CN117018169A - 一种预防呼吸道病毒感染的营养组合物制剂 - Google Patents
一种预防呼吸道病毒感染的营养组合物制剂 Download PDFInfo
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Abstract
本发明提供一种预防呼吸道病毒感染的营养组合物制剂,由乳铁蛋白和双歧杆菌组成,乳铁蛋白和双歧杆菌的配比为1g:0.25×1013CFU‑1g:0.14×1010CFU。本发明可有效拟制甲流H1N1病毒侵袭导致的炎症对人体的侵害,可长期服用,也可在感染后使用。制剂形态可以是液体,也可以是固体,可通过口服、喷雾、口含等多种形式服用,可有效预防和治疗甲流H1N1病毒引起的呼吸道感染疾病,无任何毒副作用。
Description
技术领域
本发明涉及包括类母乳成分功能活性成分乳铁蛋白(LF)和益生菌的组合物,具有预防或治疗H1N1病毒感染性呼吸道疾病的作用。
背景技术
流行性感冒(简称流感),是一种随季节规律发作的急性呼吸道传染病,主要由流感病毒引起,具有传播速度快速、传染性强和致病性重的特点。流感的病原体为正黏病毒科RNA病毒,根据基质蛋白抗原以及病毒核蛋白差异,分为甲、乙、丙型(或A、B、C型)3种,血凝素(HA)与神经氨酸酶(NA)是甲型流感病毒囊膜上的主要纤突蛋白。HA的变异没有新亚型的出现,多引起阶段性或季节性的小型流感;NA的变异则会有新亚型的产生,往往在防不胜防之时爆发传染迅速、波及范围极广的大型流感。甲型H1N1病毒是目前流行范围最广的一类病毒,这种病毒是一个三重排甲型流感病毒复合体,其中基因片段PB2和PA的母体为禽流感病毒,MP,NP,HA,NA和NS来源于猪流感病毒,参与重排的PB1是人流感病毒基因片段,由于甲型H1N1流感病毒集聚了禽流感、猪流感和人流感病毒的相关基因片段,在遗传物质上发生了质变,使其入侵宿主细胞的毒力和能力明显高于猪流感病毒亲本,引发传播迅速、致死率高的全球性大流感在所难免。
人类感染H1N1病毒后,病毒主要在呼吸道和肺中复制,流感病毒能够特异性地结合人类上呼吸道中的α-2,6-半乳糖唾液酸受体(SAα2和6Gal)和禽类细胞中的α-2,3-半乳糖唾液酸受体(SAα2和3Gal),引发病毒不断的转录、复制,造成流感在人类或禽类之间的传播。婴幼儿/儿童/孕产妇/老年人等易感人群在病毒感染后可引起严重的并发症和后遗症,对生命健康造成严重的威胁。
接种流感疫苗是目前预防流感的重要手段之一,但是流感疫苗尚有其不足之处,其一为物质限制,制备流感疫苗须能在鸡胚中高效复制方可,且用鸡胚生产流感疫苗的过程耗时长,操作复杂,无法满足流感爆发需求。其二是疫苗抗同亚型病毒感染效果较为显著,但对不同亚型病毒抗感染效果较差。抑制病毒复制的药物有奥司他韦、扎那米韦和金刚烷胺等;中药复方在提高机体免疫力,以增强自身对病毒的免疫应答能力方面一定疗效。药物制剂在临床上表现出不同程度的治疗效果,但大多数药物仍然存在药物安全问题和不良反应。特别是针对婴幼儿、儿童的有效抗病毒药物较少,且存在诸多不规范使用问题。医生在用药时对用药剂量、给药途径等进行严格把控,患儿家属也对药物使用保持谨慎态度。其中,IFN-a目前尚无雾化吸入用制剂,仅将注射用IFN-a作为雾化吸入制剂使用,该药品目前属于“超说明书用药”;奥司他韦不良反应较轻,但也会对胃肠道、肝脏、中枢神经系统、呼吸系统、血液、皮肤等发生不良副作用;阿比多尔主要不良反应包括恶心、腹泻、头晕和血清转氨酶增高等;利巴韦林具有严重的不良反应,由于其在红细胞内发生反应,主观严重不良反应是溶血性贫血,口服1-2周内幼儿/儿童可能会出现血红蛋白下降、红细胞下降、白细胞下降,严重影响孩子自身免疫力。
