2017, Vol. 52
2. 浙江工业大学药学院, 浙江 杭州 310014
, LIN Sheng1
2. School of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China
植物内生真菌是指生活在活体植物组织中, 并对宿主植物体产生无害或有益作用的真菌。内生真菌往往与药用植物存在“协同进化”的关系, 因此, 某些内生真菌也具有了产生与宿主药用植物相同或相似的次生代谢产物[1]。从1904年Freeman从毒麦中分离得到第一批植物内生真菌至今, 一大批结构新颖独特的生物活性成分被相继报道。植物内生真菌已成为天然抗菌活性成分的重要来源宝库[2]。随着近几年多重耐药菌数量的增加, 寻找新型抗菌药物尤为急迫。
瑞香科结香属植物结香Edgeworthia chrysantha, 全株入药能消肿止痛, 可治跌打损伤、风湿疼痛; 其花蕾有养阴安神的功效, 主治目赤肿痛、多泪、目翳、梦遗、白带等症[3, 4]。近年来, 对其抗菌活性的研究逐渐深入[5, 6]。Zhang等[7]在对结香的内生真菌分离的过程中, 发现了多株具有抗菌活性的内生真菌, 其中芬芳镰刀菌Fusarium redolens为丛赤壳科镰刀菌属, 别名: F. oxysporum var. redolens, 是常见的药用植物内生真菌之一。形态上为白色菌落, 菌落生长后期发红或发紫, 子囊中孢子分裂成2个近圆形的细胞[8]。其在形态学上与镰刀菌属的其他菌种较难辨别, 而用分子生物学的方式进行鉴定具有较高的灵敏度和准确性[9−11]。现有研究报道, 芬芳镰刀菌的多种次生代谢产物都表现出较好的抗菌活性[12−16], 但对其固体发酵产物的化学成分缺少系统性研究。为了从药用植物内生真菌中寻找结构多样性的活性天然产物, 本研究对结香内生真菌芬芳镰刀菌的大米固体发酵产物的乙酸乙酯部位的化学成分进行研究, 并通过多种色谱技术和波谱学方法, 对单体化合物进行结构鉴定。
结果与讨论本研究从结香内生真菌芬芳镰刀菌的大米固体发酵产物的乙酸乙酯部位, 分离鉴定了10个化合物, 分别为 (+)-7S-4-脱氧-9-羟基茎点菌吡喃酮C [(+)-7S-4-deoxy-9-hydroxyphomapyrone C] (1)、尿嘧啶 (2)、尿苷 (3)、2'-去氧尿苷 (4)、腺苷 (5)、2'-去氧腺苷 (6)、cordysinin B (7)、麦角甾醇 (8)、麦角甾-5α, 8β-环二氧-6, 22-二烯-3β-醇 (9) 和 (22E, 24S)-3α-羟基-24-甲基胆甾-5, 8, 22-三烯-7-酮 (10), 其中化合物1为新化合物。
化合物1 黄色油状物, [α]D20+5.05 (c 0.184, MeOH); HR-ESI-MS, [M+H]+ m/z 183.101 20 (计算值183.101 57), 确定其分子式为C10H15O3, 不饱和度为4; IR (vmax) 3 394, 3 187, 2 919, 2 850, 1 646, 1 468, 1 420, 1 261, 1 105, 1 033, 802, 721, 648, 548 cm−1, 表示结构中存在羟基、碳基和双键基团。UV (MeOH) λmax (log ε) 202 (3.53), 220 (3.40), 299 (3.66) nm, 为典型的α-吡喃酮母核吸收特征[17]。
化合物1的1H NMR谱中 (CD3OD, 600 MHz) 显示出2个相互偶合的烯烃质子信号δH 7.28 (1H, dq, J = 6.6, 1.2 Hz, H-4)、6.13 (1H, d, J = 6.6 Hz, H-5), 结合13C NMR中166.3 (C-2)、123.4 (C-3)、142.4 (C-4)、103.8 (C-5)、168.0 (C-6), 以及紫外吸收特征, 提示结构中含有共轭的α-吡喃酮结构[17]。此外, 1H NMR谱中还显示出2个甲基质子信号δH 1.25 (3H, d, J = 6.6 Hz, H3-10) 和2.03 (3H, J = 1.2 Hz, H3-11), 2个亚甲基信号δH 1.90, 1.70 (各1H, m, H2-8) 和3.54 (2H, m, H2-9), 以及一个次甲基质子信号δH 2.70 (1H, m, H-7)。通过HMQC对化合物1的1H和13C NMR进行准确归属后 (表 1), 其结构通过1H-1H COSY和HMBC进行确证 (图 1)。在化合物1的1H-1H COSY谱中, 除了显示出H-4与H-5的相关信号外, 还显示出H3-11与H-4的远程相关和H3-10/H-7/H2-8/H2-9的偶合片段, 结合HMBC谱中H3-11与C-2、C-4的相关, H-4与C-2、C-6、C-11的相关, H-5与C-3、C-7的相关, H-7与C-5、C-9的相关, H2-8与C-6、C-10的相关, H2-9与C-7的相关, H3-10与C-6、C-8的相关, 结合这些质子与碳的化学位移以及分子组成, 确定化合物1结构为6-(3-正丁醇基)-二氢-吡喃-2-酮[6-(3-butanol)-2H-pyran-2-one], 为4-deoxyphomapyrone C的9-羟基衍生物[18]。
