CN113854574B - 一种新型复方骨肽及其应用 - Google Patents

一种新型复方骨肽及其应用 Download PDF

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CN113854574B
CN113854574B CN202111182578.XA CN202111182578A CN113854574B CN 113854574 B CN113854574 B CN 113854574B CN 202111182578 A CN202111182578 A CN 202111182578A CN 113854574 B CN113854574 B CN 113854574B
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bone
peptide
novel compound
bone peptide
vitamin
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CN113854574A (zh
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余钧
周飒
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Hangzhou Baibeiyou Biotechnology Co ltd
Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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Abstract

本发明提供一种新型复方骨肽，原料中包括：牛骨多肽、Ⅱ型胶原蛋白肽、酪蛋白磷酸肽、乳清蛋白、大豆分离蛋白、酶解骨粉、麦芽糊精、维生素A、维生素D、维生素C、低聚果糖、接骨木莓果汁粉、姜黄、黑胡椒提取物、香精、黄原胶、三氯蔗糖。本发明的新型复方骨肽可应用于促进骨骼生长、促进钙质吸收、增强骨密度、改善骨质疏松。该复方骨肽吸收速率和吸收率均属于高水平，且临床施用量少，有广泛的应用前景。

Description

一种新型复方骨肽及其应用
技术领域
本发明属于生物医药领域，具体涉及一种新型复方骨肽及其应用。
背景技术
胶原蛋白是构成细胞外基质的主要大分子之一，牛骨肽是牛骨胶原蛋白的酶解法获得的活性肽，是将牛骨胶原蛋白的三螺旋结构彻底打开，肽链被降解成为短的肽链，得到分子质量在几千道尔顿之间的多肽混合物，这些小分子肽与生物体相容性好，具有优于蛋白质和氨基酸的营养功能。同时，其具有良好的溶解性，物理性质稳定，不同来源和不同酶解工艺得到的小分子肽具有不同的生物活性，如免疫调节、降血压、降血糖、降血脂、抗衰老、抗氧化、抗癌、抗微生物、抗毒素、增加骨密度、改善骨骼韧性等。
牛骨肽富含多种非必需氨基酸，如甘氨酸(Gly)、脯氨酸(Pro)和羟脯氨酸(Hyp)，羟脯氨酸(hydroxyproline，HYP)是胶原组织主要成分之一，且为胶原中特有的氨基酸，约占胶原氨基酸总量的13％。利用血液中的羟脯氨酸为多种胶原肽的降解产物，其作为骨吸收标志物这一特点，通过对血清中羟脯氨酸的含量进行测定，可以衡量样品给药后小鼠对骨肽的吸收和生物利用度情况。此外，文献研究表明小鼠可直接吸收骨肽样品多肽混合物中的胶原三肽Gly-Pro-Hyp，因此对小鼠体内的此三肽的测量也是评价骨肽吸收和生物利用情况的另一关键指标。
