CN112791171A - 姜或其提取物用于防治骨质疏松的用途 - Google Patents
姜或其提取物用于防治骨质疏松的用途 Download PDFInfo
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Abstract
本发明涉及姜(Zingiber officinale Rosc.)或其提取物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途。
Description
技术领域
本发明涉及姜(Zingiber officinale Rosc.)或其提取物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途。
背景技术
骨质疏松是一种常见的与年龄密切相关的骨骼疾病，其主要表现为骨骼密度和强度下降。如未得到有效治疗，骨质疏松可极大地增加中老年人骨折风险、影响行动能力，进而导致生活质量下降。引发骨质疏松的主要原因之一是骨重建受到影响。骨重建主要由骨分泌细胞(成骨细胞)和骨吸收细胞(破骨细胞)的相互作用完成，是维持骨骼系统稳定性和骨组织结构完整性的重要过程。由于雌激素水平下降可以导致破骨细胞的过度分化和功能亢进，因此，骨重建紊乱通常是更年期后女性发生骨质疏松的主要原因。此外，在正常的衰老的过程中，骨质疏松往往与破骨细胞的数量增加和功能增强密切有关，从而导致骨破坏的加快和骨量的流失。因此，破骨细胞是临床治疗骨质疏松的重要靶点。
许多信号分子在破骨细胞形成、分化、活化、存活、及骨吸收功能发挥的过程中起着重要的积极或消极的调节作用，其中最关键的2个因子：巨噬细胞集落刺激因子与核因子κB受体激活蛋白配体(Receptor activator of NF-κB ligand，RANKL)贯穿破骨细胞形成、分化、活化、发挥功能全过程。巨噬细胞集落刺激因子在骨微环境中主要由成骨细胞、骨髓间质细胞和骨细胞表达和分泌。巨噬细胞集落刺激因子的受体为前体细胞上的c-Fms，是一种酪氨酸激酶受体，其胞内段含多个酪氨酸残基。巨噬细胞集落刺激因子对破骨细胞分化的关键作用是促进核因子κB受体激活蛋白(receptor activator of NF-κB，RANK)在破骨细胞前体细胞膜上的表达。RANKL属肿瘤坏死因子蛋白超家族成员，可由成骨细胞谱系细胞，软骨及骨细胞，T、B淋巴细胞，内皮细胞表达。巨噬细胞集落刺激因子作用的发挥依赖于RANKL的存在。破骨细胞的分化过程主要由RANKL直接控制。RANKL与破骨细胞上的受体RANK相结合，RANKL介导的信号分子便通过RANK的胞内信号通路诱导释放核因子，直接调控破骨细胞分化相关基因的表达。在体内，RANKL有两种形式，分别是游离型与膜结合型。RANK是RANKL的受体，可由骨髓来源的树突状细胞、活化的T细胞和破骨细胞前体表达；RANK是破骨细胞分化、淋巴结形成、B细胞发育的关键分子。RANK缺乏内源性激酶活性，不能磷酸化并激活细胞内信号分子，需通过结合肿瘤坏死因子受体激活因子6(TRAFs)发挥功能，从而诱导破骨细胞分化和活化下游基因表达的相关信号通路蛋白。RANKL-RANK结合后可激活6条主要信号通路(核因子кB、JNK、ERK、p38、NFATc1、Akt)，调节破骨细胞形成、功能和存活。
破骨细胞对骨质的吸收过程，首先在骨的表面形成一个相对封闭的酸性化的微环境，褶皱缘(ruffled borders)的液泡H+-ATP酶将质子分泌到胞外的骨质吸收区，同时膜上的氯通道蛋白7氯离子通道在细胞外的酸性条件下分泌Cl-，H+和Cl-形成HCl，继而溶解羟基磷灰石晶体；同时也为蛋白溶解酶降解骨基质蛋白提供酸性条件，其中破骨细胞产生的组织蛋白酶K溶解骨基质的主要蛋白酶。转录因子如激活T细胞核因子1(Nuclear factor ofactivated T-cells，cytoplasmic 1，NFATc1)、核因子κB、MITF、c-Fos可激发特定基因段的转录，是破骨细胞分化相关的重要因子。NFATc1是破骨细胞分化的关键调节分子，破骨细胞前体对RANKL的最早反应即是召集RelA/p50和NFATc2至NFATc1，在钙离子的刺激下，诱导NFATc1短时的大量自我增殖、RANK活化抑制分子表达的减少，使破骨细胞生成得以发生。NFATc1可诱导积极调节子如组织蛋白酶K、抗酒石酸酸性磷酸酶、降血钙素受体、液泡ATP酶(v-ATPase)、破骨细胞相关受体(OSCAR)、β3整合素等基因的表达，也可诱导消极调节子抑制子如B淋巴细胞诱导成熟蛋白1的表达，促进成熟破骨细胞的形成。MITF可激活巨噬细胞集落刺激因子受体表达。RANK诱导c-Fos的表达可活化AP-1蛋白，诱导破骨细胞的形成，c-Fos基因的缺失会造成破骨细胞和巨噬细胞间细胞系分化的转变。
