CN112791126A - 牡丹花蕊提取物及其制备方法与应用 - Google Patents
牡丹花蕊提取物及其制备方法与应用 Download PDFInfo
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- CN112791126A CN112791126A CN202110187939.3A CN202110187939A CN112791126A CN 112791126 A CN112791126 A CN 112791126A CN 202110187939 A CN202110187939 A CN 202110187939A CN 112791126 A CN112791126 A CN 112791126A
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- peony
- peony pistil
- pistil
- ethyl acetate
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Abstract
本发明公开了牡丹花蕊提取物及其制备方法与应用。本发明所提供的牡丹花蕊提取物的制备方法,包括以下步骤:(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;(2)使用水性醇液对所述牡丹花蕊粉末进行超声提取,收集并合并提取液,得到牡丹花蕊醇提取物水溶液;(3)向步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,用乙酸乙酯萃取,得到牡丹花蕊乙酸乙酯萃取物;(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物用D101大孔吸附树脂柱进行纯化,收集洗脱液,浓缩至浸膏状态,得到牡丹花蕊目标提取物。
Description
技术领域
本发明涉及生物技术领域,特别地涉及牡丹花蕊提取物及其制备方法与应用。
背景技术
牡丹(PaeoniasuffruticosaAndr.)是芍药科(Paeoniaceae)、芍药属(PaeoniaL.)植物,为多年生落叶灌木。牡丹不仅是中国特有的木本名贵花卉,有数千年的自然生长和2000多年的人工栽培历史,而且是一种传统的药食两用的植物资源。牡丹花蕊是一个潜在的功能性食品。牡丹花蕊中碳水化物、蛋白质、不饱和脂肪酸和矿物质含量丰富,能够补充人体所需营养。
目前关于牡丹花蕊的相关研究多集中在食品领域。CN103190504B披露了一种牡丹花蕊茶的加工方法,包括下述的步骤:原料选择、晾制、干燥、大杂及花粉分离、花茶筛选、成型、灭菌。CN104524322B披露了一种牡丹花蕊前列腺袋泡茶及生产方法,其生产方法步骤如下:步骤如下:(1)牡丹花蕊晒干或烘干,筛去外粘花粉,含水量在9-13%;(2)山楂片焙炒,焙炒的火候掌握为160-190℃,2-5min,至外面焦褐色里面黄褐色即可;(3)然后将焦山楂片与薏苡仁、茯苓分别用破碎机打成颗粒状,筛除不能通过10目的大颗粒和能通过100目筛孔的细粉;(4)按照配方比例,将上述四种物料混合均匀。CN110679819A披露了一种牡丹花蕊固体饮料及其制备方法,其制备方法包括以下步骤:将喷雾干燥粉末、牡丹花蕊干粉、固体饮料添加剂混合均匀,即得。CN110507622A披露了一种牡丹花蕊片的制作工艺,其制作步骤如下:第一步:采摘含苞待放的牡丹花蕊,将牡丹花蕊进行干燥挑选;第二步:将第一步处理过的牡丹花蕊取出3-8份,使用超临界二氧化碳进行萃取,制成膏;第三步:将第一步处理过的牡丹花蕊取出1-5份,采用水热回流提取法,经过浓缩、喷雾干燥后,制成粉;第四步:取中期蕊1-3份,经过低温粉碎成粉;第五步:将第三步制作出来的喷雾干燥粉和第四步的低温粉碎粉混合搅拌均匀;第六步:将第五步混合均匀后的物质加入到第二步的膏中,然后进行彻底均质,制成粒,然后沸腾干燥;第七步:将第六步沸腾干燥后的混合物内加入硬脂酸镁,然后进行压片、凉片后包装。
以上所公开的牡丹花蕊产品或制剂,主要是基于牡丹花蕊的传统认识和功效,产品以花蕊茶为主,制备方法也主要是简单干燥、水提,并缺少对提取物的药用价值分析。
发明内容
有鉴于此,本发明的主要目的是提供牡丹花蕊提取物及其制备方法与应用。
为实现上述目的,根据本发明的一个方面,提供了牡丹花蕊提取物的制备方法。
