Antifungal Activities of Compounds Produced by Newly Isolated Acrocarpospora Strains

In our continued search for bioactive metabolites from cultures of rare Actinobacteria resources from all over Taiwan and various natural ecological environments, an active antimicrobial strain of Acrocarpospora punica 04107M was collected in Taitung County in Taiwan and prepared from soil. The bioassay-guided fractionation of the BuOH extract of a culture broth from A. punica 04107M led to the isolation of five previously undescribed compounds: Acrocarposporins A–E (Compounds 1–5). All the constituents were confirmed by HRESIMS and 1D- and 2D-NMR spectroscopy. Their antifungal activity was also evaluated. Our results showed that four constituents (Compounds 1, 2, 4, and 5) possessed mild antifungal activity against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of Acrocarpospora punica 04107M has never been studied. This is the first report on diterpenoid metabolites from the genus Acrocarpospora.


Introduction
Actinobacteria are well known as excellent producers of prolific compounds with antimicrobial, insecticidal, and many other biological activities, and almost half of the active molecules discovered from natural sources belongs to this group. They are G(+), free-living saprophytic bacteria that are widely distributed in soil, water, and colonizing plants. Actinomycete inhabitants have been identified as one of the major groups in soil populations, which may vary by soil type. Streptomycetes produce a number of antibiotics and other bioactive compounds used in clinics. Their properties are prolific, and they can produce large quantities of antibiotics and various biologically active secondary metabolites [1][2][3][4][5][6][7]. However, the active metabolites of many Taiwanese neo-actinomycetes and their mechanisms of action remain unknown. It is necessary to study the active compounds from these new species of actinomycetes through scientific methods.
We recently isolated an unpublished novel strain, named 04107M, from the soil of Taitung County, which had a unique morphology and possessed antimicrobial activities as determined by our preliminary screening. This strain was determined to be Acrocarpospora punica 04107M based on its phenotypic and genotypic data.
The genus Acrocarpospora was first described by Tamura et al. [8] and is composed of the following three species: A. corrugatum, A. macrocephala, and A. pleiomorpha [8]. However, the microorganism discussed herein was isolated and identified by our research team. There have not been many chemical investigations of the genus Acrocarpospora, and only a few articles have reported on the classification of its molecular biology [9][10][11]. Currently, However, the microorganism discussed herein was isolated and identified by our research team. There have not been many chemical investigations of the genus Acrocarpospora, and only a few articles have reported on the classification of its molecular biology [9][10][11]. Currently, more than 300 microorganisms have been screened for in vitro antimicrobial activity and A. punica 04107M was found to be one of the active ones. In the course of our continuous investigation of the biologically active metabolites present in Actinobacteria, we report herein the isolation of a new strain, Acrocarpospora punica 04107M (Figure 1), and the chemical investigation of the butanol extract of A. punica 04107M culture broths, concluding our investigation with the isolation of five new diterpenoids (Compounds 1-5) ( Figure 2) and an evaluation of their antifungal activity.  ; and a resonance at δ H 2.28 (1H, br d, and J = 13.2 Hz) that was characteristic for the Hβ-1 signal of a dehydroabietane [12]. By comparing the 1 H-NMR of Compound 1 with that of the known compound ferruginol [12], it was deduced that Compound 1 was indeed an abietane diterpene ( Figure 2). Compound 2 had a molecular ion peak at m/z 314.2250 (HR-EI-MS) and was analyzed for the presence of C21H30O2. The IR spectrum of Compound 2 exhibited the presence of an OH group at 3389 cm −1 and aromatic moieties at 1610 and 1490 cm −1 . The UV absorptions (λmax 232 nm) confirmed the presence of a conjugated C=C bond and an aromatic system. Once again, seven IHD were determined from the molecular formula, the 13 C-NMR (Table 2), and the DEPT (DEPT-90 and DEPT-135) spectra. Further spectral data (Table 2 and Figure 1) and a comparison with similar compounds [13] established the structure of Compound 2 to be a 20(10→5)-abeo-4,5-seco-abietane naphthalene-type compound. The 1 H-NMR data indicated the presence of olefinic protons giving rise to AB-type resonances (δH 7.09 (1H, d, J = 8.4 Hz, and H-6) and 7.48 (1H, d, J = 8.4 Hz, and H-7)), an iPr group (δH 3.31 (1H, sep, J = 6.8 Hz, and H-15) and 1.33 (6H, d, J = 6.8 Hz, and Compound 1 was further confirmed to be a diterpene by the discovery of twenty 13 C-NMR signals, with five methyls, three methylenes, six methines, and six quaternary carbons being present. The quaternary carbons could be further divided into one oxygen-bearing carbon at δ H 153.5 (C-12) and three olefinic carbons at δ C 149.1 (C-9), 132.8 (C-13), and 126.9 (C-8). An inspection of the 13 C-NMR spectrum and the signals at δ C 153.5, 149.1, 132.8, 126.9, 128.1 (C-14), and 109.1 (C-11) supported the presence of a tetrasubstituted benzene ring. Resonances at δ C 76.9 (H-7) and δ C 43.9 (H-6) were attributable to oxygen-bearing aliphatic tertiary carbons, and C-6 appeared up-field due to a γ-gauche effect (δ C 43.9). Based on the above analysis, six of the seven IHD were consumed by one aromatic ring and two cyclohexane rings, while the remaining IHD could be attributed to an epoxy bridge (between C-6 and C-7), and this was also supported by correlations in the HMBC spectrum ( Figure 3). The base peak in the mass spectrum of Compound 1 at m/z 284 was [M + −16], which also confirmed the existence of an epoxide moiety. The epoxy moiety was further deduced from HMBC correlations of H-6/C-10, H-7/C-5, H-14/C-7, and H-5/C-6 and C-7; COSY correlations between H-6 and H-7; and finally NOESY couplings between H-5 and H 3 -18, H-6 and H 3 -20, H-16 and H-11, and H-14 and H-7. The NOESY spectrum ( Figure 4) established a β-quasi-equatorial orientation of the two epoxide protons, which was confirmed by their coupling constant J(H-6/7) = 4.2 Hz. Additionally, a lower chemical shift at δ H 7.27 for H-14 (1H, s) was due to deshielding by the epoxy group at positions C-6 and C-7. Thus, the structure of Compound 1 was determined to be 6α,7α-epoxyferruginol and given the trivial name Acrocarposporin A.

