CN113633705A - 一种具有抗氧化活性的三叶青根茎多酚提取物及其质量控制方法和用途 - Google Patents
一种具有抗氧化活性的三叶青根茎多酚提取物及其质量控制方法和用途 Download PDFInfo
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- CN113633705A CN113633705A CN202110896253.1A CN202110896253A CN113633705A CN 113633705 A CN113633705 A CN 113633705A CN 202110896253 A CN202110896253 A CN 202110896253A CN 113633705 A CN113633705 A CN 113633705A
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- extract
- rhizome
- radix tetrastigme
- ethanol
- polyphenol extract
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Abstract
本发明公开了一种具有抗氧化活性的三叶青根茎多酚提取物及其质量控制方法和用途，所述三叶青根茎多酚提取物是由以下步骤制备所得：(1)以乙醇‑水混合溶液为提取剂，对三叶青根茎进行回流提取，提取液浓缩，干燥，得粗提物；所述乙醇‑水混合溶液的体积百分数为50％至70％，料液比为1：(5‑20)；(2)将步骤(1)所得粗提取物溶解于水中，采用有机溶剂进行萃取，萃取液浓缩，得萃取浸膏；(3)将上述萃取浸膏溶解，上样于大孔吸附树脂色谱柱，依次用不同体积百分数的乙醇‑水混合溶液梯度洗脱，收集40％至90％乙醇‑水混合溶液洗脱物，减压浓缩，即得。所述三叶青根茎多酚提取物表现出显著的体外抗氧化作用，能够应用于制药、食品及保健品领域。
Description
技术领域
本发明属于天然药物化学领域，具体涉及一种具有抗氧化活性的三叶青根茎多酚提取物及其质量控制方法和用途。
背景技术
自由基是人体进行生命活动时所产生的一种活性分子，具有调节细胞间信号传递与细胞生长、抑制病毒和细菌的作用。当自由基过量时，会诱发各种疾病，并加速机体衰老。炎症、肿瘤、衰老、心血管等疾病的发生机理与体内自由基产生过多或清除自由基能力下降有着密切的关系。清除多余自由基有益于某些疾病的预防和治疗，对人体健康意义重大。抗氧化物质是指能够清除氧自由基，抑制或消除以及减缓氧化反应的一类物质。天然抗氧化剂主要是指在生物体内(动物、植物、微生物)合成的具有抗氧化作用或诱导抗氧化剂产生的一类物质，有安全性高、无副作用的特点。天然抗氧剂主要来自植物，植物提取物，尤其是中草药提取物市场前景广阔，发展潜力巨大。
三叶青是葡萄科崖爬藤属植物三叶崖爬藤(Tetrastigmatis hemsleyani Dielset Gilg.)异名：石猴子、石抱子、拦山虎、破石珠、土经丸三叶对、小扁藤、阴灵子、三叶扁藤、金线吊葫芦、丝线吊金钟、金线吊马铃薯，主要分布于江南的浙江、安徽、江西、湖南、湖北及西南等省。目前对于三叶青的活性提取物研究报道较少，其药效作用物质基础尚不明确。这些都严重限制了三叶青作为药品、化妆品、功能性食品原料的开发与利用。
发明内容
