Study On The Main Chemical Components Of Huarong Large-leaf Mustard And The Anti-colitis Mechanism Of Its Ethanol Extract

In recent years,the research on the phytochemical constituents and functional effects of mustard has attracted great attention from the global scientific community,especially the anti-cancer,anti-inflammatory,and anti-oxidant biological activities of glucosinolates(GLS)in mustard.Huarong large-leaf mustard is the preferred variety in our province,with an annual planting area of over 500,000 mu,but there was no report on its chemical composition and functional effects,which restricted its high-value utilization.In this paper,HPLC-Q-TOF-MS technology was used to study the chemical components of fresh and fermented mustard and its ethanol extract.Using fresh mustard and fermented mustard ethanol extract as raw materials,the effect,mechanism and structur-activity relationship of preventing colon cancer and colitis were discussed.The main results were as follows:(1)Study on the main chemical components of Huarong large-leaf mustard and its ethanol extractPrimary nutrients and glucosinolates of fresh and fermented mustard were analyzed.The results show that Huarong large-leaf mustard consisted of protein,carbohydrates,dietary fiber,vitamins,minerals,glucosinolates,etc.The content of total glucosinolates in different parts of fresh mustard was different,The total glucosinolates content from high to low was ordered as: stem > petiole > stem > leaves,and the stem was the highest(53.58mol/g),while the leaf was the lowest(38.26 mol/g).During the natural fermentation process,the soluble total sugar,dietary fiber,protein,fat,vitamin C and total glucosinolate content of Huarong large-leaf mustard all decreased with fermentation time,and the total acid and amino nitrogen content increased with the fermentation time.The content of total soluble sugar,fat,protein and total glucosinolates in fresh mustard ethanol extract was significantly higher than that of fermented mustard ethanol extract.While the content of amino acid nitrogen,total polyphenols and the number of volatile components in fresh mustard ethanol extract were significantly lower than those in fermented mustard ethanol extract,anf there was no significant difference in the total flavonoid content between the two.GC-MS was used to identify 7 main volatile components in fresh Huarong large-leaf mustard,among which the relative content of allyl isothiocyanate was 92.83%.After fermentation,a large number of volatile components were produced,and a total of 70 compounds in 10 categories were identified in fermented mustard.40 volatile components were identified in the ethanol extract of fresh Huarong large-leaf mustard and 46 volatile components were identified in the ethanol extract of fermented mustard.A total of 45 compounds were identified from fresh and fermented ethanol extracts of Huarong large-leaf mustard by HPLC-Q-TOF-MS technology.Among them,there were 20 glucosinolates,including 2-propenyl GSL,3-butenyl GSL,5-hexenyl GSL,etc.,20 GSLs in fresh mustard ethanol extract,10 GSLs in fermented mustard ethanol extract,the amount of GSLs of fresh mustard ethanol extract was significantly higher than that of fermented mustard ethanol extract;20 flavonoids and their derivatives were found in the two mustard ethanol extracts,including quercetin and its derivatives,kaempferol and its derivatives,etc.,there were 8 of fresh mustard ethanol extract and 12 of fermented mustard ethanol extract.Organic acids were not identified in fresh mustard extrct,and 5 organic acid components were found in fermented mustard ethanol extract.In summary,during the fermentation of fresh mustard,not only the nutritional components have changed,but also the glucosinolates degradated during the fermentation process to produce esters,sulfur-containing,and nitrile compounds,as well as the flavonoids.For example,the isorhamnetin-3-O-diglucoside in the fresh mustard ethanol extract lost two glucose molecules to produce methylquercetin in the fermented mustard ethanol extract.The above indicated that the fermentation process of mustard was a process of continuously degrading macromolecular compounds and producing degradation products such as flavor,taste and biological active components.(2)The inhibitory effect and mechanism of Huarong large-leaf mustard ethanol extract on HCT116 human colon cancer cellsThe effects and mechanisms of fresh and fermented ethanol extracts of Huarong large-leaf mustard on CCD-18 Co and HCT116 were studied by MTT assay,flow cytometry,western blot and real-time quantitative polymerase chain reaction(q RT-PCR).The results showed that fresh mustard and fermented mustard ethanol extracts had no inhibitory effect on normal human colon myofibroblast CCD-18 Co cells,but both induced cell cycle arrest and led to extensive cellular apoptosis in human colon cancer cells.The corresponding mechanisms were downregulation of the cell cycle-related genes and signaling protein,e.g.cyclin B,cyclin D1,and cyclin E,and the upregulation the apoptosis-related genes and signaling protein(caspase-3 and cleaved caspase-3).Interesting,the fermented mustard ethanol extract displayed stronger inhibitory effect than fresh mustard extract on HCT116 cells.