CN106420715A - 源于桔绿木霉的青霉烯醇e1在制备抗淋巴瘤药物的应用 - Google Patents
源于桔绿木霉的青霉烯醇e1在制备抗淋巴瘤药物的应用 Download PDFInfo
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Abstract
本发明涉及一种源于桔绿木霉的青霉烯醇E1在制备抗淋巴瘤药物的应用。该化合物结构特征是：含有一个五环酰胺的分子骨架、含有一个羰基连接一条十碳饱和脂肪长链、N上连接一个甲基、分子存在两个羟基基团。经实验证实，所述生物碱类化合物对淋巴瘤具有较好的抑制活性。可作为制备淋巴瘤细胞增殖抑制药物或抗淋巴瘤药物用于抗肿瘤的研究。
Description
技术领域
本发明属于医药化学领域，涉及一种源于桔绿木霉的青霉烯醇E1在制备抗淋巴瘤药物的应用。
背景技术
生物碱是一类由生物次级代谢产生的含氮有机化合物，自然界中的生物碱种类较多，大多来源于植物，因此又有植物碱之称。生物碱对人和动物有重要的生理作用，包括平喘镇咳、降血糖、降血脂、抗菌、抗肿瘤、镇痛等，其中以抗菌、抗肿瘤活性最为突出。天然结构生物碱是创新药物研究中发现先导化合物的重要来源，目前应用于临床的生物碱药物已经近百种。研究发现，一些海洋真菌能够在次级代谢过程中产生结构新颖、活性好的生物碱，具有很好的药用和产业化前景。
本发明人研究得知，桔绿木霉（Trichoderma citrinoviride.）IBPT-4 (已于2013年1月25日保藏在中国典型培养物保藏中心，地址: 武汉 武汉大学，保藏编号是：CCTCCNO：M 2013055) 的发酵产物的粗提取物有很好的肿瘤细胞增殖抑制活性，遂对其活性成分进行研究。研究发现所示生物碱类化合物针对淋巴瘤具有抗肿瘤活性，目前尚未见该化合物的化学结构及针对淋巴瘤细胞增殖抑制活性的报道，因此市场上也尚未见有与此相关的药物。
发明内容
本发明的目的在于提供一种源于桔绿木霉的青霉烯醇E1在制备抗淋巴瘤药物的应用。
本发明首先涉及到一株桔绿木霉（Trichoderma citrinoviride.）IBPT-4，该菌株已于2013年1月25日保藏在中国典型培养物保藏中心，地址: 武汉 武汉大学，保藏编号是：CCTCC NO：M 2013055。
所述菌株的用途在于，将桔绿木霉（Trichoderma citrinoviride.）IBPT-4进行发酵培养，菌丝体和发酵液提取物经分离得到具有肿瘤细胞增殖抑制活性的化合物。
该化合物结构式为：
。
其结构特征是：含有一个五环酰胺的分子骨架、含有一个羰基连接一条十碳饱和脂肪长链、N上连接一个甲基、分子存在两个羟基基团。
本发明还保护了所述的化合物在制备针对淋巴瘤细胞增殖抑制药物中的用途，及该化合物在制备抗淋巴瘤药物中的用途。
本发明的显著优点：研究所示该化合物是一个结构新颖的生物碱，所述生物碱类化合物具有显著的抗淋巴瘤活性，目前尚未见该化合物的化学结构及针对淋巴瘤细胞增殖抑制活性的报道，因此市场上也尚未见有与此相关的药物。
附图说明
图1主要的COSY和HMBC信号。
具体实施方式
在如下的实施例中所指的化合物的化学结构：
。
实施例1该化合物的发酵生产及分离精制
1发酵生产
生产菌的发酵培养：按培养微生物的常规方法，取桔绿木霉（Trichoderma citrinoviride.）IBPT-4 (已于2013年1月25日保藏在中国典型培养物保藏中心，地址: 武汉 武汉大学，保藏编号是：CCTCC NO：M 2013055) 适量，接种到PDA固体斜面培养基上在28℃培养箱中培养4天。
取斜面培养4天的桔绿木霉（Trichoderma citrinoviride.）IBPT-4适量，接种到装有400mL培养液 [ 培养液组成(克/升)：甘露醇20.0，酵母膏3.0，麦芽糖20.0，味精10.0，葡萄糖10.0，KH2PO4 0.5 ，MgSO4 0.3，NaCL 6.0 ，定容到1000mL锥形瓶中]，28℃静止培养30天后，获得菌丝体和发酵液。
2 浸膏的获得
用纱布将菌丝体和发酵液分离。将发酵液与乙酸乙酯1:2（v/v）萃取两次，萃取液减压蒸馏至干，得到发酵液的乙酸乙酯浸膏。菌丝体则以含70%-80%丙酮的水溶液超声破碎3次，除去残渣将清液合并后减压浓缩去除丙酮，用按体积比为1:2加入乙酸乙酯萃取两次，减压浓缩至干得菌丝体浸膏。将菌丝体和发酵液浸膏合并后得到提取物总浸膏。
