CN117143192A - 一种具有抗组胺作用的蜂王浆蛋白肽及其制备方法和用途 - Google Patents
一种具有抗组胺作用的蜂王浆蛋白肽及其制备方法和用途 Download PDFInfo
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- CN117143192A CN117143192A CN202311283880.3A CN202311283880A CN117143192A CN 117143192 A CN117143192 A CN 117143192A CN 202311283880 A CN202311283880 A CN 202311283880A CN 117143192 A CN117143192 A CN 117143192A
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- jelly protein
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于医药技术领域,具体涉及一种具有抗组胺作用的蜂王浆蛋白肽及其制备方法和用途。所述蜂王浆蛋白肽是采用生物酶解技术水解蜂王浆蛋白获得,工艺简单。采用本发明方法制备得到的蜂王浆蛋白肽可用于预防和缓解组胺释放,还可提高机体对组胺的耐受力,作用效果显著。本发明提供的蜂王浆蛋白肽安全有效、分子量小易吸收,通过实验研究确证能够降低肥大细胞释放的组胺水平,有望抑制机体不良状态下组胺的释放,进而缓解和治疗组胺大量释放导致的过敏性组织损伤。
Description
技术领域
本发明属于医药技术领域,具体涉及一种具有抗组胺作用的蜂王浆蛋白肽及其制备方法和用途。
背景技术
过敏反应的发生一般开始于致敏阶段,经过激发而产生效应。过敏原进入机体后诱导过敏原特异性B细胞产生抗体应答,此类抗体与肥大细胞和嗜碱性粒细胞的表面相结合,而使机体处于对该过敏原的致敏状态。相同的过敏原再次进入机体时,能与致敏的肥大细胞和嗜碱性粒细胞表面的抗体特异性结合,使其释放组胺、前列腺素D、白三烯等生物活性物质,作用于效应组织或器官,引起局部甚至全身的过敏反应。过敏性疾病又被称为变态反应性疾病,是一类临床常见的免疫性疾病。近年来我国过敏性疾病患病率呈持续上升趋势,常见的过敏性疾病主要包括过敏性鼻炎、过敏性哮喘及荨麻疹等。
组胺是自体活性物质之一,由组氨酸在脱羧酶的作用下产生。组织中的组胺以无活性的结合型存在,在皮肤、支气管黏膜、肠黏膜和神经系统中含量较多。当机体受到理化刺激或发生过敏反应时,引起肥大细胞和嗜碱性粒细胞脱颗粒,导致组胺释放,组胺与受体结合产生多种生物效应,在全身或局部体现为不同类型的过敏症状。因此,使用抑制肥大细胞和嗜碱性粒细胞释放组胺的抗组胺药物为过敏反应的主要治疗手段。虽然抗组胺药物的研发和不断完善使其功效与选择性增强,但其临床疗效依然难以达到很好的综合治疗效果,存在程度各异的不良反应,对胃肠道,面部可能造成一定的损伤。因此,研究新的组胺抑制剂或组胺释放抑制剂,对缓解和治疗过敏反应具有重要意义。
蜂王浆也称蜂皇浆、蜂乳或蜂王乳,是青年工蜂的咽头腺和上颚腺等腺体分泌的混合物,专用于供给蜂王终身所需的食物。蜂王浆中蛋白质含量丰富,营养价值高,并具有免疫调节、抗炎、抗菌、抗肿瘤等多种活性,在食品和医疗领域得到广泛应用。通过蛋白酶水解或微生物发酵等技术从蜂王浆蛋白中获得的低聚肽具备多样的生物学功能,有助于调节机体功能、实现疾病预防,相关产品的研究开发具有良好的发展前景。
发明内容
本发明的目的在于克服现有技术的不足之处,提供一种安全有效的组胺释放抑制剂及其制备方法。该物质可以抑制肥大细胞释放组胺,还可以提高磷酸组胺所致瘙痒的致痒阈值,提高机体对组胺的耐受力,以缓解局部或全身的过敏反应。
