黄蜀葵茎叶多糖的化学修饰及活性研究
发布时间：2019-05-18 12:15
【摘要】：黄蜀葵(Abelmoschus Manihot(L.)Medicus)为锦葵科秋葵属植物,载于《嘉佑本草》,全株均可入药。黄蜀葵在全国范围内均有分布,目前只有其花被开发为中药及中成药,而茎、叶一般会被废弃或焚烧,直接造成了黄蜀葵生物资源的浪费。国内外关于黄蜀葵茎叶活性成分,尤其是多糖类成分的研究较少。本课题的前期研究发现黄蜀葵茎叶中多糖类成分含量较高,故本研究以废弃的黄蜀葵茎叶为原料,优化多糖的水提工艺,应用化学修饰方法对多糖进行改性,并对多糖的结构特征、免疫调节活性及抗肿瘤活性进行研究,以期为黄蜀葵茎叶资源的开发利用提供科学依据。1.黄蜀葵茎叶多糖的提取分离及组成分析考察液料比、提取时间、提取温度以及提取次数对黄蜀葵茎叶多糖提取率的影响,并应用正交设计优化粗多糖的水提工艺,确定了最优工艺:液料比30:1、提取时间1h、提取温度80℃、提取3次。以蛋白脱除率及多糖保留率为指标,确定Sevag法最佳脱蛋白次数为4次。黄蜀葵茎叶粗多糖(SLAMP)中的总糖含量为39.94%,糖醛酸含量为15.94%,还有5%左右的硫酸基,单糖组成分析表明SLAMP由甘露糖、葡萄糖醛酸、鼠李糖、葡萄糖、半乳糖以及阿拉伯糖组成。应用DEAE-52对SLAMP进行分级,获得了两个均一组分SLAMP-c、SLAMP-d,以及两个非均一组分SLAMP-a、SLAMP-b。SLAMP-a总糖含量达99.76%,分子量分布宽广,几乎不溶于水,单糖组成分析表明其主含葡萄糖,以及少量的甘露糖、半乳糖、阿拉伯糖。SLAMP-b含有66.17%的总糖以及6%左右的蛋白质,其分子量主要分布在1474.5 kDa左右,几乎不溶于水,由六种单糖组成(同SLAMP),其中葡萄糖的比例最高,鼠李糖的比例最低。SLAMP-c与SLAMP-d水溶性较好,均具有三螺旋结构,理化组成基本相似,总糖含量在50%左右,糖醛酸含量为40%左右,含有少量的硫酸基以及蛋白质,SLAMP-c的糖醛酸含量较高。SLAMP-c的分子量为477.8 kDa,SLAMP-d的分子量为264.2 kDa。SLAMP-c与SLAMP-d均由六种单糖组成(同SLAMP),摩尔比较为接近,均主要含葡萄糖醛酸,而甘露糖以及阿拉伯糖的比例较少。2.黄蜀葵茎叶中性多糖的硫酸化、乙酰化修饰及结构解析以几乎不溶于水的SLAMP-a为研究对象,采用氨基磺酸法以及乙酸酐法分别制备了三种硫酸化修饰产物以及三种乙酰化修饰产物。分子量分布表明六种修饰产物中,只有S-SLAMP-a3为均一多糖。S-SLAMP-a3水溶性较好,分子量为1044.2 kDa,DS为0.57,得率为126.57%,主要由葡萄糖组成,含有少量的甘露糖、半乳糖以及阿拉伯糖。S-SLAMP-a1以及S-SLAMP-a2微溶于水,分子量主要分布在1000～1300 kDa左右,均含有少量的低分子多糖,DS为0.1左右,红外光谱中无硫酸酯的特征峰,单糖组成与S-SLAMP-a3接近。此外,只有S-SLAMP-a1具有三螺旋结构。三种乙酰化修饰产物均几乎不溶于水,分子量分布相似,均由较均一的高分子区以及低分子区多糖组成。三种产物的DS分布在0.62～0.85,得率为40～50%,单糖组成分析表明,修饰产物中的甘露糖、半乳糖以及阿拉伯糖的比例均大幅降低,葡萄糖的比例大幅增加。刚果红实验分析表明,Ac-SLAMP-a1与Ac-SLAMP-a2具有三螺旋结构。3.黄蜀葵茎叶多糖、修饰多糖的免疫调节及抗肿瘤活性体外免疫调节活性研究表明,SLAMP以及SLAMP-c与SLAMP-d均显著刺激脾淋巴细胞的增殖,激活RAW264.7释放NO、TNF-α以及IL-6,而SLAMP-a无免疫调节活性,SLAMP-b具有一定的免疫抑制作用。六种修饰产物中,只有S-SLAMP-a3以及Ac-SLAMP-a1显著刺激脾淋巴细胞增殖以及激活RAW264.7产生NO、TNF-α以及IL-6,SLAMP-a经修饰后,免疫调节活性显著增强。根据多糖对肿瘤细胞的敏感性以及给药时间对抑制率的影响,确定肿瘤细胞为A549,给药时间为72h。SLAMP-c与SLAMP-d具有较好的抗肿瘤活性,SLAMP抗肿瘤活性较弱。SLAMP-a抗肿瘤活性很弱,修饰产物S-SLAMP-a3与Ac-SLAMP-a1均可显著提升其抗肿瘤活性,其中,Ac-SLAMP-a1的抗肿瘤活性较强。
[Abstract]:Abelmoschus Maniot (L.) Medicus is a plant of the genus Abelmoschus of the family of Abelmoschus, and can be used as a whole plant. The okra is distributed throughout the country, and only the flower is developed as a traditional Chinese medicine and a Chinese patent medicine, and the stems and the leaves are generally discarded or incinerated, which directly causes the waste of the biological resources of the anemone. The research on the active components of the stem and leaf of the okra, in particular the polysaccharide component, is less. In this study, it was found that the content of polysaccharide in the stem and leaf of Abelmoschus manihot was high, so this study was used as the raw material to optimize the water extraction process of the polysaccharide, and the polysaccharide was modified by chemical modification, and the structural characteristics of the polysaccharide were studied. The immunoregulation activity and the anti-tumor activity were studied with a view to providing a scientific basis for the development and utilization of the stem and leaf resources of Abelmoschus manihot. The extraction and separation of the polysaccharide from the stem and leaf of the okra, the influence of the extraction time, the extraction temperature and the number of times of extraction on the extraction rate of the polysaccharide from the stem and leaf of the okra are investigated. The optimum process is determined by using the orthogonal design to optimize the water extraction process of the crude polysaccharide. The optimal process is as follows: the ratio of liquid to liquid is 30:1, the extraction time is 1 h, Extracting the temperature at 80 & deg; C for 3 times. The protein removal rate and the retention rate of the polysaccharide were the index, and