Shao-Hua Wu

Chemical Constituents and Bioactivities of Plants from the Genus Paeonia

National Natural Science Foundation of China [20772105]; Fund of Yunnan Province for Young and Middle-aged Talents in Science and Technology [2008PY028]

Ten-Membered Lactones from Phomopsis sp., an Endophytic Fungus of Azadirachta indica

Four new 10-membered lactones ( 1- 4) and one known one ( 5) were isolated from the broth extract of an endophytic fungus, Phomopsis sp., obtained from the stem of Azadirachta indica. Their structures were assigned by analysis of spectroscopic data, and the structures of 1 and 4 were also confirmed by X-ray analysis. Compounds 1- 5 were tested for antifungal activity against several plant pathogens. Compound 4 demonstrated antifungal activity in the MIC value range 31.25-500 microg/mL.

Two New Solanapyrone Analogues from the Endophytic Fungus Nigrospora sp. YB-141 of Azadirachta indica

Two new solanapyrone analogues, solanapyrones N and O (1 and 2, resp.), and three known compounds, solanapyrone C (3), nigrosporalactone (4), and phomalactone (5), were isolated from the fermentation culture of Nigrospora sp. YB-141, an endophytic fungus isolated from Azadirachta indica A. Juss. The structures of the new compounds were elucidated on the basis of spectroscopic analysis. The antifungal activities of 1-5 towards seven phytopathogenic fungi were tested. Most of the compounds exhibited no or only weak antifungal activities.

A new sesquiterpenoid from Ligusticum chuanxiong Hort

A new sesquiterpenoid named (-)-alloaromadendrane-4β,10α,13,15-tetrol was isolated from the rhizome of Ligusticum chuanxiong Hort., together with two known compounds, campest-4-en-3-one and 3-carboxyethyl-phthalide. Their structures were elucidated on the basis of spectroscopic and chemical analysis. All the compounds showed mild antimicrobial activity.

Sesquiterpenoids from the endophytic fungus Trichoderma sp. PR-35 of Paeonia delavayi.

A new bisabolane‐type sesquiterpene, trichoderic acid (1), and a new acorane‐type sesquiterpene, 2β‐hydroxytrichoacorenol (2), along with three known compounds, cyclonerodiol (3), cyclonerodiol oxide (4), and sorbicillin (5), were isolated from the culture broth of Trichoderma sp. PR‐35, an endophytic fungus isolated from Paeonia delavayi. Their structures were elucidated on the basis of their IR, MS, and 1D‐ and 2D‐NMR analyses. The antibacterial and antifungal activities of 1–5 towards various types of bacteria and fungi were tested. Most of the compounds showed moderate or weak antimicrobial activities in an agar‐diffusion assay.

