CN105687319A - 一种抗骨质疏松啤酒花提取物及其制备方法和应用 - Google Patents
一种抗骨质疏松啤酒花提取物及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种抗骨质疏松啤酒花提取物及其制备方法和应用,该提取物经药理学试验证实,具有确切显著的抗骨质疏松的作用,因而可用于防治骨质疏松的药物、医疗食品或保健品。本发明还提供了啤酒花提取物的制备方法,该方法所用试剂安全,制备工艺简便,成本较低,安全性高,经由该制备工艺制备的啤酒花提取物,得率高,溶剂残留少,品质高,药效好。
Description
技术领域
本发明涉及医药技术领域,具体地说,具体涉及一种富含黄酮类成分的啤酒花提取物及其制备方法和在抗骨质疏松中的应用。
背景技术
骨质疏松是一种以骨量减少和骨微结构破坏为特征,导致骨脆性增加和易于发生骨折的代谢性骨病。该病多发于老人和绝经后妇女,随着老龄化社会的到来,该病发病率逐年上升。目前,我国的骨质疏松患者约有9000万人,社会和家庭负担非常严重,骨质疏松药物的研发是新药研发的热点之一,具有非常广阔的市场前景。迄今,已经在临床应用的骨质疏松治疗剂包括,维生素D类制剂、降钙素类制剂、***类制剂、异丙氧黄酮类制剂、维生素K2制剂、促蛋白合成甾类制剂,以及双磷酸盐类制剂。但这些药物的临床疗效有限,且有一定的副作用。如***有诱发子宫内膜癌及乳腺癌的危险,使其临床应用受到限制。目前,一般认为由于植物药疗效可靠,毒副作用低,从植物或者中药中开发抗骨质疏松药物是目前研发的重要方向。现有研究表明,骨的形成过程中,以清除受损碎片,形成骨洞为主要作用的破骨细胞,与以沉积钙质、填充骨洞为主要作用的成骨细胞相互协调,使骨质的形成与溶解处于一种动态平衡,维持骨质的不断更新。当此动态平衡被打破,骨溶解超过骨形成时,即导致骨质疏松症的发生。因此,任何能够直接或间接作用于成骨细胞和或破骨细胞,有助于保持其动态平衡的药物,均可能有助于防治骨质疏松。因此,成骨细胞、破骨细胞模型常用于抗骨质疏松药物的筛选。
啤酒花别名忽布、蛇麻花、香蛇麻花、酒花、野酒花,为桑科(Moraceae)薄草属植物啤酒花HumuluslupulusL.的雌花序,原产欧洲,我国新疆有野生,东北、华北、山东等地有栽培。人类利用啤酒花的历史已有2000多年,最初是作为助消化的药品、香料和观赏植物。公元8世纪初,德国开始将啤酒花用十啤酒生产,能赋予啤酒特有的香味、爽快的苦味,并能增加啤酒的防腐能力和非生物稳定性。除作为食品原料应用,啤酒花亦可作为药用,其味苦、性平,有健胃、消食、利尿、安神、止咳化痰之功效,用于治疗食欲不振、腹胀、肺结核、胸膜炎、失眠、癔病、浮肿、膀胱炎等。啤酒花主要含有树脂类、黄酮类及多酚类成分。特别是其所含的黄酮类成分黄腐酚,具有抗艾滋病毒、抗癌、抗氧化、抗炎、抗菌、抑制骨质增生、抑制甘油二酯酰基转移酶的活性,及预防动脉硬化和糖尿病和***样作用,并且其食用安全(杨小兰,食品科学,2007)。啤酒花含有的α-酸及β-酸也是重要化学成分,具有抗菌、抗肿瘤、抗氧化作用(马永花,时珍国医国药,2007)。当然,由于植物化学成分复杂,更多可能的有效成分还有待通过深入研究发现。