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Table 1 1H (600 MHz) and 13C (150 MHz) NMR data of compound 1(CD3OD) |
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Figure 1 The structure (left) and key COSY (right, bold lines) and HMBC (right, arrows) correlations of compound 1 |
化合物1显示出正的旋光值[α]D20 +5.05 (c 0.184, MeOH), 其CD [CD (MeOH) 233 (Δε +0.058), 291 (Δε +0.112)]谱在291 nm处呈正的Cotton效应, 通过与4-deoxyphomapyrone C对比, 确定化合物1 C-7的绝对构型为7S构型[18]。因此, 最后确定化合物1的结构为 (+)-7S-4-脱氧-9-羟基茎点菌吡喃酮C [(+)-7S-4-deoxy-9-hydroxyphomapyrone C], 为一新化合物。
采用平板纸片法, 以耻垢分枝杆菌为检测菌对化合物1~10的抗菌活性进行初步筛选。待测样品均未产生明显的抑菌斑。待测样品对其他真菌、细菌和病毒的相关活性还有待进一步研究。
实验部分Nicolet 5700型傅里叶变换红外光谱仪, Perkin-Elmer 343型旋光仪, AV-Ⅲ-500型、Inova-500型和Inova-600型核磁共振光谱仪, Agilent 1000 Series LC-MSD-Trap-SL型质谱仪, JASCO J-815型圆二色散光谱仪, Agilent HP1100型高效液相色谱仪, LC-UV 100 PLUS紫外检测器 (上海伍丰科学仪器有限公司), LC-3000高压输液泵 (创新通恒公司), Rp C18 (5 μm, 250 mm × 10 mm) (美国ES公司), Alltima Silica 10 μm (GRACE公司), Ultimate XB-CN (5 μm, 250 mm × 10 mm), Búchi Gradient Former B-687中压液相色谱仪, Rp C18, 43~60 μm (Pharmacia公司), CombiFlash Rp C18 (40~60 μm Micron Silicag Gel) 组合闪式色谱仪 (Isco公司), 羟丙基葡聚糖凝胶Sephadex LH-20 (瑞典Amersham Biosciences公司生产), CHP20/P120 MCI GEL, 硅胶柱色谱:硅胶 (200~300目) 和薄层色谱用硅胶GF254 (青岛海洋化工厂生产)。所用试剂和溶剂若无特别说明均购自北京化工厂, 级别为分析纯或色谱纯, 未经处理直接使用。
健康结香的采集鉴定以及芬芳镰刀菌纯菌种的分离由浙江工业大学章华伟教授完成[7], 菌种采用液氮保藏法保藏。
1 菌种的分子生物学鉴定菌种的鉴定采用ITS 4通用引物, 用MEGA 4构建分子进化树, 采用Neighbor Joining法的Complete Deletion模式建树, Bootstrap进行检验, 并重复1 000次。由北京三博远志生物技术有限公司提供技术支持。
2 菌种的固体发酵 2.1 液体种子制备培养液配方为马铃薯200 g、酵母膏10 g、蛋白胨6 g、葡萄糖30 g、KHP2O4 2 g、MgSO4 0.5 g, 定容至1 L, 调pH 6.8。分装至500 mL三角瓶, 每瓶约装150 mL, 115 ℃、68 kPa灭菌30 min。接种后于28 ℃、155 r·min−1培养3天。培养液稀释100倍后做种子液备用。
2.2 固体发酵方法每1 L三角瓶装大米150g、纯净水180 mL。115 ℃、68 kPa灭菌30 min。每瓶加种子液20 mL, 室温培养40天。