本发明所提供的牡丹花蕊提取物的制备方法,包括以下步骤:
(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;
(2)使用体积百分比浓度为40%-60%的水性醇液对所述牡丹花蕊粉末进行1次或多次超声提取,收集并合并提取液,从所述提取液中除去所述醇,得到牡丹花蕊醇提取物水溶液;
(3)向步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,至相对密度为1.10-1.20,用乙酸乙酯萃取1次或多次,收集并合并乙酸乙酯萃取液,浓缩至干浸膏,得到牡丹花蕊乙酸乙酯萃取物;
(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物用10%-30%体积的乙醇溶解后,用D101大孔吸附树脂柱进行纯化,其中所述纯化依次使用体积百分比浓度为10%-30%的水性醇溶液作为第一洗涤液和体积百分比浓度为50%-60%的水性醇溶液作为第二洗涤液进行洗脱,分别得到第一洗脱液和第二洗脱液,收集第二洗脱液,浓缩至浸膏状态,得到牡丹花蕊目标提取物。
步骤(4)中第一洗脱液主要目的是去除杂质,第二洗脱液主要目的是富集有效成分。步骤(4)中影响目标提取物的最关键因素是洗脱液的水性醇浓度。
所述步骤(2)中所述牡丹花蕊粉末与所述水性醇液的料液比为1g:(10-15)ml,所述超声提取是在22℃-27℃下超声提取2次-3次,每次15分钟-30分钟。
所述步骤(3)中乙酸乙酯为水饱和的乙酸乙酯溶液,以乙酸乙酯:水按体积比1:0.8-1.5加入。所述乙酸乙酯为纯的乙酸乙酯溶液,“水饱和的乙酸乙酯”,即先把乙酸乙酯和水混合、振荡,再静置,待分层以后,取乙酸乙酯层(上层)。
步骤(3)中选择乙酸乙酯做萃取溶剂,是因为一方面乙酸乙酯的极性适中,适合萃取富集牡丹花蕊中的有效成分,另一方面乙酸乙酯毒性小,与水的分层充分,不容易乳化,很适合萃取操作。萃取操作可以瞬间或短时间完成。
所述步骤(4)中所述水性醇溶液为乙醇-水混合溶剂。
所述步骤(3)中所述浓缩至干浸膏是在温度为50℃、压力为0.05MPa的条件下减压浓缩至干浸膏。
由上述牡丹花蕊提取物的制备方法制备得到牡丹花蕊提取物也属于本发明的保护范围。
根据本发明的另一个方面,提供了一种脂肪酸合酶抑制剂。
本发明所提供的脂肪酸合酶抑制剂,其活性成分为上述方法制备得到的牡丹花蕊提取物。
上述方法制备得到的牡丹花蕊提取物在制备诱导癌症细胞凋亡的产品或药物中的应用也属于本发明的保护范围。
所述癌症细胞为乳腺癌细胞MDA-MB-231。
上述方法制备得到的牡丹花蕊提取物在制备具有减肥或抗癌功效的药物、化妆品或食品中的用途也属于本发明的保护范围。
本发明提供牡丹花蕊提取物的制备方法具有以下特点或优势:
(1)不需要进行脱脂处理,即没有使用石油醚或己烷除去原料中的脂溶性杂质;不需要使用己烷或石油醚(主要成分是己烷、庚烷等烷烃),因此大大增加了产品的安全性,并可节约成本,简化工艺,减少废液排放。由于本发明所用的步骤中包含有大孔树脂吸附环节,可以有效去除脂溶性杂质,因此不必单独脱脂处理。由于本发明产品主要目的之一是得到对脂肪酸合酶抑制能力强的提取物,因此某些脂溶性成分是不必去除的。
(2)选用大孔吸附树脂进行分离,样品处理量大,工艺简单,操作方便;价格低廉,所用的有机溶剂安全无毒;两次洗脱即可实现除杂和富集有效成分的目的。
(3)分段洗脱:第一洗脱液是体积百分比浓度为10%-30%的乙醇水溶液,第二洗脱液是体积百分比浓度为40%-60%的乙醇水溶液。第一洗脱液主要是去除亲水性杂质,洗脱液弃去;第二洗脱液主要是富集有效成分。
附图说明
为了说明而非限制的目的,现在将根据本发明的优选实施例、特别是参考附图来描述本发明,其中:
图1为本发明牡丹花蕊提取物与三种目前已知的抑制剂对于脂肪酸合酶活性的抑制能力的对比结果;其中1为实施例1的本发明牡丹花蕊提取物A、2为实施例2的本发明牡丹花蕊提取物B、3为实施例3的本发明牡丹花蕊提取物C、4为cerulenin、5为EGCG、6为白藜芦醇;纵座标单位为微克/毫升。
图2为本发明牡丹花蕊提取物对乳腺癌细胞MDA-MB-231活力的影响结果。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1、制备牡丹花蕊提取物A
一、牡丹花蕊提取物A的制备方法包括以下步骤:
(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;
