CN109200057A - 红豆杉果实多糖在美白及抗衰老中的应用 - Google Patents
红豆杉果实多糖在美白及抗衰老中的应用 Download PDFInfo
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Abstract
本发明公开了一种红豆杉果实多糖在美白及抗衰老中的应用。本发明通过对酪氨酸酶的抑制实验、黑色素合成抑制实验、对双氧水诱导损伤人永生化表皮细胞的保护实验、对紫外线诱导损伤人永生化表皮细胞的保护实验,证实红豆杉果实多糖具有美白、淡斑、抗衰老、抗紫外线功效,红豆杉果实多糖可被应用于日化领域,如护肤品和/或化妆品,也可用作药品和保健品。
Description
技术领域
本发明涉及红豆杉果实多糖的新用途,具体地说,是红豆杉果实多糖在美白、抗衰老中的应用。
背景技术
红豆杉为裸子植物门(Gymnospermae)红豆衫科(Taxaceae)红豆杉属(Taxus)植物,为我国Ⅰ级珍稀保护植物,被誉为“植物黄金”、“黄金树”。我国红豆杉有4种和1变种:东北红豆杉、云南红豆杉、西藏红豆杉、中国红豆杉和其变种南方红豆杉,本专利优选南方红豆杉为实验材料。南方红豆杉(Taxus chinensisvar.mairei)又名美丽红豆杉,是我国红豆杉属中分布最广的变种。红豆杉中主要含有紫杉碱二萜化合物、紫杉宁、金松黄酮、坡那留酮、蜕皮留酮、金松双黄酮等。此外,红豆杉中所含的紫杉酚具有抗白血病及肿瘤作用,紫杉碱对高血糖肾上腺素或食物性的有降血糖的作用。其叶、枝入药都有通经、利尿、抑制糖尿病及治疗心脏病的功能。
1971年,Wani等首先从红豆杉树皮中分离到紫杉醇,据美国国家肿瘤研究所统计的临床实验结果表明,紫杉醇具有显著的抗癌作用(Wani M C,Taylor H L,Wall M E,etal.Plant antitumor agents.VI.The isolation and structure of taxol,a novelantileukemic and antitumor agent from Taxus brevifolia.[J].Journal of theAmerican Chemical Society,1971,93(9):2325.)。法国进行的临床试验证明,紫杉醇可用于晚期乳腺癌、晚期卵巢癌、膀胧癌、鼻咽癌、淋巴癌、前列腺癌、恶性黑色素瘤和头颈部肿瘤、上胃肠道癌、小细胞性和非小细胞性肺癌的治疗,被称为“晚期癌症的最后一道防线”,被认为是近十年来发现的一种最有希望的抗癌药物。
但紫杉醇主要集中在红豆杉的树皮,对红豆杉其他部位和其他成分的研究和利用均较少,造成了严重的资源浪费。为了充分利用红豆杉这一宝贵资源,应重视对红豆杉中其他成分和其他部位的研究。多糖作为自然界中含量最丰富的自然聚合物,不仅是动植物机体的重要组成成分,也是维持动植物生理活动的重要物质。进年来,随着人们对多糖的研究越来越深入,其抗肿瘤、免疫调节、抗感染、抗病毒等药理活性逐渐被发现。现有报道红豆杉枝叶多糖具有抗肿瘤作用,对多种癌症具有缓和和治疗作用。但未见对于红豆杉多糖在日化方面的研究和报道。
因此,开发红豆杉果实多糖及其药用和其他新领域,如日化方面的应用十分必要。本发明以红豆杉果实为对象,涉及红豆杉果实多糖的药理活性及其新用途。
发明内容
本发明的目的是提供一种红豆杉果实多糖的新用途。
本发明的目的是通过以下方式实现的:
红豆杉果实多糖在制备具有美白功效的药品、保健品或日化用品中应用。
红豆杉果实多糖在制备具有淡斑功效的药品、保健品或日化用品中应用。
红豆杉果实多糖在制备具有抗衰老功效的药品、保健品或日化用品中应用。
红豆杉果实多糖在制备具有抗紫外线功效的药品、保健品或日化用品中应用。
上述红豆杉果实多糖是从红豆杉属植物中分离得到的,优选红豆杉属植物为南方红豆杉(Taxus chinensis)。
优选上述红豆杉果实多糖的分子量分布范围为17.9kDa-979.2kDa。最优选红豆杉果实多糖的分子量分布范围为27.9kDa-736.3kDa。
上述红豆杉果实多糖可以采用常规方法制备得到,优选上述红豆杉果实多糖采用水提醇沉法或者发酵法制备得到。
水提醇沉法制备红豆杉果实多糖中主要包括原料的处理、脱脂、水提、浓缩、醇沉、除蛋白、再次醇沉步骤。
