长白山药用植物和食用菌的谷胱甘肽含量分析研究
|
摘要
谷胱甘肽是生物体自合成的内源性活性三肽,分为还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)两种类型。谷胱甘肽具有抗氧化、免疫,解毒、参与氨基酸的吸收和DNA的合成等重要的生物活性功能。近年来,外源性谷胱甘肽(主要是针剂和片剂等)应用于防病、治病中的报道渐多,且作为保健品、食品添加剂等领域的应用备受人们的瞩目。但外源性谷胱甘肽应用中,其有效活性功能普遍受到质疑。质疑的主要原因是;化学方法制备的外源性谷胱甘肽生物兼容性较差,因此,对其能否有效地被生物细胞所吸收来发挥其生物活性作用普遍表示怀疑。人们认为,最有效的作用机制是筛选生物兼容性好的富含谷胱甘肽和富含谷胱甘肽生物合成前驱物质的天然物作为谷胱甘肽的来源,可使生物体有效地吸收谷胱甘肽和加快细胞生物合成谷胱甘肽的能力来发挥其生物学功能。生物兼容性较好的这种外源性谷胱甘肽的主要来源是动、植物(包括菌类)。因此,分析和筛选富含谷胱甘肽的动、植物对谷胱甘肽在防病、治病中的应用都具有重要的意义。
本文在文献综述的基础上,选择长白山区出产的野生和栽培的食用菌和药用植物作为分析材料,利用高效液相色谱法同时测定氧化型和还原型谷胱甘肽的含量。结果表明;在红景天,人参,五味子三种药用植物中,五味子的谷胱甘肽总含量最高,其中野生植物的还原型谷胱甘肽和氧化型谷胱甘肽比率(GSH/GSSH)明显高于栽培的样品。在所选的食用菌供试品中,野生元蘑的谷胱甘肽的含量明显高于香菇、松蘑,榛蘑、木耳中的含量,其中野生木耳中的GSH/GSSG比率最高。同种类的食用菌供试品种,野生样品的GSH/GSSG比率高于栽培样品的比率。本研究在色谱测定供试品的制备过程中,对植物样品除蛋白处理,选择不同流动相及其配比来优化色谱分离效果等方面作了有益的探索。本实验结果对富含谷胱甘肽的药食两用的植物和菌类的筛选、GSH/GSSG比率对植物抗逆性的相关性研究,为长白山经济植物和菌类开发提供了重要的参考数据。
Glutathione is self-synthetic and endogenous active tripeptide (Z-glutamyl-Z-cysteinyl-L-glycine) in organism, it classified into reduced glutathione (GSH) and oxidized glutathione (GSSG). There are biological functions of glutathione, such as participating in absorption and transportation of amino acids, influencing the synthesis of proteins and DNA, scavenging peroxide and free radical in vivo, maintaining morphology of erythrocytes, carrying oxygen ability and detoxification, etc. Recently, the report of exogenous glutathione (mainly injection, tablet form) preventing and curing diseases gradually become increased; the application of exogenous glutathione as food additives and health products is attracting worldwide industries' attention. However, effective active function of exogenous glutathione is always common subjected to query. The main reason for the question is the worse biological compatibility of chemosynthtic glutathione. Thus, an effective measure screening of the rich and the better biological compatibility glutathione and its precursors in natural product became essential. Glutathione is widely distributed in animal and plants (including edible fungi).
Based on the progress of the research of glutathione, medical plant and edible fungi (wild and cultivated) on Changbai Mountain area can be used target material to screen the glutathione. The content of GSH and GSSG was determined simultaneously by means of high performance liquid chromatography (HPLC). The results showed that the highest total content of glutathione in Schisandra chinensis (Turcz.) Baill , followed Rhodiola sachlinensis A.Bor and Panax ginseng C.A.Mey, moreover,the radio of GSH/GSSG of the wild species is significantly higher than the cultivated. Furthermore, the content of glutathione in the wild Pleurotus ostrcatus is significantly higher than Lentinus edodes, Tricholoma matsutake,Arimillaria mellea and Auricularia auricular. The radio of GSH/GSSG of the wild species is also significantly higher than the cultivated, among the radio of GSH/GSSG of the wild Auricularia auricular is the highest. In this thesis, the optimum experimental conditions for the determination of glutathione and the separations of reversed phase ion pair chromatography of the GSH and GSSG were investigated, such as method of removing protein, selection and proportion of various flowing phase, etc. In addition, the result will offer important reference data for exploitation of economic plant and fungi on Changbai Mountain area, for screening the rich glutathione of plant and fungi having medicine and food function, for discussion about studies of plant stress resistance to the radio of GSH/GSSG.