(3)Study on the regulation and mechanism of Huarong large-leaf mustard ethanol extract on RAW264.7 cell inflammationMTT,Western blot and real-time quantitative polymerase chain reaction(q RT-PCR)were used to study the regulation and mechanism of fresh and fermented Huarong large-leaf mustard on LPS-induced inflammation in mouse macrophages RAW 264.7.The results showed that the ethanol extracts of fresh mustard and fermented mustard have no toxicity to RAW264.7 cells,but both ethanol extracts can effectively inhibit the inflammatory RAW264.7 inflammatory cells induced by LPS.Compared with the model group cells,after the intervention of the two ethanol extracts,the levels of NO,cytokines TNF-?,IL-1?,IL-6and MDA in the inflammatory cells were significantly reduced,and the activities of SOD and GSH-PX in the cells were significantly increased.The expression levels of TLR4 and its downstream NF-?B p65,COX-2,i NOS m RNA and protein in the cell lysate have all been restored.Among them,The expression levels of NF-?B P65 and COX-2 in the fresh mustard ethanol extract group were returned to the level in the control group,indicating that the fresh mustard ethanol extract had a better inhibitory effect on the inflammatory mouse macrophages RAW 264.7.(4)Study on the activity and mechanism of Huarong large-leaf mustard ethanol extract in interfering with colitis in miceUlcerative colitis model C57BL/6 mice were induced by DSS,The activities and mechanism of Huarong large-leaf mustard ethanol extract in intervention of mouse colitis were studied from the aspects of mouse signs,histopathology,metabolomics and intestinal microbial diversity.The results showed that both mustard ethanol extracts can improve the enteritis symptoms of model mice from the aspects of mouse physical signs,disease activity index and colonic histopathology,meanwhile restored MPO,SOD activity,NO,inflammatory factors(TNF-?,IFN-?,IL-6,IL-1?,IL-17,IL-10,IL-4 and IL-13)and short-chain fatty acids(acetic acid,propionic acid,butyric acid,valeric acid,Isovaleric acid,caproic acid and isobutyric acid)content in the colon tissue of inflamed mice.In addition,Illumina Miseq platform analysis showed that both fresh and fermented mustard ethanol extracts can regulate the intestinal flora imbalance.After the inflamed mice are intervened by the mustard ethanol extract,the structure of the intestinal flora in the mice of the model group returned to the intestinal flora structure of the control group.The abundance of some controversial flora decreased,especially Prevotellaceae,Bacteroides,Akkermansia,etc.,and the abundance of some beneficial microorganisms increased,especially Lactobacillus,Bifidobacteria,Blautia,etc.The two ethanol extracts showed great differences in the regulation of some bacteria such as norank?f?Muribaculaceae,norank?f?Lachnospiraceae,norank?f?Muribaculaceae,Bifidobacterium,Blautia,Prevotellaceae?UCG?001,Akkermansia,Ruminiclostridium?5.UPLC-Q-TOF/MS method was used to analysis the fecal metabolites in mice.The results showed that a total of 40 potential metabolites with significant differences were found in the fecal samples of the control and model mice.Except for deoxyadenosine in the fermented mustard ethanol extract group,other metabolites were adjusted to the control group mice's fecal metabolite level under the regulation of the two mustard ethanol extracts.Through KEGG topology analysis,five pathways with greater impact were finally obtained,which were lysine biosynthesis,steroid hormone biosynthesis,glycerophospholipid metabolism,purine metabolism,and lactam biosynthesis.Finally,m RNA and protein levels were tested by q RT-PCR and Western blot respectively,the results showed that the two mustard ethanol extracts blocked the TLR4-NF-?Bp65-COX-2 / i NOS pathway,thereby reducing the colitis in mice Inflammation,which is consistent with the regulation mechanism of LPS-induced RAW264.7 cell inflammation in this article.The results of the R language correlation analysis showed that the intestinal flora and metabolites,growth factors and enzyme activities interact with each other.For example,Bifidobacteria significantly promote isobutyric acid,isovaleric acid and deoxyadenosine with anti-inflammatory effects.Lactobacillus was significantly positively correlated with anti-inflammatory factors IL-13,SOD,and dodecanedioic acid,Blautia was significantly positively correlated with anti-inflammatory propionic acid,and the anti-inflammatory metabolite ?-tocotrienol significantly positively correlated with valeric acid,hexanoic acid and propionic acid,Meanwhile,the blockade of TLR4-NF-?Bp65-COX-2 / i NOS pathway reduced the release of pro-inflammatory factors and NO,thereby reducing inflammation symptoms.On the whole,fresh mustard ethanol extract had a better anti-inflammatory effect on colitis than fermented mustard ethanol extract,which may be related to the different regulation of intestinal flora and metabolites by their different bioactive components.In summary,the results of this article revealed the anti-inflammatory mechanism of fresh and fermented mustard ethanol extracts on inflammatory mice,which is related to the regulation of the intestinal flora,metabolites and TLR4-NF-?Bp65-COX-2/i NOS inflammatory pathways in mice,this is essential for the rational use of mustard to prevent diseases caused by inflammation.