Skip to content
Publicly Available Published by De Gruyter December 28, 2016

New oxaphenalene derivative from marine-derived Streptomyces griseorubens sp. ASMR4

  • Abdelaaty Hamed , Ahmed S. Abdel-Razek , Marcel Frese , Daniel Wibberg , Atef F. El-Haddad , Tarek M. A. Ibrahim , Jörn Kalinowski , Norbert Sewald and Mohamed Shaaban EMAIL logo

Abstract

During our search for novel bioactive compounds from extremophilic actinomycetes, the new Streptomyces griseorubens sp. ASMR4 was isolated from a soft coral collected in the Red Sea at the Hurghada coast, Egypt, and characterized taxonomically. It was fermented on large scale using a modified solid rice medium as the first example for actinomycetes so far. Work-up and purification of the strain extract using different chromatographic techniques afforded the new oxaphenalene derivative, 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a), together with seven known metabolites: ferulic acid (2), glycerol linoleate, linoleic acid methyl ester, (3R,4R)-3,4-dihydroxy-3-methylpentan-2-one/(3S,4R)-3,4-dihydroxy-3-methylpentan-2-one, anthranilic acid, phenylacetic acid, and benzoic acid. The chemical structure of the new compound (1a) was confirmed by extensive 1D and 2D NMR spectroscopy, high-resolution electron impact mass measurements, and by comparison with literature data. The antimicrobial activity of the strain extract and compounds 1a and 2 were studied using a panel of pathogenic microorganisms. The in vitro cytotoxicity of the bacterial extract was studied against the human cervix carcinoma cell line (KB-3-1) and its multi-drug-resistant subclone (KB-V1).

1 Introduction

Actinomycetes represent the most valuable microorganisms for the production and synthesis of economically important therapeutic compounds [1]. Until now, more than 10000 antibiotics have been isolated from actinomycetes [2]. They are responsible for the production of almost 50% of discovered bioactive compounds [3], including antitumor agents [4], [5], antivirals [6], antimycotics [7], antibiotics [8], [9], enzymes [10], [11], immunosuppressive agents [12], and other pharmacologically active agents [13], [14], [15].

2 Results and discussion

In the course of our ongoing search for new bioactive compounds from microorganisms, a new marine actinomycete strain was isolated and classified as Streptomyces griseorubens sp. ASMR4, on the basis of its 16S rRNA gene sequencing, cultivation behavior, and morphological and physiological characteristics. The strain ASMR4 was isolated from a soft coral collected in the Red Sea at the coast of Hurghada, Egypt. Cultivation of the strain on a solid rice medium was performed as a first example for actinomycetes. Chemical studies on an extract obtained from the culture broth of this strain using different chromatographic techniques afforded the novel oxaphenalene derivative, 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a), besides seven known metabolites: ferulic acid (2), glycerol linoleate, linoleic acid methyl ester, (3R,4R)-3,4-dihydroxy-3-methylpentan-2-one/(3S,4R)-3,4-dihydroxy-3-methyl-pentan-2-one, anthranilic acid, phenyl acetic acid, and benzoic acid. The chemical structure of the new metabolite (1a) was assigned on the basis of 1D and 2D NMR spectroscopy and high-resolution electron impact mass spectroscopy (HREIMS).

2.1 Morphological, cultural, and phenotypic characteristics

Morphological observations of the isolated strain grown on different International Streptomyces Project (ISP) media (see Table S1, Supplementary Information) revealed characteristics typical to members of the genus Streptomyces. Cultures of the strain ASMR4 grew well on all ISP media used (ISP2-ISP5). The color of the aerial mycelium is mainly grayish white, whereas the reverse side was yellow on ISP2 and green on ISP5, and showed a pale yellow pigment on Czapek Dox agar (Table S1, Supplementary Information). Based on the microscopic examinations (Fig. 1a and b), spores of the strain ASMR4 are short, spiral, and have smooth surface. However, flexuous sporophore is not dominant. Physiologically, the strain ASMR4 produces H2S and utilizes d-glucose, d-xylose, d-mannose, l-arabinose, and sucrose for growth (Table S2, Supplementary Information).

Fig. 1: Spore chain structure of the strain ASMR4 grown on an ISP4 medium (50% sea water, 10 days, 37°C). (a) Light microscopy of sporulating mycelium (1200×) showing spiral spore chains; (b) transmission electron micrography of the strain showing a spore chain with a smooth surface (bar=2 μm).
Fig. 1:

Spore chain structure of the strain ASMR4 grown on an ISP4 medium (50% sea water, 10 days, 37°C). (a) Light microscopy of sporulating mycelium (1200×) showing spiral spore chains; (b) transmission electron micrography of the strain showing a spore chain with a smooth surface (bar=2 μm).

For taxonomic classification of the strain ASMR4, its sequence was amplified by applying polymerase chain reaction (PCR), sequenced and analyzed in a BLAST-based approach. An 870 bp long DNA sequence was obtained, where the phylogenetic tree indicates the existence of an inter-relationship between several related sequences to the strain ASMR4 [16]. Consequently, phylogenetic analysis of 16S rRNA gene sequence confirmed the highly closed relation of the isolate ASMR4 to S. griseorubens (99%) and Streptomyces olivaceus (99%). The results were supported by neighbor-joining-based phylogenetic tree (Fig. 2). However, the differential characteristics between the strain ASMR4 and its closest phylogenetic neighbors using a combination of phenotypic tests (Table 1) confirmed its novelty. Therefore, we have assigned its name as S. griseorubens sp. ASMR4 (GenBank accession no. KU740035).

