HPLC-DAD-FLD同时测定柑橘果汁中12种多甲氧基黄酮和香豆素类物质
|
摘要
建立高效液相色谱-串联二极管阵列检测器和荧光检测器(high performance liquid chromatography-diode array detector-fluorescence detector,HPLC-DAD-FLD)同时检测柑橘果汁中12种多甲氧基黄酮、香豆素及呋喃香豆素类物质的方法。以0.01%磷酸、甲醇、乙腈组成三元流动相进行梯度洗脱,12种物质在30 min内实现基线分离。利用DAD和FLD获得各物质的紫外和荧光光谱信息,将柑橘果汁样品组分的光谱与之比对,并结合色谱保留时间,实现样品成分的定性分析。分别以320 nm和450 nm为紫外和荧光的检测波长,研究该方法的定量性能。结果显示:标准曲线线性关系良好;荧光定量限低至μg/L级,可作为紫外定量的重要补充手段;果汁回收率的紫外和荧光检测值分别为95.2%~104.8%和94.5%~103.5%。该方法对样品定性、定量分析准确,适合柑橘多甲氧基黄酮、香豆素等能发射荧光物质的检测。
A method using high performance liquid chromatography with diode array detection and fluorescence detection(HPLC-DAD-FLD) was developed to simultaneously analyze 12 compounds of polymethoxyflavones(PMFs), coumarins and furocoumarins in citrus juice samples. Baseline separation of twelve compounds was achieved within 30 min using gradient elution with a ternary mobile phase composed of 0.01% phosphoric acid, methanol and acetonitrile. Qualitative identification of the components in citrus juices was confirmed by comparing their UV and fluorescence(FL) spectra and retention times with those of the reference standards. Quantitative analysis of the juice components was operated at a UV wavelength of 320 nm(OD320 nm) and a fluorescence emission of 450 nm. The calibration curves showed good linearity. The limit of quantitation(LOQ) with FLD was at as low as μg/L level so that FLD could be an important complementary tool to UV detection. The recovery tests showed a good accuracy for both UV(95.2%–104.8%) and FL(94.5%–103.5%) examination. In general, this method is accurate and reliable for qualitative and quantitative analysis of citrus juice components, especially the ones having fluorescence emission like PMFs and coumarins.
A method using high performance liquid chromatography with diode array detection and fluorescence detection(HPLC-DAD-FLD) was developed to simultaneously analyze 12 compounds of polymethoxyflavones(PMFs), coumarins and furocoumarins in citrus juice samples. Baseline separation of twelve compounds was achieved within 30 min using gradient elution with a ternary mobile phase composed of 0.01% phosphoric acid, methanol and acetonitrile. Qualitative identification of the components in citrus juices was confirmed by comparing their UV and fluorescence(FL) spectra and retention times with those of the reference standards. Quantitative analysis of the juice components was operated at a UV wavelength of 320 nm(OD320 nm) and a fluorescence emission of 450 nm. The calibration curves showed good linearity. The limit of quantitation(LOQ) with FLD was at as low as μg/L level so that FLD could be an important complementary tool to UV detection. The recovery tests showed a good accuracy for both UV(95.2%–104.8%) and FL(94.5%–103.5%) examination. In general, this method is accurate and reliable for qualitative and quantitative analysis of citrus juice components, especially the ones having fluorescence emission like PMFs and coumarins.
引文
[1]吴厚玖,孙志高,王华.试论我国柑橘加工业发展方向[J].食品与发酵工业,2006,32(4):85-89.DOI:10.3321/j.issn:0253-990X.2006.04.024.
[2]杨子玉,唐颂,任婧楠,等.不同品种柑橘汁胞的品质比较研究[J].食品工业科技,2015,36(13):121-125.DOI:10.13386/j.issn1002-0306.2015.13.017.
[3]夏涛,高丽萍.类黄酮及茶儿茶素生物合成途径及其调控研究进展[J].中国农业科学,2009,42(8):2899-2908.DOI:10.3864/j.issn.0578-1752.2009.08.031.
[4]乔小燕,马春雷,陈亮.植物类黄酮生物合成途径及重要基因的调控[J].天然产物研究与开发,2009,21(2):354-360.DOI:10.3969/j.issn.1001-6880.2009.02.040.
[5]MABRY T J,ULUBELEN A.Chemistry and utilization of phenylpropanoids including flavonoids,coumarins,and lignans[J].Journal of Agricultural and Food Chemistry,1980,28(2):188-196.DOI:10.1021/jf60228a02.
