CN102050862A - 一种新忍冬绿原酸酯皂苷及其制备方法和用途 - Google Patents
一种新忍冬绿原酸酯皂苷及其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及天然药物化学领域,公开了从灰毡毛忍冬花蕾中提取分离的一种新忍冬绿原酸酯皂苷类化合物的化学结构,该化合物具有右式结构。以及该化合物的制备方法和在医药领域中的用途,尤其在制备环氧化酶II抑制剂、基质金属蛋白酶-9抑制剂或抗肿瘤药物的用途。
Description
一、技术领域:
本发明涉及天然药物化学领域,公开了从灰毡毛忍冬花蕾中提取分离的一种新忍冬绿原酸酯皂苷类化合物,以及该化合物的制备方法和在医药领域中的用途,尤其在制备环氧化酶II抑制剂、基质金属蛋白酶-9抑制剂或抗肿瘤药物的用途。
二、技术背景:
忍冬属植物中富含绿原酸类和齐墩果酸型皂苷类成分。绿原酸具有抗菌、抗病毒、增高白血球、保肝利胆、抗肿瘤、降血压、降血脂、清除自由基和兴奋中枢神经系统等作用;近年有报道表明绿原酸有较强的抑制基质金属蛋白酶-9(MMP-9)的活性(Un-Ho,J.,et al.Life Sciences,2005,77:2760-2769)。齐墩果酸型皂苷类成分也是一种重要的生物活性物质,这些皂苷报道有抑制环氧化酶II(COX-2)活性(Shepo,S.,et al.J.Nat.Prod.,2006,69:15911595)等活性。灰毡毛忍冬(Lonicera macranthoides Hand.-Mazz.)为忍冬科忍冬属植物,具有清热解毒、抗菌消炎的功效,在中医临床及民间广泛应用于痈肿疔疮,喉痹,丹毒,热毒血痢,风热感冒,温热发病等疾病的治疗。2005年版《中国药典》收载,将其与红腺忍冬、华南忍冬一同列入山银花顶下。江苏省中国科学院植物研究所在对灰毡毛忍冬花蕾中皂苷类成分的研究过程中,得到一种新的绿原酸酯皂苷类化合物,命名为Lonimacranthoide I,是迄今为止发现的第一个绿原酸酯皂苷;同时具有很强的抑制环氧化酶II(COX-2)(IC50=2.2μM)和基质金属蛋白酶9(MMP-9)(IC50=11.2μM)活性;在体内试验中,当Lonimacranthoide I剂量为2.5、5和7.5mg/kg时对小鼠K111细胞静脉注射肺转移的抑制作用分别为32.3%、42.5%和47.3%。有望开发成一类新的防治肿瘤生长和转移的抗癌药物。
环氧化酶(cyclooxygenases,COXs),又称前列腺素内过氧化物合成酶(PGHS),是前列腺素(PGs)合成过程中一个主要的限速酶,可将花生四烯酸(AA)代谢成各种前列腺素产物,从而参与机体的多种病理生理过程,如炎症、发热、出凝血机制等。研究发现COXs至少存在2种同工酶,即COX-1和COX-2。COX-1为组成型酶,被称为管家酶,在维持人体正常生理功能中起着重要的作用。COX-2是一种诱导酶,除了在上述炎症等过程中发挥重要作用外,还通过多种途径参与肿瘤的发生、发展和转移,如促进细胞增殖、抑制肿瘤细胞凋亡、诱导侵袭性、调节免疫抑制等。因此寻找COX-2选择性抑制剂来防治肿瘤已成为近年肿瘤学研究的热点之一。由于心血管副作用使得合成性COX-2抑制剂不能进入临床癌症化疗。近年从植物中发现的植物病原氧化酶与人体COX-2有极高的同源性,植物在受到病原体(病毒、细菌、真菌)的攻击时,其分子的防御和应激方式与哺乳动物相似(Bergey,D.R.,et al.Proc Natl Acad Sci USA,1996,93:12053-8)。因此,从天然植物中寻找COX-2抑制剂及其他药物具有很大潜力。初步纯化的天然COX-2抑制剂已经广泛用作癌症化疗中的辅助药物。如从车前草来的乌果酸初提取物占据美国很大市场。
