Streptomyces zerumbet W14, a novel species of the endophyte genus Streptomyces was isolated from the rhizome tissue of Zingiber zerumbet (L.) Smith. Identification of strain W14 was based on its morphology, chemotaxonomy and phylogenetic analysis using 16S rDNA sequence. It was classified as the secondary meabolites of the culture extract were studied. The major active ingredients from the crude extract were purified by silica gel column chromatography and identified by spectroscopic data. The crude extract and purified compounds were tested for their biological activities on antibacterial and anti-inflammatory properties. The crude extract showed inhibition on the growth of Gram-positive bacteria with the MIC and MBC values of 8 - 32 μg/ml and 32 - 128 μg/ml, respectively. The isolated compounds were identified to be methyl 5-(hydroxymethyl)furan-2-carboxylate (1) and geldanamycin (2). Bioassay studies showed that compound 1 had antibacterial activity against Staphylococus aureus ATCC 25923 and Methicillin Resistant S. aureus strain Sp6 (clinical isolate) with the MIC and MBC values of 1 μg/ml and 16 - 64 μg/ml, respectively, and also showed activity against Bacillus Calmette-Guérin (vaccine strain) with MIC and MBC values of 128.00 μg/ml and 128.00 μg/ml, respectively. The compound 2 at the concentration of 1 - 5 μg/ml had in vitro anti-inflammatory activity on LPS-induced RAW 264.7 cells by inhibition of mRNA expression and production of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF- α), interleukin-1 β (IL-1 β), and interleukin-6 (IL-6). These results suggest that compounds 1 and 2 produced by S. zerumbet W14 (an endophyte of Z. Zerumbet) have antibacterial and anti-inflammatory activities, respectively. Therefore, the future studies on these compounds could be useful for the management of bacterial infections and inflammatory diseases.
Inside the tissue of nearly all the healthy plants, there are many endophytic microorganisms. Endophytes are synergistic to their host, some of them produced secondary metabolites which exhibit plant growth promoters or prevent phytopathogen attacking. During our investigations of the endophytes, we isolated an endophytic actinomycete from the root tissues of Zingiber officinale Rosc. and it was identified as Streptomyces aureofaciens CMUAc130; it showed the most effective antifungal activity [
Leaf, stem, rhizome and root tissues of Zingiber zerumbet (L.) Smith were used to isolate the endophytic actinomycetes by surface-sterilization technique and validation of surface sterilization was performed as described in our previous studies [
An in vitro plate technique was used to test the inhibitory effects of strain W14 on the tested bacteria. For screening of antibacterial activity, crude extract and purified compounds were tested against S. aureus ATCC 25932, B. cereus ATCC 7064, B. subtilis ATCC 6633, Escherichia coli ATCC 10536, Salmonella Typhi ATCC 19430, Pseudomonas aeruginosa ATCC 27853, Serratia marcescens ATCC 8100, Methicillin Resistant S. aureus strain Sp6 (clinical isolate) and Bacillus Calmette-Guérin (vaccine strain) using the paper disk method of National Committee for Clinical Laboratory Standards [
The MICs of the crude extract and purified compounds were determined by NCCLS microbroth dilution methods [
HeLa cell line, L929 murine fibroblast cell line, and RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). Human peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation with Ficoll-Paque [
Cytotoxicity studies were performed on a 96-well plate. The cells were mechanically scraped and plated 2 × 105 per well containing 100 µl of DMEM medium with 10% FBS and incubated overnight. The crude extact and purified compounds 1 and 2 were dissolved in dimethylsulfoxide (DMSO) for stock solution. Cells were incubated with crude extract and purified compounds 1 and 2 at increasing concentrations and stimulated with LPS 1 µg/ml for 24 h. The DMSO concentrations in all assays did not exceed 0.1%. Twenty-four h after seeding, 100 µl new media or test compound was added, and the plates were incubated for 24 h. Cells were washed once before adding 50 µl FBS-free medium containing 5 mg/ml MTT. After 4 h of inoculation at 37˚C, the medium was discarded and the formazan blue, which formed in the cells, was dissolved in 50 µl DMSO. The optical density was measured at 450 nm. The concentration required for reducing the absorbance by 50% (IC50) compared to the control cells was determined.