本文在文献综述的基础上,选择长白山区出产的野生和栽培的食用菌和药用植物作为分析材料,利用高效液相色谱法同时测定氧化型和还原型谷胱甘肽的含量。结果表明;在红景天,人参,五味子三种药用植物中,五味子的谷胱甘肽总含量最高,其中野生植物的还原型谷胱甘肽和氧化型谷胱甘肽比率(GSH/GSSH)明显高于栽培的样品。在所选的食用菌供试品中,野生元蘑的谷胱甘肽的含量明显高于香菇、松蘑,榛蘑、木耳中的含量,其中野生木耳中的GSH/GSSG比率最高。同种类的食用菌供试品种,野生样品的GSH/GSSG比率高于栽培样品的比率。本研究在色谱测定供试品的制备过程中,对植物样品除蛋白处理,选择不同流动相及其配比来优化色谱分离效果等方面作了有益的探索。本实验结果对富含谷胱甘肽的药食两用的植物和菌类的筛选、GSH/GSSG比率对植物抗逆性的相关性研究,为长白山经济植物和菌类开发提供了重要的参考数据。
Glutathione is self-synthetic and endogenous active tripeptide (Z-glutamyl-Z-cysteinyl-L-glycine) in organism, it classified into reduced glutathione (GSH) and oxidized glutathione (GSSG). There are biological functions of glutathione, such as participating in absorption and transportation of amino acids, influencing the synthesis of proteins and DNA, scavenging peroxide and free radical in vivo, maintaining morphology of erythrocytes, carrying oxygen ability and detoxification, etc. Recently, the report of exogenous glutathione (mainly injection, tablet form) preventing and curing diseases gradually become increased; the application of exogenous glutathione as food additives and health products is attracting worldwide industries' attention. However, effective active function of exogenous glutathione is always common subjected to query. The main reason for the question is the worse biological compatibility of chemosynthtic glutathione. Thus, an effective measure screening of the rich and the better biological compatibility glutathione and its precursors in natural product became essential. Glutathione is widely distributed in animal and plants (including edible fungi).
Based on the progress of the research of glutathione, medical plant and edible fungi (wild and cultivated) on Changbai Mountain area can be used target material to screen the glutathione. The content of GSH and GSSG was determined simultaneously by means of high performance liquid chromatography (HPLC). The results showed that the highest total content of glutathione in Schisandra chinensis (Turcz.) Baill , followed Rhodiola sachlinensis A.Bor and Panax ginseng C.A.Mey, moreover,the radio of GSH/GSSG of the wild species is significantly higher than the cultivated. Furthermore, the content of glutathione in the wild Pleurotus ostrcatus is significantly higher than Lentinus edodes, Tricholoma matsutake,Arimillaria mellea and Auricularia auricular. The radio of GSH/GSSG of the wild species is also significantly higher than the cultivated, among the radio of GSH/GSSG of the wild Auricularia auricular is the highest. In this thesis, the optimum experimental conditions for the determination of glutathione and the separations of reversed phase ion pair chromatography of the GSH and GSSG were investigated, such as method of removing protein, selection and proportion of various flowing phase, etc. In addition, the result will offer important reference data for exploitation of economic plant and fungi on Changbai Mountain area, for screening the rich glutathione of plant and fungi having medicine and food function, for discussion about studies of plant stress resistance to the radio of GSH/GSSG.
