CN115448826A - 一种天然冰片代谢物的分离与鉴定 - Google Patents
一种天然冰片代谢物的分离与鉴定 Download PDFInfo
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- CN115448826A CN115448826A CN202211253571.7A CN202211253571A CN115448826A CN 115448826 A CN115448826 A CN 115448826A CN 202211253571 A CN202211253571 A CN 202211253571A CN 115448826 A CN115448826 A CN 115448826A
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- silica gel
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
本发明涉及新的药物化合物,特别涉及一种天然冰片代谢物,它们的分离与鉴定,所述化合物,其制备方法如下:天然冰片服用后,收集含药尿液,浓缩后得到浸膏,浸膏溶于2L甲醇中浸泡搅拌6h后,静置取上清,重复3次,得到浸膏218.6g,剩余残渣41.1g。首先使用硅胶柱对提取后得到的浸膏218.6g进行分离,使用20:1‑1:1的氯仿/甲醇梯度洗脱,根据薄层色谱结果合并流份,共得到6个部分(Fr.1‑Fr.6),进一步分离得到本发明的5种新化合物。
Description
技术领域:
本发明涉及新的药物化合物,特别涉及一种天然冰片大鼠体内代谢产物的分离与鉴定。
背景技术:
天然冰片:《中国药典》(2020年版)中天然冰片(右旋龙脑)条:本品为樟科植物樟Cinnamomum camphora(L.)Presl的新鲜枝、叶经提取加工制成。
本品含右旋龙脑(C10H18O)不得少于96.0%。右旋龙脑:为天然冰片中的主要成分,别名:右旋莰醇、右旋2-莰醇,(+)-Borneol,(+)-2-莰醇,camphol,CAS:464-43-7,性状:为叶状或六方形片状晶体;熔点208℃,沸点212℃,相对密度1.011(20/4℃),比旋光度+37.7(乙醇);溶于乙醇、乙醚和苯。
结构式:
天然冰片是一种天然的促进剂,可以有效的促进药物吸收,同时还具有止痛、抗菌以及防腐的作用
性凉,味辛、苦,。归心、脾、肺经。具有开窍醒神,清热止痛的功效。用于热病神昏,惊厥,中风痰厥,气郁暴厥,中恶昏迷,胸痹心痛,目赤,口疮,咽喉肿痛,耳道流脓。
冰片是临床常用的中药,中医多取其醒脑开窍、清热止痛、防腐生肌的作用,现代药理研究表明冰片具有多种药用功效。
冰片易透过血脑屏障且可促进血脑屏障的开放。研究表明,冰片能明显松弛血脑屏障的胞间闭锁小带,从而加速物质胞间转移。
促进药物吸收作用:①促进透皮吸收作用。龙脑是一种有效的透皮促进剂,可促进外用皮质激素、双氯灭痛等药的透皮吸收。②促进药物透过眼角膜,提高眼部用药的生物利用度。③促进药物透过鼻黏膜,提高药物经鼻腔吸收入脑的速度,增加了脑组织药物的吸收量,是治疗缺血性脑血管机能不全急性期的一种速效途径。
对抗循环系统缺血与损伤:冰片有利于冠脉痉挛的防治,并可减轻缺血引起的心肌损伤。实验发现:单味冰片对急性心肌梗死的麻醉犬产生与冠心苏合丸类似的作用,能使冠状窦血流量回升,减慢心率,降低心肌氧耗量。
对中枢神经系统有双向调节和保护作用:一是对中枢的双向调节作用。冰片对中枢神经兴奋性有双向调节作用,既能“镇静安神”,又有醒脑作用。二是对中枢神经系统的保护作用。冰片对大脑还有保护作用,利于脑水肿恢复,保护脑缺血后继发损伤,改善受损觉醒能力。此外,冰片还有促进神经胶质细胞分裂作用。抗炎、镇痛、抗病毒作用:天然冰片能明显抑制醋酸引起的小鼠腹腔毛细血管通透性增高,具有抗炎作用;能明显延长热刺激引起小鼠痛反应时间及痛反应,具有镇痛作用;体外直接抗病毒试验显示具有抑制流感病毒的作用。
