CN109320487A - 一种以复苏植物为原料的提取物及其制备方法和应用 - Google Patents

一种以复苏植物为原料的提取物及其制备方法和应用 Download PDF

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CN109320487A
CN109320487A CN201710645482.XA CN201710645482A CN109320487A CN 109320487 A CN109320487 A CN 109320487A CN 201710645482 A CN201710645482 A CN 201710645482A CN 109320487 A CN109320487 A CN 109320487A
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邓馨
孙润泽
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Abstract

本发明公开了一种以复苏植物为原料的提取物及其制备方法和应用。本发明提供了一种以复苏植物为原料的提取物的制备方法，包括如下步骤：(1)取复苏植物的全植株或叶片，通过研磨得到粉末；(2)取粉末，采用乙醇溶液进行提取，收集上清液；(3)完成步骤(2)后，剩余沉淀中再次加入乙醇溶液进行提取，收集上清液；(4)将步骤(2)得到的上清液和步骤(3)得到的上清液混合，进行干燥，获得干粉状提取物。本发明提供的提取物可作为添加剂或辅料，按重量比添加至保健品、药品、食品、饮品、茶、化妆品等。针对现有野生植物资源的理化特性，本发明充分利用了我国复苏植物的天然资源，可以获得明显的经济效益和社会效益。

Description

一种以复苏植物为原料的提取物及其制备方法和应用
技术领域
本发明属于稀缺生物资源的利用技术领域，涉及一种以复苏植物为原料的提取物及其制备方法和应用。
背景技术
随着全球水资源的缺乏和极端气候事件的增长，干旱对植物的生长、产量和品质的影响日趋显著。生物体受到干旱胁迫或其它不良环境因素后，会通过不同途径抵抗不良胁迫，包括耐旱相关蛋白的合成与积累、保护物质的产生、有害物质的清除等。
复苏植物因其可以忍受极度干旱的条件，待水份充足时又能很快恢复正常生命活动的特性而被认为是研究抗逆机制和提供抗逆基因的极好的植物资源。苦苣苔科有多种植物具有耐脱水复苏的特性，如旋蒴苣苔(Boeahygrometrica)和地胆旋蒴苣苔(Boeaphilippensis)等，这类植物的叶片具有很强的耐旱复苏能力，具体体现为：在室温、相对空气湿度为0的条件下生长72小时后，叶片相对含水量降为3％左右，叶片面积皱缩至原叶片面积的1/3以下，光合作用基本停止；只要重新给水，叶片就可以吸水伸展，并恢复成未处理前的叶片表观状态和生理状态(包括光合作用的恢复)。
发明内容
本发明的目的是提供一种以复苏植物为原料的提取物及其制备方法和应用。
本发明提供了一种以复苏植物为原料的提取物的制备方法，包括如下步骤：
(1)取复苏植物的全植株或叶片，通过研磨得到粉末；
(2)取步骤(1)得到的粉末，采用乙醇溶液进行提取，收集上清液；
(3)完成步骤(2)后，剩余沉淀中再次加入乙醇溶液进行提取，收集上清液；
(4)将步骤(2)得到的上清液和步骤(3)得到的上清液混合，进行干燥，获得干粉状提取物。
所述步骤(1)中，所述研磨为液氮研磨。
所述步骤(2)中，所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％。所述步骤(2)中，所述提取的条件为：25℃静置提取48h。所述步骤(2)中，通过离心收集上清液；所述离心的条件为：10000rpm离心10min。
