CN117618481A - 傣百解提取物及其制剂在解酒护肝等相关疾病中的应用 - Google Patents
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Abstract
本发明公开了傣百解提取物及其相关制剂在解酒护肝等相关疾病中的应用。本发明将制备得到的傣百解醇提物应用于解酒护肝等相关疾病的药物或保健品的开发,发挥其对酒精性肝损伤的保护作用。针对酒精性肝损伤,傣百解醇提物可减少丙氨酸氨基转移酶与天门冬氨酸氨基转移酶的分泌,减轻肝脏损伤,提高肝脏活力,缓解乙醇与酒精引起的氧化应激,提高抗氧化酶活力,减轻炎症反应,从而发挥保护作用。
Description
技术领域:
本发明涉及植物提取物应用治疗领域,具体涉及傣百解提取物及其相关制剂在解酒护肝等相关疾病中的应用。
背景技术:
酒在人类生活中扮演着不可或缺的角色,由饮酒引起的健康问题和社会问题非常严重。酒精可通过产生毒性代谢产物、引起代谢相关酶及辅酶的变化、引起微循环障碍及肝内低氧血症、肝纤维化、产生大量活性氧基团以及引起细胞免疫异常等机制导致肝脏损伤。随着生活水平的不断提升,酒精消费的人群不断年轻化,对于肝脏的损伤也引起人们的关注度,在有效预防早期酒精性肝病损伤上还缺乏有效治疗药物。
酒精性肝病,是指人体大量饮酒超过机体的代谢能力时,会产生大量对机体器官有损伤的自由基,从而造成肝脏损伤的一类病症。主要包括酒精性脂肪肝、酒精性肝炎、酒精性肝硬化及酒精性肝纤维化,其在早期的临床表现为脂肪肝,若持续酗酒可能会发展为肝炎、肝纤维化,肝硬化,严重者甚至会恶化为肝癌,严重危害身体健康。
目前,全球每年约有330万人的死亡与过量饮酒有关,酒精依赖已然成为了当今社会的重要问题之一。因此,开发出有效的解酒保肝的保健食品及药物对缓解这一疾病的发生具有重要意义。
傣百解作为傣族的传统用药,又名大百解,傣语称为“雅解先打”,意为解百毒的药,其味苦,性凉,在傣医传统理论体系中入火、土、风塔。具有清火解毒、消肿止痛之功效,常用于咽喉肿痛、口舌生疮、疗疡斑疹、肺热咳嗽、胃脘痛、尿痛、解药食毒,用药具有广阔的应用前景。
发明内容:
(1)本发明的目的在于公开一种具有解酒护肝作用的傣百解提取物及其相关制剂。
(2)本发明解酒作用是通过傣百解提取物对饮酒后雄性C57bl/6J小鼠行为活动的研究来实现的,如醉酒耐受时间、醉酒持续时间、攀爬时间。
(3)本发明预防/治疗急性酒精性肝损伤是通过对饮酒后雄性C57bl/6J各项生化指标来实现的,如血清生化指标、氧化指标、炎症指标。
附图说明:
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中
图1:雄性小鼠行为学指标变化结果;1-1.醉酒持续时间;1-2.醉酒耐受时间;1-3.攀爬时间;以mean±SD(n=12)表示;*与模型组比较P<0.05;**P<0.01
图2:雄性小鼠急性酒精肝损伤模型肝脏病理切片;2-1.对照组;2-2.红星二锅头15ml/kg组;2-3.水飞蓟宾胶囊60mg/kg组;2-4.DBJ 100mg/kg组;2-5.DBJ 300mg/kg组;2-6.DBJ 600mg/kg组(x40,x200)
图3:雄性小鼠血清生化指标变化结果;3-1.丙氨酸氨基转移酶(ALT);3-2.天门冬氨酸氨基转移酶(AST);3-3.乳酸脱氢酶(LDH);3-4.低密度脂蛋白胆固醇(LDL-C);3-5.总胆固醇(TC);3-6.甘油三酯(TG);以mean±SD(n=12)表示;
*与模型组比较P<0.05;**P<0.01;##与对照组比较P<0.05
图4:雄性小鼠肝脏氧化指标变化;4-1.超氧化物歧化酶(SOD);4-2.丙二醛(MDA);4-3.过氧化氢酶(CAT);4-4.谷胱甘肽硫基转移酶(GST);4-5.活性氧簇(ROS);以mean±SD(n=12)表示;
