新疆多花蔷薇根中熊果酸的提取及分离纯化工艺研究
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摘要
通过超声-微波辅助法提取了多花蔷薇根中的熊果酸,并对其进行分离、纯化,确定了最优提取及分离纯化条件.采用响应面法优化熊果酸的提取工艺,经脱脂、脱色、酸碱沉淀、凝胶柱层析等方法得到了分离纯化熊果酸的最佳条件,用紫外光谱(UV),红外光谱(IR),核磁共振氢谱(~1H NMR),核磁共振碳谱(13C NMR)对产品进行鉴定.结果表明:在超声功率50 W,微波功率310 W,提取时间2.4 min,液固比20:1 mL/g,微波浸提时间15 min,浸提温度55℃的提取条件下,熊果酸的得率可达2.64%.当石油醚用量为提取液体积的0.2倍,脱脂2次,活性炭用量0.04 g/mL,脱色3次,碱沉淀p H值为12,酸沉淀p H值为4,熊果酸粗品用葡聚糖凝胶LH-20分离洗脱2次时,重结晶后的熊果酸纯度在97%以上,达到较好的分离效果.
In this paper, ursolic acid was extracted from the roots of Rosa multiflora Thunb by ultrasonic-microwave-assisted extraction, and the optimum extraction and purification conditions were determined. The extraction process of ursolic acid was optimized by response surface methodology. The best conditions for separation and purification of ursolic acid were obtained by defatting, decolorizing, acid base precipitation and gel column chromatography. The products were identified by ultraviolet spectroscopy(UV),infrared spectroscopy(IR), ~1H NMR and ~(13)C NMR. The results showed that the yield of ursolic acid could reach2.64% under the conditions of ultrasonic power 50 W, microwave power 310 W, extraction time 2.4 min,liquid-solid ratio 20∶1 mL/g, microwave extraction time 15 min and extraction temperature 55℃. When the amount of petroleum ether was 0.2 times of the volume of extract, 2 times of degreasing, 0.04 g/mL of active carbon, 3 times of decolorization; the pH value of alkali precipitation was 12; the acid precipitation pH value was4; when ursolic acid crude product was separated from dextran gel LH-20 for two times, the purity of ursolic acid after recrystallization was over 97%, achieving a better separation effect.
In this paper, ursolic acid was extracted from the roots of Rosa multiflora Thunb by ultrasonic-microwave-assisted extraction, and the optimum extraction and purification conditions were determined. The extraction process of ursolic acid was optimized by response surface methodology. The best conditions for separation and purification of ursolic acid were obtained by defatting, decolorizing, acid base precipitation and gel column chromatography. The products were identified by ultraviolet spectroscopy(UV),infrared spectroscopy(IR), ~1H NMR and ~(13)C NMR. The results showed that the yield of ursolic acid could reach2.64% under the conditions of ultrasonic power 50 W, microwave power 310 W, extraction time 2.4 min,liquid-solid ratio 20∶1 mL/g, microwave extraction time 15 min and extraction temperature 55℃. When the amount of petroleum ether was 0.2 times of the volume of extract, 2 times of degreasing, 0.04 g/mL of active carbon, 3 times of decolorization; the pH value of alkali precipitation was 12; the acid precipitation pH value was4; when ursolic acid crude product was separated from dextran gel LH-20 for two times, the purity of ursolic acid after recrystallization was over 97%, achieving a better separation effect.
引文
[1]刘士侠,丛者福.新疆蔷薇[M].乌鲁木齐:新疆科技卫生出版社,2000.Liu S X,Cong Z F.Xinjiang rose[M].Urumchi:Xinjiang Science and Technology Health Press,2000.
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[3]Wang Y W,Suo Y R,Sun Y A,et al.Determination of triterpene acids from 37 different varieties of Raspberry using pre-column cerivatization and HPLC fluorescence detection[J].Chromatographia,2016,79(21-22):1 515-1 525.DOI:10.1007/s10337-016-3174-1.
