运用多维在线高效液相色谱-生物化学检测方法筛选银杏叶提取物治疗老年痴呆疾病的药效成分
|
摘要
目的建立高效液相色谱-生物化学检测(HPLC-BCD)多维在线联用分析方法,筛选银杏叶提取物治疗老年痴呆疾病(Alzheimer disease,AD)的药效成分。方法运用HPLC-UV(紫外)-1,1-二苯基-2-三硝基苯肼/2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(DPPH/ABTS)、HPLC-UV-乙酰胆碱酯酶(Ach E)方法快速筛选银杏叶提取物中的自由基清除剂和Ach E抑制剂。以加兰他敏为阳性对照,考察在线HPLC-UV-Ach E方法的稳定性和重现性。运用超高效液相色谱(UPLC)-LTQ/线性离子阱/静电场轨道阱高分辨质谱(orbitrap MS)(UPLC-LTQ/orbitrap MS)方法鉴定主要活性成分。结果 HPLC-UV-DPPH/ABTS方法检测到银杏叶提取物中的14个强抗氧化成分,而运用HPLC-UV-Ach E方法发现了3个Ach E抑制剂,通过UPLCLTQ/orbitrap MS方法鉴定了3个酶抑制剂分别为4-O-beta-glucopyranosyl-cis-coumaric acid、ginkgolide A和3-O-[2-O-(β-D-glucosyl)-α-L-rhamnosyl]quercetin。结论实验建立的在线HPLC-BCD方法具有良好的灵敏度、重现性和稳定性,能够快速筛选银杏叶提取物中的抗氧化剂和Ach E抑制剂。为快速发现中药治疗相关疾病的有效成分提供一种有效、快速、便捷的分析手段。
OBJECTIVE To develop on-line high performance liquid chromatography-biochemical detection( HPLC-BCD) methods to screen effective components in Ginkgo biloba extract for treatment of Alzheimer's disease. METHODS Radical scavengers and Ach E inhibitors in G. biloba leave extract were screened by HPLC-UV-DPPH/ABTS and HPLC-UV-Ach E methods,respectively. The stability and repeatability of the on-line HPLC-UV-Ach E method were investigated using galanthamine as a positive reference. The main active ingredients in the extract were identified by ultra high performance liquid chromatography linear ion trap/orbitrap high resolution mass spectrometry( UPLC-LTQ/orbitrap MS) method. RESULTS Fourteen antioxidants in G. biloba extract were detected by the HPLC-UV-DPPH/ABTS method,while three Ach E inhibitors were found by the HPLC-UV-Ach E method. The three Ach E inhibitors were tentatively identified by UPLC-LTQ/orbitrap MS as 4-O-beta-glucopyranosyl-cis-coumaric acid、ginkgolide A and 3-O-[2-O-( β-DGlucosyl)-α-L-rhamnosyl]quercetin,respectively. CONCLUSION The proposed on-line HPLC-BCD method shows high sensitivity,good repeatability and stability. It can be used to screen antioxidants and Ach E inhibitors in G. biloba extract. This will provide an effective,quick and convenient analysis tool to quickly find bioactive ingredients of traditional Chinese medicines for treating related diseases.
OBJECTIVE To develop on-line high performance liquid chromatography-biochemical detection( HPLC-BCD) methods to screen effective components in Ginkgo biloba extract for treatment of Alzheimer's disease. METHODS Radical scavengers and Ach E inhibitors in G. biloba leave extract were screened by HPLC-UV-DPPH/ABTS and HPLC-UV-Ach E methods,respectively. The stability and repeatability of the on-line HPLC-UV-Ach E method were investigated using galanthamine as a positive reference. The main active ingredients in the extract were identified by ultra high performance liquid chromatography linear ion trap/orbitrap high resolution mass spectrometry( UPLC-LTQ/orbitrap MS) method. RESULTS Fourteen antioxidants in G. biloba extract were detected by the HPLC-UV-DPPH/ABTS method,while three Ach E inhibitors were found by the HPLC-UV-Ach E method. The three Ach E inhibitors were tentatively identified by UPLC-LTQ/orbitrap MS as 4-O-beta-glucopyranosyl-cis-coumaric acid、ginkgolide A and 3-O-[2-O-( β-DGlucosyl)-α-L-rhamnosyl]quercetin,respectively. CONCLUSION The proposed on-line HPLC-BCD method shows high sensitivity,good repeatability and stability. It can be used to screen antioxidants and Ach E inhibitors in G. biloba extract. This will provide an effective,quick and convenient analysis tool to quickly find bioactive ingredients of traditional Chinese medicines for treating related diseases.
