CN111248445A - 一种增加骨密度的含枸杞多糖的组合物 - Google Patents
一种增加骨密度的含枸杞多糖的组合物 Download PDFInfo
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- CN111248445A CN111248445A CN202010167567.3A CN202010167567A CN111248445A CN 111248445 A CN111248445 A CN 111248445A CN 202010167567 A CN202010167567 A CN 202010167567A CN 111248445 A CN111248445 A CN 111248445A
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- lycium barbarum
- bone
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- barbarum polysaccharide
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Abstract
本发明涉及一种增加骨密度的含枸杞多糖的组合物，其特征在于由以下原料按重量份数比制成：钙20‑30份、氨基葡萄糖盐酸盐50‑70份、硫酸软骨素钠20‑40份、初乳碱性蛋白2‑6份、枸杞多糖10‑20份。
Description
技术领域
本发明涉及一种增加骨密度的含枸杞多糖的保健组合物及检测方法，属于保健食品技术领域。
背景技术
随着中国人口老龄化日益明显，骨质疏松愈来愈成为困扰中老年人的重大问题。骨质疏松表现为以骨量减少、骨组织显微结构退化为特征，并导致骨的脆性增高、骨折危险性增加。其骨量减少包括骨矿物质和基质等比例的减少；骨微结构退变是指由于骨组织吸收和形成骨代谢失衡等原因所致，表现为骨小梁结构破坏、变细和断裂；骨的脆性增高、骨力学强度下降、骨折危险度增加，其载荷承受力降低从而容易发生微细骨折或完全骨折。
由于人体骨骼的生长主要依靠骨细胞，成骨细胞和破骨细胞。成骨细胞负责建立新骨；破骨细胞负责代谢旧骨。成骨细胞是骨形成细胞，对骨组织的生长、发育、损伤修复、骨代谢平衡与骨量维持起关键作用。成骨细胞的增殖速度反映了成骨细胞的活性，而成骨细胞的活性决定着新增骨量的多少。单位体积内骨量的多少决定了骨密度的大小，因此，成骨细胞的增殖速度和活性在一定程度上影响着骨密度的大小。
目前现有的增加骨密度的保健食品多以补钙为主，复配一些促进钙吸收的原料，这类产品不能从根本上解决降低骨质流失和促进成骨细胞增殖的问题，因此，开发一种安全有效的增加骨密度的保健食品，促进骨骼生长，修复骨质，具有重大的意义。
钙是人体中重要的营养元素，是维持骨骼强壮的前提条件。补钙被公认为是防治骨质疏松的有效措施，是骨质疏松症的一个基础治疗。其机理主要是降低骨转换，抑制甲状旁腺的过度分泌，对骨密度有保护作用，并可促进骨的形成和保持骨骼强度。
氨基葡萄糖能降低促炎症转录因子的浓度，增强软骨特定部位的韧性，并且通过抑制水解酶的活性和减少脂质、蛋白质的氧化来阻止胶原蛋白变性。氨基葡萄糖通过刺激黏多糖的生化合成及增加骨骼钙质的摄取量来提高骨与软骨组织的代谢功能与营养吸收，亦能改善及增强滑膜液的黏稠度，同时氨基葡萄糖也是蛋白多糖合成的基本物质，可以特异性地作用于关节软骨，恢复软骨细胞正常的代谢功能，刺激软骨细胞产生具有正常多聚体结构的蛋白多糖，亦可抑制损伤软骨的酶如胶原蛋白酶和磷脂酶A2的活性。
