CN111773232B - 新黄酮类化合物Hip A的用途 - Google Patents
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Abstract
本发明公开了新黄酮类化合物Hip A的用途,对沙棘籽粕浸膏通过MCI凝胶柱洗脱,再依次经C18色谱柱、二醇基色谱柱、C18色谱柱半制备分离后,得到本发明新黄酮类化合物Hip A,并对化合物Hip A进行进行活性研究,验证了化合物Hip A能够降低心肌细胞中乳酸脱氢酶和NO的含量,并通过降低细胞中Bcl‑2、Bax的蛋白表达水平,从而延缓心肌细胞凋亡,可用于制备预防或治疗心肌损伤相关疾病的药物。本发明还提供了Hip A在制备具有神经保护和/或神经修复作用的产品中的应用,验证了化合物Hip A能够改善神经突触萎缩、降低tau蛋白过度磷酸化,可用于制备预防或治疗神经退行性疾病的药物。
Description
技术领域
本发明涉及黄酮类化合物的医药用途领域,特别是涉及新黄酮类化合物Hip A 的用途。
背景技术
沙棘(拉丁学名:Hippophae rhamnoides Linn.),是一种胡颓子科、沙棘属落叶性灌木,其特性是耐旱、抗风沙,可以在盐碱化土地上生存,因此被广泛用于水土保持。中国西北部大量种植沙棘,用于沙漠绿化。
沙棘营养丰富,多种维生素、黄酮类化合物、三萜类化合物、油和脂肪酸、酚类、挥发油类、微量元素、磷脂类、5-羟色胺等活性物质和人体所需的各种氨基酸和蛋白质。
沙棘种籽常用来榨油制成沙棘油,沙棘油中含有206种对人体有益的活性物质,其中有46种生物活性物质,含有大量的维生素E、维生素A、黄酮等。榨油后会产生大量的沙棘籽粕,往往会被当作废料丢弃,或生产成干粉当作饲料,价格非常低廉。倘若能更多的开发出沙棘籽粕的利用价值,将具有极大的经济意义。
另外,沙棘中含有的丰富活性物质,仍有许多未被分离、鉴定,若能对沙棘中新的化学成分及其药理作用进行更加深入、细微的研究与活性机制探讨,则有望开发出更多可治疗疾病且安全有效的天然植物来源药物。
发明内容
本发明提供从沙棘籽粕中提取得到新的黄酮类化合物Hip A在制备预防和/或治疗组织损伤或细胞损伤的产品中的用途,所述化合物具有降低心肌细胞中乳酸脱氢酶和/或NO的含量、延缓心肌细胞凋亡、改善神经突触萎缩、降低tau蛋白过度磷酸化的功效。
本发明提供化合物XVII以及其药学上可接受的盐、水合物或溶剂合物,在制备预防和/或治疗组织损伤或细胞损伤的产品中的应用,所述组织选自心肌组织和/ 或神经组织,所述细胞选自心肌细胞和/或神经细胞,所述化合物XVII(本发明中亦称为Hip A)结构如下:
进一步地,所述产品为具有心肌保护作用的产品。
进一步地,所述产品为降低心肌细胞中乳酸脱氢酶和/或NO的含量的产品。
进一步地,所述产品为延缓心肌细胞凋亡的产品。
进一步地,所述产品为Bcl-2抑制剂和/或Bax抑制剂。
所述Bcl-2抑制剂、Bax抑制剂为降低细胞中Bcl-2、Bax的蛋白表达水平的药物。
进一步地,所述产品为预防和/或治疗心律失常、室颤、心肌缺血、心肌梗死中至少一种病症的产品。
进一步地,所述产品为具有神经保护和/或神经修复作用的产品。
进一步地,所述产品为改善神经突触萎缩的产品。
进一步地,所述产品为降低tau蛋白过度磷酸化的产品。
进一步地,所述产品为预防和/或治疗神经退行性疾病的产品;
进一步地,所述神经退行性疾病的选自脑缺血(CI)、脑损伤(BI)、癫痫、阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、不同类型脊髓小脑共济失调(SCA)、Pick病中的一种或几种;更进一步地,所述神经退行性疾病选自阿尔茨海默病。
本发明中所述产品包括但不限于药物、保健品、食品等。
在本发明中,甲醇水溶液、乙醇水溶液前的百分数均指体积分数,如“40%甲醇水溶液”是指甲醇的体积分数为40%的甲醇水溶液,其余情况同理。
本发明的有益效果是:
(1)本发明对从沙棘籽粕中提取了新的黄酮类化合物Hip A,并对其进行活性研究,结果表明,本发明化合物Hip A能够降低心肌细胞中乳酸脱氢酶和NO的含量,并通过降低细胞中Bcl-2、Bax的蛋白表达水平,从而延缓心肌细胞凋亡,为研究用于心肌损伤相关疾病如心律失常、室颤、心肌缺血、心肌梗死等提供有力方向。
(2)本发明化合物Hip A能够改善神经突触萎缩、降低tau蛋白过度磷酸化,具有神经保护和神经修复作用,可用于制备预防和/或治疗神经退行性疾病的药物。
(3)本发明更加全面挖掘了沙棘的药用价值、拓展了其临床应用,为开发治疗心肌损伤相关疾病的潜在植物来源的药物提供更多的参考依据,同时实现了沙棘籽粕的废物利用,变废为宝。
附图说明
图1是沙棘籽粕各组分样品分析色谱图;
图2是沙棘籽粕SH5组分样品一维制备图;
图3是SH5中的各组分样品二维分析图;
图4是沙棘籽粕中峰1~19化合物的纯度分析;
图5是化合物Hip A的1H NMR谱图;
图6是化合物Hip A的13C NMR谱图;
图7是化合物Hip A的HSQC图;
图8是化合物Hip A的HMBC图;
图9是化合物Hip A的HHCOSY图;
图10是化合物Hip A的NOESY图;
图11是DoX诱导H9c2心肌细胞形态显微图(100×,n=3);
图12是DoX对H9c2心肌细胞活力的影响(n=3);
图13是DoX对H9c2心肌细胞培养上清液NO含量水平(n=3);
图14是DoX对H9c2心肌细胞培养上清液LDH含量水平(n=3);
图15是化合物Hip A对H9c2心肌细胞活力的影响(n=3);
图16是化合物Hip A对DoX诱导H9c2心肌细胞损伤LDH水平(n=3);
图17是化合物Hip A对DoX诱导H9c2心肌细胞损伤NO水平(n=3);
图18是化合物Hip A对DoX诱导H9c2心肌细胞凋亡蛋白的影响(n=3);
图19是5μM ATRA和40nM OA对SH-SY5Y细胞突触长度的影响;
图20是化合物Hip A对SH-SY5Y细胞活力的影响;
图21是化合物Hip A对AD细胞模型形态学的影响;
图22是沙棘新黄酮类化合物对SH-SY5Y细胞中tau蛋白磷酸化的影响。
具体实施方式
下面通过具体实施例和具体实验对本发明黄酮类新化合物的制备及应用做进一步说明,并对所述黄酮类新化合物对心肌细胞损伤、神经损伤的保护作用进行验证和说明。
本发明实施例中,所用沙棘(Hippophae rhamnoides Linn.)籽粕材料为青海省西宁市大通县产沙棘种籽利用超临界CO2提取籽油后的籽粕。
实施例1黄酮类新化合物的制备