[4]Zhang C Y,Wang C,Li W J,et al.Pharmacokinetics and pharmacodynamics of the triterpenoid ursolic acid in regulating the antioxidant,anti-inflammatory,and epigenetic gene responses in rat leukocytes[J].Molecular Pharmaceutics,2017,14(11):3 709-3 717.DOI:10.1021/acs.molpharmaceut.7b00469.
[5]Velasco J,Holgado F,Márquez-Ruiz G,et al.Concentrates of triterpenic acids obtained from crude olive pomace oils:characterization and evaluation of their potential antioxidant activity[J].J Sci Food Agric,2018,98(13):4 837-4 844.DOI:10.1002/jsfa.2018.98.issue-13.
[6]Wang C M,Jhan L Y,Tsai J S,et al.The pleiotropic antibacterial mechanisms of ursolic acid against methicillin-resistant staphylococcus aureu(MRSA)[J].Molecules,2016,21(7):884.DOI:10.3390/molecules21070884.
[7]Gossan D,Magit A,Kouassi P,et al.Antibacterial and cytotoxic triterpenoids from the roots of Combretum racemosum[J].Fitoterapia,2016,110:89-95.DOI:10.1016/j.fitote.2016.03.002.
[8]Liu B H,Piao X H,Guo L Y,et al.Ursolic acid protects against ulcerative colitis via anti-inflammatory and antioxidant effects in mice[J].Mol Med Rep,2016,13(6):4 779-4 785.DOI:10.3892/mmr.2016.5094.
[9]Li J,Li N,Yan S T,et al.Ursolic acid alleviates inflammation and against diabetes-induced nephropathy through TLR-mediated inflammatory pathway[J].Molecular medicine reports,2018,18(5):4 675-4 687.
[10]Angel R,Smith S,Glickman R,et al.Antitumor effects of ursolic acid in a mouse model of postmenopausal breast cancer[J].Nutrition and Cancer,2010,62(8):1 074-1 086.DOI:10.1080/01635581.2010.492092.
[11]Gao Y S,Yuan Y,Song G,et al.Inhibitory effect of ursolic acid and oleanolic acid from Eriobotrya fragrans on A549 cell viability in vivo[J].Genet Mol Res,2016,15(2):1-8.
[12]Sohn E J,Won G,Lee J,et al.Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell[J].Int J Biol Sci,2016,12(11):1 279-1 288.DOI:10.7150/ijbs.13453.
[13]Gutierrez G,Silrdia A,Meckes M,et al.Hepatoprotective properties of oleanolic and ursolic acids in antitubercular drug-induced liver damage[J].Asian Pac JTrop Med,2016,9(7):625-632.
[14]Shukla B,Visen P,Patnaik G,et al.Hepatoprotective activity in the rat of ursolic acid isolated from eucalyptus hybrid[J].Phytotherapy Research,2010,6(2):74-79.
[15]He K,Sing S H,Zou Z Y,et al.The hypoglycemic and synergistic effect of loganin,morroniside,and ursolic acid isolated from the fruits of Cornus officinalis[J]. Phytotherapy Research,2016,30(2):283-291.DOI:10 .1002/ptr.v30.2.
[16]Gao D,Li N,Li Q W,et al.Study of the extraction,purification and antidiabetic potential of ursolic acid from Cornus officinalis Sieb.et Zucc[J].Therapy,2008,5(5):697-705.DOI:10.2217/14750708.5.5.697.
[17]付红军,彭湘莲.枇杷叶中熊果酸的索氏提取工艺优化[J].食品与机械,2011,27(2):59-61.Fu H J,Peng X L.Optimization of extraction technology of ursolic acid from loquat leaves with soxhlet extraction[J].Food&Machinery,2011,27(2):59-61.
[18]Tarvainen M,Soumela J,Kallio H,et al.Triterpene acids in plantago major:identification,quantification and comparison of different extraction methods[J].Chromatographia,2010,71(3-4):279-284.DOI:10.1365/s10337-009-1439-7.
