CN113121475B - 不饱和脂肪酸类化合物及其制备方法和应用 - Google Patents
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Abstract
本发明涉及一种不饱和脂肪酸类化合物及其制备方法和应用,本发明采用多种色谱分离手段、光谱波谱技术,从于红背山麻杆干燥根的乙酸乙酯冷浸提取物中分离鉴定脂肪酸类化合物,并通过PC12细胞(大鼠肾上腺髓质嗜铬瘤分化细胞株)模型进行活性筛选,得到具有加强神经细胞分化功能的新不饱和脂肪酸类化合物。
Description
技术领域
本发明涉及医药领域,更特别地,涉及一种大戟科山麻杆属植物红背山麻杆Alchornea trewioides(Benth.)Muell.Arg.干燥根中分离得到一种新的不饱和脂肪酸类化合物,该化合物具有促进Neurofilament 200启动子的luciferase活性和PC12细胞分化且与神经生长因子(NGF)有显著协同作用,并提供该化合物的制备方法及应用。
背景技术
神经退行性疾病以由神经元和/或其髓鞘的丧失为主要特征,是一类严重危害人类健康的复杂疾病。阿尔茨海默病(Alzheimer Disease,AD)是其中一种最为典型的神经退行性疾病,具有记忆与认知功能障碍的特征,是老年痴呆病中最常见的类型,其发病机制十分复杂,目前尚无特效药。随着对AD发病机理不断深入研究,涌现了大量治疗方法和药物。其中,神经生长因子(nerve growth factor,NGF)的发现为治疗AD带来新的曙光。NGF是一种神经营养因子,能促进胆碱能神经元的生长和分化,在维持中枢胆碱能神经元功能中具有重要作用。现代研究表明,中枢胆碱能神经系统在学习、记忆等活动中具有重要作用,NGF对中枢胆碱能神经元营养支持作用的减弱,会导致其功能与结构发生退行性改变,而使认知功能降低。但是,NGF是生物大分子,无法自由通过血脑屏障,所以无法直接通过外源补充NGF治疗AD,为其应用于AD的治疗带来了巨大的挑战。
大量研究发现,不饱和脂肪酸在维护神经细胞的正常结构和功能中扮演重要角色,具有促进神经元及其突触生长、维持神经元正常形态、防止神经元凋亡等功能,对防止老年期神经系统功能的衰退具有重要作用。此外,不饱和脂肪酸具有脂溶性强的化学特性,容易透过血脑屏障,可以弥补NGF无法通过血脑屏障的不足。因此,寻找具有与NGF相似神经营养作用或与NGF具有协同作用的脂溶性强的天然产物小分子化合物在AD等神经退行性疾病治疗方面具有可行的研究价值。
红背山麻杆Alchornea trewioides(Benth.)Muell.Arg.是一种大戟科山麻杆属半落叶灌木,具有祛风发散、祛湿清热、杀虫止痒等功效,主要包含黄酮、酚酸、奎宁酸等化学成分。现代药理研究表明,红背山麻杆具有抗炎、抗肿瘤和抗酒精性肝纤维化等药理活性,但目前尚未发现其在加强神经细胞分化活性的报道。
发明内容
本发明的目的是从大戟科山麻杆属植物红背山麻杆Alchornea trewioides(Benth.)Muell.Arg.干燥根中分离得到具有能够提高Neurofilament 200编码基因启动子活性和促进PC12细胞分化的不饱和脂肪酸类化合物trewioidesine A。
为实现上述目的,本发明提供一种具有加强神经细胞分化的不饱和脂肪酸类化合物,所述不饱和脂肪酸类化合物的化学结构如下所示:
在本发明提供的不饱和脂肪酸类化合物中,不饱和脂肪酸类化合物可促进Neurofilament 200启动子的荧光素酶(luciferase)活性,并与神经生长因子(NGF)具有协同作用。
在本发明提供的不饱和脂肪酸类化合物中,所述不饱和脂肪酸类化合物具有促进PC12细胞分化的作用,与神经生长因子(NGF)连用时可增强PC12细胞的分化。
本发明还提供一种用于制备如上所述的不饱和脂肪酸类化合物的制备方法,包括以下步骤:
步骤S1、将红背山麻杆干燥根粉碎得到粒径小于3mm的粗粉,加入乙酸乙酯进行冷浸提取,过滤,室温减压回收乙酸乙酯,得到浸膏;
步骤S2、将所述浸膏经正相硅胶柱色谱、Sephadex LH-20柱色谱和半制备HPLC液相色谱进行分离纯化,得到所述不饱和脂肪酸类化合物。
在本发明提供的制备方法中,在步骤S1中,所述粗粉冷浸时所用乙酸乙酯的量为粗粉的1-10倍重量份,冷浸时间大于24h。
在本发明提供的制备方法中,在步骤S2中,所述正相硅胶柱色谱以石油醚/乙酸乙酯、二氯甲烷/甲醇两种溶剂系统进行梯度洗脱。
在本发明提供的制备方法中,在步骤S2中,所述Sephadex LH-20柱色谱以二氯甲烷/甲醇进行等度洗脱。
