CN116606269B - 米团花二倍半萜化合物与提取物l01及其在制药中的应用 - Google Patents
米团花二倍半萜化合物与提取物l01及其在制药中的应用 Download PDFInfo
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Abstract
本发明提供了米团花中二倍半萜化合物与提取物L01及其在制备抗炎药物与免疫抑制药物中的应用,属于天然药物化学技术领域。本发明提供的米团花中二倍半萜类化合物具有1~12所示结构中的任意一种,骨架类型非常罕见,具有抗炎和免疫抑制活性,能够应用于抗炎药物和免疫抑制药物的制备。本发明还提供了一种以化合物1~12为特征成分的米团花提取物L01及其在制备抗炎药物和免疫抑制药物中的应用。试验结果表明,本发明提供的二倍半萜类化合物对CD3/CD28单抗刺激小鼠T细胞中促炎细胞因子IFN‑γ的分泌有显著抑制作用。通过本发明技术方案获得的化合物6及米团花提取物L01在体内实验中能够显著改善银屑病临床症状。
Description
技术领域
本发明属于天然药物化学技术领域,具体涉及米团花中二倍半萜化合物与提取物L01及其制备方法和在制备抗炎药物与免疫抑制药物中的应用。
背景技术
自身免疫性疾病是机体免疫系统对自身抗原产生局部或全身性异常免疫反应而导致自身组织损伤引起的一类疾病,包括银屑病、类风湿性关节炎、系统性红斑狼疮、炎症性肠病、多发性硬化症等。目前报道的自身免疫性疾病多达80余种,临床表现多样,发病率不断提高,对人类健康造成严重影响。银屑病,又称“牛皮癣”,是一种慢性自身免疫疾病,全球患病率为2~3%。银屑病分为斑块型银屑病、滴状银屑病、脓疱状银屑病、红皮病型银屑病等,其中斑块型银屑病最为常见,临床表现多为红斑、鳞屑,全身均可发病。银屑病患者常并发银屑性关节炎、淋巴癌、心血管疾病、高血压,糖尿病以及忧郁症。目前临床治疗自身免疫性疾病主要使用免疫抑制药物,尽管已有多种疗效不错的免疫抑制药物供临床使用,但仍然存在各种各样的问题如毒副作用大、疗效不满意及价格昂贵等,亟需研发新型的免疫抑制药物。
天然产物由于其作用机制独特、毒副作用小等特点,在自身免疫性疾病的治疗中发挥着重要作用。许多中草药来源的天然产物具有显著的抗炎免疫抑制作用,尤其植物萜类化合物的抗炎和免疫抑制活性引人注目,是发现新型天然抗炎和免疫抑制药物的重要来源。米团花(Leucosceptrum canum)为唇形科米团花属的单种属植物,多年生灌木或小乔木。米团花叶和树皮有较高的药用价值,云南民间用于舒筋、止血、消炎,治疗发热、胃痛、外伤出血、闭合性骨折、黄水疮等病症。目前从米团花中发现了一些二倍半萜类化学成分,然而尚未有关于米团花中二倍半萜抗炎活性及免疫抑制活性的报道。
发明内容
有鉴于此,本发明目的在于提供米团花二倍半萜化合物与提取物L01及其制备方法和在制备抗炎药物和免疫抑制药物中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
如下结构式所示的米团花中二倍半萜化合物1~12,
本发明提供了以上方案所述二倍半萜化合物1~12的制备方法,包括以下步骤:
(1)用有机溶剂对米团花进行提取,得到提取液;
(2)将所述提取液进行减压蒸馏,浓缩得到浸膏;
(3)将所述浸膏溶解后依次进行柱层析分离和高效液相色谱分离,得到所述二倍半萜类化合物。
所述柱层析分离包括依次进行的硅胶柱层析、MCI柱层析或反相C18柱层析、葡聚糖凝胶柱层析;所述高效液相色谱分离采用制备或半制备高效液相色谱。
