霍山铁皮石斛多糖DOPA-1的结构特征及对HepG2细胞凋亡的影响
发布时间：2018-08-22 18:17
【摘要】：铁皮石斛为兰科植物铁皮石斛(Dendrobium officinale Kimura et Migo)的干燥茎,是我国药食两用名贵中药材之一,具有益胃生津,滋阴清热之功效,享有“中华九大仙草之首”之美誉。铁皮石斛在我国主要分布于安徽、浙江、湖南、云南等地,其中安徽霍山铁皮石斛又名万丈须,生长条件极为苛刻,作为安徽省道地药材一直倍受社会各界的关注,被历代研究者奉为是石斛中的上品,近年来关于霍山铁皮石斛的研究渐为盛行。铁皮石斛中含有多糖、生物碱、联苄类及氨基酸等多种活性成分,多糖作为其主要有效成分之一,在抗肿瘤、抗衰老和抗氧化等方面具有显著疗效。本论文以霍山铁皮石斛为原料,优选霍山铁皮石斛多糖脱蛋白工艺,采用DEAE-52纤维素色谱柱和Sephadex G-100凝胶色谱柱分离纯化石斛多糖,运用现代色谱分析法及化学法对其结构特征进行初步分析,并研究其对人肝癌细胞(HepG2)增殖的抑制作用。主要研究内容如下:1.霍山铁皮石斛多糖提取工艺研究。以霍山铁皮石斛鲜条为原料,以水提醇沉法提取石斛水溶性粗多糖组分,冷冻干燥后测其得率为4.2%。并以蛋白脱除率和多糖损失率为指标,对Sevag法、酶法及酶-Sevag法的脱蛋白效果进行比较,确定最佳方法为酶-Sevag法,最佳条件为:酶添加量0.5 mL,酶解温度35 oC,酶解时间40 min,再结合Sevag法萃取3次,此条件下蛋白脱除率、多糖损失率分别为81.58%、15.63%。2.霍山铁皮石斛多糖分离纯化及组成分析。霍山铁皮石斛粗多糖经DEAE-52纤维素柱和Sephadex G-100凝胶柱分离纯化,得多糖组分DOPA-1;采用紫外光谱法对DOPA-1进行全波长扫描,图谱在260 nm、280 nm处无紫外特征吸收,表明DOPA-1不含核酸和蛋白质;采用HPLC法对DOPA-1进行纯度鉴定,HPLC图谱只显示单一对称峰,表明DOPA-1为均一多糖;采用GC法分析DOPA-1的单糖组成,表明DOPA-1主要由甘露糖、葡萄糖和半乳糖组成,摩尔比为1:0.42:0.27;采用苯酚硫酸法和硫酸咔唑法分别测得DOPA-1的糖含量为83.1%,糖醛酸含量为2.31%。3.霍山铁皮石斛多糖DOPA-1理化性质及结构特征分析。运用HPLC、IR、NMR、高碘酸氧化-smith降解等现代分析方法,对DOPA-1的理化特性及结构特征进行研究,结果表明DOPA-1为一种白色絮状物,无味,易溶于水,不溶于有机溶剂,比旋光度为+128.19°,且不含淀粉、氨基酸及蛋白质,分子量为2.29×105Da;推测DOPA-1中甘露糖主要由1→3糖苷键连接,葡萄糖和半乳糖由1→2及1→6糖苷键连接;DOPA-1有典型的糖类特征吸收峰,存在β-D-甘露吡喃糖苷,且具有三股螺旋构象。4.霍山铁皮石斛多糖DOPA-1对HepG2细胞凋亡的影响。研究霍山铁皮石斛多糖DOPA-1对体外培养的HepG2细胞增殖的抑制作用,并探究其可能的作用机理。MTT法检测细胞活性,表明DOPA-1可在体外抑制HepG2细胞的生长,呈一定浓度依赖性;细胞划痕实验显示DOPA-1对HepG2细胞的迁移能力有一定的抑制作用;DCFH-DA探针法检测细胞内活性氧(ROS)水平,分析发现DOPA-1通过诱导ROS的产生损伤线粒体功能,从而诱导HepG2细胞凋亡;JC-1染色结果证明随着DOPA-1浓度的增加,线粒体膜通透性增大,膜电位不断降低,最终导致HepG2细胞的凋亡;从流式细胞术检测结果分析,HepG2细胞凋亡率随DOPA-1浓度的增加而升高,表明DOPA-1可诱导HepG2细胞凋亡;Western blot检测Bcl-2、Bax的表达,发现DOPA-1诱导HepG2细胞凋亡的分子机制可能与下调Bcl-2的表达水平,上调Bax的表达水平有关。
[Abstract]:Dendrobium officinale Kimura et Migo is the dried stem of Dendrobium officinale. It is one of the precious medicinal herbs for both medicinal and edible purposes in China. Dendrobium officinale Kimura et Migo has the effect of nourishing stomach, nourishing yin and clearing away heat. It enjoys the reputation of "the first of nine Chinese celestial plants". Dendrobium officinale is mainly distributed in Anhui, Zhejiang, Hunan, Yunnan and other places in China. Dendrobium candidum Huoshan is also known as Wanzhangxu, and its growth conditions are extremely harsh. As a genuine medicinal material in Anhui Province, it has attracted much attention from all walks of life. It has been regarded as the top grade of Dendrobium candidum by researchers of all ages. In recent years, the research on Dendrobium candidum huoshanense has become popular. Dendrobium candidum candidum candidum contains polysaccharides, alkaloids, bibenzyls and amino acids and other activities. Dendrobium officinale polysaccharides as one of its main active ingredients, has significant effects in anti-tumor, anti-aging and anti-oxidation. In this paper, Dendrobium officinale polysaccharides deproteinization process was optimized with Dendrobium officinale as raw material. Dendrobium officinale polysaccharides were separated and purified by DEAE-cellulose chromatography column and Sephadex G-100 gel chromatography column. The main research contents are as follows: 1. Extraction technology of Polysaccharides from Dendrobium huoshanense was studied. The crude polysaccharides from Dendrobium huoshanense fresh strips were extracted by water extraction and ethanol precipitation, and then lyophilized. The yield was 4.2%. The effects of Sevag, Enzyme and Enzyme-Sevag methods on protein removal and polysaccharide loss were compared. The optimum conditions were as follows: enzyme dosage 0.5 mL, enzymatic hydrolysis temperature 35 oC, enzymatic hydrolysis time 40 min, and combined with Sevag method for three times. Under these conditions, the protein removal rate was more than that by enzyme-Sevag method. The loss rate of sugar was 81.58% and 15.63% respectively. 