CN113041271B - 具有预防和抗过敏功效的组合物 - Google Patents
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Abstract
本发明涉及一种具有预防和抗过敏功效的组合物,其包含不包含桦树花粉变应原Bet v1的经处理的桦树汁。所述组合物包括饮品、保健食品、药品和皮肤外用组合物。
Description
技术领域
本发明涉及一种具有预防和抗过敏功效的组合物,其包含不含桦树花粉变应原Bet v1的经处理的桦树汁。所述组合物包括饮品、保健食品、药品和皮肤外用组合物。
背景技术
过敏是由变应原(亦称过敏原)诱发的I型变态反应。过敏原通过皮肤或黏膜接触、吸入、注射及食入等途径首次进入体内后,可造成机体的致敏状态;当机体再次接触该类过敏原时,激活免疫系统的异常活动,最终导致一系列的过敏性损伤。
临床上将其大致分为过敏反应和过敏性疾病。前者是机体对致敏原作出异常反应的全身综合征。后者则是过敏累及某特定器官及组织,导致了某种疾病的发生。常见的过敏性疾病有过敏性哮喘、过敏性鼻炎、花粉症、荨麻疹等。
花粉症是一种由花粉导致的过敏性疾病,可累及鼻黏膜、眼结膜、支气管及皮肤等部位,花粉颗粒可黏附在皮肤和眼结膜,沉积在鼻黏膜,花粉碎片可被吸入下呼吸道,花粉中的某些特殊蛋白分子浸出,透过皮肤/黏膜屏障与抗原提呈细胞结合,从而导致一系列过敏反应。花粉过敏患者通常出现明显的呼吸道症状,包括鼻痒、鼻塞、打喷嚏和流清涕、咳嗽、喘息、胸闷、咳痰和呼吸困难等,并伴有眼痒、眼结膜、眼睑红肿,以及面部、四肢等暴露部位的皮肤丘疹、红斑、风团或瘙痒等过敏症状。引发花粉症的常见致敏花粉包括春季树木花粉(如杨树、柳树、桦树花粉等)、夏季牧草花粉和秋季杂草花粉。
桦树花粉是我国北方地区春季主要致敏花粉之一。现已鉴定的桦树花粉变应原包括Bet v1、Bet v2、Bet v3、Bet v4、Bet v6、Bet v7、Bet v8,其中Bet v1是桦树花粉的主要变应原。此外,有研究证实,对桦树花粉过敏的受试者进行皮肤划痕试验,其中有39%的受试者对桦树汁表现为阳性反应,表明桦树汁有一定的致敏风险。
我们经过研究发现,经处理的桦树汁不仅没有致敏风险,而且对花粉症、过敏性鼻炎等过敏性疾病具有显著的改善作用。
发明内容
一方面,本发明涉及一种经处理的桦树汁,其不包含桦树花粉变应原Bet v1。
再一方面,本发明涉及经处理的桦树汁在具有预防和抗过敏功效的组合物中的用途,所述经处理的桦树汁不包含桦树花粉变应原Bet v1。
另一方面,本发明涉及一种具有预防和抗过敏功效的组合物,其包含经处理的桦树汁,所述经处理的桦树汁不包含桦树花粉变应原Bet v1。
又一方面,本发明涉及一种处理桦树汁原液的方法,其包括除去桦树汁原液中的桦树花粉变应原Bet v1的步骤,以获得不包含桦树花粉变应原Bet v1的经处理的桦树汁。
意料不到地,本发明人发现,与未经处理的桦树汁原液容易诱发过敏风险不同,经处理的桦树汁非但不会致敏,反而具有显著的预防过敏和抗过敏效果,在内服和/或外用时均可抑制血液中免疫球蛋白E(IgE,确诊过敏的唯一临床指标)的表达,预防和治疗花粉、尘螨、海鲜等过敏导致的鼻痒、鼻塞、打喷嚏、流清涕、咳嗽、胸闷、流泪、眼睑红肿、皮肤出现红斑、丘疹、风团等症状,这是基于现有技术所不能预料到的。
本发明中所涉及的桦树汁原液得自桦木科桦树属,其可来自白桦(Betula alba)、柔毛桦(Betula pubescens)、垂枝桦(Betula Pendula)和亚洲白桦(Betula platyphylla)这四个品种。所述桦树汁原液为在土壤解冻至早春发叶之间,人工在桦树的树干基部钻孔收集而得的无色透明、无沉淀、无杂物,具有桦树清香营养丰富的汁液。所述桦树汁原液可商购获得,例如可购自大兴安岭超越野生浆果开发有限责任公司。
