CN117122563A - 一种五倍子酸注射剂及其应用 - Google Patents
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
本发明属于五倍子酸应用技术领域,具体涉及一种五倍子酸注射剂及其应用。所述注射剂由五倍子酸和二甲基亚砜制备;将五倍子酸溶于二甲基亚砜,搅拌均匀,配制成储存液以制备注射剂,其中,五倍子酸的浓度为2.0~3.0mg/mL,尤其是五倍子酸的浓度为2.5mg/mL时,在预防和治疗缺血性脑卒中药物的应用中,五倍子酸用量为0.5mg/kg,可以降低小鼠缺血性脑梗死的体积。五倍子酸注射剂抗脑缺血作用的发现,使其被用于开发研制新的抗缺血性脑卒中的药物成为可能。本发明的研究中采用手术前、手术后1h和手术后每日给药相结合的给药方式,能够充分发挥药物的作用,而且应用方法简单。
Description
技术领域
本发明属于五倍子酸应用技术领域,具体涉及一种五倍子酸注射剂及其应用。
背景技术
缺血性脑卒中(脑梗死)是由于各种原因引起的脑部血液供应障碍,致使局部脑组织缺血缺氧性坏死而出现功能损害的一类脑血管疾病。它具有高发病率、高死亡率、高致残率和高复发率的特点,严重威胁我国人民的身体健康甚至生命安全,给家庭和社会带来沉重负担。缺血性脑卒中病人若在发病早期能够得到有效诊治,可以很大程度上改善患者的预后,减轻家庭和国家的经济负担。缺血性脑卒中的治疗原则是首先应尽快恢复脑血流,减轻脑损伤。临床上,针对缺血性脑卒中病因治疗的有效措施主要是通过静脉溶栓的方法和血管取栓。常用的静脉溶栓方法包括组织型纤溶酶原激活剂(tPA)溶栓和尿激酶静脉溶栓。但是,这些溶栓剂的时间窗仅为4-6h。事实上,多数病人从发病到就诊的时间已超过药物的时间窗而错失最佳治疗时机。另外,静脉溶栓和血管介入取栓的治疗措施也存在导致颅内出血,进一步加重病情的危险。因此,迫切需要寻找和研发一种能够有效地预防和治疗缺血性脑卒中,且不受时间窗限制的药物用于缺血性脑卒中的预防和治疗,将对积极有效预防缺血性脑卒中的发生,减少和降低该病的发病率及死亡率,保护神经功能,促进受损脑功能恢复,进而提高缺血性脑卒中病人康复期的生活质量具有重要意义。
五倍子酸(gallicacid,GA)又称没食子酸,在明代的《医学入门》和《本草纲目》中均有相关记载。GA的化学结构已明确,化学名3,4,5-三羟基苯甲酸,分子式C7H6O5。它广泛存在于掌叶大黄、山茱萸、大叶桉等植物中,除了具有抗菌、抗病毒、杀锥虫、抗肿瘤、抗氧化等多种生物学活性外,还具有焰火稳定、紫外吸收、调控动植物生长和照相显影等作用,被广泛用在生物、医药、食品、化工等领域。
本发明人长期从事缺血性脑卒中的研究。通过实验研究五倍子酸的作用,实验结果表明五倍子酸具有明显的改善缺血性脑卒中的症状和显著的神经保护作用,具有良好的应用前景和巨大的开发应用价值。
发明内容
本发明所要解决的技术问题就是提供一种五倍子酸注射剂及其应用,使用方法简单有效。采用的技术方案为:
一种五倍子酸注射剂,所述注射剂由五倍子酸和二甲基亚砜制备;
将五倍子酸溶于二甲基亚砜,搅拌均匀,配制成储存液以制备注射剂,其中,五倍子酸的浓度为2.0~3.0mg/mL。
作为进一步的优选,所述五倍子酸的浓度为2.5mg/mL。
上述的一种五倍子酸注射剂在预防和治疗缺血性脑卒中药物的应用。采用所述一种五倍子酸注射剂可以降低缺血性脑梗死的体积。促进脑梗死后脑神经功能的康复,为五倍子酸用于缺血性脑卒中的药物研发提供了保障。
作为进一步的优选,所述一种五倍子酸注射剂在应用时,五倍子酸的用量为0.1~1.0mg/kg。