为实现上述目的,本发明采用的技术方案为:
提供一种蜂王浆蛋白肽作为组胺释放抑制剂,所述蜂王浆蛋白肽是由蜂王浆蛋白通过生物酶酶解制备。
所述蜂王浆蛋白肽的制作步骤如下:1)蜂王浆蛋白的纯化:向蜂王浆冻干粉中添加5倍去离子水,搅拌溶解。用2M的NaOH溶液调节pH为10.0,继续搅拌提取30min,然后5000rpm离心20min,取上清液;向上清中加入2M的HCl溶液调节pH为3.5,置于4℃冰箱沉淀24h,5000rpm离心20min,弃去上清,收集沉淀,干燥即得纯化的蜂王浆蛋白。
酶解:称取纯化的蜂王浆蛋白,以1:30的料液比加入去离子水,搅拌均匀,调节pH为6.0-9.0,加入0.5-3.0%的蛋白酶进行生物酶解,于30-60℃下恒温搅拌反应3-8h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后使用高速离心机10000rpm离心20min,收集上清液,冷冻干燥即得蜂王浆蛋白肽。
蜂王浆蛋白通过碱提酸沉的方法进行纯化,操作简单,成本低廉,能去除水溶性糖分、淀粉、纤维素等成分。保证蛋白质质量,避免杂质对酶解优化产物造成影响,同时提高蜂王浆蛋白肽的纯度和产率。
通过酶解制备的肽产物的组胺释放抑制活性高于原蜂王浆蛋白,由此可见酶解可有效释放蜂王浆蛋白中的活性氨基酸序列。此外所述蜂王浆蛋白肽还可以提升对组胺的耐受,与降低组胺受体的表达有关。所述蛋白酶为中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、复合蛋白酶、风味蛋白酶中的一种或多种,优选为碱性蛋白酶。
值得说明的是,酶解条件的改变对于酶解产物的活性影响很大。因此,发明人基于产物的组胺释放抑制活性,优化了酶解工艺。
优选地,碱性蛋白酶的添加量为以重量百分比计1.0-2.0%。
优选地,酶解温度为40-50℃。
优选地,酶解pH为7.0-8.0。
优选地,酶解时间为3-5h。
进一步地,发明人采用不同分子量超滤膜对酶解物进行分离,通过活性比较实验证明分子量<3kDa的活性肽相对于其他分子量的片段具有更强的组胺释放抑制作用。
随后发明人采用LC-MS/MS技术从分子量低于3kDa的肽组分中鉴定了7个氨基酸序列,经活性评估发现氨基酸序列Leu-Pro-Asp-Glu-Val-Ser-Gly或His-Tyr-Val-Pro-Val(缩写为LPDEVSG或HYVPV)的组胺释放抑制活性可达80%以上,是活性较好的氨基酸序列。
本发明还提供一种所述蜂王浆蛋白肽在制备组胺释放抑制剂中的应用,可用于预防、治疗和修复机体的过敏反应及其并发症。
所述过敏性疾病,包括但不限于过敏性鼻炎、过敏性哮喘、荨麻疹、过敏性结膜炎、过敏性皮炎等。过敏反应症状包括但不限于平滑肌收缩、毛细血管扩张、血管通透性增强、腺体分泌物增多、皮肤过敏症状等。过敏反应的诱导包括但不限于摄入性过敏、接触性过敏等、情绪性过敏、自身免疫性过敏。
本发明还提供了蜂王浆蛋白肽在制备预防、改善或治疗组胺释放导致组织损伤产品中的应用,可与适宜的载体或辅料开发为口服剂型、外用剂型或注射剂型。
作为可选的方式,在上述制剂中,所述口服剂型包括但不限于片剂、胶囊剂、口服液、肠溶片、颗粒剂、糖浆剂、滴丸、水蜜丸、散剂、合剂、煎膏剂、酒剂、露剂、酊剂、舌下含片等。
所述外用剂型包括但不限于软膏、栓剂、硬质软膏、搽剂、滴眼液、油剂、乳膏、气雾剂、喷雾剂等。
注射给药方式包括但不限于皮下注射、静脉注射、肌肉注射或腹腔注射等。
或者,可根据化妆品、护肤品、日常洗护用品的常规工艺将本发明提供的蜂王浆蛋白肽制备成相应的爽肤水、保湿乳、面膜、面霜、粉饼、沐浴露、香皂、粉底液、粉底霜、精华液、护手霜、身体乳、洗手液、洗发水、护发素、洁面膏、洗面奶、防晒霜、润肤膏、雪花膏、营养液、香水等。
