CN108697753A - 含有芦根提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物 - Google Patents
含有芦根提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物 Download PDFInfo
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- CN108697753A CN108697753A CN201780011468.2A CN201780011468A CN108697753A CN 108697753 A CN108697753 A CN 108697753A CN 201780011468 A CN201780011468 A CN 201780011468A CN 108697753 A CN108697753 A CN 108697753A
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Abstract
本发明涉及含有芦根(Phragmitis Rhizoma)提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物,用芦根提取物处理小鼠的骨髓细胞的结果,显著地恢复由抗癌剂诱发的造血干细胞集落群的减少,并且在腹腔注射抗癌剂而诱导骨髓抑制的动物模型中,确认了具有显著地恢复由抗癌剂引起的骨髓抑制的效果。因此,本发明的芦根提取物可以有效地用作预防、改善及治疗由抗癌剂的副作用引起的疾病的组合物,并且以抗癌助剂与抗癌剂联合处方,从而具有提高抗癌剂的效果的可能性。
Description
技术领域
本发明涉及含有芦根(Phragmitis Rhizoma)提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物。
背景技术
癌症占据韩国死亡原因的1~2位,韩国50、60年龄段的死者中的30%死于癌症。这种癌症的治疗中最常用的是外科手术、放射疗法、化学疗法。其中,对作为化学疗法的抗癌剂的开发进行了各种尝试,但到目前为止还处于尚未开发出真正的作为治疗剂的抗癌剂的状态,并且辅助治疗剂仅仅是帮助延长短时间的生命的程度而已。
用作化学疗法的抗癌剂介入癌细胞的代谢途径,与DNA直接作用而阻断DNA的复制、转录、转译的过程,或者阻碍核酸前体的合成,并且阻碍细胞的分裂,因此显示出对细胞的细胞毒性。但是,现有的抗癌剂对癌症没有特异性的选择性,从而显示出同时产生治疗效果和毒性效果的特性,并且施用抗癌剂所引起的毒性会导致如下的多种副作用,即,骨髓的损伤所引起的白细胞、血小板、红细胞等血细胞减少症,毛囊细胞的损伤所引起的脱毛症状,对卵巢和睾丸的副作用所引起的月经不调及男性不育的原因,消化道黏膜细胞的损伤所引起的副作用为口腔炎、恶心呕吐及食物吞咽障碍和消化障碍、腹泻症状,肾小管坏死所引起的肾脏毒性,因神经系统障碍而产生的末梢神经炎和虚弱感,血管痛症及皮疹等血管障碍,皮肤及手指甲、脚指甲变色等。由此,迫切需要开发能够最小化由抗癌剂引起的副作用的同时提高抗癌治疗效果的药剂。
另外,芦根(Phragmitis Rhizoma)也称为苇根,其是经过干燥的禾本科植物芦苇(Phragmites communis Trin.)的根。芦苇生长在各地的沼泽、江边、湿地、海岸边,在春天或秋天采挖根茎,整理须根并丢弃,用水清洗后在阳光下晒干。晒干的芦根是扁平的圆柱形状,表面具有光泽且为黄白色,节部较硬且为鲜明的黄红色,节间有纵向皱纹,在晚春、初夏或秋天采集,去除泥土和须根并剥去皮膜,在新鲜时使用或在阳光下晒干并使用。
