CN108902985A - 25-羟基维生素d3在制备保健食品中的应用 - Google Patents
25-羟基维生素d3在制备保健食品中的应用 Download PDFInfo
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- CN108902985A CN108902985A CN201810584277.1A CN201810584277A CN108902985A CN 108902985 A CN108902985 A CN 108902985A CN 201810584277 A CN201810584277 A CN 201810584277A CN 108902985 A CN108902985 A CN 108902985A
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- 239000011782 vitamin Substances 0.000 title claims abstract description 19
- 229940088594 vitamin Drugs 0.000 title claims abstract description 16
- 235000021318 Calcifediol Nutrition 0.000 claims abstract description 32
- JWUBBDSIWDLEOM-XHQRYOPUSA-N (3e)-3-[(2e)-2-[1-(6-hydroxy-6-methylheptan-2-yl)-7a-methyl-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol Chemical compound C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2\C1=C\C=C1/CC(O)CCC1=C JWUBBDSIWDLEOM-XHQRYOPUSA-N 0.000 claims abstract description 28
- 230000037182 bone density Effects 0.000 claims abstract description 24
- 230000036039 immunity Effects 0.000 claims abstract description 8
- 230000003908 liver function Effects 0.000 claims abstract description 6
- 239000011647 vitamin D3 Substances 0.000 claims description 35
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 26
- 239000011575 calcium Substances 0.000 claims description 21
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- 229910052791 calcium Inorganic materials 0.000 claims description 20
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
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- 201000011510 cancer Diseases 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
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- 210000000987 immune system Anatomy 0.000 claims description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 4
