CN112168849B - 一种水龙提取物的制备方法及用途 - Google Patents
一种水龙提取物的制备方法及用途 Download PDFInfo
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- CN112168849B CN112168849B CN202011057272.7A CN202011057272A CN112168849B CN 112168849 B CN112168849 B CN 112168849B CN 202011057272 A CN202011057272 A CN 202011057272A CN 112168849 B CN112168849 B CN 112168849B
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Abstract
本发明公开了一种水龙提取物的制备方法，包括低温提取步骤、过滤步骤、脱色步骤和低温干燥步骤，采用本发明的制备方法制备的水龙提取物清除DPPH自由基的IC50值小于100μg/mL；清除ABTS自由基的IC50值小于100μg/mL；可以抑制由LPS刺激单核巨噬细胞引起的NO分泌；可以抑制胶原蛋白酶的活性，具有较强的抗氧化能力和抗炎能力，可以作为抗炎、抗衰成分用在功效性护肤品或功能性食品中。
Description
技术领域
本发明涉及到柳叶菜科水龙属植物水龙Jussiaea repens L提取物，具体涉及一种水龙提取物的制备方法及用途。
背景技术
水龙(Jussiaea repens L)为中国西南省份常用中草药，别名水瓮菜、枇杷菜、过江龙、水芥菜、过江藤、假蕹菜等。为多种中成药的原料药，主要分布于我国西南部至东部。具有清热、解毒、利尿、消肿之功效，用于治疗燥热咳嗽、酒疸、淋病、麻疹、丹毒疔疮等。水龙可食用，每逢3月份萌芽采摘，食幼芽叶，口感清润、回味好，常佐以高汤蚕豆米，老幼喜食。
目前，对水龙的应用主要集中在利用其净化富营养化水体的能力，将其作为一种净化水源的植物(何芳芳，六种水生植物对三种富营养化水体藻生长的抑制作用研究[D].广东:华南师范大学，2010)。对于水龙有效部位的提取，中药大辞典记载过塘蛇的主要化学成分类型有酚性成分、黄酮类、氨基酸、糖类等成分；黄海兰等先经乙酸乙酯萃取，再利用硅胶、RP-18和Sephadex LH-20对水龙提取物进行活性追踪分离，得到抗氧化活性成分，分离得到三个化合物，确定为槲皮素-3-O-葡萄糖甙、山奈酚-3-O-葡萄糖甙、迷迭香酸；卢如梅等采用硅胶色谱和Sephadex LH-20色谱方法，从水龙中分离得到9个化合物；黄海兰等在室温下用1:2(V/V)的氯仿：甲醇，超声提取，过滤等步骤，得到了水龙中的抗食用油脂氧化活性成分(食品科学29.8(2008):80-82.)。这些提取方式均是通过氯仿、乙酸乙酯等危险性较大的试剂来得到目标水龙成分，难以放大生产，不利于环境和人身安全。
因此，开发一种简便、高效、安全、环保的提取方法，来得到质量可控的水龙活性部位意义重大。
发明内容
本发明的目的是克服现有技术的缺陷，提供一种水龙提取物的制备方法，可以确保水龙中的化学成分不被破坏，从而最大程度的保持其生物活性。
本发明的另外一个目的是提供一种水龙提取物的用途，采用本发明的制备方法所得的水龙提取中的没食子酸和没食子酸乙酯含量稳定，具有显著的抗氧化能力和抗炎活性，可以作为功效性护肤品或功能性食品中的活性成分，起到抗炎、抗衰老等作用。
实现上述目的一种技术方案是：一种水龙提取物的制备方法，包括以下步骤：
