CN115054621A - 盐肤木果实或其提取物防治骨质疏松的用途 - Google Patents
盐肤木果实或其提取物防治骨质疏松的用途 Download PDFInfo
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Abstract
本发明公开了盐肤木果实或其提取物在制备治疗和/或预防骨质疏松的药物或保健食品中的应用;本发明通过细胞实验对盐肤木果实的提取物预防核因子κB受体活化因子配体诱导的破骨细胞生成进行了验证;实验结果证实盐肤木果实的乙醇提取物对核因子κB受体活化因子配体诱导引起的破骨细胞分化有显著的抑制作用;且盐肤木果实是食用植物调味料,安全性高,且分布较为广泛、原料易得、生长要求低;综合这些原因,盐肤木果实或其提取物在制备防治骨质疏松的药物和保健食品方面拥有较好的开发利用前景。
Description
技术领域
本发明涉及盐肤木或其提取物在制备治疗和/或预防骨质疏松的保健食品或药品中的用途,属于食品或医药技术领域。
背景技术
人类的骨骼是不断更新的,成骨细胞和破骨细胞的动态平衡是维持人类骨骼健康的重要因素。然而,当这一稳态发生失衡,会导致骨溶性疾病的发生,例如骨质疏松症、骨硬化等。骨质疏松症的主要表现有骨组织微结构恶化、骨密度降低、脆性骨折易感性增加。骨质疏松症主要分为原发性骨质疏松和继发性骨质疏松,其潜在作用机制是骨丢失快于骨形成。该疾病多发于绝经期妇女和老年人,随着医疗技术的发展,人们的平均寿命延长,老龄化社会进程加快,骨质疏松症患病率快速上升,这一情况已在世界范围内成为一个严重的公众健康问题。由于破骨细胞的过度激活在骨质疏松症病理中起到重要作用。因此,对于骨质疏松症的治疗,可在不引起毒副作用的前提下,抑制破骨细胞的生成。核因子κB受体活化因子配体(RANKL)通过募集肿瘤坏死子受体相关因子6(TRAF6),激活一系列下游级联反应,进而促进形成破骨细胞。目前,治疗骨质疏松较为常见的代表性药物有双膦酸盐和雌激素等,但是这些物质经使用后,人体可能会出现不同程度的副作用和并发症,如颌骨坏死和非典型股骨骨折。因此,开发副作用较低且能够预防或治疗骨质疏松的新产品是十分重要的。
盐肤木(Rhus chinensis Mill.)是漆树科盐肤木属的一种,是中国主要经济树种,可供制药和作工业染料的原料。其果实常被用作饮料、香料和食用果油的天然来源。根据民间记载,盐肤木果实还具有良好的药用价值,其根、叶、花及果均可入药,有清热解毒、舒筋活络、散瘀止血、涩肠止泻之效。但是,目前尚未有关于盐肤木果实或其提取物对核因子κB受体活化因子配体诱导的破骨细胞分化的报道。
发明内容
本发明提供了盐肤木果实或其提取物在医药和保健食品方面的新用途,具体而言就是将盐肤木果实或其提取物应用在制备预防和/或治疗骨质疏松的药物和保健食品中。
本发明通过核因子κB受体活化因子配体诱导RAW264.7细胞向破骨细胞分化的细胞模型,发现盐肤木果实对核因子κB受体活化因子配体诱导的破骨细胞分化具有较好的预防作用,本发明为进一步开发利用盐肤木果实作为防治骨质疏松的药物或功能食品提供了新的用途。
本发明所述的治疗和/或预防骨质疏松的药物或食品的成分(或有效成分)为盐肤木果实或其提取物,还可以加入一种或多种药物或食品上可接受的辅料,以改善药物或食品吸收效果或便于服用,如制成胶囊或丸剂、粉剂、片剂、粒剂、口服液和注射液等,即制成药剂学上适宜的使用剂型,或食品领域适宜的食用方式。
本发明中盐肤木果实提取物是按常规提取方法制得,包括但不限于是80%乙醇的提取物。
与现有技术相比,本发明具有如下优点:
1、本发明为盐肤木果实发掘了新的保健食品或医疗用途,开拓了一个新的研究领域;对盐肤木果实防治骨质疏松进行的系列研究,证实了盐肤木果实提取物具有抑制破骨细胞生成的功能,盐肤木果实80%乙醇提取物在较低剂量下可以有效地抑制核因子κB受体活化因子配体诱导的RAW264.7细胞向破骨细胞分化;因此可用于防治骨质疏松的药物和保健食品的制备与开发;
2、本发明中盐肤木果实是可食用的天然植物原材料,盐肤木植株生命力顽强,适应力强,在我国的分布广泛,原料易得;同时在日常生活中,盐肤木果实常作为食用调味料;此外,盐肤木果油也是新食品原料,具有较高的安全性;同时,其提取和制备的工艺流程简单、具有较好的市场前景、蕴藏着较好的社会和经济效应。
附图说明
