CN111991423A - 一种鹿茸醇提取物的制备方法及其应用 - Google Patents
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Abstract
本发明属于医药技术领域,提供了一种鹿茸醇提取物的制备方法及其应用。为了研究鹿茸醇提取物对MPTP诱导的帕金森病(PD)小鼠神经变性和神经炎症反应的疗效,本发明采用鹿茸醇提取物对PD模型小鼠进行治疗,通过实验证明,鹿茸醇提取物能够改善PD模型小鼠的行为障碍,可起到抑制纹状体中磷酸化Akt、p65和p38信号通路,降低星形胶质细胞的活化,保护黑质(SN)和纹状体神经元免受损伤的作用。由此可见,本发明所述的鹿茸醇提取物对PD动物模型具有很好的治疗效果,在治疗PD和其他炎症及氧化应激相关神经元疾病方面具有广阔的应用前景,为治疗帕金森病的新药研发提供了实验依据。
Description
技术领域
本发明属于医药技术领域,涉及一种鹿茸醇提取物的制备方法及其应用。
背景技术
帕金森病(PD)是一种常见的神经退行性疾病,影响了全球近1000万人。PD的特征是脑内多巴胺能神经元进行性丢失和路易小体形成,主要表现为远动迟缓、肌强直、震颤、姿势步态异常等运动障碍,常伴有嗅觉减退、睡眠障碍等多种非运动症状,患者多为散发病例,只有不到10%存在家族史。帕金森病的确切病因目前仍不清楚,遗传因素、环境因素、年龄老化、氧化应激等均可能参与PD多巴胺能神经元的变性死亡过程。虽然左旋多巴和多巴胺激动剂等药物已经暂时缓解了患者的症状,但到目前为止还没有有效的疗法或药物可以阻止或延缓PD的神经退行性进程。
鹿茸是一种传统中药,具有恢复免疫反应,改善学习和记忆,抗炎、抗应激和抗衰老等作用。鹿茸多肽通过抑制NF-κB通路调节LPS诱导的原代髓核细胞的促炎细胞因子(如TNF-α和IL-1β)。鹿茸水提取物(AAE)通过调节胆碱能标记酶活性,恢复东莨菪碱(SCOP)诱导的记忆缺陷小鼠的记忆障碍。此外,鹿茸分泌的分子可影响突起的生长和轴突的生长。
发明内容
为了研究鹿茸醇提取物对MPTP诱导的帕金森病(PD)小鼠神经变性和神经炎症反应的疗效,本发明公开了一种鹿茸醇提取物的制备方法及其应用。
具体技术方案如下:
鹿茸醇提取物的制备方法如下:
1)将鹿茸冻干粉与无水乙醇按照1g:5ml~1g:20ml的料液比混合,经80℃提取1h,得到提取液;所述冻干粉的制备方法为:将鹿茸在-80~-60℃,0.04bar条件下冷冻干燥48h,再将冷冻后的鹿茸粉碎成鹿茸冻干粉;
2)将步骤1)得到的提取液过滤,收集上清液,残渣备用;
3)将所述残渣重复提取1-3次,对得到的提取液进行过滤,收集上清液;
4)合并步骤2)与步骤3)得到的上清液,经浓缩后干燥得到鹿茸醇提取物。
在本发明的一个实施例中,所述鹿茸冻干粉在冻干之前进行了切片处理,所得鹿茸片的厚度为2-3mm。
在本发明的一个实施例中,所述鹿茸醇提取物的制备方法,具体如下:
1)将无水乙醇与鹿茸冻干粉按照1g:5mL的料液比混合,80℃下回流1h得到提取液;
2)将步骤1)得到的提取液过滤,收集上清液,残渣备用;
3)将步骤2)得到的上清液浓缩,进行干燥,得到鹿茸醇提取物。
在本发明的一个实施例中,鹿茸醇提取物的制备方法步骤3)所述干燥为放入60℃烘箱中烘干。
在本发明的一个实施例中,鹿茸醇提取物的制备方法步骤3)所述浓缩,以每克鹿茸冻干粉质量计,浓缩至液体体积为0.5mL。
进一步地限定上述制备方法获得的鹿茸醇提取物不含有蛋白质,含有萜类化合物、苯酚、类固醇、脂肪酸、脂类和糖苷。
上述的制备方法获得的鹿茸醇提取物在制备治疗和/或预防帕金森病的产品中的应用。
进一步地限定,上述应用所述产品具有下述10种功能中的一种或两种以上:
1)改善帕金森病造成的行为缺陷;