在本发明提供的制备方法中,在步骤S2中,所述半制备HPLC液相色谱以甲醇/水溶液为洗脱剂进等梯度洗脱。
本发明还提供一种如上所述的不饱和脂肪酸类化合物用于治疗神经退行性疾病的应用。
在本发明提供的应用中,单独使用所述不饱和脂肪酸类化合物或将所述不饱和脂肪酸类化合物与神经生长因子(NGF)联合使用。
本发明的不饱和脂肪酸类化合物及其制备方法和应用,具有以下有益效果:本发明采用多种色谱分离手段、光谱波谱技术,从于红背山麻杆干燥根的乙酸乙酯冷浸提取物中分离鉴定脂肪酸类化合物,并通过PC12细胞(大鼠肾上腺髓质嗜铬瘤分化细胞株)模型进行活性筛选,得到具有加强神经细胞分化功能的新不饱和脂肪酸类化合物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图:
图1所示为trewioidesine A主要HMBC、1H-1H COSY和NOESY相关图;
图2所示为trewioidesine A的ECD图谱和计算ECD图;
图3所示为trewioidesine A的紫外光谱图;
图4所示为trewioidesine A的红外光谱图;
图5所示为trewioidesine A的1H NMR谱图(CDCl3,600MHz);
图6所示为trewioidesine A的13C NMR(CDCl3,150MHz);
图7所示为trewioidesine A的HSQC谱图;
图8所示为trewioidesine A的HMBC谱图;
图9所示为trewioidesine A的1H-1H COSY谱图;
图10所示为trewioidesine A的NOESY谱图;
图11所示为trewioidesine A高分辨质谱图;
图12所示为不同浓度trewioidesine A促进Neurofilament-200启动子的luciferase活性(与控制组相比,*P<0.05,**P<0.01);
图13所示为trewioidesine A促进PC12细胞分化的活性(A:PC12细胞神经突起的生长情况;B:PC12细胞分化的比例;与控制组相比,*P<0.05,**P<0.01,***P<0.001)。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的典型实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
为了更好的理解上述技术方案,下面将结合说明书附图以及具体的实施方式对上述技术方案进行详细的说明,应当理解本发明实施例以及实施例中的具体特征是对本申请技术方案的详细的说明,而不是对本申请技术方案的限定,在不冲突的情况下,本发明实施例以及实施例中的技术特征可以相互组合。
本发明从大戟科山麻杆属植物红背山麻杆Alchornea trewioides(Benth.)Muell.Arg.干燥根中分离得到具有能够提高Neurofilament 200编码基因启动子活性和促进PC12细胞分化的不饱和脂肪酸类化合物trewioidesine A,其结果如下:
trewioidesine A的理化、波谱数据如下:
白色粉末,正离子模式HRESIMS给出准分子离子峰m/z 333.2028[M+Na]+,分子式为C18H30O4。比旋光度-10.53(c 0.1,MeOH)。UVλMeOH max:195,233nm。IR(KBr)νmax3138,2958,2931,2852,1720,1690,1640,1579,1463,1446,1379,1339,1310,1244,1231,1163,1114,972,874,849cm-1;1H和13C NMR数据见表1。
表1trewioidesine A1H(600MHz)和13C(150MHz)NMR数据(δin ppm;in CDCl3)
另一方面,trewioidesine A的制备方法如下:红背山麻杆干燥根10.0kg,粉碎得到粒径小于3mm粗粉,加入80L乙酸乙酯冷浸提取3次,每次1周,期间不时搅拌,过滤,室温减压回收乙酸乙酯,得到浸膏70.5g。将所得浸膏经正相硅胶柱色谱、Sephadex LH-20柱色谱和半制备HPLC液相色谱进行分离纯化,得到trewioidesine A。
具体地,trewioidesine A的结构鉴定过程如下:采用紫外光谱(UV)、红外光谱(IR)、一维NMR谱(1H NMR、13C NMR)、二维NMR谱(HSQC、HMBC、1H-1H COSY、NOESY)、高分辨质谱(HRESIMS)等技术鉴定trewioidesine A的平面结构,通过计算ECD的方法鉴定trewioidesine A的绝对构型。
