characters existence in all the bud products.The MS/MS fragments pattern of phenylethanoid glycosides and iridoid glycosides were analyzed and the typical samples were detected by LC-ESI/MS under positive and negative ion mode.The major constituents in L.robustum were analyzed through comparing with the standard compounds,fragment pattern and looked up in the literature.A HPLC-UV method was established to simultaneously quantification of 3 major phenylethanoid glycosides:acteoside(1),ligupurpuroside A(2) and ligupurpuroside B(3) in 84 batches samples.It is considered to be suitable for quality control of the tea products and be helpful to classify the species the small leaves Ku-Ding-Cha.The chemical compositions of essential oils from 7 batches of small leaves have been studied by GC-MS method,65 compounds were identified and quantified.35 compounds were isolated from the leaves of Ehretia thyrsiflora with silica gel, ODS,sephadex-LH-20,et al.Among them,27 compounds were elucidated on the basis of chemical and spectral evidence.They were quercetin-3-O-α-D-arabinoside(1), kaempferol-3-O-α-D-arabinoside(2),quercetin-3-O-β-D-glucopyranoside(3), hyperoside(4),kampferol-3-O-β-D-galactopyranoside(5),kampferol-3-O-β-D-gl ucopyranoside(6),kaempferol-3-O-arabinosylgalactoside(7),quercetin-3-O-arabin osylgalactoside(8),quercetin(9),kampferol(10),trans-ferulic acid(11),caffeic acid (12),p-hydroxy benzoic acid(13),methyl rosmarinate(14),rosemarinic acid(15), lithospermic acid B(16),danshensu(17),ciunamic acid(18),icariside E₅(19),(E)-ethyl caffeate(20),di(2-ethylhexyl) phthalate(21),2-methoxyl benzoic acid octyl ester(22), di(octadecyl) phthalate(23),β-Sitosterol(24),daucosterol(25),allantoin(26), [tetradecenoic acid,2,3-dihydroxypropyl ester(27)],respectively.All of the compounds were isolated from this species for the first time.Compounds 1-13,16-18,20-23,26,27 were isolated from this genus for the first time.A RP-HPLC method was firstly established for determination of 7 major polyphenols(quercetin 3-O-arabinosylgalactoside,isoquercetrin,quercetin-3-O-α-D-arabinoside, astragalin,kaempferol-3-O-α-D-arabinoside,caffeic acid and rosmarinic acid) in E.thyrsiflora.All the compounds were analyzed within 35 min.Validation statistics showed the sensitivity,precision,repeatability,stability and the recovery of the method were good.In the same time,the method was used to evaluate the content of these seven compounds in other species of family Boraginaceae which provided the evidences of resource utilization.Through the literature and chemical investigation of E.thyrsiflora,we discussed the systematic position of the genus Ehretia and the subfamily Ehretiaceae in the family Boraginaceae which has been doubtful and controversial for a long time.