【作者】 刘晓宇；

【导师】 台桂花；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 黄药子（Rhizoma Dioscorea Bulbiferac），是薯蓣科植物黄独（Dioscorea bulbifera L.）的块茎，作为中药在我国已有2000多年的历史，在中药处方中用于治疗咽喉肿痛、癌肿、咳血、疮疡肿毒等。多糖是黄药子的重要活性成分，已有文献报道黄药子多糖具有调节免疫、抗肿瘤等活性，但有关黄药子多糖的结构研究还未见报道，缺乏对黄药子多糖的全面系统的认识。本论文对黄药子多糖进行分级纯化，明确了黄药子中性多糖的组成及结构，并从中获得主要成分进行硫酸化和羧甲基化修饰，再通过对肿瘤细胞增殖和半乳凝素-3抑制活性的测定，比较多糖在修饰前后的抗癌活性，从而为黄药子多糖的进一步研究与开发提供理论基础。本论文通过热水煮提和乙醇沉淀得到黄药子水溶性总多糖DBP，将DBP用DEAE-纤维素柱层析进行分级纯化。分别利用浓度为0、0.1、0.3和0.5M的氯化钠水溶液进行分步洗脱，得到相应的四个级分：中性级分DBPN和三个酸性级分DBPA-1、DBPA-2和DBPA-3。通过酶学方法、凝胶渗透色谱和高效液相色谱法分析黄药子多糖各级分的基本结构特征：中性糖DBPN主要由半乳聚糖和葡聚糖组成；DBPA-1含有同聚半乳糖醛酸（homogalacturonan，HG）和半乳聚糖结构；DBPA-2含有HG结构，并可能含有少量I型聚鼠李半乳糖醛酸（rhamnogalacturonan I，RG-I）结构；DBPA-3以典型的HG结构为主。利用分级醇沉法，将DBPN进一步分级纯化得到分子量均一的6个级分DBPN-1~DBPN-6，重均分子量依次为22.4、1.0、0.73、0.54、0.36和0.26kDa，这6个级分主要由半乳糖和葡萄糖构成。通过对六个级分酶解特征的分析验证了黄药子中性多糖中存在独立的半乳聚糖和葡聚糖。应用高碘酸氧化、Smith降解、甲基化、傅立叶红外光谱和核磁共振等方法确定了半乳聚糖的结构为没有分支的线性β-1,4-半乳聚糖（DBPN-UD），葡聚糖为以α-1,4-Glcp为主链，并存在少量6位分支的淀粉样多糖。线性β-1,4-半乳聚糖是DBPN的主要成分，这是首次从黄药子中提取得到天然的β-1,4-半乳聚糖。因为β-1,4-半乳聚糖在黄药子中性多糖中含量高并且制备方法简单，所以黄药子是制备β-1,4-半乳聚糖的优良选材。以DBPN-UD为底物，利用三氧化硫-吡啶法制备得到6个不同取代度的硫酸化半乳聚糖， S-DBPN-UD-0.1、 S-DBPN-UD-0.25、 S-DBPN-UD-0.5、 S-DBPN-UD-1、 S-DBPN-UD-2和S-DBPN-UD-4，取代度分别为0.12、0.26、0.63、1.42、0.92和1.02；采用氢氧化钠-氯乙酸反应体系，以异丙醇为溶剂，制备得到6个不同取代度的羧甲基化半乳聚糖， CM-DBPN-UD-0.1、 CM-DBPN-UD-0.25、 CM-DBPN-UD-0.5、 CM-DBPN-UD-1、C M-DBPN-UD-2和CM-DBPN-UD-3，取代度分别为0.16、0.21、0.26、0.46、0.62和0.70。最后我们利用肿瘤细胞增殖和半乳凝素-3抑制实验比较了修饰前后多糖的抗肿瘤活性。抑制肿瘤细胞增殖实验中，我们以小鼠肉瘤细胞S180和两种人结肠癌细胞株HCT-116和HT-29为实验对象，测定了DBPN及其衍生物的活性，结果表明，硫酸化和羧甲基化修饰可以增强DBPN-UD对肿瘤细胞增殖的抑制活性，抑制活性具有浓度依赖性，并随取代度的增大而增强。对半乳凝素-3的抑制实验结果表明，DBPN-UD具有很好的抑制活性，在12.5mg/mL时可以完全抑制由半乳凝素-3引起的红细胞凝集，因此DBPN-UD具有很好的开发为半乳凝素-3抑制剂的潜力。但硫酸化和羧甲基化修饰会降低DBPN-UD对半乳凝素-3的抑制活性。本论文的研究结果为黄药子多糖的进一步研究与开发提供了理论基础。更多还原

【Abstract】 Dioscorea bulbifera L.(D. bulbifera) is a liana widely distributed throughout the tropics and temperate regions. The rhizome of D. bulbifera has been used as a traditional Chinese Medicine for over2000years to treat thyroid diseases, spasmodic, leprosy and tumors. Polysaccharides are considered as the active components of D. bulbifera. It has been reported that the polysaccharide extracts from D. bulbifera have many pharmaceutical activities. However, there is little information about D. bulbifera polysaccharides regarding their fractionation, structural characterization and biological functions. In this paper, we described the extraction, fractionation and the structural features of a neutral polysaccharide (DBPN) from D. bulbifera. Furthermore, the main fraction of DBPN, DBPN-UD, was isolated. Its sulfated and carboxymethylated derivatives were synthesized.The antitumor activities of DBPN-UD and its derivatives were evaluated. The water-soluble polysaccharides were extracted from the rhizome of D. bulbifera with hot water, precipitated by80%ethanol. The polysaccharide mixture, referred to DBP, was separated on a preparative DEAE-Cellulose column into four fractions: one neutral fraction (DBPN) and three acidic