【摘要】：鐵皮石斛為蘭科植物鐵皮石斛(Dendrobium officinale Kimura et Migo)的干燥莖,是我國藥食兩用名貴中藥材之一,具有益胃生津,滋陰清熱之功效,享有“中華九大仙草之首”之美譽(yù)。鐵皮石斛在我國主要分布于安徽、浙江、湖南、云南等地,其中安徽霍山鐵皮石斛又名萬丈須,生長條件極為苛刻,作為安徽省道地藥材一直倍受社會各界的關(guān)注,被歷代研究者奉為是石斛中的上品,近年來關(guān)于霍山鐵皮石斛的研究漸為盛行。鐵皮石斛中含有多糖、生物堿、聯(lián)芐類及氨基酸等多種活性成分,多糖作為其主要有效成分之一,在抗腫瘤、抗衰老和抗氧化等方面具有顯著療效。本論文以霍山鐵皮石斛為原料,優(yōu)選霍山鐵皮石斛多糖脫蛋白工藝,采用DEAE-52纖維素色譜柱和Sephadex G-100凝膠色譜柱分離純化石斛多糖,運(yùn)用現(xiàn)代色譜分析法及化學(xué)法對其結(jié)構(gòu)特征進(jìn)行初步分析,并研究其對人肝癌細(xì)胞(HepG2)增殖的抑制作用。主要研究內(nèi)容如下:1.霍山鐵皮石斛多糖提取工藝研究。以霍山鐵皮石斛鮮條為原料,以水提醇沉法提取石斛水溶性粗多糖組分,冷凍干燥后測其得率為4.2%。并以蛋白脫除率和多糖損失率為指標(biāo),對Sevag法、酶法及酶-Sevag法的脫蛋白效果進(jìn)行比較,確定最佳方法為酶-Sevag法,最佳條件為:酶添加量0.5 mL,酶解溫度35 oC,酶解時間40 min,再結(jié)合Sevag法萃取3次,此條件下蛋白脫除率、多糖損失率分別為81.58%、15.63%。2.霍山鐵皮石斛多糖分離純化及組成分析�；羯借F皮石斛粗多糖經(jīng)DEAE-52纖維素柱和Sephadex G-100凝膠柱分離純化,得多糖組分DOPA-1;采用紫外光譜法對DOPA-1進(jìn)行全波長掃描,圖譜在260 nm、280 nm處無紫外特征吸收,表明DOPA-1不含核酸和蛋白質(zhì);采用HPLC法對DOPA-1進(jìn)行純度鑒定,HPLC圖譜只顯示單一對稱峰,表明DOPA-1為均一多糖;采用GC法分析DOPA-1的單糖組成,表明DOPA-1主要由甘露糖、葡萄糖和半乳糖組成,摩爾比為1:0.42:0.27;采用苯酚硫酸法和硫酸咔唑法分別測得DOPA-1的糖含量為83.1%,糖醛酸含量為2.31%。3.霍山鐵皮石斛多糖DOPA-1理化性質(zhì)及結(jié)構(gòu)特征分析。運(yùn)用HPLC、IR、NMR、高碘酸氧化-smith降解等現(xiàn)代分析方法,對DOPA-1的理化特性及結(jié)構(gòu)特征進(jìn)行研究,結(jié)果表明DOPA-1為一種白色絮狀物,無味,易溶于水,不溶于有機(jī)溶劑,比旋光度為+128.19°,且不含淀粉、氨基酸及蛋白質(zhì),分子量為2.29×105Da;推測DOPA-1中甘露糖主要由1→3糖苷鍵連接,葡萄糖和半乳糖由1→2及1→6糖苷鍵連接;DOPA-1有典型的糖類特征吸收峰,存在β-D-甘露吡喃糖苷,且具有三股螺旋構(gòu)象。4.霍山鐵皮石斛多糖DOPA-1對HepG2細(xì)胞凋亡的影響。研究霍山鐵皮石斛多糖DOPA-1對體外培養(yǎng)的HepG2細(xì)胞增殖的抑制作用,并探究其可能的作用機(jī)理。MTT法檢測細(xì)胞活性,表明DOPA-1可在體外抑制HepG2細(xì)胞的生長,呈一定濃度依賴性;細(xì)胞劃痕實(shí)驗(yàn)顯示DOPA-1對HepG2細(xì)胞的遷移能力有一定的抑制作用;DCFH-DA探針法檢測細(xì)胞內(nèi)活性氧(ROS)水平,分析發(fā)現(xiàn)DOPA-1通過誘導(dǎo)ROS的產(chǎn)生損傷線粒體功能,從而誘導(dǎo)HepG2細(xì)胞凋亡;JC-1染色結(jié)果證明隨著DOPA-1濃度的增加,線粒體膜通透性增大,膜電位不斷降低,最終導(dǎo)致HepG2細(xì)胞的凋亡;從流式細(xì)胞術(shù)檢測結(jié)果分析,HepG2細(xì)胞凋亡率隨DOPA-1濃度的增加而升高,表明DOPA-1可誘導(dǎo)HepG2細(xì)胞凋亡;Western blot檢測Bcl-2、Bax的表達(dá),發(fā)現(xiàn)DOPA-1誘導(dǎo)HepG2細(xì)胞凋亡的分子機(jī)制可能與下調(diào)Bcl-2的表達(dá)水平,上調(diào)Bax的表達(dá)水平有關(guān)。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284;R285

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郝龍平;晏嘉澤;靳艷;郭明;;刺玫果多糖的制備及其抗氧化活性[J];中成藥;2016年12期

2 呂連杰;亢建民;;五味子多糖對人膠質(zhì)瘤U251細(xì)胞增殖的影響[J];中國老年學(xué)雜志;2016年02期

3 封燕;貢小輝;韋德群;劉英坤;趙明;于小鳳;OPEYEMI Joshua Olatunji;焦心怡;歐陽臻;;金蟬花多糖的抗氧化活性及結(jié)構(gòu)分析[J];食品科學(xué);2016年13期

4 吳月國;趙錚蓉;張萍;辛艷飛;劉驊;;鐵皮石斛復(fù)方顆粒對小鼠免疫功能影響的研究[J];中華中醫(yī)藥學(xué)刊;2015年08期

5 徐航;朱銳;劉瑋;高向東;;多糖高級結(jié)構(gòu)解析方法的研究進(jìn)展[J];藥學(xué)進(jìn)展;2015年05期

6 宓文佳;陳素紅;呂圭源;李燕;張sダ，

本文編號：2197913