发明目的：针对上述技术问题，本发明提供了一种具有抗氧化活性的三叶青根茎多酚提取物及其质量控制方法和用途，从三叶青根茎多酚提取物中鉴定了6个具有抗氧化活性的成分。该提取物可以用作预防、延缓衰老的药品、化妆品及功能性食品的原料。
技术方案：为了达到上述发明目的，本发明所采用的技术方案如下：
一种具有抗氧化活性的三叶青根茎多酚提取物，所述提取物是由以下步骤制备所得：
(1)以乙醇-水混合溶液为提取剂，对三叶青根茎进行回流提取，提取液浓缩，干燥，得粗提物；所述乙醇-水混合溶液的体积百分数为50％至70％(乙醇：水)，料液比为1：(5-20)；
(2)将步骤(1)所得粗提取物溶解于水中，采用有机溶剂进行萃取，萃取液浓缩，得萃取浸膏；
(3)将上述萃取浸膏溶解，上样于大孔吸附树脂色谱柱，依次用不同体积百分数的乙醇-水混合溶液梯度洗脱，收集40％至90％乙醇-水混合溶液洗脱物，减压浓缩，即获得所述三叶青根茎多酚提取物。
作为优选：
步骤(1)中，所述的三叶青根茎为崖爬藤属植物三叶崖爬藤(Tetrastigmatishemsleyani Diels et Gilg.)的根茎。
步骤(2)中采用有机溶剂进行萃取，萃取液浓缩的方法如下：依次采用石油醚和乙酸乙酯进行萃取，乙酸乙酯萃取液经减压浓缩后，获得乙酸乙酯萃取浸膏。
步骤(3)中，所述吸附树脂为大孔吸附树脂HPD100或D101。
所述三叶青根茎多酚提取物中：
(1)水分不超过6.0％、炽灼残渣不超过13.0％和总灰分不超过11.0％；
(2)按干燥品计，含芦丁不得少于1.50％，异槲皮苷不得少于1.90％，紫云英苷不得少于1.30％，山萘酚-3-O-芸香糖苷不得少于6.60％。
所述的三叶青根茎多酚提取物的质量控制方法，包括以下项目：
(1)鉴别：采用的薄层色谱法，以芦丁、山萘酚-3-O-芸香糖苷和山萘酚为对象辨别真伪；
(2)检查：按照《中国药典》中方法进行检测，项目有水分不得过6.0％、炽灼残渣不得过13.0％和总灰分不得过11.0％；
(3)含量测定：采用高效液相色谱法，以芦丁、异槲皮苷、紫云英苷和山萘酚-3-O-芸香糖苷为活性成分，本品按干燥品计，含芦丁不得少于1.50％，异槲皮苷不得少于1.90％，紫云英苷不得少于1.30％，山萘酚-3-O-芸香糖苷不得少于6.60％。
一种组合物，该组合物包含所述三叶青根茎多酚提取物，以及药学可接受的载体或赋形剂、食品可接受的食品添加剂或化妆品可接受的添加剂。
所述药学可接受的载体或赋形剂选自稀释剂、分散剂、助悬剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、粘合剂、润滑剂、稳定剂、水合剂、乳化加速剂、缓冲剂、吸收剂、着色剂、香味剂、甜味剂、离子交换剂、脱模剂、涂布剂、矫味剂、和抗氧化剂等。
所述食品可接受的添加剂选自酸度调节剂、抗结剂、消泡剂、漂白剂、膨松剂、着色剂、增味剂、护色剂、酶制剂、营养强化剂、防腐剂、甜味剂、抗氧化剂、增稠剂、和香料等。
所述化妆品可接受的添加剂选自酸度调节剂、表面活性剂、保湿剂、抗氧化剂、防腐和防霉剂、色素和特殊添加物等。
所述三叶青根茎多酚提取物、或所述的组合物在制备用作预防、延缓衰老的药品、化妆品或功能性食品原料的应用。
本发明的提取分离工艺特点如下：采用一定浓度的乙醇-水混合溶液对三叶青根茎进行提取---按极性进行不同有机溶剂萃取---结合大孔吸附树脂D101柱色谱富集目标化合物，Q-TOF LC/MS和分析型高效液相进行抗氧化活性成分的确认。通过这一流程，能够最大程度富集三叶青根茎中抗氧化性成分。