Chemical constituents from the stem bark of Trewia nudiflora

Trewia nudiflora L. is the only member of the genus Trewia (Euphorbiaceae), which is mainly distributed in India, Malaysia, and southwest of China. Previous studies have shown that the seed of T. nudiflora is a rich source of maytansinoid tumor inhibitors [1, 2]. Phytochemical studies are mainly focused on the seed and pericarp of the plant; however, there are only few reports on its stem bark [3, 4]. The air-dried powdered stem bark of T. nudiflora (8.8 kg) was extracted with 95% EtOH three times at room temperature. The EtOH extract was concentrated in vacuum to give a residue. The residue was suspended in water and successively treated with EtOAc. The EtOAc extract (32 g) was subjected to chromatography on silica gel eluting with CHCl3–MeOH gradient (1:0–0:1) to give nine fractions (I–IX). Fraction III was repeatedly subjected to column chromatography on RP-18 silica gel with MeOH-H2O (2:3) and Sephadex LH-20 with MeOH to give compound 1 (9 mg). Repeated chromatography of fraction IV on silica gel with petroleum ether–Me2CO gradient (4:1, 7:3, 6:4) and RP-18 silica gel with MeOH–H2O gradient (2:3, 1:1) afforded compounds 2 (6 mg) and 3 (10 mg). Fraction V was submitted to repeated column chromatography on silica gel with CHCl3–MeOH gradient (15:1, 10:1) and RP-18 silica gel with MeOH–H2O gradient (3:7, 2:3) to afford compounds 4 (13 mg), 5 (18 mg), and 6 (10 mg). Fraction VI was chromatographed on silica gel column with CHCl3–MeOH (8:1) to yield compounds 7 (16 mg) and 8 (13 mg). Trewiasine was isolated from the stem bark of T. nudiflora for the first time. Compounds 2–8 were isolated from this plant for the first time. The structures of these compounds were confirmed using a combination of spectral analyses, including NMR and mass spectrometry, and by comparison with reported spectroscopic data in the literature. Trewiasine (1). C37H52ClN3O11, colorless crystals, mp 180−182°C. ESI-MS m/z: 772 [M+Na]+. 1H NMR (500 MHz, CDCl3, δ, ppm, J/Hz): 2.19 (1H, dd, J = 14.5, 3.1, H-2a), 2.56 (1H, dd, J = 14.5, 12.0, H-2b), 4.77 (1H, dd, J = 12.0, 3.1, H-3), 0.78 (3H, s, 4-CH3), 3.02 (1H, d, J = 9.7, H-5), 1.28 (3H, d, J = 6.3, 6-CH3), 4.29 (1H, m, H-7), 3.53 (1H, d, J = 9.0, H-10), 5.74 (1H, dd, J = 15.1, 9.0, H-11), 6.46 (1H, dd, J = 15.1, 11.2, H-12), 6.99 (1H, d, J = 11.2, H-13), 1.54 (3H, s, 14-CH3), 4.87 (1H, s, H-15), 6.55 (1H, d, J = 1.5, H-17), 7.24 (1H, d, J = 1.4, H-21), 3.35 (3H, s, 10-OCH3), 3.37 (3H, s, 15-OCH3), 4.01 (3H, s, 20-OCH3), 3.18 (3H, s, 18-NCH3), 5.39 (1H, m, H-2′), 1.29 (3H, d, J = 6.8, 2′-CH3), 2.89 (3H, s, 2′-NCH3), 2.79 (1H, m, H-4′), 1.13 (3H, d, J = 6.8, 4′-CH3), 1.08 (3H, d, J = 6.5, 4′-CH3), 6.28 (1H, s, 9-NH). 13C NMR (125 MHz, CDCl3, δ, ppm): 32.3 (C-2), 78.1 (C-3), 59.9 (C-4), 67.4 (C-5), 38.8 (C-6), 74.1 (C-7), 36.0 (C-8), 80.7 (C-9), 85.3 (C-10), 129.6 (C-11), 132.5 (C-12), 127.8 (C-13), 142.0 (C-14), 86.6 (C-15), 141.3 (C-16), 120.2 (C-17), 139.2 (C-18), 119.2 (C-19), 156.2 (C-20), 108.7 (C-21), 176.7, 170.8, 168.7, 152.2 (4 × C=O), 56.3, 56.5, 56.7 (3 × OCH3), 14.5, 13.0, 11.9, 9.9 (4 × CH3), 35.2 (18-NCH3), 30.6 (2′-NCH3), 52.4 (C-2′), 30.4 (C-4′), 19.4, 18.8 (2 × 4′-CH3) [1, 2]. Balanophonin (2). C20H20O6, pale yellow oil. EI-MS (70 eV) m/z (%): 356 [M] + (82), 338 (100), 326 (55), 306 (18), 295 (7), 152 (24), 137 (22), 115 (12), 77 (14). 1H NMR (500 MHz, CD3COCD3, δ, ppm, J/Hz): 7.04 (1H, d, J = 1.9, H-2), 6.81 (1H, d, J = 8.1, H-5), 6.88 (1H, dd, J = 8.1, 1.9, H-6), 5.65 (1H, d, J = 6.7, H-7), 3.64 (1H, m, H-8), 3.85 (2H, d, J = 4.9, H-9), 7.29 (1H, d, J = 1.7, H-2′), 7.31 (1H, d, J = 1.7, H-6′), 7.58 (1H, d, J = 15.8, H-7′), 6.66 (1H, dd, J = 15.8, 7.8, H-8′), 9.63 (1H, d, J = 7.8, H-9′), 3.82 (3H, s, 3-OMe), 3.89 (3H, s, 5′-OMe). 13C NMR (125 MHz, CD3COCD3, δ, ppm): 133.7 (C-1), 110.5 (C-2), 148.4 (C-3), 147.5 (C-4), 115.7 (C-5), 119.7 (C-6), 89.4 (C-7), 54.2 (C-8), 64.1 (C-9), 128.9 (C-1′), 119.6 (C-2′), 131.2 (C-3′), 152.4 (C-4′), 145.6 (C-5′), 113.5 (C-6′), 154.1 (C-7′), 127.1 (C-8′), 193.8 (C-9′), 56.2 (3-OMe), 56.4 (5′-OMe) [5].