抗生育作用:冰片能使中晚期妊娠小鼠流产,妊娠终止率分别为100%和91%,但对早期妊娠作用不明显。
促进创伤愈合:冰片能增加肉芽组织结构和表皮细胞再生,修复皮肤附属器官而具有较强的创伤愈合作用。
本研究给40只SD大鼠连续7天灌胃天然冰片,每天灌胃两次,250mg/kg 50mg/kg50mg/kg。第一次给药后连续收集9天的尿液,将所得的尿液减压浓缩得到含药尿液浸膏后,利用多种分离手段从大鼠尿液中分离并鉴定了8种代谢产物,其中5种新化合物分别命名为B2,B3,B5,B6,B7。
本专利成果的研究工作得到了国家重点研发计划课题(编号2018YFC1705005)的部分资助。
发明内容
本发明从灌胃天然冰片的大鼠尿液中分离并鉴定代谢产物,并采用小鼠小胶质细胞BV2氧糖剥夺/复氧(OGD/R)模型,观察化合物对谷氨酸、Ca2+、NO、IL-6、TNF-α五个指标的影响。发现5种新化合物(B2,B3,B5,B6,B7),其中B3、B6和B7可显著降低OGD/R模型BV2细胞内Ca2+的水平,可减轻Ca2+超载造成的级联毒性反应。B6可显著提高OGD/R模型BV2细胞对细胞外谷氨酸的清除能力;B2、B3、B6可显著降低OGD/R模型BV2细胞内NO水平,具有抗炎作用。这些结果表明,这些化合物具有抗炎、抑制钙超载和抑制兴奋性氨基酸毒性作用,从而起到保护脑细胞的作用。
本发明所述化合物,其中,化合物B2,化学名称为[(1R,2R,3S,4S)-3-Hydroxy-5-ketoborneol,中文表示为:(1R,2R,3S,4S)-3-羟基-5-酮冰片
化学结构式:
其为淡黄色粉末,溶于甲醇。
HR-ESI-MS(m/z):207.0993[M+Na]+。
分子式为C10H16O3。
1H NMR谱(500MHz,CD3OD)中给出δ1.06(3H,s),0.97(3H,s),0.91(3H,s)是3个甲基的氢信号,δ2.43(1H,d,J=18.9Hz),1.91(1H,d,J=18.9Hz),2.51(1H,d,J=5.2Hz)是3个脂肪氢信号,4.10(1H,m)和4.49(1H,m)是2个连氧碳上的氢信号。
13C NMR谱(125MHz,CD3OD)中给出δ215.2是一个酮羰基碳信号,δ68.0、72.4是连氧碳信号,δ67.1是脂肪碳信号,δ13.2、17.8、20.4、41.1、44.2、49.6是6个脂肪碳信号,该化合物的核磁数据与文献[14]中(+)-(1R,2R,3S,4S)-3-hydroxyborneol的相似。
根据δ4.10(1H,m)与δ4.49(1H,m)具有COSY相关信号,从而确定H-2δ4.10与H-3δ4.49相邻。根据δ2.51(1H,d,J=5.2Hz)与δ4.49(1H,m)具有COSY相关信号,从而确定H-4δ2.51与H-3δ4.49相邻。根据H-2δ4.10与脂肪碳信号δ67.1、41.1、13.2具有HMBC相关信号,H-3δ4.49与酮羰基碳信号δ215.2、脂肪碳信号δ49.6具有HMBC相关信号,H-4δ2.51与酮羰基碳信号δ215.2、连氧碳信号δ72.4、脂肪碳信号δ49.6和δ20.4具有HMBC相关信号,脂肪氢信号δ2.43和δ1.90与酮羰基碳信号δ215.2、连氧碳信号δ72.4、脂肪碳信号δ49.6和δ13.2具有HMBC相关信号,甲基氢信号δ0.91与脂肪碳信号δ67.1、49.6、41.1、17.8具有HMBC相关信号,甲基氢信号δ0.97与脂肪碳信号δ67.1、49.6、41.1、20.4具有HMBC相关信号,甲基氢信号δ1.06与连氧碳信号δ72.4、脂肪碳信号49.6、44.2、41.1具有HMBC相关信号,从而确定化合物的结构。命名该化合物为(1R,2R,3S,4S)-3-hydroxy-5-ketoborneol,并给出了该化合物的俗名为(+)-Borneol-metabolite10(右旋龙脑代谢产物10)。