Fig. 2: Neighbor-joining phylogenetic tree of the strain ASMR4 based on 16S rRNA gene sequences, showing its close relationship to Streptomyces species.
Fig. 2:

Neighbor-joining phylogenetic tree of the strain ASMR4 based on 16S rRNA gene sequences, showing its close relationship to Streptomyces species.

Table 1:

Comparison between strain ASMR4 characteristics and its closely related Streptomyces species.a

CharacteristicStrain ASMR4S. griseorubens [17], [18], [19], [20], [21]S. olivaceus [17], [18], [19], [20], [21]
Spore massGrayish whiteGrayish redGray
Spore surfaceSmoothSpiny with very short spinesSmooth
Spore chain morphologyShort (3–8 spores); S.Short (3–8 or 10 spores); S. or Rf.; hooks, loops are presentLong (>50 spores); S., sometimes open spirals
Reverse colorBrown on ISP2 and green on ISP5Gray red with no distinctive pigments on ISP2 and ISP5Gray yellow on ISP2 and ISP5
Sucrose utilization+
H2S production+ND
Cellulose decomposition+ND
Highest sequence similarityS. griseorubens (99%)S. labedae (99.9%)S. pactum (100%)

a(+) positive; (−) negative; (ND) no data available; (Rf.) rectiflexibilis; (S.) spirales.

2.2 Fermentation and structure elucidation

The S. griseorubens sp. ASMR4 was cultivated on a solid rice medium; containing malt extract (1%) and yeast extract (0.4%). The methanol extract of the strain exhibited moderate activity against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Saccharomyces cerevisiae, and Candida albicans, whereas it showed high activity against Aspergillus niger (Table 4). In the chemical screening monitored by thin-layer chromatography (TLC), the bacterial extract exhibited numerous bands in a wide polarity range: some of them contained a group of non-UV-absorbing compounds detected as intensive violet to blue bands with anisaldehyde-sulfuric acid. UV absorbing bands exhibited pink coloration with anisaldehyde-sulfuric acid; others were yellow on TLC and turned dark green with this reagent. Separation of metabolites produced by the strain using a series of chromatographic techniques afforded the novel 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a) and ferulic acid (2). In addition, the bacterial extract afforded further six known compounds: glycerol monolinoleate [22], linoleic acid methyl ester, (3R,4R)-3,4-dihydroxy-3-methylpentan-2-one/(3S,4R)-3,4-dihydroxy-3-methyl-pentan-2-one as a mixture of diastereomers [23], [24], anthranilic acid, phenyl acetic acid, and benzoic acid [24], [25]. Their structures were confirmed by comparison of the spectroscopic data and chromatographic properties with the literature. The physicochemical properties of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a) are listed in Table 2.

Table 2:

Physicochemical properties of 8-hydroxy-2- (2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a).

AppearanceYellow solid
Rfa0.52b
Staining with anisaldehyde-sulfuric acidDark green
Molecular formulaC17H16O4 (284)
(+)-ESI-MS: m/z591 ([2M+Na]+)
(−)-ESI-MS: m/z283 ([M–H]+)
EIMS: m/z (%)284 [(13) M]+, 279 (16), 266 (6) [M–CO]+, 260 (22), 252 (17), 207 (47), 167 (44), 149 (100), 144 (64), 105 (64)
HREIMS: m/z
Found284.10353 [M+]
Calcd.284.10431
[a]D20−47 (c=0.1,CH3CN)
UV/VIS: λmax(log ε)(CH3CN): 412 (4.02); 390 (4.05), 285 (4.04), 229 (4.31)

aSilica gel G/UV254.

b(CH2Cl2-5% MeOH).

2.2.1 8-Hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene

Compound 1a was obtained as moderately polar yellow solid, which appeared as a yellow band on TLC with UV absorbance (254 nm), and greenish-blue fluorescence at 366 nm. On spraying with anisaldehyde-sulfuric acid, compound 1a exhibited a dark green color. In contrast, the compound did not show any color change on exposure to sodium hydroxide or conc. sulfuric acid, excluding its nature as hydroxyquinone or lactone system [26]. The UV spectrum of 1a displayed three major peaks at λmax=412,390 and 285 nm, indicating conjugated character that matched with the chromatographic behavior as described above (Table 2). The molecular weight was determined by electrospray ionization (ESI)-MS: The quasi-molecular ion peak was detected both in the positive and negative ESI-MS modes, confirming the molecular mass of 1a as m/z=284 Da. HREIMS of 1a provided the molecular formula C17H16O4, containing 10 double bond equivalents.