所述的水提醇沉法具体如下:
(1)原料的处理:红豆杉果实去核,干燥后适当粉碎,过60-80目筛;
(2)脱脂:取红豆杉果实粉末,加质量浓度为80%-100%的乙醇,料液比为1:5-20,冷浸20-24小时,过滤,取滤渣,风干;
(3)水提:取风干后的药渣,加入药渣1-15倍重量的水,50-90℃下搅拌提取1-10小时,过滤,所得滤渣再重复提取2次,合并滤液,即为红豆杉果实水提液;
(4)浓缩:将红豆杉水提液减压浓缩至原水提液体积的五分之一;
(5)醇沉:滤液浓缩,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4000-5000r/min离心10-20min,收集沉淀物,冷冻干燥得到冻干的粗提物;
(6)除蛋白:冻干的粗提物以水复溶,用Sevage法-酶法相结合的方法除蛋白;
(7)再次醇沉:除完蛋白后的多糖溶液浓缩到适当体积后,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4000-5000r/min离心10-20min,收集沉淀物,冷冻干燥即得到红豆杉果实多糖。
发酵法制备红豆杉果实多糖中主要包括原料的处理、脱脂、水提、发酵及醇沉步骤。
所述的发酵法提取红豆杉果实多糖具体如下:
(1)原料的处理:红豆杉果实去核,干燥后适当粉碎,过60目筛;
(2)脱脂:取红豆杉果实粉末,使用无水乙醇脱脂,药渣晾干;
(3)水提:药渣加水提取,料液比为1:1-7,提取温度为50-90℃,提取时间为3-8小时,过滤,滤渣再重复提取2次,合并滤液,即为红豆杉果实水提液;
(4)发酵:取高活性干酵母,加干酵母重80%的纯水在38℃条件下活化30分钟,向水提液中加入水提液重2%活化好的酵母,调节pH 4-6,25℃通风发酵2.5-3.5天;
(5)醇沉:发酵液抽滤,收集滤液,滤液浓缩到适当体积后,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4000-5000r/min离心10-20min,收集沉淀物,冷冻干燥即得红豆杉果实多糖粉末。
上述高活性干酵母优选为安琪牌高活性干酵母。
本发明所述的日化用品为化妆品、面膜或/和护肤品等。红豆杉果实多糖在日化用品中主要以添加剂的形式存在。
所述的红豆杉果实多糖在药品或保健品中可与药学可接受的辅料制成口服或外用制剂,如:片剂、胶囊剂、颗粒剂、丸剂、膏剂等。
与现有技术比较本发明的有益效果:本发明通过对酪氨酸酶的抑制实验、黑色素合成抑制实验、对双氧水诱导损伤人永生化表皮细胞的保护实验、对紫外线诱导损伤人永生化表皮细胞的保护实验证实红豆杉果实多糖具有美白、淡斑、抗衰老、抗紫外线的功效。
本发明红豆杉果实多糖具有美白、淡斑、抗衰老、抗紫外线等作用,可被应用于日化领域,如护肤品和/或化妆品;也可用作药品和保健品。
附图说明
图1为红豆杉果实多糖酪氨酸酶抑制实验结果图 *P<0.05,**P<0.01,***P<0.001vs Control。
图2为红豆杉果实多糖黑色素合成抑制实验结果图 *P<0.05,**P<0.01,***P<0.001vs Control。
图3为红豆杉果实多糖对H2O2诱导损伤人永生化表皮细胞(HaCaT细胞)的保护作用结果图 *P<0.05,**P<0.01vs Model。
图4为红豆杉果实多糖对紫外线诱导损伤人永生化表皮细胞(HaCaT细胞)的保护作用结果图 *P<0.05,**P<0.01vs Model。
具体实施方式
下面结合附图说明和具体实施案例对本发明进行进一步说明,本发明包括但不仅限于以下实施例。
实施例1:水提醇沉法提取红豆杉果实多糖
(1)原料的处理:南方红豆杉果实去核,干燥后粉碎,过60目筛。
(2)脱脂:取30g红豆杉果实粉末,加质量浓度为95%的乙醇,料液比为1:10,冷浸24小时,纱布过滤,取滤渣,自然风干。
(3)水提:风干后的药渣称重,加入药渣10倍重量的水,70℃下搅拌提取6小时,纱布过滤,所得滤渣再重复提取2次,合并滤液,即为红豆杉果实水提液。
(4)浓缩:将红豆杉水提液减压浓缩至原水提液体积的五分之一。
(5)醇沉:滤液用旋转蒸发仪浓缩到适当体积后,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4500r/min离心20min,收集沉淀物,冷冻干燥。