引文
[1]ASSOCIATION A.2012 Alzheimer's disease facts and figures[J].Alzheimers&Dementia:J Alzheimers Assoc,2012,8(2):131-168.
[2]DENNIS J S.Alzheimer's disease:genes,proteins,and therapy[J].Physiol Rev,2001,81(2):741.
[3]HUANG Y W,WANG G H,YUAN B,et al.Progress of drugs for Alzheimer's disease[J].Guangdong Chem(广东化工),2016,43(19):81-82.
[4]MOU L L,KOU J P,ZHU D N,et al.Antioxidant activities of the chemical constituents isolated from the leaves of Ginkgo biloba[J].Chin J Nat Med(中国天然药物杂志),2008,6(1):26-29.
[5]DAS A,SHNKER G,NATH C,et al.A comparative study in rodents of standardized extracts of bacopamonniera and Ginkgo biloba anticholinesterase and cognitive enchancing activities[J].Pharmacol Biolchem Behav,2002,73(4):893-900.
[6]XIE C M,ZHENG G L,ZHONG Z Y,et al.Effect of Ginkgo leaf on learning and memory in Alzheimers disease mice[J].Chin J Comp Med(中国比较医学杂志),2010,20(9):26-29.
[7]LE BARS P L,KATZ M M,BERMAN N,et al.A placebo-controlled,double-blind,randomized trial of an extract of Ginkgo biloba for dementia[J].JAMA,1997,278(16):1327-1332.
[8]REN F,GONG S Y.Ginkgo biloba extract for senile dementia[J].Chin J Clin Rehabil(中国临床康复杂志),2005,9(32):166-168.
[9]SHI S Y,ZHOU H H,ZHANG Y P,et al.Coupling HPLC to on-line,post-column(bio)chemical assays for high-resolution screening of bioactive compounds from complex mixtures[J].Trac-Trends Anal Chem,2009,28(7):865-877.
[10]DING X P,WANG X T,CHENh L L,et al.On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium[J].J Chromatogr B,2011,1218(9):1227-1235.
[11]LIU J,TIAN J,LI J,et al.The in-capillary DPPH-capillary electrophoresis-the diode array detector combined with reversed-electrode polarity stacking mode for screening and quantifying major antioxidants in Cuscuta chinensis Lam[J].Electrophoresis,2016,37(12):1632-1639.
[12]PENG W B,ZENG Q H,LI D P,et al.Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection[J].Biomed Chromatogr,2016,30(11):1854-1860.
[13]MARQUES L A,KOOL J,DE KANTER F,et al.Production and on-line acetylcholinesterase bioactivity profiling of chemical and biological degradation products of tacrine[J].J Pharm Biomed Anal,2010,53(3):609-616.
[14]DE JONG C F,DERKS R J E,BRUYNEEL B,et al.High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts[J].J Chromatogr B,2006,1112(1-2):303-310.
[15]ZHAO H,ZHOU S,ZHANG M,et al.An in vitro ACh E inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of ACh E inhibitors from roots of Coptis chinensis Franch[J].J Pharm Biomed Anal,2016,120(20):235-240.
[16]INGKANINAN K,BEST C M D,HEIJDEN R V D,et al.Highperformance liquid chromatography with on-line coupled UV,mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products[J].J Chromatogr A,2000,872(1-2):61-73.
[17]LI D Q,QIAN Z M,LI S P,et al.Inhibition of three selected beverage extracts onα-glucosidase and rapid identification of their active compounds using HPLC-DAD-MS/MS and biochemical detection[J].J Agr Food Chem,2010,58(11):6608-6613.
[18]LI D Q,ZHAO J,XIE J,et al.A novel sample preparation and on-line HPLC-DAD-MS/MS-BCD analysis for rapid screening and characterization of specific enzyme inhibitors in herbal extracts:case study ofα-glucosidase[J].J Pharm Biomed Anal,2014,88(25):130-135.
[19]KOOL J,VAN LIEMPD S,HARMSEN S,et al.Cytochrome P450 bio-affinity detection coupled to gradient HPLC:on-line screening of affinities to cytochrome P4501A2 and 2D6[J].J Chromatogr B,2007,858(1-2):49-58.
[20]KOOL J,VAN LIEMPD S,RAMAUTAR R,et al.Development of a novel cytochrome P450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography[J].J Biomol Scr,2005,10(5):427-436.
[21]JEURISSEN S M,CLAASSEN F W,HAVLIK J,et al.Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products[J].J Chromatogr A,2007,1141(1):81-89.
[22]LI P L,HUANG X,YUN S,et al.Chemical material basis of extract of Ginkgo biloba leaves injection by ultra-fast liquid chromatography/triple time-of-flight tandem mass spectrometry[J].Cent South Pharm(中南药学),2017,(5):573-579.