Nitrite accumulation, an indicator of NO synthesis, was measured in the culture medium by Griess reaction [
PGE2, TNF-α, IL-1β and IL-6 level in macrophage culture medium were quantified by ELISA kits (eBioscience, CA, USA) according to the manufacturer’s instructions.
Cellular proteins were extracted from control and test compound-treated RAW 264.7 cells as described in our previous study [
The expression of pro-inflammatory mediator and cytokine genes was investigated by reverse transcription―polymerase chain reaction (RT-PCR). RAW 264.7 cells were incubated with compounds 1 and 2 at different concentrations (1, 2.5, 5 and 10 µg/ml) and stimulated with LPS 1 µg/ml at 37˚C in FBS-free medium for 24 h. RAW 264.7 cells at for 24 h. Total RNA was extracted from RAW 264.7 cells after treatment with purified compounds, The cells were lysed with Trizol® and centrifuged at 12,000 rpm for 15 min at 25˚C, following the addition of chloroform. Isopropanol was added to the supernatant at a 1:1 ratio and the RNA pellet was obtained following centrifugation. After washing with ethanol, extracted RNA was solubilized in diethyl pyrocarbonate-treated RNase-free water and quantified by measuring the absorbance at 260 nm. Equal amounts of RNA (1 µg) were reverse transcribed in a master mix containing 1X reverse transcriptase (RT) buffer, 1 mM dNTPs, 500 ng of oligo(dT)15 primers, 140 U of murine Moloney leukaemia virus reverse transcriptase and 40 U of RNase inhibitor, for 45 min at 42˚C. PCR was carried out in an automatic thermal cycler (Perkin-Elmer Cetus) to amplify iNOS, TNF-α, IL-1β, IL-6 and β-actin mRNA. Primer sequences used to amplify the desired cDNA were shown in
Data are reported as mean ± SEM values of three independent determinations. Statistical analysis was performed by Student’s t-test.
Eighteen actinomycete isolates were obtained, 4 of which were isolated from leaves, 5 isolates from rhizomes, 8 isolates from roots and only one isolate from
Genea | Primer sequences (5’ - >3’) | PCR product size (bp) |
---|---|---|
iNOS | 5’-CCCTTCCGAAGTTTCTGGCAGCAGC-3’ (sense) 5’-GGCTGTCAGAGCCTCGTGGCTTTGG-3’ (antisense) | 496 |
TNF-α | 5’-TTGACCTCAGCGCTGAGTTG-3’ (sense) 5’-CCTGTAGCCCACGTCGTAGC-3’ (antisense) | 364 |
IL-1β | 5’-CAGGATGAGGACATGAGCACC-3’ (sense) 5’-CTCTGCAGACTCAAACTCCAC-3’ (antisense) | 447 |
IL-6 | 5’-GTACTCCAGAAGACCAGAGG-3’ (sense) 5’-TGCTGGTGACAACCACGGCC-3’ (antisense) | 308 |
β-actin | 5’-GTGGGCCGCCCTAGGCACCAG-3’ (sense) 5’-GGAGGAAGAGGATGCGGCAGT-3’ (antisense) | 603 |
aiNOS, Inducible nitric oxide synthase; TNF-α, Tumor necrosis factor-α; IL-1β, Interleukin-1β; IL-6, Interleukin-6.
