CN108546247A - 一种生物碱类化合物在制备抗肥胖药物中用途 - Google Patents
一种生物碱类化合物在制备抗肥胖药物中用途 Download PDFInfo
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Abstract
本发明涉及一种生物碱类化合物在制备抗肥胖药物中用途。所述的生物碱类化合物具有式I所示的结构，或其药学上可接受的盐；其结构类似物单独或以组合物形式在制备抗肥胖药物中的用途。将海洋来源土曲霉Aspergillus terreus ML‑44进行发酵培养，获得含有式I化合物的发酵物，将发酵物进行分离纯化，得到式I化合物。其中，土曲霉ML‑44，保藏编号为CGMCC No.15664，保藏日期为2018年04月17日，保藏地点为中国普通微生物菌种保藏管理中心（CGMCC）。本发明首次发现该化合物及其结构类似物具有抗肥胖活性，为开发新型抗肥胖药物提供先导化合物。
Description
技术领域
本发明涉及一种生物碱类化合物在制备抗肥胖药物中用途。
背景技术
随着社会经济的发展，我国肥胖发病率逐年递增，患者人数从1975年的240万增长到2014年的9000多万，占全世界肥胖人口的14.3%（NCD Risk Factor Collaboration,Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooledanalysis of 1698 population-based measurement studies with 19•2 millionparticipants. The Lancet, 2016, 387, 1377–1396）。肥胖与II型糖尿病、心脑血管疾病、癌症等一系列慢性疾病的发生密切相关（Smith and Minson, Obesity andadipokines: effects on sympathetic overactivity. J Physiol, 2012, 590, 1787–1801; Singer and Lumeng, The initiation of metabolic inflammation inchildhood obesity. J Clin Invest, 2017, 127, 65–73）。肥胖（包括儿童型肥胖）的迅猛发展不仅会给社会带来巨大的经济负担，也势必危及我国社会经济的长期可持续发展（Wolfenstetter, Juvenile obesity and comorbidity type 2 diabetes mellitus (T2DM) in Germany: development and cost-of-illness analysis. Gesundheitswesen,2006, 68, 600–612）。因此，肥胖防治已成为医学研究领域高度关注的重大问题。
治疗肥胖的方法主要包括减少能量和食物的摄入、增加能量的消耗、抑制脂肪细胞的分化和增殖、抑制脂肪的生成、增加脂肪的分解和氧化等。在药物治疗方面，国内外学者围绕肥胖的发病机理，并针对不同靶点研发出一系列药物，如运用降脂药物，抑制肠道内脂肪吸收（奥利司他），促进脂肪代谢等；或者运用肾上腺素能药物直接作用于中枢神经系统，起到抑制食欲，减少食量，降低体重的作用。但是，这些药物普遍存在副作用明显，治愈率低，停药后反弹明显等突出问题，临床应用价值有限。因此，筛选发现更理想的抗肥胖药物具有重要的现实意义。
