CN110201014A - 一种屏边三七总皂苷的制备方法及其应用 - Google Patents
一种屏边三七总皂苷的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开一种屏边三七总皂苷的制备方法及其应用,该制备方法将屏边三七粉末加入溶剂进行提取,过滤后将残渣进行2~3次再提取,合并滤液,减压浓缩得到浸膏,将浸膏采用大孔树脂进行分离,以乙醇/水溶液为溶剂体系,收集洗脱液,经浓缩、冷冻干燥得到屏边三七总皂苷;本发明的制备方法操作简单,拓展了屏边三七的应用范围,其提取物可在治疗和预防各种肝病的药物中应用。
Description
技术领域
本发明属生物医药技术领域,具体涉及一种屏边三七总皂苷的制备方法及其应用。
背景技术
屏边三七(Panax stipuleanatus H. T. Tsai et K. M. Feng)为五加科人参属植物,别称野三七、香刺、土三七等,其根具有散瘀定痛、疗伤止血、滋补等功效。目前,国内外学者对屏边三七的研究集中在其化学成分(杨崇仁, 等. 云南植物研究, 1985, (01):103-108.)、种植方法(发明专利CN201610036240.6、CN201610700439.4)、快速鉴定方法(发明专利CN201611218427.4)、保育方法(发明专利CN201610702302.2)、干燥方法(发明专利CN201510563209.3)、抗炎活性(发明专利CN201711210126.1)、抗癌活性(Liang C, et al.J Gins Res, 2013, 37(1): 74-79.)以及降血脂降血糖活性(发明专利CN201610352339.7)等方面的研究。
肝脏是人体内代谢药物的最大的腺体,也是人体重要的器官。肝脏在生物转化、清理有毒化学物质等过程中具有至关重要的作用,肝功能的损害能严重影响人类健康。肝损伤在全世界范围内被列为一种严重的健康问题,肝损伤主要由病毒感染、肝毒性药物以及有毒化学物质导致。目前临床上用于治疗急、慢性肝损伤的药物较少,且药效有限,所以急需开发新的安全的对抗各种急、慢性肝病的药物,但是对于屏边三七的保肝作用尚未有报道。
发明内容
本发明的目的是提供一种屏边三七总皂苷的制备方法及其应用,可有效大量制备屏边三七总皂苷,实现屏边三七在制备保肝药物中的应用。
为实现以上目的,本发明采用的技术方案为:
一种屏边三七总皂苷的制备方法,屏边三七粉末加入溶剂进行提取,过滤后将残渣进行2~3次再提取,合并滤液,减压浓缩得到浸膏,将浸膏采用大孔树脂进行洗脱分离,以乙醇/水溶液为溶剂体系,收集洗脱液,洗脱液经浓缩、冷冻干燥得到屏边三七总皂苷。
所述屏边三七粉末与溶剂的重量体积比g:mL为1:5~30,溶剂为水、甲醇、乙醇中的一种或多种任意比例混合。
所述提取方法为回流法、超声法、微波法处理中的任意一种。
所述回流法提取的温度为50~90°C,时间为2~4h。
所述超声法提取的功率为200~400W,频率为40~80kHz,温度为40~60°C,时间为30~60min。
所述微波法提取的功率为300~700W,温度为60~100°C,时间为20~40min。
所述屏边三七总皂苷中含有屏边三七皂苷R1和屏边三七皂苷R2,屏边三七皂苷R1为式Ⅰ所示化合物,屏边三七皂苷R2为式Ⅱ所示化合物:
本发明还提供屏边三七总皂苷在制备治疗肝脏疾病药物中的应用,在药物制备时可加入药学上可接受的载体、稀释剂或赋形剂。
本发明提供的药物剂型可以为固体制剂或液体制剂,主要包括片剂、散剂、颗粒剂、胶囊剂、滴丸剂、注射剂、栓剂、气雾剂等。
本发明的优点是:
1、 本发明的制备方法简单,获得的屏边三七总皂苷溶于水,且总皂苷中含有大量的屏边三七皂苷R1和R2。
2、 本发明制备的屏边三七总皂苷为天然药物,毒副作用小,可在治疗和预防各种肝病的药物中应用。
附图说明
图 1 实施例1屏边三七总皂苷的高效液相色谱图。
具体实施方式
下面通过附图和实施例对本发明方法作进一步详细说明,但本发明保护范围不局限于所述内容。
