Plant pathogens, particularly fungi, are responsible for yield reductions in food and crops throughout the world. Although these losses may be attenuated by the use of resistant cultivars, crop rotation, or sanitation practices, fungicides are often also needed to maximize crop yields (Knight et al., 1997). These antifungal chemicals also contribute substantially to the quality of food and human health by controlling many of the fungi that produce mycotoxins (Dmello et al., 1999) or by interfering in their biosynthesis (Hasan, 1999). Despite these significant benefits, synthetic fungicides are also responsible for the generation of toxic residues (Hall, 1984) and the development of resistance in pathogens (Wilson et al., 1997). These is therefore a continuing need to develop and release new fungitoxic chemicals that preserve the environment and enable a more efficient control of pathogenic fungi, improving crop yield and quality.
In earlier study the organism Streptomyces aureofaciens CMUAc130 was isolated from root tissue of Zingiber officinale Rosc. (Zingiberaceae). Cultures produced 5, 7, 4-Trimethoxy-4-phenylcoumarin 1 and 4-Hydroxy-5,7-dimethoxy-4-phenylcoumarin 2 which had antifungal activities (Taechowisan et al., 2005). In the present paper, the other 4-arylcoumarins were isolated, 3-hydroxy-5,7,4-trimethoxy-4-phenylcoumarin 3, 5,7,3,4-tetramethoxy-4-phenylcoumarin 4 and 4-hydroxy-5,7,3-trimethoxy-4-phenylcoumarin 5, we report the fungistatic and fungicidal activity exhibited by 4-arylcoumarins on several phytopathogenic fungi, among them, Aspergillus flavus, Colletotrichum musae, Fusarium oxysporum, Pythium ultimum, Rhizoctonia solani and Sclerotium rolfsii. These fungi were selected because of their importance in causing major plant diseases affecting crop yields and quality. The inhibitory activity of each isolated compound and their combinations on the fungal growth are also described.
MATERIALS AND METHODS
Organisms and Media
S. aureofaciens CMUAc130 was isolated from root tissues of Z.
officinale by the surface-sterilization technique (Taechowisan et al.,
2003). Identification of the isolate to species level was based on morphological,
cultural, physiological and biochemical characteristics and also 16S rDNA gene
sequencing as described by Taechowisan and Lumyong (2003). Spores of S. aureofaciens
CMUAc130 were used to inoculate 200 plates of International Streptomyces
Project (ISP) Medium 2 and incubated for 14 days at 28°C (Shirling and Gottlieb,
1966). The culture medium was then cut into small pieces that were extracted
with organic solvent (3x500 mL); hexane, ethyl acetate and 10% methanol in ethyl
acetate, respectively. Each of organic solvent was pooled and then taken to
dryness under rotary evaporation to give a dark brown oil (hexane extraction,
658.6 mg) and a dark brown solid (ethyl acetate extraction, 852.4 mg and 10%
methanol in ethyl acetate extraction, 583.5 mg). Isolates of A. flavus
obtained from peanut, C. musae from banana, P. ultimum from
potato, R. solani from tomato, F. oxysporum from wheat and S.
rolfsii from maize were used. All fungi were cultured on Potato Dextrose
Agar (PDA) medium.
Fractionation and Purification of the Compounds
The mixture residue of 350 mg of ethyl acetate and 10% methanol in ethyl
acetate extraction was dissolved in 10 mL methanol and fractionated on a reverse
phase column (Li Chroprep RP-18, Merck, Germany) with increase the concentration
of methanol as eluent (50, 70 and 100%). The fractions were combined on the
basis of TLC results and concentrated under the vacuum to produce the black
gum (180 mg). The black gum was mixed with 2.0 g of silica gel (Walk gel). This
mixture was then subjected to bioassay-guided separation through column (50x7.5
cm) chromatography over silica gel (50 g) eluted with chloroform-methanol (100:0,
95:5, 90:10, 85:15, 80:20, 70:30, 60:40, 50:50, 0:100). About 50 mL of each
fraction was collected. Purifications were performed by column chromatography
over silica gel (finer than 200 mesh). The fractions were combined and made
into four pooled fractions on the basis of their TLC results. The fractions,
A-1 (eluted with chloroform and methanol 95:5 to 90:10), A-2 (eluted with chloroform
and methanol 90:10 to 80:20), A-3 (eluted with chloroform and methanol 80:20
to 60:40) and A-4 (eluted with chloroform and methanol 60:40 to 30:70) were
screened again for their antifungal activity (Taechowisan et al., 2005).
