Preliminary Study On Chemical Constituents And Quality Standard Of Clerodendranthus Spicatus

Clerodendranthus spicatus is a genus of Clerodendranthus plant in the labiaceae family,and mainly produced in Guangdong,Hainan,Fujian,Taiwan,Guangxi,Yunnan and other provinces.Clerodendranthus spicatus is a traditional medicine of Dai nationality in China.It has the functions of clearing heat and detoxifying,diuresis and stone removal,cooling blood and stopping bleeding,etc.It is used for the treatment of acute and chronic nephritis,diabetes,urocystitis,urolithiasis,rheumatism and other diseases.Modern pharmacological studies have shown that Clerodendranthus spicatus has a wide range of anti-inflammatory,diuretic,antibacterial,improve renal function,anti-hyperglycemia,anti-gout,decompression,anti-tumor,improve immunity and other effects.It has been reported that Clerodendranthus spicatus contains flavonoids,phenolic acids,terpenoids,volatile oils and other chemical components.In order to further explore the pharmacodynamic substance basis and quality evaluation method of Clerodendranthus spicatus,the chemical composition and quality standard of Clerodendranthus spicatus were preliminarily studied in this paper.1.Analysis of chemical constituents of Clerodendranthus spicatus by UHPLC-QTOF-MS/MSIn order to clarify the chemical composition of Clerodendranthus spicatus,UHPLC-QTOF-MS/MS technology was used to analyze the overall chemical composition of the extract of Clerodendranthus spicatus,and diagnostic ion and neutral loss filtration strategies were established according to different structural types of compounds,which were used for chemical composition identification of Clerodendranthus spicatus.A total of 118 compounds were identified from Clerodendranthus spicatus,including 85 polyphenolic acids and 33 flavonoids.The chemical constituents of Clerodendranthus spicatus were analyzed in this experiment,which provided the basis for the extraction,separation and quality standard of the chemical constituents of Clerodendranthus spicatus.2.Study on separation and purification of chemical components from Clerodendranthus spicatusIn this study,the chemical components of chloroform and ethyl acetate extract of Clerodendranthus spicatus were separated and identified,and systematically separated and purified by silica gel column chromatography,Sephadex LH-20 gel column chromatography,ODS column chromatography,preparative HPLC and other methods.10 compounds were identified by ¹H-NMR,¹³C-NMR and other spectral techniques combined with literature analysis.They were rac syn-2,3-dihydroxy-3-（3,4-dihydroxyphenyl）-propionic acid,luteolin,3-（3,4-dihydro xyphenyl）-acrylic acid,1-（3,4-dihydroxyphenyl）-2-methoxycarbonylethyl ester,rosmarinic acid methyl ester,rosmarinic acid,oresbiusin A,kaempferol-3-O-（2’’-O-β-D-glycopyranosyl）-β-D-glucopyranoside,orthosiphoic acid A,oleanolic acid,ursolic acid.3.Identification of Clerodendranthus spicatus by thin layer chromatographyIn this study,TLC was used to establish the identification of tanshusu,rosmarinic acid and ursolic acid in Clerodendranthus spicatus.Methylbenzene-ethyl acetate-formic acid（5:3:0.8）was used as the developing agent.Under ultraviolet light（365nm）,10%sulfuric acid ethanol solution was used as the color developing agent.The results showed that thin layer chromatography could quickly identify danshinsu,rosmarinic acid and ursolic acid in Clerodendranthus spicatus.The chromatograms of13 batches of Clerodendranthus spicatus samples showed the same color spots at corresponding positions with the control samples.The results showed that the established TLC method could be used for the identification of Clerodendranthus spicatus.4.Study on chemical fingerprint of Clerodendranthus spicatusHPLC method was used to establish the fingerprint of Clerodendranthus spicatus,and 12 common peaks were identified.Two common peaks,danshensu and rosmarinic acid,were identified by comparison with control products.The similarity analysis results showed that the similarity of 13 batches of Clerodendranthus spicatus was between 0.698 and 0.997.The similarity between S4 and the control fingerprint was less than 0.970,the similarity between the other sources of Clerodendranthus spicatus and the control fingerprint was greater than 0.970.The results showed that the overall stability of Clerodendranthus spicatus was high.Through the study of HPLC fingerprint of Clerodendranthus spicatus,the quality evaluation method system was improved,which provided a strong guarantee for the quality control of Clerodendranthus spicatus.5.Study on the content determination of Clerodendranthus spicatusThe contents of danshensu and rosmarinic acid in Clerodendranthus spicatus were determined by HPLC.The methods were as follows:Venusil C₁₈（4.6 mm×250mm,5μm）,acetonitrile-0.1%phosphoric acid solution as mobile phase with gradient elution,flow rate 1 m L/min,detection wavelength 210 nm,column temperature 30℃,sample injection volume 5.0μL.Danshansu and rosmarinic acid showed good linear relationships in the range of 1.75-56μg and 15-480μg,respectively.The established HPLC method was used to determine the contents of danshensu and rosmarinic acid in Clerodendranthus spicatus from different regions.The results showed that the method was simple,stable and reliable,and provided an important reference for the quality control of Clerodendranthus spicatus.In a word,this paper carried out preliminary extraction and separation,structural identification and comprehensive composition prediction of Clerodendranthus spicatus,and carried out quality evaluation methods of Clerodendranthus spicatus from different regions,such as thin layer identification,fingerprint and multi-index content determination,which provided a reference for the quality control of Clerodendranthus spicatus medicinal materials.