Secondary metabolites of endophytic fungus Xylaria sp. YC-10 of Azadirachta indica

0009-3130/11/4705-0858 2011 Springer Science+Business Media, Inc. Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, P. R. China, e-mail: shwu123@126.com. Published in Khimiya Prirodnykh Soedinenii, No. 5, pp. 749–751, September–October, 2011. Original article submitted June 10, 2010. Chemistry of Natural Compounds, Vol. 47, No. 5, November, 2011 [Russian original No. 5, September–October, 2011]

Secondary Metabolites of Endophytic Fungus Trichoderma sp. YM 311505 of Azadirachta indica

Endophytic fungi colonize internal tissues of plants without resulting in overt damage while the tissue is alive. They are important sources to provide a wide variety of structurally unique and biologically potent natural products [1]. In our previous search for secondary metabolites of endophytic fungi from Azadirachta indica, some bioactive and/or new compounds were isolated [2–4]. Trichoderma species have long been studied as biological control agents of phytopathogens and are well known for their ability to produce a wide range of antibiotic substances and for their ability to parasitize other fungi [5]. In our ongoing work, we have studied the secondary metabolites of Trichoderma sp. YM 311505 from A. indica and obtained nine compounds. The fungal strain Trichoderma sp. YM 311505 was isolated from the fruit of A. indica collected in Yuanjiang Country, Yunnan Province, P. R. China. It was classified as a Trichoderma species by its morphological characteristics and ITS rDNA sequence analysis. The strain was deposited in Yunnan Institute of Microbiology, Kunming, P. R. China. The fungus was cultured in 500 mL Erlenmeyer flasks ( 750) containing 200 mL of PDB medium at 200 rpm at 28 C for 6 days on a rotary shaker. The culture broth was filtered to remove mycelia. The filtrate was concentrated in vacuum to 30 L and then extracted with EtOAc (5 30 L). The combined extracts were evaporated to yield 164.5 g of the crude extract, which was subjected to column chromatography on silica gel eluting with petroleum–acetone (9:1–3:7) to afford nine fractions (I–IX). Repeated chromatography of fraction II on silica gel with petroleum ether–EtOAc (5:1) and CHCl3–acetone (95:5) afforded compounds 1 (30 mg), 5 (50 mg), and 6 (4 mg). Fraction III was then subjected to column chromatography on RP-18 silica gel with acetone–H2O (8:2) to give compound 2 (28 mg). Fraction IV was subjected to repeated column chromatography on silica gel with petroleum ether–EtOAc (2:1) and RP-18 silica gel with acetone–H2O (8:2) to afford compound 9 (14 mg). Fraction V was submitted to column chromatography on silica gel with CHCl3–acetone (3:1) to give compound 3 (18 mg) and 8 (11 mg). Fraction VI was subjected to column chromatography on silica gel with petroleum ether–acetone (3:1) and RP-18 silica gel with acetone–H2O (1:1) to yeild compound 4 (10 mg). Fraction VIII was chromatographed on silica gel with CHCl3–MeOH (9:1) to give compound 7 (5 mg).

Secondary Metabolites from an Endophytic Fungus Nigrospora sp.

