CN114702388B - 一种淡黄香茶菜中的化合物及其提取分离方法和应用 - Google Patents
一种淡黄香茶菜中的化合物及其提取分离方法和应用 Download PDFInfo
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Abstract
本发明涉及一种淡黄香茶菜中的化合物Fladinols B及其提取分离方法和应用,通过对贵州道真产淡黄香茶菜进行化学成分及抑菌活性研究,从淡黄香茶菜中分离得到了化合物Fladinols B,并通过抑菌活性研究证实其具有良好的抑菌作用。
Description
技术领域
本发明涉及化合物领域,具体涉及一种淡黄香茶菜中的化合物Fladinols B及其提取分离方法和应用。
背景技术
香茶菜属植物(Isodon)分布广泛,主要分布于亚洲东部和非洲西部,约150 种,我国有90种,21个变种,其中约有30种为民间用药,以西南各省种数最多[1]。科研工作者们发现该属植物富含二萜类化合物,具有显著的抗炎、抗肿瘤、抗菌、抗病毒、降酶保肝等作用[2-6]。
淡黄香茶菜Isodon flavidus(Hand.-Mazz.)Hara主要分布于贵州兴义,大方、雷山、道真等地以及云南的中部和西部[5,8]。文献研究发现:关于淡黄香茶菜的研究主要集中在国内,国外尚未见到相关报道。国内对淡黄香茶菜的研究始于 1988年,从该植物分离得到的二萜类化合物中公开发表的有20个,结构类型涉及二环二萜、三环二萜及四环二萜等,分别为6,12,15-trihydroxy-5,8,11, 13-rosin-tetraene-7-one、8(17),13-ent-labdadien-15→16-lactone-19-oic acid、 glutinosin、lophanic acid、flavidusin A、16-hydroxy-feruginol、fladin A、fladin B、 ID-3、ferruginol、hinokiol、ent-kaurane-16β,17-diol、flavidusin B、(+)-isopimara-8(9),15-diene、rubesanolide D、8,9-dihydroxy-13-en-20-oic acid、siegesbeckiol、 isopimara-7,15-dien-19-oic acid、3,4-seco isopimara-7,15-dien-3,9-olide、isopimara-7,15-dien-3β-ol[5,9-12]。通过对以上化合物及相关文献分析:从淡黄香茶菜中发现的二萜成分虽涉及多种结构类型,但化合物的总个数并不多。因此,继续对淡黄香茶菜的化学成分进行研究,以期发现更多结构新颖和活性良好的化合物。
发明团队通过对贵州道真产淡黄香茶菜进行化学成分及抑菌活性研究,从淡黄香茶菜中分离得到了化合物Fladinols B,并通过抑菌活性研究证实其具有良好的抑菌作用。
发明内容
本发明的目的是提供一种淡黄香茶菜中的化合物Fladinols B。
本发明的另一目的是提供一种淡黄香茶菜中的化合物Fladinols B的提取分离方法。
本发明的另一目的是提供一种淡黄香茶菜中的化合物Fladinols B在制备抑菌药物中的应用。
本发明所述的淡黄香茶菜中的化合物为Fladinols B,化合物结构式为:
Fladinols B 1H-1H COSY,HMBC,and NOESY归属
Fladinols B物理常数:
Fladinols B:无色结晶(甲醇),[α]18 D-68.3(c 0.151,MeOH);IR(KBr): 3324,2952,2917,1745,cm-1;HR-ESIMS:m/z[M+Na]+=615.4384,(calcd for C39H60O4Na+,615.4384).
Fladinols B晶体数据:
Fladinols B:C39H60O4,M=592.86, α=90°,β=90°,γ=90°,/>T=100(2) K,空间群:P212121,Z=4,μ(CuKα)=0.53mm-1,Rint=0.0302,R1=0. 0293,wR(F2)=0.0758,F2=1.036.Flack=-0.01(2).
Fladinols B晶体结构式:
Fladinols B 1H NMR、13C NMR数据.
Recorded at 600MHz(1H NMR)and 150MHz(13C NMR).
