Introduction

The compound curvularin (1) is a macrolide, which shows antibiotic activity towards some fungi as a non-specific phytotoxin [1]. Curvularin and its derivatives could be potential nematicides against the root-lesion nematode, Pratylenchus penetrans [2]. More interestingly, some of these derivatives show to act as inhibitors in blocking the cell division [3] by specifically disordering the microtubule centers [4] and inducing barrel-shaped spindles [5]. It was reported that curvularin and αβ-dehydrocurvularin (2), as well as other derivates, are produced by some fungal species, such as Curvularia [6], Aspergillus [7], Alternaria [8], and Penicillium [9]. In this paper, we describe the bioassay-guided isolation of curvularin and αβ-dehydrocurvularin (see Scheme 1) from Eupenicillium sp., associated with marine sponge Axinella sp., and their structure elucidation. Using anti-microbial and anti-tumor composite screening models, we found that curvularin and αβ-dehydrocurvularin have slightly different bioactivity profiles.

Scheme 1
scheme 1

Structure of curvularin and αβ-dehydrocurvularin

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Materials and Methods

General Experimental Procedures

Melting points were determined on a XRS-1 digital melting point apparatus. The 1H and 13C NMR data were collected on an Bruker AVANCE-500 (Bruker, Switzerland) spectrometer at 500 MHz, and the chemical shifts were recorded in δ (ppm) relative to Si(Me)4 with coupling constants J in Hz. Electron ionization-mass spectrometry (EI-MS) was performed using a MAT95XP mass spectrometer (Thermo, USA). Silica gel (100–200 mesh) for open column chromatography and prepared silica gel (300 mesh) plates for TLC were produced by the Qingdao Marine Chemical Factory (Qingdao, China). Sephadex™ LH-20 was from GE Healthcare (GE, USA). Further purification of compounds was performed on a HITACHI L-2000 preparative high performance liquid chromatography (PHPLC) (HITACHI, Japan) with one YMC semi-preparative ODS column. All other chemicals used in this study are of analytical grade.

Isolation and Identification of Strain Eupenicillium sp.

The fungi isolate was obtained by serial dilution method [10] from sponge samples collected from South China Sea (18° 13′ N; 109° 29′ E) near Sanya, Hainan, China in July 2005. The sponge sample was later identified as Axinella sp. by Dr. K. J. Lee (Department of Biology, Hannam University, 133 Ojungdong, Daedukgu, Daejeon, Korea) through personal communication. The bioactive fungal strain was identified to be Eupenicillium sp. by comparing its morphological features with the reference description [11] and by online BLAST analysis of its 18S rDNA sequence with those submitted sequences on GenBank database.

Culture Media

The strain isolation was performed on the starch casein KNO3 agar (SCKA) medium composed of 20 g starch, 2 g KNO3, 2 g K2HPO4, 2 g NaCl, 0.3 g casein, 0.05 g MgSO4·7H2O, 0.02 g CaCO3, 0.01 g FeSO4·7H2O, and 18 g agar in 500 ml sterile distilled water premixed with 500 ml filtered sea water.

The germination and growth of the marine fungal isolate was undertaken on the Gauze’s No. 1 sea water (GSW) medium, which was composed of 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, and 30 g agar in 500 ml sterile distilled water premixed with 500 ml filtered sea water. All above inorganic chemicals were from Guangzhou Chemical Reagent Factory, and the organic reagents from Amresco Inc.

Composite Screening Model

Indicator Strains

The initial bioactivity screening procedure involved indicator organisms including two fungi, two Gram-positive (G+), and two Gram-negative (G−) bacteria. The two indicator G− strains, Escherichia coli (GIM 1.115) and Pseudomonas aeruginosa (GIM 1.46), two G+ strains, Bacillus subtilis (GIM 1.181) and Staphylococcus aureus (GIM 1.178), and a fungus, Saccharomyces cerevisiae Hansen (GIM 2.86), were obtained from Microbial Culture Collection Center, Guangdong Institute of Microbiology (GIMCC). Another indicator fungus Sclerotinia sclerotiorum (ACCC 30046), one plant pathogenic fungus, were from Agricultural Culture Collection of China (ACCC) through professor Zanmin Hu of Institute of Genetics and Developmental Biology, CAS.

Tumor Cell Lines

The further anti-tumor screening for ethyl acetate (EtOAc) extract of fermentation (see ‘Separation and Purification of Metabolites’) referred to four types of tumor cell including human lung adenocarcinoma epithelial cell line (A549), human Henrietta Lacks cervical cancer cell line (HeLa), mice Ehrlich ascites carcinoma cell line (Ehrlich), and human breast adenocarcinoma cell line (MCF-7) that were carried out at the Medical Science College of China Three Gorges University.

