CN100374111C - 利用黑米提取物预防或治疗过敏性疾病的组合物及其治疗用途 - Google Patents
利用黑米提取物预防或治疗过敏性疾病的组合物及其治疗用途 Download PDFInfo
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- CN100374111C CN100374111C CNB2004800118421A CN200480011842A CN100374111C CN 100374111 C CN100374111 C CN 100374111C CN B2004800118421 A CNB2004800118421 A CN B2004800118421A CN 200480011842 A CN200480011842 A CN 200480011842A CN 100374111 C CN100374111 C CN 100374111C
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- pelargonidin
- black rice
- anthocyanin
- rice extract
- mice
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Abstract
本发明涉及利用黑米提取物预防或治疗过敏性疾病的组合物及该组合物的治疗用途。更确切地说，本发明涉及预防或治疗过敏性疾病的组合物，该组合物含有下述物质作为有效成分：花葵素(pelargonidin)、花青素苷(cyanidinglycoside)或含有花葵素和花青素苷的黑米提取物；本发明还涉及所述组合物的治疗用途。花葵素、花青素苷或含有花葵素和花青素苷黑米提取物抑制组织内嗜酸粒细胞的聚集，并由此对过敏性炎症具有抑制作用，因而可将上述物质有效地用于预防或治疗与炎症和嗜酸粒细胞聚集有关的过敏性疾病，所述的过敏性疾病例如过敏性鼻炎、过敏性结膜炎、哮喘、慢性梗阻性肺部疾病、特应性皮炎、过敏性腹泻等。
Description
技术领域
本发明涉及利用黑米提取物预防或治疗过敏性疾病的组合物及该组合物的治疗用途。更确切地说，本发明涉及预防或治疗过敏性疾病的组合物，该组合物含有下述物质作为有效成分：花葵素(pelargonidin)、花青素苷(cyanidin glycoside)或含有花葵素和花青素苷的黑米提取物；本发明还涉及所述组合物的治疗用途。
背景技术
通常地，已知过敏性疾病是由气道或支气管等组织中的过敏性炎症引起的。特别地，过敏性疾病由以下途径引起：诸如尘土、花粉、真菌、各种食物和药物等过敏原(抗原)通过呼吸器官、消化器官或皮肤进入个体，然后与附着于组织内的肥大细胞表面的IgE(免疫球蛋白E)抗体结合，随后，肥大细胞分泌组胺。组胺是在鼻粘膜中引起过敏反应的最重要的化学介质，其增加血管透过性而引起鼻粘膜水肿，并刺激感觉神经末梢，诱发诸如流泪、流鼻涕、瘙痒等早期过敏反应。除了组胺，组织中的肥大细胞还分泌出诸如嗜酸粒细胞趋化因子和白三烯等具有趋化性的化学介质。嗜酸性细胞被上述的趋化性因子移动到过敏形成部位(趋化作用)，引起诸如组织损伤、炎症反应和超敏反应等后期过敏反应。
哮喘、过敏性鼻炎和特应性皮炎是过敏性疾病的例子，它们由于烟尘等引起的空气污染而变得越发严重。但是，还没有研制出令人满意的用于治疗过敏性疾病的任何有效治疗剂。一旦治疗停止，症状在几天或几周内复发，这就需要改善常规治疗剂的安全性和有效性。
迄今为止，治疗过敏性疾病的主要治疗剂是只能用于减轻症状的皮质类固醇，其不仅远未达到消除病因的根本性治疗，而且还带有严重的副作用(Rabe KF等，Eur Respir J Suppl.，34：34s～40s，2001)。大部分治疗过敏性疾病的常规治疗剂只具有抑制组胺的功能，因此它们不能抑制由组织中嗜酸粒细胞聚集引起的后期反应(这是引起炎症的主要原因)，导致慢性过敏症状。因此，迫切需要研制出克服治疗过敏性疾病的常规治疗剂的缺陷的新的抗过敏药物。