本发明用有机溶剂对米团花进行提取,得到提取液。在提取之前,本发明优选先将所述米团花粉碎至30目。在本发明中,所述米团花可以取自米团花地上部位或任一组织,如米团花的叶、花或茎阴干;本发明对所述米团花的含水率没有特别的要求,新鲜或干燥的米团花均可。
在本发明中,所述有机溶剂优选包括甲醇、乙醇、丙酮、氯仿、二氯甲烷和石油醚中的一种或几种;所述有机溶剂在每千克干重的米团花中的加入量优选为5L。在本发明中,所述提取优选为冷浸提取或热回流提取。在本发明中,所述冷浸提取的温度优选为室温,所述冷浸提取的次数优选为2~5次,更优选为3~4次;每次提取的时间优选为24~48h,更优选为36~48h;每次冷浸提取后,对所得提取体系进行过滤,并将每次过滤后的滤液合并。在本发明中,所述热回流提取的温度优选为40~50℃,更优选为45~50℃;所述热回流提取的时间优选为4~6h,更优选为4~5h。
得到提取液后,本发明将所述提取液进行减压蒸馏,浓缩得到浸膏。在本发明中,所述减压蒸馏的温度优选为35~45℃,时间优选为60~100min;所述减压蒸馏的浓缩倍数优选为50倍。经过减压蒸馏后,所述提取液中的有机溶剂被去除,提取液同时得到浓缩。
得到浸膏后,本发明将所述浸膏溶解后依次进行柱层析分离和高效液相色谱分离,得到所述二倍半萜类化合物。在本发明中,所述溶解浸膏用有机溶剂优选为氯仿,所述溶解浸膏用有机溶剂为浸膏质量的1.5~3倍。在本发明中,所述柱层析分离优选包括依次进行的硅胶柱层析、MCI柱层析或反相C18柱层析、葡聚糖凝胶柱层析。在本发明中,所述硅胶柱层析的装柱过程优选为:用200~300目硅胶装柱;将所述浸膏溶解后,拌以1~1.5倍(质量倍数)的200~300目硅胶,拌和后的试样晾干并研细过筛后转移至硅胶柱中进行柱层析。在本发明中,所述硅胶柱层析优选采用梯度洗脱,所述梯度洗脱采用的流动相优选为由溶剂A和溶剂B按体积比1:0~0:1组成的混合溶剂;所述溶剂A优选为二氯甲烷、氯仿和石油醚中任意一种,所述溶剂优选B为二氯甲烷、氯仿、丙酮、乙酸乙酯和甲醇中任意一种。本发明优选将所述梯度洗脱所得流分依次进行减压浓缩和薄层层析检测,然后合并相同流分。在本发明中,所述硅胶柱层析的具体洗脱程序优选为:以PE:CH2Cl2(1:0,1:1,0:1,v/v)和CH2Cl2:Me2CO(9:1,4:1,1:1)进行梯度洗脱,将所得流分经薄层层析检测合并后共得到6个待分离流分,即Fr.1(PE)、Fr.2(PE:CH2Cl2=1:1)、Fr.3(CH2Cl2)、Fr.4(CH2Cl2:Me2CO=9:1)、Fr.5(CH2Cl2:Me2CO=4:1)、Fr.6(CH2Cl2:Me2CO=1:1)。
Fr.4进行MCI柱层析或反相C18柱层析后,本发明将所得流分Fr.4-2进行葡聚糖凝胶柱层析。在本发明具体实施例中,所述葡聚糖凝胶的型号为LH-20。在本发明中,所述葡聚糖凝胶柱层析采用的流动相优选为二氯甲烷和甲醇以体积比1:1组成的混合溶剂;经过所述葡聚糖凝胶柱层析,得到4个亚流分即Fr.4-2-1、Fr.4-2-2、Fr.4-2-3、Fr.4-2-4。
经过上述柱层析以后,本发明将所得流分Fr.4-2-1、Fr.4-2-2、Fr.4-2-3、Fr.4-2-4进行高效液相色谱分离,得到所述二倍半萜类化合物。在本发明中,所述高效液相色谱分离采用制备或半制备高效液相色谱;所述高效液相色谱的色谱条件优选为:以C18色谱柱、C8色谱柱、苯基色谱柱或硅胶色谱柱为色谱柱,柱温为35℃;采用梯度洗脱,流动相是由乙腈和水按体积比100:0~5:95组成的混合溶剂;流动速度为3mL/min。