CN109125704A - 蜂王浆牦牛骨小分子肽及其制备方法和应用 - Google Patents
蜂王浆牦牛骨小分子肽及其制备方法和应用 Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种蜂王浆牦牛骨小分子肽及其制备方法和应用,其技术要点是所述蜂王浆牦牛骨小分子肽包括以重量份数计的蜂王浆冻干粉30‑50份、牦牛骨小分子肽30‑50份、水解蛋黄粉5‑15份、水解II型胶原蛋白5‑15份。所述蜂王浆牦牛骨小分子肽的制备方法,将蜂王浆冻干粉、牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白混合均匀即得蜂王浆牦牛骨小分子肽。所述蜂王浆牦牛骨小分子肽在制备防治骨病的药品或保健品中的应用。本发明提供的蜂王浆牦牛骨小分子肽从营养骨细胞开始,从根本上解决细胞营养以及细胞功能的问题,从源头上使骨病患者得到康复。
Description
技术领域
本发明涉及蜂产品技术领域,更具体的说,它涉及一种蜂王浆牦牛骨小分子肽及其制备方法和应用。
背景技术
骨骼系统包括身体的各种骨骼、关节与韧带,有支持躯体、保护体内重要器官、供肌肉附着、作运动杠杆等作用,部分骨骼还有造血、维持矿物质平衡的功能。
常见的骨病一般包括:风湿性关节炎(根据症状、流行病学及免疫学分析,认为风湿性关节炎与人体溶血性链球菌感染密切相关,且感染途径至关重要,咽部链球菌感染是发病的必要条件)、类风湿性关节炎(是一种以慢性侵蚀性关节炎为特征的全身性自身免疫病。)、退行性关节炎、强直性脊柱炎(属风湿病范畴,病因尚不明确,是以脊柱为主要病变部位的慢性病,累及骶髂关节,引起脊柱强直和纤维化,造成不同程度眼、肺、肌肉、骨骼病变,是自身免疫性疾病。)、颈椎病、腰椎病、肩周炎、骨质增生、股骨头坏死、骨肿瘤、骨痈疽、骨结核、骨关节痹证、痿证、筋挛等。
近年来,由于生活压力的增大、工作任务繁重、环境污染、人口老龄化等等诸多因素的影响,我国骨病发病率成逐年上升的趋势,最新统计数据显示:约2.3亿人患有关节骨病。患颈腰椎增生、股骨头坏死、滑膜炎、肩周炎及风湿性关节炎患者占全国总人口的18%,其发病率高达20%,其中8000万为重病患者。
多数骨病属于退行性病,也就是说其发病时由于年龄的增加,自然衰老而导致的。世界卫生组织统计:50岁,骨病发病率为60%,65岁为70%,75岁以上为80%。(1)关节炎的发病最为普遍,上了年纪的人由于长时间运动使关节软骨受到磨损,关节便发生了软骨退变。关节软骨的退变会令软骨剥脱,会导致软骨下骨质的破坏,形成关节炎症。人过了40岁,退行性膝关节炎随时可能会发生,且年纪越大,发生率就越高,50岁后的发生率约20%至30%,到了70~80岁时,可高达70%左右。(2)我国60岁以上老人中,有55%的人患有关节疾病,有56%的人患有骨质疏松症,女性发病率高达60%至70%;其中,椎间盘突出症是引起腰腿痛最常见的疾病,在西方总发病率15.2%~30%,国内统计达到18%。全国大约有7%-10%的人患上了颈腰椎病,50-60岁年龄段颈腰椎病的发病率约20%-30%;60-70岁年龄段达50%。
骨病与地域因素关系密切,全国有5个骨病重灾区(河南:喝酒猛,股骨头坏死;重庆:爬坡劳损,脚踝受损;青岛:阴冷潮湿,类风湿高发;贵州:难见阳光,骨质疏松高发),东北地区由于天寒地冻,膝关节受损。在东北地区,冬季气温低,寒冷时间长(甚至半年)若忽视了关节的保暖,久而久之,很容易发生骨关节疾病。骨关节炎全国平均发病率13%,东北地区最高,女性一般从40岁开始发病,60岁以上发病超过50%,骨关节炎患者占门诊的1/3,骨性关节炎、风湿性关节炎、急性滑膜炎等。