作为可选的方式,在上述用途中,所述产品是药物、保健品、功能性食品或化妆品。
相比于现有技术,本发明的有益效果为:
本发明通过酶解技术制备了蜂王浆蛋白肽,并通过工艺优化得到了高活性的组胺释放抑制产物。所述活性肽疗效显著确切,工艺简单,易于制备,无毒副作用,分子量小易吸收,其能够显著抑制组胺的释放,同时还能提升机体对组胺的耐受力,有利于易敏体质人群的脱敏治疗。所述活性肽有望抑制机体不良状态与疾病状态下组胺的释放,进而缓解和治疗组胺大量释放导致的过敏性组织损伤。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施实例对本发明作进一步说明。
实施例1:蜂王浆蛋白肽的制备
蜂王浆蛋白的纯化:蜂王浆冻干粉中加入5倍去离子水,搅拌溶解。使用2M的NaOH调节pH为10.0,继续搅拌提取30min,搅拌完成后5000rpm离心20min,收取上清液;用2M的HCl调节pH为3.5,并置于4℃冷却沉淀24h,5000rpm低温离心20min,弃去上清,收集沉淀,干燥即得纯化的蜂王浆蛋白。
蜂王浆蛋白的生物酶解:准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,调节pH为7.0,加入1%的碱性蛋白酶进行水解,于40℃下恒温搅拌3h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
实施例2:大鼠肥大细胞组胺释放实验
将7-9周龄的雄性SD大鼠麻醉后脱颈,向腹腔内注射50mL预冷的MCM溶液(150mMNaCl,3.7mM KCl,3mM Na2HPO4,5.6mM葡萄糖,0.1wt.%牛血清白蛋白,0.1wt.%明胶,pH6.8),轻轻揉动腹部5min,收集腹腔液。在4℃低温条件下、800rpm离心5min,收集腹腔细胞沉淀。弃去上清后,用MCM溶液清洗细胞一次。将肥大细胞用MCM溶液稀释成1×105/mL的细胞悬液。
组胺释放抑制活性的检测步骤如下:取500μL细胞悬液,37℃孵化培养5min,加入4μL氯化钙溶液(250mM)。于37℃培养箱继续培养5min,加入5μL供试样品(1μg/μL),10min后加入5μL的Compound 48/80溶液(0.05mg/mL)刺激细胞,反应10min后冷却中止。在4℃条件下,6000rpm离心3min,收集上清液。取400μL上清液加20μL的60wt.%高氯酸除蛋白。室温放置20min后,添加400μL生理盐水,然后13000rpm离心3min,收集上清液。
上清液中组胺含量采用HPLC法测定:分析柱:TSKgel Histamine pak(4.0mm ID×6.0cm L);前处理柱:TSK precolumn his(4.0mm ID×6.0cm L);反应温度:45℃;流动相流速:0.6mL/min;前处理液流速:0.6mL/min;反应液流速:0.1wt.%邻苯二甲醛溶液0.2mL/min;4N NaOH溶液0.2mL/min;25wt.%H3PO4溶液0.3mL/min;检测波长EM:350nm、EX:460nm。HPLC条件参照自文献(Biological and Pharmaceutical Bulletin,2003,26(10):1393-1397)。
通过如下公式计算:组胺释放抑制率(%)=(1-(样品-空白))/(Comp48/80-空白)×100。
实施例3:不同蛋白酶对蜂王浆蛋白肽组胺释放抑制活性的影响
蛋白水解酶对底物蛋白具有特定的选择性,这是由蛋白酶的活性位点和底物蛋白的结构决定的,特定的氨基酸序列或结构域更易与酶发生相互作用。不同的蛋白酶会使得水解产物具有差异性,进而导致其生物活性的不同。因此,发明人筛选了制备蜂王浆蛋白肽的生物酶。
