Establishment Of A System For In Vitro And In Vivo Studies On Digestion And Fermentation Of Polysaccharide From Seeds Of Plantago Asiatica L. With Its Beneficial Effects On Intestinal Health

Plantago, including Plantago asiatica L. and Plantago depressa Willd., waswidely used in traditional Chinese medicine. The seeds of Plantago asiatica L. fromJi’an were subjected for study in this research. Digestion, fermentation and intestinalfunctions of polysaccharide from seeds of P. asiatica L. were investigated in vitroand in vivo. The saliva, gastric and intestinal digestion, and fermentation of thepolysaccharide from P. asiatica L. were simulated and analyzed. Effects of thepolysaccharide on intestinal function were evaluated. Effects of physical treatmentson physiological activities of the polysaccharide in simulated gastrointestinalenvironment were also studied. In addition, mice were given oral administration ofpolysaccharide from P. asiatica L. by gavage to investigate the effects of thepolysaccharide on mouse colon, nutrient metabolism and colon microbiota. Effectsof physical treatments on mouse physiological activities of the polysaccharide werealso studied. Researches were also focus on whether oral administration andpre-administration of the polysaccharide could acute or prevent development andcolon microbiota changes in mice with dextran sodium sulfate (DSS)-inducedexperimental colitis. Main conclusions are summarized as follows:1. The saliva, gastric and intestinal digestion of polysaccharide from P.asiatica L. seeds was investigated in vitro. It was found that salivary amylase had noeffect on the polysaccharide; however, the polysaccharide was influenced in latergastrointestinal digestion. A steady decrease in molecular weight (Mw) of thepolysaccharide from (1903.1±93.0) x103to (4.7±0.2) x103was observed asdigestion time increased. Meanwhile, the reducing ends were increased from0.157±0.009to0.622±0.026mM, indicating the decrease of Mw may due to thebreakdown of glycosidic bonds. In addition, there was no monosaccharide releasedthroughout the whole digestion period, suggesting that the gastrointestinal digestiondid not result in a production of free monosaccharide. These results may providesome information on the digestion of polysaccharide from P. asiatica L. in vitro, andmay contribute to the methods of studying the digestion of other carbohydrates. 2. In vitro fermentation of the polysaccharide from seeds of P. asiatica L. andthe contribution of its carbohydrates to the fermentation were investigated in thisstudy. The polysaccharide was characterized by high contents of xylose, arabinoseand glucuronic acid, and it was subjected to human fecal cultures to be fermented invitro for24h. During fermentation, pH in fecal cultures decreased from6.1to5.1and the levels of total short-chain fatty acid (SCFA), acetic, propionic and n-butyricacids all significantly increased. Xylanase, arabinofuranosidase, xylosidase andglucuronidase activities were also improved. After24h incubation,47.2±1.6%oftotal carbohydrate in polysaccharide, including42.9±1.5%of arabinose,53.2±1.6%of xylose and76.4±1.2%of glucuronic acid, were consumed. In addition,relationship between carbohydrate consumption of the polysaccharide and SCFAproduction was also evaluated. It was found that the increase of acetic and n-butyricacid productions mainly resulted from the fermentation of glucuronic acid andxylose in polysaccharide, while the increase of propionic acid production wasprimarily due to the fermentation of arabinose and xylose. These results showed thatthe polysaccharide was physiologically active for human large bowel, and itscarbohydrate composition determined its SCFA production.3. Effects of polysaccharide from the seeds of P. asiatica L. on intestinalfunction were investigated in vitro. Results showed that the polysaccharide hadnotable influence on slowing down glucose diffusion and inhibiting α-amylaseactivity. These might help prolong blood glucose response and hence control thepostprandial glucose concentration. The polysaccharide could also decreasepancreatic lipase and protease activities, which may help lower the levels of serumlipids and modify protein digestibility. In addition, the polysaccharide was able tobind bile acids and may reduce cholesterol level. These results suggested that thepolysaccharide may have potential benefits for human intestinal function and mightbe used as a potential ingredient in functional food applications.4. Mice were given30days oral administration of polysaccharide from P.asiatica L. at the dose of0.4g/kg body weight by gavage to investigate the effects ofthe polysaccharide on mouse colon. Results showed that the concentrations of totalSCFA, acetic, propionic, and n-butyric acids in mouse colonic content of polysaccharide treated group were all significantly higher than that of control group(water)(p <0.05). In addition, moisture of mouse colonic content of polysaccharidetreated group was also notably higher than that of the control group (p <0.05)indicating the intake of polysaccharide from P. asiatica L. resulted in a strongerwater-holding capacity for colonic content throughout the experimental period.Furthermore, a decreased pH (from7.5±0.1to7.2±0.1) was observed in mousecolon of the polysaccharide treated group compared with the control group (pH from7.5±0.1to7.5±0.1). These results suggested that the intake of the polysaccharidefrom P. asiatica L. might be beneficial for the colon health.5. Polysaccharide from the seeds of P. asiatica L. was given via oraladministration to mice (0.4g/kg body weight,30days) to observe its effects onmouse nutrient metabolism and colon microbiota. It was found the polysaccharideintake could lower the apparent absorption of lipid. Total triglyceride, cholesterol,and atherogenic index in blood serum with total lipid and cholesterol levels in liverof polysaccharide group mice were all significantly lower than those of the controlgroup (p <0.05). Furthermore, the effect of the polysaccharide intake on mousecolon bacterial communities was investigated. Mice from the polysaccharide groupshowed a higher colon bacterial diversity than the control group. Bacteroides sp.,Eubacterium sp., butyrate-producing bacteria Butyrivibrio sp., and probioticsBifidobacterium bifidum, Lactobacillus fermentum, and Lactobacillus reuteriin inmouse colon were all increased after polysaccharide intake. These indicated that theintake of polysaccharide from P. asiatica L. could be beneficial for lipid metabolismand colon microbiota.6. Effects of microwave irradiation on microbial SCFA production and theactivities of extracellular enzymes during in vitro fermentation of the polysaccharidefrom P. asiatica L. were investigated. It was found that the apparent viscosity,average molecular weight, and particle size of the polysaccharide decreased aftermicrowave irradiation. Reducing sugar amount increased with molecular weightdecrease, suggesting the degradation may derive from glycosidic bond rupture. Thepolysaccharide surface topography was changed from large flake like structure tosmaller chips. FT-IR showed that microwave irradiation did not alter the primary functional groups in the polysaccharide. However, SCFA productions of thepolysaccharide during in vitro fermentation significantly increased after microwaveirradiation. Activities of microbial extracellular enzymes xylanase,arabinofuranosidase, xylosidase, and glucuronidase in fermentation culturessupplemented with microwave irradiation treated polysaccharide were also generallyhigher than those of untreated polysaccharide. In addition, physiological propertiesof homogenized and non-homogenized polysaccharide from the seeds of P. asiaticaL., such as the SCFA production were compared. High pressure homogenizationdecreased particle size of the polysaccharide, and changed the surface topographyfrom large flake-like structure to smaller porous chips. FT-IR showed that highpressure homogenization did not alter the primary structure of the polysaccharide.However, the production of total SCFA, propionic acid and n-butyric acid in cecaand colons of mice significantly increased after dieting supplementation withhomogenized polysaccharide. These results showed that microwave irradiation andhigh pressure homogenization treatment could be promising approaches for theproduction of value-added polysaccharides in the food industry.7. Colonic inflammation was induced in female BALB/c mice by feedingdextran sulfate sodium DSS in drinking water. Along with or after DSSadministration, mice were given polysaccharide (0.2or0.4g/kg body weight) bygavage daily. Polysaccharide administration attenuated development of symptoms(body weight loss, fecal occult blood and diarrhea) associated with colitis.Polysaccharide also blocked colon shortening, increased superoxide dismutase butsuppressed myeloperoxidase and endothelial nitric oxide synthase activities, andimproved macroscopic scores. Histological damage of colon, decrease of thymusorgan index, and increase of C-reactive protein level in mice were reduced bypolysaccharide. Increase of colonic tissue levels of TNF-α, IL-1β, IL-6, macrophageinflammatory protein2and leukotriene B4resulting from colitis were significantlydecreased after polysaccharide administration. Colitis induced decrease of fecalshort-chain fatty acid concentration was also attenuated by polysaccharide.Quantitative PCR (QPCR) results showed that polysaccharide administration couldreduce the decrease of Bifidobacterium, Lactobacilli, Bacteroidaceae and Enterobacteriaceae population sizes in colon of mice with colitis. The putativeclinical utility of the polysaccharide suggests its potential application in colitisattenuation.8. It was investigated whether pre-administration of polysaccharide from P.asiatica L. could prevent development and colon microbiota changes in mice withexperimental colitis. Colitis was induced in female BALB/c mice by feeding DSS indrinking water.7days before DSS administration, mice were given polysaccharidepre-administration (0.2or0.4g/kg body weight) daily. Polysaccharidepre-administration prevented colitis-induced body weight loss, fecal occult blood anddiarrhea. Polysaccharide blocked colon shortening, suppressed myeloperoxidase, andimproved macroscopic scores. Colon histological damage, decrease in thymus organindex, increase in colon organ index and serum CRP level due to colitis were allreduced by polysaccharide. The increase of colon tissue levels of pro-inflammatorycytokines TNF-α, IL-1β, IL-6, and IL-12, and macrophage inflammatory proteinMIP-2resulting from colitis were all significantly decreased, while level ofanti-inflammatory cytokine IL-4was increased. Furthermore, colitis-induced decreaseof fecal SCFA concentration was attenuated by polysaccharide. QPCR results showedpolysaccharide pre-administration could reduce the decrease of Bifidobacterium,Lactobacilli, Bacteroidaceae and Enterobacteriaceae population sizes and theincrease of Clostridium population size in colon of mice with colitis. The putativeclinical utility of the polysaccharide suggests its potential application in colitisprevention.