- VRIVJOXICYMTAG-IYEMJOQQSA-L iron(ii) gluconate Chemical compound [Fe+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O VRIVJOXICYMTAG-IYEMJOQQSA-L 0.000 claims description 4
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
- A23L33/155—Vitamins A or D
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
本发明提供了25‑羟基维生素D3在制备保健食品中的应用,添加25‑羟基维生素D3的保健食品有助于增加骨密度、有利于提高免疫力、有助于保护因肝功能低下造成的骨密度降低,具有开发为新一代保健食品的潜力。
Description
技术领域
本发明属于保健食品领域,具体涉及25-羟基维生素D3在制备保健食品中的应用。
背景技术
随着社会经济的进步,生活节奏的加快,人们对自身健康和食品认识不断提高,对于食品的追求不在局限于是提供人体新陈代谢和机体生长发育所必需的营养物质,而是具有调节身体机能,具备合理营养要素且能促进身体健康等方面的功效。因此,具有合理营养和保健功能的功能性食品越来越受到人们的亲睐。
保健食品是指声称具有特定保健功能或者以补充维生素、矿物质为目的的食品,即适宜于特定人群食用,具有调节机体功能,不以治疗疾病为目的,并且对人体不产生任何急性、亚急性或者慢性危害的食品。
第一代产品是针对食品的营养要素和其他有效成分的功能来推断该保健食品的功能,由于这些功能没有经过任何毒理药理等实验进行验证,《保健食品管理办法》实施后,此类食品已经归入普通食品来管理。另外,以补充维生素、矿物质,而不以提供能量为目的的“营养素补充剂”也属于保健食品的范畴,其作用是补充膳食供给的不足,预防营养缺乏和降低发生某些慢性退行性疾病的危险性。例如,补充钙、镁、碘、铁、锌、硒、VA、VC、VD、VE、B族维生素、叶酸、氨基酸、膳食纤维等营养物质的保健食品。
第二代保健食品是必须经过动物和人体实验,证明具有某项生理调节功能,目前经审批合格的保健食品中这类产品在市场上占绝大多数。
第三代保健食品是指产品不仅需要经过动物实验和人体临床实验来证明其具有某项生理调节功能,还需要进一步明确是何种成分或功能因子具有该项保健功能,明确该因子的化学结构、含量、作用机制和在食品中的稳定形态。我国目前此类产品较少,欧美、日本等国都在大力开发第三代保健产品。目前,我国大多数属于第一代或第二代产品,第三代保健食品仅占不到10%。
维生素D是甾醇类衍生物,人体皮下存在的7-脱氢胆固醇经紫外线照射产生的维生素 D3,麦角固醇紫外线照射后产生维生素D2;二者来源不同,功能一致,在人类营养中维生素 D的重要性在于调节钙和磷的代谢作用。维生素D促进钙和磷在肠道吸收,影响骨骼钙化过程,缺乏维生素D会引起儿童佝偻病,成人软骨和骨质疏松。维生素D的数量单位,以往用国际单位(I.U)表示,近来用重量表示,二者之间的关系如下:1I.U=0.025μg。
目前用于保健食品的维生素D只有维生素D2和维生素D3。维生素D2和维生素D3脂溶性比较强,吸收需要借助脂肪微乳,吸收较差,不同个体的差异性较大。吸收后的维生素D3或维生素D2大部分存储于脂肪中,使用效率极低,体重超标者使用效率更低。少部分进入血液中的维生素D3或维生素D需要运输到肝脏,再经肝脏代谢后才能变成能被人体利用的25-羟基维生素D,肝脏功能低下人群不能产生足够的维生素D储存形式的维生素D,导致体内的25-羟基维生素D的浓度偏低。肾脏、胰岛细胞、巨噬细胞、成骨细胞、破骨细胞及全身的普通细胞均需要25-羟基维生素D来参与和介导正常的细胞增殖、分化、凋亡及正常的细胞功能。我国保健食品中维生素D3的推荐剂量每天不高于600I.U,但是国外发达国家的产品用量已经超过1000I.U每天,低推荐量的维生素D3已经不能满足人体每天的需求。然而,每天食用过多的维生素D3和维生素D2,因为维生素D3和维生素D2半衰期较长为40天左右,大部分又储存于脂肪等组织中,突然的释放或转运到肝脏,会造成积蓄中毒,中毒表现主要有恶心、食欲下降、多尿、皮肤搔痒、肾衰竭,继而造成心血管系统的异常。
25-羟基维生素D3,化学名:(3β,5Z,7E)-9,10-开环胆甾-5,7,10(19)-三烯-3,25-二醇,其结构式如下:
维生素D缺乏症是全世界最常见的健康状况之一。据估计,在美国、加拿大、墨西哥、欧洲、亚洲、新西兰和澳大利亚,孩子和成人中超过30-50%都患维生素D缺乏症。即便在世界上阳光照射最强烈的地区也非常常见,这些地区包括中东地区包括沙特阿拉伯、卡塔尔、阿拉伯联合酋长国和印度。
据文献J ClinEndocrinol Metab.2005;90:3215–24报道,公认的血浆中的25-羟基维生素D (25-(OH)D)的含量低于5ng/ml的人被认为维生素D严重缺乏;血浆中的25-羟基维生素D 的含量5~20ng/ml的人被认为维生素D缺乏;血浆中的25-羟基维生素D的含量20~30ng/ml 的人被认为维生素D不足;血浆中的25-羟基维生素D的含量30~150ng/ml的人被认为维生素D正常;推荐的有利于身体健康的含量为40~100ng/ml;血浆中的25-羟基维生素D的含量高于150ng/ml的人有可能维生素D中毒。
最新研究发现维生素D缺乏症即人体血液中的25-羟基维生素D含量低于30ng/ml,会显著增加了慢性疾病的风险,包括骨密度降低、自身免疫病比如I型糖尿病、多发性硬化症和类风湿性关节炎;致命的癌症包括前列腺癌、结肠癌和乳腺癌;心血管疾病,还有中风,这些将调节免疫系统并对抗包括肺结核、上呼吸道感染的传染病。
25-羟基维生素D的血浓度大于30ng/ml被建议对健康有益,更优的是高于40ng/ml。研究发现身体中的许多组织和细胞有能力局部产生1,25-二羟基维生素D。局部产生的1,25-二羟基维生素D被认为去直接或间接控制负责细胞生长、血管生成、免疫调节、血管平滑肌和心肌细胞增殖、炎症活动的2000个基因。
维生素D对于人体健康意义重大,目前维生素D补充剂只有维生素D3和维生素D2,根据我国《保健食品原料目录(第一批)营养素补充剂原料目录(征求意见稿)》中对维生素 D3和维生素D2日最高限量为600I.U每天。文献J Clin Endocrinol Metab 93:677–681,2008报道,基于血液中25-羟基维生素D为19ng/ml的人群,连续11周每天服用1000I.U的维生素D2或维生素D3,仍然未能将血液中的25-羟基维生素D的浓度提高到30ng/ml以上。
25-羟基维生素D3少量存在于食物中,主要存在于蛋的蛋黄中和动物的血液中。其中鸡蛋的含量只有1~65μg/kg,吃鸡蛋获取足够的25-羟基维生素D3是不可取的,文献American Journal of Clinical Nutrition,2016,104(3):629报道,每天食用2枚25-羟基维生素D3加强的鸡蛋只能维持冬天光照降低导致的体内25-羟基维生素D浓度下降,而不能将体内的25-羟基维生素D3提高到30ng/ml及以上。每个鸡蛋中含量都不一样,食用过量的鸡蛋来补充25-羟基维生素D3会造成其他营养物质过剩,如胆固醇和卵磷脂过剩。
动物血液中的25-羟基维生素D3的含量极低,含量仅为5~50μg/kg,食用动物血液或血液制品补充25-羟基维生素D3也是不可取。
而将活性更强的1,25-二羟基维生素D3(骨化三醇)和1α-羟基维生素D3(阿法骨化醇) 用着保健食品日常服用,会造成高血钙和高血磷的副作用,造成维生素D中毒,中毒表现主要有恶心、食欲下降、多尿、皮肤搔痒、肾衰竭,继而造成心血管系统的异常。
发明内容
本发明要解决的技术问题是:为了解决现有技术中维生素D食物来源不足,而直接补充骨化三醇和阿法骨化醇会带来高血钙和高血磷的副作用,提供了25-羟基维生素D3在制备保健食品中的应用。
本发明的第一目的在于提供25-羟基维生素D3在制备保健食品中的应用。
优选的,所述的保健食品有助于增加骨密度、有利于提高免疫力、有助于保护因肝功能低下造成的骨密度降低、有助于I型糖尿病的好转、有助于多发性硬化症的保养、有助于类风湿性关节炎的保健、有助于防治致命的癌症、有助于心血管疾病的预防、有助于中风的预防,或能辅助调节免疫系统。
进一步的,所述的保健食品有助于增加骨密度。
更进一步的,所述的保健食品有助于保护因肝功能低下造成的骨密度降低。
优选的,所述的保健食品有助于提高免疫力。
优选的,所述的保健食品包括口服制剂、注射剂或气雾剂。
进一步的,所述的口服制剂包括软胶囊、片剂、硬胶囊、颗粒剂、粉剂、软糖或口服液。
优选的,所述的保健食品中25-羟基维生素D3每人每日的服用量为0~2000I.U/d。
进一步的,所述的保健食品中25-羟基维生素D3每人每日的服用量为10~1000I.U/d。
