CN109078047B - 桑白皮中具酪氨酸酶抑制作用的有效部位的制备及应用 - Google Patents
桑白皮中具酪氨酸酶抑制作用的有效部位的制备及应用 Download PDFInfo
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- CN109078047B CN109078047B CN201811089173.XA CN201811089173A CN109078047B CN 109078047 B CN109078047 B CN 109078047B CN 201811089173 A CN201811089173 A CN 201811089173A CN 109078047 B CN109078047 B CN 109078047B
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Abstract
本发明公开了桑白皮中具酪氨酸酶抑制作用的有效部位的制备及应用，属于医药、护肤品和食品技术领域。本发明提供的桑白皮中具酪氨酸酶抑制作用有效部位，包括50%乙醇洗脱部位和70%乙醇洗脱部位，所述50%乙醇洗脱部位的黄酮类化合物是桑根酮T、桑皮酮L、桑皮酮G、桑根酮G、桑皮酮O，所述70%乙醇洗脱部位的黄酮类化合物是桑皮酮L、桑皮酮O、桑根酮G、桑皮酮E、桑皮酮T、5'‑香叶草基‑5,7,2',4'‑四羟基黄酮、桑根酮V。所述具酪氨酸酶抑制作用的有效部位能够有效用于果蔬的酶促褐变的抑制，化妆品中的美白，以及用于预防和治疗黑色素过多导致的人体色素沉着性疾病、黑色素瘤以及其他需要抑制酪氨酸酶活性的病症药物的制备。
Description
技术领域
本发明属于食品、护肤品和医药技术领域，具体涉及桑白皮中具酪氨酸酶抑制作用的有效部位的制备及应用。
背景技术
酪氨酸，广泛应用于微生物、植物和哺乳动物中，又名单酚氧化酶，是一种含铜离子的金属氧化酶，是多酚氧化酶的一种，是动植物体内黑色素合成的关键酶。酪氨酸酶通过催化作用，先使单酚羟基氧化成邻二酚，然后邻二酚在其催化下进一步被氧化成邻二醌类，紧接着再经过一系列复杂的非酶反应，最终生成黑色素；在整个过程中，酪氨酸酶起着至关重要的作用。人的体内产生的黑色素能够阻挡太阳紫外线对皮肤的伤害，而且皮肤中黑色素的种类不同也造就了人类不同颜色的头发和皮肤，但分泌的黑色素太多则会引起不同的斑类问题，如色斑、黑斑、老年斑等。在食品工业上，酪氨酸酶催化反应导致新鲜水果、蔬菜和饮料产生褐变，降低其营养价值、市场价值和贮存周期。酪氨酸酶为黑色素合成的关键酶，其表达和活性与果蔬的褐变和人体色素沉着性疾病密切相关。因此，酪氨酸酶抑制剂可作为黑色素抑制剂应用于食品、化妆品、医药等领域。到目前为止，受限于有效性、安全性及成本等因素，只有极少数酪氨酸酶抑制剂被用于商业上。因此，寻找天然无毒高效的酪氨酸酶抑制剂应用于化妆品领域显得尤为迫切。植物天然酪氨酸酶抑制剂具有安全系数高、低毒副作用、材料易得、独特的效果等优点，受到越来越多关注。
桑白皮别名桑根皮、白桑皮、根皮，为桑科桑属植物桑(Morus alba L.)除去栓皮的根皮。桑在我国大部分地方广泛分布和种植，主产江苏、浙江、安徽、湖南、河北、四川等地。其性甘微苦、寒，归肺经，具有清肺平喘、利水消肿功能，主治肺热喘咳、水肿胀满、尿少、面目肌肤浮肿。民间常用于消炎、利尿、解热、镇咳、怯痰等。现代药理学研究表明，桑白皮具有降血糖、降血压、抗肿瘤、抗炎抑菌、抗病毒等作用。现代研究表明桑白皮中含有黄酮、生物碱、多糖、香豆素、多糖、挥发油等成分。其中黄酮类成分受到广泛关注，其具有减肥、抗糖尿病、抗氧化、降糖、抗HIV等作用。然而，对桑白皮具体美白活性成分的研究很少，现有研究还停留在粗提取物对酪氨酸酶测试(《桑提取物对酪氨酸酶的抑制作用研究》，北京化工大学，硕士论文)，或用简单薄层板进行追踪(申请号：CN201110425854.0)，依然无法探知其中具体对酪氨酸酶有抑制作用的物质。因此，探讨桑白皮中酪氨酸酶抑制作用的有效部位和成分，具有重要的研究意义。
发明内容