牛骨肽自身的功效在与其他成分复配后可能起到更好的作用。
中国专利201710938888.7中公开的一种牛骨肽固体饮料，包含：牛骨肽、玛卡粉、蛹虫草粉剂、维生素B、维生素C、酸枣粉、金银花粉、茯苓粉、党参粉、雪莲果粉、食用香精。该发明可以替代传统的煲牛骨汤的工艺，直接加温水就可以饮用，并且喝完这种牛骨肽固体饮料要比传统要比传统牛骨汤容易吸收和消化，而且具有抗疲劳、增强精力、平衡荷尔蒙、增强免疫、抗疲劳、抗贫血、安神、健脾胃、补气兼能养血、促进消化和调理胃肠道的功效，适合不同年龄段的人群食用，且生产工艺简单、卫生、安全、生产成本低，适合大范围推广普及。
中国专利202010293977.2中提供了具有改善骨胳状态、补充骨胳营养、缓解骨胳疼痛的一种改善骨胳的牛骨肽饮品及其制备方法，以牛骨胶原蛋白肽、大豆肽、鲨鱼软骨胶原蛋白肽、燕麦粉、奶粉、山药粉、葛根粉、当归粉、平卧菊三七、酵母葡聚糖、魔芋精粉、针叶樱桃、维生素D和维生素E加工制备成一种改善骨胳健康的牛骨肽饮品。这种牛骨肽饮品可明显改善骨质疏松，增强骨密度，补充骨胳钙，增强钙吸收活性，补充骨胳营养，易吸收，效果显著，起效周期短，制备工艺成熟，可大规模生产以用于人们的日常保健。
但现有技术中的牛骨肽复合产品在吸收速率，人体利用率和施用量上都有很大的优化空间。
发明内容
为了解决上述问题，本发明提供了一种新型复方骨肽，可应用于促进骨骼生长、促进钙质吸收、增强骨密度、改善骨质疏松。该复方骨肽吸收速率和吸收率均属于高水平，且临床施用量少，有广泛的应用前景。
一方面，本发明提供了一种新型复方骨肽。
所述的新型复方骨肽配方如下：
原料 配比％
牛骨多肽 12-16％
Ⅱ型胶原蛋白肽 0.5-1.2％
酪蛋白磷酸肽 0.1-0.2％
乳清蛋白 35-45％
大豆分离蛋白 4-8％
酶解骨粉 3-3.5％
麦芽糊精 20-24％
维生素A、D、C 0.1-0.4％
低聚果糖 8-12％
接骨木莓果汁粉 0.75-1.2％
姜黄 0.05-1％
黑胡椒提取物 0.005-0.2％
香精 0.05-1％
黄原胶 0.5-0.8％
三氯蔗糖 0.005-0.01％
。
优选地，所述的新型复方骨肽配方如下：
原料 配比％
牛骨多肽 15.000
Ⅱ型胶原蛋白肽 0.850
酪蛋白磷酸肽 0.150
乳清蛋白 40.000
大豆分离蛋白 5.000
酶解骨粉 3.500
麦芽糊精 22.560
维生素A、D、C 0.375
低聚果糖 10.000
接骨木莓果汁粉 0.750
姜黄 0.050
黑胡椒提取物 0.005
香精 1.000
黄原胶 0.750
三氯蔗糖 0.01
。
另一方面，本发明提供了前述的新型复方骨肽在制备药物或保健品中的应用。
所述的药物或保健品用于促进骨骼生长、促进钙质吸收、增强骨密度、改善骨质疏松。
再一方面，本发明提供了一种用于促进骨骼生长的药物或保健品。
所述的药物或保健品中包括前述的新型复方骨肽。
所述的药物或保健品中新型复方骨肽的含量为50-100％，优选为100％。
又一方面，本发明提供了一种新型复方骨肽的制备方法。
所述的制备方法用于制备前述的新型复方骨肽。
所述的制备方法中包括牛骨肽的制备。
所述的制备方法中包括以下步骤：
(1)牛骨脱脂；
(2)酶解底物制备；
(3)酶解；
(4)浓缩干燥。
具体地，所述的步骤(1)中采用高温蒸煮的方法进行脱脂；高温蒸煮采用脱脂罐为容器。
进一步地，所述的步骤(1)中脱脂罐中原料比例为牛骨：纯化水＝1：1；分离油脂的条件为温度70℃，真空度为-0.05MPa至-0.1MPa。