S1，低温提取步骤：采用醇/水混合液对水龙进行低温提取得到提取液，所述醇/水混合液中醇的体积分数为10％～40％，所述醇/水混合液的使用重量为所述水龙细粉重量的3～16倍；所述低温提取的温度为0～25℃；所述低温提取的时间为12～96h；
S2，过滤步骤：将所述提取液采用孔径为0.22μm～10μm的滤膜进行过滤，得澄清透明液体；
S3，脱色步骤：将经过过滤得到的澄清透明液体采用脱色剂进行脱色处理，所述脱色剂的使用量为所述澄清透明液体质量的0.1％～2％；脱色处理时间为0.5小时～2小时；然后过滤除去脱色剂后得到水龙提取液；
S4，低温干燥步骤：将所述水龙提取液在不超过40℃的温度下进行低温干燥即得水龙提取物。
上述的一种水龙提取物的制备方法，步骤S1中，所述水龙采用柳叶菜科水龙属植物水龙Jussiaea repens L的茎叶。
上述的一种水龙提取物的制备方法，步骤S1中，所述醇/水混合液中的醇采用甲醇、乙醇或者1，3-丁二醇，或者采用甲醇和乙醇的混合液。
上述的一种水龙提取物的制备方法，步骤S1中，低温提取过程中采用低速搅拌，所述低速搅拌的搅拌速度低于100r/min。
上述的一种水龙提取物的制备方法，步骤S2中，所述过滤采用负压抽滤或者正压压滤。
上述的一种水龙提取物的制备方法，步骤S3中，所述脱色剂采用活性炭。
上述的一种水龙提取物的制备方法，步骤S4中，所述低温干燥采用冷冻干燥、低温烘干、减压浓缩干燥或喷雾干燥。
上述的一种水龙提取物的制备方法，采用高效液相色谱法对步骤S4得到的水龙提取物中的没食子酸含量进行测定，所述水龙提取物中没食子酸含量大于5％，没食子酸乙酯含量大于2％。
本发明还提供了一种水龙提取物的用途，所述水龙提取物采用上述的一种水龙提取物的制备方法制得，所述水龙提取物清除DPPH自由基的IC50值小于100μg/mL，清除ABTS自由基的IC50值小于100μg/mL；所述水龙提取物通过抑制由LPS刺激单核巨噬细胞引起的NO分泌起到抗炎作用；所述水龙提取物用于抑制胶原蛋白酶的活性。
上述的一种水龙提取物的用途，所述水龙提取物作为抗炎、抗衰成分加入护肤品或功能性食品配方中。
采用本发明的水龙提取物的制备方法的技术方案，积极进步效果在于：
(1)本发明首次采用低温提取方法，得到水龙提取物；
(2)本发明的水龙提取物的制备方法能有效的得到水龙药材中的没食子酸和没食子酸乙酯等抗氧化成分，并且可以对其中的大分子物质和灰尘、杂质等进行去除；
(3)采用本发明的制备方法所得的所得不同批次水龙提取物中的没食子酸和没食子酸乙酯等成分含量稳定，可以保证其质量的可靠性，易于用于商业化；
(4)采用本发明的制备方法所得的所得水龙提取物，具有较强的抗氧化活性和抗炎活性，可以作为活性物用于护肤品及医药辅助用品中。
附图说明
图1为本发明的水龙提取物的制备方法的流程图；
图2为采用本发明的制备方法制得的水龙提取物的HPLC图；
图3为采用本发明的制备方法制得的水龙提取物的DPPH自由基清除作用试验结果图；
图4为采用本发明的制备方法制得的水龙提取物的ABTS自由基清除作用试验结果图；
图5为采用本发明的制备方法制得的水龙提取物抗炎活性检测试验结果图；
图6为采用本发明的制备方法制得的水龙提取物对胶原酶抑制活性检测结果图。
具体实施方式
为了使本技术领域的技术人员能更好地理解本发明的技术方案，下面结合附图对其具体实施方式进行详细地说明：
请参阅图1实施，下列实施例中的溶剂配制均为体积比，所用试剂和原料均市售可得。水龙为柳叶菜科水龙属植物水龙Jussiaea repens L的茎叶，较佳的是采用产地为西双版纳的水龙；较佳的采收季节为3-7月。
低温提取步骤中醇/水混合液的配制方法为：
A，10％＜甲醇：水(V:V)＜40％
B，10％＜乙醇：水(V:V)＜40％
C，10％＜1，3-丁二醇：水(V:V)＜40％
或者醇/水混合液采用A与B的混合溶液。
实施例1：
