CN108450939A - 一种具有延缓肌肉衰减、增强免疫力功效的组合物 - Google Patents
一种具有延缓肌肉衰减、增强免疫力功效的组合物 Download PDFInfo
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Abstract
一种具有延缓肌肉衰减、增强免疫力功效的组合物,涉及营养食品领域。组成:浓缩乳清蛋白10‑40;中链甘油三酯粉(MCT粉)15‑30;伊代欣糖10‑30;低聚果糖10‑30;小麦低聚肽粉1‑6;聚葡萄糖1‑30;水苏糖3‑10;复合矿物质1‑10;燕麦纤维粉0.5‑5;圆苞车前子壳粉1‑10;复合维生素0.1‑5;苹果纤维粉1‑10;三氯蔗糖0‑1;雪莲培养物0.001‑1;将上述配方原料粉碎成粉末,然后均匀混合制成。本发明具有改善人体肌肉状况,增强机体免疫力,改善肠道菌群和促进胆固醇代谢的功能,可以作为肌肉减少症人群的营养补充;同时产品含有均衡配比的膳食纤维,也是一款优秀的改善便秘的营养食品。
Description
技术领域
本发明涉及营养食品领域,具体涉及一种具有延缓肌肉衰减、增强免疫力功效的组合物。
背景技术
一般而言,在30岁以后,如果不常运动锻炼,人体肌肉量每年会减少1%-2%;60岁之后肌肉加速衰减。这种与增龄相关的进行性骨骼肌减少、伴随肌肉力量和(或)肌肉功能减退的症状,其实质是肌纤维数量减少及肌细胞体积缩小,会导致肌肉衰减人群生活质量下降,增加跌倒、失能、关节炎、骨质疏松和糖尿病等风险。
蛋白质是构成肌纤维的物质基础。随着年龄的增长,体内蛋白酶活性逐渐降低,导致食物蛋白的吸收利用率降低。此外,老年人群中患有三高等慢性疾病的比例较高,老年人饮食普遍过于清淡,膳食蛋白摄入不足,尤其是优质动物蛋白。综上,老年人机体的蛋白质代谢以分解代谢为主,易出现负氮平衡。一般认为,老年男性每人每天75克,女性为65克,其中优质蛋白质应占总膳食蛋白质的50%以上,同时还应补充维生素D、维生素C及钙、锌等矿物质。
目前,市场上蛋白类营养补充产品琳琅满目,如乳清蛋白粉、支链氨基酸和肌酸等,但大部分产品是为健身爱好者设计,并不是针对易出现肌肉衰减的中老年人群。
发明内容
本发明为克服上述问题,提供一种具有延缓肌肉衰减、增强免疫力功效的组合物。
本发明采用如下技术方案:一种具有延缓肌肉衰减、增强免疫力功效的组合物,由以下重量份数比的配方原料组成:浓缩乳清蛋白10-40;MCT粉15-30;伊代欣糖10-30;低聚果糖10-30;小麦低聚肽粉1-6;聚葡萄糖1-30;水苏糖3-10;复合矿物质1-10;燕麦纤维粉0.5-5;圆苞车前子壳粉1-10;复合维生素0.1-5;苹果纤维粉1-10;三氯蔗糖0-1;雪莲培养物0.001-1;
制备方法:将所有原料加入到卧式搅拌机中,混合均匀后即得到本发明。
在使用时,取本品20-30g,直接加入牛奶,豆浆或温水(60℃左右),搅拌均匀后即可饮用,简单方便。
注:(1)不适宜人群:婴幼儿、孕妇;
(2)本食品摄入量≤150g/d;
服用时间:本食品主要效果是延缓肌肉衰减,因此辅以抗阻运动效果更佳。本品推荐服用时间为抗组运动前后30min左右。
本发明的食品可以针对不同人群的不同需求,组合配伍,达到延缓肌肉衰减、增强免疫力的效果。
本发明的组合物有较好的提高特异性和非特异性免疫调节作用;可以促进胆固醇代谢,阻碍其在机体内吸收利用;可以有效的改善肠道益生菌群数,提高益生菌的黏附能力;能够通过提高肌小管形成和提高结蛋白、肌球蛋白、生肌素和成肌分化抗原来刺激肌前体细胞向成熟肌细胞分化,减少成熟时间和细胞数量来达到增肌的效果。下面通过实验数据对本发明的有益效果进行阐述。
本发明的用法用量:在使用时,取本发明产品20-30g,直接加入牛奶,豆浆或温水(60℃左右),搅拌均匀后即可饮用,简单方便。注:(1)不适宜人群:婴幼儿、孕妇;(2)本食品摄入量≤150g/d。
服用时间:本发明主要效果是延缓肌肉衰减,因此辅以抗阻运动效果更佳。本品推荐服用时间为抗阻运动前后30min左右。本发明的人群试验课题,在本市挑选了100例年龄集中在60-80岁的老年人,身体偏瘦,随机分成A、B两组,每组50人。A组为对照组,正常的吃早餐,加有一份米粥;B组正常吃早饭后,使用本产品25g(热量约等于一份米粥)温水冲饮代替米粥。早饭后30-60min内,A组和B组根据个人身体状况和意愿,做一定强度的抗阻运动,如弹力带,液压循环训练器等。一个月后,体重变化情况见表1.
