CN115120622B - 树舌灵芝乙醇提取物在缓解沙门氏菌感染的应用 - Google Patents
树舌灵芝乙醇提取物在缓解沙门氏菌感染的应用 Download PDFInfo
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Abstract
本发明公开了树舌灵芝乙醇提取物在缓解沙门氏菌感染的应用,属于生物医药领域。本发明首次证实树舌灵芝乙醇提取物通过降低沙门氏菌毒力、运动性以及生物膜形成,达到体内缓解沙门氏菌感染的作用。具体地,树舌灵芝乙醇提取物可以缓解小鼠沙门氏菌感染,具有降低沙门氏菌肝脏迁移量、降低炎症因子表达、维持肠道屏障稳态,从而达到缓解沙门氏菌感染的作用。
Description
技术领域
本发明涉及树舌灵芝乙醇提取物在缓解沙门氏菌感染的应用,属于生物医药领域。
背景技术
肠道沙门氏菌是一种革兰氏阴性细菌病原体,能够感染广泛的宿主。感染不同血清型的沙门氏菌可导致不同的临床结果,从无症状感染、急性自限性胃肠炎、侵入性系统疾病和菌血症,到肠道(伤寒)热。与腹泻和急性炎症有关的肠道沙门氏菌可以破坏胃肠道屏障,通过三型分泌系统转移到胆囊、脾脏、肝脏和骨髓,从而持续较长时间,导致慢性感染和携带。抗生素的使用是各种细菌感染的主要临床治疗药物,而抗生素的使用会削弱肠道屏障,增加微生物群与肠道免疫细胞接触的可能性,并诱发炎症。
食用真菌作为一种营养食品在世界范围内越来越受欢迎,而且因其具有丰富的活性物质而具有抗糖尿病、化学预防、抗氧化、抗炎和抗菌等作用,因此从安全营养食品中寻找缓解致病菌感染的膳食成分具有一定的意义与价值。树舌灵芝属于担子菌门,非褶菌目,灵芝菌科,灵芝属。树舌灵芝自古以来就是一种药用真菌,其味微苦、平,归脾、胃经,药用价值高,《中国药用真菌图鉴》和《长白山植物药志》中均有记载,能祛风除湿,清热,止痛,化积,止血,化痰。在临床多用于治疗乙型肝炎、食道癌、肺结核、神经衰弱等。也有文章报道用于改善痛风和改善肠癌。
发明内容
本发明主要针对树舌灵芝乙醇提取物通过降低沙门氏菌毒力、运动性以及生物膜形成,达到体内缓解沙门氏菌感染的作用。本发明证实了树舌灵芝乙醇提取物可以缓解小鼠沙门氏菌感染,具有降低沙门氏菌肝脏迁移量、降低炎症因子表达、维持肠道屏障稳态的作用。
本发明提供了树舌灵芝乙醇提取物的制备方法,所述方法为将树舌灵芝子实体磨粉后溶于乙醇进行处理,重复多次,收集上清液,浓缩并真空冷冻干燥。
在一种实施方式中,乙醇的添加量是树舌灵芝子实体的5-10倍体积。
在一种实施方式中,所述处理的条件为在40~50℃轻微震荡10~15h。
在一种实施方式中,所述乙醇的浓度为70~75%。
在一种实施方式中,所述方法为将树舌灵芝子实体磨粉后溶于5倍体积的75%乙醇,40℃轻微震荡12h,重复3次,收集上清液,旋转蒸发浓缩,真空冷冻干燥。
在一种实施方式中,旋转蒸发浓缩的温度≤40℃。
在一种实施方式中,真空冷冻干燥的条件为-80℃,-0.06kPa,48h。
本发明提供了树舌灵芝乙醇提取物在制备预防和/或缓解沙门氏菌感染的产品中的应用。
在一种实施方式中,所述应用为(a)~(h)至少一方面:
(a)降低沙门氏菌毒力;
(b)降低沙门氏菌运动性;
(c)降低沙门氏菌生物膜形成;
(d)提高沙门氏菌感染后的存活率;
