白术多糖的分子量及其单糖组成分析
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摘要
为了建立一种对白术多糖的分子量及其单糖组成测定的方法。采用水提醇沉法提取多糖,提取条件为提取温度90℃、料液比1∶16、提取时间1.5 h,重复提取3次; AB-8大孔树脂纯化。采用高效凝胶色谱法测定白术多糖的分子量,流动相:超纯水,流速:0.7 m L/min,柱温:35℃,示差折光检测器检测;柱前衍生化HPLC法检测白术多糖的单糖组成,多糖样品用三氟乙酸水解,1-苯基-3-甲基-5-吡唑啉酮柱前衍生化,经C18色谱柱分离,以磷酸盐缓冲溶液-乙腈(85∶15)洗脱,柱温40℃,流速1.0 m L/min,进样量20μL检测波长254 nm。结果显示白术多糖中95%为Mw≈3500的小分子多糖;经柱前衍生化HPLC法检测,白术多糖由D-甘露糖、D-葡萄糖、D-半乳糖、阿拉伯糖组成,其物质的量比为1∶239∶18.6∶19.3。结果表明试验建立的白术多糖单糖组成分析方法稳定、可靠,可作为白术多糖质量控制方法的参考。
A method for the determination of molecular weight and monosaccharide composition of polysaccharide from atractylodes atractyloides was established. Polysaccharides were extracted by water extraction and alcohol precipitation,the extraction conditions were as follows: extraction temperature 90 ℃ 、material-liquid ratio 1 ∶ 16,extraction time 1.5 h,repeated extraction 3 times; Purification of Polysaccharides by AB-8 Macroporous Resin.Determination of molecular weight of Atractylodes macrocephala polysaccharides by high performance gel chromatography,mobile phase: ultrapure water,flow velocity: 0. 7 m L/min,column temperature: 35 ℃,detection using differential refractive index detector. Using pre-column derivatization HPLC to measure the monosaccharide composition of polysaccharides. The polysaccharide samples were hydrolyzed in trifluoroacetic acid solution,then precolumn derivatizated with 1-phenyl-3-methyl-5-pyrazolone,The chromatographic separation was carried out on Wondasil C18( 250 mm×4.6 mm,5 μm) using phosphoric acid aqueous solution-acetonitrile( p H = 6.9) and acetonitrile( 85 ∶ 15) as mobile phases,at a flow rate of 1 m L/min,40 ℃,with 254 nm detected wavelength. The results showed: Through HPLC-GPC,95% of Polysaccharide from Atractylodes macrocephala was Mw≈3500 small molecule polysaccharide. By pre-column derivatization HPLC,Atractylodes macrocephala polysaccharides are composed of 4 kinds of monosaccharides,D-mannose,D-glucose,D-galactose and Arabia sugar. The monosaccharide composition analysis method of Atractylodes macrocephala polysaccharides established in this experiment is stable and reliable,and can be used as a reference for the quality control of Atractylodes macrocephala polysaccharides.
A method for the determination of molecular weight and monosaccharide composition of polysaccharide from atractylodes atractyloides was established. Polysaccharides were extracted by water extraction and alcohol precipitation,the extraction conditions were as follows: extraction temperature 90 ℃ 、material-liquid ratio 1 ∶ 16,extraction time 1.5 h,repeated extraction 3 times; Purification of Polysaccharides by AB-8 Macroporous Resin.Determination of molecular weight of Atractylodes macrocephala polysaccharides by high performance gel chromatography,mobile phase: ultrapure water,flow velocity: 0. 7 m L/min,column temperature: 35 ℃,detection using differential refractive index detector. Using pre-column derivatization HPLC to measure the monosaccharide composition of polysaccharides. The polysaccharide samples were hydrolyzed in trifluoroacetic acid solution,then precolumn derivatizated with 1-phenyl-3-methyl-5-pyrazolone,The chromatographic separation was carried out on Wondasil C18( 250 mm×4.6 mm,5 μm) using phosphoric acid aqueous solution-acetonitrile( p H = 6.9) and acetonitrile( 85 ∶ 15) as mobile phases,at a flow rate of 1 m L/min,40 ℃,with 254 nm detected wavelength. The results showed: Through HPLC-GPC,95% of Polysaccharide from Atractylodes macrocephala was Mw≈3500 small molecule polysaccharide. By pre-column derivatization HPLC,Atractylodes macrocephala polysaccharides are composed of 4 kinds of monosaccharides,D-mannose,D-glucose,D-galactose and Arabia sugar. The monosaccharide composition analysis method of Atractylodes macrocephala polysaccharides established in this experiment is stable and reliable,and can be used as a reference for the quality control of Atractylodes macrocephala polysaccharides.
