CN113694047A - β-石竹烯在制备抗肿瘤药物中的应用 - Google Patents
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Abstract
本发明公开β‑石竹烯在制备抗肿瘤药物中的应用。本发明提出β‑石竹烯对肿瘤细胞生长的抑制作用，并且表明β‑石竹烯能够逆转NSCLC吉非替尼耐药。本发明提出吉非替尼与β‑石竹烯联用能产生较好的协同增敏效果，能够极大程度上克服肿瘤细胞对化疗药物的耐药性。本发明对于如何减缓或逆转吉非替尼对NSCLC细胞的耐药性，减缓肿瘤的生长增值速度，减少甚至逆转吉非替尼药物的不良反应，改善NSCLC患者的临床均具有重要的意义。
Description
技术领域
本发明属于制药领域，涉及β-石竹烯在制备抗肿瘤药物中的应用，以及β-石竹烯和吉非替尼药物配伍组合物在制备抗肿瘤药物中的应用。
背景技术
癌症被认为是世界上每个国家的主要死亡原因和延长预期寿命的重要障碍。据世界卫生组织(WHO)2019年的估计，在183个国家中的112个国家中年龄为70岁之前癌症是人类第一或第二大死亡原因。总体而言，世界范围内的癌症发病率和死亡率的负担正在迅速增长。预计到2040年全球将发生2840万例新癌症病例，比2020年的1930万例增加47％。总体而言，世界范围内癌症发病率和死亡率的负担正在迅速增长。
β-石竹烯(BCP)是一种双环倍半萜型天然化合物，有α-，β-，γ-三种异构体，β-石竹烯的双键顺式异构体称为异石竹烯(isocarophyllene),国家标准GB2760-2014批准石竹烯为允许使用的食品香料。β-石竹烯英文通用名称β-Caryophyllene，分子量为204.36，其性状为无色至微黄油状液体，具有淡的丁香似香味。沸点119-121℃。β-石竹烯(BCP)是罗勒、黑胡椒、薰衣草、迷迭香、牛至、啤酒花、大麻、肉桂叶油、丁香叶油、牡荆油、陈皮油等的挥发油中主要活性成分之一。β-石竹烯的化学结构式如下：
β-石竹烯具有多种药理作用，其可减少炎症过程中促炎性细胞因子形成、降低Akt、环氧化酶的蛋白表达产生抗炎作用；可通过兴奋CB2受体及激活内源性阿片镇痛系统发挥镇痛作用；通过激动脊髓等中枢系统的CB受体,减轻脑缺血再灌注损伤，对神经变性疾病具有神经保护作用；可通过抑制肝HMG-CoA还原酶活性、下调TLR4和RAGE信号等途经减轻肝损伤、保护肝脏免受脂质损伤。BCP对多种肿瘤细胞具有细胞毒性，但关于BCP抗肿瘤作用机制尚不明确。
现有研究提示，石竹烯的药理作用具有抗炎，镇痛，神经保护，防止肝损伤，抗血糖，调节血脂等方面的应用，尚未见有抗肿瘤方面的报道。
表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)是一种特异性的EGFR酪氨酸激酶抑制剂，通过竞争性地抑制ATP与受体胞内区结合，从而抑制酪氨酸激酶活化，阻断EGFR的传导通路，达到抗肿瘤治疗的目的。
吉非替尼作为一种选择性表皮生长因子受体酪氨酸激酶抑制剂类靶向药物，被广泛应用于NSCLC的临床治疗并取得了良好的疗效，但频繁出现的耐药现象限制了其进一步的发展。其中，EGFR T790M突变是最常见的获得性耐药机制。在临床肺癌治疗中，EGFR活化突变的NSCLC患者中可检测到多达50％的EGFR T790M突变。因此，在体外进行肺癌细胞耐药机制的探索，乃至寻找能够逆转肿瘤细胞耐药的药物是目前研究的要点。
发明内容
本发明的第一个目的是针对现有技术的不足，提供β-石竹烯在制备抗肿瘤药物中的应用。
本发明的第二个目的是针对现有技术的不足，提供β-石竹烯与吉非替尼配伍的药物组合物在制备抗肿瘤药物中的应用。
本发明的第三个目的是一种药物组合物，包括β-石竹烯、吉非替尼、药剂学上可接受的辅料。
作为优选，β-石竹烯与吉非替尼的质量比为75～100：10。
所述的β-石竹烯可以采用常规的方法从天然植物中提取，也可以由合成或其他方法制得。β-石竹烯的纯度为90％。
本发明的有益效果：
本发明提出β-石竹烯对肿瘤细胞生长的抑制作用，并且表明β-石竹烯能够逆转NSCLC吉非替尼耐药。本发明提出吉非替尼与β-石竹烯联用能产生较好的协同增敏效果，能够极大程度上克服肿瘤细胞对化疗药物的耐药性。本发明对于如何减缓或逆转吉非替尼对NSCLC细胞的耐药性，减缓肿瘤的生长增值速度，减少甚至逆转吉非替尼药物的不良反应，改善NSCLC患者的临床均具有重要的意义。
附图说明
图1是HCC827细胞对不同浓度石竹烯药物的抑制率。
图2是SW-1990细胞对不同浓度石竹烯药物的抑制率。
图3是MDA-231细胞对不同浓度石竹烯药物的抑制率。
图4是HCT-116细胞对不同浓度石竹烯药物的抑制率。
图5是U251细胞对不同浓度石竹烯药物的抑制率。
图6是PC9细胞对不同浓度石竹烯药物的抑制率。
图7是对照组、吉非替尼处理组、β-石竹烯处理组和吉非替尼和β-石竹烯联合处理组下异种移植瘤的肿瘤生长曲线。
图8是四组处理组检测凋亡相关蛋白PARP,Bcl-2,mTOR蛋白，以及DNA损伤相关蛋白的表达，其中对照组记为组A、吉非替尼处理组记为组B、β-石竹烯处理组记为组C、吉非替尼和β-石竹烯联合处理组记为组D。
具体实施方式
下面结合具体实施例对本发明做进一步的分析。