The fractions A-2 and A-3 were most active against those fungi and they showed
one major spot having the same Rf value (0.63, chloroform and methanol 20:1)
in TLC. Since they were identical, we combined these fractions and by purified
them by repeated chromatography over a silica gel column, followed by recrystallization
in a mixture of hexane and chloroform (9:1). This yielded the active compounds
1 and 2. Purification of A-1 resulted respectively in compounds 3 and 4. Purification
of A-4 resulted in compound 5. The structures of the active compounds have been
identified using NMR and mass spectral data.
Structure Elucidation of the Compounds
The melting point of the compounds was determined on a Buchi-540 melting
point apparatus. Optical rotations were measured on a Perkin-Elmer 241 polarimeter,
IR spectra on a Perkin-Elmer 1 spectrometer, 1H and 13C
NMR spectra on a Bruker DRX 500 spectrometer and EI-MS and FAB-MS respectively
on a Hewlett-Packard 5989 B and a Finnigan/Thermo Quest Mat 95 XL mass spectrometer.
Data of antifungal activity assay and minimum inhibitory concentration of
CMUAc130, were analyzed by SPSS for window 11.01 (SPSS Inc., Chicago, USA).
Means of treatments for each experiment were compared using Duncans multiple
range test (p≤0.05).
The crude extracts and the purified compounds were evaluated for their antifungal
activity, using an agar serial dilution method according to Shadomy et al.
(1985). Inocula were prepared by suspending conidia from 48-96 h old cultures
(1x106 conidia mL-1) in physiological saline solution.
Similar size (1-2 mm) mature sclerotia of S. rolfsii weighing 5.80-14.3x10-4
g were also used for the assays.
Bioassay were conducted by adding the appropriate amounts of the crude extract or the purified compounds, to 5 mL of 10% glucose medium, to obtain a final concentration of 0.25-200 mg mL-1. The fungicide MCZ (ethylenebis(dithiocarbamic acid) manganese zinc complex) was used for the antifungal assays. Dilutions of the crude extract were prepared by previously dissolving in methanol (final concentration of methanol < 10%), while the purified compounds were dissolved in chloroform (final concentration of CHCl3 < 4%) before mixing with the glucose media. Plates containing culture medium, with or without addition of the above-mentioned solvents, were also used as controls for each fungus.
A broth micro dilution method (Wilson et al., 1997) was used to determine the MIC of the purified compounds individually or in different combinations. Four day old conidia from a monosporic culture of F. oxysporum were added to glucose-mineral salts medium to reach 1x105 conidia mL-1. Purified compounds dissolved in dimethyl sulfoxide (DMSO) were incorporated into each well, by duplicate, containing the spore suspension, resulting in concentrations ranging from 0.025 to 1500 mg mL-1. The final concentration of DMSO did not exceed 2-4%. Wells containing spore suspension with or without addition of DMSO were simultaneously made as controls. The percentage of fungi growth (recorded in percentage from 0 to 100) was visually determined with an inverted light microscope for each concentration of the compounds. The measurements were always done by the same operator. The inhibitory concentration (IC50) values were calculated by Probit analysis on the basis of percentage of inhibition obtained at each concentration of the samples.
Synergism among the pure compounds was also measured, adding to each well different combinations of different concentrations of each compound to be tested. In all cases, the final concentration of DMSO was lower than 8%.
