CN106188179B - 具有止泻作用的尖叶假龙胆提取物、化合物及药物组合物 - Google Patents
具有止泻作用的尖叶假龙胆提取物、化合物及药物组合物 Download PDFInfo
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Abstract
本发明实施例公开了一种通式(Ⅰ)的化合物在制备止泻药物中的用途,通式(Ⅰ)的化合物,以及包含所述化合物的尖叶假龙胆提取物,和包含所述化合物或尖叶假龙胆提取物的药物组合物。经研究表明,本发明从尖叶假龙胆中所得到的化合物具有不同程度的止泻作用,可以预期本发明所得到的化合物或含有它的尖叶假龙胆提取物、药物组合物可以用于止泻,进一步地,可以用于制备止泻药物。
Description
技术领域
本发明涉及植物化学领域,特别涉及具有止泻作用的尖叶假龙胆提取物、化合物及药物组合物。
背景技术
腹泻是一种常见症状,是指排便次数明显超过平日习惯的频率,粪质稀薄,水分增加,每日排便量超过200g,或含未消化食物或脓血、粘液。腹泻常伴有排便急迫感、***不适、失禁等症状。腹泻分急性和慢性两类,急性腹泻发病急剧,病程在2~3周之内;慢性腹泻指病程在两个月以上或间歇期在2~4周内的复发性腹泻。对于剧烈的急性腹泻或长期慢性腹泻,需要服用止泻药物以防止机体过度脱水、水盐代谢失调、消化及营养障碍。
尖叶假龙胆(Gentianella acuta(Michx.)Hulten),为龙胆科(Gentiananceae)假龙胆属(Gentianella Moench.)植物,又名苦龙胆,药名为阿古特-其其格,为龙胆科一年生草本植物,全草入药。广泛分布于我国河北、东北、内蒙古、山西、山东等地区,苏联、蒙古、北美洲也有分布。多用于蒙、藏医学中,蒙药中称为“阿古特-其其格”,具有清热解毒的功效,是治疗黄疸性肝炎、头痛、发烧等的主药。鄂温克族和鄂伦春族的猎民长期用其治疗心律失常等心脏病且效果显著,是该属唯一一种具有治疗心脏病作用的植物,在蒙医药中有着不可替代的地位。
发明内容
发明人对尖叶假龙胆进行了提取和分离,得到了尖叶假龙胆的提取物和一系列化合物,并出人意料地发现,所得到的提取物和/或化合物具有止泻作用。并基于此,完成了本发明。
本发明的第一方面提供了通式(Ⅰ)的化合物在制备止泻药物中的用途,
其中,R1、R2、R3、R4、R5各自独立的代表-H、-OH、-OCH3、 在本文中,基团结构式中的表示该基团与分子其它部分的连接点。
在本发明第一方面的一些优选实施方式中,R1代表-OH、-OCH3或R2代表-OH或R3代表-H、-OH或R4代表-H或-OH;R5代表-H或-OH。
在本发明第一方面的另一些优选的实施方式中,通式(Ⅰ)的化合物选自:
本发明的第二方面提供了通式(Ⅰ)的化合物,
其中,
R1代表-OH或R2代表-OH或R3代表-OH、R4代表-H,R5代表-OH。
本发明第二方面的一个优选实施方式中,通式(Ⅰ)的化合物选自:
本发明的第三方面提供了包含前述通式(Ⅰ)化合物的尖叶假龙胆提取物。
本发明的第四方面提供了一种用于止泻的药物组合物,其中所述药物组合物包含有药物活性成分和药学上可接受的载体或赋形剂;所述药物活性成分选自前述的通式(Ⅰ)化合物或前述的尖叶假龙胆提取物。
本发明第四方面的一个优选实施方式中,通式(Ⅰ)化合物从尖叶假龙胆中提取得到。
本发明第四方面的另一个优选实施方式中,其中所述药学可接受的载体或赋形剂选自溶剂、稀释剂、分散剂、助悬剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、粘合剂、润滑剂、稳定剂、水合剂、乳化加速剂、缓冲剂、吸收剂、着色剂、香味剂、甜味剂、离子交换剂、脱模剂、涂布剂、矫味剂、和抗氧化剂。
本发明第四方面的再一个优选实施方式中,本文所述药物组合物优选制成以下剂型:片剂、胶囊剂、粉剂、颗粒剂、锭剂、丸剂、溶液剂、混悬剂、乳剂、糖浆剂、散剂、细粒剂、小丸剂、酏剂、注射剂、药用滴剂、软膏剂、洗剂、凝胶剂、乳膏、喷雾剂、栓剂、或贴剂。