实施例1：Cell Counting Kit 8(CCK-8)检测细胞增殖
(1)细胞株的选择：
选取6种不同的细胞，分别为PC9,HCC827,HCT116,SW-1990,U251,MDA-231。上述细胞中PC9,HCC827为肺癌细胞；HCT116为结直肠癌细胞；U251为胶质瘤细胞；MDA-231为乳腺癌细胞；SW-1990为胰腺癌细胞。
选择细胞生长状态良好且处于对数生长期的六株细胞，用胰酶进行消化，加入含10％FBS的完全培养基终止，吹打成细胞悬液后转至EP管，800rpm,弃上清，加入完全培养基制成细胞悬液；
(2)使用细胞计数板计数，以每组细胞5个复孔，每孔100μL体积，不同的细胞株按照不同的细胞数接种至96孔板，并在四周边缘孔加入等量的PBS溶液，防止边缘效应。HCT116,PC9,U251按照3000个细胞接种接种至96孔板，MDA-231,SW-1990按照4000个细胞接种接种至96孔板，HCC827按照5000个细胞接种接种至96孔板。
(3)37℃、5％CO2培养箱中常规培养过夜；吸去每个孔中的液体培养基，避光条件下配制不同浓度的石竹烯药物的培养液，加入每个孔中继续培养；
(4)培养24h后，避光条件下吸去孔板中的培养液，配制实验所需要的CCK8溶液，每孔加入100μL体积(加液过程中防止气泡产生，避免酶标仪测量不准确)，培养箱中孵育1-2h；
(5)将全自动酶标仪波长调整为450nm，测定每孔的OD值；
(6)根据OD值绘制各组细胞的生长曲线，采用Graphpad 7.0计算并分析细胞增殖情况。
图1是HCC827细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为54.68μg/mL。
图2是SW-1990细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为190.2μg/mL。
图3是MDA-231细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为203.1μg/mL。
图4是HCT-116细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为104.9μg/mL。
图5是U251细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为120.1μg/mL。
图6是PC9细胞对不同浓度石竹烯药物的抑制率，其中IC50浓度为165.1μg/mL。
另外通过cck-8检测β-石竹烯与吉非替尼的协同指数。结果表明：10μM吉非替尼联合75μMβ-石竹烯时，对HCC827GR细胞有较好的抑制效果；10μM吉非替尼联合100μMβ-石竹烯时，对PC9GR细胞有较好的抑制效果。
实施例2：β-石竹烯对裸鼠皮下瘤生长的影响
选择皮下种植耐药细胞系HCC827GR，将裸鼠分为四组(每组4只)设置对照组(PBS用药剂量50mg/Kg)、吉非替尼处理组(吉非替尼的用药剂量是50mg/Kg)、β-石竹烯处理组(β-石竹烯的用药剂量是50mg/Kg)、吉非替尼和β-石竹烯联合处理组(用药剂量是50mg/Kg，其中吉非替尼与β-石竹烯的质量比为10:75)进行实验。待裸鼠成瘤5天后，进行腹腔给药以肿瘤生长天数为横坐标，肿瘤体积为纵坐标，绘制生长曲线。图7实验结果发现，吉非替尼和β-石竹烯联合处理组肿瘤体积显著小于吉非替尼处理组。因此，从体内生物学功能实验结果可以表明β-石竹烯可以逆转非小细胞肺癌细胞对吉非替尼的耐药性。
实施例3:不同药物处理组对NSCLC耐药细胞蛋白表达水平的影响
选择皮下种植耐药细胞系HCC827GR，将裸鼠分为四组(每组4只)设置对照组(PBS用药剂量50mg/Kg)、吉非替尼处理组(吉非替尼的用药剂量是50mg/Kg)、β-石竹烯处理组(β-石竹烯的用药剂量是50mg/Kg)、吉非替尼和β-石竹烯联合处理组(用药剂量是50mg/Kg，其中吉非替尼与β-石竹烯的质量比为10:75)进行实验。在20天后取瘤体提取蛋白，进行Western blot检测。
通过检测凋亡相关蛋白PARP,Bcl-2,mTOR蛋白，以及DNA损伤相关蛋白的表达，见图8。结果表明，联合加药处理组凋亡蛋白及DNA损伤均有显著的变化。其中，抗凋亡蛋白Bcl-2表达减少；凋亡相关蛋白PARP，mTOR表达增多；DNA损伤蛋白p-H2AX表达减少。以上结果表明，β-石竹烯可以通过诱导细胞凋亡逆转非小细胞肺癌细胞对吉非替尼的耐药。
上述实施例并非是对于本发明的限制，本发明并非仅限于上述实施例，只要符合本发明要求，均属于本发明的保护范围。
Claims (5)
1.β-石竹烯在制备抗肿瘤药物中的应用。
2.β-石竹烯与吉非替尼配伍的药物组合物在制备抗肿瘤药物中的应用。
3.如权利要求2所述的应用，其特征在于β-石竹烯与吉非替尼的质量比为75～100：10。
4.一种药物组合物，其特征在于包括β-石竹烯、吉非替尼、药剂学上可接受的辅料。
5.如权利要求4所述的应用，其特征在于β-石竹烯与吉非替尼的质量比为75～100：10。
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