Trichoharzianol, a New Antifungal from Trichoderma harzianum

Article pubs.acs.org/JAFC

Trichoharzianol, a New Antifungal from Trichoderma harzianum F031 Chotika Jeerapong,† Worrapong Phupong,*,† Phuwadol Bangrak,† Warin Intana,‡ and Patoomratana Tuchinda§ †

School of Science, and ‡School of Agricultural Technology, Walailak University, Tha Sala, Nakhon Si Thammarat 80161, Thailand Department of Chemistry and Center of Excellence for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

§

S Supporting Information *

ABSTRACT: A new decalin derivative, trichoharzianol (1), together with three known compounds, eujavanicol A (2), 5hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (3), and 4,6-dihydroxy-5-methylphthalide (4), were isolated from Trichoderma harzianum F031. For the first time, compounds 2−4 were reported from the Trichoderma species. Their structures were characterized by spectroscopic methods. Trichoharzianol (1) showed the highest antifungal activity against Colletotrichum gloeosporioides, with a minimum inhibitory concentration (MIC) of 128 μg/mL. KEYWORDS: Trichoderma harzianum, decalin derivatives, antifungal activity, Colletotrichum gloeosporioides, chromone, phthalide

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INTRODUCTION

General Experimental Procedures. Melting points (uncorrected) were recorded on a digital electrothermal apparatus. Optical rotations were determined on a JASCO DIP 370 digital polarimeter, with the use of a 50 mm microcell (1 mL). Ultraviolet (UV) and infrared (IR) (neat) spectra were recorded on JASCO V-630 and Bruker Tensor 27 spectrophotometers, respectively. The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Bruker Ascend 400 MHz and AV 500 MHz spectrometers in either CDCl3 or acetone-d6, with tetramethylsilane (TMS) as an internal standard. The high-resolution time-of-flight mass spectrometry (HR-TOF-MS) was recorded on a Bruker micromass model VQ-TOF-2 spectrometer. Silica gel 60H (SiO2, Merck, 70−230 mesh ASTM) and Sephadex LH20 (Amersham Biosciences) were used for column chromatography (CC). The thin-layer chromatography (TLC) analysis was performed on silica gel (SiO2, Kieselgel 60 F254), with spots visualized by UV light at 254 or 366 nm and/or stained with p-anisaldehyde solution in 2% H2SO4/EtOH. All solvents used for extraction and isolation were distilled at their boiling point ranges prior to use. Fungal Strains. T. harzianum F031, isolated from soil collected in Suphanburi, Thailand, was deposited at the Tropical Fruit Research Unit, Walailak University. This strain was identified on the basis of its morphological characteristics and the analyses of its nuclear ribosomal internal transcribed spacer (ITS) regions. The 3-day-old fungal mycelia grown in potato dextrose agar (PDA) were collected, and the fungal genomic DNA was extracted with a lysis buffer containing 250 mM Tris−HCl (pH 8.0), 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 100 mM NaCl, and 2% sodium dodecyl sulfate (SDS) according to the protocol developed by Chakraboty et al.29 The ITS regions were amplified from genomic DNA, using a pair of universal primers, ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′), for the region containing ITS1, ITS2, and the 5.8S rDNA.30 The polymerase chain reaction (PCR) was performed through the following cycle: initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C denaturation for 1

Anthracnose is one of the most economically threatening plant diseases worldwide.1 It occurs in a wide range of fruits and is caused by several Colletotrichum species. In particular, anthracnose caused by Colletotrichum gloeosporioides can damage a variety of fruits, such as mango,2 banana,3 papaya,4 and chilli.3 C. gloeosporioides can normally be controlled by treatment with fungicide, but the residues can have drastic effects on the environment and consumers. The use of antagonistic fungi, such as Trichoderma, is an alternative to control this fungus. In addition, it is particularly effective against pathogenic fungi, such as Sclerotinia sclerotiorum,5 Rhizoctonia solani,6 and Sclerotium rolfsii.7 The mechanisms8−10 of action of the antagonistic fungi from the Trichoderma species are competition, antibiosis, mycoparasitism, and induction of systemic resistance to pathogens in plants. Trichoderma species are also well-known producers of secondary metabolites with antibiotic activity.6,7,11−18 In previous studies, the bioactive secondary metabolites from Trichoderma have been found to be anthaquinones, 19 pyrones,20 sesquiterpenoids,21 diterpenoids,22 butenolides,23 alkaloid,24 etc. Isoharziandione25,26 and 6-pentyl-α-pyrone7 are well-studied bioactive metabolities and are commonly isolated from culture broths of Trichoderma spp. Isoharziandione was found to inhibit Colletotrichum capsici26 and S. rolfsii,25 while 6-pentyl-α-pyrone inhibits Pythium ulttimun27 and Armillaria mellea.28 Moreover, 6-pentyl-α-pyrone has also been found to be a plant growth promoter.9 Preliminary studies from our research group indicate that the crude extract of Trichoderma harzianum F031 can inhibit the growth of C. gloeosporioides. Therefore, it would be of interest to phytochemically investigate this extract. This paper presents the isolation of secondary metabolites from T. harzianum F031 and their antifungal activity against C. gloeosporioides. © XXXX American Chemical Society

