CN116139258B - 刺梨sod在制备用于预防或治疗肠道疾病药物中的用途 - Google Patents
刺梨sod在制备用于预防或治疗肠道疾病药物中的用途 Download PDFInfo
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- CN116139258B CN116139258B CN202310119234.7A CN202310119234A CN116139258B CN 116139258 B CN116139258 B CN 116139258B CN 202310119234 A CN202310119234 A CN 202310119234A CN 116139258 B CN116139258 B CN 116139258B
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Abstract
本发明提出了刺梨SOD在制备预防或治疗肠道疾病药物中的应用,对于刺梨SOD的推广应用具有重要意义。
Description
技术领域
本发明涉及食医药领域。具体地,本发明涉及刺梨SOD在制备用于预防或治疗肠道疾病药物中的用途。
背景技术
刺梨为蔷薇科植物缫丝花的果实,又名茨梨、木梨子,果皮上密生小肉刺。贵州、广西等地的山区盛产刺梨,年产高达数十万吨,目前刺梨以野生为主,有少量引种栽培,刺梨果中含有氨基酸、维生素、蛋白质及钙、钾、锌、镁、硒等矿物质,超氧化物歧化酶SOD、VC含量极高。
肠道疾病是多种肠道炎症性疾病的总称,临床表现为反复的腹痛腹胀、呕血便血、腹泻便秘、食欲降低、营养不良及各种全身并发症,严重时可能导致死亡的发生。
目前,鲜有报道刺梨SOD与肠道疾病之间具有相关性,有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此,本发明提出了刺梨SOD提取物在制备药物中的用途,该药物用于预防或治疗肠道疾病,对于刺梨SOD的推广应用具有重要意义。
在本发明的一个方面,本发明提出了刺梨SOD提取物在制备药物中的用途。根据本发明的实施例,所述药物用于预防或治疗肠道疾病。本发明的发明人发现,刺梨SOD提取物可以改善小鼠结肠状态和肠屏障功能,降低氧化应激和炎症反应,有助于预防或治疗肠道疾病。
根据本发明的实施例,所述肠道疾病选自炎症性肠病。
炎症性肠病(Inflammatory bowel disease,IBD)是一种临床表现腹泻、腹痛、血便且病发于回肠、直肠、结肠的特发性肠道炎症性疾病,包括溃疡性结肠炎(UlcerativeColitis, UC)和克罗恩病(Crohn Disease, CD)等。
根据本发明的实施例,所述炎症性肠病选自溃疡性结肠炎。发明人发现,刺梨SOD提取物对于溃疡性结肠炎具有较好的预防或治疗作用。
根据本发明的实施例,所述药物用于下列至少之一:缓解腹泻或便血、改善结肠长度变短和体重降低、抑制脾脏肿胀、保护结肠组织完整性、促进肠上皮细胞增生、促进肠粘液增加、促进血浆中抑炎因子的生成、抑制血浆中促炎因子的生成、改善肠屏障、促进结肠上皮细胞肠屏障蛋白生成和提高抗氧化酶含量。
根据本发明的实施例,所述促炎因子选自TNF-α、IL-6和/或IL-1β;所述抑炎因子选自IL-4和/或IL-10;所述抗氧化酶选自谷胱甘肽、超氧化物歧化酶、过氧化氢酶和/或谷胱甘肽过氧化物酶。
根据本发明的实施例,所述刺梨SOD提取物的获得方法包括:将携带有编码刺梨SOD的核酸的表达载体转入酵母菌中,进行发酵培养,得到发酵液;将所述发酵液进行第一离心处理,收集上清液,将所述上清液再进行第二离心处理,收集上清液,得到粗酶;将所述粗酶进行过滤,收集滤液,将所述滤液上样至琼脂糖凝胶层析柱中,用不同浓度的氯化钠溶液洗脱所述琼脂糖凝胶层析柱,收集目标蛋白的流出液,浓缩,得到所述刺梨SOD提取物。其中,编码所述刺梨SOD的核酸序列选自申请号为202111023920.1专利申请中的SEQ ID NO:2所示核苷酸序列。
