| This paper was composed of two parts.The first part was about the study on the quality analysis of Zhenyuan capsule,and the second part was about the synthesis of Beesioside I derivatives and the effect research.(Part 1)Due to the unique pharmacological activity and extensive clinical application,ginsenosides received much contention.The root and rhizome of ginseng is a traditional medicinal part containing ginsenosides,while the mature fruit of ginseng has become a research hotspot in recent years because of its higher content of ginsenosides.Zhenyuan capsule using ginseng fruit total saponins as raw materials has a good clinical efficacy in the treatment of coronary heart disease,angina pectoris and arrhythmia.However,total saponin content was determined in the standard of Zhenyuan capsule issued by ministry(WS3-B-2765-97-1)using the combination of TLC and UV-Visible spectrophotometry.And ginsenoside Re was determined as the index component to control the content.Because of the big error and low accuracy,and the control of single component cannot comprehensive response its internal quality,this method is still not perfect.In view of the wide application of Zhenyuan capsule in cardiovascular diseases,an analytical method was established to determine the contents and types of ginsenosides in raw materials,extracts and preparations,as well as the similarity degree between different batches,combined with thin-layer identification and determination to evaluate its quality,in the hope of providing the reference for Zhenyuan capsule’s quality control.1.Study on the quality standard(1)TLC identificationThe thin-layer identification method was established according to the methods of ginseng medicinal materials,extracts and preparations in Chinese Pharmacopoeia(2020 edition).The established method was stable and reliable through the investigation of different temperature,humidity and thin-layer plates from different manufacturers.The results of 40 batches of ginseng fruits,30 batches of ginseng fruit saponins and 30 batches of Zhenyuan capsule showed that the method had good reproducibility and simple operation.(2)DeterminationThe determinations referred to relevant standards and general requirements of Chinese Pharmacopoeia(2020 Edition)were carried out.According to the results of measurement,it is recommended that the determination parameter of corresp in ginseng fruit:total ash is not more than 15.0%;heavy metals and harmful elements are not more than 5 mg/Kg of lead,0.5 mg/Kg of cadmium,0.5 mg/Kg of arsenic,0.3 mg/Kg of mercury,20 mg/Kg of copper;pesticide residues are not more than 0.2 mg/Kg of hexachlorocyclohexane,0.2 mg/Kg of chlorophenothane,and 0.1 mg/Kg of pentachloronitrobenzene;ethanol-soluble extractives are not less than 40.0 per cent.(3)AssayThe contents of 7 kinds of ginsenosides(Rgi,Re,F3,Re,Rb2,Rd and F2)in ginseng fruits,ginseng fruit saponins and Zhenyuan capsules were determined simultaneously by using HPLC-DAD.The total contents of 7 kinds of ginsenosides in ginseng fruits,ginseng fruit saponins and Zhenyuan capsules were in the ranges of 8.96-13.51%,50.84-54.50%and 6.38-7.36%,respectively.The developed assay method was validated in terms of linear range and coefficients of determination(R2 0.995-1.000),intra-day precision and inter-day precision(RSD 0.44-1.24%,2.06-4.31%),repeatability(RSD 0.89-2.00%),stability(RSD 0.57-1.82%)and accuracy(recovery 95.38-102.09%).And it can be used to evaluate the quality of Zhenyuan capsule.(4)FingerprintHPLC fingerprints of raw materials,extracts and preparations were established under the same chromatographic system through the similarity evaluation system of chromatographic fingerprints of traditional Chinese medicine.Fingerprints of 40 batches of ginseng fruits,30 batches of ginseng fruit total saponins and 30 batches of Zhenyuan capsule were established,and 11 common peaks were identified.The similarity ranges with the control fingerprint were 0.905-1.000,0.998-1.000 and 0.998-1.000,respectively,which could reflect the advantages of consistency and the typical characteristics of chemical composition.2.Qualitative analysisUHPLC-Q-Orbitrap-MS/MS was used to analyze the types of ginsenosides in ginseng fruit,ginseng fruit total saponin and Zhenyuan capsule.A total of 108 ginsenosides in the samples were identified and can be found that types of ginsenosides in ginseng fruit sapoinin and Zhenyuan capsule were almost the same as those in raw materials,which could provide reference for the effective control of Zhenyuan capsule.(Part 2)The acquired immune deficiency syndrome(AIDS)is one of the most dangerous infectious diseases which is caused by Human immunodeficiency virus(HIV).The HAART drugs have great side effects and are easy to produce drug resistance.Looking for new target and mechanism as well as drugs with high efficiency and low toxicity without drug resistance is a hotspot of current research on development of anti-AIDS drugs.Cycloartane triterpenoids have extensive biological activities and Beesioside I with antiHIV activity was isolated from Actaea vaginata in previous studies.This part draws on the structural modification of betulinic acid,an anti-HIV agent with similar structure,then a series of derivatives were synthesized with Beesioside I as the lead compound,in hope of finding anti-HIV compounds with new targets and mechanism as well as high efficiency and low toxicity,which laid a foundation for the research of new anti-HIV drugs.Eleven derivatives including seven novel compounds were synthesized by structural modification of C-3 hydroxyl group and C-15,16 acetyl group of Beesioside I through hydrolysis and acylation,and the structures were confirmed by MS and NMR analysis.The anti-HIV activity of these compounds was determined,and the structure-activity relationship analysis suggested that appropriate acylation modification of C-3 could significantly improve its activity.The anti-HIV activity of compound 5a obtained by acylation reaction of aglycone was significantly improved,with EC50 value of 0.025 μM and TI of more than 800,which was worthy of further study. |
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