酶-超声辅助提取杨树皮总黄酮及其表征
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摘要
为优化杨树皮总黄酮的提取方法,并对提取出的黄酮单体进行结构鉴定。笔者通过单因素试验对影响杨树皮总黄酮得率的各因素进行优化,在此基础上采用Design-Expert 8.0.6软件Box-Behnken模块进行响应面试验条件优化,分析酶量、浸提液p H、酶解时间、酶解温度4个因素及各因素间交互作用对总黄酮得率的影响,优化酶-超声辅助提取其总黄酮的工艺条件,提取液经萃取分离纯化得到质量分数为95%以上的单体,同时鉴定其结构。结果表明,经过优化后的最佳工艺条件为:原料杨树皮1 g,每克杨树皮在加酶量37 mg、p H 4.6、酶解时间1.75 h、酶解温度56℃下浸提后再于40℃、功率160 W下超声处理30 min,此条件下黄酮类化合物得率可达4.56%。提取液萃取后经AB-8大孔树脂初步纯化,由乙醇/水梯度洗脱,取乙醇和水体积比为1∶1时分离得到的组分进行葡聚糖凝胶LH-20分离纯化。在葡聚糖凝胶LH-20中用质量分数95%乙醇等度洗脱,合并浓缩相同组分,并结合制备液相色谱仪进一步纯化。采用C_(18)色谱柱,流动相为甲醇-水(含体积分数0.01%甲酸),梯度洗脱,紫外检测波长为280 nm,流速1 m L/min,进样量5μL,建立杨树皮总黄酮的高效液相色谱(HPLC)分析方法。通过液质联用(HPLC-MS)分析、核磁共振(~1H NMR、~(13)C NMR)鉴定该组分为5,7,4',5'-四羟基-3'-甲氧基黄烷醇。本研究结果既可为杨树皮的基础研究提供帮助,也可为杨树皮中功能物质的开发提供借鉴。
In order to optimize the extraction method of total flavonoids from poplar bark and identify the structure of flavone monomers extracted from it,single factor experiment was used to optimize the factors affecting the extraction yield of total flavonoids from poplar bark in this study. The optimum reaction conditions were the weight of cellulase40 mg per gram of poplar bark,pH 5 of extract,50 ℃ of enzymatic temperature and 1.5 h of enzymatic time. The response surface experiment was then optimized by using the Box-Behnken module of the Design-Expert 8.0.6 software.The influence of the weight of cellulase,pH,enzymolysis time,enzymolysis temperature and the interaction between these factors on the yield was investigated. The compound was extracted,separated and purified from the extract with content of 95% which was identified chemical structure. The optimal parameters of total flavonoids were matured poplar bark 1 g,weight of cellulase 37 mg per gram of poplar bark,pH 4.6, enzymolysis time 1.75 h,enzymolysis temperature56 ℃,ultrasonic temperature 40 ℃,ultrasonic time 30 min and ultrasonic power 160 W. The maximum extraction yield of total flavonoids was 4.56%. The extract was purified by AB-8 macroporous resin and got gradient elution with ethanol/water. When the volume ratio of ethanol to water was 1 ∶1,the separated components were separated and purified by Sephadex LH-20. In the Sephadex LH-20,95% ethanol was used to elute and then the same components were consolidated and concentrated,and it was further purified by preparative liquid chromatograph. It was carried out with column C_(18) and MeOH-water( with 0.01% formic acid,volume ratio) as gradient elution at flow rate 1 mL/min, injection volume 5 μL and monitored at 280 nm to establish a method for the determination of total flavonoids in poplar bark by high performance liquid chromatography( HPLC). The compound was identified as 5,7,4',5'-tetrahydroxy-3'-methoxyflavanonol by HPLC-MS,~1 H NMR and ~(13)C NMR. This study not only provides help forthe basic research of poplar bark,but also provides a reference for the development of functional substances in poplar bark.