2)增加脑黑质中TH阳性神经元的数量;
3)逆转帕金森病造成的神经元丢失;
4)逆转帕金森病造成的TH表达降低;
5)降低α-syn蛋白水平;
6)抑制星形胶质细胞数量增加及抑制星形胶质细胞激活;
7)抑制胶质细胞激活诱导的炎症反应;
8)保护多巴胺能神经元;
9)逆转纹状体中p-Akt的增加;
10)抑制纹状体p38和p65的磷酸化。
有益效果
在1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的PD模型小鼠中,鹿茸醇提物能够改善PD模型小鼠的行为障碍,抑制纹状体中磷酸化Akt、p65和p38信号通路,降低星形胶质细胞的活化,保护黑质(SN)和纹状体神经元免受损伤。综上所述,鹿茸醇提物在治疗PD和其他炎症及氧化应激相关神经元疾病方面具有广阔的应用前景,这将为鹿茸醇提取物在抗PD新药开发中的应用提供了理论依据,也为其在生物和医药领域的应用开辟了一条新的途径。
附图说明
图1.鹿茸醇提物粗分离过程;
图2.鹿茸醇提物的SDS-PAGE图;
图3.A为帕金森动物模型的建立和鹿茸醇提物给药时间图;B为MPTP;诱导的PD小鼠在鹿茸醇提物治疗后的行为表现实验的结果图;
图4.A为黑质中TH的免疫组织化学分析图;B为纹状体中GFAP和TH的免疫组织化学分析图;
图5.纹状体中α-syn和TH的蛋白水平;
图6.Western blot分析纹状体中p-Akt、p-p65和p-p38蛋白表达;
图7.鹿茸醇提物鹿茸醇提物对黑质和不同器官(心、肝、脾、肺和肾)的苏木素-伊红(HE)染色结果图。
图3-图7中的DAE-100均是指本发明中所获得的鹿茸醇提取物。
具体实施方式
以下结合具体实施例和附图,对本发明作进一步的详细说明,本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等,除以下专门提及的内容之外,均为本领域的普遍知识和公知常识,本发明没有特别限制内容。
实施例1、具有预防和/或治疗帕金森病作用的鹿茸醇提取物的制备方法。
1)将无水乙醇与鹿茸冻干粉混合,提取,得到提取液;具体方法如下:
将购自吉林省左家特产研究所的新鲜梅花鹿二杠鹿茸用切片机切成2-3mm厚的片状,并将其置于在-80℃下,0.04bar条件下进行冷冻干燥48h,然后将冷冻干燥后的鹿茸片用低温研磨机粉碎成鹿茸冻干粉;将10g鹿茸冻干粉与50mL无水乙醇混合,使得到的混合物在水浴温度80℃下回流1h,得到提取液。
2)将步骤1)得到的提取液冷却后过滤,收集上清液,残渣备用;
3)用旋转蒸发器将步骤2)得到的上清液浓缩至液体体积约为5mL后,置于60℃的烘箱中烘干。
实施例2、鹿茸醇提取物的粗分离及粗组分成分鉴定
将鹿茸醇提取物与石油醚按照23.91g:1000mL的料液比进行混合,静置1天后,将其过滤,用甲醇洗涤沉淀得鹿茸无机盐组分,其含量为6.4%;剩余石油醚溶液部分通过中压制备色谱,以二氯甲烷:甲醇=50:50(v:v)和二氯甲烷:甲醇=0:100(v:v)为洗脱剂洗脱分别得到MEs-1、MEs-2和MEs-3组分,含量分别为46.2%、16.7%和14.9%,分离过程如图1所示。
MEs-1和MEs-2通过气相色谱测定其中游离脂肪酸含量,MEs-1含3.96%脂肪酸,MEs-2含2.34%脂肪酸,其中十五烷酸、棕榈酸、硬脂酸、油酸、花生四烯酸含量较高(如表1所示)。
表1鹿茸粗组分脂肪酸种类和含量
(转下页)
(接上页)
实施例3、鹿茸醇提取物化学组成分析
利用聚丙烯酰胺凝胶电泳(SDS-PAGE)检测鹿茸醇提物中是否含有蛋白质:鹿茸醇的上样量为50μg、上样体积为20μL。采用5%浓缩胶,12%分离胶,100V电压下进行SDS-PAGE电泳90min。电泳结束后,经考马斯亮蓝染色后,观察蛋白条带。如图2所示,经上述制备方法得到的鹿茸醇提物中没有蛋白质。
