赭曲霉次生代谢产物研究

Study on the Secondary Metabolites of Aspergillus ochraceus

摘要: 目的：研究赭曲霉次生代谢产物。方法：利用多种色谱层析方法分离出次生代谢产物，通过ESI-MS/MS及NMR等波谱方法鉴定结构。结果：共分离鉴定出9种化合物，依次是赭曲霉毒素A(1)、赭曲霉毒素B(2)、neohydroxyaspergillic acid(3)、(3R)-5-hydroxymellein(4)、5,6-dihydropenicillic acid(5)、mesaconic acid(6)、p-hydroxybenzoic acid(7)、circumdatin G(8)、(22E,24R)-ergosta-7,22-diene-3β,5α,6β- triol(9)。结论：化合物3~9为首次从赭曲霉中分离得到的化合物。

Abstract: Objective: To study the secondary metabolites from Aspergillus ochraceus. Methods: The constit-uents were isolated and purified by column chromatography and preparative TLC. Their structures were identified on the basis of comprehensive spectroscopic methods including ESI-MS and spectral data (1H, 13C-NMR). Results: Nine compounds were isolated and identified as ochratoxin A (1), ochratoxin B (2), neohydroxyaspergillic acid (3), (3R)-5-hydroxymellein (4), 5,6-dihydro- penicillic acid (5), mesaconic acid (6), p-hydroxybenzoic acid (7), circumdatin G (8), (22E,24R)- ergosta-7,22-diene-3β,5α,6β-triol (9). Conclusion: The compounds 3~9 were isolated from Asper-gillus ochraceus for the first time.

1. 引言

赭曲霉毒素具有肾毒、致癌、致畸、免疫抑制等毒性，主要由赭曲霉和青霉菌产生，包括赭曲霉毒素A、B、C、D等，其中赭曲霉毒素A的毒性最强 [1] [2] [3] [4] 。我们筛选了赭曲霉高产菌株Aspergillus ochraceus 53216，通过大米发酵培养获得培养物，应用各种硅胶柱层析、反相中压色谱和半制备高效液相色谱分离得到15个化合物，采用现代波谱方法鉴定了其中的9个化合物，包括赭曲霉毒素A(1)、赭曲霉毒素B(2)、neohydroxyaspergillic acid(3)、(3R)-5-hydroxymellein(4)、5,6-dihydropenicillic acid(5)、mesaconic acid(6)、p-hydroxybenzoic acid(7)、circumdatin G(8)、(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol(9)。结构式见图1。

2. 结果与讨论

化合物结构鉴定

赭曲霉毒素A(Ochratoxin A, OTA)(1)ESI-MS: m/z 426 [M + Na]⁺, 分子式为C₂₀H₁₈ClNO₆; ¹H NMR (CDCl₃, 600MHz) δ: 8.38 (1H, s, H-6), 7.22-7.31 (5H, m, H-16~20), 4.88 (1H, m, H-13), 4.76 (1H, m, H-3), 3.30-3.34 (1H, m, H-14a), 3.30 (1H, m, H-4a), 3.16-3.20 (1H, m, H-14b), 2.82 (1H, m, H-4b), 1.58 (3H, d, J = 6.0 Hz, H-21); ¹³C NMR (CDCl₃, 150MHz) δ: 174.4 (C-22), 169.8 (C-1), 162.7 (C-11), 159.0 (C-8), 140.8 (C-5), 138.8 (C-6), 136.8 (C-15), 129.5 (C-16,20), 128.5 (C-17,19), 127.0 (C-18), 123.2 (C-10), 120.8 (C-7), 110.2 (C-9), 76.0 (C-3), 55.1 (C-14), 37.8 (C-14), 32.4 (C-4), 20.7 (C-21)。以上数据与文献 [5] 报道的一致，故确定该化合物为赭曲霉毒素A。