本发明所述化合物提取分离方法为:
将风干和粉碎过80-100目样品5~15kg,经20~100%工业甲醇进行室温浸泡提取2~6次(7日/次),减压回收甲醇,最终得到总浸膏约;再依次用石油醚、二氯甲烷、乙酸乙酯(1:1)萃取,回收溶剂,得石油醚部位,二氯甲烷部位,乙酸乙酯萃部位;用220mL的乙酸乙酯将石油醚部位A溶解,称取硅胶200~600g拌样,挥去乙酸乙酯;先以石油醚19.5L洗脱,再以石油醚-乙酸乙酯***(100:1,50:1, 25:1,10:1,5:1,1:1)梯度洗脱,最后用乙酸乙酯16L及甲醇8.5L洗脱,按硅胶薄层色谱检测结果,将石油醚部位分段,得到A1~A4共4个极性段。
将A3样品,加入25ml的乙酸乙酯使溶解,称取200-300目的硅胶50g进行拌样,挥去乙酸乙酯;称取200-300目的硅胶200~300g湿法装柱,将拌样硅胶装入;先用石油醚3.5L***,再石油醚-乙酸乙酯***(30:1,20:1,10:1,5:1)梯度洗脱,最后用乙酸乙酯1.5L洗脱;经TLC检测,合并相同或相近流分,得到样品A3-1、 A3-2、A3-3、A3-4、A3-5;将A3-1继续用硅胶柱层析分离纯化,采用石油醚-乙酸乙酯***(50:1,20:1,10:1,5:1)梯度洗脱,经TLC检测,合并相同或相近流分,得到样品A3-1-1、A3-1-2、A3-1-3、A3-1-4、A3-1-5、A3-1-6;将样品A3-1-2 继续用凝胶柱色谱分离纯化,采用二氯甲烷-甲醇(1:1)洗脱,得到Fladinols B粗品,再用甲醇重结晶得到无色透明结晶化合物Fladinols B。
优选的,本发明所述化合物提取分离方法为:
将风干和粉碎过80-100目样品10kg,经95%工业甲醇进行室温浸泡提取4次(7日/次),减压回收甲醇,最终得到总浸膏约1.1kg;再依次用石油醚、二氯甲烷、乙酸乙酯(1:1)萃取,回收溶剂,得石油醚部位(441g),二氯甲烷部位(194g),乙酸乙酯萃部位(28g);用220mL的乙酸乙酯将石油醚部位A溶解,称取硅胶440g拌样,挥去乙酸乙酯;先以石油醚19.5L洗脱,再以石油醚-乙酸乙酯***(100:1,50:1, 25:1,10:1,5:1,1:1)梯度洗脱,最后用乙酸乙酯16L及甲醇8.5L洗脱,按硅胶薄层色谱检测结果,将石油醚部位分段,得到A1~A4共4个极性段;
将A3样品(50g),加入25ml的乙酸乙酯使溶解,称取200-300目的硅胶50g 进行拌样,挥去乙酸乙酯;称取200-300目的硅胶250g湿法装柱,将拌样硅胶装入;先用石油醚3.5L***,再石油醚-乙酸乙酯***(30:1,20:1,10:1,5:1) 梯度洗脱,最后用乙酸乙酯1.5L洗脱;经TLC检测,合并相同或相近流分,得到样品A3-1、A3-2、A3-3、A3-4、A3-5;将A3-1继续用硅胶柱层析分离纯化,采用石油醚-乙酸乙酯***(50:1,20:1,10:1,5:1)梯度洗脱,经TLC检测,合并相同或相近流分,得到样品A3-1-1、A3-1-2、A3-1-3、A3-1-4、A3-1-5、A3-1-6。将样品A3-1-2继续用凝胶柱色谱分离纯化,采用二氯甲烷-甲醇(1:1)洗脱,得到化合物Fladinols B粗品,再用甲醇重结晶得到无色透明结晶化合物Fladinols B(30.1mg)。
优选的,本发明所述室温浸泡提取次数为7日/次。
优选的,本发明所述采用硅胶柱层析,先用石油醚洗脱,再用石油醚-乙酸乙酯***按比例100:1,50:1,25:1,10:1,5:1,1:1梯度洗脱。
优选的,本发明所述采用石油醚,石油醚-乙酸乙酯***按30:1,20:1,10:1, 5:1比例进行梯度洗脱。
本发明所述化合物Fladinols B在制备抑菌药物中的应用。
优选的,本发明所述化合物Fladinols B在制备LPS诱导的巨噬细胞NO药物或制剂中的应用。
本发明所述制剂为加入药学上可接受的辅料按常规工艺制成药学上可接受的制剂。
本发明所述药学上可接受的制剂为固体制剂或液体制剂。
前述固体制剂为颗粒剂、丸剂、胶囊剂、片剂、散剂、冻干粉针剂。
前述液体制剂为口服液、注射制剂。
有益效果:
本发明与现有技术相比,具有以下有益效果:
1.现有技术存在的问题:
现有文献中未见淡黄香茶菜化合物Fladinols B及其提取分离方法的报道,也未见该化合物在制备抑菌药物中的应用研究报道。
2.本发明的有益效果:
本发明通过对贵州道真产淡黄香茶菜进行化学成分及抑菌活性研究,从淡黄香茶菜中分离得到了化合物Fladinols B,并通过抑菌活性筛选实验证实其具有良好的抑菌作用;尤其是对LPS诱导的巨噬细胞NO的表达有较好的抑制作用。
附图说明
图1:1H NMR谱
图2:13C NMR谱
图3:DEPT谱
图4:HSQC谱
图5:HMBC谱
图6:1H-1H COSY谱
图7:NOESY谱
图8:红外光谱
图9:高分辨质谱
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步地具体说明。
实施例1
本发明所述的淡黄香茶菜中的化合物为Fladinols B,化合物结构式为:
Fladinols B 1H-1H COSY,HMBC,and NOESY归属
Fladinols B物理常数:
Fladinols B:无色结晶(甲醇),[α]18 D-68.3(c 0.151,MeOH);IR(KBr): 3324,2952,2917,1745,cm-1;HR-ESIMS:m/z[M+Na]+=615.4384,(calcd for C39H60O4Na+,615.4384).
