松茸多糖的分离纯化及其活性研究
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摘要
本论文以松茸(Tricholoma matsutake Sing.)为研究对象,对其中的多糖组分进行了提取、分离、纯化、结构鉴定及生物活性等方面的初步研究。采用热水浸提、超滤分级、乙醇沉淀的方法从松茸子实体中提取得到粗多糖。粗多糖经过DEAE-Sepharose F.F离子交换柱层析和Sephadex G-100凝胶柱层析分级纯化,苯酚-硫酸法跟踪检测,得到二个多糖组分(TMS-1、TMS-2)。经过高效液相色谱法验证,所得的二个多糖组分为分子量均一的多糖组分,分子量分别约为TMS-1(22180 Da)、TMS-2(16450 Da)。采用GC-MS确定了这两个多糖的单糖组成及摩尔比例: TMS-1是由半乳糖和3-甲氧基半乳糖组成的半乳聚糖,其摩尔比为1.23:1.55; TMS-2由鼠李唐、甘露糖、葡萄糖组成,其摩尔比为1.25:1.51:4.85。经甲基化化学分析法,并结合核磁共振波谱技术(一维:1H NMR, 13C NMR,二维:H-H COSY谱、HMQC谱、HMBC谱、DEPT谱),确定其分子结构为: TMS-1 :l→)–α–Gal–(l→6)–3–O–CH3–α–Gal–(l→;TMS-2:主链由(l→6)–α–Glc, (l→2,4)-α–Man组成,支链带有l→)–α–Rha。通过体外抗氧化实验(DPPH自由基体系、02-自由基体系、·OH自由基体系),分别研究了松茸粗多糖提取物TMS、松茸多糖均一组分TMS-1和TMS-2的抗氧化活性。结果表明,TMS、TMS-1、TMS-2均具有一定的抗氧化活性。一定浓度范围内,随着浓度增加,抗氧化活性逐渐增强,并且成有效的量-效关系。在三种体外抗氧化体系中,TMS-1在·OH自由基体系中的活性较高,在0.4-0.6mg/mL浓度条件下的清除效果比同浓度的维生素C稍高,将其应用于抗氧化保健食品的研究,可以减少自由基对机体产生损伤或致突变作用,具有良好的开发前景。TMS-2对正常细胞株和肿瘤细胞株辐射存活率的测试实验结果表明:在X-ray辐照条件下松茸多糖TMS-2在50μg/ mL浓度时使正常细胞株的存活率提高了51.77%,而对照组肿瘤细胞株的存活率仅提高了6.33%,与对照组相比,差异性显著。说明TMS-2在显著保护正常细胞的同时对肿瘤细胞仅有轻度保护作用,并呈有效的量-效关系。如能将松茸多糖TMS-2用于治疗肿瘤的放化疗过程中,可以在杀伤肿瘤细胞的同时,保护正常的组织细胞,极大地降低放化疗的毒副作用,增强放化疗患者的体质、提高承受力、减轻患者的痛苦,这使得松茸多糖TMS-2在放射治疗肿瘤的临床应用上具有极大意义。
The object of this study is to isolate the water-soluble polysaccharide from the Tricholoma matsutake Sing.,and to further characterize its structure.The crude polysaccharides was obtained from the Tricholoma matsutake Sing.by water exteraction followed by ethanol precipitation. The crude polysaccharide purified by ion chromatograph DEAE-Sepharose Fast Flow,and gel permeation chromatograph Sephadex G-100,and two polysaccharide fractions obtained and termed TMS-1,TMS-2,respectively.The average Mw of the TMS-1and TMS-2 were 22180 Da Da,16450 Da Da using gel permeation chromatograph .Ion-chromatography detection GC-MS analysis indicated that TMS-1 was composed of D-galacctose and 3-O-CH3-galactose,in the molar ratio of 1.23:1.55; TMS-2 was composed of D-rhamnose,D-mannose, D-glucose,in the molar ratio of 1.25:1.51:4.85.Structural features of TMS-1and TMS-2 were investigated by a combination of instrumental and chemical analysis,including methylatio analysis and NMR(1H NMR,13C NMR,H-H COSY、HMQC、HMBC、DEPT Spectrum) spectroscopy. The main chain of TMS-1 was made of l→)–α–Gal–(l→6)–3–O–CH3–α–Gal–(l→; The main chain of TMS-2 was made of (l→6)–α–Glc, (l→2,4)-α–Man, and side chain was composed of (l→)–α–Rha residues.The antioxidant effects of different Tricholoma matsutake Sing. Polysaccharides were studied in vitro by different testing systems, which included DPPH radical ,superoxide anion radical, hydroxyl radical.The result showed that TMS,TMS-1 and TMS-2 all had certain antioxidant activities. The correlativity of their effects and the doses are positive, in a certain concentration range.The TMS-1 showed high antioxidant activities in the hydroxyl radical system . At 0.4-0.6 mg/mL, showed an excellent hydroxyl radical scavenging activity among all the fraction samples, also higher than VitamiC. Applying it to study health food antioxidant, can reduce free radicals on the body can damage or mutation, has good development prospects. In the X-ray irradiation conditions Tricholoma polysaccharide TMS-2 in the 50μg / mL concentration to normal cell lines increased the survival rate of 