CN116270717A - 红芪多糖的应用及含有其的药物或保健食品 - Google Patents
红芪多糖的应用及含有其的药物或保健食品 Download PDFInfo
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- CN116270717A CN116270717A CN202310108402.2A CN202310108402A CN116270717A CN 116270717 A CN116270717 A CN 116270717A CN 202310108402 A CN202310108402 A CN 202310108402A CN 116270717 A CN116270717 A CN 116270717A
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- polysaccharide
- liver
- hedysarum
- polybasic
- health
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- A—HUMAN NECESSITIES
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明属于药物领域,公开了红芪多糖在制备改善或治疗氟化物肝毒性药物或保健食品中的应用,所述药物或保健食品为抑制因氟化物暴露致肝脏氧化应激、炎症或凋亡水平升高的药物或保健食品,所述氟化物为氟化钠。本发明将红芪多糖应用于制备改善或治疗氟化物肝毒性药物或保健食品中,起到了改善氟中毒后肝脏组织病理学变化、抑制氟中毒导致的肝脏组织中活性氧增加、改善氟中毒导致的肝脏氧化损伤、减少氟中毒后肝脏细胞凋亡数、降低Cleaved Caspase‑3表达、抗炎的效果。
Description
技术领域
本发明属于药物领域,具体涉及红芪多糖的应用及含有其的药物或保健食品。
背景技术
氟虽是机体必需的微量元素,但其生理需要量与中毒剂量非常接近,很容易造成氟的过量摄入,导致氟中毒的发生。地方性氟中毒在世界范围内广泛存在,给人和动物健康造成严重危害。
地方性氟中毒是一种在特定地理环境中发生的生物地球化学性疾病,除了引起氟斑牙和氟骨症外,还引起机体多个器官和组织发生病理损害。我国是地方性氟中毒的高发地区,地方性氟中毒已经被列入我国现有地方病中的重点疾病,但是氟的毒性作用机制目前尚未完全阐明,给该病的诊断、预防以及后续治疗都带来较大困扰。
文献《慢性氟中毒性肝损伤及其发病机制》中指出,肝脏是慢性氟中毒毒性作用明显的靶器官之一。地方性氟中毒患者及慢性氟中毒动物的肝功能都受到显著地影响.肝脏组织形态及超微结构有明显改变。摄入过量氟不仅影响了肝脏内自由基的代谢,增强脂质过氧化反应,还促进了肝细胞凋亡。高氟对肝脏的毒性作用是多方面的,其毒性机制具有复杂性和多元性。其中氧化应激水平升高可能是慢性氟中毒引起肝损害的主要原因。自由基水平的增高可能是氟中毒发生机制中的一个重要的中间介导环节。过量氟如何影响Notch信号通路进而造成肝损伤具体机制有待进一步的深入研究。
大量人群流行病学、动物实验和体外肝细胞培养研究结果均表明,长期摄入高氟可引起肝脏组织结构异常、功能和代谢的变化,但慢性氟中毒致肝损伤的机制仍不完全肯定。