(2)按料液比1g:15ml向步骤(1)得到的干燥的牡丹花蕊粉末中加入体积百分比浓度为50%的乙醇-水溶液,于22℃下超声提取3次,每次分别为30分钟、15分钟、15分钟,收集合并提取液,将得到的提取液减压浓缩至无醇味,得到牡丹花蕊醇提取物水溶液;
(3)向步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,至相对密度为1.10,以乙酸乙酯:水按体积比1:0.8加入水饱和的乙酸乙酯溶液萃取,22℃下萃取3次,合并萃取液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至干浸膏,得到牡丹花蕊乙酸乙酯萃取物;
(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物溶解于体积百分比浓度为10%的乙醇-水混合溶剂,过滤去除不溶物,滤液上D101大孔吸附树脂柱,静置5小时,以体积百分比浓度为10%的乙醇-水混合溶剂为第一洗脱液洗脱5个柱体积,再以体积百分比浓度为50%的乙醇-水混合溶剂作为第二洗脱液洗脱5个柱体积,收集合并第二洗脱液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至无醇味,并继续减压浓缩挥干溶剂得到浸膏,经进一步干燥得到粉末状态的目标牡丹花蕊提取物A。
二、牡丹花蕊提取物A对脂肪酸合酶(FAS)活性的抑制作用
取适量的牡丹花蕊提取物A溶于DMSO中作为受试样品溶液,用脂肪酸合酶活性的标准测定方法进行试验,并且所需的有效剂量很低。所用的脂肪酸合酶活性测定方法为采用体外对肿瘤细胞内酶活进行测定的常规方法。即将乳腺癌细胞MDA-MB-231细胞(购买自中国科学院典型培养物保藏委员会细胞库)破碎后,采用乙酰辅酶A、丙二酸单酰辅酶A、NADPH为底物的标准测活方法进行测定(Tian W.X.et al.,1985.J.Biol.Chem.,260:11375-11387;Li P.,et al.,2014.Mol Cancer 13:138)。
脂肪酸合酶(FAS)活性被抑制的程度用IC50(半抑制浓度)来表示,其计算方法为:以底物中只加入相应提取溶剂的为对照,测定其脂肪酸合酶活性值,该值用A0表示,并设定其为100%。底物中加入脂肪酸合酶抑制剂提取液的为实验组,测得其脂肪酸合酶活性的数值为A0的50%时,抑制剂的浓度为IC50值。
用三种目前已知的抑制剂对于脂肪酸合酶抑制活性的半抑制浓度做对比,脂肪酸合酶活性测定方法如前所述,为采用体外对肿瘤细胞内酶活进行测定的常规方法。三种对照在做活性测定前不需进行前处理,只是溶解于DMSO中,测定时所用浓度根据它们活力的大小而有不同。
对照1:Cerulenin。Cerulenin是一种广泛使用的天然脂肪酸合成酶(FAS)抑制剂。Cerulenin是由真菌Cephalosporiumcaeruleus产生的。本发明所用的Cerulenin购自Sigma-Aldrich公司,产品目录号为219557。
对照2:EGCG。EGCG(Epigallocatechin gallate)即表没食子儿茶素没食子酸酯,是绿茶茶多酚的主要组成成分,是从茶叶中分离得到的儿茶素类单体。本发明所用的EGCG购自Sigma-Aldrich公司,产品目录号为93894。
对照3:白藜芦醇。白藜芦醇存在于多种植物中,特别是葡萄皮中含量较大。本发明所用的白藜芦醇购自Sigma-Aldrich公司,产品目录号为34092。
结果如图1所示,图1为本发明牡丹花蕊提取物与三种目前已知的抑制剂对于脂肪酸合酶活性的抑制能力的对比结果。其中1为实施例1的本发明牡丹花蕊提取物A、4为cerulenin、5为EGCG、6为白藜芦醇;纵座标单位为微克/毫升。实施例1的牡丹花蕊提取物A对于脂肪酸合酶的半抑制浓度(IC50)为3.73μg/mL,而对照1(Cerulenin)、对照2(EGCG)、对照3(白藜芦醇)对于脂肪酸合酶的半抑制浓度(IC50)分别为22.6μg/mL、24.9μg/mL、11.8μg/mL,说明牡丹花蕊提取物A对脂肪酸合酶的活性有强烈的抑制作用。
三、牡丹花蕊提取物A对乳腺癌细胞MDA-MB-231活力的影响