一种水龙提取物的制备方法，包括以下步骤：
S1，低温提取步骤：取西双版纳水龙100g，清洗干净，风干，切割成1cm长小段，粉碎机打碎后装入提取罐中，加入1kg 30％乙醇，控制提取温度在20℃，搅拌速度90r/min，在此状态下提取24h；
S2，过滤步骤：将提取液采用孔径为0.22μm的滤膜进行过滤，得澄清透明液体900g；
S3，脱色步骤：在经过过滤得到的澄清透明液体中加入活性炭9g，在40℃继续搅拌2小时进行脱色处理；然后板框过滤器除去活性炭后得到澄清透明的水龙提取液；
S4，低温干燥步骤：将水龙提取液减压浓缩除去乙醇，然后用冷冻干燥机进行干燥，得到固体粉末状的水龙提取物2.3g，收率为2.3％。
实施例2：
一种水龙提取物的制备方法，包括以下步骤：
S1，低温提取步骤：取西双版纳水龙200g，清洗干净，风干，切割成1cm长小段，粉碎机打碎后装入提取罐中，加入1.6kg 10％甲醇，控制提取温度在15℃，搅拌速度90r/min，在此状态下提取48h；
S2，过滤步骤：将提取液采用孔径为0.22μm的滤膜进行过滤，得澄清透明液体1.5kg；
S3，脱色步骤：在经过过滤得到的澄清透明液体中加入活性炭20g，在40℃继续搅拌2小时进行脱色处理；然后板框过滤器除去活性炭后得到澄清透明的水龙提取液；
S4，低温干燥步骤：将水龙提取液减压浓缩除去甲醇，然后用冷冻干燥机进行干燥，得到固体粉末状的水龙提取物4.3g，收率为2.15％。
实施例3：
一种水龙提取物的制备方法，包括以下步骤：
S1，低温提取步骤：取西双版纳水龙56.8g，清洗干净，风干，切割成1cm长小段，粉碎机打碎后装入提取罐中，加入250g 20％乙醇，控制提取温度在20℃，搅拌速度90r/min，在此状态下提取36h；
S2，过滤步骤：将提取液采用孔径为0.22μm的滤膜进行过滤，得澄清透明液体220g；
S3，脱色步骤：在经过过滤得到的澄清透明液体中加入活性炭3g，在44℃继续搅拌1小时进行脱色处理；然后板框过滤器除去活性炭后得到澄清透明的水龙提取液；
S4，低温干燥步骤：将水龙提取液减压浓缩除去乙醇，然后用冷冻干燥机进行干燥，得到固体粉末状的水龙提取物1.57g，收率为2.7％。
水龙提取物中的没食子酸含量测定：
请参阅图2，以没食子酸和没食子酸乙酯为对照品，用高效液相色谱法对实施例1和实施例3中的水龙提取物中的没食子酸进行定量。
精密称取没食子酸12.4mg，没食子酸乙酯6.9mg，用水定容至10mL，制备成混合标准品。用倍半稀释法对混合标准品进行稀释，得到如下表1的不同浓度混合标准品稀释液：
表1
取实施例1中的水龙提取物，配制成1.20mg/mL的溶液(待测样)。利用LC-MS对标准品和待测样分别进行分析，用MS和保留时间确定没食子酸和没食子酸乙酯，用积分面积和没食子酸和没食子酸乙酯浓度绘制标准曲线：
没食子酸：y＝8678x-47.473，R2＝0.9997(Y：积分面积，X:浓度)
没食子酸乙酯：y＝7714x-33.883，R2＝0.9993(Y：积分面积，X:浓度)
经计算，实施例1中的水龙提取物中没食子酸的含量为9.13％；没食子酸乙酯的含量为：3.96％。
参照上述步骤，测得实施例3中的水龙提取物中没食子酸含量为8.72％；没食子酸乙酯含量为3.15％。
水龙提取物的DPPH自由基清除作用试验：
请参阅图3，以维生素C为对照，对实施例1和实施例3中的水龙提取物的DPPH自由基清除作用进行测定。