根据本发明实施例的方法,发酵培养过程中,酵母菌可高表达刺梨SOD,得到含有刺梨SOD的发酵液。第一离心处理的目的主要是去除菌体,收集上清液,再进行第二离心处理以去除杂质,得到粗酶。将上清液进行过滤以去除杂质,将所得滤液上样至琼脂糖凝胶层析中进行梯度洗脱,可以进一步纯化刺梨SOD,获得的刺梨SOD提取物SOD收率高、纯度高、酶活高,更重要的是具有较好的预防或治疗肠道疾病的功效。
根据本发明的实施例,所述第一离心处理的转速为6000~8000 rpm,时间为8~15min。由此,以便去除菌体。
根据本发明的实施例,所述第二离心处理的转速为11000~13000 rpm,时间为8~15min。由此,以便进一步去除杂质,得到粗酶。
根据本发明的实施例,所述过滤是采用0.22 μm滤膜进行的。由此,以便进一步去除杂质,有助于后续的层析处理。
根据本发明的实施例,所述氯化钠溶液的浓度为0.15~1 mol/L,所述洗脱的流速为0.3~0.8 mL/min。发明人经过大量实验得到上述较优洗脱条件,由此,获得的刺梨SOD提取物SOD收率高、纯度高、酶活高,更重要的是具有较好的预防或治疗肠道疾病的功效。
根据本发明的实施例,采用蛋白纯化仪检测洗脱过程中流出液的组成,收集开始出现最大峰面积时的流出液直至峰消失,收集的流出液即为目标蛋白的流出液。
根据本发明的实施例,所述药物还含有药学上可接受的载体或辅料,例如载体、溶剂、悬浮剂或赋形剂。示例性的辅料可以是液体或固体,包括但不限于:pH调节剂、表面活性剂、碳水化合物、佐剂、抗氧化剂、螯合剂、离子强度增强剂、防腐剂、载剂、助流剂、甜味剂、染料/着色剂、增味剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂、乳化剂、喷雾剂、压缩空气或其它适宜的气体,或其它适宜的与药效化合物合用的非活性成分。辅料的示例包括各种乳糖,甘露醇,油类如玉米油,缓冲剂如PBS,盐水,聚乙二醇,甘油,聚丙二醇,二甲亚砜,酰胺如二甲基乙酰胺,蛋白质如白蛋白,单糖和低聚多糖如葡萄糖、乳糖、环糊精和淀粉。
术语“治疗”用于指获得期望的药理学和/或生理学效果,例如改善疾病或病症。所述效果就完全或部分预防疾病或其症状而言可以是预防性的,和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病的治疗,包括:(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生;(b)抑制疾病,例如阻滞疾病发展;或(c)缓解疾病,例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物给药至个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药,包括但不限于将含本文所述刺梨SOD提取物的药物给予有需要的个体。
在本文中所使用的术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的药物可以通过任何常见的途径被给药,只要它可以到达预期的组织。给药的各种方式是可以预期的,包括腹膜、静脉、肌肉、皮下、皮层、口服、局部、鼻腔、肺部和直肠,但是本发明不限于这些已举例的给药方式。然而,由于口服给药时,肽被消化,肽键断裂,因此口服给药的药物的活性成分应该被包被或被配制以防止其在胃部被降解或破坏。优选地,本发明的药物可以注射制剂被给药。此外,本发明的药物可以使用将活性成分传送到靶细胞的特定器械来给药。
本发明的药物的给药频率和剂量可以通过多个相关因素被确定,该因素包括要被治疗的疾病类型、给药途径、病人年龄、性别、体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例,日剂量可分为适宜形式的1剂、2剂或多剂,以在整个时间段内以1次、2次或多次给药,只要达到治疗有效量即可。
术语“治疗有效量”是指化合物足以显著改善某些与疾病或病症相关的症状的量,也即为给定病症和给药方案提供治疗效果的量。例如,在癌症治疗中,减少、预防、延缓、抑制或阻滞疾病或病症的任何症状的药物或化合物应当是治疗有效的。治疗有效量的药物或化合物不需要治愈疾病或病症,但将为疾病或病症提供治疗,使得个体的疾病或病症的发作被延缓、阻止或预防,或者疾病或病症的症状得以缓解,或者疾病或病症的期限被改变,或者例如疾病或病症变得不严重,或者加速康复。
