CN112552155A - 一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮及其应用 - Google Patents
一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮及其应用 Download PDFInfo
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Abstract
本发明公开了一种高良姜中分离的1,7‑二苯基‑4‑庚烯‑3‑酮及其应用,分离步骤如下:高良姜42kg粉碎,80%乙醇加热回流3次,分别取上层液和下层液点板,若下层液在板上无点,且上层液有点跑出,则萃取完成;萃取好后,取上层液合并旋蒸挥干,得到高良姜浸膏;得到浸膏,按1:7上柱子,硅胶上样量385g,硅胶保护柱55g,分别接洗脱剂比例为石油醚:乙酸乙酯:(30:1)、(25:1)、(20:1)、(15:1)、(10:1)、(5:1)、(5:3)冲洗来的流分,经制备液相制备,甲醇与水比例为85:15得到。本发明高良姜提取物改善IR作用显著,该化合物可上调Nrf2mRNA及下游靶基因的转录水平。
Description
技术领域
本发明涉及天然药物技术领域,特别涉及一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮及其应用。
背景技术
高良姜是姜科山姜属植物高良姜(Alpinia officinarum Hance)的干燥根茎,具有温中散寒,温通止痛,和胃平逆,行气导滞的功效,临床常用于治疗脘腹冷痛、胃寒呕吐等病症。化学研究表明高良姜主要含黄酮类、挥发油和二苯庚烷化合物。药理研究发现,其具有抗糖尿病、抗溃疡、止呕、抗炎等多方面的药理作用。据报道,高良姜提取物具有显著降糖作用。国内亦有研究报道高良姜提取物及二苯庚烷类化合物改善胰岛素抵抗作用显著。但是,目前的报道缺乏更深层次的研究,不能很好的确定高良姜分离提取的1,7-二苯基-4-庚烯-3-酮对糖尿病的具体影响。
发明内容
本发明的目的在于提供一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮及其应用,高良姜提取物改善IR作用显著,所得成分之一1,7-二苯基-4-庚烯-3-酮对高糖作用下肝脏细胞对Nrf2/ARE通路的影响,该化合物可上调Nrf2mRNA及下游靶基因的转录水平,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮,分离方法包括如下步骤:
步骤1:高良姜提取物提取:高良姜42kg粉碎,80%乙醇加热回流3次,每次2h,料液比1:10,合并滤液,浓缩至10L得到;
步骤2:分离纯化:少量高良姜提取物于分液瓶中,加入大量的乙酸乙酯,充分振摇后,静置1小时以上,出现明显分层即可,分别取上层液和下层液点板,在254nm下检视,若下层液在板上无点,且上层液有点跑出,则萃取完成;
步骤3:萃取好后,取上层液合并旋蒸挥干,得到高良姜浸膏;
步骤4:所得浸膏55g,按1:7上柱子,硅胶上样量385g,硅胶保护柱55g,分别接洗脱剂比例为石油醚:乙酸乙酯:(30:1)、(25:1)、(20:1)、(15:1)、(10:1)、(5:1)、(5:3)冲洗来的流分,A8在20:1流分的混合物中,经制备液相制备,甲醇与水比例为85:15得到。
进一步地,步骤2的点板为展开剂石油醚:乙酸乙酯=5:2。
进一步地,步骤3的上层液为乙酸乙酯层。
进一步地,包括一种药物组合物,该药物组合物含有1,7-二苯基-4-庚烯-3-酮或药学上可接受衍生物及药学上可接受的载体。
进一步地,1,7-二苯基-4-庚烯-3-酮或其药学上可接受衍生物用于制备治疗胰岛素抵抗引发疾病的药物。
进一步地,其中胰岛素抵抗引发疾病包括糖尿病、高尿酸血症、高脂血症、代谢综合征、肥胖病、心血管疾病。