从自然界中筛选具有调节脂质生成和代谢的小分子物质是目前肥胖领域研究的热点之一，天然产物提供了丰富多彩的化学多样性，可以为开发新的天然减肥药提供先导化合物（Fu et al., Natural Products with Anti-obesity Effects and DifferentMechanisms of Action. J Agric Food Chem, 2016, 64, 9571–9585; Sun et al.,Natural Dietary and Herbal Products in Anti-Obesity Treatment. Molecules,2016, 21, 1351–1365）。微生物次级代谢产物是新药及其先导结构的重要来源，而从微生物中筛选获得调节脂质代谢有关活性天然产物的研究鲜见报道。以多潜能间充质干细胞C3H10T1/2为细胞模型进行筛选，发现三株海洋来源真菌的发酵物具有抑制C3H10T1/2成脂分化的活性，其中土曲霉ML-44（Aspergillus terreus ML-44）的发酵物具有较低的最低有效浓度。进一步的活性追踪分离从ML-44大量发酵产物中分离鉴定了一个生物碱类化合物methyl 3,4,5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate（式I）。目前已知报道的该类化合物共有五个，由两分子的邻氨基苯甲酸或其衍生物和一分子的3-吡啶甲酸（烟酸）通过酰胺键连接，或进一步分子内成环而成（Arai et al., Metabolic productsof Aspregillus terreus. VI. Metabolites of the strain IFO 8835. (3). Theisolation and chemical structures of colorless metabolites. Chem Pharm Bull,1981, 29, 1005–1012.; Wang et al., Three new compounds from Aspergillus terreus PT06-2 grown in a high salt medium, Mar Drugs, 2011, 9, 1368–1378; Heet al., Asperterrestide A, a Cytotoxic Cyclic Tetrapeptide from the Marine-Derived Fungus Aspergillus terreus SCSGAF0162. J Nat Prod, 2013, 76, 1182–1186; Chen et al., Structurally diverse secondary metabolites from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001 and their biologicalevaluation. Fitoterapia, 2017, 117: 71–78）。目前有关该类化合物生物活性的文献报道很少，尤其是在调节脂质代谢、抗肥胖方面的生物活性尚无研究报道。
发明内容
本发明的目的是提供一种生物碱类化合物在制备抗肥胖药物中用途。该生物碱类化合物具有显著的体外抑制脂肪细胞分化和体内抗肥胖作用，即本发明的化合物具有良好的抗肥胖功能。
本发明提供的生物碱类化合物的结构如式I所示：
I
本发明还提供式I化合物结构类似物：terremide A、terremide B、terremide C、terremide D，或其药学上可接受的盐单独或以组合物形式在制备抗肥胖药物中的用途。
本发明还涉及土曲霉（Aspergillus terreus）ML-44，其保藏编号为CGMCC No.15664，保藏日期为2018年04月17日，保藏单位为中国普通微生物菌种保藏管理中心（CGMCC），地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101。该菌株是从太平洋牡蛎的消化道样品中分离，经分类学研究鉴定为土曲霉（Aspergillus terreus）。
本发明提供的式I化合物的制备方法包括如下步骤：
将上述的土曲霉（Aspergillus terreus）ML-44进行发酵培养，获得含有式I化合物的发酵物，将发酵物进行分离纯化，得到所述化合物。也可以使用曲霉属的其它能够产生式I化合物的生产菌进行发酵培养。
所述分离纯化包括利用本领域技术人员熟知的天然产物分离纯化的常规方法，如萃取、柱层析、高效液相制备等。