赭曲霉毒素B(Ochratoxin B, OTB)(2)ESI-MS: m/z 370 [M + H]⁺, 392 [M + Na]⁺, 761 [2M + Na]⁺, 分子式为C₂₀H₁₉NO₆; ¹H NMR (CDCl₃，600 MHz) δ: 8.32 (1H, d, J = 7.8 Hz, H-7), 7.21~7.29 (5H, m, H-17~21), 6.83 (1H, d, J = 8.4 Hz, H-6), 4.99 (1H, m, H-3), 4.76 (1H, td, J = 6.0, 4.2 Hz, H-14), 3.32 (1H, dd, J = 14.4, 5.4 Hz, H-15b), 3.19 (1H, J = 13.8, 6.6 Hz, H-15a), 2.99 (1H, m, H-4b), 2.97 (1H, m, H-4a), 1.55 (3H, d, J = 6.0 Hz, H-11); ¹³C NMR (CDCl₃，150MHz) δ: 173.4 (C-22), 170.3 (C-1), 164.2 (C-12), 160.4 (C-9), 143.9 (C-5),

Figure 1. Compounds isolated from ferment of Aspergillus ochraceus 53216

图1. 从Aspergillus ochraceus 53216发酵培养物中分离得到的化合物

138.9 (C-16), 136.4 (C-7), 129.5 (C-17,21), 128.6 (C-18,20), 127.1 (C-19), 119.2 (C-6), 118.7 (C-8), 108.8 (C-10), 76.4 (C-3), 54.3 (C-14), 37.7 (C-15), 34.7 (C-4), 20.7 (C-11)。以上数据与文献 [6] 报道的一致，故确定该化合物为赭曲霉毒素B。

neohydroxyaspergillic acid(3)ESI-MS: m/z 241 [M + H]⁺，263 [M +Na]⁺，239 [M – H]^–，479 [2M – H]^– , 分子式C₁₂H₂₀N₂O₃; ¹H NMR (CDCl₃, 600MHz) δ: 7.56 (1H, s, H-5), 4.61 (1H, d, J = 6.6 Hz, H-1′′), 2.74 (2H, d, J = 7.2 Hz, H-1′), 2.28 (1H, m, H-2′′), 2.22 (1H, m, H-2′), 1.04 (3H, d, J = 7.2 Hz, H-3′′), 0.95 (9H, d, J = 6.6 Hz, H-3′, 4′, 4′′); ¹³C NMR (CDCl₃, 150MHz) δ: 152.9 (C-2), 151.5 (C-3), 136.7 (C-6), 124.4 (C-5) , 73.8 (C-1′′), 42.0 (C-1′), 32.4 (C-2′′), 27.3 (C-2′), 22.7 (C-3′,4′), 19.6 (C-3′′), 17.5 (C-4′′). 以上数据与文献 [7] [8] 报道的一致，故确定该化合物为neohydroxyaspergillic acid。

(3R)-5-hydroxymellein(4)ESI-MS: m/z 195 [M + H]⁺, 分子式C₁₀H₁₀O₄；¹H NMR (DMSO-d₆, 600MHz) δ: 10.39 (1H, s, 5-OH), 9.33 (1H, s, 8-OH), 7.06 (1H, d, J = 9.0 Hz, H-7), 6.72 (1H, d, J = 8.4 Hz, H-6), 4.73 (1H, m, H-3), 3.07 (1H, dd, J = 16.8, 3.6 Hz, H-4a), 2.60 (1H, dd, J = 16.8, 11.4 Hz, H-4b), 1.42 (3H, d, J = 6.6 Hz, 3-CH₃); ¹³C NMR (DMSO-d₆, 150MHz) δ: 169.5 (C-4), 153.9 (C-5), 145.5 (C-8), 124.5 (C-9), 123.9 (C-6), 115.1 (C-7), 108.0 (C-10), 75.9 (C-2), 28.0 (C-1), 20.4 (2-CH₃). 以上数据与文献 [9] [10] 报道的一致，故确定该化合物为3R-5-Hydroxymellein。