图1为盐肤木果实乙醇提取物对核因子κB受体活化因子配体诱导破骨细胞生成的TRAP染色结果;其中K是对照组,M是核因子κB受体活化因子配体诱导的模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL);
图2为盐肤木果实乙醇提取物对核因子κB受体活化因子配体诱导破骨细胞生成的TRAP活力测定结果;其中A图为图1中各组TRAP阳性细胞的相对定量结果,B图为TRAP活性检测结果;图中K是对照组,M是核因子κB受体活化因子配体诱导的模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL);
图3为盐肤木果实乙醇提取物通过NF-κB信号通路抑制核因子κB受体活化因子配体诱导的破骨细胞生成的蛋白免疫印迹结果;其中A图为β-Actin、p-NF-κB、NF-κB、p-IκBα、IκBα的免疫印迹条带结果;B图为p-NF-κB/NF-κB的灰度相对定量结果;C图为p-IκBα/IκBα的灰度相对定量结果;图中K是对照组,M是核因子κB受体活化因子配体诱导的模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL);
图4为盐肤木果实乙醇提取物通过MAPKs和Akt信号通路抑制核因子κB受体活化因子配体诱导的破骨细胞生成的蛋白免疫印迹条带结果;图中K是对照组,M是核因子κB受体活化因子配体诱导模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL);
图5为盐肤木果实乙醇提取物通过MAPKs和Akt信号通路抑制核因子κB受体活化因子配体诱导的破骨细胞生成的蛋白免疫印迹灰度相对定量结果;其中A图为p-ERK/ERK的灰度相对定量结果;B图为p-JNK/JNK的灰度相对定量结果;C图为p-p38/p38的灰度相对定量结果;D图为p-Akt/ Akt的灰度相对定量结果;图中K是对照组,M是核因子κB受体活化因子配体诱导模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL);
图6盐肤木果实乙醇提取物抑制破骨细胞生成过程中c-Fos和NFATc1蛋白的表达,其中A图为c-Fos和NFATc1的免疫印迹条带结果;B图为c-Fos/β-Actin的灰度相对定量结果;C图为NFATc1/β-Actin的灰度相对定量结果;图中K是对照组,M是核因子κB受体活化因子配体诱导模型组,RL是低剂量组(50µg/mL),RH是高剂量组(100µg/mL)。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂,如无特殊说明,均为常规市售试剂或按常规方法配制的试剂,实施例中使用方法,如无特殊说明均为常规实验方法。
实施例1:盐肤木果实提取物的制备
盐肤木果实提取物是采用质量浓度80%的乙醇溶液进行超声提取,提取温度为25℃,料液比g:mL为1:5,提取时间30min,过滤,滤渣再添加80%的乙醇溶液提取一次,收集合并滤液,滤液浓缩冻干制得。
实施例2:盐肤木果实提取物对核因子κB受体活化因子配体诱导的破骨细胞分化的影响实验
应用核因子κB受体活化因子配体体外刺激RAW264.7细胞向破骨细胞分化的方法,建立破骨细胞生成的模型,通过细胞模型实验观察盐肤木果实的80%乙醇提取物对核因子κB受体活化因子配体诱导的破骨细胞生成的防治作用;RAW264.7细胞,购自昆明动物所细胞库;
1、破骨细胞生成的模型构建
将RAW264.7细胞接种在含10%胎牛血清、1%青霉素/链霉素和50ng/mL核因子κB受体活化因子配体的DMEM培养基中,在37℃、5%CO2的培养箱里培养,每隔2d用上述新鲜培养液置换一次培养基;5天后,RAW264.7细胞分化为破骨细胞,完成模型构建。
2、80%盐肤木果实提取物对破骨细胞生成的抑制作用
空白组(K组)为将RAW264.7细胞培养在含10%胎牛血清、1%青霉素/链霉素的DMEM培养基中;模型组(M组)为将RAW264.7细胞培养置在含10%胎牛血清、1%青霉素/链霉素和50ng/mL核因子κB受体活化因子配体的DMEM培养基中;低剂量组(RL组)是在模型组的基础上,培养基中加入50µg/mL的80%盐肤木果实乙醇提取物,对RAW264.7细胞进行培养;高剂量组(RH组)是在模型组的基础上,培养基中加入100µg/mL的80%盐肤木果实乙醇提取物,对RAW264.7细胞进行培养;以上各组均培养5天,隔天换液。
(1)TRAP染色
