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Endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, SW1116 cell and some microbial growths

Y.C. Song , H. Li , Y.H. Ye , C.Y. Shan , Y.M. Yang , R.X. Tan
DOI: http://dx.doi.org/10.1016/j.femsle.2004.10.005 67-72 First published online: 1 December 2004

Abstract

Fractionation of the extract of Aspergillus niger. IFB-E003, an endophyte in Cyndon dactylon, gave four known compounds naphtho-γ-pyrones rubrofusarin B, fonsecinone A, asperpyrone B and aurasperone A, which were further investigated biologically. Rubrofusarin B was shown to be cytotoxic to the colon cancer cell line SW1116 (IC50: 4.5 µg ml-1), and aurasperone A inhibitory on XO (xanthine oxidase) (IC50: 10.9 µmol l-1). Moreover, the four naphtho-γ-pyrones exhibited growth inhibitions against the five test microbes with MICs ranging in between 1.9 and 31.2 µg ml-1. The present recognition of rubrofusarin B and aurasperone A as strong co-inhibitors on XO, colon cancer cell and some microbial pathogens is of significance for the imperative discovery of new relevant therapeutic agents.

Keywords
  • Endophyte
  • Naphthopyrone
  • Xanthine oxidase
  • Antimicrobial
  • Cytotoxic

1 Introduction

With the accelerating knowledge accumulation concerning symbionts [1], endophytes have been demonstrated to be a rich and reliable source of biological active and/or chemically novel compounds that may foster great medicinal or agricultural potentials [2]. This notion has recently received an additional persuasion from our discovery of bioactive endophytic metabolites with new frameworks [3, 4] and immuno-modulating function [5]. In continuation of our endeavor regarding the topic, the substantially antimicrobial and XO (xanthine oxidase) inhibitory actions were discerned with the extract derived from the culture of Aspergillus niger. IFB-E003, an endophytic fungus harboring in the well growing leaves of Cynodon dactylon. The subsequent fractionation of the active fraction resulted in the isolation of one known mono- (rubrofusarin B 1) and three known dimeric naphtho-γ-pyrones (fonsecinone A 2, asperpyrone B 3 and aurasperone A 4).

Previously mono- and dimeric naphtho-γ-pyrones were found as yellow pigments to be secondary metabolites of the fungi such as Aspergillus sp. [6] and Fusarium sp. [6], and some plants [7, 8]. Some naphthopyrones were demonstrated to be anticancer [7, 8], antibacterial [9], antifungal [10], anti-signal transduction [11], antiallergic [12] and HMG-CoA reductase inhibitory [13]. As to the four naphtho-γ-pyrones also obtainable from the culture of the title endophyte, rubrofusarin B (1) was ascertained to be able to reverse multi-drug resistance of human epidermal KB carcinoma cells [14] and to inhibit the calmodulin-dependent activity of cAMP phosphodiesterase and NAD-kinase in the presence of CaM [15] while aurasperone A (4) was shown to be a Taq DNA polymerase inhibitor [16]. However, few communications were previously dedicated to the inhibition of any of the four naphtho-γ-pyrones on the growth of SW1116 cell and microbial pathogens Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Trichophyton rubrum and Candida albicans, and neither on activity of XO which plays multiple roles in the metabolism of xanthine and in the incidence of hyperuricemia such as gout [17]. We therefore investigated the cytotoxic, antimicrobial and XO inhibitory actions of the fungal secondary metabolites (14) with an intention to better understand the biological significance of the naphtho-γ-pyrones.