硫酸软骨素是一种结构较复杂、硫酸化的糖胺聚糖，是蛋白多糖的重要组成成分，是由己糖羧酸和己糖氨基交替组成的带有负电荷的直链碳水化合物。硫酸软骨素的功能主要通过所组成的蛋白聚糖来体现，大致分为结构功能和调节功能，其分子中含有多个硫酸基和羧基，故呈酸性，可与钠、钾、钙离子等结合成盐；可促进细胞粘附，提高骨组织的强度和韧性；可键合和调节细胞因子和生长因子，抑制蛋白酶分解，并且可影响细胞的粘附、迁移、增殖和分化，具有生物活性。
牛初乳中含有大量的促进成骨细胞生长、成熟、分化的生长因子,如维生素D3、乳铁蛋白、酪蛋白磷酸肽、核糖核酸酶等,能促进成骨细胞由不成熟向成熟分化,诱导成骨细胞合成胶原和基质蛋白质,从而促进骨基质的形成、促进钙化、加速骨折愈合。
初乳碱性蛋白粉以牛初乳为原料,经杀菌、脱脂、离心分离、去除酪蛋白、α-乳白蛋白、β-乳球蛋白,微滤、超滤、冷冻干燥等工艺而制得,为活性蛋白，分子量小，仅有30KD；可直接作用于骨骼细胞，具有良好的促进骨骼生长、增加骨密度和骨量的作用,具有促进成骨细胞增殖和调节破骨细胞的功能,保持两者的动态平衡，促进骨骼生长，修复骨质。采用初乳碱性蛋白作为入骨成骨的吸附剂，是从根本上解决了补钙利用率低的问题。
高春燕等研究了采用混合酶法对枸杞多糖进行提取，提取的最佳条件为：液料比15:1，pH5.0，提取温度50℃，提取时间2h，酶添加量为0.1％纤维素酶+0.1％木瓜蛋白酶+0.1％果胶酶，枸杞多糖的提取率为6.2％。
邹东恢等研究了采用双酶法提取枸杞多糖，最佳的提取工艺为：液料比25:1，pH6.0，提取温度55℃，提取时间2h，酶的总添加量为0.4％，其中纤维素酶与木瓜蛋白酶的比例为2:1，枸杞多糖的提取率为16.5％。
另外，本产品中特别添加了枸杞多糖，枸杞多糖对骨髓间充质干细胞具有促进增殖作用，能促进骨髓间充质干细胞向成骨细胞转化。骨髓间充质干细胞是成骨细胞的前体细胞，有较强的增殖分化成骨能力，因此，促进骨髓间充质干细胞的增殖及向成骨细胞方向转化在骨损伤修复成骨的过程中具有重要意义。
本发明提出的增加骨密度的组合物，各成分协同作用，促进钙吸收和成骨细胞增殖，抑制骨质流失，增加骨密度的效果显著。
发明内容
为解决本领域存在的上述问题，本发明提出了一种增加骨密度的含枸杞多糖的组合物，本组合物中含有多种原料做为功效成分，其中各组分之间在增加骨密度方面有显著的协同作用。
本发明所述一种增加骨密度的含枸杞多糖的组合物，包括钙、氨基葡萄糖盐酸盐、硫酸软骨素钠、初乳碱性蛋白、枸杞提取物。
一种增加骨密度的含枸杞多糖的组合物，其特征在于由以下原料按重量份数比制成：钙20-30份、氨基葡萄糖盐酸盐50-70份、硫酸软骨素钠20-40份、初乳碱性蛋白2-6份、枸杞多糖10-20份、枸杞多肽0.1-0.5份。
优选地，一种增加骨密度的含枸杞多糖的组合物，其特征在于由以下原料按重量份数比制成：钙25份、氨基葡萄糖盐酸盐60份、硫酸软骨素钠30份、初乳碱性蛋白4份、枸杞多糖15份、枸杞多肽0.1份。
其中所述钙是以生物碳酸钙、葡萄糖酸钙、乳酸钙、柠檬酸钙中的一种或多种复合形式添加。
其中，所述组合物通过以下方法制备：将各组分混匀制成软材，然后制粒、干燥和整粒，制成固体制剂，所述固体制剂选自片剂、胶囊或颗粒剂。
其中，所述固体制剂中还包括制剂成型用辅料，包括淀粉、糊精、水、微晶纤维素、羟丙甲基纤维素、硬脂酸镁中的一种或多种。
枸杞多糖的提取：
(1)取适量的枸杞粉，加入20倍的纯化水，混合均匀；
(2)置于50℃的水浴锅中，水浴20min，调节pH值为4.5；
(3)加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，混合均匀，酶解2h；
(4)将酶提取液置于沸水浴10min灭酶，冷却至室温，离心，得到离心液；
(5)向离心液中加入95％乙醇(体积分数)，到85％乙醇(体积分数)，静置，离心10000rpm，30分钟，沉淀备用，冷冻干燥，得到枸杞多糖A，提取率为17.6％。
(6)回收离心10000rpm的上清液中的乙醇，用80％乙醇通过活性炭对枸杞多肽的混合物洗脱，脱色脱腥，冷冻干燥，得到枸杞多肽A。