有益效果：相对于现有技术，本发明首次提供了以三叶青根茎为原料，以乙醇-水混合溶液为提取剂，结合大孔吸附树脂，富集抗氧化活性成分，得到了含有多个抗氧化活性成分的提取物，通过实验证实，该提取物能够有效清除自由基，抗氧化性能优异。同时，本发明还建立了该提取物的质量控制方法，并阐明了其在药物、化妆品及食品中的应用。
附图说明
图1为三叶青根茎多酚提取物的未加任何试剂的液相色谱图；
图2为三叶青根茎多酚提取物的抗氧化性成分在线抗DPPH的液相色谱图；
图3为三叶青根茎多酚提取物的抗氧化性成分在线抗羟自由基的液相色谱图；
图4为三叶青根茎多酚提取物的总离子流图；
图5为三叶青根茎多酚提取物的抗氧化性成分的液相色谱图，其中，1.芦丁；2.异槲皮苷；3.紫云英苷；4.槲皮素；5.山奈酚；6.山奈酚-3-O-芸香糖苷。
图6三叶青根茎多酚提取物的实物图片。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明，但有必要指出以下实施例只用于对发明内容的描述，并不构成对本发明保护范围的限制。
下述制备例中，所涉及主要仪器设备为安捷仑1260型高效液相色谱仪，DAD检测器，6520型Q-TOF质谱检测器；岛津UV-1800型紫外-可见分光光度计；梅特勒十万分之一天平，密理博Milli-Q TM Integral超纯水机，宁波新芝SB-120DT-超声波清洗机；所用填料：大孔树脂(上海源叶生物公司)，液相色谱柱C18(日本岛津)；所用试剂，DPPH、ABTS、TPTZ，Trolox均购自Sigma公司，其他试剂均为分析纯，购自国药集团化学试剂有限公司。所用药材，三叶青购自安徽亳州药材市场，经浙江医药高等专科学校夏苗芬教授鉴定为三叶青的根茎(Tetrastigmatis hemsleyani Diels et Gilg.Rhizome)。
实施例1：制备三叶青根茎多酚提取物
1.制备三叶青根茎粗提物。
(1)取1.0kg干燥的三叶青根茎，以体积百分数为70％的乙醇-水混合溶液为提取溶剂，料液比为1:10，回流提取三次，每次1h。合并提取液，减压浓缩即得所述粗提取物。
(2)取1.0kg干燥的三叶青根茎，以体积百分数为60％的乙醇-水混合溶液为提取溶剂，料液比为1:10，回流提取三次，每次1h。合并提取液，减压浓缩即得所述粗提取物；
(3)取1.0kg干燥的三叶青根茎，以体积百分数为50％的乙醇-水混合溶液为提取溶剂，料液比为1:10，回流提取三次，每次1h。合并提取液，减压浓缩即得所述粗提取物；
2.制备具有抗氧化活性的三叶青根茎多酚提取物。
取步骤1中粗提物，溶于水中，依次用石油醚和乙酸乙酯各萃取三次，水溶液和萃取液的体积比为1:1。乙酸乙酯萃取部分，减压浓缩后，水溶解，上样于大孔吸附树脂D101柱色谱，依次用水、体积百分数为20％、40％、60％和90％的乙醇-水梯度洗脱，收集40％至90％乙醇-水混合溶液洗脱物，减压浓缩，获得具有抗氧化活性的三叶青根茎多酚提取物，如图6所示。
3.三叶青根茎多酚提取物中抗氧化活性成分的确认
(1)优选色谱条件：C18色谱柱(4.6mm*150mm,5μm)，以乙腈(A)－0.1％三氟乙酸水溶液(B)为流动相，梯度洗脱(0～5min,18％A→25％A；5～10min,35％A；10～15min,60％A；15～20min,90％A；20～30min,18％A)，流速1.0ml/min，检测波长254nm，进样量20μL。