经研究表明,本发明从尖叶假龙胆中所得到的化合物具有不同程度的止泻作用,可以预期本发明所得到的化合物或含有它的尖叶假龙胆提取物、药物组合物可以用于止泻,进一步地,可以用于制备止泻药物。
具体实施方式
下面将结合具体实施例对本发明的技术方案进行描述,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1尖叶假龙胆提取物及化学成分的制备
尖叶假龙胆全草(3.0kg)经14倍量70%乙醇加热回流3次(回流的时间分别为3小时、2小时、2小时),合并滤液,减压回收溶剂,得浸膏1053.4g。取上述浸膏868.5g,用CHCl3-H2O(1:1,v/v)进行萃取,分别得CHCl3和H2O萃取物64.5g和804.0g。取H2O萃取物(670.0g)经D101大孔吸附树脂处理(洗脱液依次为H2O→95%乙醇→丙酮),得到H2O、95%乙醇和丙酮洗脱物分别为332.4g、294.9g和5.1g。
上述95%乙醇洗脱物(200g)经硅胶柱层析[CHCl3→CHCl3-甲醇(100:1→100:5,v/v)→CHCl3-甲醇-H2O(10:3:1→7:3:1→6:4:1,下层,v/v/v)],分离得到16个组分(组分1~组分16,按流出顺序命名,第一个流出的命名为组分1,最后流出的命名为组分16,下面的组分命名原则均与此相同)。组分7(25.7g)经离心得到1,3,5,8-四羟基氧杂蒽酮(化合物4,15.9g)和组分7-2(9.8g),组分7-2(9.8g)经制备型高效液相色谱法(PHPLC)分离制备[CH3CN-H2O(18:82→35:65→42:58,v/v)+1%醋酸],得到27个组分(组分7-2-1~组分7-2-27)。组分7-2-22(54.4mg)经Sephadex LH-20凝胶柱层析分离(甲醇),得到1,3,5-三羟基氧杂蒽酮(化合物3,25.6mg)。
组分11(20.0g)经PHPLC分离制备[CH3CN-H2O(15:85→25:75→42:58,v/v)+1%醋酸],得到29个组分(组分11-1~组分11-29)。组分11-17(790.7mg)经PHPLC分离制备[甲醇-H2O(42:58,v/v)+1%醋酸],得到蔓龙胆酮甙A(化合物7,14.6mg)。
组分13(20.0g)经PHPLC离制备[甲醇-H2O(35:65→45:55→55:45,v/v)+1%醋酸],得到20个组分(组分13-1~组分13-20)。组分13-17(504.0mg)经PHPLC分离制备[CH3CN-H2O(22:78,v/v)+1%醋酸],得到9个组分(组分13-17-1~组分13-17-9),组分13-17-5(35.4mg)经PHPLC分离制备[甲醇-H2O(50:50,v/v)+1%醋酸],得到化合物1,(将化合1命名为尖叶假龙胆酮A1,7.1mg)。
组分14(15.3g)经PHPLC离制备[CH3CN-H2O(15:85→25:75,v/v)+1%醋酸],得到20个组分(组分14-1~组分14-20)。组分14-17(72.6mg)经PHPLC分离制备[甲醇-H2O(50:50,v/v)+1%醋酸],得到当药醇甙(化合物6,27.9mg)。
组分15(14.4g)经Sephadex LH-20凝胶柱层析分离[甲醇-H2O(1:1,v/v)],得到9个组分(组分15-1~组分15-9)。组分15-4(2.4g)经PHPLC分离制备[甲醇-H2O(40:60,v/v)+1%醋酸],共得到17个组分(组分15-4-1~组分15-4-17)。组分15-4-6(215.7mg)经PHPLC分离制备[CH3CN-H2O(13:87,v/v)+1%醋酸],得到化合物2(将化合2命名为尖叶假龙胆酮A2,19.4mg)。组分15-4-7(284.5mg)经PHPLC分离制备[CH3CN-H2O(15:85,v/v)+1%醋酸],得到化合物去甲当药醇甙(化合物5,139.8mg)。
需要说明的是,本文中,涉及乙醇百分数时,无特说明,均是指体积百分数,例如,70%乙醇指的是体积百分数为70%的乙醇水溶液。