枸杞多糖的优选提取方法：
取适量的枸杞粉，加入20倍的氨基葡萄糖盐酸盐的水溶液(浓度3％)，混合均匀；置于45℃的水浴锅中，水浴30min，调节pH值为5；加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，混合均匀，酶解2h；将酶提取液置于沸水浴10min灭酶，冷却至室温，离心，得到离心液；向离心液中加入95％乙醇(体积分数)，到85％乙醇(体积分数)，静置，离心10000rpm，30分钟，沉淀备用，冷冻干燥，得到枸杞多糖B，提取率为20.3％。回收离心10000rpm的上清液中的乙醇，用80％乙醇通过活性炭对枸杞多肽的混合物洗脱，脱色脱腥，冷冻干燥，得到枸杞多肽B。
实验例：枸杞多糖B的制备的优选过程
氨基葡萄糖盐酸盐的水溶液(浓度3％)的优选过程
将适量枸杞粉，分别加入20倍的不同浓度的氨基葡萄糖盐酸盐水溶液(1％、2％、3％、4％、5％)，混合均匀，温度50℃水浴20min，调节pH4.5，加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，酶解2h，结果如图1所示。
由图1可知，浓度为3％时，枸杞多糖的提取率最高。
水浴温度的优选过程
将适量枸杞粉，分别加入20倍的3％氨基葡萄糖盐酸盐水溶液，混合均匀，分别在40、45、50、55、60℃下水浴20min，调节pH4.5，加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，酶解2h，结果如图2所示。
由图2可知，水浴温度为45℃时，枸杞多糖的提取率最高。
水浴时间的优选过程
将适量枸杞粉，分别加入20倍的3％氨基葡萄糖盐酸盐水溶液，混合均匀，温度50℃分别水浴5、10、20、30、40min，调节pH4.5，加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，酶解2h，结果如图3所示。
由图3可知，随着水浴时间的延长，枸杞多糖的提取率在逐渐提高，30min和40min的提取率相差不大，故选择水浴30min。
将适量枸杞粉，分别加入20倍的3％氨基葡萄糖盐酸盐水溶液，混合均匀，温度50℃水浴20min，调节pH分别为4、4.5、5、5.5、6，加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，酶解2h，结果如图4所示。
由图4可知，pH为5时，枸杞多糖的提取率最高。
由图1、2、3、4总结得出，枸杞多糖B的提取工艺为：20倍3％的氨基葡萄糖盐酸盐水溶液，45℃水浴30min，调节pH为5，测得枸杞多糖B得提取率为20.3％。
枸杞多糖B比枸杞多糖A的提取工艺更优选。
常规的提取方法所提取出的枸杞多糖中会掺杂有大量的蛋白质和多肽、氨基酸，导致枸杞多糖的纯度偏低，本实验采用酶解的方法提取枸杞多糖，然后用紫外分光光度计在200-1000nm范围内对枸杞多糖B进行紫外光谱扫描，如图5所示，可以看出在280nm处的曲线未出现剧烈波动或峰值，说明此方法提取的枸杞多糖B中蛋白质和多肽、氨基酸含量小，保证了枸杞多糖的纯度，提高了多糖多肽的分离效率。
附图说明
图1：氨基葡萄糖水溶液的浓度对枸杞多糖提取率的影响
图2：水浴温度对枸杞多糖提取率的影响
图3：水浴时间对枸杞多糖提取率的影响
图4：pH值对枸杞多糖提取率的影响
图5：枸杞多糖B紫外吸收图谱
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围
实施例1
一种增加骨密度的含枸杞多糖的组合物，由钙、氨基葡萄糖盐酸盐、硫酸软骨素钠、初乳碱性蛋白、枸杞多糖A组成，各组分按重量份计，钙23份(碳酸钙)、氨基葡萄糖盐酸盐65份、硫酸软骨素钠32份、初乳碱性蛋白3份、枸杞多糖A 15份、枸杞多肽A 0.1份。
