黃蜀葵莖葉多糖的化學(xué)修飾及活性研究
發(fā)布時間：2019-05-18 12:15
【摘要】：黃蜀葵(Abelmoschus Manihot(L.)Medicus)為錦葵科秋葵屬植物,載于《嘉佑本草》,全株均可入藥。黃蜀葵在全國范圍內(nèi)均有分布,目前只有其花被開發(fā)為中藥及中成藥,而莖、葉一般會被廢棄或焚燒,直接造成了黃蜀葵生物資源的浪費(fèi)。國內(nèi)外關(guān)于黃蜀葵莖葉活性成分,尤其是多糖類成分的研究較少。本課題的前期研究發(fā)現(xiàn)黃蜀葵莖葉中多糖類成分含量較高,故本研究以廢棄的黃蜀葵莖葉為原料,優(yōu)化多糖的水提工藝,應(yīng)用化學(xué)修飾方法對多糖進(jìn)行改性,并對多糖的結(jié)構(gòu)特征、免疫調(diào)節(jié)活性及抗腫瘤活性進(jìn)行研究,以期為黃蜀葵莖葉資源的開發(fā)利用提供科學(xué)依據(jù)。1.黃蜀葵莖葉多糖的提取分離及組成分析考察液料比、提取時間、提取溫度以及提取次數(shù)對黃蜀葵莖葉多糖提取率的影響,并應(yīng)用正交設(shè)計(jì)優(yōu)化粗多糖的水提工藝,確定了最優(yōu)工藝:液料比30:1、提取時間1h、提取溫度80℃、提取3次。以蛋白脫除率及多糖保留率為指標(biāo),確定Sevag法最佳脫蛋白次數(shù)為4次。黃蜀葵莖葉粗多糖(SLAMP)中的總糖含量為39.94%,糖醛酸含量為15.94%,還有5%左右的硫酸基,單糖組成分析表明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組成。應(yīng)用DEAE-52對SLAMP進(jìn)行分級,獲得了兩個均一組分SLAMP-c、SLAMP-d,以及兩個非均一組分SLAMP-a、SLAMP-b。SLAMP-a總糖含量達(dá)99.76%,分子量分布寬廣,幾乎不溶于水,單糖組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%的總糖以及6%左右的蛋白質(zhì),其分子量主要分布在1474.5 kDa左右,幾乎不溶于水,由六種單糖組成(同SLAMP),其中葡萄糖的比例最高,鼠李糖的比例最低。SLAMP-c與SLAMP-d水溶性較好,均具有三螺旋結(jié)構(gòu),理化組成基本相似,總糖含量在50%左右,糖醛酸含量為40%左右,含有少量的硫酸基以及蛋白質(zhì),SLAMP-c的糖醛酸含量較高。SLAMP-c的分子量為477.8 kDa,SLAMP-d的分子量為264.2 kDa。SLAMP-c與SLAMP-d均由六種單糖組成(同SLAMP),摩爾比較為接近,均主要含葡萄糖醛酸,而甘露糖以及阿拉伯糖的比例較少。2.黃蜀葵莖葉中性多糖的硫酸化、乙�；揎椉敖Y(jié)構(gòu)解析以幾乎不溶于水的SLAMP-a為研究對象,采用氨基磺酸法以及乙酸酐法分別制備了三種硫酸化修飾產(chǎn)物以及三種乙�；揎棶a(chǎn)物。分子量分布表明六種修飾產(chǎn)物中,只有S-SLAMP-a3為均一多糖。S-SLAMP-a3水溶性較好,分子量為1044.2 kDa,DS為0.57,得率為126.57%,主要由葡萄糖組成,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為0.1左右,紅外光譜中無硫酸酯的特征峰,單糖組成與S-SLAMP-a3接近。此外,只有S-SLAMP-a1具有三螺旋結(jié)構(gòu)。三種乙�；揎棶a(chǎn)物均幾乎不溶于水,分子量分布相似,均由較均一的高分子區(qū)以及低分子區(qū)多糖組成。三種產(chǎn)物的DS分布在0.62～0.85,得率為40～50%,單糖組成分析表明,修飾產(chǎn)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。剛果紅實(shí)驗(yàn)分析表明,Ac-SLAMP-a1與Ac-SLAMP-a2具有三螺旋結(jié)構(gòu)。3.黃蜀葵莖葉多糖、修飾多糖的免疫調(diào)節(jié)及抗腫瘤活性體外免疫調(diào)節(jié)活性研究表明,SLAMP以及SLAMP-c與SLAMP-d均顯著刺激脾淋巴細(xì)胞的增殖,激活RAW264.7釋放NO、TNF-α以及IL-6,而SLAMP-a無免疫調(diào)節(jié)活性,SLAMP-b具有一定的免疫抑制作用。六種修飾產(chǎn)物中,只有S-SLAMP-a3以及Ac-SLAMP-a1顯著刺激脾淋巴細(xì)胞增殖以及激活RAW264.7產(chǎn)生NO、TNF-α以及IL-6,SLAMP-a經(jīng)修飾后,免疫調(diào)節(jié)活性顯著增強(qiáng)。根據(jù)多糖對腫瘤細(xì)胞的敏感性以及給藥時間對抑制率的影響,確定腫瘤細(xì)胞為A549,給藥時間為72h。SLAMP-c與SLAMP-d具有較好的抗腫瘤活性,SLAMP抗腫瘤活性較弱。SLAMP-a抗腫瘤活性很弱,修飾產(chǎn)物S-SLAMP-a3與Ac-SLAMP-a1均可顯著提升其抗腫瘤活性,其中,Ac-SLAMP-a1的抗腫瘤活性較強(qiáng)。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284;R285
本文編號：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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284;R285
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1 潘欣欣;黃蜀葵莖葉多糖的化學(xué)修飾及活性研究[D];南京中醫(yī)藥大學(xué);2017年
，本文編號：2479989
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