CN110354154A - 一种可缓解干燥性皮肤炎症的沙枣多糖提取物及制备方法 - Google Patents
一种可缓解干燥性皮肤炎症的沙枣多糖提取物及制备方法 Download PDFInfo
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- CN110354154A CN110354154A CN201910594523.6A CN201910594523A CN110354154A CN 110354154 A CN110354154 A CN 110354154A CN 201910594523 A CN201910594523 A CN 201910594523A CN 110354154 A CN110354154 A CN 110354154A
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- A—HUMAN NECESSITIES
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- Medicines Containing Plant Substances (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明涉及一种可缓解干燥性皮肤炎症的沙枣多糖提取物及制备方法。本发明涉及的沙枣多糖是采用沙枣树胶经水提醇沉工艺进行制备，研究表明含有沙枣多糖的提取物有缓解干燥性皮肤炎症的作用，沙枣多糖提取物可以根据需要制成凝胶、液体、乳液、软膏霜、固体颗粒等多种剂型，应用于化妆品及皮肤病辅助治疗药物等方面。
Description
技术领域
本发明涉及一种具有抗炎保湿功效的沙枣多糖提取物，该提取物可以有效的促进保湿因子丝聚蛋白（FilaggrinFLG）、兜甲蛋白（loricrin LOR）的表达同时抑制皮肤炎症因子IL-6及TNFα的表达，可作为功效活性物质添加到化妆品中使用，用于皮肤护理。
背景技术
皮肤覆盖全身表面，是人体最大的器官之一，直接与外界环境相接触的组织，是机体天然的皮肤屏障，皮肤屏障及物理性屏障，主要由皮脂膜、角质层角蛋白、脂质、粘多糖类等共同构成, 在抵御外界有害、刺激物、日光进入, 同时具有保湿及调节抗炎作用。
人体皮肤中的天然保湿系统主要由水、脂类、天然保湿因子（NaturalMoisturizing Factor， NMF）等组成。脂类呈层状填充于角质层细胞之间，主要作用是形成水屏障，止水分流失。大多脂类为非极性物质，可以限制水分在细胞内外及细胞间流动。与皮肤屏障功能相关的脂质包括神经鞘脂、游离胆固醇和游离脂肪酸。在有活性的表皮中磷脂含量丰富，然而由基底层向角质层分化过程中，神经酸胺、胆固醇、脂肪酸的含量渐增高，脂质分布也发生了变化，颗粒层中脂质聚集于板层颗粒中，颗粒层细胞转化为角质层细胞时，这些脂质被排出并填充于细胞间隙，形成了防止水分丢失的屏障。当皮肤屏障受损时，其水屏障作用减弱，经表皮水分丢失（transepidermal water loss，TEWL）就会增多，从而出现皮肤干燥脱屑、敏性皮炎，银屑病等多种皮肤疾病。皮肤屏障的受损有多种原因，除了外在物理性因素以及年龄的影响，皮肤炎症造成皮肤屏障的受损也是值得重点关注的因素。