引文
[1]曹岗,张晓炎,丛晓东,等.白术多糖的研究进展[J].北京联合大学学报(自然科学版),2009,23(03):14-18.Cao G,Zhang X Y,Cong X D,et al.The research progress of polysaccharides from atractylodes macrocephala koidz[J].Journal of Beijing Union University(Natural Sciences),2009,23(03):14-18.
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[7]单俊杰,田庚元.白术糖复合物AMP-B的理化性质及降血糖活性的研究[J].药学学报,2003,(06):438-441.Shan J J,Tian G Y.Studies on Physico-chemical properties and hypoglycemic activity of complex polysaccharide AMP-B from atractylodes macrocephala koidz[J].Acta Pharmaceutica Sinica,2003,(06):438-441.
[8]李燕,陈素红,吉星,等.白术多糖对自发性2型糖尿病小鼠血糖及相关指标的影响[J].中国试验方剂学杂志,2015,21(10):162-165.Li Y,Chen S H,Ji X,et al.Effects of polysaccharide from atractylodes macrocephala on blood glucose and related indicators in spontaneous type 2 diabetes db/db mice[J].Chinese Journal of Experimental Traditional Medical Formulae,2015,21(10):162-165.
[9]Zhang Y Q,Xu S B,Lin Y C,et al.Antagonistic effects of 3sesquiterpene lactones from atractylodes macrocephala koidz on rat uterine contraction in vitro[J].Acta Pharmacol Sin,2000,21(1):91-96.
[10]周剑,苏德春,宋国权.白术多糖对H22肝癌小鼠抗肿瘤作用试验研究[J].亚太传统医药,2015,11(17):9-10.Zhou J,Su D C,Song G Q.Anti-Tumor effect of polysaccharide of atractylodes macrocephala on H22 liver cancer mice[J].AsiaPacific Traditional Medicine,2015,11(17):9-10.
[11]宿廷敏,王敏娟,阮时宝.白术的化学成分及药理作用研究概述[J].贵阳学院学报(自然科学版),2008,(02):32-35.Su T M,Wang M J,Ruan S B.A research review of chemical constituents and pharmacological action of atractylodes macrocephala[J].Journal of Guiyang College Natural Sqences,2008,(02):32-35.
[12]汤新慧.白术多糖对小鼠免疫功能的影响[J].中医研究,1998,(02):9-11.Tang X H.Effects of atractylodes macrocephala polysaccharides on immune function in mice[J].Traditional Chinese Medicinal Research,1998,(02):9-11.
[13]Mori H,Xu Q,Sakamoto O,Uesugi Y.Mechanisms of antitumor activity of aqueous extracts from Chinese herbs and their immunopharmacology properties[J].Japanese Journal of Pharmacology.1989,49(3):423-432.
[14]Lee J C,Lee K Y,Son Y O,et al.Stimulating effects on mouse splenocytes of glycoproteins from the herbal medicine Atractylodes macrocephala Koidz[J].Phytomedicine,2007,14(6):390-395.
[15]徐叔云,卞如濂,陈修.药理试验方法学[M].3版.北京:人民卫生出版社,2002,220-228.Xu S Y,Bian R L,Chen X.Pharmacological Test Methodology[M].3 Edition.Beijing:People's Health Publishing House,2002,220-228.
[16]王少杰,巴娟,张勇军,等.柱前衍生化HPLC法测定玉屏风复合多糖的单糖组成[J].中国兽医杂志,2018,54(04):83-86.Wang S J,Ba J,Zhang Y J,et al.Analysis of monosaccharide compositions of Yupingfeng polysaccharides by HPLC pre-column derivatization[J].Chinese Journal of Veterinary Medicine,2018,54(04):83-86.
[17]梁军,夏永刚,杨炳友,等.柱前衍生化-HPLC法分析麻黄多糖ESP-B1的单糖组成[J].中草药,2011,42(10):1985-1988.Liang J,Xia Y G,Yang B Y,et al.Analysis of monosaccharide compositions of ESP-B1 from ephedrae herba by HPLC pre-column derivatization[J].Chinese Traditional and Herbal Drugs,2011,42(10):1985-1988.
[2]张利平,梁晓美,叶伟霞.白术提高机体免疫功能的研究进展[J].中国中医药科技,2014,21(05):598-599[J].Zhang L P,Liang X M,Ye W X,et al.Advances in the study of atractylodes macrocephala on improving immune function Chinese[J].Journal of Traditional Medical Science and Technology,2014,21(05):598-599[J].
[3]常云亭,孙吉兰,邱世翠,等.白术对小白鼠免疫功能的影响[J].滨州医学院学报,2003,(05):350-351.Chang Y T,Sun J L,Qiu S C,et al.Effects of atractylodes macrocephala on the immune function of mice[J].Journal of Binzhou Medical University,2003,(05):350-351.