引文
[1]程时,丁海勤.谷胱甘肽及其抗氧化作用今日谈.生理科学进展,2002,33(1):85-90
[2]刘振玉.谷胱甘肽的研究与应用.生命的化学,1995,15(1):19-21
[3]周秀琴编译.特殊的氨基酸衍生物-谷胱甘肽.发酵科技通讯,2007,36(2):50-51
[4]卢薇.谷胱甘肽的化学与医疗作用.化学教育,2004(1):7-12
[5]郑云郎.谷胱甘肽的生物学功能.生物学通报,1995,30(5):22-24
[6]麦维军,张明永.高等植物体内的谷胱甘肽.生物学通报,2005,40(6):4-5
[7]麦维军,王颖,梁承邺,等.谷胱甘肽在植物抗逆中的作用.广西植物,2005,25(6):570-575
[8]陈坤明,宫海军,王锁民.植物谷胱甘肽代谢与环境胁迫.西北植物学报,2004,24(6):1119-1130
[9]范崇东,王森,卫功元,等.谷胱甘肽测定方法研究进展.生物技术,2004,14(1):68-70
[10]樊跃平,于健春,余跃,等.谷胱甘肽的生理意义及其各种测定方法比较、评价.中国临床营养杂志,2003,11(2):136-139
[11]杨培慧,齐剑英,冯德雄,等.谷胱甘肽的应用及其检测方法.中国生化药物杂志,2002,23(1):52-54
[12]Maura Floreani,Marcella Petrone,Patrizia Debetto,et al.A Comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues.Free Radical Research,1997,26(5):449-455
[13]廖飞,杨晓,康格非,等.紫外吸收碘量法测定微量维生素C和还原型谷胱甘肽.重庆医科大学学报,2003,28(3):372-373
[14]翟文慧,王爱月.紫外分光光度法测定保健食品中谷胱甘肽.预防医学论坛,2005,11(5):560-561
[15]郭黎平,刘国良,张卓勇,等.荧光光度法测定大豆提取液中还原型谷胱甘肽.东北师大学报自然科学版,2001,33(1):34-38
[16]黄彦生,智艳芳,孔沈燕,等.用荧光分光光度法测量冠心病患者血 浆谷胱甘肽.光谱学与光谱分析,2006,26(5):936-940
[17]Frank Tieze Enzymic.Method for Quantitative determination of nanogram amounts of total and oxidized glutathione:applications to mammalian blood and other tissues.Anal.Biochem.,1969,27:502-522
[18]陈剑,何忠于,查全性,等.谷胱甘肽在粉末微电极上的电化学行为及其检测.电化学,2004,10(3):254-259
[19]Elizabeth Jayatilleke,Spencer Shaw.A high-performance liquid chromatographic assay for reduced and oxidized glutathione in biological samples.Analytical Biochemistry,1993,213:452-457
[20]Jitka Petrlova,Radka Mikelova,Karel Stejska,et al.Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with coupled-array detection.J.Sep.Sci.,2006,29:1166-1173
[21]Wen Zhang,Fangli Wan,Wei Zhu,et al.Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes.Journal of Chromatography B,2005,818:227-232
[22]陈国民,孙航,单幼兰,等.谷胱甘肽的快速荧光检测法.临床检验杂志,2001,19(1):11-12
[23]王清江,陈相,丁飞,等.毛细管电泳.电化学检测法测定血浆中水溶性小分子抗氧化剂.分析化学,2005,33(7):969-971
[24]Mohammad A.Mansoor,Asbjerrn M.Svardal,Per M.Ueland.Determination of the in vivo redox status of cysteine,cysteinylglycine,homocysteine,and glutathione in human plasma.Analytical Biochemistry.,1992,200:218-229
[25]Marjan Gucek,Simon Makuc,Anita Mlakar,et al.Determination of glutathione in spruce needles by liquid chromatography/tandem mass spectrometry.Rapid Communications in Mass Spectrometry,2002,16:1186-1191
[26]J(?)r(?)me Bouligand,Alain Deroussent,Angelo Paci,et al.Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. Journal of Chromatography B,2006,832:67-74
[27] Xiangming Guan, Brianna Hoffman, Chandradhar Dwivedi, et al. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. Journal of Pharmaceutical and Biomedical Analysis,2003,31:251-261
[28] A.M. Di Peetra, R. Gotil, D. Bonazzi, et al. HPLC determination of glutathione and L-cysteine in pharmaceuticals after derivatization with ethacrynicacid. Journal of Pharmuceurical & Biomedical Analysis,1994,12(1):91-98
