CN116370533A - 玫瑰花在制备抗骨质疏松制品中的应用 - Google Patents
玫瑰花在制备抗骨质疏松制品中的应用 Download PDFInfo
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Abstract
本发明涉及用于治疗骨质疏松的天然药物技术领域,具体涉及玫瑰花在制备抗骨质疏松制品中的应用。实验证明:玫瑰花粉连续口服13周,可明显增加双侧卵巢切除术制备的经典骨质疏松症模型大鼠的全身骨密度,增加股骨的断裂挠度和最大弯曲力,增加骨皮质厚度和骨小梁面积,增加血清骨钙素和E2水平,提示其具有明显预防和治疗骨质疏松症的作用。此外,小鼠口服玫瑰花粉的MTD大于24g/kg,连续灌服30天的无毒性剂量为4.0g/kg,表明其具有较高的安全性。玫瑰花在防治各种因素所引起的骨质疏松症具有良好应用前景与开发潜质。
Description
技术领域
本发明涉及用于治疗骨质疏松的天然药物技术领域,具体涉及玫瑰花在制备抗骨质疏松制品中的应用。
背景技术
骨质疏松症(Osteoporosis,OP)是一种全身代谢性骨病,因骨组织微结构受损,骨量低下,骨基质和骨矿物等比例的不断减少,导致骨强度降低,骨脆性增加,易发生骨折。骨质疏松症可发生于任何年龄,但多见于绝经后女性和老年男性。骨质疏松症分为原发性和继发性两大类。原发性骨质疏松症包括绝经后骨质疏松症(Ⅰ型)、老年骨质疏松症(Ⅱ型)和特发性骨质疏松症(包括青少年型)。继发性骨质疏松症指由任何影响骨代谢的疾病和/或药物及其他明确病因导致的骨质疏松。骨质疏松症是受多因素影响的复杂疾病,被WHO视为三大老年病之一。骨质疏松性骨折会增加老年患者的致残率或致死率,给患者和家庭带来无限痛苦和沉重的经济负担。随着人口老龄化日趋严重,骨质疏松症已成为我国面临的重要公共健康问题。如何采取必要的措施减少老年人的脆性骨折发生率,已是我们所面临的一项极其紧迫的任务。
能有效治疗骨质疏松症的药物,均可以增加骨密度,改善骨质量,显著降低骨折的发生风险。目前,按作用机制可将抗骨质疏松症药物分为骨吸收抑制剂(双膦酸盐、降钙素、雌激素、选择性雌激素受体调节剂、RANKL)、骨形成促进剂(甲状旁腺激素类似物)、其他机制类(维生素D及K2等)药物及传统中药(骨碎补、淫羊藿制剂)。临床研究表明,尽管现有药物对骨质疏松症的治疗发挥了重要作用,但仍存在较大局限,如长期使用激素替代疗法有增加患乳腺癌、冠心病等疾病的几率;某些制剂疗效仍存在争议或副作用过大;单克隆抗体制剂价格昂贵,且国内尚未上市。骨骼强壮是维持人体健康的关键,骨质疏松症的防治应贯穿于生命全过程,因此骨质疏松症的预防与治疗同等重要。化学药具有靶点明确,作用单一和高选择性的特点,而维生素类预防药的剂量难以把握,过量仍危害健康。如何从天然植物中探寻多靶点、多环节、安全高效、标本兼治的预防和治疗骨质疏松症的健康产品,为广大骨质疏松患者提供更安全、价廉、简便的治疗选择,一直是医药界关注的热点之一。
发明内容
本发明意在提供玫瑰花在制备抗骨质疏松制品中的应用,以解决现有技术中缺少能够预防和治疗骨质疏松症的天然药物的技术问题。
为达到上述目的,本发明采用如下技术方案:
玫瑰花在制备抗骨质疏松制品中的应用。
本方案的原理及优点是:
玫瑰花(Rosa rugosa),是日常生活中作为观赏和食用的一类花卉,又名刺玫、穿心玫瑰、徘徊花等,是蔷薇科蔷薇属直立灌木,丛生、枝密有针刺,被绒毛。玫瑰现在我国大面积栽培,尤其在云南、贵州成为重要的食用和观赏花卉。玫瑰花具有抗炎、抗氧化、抗肿瘤、抗疲劳、降血糖、降血压等多种活性,而关于其在抗骨质疏松方面的作用和用途未见报道,其在防治骨质疏松症上具有巨大的社会效益和经济效益。
