CN112195216B - 一种基于细菌模型评价活性化合物抗氧化应激效益的方法 - Google Patents
一种基于细菌模型评价活性化合物抗氧化应激效益的方法 Download PDFInfo
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Abstract
本发明公开一种基于细菌模型评价活性化合物抗氧化应激效益的方法,包括以下步骤:(1)大肠杆菌的培养;(2)分组、氧化应激诱导、抗氧化物处理,建立空白对照组、模型组以及测试组,孵育后离心收集大肠杆菌;(3)建立甲酰赖氨酸的液质联用检测方法;(4)测定上述各组的甲酰赖氨酸含量;(5)测试组的抗氧化应激评价。本发明通过建立大肠杆菌的氧化应激模型,利用甲酰赖氨酸的含量评价氧化应激水平。与现有的技术相比,大肠杆菌培养操作简单、价格低廉,降低了测试成本,液质联用技术检测甲酰赖氨酸可直接反应氧化应激水平,并且该技术能够实现高通量检测,大大降低人力成本,结果也更准确可靠。
Description
技术领域
本发明涉及活性化合物药效及副作用评估技术领域,特别是涉及一种基于细菌模型评价活性化合物抗氧化应激效益的方法。
背景技术
细胞呼吸、酶的催化过程、巨噬细胞吞噬等一系列生物过程都会产生活性氧,低浓度的活性氧在灭杀侵入性病原体、伤口愈合以及组织修复等方面发挥着积极的作用。然而,如果活性氧超负荷生成将打破生物体内的氧化物质和还原物质之间的平衡,形成氧化应激的环境,最终导致氧化性组织损伤。维护体内氧化物质和还原物质之间的平衡,增加体内抗氧化物质水平,是对抗氧化应激的有效途径。
在“大健康”领域中,提倡通过健康饮食的生活方式预防慢性疾病。“健康食品”一般富含天然抗氧化成分,能够对抗体内氧化应激,改善体内的组织损伤情况。虽然大部分“健康食品”体外检测表明有抗氧化作用,但尚需要有效的评价方法。目前对抗氧化剂的健康评价基本上是基于“衰老及慢性病的自由基学说”,采用整体动物血液或肝脏抗氧化酶活性分析法,或者用体外肝癌细胞(例如HeGp2)或者红细胞做模型,考察抗氧化剂对化学自由基引发剂所产生的活性氧自由基(ROS)的清除及对细胞膜的保护能力。但这些方法需要进行细胞培养,或者进行动物实验,使用细胞模型或者动物模型,建模所需时间较长,费用较高,对实验技能要求高,需要较多的时间和人力成本。
发明内容
本发明的目的是提供一种基于细菌模型评价活性化合物抗氧化应激效益的方法,以解决上述现有技术存在的问题。
为实现上述目的,本发明提供一种基于细菌模型评价活性化合物抗氧化应激效益的方法,包括以下步骤:
(1)大肠杆菌的培养:将大肠杆菌菌种加入无菌液体培养基中培养,待大肠杆菌生长进入对数期,离心后收集大肠杆菌,等渗溶液清洗后重悬于等渗溶液中,测定大肠杆菌死亡率小于20%,得到大肠杆菌悬浮液;
(2)分组、氧化应激诱导以及测试组给药处理:将步骤(1)所述大肠杆菌悬浮液均分为空白对照组、氧化应激诱导组、氧化应激诱导并给药测试组后,分别在37℃孵育2小时,测定大肠杆菌死亡率小于70%,离心后去上清,收集下层大肠杆菌;
(3)大肠杆菌中甲酰赖氨酸的提取:将步骤(2)中收集的各组大肠杆菌重悬后制样处理,得到甲酰赖氨酸提取液样本;
(4)测试品的抗氧化应激效果判别:测量步骤(3)中提取的各组甲酰赖氨酸提取液样本中的甲酰赖氨酸的含量并比较,进行统计学分析,若测试组比氧化应激诱导组中甲酰赖氨酸含量明显降低,则测试组的活性化合物具有抗氧化应激功能。