(e)降低沙门氏菌肝脏转移量;
(f)降低沙门氏菌感染引起的肠道炎症
(g)降低沙门氏菌感染引起的炎症因子表达;
(h)维持肠道屏障稳态。
在一种实施方式中,所述降低沙门氏菌毒力包括降低沙门氏菌毒力基因fimF、invG、sipB、invE、flgE和/或flil的表达。
在一种实施方式中,所述运动性为泳动运动能力。
在一种实施方式中,降低炎症因子表达包括抑制促炎因子、促进抗炎因子的表达,以及抑制结肠炎相关酶的活性。
在一种实施方式中,所述抑制炎症因子表达包括降低小肠和结肠L-1β、IL-6、IL-17和TNF-α。
在一种实施方式中,所述促进抗炎因子的表达为提升小肠和结肠抗炎细胞因子IL-10。
在一种实施方式中,所述抑制炎症相关酶的活性包括抑制小肠和结肠COX-2、iNOS、MPO的浓度。
在一种实施方式中,所述维持肠道屏障稳态为防止沙门氏菌对小肠和结肠肠道屏障的破坏,具体表现为防止MUC2、Occludin和ZO-1浓度的降低。
在一种实施方式中,所述产品为药品、兽药或者饲料添加剂。
在一种实施方式中,所述树舌灵芝乙醇提取物的含量≥0.3mg/mL,或≥0.3mg/mg。
在一种实施方式中,所述药品还包括药学上可接受的赋型剂;所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
在一种实施方式中,当产品为兽药或者饲料添加剂时,有效剂量不低于100mg/Kg。
在一种实施方式中,所述兽药或者饲料添加剂中还含有常规辅料。
在一种实施方式中,所述常规辅料包括填充剂、矫味剂、粘合剂、崩解剂、润滑剂、以及抗酸剂中的一种或多种。
本发明还提供一种预防和/或缓解沙门氏菌感染的产品,所述产品中含有树舌灵芝乙醇提取物。
在一种实施方式中,所述树舌灵芝乙醇提取物的含量≥0.3mg/mL,或≥0.3mg/mg。
在一种实施方式中,所述产品为药品、兽药或者饲料添加剂。
本发明的有益效果:
本发明首次证实树舌灵芝乙醇提取物通过降低沙门氏菌毒力、运动性以及生物膜形成,达到体内缓解沙门氏菌感染的作用。具体地,树舌灵芝乙醇提取物可以缓解小鼠沙门氏菌感染,具有降低沙门氏菌肝脏迁移量、降低炎症因子表达、维持肠道屏障稳态,从而达到缓解沙门氏菌感染的作用。
附图说明
图1为树舌灵芝乙醇提取物对沙门氏菌毒力、运动性和生物膜形成;(A)树舌灵芝乙醇提取物对沙门氏菌毒力的影响,(B)树舌灵芝乙醇提取物对沙门氏菌涌动能力的影响,(C)树舌灵芝乙醇提取物对沙门氏菌生物膜形成的影响,(D)树舌灵芝乙醇提取物处理的沙门氏菌扫描电子显微照片。
图2为树舌灵芝乙醇提取物缓解沙门氏菌感染;(A)沙门氏菌感染存活率,(B)沙门氏菌感染结肠沙门存活量,(C)沙门氏菌感染肝脏移位,(D)十二指肠组织学损伤,I,对照组,II,模型组,III,树舌灵芝乙醇提取物组,(E)空肠组织学损伤,I,对照组,II,模型组,III,树舌灵芝乙醇提取物组,(F)结肠组织学损伤,I,对照组,II,模型组,III,树舌灵芝乙醇提取物组。
图3为树舌灵芝乙醇提取物对结肠肠道屏障的保护效果图;(A)MUC2,(B)Occludin,(C)ZO-1;每组6只小鼠(n=6),平均值±SD,P值使用非配对T检验计算,*P<0.05。