关节炎导致活动量减少,久坐成病,更易诱发高血压、脑中风、糖尿病等并发症。因此,骨关节炎被世界卫生组织(WHO)称为“不死的癌症”,与心脑血管疾病、癌症并列为危害人类健康的三大类重要疾病。骨病对人的危害从难以忍受的疼痛到骨骼变形、肌肉萎缩乃至活动受限,影响正常生活,严重时还会引发其他疾病。如:(1)骨质增生压迫血管会影响血液循环,特别是压迫供应脑部血液的椎动脉,会造成眩晕、走路不稳、语言不清等症状,如果骨质增生伸向椎管内压迫了脊髓,还会导致瘫痪、麻木、大小便失禁等严重后果;(2)退行性骨质疏松症最常见和最严重的并发症如胸、腰椎压缩性骨折,脊椎后弯,胸廓畸形,可使肺活量和最大换气量显著减少,患者往往可出现胸闷、气短、呼吸困难等症状;(3)关节的强直和畸形,肌肉的僵硬和萎缩,均可造成不同程度的残废,终至关节活动度受到限制,以致关节不能活动,不只影响患者的日常休息,最终使患者完全丧失劳动能力,而且使患者生活不能自理,大大降低了生活质量,同时也给患者的家眷带来诸多不便。
骨病产生的原因主要以下几个方面:(1)肝肾亏损:中医认为,肾藏精,主骨;肝藏血,主筋。肾精充足,肝血盈满,则筋骨劲强,关节灵活,人到中老年,生理机能减退,肝肾精血不足,致使筋骨失养,久而久之,容易发生骨关节病。(2)风、寒、湿邪侵蚀体内:这些燥热外邪可以10天入皮肤、30天入肌肉、90天入骨髓,侵蚀骨骼,阻塞经络,导致气血不畅,对骨骼造成伤害,引发疼痛,日久产生骨骼病变。(3)退行性病变:长期软骨磨损,钙、镁、锌等矿物质和胶原蛋白等有机质的流失,造成骨质疏松,骨密度下降,降低了矿物质的吸收、利用、转化,骨化程度降低,这方面有老年性自然老化,有营养素摄入不足,有营养素吸收不好等原因。
但是,骨病归结到根本在于成骨细胞(骨形成的主要功能细胞,负责骨基质的合成、分泌和矿化)和破骨细胞(骨组织成分的一种,行使骨吸收的功能)二者协同作用破坏,导致构成骨关节的各种细胞自身营养、再生、修复能力缺失或者降低,导致由这些细胞所组成的组织和器官功能降低或者缺失。通俗讲,就是说自身的坚守、抵抗能力不行了,当有外界伤害因素入侵人体的时候,自身难以抵挡,使危害因素得逞,从而对人体造成伤害。
但是,很多人意识不到这一点,所以很多骨病患者虽然一直没有停止过治疗或者用药,但是病情改善却不理想,甚至因为长期不合适的治疗手段或者用药,不但旧病未除,还引发其他疾病,如风湿性关节炎病人长期使用激素类药物,会导致骨质疏松。究其原因,忽视了构建骨关节的根基——骨细胞,没有真正使骨细胞强大起来。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种蜂王浆牦牛骨小分子肽,其从营养骨细胞开始,从根本上解决细胞营养以及细胞功能的问题,从源头上使骨病患者得到康复。
本发明的上述技术目的是通过以下技术方案得以实现的:
蜂王浆牦牛骨小分子肽,包括以重量份数计的蜂王浆冻干粉30-50份、牦牛骨小分子肽30-50份、水解蛋黄粉5-15份、水解II型胶原蛋白5-15份。
进一步,所述牦牛骨小分子肽采用下述方法制备:耗牛骨经清洗、破碎、蒸煮工序后,使用源自耐辐射奇球菌的蛋白质水解酶直接对蒸煮液进行水解所得到的物质,再经纯化或分离、干燥工序即得牦牛骨小分子肽。
