CN111840149A - 一种黄芩美白抗敏提取液的制备工艺及应用 - Google Patents
一种黄芩美白抗敏提取液的制备工艺及应用 Download PDFInfo
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Abstract
本发明提供一种黄芩美白抗敏提取液的制备工艺及应用，包括以下步骤：将原料黄芩根超微粉碎，然后采用乙醇水溶液进行超声提取，过滤，浓缩后得到浓缩液；将浓缩液用石油醚进行萃取，弃掉上层石油醚相，得到下层水相；将下层水相，用乙酸乙酯进行萃取，弃掉下层水相，将上层的乙酸乙酯相减压浓缩至浸膏；将浸膏采用中压制备技术分离黄芩素和黄芩苷混合物；将黄芩素和黄芩苷混合物冷冻干燥成固体粉末，配制成一定浓度的溶液，过滤除菌，即得黄芩美白抗敏提取液。本发明的工艺简便、纯度高、生产效率高，美白抗敏功效显著，安全性高，进一步拓展了黄芩美白抗敏提取液在化妆品及护肤品中的应用。
Description
技术领域
本发明涉及一种黄芩美白抗敏提取液的制备工艺，以及该黄芩美白抗敏提取液在化妆品及护肤品中的应用。
背景技术
美白主要是减少黑色素合成。黑色素广泛存在于人的皮肤、粘膜、视网膜、软脑膜及胆囊与卵巢等处。黑色素的产生多与内分泌失调、日光照射有关，黑色素细胞的多少主要取决于遗传，另外，还与内分泌激素及营养状况有关，由黑色素导致的疾病如:雀斑、黄褐斑、黑变病等，成为美容界研究的课题。中医认为:凡七情内伤、饮食不调、劳倦失宜、肾水不足、妇人经血不调等，均可致病。黑色素是由黑色素细胞合成的。黑色素细胞能合成并分泌黑色素，因此是一种腺细胞。然而黑色素的生物合成非常复杂，是通过色体(未成熟的黑色素)内酪氨酸-酪氨酸酶反应形成的。
由于审美的不同，美白成为亚洲人永不褪色的时尚话题，而各大公司大力研发美白类产品也足以证明这一点。目前多数美白产品是通过物理和化学的手段减少紫外线对皮肤的的损伤，降低对黑色素吸收的诱导，抑制黑色素的形成。但过度的使用化妆品对我们的皮肤造成负担，使皮肤出现各种过敏的症状，粉刺、痤疮、色斑等等。因而我们开发了一款美白抗敏双重功效的原料应用于化妆品中。
黄芩(Scutellaria baicalensis Georgi)，别名山茶根、土金茶根，是唇形科黄芩属多年生草本植物，黄芩以根入药，味苦、性寒，有清热燥湿、泻火解毒、止血、安胎等功效。主治温热病、上呼吸道感染、肺热咳嗽、湿热黄胆、肺炎、痢疾、咳血、目赤、胎动、高血压、痈肿疖疮等症。黄芩的临床抗菌性比黄连好，而且不产生抗药性。
目前，中国专利CN 101693001 A《黄芩提取液的生产工艺》通过粉碎加热浸泡，粉碎打浆、压滤、罐装。该工艺传统、成分复杂、杂质较多、功效不明确，未涉及主要成分的纯化。
专利CN10566981A《一种黄芩苷粗品提取工艺》主含黄芩苷提取方法主要是酸碱沉法，实验要求环境比较高，酸碱调节比例、酸碱中和程度比较难以控制，而且对环境污染比较大。
专利CN108853192A《一种黄芩提取物及其制备方法、应用及保健酒》直接酒精提取、大孔树脂分离技术，分离方法比较传统，粗提物溶解容易堵塞树脂柱，影响分离效果。大孔树脂富集的是混合化合物，成分比较复杂，不能分离单一成分，且大孔树脂用完需进行酸碱冲洗再生，环境污染比较大。我们采用中压色谱制备能够精准分离所需化合物，纯度高、生产效率高。
发明内容
本发明的目的之一是提供一种美白抗敏双重功效的黄芩美白抗敏提取液。
本发明的目的之二是提供一种简便高效、环境污染低的黄芩美白抗敏提取液的制备工艺。
本发明的目的之三是提供了一种黄芩美白抗敏提取液在制备化妆品及护肤品的应用。
本发明的目的之一通过如下技术方案实现：一种黄芩美白抗敏提取液，所述黄芩美白抗敏提取液包含：黄芩素、黄芩苷、丁二醇和水。
上述黄芩素的重量份数为1-5份，上述黄芩苷的重量份数为1-5份，上述丁二醇的重量份数为65-75份，上述水的重量份数为15-25份。
