CN109091530B - 紫苏叶提取物在预防或治疗再生障碍性贫血中的应用 - Google Patents
紫苏叶提取物在预防或治疗再生障碍性贫血中的应用 Download PDFInfo
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Abstract
本发明公开了紫苏叶提取物在制备治疗和/或预防再生障碍性贫血产品中的应用。本发明所述的紫苏叶提取物对模型小鼠的体重下降、进食量和脏器指数下降具有逆转作用；可改善外周血及骨髓造血功能的各项指标：显著提高外周血红细胞、血红蛋白、白细胞、淋巴细胞、中性粒细胞以及血小板的含量，显著增加外周血网织红细胞数，显著增加骨髓细胞数和CD34+细胞比例，改善造血功能。
Description
技术领域
本发明涉及一类紫苏叶提取物及其药物组合物用于预防和/或治疗再生障碍性贫血药物或食物中的应用，属于医药或食品技术领域。
背景技术
再生障碍性贫血(简称再障，aplastic anemia，AA)是一种化学、物理、生物因素或不明原因所致的获得性骨髓造血功能衰竭性疾病，表现为骨髓造血功能低下，全血细胞减少和贫血、出血、感染综合征。其发病原因或机制十分复杂，目前认为与造血干细胞内在缺陷、造血微环境异常及机体免疫功能紊乱有关。虽然未能明确具体的发病机制，但是对于免疫介导在发病过程中的关键作用，已经取得了比较广泛的共识。目前免疫抑制剂是主要的治疗手段，常用的免疫抑制剂包括抗胸腺球蛋白/抗淋巴细胞球蛋白、环孢霉素和甲基泼尼松龙等。这些药物价格昂贵，副作用大，复发率高，其治疗效果往往不尽人意。因此寻找治疗的其他途径显得尤为重要和紧迫。
紫苏Perilla frutescens L.是唇形科紫苏属一年生草本植物，为临床常用中药，味辛，性温，具有解表散行气和胃等功效，用于风寒感冒，咳嗽呕恶，妊娠呕吐，鱼蟹中毒。紫苏叶含有的挥发油，黄酮，酚酸等活性成分，具有良好的抗炎、抗氧化、抗过敏、止血、降血脂等药理活性。目前尚未见关于紫苏叶提取物及其治疗再生障碍性贫血的相关研究报道，为有效阐释传统中药治疗再生障碍性贫血的作用，并为寻找活性更好的治疗药物提供理论依据和实践经验，我们制备了紫苏叶提取物，并对其抗再生障碍性贫血的疗效进行了评价。
发明内容
为了克服现有技术的不足，本发明解决的技术问题是提供紫苏叶提取物在治疗再生障碍性贫血中的应用。
为解决本发明的技术问题，本发明提供如下技术方案：
本发明技术方案是提供了一种紫苏叶提取物在制备治疗和/或预防再生障碍性贫血相关产品中的应用，其特征在于，紫苏叶提取物的制备步骤如下：
1)以紫苏叶药材为原料，水或C1～C5低级醇水溶液为溶剂进行提取，回收含水低级醇溶剂，回收后的溶液浓缩，制得浸膏；
2)将浸膏加水分散，加入低极性有机溶剂萃取，剩余水溶液浓缩得到得浸膏；
3)所得浸膏以C1～C5低级醇水溶液醇沉，过滤，沉淀物干燥、粉碎得到紫苏叶提取物。
在上述制备方法的步骤1)中，提取溶剂为水或C1～C5低级醇水溶液，优选C1～C3低级醇水溶液，进一步优选为乙醇水溶液；C1～C5低级醇水溶液的低级醇浓度为0-95％(体积/体积)，优选为50-80％(体积/体积)，进一步优选为65％-75％(体积/体积)；提取方法为浸渍、渗漉、煎煮或加热回流，优选煎煮或加热回流；提取次数为1-5次，优选2-3次；浓缩为常压浓缩或减压浓缩，优选减压浓缩。
