CN114652786A - 一种黄花油点草提取物以及从黄花油点草提取物中分离丁香树脂醇的方法 - Google Patents
一种黄花油点草提取物以及从黄花油点草提取物中分离丁香树脂醇的方法 Download PDFInfo
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Abstract
本发明属于中医药技术领域,具体涉及一种黄花油点草提取物以及从黄花油点草提取物中分离丁香树脂醇的方法。本发明提供一种具有抗氧化、舒张血管功效的提取物。通过柱层析分离的方法,能够进一步从该提取物中分离得到活性药物成分丁香树脂醇。由于其抗氧化作用和舒张血管的作用,本发明的提取物和丁香树脂醇具有作为治疗或预防高血压、高血脂、肺癌及相关癌症、血管性痴呆、糖尿病勃起功能障碍、急性高尿酸血症或血栓性疾病的药物的潜力。本发明为上述新药研发提供了一种新的思路,丰富了黄花油点草的物质基础,有助于中药材黄花油点草的科学、高效利用,具有较高的应用潜力。
Description
技术领域
本发明属于中医药技术领域,具体涉及一种黄花油点草提取物以及从黄花油点草提取物中分离丁香树脂醇的方法。
背景技术
黄花油点草(Tricyrtismaculata(D.Don)Machride)又名羊角七,是百合科油点草属的干燥全草。黄花油点草全草及根入药,被民间广泛用于治疗偏头疼、发热、咳嗽、胃痛、肺痨咳嗽以及脑缺血性疾病,显著疗效。例如,黄花油点草是陕西盘龙制药生产销售的活血类中成药盘龙七片的重要成分之一。
对于黄花油点草的研究表明(食品工业科技.v.39;No.402(10):196-202. 中药材.2016.39(2):419-421.中国发明专利CN103356861A),黄花油点草的提取物具有抗氧化,可显著改善血瘀小鼠血液微循环,增加动、静脉管径及血流速度变化值,显著延长血瘀小鼠凝血时间,并且抑制血瘀大鼠血栓的形成。
然而,黄花油点草虽已被证实具有活血化瘀的作用,但是,前期的研究所用的提取物均是对黄花油点草初步萃取后的混合总馏分,其产生作用的物质基础及其作用机制尚不清楚。这使得现有技术中缺少对黄花油点草中产生活血化瘀效果有效成分的提取方法,这限制了黄花油点草进一步的科学利用。
丁香树脂醇结构式如下:
丁香树脂醇是一种从人参浆果中提取得到的中药活性成分。现有的研究表明,其具有抗血管老化(CN104023716A)和改善皮肤(CN103889413A) 等功效。关于丁香树脂醇是否存在于黄花油点草中,目前尚未有相关研究和报道。
发明内容
针对现有技术中的缺陷,本发明提供一种黄花油点草提取物以及从黄花油点草提取物中分离丁香树脂醇的方法,目的在于从黄花油点草中分离出具有活血化瘀效果的有效成分,实现了对中药材的科学利用。
一种黄花油点草乙酸乙酯提取物,包括如下步骤:
(a)用甲醇-水提取黄花油点草,浓缩制成浸膏;
(b)用乙酸乙酯萃取步骤(a)得到的浸膏,得到乙酸乙酯相,浓缩制成浸膏,即得。
优选的,步骤(a)中,所述甲醇与水体积比为(70-50):(30-50),所述提取方法为浸泡提取3-5次,每次浸泡提取采用的甲醇-水的用量为所述黄花油点草重量的4-5倍量;
和/或,步骤(b)中,乙酸乙酯萃取的次数为4-6次。
本发明还提供一种制备上述黄花油点草乙酸乙酯提取物的制备方法,其特征在于,包括如下步骤:
(a)用甲醇-水提取黄花油点草,浓缩制成浸膏;
(b)用乙酸乙酯萃取步骤(a)得到的浸膏,得到乙酸乙酯相,浓缩制成浸膏,即得。
优选的,步骤(a)中,所述甲醇与水体积比为(70-50):(30-50),所述提取方法为浸泡提取3-5次,每次浸泡提取采用的甲醇-水的用量为所述黄花油点草重量的4-5倍量;
和/或,步骤(b)中,乙酸乙酯萃取的次数为4-6次。
本发明还提供上述黄花油点草乙酸乙酯提取物在制备具有抗氧化、舒张血管功效药物中的用途。
本发明还提供一种药物组合物,它是以上述黄花油点草乙酸乙酯提取物为活性成分,加上药学上可接受的辅料或辅助性成分制成的。
本发明还提供丁香树脂醇的分离纯化方法,它是由权利要求1或2所述的黄花油点草乙酸乙酯提取物,经过分离、纯化得到。
优选的,包括如下步骤:
(1)将所述黄花油点草提取物以甲醇-水体积比50%和75%的甲醇经大孔树脂洗脱,收集体积比75%的甲醇洗脱的馏分Fr.2;
(2)将步骤(1)所得馏分Fr.2采用中压分离系统,以50%、75%和85%的甲醇在MCI柱上分离,收集体积比85%的甲醇洗脱的馏分Fr.2C;
(3)将步骤(2)所得馏分Fr.2C,以氯仿-甲醇体系分别采用体积比40:1、 20:1和10:1在硅胶柱上洗脱,收集氯仿-甲醇体积比10:1的馏分Fr.2C3;
(4)将步骤(3)所得馏分Fr.2C3,以石油醚-乙酸乙酯体系体积比4:1、 3:1、2:1、1:1在硅胶柱上洗脱,收集石油醚-乙酸乙酯1:1的馏分Fr.2C3d;
(5)将步骤(4)所得馏分Fr.2C3d,以乙腈-水体系为流动相在半制备高效液相色谱仪上进行纯化,得到丁香树脂醇。
优选的,步骤(1)中,所述黄花油点草提取物经甲醇溶解,干法拌样后,加入大孔树脂上方;和/或,所述大孔树脂采用Diaion HP-20填料;
和/或,所述MCI柱为MCI GEL CHP20P;
和/或,步骤(3)和步骤(4)中,所述硅胶柱的填料为300-400目的硅胶粉;和/或,所述硅胶柱的填料为Silica gel 60;
和/或,步骤(4)中石油醚的沸程为60℃-90℃;
和/或,步骤(5)中所述乙腈-水体系中乙腈与水的体积比为36:64。
采用本发明的技术方案,能够从黄花油点草中提取得到具有活血化瘀功效的提取物。通过柱层析分离的方法,能够进一步从该提取物中分离得到活性药物成分丁香树脂醇。这为活血化瘀方面的新药研发提供了一种新的思路,丰富了黄花油点草的物质基础,有助于中药材黄花油点草的科学、高效利用。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为实施例2中丁香树脂醇分离流程图的分离流程图;
图2为血管舒张剂Ach和SNP及丁香树脂醇的舒张反应曲线。
具体实施方式
以下实施例和实验例所用的试剂和材料均为市售品。
实施例1黄花油点草提取物
本实施例提供一种黄花油点草的提取物。
其制备方法如下:
干燥粉碎的黄花油点草(2018年采摘于陕西省柞水县,10.0kg)粗粉,室温下用4.5倍量的80%甲醇浸渍法提取4次,依次间隔时间为3,5,7,7 天,合并滤液,减压回收甲醇,浓缩,得到甲醇提取浸膏495.0g。将提取浸膏分散于水中,用乙酸乙酯萃取5次得到乙酸乙酯部分,浓缩制成浸膏。
实施例2丁香树脂醇的分离
本实施例从实施例1的黄花油点草提取物中进一步分离得到丁香树脂醇,如图1所示,具体步骤如下:
(1)将所述提取物的乙酸乙酯浸膏用甲醇溶解,干法拌样,以体积比 50%、75%、85%和100%的甲醇经大孔树脂(Diaion HP-20填料,北京慧德易科技有限责任公司,10×60cm,5×6L)洗脱,分别得到四个馏分Fr.1-4,收集体积比75%的甲醇洗脱的馏分Fr.2(24.0g);
(2)将步骤(1)所得馏分Fr.2采用中压分离系统,以50%、75%和85%的甲醇在MCI柱(MCI GEL CHP20P,日本三菱化学株式会社,6×20cm,6×180ml)上分离,得到三个馏分Fr.2A-2C,收集体积比85%的甲醇洗脱的馏分Fr.2C(1.8g);
(3)将步骤(2)所得馏分Fr.2C,以氯仿-甲醇体系体积比 40:1→20:1→10:1→5:1→2:1在硅胶柱(Silica gel 60,青岛海洋化学有限公司, 5×6L)上洗脱,所述硅胶柱的填料为300-400目的硅胶粉,收集其中氯仿: 甲醇=10:1的馏分Fr.2C3(0.6g);
(4)将步骤(3)所得馏分Fr.2C3,以石油醚-乙酸乙酯体系体积比 4:1→3:1→2:1→1:1在硅胶柱上洗脱,所述石油醚的沸程为60℃-90℃,所述硅胶柱的填料为300-400目的硅胶粉,收集石油醚-乙酸乙酯1:1的馏分 Fr.2C3d(27.8mg);
(5)将步骤(4)所得馏分Fr.2C3d,以乙腈-水体系(体积比36:64)为流动相在半制备高效液相色谱上进行纯化,在该体系下洗脱22min,根据仪器显示用圆底烧瓶收集洗脱液,采用旋转蒸发仪蒸干乙腈及水,可得到丁香树脂醇(9.2mg,保留时间tR=22min)。