Fladinols B晶体数据:
Fladinols B:C39H60O4,M=592.86, α=90°,β=90°,γ=90°,/>T=100(2) K,空间群:P212121,Z=4,μ(CuKα)=0.53mm-1,Rint=0.0302,R1=0. 0293,wR(F2)=0.0758,F2=1.036.Flack=-0.01(2).
Fladinols B晶体结构式:
Fladinols B 1H NMR、13C NMR数据.
Recorded at 600MHz(1H NMR)and 150MHz(13C NMR).
4.抑菌活性测试1
筛选模型:巨噬细胞(RAW264.7)活化抑制模型
筛选方法:酶联免疫吸附测定法(ELISA法)检测巨噬细胞肿瘤坏死因子(TNF-a)的分泌表达。
模型原理:巨噬细胞在相关因素刺激下会活化,并分泌表达TNF-a。将样品加入巨噬细胞培养中的含量,判断样品对细胞的活化抑制作用。
计算公式:活化抑制率(%)=[(LPS组TNF-a含量-样品组TNF-a含量)/LPS组TNFTNF-a含量]x100
筛选结果:
说明:
1.脂多糖为活化剂,其活化指数为36.78+9.59。
2.扁塑藤素为阳性对照,分子量:464.645.
3.样品用DMSO溶解。
4.“一”表示样品在该浓度下对细胞活力抑制较强,未进行TNF-a含量检测。评价:
化合物Fladinols B的较低浓度仍对细胞活力抑制,对LPS诱导的巨噬细胞活化TNF-a表达可能有抑制作用。
5.抑菌活性测试2
筛选模型:巨噬细胞(RAW264.7)活化抑制模型
筛选方法:Griess法检测巨噬细胞一氧化氮(NO)的释放。
模型原理:巨噬细胞在相关因素刺激下会活化,并分泌表达NO。将样品加入巨噬细胞培养孔中一定时间后,再加入脂多糖(LPS),孵育培养一定时间后,取细胞上清液测定NO的含量,判断样品对细胞的活化抑制作用。
计算公式:活化抑制率(%)=[(LPS组NO含量~样品组NO含量)/LPS组NO 含量]x100。
筛选结果:
说明:
1.脂多糖为活化剂,其活化指数为9.31±3.37。
2.PDTC为阳性对照,分子量:164.29
3.样品用DMSO溶解。
4.“一”表示样品在该浓度下对细胞活力抑制较强,未进行NO含量检测。
评价:
淡黄香茶菜中的化合物Fladinols B对LPS诱导的巨噬细胞NO的表达有较好的抑制作用。
总结:淡黄香茶菜中的化合物Fladinols B其具有良好的抑菌作用;尤其是对 LPS诱导的巨噬细胞NO的表达有较好的抑制作用。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作出一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
参考文献
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[2]章智荣.同时多原发性非小细胞肺癌临床特征与外科治疗疗效分析PH17是新的具有抗肿瘤活性的香茶菜属二萜类化合物[D].北京协和医学院,2016:10-15.
[3]杨一兵.柄叶香茶菜及其甲素体外抗菌的研究[A].中国民族医药学会.2002全国土家族苗族医药学术会议论文专辑[C].中国民族医药学会:中国民族医药学会,2002:17-18.
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[5]刘亚华,李继新,辛来香,等.雷山产淡黄香茶菜化学成分的研究[J].中草药,2006, 47(10):1657-1660.
[6]刘明洁,孙旗,王强,等.蓝萼香茶菜总二萜预处理对兔心肌缺血/再灌注时心肌细胞凋亡的影响[J].中国实验方剂学杂志,2013,19(19):229-233.
[7]曹音,姜建萍,赵英,等.壮药三叶香茶菜抗乙型肝炎病毒作用有效部位的筛选研究[J]. 湖南中医杂志,2017,33(09):168-169.
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Claims (3)
1.一种淡黄香茶菜中的化合物Fladinols B在制备抑制LPS诱导的巨噬细胞NO表达的药物中的应用,所述化合物结构式为:
。
2.根据权利要求1所述的应用,其特征在于,所述药物加入药学上可接受的辅料按常规工艺制成药学上可接受的制剂。
3.根据权利要求2所述的应用,其特征在于,所述药学上可接受的制剂为固体制剂或液体制剂。
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