进一步的,在步骤(1)和步骤(2)中,所述测定大肠杆菌死亡率的方法为细胞染色法。
进一步的,在步骤(1)和步骤(2)中,所述空白对照组是向大肠杆菌悬浮液中加入大肠杆菌等渗溶液;所述氧化应激诱导组是向大肠杆菌悬浮液中加入氧化应激诱导剂,所述氧化应激诱导剂为诱导剂与大肠杆菌等渗溶液的混合物;所述氧化应激诱导并给药测试组是向大肠杆菌悬浮液中加入氧化应激诱导剂和活性化合物混合溶液。
进一步的,在步骤(2)中,所述空白对照组、氧化应激诱导组、氧化应激诱导并给药测试组的任意一组设置平行样品,所述平行样品数量大于两个,大肠杆菌在任意一个所述平行样品中的体积比为 1%-5%。
进一步的,在步骤(2)中,所述活性化合物是从植物或微生物中提取的一种或几种天然产物;所述大肠杆菌等渗溶液为无菌PBS缓冲液;所述诱导剂与大肠杆菌等渗溶液的混合物为过氧化氢溶液与无菌PBS缓冲液的混合溶液或次氯酸溶液与无菌PBS缓冲液的混合溶液。
进一步的,在步骤(3)中,所述制样处理过程具体步骤为:
(a)将所述步骤(2)中收集到的大肠杆菌样品重悬于无菌PBS 缓冲液中,在-80℃和常温之间反复冻融3次,然后在4℃超声,使大肠杆菌细胞完全破碎,得到细胞破碎液;
(b)向步骤(a)中的细胞破碎液中加入4倍体积的-20℃预冷提取液,所述提取液是乙腈、丙酮与水按照体积比4:4:2混合得到的混合液,然后涡旋振荡30秒,充分混匀得到的细胞破碎液的混合提取液;
(c)将步骤(b)中的混合提取液在4℃,10000-12000rpm的条件下离心10分钟,将上清液真空浓缩冷冻干燥得到粗提物;
(d)向步骤(c)中的粗提物中加入乙腈水溶液,充分溶解后加入氨水溶液和FMOC乙腈溶液,室温震荡后,在4℃10000-12000rpm 离心10分钟,取上清并用水相滤膜过滤得到滤液,所述滤液用于分析甲酰赖氨酸含量。
进一步的,在步骤(4)中,所述统计学分析为判断给药后甲酰赖氨酸的降低是否具有显著性,对测试样品的抗氧化应激效果进行判别,各个平行样品之RSD小于20%。
本发明公开了以下技术效果:本发明公开的一种基于细菌模型评价活性化合物抗氧化应激效益的方法,针对现有技术费时费力、人力成本高的缺陷,建立了一种体外评价活性化合物抗氧化应激效用的方法体系,该体系的方法可高通量进行,该体系的定量指标可广泛用于植物、微生物中提取的天然产物的抗氧化应激的效用分析,从而达到指导人民通过膳食、“健康食品”预防、缓解慢性疾病的目的。
本发明一种基于细菌模型评价活性化合物抗氧化应激效益的方法,相关领域的缺陷在于,使用细胞模型或者动物模型,建模所需时间较长,费用较高,对实验技能要求高。本发明采用大肠杆菌为模式生物,大肠杆菌培养操作简单、价格低廉,降低了测试成本,液质联用技术检测甲酰赖氨酸可直接反应氧化应激水平,并且该技术能够实现高通量检测,大大降低人力成本,结果也更准确可靠。
具体实施方式
下面将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。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施方式对本发明作进一步详细的说明。
实施例1噻唑脯氨酸的抗氧化应激效益评价
1.1大肠杆菌悬浮液的制备
配置大肠杆菌无菌液体培养基,按照1:100000的体积比进行接种,37℃、220rpm恒温振荡培养,待含大肠杆菌培养液的OD值(600nm) 大于0.4,收集培养的大肠杆菌,用无菌PBS洗涤3遍,之后大肠杆菌重悬于无菌PBS溶液中,得到含大肠杆菌1-5%的悬浮液,细胞染色法测定大肠杆菌死亡率低于20%。