红芪为豆科植物多序岩黄芪的干燥根,是甘肃大宗道地药材,系我国独有品种。一直以来在医学药用方面对于红芪的药用价值缺乏研究,医学家在治疗疾病时总是认为红芪和黄芪的功效相似,没有太大的区别,如:2021年4月中华中医药学刊的一篇文章《红芪与黄芪免疫调节和抗氧化作用对比研究进展》在文中结论部分指出:“由红芪和黄芪抗氧化作用对比分析结果表明,红芪水提物抗氧化作用略强于黄芪水提物,但含红芪和黄芪的补益类复方抗氧化作用无显著差异,提示二者在抗氧化作用方面临床上可以互换使用。”;从而使红芪这味中药往往被忽视,在中医的医疗处方中几乎看不到红芪的影子,在医院的药房中也只能找到各种各样的黄芪饮片,而红芪却慢慢的淡出了医学家们视野,同时红芪的栽培面积也趋于减少。人们对于红芪的认识缺乏,导致红芪犹如秋风中的枯叶岌岌可危。
然而,从分子结构上来看,红芪和黄芪存在很大差异,如:红芪多糖由鼠李糖、木糖、阿拉伯糖、半乳糖及葡萄糖五种单糖组成,而黄芪多糖由己糖醛酸、葡萄糖、果糖、鼠李糖、阿拉伯糖、半乳糖醛酸和葡萄糖醛酸等组成,可知两者是不同的物质,则其性质也必然存在差异,只是目前相关研究不足。
红芪多糖作为红芪药材的主要活性成分之一,是一种高效抗氧化剂。有研究报道了红芪多糖对四氯化碳(CCl4)、D-氨基半乳糖等造成的肝脏氧化损伤有着良好的保护作用。
然而,在氟中毒造成肝损伤的具体机制有待进一步的深入研究以及大众对红芪多糖功效认知不足的当下,红芪多糖是否对氟化物如氟化钠(NaF)引起的肝脏损伤有保护作用尚未见相关文献和专利报道。
发明内容
针对现有技术的不足,本发明的目的在于提供红芪多糖在制备改善或治疗氟化物肝毒性药物或保健食品中的应用。
本发明的第二个目的是提供包含上述红芪多糖的改善或治疗氟化物肝毒性的药物或保健食品。
为了解决上述的技术问题,本发明采用的第一个技术方案如下:
红芪多糖在制备改善或治疗氟化物肝毒性药物或保健食品中的应用。
进一步的,上述的药物或保健食品为抑制因氟化物暴露致肝脏氧化应激、炎症或凋亡水平升高的药物或保健食品。
进一步的,上述的氟化物为氟化钠。
进一步的,患者使用上述的药物或保健食品摄入红芪多糖的质量为20~200mg·kg-1·d-1。
进一步的,患者使用上述的药物或保健食品摄入红芪多糖的质量为50~100mg·kg-1·d-1。
改善或治疗氟化物肝毒性药物或保健食品所含红芪多糖的用量在有效量之上,根据具体情况进行选择,一般为20~200mg·kg-1·d-1,优选为50~100mg·kg-1·d-1。
本发明提供的第二个技术方案是一种含有上述红芪多糖的改善或治疗氟化物肝毒性的药物或保健食品,所述药物或保健食品剂型为溶液剂、糖浆剂、颗粒剂、胶囊剂、散剂、丸剂、片剂、水针剂、冻干粉剂、贴剂、凝胶剂、膜剂、滴丸剂、酒剂、浸膏剂或缓控释制剂。
进一步的,上述的药物或保健食品中红芪多糖的含量为1~99wt%。
相对于现有技术,本发明具有以下有益效果:
本发明将红芪多糖应用于制备改善或治疗氟化物肝毒性药物或保健食品中,抑制了氟中毒后肝脏细胞出现空泡化、ROS含量增加、MDA含量增加、SOD活性降低、TUNEL阳性细胞数增加、Cleaved Caspase-3增加、TNF-α增加、IL-6增加和IL-1β增加,起到了改善氟中毒后肝脏组织病理学变化、抑制氟中毒导致的肝脏组织中活性氧增加、改善氟中毒导致的肝脏氧化损伤、减少氟中毒后肝脏细胞凋亡数、降低Cleaved Caspase-3表达、抗炎的效果。且通过对比发现,红芪多糖在改善或治疗氟化物肝毒性中的效果强度优于褪黑素,远远优于黄芪多糖,其中红芪多糖在MDA含量及SOD活性测定实验中表现的抑制MDA含量增加、SOD活性降低所体现的抗氧化作用其效果之强远超预期,打破了人们认为“红芪抗氧化作用略强于黄芪,二者在抗氧化作用方面临床上可以互换使用”的认知,并且红芪多糖改善或治疗氟化物肝毒性是多方面途径共同作用而不只是抗氧化作用的结果,验证了“高氟对肝脏的毒性作用是多方面的,其毒性机制具有复杂性和多元性”的现有猜想,也表明了红芪多糖在改善或治疗氟化物肝毒性这个新用途中的综合效果强大。