乳铁蛋白(Lactoferrin,LF)是一种多功能的铁结合糖基化球状蛋白,广泛存在于哺乳动物各种外分泌物中,如人乳、牛奶、唾液、眼泪、支气管、肠道等分泌物中,其中人初乳中LF含量最高,约7-9g/L,之后逐渐下降,人成熟乳中含量在1.5-4.0g/L之间。LF具有杀菌和抑菌、抗病毒、免疫调节、抗氧化、调节肠道菌群和促进双歧杆菌生长等功能。LF抗病毒的作用机制一般认为有两方面,一是LF可以通过抑制病毒与靶细胞结合,从而抑制病毒在细胞内的复制;另一方面是LF通过结合病毒受体如糖胺聚糖,特别是硫酸乙酰肝素(HS)的能力,LF与HS的结合避免了宿主细胞和病毒之间的首次接触,从而防止病毒感染。
使用益生菌是当前预防或治疗病毒的考虑的一种方案,且其在肠道病毒感染,如轮状病毒的预防或治疗中具有一定的疗效,得到临床验证。其在临床治疗肠道炎性疾病的分子和细胞机制主要涉及3个方面:1)益生菌可产生抗菌物质并竞争性与病原微生物粘附于肠上皮来阻止病原体在肠道的粘附和定植;2)益生菌可提高肠上皮细胞的存活率和肠上皮的屏障功能并刺激保护性反应来调节肠上皮细胞的动态平衡;3)益生菌可提高天然免疫并通过TLR信号途径调节病原体引起的炎性反应。而关于益生菌在呼吸道疾病的预防和治疗也有一些报道,但作用机制尚不明确。
发明内容
本发明的主要目的是筛选一种安全有效,可长期食用的包括类母乳功能活性成分乳铁蛋白(LF)和可长期食用的益生菌组合,可以有效预防或治疗H1N1病毒感染性呼吸道疾病。
为实现上述目的,本发明的实施方案为:一种预防呼吸道病毒感染的营养组合物制剂,由乳铁蛋白和双歧杆菌组成,乳铁蛋白和双歧杆菌的配比为1g:0.5×1015CFU-1g:0.5×108CFU。
作为优化的实施方式,所述乳铁蛋白和双歧杆菌的配比优选为1g:0.25×1013CFU-1g:0.14×1010CFU。
作为优化的实施方式,所述双歧杆菌进一步优选为动物双歧杆菌乳亚种Bb-12。
作为具体的实施方式,所述营养组合物制剂可单独作为普通食品或营养补充剂。
作为具体的实施方式,所述营养组合物制剂也可作为营养强化剂组合应用于普通食品、婴幼儿配方奶粉、保健食品;
作为具体的实施方式,所述营养组合物制剂可与药学上可接受的载体、赋形剂,制成治疗呼吸道病毒感染的药品。
作为具体的实施方式,所述益生菌组合物产品形态可以是液体,也可以是固体。
本发明可有效拟制甲流H1N1病毒侵袭导致的炎症对人体的侵害,可长期服用,也可在感染后使用。制剂形态可以是液体,也可以是固体,可通过口服、喷雾、口含等多种形式服用,可有效预防和治疗甲流H1N1病毒引起的呼吸道感染疾病,无任何毒副作用。
附图说明
图1:不同浓度活性蛋白和益生菌对HEP2细胞毒性;
图2:9个不同剂量组合下对2种炎症因子的抑制作用比较;
图3:9个组合对3种炎症因子的抑制作用比较。
具体实施方式
实施例1:一种预防呼吸道病毒感染的营养组合物制剂,由乳铁蛋白和双歧杆菌组成,乳铁蛋白和双歧杆菌的配比为1g:0.25×1013CFU-1g:0.14×1010CFU,双歧杆菌优选为长双歧杆菌长亚种BB536。