基质金属蛋白酶(MMPs)是细胞外降解基质的一大类酶系,是细胞外基质(ECM)的降解酶。MMPs是一组含锌离子的肽链内切酶,每个活性部位都含有一个锌离子,均能降解一种或几种ECM成分,现已鉴定出约20种MMPs,根据MMPs作用底物不同,将其分为四类:胶原酶、明胶酶、溶基质素和膜MMPs。其中明胶酶(MMP-9)是重要的一组酶,可在皮肤、甲状腺、前列腺、结肠、肺等肿瘤组织中表达。其在肿瘤的侵袭和转移中起着关键性作用,主要通过以下几种机制促进肿瘤的侵袭和转移:①蛋白酶作用使得肿瘤细胞周围由基质分子形成的物理屏障被破坏。促进癌细胞对周围正常组织的浸润,导致肿瘤的扩散和转移。②可以重塑细胞间豁附力,以便肿瘤细胞向周围生长。③作用于基质成分后激发其它的一些潜在的生物活性,如参与肿瘤的免疫过程等。④通过对细胞外基质的改建,促进肿瘤新血管的形成(Shankavaram,U.T.,et al.J.Biol.Chem,2001,276:19027-32;Stetle Stevenson,W.G.,et al.Semin.Cancer Biol.,1996,4:147-154;Roeb E.,et al.Med.Klin.,2003,98:763-70)。大量研究显示MMP-9的过度表达与人类恶性肿瘤的扩散和转移密切相。由于目前大部分化学合成性的第1、2代MMP抑制剂类抗癌药物的临床结果都令人失望,因此从天然产物中开发有选择性、体内稳定、不良反应小的高效第3代MMP抑制剂尤为重要。
三、发明内容:
本发明公开了一种绿原酸酯皂苷类化合物,简称Lonimacranthoide I,分子式为:C81H122O40,分子量:1734,
化学名为:3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 23-O-chlorogenic acyl-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester,化学结构式为:
上述化合物的制备方法,其特征在于以灰毡毛忍冬(Lonicera macranthoides Hand.-Mazz.)花蕾为原料,经水、甲醇、乙醇、甲醇-水混合溶液或乙醇-水混合溶液提取;水提取液直接过大孔树脂吸附,甲醇、乙醇、甲醇-水混合溶液或乙醇-水混合溶液的提取液浓缩后加水溶解过大孔树脂吸附;大孔树脂吸附物经柱层析分离而得。其中,大孔树脂包括D101、AB-8或HP-20;柱层析用担体选自硅胶、凝胶和反相硅胶中的一种或几种制得。
本发明的新绿原酸酯皂苷Lonimacranthoide I与医学上可接受的药用辅料组成药物组合物及其制剂。如,片剂、胶囊剂、以及注射剂。
本发明提供了新绿原酸酯皂苷Lonimacranthoide I制备环氧化酶II抑制剂、基质金属蛋白酶-9抑制剂或抗肿瘤药物的用途。在体外,Lonimacranthoide I抑制环氧化酶II(COX-2)的IC50为2.2μM,抑制基质金属蛋白酶-9的IC50为11.2μM。体内,当LonimacranthoideI剂量为2.5、5和7.5mg/kg时对小鼠K111细胞静脉注射肺转移的抑制作用分别为32.3%、42.5%和47.3%。
四、附图说明:
以下图可作为附件材料上报。
图1、新忍冬绿原酸酯皂苷Lonimacranthoide I的结构式
图2、化合物(I)的主要HMBC相关关系
图3、化合物(I)的HR-ESI-MS谱
图4、化合物(I)的1H-NMR谱
图5、化合物(I)的13C-NMR谱
图6、化合物(I)的HMQC谱
图7、化合物(I)的HMBC谱
图8、化合物(I)的HPLC图
五、具体实施方式:
结合具体实施方式对本发明作进一步说明,但本发明的内容并不仅仅限于所列举的实施方式。
实施例1.从灰毡毛忍冬中分离和鉴定化合物Lonimacranthoide I
将灰毡毛忍冬干燥花蕾10kg,用100kg95%(体积比)乙醇-水溶液回流提取3次,每次2小时,浓缩至无醇味得浸膏(干重1.7kg);所得浸膏加10倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂HP-20进行吸附,再以水,20%,70%乙醇溶液洗脱,合并70%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷860g。所得灰毡毛忍冬总皂苷进行硅胶柱层析,洗脱剂依次为氯仿-甲醇-水(17∶3∶0.2→4∶1∶0.1→7∶3∶0.5→3∶3∶0.5)、甲醇;其中氯仿-甲醇-水(3∶3∶0.5)部分进行反复反相硅胶C-18柱层析(洗脱剂为40%乙醇-水溶液)分离及凝胶Sephdex LH-20柱层析纯化(洗脱剂为35%乙醇-水溶液)得到化合物Lonimacranthoide I 1.4g,得率为0.014%,纯度为96.99%(HPLC面积归一化法)。