[2]DENNIS J S.Alzheimer's disease:genes,proteins,and therapy[J].Physiol Rev,2001,81(2):741.
[3]HUANG Y W,WANG G H,YUAN B,et al.Progress of drugs for Alzheimer's disease[J].Guangdong Chem(广东化工),2016,43(19):81-82.
[4]MOU L L,KOU J P,ZHU D N,et al.Antioxidant activities of the chemical constituents isolated from the leaves of Ginkgo biloba[J].Chin J Nat Med(中国天然药物杂志),2008,6(1):26-29.
[5]DAS A,SHNKER G,NATH C,et al.A comparative study in rodents of standardized extracts of bacopamonniera and Ginkgo biloba anticholinesterase and cognitive enchancing activities[J].Pharmacol Biolchem Behav,2002,73(4):893-900.
[6]XIE C M,ZHENG G L,ZHONG Z Y,et al.Effect of Ginkgo leaf on learning and memory in Alzheimers disease mice[J].Chin J Comp Med(中国比较医学杂志),2010,20(9):26-29.
[7]LE BARS P L,KATZ M M,BERMAN N,et al.A placebo-controlled,double-blind,randomized trial of an extract of Ginkgo biloba for dementia[J].JAMA,1997,278(16):1327-1332.
[8]REN F,GONG S Y.Ginkgo biloba extract for senile dementia[J].Chin J Clin Rehabil(中国临床康复杂志),2005,9(32):166-168.
[9]SHI S Y,ZHOU H H,ZHANG Y P,et al.Coupling HPLC to on-line,post-column(bio)chemical assays for high-resolution screening of bioactive compounds from complex mixtures[J].Trac-Trends Anal Chem,2009,28(7):865-877.
[10]DING X P,WANG X T,CHENh L L,et al.On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium[J].J Chromatogr B,2011,1218(9):1227-1235.
[11]LIU J,TIAN J,LI J,et al.The in-capillary DPPH-capillary electrophoresis-the diode array detector combined with reversed-electrode polarity stacking mode for screening and quantifying major antioxidants in Cuscuta chinensis Lam[J].Electrophoresis,2016,37(12):1632-1639.
[12]PENG W B,ZENG Q H,LI D P,et al.Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection[J].Biomed Chromatogr,2016,30(11):1854-1860.
[13]MARQUES L A,KOOL J,DE KANTER F,et al.Production and on-line acetylcholinesterase bioactivity profiling of chemical and biological degradation products of tacrine[J].J Pharm Biomed Anal,2010,53(3):609-616.
[14]DE JONG C F,DERKS R J E,BRUYNEEL B,et al.High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts[J].J Chromatogr B,2006,1112(1-2):303-310.
[15]ZHAO H,ZHOU S,ZHANG M,et al.An in vitro ACh E inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of ACh E inhibitors from roots of Coptis chinensis Franch[J].J Pharm Biomed Anal,2016,120(20):235-240.
[16]INGKANINAN K,BEST C M D,HEIJDEN R V D,et al.Highperformance liquid chromatography with on-line coupled UV,mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products[J].J Chromatogr A,2000,872(1-2):61-73.
[17]LI D Q,QIAN Z M,LI S P,et al.Inhibition of three selected beverage extracts onα-glucosidase and rapid identification of their active compounds using HPLC-DAD-MS/MS and biochemical detection[J].J Agr Food Chem,2010,58(11):6608-6613.
[18]LI D Q,ZHAO J,XIE J,et al.A novel sample preparation and on-line HPLC-DAD-MS/MS-BCD analysis for rapid screening and characterization of specific enzyme inhibitors in herbal extracts:case study ofα-glucosidase[J].J Pharm Biomed Anal,2014,88(25):130-135.
[19]KOOL J,VAN LIEMPD S,HARMSEN S,et al.Cytochrome P450 bio-affinity detection coupled to gradient HPLC:on-line screening of affinities to cytochrome P4501A2 and 2D6[J].J Chromatogr B,2007,858(1-2):49-58.
[20]KOOL J,VAN LIEMPD S,RAMAUTAR R,et al.Development of a novel cytochrome P450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography[J].J Biomol Scr,2005,10(5):427-436.
[21]JEURISSEN S M,CLAASSEN F W,HAVLIK J,et al.Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products[J].J Chromatogr A,2007,1141(1):81-89.
[22]LI P L,HUANG X,YUN S,et al.Chemical material basis of extract of Ginkgo biloba leaves injection by ultra-fast liquid chromatography/triple time-of-flight tandem mass spectrometry[J].Cent South Pharm(中南药学),2017,(5):573-579.