the optimal deproteinization times of the Sevag method were determined to be 4 times. The total sugar content in the crude polysaccharide (SLAMP) was 39.94%, the content of uronic acid was 15.94%, and about 5% of the sulfate group and the monosaccharide composition indicated that the LAMP was composed of mannose, glucuronic acid, rhamnose, glucose, galactose and arabinose. SLAMP-a, LAMP-d and two non-homogeneous components of SLAMP-a and LAMP-b were obtained by DEAE-52. The total sugar content of SLAMP-a, LAMP-b and SLAMP-a was 99.76%, the molecular weight distribution was broad, and it was almost insoluble in water. The analysis of the composition of monosaccharides showed that the main content of glucose and the small amount of mannose and galactose, Arabinose. SLAMP-b has a total sugar of 66.17% and a protein of about 6%, the molecular weight of which is mainly about 1474.5 kDa, is almost insoluble in water and consists of six monosaccharides (same as SLAMP), wherein the proportion of glucose is the highest, and the proportion of rhamnose is the lowest. The water solubility of SLAMP-c and SLAMP-d is good, which has three spiral structures, the physical and chemical composition is basically similar, the content of the total sugar is about 50%, the content of the uronic acid is about 40%, the content of the uronic acid contains a small amount of the sulfuric acid group and the protein, and the content of the uronic acid of the LAMP-c is high. The molecular weight of SLAMP-c is 477.8 kDa, and the molecular weight of SLAMP-d is 264.2 kDa. SLAMP-c and SLAMP-d are all composed of six monosaccharides (same as SLAMP), and the molar ratio is similar to that of SLAMP-d, which mainly contains glucuronic acid, while the proportion of mannose and arabinose is less. In this paper, the sulfation of the neutral polysaccharide of the stem and leaf of the okra, the modification and the structure analysis of the leaves of the stem and leaf of the okra were studied, and the three kinds of sulfated-modified products and the three kinds of modified products were prepared by the method of sulfamic acid and the acid-acid method. The molecular weight distribution indicates that only S-LAMP-a3 is a polysaccharide in the six modified products. The S-LAMP-a3 has a good water-solubility, a molecular weight of 1044.2 kDa, a DS of 0.57, a yield of 126.57%, mainly consisting of glucose, a small amount of mannose, galactose and arabinose. The S-SLAMP-a1 and S-LAMP-a2 are slightly soluble in water, and the molecular weight is mainly about 1000-1300 kDa, and contains a small amount of low molecular polysaccharide, the DS is about 0.1, the characteristic peak of the sulfate-free ester in the infrared spectrum and the composition of the monosaccharides are close to the S-SLAMP-a3. In addition, only S-SLAMP-a1 has a three-spiral structure. The three kinds of B-type modified products are almost insoluble in water and the molecular weight distribution is similar, and is composed of a higher polymer area and a low-molecular-area polysaccharide. The distribution of DS in the three products was 0.62-0.85, the yield was 40-50%, and the composition of the monosaccharides showed that the proportion of mannose, galactose and arabinose in the modified product was significantly reduced, and the proportion of glucose increased greatly. The results show that Ac-SLAMP-a1 and Ac-SLAMP-a2 have three spiral structures. The effects of SLAMP and SLAMP-c and SLAMP-d on the proliferation of splenocytes, the activation of RAW264.7 and the release of NO, TNF-1 and IL-6, while the SLAMP-a had no immunomodulatory activity. SLAMP-b has a certain immunosuppressive