Study On Quality Evaluation Of Desmodium Styracifolium

BackgroundDesmodii Styracifolii Herba,the dried aerial part of Desmodium styracifolium?Osb.?Merr.?Leguminosae?,is mainly distributed in Guangdong,Guangxi and Fujian provinces in the southeast of China.It is cool in nature,sweet and light in taste,with the effectiveness of clearing heat,dispelling dampness,promoting diuresis and relieving strangurtia.It is widely used for the treatment of pyretic stranguria,urolithiasis,edema,oliguria and jaundice,etc.D.styracifolium,the main component of the famous Guangdong herbal tea named Wanglaoji,has a wide range of applications in south China.ObjectiveOnly schaftoside was chosen as a marker constituent for assessing the quality of D.styracifolium in the Chinese Pharmacopeia?2015 version?.However,the single compound is difficult to evaluate inherent quality of Chinese Materia Medica due to their complex constituents.To provide a more scientific and comprehensive reference of quality control for D.styracifolium,multi-components qualitative and quantitative analytical methods were established for flavonoids.Simultaneously,the study on monosaccharide composition and pharmacological activities of polysaccharides were carried out.Methods1.According to the thin layer chromatography?TLC?identification method of D.styracifolium in Chinese Pharmacopeia?2015 version?,we investgated chromatographic conditions of TLC including plates,developing agent,sample preparation methods,humidity,temperature and so on.2.The analysis of D.styracifolium was performed by HPLC method.The content of these four components?schaftoside,isoorientin,isoschaftoside and isovitexin?from 51 batches of D.styracifolium samples were determined by external standard method?ESM?.At the same time,the HPLC fingerprint of D.styracifolium was established.The chemomeric techniques,including hierarchical cluster analysis?HCA?,principal component analysis?PCA?,discriminant analysis?DA?,were applied to classify 31 bathches of crude drugs.The accuracy of multi-components by single marker?QAMS?method was verified by comparing with the results calculated by ESM.3.The total polysaccharides of 20 batches of D.styracifolium samples derived from different locations were extracted through the water extraction and alcohol precipitation.To identify and quantify the monosaccharide compositions of D.styracifolium polysaccharides,a method of 1-phenyl-3-methyl-5-pyrazolone?PMP?precolumn derivatization HPLC analysis was performed.And the conditions including polysaccharide hydrolysis time,derivatization temperature and derivatization time were optimized.Besides,with acarbose as a positive control,the inhibitory effect of total polysaccharides of D.styracifolium on the activity of?-glycosidase was initially explored in vitro.Results1.The TLC identification method of D.styracifolium was optimized and established.As for preparation of the sample solution,an accurately weighed sample of 0.5 g was added into a flask with 62.5 mL of 80%methanol and placed into an ultrasonic bath for 20 min.The mixture were filtered,recycled methanol,and added equivalent ethyl acetate-butanone?1:1?for 2 times.The extracts were evaporated,then diluted to volume with 50%methanol in a 2 mL volumetric flask.The optimum conditions of TLC was chosen as followed:the silica gel GF₂₅₄ plate was used as the stationary phase with the ethyl acetate-butanone-formid acid-water?5:1:1:1?as the developing agent,1%AlCl₃ ethanol solution as the reagent and 365 nm as the detected wavelength.The established TLC fingerprint with four flavonoids?vicenin-2,schaftoside,isoorientin and isovitexin?as standard substances can be used to distinguish D.styracifolium and its adulterants.2.HPLC analysis was performed on a Phenomenex Polar C₁₈ column operated at 30°C.The mobile phase consisted of acetonitrile?solvent A?and 0.05%formid acid in water?solvent B?with a gradient elution procedure as followed:0²0 min,13%¹5%A;20³5 min,15%A;35⁶0 min,15%¹6%A.The flow rate was 0.8 mL/min and the sample injection volume was 10?L.The detection wavelength was set at 335 nm.At the same time,the HPLC fingerprint of D.styracifolium was established with 11 common peaks,and four of them were identified as schaftoside,isoorientin,isoschaftoside and isovitexin,respecivetly.The similarity values from 31 bathches of crude drugs and 20 bathches of decoction pieces from D.styracifolium were in the range of 0.850⁰.978and 0.880⁰.998,respecivetly.The results of the chemomeric techniques were consistent.Furthermore,no significant difference between ESM and QAMS method was found.3.The results showed that the polysaccharides of D.styracifolium samples were mainly composed of L-rhamnose,D-galacturonic acid,D-glucose,D-galactose,D-arabian sugar with small amounts of D-mannose and L-xylose.The chromatographic fingerprint of D.styracifolium polysaccharides by PMP precolumn derivatization was successfully established.The similarity values of these samples were ranged from 0.824 to 0.981 except S2',S10'and S19'samples.Simultaneously,the IC₅₀ values of acarbose and total polysaccharides were 7.7601 mg/mL and 0.7031 mg/mL,respectively.Conclusion1.The results showed that chromatogram had clear spots,better separation and good reproducibility,which confirmed the established method could be used for the TLC identification of D.styracifolium.2.The HPLC fingerprint of D.styracifolium was established and analysed using chemomeric techniques.Furthermore,QAMS method was successfully applied in the quality evaluation of D.styracifolium.3.The chromatographic fingerprint of D.styracifolium polysaccharides by PMP precolumn derivatization was established.The developed method was stable,simple and repeatable,which can provide sufficient theoretical basis for the quality evaluation of D.styracifolium polysaccharides.The total polysaccharides of D.styracifolium had stronger inhibition on?-glucosidase,which indicated it potentially existed hypoglycemic effect and its mechanism needed further experiments to verify.