黑米(Oryza Sativa L.)，富含花色素苷(anthocyanin)类化合物的米，是含钙、维生素、尼克酸等远远超过白米的健康食品。已知黑米具有改善人体自身稳定调节功能和加强免疫功能的作用。另外，已知黑米还具有预防疾病、抗氧化、抗癌尤其是降低胆固醇的作用。
花色素苷是在植物的花或果皮的红色部分发现的色素苷。花色素苷是一类化合物，在该类化合物中，葡萄糖的特异性羟基基团通过醚键与醇、酚、醛等功能基团相连。至今已发现超过200种花色素苷，包括花翠素(delphinidin)、花青素(cyanidin)、花葵素、甲基花青素(peonidin)和二甲花翠素(malvidin)。花色素苷涉及抗炎、抗微生物和降低胆固醇等作用，特别是具有抗氧化活性，该类物质的抗氧化活性比生育酚这一天然抗氧化剂高5～7倍(Tedesco I等，J.Nutr.Biochem.，(9)：505～511，2001；Youdim KA等，Biochim.Biophys.Acta.，1523(1)：117～122，2000)。但是，花色素苷类中各化合物的具体作用还没有得到阐释。
发明内容
本发明人通过研究，开发了下述治疗剂，该治疗剂通过抑制由嗜酸粒细胞引起的炎症而被用于有效地治疗过敏性疾病，其中所述由嗜酸粒细胞引起的炎症是晚期过敏性疾病的反应之一。作为结果，本发明人证实，在所研究的很多中药和天然物质中，黑米提取物能够有效抑制哮喘(一种代表性过敏性疾病)。并且，本发明人还证实了包含在黑米提取物中的花色素苷尤其是花葵素和花青素苷能够抑制嗜酸粒细胞的聚集、并能有效抑制组织内的炎症，因此能够治疗包括哮喘在内的过敏性疾病，从而完成本发明。
本发明的一个目的是提供利用黑米提取物预防或治疗过敏性疾病的方法。
本发明的另外一个目的是提供黑米提取物的新的治疗用途。
本发明进一步的目的是提供利用花葵素或花青素苷预防或治疗过敏性疾病的方法。
本发明的另外一个目的是提供利用花葵素或花青素苷对细胞、组织或机体内的嗜酸粒细胞的聚集进行抑制的方法。
本发明的另外一个目的是提供花葵素或花青素苷的新的治疗用途。
本发明进一步的目的是提供预防或治疗过敏性疾病的组合物，该组合物包含选自由黑米提取物、花葵素和花青素苷组成的组中的一种或多种。
为达到上述目的，本发明提供预防或治疗过敏性疾病的方法，该方法包括对需要的个体施用有效量的黑米提取物、花葵素或花青素苷。
为达到本发明的另外一个目的，本发明提供抑制细胞、组织或机体中嗜酸粒细胞聚集的方法，该方法包括对需要的个体施用花葵素或花青素苷。
为达到本发明的另外一个目的，本发明提供了黑米提取物、花葵素或花青素苷在制备预防或治疗过敏性疾病的治疗剂中的用途。
为进一步实现本发明的另外一个目的，本发明提供了花葵素或花青素苷在制备抑制嗜酸粒细胞聚集的治疗剂中的用途。
以下将对本发明进行描述。
本发明提供预防或治疗过敏性疾病的组合物，该组合物包含黑米提取物。
本发明还提供预防或治疗过敏性疾病的组合物，该组合物包含由式1表示的花葵素(3，5，7-三羟基-2-(4-羟基苯基)-1-苯吡喃氯)(3，5，7-trihydroxy-2-(4-hydroxyphenyl)-1-benzopyrylium chloride)或其药学上可接受的盐或糖苷作为有效成分。
<式1>
本发明还提供预防或治疗过敏性疾病的组合物，该组合物包含由式2表示的花青素苷(花青素3-O-β-吡喃葡糖苷)(cyanidin 3-O-β-glucopyranoside)或其药学上可接受的盐作为有效成分。
<式2>
本发明还提供预防或治疗过敏性疾病的组合物，该组合物包含选自由黑米提取物、花葵素和花青素苷组成的组中的一种或多种作为有效成分。