3 提取分离每瓶固体发酵物加500 mL乙酸乙酯浸泡后超声30 min, 重复3遍, 得总浸膏54 g。总浸膏经反相中压柱色谱, 甲醇 (5%~95%) 梯度洗脱得组分A~D。组分A经Sephadex LH-20柱色谱 (30%甲醇−水) 洗脱, 得组分A1~A4, 组分A3经HPLC半制备色谱纯化 (ES-C18色谱柱, 2.5%甲醇−水, 254 nm), 得化合物2 (2.0 mg, 14 min)、3 (1.5 mg, 25 min) 和4 (0.5 mg, 29 min)。组分B经MCI梯度洗脱 (10%、30%、50%、100%甲醇−水), 得组分B1~B4。组分B2经Sephadex LH-20柱色谱 (30%甲醇−水) 洗脱, 得组分B2A~B2E, 组分B2E经HPLC半制备色谱纯化 (Ultimate XB-CN, 6.0%甲醇−水, 254 nm), 得组分B2E1~B2E5, B2E1~B2E3分别经HPLC再纯化得化合物5 (ES-C18色谱柱, 5%乙腈−水, 18 min, 2.4 mg, 254 nm)、6 (Ultimate XB-CN, 5 μm, 2%乙腈−水, 19 min, 2.2 mg, 254 nm) 和7 (Ultimate XB-CN, 5%乙腈−水, 22 min, 1.3 mg, 254 nm)。组分B3经Flash柱色谱 (C18, 10%~18%甲醇−水梯度洗脱), 得组分B3A~B3D。B3D经HPLC制备色谱纯化 (ES-C18色谱柱, 26%甲醇−水, 280 nm), 得化合物1 (2.3 mg, 35 min)。组分D中析出针状结晶, 过滤后用色谱纯甲醇重结晶得化合物8 (80 mg)。组分D经硅胶柱色谱 (20:1, 10:1, 8:1, 5:1, 3:1石油醚−丙酮梯度洗脱), 得组分D1~D11。组分D9中析出白色固体, 过滤后用色谱纯甲醇重结晶得化合物9 (20 mg)。D9母液经HPLC半制备色谱纯化 (Ultimate XB-CN, 6.0%甲醇−水, 254 nm), 得化合物10 (2.5 mg, 44 min)。
4 结构鉴定化合物1 黄色油状物, [α]D20+5.05 (c 0.184, MeOH); IR (vmax) 3 394, 3 187, 2 919, 2 850, 1 646, 1 468, 1 420, 1 261, 1 105, 1 033, 802, 721, 648, 548 cm−1; UV (MeOH) λmax (log ε) 202 (3.53), 220 (3.40), 299 (3.66) nm; HR-ESI-MS, [M+H]+ m/z 183.101 20 (计算值183.101 57); 1H NMR、13C NMR数据见表 1。
化合物2 白色粉末, ESI-MS m/z 113 [M+H]+; 1H NMR (DMSO-d6, 500 MHz) δ: 10.89 (2H, br s, H-1, 3), 7.37 (1H, d, J = 7.6 Hz, H-6), 5.44 (1H, d, J = 7.6 Hz, H-5)。以上数据与文献[19]报道的尿嘧啶的数据一致。
化合物3 白色粉末, ESI-MS m/z 225 [M+H]+; 1H NMR (DMSO-d6, 600 MHz) δ: 7.89 (1H, d, J = 8.1 Hz, H-6), 5.78 (1H, d, J = 5.4 Hz, H-1'), 5.64 (1H, d, J = 8.1 Hz, H-5), 4.02 (1H, m, H-2'), 3.96 (1H, m, H-3'), 3.84 (1H, m, H-4'), 3.62 (1H, dd, J = 12.0, 3.1 Hz, H-5'a), 3.55 (1H, dd, J = 12.0, 3.1 Hz, H-5'b)。以上数据与文献[19]报道的尿苷的数据一致。
化合物4 白色粉末, ESI-MS m/z 229 [M+H]+; 1H NMR (DMSO-d6, 600 MHz) δ: 7.85 (1H, d, J = 8.1 Hz, H-6), 6.15 (1H, dd, J = 6.0, 8.0 Hz, H-1'), 5.63 (1H, d, J = 8.1 Hz, H-5), 4.23 (1H, m, H-3'), 3.78 (1H, m, H-4'), 3.57 (1H, dd, J = 12.0, 3.6 Hz, H-5'a), 3.53 (1H, dd, J = 12.0, 3.6 Hz, H-5'b), 2.26 (1H, m, H-2'a), 2.08 (1H, m, H-2'b); 13C NMR (DMSO-d6, 150 MHz) δ: 150.4 (C-2), 163.1 (C-4), 101.8 (C-5), 140.5 (C-6), 84.1 (C-1'), 40.1 (C-2'), 70.4 (C-3'), 87.4 (C-4'), 61.3 (C-5')。以上数据与文献[20]报道的2'-去氧尿苷数据一致。
