毛细管区带电泳法测定不同虫草菌粉制剂中多糖的单糖组成
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摘要
目的采用毛细管区带电泳法(CZE)测定不同虫草菌粉制剂中多糖的单糖组成。方法设计正交试验优化制剂中所提取多糖的全降解条件;以1-苯基-3-甲基-5-吡唑啉酮(PMP)为衍生试剂,采用CZE法测定其单糖组成;电泳条件:未涂层弹性石英毛细管柱(70 cm×50μm,有效长度61 cm);运行缓冲液为40 mmol/L硼砂缓冲液(pH 10.1);检测波长245nm;运行电压13 kV;重力进样(10 cm×8 s)。结果多糖全降解的最优条件为硫酸浓度1.0 mol/L,温度100℃,时间5 h;心肝宝胶囊、百令胶囊、至灵菌丝胶囊和金水宝胶囊的多糖中均含有木糖、葡萄糖、甘露糖和半乳糖4种单糖,但含量各有差异;4种制剂中多糖的单糖组成与冬虫夏草多糖中的单糖组成基本一致,且检测出了冬虫夏草中未见报道的木糖、鼠李糖和葡萄糖醛酸等单糖。对所建立的CZE法进行方法学验证,结果显示各单糖在0.01～0.20mg/m L线性关系良好,检测限0.205～0.397μg/m L,定量限0.684～1.323μg/m L,精密度RSD为0.8%～3.6%,重复性RSD为2.7%～4.7%,稳定性RSD为2.8%～4.7%。葡萄糖和半乳糖的平均回收率分别为96.1%～99.8%和95.1%～99.6%,其RSD分别为1.0%～2.0%和1.5%～1.9%。结论该方法快速高效,为虫草菌粉制剂质量标准的完善和虫草菌粉替代冬虫夏草的深入研究提供参考。
Objective To determine the monosaccharide composition of polysaccharides in different Cordyceps powder preparations by capillary zone electrophoresis(CZE). Methods The orthogonal experiment was used to optimize the total degradation conditions of the extracted polysaccharides; The monosaccharide composition was determined by CZE method using 1-phenyl-3-methyl-5-pyrazolone(PMP) as a derivatizing reagent. The electrophoresis conditions were as follows: uncoated fused silica capillary column(70 cm × 50 μm i.d., effective length was 61 cm); 40 mmol/L borax solution(pH 10.1) was used as the running buffer; The detection wavelength was set at 245 nm; The operation voltage was 13 kV; Hydrodynamic pressure injection was employed(10 cm × 8 s). Results The optimum hydrolysis conditions were as follows: 1.0 mol/L sulfuric acid solution, hydrolysis temperature 100 ℃, and hydrolysis time 5 h. The common monosaccharides of polysaccharides in Xinganbao Capsule, Bailing Capsule, Zhiling Junsi Capsule, and Jinshuibao Capsule were xylose, glucose, mannose, and galactose, but the content was different. The monosaccharide components contained in the polysaccharides of the four preparations were basically the same as those of the Cordyceps sinensis, and monosaccharides included xylose, rhamnose and glucuronic acid which were not reported in Cordyceps sinensis were detected. Each monosaccharide had a good linear relationship with the concentration range of 0.01 to 0.20 mg/mL, the limits of detection(LOD, S/N = 3) and the limits of quantification(LOQ, S/N = 10) ranged from 0.205 to 0.397 μg/m L and 0.684 to 1.323 μg/mL, respectively. RSDs of the precision test were 0.8%—3.6%, RSDs of the repeatability test were 2.7%—4.7%, and 2.8%—4.7% in the stability test. The recovery rate of the method showed that the mean recoveries of glucose and galactose ranged from 96.1% to 99.8% and 95.1% to 99.6% respectively, and RSD values fell within 1.0%—2.0% and 1.5%—1.9%, respectively. Conclusion The method is fast and efficient, and provides a reference for the improvement of quality standard of Cordyceps powder preparations and the intensive study on the substitution of Cordyceps powder for Cordyceps sinensis.
