CN106975014A - 一种人参果花青素的制备方法及用途 - Google Patents
一种人参果花青素的制备方法及用途 Download PDFInfo
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- CN106975014A CN106975014A CN201710110056.6A CN201710110056A CN106975014A CN 106975014 A CN106975014 A CN 106975014A CN 201710110056 A CN201710110056 A CN 201710110056A CN 106975014 A CN106975014 A CN 106975014A
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- ginseng fruit
- panax ginseng
- anthocyanidin
- liver
- gfa
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Abstract
本发明公开了人参果花青素(Ginseng Fruit Anthocyanins,GFA)的制备方法及用途。通过将成熟的人参果挤压去除种子后,将果浆低温提取后,通过大孔树脂柱色谱,收集低浓度乙醇洗脱部分,浓缩至干燥即得在制备保护扑热息痛(APAP)诱导的急性肝损伤药物中的应用,属于医药产品研究开发领域。同时发现GFA能够有效减少对乙酰基氨基酚致小鼠肝损伤引起的血清ALT和AST水平上升,减少肝脏组织GSH和降低肝脏组织MDA含量。因此,可以把该花青素制备成预防急性肝损伤的药物。其药物形式可以是现有的任何剂型,如片剂,胶囊剂,粉针剂,注射剂,丸剂,软胶囊,颗粒剂和贴剂等。
Description
技术领域
本发明公开了人参果花青素在制备预防急性肝损伤药物的新用途,属于中药中化合物的新生物活性的医药技术领域。
背景技术
对乙酰氨基酚(acetaminophen, APAP),又称扑热息痛,在临床上广泛用于镇痛和退热,APAP在治疗范围内是安全的,但过量摄入APAP会导致肝损伤。摄入过量APAP时,细胞中大量产生的NAPQI与GSH结合,导致GSH水平下降,大量产生的活性氧ROS以及硝基酪氨酸,从而影响细胞的能量生成和脂质代谢以及细胞的增殖、分化与凋亡。
肝脏疾病是人类最常见的疾病之一,严重威胁人类的健康。急性肝损伤是肝病发生、发展以及恶化的基本途径,情况严重者甚至引发肝硬化、肝癌,因此开发治疗和预防肝脏疾病的药物显得尤为重要。保肝类西药治疗强、见效快,但很多化学药常有的严重不良反应和耐药性,植物药为植物或其提取物制成的药物,具有整体和多靶点治疗观念,在人类历史上被广泛用来预防、保健和治疗疾病,且植物药很少发生单一化学药物所常有的严重不良反应和耐药性,因此天然植物药对治疗肝病具有广阔的研究前景。
花青素是一类广泛存在于植物中的水溶性天然色素,具有抗氧化、抗炎,抑菌,抗衰老、抗癌以及对肝脏,对心脑血管和视力的保护作用。人参来源于五加科植物人参(Panax ginseng C. A. Meyer)的干燥根及根茎,是我国传统的名贵中药材[11]。人参果是人参的成熟果实,含有人参皂苷、黄酮、多糖、生物碱、甾醇和挥发油等多种化学成分,其中皂苷及多糖含量最多且被人们深入研究,人参大部分化学成分具都有显著的药理活性,在对肝保护作用方面均有显著影响。