破骨细胞是体内唯一具有骨吸收功能的细胞，破骨细胞的数目增多或活性增强，将破坏正常的骨代谢平衡，导致骨量的丢失，从而导致骨质疏松和骨折的风险增加。目前临床上治疗骨质疏松的药物主要有基本补充剂、抑制骨吸收药物和促骨形成药物，这些药物存在长期服用副作用大、治疗效果不佳等局限性。因此开发价格低廉、副作用小、治疗效果好的骨质疏松治疗药物具有十分重要的意义。
发明内容
本发明人意外地发现姜或其提取物能够抑制破骨细胞的分化，从而可以抑制破骨细胞引起的骨量丢失。
因此，在一个方面，本发明提供姜(Zingiber officinale Rosc.)或其提取物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途。在某些实施方案中，所述的姜或其提取物作为唯一活性成分用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症。在某些实施方案中，所述的姜或其提取物也可以和维生素D和/或钙联合使用，用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症。
在另一个方面，本发明提供组合物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途，其中所述组合物由组分a)和组分b)组成，其中：
组分a)为姜(Zingiber officinale Rosc.)或其提取物，
组分b)为为维生素D和/或钙。。
在某些实施方案中，本发明所述产品为食品或药品。
在某些实施方案中，本发明所述的食品为液体饮料、固体饮料、奶粉、液态奶、发酵乳或奶片。
在某些实施方案中，本发明所述的提取物为姜的水提取物。
在某些实施方案中，本发明所述的提取物为姜粉。
在某些实施方案中，本发明所述的姜粉是姜的水提取物经干燥后获得的粉状物。
在某些实施方案中，本发明所述姜的水提取物是经一种提取方法提取获得，所述的提取方法包括以下操作：
将姜破碎，获得破碎的姜；
用水提取破碎的姜，获得姜的水提取物。
在某些实施方案中，本发明所述的提取方法还包括以下一种或多种操作：
a)浓缩；
b)杀菌；
c)干燥。
在某些实施方案中，用水提取破碎的姜时，姜和水的重量比优选为1:15～20，提取温度优选为60～90℃，提取时间优选为30～60分钟。
在某些实施方案中，所述的杀菌为UHT杀菌。杀菌温度优选为130～150℃，例如137±3℃，杀菌时间优选为1～5秒，例如3秒。
在某些实施方案中，所述的干燥为喷雾干燥。
在另一方面，本发明还提供一种组合物，由组分a)和组分b)组成，其中：
组分a)为姜(Zingiber officinale Rosc.)或其提取物，
组分b)为为维生素D和/或钙。
本发明中所述的“组分b)为维生素D和/或钙”表示组分b)可以为维生素D，也可以是钙，还可以是维生素D和钙二者的组合。
本发明中，所述的姜包括生姜和干姜。
本发明中，所述的生姜为姜科植物姜Zingiber officinale Rosc.的新鲜根茎。将生姜晒干或低温干燥后即为干姜。
本发明中，所述的食品包括人类或动物食用或者饮用的成品和原料以及按照传统既是食品又是中药材的物品。
本发明中，本发明所述的姜提取物可以商购获得，也可以依照本领域常规方法制备获得。例如，本发明所述姜提取物可以是黄山华绿园生物科技公司生产的姜速溶粉，也可以是湖南朗林生物资源股份有限公司生产的生姜提取物。
在某些实施例方案中，本发明所述的姜粉可以通过下述方法制备得到：
1)前处理：
选取优质的鲜姜，将鲜姜放入清洗机进行清洗即通过气泡清洗段，刷洗生姜表面的大部分泥沙，高压清洗环节，进行生姜的彻底清洗。对鲜姜切片，姜片的厚度应不超过5mm，对鲜姜切片进行烘干干燥，烘房温度控制在60～70℃为宜。
2)破碎匀浆：待纯水进入破碎机后；将干姜和水按质量比为1:15～20混合后共同粉碎，粉碎后料液进入胶体磨中进行匀浆，控制干姜破碎匀浆进料速度200-220Kg/H，得到水和干姜的混合浆液。
3)提取：将水和干姜的混合浆液混合，在温度≥80℃的条件下，搅拌30～60分钟，得到提取液。
4)离心：对提取液进行离心，转速为2000rpm，离心25分钟。
5)微滤：用0.2微米的微滤膜对离心后的料液进行微滤，得到微滤渗透液。
6)浓缩：对微滤渗透液进行RO膜浓缩，浓缩至料液固形物含量15-20％，得到浓缩液。
7)UHT杀菌：对浓缩液进行杀菌，杀菌温度137±3℃，时间3秒。
