HPLC-UV-FLD与显微注射法筛选恭城油茶中的抗氧化成分
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摘要
目的建立高效液相紫外荧光(HPLC-UV-FLD)衍生系统,利用显微注射荧光探针技术对恭城油茶提取物中抗氧化活性成分进行初步评价。方法采用过氧化氢急性损伤实验及羟自由基清除能力实验对恭城油茶的抗氧化活性进行检测,利用HPLC-UV-FLD衍生系统对其中的抗氧化活性成分进行筛选,并采用超高压液相色谱-四级杆-飞行时间质谱技术(UPLC-Q/TOF-MS)对收集得到的活性成分峰进行定性分析。同时通过显微注射过氧化氢荧光探针分子(NBCD)检测活体果蝇体内过氧化氢水平,探究恭城油茶及其中筛选出的活性成分在体内清除过氧化氢的作用。结果该方法方便快速,在恭城油茶提取物中筛选出了表没食子儿茶素(EGC)、表没食子儿茶素没食子酸酯(EGCG)和表儿茶素没食子酸酯(ECG)3种主要的抗氧化活性成分。同时检测出恭城油茶及其抗氧化活性成分能够显著降低果蝇体内过氧化氢水平。结论为进一步研究恭城油茶抗氧化提供物质基础,并且为天然产物中的抗氧化活性成分的筛选方法提供参考。
Objective A high performance liquid chromatography-ultraviolet-fluorescence(HPLC-UV-FLD) derivative system was established and the anti-oxidant activity of the extract of Gongcheng Tea was preliminarily evaluated by using a micro-injector imaging system combined with a fluorescent probe. Methods Hydrogen peroxide assay and hydroxyl radical scavenging ability experiments were carried out on the anti-oxidant activity of Gongcheng Tea. The anti-oxidant components of Gongcheng Tea were screened by HPLC-UV-FLD post column derivation system and analyzed by an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF MS). At the same time, the hydrogen peroxide scavenging activity of tea and active components were tested by a micro-injector imaging system combined with a hydrogen peroxide fluorescent probe(NBCD) in living Drosophila melanogaster. Results The method was simple and rapid, and three main anti-oxidant compounds, epigallocatechin,epigallocatechin gallate, and epicatechin gallate were found in Gongcheng Tea. And it was found that they could significantly reduce the level of hydrogen peroxide in D. melanogaster. Conclusion It laid a material basis for the further study of the anti-oxidant activity of Gongcheng Tea, and provided a reference for the screening of anti-oxidants in natural products.
Objective A high performance liquid chromatography-ultraviolet-fluorescence(HPLC-UV-FLD) derivative system was established and the anti-oxidant activity of the extract of Gongcheng Tea was preliminarily evaluated by using a micro-injector imaging system combined with a fluorescent probe. Methods Hydrogen peroxide assay and hydroxyl radical scavenging ability experiments were carried out on the anti-oxidant activity of Gongcheng Tea. The anti-oxidant components of Gongcheng Tea were screened by HPLC-UV-FLD post column derivation system and analyzed by an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF MS). At the same time, the hydrogen peroxide scavenging activity of tea and active components were tested by a micro-injector imaging system combined with a hydrogen peroxide fluorescent probe(NBCD) in living Drosophila melanogaster. Results The method was simple and rapid, and three main anti-oxidant compounds, epigallocatechin,epigallocatechin gallate, and epicatechin gallate were found in Gongcheng Tea. And it was found that they could significantly reduce the level of hydrogen peroxide in D. melanogaster. Conclusion It laid a material basis for the further study of the anti-oxidant activity of Gongcheng Tea, and provided a reference for the screening of anti-oxidants in natural products.
引文
[1]王者嵩,Tracy.恭城油茶户户香[J].中国烹饪,2013,3:57-57.
[2]姜伟.恭城油茶香满天[J].茶:健康天地,2011,1:34-35.
[3]何顺.少数民族饮食文化在现代都市中的传承探讨--以广西桂林恭城油茶为例[J].南宁职业技术学院学报,2017,22(5):20-23.