本发明的目的首先在于提供一种桑白皮中具酪氨酸酶抑制作用的有效部位，是从桑白皮中提取的具有较强酪氨酸酶抑制活性的含有9个黄酮类化合物的有效部位，所述有效部位是将桑白皮用乙醇浸提，所得提取液减压浓缩后用乙醇溶解，然后采用大孔树脂吸附，用不同浓度的乙醇进行洗脱，分别收集不同浓度的乙醇洗脱物，然后减压浓缩即得，所述有效部位包括50％乙醇洗脱部位和70％乙醇洗脱部位，所述50％乙醇洗脱部位的黄酮类化合物是桑根酮T(sanggenon T)、桑皮酮L(kuwanon L)、桑皮酮G(kuwanon G)、桑根酮G(sanggenon G)、桑皮酮O(kuwanon O)，所述70％乙醇洗脱部位的黄酮类化合物是桑皮酮L(kuwanon L)、桑皮酮O(kuwanon O)、桑根酮G(sanggenon G)、桑皮酮E(kuwanon E)、桑皮酮T(kuwanon T)、5'-香叶草基-5,7,2',4'-四羟基黄酮(5'-geranyl-5,7,2',4'-tetrahydroxyflavone)、桑根酮V(sanggenon V)。
所述有效部位的黄酮类化合物的质量分数为50％～66％。
本发明的另外一个目的是提供所述桑白皮中具酪氨酸酶抑制活性的有效部位的制备方法，通过以下步骤实现：
(1)将桑白皮粉碎至50-100目后用2倍质量的90-95％乙醇冷浸提取2次，每次24-28小时，所得提取液减压浓缩至干燥物；
(2)将干燥物用90-95％乙醇溶解，过滤，收集过滤液；
(3)将上述过滤液与大孔树脂D101混合拌样得到搅拌物，自然晾干或减压浓缩至干；其中，过滤液的用量按过滤液中所含固体提取物的质量与大孔树脂D101的质量比例为1:2来设定；
(4)将搅拌物上D101大孔树脂层析柱，分别用10倍柱体积量的10％、30％、50％、70％、95％乙醇洗脱，分别收集10％、30％、50％、70％、95％乙醇洗脱部位，减压浓缩至干燥物，所得50％乙醇洗脱部位和70％乙醇洗脱部位的干燥物，即为桑白皮具酪氨酸酶抑制作用的有效部位。
(5)将各浓度乙醇洗脱部位的干燥物进行酪氨酸酶抑制活性测试，并以对酪氨酸酶抑制活性为指标，以曲酸为阳性对照，确定酪氨酸酶抑制活性高的部位为50％乙醇洗脱部位和70％乙醇洗脱部位，即为本发明所述具酪氨酸酶抑制活性的有效部位。
步骤(5)确定的有效部位，进一步采用UPLC-MS进行分析鉴定，确定50％乙醇洗脱部位的黄酮类化合物为sanggenon T、kuwanon L、kuwanon G、sanggenon G、kuwanon O，70％乙醇洗脱部位的黄酮类化合物为kuwanon L、kuwanon O、sanggenon G、kuwanon E、kuwanon T、5'-geranyl-5,7,2',4'-tetrahydroxyflavone、sanggenon V。
本发明的第三个目的是提供所述的具有酪氨酸酶抑制作用的化合物及其有效部位在制备抗果蔬酶促褐变抑制剂、化妆品美白剂、洗浴用品、预防和治疗黑色素异常导致的人体色素沉着性疾病、黑色素瘤以及其他需要抑制酪氨酸酶活性的病症的药物中的应用。
本发明从桑白皮中提取的具有酪氨酸酶抑制活性的黄酮类化合物及其有效部位可以作为活性成分，不加或加入食品添加剂辅料，化妆品辅料或药剂上接受的辅料，按照相应剂型的制备方法制成制剂。
所述的剂型包括固体粉末、水溶液、醇溶液、乳液、面膜、巴布剂、注射液、滴注液、粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、口含剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、口崩剂、微丸等。
本发明采用以上技术方案，通过大孔树脂进行分段分离，通过酪氨酸酶抑制测试确定桑白皮中具酪氨酸酶抑制作用的有效部位，再应用现代分析检测技术HPLC-MS确定有效部位的主要成分。本发明的有益之处在于：提供从桑白皮中提取的具有酪氨酸酶抑制作用的黄酮类化合物的有效部位，具有比桑白皮提取物更强的活性，制成相应制剂易于质量控制，可应用于抗果蔬酶促褐变抑制剂、化妆品美白剂、预防和治疗黑色素异常导致的人体色素沉着性疾病、黑色素瘤以及其他需要抑制酪氨酸酶活性的病症的药物中。
附图说明
图1为桑白皮具有酪氨酸酶抑制作用的50％乙醇洗脱部位的UPLC/Q-TOF-MS紫外及总离子流谱图；(a)：UV色谱图；(b)：总离子流图；(c)～(g)：主要酪氨酸酶抑制剂的MS质谱图。