实施例2:一种预防呼吸道病毒感染的营养组合物制剂,由乳铁蛋白和双歧杆菌组成,乳铁蛋白和双歧杆菌的配比为最佳比例为1g:0.4×1012CFU,双歧杆菌动物双歧杆菌乳亚种Bb-12。
实施例3:将乳铁蛋白和双歧杆菌添加到婴幼儿奶粉中,乳铁蛋白和双歧杆菌的配比为1g:0.25×1013CFU-1g:0.14×1010CFU,乳铁蛋白和双歧杆菌的添加量分别>0.04%和106CFU/100g。
实施例4:将乳铁蛋白和双歧杆菌安配比1g:0.25×1013CFU-1g:0.14×1010CFU与药学上可接受的载体(如淀粉等)压制成可预防和治疗呼吸道病毒感染的片剂。也可与多孔淀粉等壁材制成胶囊剂。
实施例
为筛选上述营养组合物制剂的组合原料和配比,验证拟制流感病毒H1N1效果,本发明进行了深入研究。
本发明中的制剂组合包括一种乳源乳铁蛋白或利用酶法、微生物发酵等合成生物学手段制备获得的乳铁蛋白;
益生菌选自双歧杆菌属,包括青春双歧杆菌、动物双歧杆菌动物亚种、动物双歧杆菌乳亚种、两歧双歧杆菌、短双歧杆菌、长双歧杆菌长亚种、长双歧杆菌婴儿亚种一种或多种,进一步优选为长双歧杆菌长亚种BB536,优选为动物双歧杆菌乳亚种Bb-12。
2.1细胞和病毒
细胞株(HEP-2)购自ATCC。甲型H1N1流感病毒是由中国疾病预防控制中心提供,-80 ℃冷藏。
2.2 实验原料与试剂
实验原料:乳铁蛋白LF, 益生菌Bb-12和益生菌BB536。称取各物质适量,用DMSO溶解成20 mM的母液。H1N1流感病毒液鸡胚传代2次,测定病毒效价为2-7。
96孔板购自 Corning Costar(Cambridge, MA, USA)公司;DMEM培养基和胎牛血清(fetal bovine serum, FBS)购自美国 Gibco 公司;双抗(10,000 U/mL 青霉素-10,000g/mL 链霉素)(10,000 U/mL Penicillin-10,000 U/mL Streptomycin))购自北京索莱宝生物科技有限公司。磷酸盐缓冲液(phosphate buffered saline, PBS)、 二甲基亚砜(dimethyl sulfoxide, DMSO)购自美国 Sigma 公司。
细胞培养液:DMEM培养基含10%FBS和1%双抗;
细胞维持液:DMEM培养基含2%FBS和1%双抗。
2.3 仪器
细胞培养箱;光学显微镜;超净工作台,水浴锅,RT-PCR检测仪,Western Blot检测系统。
2.4 实验方法
1) H1N1流感病毒TCID50的测定
用不含血清的DMEM培养基对流感病毒H1N1冻存液进行10倍递次稀释,将稀释好的各个浓度的病毒液依次接种到铺满单层细胞的96孔板中,100 μL/孔,每个浓度6复孔,设正常细胞对照组。将加入病毒液的96孔板在37 ℃,5 %的CO2培养箱中吸附2 h,之后用细胞维持液替换掉病毒稀释液,再置于培养箱中继续培养48 h后观察细胞形态,记录孔数和病变程度,当细胞对照组接近于正常形态,被病毒感染的细胞病变率≥50%的孔作为病变孔,按Reed-Muench法计算病毒的半数细胞培养物感染量(TCID50)。
2)营养物质对细胞毒性实验
