CN113712858B - 一种抑菌抗炎植物提取物组合物的制备及其应用 - Google Patents
一种抑菌抗炎植物提取物组合物的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑菌抗炎植物提取物组合物及其应用。本发明以罗勒、绿原酸及药用大黄三者复配比例,对组合物的抑菌性及添加量为切入点进行相关测试,最终获得一种加量少、抑菌及抗炎效果强的植物提取组合物。其中,所述罗勒:绿原酸:大黄原料的质量比为(1‑3):(0.05‑0.2):(1‑3)。本发明组合物能够有效抑制皮肤致病菌的滋生,通过降低活性氧自由基,清除炎症因子,改善角质层水分流失等减弱炎症反应。
Description
技术领域
本发明属于化妆品领域,涉及一种抑菌抗炎植物提取物组合物的制备及其应用。
背景技术
由于人们生活方式的不断改善,生活环境的不断变化以及年龄、心理压力、环境污染、四季变化、化学品滥用等影响,也给我们的皮肤和皮肤黏膜带来了众多的伤害,皮肤疾病的发生率呈现不断上升趋势。现代医学研究表明,人体菌群失调,致病菌滋生,会导致内分泌功能紊乱,反应在皮肤上常常会出现皮脂分泌旺盛,同时并发炎症、粉刺发红、顶部发生小脓疱等。
罗勒(Ocimum basilicum),是唇形目、唇形科、罗勒属植物,别名熏草、燕草、蕙草、西王母菜,为药食两用芳香植物。味似茴香,全株小巧,叶色翠绿,花色鲜艳,芳香四溢。罗勒为主要的芳香油植物之一,挥发油主要存在于其茎、叶和花穗中,一般含油0.1~0.12%。罗勒全草均可入药,其性温味辛,具有疏风行气、活血、化湿、消食、解毒等多种功效,纯罗勒精油杀菌率百分百之九十九。
绿原酸(Chlorogenic acid)是一种苯丙酸类物质,是由咖啡酸和奎尼酸形成的酯,它存在于茵陈、金银花等常见中草药中,具有较强的广谱抗菌作用,现代科学对绿原酸生物活性的研究已深入到食品、保健、医药和日用化工等多个领域。绿原酸是一种重要的生物活性物质,具有抗菌、抗病毒、增高白血球、保肝利胆、抗肿瘤、降血压、降血脂、清除自由基和兴奋中枢神经***等作用。
大黄,一般为蓼科植物掌叶大黄(Rheum palmatum L.)、药用大黄(Rheumoffcinale Baill.)、唐古特大黄(Rheum tanguticum Maxim.ex Balf.)的干燥根和根茎。功能主治为:泻热毒,破积滞,行瘀血等。所含蒽醌类化合物有大黄素、大黄素甲醚、芦荟大黄素、大黄酸、大黄酚等。大黄抗菌谱广,对金黄色葡萄球菌、溶血性链球菌、伤寒杆菌、痢疾杆菌及幽门螺旋菌等多种革兰氏阳性菌和阴性菌有抑制作用;对多种真菌、流感病毒、肝炎病毒及人免疫缺陷病毒有抑制作用。
目前针对罗勒、绿原酸、大黄三者的应用主要集中于药品以及食品保健品领域,在化妆品领域中的应用不多。Singh S发现罗勒挥发油对大鼠角叉菜胶引起的足跖肿胀以及花生四烯酸和白三烯引起的水肿都有明显的抑制作用,同时罗勒(全草)提取物可减轻大鼠耳肿胀度,对角叉菜胶所致大鼠气囊炎模型中白细胞游出具有抑制作用,使血清中NO,MDA含量降低,SOD活性增强;近些年,关于绿原酸新的抗菌机制被报道,通过降低细菌细胞壁的硬度,减慢细菌的迁移,影响细菌细胞膜的稳定性和诱导活性氧(reactive oxygenspecies,ROS)的产生来发挥作用;Agarwal SK等研究表明,大黄中的有效成分大黄酸、芦荟大黄素、大黄酚、大黄素甲醚对新型隐球菌,须疮廯菌、白色念珠菌、烟曲菌都有抗菌作用。抑菌有效成分中大黄酸、大黄素、芦荟大黄素效果最为明显。
