铁皮石斛与霍山石斛中甘露糖、葡萄糖及柚皮素的含量比较
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摘要
目的:优化铁皮石斛中甘露糖与葡萄糖的柱前衍生HPLC含量测定方法以及柚皮素HPLC含量测定方法,比较铁皮石斛与霍山石斛中这3种成分的含量差异。方法:在2015年版《中国药典》铁皮石斛甘露糖柱前衍生HPLC含量测定项下色谱条件基础上,选择乙腈-0. 02 mol·L~(-1)乙酸铵溶液为流动相系统梯度洗脱,同时测定甘露糖与葡萄糖的含量,并分析甘露糖与葡萄糖的峰面积比值;采用Kromasil 100-5 C18色谱柱(4. 6 mm×250 mm,5μm);检测波长250 nm;流速1. 0 mL·min~(-1);柱温30℃。柚皮素HPLC含量测定采用Kromasil 100-5 C18色谱柱(4. 6 mm×250 mm,5μm);流动相乙腈-甲醇-0. 4%磷酸溶液,梯度洗脱;检测波长290 nm;流速0. 8 mL·min~(-1);柱温40℃。结果:甘露糖与葡萄糖在0. 15~3. 0,0. 075~2. 25μg线性关系良好(r=0. 999 9),平均加样回收率分别为99. 01%(RSD 2. 1%),101. 69%(RSD 2. 0%),重复性、耐用性等其他方法学研究符合要求。43批不同产区铁皮石斛中甘露糖、葡萄糖以及两者的含量之和分别在12. 75%~36. 40%,2. 93%~18. 39%,19. 23%~54. 58%,除极少数样品外,基本符合2015年版《中国药典》甘露糖含量限度要求,甘露糖与葡萄糖含量之和也接近总多糖含量限度要求;含量与产区相关性不显著。12批霍山石斛的总糖则分别在14. 33%~29. 47%,6. 64%~15. 20%,25. 73%~44. 37%,其含量以及峰面积比值基本落在铁皮石斛范围期间,多批次的平均含量也基本与铁皮石斛一致(约33%左右)。柚皮素在0. 020 8~0. 832 0μg线性关系良好(r=0. 999 9),平均加样回收率为101. 96%(RSD 1. 8%)。11批铁皮石斛与7批霍山石斛的柚皮素含量分别为0. 053 2~0. 122 4 mg·g~(-1)(均值为0. 081 0 mg·g~(-1)),0. 040 3~0. 090 0 mg·g~(-1)(均值为0. 068 3 mg·g~(-1)),铁皮石斛含量稍高于霍山石斛,但含量均亦未达到0. 02%的质量标准下限的常规要求。结论:铁皮石斛中甘露糖与葡萄糖HPLC含量测定方法重复性较好,用两者含量之和替代具有较大误差的总多糖含量作为测定指标具有可行性;单糖含量测定可应用于霍山石斛的定量质控指标;但依据2种石斛的总多糖含量、水解后的单糖含量与峰面积比值以及柚皮素含量,无法区分铁皮石斛与霍山石斛,需结合其他专属性方法方能对两种石斛进行区别。
Objective: To optimize the pre-column derivation high performance liquid chromatography(HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense,and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch. P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0. 02 mol·L~(-1) ammonium acetate solution as mobile phase for gradient elution.Kromasil 100-5 C18 was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min~(-1); column temperature was 30 ℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C18 with the acetonitrile-methanol-0. 4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0. 8 mL·min~(-1),and column temperature was 40 ℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0. 15-3. 0 μg and 0. 075-2. 25 μg(r = 0. 999 9);and their average recoveries were 99. 01%(RSD 2. 1%) and 101. 69%(RSD 2. 0%) respectively. In addition,the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them(Cm+ Cg) were 12. 75%-36. 40%,2. 93%~(-1)8. 39% and19. 23%-54. 58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch. P 2015,and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch. P. The correlation between content and origin was not significant. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them(Cm+ Cg) were 14. 33%-29. 47%,6. 64%~(-1)5. 20%,and 25. 73%-44. 37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose(Am/Ag) were within the scope of D. officinale's; in addition,their average contents were basically the same with those in D. officinale(about 33%). Next,naringenin showed a good linear relationship within the range of 0. 020 8-0. 832 0 μg(r = 0. 999 9),and its average recovery was 101. 96%(RSD 1. 8%). The content of naringenin was 0. 053 2-0. 122 4 mg·g~(-1)(average value of 0. 081 0 mg·g~(-1)) in 11 batch of D. officinale,slightly higher than 0. 040 3-0. 090 0 mg · g~(-1)(average value of 0. 068 3 mg · g~(-1)) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch. P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible,and their sum content is possible to substitute the total polysccaride determination(with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However,it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin,and shall be combined with other specificity methods for further identification.
Objective: To optimize the pre-column derivation high performance liquid chromatography(HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense,and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch. P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0. 02 mol·L~(-1) ammonium acetate solution as mobile phase for gradient elution.Kromasil 100-5 C18 was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min~(-1); column temperature was 30 ℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C18 with the acetonitrile-methanol-0. 4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0. 8 mL·min~(-1),and column temperature was 40 ℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0. 15-3. 0 μg and 0. 075-2. 25 μg(r = 0. 999 9);and their average recoveries were 99. 01%(RSD 2. 1%) and 101. 69%(RSD 2. 0%) respectively. In addition,the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them(Cm+ Cg) were 12. 75%-36. 40%,2. 93%~(-1)8. 39% and19. 23%-54. 58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch. P 2015,and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch. P. The correlation between content and origin was not significant. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them(Cm+ Cg) were 14. 33%-29. 47%,6. 64%~(-1)5. 20%,and 25. 73%-44. 37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose(Am/Ag) were within the scope of D. officinale's; in addition,their average contents were basically the same with those in D. officinale(about 33%). Next,naringenin showed a good linear relationship within the range of 0. 020 8-0. 832 0 μg(r = 0. 999 9),and its average recovery was 101. 96%(RSD 1. 8%). The content of naringenin was 0. 053 2-0. 122 4 mg·g~(-1)(average value of 0. 081 0 mg·g~(-1)) in 11 batch of D. officinale,slightly higher than 0. 040 3-0. 090 0 mg · g~(-1)(average value of 0. 068 3 mg · g~(-1)) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch. P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible,and their sum content is possible to substitute the total polysccaride determination(with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However,it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin,and shall be combined with other specificity methods for further identification.
