CN102161649B - 桑叶中提取的多酚类衍生物及制备方法与用途 - Google Patents
桑叶中提取的多酚类衍生物及制备方法与用途 Download PDFInfo
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Abstract
本发明提供一种从桑叶中提取的多酚类衍生物,包括消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2',4'-二羟基黄烷和差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2',4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷。将桑叶通经乙醇水溶液加热提取,浓缩,硅胶柱分离,洗脱,洗脱液浓缩干燥,再用制备液相色谱继续分离,收集溶液,溶液浓缩干燥后得到样品并进行结构鉴定。本发明提供的二种多酚类衍生物具有较强的酪氨酸酶抑制活性,可在制备预防和治疗黑色素合成异常导致的人体色素沉着性疾病、黑色素瘤以及其它需要抑制酪氨酸酶活性的病症药物中的应用。
Description
技术领域
本发明属于中药领域,更具体地说涉及从中药桑叶中提取的具有酪氨酸酶抑制活性的多酚类衍生物及其制备方法与制药用途。
背景技术
桑叶,又名“铁扇子”,属于桑科桑属植物桑(Morus alba L.)。桑叶作为药用始载于《神农本草经》,其味苦、性寒,具有补血、疏风、散热、益肝通气、降压利尿等功效。现代大量的药理学研究证明了桑叶具有降血糖、降血压、降血脂、清除自由基、抗衰老、抗炎、抗菌、抗病毒和抗癌等诸多药理作用。桑叶化学成分有黄酮、甾体、香豆素、挥发油、生物碱、氨基酸、有机酸、维生素和多糖等。
酪氨酸酶作为黑色素合成的关键酶,其异常过量表达可导致人体的色素沉着性疾病及黑色素瘤等。因此,酪氨酸酶抑制剂可作为黑色素抑制剂应用于医药等领域。
本发明涉及从桑叶中提取分离具有显著酪氨酸酶抑制活性的衍生物及其制剂,能够有效预防和治疗黑色素合成异常导致的人体色素沉着性疾病、黑色素瘤以及其它需要抑制酪氨酸酶活性的病症。
发明内容
本发明的目的在于提供一种从桑叶中提取的多酚类衍生物,主要包括:消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷[(2R)/(2S)-7-methoxyl-8-hydroethyl-2', 4'-dihydroxylflavane](化合物A)和 差向异构体[(2R) /(2S) -7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷[(2R) / (2S)- 7-methoxyl-8-ethyl-2', 4'-dihydroxylflavane-2''-O-β-D-glucopyranoside](化合物B)。其结构如下:
本发明的另一个目的是提供所述多酚类衍生物的制备方法,通过以下步骤实现:
(1)将桑叶用70%乙醇溶液回流提取,提取物减压浓缩至浸膏;
(2)将浸膏混悬于适量水中,用石油醚脱脂,乙酸乙酯萃取;
(3)将乙酸乙酯萃取部分浓缩至浸膏,用正相硅胶分离,洗脱剂为体积比为100:0~ 90:10的石油醚和乙酸乙酯;体积比为82:18的石油醚和乙酸乙酯;体积比为78:22~65:35的石油醚和乙酸乙酯;体积比为60:40的石油醚和乙酸乙酯及乙酸乙酯;
(4)分别收集体积比为60:40的石油醚和乙酸乙酯洗脱液及乙酸乙酯洗脱液浓缩干燥后得样品,用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为制备柱,流动相为水和甲醇,梯度洗脱。
步骤(4)收集体积比为60:40的石油醚和乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品,制备色谱的分离条件:色谱柱为制备柱Agilent Zorbax SB-C18柱 250×21.2 mm, 7 μm,流动相为水A和甲醇B,梯度洗脱程序如下:0 min,30%B;20 min,50%B;27 min,50%B;28 min,60%B;35 min,60%B;36 min,70%B;50 min,70%B;60 min,80%B;流速:10ml/min,检测波长:210nm。收集47.5 min的色谱峰,减压回收溶剂,得化合物A。
