CN106008172B - 桑枝中具酪氨酸酶抑制作用有效部位制备方法及其应用 - Google Patents
桑枝中具酪氨酸酶抑制作用有效部位制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了桑枝中具酪氨酸酶抑制作用有效部位制备方法及其应用,属于医药和食品技术领域。本发明提供的桑枝中具酪氨酸酶抑制作用有效部位,包括五种从桑枝中提取的具有酪氨酸酶抑制活性的多酚类化合物,包括:氧化白藜芦醇,草大戟素,桑辛素M,2,4,2',4'‑四羟基查尔酮,摩查尔酮A。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各种制剂。
Description
技术领域
本发明涉及桑枝中具酪氨酸酶抑制作用有效部位制备方法及其应用,属于医药和食品技术领域。
背景技术
桑枝,为桑树的枝叶、桑枝、桑条、嫩桑枝的总称,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。现代药理学研究表明,从该植物中获得的一些成分具有抗炎、抗氧化、抗癌等诸多药理作用。桑枝化学成分有黄酮,萜类,二苯乙烯类,长链脂肪酸类等。桑枝提取物对酪氨酸酶和B-16黑色素细胞中黑色素的合成具有抑制作用,但到目前为止,桑枝中对酪氨酸酶具有抑制作用的成分一直不清楚。
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。
发明内容
本发明的目的首先在于提供一种具酪氨酸酶抑制活性的有效部位,是从桑枝中提取的具有较强酪氨酸酶抑制活性的含有五种多酚类化合物的有效部位,所述五种多酚类化合物是氧化白藜芦醇、草大戟素、桑辛素M,以及2,4,2',4'-四羟基查尔酮,摩查尔酮A,其结构分别如下:
本发明的另外一个目的是提供所述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)桑枝粉碎后用2倍质量的70%乙醇浸没超声提取3次,每次1小时,提取液减压浓缩至干燥物,称重;
(2)干燥物用95%乙醇溶解,过滤;
(3)步骤(2)所得过滤液与大孔树脂D101混合拌样,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)将上述搅拌物上D101大孔树脂,分别用8倍柱体积量的20%、40%、60%、80%、95%乙醇洗脱。
步骤(4)收集40%乙醇洗脱部分8个体积,浓缩得干燥样品,称重,干燥样品进一步用硅胶柱层析分离,先后分别用30倍柱体积的体积比30:1的二氯甲烷-甲醇和体积比50:1的二氯甲烷-甲醇洗脱,先后分别得到组分A和组分B,组分A用凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得氧化白藜芦醇和2,4,2',4'-四羟基查尔酮;组分B用凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得草大戟素、桑辛素M、摩查尔酮A。
本发明的另外一个目的是提供所述的具有酪氨酸酶抑制活性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、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。
本发明的五种从桑枝中提取的具有酪氨酸酶抑制活性的多酚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。
本发明的有益之处在于:提供含有五种从桑枝中提取的具有酪氨酸酶抑制活性的多酚类化合物的有效部位,具有比桑枝提取物具有更强的活性,制成相应制剂易于质量控制,可在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的应用。
附图说明
图1为桑枝粗提取物和有效部位的高效液相色谱对照图
图2为苹果切片用各种溶液处理后抗褐变的a*值(初始用5%乙醇溶解)
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10公斤桑枝用2倍量70%乙醇超声波提取3次,每次1h,减压浓缩至干燥,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8个柱体积的20%、40%、60%、80%、95%乙醇洗脱,收集洗脱液,分别减压回收乙醇至干,40%洗脱部分用高效液相色谱检测,含有五个化合物氧化白藜芦醇、草大戟素、桑辛素M、2,4,2',4'-四羟基查尔酮、摩查尔酮A的组分为有效部位部分(图1)。
高效液相检测条件:
仪器:Waters 1525液相色谱仪配以紫外检测器(2487)。
色谱柱:Alltima C18(250×4.6mm,5μm)。
流动相:A相:含0.1%甲酸的去离子水;B相:甲醇。线性洗脱梯度:0min,10%B;20min,30%B;30min,50%B;40min,70%B;50min,100%B。流速:1.0mL/min,检测波长:280nm。
实施例2 氧化白藜芦醇和2,4,2',4'-四羟基查尔酮的制备
桑枝的提取,大孔树脂分离过程同实施例1,收集40%乙醇洗脱部分8个柱体积,浓缩干燥得样品,称重,用200-300目硅胶柱层析分离,分别用体积比为50:1和30:1的氯仿-甲醇洗脱,收集体积比为30:1的氯仿-甲醇洗脱得到的洗脱液,再用Sephadex LH-20凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得。