在一些实施方案中,除去桦树汁原液中的桦树花粉变应原Bet v1通过用超滤、层析、透析、等电点沉淀、盐析和/或密度梯度层析法进行,得到本发明的经处理的桦树汁。
在一个优选的方法中,通过亲和层析法,利用Bet v1抗体与抗原之间的特异性相互作用,将Bet v1从桦树汁原液中特异性分离。所述亲和层析法包括以下步骤:
(1)将能与桦树花粉Bet V1抗体结合的填料装入蛋白纯化柱中,然后用pH缓冲液进行平衡;
(2)将桦树花粉Bet V1抗体加入所述纯化柱,使所述填料捕获所述抗体,然后再用pH缓冲液平衡;
(3)将桦树汁原液加入所述纯化柱,使所述抗体捕获桦树汁原液中的Bet v1蛋白,和
(4)收集流穿液,得到不含Bet v1的经处理的桦树汁。
在上述方法中,所采用的蛋白纯化系统是本领域已知的,例如可以是AKTA Avant全自动智能蛋白纯化柱系统。所采用的能与桦树花粉Bet V1抗体结合的填料是本领域已知的,例如包括protein A、protein G等,其可商购获得,例如protein A可购自Cytiva。所采用的pH缓冲液是本领域已知的,pH范围为5.5-9,优选6-8,所述pH缓冲液包括例如磷酸盐缓冲液1xPBS。使用pH缓冲液进行平衡,以获得适于抗体结合填料及Bet V1蛋白与抗体结合的环境。所采用的桦树花粉Bet v1抗体是本领域已知的能够结合Bet v1蛋白的抗体,其中包括单抗、多抗或通过Bet v1蛋白免疫获得的动物血清等,例如可以是使用Bet v1蛋白自行免疫兔子获得动物血清,也可采用例如购自Novus的Birch pollen Bet v1抗体。在上述方法中,所采用的缓冲液pH、压力、流速、柱体积、进样量及其浓度等条件是本领域技术人员根据具体情况来定。
在另一个优选的方法中,通过两步超滤法,使用不同分子量的超滤膜来去除分子量为约17kDa附近的蛋白质,从而达到去除致敏蛋白Bet v1的目的。
具体地,所述两步超滤法包括以下步骤:
采用分子量为30-100kDa,优选30-50kDa的膜,将桦树汁原液进行第一次超滤,得到分子量较大的截留部分a1和超滤液b1;
采用分子量为1-10kDa,优选5-10kDa的膜,将超滤液b1进行第二次超滤,得到截留部分a2和超滤液b2;和
将截留部分a1和超滤液b2合并,即得到去除了致敏蛋白Bet v1的经处理的桦树汁。
在上述方法中,可采用的超滤膜是本领域已知的,其中包括中空膜、卷式膜、平板膜和陶瓷膜等,例如可采用购自美国科氏滤膜系统公司的超滤膜。可采用本领域已知的膜分离系统,例如购自三达膜科技(厦门)有限公司的膜分离系统。在上述方法中,可采用的压力为约0-5bar,优选约0.5-2bar,和温度通常为在4-30℃,优选4-10℃。
所述具有预防和抗过敏功效的组合物可以是饮品、保健食品、药品或皮肤外用组合物。所述皮肤外用组合物包括药物组合物和化妆品组合物。
所述经处理的桦树汁在具有预防和抗过敏功效的组合物中的含量可以为约10-100%重量,优选20-95%重量,更优选22-90%重量,最优选30-90%重量,基于所述具有预防和抗过敏功效的组合物的重量。
除所述经处理的桦树汁外,所述具有预防和抗过敏功效的组合物还可以任选地包含饮品或保健食品组合物或药品或皮肤外用组合物中常用的成分。所述常用成分是本领域已知的,包括例如媒介物、活性成分和辅料等,且本领域技术人员可根据需要选择所述常用成分的类型和用量。例如,所述媒介物包括例如稀释剂、分散剂或载体等,所述活性成分包括例如润肤剂、保湿剂、其他抗炎活性成分等,所述辅料包括例如乳化剂、增稠剂、防腐剂、香料等。