表1化合物B2的1HNMR和13CNMR数据
其中,化合物B3,化学名称为[(+)-(1R,2S,4S,5R)-5,10-Dihydroxyborneol,中文表示为:(+)-(1R,2S,4S,5R)-5,10-二羟基冰片
其为黄褐色粉末,溶于甲醇,
HR-ESI-MS(m/z):209.1148[M+Na]+,
分子式为C10H18O3。
1H NMR谱(500MHz,CD3OD)中给出δ0.99(3H,s),1.01(3H,s)是2个甲基氢信号,δ2.01(1H,m),1.62(1H,t,J=4.5Hz),1.79(1H,m),1.80(2H,m)是5个脂肪氢信号,δ3.53(2H,d,J=2.9Hz),4.34(1H,m),4.39(1H,m)是4个连氧碳上的氢信号。
13C NMR谱(125MHz,CD3OD)中给出δ63.0,71.1,74.2是3个连氧碳信号,δ20.6,21.2,28.9,32.1,49.2,53.3,55.3是7个脂肪碳信号,该化合物的核磁数据与文献[14]中的(+)-(1R,2S,4S,5R)-5-hydroxyborneol的相似,其区别在于该化合物多一个-CH2OH(δ63.0,3.53(2H,d,J=2.9Hz))基团,少一个-CH3基团,提示有一个-CH3的氢被-OH取代形成了-CH2OH基团。根据δ3.53与δ74.2,55.3,49.2,32.1具有HMBC相关信号,从而确定-CH2OH基团是在C-10位。命名该化合物为(+)-(1R,2S,4S,5R)-5,10-dihydroxyborneol,并给出俗名为(+)-Borneol-metabolite 11(右旋龙脑代谢产物11)。
表2化合物B3的1HNMR和13CNMR数据
其中,化合物B5,化学名称为[(+)(+)-(1R,2S,4R)-8-Hydroxy-5-ketoborneol,中文表示为:(+)-(1R,2S,4R)-8-羟基-5-酮冰片
化学结构式
其为白色粉末,溶于甲醇。
HR-ESI-MS(m/z):207.0992[M+Na]+。
分子式为C10H16O3。
1H NMR谱(500MHz,CD3OD)中给出δ1.00(3H,s),1.04(3H,s)是2个甲基氢信号,δ1.21(1H,m),1.91(1H,d,J=18.4Hz),2.29(1H,d,J=5.3Hz),2.54(1H,m),2.60(1H,d,J=18.4Hz)是5个脂肪氢信号,δ3.69(1H,d,J=11.4Hz),3.44(1H,d,J=11.4Hz),4.21(1H,ddd,J=9.7,3.6,1.7Hz)是3个连氧碳上的氢信号。
13C NMR谱(125MHz,CD3OD)中给出δ219.4是一个酮羰基碳信号,δ63.0,75.3是2个连氧碳信号,δ13.6,15.6,34.2,42.5,51.5,53.7,58.7是7个脂肪碳信号,该化合物的核磁数据与文献[14]中的(+)-(1R,2S,4R,5S)-5-hydroxyborneol的相似,其区别是该化合物多一个酮羰基(δ219.4)和一个-CH2OH(δ63.0,3.69(1H,d,J=11.4Hz),3.44(1H,d,J=11.4Hz))基团,少一个-CH3基团,少一个-CHOH基团,提示有一个-CH3的氢被-OH取代形成了-CH2OH基团,有一个-CHOH基团被氧化成酮羰基,根据δH-42.29,和δH-6 2.60,1.91与酮羰基碳信号δ219.4具有HMBC相关信号,从而确定δ219.4酮羰基在C-5位。根据δH-8 3.44,3.69与脂肪碳信号δ58.7,53.7,51.5,15.6具有HMBC相关信号,从而确定-CH2OH基团在C-8位。B5命名该化合物为(+)-(1R,2S,4R)-8-hydroxy-5-ketoborneol,并给出俗名为(+)-Borneol-metabolite 12(右旋龙脑代谢产物12)。