Structure Elucidation of Compounds
Compound 2 had a molecular ion peak at m/z 314.2250 (HR-EI-MS) and was analyzed for the presence of C 21 H 30 O 2 . The IR spectrum of Compound 2 exhibited the presence of an OH group at 3389 cm −1 and aromatic moieties at 1610 and 1490 cm −1 . The UV absorptions (λ max 232 nm) confirmed the presence of a conjugated C=C bond and an aromatic system. Once again, seven IHD were determined from the molecular formula, the 13 C-NMR (Table 1), and the DEPT (DEPT-90 and DEPT-135) spectra. Further spectral data (Table 1 and Figure 1) and a comparison with similar compounds [13]        The 13 C-NMR and DEPT spectra showed two tetrasubstituted benzene rings, an iPr group, and a 4-methoxy-4-methylpentyl side chain. From the seven IHD, it could be deduced that Compound 2 contained one naphthalene group. With the presence of one benzylic Me group and a side chain (4-methoxy-4-methylpentyl), Compound 2 could be assumed to be a 20(10→5)abeo-4,5-secoabieta-5(10),6,8,11,13-pentaen-4,13-diol compound but with its C-4 bearing a OMe group instead of an OH group. The following HMBC correlations were observed, which supported this assumption: OMe-4 with C-4 (δ C 74.8) and Me-18/19 with C-4 and C-3 (δ C 40.6).
Compound 3, isolated as an oil, exhibited a molecular ion (M + ) peak at m/z 318.2209 for C 20 H 30 O 3 , corresponding to six indices of hydrogen deficiency (IHD). The IR spectrum of Compound 3 displayed an absorption for an OH group at 3410 cm −1 of 1709 (C=O) and bands at 1611 and 1508 (aromatic). Our interpretation of the 1 H-NMR spectrum of Compound 3 (Table 2) revealed the signals for two methyl groups (δ H 0.71 (3H, s, and Me-19) and 1.08 (3H, s, and Me-18)), an iPr group (δ H 1.15 (3H, d, J = 7.0 Hz, and H 3 -16), 1.17 (3H, d, J = 7.0 Hz, H 3 -17) and 3.17 (1H, sept, J = 7.0 Hz, and H-15)), one OMe attached to an aromatic ring (δ H 3.79 (3H, s, and OMe-13)), and one tetrasubstituted aromatic system with two singlet aromatic protons (δ H 6.51 (1H, s, and H-14); 6.82 (1H, s, and H-11)) in addition to a phenolic hydrogen signal (δ H 7.64, exchangeable) and associated with the carbon to which it was attached (δ C 154.1, C-9), which were all in accordance with the presence of an abietane diterpenoid [14]. However, diterpenoids of the abietane class, in addition to the methyls of the iPr group, should have three singlet methyls; however, Compound 3 had only two present in its 1 H-NMR spectrum. It can be speculated that one of the singlet methyl groups was absent due to oxidation and decarboxylation. A carbon signal at δ C 216.9 (C-10) was presumed to be a ketone, while from the 1 H-NMR three protons at δ H 2.35 (m; H1-α), 2.46 (br d, J = 15.0 Hz, and H-1β), and 2.21 (m; H-5) were all affected by the ketone group and shifted to a lower magnetic field. Further evidence came from the IHD analysis, which showed that five of the six IHD were consumed by one aromatic ring and one cyclohexane ring, and the remaining IHD were due to the presence of a C=O group. Compound 3 could, therefore, be a 20-norabietane derivative (a 9,10-secoabietane-type compound).
Compound 4 was obtained as a colorless oil. Its molecular formula was determined as C 21 H 28 O 3 on the basis of an M + peak at m/z 328.2034 (calculated 328.2033 for C 21 H 28 O 3 ) in its HREIMS. UV absorptions (λ max 223 and 281 nm) again confirmed the presence of a benzenoid moiety [15]. The IR spectrum of Compound 4 exhibited the presence of an ester at 1745 cm −1 and an aromatic moiety at 1633 and 1514 cm −1 . Eight indices of hydrogen deficiency (IHD) were determined from the molecular formula, 13 C-NMR (Table 2), and DEPT spectra. The 1 H-NMR spectrum of Compound 4 indicated the presence of an iPr group (δ H 1.21 (6H, d, J = 7.2 Hz, and H-16/17) and δ H 3.19 (1H, sep, J = 7.2 Hz, and H-15)), two Me groups (δ H 1.06 (3H, s, and Me-19) and 1.15 (3H, s, and Me-18)) attached to a quaternary C-atom, one OMe (δ H 3.79 (3H, s, and OMe-13)), one methine (δ H 1.78 (1H, d, J = 13.0 Hz, and H-5)), two singlet aromatic protons at δ H 6.65 (1H, s, and H-14) and 6.89 (1H, s, and H-11) of a tetrasubstituted benzene, and four aliphatic methylenes. The 13 C-NMR and DEPT spectra showed signals of an iPr group; two Me groups; one oxygenated methine (δ C 84.2), which was associated with an OC=O group (δ C 177.1); two aromatic C-atoms; and four CH 2 and two CH groups. The 21 C-signals including two Me and an iPr group were again in accordance with an abietane derivative. However, as with Compound 3, abietane diterpenes should normally possess three singlet methyl groups, and Compound 4 only had two in its 1 H-NMR spectrum. It was speculated that one of the methyl groups may be oxidized to a lactone, which was supported by the presence of an IR absorption band at 1745 cm −1 and a 13 C-NMR signal at δ C 177.1.
A signal in the carbon spectrum at δc 177.1 was presumed to be that of an ester carbonyl group, and, due to HMBC correlations with this carbon between H 2 -1 and H-5, it could be placed at C-20. Further HMBC correlations between an oxymethine signal at δ 4.20 (d, H-3) with C-18, C-19, and C-20 supported the notion that a lactone bridge was present between C-3 and C-10. Therefore, Compound 4 was confirmed as a new lactonic abietane diterpene and was named Acrocarposporin D.  Table 2) displayed signals of one triple-substituted double bond conjugated with carbonyl groups (δ H 6.60 (1H, t, and J = 6.0 Hz)), exomethylene H-atoms (δ H 4.70 (1H; br s) and 4.22 (1H; br s)), and signals of methylene and methyl groups on acetyloxymethylene (CH 2 OAc) (δ H 4.80 (2H, br d, and J = 1.2 Hz) and 2.09 (3H, s)). Signals of one C=O C-atom (δ C 200.9 (C-12), 181.4 (C-19), and 171.0 (C-21)), one trisubstituted double bond (δ C 139.6 (C-14) and 133.5 (C-13)), and an exoethylene group (δ C 148.9 (C-8); 106.3 (C-17)) were observed in the 13 C-NMR and DEPT (DEPT90 and DEPT135) spectra (Table 1), which were responsible for five out of seven unsaturation degrees. The remaining two were ascribed to the presence of two rings. It was inferred to be a bicyclic structure with two six-membered rings; thus, it was deduced to be a labdane diterpenoid.
From the HMBC plot, one oxymethylene at δ H 4.80 (2H, br d, and J = 1.2 Hz) is correlated with δ C 170.7, 140.1 (C-14), and 134.6 (C-13), so it was inferred that the signal at δ H 4.80 (2H, br d, and J = 1.2 Hz) is between acetoxy and trisubstituted double bonds. Whereas the carbon signal at δ C 200.4 is correlated with H-11 and δ H 6.60 (1H, br d, and J = 1.2 Hz); thus, it can be inferred from the above that the ketone group is located at C-12 and δ H 6.60 (1H, br d, and J = 1.2 Hz) corresponded to H-13.
According to the NOESY spectrum, H-13/15 were both correlated with H-16 and H-13 was correlated with H-11, which confirms that the double bond is a Z-form. The correlation of H-18 with H-5 could be used to determine the equatorial orientation of CH 3 -18. Compound 5 was confirmed to be 12-oxo-15-acetoxylabda-8(17),13(Z)-dien-19-oic acid via HSQC and COSY and was named Acrocarposporin E.