所述具有预防和抗过敏功效的组合物可以通过本领域已知的任何合适的方法制备。例如,使用本领域中常用的溶解槽、乳化锅、分散器、输送泵等设备制备。制备时先将水溶性物质投入水相溶解釜,将油溶性物质投入油相溶解釜,将两个釜的温度加热至约80℃,其中对于易结块的原料,可先用分散器将其预分散。待溶解完成后将油相和水相输送至乳化锅中,均质乳化约5-15分钟。乳化完成后将料体温度降至常温,加入任选的香精、防腐剂等,并视需要调节产物的pH。相关检测指标都合格后方可灌装出货。
以上制备方法可根据剂型要求进行删减或调整。根据需要,可将所述具有预防和抗过敏功效的组合物制成口服液、片剂、膏剂、霜、乳液、精华液、喷剂、凝胶等各种剂型。
实施例
以下结合实施例,对本发明进行进一步详细说明。但是,应当理解为,这些实施例、对比例仅仅是用于更详细地说明本发明,而不应理解为用于以任何形式限制本发明所附权利要求书的范围。
实施例1:亲和层析法去除致敏蛋白Bet v1
1.实验材料
填料protein A(购自Sigma-Aldrich)、捕获抗体Birch pollen Bet V1抗体(购自Novus)、桦树汁原液(购自大兴安岭超越野生浆果开发有限责任公司)、缓冲液1×PBS、100mM醋酸pH 3.0、20%乙醇。
仪器:AKTA Avant全自动智能蛋白纯化柱系统
2.操作步骤
2.1装柱:用100ml protein A填料装柱。
2.2抗体处理:室温下(22-25℃,以下步骤均在室温下进行),将抗体所在缓冲液的pH调节至6.2,可以用1×PBS直接加入稀释。
2.3柱平衡:用1×PBS平衡protein A柱,压力控制在0.3MPa以内,缓冲液保留时间为10min,待所有pH值恒定至6.2后结束。
2.4抗体上样:将抗体样品上到protein A柱中,让protein A填料捕获抗体,压力及液体保留时间同上。
2.5再平衡:上样结束后用1×PBS平衡柱子,平衡3个柱体积,压力及液体保留时间同上。
2.6桦树汁原液上样:将桦树汁原液上到protein A柱,让柱子内的抗体来捕获桦树汁中的抗原,压力及液体保留时间同上。同时收集样品流穿液。
2.7再平衡:上样结束后用1×PBS平衡柱子,平衡3个柱体积,压力及液体保留时间同上。
2.8柱子再生:用100mM醋酸pH 3.0再生柱子,再生5个柱体积。之后用1×PBS平衡柱子,压力及液体保留时间同上。
2.9柱子保存:用20%乙醇,4℃下,保存柱子。
实验结果:在上述2.6步骤中收集的流穿液即为不含Bet V1的经处理的桦树汁。
实施例2:两步超滤法去除致敏蛋白Bet v1
1.实验材料
超滤膜:截留分子量(MWCO)10kDa和50kDa(购自美国科氏滤膜系统公司)、桦树汁原液(购自大兴安岭超越野生浆果开发有限责任公司)、2%氢氧化钠溶液。
仪器:膜分离系统(购自三达膜科技(厦门)有限公司)
2.操作步骤
2.1安装超滤膜:将MWCO 50kDa的超滤膜安装在膜分离系统。
2.2设备CIP清洗:先用纯水清洗10min,然后用2%的氢氧化钠溶液进行碱洗30min,排空碱洗液,水冲洗至pH与水接近,电导率一致。排空设备内的水。
2.3第一次超滤:将商购得到的桦树汁原液投入超滤系统循环罐,开启循环泵,调节压力为2bar。待运行稳定后,收集截留部分a1和超滤液b1。第一次超滤结束后进行设备CIP清洗(如2.2所述)。超滤过程中保证整个循环罐内液体温度为4-6℃。
2.4安装超滤膜:将MWCO 10kDa的超滤膜安装在膜分离系统,并进行设备CIP清洗(如2.2所述)。
2.5第二次超滤:将2.3获得的超滤液b1投入超滤系统循环罐,开启循环泵,调节压力为1bar。待运行稳定后,收集截留部分a2和超滤液b2。超滤过程中保证整个循环罐内液体温度为4-6℃。
