CN110711208B - 苋菜提取物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种苋菜提取物及其制备方法和应用,属于天然产物活性成分技术领域。该苋菜提取物的制备方法包括以下步骤:提取:取苋菜,以体积百分浓度为80%‑100%的乙醇水溶液为溶剂,加热提取,得提取液;萃取:以石油醚萃取上述提取液;取下层再以乙酸乙酯萃取,保留水层,水层回收溶剂后得水层提取物;纯化:将上述水层提取物以大孔树脂进行纯化,得生物碱类物质,即为所述苋菜提取物。该苋菜提取物经小鼠实验证实,具有较好的口服降低血尿酸的作用。
Description
技术领域
本发明涉及天然产物活性成分技术领域,特别是涉及一种苋菜提取物及其制备方法和应用。
背景技术
痛风是长期嘌呤代谢障碍、血尿酸增高引起组织损伤的一组异质性疾病。临床特点是高尿酸血症、痛风性急性关节炎反复发作、痛风石沉积、特征性慢性关节炎和关节畸形,常累及肾脏引起慢性间质性肾炎和肾尿酸结石形成。痛风的生化标志是高尿酸血症,指细胞外液的尿酸盐呈超饱和状态,一般认为血尿酸>416umol/L时为高尿酸血症。约5%-12%高尿酸血症患者会发展成为痛风。痛风的急性发作是尿酸钠,在关节及关节周围组织以结晶形式沉积引起的急性炎症反应。痛风不仅可以侵犯骨和关节,而且还容易累及肾脏和心血管系统。高尿酸血症及原发性痛风与肥胖症、高脂血症、高血压病、糖尿病、动脉粥样硬化等疾病呈显著正相关。因此,痛风是危害人类健康的一种严重的代谢性疾病。
近年来,随着人们生活水平的提高,饮食结构发生变化,糖、脂肪、蛋白质的摄入量明显增加,高尿酸血症和痛风的发病率日益增高,己成为一种常见病。为了避免关节炎反复发作和肾脏病变形成,痛风患者需要长期服用有降低血尿酸作用的药物。但目前安全、有效、经济的降药物较少。
我国用中草药治疗痛风病有悠久的历史,但迄今尚无能够有效降低血的中药制剂用于临床。因此,寻找新型降药物,特别是借鉴中医经验开发有降作用的中草药制剂具有重要的意义。
发明内容
本发明的目的,就是克服现有技术的不足,提供一种苋菜提取物及其制备方法和应用,该苋菜提取物具有较好的降低血中尿酸的作用。
为了达到上述目的,采用如下技术方案:
一种苋菜提取物的制备方法,包括以下步骤:
提取:取苋菜,以体积百分浓度为80%-100%的乙醇水溶液为溶剂,加热提取,得提取液;
萃取:以石油醚萃取上述提取液;取下层再以乙酸乙酯萃取,保留水层,水层回收溶剂后得水层提取物;
纯化:将上述水层提取物以大孔树脂进行纯化,得生物碱类物质,即为所述苋菜提取物。
上述苋菜(学名:Amaranthus tricolor L.)为苋科,苋属植物,具有抗氧化、抗菌、抗癌、肝保护等作用。
在其中一个实施例中,所述提取步骤中,每次按照苋菜:溶剂为1g:8-12ml的料液比加入90%-100%的乙醇溶液回流提取2-4次,每次1-3小时。
在其中一个实施例中,所述萃取步骤中,先将所述提取液浓缩至原体积的1/20-1/30,得浓缩液,再加入石油醚,按照体积比为1ml浓缩液:0.5-1.5ml石油醚进行萃取,随后再加入乙酸乙酯,按照体积比为1ml浓缩液:0.5-1.5ml乙酸乙酯进行萃取。
在其中一个实施例中,所述纯化步骤中,所述大孔树脂为HP-20型大孔树脂,所述纯化步骤为:将所述水层提取物湿法上样,以水洗脱,弃去水洗脱液;再以30%-50%的甲醇水溶液洗脱,收集甲醇洗脱液,回收溶剂,即得所述苋菜提取物。
在其中一个实施例中,所述纯化步骤中,以20柱体积的水洗脱,并以16柱体积的40%的甲醇水溶液洗脱。
在其中一个实施例中,所述纯化步骤中,还包括制备液相纯化步骤,具体为:将所述苋菜提取物上样至制备液相色谱,以下述条件精制:
固定相:C18反相柱
流动相:A相为甲醇,B相为水;
流速:5ml/min;
检测波长:252-256nm;
收集其中含有生物碱的部分洗脱液,回收溶剂,即得苋菜精提物。
上述C18反相柱优选20mm×250mm,3.5μm规格。