51.77 percent, while the control group, the survival rate of tumor cell lines increased only 6.33%, with a control group , the difference is significant..If use the TMS-2 for the treatment of cancer during chemotherapy, can kill tumor cells while protecting normal cells, greatly reducing the toxic side effects of chemotherapy, radiotherapy and chemotherapy patients enhance health and improve bear force patients to relieve their suffering, which makes the polysaccharide TMS-2 in the clinical application of radiotherapy for cancer that has great significance.
The object of this study is to isolate the water-soluble polysaccharide from the Tricholoma matsutake Sing.,and to further characterize its structure.The crude polysaccharides was obtained from the Tricholoma matsutake Sing.by water exteraction followed by ethanol precipitation. The crude polysaccharide purified by ion chromatograph DEAE-Sepharose Fast Flow,and gel permeation chromatograph Sephadex G-100,and two polysaccharide fractions obtained and termed TMS-1,TMS-2,respectively.The average Mw of the TMS-1and TMS-2 were 22180 Da Da,16450 Da Da using gel permeation chromatograph .Ion-chromatography detection GC-MS analysis indicated that TMS-1 was composed of D-galacctose and 3-O-CH3-galactose,in the molar ratio of 1.23:1.55; TMS-2 was composed of D-rhamnose,D-mannose, D-glucose,in the molar ratio of 1.25:1.51:4.85.Structural features of TMS-1and TMS-2 were investigated by a combination of instrumental and chemical analysis,including methylatio analysis and NMR(1H NMR,13C NMR,H-H COSY、HMQC、HMBC、DEPT Spectrum) spectroscopy. The main chain of TMS-1 was made of l→)–α–Gal–(l→6)–3–O–CH3–α–Gal–(l→; The main chain of TMS-2 was made of (l→6)–α–Glc, (l→2,4)-α–Man, and side chain was composed of (l→)–α–Rha residues.The antioxidant effects of different Tricholoma matsutake Sing. Polysaccharides were studied in vitro by different testing systems, which included DPPH radical ,superoxide anion radical, hydroxyl radical.The result showed that TMS,TMS-1 and TMS-2 all had certain antioxidant activities. The correlativity of their effects and the doses are positive, in a certain concentration range.The TMS-1 showed high antioxidant activities in the hydroxyl radical system . At 0.4-0.6 mg/mL, showed an excellent hydroxyl radical scavenging activity among all the fraction samples, also higher than VitamiC. Applying it to study health food antioxidant, can reduce free radicals on the body can damage or mutation, has good development prospects. In the X-ray irradiation conditions Tricholoma polysaccharide TMS-2 in the 50μg / mL concentration to normal cell lines increased the survival rate of 51.77 percent, while the control group, the survival rate of tumor cell lines increased only 6.33%, with a control group , the difference is significant..If use the TMS-2 for the treatment of cancer during chemotherapy, can kill tumor cells while protecting normal cells, greatly reducing the toxic side effects of chemotherapy, radiotherapy and chemotherapy patients enhance health and improve bear force patients to relieve their suffering, which makes the polysaccharide TMS-2 in the clinical application of radiotherapy for cancer that has great significance.
引文
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