采用CCK-8法测定细胞活力。将乳腺癌细胞MDA-MB-231接种于96孔板,贴壁过夜后使细胞密度为70%-80%,移去含血清的培养基,加入含不同药物浓度的无血清培养基,每个浓度的药物设置6个复孔。待药物处理细胞24h后,吸去培养基,每孔加入100μL新鲜无血清培养基和10μL CCK-8溶液,在37℃下孵育1-2h,然后用酶标仪检测450nm下的吸光值。数据为6个复孔的平均值,所有实验重复三次以上。以DMSO处理组细胞活力计为100%,计算牡丹花瓣提取物A处理组细胞的相对活力。
结果如图2所示,图2纵座标为MDA-MB-231细胞相对活力,横座标为不同浓度的本发明牡丹花蕊提取物A。结果显示,本发明牡丹花蕊提取物可以显著降低MDA-MB-231细胞活力(p<0.05)。
实施例2、制备牡丹花蕊提取物B
一、牡丹花蕊提取物B的制备方法包括以下步骤:
(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;
(2)按料液比1g:12ml向步骤(1)得到的干燥的牡丹花蕊粉末中加入体积百分比浓度为60%的乙醇-水溶液,于25℃下超声提取2次,每次分别为30分钟、15分钟,收集合并提取液,将得到的提取液减压浓缩至无醇味,得到牡丹花蕊醇提取物水溶液;
(3)将步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,至相对密度为1.20,以乙酸乙酯:水按体积比1:1加入水饱和的乙酸乙酯溶液萃取,25℃下萃取3次,合并萃取液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至干浸膏,得到牡丹花蕊乙酸乙酯萃取物;
(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物溶解于体积百分比浓度为30%的乙醇-水混合溶剂,过滤去除不溶物,滤液上D101大孔吸附树脂柱,静置8小时,以体积百分比浓度为30%的乙醇-水混合溶剂为第一洗脱液洗脱5个柱体积,再以体积百分比浓度为60%的乙醇-水混合溶剂作为第二洗脱液洗脱5个柱体积,收集合并第二洗脱液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至无醇味,并继续减压浓缩挥干溶剂得到浸膏,经进一步干燥得到粉末状态的目标牡丹花蕊提取物B。
二、牡丹花蕊提取物B对脂肪酸合酶(FAS)活性的抑制作用
取适量的牡丹花蕊提取物B于DMSO中作为受试样品溶液,用上述实施例1脂肪酸合酶活性的标准测定方法进行试验。
结果如图1所示,图1为本发明牡丹花蕊提取物与三种目前已知的抑制剂对于脂肪酸合酶活性的抑制能力的对比结果。其中2为实施例2的牡丹花蕊提取物B、4为cerulenin、5为EGCG、6为白藜芦醇;纵座标单位为微克/毫升。实施例2的牡丹花蕊提取物B对于脂肪酸合酶的半抑制浓度(IC50)为8.96μg/mL,而对照1(Cerulenin)、对照2(EGCG)、对照3(白藜芦醇)对于脂肪酸合酶的半抑制浓度(IC50)分别为22.6μg/mL、24.9μg/mL、11.8μg/mL,说明牡丹花蕊提取物B对脂肪酸合酶的活性有强烈的抑制作用,并且所需的有效剂量很低。
三、牡丹花蕊提取物B对乳腺癌细胞MDA-MB-231活力的影响
采用CCK-8法测定细胞活力,具体方法同实施例1。
结果与实施例1无显著差异,即本发明牡丹花蕊提取物可以显著降低MDA-MB-231细胞活力(p<0.05)。
实施例3、制备牡丹花蕊提取物C
一、牡丹花蕊提取物C的制备方法包括以下步骤:
(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;
(2)按料液比1g:15ml向步骤(1)得到的干燥的牡丹花蕊粉末中加入体积百分比浓度为40%的乙醇-水溶液,于27℃下超声提取1次,30分钟,收集提取液,将得到的提取液减压浓缩至无醇味,得到牡丹花蕊醇提取物水溶液;
(3)将步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,至相对密度为1.10,以乙酸乙酯:水按体积比1:1.5加入水饱和的乙酸乙酯溶液萃取,27℃下萃取3次,合并萃取液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至干浸膏,得到牡丹花蕊乙酸乙酯萃取物;