1.2分组、氧化应激诱导及供试品给药处理
将大肠杆菌悬浮液分为空白对照组、氧化应激诱导组、氧化应激诱导并给药测试组,每组三个平行样本,每个样本体积为25ml,以过氧化氢终浓度为1uM的PBS溶液为氧化应激诱导剂,不加诱导剂的为空白对照组,加入诱导剂的为氧化应激诱导组,同时加入诱导剂和噻唑脯氨酸的为氧化应激诱导并给药测试组。噻唑脯氨酸溶解在50%的DMSO水溶液中,再用PBS等渗溶液稀释后添加到大肠杆菌悬浮液,使得噻唑脯氨酸的最终浓度为1mg/ml;无菌条件下孵育2小时,细胞染色法测定大肠杆菌死亡率低于70%,然后4000rpm、4℃离心15分钟,去上清,下层大肠杆菌收集用于下一步分析。
1.3大肠杆菌生化分析和样品制备
1.3.1甲酰赖氨酸的提取和样品制备
(1)收集的大肠杆菌重悬于200uLPBS溶液中,在-80℃和常温之间反复冻融3次,接着在4℃超声30秒,使大肠杆菌完全破碎;
(2)向大肠杆菌破碎液中加入4倍体积的预冷提取液,该提取液的组成为乙腈:丙酮:水(4:4:2);然后涡旋振荡30秒,充分混匀;
(3)在4℃温度下11000rpm离心10min,取上清,上清利用超低温真空浓缩仪真空冷冻干燥得到粗提物;
(4)向粗提物中加入100uL乙腈水溶液(50%),充分溶解后,加入5uL的氨水溶液,20uL的50mM的FMOC乙腈溶液,室温震荡2 分钟后,在4℃、11000rpm离心10分钟,取出上清并用0.45μm水相滤膜过滤,滤液用于采用UPLC-MS/MS方法分析甲酰赖氨酸含量。
1.3.2甲酰赖氨酸浓度的精确测定
可以利用UPLC-MS/MS方式进行,检测系统可以选择AB Sciex Triple QuadTM5500LC-MS分析系统(AB Sciex Pte.Ltd.),UPLC色谱柱可以为ACQUITY UPLC Peptide HSS T3C18 Column:2.1×50mm, 1.8μm;选择0.1%甲酸水溶液作为A相,B相选择乙腈,洗脱程序为: 0min 90%A 10%B;1.5min 60%A 40%B;3.1min 10%A 90%B;4min 90%A 10%B;5min 90%A 10%B,进样体积5μL,流速设定为 0.6mL/min。用MS/MS的多反应监测正离子模式检测甲酰赖氨酸,定量的条件是甲酰赖氨酸衍生物(FL-FMOC)母离子m/z 397,子离子 m/z 112。
1.4测试品的抗氧化应激效果判别
比较氧化应激有道并给药测试组的甲酰赖氨酸的含量与氧化应激诱导组的甲酰赖氨酸的含量是否有所降低,并通过统计学判断降低是否具有显著性,对测试品的抗氧化应激效果进行判别。
1.5实验结果
1.5.1甲酰赖氨酸UPLC-MS/MS方法的线性区间研究
表1甲酰赖氨酸的标准曲线结果
由表1可以看出,甲酰赖氨酸的UPLC-MS/MS方法在0.1到 500ng/ml有较好的线性区间,落在该区间的检测结果准确、可靠。
1.5.2甲酰赖氨酸浓度及比例分析结果
表2噻唑脯氨酸对大肠杆菌甲酰赖氨酸含量及比例的影响结果
1.5.3测试品的抗氧化应激效果判别
表3噻唑脯氨酸抗氧化应激效益评价结果
由表2、表3可以看出,实验各分组组内差异较小,重现性好;噻唑脯氨酸能有效降低氧化应激水平,且效果显著(p值小于0.05)。