Antagonistic Test

For fungal inhibition test, an inoculum (5 mm in diameter) of each isolate, prepared by punching the growth agar gel of 72-h Petri dish culture, was transferred into the four corners of a 9-cm-diameter Petri dish containing PDA medium. A fungal indicator organism (5 mm in diameter) was put in the middle of the same Petri dish. The inoculated Petri dish was cultured at 28 °C for 48 h. The antagonistic action [12] was evaluated by measuring the diameter of the inhibition zones with penicillin as a positive control. For bacterial inhibition test, an indicator organism was spread on top of the Petri dish containing LB agar. An inoculum (5 mm in diameter) of each isolate, prepared by punching the growth agar gel of 72-h Petri dish culture, was transferred into the four corners of the Petri dish with indicator bacteria. The inoculated Petri dish was cultured at 28 °C for 48 h. The antagonistic action was evaluated by measuring the diameter of the inhibition zones with penicillin as a positive control. Each antagonistic test was performed in triplicate.

Fermentation Condition

The fresh mycelium growing on GSW agar at 28 °C for 5 days was inoculated into 500-ml Erlenmeyer flasks containing 150 ml GSW medium. After 2 days of incubation at 28 °C on rotary shaker at 200 rpm, a 40-ml seed culture liquid was transferred into each 1,000-ml Erlenmeyer flask containing 300 ml GSW medium. The flasks were incubated for 10 days at 28 °C and 200 rpm on a rotary shaker.

Bioassay of Fermentation Extract and Fractions

One liter culture filtrate was extracted four times with the same volume of EtOAc. After evaporation of the solvent from the combined extract in vacuo, 10 μl of the residue in serial concentration was used in an in vitro bioassay against anti-microbial screening model according to the filter paper disk diffusion methods [13] with the paper disk 5 mm in diameter. In bioassay-guided fractionation [14], the bioassay of fractions was conducted mainly against the sensitive test strains, S. aureus and S. cerevisiae Hansen, with other same parameters as above. Each fraction was tested in triplicate with dimethyl sulfoxide (DMSO) as a negative control.

Separation and Purification of Metabolites

Eupenicillium sp. was cultured in GSW medium at 28 °C and 200 rpm in a rotary shaker for 10 days. The culture broth (30 l) was filtered before being extracted with EtOAc for four times. The combined filtrates were concentrated in vacuo, and the obtained residue (9.8 g) was first fractionated by open column chromatography on silica gel (CHCl3–MeOH gradient). The activity tracking method or the bioassay-guided fractionation was applied throughout the separation and purification procedure. One bioactive fraction (0.6 g), obtained by elution with 98% CHCl3–MeOH on silica gel column, was further purified on a PHPLC equipped with a 10 mm × 250 mm ODS column (MeOH–H2O, 60:40). This fraction yielded 36 mg of compound 1 as colorless plate from MeOH. Another bioactive fraction (1.1 g), obtained by elution with 95% CHCl3–MeOH on silica gel column, was further purified on a Sephadex LH-20 column (CHCl3–MeOH, 1:1) and recrystallized from MeOH to yield 126 mg of compound 2 as a yellowy plate.

Bioassays and MIC Detections of the Purified Compounds

Anti-microbial activities and minimal inhibitory concentration (MIC) values of compound 1 and 2 (dissolved in DMSO) were assessed in vitro using anti-microbial screening strains as above by the agar serial dilution method described by Ter Laak et al. [15], with DMSO as a negative control and penicillin as a positive control. Each isolated compound of different concentration was performed in triplicate.

Using above anti-tumor screening cells, IC50 value of compounds 1 and 2 (in DMSO) against the four types of tumor cell lines was tested in triplicate according to the MTT method described in the literature [16], with DMSO as a negative control and Taxol™ as a positive control.

Characterizations and Structure Elucidations of Metabolites

Curvularin (Compound 1, See Scheme 1)

Colorless plate (MeOH), mp. 203–207 °C. EI-MS: 293 [M + H]+, MF: C16H20O5; 1H NMR data (acetone-D6, 500 MHz): 9.11(1 H, s, 16-OH), 8.69(1 H, s, 14-OH), 6.39(1 H, s, H-15), 6.34(1 H, d, J = 1.6 Hz, H-13), 4.91(1 H, m, H-8), 3.79(1 H, d, J = 15.7 Hz, H-11a), 3.70(1 H, d, J = 15.7 Hz, H-11b), 3.08(1 H, m, H-3a), 2.74(1 H, d, m, H-3b), 1.74(1 H, m, H-4a), 1.60(1 H, m, H-7b), 1.52(1 H, m, H-4b), 1.46(1 H, m, H-6a), 1.43(1 H, m, H-7a), 1.40(1 H, m, H-5a), 1.29(1 H, m, H-6b), 1.25(1 H, m, H-5b), 1.11(3 H, d, J = 6.3 Hz, 8-CH3); 13C NMR data (acetone-D6, 500 MHz): 207.3(C-2), 171.5(C-10), 160.6(C-16), 158.8(C-14), 137.5(C-12), 121.8(C-1), 112.8(C-13), 103.0(C-15), 73.1(C-8), 44.2(C-3), 40.2(C-11), 33.4(C-7), 28.0(C-5), 25.1(C-6), 23.9(C-4), 21.1(8-CH3). These data are identical to those described before elsewhere by Ghisalberti et al. [3, 17].