[4]年立全.益气健脾中药提取物对大鼠应激性溃疡及多胺影响的研究[D].广州中医药大学,2013.Nian L Q.Study on the effect of Yiqi Jianpi herb extract on stress ulcer in rats and polyamine[D].Traditional Chinese Medicine University Of Guangzhou,2013.
[5]石娜,苏洁,杨正标,等.白术多糖对D-半乳糖致衰老模型小鼠的抗氧化作用[J].中国新药杂志,2014,23(05):577-581,584.Shi N,Su j,Yang Z B,et al.Antoxidantive effect of polysaccharides from Atractylodes macrocephala in D-galactose-induced aging mice[J].Chinese Journal of New Drugs,2014,23(05):577-581,584.
[6]陈冰冰.白术的药理学研究进展[J].内蒙古中医药,2012,31(10):101-102.Chen B B.Progress in pharmacological research of rhizome atractylodis macrocephala[J].Nei Mongol Journal of Traditional Chinese Medicine,2012,31(10):101-102.
[7]单俊杰,田庚元.白术糖复合物AMP-B的理化性质及降血糖活性的研究[J].药学学报,2003,(06):438-441.Shan J J,Tian G Y.Studies on Physico-chemical properties and hypoglycemic activity of complex polysaccharide AMP-B from atractylodes macrocephala koidz[J].Acta Pharmaceutica Sinica,2003,(06):438-441.
[8]李燕,陈素红,吉星,等.白术多糖对自发性2型糖尿病小鼠血糖及相关指标的影响[J].中国试验方剂学杂志,2015,21(10):162-165.Li Y,Chen S H,Ji X,et al.Effects of polysaccharide from atractylodes macrocephala on blood glucose and related indicators in spontaneous type 2 diabetes db/db mice[J].Chinese Journal of Experimental Traditional Medical Formulae,2015,21(10):162-165.
[9]Zhang Y Q,Xu S B,Lin Y C,et al.Antagonistic effects of 3sesquiterpene lactones from atractylodes macrocephala koidz on rat uterine contraction in vitro[J].Acta Pharmacol Sin,2000,21(1):91-96.
[10]周剑,苏德春,宋国权.白术多糖对H22肝癌小鼠抗肿瘤作用试验研究[J].亚太传统医药,2015,11(17):9-10.Zhou J,Su D C,Song G Q.Anti-Tumor effect of polysaccharide of atractylodes macrocephala on H22 liver cancer mice[J].AsiaPacific Traditional Medicine,2015,11(17):9-10.
[11]宿廷敏,王敏娟,阮时宝.白术的化学成分及药理作用研究概述[J].贵阳学院学报(自然科学版),2008,(02):32-35.Su T M,Wang M J,Ruan S B.A research review of chemical constituents and pharmacological action of atractylodes macrocephala[J].Journal of Guiyang College Natural Sqences,2008,(02):32-35.
[12]汤新慧.白术多糖对小鼠免疫功能的影响[J].中医研究,1998,(02):9-11.Tang X H.Effects of atractylodes macrocephala polysaccharides on immune function in mice[J].Traditional Chinese Medicinal Research,1998,(02):9-11.
[13]Mori H,Xu Q,Sakamoto O,Uesugi Y.Mechanisms of antitumor activity of aqueous extracts from Chinese herbs and their immunopharmacology properties[J].Japanese Journal of Pharmacology.1989,49(3):423-432.
[14]Lee J C,Lee K Y,Son Y O,et al.Stimulating effects on mouse splenocytes of glycoproteins from the herbal medicine Atractylodes macrocephala Koidz[J].Phytomedicine,2007,14(6):390-395.
[15]徐叔云,卞如濂,陈修.药理试验方法学[M].3版.北京:人民卫生出版社,2002,220-228.Xu S Y,Bian R L,Chen X.Pharmacological Test Methodology[M].3 Edition.Beijing:People's Health Publishing House,2002,220-228.
[16]王少杰,巴娟,张勇军,等.柱前衍生化HPLC法测定玉屏风复合多糖的单糖组成[J].中国兽医杂志,2018,54(04):83-86.Wang S J,Ba J,Zhang Y J,et al.Analysis of monosaccharide compositions of Yupingfeng polysaccharides by HPLC pre-column derivatization[J].Chinese Journal of Veterinary Medicine,2018,54(04):83-86.
[17]梁军,夏永刚,杨炳友,等.柱前衍生化-HPLC法分析麻黄多糖ESP-B1的单糖组成[J].中草药,2011,42(10):1985-1988.Liang J,Xia Y G,Yang B Y,et al.Analysis of monosaccharide compositions of ESP-B1 from ephedrae herba by HPLC pre-column derivatization[J].Chinese Traditional and Herbal Drugs,2011,42(10):1985-1988.