Preparation Of Polysaccharides From Fibrous Roots Of Ophiopogon Japonicus And Its Preventive Effect On Stressed Gastric Ulcer

Ophiopogon japonicus（L.f）Ker-Gawl.is the dried root tuber of Ophiopogon japonicus（L.f）Ker-Gawl and is a local medicinal herb in Sichuan Province.It has sweet taste,slightly bitter and slightly cold,and belongs to the heart,lung and stomach.Ophiopogon japonicus has the effect of nourishing Yin and moistening the lung,benefiting the stomach and promoting the production of body fluid,clearing the heart and removing irritation.Ophiopogon japonicus is used as medicine in root tuber,and its fibrous root was once used as waste,which caused great waste.At present,there have been a few reports on the composition of the major herbal components in the fibrous root of Ophiopogon japonicus,and studies have shown that polysaccharide is the main component of the fibrous root of Ophiopogon japonicus,but its pharmacological activitives has not been reported.In this study,the polysaccharides from the fibrous roots of Ophiopogon japonicus were extracted by a complex enzyme,and the enzymatic hydrolysis process was optimized by response surface methodology.Purified polysaccharides were obtained by the separation and purification of crude polysaccharides.Analysis of molecular weight,monosaccharide composition and functional groups of the purified polysaccharides with the highest yields.Finally,the stress gastric ulcer model of rats was established by the water-immersion-restraint method.The preventive effects of polysaccharides from fibrous roots of Ophiopogon japonicus on stress gastric ulcer in rats were evaluated by gastric histomorphological analysis,oxidative stress factors,gastric mucosal cytoprotective factors,and pro-gastric acid secretion profile.1.Optimization of enzymatic hydrolysis of polysaccharides from fibrous roots of Ophiopogon japonicusThe fibrous root of Ophiopogon japonicus was hydrolyzed by neutral cellulase,neutral pectinase,and papain,and the polysaccharide was obtained by water extraction and alcohol precipitation.Firstly,the compound enzymatic process was optimized by screening the amount of compound enzyme added,the ratio of compound enzyme,the ratio of enzymatic material to liquid,and the enzymatic process time with the index of polysaccharide extraction rate,and then the enzymatic process was optimized by response surface method.The results showed that the single-factor fitting degree was good.The optimum compound enzymatic hydrolysis process of polysaccharides from fibrous roots of Ophiopogon japonicus was:the additional amount of the complex enzyme is 1.8%,and the proportion of the mixed enzyme is neutral cellulase:neutral pectinase:papain=5:5:6,solid-liquid ratio 1:45（W/V）,enzymolysis time 65 min.The extraction rate of polysaccharides from fibrous roots of Ophiopogon japonicus was 31.57%,which was increased by 12.68%compared with those without enzymatic hydrolysis.2.Isolation,purification,and structure analysis of polysaccharides from fibrous roots of Ophiopogon japonicus.The contents of polysaccharides,polyphenols,and protein in crude polysaccharides from fibrous roots of Ophiopogon japonicus were determined by the sulfuric acid-phenol method,Folin-phenol method,and Coomassie brilliant blue method.The results showed that the polysaccharide was light brown and fluffy,and the content of polysaccharide,polyphenol and protein was 58%,2%,and 11%,respectively.The crude polysaccharide from the fibrous roots of Ophiopogon japonicus was separated by the DEAE-Sepharose FF fiber column.The results showed that there were one neutral polysaccharide and two acidic polysaccharides in the crude polysaccharide of fibrous roots of Ophiopogon japonicus,the highest yield of neutral polysaccharide was 85%,while the acidic polysaccharides were 9%and 4%respectively.The neutral polysaccharide was purified by the DEAE-Sepharose 6FF fiber column,the content of neutral polysaccharide was 83.11%,the content of polyphenol was 0.23%,and the content of protein was 0.34%.The relative molecular weight and structure of neutral sugar were determined by HPLC and FT-IR respectively.The results showed that the molecular weight of the neutral polysaccharide of fibrous