[19]黄超华,吴杰,骆妃静.山楂中熊果酸的提取与分离[J].西北药学杂志,2009,24(6):459-460.DOI:10.3969/j.issn.1004-2407.2009.06.016.Huang C H,Wu J,Luo F J.Extraction and separation of ursolic acid from Hawthorn[J].Northwest Pharmaceutical Journal,2009,24(6):459-460.
[20]Fang X S,Wang J H,Yu X L,et al.Optimization of microwave-assisted extraction followed by RP-HPLC for the simultaneous determination of oleanolic acid and ursolic acid in the fruits of Chaenomeles sinensis[J].JSEP SCI,2010,33(8):1 147-1 155.
[21]Fana J P,Liao D D,Zhang X H.Ultrasonic assisted extraction of ursolic acid from apple pomace:A novel and facile technique[J].Separation Science and Technology,2016,51(8):1 344-1 350.DOI:10.1080/01496395.2016.1165253.
[22]王文俊,白玮玮,李军超,等.一种测定女贞子中齐墩果酸和熊果酸的新方法[J].西北植物学报,2011,31(9):1 894-1 899.Wang W J,Bai W W,Li J C,et al.Determination of oleanolic acid and ursolic acid in ligustrum iucidum ait using accelerated aolvent extraction combined with capillary zone electrophoresis[J].Acta Botanica Boreall-Occidentalia Sinica,2011,31(9):1 894-1 899.
[23]Li K Q,Chen W,Li X Y.Production process for ursolic acid[J].Journal of Chemical Industry and Engineering,2006,57(2):380-381.
[24]Li X,Fan Y,Guo W,et al.Separation and Purification of ursolic acid from Forsythia suspense leaves[J].Chinese Journal of Pharmaceuticals,2013,44(1):32-35.
[25]Park C E,Park C H.Ursolic acid isolated from Mume fructus inhibits urease activity of Helicobacter pylori[J].Korean Chemical Engineering Researc,2013,51(5):591-596.DOI:10.9713/kcer.2013.51.5.591.
[26]Fan J P,He C H.Single-step preparative separation of barbinervic acid and its epimer(rotungenic acid),along with two other pentacyclic triterpene acids from the leaves of Diospyros kaki using HSCCC[J].J Liq Chromatogr R T,2006,29(5-8):815-826.
[27]Hu H G,Zhao Q L,Pang Z C,et al.Optimization extraction,characterization and anticancer activities of polysaccharides from mango pomace[J].INT J Biol Macromol,2018,117:1 314-1 325.DOI:10.1016/j.ijbiomac.2018.05.225.
[28]廖晓峰,陈正行,于荣.栀子中三萜酸的提取及结构鉴定[J].林产化学与工业,2006,26(4):110-112.DOI:10.3321/j.issn:0253-2417.2006.04.027.Liao X F,Chen Z X,Yu R.Study on isolation and identification of triterpene acid from Gardenia jasm indose eills[J].Chemistry and Industry of Forest Products,2006,26(4):110-112.
[29]Ren Y L,Eugenio G,Czarnecki A,et al.Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives[J].Bioorgan Med Chem,2018,26(15):4 452-4 460.DOI:10.1016/j.bmc.2018.07.025.
[30]Bag B,Das S,Hasan S,et al.Nanoarchitectures by hierarchical self-assembly of ursolic acid:entrapment and release of fluorophores including anticancer drug doxorubicin[J].RSC Advances,2017,7(29):18 136-18 143.DOI:10.1039/C7RA02123B.
[2]古力齐曼·阿布力孜,阿吾提·艾买尔,毛居代·亚尔买买提,等.HPLC法测定野蔷薇中齐墩果酸和熊果酸的含量[J].食品工业科技,2016,37(6):49-51+57.Abliz·G,Amar·A,Yaermaimaiti·M,et al.Determination of oleanolic acid and ursolic acid in Rosa Multiflora Thunb by HPLC[J].Science and Technology of Food Industry,2016,37(6):49-51+57.