5,6-dihydropenicillic acid(5)ESI-MS: m/z 195 [M + Na]⁺, 155 [M + H–H₂O]⁺, 分子式C₈H₁₂O₄; ¹H NMR (CD₃OD, 600MHz) δ: 5.02 (1H, s, H-2), 4.83 (3H, 3-OCH₃), 2.12 (1H, m, H-5), 1.02, 0.91 (6H, d, J = 7.2 Hz, 2×CH₃); ¹³C NMR (CD₃OD, 150MHz) δ: 182.4 (C-3), 173.7 (C-1), 107.5 (C-4), 90.3 (C-2), 60.6 (3-OCH₃), 49.6 (C-5), 34.9 (C-6,7). 以上数据与文献 [11] [12] 报道的一致，故确定该化合物为5,6-dihydropenicillic acid。

Mesaconic acid(6)¹H NMR (DMSO-d₆, 600MHz) δ: 10.98 (-OH), 10.56 (OH), 7.24 (1H, s), 1.72 (3H, s, CH₃); ¹³C NMR (DMSO-d₆, 150MHz) δ: 164.9, 151.4, 137.6, 107.6, 11.7. 以上数据与文献 [13] 报道的Mesaconic acid一致，故确定该化合物为Mesaconic acid。

对羟基苯甲酸(p-hydroxybenzoic acid)(7) ¹H NMR (CD₃OD, 600MHz) δ: 7.88 (2H, d, J = 8.4 Hz, H-2, 6), 6.82 (2H, d, J = 8.4 Hz, H-3, 5); ¹³C NMR (CD₃OD, 150MHz) δ: 170.4 (C-7), 163.5 (C-4), 133.2 (C-2, 6), 123.0 (C-1), 116.2 (C-3, 5). 以上数据与文献 [14] 报道的对羟基苯甲酸一致，故确定该化合物为对羟基苯甲酸。

circumdatin G(8)ESI-MS: m/z 308 [M + H]⁺, 330 [M + Na]⁺, 346 [M + K]⁺, 637 [2M + Na]⁺, 653 [2M + K]⁺, 306 [M – H]^–, 613 [2M – H]^–, 分子式C₁₇H₁₃N₃O₃; ¹H NMR (DMSO-d₆, 600MHz) δ: 8.75 (1H, d, J = 6.0 Hz, NH), 7.77 (1H, dd, J = 7.8, 1.2 Hz, H-4), 7.63 (1H, m, H-5), 7.56 (3H, m, H-6, 7, 12), 7.39 (1H, dd, J = 7.8, 7.8 Hz, H-13), 7.29 (1H, dd, J = 7.8, 1.2 Hz, H-14), 4.27 (1H, m, H-19), 1.57 (3H, d, J = 6.6 Hz, H-20); ¹³C NMR (DMSO-d₆, 150MHz) δ: 166.7 (C-2), 131.3 (C-3), 128.9 (C-4), 130.6 (C-5), 128.7 (C-6), 128.7 (C-7), 133.2 (C-8), 161.1 (C-10), 121.9 (C-11), 116.6 (C-12), 128.0 (C-13), 119.4 (C-14), 152.9 (C-15), 134.8 (C-16), 154.8 (C-18), 49.7 (C-19), 14.9 (C-20). 以上数据与文献 [15] 报道的一致，故确定该化合物为circumdatin G.