表3化合物B5的1HNMR和13CNMR数据
其中,化合物B6,化学名称为(-)-(1R,2R,3R,4S)-3-hydroxy-6-ketoborneol,中文表示为:(-)-(1R,2R,3R,4S)-3-羟基-6-酮冰片
化学结构式
其为白色粉末,溶于甲醇。
HR-ESI-MS(m/z):207.0993[M+Na]+。
分子式为C10H16O3。
1H NMR谱(500MHz,CD3OD)中给出δ0.80(3H,s),0.93(3H,s),1.20(3H,s)是3个甲基氢的信号,δ1.78(1H,d,J=18.4Hz),2.10(1H,dd,J=5.4,1.6Hz,),2.35(1H,dd,J=18.4,5.4Hz)是3个脂肪氢信号,δ3.71(1H,d,J=2.0Hz),3.96(1H,t,J=1.8Hz)是2个连氧碳上的氢信号。
13C NMR谱(125MHz,CD3OD)中给出δ218.5是一个酮羰基碳信号,δ83.7,84.2是2个连氧碳信号,δ7.5,21.0,22.2,41.2,48.8,49.7,65.6是7个脂肪碳信号,该化合物的核磁数据与文献[14]中的(-)-(1S,4S,5S)-5-hydroxycamphor化合物的相似,其区别在于该化合物多一个-CHOH(δ84.2,3.96(1H,t,J=1.8Hz))基团,根据δ3.72与3.96具有COSY相关信号,δ3.96与δ218.5,83.7,65.6,7.5具有HMBC相关信号,从而确定羟基在C-2位,命名该化合物为(-)-(1R,2R,3R,4S)-3-hydroxy-6-ketoborneol,并给出俗名为(+)-Borneol-metabolite 13(右旋龙脑代谢产物13)。
表4化合物B6的1HNMR和13CNMR数据
其中,化合物B7,化学名称为[(+)(+)-(1R,2S,4R,5S)-5,9-Dihydroxyborneol,中文表示为:(+)-(1R,2S,4R,5S)-5,9-二羟基冰片。
化学结构式
其为白色粉末,溶于甲醇。
HR-ESI-MS(m/z):209.1146[M+Na]+。
分子式为C10H18O3。
1H NMR谱(500MHz,CD3OD)中给出δ0.79(s,3H),1.00(s,3H)是2个甲基氢信号,δ1.65(m,1H),1.94(m,1H),1.81(m,1H),1.84(m,1H),1.94(m,1H)是5个脂肪氢信号,δ3.29(m,1H),3.70(d,J=11.3Hz,1H),3.97(m,1H),4.39(m,1H)是4个连氧碳上的氢信号。
13C NMR谱(125MHz,CD3OD)中给出δ65.4,70.9,77.4是3个连氧碳信号,δ14.1,14.5,28.8,36.2,48.5,51.4,54.4是7个脂肪碳信号,该化合物的核磁数据与文献[14]中的(+)-(1R,2S,4R,5S)-5-hydroxyborneol的相似,其区别在于该化合物多一个-CH2OH(δ65.4,3.71(d,J=11.3Hz,1H),3.29(m,1H))基团,少一个-CH3基团,提示有一个-CH3的氢被-OH取代形成了-CH2OH基团,根据δ3.70,3.29与δ54.4,51.4,48.5,14.5具有HMBC相关信号,从而确定-CH2OH基团在C-9位,命名该化合物为(+)-(1R,2S,4R,5S)-5,9-dihydroxyborneol,并给出俗名为(+)-Borneol-metabolite 14(右旋龙脑代谢产物14)。
表5化合物B7的1HNMR和13CNMR数据
本发明所述化合物,其制备方法如下:
天然冰片服用后,收集含药尿液,浓缩后得到浸膏,将浸膏溶于2L甲醇中浸泡搅拌6h后,静置取上清,重复3次,得到浸膏218.6g,剩余残渣41.1g。首先使用硅胶柱对提取后得到的浸膏218.6g进行分离,使用20:1-1:1的氯仿/甲醇梯度洗脱,根据薄层色谱结果合并流份,共得到6个部分(Fr.1-Fr.6)。
Fr.1:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.1-1、Fr.1-2和Fr.1-3。其中,Fr.1-1再次经凝胶柱分离后,在硅胶柱上经氯仿/甲醇(30:1,每200mL再加入1mL氨水)恒流分离,得到化合物Fr.1-1-B2;Fr.1-2经同样的方法进行分离,得到化合物Fr.1-2-B1;Fr.1-3经凝胶柱分离后,在硅胶柱上使用氯仿/甲醇(30:1)恒流分离,得到化合物Fr.1-3-B4。
Fr.2:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.2-1、Fr.2-2和Fr.2-3。其中,Fr.2-1经氯仿作为溶剂进行重结晶,得到化合物Fr.2-1-U1;Fr.2-2经氯仿/丙酮(4:1-2:1)溶剂在硅胶柱上梯度洗脱后,使用凝胶柱和微孔树脂柱MCI进行分离,得到化合物Fr.2-2-B6;Fr.2-3流份经硅胶柱分离后,得到Fr.2-3-1和Fr.2-3-2,其中Fr.2-3-1经凝胶柱和硅胶柱依次分离后得到流份Fr.2-3-B5,Fr.2-3-2经凝胶柱分离后得到Fr.2-3-B3。
Fr.3:该部分先经凝胶柱分离后再用硅胶柱进行分离得到Fr.3-1和Fr.3-2。其中Fr.3-1中发现析出粉末,使用氯仿/甲醇(100:1)洗涤粉末,得到Fr.3-1-U2;Fr.3-2经甲醇重结晶得到Fr.3-2-U3。
Fr.4:该部分依次经凝胶柱分离、氯仿/甲醇(30:1-15:1)在硅胶柱上梯度洗脱、氯仿/甲醇(20:1,每200mL再加入1mL氨水)在硅胶柱上等度洗脱、再次过凝胶柱分离,最终得到Fr.4-B7。
Fr.5:该部分使用甲醇重结晶得到Fr.5-U4。
Fr.6:该部分使用氧仿/甲醇(1:1)经凝胶柱洗脱后,再用甲醇在凝胶柱上洗脱。之后使用甲醇/水(30%-100%)于微孔树脂MCI上梯度洗脱下来后,旋干后使用氯仿洗涤得到Fr.6-B8。
含药尿液中化合物的分离实验由云南西力生物技术有限公司帮助完成。
本发明化合物,其药效学结果如下:
对本发明4种新化合物B2,B3,B6,B7进行初步药效学试验,发现它们具有抗炎、抑制钙超载、抗兴奋性氨基酸毒性等活性,从而起到保护脑细胞的作用。
表6化合物对BV2细胞外谷氨酸水平的影响
注:与Control组相比,####P<0.0001;与Model组相比,*P<0.05,**P<0.01,***P<0.001)
B6可显著提高OGD/R模型BV2细胞对细胞外谷氨酸的清除能力。
表7醒脑静注射液潜在活性化合物和已知活性化合物对BV2细胞内Ca2+水平的影响
注:与Control组相比,####P<0.0001;与Model组相比,*P<0.05,**P<0.01,****P<0.0001)
B3、B6和B7可显著降低OGD/R模型BV2细胞内Ca2+的水平,可减轻Ca2+超载造成的级联毒性反应。
表8化合物对BV2细胞NO水平的影响
注:与Control组相比,####P<0.0001;与Model组相比,*P<0.05,**P<0.01,***P<0.001,****P<0.0001)
B2、B3、B6可显著降低OGD/R模型BV2细胞内NO水平,具有抗炎作用。
本发明进一步包括含有本发明化合物或其药用盐的药物组合物。其剂型选自片剂,胶囊剂、颗粒剂、口服液、栓剂、透皮制剂、注射剂。
附图说明
图1为本发明化合物分离流程图。
具体实施方式
实施例1
一种天然冰片代谢物的分离与鉴定
一、实验材料
1.仪器