The 1H NMR spectra (Table 3) of 1a revealed several spin systems: one singlet of 1H signal at δ=14.49 ppm being for a peri-hydroxy group; five aromatic signals each of 1H, among which three signals of protons of a 1,2,3-trisubstituted aromatic residue were visible at δ=7.67 (d), 7.45 (t), and 6.83 (d) ppm. Two further signals were observed as two singlets at δ=6.43 and 6.08 ppm, which are attributed mostly to further two penta-substituted aromatic residues. Three signals in the aliphatic region correspond to an oxymethine (δ=4.24 ppm, m), an sp2-bound methylene (δ=2.57 ppm), and a methyl group (δ=1.35 ppm, d). Based on the H,H correlation spectroscopy (COSY) correlations (Fig. 3), the latter three aliphatic signals are assigned to an isopropanol partial structure. An additional methyl group (δ=2.77 ppm, s) was assigned to an acetyl group bound to an aromatic residue. Based on the 1H NMR features and molecular formula, compound 1a bears three aromatic rings (1,2,3-trisubstituted ring and two penta-substituted rings), together with an acetyl and a 2-hydroxypropyl group.

Table 3:

13C NMR (125 MHz) and 1H NMR (500 MHz) data of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a) in CDCl3.

No.δC (ppm)δH(J in Hz)No.δC (ppm)δH(J in Hz)
2152.78-OH14.49 (s)
3106.26.08 (s)999.66.43 (s)
3a130.59a159.5
4115.76.83 (d, 7.9)9b117.8
5130.57.45 (t,7.9)1042.92.57 (m)
6121.87.67 (d, 7.9)1165.74.24 (m)
6a134.21223.21.35 (d, 6.2)
7110.013201.8
8168.41431.82.77 (s)
Fig. 3: H,H COSY (↔,▬) and HMBC (→) correlations of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a).
Fig. 3:

H,H COSY (↔,) and HMBC (→) correlations of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a).

According to the 13C NMR and heteronuclear multiple-quantum coherence (HMQC) data, compound 1a exhibited 17 carbon signals, among them 13 of sp2 and 4 sp3 carbon atoms. The aromatic carbons were classified into five aromatic CH signals (δ=130.5, 121.8, 115.7, 99.6 ppm) and eight quaternary carbons; one of the latter is characteristic to a carbonyl of acetophenone (δ=201.8 ppm), in addition to further three signals (δ=168.4, 159.5, 152.7 ppm) assigned to phenolic carbons. In the aliphatic region, signals of two methyl groups (δ=31.8, 23.2 ppm), one methylene group (δ=42.9 ppm), and one oxymethine group (δ=65.7 ppm) were found, confirming the existence of an acetophenone moiety, and a 2-hydroxypropyl partial structure as mentioned above.

A search in different databases (AntiBase [1], Dictionary of Natural Products [27], and Scifinder [https://scifinder.cas.org/scifinder]) considering the NMR results and the molecular formula of compound 1a confirmed its structural novelty. Consequently, an intensive study for the NMR data based on heteronuclear multiple-bond correlation (HMBC) and H,H COSY correlations was carried out. Based on the HMBC connectivities, the acetyl group is attached to C-7 (δ=110.0 ppm) as the existence of a 3J coupling from the singlet methyl group (H3-14, δ=2.77 ppm) at it, along with a direct 2J correlation to the vicinal carbonyl C-13 (δ=201.8 ppm). The peri-hydroxyl group (δH=14.49 ppm) displayed three correlations atδ=168.4 ppm (2J, C-8), 110.0 ppm (3J, C-7), and 99.6 ppm (3J, C-9), confirming its location at C-8. Furthermore, the neighboring methine proton (CH-9, δH=6.43 ppm) exhibited four 3J correlations versus C-7, the phenolic carbon C-9a (δH=159.5 ppm) and C-9b (δH=117.8 ppm) in addition to a 2J coupling at C-8 (δH=168.4 ppm), establishing the first penta-substituted aromatic residue (A), except those of C-6a (δH=130.5 ppm). On the other hand, the three aromatic protons (H-6, H-5, H-4) of aromatic ring B displayed the following assignments: H-6 (δ=7.67 ppm) exhibited four essential HMBC correlations at C-7 (δ=110.0 ppm), C-9b (δ=117.8 ppm) and C-4 (δ=115.7 ppm), and C-6a (δ=134.2 ppm), establishing the fusion of both aromatic residues A and B across C-9b and C-6a. The triplet aromatic proton H-5 (δ=7.45 ppm) showed an additional 3J coupling at C-6 in addition to C-3a (δ=130.5 ppm), whereas the doublet aromatic proton H-4 (δ=6.83 ppm) showed two rather essential correlations at C-9b and the singlet aromatic methine CH-3 (δ=106.2 ppm), confirming the direct attachment of C-3 to C-3a. Consequently, H-3 (δ=6.08 ppm) exhibited multiple HMBC correlations, including 3J with C-9b, C-4, and the methylene carbon CH2-10 (δ=42.9 ppm) along with a 2J correlation to the third phenolic carbon C-2 (δ=152.7 ppm). According to these correlations, the methylene group CH2-10 is directly attached to C-2 via a 2J HMBC cross section along with a 3J correlation to CH-3. In addition, CH2-10 was proved by an H,H COSY correlation to be directly connected to the sp3-oxygenated methine carbon C-11 (δ=65.7 ppm); and the latter is attached to the terminal doublet methyl CH3-13 (δC=23.2, δH=1.35 ppm), confirming the direct attachment of the 2-(2-hydroxypropyl) group (via CH2-10) to the phenolic carbon C-2.