本发明的目的之二通过如下技术方案实现：一种黄芩美白抗敏提取液的制备工艺，包括以下步骤：
步骤1 将原料黄芩根进行超微粉碎，然后采用乙醇水溶液进行超声提取，再将提取液用80目筛进行过滤，浓缩后得到浓缩液；
步骤2 将步骤1所得的浓缩液用石油醚进行萃取，弃掉上层石油醚相，得到下层水相；
步骤3 将步骤2所得的下层水相，用乙酸乙酯进行萃取，弃掉下层水相，将上层的乙酸乙酯相减压浓缩至浸膏；
步骤4 将步骤3所得的浸膏采用甲醇溶解，拌样，挥干，然后用甲醇水洗脱，浓缩得到黄芩素和黄芩苷混合物；
步骤5 将步骤4所得的黄芩素和黄芩苷混合物冷冻干燥为固体粉末，采用溶剂1,3-丁二醇:去离子水=3:1将固体粉末配置成浓度为8-15mg/ml的溶液，0.22μm过滤除菌，即得黄芩美白抗敏提取液，将所得提取液标记为SGB-E。
优选的，上述步骤1具体是：将原料黄芩根除杂，进行超微粉碎，然后采用质量浓度为95%的乙醇水溶液进行超声提取1h，其中提取的料液比为1:8，再将提取液用80目筛进行过滤，在48-68℃温度下，浓缩后得到浓缩液，所述浓缩液的热比重为1.1-1.3。
优选的，上述步骤1中的超声提取的次数为3次。
优选的，上述步骤2具体是：将步骤1所得的浓缩液用体积为2-3倍量的石油醚进行萃取3-4次，静置3-5h，弃掉上层石油醚相，得到下层水相。
优选的，上述步骤3具体是：将步骤2所得的下层水相，用体积为2-3倍量的乙酸乙酯进行萃取3-4次，静置3-5h，弃掉下层水相，在48-68℃温度下，将上层的乙酸乙酯相减压浓缩至浸膏。
优选的，上述步骤4具体是：采用中压分离技术，将步骤3所得的浸膏采用甲醇溶解，采用反相C18填料拌样，挥干甲醇后装入预备柱中，然后先后采用质量浓度为10%、30%、60%的甲醇水洗脱，洗脱速率为50-100ml/min，洗脱体积为3-4柱体积，浓缩得到黄芩素和黄芩苷混合物，所述反相C18填料的质量为浸膏质量的3-5倍量。
优选的，上述步骤5具体是：将步骤4所得的黄芩素和黄芩苷混合物冷冻干燥程固体粉末，冷冻干燥温度为-78℃，冷冻时间为48-72h，采用溶剂1,3-丁二醇:去离子水=3:1将固体粉末配置成浓度为10mg/ml的溶液，于十万级制备车间0.22μm微孔滤膜过滤除菌，即得黄芩美白抗敏提取液。
本发明的目的之三通过如下技术方案实现：一种使用上述的制备工艺制备的黄芩美白抗敏提取液在制备化妆品及护肤品的应用。
本发明的优点：
一、原料进行超微粉碎技术，保持了原料生物活性及营养成分，粒径变小，样品比表面积增加样品的溶解性增强，提取率变高，超微粉碎是在封闭系统中进行，避免了对环境的污染，绿色环保。
二、工艺简单、易操作、精准富集；采用中压制备分离，仪器全程监控图谱，收集需要的部位，精准分离，持续制备，生产效率高。
三、冷冻技术干燥、十万级制备车间配制，天然绿色无污染；采用-78℃低温冷冻技术，保持样品原有活性，利用率更高。
四、本发明的黄芩美白抗敏提取液美白抗敏功效显著，安全性高，进一步拓展了黄芩美白提取液在化妆品及护肤品中的应用。
附图说明
图1是本发明所制备的黄芩美白抗敏提取液对作用24h后美白相关基因的变化；
图2是本发明所制备的黄芩美白抗敏提取液对酪氨酸酶活性抑制实验；
图3是本发明所制备的黄芩美白抗敏提取液作用24h后抗敏相关基因的变化；
图4是本发明所制备的黄芩美白抗敏提取液对透明质酸酶抑制实验。
具体实施方式
本实施例提供一种黄芩美白抗敏提取液，包括黄芩素、黄芩苷、丁二醇和水。
本发明所述黄芩素的重量份数为1-5份，所述黄芩苷的重量份数为1-5份，所述丁二醇的重量份数为65-75份，所述水的重量份数为15-25份。
本发明优选实施例中，所述黄芩素的重量份数为1份、3份或5份，所述黄芩苷的重量份数为1份、3份或5份，所述丁二醇的重量份数为65份、68份或75份，所述水的重量份数为10份、15份或25份。
实施例一