Endophytic microorganisms are found in virtually every plant on earth. Natural products from endophytic fungi have been observed to inhibit or kill a wide variety of harmful microorganisms including, but not limited to, phytopathogens, as well as bacteria, fungi, viruses, and protozoans that affect humans and animals [1]. Fungi belonging to the genus Nigrospora have been a rich source of bioactive secondary metabolites, such as plant growth-inhibiting nigrosporolide and phomalactone, phytotoxic and antibacterial nigrosporins, and phytotoxic lactones [2]. During our continuous chemical investigation of endophytic fungi, we have studied the secondary metabolites of Nigrospora sp. YE 3033 isolated from Aconitum carmichaelii and obtained nine compounds. The fungal strain Nigrospora sp. YE 3033 was isolated from the root of A. carmichaelii collected in Yunnan Province, P. R. China. It was classified as a Nigrospora species by its morphological characteristics and ITS rDNA sequence analysis. The strain was deposited in Yunnan Institute of Microbiology, Kunming, P. R. China. The fungus was cultured in 1000 mL Erlenmeyer flasks ( 100) containing 250 mL of potato dextrose broth (PDB) medium at 200 rpm at 28 C for 7 days on a rotary shaker. The culture broth was filtered to remove mycelia. The filtrate was concentrated in vacuum to 5 L and then extracted with EtOAc (5 L) five times. The combined extracts were evaporated to yield 26.0 g of the crude extract, which was subjected to column chromatography on silica gel eluted with CHCl3–MeOH gradient (1:0–0:1) to afford six fractions (Fr.1–Fr.6). Repeated chromatography of Fr. 2 on silica gel with petroleum ether–acetone gradient (95:5–1:1) afforded three subfractions: Fr. 2.1–2.3. Fraction 2.1 was chromatographed on silica gel with petroleum ether–EtOAc (8:2) to yield compound 7 (2 mg). Fraction 2.2 was subjected to column chromatography on silica gel with petroleum ether–acetone gradient (8:2, 7:3) to afford compounds 1 (2 mg) and 6 (4 mg). Fraction 2.3 was submitted to column chromatography on silica gel with CHCl3–MeOH (95:5) to give compounds 2 (5 mg), 4 (7 mg), and 5 (2 mg). Fraction 4 was subjected to repeated column chromatography on RP-18 silica gel with MeOH–H2O gradient (3:7–8:2) and silica gel with CHCl3–MeOH gradient (9:1–7:3) to yield compounds 3 (4 mg), 8 (6 mg), and 9 (12 mg). The compounds were investigated by spectroscopic methods, including NMR and mass spectrometry, and were determined as 1,2,8-trihydroxyanthraquinone (1) [3], 1,3,8-trihydroxyanthraquinone (2) [4], rheoemodin (3) [5], 1,3,6-trihydroxy8-methylanthraquinone (4) [6], aloesaponarin II (5) [7], isozyganein (6) [8, 9], 1-acetyl-4,5-dihydroxy-anthraquinone (7) [10], cis-4-hydroxyscytalone (8) [11], and cerebroside B (9) [12]. All the compounds were isolated from the fungal genus Nigrospora for the first time. The minimal inhibition concentration (MIC) values of compounds 2–4, 6, 8, and 9 were determined by the broth microdilution method in 96-well culture plates as described before [13]. The indicator bacteria and fungi included Escherichia coli, Bacillus subtilis, Pyricularia oryzae, and Candida albicans. Each compound was dissolved in a small volume of sterile DMSO and then diluted to give final concentrations ranging from 512.0 to 1 g/mL by serial twofold dilutions. The plates were incubated for 48 h at 28 C. Nystatin and kanamycin were used as positive control for fungi and bacteria, respectively. The MIC value was defined as the lowest concentration of the test compound at which the microorganism did not demonstrate visible growth. Each test was performed in triplicate.

Secondary Metabolites of an Endophytic Fungus Phomopsis sp.