更进一步的,所述的保健食品中25-羟基维生素D3每人每日的服用量为16~600I.U/d。
优选的,所述的保健食品中25-羟基维生素D3每人每日的服用量为16~600I.U/d。所述的保健食品的剂型包括保健食品可接受的制剂,包括口服制剂、注射剂及气雾剂,优选的是口服制剂。所述的口服制剂包括软胶囊、片剂、硬胶囊、颗粒剂、粉剂、软糖及口服液等,更优选的是软胶囊、片剂和软糖。
本发明的第二目的在于提供一种保健食品,其特征在于包含25-羟基维生素D3及可接受的载体和/或稀释剂。
优选的,所述保健食品还包含无机盐和/或其他维生素。
进一步的,所述无机盐包括钙盐、镁盐、钾盐、锰盐、铁盐、锌盐、硒盐或铜盐。
进一步的,所述钙盐包括碳酸钙、乙酸钙、氯化钙、柠檬酸钙、葡萄糖酸钙、乳酸钙、磷酸氢钙、磷酸二氢钙、磷酸三钙、硫酸钙、L-乳酸钙或甘油磷酸钙;优选的是碳酸钙和葡萄糖酸钙,更优选的是碳酸钙。
进一步的,所述镁盐包括碳酸镁、硫酸镁、氧化镁或氯化镁;优选的是碳酸镁。
进一步的,所述钾盐包括磷酸氢二钾、磷酸二氢钾、氯化钾、柠檬酸钾或碳酸钾;优选的是磷酸氢二钾。
进一步的,所述锰盐的包括硫酸锰或葡萄糖酸锰;优选的是葡萄糖酸锰。
进一步的,所述铁盐的包括葡萄糖酸亚铁、富马酸亚铁、硫酸亚铁、乳酸亚铁或琥珀酸亚铁;优选的是葡萄糖酸亚铁。
进一步的,所述锌盐包括硫酸锌、枸橼酸锌、柠檬酸锌(三水)、葡萄糖酸锌、氧化锌或乳酸锌;优选的是葡萄糖酸锌。
进一步的,所述硒盐包括亚硒酸钠、硒酵母或L-硒-甲基硒代半胱氨酸,优选的是硒酸钠。
进一步的,所述铜盐包括硫酸铜或葡萄糖酸铜,优选的是葡萄糖酸铜。
优选的,所述维生素包括维生素A、视黄醇乙酸酯、棕榈酸视黄酯、β-胡萝卜素、维生素D2、维生素D3、维生素B1、盐酸硫胺素、硝酸硫胺素、维生素B2、核黄素、核黄素-5’- 磷酸钠、维生素B6(盐酸吡哆醇)、维生素B12(氰钴氨酸)、烟酸、烟酰胺、叶酸(喋酰谷氨酸)、D-生物素、重酒石酸胆碱、维生素C(抗坏血酸)、抗坏血酸钠、抗坏血酸钙、 L-抗坏血酸-6-棕榈酸盐(抗坏血酸棕榈酸酯)、维生素K1、泛酸钙、天然维生素E(D-α- 生育酚)、天然维生素E(D-α-醋酸生育酚)、天然维生素E(D-α-琥珀酸生育酚)、维生素E(dl-α-醋酸生育酚)、维生素E(dl-α-生育酚)或维生素E琥珀酸钙。
优选的,所述的含25-羟基维生素D3的保健食品,每人每日的服用量为0~2000I.U/d,优选的是10~1000I.U/d,更优选的是16~600I.U/d;
具体的,对于不同年龄段的消费者,具有如下细分的推荐服用量:0~3岁15~400I.U/d, 4~6岁16~600I.U/d,7~10岁16~600I.U/d,11~13岁16~600I.U/d,14~17岁16~600I.U/d,成人16~600I.U/d,孕妇16~600I.U/d,乳母16~600I.U/d。
本发明的有益效果是在于首次发现和证实了添加25-羟基维生素D3的保健食品有助于增加骨密度、有利于提高免疫力、有助于保护因肝功能低下造成的骨密度降低、有助于I型糖尿病的好转、有助于多发性硬化症的保养、有助于类风湿性关节炎的保健、有助于防治致命的癌症、有助于心血管疾病的预防、有助于中风的预防,或能辅助调节免疫系统,具有开发为新一代保健食品的潜力。
具体实施方式
以下结合实施例对本发明作进一步作具体描述,但不局限于此。
实施例1:含25-羟基维生素D3的保健食品对骨密度影响实验
(1)实验原理:机体中的钙绝大部分储存于骨骼及牙齿中,大鼠若摄入钙量不足会影响机体和骨骼的生长发育,表现为体重、身长、骨长、骨重、骨钙含量及骨密度低于摄食足量钙的正常大鼠。生长期大鼠在摄食低钙饲料的基础上分别补充碳酸钙(对照组)或受试补充含25-羟基维生素D3的保健食品(实验组),比较两者在促进机体及骨骼的生长发育,增加骨矿物质含量和增加骨密度功能上的作用,从而对受试样品增加骨密度的功能进行评价。
(2)仪器和试剂
仪器:原子吸收分光光度计、骨密度仪(单光子骨密度仪或双能,线骨密度仪或相关仪器)、精密卡尺、动物解剖器械、动物天平、105℃烘箱
试剂
硝酸,优级纯
高氯酸,优级纯
氧化镧,2%氧化镧溶液;称取分析纯氧化镧(含量大于99.98%)25克,加75ml优级纯盐酸,加去离子水至1000ml。
钙标准溶液
称取1.2486克分析纯碳酸钙(纯度大于99.99%),加50ml去离子水,加盐酸使之溶解。移入1000ml容量瓶中,加2%氧化镧溶液至刻度。此溶液1.00ml相当于500μg钙。储于聚乙烯瓶中,4℃保存,临用时稀释。