表1
附图说明
图1是本发明对ConA(刀豆素)/LPS(脂多糖)诱导的小鼠T/B淋巴细胞增殖的影响表;
图2是本发明对小鼠腹腔巨噬细胞吞噬中性红能力的影响表;
图3是本发明对LPS(脂多糖)诱导的小鼠腹腔巨噬细胞吞噬中性红能力的影响表;
图4是本发明对HepG2细胞增殖的影响表;
图5是本发明对CYP7A1基因表达表;
图6是本发明对Caco-2细胞增殖的影响表;
图7是本发明对三株肠道菌群粘附的影响表;
图8是本发明对C2C12细胞肌小管分化形成的影响表;
图9,是本发明对C2C12细胞结蛋白和成肌分化抗原的影响表;
图10是本发明对C2C12细胞生肌素和肌球蛋白的影响表。
具体实施方式
下面用最佳的实施例对本发明做详细的说明。
一种具有延缓肌肉衰减、增强免疫力功效的组合物,由以下重量份数比的配方原料组成:浓缩乳清蛋白10-40;MCT粉15-30;伊代欣糖10-30;低聚果糖10-30;小麦低聚肽粉1-6;聚葡萄糖1-30;水苏糖3-10;复合矿物质1-10;燕麦纤维粉0.5-5;圆苞车前子壳粉1-10;复合维生素0.1-5;苹果纤维粉1-10;三氯蔗糖0-1;雪莲培养物0.001-1;将所有原料加入到卧式搅拌机中,混合均匀后即得到本发明。
复合矿物质包括L-乳酸钙、硫酸镁、硫酸亚铁、乳酸锌。
复合维生素包括维生素A、维生素D、维生素E、维生素B1、维生素B2、维生素B6、维生素B12、维生素C、烟酸、叶酸、泛酸。
本发明的实验报告:
1、增强免疫力的研究
免疫系统的紊乱不仅会产生多种疾病,而且与人体衰老、肿瘤、高血压、糖尿病等疾病的发生均有密切关。本发明研究了不对Balb/c小鼠脾淋巴细胞增殖活性的调节作用,对小鼠腹腔巨噬细胞吞噬活性以及NO生成量等免疫调节功能的影响。从细胞免疫、体液免疫和非特异性免疫三方面综合评价本发明的免疫调节作用。
1.1试验方法
1.1.1小鼠脾细胞悬液的制备
将小鼠(Balb/c)拉颈处死,于75%乙醇中浸泡5min进行消毒处理,无菌分离完整脾脏,用PBS(磷酸盐缓冲液)冲去浮血,剥除结缔组织及脂肪成分。注射器吸取约5mLPBS(磷酸盐缓冲液),轻轻插入脾内吹出细胞,重复操作数次至脾外膜透明,剩余脾组织剪成大小1mm 3小块,剪碎后置于小烧杯上的200目不锈钢滤网上,用注射器针芯研磨,PBS(磷酸盐缓冲液)液洗涤,收集冲洗液至离心管,1200rpm离心5min,弃上清液,加入3mL红细胞裂解液,静置2min,加入10mL PBS(磷酸盐缓冲液)终止反应,离心(1200rpm,5min)弃上清,用PBS(磷酸盐缓冲液)液洗两次,离心(1200rpm,5min),用RPMI-1640不完全培养基洗一次,离心(1200rpm,5min),弃上清。加适量RPMI-1640完全培养基(含10%胎牛血清、100U/mL青霉素、100μg/mL链霉素和10mMHepes)。调整细胞浓度至2×106个细胞/mL,台盼兰染色检测细胞存活率大于95%。
1.1.2T/B淋巴细胞增殖实验
将脾细胞悬液(2×106个细胞/mL)按100μL/孔加入到96孔培养板中,再添加10μLConA/LPS(终浓度为10μg/ml)或不添加ConA(刀豆素)/LPS(脂多糖),10μL样品溶液,每个浓度做6次重复,将培养板置于37℃、5%CO2的培养箱中培养72h。
正常对照组:100μL脾细胞悬液+10μL完全培养基+10μLPBS(磷酸盐缓冲液);
模型组:100μL细胞悬液+10μLConA(终浓度为10μg/mL)+10μLPS(脂多糖);
样品组:100μL细胞悬液+10μLConA/LPS(终浓度为10μg/mL)/10μLPBS(磷酸盐缓冲液)+10μL样品。
本实验选用CCK-8试剂盒(碧云天)测定T/B淋巴细胞增殖影响,结果用刺激指数(SI)表示,计算如下:
1.1.3小鼠腹腔巨噬细胞的制备
