CN105884790A - D-叔亮氨酸的药物组合物及其生物医药用途 - Google Patents
D-叔亮氨酸的药物组合物及其生物医药用途 Download PDFInfo
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Abstract
本发明公开了D‑叔亮氨酸的药物组合物及其生物医药用途,本发明提供的D‑叔亮氨酸的药物组合物中含有D‑叔亮氨酸和一种从半夏的干燥块茎中分离得到的结构新颖的天然产物化合物(Ⅰ),D‑叔亮氨酸、化合物(Ⅰ)单独作用时,具有促进急性缺血心肌血管再生的作用;D‑叔亮氨酸和化合物(Ⅰ)联合作用时,促进急性缺血心肌血管再生的作用更为明显,可以开发成治疗缺血性心脏病的药物,用于促进急性缺血心肌血管再生,与现有技术相比具有突出的实质性特点和显著的进步。
Description
技术领域
本发明属于生物医药领域,涉及D-叔亮氨酸的新用途,具体涉及D-叔亮氨酸的药物组合物及其在生物医药中的应用。
背景技术
迄今为止,尚未见D-叔亮氨酸及其药物组合物与缺血性心脏病的相关性报道。
发明内容
本发明的目的在于提供一种D-叔亮氨酸的药物组合物,该药物组合物中含有D-叔亮氨酸和一种结构新颖的天然产物,D-叔亮氨酸和该天然产物可以协同治疗缺血性心脏病。
本发明的上述目的是通过下面的技术方案得以实现的:
一种具有下述结构式的化合物(Ⅰ),
一种D-叔亮氨酸的药物组合物,包括D-叔亮氨酸、如权利要求1所述的化合物(Ⅰ)和药学上可以接受的载体,制备成需要的剂型。
进一步地,药学上可以接受的载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体或润滑剂。
进一步地,所述剂型包括片剂、胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、喷雾剂、滴剂或贴剂。
上述化合物(Ⅰ)的制备方法,包含以下操作步骤:(a)将半夏的干燥块茎粉碎,用65~75%乙醇热回流提取,合并提取液,浓缩至无醇味,依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇取物用大孔树脂除杂,先用20%乙醇洗脱9个柱体积,再用65%乙醇洗脱10个柱体积,收集65%洗脱液,减压浓缩得65%乙醇洗脱浓缩物;(c)步骤(b)中65%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为65:1、35:1、15:1和7:1的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分4用正相硅胶进一步分离,依次用体积比为12:1、7:1和1:1的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为58%的甲醇水溶液等度洗脱,收集11~15个柱体积洗脱液,洗脱液减压浓缩得到化合物(Ⅰ)。
进一步地,化合物(Ⅰ)的制备方法中,步骤(a)用70%乙醇热回流提取,合并提取液。
进一步地,化合物(Ⅰ)的制备方法中,所述大孔树脂为D101型大孔吸附树脂。
进一步地,化合物(Ⅰ)的制备方法中,步骤(a)中用二氯甲烷代替乙酸乙酯进行萃取,得到二氯甲烷萃取物。
上述化合物(Ⅰ)在制备治疗缺血性心脏病的药物中的应用。
上述D-叔亮氨酸的药物组合物在制备治疗缺血性心脏病的药物中的应用。
本发明的优点:本发明提供的D-叔亮氨酸的药物组合物中含有D-叔亮氨酸和一种从半夏的干燥块茎中分离得到的结构新颖的天然产物,D-叔亮氨酸和该天然产物单独作用时,对缺血性心脏病具有治疗作用;二者联合作用时,对缺血性心脏病的治疗效果进一步提高,可以开发成治疗缺血性心脏病的药物。
具体实施方式
下面结合实施例进一步说明本发明的实质性内容,但并不以此限定本发明保护范围。
实施例1:化合物(Ⅰ)分离制备及结构确证
