高效液相色谱-紫外/质谱评价海洋天然产物体外抑制环氧合酶2活性方法研究
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摘要
目的建立基于高效液相色谱-紫外(HPLC-UV)和高效液相色谱-多级离子阱质谱分析(HPLC-ITMS/MS)的海洋天然产物体外抑制COX-2酶的活性评价方法。方法采用Agilent Poroshell 120C18色谱柱,以乙腈-0.05%磷酸水溶液为高效液相色谱流动相,二元等度洗脱,流速为0.4 mL·min~(-1),检测波长210nm,进样量10μL;质谱条件下,流动相改为乙腈-0.05%乙酸水溶液,进样量为1μL,选用ESI离子源,负离子电离模式,喷雾气压35psi,干燥气(N2)流速12L·min~(-1),干燥气温度350℃。结果建立的高效液相色谱-紫外和高效液相色谱-多级离子阱质谱法精密度和稳定性良好,对比两者测定酶底物花生四烯酸的适用性显示,基于HPLC-UV的评价方法适用极性较大的天然产物粗提物;HPLC-IT-MS/MS法则因其抗干扰能力强,适用于复杂体系中极性较弱的活性物质的检测。以建立的HPLC-UV和HPLC-IT-MS/MS方法对海洋天然产物的COX-2酶抑制活性进行评价,天然产物粗提物中黄海葵Anthopleura lanthogrammica Berkly乙醇粗提物和弯孢菌Curvularia lunata strain L2744发酵液粗提物对COX-2酶抑制作用较为明显,抑制率分别为70.1%和25.48%。结论建立的评价方法具有适用性强、结果可靠等优点,有望为天然产物粗提物中COX-2酶抑制剂体外筛选提供技术参考。
Objective To establish methods for evaluating the cyclooxygenase 2(COX-2)inhibitory activity of marine natural products in vitro based on HPLC-UV and HPLC-IT-MS/MS.Methods Arachidonic acid(AA)which was the substrate of COX-2 was determined by injecting 10μL of the samples on an Agilent Poroshell 120 C18 column with the isocratic elution solvent system composed of 0.05% phosphoric acid aqueous solution and acetonitrile.The flow rate was 0.4 mL·min~(-1) and the signal was acquired at 210 nm.The mobile phase was changed into 0.05%acetic acid aqueous solution and acetonitrile in IT-MS/MS condition with injection volume of 1μL.ESI was used as ion source in negative mode with ion spray pressure 35 psi,dry gas flow rate 12 L·min~(-1) and drying gas temperature 350℃.Results The established HPLC-UV and HPLC-IT-MS/MS methods had good precision and stability.The comparisons of different applicability of two methods showed that the evaluation method based on HPLC-UV was suitable for crude extracts of natural products with high polarity,and the HPLC-ITMS/MS method was suitable for the detection of active substances with weak polarity in complex systems because of its strong anti-interference ability.The COX-2 inhibitory activity of marine natural products was evaluated using the established HPLC-UV and HPLC-IT-MS/MS methods.The ethanol crude extract of Anthopleura lanthogrammica Berkly and crude extract of fermentation broth of Curvularia lunata strain L2744 had obvious inhibitory effect on COX-2,and the inhibition rates were 70.1%and 25.48%,respectively.Conclusion The established evaluation methods had the advantages of strong applicability and reliable results.It was expected to provide a technical reference for in vitro screening of COX-2 inhibitors from crude extracts of natural products.
