CN109223878A - 一种苹果枝条活性提取物及其提取方法与应用 - Google Patents
一种苹果枝条活性提取物及其提取方法与应用 Download PDFInfo
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Abstract
本发明涉及一种苹果树枝条活性提取物及其提取方法与应用，属于天然药物化学领域，所述方法包含如下步骤：将苹果树枝条粉碎过筛，用醇溶液提取，过滤，得到醇总提取液，浓缩，得到苹果树枝条活性提取物；所述的醇溶液加入的体积为相当于被提取物的3～5倍体积的提取溶剂；所述的提取的方式为超声、渗漉或加热回流中的一种。本发明以苹果树枝条为原料提取一种具有抑菌抗氧化作用的含多酚和黄酮提取物，变费为宝，对资源进行了充分利用，同时提供了一种新的具有抑菌抗氧化作用的化合物。
Description
技术领域
本发明属于天然药物化学领域，特别涉及一种以苹果树枝条为原料得到的具有抑菌抗氧化作用的含多酚和黄酮的苹果树枝条活性提取物及提取方法与应用。
背景技术
中国是世界上最大的苹果生产国，在东北、华北、华东、西北和西南等地均有种植，种植面积近189万平方公里，我国苹果的种植面积约占世界总面积的37％，苹果产量约占世界总产量的40％。为了调节果树的生长和果实的产量，在苹果树生长过程中，每年果农都会对果树进行修剪。据估计，我国每年修剪的苹果树枝条约为190万吨，这些废弃的枝条只能被当做柴火烧掉，既浪费了资源，又污染了环境。
发明内容
本发明要解决的技术问题在于提供一种充分利用苹果树枝的方法，所述方法以苹果树枝条为原料提取一种具有抑菌抗氧化作用的含多酚和黄酮提取物以及具体的提取方法和应用。本发明充分利用现有的废弃物，变费为宝，对资源进行了充分利用，同时提供了一种新的具有抑菌抗氧化作用的化合物。
本发明是通过如下技术方案来实现的：
一种苹果树枝条活性提取物，所述的活性提取物优选为多酚含量不低于 10％，黄酮的含量不低于20％。
本发明所述苹果树枝条活性提取物的具体提取方法，包含如下步骤：将苹果树枝条粉碎过筛，用醇溶液提取，过滤，得到醇总提取液，浓缩，得到苹果树枝条活性提取物；
所述的醇溶液加入的体积为相当于被提取物的3～5倍体积的提取溶剂；
所述的提取的方式为超声、渗漉或加热回流中的一种；
所述的醇溶液优选为乙醇溶液或甲醇溶液；
所述的乙醇溶液优选的浓度为体积百分比为60％～80％；
所述的甲醇溶液优选的浓度为体积百分比为60％～80％；
所述加热回流的条件优选为在40～60℃进行。
所述的提取优选至少提取3次，每次提取4～6h。
所述的苹果树枝条活性提取物作为抑菌剂的应用，所述的菌为：金黄色葡萄球菌、沙门氏菌或大肠杆菌。
所述的苹果树枝条活性提取物作为抗氧化剂的应用。
本发明还提供一种含有所述苹果树枝条活性提取物的抑菌剂。
本发明还提供一种含有所述苹果树枝条活性提取物的抗氧化剂。
本发明与现有技术相比的有益效果：
(1)我国每年修剪的苹果树枝条约为190万吨，这些废弃的枝条只能被当做柴火烧掉，既浪费了资源，又污染了环境，本发明提供的苹果树枝条活性提取物的制备工艺能有效地富集总多酚和总黄酮。本发明所述的苹果树枝条活性提取物制备方法简单，成本较低；
(2)本发明得到苹果树枝条活性提取物可以通过紫外分光光度法和高效液相法进行多酚和黄酮的含量测定和指纹图谱分析，该分析方法简单易控；
(3)本发明所提取的苹果树枝条活性提取物具有较好的抑菌作用，特别是对沙门氏菌、金黄色葡萄球菌、大肠杆菌有很好的抑制作用；
(4)本发明所提取的苹果树枝条活性提取物有显著的抗氧化作用；
(5)将苹果树枝条活性提取物应用于制备抑菌抗氧化的添加剂或药物，高效低毒，生产成本低，适于工业化生产。
附图说明
图1是对比实施例1得到的苹果树枝条甲醇活性提取物的HPLC谱图；
图2是对比实施例1得到的苹果树枝条乙醇活性提取物的HPLC谱图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
一种苹果树枝条活性提取物，所述提取物的具体提取方法如下：
(1)苹果树枝条抑菌抗氧化活性提取物的制备