3 化合物的分离精制
该浸膏通过100-200目硅胶拌样后，以石油醚：二氯甲烷：甲醇为洗脱液用减压硅胶色谱柱梯度洗脱。洗脱液经过活性跟踪，得到活性组分B (二氯甲烷-甲醇v/v 50:1洗脱物)，然后以石油醚：二氯甲烷：甲醇梯度组分为洗脱剂，进一步通过加压硅胶柱层析梯度洗脱，得到的活性亚组分B1(二氯甲烷-甲醇为20:1的洗脱物)以氯仿-甲醇(v/v 1:2)为溶剂进行Sephadex LH-20凝胶柱层析，最后通过半制备液相色谱(1010型ODS-A，10×250 mm，5μm)：分离流速为5 mL/min，流动相为85%乙腈含0.1% TFA，得到所示化合物（32.1 mg，t R20.7min）。
化合物 淡黄色油状物，高分辨质谱HRESI-MS在m/z 324.2161处给出分子离子峰[M – H]–，(Calcd for C18H30NO4, 324.2175)，提示分子量为325，结合波谱信息推测分子式为C18H31NO4。 1H和13C-NMR等NMR数据见表1。
表1化合物的1H和13C-NMR数据(500 MHz，in CDCl3)a)
a) 本表信号归属基于DEPT、HMQC及HMBC图谱解析结果。碳信号的种类利用DEPT方法确定。
实施例2 体外抗肿瘤活性的测试
1 实验样品及实验方法
被测样品溶液的配制 测试样品为上述实施1中分离精制的化合物纯品。精密称取适量样品，用甲醇配制成所需浓度的溶液，供测活性。
细胞系及细胞的继代培养采用淋巴瘤细胞系，细胞用含10% FBS的RPMI-1640培养基，在37℃于通入5% 二氧化碳的培养箱中继代培养。
细胞增殖抑制活性测试方法
四氮唑盐（MTT）法 取对数生长期的细胞，将细胞密度调至每毫升2×105个细胞，按每孔200微升接种于96孔细胞培养板中，于37℃通入5% CO2的培养箱中培养4小时。每孔加入2微升样品液或空白溶液，培养24小时后，每孔加MTT液（MTT的每毫升5毫克生理盐水溶液）10微升，继续培养4小时,37℃、2000转/分钟离心8分钟，吸去上清。每孔加入DMSO各100微升，在微量振荡器上振荡15分钟，至结晶完全溶解后，利用MD公司产SPECTRAMAX Plus 型酶标仪测定每孔在570nm处的吸光值（OD）值。在同一块96孔板中样品的每个浓度均设置三孔，另设三孔空白对照和无细胞调零孔（如果药物有颜色要做相应药物浓度无细胞调零）。各孔OD值先做相应无细胞调零，再取三孔平均OD值按IR (%) = (OD空白对照-OD样品)/OD空白对照 × 100%式计算每个浓度下细胞增殖抑制率 (IR%)。
2. 实验结果
细胞增殖抑制活性测试结果
在MTT法测试中，根据不同浓度的该化合物的淋巴瘤细胞增殖抑制率，应用SPSS16.0软件进行数据处理并计算半数抑制浓度IC50值。结果见表2。
表2 化合物对淋巴瘤细胞增殖的抑制活性
3. 结论
化合物具有明显的淋巴瘤细胞增殖抑制作用，可作为制备淋巴瘤细胞增殖抑制剂或抗淋巴瘤药物用于抗肿瘤的研究。
Claims (2)
1.一种源于桔绿木霉的青霉烯醇E1在制备淋巴瘤细胞增殖抑制药物中的应用，其特征在于：所述的青霉烯醇E1化学结构为
。
2.权利要求1所述的青霉烯醇E1在制备抗淋巴瘤药物中的应用。
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| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865809A (zh) * | 2014-03-10 | 2014-06-18 | 福州大学 | 一种源于桔青霉的青霉烯醇b1的抗肿瘤新用途 |
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| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865809A (zh) * | 2014-03-10 | 2014-06-18 | 福州大学 | 一种源于桔青霉的青霉烯醇b1的抗肿瘤新用途 |
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