CN115417844A - 一种环己乙醇化合物rengyolone及其制备方法与应用 - Google Patents
一种环己乙醇化合物rengyolone及其制备方法与应用 Download PDFInfo
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- 230000001052 transient effect Effects 0.000 description 1
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- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
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Abstract
Description
技术领域
本发明属于医药制备技术领域,具体地说,涉及一种环己乙醇化合物rengyolone及其制备方法与应用。
背景技术
紫葳科角蒿属(Incarvillea L.)植物,全球共有15种,我国有11种,多为我国特有种,主要分布于西南地区,东北、华北、西北等地区也有分布,在民间主要用于治疗肝炎、菌痢、贫血等病。其化学成分主要为一系列骨架新颖、结构多样、类型不同的单萜生物碱、环己乙醇类及苯乙醇苷类化合物等(Su Y Q,Shen Y H,Lin S,Tang J,Tian J M,Liu X H,Zhang W D.Two new alkaloids fromIncarvillea mairei vargrandiflora[J].Helvetica ChimicaActa,2009,29: 165-169.),部分骨架结构新颖的生物碱及环己乙醇化合物发表后(OrganicLetters,14(8),1954- 1957,2012;RSCAdvances,2,4175-4180,2012;RSCAdvances,6,65885-65888,2016;Chemistry &Biodiversity,6,779-783,2009),即被著名评述杂志NaturalProductReports评为国际热点化合物(Natural ProductReports,26,973-976,2009;28,659-662,2011;33,1126-1130,2016),引发了人们的广泛关注,以角蒿和毛子草的研究较多(Fu J J,Jin H Z,ShenY H,Zhang W D,Xu W Z.Two novelmonoterpene alkaloid dimers from Incarvillea arguta[J].Helvetica ChimicaActa,2007,90:2151-2155.)。同时角蒿属植物中的化学成分具有较好的抗炎、镇痛及细胞毒等活性。从大花鸡肉参(Incarvillea matrei var.granditlora(Wehrhahn)Grierson)中分离得到的环己酮类化合物(+)-2-(1-hydroxyl-4-oxocyclohexyl)ethyl caffeate(HOEC)抗炎活性显著(Li L,Zeng H W, Liu F,Zhang J G,Yue R C,Lu W Q,Yuan X,Dai W X,Yuan H,Sun Q Y,Huang J,Li H L,Li Y S, Shan L,Zhang W D.Target identification andvalidation of(+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate,an anti-inflammatory natural product[J].European Journal ofInflammation,2012,10 (3),297-309)。