(6)除蛋白:冻干的粗提物以水复溶,用Sevage法-酶法相结合的方法除蛋白。
(7)再次醇沉:除完蛋白后的多糖溶液浓缩到适当体积后,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4500r/min离心20min,收集沉淀物,冷冻干燥即得到红豆杉果实多糖。按照苯酚-浓硫酸法测定多糖含量,每30g药材多糖提取率为1.940%;按照凯氏定氮法测得蛋白质含量为0.937%;凝胶分子排阻色谱法测得分子量分布为69.774kDa-736.291kDa。
实施例2:发酵法提取红豆杉果实多糖
(1)原料的处理:南方红豆杉果实去核,干燥后粉碎,过60目筛。
(2)脱脂:取30g南方红豆杉果实粉末,使用无水乙醇脱脂,药渣晾干,称重。
(3)水提:药渣加水提取,料液比为1:5,提取温度为70℃,提取时间为6小时。纱布过滤,滤渣再重复提取2次,合并滤液,即为红豆杉果实水提液。
(4)发酵:取安琪牌高活性干酵母,加干酵母重80%的纯水在38℃条件下活化30分钟,向水提液中加入水提液重2%活化好的酵母,调节pH 4-6,25℃通风发酵3.5天;
(5)醇沉:发酵液用布氏漏斗抽滤,收集滤液,滤液用旋转蒸发仪浓缩到适当体积后,加入适量95%乙醇使其乙醇含量达到80%,4℃低温放置过夜,可见白色絮状沉淀析出,4500r/min离心20min,收集沉淀物,冷冻干燥即得红豆杉果实多糖粉末。按照苯酚-浓硫酸法测定多糖含量,每30g药材多糖提取率为4.080%;按照凯氏定氮法测得蛋白质含量为0.415%;凝胶分子排阻色谱法测得分子量分布为27.905kDa-231.378kDa。
以下通过具体试验说明红豆杉果实多糖的效果:
试验例1:酪氨酸酶抑制实验
取处于对数生长期的B16细胞,PBS洗涤,0.25%胰酶消化,台盼蓝染色,血球计数板计数。调整细胞密度为2×104个/mL。每孔取100μL细胞接入96孔板,12h后分别添加50uL待测药物,药物终浓度设置如下:红豆杉果实多糖(按照实施例1方法制备得到),设置20μg/mL、100μg/mL、500μg/mL三个浓度,以α-熊果苷为阳性药,并设置4μg/mL、20μg/mL、100μg/mL三个浓度。每浓度设置5个复孔。对照组不加药物,空白孔不接种细胞。37℃、5%CO2孵箱中孵育72h。弃去上清液,pH 7.4的PBS洗涤2次,每孔加10mL/L的TritonX-100溶液90μL。振荡5min溶解细胞,经37℃预温后每孔加入1%的L-Dopa 10μI。,37℃孵育30min。于490nm波长处测吸光度,以空白孔调零,测各孔吸光度值,计算络氨酸酶活力。
如图1所示,红豆杉果实多糖对酪氨酸酶具有抑制作用。红豆杉果实多糖在100μg/mL时,络氨酸酶活力为34.32%,与对照组相比,具有极显著差异(P<0.001),效果优于α-熊果苷,且随着药物浓度增加,络氨酸酶活力下降,有着明显的量效关系。表明红豆杉果实多糖具有美白、淡斑等相关功效。
试验例2:黑色素合成抑制实验
取处于对数生长期的B16细胞,PBS洗涤,0.25%胰酶消化,台盼蓝染色,血球计数板计数。调整细胞密度为2×104个/mL。每孔取100μL细胞接入96孔板,12h后分别添加50uL待测药物,药物终浓度设置如下:红豆杉果实多糖(按照实施例1方法制备得到),设置20μg/mL、100μg/mL、500μg/mL三个浓度,以α-熊果苷为阳性药,并设置4μg/mL、20μg/mL、100μg/mL三个浓度。每浓度设置5个复孔。对照组不加药物,空白孔不接种细胞。37℃、5%CO2孵箱中孵育72h。弃去上清液,加胰酶消化,加培养基终止消化并吹打成单细胞悬液,各吸取细胞悬液分别置于3个离心管中,离心后重新弃去上清。加人200μLPBS缓冲液重新悬浮,然后加入1mL1:1乙醇乙醚液,在室温下放置30min,3 000r/min离心5min后弃去上清。加入1mL10%的DMSO1mol/LNaOH溶液,80℃水浴45min后于470nm波长处测定吸光度值,计算黑色素合成量。
如图2所示,红豆杉果实多糖对黑色素合成具有抑制作用。红豆杉果实多糖在100μg/mL时,黑色素含量为67.43%,与对照组相比,具有极显著差异(P<0.001),效果优于α-熊果苷,且随着药物浓度增加,黑色素合成量下降,有着明显的量效关系。表明红豆杉果实多糖具有美白、淡斑等相关功效。