所述步骤(3)中，所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％。所述步骤(3)中，所述提取的条件为：25℃静置提取48h。所述步骤(3)中，通过离心收集上清液；所述离心的条件为：10000rpm离心10min。
所述复苏植物的全植株或叶片的鲜重为n1g，所述步骤(2)中所述乙醇溶液的体积为n2mL，所述步骤(3)中所述乙醇溶液的体积为n2mL；n1:n2:n3＝1:10:10。
本发明还保护一种以复苏植物为原料的提取物的制备方法，包括如下步骤：
(1)取复苏植物的全植株或叶片，通过研磨得到粉末；
(2)取步骤(1)得到的粉末，采用乙醇溶液进行提取，收集上清液；
(3)将步骤(2)得到的上清液进行干燥，获得干粉状提取物。
所述步骤(1)中，所述研磨为液氮研磨。
所述步骤(2)中，所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％。所述提取的条件为：25℃静置提取48h。通过离心收集上清液；所述离心的条件为：10000rpm离心10min。
本发明还保护一种以复苏植物为原料的提取物的制备方法，包括如下步骤：以复苏植物的全植株或叶片为原料，采用乙醇溶液进行提取，收集提取液并干燥，获得干粉状提取物。所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％。所述提取的条件为：25℃静置提取48h。
以上任一所述方法制备得到的提取物均属于本发明的保护范围。
本发明还保护所述提取物作为添加物在制备产品中的应用。
本发明还保护一种产品，含有所述提取物。
以上任一所述产品可为保健品、药品、食品、饮品、茶或化妆品。
以上任一所述复苏植物为苦苣苔科植物，具体可为旋蒴苣苔。
以上任一所述复苏植物可为正常生长环境中的复苏植物，也可为干旱环境中的复苏植物。以上任一所述复苏植物可为正常生长状态中的复苏植物，也可为失水状态的复苏植物。
本发明还保护一种制备绿原酸和/或香草酸和/或咖啡酸和/或香豆酸和/或阿魏酸和/或儿茶酸的方法，包括如下步骤：
取所述提取物，用甲醇重溶，得到样本液；采用高效液相色谱从样本液中纯化获得绿原酸和/或香草酸和/或咖啡酸和/或香豆酸和/或阿魏酸和/或儿茶酸；
采用Agilent 1100series HPLC-MSD trap VL配备二极管阵列检测器和ZorbaxSB-C18反相色谱柱；
A液由100体积份甲醇、20体积份乙酸和880体积份水混合得到；B液由900体积份甲醇、15体积份乙酸和85体积份水混合得到；流动相为A液、B液或A液和B液的混合液；
流动相流速为1mL/min；
洗脱程序：①B液占流动相的体积分数由0％线性上升至3.6％，时间7mim；②B液占流动相的体积分数由3.6％线性上升至15％，时间19min；③B液占流动相的体积分数由15％线性上升至25.5％，时间6min；④B液占流动相的体积分数由25.5％线性上升至29.7％，时间3min；⑤B液占流动相的体积分数由29.7％线性上升至45.5％，时间10min；⑥B液占流动相的体积分数由45.5％线性下降至0％，时间8min。
绿原酸出峰对应的保留时间为4.09min。香草酸出峰对应的保留时间为4.44min。咖啡酸出峰对应的保留时间为4.31min。香豆酸出峰对应的保留时间为11.91min。阿魏酸出峰对应的保留时间为9.22min。儿茶酸出峰对应的保留时间为3.2min。
本发明还保护一种制备芦丁和/或芹黄素和/或槲皮素和/或漆黄素和/或山奈酚和/或柚皮素的方法，包括如下步骤：
取所述提取物，用甲醇重溶，得到样本液；采用高效液相色谱从样本液中纯化获得芦丁和/或芹黄素和/或槲皮素和/或漆黄素和/或山奈酚和/或柚皮素；