Balb/c小鼠,于实验前3天腹腔注射1.5mL4%肉汤淀粉溶液。拉颈处死小鼠,于无菌工作台上将其腹部皮肤剥离,用一次性注射器向腹腔内注入RPMI-1640不完全培养液4mL,轻柔腹部2~3min,用一次性注射器取其腹部液体,反复2~3次。1200rpm离心8min收集细胞,洗涤2次,在倒置显微镜下进行细胞计数,用含10%灭活胎牛血清的RPMI-1640培养液调整细胞密度达1×106个/mL,加入96孔细胞培养板中,每孔100μL,将细胞培养板放入37℃、5%CO2饱和湿度培养箱中培养2.5h,吸弃上清,用预温培养基洗去未贴壁细胞,再加入含10%灭活胎牛血清的RPMI-164培养液,制备96孔板巨噬细胞单层。每只小鼠可产生2~3×106细胞,其中90%为巨噬细胞。
1.1.4小鼠腹腔巨噬细胞体外吞噬活性和NO生成量的测定
阳性对照组每孔加入10μL LPS,使其终浓度达30μg/mL,试验组每孔加入10μL样品,试验组分为LPS与提取物共同作用组和提取物单独添加组两种。阴性对照组、阳性对照组和试验组各设6个复孔,于37℃、5%CO2饱和湿度培养箱内培养。培养24h后,收集上清液用于NO生成量的测定。
培养24h后,用预温的无血清培养液洗涤细胞3次,每孔加入200μL预温的0.1%中性红溶液,于37℃,5%CO2培养箱内继续培养1h;倾去上清液,用预温RPMI-1640培养液清洗三遍,洗去未被吞噬的中性红;每孔加入200μL细胞溶解液(含1%(v/v)乙酸的50%(v/v)乙醇溶液),室温下放置2~3h,待细胞溶解后,用酶标仪测定OD 550值。吞噬指数(Pinocytosis index,PI)计算如下:
0.1%中性红的配置:称取0.1g中性红,溶于RPMI 1640不完全培养基中,置37℃培养箱中放置过夜,0.22μm过滤除菌。4℃避光保存。
1.2实验结果
脾脏是人体重要的免疫器官,经常采用小鼠脾细胞悬液制备淋巴细胞,利用有丝分裂原ConA(刀豆素)和LPS(脂多糖)激活免疫细胞,诱导增殖反应。其中ConA(刀豆素)的靶细胞为T淋巴细胞,可以和细胞膜表面受体结合而活化细胞,诱导IL-2(白介素)的生成和T淋巴细胞增殖反应;LPS(脂多糖)的靶细胞为B淋巴细胞和巨噬细胞。通过适量的ConA(刀豆素)和LPS(脂多糖)作用于免疫细胞,主要用来了解样本对于激活的或抑制的免疫细胞功能的影响。
参见图1,是本发明对ConA(刀豆素)/LPS(脂多糖)诱导的小鼠T/B淋巴细胞增殖的影响表。本发明在六个作用浓度可以促进小鼠脾细胞的增殖,与模型相比,无显著性差异。说明本发明在这六个浓度作用下,对小鼠脾细胞无毒副作用,可以进行后续试验。
淋巴细胞在有丝分裂原如ConA(刀豆素)、LPS(脂多糖)等的刺激下,其形态和代谢会发生一系列的改变,转化为母细胞并分化增殖。其中ConA(刀豆素)刺激T细胞增殖,LPS(脂多糖)刺激B细胞增殖。因此根据非特异性促有丝分裂原激活T和B细胞增殖反应的程度,可推测T和B细胞识别特异性抗原增殖反应。本发明对ConA(刀豆素)诱导的T-淋巴细胞增殖影响见表1。与模型对照组相比,六个作用浓度对小鼠脾T-淋巴细胞增殖均有促进作用。本发明浓度≥400μg/ml时,本发明较模型组具有显著的促进ConA(刀豆素)诱导的T-淋巴细胞增殖作用。在低浓度时,除100μg/ml外其他低浓度对T淋巴细胞增殖具有促进作用,但无显著性差异。随着作用浓度的增加,ConA(刀豆素)诱导的T淋巴细胞增殖的抑制作用呈明显的量效关系。
本发明对B-淋巴细胞增殖的抑制作用与T-淋巴细胞增殖的抑制作用趋势基本一致显,浓度≥200μg/mL时具有显著的促进作用,低浓度时无显著性差异。随着作用浓度的增加,LPS(脂多糖)诱导的B淋巴细胞增殖的抑制作用呈明显的量效关系。
机体的免疫系统分为非特异性免疫系统和特异性免疫系统,非特异免疫系统在机体抵抗疾病、消除致病因子、维持内环境稳态中起着重要作用。单核巨噬细胞系统是机体非特异性免疫系统的重要组成部分,该系统能迅速清除多种致病物质,从而维持机体内环境的稳态。巨噬细胞是单核巨噬细胞系统的主要成员,在机体免疫防御、自身稳定和免疫监视中都起着重要的作用。巨噬细胞具有吞噬和直接杀伤多种病原微生物、清除凋亡细胞和突变细胞、处理和提呈抗原,其分泌细胞因子在先天性免疫防御和获得性免疫应答中起着不可忽视的作用。