MATERIALS AND METHODS

Received: March 13, 2015 Revised: March 23, 2015 Accepted: March 29, 2015

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Journal of Agricultural and Food Chemistry Table 1. NMR Spectroscopic Data of Trichoharzianol (1)a δC

DEPT

1 2 3 4 4a 5 6 7

52.7 52.3 123.3 126.9 41.5 79.7 73.6 37.0

C CH CH CH CH CH CH CH2

8 8a 9 10

32.3 40.6 215.7 41.1

CH CH C CH2

11 1′ 2′

58.1 37.1 24.4

CH2 CH CH2

12.4 107.9 26.2 28.6 19.2 22.3 19.3

CH3 C CH3 CH3 CH3 CH3 CH3

position

3′ 4′ 5′ 6′ 1-CH3 8-CH3 1′-CH3 a

δH (Jb in Hz)

COSY

HMBC (correlated C)

1.94 5.65 5.90 1.93 3.72 4.26 2.06 1.64 1.60 1.82

(1H, m) (1H, ddd, J = 10.0, 4.0, 2.0) (1H, dd, J = 10.0, 1.5) (1H, m) (1H, dd, J = 10.0, 4.0) (1H, ddd, J = 6.5, 4.0. 3.0) (1Ha, m) (1Hb, m) (1H, m) (1H, dd, J = 10.5, 9.0)

1′, 3 2, 4 3, 4a 4, 5 4a,6 5, 7a, 7b 6, 7b, 8 6, 7a, 8 7a, 7b, 8-CH3, 8a 4a, 8

2′, 1, 9, 1-CH3, 1′-CH3 1, 4a 3, 5, 2, 8a 3, 5 4′, 6, 4 4a, 8 5, 6, 8a, 8 8a, 8 8-CH3 1, 4a, 7, 8, 1-CH3

2.81 2.65 3.86 1.10 1.43 0.74 0.73

(1Ha, ddd, J = 19.0, 7.0, 4.0) (1Hb, ddd, J = 19.0, 6.0, 4.0) (2H, m) (1H, m) (1Ha, m) (1Hb, m) (3H, d, J = 4.5)

10b, 11 10a, 11 10a, 10b 2, 1′-CH3 1′, 2′b, 3′ 1′, 2′a, 3′ 2′a, 2′b

9, 11 9, 11 9, 10 1′-CH3, 3′ 1′, 1′-CH3, 3′ 1′, 1′-CH3, 3′ 1′, 2′

1.35 1.48 1.21 0.64 0.90

(3H, (3H, (3H, (3H, (3H,

8 1′

4′, 6′ 4′, 5′ 9, 1, 2, 8a 8a, 8, 7 2, 7, 2′, 3′

s) s) s) d, J = 6.5) d, J = 7.0)

Data were recorded in CDCl3 (500 MHz). bJ values of coupling were determined by a homodecoupling experiment.

