Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a saprobic fungus Nectria
Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a saprobic fungus Nectria
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来自腐生真菌 Nectria 的细胞毒性倍半萜类化合物 MBJ-0009 和 MBJ-0010
DOI：
10.1038/ja.2013.45
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Toshio Tsuchida and Kazuo Shin-ya
中科院分区：
文献类型：
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作者：
Teppei Kawahara;Masashi Itoh;Miho Izumikawa;Noriaki Sakata;Toshio Tsuchida and Kazuo Shin-ya
在我们使用被称为“MBJ的特殊选择”的先进化合物鉴定系统筛选新型微生物代谢产物的过程中，我们成功地发现了新的天然化合物。其中，与eremophilane类MBJ-0009（1）和MBJ-0010（2）相关的酯取代的倍半萜类化合物从真菌丛赤壳菌属f26111中分离（图1）。从真菌培养物中分离的这些类型的化合物已被报道为细胞毒性、3-5抗疟剂和细胞粘附抑制剂。6.本文报道了化合物1和2的发酵、分离、结构鉴定和生物活性。从日本高知县采集的土壤样品中分离到真菌Nectria sp. f26111。将菌株培养在250-ml锥形瓶中，每个锥形瓶含有25 ml由2%马铃薯淀粉组成的种子培养基（Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc.，北海道，日本）、1%葡萄糖（Junsei Chemical，东京，日本）、2%大豆粉（Honen KillPro，J-Oil米尔斯，东京，日本）、0.1%KH 2 PO 4和0.05%MgSO 4·7 H2O。将烧瓶在旋转振荡器（220 r. 251 ℃下加热3天。将肉汤的等分试样（0.5ml）转移到含有50 ml由2%马铃薯淀粉（Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc.）组成的生产培养基的500-ml锥形瓶中，1%葡萄糖（Junsei Chemical）、2%大豆粉（Honen KillPro，J-Oil米尔斯）、0.1%KH 2 PO 4和0.05%MgSO 4·7 H2O，并在旋转振荡器（220 r. 251 ℃下放置4天。作为我们的常规步骤，直接用等体积的n-BuOH提取发酵液（2l）。然后将粗提取物在EtOAc（300 ml 3）和H2O（300 ml）之间分配，得到EtOAc提取物（730 mg）。将获得的提取物施加到硅胶中压液相色谱柱（Purif-Pack SI-30; Shoko Scientific Co.，Ltd，Yokohama，Japan），并用己烷-EtOAc（0- 25%EtOAc）的梯度系统，然后用CHCl 3-MeOH（0、1、2、5、10、20、30和100%MeOH）的逐步溶剂系统洗脱。将2%MeOH水溶液（200 mg）进行十八烷基硅烷中压液相色谱（Purif-Pack octagylsilane-30; Shoko Scientific Co.，Ltd）通过使用逐步溶剂系统（50、60、70、80、90和100%Me0H水溶液）制备。使用CAPCELL PAK C18 MGII柱通过反相HPLC纯化90%MeOH级分（127 mg（5.0 mm，20 id 150 mm; Shiseido，Tokyo，Japan）与含有0.1%甲酸的70%CH3CN水溶液通过柱色谱法（流速：10 ml/min）纯化粗产物，得到1（57.4mg，保留时间（Rt）1/4 21.7min）和粗产物2（8.5mg，Rt 1/4 19.8min）。使用与1相同的程序，从含有2的粗物质中重复HPLC纯化2（3.0 mg，Rt = 19.8 min）。
During our screening program for novel microbial metabolites using the advanced compound-identification system designated as ‘MBJ’s special selection’, we succeeded in the discovery of new natural compounds. 1, 2 Among them, ester-substituted sesquiterpenoids related to the eremophilane class MBJ-0009 (1) and MBJ-0010 (2) were isolated from a fungus Nectria sp. f26111 (Figure 1). These type of compounds isolated from fungal cultures have been reported as cytotoxic, 3–5 antimalarial 5 agents and cell-adhesion inhibitors. 6 Here, we report the fermentation, isolation, structure elucidation and brief biological activity of 1 and 2. The fungus Nectria sp. f26111 was isolated from a soil sample collected in Kochi Prefecture, Japan. The strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan), 1% glucose (Junsei Chemical, Tokyo, Japan), 2% soybean powder (Honen SoyPro, J-Oil Mills, Tokyo, Japan), 0.1% KH2PO4 and 0.05% MgSO4 Á 7H2O. The flasks were shaken on a rotary shaker (220r. pm) at 251C for 3 days. Aliquots (0.5 ml) of the broth were transferred to 500-ml Erlenmeyer flasks containing 50ml of a production medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc.), 1% glucose (Junsei Chemical), 2% soybean powder (Honen SoyPro, J-Oil Mills), 0.1% KH2PO4 and 0.05% MgSO4 Á 7H2O, and cultured on a rotary shaker (220r. pm) at 251C for 4 days. As our routine step, the fermentation broth (2l) was directly extracted with an equal volume of n-BuOH. The crude extract was then partitioned between EtOAc (300 ml 3) and H2O (300ml) to yield EtOAc extract (730mg). The obtained extract was applied to a silica gel medium-pressure liquid chromatography column (Purif-Pack SI-30; Shoko Scientific Co., Ltd, Yokohama, Japan) and eluted with a gradient system of hexane–EtOAc (0–25% EtOAc) followed by the stepwise solvent system of CHCl3–MeOH (0, 1, 2, 5, 10, 20, 30 and 100% MeOH). The 2% MeOH eluate (200mg) was subjected to octadecylsilanemedium-pressure liquid chromatography (Purif-Pack octadecylsilane-30; Shoko Scientific Co., Ltd) by using the stepwise solvent system (50, 60, 70, 80, 90 and 100% aqueous MeOH). The 90% MeOH fraction (127mg) was purified by reversed-phase HPLC using a CAPCELL PAK C18 MGII column (5.0 mm, 20 id  150 mm; Shiseido, Tokyo, Japan) with 70% aqueous CH3CN containing 0.1% formic acid (flow rate: 10mlminÀ1) to yield 1 (57.4 mg, retention time (Rt) ¼ 21.7 min) and crude 2 (8.5 mg, Rt¼ 19.8 min). The repetitive HPLC purification of 2 (3.0 mg, Rt¼ 19.8 min) from the crude material containing 2 was carried out using the same procedure as 1.