本实施例的丁香树脂醇为淡黄色油状;(c 0.2,MeOH);1H-NMR(CD3OD,600MHz)δ:6.56,4.20,4.23,6.8,3.89,3.83,3.07;13C-NMR (CD3OD,150MHz)δ:147.1,147.0,146.9,147.0,134.3,134.3,131.9,132.0, 101.9,102.1,102.7,102.7,86.0,86.2,71.8,71.5,56.3,50.8。
下面对实施例2得到的丁香树脂醇进行活性实验,以进一步证明本发明技术方案的有益效果。
实验例1抗氧化实验
1、实验方法:
实验组设置:样品组使用丁香树脂醇的甲醇溶液,阳性对照组使用阳性药Trolox的甲醇溶液,空白对照组使用甲醇溶剂。称取适量化合物配制成 1.00mg·mL-1母液,梯度稀释成0.50、0.25、0.13、0.06、0.03、0.01mg·mL-1备用。
DPPH自由基清除实验:取DPPH溶解于甲醇中配制成100μM的溶液,在酶标仪下稀释至吸光度为0.7±0.1,用锡纸包裹后置于黑暗中,备用。取 50μL不同浓度下化合物(丁香树脂醇或阳性药Trolox)依次加入96孔板中,空白对照组加入同等剂量的甲醇溶剂,再向每孔中加入100μL配制好的 DPPH溶液。将96孔板外层包裹锡纸置于室温下,黑暗中反应30min,于517 nm处测定吸光值并计算IC50值。IC50为清除50%的DPPH自由基时所需要的样品浓度。
ABTS自由基清除实验:取7mM的ABTS溶液与2.45mM的过硫酸钾反应,在室温下混匀放置12-16h至稳定,此时溶液产生ABTS自由基阳离子 (ABTS·+)。取50μL不同浓度的化合物(丁香树脂醇或阳性药Trolox)与100 μL的ABTS·+混匀,空白对照组加入同等剂量甲醇,黑暗中静置30min,于 734nm下测定吸光值。所有实验平行三次。
2、实验结果:
在DPPH实验中,丁香树脂醇的IC50为0.028±0.001mM,是阳性对照药Trolox(IC50=0.073±0.001mM)的三倍之高;
在ABTS实验中,丁香树脂醇也展现了较高的清除自由基能力,其IC50值为0.011±0.001mM,而阳性药Trolox的IC50值为0.044±0.004mM。
实验证明,实施例2制备的丁香树脂醇对自由基具有极好的清除效果。
实验例2血管功能实验
1、主要溶液的配制
Krebs-Henseleit K-H(mmol/L)缓冲液:称取6.903g NaCl、0.358g KCl、 2.1gNaHCO3、0.163g KH2PO4、0.278g CaCl2、0.144g MgSO4、1.938g D- 葡萄糖,用去离子水定容到1L,pH=7.4。
高K+K-H缓冲液:称取3.674g NaCl、4.470g KCl、2.1g NaHCO3、0.163 g KH2PO4、0.278g CaCl2、0.144g MgSO4、1.938g D-葡萄糖,使用去离子水定容到1L,pH=7.4。
2、血管环的制备
处死SD大鼠后迅速取出肠系膜主动脉,浸入K-H缓冲液中,在显微镜下分离血管外包裹的脂肪以及多余结缔组织,再将分离后的血管剪至长度约 2-3mm的血管环备用。
在37℃的超级恒温槽中加入1mL的K-H缓冲液,并向其中不断通入 95%的O2与5%的CO2混合气体,模拟体内环境。并将制备好的血管环挂在两个L型的金属针上,其中一个连接张力换能器,另一个连接调节负荷张力的微调装置上。将安装好的血管环放入准备好的超级恒温槽环境中,调节张力,给予5mg的预张力,平衡60min,期间每10min更换一次K-H缓冲液。平衡完毕后用高K+K-H缓冲液检验血管环的收缩性,选取两次收缩幅度10%的血管环用于实验。用正常K-H缓冲液洗涤血管环至张力稳定在基线后开始加药检测。
3、舒张实验
实验方法:本实验采用PE预收缩血管环。舒张实验需预先给予0.5mg 的预张力,待平衡后先加入PE(1×10-2M)预收缩动脉血管环引起最大收缩张后,采用累积浓度法加入丁香树脂醇,以SNP(硝普纳)、内皮依赖性血管舒张剂Ach(乙酰胆碱)为阳性对照药,记录张力变化并计算舒张率。