Minimum Fungicide Concentration (MFC). These tests were carried out to analyze the possible fungicidal activity exhibited by the crude extract and the purified compounds, as well as studying their fungistatic activity. Fourteen days after the beginning of the MIC assay, a circle of agar around the culture center as well as sclerotia of S. rolfsii, from those plates that exhibited negative growth were obtained. Two replicates of the agar circle or the sclerotia were placed in the center of Petri dishes containing adequate medium for each fungi and then incubated for 14 days. At the end of this period, the MFC values were recorded.
RESULTS AND DISCUSSION
Isolation of the Antifungal Active Compounds
The crude extract by ethyl acetate (350 mg) of S. aureofaciens CMUAc130
was subjected to silica gel column chromatography and four pooled fractions,
A-1, A-2, A-3 and A-4, were obtained. All the fractions performed their antifungal
activity. These active fractions were then subjected to repeated chromatography,
followed by recrystallization in a mixture of hexane and chloroform (9:1) and
then afforded compound 1 (27 mg) and 2 (20 mg) in the mixture of fractions A-2
and A-3. Purification of A-1 resulted in compound 3 (21 mg) and compound 4 (9
mg). Purification of A-4 resulted in compound 5 (7 mg) (Fig. 1).
Structure Elucidation of Compound 1
5, 7, 4-Trimethoxy-4-phenylcoumarin (C18H16O5)
was a white amorphous powder: mp 151-152°C (EtOH); UV λmax
(MeOH) nm (log ε): 250 (4.07), 325 (4.29); 1H NMR (CDCl3):δ7.20
(2H, d, J = 8.5 Hz, H-2, H-6), 6.87 (2H, d, J = 8.5 Hz, H-3,
H-5), 6.50 (1H, d, J = 2.5 Hz, H-8), 6.22 (1H, d, J = 2.5 Hz, H-6), 5.96
(1H, s, H-3), 3.83 (6H, s, OMe-7, OMe-4), 3.46 (3H, s, OMe-5); IR vmax
(CHCl3) cm-1: 1710, 1610, 1595, 1510, 1158, 1111, 1052,
952, 872, 860, 830; MS m/z (rel. int.): 312 [M]+ (80), 284 [M - CO]+
(100), 269 [M - MeCO]+ (37), 241 [M - 43-CO]+ (2).
Structure Elucidation of Compound 2
was a white amorphous powder: mp 214-215°C (MeOH); UV λmax
(MeOH) nm (log ε):256 (4.04), 324 (4.22); UV λmax (NaOMe)
nm: 256, 368; 1H NMR (CD3COCD3):δ8.50
(1H, s, exchangeable D2O, OH-4), 7.14 (2H, d, J = 8.5 Hz, H-2,
H-6), 6.82 (2H, d, J = 8.5 Hz, H-3, H-5), 6.51 (1H, d, J =
2.5 Hz, H-8), 6.37 (1H, d, J = 2.5 Hz, H-6), 5.80 (1H, s, H-3), 3.91 (3H, s,
OMe-7), 3.53 (3H, s, OMe-5); Δδ = δC5D5N-δCD3COCD3
= H-2 + H-6 (+0.19), H-3 + H-5 (+0.32), H-8 (+0.11),
H-6 (+0.01), H-3 (+0.38) OMe-7 (-0.16), OMe-5 (-0.23); IR vmax (CHCl3)
cm-1: 1708, 1612, 1598, 1512, 1159, 1112, 1054, 952, 870, 860, 832;
MS m/z (rel. int.): 298 [M]+ (100), 270 [M-CO]+ (82),
255 [M-MeCO]+ (29), 227 [M-43-CO]+ (15).
