CN107245092B - 抗炎松香烷型二萜糖苷tripterycosideC - Google Patents
抗炎松香烷型二萜糖苷tripterycosideC Download PDFInfo
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Abstract
本发明涉及医药技术领域,公开了一种从雷公藤(Tripteryginum Wilfordii Hook.F)中分得的新的松香烷型二萜糖苷化合物(命名为tripterycosideC)的结构、分离和鉴定以及在制备抗炎药物中的应用。
Description
技术领域
本发明涉及医药技术领域,尤其涉及从雷公藤中分离鉴定的新的抗炎松香烷型二萜糖苷tripterycosideC。
背景技术
雷公藤( Tripteryginum Wilfordii Hook. F) 为卫矛科雷公藤属植物,又名黄檀根和断肠草,其药用部位为根。据《全国中草药汇编》记载,雷公藤味苦、性辛、凉,有大毒。具有祛风,解毒,杀虫作用。外用治风湿性关节炎,皮肤发痒,杀蛆虫、孑孓,灭钉螺,毒鼠。雷公藤主要分布于福建、湖南和江西等地区。临床常用于治疗类风湿性关节炎、慢性肾炎、红斑狼疮等难治性免疫功能亢进疾病。目前有雷公藤片、雷公藤多苷片、雷公藤内酯、雷公藤内酯软膏、雷公藤总萜片等国产药品广泛应用于临床,疗效显著。
雷公藤的抗炎有效成分主要有松香烷型二萜,目前报道的雷公藤中具有抗炎活性的松香烷型二萜基本为苷元化合物,如雷公藤甲素、雷公藤乙素、雷公藤内酯酮和雷公藤氯内酯醇等松香烷型二萜苷元。雷公藤中关于松香烷型二萜糖苷类化合物报道极少,而具有抗炎活性的松香烷型二萜糖苷还未见报道。为了进一步从雷公藤中发现新的抗炎活性成分,为新药开发奠定基础,发明人对雷公藤进行了化学成分的深入研究。结合抗炎药效活性筛选,从雷公藤中分离鉴定了一种新的抗炎松香烷型二萜糖苷tripterycosideC。本发明首次公开了tripterycosideC的结构、分离和鉴定以及在制备抗炎药物中的应用。
发明内容
本发明所涉及的从雷公藤中提取分离鉴定的一种新的抗炎松香烷型二萜糖苷,命名为tripterycosideC,化学结构如下式Ⅰ。
式Ⅰ:tripterycosideC结构。
本发明公开了tripterycosideC的结构、分离和鉴定以及在制备抗炎药物中的应用,可为临床炎症治疗提供新的药物。本发明是通过下述技术方案来实现的。
一、提取分离
1.将雷公藤根切片,加入95%乙醇水溶液,加热回流提取3次,合并提取液,减压浓缩回收乙醇得到总浸膏。将总浸膏用乙酸乙酯溶解数次至不再溶解,合并乙酸乙酯溶液,减压浓缩回收乙酸乙酯得乙酸乙酯部位。
2.将乙酸乙酯部位经中性氧化铝色谱柱层析,用石油醚-乙酸乙酯(1:0,4:1,3:2,2:3,V/V)及乙酸乙酯-甲醇(1:0,3:1,1:1,0:1,V/V) 依次梯度洗脱,依次得到8个洗脱流份,将乙酸乙酯-甲醇(1:1,V/V)洗脱流份减压浓缩得中性氧化铝色谱柱洗脱部位。
3.将中性氧化铝色谱柱洗脱部位经MCI GEL色谱柱层析,用甲醇-水(0:1,1:9,3:7,1:1,7:3,9:1,1:0,V/V)依次梯度洗脱,依次得到7个洗脱流份,将甲醇-水(7:3,V/V)洗脱流份减压浓缩得MCI GEL色谱柱洗脱部位。
4.将MCI GEL色谱柱洗脱部位经Sephadex LH-20凝胶柱分离,甲醇洗脱,TLC检测合并,减压浓缩依次得到9个流份,将第6个流份减压浓缩得Sephadex LH-20凝胶柱洗脱部位。
5.将Sephadex LH-20凝胶柱洗脱部位经制备HPLC分离(YMC-Actus ODS-AC18色谱柱,流动相:乙腈-水33:67,检测波长210nm),得化合物tripterycosideC。
二、结构鉴定