本发明提供的另一种技术方案:高良姜中分离的1,7-二苯基-4-庚烯-3-酮在制备抗氧化食品、保健品或药品中的应用。
与现有技术相比,本发明的有益效果是:本发明给予提取物干预后,小鼠的随机血糖水平显著下降,糖耐量得到了明显改善,高良姜提取物改善IR作用显著。所得成分之一1,7-二苯基-4-庚烯-3-酮对高糖作用下肝脏细胞对Nrf2/ARE通路的影响。1,7-二苯基-4-庚烯-3-酮可上调Nrf2mRNA及下游靶基因的转录水平。
附图说明
图1为本发明分离的1,7-二苯基-4-庚烯-3-酮的结构式;
图2为本发明A8活性成分对HepG2细胞存活的影响示意图;
图3为本发明高糖干预后A8对HepG2细胞15min内糖消耗情况示意图;
图4为本发明高糖干预后不同药物对HepG2细胞中糖摄取的影响示意图;
图5为本发明A8对IR-HepG2细胞中ROS的影响示意图;
图6为本发明A8对IR-HepG2细胞中SOD的影响示意图;
图7为本发明A8药物对胰岛素抵抗模型细胞内氧化损伤标记物MDA水平的影响示意图;
图8本发明A8对Nrf2的蛋白表达量的影响示意图;
图9本发明A8对HO-1蛋白表达量的影响示意图;
图10本发明A8对IRS1蛋白表达量的影响示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮,分离方法包括如下步骤:
高良姜提取物提取:高良姜42kg粉碎,80%乙醇加热回流3次,每次2h,料液比1:10,合并滤液,浓缩至10L得到;分离纯化:少量高良姜提取物于分液瓶中,加入大量的乙酸乙酯,充分振摇后,静置1小时以上,出现明显分层即可,分别取上层液和下层液(尽量加上下层液的渣)点板(展开剂石油醚:乙酸乙酯=5:2),在254nm下检视,若下层液在板上无点,且上层液有点跑出,则萃取完成。萃取好后,取上层液(乙酸乙酯层)合并旋蒸挥干,得到高良姜浸膏。所得浸膏55g,按1:7上柱子,硅胶上样量385g,硅胶保护柱55g。分别接洗脱剂比例为石油醚:乙酸乙酯:(30:1)、(25:1)、(20:1)、(15:1)、(10:1)、(5:1)、(5:3)冲洗来的流分,A8在20:1流分的混合物中,经制备液相制备,甲醇与水比例为85:15得到。
1,7-二苯基-4-庚烯-3-酮的结构式如图1所示。
药效学实验证明:
1.A8对HepG2细胞增殖的影响
如图2所示,与正常对照组相比,DH8浓度0~40μmol/L时,对HepG2细胞的存活率未见明显影响。与5.5mM相比较*:p<0.05,**:p<0.01,***:p<0.001,n=4。
2.A8对IR-HepG2细胞糖消耗的影响
如图3所示,与正常对照组相比较,通过高糖诱导产生的IR模型组,细胞对葡萄糖的消耗量显著性降低(p<0.01),表明IR模型诱导成功,该模型组细胞对胰岛素产生抵抗。加入阳性药物-罗格列酮治疗后,与正常对照组相比,葡萄糖消耗量有显著性差异;与模型组相比较,罗格列酮组(p<0.05)的葡萄糖消耗量显著升高,表明阳性药物组具有改善IR-HepG2细胞的糖消耗。
与正常对照组相比较,A8活性成分均能改善IR-HepG2细胞的糖消耗,当A8浓度在20uM和40uM时,与空白组糖消耗量比较无显著性差异,与模型组相比存在显著差异(p<0.05)。表明A8能有效改善IR-HepG2细胞对葡萄糖的消耗。
图3中,与模型组相比较药物组*:p<0.05,**:p<0.01,***:p<0.001,n=4。与空白组相比,模型组#:p<0.05,##:p<0.01,###:p<0.001,n=4。
3.A8对IR-HepG2细胞糖摄取的影响
实验中采用2-NBDG荧光探测示踪剂观察DH8药物治疗后细胞对葡萄糖的摄取情况。结果如图4所示,与正常对照组相比较,通过高糖诱导产生的胰岛素抵抗模型组,细胞对葡萄糖的摄取量显著降低(P<0.001),表明成功诱导出了IR模型,该模型组细胞产生了IR。加入阳性药物-罗格列酮治疗后,葡萄糖摄取量与正常对照组比较,无显著差异;与模型组相比较,罗格列酮组(p<0.05)细胞的葡萄糖摄取量显著升高,表明阳性药物能有效改善IR-Hep G2细胞的糖摄取。与模型组相比较,A8浓度为10μM、20μM和40μM时葡萄糖相对摄取荧光值与模型组存在显著性差异(p<0.001)。表明A8能有效改善IR-Hep G2细胞对葡萄糖的摄取。