Structure Elucidation of Compound 3
was a white amorphous powder: mp 153-154°C (EtOH); UV λmax
(MeOH) nm (log ε):252 (4.13), 329 (4.32); UV λmax (NaOMe)
nm: 250, 288 sh, 329, 400 sh; 1H NMR (CDCl3):δ5.92
(1H, s, H-3), 5.87 (1H, s, exchangeable D2O, OH-3), 3.86 and
3.78 (3H and 3H, s and s, OMe-7, OMe-4), 3.43 (3H, s, OMe-5); IR vmax
(CHCl3) cm-1: 3525, 1710, 1615, 1597, 1510, 1158, 1111,
1052, 945, 909, 859, 828; MS m/z (rel. int.): 328 [M]+ (100), 300
[M-CO]+ (51), 285 [M-MeCO]+ (31), 257 [M-43-CO]+
||Chemical structures of 5, 7, 4-trimethoxy-4-phenylcoumarin
(1), 4-Hydroxy-5,7-dimethoxy-4-phenylcoumarin (2), 3-hydroxy-5,7,4-trimethoxy-4-phenylcoumarin
(3), 5,7,3,4-tetramethoxy-4-phenylcoumarin (4) and 4-hydroxy-5,7,3-trimethoxy-4-phenylcoumarin
Structure Elucidation of Compound 4
O6) was a white amorphous powder: mp 169-170°C (MeOH);
UV λmax (MeOH) nm (log ε):248 (4.13), 328 (4.26); 1H
NMR (CDCl3):δ6.93-6.73 (3H, complex, H-2,H-5, H-6),
6.51 (1H, d, J = 2.5 Hz, H-8), 6.25 (1H, d, J = 2.5 Hz, H-6), 6.00 (1H, s, H-3),
3.91 and 3.85 (3H and 6H, s and s, OMe-7, OMe-3, OMe-4) and 3.48
(3H, s, OMe-5); IR vmax (CHCl3) cm-1: 1708,
1610, 1595, 1510, 1158, 1111, 1052, 944, 902, 855, 826; MS m/z (rel. int.):
342 [M]+ (100), 314 [M-CO]+ (40), 299 [M-MeCO]+
(9), 271 [M-43-CO]+ (2).
Structure Elucidation of Compound 5
O6) was a white amorphous powder: mp 173-174°C (Et2O);
UV λmax (MeOH) nm (log ε):251 (4.07), 329 (4.19); UV λmax
(NaOMe) nm: 250, 331, 395; 1H NMR (CDCl3):δ6.52 (1H,
d, J = 2.5 Hz, H-8), 6.26 (1H, d, J = 2.5 Hz, H-6), 6.02 (1H, s, H-3), 3.88
and 3.86 (3H and 3H, s and s, OMe-7, OMe-7), 3.36 (3H, s, OMe-5); IR vmax
(CHCl3) cm-1: 3524, 1709, 1611, 1595, 1510, 1158, 1111,
1052, 943, 903, 855, 825; MS m/z (rel. int.): 328 [M]+ (100), 300
[M-CO]+ (66), 285 [M-MeCO]+ (9), 257 [M-43-CO]+
MIC of Extracts
The crude extracts of S. aureofaciens CMUAc130 culture were assayed
for their potential antifungal activity on different phytopathogenic fungi.
All extracts, depending on the concentration, exhibited inhibition on the growth
of all of the tested i.e., fungi A. flavus, C. musae, F. oxysporum,
P. ultimum, R. solani and S. rolfsii. The ethyl acetate
extract exhibited a significant inhibition on fungal growth with a MIC ranging
from 0.25 to 25 mg mL-1 while the 10% methanol in ethyl acetate extract
showed a mild activity (MIC = 25-100 mg mL-1) (Table
1). Hexane extract showed a low antifungal activity with MIC values from
50-200 mg mL-1, which means that low hydrophobic antifungal substances
(oils) had a low potential in the antifungal activity. The presence of hydrophobic
antifungal compounds is in agreement with Hernandez et al. (2007) who
have reported antifungal activity in hydrophobic solvent-extracted compounds
from Cordia curassavica. The activities of the most extracts were less
than that of the control antifungal agent MCZ by at least 1 order of magnitude.