将用上述方式培养5天后的各组细胞采用TRAP染色试剂盒(购自上海哈灵生物科技有限公司)进行染色,参照试剂盒说明书中方法进行;
各实验组TRAP染色结果如图1和图2A所示;M组RAW264.7细胞分化生成了大量的TRAP阳性破骨细胞,而加入了50µg/mL (RL组)和100µg/mL (RH组) 盐肤木果实乙醇提取物后,TRAP阳性破骨细胞的生成受到了不同程度的抑制,与M组相比,RL组和RH组的TRAP阳性破骨细胞量均显著降低。
(2)TRAP活力测定
TRAP活性测定采用抗酒石酸酸性磷酸酶(TRAP)活性测定试剂盒(购自上海碧云天生物技术有限公司),实验参照试剂盒说明书进行;结果见图2B,从图中可以看出M组的TRAP活性较其他三组显著上升,且RH组的TRAP活力与K组相当,这与TRAP染色结果一致。这些结果表明,无论是高浓度还是低浓度,盐肤木果实乙醇提取物均能有效地抑制RAW264.7细胞向破骨细胞分化。
(3)盐肤木果实提取物通过MAPKs、NF-κB和Akt信号通路抑制核因子κB受体活化因子配体诱导的破骨细胞生成
通过蛋白免疫印迹法研究和分析了盐肤木果实乙醇提取物对核因子κB受体活化因子配体诱导破骨细胞生成的分子抑制机制;
将培养5天后的细胞用PBS清洗两次,然后向其中加入含有1mmol/L磷酸酶抑制剂(PMSF,购自上海碧云天生物技术有限公司)的高效组织裂解液(购自上海碧云天生物技术有限公司),然后吸到离心管中,用组织匀浆仪进行低温均质破碎裂解,裂解完全后,离心(4℃、10000g,10min),收集上清液。用蛋白定量检测试剂盒(购自上海碧云天生物技术有限公司)测定各组提取液中蛋白的含量,用以后续的蛋白免疫印迹,以保证各组提取液中蛋白含量相当。然后利用蛋白免疫印迹法检测以下指标:β-Actin、NF-κB、p-IκBα、IκBα、p-JNK、JNK、p-ERK、ERK、p-p38、p38、Akt、p-Akt(以上抗体均购自武汉ABclonal)、p-NF-κB(抗体购自美国Affinity )。蛋白免疫印迹的操作如下:向测定完蛋白含量的各组提取液中加入总体积1/4的5×Loading buffer,煮沸10min,冷却后储藏在-30℃,上样之前对各组样品进行稀释,使得最终上样量为10µL,上样浓度为3µg/µL;接着制胶、灌胶、上样和跑胶;结束后进行转膜;转膜完毕后进行封闭、敷一抗、敷二抗和洗膜;最后使用ECL发光进行化学显色,得到蛋白条带;
结果如图3、图4和图5所示,结果显示,M组的p-NF-κB、p-IκBα、p-ERK、p-JNK、p-p38以及p-Akt的表达量较K组显著上升,除了RL组的p-p38的表达量与M组无显著性差异以外,RL组和RH组的上述蛋白表达量较M组都有明显的下降,这说明盐肤木果实乙醇提取物通过抑制MAPKs、NF-κB和Akt信号通路来抑制核因子κB受体活化因子配体诱导的破骨细胞的形成。这些结果进一步阐述了盐肤木果实提取物抑制核因子κB受体活化因子配体诱导的破骨细胞形成的作用机制。
(4)盐肤木果实提取物抑制核因子κB受体活化因子配体诱导的破骨细胞中c-Fos和NFATc1的表达
蛋白提取方法与步骤以及蛋白免疫印迹操作步骤与步骤(3)一致,利用蛋白免疫印迹法检测以下指标:β-Actin、c-Fos和NFATc1;
在破骨细胞生成的过程中,c-Fos是RANKL重要的下游调控因子,它促进破骨细胞的形成主要通过激活下游因子NFATc1来实现。NFATc1和c-Fos是破骨细胞形成的关键转录调节因子。如图6所示,M组的c-Fos和NFATc1的表达量较K组的均显著上调。而加入了不同浓度的盐肤木果实乙醇提取物后,与M组相比,RL组和RH组的c-Fos和NFATc1的表达量均明显降低;这表明NFATc1和c-Fos也是盐肤木果实抑制破骨细胞生成的潜在作用靶点。根据这一结果,我们可得出结论:盐肤木果实乙醇提取物可降低核因子κB受体活化因子配体诱导的破骨细胞生成过程中NFATc1和c-Fos的蛋白水平;进一步表明盐肤木果实乙醇提取物能够有效的抑制破骨细胞的生成。
综上所述:可以确定盐肤木果实提取物可以有效地治疗和/或预防骨质疏松。因此,盐肤木果实或其提取物可以用来开发防治骨质疏松的药物或保健食品。
Claims (2)
1.盐肤木果实或其提取物在制备治疗和/或预防骨质疏松的药物中的应用。
2.盐肤木果实或其提取物在制备治疗和/或预防骨质疏松的保健食品中的应用。
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