当人体表皮菌群发生失调时,致病菌的滋生会引起皮肤的炎症反应,同时产生的炎症因子又反过来刺激皮肤,造成活性氧自由基的增多,产生更大的损伤,出现粉刺发红、表皮水分流失等现象。因此本发明一方面通过调节表皮菌群的生物活性,抑制有害菌的滋生降低皮肤感染的风险,一方面通过降低活性氧自由基,清除炎症因子,改善角质层水分流失等减弱炎症反应。
现有的研究几乎很少提及到罗勒、绿原酸及药用大黄三者复配在化妆品领域抑菌性的应用。
发明内容
本发明的目的是提供一种抑菌抗炎植物提取物组合物及其应用,解决现有技术中存在的问题。本发明以罗勒、绿原酸及药用大黄三者复配比例,对组合物的抑菌性及添加量为切入点进行相关测试,最终获得一种加量少、抑菌及抗炎效果强的植物提取组合物。
针对上述技术背景存在的问题,本发明旨在提供一种抗炎抑菌的植物提取组合物的制备方法,包括以下步骤:
1)将大黄原料粉碎,用去离子水、纤维素酶提取,过滤得到第一初滤液;
2)将新鲜罗勒切段,用乙醇提取得到第二初滤液;
3)将第一初滤液与第二初滤液混合,浓缩后用水稀释,再用有机相萃取,将有机相合并浓缩后得到流浸膏;
4)将流浸膏用乙醇溶解,加入绿原酸粉末溶解,浓缩得到浓缩液,超滤得到所述的抗炎抑菌的植物提取组合物;
其中,所述罗勒:绿原酸:大黄原料的质量比为(1-3):(0.05-0.2):(1-3)。
作为优选,所述的制备方法包括以下步骤:
1)将大黄原料粉碎;加入去离子水提取,大黄原料与去离子水的质量比为1:20-1:50,浸润1-3小时;水浴,温度为40-50℃时加入纤维素酶,所加入的酶总质量为大黄原料质量的10%-20%,搅拌20分钟,升温至40-60℃微波处理5-15分钟,超声水浴回流提取1-3小时,冷却至20-30℃,用100-200目纱布过滤,得第一初滤液;
2)将新鲜罗勒按质量比切为1-2cm段,用3-5倍量的95%的乙醇溶液浸泡1h后,低温,经超声振荡提取,过滤,合并滤液,得第二初滤液;
3)将第一初滤液与第二初滤液混合,减压浓缩,加入浓缩液1倍体积量的蒸馏水,再依次用石油醚、氯仿、乙酸乙酯进行萃取;重复2-3次,直至萃取液成无色,合并乙酸乙酯层减压浓缩得流浸膏;
4)用1-3倍体积量的乙醇溶解浸膏,按质量比加入绿原酸粉末,搅拌至充分溶解;过滤,加入澄清剂,静置后离心30-60分钟,用微滤膜过滤后得到滤液。减压浓缩回收乙醇,得浓缩液;用超滤膜进行过滤,得到所述抗炎抑菌的植物提取组合物;
其中所述罗勒:绿原酸:大黄原料的质量比为(1-3):(0.05-0.2):(1-3)。
进一步优选,所述罗勒:绿原酸:大黄原料的质量比为1:0.2:2或1:0.1:2或1:0.1:3。
进一步优选,所述步骤4)中的澄清剂为天然澄清剂、壳聚糖、果汁澄清剂、明胶、蛋清中的一种或几种。
进一步优选,所述步骤4)中的微滤膜孔径为0.1-1.0μm;超滤膜孔径为0.01-0.05μm。
本发明还提供了一种抗炎抑菌的植物提取组合物,由上述制备方法制备得到;
其中所述罗勒:绿原酸:大黄原料的质量比为1:0.2:2或1:0.1:2或1:0.1:3。
在某些更具体的实施例中,制备方法具体为:
(1)将大黄原料按照质量比粉碎;
(2)加入去离子水提取,原料与去离子水的质量比为1:20,浸润2小时。水浴,温度为48℃时加入纤维素酶,所加入的酶总质量占原料总质量的10%,搅拌20分钟,升温至54℃微波处理10分钟,超声水浴回流提取3小时;
(3)将步骤(2)得到的提取液冷却至室温,用200目纱布过滤,得初滤液1;
(4)将新鲜罗勒按质量比切为1-2cm段,用3倍量的95%的乙醇溶液浸泡1h后,低温,经超声振荡提取,过滤,合并滤液,得初滤液2;