具体地，所述的步骤(2)中包括盐酸浸泡、维生素C水溶液浸泡。
具体地，所述的步骤(3)中包括酸性蛋白酶酶解、中性蛋白酶酶解。
在一些实施例中，所述的制备方法包括以下步骤：
(1)牛骨脱脂：
牛骨破碎至大小1-3cm加入脱脂罐；以1∶1比例向脱脂罐加入纯化水，沸煮3h；脱脂罐温度降至70℃左右，真空度为-0.05MPa至-0.1MPa，分离上层油脂；常压蒸煮：再以1∶1比例继续向脱脂罐内加纯化水，升温至100℃后，蒸煮3h；静置后，分离上层油脂；
(2)脱脂后牛骨置于质量分数为1％的盐酸溶液中浸泡4h；牛骨与盐酸溶液的用量比例为1kg：3L；
(3)牛骨粉碎并磨成骨泥，加入质量分数2％的维生素C水溶液中，浸泡6-8h得到酶解底物；牛骨与维生素C水溶液的用量比例为1kg：3L；
(4)按质量比1％加入酸性蛋白酶酶解2-3h；调整pH至7，按质量比1％加入中性蛋白酶酶解处理1-2h；酶解结束后，升温；
(5)过滤得到水解液；
(6)浓缩：将水解液采用刮板浓缩器浓缩得到质量分数30％的浓缩提取液；
(7)包埋干燥后制成颗粒；
(8)称重复配。
又一方面，本发明提供了前述的制备方法在制备用于促进骨骼生长的药物或保健品中的应用。
所述的药物或保健品用于促进骨骼生长、促进钙质吸收、增强骨密度、改善骨质疏松。
本发明提供的新型复方骨肽具有以下优点：
1、吸收速率快；
2、临床用量少；
3、吸收效率高。
附图说明
图1为血清中羟脯氨酸随时间含量变化图；其中，A：空白组；B：低剂量组(1.6g/kg)；C：中剂量组(3.2g/kg)；D：高剂量组(4.8g/kg)；E：骨肽片组(576mg/kg)；F：注射液组(90mg/kg)。
图2为小鼠血清中羟脯氨酸药时曲线面积计算结果。
图3为血清中三肽Gly-Pro-Hyp随时间含量变化图；其中，A：空白组；B：低剂量组(1.6g/kg)；C：中剂量组(3.2g/kg)；D：高剂量组(4.8g/kg)；E：骨肽片组(576mg/kg)；F：注射液组(90mg/kg)。
图4为三肽Gly-Pro-Hyp药时曲线面积计算结果。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1一种新型复方骨肽粉
配方如下：
实施例2一种新型复方骨肽的制备方法
本实施例制备的为实施例1提供的新型复方骨肽。包括以下步骤：
(1)牛骨脱脂：
牛骨破碎至大小1-3cm加入脱脂罐；以1∶1比例向脱脂罐加入纯化水，沸煮3h；脱脂罐温度降至70℃左右，真空度为-0.05MPa至-0.1MPa，分离上层油脂；常压蒸煮：再以1∶1比例继续向脱脂罐内加纯化水，升温至100℃后，蒸煮3h；静置后，分离上层油脂；
(2)脱脂后牛骨置于质量分数为1％的盐酸溶液中浸泡4h；牛骨与盐酸溶液的用量比例为1kg：3L；
(3)牛骨粉碎并磨成骨泥，加入质量分数2％的维生素C水溶液中，浸泡6-8h得到酶解底物；牛骨与维生素C水溶液的用量比例为1kg：3L；
(4)按质量比1％加入酸性蛋白酶酶解2-3h；调整pH至7，按质量比1％加入中性蛋白酶酶解处理1-2h；酶解结束后，升温；
(5)过滤得到水解液；
(6)浓缩：将水解液采用刮板浓缩器浓缩得到质量分数30％的浓缩提取液；
(7)包埋干燥后制成颗粒即为牛骨多肽。
按照实施例1中的成分进行称重复配。
以下实验例中选用的复方骨肽均为实施例1提供的复方骨肽。
以下实验例中，骨肽片购自吉林华康药业股份有限公司，国药准字H20058927；注射复方骨肽购自南京新百药业有限公司，国药准字H20003533。均于4℃干燥保存。