Objective To establish methods for evaluating the cyclooxygenase 2(COX-2)inhibitory activity of marine natural products in vitro based on HPLC-UV and HPLC-IT-MS/MS.Methods Arachidonic acid(AA)which was the substrate of COX-2 was determined by injecting 10μL of the samples on an Agilent Poroshell 120 C18 column with the isocratic elution solvent system composed of 0.05% phosphoric acid aqueous solution and acetonitrile.The flow rate was 0.4 mL·min~(-1) and the signal was acquired at 210 nm.The mobile phase was changed into 0.05%acetic acid aqueous solution and acetonitrile in IT-MS/MS condition with injection volume of 1μL.ESI was used as ion source in negative mode with ion spray pressure 35 psi,dry gas flow rate 12 L·min~(-1) and drying gas temperature 350℃.Results The established HPLC-UV and HPLC-IT-MS/MS methods had good precision and stability.The comparisons of different applicability of two methods showed that the evaluation method based on HPLC-UV was suitable for crude extracts of natural products with high polarity,and the HPLC-ITMS/MS method was suitable for the detection of active substances with weak polarity in complex systems because of its strong anti-interference ability.The COX-2 inhibitory activity of marine natural products was evaluated using the established HPLC-UV and HPLC-IT-MS/MS methods.The ethanol crude extract of Anthopleura lanthogrammica Berkly and crude extract of fermentation broth of Curvularia lunata strain L2744 had obvious inhibitory effect on COX-2,and the inhibition rates were 70.1%and 25.48%,respectively.Conclusion The established evaluation methods had the advantages of strong applicability and reliable results.It was expected to provide a technical reference for in vitro screening of COX-2 inhibitors from crude extracts of natural products.
引文
[1]Seibert K,Zhang Y,Leahy K,et al.Pharmacological and biochemical demonstration of the role of cyclooxygenase 2in inflammation and pain[J].Proc Natl Acad Sci USA,1994,91(25):12013-12017.
[2]Samad T A,Moore K A,Sapirstein A,et al.Interleukin-1beta-mediated induction of COX-2in the CNS contributes to inflammatory pain hypersensitivity[J].Nature,2001,410(6827):471-475.
[3]Adelizzi R A.COX-1and COX-2in health and disease[J].J Am Osteopath Assoc,1999,99(11Suppl):7-12.
[4]Steinbach G,Lynch P M,Phillips R K,et al.The effect of celecoxib,a cyclooxygenase-2inhibitor,in familial adenomatous polyposis[J].Engl J Med,2000,342(26):1946-1952.
[5]Liu X,Yao S,Kirschenbaum A,et al.NS398,a selective cyclooxygenase-2inhibitor,induces apoptosis and down-regulates bcl-2expression in LNCaP cells[J].Cancer Res,1998,58(19):4245-4249.
[6]Gupta R A,Dubois R N.Colorectal cancer prevention and treatment by inhibition of COX-2[J].Nat Rev Cancer,2001,1(1):11-21.
[7]Hsu A L,Ching T T,Wang D S,et al.The cyclooxygenase-2inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2[J].J Biol Chem,2000,275(15):11397-11403.
[8]Bansal S,Bala M,Suthar S K,et al.Design and synthesis of novel 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazoles as selective COX-2inhibitors with potent anti-inflammatory activity[J].Eur J Med Chem,2014,80(1):167-174.
[9]Alzweiri M,Sallam M,Al-Zyoud W,et al.Stability study of etoricoxib a selective cyclooxygenase-2inhibitor by a new single and repid reversed phase HPLC method[J].Symmetry,2018,10:288-299.
[10]Cao H,Yu R,Tao Y,et al.Measurement of cyclooxygenase inhibition using liquid chromatography-tandem mass spectrometry[J].J Pharm Biomed Anal,2011,54(1):230-235.
[11]Xiong X,He Y N,Feng B,et al.Screening for the anti-inflammation quality markers of Xiaojin Pills based on HPLCMS/MS method COX-2inhibition test and protein interaction network[J].Sci Rep,2018,8(1):7454-7463.
[12]Deng X,Shi S,Li S,et al.Magnetic ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry to screen and characterize cyclooxygenase-2inhibitors from green tea[J].J Chromatogr B Analyt Technol Biomed Life Sci,2014,973C:55-60.
[13]Van Breemen R B,Tao Y,Li W.Cyclooxygenase-2inhibitors in ginger(Zingiber officinale)[J].Fitoterapia,2011,82(1):38-43.
[14]Ma Y,Liu J,Shi H M,et al.Isolation of chracacterization of anti-inflammatory peptides derived from whey protain[J].J Dairy Sci,2016,99(9):6902-6912.
[15]Razzaghi-Asl N,Mirzayi S,Mahnam K,et al.Identification of COX-2inhibitors via structure-based virtual screening and molecular dynamics simulation[J].J Mol Graph Model,2018,83:138-152.