[6]吴宏伟.陈皮多甲氧基黄酮有效部位研究[D].北京:北京中医药大学,2006:7;12.
[7]LI S,PAN M H,LO C Y,et al.Chemistry and health effects of polymethoxyflavones and hydroxylated polymethoxyflavones[J].Journal of Functional Foods,2009,1(1):2-12.DOI:10.1016/j.jff.2008.09.003.
[8]LI S,WANG H,GUO L,et al.Chemistry and bioactivity of nobiletin and its metabolites[J].Journal of Functional Foods,2014,6:2-10.DOI:10.1016/j.jff.2013.12.011.
[9]ZHANG C,LU Y,TAO L,et al.Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts[J].Journal of Enzyme Inhibition and Medicinal Chemistry,2007,22(1):91-98.DOI:10.1080/14756360600953876.
[10]MORLEY K L,FERGUSON P J,KOROPATNICK J.Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells[J].Cancer Letters,2007,251(1):168-178.DOI:10.1016/j.canlet.2006.11.016.
[11]MONTANARI A,CHEN J,WIDMER W.Citrus flavonoids:a review of past biological activity against disease-discovery of new flavonoids from dancy tangerine cold pressed peel oil solids and leaves[J].Flavonoids in the Living System,1998,439:103-116.
[12]MANTHEY J A,GUTHRIE N.Antiproliferative activities of citrus flavonoids against six human cancer cell lines[J].Journal of Agricultural and Food Chemistry,2002,50(21):5837-5843.DOI:10.1021/jf020121d.
[13]PAN G G,KILMARTIN P A,SMITH B G,et al.Detection of orange juice adulteration by tangelo juice using multivariate analysis of polymethoxylated flavones and carotenoids[J].Journal of the Science of Food and Agriculture,2002,82(4):421-427.DOI:10.1002/jsfa.1051.
[14]E S C R I C H E I,K A D A R M,J U A N-B O R R S M,e t a l.U s i n g flavonoids,phenolic compounds and headspace volatile profile for botanical authentication of lemon and orange honeys[J].Food Research International,2011,44(5):1504-1513.DOI:10.1016/j.foodres.2011.03.049.
[15]张韶瑜,孟林,高文远,等.香豆素类化合物生物学活性研究进展[J].中国中药杂志,2005,30(6):410-414.DOI:10.3321/j.issn:1001-5302.2005.06.002.
[16]UM M Y,MOON M K,AHN J,et al.Coumarin attenuates hepatic steatosis by down-regulating lipogenic gene expression in mice fed a high-fat diet[J].The British Journal of Nutrition,2013,109(9):1590-1597.DOI:10.1017/S0007114512005260.
[17]LEOPOLDINI M,MALAJ N,TOSCANO M,et al.On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-Co A reductase(HMGR)enzyme[J].Journal of Agricultural and Food Chemistry,2010,58(19):10768-10773.DOI:10.3168/jds.2006-897.
[18]MANDALARI G,BENNETT R N,BISIGNANO G,et al.Antimicrobial activity of flavonoids extracted from bergamot(Citrus bergamia Risso)peel,a byproduct of the essential oil industry[J].Journal of Applied Microbiology,2007,103(6):2056-2064.DOI:10.1111/j.1365-2672.2007.03456.x.
[19]TROMBETTA D,CIMINO F,CRISTANI M,et al.In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-α(TNF-α)[J].Journal of Agricultural and Food Chemistry,2010,58(14):8430-8436.DOI:10.1021/jf1008605.
[20]FUKUDA K,GUO L,OHASHI N,et al.Amounts and variation in grapefruit juice of the main components causing grapefruit-drug interaction[J].Journal of Chromatography B,Biomedical Sciences and Applications,2000,741(2):195-203.
[21]SHIM S C,CHOI K Y,SONG P S.Studies on the phototoxicity of coumarin derivatives-Ⅰ.Photocyclodimerization of5,7-dimethoxycoumarin[J].Photochemistry and Photobiology,1978,27(1):25-31.DOI:10.1111/j.1751-1097.1978.tb07560.x.
[22]KAIDBEY K H,KLIGMAN A M.Photosensitization by coumarin derivatives:structure-activity relationships[J].Archives of Dermatology,1981,117(5):258-263.DOI:10.1001/archderm.1981.01650050014012.