本发明的黑米提取物包含由式1表示的花葵素(3，5，7-三羟基-2-(4-羟基苯基)-1-苯吡喃氯)或由式2表示的花青素苷(花青素3-O-β-吡喃葡糖苷)。
本发明的黑米(Oryza sativa L.)是颜色为黑色的米，其富含花色素苷类化合物，并且易通过商购获得。
在本发明预防或治疗过敏性疾病的组合物中的黑米提取物可以采用相关领域中公知的常规提取方法从黑米(Oryza sativa L)中制备。该提取方法包括但不限于，醇提取、水提取、有机溶液提取以及超临界流体提取等。优选地，可以用水和有机溶剂中的一种，或使用水和有机溶剂的混合物进行提取，所述的有机溶剂例如C1～C4低级醇、丙酮、乙酸甲酯、乙酸乙酯、丙三醇、丙二醇、1，3-丁二醇、正己烷、二乙醚、苯和二氯甲烷。粉碎黑米，将上述溶剂之一加入其中。通过过滤弃去残余物，用真空蒸发器通过搅拌浓缩过滤后的溶液。去除溶剂，冷冻干燥浓缩液，使其粉末化。
优选的提取温度为15℃～80℃，更优选为25℃～60℃。提取时间取决于提取温度，但是一般为5小时～24小时，并且优选7小时～12个小时。如果在提取中使用振荡器，则可以提高提取效率。
在本发明的一个实施方案中，在黑米中加入乙醇，接着在35℃提取7个小时，然后，通过干燥使提取液蒸发，获得粉末状黑米提取物(见实施例1)。
虽然其具体的作用机制还未被披露，但已经确信，本发明用于预防或治疗过敏性疾病的组合物中所含有的由式1表示的花葵素具有强抗氧化活性。特别地，花葵素不但毒性小，而且更容易被吸收，使得它成为适合于对人施用的优秀治疗剂(Ross JA等，Annu Rev Nutr.，2002；22：19～34，综述)。但是，除了将其作为抗氧化剂之外，至今还未知其他用途。
本发明预防或治疗过敏性疾病的组合物中所含有的由式2表示的花青素苷(花青素3-O-β-吡喃葡糖苷)是属于花色素苷类的天然物质，据报道，该物质具有强氧化活性。
本发明预防或治疗过敏性疾病的组合物中所含有的由式1表示的花葵素和由式2表示的花青素苷既可以通过商购得到，也可以通过常规合成方法进行制备(Nakajima N等，Biosci.Biotechnol.Biochem.，61(11)：1926～1928，1997，Amorini AM等，Free Radic.Res.，35(6)：953～966)。优选地，可以从天然物质中将它们进行分离和纯化。更优选从黑米中对它们进行分离。可以通过相关领域公知的常规方法从黑米中对花葵素或花青素苷进行分离和纯化。特别地，可以用水或有机溶剂提取黑米中的有效成分。然后，用色谱层析法对该成分进行分离和纯化，据此获得纯的目标化合物。
在本发明的一个实施方案中，用卵清蛋白致敏而诱发小鼠哮喘，研究黑米提取物对其中炎症(过敏性疾病的主要症状)的抑制。结果发现，本发明的黑米提取物显著地抑制由卵清蛋白诱发哮喘的小鼠的肺中的炎症(见图1)。
在本发明的另一个实施方案中，研究了黑米提取物的主要成分花色素苷类化合物对组织中的炎症和嗜酸粒细胞的聚集(为过敏性疾病的主要症状)的抑制。将诸如花葵素、花翠素、甲基花青素和花青素苷等主要的花色素苷类化合物施用于卵清蛋白诱发的哮喘小鼠。结果证实花葵素和花青素苷显著地抑制气道内嗜酸粒细胞(炎症诱发细胞)的聚集，并显著抑制肺部的炎症(见图2～图4，表1)。在众多花色素苷类化合物中，未发现花翠素具有上述作用。甲基花青素作用较弱。但是，花葵素和花青素苷被证实对气道内嗜酸粒细胞的聚集和肺部炎症具有抑制作用，提示花葵素和花青素苷可被有效地用作预防或治疗过敏性疾病的组合物。
因此，包含本发明的黑米提取物、花葵素或花青素苷的组合物可以有效地用于预防或治疗选自下述的过敏性疾病：支气管哮喘、慢性梗阻性肺部疾病、枯草热、血管运动神经性鼻炎、肥厚性鼻炎、过敏性支气管炎、短暂性肺部浸润、过敏性胃炎、过敏性腹泻、过敏性口腔炎、肠紫癜、结节性动脉周围炎、闭塞性动脉内膜炎、心绞痛、心内膜炎、荨麻疹、血管神经性水肿、结节性红斑、紫癜、特应性皮炎、水疱、交感性眼炎、过敏性结膜炎和过敏性角膜炎，所述的过敏性疾病发生于哺乳动物中，特别是人体中。