(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol(9) ESI-MS: m/z 453 [M + Na]⁺, 分子式C₂₈H₄₆O₃；¹H NMR (Pyridine-d₅, 600 MHz) δ: 5.73 (1H, m, H-7), 5.24 (1H, dd, J = 15.0, 7.8 Hz, H-22), 5.18 (1H, dd, J = 15.0, 8.4 Hz, H-23), 4.82 (1H, m, H-3), 4.31 (1H, br s, H-6), 1.53 (3H, s, H-19), 1.05 (3H, d, J = 6.6 Hz, H-27), 0.94 (3H, d, J = 6.6 Hz, H-28), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.86 (3H, d, J = 6.6 Hz, H-21), 0.65 (3H, d, J = 6.6 Hz, H-18); ¹³C NMR (Pyridine-d₅, 150MHz) δ: 33.0 (C-1), 34.2 (C-2), 68.0 (C-3), 42.4 (C-4), 76.5 (C-5), 74.6 (C-6), 120.9 (C-7), 141.9 (C-8), 44.1 (C-9), 38.4 (C-10), 22.8 (C-11), 40.3 (C-12), 44.1 (C-13), 55.6 (C-14), 23.8 (C-15), 28.8 (C-16), 56.5 (C-17), 12.9 (C-18), 19.2 (C-19), 41.2 (C-20), 20.2 (C-21), 136.6 (C-22), 132.5 (C-23), 43.4 (C-24), 33.7 (C-25), 20.5 (C-26), 21.8 (C-27), 18.2 (C-28). 以上数据与文献 [16] 报道的一致，故确定该化合物为(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol.

3. 实验部分

3.1. 仪器与试剂

Perkin-Elmer 341 旋光仪；Shimadzu UV-160A 紫外光谱仪(Shimadzu Corporation, Japan); Shimadzu FTIR-8400S红外光谱仪；LTQ Orbitrap XL质谱仪 (Thermo Scientific, America); Bruker AV-600核磁共振仪(600 MHz for ¹H and 150 MHz for ¹³C) (Bruker Biospin Inc., Germany), tetramethylsilane (TMS)为内标。硅胶 (100-200, 200-300目, Qingdao Marine Chemistry Ltd., China)； 凝胶Sephadex LH-20 (20-100 µ, Pharmacia)；薄层硅胶 GF₂₅₄ plates (Yantai Marine Chemical Co., Ltd., China)；制备 HPLC (LUMTECH instrument with a UV detector at 210 nm and using a YMC-Pack C₁₈ column) (250 mm × 20 mm inside diameter (I.D.), 5 μm, YMC, Japan)；试剂均为分析纯(北京化工厂)。

3.2. 菌种培养

赭曲霉菌株(Aspergillus ochraceus 53216)由中科院微生物所提供。采用发酵培养，大米培养基(糙米:水 1:1)，25℃培养1个月。

3.3. 提取分离

对赭曲霉的大米培养物(8 kg)进行粉碎，用80%的乙醇溶液加热回流提取4次，每次提取时间为2小时，合并提取液减压浓缩至无醇味时，有不溶物析出、过滤，滤液转入分液漏斗中，采用等体积乙酸乙酯(含少量甲醇)进行萃取，萃取四次后，合并萃取液，回收溶剂得到乙酸乙酯萃取物(50 g)。对不溶物(40 g)进行硅胶柱层析，石油醚/乙酸乙酯系统(100:0-0:100)洗脱，薄层检测含有毒素的流份合并入乙酸乙酯相，

Figure 2. Experimental flow chart

图2. 实验流程图

氯仿/甲醇(100:0-0:100)系统洗脱，合并相同流份，得到AOY A~E5个部分。AOYB采用硅胶柱层析，石油醚/丙酮(10:1-0:1)系统洗脱，洗脱前段下来的是一些脂肪状的物质，后面洗脱的流份经薄层检查合并为 a~e 5个部分。AOYB b经过反相中压ODS柱层析，甲醇/水系统(30%~100%)洗脱，采用凝胶柱层析，甲醇洗脱得到化合物3 (5 mg)、4 (6 mg)、1和2的粗品。1和2的粗品经过甲醇重结晶得到化合物1 (4 mg)和2 (3 mg)。AOYB d经过反相中压和凝胶柱层析得到化合物5 (7 mg)、6 (10 mg)和7 (9 mg)。AOYC经过反相中压、半制备高效液相得到化合物8 (4 mg)和9 (5 mg)。流程图见图2。

基金项目

资助信息：湖南省高校产业化培育项目(项目编号：11CY013)；民族药用植物资源研究与利用湖南省重点实验室开放基金项目(项目编号：YYZW2016-11)。