人参果花青素(Ginseng fruit anthocyanins, GFA)是从人参果中提取的类黄酮类成分,是一种水溶性色素,具有抗氧化、抗自由基等作用。目前为止,尽管人参皂苷类成分的肝损伤保护活性屡见报道,但有关人参果中花青素类成分是否具有保护肝损伤的药理作用尚不明确。本文从人参果花青素的抗氧化、抑制硝化应激,减少炎症反应及抑制细胞凋亡等机制证明人参果花青素对APAP诱导的小鼠肝损伤具有保护作用,为人参果花青素的临床应用提供一定的理论参考。
发明内容
本发明提供了人参果花青素在制备预防扑热息痛(APAP)诱导的急性肝损伤药物中的应用。
本发明所述的人参果花青素在用于上述用途时,其口服或胃肠外给药,均是安全的,在口服情况下,其可以以任何常规的形式给药,如片剂,胶囊剂,粉针剂,注射剂,丸剂,软胶囊,颗粒剂和贴剂等。
本发明制备保护急性肝损伤药物是由有效成分与固体或液体的赋形剂一起构成的,这里使用的固体或液体的赋形剂在本领域是众所周知的,下面举几个例子,散剂是内服的粉末剂,它的赋形剂有乳糖,淀粉,糊精,碳酸钙,合成或天然硫酸铝,氧化镁,硬脂酸镁,碳酸氢钠,干燥酵母等;溶液剂的赋形剂有水,甘油,1,2-丙二醇,单糖浆,乙醇,乙二醇,聚乙二醇,山梨糖醇等;软膏剂的赋形剂可以使用脂油,含水羊毛脂、凡士林、甘油、蜂蜡、木蜡、液体石蜡等组合成的疏水剂或亲水剂。本发明的药物组合物可以按现有技术中已知的方法如混合、制粒、压片来制备。本发明药物组合物还可以包括各种药学使用的任何组分,如香味剂、着色剂、甜味剂等。
本发明可以通过下面的实验例进一步说明。
实验例1.人参果花青素的制备方法
1.1人参果花青素制备方法采用如下步骤:
首先,将成熟的人参果,通过机器挤压分离果肉和种子,将果肉按100:0.5加入NaCO3,充分混匀,静置72小时以上。将静置好的果肉投入提取罐,按12:1加入深井水或蒸馏水,加热至35~40℃,提取8~10小时,沉降净化10~16小时,过滤,稍浓缩至原液体积的80~90%为宜。将上述浓缩液注入大孔树脂柱进行吸附。用4倍柱体积的30~50%乙醇洗脱树脂柱,收集回收液,进一步沉降净化8~10小时,在35~45%下进行低温浓缩,分离乙醇,冷冻干燥12小时以上,取出即得人参果花青素。
NaCO3能够有效去除人参果浆中的杂质,与不添加NaCO3比较,人参果花青素的收率提高15~20%。
1.2人参果花青素成分分析
通过分析:人参果花青素以原花青素为主,主要为儿茶素、表儿茶素及形成的二聚体,此外尚含少量黄酮类成分,如人参黄酮苷(Panasenoside)
实验例2.人参果花青素对扑热息痛致小鼠肝损伤的保护作用
1材料与方法
1.1动物: 雄性ICR小鼠,SPF级,5~6周,体重20~22g,购自长春市亿斯实验动物技术有限责任公司,合格证号:SCXK(吉)2011-0004,自由饮水,适应性饲养一周后随机分为4组后进行实验。
材料仪器: 人参果花青素,来自抚松安东参业有限公司;APAP(对乙酰氨基酚)源于阿拉丁试剂有限公司;苏木素-伊红染液(H&E),Hoechst33258染色液,上海碧云天生物技术有限公司;谷丙转氨酶(ALT)、谷草转氨酶(AST)、丙二醛(MDA)及谷胱甘肽(GSH)试剂盒,购于南京建成生物工程有限公司;兔来源单克隆抗鼠诱导型一氧化氮合酶(iNOS)、诱导型环氧化酶(COX-2)和3-硝基络氨酸(3-NT),购于美国Cell Signaling Technology公司;免疫组化试剂盒(SV0002)、免疫荧光试剂盒(SA1074),购于博士德生物技术有限公司;Hoechst 33258染色试剂盒(C0003),碧云天。PL303电子天平,上海梅特勒-托利多仪器有限公司;显微镜Leica DM500和Leica DM2500荧光显微镜,德国徕卡;德国SPECTROstarNano全波长扫描式酶标仪,德国BMG LABTECH公司;高速冷冻离心机,北京昊特斯科技有限公司。