化合物5 黄色粉末, ESI-MS m/z 268 [M+H]+; 1H NMR (DMSO-d6, 600 MHz) δ: 8.34 (1H, s, H-2), 8.13 (1H, s, H-8), 5.87 (1H, d, J = 6.2 Hz, H-1'), 4.59 (1H, m, H-2'), 4.13 (1H, m, H-3'), 3.95 (1H, m, H-4'), 3.66 (1H, dd, J = 12.1, 3.6 Hz, H-5'a), 3.54 (1H, dd, J = 12.1, 3.6 Hz, H-5'b); 13C NMR (DMSO-d6, 150 MHz) δ: 152.4 (C-2), 149.1 (C-4), 119.4 (C-5), 156.2 (C-6), 139.9 (C-8), 87.9 (C-1'), 73.5 (C-2'), 70.6 (C-3'), 85.9 (C-4'), 61.7 (C-5')。以上1H NMR数据与文献[21]报道的腺苷数据一致。
化合物6 黄色粉末, ESI-MS m/z 250 [M−H]+; 1H NMR (DMSO-d6, 600 MHz) δ: 8.32 (1H, s, H-2), 8.18 (1H, s, H-8), 6.43 (1H, dd, J = 8.0, 6.0 Hz, H-1'), 4.58 (1H, m, H-3'), 4.07 (1H, m, H-4'), 3.84 (1H, dd, J = 12.3, 3.8 Hz, H-5'a), 3.74 (1H, dd, J = 12.3, 3.8 Hz, H-5'b), 2.81 (1H, m, H-2'a), 2.41 (1H, m, H-2'b); 13C NMR (DMSO-d6, 150 MHz) δ: 153.5 (C-2), 150.0 (C-4), 120.9 (C-5), 157.5 (C-6), 141.6 (C-8), 89.9 (C-1'), 41.6 (C-2'), 73.1 (C-3'), 87.2 (C-4'), 63.6 (C-5')。以上1H NMR数据与文献[22]报道的2'-去氧腺苷数据一致。
化合物7 白色固体, ESI-MS m/z 282 [M+H]+; 1H NMR (DMSO-d6, 600 MHz) δ: 8.34 (1H, s, H-2), 8.19 (1H, s, H-8), 6.06 (1H, d, J = 6.1 Hz, H-1'), 4.49 (1H, m, H-2'), 4.43 (1H, m, H-3'), 4.16 (1H, m, H-4'), 3.89 (1H, dd, J = 12.5, 2.8 Hz, H-5'a), 3.75 (1H, dd, J = 12.5, 2.8 Hz, H-5'b); 13C NMR (DMSO-d6, 150 MHz) δ: 153.7 (C-2), 150.0 (C-4), 121.0 (C-5), 157.6 (C-6), 141.9 (C-8), 89.2 (C-1'), 84.6 (C-2'), 58.8 (C-2' OCH3), 70.9 (C-3'), 88.4 (C-4'), 63.2 (C-5')。以上数据与文献[23]报道的cordysinin B数据一致。
化合物8 无色针晶, EI-MS m/z 397 [M+H]+; 1H NMR (Acetone-d6, 500 MHz) δ: 5.52 (1H, m, H-6), 5.36 (1H, m, H-7), 5.25 (1H, m, H-23), 5.21 (1H, m, H-22), 3.50 (1H, m, H-3), 1.05 (6H, d, J = 6.6 Hz, H-21, H-28), 0.94 (3H, s, H-19), 0.86 (3H, d, J = 6.8 Hz, H-27), 0.83 (3H, d, J = 6.8 Hz, H-26), 0.66 (3H, s, H-18); 13C NMR (Acetone-d6, 125 MHz) δ: 39.9 (C-1), 32.9 (C-2), 70.3 (C-3), 41.8 (C-4), 141.4 (C-5), 120.0 (C-6), 117.4 (C-7), 141.6 (C-8), 47.2 (C-9), 37.9 (C-10), 21.8 (C-11), 39.3 (C-12), 43.8 (C-13), 55.3 (C-14), 23.7 (C-15), 29.0 (C-16), 56.5 (C-17), 12.4 (C-18), 16.6 (C-19), 41.3 (C-20), 21.6 (C-21), 132.7 (C-22), 136.6 (C-23), 43.6 (C-24), 33.9 (C-25), 20.0 (C-26), 20.3 (C-27), 18.1 (C-28)。以上1H NMR和13C NMR数据与文献[24]报道的麦角甾醇数据一致。