采用Agilent 1200series HPLC-MSD trap VL配备可变波长检测器和ZorbaxSB-C18反相色谱柱；
A液由50体积份乙腈、85体积份甲酸和865体积份水混合得到；B液由250体积份乙腈、450体积份甲醇、85体积份甲酸和215体积份水混合得到；流动相为A液、B液或A液和B液的混合液；
流动相流速为1mL/min；
洗脱程序：①B液占流动相的体积分数由0％线性上升至14.2％，时间24.2min；②B液占流动相的体积分数由14.2％线性上升至15.7％，时间2.8min；③B液占流动相的体积分数由15.7％线性上升至18.8％，时间6.4min；④B液占流动相的体积分数由18.8％线性上升至23.5％，时间5.4min；⑤B液占流动相的体积分数由23.5％线性上升至26％，时间6min；⑥B液占流动相的体积分数由26％线性上升至27.4％，时间2min；⑦B液占流动相的体积分数由27.4％线性上升至32％，时间4.6min；⑧B液占流动相的体积分数由32％线性上升至40％，时间10.2min；⑨B液占流动相的体积分数由40％线性上升至100％，时间6min；⑩B液占流动相的体积分数由100％线性下降至0％，时间10.6min。
芦丁出峰对应的保留时间为12.29min。芹黄素出峰对应的保留时间为11.05min。槲皮素出峰对应的保留时间为18.57min。漆黄素出峰对应的保留时间为12.88min。山奈酚出峰对应的保留时间为22.62min。柚皮素出峰对应的保留时间为10.22min。
本发明提供了复苏植物提取物及其制备方法。本发明提供的提取物可作为添加剂或辅料，按重量比添加至保健品、药品、食品、饮品、茶、化妆品等。针对现有野生植物资源的理化特性，本发明提供了一种复苏植物的利用技术及集成，充分利用了我国苦苣苔科复苏植物的天然资源，通过有效成分的提取和应用技术可以获得明显的经济效益和社会效益。
附图说明
图1为提取物中酚酸类物质的含量。
图2为提取物中类黄酮类物质的含量。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
旋蒴苣苔(Boea hygrometrica)：参考文献：复苏植物耐脱水机制研究进展，刘杰等，生物技术通报，2016,32(10):42-51。
用于实施例1的细胞分裂素为6-苄氨基嘌呤，CAS号为1214-39-7。
用于实施例1的生长素为吲哚乙酸，CAS号为87-51-4。
实施例1、旋蒴苣苔的扩繁
1、制备外植体
取旋蒴苣苔植株的新鲜叶片，用自来水冲洗，然后用75％(体积百分含量)酒精水溶液消毒10sec，然后用消毒液消毒3min，然后无菌蒸馏水冲洗5min，然后用无菌手术刀在每个叶片上进行5-10处划伤。
消毒液：含10g/100mL NaClO和0.01％(体积百分含量)吐温-20的水溶液。
2、诱导
取步骤1得到的外植体，置于装有诱导培养基的培养皿中培养15天，再转接至装有新的诱导培养基的培养瓶中培养3周。
诱导培养基：含1mg/L生长素和3mg/L细胞分裂素的1/2MS固体培养基。
培养条件：25℃，光照16小时/黑暗8小时，光照强度3500lx。
可以观察到，在装有诱导培养基的培养皿中培养15天后叶片的伤口处开始有幼芽生成，在装有新的诱导培养基的培养瓶中培养3周后叶片的伤口处有大量幼芽生成(平均每个叶片再分化产生12-15个幼芽)。
3、生根
完成步骤2后，从叶片上剥离幼芽，将幼芽置于装有生根培养基的培养瓶中培养至根长为3-5cm(通常需要4-8周)。
生根培养基：含3mg/L生长素的1/2MS固体培养基。
培养条件：25℃，光照16小时/黑暗8小时，光照强度3500lx。
4、炼苗
完成步骤3后，取生根的幼苗，移栽至装有炼苗基质的花盆中，培养7天。
炼苗基质：8体积份中位泥炭土与3体积份蛭石混合。