Discussion
Generally speaking, actinomycetes have been developed as a large library that can be expected to provide a variety of structurally unique and pharmacologically active natural products. Secondary metabolites of the genus Acrocarpospora have been rarely studied. The A. punica-type strain 04107M-2 T had only 10 components reported by our team in the past [16].
After modifying the fermentation conditions, we obtained five new components from the BuOH active layer and the isolated backbones, including 20(10→5)abeo-4,5secoabietane naphthalene analogs, abietane, and 20-nor-9,10-secoabietane metabolites. Abietanes are naturally occurring tricyclic diterpenoids that have been isolated from multiple terrestrial plant sources, especially from conifer resins [17,18]. Abietic acid and dehydroabietic acid are the main components of pine resin obtained from Pinus sp. [19]. Colophony, the distillation residue of pine oleoresin, is the main source for abietanes, along with conifers other than pines belonging to the Araucariaceae, Cupressaceae, Phyllocladaceae, Pinaceae, and Podocarpaceae families.
Asteraceae, Celastraceae, Hydrocharitaceae, and Lamiaceae, along with some fungal species, are also known to produce abietane diterpenoids [20]. In 2016, a novel norditerpenoid, actinomadurol, was isolated from the rare actinomycete strain, Actinomadura sp. KC 191, together with the known compound JBIR-65 [21]. To the best of our knowledge, this is the first report of abietane-type metabolites from the actinomycete genus Acrocarpospora. These results demonstrate that Acrocarpospora produces unique and diverse metabolites in different fermentation conditions and soil-derived collections. Therefore, in a special ecological environment, more natural products with biological activity may be found by searching for Acrocarpospora species.