Based on the above intensive studies of HMBC and H,H COSY correlations (Fig. 3), and the molecular formula of 1a, three alternative structures have been considered according to the ring closure system: 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a), 1-[2,4-dihydroxy-5-(4-methyl-oxetan-2-ylidenemethyl)-naphthalen-1-yl]-ethanone (1b), and 1-(5,10-dihydroxy-8-methyl-8,9-dihydro-7-oxa-cycloocta[d,e]naphthalen-4-yl)-ethanone (1c). In structure 1c, a ring closure between the phenolic C-9a and hydroxymethine C-11 gives an eight-membered ring fused with the remaining naphthalene system with a second phenolic hydroxyl group at C-2. Because of the absence of an HMBC correlation between H-11 and C-9a, and the lower chemical shift of C-11 than the expected value, structure 1c was excluded. Alternatively, structure 1b is characterized by an oxetane ring, a very rare skeleton in nature due to its high strain. In addition, because of the lower chemical shift of the oxymethine carbon C-11 than the expected one for such a structure, 1b was discarded too. Structure 1a is the most and sole reasonable structure in agreement with the stability, chromatographic property, and the NMR data discussed above.

Based on the nuclear Overhauser effect spectroscopy (NOESY) spectrum, a correlation between H-4 and H-3 was confirmed and the latter exhibited connectivity with the methylene protons H2-10 at δ=2.57 ppm. However, the proton of the chiral carbon C-11 (δ=4.24 ppm) did not exhibit any coupling either with H-9 or H-3 (Fig. 4). Moreover, the CD spectrum (Fig. S12, Supplementary Information) of 1a showed no definite absorbing signals, which might be attributed to that C-11 is not directly attached to the aromatic system of 1a. On the other hand, compound 1a showed a negative optical rotation ([a]D20=47°) at 589 nm (Table 2), which, however, is insufficient to deduce the absolute configuration at C-11. A further confirmation of the configuration is unfortunately not possible as the remaining quantity of 1a has been consumed during the biological activity testing.

Fig. 4: NOESY connectivities of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a).
Fig. 4:

NOESY connectivities of 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a).

1-Oxaphenalene derivatives are very rare in nature, exhibiting a wide range of high antimicrobial activities. For example, the antibiotic biflorin was isolated from the roots of Capariabiflora L (Scrophulariaceae), exhibiting a broad spectrum of activity against diverse pathogenic microorganisms [28].

2.2.2 Ferulic acid

Ferulic acid (FA, 2), a highly antioxidant agent, is commonly found in commelinid plants (rice, wheat, oats, and pineapple), grasses, grains, vegetables, flowers, fruits, leaves, beans, seeds of coffee, artichoke, peanut, and nuts [29]. A combination of commercial enzymes that show FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes were reported to enhance the enzymatic production of FA from defatted rice bran [30]. We report herein production of FA from Streptomyces sp. without the addition of any combined enzymes, referring to the high capability of S. griseorubens sp. ASMR4 to release the corresponding enzymes, and hence to afford the free FA from rice used as the cultivation medium.

2.3 Biological activities

Antimicrobial activity testing of the crude extract of S. griseorubens sp. ASMR4 was carried out against a set of microorganisms using the agar diffusion technique. Filtrate, mycelia, and rice medium extracts of the S. griseorubens sp. ASMR4 showed similarity in their low to moderate activity against Gram-positive (9–12 mm), Gram-negative (8–12 mm) bacteria, and moderate to high activity against fungi (10–18 mm), and yeast (12 mm). Nevertheless, extracts of cells and rice medium showed a moderate activity against C. albicans (10 mm) (Table 4). Alternatively, compounds 1a and 2 were tested against Gram-positive bacteria (Micrococcus luteus DSMZ 1605, Streptococcus minor DSMZ 17118, Streptococcus ferus DSMZ 18308) and Gram-negative bacteria (Escherichia coli DSMZ 704 and Pseudomonas agarici 11810), pronouncing no activity. Compounds 1a and 2 were also investigated in an in vitro cytotoxicity assay against the human cervix carcinoma cell line (KB-3-1) and its multi-drug-resistant subclone (KB-V1), compared with cryptophycin-52 as a reference sample, showing no cytotoxicity (Table 5).

Table 4:

Antimicrobial activities of the ASMR4 extract in agar diffusion assays (in mm diameter).

S. aureusP. aeruginosaB. subtilisC. AlbicansS. cerevisiaeA. niger
Extract (filtrate)98101210
Extract (cells)121212101218
Extract (rice medium)121211101215
Table 5:

In vitro cytotoxicity against KB-3-1 and KB-V1 cell lines (range: 5×10−4–5×10−7 mol L−1).