将HEP-2细胞复苏,传代3-4次待细胞生长良好,胰酶消化,用完全培养基将细胞的浓度稀释为1.5×105个/mL,吹打混匀后接种于96孔板,100 μL/孔,37 ℃、5% CO2培养箱中孵育24 h。用培养液将待测物分别连续对倍递次稀释(最大浓度200 μM),将各浓度加入96孔板,100 μL/孔,每个浓度3复孔,设空白对照组。放入37 ℃,5 % CO2培养箱中持续培养48h,观察细胞形态,弃上清,用PBS将CCK-8配成10 %的溶液,每孔加入100 μL,作用1 h后用酶标仪于540 nm波长处测定细胞吸光度,根据公式:细胞存活率% = 给药组的吸光度值(A)/细胞对照组吸光度值(A)×100 %计算细胞存活率。
3)营养组合物抗H1N1病毒有效率检测
设置正常细胞对照(阴性对照组)、流感病毒对照组(阳性对照组)和给药组。取已长成单层细胞的培养板,吸除培养液,加入100 TCID50的病毒液(流感病毒对照组和给药组)或培养基(正常组),100 µL/孔,在37ºC、5% CO2培养箱中吸附4 h后,将流感病毒液吸除,加入适合剂量的化合物各100 µL,每浓度3个复孔,重复3次。48 h后观察细胞形态,弃上清,用PBS将(CCK-8试剂盒)试剂配成10 %的溶液,每孔加入100 μL,作用1 h后用酶标仪于540 nm波长处测定细胞吸光度,根据公式:抑制率(%)= (药物平均A值−病毒对照组平均A值) /(细胞对照平均A值−病毒对照组平均A值) ×100 %计算药物对病毒致细胞的病变抑制率。
4)营养组合物抗H1N1病毒机制研究
设置正常细胞对照(阴性对照组)、流感病毒对照组(阳性对照组)和给药组。取已长成单层细胞的培养板,吸除培养液,加入100 TCID50的病毒液(流感病毒对照组和给药组)或培养基(正常组),1.0 mL/孔,在37ºC、5% CO2培养箱中吸附4 h后,将流感病毒液吸除,加入适合剂量的实验原料或营养组合物制剂各1.0 mL,每浓度3个复孔,重复3次。48 h后取上清用ELISA试剂盒检测炎症因子肿瘤坏死因子-α(TNF-α,)、诱导型一氧化氮合成酶(iNOS)和白细胞介素-6(IL6)的含量。
设置正常细胞对照(阴性对照组)、流感病毒对照组(阳性对照组)和给药组。取已长成单层细胞的培养板,吸除培养液,加入100 TCID50的病毒液(流感病毒对照组和给药组)或培养基(正常组),1.0 mL/孔,在37ºC、5% CO2培养箱中吸附4 h后,将流感病毒液吸除,加入实验原料或营养组合物制剂各1.0 mL,每浓度3个复孔,重复3次。48 h后吸除上清液,用预冷PBS洗细胞两次,Trizol提取总RNA,逆转录后采用实时荧光定量PCR检测炎症因子TNF-α, iNOS和IL6的mRNA表达量。
本研究中首先考察了几种营养物质对HEP2细胞的毒性,结果如图1所示。从图中可以看出两种益生菌Bb-12和BB536在剂量为2000 μg/mL时,细胞的存活率小于80 %,1000 μg/mL时,细胞的存活率接近80 %,说明2000 μg/mL这个剂量对细胞具有一定的毒性,1000 μg/mL的细胞毒性次之。而功能活性蛋白乳铁蛋白浓度达到2000μg/mL时细胞存活率仍接近于100%,这说明乳铁蛋白对细胞无毒害;同时结合图1可以看出营养物质的半数毒性浓度(TC50)均大于给药的最大浓度,进一步说明这些营养物质对细胞的存活率几乎没有影响,具有足够的安全性。结合上述结果,在药物抗病毒有效率的检测实验中,益生菌和乳铁蛋白的最大浓度设置为2000 μg/mL,其中益生菌的活度为0.2×1012CFU/g,故益生菌的最大浓度也即0.4×109CFU/mL.