图4中,与模型组相比较药物组*:p<0.05,**:p<0.01,***:p<0.001,n=4。与空白组相比,模型组#:p<0.05,##:p<0.01,###:p<0.001,n=4。
4.药物对胰岛素抵抗模型细胞内ROS水平的影响
本实验使用荧光探针DCFH-DA染色后,置于流式细胞仪上检测细胞内ROS水平。经过分析,结果如图5所示,模型组与正常对照组比较,细胞中的ROS水平极显著性升高(P<0.001);与模型组相比较,阳性对照组(与模型组间均存在极显著性差异(P<0.001),当A8浓度为10uM、20uM和50uM时,与模型组间均有显著性差异(P<0.05)时,与模型组间均存在显著性差异(P<0.05),在浓度为50uM时效果最明显(P<0.001)。A8显著的降低了IR-Hep G2细胞中的ROS水平,表现出了显著的抗氧化活性。
图5中,与模型组相比较药物组*:p<0.05,**:p<0.01,***:p<0.001,n=4。与空白组相比,模型组#:p<0.05,##:p<0.01,###:p<0.001,n=4。
5.药物对胰岛素抵抗模型细胞内抗氧化酶SOD活性的影响
氧化应激发生时,机体会发生一系列的抗氧化反应来对细胞起到保护作用。SOD是机体中防御自由基损伤的重要抗氧化酶之一,在细胞中,可清除自由基对细胞起到保护作用,其活力的大小可体现细胞清除氧自由基的能力。A8对IR-Hep G2细胞中SOD活性的影响如图6,结果显示,A8可促进SOD的释放。与正常对照组相比较,模型组中的SOD活力极显著性降低(P<0.001),罗格列酮组与正常对照组间均无显著性差异,与模型组相比较,当A8浓度达到10uM、20uM时,IR-HepG2细胞中SOD活力显著性升高(P<0.05),然而在浓度为40uM时对IR-HepG2细胞中SOD活力降低没有改善作用。
图6中,与模型组相比较药物组*:p<0.05,**:p<0.01,***:p<0.001,n=4。与空白组相比,模型组#:p<0.05,##:p<0.01,###:p<0.001,n=4。
6.A8药物对胰岛素抵抗模型细胞内氧化损伤标记物MDA水平的影响
丙二醛(MDA)是细胞内氧化性损伤的重要标志物,经过12h处理后,各干预组MDA含量如图所示,正常对照组分别与罗格列酮钠组比较,均无显著差异;正常对照组与模型组相比较,二者之间存在极显著性差异(P<0.001),如图7还可看出,当A8浓度在10uM时,与模型组相比较,显著降低了MDA的含量(P<0.001),且A8各浓度间不明显差异,说明A8活性组分可有效降低IR-Hep G2细胞模型中MDA的含量,改善IR模型中的氧化应激状态。
图7中,与模型组相比较药物组*:p<0.05,**:p<0.01,***:p<0.001,n=4。与空白组相比,模型组#:p<0.05,##:p<0.01,###:p<0.001,n=4。
葡萄糖消耗实验研究表明,DH8均能升高IR-HepG2细胞的糖消耗量,对IR-HepG2细胞的糖代谢具有更好的改善作用。DH8可有效降低模型中的ROS,提高抗氧化物质的活力。
7.A8活化Nrf2/ARE信号通路实验
(1)核转录因子Nrf2具有抗氧化应激的作用,DH8给药后,Nrf2的蛋白表达量显著性升高,说明DH8可通过激活Nrf2起到抗氧化应激作用,从而改善由高糖引起的胰岛素抵抗,结果如图8。各组间比较采用单因素方差分析,P<0.05认为差异有统计学意义。与空白组比较,#p<0.05,##p<0.01,###p<0.001;与模型组相比,*p<0.05,**p<0.01,***p<0.001。