8)喷雾干燥：对杀菌液进行喷雾干燥，得干粉，过40目筛网，即得。
本发明的有益效果
本发明人发现姜或其提取物能够抑制破骨细胞的分化，从而可以抑制破骨细胞引起的骨量丢失，能够用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症。发明人还发现姜或其提取物对骨髓源性单核细胞没有明显毒性作用。姜或其提取物作为天然植物来源的功效原料可以用于防治骨质疏松，可单独食用，也可以功效成分的形式过添加到普通食品(如饮料(包括固体饮料和液体饮料)、液态奶、奶粉、发酵乳、奶片等)、保健品、药品等产品中。
附图说明
图1显示了48小时后MTT分析结果，结果显示：浓度小于32μg/ml的姜提取物下对破骨前体细胞BMMS没有细胞毒性作用；
图2显示了不同浓度的姜提取物抑制破骨细胞的分化的实验结果，其中A为抗酒石酸染液(TRAP染液)对破骨细胞的染色结果；B为破骨细胞的数量(TRAP染色3个核以上定义为破骨细胞)的定量技术结果；
图3显示了荧光素酶报告基因测定结果，结果显示姜提取物(4μg/ml和8μg/ml)能显著抑制RANKL诱导NFATc1的激活作用。
具体实施方式
下面结合本发明的具体实施例来进一步说明本发明的实质性内容，应理解，以下实施例仅用于说明本发明，但并不以此来限定本发明的保护范围。下面实施例中未注明具体条件者，按照常规条件或制造商建议的进行。所用药品或试剂未注明生产厂商者，均为可以通过市购获得的常规产品。
虽然以下实施例中所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，如果未特别说明，下面实施例中所用的材料和操作方法是本领域公知的。
以下实施例中所用姜提取物为速溶姜粉，购自黄山华绿园生物科技公司，货号20180503，使用时根据需要用水配制成不同浓度的溶液。
实施例
1.骨髓源性单核细胞培养
从6周龄C57BL/6J小鼠(购自澳大利亚动物资源中心，柏斯)中分离出胫骨和股骨，用1倍的磷酸缓冲盐溶液(phosphate buffer saline，PBS，PH＝7.4)反复冲洗骨髓，离心(1000rpm,1分钟)，用完全培养基对细胞进行重悬，并且添加重组巨噬细胞集落刺激因子(M-CSF，R&D Systems,MN,USA，终浓度为30ng/ml)，在37℃/5％CO2条件下进行培养，隔天进行培养基的更换，经过3天的培养，贴壁的细胞即为骨髓源性单核细胞(BMMs)，用来进行下一步的实验。
本实验中所用的完全培养基为含有10％胎牛血清(FBS)、2mM L-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的α-MEM培养基，其中α-MEM培养基购自Sigma。
2.MTT法检测姜提取物对骨髓源性单核细胞毒性的影响
将骨髓源性单核细胞接种于96孔板中，每孔约6×103个细胞/孔，每孔加入100μl完全培养基，在37℃/5％CO2条件下培养过夜，待细胞贴壁。第二天，除正常对照组外，给药组分别加入用不同浓度的姜提取物对骨髓源性单核细胞进行干预，使其终浓度分别为2μg/ml、4μg/ml、8μg/ml、16μg/ml和32μg/ml，然后再加MTT溶液(5mg/ml)20μl/孔，与细胞共在37℃/5％CO2条件下培养2h，小心吸去培养液，每孔加入150μl二甲基亚砜(dimethylsulfoxide，DMSO)，震荡10分钟充分溶解，用酶标仪在490nm波长检测吸光度值。
本实验中所用的完全培养基为含有10％胎牛血清(FBS)、2mM L-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的α-MEM培养基，其中α-MEM培养基购自Sigma。
3.破骨细胞分化实验
将6×103细胞/孔的BMMs种在96孔培养板中，每孔加入100μl含M-CSF(30ng/ml)和GST-rRANKL(50ng/ml，购自Abcam)的α-MEM培养基，给药组添加不同浓度的姜提取物(终浓度分别为2μg/ml、4μg/ml、8μg/ml、16μg/ml、32μg/ml)，同时设无药物对照组，无药对照组不添加姜提取物。细胞培养液每2天更换一次。5天后，吸掉培养液用PBS洗涤3次，用4％多聚甲醛的固定溶液固定15min，再用PBS洗涤3次，然后用抗酒石酸酸性磷酸酶染液(TRAP染液)在室温染色45min，把TRACP阳性多核细胞(>3核)定义为破骨细胞。
4.荧光素酶报告基因测定