(5)合并步骤(3)和步骤(4)的滤液,置于旋转蒸发仪中减压浓缩,温度为55℃,得流浸膏;用1倍量的蒸馏水溶解浸膏,依次用石油醚、氯仿、乙酸乙酯进行萃取,其体积比为1:1;用力振摇静止3h后,分离有机层,继续萃取3次,直至无色,合并乙酸乙酯层,置于旋转蒸发仪中减压浓缩,回收乙酸乙酯,温度为35℃,得流浸膏;
(6)用3倍量的乙醇溶解浸膏,按质量比加入绿原酸粉末,搅拌至充分溶解,加入澄清剂,静置后离心60分钟,用微滤膜过滤后得到滤液。过滤,减压浓缩回收乙醇,得浓缩液。
(7)其特征在于,步骤(5)所述转速为8000rpm;
(8)将步骤(6)得到的溶液用超滤膜进行过滤,收集滤液,得到一种有效抗炎抑菌的植物提取物组合物。
本发明还提供了一种上述组合物在制备具有抗炎抑菌效果的化妆品中的应用。
本发明还提供了一种化妆品,包括所述组合物以及化妆品可使用的助剂。
作为优选,所述组合物占化妆品的质量百分比为0.5-5%。
作为优选,所述化妆品为水剂、乳剂、喷雾、膏霜、凝胶剂或面膜。
本申请具有以下有益效果:
1)能够有效抑制皮肤致病菌的滋生,且对皮肤表皮正常的菌群没有影响。
2)能够通过降低活性氧自由基,清除炎症因子,改善角质层水分流失等减弱炎症反应。
3)相对于常规抑菌组合物,本发明使用天然产物作为原料,更加安全无刺激。
附图说明
图1为不同组合物对于IL1-α分泌的影响;
图2为不同组合物对于IL-6分泌的影响;
图3为不同组合物对于经皮水分散失的影响;
图4为不同组合物对于红斑的影响。
具体实施方式
以下将通过实施例来详细说明本申请的实施方式,借此对本申请如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
本申请中所用原料、设备,若无特别说明,均为本领域的常用原料、设备,均来自市售产品。本申请中所用方法,若无特别说明,均为本领域的常规方法。
本申请还存在其它多种可实施的技术方案,在此不做一一列举,本申请权利要求中要求保护的技术方案都是可以实施的。
实施例1.能够有效抗炎抑菌的植物提取组合物的制备
在本发明下述的实施例及对比例中,能够有效抗炎抑菌的植物提取组合物采用以下工艺制备。
(1)将大黄原料按照质量比粉碎;
(2)加入去离子水提取,原料与去离子水的质量比为1:20-1:50,浸润1-3小时。水浴,温度为40-50℃时加入纤维素酶,所加入的酶总质量占原料总质量的10%-20%,搅拌20分钟,升温至40-60℃微波处理5-15分钟,超声水浴回流提取1-3小时;
(3)将步骤(2)得到的提取液冷却至20-30℃,用100-200目纱布过滤,得初滤液1;
(4)将新鲜罗勒按质量比切为1-2cm段,用3-5倍量的95%的乙醇溶液浸泡1h后,低温,经超声振荡提取,过滤,合并滤液,得初滤液2;
(5)合并步骤(3)和步骤(4)的滤液,置于旋转蒸发仪中减压浓缩,温度为50-60℃,得流浸膏;用1倍量的蒸馏水溶解浸膏,依次用石油醚、氯仿、乙酸乙酯进行萃取,其体积比为1:1;用力振摇静止2-4h后,分离有机层,继续萃取2-3次,直至无色,合并乙酸乙酯层,置于旋转蒸发仪中减压浓缩,回收乙酸乙酯,温度为35-50℃,得流浸膏;
(6)用1-3倍量的乙醇溶解浸膏,加入澄清剂,静置后离心30-60分钟,用微滤膜过滤后得到滤液,加入1-3倍量的溶液回流提取3-5小时。按质量比加入绿原酸粉末,搅拌至充分溶解。过滤,减压浓缩回收乙醇,得浓缩液。
(7)其特征在于,步骤(5)所述转速为5000-10000rpm;
(8)将步骤(6)得到的溶液用超滤膜进行过滤,收集滤液,得到一种有效抗炎抑菌的植物提取物组合物。