实验方法
(3)实验动物:出生2周左右的断乳大鼠,体重约60~75克,同一性别,每组8~12只。每组10只/笼群养,共分8组。购于上海斯莱克实验动物有限责任公司,实验动物质量许可证号:SCXK(沪)2007-0005,饲养于清洁级动物房,喂养条件包括标准饲料,自来水,室温保持在(24±2)℃,湿度50-60%,每日光照与黑暗时间各12h。
基础饲料:用此基础饲料配制低钙对照组以及各剂量组。
表1基础饲料配方(Ca2+计,调整Ca2+为150mg/100g饲料)
成分 | % |
酪蛋白 | 10.0 |
黄豆粉① | 15.0 |
混合盐② | 1.0 |
小麦面粉 | 54.0 |
玉米油或花生油 | 4.0 |
纤维素 | 2.0 |
混合维生素③ | 2.6 |
氯化胆碱 | 0.2 |
dl-蛋氨酸 | 0.2 |
淀粉④ | 11.0 |
①需高压处理后用
②混合盐:每kg混合盐中各组份含量:KH2PO4501.4g;NaCl74.0g;MgCO350.2g;乳酸亚铁54g;乳酸锌4.16g;MnCO33.5g;CuSO4-5H2O0.605g;Na2SeO36.6mg;KI7.76mg; CrCl3-6H2O0.292g;加蔗糖到1kg。
③混合维生素:每kg混合维生素中各组份含量:维生素A400,000I.U;维生素D3,100,000I.U;维生素E500I.U;维生素K5mg;维生素B1600mg;维生素B2600mg;维生素B6700mg;维生素B121mg;尼克酸3g;叶酸200mg;泛酸钙1.6g;生物素20mg;加蔗糖到1kg。
④可根据各组待测受试物(或碳酸钙)的需用量调节淀粉用量;
(4)剂量分组:
共八个剂量组分组见表2:
表2实施例1试验分组和剂量
受试物给予途径:经口灌胃给受25-羟基维生素D3油剂。
(5)实验步骤
出生4周的断乳大鼠经适应期一周后,禁食12小时,称体重,按体重随机分组,分笼饲养。饮用去离子水以避免从饮水中获得钙。
(6)观察指标
体重测定:禁食12小时后,测定体重。每周一次。
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。
股骨骨密度测定:
用骨密度仪,(单光子骨密度仪或双能X线骨密度仪或其它相关仪器)测量股骨中点及股远心端的骨密度。
在股骨远心端及股骨中点画出标记以确定测量点。
股骨中点测量点的确定:测量股骨全长,通过其中点,沿横截面方向画一直线,此为股骨中点测量点(截面)。
股骨远心端测量点的确定:于股骨远心端关节凹槽最下缘处确定测量点,通过此点,画一与上述股骨中点处标记平行的直线,此为股骨远心端测量点(截面)。
测量前用骨模型对仪器进行校准。
经校准可以使用后,继续测定骨模型并与其标准值进行比较,误差不得超过3%。
单光子骨密度仪或双能X线骨密度仪参数设定:测定方式:精密
扫描次数:2次
扫描方式:自动
探测域值:99%
测量股骨中点及股骨远心端骨密度
将待测骨放置于测量台上与骨密度仪探测头移动方向垂直;移动待测骨,令其待测点标记线与探测头移动轨迹于测量台上的垂直投影重合。开始测量,每点应重复测定两次,如两次结果不平行(误差大于10%),应当重新测量。将两次结果取平均值,以BW(骨宽)除 BMC(骨矿物质含量),其结果即为BMD(骨密度)。
(7)骨钙含量及饲料含钙量测定:
用原子吸收法测量。
样品采集与制备
饲料样品经均匀混合并过20目筛,在烘箱中烘干,置干燥器中冷却后称重,磨细。注意防止在样品制备过程中的污染。
取大鼠一侧股骨在105℃烘箱中烘干至恒重后,置于三角瓶中进行消化。
样品消化
根据样品中钙的含量,准确称取0.300~1.00克,置于150ml三角瓶中,上盖小漏斗,加入混合酸(硝酸:高氯酸4:1)15~20ml,在电热板上加热消化直至冒白烟并透明无色。酸液不够时可以再加入少量混合酸。消化液透明无色后加数毫升去离子水,煮沸以赶除剩余的酸,重复两次,最后消化液的体积不超过1ml。
消化样品时应同时作空白试验,加入与样品消化时相同体积的混合酸,在相同条件下消化。
测定
按照原子吸收分光光度计仪器说明书的步骤进行。测定液、标准溶液和空白均用0.5%氧化镧溶液稀释,定容。
数据处理及结果判定
