CN110898116A - 一种鸡矢藤提取物及其分离制备方法和应用 - Google Patents
一种鸡矢藤提取物及其分离制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种鸡矢藤提取物及其分离制备方法和医药用途,所述分离制备方法为:称取鸡矢藤药材,加8‑20倍量水煎煮1‑3次,每次1‑3h,滤过,浓缩;加入乙醇,调节至含醇量为50‑80%,搅拌均匀,静置12‑24h,取上清液,回收乙醇,浓缩成稠膏;加乙醇至溶解完全,加柱层析硅胶拌样;干燥,将干燥后的样品放入层析柱中,依次用乙酸乙酯、乙酸乙酯‑甲醇溶剂洗脱至2‑6倍柱体积,收集洗脱液;回收溶剂,浓缩;合并含有鸡矢藤苷成分的洗脱液,即得所述鸡矢藤提取物。本发明所述制备方法操作简单,成本较低,安全无毒,所述提取物可显著降低次黄嘌呤诱导的高尿酸血症小鼠血清尿酸水平。
Description
技术领域
本发明属于医药技术领域,涉及天然中药的分离,具体涉及一种鸡矢藤提取物及其分离制备方法和应用。
背景技术
尿酸为机体内嘌呤代谢的终产物。体内衰老细胞和食物,尤其是富含嘌呤的食物如动物内脏、海鲜等在体内新陈代谢的过程中产生嘌呤,嘌呤在肝脏中再次氧化为尿酸。由于饮食、疾病等原因,引起嘌呤代谢紊乱,近年的研究表明,高尿酸血症不仅是痛风最重要的生化基础,而且与高血压、高脂血症、动脉粥样硬化、肥胖、胰岛素抵抗的发生密切相关,所以控制高尿酸血症对治疗痛风等严重代谢性疾病具有重要意义。
鸡矢藤为茜草科植物鸡矢藤Paederia scandens(Lour.)Merr.的干燥地上部分,壮药名为勾邓骂(Gaeudaekmaj),瑶药名为缺解美(jaih nqaiv hmei)。《广西药用植物名录》、《广西壮族自治区壮药质量标准》、《中国瑶药学》均将其收录。壮医认为其通谷道,除湿度,祛风毒,活血止痛;用于肝脾肿大、食滞、胃痛、水肿、泄泻、痢疾、风湿骨痛、跌打损伤、瘰疬、痈疮、耳鸣。瑶医认为其祛风利湿,清热解毒,消肿,化痰止咳,消食化积。研究表明,鸡矢藤具有抗高尿酸作用。
经查阅文献,鸡矢藤含苷类成分的提取物的提取分离纯化可采用多种方法来实现。例如申请号为201410266480.6、专利名称为《一种从鸡矢藤中提取鸡矢藤苷的方法》的中国专利公开了采用70-90%的乙醇提取、超滤膜超滤、大孔树脂柱洗脱等手段来提取鸡矢藤中苷类成分的方法,但是该操作方法复杂,生产成本高,不适合工业化生产。申请号为201410620341.9、专利名称为《鸡矢藤提取物及其用途》的中国专利也公开了采用乙醇提取得提取液、提取液浓缩至无醇味上大孔吸附树脂层析,再依次用水和80-90%乙醇洗脱的方法来获得鸡矢藤提取物,该法亦存在着生产成本高的问题。
发明内容
本发明要解决的其中一个技术问题是:针对上述现有技术存在的不足,提供一种操作简单、成本较低、安全、无毒且易于产业化的鸡矢藤提取物的提取制备方法。
本发明以如下技术方案解决上述技术问题:
一种鸡矢藤提取物的分离制备方法,包括以下具体步骤:
(1)称取鸡矢藤药材,以鸡矢藤药材的质量计量,加8-20倍量水煎煮1-3次,每次1-3h,滤过,滤液浓缩至浓度为每1ml相当于原药材1-2g;
(2)在浓缩滤液中加入乙醇,调节至含醇量为50-80%,搅拌均匀,静置12-24h,取上清液,回收乙醇,浓缩成稠膏;
(3)稠膏加乙醇至溶解完全,加柱层析硅胶拌样,稠膏与硅胶的比例为1g∶1g-1g∶3g;干燥,将干燥后的样品放入层析柱中,用乙酸乙酯洗脱至2-6倍柱体积;再用体积比为9∶1-7∶3 的乙酸乙酯-甲醇溶剂洗脱至2-6倍柱体积,收集洗脱液;
(4)洗脱液回收溶剂,浓缩;合并含有鸡矢藤苷成分的洗脱液,即得所述鸡矢藤提取物。
优选地,所述的步骤(3)中,所述稠膏与硅胶的比例为1g∶1g;所述乙酸乙酯-甲醇溶剂的体积比为9∶1、8∶2、7∶3;所述收集的洗脱液浓度为每1ml相当于原药材1-2g。所述的步骤(4)中,采用薄层色谱法进行洗脱液中鸡矢藤总苷类成分的监控,再按照监控结果合并含有鸡矢藤苷成分的洗脱液。
优选地,所述的鸡矢藤提取物中总苷含量≥50%。所述的总苷含量以鸡矢藤苷计量。