根据本发明的实施例,所述药物中刺梨SOD提取物的有效剂量为4~8 kU/kg鼠体重/天,或者20~40 kU/60kg人体重/天。
根据本发明的实施例,所述药物的剂型选自片剂、液体、粉末冲剂、咀嚼片剂或软凝胶。
有益效果
(1)本发明提供的刺梨SOD提取物不仅可以有效地抑制DSS诱导的小鼠体重下降、腹泻和便血等结肠炎症状;还能通过促进肠上皮细胞增生、肠粘液增加,进而改善肠屏障功能。
(2)本发明提供的刺梨SOD提取物能够为改善、治疗肠道性疾病包括炎症性肠病、腹泻和便血等、改善肠屏障功能和肠道健康开辟新的药物干预途径,这对于刺梨SOD在市场上的应用具有显著的推进作用。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1为实施例2中PBS组、DSS组与CL_SOD组实验设计、小鼠体重变化、疾病活动指数、小鼠实物图、脾脏脏器系数、结肠长度对比图;
图2为实施例3中PBS组、DSS组与CL_SOD组小鼠结肠组织HE染色病理状态对比图,其中,染色图标尺为100μm;
图3为实施例4中PBS组、DSS组与CL_SOD组小鼠结肠组织AB染色杯状细胞(Gobletcell, GCS)对比图,其中,染色图标尺为100μm;
图4为实施例5中PBS组、DSS组与CL_SOD组小鼠结肠组织PAS染色粘液对比图,其中,染色图标尺为100μm;
图5为实施例6中PBS组、DSS组与CL_SOD组小鼠血浆中抗氧化物质含量对比图;
图6为实施例7中PBS组、DSS组与CL_SOD组小鼠血浆中炎症因子含量对比图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
在该实施例中,按照下列方法获得刺梨SOD提取物:
1、参考申请号为202111023920.1的专利申请中实施例1的步骤1和2,制得含有刺梨SOD的发酵液;
2、将发酵液于7000 rpm离心15 min,收集上清液再于12000 rpm离心15 min,收集上清液,得到粗酶;
3、将粗酶通过0.22μm过滤器过滤,将滤液上样至Capto Q琼脂糖层析柱(GE医疗,瑞典),以0.5 mL/min的流速用0.15~1 mol/L的NaCl溶液线性梯度洗脱,整个洗脱过程在4℃下进行,AKTA蛋白纯化仪检测流出液,当出现峰面积最大的峰时,收集流出液直至出峰结束,将流出液浓缩,得到刺梨SOD提取物,纯度超99.99%,SOD比活力超1.3万 U/mg。
实施例2:刺梨SOD能改善小鼠的炎症性肠病
以葡聚糖硫酸钠(Dextran Sulfate Sodium, DSS)诱导的小鼠结肠炎为研究模型,模型建立如下:选取6周龄健康雄性C57BL/6J小鼠,并在标准12小时昼夜循环、22℃恒温、无特定病原体(SPF)环境下进行饲养。在适应性饲养1周后,将小鼠随机分为正常组(PBS,8只)、模型组(DSS,8只)和刺梨SOD组(CL_SOD+DSS,8只)。在适应性饲养1周后PBS组和DSS灌胃0.1 mol/L PBS(0.1ml/10g(小鼠体重)/天)、CL_SOD组灌胃实施例1获得的刺梨SOD提取物(7.5 kU/kg(小鼠体重)/天)。连续灌胃3周后,将2.5%DSS添加到3组小鼠的饮用水中,小鼠连续自由饮水7天诱导结肠炎。在诱导过程中,每日记录小鼠体重、粪便状态,便血情况;实验结束后,摘眼球取血于EDTA抗凝剂离心管中,静置离心后获取血浆,解剖小鼠,摘取结肠,脾脏。量取结肠长度,脾脏称重,拍照,收集结肠内容物,用4%的***固定液保存2 mm结肠组织样品,其余结肠组织收集于冻存管,放入液氮冻存,解剖结束后,结肠组织和肠道内容物等样品转置-80℃冰箱保存。
PBS、DSS、CL_SOD这3组不同处理小鼠的炎症性肠病体症变化如图1所示,图1的a为实验设计过程。图1的b为IBD造模后小鼠体重变化趋势,可以看出,DSS诱导3天后,与PBS组小鼠相比,DSS组小鼠体重下降显著,诱导7天后,DSS组小鼠的平均体重为初始体重的82.21±9.78%,CL_SOD组小鼠的平均体重为初始体重的88.17±4.34%,差异显著性p<0.01。由此,表明刺梨SOD可显著缓解DSS诱导的小鼠体重降低。