In order to optimize the extraction method of total flavonoids from poplar bark and identify the structure of flavone monomers extracted from it,single factor experiment was used to optimize the factors affecting the extraction yield of total flavonoids from poplar bark in this study. The optimum reaction conditions were the weight of cellulase40 mg per gram of poplar bark,pH 5 of extract,50 ℃ of enzymatic temperature and 1.5 h of enzymatic time. The response surface experiment was then optimized by using the Box-Behnken module of the Design-Expert 8.0.6 software.The influence of the weight of cellulase,pH,enzymolysis time,enzymolysis temperature and the interaction between these factors on the yield was investigated. The compound was extracted,separated and purified from the extract with content of 95% which was identified chemical structure. The optimal parameters of total flavonoids were matured poplar bark 1 g,weight of cellulase 37 mg per gram of poplar bark,pH 4.6, enzymolysis time 1.75 h,enzymolysis temperature56 ℃,ultrasonic temperature 40 ℃,ultrasonic time 30 min and ultrasonic power 160 W. The maximum extraction yield of total flavonoids was 4.56%. The extract was purified by AB-8 macroporous resin and got gradient elution with ethanol/water. When the volume ratio of ethanol to water was 1 ∶1,the separated components were separated and purified by Sephadex LH-20. In the Sephadex LH-20,95% ethanol was used to elute and then the same components were consolidated and concentrated,and it was further purified by preparative liquid chromatograph. It was carried out with column C_(18) and MeOH-water( with 0.01% formic acid,volume ratio) as gradient elution at flow rate 1 mL/min, injection volume 5 μL and monitored at 280 nm to establish a method for the determination of total flavonoids in poplar bark by high performance liquid chromatography( HPLC). The compound was identified as 5,7,4',5'-tetrahydroxy-3'-methoxyflavanonol by HPLC-MS,~1 H NMR and ~(13)C NMR. This study not only provides help forthe basic research of poplar bark,but also provides a reference for the development of functional substances in poplar bark.
引文
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[15]黄道榜,王威,陈勇花,等.基于响应面法的重组竹硅铝溶胶防霉剂研究[J].林业工程学报,2018,3(3):29-34.DOI:10.13360/j.issn.2096-1359.2018.03.005.HUANG D B,WANG W,CHEN Y H,et al.Optimization of silicaalumina sol anti-mold agent of bamboo scrimber using response surface methodology[J].Journal of Forestry Engineering,2018,3(3):29-34.
[16]张爱华,易智彪,吴红,等.响应面法优化钙基固体碱KF/CaO催化沉香籽油制备生物柴油[J].林业工程学报,2016,1(5):89-94.DOI:10.13360/j.issn.2096-1359.2016.05.016.ZHANG A H,YI Z B,WU H,et al.Optimized preparation of biodiesel from KF/CaO-catalysed Aquilaria sinensis seed oil using response surface method[J].Journal of Forestry Engineering,2016,1(5):89-94.
[17]ZHANG Y S,QUE S,YANG X W,et al.Isolation and identification of metabolites from dihydromyricetin[J].Magnetic Resonance in Chemistry,2007,45(11):909-916.DOI:10.1002/mrc.2051.
[2]WEI M Y,MA Y H,LIU Y Y,et al.Urinary metabolomics study on the anti-inflammation effects of flavonoids obtained from Glycyrrhiza[J].Journal of Chromatography B,2018,1086:1-10.DOI:10.1016/j.jchromb.2018.04.007.
[3]张英强,李多伟,智旭亮.苦荞麦黄酮和蛋白质提取工艺的优化[J].天然产物研究与开发,2016,28(2):293-299.DOI:10.16333/j.1001-6880.2016.2.023.ZHANG Y Q,LI D W,ZHI X L.Optimization of extraction of flavonoid and protein from tartarybuckwheat[J].Natural Product Research and Development,2016,28(2):293-299.
[4]LIU Y H,MOU X,ZHOU D Y,et al.Extraction of flavonoids from Chrysanthemum morifolium and antitumor activity in vitro[J].Experimental and Therapeutic Medicine,2018,15(2):1203-1210.DOI:10.3892/etm.2017.5574.
[5]HUANG P,ZHANG Q,PAN H Y,et al.Optimization of integrated extraction-adsorption process for the extraction and purification of total flavonoids from Scutellariae barbatae herba[J].Separation and Purification Technology,2017,175:203-212.DOI:10.1016/j.seppur.2016.11.031.
[6]李侠,邹基豪,王大为.响应面试验优化超声波-酶法提取绿豆皮黄酮类化合物工艺[J].食品科学,2017,38(8):206-212.DOI:10.7506/spkx1002-6630-201708032.LI X,ZOU J H,WANG D W.Optimization of ultrasonic-assisted enzymatic extraction of flavonoids from mung beancoat[J].Food Science,2017,38(8):206-212.