本发明中的“有效量”是指施用于患者后显示预防或治疗作用的化合物或提取物的量。通常，黑米提取物的施用量可以为每天1mg/kg～100mg/kg，优选为每天10mg/kg～30mg/kg。由式1表示的花葵素的施用量可以为每天0.1mg/kg～10mg/kg，优选为每天0.5mg/kg～2mg/kg。由式2表示的花青素苷的施用量可以为每天1mg/kg～30mg/kg，优选为每天5mg/kg～20mg/kg。在可能的有效范围内，化合物和提取物可以每天施用一次或数次。黑米提取物、花葵素或花青素苷的施用量范围可以随施用途径、施用对象、年龄、性别、体重、疾病的轻重程度以及患者的其他个体差异而适当变化。包含本发明的黑米提取物、花葵素或花青素苷的组合物并不限于所述的剂型、施用途径或方法，只要其保留有本发明的效果即可。
本文中的“个体”是指包括人在内的哺乳动物。所述的个体包括需要治疗的患者。
本发明的组合物能够以多种方式和多种方法进行制备和施用。例如，可采用口服施用、直肠施用、局部施用、腹膜内施用、眼内施用、肺内施用以及鼻内施用中的任何施用方式。并且，该组合物可以配成各种剂型，如片剂，锭剂、分散剂、悬浮剂、液体制剂、胶囊、乳膏、软膏以及气雾剂。
含有花葵素的本发明的组合物含有花葵素及其药学上可接受的盐或糖苷作为活性成分，还可以进一步含有药学上可接受的载体和其他治疗成份。含有花青素苷的本发明的组合物含有花青素苷及其药学上可接受的盐，并且可以进一步含有药学上可接受的载体和其他治疗成份。本文中的“药学上可接受的盐”是指由药学上可接受的无毒的碱或酸(包括无机碱或无机酸以及有机碱或有机酸)制得的盐。
包括用于口服施用，直肠施用，局部施用，皮下施用，以及包括肌内施用和静脉内施用、眼内施用、肺内施用(鼻吸入或口吸入)或鼻内施用在内的肠胃外施用的组合物在内，其中最适宜的施用途径是根据疾病的特征和严重性以及活性成分的特性从上述途径中选择的途径。根据药学领域公知的一般制备方法，可以方便地制备所述组合物的单一剂量剂型。
就吸入而言，可以通过使用压力容器或喷雾器以气雾剂喷雾的形式对本发明的化合物或提取物进行递送。也可以以粉末形式通过粉末吸入装置对本发明的化合物或提取物进行递送。优选的吸入递送装置是定量吸入(MDI)气雾剂，其可以通过将诸如碳氟化合物或碳氢化合物等推进剂中的一种与黑米提取物或式1和式2的化合物进行混合而配制成溶液或悬浮液的形式。
透皮制剂、气雾剂、乳膏、软膏、洗剂和喷雾剂是黑米提取物、花葵素或花青素苷局部施用剂型的好例子。
可以根据一般的药学施用实用技术，将作为活性成分的黑米提取物、花葵素或花青素苷与药学上可接受的载体进行混合。载体可以因施用途径(例如口服或包括静脉注射在内的肠胃外施用)而异。就用于口服的诸如悬浮剂、酏剂和溶液剂等液体剂型的制备而言，可以使用诸如水、乙二醇、油、醇、调味剂、消毒剂、着色剂等药学上的常规赋型剂。用于口服的固体剂型包括粉剂、胶囊和片剂。固体剂型可以通过混合一种或多种适宜的赋形剂(如淀粉、葡萄糖、微晶纤维素、稀释剂、粒化剂、润滑剂、粘合剂和崩解剂等)进行制备。固体剂型比液体剂型更适于口服。其中使用固体的药学载体的片剂和胶囊是口服的最方便的形式。如果需要，可以根据标准的水性技术或非水性技术来包被片剂。肠胃外施用的载体包括水、适宜的油、盐水、水溶性葡萄糖或乙二醇等。可以另外含有稳定剂和防腐剂。诸如亚硫酸氢钠，亚硫酸钠和抗坏血酸等抗氧化剂是适宜的稳定剂。防腐剂的例子包括苯扎氯铵(benzalconium chloride)、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯和氯代丁醇。还可以采用下面文献中所列的其他药学上可接受的载体(Remington’s pharmaceutical sciences，19th ed.，Mack Publishing Company，Easton，PA，1995)。