stems. The strain W14 showed promissing activities against S. aureus ATCC 25932, B. cereus ATCC 7064 and B. subtilis ATCC 6633. However the other isolates had no potential of antibacterial activity to the tested bacteria. Therefore, all subsequent experiments involved the use of the strain W14. Based on results in the presence of LL-type diaminopimelic acid in the whole-cell extracts, the strain W14 was identified as belonging to the genus Streptomyces. Direct sequencing of the PCR-amplified 16S rDNA of the strain W14 was determined from position 8 to position 1464 of the 16S rDNA gene sequence of E. coli numbering system [
to salt and grew well at pH 5.6 to 11.0, at 37˚C but was unable to grown on temperature above 45˚C. This strain utilized glucose, mannose, meso-inositol, sucrose, galactose, fructose, lactose, maltose, mannitol, mannose, raffinose, arabinose, sodium acetate and sodium citrate, but did not utilize xylose, sorbitol and CM-cellulose. It showed moderate activities of catalase, gelatinase and caseinase, hydrolysed casein, starch and tyrosine, but did not produce hydrogen sulphide. However, it also resisted ampicillin and erythromycin. A comparative analysis of the cultural and biochemical characteristics of strain W14 with respect to its phylogenetic relatives was shown in
Characteristics | W14 | DSM 41697T | M1463T |
---|---|---|---|
Antimicrobial activity against: | |||
Bacillus subtilis | + | + | + |
Escherichia coli | − | − | − |
Staphylococcus aureus | + | − | + |
Candida albicans | + | + | + |
Aspergillus flavus | + | No data | − |
Aspergillus fumigatus | + | No data | No data |
Aspergillus niger | − | No data | + |
Decomposition of: | |||
Casein | + | + | + |
Starch | + | + | + |
L-Tyrosine | + | + | − |
Xanthine | Not done | − | − |
Growth at: | |||
4˚C | − | − | − |
37˚C | + | + | + |
45˚C | − | − | − |
pH 4.0 | − | − | − |
pH 5.6 | + | + | + |
pH 11.0 | + | − | No data |
Growth in the presence of: | |||
Lysozyme (0.005%, w/v) | + | + | No data |
Sodium chloride (3%, w/v) | + | + | No data |
Sodium chloride (5%, w/v) | + | − | No data |
Resistance to antibiotics (ug): | |||
Ampicillin (10) | + | + | No data |
Chloramphenicol (30) | −(16.9)a | −(15)a | + |
Erythromycin (15) | + | −(18)a | No data |
Gentamicin sulphate (10) | −(45.6)a | −(28)a | − |
---|---|---|---|
Kanamycin sulphate (30) | −(33.0)a | −(34)a | No data |
Neomycin sulphate (30) | −(35.6)a | −(30)a | − |
Novobiocin sulphate (30) | Notdone | −(40)a | No data |
Streptomycin sulphate (10) | −(18.0)a | −(35)a | − |
Tetracycline hydrochloride (30) | −(36.3)a | −(20)a | No data |
Ampicillin (10) | + | + | No data |
Chloramphenicol (30) | −(16.9)a | −(15)a | + |
Growth on sole carbon sources (1%, w/v) | |||
L-Arabinose | + | + | + |
D-Fructose | + | + | + |
D-Galactose | + | + | + |
D-Glucose | + | + | + |
meso-Inositol | + | + | − |
D-Lactose | + | − | + |
D-Maltose | + | + | + |
D-Mannitol | + | + | + |
D-Mannose | + | + | + |
D-Raffinose | + | + | − |
α-L-Rhamnose | Not done | + | + |
D-Sorbitol | − | − | − |
Starch | + | + | No data |
D-Sucrose | + | − | − |
D-Xylose | − | + | − |
CM-cellulose (0.1%, w/v) | − | − | No data |
Sodium acetate (0.1%, w/v) | + | − | − |
Sodium citrate (0.1%, w/v) | + | − | − |
L-Arabinose | + | + | + |
D-Fructose | + | + | + |
D-Galactose | + | + | + |
D-Glucose | + | + | + |
meso-Inositol | + | + | − |
D-Lactose | + | − | + |
D-Maltose | + | + | + |
Production of: | |||
Hydrogen sulphide | − | − | No data |
aNumbers in parentheses indicate the diameters of the inhibition zones (mm). bS. malaysiensis DSM 41697T (data from Al-Tai et al. [
Streptomyces species | Similarity to strain W14 | No. nucleotide differences/Total no. nucleotides compared |
---|---|---|
S. malaysiensis NBRC 16446 | 99.65 | 5/1436 |
S. samsunensis M1463 | 99.64 | 5/1418 |
S. asiaticus NBRC 100774 | 98.54 | 21/1443 |
S. rhizosphaericus NBRC 100778 | 98.54 | 21/1443 |
S. hygroscopicus subsp. azalomyceticus NBRC 13868 | 98.54 | 21/1442 |
S. griseiniger NRRL B-1865 | 98.39 | 23/1436 |
S. cangkringensis DSM 41769 | 98.31 | 24/1422 |
S. sporoclivatus NBRC 100767 | 98.26 | 25/1442 |
S. yatensis NBRC 101000 | 98.19 | 26/1443 |
S. castelarensis subsp. enhygrus NRRL 3664 | 98.12 | 27/1443 |
Thus, based on results of the phenotypic and genotypic analysis, the strain W14 represents a novel species of the genus Streptomyces, for which we propose the name Streptomyces zerumbet sp. nov.