培养条件：25℃，光照16小时/黑暗8小时，光照强度3500lx。
5、温室栽培
完成步骤4后，将所述花盆转移至温室，正常管理16周，然后收获新鲜叶片进行实施例2。
6、温室栽培
完成步骤4后，将所述花盆转移至温室，正常管理14周，然后持续不浇水2周，然后收获新鲜叶片进行实施例3。
实施例2、提取物的制备
1、取旋蒴苣苔的新鲜叶片，按重量均分为两份(每份鲜重均为n1g)，一份烘干至恒重后称取干重，另一份液氮研磨得到粉末。
2、取步骤1得到的粉末，加入n2mL 70％(体积比)乙醇水溶液，先剧烈震荡10min使其充分混匀，然后25℃静置提取48h，然后10000rpm离心10min，收集上清液。
3、完成步骤2后，在沉淀中加入n3mL70％(体积比)乙醇水溶液，先剧烈震荡10min使其充分混匀，然后25℃静置提取48h，然后10000rpm离心10min，收集上清液。
4、将步骤2得到的上清液和步骤3得到的上清液混合，进行真空干燥，得到干粉。
n1:n2:n3＝1:10:10。
n1＝1000g时，得到0.1g干粉。将干粉命名为提取物甲。
实施例3、提取物的制备
1、取旋蒴苣苔的新鲜叶片，按重量均分为两份(每份鲜重均为n1g)，一份烘干至恒重后称取干重，另一份液氮研磨得到粉末。
2、取步骤1得到的粉末，加入n2mL 70％(体积比)乙醇水溶液，先剧烈震荡10min使其充分混匀，然后25℃静置提取48h，然后10000rpm离心10min，收集上清液。
3、完成步骤2后，在沉淀中加入n3mL70％(体积比)乙醇水溶液，先剧烈震荡10min使其充分混匀，然后25℃静置提取48h，然后10000rpm离心10min，收集上清液。
4、将步骤2得到的上清液和步骤3得到的上清液混合，进行真空干燥，得到干粉。
n1:n2:n3＝1:10:10。
将干粉命名为提取物乙。
实施例4、提取物的成分检测
取实施例2制备的提取物甲或实施例3制备的提取物乙，用甲醇重溶，得到待测溶液，然后采用高效液相色谱-质谱联用仪进行成分检测。
一、酚酸检测
液相色谱参数：采用Agilent 1100series HPLC-MSD trap VL配备二极管阵列检测器和Zorbax SB-C18(250×4mm,5μm)反相色谱柱。上样量：10μL。柱温：30℃。A液由100体积份甲醇、20体积份乙酸和880体积份水混合得到。B液由900体积份甲醇、15体积份乙酸和85体积份水混合得到。流动相为A液、B液或A液和B液的混合液。洗脱程序(流动相流速为1mL/min)：①B液占流动相的体积分数由0％线性上升至3.6％，时间7mim；②B液占流动相的体积分数由3.6％线性上升至15％，时间19min；③B液占流动相的体积分数由15％线性上升至25.5％，时间6min；④B液占流动相的体积分数由25.5％线性上升至29.7％，时间3min；⑤B液占流动相的体积分数由29.7％线性上升至45.5％，时间10min；⑥B液占流动相的体积分数由45.5％线性下降至0％，时间8min。咖啡酸和绿原酸检测波长为325nm，其它酚酸检测波长为275nm。
质谱参数：负离子模式，喷雾电压为241.3kPa，干气流量为10L/min，干气温度为350℃，阱离子电荷控制为30000单元，碰撞离子电压为1V，m/z扫描为100-1000。
如果待测溶液进行液相色谱时出现与某标准品保留时间一致的峰且相应的质谱数据与该标准品一致，说明待测溶液中含有该物质，将待测溶液中该峰的峰面积代入用标准品制作的标准曲线，计算待测溶液中该物质的含量。
在上述液相色谱参数下，绿原酸标准品出峰对应的保留时间为4.09min，香草酸标准品出峰对应的保留时间为4.44min，咖啡酸标准品出峰对应的保留时间为4.31min，香豆酸标准品出峰对应的保留时间为11.91min，阿魏酸标准品出峰对应的保留时间为9.22min，儿茶酸标准品出峰对应的保留时间为3.2min。
