鹿衔草(
对鹿衔草内生真菌
采用柱层析和制备型高效液相色谱对鹿衔草内生真菌
从鹿衔草内生真菌
化合物
To isolate and identify the secondary metabolites of endophytic
They were isolated and purified by column chromatography and preparative high performance liquid chromatography (HPLC). The structures were identified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Moreover, the anti-neuroinflammatory activity was verified in LPS-induced inflammatory BV-2 cells by reverse docking.
A total of 12 compounds were isolated and identified: cyclopenin (
Compound
植物内生真菌是指寄居在健康植物组织内部而不引起植物明显感染迹象的一类真菌[
本研究从鹿衔草中分离出内生真菌
鹿衔草于2019年10月在黑龙江省大兴安岭地区加格达奇区采集,植物样本经过干燥处理(编号191001-01)存放于武汉科技大学医学院药学系天然药化实验室。
PDA培养基(g/L):马铃薯200.0,葡萄糖20.0,琼脂20.0,加蒸馏水定容至1 L,不加琼脂为PDB培养基,0.1 MPa高压灭菌30 min,冷却后备用。大米培养基:190 mL蒸馏水,150.0 g大米,0.1 MPa高压灭菌30 min,冷却后备用。
柱层析硅胶(200−300目),青岛海洋化工厂分厂;MTT,Sigma公司;BV-2细胞,武汉Procell公司;胎牛血清和DMEM培养基,Hyclone公司;一氧化氮检测试剂盒,南京建成公司;ELISA试剂盒,Immunoway公司。超高效液质联用仪、制备型高效液相色谱仪、C18 OBD制备柱(19 mm×250 mm/10 mm×250 mm,5 μm),Waters公司;核磁共振仪,Brucker BioSpin技术有限公司;倒置相差显微镜,Olympus公司;超高分辨质谱仪,赛默飞世尔科技(中国)有限公司。
将鹿衔草洗净室温干燥,叶片用75%乙醇表面消毒后,无菌水冲洗3次,用灭菌的剪刀将其剪成1 cm左右的小方块,转移至灭菌的研钵中研碎,加入5 mL无菌水,再继续研磨均匀。使用移液枪吸取研钵中混匀的液体100 μL至PDA平板上,用无菌刮刀将菌液在平板上涂布均匀。将处理好的平板先平放于桌面30 min左右,以便菌液能够完全渗透入培养基内,再将平板翻转,28 ℃培养5 d,观察到平板上有菌落形成后,挑取单菌落至新的培养基上,28 ℃恒温培养3 d,重复操作,直至观察到单菌落状态,将纯化好的菌种转移到甘油管中,编号并低温保存。
将低温保存的菌种转移至PDA平板上活化,28 ℃培养3 d,将活化好的菌株接种至8瓶装有100 mL PDB培养基的锥形瓶中,28 ℃、150 r/min避光培养48 h获得种子液,按20%的接种量接种至30瓶大米培养基中,无菌搅拌均匀,28 ℃避光静置培养15 d。
发酵结束后每瓶加入500 mL乙酸乙酯,合并,搅拌2 h后静置,萃取3次,两层纱布过滤,合并滤液,减压浓缩得到浸膏115.3 g,进行硅胶(200−300目)柱层析,用石油醚: 乙酸乙酯: 甲醇体积比为15:1:0−0:0:1进行梯度洗脱,通过超高效液质联用仪进行组分分析,合并相同部分,共得到9组分(Fr.1−9)。Fr.5组分(8.3 g)经制备型高效液相色谱(high performance liquid chromatography,HPLC)梯度洗脱(流动相均为乙腈和水) 30 min,流速24 mL/min,得到化合物
6,
BV-2细胞用含有10%胎牛血清的高糖DMEM培养基培养,置于37 ℃、5% CO2条件下培养,待细胞密度达到80%时接种到96孔板中(1×104个/孔),在37 ℃培养24 h,待细胞贴壁。实验设置空白组、LPS (5 μg/mL)模型组、阳性药物多奈哌齐(20 μg/mL)组和化合物
实验各组设置同上,化合物
实验各组设置同上,化合物
所有数据用±SD表示,采用GraphPad Prism 6.0软件进行数据统计分析和作图,组间比较采用单因素方差分析(one-way ANOVA),
纯化后编号为LXC-15菌株的ITS序列(包括ITS1、5.8S和ITS2)长度为491 bp,将其提交至GenBank,得到登录号为ON164812.1。利用BLAST对该序列进行相似性分析,将所获与其相似的序列通过Muscle[
LXC-15菌株基于ITS序列构建的系统发育树
Phylogenetic tree of LXC-15 strain based on ITS sequence similarity.