本发明对玫瑰花进行了体内抗骨质疏松症的作用研究,结果表明:以0.5、1.0、2.0g/kg剂量给切除卵巢致骨质疏松模型大鼠连续灌胃13周,均能明显增加模型动物的股骨重量和子宫系数及血清中E2、OCN和OPG水平,降低血清中ALP,ACP、RANKL、IL-6、IL-1β水平,改善骨生物力学,增加骨密度;组织病理学检查表明玫瑰花能不同程度增加骨皮质厚度和骨小梁面积。结果表明玫瑰花对骨质疏松模型大鼠具有明显防治作用。
本发明的实验证明玫瑰花在小鼠急性毒性实验中未表现出致死性毒性,其一日内灌胃给药的最大耐受量超过24g/kg,相当于同种动物药效学有效剂量0.5g/kg的48倍。小鼠以1.0、2.0、4.0g/kg剂量连续灌服玫瑰花粉30天,其外观行为、肝肾功能及大体解剖均未见毒理学意义的改变,提示玫瑰花粉对小鼠重复给药30天的无毒性反应剂量为4.0g/kg,表明口服玫瑰花具有较高的安全性。
综上所述,玫瑰花能明显增加双侧卵巢切除大鼠的全身骨密度,增加股骨的断裂挠度和最大弯曲力,增加骨皮质厚度和骨小梁面积,其机制与提高雌激素水平和调整骨代谢有关,可用于预防和治疗临床上的原发性骨质疏松症(成年型、经绝期、老年性)和继发性骨质疏松症(内分泌性、营养性、遗传性等)。本发明的有益效果在于:本发明首次发现了玫瑰花具有改善骨生物力学及增加骨密度的作用,可应用于防治骨质疏松症的药物、食品、保健品和化妆品中。
进一步,所述制品包括药品、食品、保健品和化妆品。玫瑰花是一种可食用植物,将可其制备为药品、食品、保健品和化妆品,并达到防治骨质疏松的目的。
进一步,使用玫瑰花的花瓣制备所述制品。玫瑰花的花瓣为主要功效原料,采用花瓣入药的形式。
进一步,先将所述花瓣制备为粉末、冻干品或者提取物,再使用粉末、冻干品或者提取物制备所述制品。将玫瑰花的花瓣粉碎或者制备提取物,可以增加功效成分的生物利用度。并且特别是通过提取物的形式,可以达到充分富集功效成分的目的。
进一步,所述粉末由如下方法制备:将花瓣烘干后粉碎,获得粉末;
所述冻干品由如下方法制备:花瓣和水混合匀浆,再经冷冻干燥获得冻干品;
所述提取物由如下方法制备:使用乙醇为溶剂对花瓣进行回流提取,然后再经干燥粉碎,获得提取物。
采用上述方式均可获得具有治疗效果的来自于玫瑰花瓣的原料,并实现功效成分的富集,提升制品的生物利用度和疗效。
进一步,所述产品的原料还包括其他功效成分或者辅料。出于加强治疗作用以及调整剂型的目的,可以在玫瑰花中加入其他功效成分或者辅料。
进一步,所述辅料包括魔芋葡聚糖粉和乙醇。以魔芋葡聚糖粉和乙醇为辅料,将玫瑰花制备为颗粒剂(冲剂)的形式,便于携带和服用。
进一步,所述其他功效成分包括软骨素。玫瑰花可以单独或与其他一种或多种物质组合,达到强化或协同防治骨质疏松的作用。
进一步,所述制品为片剂、胶囊剂、冲剂、口服液。可根据实际应用的需要,将抗骨质疏松的产品制备为各种剂型,都可以实现治疗或者预防骨质疏松的目的。
进一步,玫瑰花的剂量范围为每公斤体重10mg-10000mg花瓣。玫瑰花花瓣本身是一种可使用的食品,小鼠经口灌胃途径急性毒性试验以及小鼠经口灌胃途径亚急性毒性试验结果显示玫瑰花服用安全性高,每公斤体重服用10mg-10000mg花瓣不会引起毒性反应。可根据治疗或者预防的需求,在上述范围内调整玫瑰花的用量。在基于治疗或预防而使用所述玫瑰花时,一般指日剂量在每公斤体重10mg-10000mg范围内给药,如有需要可以分剂量给药。