作为更进一步的优选,所述一种五倍子酸注射剂在应用时,五倍子酸的用量为0.5mg/kg,效果最好。
与现有技术相比,本发明的显著效果在于:
1)本发明提供了一种五倍子酸注射剂,所述注射剂采用腹腔注射,可以降低小鼠缺血性脑梗死的体积,并明确了五倍子酸的用量为0.5mg/kg;
2)本发明中明确了五倍子酸可以改善大脑中动脉栓塞(middle cerebral arteryocclusion,MCAO)病情,促进小鼠的功能康复;
3)五倍子酸注射剂抗脑缺血作用的发现,使其被用于开发研制新的抗缺血性脑卒中的药物成为可能;
4)与传统的术前或术后给药相比,本发明的研究中采用手术前、手术后1h和手术后每日给药相结合的给药方式,能够充分发挥药物的作用,而且应用方法简单。
附图说明
图1为本发明实验方法流程图。
图2为本发明实施例中对脑梗死体积测定;其中,A图为脑组织切片的TTC染色对比图,图B为统计分析A图结果。
图3为本发明实施例中五倍子酸注射剂对手术后小鼠体重的影响。
图4为本发明实施例中五倍子酸注射剂对缺血性脑卒中小鼠神经功能恢复的影响。
具体实施方式
附图仅用于示例性说明;对于本领域的技术人员来说,附图中的某些公知结构及其说明可能省略,因此,不能理解为对本发明的限制。
本发明实施例所使用的五倍子酸的结构式为:
本发明所使用的五倍子酸来源于Sigma-Aldrich,纯度≥98.5%,二甲基亚砜的来源于Sigma-Aldrich,纯度≥99.9%。
一种五倍子酸注射剂,所述注射剂包括五倍子酸和二甲基亚砜。
将五倍子酸溶于二甲基亚砜,搅拌均匀,配制成储存液以制备注射剂,其中,五倍子酸的浓度为2.5mg/mL。
上述的一种五倍子酸注射剂在预防和治疗缺血性脑卒中的应用,可以降低缺血性脑梗死的体积。促进脑梗死后脑神经功能的康复,为五倍子酸用于缺血性脑卒中的药物研发提供了保障。
为了验证本发明的五倍子酸注射剂在预防和治疗缺血性脑卒中应用时使用的用量,采用小鼠作为实验动物进行测试。
1.小鼠分组:80只雄性C57BL/6J小鼠,年龄6-8W,体重:22-24g。其中,60只小鼠被用于五倍子酸药物浓度测定,剩余小鼠被随机分为对照组和用药组,每组10只。
2.总体实验设计及实施方案
以C57BL/6J小鼠制备MCAO模型,分别于手术前后给予五倍子酸腹腔注射(IP),检测小鼠的脑梗死体积和术后神经功能恢复情况。如图1所示,为本实施例测试实验流程技术路线图。
3.缺血性脑卒中小鼠模型的制备
(1)C57BL/6J小鼠经过1.25%三溴乙醇(Sigma),0.2mL/10g麻醉后,颈部皮肤去除毛发,放置于手术台上固定。手术过程中,小鼠体温始终保持在37℃±0.5℃。将小鼠颈部伸直,局部皮肤用5%碘酊和75%酒精消毒后,取颈正中切口剪开皮肤,体视显微镜下分离皮肤下软组织,以小鼠用组织拉钩分离固定组织,暴露颈动脉鞘。
(2)以显微手术镊子小心分离出颈总动脉约1.5cm,放置动脉夹,阻断动脉血流。
(3)分离颈外侧动脉,动脉的远心端游离并用线牢固结扎。近心端穿线备用。
(4)小心分离颈内动脉,放置动脉夹,阻断血流。
(5)在颈外动脉的远心端和近心端之间用动脉剪刀剪一个小口,将线栓由颈外动脉插入颈内动脉。
(6)移除颈内动脉处固定的动脉夹,将线栓插入颈内动脉离心端,最终插入大脑中动脉起始处,阻断血流。
(7)暂时固定结扎颈外动脉近心端,防止线栓脱落,60min后小心将线栓移除,永久固定结扎颈外动脉近心端,移除颈总动脉夹,恢复小鼠脑部血流。