[3]Wang Y W,Suo Y R,Sun Y A,et al.Determination of triterpene acids from 37 different varieties of Raspberry using pre-column cerivatization and HPLC fluorescence detection[J].Chromatographia,2016,79(21-22):1 515-1 525.DOI:10.1007/s10337-016-3174-1.
[4]Zhang C Y,Wang C,Li W J,et al.Pharmacokinetics and pharmacodynamics of the triterpenoid ursolic acid in regulating the antioxidant,anti-inflammatory,and epigenetic gene responses in rat leukocytes[J].Molecular Pharmaceutics,2017,14(11):3 709-3 717.DOI:10.1021/acs.molpharmaceut.7b00469.
[5]Velasco J,Holgado F,Márquez-Ruiz G,et al.Concentrates of triterpenic acids obtained from crude olive pomace oils:characterization and evaluation of their potential antioxidant activity[J].J Sci Food Agric,2018,98(13):4 837-4 844.DOI:10.1002/jsfa.2018.98.issue-13.
[6]Wang C M,Jhan L Y,Tsai J S,et al.The pleiotropic antibacterial mechanisms of ursolic acid against methicillin-resistant staphylococcus aureu(MRSA)[J].Molecules,2016,21(7):884.DOI:10.3390/molecules21070884.
[7]Gossan D,Magit A,Kouassi P,et al.Antibacterial and cytotoxic triterpenoids from the roots of Combretum racemosum[J].Fitoterapia,2016,110:89-95.DOI:10.1016/j.fitote.2016.03.002.
[8]Liu B H,Piao X H,Guo L Y,et al.Ursolic acid protects against ulcerative colitis via anti-inflammatory and antioxidant effects in mice[J].Mol Med Rep,2016,13(6):4 779-4 785.DOI:10.3892/mmr.2016.5094.
[9]Li J,Li N,Yan S T,et al.Ursolic acid alleviates inflammation and against diabetes-induced nephropathy through TLR-mediated inflammatory pathway[J].Molecular medicine reports,2018,18(5):4 675-4 687.
[10]Angel R,Smith S,Glickman R,et al.Antitumor effects of ursolic acid in a mouse model of postmenopausal breast cancer[J].Nutrition and Cancer,2010,62(8):1 074-1 086.DOI:10.1080/01635581.2010.492092.
[11]Gao Y S,Yuan Y,Song G,et al.Inhibitory effect of ursolic acid and oleanolic acid from Eriobotrya fragrans on A549 cell viability in vivo[J].Genet Mol Res,2016,15(2):1-8.
[12]Sohn E J,Won G,Lee J,et al.Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell[J].Int J Biol Sci,2016,12(11):1 279-1 288.DOI:10.7150/ijbs.13453.
[13]Gutierrez G,Silrdia A,Meckes M,et al.Hepatoprotective properties of oleanolic and ursolic acids in antitubercular drug-induced liver damage[J].Asian Pac JTrop Med,2016,9(7):625-632.
[14]Shukla B,Visen P,Patnaik G,et al.Hepatoprotective activity in the rat of ursolic acid isolated from eucalyptus hybrid[J].Phytotherapy Research,2010,6(2):74-79.
[15]He K,Sing S H,Zou Z Y,et al.The hypoglycemic and synergistic effect of loganin,morroniside,and ursolic acid isolated from the fruits of Cornus officinalis[J]. Phytotherapy Research,2016,30(2):283-291.DOI:10 .1002/ptr.v30.2.
[16]Gao D,Li N,Li Q W,et al.Study of the extraction,purification and antidiabetic potential of ursolic acid from Cornus officinalis Sieb.et Zucc[J].Therapy,2008,5(5):697-705.DOI:10.2217/14750708.5.5.697.