化合物
化合物
化合物
化合物
化合物
化合物
化合物
化合物
化合物
化合物
化合物
化合物11的主要1H-1H COSY和HMBC相关信号
Key 1H-1H COSY and HMBC correlations of compound 11.
化合物11的1H-NMR (500 MHz)和13C-NMR (125 MHz)波谱数据(DMSO-d6)
1H-NMR(500 MHz) and 13C-NMR(125 MHz) of compound 11(DMSO-d6)
C | C | ||||
注:/:无 |
|||||
1 | / | 10.82 (1H, br s) | 7a | 136.3 | / |
2 | 122.7 | 7.16 (1H, d, |
8 | 25.1 | 2.84 (2H, t, |
3 | 111.7 | / | 9 | 39.5 | 3.35 (2H, td, |
3a | 127.2 | / | 10 | / | 8.19 (1H, t, |
4 | 118.2 | 7.54 (1H, d, |
11 | 165.8 | / |
5 | 118.3 | 6.98 (1H, t, |
12 | 42.7 | 3.15 (2H, s) |
6 | 120.9 | 7.07 (1H, t, |
13 | 169.6 | / |
7 | 111.4 | 7.34 (1H, d, |
化合物
从鹿衔草内生真菌
化合物1−12的结构
The structures of compounds 1−12.
结果显示,化合物
化合物11分子对接相关信息
Related information of molecular docking model
Proteins | PDB ID | Site sphere | −CDOCKER_ENERGY | RMSD(Å) | |||||
x | y | z | r | O.L. | Compound |
||||
注:O.L.:原配体 |
|||||||||
iNOS | 2Y37 | 4.926 0 | −73.731 4 | −22.837 9 | 7.044 7 | 21.646 6 | 32.057 4 | 1.514 5 | |
PI3K | 1E7V | 20.614 8 | 62.246 3 | 21.599 4 | 6.577 8 | 24.787 8 | 46.107 4 | 0.739 6 | |
SAPK | 3V6R | 21.950 5 | −59.127 5 | −15.840 3 | 11.000 9 | 39.067 5 | 35.194 2 | 1.625 1 | |
p38 | 3DT1 | 22.295 5 | 11.013 2 | 29.603 7 | 11.856 6 | 22.919 8 | 40.436 1 | 1.701 0 | |
CNR2 | 6KPC | 10.510 4 | 1.389 9 | −45.075 4 | 9.525 1 | 46.371 9 | 42.055 0 | 0.672 1 | |
ERK1 | 2ZOQ | 0.701 2 | −22.751 4 | 49.430 2 | 6.065 3 | 19.970 1 | 38.670 8 | 0.984 3 |
化合物11与靶点蛋白相互作用2D图
2D diagram of the interaction of compound 11 with target proteins.
BV-2细胞存活率试验结果(
化合物11对LPS诱导BV-2细胞存活率的影响(
Effect of the compound 11 on LPS-induced BV-2 cell viability (
如
化合物11对LPS诱导的BV-2细胞炎症因子NO (A)、TNF-α (B)、IL-1β (C)和IL-6 (D)水平的影响(
Effect of the compound 11 on NO (A), TNF-α (B), IL-1β (C) and IL-6 (D) level in LPS-induced BV-2 cells (
神经退行性疾病发病机制广泛,其病理过程涉及Aβ25-35、神经炎症、氧化应激和Tau蛋白等多种因素影响。神经炎症反应是中枢神经系统的一种免疫应答反应,在阿尔兹海默病、帕金森病等神经退行性疾病中发挥着重要的调节作用。小胶质细胞过度活化会释放大量炎症介质(NO)和炎症因子(IL-1β、TNF-α、IL-6),从而导致神经炎性疾病的发生,抑制小胶质细胞过度活化可以减弱各种神经系统疾病的病理进程,因此BV-2小胶质细胞常用来评价各种小分子化合物的抗神经炎症作用[
青霉属内生真菌次生代谢产物的多样性为寻找新药先导化合物开辟了道路,已经成为发现活性化合物的重要来源,在农业、生物技术和医药领域也有着广泛的应用前景[
鹿衔草作为我国传统的中草药,在我国种类较丰富且药理活性显著,但国内外相关研究报道较少,其化学成分和内生菌、共生菌次生代谢产物的研究也亟待开发。此研究丰富了鹿衔草内生真菌的成分研究,为后续鹿衔草内生真菌的药效物质基础及其活性研究提供了参考,同时为新颖神经退行性疾病药物的开发提供了先导化合物。
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