CN106892957B - 一种齐墩果烷型三萜皂苷类化合物及其制备方法和应用 - Google Patents
一种齐墩果烷型三萜皂苷类化合物及其制备方法和应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Steroid Compounds (AREA)
Abstract
本发明涉及一种齐墩果烷型三萜皂苷类化合物，该化合物的分子结构如下式(Ⅰ)所示。本发明所述的化合物由毛冬青根乙醇回流提取和柱色谱分离纯化得到。本发明所述的化合物具有抗炎活性，可用于制备预防和治疗炎症性血管疾病的药物。
Description
技术领域
本发明涉及甾族化合物，具体涉及五环三萜类化合物以及该化合物在制备治疗炎症性血管疾病药物中的应用。
背景技术
毛冬青为冬青科(Aquifoliaceae)冬青属植物Ilex pubescens Hook.et Arn.，别名乌尾丁、六月霜、细叶冬青、毛披树、水火药、火烙木等，主产于广东、江西、福建、广西等地，是我国南方常用中药。其药用部位为根，具有活血通络、消肿止痛、清热解毒之功效，临床上广泛用于治疗冠心病、心绞痛、血栓闭塞性脉管炎等。
有文献报道过毛冬青部位的抗炎作用(Wang JR,Zhou H,Jiang ZH,Wong YF,LiuL.In vivo anti-inflammatory and analgesic activities of a purified sapononfraction derived from the root of Ilex pubescens.Biol.Pharm.Bull.2008,21,643-650.)，同时有文献报道从中分离得到的三萜皂苷类化合物具有抗肿瘤(Zhou Y,Chai XY,Zeng K,Zhang JY,Li N,Jiang Y,Tu PF,llexpublesnins C-M,eleven new triterpenesaponins from the roots of Ilex pubescens.Planta.Med.2013,79,70-77.)、抗血栓(熊天琴,陈元元,李红侠.毛冬青皂苷B3的抗血栓作用研究,中草药.2012,43,1758-1788)和抑制黄嘌呤氧化酶(Zhou Z L,Feng Z C,Yin W Q,Zhang H L.A new triterpenesaponin from the leaves of Ilex pubescens and its XOD inhibitoryactivity.Chem.Nat.Compd.2013,49(4):682-684.)作用。
齐墩果烷型三萜皂苷类化合物在植物界分布较广，据文献报道从1996年至2012年期间，共从自然界发现齐墩果烷型三萜类化合物超过上千个(Dinda B,Debnath S,MohantaBC,Harigaya Y.Naturally Occurring Triterpenoid Saponins.ChemistryBiodiversity.2010,2327-2580.Hill RA,ConnollyJD.Triterpenoids.Nat.Prod.Rep.2015,32:273-327)。关于该类化合物抗炎活性的报道并不是特别多，国外学者Son(Kwak WJ,Son KH.Loniceroside C,an AntiinflammatorySaponin from Lonicera japonica.Chem.Pharm.Bull.2003,51:333-335.)从忍冬(Lonicera japonica)的地上部分分离得到两个齐墩果烷型三萜类化合物，分别是Loniceroside A和Loniceroside C，并用巴豆油诱导小鼠耳水肿实验评价其抗炎活性，结果显示在100mg/kg的剂量下，其抑制率分别为30.2％和31.0％。有学者(Wang HL,YuBY.Two New Triterpenoid Saponins Isolated from Polygalajaponica.Chem.Pharm.Bull.2006,54:1739-1742.)从远志属植物瓜子金(Polygalajaponica)中发现6个齐墩果烷型型三萜类化合物，在角叉菜胶诱导小鼠急性足跖肿胀模型中，皂苷3、4、5在0.1mol/kg的剂量下表现出较为显著的抗炎活性。有学者(Wei F,LinRC.Antiinflammatory Triterpenoid Saponins from the Seeds of Aesculuschinensis.Chem.Pharm.Bull.2004,52:1246-1248.)