CN116789526A - 真菌来源的萜类化合物及其制备方法与应用 - Google Patents
真菌来源的萜类化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了极地真菌来源的萜类化合物及其制备方法与应用,分别提供了极地真菌来源的菖蒲烷型倍半萜化合物eutyacorane以及海松烷型二萜化合物libertellenone Z,结构分别如下式I和式II所示:
Description
技术领域
本发明属于海洋生物及医药技术领域,涉及极地真菌来源的萜类化合物及其制备方法与应用,具体涉及从极地来源的弯孢聚壳属真菌次级代谢产物中分离到的一种菖蒲烷型倍半萜化合物eutyacorane和一种海松烷型二萜化合物libertellenone Z,以及它们在制备抗炎药物中的应用。
技术背景
极地真菌由于其生存环境的极端性,会导致其产生众多结构新颖的天然活性产物。Eutypella sp.D-1是一株主产萜类化合物的极地真菌,在之前的研究中,Eutypellasp.D-1的次级代谢产物被发现具有良好的生物学活性,实验表明大部分化合物都有良好的抗肿瘤细胞毒活性,尤其是萜类化合物,其中海松烷型二萜类化合物libertellenone H对胰腺癌细胞SW-1990、胃癌细胞SG7901、宫颈癌细胞HeLa及神经胶质瘤细胞U251等均表现出不同程度的细胞毒活性,IC50值范围在3-50μM[1];libertellenone O-Q对HeLa、MCF-7、HCT-116、K562、SW1990肿瘤细胞均表现出显著的细胞毒活性,IC50值范围为0.26-6.02μM[2]。
从Eutypella sp.D-1分离到的绝大部分是二萜化合物,倍半萜化合物相对较少,该类化合物表现出显著的抗菌活性,例如,桉烷型倍半萜类化合物eut-Guaianesesquiterpene对Escherichia coli、Bacillussubtilis、Staphylococcus aureus菌株表现出显著的抗菌活性[3],由此可见,Eutypella sp.D-1所产萜类次级代谢产物是抗肿瘤、抗菌等一系列药物先导化合物的重要来源。在之前的研究中,未在Eutypella sp.D-1中发现菖蒲烷型的倍半萜化合物;同时,由于libertellenone系列化合物的良好活性,继续挖掘该系列化合物也有重要意义。
近年来天然产物由于其具有的结构新颖、活性多样及副作用小等优点,也成为研发抗炎药物的新来源。到目前为止有关菖蒲烷型倍半萜类和libertellenone类化合物的抗炎活性数据如下表1所示[4],活性测定方法为在LPS诱导的小鼠巨噬细胞RAW264.7炎症模型下,在10μM药物浓度下,测定对NO释放的抑制率,地塞米松为阳性对照。
表1化合物抗炎活性数据
上表中各化合物的结构如下:
发明内容
本发明基于上述研究进行,对Eutypella sp.D-1的次级代谢产物继续进行探索,提供了两种新型的萜类结构:菖蒲烷型的倍半萜化合物以及海松烷型二萜化合物libertellenone Z。
本发明的第一目的在于提供了上述两种化合物的结构;第二目的在于提供通过发酵方法分离得到两种化合物;第三目的在于提供该两种化合物在制备抗炎药物中的用途。
为了实现上述目的,本发明采用的技术方案如下:
本发明的第一方面,提供了一种极地真菌来源的菖蒲烷型倍半萜化合物eutyacorane,其化学结构如下式I所示:
本发明的第二方面,提供了一种极地真菌来源的海松烷型二萜化合物libertellenone Z,其化学结构如下式II所示:
所述极地真菌为分离自北极仙女木根基土壤的弯孢聚壳属真菌Eutypella sp.D-1,于2013年4月12日保藏于中国典型培养物保藏中心(CCTCC),地址位于中国武汉,武汉大学,保藏编号为CCTCC NO:M 2013144。
本发明的第三方面,提供了上述菖蒲烷型倍半萜化合物eutyacorane和海松烷型二萜化合物libertellenone Z的制备方法,包括以下步骤:
a)菌株发酵
使用无菌接种环挑取适量Eutypella sp.D-1菌丝体于50mL体积的无菌PDB液体培养基(24g/L)中,封口膜密封后置于摇床内以28℃、180rpm的条件培养3d,得到种子液备用。按接种量5%(V/V)的比例将种子液加入到500mL体积的PDB培养基中,封口膜密封后置于摇床内以24℃、180rpm的条件培养9d。
b)发酵产物收集