The partial components of crude extract were observed by TLC analysis in a solvent system comprising of acetone: hexane (2:3). The analysis of the separated compounds under UV light (254 and 365 nm) and upon derivatizing the TLC with vanillin/sulfuric acid reagent, revealed at least seven major spots with Rf values of 0.07, 0.20, 0.33, 0.46, 0.67, 0.77 and 0.91. In order to find out the correlation of these spots with antimicrobial property, bioautography of the freshy developed air-dried TLC strips containing separated components of the extract was performed, using B. cereus, B. subtilis and S. aureus as tested microorganisms. The results exhibited that the strain W14 produced at least one comound with Rf values of 0.67 against tested bacteria (
Column chromatography on silica gel with acetone: hexane as the mobile phase resulted in the separation of two major compounds. Identification of each compound was carried out by 1H-NMR, 13C-NMR as following.
Compound 1: The MS gave a [M + Na]+ ion at m/z 179.0287 which corresponded to the molecular formula C7H8O4, indicating three double bond equivalents in the molecule. The 1H-NMR spectral data (CDCl3) of compound 1 showed methoxy proton at δH 3.88 (3H, s), oxygenated methine at δH 4.66 (2H, s), two furan protons at δH 6.42 (1H, d, J = 3.3 Hz) and 7.21 (1H, d, J = 3.3 Hz) ppm. The 13C-NMR spectrum exhibited 7 signals which were classified by the HMQC spectra as methyoxy carbon at δC 52.2, oxygenated methine at δC 57.7, two furan carbons at δC 109.0 (C-4) and δC 119.0 (C-3), two quarternary furan carbons at δC 144.3 (C-2) and δC 158 (C-5) and carbonyl ester at δC 159.4 ppm. The 1H-1H COSY spectrum revealed the connectivity in CDCl3 from H-3 through H-4. The HMBC spectrum showed the following long-range correlation; methoxy proton (δH 3.88) to ester carbonyl carbon C-1 (δ 159.4); oxygenated methine (δ 4.66) to quarternary furan carbon C-5 (δ 158.6) and furan carbon C-4 (δ 109.7); furan proton C-4 (δ 6.42) to quarternary furan carbon C-5 (δ 158.6), furan carbon C-3 (δ 119.1) and quarternary furan carbon C-2 (δ 144.3) and furan proton C-3 (δ 7.12) to quarternary furan carbon C-2 (δ 144.3), furan carbon C-4 (δ 109.7) and quarternary furan carbon C-5 (δ 158.6). The spectral data revealed the compound 1 to be methyl 5-(hydroxymethyl)furan-2-carboxylate (
Compound 2: The IR spectrum displayed characteristic absorption bands of NH and OH stretches at n 3478, 3440, 3336 and 3297 cm−1, CH stretch in CH3 and CH2 at n 2927 cm−1, CH stretch in methyl ether at n 2853 cm−1, C=O stretch in OCONH2 at n 1729 cm−1, C=O stretch in a,b-unsaturated amide at n 1701 cm−1 and C = O stretches in quinone at n 1675 and 1652 cm−1. The MS gave a [M+Na]+ ion at m/z 583.2571 which corresponded to the molecular formula C29H40N2O9 for the compound, indicating eleven double bond equivalents in the molecule. The structure was fully elucidated by 1H NMR, 13C NMR spectrosopy, DEPT-135, and 2D NMR spectral studies. The 