红豆越橘总三萜的纯化及体外抗炎活性
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摘要
【目的】为了综合开发利用红豆越橘果实,通过大孔吸附树脂-Sephadex LH-20纯化工艺获得纯度较高的红豆越橘总三萜化合物,并分析此三萜化合物的体外抗炎活性。【方法】以野生矮丛红豆越橘为原料,首先采用静态吸附-解析实验和动态吸附-解析实验筛选大孔吸附树脂,优化最佳工艺,确定最佳上样质量浓度、pH、上样体积、上样流速以及洗脱液浓度;然后采用Sephadex LH-20羟丙基葡聚糖凝胶二次纯化,获得高纯度的红豆越橘总三萜;采用对透明质酸酶和牛血清白蛋白的变性抑制率为抗炎评价指标,分析红豆越橘总三萜的抗炎活性。【结果】比较7种大孔树脂对红豆越橘总三萜的纯化效果,结果显示,X-5树脂最适合初级纯化,最佳纯化工艺优化结果为上样质量浓度1.5 mg/mL、样液pH 6、上样量为4/3 BV、上样流速1 BV/h、80%(体积分数)的乙醇进行洗脱,红豆越橘总三萜的纯度由原来的5.13%提高到29.46%。进一步采用Sephadex LH-20二次纯化获得纯度为(43.25±0.31)%的红豆越橘总三萜,抗炎活性结果显示,对透明质酸酶和牛血清白蛋白变性抑制率分别可达(81.5±1.37)%、(72.59±1.84)%。【结论】红豆越橘是一种营养丰富的浆果,通过二次纯化技术获得纯度较高的三萜类化合物,并初步证实红豆越橘总三萜具有一定的抗炎活性。
【Objective】 In order to further develop and utilize lingonberry(Vaccinium vitis-idaea) fruits, the total triterpenoids of lingonberry were obtained, and the purification process was optimized through using macroporous adsorption resin and secondary purification of with Sephadex LH-20. The in vitro anti-inflammatory activity of this triterpene compound was analyzed. 【Method】 The experiment was based on the wild buckwheat-hung lingonberry as the raw material. The a static adsorption-analysis experiment, and the dynamic adsorption-analysis experiment were used to select the macroporous adsorption resin, optimization of the best process, and determination of the best sample concentration, pH 6, sample volume, sample flow rate, and ethanol concentration of the eluent. The secondary purification used Sephadex LH-20 hydroxypropyl dextran gel to obtain high-purity lingonberry total triterpene. Using hyaluronidase and the inhibition of serum albumin denaturation as an anti-inflammatory evaluation index to evaluate the anti-inflammatory activity of the total triterpenes of from lingonberry. 【Result】 The purification effect of seven macroporous resins on the total triterpenes of lingonberry was compared. The results showed that the X-5 resin was most suitable for the primary purification. The optimum purification process was the sample concentration of 1.5 mg/mL, elution at pH 6, loading volume 4/3 BV, loading flow rate of 1 BV/h, and 80% ethanol as eluant. The purity of the total triterpenes of lingonberry increased from 5.13% to 29.46%. Further purification using Sephadex LH-20 obtained a purity of(43.25±0.31) %. The total anti-inflammatory activity of the lingonberry was(81.5±1.37) % for the hyaluronidase and(72.59±1.84) % for the denaturation of bovine serum albumin. 【Conclusion】 Lingonberry is a nutrient-rich berry. High-purity triterpenoids were obtained through the secondary purification technology, and it was preliminarily showed that the total triterpenes of lingonberry had significant anti-inflammatory activity. Our results provided a certain theoretical basis for the further development and utilization of lingonberry.