上述黑米提取物、花葵素或花青素苷可以以食品组合物的形式提供，用于预防和治疗过敏性疾病。本发明的食品组合物包括功能性食品、营养添加剂、健康食品和食品添加剂等所有可能的类型。可以根据相关领域公知的常规方法将这些食品组合物制成各种形式。例如，作为健康食品，本发明提取物本身可以被制成茶、汁和饮料的形式或颗粒、胶囊和粉末的形式。
为制备功能性食品，可以将本发明的提取物或化合物加入以下物质中：饮料(包括酒精饮料)，水果及其加工食品(例如：罐头水果、瓶装食品、果酱和果子酱等)，鱼、肉及其加工食品(例如：火腿、香肠和咸牛肉等)，面包和面条(小麦面条、荞麦面条、拉面(ramyun)、意大利式细面条和通心面等)，果汁，各种饮料，饼干，面筋，乳制品(例如：黄油和奶酪等)，植物油，人造奶油，植物蛋白，经高压杀菌的食品，冷冻食品和各种调味品(例如：豆酱、酱油和调味汁等)等。
为了用作食品添加剂，本发明的提取物或化合物被优选制成粉末或浓缩物形式。
在本发明的食品组合物中，本发明的提取物或化合物的优选含量为占该组合物总重量的1重量％～90重量％。更优选为10重量％～50重量％。如上面所解释的那样，黑米提取物、花葵素或花青素苷抑制嗜酸粒细胞(炎症诱发细胞)的聚集和组织炎症，因而包含上述黑米提取物、花葵素或花青素苷的健康食品组合物能有效地用作预防或治疗过敏性疾病的补充物。
本发明进一步提供黑米提取物、花葵素或花青素苷的治疗性用途。特别地，本发明提供花葵素或花青素苷在制备抑制所需的细胞、组织或个体中嗜酸粒细胞聚集的治疗剂中的用途。除了花葵素或花青素苷以外，所述的治疗剂还可以进一步包括药学上可接受的载体。所述药学上可接受的载体已在上面举例说明。
本发明还提供黑米提取物、花葵素或花青素苷在制备预防和治疗过敏性疾病的治疗剂中的用途。所述的过敏性疾病已在上面举例说明。
附图说明
图1是一组显微照片，该显微照片显示用黑米提取物处理由卵清蛋白诱发哮喘的小鼠后，对该小鼠肺部炎症的抑制。
A：野生型小鼠，
B：由卵清蛋白诱发哮喘的小鼠，
C：用黑米提取物(10mg/kg)处理的小鼠。
图2显示：用花色素苷类化合物处理由卵清蛋白诱发哮喘的小鼠后，分别对小鼠气道内嗜酸粒细胞聚集的抑制率。
1：野生型小鼠，
2：由卵清蛋白诱发哮喘的小鼠，
3：用花葵素(0.5mg/kg)处理后的小鼠，
4：用花葵素(1.25mg/kg)处理后的小鼠，
5：用甲基花青素(0.5mg/kg)处理后的小鼠，
6：用甲基花青素(1.25mg/kg)处理后的小鼠，
7：用花翠素(0.5mg/kg)处理后的小鼠，
8.用花翠素(1.25mg/kg)处理后的小鼠。
图3是一组显微照片，该显微照片显示用花葵素处理由卵清蛋白诱发哮喘的小鼠后，对小鼠肺部炎症的抑制。
A：野生型小鼠，
B：由卵清蛋白诱发哮喘的小鼠，
C：用花葵素(1.25mg/kg)处理后的小鼠，
D：用花翠素(1.25mg/kg)处理后的小鼠。
图4是一组显微照片，该显微照片显示用花青素苷处理由卵清蛋白诱发哮喘的小鼠后，对小鼠肺部炎症的抑制。
A：野生型小鼠，
B：由卵清蛋白诱发的哮喘小鼠，
C：用花青素苷(1.5mg/kg)处理后的小鼠，
D：用花青素苷(4.5mg/kg)处理后的小鼠。
具体实施方式
本发明将通过下面的实施例进行更详细的描述。但是，下面显示的实施例只是用以说明本发明；本发明的范围不应该被解释为仅限于此。
<实施例1>黑米提取物的制备
将黑米(韩国生产，京东市场，韩国汉城)粉碎，向其中加入100％乙醇，然后在35℃提取7小时。蒸发获得的提取物，冷冻干燥剩余物，获得粉末状黑米提取物。
<实施例2>黑米提取物对哮喘的抑制作用的检测