本发明提供的一种式I化合物的具体制备方法包括如下步骤：
1) 将上述的土曲霉（Aspergillus terreus）ML-44进行大米固体发酵培养，获得发酵物；
2) 将所得发酵物用50%-90%（v/v）的丙酮水溶液超声浸提、过滤，滤液经减压浓缩至不含丙酮，残余混悬液用乙酸乙酯萃取，得到发酵物的乙酸乙酯萃取物；
3) 将步骤2)得到乙酸乙酯萃取物减压浓缩至干，得到乙酸乙酯浸膏；
4) 将乙酸乙酯总浸膏依次用硅胶柱层析（石油醚‒二氯甲烷‒甲醇梯度洗脱）、Sephadex LH-20柱层析（甲醇洗脱）和ODS柱层析（水→100%甲醇梯度洗脱）分离，得到含有所述化合物的柱层析组分；
5) 将含有所述化合物的柱层析组分经HPLC分离。
其中，步骤2)中的丙酮水溶液为70%-90%（v/v），优选为75%-85%（v/v），更优选为80%（v/v）。
本发明提供了土曲霉发酵物的提取物，该提取物含有本发明的式I化合物及其结构类似物。具体地，所述提取物为土曲霉发酵物的乙酸乙酯萃取物、乙酸乙酯总浸膏、或者层析组分。所述提取物可以参照上面本发明的化合物的制备方法的相应步骤制得。具体地，所述提取物可以通过萃取、柱层析、高效液相制备得到。
本发明提供了土曲霉发酵物的提取物在制备抗肥胖药物中的用途。
本发明提供了上述土曲霉在制备式I化合物或者发酵物提取物中的用途。
总之，本发明提供了含有所述的具有式I所示的结构或其药学上可接受的盐的药物，所述药物包括药学上可接受的辅助剂；所述药物为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂等。
本发明中的术语“药学上可接受的盐”可以是药用无机或有机盐。本发明式I化合物中具有碱性基团可以与无机酸形成药用盐，例如硫酸盐、盐酸盐、氢溴酸盐、磷酸盐；也可与有机酸形成药用盐，例如乙酸盐、草酸盐、柠檬酸盐、葡萄糖酸盐、琥珀酸盐、酒石酸盐、对甲苯磺酸盐、甲磺酸盐、苯甲酸盐、乳酸盐、马来酸盐等。
本发明的式I化合物及其上述结构类似物可与各种药物上可接受的载体、赋形剂或辅料配伍制成抗肥胖药物，用于肥胖症及相关代谢综合征的治疗。
本发明化合物可单独或以药物组合物的形式给药。给药途径可以是口服、非肠道或局部给药。药物组合物可根据给药途径配成各种适宜的剂型。
本发明化合物的药物组合物可以以下面的任意方式施用：口服，喷雾吸入，直肠用药，鼻腔用药，颊部用药，局部用药，非肠道用药，如皮下，静脉，肌内，腹膜内，鞘内，心室内，胸骨内和颅内注射或输入，或借助一种外植储器用药。其中优选口服、腹膜内或静脉内给药方式。
当口服用药时，本发明化合物可制成任意口服可接受的制剂形式，包括但不限于片剂、胶囊、水溶液或水悬浮液。其中，片剂使用的载体一般包括乳糖和玉米淀粉，另外也可加入润滑剂如硬脂酸镁。胶囊制剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。水悬浮液制剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合使用。任选地，以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当皮肤局部施用时，本发明化合物可制成适当的软膏、洗剂或霜剂制剂形式，其中将活性成分悬浮或溶解于一种或多种载体中。软膏制剂可使用的载体包括但不限于：矿物油、液体凡士林、白凡士林、丙二醇、聚氧化乙烯、聚氧化丙烯、乳化蜡和水；洗剂或霜剂可使用的载体包括但不限于：矿物油、脱水山梨糖醇单硬脂酸酯、吐温60、十六烷酯蜡、十六碳烯芳醇、2-辛基十二烷醇、苄醇和水。
本发明化合物还可以无菌注射制剂形式用药，包括无菌注射水或油悬浮液或无菌注射溶液。其中，可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
另外需要指出，本发明化合物使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。优选的使用剂量介于0.01-100 mg/kg体重/天。