实施例2化合物1-7的鉴定
通过紫外-可见光谱、红外光谱、核磁共振波谱(NMR)、质谱(MS)等多种波谱技术确定化合物1-7结构;所确定的化合物1-7结构如下表1所示。
表1化合物1-7的结构式
对于化合物1-7的具体表征结果如下:
化合物1的表征结果:
黄色粉末;[α]25 D-89.5°(c 0.11,甲醇);UVλmax(甲醇)nm(logε):252(4.41),274(4.30),331(4.07);IR(KBr)νmax 3362,2922,2852,1635,1612,1587,1499,1289,1243,1209,1173,1073cm–1;高分辨Q-TOF-ESI-MS给出其准分子离子峰为m/z 421.0796[M–H]–,确定其分子式为C19H18O11(C19H17O11,计算值421.0776)。
1H NMR(DMSO-d6,500MHz)δ:6.47(1H,br.s,H-2),6.68(1H,br.s,H-4),7.29(1H,d,J=8.0Hz,H-6),6.66(1H,d,J=8.0Hz,H-7),11.09(1H,br.s,1-OH),5.14(1H,d,J=7.0Hz,H-1'),3.28(1H,dd,J=7.0、8.0Hz,H-2'),3.30(1H,dd,J=7.0、8.0Hz,H-3'),3.19(1H,dd,J=7.0、8.0Hz,H-4'),3.48(1H,m,overlapped,H-5'),[3.48(1H,m,overlapped),3.71(1H,br.d,ca.J=10Hz),H2-6']。
13C NMR(DMSO-d6,125MHz)δ:161.9(C-1),99.0(C-2),164.8(C-3),94.6(C-4),157.1(C-4a),143.4(C-4b),137.3(C-5),123.9(C-6),109.5(C-7),151.7(C-8),107.5(C-8a),102.8(C-8b),184.1(C-9),99.6(C-1'),73.0(C-2'),76.2(C-3'),69.4(C-4'),77.1(C-5'),60.5(C-6')。
化合物2的表征结果:
黄色粉末;[α]25 D-55.7°(c 0.23,甲醇);UVλmax(甲醇)nm(logε):252(4.41),274(4.30),331(4.07);IR(KBr)νmax 3356,2922,2852,1648,1610,1585,1490,1457,1294,1243,1209,1073cm–1;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 583.1325[M–H]–,确定其分子式为C25H28O16(C28H27O16,计算值583.1305)。
1H NMR(DMSO-d6,500MHz)δ:6.69(1H,br.s,H-2),6.46(1H,br.s,H-4),7.15(1H,d,J=8.5Hz,H-6),6.54(1H,d,J=8.5Hz,H-7),12.54(1H,br.s,8-OH),4.94(1H,d,J=7.5Hz,H-1'),3.42(1H,dd,J=7.5、8.5Hz,H-2'),3.33(1H,dd,J=8.5、8.5Hz,H-3'),3.28(1H,dd,J=8.5、8.5Hz,H-4'),3.62(1H,m,H-5'),[3.66(1H,m,overlapped),4.03(1H,br.d,ca.J=11Hz),H2-6'],4.25(1H,d,J=7.5Hz,H-1”),3.03(1H,dd,J=7.5、8.5Hz,H-2”),3.17(1H,dd,J=8.5、8.5Hz,H-3”),3.08(1H,m,overlapped,H-4”),3.08(1H,m,overlapped,H-5”),[3.43(1H,dd,J=5.0、11.5Hz),3.67(1H,br.d,ca.J=12Hz),H2-6”]。