αβ-Dehydrocurvularin (Compound 2, See Scheme 1)

Yellowy plate (MeOH), mp. 218–223 °C. EI-MS: 291 [M + H]+; MF: C16H18O5; 1H NMR data (acetone-D6, 500 MHz): 10.20(1 H, s, 16-OH), 9.70(1 H, s, 14-OH), 6.77(1 H, m, H-3), 6.60(1 H, m, H-4), 6.35(1 H, s, H-13), 6.31(1 H, d, H-15), 4.74(1 H, m, H-8), 4.08(1H, d, J = 17.7 Hz, H-11a), 3.62(1H, d, J = 17.7 Hz, H-11b), 2.35(1 H, m, H-5b), 2.42(1 H, m, H-5a), 1.86(1 H, m, H-6a), 1.86(1 H, m, H-7a), 1.65(1 H, m, H-6b), 1.62(1 H, m, H-7b), 1.19(3H, d, J = 6.4 Hz, 8-CH3); 13C NMR data (acetone-D6, 500 MHz): 197.7(C-2), 172.3(C-10), 166.4(C-16), 163.6(C-14), 150.0(C-4), 139.9(C-12), 133.0(C-3), 116.0(C-1), 114.2(C-13), 103.4(C-15), 73.3(C-8), 44.1(C-11), 35.2(C-7), 33.6(C-5), 25.7(C-6), 22.0(4-CH3). These data are identical to those described in the literature [8, 18].

Results and Discussion

With anti-microbial model, especially with G+ indicator strain S. aureus and two fungal indicator strains S. sclerotiorum and S. cerevisiae, we found that one isolate from marine sponge Axinella sp. collected in South China Sea near Sanya, later identified as fungus Eupenicillium sp., had notably inhibitory activity in antagonistic test. So, further study on screening of bioactive compounds focused on Eupenicillium sp. Its main morphological characteristics are its tough, dense penicilli bearing long, broad columns of conidia, and its smooth-walled, unflanged ascospores which are produced within 14 days of inoculation onto GSW medium. It was reported that the oligotrophic culture media are more suitable for growth of marine-derived microbe [19, 20]. From several culture media including LB, SCKA, 2216E, PDA, YPD, PDAS, and GSW, the GSW medium with the least nutrition in 50% natural filtered sea water was selected for germination, growth, and fermentation of the fungus isolate because Eupenicillium sp. grew faster and produced more active compounds in this medium.

The antagonistic actions of the fungus Eupenicillium sp. was shown in Table 1, although nearly no anti-bacterial activity was detected when G− strains E. coli and P. aeruginosa were used as test strains, but its anti-Staphylococcus aureus and anti-fungal actions as indicated by the antagonistic tests were fairly similar to those of EtOAc extracts derived from its corresponding cultures. The results of anti-microbial activity tests showed that Eupenicillium sp. produced the effective anti-Staphylococcus aureus metabolites. Furthermore, the anti-Staphylococcus aureus activity of the EtOAc extract of the culture was stronger than to that of penicillin, which was used as a positive control under the same mass concentration (Table 1). As we have known, S. aureus, both hospital-acquired and community-acquired, which is the most common cause of staph infections, is a dangerous pathogen that involved in an increasing number of serious infections including acute bacterial meningitis with high risk for morbidity and mortality [21, 22]. Subsequently, a scaled-up culture of Eupenicillium sp. was prepared to purify and characterize the key anti-Staphylococcus aureus metabolites.

Table 1 The anti-microbial activity of Eupenicillium sp. from marine sponge Axinella sp.
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Using the anti-fungal and anti-Staphylococcus aureus bioassay-guided fractionation, two known metabolites (compounds 1 and 2) were purified from the EtOAc extract of Eupenicillium sp. Compound 1 was identified as curvularin and compound 2 was identified as αβ-dehydrocurvularin by comparing their spectral data with those of the authentic data [3, 8, 18] after the spectral analyses including MS, 1H NMR, 13C NMR, and DEPT.