仪器Brukerav-300光谱仪(300MHz 1H,75MHz 13C),通过核磁共振(NMR)对鹿茸醇提物DAE-100中的化合物进行了鉴定。1H-NMR的溶剂为DMSO-D6,13C-NMR的溶剂为MEOD。1HNMR和13C NMR评价鹿茸醇提物的性质。化学分析表明,鹿茸醇提物中存在萜类化合物、苯酚、类固醇、脂类和糖苷。
实施例4、鹿茸醇提取物的药效学研究
本实施例中所用到的小鼠为雄性C57BL/6小鼠,小鼠的周龄为8-10周,体重为20-24g。
1)动物造模及给药
造模前,小鼠连续3天每天进行3次10min的预训练试验。该测试仪器棒直径为3厘米,转速设为40rpm。将小鼠放在旋转杆上,记录每只小鼠在杆上保持平衡的时间。每只小鼠测试三次,间隔20min。平均时间被认为是最后的分数,仅选择行为一致的小鼠进行后续实验。小鼠称重后随机分为3组,每组6只。分为生理盐水组(Saline)、模型组(30mg/kg MPTP)、治疗组(MPTP+30mg/kg鹿茸醇提物)。模型组腹腔注射30mg/kg MPTP,每天一次,连续五天。生理盐水组小鼠每天腹腔注射相同剂量的生理盐水。治疗组(MPTP+30mg/kg鹿茸醇提物),先腹腔注射30mg/kg鹿茸醇提物,半小时后注射30mg/kg MPTP,连续五天。图3中的A总结了实验方案。
转棒实验结果如图3中的B所示,盐水组小鼠在旋转棒上停留时间为918s,模型组小鼠停留时间(145s)明显缩短。经鹿茸醇提物治疗后,在旋转棒上的停留时间(776s)比MPTP组长得多。治疗组小鼠的性能与盐水组小鼠大致相同,表明其PD行为有显著改善。结果表明鹿茸醇提物能够改善PD小鼠的行为缺陷。
2)免疫组化染色和Western blot分析
末次腹腔注射后,第二天将小鼠断颈,取脑,用PBS将脑组织清洗两次,并在冰上迅速从脑组织中分离出脑黑质。其中,每组6只脑组织放入事先配好的4%多聚甲醛溶液中固定24h后,进行石蜡包埋,选取纹状体区和黑质区连续做冠状切片,厚度为5μm。将制作好的切片用免疫组织化学染色、苏木素和伊红染色后,于显微镜下观察纹状体区和黑质区内神经元细胞数量和形态并拍照。另外6只小鼠的脑组织放入-80℃冰箱保存,将纹状体与RIPA裂解液按照的100mg:1mL的比例进行匀浆,得到匀浆液。将匀浆液于4℃,12000rpm条件下离心10min,收集上清液,用于后续蛋白免疫印迹检测。
如图4中A所示,模型组黑质(SN)中的TH阳性神经元明显减少。治疗组TH阳性神经元数量明显增加。另一方面,如图4中B所示,模型组纹状体中TH阳性神经元的数量明显减少,鹿茸醇提物治疗可显著抑制MPTP诱导的PD模型小鼠纹状体TH阳性神经元的减少。
Western blot结果表明,模型组纹状体中TH的表达下调,鹿茸醇提取物逆转了纹状体中TH的减少(如图5所示)。这些结果表明,将鹿茸醇提取物用于治疗PD模型小鼠,可显著逆转MPTP诱导的PD模型小鼠多巴胺神经元的丢失和TH表达的降低。
如图5所示,模型组MPTP诱导α-syn明显上调,鹿茸醇提物治疗后α-syn蛋白水平明显下降。TH的上调和α-syn的下调表明,鹿茸醇提物具有防止多巴胺神经元变性的潜力。
如图4中B所示,模型组GFAP表达较高,表明星形胶质细胞数量增加,具有星形胶质细胞肥大的炎症反应特征。鹿茸醇提物治疗后,显著阻止MPTP诱导的星形胶质细胞数量增加及其激活。结果表明鹿茸醇提物抑制了胶质细胞激活诱导的炎症反应,保护多巴胺能神经元。
如图6所示,MPTP诱导p-AKT明显上调,鹿茸醇提物逆转了纹状体中p-AKT的增加。因此,鹿茸醇提物通过逆转AKT信号有效治疗MPTP诱导的PD小鼠模型。模型组p-p65和p-p38蛋白水平明显高于生理盐水注射的对照组小鼠。鹿茸醇提物治疗组p65和p38磷酸化明显降低。鹿茸醇提物抑制了MPTP诱导的动物模型中纹状体p38和p65的磷酸化。