在本发明具体实施例中,所述高效液相色谱的分离程序具体优选为:采用安捷伦1200半制备液相色谱仪,ZorbaxSB-C18 column(5μm,9.4×250mm,3mL/min),柱温35℃;在MeCN-H2O(75:25,v/v)的洗脱条件下从Fr.4-2-1得到化合物3(tR 14.5min)、化合物4(tR 17.2min)、化合物6(tR 21.4min);在MeCN-H2O(70:30,v/v)的洗脱条件下从Fr.4-2-2得到化合物1(tR 11.6min)、化合物8(tR13.5min)、化合物10(tR 14.8min);在MeCN-H2O(80:20,v/v)的洗脱条件下从Fr.4-2-3得到化合物7(tR 9.7min)、化合物9(tR 10.5min)、12(tR 13.8min);在MeCN-H2O(60:40,v/v)的洗脱条件下从Fr.4-2-4得到11(tR 11.5min)、化合物2(tR 14.4min)、化合物5(tR28.6min)。
本发明提供了以上方案所述米团花提取物L01及其制备方法,其制备方法包括如下步骤:
(1)将粉碎的米团花与有机溶剂混合,超声处理后,收集提取液:所述有机溶剂包括甲醇、乙醇、丙酮、氯仿、二氯甲烷和石油醚中的一种或几种;所述粉碎的米团花地上部分与有机溶剂的料液比为1:(3~8);所述超声处理时间为10~40min;
(2)将上述提取液经浓缩处理后得到粗提物:所述浓缩处理通过旋转蒸发方式实现,温度为30~60℃;
(3)将上述粗提物进行硅胶柱层析分离洗脱,收集丙酮洗脱相浓缩得到米团花提取物L01:所述硅胶柱层析洗脱过程使用的洗脱剂依次为石油醚和丙酮;所述浓缩处理通过旋转蒸发方式实现,温度为30~60℃。
具体地,米团花提取物L01的制备方法为:
(1)采自米团花地上部位阴干粉碎到30目,在室温下用10L石油醚充分混合,超声提取三次,每次30min,过滤后合并提取液;
(2)将上述提取液通过旋转蒸发仪在45℃下浓缩处理后得到浸膏;
(3)将上述浸膏经0.5L氯仿溶解,拌以硅胶200~300目,放置晾干并研细过筛,用硅胶200~300目装柱进行柱层析,依次以石油醚和丙酮进行洗脱,浓缩处理丙酮洗脱液得到米团花提取物L01。
本发明还提供了米团花二倍半萜化合物1~12及提取物L01在制备抗炎药物或免疫抑制药物中的应用,以及其在制备T细胞增殖抑制剂或细胞因子抑制剂及抗银屑病药物中的应用。
此外,本发明同时提供了一种药物组合物,包括药学上可接受的载体和所述二倍半萜类化合物1~12中的一种或几种。
以及,一种药物组合物,包括药学上可接受的载体和所述的米团花提取物L01。
在本发明中,所述药物组合物中化合物1~12或提取物L01的质量百分含量优选为0.1~99%,更优选为0.5~90%;所述药物组合物中药用载体的总质量百分含量优选为1~99.9%,更优选为10~99.5%。
本发明对于所述药用载体没有特殊限定,采用本领域熟知的药用载体即可,具体如固体、半固体或液体稀释剂,填料或药物制品辅剂中的一种或多种。在本发明中,所述药物组合物的制剂种类优选包括液体制剂、固体制剂、喷剂或雾剂;所述液体制剂优选包括注射剂、混悬剂、乳剂、溶液剂或糖浆剂;所述固体制剂优选包括片剂、胶囊剂、颗粒剂或冲剂。本发明中对于所述药物组合物的制备方法没有特殊限定,采用本领域熟知的制备方法即可。
在本发明中,所述药物组合物及提取物L01的给药方式优选为注射、口服、舌下给药或粘膜透析;所述注射优选包括静脉注射、静脉滴注、肌肉注射、腹腔注射或皮下注射。
在本发明中,所述药物组合物及提取物L01优选以单位体重服用量的形式使用。在本发明中,所述单位体重服用量优选为0.5~500mg/kg。
