CN1981821A - 一种丹皮有效组分、制剂及其制备方法与用途 - Google Patents
一种丹皮有效组分、制剂及其制备方法与用途 Download PDFInfo
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- CN1981821A CN1981821A CN 200510122351 CN200510122351A CN1981821A CN 1981821 A CN1981821 A CN 1981821A CN 200510122351 CN200510122351 CN 200510122351 CN 200510122351 A CN200510122351 A CN 200510122351A CN 1981821 A CN1981821 A CN 1981821A
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Abstract
本发明涉及一种丹皮有效组分,制剂及其制备方法与用途。以重量百分比计,本发明的丹皮有效组分含有75~85%的氧化苯甲酰芍药苷。本发明的丹皮有效组分的制备方法包括下列步骤:将丹皮药材粉碎后加入乙酸乙酯和乙醇,加热提取得提取液I,弃之。药渣加入乙醇,加热提取得提取液II,将提取液II浓缩成浸膏,用甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用低浓度甲醇作为流动相,得洗脱液I,然后改换高浓度甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品,即得。本发明提供的丹皮有效组分化学成分简单明确,在药理研究上更易于阐明其作用机制,在生产中更易于药物的质量控制。
Description
技术领域
本发明涉及一种治疗心血管疾病的中药提取物,具体地说涉及从丹皮中提取的有效组分,制剂及其制备方法与用途,该有效组分主要含有氧化苯甲酰芍药苷(Benzoyloxypaeoniflorin)。
背景技术
冠心病和心绞痛是危害人类健康的“第一杀手”,近年来,随着我国人口老年化以及人们工作、生活、饮食结构及环境等的变化,冠心病等心脑血管疾病的发生率也逐年增多,严重威胁着人民的身心健康。天然产物中许多活性物质具有抗心肌缺血、缺氧作用,其中一些已开发成治疗冠心病和心绞痛的新药,如治疗冠心病的药物地奥心血康,它是由从中药中提取的8种甾体皂甙制成的纯中药制剂;心达康是以沙棘为原料提取加工而成的纯天然药物,其有效成分为沙棘总黄酮。因而从天然产物中寻找具有抗心肌缺血、缺氧生理活性的有效物质,是发现、开发新药的有效途径之一。我国药用生物资源十分丰富,其生理活性物质是研究和发现新药先导化学物,开发新药的天然宝库。目前,我国从天然产物中提取活性物质,用于开发成治疗冠心病、安全性好、毒性低的新药还很少,从天然产物中提取活性物质,开发成具有抗心肌缺血,用于治疗冠心病和心绞痛的新药,具有重要应用价值和广阔发展前景。
丹皮又名牡丹皮,系双子叶植物药毛茛科植物牡丹(Paeonia suffruticosa Andr.)的根皮。分布河北、河南、山东、四川、陕西、甘肃等地。全国各地均有栽培,药材主产安徽、四川、甘肃、陕西、湖北、湖南、山东、贵州等地,此外,云南、浙江亦产。其性微寒,味辛苦,凉,入心、肝、肾经,具有清热,凉血,和血,消瘀的功效。丹皮炮制最早见于我国梁代陶宏景所撰《集注》中,认为:“皆促破,去心。”《本草纲目》等医书中记载“丹皮木心不入药,需去之。”研究表明:丹皮含丹皮酚(Paeonol)、芍药甙(Paeoniflorin)、羟基芍药甙、苯甲酰芍药甙(Benzoylpaeoniflorin)、苯甲酰羟基芍药甙、芍药苷元(Paeoniflorigenone)、6-羟基香豆素(6-hydroxycoumarin)和没食子酸(GallicAcid),以及苯甲酸、植物甾醇、蔗糖、葡萄糖、阿拉伯糖等。挥发油中含有苯甲酸(Benzoic Acid)、十四烷烃(Tetradecane)、2-羟基-4-甲氧基-苯乙酮(2-hydroxy-4-methoxy acetophenone)、2,6-双特丁基对苯醌(2.6-ditert-butyl-p-Benzoquinone)、邻苯二甲酸二乙酯(Diethyl Phthalate)、十四酸异丙酯(Isopropyl Myristate)等化合物(陈悦娇等,广州食品工业科技,2002,18(4):36-37)。