上述各组分混合混匀后制成软材，然后制粒，干燥和整粒，获得组合物颗粒1。
枸杞多糖A、枸杞多肽A的制备：
取适量的枸杞粉，加入20倍的纯化水，混合均匀；
置于50℃的水浴锅中，水浴20min，调节pH值为4.5；
加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，混合均匀，酶解2h；
将酶提取液置于沸水浴10min灭酶，冷却至室温，离心5000rpm，得到离心液；
向离心液中加入95％乙醇(体积分数)，到85％乙醇(体积分数)，静置，离心10000rpm，30分钟，沉淀备用，冷冻干燥，得到枸杞多糖A；
回收离心10000rpm的上清液中的乙醇，用80％乙醇通过活性炭对枸杞多肽的混合物洗脱，脱色脱腥，冷冻干燥，得到枸杞多肽A。
实施例2
一种增加骨密度的含枸杞多糖的组合物，由钙、氨基葡萄糖盐酸盐、硫酸软骨素钠、初乳碱性蛋白、枸杞多糖B组成，各组分按重量份计，钙23份(碳酸钙)、氨基葡萄糖盐酸盐20份、硫酸软骨素钠32份、初乳碱性蛋白3份、枸杞多糖B 15份、枸杞多肽B 0.1份；
上述各组分混合混匀后制成软材，然后制粒，干燥和整粒，获得组合物颗粒2；
枸杞多糖B的提取：
取适量的枸杞粉，加入20倍的氨基葡萄糖盐酸盐的水溶液(浓度3％)，混合均匀；置于45℃的水浴锅中，水浴30min，调节pH值为5；加入溶解好的0.26％的木瓜蛋白酶和0.14％的果胶酶，混合均匀，酶解2h；
将酶提取液置于沸水浴10min灭酶，冷却至室温，离心5000rpm，得到离心液；
向离心液中加入95％乙醇(体积分数)，到85％乙醇(体积分数)，静置，离心10000rpm，30分钟，沉淀备用，冷冻干燥，得到枸杞多糖B；
回收离心10000rpm的上清液中的乙醇，用80％乙醇通过活性炭对枸杞多肽的混合物洗脱，脱色脱腥，冷冻干燥，得到枸杞多肽B。
实施例3 实施例1中枸杞多糖A的分析方法
1、对照品溶液的配制
精密称取无水葡萄糖对照品10mg，置于100ml容量瓶中，加水溶解，稀释至刻度，摇匀，即得对照品储备液。
精密吸取对照品储备液0.2、0.4、0.6、0.8、1.0ml，分别置于具塞试管中，分别加水补至2ml，各精密加入5.5％苯酚溶液1ml，摇匀，迅速精密加入硫酸5ml，50℃水浴10min，快速冷却至室温，以相应的试剂做空白，487nm处测定吸光度，以吸光度为纵坐标，浓度为横坐标，绘制标准曲线。
2、样品测定
称取实施例1的组合物颗粒样品适量，置于100ml容量瓶中，加水适量于沸水浴加热15min，冷却至室温补水至刻度，混匀后过滤，弃去初滤液，收集余下滤液。吸取滤液5ml，置于具塞试管中，加入无水乙醇30ml，混匀，于4℃冰箱中静置6h，离心，弃去上清液，残渣用80％乙醇洗涤数次，残渣用水溶解并定容至10ml，得样品溶液。
精确吸取实施例1的样品溶液0.5ml至具塞试管中，加水至2ml，加入5.5％苯酚溶液1ml，摇匀，迅速精密加入硫酸5ml，50℃水浴10min，快速冷却至室温，487nm处测定吸光度，根据标准曲线计算葡萄糖含量，根据系数折算粗多糖含量。
实施例4 实施例2中枸杞多糖B的分析方法
称取实施例2的组合物颗粒样品适量，其余步骤同实施例3。
实施例5：增加骨密度实验
1.样品：本发明增加骨密度的组合物2。
2.增加骨密度实验的实验方法：参照《保健食品检验与评价技术规范》中的增加骨密度功能检验方法。
3.实验动物：出生4周左右的断乳大鼠，体重约60-75g，同一性别，每组8-12只。
4.动物分组：本品人体推荐量为0.1g/kg.BW。实验设三个剂量组，以人体推荐量的5倍为其中一个剂量组，即0.5g/kg.BW,另外两个剂量组分别为1g/kg.BW、3g/kg.BW，同时，设一个低钙对照组和与高剂量水平相同的碳酸钙对照组。用低钙对照组饲料配制受试样品各剂量组和碳酸钙对照组。受试样品给予时间3个月。