[4]杨成林.六大因素造就长寿之乡--介绍广西恭城县[J].长寿,2016,4:10-11.
[5]Clancy D J,Gems D,Hafen E,et al.Dietary restriction in long-lived dwarf flies[J].Science,2002,296(5566):319.
[6]Decabo R,Carmona-Gutierrez D,Bernier M,et al.The search for antiaging interventions:From elixirs to fasting regimens[J].Cell,2014,157(7):1515-1526.
[7]D’Adda D F F,Reaper P M,Clayfarrace L,et al.A DNAdamage checkpoint response in telomere-initiated senescence[J].Nature,2003,426(6963):194-198.
[8]Wang M C,Bohmann D,Jasper H.JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila[J].Dev Cell,2003,5(5):811-816.
[9]Christina C,Landis G N,Donna F,et al.Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes[J].Genome Biol,2007,8(12):R262.
[10]郭育.太白楤木皂苷抗衰老作用及机制研究[D].西安:陕西师范大学,2013.
[11]Cui H,Kong Y,Zhang H.Oxidative stress,mitochondrial dysfunction,and aging[J].J Signal Transduction,2012,doi:10.1155/2012/646354.
[12]宋朝春,魏冉磊,樊晓兰,等.衰老及抗衰老药物的研究进展[J].中国生化药物杂志,2015,35(1):163-170.
[13]Li N,Liu Y,Li S,et al.High-precision,pressure-driven pump for sub-picoliter scale quantitative injection[J].Modern Physics Lett B,2017,31(13):1750148.
[14]Gao J,Tao J,Zhang N,et al.Formula optimization of the Jiashitang scar removal ointment and antiinflammatory compounds screening by NF-κB bioactivity-guided dual-luciferase reporter assay system[J].Phytother Res,2015,29(2):241-250.
[15]徐金荣,陈忠明.高效液相色谱-荧光检测法测定环境样品中的过氧化物[J].色谱,2005,23(4):366-369.
[16]Peng C,Zuo Y,Kwan K M,et al.Blueberry extract prolongs lifespan of Drosophila melanogaster[J].Exp Gerontol,2012,47(2):170-178.
[17]段丹丹,高丽,王珂欣,等.黄芩素通过抗氧化应激延长果蝇寿命的机制[J].药学学报,2016,51(9):1401-1406.
[18]CocheméH M,Logan A,Prime T A,et al.Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila[J].Nat Protocols,2012,7(5):946-958.
[19]Cheignon C,Tomas M,Bonnefontrousselot D,et al.Oxidative stress and the amyloid beta peptide in Alzheimer’s disease[J].Redox Biol,2018,14:450-464.
[20]Hong J,Maguhn J,Freitag D,et al.Determination of H2O2 and organic peroxides by high-performance liq(1)uid chromatography with post-column UVirradiation,derivatization and fluorescence detection[J].Fresenius J Anal Chem,1998,361(2):124-128.
[21]Wang K,Glaze W H.High-performance liquid chromatography with postcolumn derivatization for simultaneous determination of organic peroxides and hydrogen peroxide[J].J Chromatogr A,1998,1083(1/2):219-222.
[22]Chu G,Cheng D,Liu W,et al.Screening and evaluation of antioxidants from lees by micro-injector systems combined with a fluorescent probe,N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine,in living Drosophila[J].Chin Chem Lett,2018,29(10):1521-1527.
[23]Miwa S,Treumann A,Bell A,et al.Carboxylesterase converts amplex red to resorufin:Implications for mitochondrial H2O2 release assays[J].Free Rad Biol Med,2015,90:173-183.
[24]Han Z,Liang X,Ren X,et al.A novel 3,7-dihydroxyphenoxazine-based fluorescent probe for selective detection of intracellular hydrogen peroxide[J].Chem Asian J,2016,11(6):818-822.
[25]丁阳平,陆昌琪,候宏晓,等.儿茶素氧化产物及形成机制研究[J].中国中药杂志,2017,42(2):239-253.
[26]张心亚,禄子薇,张双琳,等.普洱茶多酚的提取及抗氧化作用[J].当代教育实践与教学研究:电子版2017(3):241-242.