准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,调节pH为7.0,分别加入1%的中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、复合蛋白酶、风味蛋白酶进行水解,于40℃下恒温搅拌反应3h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
通过实施例2所述的方法分别评价所制备蜂王浆蛋白肽的组胺释放抑制活性,结果如表1所示。比较发现,蜂王浆蛋白本身具有组胺释放抑制活性,但活性非常微弱,抑制率仅为10.2%,而经蛋白酶水解后活性得到显著增强,表明酶解技术有利于释放蛋白中的活性肽段,提升其抑制组胺释放的活性。但不同蛋白酶制备的蜂王浆肽活性有所差异,碱性蛋白酶和复合蛋白酶制备的肽活性较强,其组胺释放抑制率可达到50%以上。
表1.不同蛋白酶对蜂王浆蛋白肽组胺释放抑制活性的影响
酶 | 组胺释放抑制率(%) |
中性蛋白酶 | 42.3 |
碱性蛋白酶 | 56.4 |
胃蛋白酶 | 35.4 |
胰蛋白酶 | 47.2 |
木瓜蛋白酶 | 29.6 |
菠萝蛋白酶 | 33.8 |
复合蛋白酶 | 50.7 |
风味蛋白酶 | 41.3 |
/ | 10.2 |
实施例4:不同酶用量对蜂王浆蛋白肽组胺释放抑制活性的影响
酶用量决定了蛋白水解反应的速率,更多的酶分子参与催化反应,能够提高底物与酶的碰撞频率。在低于最优酶用量时,反应速率可能受限,限制了反应的进行;而超过一定酶饱和点后,进一步增加酶用量对反应速率的增加效果不大。基于实施例3中碱性蛋白酶制备蜂王浆肽的最优活性,发明人优化了碱性蛋白酶的用量。
准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,调节pH为7.0,分别加入0.2%、0.5%、1.0%、1.5%、2.0%、2.5%、3.0%的碱性蛋白酶进行水解,于40℃下恒温搅拌反应3h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
发明人比较了不同碱性蛋白酶用量制备的蜂王浆蛋白肽的组胺释放抑制活性,结果如表2所示。当酶用量在0.2-2.0%范围内,随着酶用量的增加产物的活性也递增;但当酶用量大于2.0%时,产物的活性并没有得到进一步的提升。基于产物活性与工艺成本考虑,碱性蛋白酶的用量为1.0-2.0%较为合适。
表2.不同碱性蛋白酶用量对蜂王浆蛋白肽组胺释放抑制活性的影响
碱性蛋白酶(%) | 组胺释放抑制率(%) |
0.2 | 24.4 |
0.5 | 34.6 |
1.0 | 55.9 |
1.5 | 62.7 |
2.0 | 67.4 |
2.5 | 65.3 |
3.0 | 66.7 |
实施例5:不同温度对蜂王浆蛋白肽组胺释放抑制活性的影响
酶活性受到温度的影响,在一定温度范围内,温度增加有利于酶与底物的碰撞,进而加速催化反应。然而,超过最适温度范围,酶会发生热失活;而低温也不利于酶活性。因此,基于产物的组胺释放抑制活性,发明人优化了酶解反应的温度。
准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,调节pH为7.0,加入1%的碱性蛋白酶进行水解,分别于25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃下恒温搅拌反应3h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