2 Materials and methods

2.1 General

EI-MS spectra were recorded on a VG-ZAB-HS mass spectrometer and HR-ESI-MS spectra on a Mariner Mass 5304 instrument. The 1H- and 13C NMR (nuclear magnetic resonance) measurements including two-dimensional experiments HMQC (heteronuclear multiple quantum coherence), HMBC (heteronuclear multiple-bond correlation) and NOESY (nuclear overhauser effect spectroscopy) were performed on a Bruker DRX-500 NMR spectrometer co-using TMS and solvent signals as the internal standards. The U-3000 Spectrometer for absorbance measurements was from Hitachi, Japan and ELISA plate reader was from Sunrise, USA. Silica gel (200–300 mesh) for CC (column chromatography) and silica GF254 (10–20 mm) for TLC (thin-layer chromatography) were products of Qingdao Marine Chemical Factory, China. Sephadex LH-20 and ODS C18 were from Pharmacia Biotech, Sweden. All chemicals used in the study were of analytical grade.

2.2 Endophyte A. niger IFB-E003

A. niger IFB-E003 was an endophytic fungus strain isolated from the normally growing leaves of C. dactylon (L.) collected in early November 2001 from Yancheng Biosphere Reserve, Jiangsu Province, PR China. Fungal identification was based on the morphology, pigmentation, texture and growth rate of the colony, as well as microscopic characteristics such as pattern of sporulation and type of conidiophores as specified elsewhere [18, 19].

2.3 Culture

Liquid cultivation of the isolated fungal endophytes was accomplished on Czapek's medium (30 g sucrose, 3 g NaNO3, 1 g K2HPO4, 1 g yeast extract, 0.5 g KCl, 0.5 g MgSO4· 7H2O, 0.01 g FeSO4 and 1000 ml H2O). To make the inoculum for solid fermentation, the fresh mycelia and spores grown on PDA medium at 28 °C for 5 days were dug down 10 dollops (5 mm diameter) and inoculated into 1000 ml flasks containing 400 ml of the broth (200 g potato, 20 g dextrose and 1000 ml H2O), followed by shaking incubation rotating at 140 rpm for 5 days at 28 °C. The subsequent solid culture was accomplished by adding 15 ml each above-made inoculum into 400 bottles (6.5 cm in diameter and 9 cm in height) for cannery each containing 15 ml water, 7.5 g millet, 7.5 g bran, 0.5 g yeast extract, 0.1 g tartrate sodium, 0.1 g glutamine sodium, 0.01 g copperas and 0.1 ml pure corn oil as substrate. Culture temperature was 28 ± 1 °C, relative humidity was 60–70%, initial water content was 66.7% and cultivation time was 30 days. Moreover 20 bottles without inoculum were prepared as above for comparison to prove all compounds to be the endophytic fungal metabolites by TLC and LC–MS examinations.

2.4 Extraction and fractionation

The crude solid fermentation product (ca. 2.5 kg, not completely dried) collected after the desiccation and grinding of 400 bottles of the cultures was exhaustively extracted with CHCl3/MeOH (1:1, v/v) mixture (15 L × 3). Evaporation of the solvent in vacuo yielded a black residue (ca. 300 g, not totally dried), which was dissolved in possibly less amount of MeOH at around 45 °C and the resultant solution was kept overnight at −20 °C, followed by filtration to eliminate waxy substances as the precipitate that formed. Removal of MeOH from the filtrate under reduced pressure gave a brown lump, to which MeOH was added until it dissolved completely. To the afforded solution, acetone was added drop by drop with acetone finally at 15% (v/v). The acetone-containing liquor was left overnight at −20 °C followed by filtration to get rid of salts and saccharides that accompanied. In vacuo evaporation of the solvent from the filtrate afforded a residue (ca. 100 g, not entirely dried), which was then chromatographed over silica gel (1500 g) column (diameter: 8 cm) eluted with polarity growing CHCl3/MeOH mixtures (100:0 → 0:100, v/v). CC eluates (500 ml each) were combined according to TLC monitoring to give 10 fractions. The antimicrobial and XO inhibitory fraction (Fr. 3) was subjected again to CC over silica gel (220 g) column (diameter: 2.5 cm) using CHCl3/MeOH gradients (50:1 → 25:1, v/v) to give Fr. 3.1 (2.1 g), which was further separated over silica gel column with CHCl3–MeOH gradients (50:1 → 25:1, v/v) to yield Fr. 3.1.1 (0.2 g) and Fr. 3.1.2. Another bioactive part Fr. 2 was separated by silica gel CC with CHCl3/MeOH mixtures (100:1 → 25:1, v/v) to afford two factions (Fr. 2.1: 0.6 g and Fr. 2.2: 0.4 g). Successive CC separations of Fr. 2.2 over silica gel with CHCl3/MeOH (100:1 → 25:1, v/v) and over ODS C18 with H2O/MeOH (1:1 → 0:100, v/v) gave two parts: Fr. 2.2.1 (0.2 g) and Fr. 2.2.2 (0.156 g). The combined mixture of Fr. 2.2.2 and Fr. 3.1.1 was subjected to the repeated gel filtration over Sephadex LH-20 with CHCl3/MeOH (1:1, v/v) to yield 1 (15 mg). Further CC fractionation of Fr. 2.1 was accomplished over silica gel with CHCl3/MeOH gradient (100:1 → 10:1, v/v) and then over ODS C18 with H2O/MeOH (1:1 → 0:100, v/v) to afford five fractions: Fr. 2.1.1 (58 mg), Fr. 2.1.2 (36 mg), Fr. 2.1.3 (30 mg), Fr. 2.1.4 (30 mg) and Fr 2.1.5 (470 mg). Repeated CC fractionation over silica gel of Fr. 2.1.2, Fr 2.1.3 and Fr. 2.1.4 gave metabolites 2 (7 mg), 3 (5 mg) and 4 (4 mg), respectively.