在其中一个实施例中,所述制备液相纯化步骤中,采用梯度洗脱,所述梯度洗脱的具体参数为:0—10min,A相浓度由0%升至10%;10—20min,A相浓度由10%升至20%;20—30min,A相浓度由20%升至30%;20—30min,A相浓度由30%升至100%;
收集保留时间为28-32min的色谱峰,回收溶剂,即得苋菜精提物。
本发明还公开了述的苋菜提取物的制备方法制备得到的苋菜提取物。
本发明还公开了上述的苋菜提取物在制备用于预防和治疗高尿酸血症的药物中的应用。
本发明还公开了一种药物组合物,包括作为活性成分的上述的苋菜提取物,以及药学上可接受的辅料。
与现有技术相比,本发明的有益效果在于:
本发明的苋菜提取物,通过乙醇水溶液提取后,以石油醚去除脂类成分,再以乙酸乙酯去除中等极性成分,得到的水层提取物以大孔树脂纯化后,得到生物碱类物质,即为苋菜提取物。该提取物经小鼠实验证实,具有较好的口服降低血尿酸的作用。
具体实施方式
下面将结合具体实施方法来详细说明本发明,在本发明的示意性实施及说明用来解释本发明,但并不作为对本发明的限定。
以下实施例和实验例中所用原料,如无特别说明,均为市售购得。
实施例1
一种苋菜提取物,通过以下制备方法得到:
一、提取。
取1000g苋菜叶(鲜品),先将苋菜超微粉碎,再用10倍95%乙醇(10L)80℃提取2h,共提取3次;合并提取液。
二、萃取。
将上述提取液用旋转蒸发60℃浓缩至400ml左右,先用石油醚按体积比1:1萃取,上层为石油醚层,取下层,继续用乙酸乙酯按照体积比为1:1萃取,分层后下层即为为水层,上层为乙酸乙酯层。
回收溶剂,得到石油醚层提取物、乙酸乙酯层提取物和水层提取物。
三、纯化。
1、大孔树脂纯化。
取上述水层提取物20g,采用湿法上样,柱子直径约为10cm,长50cm,充分吸附在HP-20大孔树脂(日本三菱化工))中12h以上;之后用蒸馏水冲洗,流速约2ml/min洗脱20柱体积,洗脱液至接近无色,再用40%甲醇洗脱,流速约2ml/min,洗脱16柱体积,收集40%甲醇洗脱液,用旋转蒸发仪60℃浓缩,再使其烘干,称重。得到40%甲醇洗脱部分为2g,即为苋菜提取物。
2、水层提取物的分离
将所述苋菜提取物上样至制备液相色谱,以下述条件精制:
固定相:C18反相柱(20mm×250mm,3.5μm)
流动相:A相为甲醇,B相为水;梯度洗脱35min:0—10min,A相浓度由0%升至10%;10—20min,A相浓度由10%升至20%;20—30min,A相浓度由20%升至30%;20—30min,A相浓度由30%升至100%;
流速:5ml/min;
检测波长:252-256nm;
收集保留时间为28-32min的色谱峰,回收溶剂,得苋菜精提物。
实验例1
将实施例1制备得到的苋菜精提物进行鉴别测试。
经测试,上述苋菜精提物经碘化铋钾显色,有棕红色沉淀生成,证明其主要成分为生物碱类物质。
实验例2
苋菜对小鼠高尿酸血症的作用。
1、高尿酸血症小鼠模型建立
将昆明种小鼠随机分组,每组8只。分为:正常对照组、模型组、石油醚层提取物组、乙酸乙酯层提取物组、水层提取物组、苋菜提取物组、苋菜精提物组、别嘌呤醇组及苯溴马隆组。
其中,石油醚层提取物组、乙酸乙酯层提取物组、水层提取物组、苋菜提取物组、苋菜精提物组均按照以下剂量口服给药:浓度280mg/ml,10ml/kg灌胃,每日1次,连续7天。
别嘌呤醇组按40mg/Kg灌胃给药,苯溴马隆组按20mg/Kg灌胃给药,每日1次,连续7天。第7天,除对照组外,余各组在末次给药前1时氧嗪酸钾盐小鼠腹腔注射,1小时后模型组及各实验组灌胃给药,灌胃后1小时尾取血测定血尿酸,后摘眼球取血,离心分离血清,测定血尿酸(采用市售尿酸试剂盒测定)。
结果如下表所示。
表1.苋菜对高尿酸血症小鼠血尿酸水平的影响
注:*表示与对照组相比,具有统计学上的差异,P≤0.05;#表示与模型组相比,具有统计学上的差异,P≤0.01;##*表示与模型组相比,具有统计学上的差异,P≤0.05.