实验数据采用方差分析,但需按方差分析的程序先进行方差齐性检验,方差齐,计算F 值,F值<F0.05,结论:各组均数间差异无显著性;F值≥F0.05,P≤0.05,用多个实验组和一个对照组间均数的两两比较方法进行统计;对非正态或方差不齐的数据进行适当的变量转换,待满足正态或方差齐要求后,用转换后的数据进行统计;若变量转换后仍未达到正态或方差齐的目的,改用秩和检验进行统计。
结果判定
骨钙含量或骨密度显著高于低钙对照组且不低于相应剂量碳酸钙对照组,钙的吸收率不低于碳酸钙对照组,可判定受试物具有增加骨密度功能的作用。
试验结果:
试验组3、4、5、6、7、8组F值均大于等于F0.05,食用25-羟基维生素D3有助于增加骨密度。
实施例2:含25-羟基维生素D3的保健食品提高免疫力验证
原理:用小鼠腹腔巨噬细胞吞噬鸡红细胞实验(半体内法),在体内腹腔巨噬细胞能吞噬鸡红细胞。据此判断巨噬细胞的吞噬功能。如果两个剂量组为阳性,则判断本品有提高免疫力的功能。
仪器和材料
显微镜、鸡红细胞、丙酮、甲醇、生理盐水、Giemsa染液。
实验步骤:鸡红细胞悬液制备取鸡血置于有玻璃珠的锥形瓶中,朝一个方向充分摇动,以脱纤维。用生理盐水洗涤2~3次,离心(2000r/min,10min),去上清,用生理盐水配成20% (v/v)的鸡红细胞悬液。
实验动物:出生2周左右的断乳大鼠,体重约60~75克,同一性别,每组8~12只。每组10只/笼群养,共分8组。购于上海斯莱克实验动物有限责任公司,实验动物质量许可证号:SCXK(沪)2007-0005,饲养于清洁级动物房,喂养条件包括标准饲料,自来水,室温保持在(24±2)℃,湿度50-60%,每日光照与黑暗时间各12h。
试验分组:
共7个组喂养基础食量加经胃部灌食25-羟基维生素D3油剂,喂养8周后,用于试验见表3:
表3实施例2试验分组和剂量
吞噬功能测定
每鼠腹腔注射20%鸡红细胞悬液1ml。间隔30min,颈椎脱臼处死动物,将其仰位固定于鼠板上,正中剪开腹壁皮肤,经腹腔注入生理盐水2ml,转动鼠板1min。然后吸出腹腔洗液1ml,平均分滴于2片载玻片上,放入垫有湿沙布的搪瓷盒内,移置37℃孵箱温育30min。孵毕,于生理盐水中漂洗,以除去未贴片细胞。晾干,以1:1丙酮甲醇溶液固定,4%(v/v)Giemsa磷酸缓冲液染色3min,再用蒸馏水漂洗晾干。
油镜下计数巨噬细胞,每张片计数100个,按下式计算吞噬百分率和吞噬指数。
吞噬鸡红细胞的巨噬细胞数:
在计数时,同时观察鸡红细胞被消化的程度。借以判定巨噬细胞吞噬与消化功能,通常分为4级:
1级:未消化。被吞噬的鸡红细胞完整,胞质浅红或浅黄带绿色,胞核浅紫色。
2级:轻度消化。胞质浅黄绿色、胞核固缩呈紫蓝色。
3级:重度消化。胞质淡染,胞核淡浅灰色。
4级:完全消化。巨噬细胞内仅见形态类似鸡红细胞大小的空泡,边缘整齐,胞核隐约可见。
25-羟基维生素D含量测定:
取眼球血,-70℃液氮保存,以25-羟基维生素D2为内标,用LC-MS/MS测量,25-羟基维生素D包含25-羟基维生素D2和25-羟基维生素D3。测定值如表4所示:
表4各组大鼠体内25-羟基维生素D含量
数据处理及结果判定
以吞噬百分率或吞噬指数表示小鼠巨噬细胞的吞噬能力。吞噬百分率需进行数据转换,式中P为吞噬百分率,用小数表示。在进行方差分析时,需按方差分析的程序先进行方差齐性检验,方差齐,计算F值,F值<F0.05,结论:各组均数间差异无显著性;F值≥F0.05,P≤0.05,用多个实验组和一个对照组间均数的两两比较方法进行统计;对非正态或方差,不齐的数据进行适当的变量转换,待满足正态或方差齐要求后,用转换后的数据进行统计:若变量转换后仍未达到正态或方差齐的目的,改用秩和检验进行统计。受试样品组的吞噬百分率或吞噬指数与对照组比较,差异均有显著性,方可判定该项实验结果阳性。
试验结果:
试验结果表明,食用25-羟基维生素D3的2~7组大鼠巨噬细胞的吞噬能力显著区别于对照组。
实施例3:软胶囊剂型的25-羟基维生素D3的制备
在氩气保护下,将含量99.8%的一水合25-羟基维生素D30.157g溶于300g无水乙醇中,加1.0克BHT制备得到重量比为0.05088%的乙醇溶液,用高效液相检测溶液含量,根据含量进行调整。再向其中加入约29692.872g椰子油分馏甘油三酯,制备得到含量重量比为0.00052%的一水合25-羟基维生素D3的油性溶剂,并用高效液相检测含量,相对偏差控制在3%以内, pH=4.5。
另外,将明胶7600g、甘油2200g、纯化水10000g、氧化钛200g及红色氧化铁60g加热搅拌调制成明胶溶液。