*与模型组比较P<0.05;**P<0.01;##与对照组比较P<0.05
图5:雄性小鼠肝脏炎症指标变化;5-1.肿瘤坏死因子α(TNF-α);5-2.白介素6(IL-6);5-3.白细胞介素1β(IL-1β);以mean±SD(n≡12)表示;
*与模型组比较P<0.05;**P<0.01;
##与对照组比较P<0.05
具体实施方案:
使本发明容易理解,下面将详细说明本发明。但在详细描述本发明前,应当理解本发明不限于描述的具体实施方式。还应当理解,本文中使用的术语仅为了描述具体实施方式,而并不表示限制性的。
除非另有定义,本文使用的所有术语与本发明所属领域的普通技术人员的通常理解具有相同的意义。虽然与本文中描述的方法和材料类似或等同的任何方法和材料也可在本发明的实施或测试中使用,但是现在描述了优选的方法和材料。
1.本发明所述用语“醉酒耐受时间”:雄性小鼠从灌酒至醉酒(翻正反射消失)的时间。
2.本发明所述用语“醉酒持续时间”:雄性小鼠从醉酒到醒酒(翻正反射恢复)的时间。
3.本发明所述用语“攀爬时间”:给予酒精后立即将小鼠放至在水平悬空的金属网中央,然后立即翻转金属网,使小鼠呈倒挂状态。观察小鼠的攀附运动情况,并记录下各组小鼠的攀附时间。以掉落3次为终止,计算3次攀附时间的均值作为其最终实验结果随即将小鼠放在垂直的金属网上,记录各小鼠在网上的攀附时间。
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例:
一.傣百解提取物解酒作用研究
1.1试验分组
雄性C57BL/6J小鼠,在12小时明暗循环、温度22±3℃、相对湿度40-60%的房间中驯化一周,按照表中的给药剂量给药。
在连续给予傣百解提取物或生理盐水灌胃的第7天,给药1h后,给予酒精造模一次,空白组按照15ml/kg体重给予生理盐水,其他组按照15ml/kg体重灌胃56°红星二锅头,并在造模后6h内禁食不禁水。
1.2行为学观察
灌胃白酒后小鼠会出现,眼睛紧闭、毛发松散、凌乱,身体瘫软,步姿不稳,左右摇晃,后腹拖地爬行,活动量减少,嗜睡的现象,刺激身体反应不灵敏、失去知觉,翻正反射现象消失等现象。每5或10分钟对行为变化进行评分一次。记录并计算灌胃给酒时间、醉酒时间(翻正反射消失时间)、醒酒时间(翻正反射恢复时间)醉酒耐受时间、持续时间。
1.3攀爬实验
灌服白酒后常用攀爬实验来评价动物肢体肌力与协调力。给予酒精后立即将小鼠放至在水平悬空的金属网中央,然后立即翻转金属网,使小鼠呈倒挂状态。观察小鼠的攀附运动情况,并记录下各组小鼠的攀附时间。以掉落3次为终止,计算3次攀附时间的均值作为其最终实验结果随即将小鼠放在垂直的金属网上,记录各小鼠在网上的攀附时间。
如图1所示,乙醇与酒精诱导醉酒后,模型组醉酒耐受时间较短,醉酒持续时间较长,且攀爬时间较短与给药组有较大差异,傣百解剂量组对醉酒有一定改善作用,醉酒耐受时间较长,醉酒持续时间较短,且攀爬时间显著性增加。
二.傣百解预防急性酒精性肝损伤作用研究
2.1试验分组
雄性C57BL/6J小鼠,在12小时明暗循环、温度22±3℃、相对湿度40-60%的房间中驯化一周,按照表中的给药剂量给药。
在连续给予傣百解提取物或生理盐水灌胃的第18天,给药1h后,给予酒精造模,连续4天,空白组按照15ml/kg体重给予生理盐水,其他组按照15ml/kg体重灌胃56°红星二锅头,并在第21天造模后6h内禁食不禁水。
2.2肝组织切片观察
(1)石蜡切片脱蜡至水:依次将3-5μm厚的肝脏组织切片放入二甲苯I 20min-二甲苯II 20min-无水乙醇I 5min-无水乙醇II 5min-75%酒精5min,自来水洗;(2)苏木素染色:切片入苏木素染液染3-5min,自来水洗,分化液分化,自来水洗,返蓝液返蓝,流水冲洗;(3)伊红染色:切片依次入85%、95%的梯度酒精脱水各5min,入伊红染液中染色5min;(4)脱水封片:切片依次放入无水乙醇I 5min-无水乙醇II 5min-无水乙醇III 5min-二甲I5min-二甲苯II 5min透明,中性树胶封片。