实验结果:如图2所示,红色曲线为阳性对照药Ach,最大舒张值为69.1%±4.14%;绿色曲线为阳性对照药SNP,最大舒张值为74.0%±10.23%。丁香树脂醇对PE诱导的肠系膜动脉血管环收缩具有较强的舒张作用,与阳性药 SNP作用的最终结果近乎相等,最大舒张率为69.04%±4.07%。
实验证明,实施例2制备的丁香树脂醇具有舒张血管的作用。
通过上述实施例和实验例可以看出,本发明从黄花油点草中分离出了一种具有活性的提取物,并进一步分离得到了丁香树脂醇。实验证明,本发明得到的丁香树脂醇具有抗氧化作用和舒张血管的作用,这说明丁香树脂醇是黄花油点草产生活血化瘀功效的活性成分。由于其抗氧化作用和舒张血管的作用,发明的提取物和丁香树脂醇具有作为治疗或预防高血压、高血脂、肺癌及相关癌症、血管性痴呆、糖尿病勃起功能障碍、急性高尿酸血症或血栓性疾病的药物的潜力。本发明为上述新药研发提供了一种新的思路,丰富了黄花油点草的物质基础,有助于中药材黄花油点草的科学、高效利用,具有较高的应用潜力。
Claims (9)
1.一种黄花油点草乙酸乙酯提取物,其特征在于:包括如下步骤:
(a)用甲醇-水提取黄花油点草,浓缩制成浸膏;
(b)用乙酸乙酯萃取步骤(a)得到的浸膏,得到乙酸乙酯相,浓缩制成浸膏,即得。
2.根据权利要求1所述的黄花油点草乙酸乙酯提取物,其特征在于:步骤(a)中,所述甲醇与水体积比为(70-50):(30-50),所述提取方法为浸泡提取3-5次,每次浸泡提取采用的甲醇-水的用量为所述黄花油点草重量的4-5倍量;
和/或,步骤(b)中,乙酸乙酯萃取的次数为4-6次。
3.一种制备权利要求1或2所述的黄花油点草乙酸乙酯提取物的制备方法,其特征在于,包括如下步骤:
(a)用甲醇-水提取黄花油点草,浓缩制成浸膏;
(b)用乙酸乙酯萃取步骤(a)得到的浸膏,得到乙酸乙酯相,浓缩制成浸膏,即得。
4.根据权利要求3所述的黄花油点草乙酸乙酯提取物,其特征在于:步骤(a)中,所述甲醇与水体积比为(70-50):(30-50),所述提取方法为浸泡提取3-5次,每次浸泡提取采用的甲醇-水的用量为所述黄花油点草重量的4-5倍量;
和/或,步骤(b)中,乙酸乙酯萃取的次数为4-6次。
5.权利要求1或2所述的黄花油点草乙酸乙酯提取物在制备具有抗氧化、舒张血管功效药物中的用途。
6.一种药物组合物,其特征在于:它是以权利要求1或2所述的黄花油点草乙酸乙酯提取物为活性成分,加上药学上可接受的辅料或辅助性成分制成的。
7.丁香树脂醇的分离纯化方法,其特征在于:它是由权利要求1或2所述的黄花油点草乙酸乙酯提取物,经过分离、纯化得到。
8.根据权利要求7所述的分离纯化方法,其特征在于:包括如下步骤:
(1)将所述黄花油点草提取物以甲醇-水体积比50%和75%的甲醇经大孔树脂洗脱,收集体积比75%的甲醇洗脱的馏分Fr.2;
(2)将步骤(1)所得馏分Fr.2采用中压分离系统,以50%、75%和85%的甲醇在MCI柱上分离,收集体积比85%的甲醇洗脱的馏分Fr.2C;
(3)将步骤(2)所得馏分Fr.2C,以氯仿-甲醇体系分别采用体积比40:1、20:1和10:1在硅胶柱上洗脱,收集氯仿-甲醇体积比10:1的馏分Fr.2C3;
(4)将步骤(3)所得馏分Fr.2C3,以石油醚-乙酸乙酯体系体积比4:1、3:1、2:1、1:1在硅胶柱上洗脱,收集石油醚-乙酸乙酯1:1的馏分Fr.2C3d;
(5)将步骤(4)所得馏分Fr.2C3d,以乙腈-水体系为流动相在半制备高效液相色谱仪上进行纯化,得到丁香树脂醇。
9.按照权利要求8所述的方法,其特征在于:步骤(1)中,所述黄花油点草提取物经甲醇溶解,干法拌样后,加入大孔树脂上方;和/或,所述大孔树脂采用Diaion HP-20填料;
和/或,所述MCI柱为MCI GEL CHP20P;
和/或,步骤(3)和步骤(4)中,所述硅胶柱的填料为300-400目的硅胶粉;和/或,所述硅胶柱的填料为Silica gel 60;
和/或,步骤(4)中石油醚的沸程为60℃-90℃;
和/或,步骤(5)中所述乙腈-水体系中乙腈与水的体积比为36:64。
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