fractions (DBPA-1, DBPA-2and DBPA-3) corresponding to0.0,0.1,0.3and0.5M NaCl elution, respectively. The major structural features of D. bulbifera polysaccharide fractions were elucidated by using high performance liquid chromatography and enzymolysis. The results showed that DBPN was heterogeneous and contained galactan and glucan. DBPA-1might be composed of homogalacturonan (HG) and galactan domains. DBPA-2might contain both homogalacturonan (HG) and small amount of type-I rhamnogalacturonan (RG-I) domains and DBPA-3might be composed of homogalacturonan (HG) domain.DBPN was completely fractionated by stepwise ethanol precipitation, which produced six homogenous fractions: DBPN-1(26.0%), DBPN-2(1.8%), DBPN-3(3.0%), DBPN-4(2.0%), DBPN-5(3.2%), and DBPN-6(2.6%) corresponding to final ethanol concentrations of40%,45%,50%,55%.65%, and75%with molecular weight of22.4,1.0,0.73,0.54,0.36and0.26kDa, respectively. Their structural features were elucidated by high performance liquid chromatography, enzymolysis, Fourier transform infrared spectroscopy and13C-nuclear magnetic resonance spectroscopy. The results indicated that DBPN contained galactan and glucan. The galactan was a linear β-(1,4)-D-galactan without side chains and contained galactose (96.9%). The glucan was a starch-like molecule. This is the first report of a linear β-(1,4)-D-galactan without side chains from D. bulbifera. D. bulbifera would be a suitable source for linear β-1,4-D-galactan in good yield and by simple preparation method. Sulfation with SO3-pyridine yielded six DBPN-UD sulfates: S-DBPN-UD-0.1, S-DBPN-UD-0.25, S-DBPN-UD-0.5, S-DBPN-UD-1, S-DBPN-UD-2and S-DBPN-UD-4with DS of0.12,0.26,0.63,1.42,0.92and1.02；Carboxymethylation with sodium salt of monochloro acetic acid (SMCA) yielded six DBPN-UD carboxymethylates: CM-DBPN-UD-0.1, CM-DBPN-UD-0.25, CM-DBPN-UD-0.5, CM-DBPN-UD-1, CM-DBPN-UD-2and CM-DBPN-UD-3with DS of0.16、0.21、0.26、0.46、0.62and0.70.The anticancer activities of DBPN and its derivatives were determined using an MTT assay and three cancer cell lines including sarcoma180and human colon cancer cells HCT-116and HT-29.. The results showed that sulfation and carboxymethylation of DBPN enhanced its inhibitory activityin a DS dependent manner. DBPN-UD and its derivatives were also tested for their anti-galectin-3activities using a galectin-3-mediated hemagglutination assay. DBPN-UD showed potent inhibition to the agglutination with an MIC of12.5μg/ml, similar to lactose, a standard galectin-3inhibitor. Sulfation and carboxymethylation significantly decreased the anti-galectin-3activity of DBPN-UD. The discovery that DBPN-UD had anti-galectin-3activity is valuable in guiding the preparation of effective pharmaceuticals from D. bulbifera for cancer prevention and treatment.The results of this paper will provide the theoretical basis for further research and development of D. bulbifera polysaccharides and improve the application of D. bulbifera polysaccharides.更多还原