从红波罗花(I.delavayi)中分离得到的新型苯并呋喃酮二聚体类化合物incarviditone对白血病细胞HL-60和T淋巴细胞白血病6T-CEM细胞具有较好的细胞毒性,IC50值分别为14.8和22.2μg/mL(Chen Y Q,Shen Y H,Su Y Q,Kong L Y,Zhang W D.Incarviditone:A novel cytotoxic benzofuranone dimer from Incarvillea delavayiBureau et Franchet[J].Chemistry&Biodiversity,2009,6(5):779-783.)。同时还发现从红波罗花中分离得到的含有异喹啉并环烷新型骨架的生物碱类化合物Delavatine A能显著的抑制LPS诱导的 BV2细胞NO的产生从而发挥神经保护作用(Xie Q,Wu G Z,Yang N,Shen YH,Tang J,Zhang, W D.Delavatine A,an unusual isoquinoline alkaloidexerts anti-inflammation on LPS-induced proinflammatory cytokines productionby suppressing NF-kB activation in BV-2microglia[J]. Biochemical andBiophysical Research Communications,2018,144(5):1-7.)。前人的研究表明角蒿属植物中的化学成分在开发抗炎,镇痛及治疗中枢神经系统疾病药物上具有较大的应用前景。
脑卒中(Cerebral Stroke)又称“中风”和“脑血管意外”(cerebralvascularaccident,CVA),是一种脑组织损伤的急性脑血管疾病,疾病产生的原因是脑部血管阻塞导致血液不能流入大脑。脑卒中包括缺血性和出血性卒中,缺血性卒中的发病率高于出血性卒中,占脑卒中总数的60%~70%,具有发病率高、死亡率高、致残率高的特点,永久性或一过性脑功能障碍是脑卒中在临床上的主要症状。目前美国FDA批准的对缺血性脑卒中具有确切治疗效果的药物仅有重组组织型纤溶酶原激活剂,但也存在治疗窗窄,并发症频发等缺点,且临床研究己进一步证实钙离子拮抗剂、谷氨酸受体抑制剂等脑损伤保护药物对脑梗死患者无效。因此发现新作用途径的中枢神经系统疾病的药物对缺血性脑卒中的治疗非常迫切。
鉴于角蒿属植物化学成分结构独新颖,其中的生物碱类及环己乙醇类成分有较好的清除自由基及神经保护活性,且鸡肉参的化学成分研究较少,生物活性未见报道,所以亟需对鸡肉参进行系统化学成分研究,以期从中分离得到有较好的治疗中枢神经系统疾病的化合物。
发明内容
本发明的第一个目的是提供一种从角蒿属植物鸡肉参中分离得到活性显著的环己乙醇化合物rengyolone。
本发明的第二个目的是提供一种所述环己乙醇化合物rengyolone的制备方法。
本发明的第三个目的是提供一种所述环己乙醇化合物rengyolone在制备治疗中枢神经系统疾病的药物中的应用。
为了实现上述目的,本发明采用的技术方案如下:
本发明的第一方面提供了一种环己乙醇化合物rengyolone,化学结构如式I所示:
所述环己乙醇化合物rengyolone是从紫葳科角蒿属植物鸡肉参(Incarvilleamairei(Lévl.) Grierson)中分离制备得到。
本发明的第二方面提供了一种所述环己乙醇化合物rengyolone的制备方法,包括以下步骤:
将干燥的鸡肉参全草粉碎后用80%乙醇回流提取至少三次,合并提取液,浓缩得到醇提物,上述醇提物加水混悬后以乙酸乙酯进行液-液萃取至少三次,合并萃取液,浓缩后得到乙酸乙酯部位,分离并纯化得到所述环己乙醇化合物rengyolone。
所述分离并纯化包括以下步骤:
乙酸乙酯部位过大孔树脂(CPH20/P120,50-75μΜ,三菱化学)以甲醇水洗脱(比例为0、 30%、70%、100%),得到甲醇部位,经中压制备液相色谱,以ODS和Sephadex LH-20作为固定相,以MeOH-H2O 10%-100%按洗脱梯度的不同得到13个组分:E1-E310%-30%;E4-E730%-50%;E8-E1050%-80%;E11-E1280%-100%;E13100%;组分E11经Sephadex LH-20以MeOH-H2O 70%等度洗脱收集得到5个组分E11-1-E11-5;其中,组分E11-3经硅胶柱色谱层析以体积比为100:0-0:100的石油醚:乙酸乙酯梯度洗脱后得到所述环己乙醇化合物rengyolone。