具体实施方式
本发明公开了所述玫瑰花具有良好的预防和治疗骨质疏松的作用,其应用于药物、食品、保健品、化妆品中具有巨大的经济价值。本领域技术人员可以借鉴本文内容,以玫瑰花(或提取物、精制物)为核心原料的组合物开发预防和治疗骨质疏松的药物、保健品、食品和化妆品,需要特别指出的是,所有这些类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合具体实施例对本发明作进一步的详细说明,但并不以此来限定本发明的内容。
实施例1:玫瑰花粉的制备
取新鲜玫瑰花20kg,摘取花瓣,45℃烘干(水分小于7%),用粉碎机粉碎(过120目筛),再用破壁粉碎机粉碎成破壁粉(500目),得1.65kg。
实施例2:玫瑰花冻干粉的制备
取新鲜玫瑰花20kg,摘取花瓣,按花﹕水(2:1)混合,匀浆,冷冻至-40℃,放入冷冻干燥机干燥,得冻干粉1.36kg。
实施例3:玫瑰花提取物制备
取新鲜玫瑰花20kg,摘取花瓣,加入75%乙醇200kg,80℃加热回流2小时,过滤,80℃减压浓缩,至无醇味,放入烘箱,80℃减压干燥,粉碎,得1.52kg。
实施例4:玫瑰花颗粒(冲剂)
取玫瑰花提取物1kg,加入魔芋葡聚糖粉4kg,用50%乙醇制粒,烘干,整粒,过筛,得4.6kg,分装(每袋5g)。
实施例5:复方玫瑰花胶囊
取玫瑰花提取物1kg、软骨素1kg,混合,制粒,干燥,整粒,填装胶囊(每粒约0.5g)。
实验例1:玫瑰花对切除卵巢致骨质疏松模型大鼠的影响
1.试验材料
1.1受试物
玫瑰花超微粉(RO),紫色粉末,批号20160501,采用实施例1的方法制备。
1.2动物
SPF级雌性SD大鼠,7-9周龄,体重220-250g,由昆明医科大学实验动物中心提供,生产许可证号:SCXK(滇)2015-0002,发证机关:昆明市科学技术局。
2 实验方法
2.1 模型制备及分组给药
参照文献的方法,选雌性SD大鼠70只,适应性饲养7天后,除随机抽取10只为假手术组外,其余均用10%水合氯醛腹腔注射麻醉,四肢及头部固定于手术台上,用碘酒及酒精棉球擦拭下腹部皮肤消毒备皮,于无菌条件下经腹部手术摘除双侧卵巢。假手术组除不切除卵巢外,其余操作均与去势动物相同。去势大鼠恢复饲养一周后,剔除体重偏差较大或状况不佳者,按体重随机分为5组:模型对照组;化药阳性对照戊酸雌二醇片1.0mg/kg组,玫瑰花超微粉低、中、高剂量(0.5、1.0、2.0g/kg)组,每组11只。各给药组每日按剂量分别灌胃给予不同药物,模型对照组和假手术组给予纯水,各组给药容积均为10mL/kg,连续给药13周。
(模型制备的参考文献具体为:F.Li,X.Yang,J.Bi,Z.Yang and C.Zhang,Antiosteoporotic activity of Du-Zhong-Wan water extract in ovariectomizedrats,Pharm Biol,2016,54,1857-1864.;宋敏,李晶,李志锋.淫羊藿总黄酮抑制去势大鼠骨吸收的研究.中医研究,2010,23(5):19.;刘晓青,崔燎,吴铁,等.人参水煎剂防治去卵巢大鼠骨量丢失的骨形态计量学观察.中国骨质疏松杂志,2003,9(4):27-30.;刘钰瑜,崔燎,吴铁,等.雌孕激素合用对去卵巢大鼠骨质疏松的预防作用.中国药理学通报,2003,19(11):1303-1306.;崔燎,吴铁,刘晓青,等.人参茎叶皂苷与小剂量雌激素联合用药防治去卵巢大鼠骨丢失.药学学报,2002,37(7):501-505.)