1、组分的制备
(1)将20.00kg沙棘(Hippophae rhamnoides Linn.)籽粕采用70%乙醇溶液,料液比为1:20kg/L,每次提取2h,温度为60℃,提取3次,合并3次提取液后减压浓缩,得到3.50kg的沙棘籽粕醇提物。
(2)将醇提物冷冻干燥,粉碎过120目筛,加少量甲醇,与200~300目正相硅胶1:1混匀拌样,挥发至无醇味。在玻璃层析柱中加入7kg 200~300目正相硅胶,用二氯甲烷:甲醇:水=7:3:0.5做流动相进行洗脱,洗脱体积为20个柱体积,对洗脱液再减压浓缩,得到浸膏。
(3)将浸膏溶入13L的50%甲醇溶液中,取2L减压浓缩至0.5L,利用MCI 凝胶柱进行洗脱,依次用20%、40%、60%、80%和100%甲醇溶液进行洗脱,每个浓度各洗脱5个柱体积,重复取样2L减压浓缩3次并洗脱,得到20%甲醇洗脱部位质量为59.12g,40%甲醇洗脱部位质量为71.86g,60%甲醇溶液洗脱部位质量为 22.35g,80%甲醇溶液洗脱部位质量为20.81g,100%甲醇溶液洗脱部位质量为 35.54g。
(4)将22.35g的60%甲醇溶液洗脱部位溶解于110mL的60%的甲醇溶液中,进行HPLC上分析,色谱条件为:色谱柱Dubhe C18(4.6×250mm,5μm),进样量 20μL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,采用如下程序进行梯度洗脱: 0min,90%流动相A-10%流动相B;60min,80%流动相A-20%流动相B,流速为 1mL/min,波长为254nm。采用汉邦N7010半制备型液相色谱仪对样品进行制备,制备条件为:色谱柱Dubhe C18(20×250mm,5μm),进样量1.5mL,流动相A: 0.1%甲酸水溶液,流动相B:乙腈,采用如下程序进行梯度洗脱:0min,90%流动相A-10%流动相B;60min,80%流动相A-20%流动相B,流速为19mL/min,波长 254nm,最后得到6个组分,分别命名为SH1~SH6,SH1组分为0~14min,SH2组分为14~23min,SH3组分为23~29min,SH4组分为29~37min,SH5组分为37~46min,SH6组分为46~60min,如图1,其中,组分SH5的质量为3.34g。
(5)将3.34g的SH5组份溶解于8mL的二甲基亚砜(DMSO)中,采用 Agilent1260进行HPLC分析,色谱条件为:色谱柱Kromasil 60-5-Diol(4.6×250mm, 5μm),进样量0.1μL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,采用如下程序进行梯度洗脱:0min,100%流动相B;60min,20%流动相A-80%流动相B,流速为1mL/min,波长为254nm。采用汉邦N7010半制备型液相色谱仪对样品进行制备,制备条件为:Kromasil 60-5-Diol(21×250mm,5μm),进样量0.4mL,流动相 A:0.1%甲酸水溶液,流动相B:乙腈,采用如下程序进行梯度洗脱:0min,100%流动相B;60min,20%流动相A-80%流动相B,流速为21mL/min,波长为254nm,最后得到9个亚组分(SH5-1~SH5-9),SH5-1~SH5-9的9个亚组分的保留时间分别为0~6min,6~8min,8~22min,22~27min,27~37min,37~42min,42~55min, 55~57min,57~60min,如图2。
2、单体化合物的制备
(1)SH5-3组分的分离
将0.107g的SH5-3组分溶解于5mL甲醇中,加DMSO助溶,采用Agilent1260 进行HPLC分析,色谱条件为:色谱柱Kromasil 100-5-C18(4.6×250mm,5μm)分析柱,进样量0.1μL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,时间60min,流速为1mL/min,波长为254nm。用汉邦N7010半制备型液相色谱仪对样品进行制备,制备条件为:色谱柱Kromasil 100-5-C18(21×250mm,5μm)制备柱,进样量0.2mL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,流速为21mL/min,波长为254nm。得到SH5-3组分下的3个峰为峰2、峰3、峰4,峰4的保留时间为22.707min。
(2)SH5-8组分的分离
将0.158g的SH5-8组分溶解于2mL甲醇中,加400μLDMSO助溶,采用 Agilent1260进行HPLC分析,色谱条件为:Kromasil 100-5-C18(4.6×250mm,5μm),进样量0.5μL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,时间60min,流速为1mL/min,波长为254nm。用汉邦N7010半制备型液相色谱仪进行制备,制备条件:Kromasil 100-5-C18(21×250mm,5μm),进样量0.3mL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,时间60min,流速为 21mL/min,波长为254nm。得到SH5-8下的1个峰为峰17,保留时间为26.641min。
(3)SH5-9组分的分离
将0.153g的SH5-9组分溶解于2mL甲醇中,加400μLDMSO助溶,采用 Agilent1260进行HPLC分析,色谱条件为:Kromasil 100-5-C18(4.6×250mm,5μm),进样量0.5μL,流动相A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,时间60min,流速为1mL/min,波长为254nm。用汉邦N7010半制备型液相色谱仪制备,制备条件为:Kromasil 100-5-C18(21×250mm,5μm),进样量0.2mL,流动相 A:0.1%甲酸水溶液,流动相B:乙腈,洗脱条件15%B,时间60min,流速为21mL/min,波长为254nm,得到SH5-9下的峰18和峰19,保留时间分别为53.891min和 57.300min。
峰2、3、4、17、18、19的制备分离图谱见图3。
将峰2、3、4、17、18、19得到的化合物进行纯度检测,每个化合物的纯度都大于92%,纯度检测图见图4。
2、单体化合物的结构鉴定