试验例3:红豆杉果实多糖对H2O2诱导损伤人永生化表皮细胞(HaCaT细胞)的保护作用
HaCaT细胞接种于含10%胎牛血清和1%双抗(青-链霉素)的DMEM高糖型细胞培养液中,每3d更换培养基一次,待用。取指数生长期细胞,按2×104/mL浓度,每孔100μL接种于96孔细胞培养板,37℃,5%CO2湿化培养,待细胞完全贴壁后,更换含250μmol/L过氧化氢的DMEM培养液继续培养4h后,弃上清液,PBS洗涤三遍,加入20μg/mL、100μg/mL、500μg/mL红豆杉果实多糖(按照实施例1方法制备得到)连续培养24h,以4μg/mL维生素C为阳性组。待培养结束后弃上清液,每孔加入100μLMTT溶液(终浓度0.5mg/mL)混合液避光振荡30min。490nm处以多孔酶标仪测定OD值计算细胞存活率。
如图3所示,红豆杉果实多糖对H2O2诱导损伤的HaCaT细胞具有较好的保护作用。红豆杉果实多糖在100μg/mL时,细胞活力为87.02%,与模型组相比,具有显著差异(P<0.05),效果优于维生素C,并具有明显的量效关系。说明其具有一定的抗衰老护肤活性。
试验例4:红豆杉果实多糖对紫外线诱导损伤人永生化表皮细胞(HaCaT细胞)的保护作用
HaCaT细胞用完全培养基,置于37℃,5%CO2的培养箱中常规培养。传代时以2.5%胰酶和0.02%EDTA按1∶1(V/V)消化。将指数生长期细胞随机分为6组,对照组为正常培养细胞;模型组细胞以紫外线辐射损伤细胞5min;红豆杉果实多糖高、中、低浓度组细胞在含有不同浓度的红豆杉果实多糖(按照实施例2方法制备得到)(20μg/mL、100μg/mL、500μg/mL)的培养液中孵育24h后,以PBS替换培养基,紫外线辐射损伤5min;阳性对照组维生素C组细胞在含有5.68mmol/L维生素C的培养液中预孵育24h后,紫外线辐射损伤5min。弃PBS后,加入培养基培养24h,然后分别在每孔加入15μl MTT(5mg/ml),在细胞培养箱中培养4h,弃上清液,每孔加150μl二甲基亚砜,常温下振荡10min,酶标仪测490nm波长处的吸光度(OD)值,计算细胞存活率。
如图4所示,红豆杉果实多糖对紫外线诱导损伤的HaCaT细胞具有较好的保护作用。红豆杉果实多糖在100μg/mL时,细胞活力为81.10%,与模型组相比,具有极显著差异(P<0.05),效果优于维生素C,并具有明显的量效关系。说明其具有一定的抗紫外线护肤活性。
综上所述,本发明所得的红豆杉果实多糖具有美白、淡斑、抗衰老、抗紫外线功效,不仅可用作化妆品或/和护肤品的添加剂,也可用于其他日化用品中。
以上实施例是对本发明进行的具体阐述的范例,但本发明并不仅限制于以上实施例,任何对本发明的等同修改和替代也都在本发明所包含的范围之内。因此,在不脱离本发明的精神和范围下所做的均等变换和修改,都应涵盖在本发明的范畴之内。
Claims (10)
1.红豆杉果实多糖在制备具有美白功效的药品、保健品或日化用品中的应用。
2.红豆杉果实多糖在制备具有淡斑功效的药品、保健品或日化用品中的应用。
3.红豆杉果实多糖在制备具有抗衰老功效的药品、保健品或日化用品中的应用。
4.红豆杉果实多糖在制备具有抗紫外线功效的药品、保健品或日化用品中的应用。
5.根据权利要求1-4任意一项权利要求所述的应用,其特征在于,所述红豆杉果实多糖是从红豆杉属植物中分离得到的。
6.根据权利要求5所述的应用,其特征在于,所述的红豆杉属植物为南方红豆杉(Taxuschinensis)。
7.如权利要求1-4任意一项权利要求所述的应用,其特征在于所述红豆杉果实多糖的分子量分布范围为17.9kDa-979.2kDa。
8.如权利要求1-4任意一项权利要求所述的应用,其特征在于所述红豆杉果实多糖采用水提醇沉法或者发酵法制备得到。
9.根据权利要求1-4任意一项权利要求所述的应用,其特征在于,所述的日化用品为化妆品或/和护肤品。
10.根据权利要求1-4任意一项权利要求所述的应用,其特征在于,红豆杉果实多糖在日化用品中以添加剂的形式存在。
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