CN112076247B - 紫苏叶提取物在制备治疗慢性阻塞性肺疾病药物中的应用 - Google Patents
紫苏叶提取物在制备治疗慢性阻塞性肺疾病药物中的应用 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/或预防慢性阻塞性肺病(COPD)药物中的应用。本发明所述的紫苏叶提取物对香烟诱导COPD模型小鼠肺部炎症病理损伤有逆转作用;可显著抑制模型小鼠肺泡灌洗液中炎性细胞因子TNF‑α、IL‑1β的生成;显著抑制模型小鼠肺泡灌洗液中白细胞总数、淋巴细胞和嗜酸性粒细胞的募集。
Description
技术领域
本发明涉及一类紫苏叶提取物,其制备方法及其在制备用于预防和/或治疗慢性阻塞性肺疾病药物或食物中的应用,属于医药或食品技术领域。
背景技术
慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)在世界范围内的患病率和死亡率呈逐年上升趋势,已成为严重威胁公众健康的慢性疾病。COPD是一种以气道受限为特征的疾病,气流受限不完全可逆,呈进行性发展,主要累及肺部,其慢性炎症反应遍及气道、肺实质和肺血管,其发病机制与气道和肺对香烟烟雾等有害气体或有害颗粒的慢性炎症反应增强有关。气道炎症是COPD的重要病理基础,然而,目前尚无药物可以彻底有效的控制COPD气道炎症,临床多依靠皮质激素类抗炎药的吸入疗法,但是,研究发现皮质激素吸入用于COPD气道炎症治疗存在抗药性,而且,长期使用皮质激素显著增加感染性肺炎等不良反应风险。此外,非甾体类抗炎药临床应用于COPD治疗疗效有限,也存在引发心脏病或全身凝血障碍性疾病等严重不良反应问题。
我国传统中医药以其毒副作用小、良好的治疗效果已突显其优势,也逐渐的被世界医学界所认可。紫苏(Perilla frutescens L.)是唇形科紫苏属一年生草本植物,其叶为临床常用中药,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,鱼蟹中毒等治疗。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COPD气道炎症的相关研究报道,为有效阐释传统中药治疗COPD的作用,并为寻找活性更好的抗COPD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COPD的活性进行了评价。
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一种紫苏叶提取物在制备抗COPD药物中的应用。
为解决本发明的技术问题,本发明提供如下技术方案:
本发明技术方案提供了一种紫苏叶提取物在制备预防和/或治疗慢性阻塞性肺疾病药物中的应用,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,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)将步骤1)得到的浸膏加水分散,加入低极性有机溶剂萃取,剩余水溶液浓缩得到浸膏;
3)将步骤2)得到的浸膏以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。
长期吸烟是最常见的人类COPD致病因素,以香烟诱导模拟小鼠的COPD模型。紫苏叶提取物在剂量50、100mg/kg下灌胃给药,对香烟诱导的COPD小鼠肺泡灌洗液(BALF)中炎性细胞因子TNF-α、IL-1β的生成有显著抑制作用;对BALF中白细胞总数、中性粒细胞和巨噬细胞的募集有抑制作用;对小鼠肺部炎症病理损伤有逆转作用。
本发明技术方案的第二方面是提供了一种药物组合物在制备预防和/或治疗慢性阻塞性肺疾病药物中的应用,所述的药物组合物含有第一方面所述紫苏叶提取物以及药学上可接受的载体或赋形剂。其中紫苏叶提取物的制备方法如本发明第一方面所述。
用于此目的时,如果需要,可将活性成分与一种或多种固体或液体药物赋形剂和/或辅剂结合,制成可作为人药使用的适当施用形式或剂量形式。
本发明的药物组合物可以单位剂量形式给药,给药途径可为肠道或非肠道,如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。
本发明的药物组合物的给药途径可为注射给药。注射包括静脉注射、肌肉注射、皮下注射、皮内注射和穴位注射等。给药剂型可以是液体剂型、固体剂型。如液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂等。
本发明的组合物可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将单位给药剂型制成片剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如稀释剂与吸收剂,如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化纳、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等;湿润剂与粘合剂,如水、甘泊、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素纳、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等;崩解剂,例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢纳与枸橼酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等;崩解抑制剂,例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等;吸收促进剂,例如季铵盐、十二烷基硫酸纳等;润滑剂,例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片,例如糖包衣片、薄膜包衣片、肠溶包衣片,或双层片和多层片。