具体地,trewioidesine A的活性评价如下:于采用PC12细胞模型,通过检测NF200启动子表达luciferase的诱导活性反应trewioidesine A对神经细胞分化的影响,同时采用光学显微镜拍照分析神经突起的生长情况,考察trewioidesine A以及与NGF共同作用于PC12细胞分化作用。
实施例1:trewioidesine A的提取分离和结构鉴定
1.1植物原料及来源:大戟科山麻杆属植物红背山麻杆(拉丁学名:Alchorneatrewioides(Benth.)Muell.Arg.)干燥的根。红背山麻杆的根2020年1月采自广东省湛江市廉江市和寮镇,样品存放于香港科技大学深圳研究院中药研发中心。
1.2提取分离的试剂:提取分离所用石油醚(广东光华科技有限公司)、二氯甲烷(西陇科学股份有限公司)、乙酸乙酯(天津市永大化学试剂有限公司)、甲醇(西陇科学股份有限公司)等均为分析纯;超纯水(Merck Direct-Q超纯水系统)。
1.3色谱柱及填料
色谱柱:YMC-Pack ODS-A(250×20mm,5μm;柱I),YMC-Pack ODS-A(250×10mm,5μm;柱II);色谱填料:硅胶(200~300目,青岛海洋化工厂),Sephadex LH-20填料(瑞典,Amersham Bioscience);薄层色谱GF254硅胶板(青岛海洋化工厂)。
1.4仪器
AVANCE III 600MHZ超导核磁共振波谱仪(瑞士Bruker公司);maXis impact高分辨电喷雾四级杆-飞行时间质谱仪(美国Bruker Daltonics公司);汉邦科技半制备液相(NP7005C液相输液泵,NU3000C紫外可变波长检测器,EasyChrom-1000工作站,中国汉邦科技有限公司);UV-2401PC紫外可见分光光度仪(日本岛津公司);Nexus470傅立叶红外光谱仪(美国热电尼高力公司);ZF-1型三用紫外分析仪(江苏海门市其林贝耳仪器制造有限公司);Adam SAB225i半微量分析天平(Max 220g,d=0.01mg,英国Adam公司);Merck Direct-Q超纯水系统(德国Merck Millipore公司);旋转蒸发仪(瑞士BUCHI公司);DLSB-5/20低温冷却液循环泵(郑州长城科工贸有限公司);DZF-6020真空干燥箱(上海一恒科学仪器有限公司)。
1.5实验方法
红背山麻杆干燥根10.0kg,粉碎得到粒径小于3mm粗粉,加入80L乙酸乙酯冷浸提取3次,每次1周,期间不时搅拌,过滤,室温减压回收乙酸乙酯,得到浸膏70.5g。
称取乙酸乙酯部位135.0g,经硅胶(200~300目)柱色谱分离,采用石油醚-乙酸乙酯(10:1→10:4,v/v)、二氯甲烷-甲醇(5:0.1→5:3,v/v))洗脱,得到62个流分,通过TLC薄层检识,合并成分相似流分,得到12个流分(Fr.A~L)。流分Fr.G(1.1g)经Sephadex LH-20柱色谱分离,二氯甲烷-甲醇(1:1)洗脱,得到5个流分(Fr.G1~G5)。Fr.G3(0.78g)经半制备液相(甲醇:水=80:20,吸收波长210nm,柱I)分离,得到4个流分(Fr.G3a~Fr.G3d)。Fr.G3b经半制备液相(甲醇:水=70:30,吸收波长210nm,柱II)纯化,得到trewioidesine A(2.1mg,tR 53.13min)。
1.6结构解析
该化合物为白色粉末,正离子模式HRESIMS给出准分子离子峰m/z333.2028[M+Na]+(计算值333.2036),推断该化合物的分子式C18H30O4,计算不饱和度为4。红外数据显示(图4),分子结构中含有羟基(2931cm–1)和羰基(1690cm–1)等官能团。在1H NMR中(图5),观察到一组反式烯烃质子信号[δH6.51(1H,dd,J=15.6,7.2Hz),6.38(1H,d,J=15.6Hz)],两个次甲基信号[δH3.21(1H,dd,J=7.2,1.8Hz),2.91(1H,td,J=5.4,1.8Hz)],十一个亚甲基信号[δH2.53(2H,td,J=7.2,2.4Hz),2.34(2H,t,J=7.2Hz),1.61(6H,m),1.46(2H,m),1.32(10H,m)]和一个甲基信号[δH0.90(3H,t,J=7.2Hz)]。在13C NMR中(图6)观察到18个碳信号,包括1个酮羰基碳信号(δC 199.8),1个羧羰基碳信号(δC 177.3),2个烯碳信号(δC142.7,131.5),2个次甲基碳信号(δC 61.8,56.8),11个亚甲基碳信号(δC 40.6,33.7,32.0,31.7,29.1×2,28.9,25.6,24.7,24.0,22.7)以及一个甲基碳信(δC14.1)。综合以上分析,推断该化合物为不饱和脂肪酸类化合物。其NMR数据与化合物(10E)-9-oxohexadec-10-enoic acid相似,主要不同之处是该化合物多出两个次甲基和两个亚甲基。