用连续式软胶囊制造机对上述一水合25-羟基维生素D3的油性溶液进行包囊以制备软胶囊。得到的软胶囊每个平均重量1.0g,内含溶剂600mg。每个胶囊含一水合25-羟基维生素 D3125I.U,装量差异±10%。
含量测定方法:
样品处理:
取10个软胶囊精确去壳。用尼龙0.45μm过滤其中部分用于检测。
液相条件:
装置:高相液相,配UV检测器。
色谱柱:waters C18,4.6mm×150mm,5μm。
流动相:甲醇:水=800:200(V:V)。
流速:1.5ml/min。
检测波长:265nm。
进样量:500uL。
计算:
F:前-25-羟基维生素D3校正因子;
As,pre:本品中预25-羟基维生素D3峰面积;
As:中25-羟基维生素D3的峰面积;
A:标准溶液中25-羟基维生素D3的峰面积;
Apre:标准溶液中预25-羟基维生素D3的峰面积;
m:称取试料的质量,单位为克(g);
c:标准溶液的浓度,单位为I.U每毫升(I.U/mL);
n:试料的稀释倍数。
实施例4:含25-羟基维生素D3和碳酸钙的片剂的制备
处方(10000片量,规格:4±0.4g)
表5实施例4配方表
将碳酸钙和淀粉过80目筛;
将0.1kg苯甲酸钠溶解于到20kg蒸馏水中,再加入10kg淀粉分散均匀,加热至约80℃使糊化,冷却至温浆后使用。
精密称取62.50mg的25-羟基维生素D3,避光下用无水乙醇100ml溶解,加入0.5gBHT。将25-羟基维生素D3的乙醇溶液加入到糊化淀粉中,搅拌30min直到均一。
分批加入淀粉10kg,搅拌均匀后过20目尼龙筛制湿颗粒;将湿颗粒于50~60℃烘箱干燥30min,用20目筛进行整粒;整粒后颗粒与1.5kg滑石粉和3.5kg淀粉混合均匀,以φ8mm冲模进行冲模压片,片重差异不大于10%,每个单位制剂含25-羟基维生素D3含量为250I.U±10%,含钙600mg。
片重差异:取20片精密称定重量,求得平均片重,再称定各片的重量,按下式计算片重差异:
片重差异=(单片重-平均片重)/平均片重×100%
含量测定方法:
样品处理:
称取单个片剂,于100mL棕色容量瓶中,加10mL水于45-50℃水浴中2分钟,确保片剂分解。如果4-5分钟后仍有粉末未完全溶解,使用超声震荡使样品完全溶解。将容量瓶从超声震荡仪中取出,加入40mL甲醇,并轻晃至完全溶解。晃动时再加入40mL甲醇(A.7.1) 再晃动。将容量瓶放置至室温,甲醇定容并混合均匀。用尼龙0.45μm过滤其中部分用于检测,弃用最初1mL滤液。
液相条件:
装置:高相液相,配UV检测器。
色谱柱:waters C18,4.6mm×150mm,5μm。
流动相:甲醇:水=800:200(V:V)。
流速:1.5ml/min。
检测波长:265nm。
进样量:500uL。
计算:
F:前-25-羟基维生素D3校正因子;
As,pre:本品中预25-羟基维生素D3峰面积;
As:中25-羟基维生素D3的峰面积;
A:标准溶液中25-羟基维生素D3的峰面积;
Apre:标准溶液中预25-羟基维生素D3的峰面积;
m:称取试料的质量,单位为克(g);
c:标准溶液的浓度,单位为I.U每毫升(I.U/mL);
n:试料的稀释倍数。
实施例5:含25-羟基维生素D3和碳酸钙的复合维生素片剂的制备
处方(10000片量,规格:4±0.4g)
表6实施例5处方信息
将碳酸钙和淀粉过80目筛;
将0.1kg苯甲酸钠、维生素C、维生素K、生物素、维生素B12、烟酸、维生素B6、维生素B1、核黄素、叶酸、葡萄糖酸亚铁、泛酸钙、磷酸氢钙、碘酸钾、氧化镁、葡萄糖酸锌、 L-硒-甲基硒代半胱氨酸、葡萄糖酸铜、碳酸镁溶解于到20kg蒸馏水中,加柠檬酸调节pH=5~6,再加入10kg淀粉分散均匀,加热至约80℃使糊化,冷却至温浆后使用。
精密称取62.50mg的25-羟基维生素D3,避光下用无水乙醇100ml溶解,加入0.5gBHT,加入维生素A和维生素E,搅拌溶解清。
将25-羟基维生素D3的乙醇溶液加入到糊化淀粉中,搅拌30min直到均一。
分批加入淀粉10kg,搅拌均匀后过20目尼龙筛制湿颗粒;将湿颗粒于50~60℃烘箱干燥30min,用20目筛进行整粒;整粒后颗粒与1.5kg滑石粉和3.5kg淀粉混合均匀,以φ8mm 冲模进行冲模压片,片重差异不大于10%,25-羟基维生素D3含量为250I.U±10%。
实施例6含25-羟基维生素D3的软糖的制备
表7实施例6处方信息