化合物Lonimacranthoide I的鉴定,白色粉末,TLC香草醛-浓硫酸试液加热显紫红色,放置后变蓝。Molish反应和Liebermann-Burchard反应阳性。难溶于氯仿,易溶于水-甲醇混合溶液。以上信息提示该化合物为皂苷类化合物。由HR-ESI-MS:[M+Na]+at m/z 1757.7274(calculated m/z 1757.7404)(见附图3)以及DEPT谱确定其分子式为C81H122O40,分子量1734。由1H、13C-NMR谱(C5D5N,500MHz)(见附图4、5)以及完全酸水解后co-TLC实验结果表明其皂苷元为常春藤皂苷元。13C-NMR:C-3(δ81.7)和C-28(δ176.5)表明该化合物在C-3、28位都接有糖链。1H-NMR谱及HMQC谱(见附图6)中六个糖端基信号:δ4.82(1H,d,J=6.6Hz),4.99(1H,d,J=7.6Hz),5.15(1H,d,J=7.7Hz),5.58(1H,d,J=7.1Hz),6.20(1H,d,J=8.4Hz),and6.23(1H,br s);以及13C-NMR:δ95.6,101.5,105.0,105.3,105.4和106.6显示化合物在C-28与C-3位上共有六个糖取代。糖的种类和比例由GC和2D-NMR确定为阿拉伯糖、鼠李糖、葡萄糖(1∶1∶4)。糖链的连接顺序由HMBC谱(见附图2、7)确定:由Glc II的H-1(δ5.15)与Glc I的C-4(δ81.3)相关,Glc I的H-1(δ5.58)与Rha的C-3(δ84.1)相关,Rha的H-1(δ6.23)与Ara的C-2(δ75.1)相关,Ara的H-1(δ4.82)与苷元的C-3(δ81.7)相关,确定C-3的糖链3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl;由Glc IV的H-1(δ4.99)与Glc III的C-6(δ69.4)相关,Glc III的H-1(δ6.20)与C-28(δ176.5)相关,确定C-28的糖链28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl。1H-NMR中除了苷元和糖的氢信号还有:反式双键氢信号[δ6.50(1H,d,J=15.9Hz,H-8″),7.91(1H,d,J=15.9Hz,H-7″)],1、3、4-三取代苯氢信号[δ7.01(1H,dd,J=2.3,8.3Hz,H-6″),7.15(1H,d,J=8.3Hz,H-5″)和7.54(1H,d,J=2.3Hz,H-2″)],三个接氧甲基质子[δ4.65(1H,m,H-3′),δ4.17(1H,m,H-4′)和δ6.21(1H,m,H-5′)],四个亚甲基质子[δ2.68,2.91(2H,m,H-2′)和2.70,2.91(2H,m,H-6′)],这些信号结合碳谱信号表明该皂苷结构存在绿原酸酯。由HMBC谱确定绿原酸酯接在苷元的C-23位。综合各数据及与文献对比鉴定化合物Lonimacranthoide I为:3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 23-O-chlorogenic acyl-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester。经CA网络版检索为新化合物。
Table 1化合物Lonimacranthoide I的苷元与糖链部分的NMR数据(δ,ppm,0=TMS,C5D5N)
aValues in parantheses are1H-1H splittings in cases where these are clearly resolved.
bAra=α-L-arabinopyranose;cRha=α-L-rhamnopyranose;dGlc=β-D-glucopyranose.