芦根味甜且性寒,已知其作用于肺经及胃经。就芦根的药效而言,其具有降热并生成津液而止呕的功效,并且一直以来用于治疗热病引起的呕吐、食道癌、肺脓肿,并用于解河豚毒。此外,已有报道称其具有阻断紫外线的作用,并促进黑色素细胞的增殖,使头发乌黑并具有润泽,在用作美白霜时,对皮肤提供美白作用及营养。对芦根的生理活性的研究也有报道称芦根提取物对血清中的葡萄糖、胰岛素及脂质的合成产生影响,并且还有报道称芦根提取物具有抑制由于施用链脲霉素(streptozotocin)而引起的胰岛A细胞内胰高血糖素颗粒的增加的作用和抑制B细胞内胰岛素脱粒的效果。此外,已知芦根甲醇提取物具有降低血液中胆固醇和葡萄糖的效果。已知芦根的根茎甲醇提取物显著地减少高甘油三酯血症和高胆固醇血症及糖尿病性高血脂症的白鼠的中性脂肪的浓度,在甲醇提取物中分离的β-谷甾醇(β-sitosterol)和对香豆酸(p-coumaric acid)具有改善血液中脂肪浓度的效果。
就芦根提取物相关技术而言,已知有韩国授权专利第1098875号中公开的具有增强免疫功能及抗癌功能的芦根提取物的制备方法,韩国授权专利第1151718号中公开的包含芦根提取物作为有效成分的用于皮肤保湿、用于治疗皮肤炎或特应性皮炎的组合物,但没有公开本发明的含有芦根提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物。
发明内容
要解决的技术问题
本发明是根据如上所述的要求而提出的,本发明涉及含有芦根提取物作为有效成分的用于预防、改善或治疗由抗癌剂的副作用引起的疾病的组合物,更详细地,用本发明的芦根提取物处理由于施用抗癌剂而导致造血干细胞减少的小鼠的骨髓细胞,从而显著地恢复造血干细胞的存活,并且在腹腔注射抗癌剂而诱导骨髓抑制的动物模型中也确认了显著地恢复由抗癌剂引起的骨髓抑制,从而完成了本发明。
技术方案
为了实现上述目的,本发明提供用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其含有芦根提取物作为有效成分。
此外,本发明提供用于预防或改善由抗癌剂的副作用引起的疾病的健康功能食品,其含有芦根提取物作为有效成分。
此外,本发明提供抗癌助剂,其含有芦根提取物作为有效成分。
发明效果
本发明涉及含有芦根提取物作为有效成分的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其不仅能够显著地恢复由抗癌剂诱发的造血干细胞集落的减少,而且还能够显著地恢复由抗癌剂引起的骨髓抑制,因此本发明的芦根提取物能够用作预防及治疗由抗癌剂的副作用引起的疾病的药学组合物、健康功能食品或抗癌助剂。
附图说明
图1为通过CFU(集落形成单位,colony forming unit)分析确认的用不同浓度的抗癌剂(多西他赛、阿霉素、伊立替康、紫杉醇或柔红霉素)进行处理时对从小鼠的股骨(femur)中分离的骨髓细胞的生长及分化所产生的影响的结果。*、**、***表示与没有用抗癌剂进行处理的对照组的CFU(集落形成单位,colony forming unit)相比,用抗癌剂进行处理的组在统计上具有显著的差异,*表示p值小于0.05,**表示p值小于0.01,***表示p值小于0.001。
图2示出对C5BL/6小鼠进行50mg/kg、100mg/kg、150mg/kg的多西他赛腹腔注射的3天后(A)和7天后(B)在股骨(femur)中确认骨髓细胞数量的结果。**、***表示与没有用多西他赛进行处理的对照组(Veh)相比,用多西他赛进行处理的组的细胞存活率在统计上具有显著的差异,**表示p值小于0.01,***表示p值小于0.001。
图3为确认用抗癌剂(多西他赛、阿霉素、伊立替康、紫杉醇或柔红霉素)进行处理时的小鼠的骨髓细胞的减少,以及用上述抗癌剂和25μg/ml、50μg/ml及100μg/ml的本发明的芦根提取物联合处理时抑制骨髓细胞减少的效果的结果。##、###、####表示与没有用抗癌剂进行处理的对照组(Control)相比,用抗癌剂进行处理的组的CFU在统计上具有显著的差异,##表示p值小于0.01,###表示p值小于0.001,####表示p值小于0.0001,*、**、***表示抗癌剂处理组和抗癌剂及芦根提取物的联合处理组的CFU在统计上具有显著的差异,*表示p值小于0.05,**表示p值小于0.01,***表示p值小于0.001。