在该实施例中，进行实验以研究黑米提取物是否能够通过抑制由卵清蛋白诱发哮喘的小鼠的炎症从而抑制过敏性哮喘。
首先，为制备哮喘动物模型，将200μl卵清蛋白溶液(将200μg的卵清蛋白和1000μg的氧化铝凝胶溶解于生理盐水中)分别注入20只10周龄的雌鼠(C57BL/6，Damul Science，大田，韩国)的腹腔中。两周后，用200μl卵清蛋白溶液(2％w/v(重量/体积))喷洒每只小鼠使其致敏。在第21天、22天、23天再用200μl的1％卵清蛋白溶液喷射，在第25天再用10％卵清蛋白溶液喷洒以进行致敏。
用10％卵清蛋白致敏诱发哮喘的上述小鼠被分成两组，一组用作不进行任何试剂处理的阴性对照，另一组用10mg/kg黑米提取物进行处理。通过腹膜内注射来施用黑米提取物，在第24天和第25天对卵清蛋白处理后的小鼠进行两次注射。
用黑米提取物处理后48小时，用醚(ether)杀死小鼠。研究每只小鼠肺部的炎症。结果如图1所示，与野生型的正常小鼠(见图1A)相比，由卵清蛋白(哮喘诱发抗原)诱发哮喘的小鼠的支气管炎症明显加重(见图1B)。但是，黑米提取物的施用明显减轻支气管炎症(见图1C)。因此，证实了黑米提取物能够有效地抑制哮喘。
<实施例3>黑米提取物中所含有的花葵素对气道内嗜酸粒细胞聚集的作用
具有上述哮喘抑制作用的黑米提取物的主要成分是花色素苷类化合物。在该实施例中，通过实验，研究了黑米提取物的主要活性成分花色素苷类化合物是否能够抑制哮喘，以及明确是哪种花色素苷确实可以抑制哮喘。
采用与上述实施例2相同的方法诱发小鼠哮喘，然后将所述小鼠分成四组，一组用作阴性对照，其他三组用作实验组，分别进行甲基花青素、花翠素和花葵素(0.5mg/kg，1.25mg/kg)处理。通过腹膜腔内注射来施用每组化合物，在用卵清蛋白处理后的第24天和第25天，对小鼠进行两次注射。
处理后的第二天，用醚杀死每只小鼠。然后，将微型管与气管相连。通过所述微型管注射PBS(磷酸盐缓冲盐水，0.8ml)，并通过该微型管进行回收，重复两次，获得支气管肺泡冲洗液(BALF)。离心该冲洗液，以分离气道内腔细胞以及从细胞和肺部分泌的各种蛋白。
用细胞离心涂片器(cytospin)将分离的细胞固定在载玻片上，并用Diff-quick染色溶液染色。用连接在Carzeiss显微镜(型号：AXIOVERT25-CEL)上的数码相机拍照。每个样本挑选5个随机区域，进行嗜酸粒细胞计数，在图2中显示每个样本中的嗜酸粒细胞百分比。
如图2所示，接触卵清蛋白的小鼠气道内的嗜酸粒细胞百分比为58％。相反地，在采用花葵素、甲基花青素和花翠素等花色素苷处理过的各组小鼠中，其气道内的嗜酸粒细胞聚集则受到抑制。特别地，在施用0.5mg/kg和1.25mg/kg的花葵素的情况中，其气道内嗜酸粒细胞百分比分别降至30％和20％。证实了花葵素与其它花色素苷类化合物相比能更有效地抑制气道内嗜酸粒细胞的聚集。
<实施例4>花葵素对哮喘炎症的抑制作用
在该实施例中，通过实验，研究了先前已被证实大量地抑制气道内嗜酸粒细胞聚集的花葵素是否能够通过抑制肺部的炎症来抑制过敏性哮喘。
将如上述实施例2的由10％卵清蛋白诱发哮喘的小鼠分成三组；第一组用作不进行任何试剂处理的阴性对照，第2组用作阳性对照，用1.25mg/kg花翠素进行处理，第3组用1.25mg/kg花葵素进行处理。在卵清蛋白处理后的第24天和第25天，通过腹膜腔内注射对小鼠施用两次上述每种化合物。
用每种化合物处理后48小时，用醚杀死小鼠。研究每只小鼠肺部的炎症。结果如图3所示，与正常的野生型小鼠相比(见图3A))，在由卵清蛋白(哮喘诱发抗原)诱发的小鼠中，支气管炎症显著加重(见图3B))。另一方面，花葵素的施用显著减轻炎症(见图3C)。在用1.25mg/kg和0.5mg/kg的花葵素处理过的两组中，花葵素的炎症抑制作用是显著的(数据未显示)。相反地，花翠素(另外一种花色素苷)(1.25mg/kg)没有明显地抑制支气管炎症(图3D)。