【Objective】 In order to further develop and utilize lingonberry(Vaccinium vitis-idaea) fruits, the total triterpenoids of lingonberry were obtained, and the purification process was optimized through using macroporous adsorption resin and secondary purification of with Sephadex LH-20. The in vitro anti-inflammatory activity of this triterpene compound was analyzed. 【Method】 The experiment was based on the wild buckwheat-hung lingonberry as the raw material. The a static adsorption-analysis experiment, and the dynamic adsorption-analysis experiment were used to select the macroporous adsorption resin, optimization of the best process, and determination of the best sample concentration, pH 6, sample volume, sample flow rate, and ethanol concentration of the eluent. The secondary purification used Sephadex LH-20 hydroxypropyl dextran gel to obtain high-purity lingonberry total triterpene. Using hyaluronidase and the inhibition of serum albumin denaturation as an anti-inflammatory evaluation index to evaluate the anti-inflammatory activity of the total triterpenes of from lingonberry. 【Result】 The purification effect of seven macroporous resins on the total triterpenes of lingonberry was compared. The results showed that the X-5 resin was most suitable for the primary purification. The optimum purification process was the sample concentration of 1.5 mg/mL, elution at pH 6, loading volume 4/3 BV, loading flow rate of 1 BV/h, and 80% ethanol as eluant. The purity of the total triterpenes of lingonberry increased from 5.13% to 29.46%. Further purification using Sephadex LH-20 obtained a purity of(43.25±0.31) %. The total anti-inflammatory activity of the lingonberry was(81.5±1.37) % for the hyaluronidase and(72.59±1.84) % for the denaturation of bovine serum albumin. 【Conclusion】 Lingonberry is a nutrient-rich berry. High-purity triterpenoids were obtained through the secondary purification technology, and it was preliminarily showed that the total triterpenes of lingonberry had significant anti-inflammatory activity. Our results provided a certain theoretical basis for the further development and utilization of lingonberry.
引文
[1] BUJOR O C,GINIES C,POPA V I,et al.Phenolic compounds and antioxidant activity of lingonberry (Vaccinium vitis-idaea L.) leaf,stem and fruit at different harvest periods[J].Food Chemistry,2018,252:356-365.DOI:10.1016/j.foodchem.2018.01.052.
[2] SZAKIEL A,PACZKOWSKI C,KOIVUNIEMI H,et al.Comparison of the triterpenoid content of berries and leaves of lingonberry Vaccinium vitis-idaea from Finland and Poland[J].Journal of Agricultural and Food Chemistry,2012,60(19):4994-5002.DOI:10.1021/jf300375b.
[3] EK S,KARTIMO H,MATTILA S,et al.Characterization of phenolic compounds from lingonberry (Vaccinium vitis-idaea)[J].Journal of Agricultural and Food Chemistry,2006,54(26):9834-9842.DOI:10.1021/jf0623687.
[4] KURKIN V A,RYAZANOVA T K,PLATONOV I A,et al.Determination of arbutin in Vaccinium vitis-idaea L.leaves[J].Pharmaceutical Chemistry Journal,2017,51(4):281-284.DOI:10.1007/s11094-017-1598-z.
[5] FAN Z L,WANG Z Y,LIU J R.Cold-field fruit extracts exert different antioxidant and antiproliferative activities in vitro[J].Food Chemistry,2011,129(2):402-407.DOI:http://dx.doi.org/10.1016/j.foodchem.2011.04.091.
[6] ISAAK C K,PETKAU J C,O K,et al.Manitoba lingonberry (Vaccinium vitis-idaea) bioactivities in ischemia-reperfusion injury[J].Journal of Agricultural and Food Chemistry,2015,63(23):5660-5669.DOI:10.1021/acs.jafc.5b00797.
[7] ALY E M,ALI M A.Effects of bilberry on deoxyribonucleic acid damage and oxidant-antioxidant balance in the lens,induced by ultraviolet radiation[J].The Malaysian Journal of Medical Sciences,2014,21(1):11.
[8] 樊梓鸾,王振宇.红豆越橘体外抗氧化和抗细胞增殖活性研究[J].现代食品科技,2010,26(10):1081-1086.DOI:10.13982/j.mfst.1673-9078.2010.10.032.FAN Z L,WANG Z Y.Antioxidant and antiproliferative activities of lingonberries in vitro[J].Modern Food Science and Technology,2010,26(10):1081-1086.
[9] KIVIMAKI A S,SILTARI A,EHLERS P I,et al.Lingonberry juice lowers blood pressure of spontaneously hypertensive rats (SHR)[J].Journal of Functional Foods,2013,5(3):1432-1440.DOI:10.1016/j.jff.2013.05.012.
[10] TORRONEN R,KOLEHMAINEN M,SARKKINEN E,et al.Postprandial glucose,insulin,and free fatty acid responses to sucrose consumed with blackcurrants and lingonberries in healthy women[J].The American Journal of Clinical Nutrition,2012,96(3):527-533.DOI:10.3945/ajcn.112.042184.
[11] ZAMORA-ROS R,KNAZE V,LUJAN-BARROSO L,et al.Estimation of the intake of anthocyanidins and their food sources in the European Prospective Investigation into Cancer and Nutrition (EPIC) study[J].British Journal of Nutrition,2011,106(7):1090-1099.DOI:10.1017/S0007114511001437.
[12] GUTIERREZ R M P.Anti-inflammatory effect of birsonimadiol from seeds of Byrsonima crassifolia[J].Food Science and Biotechnology,2016,25(2):561-566.DOI:10.1007/s10068-016-0078-7.