[27]刘大美,毕宏生,解孝锋,等.茶多酚体内抗氧化作用研究进展[J].山东医药,2013,53(16):82-84.
[28]Lee L S,Kim S H,Kim Y B,et al.Quantitative analysis of major constituents in green tea with different plucking periods and their antioxidant activity[J].Molecules,2014,19(7):9173-9186.
[29]Shrestha R,Lama J P,Shrestha K.Total polyphenols content and antioxidant activity of different tea commercially produced in nepal[J].J Food Sci Technol Nepal,2010,6:73-79.
[30]Ahmad R S,Butt M S,Huma N,et al.Quantitative and qualitative portrait of green tea catechins(Gtc)through HPLC[J].Inter J Food Properties,2014,17(7):1626-1636.
[31]Sroka Z,Cisowski W.Hydrogen peroxide scavenging,antioxidant and anti-radical activity of some phenolic acids[J].Food Chem Toxicol,2003,41(6):753-758.
[32]Hou Y,Zhou J,Gao Z,et al.A Protease-activated ratiometric fluorescent probe for pH-mapping of malignant tumor[J].Adv Sci Technol,2016,102(3):51-56.
[33]Wu Y T,Shanmugam C,Tseng W B,et al.A gold nanocluster-based fluorescent probe for simultaneous pHand temperature sensing and its application to cellular imaging and logic gates[J].Nanoscale,2016,8(21):11210-11216.
[34]Zhang S,Sun M,Yan Y,et al.A turn-on fluorescence probe for the selective and sensitive detection of fluoride ions[J].Anal Bioanal Chem,2017,409(8):2075-2081.
[35]Zhao Y J,Miao K,Zhu Z,et al.Fluorescence quenching of a conjugated polymer by synergistic amine-carboxylic acid andπ-πinteractions for selective detection of aromatic amines in aqueous solution[J].ACS Sensors,2017,2(6):842-847.
[2]姜伟.恭城油茶香满天[J].茶:健康天地,2011,1:34-35.
[3]何顺.少数民族饮食文化在现代都市中的传承探讨--以广西桂林恭城油茶为例[J].南宁职业技术学院学报,2017,22(5):20-23.
[4]杨成林.六大因素造就长寿之乡--介绍广西恭城县[J].长寿,2016,4:10-11.
[5]Clancy D J,Gems D,Hafen E,et al.Dietary restriction in long-lived dwarf flies[J].Science,2002,296(5566):319.
[6]Decabo R,Carmona-Gutierrez D,Bernier M,et al.The search for antiaging interventions:From elixirs to fasting regimens[J].Cell,2014,157(7):1515-1526.
[7]D’Adda D F F,Reaper P M,Clayfarrace L,et al.A DNAdamage checkpoint response in telomere-initiated senescence[J].Nature,2003,426(6963):194-198.
[8]Wang M C,Bohmann D,Jasper H.JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila[J].Dev Cell,2003,5(5):811-816.
[9]Christina C,Landis G N,Donna F,et al.Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes[J].Genome Biol,2007,8(12):R262.
[10]郭育.太白楤木皂苷抗衰老作用及机制研究[D].西安:陕西师范大学,2013.
[11]Cui H,Kong Y,Zhang H.Oxidative stress,mitochondrial dysfunction,and aging[J].J Signal Transduction,2012,doi:10.1155/2012/646354.
[12]宋朝春,魏冉磊,樊晓兰,等.衰老及抗衰老药物的研究进展[J].中国生化药物杂志,2015,35(1):163-170.
[13]Li N,Liu Y,Li S,et al.High-precision,pressure-driven pump for sub-picoliter scale quantitative injection[J].Modern Physics Lett B,2017,31(13):1750148.
[14]Gao J,Tao J,Zhang N,et al.Formula optimization of the Jiashitang scar removal ointment and antiinflammatory compounds screening by NF-κB bioactivity-guided dual-luciferase reporter assay system[J].Phytother Res,2015,29(2):241-250.
[15]徐金荣,陈忠明.高效液相色谱-荧光检测法测定环境样品中的过氧化物[J].色谱,2005,23(4):366-369.