Objective To determine the monosaccharide composition of polysaccharides in different Cordyceps powder preparations by capillary zone electrophoresis(CZE). Methods The orthogonal experiment was used to optimize the total degradation conditions of the extracted polysaccharides; The monosaccharide composition was determined by CZE method using 1-phenyl-3-methyl-5-pyrazolone(PMP) as a derivatizing reagent. The electrophoresis conditions were as follows: uncoated fused silica capillary column(70 cm × 50 μm i.d., effective length was 61 cm); 40 mmol/L borax solution(pH 10.1) was used as the running buffer; The detection wavelength was set at 245 nm; The operation voltage was 13 kV; Hydrodynamic pressure injection was employed(10 cm × 8 s). Results The optimum hydrolysis conditions were as follows: 1.0 mol/L sulfuric acid solution, hydrolysis temperature 100 ℃, and hydrolysis time 5 h. The common monosaccharides of polysaccharides in Xinganbao Capsule, Bailing Capsule, Zhiling Junsi Capsule, and Jinshuibao Capsule were xylose, glucose, mannose, and galactose, but the content was different. The monosaccharide components contained in the polysaccharides of the four preparations were basically the same as those of the Cordyceps sinensis, and monosaccharides included xylose, rhamnose and glucuronic acid which were not reported in Cordyceps sinensis were detected. Each monosaccharide had a good linear relationship with the concentration range of 0.01 to 0.20 mg/mL, the limits of detection(LOD, S/N = 3) and the limits of quantification(LOQ, S/N = 10) ranged from 0.205 to 0.397 μg/m L and 0.684 to 1.323 μg/mL, respectively. RSDs of the precision test were 0.8%—3.6%, RSDs of the repeatability test were 2.7%—4.7%, and 2.8%—4.7% in the stability test. The recovery rate of the method showed that the mean recoveries of glucose and galactose ranged from 96.1% to 99.8% and 95.1% to 99.6% respectively, and RSD values fell within 1.0%—2.0% and 1.5%—1.9%, respectively. Conclusion The method is fast and efficient, and provides a reference for the improvement of quality standard of Cordyceps powder preparations and the intensive study on the substitution of Cordyceps powder for Cordyceps sinensis.
引文
[1]邓勇,张杰良,王兰英,等.薄层色谱法分析不同虫草多糖的单糖组成[J].药物分析杂志,2018,38(1):13-21.
[2]中国药典[S].一部.2015.
[3]Yan J K,Wang W Q,Wu J Y.Recent advances in Cordyceps sinensis polysaccharides:Mycelial fermentation,isolation,structure,and bioactivities:Areview[J].J Funct Foods,2014,6(1):33-47.
[4]Li S P,Zhao K J,Ji Z N,et al.A polysaccharide isolated from Cordyceps sinensis,a traditional Chinese medicine,protects PC12 cells against hydrogen peroxide-induced injury[J].Life Sci,2003,73(19):2503-2513.
[5]黄慧莲,杨敏娟,管咏梅,等.近5年发酵虫草菌粉的化学成分和临床应用研究进展[J].世界科学技术-中医药现代化,2014,16(10):2242-2245.
[6]张薇薇,龚韬,韩东河,等.人工虫草与冬虫夏草成分的比较研究[J].北京中医药,2016,35(1):87-91.
[7]邹秦文,肖新月,林瑞超.冬虫夏草液体深层发酵菌丝体相关制剂的研究现状[J].药物分析杂志,2009,29(4):680-687.
[8]邱霞.心肝宝胶囊治疗肺心病的临床疗效[J].中国实用医药,2013,8(36):173-174.
[9]许慧娟,李时悦.百令胶囊的药理作用及其在肺部疾病的研究进展[J].中国中药杂志,2010,35(20):2777-2781.
[10]刘燕霞.至灵胶囊联合缬沙坦分散片治疗早期糖尿病肾病临床效果分析[J].内蒙古中医药,2017,36(10):7-8.
[11]李兴高,陈奇,黄梦雨,等.金水宝胶囊药理研究进展[J].江西中医学院学报,2000,12(3):143-144.
[12]马淑梅,余伯成,唐亮,等.虫草素制剂对百草枯致大鼠肺纤维化的作用研究[J].药物评价研究,2016,39(5):753-757.
[13]李蓉,孙书娟,江晓路.虫草多糖的分离纯化及结构鉴定[J].中国药师,2015,18(3):407-419.
[14]杨威,殷茵,宋丽艳,等.高效阴离子交换-脉冲安培检测色谱法测定人工培养蛹虫草多糖的单糖组成[J].中草药,2008,39(4):531-535.
[15]郭思维.虫草多糖脱色脱蛋白工艺研究及抗NCI-H446肿瘤细胞活性检测[D].长沙:湖南师范大学,2014.
[16]宗雯雯,沈照鹏,卜义明,等.蛹虫草多糖单糖组成的方法学研究[J].菌物学报,37(3):395-404.
[17]陈丽华,张清华,欧阳胜,等.虫草发酵液多糖含量的测定[J].时珍国医国药,2015,26(12):2880-2882.
[18]崔立勋,张爱华,孙守英.虫草多糖的分离纯化及含量测定[J].中国医药科学,2012,2(21):95-96.
[19]王建壮,安洁,吕华冲.蛹虫草子实体中多糖含量测定方法的研究[J].中国现代药物应用,2008,2(15):19-20.