巨噬细胞有3种活化状态:静息状态、致敏状态和亢奋(活化)状态。当机体或局部未受到病原体等的刺激时,巨噬细胞常处于静息状态,胞体较小,细胞器较不发达,几乎不运动,但寿命较长而更新率低。在炎症或其它因子的刺激下,巨噬细胞从静息转向活化,细胞增大,代谢增强,溶酶体增多,细胞的变形运动及吞噬能力均增强。经LPS诱导后,小鼠腹腔巨噬细胞被激活,吞噬中性红的能力显著增加,说明造模成功。
研究发现,本发明浓度除200μg/mL外,其他作用浓度下均可以促进活化后巨噬细胞吞噬中性红能力;在800μg/mL时,具有显著地促进作用,PI值高达:1.26(P<0.05)。参见图2,是本发明对小鼠腹腔巨噬细胞吞噬中性红能力的影响表。
当用LPS(脂多糖)激活巨噬细胞时,对照组NO合成量可达未被激活的巨噬细胞的4~6倍,说明经诱导后的巨噬细胞处于激活状态。除400μg/mL和800μg/mL外,其他四个作用浓度对激活后巨噬细胞NO生成作用与空白对照比较变化不明显。本发明浓度在400μg/mL和800μg/mL时显著促进活化的巨噬细胞生成NO。此结果与LPS(脂多糖)诱导后巨噬细胞吞噬中性红能力趋势基本保持一致,说明活化后的腹腔巨噬细胞吞噬能力与NO生成量呈正相关。参见图3,是本发明对LPS(脂多糖)诱导的小鼠腹腔巨噬细胞吞噬中性红能力的影响表。
2.降胆固醇活性的研究
高胆固醇血症是动脉粥样硬化及心血管疾病的危险因子,对人类身心健康和生命造成很大威胁,改善高胆固醇血症的最好办法是抑制肝脏胆固醇合成和促进胆固醇代谢。本发明调节肝脏中胆固醇代谢酶的作用,阻碍胆固醇的吸收,起到降低血液胆固醇的作用。
2.1试验方法
2.1.1细胞培养
使用含10%胎牛血清的DMEM培养基,在37℃、5%CO2条件下培养HepG2细胞。每2天换一次液,细胞融合至培养皿的80%左右时,进行传代。3次传代后取处于对数期、生长状态良好的细胞用于实验。
2.1.2Real-Time PCR分析
(1)细胞总RNA提取
将细胞接种于6孔板中,每孔加3mL,约5×104个/mL,置于37℃、5%CO2培养箱中培养,待细胞融合至60%后,吸除培养液,用PBS(磷酸盐缓冲液)冲洗,加入3mL不含血清的DMEM培养基,用不同浓度的低聚肽培养液处理细胞,同时设不加低聚肽的对照孔,每组6个平行样本。在培养箱中孵育24h后,提取细胞总RNA,按照RNA提取试剂盒说明书进行,利用核酸蛋白测定仪测定RNA的浓度,取0.5μgRNA溶液用于Real-TimePCR反应,剩余RNA溶液放于-80℃冰箱中保存。
(2)cDNA合成
按反转录试剂盒说明书进行,20μL体系中含有5×PrimeButter 4μL,OligodT Primer 1μL,Random Primer 5μL,dNTP Mixture 2μL,RNase Inhibitor0.5μL,RTase 2μL,DEPC水以及RNA溶液共5.5μL。反应条件:25℃,10min;42℃,60min;4℃,∞。合成好的cDNA 4℃保存待测。
(3)Real-TimePCR反应分析CYP7A1基因表达的变化
利用Primer Premier 5.0软件设计PCR引物,委托公司合成。以β肌动蛋白(β-actin)为看家基因。按照PCR试剂盒说明书上机进行PCR扩增。实验重复3次。
2.2实验结果
肝脏特异性酶胆固醇7α-羟化酶(CYP7A1)是在肝脏合成并促使胆固醇转化成胆酸的限速酶,对维持体内胆固醇代谢平衡起到关键作用。可通过提高CYP7A1基因的表达,来抑制胆固醇的合成。
为了保证实验结果的真实性和可比性,样品的处理后的体系应对HepG2细胞的增殖没有明显的影响,维持细胞的正常成。因此,实验中样品孵育浓度应在其对细胞产生毒性浓度之下。因此,实验中研究了本发明不同浓度下对细胞增殖的影响,结果见图4。与空白对照组相比,本发明六个浓度对HepG2细胞增殖无显著性影响,可应用于后续试验中。参见图4,是本发明对HepG2细胞增殖的影响表。