在上述制备方法的步骤2)中，萃取所用低极性有机溶剂为环己烷、石油醚、二氯甲烷、氯仿、乙酸乙酯、正丁醇、异戊醇或它们的混合溶剂，优选乙酸乙酯或正丁醇溶剂，进一步优选为正丁醇溶剂；萃取次数为1-5次，优选2-3次。浓缩为常压浓缩或减压浓缩，优选减压浓缩。
在上述制备方法的步骤3)中，醇沉所用C1～C5低级醇水溶液优选C2～C3低级醇水溶液，进一步优选为乙醇水溶液；C1～C5低级醇浓度为50-90％(体积/体积)，优选为60-85％(体积/体积)，进一步优选为75％-85％(体积/体积)；醇沉时间为8-72h,优选为12-24h；干燥方法为常压干燥、真空干燥、冷冻干燥、喷雾干燥，优选真空干燥、冷冻干燥。
以上所述的产品包括药品或保健品。
再生障碍性贫血是一种血液科常见疾病。以钴-60γ射线+胸腺免疫细胞接种制备小鼠再生障碍性贫血模型，紫苏叶提取物在0.1，0.2g/kg剂量下灌胃给药。结果显示，紫苏叶提取物对模型小鼠的体重下降、进食量和脏器指数下降具有逆转作用；可改善外周血及骨髓造血功能的各项指标：显著提高外周血红细胞、血红蛋白、白细胞、淋巴细胞、中性粒细胞以及血小板的含量，显著增加外周血网织红细胞数，显著增加骨髓细胞数和CD34+细胞比例，改善造血功能。
本发明的优点在于：(1)本发明属于中药制剂，成分纯天然，毒副作用低；(2)本发明能有效预防和/或治疗再生障碍性贫血；(3)本品原料易得，制备工艺简单，易于规范化生产。
具体实施方式
以下实施例对本发明做进一步的说明。但本发明并不限于这些实施例。
实施例1：紫苏叶提取物的制备
取干燥的紫苏叶1.5kg，加入70％乙醇5升，加热回流1.5小时，趁热过滤。滤渣以同样的方法再处理一次；合并两次所得滤液，减压回收乙醇，得浸膏65g。该浸膏加入300mL水搅拌制成混悬液，分别以150mL正丁醇萃取2次。剩余水部分加入1580mL95％乙醇溶解，室温沉淀24h，减压抽滤，沉淀物以50ml水溶解，冷冻干燥、粉碎得紫苏叶提取物(22g)。
实施例2：剂型制备
胶囊：紫苏叶提取物100g，加入糊精147.5g，硬脂酸镁2.5g，充分拌均匀后，按胶囊制备法制成1000粒，填装于250mg胶囊，每粒含紫苏叶提取物100mg，其总量不低于50％。
片剂：取紫苏叶提取物100g，加入淀粉100g，糊精80g，蔗糖15g，充分搅拌混匀，制成颗粒，在60℃以下干燥，加入适量硬脂酸镁、滑石粉，混匀，压制成1000片，包糖衣或薄膜衣，即得。
颗粒剂：取紫苏叶提取物100g，加入适量蔗糖和糊精，按颗粒剂制法制成颗粒，干燥，制成1000g。
口服液：取紫苏叶药材，70％乙醇加热回流1.5小时提取2次，滤过，合并滤液，减压回收乙醇；浸膏加入等量水搅拌制成混悬液，正丁醇萃取2次；水部分加入95％乙醇适量溶解，室温沉淀24h，减压抽滤，沉淀物以40％氢氧化钠调节PH值至7.0，搅匀，4-8℃冷藏48小时，滤过，滤液加入蔗糖300g，搅匀使溶解，再加入适量香精并调节PH值至7.0，加水制成1000ml，搅匀，静置12小时，滤过，灌装，灭菌，即得。
药理实验
实验例1：紫苏叶提取物对再生障碍性贫血的治疗作用
实验方法：