在HMBC谱中(图8),观察到H-9/H-10和C-8有远程相关信号,证明该化合物的结构中存在α,β-不饱和酮片段;H-9和亚甲基(δC40.6)远程相关信号,表明C-7和C-8相连接;H2-6和C-8,H2-7和C-5有远程相关信号,表明C-6和C-5/C-7相连接,形成C-5/C-6/C-7碳链结构片段;H2-2/H2-3和C-1,H2-2和C-4有远程相关信号,表明C-2和羧基相连接,C-3和C-2/C-4相连接,从形成C-1/C-2/C-3/C-4结构片段。此外,在HMBC谱中(图8),还观察到H2-14和C-12/C-16,H2-15和C-13,H3-18和C-16/C-17有远程相关信号,以及在1H-1H COSY谱(图9)中H-10/H-11/H-12有相关信号,表明化合结构中的C-12/C-13/C-14/C-15/C-16/C-17/C-18碳链结构片段。综合以上分析,根据分子式C18H30O4可推断,C-11和C-12被氧化成环氧化合物。通过NOESY实验确定了该化合物的相对构型(图1),进一步通过计算ECD法确定了其绝对构型。该化合物的实验ECD图谱显示在230nm出现负的Cotton效应,这一结果与计算的构型为(11R,12R)的ECD图谱基本一致(图2)。因此,确定该化合物的绝对构型为(11R,12R),结构上所示,命名为trewioidesine A。
实施例2:trewioidesine A诱导NF 200启动子表达luciferase的活性检测
测试的实验包括以下步骤:
(1)培养PC12细胞(大鼠肾上腺髓质嗜铬瘤分化细胞株):在37℃,5%CO2及饱和湿度条件下,将细胞置于DMEM培养基(含6%胎牛血清,6%马血清,100U/mL青霉素及100μm/mL链霉素)。
(2)将PC12细胞以8×104/mL接种于24孔板中,24h后,将含有Neurofilament200编码基因基因启动子的荧光素酶报告基因载体pNF200-Luc转染到PC12细胞中。4h后,换成低血清培养基(DMEM培养基,1%胎牛血清+1%马血清),随后加入不同终浓度的trewioidesine A。
(3)48h后,移除细胞培养液,并用PBS冲洗。裂解液(100mM PBS,1mM DTT,0.2%Triton X-100,pH 7.8)裂解后,12000g高速离心15分钟得到含有荧光素酶的细胞裂解液。
(4)将50μL细胞裂解液转移到不透光的96孔酶标板上,加入荧光素酶的底物,用化学发光仪(Promega Glomax 96孔化学发光仪)检测荧光素酶活性。各样本活性用总蛋白浓度做归一化处理,得出实验结果。
(5)实验结果如图12所示,trewioidesine A具有促进Neurofilament 200启动子的luciferase活性,且呈浓度依赖性,与NGF具有显著协同作用。
实施例3:trewioidesine A对神经细胞分化作用测试
测试的实验包括以下步骤:
(1)将PC12细胞接种(2×104cells/mL)到6孔板中,24小时后,将培养基更换为低血清培养基(DMEM培养基,1%胎牛血清+1%马血清)继续培养24小时。
(2)随后加入10μM的trewioidesine A处理48小时,观察细胞分化和突起生长情况。以终浓度20ng/mL NGF的处理组作为阳性对照,同时考察和终浓度1ng/mL NGF联用处理的效果。
(3)用光学显微镜拍照分析神经突起的生长情况,每孔随机选择5个视野,每个视野观察至少100个细胞。细胞的一个或多个神经突起长度超过胞体直径则归类为分化细胞。
(4)实验结果如图13所示,trewioidesine A具有促进PC12细胞分化活性,与少量NGF连用能显著提高分化细胞的比例。
Claims (2)
2.根据权利要求1所述的制备方法,其特征在于,在步骤S1中,所述粗粉冷浸时所用乙酸乙酯的量为粗粉的1-10倍重量份,冷浸时间大于24h。
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