本发明要解决的另一个技术问题是:提供按照上述方法制备得到的鸡矢藤提取物及其医药用途。
本发明解决上述技术问题所采取的技术方案是:按照上述方法制备得到的鸡矢藤提取物,及该提取物用于制备抗高尿酸血症医药的用途。
与现有技术相比较,本发明具有以下优点:
1.本发明所述的分离制备方法,采用的提取液主要为水,溶剂安全,无毒,也不会像大孔树脂类分离手段有可能带来有害的有机残留物;且操作简单,成本较低,易于产业化。
2.本发明所得的鸡矢藤提取物可显著降低次黄嘌呤诱导的高尿酸血症小鼠血清尿酸水平 (P<0.05或P<0.01),具有很好的医药价值。
具体实施方式
下面将使用本领域技术人员向本领域其它的技术人员传达他们工作的实质所通常使用的术语来描述本方案的发明理念。然而,这些发明理念可体现为许多不同的形式,因而不应视为限于本方案中所述的实施例。
本发明所使用的材料与仪器如下:
1.实验动物
SPF级昆明种小鼠,体重18-22g,由广西医科大学实验动物中心提供,动物质量合格证号:SCXK桂2014-0002。
2.药物与试剂
鸡矢藤药材,购自南宁水街药材市场,经广西中医药研究院鉴定为茜草科鸡矢藤属植物鸡矢藤Paederia scandens(Lour.)Merr;
鸡矢藤提取物,按照本发明提取实例一制备所得;
别嘌醇片,上海信宜万象药业股份有限公司,批号:05161002;
脂多糖(LPS),北京索莱宝科技有限公司,批号818E035;
胎牛血清,澳洲AusGeneX公司,批号FBS00315-1;
DMEM培养基,美国Hyclone公司,批号AD1285463;
尿酸检测测试盒,南京建成生物工程研究所,批号:20170408;
柱层析硅胶,青岛海洋化工厂,批号0110227;
乙腈、甲醇为色谱纯(德国Merck公司);无水乙醇、乙酸乙酯、甲醇等试剂均为分析纯;水为超纯水。
3.主要仪器
TDL-5000B型低速冷冻离心机(上海安亭科学仪器厂);FA1004电子天平(上海精科天平仪器厂)。
一、鸡矢藤提取物提取实施例
1.提取实例一
称取鸡矢藤药材200g,加8倍量水煎煮2次,每次1.5h,滤过,滤液浓缩至200ml,加入乙醇,调节至含醇量为50-80%,搅拌均匀,静置12-24h,取上清液,回收乙醇,浓缩得稠膏30.6g,稠膏加乙醇至溶解完全,加柱层析硅胶拌样(1g稠膏∶2g硅胶),干燥,将干燥后的样品放入层析柱中,用乙酸乙酯洗脱至3倍柱体积,再用乙酸乙酯-甲醇(9∶1)洗脱至3 倍柱体积,收集洗脱液,收集的洗脱液浓度为每1ml相当于原药材1-2g,洗脱液回收溶剂,浓缩,采用薄层色谱法进行监控,合并含有鸡矢藤苷成分的洗脱液,即得鸡矢藤提取物4.5g (总苷含量>50%)。
2.提取实例二
称取鸡矢藤药材1000g,加8倍量水煎煮2次,每次1.5h,滤过,滤液浓缩至1000ml,加入乙醇,调节至含醇量为50-80%,搅拌均匀,静置12-24h,取上清液,回收乙醇,浓缩得稠膏144.7g,稠膏加乙醇至溶解完全,加柱层析硅胶拌样(1g稠膏∶1g硅胶),干燥,将干燥后的样品放入层析柱中,用乙酸乙酯洗脱至4倍柱体积,再用乙酸乙酯-甲醇(7∶3)洗脱至4倍柱体积,收集洗脱液,收集的洗脱液浓度为每1ml相当于原药材1-2g,洗脱液回收溶剂,浓缩,采用薄层色谱法进行监控,合并含有鸡矢藤苷成分的洗脱液,即得鸡矢藤提取物 21.3g(总苷含量>50%)。
3.提取实例三
称取鸡矢藤药材3000g,加8倍量水煎煮2次,每次1.5h,滤过,滤液浓缩至3000ml,加入乙醇,调节至含醇量为50-80%,搅拌均匀,静置12-24h,取上清液,回收乙醇,浓缩得稠膏435.1g,稠膏加乙醇至溶解完全,加柱层析硅胶拌样(1g稠膏∶1g硅胶),干燥,将干燥后的样品放入层析柱中,用乙酸乙酯洗脱至5倍柱体积,再用乙酸乙酯-甲醇(8∶2)洗脱至5倍柱体积,收集洗脱液,收集的洗脱液浓度为每1ml相当于原药材1-2g,洗脱液回收溶剂,浓缩,采用薄层色谱法进行监控,合并含有鸡矢藤苷成分的洗脱液,即得鸡矢藤提取物 59.8g(总苷含量>50%)。
二、鸡矢藤提取物含量测定
1.对照品溶液的制备
精密称取鸡矢藤苷对照品5mg,置于10mL容量瓶中,加甲醇至刻度,摇匀,制成0.50mg/mL 的对照品溶液。