取18-20g雄性BALB/c小鼠，随机分为正常对照组、模型对照组、阳性对照组(再造生血片1.94g/kg，灌胃给药)、紫提取物组(0.1g/kg、0.2g/kg，灌胃给药)，每组12只。除空白对照组外，各组动物采用钴-60γ射线(5.4Gy钴-60γ射线一次全身照射)+DBA/2小鼠胸腺：脾免疫细胞(2：1)静脉注射接种制备再生障碍性贫血模型；从造模当日开始灌胃给药，每天1次，连续14天。于末次给药1小时后，小鼠眶静脉采血，EDTA-K2抗凝，五分类血细胞分析仪检测血液常规以及流式细胞仪检测网织红细胞数；摘取小鼠胸腺和脾脏，称其湿重，并计算脏器指数；取小鼠右侧股骨骨髓，流式细胞仪检测骨髓细胞计数和CD34+细胞比例。
实验结果：
(1)紫苏叶提取物对再生障碍性贫血小鼠生长情况和进食量的影响
体重是衡量患者生存质量的重要指标之一。再障患者由于长期处于贫血、出血、感染等症状中，极易出现由于消化、吸收等功能紊乱而引起的体重降低。本实验通过称量再障小鼠体重和进食量来初步观察紫苏叶提取物对再生障碍性贫血小鼠一般生长情况的影响。结果如表1.1和1.2所示。表1.1结果显示，与正常对照组比较，再障模型组小鼠7天、14天体重显著降低，而紫苏叶提取物可减少再障小鼠的体重降低，其中0.2g/kg组第7天体重与模型组比较差异显著。表1.2结果显示，与正常对照组比较，再障模型小鼠7天、14天进食量显著降低，而紫苏叶提取物及阳性对照药可显著增加再障小鼠的进食量，提示，紫苏叶提取物及阳性对照药具有改善再障引起的小鼠消化功能损伤。
表1.1紫苏叶提取物对再生障碍性贫血小鼠体重生长的影响(Mean±Std,n＝10-12)
备注：与空白对照组比较，#p＜0.05，##p＜0.01；与模型对照组比较，*p＜0.05，**p＜0.01
表1.2紫苏叶提取物对再生障碍性贫血小鼠进食量的影响(Mean±Std,n＝10-12)
备注：与空白对照组比较，#p＜0.05，##p＜0.01；与模型对照组比较，*p＜0.05，**p＜0.01
(2)紫苏叶提取物对再生障碍性贫血小鼠胸腺指数和脾脏指数的影响
胸腺、脾脏脏器指数可反映动物的免疫功能状态，本实验于末次给药1小时后，摘取各组小鼠的胸腺和脾脏，称其湿重，并计算脏器指数(脏器指数＝脏器重量/当日体重×100％)。结果见表2。表2结果显示，紫苏叶提取物可增加再生障碍性贫血小鼠的胸腺指数，其中0.2g/kg剂量与模型组比较差异具有显著性，说明其具有改善再障小鼠免疫功能低下的作用。
表2紫苏叶提取物对再生障碍性贫血小鼠脏器指数的影响(Mean±Std,n＝10-12)
备注：与空白对照组比较，#p＜0.05，##p＜0.01；与模型对照组比较，*p＜0.05，**p＜0.01
(3)紫苏叶提取物对再生障碍性贫血小鼠血液常规检查结果的影响
外周血全血细胞的减少是贫血、出血、感染等临床症状发生的主要原因，同时也是反映骨髓衰竭的间接指标。外周血各项指标尤其是红细胞、血红蛋白、白细胞、淋巴细胞和血小板的改善是再障得到有效治疗最直接的证据。本实验于末次给药1小时后，小鼠眼球取血，采用五分类血细胞分析仪检测小鼠外周血的各项指标。结果见表3A、3B、3C。
①紫苏叶提取物对再生障碍性贫血小鼠外周血中红细胞数和血红蛋白含量的影响