发明人比较了不同温度下制备的蜂王浆蛋白肽的组胺释放抑制活性,结果如表3所示。当温度为25-50℃时,产物的组胺释放抑制率逐步提升至63.7%;当温度继续升高时,产物的活性反而有所降低,可能与高温破坏了酶的结构有关,降低了酶解的效率。因此,基于产物活性与能耗成本,酶解的温度采用40-50℃较为合适。
表3.不同温度对蜂王浆蛋白肽组胺释放抑制活性的影响
温度(℃) | 组胺释放抑制率(%) |
25 | 36.8 |
30 | 40.5 |
35 | 44.7 |
40 | 55.1 |
45 | 60.4 |
50 | 63.7 |
55 | 61.3 |
60 | 55.7 |
实施例6:不同pH对蜂王浆蛋白肽组胺释放抑制活性的影响
pH是影响酶活性的另一个关键因素。酶活性对pH值的变化非常敏感,因为酶分子的三维结构和电荷状态都与其活性密切相关。每种酶都有其最适pH值范围,超出最适pH范围,酶的活性将下降,降低酶解反应的效率与限度。因此,基于产物的组胺释放抑制活性,发明人优化了酶解反应的起始pH值。
准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,分别调节pH为6.0、6.5、7.0、7.5、8.0、8.5、9.0,加入1%的碱性蛋白酶进行水解,在40℃下恒温搅拌反应3h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
pH值对酶解产物组胺释放抑制率的影响如表4所示,pH在7.0-8.0之间可保证酶解产物较好的组胺释放抑制活性。
表4.不同pH对蜂王浆蛋白肽组胺释放抑制活性的影响
pH | 组胺释放抑制率(%) |
6.0 | 44.8 |
6.5 | 51.1 |
7.0 | 56.4 |
7.5 | 61.2 |
8.0 | 63.5 |
8.5 | 60.4 |
9.0 | 58.7 |
实施例7:不同酶解时间对蜂王浆蛋白肽组胺释放抑制活性的影响
酶解时间也会影响产物的组分与活性,随着反应时间的延长,更多的底物分子与酶发生反应,从而生成更多的产物,但可能会引起非特异性反应或副反应。因此,基于产物的组胺释放抑制活性,发明人优化了酶解反应的时间。
准确称取纯化的蜂王浆蛋白10g溶解到300mL去离子水中,搅拌均匀,调节pH为7.0,加入1%的碱性蛋白酶进行水解,在40℃下恒温分别搅拌反应1、2、3、4、5、6、7、8h。反应结束后于90℃下水浴15min使酶灭活,冷却至室温后10000rpm离心20min,收集上清液,冷冻干燥后即得蜂王浆蛋白肽。
酶解时间对产物组胺释放抑制率的影响如表5所示,组胺释放抑制率在3-5h内随着酶解时间的增加而增加,而更长的酶解时间不利于保持产物的活性。因此,酶解时间选定3-5h为较为合适。
表5.不同酶解时间对蜂王浆蛋白肽组胺释放抑制活性的影响
时间(h) | 组胺释放抑制率(%) |
1 | 31.8 |
2 | 40.7 |
3 | 54.8 |
4 | 57.2 |
5 | 63.2 |
6 | 61.4 |
7 | 57.3 |
8 | 56.5 |
实施例8:蜂王浆蛋白肽不同分子量片段的活性比较
蛋白质经过酶解后产生的复杂多肽具有不同的分子量,而这些肽段的活性也有差异。发明人将实施例1中获得的蜂王浆蛋白肽采用超滤膜进行分离,收集如下分子量片段的组分:<3kDa、3-5kDa、5-10kDa和>10kDa,冷冻干燥后即得相应分子量范围的生物活性肽。经活性评估发现,结果见表6,不同分子量蜂王浆肽的组胺释放抑制活性不同,分子量越小活性越强,分子量<3kDa的蜂王浆蛋白肽,有更强的组胺释放抑制活性,抑制率高达71.4%。
表6.不同分子量蜂王浆肽的组胺释放抑制率
分子量 | 组胺释放抑制率(%) |
<3kDa | 71.4 |
3-5kDa | 52.1 |