分离方法:(a)将半夏的干燥块茎(2kg)粉碎,用70%乙醇热回流提取(15L×3次),合并提取液,浓缩至无醇味(3L),依次用石油醚(3L×3次)、乙酸乙酯(3L×3次)和水饱和的正丁醇(3L×3次)萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中乙酸乙酯萃取物用D101型大孔树脂除杂,先用20%乙醇洗脱9个柱体积,再用65%乙醇洗脱10个柱体积,收集65%洗脱液,减压浓缩得65%乙醇洗脱浓缩物;(c)步骤(b)中65%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为65:1(9个柱体积)、35:1(10个柱体积)、15:1(8个柱体积)和7:1(8个柱体积)的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分4用正相硅胶进一步分离,依次用体积比为12:1(6个柱体积)、7:1(7个柱体积)和1:1(6个柱体积)的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为58%的甲醇水溶液等度洗脱,收集11~15个柱体积洗脱液,洗脱液减压浓缩得到化合物(Ⅰ)(325mg,HPLC归一化纯度大于98%)。
结构确证:无定型粉末,HR-ESI-MS显示[M+H]+为m/z 279.1178,结合核磁特征可得分子式为C15H18O5,不饱和度为7。核磁共振氢谱数据δH(ppm,CDCl3,500MHz):H-2(3.14,d,J=2.7Hz),H-3(3.27,d,J=2.7Hz),H-5(2.09,d,J=11.7Hz),H-6(3.85,dd,J=11.7,10.6Hz),H-7(2.54,dt,J=3.5,10.6Hz),H-8α(2.05,ddt,J=13.3,3.6,3.2Hz),H-8β(1.65,ddt,J=13.3,3.2,13.2Hz),H-9α(2.12,dt,J=3.2,13.2Hz),H-9β(1.94,dt,J=13.2,3.2Hz),H-13(5.79,d,J=3.1Hz),H-13(6.08,d,J=3.1Hz),H-14(1.05,s),H-15(1.41,s);核磁共振碳谱数据δC(ppm,CDCl3,125MHz):211.4(C,1-C),45.5(CH,2-C),62.9(CH,3-C),69.5(C,4-C),56.7(CH,5-C),73.6(CH,6-C),50.2(CH,7-C),20.2(CH2,8-C),31.7(CH2,9-C),39.6(C,10-C),138.3(C,11-C),170.3(C,12-C),117.9(CH2,13-C),14.1(CH3,14-C),23.2(CH3,15-C)。红外波谱中的1716cm-1吸收带与UV谱中的236nm吸收带表明该化合物含有γ-内酯结构,进一步并通过分析13C-NMR谱中的δC73.6,50.2,138.3,170.3,117.9的碳信号可知存在环外亚甲基γ-内酯结构。13C-NMR、DEPT和HSQC谱中显示有15个碳信号,包括两个甲基,三个亚甲基(一个烯烃碳),五个次甲基(三个连氧碳),以及五个季碳(一个烯烃碳,两个羰基碳和一个连氧季碳)。以上功能结构再结合不饱和数表明该化合物为四环结构。1H-NMR谱结合HSQC谱显示两个甲基质子信号δH 1.05(3H,s)、1.41(3H,s),一对端烯烃质子信号δH 6.08(1H,d,J=3.1Hz)与5.79(1H,d,J=3.1Hz),三个连氧次甲基质子信号δH 3.14(1H,2.7Hz)、3.27(1H,d,J=2.7Hz)与3.85(1H,dd,J=11.7,10.6Hz)。NMR谱数据δH 3.14(1H,dd,J=2.7Hz)、3.27(1H,d,J=2.7Hz)与δC 45.5和62.9可知C-2和C-3形成环氧结构。通过1H-1H COSY谱中H-2/H-3与H-5/H-6/H-7/H2-8/H2-9相关信号,以及HMBC谱中显示的H-2与C-1和C-3,H-3与C-1、C-2、C-4和C-5,H-5与C-6和C-10,H-6与C-7、C-8、C-11和C-12,H-7与C-6、C-11、C-12和C-13,H2-13与C-7和C-12,H3-14与C-10相关信号,可以构建该化合物的连接方式,并且上述波谱数据表明该化合物为桉叶烷型倍半萜,并且存在环外亚甲基γ-内酯结构。该化合物中的C-2、C-3、C-4、C-5、C-6、C-7和C-10为手性碳,通过NOESY试验与H-H偶合常数确认了相对构型。NOESY谱中H-5/H-7、H-5/H-8α、H-7/H-8α、H-7/H-9α、H-6/H-8β以及H-6/H-14相关信号表明A/B与B/C环反式连接。