Endophytic fungi are eukaryotic organisms that live in the internal tissues of living plants [1]. They are relatively unstudied potential sources of novel and biologically active natural products [2]. Endophytic Phomopsis strains have gained attention in most cases involving metabolite research, which has led to the discovery of various bioactive secondary metabolites [3]. In the course of our studies on secondary metabolites of endophytic fungi from medicinal plants, two new ten-membered lactones had been reported from a Phomopsis strain [4]. During our ongoing work on endophytic fungi, a fungal strain of Phomopsis sp. YM 355364 was isolated from the traditional Chinese medicinal plant Aconitum carmichaelii Debeaux. We have studied the secondary metabolites of this strain and obtained 17 compounds (1–17). The fungal strain Phomopsis sp. YM 355364 was isolated from the stem of A. carmichaelii collected in Yunnan Province, P. R. China. It was classified as a Phomopsis species by its morphological characteristics and ITS rDNA sequence analysis. The strain was deposited in Yunnan Institute of Microbiology, Kunming, P. R. China. The fungus was cultured in 1000 mL Erlenmeyer flasks ( 200) containing 300 mL of potato dextrose broth (PDB) medium at 200 rpm at 28 C for 7 days on a rotary shaker. The culture broth was filtered, and the filtrate was concentrated in vacuum to 20 L and then extracted with EtOAc (20 L) four times. The combined extracts were evaporated to yield 85.2 g of the crude extract, which was subjected to column chromatography on silica gel eluting with CHCl3–MeOH (10:0–0:10) to afford ten fractions. Fraction 2 was then chromatographed on a silica gel column with petroleum ether–EtOAc (4:1) to afford compounds 5 (8 mg) and 17 (12 mg). Fraction 3 was subjected to column chromatography on RP-18 silica gel with acetone–H2O (45:55–95:5) and silica gel with petroleum ether–EtOAc (85:15–1:1) to afford compounds 1 (8 mg), 2 (2 mg), and 9 (6 mg). Fraction 4 was repeatedly subjected to column chromatography on silica gel with petroleum ether–acetone (8:2–1:1) to yield compounds 6 (4 mg), 7 (8 mg), and 15 (11 mg). Fraction 5 was submitted to column chromatography on silica gel with petroleum ether–acetone (8:2–1:1) and Sephadex LH-20 with MeOH to afford compounds 4 (8 mg) and 8 (13 mg). Fraction 6 was submitted to column chromatography on silica gel with CHCl3–acetone (95:5–7:3) and RP-18 silica gel with MeOH–H2O (1:1–7:3) to give compounds 10 (8 mg) and 13 (5 mg). Fraction 7 was chromatographed on a RP-18 silica gel column eluting with MeOH–H2O (4:6–8:2) to yield compound 11 (12 mg). Fraction 8 was submitted to column chromatography on silica gel with CHCl3–acetone (9:1–1:1) and recrystallized to afford compound 16 (7 mg). Fraction 9 was chromatographed repeatedly on a silica gel column with petroleum ether–acetone (1:1) and Sephadex LH-20 with MeOH to afford compounds 3 (3 mg) and 14 (8 mg). Fraction 10 was submitted to column chromatography on silica gel with CHCl3–MeOH (9:1) to yield compound 12 (8 mg). The structures of compounds were identified by spectroscopic methods, including NMR and mass spectrometry. They were determined to be cyathisterol (1) [5], (24R)-6 -hydroxy-24-ethylcholest-4-en-3-one (2) [6], hydroxyvertixanthone (3) [7], isosclerone (4) [8], 7-methoxy-4,6-dimethylphthalide (5) [9], convolvulol (6) [10], 4-hydroxybenzaldehyde (7) [11], phenylacetic acid (8) [12], 1H-indole-3-carbaldehyde (9) [13], 3-hydroxyindole (10) [14], nicotinic acid (11) [15], adenosine (12) [16], N-hexadecanoylsphinganane (13) [17], 9,10,11-trihydroxy-(12Z)-octadecenoic acid (14) [18], 8-hydroxy-(9E)octadecenoic acid (15) [19], 1-monopalmitin (16) [20], and (2E)-nonadecenoic acid (17) [21]. Compounds 1–5 and 13–15 were isolated from the fungal genus Phomopsis for the first time.