[20]葛新,李云兰,白小红,等.比色法测定人工虫草菌丝体多糖含量[J].山西医科大学学报,2001,32(5):418-419.
[21]朱卫丰,赵加茜,刘小林,等.金水宝胶囊中发酵虫草菌粉多糖的指纹图谱研究[J].中草药,2017,48(16):3322-3326.
[22]张利沙,孙玮玮,徐自慧,等.复方南板蓝根颗粒中多糖的含量测定及单糖组成分析[J].药物分析杂志,2017,37(10):1845-1850.
[23]张颖,曾艳,张丽姣,等.蛹虫草菌糠多糖的分离纯化及结构组成分析[J].食品科学,2014,35(13):54-58.
[24]彭彦峰,吴兆亮,李英杰.分光光度法测定微生物发酵液中D-核糖浓度及其机理[J].分析化学,2002,30(8):975-977.
[2]中国药典[S].一部.2015.
[3]Yan J K,Wang W Q,Wu J Y.Recent advances in Cordyceps sinensis polysaccharides:Mycelial fermentation,isolation,structure,and bioactivities:Areview[J].J Funct Foods,2014,6(1):33-47.
[4]Li S P,Zhao K J,Ji Z N,et al.A polysaccharide isolated from Cordyceps sinensis,a traditional Chinese medicine,protects PC12 cells against hydrogen peroxide-induced injury[J].Life Sci,2003,73(19):2503-2513.
[5]黄慧莲,杨敏娟,管咏梅,等.近5年发酵虫草菌粉的化学成分和临床应用研究进展[J].世界科学技术-中医药现代化,2014,16(10):2242-2245.
[6]张薇薇,龚韬,韩东河,等.人工虫草与冬虫夏草成分的比较研究[J].北京中医药,2016,35(1):87-91.
[7]邹秦文,肖新月,林瑞超.冬虫夏草液体深层发酵菌丝体相关制剂的研究现状[J].药物分析杂志,2009,29(4):680-687.
[8]邱霞.心肝宝胶囊治疗肺心病的临床疗效[J].中国实用医药,2013,8(36):173-174.
[9]许慧娟,李时悦.百令胶囊的药理作用及其在肺部疾病的研究进展[J].中国中药杂志,2010,35(20):2777-2781.
[10]刘燕霞.至灵胶囊联合缬沙坦分散片治疗早期糖尿病肾病临床效果分析[J].内蒙古中医药,2017,36(10):7-8.
[11]李兴高,陈奇,黄梦雨,等.金水宝胶囊药理研究进展[J].江西中医学院学报,2000,12(3):143-144.
[12]马淑梅,余伯成,唐亮,等.虫草素制剂对百草枯致大鼠肺纤维化的作用研究[J].药物评价研究,2016,39(5):753-757.
[13]李蓉,孙书娟,江晓路.虫草多糖的分离纯化及结构鉴定[J].中国药师,2015,18(3):407-419.
[14]杨威,殷茵,宋丽艳,等.高效阴离子交换-脉冲安培检测色谱法测定人工培养蛹虫草多糖的单糖组成[J].中草药,2008,39(4):531-535.
[15]郭思维.虫草多糖脱色脱蛋白工艺研究及抗NCI-H446肿瘤细胞活性检测[D].长沙:湖南师范大学,2014.
[16]宗雯雯,沈照鹏,卜义明,等.蛹虫草多糖单糖组成的方法学研究[J].菌物学报,37(3):395-404.
[17]陈丽华,张清华,欧阳胜,等.虫草发酵液多糖含量的测定[J].时珍国医国药,2015,26(12):2880-2882.
[18]崔立勋,张爱华,孙守英.虫草多糖的分离纯化及含量测定[J].中国医药科学,2012,2(21):95-96.
[19]王建壮,安洁,吕华冲.蛹虫草子实体中多糖含量测定方法的研究[J].中国现代药物应用,2008,2(15):19-20.
[20]葛新,李云兰,白小红,等.比色法测定人工虫草菌丝体多糖含量[J].山西医科大学学报,2001,32(5):418-419.
[21]朱卫丰,赵加茜,刘小林,等.金水宝胶囊中发酵虫草菌粉多糖的指纹图谱研究[J].中草药,2017,48(16):3322-3326.
[22]张利沙,孙玮玮,徐自慧,等.复方南板蓝根颗粒中多糖的含量测定及单糖组成分析[J].药物分析杂志,2017,37(10):1845-1850.
[23]张颖,曾艳,张丽姣,等.蛹虫草菌糠多糖的分离纯化及结构组成分析[J].食品科学,2014,35(13):54-58.
[24]彭彦峰,吴兆亮,李英杰.分光光度法测定微生物发酵液中D-核糖浓度及其机理[J].分析化学,2002,30(8):975-977.