疾病活动指数(Disease activity index,DAI)是结合小鼠体重减轻、粪便稀疏度和便血等参数评价结肠炎模型严重程度的重要指标。3组小鼠疾病活动指数(DAI)对比数据如图1的c所示,DAI评分越高,说明小鼠腹泻和便血越严重。图1的d为小鼠解剖前拍照对比图,结合两图可以看出:与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠DAI明显降低,即小鼠结肠炎症状得到明显改善,表明刺梨SOD能够显著改善DSS诱导的小鼠疾病活动指数。
脾脏是小鼠的最大免疫器官,脾脏系数(脾脏的重量与体重的比值)能有效反映小鼠对外来物质的免疫反应和减毒功能,同时也是毒理实验中常用的指标。3组小鼠脾脏系数对比如图1的e所示,结合小鼠脾脏照片与数据统计图可以看出:与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠DAI即小鼠脾脏系数明显降低,表明刺梨SOD能够显著改善DSS诱导的小鼠免疫反应和减毒功能。
结肠长度是评价结肠炎症严重程度的另一重要参考指标。如图1的f,结合小鼠结肠长度照片和结肠数据对比图可以看出:与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠结肠长度较长,即刺梨SOD能够显著改善DSS诱导引起的结肠长度变短、充血和水肿等炎性症状。
综合图1可以得知,刺梨SOD能够有效抑制DSS诱导的小鼠体重下降、腹泻和便血,缓解结肠缩短和脾脏肿胀等症状。
实施例3:刺梨SOD能够促进健康小鼠的结肠病理状态
以实施例2中DSS诱导的小鼠结肠炎为研究模型,探究刺梨SOD对小鼠的结肠组织病理状态的影响。利用苏木精/伊红(Hematoxylin&Eosin,H&E)染色法检测小鼠结肠病理状态。具体如图2所示,图2的a、b、c为3组小鼠肠上皮组织对比图,图2的d、e、f为3组小鼠结肠粘膜层厚度、结肠隐窝深度、结肠组织病理学评分的数据对比图。从图2可以看出,与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠表现出明显改善结肠病理状态。
实施例4:刺梨SOD能够促进健康小鼠的肠上皮杯状细胞增殖
杯状细胞是一类特殊的肠上皮细胞,它能够产生和分泌粘蛋白到肠腔中形成粘液层维持肠道屏障功能,防止过度免疫激活。以实施例2中DSS诱导的小鼠结肠炎为研究模型,探究刺梨SOD对小鼠的肠上皮杯状细胞增殖作用。利用过碘酸雪夫(Periodic acid-Schiff,PAS)染色法检测小鼠结肠上皮杯状细胞的形态学变化。使用Image J对PAS染色的结果进行GCs定量分析。
具体如图3所示,图3的a、b、c为3组小鼠肠上皮杯状细胞对比图,图3的d为3组小鼠肠上皮杯状细胞面积占比的数据对比图。从图3可以看出,与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠表现出明显促进肠上皮杯状细胞增殖。
实施例5:刺梨SOD能够促进健康小鼠的肠粘液产生
粘液层具有防止肠道内细菌及其它致病性抗原进入机体血液循环及其它组织、器官的重要功能。以实施例2中DSS诱导的小鼠结肠炎为研究模型,探究刺梨SOD对小鼠的肠粘液产生。利用阿利新蓝(Alcian Blue,AB)染色法分析了小鼠的结肠粘液产生变化。
具体如图4所示。图4的a、b、c为3组小鼠结肠粘液屏障实物对比图,图4的d为3组小鼠结肠粘液层面积占比数据对比图。从图4可以看出,与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠表现出明显增多的粘液。
因此,刺梨SOD具有促进肠上皮细胞增生、肠粘液增加的作用,对于机体肠道的抗感染作用和免疫作用具有重要影响,进而在对于炎症性肠病、囊性纤维化、肠道肿瘤等多种肠道疾病的发生发展中起到重要的作用。
实施例6:刺梨SOD能增强抗氧化能力,缓解氧化损伤
