Development And Application Of Active Component In Nostoc Commune On Anti-ulcerative Colitis

Objective:Nostoc commune is a kind of nitrogen-fixing cyanobacteria.Several studies have shown that cyanobacteria can prevent and treat intestinal inflammation.The active ingredients of Nostoc commune such as polysaccharides and water stress proteins（WSP）have also proven useful in treating colon cancer.These results greatly imply that the Nostoc commune may relieve ulcerative colitis（UC）,however,the main active ingredients and their mechanism of action in Nostoc commune against UC are still unclear.Therefore,this study intends to investigate the therapeutic effects of the Nostoc commune on UC from both in vivo and in vitro,further identify the main active components of Nostoc commune against UC,and elucidate its possible molecular mechanism,to lay the foundation for the development of its main active ingredients into a new drug.Methods:The Nostoc commune were collected,and the filtrate was obtained by reflux extraction with 95%ethanol,which was concentrated to obtain the Nostoc commune alcohol exact（AENC）.The extract was purified on silica gel column by stages,and then gradient eluted with petroleum ether-ethyl acetate.The ethyl acetate and methanol were discharged from the column,and the TLC point plate was merged into A-I sections.Semi-preparative high-performance liquid chromatography（HPLC）was used for purification,and the A,B,C,D,E,F,G,H,I fractions of the Nostoc commune extract were obtained.The activity test found that the active ingredient was mainly in the ethyl acetate unloading column.HPLC analysis showed that there was a main peak in the large polar part of the segment.The position of the peak（G,H and ethyl acetate unloading section I）was combined and purified by a macroporous resin column,and eluted with methanol.The target compounds were combined and purified using semi-preparative HPLC（25%methanol+0.1%formic acid）to obtain monomer components,and HPLC analysis was used to verify the purity.The structure was identified by carbon nuclear magnetic resonance spectrum(¹³C-NMR)and hydrogen spectrum（¹H-NMR）.In order to determine the anti-UC active ingredients in the Nostoc commune,animal experiments and cell experiments were used to further evaluate its efficacy and explore its mechanism.In vivo,C57BL/6 mice induced experimental colitis by freely drinking 2.5%dextran sodium sulfate（DSS）for 7 days,using disease activity index（DAI）score,myeloperoxidase（MPO）activity measurement,Hematoxylin and eosin staining（H&E）,real-time fluorescent quantitative PCR（q PCR）,enzyme-linked immunosorbent assay（ELISA）and other methods were used to investigate 20 mg/kg and 100 mg/kg alcohol extract of Nostoc commune（AENC）,50 mg/kg of the various fractions of AENC（B,C,D,E,F,G,H,I）,20mg/kg of the I fraction（AI former/later paragraph）,25 mg/kg of p-hydroxybenzene and 200 mg/kg positive control（mesalazine）on mice with colitis.In vitro,the MTT method is used to detect the effects of different concentrations of HD and experimental concentrations of lipopolysaccharide（LPS）on the viability of HT-29 cells;the selected HD concentrations（1,3,and 10μM）are combined with 1μg/mL LPS HT-29 cells,and a positive control group is preset;tumor necrosis factorα（TNF-α）,inflammatory factor 6（IL-6）,inflammatory factor 1-beta（IL-1β）and E-cadherin are detected by Q-PCR and ELISA methods to preliminarily explore the molecular mechanism of HD,the main active ingredient in the Nostoc commune,against UC.Results:（1）AENC（100 mg/kg）is better than AENC（20 mg/kg）in improving the symptoms of DSS-induced colitis in mice,and is equivalent to mesalazine（200 mg/kg）.The specific performance is relief Weight loss,lower DAI score,improve colon shortening,down-regulate the expression of inflammatory factors and reduce MPO activity.（2）Fraction B,C,D,E,F,G,H（50 mg/kg）had only a weak improvement effect,while Fraction I（50 mg/kg）had a more significant anti-UC effect.（3）The AI former paragraph（20 mg/kg）is better than the AI later paragraph（20 mg/kg）in improving DSS-induced colitis in mice.（4）AENC I fraction was structurally identified as p-hydroxybenzaldehyde（HD）,HD is the main active ingredient against UC in the Nostoc commune.（5）HD（25 mg/kg）significantly inhibited the symptoms of DSS-induced colitis in mice,and its anti-UC effect was superior to that mesalazine（200 mg/kg）.（6）HD（10μM）could significantly inhibit the inflammatory response of HT-29 cells induced by 1μg/mL LPS.（7）At the animal level,HD（25 mg/kg）significantly up-regulated the expression of E-cadherin mRNA in the colon tissue of DSS-induced colitis mice;at the cellular level,HD（10μM）significantly up-regulated LPS-stimulated expression of E-cadherin mRNA in HT-29 cells.Conclusions:This study provides new insights into the main active ingredients of the anti-ulcerative colitis in the Nostoc commune.In addition to the polysaccharides and water stress protein（WSP）reported in the previous study,the newly discovered active ingredients HD in the Nostoc commune could repair the intestinal mucosal damage via down-regulating the expression of pro-inflammatory cytokines and up-regulating the expression of E-cadherin,so as to play the role of resistance to UC.