目前,由于啤酒花中以上成分的含量有限,制备工艺复杂,成本较高,以单体成分为主的新药开发难度较大,而以包含上述啤酒花成分的提取物,进行新药研发则具有显著的优势。目前,已有专利技术(CN201510220128.3,CN201310024053.2,CN201310024052.8,CN201510027779.0,CN201410460743.7,CN200810037197.0,CN201410182552.9,CN201410182552.9),多采用溶剂粗提,结合大孔树脂、聚酰胺除杂工艺设计,来制备啤酒花提取物或其中有效成分,但存在工艺步骤多,成本高,所用试剂及树脂所含的有害杂质可能引入到最终提取物中等方面的弊端。另有专利技术,采用超临界流体萃取技术(CN200910089532.6,CN02815697.8),或者膜技术(CN201210412183.9,CN201110218675.X)进行啤酒花提取物的制备,但成本高,工艺复杂,提取效率低,是显而易见的问题。因此,克服现有工艺技术的缺点,开发设计一种工艺简便、成本低、提取效率高的啤酒花提取物制备工艺是十分必要的。鉴于中草药成分的高度复杂性,发明人设计本制备工艺的出发点在于,基于简便、安全、低成本的制备工艺基础上制备得到的提取物,不片面追求单一成分的高含量,而应该在所设定的药理作用方面,具有显著的效果,以方便作为药物、食品的应用。
发明内容
本发明的目的是针对现有技术中的不足,提供一种抗骨质疏松的啤酒花提取物。
本发明的再一的目的是,提供如上所述啤酒花提取物的制备方法。
本发明的另一的目的是,提供如上所述啤酒花提取物的用途。
为实现上述目的,本发明采取的技术方案是:
一种抗骨质疏松的啤酒花提取物,所述提取物的制备方法包括如下步骤:
a)将啤酒花药材用提取溶剂提取,得提取液;
b)将上述提取液减压回收至无醇味,得浓缩液;
c)将上述浓缩液冷却、分取沉淀;
d)将上述沉淀以溶剂溶解,加入脱色剂除杂脱色,得除杂后脱色液;
e)将上述脱色液浓缩,干燥,粉碎,得提取物。
其中浓缩方式可以采用40-100℃减压浓缩、烘干浓缩以及其它药学上可接受的浓缩方式。当然顾忌过高的温度可能对提取物中化学成分稳定性造成影响,选用50-75℃温度浓缩更为可行。
其中干燥方式可以采用50-100℃常压烘干、真空干燥及其他可接受的干燥方式。粉碎方式可以采用球磨机、冲击式粉碎机、流能磨粉碎以及其它可接受的粉碎方式。如将喷雾干燥、冷冻干燥技术用于本步骤,可同时实现烘干、粉碎,应用更为方便。
优选地,所述步骤a中所用的提取方法为回流提取,选用的提取溶剂为80-95%乙醇或甲醇水溶液,优选为80%的乙醇水溶液。
优选地,所述步骤a中提取溶剂选用80%的乙醇水溶液,分别用12、10、10倍量的提取溶剂提取1.5小时、1小时、1小时,合并得提取液。
优选地,所述步骤c中分取沉淀的方法为过滤或离心分离;所述离心分离的参数为:5000rpm,离心1-30分钟。
优选地,所述步骤d中的溶剂为乙醇、甲醇、丙酮或其水溶液。
优选地,所述步骤d中的溶剂为丙酮或丙酮水溶液,浓度优选大于70%浓度(V/V),特别优选大于90%浓度(V/V)。
优选地,所述步骤d中的脱色剂为活性炭、活性白土或两者的混合。
优选地,所述步骤d中的脱色剂为活性碳﹕活性白土=1﹕100-100﹕1的混合物,用量为原药材重量的100%-1000%,优选用量为原药材重量的100%。