C. musae, F. oxysporum and S. rolfsii were the highly
affected fungi (MIC = 0.25-50, 0.5-50 and 0.25-25 mg mL-1, respectively),
in contrast with A. flavus, which was the least inhibited (MIC = 50-200
mg mL-1), matching observations made by Iida et al. ( 1999).
In concentrations lower than the MIC values, a slow growth of the fungi was
observed in comparison to the controls.
MFC of Extracts
Ethyl acetate extraction was the most effective treatment, exhibiting an
MFC from 10-50 mg mL-1, while the extracts of 10% methanol in ethyl
acetate and hexane exhibited MFC from 50-100 mg mL-1 and 100- >200
mg mL-1, respectively (Table 2). These finding
indicated that ethyl acetate extracts obtained from S. aureofaciens CMUAc130
culture performed a significant fungistatic and fungicide effect on different
Antifungal Activity of Compounds 1-5
MIC values of isolated compounds 1-5 measured by the broth micro dilution
method (Table 3). Compounds 1, 2 and 3 showed a total growth
inhibition of the pathogenic fungi F. oxysporum at 0.30 (MIC 1), 1.00
(MIC 2) and 0.40 (MIC 3) mg mL-1, respectively, while the MIC of
compound 4 and 5 was observed at 10.0 (MIC 4) and 20.0 (MIC 5) mg mL-1,
respectively. The MIC value of compound 4 and 5 was high and produced low yield,
so they were excluded for synergistic assay.
|| MIC of different extracts from culture of S. aureofaciens
|a: MIC is defined as the lowest concentration providing
complete inhibition of fungal growth. Results are the average of two replicates
measured at 48 h after incubation. HE: hexane extract; EAE: ethyl acetate
extract; 10%M-EAE: 10% methanol in ethyl acetate extract; MCZ: ethylenebis(dithiocarbamic
acid) manganese zinc complex
|| MFC of different extracts from culture of S. aureofaciens
|a: MFC is defined as the lowest concentration providing
complete inhibition of fungal growth in a medium free of extract or compound.
Results, the average of two replicates, were measured at 14 days after incubation.
HE: hexane extract; EAE: ethyl acetate extract; 10%M-EAE: 10% methanol in
ethyl acetate extract; MCZ: ethylenebis (dithiocarbamic acid) manganese
|| Antifungal activity of 1-5
|a: Results, the average of two replicates, were
measured at 48 h from the beginning
At 0.20 mg mL-1, compound 1 developed 75% of growth inhibition (Fig. 2) while at 0.10 mg mL-1 showed a 25% inhibition. The same percentage of inhibition was detected when the compound 3 at 0.20 mg mL-1 was used. The fungal growth was inhibited by 20 and 60% when compound 2 at concentrations of 0.60 and 0.80 mg mL-1, respectively was applied. As observed, the significant inhibitory activity of compounds 1-3 increased while the IC50 indicated a similar performance for 1 (0.17 mg mL-1; 95% confidence interval = 0.16-0.43) and 3 (0.25 mg mL-1; 95% confidence interval = 0.08-0.74) and this effect was better than that for 2 (0.78 mg mL-1; 95% confidence interval = 0.37-1.24).
Because the resultant residue presented a higher antifungal activity (MIC and MFC = 0.50 mg mL-1) than those exhibited by pure compounds, the presence of a synergistic effect between 1, 2 and 3 was suspected. The compounds were added in different combinations of two or three that in most cases were equivalent to half or less of their MIC values.