本发明的第三方面提供了一种所述环己乙醇化合物rengyolone在制备治疗中枢神经系统疾病的药物中的应用。
所述中枢神经系统疾病选自神经退行性疾病、脑卒中、癫痫、脑外伤、休克、HIV痴呆、青光眼、抑郁、神经胶质瘤、多发性硬化症等;较优选的选自脑卒中,更优选为缺血性脑卒中。
所述治疗中枢神经系统疾病的药物是以本发明的环己乙醇化合物rengyolone作为单一活性成分的药物或与其他药用载体组合制成的药用组合物,用于制备治疗中枢神经系统疾病相关的药物;可通过口服、非肠道、脑内直接给药、鼻腔脑靶向给药、基因工程法、受体介导转运法给药或其它局部途径给药;给药剂型可以是片剂(分散片、含片、口崩片、缓释片)、颗粒剂、丸剂、胶囊剂、乳剂、溶液、悬浮液、注射剂、滴注剂、微囊微球制剂、栓剂、软膏剂、喷雾剂、靶向制剂、粉针剂或气雾剂等药学上可接受的剂型。
由于采用上述技术方案,本发明具有以下优点和有益效果:
本发明对来自于紫葳科角蒿属植物鸡肉参进行了系统研究,从中发现了一种环己乙醇化合物rengyolone。经体外抗炎活性筛选发现,该化合物对LPS诱导小鼠神经小胶质细胞BV2 细胞NO的产生有显著的抑制作用,其IC50值为11.52μM,且对BV2细胞毒性较小。以MCAO/R模型作为大鼠急性脑缺血再灌注损伤模型研究该化合物的体内神经保护作用,神经行为学评分结果显示rengyolone高剂量组对MCAO/R模型所致的急性缺血性脑卒中再灌注大鼠的神经功能缺失有明显的改善作用,同时大鼠脑梗死体积结果显示与模型组相比rengyolone能明显降低MCAO/R大鼠脑梗死体积,高剂量组效果好于阳性对照丁苯酞组。因此,本发明中的环己乙醇化合物rengyolone具有成为治疗中枢神经系统疾病药物的巨大潜力。
本发明提供的环己乙醇化合物rengyolone来自于紫葳科角蒿属植物鸡肉参,且该化合物在植物鸡肉参中大量存在。从化学角度而言,环己乙醇化合物rengyolone结构简单,可方便通过化学合成方法制备,故化合物的来源多样,且资源丰富。第二方面,该环己乙醇化合物 rengyolone能显著地抑制LPS诱导的小鼠小胶质细胞BV2产生NO,显示该化合物具有显著地抗神经炎症潜力,且对BV2细胞的毒性较低;同时采用MCAO/R大鼠急性脑缺血再灌注损伤模型研究该化合物的体内神经保护活性时还发现化合物能显著改善MCAO/R大鼠的神经行为学评分,并显著降低MCAO/R大鼠的脑梗死体积,具有较好的开发为治疗脑卒中药物的前景。
本发明的环己乙醇化合物rengyolone对脂多糖诱导的小胶质细胞NO的产生有显著的抑制作用,且对小胶质细胞毒性非常小;同时采用MCAO/R模型作为大鼠急性脑缺血再灌注损伤模型研究环己乙醇化合物rengyolone的体内神经保护作用发现,其能显著改善造模后大鼠的神经行为学评分及显著降低大鼠的脑梗死体积,故可用于制备治疗中枢神经系统疾病药物。
附图说明
图1为环己乙醇化合物rengyolone对LPS诱导BV2细胞NO产生抑制作用的示意图,浓度为6.25μM、12.5μM、25μM、50μM、100μM的环己乙醇化合物rengyolone作用24h 后对BV2细胞NO的产生的抑制作用,实验数据均以平均数±标准误表示,采用单因素方差分析(one-wayANOVA),Duncan test检验,P<0.05为差异显著(*,P<0.05;**,P<0.01vs. model)。
图2为环己乙醇化合物rengyolone对BV2细胞的细胞毒作用示意图,浓度为6.25μM、 12.5μM、25μM、50μM、100μM的环己乙醇化合物rengyolone作用24h后对BV2细胞产生的细胞毒作用,实验数据均以平均数±标准误表示,采用单因素方差分析(one-way ANOVA),Duncan test检验,P<0.05为差异显著(*,P<0.05;**,P<0.01,***,P<0.001vs. Vehicle)。