effect. In the six modified products, only S-SLAMP-a3 and Ac-SLAMP-a1 significantly stimulate the proliferation of splenic lymphocytes and activate RAW264.7 to produce NO, TNF-1, and IL-6, and the immunomodulatory activity of SLAMP-a is significantly enhanced. According to the sensitivity of the polysaccharide to the tumor cells and the effect of the administration time on the inhibition rate, the tumor cells are determined to be A549, the administration time is 72h, the LAMP-c and the LAMP-d have better anti-tumor activity, and the anti-tumor activity of the LAMP is weak. The anti-tumor activity of SLAMP-a is very weak, and the anti-tumor activity of the modified product S-LAMP-a3 and Ac-SLAMP-a1 can be remarkably improved, and the anti-tumor activity of the Ac-SLAMP-a1 is strong.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285
本文编号：2479989
[Abstract]:Abelmoschus Maniot (L.) Medicus is a plant of the genus Abelmoschus of the family of Abelmoschus, and can be used as a whole plant. The okra is distributed throughout the country, and only the flower is developed as a traditional Chinese medicine and a Chinese patent medicine, and the stems and the leaves are generally discarded or incinerated, which directly causes the waste of the biological resources of the anemone. The research on the active components of the stem and leaf of the okra, in particular the polysaccharide component, is less. In this study, it was found that the content of polysaccharide in the stem and leaf of Abelmoschus manihot was high, so this study was used as the raw material to optimize the water extraction process of the polysaccharide, and the polysaccharide was modified by chemical modification, and the structural characteristics of the polysaccharide were studied. The immunoregulation activity and the anti-tumor activity were studied with a view to providing a scientific basis for the development and utilization of the stem and leaf resources of Abelmoschus manihot. The extraction and separation of the polysaccharide from the stem and leaf of the okra, the influence of the extraction time, the extraction temperature and the number of times of extraction on the extraction rate of the polysaccharide from the stem and leaf of the okra are investigated. The optimum process is determined by using the orthogonal design to optimize the water extraction process of the crude polysaccharide. The optimal process is as follows: the ratio of liquid to liquid is 30:1, the extraction time is 1 h, Extracting the temperature at 80 & deg; C for 3 times. The protein removal rate and the retention rate of the polysaccharide were the index, and the optimal deproteinization times of the Sevag method were determined to be 4 times. The total sugar content in the crude polysaccharide (SLAMP) was 39.94%, the content of uronic acid was 15.94%, and about 5% of the sulfate group and the monosaccharide composition indicated that the LAMP was composed of mannose, glucuronic acid, rhamnose, glucose, galactose and arabinose. SLAMP-a, LAMP-d and two non-homogeneous components of SLAMP-a and LAMP-b were obtained by DEAE-52. The total sugar content of SLAMP-a, LAMP-b and SLAMP-a was 99.76%, the molecular weight distribution was broad, and it was almost insoluble in water. The analysis of the composition of monosaccharides showed that the main content of glucose and the small amount of mannose and galactose, Arabinose. SLAMP-b has a total sugar of 66.17% and a protein of about 6%, the molecular weight of which is mainly about 1474.5 kDa, is almost insoluble in water and consists of six monosaccharides (same as SLAMP), wherein the proportion of