经采用***化学成分分离、鉴定,表明其含有黄腐酚、α-酸、β-酸等化学成分。
为实现上述第二个目的,本发明采取的技术方案是:
如上任一所述啤酒花提取物的制备方法,包括如下步骤:
a)将啤酒花药材用提取溶剂提取,得提取液;
b)将上述提取液减压回收至无醇味,得浓缩液;
c)将上述浓缩液冷却、分取沉淀;
d)将上述沉淀以溶剂溶解,加入脱色剂除杂脱色,得除杂后脱色液;
e)将上述脱色液浓缩,干燥,粉碎,得提取物;
所述步骤a中所用的提取方法为回流提取,选用的提取溶剂为80-95%乙醇或甲醇水溶液,优选80%的乙醇水溶液;
所述步骤a中分别用12、10、10倍量的提取溶剂提取1.5小时、1小时、1小时,合并得提取液;
所述步骤d中的脱色剂为活性碳﹕活性白土=1﹕100-100﹕1的混合物,用量为原药材重量的100%-1000%。
为实现上述第三个目的,本发明采取的技术方案是:
如上任一所述的提取物在制备抗骨质疏松的药物、医疗食品或保健品中的应用。
为实现上述第四个目的,本发明采取的技术方案是:
一种抗骨质疏松的组合物,包括:如上任一所述的啤酒花提取物、及药学上可接受的赋形剂;其中所述赋形剂的种类不受特别限制,只要能够使药物形成容易进行给药的剂型即可。
根据本发明的一些具体示例,所述赋形剂为选自:粘合剂、填料、涂膜聚合物、增塑剂、助流剂、崩解剂和润滑剂的至少一种。所述药物呈选自胶囊剂、丸剂、片剂、颗粒剂、口服液体、内服膏剂、气雾剂以及喷雾剂的至少一种的形式。根据本发明的一些优选实施例,所述药物呈胶囊剂的形式。由此,易于进行给药。该赋形剂可以是例如乳化剂(鲸蜡硬脂醇、鲸蜡硬脂基葡糖苷、氢化蓖麻油)、流变添加剂、抗氧化剂(如维生素类、生育酚类、或本领域已知的其他抗氧化剂)。乳化剂可以是表面活性剂,通过降低界面张力而降低体系的自由能;可选地,也可以使用非表面活性物质,例如***树胶、明胶、亲水胶体或精细地细分的粉末(例如滑石)。在一个实施例中,赋形剂可以以3%(m/m)至8%(m/m)的总浓度存在,该浓度当然是参考性的,考虑到无论如何本领域技术人员也会知道怎样根据技术人员意欲进行的实施例调节必要的赋形剂的浓度,而不需要加入创造性劳动。
本发明的组合物可以处于以下形式:胶囊、片剂、锭剂、硬明胶、软明胶、颗粒剂、粉剂、糖浆、酏剂、悬浮剂、乳剂。为了口服给药,组合物可以以每日单位剂量或者每日单位剂量的级分(例如,根据主治医生的判断,一天可以服用2、3、4、5、6或更多个胶囊、片剂、锭剂、颗粒剂或粉剂单剂量,或者明胶)制备,且可以含有常规的赋形剂,包括,例如,粘合剂,如***树胶、明胶、山梨糖醇、黄蓍树胶、和/或聚乙烯吡咯烷酮;填充剂,如乳糖、糖、玉米淀粉、米淀粉、磷酸钙、山梨糖醇和/或甘氨酸;压片润滑剂,如硬脂酸镁、滑石、聚乙二醇和/或硅石;崩解剂,例如土豆淀粉;和润湿剂,如硫酸月桂酸钠。片剂可以根据标准药物实践中公知的方法涂布。组合物还可以以液体或半液体形式(如悬浮剂、乳剂、溶液)制备,用于口服给药,且可以任选地含有给予组合物可口味道的天然芳化试剂。粉剂或颗粒剂形式的组合物可以在适当的容器中预先测量且准备使用,通过摄入本身或再悬浮在适当的液体(例如水、茶等)中。同样,在这种情况下,组合物可以含有给予组合物可口味道的天然芳化试剂。明显地,所有以上指出的赋形剂可以以药学上可接受的等级使用。