本发明对CYP7A1基因表达影响结果如表5。较模型对照组比较,本发明在六个作用浓度下,随着浓度的增加促进CYP7A1基因表达的能力增加,量效关系呈线性关系。从400μg/mL起具有显著的促进作用。因此,可以推断:本发明可以促进体内胆固醇的转化,清除过多胆固醇的累积。参见图5,是本发明对CYP7A1基因表达表。
3.肠道菌粘附实验研究
人体肠道内寄生着10万亿个细菌,它们保持一定比例的动态平衡,它们能影响体重和消化能力、抵御感染和自体免疫疾病的患病风险,还能控制人体对癌症治疗药物的反应。如果肠内有益菌菌群占优,肠内黏膜呈现粉红色。肠道益生菌具有促进排便、有助于维生素合成、迅速排出有害物质和避免病原菌侵害等优点。本发明对三株益生菌:唾液乳杆菌、鼠李糖乳杆菌和副于酪乳杆菌粘附能力的影响,本发明对肠道益生菌的调节作用。
3.1细胞培养
细胞复苏及培养:用含灭活的体积分数10%FBS、1%非必须氨基酸(NEAA)、2%谷氨酰胺和1%双抗的MEM完全培养基,在37℃,5%CO2恒温培养箱中培养Caco-2细胞,待细胞长满培养瓶瓶底后,先用PBS缓冲液洗2次,再加0.25%胰酶过度消化,细胞脱落后加入MEM完全培养基终止反应,将细胞吹打成细胞悬液,1200rpm离心5min,弃上清,加入完全MEM重悬,按1∶3进行传代细胞冻存:细胞培养基悬液+20%的FBS+5%DMSO(800μL细胞培养悬液+120μL FBS+80μLDMSO)。
3.2肠道菌粘附
粘附细胞单层的制备:将细胞密度调整为105个/mL,1mL/孔接入24孔板中,隔天换液,培养至细胞形成致密单层(约2天左右),至少粘附前1h更换不完全MEM。肠道菌悬液的制备:用DMSO配制1mmol/L cFDA-SE的贮备液(称5.0mgcFDA-SE溶解于8.969mL的DMSO中),过滤除菌,-20℃储存备用。将乳酸杆菌以1%(v/v)的接种量接入MRS培养基,37℃条件下培养至培养基浑浊后(OD 600=1.5左右),在8000rpm,4℃,5min收集菌体沉淀。用无菌PBS(磷酸盐缓冲液)洗3次,然后重悬于PBS(磷酸盐缓冲液)溶液中,并调整菌体浓度至1×109CFU/mL。向乳酸菌悬浮液中加入cFDA-SE贮液(1mmol/L),至终浓度为20μmol/L,37℃避光静置20min,之后离心,并用PBS(磷酸盐缓冲液)溶液洗3次,以去除多余的荧光染料。在488nm的激发波长下进行流式细胞检测,与未标记的菌体作对比分析cFDA-SE的标记率。
粘附实验:将粘附细胞单层弃上清,PBS(磷酸盐缓冲液)洗2次,加入样品和菌悬液(菌悬液终浓度为5×108/mL),同时设置不加样品的空白对照,每组3个复孔,粘附1.5h。之后弃上清,PBS(磷酸盐缓冲液)洗2次,洗去未粘附的乳酸杆菌,胰酶消化细胞约10min,待细胞与培养板底部完全脱落,加入MEM完全培养液终止消化,收集孔内的菌体悬液,流式细胞仪检测荧光强度(FL1通道)。
粘附率=粘附乳酸菌荧光强度/初始乳酸菌荧光强度
3.3实验结果
本发明对Caco-2细胞增殖影响见表6,由表可知六个作用低浓度下均对细胞增殖无抑制作用,可以用于后续研究。参见图6,是本发明对Caco-2细胞增殖的影响表。
本发明对三株肠道益生菌和大肠杆菌粘附能力的影响见表7。由表可看出本发明具有较好促进鼠李糖乳杆菌、副干酪乳杆菌和唾液乳杆菌的粘附,在中高浓度对鼠李糖乳杆菌和副干酪乳杆菌粘附能力的促进作用较对照具有显著性的差异(p<0.05);本发明在六个作用浓度下,随着作用浓度的增加,唾液乳杆菌粘附能力呈线性增加,较模型组都具有显著性的差异,最高粘附率为36.00%,模型组仅为20.55%,提高了近15%左右。
参见图7,是本发明对三株肠道菌群粘附的影响表。
4.增加肌肉