步骤(4)收集乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品;制备色谱的分离条件: 色谱柱:Agilent Zorbax SB-C18柱 250×21.2 mm, 7 μm,流动相为水A和甲醇B,梯度洗脱程序如下:0 min,25%B;32 min,44%B;47 min,44%B;48 min,55%B;71 min,60%B;75 min,100%B。流速:10ml/min,检测波长:210nm。收集72.3min的色谱峰,减压回收溶剂,得化合物B。
本发明的再一个目的是提供所述多酚类衍生物在制备预防和治疗黑色素合成异常导致的人体色素沉着性疾病、黑色素瘤以及其它需要抑制酪氨酸酶活性的病症药物中的应用。
本发明提供的多酚类衍生物可以作为活性成分,加入药剂学上接受的辅料,按照药剂学上记载的制剂的制备方法制成制剂。
所述的制剂包括注射液、滴注液、粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂 、崩解剂、口崩片、微丸等。
本发明的有益之处是:提供的二种桑叶中提取的具有酪氨酸酶抑制活性的多酚类化合物具有比桑叶提取物更强的活性,制成制剂易于药物的质量控制,可在制备预防和治疗黑色素合成异常导致的人体色素沉着性疾病、黑色素瘤以及其它需要抑制酪氨酸酶活性的病症中应用。
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,该实施例仅用于说明本发明而非对本发明的限制。
实施例一 消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷的制备
将10 kg桑叶用9倍量70%乙醇回流提取2次,每次2h,减压浓缩至浸膏,取浸膏混悬于适量水中,用石油醚萃取脱脂,分离水层再用乙酸乙酯萃取6次。将乙酸乙酯萃取部分减压回收溶剂,得160 g浸膏,将浸膏与100-200目硅胶按重量比为1:1的比例拌匀后,加于硅胶柱中分离。洗脱剂为体积比为100:0~ 90:10的石油醚和乙酸乙酯;体积比为82:18的石油醚和乙酸乙酯;体积比为78:22~65:35的石油醚和乙酸乙酯;体积比为60:40的石油醚和乙酸乙酯及乙酸乙酯,收集体积比为60:40的石油醚和乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为制备柱,流动相为水和甲醇,梯度洗脱。
制备液相色谱分离条件:
仪器:岛津 LC-8A 制备液相色谱仪配以DAD检测器。
色谱柱:Agilent Zorbax SB-C18柱 (250×21.2 mm, 7 μm)。
流动相:A相:水;B相:甲醇。线性洗脱梯度:0 min,30%B;20 min,50%B;27 min,50%B;28 min,60%B;35 min,60%B;36 min,70%B;50 min,70%B;60 min,80%B。流速:10ml/min,检测波长:210nm。收集47.5min 的色谱峰,减压回收溶剂,得(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷。
消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷的核磁共振光谱及质谱数据如下:
1H NMR (500MHz, DMSO-d6): δ 7.04 (1H, d, H-6'), 6.88 (1H, d, H-5), 6.48 (1H, d, H-6), 6.32 (1H, d, H-3'), 6.23 (1H, dd, H-5'), 5.15 (1H, dd, H-2), 3.72 (3H, s, OCH3-7), 3.39 (1H, m, H-2''), 2.72 (1H, m, H-1''), 2.68, 2.82 (2H, 2m, H-4), 1.79, 2.05 (2H, 2m, H-3)。13C NMR (500MHz, DMSO-d6): δ 72.3 (C-2), 28.1 (C-3), 24.4 (C-4), 114.3 (C-4a), 127.1 (C-5), 103.0 (C-6), 156.4 (C-7), 113.2 (C-8), 153.7 (C-8a), 118.8 (C-1'), 154.8 (C-2'), 102.3 (C-3'), 157.5 (C-4'), 106.2 (C-5'), 126.9 (C-6'), 26.9 (C-1''), 60.0 (C-2''), 55.6 (OCH3-7)。
ESI-MS: m/z 315.21 [M-H]-。
CD谱中,在275nm处的Cotton效应为Δε =-0.0125。