[23]FEGER W,BRANDAUER H,GABRIS P,et al.Nonvolatiles of commercial lime and grapefruit oils separated by high-speed countercurrent chromatography[J].Journal of Agricultural and Food Chemistry,2006,54(6):2242-2252.DOI:10.1021/jf052267t.
[24]RUSSO M,BONACCORSI I,COSTA R,et al.Reduced time HPLC analyses for fast quality control of citrus essential oils[J].Journal of Essential Oil Research,2015,27(4):307-315.DOI:10.1080/10412905.2015.1027419.
[25]DUGO P,PIPERNO A,ROMEO R,et al.Determination of oxygen heterocyclic components in citrus products by HPLC with UV detection[J].Journal of Agricultural and Food Chemistry,2009,57(15):6543-6551.DOI:10.1021/jf901209r.
[26]冉玥,焦必宁,赵其阳,等.超高效液相色谱法同时测定柑橘中11种类黄酮物质[J].食品科学,2013,34(4):168-172.
[27]章翀玮,魏东芝,周文瑜,等.柑橘类黄酮成分的同步HPLC检测(英文)[J].中成药,2007,29(1):99-104.DOI:10.3969/j.issn.1001-1528.2007.01.029.
[28]GUO L Q,BAO H,YANG J.Correlation of furanocoumarin composition and rat cytochrome P450 3A inhibition among citrus fruit juices[J].中国临床药理学与治疗学,2004,9(9):970-977.
[29]秦锋,汪涛,蒋学华.高效液相色谱法测定大鼠血浆中水合橙皮内酯的浓度及药动学研究[J].时珍国医国药,2015,26(3):550-552.
[30]杨洁,孙婷,姚华,等.RP-HPLC-DAD法测定陈皮中3种多甲氧基黄酮的质量分数[J].吉林大学学报(理学版),2014,52(2):348-352.DOI:10.13413/j.cnki.jdxblxb.2014.02.37.
[2]杨子玉,唐颂,任婧楠,等.不同品种柑橘汁胞的品质比较研究[J].食品工业科技,2015,36(13):121-125.DOI:10.13386/j.issn1002-0306.2015.13.017.
[3]夏涛,高丽萍.类黄酮及茶儿茶素生物合成途径及其调控研究进展[J].中国农业科学,2009,42(8):2899-2908.DOI:10.3864/j.issn.0578-1752.2009.08.031.
[4]乔小燕,马春雷,陈亮.植物类黄酮生物合成途径及重要基因的调控[J].天然产物研究与开发,2009,21(2):354-360.DOI:10.3969/j.issn.1001-6880.2009.02.040.
[5]MABRY T J,ULUBELEN A.Chemistry and utilization of phenylpropanoids including flavonoids,coumarins,and lignans[J].Journal of Agricultural and Food Chemistry,1980,28(2):188-196.DOI:10.1021/jf60228a02.
[6]吴宏伟.陈皮多甲氧基黄酮有效部位研究[D].北京:北京中医药大学,2006:7;12.
[7]LI S,PAN M H,LO C Y,et al.Chemistry and health effects of polymethoxyflavones and hydroxylated polymethoxyflavones[J].Journal of Functional Foods,2009,1(1):2-12.DOI:10.1016/j.jff.2008.09.003.
[8]LI S,WANG H,GUO L,et al.Chemistry and bioactivity of nobiletin and its metabolites[J].Journal of Functional Foods,2014,6:2-10.DOI:10.1016/j.jff.2013.12.011.
[9]ZHANG C,LU Y,TAO L,et al.Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts[J].Journal of Enzyme Inhibition and Medicinal Chemistry,2007,22(1):91-98.DOI:10.1080/14756360600953876.
[10]MORLEY K L,FERGUSON P J,KOROPATNICK J.Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells[J].Cancer Letters,2007,251(1):168-178.DOI:10.1016/j.canlet.2006.11.016.
[11]MONTANARI A,CHEN J,WIDMER W.Citrus flavonoids:a review of past biological activity against disease-discovery of new flavonoids from dancy tangerine cold pressed peel oil solids and leaves[J].Flavonoids in the Living System,1998,439:103-116.
[12]MANTHEY J A,GUTHRIE N.Antiproliferative activities of citrus flavonoids against six human cancer cell lines[J].Journal of Agricultural and Food Chemistry,2002,50(21):5837-5843.DOI:10.1021/jf020121d.
[13]PAN G G,KILMARTIN P A,SMITH B G,et al.Detection of orange juice adulteration by tangelo juice using multivariate analysis of polymethoxylated flavones and carotenoids[J].Journal of the Science of Food and Agriculture,2002,82(4):421-427.DOI:10.1002/jsfa.1051.