皮脂膜可以润泽皮肤，是皮肤的保护屏障，当皮脂分泌量不足或水分不足时会导致皮肤干燥，使皮肤最外层的屏障受损，影响皮肤的健康状态，通过查阅文献人参多糖对皮脂腺细胞有一定的影响，会刺激皮脂分泌，缓解皮肤干燥，人参多糖含有吸水基团，可以利用其特点来増加皮肤的水含量，缓解干燥状况。
沙枣（Elaeagnus angustifolia L.），胡颓子科，又名银柳，在我国西北地区分布很广，作为重要的防护林树种，有着非常丰富的资源。沙枣的食用和药用价值很高，沙枣的食用和药用价值很高，果实中含有大量的糖类、蛋白质等，有固精、健胃、止泻等保健功能。但目前沙枣树的经济价值并未引起重视，只有很小一部分沙枣在民间被加工利用，大部分未被采集，利用很低，造成了很大的浪费，新疆沙枣资源丰富，沙枣中多糖的平均含量在9%～10.9%之间，多糖总含量较高，糖的部分主要含有鼠李糖、阿拉伯糖、木糖、葡萄糖、甘露糖、半乳糖，目前报道沙枣多糖主要生物活性有抗病毒、增强免疫功能、抗衰老、清除自由基的作用，通过进一步研究发现沙枣多糖在缓解干燥性皮肤炎症方面具有良好的作用，所以沙枣多糖在化妆品领域具有广阔的应用前景。
目前无报道沙枣多糖提取物可抑制皮肤炎症因子水平及提高促保湿相关因子蛋白的表达。
本申请首次以沙枣多糖提取物为研究对象，考察其对干燥性皮肤炎症的缓解作用。
发明内容
本发明的目的在于开发一种无毒、无副作用的能与人体肌肤有很好的亲和力的保湿功效成分，提供了一种具有保湿功效的沙枣多糖提取物，应用于保湿化妆品的有效成分。
技术方案。
具体实施方式
下面结合附图表和实施例对本发明做进一步说明，但是这些具体的实施例不应当理解为对本分明的限制。下述实施例中，沙枣采购于新疆，但并不限于此。
实施实例1 沙枣多糖提取物的制备及多糖含量确定
应用测试
1. 确定沙枣多糖的提取工艺测试
1.1 沙枣多糖提取物的制备
（1）以沙枣树胶为原料利用弱酸水溶液提取，具体为将用蒸馏水溶解的树胶溶液，浓度为5%，加热至温度80℃，并搅拌滴加浓度为8%的盐酸，用浓度为10%的过氧化氢处理2分钟脱色，得到多糖溶液（2）将多糖溶液中加入3-6倍体积的丙酮进行沉淀，并离心分离得到多糖沉降物（3）将多糖沉降物冷冻干燥、过滤、粉碎得到的沙枣树胶多糖白色粉末。
1.2沙枣多糖提取物中糖含量确定
（1）葡萄糖标准曲线
准确称取标准（葡萄糖）20mg于500ml容量瓶中，加水至刻度，分别吸取0.4、0.6、0.8、1.0、1.2、1.4、1.6及1.8ml,各以蒸馏水补至2.0ml，然后加入6%苯酚1.0ml及浓硫酸5.0ml,均匀冷却，沸水浴显色15min以后于490nm测光密度，以2.0ml水按同样显色操作为空白，以吸光度（Y）为纵坐标，以葡萄糖质量浓度（X）为横坐标，得标准曲线。结果见图1
葡萄糖线性关系如图1所示，线性回归方程为y=2.1307x-0.0164，相关系数R2=0.9992,表明葡萄糖浓度在0-0.5mg/ml范围内与吸光度具有良好的线性关系。
（2）苯酚硫酸法测多糖含量