图4为确认用多西他赛进行处理时的小鼠体重的减少,以及用上述多西他赛和本发明的芦根提取物联合处理时抑制体重减少的效果的结果。对照组(Control)为没有进行任何处理的小鼠,多西他赛+芦根-125及多西他赛+芦根-250表示联合施用多西他赛和125μg/ml及250μg/ml的芦根提取物的小鼠。
图5为确认用多西他赛进行处理时的小鼠的骨髓细胞的减少,以及用15nM的上述多西他赛和125μg/ml及250μg/ml的本发明的芦根提取物联合处理时抑制骨髓细胞减少的效果的结果。对照组为没有进行任何处理的组,多西他赛+芦根-125及多西他赛+芦根-250表示联合施用多西他赛和125μg/ml及250μg/ml的芦根提取物。#表示与没有用多西他赛进行处理的对照组(Control)相比,用多西他赛进行处理的组的骨髓细胞的数量在统计上具有显著的差异,p值小于0.05,*、**表示多西他赛处理组和多西他赛及芦根提取物的联合处理组的骨髓细胞的数量在统计上具有显著的差异,*表示p值小于0.05,**表示p值小于0.01。
图6为通过H&E染色确认用多西他赛进行处理的小鼠的骨髓结构发生异常(箭头部分),但是用多西他赛和125mg/kg(芦根-125)及250mg/kg(芦根-250)的本发明的芦根提取物进行联合处理后骨髓细胞几乎不会发生异常的结果。
图7为胸腺(thymus)的组织病理学分析结果,其是确认施用多西他赛时的组织收缩及功能性淋巴器官的消失(箭头标记),并且确认用多西他赛和125mg/kg(芦根-125)及250mg/kg(芦根-250)的本发明的芦根提取物联合处理时的使组织收缩及功能性淋巴器官的消失得到恢复的结果。
图8为在由抗癌剂诱导骨髓抑制的动物模型中确认芦根提取物的缓解骨髓抑制的能力的结果。多西他赛+芦根-125及多西他赛+芦根-250表示联合施用多西他赛和125mg/kg及250mg/kg的芦根。*表示多西他赛处理组和多西他赛及125mg/kg的芦根提取物的联合处理组的促进免疫的细胞因子(immune-stimulating cytokine)IL-3在统计上具有显著的差异,p值小于0.05。
图9为在小鼠的脾脏细胞中确认用芦根提取物进行处理时的促进免疫的细胞因子(immune-stimulating cytokine)的表达量的变化的结果。
最佳实施方式
本发明涉及用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其含有芦根(Phragmitis Rhizoma)提取物作为有效成分。
所述芦根提取物可以是通过包括下述步骤的方法制备的,但并不限定于此。
1)向芦根添加提取溶剂进行提取;
2)对步骤1)的提取物进行过滤;以及
3)对步骤2)的经过过滤的提取物进行减压浓缩并进行干燥,从而制备提取物。
上述步骤1)中的提取溶剂优选为水、C1~C4的低级醇或它们的混合物,但并不限定于此。
在上述制备方法中,芦根提取物的提取可以采用过滤法、热水提取、浸提、回流冷却提取及超声波提取等本领域公知的所有常规方法。优选地,添加干燥的芦根体积的1~20倍的上述提取溶剂进行提取,更优选添加3~10倍的上述提取溶剂。提取温度优选为20~50℃,但并不限定于此。此外,提取时间优选为10~100小时,更优选为24~96小时,最优选为72小时,但并不限定于此。在上述方法中,步骤3)的减压浓缩优选使用真空减压浓缩仪或真空旋转蒸发仪,但并不限定于此。此外,干燥优选进行减压干燥、真空干燥、沸腾干燥、喷雾干燥或冷冻干燥,但并不限定于此。
所述癌症优选为选自肺癌、乳腺癌、肝癌、胃癌、大肠癌、结肠癌、皮肤癌、膀胱癌、前列腺癌、卵巢癌、宫颈癌、甲状腺癌、肾癌、纤维肉瘤、黑色素瘤及血癌中的任一种癌症,但并不限定于此,只要是可以诊断的癌症,则不受限制。
