CN115109057B - 一种具有美白护肤作用的萍蓬草生物碱提取物及其制备方法 - Google Patents
一种具有美白护肤作用的萍蓬草生物碱提取物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有美白护肤作用的萍蓬草生物碱提取物及其制备方法,将干青萍蓬草置于pH为2‑3的水中回流提取,得萍蓬草水提液,过滤,滤液浓缩,得浓缩液,离心收集上清液,加入异丙醇、硫酸铵和氯化钠,得到双水相体系,调节pH至10‑11,混合均匀,在60‑80℃下萃取15‑30min,之后离心分相,收集异丙醇相,回收其中的异丙醇,得萍蓬草生物碱提取物;本发明方法简单易行,生产成本低、不存在有机溶剂存留,将所得萍蓬草生物碱提取物进行美白护肤评价,具有较好的效果。
Description
技术领域
本发明涉及一种具有美白护肤作用的萍蓬草生物碱提取物及其制备方法。
背景技术
萍蓬草Nuphar pumilum为睡莲科萍蓬草属植物,也是极具有观赏价值的水生植物,广泛分布于黑龙江、河北、江苏、浙江、江西、贵州等省市。萍蓬草种子含有丰富的淀粉,可食用;果实可以煮粥,根茎可食用也可入药,能健脾胃,可补虚止血,可用于治疗病后体弱,月经不调,刀伤等症。在日本,民间用作退热剂、止痛剂、抗炎剂。近年来又从此植物中发现多种单体成分,其中一些倍半萜类萍蓬草生物碱具有显著的免疫抑制作斥和肿瘤细胞转移抑制作用。
萍蓬草属植物的主要药效成分为生物碱,按化学分类称萍蓬草碱生物碱,也有归属于萍蓬草喹诺里西丁类或哌啶类。本发明研究表明萍蓬草生物碱有较好的美白护肤作用。现有从萍蓬草中提取萍蓬草生物碱的方法一般采用乙酸乙酯等溶剂萃取,或用大孔吸附树脂法纯化。这些方法的不足之处是,费时、提取率低、提取的萍蓬草生物碱品质不高等。
发明内容
本发明提供了一种具有美白护肤作用的萍蓬草生物碱提取物及其制备方法,该法是一种处理量大,能耗低、生产成本低、纯化得率高、产品纯度好、设备投资少、不引起萍蓬草生物碱失活或变性、分相时间短、界面张力小、不存在有机溶剂残留、分离过程更经济、易于工程放大和连续操作的双水相萃取分离纯化萍蓬草生物碱工艺。
本发明制备的萍蓬草生物碱提取物,采用体外透明质酸酶抑制评价以及酪氨酸酶抑制评价,结果表明萍蓬草生物碱提取物具有很好的美白护肤作用,该生物碱提取物可用于美白护肤领域。
本发明的技术方案如下:
一种萍蓬草生物碱提取物的制备方法,步骤如下:
(1)将干青萍蓬草置于pH为2-3的水中回流提取,得萍蓬草水提液,过滤,滤液浓缩,得浓缩液;
所述干青萍蓬草与pH为2-3的水的液料质量体积比为1:6-10kg/L;
所述回流提取的温度在80-100℃,时间为1-3h;
所述滤液在0.08-0.1MPa条件下进行浓缩,浓缩至原始体积的1/3-1/2;
(2)将步骤(1)所得浓缩液离心,收集上清液;
(3)在步骤(2)收集的上清液中加入异丙醇、硫酸铵和氯化钠,得到双水相体系,调节pH至10-11,混合均匀,在60-80℃下萃取15-30min,之后离心分相,收集异丙醇相(上相);
所述双水相体系中,异丙醇的质量分数为30-33%,硫酸铵的质量分数为16-18%,氯化钠的质量分数为0.2-0.6%;
(4)回收步骤(3)收集的异丙醇相中的异丙醇,得萍蓬草生物碱提取物;
所得萍蓬草生物碱提取物中,萍蓬草碱含量可达53%以上,回收率可达70%以上。
本发明萍蓬草生物碱提取物中萍蓬草碱生物碱的含量检测方法如下:
精密称取含生物碱对照品12mg的样液,于25mL容量瓶中用二氯甲烷定容至刻度。精密吸取上述溶液2.8,2.4,1.6,1.2,0.8,0.4mL,于50mL锥形瓶中,添加二氯甲烷使有机相为10mL,配成浓度为0.14、0.12、0.08、0.06、0.04、0.02mg/mL生物碱对照品溶液,然后每瓶各加入10mL 配置好的溴百里香酚蓝缓冲液,超声5min,静置0.5h,取下层有机相以5000rpm离心10min后,在413nm下测定吸光度值。以浓度C(mg/mL)为横坐标,吸光度值A为纵坐标,进行线性回归,确定标准曲线。