料液比60:1（v/w），80℃水浴提取4h；离心（4℃，4000 rpm 20 min），上清液加入4倍体积的95％乙醇，涡旋，4℃沉淀过夜；4℃ 4000 rpm离心20 min，弃上清，用丙醇洗涤沉淀一次，离心，复溶后冻干得多糖粗制品，定容取样，吸取0.2 ml的样品液，以蒸馏水补至2.0ml，然后加入6%苯酚1.0ml及浓硫酸5.0ml,摇匀冷却室温放置20分钟以后于490nm测光密度。每次测定取双样对照。以标准曲线计算多糖含量为22.3%，进一步洗脱沉淀后获得糖含量为95%左右的沙枣多糖提取物粉末样品。
实施实例2沙枣多糖提取物对 LPS诱导的Hacat细胞炎症模型效果实验（沙枣多糖提取物多糖含量为0.05%-95%）
应用测试
2．沙枣多糖提取物细胞毒性测定
2.1沙枣多糖提取物溶液的配置
配制沙枣多糖（EPA）提取物母液，浓度为10mg/ml（蒸馏水溶解）。利用母液配制浓度分别为10mg/ml、5mg/ml、2.5mg/ml、1.25mg/ml、0.625mg/ml、的提取物储存液（蒸馏水倍数稀释）各2ml保存待用。利用各浓度沙枣多糖（EPA）储存液与含有10%血清的完全培养基配制浓度分别为500μg/ml、250μg/ml、125μg/ml、62.5μg/ml、31.25μg/ml的待测溶液。
2.2细胞种板与给药
将处于对数生长期的人源角质生长细胞用胰酶消化，取细胞悬液离心，倾倒掉上层废液后加入新的含有10%血清的完全培养基再通过血球计数板计数。取96孔板，每孔加入100ul的悬浮细胞液，细胞密度为5000/孔，置于二氧化碳培养箱培养12h。弃去旧的培养基，将配置好的含有不同浓度药物的完全培养基加入到96孔板中，每种浓度设5个复孔，一个空白组与一个调零组。
2.3 HaCat细胞MTT测试
给药48h，弃去细胞培养液，并用PBS洗两次。将配好的MTT溶液加入到96孔板中，每孔100ul。避光孵育3h后移除MTT溶液，每孔加入100ul的二甲基亚砜，震摇15后用酶标仪测定其吸光度。实验重复3次，结果如图2所示。
2.4沙枣多糖提取物对LPS诱导的Hacat细胞炎症模型效果实验
HaCaT细胞接种于6板，每孔 2mL，密度 1×105·个/mL-1。分为空白组，LPS 组，沙枣多糖高、中、低剂量组。贴壁24h后，空白组、LPS组换新鲜的完全培养液，沙枣多糖高、中、低剂量组分别将培养基换为含沙枣多糖浓度为1000，500，125ug·mL-1的完全培养液，培养 24h，除空白组外均加入 LPS，终浓度为 5μg·mL-1，继续培养 2h后，倒去培养液，用 4 ℃预冷PBS 洗涤 2 遍，用Trizol提取总 RNA，RT-PCR定量分析丝聚合蛋白（FLG）、兜甲蛋白（LOR）、白介素6（IL-6）、肿瘤坏死因子（TNFα）mRNA表达，具体如图3所示，当多糖浓度为500μg·mL-1时具有较好的炎症抑制及促保湿相关因子表达的作用。
实施实例3沙枣多糖提取物对小鼠皮肤丙酮/乙醚皮肤干燥模型效果实验（沙枣多糖提取物多糖含量为0.05%-95%）
应用测试
3、沙枣多糖提取物对小鼠皮肤丙酮/乙醚皮肤干燥模型效果实验
3.1小鼠造模及给药方法
模型（损伤）组6只；损伤给药组6*3只；空白对照组和阳性对照组与1）共用。共24只。造模前2 d，剃毛并用1%NaS脱毛，造模当天，丙酮/乙醚-水干燥（AEW）模型：用2cm×2cm 脱脂棉蘸取丙酮∶乙醚= 1∶1混合液覆盖于脱毛处15s，然后取另一2cm×2cm脱脂棉蘸取蒸馏水覆盖于脱毛处30s，给药组于造模后1h涂抹一定量的多糖溶液；每天2次，连续7d。