实施例2.不同比例组合物复配抑菌效果评价
(1)将大黄原料按照质量比粉碎;
(2)加入去离子水提取,原料与去离子水的质量比为1:20,浸润2小时。水浴,温度为48℃时加入纤维素酶,所加入的酶总质量占原料总质量的10%,搅拌20分钟,升温至54℃微波处理10分钟,超声水浴回流提取3小时;
(3)将步骤(2)得到的提取液冷却至室温,用200目纱布过滤,得初滤液1;
(4)将新鲜罗勒按质量比切为1-2cm段,用3倍量的95%的乙醇溶液浸泡1h后,低温,经超声振荡提取,过滤,合并滤液,得初滤液2;
(5)合并步骤(3)和步骤(4)的滤液,置于旋转蒸发仪中减压浓缩,温度为55℃,得流浸膏;用1倍量的蒸馏水溶解浸膏,依次用石油醚、氯仿、乙酸乙酯进行萃取,其体积比为1:1;用力振摇静止3h后,分离有机层,继续萃取3次,直至无色,合并乙酸乙酯层,置于旋转蒸发仪中减压浓缩,回收乙酸乙酯,温度为35℃,得流浸膏;
(6)用3倍量的乙醇溶解浸膏,按质量比加入绿原酸粉末,搅拌至充分溶解,加入澄清剂,静置后离心60分钟,用微滤膜过滤后得到滤液。过滤,减压浓缩回收乙醇,得浓缩液。
(7)其特征在于,步骤(5)所述转速为8000rpm;
(8)将步骤(6)得到的溶液用超滤膜进行过滤,收集滤液,得到一种有效抗炎抑菌的植物提取物组合物。
将三种植物提取物按照下表1比例进行复配,得到组合物1-15,对比例1-3。
表1
在本发明上述的组合物及对比例中,抑菌抗炎植物提取组合物抑菌及修复效应测试方法和结果如下:
抑菌效果检测
菌种选用:金黄色葡萄球菌,铜绿假单胞菌,痤疮丙酸杆菌;菌液制备所选用铜绿假单胞菌\金黄色葡萄球菌\痤疮丙酸杆菌为所列菌种。制备:用无菌生理盐水将其稀释成约1.0x105cfu/ml~1.0x106cfu/ml的菌悬液。制备好的菌悬液应在2h内使用,或在2℃~8℃保存不超过24h。
抑菌效果检测:取实施例2步骤(8)中所得植物组合物,取无菌干燥滤纸片,每片滴加实际植物组合物20μL。试验菌接种:取1mL各试验菌悬液,在营养琼脂培养基表面均匀涂抹;抑菌试验样品贴放,每次试验贴放一个染菌平板,滤纸片为每皿4片,3片为试验样片,1片为对照样片为空白。用无菌镊子取样片贴放于平板表面,置于36±1℃恒温培养箱中,培养18h后观察结果,用游标卡尺测量抑菌环的直径。结果为3次测量数据的平均值。
实验结果:结果下表2,将组合物1-15与对比例1-3比较后,发现:
1)3种原料复配组合后的抑菌活性优于单一植物;
2)在3种植物复配组合的不同比例对应的抗菌活性不同,优选为罗勒:绿原酸:大黄=(1-3):(0.05-0.2):(1-3),更优选为罗勒:绿原酸:大黄=1:0.2:2或1:0.1:2或1:0.1:3。
表2
上述试验结果表明,测试例1-15组合物相对空白组,都具有良好的抑菌效果,但相同条件下组合物比例不同,对各种菌的抑菌强弱也会有影响,其中组合物6、8、10的抑菌效果相对较好。
实施例3.细胞炎症检测
(1)MTT细胞毒性测试
在96well multi plate(corning)中按照1X104 cells/well的密度接种含有10%牛血清的DMEM培养基和角质形成细胞(HaCaT)各100μL,培养24小时后换成无血清培养基。在无血清培养基中分别加入上述实施例中组合物6、8、10进行处理后培养24小时。之后去除培养基,用20μL的MTT溶液进行处理,在37℃下使其反应2小时。在去除MTT溶液的细胞中加入200μL异丙醇,轻轻地震荡30min,完全溶解结晶甲臜,在570nm处测定吸光度,根据如下公式计算细胞存活率。
对照组不加入样品进行试验。细胞毒性相关结果见下表3.