glucose is the highest, and the proportion of rhamnose is the lowest. The water solubility of SLAMP-c and SLAMP-d is good, which has three spiral structures, the physical and chemical composition is basically similar, the content of the total sugar is about 50%, the content of the uronic acid is about 40%, the content of the uronic acid contains a small amount of the sulfuric acid group and the protein, and the content of the uronic acid of the LAMP-c is high. The molecular weight of SLAMP-c is 477.8 kDa, and the molecular weight of SLAMP-d is 264.2 kDa. SLAMP-c and SLAMP-d are all composed of six monosaccharides (same as SLAMP), and the molar ratio is similar to that of SLAMP-d, which mainly contains glucuronic acid, while the proportion of mannose and arabinose is less. In this paper, the sulfation of the neutral polysaccharide of the stem and leaf of the okra, the modification and the structure analysis of the leaves of the stem and leaf of the okra were studied, and the three kinds of sulfated-modified products and the three kinds of modified products were prepared by the method of sulfamic acid and the acid-acid method. The molecular weight distribution indicates that only S-LAMP-a3 is a polysaccharide in the six modified products. The S-LAMP-a3 has a good water-solubility, a molecular weight of 1044.2 kDa, a DS of 0.57, a yield of 126.57%, mainly consisting of glucose, a small amount of mannose, galactose and arabinose. The S-SLAMP-a1 and S-LAMP-a2 are slightly soluble in water, and the molecular weight is mainly about 1000-1300 kDa, and contains a small amount of low molecular polysaccharide, the DS is about 0.1, the characteristic peak of the sulfate-free ester in the infrared spectrum and the composition of the monosaccharides are close to the S-SLAMP-a3. In addition, only S-SLAMP-a1 has a three-spiral structure. The three kinds of B-type modified products are almost insoluble in water and the molecular weight distribution is similar, and is composed of a higher polymer area and a low-molecular-area polysaccharide. The distribution of DS in the three products was 0.62-0.85, the yield was 40-50%, and the composition of the monosaccharides showed that the proportion of mannose, galactose and arabinose in the modified product was significantly reduced, and the proportion of glucose increased greatly. The results show that Ac-SLAMP-a1 and Ac-SLAMP-a2 have three spiral structures. The effects of SLAMP and SLAMP-c and SLAMP-d on the proliferation of splenocytes, the activation of RAW264.7 and the release of NO, TNF-1 and IL-6, while the SLAMP-a had no immunomodulatory activity. SLAMP-b has a certain immunosuppressive effect. In the six modified products, only S-SLAMP-a3 and Ac-SLAMP-a1 significantly stimulate the proliferation of splenic lymphocytes and activate RAW264.7 to produce NO, TNF-1, and IL-6, and the immunomodulatory activity of SLAMP-a is significantly enhanced. According to the sensitivity of the polysaccharide to the tumor cells and the effect of the administration time on the inhibition rate, the tumor cells are determined to be A549, the administration time is 72h, the LAMP-c and the LAMP-d have better anti-tumor activity, and the anti-tumor activity of the LAMP is weak. The anti-tumor activity of SLAMP-a is very weak, and the anti-tumor activity of the modified product S-LAMP-a3 and Ac-SLAMP-a1 can be remarkably improved, and the anti-tumor activity of the Ac-SLAMP-a1 is strong.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285
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1 潘欣欣;黄蜀葵茎叶多糖的化学修饰及活性研究[D];南京中医药大学;2017年
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