亳芍多糖的积累规律及其单糖组成GC-MS分析
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摘要
目的:揭示亳芍多糖的动态积累规律,为亳芍的科学采收、质量评价及临床用药提供依据。方法:采用蒽酮比色法,以葡萄糖为标准对照品,检测波长628 nm,测定亳芍多糖的含量;亳芍多糖溶于0.5 mol·L~(-1)盐酸甲醇溶液,80℃水浴酸解16 h,氮气吹干后加入吡啶-六甲基二硅胺烷-三甲基氯硅烷(10∶2∶1),80℃水浴反应30 min,氮气吹干,残渣溶于正己烷,采用TG-5熔融石英毛细管柱、1.0 m L·min~(-1)氦气为载气、电子电离离子源(EI)、质量扫描范围为50~350 amu、三甲基硅烷(TMS)柱前衍生化-GC-MS分析法测定单糖组成及其含量。结果:栽培年限影响亳芍多糖积累,1~7年亳芍中多糖的含量分别为(2.7±0.03)%、(3.1±0.23)%、(4.1±0.10)%、(2.2±0.33)%、(1.5±0.23)%、(1.6±0.10)%和(1.6±0.07)%;栽培1~7年亳芍多糖均为葡萄糖、果糖、甘露糖、半乳糖、半乳糖醛酸、木糖、阿拉伯糖、鼠李糖组成的杂多糖,但比例随栽培年限不同而变化。结论:从多糖含量角度,栽培3年的亳芍多糖含量最高,品质最佳;栽培年限对亳芍多糖的单糖种类没有影响,但对单糖比例及含量产生明显影响,提示不同株龄的亳芍多糖的结构可能不同。
Objective:To reveal the accumulation of polysaccharide in Bozhou Peony P. lactiflora and provide references for its scientific harvesting,quality evaluating and clinical medication. Methods:The contents of the total polysaccharide in different ages of Bozhou Peony P. lactiflora were determined by an anthrone-sulfuric acid colorimetric method at 628 nm using glucose as standard. The polysaccharide of each Dendrobium sample was treated with methanolysis reagent(Me OH containing HCl 0.5 mol·L-1) in nitrogen atmosphere at 80 ℃ for 16 h to hydrolyze them to their corresponding methyl monosaccharides,and thereafter the excess of reagent was removed using a stream of nitrogen gas. The conversion of the methyl glycosides to their trimethylsilyl(TMS) derivatives was performed by adding a mixed pyridine-hexamethyldisilazane-trimethylclorosilane(10∶2∶1,v/v) to the dried material. The reaction was carried out at 80 ℃ for 30 min and the reagent removed using a stream of nitrogen gas and the derived residues were extracted with hexane. The trimethylsilyl-ester O-methyl monosaccharides were separated with Trace 1300 Gas chromatograph coupled to ISQ mass spectrometerseries equipments including a Tri PLUS RSH autosampler on a TG-5 fused-silica capillary column. The carrier gas was Helium,1.0 m L·min~(-1). The MS transfer line and ion source were at 250 ℃ and 280 ℃,respectively. The MS mode was electron ionization(EI). The mass range scanned was 50-350 amu. Different standard carbohydrates were also converted to their corresponding TMS derivatives and analyzed by GC-MS in order to obtain patterns for identification and the standard calibration curves. Results:The accumulation of polysaccharide varied remarkably as their ages increased. The polysaccharide contents in 1-7 cultivated-years of Bozhou Peony P. lactiflora were(2.7±0.03)%,(3.1±0.23)%,(4.1±0.10)%,(2.2±0.33)%,(1.5±0.23)%,(1.6±0.10)% and(1.6±0.07)%,respectively. The polysaccharides in different ages of Bozhou Peony P. lactiflora were all heteropolysaccharides composed of glucose,fructose,mannose,galactose,galacturonic acid,xylose,arabinose and rhamnose while the ratios of the mono-saccharides changed remarkable as the age varied. Conclusion:The kinds of the mono-saccharides were the same while the ratios of them were quite different in the plants at 1-7 ages. This suggested that the structure of polysaccharide in Bozhou Peony P. lactiflora might be varied as the plant ages changes.