min, 55 °C annealing for 1 min, 72 °C extension for 2 min, and a final extension at 72 °C for 10 min. The amplified products were purified with a Gel/PCR DNA Fragment Extraction Kit (Geneaid, Taiwan) according to the guidance of the manufacturer and cloned into pGMEMT-Easy vectors (Promega Corporation, Madison, WI) for DNA sequencing. A BLASTN search was used to search for sequences of the closest match in the GenBank database. C. gloeosporioides was purchased from the Plant Health Clinic, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand. Fermentation. T. harzianum F031 from a slant culture was subcultured on PDA for 7 days. The 5 mm discs of growing colonies were cut from the margin of each plate and inoculated into an Erlenmeyer flask containing 250 mL of potato dextrose broth (PDB). After a 27-day incubation at room temperature, the stationary cultures were filtered through filter paper (Whatman No. 1) to obtain the culture filtrate. Extraction and Purification. The combined culture filtrate (20 L) was exhaustively extracted with EtOAc (3× 1 L). The organic layers were combined, dried over Na2SO4, and evaporated under reduced pressure to yield the EtOAc extract (6.10 g). This material was fractionated by the solid-phase extraction (SPE), eluted with EtOAc− hexane and MeOH−EtOAc gradients (5% increasing polarity with 20 mL each). From the basic TLC profiles, four fractions (F1−F4) were obtained. Fraction F1 (2.32 g, eluted with 5−30% EtOAc−hexane) was separated by CC (SiO2), eluted with 20% EtOAc−hexane to provide 14 subfractions (S1−S14). Subfraction S3 (24.3 mg) was further purified by CC (SiO2), eluted with 15% EtOAc−hexane to give compound 1 (12.7 mg) as a colorless amorphous solid. Subfraction S7 (38.4 mg) was separated by CC (SiO2), eluted with 10% EtOAc− CH2Cl2. The product was further purified by CC (SiO2), eluted with 10% EtOAc−hexane to afford compound 3 (4.2 mg) as a white solid. Subfraction S8 (119.2 mg) was separated by CC (SiO2), eluted with 5% acetone−CH2Cl2 to obtain eight portions (C1−C8). Portion C4 (12.7 mg) was further purified by CC (SiO2), eluted with 20% acetone−hexane to yield compound 4 (3.0 mg) as a white solid.

Subfraction S10 (541.4 mg) was separated by CC (Sephadex LH 20), eluted with 50% MeOH−CH2Cl2 to afford four subfractions (D1− D4). Purification of subfraction D2 (326.1 mg) by two consecutive CC (SiO2; first CC, 10% EtOAc−hexane; second CC, 40% EtOAc− hexane) resulted in compound 2 (8.3 mg), a colorless amorphous solid. Trichoharzianol (1): colorless amorphous solid; [α]25 D +121.5 (c 0.13, MeOH); IR νmax, 3489, 1700, 1242, 1218 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 1 and the Supporting Information; HRESIMS (m/z), 365.2686 [M + H]+ (calcd for C22H38O4, 365.2692). Antifungal Activity Evaluation. The agar dilution method20 was used to evaluate antifungal activity of the crude extract. The sample was prepared as a stock solution at 10 mg/mL in dimethyl sulfoxide (DMSO). PDA (9.8 mL) was sterilized, and the sample (200 μL) was added at 48 °C to obtain a final concentration of 200 μg/mL and then poured into a 9 cm diameter Petri dish. The dish was left at room temperature overnight. The disc (5 mm diameter), which was removed from the margin of a 3-day-old colony of C. gloeosporioides, was placed at the center of the PDA plate and inoculated at room temperature for 3 days. The diameter (in millimeters) of the mycelium growth was recorded and calculated as a percentage of growth inhibition = (Y − X)/Y × 100, where Y is the growth diameter in the untreated control and X is the growth diameter in the treatment. The results of this quadruplicate experiment were reported as the antifungal activity. The microdilution method, with 96 well microliter plates, was used to evaluate antifungal activity of the fractions and pure compounds.31 The samples were prepared as 2-fold dilutions and added (50 μL) to each well, which contained the spore suspension (50 μL) and RPMI1640 medium (100 μL), to give a final concentration of 1024−8 μg/ mL for the fractions and 256−0.125 μg/mL for the pure compounds, again performed in quadruplicate. After the mixtures were incubated for 48 h at room temperature, 0.18% resazurin (10 μL) was added to each well and incubated continuously for 3−4 h. Amphotericin B (10 μg/mL, 50 μL) and 1% DMSO (50 μL) were used as a positive control and control, respectively. A positive result was indicated by a B

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Journal of Agricultural and Food Chemistry color change from blue to pink. The lowest concentration at which the color did not change was taken to be the minimum inhibitory concentration (MIC).