此外,通过H-2/H-14、H-3/H-14、H-2/H-15、H-3/H-15、H-6/H-15以及H-14/H-15相关信号确定C-2与C-3的环氧结构为α构型。NOE差谱试验中照射H-14甲基可以发现H-3、H-6、H-8β、H-9β、H-15有明显增益,因此以上相关信号可以表明该化合物的C-2、C-3、C-4、C-5、C-6、C-7和C-10构型为2R、3R、4S、5S、6S、7S和10R。综合氢谱、碳谱、HMBC谱和NOESY谱,以及文献关于相关类型核磁数据,可基本确定该化合物如下所示,立体构型进一步通过ECD试验确定,理论值与实验值基本一致。
该化合物化学式及碳原子编号如下:
实施例2:药理作用
1、材料与方法
1.1动物
Wistar大鼠,雌雄各半,体重280~320g,由吉林大学白求恩医学院动物实验中心提供。
1.2试剂与样品
D-叔亮氨酸购自中国药品生物制品检定所。化合物(Ⅰ)自制,制备方法见实施例1。氯化三苯基四氮唑(TTC)购于中国医药上海化学试剂公司;酒石酸美托洛尔片购于阿斯利康制药有限公司;CD34、VEGF、VEGFR1和VEGFR2免疫组化试剂盒购于武汉博士德有限公司;p-Akt免疫组化试剂盒购于美国Cell Signaling公司;NO试剂盒购于南京建成研究所。
1.3大鼠分组及模型制备
急性心肌缺血模型的建立:麻醉大鼠,开胸后于左心耳与肺动脉圆锥间左心耳下1mm左右穿线结扎左冠脉前降支(LAD)。大鼠按随机抽签法分为6组:假手术组、模型对照组、阳性对照组(美托洛尔,4.5mg·kg-1)和D-叔亮氨酸组(10mg·kg-1)、化合物(Ⅰ)组(10mg·kg-1)、D-叔亮氨酸与化合物(Ⅰ)组合物组【5mg·kg-1D-叔亮氨酸+5mg·kg-1化合物(Ⅰ)】。阳性药组给予美托洛尔,假手术组左冠状动脉只穿线,不结扎。各组动物于模型制备后1h腹腔注射给药,假手术组、模型组给予等量生理盐水,每天给药1次,连续给药14d后,处死大鼠。
1.4TTC染色测量心肌梗死面积
大鼠麻醉后,开胸取出心脏,剪下右心室,将左心室心肌横向切成2~3mm薄片,置于1%TTC溶液中染色,37℃恒温水浴5~10min,取出放入10%甲醛溶液中固定,正常心肌组织染成红色,梗死区呈白色。利用BI-2000图像分析系统测量左心室心肌梗死面积和左心室全面积,梗死面积=心肌梗死面积/左心室全面积×100%。
1.5大鼠心肌梗死边缘区微血管密度的测定
取心脏,在左心室结扎线下方,略白的区域为梗死区,其周围为缺血区,取梗死周围2mm缺血区的心肌,用甲醛固定,石蜡包埋切片后,SABC法免疫组化染色,具体步骤按试剂盒说明书进行,用PBS替换一抗作阴性对照。用抗CD34多克隆抗体免疫组化染色,于光学显微镜下观察微血管密度(MVD)。具体方法为:先在40×视野下找到心肌梗死边缘区,然后在×400视野下进行大鼠心肌梗死边缘区微血管计数,每张切片随机选取5个视野,计数每个视野的微血管数,取其平均值作为该样本的MVD值,MVD以毛细血管个数/视野来表示。
1.6大鼠心肌组织NO水平测定
取心肌组织100mg,置于玻璃匀浆器中加入0.01mol/L PBS 1mL,冰浴下匀浆,4℃离心4000r/min,15min,取上清测定NO水平,具体步骤按试剂盒说明书进行。
1.7统计学方法
实验数据用均数±标准差(x±s)表示,应用SPSS18.0版统计软件进行单因素方差分析和t检验,以P<0.05为差异有统计学意义。
2、实验结果
2.1对心肌梗死面积的影响
假手术组心肌组织呈现红色,模型组和给药组部分心肌组织呈白色,即为心肌梗死区域。采用BI-2000图像分析系统测量心肌梗死面积结果表明,与模型对照组比较,D-叔亮氨酸与化合物(Ⅰ)组合物组与阳性药组心肌梗死面积明显减小(P<0.01);与模型对照组比较,D-叔亮氨酸组、化合物(Ⅰ)组心肌梗死面积减小(P<0.05)。结果见表1。
2.2对大鼠心肌梗死边缘区微血管密度的影响
与模型对照组比较,D-叔亮氨酸与化合物(Ⅰ)组合物组与阳性药组MVD明显增加(P<0.01),D-叔亮氨酸组、化合物(Ⅰ)组MVD增加(P<0.05)。结果见表1。
2.3对急性心肌缺血大鼠心肌组织NO水平的影响