如表3A结果所示，紫苏叶提取物可改善再障小鼠外周血红细胞计数的减少，与模型对照组比较差异具有显著性(p<0.05或0.01)；紫苏叶提取物对再障小鼠外周血血红蛋白含量低下具有改善作用，0.2g/kg剂量组与模型对照组比较差异具有显著性(p<0.01)；此外，紫苏叶提取物对再障小鼠外周血红细胞压积(HCT)具有显著的作用。
表3A紫苏叶提取物对再生障碍性贫血小鼠外周血中红细胞数和血红蛋白含量的影响(Mean±Std,n＝10)
备注：红细胞(RBC)，血红蛋白(HGB)，血红细胞压积(HCT)，红细胞平均体积(MCV)，平均红细胞血红蛋白含量(MCH)，平均红细胞血红蛋白浓度(MCHC)，红细胞体积分布宽度标准差(RDW-SD)，红细胞体积分布宽度变异系数(RDW-CV)；#p＜0.05，##p＜0.01vs空白对照组；*p＜0.05，**p＜0.05vs模型对照组。
②紫苏叶提取物对再生障碍性贫血小鼠外周血中白细胞数和淋巴细胞数的影响
如表3B结果显示，紫苏叶提取物在0.1、0.2g/kg剂量下对再生障碍性贫血模型小鼠灌胃给药，可显著增加小鼠外周血中白细胞计数，0.2g/kg剂量组可显著增加再障小鼠外周血淋巴细胞计数、中性粒细胞计数(p<0.05或0.01)，且效果优于等效剂量阳性对照药-再生造血片。
表3B紫苏叶提取物对再生障碍性贫血小鼠外周血中白细胞分类计数的影响(Mean±Std,n＝10)
备注：白细胞计数(WBC)，淋巴细胞计数(LYM#)，单核细胞计数(Mon#)，中性粒细胞计数(NEUT#)，嗜酸性粒细胞计数(Eos#)，嗜碱性粒细胞计数(Bas#)，淋巴细胞百分比(LYM％)，单核细胞百分比(Mon％)，中性粒细胞百分比(NEUT％)，嗜酸性粒细胞百分比(Eos％)，嗜碱性粒细胞百分比(Bas％)；#p＜0.05，##p＜0.01vs空白对照组；*p＜0.05，**p＜0.05vs模型对照组。
③紫苏叶提取物对再生障碍性贫血小鼠外周血中血小板计数的影响
如表3C结果所示，紫苏叶提取物可改善再生障碍性贫血模型小鼠外周血血小板的减少，且0.2g/kg灌胃给药剂量组与模型对照组比较差异具有显著性(p<0.05)。且与模型组比较，可0.2g/kg剂量组对再障小鼠血板体积及体积平均分布宽度的改变具有显著的作用。
表3C紫苏叶提取物对再生障碍性贫血小鼠外周血中血小板的影响(Mean±Std,n＝10)
备注：血小板计数(PLT)，平均血小板体积(MPV)，血小板体积分布宽度(PDW)；#p＜0.05，##p＜0.01vs空白对照组；*p＜0.05，**p＜0.05vs模型对照组。
(4)紫苏叶提取物对再生障碍性贫血小鼠外周血网织红细胞数的影响。
网织红细胞是尚未完全成熟的红细胞，在外周血液中的数值可反映骨髓红细胞的生成功能和骨髓造血功能，对血液病的诊断和治疗反应的观察均有重要意义。在临床对再生障碍性贫血患者进行治疗的过程中,网织红细胞参数的升高是患者的骨髓造血功能得到恢复的一种重要指标。本实验于末次给药1小时后，各组小鼠眼眶静脉采血，以噻唑橙作为RNA的荧光染料标记外周血网织红细胞，并采用流式细胞术检测外周血网织红细胞比例，并根据外周血红细胞计数计算外周血网织红细胞计数。结果见表4。表4结果显示，与正常对照组比较，再障模型组小鼠外周血网织红细胞比例及计数均显著降低，而紫苏叶提取物在0.1，0.2g/kg剂量下灌胃给药，可显著增加再生障碍性贫血小鼠外周血的网织红细胞比例和计数(p<0.05或0.01)，且效果优于等效剂量的再生造血片。提示，紫苏叶提取物对再生障碍性贫血小鼠的骨髓红细胞生成功能和骨髓造血功能具有明显的改善作用。