3)鹿茸醇提物的体内病理学评价
小鼠腹腔注射DAE-100(10和30mg/kg)7天,随后组织(黑质、心、肝、脾、肺和肾)放入事先配好的4%多聚甲醛溶液固定24h,进行石蜡包埋,做冠状切片,厚度为5μm。苏木素和伊红染色分析组织病理情况。
为探讨鹿茸醇提物的体内安全性,苏木精-伊红(HE)染色检测不同器官(心、肝、脾、肺和肾)和黑质(如图7中A)。和对照组相比,鹿茸醇提物(10和30mg/kg)处理组心(Heart)、肝(Liver)、脾(Spleen)、肺(Lung)、肾(Kidney)和黑质(Substantia nigra)没有发现损伤,小鼠无明显不良反应。MPTP处理的小鼠,黑质神经元和胶质细胞表现出明显的分离(如图7中B)。鹿茸醇提物(30mg/kg)治疗,病变组织正常。结果表明,鹿茸醇提物对PD的治疗有显著效果。
综合以上多个实施例可知,鹿茸醇提物中没有蛋白质或多肽成分,有萜类化合物、苯酚、类固醇、脂肪酸、脂类和糖苷。鹿茸醇提物改善了MPTP诱导的C57BL/6小鼠行为异常和多巴胺能神经元丢失。因此,鹿茸醇提物有可能被开发成一种有效和有用的治疗剂,对抗PD等疾病。
以上所述仅为本申请的实施例而已,并不用于限制本申请。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的权利要求范围之内。
Claims (8)
1.一种鹿茸醇提取物的制备方法,其特征在于,方法如下:
1)将鹿茸冻干粉与无水乙醇按照1g:5ml~1g:20ml的料液比混合,经80℃提取1h,得到提取液;所述冻干粉的制备方法为:将鹿茸在-80~-60℃,0.04bar条件下冷冻干燥48h,再将冷冻后的鹿茸粉碎成鹿茸冻干粉;
2)将步骤1)得到的提取液过滤,收集上清液,残渣备用;
3)将所述残渣重复提取1-3次,对得到的提取液进行过滤,收集上清液;
4)合并步骤2)与步骤3)得到的上清液,经浓缩后干燥得到鹿茸醇提取物。
2.根据权利要求1所述的制备方法,其特征在于,鹿茸冷冻前进行了切片处理,所得鹿茸片的厚度为2-3mm。
3.根据权利要求1所述的制备方法,其特征在于,具体如下:
1)将无水乙醇与鹿茸冻干粉按照1g:5mL的料液比混合,80℃下回流1h得到提取液;
2)将步骤1)得到的提取液过滤,收集上清液,残渣备用;
3)将步骤2)得到的上清液浓缩,进行干燥,得到鹿茸醇提取物。
4.根据权利要求3所述的制备方法,其特征在于,步骤3)所述干燥为60℃烘干。
5.根据权利要求3所述的制备方法,其特征在于,步骤3)所述浓缩,以每克鹿茸冻干粉质量计,浓缩至液体体积为0.5mL。
6.根据权利要求1-5任意一项所述的制备方法,其特征在于,所述获得的鹿茸醇提取物,不含有蛋白质,含有萜类化合物、苯酚、类固醇、脂肪酸、脂类和糖苷。
7.权利要求1-5任意一项所述的制备方法获得的鹿茸醇提取物在制备治疗和/或预防帕金森病的产品中的应用。
8.根据权利要求7所述的应用,其特征在于,所述产品具有下述10种功能中的一种或两种以上:
1)改善帕金森病造成的行为缺陷;
2)增加脑黑质中TH阳性神经元的数量;
3)逆转帕金森病造成的神经元丢失;
4)逆转帕金森病造成的TH表达降低;
5)降低α-syn蛋白水平;
6)抑制星形胶质细胞数量增加及抑制星形胶质细胞激活;
7)抑制胶质细胞激活诱导的炎症反应;
8)保护多巴胺能神经元;
9)逆转纹状体中p-Akt的增加;
10)抑制纹状体p38和p65的磷酸化。
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