图4为树舌灵芝乙醇提取物对小肠肠道屏障的保护效果图;(A)MUC2,(B)Occludin,(C)ZO-1;每组6只小鼠(n=6),平均值±SD,P值使用非配对T检验计算,*P<0.05。
图5为树舌灵芝乙醇提取物对结肠炎症相关酶以及炎症因子的影响;(A)COX-2,(B)iNOS,(C)MPO,(D)IL-1β,(E)IL-6,(F)IL-17,(G)TNF-α,(H)IL-10;每组6只小鼠(n=6),平均值±SD,P值使用非配对T检验计算,*P<0.05。
图6为树舌灵芝乙醇提取物对小肠炎症相关酶以及炎症因子的影响;(A)COX-2,(B)iNOS,(C)MPO,(D)IL-1β,(E)IL-6,(F)IL-17,(G)TNF-α,(H)IL-10;每组6只小鼠(n=6),平均值±SD,P值使用非配对T检验计算,*P<0.05。
具体实施方式
实施例1:树舌灵芝乙醇提取物的制备及主要成分检测
(1)树舌灵芝乙醇提取物的制备:
将干树舌灵芝子实体切成小块,磨成细粉后,添加5体积的75%乙醇,于40℃下轻微震荡12h,取上清,重复提取三次,合并上清液,旋转蒸发浓缩(温度不超过40℃),最后冷冻干燥。
(2)树舌灵芝乙醇提取物主成分分析:
冷冻干燥后的提取物溶解于甲醇中,使用Thermo Fisher ScientificUltiMateTM3000 UHPLC(Thermo Scientific,Germering,Germany)在LC-MS中检测生物活性化合物。使用带有全扫描MS/dd-MS2的Q Exactive质谱仪来获得数据,并配备了电喷雾电离源。使用UPLC Ultimate 3,000中的HSS T3柱(2.1mm×100mm×1.8μm)分离甲醇提取物,温度保持在40℃。流动相为含0.1%甲酸的水,流动相B为含0.1%甲酸的乙腈。洗脱条件监测为0-1.5分钟(5%B),1.5-4.0分钟(5%B-40%B),4.0-5.0分钟(40%B-95%B),5.0-6.0分钟(95%B),6.0-6.1分钟(95%-5%B),和6.1-8.0分钟(5%B)。将每个化合物的进口名称、分子式、分子质量、CAS号、电离方法、主要准确质量、1-2个特征二级碎片离子和保留时间插入TraceFinder软件,以建立一个准确的质量数据库。通过在mzVault谱系管理软件中收集每个化合物的质谱,建立高分辨率的质谱库。主成分见表1,树舌灵芝乙醇提取物中主要为灵芝酸类。
表1.LC-HRESIMS树舌灵芝乙醇提物主要成分
实施例2:树舌灵芝乙醇提取物对沙门氏菌毒力、运动性以及生物膜形成的影响
(1)测定树舌灵芝乙醇提取物对沙门氏菌毒力的转录影响:
为了测定树舌灵芝乙醇提取物对沙门氏菌毒力基因的表达情况,将实施例1制备的树舌灵芝乙醇提取物溶于DMSO后(终浓度0.3g/mL)与103CFU/mL沙门氏菌37℃共同培养8h,12000rpm离心收集菌体。并设置不添加树舌灵芝乙醇提取物的对照组,对照组中加入与等体积的DMSO和103CFU/mL沙门氏菌。
使用Cell/Tissue Total RNA Isolation Kit(Vazyme Biotech Co.,Ltd.)提取沙门氏菌的总RNA。使用/>ⅢAll-in-one RT SuperMix Perfect forqPCR(Vazyme Biotech Co.,Ltd)转录成cDNA,并使用Taq Pro Universal SYBR qPCRMaster Mix(Vazyme Biotech Co.,Ltd)进行qRT-PCR分析。采用2-ΔΔCt方法进行定量,定量为16S的标准化表达,并以与对照组相比的倍数变化表示。用于RT-qPCR的所有引物的序列列于表2。