表3
(2)炎症因子检测
IL1-α,IL-6为细胞炎症因子,采用ELISA法进行检测。其中以没做任何处理的HaCat细胞作为空白组,以脂多糖(LPS)诱导的HaCat细胞作为模型组。用1μg/ml LPS刺激HaCat细胞后,将1%含量的组合物6、8、10以每孔100μl加入12孔细胞板,于37℃、5%的CO2恒温培养箱培养72h,每个实验组3个复孔。收集细胞培养上清,通过ELISA检测试剂盒进行抗炎功效评价,结果取平均值。测试如下,以空白组检测的含量为参考量,即为100%。
具体结果如图1和图2所示,图1为不同组合物对于IL1-α分泌的影响;图2为不同组合物对于IL-6分泌的影响。结果表明,组合物6、8、10可以很好的抑制LPS刺激后IL1-α和IL-6炎症因子的产生,具有显著的抗炎效果,组合物10的效果最好。
实施例4.
在具体实施方式中,本发明提供了一种能够有效抗炎抑菌的面膜肌底液的配方工艺,具体配方如下表4:
表4
添加权利要求所述提取物,将组合物6、8、10加入下表5面膜肌底液配方中,所用的配方为本领域常用的配方,添加量为1%,制得面膜肌底液实施例1-3。
表5
组合物6 | 组合物8 | 组合物10 |
样品1 | 样品2 | 样品3 |
1.安全性测试(人体皮肤斑贴试验)
选取15名年龄为20-50岁之间的无皮肤病过敏史的健康受试者,斑贴方法:选用合格的斑试器,以封闭式斑贴试验方式,取约15μL样品1-3的样品滴于斑试器内,外用专用胶带贴覆于受试者背部,每个受试者贴20个斑试器,分别贴试样品1-3的肌底液样品,24小时后除去受试物,去除后分别于0.5、6、12、24、48小时观察皮肤反应,按照《护肤品卫生规范》中皮肤反应等级标准记录其结果。
试验结果:人体皮肤斑贴试验结果显示所有受试者通过斑贴试验,在0.5、6、12、24、48小时观察皮肤反应,其中0例出现皮肤红斑、丘疹、水疱等不良反应,说明本发明的产品安全,无刺激。
2.皮肤水分流失及红斑修复测试
(1)测试人数:共45人;分为5组,每组9人。
(2)受试部位:受试人员前臂屈侧柔弱的皮肤处;
(3)将要进行试验的部位用70%酒精进行消毒,待酒精完全蒸发后,测定皮肤skincolor红斑值(ERYTHEMA)和TEWL值;在受试人员前臂屈侧较为柔弱的皮肤处选定1*1cm2的区域,分别为对照组(空白)、模型组(0.075%辣椒素刺激),其余为试验组。其中试验组采用上述优选组合物6、8、10(20μL,10mg/mL)保护,再给予0.075%辣椒素刺激,30min后进行skin color红斑值和TEWL值的测定,评价修复状况。
具体结果如图3和图4所示,图3为不同组合物对于经皮水分散失的影响;图4为不同组合物对于红斑的影响。
实验结果:所述各植物提取组合物可有效缓解由于辣椒素刺激所产生的经皮水分散失(TEWL)升高和红斑(ERYTHEMA)的增加。
本申请说明书中未作详细描述的内容属于本领域技术人员的公知常识。
如在通篇说明书及权利要求当中所提及的“包含”为一开放式用语,故应解释成“包含但不限定于”。“大致”是指在可接收的误差范围内,本领域技术人员能够在一定误差范围内解决所述技术问题,基本达到所述技术效果。
还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的商品或者***不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种商品或者***所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的商品或者***中还存在另外的相同要素。