将发酵液使用8层纱布进行过滤,滤液与等体积的乙酸乙酯充分混合,待分层后收集乙酸乙酯层,此过程重复三次;使用旋转蒸发仪将乙酸乙酯蒸干;同时,将过滤后的菌丝体浸泡在二氯甲烷与甲醇(1:1)的混合溶剂中,超声破碎30min,此过程重复三次;将混合液使用8层纱布进行过滤,使用旋转蒸发仪将溶剂蒸干,将发酵产物进行合并,即可得到Eutypella sp.D-1总发酵产物。
c)发酵产物分离纯化
首先利用甲醇与水的流动相对总发酵产物进行了MPLC洗脱,对洗脱下来的化合物进行TLC分析;利用石油醚和乙酸乙酯的流动相对组分进行正相硅胶层析柱洗脱,随后利用HPLC对洗脱下来的化合物进行纯化,菖蒲烷型倍半萜化合物eutyacorane的制备条件为60%甲醇/水,流速2mL/min,保留时间52min;海松烷型二萜化合物libertellenone Z的制备条件为75%甲醇/水,流速2mL/min,保留时间32min。
本发明的第四方面,提供了上述菖蒲烷型倍半萜化合物eutyacorane和海松烷型二萜化合物libertellenone Z在制备抗炎药物中的应用。
实验结果表明,在LPS诱导的小鼠巨噬细胞RAW264.7炎症模型下,菖蒲烷型倍半萜化合物eutyacorane和海松烷型二萜化合物libertellenone Z表现出显著的NO释放抑制活性,以模型组为对照,对NO释放的抑制率分别为65%和60%,只略低于阳性药地塞米松的抑制率72%,可作为当前抗炎药物的补充用于制备抗炎药物。
本发明的有益技术效果如下:
本发明对Eutypella sp.D-1的次级代谢产物继续进行探索,提供了两种新型的萜类结构:菖蒲烷型的倍半萜化合物以及海松烷型二萜化合物libertellenone Z,实验结果显示,两种化合物对于LPS诱导的小鼠巨噬细胞RAW264.7炎症模型中NO的释放具有较佳的抑制率,为抗炎药物提供了新的种类选择。
具体实施方式
下面通过实施例对本发明做进一步阐述,其目的是为更好理解本发明的内容。所举之例并不限制本发明的保护范围。
实施例1:菌株发酵与产物收集
(1)菌株发酵
使用无菌接种环挑取适量Eutypella sp.D-1菌丝体于50mL体积的无菌PDB液体培养基(24g/L)中,封口膜密封后置于摇床内以28℃、180rpm的条件培养3d,得到种子液备用。按接种量5%(V/V)的比例将种子液加入到500mL体积的PDB培养基中,封口膜密封后置于摇床内以24℃、180rpm的条件培养9d。
(2)发酵产物收集
将发酵液使用8层纱布进行过滤,滤液与等体积的乙酸乙酯充分混合,待分层后收集乙酸乙酯层,此过程重复三次;使用旋转蒸发仪将乙酸乙酯蒸干;同时,将过滤后的菌丝体浸泡在二氯甲烷与甲醇(1:1)的混合溶剂中,超声破碎30min,此过程重复三次;将混合液使用8层纱布进行过滤,使用旋转蒸发仪将溶剂蒸干,将发酵产物进行合并,即可得到Eutypella sp.D-1总发酵产物。
实施例2:菖蒲烷型倍半萜化合物eutyacorane的分离纯化
首先利用甲醇与水的流动相对总发酵产物进行了MPLC洗脱,对洗脱下来的化合物进行TLC分析;利用石油醚和乙酸乙酯的流动相对组分进行正相硅胶层析柱洗脱,随后利用HPLC对洗脱下来的化合物进行纯化,条件为60%甲醇/水,流速2mL/min,保留时间52min,最终得到一种新的菖蒲烷型的倍半萜化合物eutyacorane,结构如下所示:
eutyacorane,无色油状物,分子式C15H26O2,分子量238;高分辨质谱261.18
[M+Na]+,eutyacorane的结构母核属于菖蒲烷型倍半萜。1H和13C核磁共振谱数据见表2:
表2eutyacorane的核磁共振数据
实施例3:海松烷型二萜化合物libertellenone Z的分离纯化
首先利用甲醇与水的流动相对总发酵产物进行了MPLC洗脱,对洗脱下来的化合物进行TLC分析;利用石油醚和乙酸乙酯的流动相对组分进行正相硅胶层析柱洗脱,随后利用HPLC对洗脱下来的化合物进行纯化,条件为75%甲醇/水,流速2mL/min,保留时间32min,最终得到一种新的海松烷型的二萜化合物libertellenone Z。
libertellenone Z,黄色油状物,分子式C20H28O4,分子量332;高分辨质谱355.18[M+Na]+,libertellenone Z的结构母核属于海松烷型二萜。1H和13C核磁共振谱数据见表3。
表3libertellenone Z的核磁共振数据
实施例4:化合物的抗炎活性实验
