CN102161650B - 桑叶中多酚类次生代谢产物及制备方法与用途 - Google Patents
桑叶中多酚类次生代谢产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次生代谢产物,包括化合物(2S)-Euchrenonea7和消旋化合物(2R)/(2S)-7-甲氧基-8-羟乙基-2',4'-二羟基黄烷。将药材通过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。本发明提供的多酚类次生代谢产物具有比桑叶提取物更强的α-葡萄糖苷酶抑制活性,可在预防和治疗餐后高血糖症、糖尿病综合症药物中的应用。
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分离具有α-葡萄糖苷酶抑制活性的多酚类次生代谢产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。
糖尿病是一种慢性的内分泌代谢紊乱性以高血糖为特征的疾病。是由于胰岛素分泌减少或相对不足,引起糖、脂肪及蛋白质的代谢紊乱,促使血糖增高。糖尿病的发病率在全球范围内都呈升高趋势。其中2型糖尿病约占80%~90%。对于2型糖尿病的治疗,主要是依靠现有的口服降糖药物进行治疗。
α-葡萄糖苷酶抑制剂能抑制小肠内的α-葡萄糖苷酶活性,抑制寡糖水解为可吸收的单糖,进而抑制或延缓葡萄糖在肠道内的吸收,有效降低餐后高血糖。因此,α-葡萄糖苷酶抑制剂能够有效预防和治疗餐后高血糖症、糖尿病综合症中的高血糖症、肥胖症、以及其它需要抑制α-葡萄糖苷酶活性的病症。
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次生代谢产物,所述的多酚类次生代谢产物包括(2S)-Euchrenone a7(化合物A)和 (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](消旋化合物B)。化合物结构如下:
本发明的另一个目的是提供所述桑叶中多酚类次生代谢产物的制备方法,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~ 60:40的石油醚和乙酸乙酯;
(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次生代谢产物;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。
步骤(4)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48.5min的色谱峰,减压回收溶剂,得化合物A。
步骤(4)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47.5min的色谱峰,减压回收溶剂,得消旋化合物B。
本发明的再一个目的是提供所述桑叶中多酚类次生代谢产物在制备预防和治疗餐后高血糖症、糖尿病综合症中的药物中的应用。所述的疾病包括高血糖症、肥胖症、以及其它需要抑制α-葡萄糖苷酶活性的病症。经实验证实,本发明提供的两种从桑叶中提取的多酚类次生代谢产物具有α-葡萄糖苷酶抑制活性。
以本发明提供的两种多酚类次生代谢产物作为活性成分,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。
本发明从桑叶中提取分离具有显著α-葡萄糖苷酶抑制活性的多酚类次生代谢产物,具有比桑叶提取物更强的α-葡萄糖苷酶抑制活性,制成的制剂易于药物的质量控制,可在制备预防和治疗餐后高血糖症、糖尿病综合症中的高血糖症、肥胖症、或其它需要抑制α-葡萄糖苷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B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。洗脱液依次为石油醚;体积比为99:1~65:35的石油醚和乙酸乙酯;体积比为60:40的石油醚和乙酸乙酯,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,得消旋化合物B。
消旋化合物B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 化合物A的制备
桑叶的提取、萃取、硅胶分离过程以及制备液相色谱条件如实施例一,所不同的是收集48.5min的色谱峰,减压回收溶剂,得化合物A。
化合物A的核磁共振光谱及质谱数据如下。
1H NMR (500MHz, DMSO-d6): δ 7.50 (1H, d, H-5), 7.21 (1H, d, H-6'), 6.55 (1H, d, H-6), 6.34 (1H, d, H-3'), 6.25 (1H, dd, H-5'), 5.23 (1H, dd, H-2), 5.15 (1H, dd, H-2''), 3.18 (1H, d, H-1''), 3.00, 2.58 (2H, 2m, H-4), 1.58 (6H, s, CH3-4', 5')。13C NMR (500MHz, DMSO-d6): δ 74.4 (C-2), 42.4 (C-3), 191.1 (C-4), 113.4 (C-4a), 125.3 (C-5), 109.7 (C-6), 162.1 (C-7), 114.9 (C-8), 161.2 (C-8a), 116.3 (C-1'), 155.6 (C-2'), 102.4 (C-3'), 158.3 (C-4'), 106.3 (C-5'), 126.7 (C-6'), 21.8 (C-1''), 122.4 (C-2''), 130.5 (C-3''),17.7 (C-4''), 25.5 (C-5'')。