CompoundMolecular weight (g mol−1)IC50 KB-3-1 (mol L−1)IC50 KB-V1 (mol L−1)
1a284
2194
Cryptophycin-526751.3×10−112.3×10−10

3 Experimental section

3.1 General experimental procedure

NMR spectra (1H NMR, 13C NMR, DEPT, COSY, HMQC, and HMBC) were measured on Bruker Avance DRX 500 and DRX 600 MHz spectrometers using standard pulse sequences and referenced to residual solvent signals. HREIMS was determined using a GCT Premier Spectrometer. The ultraviolet and visible (UV–Vis) spectra were measured on Spectro UV–Vis Double Beam PC8 scanning auto Cell UVD-3200 (Labomed, Inc.). Column chromatography was carried out on silica gel 60 (0.040–0.063 mm, Merck) and Sephadex LH-20 as the stationary phases. Preparative TLC (0.5 mm thick) and analytical TLC were performed with pre-coated Merck silica gel 60 PF254+366. Rf values of the bioactive compounds and visualization of their chromatograms was carried out under UV light (254 and 366 nm) and further by spraying with anisaldehyde-sulfuric acid followed by heating.

3.2 Isolation of the producing strain

The marine strain ASMR4 was obtained from a soft coral collected in the Red Sea at the coast of Hurghada, Egypt, in 30 m depth. It was cultivated ina starch nitrate medium (g L−1) (starch, 20.0 g; KNO3, 2.0 g; K2HPO4, 1.0 g; MgSO4·7 H2O, 0.5 g; NaCl, 0.5 g; FeSO4·7 H2O, 0.01 g; CaCO3, 3.0 g; agar, 20.0 g; and 1000 mL of 50% sea water). The pH was adjusted to 7.2. The samples were washed three times with sterilized water to remove all loosely attached bacteria, and then cut with a sterile scalpel to reach the inner tissue surface. Next, 5 mL of sterilized sea water was added to each sample and incubated for 30 min into a reciprocal water bath at 30°C. A series of tenfold dilution was made with sterile sea water and platted (100 μL) on prepared media. The plates were then incubated at 28°C for 6–8 weeks. The colonies with distinct morphological characteristics were selected and transferred onto freshly prepared solid media and stored in a refrigerator at 4°C. The strain was deposited in the collection of the Microbial Chemistry Department, National Research Centre (NRC), Egypt.

3.2.1 DNA isolation and 16S rRNA sequencing

The strain was inoculated in 100 mL Erlenmeyer flasks each containing 50 mL of ISP2 medium composition (g L−1): malt extract, 10; yeast extract, 4; glucose 4 (pH 7.2) at 28°C for 3 days. Then, 3 mL of culture were centrifuged at 5000×g. The cell pellet was used for isolation of DNA. Genomic DNA of the strain was isolated using a bacterial Genome DNA isolation kit (Qiagen-kit: DNeasy Blood & Tissue Cat. No: 69504) following the manufacturer’s manual. The DNA concentration was 29 ng μL−1. DNA amplification was carried out with a primer set (9F/1541R). The following amplification profile was used: an initial denaturation step at 94°C for 2 min was followed by 30 amplification cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min and a final extension step of 72°C for 2 min. After agarose gel electrophoresis, the PCR product was detected and visualized by UV fluorescence after ethidium bromide staining [31]. PCR cleanup was performed according to the description manual in Qiagen-kit: 28104. DNA sequences were determined by the CeBiTec DNA sequencing core facility on an Applied Biosystems AbiPrism 3730 sequencer using BigDye-terminator chemistry. Premixed reagents were used from Applied Biosystems. The results of 16S rRNA sequence were compared with the available database at GenBank by using BLAST software (blastn) on National Centre Biotechnology Information [32]. The phylogenetic tree was constructed using the neighbor-joining tree method.

3.3 Morphological, cultural, and physiological characterization of the strain

The cultivation characteristics of the strain ASMR4 were studied according to the guidelines established in the ISP [33]. The aerial mycelium, pigmentation, and morphological features of the strain ASMR4 were examined after its growth on different ISP media, starch nitrate medium, and Czapek Dox agar medium for 2, 3, and 4 weeks, and the observations were recorded weekly. The micromorphology was studied on a culture grown at 37°C for 10 days on an ISP4 medium [34]. The cells were examined for spore chain morphology under a light microscope (Olympus CH-2) at a magnification of 1200×. Electron micrographs were made with a transmission electron microscope at NRC, Egypt. Physiological and biochemical characteristics were studied by hydrogen sulfide production [35], cellulose decomposition [36], NaCl tolerance [37], and utilization of different carbon sources [33].

3.3.1 Fermentation A (culture broth medium), working-up, and isolation

One milliliter of prepared spore suspension (OD550 0.2) of the strain ASMR4 was used to inoculate 18 of 1 L Erlenmeyer flasks containing 300 mL of ISP2 medium. The flasks were incubated in a shaking incubator (100 rpm) at 30°C for 7 days. The culture was harvested and centrifuged (4500 rpm at 5°C) to separate cells. The medium was extracted by ethyl acetate and the cells were extracted with acetone. Acetone and water were evaporated in vacuo. Finally, the residue was re-extracted with ethyl acetate. The obtained ethyl acetate extracts were finally combined, in vacuo concentrated to dryness affording a reddish brown crude extract (3.2 g), which was then applied to working-up stages. An application of the extract to a series of purification techniques starting with silica gel column eluted with petroleum ether–ethylacetate gradient afforded three fractions I (1.2 g), II (0.75 g), and III (0.21 g) based on TLC monitoring. Purification of fraction II using Sephadex LH-20 (DCM-40% MeOH) delivered the diastereomeric mixture: (3R,4R)-3,4-dihydroxy-3-methylpentan-2-one/(3S,4R)-3,4-dihydroxy-3-methylpentan-2-one as colorless oil (7 mg). By a similar way, fraction III was purified to give benzoic acid (6 mg) as colorless solid.