2.5 Antimicrobial assay

Antimicrobial actions were assessed using as test microbes three bacteria (B. subtilis, E. coli and P. fluorescence) and two fungi (T. rubrum and C. albicans). MICs (minimal inhibitory concentrations) were evaluated by a liquid dilution method as described elsewhere [9, 20]. The test compounds were initially dissolved in 5% DMSO (in water) to give the corresponding stock solutions at 500 μg ml−1. Penicillin, amikacin sulfate and ketoconazole were co-assayed as positive antimicrobial references with 5% DMSO as negative control. Mixtures containing aliquots of precultured bacterial or fungal solution with the final concentration of about 105 CFU ml−1 and serial twofold dilution of test compounds were incubated at 28 °C for 24 h on 96-well plates. The lowest concentration of a test compound at which no visual turbidity arising from any microbial growth could be detected, was designated as MIC.

2.6 XO inhibition

The XO inhibition of the compounds was evaluated as described earlier [21, 22]. The reaction mixture contained 50 mmol l−1 K2HPO4 (pH 7.8), 120 μmol l−1 xanthine and 2.5 mU xanthine oxidase. The test compounds, initially dissolved in DMSO at 500 μg ml−1, were then incorporated in the reaction mixture to assay the inhibitory activity with allopurinol co-tested as positive control. The absorption increment at 295 nm at 25 °C indicated the formation of uric acid and the product was expressed as μmol uric acid per minute. The reaction kinetics was linear during the first 6 min of reaction. All determinations were performed in triplicate. The inhibition of XO was expressed as IC50, the concentration at which only a half-maximal enzyme activity was discerned.

2.7 Cytotoxic assay

The cytotoxicity was measured as detailed elsewhere [23]. Thus, SW1116 cell was incubated in 1640 medium with 10% fetal bovine serum until cells were in their logarithmic growth phase. Cell suspension was made to reach approximately 2 × 104 cells ml−1. They were then seeded into 96-well plates with 100 μl in each well, followed by incubating in CO2 incubator at 37 °C for 24 h. The stock solutions of the four compounds were diluted with the vehicle and a possibly less amounts of DMSO (the final concentration of DMSO less than 1%) were added wherever necessary for getting the uniform test solutions at given concentrations. The solution of each compound at each preset concentration, along with the positive control (5-FU), was put to five wells in the 96-well plate followed by cultivation at 37 °C for 48 h. Then 20 μl of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) at 5 mg ml−1 was added into each well on the plate. After 4 h, the 1640 medium was removed and 150 μl DMSO was added. Finally the absorbance at 570 nm was measured on an ELISA plate reader. The IC50 value was defined as the concentration at which 50% survival of cells was allowed only.

