Comparison Of Changes In Chemical Components Of Sophora Flavescens Before And After Processing And Its Effect On Mice With Damp-heat Ulcerative Coliti

Purpose:Regarding the differences in the use of Sophora flavescens,which modernly uses sophora flavescens,anciently uses processed products of Sophora flavescens.Based on previous research,the Stir-fried sophora flavescens with wheat bran and Processed sophora flavescens with rice swill,which was selected from eight kinds of Sophora flavescens concoctions recorded in ancient texts,were further studied.By investigating the changes of chemical components before and after processing of Sophora flavescens,its effect on damp-heat ulcerative colitis(UC)mice and its mechanism,as well as its effect on the intestinal microflora of damp-heat UC mice,the processing principle of Sophora flavescens was analyzed to provide the basis for the clinical application of Sophora flavescens processed products.The optimized processing technology of Sophora flavescens made from rice swill was optimized,and the quality standards of Sophora flavescens and Processed sophora flavescens with rice swill were established to provide a theoretical basis for the processing technology,quality standard research,and rational clinical application of Processed sophora flavescens with rice swill.Material and method:Material:Sophora flavescens Ait.,a legume plant,was identified by Professor Yin Haibo of Liaoning University of Traditional Chinese Medicine as the dried root of Sophora flavescens Ait;Waters Acquire TM I-class ultra-high performance liquid chromatograph,equipped with Masslynx 4.1 mass spectrometry workstation(Waters,USA);Waters e2695 high performance liquid chromatograph;Multiskan MK3 enzyme marker;Mass spectrum grade methanol,acetonitrile;Milli-Q water purifier,etc;TLR4、My D88、NF-κBp65 antibody was purchased from Cell Signaling Biotechnology Co.,Ltd;C57BL/6 mouse,purchased from Liaoning Changsheng Biotechnology Co.,Ltd.Method:1.Differences in chemical components of before and after processing of Sophora flavescens based on UPLC-Q-TOF-MS~EanalysisUltra high-performance liquid chromatography quadrupole time of Flight-Mass spectrometry(UPLC-Q-TOF-MS~E)was used to scan in positive and negative ion modes,and qualitative analysis of the chemical components of Sophora flavescens,Stir-fried sophora flavescens with wheat bran,and Processed sophora flavescens with rice swill was conducted by combining the total chemical composition database of Sophora flavescens and the built-in chemical composition database of UNIFI information platform as a screening database.At the same time,using SIMCA-P software and multivariate statistical analysis method,through principal component analysis(PCA)and orthogonal partial least-squares discriminant analysis(OPLS-DA),we searched for the markers of chemical composition differences between Sophora flavescens and Stir-fried sophora flavescens with wheat bran,and Sophora flavescens and Processed sophora flavescens with rice swill.2.Effects of Sophora flavescens and its processed products in mice with damp-heat UC,and the regulatory effects of them on TLR4/My D88/NF-κB signaling pathway.SPF-grade C57BL/6 mice were randomly divided into 8 groups:control group,model group,positive group,Sophora flavescens,Stir-fried sophora flavescens with wheat bran,and Processed sophora flavescens with rice swill,wheat bran of the processing materials group,and rice water of the processing materials group,with 6 mice in each group.The damp-heat UC mice model was established by freely drinking 3%dextran sodium sulfate(DSS)distilled water with high sugar and high-fat diet honey water and a continuous high temperature and high humidity environment.At the same time,the model was given intragastric administration for 10 consecutive days,and the general disease activity index(DAI)score was performed.On the 11th day,the mice were killed,the colon was taken and the length was measured,and the macrophage migration inhibitory factor(MIF)and tumor necrosis factor in the serum of mice were detected by enzyme-linked immunosorbent assay-α(TNF-α),Interleukin-6(IL-6),interleukin-1β(IL-1β),Interleukin-8(IL-8),interleukin-10(IL-10)and the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in colon tissue.HE staining was used to observe pathological sections,and PCR and Western Blot were used to detect TLR4,My D88,NF-κBp65 m RNA,and protein expression.3.Effect of Sophora flavescens and its processed products on Intestinal flora in mice with damp-heat UCThe damp-heat UC mice model was established by freely drinking 3%dextran sodium sulfate(DSS)distilled water with high sugar and high-fat diet honey water and a continuous high temperature and high humidity environment.At the same time,the model was given intragastric administration for 10 consecutive days,and after the last intragastric administration,the feces of mice in the control group,model group,positive group,Sophora flavescens group,Stir-fried sophora flavescens with wheat bran group,and Processed sophora flavescens with rice swill group were collected and stored in sterile EP tube,quickly put into