本发明提供了式I化合物的初步的细胞活性测试，该式I化合物能够抑制小鼠胚胎间充质C3H10T1/2细胞分化为脂肪细胞，显著抑制细胞内脂肪积聚；在体活性预实验结果显示其能明显抑制高脂饮食诱导雄性果蝇肥胖的发生，使果蝇体重维持在较低水平。因而本发明的化合物具有良好的抗肥胖功能，为开发新型抗肥胖药物提供先导化合物。
附图说明
图1 化合物I显著抑制C3H10T1/2细胞的成脂分化;a)空白对照,b)化合物I（20 μM）。
图2 化合物I显著抑制高脂诱导的雄性果蝇肥胖。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
在以下实施例中，所称化合物I即式I所示化合物，系本发明从海洋来源真菌Aspergillus terreus ML-44发酵物乙酸乙酯提取物中分离得到，并首次发现其抗肥胖功能。
阿拉伯数字表示相应标位。
I
在下面实施例的结构研究中，ESI-MS采用美国Thermo Scientific公司LCQ Fleet液质联用仪测定，NMR谱图用瑞士Bruke公司Avance III 500型超导核磁共振仪（500 MHz 1H-NMR，125 MHz 13C-NMR）测定。
实施例1：微生物发酵培养与化合物的制备
1. 发酵培养与发酵物的提取处理
1) 生产菌株
本实施例中用于发酵生产化合物I的产生菌是分离自太平洋牡蛎消化道的土曲霉菌（Aspergillus terreus）ML-44株，保藏于中国普通微生物菌种保藏管理中心（CGMCC），保藏编号为CGMCC No. 15664。
2) 发酵培养
按微生物培养的常规方法，从4℃冰箱取出曲霉菌（Aspergillus terreus）ML-44试管斜面，在无菌条件下用接种环刮取适量孢子，划线接种于新配制的5个PDA固体培养基（组成：葡萄糖2%、琼脂2%、NaCl 1.5%，用20%土豆的水煮液配制）平板上，28℃培养箱中活化培养5天，分别用10ml无菌水洗取孢子，并将全部孢子合并分散于100 ml无菌水中，得到孢子悬液。将该孢子悬液按每瓶4 ml的接种量，分别接种于装有大米固体培养基（每瓶80 g市售大米加150 ml天然海水，放置隔夜后于121℃灭菌30 min制得）的24个1000 ml三角烧瓶中，于室温静置培养35天。
2. 提取处理与乙酸乙酯浸膏的制备
将发酵物用体积分数为80％的丙酮水溶液（每瓶500 ml）浸泡混悬，超声处理1h，室温浸提24h后用4层纱布过滤，重复操作3次。合并滤液，减压浓缩至不含丙酮后，残余水层约3L用等体积乙酸乙酯萃取3次，得发酵物乙酸乙酯萃取物，减压浓缩至干，得到乙酸乙酯浸膏66.2 g。
3. 乙酸乙酯浸膏的柱层析分离与含有目标化合物I-V 的柱层析组分的制备
将上述乙酸乙酯浸膏66.2 g用二氯甲烷-甲醇（v/v 1:1）200ml溶解，滤除不溶物，加入90 g层析硅胶（200-300目）吸附拌样，减压蒸干后添加到预装硅胶玻璃减压柱（120 g硅胶，柱床4.8 × 18.0 cm）上，用石油醚-二氯甲烷-甲醇溶剂系统进行梯度洗脱层析，得到若干流分。根据硅胶薄层分析结果合并相应流分，按洗脱极性顺序得到8个组分：Fr-1（12.4 g,石油醚）、Fr-2（6.3 g，石油醚-二氯甲烷1:1洗脱）、Fr-3（4.5 g，石油醚-二氯甲烷1:2洗脱）、Fr-4（11.6 g，二氯甲烷洗脱）、Fr-5（15.8 g，二氯甲烷-甲醇99:1洗脱）、Fr-6（6.2 g，二氯甲烷-甲醇95:5洗脱）、Fr-7（1.4 g，二氯甲烷9:1洗脱）、Fr-8（1.9 g，二氯甲烷-甲醇7:3洗脱）。
将Fr-4（11.6 g）用约50 ml甲醇溶解后上Sephadex LH-20柱，甲醇洗脱，接取18个流份（各接30 ml）；根据HPLC分析结果，将组分ML-4凝胶层析流份1-18合并为8个组分：Fr-4-1-Fr-4-8。对组分Fr-4-5（约1.8 g）用甲醇溶解并用0.45μm滤膜过滤后，采用QuikSep中低压色谱系统（慧德易，北京，中国）以80%甲醇（流速10 ml/min，室温）在预装有ODS的玻璃柱（柱床1.8cm×30cm）上进行分离。根据紫外检测结果接取7个组分：ML-4-5-1-ML-4-5-7。其中，Fr-4-5-2中含有目标化合物I。