先将5kg白糖和麦芽糖浆混合后加入5kg水中进行溶糖,再用加温进行熔糖,当白糖颗粒完全熔化即可。
精密称取150mg的25-羟基维生素D3,避光下用无水乙醇100g溶解,加入0.6gBHT,加入乙基麦芽酚。
现将各种食用胶按照配料表称重后加入纯净水浸润4h,使其充分吸水溶胀,再在相应的温度60-80℃条件下水浴2h溶解。
在预冷的糖溶液中将各食用胶溶液逐一加入并不断搅拌,将25-羟基维生素D3的乙醇溶液加入到体系中。静置5h。将静置后的糖胶混合液灌注于模型中,冷却5h,凝固后脱模,得含25-羟基维生素D3的软糖。
以上述依据本发明的理想实施例为启示,通过上述的说明内容,相关工作人员完全可以在不偏离本项发明技术思想的范围内,进行多样的变更以及修改。本项发明的技术性范围并不局限于说明书上的内容,必须要根据权利要求范围来确定其技术性范围。
Claims (23)
1.25-羟基维生素D3在制备保健食品中的应用。
2.如权利要求1所述的应用,其特征在于:所述的保健食品有助于增加骨密度、有利于提高免疫力、有助于保护因肝功能低下造成的骨密度降低、有助于I型糖尿病的好转、有助于多发性硬化症的保养、有助于类风湿性关节炎的保健、有助于防治致命的癌症、有助于心血管疾病的预防、有助于中风的预防,或能辅助调节免疫系统。
3.如权利要求1所述的应用,其特征在于:所述的保健食品有助于增加骨密度。
4.如权利要求3所述的应用,其特征在于:所述的保健食品有助于保护因肝功能低下造成的骨密度降低。
5.如权利要求1所述的应用,其特征在于:所述的保健食品有助于提高免疫力。
6.如权利要求1所述的应用,其特征在于:所述的保健食品包括口服制剂、注射剂或气雾剂。
7.如权利要求6所述的应用,其特征在于:所述的口服制剂包括软胶囊、片剂、硬胶囊、颗粒剂、粉剂、软糖或口服液。
8.如权利要求1所述的应用,其特征在于:所述的保健食品中25-羟基维生素D3每人每日的服用量为0~2000I.U/d。
9.如权利要求8所述的应用,其特征在于:所述的保健食品中25-羟基维生素D3每人每日的服用量为10~1000I.U/d。
10.如权利要求8所述的应用,其特征在于:所述的保健食品中25-羟基维生素D3每人每日的服用量为16~600I.U/d。
11.一种保健食品,其特征在于包含25-羟基维生素D3及可接受的载体和/或稀释剂。
12.如权利要求11所述的保健食品,其特征在于:所述保健食品还包含无机盐和/或其他维生素。
13.如权利要求11所述的保健食品,其特征在于:所述无机盐包括钙盐、镁盐、钾盐、锰盐、铁盐、锌盐、硒盐或铜盐。
14.如权利要求13所述的保健食品,其特征在于:所述钙盐包括碳酸钙、乙酸钙、氯化钙、柠檬酸钙、葡萄糖酸钙、乳酸钙、磷酸氢钙、磷酸二氢钙、磷酸三钙、硫酸钙、L-乳酸钙或甘油磷酸钙。
15.如权利要求13所述的保健食品,其特征在于:所述镁盐包括碳酸镁、硫酸镁、氧化镁或氯化镁。
16.如权利要求13所述的保健食品,其特征在于:所述钾盐包括磷酸氢二钾、磷酸二氢钾、氯化钾、柠檬酸钾或碳酸钾。
17.如权利要求13所述的保健食品,其特征在于:所述锰盐包括硫酸锰或葡萄糖酸锰。
18.如权利要求13所述的保健食品,其特征在于:所述铁盐包括葡萄糖酸亚铁、富马酸亚铁、硫酸亚铁、乳酸亚铁或琥珀酸亚铁。
19.如权利要求13所述的保健食品,其特征在于:所述锌盐包括硫酸锌、枸橼酸锌、三水柠檬酸锌、葡萄糖酸锌、氧化锌或乳酸锌。
20.如权利要求13所述的保健食品,其特征在于:所述硒盐包括亚硒酸钠、硒酵母或L-硒-甲基硒代半胱氨酸。
21.如权利要求13所述的保健食品,其特征在于:所述铜盐包括硫酸铜或葡萄糖酸铜。
22.如权利要求12所述的保健食品,其特征在于:所述其他维生素包括维生素A、视黄醇乙酸酯、棕榈酸视黄酯、β-胡萝卜素、维生素D2、维生素D3、维生素B1、盐酸硫胺素、硝酸硫胺素、维生素B2、核黄素、核黄素-5’-磷酸钠、维生素B6、维生素B12、烟酸、烟酰胺、叶酸、D-生物素、重酒石酸胆碱、维生素C、抗坏血酸钠、抗坏血酸钙、L-抗坏血酸-6-棕榈酸盐、维生素K1、泛酸钙、D-α-生育酚、D-α-醋酸生育酚、D-α-琥珀酸生育酚、dl -α-醋酸生育酚、dl-α-生育酚或维生素E琥珀酸钙。
23.如权利要求12所述的保健食品,其特征在于:所述保健食品中25-羟基维生素D3每人每日的服用量为16~600I.U/d。
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