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Studies On The Selection Of Antirheumatic Active Components And The Preparation Of Microemulsion Of Securidaca In Appendiculata

Posted on:2021-03-15Degree:MasterType:Thesis
Country:ChinaCandidate:Y FanFull Text:PDF
GTID:2404330602967512Subject:Pharmacy
Abstract/Summary:PDF Full Text Request
Background:Securidaca inappendiculata Hassk(SI)is traditionally used to treat inflamm-ation and rheumatoid arthritis.In recent years,the research on the anti-rheum-atic effect of SI has obtained some results.The anti-inflammatory and anti-rhe-umatic effects of SI have been fully confirmed.In previous research,we also found that SIAQ has a more significant effect than SIW.SID is a potential anti-rheumatic activity by studying the different polar extraction positions of th-e alcohol extract,and it is confirmed by AA model.Through the separation of SID,various potential active ingredients represented by xanthone(XAN)were obtained.In vivo experiments have also shown that XAN has an effective the-rapeutic effect on AA.XAN is a polyphenol with a highly symmetrical struct-ure.It is not only high in species but also abundant in species.Structural ana-lysis shows that only methoxy and hydroxyl groups are substituted,which sug-gests that it is very important for scientific research.In addition to XAN,th-ere are aromatic derivatives such as phenylpropionic acid and benzophenone in SI.They have a close biological relationship with XAN and common structural characteristics ofα,β-unsaturated carbonyl groups.It is speculated that they m-ay have similar physiological activities as XAN and have a certain contribution to the therapeutic effect of SI.This study was conducted to select the act-ive ingredients of rheumatoid arthritis and further clarify the basis of its efficacy.Objective:We want to establish a polyphenol-rich component(PRF)enrichment and purification process.A model of acute inflammation induced by lipopolysaccha-ride(LPS)was used to demonstrate that PRF has better anti-inflammatory acti-vity than PDF.During this experiment,the evaluation of the difference betwee-n the anti-inflammatory effects of PRF and PDF was completed,and PRF was initially explored as a potential anti-inflammatory mechanism of anti-rheumati-c active site On this basis,we further evaluated the therapeutic effect of PRF on collagen-induced arthritis(CIA)animals,and confirmed that components are the material basis of anti-rheumatic.Finally,the PRF-SME was constructed,and by comparing therapeutic effects of the solution in rats,it was clear that it has a better therapeutic effect than the traditional administration method.Methods:1.Established the enrichment and purification process of polyphenols in SI by using macroporous resin and chemical method.Using 1,7-dihydroxy-3,4-di methoxyxanthone(S-XAN)and ferulic acid as controls,the content of polyphe-nols in each component was analyzed by HPLC-UV combined with FCR,the-n we evaluate the transfer efficiency and knockout effect.The characteristic si-gnals of aromatic protons were analyzed by 1H-NMR,and the relative intensiti-es were used to assess the distribution of polyphenols in the mixtures.2.PRF and PDF were evaluated for their therapeutic effects on lipopoly-s accharide induced acute lung injury(ALI)in rats.The treatment effect of ALI was assessed based on observation for typical pathological changes such as al-veolar edema or alveolar walls thickening,alveolar hemorrhage,neutrophil,CB-C whole blood analysis results,MDA and TNF-αlevels,etc.The research of PRF anti-inflammatory mechanism is mainly realized by analyzing the expressi-on levels of ROS,NAMPT,Sirt1,HMGB1,TLR4 and p-p65.The intraceed cells manually by the aid of Prussian blue staining.Cytotoxicity of PRF was evaluated based on the percentage of dead cells under treatments.The correlat-ion between possible signal pathways and inflammatory effects was analyzed based on the measurement results.3.The construction of PRF-SME drug-loading system is mainly completed by drawing a pseudo-ternary phase diagram combined with orthogonal experi-ments,and characterizing and identifying by measuring the encapsulation ratio,p-article size distribution,and electron scanning microscope technology.4.Immunization Grade Bovine Type II Collagen was used to establish a CIA model,which was treated with PRF solution and PRF-SME,respectively.T he therapeutic effects of PRF on experimental arthritis rats and the different tre atment effects of the two dosing regimens were studied.The main evaluation c ontents include:inflammation of affiliated tissues,degree of swelling,degree-of articular cartilage erosion,degree of hyperplasia and hypertrophy of synoviu-m,serum interleukin 1β(IL-1β),tumor necrosis factor-α(TNF-α),Matrixmetallopro teinase 9(MMP9)concentration level,TLR4,My D88,p65,JNK andother ene s in synovial membrane.Results:1.It was finally determined that HPD300 macroporous resin was selected to enrich total phenolic compounds in SI.The sample rate was 1BV·h-1,the eluent was 4BV 75%ethanol,the elution rate was 2BV·h-1,and the volume of water for elution was 3BV.FCR assay suggested total polyphenols contents in PDF and PRF were 1.53%and 12.69%,respectively.To confirm it,we as sessed polyphenols distributions with a 1H-NMR based strategy.It was found that aromatic protons contributed 0.44 and 5.51%to the total signal intensity in PDF and PRF respectively,which was consistent to results from FCR assay.HPLC analyses further confirmed chemical composition difference between PRF and PDF.The chromatograms suggested hydrophilic parts of both PRF and PDF shared the similar composition profiles,while most lipophilic compounds with intense optical absorption at 264nm were absent in PDF.2.PRF and PDF were evaluated for their therapeutic effects on lipopolys-accharide induced acute lung injury(ALI)in rats.PRF efficiently reduced mal-ondialdehyde level,suppressed NAMPT/Sirt1 expression and alleviated the sev-erity of ALI.Furthermore,PRF significantly down regulated the expressions of ROS,NAMPT,Sirt1,HMGB1,TLR4 and p-p65 in LPS treated RAW264.7cells.These effects were greatly abrogated by nicotinamide mononucleotide sti-mulus elicited up-regulation of NAMPT and overshadowed by N-acetyl-L-cyste-ine co-treatments.The results suggested that inhibition of PRF on TLR4/NF-κB pathway was mediated by NAMPT signaling,which fluctuated in accordance to intracellular oxidative stress levels.3.PRF-SME formulation is IPM-El-35-isopropanol.It was determined that the oil content in the prescription was 15%,the km value was 2,and the drug concentration was 2.5%.PRF-SME was observed under an electron microscope and was spherical or nearly spherical particles with a particle diam eter of about 20nm.4.Both the PRF solution and the PRF-SME have exact therapeutic effects on experimental arthritis animals.It has been confirmed that PRF is an impo-rtant anti-inflammatory and anti-rheumatic active ingredient in SI,which shows the relief of the swelling degree of auxiliary tissues,synovial hyperplasia sy-mptoms were effectively controlled,the levels of serum related pro-inflammato-ry factors were reduced,abnormal indicators in synovial tissue were restored,and the effect of PRF-SME was better than PRF solution.Conclusion:PRF in SI has an exact therapeutic effect on CIA model rats,and it is an important material basis for RA treatment of this ethnic medicine,and the the rapeutic effect is improved by constructing PRF-SME.During the experiment,when evaluating the anti-inflammatory effect of PRF on ALI rats,this study revealed a unique anti-inflammatory mechanism of polyphenols from SI.Inapp-endiculata through regulation of NAMPT mediated TLR4/NF-κB activation.Th-rough down-regulation of NAMPT,inhibition of TLR4/NF-κB pathway activati-on and anti-inflammatory effect.
Keywords/Search Tags:Securidaca inappendiculata, polyphenols rich fraction, rheumatoid arthritis, self-microemulsifying, anti-inflammatory mechanism
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