(8)将小鼠暴露于手术视野中的组织归回原位,缝合小鼠颈部切口。在整个手术过程中,注意无菌操作和给小鼠保温;待小鼠苏醒后放回笼内,术后严密观察小鼠的活动状态和伤口出血情况。
(9)根据小鼠综合评分来判断小鼠术后是否造模成功。
4.五倍子酸药物浓度的选择
将五倍子酸溶于二甲基亚砜(DMSO),制备2.5mg/mL的储存液,备用。小鼠于手术前16h和MCAO手术后1小时经腹腔注射不同浓度的五倍子酸。每组小鼠腹腔注射五倍子酸的浓度分别为0,0.1,0.25,0.5,1.0,2.0mg/kg。药物被注射入每只小鼠体内的最终体积为0.1mL,观察并记录小鼠的变化。在小鼠脑部缺血再灌注后24h,取小鼠的脑组织进行脑组织染色,鉴定脑梗死体积。
5.小鼠神经功能检测方法
(1)神经功能损害检测评定标准
MCAO手术后,采用Longa五分法对术后小鼠初步进行综合性评分。
①手术后小鼠无明显的神经功能损害症状:0分;
②提尾时,小鼠损伤对侧前肢不能完全伸直:1分;
③小鼠不能直行,向瘫痪侧转圈:2分;
④小鼠行走时,向瘫痪侧倾倒:3分;
⑤小鼠不能主动行走,存在意识丧失现象:4分;
所有MCAO术后的小鼠经初步评分后,评分为处于2~3分之间的小鼠用于后续实验操作。
(2)小鼠脑梗死体积的测定
红四氮唑(2,3,5-Triphenyl-2H-tetrazolium chloride,TTC)染色。TTC染色的原理为红四氮唑是呼吸链中吡啶—核苷结构酶系统的质子受体。当它与正常组织中的脱氢酶反应时,正常组织被染成深红色,而缺血组织因脱氢酶活性降低或缺失,缺血组织染色后呈现苍白色。
红四氮唑(Sigma,纯度≥99%)溶液的配制:以PBS配制成2%的溶液,2-8℃避光保存。小鼠在脑缺血再灌注后的第24h,经1.25%三溴乙醇(0.2mL/10g)麻醉后,迅速取出脑组织,置于0~4℃PBS溶液中,随后转移到-20℃冰箱冻存30min。小心取出脑组织行冠状切片,脑片切的厚度约为2mm。将切好的每只小鼠的全部脑片放在2%红四氮唑溶液中,37℃避光水浴30min,每隔5min轻轻晃动染液一次,使脑片被充分均匀染色。随后,取出脑组织切片,按照从前向后的顺序依次排好,马上拍照后置于10%的中性甲醛溶液中固定脑组织。
脑梗死体积的测量及计算采用Image J(National Institutes of Health,NIH)软件。
计算公式:梗死体积/(梗死体积+未梗死体积)×100%
(3)小鼠体重的测定
小鼠手术当日和手术后第1,3,5,7d,将小鼠放在小鼠体重称上,称量小鼠体重,分别记录对照组和用药组小鼠的实验数据,其结果见图3所示。小鼠生存期间每日给予0.5mg/kg的五倍子酸腹腔注射。
(4)小鼠神经学评分标准见表1所示。
表1为神经学评分标准
手术当日及手术后第1d,第3d,第5d和第7d对小鼠进行神经功能学评分。在此期间,所有的实验用药组小鼠均每日腹腔注射0.5mg/kg的五倍子酸注射剂,对照组小鼠注射同体积的DMSO溶于生理盐水。
6.统计分析
GraphPad Prism 8.2(Graphpad公司)绘图分析软件用于实验数据分析及绘图。两组之间的数据分析采用t检验。多于两组实验数据则用方差分析(单因素或双因素)后,各组之间的比较用Dunnett’s多因素分析检验或Sidak’s多因素分析比较。
7.实验结果
(1)对小鼠MCAO术后神经功能损害综合评分,判定手术的成模率
小鼠神经损害功能评分见表2所示。
表2小鼠分组及术后神经功能损害综合评分
组别 | 小鼠数目(n) | 综合评分/分 |
对照组 | 10只 | 2~3 |
实验组 | 50只 | 2~3 |
