红豆杉多糖纯化、结构分析和抗肿瘤活性研究
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摘要
红豆杉是一种非常稀有且具有悠久历史的植物,自从在红豆杉树皮和枝叶中提取得到紫杉醇后,人们对它的关注日益受到加强。但是按照目前的提取工艺,紫杉枝叶在被提取紫杉醇后,往往被丢弃,既浪费了资源,又污染了环境。实际上紫杉中除含有重要的天然抗肿瘤药物—紫杉醇外,还其它一系列生物活性物质—如萜类、黄酮类及生物碱等,最近我们从红豆杉叶中发现并分离纯化得到一种大分子生物活性物质—红豆杉多糖,目前尚未见文献报道。
本工作主要是从对前期所提取得到的粗多糖进行进一步的纯化、结构分析和抗肿瘤药理活性研究。在对红豆杉粗步提取后,对其进行脱色处理,主要使用双氧水、大孔吸附树脂和离子交换树脂等三种方法。通过研究发现双氧水的最佳脱色条件是在使用15%的双氧水浓度,pH9.0,温度为40℃和反应40min,在这个条件下脱色率和糖保留率分别为60.4%和84.7%。在静态树脂筛选中Purolite的阴离子交换树脂A500P、Amberlite的中性大孔吸附树脂和非极性大孔吸附树脂NKA、X5都具有较好的脱色效果,最佳脱色条件是pH5.9、温度35℃和150min。在动态脱色时A500P、XAD7HP和NKA以5BV/h具有较好的脱色效果,X5以1BV/h具有较好的脱色效果,脱色糖溶液体积均为4BV.将脱完色的多糖溶液通过使用DEAE-Sepharose FF和乙醇分级,最后通过Sephadex G-25脱盐,得到4种组分的多糖(分别命名为PCH1,PCH2,PCH3和PCH0.5)。之后多糖进行包括化学和光谱仪器分析方法在内的结构分析,发现此多糖PCH2、PCH3和PCH0.5的分子量(Mw)分别为5.8×10~3D、5.43×10~4D和1.78×10~5D,由2,4-二氧甲基-D-甘露糖,2-脱氧-D-葡萄糖,D-木糖(α型),D-木糖(β型),D-阿拉伯糖,D-甘露糖,D-半乳糖组成含量比为2:6:28:9:3:3:49,同时具有吡喃糖α-和β-端基差向异构的C-H变角振动,具有较多的支链组成。药理学实验明确了此多糖在肿瘤生长的抑制作用、对整体动物的耐受力的增强作用、对免疫低下小鼠的免疫功能提高作用和提高生命延长率等方面具有良好抗肿瘤活性。
Yew (Taxus sp.) is a rare species of plant, which has a long history. Since taxol was extracted from the barks, leaves and branches of Taxus, it has attracted more and more attention. But according to the current process, a lot of residues were left after extraction. However, many different kinds of bio-active components, such as terpenoids, flavonoids and alkaloids, remained in the residue. Recently, we also initially isolated and purified polysaccharides from the leaves of Taxus maire, which have not reported in literatures.In this work, purification, structure analysis and antitumor activity of the polysaccharides were investigated. After extracted from the leaves of Taxus mairel with hot water, the crude polysaccharides were decolorized with three methods including solution of H2O2, normal anion exchanger chromatograph and macroporous resin adsorption chromatograph. Under the optimized operating conditions for H2O2 decolorization were 15% concentration, pH 9.0, 40℃ and 40min retention time, the ratios of decolorization and polysaccharides retention were 60.4% and 84.7%, respectively. Anion exchange resin Purolite A500P, polar adsorption resin Amberlite XAD7HP and nonpolar resin NKA and X5 also showed the good results of decolorization. The optimized operating conditions for decolorization with both anion exchange and adsorption chromatography were as follows: pH 5.9, 35℃, 150 min retention time under static adsorption or flow rates: 5BV/hr both for XAD7HP and NKA, 1BV/hr for X5 under dynamic absorption. After that, 4 kinds of polysaccharides (named PCH1, PCH2,PCH3 and PCH0.5) were fractionated by ethanol precipitate, followed by DEAE-Sepharose FF anion exchange chromatography and Sephadex G-25 desalinisation. Then, the structure and chemical component of the aforementioned polysaccharides were studied by chemical and instrumental analysis. The results showed that the average Mw of the 3 kinds of polysaccharides were 5.8×10~-3D 5.43×10~4KD 和 1.78×10~5D, respectively. The PCH0.5 which consists of branched chains, is composed of 2,4-Methoxye-Man, 2--deoxyl-glc, D-Xly(a), D-Xly(p), D-Ara and D-Man with ratio of 2:6:28:9:3:3:49 and
contains both a and P deviating vibration of C-H as pyranoses. Experiments on pharmacological effects of mice indicated that the total four aforementioned polysaccharides have great activities in inhibiting the tumor growth, increasing hypoxic tolerance, improving the immune function and raising the life-prolonging rate.