实施例1
屏边三七皂苷R1和R2的提取分离方法如下:
屏边三七主根及根茎粉末(1kg),用甲醇浸提3次(每次10L),减压浓缩得到甲醇浸膏(250.6g),加入2L水溶解甲醇浸膏,依次经石油醚萃取脱脂、饱和正丁醇萃取,得到正丁醇浸膏(30g),正丁醇浸膏采用硅胶柱色谱分离,依次用二氯甲烷:甲醇(体积比例为95:5,90:10,80:20,60:40,50:50)梯度洗脱,得到屏边三七有效组分(11.7g),屏边三七有效组分(11.7g)经ODS柱色谱分离(溶剂系统按体积比为甲醇:水 = 55:45,75:25,80:20,90:10,100:0)为流动相洗脱,得到屏边三七皂苷R1(1.5g),屏边三七皂苷R1(5.6 g),经LC-MS检测屏边三七皂苷R1为式Ⅰ所示化合物,屏边三七皂苷R2为式Ⅱ所示化合物:
实施例2
采用回流提取法制备屏边三七总皂苷的方法如下:
取屏边三七主根及根茎粉末(200g),加入6L乙醇水溶液,乙醇水溶液是乙醇和水按照体积比70:100的比例混合得到,在60°C温度下,加热回流3h,过滤后将残渣重复提取3次,合并滤液,经减压浓缩干燥后得到屏边三七提取物浸膏(95.3 g)。
屏边三七提取物浸膏(95.3 g)经大孔树脂柱色谱分离(溶剂系统按体积比为乙醇:水= 0:100,20:80,40:60,60:40,70:30,80:20,90:10,100:0洗脱),收集乙醇/水溶剂体系中乙醇体积分数为70%~90%的乙醇洗脱液,洗脱液经常规浓缩、冷冻干燥得到屏边三七总皂苷粉末(62.5 g)。
经高效液相色谱法测定,屏边三七皂苷R1和屏边三七皂苷R2重量之占屏边三七总皂苷中的含量为32.4%。
实施例3
采用超声提取法制备屏边三七总皂苷的方法如下:
取屏边三七主根及根茎粉末(200g),加入2L乙醇水溶液,乙醇水溶液是乙醇和水按照体积比70:100的比例混合得到,在50°C温度下,超声提取30min,功率250W,频率50KHz,重复提取3次,过滤,合并滤液,经减压浓缩干燥后得到屏边三七提取物浸膏(82.8 g)。
屏边三七提取物浸膏(82.8 g)经大孔树脂柱色谱分离(溶剂系统按体积比为乙醇:水 = 0:100,20:80,40:60,60:40,70:30,80:20,90:10,100:0洗脱),收集乙醇/水溶剂体系中乙醇体积分数为70%~90%的乙醇洗脱液,洗脱液经常规浓缩、冷冻干燥得到屏边三七总皂苷(53.6 g)。
经高效液相色谱法测定,屏边三七皂苷R1和R2重量之和在屏边三七总皂苷中的含量为25.9%。
实施例4
采用微波提取法制备屏边三七总皂苷的方法如下:
取屏边三七主根及根茎粉末(200g),加入1L乙醇水溶液,乙醇水溶液是乙醇和水按照体积比70:100的比例混合得到,在80°C温度下,微波提取30min,功率500W,重复提取3次,过滤,合并滤液,经减压浓缩干燥后得到屏边三七提取物(95.4g)。
屏边三七提取物(82.8 g)经大孔树脂柱色谱分离(溶剂系统按体积比为乙醇:水= 0:100,20:80,40:60,60:40,70:30,80:20,90:10,100:0洗脱),收集乙醇/水溶剂体系中乙醇体积分数为70%~90%的乙醇洗脱液,经浓缩、冷冻干燥得到屏边三七总皂苷(61.9 g)。
经高效液相色谱法测定,屏边三七皂苷R1和R2重量之和在屏边三七总皂苷中的含量为33.7%。
实施例5
屏边三七总皂苷的保肝活性,本实施例研究屏边三七总皂苷对四氯化碳致小鼠急性肝损伤的保护作用。
实验方法:
(1)将108只SPF级昆明种小鼠(体重20±2g,雌雄各半)小鼠适应性喂养7天,
(2)将108只小鼠分为9组,每组12只,分别为:
空白组:常规喂养,每天给予等量蒸馏水;
模型组:常规喂养,每天给予等量蒸馏水;
联苯双酯组:常规喂食,每天给予联苯双酯喂食(100mg/kg);
屏边三七总皂苷低剂量组:常规喂食,每天给予屏边三七总皂苷喂食(100mg/kg);