表2RT-qPCR检测引物
由图1A可知,树舌灵芝乙醇提取物可以降低沙门氏菌毒力因子fimF、invG、sipB、invE、flgE和flil的表达,相比对照组,分别降低了70.12%、39.65%、64.31%、60.47%、38.46%、41.14%。
(2)测定树舌灵芝乙醇提取物对沙门氏菌运动性的影响:
分别配制含有0.5%琼脂的Serum-SAPI平板测定细菌的泳动能力,实验组平板加入树舌灵芝乙醇提取物(溶于DMSO,终浓度为3mg/mL),对照组加入等体积的DMSO。将过夜培养的沙门氏菌离心收集并用PBS重悬成1×106CFU/mL的菌悬液,取5μL点种在平板中央,28℃静置培养48h后测量菌落的直径。
如图1B所示,实验组沙门氏菌菌落的直径为1.85cm,相比对照组减少了63.51%,说明树舌灵芝乙醇提取物显著降低了沙门氏菌泳动能力。
(3)测定树舌灵芝乙醇提取物对沙门氏菌生物膜的影响:
将沙门氏菌培养至对数期,PBS洗涤3次,用Serum-SAPI培养基(含终浓度3mg/mL树舌灵芝乙醇提取物或不含树舌灵芝乙醇提取物)稀释至5×106CFU/mL,取200μL菌液于96孔板,每个样品5个重复,28℃培养48h,在600nm波长处测定样品的吸光度。倒出内容物,用300μL无菌PBS洗涤3次,200μL甲醛固定15min,去甲醛,干燥5~10min,再用200μL结晶紫溶液(1%,w/v)染色15min,双蒸水洗涤5次,干燥5~10min,最后用无水乙醇溶解,590nm波长处测定样品的吸光度。生物膜相对值=OD590(测量值)/OD600(细菌浓度值)。
结果如图1C所示,树舌灵芝乙醇提取物可以降低沙门氏菌生物膜的形成,实验组相比对照组降低了58.20%。
图1D为树舌灵芝乙醇提取物处理的沙门氏菌扫描电子显微照片。
实施例3:树舌灵芝乙醇提取物对沙门氏菌感染小鼠的保护作用
(1)实验分组及处理方法:
分别设置4组:空白对照组、沙门氏菌感染组、树舌灵芝乙醇提取物组。
雄性C57BL6/J小鼠(n=30),7周龄,体重18至22克,在江苏省血吸虫病防治研究所饲养(25±2℃,12/12小时光照/黑暗期)。在实验室条件下给予小鼠自由水和食物,至少一周时间。然后,将30只小鼠分成3组(每组n=10),喂食无菌水和标准饲料。
所有小鼠均用链霉素(每只小鼠20毫克)处理,24小时后,除对照组外,其他组均灌喂1.5×106CFU肠道沙门氏菌。将实施例1中的树舌灵芝乙醇提取物悬浮在生理盐水中,小鼠灌喂肠道沙门氏菌后,树舌灵芝乙醇提取物组灌喂100mg/(kg.bw)树舌灵芝乙醇提取物,沙门氏菌感染组灌胃相应体积的生理盐水,每天记录小鼠的存活情况。
表3实验动物分组
以7天后各组存活的小鼠计算存活率,并于CO2窒息死亡后,将小鼠十二指肠、空肠、回肠和结肠的部分进行固定和处理,进行苏木精和伊红染色,部分用于制备RNA和蛋白质,将小鼠肝脏匀浆的连续稀释液涂布在含有50μg/ml链霉素的MacConkey琼脂上。