将本实验分离得到的化合物通过UV、HR-ESI-MS、ESI-MS、1H-NMR、13C-NMR、 HMBC、HSQC、HHCOSY、TOCSY等有机波谱分析方法进行结构解析,结果如下:
化合物IV(峰4):淡黄色粉末,其质量为4mg,新化合物,命名为(6S,9R)-9-Hydrox-4,7-megastigmadien-3-one 9-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (简称Hyd),化学式为C24H38O11,通过高分辨质谱(HR-ESI-MS)分析,其质核比 (M/Z)503.2414[M+H]+。1H NMR(600MHz,CD3OD):δ:5.88(1H,br,s,H -4),5.75(1H,dd,J=15.4,6.5,H-8),5.63(1H,ddd,J=15.4,9.4,0.7,H-7), 4.98(1H,d,J=2.5Hz,H-1’),4.63(1H,m,H-9),4.31(1H,d,J=7.8Hz,H-1’),3.95(1H,d,J=9.7Hz,H-4’),3.93(1H,dd,J=11.3,1.8,H-6’),3.87(1H,d, J=2.5Hz,H-2’),3.74(1H,d,J=9.7Hz,H-4’),3.57(1H,dd,J=11.3,5.9,H-6’), 3.56(1H,d,J=11.5Hz,H-5’),3.54(1H,d,J=11.5Hz,H-5’),3.33(1H,m,H -5’),3.32(1H,m,H-3’),3.27(1H,t,J=9.2,H-4’),3.16(1H,dd,J=9.1,7.8, H-2’),2.68(1H,d,J=9.4,H-6),2.41(1H,d,J=16.7,H-2),2.06(1H,d,J =16.7,H-2),1.96(3H,d,J=1.1Hz,H-13),1.29(3H,d,J=6.4,H-10),1.02 (3H,s,H-11),0.96(3H,s,H-12);13C NMR(600MHz,CD3OD):δ:202. 1(C-3),166.1(C-5),138.3(C-8),129.1(C-7),126.1(C-4),111.0(C-1’),1 02.6(C-1’),80.5(C-3’),78.0(C-3’,C-2’),77.2(C-9),77.0(C-5’),75.2(C-2’),75.0(C-4’),71.6(C-4’),68.6(C-6’),65.5(C-5’),56.8(C-6),48.6(C-2),37. 2(C-1),28.1(C-11),27.3(C-12),24.0(C-13),21.2(C-10)。
化合物XVII(峰17):黄色粉末,质量为50mg,新化合物,命名为Hippophandine A(简称Hip A),化学式为C50H60O29。通过高分辨质谱(HR-ESI-MS)分析,其质核比(M/Z)为1123.3146[M+H]+,与C50H61O29的计算值1124.3220相符。1H NMR(600 MHz,CD3OD):δ:8.03(2H,d,J=8.8Hz,H-2′,H-6′),7.25(1H,d,J=15.9 Hz,H-7″″″),6.89(2H,d,J=8.8Hz,H-3′,H-5′),6.42(2H,s,H-2″″″, H-6″″″),6.34(1H,d,J=2.0Hz,H-8),6.30(1H,d,J=2.0Hz,H-6),5.99 (1H,d,J=15.9Hz,H-8″″″),5.51(1H,br s,H-1″),5.13(1H,d,J=7.5Hz,H-1″″),4.66(1H,d,J=7.7Hz,H-1″″′),4.63(1H,d,J=7.8Hz,H-1″′), 4.47(1H,dd,J=11.7,7.3Hz,H-6″″′),4.38(1H,dd,J=11.7,2.0Hz, H-6″″′),4.09(1H,br s,H-2″),4.05(1H,dd,J=9.4,3.4Hz,H-3″),3.85 (1H,dd,J=11.9,1.8Hz,H-6″′),3.74(6H,s,H-OCH3),3.72(1H,t,J=9.4 Hz,H-4″),3.68(1H,m,H-6″′),3.67(1H,m,H-5″″′),3.63(1H,m,H-2″″), 3.62(1H,m,H-5″),3.60(1H,m,H-6″″),3.51(1H,t,J=9.1Hz,H-3″″), 3.47(1H,t,J=9.1Hz,H-3″″′),3.45(1H,m,H-6″″),3.40(1H,dd,J=9.1, 7.7Hz,H-2″″′),3.38(1H,t,J=9.2Hz,H-3″′),3.35(1H,t,J=9.2Hz,H-4″′), 3.32(1H,t,J=9.1Hz,H-4″″),3.30(1H,m,H-4″″′),3.29(1H,m,H-5″′), 3.21(1H,dd,J=9.2,7.8Hz,H-2″′),3.07(1H,m,H-5″″),1.31(3H,d, J=6.1Hz,H-6″);13C NMR(600MHz,CD3OD):δ:179.9(C-4),168.7(C-9″″″), 163.2(C-7),162.6(C-5),161.7(C-4′),158.7(C-2),157.5(C-9),149.0(C-3″″″,C-5″″″),146.7(C-7″″″),139.1(C-4″″″),135.3(C-3),132.7(C-2′,C-6′),126.2 (C-1″″″),122.5(C-1′),116.3(C-3′,C-5′),115.3(C-8″″″),107.2(C-10),106.8 (C-1″″′),106.1(C-2″″″,C-6″″″),105.6(C-1″′),100.9(C-1″″),100.2(C-6), 99.7(C-1″),95.3(C-8),85.5(C-2″″),82.8(C-4″),78.3(C-5″″),78.2(C-3″′),78.1(C-5″′),77.8(C-3″″′),77.6(C-3″″),76.4(C-2″″′),76.1(C-2″′),75.6(C5″″′), 72.2(C-4″′),72.1(C-3″),71.6(C-4″″′),71.5(C-2″),71.1(C-4″″),69.8(C-5″), 64.8(C-6″″′),62.8(C-6″′),62.3(C-6″″),56.5(C-OCH3),18.3(C-6″)。