从上述结果可以看出,模型组与对照组相比,血尿酸值明显升高,且具有统计学上的差异,说明造模成功。
石油醚层提取物组和乙酸乙酯层提取物组降血尿酸效果不明显,该两组血尿酸值与模型组相当。
水层提取物组、苋菜提取物组、苋菜精提物组均具有一定的降低血尿酸效果,而其中苋菜提取组和苋菜精提组和模型组相比,具有统计学上的差异,且苋菜精提组具有最佳的效果。
以上对本发明实施例所提供的技术方案进行了详细介绍,本文中应用了具体个例对本发明实施例的原理以及实施方式进行了阐述,以上实施例的说明只适用于帮助理解本发明实施例的原理;同时,对于本领域的一般技术人员,依据本发明实施例,在具体实施方式以及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本发明的限制。
Claims (9)
1.一种苋菜提取物的制备方法,其特征在于,包括以下步骤:
提取:取苋菜,以体积百分浓度为80%-100%的乙醇水溶液为溶剂,加热提取,得提取液;
萃取:以石油醚萃取上述提取液,弃去石油醚;取下层再以乙酸乙酯萃取,弃去乙酸乙酯层,保留水层,水层回收溶剂后得水层提取物;
纯化:将上述水层提取物以大孔树脂进行纯化,所述大孔树脂为HP-20型大孔树脂,所述纯化步骤为:将所述水层提取物湿法上样,以水洗脱,弃去水洗脱液;再以30%-50%的甲醇水溶液洗脱,收集甲醇洗脱液,回收溶剂,得生物碱类物质,即为所述苋菜提取物。
2.根据权利要求1所述的苋菜提取物的制备方法,其特征在于:所述提取步骤中,每次按照苋菜:溶剂为1g:8-12ml的料液比加入90%-100%的乙醇溶液回流提取2-4次,每次1-3小时。
3.根据权利要求1所述的苋菜提取物的制备方法,其特征在于:所述萃取步骤中,先将所述提取液浓缩至原体积的1/20-1/30,得浓缩液,再加入石油醚,按照体积比为1ml浓缩液:0.5-1.5ml石油醚进行萃取,随后再加入乙酸乙酯,按照体积比为1ml浓缩液:0.5-1.5ml乙酸乙酯进行萃取。
4.根据权利要求1所述的苋菜提取物的制备方法,其特征在于:所述纯化步骤中,以20柱体积的水洗脱,并以16柱体积的40%的甲醇水溶液洗脱。
5.根据权利要求1-4任一项所述的苋菜提取物的制备方法,其特征在于:所述纯化步骤中,还包括制备液相纯化步骤,具体为:将所述苋菜提取物上样至制备液相色谱,以下述条件精制:
固定相:C18反相柱
流动相:A相为甲醇,B相为水;
流速:5ml/min;
检测波长:252-256nm;
收集其中含有生物碱的部分洗脱液,回收溶剂,即得苋菜精提物。
6.根据权利要求5所述的苋菜提取物的制备方法,其特征在于:所述制备液相纯化步骤中,采用梯度洗脱,所述梯度洗脱的具体参数为:0—10min,A相浓度由0%升至10%;10—20min,A相浓度由10%升至20%;20—30min,A相浓度由20%升至30%;20—30min,A相浓度由30%升至100%;
收集保留时间为28-32min的色谱峰,回收溶剂,即得苋菜精提物。
7.权利要求1-6任一项所述的苋菜提取物的制备方法制备得到的苋菜提取物。
8.权利要求7所述的苋菜提取物在制备用于预防和治疗高尿酸血症的药物中的应用。
9.一种药物组合物,其特征在于,包括作为活性成分的权利要求7所述的苋菜提取物,以及药学上可接受的辅料。
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