附图说明
图1为各组大鼠肝脏组织病理学变化图。
图2为各组大鼠ROS检测结果图。
图3为各组大鼠肝脏TUNEL阳性细胞凋亡检测结果图。
图4为各组大鼠肝脏Cleaved Caspase-3免疫荧光染色图。
图5为各组大鼠肝脏TNF-α、IL-6和IL-1β的表达图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
以下采用的:
试剂:
DHE染液来源于美国Sigma-Aldrich公司;
红芪多糖来源于兰州大学中西医结合研究所;
黄芪多糖来源于北京索莱宝科技公司;
脂质氧化(MDA)检测试剂盒、超氧化物歧化酶(SOD)检测试剂盒来源于南京建成生物工程研究所;
BCA蛋白浓度测定试剂盒、封闭液、DAPI染液、TUNEL细胞凋亡检测试剂盒来源于上海碧云天生物公司;
TNF-α、IL-6、IL-1β、Cleaved-Caspase 3(Asp175)抗体和羊抗兔IgG(H+L)Fluor594-conjugated二抗来源于江苏亲科生物;
蛋白酶K来源于武汉赛维尔生物科技有限公司;
戊巴比妥钠来源于上海上药新亚药业有限公司。
仪器:
SW-CJ-2F型超净工作台来源于苏州安泰空气技术有限公司;
分析天平来源于德国Sartorius公司;
SpectraMax M5多功能酶标仪来源于美国Molecular Devices公司;
Milli-Q超纯水仪来源于美国Millipore公司;
Cryotome E冰冻切片机来源于美国Thermo公司;
Pannoramic切片数字扫描系统来源于匈牙利3DHISTECH公司;
高速冷冻离心机来源于德国Eppendorf公司。
相关溶液的配制
NaF溶液:分析天平称取1.0g的NaF,用1L的双蒸水将其充分溶解后,添加双蒸水配制成100mg/L的NaF工作液;
红芪多糖溶液:将红芪多糖溶解于生理盐水中配制成浓度为5mg/ml的悬混液,现用现配;
黄芪多糖溶液:将黄芪多糖溶解于生理盐水中配制成浓度为5mg/ml的悬混液,现用现配;
褪黑素溶液:分析天平称取褪黑素22.5mg溶解于168μL无水乙醇中,涡旋震荡至全部溶解,然后加入生理盐水4.35ml混匀,配制成浓度为5mg/ml的混合液,分装后在-20℃保存备用。
实施例1
为每10ml上述5mg/ml红芪多糖溶液溶解于1kg生理盐水中,制备的50mg/kg红芪多糖溶液。
实施例2
红芪多糖粉,加药物制剂常用的润滑剂、崩解剂,混合压片,制成片剂,患者服用该片剂每天摄入红芪多糖的质量为50mg·kg-1。
实施例3
红芪多糖粉,加药物制剂常用的润滑剂、稀释剂,混合压片,制成颗粒剂,患者服用该颗粒剂每天摄入红芪多糖的质量为100mg·kg-1。
实施例4
红芪多糖粉,加注射用水制备成浓度为0.01-10mg/ml的溶液,然后高压灭菌,在百级净化环境中装于西林瓶中,冷冻干燥,制成用于肌肉注射或静脉注射的冻干粉针剂,患者注射该冻干粉针剂每天摄入红芪多糖的质量为20mg·kg-1。
实施例5
红芪多糖粉,与甜味剂和矫味剂,制成红芪多糖饮料,患者饮用该饮料每天摄入红芪多糖的质量为180mg·kg-1。
对比例1