C2C12细胞是由C3H小鼠骨骼肌卫星细胞永生化而来的细胞株。因为细胞形态和特性均一,可无限代培养,因此常常被用做肌系细胞发育分化研究的模板。本发明研究以C2C1成肌细胞为研究对象,采用不通浓度的配方三干预体外培养,诱导其向成熟肌细胞分化,揭示本发明促进肌发育分化过程一些肌特征性分子的表达情况。
4.1试验方法
4.1.1C2C12细胞增殖培养复苏C2C12细胞
用含100mL/L FBS血清的高糖DMEM培养基调节细胞浓度为5×103/mL,移入培养瓶中,置37℃,体积分数5%CO2的孵箱中培养,隔2d换液,观察细胞形态和生长情况。
4.1.2C2C12细胞诱导肌分化培养
用TNE消化增殖培养达90%~100%融合的C2C12细胞,用含20mL/L马血清的高糖DMEM培养基调节细胞浓度为5×104/mL,置37℃,体积分数5%CO2的孵箱中培养,观察细胞形态和生长情况,特别注意是否肌管出现。
4.1.3Gimsa染色并显微镜下观察
按四川大学华西临床医院实验中心干细胞与组织工程研究室方法进行Gimsa染色,观察细胞形态和生长情况,特别注意是否肌管出现。
4.1.4细胞免疫化学检测细胞肌性蛋白分子表达
常规制作经肌分化诱导的C2C12细胞不同时期(经10ml/L马血清培养d1和d6两个时间点)的细胞爬片,经40g/L多聚甲醛固定之后按试剂盒的使用说明行DAB免疫细胞化学染色,观察、计算阳性率。细胞在6孔板培养,每个爬片计1个样品,使用时每个样品细胞数达3×105~5×105。
4.2实验结果
C2C12肌前体细胞在含10ml/L浓度马血清的高糖DMEM中培养,约2~4d后显微镜下开始观察到细胞的融合出现,发现肌管形成,随着时间推移,细胞密度变大变长,细胞融合越来越多,可以见到许多有两个核以上象串珠似的肌管出现,到第8~10天达高峰,之后细胞逐渐衰老死亡。由表8可知,本发明可以促进肌前体细胞肌小管的形成,随着作用浓度和分化时间的增加,肌小管形成数量明显增加。
参见图8,是本发明对C2C12细胞肌小管分化形成的影响表。
免疫细胞化学检测发现,C2C12细胞未经本发明刺激(d1)时结蛋白(desmin)和成肌分化抗原(MyoD)表达较高阳性率(分别约为:30.26%和14.02%,表9),而生肌素(myogenin)和肌球蛋白(myosin)虽表达,但表达率很低(分别约为:3.22%和1.55%,表10)。经过三个浓度的刺激6d后,结蛋白(desmin)、成肌分化抗原(MyoD)、生肌素(myogenin)和肌球蛋白(myosin)表达都有明显提高(表达阳性率分别约为:79.26%、82.25%、89.53%;70.26%、86.00%、87.52%;19.56%、25.54%、30.26%;23.25%、29.59%、27.53%)。
参见图9,是本发明对C2C12细胞结蛋白和成肌分化抗原的影响表。
参见图10,是本发明对C2C12细胞生肌素和肌球蛋白的影响表。
经上述研究表明:
(1)本发明在高浓度作用下具有较好的促进T&B淋巴细胞增殖的作用,并可以提高巨噬细胞的吞噬能力和NO生成能力,本发明具有较好的提高特异性和非特异性免疫调节作用。
(2)本发明可以显著提高CYP7A1基因表达的能力,可以得出:本发明可以促进胆固醇代谢,阻碍其在机体内吸收利用。
(3)本发明可以有效的改善肠道益生菌群数,提高益生菌的黏附能力。
(4)本发明能够通过提高肌小管形成和提高结蛋白、肌球蛋白、生肌素和成肌分化抗原来刺激肌前体细胞向成熟肌细胞分化,减少成熟时间和细胞数量。
本发明的配方原料作用:
浓缩乳清蛋白选用蛋白含量为80%的浓缩乳清蛋白。浓缩乳清蛋白具有以下功效:增强免疫功能;有利于心血管系统的健康;有利于肌肉健壮和增强体力、提高运动效果;改善精神面貌、提高工作和学习效率;为乳糖不耐症、酪蛋白和谷蛋白过敏者提供优质蛋白;减少皮肤皱纹和光老化,促进伤口愈合。运动中的我们需要消耗更多的蛋白质来延缓或者构筑肌肉。乳清蛋白中特别含有丰富的支链氨基酸,而支链氨基酸具有阻止肌肉分解,促进肌肉合成的功效,对于修复运动损伤也大有禆益。