所述抗癌剂包括抗肿瘤抗生素(antitumor antibiotics)、拓扑异构酶抑制剂(topoisomerase inhibitor)或紫杉烷(taxane)类抗癌剂,但并不限定于此,其包括临床、药学、生物医学上可以使用的所有抗癌剂。
所述抗肿瘤抗生素优选为选自放线菌素D(actinomycin D)、硫酸博来霉素(bleomycin sulfate)、道诺霉素(daunomycin)、柔红霉素(daunorubicin)、阿霉素(doxorubicin)、表阿霉素(epirubicin)、伊达比星(idarubicin)、丝裂霉素(mitomycin)、丝裂霉素C(mitomycin-C)及光辉霉素(Mithramycin)中的任一种以上,但并不限定于此。
所述拓扑异构酶抑制剂优选为选自伊立替康(irinotecan)、喜树碱(camptothecin)、新生霉素(novobiocin)、表阿霉素(epirubicin)、更生霉素(dactinomycin)、安吖啶(amsacrine)、替尼泊苷(teniposide)及依托泊苷(etoposide)中的任一种以上,但并不限定于此。
所述紫杉烷类抗癌剂优选为选自紫杉醇(paclitaxel)及多西他赛(docetaxel)中的任一种或全部两种。
上述由副作用引起的疾病优选为选自造血毒性、贫血及中性粒细胞减少症中的任一种以上,但并不限定于此。
本发明的组合物中,相对于组合物的总重量,可以含有0.1~99.9重量%的本发明的芦根提取物或分馏物作为有效成分,并且可以包含药剂学上可接受的载体、赋形剂或稀释剂。
本发明的组合物可以是口服或非口服的多种剂型。在进行制剂化时,使用通常使用的填充剂、增量剂、粘合剂、润湿剂、崩解剂、表面活性剂等稀释剂或赋形剂来制备。用于口服施用的固体制剂包括片剂、丸剂、散剂、颗粒剂、胶囊剂等,这种固体制剂是通过在一种以上的化合物中混合至少一种以上的如淀粉、碳酸钙、蔗糖(sucrose)或乳糖(lactose)、明胶等赋形剂来制备。此外,除了单纯的赋形剂之外,还使用如硬脂酸镁、滑石等润滑剂。用于口服施用的液体制剂有悬浮剂、内用液剂、乳剂、糖浆剂等,除了常用的单纯稀释剂水、液体石蜡以外,可以包含多种赋形剂,例如润湿剂、甜味剂、芳香剂、保存剂等。用于非口服施用的制剂包括灭菌的水溶液、非水性溶剂、悬浮剂、乳剂、冷冻干燥制剂、栓剂。非水性溶剂及悬浮溶剂可以使用丙二醇(propylene glycol)、聚乙二醇、诸如橄榄油的植物油、诸如油酸乙酯的可注射的酯等。栓剂的基质可以使用witepsol、聚乙二醇(Macrogol)、吐温(tween)61、可可脂、月桂脂、甘油明胶等。
本发明的组合物可以通过口服或非口服进行施用,非口服施用时优选选择皮肤外用或腹腔内、直肠、静脉、肌肉、皮下、子宫内硬膜或脑血管内注射方式,最优选以皮肤外用进行使用。
本发明的组合物是以药剂学有效量进行施用。本发明中,“药剂学有效量”是指可应用于医学治疗并以合理的受益/风险比例治疗疾病所充足的量,有效剂量的水平可以根据包括患者的疾病的种类、严重程度、药物的活性、对药物的敏感度、施用时间、施用途径及排泄比率、治疗疗程、同时使用的药物在内的因素及其他医学领域中众所周知的因素来决定。本发明的组合物可以作为单独治疗剂进行施用或可以与其他治疗剂联合施用,可以依次或同时与现有的治疗剂进行施用,并且可以进行单一或多种施用。重要的是考虑所有上述因素,施用能够以最少的量获得最大效果的量而没有副作用,这可以由本领域技术人员容易地决定。
本发明的组合物的施用量的范围根据患者的体重、年龄、性别、健康状态、饮食、施用时间、施用方法、排泄率及疾病的严重程度而不同,以芦根提取物的量为基准,每天的施用量为0.01~2,000mg/kg,优选为30~500mg/kg,更优选为50~300mg/kg,每天可以施用1~6次。本发明的组合物可以单独使用,或者可以与手术、放射治疗、激素治疗、化学治疗及使用生物学反应调节剂的方法联合使用。