3.2小鼠经皮失水率测试
通过MPA仪器监测空白组（Control），模型组（EW）与沙枣多糖组（EPA-AEW）的经皮失水率，同时观察皮肤表观变化，得到结果如图4所示。相比较模型组（AEW），沙枣多糖能够较为明显的改善皮肤的失水程度。
3.3小鼠皮肤角质层病理学观察
实验结束后将小白鼠脱臼处死后，取下的皮肤组织用4%多聚甲醛块固定，固定成功后需要修剪25mm*25mm*5mm，放入包埋盒中，流水冲洗（去除组织中的固定液）30min再将组织块置于既溶于酒精，又溶于石蜡的透明剂二甲苯中透明，以二甲苯替换出组织块的中酒精，才能浸蜡包埋，将已透明的组织块置于已溶化的石蜡中，放入溶蜡箱保温，将包埋好的蜡块固定于切片机上，切成5μm薄片，放到加热的水中烫平，再贴到载玻片上，放45℃恒温箱中烘干，用二甲苯脱蜡，逐级经分级浓度的乙醇直至30%酒精，用苏木精-伊红染色法（ematoxylin-eosin staining&E）染色，苏木精染液为碱性，主要使细胞核内的染色质与胞质内的核酸着紫蓝色；伊红为酸性染料，主要使细胞质和细胞外基质中的成分着红色。H&E染色结果表明，模型组比于空白组较明显的增生、角化不全、炎症细胞渗透，沙枣多糖给药组于增生、角化不全及炎症细胞渗透有较明显的改善作用，如图5所示。
3.4小鼠皮肤组织保湿功能指标测试
实验结束后将小白鼠颈椎脱臼处死，取下的皮肤组织提取RNA，RT-PCR定量分析皮肤组织中丝聚合蛋白（FLG）、兜甲蛋白（LOR）及白介素6（IL-6）mRNA表达，具体如图5所示。
角质中间丝蛋白在博来素水解酶的催化降解下会转化生成皮肤角质层中天然的保湿因子（NMF），角质中间丝蛋白主要有丝聚合蛋白（FLG）、兜甲蛋白（LOR），所以FLG、LOR蛋白表达增加促进皮肤保湿，从图6看出，沙枣多糖组（EAP-AEW）相比模型组（AEW）LOR及FLG的mRNA表达增加，模型组（AEW）相比空白组（Control）IL-6表达增加，说明在用丙酮/乙醚/水处理时引起的皮肤炎症，沙枣多糖组（EPA-AEW）有效的降低了IL-6的表达，说明沙枣多糖不仅具有一定保湿的功效还具有缓解皮肤炎症的作用。
（1）总RNA抽提（枪头和离心管均经过湿热灭菌，无RNA酶）
1）取匀浆管，加入1ml的Trizol Reagent，置冰上预冷。
2）取100mg组织，加入到匀浆管中。
3）匀浆仪充分研磨直至无可见组织块。
4）12000rpm离心10min取上清。
5）加入250 μl三氯甲烷，颠倒离心管15s，充分混匀，静置3min。
6）4℃下12000rpm离心10min。
7）将上清转移到一新的离心管中，加入0.8倍体积的异丙醇，颠倒混匀。
8）-20℃放置15min。
9）4℃下12000rpm离心10min，管底的白色沉淀即为RNA。
10）吸除液体，加入75%乙醇1.5ml洗涤沉淀。
11）4℃下12000rpm离心5min。
12）将液体吸除干净，将离心管置于超净台上吹3min。
13）加入15μl无RNA酶的水溶解RNA。
14）55℃孵育5min。
15)使用Nanodrop 2000检测RNA浓度及纯度：仪器空白调零后取2.5μl 待测RNA溶液于检测基座上，放下样品臂，使用电脑上的软件开始吸光值检测。
16）将浓度过高的RNA进行适当比例的稀释，使其终浓度为200ng/μl.