化合物XVIII(峰18):黄色粉末,质量为41mg,新化合物,命名为Hippophandine B(简称Hip B),化学式为C51H66O29。通过高分辨质谱(HR-ESI-MS)分析,其质核比(M/Z)为1141.3614[M+H]+,与C51H67O29的计算值1142.3690相符。1H NMR(600 MHz,CD3OD):δ:8.09(2H,d,J=8.8Hz,H-2′,H-6′),7.02(1H,d,J=15.7 Hz,H-3″″′),6.88(2H,d,J=8.8Hz,H-3′,H-5′),6.71(1H,d,J=1.6Hz, H-8),6.47(1H,d,J=1.6Hz,H-6),5.63(1H,br t,J=7.0Hz,H-5″″′),5.59 (1H,br s,H-1″),5.50(1H,d,J=15.7Hz,H-2″″′),5.15(1H,d,J=7.4 Hz,H-1″′),4.70(1H,d,J=7.6Hz,H-1″″),4.36(1H,d,J=7.8Hz,H-1″″″), 4.33-4.39(2H,m,H-6″″),4.05(1H,br s,H-2″),3.97(1H,m,H-8″″′), 3.85(2H,m,H-3″,H-6″″″),3.69(1H,m,H-6″″″),3.67(1H,m,H-2″′), 3.66(1H,m,H-6″′),3.60(1H,m,H-5″),3.59(1H,m,H-5″″),3.52(1H, t,J=9.1Hz,H-3″′),3.49(1H,m,H-6″′),3.48(1H,m,H-4″),3.44(1H,t,J=9.0Hz,H-3″″),3.37(1H,m,H-2″″),3.36(1H,m,H-3″″″),3.34(3H, m,H-4″″″,H-4″″,H-4″′),3.26(1H,m,H-5″″″),3.21(1H,dd,J=9.0,7.8 Hz,H-2″″″),3.11(1H,m,H-5″′),2.72(1H,m,H-9″″′),2.16-2.25(2H, m,H-6″″′),1.52-1.59(2H,m,H-7″″′),1.51(3H,brs,H-12″″′),1.25(3H, d,J=6.1Hz,H-6″),1.12(3H,d,J=6.1Hz,H-11″″′);13C NMR(600MHz,CD3OD):δ:180.1(C-4),169.0(C-1″″′),163.4(C-7),163.0(C-5),161.8(C-4′),159.1(C-2),158.0(C-9),151.2(C-3″″′),143.8(C-5″″′),135.4(C-3),134.0 (C-4″″′),132.8(C-2′,C-6′),122.3(C-1′),116.4(C-3′,C-5′),115.5(C-2″″′), 107.5(C-10),106.2(C-1″″),103.6(C-1″″″),101.0(C-1″′),100.6(C-6),99.7 (C-1″),95.6(C-8),84.8(C-2″′),81.3(C-8″″′),78.3(C-5″′),78.0(C-3″″″), 77.9(C-5″″″),77.8(C-3″″),77.7(C-3″′),76.2(C-2″″),75.7(C-5″″),75.3(C-2″″″), 73.6(C-4″),72.0(C-3″),71.8(C-4″″″),71.7(C-2″,C-4″″),71.3(C-5″),70.9 (C-4″′),64.6(C-6″″),62.9(C-6″″″),62.4(C-6″′),45.3(C-9″″′),31.7(C-7″″′), 25.6(C-6″″′),18.2(C-6″),12.8(C-11″″′),12.1(C-12″″′)。
化合物XIX(峰19):棕黄色粉末,质量为15mg,新化合物,命名为Hippophandine C(简称Hip C),化学式为C51H66O29。通过高分辨质谱(HR-ESI-MS)分析,其质核比(M/Z)为1141.3614[M+H]+,与C51H67O29的计算值1142.3690相符。1H NMR(600 MHz,CD3OD):δ:8.06(2H,d,J=8.8Hz,H-2′,H-6′),6.96(1H,br d,J=11.5 Hz,H-3″″′),6.87(2H,d,J=8.8Hz,H-3′,H-5′),6.74(1H,d,J=1.8Hz, H-8),6.47(1H,d,J=1.8Hz,H-6),6.10(1H,dd,J=14.8,11.5Hz,H-4″″′), 5.80(1H,dt,J=14.8,7.1Hz,H-5″″′),5.59(1H,br s,H-1″),5.21(1H, d,J=7.5Hz,H-1″′),4.73(1H,d,J=7.7Hz,H-1″″),4.38(1H,dd,J=12.0, 2.1Hz,H-6″″),4.36(1H,d,J=7.8Hz,H-1″″″),4.34(1H,dd,J=12.0,5.6 Hz,H-6″″),4.06(1H,m,H-8″″′),4.05(1H,br s,H-2″),3.85(1H,m,H-3″),3.84(1H,m,H-6″″″),3.70(2H,m,H-6″″″,H-2″′),3.66(1H,dd, J=12.1,1.7Hz,H-6″′),3.60(2H,m,H-5″,H-5″″),3.54(1H,t,J=9.1Hz, H-3″′),3.50(1H,m,H-6″′),3.48(1H,t,J=9.3Hz,H-4″),3.45(1H,t, J=9.0Hz,H-3″″),3.38(1H,m,H-2″″),3.36(3H,m,H-4″″,H-3″″″,H-4″″″), 3.35(1H,m,H-4″′),3.25(1H,m,H-5″″″),3.19(1H,dd,J=9.0,7.8Hz, H-2″″″),3.12(1H,m,H-5″′),2.53(1H,dd,J=15.4,6.8Hz,H-9″″′),2.46 (1H,dd,J=15.4,4.1Hz,H-9″″′),,2.36(1H,m,H-6″″′),1.79(1H,m, H-7″″′),1.78(1H,m,H-6″″′),1.62(3H,br s,H-12″″′),1.25(3H,d,J=6.1Hz,H-6″),0.82(3H,d,J=6.1Hz,H-11″″′);13C NMR(600MHz,CD3OD):δ: 180.0(C-4),176.7(C-10″″′),169.9(C-1″″′),163.4(C-7),162.9(C-5),161.7 (C-4′),159.1(C-2),158.0(C-9),143.8(C-5″″′),140.4(C-3″″′),135.4(C-3), 