1H-NMR spectral data (DMSO-d6) of compound 2 showed four methyl groups at δH 0.75 (3HJ = 6.8 Hz), 0.97 (3H, brs), 1.6.2 (3H, s) and 1.93 (3H, s) three methoxy protons at δH 3.23 (3H, s), 3.24 (3H, s) and 3.96 (3H, s), five olefin protons at δH 5.50 (1H, d, J = 8.5 Hz), 5.81 (1H, br), 6.58 (1H, t), 6.95 (1H, d) and 7.04 (1H, s), four oxygenated methines at δH 3.09 (2H, br), 4.36 (1H, d, J = 7.6 Hz) and 4.88 (1H, br), amine hydrogen at dH 9.18 (1H, br), two methanefriyl group at δH 1.93 (1H, s)
and 2.56 (1H) and methanediyl groups at δH 1.45 (2H, br), 2.18 (1H, dd, J = 4.8 and 12.5 Hz) and 2.43 (1H, dd, J = 9.9 and 12.5 Hz) ppm. The 13C-NMR spectrum exhibited 39 signals which were classified by the DEPT-135 and HMQC spectra as four methyl carbons at δC 12.8 (2-Me), 13.0 (8-Me), 13.4 (8-Me) and 23.9 (14-Me), three methoxy carbons at δC 56.5 (12-OMe), 57.1 (6-OMe), and 61.6 (17-OMe), five olefin carbons at δC 111.3 (C-19), 126.3 (C-4), 128.7 (C-3), 132.4 (C-9) and 138.7 (C-5), five quarternary olefin carbons at δC 128.7 (C-16), 129.1 (C-8), 133.2 (C-2), 140.1 (C-20) and 156.9 (C-17), two methanetriyl carbons at δC 27.1 (C-14) and 32.6 (C-10), two methanediyl carbons at δC 31.3 (C-13) and 32.2 (C-15), four oxygenated methines at δC 72.4 (C-11), 80.7 (C-12), 81.1 (C-7) and 82.3 (C-6), four carbonyl carbons at δC 156.6 (7-OCONH2), 169.7 (C-1), 183.6 (C-21) and 184.3 (C-18). The 1H-1H COSY spectrum revealed the connectivity, in DMSO-d6 from H-3 through H-4; H-4 through H-3 and H-5; H-5 through H-4 and H-6; H-6 through H-5 and H-7; H-7 through H-6; H-9 through H-10; H-12 through H-9 and 10-Me; 10-Me through H-10; H-12 through H-13; H-13 through H-12 and H-14; H-14 through H-13, 14-Me and H-15; 14-Me through H-14 and H-15 through H-14. The HMBC spectrum showed the following long-range correlations; 2-Me (δH 1.93) to C-1 (δC 169.7), C-2 (δH 133.2) to C-3 (δC 128.7) and C-4 (δC 126.3); H-4 (δH 6.58) to C-2 (δC 133.2) and C-6 (δC 82.3); H-6 (δH 4.36) to C-4 (δC 126.3), 6-OMe (δC 57.1); 6-OMe (δH 3.24) to C-6 (δC 82.3); H-7 (δH 4.88) to C-5 (δC 138.7), 7-OCONH2 (δC 156.6), C-9 (δC 132.4) and 8-Me (δC 13.0); 8-Me (δH 1.62) to C-7 (δC 81.1) and C-9 (δC 132.4); H-9 (δH 5.50) to C-7 (δC 81.1),and 8-Me (δC 13.0); 10-Me (δH 0.75) to C-9 (δC 132.4), C-10 (δC 32.6), and C-11 (δC 72.4); H-11 (δH 3.09) to 10-Me (δC 13.4); H-12 (δH 3.09) to 12-OMe (δC 56.5); 12-OMe (δH 3.23) to C-12 (δC 80.7); H-13 (δH 1.45) to 14-Me (δC 23.9); H-14 (δH 1.93) to C-12 (δC 80.7) and C-16 (δC 128.7); 14-Me (δH 0.97) to C-14 (δC 27.1) and C-15 (δC 32.2); H-15 (δH 2.18, 2,43) to C-13 (δC 31.3), C-14 (δC 27.1), C-16 (δC 128.7), C-17 (δC 156.9) and C-21 (δC 183.6); 17-OMe (δH 3.96) to C-17 (δC 156.9) and NH (δH 9.18) to C-1 (δC 169.7), C-19 (δC 111.3) and C-21 (δC 183.6). The spectral data revealed the compound 2 to be geldanamycin (
13C-NMR spectral data of which were in good agreement with those of geldanamycin (