将新鲜苹果树枝条(200g)放入烘箱，50℃干燥，粉碎，过100目筛，用体积百分比为70％的乙醇于室温下进行渗漉提取3次，每次5小时，过滤，得到总提取液。将总提取液减压蒸馏至干燥得到总提物，将此总提物放入超低温冰箱，-80℃冷冻4h，放入冷冻干燥机干燥24h，最后得到苹果树枝条活性提取物(21.3g)。
(2)对步骤(1)得到的活性提取物多酚含量进行分析
精确称取0.5000g焦性没食子酸，置于100mL容量瓶中溶解定溶至刻度，摇匀。以此为母液配制成浓度分别为0，100、200、300、400、500μg/mL的没食子酸工作液，准确吸取1mL工作液至50mL的容量瓶中，各加入20mL水， 5mL福林酚试剂，混匀，30s～8min内各加入24mL15％的碳酸钠溶液，混合后用水定容，摇匀。将上述溶液在20℃下放置2h。然后用分光光度计在765nm 波长下，测定不同浓度溶液的吸光度，见表1，绘制标准曲线。
表1没食子酸标准样品浓度与吸光度
| 没食子酸浓度(μg/mL) | 吸光度 | 校正后 |
| 0 | 0.002 | - |
| 100 | 0.120 | 0.118 |
| 200 | 0.363 | 0.361 |
| 300 | 0.634 | 0.632 |
| 400 | 0.917 | 0.915 |
| 500 | 1.287 | 1.285 |
称取步骤(1)得到的苹果树枝条活性提取物0.3g溶于2mL乙醇中，用水定容至50mL，按上述方法测定其吸光度。带入标准曲线，求样品中的总多酚含量为10.21％。
(3)对步骤(1)得到的活性提取物黄酮含量进行分析
准确称取50mg芦丁标准品溶于100mL 60％乙醇溶液中，水浴上微热，使溶解，放冷后用60％乙醇溶液稀释至刻度，得到芦丁标准溶液(500μg/mL)。分别吸取芦丁标准溶液0、0.2、0.4、0.8、1.6、2.0mL于10mL容量瓶中，用60％乙醇溶液稀释至2mL，加入5％亚硝酸钠0.2mL摇匀，静置6min；加入10％硝酸铝0.2 mL摇匀，静置6min；加入4％氢氧化钠2mL，用水定容至刻度，静置15min，于510nm下测定吸光度，见表2，并绘制标准曲线。
表2芦丁标准样品浓度与吸光度
| 芦丁浓度(μg/mL) | 吸光度 | 校正后 |
| 0 | 0.063 | - |
| 10 | 0.173 | 0.110 |
| 20 | 0.289 | 0.226 |
| 40 | 0.516 | 0.453 |
| 80 | 0.976 | 0.913 |
| 100 | 1.181 | 1.118 |
称取步骤(1)得到的苹果树枝条活性提取物0.04g溶于2mL乙醇中，定容至10ml，取0.5ml，按照以上方法进行测。测定吸光度值，求样品中的总黄酮含量为26.74％。
(4)对步骤(1)得到的活性提取物抗氧化活性分析
绘制DPPH自由基清除能力的标准曲线：称取9.85mg DPPH以无水乙醇定容至250mL，作为DPPH工作液用无水乙醇配置0.1mmol/L的DPPH溶液。配置0.5mg/mL的Vc溶液(作为对照)。将Vc溶液对倍稀释成6个不同浓度。取 15ml离心管，将2mL Vc溶液+2mL DPPH溶液混匀后测定其吸光度Asample，同时测定2mL DPPH溶液+2mL溶剂乙醇混合后的吸光度Acontrol,2mL Vc溶液与2mL无水乙醇混合后的吸光度Ablank，加入到同一试管中，摇匀，室温下暗处静置30min，于517nm处测定吸光度。每个浓度做3个平行试验，取平均值。并根据公式计算维生素C溶液对DPPH·自由基的清除率。清除率(％)＝(1- (Asample-Ablank)/Acontrol)。以Vc溶液浓度为自变量，自由基清除率为因变量作图并进行线性拟合。