tripterycosideC:黄色固体(溶剂甲醇),根据HR-TOF-MS (见附图1)确定分子式为C26H32O10 ([M+H]+,实测值:m/z 505.2064,理论计算值:m/z 505.2068;[M-H]-,实测值:m/z 503.1902,理论计算值:m/z 503.1923)。紫外光谱图(220nm,274nm,373nm)和NMR显示结构中含有苯环。氢核磁共振谱(1H-NMR)(见附图2)和碳核磁共振谱(13C-NMR)(见附图3)中显示3个特征甲基(δ H 1.25,d,6.9Hz,H-16;δ H 1.21,d,6.9Hz,H-17;δ H 1.31,s,H-20) (δ C 22.7,C-16;δ C 22.6,C-17;δ C 17.4,C-20)和1个特征亚甲基(δ H 4.87,H-19) (δ C 72.2,C-19) (见表1),结合苯环结构说明tripterycosideC为类似雷酚内酯的松香烷型二萜类结构。13C-NMR谱中6个碳信号(δ C 103.9,C-1’;δ C 75.3,C-2’;δ C 78.5,C-3’;δ C 71.6,C-4’;δ C 78.8,C-5’;δ C 62.7,C-6’)结合1H-NMR谱中糖端基氢信号耦合常数(δ H 4.94,1H,d,J=7.6Hz,H-1’)说明结构中含有β-葡萄糖。除葡萄糖信号外,根据1H-NMR和13C-NMR结合HSQC谱(见附图4),确定化合物含有3个甲基,4个亚甲基,3个次甲基,10个季碳,其中1个苯环、另外1个双键、1个酮羰基和1个酯羰基。根据分子式C26H32O10求得不饱和度为11,扣除6个双键数,求得分子中含有5个环。分子式C26H32O10中含有10个氧原子,除1个葡萄糖和1个酯羰基的氧原子外,剩余2个氧原子分别属于1个羟基和1个羰基。以上信息说明tripterycosideC含有雷酚内酯、葡萄糖、羟基和羰基结构。葡萄糖、羟基和羰基的取代位置可由HMBC相关图谱(见式Ⅱ和附图5)确定,HMBC谱显示葡萄糖端基质子H-1’(δ H 4.94,d,7.6Hz) 与C-11 (δ C 148.8)相关,说明葡萄糖与C-11相连。苯环上H-14(δ H 7.44,s) 与C-15 (δ C 27.8),H-14(δ H 7.44,s) 与C-8 (δ C 116.7) ,H-14(δ H 7.44,s) 与C-9 (δ C 137.4) ,H-14(δ H 7.44,s) 与C-12 (δ C 158.6),H-15(δ H 3.33,hept,6.9Hz) 与C-14 (δ C 125.9)存在HMBC相关,说明苯环上C-14未被取代。结合H-15(δ H 3.33,hept,6.9Hz) 与C-12 (δ C 158.6)等其它HMBC相关信号、1H-NMR和13C-NMR化学位移数据,确定OH 位于C-12位。根据H-5(δ H 3.31) 与C-7 (δ C 205.6),H-6(δ H 3.33,t,6.9Hz) 与C-7 (δ C 205.6) 存在HMBC相关,确定羰基位于C-7位。紫外光谱图(373nm)进一步支持羰基与苯环共轭,即羰基位于C-7位。综上所述,tripterycosideC结构确定如式Ⅰ,经查文献确定tripterycosideC为新化合物。
式Ⅱ:tripterycosideC的关键HMBC相关。
综合1H-NMR、13C-NMR、HSQC和HMBC谱,tripterycosideC的碳和氢核磁信号归属见表1。
表1. tripterycosideC的1H-NMR和13C-NMR数据 (600MHz,溶剂CD3OD)
。
三、抗炎药效学评价
本发明对tripterycosideC进行了抗炎药效学评价。
1. tripterycosideC对脂多糖(LPS)诱导的大鼠膝关节滑膜原代成纤维细胞炎症因子白介素1β(IL-1β)过量表达的影响实验。
1.1 大鼠膝关节滑膜原代成纤维细胞的培养
雄性SD大鼠(180-220g)乙醚麻醉后处死,75% 酒精浸泡15-20分钟后,取出膝关节滑膜组织,剪碎,利用II型胶原酶(4mg/ml)消化2h,加入含10% 胎牛血清的RPMI Medium1640培养,3天左右细胞慢慢爬出,待细胞长至融合状态后,用0.25%胰酶(含0.1%的EDTA)消化2分钟,细胞传代继续培养,原代细胞3-6代用于下面的实验。
