【作者】 王辉；

【导师】 聂少平； 肖建波；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2022， 博士

【摘要】 膳食纤维和黄酮是食物来源的重要特殊营养素,对肠道炎症、肥胖等均有很好的改善作用。蔬菜是摄入膳食纤维和黄酮的主要途径,其健康效应已有较多报道。然而,鲜有文献报道膳食纤维和黄酮提取后同时摄入在机体生理层面的相互作用及对机体健康的影响。因此,本论文筛选两种富含黄酮类化合物的蔬菜(羽衣甘蓝和西芹),并以其为来源的膳食纤维与黄酮为研究对象,采用葡聚糖硫酸钠(Dextran sodium sulfate,DSS)诱导的小鼠结肠炎模型,考察膳食纤维和黄酮联合使用对肠道健康的影响,并进一步探讨了西芹可溶性膳食纤维影响西芹黄酮类化合物缓解结肠炎能力的潜在机制,为膳食纤维和黄酮在体内的相互作用研究提供参考和理论基础。主要研究内容如下:1、根据膳食纤维和黄酮含量,筛选出两种富含膳食纤维和黄酮的蔬菜(西芹和羽衣甘蓝)作为研究对象。采用DSS建立小鼠结肠炎模型,研究蔬菜对DSS诱导的结肠炎模型的缓解作用,并分别采用树脂法和酶法对蔬菜中的黄酮和膳食纤维进行分离纯化。结果发现:两种蔬菜对结肠炎的缓解作用有所不同。羽衣甘蓝可显著缓解小鼠的体重降低,体重变化均从90%增加至约99%,而西芹仅为95%。羽衣甘蓝的DAI指数抑制作用最优,可从最高2.2分显著降低至约0.8分,而西芹组为1.2分。两种蔬菜均可显著缓解结肠的病理学损伤。此外,NKA-9树脂可用于羽衣甘蓝黄酮类化合物(Kale flavonoids,KF)的纯化,其中黄酮类化合物主要为槲皮素和山奈酚的衍生物。KF中的黄酮含量为31.16±1.26%。羽衣甘蓝可溶性膳食纤维(Kale soluble dietary fiber,KSDF)的单糖组成主要为中性糖,且主要由Gal、Ara和Man组成。西芹黄酮类化合物(Celery flavonoids,CF)中的黄酮含量为88%,包括67.85%的Apiin、9.76%的Chrysoeriol7-O-apiosylglucoside和10.22%的Luteolin 7-O-apiosylglucoside。西芹可溶性膳食纤维(Celery soluble dietary fiber,CSDF)主要是以Gal A和Gal为主的果胶类膳食纤维,西芹不可溶性膳食纤维(Celery insoluble dietary fiber,CIDF)与羽衣甘蓝不可溶性膳食纤维(Kale insoluble dietary fiber,KIDF)类似,可能为以纤维素为骨架同时连接有少量果胶的不可溶性膳食纤维。结果表明,西芹和羽衣甘蓝均对结肠炎小鼠有改善作用,并从该两种蔬菜中分离纯化获得高纯度的膳食纤维和黄酮,可用于接下来的研究。2、为进一步探究膳食纤维与黄酮联合使用对结肠炎小鼠的影响,首先选用前期结肠炎缓解作用较优的羽衣甘蓝作为研究对象,研究其来源的膳食纤维与黄酮联合使用对结肠炎的影响。采用DSS建立小鼠结肠炎模型,研究羽衣甘蓝可溶性膳食纤维(KSDF)和羽衣甘蓝不可溶性膳食纤维(KIDF)分别与羽衣甘蓝黄酮类化合物(KF)联合使用对结肠炎的影响。结果发现:在DSS诱导的结肠炎小鼠实验中,KSDF、KIDF和KF单独给药后,三者均可不同程度的缓解体重降低(从87%增至93%、95%和91%)和结肠缩短(从5.4 cm增至6.2 cm、5.8 cm和5.8cm)、降低DAI指数(从3分降至1.7、1.5和2分)、促进黏蛋白分泌(从结肠面积的2.5%增至6%以上)。此外,单独给与KIDF和KF还可显著抑制结肠炎小鼠结肠TNF-α、IL-6、IL-1β和IL-33的m RNA表达,且均可降低至模型组的70%以下。但KSDF仅对TNF-α和IL-33的m RNA表达有显著抑制作用,而对IL-6和IL-1β无显著影响。在KSDF和KF联合使用后,组内的小鼠出现20%死亡,且原本单独给与KSDF和KF缓解结肠炎各项指标的作用均显著减弱,如DAI指数升至2.5分、结肠长度仅为5.5 cm、对TNF-α、IL-6、IL-1β和IL-33的m RNA表达均无显著抑制作用。而KIDF和KF联合使用后缓解结肠炎的作用未有显著改变。结肠炎小鼠的肠道菌群组成发生紊乱,单独给与KSDF、KIDF和KF均可显著降低有害菌Sutterella的相对丰度(从5%降低至3%以下),而KSDF和KF联合使用组Sutterella的相对丰度高达6%,显著高于KSDF组和KF组。Parabacteroides的结果与之相似,单独给与KSDF可将其丰度从模型组的7%显著降低为4%,而联合使用KSDF和KF后该菌丰度显著增加为12%,显著高于模型组、KSDF组和KF组。此外,尽管模型组的有益菌Akkermansia丰度在模型组中从低于1%显著增加为10%,但KF显著促进其丰度高达22%,远高于正常组和模型组。KSDF组的Akkermansia丰度为18%,与模型组无显著差异,但当KSDF与KF联合使用后,Akkermansia被显著抑制至约6%。结果表明,KSDF与KF联合使用后对结肠炎的缓解作用出现了明显的拮抗现象。3、为进一步探究膳食纤维与黄酮在缓解结肠炎中的拮抗是否存在于其他蔬菜,研究了西芹膳食纤维与黄酮类化合物联合使用对结肠炎的影响。采用DSS建立小鼠结肠炎模型,研究西芹可溶性膳食纤维(CSDF)和西芹不可溶性膳食纤维(CIDF)分别与西芹黄酮类化合物(CF)联合使用对结肠炎的影响。结果发现:在DSS诱导的结肠炎小鼠实验中,CSDF和CF单独给药后,CSDF和CF可显著缓解结肠炎小鼠的体重降低(从约88%增至约94%和93%)、降低便血和便稀症状(DAI指数从3.5分降至1.9分和2分)、缓解结肠缩短(从5 cm增至5.8 cm和5.7 cm)。此外,CSDF和CF还可显著缓解结肠组织损伤(组织损伤评分从9分降低为4.8分和4.7分)、增加黏液表达能力(从模型组的2.2%增加为5%和6%),并降低结肠MPO(从18μU/mg降至14μU/mg和13μU/mg)、i NOS(从0.9μU/mg降至0.75μU/mg和0.6μU/mg)、IL-6(从200 pg/mg降至180 pg/mg和130 pg/mg)和IL-1β(从250 pg/mg降至200 pg/mg和160 pg/mg)的水平。CF还对小鼠肠道通透性具有显著降低作用,血清FITC-dextran水平仅为模型组的1/4。而CSDF和CF联合使用后,对小鼠上述各项指标的改善作用均弱于单独给与CF的作用,如体重变化显著低于CSDF和CF组(为89%)、DAI指数显著高于CSDF组(约2.5分)、组织损伤评分为6.5分、黏蛋白面积为3.8%、血清FITC-dextran水平与模型组相当。另外,结肠炎小鼠肠道菌群多样性降低,Shannon指数从正常的5.5降为3.9,且有害菌Escherichia Shigella、Clostridium sensu stricto 1和Paraclostridium的相对丰度显著增加。