引文
[1]魏刚,顺庆生,杨明志.石斛求真——中国药用石斛之历史、功效、真影与特征指纹图谱[M].成都:四川科学技术出版社,2014:3-19.
[2]魏刚,顺庆生,李名海,等.中华仙草——霍山石斛[M].成都:四川科学技术出版社,2015:203.
[3]罗宇琴,肖苏萍,赵润怀,等.霍山石斛与铁皮石斛对比研究进展[J].中国现代中药,2016,18(8):1067-1070,1080.
[4]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2010:265-266.
[5]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:282.
[6]黄月纯,叶家宏,戴亚峰,等.霍山石斛与美花石斛多糖柱前衍生HPLC特征图谱比较[J].中药新药与临床药理,2014,25(6):725-730.
[7]甘小娜,徐英,徐红,等.铁皮石斛中多糖和甘露糖含量测定方法的改进及与齿瓣石斛的比较研究[J].中国药品标准,2014,15(4):276-279.
[8]陈晓梅,王芳菲,王云强,等.基于柚皮素、联苄和多糖分析的珍贵药用植物铁皮石斛鉴别分析[J].中国科学:生命科学,2012,42(12):1002-1009.
[9]周桂芬,陈素红,吕圭源,等.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].中国中药杂志,2013,38(4):520-523.
[10]周天策.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].黑龙江科技信息,2016(22):129.
[11]王少平,孙乙铭,姜艳,等.基于指纹图谱及柚皮素含量对铁皮石斛及伪品的鉴定[J].浙江农业学报,2015,27(12):2199-2205.
[12]孟海涛.安徽霍山产三种石斛HPLC化学指纹研究[D].合肥:合肥工业大学,2015.
[13]叶子,卢叶,薛亚甫,等.铁皮石斛专属性成分的分离制备及质量标准研究[J].中国中药杂志,2016,41(13):2481-2486.
[14]魏刚,顺庆生,黄月纯,等. 3种铁皮石斛种源HPLC特征图谱比较研究[J].中药新药与临床药理,2014,25(4):467-471.
[15]黄月纯,谢镇山,任晋,等. 3种种源铁皮石斛叶黄酮类成分HPLC特征图谱比较[J].中国实验方剂学杂志,2015,21(24):37-40.
[16]魏刚,顺庆生,戴亚峰,等.霍山石斛HPLC特征图谱研究[J].中成药,2014,36(12):2642-2644.
[17] WU C F,GUI S H,HUANG Y C,et al. Characteristic fingerprint analysis of Dendrobium huoshanense by ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry[J]. Anal Meth,2016,8(18):3802-3808.
[2]魏刚,顺庆生,李名海,等.中华仙草——霍山石斛[M].成都:四川科学技术出版社,2015:203.
[3]罗宇琴,肖苏萍,赵润怀,等.霍山石斛与铁皮石斛对比研究进展[J].中国现代中药,2016,18(8):1067-1070,1080.
[4]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2010:265-266.
[5]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:282.
[6]黄月纯,叶家宏,戴亚峰,等.霍山石斛与美花石斛多糖柱前衍生HPLC特征图谱比较[J].中药新药与临床药理,2014,25(6):725-730.
[7]甘小娜,徐英,徐红,等.铁皮石斛中多糖和甘露糖含量测定方法的改进及与齿瓣石斛的比较研究[J].中国药品标准,2014,15(4):276-279.
[8]陈晓梅,王芳菲,王云强,等.基于柚皮素、联苄和多糖分析的珍贵药用植物铁皮石斛鉴别分析[J].中国科学:生命科学,2012,42(12):1002-1009.
[9]周桂芬,陈素红,吕圭源,等.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].中国中药杂志,2013,38(4):520-523.
[10]周天策.高效液相色谱法测定铁皮石斛中柚皮素的含量[J].黑龙江科技信息,2016(22):129.
[11]王少平,孙乙铭,姜艳,等.基于指纹图谱及柚皮素含量对铁皮石斛及伪品的鉴定[J].浙江农业学报,2015,27(12):2199-2205.
[12]孟海涛.安徽霍山产三种石斛HPLC化学指纹研究[D].合肥:合肥工业大学,2015.
[13]叶子,卢叶,薛亚甫,等.铁皮石斛专属性成分的分离制备及质量标准研究[J].中国中药杂志,2016,41(13):2481-2486.
[14]魏刚,顺庆生,黄月纯,等. 3种铁皮石斛种源HPLC特征图谱比较研究[J].中药新药与临床药理,2014,25(4):467-471.
[15]黄月纯,谢镇山,任晋,等. 3种种源铁皮石斛叶黄酮类成分HPLC特征图谱比较[J].中国实验方剂学杂志,2015,21(24):37-40.
[16]魏刚,顺庆生,戴亚峰,等.霍山石斛HPLC特征图谱研究[J].中成药,2014,36(12):2642-2644.
[17] WU C F,GUI S H,HUANG Y C,et al. Characteristic fingerprint analysis of Dendrobium huoshanense by ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry[J]. Anal Meth,2016,8(18):3802-3808.