Crude extract and purified compounds from Streptomyces zerumbet strain W14 were tested for their cytotoxic activities by MTT-assay on L929, RAW264.7, HeLa cells and PBMC. The IC50 values were exhibited in
Purified compounds from Streptomyces zerumbet strain W14 were tested for their antibacterial activity by MIC and MBC mehthods. Results in
No. | δC compound 1 | δC CF | δH compound 1 | δH CF | HMBC (H → C) | COSY |
---|---|---|---|---|---|---|
1 | 159.4, C | 160.3, C | - | - | - | - |
2 | 144.3, C | 142.8, C | - | - | - | - |
3 | 119.1, CH | 119.0, CH | 7.12 (d, 3.3) | 7.21 (d, 3.4) | 2, 4, 5 | 4 |
4 | 109.7, CH | 109.0, CH | 6.42 (d, 3.3) | 6.44 (d, 3.4) | 2, 3, 5 | 3 |
5 | 158.6, C | 158.2, C | - | - | - | - |
6 | 57.7, CH2 | 56.1, CH2 | 4.66 (s) | 4.45 (d, 5.8) | 4, 5 | - |
1-OMe | 52.2, CH3 | 51.9, CH3 | 3.88 (s) | 3.77 (s) | 1 | - |
6-OH | - | - | - | 5.80 (t, 5.8) | - | - |
aCF, Curvularia lunata furanoid toxin (data from Liu et al., [
No. | δC compound 2 | δC GDA | δH compound 2 | δH GDA | HMBC (H → C) | COSY | NOESY |
---|---|---|---|---|---|---|---|
1 | 169.7 C | 169.1 | - | - | - | - | - |
2 | 133.2 C | 133.2 | - | - | - | - | - |
2-Me | 12.8 CH3 | 12.2 | 1.93 s | 1.91 s | 1, 2, 3, 4 | - | - |
3 | 128.7 CH | 128.4 | 6.95 d | 6.95 d | - | 4 | NH, 4, 6, 7 |
4 | 126.3 CH | 125.7 | 6.58 t | 6.56 t | 2, 6 | 3, 5 | 3, 5 |
5 | 138.7 CH | 137.8 | 5.81 br | 5.80 t | - | 4, 6 | 4, 6 |
6 | 82.3 CH | 81.6 | 4.36 d (7.6) | 4.34 d | 4, 6-OMe | 5, 7 | 3, 5, 7 |
6-OMe | 57.1 CH3 | 56.0 | 3.24 s | 3.22 s | 6 | - | - |
7 | 81.1 CH | 80.6 | 4.88 br | 4.86 br | 5, 7-OCONH2, 9, 8-Me | 6 | 3, 6, 9 |
7-OCONH2 | 156.6 C | 156.0 | - | 6.45 br | - | - | - |
8 | 129.1 C | 132.6 | - | - | - | - | - |
8-Me | 13.0 CH3 | 12.5 | 1.62 s | 1.61 s | 7, 9 | - | 10 |
9 | 132.4 CH | 131.9 | 5.50 d (8.5) | 5.51 d | 7, 8-Me | 10 | 7 |
10 | 32.6 CH | 32.1 | 2.56 | 3.61 m | - | 9, 10-Me | 8-Me, 10-Me, 11, 12 |
10-Me | 13.4 CH3 | 23.3 | 0.75 d (6.8) | 0.97 d | 9, 10, 11 | 10 | 10, 11, 12 |
11 | 72.4 CH | 71.9 | 3.09 br | 3.29 s | 10-Me | - | 10, 10-Me, 13, 14 |
11-OH | - | - | - | - | - | - | - |
12 | 80.7 CH | 80.2 | 3.09 br | 3.07 m | 12-OMe | 13 | 10, 10-Me, 13, 14 |
12-OMe | 56.5 CH3 | 56.6 | 3.23 s | 3.23 s | 12 | - | - |
13 | 31.3 CH2 | 31.0 | 1.45 br | 1.45 m | 14-Me | 12, 14 | 11, 12, 14 |
14 | 27.1 CH | 26.6 | 1.93 s | 1.91 br | 12, 16 | 13, 14-Me, 15a, 15b | 13 |
14-Me | 23.9 CH3 | 13.0 | 0.97 br | 0.76 d | 14, 15 | 14 | - |
15a | 32.2 CH2 | 31.7 | 2.43 dd (12.5, 9.9) | 2.42 m | 13, 14, 16, 17, 21 | 14, 15b | - |
15b | 31.7 | 2.18 dd (12.5, 4.8) | 13, 14, 16, 17, 21 | 14, 15a | - | ||
16 | 128.7 C | 128.1 | - | - | - | - | - |
17 | 156.9 C | 156.4 | - | - | - | - | - |
17-OMe | 61.6 CH3 | 61.0 | 3.96 s | 3.93 s | 17 | - | - |
18 | 184.3 C | 183.6 | - | - | - | - | - |
19 | 111.3 CH | 110.9 | 7.04 s | 7.02 s | - | - | - |
20 | 140.1 C | 139.6 | - | - | - | - | - |
21 | 183.6 C | 183.1 | - | - | - | - | - |
NH | - | - | 9.18 NH, br | 9.14 NH, br | 1, 19, 21 | - | 3 |
aGDA, geldanamycin (data from Ōmura et al. [