方法
1.3.1药物配制: 称取GFA,加CMC-Na混悬至所需浓度(200mg/kg、400mg/kg);称取APAP溶于热的生理盐水至所需浓度。
动物分组及处理:ICR小鼠适应性饲养一周后随机分为4组,即空白对照组(Normal)、模型组(APAP)、GFA低剂量组(200mg/kg, APAP+GFA)、GFA高剂量组(400mg/kg,APAP+GFA),每组8只,GFA采用灌胃方式给药,空白对照组和模型组以等量生理盐水灌胃,一天一次,连续给药7天。末次给药后,禁食不禁水12小时后模型组按250mg/kg腹腔注射APAP,造模12小时后禁食不禁水,断颈处死。眼球取血,血液室温凝结1h后以3000rpm/10min离心,留取上清供血清指标ALT和AST检测。取肝脏组织溶于生理盐水中制成肝匀浆用于检测GSH和MDA水平,取相同部位的肝组织固定于10%甲醛中,用于肝组织切片观察病理变化。
计算免疫器官指数:称取小鼠体质量和肝脏、脾脏质量,计算脏器指数。
肝脏(脾脏)指数=肝脏(脾脏)质量/小鼠体质量
1.3.4血清学指标:依照南京建成生物有限技术公司提供的试剂盒说明书,采用微板法检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)肝功能指标。
氧化应激指标:取冻存的肝组织,制备组织匀浆,取上清液,采用二硫代对硝基苯法和硫代巴比妥法试剂盒测定GSH和MDA的含量,按照试剂盒说明书进行操作。
染色:将肝组织固定于10%甲醛中,石蜡包埋切片,将切片放入二甲苯中脱蜡30min,再放入无水乙醇中30min,依次放入95%、70%、50%乙醇中3min水化,最后放入蒸馏水中5min,加入苏木素,1min后流水冲洗反蓝,返蓝后依次放入50%、70%、95%乙醇中各3min,滴加伊红,1min后放入无水乙醇,5min后放入二甲苯中保持30min,滴加树脂,盖玻片封片,光镜下观察肝组织切片的病理变化。
染色:取肝组织石蜡切片,脱蜡水化(与H&E相同)后,从蒸馏水中取出切片,用PBS缓冲液清洗两次,每次3min,滴加Hoechst 33258染色剂,5min后用PBS洗两次,每次3min,擦干后滴加抗荧光封片液,盖上载玻片,荧光显微镜下观察组织。显微镜下随机选取3个高倍视野,计算阳性肝细胞数和肝细胞总数。
肝细胞凋亡率=阳性肝细胞数/肝细胞总数。
免疫组织化学染色:取肝组织石蜡切片,脱蜡水化(与H&E相同)后,从蒸馏水中取出切片,加入内源性过氧化物酶37℃烘箱中保存30min,用蒸馏水洗3次,每次3min,放入抗原修复液中微波修复8min,恢复室温后滴加5%BSA封闭液,保留10min后擦掉封闭液加入抗体iNOS(1:100)、COX-2(1:100),4℃孵育过夜。次日,恢复室温后PBS冲洗三次,每次5min,擦干切片;滴加聚合HRP标记抗兔IgG置37℃烘箱保存1h,之后用PBS洗三次,每次5min;滴加DAB1min,加入苏木素1min后流水返蓝,重复H&E染色返蓝后过程,显微镜下观察组织。