吴少华等人首次从丹皮中分离得到了以下五种化合物:白桦脂酸(Betulinic Acid)、白桦脂醇(Betulin),齐墩果酸(OleanolicAcid)、3β,23-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid、3-O-methylpaeonisuffral(吴少华等,中草药,2002,33(8):679-680)。
丹皮对实验性心肌缺血有减轻损伤程度作用,并能够降低心肌耗氧量,增加冠脉流量,认为丹皮有调节血行,疏通血脉之作用,因而对心肌缺血有保护作用(马玉玲等,山西医药杂志,1984,13(4):212-214)。
发明内容
本发明的目的在于提供一种丹皮有效组分。
本发明的另一目的在于提供该丹皮有效组分的制备方法及其用途。
本发明还提供了包含丹皮有效组分的制剂。
本发明以如下技术方案予以实施:
本发明的丹皮有效组分中,主要含有氧化苯甲酰芍药苷。其中,以重量百分比计,氧化苯甲酰芍药苷的含量为75~85%。
优选的,本发明的丹皮有效组分中,以重量百分比计,氧化苯甲酰芍药苷的含量为77~83%。
最佳的,本发明的丹皮有效组分中,以重量百分比计,氧化苯甲酰芍药苷的含量为79~81%。
本发明的丹皮有效组分的制备方法,包括下列步骤:将丹皮药材粉碎后加入乙酸乙酯和乙醇,加热提取得提取液I,弃之。药渣加入乙醇,加热提取得提取液II,将提取液II浓缩成浸膏,用甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用低浓度甲醇作为流动相,得洗脱液I,然后改换高浓度甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱。
优选的,本发明的丹皮有效组分制备方法,包括下列步骤:将丹皮药材粉碎后加入乙酸乙酯和乙醇(1∶0.8~1.2),加热回流0.8~1.2小时,提取1~3次,合并滤液得提取液I,弃之。在药渣中加入60%~80%乙醇,加热回流0.8~1.2小时,提取1~3次,滤液合并得提取液II,将提取液II浓缩成浸膏,用40%~60%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用3%~8%甲醇作为流动相,得洗脱液I,然后改换50%~70%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱,流速为2.8~3.2ml/min,柱温为室温。
最佳的,本发明的丹皮有效组分制备方法,包括下列步骤:取丹皮药材,将其粉碎后加入乙酸乙酯和乙醇(1∶1),加热回流1小时,提取2次,滤液合并得提取液I。药渣加入70%乙醇,加热回流1小时,提取2次,滤液合并得提取液II,将提取液II浓缩成浸膏,用50%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用5%甲醇作为流动相,得洗脱液I,然后改换60%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品:制备色谱的分离条件:色谱柱为半制备柱(Lichrospher C18;10.0mm×250mm,5μm),流动相为水(A)和乙腈(B),梯度洗脱程序如下:0分钟时,流动相A为90%的水溶液、流动相B为10%的乙腈溶液;8分钟时,流动相A为81%的水溶液、流动相B为19%的乙腈溶液;60分钟时,流动相A为65%的水溶液、流动相B为35%的乙腈溶液;流速为3ml/min,柱温为室温。样品用100%乙醇溶解,经制备液相色谱分离,在时间段7.7~10.7min收集溶液,溶液经浓缩干燥后得到有效组分。
本发明的丹皮有效组分可以作为活性成分,加入药剂学上接受的辅料,按照药剂学上记载的制剂的制备方法制成制剂。
本发明的丹皮有效组分还可以作为活性成分之一,加入药剂学上接受的辅料,按照药剂学上记载的制剂的制备方法制成制剂。
所述的制剂包括注射液、滴注液、粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、崩解剂、口崩片、微丸等。
