CN112138057B - 杨树芽提取物在制备脂肪氧合酶抑制剂中的用途 - Google Patents
杨树芽提取物在制备脂肪氧合酶抑制剂中的用途 Download PDFInfo
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Abstract
本发明公开了杨树芽提取物在制备脂肪氧合酶抑制剂中的用途，本发明还公开了一种杨树芽提取物的制备方法。本发明首次提供了杨树芽提取物在制备脂肪氧合酶抑制剂中的用途，利用该用途，可将杨树芽提取物制成食品添加剂，用于改善果蔬等加工制品的风味口感，防止含油食物发生劣变，延长其贮藏期，另外还有可能用于制备哮喘、过敏性鼻炎等治疗药物，用于缓解过敏症状等，具有非常广泛的应用前景。
Description
技术领域
本发明涉及植物提取物的应用，具体涉及杨树芽提取物作为脂肪氧合酶抑制剂的新用途。
背景技术
脂肪氧合酶(lipoxygenase，LOX)又称脂氧化酶，是一组具有氧化还原性的非血红素铁蛋白，能够专一性地催化含顺，顺-1，4-戊二烯结构的不饱和脂肪酸(或酯)发生加氧反应，生成具有共轭双键的氢过氧化物。LOX存在于各种植物中，如谷类种子、大豆、豌豆、马铃薯中，以大豆中的含量最高。食物体系中存在脂氧合酶可造成食物营养质量的下降及增加贮藏的困难，可导致果蔬加工制品产生不良风味，油脂和含油食物在贮藏和加工过程中发生劣变等。
研究还发现，脂肪氧合酶参与的花生四烯酸-白三烯类代谢产物白三烯类、血栓素等物质，能致使机体发生疼痛、血管扩张、血管通透性增强及组织水肿等症状，甚至诱导其他炎性疾病及过敏反应的产生。因此，脂肪氧合酶被认为是引起哮喘、过敏性鼻炎等疾病发生的重要因素。
杨树芽是杨属植物尚未充分发育生长的枝条或花序，其分泌的芽脂是我国杨树型蜂胶的主要胶源物质，富含咖啡酸、对香豆酸、槲皮素、山奈酚、异鼠李素等多种黄酮、酚酸类活性化合物，但目前对于杨树芽提取物的药用和食用价值尚未得到充分利用。鉴于此，研究设计一种杨树芽提取物作为脂肪氧合酶抑制剂的用途，可以为杨树芽提取物的深层次开发利用提高理论依据。
发明内容
本发明的目的是提供一种杨树芽提取物在制备脂肪氧合酶抑制剂中的用途，本发明的另一个目的是一种杨树芽提取物的制备方法，采用该方法制备的杨树芽提取物具有更好的活性。
为了实现上述目的，本发明所采取的技术方案如下：
首先，申请人制备了杨树芽粗提取物和不同浓度乙醇的大孔树脂洗脱组分，通过对杨树芽粗提取物和各洗脱组分进行高效液相色谱分析，结果发现80％洗脱组分的成分与30％、50％差别较大，于是利用杨树芽粗提取物以及50％、80％洗脱组分作为研究对象考察了提取物对脂肪氧合酶的抑制活性，结果发现，各提取物对脂肪氧合酶均具有一定的抑制活性，其中80％洗脱组分对脂肪氧合酶的抑制效果最佳，且经由AB-8型大孔树脂纯化得到的杨树芽提取物活性高于XAD-2大孔树脂，进一步选择该组份对其抑制机理进行探究，发现该组份对脂肪氧合酶的抑制类型为可逆性、混合型抑制。
以上结果表明：杨树芽提取物可用于制备脂肪氧合酶抑制剂，可在体内或体外发挥对脂肪氧合酶的抑制作用。
本发明进一步提供了一种杨树芽提取物的制备方法，包括以下步骤：
(1)将杨树芽粉碎后石油醚脱脂，然后按照料液重量体积比1：5-20加入60-80％乙醇，超声辅助提取1-3次，每次提取时间为30-120min，过滤，将提取液减压浓缩并干燥；得到杨树芽粗提取物；
(2)将干燥后的杨树芽粗提取物溶解后上AB-8型大孔树脂柱，充分吸附后依次用蒸馏水和50％乙醇洗脱，然后用80％乙醇洗脱，收集80％乙醇洗脱液并浓缩干燥，即得杨树芽提取物。
优选地，步骤(1)中，按照料液重量体积比1：10加入75％乙醇，超声辅助提取3次，每次提取时间为60min。
采用该方法制备的杨树芽提取物具有更好的活性。
本发明中所涉及到的乙醇浓度，均为体积浓度，如80％乙醇指的是每100ml乙醇水溶液中含有乙醇80ml。
本发明的有益效果是：本发明首次提供了杨树芽提取物在制备脂肪氧合酶抑制剂中的用途，利用该用途，可将杨树芽提取物制成食品添加剂，用于改善果蔬等加工制品的风味口感，防止含油食物发生劣变，延长其贮藏期，另外还有可能用于制备哮喘、过敏性鼻炎等治疗药物，用于缓解过敏症状等，具有非常广泛的应用前景。
附图说明
图1是不同浓度乙醇洗脱组分对杨树芽乙醇粗提物的分离效果图，A-D分别代表杨树芽粗提取物、AB-8 30％洗脱物、AB-8 50％洗脱物、AB-8 80％洗脱物。
图2是杨树芽AB-8 80％洗脱物对脂肪氧合酶可逆性抑制曲线图。
图3是杨树芽AB-8 80％洗脱物对脂肪氧合酶抑制类型的试验结果。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
实施例1杨树芽乙醇粗提物和不同级分洗脱产物的制备方法