3 Results and discussion

Systematic screening for the bioactive secondary metabolites from liquid cultures of endophytes residing in the normal tissues of the stargrass C. dactylon led to the recognition of the endophytic strain IFB-E003 as an antimicrobial and XO inhibitory metabolite producer. Colonies of the producing endophytic strain grew faster to attain full plate with 9 cm in diameter after incubation on PDA at 28 °C for 5 days. The aerial mycelia, yellowish at the beginning, became black at the end of cultivation and took on black color in PDA plates. Septate hyphae were hyaline and conidiophores were produced erectly from the aerial hypha, single, upright, terminating in a globose swelling, bearing phialides at the apex. Conidia were 1-celled, globose, black, roughness surface, aggregating at the tip of the vesicle to form a globose head. The conidial heads were black. These morphological characteristics led to identification of the endophyte as A. niger[18, 19]. The CHCl3/MeOH (1:1, v/v) extract of the re-grown culture of this strain on solid medium showed as well confirmed antimicrobial and XO inhibitory activities. The subsequent scaled-up re-growth of the strain was therefore accomplished by solid culture protocol.

The CHCl3/MeOH (1:1, v/v) extract derived from the endophyte solid culture was separated by a combination of chromatographies over silica gel, ODS C18 and Sephadex LH-20 columns to afford four yellow pigments. Scrutiny of the spectral data (1H and 13C NMR, HMQC, HMBC and NOESY) acquired for each sample allowed the identification of the four metabolites as naphtho-γ-pyrones rubrofusarin B (1) [6, 24], fonsecinone A (2) [16], asperpyrone B (3) and aurasperone A (4) [6]. The mentioned spectra of the four compounds are available upon request from the corresponding author. Previously, the fungal naphtho-γ-pyrones with identical and similar carbon frameworks and substitution patterns were characterized as the secondary metabolites of Aspergillus sp. [11, 14, 16, 25] and Fusarium sp. [6]. The present first-time isolation of metabolites 14 from the endophyte culture highlighted that naphtho-γ-pyrones could be among the characteristic products of Aspergillus species [6]. And further attention to the biosynthetic pathway of naphthopyrones is desired to understand the real origin of this family of metabolites, which have been co-characterized from both fungi [6, 14] and plants [7, 8].

The antimicrobial action of naphtho-γ-pyrones 14 expressed as MICs (μg ml−1) was summarized in Table 1. In general, the inhibition order of the metabolites was 4 > 1 > 2 > 3. In particular, the inhibitory effect of compound 4 against C. albican and T. rubrum was similar to that of the positive reference ketoconazole (see Fig. 1).

View this table:
1

In vitro antimicrobial activities of compounds 14a

Test microbeMIC (μg ml−1)
Test compoundPositive control
1234PenicillinAmikacin sulfateKetoconazole
B. subtilis3.915.631.21.90.780.45b
E. coli3.97.815.67.83.9
P. fluorecens7.87.87.81.90.783.9
T. rubrum7.87.87.83.93.9
C. albicans15.615.615.61.93.9
  • a Penicillin and amikacin sulfate were the positive controls against three bacteria while ketoconazole was the positive control against two fungi.

  • b The hyphen means no substantial inhibition detected.

1

Structures of metabolites 14.