屏边三七总皂苷中剂量组:常规喂食,每天给予屏边三七总皂苷喂食(150mg/kg);
屏边三七总皂苷高剂量组:常规喂食,每天给予屏边三七总皂苷喂食(200mg/kg);
三七总皂苷低剂量组:常规喂食,每天给予三七总皂苷喂食(100mg/kg);
三七总皂苷中剂量组:常规喂食,每天给予三七总皂苷喂食(150mg/kg);
三七总皂苷高剂量组:常规喂食,每天给予三七总皂苷喂食(200mg/kg);
屏边三七总皂苷由实施例3获得,三七总皂苷购买于中国食品药品检定研究院,各给药组每天灌胃给药1次,连续给药7天,剂量为0.1mL/10g;
(3)末次给药后2h,除空白组,其它组以0.1mL/10 g剂量腹腔注射0.4%CCl4橄榄油溶液,禁食不禁水16h后,小鼠眼眶取血;脱臼处死小鼠,取肝脏,经液氮处理后,冻存于-80℃冰箱,取全血于装有0.109 mol/L柠檬酸三钠即抗凝剂的离心管中,以抗凝剂:全血为1:9的比例收集全血,在4℃下、3500 r/min离心15 min,收集上清,用于测定谷丙转氨酶(ALT)、谷草转氨酶(AST)。
肝损伤由血清中ALT、AST酶活力评价。由表1可知,与空白组相比,模型组ALT、AST酶活力显著升高,分别是43.81±7.39U/L、54.84±4.37U/L;表明CCl4致小鼠急性肝损伤模型制作成功,给药屏边三七总皂苷、三七总皂苷后,给药组相较于模型组ALT、AST酶活力均有所下降,在相同剂量下,屏边三七总皂苷比三七总皂苷更显著地降低ALT、AST酶活力,结果表明屏边三七总皂苷降低了CCl4对肝脏细胞的损害,保肝活性优于三七总皂苷。
表 1 屏边三七总皂苷、三七总皂苷对CCl4致小鼠急性肝损伤的影响()
与空白组比较,△ p<0.05,△△ p<0.01,△△△ p<0.001;与模型组比较,* p<0.05,** p<0.01,*** p<0.001
实施例2的提取温度在50~90°C取值,时间在2~4h取值,提取溶剂为水、甲醇、乙醇中的一种或多种任意比例混合,其他参数不变也可以得到屏边三七总皂苷粉末;实施例3的提取超声功率在200~400W取值,频率在40~80kHz取值,温度在40~60°C取值,时间在30~60min取值,提取溶剂为水、甲醇、乙醇中的一种或多种任意比例混合,其他参数不变也可以得到屏边三七总皂苷粉末;实施例4的提取微波功率在300~700W取值,温度在60~100°C取值,时间在20~40min取值,提取溶剂为水、甲醇、乙醇中的一种或多种任意比例混合,其他参数不变也可以得到屏边三七总皂苷粉末;且按照实施例5的方法也可以得出实施例5的结论。
Claims (8)
1.一种屏边三七总皂苷的制备方法,其特征在于,屏边三七粉末加入溶剂进行提取,过滤后将残渣进行2~3次再提取,合并滤液,减压浓缩得到浸膏,将浸膏采用大孔树脂进行洗脱分离,以乙醇/水溶液为溶剂体系,收集洗脱液,经浓缩、冷冻干燥得到屏边三七总皂苷。
2.根据权利要求1所述制备方法,其特征在于,屏边三七粉末与溶剂的质量体积比g:mL为1:5~30,溶剂为水、甲醇、乙醇中的一种或多种任意比例混合。
3.根据权利要求1所述制备方法,其特征在于,提取方法为回流法、超声法、微波法处理中的任意一种。
4.根据权利要求2所述制备方法,其特征在于,回流法提取的温度为50~90°C,时间为2~4h。
5.根据权利要求2所述制备方法,其特征在于,超声法提取的功率为200~400W,频率为40~80kHz,温度为40~60°C,时间为30~60min。
6.根据权利要求2所述制备方法,其特征在于,微波法提取的功率为300~700W,温度为60~100°C,时间为20~40min。
7.根据权利要求1所述制备方法,其特征在于,屏边三七总皂苷中含有屏边三七皂苷R1和屏边三七皂苷R2,屏边三七皂苷R1为式Ⅰ所示化合物,屏边三七皂苷R2为式Ⅱ所示化合物:
。
8.权利要求1所述屏边三七总皂苷在制备治疗肝脏疾病药物中的应用。
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