(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物溶解于体积百分比浓度为20%的乙醇-水混合溶剂,过滤去除不溶物,滤液上D101大孔吸附树脂柱,静置10小时,以体积百分比浓度为20%的乙醇-水混合溶剂为第一洗脱液洗脱5个柱体积,再以体积百分比浓度为60%的乙醇-水溶液混合溶剂作为第二洗脱液洗脱5个柱体积,收集合并第二洗脱液,在温度为50℃、压力为0.05MPa的条件下减压浓缩至无醇味,并继续减压浓缩挥干溶剂得到浸膏,经进一步干燥得到粉末状态的目标牡丹花蕊提取物C。
二、牡丹花蕊提取物C对脂肪酸合酶(FAS)活性的抑制作用
取适量的牡丹花蕊提取物C溶于DMSO中作为受试样品溶液,用上述实施例1脂肪酸合酶活性的标准测定方法进行试验。
结果如图1所示,图1为本发明牡丹花蕊提取物与三种目前已知的抑制剂对于脂肪酸合酶活性的抑制能力的对比结果。其中3为实施例3的牡丹花蕊提取物C、4为cerulenin、5为EGCG、6为白藜芦醇;纵座标单位为微克/毫升。实施例3的牡丹花蕊提取物C对于脂肪酸合酶的半抑制浓度(IC50)为5.46μg/mL,而对照1(Cerulenin)、对照2(EGCG)、对照3(白藜芦醇)对于脂肪酸合酶的半抑制浓度(IC50)分别为22.6μg/mL、24.9μg/mL、11.8μg/mL,说明牡丹花蕊提取物C对脂肪酸合酶的活性有强烈的抑制作用,并且所需的有效剂量很低。
三、牡丹花蕊提取物C对乳腺癌细胞MDA-MB-231活力的影响
采用CCK-8法测定细胞活力,具体方法同实施例1。
结果与实施例1无显著差异,即本发明牡丹花蕊提取物C可以显著降低MDA-MB-231细胞活力(p<0.05)。
上述实施例1~3中的牡丹花蕊提取物在制备具有减肥或抗癌功效的药物中的应用。
上述实施例1~3中的牡丹花蕊提取物在制备具有减肥或抗癌功效的保健食品中的应用。
上述实施例1~3中的牡丹花蕊提取物在制备具有减肥或抗癌功效的功能化妆品中的应用。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,取决于设计要求和其他因素,可以发生各种各样的修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
Claims (10)
1.牡丹花蕊提取物的制备方法,包括以下步骤:
(1)将牡丹花蕊干燥、粉碎,得到干燥的牡丹花蕊粉末;
(2)使用体积百分比浓度为40%-60%的水性醇液对所述牡丹花蕊粉末进行1次或多次超声提取,收集并合并提取液,从所述提取液中除去所述醇,得到牡丹花蕊醇提取物水溶液;
(3)向步骤(2)得到的牡丹花蕊醇提取物水溶液加水稀释,至相对密度为1.10-1.20,用乙酸乙酯萃取1次或多次,收集并合并乙酸乙酯萃取液,浓缩至干浸膏,得到牡丹花蕊乙酸乙酯萃取物;
(4)将步骤(3)得到的牡丹花蕊乙酸乙酯萃取物用体积百分比浓度为10%-30%的水性醇溶液溶解后,用D101大孔吸附树脂柱进行纯化,其中所述纯化依次使用体积百分比浓度为10%-30%的水性醇溶液作为第一洗涤液和体积百分比浓度为50%-60%的水性醇溶液作为第二洗涤液进行洗脱,分别得到第一洗脱液和第二洗脱液,收集第二洗脱液,浓缩至浸膏状态,得到牡丹花蕊目标提取物。
2.根据权利要求1所述的牡丹花蕊提取物的制备方法,其特征在于:
所述步骤(2)中所述牡丹花蕊粉末与所述水性醇液的料液比为1g:(10-15)ml,所述超声提取是在22℃-27℃下超声提取2次-3次,每次15分钟-30分钟。
3.根据权利要求1所述的牡丹花蕊提取物的制备方法,其特征在于:
所述步骤(3)中乙酸乙酯为水饱和的乙酸乙酯溶液,以乙酸乙酯:水按体积比1:0.8-1.5加入。
4.根据权利要求1所述的牡丹花蕊提取物的制备方法,其特征在于:所述步骤(4)中所述水性醇溶液为乙醇-水混合溶剂。
5.根据权利要求1所述的牡丹花蕊提取物的制备方法,其特征在于:所述步骤(3)中所述浓缩至干浸膏是在温度为50℃、压力为0.05MPa的条件下减压浓缩至干浸膏。
6.由权利要求1-5中任一所述的方法制备得到的牡丹花蕊提取物。
7.一种脂肪酸合酶抑制剂,其活性成分为权利要求1-5中任一所述方法制备得到的牡丹花蕊提取物。
8.权利要求1-5中任一所述的方法制备得到的牡丹花蕊提取物在制备诱导癌症细胞凋亡的产品或药物中的应用。
9.根据权利要求8所述的应用,其特征在于:所述癌症细胞为乳腺癌细胞MDA-MB-231。
10.权利要求1-5中任一所述的方法制备得到的牡丹花蕊提取物在制备具有减肥或抗癌功效的药物、化妆品或食品中的用途。
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