The four naphthopyrones were evaluated for XO inhibitory actions with the IC50 values given in Table 2, which demonstrated that the general order of the four compounds in the enzyme inhibition was 4 > 1 > 2 > 3. This enzyme inhibition order was something like that in their actions on the test microbes as mentioned above. It was noteworthy that both 1 and 4 displayed significant inhibitions on xanthine oxidase with their IC50 values fairly comparable to that of the positive control allopurinol. As detailed below, special attention was subsequently paid to the kinetic features of compound 1 in the enzyme inhibitory action owing to the discerned substantial activity and sufficient amount of the material. The reaction rate (v) of XO with the substrate follows a Michaelis–Menten equation [26]. As illustrated by Fig. 2, the mode of inhibition could be readily indicated by Lineweaver–Burk plots for the enzyme inhibition of 1, utilizing xanthine as substrate and following the procedure detailed elsewhere [21, 22]. The obtained Lineweaver–Burk plots showed that compound 1 inhibited the enzyme in a linear mixed-type mode. Namely, the discerned inhibition is between competitive and non-competitive type, which was similar to those of the extracts derived from some goat-treating traditional Chinese medicinal plant extracts [21]. This observation implied that compound 1 inhibited XO by binding either with the free enzyme or with the enzyme–substrate complex [27]. Accordingly, the rate (v) of uric acid generation by XO in the presence of compound 1 can be finally expressed as v=Vmax [S]/(Km+ [S])(1 + [I]/Ki) (Vmax and Km for xanthine). Calculated through above equation, Vmax and Km detected for the enzyme were 0.0151 μmol min−1and 83.1000 μmol l−1 and Ki for 1 was disclosed to be 24.1214 μg ml−1.

View this table:
2

IC50 (μmol l−1) of compoundsa14 in vitro xanthine oxidase inhibitive activities

Compound1234Allopurinol
IC50 (μmol l−1)16.819.537.710.99.8
  • a Mean values of triplicates are expressed as μmol l−1.

2

Lineweaver–Burk plots in the absence (♦) and presence of rubrofusarin B (1) at 17.5 μmol l−1 (▪) and 26.2 μmol l−1 (▲) with xanthine as the common substrate.

From the substantial inhibition discerned with the four naphtho-γ-pyrones obtained from the bioactive CC fractions, it could be concluded that they might be responsible for the antimicrobial and XO inhibitory activities initially observed with the extract of the endophytic strain.

The four naphthopyrones were additionally tested in vitro for the cytotoxicity against the colon cancer cell line SW1116 using MTT method with the results comparatively summarized in Table 3. The most active one was ascertained to be 1, which exhibited an 80% inhibition even at 10 μg ml−1, quite close to that discerned with the positive control 5-FU at the same concentration. The IC50 value of 1 against the cancer cell was measured to be 4.5 μg ml−1 (15.7 μmol l−1) and that of 5-FU 5 μg ml−1 (37.3 μmol l−1). Regarding the three dimers, the inhibition order against the cell line was 4 > 2 > 3.

View this table:
3

In vitro inhibition ratesa of compounds 14 against SW1116 cell at the concentration of 10 μg ml−1

Compound12345-FU
Inhibition rate (%)80.023.518.727.981.0
  • a Mean values of triplicates are expressed as %.

The present work described the first-time isolation of naphthopyrones from the endophyte culture. The subsequent antimicrobial attention to the four metabolites disclosed that the growth inhibitory effects of aurasperone A (4) against C. albican and T. rubrum were quite close to that of ketoconazole, an antifungal agent being prescribed in clinic. And this naphthopyrone, along with rubrofusarin B (1), displayed additionally significant inhibitions on XO with their IC50 values fairly comparable to that of the positive control allopurinol. Moreover, a significant cytotoxicity against SW1116 cell was discerned with rubrofusarin B (1). The aforementioned demonstration of rubrofusarin B (1) and aurasperone A (4) as co-inhibitors on XO, colon cancer cell line SW1116 and the human opportunistic pathogens C. albican and T. rubrum is of special values in the discovery of new anti-cancer, antifungal and anti-hyperuricuric drugs.

Acknowledgements

The work was co-supported by the Ministry of Education (Key Project No: 104195), by NSFC (30300007) and by JSNSF (BK2003410). We were grateful to W.Y. Huang and Z. Zhang for the assistance in endophyte isolations and MS measurements, respectively.

Footnotes

  • Editor: G.M. Gadd

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View Abstract