核磁共振仪:Bruker AV-400,DRX-500,Avance III 600NMR Spectrometer;高分辨液质联用仪:岛津Shimadzu HPLC-DAD-IT-TOF-MSn,数据采集和处理采用ShimadzuLCMSSolution Version 3.50.348软件,分子式和元素组成与预测采用Formula Predictor和Accurate Mass Calculator软件;
液相色谱仪:Agilent 1200 series HPLC;
色谱柱:Extend-C18 column(5μm,4.6×150mm);
R-210旋转蒸发仪(瑞士Buchi公司);
SHB-3循环水多用真空泵(河南省予华仪器有限公司);
代谢笼(DXL-DL型,苏州冯氏实验动物设备有限公司)。
2.填料
Silica Gel(200-300目,青岛海洋化工厂);Chromatorex C-18(40–75μm,FujiSilysiaChemical Ltd.,Japan);Sephadex LH-20(GE Healthcare Bio-Sciences AB,Uppsala,Sweden);
MCI(Mitsubishi Chemical Corporation,Japan)。
3.试剂
色谱级甲醇(上海星可高纯溶剂有限公司);蒸馏水(实验室经Milli-Q纯净水净化系统纯化);吐温80(药用级,罗恩试剂,RH147089);其他试剂均为工业用试剂。
4.药物
天然冰片(江苏省金叶子生物科技有限公司,C01170704,使用GC检测纯度(右旋龙脑的含量)=99.81%)。
5.实验动物
SD大鼠,雄性,体重230-250g,购自北京大学医学部实验动物科学部。所有动物实验均得到北京大学医学伦理委员会的许可,许可证号为:SYXK2016-0041。
二、实验方法
1.天然冰片给药溶液配制
将天然冰片于研钵中研碎,混悬于5%的吐温80水溶液中,浓度为60mg/mL。
2.大鼠尿液收集和处理
将40只SD大鼠(220g左右)平均随机置于代谢笼中,每笼两只,每日给食两次,适应性饲养5天,每天喂食两次。5天后给大鼠灌胃天然冰片的吐温80混悬液,给药剂量250mg/kg(相当于10倍临床剂量),每天早晚8点各给药一次,观察有无腹泻情况,连续给药7天,停药后继续收集2天尿液。接尿管中事先储有一倍量95%乙醇,给药后尿液每3-4h收集一次(但夜间收尿间隔为5-6h),尿液置于4℃下临时保存。
及时将所得尿液的乙醇溶液减压浓缩(40℃)至干得到浓稠的褐色浸膏粗品约250g,置于-80℃保存。
3.含药尿液化合物分离
将含药尿液浸膏溶于2L甲醇中浸泡搅拌6h后,静置取上清,重复3次,得到浸膏218.6g,剩余残渣41.1g。首先使用硅胶柱对提取后得到的浸膏218.6g进行分离,使用20:1-1:1的氯仿/甲醇梯度洗脱,根据薄层色谱结果合并流份,共得到6个部分(Fr.1-Fr.6)。分离流程图见附图1。
Fr.1:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.1-1、Fr.1-2和Fr.1-3。其中,Fr.1-1再次经凝胶柱分离后,在硅胶柱上经氯仿/甲醇(30:1,每200mL再加入1mL氨水)恒流分离,得到化合物Fr.1-1-B2;Fr.1-2经同样的方法进行分离,得到化合物Fr.1-2-B1;Fr.1-3经凝胶柱分离后,在硅胶柱上使用氯仿/甲醇(30:1)恒流分离,得到化合物Fr.1-3-B4。
Fr.2:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.2-1、Fr.2-2和Fr.2-3。其中,Fr.2-1经氯仿作为溶剂进行重结晶,得到化合物Fr.2-1-U1;Fr.2-2经氯仿/丙酮(4:1-2:1)溶剂在硅胶柱上梯度洗脱后,使用凝胶柱和微孔树脂柱MCI进行分离,得到化合物Fr.2-2-B6;Fr.2-3流份经硅胶柱分离后,得到Fr.2-3-1和Fr.2-3-2,其中Fr.2-3-1经凝胶柱和硅胶柱依次分离后得到流份Fr.2-3-B5,Fr.2-3-2经凝胶柱分离后得到Fr.2-3-B3。