本发明的丹皮有效组分及其制剂可在制备治疗、预防心血管疾病药物中进行应用。
本发明的有益效果为:
1.本发明的提取分离工艺中使用了正相硅胶柱,能有效地除去糖、蛋白质、氨基酸等杂质,提高有效成分的含量,同时采用了制备色谱,能快速准确的得到有效成分。
2.本发明提供的丹皮有效组分化学成分简单明确,在药理研究上更易于阐明其作用机制,在生产中更易于药物的质量控制。
附图说明
图1为丹皮有效组分的HPLC分析图。
具体实施方式
下面将结合本发明的实施例进一步详细说明本发明的实质内容,该实施例仅用于说明本发明而对本发明并没有限制。
实施例一 丹皮有效组分的制备
将200g丹皮药材粉碎后加入乙酸乙酯和乙醇,加热提取得提取液I,弃之。药渣加入乙醇,加热提取得提取液II,将提取液II浓缩成浸膏42g,用甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用低浓度甲醇作为流动相,得洗脱液I,然后改换高浓度甲醇作为流动相,得洗脱液II,浓缩干燥后得样品20.16g;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱。样品用100%乙醇溶解,经制备液相色谱分离,在时间段48.5-58min收集溶液,溶液经浓缩干燥后得到有效组分1.68g。
实施例二 丹皮有效组分的制备
将500g丹皮药材粉碎后加入乙酸乙酯和乙醇(1∶0.8~1.2),加热回流0.8~1.2小时,提取1~3次,合并滤液得提取液I,弃之。在药渣中加入60%~80%乙醇,加热回流0.8~1.2小时,提取1~3次,滤液合并得提取液II,将提取液II浓缩成浸膏106g,用40%~60%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用3%~8%甲醇作为流动相,得洗脱液I,然后改换50%~70%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品50.62g;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱,流速为2.8~3.2ml/min,柱温为室温。样品用100%乙醇溶解,经制备液相色谱分离,在时间段48.5-58min收集溶液,溶液经浓缩干燥后得到有效组分4.85g。
实施例三 丹皮有效组分的制备
取丹皮药材250g,将其粉碎后加入乙酸乙酯和乙醇(1∶1),加热回流1小时,提取2次,滤液合并得提取液I。药渣加入70%乙醇,加热回流1小时,提取2次,滤液合并得提取液II,将提取液II浓缩成浸膏55g,用50%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用5%甲醇作为流动相,得洗脱液I,然后改换60%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品26.48g。用制备液相色谱继续分离得到的样品。制备色谱的分离条件:色谱柱为汉邦半制备柱(Lichrospher C18;10.0mm×250mm,5μm),流动相为水(A)和乙腈(B),洗脱梯度见表2,流速为3ml/min,柱温为室温。样品用100%乙醇溶解,经制备液相色谱分离,在时间段48.5-58min收集溶液,溶液经浓缩干燥后得到有效组分2.23g。
实施例四 丹皮有效组分的HPLC分析
(1)丹皮提取物中氧化苯甲酰芍药苷的含量测定(高效液相色谱法)
色谱条件 色谱柱Agilent Zorbax SB-C18柱(4.6mm×150mm,5μm);采用梯度洗脱,流动相A相为0.2%冰醋酸水溶液,流动相B相为含0.2%冰醋酸的乙腈溶液;梯度洗脱程序如下:0分钟时,流动相A为90%的0.2%冰醋酸水溶液、流动相B为10%的0.2%冰醋酸的乙腈溶液;10分钟时,流动相A为70%的0.2%冰醋酸水溶液、流动相B为30%的0.2%冰醋酸的乙腈溶液;30分钟时,流动相A为50%的0.2%冰醋酸水溶液、流动相B为50%的0.2%冰醋酸的乙腈溶液;流速0.5mL·min-1;检测波长全波长;柱温30℃;ELSD条件:漂移管温度105℃;氮气流速1.8L/min