进一步对两种益生菌和乳铁蛋白在抑制病毒致细胞病变方面的作用进行了研究,结果发现益生菌Bb-12对H1N1流感病毒致HEP-2细胞病变具有明显的抑制作用,IC50值为68.63±22.48μg/mL。而益生菌BB536和乳铁蛋白的IC50值均大于1000μg/mL,二者对病毒感染的抑制作用初见不明显。进一步再次对不同浓度营养物质单体和组合物的抑制H1N1病毒感染的抑制率分析,结果如表1所示。从表中可以看出,
LF在低浓度(50μg/mL)和高浓度(200μg/mL)下对病毒的抑制率分别为25%和30%,抑制作用不明显;BB536在低浓度(40μg/mL)和高浓度(160μg/mL)时对病毒的抑制率分别为20%和35%,抑制效果同样不明显;Bb-12在低浓度(40μg/mL)和高浓度(160μg/mL)时对病毒的抑制率分别为60%和75%,抑制效果在高浓度时较好。之后将活性蛋白和两种益生菌分别组合后发现BB536和LF的组合抑制率仅达35%,而LF高浓度和Bb-12高浓度进行组合后发现抑制率可达85%,比两种单体在高浓度的情况下的抑制率更高,初步表现出二者的协同作用。
表1功能营养成分组合物对病毒致细胞病变的抑制率
基于前期的初步研究,再对LF和Bb-12抑制H1N1流感病毒的作用机制进一步研究,具体实施方案设计如表2所示。
表2 不同剂量营养组合物抑制H1N1流感病毒实验设计方案
3.2 检测方法及检测结果
流感病毒侵染机体细胞后,会通过Toll 样受体(TLR)通路传导激活NF-κB通路,引起炎症反应并导致细胞的凋亡。TLR 激活刺激抗病毒应答,并可能导致促炎介质的大量分泌。在炎症诱导的免疫反应中,促炎细胞因子TNF-α、IL- 6等起着关键的调节作用。肿瘤坏死因子-a(TNF-α)是由多种细胞在炎症反应和免疫调节刺激时产生的一种多效性细胞因子,可以诱导细胞调亡。IL-6是急性期反应的强激活剂,有助于全身和局部炎症反应。过量的IL-6可诱发多种慢性炎症疾病。NO自由基在炎症和免疫反应中同样至关重要。它由NOS(eNOS)和iNOS等酶通过l -精氨酸途径合成。在正常生理条件下,休眠细胞内iNOS处于休眠状态。然而,在病理条件下,它产生大量的NO,并在慢性感染、炎症中发挥双重作用。减少NO的产生可能是治疗多种炎症性疾病的有效策略。本研究首先利用ELISA的方法对两种促炎因子的生成进行了检测,并且进一步采用PCR对炎症因子在基因水平上的的mRNA的表达量进行再次确定。
从表3中可以看出正常对照组中促炎因子TNF-a、IL-6和iNOS的量分别为59.67±6.67pg/mL、246.39±8.24pg/mL和2.87±0.09pg/mL,而经过病毒侵染后的模型组中三者的量分别达到了144.11±1.92pg/mL、417.62±6.46pg/mL和5.14±0.25pg/mL,三种炎症因子均有显著升高(如表4所示),说明造模成功。通过营养物质LF和益生菌Bb-12低中高不同剂量干预处理发现促炎因子的量均有降低(表4):差异分析后发现两种营养物质的6个不同浓度下促炎因子IL-6和iNOS均比模型组显著(p<0.05)或极显著降低(p≤0.001);而对于TNF-a的影响,结果发现益生菌Bb-12在三个剂量下均比模型组显著(p<0.05)或极显著(p<0.0001)降低,而LF在低浓度时FL-L组比模型组有显著降低(p<0.05),中剂量组LF-M和高剂量组LF-H比模型组有降低,但差异不显著(p>0.05),这说明LF单体在抑制H1N1病毒引起的细胞炎症发生方面效果不显著。但将两种营养物质以不同剂量搭配组合后形成的9个组合中三种炎症因子TNF-a、IL-6和iNOS均比模型组产生了极显著差异(p<0.0001)。这说明益生菌Bb-12和LF组合后可以起到显著抑制H1N1病毒致细胞产生产生炎症风暴的作用。