Table 2化合物Lonimacranthoide I的绿原酸酯部分与绿原酸的NMR数据(δ,ppm,0=TMS,C5D5N)
HPLC法测定Lonimacranthoide I的纯度,色谱条件为:色谱柱C18(Agilent EclipseXDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按LonimacranthoideI峰计算应不低于1000。Lonimacranthoide I的保留时间为6.85min,按面积归一化法计算产物的纯度为96.99%(色谱图见附图8)。
实施例2、用水提法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用水加热提取三次,水用量为10升,提取时间为2小时,提取温度为100℃,提取液经大孔树脂D101吸附,再用水,15%,75%乙醇洗脱,75%乙醇洗脱液减压回收溶剂得灰毡毛忍冬总皂苷87g。所得灰毡毛忍冬总皂苷进行硅胶柱层析,流动相依次为氯仿-甲醇-水(17∶3∶0.2→4∶1∶0.1→7∶3∶0.5→3∶3∶0.5)、甲醇;其中氯仿-甲醇-水(3∶3∶0.5)进行反复反相硅胶C-8柱层析(洗脱剂为30%乙醇-水溶液)分离及凝胶柱Sephdex LH-20层析纯化(洗脱剂为35%乙醇-水溶液)得到化合物Lonimacranthoide I 0.12克,得率为0.012%,纯度为95%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.82min。
实施例3、用甲醇冷浸提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1Kg,用甲醇冷浸提取三次,甲醇用量为20升,提取时间为20天,提取温度为室温,提取液浓缩至无醇味得浸膏(干重为120g);所得浸膏加10倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂AB-8进行吸附,再以水,10%,80%乙醇溶液洗脱,合并80%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷81g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C18柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.11克,得率为0.011%,纯度为98%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.87min。
实施例4、用甲醇加热回流提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用8kg甲醇回流提取3次,每次2小时,提取液浓缩至无醇味得浸膏(干重为150g);所得浸膏加10倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂D101进行吸附,再以水,10%,75%乙醇溶液洗脱,合并75%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷89g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C18柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.14克,得率为0.014%,纯度为96%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent EclipseXDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按LonimacranthoideI峰计算应不低于1000。Lonimacranthoide I的保留时间为6.85min。
实施例5、用高浓度的甲醇-水混合溶液提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用10kg 95%(体积比)甲醇-水的混合溶液回流提取3次,每次2小时,提取液浓缩至无醇味得浸膏(干重为170g);所得浸膏加9倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂HP-20进行吸附,再以水,10%,75%乙醇溶液洗脱,合并75%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷78g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C8柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.15克,得率为0.015%,纯度为97%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.87min。
实施例6、用低浓度的甲醇-水混合溶液提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用8kg 40%(体积比)甲醇-水的混合溶液回流提取3次,每次2小时,提取液浓缩至无醇味得浸膏(干重为180g);所得浸膏加4倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂HP-20进行吸附,再以水,10%,75%乙醇溶液洗脱,合并75%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷79g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C18柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.09克,得率为0.009%,纯度为98%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.86min。
实施例7、用乙醇冷浸提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用乙醇冷浸提取三次,乙醇用量为20升,提取时间为20天,提取温度为室温,提取液浓缩至无醇味得浸膏(干重为110g);所得浸膏加10倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂D-101进行吸附,再以水,10%,75%乙醇溶液洗脱,合并75%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷75g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C18柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.11克,得率为0.011%,纯度为98%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.86min。