利用DSS构建的IBD模型,会导致结肠病变组织产生大量的自由基,进而导致结肠氧化损伤。以实施例2中DSS诱导的小鼠结肠炎为研究模型,探究刺梨SOD对缓解小鼠氧化损伤的影响。采用商用试剂盒(Solarbio,北京,中国)检测血浆中具有代表性的氧化还原酶,如总抗氧化能力(T-AOC)、丙二醇(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPX)等含量。
具体如图5所示。图5的a~f为3组小鼠血浆中T-AOC、MDA、GSH、SOD、CAT和GPX含量检测对比图,从图5可以看出,与DSS组小鼠相比,灌胃刺梨SOD的CL_SOD组小鼠血浆中抗氧化酶(谷胱甘肽、超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶)的含量得到提高,过氧化产物(丙二醇)含量得到降低。因此,刺梨SOD能增强小鼠结肠抗氧化能力,缓解氧化损伤。
实施例7:刺梨SOD能增强小鼠抗炎能力,缓解炎症损伤
炎症反应是IBD的重要指标,以实施例2中DSS诱导的小鼠IBD为模型,探究刺梨SOD对缓解小鼠炎症损伤的影响。采用ELISA酶联免疫吸附法(上海酶联生物技术有限公司)检测小鼠血浆中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)和白细胞介素-10(IL-10)的浓度。
具体如图6所示。图6的a~e分别为3组小鼠血浆中TNF-α、IL-6、IL-1β、IL-4和IL-10的浓度。从图6可以看出,与DSS组小鼠相比,灌胃CL_SOD组小鼠血浆中促进炎症因子TNF-α、IL-6和IL-1β的浓度显著降低,抑制炎症因子IL-4和IL-10的浓度显著提高。说明刺梨SOD能增强小鼠抗炎能力,缓解炎症损伤。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、 “示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (7)
1.刺梨SOD提取物在制备药物中的用途,其特征在于,所述药物用于预防或治疗肠道疾病,所述肠道疾病选自溃疡性结肠炎;
所述刺梨SOD提取物的获得方法包括:
将携带有编码刺梨SOD的核酸的表达载体转入酵母菌中,进行发酵培养,得到发酵液;
将所述发酵液进行第一离心处理,收集上清液,将所述上清液再进行第二离心处理,收集上清液,得到粗酶;
将所述粗酶进行过滤,收集滤液,将所述滤液上样至琼脂糖凝胶层析柱中,用不同浓度的氯化钠溶液洗脱所述琼脂糖凝胶层析柱,收集目标蛋白的流出液,浓缩,得到所述刺梨SOD提取物;
所述第一离心处理的转速为6000~8000 rpm,时间为8~15 min;
所述第二离心处理的转速为11000~13000 rpm,时间为8~15 min;
所述过滤是采用0.22 μm滤膜进行的;
所述氯化钠溶液的浓度为0.15~1 mol/L;
所述洗脱的流速为0.3~0.8 mL/min;
采用蛋白纯化仪检测洗脱过程中流出液的组成,收集开始出现最大峰面积时的流出液直至峰消失,收集的流出液即为目标蛋白的流出液。
2.根据权利要求1所述的用途,其特征在于,所述药物用于下列至少之一:
缓解腹泻或便血、改善结肠长度变短和体重降低、抑制脾脏肿胀、保护结肠组织完整性、促进肠上皮细胞增生、促进肠粘液增加、促进血浆中抑炎因子的生成、抑制血浆中促炎因子的生成、改善肠屏障、促进结肠上皮细胞肠屏障蛋白生成和提高抗氧化酶含量。
3.根据权利要求2所述的用途,其特征在于,所述促炎因子选自TNF-α、IL-6和/或IL-1β;
所述抑炎因子选自IL-4和/或IL-10;
所述抗氧化酶选自谷胱甘肽、超氧化物歧化酶、过氧化氢酶和/或谷胱甘肽过氧化物酶。
4.根据权利要求1所述的用途,其特征在于,所述药物还含有药学上可接受的辅料。
5.根据权利要求1所述的用途,其特征在于,所述药物中刺梨SOD提取物的有效剂量为4~8 kU/kg鼠体重/天,或者20~40 kU/60kg人体重/天。
6.根据权利要求1所述的用途,其特征在于,所述药物的剂型选自片剂、液体、粉末冲剂或软凝胶。
7.根据权利要求1所述的用途,其特征在于,所述药物的剂型选自咀嚼片剂。
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