3.3.2 Fermentation B (rice medium), working-up, and isolation

The spore suspension of the strain ASMR4 was inoculated into 100 mL of ISP2 medium and cultivated at 28°C for 3 days as seed culture. Then, 5 mL of seed culture was used to inoculate 1 L Erlenmeyer flasks (11 flasks) containing a modified rice medium composition: 100 g commercial rice; 150 mL distilled water containing 0.4% yeast extract, and 1% malt extract were allied in each flask under sterile conditions. The flasks were incubated for 14 days at 37°C. The methanol was separated from rice by filtration under vacuum. After filtration, the water–methanol fraction was evaporated to remove methanol using rotary evaporator (Heidolph). After complete evaporation of methanol, the water phase was re-extracted by ethyl acetate. The obtained ethyl acetate extracts were finally concentrated in vacuo to dryness and then applied to working-up stages.

The crude extract (3.1 g) was separated by column chromatography on silica gel (60×3 cm) using cyclohexane-EtOAc-MeOH gradient (0.5 L cyclohexane, 0.5 L cyclohexane-EtOAc [8:2], 0.5 L cyclohexane-EtOAc [6:4], 0.5 L cyclohexane-EtOAc [4:6], 0.5 L cyclohexane-EtOAc [2:8], 0.5 L EtOAc, 0.3 L EtOAc-MeOH [97:3], 0.3 L EtOAc-MeOH [95:5], 0.3 L EtOAc-MeOH [90:10], 0.3 L EtOAc-MeOH [80:20] 0.3 L MeOH). According to TLC monitoring, four fractions were obtained: FI (0.66 g), FII (0.37 g), FIII (0.11 g), and FIV (0.2 g). An application of fraction I to a further column chromatography on silica gel and elution with cyclohexane-CH2Cl2 gradient afforded methyl linoleate as colorless oil (47 mg). Similarly, fraction III was divided into the two sub-fractions IIIa (0.05 g) and IIIb (0.03 g) by application to a further column on silica gel and elution with cyclohexane-CH2Cl2-CH3OH, whereas IIIa was further purified on Sephadex LH-20 column (CH2Cl2-40% MeOH) to afford phenylacetic acid (3 mg) and anthranilic acid (12 mg). Purification of sub-fraction IIIb using an eluent gradient (CH2Cl2-MeOH) on silica gel, followed by preparative TLC (CH2Cl2-5% MeOH) and Sephadex LH-20 (CH2Cl2-40% MeOH), delivered 8-hydroxy-2-(2-hydroxypropyl)-7-acetyl-1-oxaphenalene (1a, 6 mg). Fraction FIV was subjected purification on Sephadex LH-20 (CH2Cl2-40% MeOH), which afforded three sub-fractions IVa (20 mg), IVb (70 mg), and IVc (50 mg). Purification of sub-fraction IVb using Sephadex LH-20 (CH2Cl2-40% MeOH) gave glycerol linoleate as colorless oil (20 mg), whereas sub-fraction IVc was purified on silica gel column (CH2Cl2-MeOH gradient) delivering FA (2, 16 mg) as a colorless solid.

3.4 Antimicrobial assay using the agar diffusion test

Antimicrobial activity testing of the crude extract of the marine isolate ASMR4 was carried out together with the two new compounds 1 and 2 against a set of microorganisms using the agar diffusion technique. Paper-disk diffusion assay [38] with some modifications has been followed to measure the antimicrobial activity. Twenty milliliters of medium seeded with test organism was poured into 9-cm sterile Petri dishes. After solidification, the paper disks (6 mm diameter) were placed on inoculated agar plates and allowed to diffuse the loaded substances into the refrigerator at 4°C for 2 h. The plates were incubated for 24 h at 35°C. Both bacteria and yeasts were grown on a nutrient agar medium: 3 g L−1beef extract, 10 g L−1peptone, and 20 g L−1agar. The pH was adjusted to 7.2. Fungal strain was grown on a potato dextrose agar medium (g L−1): potato extract, 4; dextrose, 20; agar No. 1, 15 (pH 6). After incubation, the diameters of inhibition zones were measured with a wide panel of test microorganisms comprising Gram-positive bacteria (B. subtilis ATCC6633 and S. aureus ATCC6538-P), Gram-negative bacteria (Pseudomonas areuginosa ATCC 27853 and E. coli), yeasts (C. albicans ATCC 10231, S. cerevisiae ATCC 9080), and the fungus (A. niger NRRL A-326).