通过采用上述技术方案,本发明具有如下有益效果:
采用本发明技术方案得到的米团花二倍半萜化合物1~12,在体外实验中,显著降低CD3/CD28单抗刺激小鼠T细胞中炎症细胞因子IFN-γ的分泌。在小鼠体内实验中,化合物6和米团花提取物L01均显著降低银屑病模型小鼠血清中促炎细胞因子IFN-γ和IL-17A的含量,明显改善体重减轻、背部皮肤鳞片和厚度等银屑病小鼠临床发病症状,减轻小鼠皮肤的病理学损伤和全身免疫反应。这为筛选安全、高效的免疫抑制剂提供候选材料,也为银屑病等自身免疫性疾病的治疗提供有效的策略。
附图说明
图1为本发明二倍半萜化合物1的单晶X-衍射结构图;
图2为本发明二倍半萜化合物2的单晶X-衍射结构图;
图3为本发明二倍半萜化合物4的单晶X-衍射结构图;
图4为本发明二倍半萜化合物1~12的结构示意图;
图5为本发明米团花提取物L01处理后各组小鼠背部皮肤的图片;
图6为本发明米团花提取物L01处理后小鼠背部皮肤H&E染色切片;
图7为本发明米团花提取物L01处理后小鼠血清中细胞因子IFN-γ和IL-17A含量变化图;
图8为本发明二倍半萜化合物6处理后各组小鼠背部皮肤的图片;
图9为本发明二倍半萜化合物6处理后小鼠背部皮肤H&E染色切片;
图10为本发明二倍半萜化合物6处理后小鼠血清中细胞因子IFN-γ和IL-17A含量变化图。
具体实施方式
下面结合附图,用本发明的实施例来进一步说明本发明米团花二倍半萜化合物与提取物L01及其制备方法和在制备抗炎药物和免疫抑制药物中的应用,但是不能把它们理解为对本发明保护范围的限定。
实施例1
具体地,本发明中二倍半萜化合物1~12的提取与分离步骤如下:
(1)采自云南的米团花地上部位阴干粉碎到30目后得到10.0kg样品,在室温下用50L石油醚浸泡提取3次,每次48h,过滤后合并提取液,减压蒸馏除去溶剂后得总浸膏230g;
(2)浸膏经2L氯仿溶解,拌以253g硅胶(200~300目),放置晾干并研细过筛,用2.0kg硅胶(200~300目)装柱进行柱层析,依次以石油醚:二氯甲烷(1:0,1:1,0:1,v/v)和氯仿:丙酮(9:1,4:1,1:1)进行梯度洗脱,将所得流分减压浓缩后经薄层层析检测合并相同流分后共得到6个待分离流分,即Fr.1(石油醚)、Fr.2(石油醚:氯仿=1:1)、Fr.3(氯仿)、Fr.4(氯仿:丙酮=9:1)、Fr.5(氯仿:丙酮=4:1)、Fr.6(氯仿:丙酮=1:1);
(3)对Fr.4(48g)进行MCI柱层析,以甲醇:水(50%,60%,70%,80%,90%,100%,v/v)进行梯度洗脱,得到4个亚流分Fr.4-1~Fr.4-4;
对流分Fr.4-2进行葡聚糖凝胶柱层析,以二氯甲烷:甲醇(1:1,v/v)进行洗脱,得到4个亚流分Fr.4-2-1~Fr.4-2-4;
对流分Fr.4-2-1~Fr.4-2-4进行高效液相色谱分离:采用安捷伦1200半制备液相色谱仪,Zorbax SB-C18 column(5μm,9.4×250mm,3mL/min),柱温35℃;在MeCN-H2O(75:25,v/v)的洗脱条件下从Fr.4-2-1得到化合物3(tR 14.5min)、化合物4(tR 17.2min)、化合物6(tR 21.4min);在MeCN-H2O(70:30,v/v)的洗脱条件下从Fr.4-2-2得到化合物1(tR11.6min)、化合物8(tR 13.5min)、化合物10(tR 14.8min);在MeCN-H2O(80:20,v/v)的洗脱条件下从Fr.4-2-3得到化合物7(tR 9.7min)、化合物9(tR 10.5min)、12(tR 13.8min);在MeCN-H2O(60:40,v/v)的洗脱条件下从Fr.4-2-4得到化合物11(tR 11.5min)、2(tR14.4min)、化合物5(tR 28.6min);