Test agents | IC50a (µg/ml) | |||
---|---|---|---|---|
L929b | PBMC | RAW 264.7 | HeLa | |
Crude extract | 108.85 | 165.31 | 4.67 | -c |
Compound 1 | 239.06 | 1097.07 | >512.00 | 64.00 |
Compound 2 | 1372.23 | 52.23 | >512.00 | >512.00 |
aIC50 values represent the concentration causing 50% growth inhibition. They were determined by linear regression analysis. bL929, murine fibroblast cell line; PBMC, human peripheral blood mononuclear cells; RAW 264.7, murine macrophage cell line; HeLa, human cervical carcinoma cell line. -c, Not determine.
Bacteria | Concentration (µg/ml) | |
---|---|---|
MIC | MBC | |
Staphylococcus aureus ATCC 25923 | 1.00 | 16.00 |
MRSA | 1.00 | 64.00 |
Bacillus Calmette-Guérin (BCG) | 128.00 | 128.00 |
LSP caused a significant increase in NO and PGE2 production when compared with the blank control, only compound 2 caused a significant reduction in NO and PGE2 production when compared with LPS-induced control group (p < 0.05). In detail, the production of NO in LPS-induced RAW 264.7 incubated with compound 2 at concentrations of 1, 2.5 and 5 µg/ml for 24 h were 48.72 ± 7.43, 36.51 ± 5.84 and 20.28 ± 4.66 µM, respectively, and the production of PGE2 were 40.74 ± 6.05, 26.62 ± 6.83 and 20.77 ± 4.48 ng/ml, respectively, while the production of NO and PGE2 in the group treated with LPS only was 52.64 ± 6.11 µM and 51.75 ± 6.56 ng/ml, respectively. Therefore, the inhibitory levels of compound 2 on NO and PGE2 production also showed a dose-dependent pattern (
The effects of compound 2 on iNOS and COX-2 production in LPS-induced RAW 264.7 cells were also carried out by Western blot analysis. Results of relative density ratio from Western blot analysis further indicated that iNOS and
COX-2 production in LPS-induced RAW 264.7 cells were significantly reduced when treated with purified compounds in different concentration (
In this study, data showed that compound 2 decreased production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in LPS-induced RAW 264.7 cells (p < 0.05) (Figures 9-11). Treatment with LPS alone in RAW 264.7 cells resulted in a significant increase of pro-inflammatory cytokine productions compared with the blank control group. The detailed results of this assay are as follows: TNF-α productions in LPS-induced RAW 264.7 cells incubated with compound 2 at concentrations of 1, 2.5 and 5 µg/ml for 24 h were 36.56 ± 5.51, 22.79 ± 4.80 and
14.73 ± 4.52 ng/ml, respectively, IL-1β productions in LPS-induced RAW 264.7 cells incubated with compound 2 at different concentrations were 29.57 ± 6.28, 15.64 ± 7.26 and 9.62 ± 4.58 ng/ml, respectively, and IL-6 productions in LPS-induced RAW 264.7 cells incubated with compound 2 at different concentrations were 25.37 ± 4.62, 17.45 ± 3.67 and 9.46 ± 3.34 ng/ml, respectively. Therefore, treatment with compound 2 (1 - 5 μg/ml) clearly inhibited the production of TNF-α, IL-1β and IL-6 in a dose-dependent manner in LPS-induced RAW 264.7 cells.