Objective:To reveal the accumulation of polysaccharide in Bozhou Peony P. lactiflora and provide references for its scientific harvesting,quality evaluating and clinical medication. Methods:The contents of the total polysaccharide in different ages of Bozhou Peony P. lactiflora were determined by an anthrone-sulfuric acid colorimetric method at 628 nm using glucose as standard. The polysaccharide of each Dendrobium sample was treated with methanolysis reagent(Me OH containing HCl 0.5 mol·L-1) in nitrogen atmosphere at 80 ℃ for 16 h to hydrolyze them to their corresponding methyl monosaccharides,and thereafter the excess of reagent was removed using a stream of nitrogen gas. The conversion of the methyl glycosides to their trimethylsilyl(TMS) derivatives was performed by adding a mixed pyridine-hexamethyldisilazane-trimethylclorosilane(10∶2∶1,v/v) to the dried material. The reaction was carried out at 80 ℃ for 30 min and the reagent removed using a stream of nitrogen gas and the derived residues were extracted with hexane. The trimethylsilyl-ester O-methyl monosaccharides were separated with Trace 1300 Gas chromatograph coupled to ISQ mass spectrometerseries equipments including a Tri PLUS RSH autosampler on a TG-5 fused-silica capillary column. The carrier gas was Helium,1.0 m L·min~(-1). The MS transfer line and ion source were at 250 ℃ and 280 ℃,respectively. The MS mode was electron ionization(EI). The mass range scanned was 50-350 amu. Different standard carbohydrates were also converted to their corresponding TMS derivatives and analyzed by GC-MS in order to obtain patterns for identification and the standard calibration curves. Results:The accumulation of polysaccharide varied remarkably as their ages increased. The polysaccharide contents in 1-7 cultivated-years of Bozhou Peony P. lactiflora were(2.7±0.03)%,(3.1±0.23)%,(4.1±0.10)%,(2.2±0.33)%,(1.5±0.23)%,(1.6±0.10)% and(1.6±0.07)%,respectively. The polysaccharides in different ages of Bozhou Peony P. lactiflora were all heteropolysaccharides composed of glucose,fructose,mannose,galactose,galacturonic acid,xylose,arabinose and rhamnose while the ratios of the mono-saccharides changed remarkable as the age varied. Conclusion:The kinds of the mono-saccharides were the same while the ratios of them were quite different in the plants at 1-7 ages. This suggested that the structure of polysaccharide in Bozhou Peony P. lactiflora might be varied as the plant ages changes.
引文
[1]李晋奇,彭成,姬洁莹.制川乌总碱与白芍总苷、白芍多糖配伍治疗类风湿性关节炎大鼠的作用机制研究[J].中国中药杂志,2009,34(22):2937LI JQ,PENG C,JI JY.Mechanism study of action on compatible using of total alkaloids of Radix Aconiti Praeparata and total glycosides or polysaccharides of Radix Paeoniae Alba therapy on rheumatoid arthritis in rats[J].China J Chin Mater Med,2009,34(22):2937
[2]汪芸,陶移文,陈法江,等.不同制备方法对生白芍和炒白芍中多糖含量及理化性质的影响[J].中药材,2013,36(7):1068WANG Y,TAO YW,CHEN FJ,et al.Influence of different preparation on the content of carbohydrate and physicochemical properties of polysaccharides from raw Paeoniae Radix Alba and stir-baked Paeoniae Radix Alba[J].J Chin Med Mater,2013,36(7):1068
[3]张晓燕,高崇凯,王金辉,等.白芍中的一种新的单萜苷[J].药学学报,2002,37(9):705ZHANG XY,GAO CK,WANG JH,et al.A new monoterpene glycosides of Radix Paeoniae Alba[J].Acta Pharm Sin,2002,37(9):705