3.5 Cytotoxicity assays

The KB-3-1 and KB-VI cells were cultivated as a monolayer in Dulbecco’s modified Eagle medium with glucose (4.5 g L−1), l-glutamine, sodium pyruvate, and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). The cells were maintained at 37°C and 5.3% CO2-humidified air. On the day before the test, the cells (70% confluence) were detached with trypsin-ethylenediamine tetraacetic acid solution [0.05%; 0.02% in Dulbecco’s phosphate buffered saline (DPBS)] and placed in sterile 96-well plates in a density of 10000 cells in 100 μL medium per well. The dilution series of the compounds were prepared from stock solutions in dimethyl sulfoxide (DMSO) of concentrations of 100 mm, 50 mm, or 25 mm. The stock solutions were diluted with a culture medium (10% FBS) down to the pm range. The dilution prepared from the stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 37°C and 5.3% CO2-humidified air, 30 μL of an aqueous resazurin solution (175 μM) was added to each well. The cells were incubated under the same conditions for 5 h. Subsequently, the fluorescence was measured. The excitation was effected at a wavelength of 530 nm, whereas the emission was recorded at a wavelength of 588 nm. The IC50 values were calculated as a sigmoidal dose–response curve using GraphPad Prism 4.03. The IC50 values equal the drug concentrations, at which vitality is 50% [39], [40].

4 Supplementary information

NMR spectra and other supplementary data associated with this article are given as Supplementary information available online (DOI: 10.1515/znb-2016-0145).

Acknowledgments

The authors are thankful to the NMR and MS Departments in Bielefeld University for the spectral measurements. They thank Carmela Michalek for biological activity testing and Marco Wißbrock for technical assistance. DNA sequences were determined by the CeBiTec DNA sequencing core facility. The bioinformatics support of the BMBF-funded project “Bielefeld-Gießen Resource Center for Microbial Bioinformatics—BiGi (Grant number 031A533)” within the German Network for Bioinformatics Infrastructure (de.NBI) is gratefully acknowledged. This research work has been financed by the German Academic Exchange Service (DAAD) with funds from the German Federal Foreign Office in the frame of the Research Training Network “Novel Cytotoxic Drugs from Extremophilic Actinomycetes” (Project ID 57166072).

References

[1] H. Laatsch, AntiBase, A Data Base for Rapid Structural Determination of Microbial Natural Products, Wiley-VCH, Weinheim (Germany) 2014 and annual updates.Search in Google Scholar

[2] P. Kekuda, K. Shobha, R. Onkarappa, J. Pharm. Res. 2010, 3, 250.Search in Google Scholar

[3] J. Berdy, J. Antibiot.2005, 58, 1.10.1038/ja.2005.1Search in Google Scholar PubMed

[4] H. W. Zhang, Y. C. Song, R. X. Tan, Nat. Prod. Rep. 2006, 23, 753.10.1039/b609472bSearch in Google Scholar PubMed

[5] G. M. Cragg, D. J. Newman, J. Ethnopharmacol. 2005, 100, 72.10.1016/j.jep.2005.05.011Search in Google Scholar PubMed

[6] S. B. Singh, D. L. Zink, Z. Guan, J. Collado, F. Pelaez, P. J. Felock, D. J. Hazuda, Helv. Chim. Acta2003, 86, 3380.10.1002/hlca.200390281Search in Google Scholar

[7] S. F. Brady, J. Clardy, J. Nat. Prod.2000, 63, 1447.10.1021/np990568pSearch in Google Scholar PubMed

[8] M. S. Butler, J. Nat. Prod. 2004, 67, 2141.10.1021/np040106ySearch in Google Scholar PubMed

[9] W. R. Strohl in Microbial Diversity and Bioprospecting (Ed.: A. T. Bull), ASM Press, Washington, DC, 2004, p. 336.10.1128/9781555817770.ch31Search in Google Scholar

[10] C. Oldfield, N. T. Wood, S. C. Gilbert, F. D. Murray, F. R. Faure, Antonie Van Leeuwenhoek1998, 74, 119.10.1023/A:1001724516342Search in Google Scholar

[11] W. Pecznska-Czoch, M. Mordarski, in Actinomycetes in Biotechnology (Eds.: M. Goodfellow, S. T. Williams, M. Mordarski), Academic Press, London, 1988, p. 219.10.1016/B978-0-12-289673-6.50011-7Search in Google Scholar

[12] J. Mann, Nat. Prod. Rep. 2001, 18, 417.10.1039/b001720pSearch in Google Scholar PubMed

[13] Y. C. Song, H. Li, Y. H.Ye, C. Y. Shan, Y. M. Yang, R. X. Tan, FEMS Microbiol. Lett.2004, 241, 67.10.1016/j.femsle.2004.10.005Search in Google Scholar PubMed

[14] M. V. Arasu, V. Duraipandiyan, S. Ignacimuthu, Chemosphere2013, 90, 479.10.1016/j.chemosphere.2012.08.006Search in Google Scholar PubMed

[15] C. Balachandran, V. Duraipandiyan, N. Emi, S. Ignacimuthu, South Indian J. Biol. Sci.2015, 1, 7.10.22205/sijbs/2015/v1/i1/100436Search in Google Scholar

[16] S. Dharni, M. Alam, A. Samad, F. Khan, Abdul-Khaliq, S. Luqman, D. D. Patra, Ind. J. Biotechnol. 2012, 11, 438.Search in Google Scholar