[13] AYATOLLAHI A M,ZAREI S M,MEMARNEJADIAN A,et al.Triterpene constituents of Euphorbia erythradenia Bioss.and their anti-HIV activity[J].Iranian Journal of Pharmaceutical Research,2016,15:19-27.
[14] PAGNING A L N,LATEEF M,TAPONDJOU L A,et al.New triterpene and new flavone glucoside from Rhynchospora corymbosa (Cyperaceae) with their antimicrobial,tyrosinase and butyrylcholinesterase inhibitory activities[J].Phytochemistry Letters,2016,16:121-128.DOI:http://dx.doi.org/10.1016/j.phytol.2016.03.011.
[15] SUDHAHAR V,KUMAR S A,SUDHARSAN P T,et al.Protective effect of lupeol and its ester on cardiac abnormalities in experimental hypercholesterolemia[J].Vascular Pharmacology,2007,46(6):412-418.DOI:10.1016/j.vph.2006.12.005.
[16] JANG H J,HAN I H,KIM Y J,et al.Anticarcinogenic effects of products of heat-processed ginsenoside Re,a major constituent of ginseng berry,on human gastric cancer cells[J].Journal of Agricultural and Food Chemistry,2014,62(13):2830-2836.DOI:10.1021/jf5000776.
[17] 朱安运,夏嫱,李小波,等.栀子与西红花中西红花苷纯化、分析及构效关系研究进展[J].药物分析杂志,2018,38(05):735-747.DOI:10.16155/j.0254-1793.2018.05.01.ZHU A Y,XIA Q,LI X B,et al.Research progress of purification,determination and structure-activity relationship of crocins from gardenia and saffron [J].Chin J Pharm Anal,2018,38(5):735-747.
[18] 赵瑜,陈波,罗旭彪,等.大孔吸附树脂分离纯化人参二醇类和三醇类皂甙[J].天然产物研究与开发,2004,16(3):235-238.DOI:10.16333/j.1001-6880.2004.03.017.ZHAO Y,CHEN B,LUO X B,et al.Study on separation and purification process of panaxdiol type ginsenosides and panaxtriol type ginsenosides with Macroporous adsorption resin[J].Natural Product Resarch and Development,2004,16(3):235-238.
[19] JIA G,LU X.Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins[J].Journal of Chromatography A,2008,1193(1):136-141.DOI:http://dx.doi.org/10.1016/j.chroma.2008.04.024.
[20] KUMARI A,KAKKAR P.Lupeol protects against acetaminophen-induced oxidative stress and cell death in rat primary hepatocytes[J].Food and Chemical Toxicology,2012,50(5):1781-1789.DOI:10.1016/j.fct.2012.02.042.
[21] JAMUNA S,KARTHIKA K,PAULSAMY S,et al.Confertin and scopoletin from leaf and root extracts of Hypochaeris radicata have anti-inflammatory and antioxidant activities[J].Industrial Crops and Products,2015,70:221-230.DOI:10.1016/j.indcrop.2015.03.039.
[22] SAMEE H,LI Z,LIN H,et al.Anti-allergic effects of ethanol extracts from brown seaweeds[J].Journal of Zhejiang University Science B,2009,10(2):147-153.DOI:10.1631/jzus.B0820185.
[23] BOUGANDOURA A,D’ABROSCA B,AMEDDAH S,et al.Chemical constituents and in vitro anti-inflammatory activity of Cistanche violacea Desf.(Orobanchaceae) extract[J].Fitoterapia,2016,109:248-253.DOI:10.1016/j.fitote.2016.01.010.
[24] 沈思,李孚杰,梅光明,等.茯苓皮三萜类物质含量的测定及其抑菌活性的研究[J].食品科学,2009,30(1):95-98..SHEN S,LI F J,MEI G M,et al.Study on content detemrination and antibacterial ecffets of trietprenes from seleortia of Poria cocos[J].Food Science,2009,30(1):95-98.
[25] 张银堂,张彦峥,陈芳,等.化学计量学-荧光光谱法研究贝诺酯和双氯芬酸钠与牛血清白蛋白的相互作用[J].分析化学,2009,37(A01):220-220.ZHANG Y T,ZHANG Y Z,CHEN F,et al.Chemometrics- fluorescence spectroscopy study of the interaction between benoxidate and diclofenac sodium and bovine serum albumin[J].Analytical Chemistry,2009,37(A01):220-220.
