【作者】 王聪；

【导师】 姜申德； 陈海霞；

【作者基本信息】 天津大学 ， 应用化学， 2018， 博士

【摘要】 桦褐孔菌(Inonotus obliquus)属担子菌亚门多孔菌科褐卧孔菌属真菌,又名白桦茸、桦茸菌等。作为一种珍贵的药食两用型真菌,桦褐孔菌在民间被广泛应用于糖尿病的预防和治疗。目前,桦褐孔菌中的降血糖成分研究主要以粗提物或者粗多糖为主体,对小分子化合物的降血糖活性考察也仅局限于α-淀粉酶和α-葡萄糖苷酶抑制等活性评价体系,缺乏对桦褐孔菌中主要降血糖活性成分的深入了解。因此,本文以桦褐孔菌菌核为研究对象,考察了桦褐孔菌多糖及其铬络合物、桦褐孔菌醇和麦角甾醇的体内外降血糖活性;采用体外模拟消化模型探讨了桦褐孔菌多糖的消化特性;研究探讨了不同pH和培养条件下桦褐孔菌菌丝的主要成分与其生物活性的变化情况,为桦褐孔菌培养条件的选择提供理论依据。采用DEAE-52阴离子色谱层析法分离纯化得到了三个离子强度不同的桦褐孔菌多糖组分IOPS-1、IOPS-2和IOPS-5。葡聚糖凝胶色谱、气相色谱表明三个多糖组分主要由L-鼠李糖、D-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖、D-半乳糖等单糖组成,分子量分别为75.94kDa、97.12kDa和114.3kDa。扫描电镜结果表明IOPS-2的表观形态特征与IOPS-1、IOPS-5差异较大。刚果红实验和圆二色谱则表明IOPS-1、IOPS-2和IOPS-5在溶液中以规则的三螺旋结构存在,且IOPS-5的高级结构要比IOPS-1和IOPS-2更为复杂。三个多糖组分均具有较好的抗氧化活性、α-淀粉酶、α-葡萄糖苷酶及胰脂肪酶抑制活性,且IOPS-2的生物学活性要优于IOPS-1和IOPS-5。此外三个多糖组分还能够有效提升过氧化氢诱导氧化损伤的L02细胞的存活率,修复受损的线粒体,维持细胞的正常形态。以桦褐孔菌多糖为配体合成多糖铬络合物,红外光谱及圆二色谱图表明多糖通过羟基、羰基与铬元素相互作用而结合。多糖络合铬元素后会改变其组成成分、单糖摩尔比以及抗氧化活性。多糖及其铬络合物还能够有效抑制非酶糖基化末端终产物的形成、α-淀粉酶、α-葡萄糖苷酶及胰脂肪酶的活性,展现出一定的餐后血糖调节能力及降脂潜力。给予2型糖尿病小鼠多糖铬络合物后,糖尿病小鼠的体重和器官指数得到恢复,血糖和胰岛素水平呈现一定程度的降低,葡萄糖耐受能力增强,肝糖原和肌糖原的含量显著升高,内源性的抗氧化能力也得到提升,组织损伤得到修复。桦褐孔菌多糖铬络合物还能够通过降低胆固醇、甘油三酯、糖化血清蛋白、游离脂肪酸、低密度脂蛋白胆固醇水平,提升高密度脂蛋白胆固醇水平来实现对糖尿病小鼠血脂的调节。此外,亚急性毒性研究证明了桦褐孔菌多糖铬络合物对正常小鼠无明显毒性。多糖在模拟消化的三个阶段(唾液、胃、小肠)均发生了不同程度的糖苷键的断裂及聚集体的破坏,进而导致其抗氧化活性、α-淀粉酶和α-葡萄糖苷酶抑制活性发生变化。而模拟消化过程中pH的变化和较长的消化时间则可能是导致多糖降解的主要原因。桦褐孔菌醇和麦角甾醇能够通过与受体蛋白BSA、α-淀粉酶和α-葡萄糖苷上的氨基酸形成氢键而相互作用,进而有效抑制α-淀粉酶、α-葡萄糖苷的活性及非酶糖基化末端产物的形成,表现出一定的降血糖潜力。不同碳源、氮源、金属离子、氨基酸及pH等影响因素对桦褐孔菌菌丝的生长量、组成成分、抗氧化活性、α-淀粉酶和α-葡萄糖苷酶抑制活性等均有不同程度的影响。其中葡萄糖、Mg2+离子、Zn2+离子及酸性环境与总三萜的累积、菌丝水提物的α-葡萄糖苷酶抑制活性有明显的正相关性。Ca2+离子和碱性环境有助于黄酮的累积,并且与提取物的DPPH自由基清除活性、铁还原力以及α-葡萄糖苷酶抑制活性显著相关。氮源和氨基酸则主要与多酚、中性糖、酸性糖的累积,提取物的DPPH自由基清除活性及α-淀粉酶抑制活性相关。更多还原