[17] E. B. Shirling, D. Gottlieb, Int. J. Syst. Bacteriol. 1968, 18, 69.10.1099/00207713-18-2-69Search in Google Scholar

[18] E. B. Shirling, D. Gottlieb, Int. J. Syst. Bacteriol.1968, 18, 279.10.1099/00207713-18-4-279Search in Google Scholar

[19] E. B. Shirling, D. Gottlieb, Int. J. Syst. Bacteriol.1972, 22, 265.10.1099/00207713-22-4-265Search in Google Scholar

[20] L. Ye, Q. Zhou, C. Liu, X. Luo, G. Na, T. Xi, Ind. J. Marine Sci.2009, 38, 14.Search in Google Scholar

[21] W. Whitman, M. Goodfellow, P. Kampfer, H.-J. Busse, M. Trujillo, W. Ludwig, K.-I. Suzuki, A. Parte, Bergey’s Manual of Systematic Bacteriology, The Actinobacteria, Vol. 5, 2nd ed., Springer, New York, 2012.10.1007/978-0-387-68233-4Search in Google Scholar

[22] M. Shaaban, H. Nasr, A. Z. Hassan, M. S. Asker, Rev. Latinoam. Quim.2013, 41, 50.Search in Google Scholar

[23] K. A. Shaaban, M. S. Abdel-Aziz, M. S. Asker, M. Shaaban, Bull NRC2009, 34, 87.Search in Google Scholar

[24] M. Shaaban, A. S. Abdel-Razik, M. S. Abdel-Aziz, A. A. AbouZied, M. Fadel, J. Appl. Sci. Res.2013, 9, 996.Search in Google Scholar

[25] M. Shaaban, Dissertation, Georg-August University, Göttingen (Germany) 2004.Search in Google Scholar

[26] M. Shaaban, K. A Shaaban, M. S Abdel-Aziz, Org. Med. Chem. Lett.2012, 2, 2.10.1186/2191-2858-2-30Search in Google Scholar PubMed PubMed Central

[27] Chapman & Hall Chemical Database, Dictionary of Natural Products on CD-ROM, 2016.Search in Google Scholar

[28] G. O. De Lima, I. L. D’Albuquerque, M. M. DE Albuquerque, E. Silva, J. A. Bandeira, Rev. Inst. Antibiot. Univ. Recife1959, No. 1–2, 3.Search in Google Scholar

[29] N. Kumar, V. Pruthi, Biotechnol. Rep.2014, 4, 86.10.1016/j.btre.2014.09.002Search in Google Scholar PubMed PubMed Central

[30] M. Uraji, M. Kimura, Y. Inoue, K. Kawakami, Y. Kumagai, K. Harazono, T. Hatanaka, Appl. Biochem. Biotechnol. 2013, 171, 1085.10.1007/s12010-013-0190-6Search in Google Scholar PubMed

[31] V. Kumar, A. Bharti, O. P. Gusain, G. S. Bisht, J. Sci. Engg. Tech. Mgt.2010, 2, 10.Search in Google Scholar

[32] http://www.ncbi.nlm.nih.gov (accessed July 2016).Search in Google Scholar

[33] E. B. Shirling, D. Gottlieb, Int. J. Syst. Bacteriol.1966, 16, 313.10.1099/00207713-16-3-313Search in Google Scholar

[34] A. F. Yassin, E. A. Galiniski, A. Wohlfarth, K. D. Jahnke, K. P. Schaal, H. G. Truper, Int. J. Syst. Bacteriol. 1993, 43, 266.10.1099/00207713-43-2-266Search in Google Scholar

[35] E. Kuster, S. T. Williams, Appl. Microbiol.1964, 12, 46.10.1128/am.12.1.46-52.1964Search in Google Scholar

[36] D. L. Crawford, E. McCoy, Appl. Microbiol. 1972, 24, 150.10.1128/am.24.1.150-152.1972Search in Google Scholar PubMed PubMed Central

[37] H. D. Tresner, J. A. Hayes, E. J. Backus, Appl. Microbiol.1968, 16, 1134.10.1128/am.16.8.1134-1136.1968Search in Google Scholar PubMed PubMed Central

[38] A. W. Baur, W. M. Kirby, J. C. Sherris, M. Truck, Am. J. Clin. Pathol.1966, 45, 493.10.1093/ajcp/45.4_ts.493Search in Google Scholar

[39] A. F. Awantu, B. N. Lenta, T. Bogner, Y. F. Fongang, S. Ngouela, J. D. Wansi, E. Tsamo, N. Sewald, Z. Naturforsch.2011, 66b, 624.10.1515/znb-2011-0610Search in Google Scholar

[40] B. Sammet, T. Bogner, M. Nahrwold, C. Weiss, N. Sewald, J. Org. Chem.2010, 75, 6953.10.1021/jo101563sSearch in Google Scholar PubMed


Supplemental Material:

The online version of this article (DOI: 10.1515/znb-2016-0145) offers supplementary material, available to authorized users.


Received: 2016-6-29
Accepted: 2016-11-3
Published Online: 2016-12-28
Published in Print: 2017-1-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 19.3.2024 from https://www.degruyter.com/document/doi/10.1515/znb-2016-0145/html
Scroll to top button