结构解析表明该化合物为:(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷。
实施例二 差向异构体混合物[(2R)/ (2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷的制备
桑叶的提取、萃取、硅胶分离过程同实施例一,所不同的是收集乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为制备柱,流动相为水和甲醇,梯度洗脱。
制备液相色谱条件:
仪器:岛津 LC-8A 制备液相色谱仪配以DAD检测器。
色谱柱:Agilent Zorbax SB-C18柱 (250×21.2 mm, 7 μm)。
流动相:A相:水;B相:甲醇。线性洗脱梯度:0 min,25%B;32 min,44%B;47 min,44%B;48 min,55%B;71 min,60%B;75 min,100%B。流速:10ml/min,检测波长:210nm。收集72.3min的色谱峰,减压回收溶剂,得差向异构体混合物(2R)/ (2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷。
(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷的核磁共振光谱及质谱数据如下:
1H NMR (500MHz, DMSO-d6): δ 7.06 (1H, d, H-6'), 6.91 (1H, d, H-5), 6.51 (1H, d, H-6), 6.33 (1H, d, H-3'), 6.24 (H, dd, H-5'), 5.15 (1H, dd, H-2), 4.12 (1H, d, H-1'''), 3.75 (3H, s, OCH3-7), 3.63, 3.45 (2H, 2m, H-6'''), 3.44 (2H, m, H-2''), 3.12 (2H, m, H-3'''), 3.06 (H, m, H-4'''), 3.04 (H, m, H-5'''), 2.93 (H, m, H-2'''), 2.84 (H, m, H-1''), 2.64, 2.83 (2H, 2m, H-4), 2.08, 1.79 (2H, 2m, H-3)。13C NMR (500MHz, DMSO-d6): δ 72.6 (C-2), 28.0/28.1 (C-3), 24.4 (C-4), 114.4 (C-4a), 127.4 (C-5), 103.1 (C-6), 156.4 (C-7), 112.3 (C-8), 153.7/153.8 (C-8a), 118.7/118.8 (C-1'), 154.9/155.0 (C-2'), 102.3 (C-3'), 157.6 (C-4'), 106.2 (C-5'), 126.7 (C-6'), 23.4 (C-1''), 67.3 (C-2''), 55.6 (OCH3-7), 102.8/103.0 (C-1'''), 73.5 (C-2'''), 76.9 (C-3'''), 70.0 (C-4'''), 76.8 (C-5'''), 61.0 (C-6''')。
ESI-MS: m/z 477.33 [M-H]-。
CD谱中,在275nm处的Cotton效应为Δε =-0.040。
结构解析表明其为:差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷。
实施例三 酪氨酸酶抑制活性的评价
将桑叶总提物配成6.25,12.5,25,50,100μg/ml浓度的溶液;将消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷和差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷别配成3.125,6.25,12.5,25,50 μmol/L浓度的溶液;将阳性药物曲酸配成6.25,12.5,25,50,100 μmol/L浓度的溶液。取96孔板,加250U/ml 酪氨酸酶20μl,各浓度待测样品溶液20μl,及100μl磷酸盐缓冲液(100 mmol/L,pH 6.8)混匀后25 °C下孵育10min。再加反应底物
3 mmol/L L-酪氨酸20μl,混匀后25 °C下孵育30min。在492nm处测定其吸光度值。其中酶溶液,样品溶液,反应底物溶液均以100 mmol/L,pH 6.8磷酸盐缓冲液配制。