【Abstract】 Inonotus obliquus(I.obliquus),commonly called Chaga or Betula,belongs to the family of Basidiomycota,Inonotus.Traditionally,I.obliquus is consumed for the treatment of diabetes mellitus(DM)for centuries owing to its medical properties.Currently,the investigations of hypoglycemic constitutes from I.obliquus were mainly focused on the crude extracts and polysaccharides.The hypoglycemic effects of chemicals were commonly evaluated by using αamylase and α-glucosidase inhibitory assay.Despite this,the hypoglycemic constitutes from I.obliquus were not fully understood.The objective of this study was to investigate the hypoglycemic effects of polysaccharides,inotodiol and ergosterol from L obliquus.The digestive properties of polysaccharides were investigated by using simulated digestion model.In addition,the relationship between the compositions and bioactivities of extracts of mycelia under different influential factors was also demonstrated,which could provide the theoretical basis for the culture of I.obliquus.IOPS-1,IOPS-2 and IOPS-5 were obtained from I.obliquus by using DEAE-52 ion exchange column chromatography.They were all composed of L-rhamnose,D-arabinose,D-xylose,Dmannose,D-galactose,and D-glucose with the molecular weight of 75.94 kDa,97.12 kDa and 114.3 kDa,separately.The morphological property of UIOPS-2 was obviously different from IOPS-1 and IOPS-5.The Congo red assay and CD spectra indicated that these three fractions had an ordered conformation(triplex).Moreover,IOPS-2 exhibited better antioxidant activity,α-amylase,α-glucosidase and pancreatic lipase inhibitory activities than other two fractions.Besides,IOPS1,IOPS-2 and IOPS-5 could attenuate the oxidative damage induced by H2O2 in hepatic L02 cells via enhancing the cell viability,inhibiting the morphology alteration and maintaining the integrity of mitochondria.A polysaccharides-chromium(Ⅲ)complex(UIOPC)was synthesized by using polysaccharides as ligand.FTIR spectra and CD spectra indicated that the Cr3+was coordinated with the hydroxyl and carboxyl groups of polysaccharides,resulted in the changes of composition,monosaccharides ratio and antioxidant activity of UIOPS.UIOPS and UIOPC could effectively inhibit the formations of AGEs and activities of digestive enzymes,suggesting great control of postprandial hyperglycemia and hyperlipidemia.UIOPC also exhibited anti-diabetic effects in T2DM.After the treatment of UIOPC,the body weight and organ index of diabetic mice were recovered.The blood glucose and insulin levels were decreased with the improvement of glucose tolerance.While,the glycogen levels were increased and intracellular antioxidant system was improved.UIOPC could alleviate the hyperlipidemia of diabetic mice by decreasing the levels of TC,TG,LDL-C,GSP,FFAs and increasing the level of HDL-C.The tissue damages were also recovered to normal.In addition,UIOPC had no obviously sub-acute toxicity on the normal mice.UIOPS-1 was degraded in the three digestion process and resulted in the changes of physicochemical property,antioxidant activity,α-amylase and α-glucosidase inhibitory activities.The breakdown of glycosidic bonds might be associated with pH and the long digestion time.Inotodiol and ergosterol could interact with their targeted proteins through forming hydrogen bonds,which lead to the inhibition of the advanced glycation end products formation,α-amylase and α-glucosidase activities.Different influential factors affected the growth,compositions and bioactivities of cultured mycelia of I.obliquus.Results of PCA indicated that glucose,Mg2+,Zn2+and the acid situation were beneficial for the accumulation of triterpenes and the α-glucosidase inhibitory activity.Ca2+and alkaline situation promoted the accumulation of flavones,antioxidant and α-glucosidase inhibitory activity.In addition,nitrogen sources and amino acids were related to the accumulation of polyphenols,neutral sugar,uronic acid,DPPH scavenging and α-amylase inhibitory activity.更多还原