From bioassay-guided fractionation procedure, it was ascertained that both purified compounds inhibited against two fungal indicator strains at a similar concentration. And more interestingly, αβ-dehydrocurvularin inhibited strongly against S. aureus, a Gram-positive low G + C bacteria that are often hospital-acquired and/or community-acquired pathogen, whereas curvularin has no activity at all (Fig. 1). Looking through their chemical structure (see Scheme 1), there is only one functional group difference between the two compounds; αβ-dehydrocurvularin has a double bond between C-3 and C-4 position but curvularin has none in the corresponding position. Zhang et al. [23] demonstrated that a double bond, the only structural difference between two flavonoids (as one counterpart), is one of the important structural properties essential for potent interaction between flavonoid and the breast cancer resistance protein, and the flavonoid compound with double bond is more active than its counterpart. It was reported that the bioactivity of some other types of chemical compounds with double bond is notably stronger than those of their corresponding hydrogenated derivates [24–26]. The presence of a 3,4-double bond in αβ-dehydrocurvularin exhibited the inhibitory activity against S. aureus whereas curvularin did not, indicating that the presence of the 3,4-double bond is critical for inhibiting S. aureus. Because αβ-dehydrocurvularin has a partial planar backbone structure due to the conjugation of π electrons in the presence of a 3,4-double bond and the carbonyl group at position C-2, in contrast to a non-planar conformation of curvularin lacking this double bond, it is very likely that this partial planar conformation may be beneficial for the binding of αβ-dehydrocurvularin to the binding site(s) of S. aureus. Respecting no anti-bacterial activity against G− strains for Eupenicillium sp., whether the mechanism of the activity against G+ bacteria is related to the bacterial cell wall or not needs further investigation.

Fig. 1
figure 1

The anti-Staphylococcus aureus activity difference between curvularin and αβ-dehydrocurvularin. 0: DMSO; 1: 500 μg/ml curvularin in DMSO; 2: 500 μg/ml αβ-dehydrocurvularin in DMSO assayed with the paper disk 5 mm in diameter on GSW medium using filter paper disk diffusing method described by de Beer and Sherwood [13]

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In addition, the two purified fungal metabolites were bioassayed against test G+ bacteria B. subtilis and S. aureus, and test fungi S. sclerotiorum and S. cerevisiae by the two-fold serial dilution method (DMSO as solvent) starting from initial concentration 1.2 × 104 μg/ml. The MIC values are shown in Table 2. The results based on bioassay-guided fractionation and MICs test suggested that curvularin and αβ-dehydrocurvularin are the two major anti-fungal components, and αβ-dehydrocurvularin is the only anti-Staphylococcus aureus component in the fungal culture of Eupenicillium sp. Both compounds were relative weakly bioactive against the two test fungi, but especially αβ-dehydrocurvularin was more strongly active against S. aureus with the MIC just one order of magnitude higher than that of penicillin used as a positive control.

Table 2 The minimal inhibitory concentration (MIC) values of curvularin and αβ-dehydrocurvularin (in μg/ml).
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Curvularin and its derivates, as cell division inhibitors [3, 27] and unique spindle poisons [5], can be used as mold compounds for research on molecular biology. Furthermore, it was reported that curvularin, an inhibitor against A549 cell, is a new transcriptionally based inhibitors of iNOS (inducible nitric-oxide synthase) acting on the Janus tyrosine kinase-STAT (the signal transducer and activator of transcription) pathway [28]. So it may represent lead structures for the development of drugs inhibiting iNOS-dependent overproduction of NO in pathophysiological situations. To fully understand the anti-tumor activity of curvularin and αβ-dehydrocurvularin, the IC50 values of the two compounds were assayed according to the MTT method. The results showed that both curvularin and αβ-dehydrocurvularin are actively against the four tumor cell lines (Table 3). The IC50 values of the two compounds against the four types of tumor cell lines, A549, HeLa, Ehrlich, and MCF-7, are listed in Table 3. The IC50 values of curvularin are almost always one order of magnitude higher than those of αβ-dehydrocurvularin against corresponding test cell lines, which might be due to the lack of the 3,4-double bond in the former compound.

Table 3 The IC50 values of compounds 1 and 2 against tumor cell lines (in μg/ml).
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In conclusion, this study demonstrated that curvularin and αβ-dehydrocurvularin isolated from Eupenicillium sp., a symbiotic fungus in marine sponge Axinella sp., were the two main structurally related secondary metabolites with slightly different antibiotic profiles. Both compounds are active against fungi and numerical cancer cell lines. But αβ-dehydrocurvularin showed bioactivity against S. aureus while curvularin did not, which indicates that the partial planar backbone structure in the presence of a 3,4-double bond plays an important role in the inhibition of S. aureus, a Gram-positive low G + C bacteria that are often the hospital-acquired and/or community-acquired pathogen.