为了将给药单元制成丸剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如稀释剂与吸收剂,如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等;粘合剂,如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等;崩解剂,如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
为了将给药单元制成栓剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酶、明胶、半合成甘油酶等。
为了将给药单元制成胶囊,将有效成分与上述的各种载体混合,并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分制成微囊剂,混悬于水性介质中形成混悬剂,亦可装入硬胶囊中或制成注射剂应用。
例如,将本发明的组合物制成注射用制剂,如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂,这种制剂可以是含水或非水的,可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、1,3一丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酶等。另外,为了制备等渗注射液,可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油,此外,还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
此外如需要,也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
本发明药用组合物的给药剂量取决于许多因素,例如所要预防或治疗疾病的性质和严重程度,患者或动物的性别、年龄、体重、性格及个体反应,给药途径、给药次数等,因此本发明的治疗剂量可以有大范围的变化。一般来讲,本发明中药学成分的使用剂量是本领域技术人员公知的。可以根据本发明药用组合物中最后的制剂中所含有的实际有效药物数量,加以适当的调整,以达到其治疗有效量的要求,完成本发明的治疗COPD的目的。通常对体重约75公斤患者,所给提取物的日剂量为0.001mg/kg体重~500mg/kg体重,优选3mg/kg体重~30mg/kg体重。上述剂量可以单一剂量形式或分成几个,例如二、三或四个剂量形式给药,这受限于给药医生的临床经验以及给药方案。
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)本发明能有效防治慢性阻塞性肺疾病;(3)本品制备工艺简单,易于规范化生产。
附图说明
图1紫苏叶提取物对COPD小鼠肺组织病理学损伤的影响;
具体实施方式
以下实施例对本发明作进一步的说明,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乙酸乙酯萃取2次。剩余水部分加入1580mL95%乙醇溶解,室温沉淀24h,减压抽滤,滤渣以50ml水溶解,冷冻干燥、粉碎得紫苏叶提取物。
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,乙酸乙酯萃取2次;水部分加入95%乙醇适量溶解,室温沉淀24h,减压抽滤,滤渣加水适量溶解,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:紫苏叶提取物对香烟诱导小鼠COPD气道炎症的抑制作用
方法:SPF级雄性BALB/c小鼠,18-20g,随机分为正常对照组、模型对照组、阳性对照组(罗氟司特,2mg/kg,灌胃给药)、药物处理组(紫苏叶提取物50,100mg/kg,灌胃给药),每组20只。除空白对照组外,各组动物于实验第1天和第12天,麻醉后气管滴入50μl/只含有40μg LPS的生理盐水溶液;第2-11天和第13-22天,将小鼠置于小动物烟气吸入微环境制备系统(香烟烟雾:260-300ppm,温度22-28℃,氧气≥18.0%)中进行熏烟,每次4根烟,60min/次,每天1次,连续3周,每日烟熏前1小时灌胃给药。小鼠于末次熏烟1小时后,取部分小鼠肺组织,进行组织病理学检测和评价。其余各组小鼠以苯巴比妥钠麻醉,用0.7mL预冷的生理盐水灌流,冲洗3次,吸出支气管肺泡灌洗液(BALF),离心,上清用于分析TNF-α及IL-1β的含量;PBS缓冲液重悬细胞,进行白细胞分类计数。
结果:
(1)紫苏叶提取物改善香烟诱导COPD小鼠肺组织炎性病理的损伤
对小鼠肺部病理观察可见(图1和表1),模型组肺泡壁变宽,肺泡壁上皮细胞肿胀、变圆、部分脱落,肺泡腔可见巨噬细胞、小支气管和细支气管周围灶性炎性细胞浸润,肺组织边缘肺泡腔扩张。紫苏叶提取物组肺泡壁上皮细胞肿胀,肺泡腔可见少量巨噬细胞和粒细胞浸润,未见淋巴细胞浸润,小鼠肺组织未见出血。