Biological Studies
Culture broth from A. punica 04107M was tested for antifungal activity against the following fungi: Aspergillus niger (BCRC-31512), Penicillium italicum (BCRC-30567), Candida albicans (BCRC-21538), and Saccharomyces cerevisiae (BCRC-20822). The antifungal data are shown in Table 3, and the clinically used antifungal drug ketoconazole was employed as a positive control. Table 3. Antifungal activity of six sufficient compounds isolated from the culture broth of A. punica 04107M (diameter of the zone of growth-inhibitory fungicidal zone is given in mm, including the diameter of the disk, which is 8 mm). Inhibitory zone diameter (mm); + inhibitory zone; positive control (STD): ketoconazole. Each value represents the mean ± SD.
The inhibitory activity of Compounds 1, 2, 4, and 5 against A. niger, P. italicum, C. albicans, and S. cerevisiae was further tested using the method described in the experimental section (Table 4). Compound 1 has inhibitory activity against A. niger, P. italicum, and C. albicans strains with MIC values of 54.87, 53.98, and 49.56 µg/mL. Compound 2 has inhibitory activity against Saccharomyces cerevisiae with an MIC value of 57.38 µg/mL. Compound 4 was found to have moderate inhibitory activity against the P. italicum, C. albicans, and S. cerevisiae strains with MIC values ranging from 38.89 to 42.78 µg/mL. Compound 5 also had MIC values of 59.78, 51.32, and 56.92 µg/mL against A. niger, C. albicans, and S. cerevisiae, respectively. They were less biologically active than the reference compound, ketoconazole, which had MIC values of 3.25, 6.72, 11.79, and 3.16 µg/mL against A. niger, Pseudomonas italia, C. albicans, and S. cerevisiae, respectively. In this bioassay, no antifungal activity (MIC > 100) was observed for Compound 3 at concentrations below 100 µg/mL.