此外,本发明涉及用于预防或改善由抗癌剂的副作用引起的疾病的健康功能食品,其含有芦根提取物作为有效成分。所述芦根提取物的提取溶剂优选为水、C1~C4的低级醇或它们的混合物,但并不限定于此。所述抗癌剂包括抗肿瘤抗生素、拓扑异构酶抑制剂或紫杉烷类抗癌剂,但并不限定于此,其包括临床、药学、生物医学上可以使用的所有抗癌剂。上述由抗癌剂的副作用引起的疾病的特征在于选自造血毒性、贫血及中性粒细胞减少症中的任一种以上。
本发明的健康食品可以直接添加芦根提取物或者与其他食品或食品成分一同使用,并且可以根据常规的方法适当地使用。所述健康食品的种类没有特别的限制。可以添加所述芦根提取物的食品的例子有肉类、香肠、面包、巧克力、糖类、点心类、饼干类、披萨、拉面、其他面类、口香糖类、包括冰淇淋类在内的乳制品、各种汤、饮料、茶、保健饮料、酒精饮料及维生素复合剂等,并且包括所有常规意义上的健康食品。包含本发明的组合物的健康饮料如同通常的饮料可以含有多种香味剂或天然碳水化合物等作为附加成分。上述的天然碳水化合物是诸如葡萄糖、果糖的单糖,诸如麦芽糖、蔗糖的二糖,以及诸如糊精、环糊精的多糖,木糖醇、山梨糖醇、赤藓糖醇等糖醇。甜味剂可以使用诸如奇异果甜蛋白、甜菊提取物的天然甜味剂,或者诸如糖精、阿斯巴甜的合成甜味剂等。每100g的本发明的组合物中,所述天然碳水化合物的比率一般为约0.01~0.04g,优选为约0.02~0.03g。
除了上述有效成分之外,本发明的健康食品还可以进一步含有各种营养剂、维生素、电解质、风味剂、着色剂、果胶酸及其盐、海藻酸及其盐、有机酸、保护性胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、酒精、碳酸饮料中使用的碳酸化剂等。除此之外,还可以含有用于制备果汁或蔬菜饮料的果肉。这些成分可以单独使用或混合使用。这种添加剂的比率并不非常重要,但是相对于100重量份的本发明的组合物,通常在0.01~2重量份的范围中选择。
此外,本发明涉及含有芦根提取物作为有效成分的抗癌助剂。所述抗癌助剂的特征在于抑制由于施用抗癌剂而诱发的选自造血毒性、贫血及中性粒细胞减少症中的一种以上的抗癌剂的副作用。
所述抗癌助剂可以在芦根提取物中进一步含有一种以上的显示出相同或相似功能的有效成分。在临床上施用时,所述抗癌助剂可以通过口服或非口服进行施用,非口服施用时可以通过腹腔内注射、直肠内注射、皮下注射、静脉注射、肌肉内注射、子宫内硬膜注射、脑血管内注射或胸部内注射进行施用,并且可以以常规的医药品制剂的形态使用。
所述抗癌助剂可以单独使用,或者可以与手术、放射治疗、激素治疗、化学治疗及使用生物学反应调节剂的方法联合使用。所述抗癌助剂的每天的施用量为约0.0001~100mg/kg,优选为0.001~10mg/kg,优选每天施用1次或分数次施用,但其范围根据患者的体重、年龄、性别、健康状态、饮食、施用时间、施用方法、排泄率及疾病的严重程度等而不同。实际在临床上施用时,本发明的抗癌助剂可以以非口服的多种剂型施用,在进行制剂化时使用通常使用的填充剂、增量剂、粘合剂、润湿剂、崩解剂、表面活性剂等稀释剂或赋形剂来制备。用于非口服施用的制剂包括灭菌的水溶液、非水性溶剂、悬浮剂、乳剂、冷冻干燥制剂、栓剂。非水性溶剂及悬浮溶剂可以使用丙二醇(propylene glycol)、聚乙二醇、诸如橄榄油的植物油、诸如油酸乙酯的可注射的酯等。栓剂的基质可以使用witepsol、聚乙二醇(Macrogol)、吐温(tween)61、可可脂、月桂脂、甘油明胶等。
下面,通过实施例对本发明进行更加详细的说明。这些实施例仅仅是用于更加具体地说明本发明,本发明的范围不受其限制,这对于本领域具有通常知识的技术人员而言是显而易见的。
实施例1.芦根提取物的制备