132.6(C-2′,C-6′),127.9(C-4″″′),125.4(C-2″″′),122.4(C-1′),116.4(C-3′, C-5′),107.5(C-10),105.8(C-1″″),103.8(C-1″″″),100.9(C-1″′),100.6(C-6), 99.8(C-1″),95.7(C-8),84.1(C-2″′),80.7(C-8″″′),78.3(C-5″′),78.1(C-3″″″), 77.8(C-5″″″,C-3″″),77.7(C-3″′),76.1(C-2″″),75.7(C-5″″),75.3(C-2″″″), 73.6(C-4″),72.1(C-3″),71.7(C-2″,C-4″″″),71.6(C-4″″),71.3(C-5″),71.1 (C-4″′),64.9(C-6″″),62.9(C-6″″″),62.4(C-6″′),38.3(C-7″″′),38.1(C-9″″′), 37.0(C-6″″′),18.1(C-6″),12.5(C-12″″′)。
下面通过试验例证明本发明新化合物Hip A的有益效果:
试验例1化合物Hip A对多柔比星致心肌细胞损伤的保护作用
1实验材料、试剂与仪器
1.1 H9c2大鼠心肌细胞
H9c2(2-1)大鼠心肌细胞购自于中国科学院上海细胞库。H9c2心肌细胞最初由B.Kimes和B.Brandt以源于BD1X大鼠胚胎心脏组织的心室细胞为细胞培养来源,通过选择性的继代培养方式而亚克隆的细胞株。它同时表现出具有骨骼肌与心肌功能的特异性心肌细胞株系。
1.2材料与试剂
(1)DoX母液配制:10mgDoX溶解于3.680mL PBS中,配置成终浓度为 5mmol/L母液,于-20℃保存,可稀释至100μmol/L分装,避免反复冻融。注意DoX 在配制过程中最好采用玻璃器皿,若采用EP管浓度不宜低于1mmol/L。
(2)5mg/mL母液配制
MTT对细菌敏感,见光易变性,配制时需要无菌,避光。于100mLPBS中充分溶解0.5gMTT粉末至MTT溶液终浓度为5mg/mL,0.22μM微孔滤膜过滤除菌后分装至1.5mLEP管,-20℃长期避光保存,避免反复冻融。注意:当MTT溶液变成灰绿色时,不可使用。
(3)Western blot所用试剂配制
1)BCA工作液配制:按试剂A比试剂B为50:1的比例配制所需体积。
2)蛋白标准液配制:蛋白标准液浓度为50mg/mL,用PBS稀释至终浓度为 0.5mg/mL,200μL分装,-20℃保存,现用现取。
3)10%(W/V)过硫酸铵(AP)溶液配制:过硫酸铵0.5g溶于5.0mL超纯水中,溶解后分装,-20℃避光保存,现用现取。
4)5×SDS-PAGE电泳液配制:称取Tris 15.1g,Glycine 94.0g加超纯水超声助溶解后,为防止起泡最后加入5.0g SDS,定容至1000mL,可常温保存。用时量取5×电泳液100mL,加超纯水定容至500mL,稀释为1×电泳液。
5)10×转膜液配制:称取Tris 30.3g,Glycine 144.0g,加超纯水溶解后,定容至1000mL。1×转膜液配制:取50mL10×转膜液(1份),加入100mL甲醇(2 份),超纯水(7份)补至500mL,即为1×转膜液。可重复使用3~5次,4℃冰箱保存。
6)10×TBS溶液配制:加超纯水溶解已称取的12.12g Tris、40.03g NaCl,调节pH至7.6,加入超纯水定容至500mL,4℃冰箱保存备用。
7)1×TBST缓冲液配制:移液枪吸取500μL的吐温20,加入至50mL的 10×TBS中混匀,加超纯水定容至500mL混匀,4℃冰箱保存待用。注意:吐温20 因很粘稠需将移液枪枪头剪开,便于吸取,把枪头整个打入缓冲液中。
8)封闭液的配制:称取0.5g脱脂奶粉(或BSA),加入10mL TBST溶液混匀即可,最好根据比例,按所需的量现用现配,可重复用2~3次,4℃冰箱保存。注意使用过程中只使用其上层清液,勿将下层颗粒物一并使用,防止封闭不彻底,膜上会出现黑色斑点。
9)一抗溶液的配制:β-actin、COX-2、Caspase-3等抗体时用抗体稀释液以1:1000稀释。常用时4℃冰箱保存,不用时-20℃冰箱保存。为延长抗体使用期限,可加入适量叠氮化钠(NaN3)。
10)二抗溶液的配制:按照山羊抗兔或山羊抗鼠抗体:抗体稀释液液=1:2000 进行稀释。抗体常用时4℃冰箱保存,不用时-20℃冰箱保存。
1.3H9c2心肌细胞体外培养
用含10%FBS及1%双抗的DMEM高糖培养基于37℃、5%CO2饱和湿度的培养箱中培养H9c2心肌细胞,隔天换液,待细胞生长密度达到70%~80%时进行传代培养。
1.4 H9c2心肌细胞冻存
选取生长密度到达70%~80%处于对数生长期的H9c2心肌细胞,细胞的消化操作见上述传代步骤。最后将收集到的细胞悬液放入无菌离心管,1000rpm,温度 25℃,离心6min,弃上清,彻底去除胰蛋白酶作用,并出去细胞杂质颗粒物的影响。用DMEM高糖培养基轻柔吹打重悬细胞后,细胞计数,并将细胞调至相应浓度。按照DMEM高糖培养基:FBS:DMSO=5:4:1比例,配成合适细胞冻存液加入已经重悬好的细胞,轻轻吹打至均匀,以适当体积分装到无菌的细胞冻存管中,每管1.0~ 1.2mL。冻存管封口膜封好后,标记细胞名称、代数、冻存日期及冻存者等信息,置于异丙醇梯度降温盒后,于-80℃存放24h后,转移至液氮罐中保存。
1.5 H9c2心肌细胞复苏
准备工作:操作前打开紫外灯,照射超净工作台约30min,用新洁尔灭喷洒擦拭工作台面后,温育DMEM高糖培养基。提前打开水浴锅提前调试温度至37℃,然后迅速将从液氮罐中取出的H9c2心肌细胞,放入37℃水浴,期间来回晃动,加速溶解,达到1min内快速溶解。将冻存管中细胞吸取到含有10%FBS的DMEM 高糖培养基中,注意要保证细胞均匀分布。在培养皿上标记细胞名称、代数和日期等信息,置7℃,5%CO2饱和湿度培养箱中培养6h左右,显微镜下观察细胞至完全贴壁后,吸弃培养基,PBS润洗2~3遍,去除残余DMSO,更换新的培养基。待细胞密度达70%~80%时,即可进行传代操作。
1.6 H9c2心肌细胞传代
培养H9c2心肌细胞融合至70%~80%左右时进行传代培养。操作前打开紫外灯,照射超净工作台约30min,用新洁尔灭喷洒擦拭工作台面。移液枪将培养皿中的旧培养基弃去,用37℃温育的PBS磷酸缓冲液洗涤2次。100mm培养皿加入含有0.25%EDTA的胰蛋白酶消化液750μL,置于培养箱中消化1~2min。在显微镜下观察,等到多数细胞间隙变大、细胞几乎变为透光圆形时,微微晃动培养皿皿壁,有少数细胞细沙状脱落,即为消化完成。真空泵吸弃胰酶,移液枪加入适量培养基终止消化,并轻柔吹打至全部脱落,制成均匀细胞悬液。按照1:2~1:4比例接种到新的培养皿,补加相应的血清和培养基,放入培养箱中继续培养。