酶活性抑制率=[A空白-( A样品-A背景)]/ A空白×100%
A空白:含底物和酶,不加待测样品反应后的吸收值;
A样品:含底物和酶,加入待测样品反应后的吸收值;
A背景:含底物和待测样品,不加酶的吸收值。
桑叶总提物的IC50值为68.04 μg/ml,消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷和差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷的IC50值分别为0.61 μmol/L (0.19μg/ml)和0.92 μmol/L (0.44μg/ml),阳性药物曲酸的IC50值为17.29 μmol/L (2.46μg/ml)。
实施例四 滴丸制剂的制备
取具有酪氨酸酶抑制活性的消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷或差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷 0.1g与10.5g聚乙二醇-20000混合均匀,加热熔融,化料后移至滴丸滴灌中,药液滴至6~8℃液体石蜡中,除油,制得滴丸400粒。
实施例五 冻干粉针剂的制备
取具有酪氨酸酶抑制活性的消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷和差向异构体混合物(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷 0.1g、葡萄糖4.5g、硫代硫酸钠0.9g和蒸馏水1000ml,上述组分混合均匀后,分装400支,冷冻干燥,即得。
Claims (4)
1.一种从桑叶中提取的多酚类衍生物,其特征在于,所述衍生物主要包括:化合物A:(2R)/(2S)-7-甲氧基-8-羟乙基-2', 4'-二羟基黄烷和化合物B:(2R/2S)-7-甲氧基-8乙基-2', 4'-二羟基黄烷-2''-O-β-D-吡喃葡萄糖苷,通过以下步骤实现:(1)将桑叶用70%乙醇回流提取,提取物减压浓缩至浸膏;(2)将浸膏混悬于适量水中,用石油醚脱脂,乙酸乙酯萃取;(3)将乙酸乙酯萃取部分用正相硅胶分离,洗脱剂为体积比为100:0~ 90:10的石油醚和乙酸乙酯,体积比为82:18的石油醚和乙酸乙酯,体积比为78:22~65:35的石油醚和乙酸乙酯,体积比为60:40的石油醚和乙酸乙酯及乙酸乙酯;(4)分别收集体积比为60:40的石油醚和乙酸乙酯洗脱液及乙酸乙酯洗脱液,浓缩干燥后得衍生物,用制备液相色谱继续分离得到衍生物;制备色谱的分离条件:色谱柱为制备柱,流动相为水和甲醇,梯度洗脱;
所述化合物A通过以下步骤获得:步骤(4)收集体积比为60:40的石油醚和乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品,制备色谱的分离条件:色谱柱为制备柱Agilent Zorbax SB-C18柱 250×21.2 mm, 7 μm,流动相为水A和甲醇B,梯度洗脱程序如下:0 min,30%B;20 min,50%B;27 min,50%B;28 min,60%B;35 min,60%B;36 min,70%B;50 min,70%B;60 min,80%B;流速:10ml/min,检测波长:210nm,收集47.5 min的色谱峰,减压回收溶剂,得化合物A;
所述化合物B通过以下步骤获得:步骤(4)收集乙酸乙酯洗脱液,浓缩干燥后得样品,用制备液相色谱继续分离得到的样品;制备色谱的分离条件: 色谱柱:Agilent Zorbax SB-C18柱 250×21.2 mm, 7 μm,流动相为水A和甲醇B,梯度洗脱程序如下:0 min,25%B;32 min,44%B;47 min,44%B;48 min,55%B;71 min,60%B;75 min,100%B。流速:10ml/min,检测波长:210nm,收集72.3min的色谱峰,减压回收溶剂,得化合物B。
2.根据权利要求1所述的一种从桑叶中提取的多酚类衍生物在制备预防和治疗黑色素合成异常导致的人体色素沉着性疾病、黑色素瘤药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述药物由化合物A或化合物B加入药剂学上可接受的辅料,按照药剂学上记载的制剂制备方法制成。
4.根据权利要求3所述的应用,其特征在于,所述的制剂包括液体制剂或固体制剂。
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