（2）反转录（枪头和PCR均经过湿热灭菌，无RNA酶）
1）取一PCR管，加入含2μg RNA的溶液。
2）加入1μl oligo(dT)18。
3）用无核糖核酸酶的去离子水补足至12μl。
4）于PCR仪上65℃保温5min，迅速置冰上冷却。
5）依次加入4μl 5× Reaction Buffer，2μl 10mM dNTP Mix，1μlRiboLockRNAase抑制剂（20U/μl）和1μl RevertAi M-MuLV 逆转录酶（200u/μl），用枪抽吸混匀。
6）于PCR仪上42℃保温60min，结束后70℃保温5min灭活反转录酶。
（3）定量PCR
1）取0.2ml PCR管，配制如下反应体系，每个反转录产物配制3管。
2× qPCR Mix 12.5μl
7.5μM基因引物 2.0μl
反转录产物 2.5μl
ddH2O 8.0μl
2）PCR扩增
预变性 95℃，10min
循环（40次） 95℃，15s→60℃，60s
熔解曲线 60℃→95℃，每15s升温0.3℃
（4）结果处理
ΔΔCT法：
A=CT（的基因，待测样本）- CT（内标基因，待测样本）
B=CT（目的基因，对照样本）- CT（内标基因，对照样本）
K=A-B
表达倍数=2-K
结果如图5所示，沙枣多糖组（EAP-AEW）。
附图表说明
1、一种可缓解干燥性皮肤炎症的沙枣多糖提取物，沙枣多糖总含量为20%-90%。
2、申请人研究发现，沙枣多糖提取物可有效的缓解丙酮/乙醚诱导的小鼠干燥性皮肤炎症，并且发现沙枣多糖提取物浓度为1000ug/ml时，对人源角质形成细胞存活率高于80%，说明沙枣多糖提取物与细胞亲和性好，基本无毒副作用。
3、一种可缓解干燥性皮肤炎症的沙枣多糖提取物，其特征在于其剂型为在化妆品或皮肤病治疗药物药学上可接受的稀释剂、体、赋形剂、辅料或媒介物。
4、所述沙枣多糖提取物制剂的剂型为固体颗粒、胶囊、片剂、凝胶、液体、乳液、软膏等多种剂型。
图1为葡萄糖标准曲线
图2为沙枣多糖提取物对Hacat细胞毒性试验
图3为沙枣多糖对LPS诱导的Hacat细胞炎症模型效果试验
图4为实施组证明沙枣多糖对丙酮/乙醚诱导的小鼠干燥皮肤炎症有一定的缓解作用及改善皮肤经皮失水率。
图5为实施组的小鼠皮肤H&E染色图及小鼠皮肤角质层厚度分析。
图6为实施组的小鼠皮肤组织丝聚蛋白（FLG）、兜甲蛋白（LOR）、白介素6（IL-6）的蛋白表达水平。
Claims (5)
1.一种可缓解干燥性皮肤炎症的沙枣多糖提取物，其特征在于，沙枣多糖提取物中多糖含量百分比为0.05%-95.0%。
2.根据权利要求1所述的沙枣多糖提取物的制备方法，其特征在于制备方法涉及如下步骤（1）以沙枣树胶为原料利用弱酸水溶液提取，具体为将用蒸馏水溶解的树胶溶液，浓度为5%，加热至温度80℃，并搅拌滴加浓度为8%的盐酸，用浓度为10%的过氧化氢处理2分钟脱色，得到多糖溶液（2）将多糖溶液中加入3-6倍体积的丙酮进行沉淀，并离心分离得到多糖沉降物（3）将多糖沉降物冷冻干燥、过滤、粉碎得到的沙枣树胶多糖白色粉末。
3.根据权利要求1和2所述的沙枣多糖提取物，其特征在于，沙枣多糖提取物具有改善干燥性皮肤炎症的作用。
4.根据权利要求1-3所述一种可缓解干燥性皮肤炎症的沙枣多糖提取物，其特征在于其剂型为在化妆品或皮肤病治疗药物药学上可接受的稀释剂，载体、赋形剂、辅料或媒介物。
5.所述沙枣多糖提取物制剂的剂型为固体颗粒、胶囊、片剂、凝胶、液体、乳液、软膏等多种剂型。
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| CN111118070A (zh) * | 2019-12-16 | 2020-05-08 | 黑龙江锦绣大地生物工程有限公司 | 一种以沙枣果实为原料生产燃料乙醇及沙枣多糖的方法 |
| RU2793349C1 (ru) * | 2022-02-01 | 2023-03-31 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Астраханский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Средство с противовоспалительной активностью |
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| CN111118070A (zh) * | 2019-12-16 | 2020-05-08 | 黑龙江锦绣大地生物工程有限公司 | 一种以沙枣果实为原料生产燃料乙醇及沙枣多糖的方法 |
| RU2793349C1 (ru) * | 2022-02-01 | 2023-03-31 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Астраханский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Средство с противовоспалительной активностью |
| RU2795979C1 (ru) * | 2022-02-01 | 2023-05-15 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Астраханский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Противоязвенное средство |
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