roots of Ophiopogon japonicus was 70.817 k Da,which was mainly composed of D-mannose,D-glucose,D-galactose,and a small amount of L-arabinose and L-fucose.The neutral polysaccharide of fibrous roots of Ophiopogon japonicus contains O-H,C-H,C=O,and C-O,which are characteristic functional groups of polysaccharides and contain pyran ring and furan ring.3.Preventive effect of polysaccharides from fibrous roots of Ophiopogon japonicus on stress gastric ulcer induced by the water-immersion-restraint method in rats.In this study,polysaccharides from fibrous roots of Ophiopogon japonicus（100mg/kg/d,200 mg/kg/d,400 mg/kg/d）as the preventive dosing group,cimetidine（36.3mg/kg/d）as the positive group,by gavage for 14 days,using water-immersion-restraint for6 hours to establish stress gastric ulcer in rats.Dissection and sampling immediately after mold making.The appearance and histology of gastric ulcer were observed employing eye observation,vernier caliper measurement,and pathological tissue section.Measurement of the p H of gastric contents by p H paper to observe changes in gastric acid secretion in the stomach;Determination of MDA concentrations in serum and tissues by the TBA method;Determination of SOD activity in serum by UV colorimetric method;ABTS method for the determination of T-AOC concentrations in serum and tissues;Determination of PCO in serum,s Ig A concentration in gastric tissue by ELISA;RT-PCR method for the determination of gastric tissue SOD,GSH-Px,IL-1β,NF-κB p65,NOS₃,ET-1,PTGS-2,EGF,Caspase-8,PGc,GAS-R,SS,Ach-M₃R,H⁺-K⁺-ATPase,H₂R gene expression.The above experimental observations were used to evaluate the preventive effects of polysaccharides from fibrous roots of Ophiopogon japonicus on stress gastric ulcer in terms of oxidative stress factors,inflammatory factors,gastric tissue blood microcirculation,gastric tissue mucosal cell protection and gastric acid secretion.The results showed that:（1）Polysaccharides from fibrous roots of Ophiopogon japonicus can significantly reduce the number of gastric ulcers,reduce the gastric ulcer index,reduce the gastric mucosal inflammatory cell infiltration and gastric mucosal bleeding to ensure the integrity of gastric mucosa.（2）Polysaccharides from fibrous roots of Ophiopogon japonicus can significantly increase the content of antioxidant enzymes（SOD,T-AOC,GSH-Px）in gastric tissue and serum,significantly reduce the content of oxidative products（MDA,PCO）and improve the oxidative stress state of stress gastric ulcer in rats.（3）Polysaccharides from fibrous roots of Ophiopogon japonicus can significantly reduce the expression of inflammatory factors IL-1β,NF-κB p65 gene,reduce the inflammation of gastric ulcer tissue.（4）Polysaccharides from fibrous roots of Ophiopogon japonicus can increase NOS₃,significantly reduce the expression of the ET-1 gene,improve the local vasoconstriction and relaxation of gastric tissue,and then improve the microcirculation of gastric tissue.（5）Polysaccharides from fibrous roots of Ophiopogon japonicus can significantly increase the expression of PTGS-2 and EGF gene,reduce the expression of Caspase-8 gene,protect the integrity of gastric mucosal cells,stimulate the growth of gastric mucosal cells,reduce the necrosis of gastric mucosal cells,and improve the integrity of gastric mucosal epithelium.（6）Polysaccharides from fibrous roots of Ophiopogon japonicus can significantly reduce the expression of GAS-R,SS,Ach-M₃R,H⁺-K⁺-ATPase,H₂R gene,and PGc gene,reduce gastric acid secretion and pepsinogen content,improve gastric acid and pepsin on gastric mucosa erosion,and then achieve the prevention of polysaccharides from fibrous roots of Ophiopogon japonicus on gastric ulcer induced by the water-immersion-restraint method in rats.Compared with cimetidine,polysaccharides from fibrous roots of Ophiopogon japonicus have less effect on reducing gastric acid secretion than cimetidine,but a better effect on antioxidation,anti-inflammation,and protecting gastric mucosa.Among them,the best comprehensive effect was obtained with the dosage of 400 mg/kg/d of polysaccharides from fibrous roots of Ophiopogon japonicus,and the second was 200 mg/kg/d of polysaccharide from the fibrous root of Ophiopogon japonicus.