更进一步,所述牦牛骨小分子肽的分子量为1000道尔以下。
进一步,所述水解蛋黄粉采用下述方法制备:生蛋黄经冷冻干燥、粉碎后得到粗制蛋黄粉;粗制蛋黄粉经脱脂工序,并顺次经过等电点沉淀法提取、冷冻干燥工序后得到蛋黄蛋白;顺次使用源自耐辐射奇球菌的蛋白质水解酶和嗜热脂肪芽孢杆菌的蛋白质水解酶对蛋黄蛋白进行二次酶解,所得酶解产物经纯化或分离、干燥工序即得水解蛋黄粉。
本发明的目的在于还提供上述蜂王浆牦牛骨小分子肽的制备方法,将蜂王浆冻干粉、牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白混合均匀即得蜂王浆牦牛骨小分子肽。
本发明的目的在于还提供上述蜂王浆牦牛骨小分子肽在制备防治骨病的药品或保健品中的应用。
进一步,所述药品或保健品为片剂、颗粒剂、散剂、丸剂或胶囊剂。
综上所述,本发明具有以下有益效果:本发明首次提出“细胞好营养,骨骼才健康,自身免疫强,疾病无处藏”的健康理念。将蜂王浆与牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白相结合,从营养骨细胞开始,从根本上解决细胞营养以及细胞功能的问题,从源头上使骨病患者得到康复。
附图说明
图1是表示使用实施例1-3和对比例1-4中得到的产品测定骨生长促进效果的结果图;图2是表示使用实施例1-3和对比例1-4中得到的产品测定钙化促进效果的结果的图。
具体实施方式
以下结合实施例对本发明作进一步详细说明。
在本发明中,作为制备牦牛骨小分子肽中使用的蛋白质水解酶,使用源自耐辐射奇球菌的蛋白质水解酶。源自耐辐射奇球菌的蛋白质水解酶随着使用的酶的不同而适当改变,但是酶和蒸煮液的质量比优选从1∶100到1∶500,其中蒸煮液中的溶质和水溶液的重量比为1∶8到1∶12。还有,酶的反应温度或反应时间也随着使用的酶的不同而不同,不过优选在40~60℃下进行水解反应2~4小时。即使反应时间过长或过短,得到的水解产物不能充分得出成骨细胞增殖促进作用、骨生长以及骨强化作用。
上述方法得到的牦牛骨小分子肽的分子量为1000道尔以下。
在本发明中,作为制备水解蛋黄粉中使用的蛋白质水解酶,顺次使用源自耐辐射奇球菌的蛋白质水解酶和嗜热脂肪芽孢杆菌的蛋白质水解酶对蛋黄蛋白进行二次酶解。其中,使用源自耐辐射奇球菌的蛋白质水解酶和蛋黄蛋白的重量比为7~9∶100,在50~60℃下进行水解反应1~3小时;使用源自嗜热脂肪芽孢杆菌的蛋白质水解酶和蛋黄蛋白的重量比为3~5∶100,在在50~60℃下进行水解反应1~3小时。在两种蛋白质水解酶相互配合的情况下,得到的水解产物能够充分得出成骨细胞增殖促进作用、骨生长以及骨强化作用。
牦牛骨小分子肽制备例:耗牛骨经清洗、破碎、蒸煮工序后,分离出蒸煮液,并控制蒸煮液的蒸煮液中的溶质和水溶液的重量比为1∶10;将蒸煮液1000g升温至55℃,再添加源自耐辐射奇球菌的蛋白质水解酶5g水解3小时,灭酶,在4000rpm下进行10分钟的离心分离得到上清液,所得上清液经喷雾干燥得到牦牛骨小分子肽。
水解蛋黄粉制备例:生蛋黄经冷冻干燥、粉碎后得到粗制蛋黄粉;粗制蛋黄粉经脱脂工序,并顺次经过等电点沉淀法提取、冷冻干燥工序后得到蛋黄蛋白;将蛋黄蛋白10g加入到100g蒸馏水中,升温至55℃,先添加0.8g源自耐辐射奇球菌的蛋白质水解酶水解2小时,再添加0.4g源自嗜热脂肪芽孢杆菌的蛋白质水解酶水解2小时,灭酶,在4000rpm下进行10分钟的离心分离得到上清液,所得上清液经喷雾干燥得到水解蛋黄粉。