单独给与CSDF或CF后,对Escherichia Shigella、Clostridium sensu stricto 1和Paraclostridium具有显著抑制作用,但当CSDF和CF联合使用后,对有害菌的抑制作用显著削弱甚至高于模型组。此外,与KF类似的,CF也可通过大量促进Akkermansia的丰度改善结肠炎,从3.2%增至20%。但与CSDF的联合使用抑制了CF对Akkermansia的富集作用,丰度仅为3.3%。结果表明,可溶性膳食纤维与黄酮联合使用对结肠炎的拮抗作用同样发生在西芹中,因此该拮抗作用可能同时存在于其他富含膳食纤维与黄酮的食物中。4、CF中黄酮含量较高、成分简单,因此本论文选择CSDF与CF作为研究对象,并对二者的拮抗作用机理进行进一步的探究。采用体外LPS诱导RAW264.7的细胞炎症模型、体内代谢和体外发酵模型以及筛菌技术,研究CSDF干扰CF结肠炎缓解作用可能的影响机制。结果发现:CF和作为CF主要成分的Apiin对RAW264.7细胞TNF-α表达无显著抑制作用,Apigenin可显著抑制LPS诱导RAW264.7细胞产生的TNF-α,糖苷的存在会抑制Apigenin的抗炎活性。体内代谢实验发现,CSDF组小鼠尿液中Apigenin水平显著低于正常组,仅为正常组的1/5。抗生素处理后小鼠的粪便中可回收60%的Apiin,而尿液中几乎不含有Apigenin。体外发酵结果发现,1.6 mg/m L CSDF即可促进Apiin和Apigenin在24h内完全脱糖苷和降解,且CSDF浓度越高促进作用越强。Apiin及其代谢产物对Akkermansia muciniphila(AKK)菌无直接促增殖作用,而Apigenin可抑制AKK以外的一些菌生长的生长,为AKK菌增殖提供有利生长环境。CSDF与CF在结肠炎小鼠中的拮抗作用的机制可能是CSDF通过促进CF的降解,尤其是Apiin和Apigenin的降解,从而抑制了黄酮体内抗炎活性和对AKK菌的促进。此外,本研究从人和小鼠粪便中筛选到四株具有芹菜糖苷酶外切酶活性的菌株(人源:Parasutterella excrementihominis和Collinsella aerofaciens;鼠源:Muribaculum sp.和Robinsoniella sp.)。以上结果表明,羽衣甘蓝和西芹中可溶性膳食纤维和黄酮的同时摄入对结肠炎的缓解作用会弱于两种营养素单独摄入时的作用,而可溶性膳食纤维可能是通过加速了黄酮的降解,从而减弱了西芹黄酮类化合物的肠道保护作用。更多还原

【Abstract】 Dietary fiber and flavonoids are important functional nutrients from food sources and have shown promising alleviating effects on intestinal inflammation,obesity,and other diseases.Vegetables are the main way for the intake of dietary fiber and flavonoids,that their health effects are significant.However,few studies were reported on the effect of the interplay between the simultaneous intake of dietary fiber and flavonoids at the physiological level on organismal health.Therefore,this paper screened two vegetables(kale and celery)with the highest flavone content,and took dietary fiber and flavone among them as the research object,using dextran sodium sulfate(DSS)induced colitis model in mice to investigate the effects of dietary fiber and flavone on intestinal health,Furthermore,the potential mechanism that soluble dietary fiber of celery could weaken the ability of celery flavonoids to alleviate colitis was discussed from the perspective of flavonoids degradation.It provides data and theoretical basis for the study of the interaction between dietary fiber and flavonoids in vivo.The main research contents are summarized as follows:1.According to the content of dietary fiber and flavonoids,two vegetables(celery and kale)rich in dietary fiber and flavonoids were selected.The mice colitis model was established by DSS,and the effects of two vegetables on DSS induced colitis model were studied,and flavonoids and dietary fibers from the two vegetables were isolated and purified by resin method and enzyme method,respectively.The results showed that two vegetables had different effects on alleviating colitis.Kale significantly alleviated the weight loss of mice,and the weight change increased from90% to about 99%,while celery was only 95%.The inhibition effect of DAI index of kale was