为了制备本发明的有效成分芦根提取物,从光明堂制药公司(蔚山,韩国)购买了芦根。将100g的所购买的上述芦根进行粉碎,然后与2L的水一同加入到圆形烧瓶中进行混合。加热与附着有冷却管的回流提取装置连接的恒温水槽(water bath),共反复提取2次,1次2小时。使用纸质滤纸(沃特曼2号(Whatman No.2))和真空泵(Vacuum pump,GAST公司),对经过提取的提取物进行减压过滤。使用旋转浓缩仪(Rotary evaporator,EYELA公司),对经过过滤的液相提取物进行减压浓缩,并对经过浓缩的提取物进行冷冻干燥,然后用研钵进行均质化,从而获得芦根提取物。将其装在密封的塑料容器中,并保管于4℃的低温储存室直至实验前。
实施例2.抗癌剂的造血毒性的确认
为了确认目前在临床上使用的抗癌剂的造血毒性效果,通过体外(ex vivo)、体内(in vivo)实验确认了不同浓度的抗癌剂对小鼠骨髓细胞中的造血干细胞的生长及分化所产生的影响。
1)在小鼠的骨髓细胞中抗癌剂的造血毒性效果的确认(体外(ex vivo))
从小鼠的股骨(femur)中分离骨髓细胞。之后,在MethoCult GF M3434培养基中培养上述骨髓细胞7~10天,从而诱导造血干细胞的生长,所述MethoCult GF M3434培养基包含以不同浓度添加多西他赛、阿霉素、伊立替康、紫杉醇及柔红霉素的源自鼠的重组干细胞因子(recombinant murine stem cell factor,rm stem cell factor)、rm IL-3、源自人的重组IL-6(recombinant human IL-6,rh IL-6)及rh红细胞生成素(rhErythropoietin)。获取生长的造血干细胞,然后实施集落形成单位分析(Colony FormingUnit assay,CFU assay),观察抗癌剂对造血干细胞的生长及分化所产生的影响。
其结果,如图1所示,在低浓度下也显示出造血毒性,尤其可以确认,多西他赛为50nM以上、阿霉素为50μM以上、伊立替康为5μM以上、紫杉醇为25nM以上、柔红霉素为100nM以上时显示出非常严重的造血毒性。
2)腹腔注射抗癌剂的小鼠中造血毒性效果的确认(体内(in vivo))
对C57BL/6小鼠进行50mg/kg、100mg/kg及150mg/kg的多西他赛腹腔注射。在3天后和7天后,从小鼠的股骨中分离骨髓细胞进行计数,并测量存活率。
其结果,如图2所示,注射50mg/kg、100mg/kg及150mg/kg的多西他赛的所有情况下,在3天后和7天后,所分离的骨髓细胞的数量显著减少,因此确认了在体内(in vivo)动物模型中显示出造血毒性。
实施例3.对于由抗癌剂诱导的骨髓毒性的芦根提取物的恢复骨髓毒性的效果的确认
为了确认芦根提取物是否具有缓解骨髓细胞中的由抗癌剂(15nM的多西他赛、100nM的阿霉素、100μM的伊立替康、50nM的紫杉醇及100nM的柔红霉素)诱导的造血毒性的效果,用15nM的多西他赛、100nM的阿霉素、100μM的伊立替康、50nM的紫杉醇或100nM的柔红霉素和芦根提取物联合处理,从而确认了缓解骨髓毒性的效果。
具体地,从小鼠的股骨中分离骨髓细胞,然后用15nM的诱发骨髓细胞的造血毒性的多西他赛等抗癌剂处理MethoCult GF M3434培养基,所述培养基包含源自鼠的重组干细胞因子、rm IL-3、源自人的重组IL-6及rh红细胞生成素。之后,分别添加上述实施例1中制备的25μg/ml、50μg/ml及100μg/ml的芦根提取物,并培养7天的同时诱导造血干细胞的生长。7天后,获取生长的造血干细胞,并实施CFU分析来确认造血干细胞的生长。作为阴性对照组,用包含0.5%的DMSO的PBS来代替芦根提取物作为溶剂(载体(vehicle))进行处理,并实施与上述相同的方法。
其结果,如图3所示,确认了通过芦根提取物由各个抗癌剂诱发的造血干细胞集落的减少在统计上得到显著的恢复(图3)。
实施例4.在由多西他赛诱导骨髓抑制的动物模型中芦根提取物的缓解骨髓抑制的能力的确认
为了确认芦根提取物是否在骨髓抑制诱导动物模型中显示出缓解造血毒性的效果,对小鼠进行多西他赛腹腔注射来诱导骨髓抑制,然后确认由芦根提取物引起的变化。