Fr.3:该部分先经凝胶柱分离后再用硅胶柱进行分离得到Fr.3-1和Fr.3-2。其中Fr.3-1中发现析出粉末,使用氯仿/甲醇(100:1)洗涤粉末,得到Fr.3-1-U2;Fr.3-2经甲醇重结晶得到Fr.3-2-U3。
Fr.4:该部分依次经凝胶柱分离、氯仿/甲醇(30:1-15:1)在硅胶柱上梯度洗脱、氯仿/甲醇(20:1,每200mL再加入1mL氨水)在硅胶柱上等度洗脱、再次过凝胶柱分离,最终得到Fr.4-B7。
Fr.5:该部分使用甲醇重结晶得到Fr.5-U4。
Fr.6:该部分使用氧仿/甲醇(1:1)经凝胶柱洗脱后,再用甲醇在凝胶柱上洗脱。之后使用甲醇/水(30%-100%)于微孔树脂MCI上梯度洗脱下来后,旋干后使用氯仿洗涤得到Fr.6-B8。
含药尿液中化合物的分离实验由云南西力生物技术有限公司帮助完成。
三、实验结果
经过对大鼠含药尿液浸膏的分离,排除动物内源性物质(Fr.2-1-U1、Fr.3-1-U2、Fr.3-2-U3、Fr.5-U4)后,共分离得到8种代谢产物,分别为Fr.1-2-B1(B1,30mg)、Fr.1-1-B2(B2,18mg)、Fr.2-3-B3(B3,9mg)、Fr.1-3-B4(B4,4mg)、Fr.2-3-B5(B5,1mg)、Fr.2-2-B6(B6,2mg)、Fr.4-B7(B7,17mg)、Fr.6-B8(B8,67mg),对这8种代谢产物进行结构鉴定。
本发明采用Silica Gel、Sephadex LH-20、MCI等多种分离手段从给药天然冰片大鼠尿液中分离并鉴定了8种代谢产物,其中5种为新化合物,1种为首次从动物体内分离并利用核磁技术确定结构的化合物,2种为已知化合物,在分离得到的8种天然冰片代谢产物中,有2种为右旋龙脑的羟基化产物,2种为右旋龙脑的二羟基化产物,3种为右旋龙脑的二羟基化-羟基氧化产物,1种为右旋龙脑的葡萄糖醛酸结合物。这8种化合物结构母核相同,均为在右旋龙脑的结构基础上发生氧化反应或结合反应,除了B8外,其余均为同分异构体。结合本课题组前期的研究基础,这些代谢产物的化学结构具有相似性,可能具有相同或相似的活性并极有可能作用于相同或类似的靶点。
Claims (4)
1.天然冰片代谢物,包括
化合物B2,化学名称为[(1R,2R,3S,4S)-3-Hydroxy-5-ketoborneol,中文表示为:(1R,2R,3S,4S)-3-羟基-5-酮冰片
化合物B3,化学名称为[(+)-(1R,2S,4S,5R)-5,10-Dihydroxyborneol,中文表示为:(+)-(1R,2S,4S,5R)-5,10-二羟基冰片
化合物B5,化学名称为[(+)(+)-(1R,2S,4R)-8-Hydroxy-5-ketoborneol,中文表示为:(+)-(1R,2S,4R)-8-羟基-5-酮冰片
化合物B6,化学名称为(-)-(1R,2R,3R,4S)-3-hydroxy-6-ketoborneol,中文表示为:(-)-(1R,2R,3R,4S)-3-羟基-6-酮冰片
化合物B7,化学名称为[(+)(+)-(1R,2S,4R,5S)-5,9-Dihydroxyborneol,中文表示为:(+)-(1R,2S,4R,5S)-5,9-二羟基冰片。
2.权利要求1所述化合物的制备方法,所述方法,步骤如下:
天然冰片服用后,收集含药尿液,浓缩后得到浸膏,将浸膏溶于2L甲醇中浸泡搅拌6h后,静置取上清,重复3次,得到浸膏218.6g,剩余残渣41.1g。首先使用硅胶柱对提取后得到的浸膏218.6g进行分离,使用20:1-1:1的氯仿/甲醇梯度洗脱,根据薄层色谱结果合并流份,共得到6个部分为Fr.1-Fr.6;
Fr.1:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.1-1、Fr.1-2和Fr.1-3,其中,Fr.1-1再次经凝胶柱分离后,在硅胶柱上经氯仿/甲醇(30:1,每200mL再加入1mL氨水)恒流分离,得到化合物Fr.1-1-B2;