各化合物所得质量分别为化合物1(2.0mg)、化合物2(3.2mg)、化合物3(9.5mg)、化合物4(3.5mg)、化合物5(2.0mg)、化合物6(35.0mg)、化合物7(2.0mg)、化合物8(4.0mg)、化合物9(3.5mg)、化合物10(13.0mg)化合物11(1.2mg)、化合物12(2.5mg)。
化合物1~12的物理和光谱数据:
化合物1:白色固体;比旋光度(c 0.30,MeOH);红外IR(KBr)νmax3429,2955,2931,2870,1742,1721,1640,1377,1164cm–1;高分辨质谱HRESIMS m/z 431.2443[M-H]-;1H和13C NMR数据见表1.
化合物2:白色固体;比旋光度(c 0.10,MeOH);红外IR(KBr)νmax3435,2973,2936,2871,1760,1716,1452,1378,1140cm–1;高分辨质谱HRESIMS m/z 455.2405[M+Na]+;1H和13C NMR数据见表1.
化合物3:无色油状液体;比旋光度(c 0.10,MeOH);红外IR(KBr)νmax3437,2973,2936,2871,1715,1452,1377,1138,1013cm–1;高分辨质谱HRESIMS m/z439.2459[M+Na]+;1H和13C NMR数据见表1.
化合物4:无色油状液体;比旋光度(c 0.13,MeOH);红外IR(KBr)νmax3470,2967,2938,2874,1684,1462,1394,1378,1010cm–1;高分辨质谱HRESIMS m/z375.2141[M+Na]+;1H和13C NMR数据见表1.
化合物5:无色油状液体;比旋光度(c 0.06,MeOH);红外IR(KBr)νmax3438,2971,2939,2871,1704,1684,1452,1379,1055cm–1;高分辨质谱HRESIMS m/z391.2091[M+Na]+;1H和13C NMR数据见表2.
化合物6:白色固体;比旋光度(c 0.10,MeOH);红外IR(KBr)νmax3429,2956,2874,1766,1661,1457,1380,1166cm–1;高分辨质谱HRESIMS m/z455.2396[M+Na]+;1H和13C NMR数据见表2.
化合物7:无色油状液体;比旋光度(c 0.05,MeOH);红外IR(KBr)νmax3435,2937,2870,1712,1674,1451,1378,1144cm–1;高分辨质谱HRESIMS m/z 469.2195[M+Na]+;1H和13C NMR数据见表2.
化合物8:无色油状液体;比旋光度(c 0.06,MeOH);红外IR(KBr)νmax3436,2970,2871,1715,1687,1452,1379,1170cm–1;高分辨质谱HRESIMS m/z 471.2353[M+Na]+;1H和13C NMR数据见表2.
化合物9:无色油状液体;比旋光度(c 0.10,MeOH);红外IR(KBr)νmax2969,2939,2871,1770,1685,1451,1380,1168cm–1;高分辨质谱HRESIMS m/z 501.2458[M+Na]+;1H和13C NMR数据见表3.