(2)将所述三叶青根茎多酚提取物溶液经0.45μm的膜过滤后注入高效液相色谱仪中，按步骤(1)的条件测定，如图1所示，图中9个色谱峰分别代表9个不同的化学成分。
(3)将所述三叶青根茎多酚提取物溶液与DPPH溶液混合均匀，避光孵化30min后,经0.45μm的膜过滤后注入高效液相色谱仪中，按步骤(1)的条件测定，如图2所示，图中9个色谱峰的峰面积和峰高均减小，可以推测这9个化学成分均能清除DPPH自由基。
(4)将所述三叶青根茎多酚提取物溶液与H2O2溶液、FeSO4溶液(1:1,v/v)混合均匀，避光孵化7min后,经0.45μm的膜过滤后注入高效液相色谱仪中，按步骤(1)的条件测定，如图3所示，图中9个色谱峰的峰面积和峰高均减小，可以推测这9个化学成分均能清除羟自由基。
(5)对三叶青根茎多酚提取物中能清除DPPH自由基和羟自由基的成分的分子量用液相色谱串联四极杆飞行时间质谱仪(Q-TOF LC/MS)测定，在负离子源下记录图谱，如图4所示。流动相系统是C18色谱柱(50×2.1mm,1.8μm)；流动相：以乙腈(A)－0.1％三氟乙酸水溶液(B)为流动相，梯度洗脱(0～5min,18％A→25％A；5～10min,35％A；10～15min,60％A；15～20min,90％A；进校体积1μL，柱温：室温，液相流量0.2mL/min，扫描范围：50～1500，毛细管电压：4000V。以流速为10mL/min，温度为350℃的氮气为干燥气体。雾化器压力为40psi。
确定抗氧化成分的分子量之后，用相关物质的标准品通过高效液相色谱法进一步去验证。按步骤(1)的条件测定，如图5所示。
最终确认上述三叶青根茎多酚提取物中抗氧化成分为芦丁、异槲皮苷、紫云英苷、槲皮素、山奈酚和山奈酚-3-O-芸香糖苷。
实施例2：建立三叶青根茎多酚提取物的质量控制方法
1.鉴别：取本品0.2g，加60％乙醇25ml，溶解后，滤过，作为供试品溶液。另取芦丁、山萘酚与山萘酚-3-O-芸香糖苷对照品，分别加60％乙醇制成每1ml含0.1mg的溶液，作为对照品溶液。照薄层色谱法(《中国药典》2020年版通则0502)试验，分别吸取芦丁、山萘酚-3-O-芸香糖苷对照品溶液与供试品溶液约5μl，分别点于同一硅胶G薄层板上，以甲苯︰乙酸乙酯︰甲酸(1︰3︰1.5)，展开，取出，晾干，喷以2％三氯化铝溶液，晾干，置365nm下检视；再吸取山萘酚对照品溶液与供试品溶液约5μl，分别点于同一硅胶G薄层板上，以甲苯︰乙酸乙酯︰甲酸(3︰1︰1)，展开，取出，晾干，喷以2％三氯化铝溶液，晾干，置365nm下检视。供试品色谱中，在与对照品色谱相应的位置上，应显相同颜色的斑点。
实施例1所得三叶青根茎多酚提取物，按照上述方法进行鉴别，在与对照品色谱相应的位置上，均能显相同颜色的斑点。
2.检查：水分按照《中国药典》2020年版通则0832第二法操作；炽灼残渣按照《中国药典》2020年版通则0841操作；总灰分按照《中国药典》2020年版通则2302第一法操作。
按照上述方法进行检查，实施例1所得三叶青根茎多酚提取物中，水分分别为5.85％、5.39％和5.94％；炽灼残渣分别为11.8％、12.3％和12.4％；总灰分分别为10.5％、10.1％和9.7％；
3.含量测定：照高效液相色谱法(《中国药典》2020年版通则0512)测定。