免疫荧光染色:取肝组织石蜡切片,脱蜡水化(与H&E相同)后,从蒸馏水中取出切片,放入抗原修复液中微波修复8min,恢复室温后滴加血清封闭液,室温放置20min,擦干后加入3-NT(1:200),4℃孵育过夜。次日,恢复室温后PBS冲洗三次,每次5min,擦干切片,滴加生物素化羊抗兔IgG置37℃烘箱保存30min,之后用PBS洗三次,每次5min,滴加CY3(试剂盒内置)置37℃烘箱保存30min,之后用PBS洗三次,每次5min;滴加DAB1min,之后用PBS洗三次,每次5min,擦干后用抗荧光猝灭PVB封片液封片,37℃烘箱保存30min,荧光显微镜下观察。
统计学方法采用SPSS18.0软件,单因素方差分析法(ANOVA)进行统计分析,组间比较各项数据以Mean±S.D.表示;Ridit分析用于组织学检查比较;采用GraphPad Prism6.0软件进行方差分析并作图,P<0.05有统计学意义。
结果
2.1GFA对APAP诱导小鼠肝损伤脏器指数的影响
给药7天后,GFA对小鼠脏器指数的影响见表1:与空白对照组相比,模型型组小鼠肝组织颜色加深且肿大,肝指数明显增高(P<0.05)。预给药7天后,花青素低、高剂量组均可明显降低小鼠的肝指数(P<0.05)。此外,与空白组相比,小鼠的脾指数可以明显下降(P<0.05),GFA两个给药组明显抑制了脾指数的下降(P<0.05)。结果表明:GFA能够改善APAP致肝损伤小鼠肝肿胀,调节机体免疫。
表1. GFA对小鼠脏器指数的影响
注:与正常对照组相比* P<0.05;与模型组相比# P<0.05
2.2 GFA对APAP诱导小鼠血清指标的影响
ALT和AST是肝损伤的主要酶标记物,当肝细胞受到损伤时,这些标记物会从肝脏渗漏到血液中从而导致二者血清浓度急剧升高。如表2所示,空白对照组比较,模型组小鼠血清中ALT和AST的水平显著升高(P<0.05),GFA低、高剂量组能够明显抑制了血清中ALT和AST水平的升高且存在一定的剂量效应关系(P<0.05)。肝脏受损时给药组与模型组相比血浆中ALT、AST水平的降低说明人参果花青素具有较好的保护肝脏受损的作用。
表2.人参果花青素对小鼠血清中ALT和AST的影响
注:与正常对照组相比* P<0.05;与模型组相比# P<0.05
2.3GFA对小鼠肝脏中GSH和MDA的影响
如表3所示,与空白组比较,肝损伤模型组小鼠肝组织匀浆中的GSH水平明显低于空白组(P<0.05),给药组显著地抑制了GSH水平的降低。MDA水平显示组织的脂质过氧化产物的多少,直接说明细胞受损程度;与空白组比较模型组的MDA含量明显高于空白组(P<0.05),GFA低、高给药明显抑制了肝组织中MDA水平的升高。以上数据说明GFA能够提高肝组织抗氧化能力以及减缓APAP对肝细胞产生的脂质过氧化作用。
表3.GFA对小鼠匀浆中GSH和MDA的影响
注:与正常对照组相比* P<0.05;与模型组相比# P<0.05
2.4GFA对小鼠肝组织病理变化影响
如表4所示,空白对照组小鼠肝组织细胞排列整齐且形态正常;APAP模型组肝组织中央静脉附近细胞大面积坏死,坏死处可见无核肝细胞残体,肝窦扩张充血,伴有部分细胞变性肿胀和细胞炎性浸润,部分凋亡细胞有明显的点状和灶状坏死;给药组细胞坏死程度减轻,炎性细胞浸润减轻,高剂量组细胞坏死显著减轻,肝细胞排列比较规律。细胞坏死程度如表2所示,APAP模型组损伤坏死较严重即模型成功,由此可直接反映出GFA低、高剂量组对缓解肝组织损伤均有一定的作用,图略。
表4 小鼠肝组织坏死评分结果