化合物9 无色针晶, EI-MS m/z 429 [M+H]+; 1H NMR (CDCl3, 500 MHz) δ: 6.50 (1H, d, J = 8.5 Hz, H-7), 6.24 (1H, d, J = 8.5 Hz, H-6), 5.22 (1H, dd, J = 15.3, 7.5 Hz, H-23), 5.14 (1H, dd, J = 15.3, 8.3 Hz, H-22), 3.97 (1H, m, H-3), 1.00 (3H, d, J = 6.6 Hz, H-28), 0.91 (3H, d, J = 6.8 Hz, H-21), 0.88 (3H, s, C-19), 0.84 (3H, s, C-18), 0.82 (3H, d, J = 4.5Hz, H-26), 0.81 (3H, d, J = 3.0 Hz, H-27); 13C NMR (CDCl3, 125 MHz) δ: 37.1 (C-1), 30.3 (C-2), 66.6 (C-3), 34.8 (C-4), 82.3 (C-5), 135.3 (C-6), 130.9 (C-7), 79.6 (C-8), 51.2 (C-9), 37.1 (C-10), 20.8 (C-11), 39.5 (C-12), 44.7 (C-13), 51.8 (C-14), 23.6 (C-15), 28.8 (C-16), 56.3 (C-17), 13.0 (C-18), 18.3 (C-19), 39.9 (C-20), 21.0 (C-21), 135.5 (C-22), 132.4 (C-23), 42.9 (C-24), 33.2 (C-25), 19.8 (C-26), 20.1 (C-27), 17.7 (C-28)。以上1H NMR和13C NMR数据与文献[24]报道的麦角甾-5α, 8β-环二氧-6, 22-二烯-3β-醇数据一致。
化合物10 无色晶体, EI-MS m/z 411 [M+H]+; 1H NMR (CD3OD, 500 MHz) δ: 6.01 (1H, br s, H-6), 5.25 (1H, dd, J = 15.4, 7.9 Hz, H-22), 5.21 (1H, dd, J = 15.4, 7.9 Hz, H-23), 3.55 (1H, m, H-3), 1.38 (3H, s, C-19), 1.08 (1H, d, J = 6.6 Hz, H-21), 0.95 (3H, d, J = 6.6 Hz, H-28), 0.87 (3H, d, J = 6.7 Hz, H-27), 0.85 (3H, d, J = 6.7 Hz, H-26), 0.69 (3H, s, C-18); 13C NMR (CD3OD, 125 MHz) δ: 35.9 (C-1), 31.3 (C-2), 72.7 (C-3), 42.8 (C-4), 166.3 (C-5), 126.6 (C-6), 188.4 (C-7), 134.6 (C-8), 165.3 (C-9), 43.7 (C-10), 25.8 (C-11), 36.7 (C-12), 43.5 (C-13), 49.7 (C-14), 25.9 (C-15), 30.6 (C-16), 54.7 (C-17), 12.3 (C-18), 24.3 (C-19), 41.7 (C-20), 21.6 (C-21), 137.0 (C-22), 133.3 (C-23), 44.4 (C-24), 34.4 (C-25), 20.1 (C-26), 20.5 (C-27), 18.2 (C-28)。以上1H NMR和13C NMR数据与文献[25]报道的 (22E, 24S)-3α-羟基-24-甲基胆甾-5, 8, 22-三烯-7-酮数据一致。
5 抗耻垢分枝杆菌活性的初步筛选采用平板纸片法 (K-B法), 将耻垢分枝杆菌按1.5%接种于分枝杆菌培养基中, 倒平板。将10 μL (10 mmol·L−1) 待测样品加于灭菌的纸片上, 风机吹干后贴于平板上, 37 ℃培养24 h后观测[26], 肉眼可见抑菌圈即为阳性样品。
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