[26] 马涛,林峰,林单,等.食源性低聚肽抗过敏活性研究[J].食品科技,2014,39(2):27-30..MA T,LIN F,LIN D,et al.Antiallergic activity of food-derived oligopeptides[J].Food Science and Technology,2014,39(2):27-30.
[27] 王维有,曹晨晨,欧赟,等.大枣中环磷酸腺苷的提取及体外抗过敏活性研究[J].食品工业科技,2013,34(11):49-52.DOI:10.13386/j.issn1002-0306.2013.11.069.WANG W Y,CAO C C,OU Y,et al.Extraction of cyclic adenosine monophosphate and antiallergic activity from jujube in vitro[J].Food Industry Technology,2013,34(11):49-52.
[2] SZAKIEL A,PACZKOWSKI C,KOIVUNIEMI H,et al.Comparison of the triterpenoid content of berries and leaves of lingonberry Vaccinium vitis-idaea from Finland and Poland[J].Journal of Agricultural and Food Chemistry,2012,60(19):4994-5002.DOI:10.1021/jf300375b.
[3] EK S,KARTIMO H,MATTILA S,et al.Characterization of phenolic compounds from lingonberry (Vaccinium vitis-idaea)[J].Journal of Agricultural and Food Chemistry,2006,54(26):9834-9842.DOI:10.1021/jf0623687.
[4] KURKIN V A,RYAZANOVA T K,PLATONOV I A,et al.Determination of arbutin in Vaccinium vitis-idaea L.leaves[J].Pharmaceutical Chemistry Journal,2017,51(4):281-284.DOI:10.1007/s11094-017-1598-z.
[5] FAN Z L,WANG Z Y,LIU J R.Cold-field fruit extracts exert different antioxidant and antiproliferative activities in vitro[J].Food Chemistry,2011,129(2):402-407.DOI:http://dx.doi.org/10.1016/j.foodchem.2011.04.091.
[6] ISAAK C K,PETKAU J C,O K,et al.Manitoba lingonberry (Vaccinium vitis-idaea) bioactivities in ischemia-reperfusion injury[J].Journal of Agricultural and Food Chemistry,2015,63(23):5660-5669.DOI:10.1021/acs.jafc.5b00797.
[7] ALY E M,ALI M A.Effects of bilberry on deoxyribonucleic acid damage and oxidant-antioxidant balance in the lens,induced by ultraviolet radiation[J].The Malaysian Journal of Medical Sciences,2014,21(1):11.
[8] 樊梓鸾,王振宇.红豆越橘体外抗氧化和抗细胞增殖活性研究[J].现代食品科技,2010,26(10):1081-1086.DOI:10.13982/j.mfst.1673-9078.2010.10.032.FAN Z L,WANG Z Y.Antioxidant and antiproliferative activities of lingonberries in vitro[J].Modern Food Science and Technology,2010,26(10):1081-1086.
[9] KIVIMAKI A S,SILTARI A,EHLERS P I,et al.Lingonberry juice lowers blood pressure of spontaneously hypertensive rats (SHR)[J].Journal of Functional Foods,2013,5(3):1432-1440.DOI:10.1016/j.jff.2013.05.012.
[10] TORRONEN R,KOLEHMAINEN M,SARKKINEN E,et al.Postprandial glucose,insulin,and free fatty acid responses to sucrose consumed with blackcurrants and lingonberries in healthy women[J].The American Journal of Clinical Nutrition,2012,96(3):527-533.DOI:10.3945/ajcn.112.042184.
[11] ZAMORA-ROS R,KNAZE V,LUJAN-BARROSO L,et al.Estimation of the intake of anthocyanidins and their food sources in the European Prospective Investigation into Cancer and Nutrition (EPIC) study[J].British Journal of Nutrition,2011,106(7):1090-1099.DOI:10.1017/S0007114511001437.
[12] GUTIERREZ R M P.Anti-inflammatory effect of birsonimadiol from seeds of Byrsonima crassifolia[J].Food Science and Biotechnology,2016,25(2):561-566.DOI:10.1007/s10068-016-0078-7.
[13] AYATOLLAHI A M,ZAREI S M,MEMARNEJADIAN A,et al.Triterpene constituents of Euphorbia erythradenia Bioss.and their anti-HIV activity[J].Iranian Journal of Pharmaceutical Research,2016,15:19-27.
[14] PAGNING A L N,LATEEF M,TAPONDJOU L A,et al.New triterpene and new flavone glucoside from Rhynchospora corymbosa (Cyperaceae) with their antimicrobial,tyrosinase and butyrylcholinesterase inhibitory activities[J].Phytochemistry Letters,2016,16:121-128.DOI:http://dx.doi.org/10.1016/j.phytol.2016.03.011.
