中药北沙参多糖与单糖组成和含量测定方法研究

【作者】 任浩娜；

【导师】 陈晓辉； 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2008， 硕士

【摘要】 中药北沙参为伞形科植物珊瑚菜（Glehnia littoralis Fr．Schmidt ex Miq．）的干燥根。本品性甘、微苦、微寒、归肺、胃经。具有滋阴清肺，益胃生津的功效。文献报道，其含有多糖类、香豆素类、磷脂、氨基酸、微量元素等多种有效成分，其中多糖类所占的比例最大，也是其主要有效成分之一。文献报道用苯酚．硫酸法对其总糖含量进行测定，关于其多糖的单糖组成的研究没有报道。本研究确定了沙参多糖的单糖组成，并对其总糖和单糖含量进行了测定，为中药北沙参多糖的质量控制标准提供了科学依据。建立了沙参多糖的提取、纯化、精制方法。采用水提取乙醇沉淀的方法提取沙参粗多糖，用石油醚除去脂类，80％乙醇除去单糖、低聚糖、苷类，Sevag法除去蛋白，对粗多糖进行纯化、精制，得到精制沙参多糖。通过Feling反应、Molish反应，紫外分光光度法等手段鉴定为不含单糖、蛋白质、核酸等物质的纯化多糖，以上结果与文献报道一致，并通过苯酚-硫酸法测定总多糖含量线性范围为1．96～6．86μg／mL（r=0．9990），平均回收率为97．6％（RSD=1．5％）。建立了确定沙参多糖的单糖组成和测定各单糖含量的柱前衍生化GC方法。用三氟乙酸水解多糖，水解产物加上衍生化试剂盐酸羟胺在吡啶中加热反应生成糖肟，加入醋酸酐为乙酰化试剂，在加热的条件下继续反应，生成具有挥发性的糖腈乙酸酯产物，采用GC色谱法，色谱柱：DM-5（30 m×0．25 mm，0．25μm）；柱温：160℃→2℃／min→195℃（6 min）→10℃／min→230℃（3 min）；载气：氮气；进样器温度：270℃；进样方式：分流（10：1）；检测器：FID 280℃（氮气体积流量30 mL／min，氢气体积流量40 mL／min,空气体积流量400mL／min）。结果表明沙参多糖由D-鼠李糖，L-（+）-阿拉伯糖，D-甘露糖，D-葡萄糖和D-半乳糖等7种单糖组成（有2种未鉴定出）。D-鼠李糖，L-（+）-阿拉伯糖，D-甘露糖，D-葡萄糖和D-半乳糖的线性范围分别为2．4～24μg／mL（r=0．9992），6～60μg／mL（r=0．9996），1．2～12μg／mL（r=0．9991），140～1400μg／mL（r=0．9991）和6～60μg／mL（r=0．9994）。回收率分别为91．0％（RSD=2．2％），91．2％（RSD=2．2％），87．3％（RSD=3．1％），94．7％（RSD=2．3％），90．5％（RSD=2．9％）。建立了确定沙参多糖的单糖组成和测定各单糖含量的柱前衍生化HPLC-UV法。用三氟乙酸水解多糖，水解产物加上衍生化试剂1-苯基-3-甲基-5-吡唑酮（PMP），采用高效液相色谱紫外检测法，色谱柱：C₁₈色谱柱（250 mm×4．6 mm，5μm）；流动相：19％（v／v）乙腈-醋酸铵缓冲液（醋酸铵-冰醋酸，pH 5．5），等度洗脱；流速：1．0 mL／min；检测器：紫外检测器；检测波长：250 nm；柱温：35℃。结果表明沙参多糖由D-甘露糖，D-鼠李糖，半乳糖醛酸，D-葡萄糖，D-半乳糖和L-（+）-阿拉伯糖等8种单糖组成（有2种未鉴定出）。D-甘露糖，D-鼠李糖，半乳糖醛酸，D-葡萄糖，D-半乳糖和L-（+）-阿拉伯糖的线性范围分别为0．72～14．4μg／mL（r=0．9990），2．18～43．6μg／mL（r=0．9994），11．87～237．4μg／mL（r=0．9990），304．2～6084μg／mL（r=0．9990），14．4～288μg／mL（r=0．9990）和9．6～192μg／mL（r=0．9992），平均回收率分别为94．0％（RSD=2．5％），88．5％（RSD=2．5％），93．9％（RSD=2．3％），95．9％（RSD=2．7％），89．2％（RSD=2．5％）和90．8％（RSD=2．8％）。更多还原