本发明所采用的各种材料均可以由商业购得,或根据现有技术的相关手段自行准备。本发明中大肠杆菌等渗溶液为PBS缓冲液。本发明中未特殊标明温度的操作均在常温下进行,常温满足常规定义,即 20℃-25℃。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.一种基于细菌模型评价活性化合物抗氧化应激效益的方法,其特征在于,包括以下步骤:
(1)大肠杆菌的培养:将大肠杆菌菌种加入无菌液体培养基中培养,待大肠杆菌生长进入对数期,离心后收集大肠杆菌,等渗溶液清洗后重悬于等渗溶液中,测定大肠杆菌死亡率小于20%,得到大肠杆菌悬浮液;
(2)分组、氧化应激诱导以及测试组给药处理:将步骤(1)所述大肠杆菌悬浮液均分为空白对照组、氧化应激诱导组、氧化应激诱导并给药测试组后,分别在37℃孵育2小时,测定大肠杆菌死亡率小于70%,离心后去上清,收集下层大肠杆菌;
所述空白对照组是向大肠杆菌悬浮液中加入大肠杆菌等渗溶液;所述氧化应激诱导组是向大肠杆菌悬浮液中加入氧化应激诱导剂,所述氧化应激诱导剂为诱导剂与大肠杆菌等渗溶液的混合物;所述氧化应激诱导并给药测试组是向大肠杆菌悬浮液中加入氧化应激诱导剂和活性化合物混合溶液;
所述活性化合物为噻唑脯氨酸;所述大肠杆菌等渗溶液为无菌PBS缓冲液;所述诱导剂与大肠杆菌等渗溶液的混合物为过氧化氢溶液与无菌PBS缓冲液的混合溶液;
(3)大肠杆菌中甲酰赖氨酸的提取:将步骤(2)中收集的各组大肠杆菌重悬后制样处理,得到甲酰赖氨酸提取液样本;
(4)测试品的抗氧化应激效果判别:测量步骤(3)中提取的各组甲酰赖氨酸提取液样本中的甲酰赖氨酸的含量并比较,进行统计学分析,若测试组比氧化应激诱导组中甲酰赖氨酸含量明显降低,则测试组的活性化合物具有抗氧化应激功能。
2.根据权利要求1所述的一种基于细菌模型评价活性化合物抗氧化应激效益的方法,其特征在于:在步骤(1)和步骤(2)中,所述测定大肠杆菌死亡率的方法为细胞染色法。
3.根据权利要求1所述的一种基于细菌模型评价活性化合物抗氧化应激效益的方法,其特征在于:在步骤(2)中,所述空白对照组、氧化应激诱导组、氧化应激诱导并给药测试组的任意一组设置平行样品,所述平行样品数量大于两个,大肠杆菌在任意一个所述平行样品中的体积比为1%-5%。
4.根据权利要求1所述的一种基于细菌模型评价活性化合物抗氧化应激效益的方法,其特征在于:在步骤(3)中,所述制样处理过程具体步骤为:
(a)将所述步骤(2)中收集到的大肠杆菌样品重悬于无菌PBS缓冲液中,在-80℃和常温之间反复冻融3次,然后在4℃超声,使大肠杆菌细胞完全破碎,得到细胞破碎液;
(b)向步骤(a)中的细胞破碎液中加入4倍体积的-20℃预冷提取液,所述提取液是乙腈、丙酮与水按照体积比4:4:2混合得到的混合液,然后涡旋振荡30秒,充分混匀得到的细胞破碎液的混合提取液;
(c)将步骤(b)中的混合提取液在4℃,10000-12000rpm的条件下离心10分钟,将上清液真空浓缩冷冻干燥得到粗提物;
(d)向步骤(c)中的粗提物中加入乙腈水溶液,充分溶解后加入氨水溶液和FMOC乙腈溶液,室温震荡后,在4℃10000-12000rpm离心10分钟,取上清并用水相滤膜过滤得到滤液,所述滤液用于分析甲酰赖氨酸含量。
5.根据权利要求1所述的一种基于细菌模型评价活性化合物抗氧化应激效益的方法,其特征在于:在步骤(4)中,所述统计学分析为判断给药后甲酰赖氨酸的降低是否具有显著性,对测试样品的抗氧化应激效果进行判别,各个平行样品之RSD小于20%。
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