提取物甲和提取物乙中，各种酚酸物质的含量见图1。
二、类黄酮检测
液相色谱参数：采用Agilent 1200series HPLC-MSD trap VL配备可变波长检测器和Zorbax SB-C18(50×3mm,1.8μm)反相色谱柱。上样量：50μL。柱温：40℃。A液由50体积份乙腈、85体积份甲酸和865体积份水混合得到。B液由250体积份乙腈、450体积份甲醇、85体积份甲酸和215体积份水混合得到。流动相为A液、B液或A液和B液的混合液。洗脱程序(流动相流速为1mL/min)：①B液占流动相的体积分数由0％线性上升至14.2％，时间24.2min；②B液占流动相的体积分数由14.2％线性上升至15.7％，时间2.8min；③B液占流动相的体积分数由15.7％线性上升至18.8％，时间6.4min；④B液占流动相的体积分数由18.8％线性上升至23.5％，时间5.4min；⑤B液占流动相的体积分数由23.5％线性上升至26％，时间6min；⑥B液占流动相的体积分数由26％线性上升至27.4％，时间2min；⑦B液占流动相的体积分数由27.4％线性上升至32％，时间4.6min；⑧B液占流动相的体积分数由32％线性上升至40％，时间10.2min；⑨B液占流动相的体积分数由40％线性上升至100％，时间6min；⑩B液占流动相的体积分数由100％线性下降至0％，时间10.6min。检测波长为360nm。
质谱参数：负离子模式，喷雾电压为30psi，干气流量为10L/min，干气温度为325℃，阱离子电荷控制为30,000单元，碰撞离子电压为1V，m/z扫描为100-1,000。
如果待测溶液进行液相色谱时出现与某标准品保留时间一致的峰且相应的质谱数据与该标准品一致，说明待测溶液中含有该物质，将待测溶液中该峰的峰面积代入用标准品制作的标准曲线，计算待测溶液中该物质的含量。
在上述液相色谱参数下，芦丁标准品出峰对应的保留时间为12.29min，芹黄素标准品出峰对应的保留时间为11.05min，槲皮素标准品出峰对应的保留时间为18.57min，漆黄素标准品出峰对应的保留时间为12.88min，山奈酚标准品出峰对应的保留时间为22.62min，柚皮素标准品出峰对应的保留时间为10.22min。
提取物甲和提取物乙中，各种类黄酮物质的含量见图2。
提取物中具有的各种酚酸物质和各种类黄酮物质对人体具有抗病原微生物、消炎、活血、止血、止咳等药用价值以及抗氧化、抗衰老、保湿等保健作用。可以将提取物(干粉)作为添加物，按照1:100-10000重量比添加到保健品、药品、食品、饮品、茶、化妆品等产品中。
上述实施例仅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，其他未背离本发明的精神实质与原理下所做的改变、替代、组合，均应为等效的置换方式，都包含在本发明的保护范围内。

Claims (10)

1.一种以复苏植物为原料的提取物的制备方法，包括如下步骤：
(1)取复苏植物的全植株或叶片，通过研磨得到粉末；
(2)取步骤(1)得到的粉末，采用乙醇溶液进行提取，收集上清液；
(3)完成步骤(2)后，剩余沉淀中再次加入乙醇溶液进行提取，收集上清液；
(4)将步骤(2)得到的上清液和步骤(3)得到的上清液混合，进行干燥，获得干粉状提取物。
2.如权利要求1所述的方法，其特征在于：所述步骤(2)中，所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％；所述步骤(3)中，所述乙醇溶液为乙醇水溶液，乙醇的体积百分含量为70％。
3.如权利要求1或2所述的方法，其特征在于：所述步骤(2)中，所述提取的条件为：25℃静置提取48h；所述步骤(3)中，所述提取的条件为：25℃静置提取48h。