[7]张翠平,胡福良.杨属植物化学成分的研究概况[J].天然产物研究与开发,2012,24(S1):165-168.DOI:10.16333/j.1001-6880.2012.s1.033.ZHANG C P,HU F L.Review on chemical constituents of Populus species[J].Natural Product Research and Development,2012,24(S1):165-168.
[8]侯莹莹,赵岩,唐国胜,等.RP-HPLC同时测定杨树花和杨树叶中3个植物甾醇含量[J].药物分析杂志,2014,34(12):2150-2153.DOI:10.16155/j.0254-1793.2014.12.009.HOU Y Y,ZHAO Y,TANG G S,et al.Determination of three phytosterols in Flos Populi Canadensis and leaves of Populus canadensis Moench by RP-HPLC[J].Chinese Journal of Pharmaceutical Analysis,2014,34(12):2150-2153.
[9]POBLOCKA-OLECH L,MIGAS P,KRAUZE-BARANOWSKAM.TLC determination of some flavanones in the buds of different genus Populus species and hybrids[J].Acta Pharmaceutica,2018,68(2):199-210.DOI:10.2478/acph-2018-0018.
[10]KUS'P M,OKIN'CZYC P,JAKOVLJEVIC'M,et al.Development of supercritical CO2 extraction of bioactive phytochemicals from black poplar(Populus nigra L.)buds followed by GC-MS and UHPLC-DAD-QqTOF-MS[J].Journal of Pharmaceutical and Biomedical Analysis,2018,158:15-27.DOI:10.1016/j.jpba.2018.05.041.
[11]张小芸.杨树皮原花色素提取与生物活性研究[D].南京:南京林业大学,2011.ZHANG X Y.Study on biological activities of proanthocyanidins extract from black poplar(Populus deltoides)bark[D].Nanjing:Nanjing Forestry University,2011.
[12]郭玉玉,王玲,罗金岳,等.超临界CO2提取油橄榄叶中橄榄苦苷及其结构鉴定[J].林产化学与工业,2015,35(2)73-78.DOI:10.3969/j.issn.0253-2417.2015.02.011.GUO Y Y,WANG L,LUO J Y,et al.Extraction and structure identification of oleuropein from olea europaea leaves by supercritical carbon dioxide extraction technology[J].Chemistry and Industry of Forest Products,2015,35(2):73-78.
[13]王新,肖凯军.纤维素酶-超声辅助提取麦麸总黄酮的工艺研究[J].食品科技,2009,34(12):230-234.DOI:10.13684/j.cnki.spkj.2009.12.075.WANG X,XIAO K J.Study on total flavones extraction from wheat bran under the intergrated treatment with ultrasonic and cellulase[J].Food Science and Technology,2009,34(12):230-234.
[14]李会端,乔爱平.酶辅助法提取透骨草中总黄酮及抗氧化性研究[J].天然产物研究与开发,2015,27(3):432-437.DOI:10.16333/j.1001-6880.2015.03.012.LI H D,QIAO A P.Enzyme-assisted extraction of total flavonoids from speranskia and study on antioxidant effects[J].Natural Product Research and Development,2015,27(3):432-437.
[15]黄道榜,王威,陈勇花,等.基于响应面法的重组竹硅铝溶胶防霉剂研究[J].林业工程学报,2018,3(3):29-34.DOI:10.13360/j.issn.2096-1359.2018.03.005.HUANG D B,WANG W,CHEN Y H,et al.Optimization of silicaalumina sol anti-mold agent of bamboo scrimber using response surface methodology[J].Journal of Forestry Engineering,2018,3(3):29-34.
[16]张爱华,易智彪,吴红,等.响应面法优化钙基固体碱KF/CaO催化沉香籽油制备生物柴油[J].林业工程学报,2016,1(5):89-94.DOI:10.13360/j.issn.2096-1359.2016.05.016.ZHANG A H,YI Z B,WU H,et al.Optimized preparation of biodiesel from KF/CaO-catalysed Aquilaria sinensis seed oil using response surface method[J].Journal of Forestry Engineering,2016,1(5):89-94.
[17]ZHANG Y S,QUE S,YANG X W,et al.Isolation and identification of metabolites from dihydromyricetin[J].Magnetic Resonance in Chemistry,2007,45(11):909-916.DOI:10.1002/mrc.2051.