以上所述的组合物可以处于药物组合物的形式,即包括药物级成分,或者组合物可以是特殊用途食品或被引入特殊用途食品或医疗设备。本发明的药物的给药剂量不受特别限制,实际应用中,可以根据给药对象的健康状况灵活选择。
本申请发明人在抗骨质疏松中药、天然药物筛选过程中,发现啤酒花的提取物具有较强的抗骨质疏松作用,进一步研究表明此提取物含黄腐酚、α-酸、β-酸等成分及其它未知成分,具有用于制备抗骨质疏松药物及食品的前景。前期试验过程中,发明人曾尝试采用背景技术中的树脂除杂脱色工艺路线,制备啤酒花的提取物,结果表明不仅工艺步骤繁琐,而且综合效益并不理想。
对于啤酒花提取物制备而言,良好的除杂脱色方法,对于去除杂质、富集有效成分、提高产品成色、品质,以及所应具有的良好药效,至关重要。在试验过程中,通过对比试验优化设计了提取物制备工艺,经由该制备工艺制备的啤酒花提取物,药理试验确证具有显著确切的抗骨质疏松作用,因而可以用于制备抗骨质疏松的药物。
本发明优点在于:
本发明提取物的制备方法具有工艺简便,成本低廉,安全有效,无有机溶剂残留等显著优点。鉴于在现代社会中,以及老龄化社会的到来,骨质疏松的发病率的急剧上升,缺乏理想、有效的治疗药物,该啤酒花提取物经药理试验证实具有显著的抗骨质疏松作用,因而可用于制备抗骨质疏松的药物及食品,具有广阔的应用前景,蕴藏着巨大的社会效益和经济效益。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1啤酒花提取物的制备(一)
啤酒花药材粗粉2kg,以80%乙醇,12倍、10倍、10倍,1.5小时、1小时、1小时,回流提取,提取液趁热过滤,合并提取液,减压回收溶剂至无醇味,蒸馏水调节浓度为1g(生药)/ml,5000rpm,离心15分钟,分离沉淀,以适量无水乙醇溶解,补加至10000ml。加入混合脱色剂(活性炭:活性白土=1:10)2000g除杂脱色,脱色后溶液呈淡黄色,将脱色后的脱色液减压浓缩,60℃真空干燥,粉碎,得提取物31.22g,产品外观淡黄色。
实施例2啤酒花提取物的制备(二)
啤酒花药材粗粉2kg,以95%乙醇作为提取溶剂,以活性炭作为脱色剂,其余同实施例1,得提取物7.34g,脱色后溶液呈淡黄绿色,产品外观黄棕色。
实施例3啤酒花提取物的制备(三)
啤酒花药材粗粉2kg,以95%乙醇作为提取溶剂,加入活性白土作为脱色剂,其余同实施例1,结果脱色后溶液呈淡绿色,得提取物22.42g,产品外观黄棕色。
实施例4啤酒花提取物的制备(四)
啤酒花药材粗粉2kg,以50倍80%乙醇作为提取溶剂渗漉提取,其余同实施例1,脱色后溶液呈淡黄绿色,得提取物24.54g,产品外观淡黄色。
实施例5啤酒花提取物的制备(五)
啤酒花药材粗粉2kg,混合脱色剂用量500g,其余同实施例1,脱色后溶液呈深黄色,得提取物42.48g,产品外观黄褐色。
实施例6啤酒花提取物的制备(六)
啤酒花药材粗粉2kg,加入活性碳1000g脱色,其余同实施例1,结果脱色后溶液淡黄色,得提取物9.71g,产品外观淡棕色。
实施例7本发明啤酒花提取物对成骨肉瘤细胞MG-63-PERE的作用