具体地,将C57BL/6小鼠分为4个组,组1为对照组(control),其是用包含5%的乙醇和2%的聚山梨醇酯80(polysorbate 80)的盐水(saline)进行处理的组,组2为腹腔内施用3次30mg/kg的多西他赛的多西他赛处理组,组3为多西他赛及125mg/kg的芦根提取物的施用组,其在施用多西他赛的前两天开始每天强制施用1次125mg/kg的芦根提取物,并且在施用多西他赛的3天期间也每天强制施用1次芦根提取物,组4为多西他赛及250mg/kg的芦根提取物的施用组,其通过与上述组3相同的方法,在施用多西他赛的前两天开始每天施用1次250mg/kg的芦根提取物,并且在施用多西他赛的3天期间也每天强制施用1次250mg/kg的芦根提取物。
之后,每天测量上述对照组、多西他赛施用组、多西他赛及125mg/kg的芦根提取物的联合施用组和多西他赛及250mg/kg的芦根提取物的联合施用组的小鼠的体重变化,然后在第6天解剖小鼠,测量骨髓内细胞数量的变化和对造血作用起重要作用的脾脏(spleen)及胸腺(thymus)的重量。
其结果,如图4及图5所示,确认了对小鼠施用多西他赛时,诱导造血毒性及骨髓抑制的同时诱导体重的显著减少,但联合施用芦根提取物时,体重的减少得到缓解(图4),并且与对照组相比,在多西他赛施用组中骨髓细胞(Bone marrow mononuclear cells)的数量显著减少,在施用芦根提取物的组中骨髓抑制得到恢复(图5)。
对上述各施用组的小鼠股骨进行组织学分析的结果,在多西他赛单独施用组中发现了造血细胞的减少所引起的细胞过少(hypocellularity)现象及骨髓内细胞结构的异常(用黄色箭头标记),但在芦根提取物的联合施用组中确认了这种现象得到减少(图6),在胸腺(Thymus)的组织病理学分析结果中也观察到联合施用芦根时,施用多西他赛而引起的组织收缩及功能性淋巴器官的消失得到恢复(图7)。
此外,就施用多西他赛的小鼠而言,由于导致参与造血作用的体内器官脾脏(spleen)和胸腺(thymus)的异常,出现体重的急剧减少所引起的器官指数(organ index)的减少,但是在联合施用多西他赛和芦根提取物的组中确认了器官指数的减少得到缓解。
器官指数(organ index,mg/g)=器官的重量(mg)/个体的重量(g)
在多西他赛施用组中观察到参与造血作用的生物器官脾脏及胸腺的收缩和重量的减少,在芦根提取物施用组中确认了这种减少得到缓解的效果(表1)。
[表1]
对多西他赛施用组、多西他赛及芦根提取物的联合施用组的脾脏和胸腺的重量变化进行确认的器官指数(organ index)
另外,从小鼠的脾脏(spleen)组织中获得mRNA,并确认参与造血作用的细胞因子的表达变化。在施用多西他赛时,深入参与造血作用(hematopoiesis),尤其深入参与髓系祖细胞(myeloid progenitor cell)的分化和增殖的IL-3的表达大幅减少,在联合施用多西他赛和芦根提取物时,确认了使减少的IL-3的表达得到恢复(图8)。
实施例5.芦根提取物对小鼠的脾脏细胞中的促进免疫的细胞因子(immune-stimulating cytokine)所产生的影响的确认(体内(in vivo))
为了确认芦根提取物在骨髓抑制诱导动物模型中对参与造血作用的细胞因子的表达所产生的影响,对小鼠进行多西他赛腹腔注射来诱导骨髓抑制,然后确认由芦根提取物引起的变化。
具体地,将C57BL/6小鼠分为5个组,组1的对照组(control)为用包含5%的乙醇和2%的聚山梨醇酯80(polysorbate 80)的盐水(saline)进行处理的组,组2为腹腔内施用3次30mg/kg的多西他赛的多西他赛处理组,组3为多西他赛及125mg/kg的芦根提取物的施用组,其在施用多西他赛的前两天开始每天强制施用1次125mg/kg的芦根提取物,并且在施用多西他赛的3天及之后的3天期间也每天强制施用1次芦根提取物,组4为通过与上述组3相同的方法施用多西他赛及250mg/kg的芦根提取物的施用组,组5为同样通过相同的方法施用多西他赛及500mg/kg的芦根提取物的施用组。