Microorganism, Cultivation, and Preparation of the Actinobacteria Strain
This strain was isolated from soil samples collected in Taitung County, Taiwan, using HV agar and cultured at 28 • C for 3 weeks. The strain was kept on oat agar and the spores or mycelial fragments of the strain were suspended in broth containing 20% (v/v) glycerol and stored at -20 • C. The medium for the inoculum contained malt extract, 3 g; yeast extract, 3 g; glucose, 5 g; agar, 1.5 g; and 1 L of distilled water. The initial pH of the medium was 8. The synthetic medium contained Glucose, 20 g; Sodium Glutamate (MSG), 10 g; K2HPO4, 5 g; KH2PO4, 5 g; MgSO4•7H2O, 1.0 g; KCl, 0.5 g; ZnSO4•7H2O, 0.01 g; FeSO4•7H2O, 0.01g; and MnSO4•H2O, 0.003 per liter of distilled water. The initial pH of the medium was adjusted to 5.5. The tilted cultures were maintained on Difco potato dextrose agar (PDA). Spores of the strain were prepared by growing them on PDA slants at 28 • C for 14 days. The spores were washed with sterile water. A 5L Erlenmeyer flask was incubated containing 2L of inoculation medium with the suspension of 107 spores for 3 days at 28 • C on a rotary shaker. This inoculum was transferred to a 100 L fermenter (B. Braun, Germany) containing 30 L of synthetic medium, which was operated at 100 rpm and 30 • C with an aeration rate of 0.3 vvm. After 14 days of culture, fermentation was stopped, and the liquid culture was separated from the mycelium by filtration.