[29] E. Bald, G. Chwatko, R. G(?)owacki, et al. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. Journal of ChromatographyA,2004,1032:109-115
[30] W. Li, S.S. Bollecker, J.D. Schofield. Glutathione and related thiol compounds. I. glutathione and related thiol compounds in flour. Journal of Cereal Science,2004,39:205-212
[31] Miguel Asensi, Juan Sastre, Federico V Pallardo, et al. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Analytical Biochemistry,1994,217:323-328
[32] Jun Qu, Yiming Wang, Guoan Luo, et al. Validated quantitation of underivatized amino acids in human blood samples by volatile ion-pair reversed-phase liquid chromatography coupled to isotope dilution tandem mass spectrometry. Analytical Chemistry,2002, 74: 2034-2040
[33]陈金水,李朕杰.谷胱甘肽一种新型饲用添加剂.安全饲料.2005,5:40-41
[34]沈亚领,李爽,迟莉丽,等.谷胱甘肽的应用与生产.工业微生物,2000,30(2):41-45
[35]席秀芳,赵秀川.谷胱甘肽的药理与临床.中国医学文摘·内科学,2004,25(1):125-127
[36]王爱月,解魁,李发生.高效液相色谱法测定保健食品中谷胱甘肽含量的方法研究.中国卫生检验杂志,2007,17(7):1181-1182
[37]黄颖,段建平,杨明灿,等.毛细管电泳法测定黄瓜和西红柿中的谷胱甘肽.色谱,2003,21(5):510-512
[38]王春,严伟民.注射用还原型谷胱甘肽质量标准分析方法HPLC法的建立与验证.中国临床药学杂志,2005,14(3):167-169
[39]王秋林,王浩毅,丁怡,等.血浆中游离还原型及氧化型谷胱甘肽的荧光测定法.光谱学与光谱分析,2005,25(11):1834-1838
[40]林丽,曹旭妮,张文,等.碳纳米管修饰电极用于高效液相色谱对全血中巯基化合物的测定.分析化学,2003,31(3):261-265
[41]任亦飞,丁劲松,彭文兴,等.HPLC-MS法测定正常人血浆和全血中还原型谷胱甘肽的浓度.中南药学,2004,2(3):154-156
[2]刘振玉.谷胱甘肽的研究与应用.生命的化学,1995,15(1):19-21
[3]周秀琴编译.特殊的氨基酸衍生物-谷胱甘肽.发酵科技通讯,2007,36(2):50-51
[4]卢薇.谷胱甘肽的化学与医疗作用.化学教育,2004(1):7-12
[5]郑云郎.谷胱甘肽的生物学功能.生物学通报,1995,30(5):22-24
[6]麦维军,张明永.高等植物体内的谷胱甘肽.生物学通报,2005,40(6):4-5
[7]麦维军,王颖,梁承邺,等.谷胱甘肽在植物抗逆中的作用.广西植物,2005,25(6):570-575
[8]陈坤明,宫海军,王锁民.植物谷胱甘肽代谢与环境胁迫.西北植物学报,2004,24(6):1119-1130
[9]范崇东,王森,卫功元,等.谷胱甘肽测定方法研究进展.生物技术,2004,14(1):68-70
[10]樊跃平,于健春,余跃,等.谷胱甘肽的生理意义及其各种测定方法比较、评价.中国临床营养杂志,2003,11(2):136-139
[11]杨培慧,齐剑英,冯德雄,等.谷胱甘肽的应用及其检测方法.中国生化药物杂志,2002,23(1):52-54
[12]Maura Floreani,Marcella Petrone,Patrizia Debetto,et al.A Comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues.Free Radical Research,1997,26(5):449-455
[13]廖飞,杨晓,康格非,等.紫外吸收碘量法测定微量维生素C和还原型谷胱甘肽.重庆医科大学学报,2003,28(3):372-373
[14]翟文慧,王爱月.紫外分光光度法测定保健食品中谷胱甘肽.预防医学论坛,2005,11(5):560-561
[15]郭黎平,刘国良,张卓勇,等.荧光光度法测定大豆提取液中还原型谷胱甘肽.东北师大学报自然科学版,2001,33(1):34-38
[16]黄彦生,智艳芳,孔沈燕,等.用荧光分光光度法测量冠心病患者血 浆谷胱甘肽.光谱学与光谱分析,2006,26(5):936-940
[17]Frank Tieze Enzymic.Method for Quantitative determination of nanogram amounts of total and oxidized glutathione:applications to mammalian blood and other tissues.Anal.Biochem.,1969,27:502-522
[18]陈剑,何忠于,查全性,等.谷胱甘肽在粉末微电极上的电化学行为及其检测.电化学,2004,10(3):254-259
[19]Elizabeth Jayatilleke,Spencer Shaw.A high-performance liquid chromatographic assay for reduced and oxidized glutathione in biological samples.Analytical Biochemistry,1993,213:452-457
