Study On Chemical Constituents Of Moringa Oleifera Seeds And Their Effect On TPA-induced Psoriasis In The Mice

Objective:Moringa oleifera L.,native to India,Arabian Peninsula,is a versatile tropical plant of the genus Moringa(Moringa Adans.).Moringa contains polysaccharides,glycosides,flavonoids,phenols,steroids,terpenes,alkaloids,etc.In recent years,Moringa has been widely studied for its hypoglycemic,anti-tumor,and anti-inflammatory effects because it contains phenolic compounds and glycosides that are particularly structurally linked to nitrile groups,isothiocyanate groups,and thiocarbamate groups.Psoriasis is one of the most common skin inflammatory diseases and affects more than2% of the population in Western countries.In recent years,several studies have shown that the IL-23/IL-17 axis plays a central role in the pathogenesis of psoriasis;of which IL-23 is particularly critical,IL-23 inhibitors are used to treat psoriasis.Disease is the focus of research in recent years and is gradually being used.Based on the research above,this project mainly isolates the chemical constituents of Moringa oleifera,and in vitro screening of its chemical constituents,establishes a mouse model of psoriasis-like mouse model induced by phorbol ester(TPA),to study the effect of in vitro anti-inflammatory active substances in Moringa oleifera seeds on TPA-induced animal skin lesions and explore its mechanism.Methods:(1)Separation and purification of compounds by silica gel column chromatography,Sephadex LH-20,and High Performance Liquid Chromatography(HPLC).Analysis and identification were performed by nuclear magnetic resonance techniques and literature comparisons,and the structure of the compound was confirmed by mass spectrometry.(2)The active ingredients were separated from the chemical constituents isolated from Moringa seed.sitosterol-3-o-β-D-glucopyranoside(C3),niazirin(C4)and marumoside A(C5)were used for LPS-induced macrophage THP-1 in vitro inflammation assay.Cell viability of THP-1 and TC50 of each monomeric compound were determined by CCK-8 assay.The expression levels of IL-1β,IL-12,IL-8,IL-23,IL-22,and TNF-α in inflammatory cytokines were measured by quantitative real-time PCR.(3)In order to investigate the in vitro anti-inflammatory activity of the chemical constituents of Moringa oleifera,a psoriasis-like skin inflammatory model induced by phorbol ester(TPA)was established,and an emulsion made of compound C3,C4 and C5 was used for intervention treatment.The morphological changes of the skin lesions of the mice before and after administration were observed,and the skin thickness of the skin tissue sections of each group of animals was measured using Image J software.Compared with the model group,the change in mouse skin thickness after administration was found.By observing the skin tissue sections taken under the 40 X objective of the Leica DMR micrographic analysis system,compared with the model group,the thickness of the epidermis and telangiectasia after administration have affected the inflammatory cell infiltration of the drug.Real-time Quantitative PCR Detecting System(QPCR)was used to detect the expression of IL-17 and IL-23 m RNA in skin lesions of mice.Results:(1)Five kinds of monomeric compounds were isolated from Moringa oleifera,which were stigmasterol(C1),long-chain hydrocarbon(C2),sitosterol-3-o-β-D-glucopyranoside(C3),niazirin(C4)and marumoside A(C5).(2)In vitro anti-inflammatory activity studies showed that sitosterol-3-o-β-D-glucopyranoside(C3),niazirin(C4)and marumoside A(C5)had a certain adjustment ability to relative m RNA levels of cytokines IL-1β,IL-8,IL-12,IL-17,IL-22,IL-23 and TNF-α.The regulation of IL-17 is the most significant(P<0.05),followed by IL-22 and IL-23(P<0.05).Then the effect of three compounds on the IL-23 protein content in culture fluid of THP-1 was tested.The results showed that compounds C3 and C4 can significantly reduce the IL-23 level in the culture fluid.Compound C5 stimulated the production and release of IL-23(P<0.05).(3)The animal experiments showed that in vitro anti-inflammatory active substances in Moringa oleifera seeds improved phorbol ester(TPA)-induced mouse psoriatic lesions;skin tissue sections also proved that sitosterol-3-o-β-D-glucopyranoside(C3),niazirin(C4)and marumoside A(C5)can effectively inhibit TPA induction.Thickening of the acanthosis,skin inflammation,and congestion changes.Quantitative real-time PCR results showed that the expression levels of IL-17 and IL-23 mRNA were significantly decreased in the C3 and C5 treatment groups,which was significantly different from the model group(P<0.05).However,the expression of IL-23 in the C4 treatment group was increased and showed a concentration-dependent manner.Conclusion:The three compounds isolated from Moringa oleifera,sitosterol-3-o-β-Dglucopyranoside(C3),niazirin(C4),and marumoside A(C5)showed anti-inflammatory activity on macrophage THP-1 inflammatory cell model in vitro induced by LPS,and ameliorated phorbol ester(TPA)-induced psoriatic lesions in mice and skin tissue sections.