为每10ml上述5mg/ml黄芪多糖溶液溶解于1kg生理盐水中,制备的50mg/kg黄芪多糖溶液。
对比例2
为每10ml上述5mg/ml褪黑素溶液溶解于1kg生理盐水中,制备的50mg/kg褪黑素溶液。
相关实验及效果验证
1.1实验模型构建
选取体重180~220g、饲养于标准的动物实验室(湿度:55%~65%,温度:22~24℃,12:12小时昼夜节律)的8周龄清洁级SD大鼠(雌雄各半)50只,分为对照组(10只)和实验组(40只),适应性喂养一周,期间对照组饮用蒸馏水,实验组饮用NaF工作液。染毒16周后,将大鼠进行如下处理:对照组灌胃给予10ml/kg的生理盐水。实验组平均分为以下四组:①NaF组:灌胃给予10ml/kg的生理盐水;②红芪多糖组:灌胃给予实施例1红芪多糖溶液;③黄芪多糖组:灌胃给予对比例1黄芪多糖溶液;④褪黑素组:灌胃给予对比例2褪黑素溶液。连续灌胃给药8周。然后腹腔注射3%的戊巴比妥钠(4ml/kg)进行麻醉。
1.2肝脏病理学观察
将各组大鼠肝脏组织用4%多聚甲醛固定,常规石蜡包埋切片,经常规HE染色后观察肝脏的组织结构变化。
1.3活性氧(ROS)检测
将各组大鼠部分新鲜肝脏组织放入OCT包埋剂中快速冷冻,通过冰冻切片机来制备冰冻切片。切片用自发荧光猝灭剂孵育5min,之后用流水冲洗。滴加DHE染液,在黑暗条件下37℃孵育30min。磷酸盐缓冲液洗涤3次后滴加DAPI染色液染核10min。抗荧光猝灭剂封片后扫描照片。
1.4丙二醛(MDA)含量及超氧化物歧化酶(SOD)测定
收集大鼠肝脏组织块(约100mg),加入9倍体积预冷的生理盐水,在冰浴中匀浆后,4℃条件下2000r/min离心20min后收集上清液。BCA法测定蛋白浓度后按MDA和SOD检测说明书进行操作。
1.5凋亡检测
石蜡切片经脱蜡至水后用蛋白酶K进行修复,之后按照TUNEL说明书进行操作。扫描照片从每个组织切片中选择5个视野,对视野内的所有细胞进行计数,并分析TUNEL阳性细胞的比例。
1.6免疫荧光和免疫组化
按照组织免疫荧光操作检测各组肝脏组织中肿瘤坏死因子(TNF-α)的表达水平,使用Image J软件对荧光强度进行分析;通过免疫组化法检测肝脏组织中白介素6(IL-6)和IL-1β的表达情况,操作步骤按照试剂盒的说明书来进行,染色结束后使用Image J软件对各视野的平均光密度值进行统计。
1.7统计学方法
采用SPSS21.0软件对数据进行统计分析,计量资料实验数据以平均值±标准差的形式表示,经方差齐性检验后,采用单因素方差分析结合LSD检验进行分析。
2.实验结果及分析
2.1肝脏组织病理学变化
各组大鼠肝脏组织病理学变化如图1所示(标尺为20μm),NaF组大鼠肝脏细胞出现大量空泡化,相比NaF组,红芪多糖组、褪黑素组、黄芪多糖组空泡状的肝细胞数量明显减少,表明红芪多糖溶液、褪黑素溶液、黄芪多糖溶液具有改善氟中毒后肝脏组织病理学变化的效果,并且红芪多糖溶液的效果优于褪黑素溶液和黄芪多糖溶液。
2.2ROS检测结果
各组大鼠肝脏DHE染色变化如图2所示(标尺为20μm),与对照组相比,NaF组、红芪多糖组、褪黑素组、黄芪多糖组大鼠肝脏组织红色荧光强度均出现不同程度增加现象。与NaF组比较,红芪多糖组、褪黑素组、黄芪多糖组大鼠肝脏组织红色荧光强度有所降低,其中红芪多糖组红色荧光强度降低程度最大,表明红芪多糖溶液对ROS的抑制效果优于褪黑素溶液优于黄芪多糖溶液,使得氟中毒肝脏组织中活性氧增加量减少。
2.3MDA含量和SOD活性变化