取黄芩根2kg，进行超微粉碎，加入16L 95%乙醇溶液，进行超声提取，提取温度为常温，提取时间1h；提取三次，混合三次提取液后总共得到提取液48L，将提取液过80目网筛过滤。将过滤后的提取液减压浓缩，浓缩温度60℃，浓缩至0.5L。
在浓缩液中加入1.5L石油醚，每隔20min充分搅拌使混合均匀，常温静置3h，吸出上层液体并弃掉，重复三次。
在下层水相中加入1.5L乙酸乙酯，每隔20min充分搅拌使混合均匀，常温静置3h，放掉下层液体并弃掉，重复三次；将乙酸乙酯萃取的部位减压浓缩，浓缩温度60℃，浓缩至浸膏。
将浸膏放入-80℃冰箱冷冻4h后移入冷冻干燥机，在-78℃下冷冻72h，得到固体粉末100g。取25g进行中压制备分离，用20ml甲醇溶解，样品与C18质量比为1:3拌样，挥干甲醇装入预备柱中，然后用10%、30%、60%的甲醇水洗脱，流速为100ml/min，柱体积为1L，冲洗3倍的柱体积，将其检测合并30%、60%，60℃浓缩蒸干，得到粉末10g。
分批制备总得到40g粉末，将40g固体粉末加入3L的丁二醇，搅拌使充分溶解，再加入1L去离子水，充分搅拌使混合均匀，于10万级制备车间，0.22μm过滤除菌，得到4L的黄芩美白抗敏提取液。
实施例二
取黄芩根10kg，进行超微粉碎，加80L 95%乙醇溶液，进行超声提取，提取温度为常温，提取时间1h；提取三次，混合三次提取液后总共得到提取液48L，将提取液过80目网筛过滤。将过滤后的提取液减压浓缩，浓缩温度60℃，浓缩至5L。
在浓缩液中加入15L石油醚，每隔20min充分搅拌使混合均匀，常温静置3h，吸出上层液体并弃掉，重复三次。
在下层水相中加入15L乙酸乙酯，每隔20min充分搅拌使混合均匀，常温静置3h，放掉下层液体并弃掉，重复三次；将乙酸乙酯萃取的部位减压浓缩，浓缩温度60℃，浓缩至浸膏。
将浸膏放入-80℃冰箱冷冻4h后移入冷冻干燥机，在-78℃下冷冻72h，得到固体粉末500g。取50g进行中压制备分离，用50ml甲醇溶解，样品与C18质量比为1:3拌样，挥干甲醇装入预备柱中，然后用10%、30%、60%的甲醇水洗脱，流速为100ml/min，柱体积为2L，冲洗3倍的柱体积，将其检测合并30%、60%，60℃浓缩蒸干，得到粉末20g。
分批制备总得到200g粉末，将200g固体粉末加入15L的丁二醇，搅拌使充分溶解，再加入5L去离子水，充分搅拌使混合均匀，于十万级制备车间，0.22μm过滤除菌，得到20L的黄芩美白抗敏提取液。
对上述制备工艺所制备的黄芩美白抗敏提取液在美白因子上进行功效评价：
以正常细胞为空白组，以添加White 377的细胞为对照，和添加黄芩提取液的细胞进行实验对比。利用RTPCR检测仪检测MITF、TRP1、TRP2、TYR对应mRNA的表达量，在基因水平对黄芩提取液进行功效评价。MITF：调节MC的生长、分化参与色素的沉着；TRP1：与TYR作用相似，使酪氨酸羟化酶和多巴酶活性增加，色素沉着；TRP2：调节TYR的催化活性，具加速黑素生成的作用；TYR:黑素生成的限速酶，决定黑素合成的速度和产量，详见图1。结果表明：SGB-E分别可显著降低MITF、TRP1、TYR2、TYR基因，保持肌肤美白。
对上述制备工艺所制备的黄芩美白抗敏提取液进行酪氨酸酶活性检测验证黄芩美白抗敏提取液具有美白的功效；以Control为空白对照组，Hyd为阳性对照（氢醌），8为White 377，和添加黄芩提取液的细胞进行实验对比，详见图2。结果表明：与空白组相比，8可明显抑制酪氨酸酶活性，抑制率为67%。与对照组和阳性对照组相比，SGB-E可抑制酪氨酸酶活性，且抑制率为65%，P<0.05 ，有统计学意义。
对上述制备工艺所制备的黄芩美白抗敏提取液在炎症因子上进行功效评价：
发明中空白组（PMA诱导，未加LPS）、模型组（PMA诱导，加LPS活化）、阳性组为地塞米松组和添加黄芩提取液的细胞进行实验对比，利用RTPCR检测仪检测IL-1B、IL-6、IL-10对应mRNA的表达量，在基因水平对黄芩提取液进行功效评价，详见图3。结果表明：SGB-E可抑制巨噬细胞分泌炎症因子IL-1B、IL-6，同时促进IL-10炎症因子分泌，且效果由于地塞米松，表明提取液具有抗炎功效。