1.7 MTT法检测细胞活力
选取生长密度到达70%~80%处于对数生长期的H9c2心肌细胞,细胞的消化操作见上述传代步骤。用含10%FBS的DMEM高糖培养基配成均匀单细胞悬液,以7500个/孔的密度接种于96孔细胞培养板中,每孔体积200μL。在37℃、5%CO2饱和湿度条件下培养待细胞贴壁完全,加入不同浓度的DoX诱导24h后,每孔加入 20μL5mg/mL的MTT溶液,培养箱孵育3h,弃去培养基终止培养。每孔加入100μL DMSO,振荡10min,使甲瓒充分溶解,用多功能酶标仪于490nm处测定吸光值。
细胞存活率(%)=(药物处理组OD/空白组OD)×100%
1.8 Griess法检测细胞上清液NO含量
(1)标准品配制:将1mol/L NaNO2标准溶液,用细胞培养液(DMEM +10%FBS)梯度稀释成0,1,2,5,10,20,40,60,100μmol/L的系列标准品溶液,用以绘制标准曲线。
(2)按50μL/孔,在96孔板中加入样品。后按50μL/孔,在各孔中加入室温 GriessReagent I。再按50μL/孔,在各孔中加入室温Griess Reagent II。最后用540nm 测定吸光度。
1.9微孔板法检测细胞上清的乳酸脱氢酶(LDH)
具体操作方法见说明书。
1.10 Westernblot实验方法
(1)细胞蛋白抽提
a.准备工作:准备碎冰浴,标记EP管,预冷PBS备用,配制裂解液(RIPA 裂解缓冲液:PMSF=100:1)。真空泵尽可能吸净培养基,培养基的残留会影响蛋白吸光度的测定,进而影响蛋白浓度测定准确性。
b.清洗:缓慢转圈并加入冷PBS,防止冲散吸走细胞。润洗2遍后,将PBS 尽量吸弃,否则会稀释蛋白的浓度,导致浓度过低,上样量过大。
c.细胞裂解:每孔加入适量裂解液,尽量使裂解液均匀平铺于皿底。置4℃冰箱或冰上裂解5min。
d.蛋白抽提:细胞刮刀用PBS润洗拭干,刮刀于冰上刮取细胞。细胞刮刀尽量沿同一方向刮取细胞,尽可能多的刮取细胞。收集皿内细胞抽提液,置于EP管内,同时吸取刮刀上的液体。细胞刮刀刮取不同组别时,应清洗和擦干。
e.离心:蛋白裂解液冰上裂解30min,每5min混弹1次。12000g,4℃,离心15min,收集上清液弃沉淀,-80℃保存,待蛋白定量。
(2)BCA法蛋白含量测定
a.准备工作:已提取的蛋白置于冰浴盒上,防止降解变性。
b.标准蛋白配制:用PBS缓冲液将50mg/mL蛋白标准品稀释至浓度为 0.5mg/mL。分别取0.5mg/mL蛋白标准液0、2、4、6、8、12、16和20μL加入到96孔微孔板中,每组设置3个平行,用PBS缓冲液补足至20μL。
c.待定量蛋白液配制:4μL蛋白待测液加入76μLPBS缓冲液,每20μL,3个重复,有剩余,这样出现误差的可能性会降低。每个EP管内先加入76μL PBS,分批融化蛋白,小型涡旋离心,混匀。加入4μL待定量蛋白液至80μL。96孔板每孔加入20μL蛋白液。
d.每孔加入200μL工作液,AB工作液配置A液:B液=50:1,工作液加入96 孔板内时,枪头略高于总体界面,以免串染。
e.微孔板振荡器,37℃孵育30min后,酶标仪562nm处测定各孔吸光值。
f.根据标准曲线计算样品中的蛋白含量。Western blot实验中采用等体积等量上样,即用PBS稀释蛋白样品至一致浓度,同时计算上样体积所需要的5×蛋白上样缓冲液(Loading buffer)的体积,调整浓度一致后,计算30μg蛋白溶液的体积即为上样体积。
g.蛋白变性:EP管封口膜封口标记后,100℃水浴10min,充分变性蛋白。加样前,将样品离心混匀后再使用,变性定量后蛋白一般-80℃存时间为1个月。
(3)SDS-PAGE电泳
a.配胶:根据目的蛋白分子量选择相应的分离胶浓度;洗净玻璃板,用超纯水冲洗,将与胶接触的一面向下倾斜置于干净的无尘纸晾干;配胶之前各溶液室温放置,防止凝胶过程中产生的热量,使低温时溶解于储存液中的气体析出而产生气泡。固定胶板后,用去离子水试漏,测试后,擦拭干净水分。
分离胶配制:确定装置不漏水后,根据目标蛋白的分子量大小,按照配胶说明选择合适的分离胶浓度,按所需体积加入各组分,TEMED要最后加入,混合均匀后灌胶。分离胶充分混匀后,移液枪吸取,于左侧抵住顶端玻璃缓慢加入凝胶,至板凸出端平齐略高。灌入2/3的分离胶后应立即封胶,异丙醇封胶后切记,勿动。一般实验室也常采用水封的方式封胶,但液面的平整性略差。待胶凝后将封胶液倒掉,如用异丙醇封胶需用超纯水冲洗干净。
浓缩胶配制:按照配胶说明配制5%的浓缩胶,沿玻璃板加满浓缩胶,避免产生气泡,梳子与玻璃呈45°贴近,快速将梳子插入浓缩胶中,在未凝固时缓慢插入,一旦产生气泡,立刻拔出,赶净气泡再重新插入。静止凝固1h左右,凝固长时间有利于胶结构的形成,因为肉眼观察胶凝时其内部分子的排列尚未完成。超净纸包裹,加入适量超纯水润湿,4℃密封保存。使用前,放入电泳液内活化10~15min再使用。使用时将玻璃板安装至电泳槽中,按量加入电泳缓冲液。小心取出梳子,准备上样。注意观察电泳槽是否漏液,漏液会导致电导率降低。
b.上样
样品放置冰箱后,蛋白会发生沉降,溶液不均一。上样前先融化离心再反复混弹或者用枪吹打,注意更换枪头。枪头或者微量进样器将样品缓慢加入孔中,其中第1孔需加2~5μL的Marker。上样完成后,需要尽快进行电泳,电泳条件为: Step1:80V,30min;Step2:120V,180min,待Loading buffer走到最下端的时候停止电泳。注意:枪头吸取蛋白,插入到凝胶样品孔内,勿伸入太多,以免枪头撑开玻璃板。
c.转膜
根据目的蛋白及样品数量,提前裁剪好PVDF膜,并置于甲醇溶液中1~3min 活化。干手套操作,勿使PVDF膜沾水。膜在使用过程中,请用镊子夹取,以免手上蛋白污染PVDF膜。PVDF膜在裁剪时,在右上方做标记,以区分正反。将转膜过程使用的―三明治结构‖浸泡在4℃预冷的转膜液中,至完全浸湿。膜在转膜液中提前润湿。自来水冲洗含有电泳液的胶板,根据Marker分子量对目标蛋白凝胶割胶。按照―黑胶白板‖的原理,将凝胶平整放置在黑板一侧,且将PVDF膜左上角缺口对准胶面。转膜液反复润湿胶板和膜。直至两者相互贴合无气泡。在膜一侧覆盖滤纸及丝绵,用试管轻轻赶压气泡,至两者之间无气泡产生。力气不要太大,以免膜胶之间发生错位。将夹板放入转膜电极槽内,黑对黑,红对红。为防止转膜时温度过高,因此需要在转膜槽的一边放上冰袋等来达到降温的目的,整个转膜过程需要在冰浴中进行。根据目标蛋白的分子量大小,确定转膜时间及转膜需要的电流大小。