liquid nitrogen,and stored at-80℃.Six samples in each group were tested for intestinal microflora diversity,and the effective sequence of 16S r RNA V3-V4 region was analyzed by fluorescence quantitative analysis using the Illumina Mi Seq sequencing platform×468 bp double-ended sequencing.4.Optimization of processing technology of Processed sophora flavescens with rice swillAccording to the processing technology records of Processed sophora flavescens with rice swill in the“Lei Gong Pao Zhi Lun”,the influence factors of steaming time,soaking time,and concentration of rice swill were investigated,and the best processing technology of Processed sophora flavescens with rice swill was optimized by orthogonal design method with the three incremental flavonoid components of Processed sophora flavescens with rice swill,such as Trifolirhizin,Isoxanthohumol,and Sophoraflavanone G are used as the investigation indicators.5.Study on quality standards of Sophora flavescens and Processed sophora flavescens with rice swillBased on the best optimization of the processing technology of Processed sophora flavescens with rice swill,the incremental components of Processed sophora flavescens with rice swill,such as Trifolirhizin and Sophoraflavanone G,are used as the detection indicators for the thin layer identification and content determination of Sophora flavescens and Processed sophora flavescens with rice swill,to check the water and ash content of Sophora flavescens and Processed sophora flavescens with rice swill,and to systematically study the extract,character identification and microscopic identification of Sophora flavescens and Processed sophora flavescens with rice swill.To establish the quality standard of Sophora flavescens and Processed sophora flavescens with rice swill.Results:1.Differences in chemical components of before and after processing of Sophora flavescens based on UPLC-Q-TOF-MS~EanalysisIn the positive ion mode,75 compounds were identified,mainly flavonoids and alkaloids,including 50 flavonoids,16 alkaloids,4 triterpenes,3 diphenylmethyl derivatives,1 glycoside,and 1 anthraquinone;In the negative ion mode,44 compounds were identified,mainly flavonoids,including 41 flavonoids,1 alkaloid,1 diphenylmethyl derivative,and 1 glycoside;The comparison between with Sophora flavescens and Stir-fried sophora flavescens with wheat Bran.Six different components were identified under the positive and negative ion modes,most of which were flavonoids.The comparison between Sophora flavescens and Processed sophora flavescens with rice swill.Under the positive and negative ion modes,9and 1 different components were identified,most of which were flavonoids and triterpenoids.2.Effects of Sophora flavescens and its processed products in mice with damp-heat UC,and the regulatory effects of them on TLR4/My D88/NF-κB signaling pathway.Compared with the control group,the DAI score of UC mice with damp-heat type increased;The length of the colon is reduced;Colon disease is serious,with a large number of lymphocyte infiltration;The content of MIF,TNF-α,IL-6,IL-1β,IL-8 in serum increased and IL-10 decreased(P<0.01);The content of MDA in colon tissue increased and the content of SOD decreased(P<0.01);The expression of TLR4,My D88,NF-κBp65 m RNA and protein in colon tissue increased significantly(P<0.01).Compared with the model group,each administration group can reduce the DAI score of damp-heat UC mice;Increase its colon length(P<0.01);Reduce the severity of colon lesions and lymphocyte infiltration;Reduce the content of MIF,TNF-α,IL-6,IL-1β,IL-8 in serum of damp-heat UC mice,increase the content of IL-10(P<0.01);Reduce the content of MDA in colon tissue and increase the level of SOD(P<0.01);Reduce TLR4,My D88,NF-κBp65 m RNA and protein expression in colon tissue(P<0.01).There was no significant difference between the wheat bran of the processing materials group,and rice water of the processing materials group compared with the model group(P>0.05).Sophora flavescens and its processed products have the effect of treating damp-heat UC,and the effect is better after Stir-fried with bran or processed with rice swill.3.Effect of Sophora flavescens and its processed products on Intestinal flora in mice with damp-heat UCAlpha diversity analysis showed that compared with the control group,the Shannon index and Simpson index of intestinal flora in model mice with damp-heat UC were significantly lower(P<0.01);Beta diversity analysis showed that there were differences in the structure and diversity of intestinal flora between the control group and the model group;At the level of phylum classification,the relative abundance of Firmicutes and Bacteroides in model mice with damp-heat UC were decreased,while the relative abundance of Proteobacteria increased significantly(P<0.01);At the level of genus classification,the relative abundance of Lactobacillus,norank＿f＿＿Muriaculaceae,Helicobacter,Bacteroides,Bifidobacterium,Prevotellaceae＿UCG-001,Paracteroides,Odoribacter,g＿＿Lachnospiraceae＿NK4A136＿Group,Candidatus＿Saccharimonas,Prevotelaceae＿Ga6A1＿group and Dubosella in model mice with damp-heat UC were