图3为不同剂量的环己乙醇化合物rengyolone作用于ICR小鼠以后,小鼠的体重变化示意图,化合物的剂量为10、20、30mg/kg,实验数据均以平均数±标准误表示,目的是筛选出环己乙醇化合物rengyolone作用于MCAO/R大鼠急性脑缺血再灌注损伤模型研究的适宜浓度范围。
图4为不同剂量的环己乙醇化合物rengyolone对MCAO/R脑缺血大鼠的保护作用示意图, A为神经行为学评分,B为大鼠脑体积梗死率统计图,C为各组脑梗死示意图。其中Sham表示假手术组,MCAO表示模型组,NBP表示阳性药丁苯酞组,给药剂量为100mg/kg,实验数据均以平均数±标准误表示,采用单因素方差分析(one-wayANOVA),Duncan test检验,P<0.05为差异显著(*,P<0.05;**,P<0.01,***,P<0.001vs.Model)。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1
环己乙醇化合物rengyolone的制备
1、样品来源
紫葳科角蒿属植物鸡肉参(Incarvillea mairei(Lévl.)Grierson)样品来自云南省大理市鹤庆县。
2、化学成分的提取分离与纯化方法
干燥的鸡肉参全草13公斤,粉碎后,以80%乙醇回流提取3次,每次1小时,合并的提取液减压真空浓缩回收溶剂,并得到5kg醇提物。上述醇提物加水混悬后加入乙酸乙酯(30L)进行液-液萃取5次,浓缩后得到700g乙酸乙酯部位。乙酸乙酯部位过大孔树脂(CPH20/P120,50-75μΜ,三菱化学)以甲醇水(比例为0、30%、70%、100%)洗脱,得到250 g30%甲醇部位,经中压制备液相色谱,以ODS和Sephadex LH-20作为固定相,以甲醇和水的不同比例作为洗脱剂,以MeOH-H2O 10%-100%按洗脱梯度的不同得到13个组分:E1-E310%-30%;E4-E730%-50%;E8-E1050%-80%;E11-E1280%-100%;E13100%;E1(17g),E2(10g),E3(15g),E4(15g),E5(20g),E6(15g),E7(10g),E8(5g),E9 (18g),E10(22g),E11(79g),E12(10g),E13(10g),组分E11经Sephadex LH-20 以MeOH-H2O 70%等度洗脱收集得到5个组分E11-1-E11-5;其中,组分E11-3主要含目标化合物rengyolone,组分E11-3继续经硅胶柱色谱层析以石油醚:乙酸乙酯(100:0-0:100)梯度洗脱后得到化合物rengyolone。
3、环己乙醇化合物rengyolone的结构确定
利用1H-NMR、13C-NMR、ESIMS得出化合物核磁和分子量信息,经碳谱和氢谱数据与已知化合物比对,确定化合物的结构如式I所示:
环己乙醇化合物rengyolone:无色油状物;ESI-MS显示准分子离子峰m/z 155.107([M+ H]+,calcd.155.016)。根据1H-NMR和13C-NMR确定分子式为C8H10O3。1H-NMR(Methanol-d4,500MHz)谱中可以观察到:δH 3.99(td,1H,J=5.84,5.81Hz,H-2a),δH 3.83(dd,1H,J=15.46,7.90Hz,H-2b),δH 2.27(m,1H,H-3a),δH 2.19(m,1H,H-3b),δH 6.79(dd,1H,J=10.17, 1.68Hz,H-5),δH 5.95(d,1H,J=10.17Hz,H-6),δH 2.76(dd,1H,J=16.76,4.08Hz,H-8a),δH 2.56(dd,1H,J=16.76,4.94Hz,H-8b),δH 4.14(td,1H,4.55,1.71Hz,H-9);13C-NMR(Methanol- d4,125MHz)谱中:δC 67.1(t,C-2),δC 40.3(t,C-3),δC 75.5(s,C-4),δC150.7(d,C-5),δC 128.8(d, C-6),δC 199.1(s,C-7),δC 40.5(t,C-8),δC 82.3(d,C-9).