[4]许源,刘培,严辉,等.白芍初加工过程中单萜苷类及多羟基化合物的变化分析[J].中药材,2014,37(5):775XU Y,LIU P,YAN H,et al.Analysis of variation of monoterpene glycosides and polyhydroxy compound in Paeoniae Radix Alba during preliminary processing[J].J Chin Med Mater,2014,37(5):775
[5]陈乃东,陈翰,陈乃富,等.HPLC法同时测定不同栽培年限亳芍中芍药苷和芍药内酯苷的含量[J].天然产物研究与开发,2015,27(6):1047CHEN ND,CHEN H,CHEN NF,et al.Simultaneously determination of paeoniflorin in different cultivated years of Bozhou peony by HPLC method[J].Nat Prod Res Dev,2015,27(6):1047
[6]陈乃东,朱旺生,杨小东.亳芍总生物碱提取及抑菌活性的研究[J].天然产物研究与开发,2015,27(5):827CHEN ND,ZHU WS,YANG XD.Extraction and antibacaterial activity of total alkaloids from Bozhou Peony Paeonia lactiflora[J].Nat Prod Res Dev,2015,27(5):827
[7]陈乃东,周飞.亳芍生物碱提取及含量测定[J].中国中医药信息杂志,2013,20(10):49CHEN ND,ZHOU F.Extraction and determination of alkaloids from Bozhou Peony Paeonia lactiflora[J].Chin J Inf Tradit Chin Med,2013,20(10):49
[8]李雪莲,来平凡.白芍炮制本草学研究及现代研究进展[J].亚太传统医药,2008,4(6):42LI XL,LAI PF.Studies and modern research progress on Radix Paeoniae Alba herbalism[J].Asia Pac Tradit Med,2008,4(6):42
[9]刘鹰翔,马玉卓.白芍的化学成分与药理研究进展[J].中草药,1995,26(8):437LIU YX,MA YZ.Chemical composition and pharmacological research progress on Radix Paeoniae Alba herbalism[J].Chin Tradit Herb Drugs,1995,26(8):437
[10]王长云,管华诗.氨基多糖研究概况与展望[J].生物工程进展,1995,15(5):2WANG CY,GUAN HS.Advance in amino polysaccharide[J].Prog Biotechnol,1995,15(5):2
[11]郭忠武,王来庵.糖化学研究进展[J].化学进展,1995,7(1):10GUO ZW,WANG LA.Progress and trends on carbohydrate research[J].Prog Chem,1995,7(1):10
[12]杨树平,温爱平,吴冬梅,等.河套向日葵茎髓多糖的提取及含量测定[J].中国中药杂志,2003,28(9):887YANG SP,WEN AP,WU DM,et al.Extraction and content determination of polysaccharide in sunflower stalk pulp in Heta[J].China J Chin Mater Med,2003,28(9):887
[13]李金花,农石生,韦柳培,等.芒果叶多糖的提取及含量测定[J].食品研究与开发,2012,33(3):135LI JH,NONG SS,WEI LP,et al.Determination for the content of polysaccharide and extract of Mangifera indica Linn.leaves[J].Food Res Dev,2012,33(3):135
[14]王德才,李同德,高丽君.杭白芷多糖的提取分离及其含量测定[J].中国中医药科技,2008,15(4):283WANG DC,LI TD,GAO LJ.Extraction,separation and its content determination of polysaccharide from Hangzhou Angelica dahurica[J].Chin J Tradit Med Sci Technol,2008,15(4):283
[15]DOCO T,O’NEIL MA,PELLERIN P.Determination of the neutral and acidic glycosyl-residue compositions of plant polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glycoside derivatives[J].Carbohydr Polym,2001,46(3):249
[16]ZENAIDA G,OLGA M,áLVAROG,et al.Quantitative determination of wine polysaccharides by gas chromatography-mass spectrometry(GC-MS)and size exclusion chromatography[J].Food Chem,2012,131(1):367
[17]CHEN ND,CHEN NF,LI J,et al.Similarity evaluation of different origins and species of Dendrobiums by GC-MS and FTIR analysis of polysaccharides[J].Int J Anal Chem,2015,2015(1):8
[18]陈乃东,李俊,孟云飞,等.柱前衍生化HPCE法测定不同种源石斛中多糖的单糖组成[J].中药材,2015,27(8):1608CHEN ND,LI J,MENG YF,et al.Comparative study on polysaccharide from tissue-cultured and wild Dendrobium huoshanese and Dendrobium moniliforme as well as wild Dendrobium henanese by PMP-HPCE fingerprint[J].J Chin Med Mater,2015,27(8):1608
[2]汪芸,陶移文,陈法江,等.不同制备方法对生白芍和炒白芍中多糖含量及理化性质的影响[J].中药材,2013,36(7):1068WANG Y,TAO YW,CHEN FJ,et al.Influence of different preparation on the content of carbohydrate and physicochemical properties of polysaccharides from raw Paeoniae Radix Alba and stir-baked Paeoniae Radix Alba[J].J Chin Med Mater,2013,36(7):1068