2. The polysaccharide of Dendrobium candidum Huoshan was separated and purified by DEAE-52 cellulose column and Sephadex G-100 gel column, and the polysaccharide component DOPA-1 was obtained. The results showed that DOPA-1 contained no nucleic acid and protein; the purity of DOPA-1 was identified by HPLC, and the chromatogram showed only a single symmetrical peak, indicating that DOPA-1 was a homogeneous polysaccharide; the monosaccharide composition of DOPA-1 was analyzed by GC, indicating that DOPA-1 was mainly composed of mannose, glucose and galactose, the molar ratio was 1:0.42:0.27; phenol sulfuric acid method and carbazole sulfate method were used. The sugar content of DOPA-1 was 83.1% and the content of glucuronic acid was 2.31%. 3. Physicochemical properties and structural characteristics of polysaccharide DOPA-1 from Dendrobium candidum Huoshanense were analyzed. The physicochemical properties and structural characteristics of DOPA-1 were studied by modern analytical methods such as HPLC, IR, NMR and oxidation-smith degradation of periodate. The results showed that DOPA-1 was a white flocculent without any floc. Taste, soluble in water, insoluble in organic solvents, specific optical rotation is + 128.19 degrees, and does not contain starch, amino acids and proteins, molecular weight is 2.29 *105Da; It is speculated that mannose in DOPA-1 is mainly linked by 1_3 glycoside bond, glucose and galactose are linked by 1_2 and 1_glycoside bond; DOPA-1 has typical absorption peaks of carbohydrates, and exists beta-D-mannose. The effect of polysaccharide DOPA-1 from Dendrobium huoshanense on the apoptosis of HepG2 cells was studied. The inhibitory effect of polysaccharide DOPA-1 from Dendrobium huoshanense on the proliferation of HepG2 cells in vitro was studied and its possible mechanism was explored. MTT assay showed that DOPA-1 could inhibit the growth of HepG2 cells in vitro. Cell scratch assay showed that DOPA-1 inhibited the migration of HepG2 cells to a certain extent; DCFH-DA probe assay was used to detect the level of reactive oxygen species (ROS) in HepG2 cells, and it was found that DOPA-1 could induce apoptosis of HepG2 cells by inducing the production of ROS. Mitochondrial membrane permeability increased, membrane potential decreased, and ultimately led to apoptosis of HepG2 cells; flow cytometry analysis showed that the apoptosis rate of HepG2 cells increased with the increase of DOPA-1 concentration, indicating that DOPA-1 could induce apoptosis of HepG2 cells; Western blot detection of Bcl-2, Bax expression, found that DOPA-1 induced apoptosis of HepG2 cells. It may be related to down regulating the expression level of Bcl-2 and up regulating the expression level of Bax.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285
[Abstract]:Dendrobium officinale Kimura et Migo is the dried stem of Dendrobium officinale. It is one of the precious medicinal herbs for both medicinal and edible purposes in China. Dendrobium officinale Kimura et Migo has the effect of nourishing stomach, nourishing yin and clearing away heat. It enjoys the reputation of "the first of nine Chinese celestial plants". Dendrobium officinale is mainly distributed in Anhui, Zhejiang, Hunan, Yunnan and other places in China. Dendrobium candidum Huoshan is also known as Wanzhangxu, and its growth conditions are extremely harsh. As a genuine medicinal material in Anhui Province, it has attracted much attention from all walks of life. It has been regarded as the top grade of Dendrobium candidum by researchers of all ages. In recent years, the research on Dendrobium candidum huoshanense has become popular. Dendrobium candidum candidum candidum contains polysaccharides, alkaloids, bibenzyls and amino acids and other activities. Dendrobium officinale polysaccharides as one of its main active ingredients, has significant effects in anti-tumor, anti-aging and anti-oxidation. In this