各组大鼠肝脏DHE染色变化如表1所示,与对照组相比,实验组大鼠肝脏组织中MDA含量增加,SOD活性降低,其中NaF组差异最显著(P<0.05)。与NaF组相比,红芪多糖组、褪黑素组和黄芪多糖组大鼠肝脏MDA含量降低,SOD活性升高,且差异显著(P<0.05)。其中红芪多糖组与NaF组差异最显著,表明红芪多糖溶液在改善染氟大鼠肝脏氧化损伤中的效果最佳,优于褪黑素溶液,远远优于黄芪多糖溶液。
表1
*表示与对照组比较P<0.05;#表示与NaF组比较P<0.05
2.4凋亡检测结果
各组大鼠肝脏TUNEL阳性细胞数如图3所示(标尺为20μm),白色箭头表示TUNEL阳性细胞,A为对照组;B为NaF组;C为红芪多糖组;D为褪黑素组;E为黄芪多糖组。与对照组比较,实验组大鼠肝脏组织中TUNEL阳性细胞数显著增加,其中NaF组大鼠肝脏组织中TUNEL阳性细胞数显著增加,相比NaF组,红芪多糖组、褪黑素组和黄芪多糖组不同程度降低了NaF诱导的肝脏组织TUNEL阳性细胞数增多,其中红芪多糖组降低程度最大,表明红芪多糖溶液在减弱染氟大鼠肝脏细胞凋亡中的效果优于黄芪多糖溶液和褪黑素溶液。
2.5免疫荧光和免疫组化结果
各组大鼠肝脏Cleaved Caspase-3免疫荧光染色如图4所示(标尺为20μm),与对照组相比,实验组红色荧光强度明显增强;与NaF组相比,红芪多糖组、褪黑素组和黄芪多糖组红色荧光强度有所下降,其中红芪多糖组降低程度最大,表明红芪多糖溶液较褪黑素溶液和黄芪多糖溶液,在降低Cleaved Caspase-3表达中的作用更优。
各组大鼠肝脏TNF-α、IL-6和IL-1β的表达如图5所示(标尺为20μm),图左表示TNF-α免疫荧光检测,图中和图右表示IL-6和IL-1β的免疫组化检测,A为对照组;B为NaF组;C为红芪多糖组;D为褪黑素组;E为黄芪多糖组。TNF-α的免疫荧光结果表明,红芪多糖溶液、褪黑素溶液和黄芪多糖溶液处理后降低了NaF诱导的红色荧光强度增强,较NaF组差异显著(P<0.05)。IL-6和IL-1β的免疫组化结果表明,与NaF组比较,红芪多糖组、褪黑素组和黄芪多糖组的平均光密度值降低(P<0.05),表明红芪多糖溶液的抗炎作用要优于褪黑素溶液和黄芪多糖溶液。
申请人声明,本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (7)
1.红芪多糖在制备改善或治疗氟化物肝毒性药物或保健食品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物或保健食品为抑制因氟化物暴露致肝脏氧化应激、炎症或凋亡水平升高的药物或保健食品。
3.根据权利要求1所述的应用,其特征在于,所述氟化物为氟化钠。
4.根据权利要求1所述的应用,其特征在于,患者使用所述药物或保健食品摄入红芪多糖的质量为20~200mg·kg-1·d-1。
5.根据权利要求4所述的应用,其特征在于,患者使用所述药物或保健食品摄入红芪多糖的质量为50~100mg·kg-1·d-1。
6.一种含有如权利要求1~5所述红芪多糖的改善或治疗氟化物肝毒性的药物或保健食品,其特征在于,所述药物或保健食品剂型为溶液剂、糖浆剂、颗粒剂、胶囊剂、散剂、丸剂、片剂、水针剂、冻干粉剂、贴剂、凝胶剂、膜剂、滴丸剂、酒剂、浸膏剂或缓控释制剂。
7.根据权利要求6所述的一种药物或保健食品,其特征在于,所述药物或保健食品中红芪多糖的含量为1~99wt%。
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