将杨树芽用粉碎机粉碎后，取10g粉碎后的杨树芽于烧杯，加入适量的石油醚进行超声脱脂，重复3次，随后将脱脂后的杨树芽置于通风橱过夜放置，使残留的石油醚挥发。按照1:10的料液重量体积比加入100mL 75％乙醇，超声辅助提取3次，每次60min，抽滤得到澄清的乙醇粗提液，并将其进行旋蒸浓缩、冷冻干燥获得杨树芽粗提取物。称取一定量的杨树芽粗提取物，用乙醇溶解配制成浓度为10mg/mL的粗提液，将其与AB-8大孔树脂混合并磁力搅拌2h使其充分吸附，随后用蒸馏水冲洗去除糖类和其他水溶性物质，接着依次用30％、50％、80％乙醇进行洗脱，分别收集三种浓度的洗脱液并将其旋蒸浓缩，冻干得到杨树芽30％、50％、80％三个洗脱组分。利用高效液相色谱对3个洗脱组分进行分离效果的分析，结果如图1所示。从图中可以看出，30％、50％两个组分的色谱图比较接近，说明二者所含化合物的极性相当，而80％洗脱组分的色谱图与30％、50％差别较大，说明所含化合物是与前两组极性完全不同的片段，因此在后续脂氧氧合酶抑制实验中选择杨树芽粗提取物以及50％、80％洗脱组分作为研究对象。
实施例2
为了验证杨树芽提取物对脂肪氧合酶的抑制效果，并对杨树芽中抑制脂肪氧合酶的高活性片段进行筛选，以亚油酸为反应底物，采用Fe2+氧化-二甲酚橙显色法测定杨树芽提取物对脂肪氧合酶的半抑制浓度。为进一步研究杨树芽提取物对脂肪氧合酶的抑制机理，利用酶促反应动力学对脂肪氧合酶抑制可逆性和抑制类型进行判断，具体如下。
(1)将本发明提取得到的杨树芽提取物和不同洗脱组分进行脂肪氧合酶半抑制浓度测定，同时对比分析了前人利用XAD-2大孔树脂分离纯化得到的分离物对脂肪氧合酶的抑制活性。测定结果如表1所示：可以发现，经由AB-8大孔树脂吸附，80％乙醇洗脱得到的洗脱组分，在所有测试对象中具有最低的脂肪氧合酶半抑制浓度，说明该组份对脂肪氧合酶的抑制效果最佳。
表1不同方式得到的杨树芽提取物对脂肪氧合酶的IC50
| 样品名称 | IC<sub>50</sub>(μg/ml) |
| 杨树芽粗提取物 | 69.40±3.31<sup>b</sup> |
| AB-8 50％ | 75.84±1.14<sup>c</sup> |
| AB-8 80％ | 56.24±3.16<sup>a</sup> |
| XAD-2 50％ | 64.10±0.70<sup>d</sup> |
| XAD-2 80％ | 61.48±1.84<sup>d</sup> |
注：每一行中不同字母代表在统计学上具有显著性差异(p＜0.05)。
(2)固定底物亚油酸的浓度，改变反应体系中杨树芽提取物和脂肪氧合酶的浓度，测定酶浓度与其活力之间的关系。由图2可知，不同浓度的杨树芽80％洗脱组分与不同浓度的脂肪氧合酶酶促反应初速率呈线性关系，并交于原点，且随杨树芽80％洗脱组分浓度的增大，反应初速率逐渐降低，说明杨树芽80％洗脱组分对脂肪氧合酶的抑制作用为可逆性抑制。
(3)固定脂肪氧合酶浓度，改变反应体系中杨树芽80％洗脱组分和底物亚油酸的浓度，通过测定单位时间内OD234nm的变化，得到脂肪氧合酶的酶促反应初速率，并根据Lineweaver-Burk双倒数法，以底物亚油酸浓度的倒数1/[S]为横坐标，反应初速率1/[V]为纵坐标作图，判断脂肪氧合酶抑制类型。由图3可知，三条直线相交于第二象限，并且随着80％洗脱组分浓度的增加，米氏常数(Km)和最大反应速率(Vmax)均发生了变化，抑制类型表现为混合型抑制。
结论分析：
杨树芽粗提取物及其不同浓度乙醇洗脱组分均具有较好的脂肪氧合酶抑制活性，其中经由AB-8大孔树脂吸附，80％乙醇洗脱得到的组分对脂肪氧合酶的抑制效果最佳，并且优于经由XAD-2大孔树脂纯化得到的杨树芽提取物，说明80％组份是杨树芽提取物发挥其脂肪氧合酶抑制活性的主要部位，故而可将该组份作为脂肪氧合酶抑制剂进行深入开发。同时选择该组份对其抑制机理进行探究，发现该组份对脂肪氧合酶的抑制类型为可逆性、混合型抑制。
以上所述，仅为本发明较佳实施例，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
Claims (2)
1.杨树芽提取物在制备非治疗目的的脂肪氧合酶抑制剂中的用途，所述杨树芽提取物的制备方法包括以下步骤：
（1）将杨树芽粉碎后石油醚脱脂，然后按照料液重量体积比1：5-20加入60-80%乙醇，超声辅助提取1-3次，每次提取时间为30-120min，过滤，将提取液减压浓缩并干燥；得到杨树芽粗提取物；
（2）将干燥后的杨树芽粗提取物溶解后上AB-8型大孔树脂柱，充分吸附后依次用蒸馏水和50%乙醇洗脱，然后用80%乙醇洗脱，收集80%乙醇洗脱液并浓缩干燥，即得杨树芽提取物。
2.如权利要求1所述的用途，其特征在于：步骤（1）中，按照料液重量体积比1：10加入75%乙醇，超声辅助提取3次，每次提取时间为60min。
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