[16]Li Y D,Christopher M F,Chen M H,et al.Primary virtual and in vitro bioassay screening of natural inhibitors from flavonoids against COX-2[J].Chin J Nat Med,2011,9(2):156-160.
[17]Van der Ouderaa F J,Buytenhek M.Purification of PGH synthase from sheep vesicular glands[J].Methods Enzymol,1982,86:60-68.
[18]Kulmacz R J.Prostaglandin G2levels during reaction of prostaglandin H synthase with arachidonic acid[J].Prostaglandins,1987,34(2):225-240.
[19]White H L,Glassman A T.A simple radiochemical assay for prostaglandin synthetase[J].Prostaglandins,1974,7(2):123-129.
[20]Laufer S,Zechmeister P,Klein T.Development of an invitro test system for the evaluation of cyclooxygenase-2inhibitors[J].Inflamm Res,1999,48(3):133-138.
[21]Fumio Y,Kimihiro Y,Katsuaki K,et al.Substrate probe,enzyme-activity detection method by a multi-dimensional nuclear magnetic resonance method and enzyme-activity imaging method:USA,US2010248279[P].2010-09-30.
[22]徐斌,董英,胡庆松,等.紫外分光光度发快速测定小麦胚芽脂肪氧化酶活力[J].分析化学,2009,37(8):1252-1252.
[23]牛宗亮,王荣镇,董新伟,等.马粪海胆生殖腺营养成分的含量测定[J].中国海洋药物,2009,28(6):26-30.
[24]Hall T C,Bilku D K,Neal C P,et al.The impact of an omega-3fatty acid rich lipid emulsion on fatty acid profiles in critically ill septic patients[J].Prostaglandins Leukot Essent Fatty Acids,2016,112:1-11.
[25]王燕华,金春爱,孙印石,等.不同加工方式的鹿茸脂肪酸的气相色谱分析[J].中草药,2017,48(12):2431-2441.
[26]Shi S,Yi L Z,Yun Y H,et al.A combination of GC-MS and chemometrics reveals metabolic differences between serum and plasma[J].Anal Methods,2015,7(5):1751-1757.
[27]Kayacelebi A A,Schauerte C,Kling K,et al.Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase(MAGL)activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol[J].J Chromatogr B Analyt Technol Biomed Life Sci,2017,1047:151-159.
[28]Banerjee D,Mazumder S,Kumar Sinha A.Involvement of Nitric Oxide on Calcium Mobilization and arachidonic acid pathway activation during platelet aggregation with different aggregating agonists[J].Int J Biomed Sci,2016,12(1):25-35.
[29]王桂花,谢树莲.刺叶提灯藓花生四烯酸提取与含量测定[J].食品科学,2010,31(6):52-54.
[30]钟宇,陈滨,李建,等.LC-MS/MS分析血浆中脂肪酸及代谢产物[J].质谱学报,2018,39(3):310-315.
[31]谷艳,石先哲,赵素敏,等.高效液相色谱-电喷雾串联四级杆质谱法检测小鼠组织中花生四烯酸及其代谢产物[J].分析化学,2010,38(8):1089-1094.
[32]Najera A I,Renobales M D,Barron L.Effects of pH,temperature,CaCl2and enzyme concentrations on the rennet-clotting properties of milk:a multifactorial study[J].Food Chem,2003,80(3):345-352.
[33]Su J Y,Storey K B.Fish muscle phosphofructokinase:Influences of protein concentration on enzyme kinetic behaviour[J].Int J Biochem Cell Biol,1995,27(27):1277-1283.
[34]Santos N C,Figueira-Coelho J,Martins-Silva J,et al.Multidisciplinary utilization of dimethyl sulfoxide:pharmacological,cellular,and molecular aspects[J].Biochem Pharmacol,2003,65(7):1035-1041.
[35]贾卓美,唐旭利,乔丹,等.中国西沙海绵Dactylospongia elegans化学成分研究[J].中国海洋药物,2018,37(1):16-19.
[36]黄娣,徐永健.不同方法提取大海马脂类及脂肪酸组成分析[J].中国海洋药物,2016,35(2):35-40.