如图2所示,肝脏组织被膜由厚薄均匀的富含弹性纤维致密结缔组织构成。通过H&E染色观察小鼠肝脏组织病理变化显示,空白组细胞形态正常,肝小叶分界明显,排列规则,肝板排列规则、整齐,肝窦无明显扩张或挤压;相邻肝小叶之间的门管区无明显异常;未见明显的炎性改变。与空白组相比,模型组显示肝组织边缘可见较多肝细胞轻度水样变性,细胞肿胀,胞质疏松淡染(黑色箭头);少量汇管区周围有的结缔组织增生。傣百解低、中、高剂量给药组肝脏病理变化显著改善,呈放射状排列的肝细胞和肝血窦,肝细胞圆润、饱满;肝板排列规则、整齐,肝窦无明显扩张或挤压;相邻肝小叶之间的门管区无明显异常;未见明显的炎性改变。
2.3血清生化指标检测
血清样本被适当稀释和处理,血清中生化指标测定按照说明书进行试验,所用试剂盒为上海酶联Elisa试剂盒。
酒精诱导肝脏损伤后,血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、甘油三酯(TG)、乳酸脱氢酶(LDH)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)水平显著升高,与模型损伤组相比,傣百解高剂量给药组血清中丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、甘油三酯、乳酸脱氢酶、总胆固醇、低密度脂蛋白胆固醇水平含量均降低(图3)。
2.4肝脏组织抗氧化能力的测定
将肝脏组织样本准确称重20mg,用200μL预冷的PBS均质。然后在4℃下,14000rpm离心15min,收集组织上清。使用商业试剂盒评估氧化/抗氧化状态,以确定抗氧化酶的活性,包括SOD、CAT、GST、CAT和ROS的水平。
如图4所示,乙醇与酒精诱导损伤后,肝脏组织内会产生过量ROS积累,最终导致MDA过量产生,给药组均下调了ROS和MDA的水平。模型组的SOD、CAT、GST抗氧化酶的活性显著降低,傣百解高剂量给药组的SOD、GST和CAT活性显著升高。
2.5肝脏组织TNF-α、IL-6和IL-1β含量
将肝脏组织样本准确称重20mg,用200μL预冷的PBS均质。然后在4℃下,14000rpm离心15min,收集组织上清。使用商业试剂盒评估炎症/抗炎状态,以确定抗炎的活性,包括TNF-α、IL-6和IL-1β的水平。
如图5所示,乙醇与酒精诱导损伤后,肝脏组织内TNF-α、IL-6、IL-1β含量均会提高,模型组的TNF-α、IL-6、IL-1β酶的含量显著上升,给药组均下调了TNF-α、IL-6、IL-1β的水平,傣百解高剂量给药组的TNF-α、IL-6和IL-1β活性显著升高。
应当注意的是,以上所述的实施例仅用于解释本发明,并不构成对本发明的任何限制。通过参照典型实施例对本发明进行了描述,但应当理解为其中所用的词语为描述性和解释性词汇,而不是限定性词汇。可以按规定在本发明权利要求的范围内对本发明作出修改,以及在不背离本发明的范围和精神内对本发明进行修订。尽管其中描述的本发明涉及特定的提取方法,但是并不意味着本发明限于其中公开的特定例,相反,本发明可扩展至其他所有具有相同功能的方法和应用。
Claims (3)
1.傣百解提取物及其制剂在解酒护肝等相关疾病中的应用。
2.如权利要求1所述的用途,其特征在于,所述的乙醇和或酒精致肝脏损伤,氧化应激,产生炎症等方面。
3.如权利要求1所述的用途,其特征在于傣百解提取物与药学上可接受的载体或辅料制备成颗粒剂、片剂、胶囊剂、缓控释制剂、丸剂、酒剂、混悬剂、冻干粉等剂型的用于解酒护肝的药物或保健品。
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