实施例1:将蜂王浆冻干粉300g、由牦牛骨小分子肽制备例提供的牦牛骨小分子肽500g、由水解蛋黄粉制备例提供的水解蛋黄粉50g、水解II型胶原蛋白150g混合均匀即得蜂王浆牦牛骨小分子肽。
实施例2:将蜂王浆冻干粉400g、由牦牛骨小分子肽制备例提供的牦牛骨小分子肽400g、由水解蛋黄粉制备例提供的水解蛋黄粉100g、水解II型胶原蛋白100g混合均匀即得蜂王浆牦牛骨小分子肽。
实施例3:将蜂王浆冻干粉500g、由牦牛骨小分子肽制备例提供的牦牛骨小分子肽300g、由水解蛋黄粉制备例提供的水解蛋黄粉150g、水解II型胶原蛋白50g混合均匀即得蜂王浆牦牛骨小分子肽。
对比例1:与实施例2的区别仅在于未添加蜂王浆冻干粉。
对比例2:与实施例2的区别仅在于未添加牦牛骨小分子肽。
对比例3:与实施例2的区别仅在于未添加水解蛋黄粉。
对比例4:与实施例2的区别仅在于水解II型胶原蛋白。
采用Sprague-Dawley系大鼠评价对实施例1-3和对比例1-4所得产品进行骨生长效果实验。具体来讲,给作为荧光物质的四环素,利用四环素和钙形成螯合物沉积于骨的现象,观察形成骨的荧光带,由此来测定胫骨的生长。即,用胃探针(sonde)Sprague-Dawley系大鼠经口服用(每天,每1kg体重给10mg,20mg或者50mg)溶解于蒸馏水而得到的试料(浓度20mg/mL)。然后,在试验期间中的第3天和第5天以注射的方式给荧光物质(四环素10mg/kg),第7天摘出胫骨,测定在荧光显微镜下观察到的2条荧光带的间隔(从给四环素的第3天到第5天的2天间的胫骨骨端的伸长)。其结果显示在图1。从图1中可知,骨生长促进效果不仅与投加量有关,而且还与配方配比有关。本发明将蜂王浆冻干粉与牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白相结合,对成骨细胞的活化作用具有相互促进的作用,并且使用采用本发明提供的牦牛骨小分子肽和水解蛋黄粉可进一步提高对成骨细胞的活化作用。
对实施例1-3和对比例1-4所得产品进行钙化促进效果实验。把6×105个MC3T3-E1细胞接种于(α-MEM培养基,10%FCS,2mmol/L甘油磷酸)3.5cm的培养皿中,加入浓度100μg/mL的实施例1-3和对比例1-4所得产品,每隔两天更换一次培养基,连续培养4周,用PBS(磷酸缓冲液)冲洗细胞,加入5(v/v)%高氯酸,用超音波处理,把沉积在细胞中的钙洗脱,测定洗脱的钙量作为钙化活性。其结果显示在图2中。由图2可知,通过添加本发明提供的产品,钙化活性显著上升。本发明将蜂王浆冻干粉与牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白相结合,对钙化活性具有相互促进的作用,并且使用采用本发明提供的牦牛骨小分子肽和水解蛋黄粉可进一步提高钙化活性。
对实施例1-3和对比例1-4所得产品进行破骨细胞的分化以及增殖抑制效果实验。从小鼠的骨髓中分离出骨髓细胞,测定向破骨细胞的分化以及增殖。首先,把小鼠大腿骨的骨髓细胞,用含有作为分化诱导因子的RANKL(receptoractivatorofNF-κBligand:NF-kB活化受体配体)的α-MEM培养基培养3天后,用样品处理,再培养4天。然后,用固定液把细胞固定,应用TRAP染色试剂盒进行TRAP染色,在显微镜下观察染成红色的破骨细胞的个数。对于添加实施例1-3和对比例1-4所得产品25μg/mL、50μg/mL以及100μg/mL而培养的细胞和未添加来培养的细胞,测定破骨细胞的产生频度,结果见表1。