5-10kDa | 34.6 |
>10kDa | 29.5 |
未分离 | 55.7 |
实施例9:蜂王浆蛋白肽对致敏小鼠血浆中组胺水平的影响
为验证本发明所述蜂王浆蛋白肽的效果,发明人对实施例1制备蜂王浆蛋白与蜂王浆蛋白肽以及实施例8分离的分子量<3kDa的蜂王浆蛋白肽的体内活性进行了评估。
将40只小鼠随机分为对照组,模型组,蜂王浆蛋白组(200mg/kg),蜂王浆蛋白肽组(200mg/kg)与蜂王浆蛋白肽(<3kDa)组(200mg/kg),每组8只。除对照组外,各组小鼠于右耳皮内注射100mg的粉尘螨粗提浸液,一周2次,共注射4次。蜂王浆蛋白及其活性肽提前灌胃给药1周,每天1次,致敏实验期间维持给药,正常组和模型组给与等体积的生理盐水。给药结束后,麻醉小鼠心脏取血。
小鼠血浆中组胺的检测步骤如下:心脏取血置于抗凝剂处理的离心管中,室温4000rpm离心20min,收集上清血浆,使用小鼠组胺酶联免疫分析试剂盒检测小鼠血浆中组胺的水平。
结果如表7所示,致敏后小鼠血浆中的组胺水平显著增加,而蜂王浆蛋白、蜂王浆蛋白肽、蜂王浆蛋白肽(<3kDa)均显著降低了组胺的水平。而且,它们对组胺释放抑制活性的强弱依次为蜂王浆蛋白肽(<3kDa)>蜂王浆蛋白肽>蜂王浆蛋白,这与体外的结果相近。
表7.小鼠血浆中的组胺含量
组别 | 组胺(ng/mL) |
正常组 | 297.7±42.7 |
模型组 | 1135.7±118.4* |
蜂王浆蛋白 | 979.9±81.4# |
蜂王浆蛋白肽 | 744.6±103.7# |
蜂王浆蛋白肽(<3kDa) | 489.5±156.9# |
与正常组相比,*P<0.05;与模型组相比,#P<0.05。
实施例10:蜂王浆蛋白肽对致痒阈值的影响
将32只豚鼠随机分为正常组和实施例1制备蜂王浆蛋白肽100mg/kg、200mg/kg和400mg/kg三个剂量组,每组8只。给药组连续灌胃给药2周,每天1次,正常组给与等体积的生理盐水。给药结束后,各组小鼠均脱去右后足背毛,并用粗砂纸擦伤脱毛区,面积约1cm2,向创面滴加50μL的磷酸组胺溶液,质量浓度依次递增,分别为0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%等,梯度之间相隔3min,直至豚鼠回舔右后足,最后出现舔足时所用磷酸组胺的总量(μg)即为致痒阈值。
结果表8可知,本发明提供蜂王浆蛋白肽对磷酸组胺所致瘙痒有一定的抑制作用,可显著提高豚鼠的致痒阈值,且具有剂量依赖性。
表8.豚鼠的致痒阈
组别 | 致痒阈(μg) |
正常组 | 40.6±34.3 |
蜂王浆蛋白肽(100mg/kg) | 97.5±45.1 |
蜂王浆蛋白肽(200mg/kg) | 140.0±53.6* |
蜂王浆蛋白肽(400mg/kg) | 175.6±66.7* |
与正常组相比,*P<0.05。
实施例11:蜂王浆蛋白肽对豚鼠足底皮肤HRH1水平的影响
组胺受体类型1(histamine receptor H1,HRH1)广泛分布在中枢神经系统和外周组织中,在过敏反应中起着重要作用。抗组胺药物包括氯雷他定、苯海拉明和依巴斯汀等都属于选择性HRH1受体拮抗剂,它们能够结合到HRH1受体上,阻止组胺与该受体的结合。因此,发明人采用qPCR技术检测了豚鼠皮肤组织中HRH1受体的表达水平。结果如表9所示,与正常组相比,蜂王浆蛋白肽显著降低小鼠足部皮肤中HRH1基因的表达水平。
表9.豚鼠足部皮肤HRH1基因表达水平
与正常组相比,*P<0.05。
实施例12:蜂王浆蛋白肽活性序列分析评估