采用中国人民解放军第二军医大学药学院提供的稳定转染以***反应原件为启动子以萤火虫荧光素酶为报告基因的成骨肉瘤细胞MG-63-PERE。实验过程:在96孔板上以5000个细胞每孔铺板,细胞贴壁后,加入啤酒花提取物溶液(DMSO浓度<2%)(提取物按实施例1方法制备),在培养箱中培养6小时后去除上层液,冰冷PBS清洗,每孔加入20μl细胞裂解液待用。检测方法:在化学发光检测仪上测量,使用双报告基因检测***,先测量萤火虫荧光素酶的发光量,接着淬灭,测量内标海肾荧光素酶的发光量。实验结果:结果以萤火虫荧光素酶量/海肾荧光素酶量的百分数来表示:效果(%)=样品组发光量/本底发光量×100%。根据数据分析结果(表1),结果>150%可视为有***样作用,可能会有抗骨质疏松的作用。结果表明啤酒花提取物具有***样作用,此可能是其发挥抗骨质疏松作用机制之一方面。
表1啤酒花提取物对成骨肉瘤细胞MG-63-PERE的影响
实施例8本发明啤酒花提取物对成骨细胞增殖的影响
取新生Wistar大鼠,在无菌条件下,分离颅顶盖骨,剔除骨膜及其它组织,用D-Hank's液冲洗2-3次,用眼科剪将骨片剪碎,加入0.25%的胰酶消化5min左右(37℃,5%CO2培养箱内),弃上清液,D-Hank's冲洗2-3次,再用含0.25%胰酶的3mg/ml的I型胶原酶的消化液消化1h(37℃,5%CO2培养箱内);吸取消化液,经140目尼龙网过滤,再用DMEM培养液冲洗,收集冲洗液,过滤后与消化液混合,以1000r/min离心10min,收集细胞,即为新鲜的成骨细胞。加入含10%小牛血清的DMEM培养基,调整密度为1×105细胞/ml,接种于100ml的培养瓶中,置37℃,5%CO2的培养箱中培养。24小时贴壁后换液。以后视细胞生长情况2-3天换液一次。将培养的第二代大鼠颅盖骨成骨细胞,用含10%新生牛血清的DMEM培养液配制成密度为2×104/ml的细胞悬液,以100μl每孔的体积接种于96孔培养板中,培养24h后,给药,对照组只加10%血清DMEM培养液,其余各组分别加入含不同浓度啤酒花提取物(提取物由实施例1方法制备)的含10%血清DMEM培养液,继续放入培养箱培养48h,取出培养板,各孔加入20μl四氮唑蓝(MTT,用PBS液配制5mg/ml的MTT液),放入培养箱中孵育4h,取出培养板,在倒置相差显微镜下可见细胞内有条状黑色沉淀,弃去上清液,每孔加入DMSO(二甲基亚砜)150μl,DMSO的颜色变成紫色,把96孔培养板放于酶标仪上震荡2min以便沉淀完全溶解于DMSO,检测各孔OD值(λ=540nm),以公式“成骨细胞生长促进率(%)=(实验组OD-对照组OD值/对照组OD值)×100%”计算啤酒花提取物对成骨细胞增殖的促进作用,结果见表2。表明啤酒花提取物具有浓度依赖性的促进成骨细胞增殖的作用。
表2啤酒花提取物对成骨细胞增殖的影响
实施例9本发明啤酒花提取物对破骨细胞增殖的影响
选取2-3天的新生SD大鼠,颈椎拉断处死,以体积分数为75%的乙醇浸泡5分钟,在无菌条件下分离股骨,用5ml含10-8M的1,25-(OH)2VitaminD3和10-7M***及10%胎牛血清的α-MEM培养基冲洗骨髓腔,将骨髓内的细胞冲出。用PBS缓冲液洗细胞2次。即得新鲜的骨髓细胞。