[3]张晓燕,高崇凯,王金辉,等.白芍中的一种新的单萜苷[J].药学学报,2002,37(9):705ZHANG XY,GAO CK,WANG JH,et al.A new monoterpene glycosides of Radix Paeoniae Alba[J].Acta Pharm Sin,2002,37(9):705
[4]许源,刘培,严辉,等.白芍初加工过程中单萜苷类及多羟基化合物的变化分析[J].中药材,2014,37(5):775XU Y,LIU P,YAN H,et al.Analysis of variation of monoterpene glycosides and polyhydroxy compound in Paeoniae Radix Alba during preliminary processing[J].J Chin Med Mater,2014,37(5):775
[5]陈乃东,陈翰,陈乃富,等.HPLC法同时测定不同栽培年限亳芍中芍药苷和芍药内酯苷的含量[J].天然产物研究与开发,2015,27(6):1047CHEN ND,CHEN H,CHEN NF,et al.Simultaneously determination of paeoniflorin in different cultivated years of Bozhou peony by HPLC method[J].Nat Prod Res Dev,2015,27(6):1047
[6]陈乃东,朱旺生,杨小东.亳芍总生物碱提取及抑菌活性的研究[J].天然产物研究与开发,2015,27(5):827CHEN ND,ZHU WS,YANG XD.Extraction and antibacaterial activity of total alkaloids from Bozhou Peony Paeonia lactiflora[J].Nat Prod Res Dev,2015,27(5):827
[7]陈乃东,周飞.亳芍生物碱提取及含量测定[J].中国中医药信息杂志,2013,20(10):49CHEN ND,ZHOU F.Extraction and determination of alkaloids from Bozhou Peony Paeonia lactiflora[J].Chin J Inf Tradit Chin Med,2013,20(10):49
[8]李雪莲,来平凡.白芍炮制本草学研究及现代研究进展[J].亚太传统医药,2008,4(6):42LI XL,LAI PF.Studies and modern research progress on Radix Paeoniae Alba herbalism[J].Asia Pac Tradit Med,2008,4(6):42
[9]刘鹰翔,马玉卓.白芍的化学成分与药理研究进展[J].中草药,1995,26(8):437LIU YX,MA YZ.Chemical composition and pharmacological research progress on Radix Paeoniae Alba herbalism[J].Chin Tradit Herb Drugs,1995,26(8):437
[10]王长云,管华诗.氨基多糖研究概况与展望[J].生物工程进展,1995,15(5):2WANG CY,GUAN HS.Advance in amino polysaccharide[J].Prog Biotechnol,1995,15(5):2
[11]郭忠武,王来庵.糖化学研究进展[J].化学进展,1995,7(1):10GUO ZW,WANG LA.Progress and trends on carbohydrate research[J].Prog Chem,1995,7(1):10
[12]杨树平,温爱平,吴冬梅,等.河套向日葵茎髓多糖的提取及含量测定[J].中国中药杂志,2003,28(9):887YANG SP,WEN AP,WU DM,et al.Extraction and content determination of polysaccharide in sunflower stalk pulp in Heta[J].China J Chin Mater Med,2003,28(9):887
[13]李金花,农石生,韦柳培,等.芒果叶多糖的提取及含量测定[J].食品研究与开发,2012,33(3):135LI JH,NONG SS,WEI LP,et al.Determination for the content of polysaccharide and extract of Mangifera indica Linn.leaves[J].Food Res Dev,2012,33(3):135
[14]王德才,李同德,高丽君.杭白芷多糖的提取分离及其含量测定[J].中国中医药科技,2008,15(4):283WANG DC,LI TD,GAO LJ.Extraction,separation and its content determination of polysaccharide from Hangzhou Angelica dahurica[J].Chin J Tradit Med Sci Technol,2008,15(4):283
[15]DOCO T,O’NEIL MA,PELLERIN P.Determination of the neutral and acidic glycosyl-residue compositions of plant polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glycoside derivatives[J].Carbohydr Polym,2001,46(3):249
[16]ZENAIDA G,OLGA M,áLVAROG,et al.Quantitative determination of wine polysaccharides by gas chromatography-mass spectrometry(GC-MS)and size exclusion chromatography[J].Food Chem,2012,131(1):367
[17]CHEN ND,CHEN NF,LI J,et al.Similarity evaluation of different origins and species of Dendrobiums by GC-MS and FTIR analysis of polysaccharides[J].Int J Anal Chem,2015,2015(1):8
[18]陈乃东,李俊,孟云飞,等.柱前衍生化HPCE法测定不同种源石斛中多糖的单糖组成[J].中药材,2015,27(8):1608CHEN ND,LI J,MENG YF,et al.Comparative study on polysaccharide from tissue-cultured and wild Dendrobium huoshanese and Dendrobium moniliforme as well as wild Dendrobium henanese by PMP-HPCE fingerprint[J].J Chin Med Mater,2015,27(8):1608