1.2 测定tripterycosideC对LPS诱导的膝关节滑膜原代成纤维细胞IL-1β表达的影响
tripterycosideC用DMSO溶解,溶解后母液浓度为10mM,临用前用磷酸盐缓冲液(PBS)稀释到1mM,实验终浓度为10μM。将细胞以2×106个/ml的浓度接种于48孔板,37℃,5%CO2条件下培养至80% 的融合状态,PBS洗涤两次。换成无血清培养基,加入tripterycosideC溶液,使最终浓度为10μM,0.5h后,加入刺激剂LPS(10ng/ml)。实验分为空白组,模型组,tripterycosideC给药组,阳性对照药泼尼松龙给药组(实验终浓度为10μM)。模型组用刺激剂LPS(10ng/ml)造模,给药组加入对应化合物,每组6个复孔,37℃,5% CO2条件下孵育24小时后,按大鼠IL-1β ELISA试剂盒说明要求进行测定OD值,检测波长450nm。
1.3 实验结果
结果(见表2)表明,模型组IL-1β表达与空白组比较有极显著性升高(P<0.001),说明造模成功。tripterycosideC、泼尼松龙给药组与模型组比较均有极显著性差异(P<0.001),说明tripterycosideC能显著抑制LPS诱导的膝关节滑膜原代成纤维细胞炎症因子IL-1β的过量表达,抑制效果与阳性药泼尼松龙相当,说明tripterycosideC具有明显的抗炎活性。
表2. tripterycosideC对LPS诱导的滑膜原代成纤维细胞IL-1β表达的影响(±S,n=6)
*** 与模型组比较P<0.001。
2. tripterycosideC对二甲苯引起的小鼠耳肿胀模型影响实验
tripterycosideC用2 %丙二醇水溶液溶解。取昆明种小鼠,雌雄各半,体重18-22g,随机均分成模型组,tripterycosideC给药组,阳性对照药吲哚美辛给药组,每组10只。各给药组按10mg/kg剂量灌胃给药,模型组给等体积2%丙二醇水溶液。连续给药3天,末次给药后1小时,将20μl二甲苯涂于小鼠右耳廓两面致炎, 左耳不涂为对照。1小时后将小鼠处死,剪下双耳, 用直径6mm 的打孔器分别在同一部位打下圆耳片, 称重。以左右耳片重量差作为肿胀度, 计算抑制率。实验结果见表3,各给药组的平均耳肿胀度与模型组相比均有显著性( P < 0.01) 差异,tripterycosideC抑制率为57.7%,其抑制率高于阳性药吲哚美辛。实验结果表明tripterycosideC具有显著的抗炎活性,优于阳性药吲哚美辛。
表3. tripterycosideC对二甲苯引起的小鼠耳肿胀模型影响实验结果(±S,n=10)
* 与模型组比较P < 0.01。
四、抗炎药物制剂制备
tripterycosideC或以其为基本活性的药物组合物可以与药学上合适的辅料或载体结合,采用公知方法制成注射剂、片剂、胶囊剂、滴丸剂、控释制剂、缓释制剂、纳米制剂等制剂抗炎药物。
附图说明
附图1为tripterycosideC的HR-TOF-MS(负离子模式)谱图。
附图2为tripterycosideC的1H-NMR谱图。
附图3为tripterycosideC的13C-NMR谱图。
附图4为tripterycosideC的HSQC谱图。
附图5为tripterycosideC的HMBC谱图。
具体实施方式
下面通过实施例作进一步描述,但本发明并不限于实施例所述范围。
将雷公藤干燥根119千克切片,加入95%乙醇400升,于70℃加热回流提取3次(1.5×1.5×1小时)。合并提取液,减压浓缩回收溶剂得浸膏12Kg。浸膏用乙酸乙酯溶解多次,合并乙酸乙酯溶液,减压浓缩回收溶剂得乙酸乙酯部位1.87Kg。