d.封闭及杂交
封闭:此时注意区分膜的正反面,与凝胶接触的一面是正面,此面朝上与抗体结合孵育。将膜取出,TBST稍加漂洗,浸没于5%脱脂奶粉中封闭1h。脱脂奶粉封闭PVDF膜上非特异性的位点。
洗膜:TBST溶液每次洗涤5~10min,共3次。
孵育一抗:按照比例稀释一抗,一般为1:1000~1:10000。PVDF膜正面朝上加入一抗,使一抗尽量没过膜,4℃静置过夜。孵育完成后回收一抗溶液。
洗膜:TBST溶液每次洗涤5~10min,共3次。孵育二抗:根据一抗选择合适的二抗,按1:1000比例稀释二抗,室温轻摇孵育1~2h。孵育结束后,回收二抗,重复使用。
洗膜:TBST溶液每次洗涤5~10min,共3次。
e.显影
将PVDF膜放在显影黑板上,水平放置,赶走气泡。等体积混匀A液和B液,将混合好的ECL试剂轻柔滴加在PVDF膜上,并用凝胶成像系统进行拍照。显影仪器拍摄,延长拍摄时间等。进行分析与扫描。拍摄后,回收膜,浸润至TBST溶液中,可保留反复使用。必要时可先用丽春红染色(2%乙酸,0.5%丽春红的水溶液) 观察蛋白条带,再用去离子水和TBST将丽春红洗去。
2.实验结果
2.1 H9c2心肌细胞形态学观察
形态学实验观察时细胞被分成了6个组:正常对照组、1μmol/L、2μmol/L、3 μmol/L、4μmol/L和5μmol/L DoX处理组。DoX刺激心肌细胞24h后,倒置显微镜下观察心肌细胞。结果如图11所示,发现正常对照组H9c2心肌细胞成纺锤形、三角形或者是多角形,胞体饱满,且有长度不一的突起。与正常对照组相比不同浓度的DoX处理组心肌细胞胞体皱缩,折光率降低,细胞的体积明显减小,细胞密度明显减小。
2.2 MTT检测H9c2心肌细胞存活率
各浓度DoX处理组及正常对照组心肌细胞处理作用2h后,进行MTT细胞存活率实验,结果如图12所示,1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L DoX处理组与正常对照组相比较对细胞活力有明显杀伤抑制作用,各组之间存在统计学差异P<0.01。通过计算H9c2细胞活力的半数致死率,DoX的浓度为IC50=2.46 μM。
2.3 H9c2心肌细胞培养上清液中NO含量
Griess试剂中的磺胺酸通常溶解在酸性液体溶液中,其与亚硝酸盐接触时,会形成重氮化合物。Griess试剂中的亚乙基乙二胺使重氮化合物变成在540nm处有最大吸收的粉红色产物,该反应生成的产物浓度与NO浓度具有线性关系。结果如图 13所示,发现1μmol/L、2μmol/L、3μmol/L、4μmol/L和5μmol/LDoX处理组NO 含量水平与正常对照组相比无显著差异。
2.4 H9c2心肌细胞上清液中LDH含量
细胞凋亡或坏死而造成的细胞膜结构的破坏会导致细胞浆内的乳酸脱氢酶释放到培养基里,在乳酸脱氢酶的作用下,NAD+被氧化还原成NADH,NADH和INT 被硫辛酰胺脱氢酶催化反应生成NAD+和强生色物甲臜,在490nm波长下产生吸收峰,从而可以通过闭塞来定量乳酸脱氢酶的活性。结果如图14所示,3μmol/L、4 μmol/L和5μmol/LDoX处理组与正常对照组相比较对细胞培养液中LDH含量的水平有升高的趋势但没有显著差异,各组之间无统计学差异。
2.5化合物Hip A对H9c2心肌细胞细胞活力的影响
通过MTT法检测化合物Hip A对H9c2心肌细胞活力的影响。化合物Hip A 设置了6组浓度梯度(0、0.1、1、10、50和100μmol/L)从图15可以看出,化合物Hip A在浓度0~10μmol/L对H9c2心肌细胞没有毒性作用,因此在选择化合物 Hip A与DoX联用时,化合物Hip A的选择10μmol/L。
2.6化合物Hip A对DoX诱导H9c2心肌细胞的LDH水平
乳酸脱氢酶(LDH)是检测细胞凋亡或坏死的重要指标,如图16所示,Hip A 化合物对DoX诱导的H9c2心肌损伤时有显著的保护作用(P<0.05)。
2.7化合物Hip A对DoX诱导H9c2心肌细胞的NO水平
细胞培养上清液中NO的含量水平是检测心肌细胞损伤与否的一个重要指标。如图17所示,化合物Hip A能降低损伤的心肌细胞中NO水平,表明Hip A对DoX 诱导的H9c2心肌细胞损伤具有显著的保护作用(P<0.05)。
2.8化合物Hip A对DoX诱导H9c2心肌细胞凋亡蛋白的影响
Bcl-2和Bax通过控制线粒体膜的通透性来调节凋亡激活物,通过Bcl-2和Bax 的比值确定凋亡的发生。如图18所示,新黄酮类化合物Hyd、Hip A、Hip B和Hip C都表现出了对DoX诱导H9c2心肌细胞凋亡的保护作用。
试验例2化合物Hip A的神经保护作用
1.实验材料、试剂与仪器
1.1主要试剂配制
ATRA溶液配制:称取15.0mg全反式维甲酸粉末,置于灭菌1.5mL离心管中,加1mLDMSO震荡溶解,即为50mM的ATRA溶液,用200μL离心管分装,-20℃冰箱保存备用,临用前用DMEM培养基稀释到所需浓度。
OA溶液配制:每支冈田酸含量为50μg,加入593μL的DMSO将其充分溶解,成100μM的母液,用200μL离心管分装,-20℃冰箱保存备用。
Giemsa溶液配制:
(1)吉姆萨储备液的配制:Giemsa粉0.8g,甘油50mL,甲醇50mL。将吉姆萨粉溶入少量甘油中,在研钵中充分研磨后加入甲醇,混合摇匀,56℃恒温2 h后置于棕色瓶中,4℃冰箱储备。
(2)工作液的配置:A液:用天平称取11.93g磷酸氢二钠(Na2HPO4.12H2O) 溶解到500mL超纯水中;B液:用天平称4.535g磷酸二氢钾(KH2PO4)溶解到500mL超纯水中;用时将A液:B液按1:1混合,即为pH 7.0的工作液。
(3)Giemsa储备液:工作液=1:4,混合均匀即为Giemsa染色液,现配现用。
MTT溶液配制:称取25.0mg MTT粉末,加入5mL PBS使其充分溶解,得到浓度为5mg/mL的MTT溶液,分装后-20℃冰箱避光保存。
1.2实验方法
(1)细胞的复苏、传代及培养