图2为桑白皮具有酪氨酸酶抑制作用的50％乙醇洗脱部位中主要酪氨酸酶抑制剂的MS/MS质谱图。
图3为桑白皮具有酪氨酸酶抑制作用的70％乙醇洗脱部位的UPLC/Q-TOF-MS紫外及总离子流谱图；(a)：UV色谱图；(b)：总离子流图；(c)～(i)：主要酪氨酸酶抑制剂的MS/MS质谱图。
图4为桑白皮具有酪氨酸酶抑制作用的70％乙醇洗脱部位的主要酪氨酸酶抑制剂的MS/MS质谱图。
图5为总黄酮测定芦丁标准曲线。
具体实施方式
下面将结合实施例进一步详细说明本发明的实质内容和有益效果，这些实施例仅用于说明本发明而非对本发明的限制。此外，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1 有效部位的制备
将20公斤桑白皮粉碎成50-200目粒度后，用2倍量95％乙醇浸泡提取2次，每次24h，所得提取液减压浓缩至干燥物(即桑白皮粗提取物)，称重。取适量干燥物用95％乙醇溶解，过滤，加入大孔树脂D101中，自然晾干或减压蒸干，然后上大孔树脂D101层析柱，分别用10个柱体积的10％、30％、50％、70％、95％乙醇洗脱，收集洗脱液，分别减压回收乙醇至干(分别得到集10％、30％、50％、70％、95％乙醇洗脱部位)，称重，待进行酪氨酸酶抑制活性测试。
将桑白皮粗提取物和各浓度乙醇洗脱部位溶于DMSO，先配成1mg/mL溶液，使用时稀释到需要浓度。检测10μg/mL粗提取物和有效部位抑制酪氨酸酶的活性。阳性对照曲酸配成25、20、10、7.5、5μg/mL浓度的溶液。将上述三种溶液各30μL，用970μL磷酸盐缓冲溶液配成1mL，加进0.1mg/mL的酪氨酸1mL，然后加入由磷酸盐缓冲溶液配成的1mL酪氨酸酶(200U/mL)，在37℃下孵育20min，于492nm处测定吸光值。
酶活性抑制率＝[(A2-A1)-(B2-B1)]/(A2-A1)×100％
A1为0min时候未加抑制剂的吸收值；A2为20min后未加抑制剂的吸收值；
B1为0min时候加抑制剂的吸收值；B2为20min后加了抑制剂的吸收值。
10μg/mL桑白皮粗提取物、50％乙醇洗脱部位和70％乙醇洗脱部位对酪氨酸酶抑制率分别为31.08％、90.16％和98.03％，阳性对照曲酸10μg/mL对酪氨酸酶抑制率为71.65％。
10％、30％、95％乙醇洗脱部位对酪氨酸酶抑制率分别为14.70％、67.59％、46.50％，相比桑白皮粗提取物，这3个浓度的乙醇洗脱部位对酪氨酸酶抑制率有所提高，但酪氨酸酶抑制率低于50％乙醇洗脱部位和70％乙醇洗脱部位。因此，本发明确定的桑白皮中具酪氨酸酶抑制活性的有效部位为50％乙醇洗脱部位和70％乙醇洗脱部位。
实施例2 桑白皮中具酪氨酸酶抑制作用的有效部位中主要酪氨酸酶抑制成分的分析鉴定
样品配置：分别称取样品(50％乙醇洗脱部位、70％乙醇洗脱部位)5.0毫克，置于1.5毫升离心管中，用1.0毫升甲醇溶解。10000rps离心2分钟，取清液用甲醇稀释，置于1.5毫升进样瓶，进样2ul。
超高效液色谱-四级杆飞行质谱联用检测条件：仪器：Waters Maldi Synapt Q-TOF MS；色谱仪UHPLC：Waters Acquity UPLC；检测器UV detector：Waters Acquity PDA；色谱柱：Acquity BEH C18(1.7μm；50×2.1mm I.D.)。
流动相：流动相A相为乙腈，B相为0.1mol/L甲酸；洗脱梯度：0min，5％A；8min，30％A；10min，80％A；13min，100％A；14min，100％A；14.1min，5％A。流速，0.3mL/min。
质谱条件：电喷雾离子源，负离子扫描模式；锥孔电压，30V；毛细管电压，3.0kV；通道1碰撞能量，6eV；通道2碰撞能量，20eV；离子源温度，100℃；脱溶剂气温度，400℃；扫描范围，50-1500Da。
表1 50％乙醇洗脱部位超高效液色谱-质谱联用检测结果
如图1(c)和图2(a)所示，7.00min处峰负离子的m/z为711[M-H]-，表明其分子量为712，其主要碎片为549.1、439.1、313.1、287.0、277.1、125.0，根据分子量和分子碎片断裂方式确定该化合物为sanggenon T。