(2)血红素氧合酶-1(HO-1),是Nrf2下游相关的蛋白,HO-1路径是典型的细胞对抗氧化应激的防御系统,给药后,HO-1蛋白表达量显著升高,表明A8可通过激活Nrf2的下游HO-1蛋白起到抗氧化应激作用,从而改善由高糖引起的胰岛素抵抗,结果如图9。各组间比较采用单因素方差分析,P<0.05认为差异有统计学意义。与空白组比较,#p<0.05,##p<0.01,###p<0.001;与模型组相比,*p<0.05,**p<0.01,***p<0.001。
(3)当糖代谢紊乱时,使得胰岛素受体底物磷酸化水平提高,导致IRS1被泛素化蛋白酶系统降解,加重胰岛素抵抗。实验结果表明给A8后,IRS1的蛋白表达量增加,意味着提高了IRS1的生物活性,改善了葡萄糖摄取与利用,并且减轻胰岛素抵抗,结果如图10。
基于氧化应激建立胰岛素抵抗的HepG2细胞模型(IR-HepG2细胞),并利用此模型研究1,7-二苯基-4-庚烯-3-酮改善胰岛素抵抗作用的具体机制和作用的信号途径。研究高良姜的活性成分DHs是否能通过Nrf2-AER信号改善IR-HepG2细胞中的氧化应激和糖代谢,阐明高良姜活性成分通过Nrf2-AER信号通路改善胰岛素抵抗作用的网络药理机制,为开发改善胰岛素抵抗的高良姜成分药物提供帮助。
通过研究发现高良姜1,7-二苯基-4-庚烯-3-酮的改善胰岛素抵抗作用(IR)与激活Nrf2/ARE通路密切相关。所得成分1,7-二苯基-4-庚烯-3-酮对高糖作用下肝脏细胞对Nrf2/ARE通路的影响。1,7-二苯基-4-庚烯-3-酮可上调Nrf2mRNA及下游靶基因的转录水平。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (7)
1.一种高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,分离方法包括如下步骤:
步骤1:高良姜提取物提取:高良姜42kg粉碎,80%乙醇加热回流3次,每次2h,料液比1:10,合并滤液,浓缩至10L得到;
步骤2:分离纯化:少量高良姜提取物于分液瓶中,加入大量的乙酸乙酯,充分振摇后,静置1小时以上,出现明显分层即可,分别取上层液和下层液点板,在254nm下检视,若下层液在板上无点,且上层液有点跑出,则萃取完成;
步骤3:萃取好后,取上层液合并旋蒸挥干,得到高良姜浸膏;
步骤4:所得浸膏55g,按1:7上柱子,硅胶上样量385g,硅胶保护柱55g,分别接洗脱剂比例为石油醚:乙酸乙酯:(30:1)、(25:1)、(20:1)、(15:1)、(10:1)、(5:1)、(5:3)冲洗来的流分,A8在20:1流分的混合物中,经制备液相制备,甲醇与水比例为85:15得到。
2.如权利要求1所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,步骤2的点板为展开剂石油醚:乙酸乙酯=5:2。
3.如权利要求1所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,步骤3的上层液为乙酸乙酯层。
4.如权利要求1所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,包括一种药物组合物,该药物组合物含有1,7-二苯基-4-庚烯-3-酮或药学上可接受衍生物及药学上可接受的载体。
5.如权利要求1所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,1,7-二苯基-4-庚烯-3-酮或其药学上可接受衍生物用于制备治疗胰岛素抵抗引发疾病的药物。
6.如权利要求5所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮,其特征在于,其中胰岛素抵抗引发疾病包括糖尿病、高尿酸血症、高脂血症、代谢综合征、肥胖病、心血管疾病。
7.如权利要求1所述的高良姜中分离的1,7-二苯基-4-庚烯-3-酮在制备抗氧化食品、保健品或药品中的应用。
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