色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以0.1％三氟乙酸的水为流动相A，以0.1％三氟乙酸的乙腈为流动相B；检测波长为254nm。理论板数按芦丁、异槲皮苷、紫云英苷和山萘酚-3-O-芸香糖苷峰计算均应不低于3000。
对照品溶液的制备 分别取芦丁、异槲皮苷和紫云英苷适量，加70％乙醇制成每1mL中含有芦丁和紫云英苷20μg、异槲皮苷30μg的混合对照品溶液(Ⅰ)；再取山萘酚-3-O-芸香糖苷对照品适量，加70％乙醇制成每1mL中含山萘酚-3-O-芸香糖苷50μg的对照品溶液(Ⅱ)。
供试品溶液的制备 取本品约0.2g，精密称定，置50mL量瓶中，加70％乙醇溶解，并稀释至刻度，摇匀，即得。
测定法 分别精密吸取对照品溶液(Ⅰ)、对照品溶液(Ⅱ)与供试品溶液各10μl，注入液相色谱仪，测定，即得。
按照上述方法进行含量测定，实施例1所得三叶青根茎多酚提取物，按干燥品计，含芦丁分别为1.69％、1.58％和1.80％；含异槲皮苷分别为2.63％、2.27％和2.05％；含紫云英苷分别为1.43％、1.38％和1.56％；含山萘酚-3-O-芸香糖苷分别为7.82％、7.26％和6.84％。
实施例3：三叶青根茎多酚提取物的的抗氧化活性测试
1.DPPH测定：将DPPH 80％乙醇溶液(0.0395mg/mL)3.0mL与0.1mL不同浓度样品溶液(0.2mg/mL-4mg/mL)混合，混匀，室温、暗处放置30min，在517nm处测吸光度(A1)，同时测定0.1mL 80％乙醇与3.0ml DPPH 80％乙醇溶液混合液的测吸光度(A0)。计算公式：清除率％＝[1-(A1-A2)/A0]×100％。重复测定3次，结果用平均值±SD表示。
2.TEAC法(Trolox equivalent antioxidant capacity assay)
将7.0mM ABTS与4.95mM过硫酸钾溶液等体积混合，于室温、暗处反应1 2h，形成蓝绿色的ABTS·+溶液，经适当稀释并在734nm处的吸光度调至0.70±0.02。取3.0mL上述ABTS·+溶液与20μL样品溶液混合，混匀，30℃水浴反应10min，于734nm波长处测定吸光度(A1)，同时测定3.0mLABTS·+与20μL溶剂混合溶液的吸光度(A2)，3.0mL溶剂与20μL样液混合溶液的吸光度(A0)。计算吸光度减少值ΔA[ΔATrolox＝(A2-A1)-A0]，根据Trolox反应标准曲线计算样液抗氧化能力相当于标准品Trolox清除ABTS·+能力的比值，即TEAC值。重复测定3次，结果用平均值±SD表示。
3.FRAP法
取样品溶液各20μL，加3.0mL FRAP试剂[按照300mM醋酸盐缓冲液(pH 3.6)︰10mMTPTZ溶液︰20mM FeCl3·6H2O溶液(10︰1︰1)的比例将三种溶液混合，摇匀，即得]，混匀后，37℃水浴反应30min，用蒸馏水做空白，于593nm波长处测定吸光度。以系列浓度FeSO4(0.1-1.0mM)作标准曲线，样品溶液抗氧化能力以达到同样吸光度所需FeSO4的物质的量计算FRAP值。重复测定3次，结果用平均值±SD表示。
4.羟基自由基清除法