[20]Jitka Petrlova,Radka Mikelova,Karel Stejska,et al.Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with coupled-array detection.J.Sep.Sci.,2006,29:1166-1173
[21]Wen Zhang,Fangli Wan,Wei Zhu,et al.Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes.Journal of Chromatography B,2005,818:227-232
[22]陈国民,孙航,单幼兰,等.谷胱甘肽的快速荧光检测法.临床检验杂志,2001,19(1):11-12
[23]王清江,陈相,丁飞,等.毛细管电泳.电化学检测法测定血浆中水溶性小分子抗氧化剂.分析化学,2005,33(7):969-971
[24]Mohammad A.Mansoor,Asbjerrn M.Svardal,Per M.Ueland.Determination of the in vivo redox status of cysteine,cysteinylglycine,homocysteine,and glutathione in human plasma.Analytical Biochemistry.,1992,200:218-229
[25]Marjan Gucek,Simon Makuc,Anita Mlakar,et al.Determination of glutathione in spruce needles by liquid chromatography/tandem mass spectrometry.Rapid Communications in Mass Spectrometry,2002,16:1186-1191
[26]J(?)r(?)me Bouligand,Alain Deroussent,Angelo Paci,et al.Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. Journal of Chromatography B,2006,832:67-74
[27] Xiangming Guan, Brianna Hoffman, Chandradhar Dwivedi, et al. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. Journal of Pharmaceutical and Biomedical Analysis,2003,31:251-261
[28] A.M. Di Peetra, R. Gotil, D. Bonazzi, et al. HPLC determination of glutathione and L-cysteine in pharmaceuticals after derivatization with ethacrynicacid. Journal of Pharmuceurical & Biomedical Analysis,1994,12(1):91-98
[29] E. Bald, G. Chwatko, R. G(?)owacki, et al. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. Journal of ChromatographyA,2004,1032:109-115
[30] W. Li, S.S. Bollecker, J.D. Schofield. Glutathione and related thiol compounds. I. glutathione and related thiol compounds in flour. Journal of Cereal Science,2004,39:205-212
[31] Miguel Asensi, Juan Sastre, Federico V Pallardo, et al. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Analytical Biochemistry,1994,217:323-328
[32] Jun Qu, Yiming Wang, Guoan Luo, et al. Validated quantitation of underivatized amino acids in human blood samples by volatile ion-pair reversed-phase liquid chromatography coupled to isotope dilution tandem mass spectrometry. Analytical Chemistry,2002, 74: 2034-2040
[33]陈金水,李朕杰.谷胱甘肽一种新型饲用添加剂.安全饲料.2005,5:40-41
[34]沈亚领,李爽,迟莉丽,等.谷胱甘肽的应用与生产.工业微生物,2000,30(2):41-45
[35]席秀芳,赵秀川.谷胱甘肽的药理与临床.中国医学文摘·内科学,2004,25(1):125-127
[36]王爱月,解魁,李发生.高效液相色谱法测定保健食品中谷胱甘肽含量的方法研究.中国卫生检验杂志,2007,17(7):1181-1182
[37]黄颖,段建平,杨明灿,等.毛细管电泳法测定黄瓜和西红柿中的谷胱甘肽.色谱,2003,21(5):510-512
[38]王春,严伟民.注射用还原型谷胱甘肽质量标准分析方法HPLC法的建立与验证.中国临床药学杂志,2005,14(3):167-169
[39]王秋林,王浩毅,丁怡,等.血浆中游离还原型及氧化型谷胱甘肽的荧光测定法.光谱学与光谱分析,2005,25(11):1834-1838
[40]林丽,曹旭妮,张文,等.碳纳米管修饰电极用于高效液相色谱对全血中巯基化合物的测定.分析化学,2003,31(3):261-265
[41]任亦飞,丁劲松,彭文兴,等.HPLC-MS法测定正常人血浆和全血中还原型谷胱甘肽的浓度.中南药学,2004,2(3):154-156