实施例2
细胞毒活性测试
1、实验材料
小鼠小胶质细胞(BV2),中科院细胞所提供;DMEM培养基、胎牛血清、胰蛋白酶、PBS缓冲液、Cell Counting Kit-8(CCK-8试剂盒)、Griess试剂盒、CO2细胞培养箱、96孔板、低温超速离心机、倒置相差显微镜、微量移液器。
环己乙醇化合物rengyolone是由实施例1制备得到。
2、实验方法
(1)BV2细胞体外培养
将复苏后的小鼠小胶质细胞(BV2)接种于培养皿,每皿加入10ml添加了10%(v/v)胎牛血清、100U/mL青霉素以及100U/mL链霉素的DMEM培养基,在温度37℃、5%CO2条件下进行培养。待细胞生长至80%-90%汇合时进行细胞传代。倾倒去除培养器皿内的培养基,用PBS洗涤细胞1次,弃去PBS,根据培养面积加入适量0.1%胰酶溶液消化,37℃放置1min,并在倒置相差显微镜下观察,待到绝大部分细胞变圆并悬浮后弃去胰酶并加入培养基终止消化,吹吸几次以离散成团的细胞,按1:2比例进行细胞传代,传代后的细胞24h 后换液一次。
(2)细胞毒活性实验
CCK-8法检测细胞毒活性实验
收集对数期生长的小鼠小胶质细胞(BV2),细胞经计数后接种于96孔培养板,8000细胞/ 孔,每孔加入90μL DMEM培养液(含血清、100U/mL青霉素以及100U/mL链霉素),37℃,5%CO2条件下培养,待细胞贴壁后加入10μL给药浓度为100μM、50μM、25μM、12.5μM、 6.25μM的待测环己乙醇化合物rengyolone继续培养24h。培养终止后,每孔加入CCK-8试剂 10μL,37℃恒温孵育30-45min后在酶标仪上测定波长为450nm处的吸光度。
(3)LPS刺激BV2细胞释放实验
将处于生长对数期的小胶质细胞(BV2)铺于96孔细胞板中,细胞数目为4×104/100μL。继续培养24h后将2μg/mL LPS(DMEM稀释)加入浓度为4×104/100μL的细胞悬液中,同时加入待测化合物和阳性药氨基胍。待测环己乙醇化合物rengyolone和阳性药氨基胍的给药浓度为100μM、50μM、25μM、12.5μM、6.25μM,同时加完待测化合物和LPS后继续恒温培养24h,吸取100ml细胞培养基上清液以Griess法通过测定OD值来反映细胞培养基中亚硝酸盐的含量间接换算细胞中产生NO的浓度。实验步骤为吸取100μL细胞培养基上清液于另一96孔细胞板,先加入50μL Griess试剂1,再加入50μL Griess试剂2,在570nm 波长处测吸光度,同时以NaNO3标准溶液绘制标准曲线,计算细胞产生NO的浓度。
3.实验结果
结果如图1和图2所示,图1为环己乙醇化合物rengyolone对LPS诱导BV2细胞NO 产生抑制作用的示意图,浓度为6.25μM、12.5μM、25μM、50μM、100μM的环己乙醇化合物rengyolone作用24h后对BV2细胞NO的产生的抑制作用,实验数据均以平均数±标准误表示,采用单因素方差分析(one-wayANOVA),Duncan test检验,P<0.05为差异显著 (*,P<0.05;**,P<0.01vs.model)。图2为环己乙醇化合物rengyolone对BV2细胞的细胞毒作用示意图,浓度为6.25μM、12.5μM、25μM、50μM、100μM的环己乙醇化合物 rengyolone作用24h后对BV2细胞产生的细胞毒作用,实验数据均以平均数±标准误表示,采用单因素方差分析(one-wayANOVA),Duncan test检验,P<0.05为差异显著(*,P<0.05; **,P<0.01,***,P<0.001vs.Vehicle)。
从图1中可以看出,待测化合物环己乙醇化合物rengyolone在100μM浓度时对BV2细胞有毒性;但是在50μM内对BV2细胞几乎没有毒性;从图2中可以看出,环己乙醇化合物rengyolone在浓度6.25μM-50μM之间,均可以显著降低LPS诱导的BV2细胞NO的释放,与阳性药氨基胍相比,IC50值为11.52±0.48μM,效果显著优于阳性药氨基胍(IC50值为26.13±0.24μM),结果说明环己乙醇化合物rengyolone有较强的体外抗神经炎活性。
实施例3
环己乙醇化合物rengyolone作用于SD大鼠的适宜浓度范围
1、仪器与试剂:
ICR小鼠20g±2g,灌胃针,环己乙醇化合物rengyolone(纯度>98%),生理盐水。
2、实验方法:
(1)药物配制
环己乙醇化合物rengyolone用生理盐水配置成10mg/kg、20mg/kg、30mg/kg的工作液。
(2)实验分组和给药方案
20g±2g的ICR小鼠32只随机分为四组,每组8只,分别为对照组、10mg/kg组、20mg/kg组、30mg/kg组,小鼠适应性喂养一周,期间正常喂食,饮水。一周后开始给药,每天灌胃方式给药一次,给药前后记录小鼠的体重及观察小鼠的精神状态,给药一周。
3、实验结果:
结果如图3所示,图3为不同剂量的环己乙醇化合物rengyolone作用于ICR小鼠以后,小鼠的体重变化示意图,化合物的剂量为10、20、30mg/kg,实验数据均以平均数±标准误表示,目的是筛选出环己乙醇化合物rengyolone作用于MCAO/R大鼠急性脑缺血再灌注损伤模型研究的适宜浓度范围。从图3中可以看出,实验7天后,与空白组相比,给药浓度为10、20、30mg/kg的环己乙醇化合物rengyolone对小鼠的精神状态和体重变化无显著性差异,说明在这三个剂量下该化合物对小鼠没有明显的毒性,通过公式(V1=0.7102v2)换算成大鼠的适宜给药浓度,筛选出了MCAO/R SD大鼠急性脑缺血再灌注损伤的研究的适宜给药浓度,即0-20mg/kg的环己乙醇化合物rengyolone可作为SD大鼠的给药剂量,因此,选取了10 mg/kg、20mg/kg两个剂量进行后面的研究。
实施例4
环己乙醇化合物rengyolone对MCAO/R脑缺血再灌注大鼠的保护作用
1、仪器与试剂:
MCAO线栓(230-260g),手术器材(持针器、弯剪、直剪、眼科镊、缝合针、干棉球、酒精棉球、10%水合氯醛、注射器、碘伏、棉签、记号笔),自制拉钩(皮筋+曲别针+大头针)。 SD雄性大鼠250±5g,环己乙醇化合物rengyolone(纯度>98%),丁苯酞(阳性药),生理盐水,注射用植物油。
2、实验方法:
(1)药物配制
环己乙醇化合物rengyolone用生理盐水配置成10mg/kg、20mg/kg的工作液,丁苯酞按照药物说明书,加注射用植物油配制成100mg/mL的工作液。
(2)实验分组和给药方案
250±5g SD雄性大鼠48只随机分为6组,每组8只,分别为假手术组、对照组、模型组、丁苯酞组(阳性药,浓度为100mg/kg)、10mg/kg rengyolone组、20mg/kg rengyolone组。大鼠适应性喂养一周,期间正常喂食,饮水,于造模前12h禁食不禁水,造模前称量体重为250±5g。
(3)大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型
用10%水合氯醛(0.3mL/100g)腹腔注射麻醉。于颈前正中位置剃毛、消毒,切开2cm左右切口,钝性分离右侧颈总动脉(common carotid arteries,CCA)、颈外动脉(external carotidartery,ECA)、颈内动脉(internal carotid artery,ICA),用5-0手术线分别结扎ECA 的远心端,CCA的近心端,并于CCA的近心端与ECA和ICA交叉处之间进行活结结扎;以与大鼠体重匹配的栓线自CCA结扎处经CCA与ICA分叉部插入ICA颅内段,使栓线上标记过 ECA和ICA交叉处,到达大脑中动脉环,扎紧活结以防止出血和线栓移动。逐层缝合颈部皮肤。阻断2h后,拔出线栓进行再灌注22h。假手术组仅将CCA、ECA、ICA游离出来,不做结扎、插线处理,其他操作同模型组。
(4)神经功能缺失体征评分:
采用Longa法双盲评估每只大鼠的神经功能:
0分:无神经功能损伤;
1分:不能完全伸展对侧前肢;
2分:不能伸展对侧前肢;
3分:轻度向对侧转圈;
4分:严重向对侧转圈;
5分:对侧肢体偏瘫;
分值越高,说明动物行为障碍越严重。
(5)四氮唑(2,3,5-triphenyltetrazoliumiides,TTC)法染色:
TTC(2,3,5-氯化三苯基四氮唑)是一种脂溶性光敏感的复合物。1894年,首次合成后用来检测种子的生存能力;从1958年开始,可以用来染色后检测哺乳动物组织的缺血性梗塞,是一种呼吸链中吡啶核苷结构酶系统的质子受体,与正常组织中的脱氢酶反应而呈现红色,而缺血的组织内脱氢酶的活性下降,不能进行反应,故不会产生变化而呈现白色。