2.6设备CIP清洗:第二次超滤结束后进行设备CIP清洗(如2.1所述)。
实验结果:将2.3步中收集的截留部分a1和2.5步中收集的超滤液b2合并,即得到不含Bet V1的经处理的桦树汁。
实施例3:经处理的桦树汁中致敏蛋白含量的变化
利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对桦树汁原液、实施例1和2中获得的经处理的桦树汁中的蛋白质含量的进行定性分析。
结果显示,桦树汁原液在分子量为约17kDa处有明显的条带,因为桦树花粉中的主要变应原Bet v1为分子量为约17kDa的蛋白质,因此可推测桦树汁中含有与桦树花粉相同的致敏蛋白Bet v1。而实施例1和2中获得的经处理的桦树汁在分子量为约17kDa处没有明显条带,表明其中不再有致敏蛋白Bet v1。
实施例4:经处理的桦树汁的抗过敏功效
本实施例利用杨树花粉粗提物致敏和激发建立小鼠过敏模型,考察桦树汁内服的抗过敏功效。
实验方法如下。
1.实验动物:雄性豚鼠。
2.实验材料:杨树花粉粗提物,总lgE、IL-4、IFN-γ酶联免疫吸附反应检测(ELISA)试剂盒、实施例2的经处理的桦树汁、桦树汁原液、30%的实施例2的经处理的桦树汁。
3.实验过程如下:
3.1实验设计
3.2动物致敏及激发方法
将杨树花粉粗提物混悬于含5%氢氧化铝免疫佐剂的生理盐水中,配成浓度为3.5mg/ml的溶液,正常组采用生理盐水溶液,每只豚鼠腹腔注射1ml溶液。每间隔5天重复致敏1次,共3次。第3次致敏后的第5天进行激发试验,按组分别将单个豚鼠放入50cm x 40cmx 30cm密闭玻璃盒内(上部用保鲜膜封住,留出气孔),用超声雾化器向密闭盒内喷雾生理盐水或抗原。模型组及各实验组喷杨树花粉粗提物(3.5mg/ml)20min进行激发,正常对照组喷雾生理盐水20min,激发每天1次,连续5天。
4.考察指标:酶联免疫吸附试验测血清中的总IgE、IL-4及IFN-γ水平。
得到的结果如下表所示:
*与#均代表与模型组相比的显著性差异,不同的是*代表正向结果,#代表负向结果。****(P<0.0001);#(P<0.05);NS代表与模型组相比无显著性差异
上表中的结果表明,与正常组相比模型组均可诱导血清中总IgE,IL-4高于正常组,血清中IFN-r则显著低于正常组。而A、B、C组中实施例2的经处理的桦树汁组和30%的实施例2的经处理的桦树汁组均可以使得血清中IgE、IL-4、IFN-r均趋于正常,且A组的效果最为显著;但是A、B、C组中桦树汁原液组作用到小鼠后却会使得血清中IgE、IL-4的含量轻微高于模型组、IFN-r的含量轻微低于模型组或者与模型组相比无显著性差异,说明桦树汁原液存在进一步激发过敏反应的风险。
实施例5:经处理的的桦树汁内服与外用抗过敏人体临床功效
在春季,选择45名被诊断对花粉过敏且往年在春季容易过敏的患者和45名目前正处于过敏阶段的中轻度患者,其血清中IgE含量大于165.3IU/毫升并伴有鼻痒、鼻塞、打喷嚏、流清涕、咳嗽、胸闷、流泪、眼睑红肿、皮肤出现红斑、丘疹、风团等症状中的一项或多项,进行抗过敏或预防过敏的临床测试,其中实验中预防和过敏后处理组所用的桦树汁为实施例1的经处理的桦树汁。
1.分组及实验方法如下所示:
2.检测指标:
效果判断标准:
痊愈:呼吸道、眼部或皮肤相关过敏症状全部消退,疗效率达100%;
显效:呼吸道、眼部或皮肤相关过敏症状明显或大部分消退,疗效率达70%以上;
有效:呼吸道、眼部或皮肤相关过敏症状部分消退,疗效率达30%以上;
无效:呼吸道、眼部或皮肤相关过敏症状未见消退甚至恶化,疗效率未达30%;
3.实验结果