4. 化合物I的HPLC制备
将含有化合物I的组分Fr-4-5-2（180 mg）用5 ml甲醇溶解，用0.45μm滤膜过滤后，用Waters 2545型HPLC系统，利用Gemini C18制备柱（21.2 mm × 250 mm）进行HPLC分离（柱温26℃，以68%甲醇为流动相，流速10 ml/min，每次进样0.5 ml，检测波长为210和254 nm)，制得化合物I（62 mg，t R = 19.6 min）。
化合物I的理化常数与波谱数据
化合物I为无色针状结晶（MeOH），m.p. 137–139 oC。正离子ESI-MS：m/z 466 [M + H]+，488 [M + Na]+，931 [2M + H]+；负离子ESI-MS：m/z 464 [M - H]-，490 [M + Cl]-。1HNMR（500 MHz，CDCl3）δ：12.20 (1H, s, 2-NH), 9.20 (1H, br s, 2ʹʹ-NH), 9.19 (1H,d, J = 1.90 Hz, H-2ʹ), 8.79 (1H, br d, J = 7.85, H-3), 8.67 (1H, dd, J =4.80, 1.90 Hz, H-4ʹ), 8.21 (1H, dt, J = 7.90, 1.90 Hz, H-6ʹ), 7.87 (1H, dd, J= 7.85, 1.10 Hz, H-6), 7.56 (1H, td, J = 7.85, 1.10 Hz, H-4), 7.33 (1H, dd, J= 7.90, 4.80 Hz, H-5ʹ), 7.24 (1H, s, H-6ʹʹ), 7.19 (1H, br t, J = 7.85 Hz, H-5), 3.94 (3H, s, 5ʹʹ-OCH3), 3.874 (3H, s, 4ʹʹ-OCH3), 3.867 (3H, s, 3ʹʹ-OCH3),3.78 (3H, s, 7ʹʹ-OCH3)。13C NMR（125 MHz，CDCl3）δ：168.2 (C-7ʹʹ), 167.2 (C-7),164.0 (C-7ʹ), 152.6 (C-4ʹ), 151.5 (C-5ʹʹ), 149.2 (C-4ʹʹ), 148.9 (C-2ʹ), 146.9(C-3ʹʹ), 140.4 (C-2), 135.2 (C-6ʹ), 133.6 (C-4), 130.6 (C-1ʹ), 127.9 (C-6),125.7 (C-2ʹʹ), 123.8 (C-5), 123.6 (C-5ʹ), 121.8 (C-3), 120.4 (C-1), 119.1 (C-1ʹʹ), 108.8 (C-6ʹʹ), 61.24 (4ʹʹ-OCH3), 61.23 (3ʹʹ-OCH3), 56.5 (5ʹʹ-OCH3), 52.7(7ʹʹ-OCH3)。以上NMR数据经与文献数据对照予以归属。
实施例2：化合物I抑制C3H10T1/2细胞成脂分化
1）化合物I对C3H10T1/2细胞的生长抑制作用
采用MTT法检测化合物I对C3H10T1/2细胞活力的影响。取对数生长期的C3H10T1/2细胞，用新鲜DMEM培养基配制细胞密度为2×105个/ml的细胞悬液，接种于96孔板中，每孔200μl，37℃培养12 h后样品组每孔加样品液各2 μl，空白对照组则每孔加甲醇各2 μl，于37℃处理48 h。每孔加预冷的5mg/ml的MTT溶液（用PBS溶液配制）各20 μl，37℃孵育4 h后，于4℃、2000 rpm离心10 min，吸去上清液，每孔各加150 μl DMSO，置于酶标仪上充分振荡使MTT紫色产物完全溶解，测量每孔570 nm处的OD值。实验中样品和空白对照组均分别设三个平行孔，取OD平均值，按IR%＝(OD空白-OD样品)/OD空白×100%公式，计算样品对C3H10T1/2细胞的抑制率（IR%）。在MTT法测试中，结合显微镜观察检查，化合物I在100 μM作用浓度下对C3H10T1/2细胞均未显示出任何细胞毒性和生长抑制作用。