本工作主要是从对前期所提取得到的粗多糖进行进一步的纯化、结构分析和抗肿瘤药理活性研究。在对红豆杉粗步提取后,对其进行脱色处理,主要使用双氧水、大孔吸附树脂和离子交换树脂等三种方法。通过研究发现双氧水的最佳脱色条件是在使用15%的双氧水浓度,pH9.0,温度为40℃和反应40min,在这个条件下脱色率和糖保留率分别为60.4%和84.7%。在静态树脂筛选中Purolite的阴离子交换树脂A500P、Amberlite的中性大孔吸附树脂和非极性大孔吸附树脂NKA、X5都具有较好的脱色效果,最佳脱色条件是pH5.9、温度35℃和150min。在动态脱色时A500P、XAD7HP和NKA以5BV/h具有较好的脱色效果,X5以1BV/h具有较好的脱色效果,脱色糖溶液体积均为4BV.将脱完色的多糖溶液通过使用DEAE-Sepharose FF和乙醇分级,最后通过Sephadex G-25脱盐,得到4种组分的多糖(分别命名为PCH1,PCH2,PCH3和PCH0.5)。之后多糖进行包括化学和光谱仪器分析方法在内的结构分析,发现此多糖PCH2、PCH3和PCH0.5的分子量(Mw)分别为5.8×10~3D、5.43×10~4D和1.78×10~5D,由2,4-二氧甲基-D-甘露糖,2-脱氧-D-葡萄糖,D-木糖(α型),D-木糖(β型),D-阿拉伯糖,D-甘露糖,D-半乳糖组成含量比为2:6:28:9:3:3:49,同时具有吡喃糖α-和β-端基差向异构的C-H变角振动,具有较多的支链组成。药理学实验明确了此多糖在肿瘤生长的抑制作用、对整体动物的耐受力的增强作用、对免疫低下小鼠的免疫功能提高作用和提高生命延长率等方面具有良好抗肿瘤活性。
Yew (Taxus sp.) is a rare species of plant, which has a long history. Since taxol was extracted from the barks, leaves and branches of Taxus, it has attracted more and more attention. But according to the current process, a lot of residues were left after extraction. However, many different kinds of bio-active components, such as terpenoids, flavonoids and alkaloids, remained in the residue. Recently, we also initially isolated and purified polysaccharides from the leaves of Taxus maire, which have not reported in literatures.In this work, purification, structure analysis and antitumor activity of the polysaccharides were investigated. After extracted from the leaves of Taxus mairel with hot water, the crude polysaccharides were decolorized with three methods including solution of H2O2, normal anion exchanger chromatograph and macroporous resin adsorption chromatograph. Under the optimized operating conditions for H2O2 decolorization were 15% concentration, pH 9.0, 40℃ and 40min retention time, the ratios of decolorization and polysaccharides retention were 60.4% and 84.7%, respectively. Anion exchange resin Purolite A500P, polar adsorption resin Amberlite XAD7HP and nonpolar resin NKA and X5 also showed the good results of decolorization. The optimized operating conditions for decolorization with both anion exchange and adsorption chromatography were as follows: pH 5.9, 35℃, 150 min retention time under static adsorption or flow rates: 5BV/hr both for XAD7HP and NKA, 1BV/hr for X5 under dynamic absorption. After that, 4 kinds of polysaccharides (named PCH1, PCH2,PCH3 and PCH0.5) were fractionated by ethanol precipitate, followed by DEAE-Sepharose FF anion exchange chromatography and Sephadex G-25 desalinisation. Then, the structure and chemical component of the aforementioned polysaccharides were studied by chemical and instrumental analysis. The results showed that the average Mw of the 3 kinds of polysaccharides were 5.8×10~-3D 5.43×10~4KD 和 1.78×10~5D, respectively. The PCH0.5 which consists of branched chains, is composed of 2,4-Methoxye-Man, 2--deoxyl-glc, D-Xly(a), D-Xly(p), D-Ara and D-Man with ratio of 2:6:28:9:3:3:49 and
contains both a and P deviating vibration of C-H as pyranoses. Experiments on pharmacological effects of mice indicated that the total four aforementioned polysaccharides have great activities in inhibiting the tumor growth, increasing hypoxic tolerance, improving the immune function and raising the life-prolonging rate.