[15] SUDHAHAR V,KUMAR S A,SUDHARSAN P T,et al.Protective effect of lupeol and its ester on cardiac abnormalities in experimental hypercholesterolemia[J].Vascular Pharmacology,2007,46(6):412-418.DOI:10.1016/j.vph.2006.12.005.
[16] JANG H J,HAN I H,KIM Y J,et al.Anticarcinogenic effects of products of heat-processed ginsenoside Re,a major constituent of ginseng berry,on human gastric cancer cells[J].Journal of Agricultural and Food Chemistry,2014,62(13):2830-2836.DOI:10.1021/jf5000776.
[17] 朱安运,夏嫱,李小波,等.栀子与西红花中西红花苷纯化、分析及构效关系研究进展[J].药物分析杂志,2018,38(05):735-747.DOI:10.16155/j.0254-1793.2018.05.01.ZHU A Y,XIA Q,LI X B,et al.Research progress of purification,determination and structure-activity relationship of crocins from gardenia and saffron [J].Chin J Pharm Anal,2018,38(5):735-747.
[18] 赵瑜,陈波,罗旭彪,等.大孔吸附树脂分离纯化人参二醇类和三醇类皂甙[J].天然产物研究与开发,2004,16(3):235-238.DOI:10.16333/j.1001-6880.2004.03.017.ZHAO Y,CHEN B,LUO X B,et al.Study on separation and purification process of panaxdiol type ginsenosides and panaxtriol type ginsenosides with Macroporous adsorption resin[J].Natural Product Resarch and Development,2004,16(3):235-238.
[19] JIA G,LU X.Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins[J].Journal of Chromatography A,2008,1193(1):136-141.DOI:http://dx.doi.org/10.1016/j.chroma.2008.04.024.
[20] KUMARI A,KAKKAR P.Lupeol protects against acetaminophen-induced oxidative stress and cell death in rat primary hepatocytes[J].Food and Chemical Toxicology,2012,50(5):1781-1789.DOI:10.1016/j.fct.2012.02.042.
[21] JAMUNA S,KARTHIKA K,PAULSAMY S,et al.Confertin and scopoletin from leaf and root extracts of Hypochaeris radicata have anti-inflammatory and antioxidant activities[J].Industrial Crops and Products,2015,70:221-230.DOI:10.1016/j.indcrop.2015.03.039.
[22] SAMEE H,LI Z,LIN H,et al.Anti-allergic effects of ethanol extracts from brown seaweeds[J].Journal of Zhejiang University Science B,2009,10(2):147-153.DOI:10.1631/jzus.B0820185.
[23] BOUGANDOURA A,D’ABROSCA B,AMEDDAH S,et al.Chemical constituents and in vitro anti-inflammatory activity of Cistanche violacea Desf.(Orobanchaceae) extract[J].Fitoterapia,2016,109:248-253.DOI:10.1016/j.fitote.2016.01.010.
[24] 沈思,李孚杰,梅光明,等.茯苓皮三萜类物质含量的测定及其抑菌活性的研究[J].食品科学,2009,30(1):95-98..SHEN S,LI F J,MEI G M,et al.Study on content detemrination and antibacterial ecffets of trietprenes from seleortia of Poria cocos[J].Food Science,2009,30(1):95-98.
[25] 张银堂,张彦峥,陈芳,等.化学计量学-荧光光谱法研究贝诺酯和双氯芬酸钠与牛血清白蛋白的相互作用[J].分析化学,2009,37(A01):220-220.ZHANG Y T,ZHANG Y Z,CHEN F,et al.Chemometrics- fluorescence spectroscopy study of the interaction between benoxidate and diclofenac sodium and bovine serum albumin[J].Analytical Chemistry,2009,37(A01):220-220.
[26] 马涛,林峰,林单,等.食源性低聚肽抗过敏活性研究[J].食品科技,2014,39(2):27-30..MA T,LIN F,LIN D,et al.Antiallergic activity of food-derived oligopeptides[J].Food Science and Technology,2014,39(2):27-30.
[27] 王维有,曹晨晨,欧赟,等.大枣中环磷酸腺苷的提取及体外抗过敏活性研究[J].食品工业科技,2013,34(11):49-52.DOI:10.13386/j.issn1002-0306.2013.11.069.WANG W Y,CAO C C,OU Y,et al.Extraction of cyclic adenosine monophosphate and antiallergic activity from jujube in vitro[J].Food Industry Technology,2013,34(11):49-52.