精密称取1.00g萍蓬草细粉或者萍蓬草提取物,加入10mL二氯甲烷,超声10min后,分液得到有机相;移取1mL有机相入50mL锥形瓶,添加二氯甲烷使有机相为10mL,然后加入10mL 配置好的溴百里香酚蓝缓冲液,超声5min,静置0.5h,取下层有机相以5000rpm离心10min后,在413nm下测定吸光度值。根据标准曲线计算萍蓬草提取物中生物碱浓度。
总生物碱(%)=(生物碱浓度×体积×稀释倍数)/提取物重量×100%
本发明还涉及上述制备方法制备的萍蓬草生物碱提取物,所述萍蓬草生物碱提取物可应用于美白产品的制备。具体应用的方法为:
将萍蓬草生物碱提取物添加到普通的美白保湿液中混匀,将起到良好的美白护肤效果;
所述萍蓬草生物碱提取物的添加量在0.05-0.2wt%。
本发明的有益效果在于:
本发明经热回流提取、离心、双水相萃取后,使萍蓬草生物碱得以浓缩纯化,获得高品质的萍蓬草生物碱。本发明利用萍蓬草生物碱在异丙醇/硫酸铵双水相系统中的选择性分配,在萍蓬草生物碱进入双水相体系后,在两相间进行选择性分配,表现出一定的分配系数。萍蓬草生物碱对异丙醇具有亲和性,硫酸铵对杂质具有一定的亲和性,使萍蓬草生物碱能较容易地溶解在上相异丙醇中,而一些杂质分配在下相。
本发明用于从萍蓬草中提取纯化萍蓬草生物碱,处理量大,能耗低、生产成本低、纯化得率高、产品纯度好、设备投资少、不引起萍蓬草生物碱失活或变性、分相时间短、界面张力小、不存在有机溶剂残留、分离过程经济、易于工程放大和连续操作。
附图说明
图1:Nupharidine萍蓬草碱质谱图。
具体实施方式
下面通过具体实施例进一步描述本发明,但本发明的保护范围并不仅限于此。
实施例1
(1)在去杂、清洗、烘干的干青萍蓬草10kg中,加入料液比为8的水,用10wt%盐酸调pH值至2,90℃回流提取2次,每次1h,过100目筛,合并滤液,于0.09MPa浓缩滤液至1/2体积,得浓缩液。
(2)将浓缩液至离心机中,在3000rpm的转速下离心10min,收集上清液备用。
(3)在该上清液中加入异丙醇、硫酸铵和氯化钠,使整个双水相体系中32%(w/w)的异丙醇, 17%(w/w)的硫酸铵0.4%(w/w)的氯化钠,并采用10wt%NaOH水溶液调节体系pH至10,混合均匀,萃取温度80℃条件下,萃取30min,离心(3000rpm,10min)分相,收集异丙醇相(上相)。
(4)用所述步骤(3)的异丙醇相,回收异丙醇,得萍蓬草生物碱提取物115g。
实施例2
(1)在去杂、清洗、烘干的干青萍蓬草1kg中,加入料液比为10的水,用10wt%盐酸调pH值至3,90℃回流提取2次,每次1h,过100目筛,合并滤液,0.09MPa浓缩至1/3体积,得浓缩液。
(2)将浓缩液至离心机中,在3000rpm的转速下离心10min,收集上清液备用。
(3)在该上清液中加入一定量异丙醇、硫酸铵和氯化钠,使整个双水相体系中30%(w/w)的异丙醇,18%(w/w)的硫酸铵0.2%(w/w)的氯化钠,并采用10wt%NaOH水溶液调节体系pH至11,混合均匀,萃取温度70℃条件下,萃取30min,离心(3000rpm,10min)分相,收集异丙醇相(上相)。
(4)用所述步骤(3)的异丙醇相,回收异丙醇,得萍蓬草生物碱提取物12g。
(5)萍蓬草生物碱提取物12g用20mL丙酮分散溶解,过滤,滤饼减压干燥得不溶解部分5.4g。进一步用10mL甲醇-丙酮(1:1)溶解,放置于40度环境蒸发结晶重复三次得样品3.1g。经核磁和质谱检测为萍蓬草碱。
Nupharidine萍蓬草碱碳谱数据
C1:30.66 | C9:21.32 |
C2:32.26 | C10:78.06 |
C3:27.58 | C11:19.06 |
C4:73.11 | C12:19.09 |
C13:118.65 | |
C6:57.42 | C14:142.96 |