[37]Nikolic D,Habibi-Goudarzi S,Corley D G,et al.Evaluation of cyclooxygenase-2inhibitors using pulsed ultrafiltration mass spectrometry[J].Anal Chem,2000,72(16):3853-3859.
[38]刘伟,王娟,姜孝玉,等.海葵溶细胞素的研究进展[J].海洋科学,2005,29(4):56-62.
[39]Ko S C,Jeon Y J.Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7macrophages and in vivo zebrafish model[J].Nutr Res Pract,2015,9(3):219-226.
[2]Samad T A,Moore K A,Sapirstein A,et al.Interleukin-1beta-mediated induction of COX-2in the CNS contributes to inflammatory pain hypersensitivity[J].Nature,2001,410(6827):471-475.
[3]Adelizzi R A.COX-1and COX-2in health and disease[J].J Am Osteopath Assoc,1999,99(11Suppl):7-12.
[4]Steinbach G,Lynch P M,Phillips R K,et al.The effect of celecoxib,a cyclooxygenase-2inhibitor,in familial adenomatous polyposis[J].Engl J Med,2000,342(26):1946-1952.
[5]Liu X,Yao S,Kirschenbaum A,et al.NS398,a selective cyclooxygenase-2inhibitor,induces apoptosis and down-regulates bcl-2expression in LNCaP cells[J].Cancer Res,1998,58(19):4245-4249.
[6]Gupta R A,Dubois R N.Colorectal cancer prevention and treatment by inhibition of COX-2[J].Nat Rev Cancer,2001,1(1):11-21.
[7]Hsu A L,Ching T T,Wang D S,et al.The cyclooxygenase-2inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2[J].J Biol Chem,2000,275(15):11397-11403.
[8]Bansal S,Bala M,Suthar S K,et al.Design and synthesis of novel 2-phenyl-5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazoles as selective COX-2inhibitors with potent anti-inflammatory activity[J].Eur J Med Chem,2014,80(1):167-174.
[9]Alzweiri M,Sallam M,Al-Zyoud W,et al.Stability study of etoricoxib a selective cyclooxygenase-2inhibitor by a new single and repid reversed phase HPLC method[J].Symmetry,2018,10:288-299.
[10]Cao H,Yu R,Tao Y,et al.Measurement of cyclooxygenase inhibition using liquid chromatography-tandem mass spectrometry[J].J Pharm Biomed Anal,2011,54(1):230-235.
[11]Xiong X,He Y N,Feng B,et al.Screening for the anti-inflammation quality markers of Xiaojin Pills based on HPLCMS/MS method COX-2inhibition test and protein interaction network[J].Sci Rep,2018,8(1):7454-7463.
[12]Deng X,Shi S,Li S,et al.Magnetic ligand fishing combination with high-performance liquid chromatography-diode array detector-mass spectrometry to screen and characterize cyclooxygenase-2inhibitors from green tea[J].J Chromatogr B Analyt Technol Biomed Life Sci,2014,973C:55-60.
[13]Van Breemen R B,Tao Y,Li W.Cyclooxygenase-2inhibitors in ginger(Zingiber officinale)[J].Fitoterapia,2011,82(1):38-43.
[14]Ma Y,Liu J,Shi H M,et al.Isolation of chracacterization of anti-inflammatory peptides derived from whey protain[J].J Dairy Sci,2016,99(9):6902-6912.
[15]Razzaghi-Asl N,Mirzayi S,Mahnam K,et al.Identification of COX-2inhibitors via structure-based virtual screening and molecular dynamics simulation[J].J Mol Graph Model,2018,83:138-152.
[16]Li Y D,Christopher M F,Chen M H,et al.Primary virtual and in vitro bioassay screening of natural inhibitors from flavonoids against COX-2[J].Chin J Nat Med,2011,9(2):156-160.
[17]Van der Ouderaa F J,Buytenhek M.Purification of PGH synthase from sheep vesicular glands[J].Methods Enzymol,1982,86:60-68.
[18]Kulmacz R J.Prostaglandin G2levels during reaction of prostaglandin H synthase with arachidonic acid[J].Prostaglandins,1987,34(2):225-240.