Isolation and Characterization of Secondary Metabolites
The culture filtrate (20 l) was repeatedly extracted with BuOH five times. The pooled BuOH fraction was evaporated under vacuum to yield 78 gm of dark brown residue; the extract was then applied to normal-phase column chromatography packed with silica gel and eluted with methylene chloride/ethyl acetate/acetone/methanol in a stepwise gradient mode to yield 9 fractions (1 to 9). Fraction 3 (2897 mg) was purified by normalphase MPLC, which was eluted with CH2Cl2-EtOAc (5:1) to afford Acrocarposporin B (Compound 2) (4.2 mg).

Antifungal Activity Assays
The assays tested for the presence of microorganisms. The in vitro antifungal activity of Compounds 1-5 was tested against a panel of laboratory control strains belonging to the Bioresource Collection and Research Center (BCRC) in Hsinchu, Taiwan, namely, the fungal organisms Aspergillus niger (BCRC-31512), Penicillium italicum (BCRC-30567), Candida albicans (BCRC-21538), and Saccharomyces cerevisiae (BCRC-20822).

By Disk Diffusion Assay
Antifungal susceptibility testing of the isolated compounds was performed with the following strains: Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae by the disk diffusion method and the following CLSI guidelines were applied: M44-A and M44-S2 for yeasts [22,23] and M-51P for filamentous fungi [24]. A standard disk of ketoconazole was used as a positive control, while a disk imbued with 50 µL of pure DMSO was used as a negative control. The diameters of the inhibition zones were measured in millimeters by means of a slide caliper. Each test was performed in triplicate, and the results were analyzed for statistical significance [22][23][24].

By Broth Dilution Assay
The MIC determination for the antifungal assay was performed according to the Clinical and Laboratory Standard Institute (CLSI) using the broth dilution assay method [25][26][27]. Extract stock solutions and partitions were prepared in 5% DMSO, and twofold serial dilutions were prepared in RPMI in 96-well microtiter plates (Corning Incorporated, Corning, NY, USA). The final concentrations ranged from 0.98 to 2.000 g mL −1 . Test organisms (100 µL) were added to each well in microtiter plates. The growth control contained medium and inoculum. Blank controls contained medium only. The microtiter plates were then incubated at 35 • C and the endpoints were read after 48 h. The lowest concentration for each test compound at which color change occurred was recorded as its primary MIC value. The average of primary values from three individual tests were calculated, and the average was taken as the final MIC value for each of the test compounds.

Conclusions
Actinomycetes have potential economic and biotechnological value and have long been recognized as major microorganisms in the medical industry. To date, there are tens of thousands of antibiotics produced by microorganisms, of which more than 70% are derived from Actinobacteria [28]. The secondary metabolites of Actinobacteria have various structures and biological activities, including antibacterial, antifungal, antitumor, insecticidal, and herbicidal properties; enzyme inhibitory activity; and immune regulatory activity [29,30], indicating that Actinobacteria have great potential with respect to the development of new medicine. As part of our investigations aimed at exploring structurally novel, bioactive secondary metabolites from actinomycetes, the chemical research on the fermentation extract of Acrocarpospora punica led to the isolation of five previously undescribed compounds, namely, Acrocarposporins A-E (Compounds 1-5) (Figure 2). The structures of these isolates were determined by spectroscopic experiments. The BuOH soluble fraction from the A. punica fermentation broth was tested for antifungal activities. Our results indicated that Compounds 1, 2, 4, and 5 displayed moderate antifungal activities against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of Acrocarpospora punica 04107M has never been studied. This is the first report on diterpenoid metabolites from the genus Acrocarpospora.