将骨髓细胞和成骨细胞共同悬浮在含10-8M的1,25-(OH)2VitaminD3,10-7M的***,以及10%胎牛血清的α-MEM培养基中,使成骨细胞浓度为1×105个细胞/ml,骨髓单核细胞浓度为1×106细胞/ml,将细胞悬浮液接种于6孔板内,每孔100μl,于37℃、5%CO2的培养箱中培养,24小时后,更换新鲜的培养基,以后每3天换培养基1次,培养8天后,破骨细胞分化成熟,换含不同浓度啤酒花提取物(提取物由实施例1方法制备)的α-MEM培养基100μl,继续培养48h,弃上清液,用PBS洗涤两次,用10μl0.1%的区拉通破碎细胞,15分钟后加入反应液(反应液的配制:称取对硝基苯基磷酸二钠0.4g,用去离子水溶解,加入酒石酸钾2.0g,加去离子水水溶解至150ml,用1NHCl调节pH至3.5,再补加去离子水至200ml)100μl,于37℃反应30分钟,迅速加入100μl1N的氢氧化钠溶液终止反应,于波长410nm处测定其OD值,此OD值经标准曲线(以对硝基苯酚溶液浓度为横坐标,其在405nm的OD值为纵坐标建立标准曲线)换算为以每孔生成的对硝基苯酚(p-nitrophenol)的纳摩尔数,此纳摩尔数即可表示抗酒石酸酸性磷酸酶的活性。抗酒石酸酸性磷酸酶活性(TRAP)是破骨细胞分化的主要标志之一。结果见表3。以公式“破骨细胞增殖抑制率(%)=(对照组TRAP值-实验组TRAP值/对照组TRAP值)×100%”计算啤酒花提取物对破骨细胞增殖的抑制作用,结果见表3。表明啤酒花提取物具有浓度依赖性的抑制破骨细胞增殖的作用。
表3啤酒花提取物对破骨细胞增殖的影响
实施例10本发明啤酒花提取物对去卵巢骨质疏松大鼠的作用
1动物、材料与试药
SD清洁级大鼠,雌性,3月龄,购于上海西普尔比凯实验动物有限公司;尼尔雌醇,上海华联制药有限公司(批号:031008);DPD(脱氧吡啶)ELISA试剂盒为美国RapidBio公司产品,购于上海轩昊科技发展有限公司;抗酒石酸酸性磷酸酶测定试剂盒购于南京建成生物工程研究所。自动生化分析仪、外周骨定量计算机断层扫描仪(pQCT,peripheralQuantitativeComputedTomography,XCTReasearchSA)德国Stratec公司生产,酶标仪(美国BIOTECK);啤酒花提取物1、啤酒花提取物2、啤酒花提取物3、啤酒花提取物4(分别由实施例1-4的方法制备)。
2实验方法
动物分组:将大鼠随机分为6组,假手术组、模型组、阳性对照组和啤酒花提取物低、中和高剂量组,每组10只大鼠。假手术组保留卵巢,其余各组均摘除卵巢。
药液配制:将羧甲基纤维素钠(CMC-Na)用蒸馏水配成0.5%CMC-Na溶液,作为溶剂分别配制0.1mg/ml的尼尔雌醇以及5mg/ml、10mg/ml和20mg/ml的啤酒花提取物溶液(分别对应低、中、高剂量),尼尔雌醇为阳性对照液。
动物试验:所有动物在24~28℃,通风良好的条件下饲养,每周称体重一次。各组动物口服给药,每鼠每次按体重1ml/100g,阴性对照组口服0.5%CMC-Na,每天1次,每周6次;阳性对照组口服尼尔雌醇,每周1次;啤酒花提取物低、中、高剂量组各每天1次,每周6次。12周后,将大鼠用10%水合氯醛麻醉,腹部切开皮肤、肌肉,暴露腹腔,腹动脉取血,分离血清,迅速剥离其右侧胫骨。胫骨以外周骨定量计算机断层扫描仪(pQCT,德国Stratec公司)测定其骨密度、骨含量。
3实验结果