苹果树枝条活性提取物自由基清除能力计算：配置2mg/mL的样品溶液。将测试样品稀释至不同浓度，对倍稀释成6个浓度。取15ml离心管，将2mL测试样品溶液+2mL DPPH溶液测定其吸光度Asample，同时测定2mL DPPH溶液+2mL 溶剂乙醇混合后的吸光度Acontrol，2mL测试样品溶液与2mL无水乙醇混合后的吸光度Ablank，加入到同一试管中，摇匀，室温下暗处静置30min，于517nm处测定吸光度。每个样品做3个平行试验，取平均值。并根据公式计算出样品对 DPPH·自由基的清除率。以样品浓度为自变量，自由基清除率为因变量作图并进行线性拟合，计算IC50值，其中IC50值定义为清除率为50％时所需抗氧化剂的浓度，所需浓度越低，表明该物质抗氧化性越强。根据计算，VC的半抑制浓度(IC50)为0.1085mg/ml，苹果树枝条提取物的半抑制浓度(IC50)为0.9254mg/ml，具有较强的抗氧化活性。
(5)对步骤(1)得到的活性提取物抑菌活性分析
样品溶液配制：称取50mg的苹果树枝条提取物粉末于(2mL)EP管中，加入10％DMSO作为助溶剂，震荡使药物溶解，在超净工作台里加入灭菌生理盐水，配成浓度为50mg·mL-1药液。
菌液培养：将金黄色葡萄球菌、沙门氏菌、大肠杆菌三株菌中接种于LB琼脂培养基，置于37℃恒温箱中培养20h，管配置细菌浓度为1.5×108CFU·mL-1，用于做药物敏感性和琼脂扩散法测量抑菌圈。细菌300倍稀释成5×105CFU·mL -1，用于最低抑菌浓度(MIC)的测定。
体外抑菌活性的测定：采用微量液体培养基倍比稀释法。取15mL离心管，加入1mL细菌浓度为5×105CFU·mL-1的菌液。每个离心管分别加入50μL浓度0、 5、2.5、1.25、0.625、0.3125mg·mL-1药液，37℃恒温振荡培养20h。分别取100μL 菌液，均匀涂布于LB琼脂平板，37℃恒温培养箱培养20h，计数，计算MIC值，如表3所示：
表3苹果树枝条活性提取物对细菌的最低抑菌浓度(MIC)
| 菌种 | MIC(mg/mL) | 空白对照 |
| 金黄色葡萄球菌 | 2.78 | 长菌 |
| 沙门氏菌 | 3.12 | 长菌 |
| 大肠杆菌 | 1.56 | 长菌 |
对比实施例1
苹果树枝条乙醇提取物和甲醇活性提取物的比较
(1)样品制备
取2份苹果树枝条各10g，粉碎，过100目筛，分别用40ml甲醇和乙醇超声提取1小时，提取液用0.22μm微孔滤膜过滤，滤液取20μL注入HPLC进行分析。
(2)分析条件
采用Alliance仪器系统，反相色谱柱(150×4.6mm，3μm；waters，USA)，流动相：水(A)和甲醇(B)。梯度洗脱系统：0～20min：10％→30％B；20～ 50min：30％→100％B；50～55min：100％B；55～60min:100％→10％B。流速： 1ml/min，检测波长：210nm。
(3)分析结果
结果如图1、图2所示，苹果树枝条甲醇活性提取物和乙醇活性提取物的主要色谱峰保留时间基本一致，说明它们的化学成分非常接近。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.一种苹果树枝条活性提取物，其特征在于所述的活性提取物中多酚含量不低于10％，黄酮的含量不低于20％。
2.权利要求1所述苹果树枝条活性提取物的具体提取方法，其特征在于所述方法包含如下步骤：将苹果树枝条粉碎过筛，用醇溶液提取，过滤，得到醇总提取液，浓缩，得到苹果树枝条活性提取物；
所述的醇溶液加入的体积为相当于被提取物的3～5倍体积的提取溶剂；
所述的提取的方式为超声、渗漉或加热回流中的一种。
3.根据权利要求2所述的方法，其特征在于所述的提取至少提取3次，每次提取4～6h。
4.根据权利要求2所述的方法，其特征在于所述的醇溶液为乙醇溶液或甲醇溶液。
5.根据权利要求4所述的方法，其特征在于所述的乙醇溶液的浓度为体积百分比为60％～80％；甲醇溶液的浓度为体积百分比为60％～80％。
6.根据权利要求2所述的方法，其特征在于所述加热回流的条件为在40～60℃进行。
7.权利要求1-6任何一项所述的苹果树枝条活性提取物作为抑菌剂的应用，所述的菌为：金黄色葡萄球菌、沙门氏菌或大肠杆菌。
8.权利要求1-6任何一项所述的苹果树枝条活性提取物作为抗氧化剂的应用。
9.一种含有权利要求1-6任何一项所述苹果树枝条活性提取物的抑菌剂。
10.一种含有权利要求1-6任何一项所述苹果树枝条活性提取物的抗氧化剂。
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