5.股骨重量测定：动物喂养3个月后处死，剥离出右侧股骨，于105℃烤箱中烤至恒重，称量骨干重。
6.股骨骨密度测定：应用骨密度仪测量股骨中点及股骨远心端骨密度。
7.骨钙含量及饲料含钙量测定：用原子吸收法测定骨钙含量。
8.钙吸收试验：试验3周后将动物进行3天钙代谢试验。记录3天进食量，收集72小时粪便，鼠粪样品在80℃烘箱中烘干，置干燥器中冷却后磨细。原子吸收法测定饲料、粪便中钙含量。摄入钙(mg/d)＝饲料中钙含量(mg/g)×饲料消费量(g/d)，粪钙(mg/d)＝粪便中钙含量(mg/g)×粪便排出量(g/d)，钙表现吸收率＝(摄入钙-粪钙)/摄入钙×100％。
9.试验结果表1对大鼠体重、股骨的影响
备注：**与低钙对照组比较，P<0.01；★与高剂量碳酸钙组比较P<0.05。
由表1可见，三个剂量组和高剂量碳酸钙对照组的实验前、中、后体重与低钙对照组之间无显著性差异(P＞0.05)，三个剂量组和高剂量碳酸钙对照组的右股骨骨重均较低钙对照组极显著增加(P＜0.01)。受试物各剂量组与高剂量碳酸钙对照组比较，右股骨重均无显著性差异(P＞0.05)。
表2对大鼠骨密度及骨钙含量的影响
| 组别 | 动物数(只) | 左股骨中心点骨密度 | 左股骨远心端骨密度 | 右股骨骨钙含量 |
| 低钙对照组 | 12 | 0.2236±0.0135 | 0.3658±0.0267 | 234.65±56.24 |
| 低剂量组 | 12 | 0.2587±0.0125<sup>*</sup> | 0.3926±0.0192<sup>*</sup> | 241.36±68.45 |
| 中剂量组 | 11 | 0.2734±0.0097<sup>**</sup> | 0.4211±0.0185<sup>**</sup> | 246.37±70.12<sup>*</sup> |
| 高剂量组 | 11 | 0.2745±0.0112<sup>**</sup> | 0.4223±0.0174<sup>**</sup> | 251.72±65.27<sup>**</sup> |
| 高剂量碳酸 | 12 | 0.2697±0.0154<sup>**</sup> | 0.4169±0.0243<sup>**</sup> | 252.48±57.39<sup>**</sup> |
备注：*与低钙对照组比较，P＜0.05；**与低钙对照组比较，P＜0.01；★与高剂量碳酸钙组比较P＜0.05。
由表2可见，低剂量组的左股骨中心点骨密度和远心端骨密度均显著高于低钙对照组(P＜0.05)，高剂量碳酸钙对照组与中、高剂量组的左股骨中心点骨密度和远心端骨密度均极显著高于低钙对照组(P＜0.01)。中剂量组的右股骨骨钙含量显著高于对照组(P＜0.05)，高剂量组和高剂量碳酸钙对照组的右股骨骨钙含量极显著高于对照组(P＜0.01)。受试物各剂量组与高剂量碳酸钙对照组比较，骨密度及骨钙含量均无显著性差异(P＞0.05)。
表3对大鼠钙吸收的影响
由表3可见，钙吸收实验开始时，三个剂量组的体重和身长与低钙对照组以及高剂量碳酸钙对照组比较均无显著差异(P＞0.05)；实验结束时，受试物各组的体重、身长和钙表现吸收率与低钙对照组及高剂量碳酸钙对照组比较均无显著性差异(P＞0.05)。
由表1、表2、表3综合可知，本组合物具有增加骨密度的功能。
Claims (2)
1.一种增加骨密度的含枸杞多糖的组合物，其特征在于由以下原料按重量份数比制成：钙20-30份、氨基葡萄糖盐酸盐50-70份、硫酸软骨素钠20-40份、初乳碱性蛋白2-6份、枸杞多糖10-20份。
2.一种增加骨密度的含枸杞多糖的组合物，其特征在于由以下原料按重量份数比制成：钙25份、氨基葡萄糖盐酸盐60份、硫酸软骨素钠30份、初乳碱性蛋白4份、枸杞多糖15份。
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