Fr.2:该部分先经凝胶柱分离后再用硅胶柱分离得到Fr.2-1、Fr.2-2和Fr.2-3,其中,Fr.2-1经氯仿作为溶剂进行重结晶,得到化合物Fr.2-1-U1;Fr.2-2经氯仿/丙酮(4:1-2:1)溶剂在硅胶柱上梯度洗脱后,使用凝胶柱和微孔树脂柱MCI进行分离,得到化合物Fr.2-2-B6;Fr.2-3流份经硅胶柱分离后,得到Fr.2-3-1和Fr.2-3-2,其中Fr.2-3-1经凝胶柱和硅胶柱依次分离后得到流份Fr.2-3-B5,Fr.2-3-2经凝胶柱分离后得到Fr.2-3-B3,
Fr.4:该部分依次经凝胶柱分离、氯仿/甲醇(30:1-15:1)在硅胶柱上梯度洗脱、氯仿/甲醇(20:1,每200mL再加入1mL氨水)在硅胶柱上等度洗脱、再次过凝胶柱分离,最终得到Fr.4-B7。
3.含有权利要求1所述化合物或其药用盐的药物组合物。
4.根据权利要求3所述的药物组合物,其剂型选自片剂,胶囊剂、颗粒剂、口服液、栓剂、透皮制剂、注射剂。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1823922A (zh) * | 2005-12-26 | 2006-08-30 | 深圳市生物谷科技有限公司 | 含有冰片的药物组合物 |
CN110038056A (zh) * | 2019-05-29 | 2019-07-23 | 安徽农业大学 | 一种治疗宠物肢体炎性肿胀的复方五倍子软膏及其制备方法 |
CN110627759A (zh) * | 2019-09-26 | 2019-12-31 | 中央民族大学 | 倍半萜类化合物及其制备方法和用途 |
CN113603570A (zh) * | 2021-09-28 | 2021-11-05 | 潍坊科技学院 | 一种益母草碱冰片衍生物、制备方法及其应用 |
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1823922A (zh) * | 2005-12-26 | 2006-08-30 | 深圳市生物谷科技有限公司 | 含有冰片的药物组合物 |
CN110038056A (zh) * | 2019-05-29 | 2019-07-23 | 安徽农业大学 | 一种治疗宠物肢体炎性肿胀的复方五倍子软膏及其制备方法 |
CN110627759A (zh) * | 2019-09-26 | 2019-12-31 | 中央民族大学 | 倍半萜类化合物及其制备方法和用途 |
CN113603570A (zh) * | 2021-09-28 | 2021-11-05 | 潍坊科技学院 | 一种益母草碱冰片衍生物、制备方法及其应用 |
Non-Patent Citations (2)
Title |
---|
RICHARDA. HARTLINE等: "Induction Specificity and Catabolite Repression of the Early Enzymesin Camphor Degradation by Pseudomonas putida", JOURNAL OF BACTERIOLOGY, vol. 106, no. 2, pages 468 - 478 * |
S.S.BHOSALE: "a new approach to the synthesis of optically active (+)-1R-trans–Pyrethroids", INDIAN JOURNAL OF CHEMISTRY, vol. 24, pages 543 - 546 * |
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