化合物10:无色油状液体;比旋光度(c 0.06,MeOH);红外IR(KBr)νmax3451,2969,2939,2871,1683,1449,1380,1371cm–1;高分辨质谱HRESIMS m/z 517.2775[M+Na]+;1H和13C NMR数据见表3.
化合物11:无色油状液体;比旋光度(c 0.11,MeOH);红外IR(KBr)νmax3446,2960,2833,1730,1690,1422,1381,1200cm–1;高分辨质谱HRESIMS m/z 471.2353[M+Na]+;1H和13C NMR数据见表3.
化合物12:无色油状液体;比旋光度(c 0.06,MeOH);红外IR(KBr)νmax3433,2966,2835,1718,1688,1441,1377,1175cm–1;高分辨质谱HRESIMS m/z 471.2353[M+Na]+;1H和13C NMR数据见表3.
图1~3分别为化合物1、化合物2、化合物4的单晶X-衍射结构图。
由化合物1~12的物理和光谱数据可知,化合物1~12符合图4所示结构。
表1化合物1~4的1H和13C NMR数据(acetone-d6,J in Hz)
表2化合物5~8的1H和13C NMR数据(acetone-d6,J in Hz)
表3化合物9~12的1H和13C NMR数据(acetone-d6,J in Hz)
实施例2
具体地,本发明中米团花提取物L01的制备:
(1)采自云南的米团花地上部位阴干粉碎到30目后得到2.0kg样品,在室温下用10L石油醚充分混合,超声提取三次,每次30min,过滤后合并提取液;
(2)将上述提取液通过旋转蒸发仪在45℃下浓缩处理后得到浸膏30g;
(3)将上述浸膏经0.5L氯仿溶解,拌以33g硅胶(200~300目),放置晾干并研细过筛,用240g硅胶(200~300目)装柱进行柱层析,依次以100%石油醚和100%丙酮进行洗脱,浓缩处理丙酮洗脱液得到米团花提取物L01(18g)。
实施例3
对本发明的化合物1~12进行体外免疫抑制活性测试:
CD3、CD28单抗、IFN-γ检测试剂盒购买于BD Bioscience公司,CCK-8试剂盒购买于美仑公司,RPMI-1640培养基和胎牛血清购买于Biological Industries公司。
配制待测样品:将实施例1的12个化合物分别溶解于DMSO配制成20mM的储存液。
具体方法:从6~8周雌性C57BL/6小鼠无菌分离脾细胞,并用尼龙毛柱法分离获得T细胞,然后接种T细胞(4×105/well)于96孔板(包被有5μg/mL CD3单抗),同时各组分别加入配制好的40、20、10、5、2.5、1.25μM化合物及2μg/mL CD28单抗,置于37℃/5%CO2培养箱中培养。48h后收集细胞上清,酶联免疫吸附测定法(Enzyme-Linked Immuno-SorbentAssay,ELISA)检测上清中IFN-γ分泌情况;每孔细胞中加入10μL CCK-8试剂,37℃孵育4h后测定OD450,以评价细胞增殖情况。实验同时设置环孢菌素(Cyclosporine A,CsA)阳性对照组、DMSO阴性对照组、未刺激组及空白对照组。计算化合物对IFN-γ的抑制活性,并应用GraphPad Prism软件分析半数抑制浓度IC50,结果如表3所示。
表3化合物1~12抑制CD3/CD28单抗刺激小鼠T细胞中促炎细胞因子IFN-γ的分泌
aCsA:环孢菌素(cyclosporine A,CsA),阳性对照。