如图1(d)和图2(b)所示，7.83min处峰负离子的m/z为625[M-H]-，表明其分子量为626，其主要碎片为499.1、389.1、279.0、227.0、175.1、125.0、109.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon L。
如图1(e)和图2(c)所示，10.10min处峰负离子的m/z为691[M-H]-，表明其分子量为692，其主要碎片为581.1、419.1、353.1，根据分子量和分子碎片断裂方式确定该化合物为kuwanon G。
如图1(f)和图2(d)所示，10.66min处峰负离子的m/z为693[M-H]-，表明其分子量为694，其主要碎片为563.1、437.1，根据分子量和分子碎片断裂方式确定该化合物为sanggenon G。
如图1(g)和图2(e)所示，11.98min处峰负离子的m/z为693[M-H]-，表明其分子量为694，其主要碎片为581.1、531.1、421.1、405.1、287.0、259.1、161.0、125.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon O。
表2 70％乙醇洗脱部位超高效液色谱-质谱联用检测结果
如图3(c)和图4(a)所示，9.45min处峰负离子的m/z为625[M-H]-，表明其分子量为626，其主要碎片为499.1、389.1、285.0、227.1、125.0、109.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon L。
如图3(d)和图4(b)所示，13.16min处峰负离子的m/z为693[M-H]-，表明其分子量为694，其主要碎片为567.1、457.1、389.1、347.1、279.1、177.1、125.0，根据分子量和分子碎片断裂方式确定该化合物为sanggenon G。
如图3(e)和图4(c)所示，14.91min处峰负离子的m/z为693[M-H]-，表明其分子量为693，其主要碎片为583.1、531.1、421.1、405.1、287.1、259.1、161.0、125.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon O。
如图3(f)和图4(d)所示，17.44min处峰负离子的m/z为423[M-H]-，表明其分子量为424，其主要碎片为297.1、125.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon E。
如图3(g)和图4(e)所示，18.02min处峰负离子的m/z为421[M-H]-，表明其分子量为422，其主要碎片为295.1、126.0、125.0，根据分子量和分子碎片断裂方式确定该化合物为kuwanon T。
如图3(h)和图4(f)所示，18.43min处峰负离子的m/z为421[M-H]-，表明其分子量为422，其主要碎片为295.1,125.0，根据分子量和分子碎片断裂方式确定该化合物为5'-geranyl-5,7,2',4'-tetrahydroxyflavone。
如图3(i)和图4(g)所示，19.46min处峰负离子的m/z为419[M-H]-，表明其分子量为420，其主要碎片为297.1、255.2、121.0，根据分子量和分子碎片断裂方式确定该化合物为sanggenon V。
实施例3 桑白皮中具酪氨酸酶抑制作用的有效部位提取物总黄酮含量测定
总黄酮测定：分别移取0.2mg/mL的芦丁标准溶液0.0、1.0、2.0、4.0、6.0、8.0和10.0mL于25mL容量瓶中，加入60％乙醇溶液稀释至15mL，加入5％(w/v)亚硝酸钠溶液0.3mL，摇匀，静置5min，加入10％(w/v)硝酸铝溶液0.3mL，摇匀，再加入1mol/L氢氧化钠溶液2mL，用60％乙醇溶液稀释至刻度线处，室温静置15min。以第一管为空白对照，于波长510nm处测定吸光度值，绘制芦丁标准曲线。芦丁绘制标准曲线如图5所示：回归方程y＝7.2432x-0.0111，相关性系数R2＝0.9989。