依次加入1.0mL磷酸盐缓冲液(PBS，pH7.4)、0.5mL(0.148mg·mL-1)邻二氮菲溶液、0.5mL(0.208mg·mL-1)硫酸亚铁溶液、1.0mL样品溶液，立即混匀后，加入1.0mL(0.01％,v/v)过氧化氢溶液，立即混匀，37℃水浴反应60min，在536nm波长下测吸光度。以1.0mL 60％甲醇替代样品溶液，迅速混入1.0mL过氧化氢溶液，待反应结束后测其吸光度(A损)。以1.0mL60％甲醇替代样品溶液与过氧化氢溶液，立即混匀，待反应结束后测其吸光度(A未损)。计算公式：清除率％＝[(A样-A损)/(A损-A未损)]×100％。重复测定3次，结果用平均值±SD表示。
结果如表1所示。
表1三叶青根茎多酚提取物抗氧化能力
Claims (9)
1.一种具有抗氧化活性的三叶青根茎多酚提取物，其特征在于，所述三叶青根茎多酚提取物是由以下步骤制备所得：
(1)以乙醇-水混合溶液为提取剂，对三叶青根茎进行回流提取，提取液浓缩，干燥，得粗提物；所述乙醇-水混合溶液的体积百分数为50％至70％，料液比为1：(5-20)；
(2)将步骤(1)所得粗提取物溶解于水中，采用有机溶剂进行萃取，萃取液浓缩，得萃取浸膏；
(3)将上述萃取浸膏溶解，上样于大孔吸附树脂色谱柱，依次用不同体积百分数的乙醇-水混合溶液梯度洗脱，收集40％至90％乙醇-水混合溶液洗脱物，减压浓缩，即获得所述三叶青根茎多酚提取物。
2.根据权利要求1所述的三叶青茎多酚提取物，其特征在于，步骤(1)中，所述的三叶青根茎为崖爬藤属植物三叶崖爬藤(Tetrastigmatis hemsleyani Diels et Gilg.)的根茎。
3.根据权利要求1所述的三叶青根茎多酚提取物，其特征在于，步骤(2)中采用有机溶剂进行萃取，萃取液浓缩的方法如下：依次采用石油醚和乙酸乙酯进行萃取，乙酸乙酯萃取液经减压浓缩后，获得乙酸乙酯萃取浸膏。
4.根据权利要求1所述的三叶青根茎多酚提取物，其特征在于，步骤(3)中，所述大孔吸附树脂为大孔吸附树脂HPD100或D101。
5.根据权利要求1所述的三叶青根茎多酚提取物，其特征在于，所述三叶青根茎多酚提取物中：
(1)水分不超过6.0％、炽灼残渣不超过13.0％和总灰分不超过11.0％；
(2)按干燥品计，含芦丁不得少于1.50％，异槲皮苷不得少于1.90％，紫云英苷不得少于1.30％，山萘酚-3-O-芸香糖苷不得少于6.60％。
6.权利要求1-5任一项所述的三叶青根茎多酚提取物的质量控制方法，其特征在于，包括以下项目：
(1)鉴别：采用的薄层色谱法，以芦丁、山萘酚-3-O-芸香糖苷和山萘酚为对象辨别真伪；
(2)检查：按照《中国药典》中方法进行检测，项目有水分不得过6.0％、炽灼残渣不得过13.0％和总灰分不得过11.0％；
(3)含量测定：采用高效液相色谱法，以芦丁、异槲皮苷、紫云英苷和山萘酚-3-O-芸香糖苷为活性成分，本品按干燥品计，含芦丁不得少于1.50％，异槲皮苷不得少于1.90％，紫云英苷不得少于1.30％，山萘酚-3-O-芸香糖苷不得少于6.60％。
7.一种组合物，其特征在于，该组合物包含权利要求1-5任一项所三叶青根茎多酚提取物，以及药学可接受的载体或赋形剂、食品可接受的食品添加剂或者化妆品可接受的添加剂。
8.权利要求1-5任一项所述三叶青根茎多酚提取物、或权利要求7所述的组合物在制备预防、延缓衰老的药品、化妆品或功能性食品原料中的应用。
9.根据权利要求8所述的应用，其特征在于，所述预防、延缓衰老包括清除自由基、预防由自由基引起的衰老，改善面色黯淡、提亮肤色、肌肤松驰、淡化细纹。
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