发明人通过LC-MS/MS质谱技术从分子量低于3kDa的蜂王浆蛋白肽鉴定了7个氨基酸序列(表10)。具体条件如下:赛默飞(Thermo Fisher Scientific)双三元高效液相色谱(DGLC)串联四极杆轨道阱高分辨质谱仪(Q-Exactive)进行LC-MS/MS分析。液相采用反相系统分离,以0.1%甲酸水为洗脱A相,乙腈为洗脱B相,色谱柱型号为:HSS T3 1.8μm,C18,100×2.1mm,Waters Acquity,洗脱程序如下:0-5.5min:5%-95% B;9.0-9.5min:95%-5%B;13.0min:5% B,流速为0.3mL/min,柱温为35℃,进样量2μL,质谱扫描模式为正离子,一级质谱扫描质量范围为150-1500m/z,分辨率为35,000,最大注射时间为100ms,MS2扫描分辨率为17,500,最大注射时间为50ms,碰撞能为35eV,毛细管喷雾电压为3.0kV,毛细管温度为320℃,辅助器加热温度为350℃,S-lens Rf设置为60。
发明人合成了7个氨基酸序列并评估了其组胺释放抑制作用。结果如表10所示,其中LPDEVSG和HYVPV的组胺释放抑制率可达80%以上,活性较强。
表10. 7个氨基酸序列的组胺释放抑制活性
氨基酸序列 | 组胺释放抑制率(%) |
LPDEVSG | 85.7 |
MK | 31.1 |
HYVPV | 80.4 |
ITTNP | 61.2 |
MNPPK | 55.9 |
YYRA | 64.1 |
LKA | 33.4 |
需要进一步说明的是,以上实施例仅用以说明本发明提供的技术方案,但本发明并不局限于以上实施例。因此,对上述实施例所记载的技术方案进行修改,或对其中部分技术特征进行等同替换,并不使相应技术方案的本质脱离本发明实施例所述技术方案的范围。
Claims (10)
1.一种蜂王浆蛋白肽,其特征在于,所述蜂王浆蛋白肽是由蜂王浆蛋白通过生物酶解获得,所述蜂王浆蛋白肽是一种安全有效的组胺释放抑制剂。
2.根据权利要求1所述的蜂王浆蛋白肽,其特征在于,所述蜂王浆蛋白肽是小分子蜂王浆肽,所述小分子蜂王浆肽的氨基酸序列为Leu-Pro-Asp-Glu-Val-Ser-Gly或His-Tyr-Val-Pro-Val,缩写为LPDEVSG或HYVPV。
3.根据权利要求1所述的蜂王浆蛋白肽的制备方法,其特征在于,所述制备方法包括如下步骤:
步骤1):通过碱溶酸沉法提纯蜂王浆蛋白;
步骤2):使用蛋白酶将蜂王浆蛋白水解为多肽;
步骤3):利用超滤技术分离不同分子量片段的蜂王浆蛋白肽。
4.根据权利要求3所述的蜂王浆蛋白肽的制备方法,其特征在于,步骤2)中所述蛋白酶选自以下一种或多种:中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、复合蛋白酶或风味蛋白酶,优选地,所述蛋白酶为碱性蛋白酶。
5.根据权利要求3所述的蜂王浆蛋白肽的制备方法,其特征在于,步骤2)中碱性蛋白酶的添加量以重量百分比计为1.0-2.0%;酶解温度为40-50℃;酶解pH为7.0-8.0;酶解时间为3-5小时。
6.根据权利要求3所述的蜂王浆蛋白肽的制备方法,其特征在于,步骤3)中采用超滤膜的截留分子量为<3kDa。
7.权利要求1或权利要求2所述的蜂王浆蛋白肽或者采用权利要求3至6中任一项所述的制备方法制备得到的蜂王浆蛋白肽在制备组胺释放抑制剂中的用途。
8.根据权利要求7所述的用途,其特征在于,所述组胺释放抑制剂为护肤品、功能性食品、保健品或药品。
9.根据权利要求7所述的用途,其特征在于,所述用途为预防或修复机体过敏反应及其并发症。
10.根据权利要求9所述的用途,其特征在于,所述过敏反应的症状选自以下一种或多种:平滑肌收缩、毛细血管扩大、通透性增强、腺体分泌物增多、过敏性鼻炎、支气管哮喘或荨麻疹。
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