ESI-MS: m/z 339.19 [M-H]-。
CD谱中,在275nm处的Cotton效应为Δε =-0.0345。
结构解析表明该化合物为:(2S)-Euchrenone a7。
实施例三 化合物A的α-葡萄糖苷酶抑制活性的评价
将桑叶总提物制成12.5~200 μg/ml浓度的溶液;将化合物A制成6.25~100μM浓度的溶液;将阳性药物阿卡波糖制成25~400 μM浓度的溶液。取96孔板,加0.08U/ml α-葡萄糖苷酶20μl,各浓度待测样品溶液20μl,混匀后37℃下孵育10 min。再加反应底物0.375 mM 4-硝基苯-α-D-吡喃葡萄糖苷40 μl,混匀后37 ℃下孵育30min。加1M Na2CO3 80μl终止反应,在405nm处测定其吸光度值。计算各样品的α-葡萄糖苷酶活性抑制率及IC50值。其中酶溶液,样品溶液,反应底物溶液均以250 mM,pH 6.5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
桑叶总提物在200 μg/ml浓度下抑制率为46.04%,化合物A在50 μM (17.00 μg/ml)浓度下抑制率为98.50%,阳性药物阿卡波糖在200 μM (129.12 μg/ml)浓度下抑制率为52.77%。桑叶总提物的IC50值为226.50 μg/ml,化合物A的IC50值为6.16 μM(2.09 μg/ml),阳性药物阿卡波糖的IC50值为174.03 μM(112.4 μg/ml)。
实施例四 消旋化合物B的α-葡萄糖苷酶抑制活性的评价
将桑叶总提物制成200 μg/ml浓度的溶液;将消旋化合物B 制成200μM(63.20 μg/ml)浓度的溶液;将阳性药物阿卡波糖配成200 μM(129.12 μg/ml)浓度的溶液。按实施例三同样方法操作,计算各样品的α-葡萄糖苷酶活性抑制率。结果:桑叶总提物在200 μg/ml浓度下抑制率为46.04%, 消旋化合物B在200 μM (63.20 μg/ml)浓度下抑制率为75.71%,阳性药物阿卡波糖在200 μM (129.12 μg/ml)浓度下抑制率为52.77%。
实施例五 滴丸制剂的制备
取具有α-葡萄糖苷酶抑制活性的化合物A或 消旋化合物B 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。
实施例六 冻干粉针剂的制备
取具有α-葡萄糖苷酶抑制活性的化合物A或 消旋化合物B 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 (5)
1.一种从桑叶中提取的多酚类次生代谢产物,其特征在于,所述次生代谢产物为消旋化合物B:(2R)/(2S)-7-甲氧基-8-羟乙基-2',4'-二羟基黄烷,通过以下步骤制备获得:将桑叶用70%乙醇回流提取,提取物减压浓缩至浸膏;将浸膏混悬于适量水中,用石油醚脱脂,乙酸乙酯萃取;将乙酸乙酯萃取部分用正相硅胶分离,洗脱剂为体积比为100:0~60:40的石油醚和乙酸乙酯;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多酚类次生代谢产物;其中制备色谱的分离条件:色谱柱为制备柱Agilent ZorbaxSB-C18柱250×21.2mm,7μm,流动相为水A和甲醇B,梯度洗脱程序如下:0min,30%B;20min,50%B;27min,50%B;28min,60%B;35min,60%B;36min,70%B;50min,70%B;60min,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,得消旋化合物B。
2.一种从桑叶中提取多酚类次生代谢产物的方法,所述多酚类次生代谢产物为化合物A:(2S)-Euchrenone a7,其特征在于,通过以下步骤实现:将桑叶用70%乙醇回流提取,提取物减压浓缩至浸膏;将浸膏混悬于适量水中,用石油醚脱脂,乙酸乙酯萃取;将乙酸乙酯萃取部分用正相硅胶分离,洗脱剂为体积比为100:0~60:40的石油醚和乙酸乙酯;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多酚类次生代谢产物;其中制备色谱的分离条件:色谱柱为制备柱Agilent Zorbax SB-C18柱250×21.2mm,7μm,流动相为水A和甲醇B,梯度洗脱程序如下:0min,30%B;20min,50%B;27min,50%B;28min,60%B;35min,60%B;36min,70%B;50min,70%B;60min,80%B,流速:10ml/min,检测波长:210nm,收集48.5min的色谱峰,减压回收溶剂,得化合物A。
3.根据权利要求1所述的一种从桑叶中提取的多酚类次生代谢产物消旋化合物B在制备预防和治疗餐后高血糖症、糖尿病综合症的药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述药物由消旋化合物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。
5.根据权利要求4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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