以下实验例中，血清的制备方法如下：小鼠眼球主动脉取出血液，置于具塞离心管中，常温静置30min后，4000rpm离心10min。用移液器缓慢取出上部血清置于另一具塞离心管中，用于血清指标的测定。
以下实验例中，新型复方骨肽用药量低剂量组为1.6g/kg；中剂量组为3.2g/kg；高剂量组为4.8g/kg。骨肽片组用药量为576mg/kg；注射液组用药量为90mg/kg。
实验例1检测血清中羟脯氨酸
选用健康的C57/BL小鼠，设置空白对照组，阳性药物对照组1(骨肽注射液组)、阳性药物对照组2(骨肽片组)、复方骨肽低、中、高剂量共6组，每组20只，均经适应性饲养一周后，于清晨分别给药一次。给药后，分别于1、3、6、12、24h后每组处死4只小鼠，取得每只小鼠的血浆，测定血清内羟脯氨酸含量(ELISA试剂盒)，比较样品的生物利用度情况。计算血清内羟脯氨酸药时曲线下面积AUC，比较样品药物吸收的有效暴露量。
结果如图1所示。
由图1可以得出实验组小鼠血清中羟脯氨酸的含量均有明显的增加。随着骨肽样品剂量的提高，血清中羟脯氨酸的含量水平也随之提高；且骨肽样品灌胃小鼠血清中羟脯氨酸含量在灌胃3h后，就基本达到了最高水平，而且随着时间的延长羟脯氨酸含量一直保持较高水平，在24h后都未发生明显下降。说明骨肽样品吸收速率明显高于骨肽片和骨肽注射液。
实验例2三肽Gly-Pro-Hyp检测
阳性药物对照组和实验组饲养给药同实验例1。给药(注射或灌胃)后，分别于1、3、6、12、24h后每组处死4只小鼠，取得每只小鼠的血清，测定血清中三肽Gly-Pro-Hyp(甘氨酸-脯氨酸-羟脯氨酸)的含量(采用标品Pro-Hyp-Gly高效液相色谱法测定含量)，比较样品的生物利用度情况。计算血清内三肽Gly-Pro-Hyp药时曲线下面积AUC，比较样品药物吸收的有效暴露量。
小鼠血清中羟脯氨酸药时曲线面积如图2所示。药时曲线下面积AUC是以给药后血药浓度为纵坐标，以时间为横坐标所得曲线下的面积。本实验中使用积分法求得，其可以表示服用某一剂量药物后一定时间内药物吸收入血的相对分量，与生物体吸收循环的药量成正比。通过图2可以看出，随骨肽样品剂量的提高，其羟脯氨酸AUC值随之提高。且低剂量、中剂量、高剂量骨肽样品的羟脯氨酸AUC远高于临床剂量骨肽注射液组与骨肽片组，证明了骨肽样品有效吸收的优势。说明，临床使用剂量——低剂量组的有效暴露明显高于骨肽注射液和骨肽片。
血清中三肽Gly-Pro-Hyp随时间含量变化图如图3所示。由图3可以得出各实验组小鼠血清中三肽Gly-Pro-Hyp的含量均有明显的增加。随着骨肽样品剂量的提高，血清中Gly-Pro-Hyp的含量水平也随之提高；且骨肽样品灌胃小鼠血清中三肽Gly-Pro-Hyp在灌胃3h后，就达到了最高水平；而且随着时间的延长，Gly-Pro-Hyp含量一直保持较高水平，在24h都未发生明显下降。说明骨肽样品吸收率明显高于骨肽片和骨肽注射液。
三肽Gly-Pro-Hyp药时曲线面积如图4所示。本实验中使用积分法求得Gly-Pro-Hyp含量的药时曲线下面积，从图4中可以看出，随骨肽样品剂量的提高，其Gly-Pro-Hyp的AUC值随之提高。且低剂量、中剂量、高剂量骨肽样品远高于临床剂量骨肽注射液组与骨肽片组，证明了骨肽样品有效吸收的优势。说明，临床使用剂量——低剂量骨肽样品的药物暴露明显高于骨肽注射液和骨肽片。
由实验例1-2结果可知，小鼠骨肽样品吸收速率明显高于骨肽片和骨肽注射液，且骨肽样品发挥作用的时间明显长于骨肽片和骨肽注射液。