[19]White H L,Glassman A T.A simple radiochemical assay for prostaglandin synthetase[J].Prostaglandins,1974,7(2):123-129.
[20]Laufer S,Zechmeister P,Klein T.Development of an invitro test system for the evaluation of cyclooxygenase-2inhibitors[J].Inflamm Res,1999,48(3):133-138.
[21]Fumio Y,Kimihiro Y,Katsuaki K,et al.Substrate probe,enzyme-activity detection method by a multi-dimensional nuclear magnetic resonance method and enzyme-activity imaging method:USA,US2010248279[P].2010-09-30.
[22]徐斌,董英,胡庆松,等.紫外分光光度发快速测定小麦胚芽脂肪氧化酶活力[J].分析化学,2009,37(8):1252-1252.
[23]牛宗亮,王荣镇,董新伟,等.马粪海胆生殖腺营养成分的含量测定[J].中国海洋药物,2009,28(6):26-30.
[24]Hall T C,Bilku D K,Neal C P,et al.The impact of an omega-3fatty acid rich lipid emulsion on fatty acid profiles in critically ill septic patients[J].Prostaglandins Leukot Essent Fatty Acids,2016,112:1-11.
[25]王燕华,金春爱,孙印石,等.不同加工方式的鹿茸脂肪酸的气相色谱分析[J].中草药,2017,48(12):2431-2441.
[26]Shi S,Yi L Z,Yun Y H,et al.A combination of GC-MS and chemometrics reveals metabolic differences between serum and plasma[J].Anal Methods,2015,7(5):1751-1757.
[27]Kayacelebi A A,Schauerte C,Kling K,et al.Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase(MAGL)activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol[J].J Chromatogr B Analyt Technol Biomed Life Sci,2017,1047:151-159.
[28]Banerjee D,Mazumder S,Kumar Sinha A.Involvement of Nitric Oxide on Calcium Mobilization and arachidonic acid pathway activation during platelet aggregation with different aggregating agonists[J].Int J Biomed Sci,2016,12(1):25-35.
[29]王桂花,谢树莲.刺叶提灯藓花生四烯酸提取与含量测定[J].食品科学,2010,31(6):52-54.
[30]钟宇,陈滨,李建,等.LC-MS/MS分析血浆中脂肪酸及代谢产物[J].质谱学报,2018,39(3):310-315.
[31]谷艳,石先哲,赵素敏,等.高效液相色谱-电喷雾串联四级杆质谱法检测小鼠组织中花生四烯酸及其代谢产物[J].分析化学,2010,38(8):1089-1094.
[32]Najera A I,Renobales M D,Barron L.Effects of pH,temperature,CaCl2and enzyme concentrations on the rennet-clotting properties of milk:a multifactorial study[J].Food Chem,2003,80(3):345-352.
[33]Su J Y,Storey K B.Fish muscle phosphofructokinase:Influences of protein concentration on enzyme kinetic behaviour[J].Int J Biochem Cell Biol,1995,27(27):1277-1283.
[34]Santos N C,Figueira-Coelho J,Martins-Silva J,et al.Multidisciplinary utilization of dimethyl sulfoxide:pharmacological,cellular,and molecular aspects[J].Biochem Pharmacol,2003,65(7):1035-1041.
[35]贾卓美,唐旭利,乔丹,等.中国西沙海绵Dactylospongia elegans化学成分研究[J].中国海洋药物,2018,37(1):16-19.
[36]黄娣,徐永健.不同方法提取大海马脂类及脂肪酸组成分析[J].中国海洋药物,2016,35(2):35-40.
[37]Nikolic D,Habibi-Goudarzi S,Corley D G,et al.Evaluation of cyclooxygenase-2inhibitors using pulsed ultrafiltration mass spectrometry[J].Anal Chem,2000,72(16):3853-3859.
[38]刘伟,王娟,姜孝玉,等.海葵溶细胞素的研究进展[J].海洋科学,2005,29(4):56-62.
[39]Ko S C,Jeon Y J.Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7macrophages and in vivo zebrafish model[J].Nutr Res Pract,2015,9(3):219-226.