4.如权利要求1至3中任一所述的方法，其特征在于：
所述步骤(2)中，通过离心收集上清液；所述离心的条件为：10000rpm离心10min；
所述步骤(3)中，通过离心收集上清液；所述离心的条件为：10000rpm离心10min。
5.一种以复苏植物为原料的提取物的制备方法，包括如下步骤：
(1)取复苏植物的全植株或叶片，通过研磨得到粉末；
(2)取步骤(1)得到的粉末，采用乙醇溶液进行提取，收集上清液；
(3)将步骤(2)得到的上清液进行干燥，获得干粉状提取物。
6.一种以复苏植物为原料的提取物的制备方法，包括如下步骤：以复苏植物的全植株或叶片为原料，采用乙醇溶液进行提取，收集提取液并干燥，获得干粉状提取物。
7.权利要求1至6中任一所述方法制备得到的提取物。
8.一种应用或一种产品；
所述应用为权利要求7所述提取物作为添加物在制备产品中的应用；
所述产品为含有权利要求7所述提取物的产品。
9.一种制备绿原酸和/或香草酸和/或咖啡酸和/或香豆酸和/或阿魏酸和/或儿茶酸的方法，包括如下步骤：
取权利要求7所述提取物，用甲醇重溶，得到样本液；采用高效液相色谱从样本液中纯化获得绿原酸和/或香草酸和/或咖啡酸和/或香豆酸和/或阿魏酸和/或儿茶酸；
采用Agilent 1100series HPLC-MSD trap VL配备二极管阵列检测器和Zorbax SB-C18反相色谱柱；
A液由100体积份甲醇、20体积份乙酸和880体积份水混合得到；B液由900体积份甲醇、15体积份乙酸和85体积份水混合得到；流动相为A液、B液或A液和B液的混合液；
流动相流速为1mL/min；
洗脱程序：①B液占流动相的体积分数由0％线性上升至3.6％，时间7mim；②B液占流动相的体积分数由3.6％线性上升至15％，时间19min；③B液占流动相的体积分数由15％线性上升至25.5％，时间6min；④B液占流动相的体积分数由25.5％线性上升至29.7％，时间3min；⑤B液占流动相的体积分数由29.7％线性上升至45.5％，时间10min；⑥B液占流动相的体积分数由45.5％线性下降至0％，时间8min。
10.一种制备芦丁和/或芹黄素和/或槲皮素和/或漆黄素和/或山奈酚和/或柚皮素的方法，包括如下步骤：
取权利要求7所述提取物，用甲醇重溶，得到样本液；采用高效液相色谱从样本液中纯化获得芦丁和/或芹黄素和/或槲皮素和/或漆黄素和/或山奈酚和/或柚皮素；
采用Agilent 1200series HPLC-MSD trap VL配备可变波长检测器和Zorbax SB-C18反相色谱柱；
A液由50体积份乙腈、85体积份甲酸和865体积份水混合得到；B液由250体积份乙腈、450体积份甲醇、85体积份甲酸和215体积份水混合得到；流动相为A液、B液或A液和B液的混合液；
流动相流速为1mL/min；
洗脱程序：①B液占流动相的体积分数由0％线性上升至14.2％，时间24.2min；②B液占流动相的体积分数由14.2％线性上升至15.7％，时间2.8min；③B液占流动相的体积分数由15.7％线性上升至18.8％，时间6.4min；④B液占流动相的体积分数由18.8％线性上升至23.5％，时间5.4min；⑤B液占流动相的体积分数由23.5％线性上升至26％，时间6min；⑥B液占流动相的体积分数由26％线性上升至27.4％，时间2min；⑦B液占流动相的体积分数由27.4％线性上升至32％，时间4.6min；⑧B液占流动相的体积分数由32％线性上升至40％，时间10.2min；⑨B液占流动相的体积分数由40％线性上升至100％，时间6min；⑩B液占流动相的体积分数由100％线性下降至0％，时间10.6min。
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