伊代欣糖甜味纯正,易溶于水,不会被唾液、胃酸和胰液消化,一直到小肠才被蔗糖酶-异麦芽糖酶(S-Icomplex)复合水解,成葡萄糖和果糖吸收,持续缓慢供能,提高抗疲劳能力,血糖上升速率显著低于葡萄糖,不致龋齿。
低聚果糖又称蔗果低聚糖,除具有一般功能性低聚糖的物理化学性质外,还能明显改善肠道内微生物种群比例。它是肠内双歧杆菌的活化增殖因子,可减少和抑制肠内腐败物质的产生,抑制有害细菌的生长,调节肠道微生物平衡;能促进微量元素铁、钙的吸收与利用,以防止骨质疏松症;且口味纯正香甜可口,具有类似脂肪的香味和爽口的滑腻感。
小麦低聚肽粉是从天然食品——小麦蛋白粉中提取的蛋白质,再经过定向酶切及特定小肽分离技术获得的小分子多肽物质。小麦低聚肽粉具有以下作用:(1)有抑制胆固醇上升的作用。小麦低聚肽能促进胰岛素分泌作用,其功能物质是低聚蛋氨酸,可用于调节人的血糖,改善糖尿病症状。小麦肽能阻碍血管紧张素酶的作用,因而有降血压作用。小麦低聚肽的特点之一是含高谷酰胺,能够有效调节神经,也可作肠功能障碍时的特殊营养物质。(2)具有免疫调节、抗氧化等多种生物活性,能够刺激机体淋巴细胞增殖,增强巨噬细胞吞噬功能,提高机体抵御外界病原体感染的能力,降低机体发病率等。(3)小麦低聚肽是小麦蛋白酶解产物,能抑制血管紧张素转化酶的活性,使血管紧张素原不能转变成能使血压升高的血管紧张素II,从而生理地降低血压,而对正常血压不起用。
聚葡萄糖是一种水溶性的膳食纤维,是一种具有保健功能性的食品组分,可以补充人体所需的水溶性膳食纤维。聚葡萄糖具有以下功能特性:1、调节脂类代谢:聚葡萄糖可在小肠内造成一层膜,并缠裹部分食物脂肪,能有效限制消化道内脂肪的吸收,促进类脂化合物的排泄,增加饱腹感,减少进食量,从而达到调节血脂,减少脂肪堆积,预防肥胖等功效。2、降低胆固醇:聚葡萄糖能吸附胆汁酸、胆固醇变异原等有机分子,抑制总胆固醇(TC)浓度升高,降低胆酸及盐类的合成与吸收,降低人体血浆和肝脏胆固醇水平,防治冠状动脉硬化,胆石症和预防心脑血管疾病等。3、减少糖量吸收:聚葡萄糖可阻碍食物与消化液充分接触,抑制增血糖素分泌,促使葡萄糖吸收减慢,从而降低餐后血糖水平,充分发挥胰岛素的作用,防止糖尿病。4、预防治便秘:聚葡萄糖能在人体肠道中吸水膨胀并保持水分,增加粪便体积,刺激肠道蠕动,加速排便频率,起到润肠通便和预防便秘的作用。
水苏糖是天然存在的一种四糖,对人体胃肠道内的双歧杆菌、乳酸杆菌等有益菌群有着极明显的增殖作用,能迅速改善人体消化道内环境,调节微生态菌群平衡。它能促进形成有益菌在消化道内的优势菌地位,抑制产气产酸梭状芽孢杆菌等腐败菌的生产,另外产生大量生理活性物质,调节肠道pH值、灭杀致病菌,阻遏腐败产物生成,抑制内源致癌物的产生和吸收,并且分解衍生出多重免疫功能因子。俗称:有益菌的食物。
燕麦纤维粉以燕麦膳食纤维为原料,采用专有技术特殊工艺加工而成,大幅度提高了可溶性膳食纤维的比例,尤其是可溶性的β-葡聚糖。
圆苞车前子壳粉是人工种植的车前科车前属圆苞车前种子的外壳,有以下功效:1、心血管疾病。可溶性圆苞车前子纤维具有降低血胆固醇的功效。在结肠内益生菌的作用下,圆苞车前子纤维中的糖类物质经发酵分解为短链脂肪酸,从而阻止胆固醇的合成。2、润肠通便。圆苞车前子纤维可用于防止/治疗便秘或腹泻。它可充分吸收水分,增大体积至可排泄团块,增加肠道蠕动,减少粪便在肠内运送时间,并逐渐变得有规律。3、预防结肠癌及其他肠胃疾病的罹患机率。另外,圆苞车前子纤维还发挥解毒剂的作用。通过大量吸收水分和其他有害物质,增大粪便体积,螯合重金属和其他有害化学毒素,从而降低致癌物或有害物质之浓度;同时,促进肠道蠕动,加速其排出,减少与肠壁粘膜接触的时间。因而可预防或降低结肠癌、大肠炎等肠道疾患之罹患机率。4、糖尿病-血糖控制。圆苞车前子纤维有助于控制饭后的血糖上升。同时,通过对糖尿病的干预,圆苞车前草有助于减少糖尿病患者对胰岛素的需求量。5、控制体重。由于圆苞车前子壳含有丰富的水溶性纤维,遇水会膨胀形成数十倍的凝胶团,能增加饱足感,却不会提供热量,而且降低热量摄取。