[17]付红军,彭湘莲.枇杷叶中熊果酸的索氏提取工艺优化[J].食品与机械,2011,27(2):59-61.Fu H J,Peng X L.Optimization of extraction technology of ursolic acid from loquat leaves with soxhlet extraction[J].Food&Machinery,2011,27(2):59-61.
[18]Tarvainen M,Soumela J,Kallio H,et al.Triterpene acids in plantago major:identification,quantification and comparison of different extraction methods[J].Chromatographia,2010,71(3-4):279-284.DOI:10.1365/s10337-009-1439-7.
[19]黄超华,吴杰,骆妃静.山楂中熊果酸的提取与分离[J].西北药学杂志,2009,24(6):459-460.DOI:10.3969/j.issn.1004-2407.2009.06.016.Huang C H,Wu J,Luo F J.Extraction and separation of ursolic acid from Hawthorn[J].Northwest Pharmaceutical Journal,2009,24(6):459-460.
[20]Fang X S,Wang J H,Yu X L,et al.Optimization of microwave-assisted extraction followed by RP-HPLC for the simultaneous determination of oleanolic acid and ursolic acid in the fruits of Chaenomeles sinensis[J].JSEP SCI,2010,33(8):1 147-1 155.
[21]Fana J P,Liao D D,Zhang X H.Ultrasonic assisted extraction of ursolic acid from apple pomace:A novel and facile technique[J].Separation Science and Technology,2016,51(8):1 344-1 350.DOI:10.1080/01496395.2016.1165253.
[22]王文俊,白玮玮,李军超,等.一种测定女贞子中齐墩果酸和熊果酸的新方法[J].西北植物学报,2011,31(9):1 894-1 899.Wang W J,Bai W W,Li J C,et al.Determination of oleanolic acid and ursolic acid in ligustrum iucidum ait using accelerated aolvent extraction combined with capillary zone electrophoresis[J].Acta Botanica Boreall-Occidentalia Sinica,2011,31(9):1 894-1 899.
[23]Li K Q,Chen W,Li X Y.Production process for ursolic acid[J].Journal of Chemical Industry and Engineering,2006,57(2):380-381.
[24]Li X,Fan Y,Guo W,et al.Separation and Purification of ursolic acid from Forsythia suspense leaves[J].Chinese Journal of Pharmaceuticals,2013,44(1):32-35.
[25]Park C E,Park C H.Ursolic acid isolated from Mume fructus inhibits urease activity of Helicobacter pylori[J].Korean Chemical Engineering Researc,2013,51(5):591-596.DOI:10.9713/kcer.2013.51.5.591.
[26]Fan J P,He C H.Single-step preparative separation of barbinervic acid and its epimer(rotungenic acid),along with two other pentacyclic triterpene acids from the leaves of Diospyros kaki using HSCCC[J].J Liq Chromatogr R T,2006,29(5-8):815-826.
[27]Hu H G,Zhao Q L,Pang Z C,et al.Optimization extraction,characterization and anticancer activities of polysaccharides from mango pomace[J].INT J Biol Macromol,2018,117:1 314-1 325.DOI:10.1016/j.ijbiomac.2018.05.225.
[28]廖晓峰,陈正行,于荣.栀子中三萜酸的提取及结构鉴定[J].林产化学与工业,2006,26(4):110-112.DOI:10.3321/j.issn:0253-2417.2006.04.027.Liao X F,Chen Z X,Yu R.Study on isolation and identification of triterpene acid from Gardenia jasm indose eills[J].Chemistry and Industry of Forest Products,2006,26(4):110-112.
[29]Ren Y L,Eugenio G,Czarnecki A,et al.Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives[J].Bioorgan Med Chem,2018,26(15):4 452-4 460.DOI:10.1016/j.bmc.2018.07.025.
[30]Bag B,Das S,Hasan S,et al.Nanoarchitectures by hierarchical self-assembly of ursolic acid:entrapment and release of fluorophores including anticancer drug doxorubicin[J].RSC Advances,2017,7(29):18 136-18 143.DOI:10.1039/C7RA02123B.