从七叶树(Aesculus chinensis)的种子中发现四个齐墩果烷型三萜类化合物，它们分别是escin 1a、escin 1b、isoescin 1a和isoescin 1b，在二甲苯诱导小鼠耳水肿模型中，在30mg/kg的剂量下抑制率为67.3％-79.3％。有学者(Shi SP,Tu PF.Triterpene Saponins from Clematismandshurica.J.Nat.Prod.2006,69:1591-1595)从辣蓼铁线莲的根茎中发现两个齐墩果烷型三萜类化合物，分别是clematomandshurica saponins A和B，在LPS诱导小鼠腹腔巨噬细胞模型中，它们抑制COX-2的IC50值分别为2.66和2.58μM。有学者(Wu C,LiYL.Triterpenoid Saponins from the stem Barks of Schefflera heptaphylla.PlantaMed.2013,79:1348-1355)从鸭脚木的树皮中发现两个齐墩果烷型三萜类化合物Heptoleoside B和C，在LPS诱导RAW264.7细胞模型中，在40μM的浓度下，对NO的抑制率分别为22.5％和45.2％，但是所得两个齐墩果烷型三萜皂苷化合物的抗炎活性仍不理想。
发明内容
本发明要解决的技术问题提供一种齐墩果烷型三萜皂苷类化合物，该化合物治疗炎症性血管疾病的效果显著。
本发明要解决上述问题的技术方案是：
一种齐墩果烷型三萜皂苷类化合物，其分子结构如式Ⅰ所示：
本发明所述的齐墩果烷型三萜皂苷类化合物的分子式C41H64O14，HR-ESI-MS m/z[M+Na]+:803.4190，化学名称为3-O-β-D-吡喃葡萄糖基-3β,19α-二羟基齐墩果烷-24-醛基-12-烯-28-酸-O-β-D-吡喃葡萄糖苷。
本发明所述的齐墩果烷型三萜皂苷类化合物可以通过化学合成的方法。本领域的技术人员可以通过本领域的知识确定合成步骤和工艺条件。本发明所述的齐墩果烷型三萜皂苷类化合物还可以从毛冬青(Ilex pubescens Hook et Arn.)中分离得到，具体分离方法由下步骤组成：
(1)取毛冬青根，依次用12、10和8倍浓度为70％的乙醇回流提取2h，合并乙醇提取液，回收乙醇并减压浓缩至无醇味，得总浸膏；
(2)将总浸膏溶解在水中，过D‐101型大孔树脂柱，依次用浓度为30％的乙醇、浓度为60％的乙醇和浓度为95％的乙醇洗脱；收集浓度为60％的乙醇洗脱液过200～300目的硅胶柱，以氯仿-甲醇为溶剂按氯仿︰甲醇为100︰1～0︰1的梯度进行洗脱；收集氯仿︰甲醇为9︰1的洗脱液过Sephadex LH-20柱，以甲醇为洗脱剂洗脱，收集洗脱液浓缩后过ODS柱，收集甲醇：水为52：48的洗脱液，浓缩后得到白色粉末状结晶即可。
上述分离方法中所述的乙醇的浓度均为体积浓度。
上述分离方法中所述的毛冬青是冬青科(Aquifoliaceae)冬青属(Ilex)植物毛冬青的干燥根。
本发明所述的齐墩果烷型三萜皂苷类化合物，具有抗炎作用，可用于制备预防和治疗治疗炎症性血管疾病的药物。所述的药物是由(Ⅰ)式所示齐墩果烷型三萜皂苷类化合物和药学上可以接受的辅料组成的常用的剂型，如，注射剂、片剂或胶囊剂等。所述制剂中(I)式所示的化合物的重量百分含量为10％～50％。
下面通过实验来证明(Ⅰ)式所示齐墩果烷型三萜皂苷类化合物所具有的技术效果。采用脂多糖(LPS)诱导的RAW264.7细胞模型，研究化合物对细胞中一氧化氮合成酶(iNOS)和环氧酶-2(COX-2)蛋白表达量的影响，考察本发明所述化合物的抗炎作用，具体实验方法如下所述。
材料和方法
1、样品1来源于具体实施例1所示制备方法。对照品clematomandshuricasaponins B为文献(Shi SP,Tu PF.Triterpene Saponins from Clematismandshurica.J.Nat.Prod.2006,69:1591-1595)所报道化合物，具体结构式如下式(Ⅱ)所示。阳性对照药DEX为地塞米松。
clematomandshurica saponins B
2、鼠源巨噬细胞RAW264.7购自美国标准菌库，保存在含有10％FBS、100units/ml盘尼西林G、100mg/ml链霉素及2mM L-左旋谷酰胺培养液中，在饱和湿度、37℃、5％CO2条件下培养。
3、化合物溶解在DMSO中，母液浓度配成30mM，工作浓度为30μM。