<实施例5>黑米提取物中的花青素苷对气道内嗜酸粒细胞聚集和对哮喘炎症的抑制作用
在该实施例中，研究了黑米提取物中含有的花青素苷对气道内嗜酸粒细胞聚集和对哮喘的抑制能力。
为制备哮喘动物模型，在实验第1天和第10天，将0.5ml的卵清蛋白溶液(将500μg的卵清蛋白和10mg的氧化铝凝胶溶解于1ml的PBS溶液中)分别注入20只5周龄的雌鼠(Balb/c，Orient，汉城)腹腔内。在第21、22、23天，用所述卵清蛋白溶液喷洒每只小鼠以诱发哮喘。
将采用卵清蛋白诱发哮喘的小鼠(n＝15)被分成三组，一组用作不进行任何试剂处理的阴性对照，另两组用作实验组，每组分别用1.5mg/kg和4.5mg/kg花青素苷进行处理。从第2天到第23天，一天一次连续口服施用花青素苷。
最后一次致敏后24小时，用醚杀死每只小鼠，然后，将微型管与气管相连。通过所述微型管注射0.8ml的PBS，并由该微型管进行回收，重复两次，将获得的支气管肺泡冲洗液(BALF)离心，以分离气道内腔细胞和从细胞及肺部分泌的各种蛋白。
用细胞离心涂片器将分离的细胞固定在载玻片上，并用Diff-quick染色溶液染色。用连接在Carzeiss显微镜(型号：AXIOVERT 25-CEL)上的数码相机拍照。每个样本挑选5个随机区域，进行嗜酸粒细胞计数，每个样本中的嗜酸粒细胞百分比显示在表1中。
<表1>
| 处理组 | 浓度 | 动物数量 | 嗜酸粒细胞百分比 |
| 野生型小鼠组 | - | 5 | 0.1±0.0 |
| 阴性对照组 | - | 5 | 62.7±4.3 |
| 花青素苷处理组 | 1.5mg/kg | 5 | 49.5±5.8* |
| 花青素苷处理组 | 4.5mg/kg | 5 | 38.2±6.2* |
| *：显著差异(p＜0.05) | |||
如表1所示，接触卵清蛋白的小鼠的气道内嗜酸粒细胞百分比(阴性对照)是63％，而嗜酸粒细胞在气道内的聚集被证实受到所施用的花青素苷的抑制。准确地，随着花青素苷浓度的增加，嗜酸粒细胞在气道内的百分比与阴性对照相比，分别降到49％和38％。证实了花青素苷有效地抑制气道内嗜酸粒细胞的聚集。还分析了被杀死小鼠的肺细胞中的炎症，结果在图4中显示。如图4所示，与正常的野生型小鼠相比(图4A)，在由卵清蛋白(哮喘诱发抗原)(图4B)诱发的小鼠中，支气管炎症显著加重。但是，花青素苷的施用明显减轻了所述炎症(图4C和图4D)。
<实施例6>含有本发明的黑米提取物的饮料组合物的制造
通过将在上面实施例1中获得的黑米提取物(25％)、维生素A(0.15％)、维生素D(0.2％)、维生素B2(0.15％)，维生素C(2.0％)，牛磺酸(3.0％)，聚葡萄糖(2.5％)和纯化水混合在一起，制备饮料组合物。
工业实用性
如上所述，已经证实了花葵素和花青素苷或包含花葵素和花青素苷的黑米提取物能够抑制组织内嗜酸粒细胞的聚集并由此对过敏性炎症具有抑制作用。因此，本发明的花葵素、花青素苷或黑米提取物能有效地用于预防或治疗伴有组织炎症和嗜酸粒细胞聚集的过敏性疾病。所述疾病例如，过敏性鼻炎、过敏性结膜炎、哮喘、慢性梗阻性肺部疾病、特应性皮炎和过敏性腹泻等。
Claims (1)
1.黑米提取物在制备预防或治疗支气管哮喘的治疗剂中的用途。
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| BRPI0409199A (pt) | 2006-05-02 |
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| JP2006515372A (ja) | 2006-05-25 |
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