13C NMR(DMSO-d6,125MHz)δ:159.6(C-1),101.5(C-2),167.1(C-3),97.0(C-4),158.6(C-4a),142.7(C-4b),136.5(C-5),122.3(C-6),108.7(C-7),152.6(C-8),108.6(C-8a),103.3(C-8b),180.2(C-9),101.6(C-1'),73.3(C-2'),76.0(C-3'),69.4(C-4'),75.9(C-5'),68.2(C-6'),103.4(C-1”),73.5(C-2”),76.5(C-3”),70.0(C-4”),76.8(C-5”),60.9(C-6”)。
化合物3的表征结果:
黄色粉末;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 243.0295[M–H]–,确定其分子式为C13H8O5(C13H7O5,计算值243.0299)。
1H NMR(DMSO-d6,500MHz)δ:6.22(1H,d,J=2.0Hz,H-2),6.44(1H,d,J=2.0Hz,H-4),7.32(1H,dd,J=1.5、7.5Hz,H-6),7.25(1H,dd,J=7.5、7.5Hz,H-7),7.56(1H,dd,J=1.5、7.5Hz,H-8),12.91(1H,br.s,1-OH)。
13C NMR(DMSO-d6,125MHz)δ:162.8(C-1),98.1(C-2),165.9(C-3),94.0(C-4),157.2(C-4a),144.8(C-4b),146.1(C-5),120.9(C-6),124.0(C-7),114.4(C-8),120.5(C-8a),102.0(C-8b),180.0(C-9)。
化合物4的表征结果:
黄色粉末;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 259.0258[M–H]–,确定其分子式为C13H8O6(C13H7O6,计算值259.0248)。
1H NMR(DMSO-d6,500MHz)δ:6.24(1H,d,J=1.5Hz,H-2),6.41(1H,d,J=1.5Hz,H-4),7.24(1H,d,J=8.5Hz,H-6),6.60(1H,d,J=8.5Hz,H-7),11.14(1H,br.s,8-OH),11.90(1H,br.s,1-OH)。
13C NMR(DMSO-d6,125MHz)δ:162.3(C-1),98.5(C-2),166.6(C-3),94.3(C-4),157.4(C-4a),143.3(C-4b),137.2(C-5),123.5(C-6),109.3(C-7),151.9(C-8),107.3(C-8a),101.1(C-8b),183.7(C-9)。
化合物5的表征结果:
黄色粉末;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 421.0795[M–H]–,确定其分子式为C19H18O11(C19H17O11,计算值421.0776)。
1H NMR(DMSO-d6,500MHz)δ:6.23(1H,d,J=2.0Hz,H-2),6.43(1H,d,J=2.0Hz,H-4),7.30(1H,d,J=9.0Hz,H-6),7.20(1H,d,J=9.0Hz,H-7),13.11(1H,s,1-OH),4.87(1H,d,J=7.5Hz,H-1'),3.48(1H,dd,J=7.5、8.5Hz,H-2'),3.41(1H,dd,J=8.5、9.0Hz,H-3'),3.31(1H,dd,J=9.0、9.0Hz,H-4'),3.44(1H,m,H-5'),[3.63(1H,dd,J=5.0、11.0Hz),3.84(1H,br.d,ca.J=11Hz),H2-6']。