表3 营养物质及其不同组合在抗H1N1病毒过程中炎症因子生成量
同时本研究进一步地比较了9个组合物抗炎症因子产生的功效,结果发现9个组合使得iNOS的产生量均未达到对照组的水平(p<0.05),但在炎症因子TNF-a和IL-6的产生过程中,LF以中和高剂量与Bb-12的低、中、高剂量分别组合后均可达到对照组的水平(p>0.05),而在IL-6的产生中,两种营养物质的高剂量组合LF-BB12(HH)组可达到对照组的水平(p>0.05),结果如图2所示。这说明LF-BB12(HH)组合具有最佳的抑制H1N1甲流病毒引起的上呼吸道感染的作用。
进一步地,本研究采用PCR检测对炎症因子的mRNA的表达量进一步分析,以验证这一结果,结果如图3所示。从图中可以看出,LF-BB12(HH)组的三个炎症因子TNF-a、IL-6和iNOS的mRNA表达量最低,经过差异分析后对于TNF-a因子,除了LF-BB12(LM)组、LF-BB12(LH)组和LF-BB12(MM)组与LF-BB12(HH)组无显著差异(p>0.05)以外,其余几个组均显著低于LF-BB12(HH)组(p<0.05);而对于IL-6,除了LF-BB12(MM)和LF-BB12(HL)组与LF-BB12(HH)组无显著差异(p>0.05)以外,其他几个组均显著低于LF-BB12(HH)组(p<0.05);而对于iNOS,除了LF-BB12(LH)组与LF-BB12(HH)组无显著差异(p>0.05)以外,其余几个组均显著低于LF-BB12(HH)组(p<0.05)。
因此,本研究认为乳铁蛋白LF和益生菌Bb-12在高剂量组合(LF/Bb-12为1g:0.4×1012CFU)下具有最优的抑制H1N1病毒致细胞感染产生的炎症风暴,进而抑制病毒对人体造成的伤害。研究结果发现使用两种营养制剂干预经过H1N1病毒侵染后的细胞,三种炎症因子的生成量和基因水平上的mRNA的表达量均显著降低,说明该组合物不仅安全,对人体无任何毒副作用外,还可有效抑制甲流H1N1病毒感染人体后引发的炎症爆发对人体的严重伤害。
Claims (7)
1.一种预防呼吸道病毒感染的营养组合物制剂,其特征在于,由乳铁蛋白和双歧杆菌组成,乳铁蛋白和双歧杆菌的配比为1g:0.5×1015CFU-1g:0.5×108CFU。
2.根据权利要求1所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,所述乳铁蛋白和双歧杆菌的配比为1g:0.25×1013CFU-1g:0.14×1010CFU。
3.根据权利要求1或2所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,所述双歧杆菌进为动物双歧杆菌乳亚种Bb-12。
4.根据权利要求1所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,可单独作为普通食品或营养补充剂。
5.根据权利要求1所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,可作为营养强化剂组合应用于普通食品、婴幼儿配方奶粉、保健食品。
6.根据权利要求1所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,可与药学上可接受的载体、赋形剂,制成治疗呼吸道病毒感染的药品。
7.根据权利要求1所述的预防呼吸道病毒感染的营养组合物制剂,其特征在于,其产品形态可以是液体,也可以是固体。
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