CN103690883B - 一种姜黄的乙酸乙酯提取物及其制备、检测方法和用途 - Google Patents
一种姜黄的乙酸乙酯提取物及其制备、检测方法和用途 Download PDFInfo
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- CN103690883B CN103690883B CN201310677128.7A CN201310677128A CN103690883B CN 103690883 B CN103690883 B CN 103690883B CN 201310677128 A CN201310677128 A CN 201310677128A CN 103690883 B CN103690883 B CN 103690883B
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Abstract
本发明涉及一种姜黄的乙酸乙酯提取物及其制备、检测方法和用途,所述乙酸乙酯提取物由以下方法制备:姜黄,用石油醚浸泡,石油醚渗滤,回收残渣,挥干残渣中的石油醚,然后用乙醇回流提取,提取液浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干,即得乙酸乙酯提取物。经过体外试验,发现本发明提供的姜黄的乙酸乙酯提取物可抑制EGFR活性,效果明显。
Description
[技术领域]
本发明涉及姜黄提取物及其制备、检测方法和用途,具体涉及一种姜黄的乙酸乙酯提取物及其制备、检测方法和用途。
[背景技术]
表皮生长因子受体(EGFR)是EGFR酪氨酸激酶家族的成员之一,它包含有一个细胞外配体结合区、单一的跨膜区和一个含酪氨酸激酶结构的胞内区。EGFR与配体结合导致重要的构象变化,暴露受体中的二聚环结构,引发自身磷酸化,转导下游信号,调节细胞的增殖、凋亡、迁移、存活和一系列复杂的过程。EGRF在肿瘤细胞中常处于过度激活状态,使细胞周期失去调控,导致肿瘤细胞无限制生长,如神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、前列腺癌、肝癌、结肠癌、胃癌等。EGFR是目前抗肿瘤分子靶向治疗中,最成功也是最受关注的治疗靶点之一。FDA已批准两种靶向EGFR的小分子抑制剂,gefitinib(吉非替尼,易瑞沙)和erlotinib(厄洛替尼,特罗凯),随着时间的推移,在治疗中出现了耐药现象。而国内迄今为止,尚未有特异性EGFR抑制剂出现。
姜黄,来源于姜科植物姜黄Curcuma longa L.的干燥根茎,具有破气行血、通经止痛之功效,姜黄素为姜黄根茎中的一种天然有效成分,具有如下药理作用:。
1、抗肿瘤作用:姜黄素可以预防和治疗多种肿瘤,结肠癌、胃癌、肝癌、乳腺癌、前列腺癌、皮肤癌、白血病等,可以作为抗突变剂和抗癌剂,美国国立肿瘤研究所已将其列为第3代癌化学预防药。
2、对心血管系统的作用:姜黄乙醇提取物具有扩血管作用;姜黄的热水提取物有强的血小板聚集抑制作用。
3、抗氧化作用:姜黄素是一种新型的抗氧化剂,可阻止丙酮痊/赖氨酸诱导的氧化应激和DNA损害,并且可抑制丙酮醛诱导的人单核细胞凋亡和活性氧的产生。
4、抗炎抗病毒作用:姜黄素对花生四烯酸代谢中的环氧合酶和脂氧合酶均有拮抗作用。
5、对心脏功能的作用:姜黄在治疗心衰的过程中,既能够改善心室收缩功能,又能够改善其舒张功能,且这种作用与药物的治疗时间和给药剂量有关,这可能有提高患者远期生存率的作用。
6、对肝脏疾病的作用:肝细胞的损伤是肝纤维化发生的重要始动因素,大量的研究证实姜黄素具有抗脂质过氧化作用和抗氧自由基效应。可有效预防乙醇和多不饱和脂肪酸所致的肝毒性损伤,阻止肝损伤进一步向肝纤维化方向发展。