4、RAW264.7细胞以8×104个/孔接种于24孔板中培养24h，细胞经过测试样品和阳性对照药地塞米松(0.5μM)预处理1h，然后用100ng/ml的LPS刺激18h，设空白组合正常组。
5、Western Blot方法：LPS刺激之后，用含有蛋白酶抑制剂的RIPA裂解液将细胞总蛋白提取出来，通过Bio-Rad蛋白质定量法测得浓度，凝胶电泳之后转膜，5％脱脂牛奶进行封闭，然后用iNOS、COX-2一抗和鼠特异性抗体在4℃条件下孵育过夜，室温二抗孵育，蛋白表达含量用Odyssey v3.0软件进行分析。
6、统计分析：上述检测结果用X±S表示，样品组合空白对照组数据进行统计数据学分析，P＜0.05表示具有显著性差异。
表1 iNOS和COX-2蛋白表达抑制率
以上实验结果表明化合物1能够显著性抑制LPS诱导的RAW264.7细胞中iNOS和COX-2蛋白的表达。表明该化合物均具有较好的节抗炎作用。同时与已报道化合物clematomandshurica saponins B相比，虽然两个化合物对COX-2抑制率相差不大，但是化合物1对iNOS的抑制率明显要大于clematomandshurica saponins B，也就是说化合物1具有多靶标的特点。
附图说明
图1本发明所述化合物的1H-NMR图(400MHz)。
图2本发明所述化合物的13C-NMR图(100MHz)。
图3本发明所述化合物的DEPT图。
图4本发明所述化合物的COSY NMR图。
图5本发明所述化合物的HSQC NMR图。
图6本发明所述化合物的HMBC NMR图。
图7本发明所述化合物的NOESY NMR图。
具体实施方式
下述实验用毛冬青(Ilex pubescens Hook et Arn.)根采自广东省从化市。
例1：
一、化合物的制备
(1)取毛冬青根，依次用12、10和8倍的浓度为70％的乙醇回流提取2h，合并乙醇提取液，过滤后回收乙醇并减压浓缩至无醇味，得总浸膏2.2kg；
(2)将总浸膏溶解在水中，过D‐101型大孔树脂柱，依次用浓度为30％的乙醇、浓度为60％的乙醇和浓度为95％的乙醇洗脱；收集浓度为60％的乙醇洗脱液减压回收溶剂，得稠浸膏566g，该稠浸膏过200～300目的硅胶柱，以氯仿-甲醇为溶剂按氯仿︰甲醇为100︰1～0︰1的梯度进行洗脱；收集氯仿︰甲醇为9︰1的洗脱液浓缩后过Sephadex LH‐20柱，以甲醇为洗脱剂洗脱，收集洗脱液浓缩后过ODS柱，收集甲醇：水为52：48的洗脱液，浓缩后得到白色粉末状结晶16mg。
二、化合物的鉴定
所得到的白色粉末状结晶Liebermann-Burchard反应呈阳性，最终显示紫红色，说明其为三萜类化合物。
参见图1～5，IR显示有羟基吸收(3429cm-1)，羰基吸收(1705cm-1)，双键吸收(1632cm-1)。高分辨质谱给出分子式C41H64O14，[M+Na]+:803.4190，根据分子式计算出该分子的不饱和度为9。该化合物通过酸水解，衍生化后用TLC和HPLC分析并与糖标准品的衍生物对照，证明其糖单元为D-木糖和D-葡萄糖。参见表1，13C-NMR谱中共显示41个碳信号，其中11个归属为糖，剩下30个碳信号提示可能为三萜苷元。参见表1，1H-NMR谱中显示有6个角甲基信号[δH 0.79,0.96,1.08,1.16,1.60,1.63]。一组三取代双键(δH 5.51,δC 123.6,C-12；δC144.2,C-13)，一个醛基(δH 10.38,δC 206.4,CHO-24)，一个酯羰基(δC 177.6,COOR-28)，一个木糖的端基质子信号(δH 4.84,δC 108.2,CH-Xyl-1)和一个葡萄糖的端基质子信号(δH6.36,δC 96.2,CH-Glc-1)，其耦合常数为7.2和8.0提示D-木糖和D-葡萄糖均为β-构型。以上信息提示该化合物可能为同时具有羧基、醛基和羟基的齐墩果烷型三萜皂苷。与已知化合物ilexpublesnin L比较碳谱数据，两者化学位移很相似，区别在于3位不同的糖单元。
参见图6，HMBC谱显示烯氢δH 5.51(1H,br s,H-12)与δC 24.9(C-11)、δC 42.4(C-14)和δC 45.0(C-18)均有远程相关，提示双键处于C-12与C-13之间。HMBC谱显示连氧氢δH3.59(1H,m,H-19)与δC 46.8(C-17)和δC 29.1(C-29)均有远程相关，提示羟基处于C-19位。HMBC谱显示δH 4.84(d,J＝7.2Hz,CH-xyl-1)与δC 87.0(C-3)有远程相关确定木糖连接在C-3位上。