操作前打开紫外灯照射超净工作台15min,用酒精擦拭工作台面;把从液氮罐中取出的细胞迅速放入37℃水浴中,1min内快速化开,切忌来回取出观看是否化开;将冻存管里的细胞转移到含有10%FBS的DMEM培养基中;在培养皿上标记细胞名称、时间和代数,置于37℃,5%CO2培养箱中培养。细胞贴壁后,换新的培养液继续培养,注意观察细胞形态,待细胞生长融合达80%左右时,即可进行传代操作。弃去培养皿中的旧培养液,PBS磷酸盐缓冲液洗涤1次;加入含有0.25%EDTA的胰蛋白酶消化液,培养箱中消化30s~1min,显微镜下观察,等大部分细胞几乎为圆形、细胞间隙变大时,加入DMEM培养基终止消化,用移液枪反复几次轻柔吹打贴壁细胞,待细胞全部脱落得到细胞悬液;将混匀的细胞按所需比例稀释,接种到新的培养基中,放入培养箱继续培养。
(2)细胞冻存
选取对数生长期的细胞,按上述步骤传代,将收集到的细胞转移到无菌离心管中,860rpm常温离心6min;弃去上清液,将DMEM培养基:FBS:DMSO按5:4:1 配成的细胞冻存液加入,轻轻吹打混匀,分装到无菌细胞冻存管中,每管1.2mL;在4℃冰箱放置10min,-20℃冰箱放置1h,-80℃冰箱过夜后,将细胞转移到液氮罐中保存。
2.实验结果
2.1 AD体外细胞模型的建立
如图19所示,取对数生长期的SH-SY5Y细胞,以3×104个/mL接种于6孔板中,每孔加入细胞悬液3mL。用含10%FBS的DMEM培养基在37℃、 5%CO2的培养箱中培养24h,换为终浓度为5μM的ATRA、2%FBS的DMEM 培养基继续培养。每天观察形态,隔天换液,诱导4天,使其成为分化成熟的神经细胞。吸弃原培养基,加入终浓度为40nM的OA、2%FBS的DMEM培养基作用6h。然后通过Giemsa染色法和相差显微镜进行形态学观察,采用Image proplus 6.0测量细胞突触长度与胞体长度,计算比值,评价模型的成功度(Wang YY, Song,XY,Liu,DD,etal.IMM-H004 reduced okadaic acid-induced neurotoxicity byinhibiting Taupathology in vitro and in vivo.Neurotoxicology 2019,75:221-232.;Medeiros LM,Bastiani MD,Rico,EP,et al.Cholinergic Differentiation of Human NeuroblastomaSH-SY5Y Cell Line and Its Potential Use as an In Vitro Model for Alzheimer'sDisease Studies.Mol Neurobiol2019,56:7355-7367.;ZhangZ,Simpkins,JW. OkadaicAcid Induces Tau Phosphorylation in SH-SY5Y Cells in an Estrogen-PreventableManner.Brain Res 2010,1345:176-181.)。
2.2化合物Hip A对SH-SY5Y细胞活力的影响
MTT检测细胞活力原理:活细胞线粒体中的琥珀酸脱氢酶能使外源性的MTT 还原为水不溶性的蓝紫色甲瓒,并沉积在细胞中,而死细胞无此功能。DMSO能溶解沉积在细胞中的甲瓒,使用酶标仪在490nm处测定其吸光度值,根据此来判断活细胞的数量。OD值越大,说明细胞活性越强,即药物毒性越小。当细胞存活率高于80%时,即认为药物对细胞没有毒性作用,从而确定药物作用的最佳浓度范围。由图20可以看出,化合物Hip A在100μM时细胞活力显著降低。因此,本实验选用10μM作为最适浓度,并进行后续研究。
2.3化合物Hip A对AD细胞模型形态学的影响
Giemsa染液为天青色素和伊红的混合物,可将细胞核染成紫红色或蓝紫色,胞浆染成粉红色,在显微镜下呈现出清晰的细胞图像。本实验利用此方法对SH-SY5Y 细胞突触变化情况进行研究,实验分为ATRA组(正常组)、OA组(损伤组)和药物处理组。在诱导4d后,ATRA组用含2%血清的正常培养液继续培养;OA组在正常培养液中加入终浓度为40nM的OA培养;药物处理组将终浓度40nM的OA 和终浓度为10μM的化合物Hip A同时加入培养液中培养。6h后进行Giemsa染色,观察细胞突触长度的变化,实验结果如图21所示。
由图21可知,OA处理后细胞变圆,皱缩,突触比明显缩短。Hip A可显著改善OA所致的突触萎缩,对被损伤的神经具有修复作用,以维持细胞的正常形态。
2.4 Western Blotting检测tau蛋白磷酸化相关蛋白的表达
AD患者脑中tau蛋白磷酸化水平显著升高,针对tau蛋白靶点研发预防治疗AD 的药物具有巨大的前景,因此,本实验通过Western Blooting方法研究了黄酮类化合物对p-tau Ser 262、p-tau Ser 396及tau 5的表达的影响。其中p-tau Ser 262、p-tau Ser 396与tau 5比值分别代表tau蛋白在丝氨酸262、396位点的磷酸化程度,比值越高说明磷酸化程度越高。由图22可知,本发明化合物Hip A可显著降低tau蛋白在Ser 262及Ser 396位点的磷酸化水平,且优于其它化合物,表明化合物Hip A具有较好的改善tau蛋白过度磷酸化的作用,提示其具有治疗AD的潜力。
综上所述,本发明提取得到的新黄酮类化合物具有修复心肌损伤、保护心肌细胞、神经保护的作用,为开发预防和/或治疗相关疾病的潜在植物来源的药物提供更多的参考依据。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (9)
1.化合物XVII以及其药学上可接受的盐,在制备预防和/或治疗多柔比星诱导的心肌细胞损伤或冈田酸诱导的神经细胞损伤的产品中的应用,所述化合物XVII结构如下:
2.根据权利要求1所述的应用,其特征在于,所述产品为具有心肌保护作用的产品。
3.根据权利要求1或2所述的应用,其特征在于,所述产品为降低心肌细胞中乳酸脱氢酶和/或NO的含量的产品。
4.根据权利要求1或2所述的应用,其特征在于,所述产品为延缓心肌细胞凋亡的产品。
5.根据权利要求4所述的应用,其特征在于,所述产品为Bcl-2抑制剂和/或Bax抑制剂。
6.根据权利要求1所述的应用,其特征在于,所述产品为具有神经保护和/或神经修复作用的产品。
7.根据权利要求6所述的应用,其特征在于,所述产品为改善神经突触萎缩的产品。
8.根据权利要求6所述的应用,其特征在于,所述产品为降低tau蛋白过度磷酸化的产品。
9.根据权利要求6所述的应用,其特征在于,所述产品为预防和/或治疗阿尔兹海默病的产品。
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