7.其它作用:提高免疫力的作用。
迄今为止,尚未有关于姜黄的乙酸乙酯提取物用于抑制EGFR的姜黄乙酸乙酯提取物的相关报道。
[发明内容]
为了弥补现有技术中尚无用于制造抑制EGFR的姜黄乙酸乙酯提取物的空白,本发明提供了姜黄的乙酸乙酯提取物及其制备、检测方法和用途。
为实现上述目的,设计一种姜黄的乙酸乙酯提取物的制备方法,该方法由以下步骤组成:
a.将姜黄加入到石油醚中浸泡12~48小时,每100g姜黄需要300~500毫升的石油醚,然后渗滤回收残渣;
b.所述的残渣用浓度为60%~90%的乙醇分2~3次提取,每100克残渣每次需要300~500毫升的乙醇,每次提取的时间是1~3小时,收集得到提取液;
c.所述的提取液进行浓缩得到浓缩液;
d.用与浓缩液等体积的乙酸乙酯对所述的浓缩液进行萃取,收集乙酸乙酯的萃取液;
e.蒸干所述的萃取液得到提取物。
上述制备方法还具有如下优化方案:
每100g姜黄需要300毫升的石油醚浸泡。
所述的浸泡时间为18~36小时。
所述的乙醇浓度为70~90%。
所述的浸泡时间为24小时,
所述的乙醇浓度为80%,
每100克残渣每次提取优选需要300毫升的乙醇,
所述的步骤(b)中的提取次数为2次。
本发明还一种用上述述方法提取的姜黄的乙酸乙酯提取物,其特征在于该提取物含有主要成分:香豆酸,姜黄酮,双去甲氧基姜黄素,去甲基姜黄素,姜黄素,1-(4-羟基-3,5-二甲氧基苯基)-7-(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮。
本发明还包括一种药物制剂,由所述姜黄的乙酸乙酯提取物和药学上可接受的载体组成。
所述制剂为片剂、胶囊、颗粒或丸剂。
所述药学上可接受的载体或稀释剂是指药学领域常规的药物载体,选自填充剂、粘合剂、崩解剂、润滑剂、助悬剂、润湿剂、溶剂、表面活性剂或矫味剂中的一种或几种。
所述填充剂选自淀粉、蔗糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素或葡萄糖等;
所述粘合剂选自纤维素衍生物、藻酸盐、淀粉、糊精、明胶或聚乙烯吡咯烷酮等;
所述崩解剂选自微晶纤维素、羧甲基淀粉钠、交联聚乙烯吡咯烷酮、低取代羟丙基纤维素或交联羧甲基纤维素钠;
所述润滑剂选自硬脂酸、聚乙二醇、碳酸钙、碳酸氢钠、微粉硅胶、滑石粉或硬脂酸镁;
所述助悬剂选自微粉硅胶、蜂蜡、纤维素、固态聚乙二醇;
所述润湿剂选自甘油、吐温-80、乙氧基氢化蓖麻油或卵磷脂;
所述溶剂选自乙醇、液态聚乙二醇、异丙醇、吐温-80、甘油、丙二醇或植物油,所述植物油选自大豆油、蓖麻油、花生油、调和油等;
所述表面活性剂选自十二烷基苯磺酸钠、硬脂酸、聚氧乙烯-聚氧丙烯共聚物、脂肪酸山梨坦或聚山梨酯(吐温)等;
所述矫味剂选自阿斯巴甜、蔗糖素、香精、柠檬酸或糖精钠。
本发明还提供了姜黄的乙酸乙酯提取物的在制备抗EGFR活性的应用。
在抑制EGRF活性时,姜黄的乙酸乙酯提取物的用量(即每日服用量限定为0.1mg-3g)。
本发明的姜黄的乙酸乙酯提取物可用于制备抑制EGFR的药物。
一种检测上述姜黄的乙酸乙酯提取物的方法,用超高效液相色谱-电喷雾串联质仪对所述姜黄提取物进行分析,以获得乙酸乙酯提取物的UPLC-Q-TOF-MS图谱,其中:
a.质谱条件为:ESI离子源,正离子模式检测;雾化器N2压力为50psi;干燥气N2流速为15L/min;质量扫描范围50--1200;自动3级质谱;
b.色谱分析条件为:色谱柱:Waters ACQUITY UPLC TM BEH C18Column(50mm×2.1mm.i d.,1.7μm);二元梯度洗脱,A相为0.1%甲酸水溶液;B相为乙腈,流速为0.35mL/min,梯度洗脱的程序如下:
本发明提供的姜黄的乙酸乙酯提取物具有以下优点:
1、本发明提供了一种姜黄的乙酸乙酯提取物,该提取物是姜黄经过石油醚浸泡,然后渗滤,回收的残渣经过醇提,乙酸乙酯萃取得到的,经过检测,其含香豆酸,姜黄酮,双去甲氧基姜黄素,去甲基姜黄素,姜黄素,1-(4-羟基-3,5-二甲氧基苯基)-7-(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮。
2、本发明应用超高效液相色谱-质谱联机分析技术及结果构建姜黄提取物指纹图谱的方法。该方法包括姜黄的乙酸乙酯提取物的UPLC-MS分析条件。色谱峰的特征、色谱组分的质谱提供的化合物结果信息等。本发明公开的指纹图谱及其技术可用于姜黄的品种鉴别及姜黄提取物的质量监控。
3、本发明首次提供了姜黄的乙酸乙酯提取物在制备抗EGRF的药物中的应用,经过体外试验,发现本发明提供的姜黄的乙酸乙酯提取物可抑制EGFR,效果明显。
[附图说明]
图1为姜黄的乙酸乙酯提取物的UPLC-Q-TOF-MS正源总离子流图;
图2为姜黄的乙酸乙酯提取物的UPLC色谱图。
[具体实施方式]
为了使本发明的目的、技术方案及优点更加清楚明白,对本发明进行进一步详细说明。本申请中的生产设备都是本领域的常用设备,应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1:
姜黄的乙酸乙酯提取物
姜黄粉碎成粗粉,过35目筛,称取500g姜黄粗粉,然后用1500ml的石油醚浸泡24h,然后用石油醚渗滤,至渗漉液颜色变浅(约收取渗滤液2500ml),回收残渣,挥干残渣中的石油醚,然后用80%乙醇回流提取2次,每次乙醇的用量为2500ml,每次提取时间为2小时,合并提取液,将其减压浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干得乙酸乙酯提取物4.47g(收率为1.49%)。
实施例2:
姜黄的乙酸乙酯提取物
姜黄粉碎成粗粉,过20目筛,称取500g姜黄粗粉,然后用2500ml的石油醚浸泡48h,然后用石油醚渗漉至渗漉液颜色变浅(约收取渗滤液3000ml),回收残渣,挥干残渣中的石油醚,然后用75%乙醇回流提取2次,每次用2000ml的乙醇,每次提取时间为2小时,合并提取液,然后浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干得乙酸乙酯提取物7.45g(收率为2.48%)。
实施例3:
姜黄的乙酸乙酯提取物
姜黄粉碎成粗粉,过25目筛,称取500g姜黄粗粉,然后用2000ml的石油醚浸泡24h,然后用石油醚渗漉,至渗漉液颜色变浅(约收取渗滤液5000ml),回收残渣,挥干残渣中的石油醚,然后用70%的乙醇回流提取2次,每次用1500ml的乙醇,每次提取时间为2小时,合并提取液,然后浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干得乙酸乙酯提取物9.92g(收率为1.99%)。
实施例4:
姜黄的乙酸乙酯提取物
姜黄粉碎成粗粉,过30目筛,称取500g姜黄粗粉,然后用2500ml的石油醚浸泡36h,然后用石油醚渗漉,至渗漉液颜色变浅(约收取渗滤液3500ml),回收残渣,挥干残渣中的石油醚,然后用90%乙醇回流提取2次,每次用2500ml的乙醇,每次提取时间为2小时,合并提取液,然后浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干得乙酸乙酯提取物9.07g(收率为2.12%)。
实施例5:
姜黄的乙酸乙酯提取物