As shown in Table 4 , when compound 1 at 0.15 mg mL-1
(0.5 MIC 1) was combined with 3 at 0.10 mg mL-1 (0.25 MIC 3) or with
2 at 0.50 mg mL-1 (0.5 MIC 2), a 100% inhibition in F. oxysporum
growth was observed. The same results were obtained when the same concentration
of 1, concentrations of 2 or 3 were tested and decreased to 0.25 mg mL-1
(0.25 MIC 2) and 0.04 mg mL-1 (0.1 MIC 3), respectively. When compound
1 was added at 0.075 mg mL-1, equivalent to 0.25 of its MIC value
and 2 at 0.50 mg mL-1, a 90% inhibition was detected.
|| Synergism among different concentrations of 1-3 combined
|aResults, the average of two replicates, were obtained
at 48 h from the beginning
||Effect on fungi growth inhibition of different concentrations
of 1 (♦), 2(■) and 3(▲)
When 1 was added at a concentration 10 times lower than the value of MIC 1
(0.03 mg mL-1) and 2 at 0.50 mg mL-1 (0.5 MIC 2) or 3
at 0.20 mg mL-1 (0.5 MIC 3), a 75% and 80% inhibition were observed,
respectively. When decreased the concentration of 2 to 0.25 mg mL-1
(0.25 MIC 2), a significant inhibition (50%) was still observed. When the same
concentration of 1 was combined with 3 at 0.10 mg mL-1 (0.25 MIC
3), a 60% growth inhibition was shown when 3 was added at 0.04 mg mL-1
(0.1 MIC 3), the inhibition was 45%. These results indicated that the combination
between 1 and 3 presented a poor synergistic effect than 1 and 2. Adding 2 and
3 at 0.50 (0.5 MIC 2) and 0.10 mg mL-1 (0.25 MIC 3), respectively,
59% inhibition was observed, reaching 85% when the concentration of 3 was 0.20
mg mL-1 (0.5 MIC 3). Lower concentrations of both compounds exhibited
inhibition values ranging from 3 to 45%, meaning that the synergism between
2 and 3 is lower than that observed between 1 and 2 or between 1 and 3.
When all three compounds were combined together, each at a concentration of about 0.25 of its MIC, 100% control of the growth of F. oxysporum was obtained, demonstrating a better control than that observed when pairs of compounds were combined at approximately 0.25 of their MIC values. There was 63% inhibition on the fungal growth in 1 and 3 treatments at 0.03 (0.1 MIC 1) and 0.04 mg mL-1 (0.1 MIC 3), respectively and 2 at concentration of 0.10 mg mL-1 (0.1 MIC 3). If the concentration of at least one of the compounds was increased, the inhibition also increased, obtaining 88% inhibition when compounds 1 and 3 remained at the same concentration (0.1 MIC 1 and 3) and 2 was increased to 0.50 mg mL-1 (0.5 MIC 2) (Table 5).
In summary, when the antifungal active principles 1-3 were combined in pairs
or all three together in concentrations below their MIC values, an unexpected
fungal growth inhibition almost as high as that obtained with the synthetic
compounds MCZ was observed. This result showed a clear synergistic effect between
them, which was not observed till now.
|| Synergism among different concentrations of 1-3 combining
|a: Results, the average of two replicates, were
obtained at 48 h from the beginning
This effect enabled the quantities of each compound needed for a total inhibition
to be decreased at least 4-fold. The synergistic effect is one of the most important
characteristics exhibited by natural extracts, increasing their efficacy in
contrast to that which could be obtained with the equivalent amount of the active
constituents alone (Amiguet et al., 2005; Cassella et al., 2002;
Gershon et al., 2004; Hsu et al., 2007; Schultz and Nicholas,
2002; Tallarida, 2001; Yen and Chang, 2006 ).
In conclusion, this study demonstrates that extracts from the culture of S. aureofaciens CMUAc130 and their respective bioactive compounds showed an important fungitoxic effect against the microorganisms studied, increasing the antifungal potency of the active principles when they are combined.
This study was supported of Faculty of Science, Silpakorn University, Nakorn Pathom, Thailand, as well as the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany (KIB), Chinese Academy of Sciences. The authors are grateful to Mr. Y.-N. He and Ms. H.-L. Liang in KIB for measuring NMR and MS data, respectively.