the best,which could be significantly reduced from 2.2 points to about 0.8points,while that of celery group was 1.2 points.Both vegetables can significantly alleviate the pathological damage of colon.In addition,NKA-9 resin can be used for the purification of kale flavonoids(KF),among which flavonoids are mainly derivatives of quercetin and kaempferol.The flavonoid content in KF is 31.16 ±1.26%.Kale soluble dietary fiber(KSDF)is a neutral sugar,mainly composed of Gal,Ara and Man.Kale insoluble dietary fiber(KIDF)may be an insoluble dietary fiber with cellulose skeleton and a small amount of pectin.The content of flavonoids in celery flavonoids(CF)is 88%,including 67.85% Apiin,9.76% Chrysoerol7-O-apiosylglucoside and 10.22% Luteolin 7-O-apiosylglucoside.Celery soluble dietary fiber(CSDF)is mainly composed of Gal A and Gal.Celery insoluble dietary fiber(CIDF)is similar to KIDF,which may be an insoluble dietary fiber with a small amount of pectin connected to cellulose as the skeleton.The results showed that both celery and kale could improve colitis in mice,and high-purity dietary fiber and flavonoids were obtained from the two vegetables,which could be used for further research.2.To further explore the effects of dietary fiber combined with flavonoids on colitis mice,kale was selected as the research object,and to explore the effects of kale dietary fiber combined with kale flavonoids on colitis.A mouse colitis model was established by DSS to study the effects of KSDF and KIDF combined with KF on colitis.The results showed that in the DSS induced colitis mice,KSDF,KIDF and KF can relieve body weight loss(from 87% to 93%,95% and 91%)and colon shortening(from 5.4 cm to 6.2 cm,5.8 cm and 5.8 cm respectively),reduce DAI index(from 3points to 1.7,1.5 and 2 points),and promote mucin secretion(from 2.5% of the colon area to more than 6%)to varying degrees after single administration.In addition,giving KIDF and KF alone can also significantly inhibit colon TNF-α,IL-6,IL-1β and IL-33 m RNA expression in colitis mice,which was decreased to less than 70% of the model group.But KSDF only inhibited TNF-α and IL-33 m RNA expression,but not decreased IL-6 and IL-1β m RNA levels.After the combined use of KSDF and KF,20% of mice in the group died,and the effect of KSDF and KF alone on alleviating colitis was significantly weakened,such as DAI index rising to 2.5 points,colon length only 5.5 cm,and TNF-α,IL-6,IL-1 β and IL-33 m RNA expression was not significantly inhibited.However,the combined use of KIDF and KF did not significantly change the effect of alleviating colitis.Colitis mice have a disturbed composition of the gut flora.KSDF,KIDF or KF significantly decreased the relative abundance of the harmful bacterium(Sutterella,from 5% to below 3%).However,the relative abundance of Sutterella in the KSDF+KF group was up to 6%,which was significantly higher than that in the KSDF or KF groups.The relative abundance