在上述动物中获取脾脏组织,并提取mRNA,然后确认参与造血作用的细胞因子的表达变化的结果,确认了促进造血干细胞的分化和增殖的细胞因子IL-3、IL-6、SCF(干细胞因子(stem cell factor))及GM-CSF(粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor))的表达得到增加(图9)。
实施例6.在由多西他赛诱导骨髓抑制的动物模型中芦根提取物所产生的影响的确认
为了确认芦根提取物对骨髓抑制诱导动物模型的血液指标所产生的影响,对小鼠进行多西他赛腹腔注射来诱导骨髓抑制,然后确认了由芦根提取物引起的变化。
具体地,将C57BL/6小鼠分为5个组,组1的对照组(control)为用包含5%的乙醇和2%的聚山梨醇酯80(polysorbate 80)的盐水(saline)进行处理的组,组2为腹腔内施用3次30mg/kg的多西他赛的多西他赛处理组,组3为多西他赛及30mg/kg的芦根提取物的施用组,其在施用多西他赛的前两天开始每天强制施用1次30mg/kg的芦根提取物,并且在施用多西他赛的3天及之后的3天期间也每天强制施用1次芦根提取物,组4为通过与上述组3相同的方法施用多西他赛及100mg/kg的芦根提取物的施用组,组5为同样通过相同的方法施用多西他赛及300mg/kg的芦根提取物的施用组。
从上述动物中采集血液并确认血细胞数量变化的结果,在多西他赛施用组中确认了白细胞、中性粒细胞、淋巴细胞及红细胞的减少,在芦根提取物的联合施用组中观察到其的恢复。
[表2]
所述表2的数据表示从10只小鼠中获得的值的平均值±标准误差(SEM),并且相对于多西他赛处理组,#表示p<0.05,##表示p<0.01。
Claims (10)
1.用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其含有芦根(Phragmitis Rhizoma)提取物作为有效成分。
2.根据权利要求1所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述提取物是将水、C1~C4的低级醇或它们的混合物作为溶剂来进行提取。
3.根据权利要求1所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述抗癌剂为抗肿瘤抗生素、拓扑异构酶抑制剂或紫杉烷类抗癌剂。
4.根据权利要求3所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述抗肿瘤抗生素为选自放线菌素D、硫酸博来霉素、道诺霉素、柔红霉素、阿霉素、表阿霉素、伊达比星、丝裂霉素、丝裂霉素C及光辉霉素中的任一种以上。
5.根据权利要求3所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述拓扑异构酶抑制剂为选自伊立替康、喜树碱、新生霉素、表阿霉素、更生霉素、安吖啶、替尼泊苷及依托泊苷中的任一种以上。
6.根据权利要求3所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述紫杉烷类抗癌剂为选自紫杉醇及多西他赛中的任一种以上。
7.根据权利要求1所述的用于预防或治疗由抗癌剂的副作用引起的疾病的药学组合物,其特征在于,所述由抗癌剂的副作用引起的疾病为选自造血毒性、贫血及中性粒细胞减少症中的一种以上。
8.用于预防或改善由抗癌剂的副作用引起的疾病的健康功能食品,其含有芦根提取物作为有效成分。
9.抗癌助剂,其含有芦根提取物作为有效成分。
10.根据权利要求9所述的抗癌助剂,其特征在于,所述抗癌助剂改善由于施用抗癌剂而诱发的选自造血毒性、贫血及中性粒细胞减少症中的一种以上的由抗癌剂的副作用引起的疾病。
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