【作者】 程安媛；

【导师】 朱照静；

【作者基本信息】 重庆医科大学 ， 药物化学， 2008， 硕士

【摘要】 白芨多糖是中药白芨治疗胃溃疡的主要成分,是一种高分子粘性化合物,由葡萄糖与甘露糖以1:4的比例聚合而成,可粘附于体内粘膜、组织相容性好,在胃粘膜表面可形成一层保护膜,起到保护和修复溃疡面的作用。本课题对白芨多糖的提取、精制、质量评价进行了研究,通过体内外粘附性能考察,为白芨多糖作为胃粘膜粘附性材料奠定基础。本课题研究分为四部分:第一部分:白芨多糖的制备与鉴定白芨采用水提醇沉法提取和有机溶剂洗涤、透析等精制处理,可获得纯度好的白芨多糖,经正交设计筛选出的最佳提取条件,制备的白芨多糖的平均收率为13.8%。对白芨多糖进行了理化鉴定和红外光谱鉴定表明白芨多糖由葡萄糖和甘露糖组成,以β糖苷键连接。第二部分:白芨多糖的质量研究1.采用硫酸-咔唑法检测白芨多糖的酸性糖成分,并进行pH值测定。硫酸-咔唑法测得白芨多糖不含酸性糖成分,pH值测定结果表明纯度越高,pH值越接近中性,测得最佳工艺制备的白芨多糖pH值为7.3。2.采用蒽酮-硫酸法,以葡聚糖T-20为对照品,建立了紫外比色测定白芨多糖含量的方法,在15 mg·L^-1-50 mg·L^-1范围内葡聚糖T-20的浓度与吸光度呈良好的线性关系,线性回归方程:y=48.383x+1.6295（r=0.9992,n=6）;通过影响显色因素考察,确定最佳测定条件:蒽酮试剂与白芨多糖或葡聚糖T-20溶液的体积比2:1、水解时间15min、水解温度100℃,在该条件下测得白芨多糖平均含量为63.70%。3.建立了高效毛细管电泳（HPCE）测定白芨多糖的单糖组分的方法,以葡萄糖和甘露糖为标准对照品,水解后柱前衍生化,硼砂缓冲液50 mmol/L-1 （pH=10.5）测得白芨多糖中的单糖组分葡萄糖与甘露糖的相对比例为1:4.6。4.采用FASCO全自动流变血液快测仪,高中低三种剪切速率测定白芨多糖的粘度。测定结果表明白芨多糖是一种典型的假塑性流体,其粘度随着剪切速率的增加、温度的增高而降低;粘度的测定值误差,随剪切速率增加而增加。第三部分:白芨多糖体外粘附性评价1.采用无水乙醇灌胃,成功建立了KM小鼠胃溃疡模型。2.采用自制的分离力测定仪,建立了测定分离力方法和筛选出了最佳测定条件:组织润湿时间为2 h、外加压力100 g、施压时间为15 min、加水速率为10 ml/min。测得白芨多糖对正常组织与模型组织的分离力为50.79 g/cm2、50.72 g/cm2,二两者之间不存在显著性差异（P=0.92）,与Carbopol的分离力（正常29.96 g/cm2、模型29.52 g/cm2）存在显著性差异（P<0.01） ,表明白芨多糖的粘附性优于Carbopol。3.采用自制的组织留存量测定仪,建立了组织留存量测定方法并筛选出了最佳条件:组织润湿时间为2 h、盐酸冲洗速率为20 ml/min。采用蒽酮-硫酸法、以葡聚糖T-20为对标准照品,建立了多糖组织留存量测定方法,在15 mg·L^-1-50 mg·L^-1范围内葡聚糖T-20的浓度与吸光度呈良好的线性关系,线性回归方程:y=0.0199x-0.0052（r=0.9990,n=6）。最佳条件下测得白芨多糖对正常组织及模型组织的留存量分别为60.64%、61.16%,二者之间不存在显著性差异（P=0.68）。4.采用分离力和组织留存量测定的最佳条件,考察pH为6.8和pH为1.2的润湿介质对白芨多糖的分离力和组织留存量的影响。结果表明润湿介质pH为6.8时,白芨多糖对正常组织和模型组织的分离力分别为52.13 g/cm²、53.12 g/cm²,二者之间不存在显著性差异（P=0.40）;白芨多糖对正常组织和模型组织的留存量分别为62.36%、63.05%,二者之间不存在显著性差异（P=0.52）。润湿介质pH为1.2时,白芨多糖对正常组织和模型组织的分离力分别为44.57 g/cm2、46.64 g/cm²,二者之间存在显著差异（P=0.039）;白芨多糖对正常组织和模型组织的留存量分别为57.76%、60.43%,二者之间存在显著性差异（P=0.046）。随润湿介质pH的降低,白芨多糖对正常粘膜与模型粘膜的分离力降低,组织留存量也降低。第四部分:白芨多糖膜粘附性体内评价以硫酸钡试剂造影、X射线检测,建立了白芨多糖体内粘附性评价方法,考察白芨多糖在正常组及模型组大鼠的胃内滞留情况。结果表明白芨多糖在正常大鼠及模型大鼠体内均有较强的粘附性,在模型组织内的滞留时间（>24h）比正常组织内的滞留时间（<24h）长,且随浓度增加,粘附性增强。白芨多糖作为一种新型的膜粘附材料方面的研究尚未见报道。本课题首次对白芨多糖的体内外粘附性能进行了系统的研究,证实白芨多糖对胃粘膜有很强的粘附性,在损伤的模型大鼠胃内的滞留时间较正常大鼠胃内滞留时间长,其粘附性与白芨多糖的浓度呈正相关,从而为白芨多糖作为一种新型的胃粘附载体材料提供了理论依据。更多还原

【Abstract】 Bletilla striata polysaccharide is the essential ingredient of Bletilla Striata （Thunb.） Reichb.f., which is a high polymer compound. It is composed of glucose and mannose with the ratio of 1:4. Bletilla striata polysaccharide could be an adhesive material to mucosa with a good histocompatibility, as a great membrana protectiva to gastric mucosa, restoring the gastric ulcers and protecting them from harmful factors.The study is mainly test on four topics:Part I: Preparation of Bletilla striata polysaccharideWith water as solvent extraction and absolute alcohol as ethanol precipitation and extraction is purified by organic solvents and then to be dialyzed, we can get a polysaccharide with a good quality and the yield is 13.8%. The physical and chemical identification and IR detection showed that Bletilla striata polysaccharide is composed of glucose and mannose, which is linked by the glycosidic link.Part II: Quality studies of Bletilla striata polysaccharide1. By