上述说明示出并描述了本申请的若干优选实施例,但如前所述,应当理解本申请并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述发明构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本申请的精神和范围,则都应在本申请所附权利要求的保护范围。
Claims (11)
1.一种抗炎抑菌的植物提取组合物的制备方法,其特征在于,包括以下步骤:
1)将大黄原料粉碎,用去离子水、纤维素酶提取,过滤得到第一初滤液;
2)将新鲜罗勒切段,用乙醇提取得到第二初滤液;
3)将第一初滤液与第二初滤液混合,浓缩后用水稀释,再依次用石油醚、氯仿、乙酸乙酯进行萃取,将乙酸乙酯层合并浓缩后得到流浸膏;
4)将流浸膏用乙醇溶解,加入绿原酸粉末溶解,浓缩得到浓缩液,超滤得到所述的抗炎抑菌的植物提取组合物;
其中,所述罗勒:绿原酸:大黄原料的质量比为1:0.2:2或1:0.1:2或1:0.1:3。
2.如权利要求1所述的制备方法,其特征在于,包括以下步骤:
1)将大黄原料粉碎;加入去离子水提取,大黄原料与去离子水的质量比为1:20-1:50,浸润1-3小时;水浴,温度为40-50℃时加入纤维素酶,所加入的酶总质量为大黄原料质量的10%-20%,搅拌20分钟,升温至40-60℃微波处理5-15分钟,超声水浴回流提取1-3小时,冷却至20-30℃,用100-200目纱布过滤,得第一初滤液;
2)按质量比,将新鲜罗勒切为1-2cm段,用3-5倍量的95%的乙醇溶液浸泡1h后,低温,经超声振荡提取,过滤,合并滤液,得第二初滤液;
3)将第一初滤液与第二初滤液混合,减压浓缩,加入浓缩液1倍体积量的蒸馏水,再依次用石油醚、氯仿、乙酸乙酯进行萃取;重复2-3次,直至萃取液成无色,合并乙酸乙酯层减压浓缩得流浸膏;
4)用1-3倍体积量的乙醇溶解流浸膏,按质量比加入绿原酸粉末,搅拌至充分溶解,加入澄清剂,静置后离心30-60分钟,用微滤膜过滤后得到滤液;减压浓缩回收乙醇,得浓缩液;用超滤膜进行过滤,得到所述抗炎抑菌的植物提取组合物;
其中所述罗勒:绿原酸:大黄原料的质量比为1:0.2:2或1:0.1:2或1:0.1:3。
3.如权利要求2所述的制备方法,其特征在于,所述步骤4)中的澄清剂为天然澄清剂。
4.如权利要求2所述的制备方法,其特征在于,所述步骤4)中的澄清剂为果汁澄清剂。
5.如权利要求2所述的制备方法,其特征在于,所述步骤4)中的澄清剂为壳聚糖、明胶、蛋清中的一种或几种。
6.如权利要求2所述的制备方法,其特征在于,所述步骤4)中的微滤膜孔径为0.1-1.0μm;超滤膜孔径为0.01-0.05μm。
7.一种抗炎抑菌的植物提取组合物,其特征在于,由权利要求1至6任一所述制备方法制备得到;
其中所述罗勒:绿原酸:大黄原料的质量比为1:0.2:2或1:0.1:2或1:0.1:3。
8.一种如权利要求7所述组合物在制备具有抗炎抑菌效果的化妆品中的应用。
9.一种化妆品,其特征在于,包括权利要求7所述组合物以及化妆品可使用的助剂。
10.如权利要求9所述化妆品,其特征在于,所述组合物占化妆品的质量百分比为0.5-5%。
11.如权利要求9所述化妆品,其特征在于,所述化妆品为水剂、乳剂、喷雾、膏霜、凝胶剂或面膜。
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