change of Parabacteroides was similar to it.KSDF can significantly reduce its abundance from 7% of the model group to 4%,while KSDF+KF can significantly increase its abundance to 12%,which was significantly higher than the model group,KSDF group and KF group.In addition,although the relative abundance of Akkermania in the model group increased significantly from less than 1% to 10%,but KF significantly promoted its relative abundance up to 22%,much higher than the normal group and the model group.The Akkermania relative abundance of KSDF group was 18%,which has no significant difference with the model group,but when KSDF and KF were used together,Akkermania was significantly inhibited to about6%.The results showed that the combination of KSDF and KF had an obvious antagonistic effect on colitis.3.In order to further explore whether the antagonism of dietary fiber and flavonoids in alleviating colitis exists in other vegetables,this paper studied the effect of celery dietary fiber and celery flavonoids combined use on colitis.A mouse colitis model was established using DSS to investigate the effects of CSDF and CIDF.The effects of the combination of CF and CSDF on colitis,respectively.In DSS-induced colitis mice,CSDF and CF,when administered alone,significantly reduced weight loss(from approximately 88% to approximately 94% and 93%),decreased blood and loose stools(DAI score from 3.5 to 1.9 and 2),and shortened colon(from 5 cm to 5.8cm and 5.7 cm)in mice with colitis.In addition,CSDF and CF significantly alleviated colonic tissue damage(tissue damage score decreased from 9 to 4.8 and 4.7),increased mucus expression capacity(from 2.2% to 5% and 6% in the model group),and decreased colonic MPO(from 18 μU/mg to 14 μU/mg and 13 μU/mg),i NOS(from 0.9 μU/mg to 0.75 CF also had a significant reduction in intestinal permeability in mice,with serum FITC-dextran levels only 1/4 of those in the model group.The combination of CSDF and CF had a weaker effect on the improvement of the above indices than the administration of CF alone,such as the change in body weight was significantly lower than that of the CSDF and CF groups(89%),the DAI index was significantly higher than that of the CSDF group(about 2.5 points),the tissue damage score was 6.5 points,the mucin area was 3.8%,and the serum FITC-dextran levels were comparable to those of the model group.In addition,the intestinal flora diversity of mice with colitis decreased,with the Shannon index decreasing from the normal5.5 to 3.9,and the relative abundance of harmful bacteria Escherichia Shigella,Clostridium sensu stricto 1 and Paraclostridium significantly increased.The administration of CSDF or CF alone had a significant effect on Escherichia Shigella,Clostridium sensu stricto 1 and Paraclostridium inhibition,but when CSDF and CF were combined,the inhibition of harmful bacteria was significantly weaker or even higher than in the model group.In addition,like