Code of point (n=8): Level 0 calculated 0 mark; Level I calculated 1mark; Level II calculated 2 marks; Level III calculated 3 marks; Level IVcalculated 4 marks;Values are expressed as the Mean ± S.D,n= 8. *P<0.05vsNormal group; # P<0.05vs APAP group
2.5GFA对小鼠肝组织细胞凋亡的影响
凋亡细胞通过Hoechst33258染色后细胞呈阳性表达,空白对照组肝细胞排列整齐且细胞核清晰清晰可见,几乎没有蓝色荧光细胞;模型组中央静脉附近细胞大面积坏死,细胞核固缩,有大面积蓝色细胞核且荧光较强;给药组坏死面积减小,高剂量组损伤显著缓解。推测GFA对抗APAP肝损伤的保护作用可能是通过诱导细胞凋亡实现的,图略。
对小鼠肝组织COX-2和iNOS蛋白表达的影响
空白组小鼠细胞结构正常,COX-2主要在胞浆中表达,模型组小鼠中央静脉附近呈阳性表达,细胞核排列杂乱,部分无完整细胞结构;给药剂量组呈现不同程度阳性表达,其中高剂量组较低剂量组细胞形态健康整齐且阳性表达较少。iNOS主要在细胞的细胞核上阳性表达,模型组细胞核排列紊乱阳性表达非常明显,细胞结构损伤严重,低剂量组细胞核阳性表达明显减少,高剂量组表达情况基本与对照组小鼠相同。COX-2与iNOS在受到炎性因子刺激的时候呈高表达状态,推测GFA可能有抗炎症作用,图略。
对小鼠肝组织3-NT蛋白表达的影响
肝组织内的活性氮族与活性氧族发生作用使蛋白质中的酪氨酸硝化成3-硝基络氨酸(3-NT),引起细胞坏死或者凋亡。细胞核经DAPI染色后表现为蓝色亮点,3-NT细胞阳性表达为红色荧光亮点,空白对照组小鼠肝细胞结构排列整齐中央静脉清晰可见且无荧光表达;APAP组小鼠中央静脉附近大面积荧光表达明显,细胞严重损伤;给药低高剂量组阳性表达明显减少,根据光密度软件分析显示,GFA低高剂量组3-NT表达率分别减少35%和42%。该结果显示GFA保护肝损伤可能是抑制硝化应激而实现的,图略。
讨论
研究结果显示,250mg/kg的APAP诱导小鼠血清中AST和ALT水平显著升高,肝组织中GSH水平的降低和MDA水平的升高,模型组肝细胞肿胀,排列疏松,细胞核部分消失或坏死,说明造模成功;GFA给药组与模型组相比血清中AST、ALT水平升高,肝组织中GSH升高和MDA水平降低,结果显示人参果花青素可以缓解对乙酰氨基酚造成的肝损伤。
肝组织损伤时,细胞中大量产生NAPQI与GSH结合,导致GSH含量下降,细胞中超氧负离子歧化作用导致大量活性氧生成,对细胞产生氧化应激作用。结果表明:GFA给药组中GSH含量升高,MDA含量降低,说明GFA缓解了氧化应激而发挥保护作用。正常肝组织细胞中几乎炎症因子iNOS无表达,肝组织受损时组织中大量表达的iNOS催化产生的NO与大量的活性氧反应产生的氧化亚硝酸盐进而与酪氨酸反应生成硝基酪氨酸。从免疫荧光结果可以看出GFA给药组3-NT阳性表达明显减少,推测这与GFA能够抑制炎症因子表达、减缓硝化应激作用有关。除此之外,H&E和Hoechst33258染色结果显示模型组肝细胞坏死、肿胀,部分细胞发生炎性浸润,说明GFA具有抗凋亡作用。
目前对GFA的活性研究报道相对较少,本实验首次证明GFA对APAP诱导肝损伤具有保护作用,其可能的机制包括抑制氧化应激和硝化应激,减少炎症反应及抑制细胞凋亡,为GFA的深入研究及临床应用提供一定的理论依据。
实施例药物的实施例