氧化白藜芦醇的核磁共振光谱数据如下:
1H NMR(400MHz,CD3OD)δ:7.32(1H,d,J=9.1Hz,H-6),7.26(1H,d,J=16.4Hz,H-7),6.81(1H,d,J=16.4Hz,H-8),6.45(2H,d,J=2.0Hz,H-2',6'),6.31(2H,overlap,H-3,5),6.14(1H,t,J=2.0Hz,H-4').13C NMR(100MHz,CD3OD)δ:159.8(C,C-3',5'),159.4(C,C-4),157.6(C,C-2),142.5(C,C-1'),128.7(CH,C-6),126.8(CH,C-8),125.1(CH,C-7),118.1(C,C-1),108.7(CH,C-5),106.0(CH,C-2',6'),103.9(CH,C-3),102.6(CH,C-4')。
ESI-MS:m/z 243.1[M-H]-(C14H11O4)。
结构解析表明该化合物为氧化白藜芦醇。
2,4,2',4'-四羟基查尔酮的核磁共振光谱数据如下:
1H NMR(Acetone-d6,400MHz)δ:13.797(1H,s,OH-2'),9.690(1H,OH-4),9.490(1H,s,OH-2),9.163(1H,OH-4'),8.211(1H,d,J=15.6Hz,H-α),8.007(1H,d,J=9.2Hz,H-6),7.775(1H,d,J=15.4Hz,H-β),7.667(1H,d,J=8.8Hz,H-6'),6.521(1H,d,J=2.0Hz,H-3),6.441(1H,dd,J=8.8,2.4Hz,H-5),6.431(1H,dd,J=8.4,2.4Hz,H-5'),6.345(1H,d,J=2.4Hz,H-3');13C NMR(Acetone-d6,100MHz)δ:193.31(C=O),167.60(C,C-4'),165.62(C,C-2'),162.63(C,C-4),160.30(C,C-2),141.25(CH,C-β),132.96(CH,C-6),131.79(CH,C-6'),117.24(CH,C-α),115.17(C,C-1'),114.61(C,C-1),109.21(CH,C-5),108.70(CH,C-5'),103.86(CH,C-3'),103.76(CH,C-3)。
ESI-MS:m/z 271.1[M-H]-(C15H11O5)。
结构解析表明该化合物为2,4,2',4'-四羟基查尔酮。
实施例3 草大戟素,桑辛素M和摩查尔酮A的制备
桑枝的提取,大孔树脂分离过程同实施例1,收集40%乙醇洗脱部分8个柱体积,浓缩干燥得样品,称重,用200-300目硅胶柱层析分离,分别用体积比为50:1和30:1的氯仿-甲醇洗脱,收集以体积比为50:1的氯仿-甲醇洗脱得到的洗脱液,再用Sephadex LH-20凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得草大戟素、桑辛素M和摩查尔酮A。
草大戟素的核磁共振光谱数据如下:
1H NMR(Acetone-d6,400MHz)δ:12.210(1H,OH-5),9.019(2H,OH),7.290(1H,d,J=8.4Hz,H-6'),6.465(1H,d,J=2.4Hz,H-2'),6.415(1H,dd,J=8.4,2.4Hz,H-5'),5.955(1H,d,J=2.0Hz,H-8),5.936(1H,d,J=2.0Hz,H-6),5.692(1H,dd,J=13.2,2.8Hz,H-2),3.163(1H,dd,J=17.2,13.2Hz,H-3),2.703(1H,dd,J=17.2,2.8Hz,H-3);13C NMR(Acetone-d6,100MHz)δ:197.8(C=O,C-4),167.6(C,C-7),165.3(C,C-5),164.9(C,C-9),159.7(C,C-2'),156.5(C,C-4'),129.0(CH,C-6'),117.3(C,C-1'),107.9(CH,C-5'),103.6(CH,C-3'),103.1(C,C-10),96.8(CH,C-6),95.9(CH,C-8),75.4(CH,C-2),42.7(CH2,C-3)。
ESI-MS:m/z[M-H]-287.1(C15H11O6)。
结构解析表明该化合物为草大戟素。
桑辛素M的核磁共振光谱数据如下:
1H NMR(Acetone-d6,400MHz)δ:8.60(3H,br s,OH),7.40(1H,d,J=8.4Hz,H-4),7.20(1H,s,H-3),6.99(1H,d,J=2.0Hz,H-7),6.86(2H,d,J=2.0Hz,H-2',6'),6.81(1H,dd,J=8.4,2.0Hz,H-5),6.37(1H,t,J=2.0Hz,H-4');13C NMR(Acetone-d6,100MHz)δ:159.83(C,C-3',5'),156.78(C,C-7a),156.71(C,C-2),155.58(C,C-6),133.47(C,C-1'),100.8(CH,C-3),122.65(C,C-3a),122.09(CH,C-4),113.31(CH,C-5),98.51(CH,C-3),103.92(CH,C-2',6'),103.61(CH,C-7),102.42(CH,C-4')。
ESI-MS:m/z 241.0[M-H]-(C14H9O4)。