实验结果表明大鼠去卵巢后,骨含量和骨密度显著下降,以骨小梁骨含量和密度降低最为显著。尼尔雌醇能显著增加去卵巢大鼠的骨含量和骨密度,不同剂量啤酒花提取物均能不同程度的提高去卵巢大鼠的骨含量和骨密度(表4、5)。说明本发明的啤酒花提取物1可以提高去卵巢大鼠的骨小梁的骨含量和骨密度,故具有防治骨质疏松的作用。
表4啤酒花提取物对去卵巢大鼠骨密度的影响(n=10,Mean±SD)
▲:与假手术组比较,P<0.05;△:与阴性对照组比较,P<0.05
表5啤酒花提取物对去卵巢大鼠骨含量的影响(n=10,Mean±SD)
▲:与假手术组比较,P<0.05;△:与阴性对照组比较,P<0.05
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种抗骨质疏松的啤酒花提取物,其特征在于,所述提取物的制备方法如下:
a)将啤酒花药材用提取溶剂提取,得提取液;
b)将上述提取液减压回收至无醇味,得浓缩液;
c)将上述浓缩液冷却、分取沉淀;
d)将上述沉淀以溶剂溶解,加入脱色剂除杂脱色,得除杂后脱色液;
e)将上述脱色液浓缩,干燥,粉碎,得提取物。
2.根据权利要求1所述的提取物,其特征在于,所述步骤a中所用的提取方法为回流提取,选用的提取溶剂为80-95%乙醇或甲醇水溶液,优选为80%的乙醇水溶液。
3.根据权利要求2所述的提取物,其特征在于,所述步骤a中提取溶剂选用80%的乙醇水溶液,分别用12、10、10倍量的提取溶剂提取1.5小时、1小时、1小时,合并得提取液。
4.根据权利要求1所述的提取物,其特征在于,所述步骤c中分取沉淀的方法为过滤或离心分离;所述离心分离的参数为:5000rpm,离心1-30分钟。
5.根据权利要求1所述的提取物,其特征在于,所述步骤d中的溶解沉淀所用的溶剂为乙醇、甲醇、丙酮或其水溶液。
6.根据权利要求1所述的提取物,其特征在于,所述步骤d中的脱色剂为活性炭、活性白土或两者的混合。
7.根据权利要求6所述的提取物,其特征在于,所述步骤d中的脱色剂为活性碳﹕活性白土=1﹕100-100﹕1的混合物,用量为原药材重量的100%-1000%,优选用量为原材料重量的100%。
8.权利要求1-7任一所述啤酒花提取物的制备方法,其特征在于,所述制备方法包括如下步骤:
a)将啤酒花药材用提取溶剂提取,得提取液;
b)将上述提取液减压回收至无醇味,得浓缩液;
c)将上述浓缩液冷却、分取沉淀;
d)将上述沉淀以溶剂溶解,加入脱色剂除杂脱色,得除杂后脱色液;
e)将上述脱色液浓缩,干燥,粉碎,得提取物;
所述步骤a中所用的提取方法为回流提取,选用的提取溶剂为80-95%乙醇或甲醇水溶液,优选80%的乙醇水溶液;
所述步骤a中分别用12、10、10倍量的提取溶剂提取1.5小时、1小时、1小时,合并得提取液;
所述步骤d中的脱色剂为活性碳﹕活性白土=1﹕100-100﹕1的混合物,用量为原药材重量的100%-1000%。
9.权利要求1-7任一所述的提取物在制备抗骨质疏松的药物、医疗食品或保健品中的应用。
10.一种抗骨质疏松的组合物,其特征在于,包括:权利要求1-7任一所述的啤酒花提取物、及药学上可接受的赋形剂;所述的赋形剂选自:粘合剂、填料、涂膜聚合物、增塑剂、助流剂、崩解剂或润滑剂中的一种或多种。
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