供试品溶液的制备 称取本品约1.53mg,用甲醇溶液溶解1ml容量瓶中,稀释至刻度,摇匀,即得。
测定方法 精密吸取供试品溶液5ul,注入液相色谱仪,测定,即得。
(2)上述丹皮提取物HPLC-ELSD指纹图谱
测定方法 参见(1)上述丹皮提取物中的氧化苯甲酰芍药苷含量测定(高效液相色谱法)。记录色谱时间为30分钟。
分析结果 丹皮有效组分的HPLC-ELSD分析谱图见图1。图1中的1号峰为氧化苯甲酰芍药苷。
氧化苯甲酰芍药苷结构式:
实施例五 滴丸的制备
取实施例三的丹皮有效组分1g与10.5g聚乙二醇-6000混合均匀,加热熔融,化料后移至滴丸滴灌中,药液滴至6~8℃液体石蜡中,除油,制得滴丸800粒。
实施例六 冻干粉针剂的制备
取实施例三的丹皮有效组分1g、葡萄糖5.5g、硫代硫酸钠0.9g和蒸馏水1ml,上述组分混合均匀后,冷冻干燥,分装600支,即得。
实施例七 冻干粉针剂的制备
取降香油1.5g,加入到13ml饱和的羟丙基β-环糊精中,搅拌溶解,滤过,滤液低温干燥得降香油和羟丙基β-环糊精的包合物粉末。除上述降香油合羟丙基β-环糊精的包合物粉末外,再取实施例三的丹皮提取物1g、甘露醇5.5g、依地酸钙钠0.9g和蒸馏水2ml,上述组分混匀后,冷冻干燥,分装500支,即得。
实施例八 丹皮有效组分的药理实验
1药理模型:乳鼠心肌细胞缺血缺氧模型
原代心肌细胞培养 出生1~3天SD乳鼠处死后,取心脏,经PBS液清洗后减成1mm3左右的碎块。用0.125%胰蛋白酶(Sigma,USA)+0.05%胶原酶II(Gibco,USA)于37度消化3~4次。差速贴壁法分离成纤维细胞1.5小时后,细胞悬液转移至48孔板中(每孔约4×105细胞)。经DMEM(Gibco,USA)加10%胎牛血清(Falcon,USA)培养3-6天的细胞供药效筛选。筛选前1天替换为无血清培养液。培养3~6天的心肌细胞,PBS洗涤后加入含药培养液。置于95%N2和5%CO2培养箱中缺氧6小时。然后在CO2培养箱中复氧3小时。取细胞上清液测定LDH含量。
给药方案 提取得到的丹皮有效组分用无糖Hanks液配成10-4g/mL供试液,脂溶性成分可加入少量DMSO助溶(DMSO终浓度控制在0.2%以下)。各组分在培养液中的终浓度控制在10-5g/mL。
乳酸脱氢酶含量测定 细胞培养液中乳酸脱氢酶含量能够表示细胞损伤程度。漏入上清液中的乳酸脱氢酶含量越高,表明细胞损伤也显著。乳酸脱氢酶测定试剂盒购自南京建成生物工程研究所。测定方法为:取30微升细胞上清液,加入50微升基质缓冲液及10微升乳酸脱氢酶辅酶,37度振荡15分钟,再加入50微升2,4-二硝基苯肼,37度振荡15分钟。平行空白。取反应液50微升加入200微升0.4mol/L氢氧化钠溶液,静置3~4分钟后,用酶标仪于450nm测定光度值。
药效结果
所有数据用均值±方差表示。采用单边方差分析和T检验进行统计学分析。与模型组相比,P<0.05认为显著有效。药效结果见表1。根据心肌细胞乳酸脱氢酶(LDH)检测结果,丹皮有效组分对降低LDH释放有非常显著效果。
表1
Mean | Std | P | |
丹皮有效组分模型组 | 0.18730.152333 | 0.020.01823 | 0.02 |
2药理模型:脐静脉内皮细胞缺氧复氧模型
原代脐静脉内皮细胞培养 取健康新生儿脐带,用30~50mlHanks液洗净静脉血管内的残留血迹。视脐带长度加入相应量的0.1%胶原酶I,转入培养箱消化15~20分钟。随后将脐带内消化液小心收集到烧杯中,并用Hanks液将烧杯润洗两次,一并收集到烧杯中分装于离心管,900rpm离心10分钟。吸去上清液,用M199培养液(含10%胎牛血清,100U/ml双抗,0.15mg/ml Heparin,10uM VC)充分溶解沉淀。将所得细胞悬液分装于5cm2培养瓶,置于培养箱内培养。第二天换成采用含30ug/ml ECGS的M199培养液,之后每三天换一次培养液直至细胞铺满底面。所用细胞均为原代内皮细胞。造模前将培养瓶内细胞接种至96孔板并培养一天,所用细胞量为3.5~4万/孔。造模时,吸弃旧的M199培养液,加入含药无酚红M199培养液,每孔总量为100ul。置于95%N2和5%CO2培养箱中缺氧12小时,然后在CO2培养箱中复氧2小时。取细胞上清液测定LDH含量和NO含量。
