| PurposeTo elucidate the pharmacological substances that exert anti-inflammatory,antioxidant and hypoglycemic activities in Radix rosea;to isolate and purify the tannins,which are rarely studied in the literature,and to identify their structures in order to elucidate the composition of tannins in Radix rosea.To explore the anti-inflammatory mechanism of the main active components(triterpenes and tannins)of Radix rosea by using network pharmacological methods;to analyze the major classes of components in Radix et caulis rosea from different base sources and different origins,and to conduct a comparative study on their fingerprint profiles.Methods1、The anti-inflammatory activity of ethanol extract of Radix rosea and its different polar extracted parts were investigated using croton oil to cause auricular swelling in mice;the antioxidant activity of each part in vitro was determined by DPPH,ABTS and FRAP methods;the hypoglycemic activity of each part was investigated using α-glucosidase inhibition.The content of total triterpenes,total phenols,total tannins and condensed tannins in each part of Radix rosea was determined by UV spectrophotometry;the chemical composition of each part was analyzed by UPLC-Q-TOF-MS/MS technique,and the structure of the main peaks was inferred to identify the active components that exert anti-inflammatory,antioxidant and hypoglycemic activities.2、The active components of the n-butanol extracted parts of Radix rosea were systematically separated using modern separation and purification methods such as macroporous resin column chromatography,silica gel column chromatography,MCI chromatography,reversed-phase ODS column chromatography,preparative HPLC and semi-preparative HPLC.3、Through extensive literature mining and collation and combined with the previous research foundation of our laboratory,we obtained the reported triterpenes or tannins of Radix rosea.The active ingredients and potential targets were predicted by Swiss ADME;the main anti-inflammatory targets were obtained by Gencards and OMIM databases,and the active ingredient-anti-inflammatory target network was constructed by Cytoscape 3.7.2 software.Protein interaction analysis was performed using String platform,and KEGG pathway analysis was performed utilizing Metascape platform.4、The contents of total triterpenes and total tannins in Radix et caulis rosea were determined by UV spectrophotometric method.The orthogonal test was utilized to investigate the effects of reagent concentration,material-to-liquid ratio,extraction time and extraction temperature on the extraction process and to determine the best extraction process.According to the best extraction process,the contents of total triterpenes and total tannins in Radix et caulis rosea from different basic sources and different origins were compared.5、The fingerprints of the HPLC of Radix et caulis rosea were established on a Kromasil C8 column(4.6×250 mm,5 μm)with the mobile phase gradient elution of methanol-0.05%phosphoric acid water,the volume flow rate of 1 mL·min-1,the detection wavelength of 280 nm and the column temperature of 30℃,and the clustering analysis and principal component analysis were performed on the fingerprints.Results1、The ethyl acetate extracted part had the best anti-inflammatory and antioxidant activity,and the n-butanol extracted part had the best hypoglycemic activity.From the results of chemical composition determination of each part,the contents of total triterpenes,total phenols,total tannins and condensed tannins were 3.81%,50.33%,3.32%and 39.79%in the ethyl acetate extracted part,and 0.88%,41.42%,2.25%and 23.55%in the n-butanol extracted part in that order,and by LC-MS/MS analysis,26 compounds,including 11 condensed tannins,4 hydrolysable tannins,6 triterpenoids,3 flavonoids,1 benzoic acid derivative and 1 chlorogenic acid compound,were inferred from the ethyl acetate extraction site;19 compounds,including 4 condensed tannins,5 hydrolyzable tannins,3 triterpenoids,5 flavonoids,1 benzoic acid derivative and 1 chlorogenic acid compound,were inferred from the n-butanol extraction site.2、Nine compounds were isolated from n-butanol extraction site,five compounds of them were identified,including 2 flavonoids,3 hydrolyzable tannins,namely(1)epicatechin,(2)catechin,(3)ellagic acid-4’-O-xyloside,(4)ellagic acid,(5)3-methyl ellagic acid-4’-O-xyloside,5 of them was isolated from Radix rosea for the first time.3、The core targets of anti-inflammatory of the main components of Radix rosea are IL-6,TNF,MAPK3,PTGS2,EGFR,AKT1,VEGFA,SRC,etc.The anti-inflammatory targets mainly involve Pathways in cancer,IL-17 signaling pathway,Proteoglycans in cancer,Endocrine resistance,Serotonergic synapse,EGFR tyrosine kinase inhibitor resistance,bladder cancer,MicroRNAs in cancer,Ovarian steroidogenesis,etc.4、The best extraction process for total triterpenes was determined by the orthogonal design with the solvent of trichloromethane,the material-liquid ratio of 1:40,the reflux extraction time of 60 min,and the reflux temperature of 60℃;the best extraction process for total tannins was the ethanol concentration of 70%,the material-liquid ratio of 1:10,and the ultrasonic extraction time of 30 min.The contents of total triterpenes in Radix rosea were 0.70%～1.60%,and the contents of total tannins were 5.17%～10.92%;the contents of total triterpenes in Caulis rosea were 0.53%-0.90%,and the contents of total tannins were 2.50%-9.58%.5、HPLC was used to establish the fingerprint profiles of 21 batches in Radix et caulis rosea,and 25 common peaks were selected.6 of the common peaks were identified by control comparison,including gallic acid,catechin,epicatechin,ellagic acid-4’-O-xyloside,ellagic acid-4’-O-arabinofuranoside and ellagic acid.21 batches of herbs had some differences,with similarity of 0.408～0.978.Cluster analysis clustered the fingerprints of the 21 batches of Radix et caulis rosea herbs into five classes,with lot number JY6 as class 1,lot number JY5 as class 2,lot numbers JY1 and JY2 as class 3,lot number JY7 as class 4,and the rest of the samples clustered into one class.The principal component analysis showed that JY1 from Xing’an County,Guilin,Guangxi,JY6 from Xupu,Huaihua,Hunan,JY7 from Jishou,Hunan and JY17 from Hunan were in the top 4 of all samples,indicating that the quality of the four batches of Radix rosea were better.Conclusion1、Ethyl acetate extraction site is the main active site of anti-inflammatory and antioxidant of Radix rosea,n-butanol extraction site is the main active site of hypoglycemia,and phenolic components mainly tannins may be the main active components of anti-inflammatory,antioxidant and hypoglycemia of Radix rosea.2、Preliminary revealed the anti-inflammatory targets and action pathways of the main components of Radix rosea,providing the basis for the clinical development and utilization of Radix rosea.3、Preliminary establishment of the quality evaluation method of Radix rosea,laying the foundation for the subsequent development of quality standards.4、The content of triterpenes and tannins in Radix rosea are generally higher than that of Caulis rosea,and there are some differences in the ratio and type of chemical composition of Radix et caulis rosea,whether Caulis rosea can be mixed into Radix rosea for use still needs subsequent in-depth research,so as not to affect the clinical efficacy of Radix rosea. |
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