13C NMR(DMSO-d6,125MHz)δ:163.0(C-1),98.3(C-2),165.5(C-3),93.6(C-4),156.5(C-4a),144.9(C-4b),141.1(C-5),120.9(C-6),112.7(C-7),149.4(C-8),111.9(C-8a),102.7(C-8b),180.8(C-9),103.5(C-1'),73.6(C-2'),76.0(C-3'),69.8(C-4'),77.4(C-5'),60.9(C-6')。
化合物6的表征结果:
黄色粉末;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 435.0935[M–H]–,确定其分子式为C20H20O11(C20H19O11,计算值435.0933)。
1H NMR(DMSO-d6,500MHz)δ:3.89(3H,s,3-OCH3),6.35(1H,s,H-2),6.56(1H,s,H-4),7.27(1H,d,J=9.0Hz,H-6),7.14(1H,d,J=9.0Hz,H-7),13.08(1H,br.s,1-OH),4.82(1H,d,J=7.5Hz,H-1'),3.37(1H,dd,J=7.5、8.0Hz,H-2'),3.31(1H,dd,J=8.0、9.5Hz,H-3'),3.21(1H,dd,J=9.0、9.5Hz,H-4'),3.34(1H,m,H-5'),[3.53(1H,dd,J=6.0、12.0Hz),3.75(1H,br.d,ca.J=12Hz),H2-6']。
13C NMR(DMSO-d6,125MHz)δ:162.6(C-1),97.1(C-2),166.2(C-3),92.1(C-4),156.3(C-4a),144.9(C-4b),140.9(C-5),121.0(C-6),112.3(C-7),149.4(C-8),111.8(C-8a),103.5(C-8b),181.0(C-9),56.0(C-OCH3),103.1(C-1'),73.5(C-2'),76.0(C-3'),69.7(C-4'),77.4(C-5'),60.8(C-6')。
化合物7的表征结果:
黄色粉末;高分辨Q-TOF-ESI-MS给出其准分子离子峰m/z 421.0775[M–H]–,确定其分子式为C19H18O11(C19H17O11,计算值421.0776)。
1H NMR(DMSO-d6,500MHz)δ:7.39(1H,d,J=9.0Hz,H-3),7.28(1H,d,J=9.0Hz,H-4),6.19(1H,s,H-5),6.06(1H,s,H-7),12.97(1H,br.s,8-OH),4.96(1H,d,J=7.5Hz,H-1'),3.48(1H,m,H-2'),3.30(1H,m,H-3'),3.23(1H,dd,J=8.5、9.0Hz,H-4'),3.30(1H,m,H-5'),[3.48(1H,m),3.68(1H,br.d,ca.J=12Hz),H2-6']。
13C NMR(DMSO-d6,125MHz)δ:141.8(C-1),146.1(C-2),124.6(C-3),114.3(C-4),149.3(C-4a),156.9(C-4b),93.8(C-5),169.0(C-6),98.9(C-7),162.8(C-8),101.4(C-8a),114.0(C-8b),179.4(C-9),105.0(C-1'),73.6(C-2'),75.8(C-3'),69.3(C-4'),77.5(C-5'),60.5(C-6')。
实施例3尖叶假龙胆提取物及单体成分对小鼠离体小肠平滑肌自发收缩活动的影响
1.小鼠离体小肠平滑肌标本的制备
利用体重25~30g的健康雄性小鼠,小鼠禁食24小时后,颈椎脱臼处死,迅速沿腹正中线开腹部,取近回盲部小肠10cm。置于盛有室温(约25℃)的台氏液(NaCl 8.0g/L,CaCl2 0.2g/L,KCl 0.2g/L,MgCl2 0.1g/L,NaHCO3 1.0g/L,KH2PO4 0.05g/L,葡萄糖1.0g/L,pH 7.4)的培养皿中,洗净肠内容物。将其剪成1cm的肠段作为实验标本。
2.收缩活动的记录方法