the carzapol-sulfuric acid method, we detected wether the uronic acid was a component of the Bletilla striata polysaccharide and determined the polysaccharide’s pH value. The carzapol-sulfuric acid method confirmed that there is no uronic acid component of the Bletilla striata polysaccharide. The pH determination showed that the higher the purity of polysaccharide, the closer the pH value to neutral status, and the pH value of the purified one is 7.3.2. Developed the determination of the polysaccharide by UV, with the anthrone-sulfuric acid method and dextrin T-20 as the standard reference substance, the concentration is linear with the UV absorbance very well between 15 mg·L^-1-50 mg·L^-1, and the linear regression equation is y=48.383x+1.6295,（r=0.9992,n=6）. Investigating the color affected factors, we identified the best determination conditions: the volume ratio of anthrone-reagents and Bletilla striata polysaccharide or dextrin T-20 solution is 2:1, reaction time is 10 min, hydrolysis temperature is 100℃. In this condition, the average content of Bletilla striata polysaccharide is 63.70%.3. Established the method to determin the monosaccharides of Bletilla striata polysaccharide, with glucose and mannose as the standard reference substances, pre-column derivatization, and borax buffer of 50mmol/L-1 （pH = 10.5）, the result showed that the relative ratio of glucose and mannose is 1:4.6.4. By the FASCO automatic blood flow measurement device, with three different degrees of the Shear rate, we detected the viscosity of Bletilla striata polysaccharide.The result showed that Bletilla striata polysaccharide is a typical pseudoplastic fluid, which was shear thinning. The viscosity of Bletilla striata polysaccharide decreased with the increasing of the shear rate and temperature, the RSD increased with the increasing of shear rate.Part III: Bletilla striata polysaccharide adhesion in vitro evaluation1. Built the animal model soccsessfully by absolute alcohol on KM rat.2. With the self-made separation detector, developed a method for the determination of separation, The best detective conditions was: tissues wetting time for 2 h, pressure plus on the tissues is 100 g, time of pressing was 15 min, water flow rate was 10 ml/min.The result showed that the separation of Bletilla striata polysaccharide on normal tissues and the model tissues were 50.79 g/cm², 50.72 g/cm², and between the two there was no significant difference （P=0.92）, which was significant different from the separation of Carbopol （normal 29.96 g/cm², Model 29.52 g/cm²） （P<0.01）,which showed that the adhesion of Bletilla striata polysaccharide is superior to that of Carbopol.3. With the self-made detector for the adhesion of Bletilla striata polysaccharide to the nomal tissues and model tissues, screened the the best detective conditions: tissues wetting time is 2 h, hydrochloric acid washing rate is 20 ml/min. Anthrone-sulfuric acid, dextron T-20 as the standard reference substance, established the determination of polysaccharide of the washing water, the concentration