表1
产品添加量 | 0ug/mL | 25ug/mL | 50ug/mL | 100ug/mL |
实施例1 | 22.5% | 15 | 1.5 | 0 |
实施例2 | — | 12 | 0.9 | 0 |
实施例3 | — | 14 | 1.4 | 0 |
对比例1 | — | 25 | 18 | 2.2 |
对比例2 | — | 24 | 17.5 | 2.0 |
对比例3 | — | 26 | 19 | 2.4 |
对比例4 | — | 25 | 17 | 2.5 |
从表1可知,破骨细胞的发生频度的受抑制依赖于产品的浓度。通过对比实施例1-3和对比例1-4测定数据可知,本发明将蜂王浆与牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白相结合,对破骨细胞的抑制作用具有相互促进的作用,并且使用采用本发明提供的牦牛骨小分子肽和水解蛋黄粉可进一步提高对破骨细胞的抑制作用。本发明提供的蜂王浆牦牛骨小分子肽从营养骨细胞开始,从根本上解决细胞营养以及细胞功能的问题,从源头上使骨病患者得到康复。
采用实施例2提供的蜂王浆牦牛骨小分子肽进行临床应用。
案例1:张淑云,女,64岁,吉林市人,腰椎盘突出10多年,不能自己起床,需要别人扶着起床,右侧腰部肌肉萎缩严重,有明显凹陷,不能自由蹲起,上楼费力。
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一个月,右侧腰部肌肉萎缩处肌肉再生,可以自己起床,不需要别人搀扶,可自行蹲起,腿有力量,可以自如爬6楼来回,不觉得累。
案例2:裴毅,男,64岁,吉林市人,肩周炎,胳膊抬举困难,摸不着后脑勺。
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一个月,胳膊能抬起,可以摸到后脑勺。
案例3:王桂华,女,66岁,吉林市人,20多年网球肘,不能拎重物,不能干家务,一直从肩到手贴膏药,不见好。
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一周左右,胳膊灵活,不再疼,能拎菜上楼,能做家务。
案例4:李宗海,男,70岁,吉林市人,腿关节疼,怕冷畏寒,迈步困难。
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约15天左右,腿发热,走路灵活,屈伸自如。
案例5:薛立霞,女,70岁,吉林市人,腰疼、肩膀疼
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一周左右,腰、间疼痛消失,运动自如。
案例6:朱继伟,男,67岁,吉林市人,腰椎盘突出30多年,颈椎病
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一个月左右,腰疼痛消失,颈椎病缓解。
案例7:李秀,女,67岁,吉林市人,足跟痛
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,约一周左右,足跟痛消失。
