高效液相色谱法测定天然冬虫夏草和蛹虫草及其人工培养物中3个核苷类化合物含量
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摘要
目的:建立快速分析天然冬虫夏草、蛹虫草及其发酵制品中核苷类化合物成分的高效液相色谱法,比较天然冬虫夏草和蛹虫草及其发酵制品中核苷类化合物的含量。方法:采用超声提取的方法,制备供试品溶液。色谱柱为Hypersil C18柱(200 mm×4.6 mm,5μm),流动相为pH6.5磷酸盐缓冲液-甲醇(85∶15),检测波长为260 nm。结果:天然冬虫夏草和蛹虫草及其人工培养物中的3种核苷类成分在25 min内达到基线分离;腺苷在5.0~80.0μg·ml-1范围内与峰面积呈现良好的线性关系,相关系数为0.999 3,样品回收率为99.4%;虫草素在4.37~70.0μg·ml-1范围内与峰面积呈现良好的线性关系,相关系数为0.999 7,样品回收率为98.3%;腺嘌呤在6.75~108.0μg·ml-1范围内与峰面积呈现良好的线性关系,相关系数为0.999 7,样品回收率为98.9%。结论:天然虫草与人工虫草培养物有相似的核苷类化合物,但在含量上有较大差异。此方法简便、准确,重复性好,可作为判定天然虫草及其培养物质量的一种方法。
OBJECTIVE To establish a high performance liquid chromatography(HPLC)method for determination of nucleosides from natural and cultured Cordyceps sinensis and Cordyceps militaris,and compare their contents.METHODS The contents of nucleosides were determined using HPLC on Hypersil C18 column with pH6.0phosphate-methanol(85∶15)as mobile phase and detected at 260 nm.RESULTS Under the optimized conditions,the three nucleosides reached baseline separation in 25 minutes.Adenosine,Cordycepin and Adenine showed good linear relationship in the range of 5.0-80.0μg·ml-1(r=0.999 3),4.37-70.0μg·ml-1(r=0.999 7)and 6.75-108.0μg·ml-1(r=0.999 7),respectively.CONCLUSION Similar nucleosides have been obtained from natural Cordyceps sinensis and cultured Cordyceps militaris,but with relatively great differences in their contents.This method can be utilized for quantitative analysis of nucleosides in natural and cultured Cordyceps and its substitutes,and it is helpful to control their quality.The method is accurate,fast,and convenient.
OBJECTIVE To establish a high performance liquid chromatography(HPLC)method for determination of nucleosides from natural and cultured Cordyceps sinensis and Cordyceps militaris,and compare their contents.METHODS The contents of nucleosides were determined using HPLC on Hypersil C18 column with pH6.0phosphate-methanol(85∶15)as mobile phase and detected at 260 nm.RESULTS Under the optimized conditions,the three nucleosides reached baseline separation in 25 minutes.Adenosine,Cordycepin and Adenine showed good linear relationship in the range of 5.0-80.0μg·ml-1(r=0.999 3),4.37-70.0μg·ml-1(r=0.999 7)and 6.75-108.0μg·ml-1(r=0.999 7),respectively.CONCLUSION Similar nucleosides have been obtained from natural Cordyceps sinensis and cultured Cordyceps militaris,but with relatively great differences in their contents.This method can be utilized for quantitative analysis of nucleosides in natural and cultured Cordyceps and its substitutes,and it is helpful to control their quality.The method is accurate,fast,and convenient.
引文
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[7]Zhou X,Meyer CU,Schmidtke P et al.Effect of cordycep in on interleukin-10 production of human peripheral blood mononuclear cells[J].Eur J Pharm acol,2002,453(2-3):309.
[8]Ahn YJ,Park SJ,Lee SG.Cordycep in:Selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp[J].J Agric Food Chem,2000,48(7):2744.
[9]Kodama EN,Mc Caffrey RP,Yusa K,et al.Antileukemic activity and mechanism of action of cordycep in against terminal deoxynucleotidyl transferase-positive(TdT+)leukemic cells.B iochem Pharm acol,2000,59(3):273.