与模型对照组比较,药物组大鼠心肌组织NO水平均具有升高趋势;与模型对照组比较,D-叔亮氨酸与化合物(Ⅰ)组合物组与阳性药组心肌组织的NO水平明显升高,D-叔亮氨酸组、化合物(Ⅰ)组心肌组织的NO水平升高(P<0.05)。结果见表1。
表1对大鼠心肌梗死面积、梗死边缘区微血管密度及心肌组织NO水平的影响
组别 | 心肌梗死面积(%) | MVD(个/视野) | NO(μmol/g) |
假手术组 | 0 | 16.08±0.58 | 1.81±0.25 |
模型对照组 | 21.00±6.92 | 7.23±0.63 | 0.76±0.22 |
阳性对照组 | 8.14±2.64 | 17.81±1.38 | 1.95±0.25 |
D-叔亮氨酸组 | 12.81±4.27 | 13.63±1.77 | 1.33±0.20 |
化合物(Ⅰ)组 | 11.05±3.12 | 13.16±0.76 | 1.42±0.49 |
D-叔亮氨酸与化合物(Ⅰ)组合物组 | 6.82±2.21 | 17.19±0.89 | 2.09±0.66 |
心肌缺血后的心肌修复过程与血管再生密切相关,缺血区内是否有血管迅速再生对心肌缺血的预后有十分重要的作用。微血管密度是直接反映血管再生的较可靠的解剖学指标,梗死范围可间接反映血管再生情况,梗死面积越小,说明血管再生越丰富。采用这两个指标,基本上能反映缺血心肌内血管新生情况。
上述试验结果表明,D-叔亮氨酸、化合物(Ⅰ)单独作用时,具有促进急性缺血心肌血管再生的作用;D-叔亮氨酸和化合物(Ⅰ)联合作用时,促进急性缺血心肌血管再生的作用更为明显,可以开发成治疗缺血性心脏病的药物,用于促进急性缺血心肌血管再生。
上述实施例的作用在于说明本发明的实质性内容,但并不以此限定本发明的保护范围。本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和保护范围。
Claims (10)
1.一种具有下述结构式的化合物(Ⅰ),
2.一种D-叔亮氨酸的药物组合物,其特征在于:包括D-叔亮氨酸、如权利要求1所述的化合物(Ⅰ)和药学上可以接受的载体,制备成需要的剂型。
3.根据权利要求2所述的D-叔亮氨酸的药物组合物,其特征在于:药学上可以接受的载体包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体或润滑剂。
4.根据权利要求2所述的D-叔亮氨酸的药物组合物,其特征在于:所述剂型包括片剂、胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、喷雾剂、滴剂或贴剂。
5.权利要求1所述的化合物(Ⅰ)的制备方法,其特征在于,包含以下操作步骤:(a)将半夏的干燥块茎粉碎,用65~75%乙醇热回流提取,合并提取液,浓缩至无醇味,依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇取物用大孔树脂除杂,先用20%乙醇洗脱9个柱体积,再用65%乙醇洗脱10个柱体积,收集65%洗脱液,减压浓缩得65%乙醇洗脱浓缩物;(c)步骤(b)中65%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为65:1、35:1、15:1和7:1的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分4用正相硅胶进一步分离,依次用体积比为12:1、7:1和1:1的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为58%的甲醇水溶液等度洗脱,收集11~15个柱体积洗脱液,洗脱液减压浓缩得到化合物(Ⅰ)。
6.根据权利要求5所述的化合物(Ⅰ)的制备方法,其特征在于:步骤(a)用70%乙醇热回流提取,合并提取液。
7.根据权利要求5所述的化合物(Ⅰ)的制备方法,其特征在于:所述大孔树脂为D101型大孔吸附树脂。
8.根据权利要求5所述的化合物(Ⅰ)的制备方法,其特征在于:步骤(a)中用二氯甲烷代替乙酸乙酯进行萃取,得到二氯甲烷萃取物。
9.权利要求1所述的化合物(Ⅰ)在制备治疗缺血性心脏病的药物中的应用。
10.权利要求2~4任一所述的D-叔亮氨酸的药物组合物在制备治疗缺血性心脏病的药物中的应用。
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