结果如图2所示,树舌灵芝乙醇提取物提高了沙门氏菌感染后小鼠的存活率,相比没有灌胃树舌灵芝乙醇提取物的沙门氏菌感染组,存活率达到90%以上(图2A)。树舌灵芝乙醇提取物降低沙门氏菌肝脏转移量以及降低结肠沙门氏菌的存活量(图2B和图2C)。
通过组织病理学显示,树舌灵芝乙醇提取物降低了十二指肠、空肠和结肠因沙门氏菌感染引起的炎症浸润(图2D)。
实施例4:树舌灵芝乙醇提取物对沙门氏菌感染引起的炎症以及肠道屏障的影响
将小鼠空肠和结肠组织样本匀浆制备成的混合物I和结肠和小肠组织样本匀浆制备成的混合物II分别在4℃下以12000rpm离心15分钟,得到上清液。使用ELISA试剂盒(南京森贝佳生物技术有限公司,江苏南京,中国)评估结肠和空肠组织样本的COX-2、iNOS、MPO、MUC2、ZO-1和Occludin的变化,R&D ELISA试剂盒评估结肠和小肠组织样本的IL-6、IL-1β、IL-17、TNF-α和IL-10。
通过图3和图4发现,与沙门氏菌感染组相比,树舌灵芝乙醇提取物显著提高了小鼠结肠和空肠的肠道屏障相关蛋白MUC2、ZO-1和Occludin浓度。
通过图5和图6发现,树舌灵芝乙醇提取物显著降低了小鼠结肠和空肠的炎症相关酶COX-2、INOS和MPO的浓度,同时降低了结肠和小肠的促炎因子IL-6、IL-1β、IL-17和TNF-α的浓度,增加了结肠和小肠的抑炎因子IL-10浓度。
综上所述,树舌灵芝乙醇提取物通过降低沙门氏菌毒力、运动性以及生物膜形成,达到体内缓解沙门氏菌感染的作用。具体地,树舌灵芝乙醇提取物可以缓解小鼠沙门氏菌感染,具有降低沙门氏菌肝脏迁移量、降低结肠沙门氏菌存活量、降低炎症相关酶浓度、降低促炎因子浓度、增加抑炎因子浓度和维持肠道屏障稳态,从而达到缓解沙门氏菌感染的作用。
综上所述,树舌灵芝乙醇提取物通过降低沙门氏菌毒力、运动性以及生物膜形成,达到体内缓解沙门氏菌感染的作用。具体地,树舌灵芝乙醇提取物可以缓解小鼠沙门氏菌感染,具有降低沙门氏菌肝脏迁移量、降低结肠沙门氏菌存活量、降低炎症相关酶浓度、降低促炎因子浓度、增加抑炎因子浓度和维持肠道屏障稳态,从而达到缓解沙门氏菌感染的作用。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (5)
1.树舌灵芝乙醇提取物在制备预防和/或缓解沙门氏菌感染的产品中的应用;
所述树舌灵芝乙醇提取物的制备方法为将树舌灵芝子实体磨粉后溶于乙醇进行处理,重复多次,收集上清液,浓缩并真空冷冻干燥;
所述乙醇的浓度为70~75%,乙醇的添加量是树舌灵芝子实体的5-10倍体积;
所述产品为药品或者饲料添加剂。
2.根据权利要求1所述的应用,其特征在于,所述树舌灵芝乙醇提取物的含量≥0.3mg/mL,或≥0.3 mg/mg。
3.根据权利要求1所述的应用,其特征在于,所述药品还包括药学上可接受的赋型剂。
4.根据权利要求1所述的应用,其特征在于,所述饲料添加剂中还含有常规辅料。
5.根据权利要求4所述的应用,其特征在于,所述常规辅料选自填充剂、矫味剂、粘合剂、崩解剂、润滑剂、以及抗酸剂中的一种或多种。
Priority Applications (1)
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