C7:26.15 | C15:111.69 |
C8:25.67 | C16:144.56 |
实施例3透明质酸酶抑制实验
透明质酸酶抑制实验与护肤美白有着直接联系。对实施例1所获得的萍蓬草生物碱提取物进行实验表明其对透明质酸酶有一定的抑制活性。具体过程与结果如下:每个样品均采用Elson-Morgan 改良法测定提取物与透明质酸酶作用,1、2号试管(0.5mL样溶液和0.5mL透明质酸酶),3、4号试管(0.5mL蒸馏水和0.5mL pH=5.4缓冲溶剂),37℃保温20min;加入0.1mL(2.5mol/L)氯化钙溶液,37℃保温20min;1、3号试管中加入0.5mL透明质酸钾,2、4号试管中加入0.5mL pH=5.4 缓冲溶剂,37℃保温40min;室温放置10min,加入0.5mL乙酰丙酮溶液(3.5mL乙酰丙酮溶于 50mL 0.1mol/L碳酸钠溶液中)、0.1mL NaOH溶液(5mol/L)和0.5mL蒸馏水,放入沸水浴中15min;冷却5min,放入冰水浴10min;室温放置10min,加入1mL P-DAB显色剂(0.8g DBMA与15mL 浓HCl以及无水乙醇混合均匀);充分振荡后,加入无水乙醇3.5mL,室温放置30min,显色,于 530nm处测定OD值。
透明质酸酶抑制率(%)=[(C-D)-(A-B)]/(C-D)×100%
式中:
A为1号试管中溶液的OD值;
B为2号试管中溶液的OD值;
C为3号试管中溶液的OD值;
D为4号试管中溶液的OD值。
表1萍蓬草生物碱提取物对透明质酸酶抑制效果
*:鸢尾苷为阳性对照
实施例4酪氨酸酶抑制率测定方法
在多只试管中各自加入2mL 1mg/mL的左旋多巴溶液,再分别加入1mL 5种不同的浓度的实施例 1获得的萍蓬草生物碱提取物水溶液(0.05mg/mL,0.1mg/mL,0.2mg/mL,0.4mg/mL),采用阳性对照(0.14mg/mL曲酸溶液)后,加入pH 6.8磷酸氢二钠-柠檬酸缓冲液补齐至反应体系总体积为3.5mL,每组设置3个平型管,于37℃条件下保持10min后,加入比活力为120U/mL的酪氨酸酶溶液0.5mL,继续保持5min后,在分光光度计475nm处测定酪氨酸酶的抑制率,测试结果如表2所示:
表2萍蓬草生物碱提取物对酪氨酸酶抑制效果
*:曲酸为阳性对照。
Claims (7)
1.一种萍蓬草生物碱提取物的制备方法,其特征在于,所述制备方法的步骤如下:
(1)将干青萍蓬草置于pH为2-3的水中回流提取,得萍蓬草水提液,过滤,滤液浓缩,得浓缩液;
(2)将步骤(1)所得浓缩液离心,收集上清液;
(3)在步骤(2)收集的上清液中加入异丙醇、硫酸铵和氯化钠,得到双水相体系,调节pH至10-11,混合均匀,在60-80℃下萃取15-30min,之后离心分相,收集异丙醇相;
所述双水相体系中,异丙醇的质量分数为30-33%,硫酸铵的质量分数为16-18%,氯化钠的质量分数为0.2-0.6%;
(4)回收步骤(3)收集的异丙醇相中的异丙醇,得萍蓬草生物碱提取物。
2.如权利要求1所述的制备方法,其特征在于,步骤(1)中,所述干青萍蓬草与pH为2-3的水的液料质量体积比为1:6-10kg/L。
3.如权利要求1所述的制备方法,其特征在于,步骤(1)中,所述回流提取的温度在80-100℃,时间为1-3h。
4.如权利要求1所述的制备方法,其特征在于,步骤(1)中,所述滤液在0.08-0.1MPa条件下进行浓缩,浓缩至原始体积的1/3-1/2。
5.如权利要求1所述的制备方法制备的萍蓬草生物碱提取物。
6.如权利要求5所述的萍蓬草生物碱提取物在美白产品的制备中的应用。
7.如权利要求6所述的应用,其特征在于,所述应用的方法为:
将萍蓬草生物碱提取物添加到普通的美白保湿液中混匀,将起到良好的美白护肤效果;
所述萍蓬草生物碱提取物的添加量在0.05-0.2wt%。
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