2.2血液生化指标检测
末次给药后禁食给水过夜,股动脉取血,4℃、3,000rpm离心10min,收集血清于-20℃冻存,按试剂盒说明书测定大鼠血清中ALP、ACP、Ca、P、OCN和E2水平;另取一部分全血,用EDTA抗凝,分离血浆,测定大鼠血浆中的OPG、RANKL、IL-1β和IL-6水平。
2.3脏器系数测定
取血后剖取大鼠子宫和左侧股骨并称重,按下式计算脏器系数:
股骨湿重(干重)系数=股骨湿重(干重)/体重;
子宫系数=子宫湿重/体重。
2.4骨生物力学检测
采用美国Lunar-DPXIQ双能X线吸收骨密度仪(DEXA)测定骨密度。取大鼠左侧股骨并用纱布去除肌肉等组织,采用HH6005型单光子股矿物密度测定仪(北京核海技术开发公司)测定骨密度(BMD),将股骨置于仪器测试台面上,将其近心端中上1/3切面处与仪器测定标线对齐,启动仪器开始扫描,定好预扫左右边界线,正式扫描测量。取大鼠左侧股骨,在CSS-44100生物力学测试仪上测量其最大弯曲力(Maximum load)、断裂挠度(Maximumdeflection)等生物力学指标,实验条件:跨距20mm,速度5mm/秒。
2.5股骨病理组织学分析
快速收集大鼠右侧新鲜股骨,固定在10%中性福尔马林溶液中72小时,再将股骨块于流水下冲洗2-3小时以充分去除组织中残留的福尔马林,剪取大小适中的组织,通过石蜡包埋、切片(4m)及H&E染色,在显微镜下观察股骨切片。使用医学图像分析系统(BI-2000,成都泰盟科技有限公司)获得股骨骨皮质和骨小梁结构的静态图像,并通过计算机辅助软件测量股骨骨皮质厚度和骨小梁面积。
3实验结果
3.1对模型动物一般情况的影响
实验期间,假手术组动物被毛、精神、食欲、活动正常;模型组动物部分出现体毛稀疏无光泽、畏寒倦卧、精神萎靡、反应迟钝、活动迟缓、食欲下降、身体肥胖、嗜睡等症状;玫瑰花组动物进食、饮水正常,皮毛有光泽,状态较模型组有明显改善,至实验结束无动物死亡。
3.2对模型动物体重的影响
由表1可见,给药前各模型组动物因切除卵巢后基础体重与假手术组相比有显著差异(P<0.01);给药期间,各组动物体重均有增长,其中模型组的增长幅度较大,与假手术组比较差异有统计学意义(P<0.01);阳性对照戊酸雌二醇片组自给药第2周起体重增加幅度低于模型组,其中第6至13w均有统计学差异(P<0.05/0.01);RO各组动物体重增加幅度自第6周起较模型组低,其中高剂量组在第7、8、12、13w的体重与模型对照组相比差异有统计学意义(P<0.05/0.01)。
3.3对模型动物脏器系数的影响
由表2可见,与假手术组相比,模型组子宫和股骨干/湿重系数明显降低(P<0.01);与模型对照组相比,各给药组均能不同程度增加子宫和股骨系数,其中以阳性对照和RO高剂量组作用最为显著(P<0.05/0.01)。
3.4对血清生化指标的影响
由表3和表4可见,与假手术组相比,模型对照组动物血清OCN、ALP、ACP、P含量明显升高(P<0.05/0.01)、E2含量明显降低(p<0.01);各给药组对上述指标的增加和降低有不同程度的对抗作用,其中阳性对照和RO高剂量组作用明显(P<0.05/0.01)。
3.5对血浆OPG、RANKL含量的影响
由表5可见,与假手术组相比,模型对照组动物血清OPG明显降低(P<0.01)、RANKL含量明显增加(P<0.01);各给药组对上述指标的降低和增加有不同程度的对抗作用,其中以阳性对照和RO中、高剂量组作用为显著(P<0.05/0.01)。
3.6对血浆IL-6、IL-1β水平的影响
由表6可见,与假手术组相比,模型对照组动物血清IL-6、IL-1β水平均明显升高(P<0.01);各给药组对上述两个指标的增加有不同程度的对抗作用,其中以阳性对照和RO高剂量组作用最显著(P<0.01)。
3.7对大鼠骨密度、骨生物力学指标的影响
由表7可见,与假手术组相比,模型对照组骨密度、断裂挠度、最大弯曲力明显下降(P<0.01);各给药组对上述指标有不同程度的改善作用,其中阳性对照和RO中、高剂量组作用明显(P<0.05/0.01)。提示其可显著增加骨质疏松症大鼠股骨的断裂挠度和最大弯曲力、增加骨头的韧性、有效地提高骨的结构力学和材料力学性能,从而有效地防治骨质疏松。
3.8对大鼠骨皮质厚度、骨小梁面积的影响
由表8可见,与假手术组相比,模型对照组骨皮质厚度和骨小梁面积明显下降(P<0.01);各给药组对上述指标有不同程度的改善作用,其中以阳性对照和RO中、高剂量组作用为显著(P<0.05/0.01)。
4实验结论
玫瑰花以0.5、1.0、2.0g/kg剂量连续灌胃13周,可明显增加双侧卵巢切除致骨质疏松大鼠的全身骨密度,增加股骨断裂挠度和最大弯曲力,增加骨皮质厚度和骨小梁面积,其机制与提高雌激素水平和调整骨代谢有关。
实验例2:玫瑰花急性毒性试验
1试验操作及结果