paper, Dendrobium officinale polysaccharides deproteinization process was optimized with Dendrobium officinale as raw material. Dendrobium officinale polysaccharides were separated and purified by DEAE-cellulose chromatography column and Sephadex G-100 gel chromatography column. The main research contents are as follows: 1. Extraction technology of Polysaccharides from Dendrobium huoshanense was studied. The crude polysaccharides from Dendrobium huoshanense fresh strips were extracted by water extraction and ethanol precipitation, and then lyophilized. The yield was 4.2%. The effects of Sevag, Enzyme and Enzyme-Sevag methods on protein removal and polysaccharide loss were compared. The optimum conditions were as follows: enzyme dosage 0.5 mL, enzymatic hydrolysis temperature 35 oC, enzymatic hydrolysis time 40 min, and combined with Sevag method for three times. Under these conditions, the protein removal rate was more than that by enzyme-Sevag method. The loss rate of sugar was 81.58% and 15.63% respectively. 2. The polysaccharide of Dendrobium candidum Huoshan was separated and purified by DEAE-52 cellulose column and Sephadex G-100 gel column, and the polysaccharide component DOPA-1 was obtained. The results showed that DOPA-1 contained no nucleic acid and protein; the purity of DOPA-1 was identified by HPLC, and the chromatogram showed only a single symmetrical peak, indicating that DOPA-1 was a homogeneous polysaccharide; the monosaccharide composition of DOPA-1 was analyzed by GC, indicating that DOPA-1 was mainly composed of mannose, glucose and galactose, the molar ratio was 1:0.42:0.27; phenol sulfuric acid method and carbazole sulfate method were used. The sugar content of DOPA-1 was 83.1% and the content of glucuronic acid was 2.31%. 3. Physicochemical properties and structural characteristics of polysaccharide DOPA-1 from Dendrobium candidum Huoshanense were analyzed. The physicochemical properties and structural characteristics of DOPA-1 were studied by modern analytical methods such as HPLC, IR, NMR and oxidation-smith degradation of periodate. The results showed that DOPA-1 was a white flocculent without any floc. Taste, soluble in water, insoluble in organic solvents, specific optical rotation is + 128.19 degrees, and does not contain starch, amino acids and proteins, molecular weight is 2.29 *105Da; It is speculated that mannose in DOPA-1 is mainly linked by 1_3 glycoside bond, glucose and galactose are linked by 1_2 and 1_glycoside bond; DOPA-1 has typical absorption peaks of carbohydrates, and exists beta-D-mannose. The effect of polysaccharide DOPA-1 from Dendrobium huoshanense on the apoptosis of HepG2 cells was studied. The inhibitory effect of polysaccharide DOPA-1 from Dendrobium huoshanense on the proliferation of HepG2 cells in vitro was studied and its possible mechanism was explored. MTT assay showed that DOPA-1 could inhibit the growth of HepG2 cells in vitro. Cell scratch assay showed that DOPA-1 inhibited the migration of HepG2 cells to a certain extent; DCFH-DA probe assay was used to detect the level of reactive oxygen species (ROS) in HepG2 cells, and it was found that DOPA-1 could induce apoptosis of HepG2 cells by inducing the production of ROS. Mitochondrial membrane permeability increased, membrane potential decreased, and ultimately led to apoptosis of HepG2 cells; flow cytometry analysis showed that the apoptosis rate of HepG2 cells increased with the increase of DOPA-1 concentration, indicating that DOPA-1 could induce apoptosis of HepG2 cells; Western blot detection of Bcl-2, Bax expression, found that DOPA-1 induced apoptosis of HepG2 cells. It may be related to down regulating the expression level of Bcl-2 and up regulating the expression level of Bax.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285
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