取维生素C标准品，配制成浓度为0.54mg/mL，0.27mg/mL，0.135mg/mL，0.068mg/mL，0.034mg/mL，0.017mg/mL，0.0085mg/mL的溶液，各取150μL加入96孔板中；取实施例1中的水龙提取物配制成浓度为0.88mg/mL的溶液，倍半稀释法将其稀释成0.44mg/mL，0.22mg/mL，0.11mg/mL，0.055mg/mL，0.028mg/mL，0.014mg/mL，0.007mg/mL的溶液；将配制好的溶液各取150μL加入96孔板中；取DPPH，配制成0.51mg/mL的溶液后，向待测样和对照样中各加入150μL，反应30分钟后测定417nm吸光度，以吸光度和样品浓度绘制曲线，结果如图3所示。
图3中的结果显示，在该条件下，维生素C对DPPH自由基清除作用IC50为0.024mg/mL，实施例1中的水龙提取物对DPPH自由基清除作用IC50为0.039mg/mL。
参照上述步骤，测得实施例3中的水龙提取物对DPPH自由基清除作用IC50为0.028mg/mL。
水龙提取物的ABTS自由基清除作用试验：
请参阅图4，选用碧云天总抗氧化能力检测试剂盒对水龙提取物进行测定，以维生素C为对照，对实施例1和实施例3中的水龙提取物的ABTS自由基清除作用进行测定。
取维生素C标准品，配制成浓度为1.08mg/mL，0.54mg/mL，0.27mg/mL，0.135mg/mL，0.068mg/mL，0.034mg/mL，0.017mg/mL，0.0085mg/mL的溶液；取实施例1中的水龙提取物配制成浓度为0.88mg/mL的溶液，倍半稀释法将其稀释成1.08mg/mL，0.54mg/mL，0.27mg/mL，0.135mg/mL，0.068mg/mL，0.034mg/mL，0.017mg/mL，0.0085mg/mL的溶液。将配制好的维生素C和水龙提取物溶液各取10μL加入96孔板中；取ABTS工作液200μL加入到各样品孔中，震荡混匀，反应6min后测定734nm吸光度值。以吸光度和样品浓度绘制曲线，结果如图4所示。
图4中结果显示，在该条件下，维生素C对ABTS自由基清除作用IC50为0.056mg/mL，实施例1中的水龙提取物对ABTS自由基清除作用IC50为0.087mg/mL。
参照上述步骤，测得实施例3中的水龙提取物对ABTS自由基清除作用IC50为0.078mg/mL。
水龙提取物抗炎活性检测试验：
请参阅图5，以醋酸地塞米松为对照，通过LPS刺激后的RAW264.7细胞中NO分泌量评价水龙提取物的抗炎作用，测定实施例1所得水龙提取物对LPS诱导的RAW264.7细胞NO释放的影响。
取对数生长期的RAW264.7细胞，接种于96孔板中，细胞培养箱中培养24小时。实验设置正常对照组(不加LPS和样品)，LPS组(10μg/mL)，LPS+水龙提取物(终浓度120μg/mL、240μg/mL)，LPS+地塞米松组(地塞米松终浓度10μg/mL)，每组三个复孔。以Griess法测定NO的生成量，水龙提取物抗炎活性检测试验结果如图5所示。
图5中结果显示，水龙提取物在120μg/mL、240μg/mL时，可以显著抑制LPS刺激引起的NO分泌；用CCK8法测定水龙提取物对RAW264.7细胞的生长抑制作用，结果发现在120μg/mL、240μg/mL时不抑制细胞的生长。水龙提取物通过抑制由LPS刺激单核巨噬细胞引起的NO分泌起到抗炎作用。
水龙提取物对胶原酶抑制活性检测试验：
请参阅图6，以四环素为对照，测定实施例1所得水龙提取物对胶原酶的抑制作用。
以N-(3-[2-furyl]Acryloyo)-Leu-Gly-Pro-Ala(FALGPA)为底物，用50mmol/mLTricine，0.4mol/L NaCl，10mmol/mL CaCl2 pH＝7.5的缓冲液调节底物FALGPA的浓度，使其在波长305nm处的吸光值在0.7左右。取96孔板，每孔加入0.2mg/mL的胶原酶液140uL，分别加入不同浓度的样品溶液以及四环素溶液60ul，37℃保温20min后，加入底物溶液40uL，反应总体积为240ul，同时设100％酶活性对照孔，即用60uL PBS代替样品溶液，测定335nm的吸光度变化情况。