由表3可以看出,本发明的化合物1~12明显抑制CD3/CD28单抗刺激小鼠T细胞中促炎细胞因子IFN-γ的分泌,说明这类化合物具有抗炎作用及免疫抑制作用。同时,这类化合物与活性之间具有一定的构效关系,为今后的化合物结构优化及改造提供了依据。
实施例4
对米团花提取物L01进行体内抗银屑病活性测试:
BABL/6小鼠购自北京维通利华实验动物技术有限公司,咪喹莫特乳膏购自四川明欣药业有限责任公司,凡士林购自天津市致远化学试剂有限公司。
咪喹莫特诱导银屑病小鼠模型的建立及药效学研究:小鼠随机分为正常组,模型组,甲氨蝶呤(MTX)治疗组(1mg/kg),米团花二倍半萜治疗组(50,150,450mg/kg),每组6只小鼠。在实验开始前对小鼠进行背部除毛,第1天开始在背部均匀涂抹62.5mg的0.5%咪喹莫特软膏,正常对照组小鼠的背部涂抹等量的凡士林,每天涂抹一次,持续6天。第1天至第6天,各治疗组分别通过灌胃给药。实验期间对小鼠称重,并按照以下标准对小鼠的银屑病面积和严重程度指数(PASI)进行评分:0分,无红斑、鳞片,皮肤正常;1分,轻度红斑,有少许细鳞片,皮损处皮肤略高于正常皮肤;2分,中度红色斑块,皮损表面覆盖鳞片,皮损呈片状或中度隆起;3分,重度深红色斑块,几乎所有皮损表面覆盖,有较厚的鳞片或明显的皮损;4分,非常严重和非常深的红色斑块,所有皮损均被很厚的层状鳞片覆盖,皮损增厚突起非常明显。试验结束后取小鼠背部皮肤制成病理切片。ELISA方法检测小鼠血清中促炎细胞因子IFN-γ和IL-17A的含量。
由图5可以看出米团花提取物L01明显改善银屑病小鼠发病症状。图6实验结果表明米团花提取物L01显著抑制银屑病小鼠皮肤组织的病理学损伤。皮肤病理切片HE染色发现,银屑病小鼠皮肤表皮层增厚,角质层出现角化不全和表皮突,真皮层增厚且出现淋巴细胞浸润,而治疗组小鼠的上述症状得到明显改善。图7实验结果表明米团花提取物L01显著降低银屑病小鼠血清中促炎性细胞因子IFN-γ和IL-17A的含量。这些研究证实米团花提取物L01具有较好的体内抗银屑病效果,在临床治疗银屑病等自身免疫疾病方面具有良好的应用前景。
实施例5
对本发明的二倍半萜化合物6进行体内抗银屑病活性测试:
咪喹莫特诱导银屑病小鼠模型的建立方法同实施例5。小鼠随机分为正常组,模型组,甲氨蝶呤(MTX)治疗组(1mg/kg),化合物6治疗组(25和50mg/kg),每组6只小鼠。第1天至第6天,各治疗组分别通过腹腔注射给药。实验期间对小鼠称重,并进行PASI评分。试验结束后取小鼠背部皮肤制成病理切片。ELISA方法检测小鼠血清中促炎细胞因子IFN-γ和IL-17A的含量。
图8实验结果表明化合物6明显改善银屑病小鼠发病症状。图9表明化合物6显著抑制银屑病小鼠皮肤组织的病理学损伤。皮肤病理切片HE染色发现,银屑病小鼠皮肤表皮层增厚,角质层出现角化不全和表皮突,真皮层增厚且出现淋巴细胞浸润,而治疗组小鼠的上述症状得到明显改善。图7实验结果表明化合物6显著降低银屑病小鼠血清中促炎性细胞因子IFN-γ和IL-17A的含量。这些结构表明化合物6具有较好的体内抗银屑病效果,可用于银屑病等自身免疫疾病等自身免疫疾病的治疗。
制剂实施例
在以下制剂实施例中,选择常规试剂,并按照现有常规方法进行制剂制备,本应用例仅体现本发明所述米团花提取物L01或化合物1~12中的至少一种能够制备成不同的制剂,对具体试剂和操作不作具体限定:
(1)本发明米团花提取物L01或化合物1~12制备成片剂:
片剂原料:取米团花提取物L01或化合物1~12其中一种或任意几种混合10mg,乳糖180mg,淀粉55mg,硬脂酸镁5mg;