姜黄粉碎成粗粉,过20目筛,称取500g姜黄粗粉,然后用2000ml的石油醚浸泡48h,然后用石油醚渗漉,至渗漉液颜色变浅(约收取渗滤液3000ml),回收残渣,挥干残渣中的石油醚,然后用60%乙醇回流提取2次,每次用2000ml的乙醇,每次提取时间为2小时,合并提取液,然后浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层清液,蒸干得乙酸乙酯提取物9.81g(收率为2.02%)。
实施例6:
含姜黄的乙酸乙酯提取物的胶囊
1、组成:姜黄的石油醚提取物(实施例1的方法制备)50g,淀粉15g,硬脂酸镁5g。
2、制备方法:按照配比称取原辅料,混合均匀,分装胶囊。
实施例7:
含姜黄的乙酸乙酯提取物的片剂
1、组成:姜黄的乙醇提取物(实施例2的方法制备)50g,淀粉25g,微晶纤维素20g,硬脂酸镁5g。
2、制备方法:按照配比称取原辅料,混合均匀,分装胶囊。
实施例8:
含姜黄的乙酸乙酯提取物的颗粒剂
1、组成:姜黄的乙酸乙酯提取物(实施例3的方法制备)50g,淀粉15g,微晶纤维素20g,硬脂酸镁6g。
2、制备方法:按照配比称取原辅料,按照等量递加法混合均匀,直接压片。
实验例9:
药效实验
1、实验材料:
EGFR激酶(Invitrogen,USA);HTRF KinEASE-TK(Cisbio,USA);ATP、MgCl2、MnCl2、DTT(国产)。
姜黄的乙酸乙酯提取物溶液:将实施例1制备得到的姜黄乙酸乙酯提取物用蒸馏水溶解,浓度为10ug/mL;
阳性对照药:Staurosporine(十字孢碱),其浓度为10ug/mL。
2、实验分组:
阳性对照组:十字孢碱,浓度为10μg/mL;
实验组:实施例1-5组,浓度为10μg/mL。
3、实验模型:
以384孔板为实验容器每孔加入20μL缓冲液,10μL反应底物,10μLVEGFR-2激酶,10μL ATP。在37℃孵箱中孵育30min。然后,依次加入25μL链激酶素标记的XL-665及25μL EuK标记的抗磷酸化的酪氨酸激酶抗体,室温反应用60min。荧光信号参数设置如下:Em=670nm,Ex1=670nm.Ex2=612nm,lagtime=150s,integration time=500s,gain=150。反应设3复孔,检测时每孔读数10次设阴性对照及空白对照。抑制率计算:抑制率/%=(A标准-A样品)/(A标准-A空白)×100。EGRF对照组用去离子水代替待评价样品,空白对照组用去离子水代替评价样品并且用反应缓冲液代替EGRF。
4、实验结果见表2,表3:
表2姜黄各提取部位对EGFR的抑制率及IC50值
表3:半数抑制浓度IC50LogCon(ug/mL)
表2结果显示:半数抑制浓度越低,其抗体的灵敏度越高,表3中实施例4的IC50为3.621,为五个实例中IC50最小的一种,说明其抗EGFR效果较好)。
实验例2:
姜黄的乙酸乙酯提取物超高效液相色谱-四级杆飞行时间质谱联用分析系统(即UPLC-Q-TOF-MS)分析
1、实验部分
1.1试剂与样品
乙腈为色谱纯,美国Fisher公司;
超纯水,实验室ELGA PURELAB Classic-UVF纯水机,英国;
其他试剂均为市售国产分析纯。
1.2样品处理
姜黄粉碎成粗粉,过35目筛,称取500g姜黄粗粉,然后用1500ml的石油醚浸泡24h,用石油醚渗滤至渗漉液颜色变浅(约收取渗滤液2500ml),回收残渣,将残渣挥干石油醚,然后用80%乙醇回流提取2次,每次乙醇的用量为2500ml,每次提取时间为2小时,合并两次醇提液,将其减压浓缩至无醇味,然后用与浓缩液等体积的乙酸乙酯萃取,收集上层提取液,蒸干得乙酸乙酯提取物。
1.3仪器与条件