参见图7，ROESY谱显示其相关峰有H-C(3)/H-C(5)、H-C(5)/H-C(9)、H-C(9)/H-C(27)、H-C(20)/H-C(27)提示19位氧取代、23-Me和27-Me处于α位，相关峰H-C(24)/H-C(25)、H-C(25)/H-C(26)、H-C(19)/H-C(30)提示3位氧取代、24-CHO、25-Me、26-Me和30-Me处于β位。综上确定新化合物的结构为3-O-β-D-吡喃葡萄糖基-3β,19α-二羟基齐墩果烷-24-醛基-12-烯-28-酸-O-β-D-吡喃葡萄糖苷。
表2化合物Ⅰ的1H NMR和13C NMR数据(Pyridine-d5,J＝Hz)
例2：(注射剂)
取采用上述实施例1所述方法得到的化合物1000mg，加1000ml的注射用水，用碳酸钠调pH值至7～7.5，搅拌使溶解，除菌滤过，灌封，经100℃15分钟流通蒸汽灭菌，制成每支2mg/2ml的注射液供注射使用。
例3：(胶囊剂)
取采用上述实施例1所述方法得到的化合物5000mg与4000mg微晶纤维素、500mg羧甲基淀粉钠、400mg十二烷基硫酸钠等辅料充分混合，采用辊压法进行干法制粒，再与适量硬脂酸镁混匀，填充入3#空心胶囊，制成规格为100mg/粒的胶囊剂供口服使用。
例4：(片剂)
取采用上述实施例1所述方法得到的化合物5000mg与4000mg淀粉、200mg交联PVP、300mg羧甲基淀粉钠混合均匀，用5％PVP的75％乙醇溶液作为粘合剂，制软材，以18目筛制粒，60℃干燥后1h，20目整粒后加入适量滑石粉，混匀，压片，制成规格为100mg/片的片剂供口服使用。
Claims (5)
1.一种齐墩果烷型三萜皂苷类化合物，其分子结构如下式(Ⅰ)所示：
2.权利要求1所述的齐墩果烷型三萜皂苷类化合物的制备方法，该方法由以 下步骤组成：
(1)取毛冬青根，依次用12、10和8倍浓度为70％的乙醇回流提取2h，合并乙醇提取液，回收乙醇并减压浓缩至无醇味，得总浸膏；
(2)将总浸膏溶解在水中，过D-101型大孔树脂柱，依次用浓度为30％的乙醇、浓度为60％的乙醇和浓度为95％的乙醇洗脱；收集浓度为60％的乙醇洗脱液减压回收溶剂，得稠浸膏，该稠浸膏过200～300目的硅胶柱，以氯仿-甲醇为溶剂按氯仿︰甲醇为100︰1～0︰1的梯度进行洗脱；收集氯仿︰甲醇为9︰1的洗脱液，浓缩后过Sephadex LH-20柱，以甲醇为洗脱剂洗脱，收集洗脱液浓缩后过ODS柱，收集甲醇：水为52：48的洗脱液，浓缩后得到白色无定形粉末即可。
3.权利要求1所述的齐墩果烷型三萜皂苷类化合物在制备预防和治疗炎症性血管疾病药物中的应用。
4.一种预防和治疗炎症性血管疾病的药物，该药物由权利要求1所述的齐墩果烷型三萜皂苷类化合物和药学上可以接受的辅料组成，其中所述的齐墩果烷型三萜皂苷类化合物的重量百分含量为10％～50％。
5.根据权利要求4所述的预防和治疗炎症性血管疾病的药物，其特征在于，所述的药物是注射剂、口服片剂或胶囊剂。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709903A (zh) * | 2005-06-17 | 2005-12-21 | 中国人民解放军第二军医大学 | 皂苷类化合物及其苷元在制备治疗神经元损伤药物中的应用 |
| CN101590134A (zh) * | 2009-06-30 | 2009-12-02 | 沈阳药科大学 | 具有抗炎镇痛作用的地榆总三萜及其制备方法 |
-
2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709903A (zh) * | 2005-06-17 | 2005-12-21 | 中国人民解放军第二军医大学 | 皂苷类化合物及其苷元在制备治疗神经元损伤药物中的应用 |
| CN101590134A (zh) * | 2009-06-30 | 2009-12-02 | 沈阳药科大学 | 具有抗炎镇痛作用的地榆总三萜及其制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| Two New Antiinflammatory Triterpene Saponins from the Egyptian Medicinal Food Black Cumin (Seeds of Nigella sativa);Mohamed Elbandy et al.;《Bull.Korean Chem.Soc.》;20091231;第30卷(第8期);1811-1816 * |
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