结构解析表明该化合物为桑辛素M。
摩查尔酮A的核磁共振光谱数据如下:
1H NMR(Acetone-d6,400MHz)δ:14.155(1H,br s,OH-2'),9.381,9.081(3H,OH-4',2,4),8.213(1H,d,J=15.6Hz,H-α),7.875(1H,d,J=9.2Hz,H-6'),7.785(1H,d,J=15.2Hz,H-β),7.667(1H,d,J=8.4Hz,H-6),6.525(1H,d,J=2.4Hz,H-3),6.522(1H,d,J=8.8Hz,H-5'),6.444(1H,dd,J=8.4,2.4Hz,H-5),5.276(1H,m,H-2″),3.363(2H,d,J=7.2Hz,H-1″),1.775,1.638(6H,br s,H-4″,5″);13C NMR(Acetone-d6,100MHz)δ:193.54(C,C=O),165.19(C,C-4'),162.64(C,C-2'),162.45(C,C-4),160.13(C,C-2),140.97(CH,C-β),131.79(CH,C-6'),131.44(C,C-3″),129.98(CH,C-6),123.53(CH,C-2″),117.56(CH,C-α),116.15(C,C-1),115.29(C,C-3'),114.56(C,C-1'),109.23(CH,C-5),108.00(CH,C-5'),103.76(CH,C-3),25.96(CH2,C-1″),22.40(CH3,C-4″),18.01(CH3,C-5″)。
ESI-MS:m/z 339.1[M-H]-(C20H19O5)。
结构解析表明该化合物为摩查尔酮A。
实施例4 桑枝提取物和有效部位酪氨酸酶抑制活性评价
将桑枝粉碎后乙醇浸提得到的桑枝粗提取物和有效部位溶于DMSO,先配成1mg/mL溶液,使用时稀释到需要浓度。检测三种浓度的粗提取物和有效部位抑制酪氨酸酶的活性,三种浓度为50、30、10μg/mL。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。
桑枝粗提取物和有效部位在3个浓度的抑制率分别为20.2%、11.5%、9.6%和35.8%、22.7%、15.1%,阳性对照曲酸的IC50为7.01μg/mL。
实施例5 五个活性化合物酪氨酸酶抑制活性评价
将氧化白藜芦醇、草大戟素、桑辛素M分别配成3.33、1.67、0.84、0.33、0.17、0.03μg/mL的浓度的溶液,按上述方法测定IC50。得到活性化合物抑制酪氨酸酶的IC50分别为0.10±0.01μM、0.98±0.01μM、8.00±0.22μM。将2,4,2',4'-四羟基查尔酮和摩查尔酮A分别配成1.67、0.84、0.33、0.17、0.03、0.01、0.005μg/mL的浓度的溶液,按上述方法测定IC50。得到活性化合物抑制酪氨酸酶的IC50分别为0.07±0.02μM和0.08±0.02μM,曲酸的IC50为49.3μM。
实施例6 有效部位提取物对苹果切片的抗褐变效果评价
将苹果切成4毫米厚薄片,分别浸入水,0.01%4-己基间苯二酚,0.01%桑枝有效部位,0.1%维生素C,0.01%桑枝有效部位+0.1%维生素C,0.01%的4-己基间苯二酚+0.1%维生素C,然后在0h,3h,6h,12h,24h时分别用色差仪检测其颜色变化程度,结果表明,0.01%桑枝有效部位+0.1%维生素与0.01%4-己基间苯二酚的抗褐变效果在24小时内没有显著性差别。
实施例7 含桑枝有效部位提取物的美白乳液配制
配方如表1。
表1
制备方法:将A相中提取物混合物加入到70℃加热的聚乙二醇400中,然后加入甘油、去离子水,混合均匀;B相80℃溶解混合均匀;将A相加入B相并均质10分钟,降温至50℃时加入C相并均质5分钟,陈化24h后灌装,即可。
Claims (1)
1.一种具酪氨酸酶抑制活性的有效部位的制备方法,其特征在于,具酪氨酸酶抑制活性的有效部位是从桑枝中提取的具有酪氨酸酶抑制活性的含有五种多酚类化合物的有效部位,所述五种多酚类化合物是氧化白藜芦醇、草大戟素、桑辛素M,以及2,4,2',4'-四羟基查尔酮,摩查尔酮A,其结构分别如下:
所含的五种多酚类化合物的质量分数达10%~15%;
其制备方法通过以下步骤实现:
(1)桑枝粉碎后用2倍质量的70%乙醇浸没超声提取3次,每次1小时,提取液减压浓缩至干燥物,称重;
(2)干燥物用95%乙醇溶解,过滤;
(3)步骤(2)所得过滤液与大孔树脂D101混合拌样,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)将上述搅拌物上D101大孔树脂,分别用8倍柱体积量的20%、40%、60%、80%、95%乙醇洗脱,收集40%乙醇洗脱部分;
步骤(4)收集40%乙醇洗脱部分8个体积,浓缩得干燥样品,称重,干燥样品进一步用硅胶柱层析分离,先后分别用30倍柱体积的体积比30:1的二氯甲烷-甲醇和体积比50:1的二氯甲烷-甲醇洗脱,先后分别得到组分A和组分B,组分A用凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得氧化白藜芦醇和2,4,2',4'-四羟基查尔酮;组分B用凝胶柱层析进行分离,体积比1:1的甲醇-水洗脱,即得草大戟素、桑辛素M、摩查尔酮A。
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