案例8:杨双秀,女,66岁,吉林市人,髋关节肌肉萎缩、腰部发硬、疼痛;肩周炎。
服用蜂王浆牦牛骨小分子肽,3次/天,每次6g,一个月,腰部灵活,肌肉变软,肩周炎疼痛减轻。
案例9:邵玉方,男,70岁,吉林市人,颈椎病20多年。
服用蜂王浆牦牛骨小分子肽,1次/天,每次6g,约一个月左右,颈椎病症状明显改善。
案例10:于智伟,男,76岁,吉林市人,髋关节疼痛,走路困难,需要拄拐。
服用蜂王浆牦牛骨小分子肽,4次/天,每次6g,约2个月,走路轻松,不拄拐可以自行走路,可以爬楼。
案例11:王风清,女,79岁,吉林市人,腰疼累及到腿,腿发沉,走路慢,不能走长路。
服用蜂王浆牦牛骨小分子肽,4次/天,每次6g,约2个月,腰不再疼,走路轻松,腿不再发沉。
案例12:陈玉兰,女,77岁,吉林市人,腰椎盘突出,腰疼厉害,早晨起床极其费力,需要缓解1个小时才能由别人扶助起床;不能久坐,坐下起不来,需要别人扶起。
服用蜂王浆牦牛骨小分子肽,4次/天,每次6g,约2个月,腰疼缓解,起床轻松,缓解10分钟左右就能起床,大大缩减起床时间;坐下后能自行起来,不再需要别人扶着才能起来。
案例13:王淑兰,女,66岁,吉林市人,严重腰椎盘突出,不敢外出,不能长时间坐着。
服用蜂王浆牦牛骨小分子肽,4次/天,每次6g,约2.5个月,腰椎盘突出明显改善,可坐车外出旅游。
案例14:车淑琴,女,79岁,吉林市,腿疼、腿无力,髋关节不灵活,走路费力。
服用蜂王浆牦牛骨小分子肽,4次/天,每次6g,近3个月,腰腿有力、灵活,走路轻松,不累,可外出旅游,2018年10月14日还能爬长城。
应该理解的是,本发明实施例所述制备方法仅仅是用于说明本发明,而不是对本发明的限制,在本发明的构思前提下对本发明制备方法的简单改进都属于本发明要求保护的范围。
Claims (7)
1.蜂王浆牦牛骨小分子肽,其特征在于,包括以重量份数计的蜂王浆冻干粉30-50份、牦牛骨小分子肽30-50份、水解蛋黄粉5-15份、水解II型胶原蛋白5-15份。
2.根据权利要求1所述的蜂王浆牦牛骨小分子肽,其特征在于,所述牦牛骨小分子肽采用下述方法制备:耗牛骨经清洗、破碎、蒸煮工序后,使用源自耐辐射奇球菌的蛋白质水解酶直接对蒸煮液进行水解所得到的物质,再经纯化或分离、干燥工序即得牦牛骨小分子肽。
3.根据权利要求1或2所述的蜂王浆牦牛骨小分子肽,其特征在于,所述牦牛骨小分子肽的分子量为1000道尔以下。
4.根据权利要求1所述的蜂王浆牦牛骨小分子肽,其特征在于,所述水解蛋黄粉采用下述方法制备:生蛋黄经冷冻干燥、粉碎后得到粗制蛋黄粉;粗制蛋黄粉经脱脂工序,并顺次经过等电点沉淀法提取、冷冻干燥工序后得到蛋黄蛋白;顺次使用源自耐辐射奇球菌的蛋白质水解酶和嗜热脂肪芽孢杆菌的蛋白质水解酶对蛋黄蛋白进行二次酶解,所得酶解产物经纯化或分离、干燥工序即得水解蛋黄粉。
5.如权利要求1至4中任意一项所述的蜂王浆牦牛骨小分子肽的制备方法,其特征在于,将蜂王浆冻干粉、牦牛骨小分子肽、水解蛋黄粉、水解II型胶原蛋白混合均匀即得蜂王浆牦牛骨小分子肽。
6.如权利要求1至4中任意一项所述的蜂王浆牦牛骨小分子肽在制备防治骨病的药品或保健品中的应用。
7.根据权利要求6所述的蜂王浆牦牛骨小分子肽在制备防治骨病的药品或保健品中的应用,其特征在于,所述药品或保健品为片剂、颗粒剂、散剂、丸剂或胶囊剂。
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