预试表明小鼠口服玫瑰花无法测出LD50,故以一日内3次灌胃的最大给药量来反映其急性毒性情况。取18~20g小鼠40只,按体重和性别随机分为对照组及玫瑰花粉组,每组20只,雌雄各半。禁食不禁水8小时后,以0.2g/mL的最大浓度、40mL/kg的最大容积1日内给小鼠灌胃3次,每次间隔6小时,对照组给予同容积的纯水。连续观察14d,记录中毒症状和死亡情况。观察期结束脱臼处死动物,解剖,肉眼观察各组织和脏器的体积、颜色、质地等有无明显异常。结果如下:
表9:玫瑰花粉对小鼠最大耐受量试验体重影响(与对照组比较,P>0.05)
由表9可见,小鼠一日内以0.2g/mL的最大浓度、40mL/kg的最大体积间隔6小时3次灌服玫瑰花粉,在连续14天的观察期内未见动物发生明显毒性,也未出现死亡,且两组体重增长无明显差异;对观察期结束处死动物的尸检亦未发现组织或脏器有明显异常。
2试验结论
玫瑰花在小鼠急性毒性实验中未表现出致死性毒性,其一日内灌胃给药的最大耐受量超过24g/kg,相当于同种动物药效学有效剂量0.5g/kg的48倍。
实验例3:玫瑰花亚急性毒性试验
1试验操作及结果
取19-21g小鼠48只,雌雄各半,按性别和体重随机分为4组:溶媒对照组;玫瑰花粉1.0、2.0、4.0g/kg组,每组12只。各组动物每日按剂量灌胃给药一次,对照组给予纯水,容积均为20mL/kg,连续30天。每日观察动物的一般行为、中毒反应及死亡情况,每周称取体重一次以调整给药剂量。末次给药后禁食不禁水12h,称取动物体重后,摘眼球取血分离血清,测定ALT、AST、Cre、BUN、UA;剖取心、肝、脾、肺、肾、胸腺于分析天平上称重,计算脏器系数。结果见表10-12。
由表10可见,试验期间各组动物活动自如、皮毛光泽,一般状况良好,但玫瑰花三个剂量组的体重增长均明显低于对照组,并有明显量-效及一定的时-效趋势。除4.0g/kg组于给药第20天灌胃失误致4号小鼠死亡(解剖证实)外,其余各组无一死亡。试验结束处死解剖动物,肉眼观察心、肝、脾、肺、肾等内脏组织,未见明显异常。
由表11可见,小鼠以1.0、2.0、4.0g/kg剂量连续灌服玫瑰花30天,除高剂量组血清BUN明显降低外,其余肝、肾功能指标与对照组相比无明显差异。
由表12可见,小鼠以1.0、2.0、4.0g/kg剂量连续灌服玫瑰花30天,除高剂量组胸腺及脾脏系数明显低于对照组外,其余脏器系数与对照组相比无无统计学差异。
2试验结论
小鼠以1.0、2.0、4.0g/kg剂量连续灌服玫瑰花粉30天,其外观行为、肝肾功能及大体解剖均未见毒理学意义的改变,表明口服玫瑰花具有较高的安全性。
以上所述的仅是本发明的实施例,方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出,对于本领域的技术人员来说,在不脱离本发明技术方案的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (10)
1.玫瑰花在制备抗骨质疏松制品中的应用。
2.根据权利要求1所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:所述制品包括药品、食品、保健品和化妆品。
3.根据权利要求1所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:使用玫瑰花的花瓣制备所述制品。
4.根据权利要求3所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:先将所述花瓣制备为粉末、冻干品或者提取物,再使用粉末、冻干品或者提取物制备所述制品。
5.根据权利要求4所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:
所述粉末由如下方法制备:将花瓣烘干后粉碎,获得粉末;
所述冻干品由如下方法制备:花瓣和水混合匀浆,再经冷冻干燥获得冻干品;
所述提取物由如下方法制备:使用乙醇为溶剂对花瓣进行回流提取,然后再经干燥粉碎,获得提取物。
6.根据权利要求1所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:所述产品的原料还包括其他功效成分或者辅料。
7.根据权利要求6所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:所述辅料包括魔芋葡聚糖粉和乙醇。
8.根据权利要求6所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:所述其他功效成分包括软骨素。
9.根据权利要求1所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:所述制品为片剂、胶囊剂、冲剂、口服液。
10.根据权利要求1所述的玫瑰花在制备抗骨质疏松制品中的应用,其特征在于:玫瑰花的剂量范围为每公斤体重10mg-10000mg。
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