实施例8、用低浓度的乙醇-水混合溶液提取法从灰毡毛忍冬中制备Lonimacranthoide I
灰毡毛忍冬干燥花蕾1kg,用10kg 30%(体积比)乙醇-水的混合溶液回流提取3次,每次2小时,提取液浓缩至无醇味得浸膏(干重约为170g);所得浸膏加4倍体积的水溶解,滤纸过滤除去水不溶物,滤液用大孔树脂HP-20进行吸附,再以水,10%,75%乙醇溶液洗脱,合并75%乙醇溶液洗脱液,浓缩得灰毡毛忍冬总皂苷8.2g。所得灰毡毛忍冬总皂苷再经柱层析(硅胶柱层析:氯仿-甲醇-水梯度洗脱系统,RP-C18柱层析:水-甲醇系统,凝胶柱层析:水-甲醇系统)分离后,得到化合物Lonimacranthoide I 0.13克,得率为0.013%,纯度为97%(外标法)。本实施例中所得产物的鉴定及纯度检查,是以实施例1所得Lonimacranthoide I为对照品采用HPLC法检测计算得到。HPLC测定方法的色谱条件为:色谱柱C18(Agilent Eclipse XDB-C18,4.6×250mm,5μm);柱温:35℃;流动相:甲醇-水(含0.1%磷酸)(45∶55)溶液洗脱;流速:1.0mL/min;进样量5μl;UV检测波长:332nm;理论塔板数按Lonimacranthoide I峰计算应不低于1000。Lonimacranthoide I的保留时间为6.87min。
实施例9、化合物Lonimacranthoide I体外抑制环氧化酶II(COX-2)的活性测试
以每孔1×105个细胞接种于96孔培养板,24h后换培养基。设不加药物的细胞为对照组,加入不同浓度Lonimacranthoide I培养24h后收集上清,存于-20℃备用。取上述细胞培养上清按放射免疫试剂盒说明测定前列腺素E(prostaglandin E2,PGE2)。
表3不同浓度Lonimacranthoide I对COX-2的作用
结果显示,随着化合物浓度增加,环氧化酶II(COX-2)活性逐渐降低,IC50=2.2μM。实施例10、化合物Lonimacranthoide I体外抑制基质金属蛋白酶9(MMP-9)的活性测试
收集细胞,以105个/孔接种24孔板,培养过夜。PBS洗两遍,换无血清培养基培养1h,去培养基后,PBS洗2遍,加200μl的不同浓度Lonimacranthoide I和溶剂对照的无血清培养基培养24h。离心收集细胞上清液,SDS-PAGE电泳。100ml 2.5%Triton-X-100洗脱半小时,然后加入100ml明胶酶缓冲液,37度恒温摇床中温育18-24h。染色,脱色直至条带清晰。
表4明胶酶谱法检测MMP-9的活性
结果显示,相对未给药对照组,给药后MMP-9被抑制,IC50=11.2μM。
实施例11、化合物Lonimacranthoide I对小鼠黑色素瘤试验性转移的抑制作用
取对数生长期的小鼠肺高转移黑色素瘤细胞K111,用PBS稀释至2.5×105个/ml,每只小鼠静脉注射0.2ml后随机分为5组,每组8只,次日给药。设3个给药组,分别每日灌胃给予Lonimacranthoide I 2.5、5和7.5mg/kg,连续给药14d;阴性对照组为溶剂对照组,每日灌胃给予0.5%CMCNa 0.5ml,连续14d;环磷酰胺为阳性对照组,每日皮下注射(30mg/kg,7d)。第21天所有动物拉颈处死,取出肺,计数肺上黑色素瘤斑点数,记为转移灶数,按下式计算转移抑制率。
转移抑制率(%)=(阴性对照组平均转移灶数一给药组平均转移灶数)/阴性对照组平均转移灶数×100%
结果表明,化合物Lonimacranthoide I剂量为2.5、5和7.5mg/kg时对小鼠K111细胞静脉注射肺转移的抑制作用分别为32.3%、42.5%和47.3%。高剂量组与阴性对照组相比,肺转移灶数显著减少(P<0.05)。
实施例12、含本发明新皂苷单体Lonimacranthoide I的片剂
取实施例1制得的Lonimacranthoide I10g与微晶纤维素50g及糊精5g混合,用适量30%乙醇做湿润剂,制成软材,常规方法制粒,加入适量硬脂酸镁混合,制成片剂。本片剂中每片含Lonimacranthoide I 50mg。
实施例13、含本发明新皂苷单体Lonimacranthoide I的胶囊剂
取Lonimacranthoide I 5g与淀粉7g,糊精10g,糖粉10g混合,用适量30%乙醇做湿润剂,制成软材,常规方法制粒,装入硬胶囊中。本胶囊剂中每粒含Lonimacranthoide I70mg。
实施例14、含本发明新皂苷单体Lonimacranthoide I的注射剂
取Lonimacranthoide I 5g以普通注射剂制备方法,用250ml 60℃注射用蒸馏水溶解,用NaCl调节等张,封入安瓶。本注射剂10ml含Lonimacranthoide I 200mg。
Claims (6)
2.制备权利要求1所述化合物的方法,其特征在于以灰毡毛忍冬花蕾为原料,经水、甲醇、乙醇或者甲醇-水、乙醇-水的混合溶液提取;水提取液直接过大孔树脂吸附,甲醇、乙醇或者甲醇-水、乙醇-水的混合溶液的提取液浓缩后加水溶解过大孔树脂吸附;大孔树脂吸附物经柱层析分离而得。
3.根据权利要求2所述的制备方法,其特征在于所述的大孔树脂为D101、AB-8、HP-20中的一种;柱层析用担体选自硅胶、凝胶、反相硅胶中的一种或几种。
4.权利要求1所述的化合物与医学上可接受的药用辅料组成药物制剂。
5.根据权利要求4所述的制剂剂型是片剂、胶囊剂和注射剂。
6.权利要求1所述的化合物在制备环氧化酶II抑制剂、基质金属蛋白酶-9抑制剂或抗肿瘤药物的用途。
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CN102424699A (zh) * | 2011-09-23 | 2012-04-25 | 江苏省中国科学院植物研究所 | 一种新灰毡毛忍冬皂苷及其制备方法和用途 |
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CN102424699A (zh) * | 2011-09-23 | 2012-04-25 | 江苏省中国科学院植物研究所 | 一种新灰毡毛忍冬皂苷及其制备方法和用途 |
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CN102424699B (zh) * | 2011-09-23 | 2016-02-17 | 江苏省中国科学院植物研究所 | 一种新灰毡毛忍冬皂苷及其制备方法和用途 |
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