表4紫苏叶提取物对再生障碍性贫血小鼠外周血中网织红细胞数的影响(Mean±Std,n＝10)
备注：#p＜0.05，##p＜0.01vs空白对照组；*p＜0.05，**p＜0.05vs模型对照组
(5)紫苏叶提取物对再生障碍性贫血小鼠骨髓细胞数和CD34+细胞比例的影响。
CD34+细胞主要表达于早期造血干细胞、祖细胞,可能在造血干细胞、祖细胞于骨髓的粘附和归巢过程中起作用。骨髓CD34+细胞可以直接反映出骨髓造血细胞的生成情况。再障患者骨髓CD34+细胞较正常显著减少，因此，骨髓CD34+细胞对再生障碍性贫血的发病机制研究具有重要的意义，亦可作为再生障碍性贫血的诊断、鉴别诊断和疗效判定的重要指标。本实验于末次给药1小时后，取各组小鼠的右侧股骨，冲洗并收集骨髓细胞，采用流式细胞术对骨髓中细胞数量及CD34+细胞比例进行测定。实验结果见表5。如表5结果显示，与正常对照组比较，再障模型小鼠骨髓细胞计数及CD34+细胞比例均显著降低，紫苏叶提取物在0.1，0.2g/kg剂量下灌胃给药，可升高再生障碍性贫血小鼠的骨髓细胞计数；与模型组比较，紫苏叶提取物可显著升高CD34+细胞比例(p<0.05或0.01)。结果提示，紫苏叶提取物对再生障碍性贫血小鼠骨髓造血功能衰竭具有改善作用。
表5紫苏叶提取物对再生障碍性贫血小鼠骨髓细胞数和CD34+细胞比例的影响(Mean±Std,n＝10)
备注：#p＜0.05，##p＜0.01vs空白对照组；*p＜0.05，**p＜0.05vs模型对照组
综上研究结果显示，紫苏叶提取物可以改善再障小鼠的饮食情况，减少再障小鼠的体重降低，增强再障小鼠的消化、吸收功能，增加胸腺指数；对小鼠外周血血常规和网织红细胞的检测表明，紫苏叶提取物可显著改善再生障碍性贫血小鼠外周血红细胞计数、血红蛋白含量、白细胞计数和血小板计数，从而减少再障的贫血、出血、感染等各种临床症状的发生，减轻的外周血损伤；同时，紫苏叶提取物可显著增加外周血网织红细胞计数、骨髓细胞计数以及骨髓CD34+细胞比例，从而改善再生障碍性贫血的造血功能，对其发挥明显的治疗作用。而且，中药来源的紫苏叶提取物尚未发现有任何毒副作用，可见其在再生障碍性贫血的治疗中具有十分广阔的应用前景。
Claims (1)
1.一种紫苏叶提取物在制备治疗和/或预防再生障碍性贫血药品中的应用，其特征在于，所述紫苏叶提取物制备方法为如下步骤：
1)以紫苏叶为原料，70％乙醇溶液为溶剂进行提取，回收乙醇溶剂，回收后的溶液浓缩，制得浸膏；
2)将步骤1)得到的浸膏加水分散，加入正丁醇萃取，剩余水溶液浓缩得到浸膏；
3)将步骤2)得到的浸膏以95％乙醇醇沉，过滤，沉淀物干燥、粉碎得到紫苏叶提取物；
所述步骤1)中的提取的方法为浸渍、渗漉、煎煮或加热回流；
所述步骤2)中，萃取次数为1-5次。
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