is linear with the UV absorbance very well between 15 mg·L^-1-50 mg·L^-1, and the linear regression equation is: y= 0.0199x-0.0052 （r=0.9990,n=6）. The result showed that the remains weight percentage of Bletilla striata polysaccharide on the normal tissues and model tissues were 60.64%, 61.16%. Between the two there was no significant difference （P=0.68）.4. With the best detective conditions, investigated the affected factors of defferent pH value of wetting media, determined the adhesive of Bletilla striata polysaccharide to normal tissues and model tissues with the pH value of wetting media of 6.8 and 1.2. The result showed that when the pH of the wetting medium is 6.8,the separation of Bletilla striata polysaccharide on normal tissue and model tissues were 52.13 g/cm², 53.12 g/cm2, there was no significant difference（P=0.40）. The weight percentage of Bletilla striata polysaccharide remained on normal tissues and the model tissues were 62.36%, 63.05%, there was no significant difference （P=0.52）. When the pH of the wetting medium is 1.2, the separation of Bletilla striata polysaccharide on normal tissue and model tissues were 44.57 g/cm², 46.64g/cm², there was no significant difference between the two （P=0.039）; The weight percentage of Bletilla striata polysaccharide remained on normal tissues and the model tissues were 57.76%, 60.43%, there was significant difference between the two （P=0.046）. The result showed that the adhesion of Bletilla striata polysaccharide is defferent when they are wetted by the wetting media of defferent pH value, the lower of pH value was, the lower separation and weight percentage remained on normal tissues and model tissues.Part IV: Bletilla striata polysaccharide selective adhesion in vivo evaluationDeveloped the method of barium sulfate reagent in two degrees concentration polysaccharide treated on SD rats, we detected at the time of 1.5, 3, 6, 12, 24 h by X-ray, contrasted the adhesion of the two groups of Bletilla striata polysaccharide on the normal group and the model group, and with two blank groups each. The results showed that the adhesion of Bletilla striata polysaccharide is very well both in model rats and normal rats, the adhesion of the polysaccharide in model group is much stronger （>24 h） than that in normal one （<24 h）.And higher the concentration is, the stronger the ashesion is.It has not been reported that Bletilla striata polysaccharide is as a new type of membrane adhesion to tissues. The theme studied firstly on this issue, which is primarily evaluated and investigated the adhesion of Bletilla striata polysaccharide in vitro and in vivo.The study is confirmed the great adhesion of Bletilla striata polysaccharide to gastric mucosa. The result showed that the remained time in model rats is much longer than that in the normal rats, and the adhesion of Bletilla striata polysaccharide is positively correlated with the concentration, so as to provide a theoretical basis to the further research on exploring Bletilla striata polysaccharide as a new type of adhesive material.更多还原