结论:紫苏叶提取物在剂量50,100mg/kg下灌胃给药,对香烟诱导的小鼠慢性阻塞性肺淤血、炎性细胞浸润等有显著的治疗作用。
表1紫苏叶提取物对COPD小鼠肺组织病理炎症评价的影响(Mean±Std,n=4)
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COPD小鼠肺泡灌洗液(BALF)中炎性细胞募集的抑制作用
①紫苏叶提取物对COPD小鼠BALF中白细胞总数的影响
COPD以遍及气道、肺实质和肺血管的慢性炎症为特征,这种炎症表现为中性粒细胞、T淋巴细胞和巨噬细胞在肺内增多。激活的炎症细胞释放一系列炎性介质破坏肺结构或维持中性粒细胞炎症的介质。本实验目的在于考察紫苏叶提取物对COPD小鼠BALF中总炎性细胞募集的影响,结果见表2。
结果:紫苏叶提取物在剂量50,100mg/kg下对COPD模型小鼠灌胃给药,可降低COPD小鼠BALF中总炎性细胞募集。
表2紫苏叶提取物对COPD小鼠BALF中白细胞总数的影响(Mean±Std,n=10)
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COPD小鼠BALF中中性粒细胞分类计数的影响
中性粒细胞在COPD发病中具有重要作用,它可以释放丝氨酸蛋白酶并诱导动物产生人类肺气肿样的病理变化。中性粒细胞生命短暂,它循环招募到气道以及穿越间隙腔的过程十分迅速。病理研究证明有些COPD患者支气管组织内中性粒细胞数目增加与气流阻塞得程度有关。吸烟的COPD患者气道内中性粒细胞数目增加,特别是那些伴有慢性支气管炎的患者。本实验目的在于考察紫苏叶提取物对COPD小鼠BALF中中性粒细胞募集的影响,结果见表3。
结果:紫苏叶提取物在剂量50,100mg/kg下对COPD模型小鼠灌胃给药,可降低小鼠BALF中中性粒细胞募集。
表3紫苏叶提取物对COPD小鼠BALF中中性粒细胞分类计数的影响(Mean±Std,n=10)
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COPD小鼠BALF中巨噬细胞分类计数的影响
吸烟者和COPD患者肺内巨噬细胞较正常人群水平增加,多聚集在肺泡、细支气管和小气道。肺泡壁巨噬细胞数目和轻中度肺气肿以及COPD患者的小气道疾病程度呈正相关。组织病变和损伤部位可见到COPD缓慢进展和慢性病变与巨噬细胞长期增加平行。巨噬细胞可能通过释放基质金属蛋白酶导致弹性组织降解能力异常增高,诱发肺气肿的发生。本实验目的在于考察紫苏叶提取物对COPD小鼠BALF中巨噬细胞募集的影响,结果见表4。
结果:紫苏叶提取物在剂量50,100mg/kg下对COPD模型小鼠灌胃给药,可降低小鼠BALF中巨噬细胞募集。
表4紫苏叶提取物对COPD小鼠BALF中巨噬细胞分类计数的影响(Mean±Std,n=10)
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COPD小鼠肺泡灌洗液(BALF)中炎症介质生成的抑制作用
①紫苏叶提取物对COPD小鼠BALF中TNF-α生成的影响
前炎性细胞因子TNF-α是COPD的发病过程中的启动因子。COPD患者的TNF-α水平高于正常人,培养的支气管上皮细胞与香烟的烟雾接触可分泌TNF-α。TNF-α可促使中性粒细胞脱颗粒,诱导起到粘膜细胞增生和高分泌,增加上皮细胞IL-8生成,增加巨噬细胞基质金属蛋白酶生成,促进气道高反应性。本实验目的在于考察紫苏叶提取物对COPD小鼠BALF中TNF-α生成的影响,结果见表5。
结果:紫苏叶提取物在剂量50,100mg/kg下对COPD模型小鼠灌胃给药,可降低小鼠BALF中TNF-α的生成。
表5紫苏叶提取物对COPD小鼠BALF中TNF-α生成的影响(Mean±Std,n=10)
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COPD小鼠BALF中IL-1β生成的影响
IL-1β能激活巨噬细胞等炎症细胞释放炎症介质,在肺内可引起以巨噬细胞、粒细胞浸润为特征的肺部炎症。IL-1β亦可诱导巨噬细胞产生基质金属蛋白酶-9,降解肺实质中细胞外基质成分,引起肺泡隔弹性纤维的破裂导致气道重塑及气道纤维化。本实验目的在于考察紫苏叶提取物对COPD小鼠BALF中IL-1β生成的影响,结果见表6。
结果:紫苏叶提取物在剂量50,100mg/kg下对COPD模型小鼠灌胃给药,可降低小鼠BALF中IL-1β生成。
表6紫苏叶提取物对COPD小鼠BALF中IL-1β生成的影响(Mean±Std,n=10)
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。
Claims (1)
1.一种紫苏叶提取物在制备预防和/或治疗慢性阻塞性肺疾病药物中的应用,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%乙醇水溶液为溶剂进行加热回流提取,回收溶剂,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,加入乙酸乙酯萃取,萃取次数为2次,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;
所述紫苏叶提取物应用于由香烟诱导的COPD气道炎症。
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