引文
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[2] 许新得,高荫榆,陈才水等.茶叶多糖的纯化及组分研究.食品科学,2000,21(8):13～15.
[3] 魏宝东,陈留勇,孟宪军等.黄桃水溶性多糖的化学结构分析.食品工业科技,2005,26(6):86～88.
[4] 王述声,刘丹,梁忠岩等.榆耳水溶性多糖的结构特征.分子科学学报,2005,21(1):36～38.
[5] 刘成梅,付桂明,游海等.百合多糖的纯化与化学结构鉴定研究.食品科学,2002,23(5):114～116.
[6] 雷德柱,于淑娟,曹珍年.灰树花胞外多糖的结构分析.菌物系统,2002,21(2):280～282
[7] 白日霞.鬼毛针中碱溶多糖的结构和生物活性.应用化学,2001,18(6):487～489.
[8] 王展,方积年.高场核磁共振波谱在多糖结构研究中的应用.分析化学,200028(2):240～247
[9] 乔善义王立岩赵毅民等.山药多糖的提取分离和结构测定.中国天然药物,2003,1(3):155～156.
[10] Reinhold VN, ReinholdBB, CostelloCE. Anal Chem, 1995, 67: 1772.
[11] 来鲁华,杨昱婷,寡糖的构象分析.生物化学与生物物理进展,1995,22(4):290～294.
[12] 高娃,张欣,孔祥辉等.高压液相色谱法分析常见单糖、双糖.生物技术,1998,8(8):44～46.
[13] 胡亚男,王义明,罗国安.质谱在糖分析中的应用.药学学报,2000,35(5):397～400.
[14] J. M. Luk, P. Tam, S. T. Fan, and M. W. L. Koo Immunosuppressive Effects of Tripterygium wilfordii Polysaccharide on LPS-Stimulated Human Monocytes. Transplantation Proceedings, 2000, 32(7), 2013～2015
[15] 汪积慧,李鸿梅 枸杞多糖免疫调节作用的研究.齐齐哈尔医学院学报, 2002,11(23):1204～1205.
[16] 苗明三.大枣多糖对免疫抑制小鼠腹腔巨噬细胞产生IL—1α及脾细胞体外增殖的影响.中药药理与临床,2004:20(4),21～23.
[17] 袁学千,王淑梅,高权国.刺五加多糖增强小鼠免疫功能的实验研究.中医药学报,2004,4(32),48～50.
[18] 刘俊英,曾广仙,熊金蓉等.黄芪多糖对创伤应激小鼠细胞免疫功能影响的形态计量学研究.中国体视学与图像分析,2004,9(2)91～94
[19] H. ERamesh, K. Yamaki, T. Tsushida Effect of fenugreek(Trigonella foenum-graecum L.) galactomannan fraction on phagocytosis in rat macrophages and on proliferation and Igm secretion in HB4C5 cells. Carbohydrate Polymers, 2002, 50: 79～83.
[20] 翁玲,刘彦,刘学英等.黄芩多糖粉针剂对小鼠脾细胞分泌细胞因子及NK杀伤能力的影响.中医药学刊,2003,21(9):1522～1524
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