decreased significantly(P<0.01),while the relative abundance of Enterobacter and Escherichia-Shigella were increased significantly(P<0.01);Compared with the model group,the Shannon and Simpson indexes of each administration group were significantly increased(P<0.01);The structure and diversity of intestinal microflora of mice in each administration group were close to those in the control group;Each administration group could significantly increase the abundance of Bacterobacter and Bacteroides,and decrease the abundance of Proteus(P<0.05,P<0.01);Compared with Sophora flavescens group,the Stir-fried sophora flavescens with wheat bran group had a more significant effect on up-regulating Bacteroides(P<0.05),and Processed sophora flavescens with rice swill group had a more significant effect on up-regulating Bacteroides(P>0.05);The administration group of sophora flavescens,Stir-fried sophora flavescens with wheat bran,and Processed sophora flavescens with rice swill can significantly increase the abundance of Lactobacillus,norank＿f＿＿Muriaculaceae,Helicobacter,Bacteroides,Bifidobacterium,Prevotellaceae＿UCG-001,Paracteroides,Odoribacter,g＿＿Lachnospiraceae＿NK4A136＿Group,Candidatus＿Saccharimonas,Prevotelaceae＿Ga6A1＿group and Dubosella,significantly decreased the abundance of enterobacter,Escherichia-Shigella,and each species had significant differences(P<0.05,P<0.01).A large number of Prevotellaceae＿Ga6A1＿Group,Dubosella,and Bifidobacterium appeared in the Stir-fried sophora flavescens with wheat bran group.Processed sophora flavescens with rice swill group has a large number of Prevotelaceae＿Ga6A1＿group which was significantly different from the model group and the Sophora flavescens group(P<0.05,P<0.01).4.Optimization of processing technology of Processed sophora flavescens with rice swillThe optimum processing technology of Processed sophora flavescens with rice swill was selected by orthogonal test as follows:Take the Sophora flavescens slices,add the rice swill with the concentration of 2%,soak it for 6 hours,and rinse 2 times with water,steam it at atmospheric pressure for 6 hours,and dry it in the sun.(Preparation of rice swill:take Rice noodles with No.4 sieve,mix it with water,stir it to get a suspension,and get it.1000 m L water is used for every 2 g of Rice noodles,and the amount of rice swill is based on the amount of Sophora flavescens slices)5.Study on quality standards of Sophora flavescens and Processed sophora flavescens with rice swillThe tentative quality standard of Sophora flavescens and Processed sophora flavescens with rice swill are as follows:The microscopic identification of sophora flavescens was the same as the 2020 edition of Pharmacopoeia(I),compared with raw bitter ginseng,the microscopic identification of Processed sophora flavescens with rice swill should be free of starch grains;Under 254 nm and 365 nm ultraviolet light,the test samples of the Sophora flavescens and Processed sophora flavescens with rice swill show the same color spots on the corresponding positions of the standard samples of Trifolirhizin and Sophoraflavanone G;Sophora flavescens,Processed sophora flavescens with rice swill’s moisture shall not exceed11.0%;Sophora flavescens,Processed sophora flavescens with rice swill’s total ash shall not exceed 8.0%;Sophora flavescent’s water-soluble leachate shall not be less than 20.0%,Processed sophora flavescens with rice swill’s water-soluble leachate shall not be less than13.7%.The amount of Sophora flavescens containing Trifolirhizin must not be less than0.09%,the amount of Processed sophora flavescens with rice swill containing Trifolirhizin must not be less than 0.14%,the amount of Sophora flavescens containing locust Sophoraflavanone G must not be less than 0.09%,the amount of Processed sophora flavescens with rice swill containing locust Sophoraflavanone G must not be less than 0.14%.Conclusion:1.Flavonoids and triterpenoids were changed after Sophora flavescens were fried and processed in wheat bran and rice swill,which are different components among Sophora flavescens and its products.2.Sophora flavescens and its processed products could inhibit TLR4/My D88/NF-κB pathway,thus exerting the effect of treating damp-heat type UC,and the effect is better after being fried and processed in wheat bran or rice swill.3.Sophora flavescens and its processed products could regulate the intestinal flora structure of damp-heat UC mice,thus achieving the therapeutic effect of alleviating the intestinal inflammatory response,among which the effects of Stir-fried sophora flavescens with wheat bran,and Processed sophora flavescens with rice swill showed are better than Sophora flavescens.4.The best concoction process of Processed sophora flavescens with rice swill,was obtained by an orthogonal test,and the method was stable and feasible.5.The quality standard developed for the determination of the incremental components Trifolirhizin and Sophoraflavanone G in Processed sophora flavescens with rice swill can be used as a reference for the evaluation and quality control of the concoction process of Processed sophora flavescens with rice swill.