制备药剂的实施例一
胶囊剂的制备200g人参果花青素,药用淀粉适量,两者充分混匀,装胶囊,制成1000粒胶囊,每粒重0.25g,每粒人参果花青素 200mg。口服,每次4粒,每日三次。
制备药剂的实施例二
片剂的制备 200g人参果花青素,药用淀粉适量,两者充分混匀,用乙醇做粘合剂制湿颗粒,干燥,过120目筛整粒,装胶囊,每粒200 mg,每次口服1-2粒,每日2次。
制备药剂的实施例三
滴丸剂的制备聚乙二醇4000 300g,在水浴上熔化,加入人参果花青素原料200g,搅拌均匀,倾入保温管中,调节恒温装置,使药液在80-90℃下滴入冷却过的液体石蜡中(温度±4℃),滴完后,将药丸倾入滤纸上吸干石蜡油,再加入少量滑石粉,混匀,得人参果花青素滴丸1000粒。口服,一次4粒,每日三次,饭后服用。
以上根据上述实施方式对本发明的人参果花青素在制备预防急性肝损伤药物的应用进行了说明,但本发明并不限于上述实施方式,在不脱离其要旨的范围内,可在各种方式中实施本发明。除了上述实施方式之外,其它等同技术方案也应当在其保护范围之内,在此不再一一叙述。
实施例药物的实施例
制备药剂的实施例一
胶囊剂的制备200g人参果花青素,药用淀粉适量,两者充分混匀,装胶囊,制成1000粒胶囊,每粒重0.25g,每粒人参果花青素 200mg。口服,每次4粒,每日三次。
制备药剂的实施例二
片剂的制备 200g人参果花青素,药用淀粉适量,两者充分混匀,用乙醇做粘合剂制湿颗粒,干燥,过120目筛整粒,装胶囊,每粒200 mg,每次口服1-2粒,每日2次。
制备药剂的实施例三
滴丸剂的制备聚乙二醇4000 300g,在水浴上熔化,加入人参果花青素原料200g,搅拌均匀,倾入保温管中,调节恒温装置,使药液在80-90℃下滴入冷却过的液体石蜡中(温度±4℃),滴完后,将药丸倾入滤纸上吸干石蜡油,再加入少量滑石粉,混匀,得人参果花青素滴丸1000粒。口服,一次4粒,每日三次,饭后服用。
以上根据上述实施方式对本发明的人参果花青素在制备预防急性肝损伤药物的应用进行了说明,但本发明并不限于上述实施方式,在不脱离其要旨的范围内,可在各种方式中实施本发明。除了上述实施方式之外,其它等同技术方案也应当在其保护范围之内,在此不再一一叙述。
Claims (3)
1.一种人参果花青素(Ginseng Fruit Anthocyanins)的制备方法,主要包括如下步骤:首先,将成熟的人参果,通过机器挤压分离果肉和种子,将果肉按100:0.5加入NaCO3,充分混匀,静置72小时以上,将静置好的果肉投入提取罐,按12:1加入深井水或蒸馏水,加热至35~40℃,提取8~10小时,沉降净化10~16小时,过滤,稍浓缩至原液体积的80~90%为宜,将上述浓缩液注入大孔树脂柱进行吸附,用4倍柱体积的30~50%乙醇洗脱树脂柱,收集回收液,进一步沉降净化8~10小时,在35~45%下进行低温浓缩,分离乙醇,冷冻干燥12小时以上,取出即得人参果花青素。
2.人参果花青素能够有效减少对乙酰基氨基酚所致的氧化应激损伤,同时能够抑制硝化应激,减少炎症反应及抑制细胞凋亡等。
3.根据权利要求1所述的应用,其特征在于:将人参果花青素作为活性成份或与其它药物组合,与制药中可以接受的辅助和/或添加成份混合后,按常规的制药方法和工艺要求,制成用于治疗或预防急性肝损伤的制药中可接受的任何剂型,如片剂,胶囊剂,粉针剂,注射剂,丸剂,软胶囊,颗粒剂和贴剂等。
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