MCAO术后的小鼠经过综合评分。仅评分结果高于2分,低于3分的小鼠用于后续研究。
(2)五倍子酸导致小鼠MCAO后脑梗死的体积减小
小鼠于手术后24h取脑组织进行TTC染色,测定脑梗死体积。实验结果如图2所示。左边的A图是脑组织切片的TTC染色。其中,每一行代表不同小鼠脑组织的同一解剖位置的染色。每一列的5张脑片代表一只小鼠的全部脑组织切片。这样,图中共6只小鼠,每只小鼠分别切了5张脑片,计30个小图。
右面的B图是统计分析A图结果。数值为(均数±标准差),n=10,与实验对照组相比,n.s:无统计学差异。统计结果显示,与实验对照组小鼠(0mg/kg五倍子酸)相比,小鼠经过0.5mg/kg的五倍子酸处理后,脑梗死的体积明显变小。若五倍子酸的浓度低于0.5mg/kg,则对脑梗死的体积无明显作用(P>0.05),若药物浓度继续升高而高于0.5mg/kg时,如1.0mg/kg和2.0mg/kg时,高浓度的五倍子酸的应用并没有降低小鼠脑梗死的体积(P>0.05)。因此在本发明中,用于小鼠MCAO损伤后神经康复功能检测的小鼠,于MCAO手术前后分别注射了0.5mg/kg五倍子酸。
(3)五倍子酸对小鼠体重的影响
如图3所示,五倍子酸注射剂对手术后小鼠体重的影响。小鼠经过MCAO后,分别在手术后第0,1,3,5,7d测定小鼠体重的变化。实验数据为(均数±标准差),n=10,n.s:无统计学差异。小鼠手术后的体重有所降低,但与对照组相比,五倍子酸没有对小鼠的术后体重产生明显的影响,两组小鼠之间的体重无明显统计学差异(P>0.05)。
(4)五倍子酸注射剂促进MCAO术后小鼠神经功能恢复
如图4所示,五倍子酸注射剂对缺血性脑卒中小鼠神经功能恢复的影响。在小鼠MCAO手术后的第0,1,3,5,7d,分别对小鼠进行神经学评分。实验数据为(均数±标准差),n=10,*P<0.05,**P<0.01,****P<0.0001。
小鼠在手术前16h经腹腔注射0.5mg/kg五倍子酸,并于MCAO手术后1h,经腹腔注射0.5mg/kg五倍子酸。小鼠在脑缺血再灌注后的第1d,第3d、第5d和第7d分别进行神经学评分标准判定。与对照组相比,如图3所示五倍子酸在术后1d就能够明显促进术后小鼠脑神经功能的恢复(*P<0.05)。在手术后的第3d,第5d和第7d同样检测到五倍子酸可以显著降低小鼠的神经学评分(**P<0.01,****P<0.0001),促进小鼠的神经功能康复。
上述测试结果证明,采用腹腔注射的方法,五倍子酸注射量为0.5mg/kg时,五倍子酸注射剂可以减小脑梗死的体积,促进脑梗死后脑神经功能的康复,为五倍子酸注射剂用于缺血性脑卒中的药物研发提供了保障。
本发明实施例中,统计学P值即概率,代表某一事件发生的可能性大小。P≤0.05被认为是统计学的边界线。
当然,上述说明并非是对本发明的限制,本发明也并不仅限于上述举例,本技术领域的技术人员在本发明的实质范围内所做出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (3)
1.一种五倍子酸注射剂,其特征在于,所述注射剂由五倍子酸和二甲基亚砜制备;
将五倍子酸溶于二甲基亚砜,搅拌均匀,配制成储存液以制备注射剂,其中,五倍子酸的浓度为2.0~3.0mg/mL。
2.根据权利要求1所述的一种五倍子酸注射剂,其特征在于,所述五倍子酸的浓度为2.5mg/mL。
3.如权利要求1或2所述的一种五倍子酸注射剂在预防和治疗缺血性脑卒中药物的应用。
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