给药方案 提取得到的三七有效组分用无糖Hanks液配成10-4g/mL供试液,脂溶性成分可加入少量DMSO助溶(DMSO终浓度控制在0.2%以下)。各组分在无酚红M199培养液中的终浓度控制在10-5g/mL。
NO含量测定 采用Greiss试剂法测定,取90ul细胞上清液,加入等量Greiss试剂,室温下静置10分钟,用酶标仪于550nm测定吸光度值。
药效结果 所有数据用均值±方差表示。采用单边方差分析和T检验进行统计学分析。与模型组相比,P<0.05认为显著有效。药效结果见表2。根据内皮细胞NO含量检测结果,枳壳有效组分对抑制内皮细胞缺氧-复氧后NO过度表达有非常显著效果。
表2
组分 | Mean | Std | P |
丹皮有效组分模型组 | 0.0570.056 | 0.0020.003 | 0.004 |
Claims (10)
1.一种丹皮有效组分,其特征在于,以重量百分比计,氧化苯甲酰芍药苷的含量为75~85%。
2.如权利要求1所述的丹皮有效组分,以重量百分比计,氧化苯甲酰芍药苷的含量为77~83%。
3.如权利要求1所述的丹皮有效组分,以重量百分比计,氧化苯甲酰芍药苷的含量为79~81%。
4.权利要求1-3任一权利要求所述的丹皮有效组分的制备方法,包括下列步骤:将丹皮药材粉碎后加入乙酸乙酯和乙醇,加热提取得提取液I,弃之。药渣加入乙醇,加热提取得提取液II,将提取液II浓缩成浸膏,用甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用低浓度甲醇作为流动相,得洗脱液I,然后改换高浓度甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱。
5.如权利要求书4所述的丹皮有效组分的制备方法,包括下列步骤:将丹皮药材粉碎后加入乙酸乙酯和乙醇,加热回流0.8~1.2小时,提取1~3次,合并滤液得提取液I,弃之。在药渣中加入60%~80%乙醇,加热回流0.8~1.2小时,提取1~3次,滤液合并得提取液II,将提取液II浓缩成浸膏,用40%~60%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用3%~8%甲醇作为流动相,得洗脱液I,然后改换50%~70%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品;制备色谱的分离条件:色谱柱为半制备柱,流动相为水和乙腈,梯度洗脱,流速为2.8~3.2ml/min,柱温为室温。
6.如权利要求5所述的丹皮有效组分的制备方法,其中所述的乙酸乙酯和乙醇的体积比为1∶0.8~1.2。
7.如权利要求书4所述的丹皮有效组分的制备方法,包括下列步骤:取丹皮药材,将其粉碎后加入乙酸乙酯和乙醇,加热回流1小时,提取2次,滤液合并得提取液I。药渣加入70%乙醇,加热回流1小时,提取2次,滤液合并得提取液II,将提取液II浓缩成浸膏,用50%甲醇溶解浸膏,采用ODS-C18柱对其进行分离,首先用5%甲醇作为流动相,得洗脱液I,然后改换60%甲醇作为流动相,得洗脱液II,浓缩干燥后得样品;用制备液相色谱继续分离得到的样品:制备色谱的分离条件:色谱柱为半制备柱(LichrospherC18;10.0mm×250mm,5μm),流动相为水(A)和乙腈(B),梯度洗脱程序如下:0分钟时,流动相A为90%的水溶液、流动相B为10%的乙腈溶液;8分钟时,流动相A为81%的水溶液、流动相B为19%的乙腈溶液;60分钟时,流动相A为65%的水溶液、流动相B为35%的乙腈溶液;流速为3ml/min,柱温为室温;样品用100%乙醇溶解,经制备液相色谱分离,在时间段48.5~58.0min收集溶液,溶液经浓缩干燥后得到有效组分。
8.如权利要求7所述的丹皮有效组分的制备方法,其中所述的乙酸乙酯和乙醇的体积比为1∶1。
9.含有权利要求1-3任一权利要求所述的丹皮有效组分的药用制剂,其特征是,按照药剂学允许的制备方法制成药剂学接受的任一药用制剂。
10.权利要求1-3任一权利要求所述的丹皮有效组分在制备治疗和预防心血管疾病药物中的应用。
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