2）化合物I抑制C3H10T1/2细胞分化脂肪细胞
接种C3H10T1/2细胞至6孔板中，培养至细胞铺满80%皿底后，加入新鲜的诱导培养基（DMEM培养基+10%胎牛血清+0.5 mM异丁基甲基黄嘌呤+0.1 mM吲哚美辛+1.0 μM地塞米松+10μg/mL胰岛素+1%青链霉素双抗溶液）培养2天；换分化培养基（DMEM培养基+10%胎牛血清+1.0 μg/mL胰岛素+1%青链霉素双抗溶液）继续培养2天；换生长培养基（DMEM培养基+10%胎牛血清+1%青链霉素双抗溶液）继续培养4天（每2天换次新鲜培养基）。处理组各培养基中均加入样品溶液各2 μl，对照组不加同法操作。在培养第8天，分别进行油红O染色、细胞镜检。染色结果显示空白组大部分细胞成功分化为成熟脂肪细胞，细胞内油滴多而大，而处理组细胞内几乎没有油滴（图1），表明化合物I能够极显著地抑制C3H10T1/2细胞的成脂分化。
实施例3：化合物I抑制高脂诱导雄性果蝇肥胖的发生
取3天龄雄性果蝇，分为3组，对照组以基本培养基进行培养，HFD诱导组给予高脂培养基（基本培养基添加15%猪油），处理组则给予添加化合物I的高脂培养基（终浓度0.1 mg/ml），连续处理20天，对各组果蝇体重进行检测。由图2中检测结果可见，以高脂培养基培养可使果蝇体重显著增加，形成肥胖果蝇；同时给予化合物I处理可明显抑制HFD诱导的果蝇肥胖。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (10)
1.一种用于制备抗肥胖药物的生物碱类化合物，其特征在于，所述的生物碱类化合物具有式I所示的结构，或其药学上可接受的盐：
I。
2.权利要求1所述的式I化合物及其结构类似物terremide A、terremide B、terremideC、terremide D，或其药学上可接受的盐单独或以组合物形式在制备抗肥胖药物中的用途。
3.土曲霉ML-44，其保藏编号为CGMCC No.15664。
4.权利要求1所述的式I化合物的制备方法，其特征在于包括如下步骤：将海洋来源土曲霉Aspergillus terreus ML-44进行发酵培养，获得含有式I化合物的发酵物，将发酵物进行分离纯化，得到式I化合物。
5.根据权利要求4所述的制备方法，其特征在于所述分离纯化包括萃取、柱层析和高效液相制备。
6.一种权利要求1所述的式I化合物的制备方法，其特征在于包括如下步骤：
1) 将土曲霉（Aspergillus terreus）ML-44进行大米固体发酵培养，获得发酵物；
2) 将所得发酵物用50%-90%（v/v）的丙酮水溶液超声浸提、过滤，滤液经减压浓缩至不含丙酮，残余混悬液用乙酸乙酯萃取，得到发酵物的乙酸乙酯萃取物；
3) 将步骤2)得到乙酸乙酯萃取物减压浓缩至干，得到乙酸乙酯浸膏；
4) 将乙酸乙酯总浸膏依次用硅胶柱层析，石油醚‒二氯甲烷‒甲醇梯度洗脱、SephadexLH-20柱层析，甲醇洗脱，和ODS柱层析，水→100%甲醇梯度洗脱，分离，得到含有所述化合物的柱层析组分；
5) 将含有所述化合物的柱层析组分经HPLC分离；
其中，步骤2)中的丙酮水溶液优选为75%-85%（v/v），更优选为80%（v/v）。
7.一种土曲霉（Aspergillus terreus）ML-44发酵物的提取物，其特征在于所述提取物含有权利要求1所述的式I化合物。
8.权利要求7所述的提取物在制备抗肥胖药物中的用途。
9.权利要求3所述的土曲霉（Aspergillus terreus）ML-44在制备权利要求1所述的式I化合物或者权利要求7所述的提取物中的用途。
10.一种抗肥胖症药物，其特征在于含有权利要求1所述的具有式I所示的结构，或其药学上可接受的盐，以及药学上可接受的辅助剂；所述药物为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂。
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