取对数生长期的小鼠巨噬细胞RAW264.7,用加10%血清的DMEM高糖培养基稀释成1~2×105个/mL的浓度接种到96孔板中,每孔100μL,置于细胞培养箱中培养24h。将待测化合物用DMSO溶解,并使用DMEM培养基配制成10μM的溶液,将LPS(TLR4激活剂)加入到上述浓度的溶液中,使LPS终浓度为1μg/mL,并设置阳性对照地塞米松组、模型组、空白对照组。将96孔板中原有培养基吸出,加入配制的样品溶液100μL,置于细胞培养箱中培养24h。吸取96孔板中的上清液50μL,按照NO测定试剂盒说明书的方法,使用酶标仪测定上清液540nm处的吸光度。NO释放量抑制率的计算公式为:抑制率=(模型组NO释放量-化合物组NO释放量)/模型组NO释放量×100%。
结果表明,在LPS诱导的小鼠巨噬细胞RAW264.7炎症模型下,菖蒲烷型倍半萜化合物eutyacorane和海松烷型二萜化合物libertellenone Z表现出显著的NO释放抑制活性,以模型组为对照,对NO释放的抑制率分别为65%和60%,只略低于阳性药地塞米松的抑制率72%,可用于制备抗炎药物。
以上所述仅是本发明的较佳实施例而已,并非对本发明做任何形式上的限制。虽然本发明已经以上述的实施例予以披露,但并不限制本发明的使用范围。任何熟悉本专利的技术人员在不脱离本发明技术方案范围内,可以利用上述提示的技术内容做出一些变动或修饰,应该视为等效实施例。凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例做出任何的简单修改、等同变化与修饰,仍然属于本发明方案的范围。
Claims (9)
1.一种极地真菌来源的菖蒲烷型倍半萜化合物eutyacorane,其特征在于,其化学结构如下式I所示:
2.一种极地真菌来源的海松烷型二萜化合物libertellenone Z,其特征在于,其化学结构如下式II所示:
3.根据权利要求1或2所述的化合物,其特征在于:
其中,所述极地真菌为分离自北极仙女木根基土壤的弯孢聚壳属真菌Eutypellasp.D-1,保藏编号为CCTCC NO:M 2013144。
4.权利要求1或2所述的化合物的制备方法,其特征在于,包括如下步骤:将Eutypellasp.D-1种子液经PDB培养基摇床培养后,将发酵液与菌丝体裂解液的过滤液合并后得到总发酵产物;产物依次经MPLC洗脱、正相硅胶层析柱洗脱后利用HPLC对洗脱下来的化合物进行纯化,菖蒲烷型倍半萜化合物eutyacorane的制备条件为60%甲醇/水,流速2mL/min,保留时间52min;海松烷型二萜化合物libertellenone Z的制备条件为75%甲醇/水,流速2mL/min,保留时间32min。
5.根据权利要求4所述的化合物的制备方法,其特征在于:
其中,Eutypella sp.D-1种子液的制备方法如下:使用无菌接种环挑取适量Eutypellasp.D-1菌丝体于24g/L的无菌PDB液体培养基中,封口膜密封后置于摇床内以28℃、180rpm的条件培养3d,得到种子液备用。
6.根据权利要求4所述的化合物的制备方法,其特征在于:
其中,种子液的发酵方法如下:按体积比将接种量5%种子液加入到PDB培养基中,封口膜密封后置于摇床内以24℃、180rpm的条件培养9d。
7.根据权利要求4所述的化合物的制备方法,其特征在于:
其中,发酵产物收集的方法如下:将发酵液使用8层纱布进行过滤,滤液与等体积的乙酸乙酯充分混合,待分层后收集乙酸乙酯层,此过程重复三次,并使用旋转蒸发仪将乙酸乙酯蒸干;同时,将过滤后的菌丝体浸泡在体积比为1:1的二氯甲烷与甲醇的混合溶剂中,超声破碎30min,过程重复三次后将混合液使用8层纱布进行过滤,并使用旋转蒸发仪将溶剂蒸干;将发酵产物进行合并,即可得到Eutypella sp.D-1总发酵产物。
8.根据权利要求4所述的化合物的制备方法,其特征在于:
其中,产物分离的方法如下:首先利用甲醇与水的流动相对总发酵产物进行了MPLC洗脱,对洗脱下来的化合物进行TLC分析;然后利用石油醚和乙酸乙酯的流动相对组分进行正相硅胶层析柱洗脱;随后利用HPLC对洗脱下来的化合物进行纯化,菖蒲烷型倍半萜化合物eutyacorane的制备条件为60%甲醇/水,流速2mL/min,保留时间52min;海松烷型二萜化合物libertellenone Z的制备条件为75%甲醇/水,流速2mL/min,保留时间32min。
9.权利要求1所述的菖蒲烷型倍半萜化合物eutyacorane或权利要求2所述的海松烷型二萜化合物libertellenone Z在制备抗炎药物中的应用。
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