对上述制备工艺所制备的黄芩美白抗敏提取液进行透明质酸酶实验，验证黄芩提取液具有抗敏的作用；发明中对照组为甘草酸二钾，样品为黄芩提取液的三个梯度分为高中低，进行透明质酸酶对比试验，详见图4。结果表明：与空白组相比，甘草酸二钾可明显抑制透明质酸酶活性，抑制率为26.72%。黄芩低、中、高三个剂量组都能抑制透明质酸酶活性，抑制率分别为：92.99%，94.08%，95.64%, 且随着浓度的增高抑制率增大，表明其具有抗敏抗炎作用。
Claims (10)
1.一种黄芩美白抗敏提取液，其特征在于，所述黄芩美白抗敏提取液包含：黄芩素、黄芩苷、丁二醇和水。
2.根据权利要求所述的黄芩美白抗敏提取液，其特征在于，所述黄芩素的重量份数为1-5份，所述黄芩苷的重量份数为1-5份，所述丁二醇的重量份数为65-75份，所述水的重量份数为15-25份。
3.一种黄芩美白抗敏提取液的制备工艺，其特征在于，包括以下步骤：
步骤1 将原料黄芩根进行超微粉碎，然后采用乙醇水溶液进行超声提取，再将提取液用80目筛进行过滤，浓缩后得到浓缩液；
步骤2 将步骤1所得的浓缩液用石油醚进行萃取，弃掉上层石油醚相，得到下层水相；
步骤3 将步骤2所得的下层水相，用乙酸乙酯进行萃取，弃掉下层水相，将上层的乙酸乙酯相减压浓缩至浸膏；
步骤4 将步骤3所得的浸膏采用甲醇溶解，拌样，挥干，然后用甲醇水洗脱，浓缩得到黄芩素和黄芩苷混合物；
步骤5 将步骤4所得的黄芩素和黄芩苷混合物冷冻干燥为固体粉末，采用溶剂1,3-丁二醇:去离子水=3:1将固体粉末配置成浓度为8-15mg/ml的溶液，0.22μm过滤除菌，即得黄芩美白抗敏提取液。
4.根据权利要求3所述的黄芩美白抗敏提取液的制备工艺，其特征在于，所述步骤1具体是：将原料黄芩根除杂，进行超微粉碎，然后采用质量浓度为95%的乙醇水溶液进行超声提取1h/次，其中提取的料液比为1:5-1:10，再将提取液用80目筛进行过滤，在48-68℃温度下，浓缩后得到浓缩液，所述浓缩液的热比重为1.1-1.3。
5.根据权利要求3或4所述的黄芩美白抗敏提取液的制备工艺，其特征在于：所述步骤3中的超声提取的次数为3次。
6.根据权利要求3所述的黄芩美白抗敏提取液的制备工艺，其特征在于，所述步骤2具体是：将步骤1所得的浓缩液用体积为2-3倍量的石油醚进行萃取3-4次，静置3-5h，弃掉上层石油醚相，得到下层水相。
7.根据权利要求3所述的黄芩美白抗敏提取液的制备工艺，其特征在于，所述步骤3具体是：将步骤2所得的下层水相，用体积为2-3倍量的乙酸乙酯进行萃取3-4次，静置3-5h，弃掉下层水相，在48-68℃温度下，将上层的乙酸乙酯相减压浓缩至浸膏。
8.根据权利要求3所述的黄芩美白抗敏提取液的制备工艺，其特征在于，所述步骤4具体是：将步骤3所得的浸膏采用甲醇溶解，采用反相C18填料拌样，挥干甲醇后装入预备柱中，然后先后采用质量浓度为10%、30%、60%的甲醇水洗脱，洗脱速率为50-100ml/min，洗脱体积为3-4柱体积，浓缩得到黄芩素和黄芩苷混合粉末，所述反相C18填料的质量为浸膏质量的3-5倍量。
9.根据权利要求3所述的黄芩美白抗敏提取液的制备工艺，其特征在于：所述步骤5具体是：将步骤4所得的黄芩素和黄芩苷混合粉末冷冻干燥程固体粉末，冷冻干燥温度为-78℃，冷冻时间为48-72h，采用溶剂1,3-丁二醇:去离子水=3:1将固体粉末配置成浓度为10mg/ml的溶液，于十万级制备车间0.22μm微孔滤膜过滤除菌，即得黄芩美白抗敏提取液。
10.一种使用权利要求3-9任一项的制备工艺制备的黄芩美白抗敏提取液在制备化妆品及护肤品的应用。
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