上述结果表明,经处理的桦树汁无论是内服还是外用均有预防换季花粉过敏的效果,而内服加外用联合用药效果最佳。此外,过敏期患者的临床测试表明,经处理的桦树汁内服、外用均有较好的抗过敏功效,同样的,内服加外用联合用药抗过敏效果最佳。
实施例6:具有预防过敏功效的喷雾组合物
所述喷雾组合物的配方如下表(%):
序号 | 成分 | A1 | A2 |
1 | 实施例1的桦树汁 | 94.9 | 0 |
2 | 水 | 0 | 94.9 |
3 | 透明质酸钠 | 0.15 | 0.15 |
4 | 泛醇 | 0.5 | 0.5 |
5 | 尼泊金乙酯 | 0.15 | 0.15 |
6 | PPG-10甲基葡糖醚 | 1 | 1 |
7 | 甘油 | 3 | 3 |
8 | 苯氧乙醇 | 0.3 | 0.3 |
上述喷雾组合物如下制备:
1.将原料1、2、3、4、5、6、7投入乳化锅,边搅拌边升温至80℃,保温10分钟;
2.边搅拌边降温至45℃;
3.加入原料8,搅拌10分钟;
4.检验合格后出料。
在春季,选择30名被诊断为对花粉过敏且往年在春季容易过敏的患者进行预防过敏功效的临床测试。让其中的15人每天在皮肤裸露部位喷涂所述喷雾组合物A1,让另外15人喷涂对照喷雾A2,每天至少喷3次,出门必喷,持续整个春季(3-5月)。
结果发现,喷涂所述喷雾组合物的15人中有13人整个春季均未出现过敏反应,有2人出现轻微的花粉过敏反应,但是症状较往年都有所减轻。然而,喷涂对照喷雾的15人中有12人均出现了往年的过敏反应。这表明,含有本实施例的喷雾组合物具有显著的预防过敏的功效。
实施例7:抗过敏功能性饮品
序号 | 成分 | 含量,% |
1 | 实施例2的桦树汁 | 99.4 |
2 | 水 | 0 |
3 | 透明质酸钠 | 0.5 |
4 | 食用香精 | 0.1 |
上述抗过敏功能性饮品如下制备:
1.将原料1、2、3、4投入调配桶中,搅拌10分钟;
2.调配液检验合格后经1μm布袋过滤,去除杂质;
3.滤液经超高温瞬时灭菌(UHT)20s,加热温度为120℃;
4.灭菌后无菌罐装。
在春季,选择15名目前正处于过敏阶段的中轻度患者,其血清中IgE含量大于165.3IU/毫升并伴有鼻痒、鼻塞、打喷嚏、流清涕、咳嗽、胸闷、流泪、眼睑红肿、皮肤出现红斑、丘疹、风团等症状中的一项或多项。让所述患者每天服用所述抗过敏功能性饮品500ml,持续20天。20天后,抽血检测IgE、以及评估过敏症状效果,其中效果判断标准同上。
结果表明,15人中有14人的IgE小于165.3IU/毫升;其中,痊愈11人、显效4人,这表明所述抗过敏功能性饮品具有显著的抗过敏功效。
Claims (4)
1.一种处理桦树汁原液的方法,其包括除去桦树汁原液中的桦树花粉变应原Bet v1的步骤,以获得不包含桦树花粉变应原Bet v1的经处理的桦树汁;
其中通过两步超滤法来除去桦树汁原液中的桦树花粉变应原Bet v1,所述两步超滤法包括如下步骤:
采用分子量为30-100kDa的膜,将桦树汁原液进行第一次超滤,得到分子量较大的截留部分a1和超滤液b1;
采用分子量为1-10kDa的膜,将超滤液b1进行第二次超滤,得到截留部分a2和超滤液b2;和
将截留部分a1和超滤液b2合并,得到不含Bet v1的经处理的桦树汁。
2.通过权利要求1的方法获得的不含Bet v1的经处理的桦树汁。
3.一种具有预防和抗过敏功效的组合物,其包含权利要求2所述的经处理的桦树汁。
4.根据权利要求3所述的组合物,其中所述经处理的桦树汁的含量为10-100%重量,基于所述具有预防和抗过敏功效的组合物的重量。
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