3、实验结果:
结果如图4所示,图4为不同剂量的环己乙醇化合物rengyolone对MCAO/R脑缺血大鼠的保护作用示意图,A为神经行为学评分,B为大鼠脑体积梗死率统计图,C为各组TTC染色脑组织梗死体积示意图。其中Sham表示假手术组,MCAO表示模型组,NBP表示阳性药丁苯酞组,给药剂量为100mg/kg,实验数据均以平均数±标准误表示,采用单因素方差分析 (one-wayANOVA),Duncan test检验,P<0.05为差异显著(*,P<0.05;**,P<0.01,***,P <0.001vs.Model)。
从图中可以看出,A中可以看出与模型组相比,阳性药丁苯酞(浓度为100mg/kg)、环己乙醇化合物rengyolone的给药剂量为10mg/kg、20mg/kg组均可改善MCAO/R大鼠的神经功能缺失体征评分,且化合物的给药剂量为20mg/kg组改善MCAO/R大鼠神经行为学评分的效果与剂量为100mg/kg的阳性药丁苯酞的效果相当;B和C中可以得出,相对于假手术组(Sham),模型组(MCAO)大鼠缺血再灌注后脑缺血体积为30.80±1.98%(p<0.01vs.Sham),阳性对照NBP组、给药剂量为10mg/kg和20mg/kg的环己乙醇化合物rengyolone均可显著降低造模后大鼠脑梗死体积,大鼠脑梗死体积分别减少至24.74±0.34%(p≤0.01vs MCAO)、20.96±4.97%(p≤0.01vs MCAO)、22.28±2.76%(p≤0.01vs MCAO)。结果表明, 10mg/kg和20mg/kg的环己乙醇化合物rengyolone均可显著降低造模后大鼠脑梗死体积,具有较好的神经保护活性,且给药剂量为10mg/kg和20mg/kg的环己乙醇化合物 rengyolone降低造模后大鼠脑梗死体积的效果与100mg/kg的阳性药丁苯酞效果相当甚至略优于阳性药。因此,本发明的环己乙醇化合物rengyolone可用于制备治疗缺血性脑卒中的药物。
以上所述仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专利的技术人员在不脱离本发明技术方案范围内,当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明方案的范围内。
Claims (6)
2.根据权利要求1所述的环己乙醇化合物rengyolone,其特征在于,所述环己乙醇化合物rengyolone是从紫葳科角蒿属植物鸡肉参中分离制备得到。
3.一种权利要求1或2所述的环己乙醇化合物rengyolone的制备方法,其特征在于,包括以下步骤:
将干燥的鸡肉参全草粉碎后用80%乙醇回流提取至少三次,合并提取液,浓缩得到醇提物,上述醇提物加水混悬后以乙酸乙酯进行液-液萃取至少三次,合并萃取液,浓缩后得到乙酸乙酯部位,分离并纯化得到所述环己乙醇化合物rengyolone;
所述分离并纯化包括以下步骤:
乙酸乙酯部位过大孔树脂以甲醇水洗脱,得到甲醇部位,经中压制备液相色谱,以ODS和Sephadex LH-20作为固定相,以MeOH-H2O 10%-100%按洗脱梯度的不同得到13个组分:E1-E310%-30%;E4-E730%-50%;E8-E1050%-80%;E11-E1280%-100%;E13100%;组分E11经Sephadex LH-20以MeOH-H2O 70%等度洗脱收集得到5个组分E11-1-E11-5;其中,组分E11-3经硅胶柱色谱层析以体积比为100:0-0:100的石油醚:乙酸乙酯梯度洗脱后得到所述环己乙醇化合物rengyolone。
4.一种权利要求1或2所述的环己乙醇化合物rengyolone在制备治疗中枢神经系统疾病的药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述中枢神经系统疾病选自神经退行性疾病、脑卒中、癫痫、脑外伤、休克、HIV痴呆、青光眼、抑郁、神经胶质瘤、多发性硬化症。
6.根据权利要求5所述的应用,其特征在于,所述中枢神经系统疾病选自缺血性脑卒中。
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