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RESULTS AND DISCUSSION Extraction and Purification. The crude EtOAc extract (6.10 g) of broth culture was fractionated by SEP to provide four combined fractions (F1−F4) based on their TLC characteristics. Subfractions (S1−S14) were obtained from fraction F1 by CC (SiO2). Subfractions S3, S10, S7, and S8 were further purified to afford compounds 1, 2, 3, and 4, respectively. Structural Elucidations. Trichoharzianol (1) was shown to possess a molecular formula C22H37O4 as determined by HRTOF-MS, exhibiting a [M + H]+ peak at m/z 365.2686 (calcd for C22H38O4, 365.2692). Its IR spectrum displayed the absorption bands for hydroxyl (3489 cm−1) and carbonyl (1700 cm−1) groups. The 1H NMR spectroscopic data (Table 1) displayed characteristics resembling those of eujavanical A.32,33 However, the two methine protons (H-5 and H-6) were shifted downfield to δH 3.71 and 4.26, because they are connected to oxygen atoms of the acetonide group at C5−C6. The gem-dimethyl groups of acetonide appeared at δ 1.48 and 1.35. The significant downfield shifts of C-5 and C-6 (δC 79.7 and 73.6) supported the presence of an acetonide group at C5− C6. The substituents were designated to be a 3-hydroxypropionyl group at C-1, a 1-methylpropyl group at C-2, two methyl groups at C-1 and C-8, and an acetonide group at C-5 and C-6. The assignments of the 1H and 13C NMR spectra were based on the correlation spectroscopy (COSY), heteronuclear singlequantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) data (see Table 1). The attachments of side chains on the decalin moiety were established by the analysis of the HMBC data. The location of the 3hydroxylpropyl group at C-1 was suggested by the correlations of 1-CH3 (δH 1.21) to C-9. H-2 (δ 1.94) showed correlations to C-2′ and 1′-CH3, confirming the attachment of a 1methylpropyl group at C-2. The correlations of a methyl (δH 1.21) to C-8a and C-2, together with another methyl (δH 0.64) to C-8a, C-8, and C-7, indicated the connections of two methyls at C-1 and C-8, respectively. Furthermore, the correlations between H-5 (δH 3.72) and H-6 (δH 4.26) to C4′ indicated the presence of an acetonide group at C-5−C-6. The relative configuration of compound 1 was determined by the nuclear Overhauser effect spectrometry (NOESY) correlations as shown in Figure 2. The above data confirmed that compound 1 was an acetonide derivative eujavanicol A,33 and the structure of trichoharzianol (1) was established as shown in Figure 1. Eujavanicol A (2) was reported previously from Eupenicillium javanicum,33 whereas 5-hydroxy-3-hydroxymethyl-2-methyl-7methoxychromone (3) and 4,6-dihydroxy-5-methylphthalide (4) were found in the lichen Graphis scripta34 and Talaromyces flavus,35 respectively. Their structures were identified with spectroscopic data (UV, IR, NMR, and MS) and compared to those reported in the literature. Antifungal Activity. The crude extract of T. harzianum F031 showed the antifungal activity against C. gloeosporioides with 76.6% inhibition by an agar dilution assay at a concentration of 0.2 mg/mL. After further fractionation and purification, the fractions and all the pure compounds were evaluated for their antifungal activity and their MIC values were

Figure 1. Structure of trichoharzianol (1), eujavanicol A (2), 5hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (3), and 4,6dihydroxy-5-methylphthalide (4).

Figure 2. Selective NOESY correlation of trichoharzianol (1).

determined by microdilution. Fraction A showed the highest antifungal activity against C. gloeosporioides with a MIC of 512 μg/mL, while fractions B−D exhibited MIC values of >1024 μg/mL. Trichoharzianol (1) showed the highest antifungal activity against C. gloeosporioides with MIC of 128 μg/mL (Table 2), followed by 5-hydroxy-3-hydroxymethyl-2-methyl-7Table 2. Antifungal Activity against C. gloeosporioides of Compounds 1−4 compound

MIC (μg/mL)

trichoharzianol (1) eujavanical A (2) 5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (3) 4,6-dihydroxy-5-methylphthalide (4)

128 >256 256 >256

methoxychromone (3) (MIC of 256 μg/mL). It should be noted here that eujavanicol A (2) and 4,6-dihydroxy-5methylphthalide (4) showed antifungal activity of >256 μg/mL. Trichoharzianol (1) is found to be a new and major component from T. harzianum F031 strain and shows the highest antifungal activity against C. gloeosporioides. In addition, eujavanicol A (2), 5-hydroxy-3-hydroxymethyl-2-methyl-7methoxychromone (3), and 4,6-dihydroxy-5-methylphthalide (4) are reported from this strain for the first time.

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of trichoharzianol (1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +66-7567-2989 and/or +66-7567-2090. Fax: +667567-2004. E-mail: [email protected]. Funding

This work was supported by Walailak University Fund and IFSNRCT Scientific Collaboration on Establishment of the International Research Network in Thailand’s Competent Fields. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Utilization of Natural Products Research Unit and the Tropical Fruit Research Unit at Walailak University and the Center of Excellence for Innovation in Chemistry (PERCH-CIC) at Mahidol University for access to laboratory facilities.

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REFERENCES

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DOI: 10.1021/acs.jafc.5b01258 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b01258 J. Agric. Food Chem. XXXX, XXX, XXX−XXX