[16]Peng C,Zuo Y,Kwan K M,et al.Blueberry extract prolongs lifespan of Drosophila melanogaster[J].Exp Gerontol,2012,47(2):170-178.
[17]段丹丹,高丽,王珂欣,等.黄芩素通过抗氧化应激延长果蝇寿命的机制[J].药学学报,2016,51(9):1401-1406.
[18]CocheméH M,Logan A,Prime T A,et al.Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila[J].Nat Protocols,2012,7(5):946-958.
[19]Cheignon C,Tomas M,Bonnefontrousselot D,et al.Oxidative stress and the amyloid beta peptide in Alzheimer’s disease[J].Redox Biol,2018,14:450-464.
[20]Hong J,Maguhn J,Freitag D,et al.Determination of H2O2 and organic peroxides by high-performance liq(1)uid chromatography with post-column UVirradiation,derivatization and fluorescence detection[J].Fresenius J Anal Chem,1998,361(2):124-128.
[21]Wang K,Glaze W H.High-performance liquid chromatography with postcolumn derivatization for simultaneous determination of organic peroxides and hydrogen peroxide[J].J Chromatogr A,1998,1083(1/2):219-222.
[22]Chu G,Cheng D,Liu W,et al.Screening and evaluation of antioxidants from lees by micro-injector systems combined with a fluorescent probe,N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine,in living Drosophila[J].Chin Chem Lett,2018,29(10):1521-1527.
[23]Miwa S,Treumann A,Bell A,et al.Carboxylesterase converts amplex red to resorufin:Implications for mitochondrial H2O2 release assays[J].Free Rad Biol Med,2015,90:173-183.
[24]Han Z,Liang X,Ren X,et al.A novel 3,7-dihydroxyphenoxazine-based fluorescent probe for selective detection of intracellular hydrogen peroxide[J].Chem Asian J,2016,11(6):818-822.
[25]丁阳平,陆昌琪,候宏晓,等.儿茶素氧化产物及形成机制研究[J].中国中药杂志,2017,42(2):239-253.
[26]张心亚,禄子薇,张双琳,等.普洱茶多酚的提取及抗氧化作用[J].当代教育实践与教学研究:电子版2017(3):241-242.
[27]刘大美,毕宏生,解孝锋,等.茶多酚体内抗氧化作用研究进展[J].山东医药,2013,53(16):82-84.
[28]Lee L S,Kim S H,Kim Y B,et al.Quantitative analysis of major constituents in green tea with different plucking periods and their antioxidant activity[J].Molecules,2014,19(7):9173-9186.
[29]Shrestha R,Lama J P,Shrestha K.Total polyphenols content and antioxidant activity of different tea commercially produced in nepal[J].J Food Sci Technol Nepal,2010,6:73-79.
[30]Ahmad R S,Butt M S,Huma N,et al.Quantitative and qualitative portrait of green tea catechins(Gtc)through HPLC[J].Inter J Food Properties,2014,17(7):1626-1636.
[31]Sroka Z,Cisowski W.Hydrogen peroxide scavenging,antioxidant and anti-radical activity of some phenolic acids[J].Food Chem Toxicol,2003,41(6):753-758.
[32]Hou Y,Zhou J,Gao Z,et al.A Protease-activated ratiometric fluorescent probe for pH-mapping of malignant tumor[J].Adv Sci Technol,2016,102(3):51-56.
[33]Wu Y T,Shanmugam C,Tseng W B,et al.A gold nanocluster-based fluorescent probe for simultaneous pHand temperature sensing and its application to cellular imaging and logic gates[J].Nanoscale,2016,8(21):11210-11216.
[34]Zhang S,Sun M,Yan Y,et al.A turn-on fluorescence probe for the selective and sensitive detection of fluoride ions[J].Anal Bioanal Chem,2017,409(8):2075-2081.
[35]Zhao Y J,Miao K,Zhu Z,et al.Fluorescence quenching of a conjugated polymer by synergistic amine-carboxylic acid andπ-πinteractions for selective detection of aromatic amines in aqueous solution[J].ACS Sensors,2017,2(6):842-847.