KF,CF improved colitis by substantially promoting the abundance of Akkermansia,from 3.2% to 20%.However,the combination with CSDF inhibited the enrichment effect of CF on Akkermansia,with an abundance of only 3.3%.The results suggest that the antagonistic effect of soluble dietary fiber in combination with flavonoids on colitis also occurred in celery,so the antagonistic effect may be a general phenomenon.4.Because of the high flavonoid content and simple composition in CF,CSDF was selected with CF,and the mechanism of antagonism between them was further explored.The cellular inflammation model of LPS induced RAW264.7 in vitro,metabolic in vivo and fermentation models in vitro as well as the sieving technique were used to investigate the possible influencing mechanisms of CSDF interfering with the alleviating effect of CF colitis.It was found that CF and Apiin,a major component of CF,did not significantly inhibit TNF-α expression in RAW264.7 cells,whereas apigenin could significantly inhibit TNF-α production induced by LPS in RAW264.7 cells,and the presence of glycosides impaired the anti-inflammatory activity of apigenin.In vivo metabolic assay revealed that the urine apigenin level of mice in CSDF group was significantly lower than that in normal group,which was only 1/5 of that in normal group.60% of Apiin could be recovered in the feces of mice after antibiotic treatment,while little apigenin was contained in the urine.In vitro fermentation results found that 1.6 mg/ml CSDF could promote the complete deglycosylation and degradation of Apiin and apigenin within 24 h,and the higher the concentration of CSDF,the stronger the promotion.It is not that Apiin itself and its metabolites directly promote the proliferation of Akkermansia muciniphila(AKK),but that Apigenin inhibits the growth of bacteria other than AKK,providing a favorable growth environment for the proliferation of AKK.The mechanism of antagonism between CSDF and CF in colitis mice may be that CSDF inhibits the promotion of Akkermansia in vivo by Apiin through promoting the degradation of CF,especially the degradation of Apiin and Apigenin.In addition,four strains with apigenin exo-apiosidase activity(Human: Parasutterella excitatihominis and Collinsella aerofaciens;murine: Muribaculum sp.and Robinsoniella sp.)were selected from human and mouse feces in this study.The results indicated that the anti-inflammatory activity of CF exerted a deglycosylation dependent on gut flora,and CSDF attenuated the gut protective effect of CF,probably by accelerating the degradation of CF.The above results indicated that the simultaneous intake of soluble dietary fiber and flavonoids in kale and celery will have a weaker effect on the alleviation of colitis than the single intake of the two nutrients,and the celery soluble dietary fiber may accelerate the degradation of flavonoids,thereby weakening the intestinal protection of celery flavonoids.更多还原