制备方法:将原料、乳糖和淀粉混合,用丙二醇均匀湿润,把湿润后的混合物过筛并干燥,再过筛,加入硬脂酸镁,然后将混合物压片,每片重250mg,原料含量为10mg。
(2)本发明米团花提取物L01或化合物1~12制备成胶囊剂:
胶囊剂原料:取米团花提取物L01或化合物1~12其中一种或任意几种混合10mg,乳糖187mg,硬脂酸镁3mg;
制备方法:将原料与助剂混合,过筛,均匀混合,把得到的混合物装入硬明胶胶囊,每个胶囊重200mg,原料含量为10mg。
(3)取米团花提取物L01或化合物1~12其中一种或任意几种溶于无菌注射用水中,搅拌至原料溶解,经无菌抽滤漏斗过滤和无菌精滤后分装于安瓿中,然后低温冷冻干燥后无菌熔封,得到药物粉针剂。
(4)将米团花提取物L01或化合物1~12其中一种或任意几种,用DMSO溶解后,按常规方法加注射用水,精滤,灌封灭菌制成注射液,所述注射液的浓度为0.5~5mg/mL。
(5)将米团花提取物L01或化合物1~12其中一种或任意几种,按常规口服液制备方法制成口服液
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.如下结构式所示的米团花中二倍半萜化合物1~12,
2.权利要求1所述的米团花中二倍半萜化合物1~12的制备方法,其特征在于,该方法包括以下步骤:
(1)采自云南的米团花地上部位阴干粉碎到30目后得到样品,在室温下用50L石油醚浸泡提取3次,每次48h,过滤后合并提取液,减压蒸馏除去溶剂后得总浸膏;
(2)总浸膏经2L氯仿溶解,拌以200~300目硅胶,放置晾干并研细过筛,用硅胶装柱进行柱层析,依次以石油醚:二氯甲烷1:0,1:1,0:1,v/v和氯仿:丙酮9:1,4:1,1:1进行梯度洗脱,将所得流分减压浓缩后经薄层层析检测合并相同流分后共得到6个待分离流分,即石油醚部分Fr.1、石油醚:氯仿=1:1部分Fr.2、氯仿部分Fr.3、氯仿:丙酮=9:1部分Fr.4、氯仿:丙酮=4:1部分Fr.5、氯仿:丙酮=1:1部分Fr.6;
(3)对Fr.4进行MCI柱层析,以甲醇:水50%,60%,70%,80%,90%,100%,v/v进行梯度洗脱,得到4个亚流分Fr.4-1~Fr.4-4;
对流分Fr.4-2进行葡聚糖凝胶柱层析,以二氯甲烷:甲醇1:1,v/v进行洗脱,得到4个亚流分Fr.4-2-1~Fr.4-2-4;
对流分Fr.4-2-1~Fr.4-2-4进行高效液相色谱分离:采用安捷伦1200半制备液相色谱仪,Zorbax SB-C18 column:5μm,9.4×250mm,3mL/min,柱温35℃;在MeCN-H2O 75:25,v/v的洗脱条件下从Fr.4-2-1得到化合物3、4、6;在MeCN-H2O 70:30,v/v的洗脱条件下从Fr.4-2-2得到化合物1、8、10;在MeCN-H2O 80:20,v/v的洗脱条件下从Fr.4-2-3得到化合物7、9、12;在MeCN-H2O 60:40,v/v的洗脱条件下从Fr.4-2-4得到化合物11、2、5。
3.权利要求1所述的米团花中二倍半萜化合物1~12在制备抗炎药物或免疫抑制药物中的应用。
4.如下结构式所述的米团花中二倍半萜类化合物6在制备炎症细胞因子抑制剂或抗银屑病药物中的应用,
5.一种药物组合物,包括药学上可接受的载体和权利要求1所述的米团花中二倍半萜化合物1~12中的一种或几种。
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