将乙酸乙酯部位浸膏1.87kg,经中性氧化铝柱色谱层析(200-300目,22.5Kg),石油醚-乙酸乙酯(1:0,4:1,3:2,2:3,V/V)及乙酸乙酯-甲醇(1:0,3:1,1:1,0:1,V/V)梯度洗脱,每个流份收集50L,依次得到8个洗脱流份(Fr.1~Fr.8)。
将Fr.7流份(即:上述乙酸乙酯-甲醇 1:1洗脱流份)(15.6g)经MCI GEL-CHP 20P色谱柱分离,甲醇-水(0:1,1:9,3:7,1:1,7:3,9:1,1:0,V/V)梯度洗脱,依次得7个洗脱流份(Fr.7.1~Fr.7.7)。
将Fr.7.5(即:上述甲醇-水7:3洗脱流份)经Sephadex LH-20凝胶柱分离,甲醇洗脱,TLC检测合并依次得到9个流份(Fr.7.5.1~Fr.7.5.9)。
取Fr.7.5.6(300mg)经制备HPLC分离 (YMC-Actus ODS-AC18色谱柱,流动相:乙腈-水33:67,检测波长210nm),得化合物tripterycosideC(保留时间13.52min)(6mg)。
tripterycosideC的片剂制备:取50mg化合物与淀粉25mg,糊精25mg混合,用适量30%乙醇湿润,常规制粒,加入适量硬脂酸镁混合,压片,即得。
tripterycosideC的胶囊剂制备:取50mg化合物与淀粉60mg,糊精10mg,糖粉10mg混合,用适量30%乙醇湿润,常规制粒,装入硬胶囊中,即得。
Claims (3)
1.一种结构如下式Ⅰ所示的松香烷型二萜糖苷化合物,命名为tripterycosideC:
式Ⅰ。
2.如权利要求1所述的化合物tripterycosideC的制备过程如下:将雷公藤Tripteryginum Wilfordii Hook. F根切片,加入95%乙醇水溶液,加热回流提取,减压浓缩回收乙醇得到总浸膏;将总浸膏用乙酸乙酯溶解数次至不再溶解,合并乙酸乙酯溶液,减压浓缩回收乙酸乙酯得乙酸乙酯部位;将乙酸乙酯部位经中性氧化铝色谱柱层析,用石油醚-乙酸乙酯以及乙酸乙酯-甲醇依次梯度洗脱,将体积比1:1的乙酸乙酯-甲醇洗脱流份减压浓缩得中性氧化铝色谱柱洗脱部位;将中性氧化铝色谱柱洗脱部位经MCI GEL色谱柱层析,用甲醇-水依次梯度洗脱,将体积比7:3的甲醇-水洗脱流份减压浓缩得MCI GEL色谱柱洗脱部位;将MCI GEL色谱柱洗脱部位经Sephadex LH-20凝胶柱分离,甲醇洗脱,TLC检测合并,减压浓缩得Sephadex LH-20凝胶柱洗脱部位;将Sephadex LH-20凝胶柱洗脱部位经制备HPLC分离得化合物tripterycosideC。
3.如权利要求1所述的化合物tripterycosideC在制备抗炎药物中的应用。
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A Novel Diterpenoid,11-O-b -D-glucopyranosylneo-triptophenolide,from Tripterygium wilfordii Hook.f;Song ZHAO 等;《Chinese Chemical Letters》;20020731;第13卷(第7期);第641-644页 |
Oxidation and glucose conjugation of synthetic abietane diterpenes by Cunninghamella sp. II. Novel routes to the family diterpenes from Tripterygium wilfordii;R. Milanova 等;《J. Nat. Prod.》;19950228;第58卷(第1期);第68-73页,尤其是文章摘要,第69页流程图1以及第70页流程图2 |
雷酚内酯的抗炎免疫药理作用;杨峻 等;《中草药》;19960228;第26卷(第1期);第24-27页,尤其是文章摘要以及第27页3.讨论内容 |
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