美国Waters AcquityTM UPLC/Q-TOF Premier;MassL-ynxV4.1工作站。
色谱条件同表1,即:
质谱条件:ESI离子源,正离子模式检测;雾化器N2压力50psi;干燥气N2,流速15L/min;质量扫描范围50--1200;自动3级质谱。(自动选择最强离子进行源内诱导裂解)。
2、结果与分析:结果见图1-2和表4
图1为姜黄乙酸乙酯提取物UPLC-Q-TOF-MS总离子流图。
针对图1即姜黄的乙酸乙酯提取物的UPLC-Q-TOF-MS(﹢)分析的总离子流图,分析其色谱峰组分指纹信息,结果见图2和表4
表4:姜黄乙酸乙酯提取物中活性组分分析
表3内容显示:经查阅中外文献,分析鉴定出姜黄乙酸乙酯提取物中的9个色谱峰,其中6个峰的峰号分别代表:1为香豆酸、2为姜黄酮、3为1-(4-羟基-3,5-二甲氧基苯基)-7-(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮、4为双去甲氧基姜黄素、5为去甲基姜黄素、6为姜黄素,其余为3个未知成分。
结果表明:姜黄的乙酸乙酯提取物含有姜黄素类化合物,也是抗EGFR活性的主要成分。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (8)
1.一种姜黄的乙酸乙酯提取物,其特征在于该提取物含有主要成分:香豆酸,姜黄酮,双去甲氧基姜黄素,去甲基姜黄素,姜黄素,1-(4-羟基-3,5-二甲氧基苯基)-7-(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮,所述的提取物的提取方法由以下步骤组成:
a.将姜黄加入到石油醚中浸泡12~48小时,每100g姜黄需要300~500毫升的石油醚,然后渗滤回收残渣;
b.所述的残渣用浓度为60%~90%的乙醇分2~3次提取,每100克残渣每次需要300~500毫升的乙醇,每次提取的时间是1~3小时,收集得到提取液;
c.所述的提取液进行浓缩得到浓缩液;
d.用与浓缩液等体积的乙酸乙酯对所述的浓缩液进行萃取,收集乙酸乙酯的萃取液;
e.蒸干所述的萃取液得到提取物。
2.如权利要求1所述的一种姜黄的乙酸乙酯提取物,其特征在于每100g姜黄需要300毫升的石油醚浸泡。
3.如权利要求1所述的一种姜黄的乙酸乙酯提取物,其特征在于所述的浸泡时间为18~36小时。
4.如权利要求1所述的一种姜黄的乙酸乙酯提取物,其特征在于所述的乙醇浓度为70~90%。
5.如权利要求1所述的一种姜黄的乙酸乙酯提取物,其特征在于所述的浸泡时间为24小时,所述的步骤(b)中的提取次数为2次,所述的乙醇浓度为80%,每100克残渣每次需要300毫升的乙醇。
6.一种药物制剂,由权利要求1所述的姜黄的乙酸乙酯提取物和药学上可接受的载体组成。
7.权利要求1所述姜黄的乙酸乙酯提取物在制备抑制EGFR药物中的应用。
8.一种检测权利要求1所述姜黄的乙酸乙酯提取物的方法,用超高效液相色谱-电喷雾串联质仪对所述姜黄提取物进行分析,以获得乙酸乙酯提取物的UPLC-Q-TOF-MS图谱,其特征在于:
a.质谱条件为:ESI离子源,正离子模式检测;雾化器N2压力为50psi;干燥气N2流速为15L/min;质量扫描范围50--1200;自动3级质谱;
b.色谱分析条件为:色谱柱:Waters ACQUITY UPLC TM BEH C18 Column 50mm×2.1mm.i d.,1.7μm;二元梯度洗脱,A相为0.1%甲酸水溶液;B相为乙腈,流速为0.35mL/min,梯度洗脱的程序如下:
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