为了研究姜提取物对NFATc1转录激活，用NFATc1应答的荧光素酶报告构建体稳定转染RAW264.7细胞(American Type Culture Collection(ATCC)；TIB-71TM；Manassas,VA,USA)，将转染的细胞以1.5×105细胞/孔的密度加入到48孔板中，并向每孔中加入姜提取物(终浓度为4μg/ml、8μg/ml)，在37℃/5％CO2条件下培养1小时。预处理后，向每孔中加入GST-RANKL(终浓度100ng/ml)，刺激细胞24小时。待刺激结束后，根据试剂盒(Promega,Sydney,NSW,Australia)说明书先溶解RAW264.7细胞，然后使用荧光素酶报告基因测定系统(BMG LABTECH,Ortenberg,Germany)测定NFATc1应答的荧光素酶的活性。
5.数据分析
本发明所有的数据均以平均数±标准差的形式表示。数据分析的方法为单因素方差分析，然后使用Dunnett检验进行多项对比。在*p<0.05时，视为差别具有统计学意义。
6.实验结果
MTT法是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中，而死细胞无此功能。通过MTT实验(参见图1)，发现32μg/ml以下各浓度的姜提取物对破骨前体细胞骨髓源性单核细胞(BMMs)的生长和存活没有显著性影响，即32μg/ml以下浓度的姜提取物对骨髓源性单核细胞没有明显毒性作用。
不同浓度的姜提取物(2μg/ml、4μg/ml、8μg/ml、16μg/ml和32μg/ml)对破骨细胞分化的影响如图2所示。结果显示：经过5天RANKL作用下，三个细胞核以上的抗酒石酸染液阳性的破骨细胞形成。姜提取物在没有毒性作用的条件下，2μg/ml姜的提取物对破骨细胞分化没有显著性的抑制作用。但其他各个浓度的姜提取物均可以抑制破骨细胞分化，三个细胞核以上的抗酒石酸染液阳性的破骨细胞计数显著性的降低，姜的提取物抑制破骨细胞分化是浓度依赖性的，姜提取物的最低抑制浓度是4μg/ml。
NFATcl是破骨细胞形成和分化的关键调节因子，NFATcl的激活是破骨细胞最终形成的标志性事件。为了进一步研究姜提取物抑制破骨细胞分化的分子机理，发明人采用荧光素酶报告法研究和分析了姜提取物对RANKL作用下NFATc1活性的影响。如图3所示，在RANKL的刺激作用下能极大的提高NFATc1的荧光素酶的活性，提示破骨前体细胞的NFATc1信号通路被RANKL激活，而姜提取物(4μg/ml和8μg/ml)能显著地抑制RANKL诱导NFATc1的激活作用。
以上实验结果证实：在安全浓度下的姜提取物可以通过抑制破骨细胞分化的关键信号通路--NFATc1信号通路，从而抑制破骨细胞的分化。破骨细胞是体内唯一具有骨吸收功能的细胞，破骨细胞的数目增多或活性增强，将破坏骨代谢平衡，导致骨量的丢失，从而导致骨质疏松和骨折的风险增加。本发明人发现姜提取物能够抑制破骨细胞的分化，从而可以抑制破骨细胞引起的骨量丢失。因此本发明提供了一种可防治骨质疏松的天然植物来源的功效原料。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (11)
1.姜(Zingiber officinale Rosc.)或其提取物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途。
2.组合物在制备用于预防、治疗、辅助治疗、减轻或缓解骨质疏松症的产品中的用途，其中所述组合物由组分a)和组分b)组成，其中组分a)为姜(Zingiber officinale Rosc.)或其提取物，组分b)为维生素D和/或钙。
3.权利要求1所述的用途，其中所述的姜或其提取物作为唯一活性成分。
4.权利要求1或2所述的用途，其中所述产品为食品或药品。
5.权利要求4所述的用途，其中所述的食品为液体饮料、固体饮料、奶粉、液态奶、发酵乳或奶片。
6.权利要求1或2所述的用途，其中所述的提取物为姜的水提取物。
7.权利要求1或2所述的用途，其中所述的提取物为姜粉。
8.权利要求7所述的用途，其中所述姜粉是姜的水提取物经干燥后获得的粉状物。
9.权利要求6所述的用途，其中所述姜的水提取物是经一种提取方法提取获得，所述的提取方法包括以下操作：
将姜破碎，获得破碎的姜；
用水提取破碎的姜，获得姜的水提取物。
10.权利要求9所述的用途，所述的提取方法还包括以下一种或多种操作：
a)浓缩；
b)杀菌；
c)干燥。
11.一种组合物，由组分a)和组分b)组成，其中：
组分a)为姜(Zingiber officinale Rosc.)或其提取物，
组分b)为为维生素D和/或钙。
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