Pharmacognostical Studies On Aster Flaccidus,A.Farreri And A.himalaicus

Aster.flaccidus,A.farreri.and A.himalaicus.are perennial herbs of genuses Aster in Asteraceae family,which are the main sources of Traditional Tibetan Medicine Meiduoluomi.All three kinds are rich in source and mainly distributed in Qinghai-Tibet Plateau in China.Tibetan medicine Asteris Flos has the efficacy of heat-clearing and detoxifying,eliminating phlegm and relieving cough,and is mainly used for the treatment of plague,cold"Long"and"Huangshui" disease,poisoning,bronchitis,cough and asthma,cough purulent blood,etc in Tibetan clinic.At present,the most commonly used origins have not been included in the national or local standards of medicinal materials.in this paper,a systematic pharmacognostical study was carried out on Aster flaccidus,A.farreri and A.himalaicus in order to lay a foundation for improving and promoting the quality evaluation system of the multi-origin Tibetan medicine "Meiduoluomei".The main research contents are as follows:(1)The morphological identification and microscopic identification of the three kinds of asters were studied to obtain thier main identification characteristics.Aster flaccidus,with slender and purple stems,its involucre densely covered with white long pubescence A fareri,with thick stems and slightly drooping near flower end,its leaves are extremely leptophyllous.A.himalaicus,its stems are grown slanting sideways at base and are curved lower part.In microscopic characters,the distribution of phloem fibers and wood fibers of the root,the characteristics of stem pith and the differentiated degree of mesophyll tissues are the main distinctive features of the three species of aster.(2)The DNA barcode of the three kinds of asters were established.ITS sequence and rbcL sequence were used to distinguish the three asters.The results showed that the maximum intraspecific genetic distance of the two DNA barcodes was less than the interspecific genetic distance,and the PCR efficiency and sequencing success rate are reached 100%.the Nj phylogenetic tree showed that ITS sequence was better than rbcL sequence in distinguishing the three asters at species level.(3)The UPLC fingerprints of the three kinds of asters were established.Taking the common peaks of three kinds of asters as indicators,the similarity of fingerprints of three kinds of asters was evaluated by the similarity evaluation system(2004 edition),and the fingerprint data of three kinds of asters were analyzed by cluster analysis(HCA)and principal component analysis(PCA).There were 11 common peaks confirmed individually in Aster flaccidus.and Afarreri,and A.himalaicus has 12 common peaks.Compared with the reference substance,three component peaks of the three kinds asters were identified,including chlorogenic acid(peak2),isochlorogenic acid B(peak 9)and isochlorogenic acid A(peak A(10).The results of similarity showed that the similarity among different batches of the three kinds of Asters was good,but there were big differences among individual batches.The results of cluster analysis(HCA)show that the three kinds of aster can be clustered into 9 categories when the euclidean distance is 5,and the three kinds of asters can be clustered into one category in each other,which is confirmed by the results of principal component analysis(PCA).Indicating that the internal components of the three kinds of aster are similar,and it is difficult to cluster by the common peak area.(4)Three qualitative and quantitative analysis methods of the three Asters UPLC-DAD-QTOF-MS/MS were established.Chromatographic conditions:Acquity UPLC HSS T3 C18 column(100 mm × 2.1 mm,1.8 μm),acetonitrile(A)-0.1%formic acid aqueous solution(B)as mobile phase,gradient elution:10-20%A(0-0.8min),20-28%A(0.8-28-38%A(4.0-4.5min),38-60%A(4.5-5.5min),60-92%A(5.5-10.0min),95-95%A(10.0-12.5min),95-10%A(12.5-13.0min),and 10%A(13.0-15.0min).Column temperature is 35℃,volume flow rate is 0.4 mL/min,and injection volume is 2L.The mass spectrometry conditions are as follows:the ion source is electrospray ion source negative ion mode(ESI-),capillary voltage is 2.8 kV,cone voltage is 40 V,ion source temperature is 100℃,desolvation gas temperature is 400℃,desolvation gas flow rate is 800L/h,collision energy low energy is 6V,gradient high energy is 35?55 V,collision gas is argon,scanning time is 0.2 s,and the mass scanning range is m/z 100～1500,and leucine enkephalin is used for real-time mass calibration during acquisition.The results showed that the contents of chlorogenic acid,rutin and isochlorogenic acid B in the three kinds of asters were all over 0.1%,which were the main components of the three asters.However,the contents of six components are different among the three Aster species.The average relative contents of chlorogenic acid(0.343%)and isoquercitrin(0.044%)in Aster.flaccidus.are higher than those in Afarreri and A.himalaicus.The average relative content of isochlorogenic acid B(0.236%)in A.farreri was significantly higher than that in Aster flaccidus.and A.himalaicus.However,the average relative contents of rutin(0.210%)and 1,3-dicaffeoyl quinic acid(0.105%)in A.himalaicus.were higher than those in Aste.flaccidus.and A.farreri.24 compounds were inferred and identified from the three species of aster,including quinic acid,citric acid,neochlorogenic acid,chlorogenic acid,1-caffeoylquinic acid,3-p-hydroxycinnamic acid,3-O-ferulylquinic acid,3,4-dicaffeoylquinic acid(isochlorogenic acid B),3,5-dicaffeoylquinic acid(isochlorogenic acid A)and 1,3-dicaffeoylquinic acid,rutin,aringenin-7-O-glucuronide,quercetin-3-O-glucuronide,scutellarin,isoquercit rin,kaempferol-3-O-rutinoside,6-O-Acetylisoquercitrin,isorhamnetin-3-O-neohesperid in,apigenin-7-O-glucuronide,glycerin-7-O-glucoside,scutellarin,quercetin Vegetarian,apigenin.(5)On the basis of Aster souliei Franch.used as Tibetan Aster,drafting the draft standard of the three origins of Tibetan Aster,All the three Aster will be included in the Quality Standard of Tibetan Medicinal Materials in Sichuan Province(2020 edition).