测得的桑白皮粗提取总黄酮含量为7.8％，50％乙醇洗脱有效部位提取物中总黄酮含量为58％，70％乙醇洗脱有效部位总黄酮含量为66％。
桑白皮中具酪氨酸酶抑制作用的有效部位提取物中总黄酮含量高于桑白皮粗提取物，说明其酪氨酸酶抑制活性主要是这些黄酮起作用的。
实施例4 含桑白皮具酪氨酸酶抑制作用的有效部位提取物美白面膜的制备
桑白皮提取物0.6％，甘草提取物0.3％，尿囊素0.1％，D-泛醇1％，尼泊金甲酯0.1％，1，2-已二醇0.6％，植物抗敏剂0.3％，香料适量，去离子水加至100％。
其中桑白皮提取物是指桑白皮50％或70％乙醇洗脱部位的提取物。
制法：准确称量各物料，用干净并消毒好的器皿盛放；先把称好量的水加入到搅拌锅中，再加入提取物，慢慢搅拌至升温到85℃；恒温搅拌30min，溶解至无颗粒，降温30℃备用；把过滤好的水倒入乳化锅搅拌加热至90℃，加入以上各物料恒温搅拌均质3min待溶解无颗粒；降温至35℃取料送检确认合格后出料。
实施例5 含桑白皮具酪氨酸酶抑制作用的有效部位提取物的沐浴露配制
桑白皮提取物0.2％，甘草提取物0.1％，EDTA-2Na0.1％，尼泊金甲酯0.1％，AES70％15％，MES 8％，CAB-35 5％，食盐1％，A5356金盏菊0.2％，香料适量，去离子水加至100％。
其中桑白皮提取物是指桑白皮50％或70％乙醇洗脱部位的提取物。
制法：准确称量各物料，用干净并消毒好的器皿盛放；先把称好量的水加入到搅拌锅中，再加入提取物，慢慢搅拌至升温到85℃；恒温搅拌30min，溶解至无颗粒，降温30℃过滤备用；把过滤好的水倒入搅拌锅中搅拌，顺序加入原料，并加热到85℃；搅拌至溶解无颗粒，保温30min，然后降温，搅拌均匀，加入食盐调粘度；取料送检确认合格出料，静置。
Claims (7)
1.一种制备桑白皮中具酪氨酸酶抑制作用的有效部位的方法，其特征在于，通过以下步骤实现：
（1）将桑白皮粉碎至50-100目后用2倍质量的90-95%乙醇冷浸提取2次，每次24-28小时，所得提取液减压浓缩至干燥物；
（2）将干燥物用90-95%乙醇溶解，过滤，收集过滤液；
（3）将上述过滤液与大孔树脂混合拌样得到搅拌物，自然晾干或减压浓缩至干；其中，过滤液的用量按过滤液中所含固体提取物的质量与大孔树脂的质量比例为1:2来设定；
（4）将搅拌物上大孔树脂层析柱，分别用10倍柱体积量的10%、30%、50%、70%、95%乙醇洗脱，分别收集10%、30%、50%、70%、95%乙醇洗脱部位，减压浓缩至干燥物，所得50%乙醇洗脱部位和70%乙醇洗脱部位的干燥物，即为桑白皮具酪氨酸酶抑制作用的有效部位；
所述有效部位的黄酮类化合物的质量分数为50%~66%，其中50%乙醇洗脱部位的黄酮类化合物是桑根酮 T、桑皮酮L、桑皮酮G、桑根酮G、桑皮酮 O， 70%乙醇洗脱部位的黄酮类化合物是桑皮酮 L、桑皮酮 O、桑根酮 G、桑皮酮 E、桑皮酮 T、5'-香叶草基-5,7,2',4'-四羟基黄酮、桑根酮 V。
2.根据权利要求1所述的制备桑白皮中具酪氨酸酶抑制作用的有效部位的方法，其特征在于，确定桑白皮具酪氨酸酶抑制作用的有效部位的方法为：将各浓度乙醇洗脱部位进行酪氨酸酶抑制活性测试，并以对酪氨酸酶抑制活性为指标，以曲酸为阳性对照，确定酪氨酸酶抑制活性高的部位为有效部位，即50%乙醇洗脱部位和70%乙醇洗脱部位。
3.根据权利要求1所述的制备桑白皮中具酪氨酸酶抑制作用的有效部位的方法，其特征在于，所述有效部位的成分采用UPLC-MS检测鉴定。
4.根据权利要求1-3任一项所述的方法得到的桑白皮中具酪氨酸酶抑制作用的有效部位。
5.如权利要求4所述的桑白皮中具酪氨酸酶抑制作用的有效部位在制备抗果蔬酶促褐变抑制剂中的应用。
6.如权利要求4所述的桑白皮中具酪氨酸酶抑制作用的有效部位在制备美白化妆品或洗浴用品中的应用。
7.如权利要求4所述的桑白皮中具酪氨酸酶抑制作用的有效部位在制备预防和治疗黑色素异常导致的人体色素沉着性疾病、黑色素瘤以及其他需要抑制酪氨酸酶活性的病症的药物中的应用。
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