[14]E S C R I C H E I,K A D A R M,J U A N-B O R R S M,e t a l.U s i n g flavonoids,phenolic compounds and headspace volatile profile for botanical authentication of lemon and orange honeys[J].Food Research International,2011,44(5):1504-1513.DOI:10.1016/j.foodres.2011.03.049.
[15]张韶瑜,孟林,高文远,等.香豆素类化合物生物学活性研究进展[J].中国中药杂志,2005,30(6):410-414.DOI:10.3321/j.issn:1001-5302.2005.06.002.
[16]UM M Y,MOON M K,AHN J,et al.Coumarin attenuates hepatic steatosis by down-regulating lipogenic gene expression in mice fed a high-fat diet[J].The British Journal of Nutrition,2013,109(9):1590-1597.DOI:10.1017/S0007114512005260.
[17]LEOPOLDINI M,MALAJ N,TOSCANO M,et al.On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-Co A reductase(HMGR)enzyme[J].Journal of Agricultural and Food Chemistry,2010,58(19):10768-10773.DOI:10.3168/jds.2006-897.
[18]MANDALARI G,BENNETT R N,BISIGNANO G,et al.Antimicrobial activity of flavonoids extracted from bergamot(Citrus bergamia Risso)peel,a byproduct of the essential oil industry[J].Journal of Applied Microbiology,2007,103(6):2056-2064.DOI:10.1111/j.1365-2672.2007.03456.x.
[19]TROMBETTA D,CIMINO F,CRISTANI M,et al.In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-α(TNF-α)[J].Journal of Agricultural and Food Chemistry,2010,58(14):8430-8436.DOI:10.1021/jf1008605.
[20]FUKUDA K,GUO L,OHASHI N,et al.Amounts and variation in grapefruit juice of the main components causing grapefruit-drug interaction[J].Journal of Chromatography B,Biomedical Sciences and Applications,2000,741(2):195-203.
[21]SHIM S C,CHOI K Y,SONG P S.Studies on the phototoxicity of coumarin derivatives-Ⅰ.Photocyclodimerization of5,7-dimethoxycoumarin[J].Photochemistry and Photobiology,1978,27(1):25-31.DOI:10.1111/j.1751-1097.1978.tb07560.x.
[22]KAIDBEY K H,KLIGMAN A M.Photosensitization by coumarin derivatives:structure-activity relationships[J].Archives of Dermatology,1981,117(5):258-263.DOI:10.1001/archderm.1981.01650050014012.
[23]FEGER W,BRANDAUER H,GABRIS P,et al.Nonvolatiles of commercial lime and grapefruit oils separated by high-speed countercurrent chromatography[J].Journal of Agricultural and Food Chemistry,2006,54(6):2242-2252.DOI:10.1021/jf052267t.
[24]RUSSO M,BONACCORSI I,COSTA R,et al.Reduced time HPLC analyses for fast quality control of citrus essential oils[J].Journal of Essential Oil Research,2015,27(4):307-315.DOI:10.1080/10412905.2015.1027419.
[25]DUGO P,PIPERNO A,ROMEO R,et al.Determination of oxygen heterocyclic components in citrus products by HPLC with UV detection[J].Journal of Agricultural and Food Chemistry,2009,57(15):6543-6551.DOI:10.1021/jf901209r.
[26]冉玥,焦必宁,赵其阳,等.超高效液相色谱法同时测定柑橘中11种类黄酮物质[J].食品科学,2013,34(4):168-172.
[27]章翀玮,魏东芝,周文瑜,等.柑橘类黄酮成分的同步HPLC检测(英文)[J].中成药,2007,29(1):99-104.DOI:10.3969/j.issn.1001-1528.2007.01.029.
[28]GUO L Q,BAO H,YANG J.Correlation of furanocoumarin composition and rat cytochrome P450 3A inhibition among citrus fruit juices[J].中国临床药理学与治疗学,2004,9(9):970-977.
[29]秦锋,汪涛,蒋学华.高效液相色谱法测定大鼠血浆中水合橙皮内酯的浓度及药动学研究[J].时珍国医国药,2015,26(3):550-552.
[30]杨洁,孙婷,姚华,等.RP-HPLC-DAD法测定陈皮中3种多甲氧基黄酮的质量分数[J].吉林大学学报(理学版),2014,52(2):348-352.DOI:10.13413/j.cnki.jdxblxb.2014.02.37.