胶原酶抑制率＝(空白组吸光度变化值-样品组吸光度变化值)/空白组吸光度变化值。
水龙提取物对胶原酶抑制活性检测试验结果如图6所示，水龙提取物在0.65mg/mL到2.6mg/mL区间内，对胶原酶具有抑制作用，随着浓度的增加而增强。
采用本发明的制备方法制得的水龙提取物，在维生素C清除DPPH自由基的IC50值为30μg/mL时，水龙提取物对DPPH自由基清除的IC50值低于100μg/mL，与维生素C的DPPH自由基清除能力相当；且在维生素C清除ABTS自由基的IC50值为100μg/mL时，水龙提取物对ABTS自由基清除的IC50值低于200μg/mL，与维生素C的ABTS自由基清除能力相当。水龙提取物在120μg/mL、240μg/mL时，可以显著抑制LPS刺激引起的NO分泌；水龙提取物在0.65mg/mL到2.6mg/mL区间内，对胶原酶具有抑制作用，随着浓度的增加而增强。
采用本发明的制备方法制得的水龙提取物，具有较强的抗氧化作用和抗衰作用，可以作为抗炎、抗衰成分加入护肤品或功能性食品配方中。
综上所述，本发明的水龙提取物的制备方法，可以确保水龙中的化学成分不被破坏，从而最大程度的保持其生物活性；采用本发明的制备方法所得的水龙提取中的没食子酸和没食子酸乙酯含量稳定，具有显著的抗氧化能力和抗炎活性，可以作为功效性护肤品或功能性食品中的活性成分，起到抗炎、抗衰老等作用。
本技术领域中的普通技术人员应当认识到，以上的实施例仅是用来说明本发明，而并非用作对本发明的限定，只要在本发明的实质精神范围内，对以上所述实施例的变化、变型都将落在本发明的权利要求书范围内。
Claims (3)
1.一种水龙提取物的制备方法，其特征在于，包括以下步骤：
S1，低温提取步骤：采用醇/水混合液对水龙进行低温提取得到提取液，所述醇/水混合液中醇的体积分数为10％～40％，所述醇/水混合液的使用重量为所述水龙细粉重量的3～16倍；所述低温提取的温度为0～25℃；所述低温提取的时间为12～96h；所述水龙采用柳叶菜科水龙属植物水龙Jussiaea repens L的茎叶；所述醇/水混合液中的醇采用甲醇、乙醇或者1，3-丁二醇，或者采用甲醇和乙醇的混合液；低温提取过程中采用低速搅拌，所述低速搅拌的搅拌速度低于100r/min；
S2，过滤步骤：将所述提取液采用孔径为0.22μm～10μm的滤膜进行过滤，得澄清透明液体；所述过滤采用负压抽滤或者正压压滤；
S3，脱色步骤：将经过过滤得到的澄清透明液体采用脱色剂进行脱色处理，所述脱色剂的使用量为所述澄清透明液体质量的0.1％～2％；脱色处理时间为0.5小时～2小时；然后过滤除去脱色剂后得到水龙提取液；所述脱色剂采用活性炭；
S4，低温干燥步骤：将所述水龙提取液在不超过40℃的温度下进行低温干燥即得水龙提取物；所述低温干燥采用冷冻干燥、低温烘干、减压浓缩干燥或喷雾干燥；
采用高效液相色谱法对步骤S4得到的水龙提取物中的没食子酸含量进行测定，所述水龙提取物中没食子酸含量大于5％，没食子酸乙酯含量大于2％。
2.一种如权利要求1所述的水龙提取物的制备方法制得的水龙提取物在制备护肤品或功能性食品中的用途。
3.如权利要求2所述的一种水龙提取物的用途，其特征在于，所述水龙提取物清除DPPH自由基的IC50值小于100μg/mL，清除ABTS自由基的IC50值小于100μg/mL；所述水龙提取物通过抑制由LPS刺激单核巨噬细胞引起的NO分泌起到抗炎作用；所述水龙提取物用于抑制胶原蛋白酶的活性。
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