苹果纤维是褐色或红褐色干燥粉末,有苹果特有的甜味、香味和酸味。苹果纤维具有以下功能:1、调节肠道功能:增加排便量及大便次数,改善便秘,曾对透析患者试验,效果良好。2、调节血脂:能增加胆汁酸的排泄量,降低血清胆固醇。
复合维生素用于医治疲劳,预防因饮食不平衡所引起的维生素缺乏。体育用途长时间运动或训练时的预防维生素的“流失”。
雪莲培养物,是在无菌条件下,将雪莲离体的植物器官、组织、细胞、胚胎或原生质体培养在人工配制的培养基上,在人工控制的条件下进行培养,使其组织或细胞增值进而按照需要进行培养的技术;或使其脱分化产生愈伤组织,并再逐步分化出器官并长成完整的植株而生产的培养物。中医认为,雪莲温肾助阳,祛风胜湿,通经活血。雪莲的独特功效越来越受到重视,临床主要用于素体阳虚、血瘀阻络之风寒湿痹痛、小腹疼痛、月经不调等病证的预防和治疗,亦在抗早孕方面。经系统研究表明,雪莲培养物不仅含有与天然雪莲相近的活性成分,且亦具有与之相当的多方面的药理活性。其主要药理作用镇痛、抗炎、抗风湿;抑制血小板聚集、降血脂、改善血液循环;对免疫系统具有调节作用;同时还有抗氧化、抗辐射、和抗疲劳等多方面的药效学作用。
最后应说明的是:显然,上述实施例仅仅是为清楚地说明本发明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围之中。
Claims (6)
1.一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征在于,由以下重量份数比的配方原料组成:浓缩乳清蛋白10-40;MCT粉15-30;伊代欣糖10-30;低聚果糖10-30;小麦低聚肽粉1-6;聚葡萄糖1-30;水苏糖3-10;复合矿物质1-10;燕麦纤维粉0.5-5;圆苞车前子壳粉1-10;复合维生素0.1-5;苹果纤维粉1-10;三氯蔗糖0-1;雪莲培养物0.001-1;将所有原料加入到卧式搅拌机中,混合均匀后即可。
2.如权利要求1所述一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征在于,由以下重量份数比的原料组成:浓缩乳清蛋白10-20;MCT粉15-22.5;伊代欣糖10-15;低聚果糖10-15;小麦低聚肽粉1-3;聚葡萄糖1-15;水苏糖3-6.5;复合矿物质5-10;燕麦纤维粉2.5-5;圆苞车前子壳粉5-10;复合维生素2.5-5;苹果纤维粉5-10;三氯蔗糖0.5-1;雪莲培养物0.5-1;将所有原料加入到卧式搅拌机中,混合均匀后即可。
3.如权利要求1所述一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征在于,由以下重量份数比的原料组成:浓缩乳清蛋白20-40;MCT粉22.5-30;伊代欣糖15-30;低聚果糖15-30;小麦低聚肽粉3-6;聚葡萄糖15-30;水苏糖6.5-10;复合矿物质1-5;燕麦纤维粉0.5-2.5;圆苞车前子壳粉1-5;复合维生素0.1-2.5;苹果纤维粉1-5;三氯蔗糖0-0.5;雪莲培养物0.001-0.5;将所有原料加入到卧式搅拌机中,混合均匀后即可。
4.根据权利要求1、2或3所述的一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征为:所述复合维生素包括维生素A、维生素D、维生素E、维生素B1、维生素B2、维生素B6、维生素B12、维生素C、烟酸、叶酸、泛酸。
5.根据权利要求1、2或3所述的一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征为:所述复合矿物质包括L-乳酸钙、硫酸镁、硫酸亚铁、乳酸锌。
6.根据权利要求1、2或3所述的一种具有延缓肌肉衰减、增强免疫力功效的组合物,其特征是:所述组合物的剂型包括粉末、颗粒剂、饮料和能量棒。
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