【Abstract】 This paper is about the content of polysaccharide in Radix Glehniae and the contents of monosaccharide compositions in polysaccharide .Phenol-sulfuric acid method was used for determination of the amount of total GLP. HPLC and GC were used for determination of contents of monosaccharide compositions in polysaccharide.Hot water extracting and ethanol precipitating method were employed to isolate polysaccharide. Protein was removed by Sevag method. The amount of total GLP was measured with phenol-sulfuric acid method. UV-spectrophotometer was used to determine the characteristic absorption and concentration of GLP. The content of GLP was good linearity over the range of 1.96⁶.86μg/mL （r = 0.9990） . The average recovery was 97.6%（ RSD =1.5%）.GLP sample was hydrolyzed with trifluoroacetic acid. Hydroxylamine hydrochloride, pyridine and acetic anhydride were added as derivative agents. The hydrolyzed monodaccharides were converted into corresponding aldoononitrile zcetates. The derivatives were determined by GC. DM-5 column （30 m×0. 25 mm, 0. 25μm） was used. The inlet temperature was maintained at 270℃. The column oven was begun at 160℃, then programmed from 160℃to 195℃at 2℃/min, hold for 6 min ,then programmed from 195℃to 230℃at 10℃/min, and finally, hold for 3 min. Helium at a constant flow rate of 30 mL/min was used as carrier gas. Concentrations of seven monosaccharides mannose, rhamnose, glucose, galactose, arabinose, and two unidentifiable monosaccharides were determined in GLP sample. The contents of mannose rhamnose, galacturonic-acid, glucose, galactose and arabinose were good linearity over the range of 2.4-24μg/mL（r = 0.9992）, 6-60μg/mL （r = 0.9996）, 1.2-12μg/mL （r = 0.9991）, 140¹400μg/mL （r = 0.9991）, 6-60μg/mL （r = 0.9994）. The average recoveries were 91.0%（ RSD =2.2%）, 91.2%（RSD=2.2%）, 87.3%（RSD=3.1%）, 94.7%（ RSD =2.3%）, 90.5%（ RSD =2.9%）.GLP sample was hydrolyzed with trifluoroacetic acid.1-pheny-3-methyl-5-pyrazolone （PMP） was added as derivative agent. The hydrolyzed monodaccharides were converted into derivatives. The derivatives were determined by HPLC. The analytical column was C₁₈ （250 mm×4.6 mm, 5μm）.The mobile phase was 19% （v/v） acetonitrle- ammoniumacetact （ammoniumacetact - glacial acetic acid pH 5.5）, isocratic elution prosess. The flow rate was 1.0 mL /min and the column temperature was 35℃. The detector was ultriolet detector. The detection wavelength was 250 nm. Concentrations of eight monosaccharides-mannose, rhamnose, galacturonic-acid, glucose, galactose, arabinose and two unidentifiable monosaccharides were determined in GLP sample. The contents of mannose rhamnose, galacturonic-acid, glucose, galactose and arabinose were good linearity over the range of 0.72-14.4μg/mL （r = 0.9990）, 2.18-43.6μg/mL （r = 0.9990）, 11.87-237.4μg/mL （r = 0.9994）, 304.2-6084μg/mL （r = 0.9990）, 14.4-288μg/mL （r = 0.9990）, 9.6-192μg/mL （r = 0.9992）. The average recoveries were94.0%（ RSD =2.5%）, 88.5%（ RSD =2.5%）, 93.9%（ RSD =2.3%）, 95.9%（ RSD =2.7%）, 89.2%（ RSD =2.5%）和P 90.8%（ RSD =2.8%）.更多还原