2.供试品溶液的制备
分别精密称取上述三个提取实施例所得的鸡矢藤提取物样品0.2377g置于25ml容量瓶中,加甲醇至刻度,摇匀,制成4.754mg/mL的供试品溶液。
3.显色条件
精密吸取对照品溶液、供试品溶液和甲醇0.5mL于10mL试管中,水浴蒸干,冷却至室温后,加入0.2mL的5%香草醛冰醋酸溶液和0.8mL的高氯酸,经60℃水浴加热15min,取出立即置冰水浴中3min,用冰醋酸定容至刻度,摇匀,以甲醇管为空白,用紫外-可见分光光度计于620nm处测定吸光度。
4.吸收波长的选择
精密吸取对照品溶液,供试品溶液和甲醇0.5mL,按显色条件显色,用紫外-可见分光光度计于200~700nm范围内扫描,结果对照品和供试品溶液再在620nm处具有最大吸收,故选择620nm为测定波长。
5.样品测定
配制鸡矢藤提取物样品溶液,以甲醇管为空白,用紫外-可见分光光度计于620nm处测定吸光度。以吸光度为纵坐标(y),浓度为横坐标(x),绘制标准曲线,得标准曲线及回归方程y=2.016x+0.01(r2=0.9974)。根据研究建立的测定方法和条件,对鸡矢藤提取物进行总苷含量的测定,根据标准曲线计算得出鸡矢藤提取物中总苷(以鸡矢藤苷计)含量。结果表明,鸡矢藤提取物中总苷(以鸡矢藤苷计)含量>50%。
三、鸡矢藤提取物对次黄嘌呤诱导的高尿酸血症小鼠尿酸水平影响研究
1.试验方法
小鼠60只,雄性,随机分为6组,每组10只,即正常对照组、模型对照组、阳性对照组(别嘌醇片0.05g.kg-1)、鸡矢藤水提样品高、中、低剂量组(20、10、5mg.kg-1,下同)。灌胃给药,1次/d,正常对照组和模型对照组灌胃给予等容量生理盐水,连续10d。末次给药1h后腹腔注射次黄嘌呤600mg.kg-1,1h后眼球取血,3000rpm离心10min,取上清液,磷钨酸比色法测血清尿酸水平。
2.实验结果
和正常对照组比较,模型组小鼠血清尿酸水平显著增高,差异有统计学意义(P<0.01)。和模型组比较,鸡矢藤提取物高、中、低(20、10、5mg.kg-1)剂量组小鼠血清尿酸水平显著降低,差异有统计学意义(P<0.05或P<0.01)。见表1。
与模型组比较1)P<0.05,2)P<0.01 。
Claims (8)
1.一种鸡矢藤提取物的分离制备方法,其特征在于,包括以下具体步骤:
(1)称取鸡矢藤药材,以鸡矢藤药材的质量计量,加8-20倍量水煎煮1-3次,每次1-3h,滤过,将滤液浓缩至浓度为每1ml相当于原药材1-2g;
(2)在浓缩滤液中加入乙醇,调节至含醇量为50-80%,搅拌均匀,静置12-24h,取上清液,回收乙醇,浓缩成稠膏;
(3)稠膏加乙醇至溶解完全,加柱层析硅胶拌样,稠膏与硅胶的比例为1g∶1g-1g∶3g;干燥,将干燥后的样品放入层析柱中,用乙酸乙酯洗脱至2-6倍柱体积;再用体积比为9∶1-7∶3的乙酸乙酯-甲醇溶剂洗脱至2-6倍柱体积,收集洗脱液;
(4)洗脱液回收溶剂,浓缩;合并含有鸡矢藤苷成分的洗脱液,即得所述鸡矢藤提取物。
2.根据权利要求1所述的鸡矢藤提取物的分离制备方法,其特征在于,所述的步骤(3)中,所述稠膏与硅胶的比例为1g∶1g。
3.根据权利要求1所述的鸡矢藤提取物的分离制备方法,其特征在于,所述的步骤(3)中,所述乙酸乙酯-甲醇溶剂的体积比为9∶1、8∶2或7∶3。
4.根据权利要求1所述的鸡矢藤提取物的分离制备方法,其特征在于,所述的步骤(3)中,收集到的洗脱液浓度为每1ml相当于原药材1-2g。
5.根据权利要求1所述的鸡矢藤提取物的分离制备方法,其特征在于,所述的步骤(4)中,采用薄层色谱法进行洗脱液中鸡矢藤总苷类成分的监控,再按照监控结果合并含有鸡矢藤苷成分的洗脱液。
6.根据权利要求1所述的鸡矢藤提取物的分离制备方法,其特征在于,所述的鸡矢藤提取物中总苷含量≥50%;所述的总苷含量以鸡矢藤苷计量。
7.根据权利要求1-6任一项所述的分离制备方法制备得到的鸡矢藤提取物。
8.权利要求7所述的鸡矢藤提取物用于制备抗高尿酸血症医药的用途。
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