[10]Shiao MS,Wang ZN,Lin LJ,et al.Profiles of nucleosides and nitrogen bases in Chinese medicinal fungus Cordyceps sinensis and species[J].Botanical Bulletin of Academia Sinica(Taipei),1994,35(4);261-267.
[11]汪宇,于荣敏,杨光照.HPCE法测定蛹虫草及其培养物中核苷类化合物的含量[J].解放军药学学报,2003,19(5):331.
[12]刘玉军,徐芳,陈波,等.毛细管区带电泳法测定冬虫夏草及人工蛹虫草子实体中核苷及碱成分的含量[J].药物分析杂志,2010,30(1):24-29.
[13]Li SP,Li P,Lai CM.Simultaneous determination of ergosterol,nucleosides and their bases from natural andcultured Cordycep s by p ressurised liquid extraction and high-performance liquid chromatography[J].J ChromatogrA,2004,1036(2):239.
[14]米莉莉,王智华,洪筱坤.冬虫夏草及人工菌丝体的核苷类成分的HPLC研究[J].中草药,2003,34(6):573.
[15]Fan H,Li SP,Xiang JJ.Qualitative and quantitative determination of nucleosides,bases and their analogues in natural and cultured Cordyceps by p ressurized liquid extraction and high performance liquid chromatography electrosp rayionization tandem mass spectrometry(HPLC-ESI-MS/MS)[J].Anal Chim Acta,2006,567(2):218.
[2]Toda N,Okunishi H,Taniyama K,et al.Responses to adenosine nucleotides and related compounds of isolated dog cerebral,coronary and mesenteric arteries[J].Blood Vessels,1982,19(5):226.
[3]Pelleg A,Porter RS.The pharmacology of adenosine[J].Pharmacotherapy,1990,10(3):157.
[4]Ribeiro JA.Purinergic inhibition of neurotransmitterrelease in the central nervous system[J].Pharmacol Toxicol,1995,77(5):299-305.
[5]中国药典.一部[S].2010:86.
[6]Cunningham KG,Hutchinson SA,Manson W.Cordycepin,a metablic Product from Cultures of Cordyceps militaris(Linn.)Link[J].Nature,1950,166(4231):949.
[7]Zhou X,Meyer CU,Schmidtke P et al.Effect of cordycep in on interleukin-10 production of human peripheral blood mononuclear cells[J].Eur J Pharm acol,2002,453(2-3):309.
[8]Ahn YJ,Park SJ,Lee SG.Cordycep in:Selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp[J].J Agric Food Chem,2000,48(7):2744.
[9]Kodama EN,Mc Caffrey RP,Yusa K,et al.Antileukemic activity and mechanism of action of cordycep in against terminal deoxynucleotidyl transferase-positive(TdT+)leukemic cells.B iochem Pharm acol,2000,59(3):273.
[10]Shiao MS,Wang ZN,Lin LJ,et al.Profiles of nucleosides and nitrogen bases in Chinese medicinal fungus Cordyceps sinensis and species[J].Botanical Bulletin of Academia Sinica(Taipei),1994,35(4);261-267.
[11]汪宇,于荣敏,杨光照.HPCE法测定蛹虫草及其培养物中核苷类化合物的含量[J].解放军药学学报,2003,19(5):331.
[12]刘玉军,徐芳,陈波,等.毛细管区带电泳法测定冬虫夏草及人工蛹虫草子实体中核苷及碱成分的含量[J].药物分析杂志,2010,30(1):24-29.
[13]Li SP,Li P,Lai CM.Simultaneous determination of ergosterol,nucleosides and their bases from natural andcultured Cordycep s by p ressurised liquid extraction and high-performance liquid chromatography[J].J ChromatogrA,2004,1036(2):239.
[14]米莉莉,王智华,洪筱坤.冬虫夏草及人工菌丝体的核苷类成分的HPLC研究[J].中草药,2003,34(6):573.
[15]Fan H,Li SP,Xiang JJ.Qualitative and quantitative determination of nucleosides,bases and their analogues in natural and cultured Cordyceps by p ressurized liquid extraction and high performance liquid chromatography electrosp rayionization tandem mass spectrometry(HPLC-ESI-MS/MS)[J].Anal Chim Acta,2006,567(2):218.
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