通过对羟脯氨酸及三肽Gly-Pro-Hyp进行药代数学模型的模拟，利用积分法求得药时曲线下面积(AUC)。对比结果发现，随着新型骨肽配方样品剂量的提高，其AUC值也随之提高，证明小鼠吸收骨肽样品的有效暴露量增加。且低剂量骨肽样品组羟脯氨酸的AUC和三肽Gly-Pro-Hyp的AUC明显高于临床剂量骨肽注射液与骨肽片组。说明，临床使用剂量——低剂量骨肽样品的药物暴露明显高于骨肽注射液和骨肽片。
结果表明：
1、采用复配的骨肽可以使标志性营养活性成分羟脯氨酸和三肽达到高浓度，效果等同或优于同类药品效果。
2、合适的复方成分比例高效率综合解决骨骼等多种综合性问题。
3、配方口感顺滑，依从性好，适合持续使用。
对比例
设置对比例并参考实验例1-2的方法对对比例和实施例1进行检测，检测时均参考高剂量组进行操作。
对比例设置如下：
实验结果如下：

Claims (6)

1.一种新型复方骨肽，其特征在于，配方如下：
原料 配比％ 牛骨多肽 12-16％ Ⅱ型胶原蛋白肽 0.5-1.2％ 酪蛋白磷酸肽 0.1-0.2％ 乳清蛋白 35-45％ 大豆分离蛋白 4-8％ 酶解骨粉 3-3.5％ 麦芽糊精 20-24％ 维生素A、D、C 0.1-0.4％ 低聚果糖 8-12％ 接骨木莓果汁粉 0.75-1.2％ 姜黄 0.05-1％ 黑胡椒提取物 0.005-0.2％ 香精 0.05-1％ 黄原胶 0.5-0.8％ 三氯蔗糖 0.005-0.01％
；
所述的新型复方骨肽制备方法包括以下步骤：
S1、牛骨脱脂：牛骨破碎至大小1-3cm加入脱脂罐；以1∶1比例向脱脂罐加入纯化水，沸煮3h；脱脂罐温度降至70℃，真空度为-0.05MPa至-0.1MPa，分离上层油脂；常压蒸煮：再以1∶1比例继续向脱脂罐内加纯化水，升温至100℃后，蒸煮3h；静置后，分离上层油脂；
S2、脱脂后牛骨置于质量分数为1％的盐酸溶液中浸泡4h；牛骨与盐酸溶液的用量比例为1kg：3L；
S3、牛骨粉碎并磨成骨泥，加入质量分数2％的维生素C水溶液中，浸泡6-8h得到酶解底物；牛骨与维生素C水溶液的用量比例为1kg：3L；
S4、按质量比1％加入酸性蛋白酶酶解2-3h；调整pH至7，按质量比1％加入中性蛋白酶酶解处理1-2h；酶解结束后，升温；
S5、过滤得到水解液；
S6、浓缩：将水解液采用刮板浓缩器浓缩得到质量分数30％的浓缩提取液；
S7、包埋干燥后制成颗粒即为牛骨多肽。
2.根据权利要求1所述的新型复方骨肽，其特征在于，配方如下：
原料 配比％ 牛骨多肽 15.000 Ⅱ型胶原蛋白肽 0.850 酪蛋白磷酸肽 0.150 乳清蛋白 40.000 大豆分离蛋白 5.000 酶解骨粉 3.500 麦芽糊精 22.560 维生素A、D、C 0.375 低聚果糖 10.000 接骨木莓果汁粉 0.750 姜黄 0.050 黑胡椒提取物 0.005 香精 1.000 黄原胶 0.750 三氯蔗糖 0.01
。
3.权利要求1-2任一项所述的新型复方骨肽在制备药物或保健品中的应用，其特征在于，所述的药物或保健品用于促进骨骼生长、促进钙质吸收、增强骨密度、改善骨质疏松。
4.一种用于促进骨骼生长的药物或保健品，其特征在于，所述的药物或保健品中包括权利要求1-2任一项所述的新型复方骨肽。
5.根据权利要求4所述的药物或保健品，其特征在于，所述的药物中新型复方骨肽的含量为50％-100％。
6.根据权利要求5所述的药物或保健品，其特征在于，所述的药物中新型复方骨肽的含量为100％。
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