将标本的一端固定于L形钩上,置于盛有10mL台氏液的平滑肌槽中;另一端连接张力换能器,持续通混合气体(95%O2+5%CO2,每秒1~2个气泡),温度(37.0±0.5℃),张力负荷1g,平衡15分钟,待***稳定后,开始实验。
3.实验分组
制备小鼠离体小肠平滑肌标本,收缩活动稳定后,向平滑肌槽中分别加入阳性对照药盐酸洛哌丁胺(终浓度为10μM,二甲基亚砜含量0.1%),尖叶假龙胆总提取物、D101大孔吸附树脂95%乙醇洗脱物(终浓度为150μg/mL),化合物1-7(终浓度为40μM,二甲基亚砜含量0.1%,体积百分数),于加药前1分钟及加药后4分钟观察记录小鼠小肠平滑肌张力、频率等的活动变化。
采用labchart 7软件记录各时间段内收缩张力和收缩频率,收缩张力计算平均值,将正常组的收缩张力平均值和收缩频率设定为100%,采用以下公式计算相对收缩张力和相对频率,实验结果记录于表2中。
相对收缩张力(%)=(给药组张力平均值/正常组张力平均值)×100
相对频率(%)=(给药组频率/正常组频率)×100
表2尖叶假龙胆各提取物及单体成分对小鼠离体小肠平滑肌自发收缩活动的影响
数据以平均值±标准偏差表示,与空白组比较:*p<0.05,**p<0.01,***P<0.001,n=6。正常组:空白小鼠离体小肠平滑肌;阳性对照组:盐酸洛哌丁胺,终浓度10μM;尖叶假龙胆70%乙醇提取物、D101大孔吸附树脂95%乙醇洗脱物:终浓度150μg/mL;化合物1–7,终浓度40μM。以正常组的收缩张力及频率为100%计,计算各给药组的相对收缩张力及频率。
从表2中可以看出,尖叶假龙胆70%乙醇提取物、D101大孔吸附树脂95%乙醇洗脱物及化合物1–7对离体小肠平滑肌自发收缩频率变化无显著影响,但它们均能显著影响小肠平滑肌的张力变化,其水平与阳性药组相当或优于阳性药组,具有止泻作用。
需要说明的是,一般的止泻药例如盐酸洛哌丁胺既在显著影响小肠平滑肌的张力变化的同时又对小肠平滑肌自发收缩频率变化有显著影响,而对小肠平滑肌自发收缩频率变化有显著影响会对食物营养的正常吸收产生不利的影响,对人体是不利的,而本发明中所分离得到的尖叶假龙胆70%乙醇提取物、D101大孔吸附树脂95%乙醇洗脱物及化合物1–7对离体小肠平滑肌自发收缩频率变化无显著影响,可以避免此问题,由此也可以推断,包含有尖叶假龙胆70%乙醇提取物、D101大孔吸附树脂95%乙醇洗脱物或化合物1–7的药物组合物也可以避免此问题。
以上对本发明所提供的具有止泻作用的尖叶假龙胆提取物、化合物及药物组合物进行了详细介绍。本文中应用了具体实施例对本发明的原理及实施方式进行了阐述,以上实施例的说明只是用于帮助理解本发明的方法及其中心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护。
Claims (6)
1.通式(Ⅰ)的化合物在制备止泻药物中的用途,
其中,
R1代表-OH、-OCH3或R2代表-OH或R3代表-H、-OH或R4代表-H或-OH;R5代表-H或-OH。
2.如权利要求1所述的通式(Ⅰ)的化合物的用途,其中,通式(Ⅰ)的化合物选自:
3.如下式所示的化合物:
4.一种用于止泻的药物组合物,其中所述药物组合物包含有药物活性成分和药学上可接受的载体或赋形剂;所述药物活性成分选自权利要求1-3中任一项所述的化合物。
5.如权利要求4所述的药物组合物,其中所述药学可接受的载体或赋形剂选自稀释剂、分散剂、助悬剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、粘合剂、润滑剂、稳定剂、水合剂、乳化加速剂、缓冲剂、吸收剂、着色剂、离子交换剂、脱模剂、涂布剂、矫味剂、和抗氧化剂。
6.根据权利要求4或5所述的药物组合物,其中所述药物组合物的剂型为片剂、胶囊剂、粉剂、颗粒剂、锭剂、丸剂、溶液剂、混悬剂、乳剂、糖浆剂、散剂、酏剂、注射剂、药用滴剂、软膏剂、洗剂、凝胶剂、乳膏、喷雾剂、栓剂、或贴剂。
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