Since compound 2 inhibited the production of NO and proinflammatory cytokines, the correlation between the concentration of compound 2 and the mRNA expression of iNOS and proinflammatory cytokines was investigated. RAW 264.7 cells were pretreated with different concentrations (1 - 10 μg/ml) of compound 2 for 2 h and then incubated with or without 1 μg/ml of LPS for 6 h, total mRNA was isolated, and the mRNA levels of iNOS and proinflammatory cytokines were examined by RT-PCR. Treatment with LPS also significantly increased the mRNA expression levels of iNOS and proinflammatory cytokines (
Strain W14 was isolated from the rhizome tissue of Zingiber zerumbet (L.) Smith, a medicinal plant containing several compounds, for example, polyphenols, alkaloids and terpenes [
Two major compounds were isolated from the Streptomyces zerumbet W14 crude extract, and were identified to be methyl 5-(hydroxymethyl)furan-2-carboxylate (1) and geldanamycin (2). To the best of our knowledge, compound 1 is furanoid toxin, which had been isolated from Curvularia lunata, the pathogen that causes Curvularia leaf spot on maize [
pyogenes, Proteus vulgari, and E. coli [
During our investigations of the anti-inflammatory compounds, biphenyls were isolated from Streptomyces sp. BO-07 which had anti-inflammatory activities [
Our results indicate that compound 2 inhibits the TNF-α, IL-1β and IL-6 mRNA transcription. The results were in agreement with geladanamycin inhibits the production of TNF-α in taxol, LPS, or CpG DNA-activated RAW 264.7 cells [
In addition, previous work by Igarashi et al. [
The results obtained here demonstrate that Streptomyces zerumbet, a novel species isolated from Zingiber zerumbet (L.) Smith could produce methyl 5-(hydroxymethyl)furan-2-carboxylate (1) and geldanamycin (2). The compound 1 had antibacterial activity against Staphylococus aureus ATCC 25923 and Methicillin-resistant S. aureus with the MIC and MBC values of 1 µg/ml and 16 - 64 µg/ml, respectively, while the compound 2 at the concentration of 1 - 5 µg/ml had in vitro anti-inflammatory activity on LPS-induced RAW 264.7 cells by inhibition of mRNA expression and production of inducible NO synthase (iNOS), nitric tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). These results indicate that the compounds 1 and 2 exhibited promising antibacterial and anti-inflammatory activities, respectively. In conclusion we suggest that the future studies on these compounds could be useful for the management of bacterial infections and inflammatory diseases. And the compound relationship between endophyte and host plants should be also further studied in the future.
The authors are grateful to Ms Sopita Rattanopas and Ms Siwaporn Inpang in the Department of Chemistry, Faculty of Science, Silpakorn University, Thailand, for measuring NMR and MS data, respectively. This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Thailand.
The authors declare no conflicts of interest regarding the publication of this paper.
Taechowisan, T., Puckdee, W. and Phutdhawong, W.S. (2019) Streptomyces zerumbet, a Novel Species from Zingiber zerumbet (L.) Smith and Isolation of Its Bioactive Compounds. Advances in Microbiology, 9, 194-219. https://doi.org/10.4236/aim.2019.93015