Saccharpiscinols A–C: Flavans with Potential Anti-Inflammatory Activities from One Actinobacteria Saccharomonospora piscinae

Phytochemical investigation and chromatographic separation of extracts from the actinobacteria strain Saccharomonospora piscinae that was isolated from dried fishpond sediment of Kouhu township, in the south of Taiwan, led to the isolation of three new compounds, saccharpiscinols A–C (1–3, respectively), and three new natural products, namely (2S)-5,7,3′,4′-tetrahydroxy-6,8-dimethylflavanone (4), methyl-4-hydroxy-2-methoxy-6-methylbenzoate (5), and (±)-7-acetyl-4,8-dihydroxy-6-methyl-1-tetralone (6). Compounds 4–6 were reported before as synthesized products, herein, they are reported from nature for the first time. The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic data analysis (1D- and 2D-NMR, MS, and UV) and comparison with literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Saccharpiscinol A showed inhibitory activities against LPS-induced NO production.


Introduction
Actinobacteria are widely distributed in nature, and they are very useful in the pharmaceutical industry due to their seemingly unlimited capacity to produce secondary metabolites with diverse chemical structures and biological activities [1][2][3]. They are gram positive, free-living saprophytic bacteria that are found widely distributed in soil, water, and colonizing plants. Actinobacteria inhabitants have been identified as one of the major groups of the soil population [2,3], which may vary with the soil type. In the process of investigation on the diversity of cultivable actinobacteria associated with soil from Taiwan, we isolated a strain named 06168H-1 T , which was isolated from a fishpond sediment sample collected from the southern area of Taiwan and had a unique morphology [4]. This strain was determined to be Saccharomonospora piscinae (Family: Pseudonocardiaceae), based on their phenotypic and genotypic data [4]. In order to find new bioactive compounds, the rare genera in this group have been identified and studied, including the genus Saccharomonospora, which is a genus of bacteria that was only discovered by Nonomura and Ohara, and contained 12 species until now [4]. Saccharomonospora strains are widely distributed in nature, including in soil, compost, plant bodies, marine sediments, sponges, and other environments. Therefore, in addition to mesophilic bacteria, there are also thermophilic strains. The spores of some thermophilic strains (Saccharomonospora viridis) can cause allergic pneumonia (farmer's lung disease). In addition, research reports indicate that strains of this genus can decompose many natural or synthetic compounds, such as polyester, rice straw, mushroom compost, food waste compost, protein, and starch. S. piscinae was isolated from the bottom of the southern fishpond. The colony is blue and performs aerobic growth. The aerial hyphae can produce non-moving short spore chains. It has high salt tolerance and is a salt-tolerant strain. More specialised physiological characteristics can produce special metabolites. It belongs to a special environment strain and is worthy of further investigation and study of its metabolites. However, investigation on the secondary metabolite of Saccharomonospora sp. is still very limited.
Nitric oxide (NO) is a mediator in the inflammatory response involved in host defense. In our effort to search for structurally interesting and bioactive natural products from microbes, we had isolated and identified over 300 microbial strains from Formosan plants and special environment research materials, and the crude EtOAc and BuOH extracts from those were screened for their inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) release production in RAW 264.7 murine macrophages.
In the course of our search for potential diverse secondary metabolites from natural microbial sources, and to further understand the minor metabolites of the genus Saccharomonospora, we examined the EtOAc extract of S. piscinae, which showed rich metabolites according to the fingerprint analysis and inhibitory activity on LPS-induced NO release in RAW 264.7 murine macrophages, as determined by our primary screening (approximately 95% inhibition at a concentration of 10 µg/mL). Investigation of the bioactive metabolites of the active EtOAc extract from the microbe S. piscinae, fermented by solid rice medium, was investigated. The metabolites investigation guided by the HPLC profile analysis and 1D NMR spectra prescreening led to the isolation of three new metabolites, saccharpiscinols A-C (1-3, respectively), and three metabolites isolated for the first time from nature sources, (2S)-5,7,3 ,4 -tetrahydroxy-6,8-dimethylflavanone (4), methyl-4-hydroxy-2-methoxy-6-methylbenzoate (5), and (±)-7-acetyl-4,8-dihydroxy-6methyl-1-tetralone (6).The structures of these isolates were established by means of spectral experiments. The isolation, structural elucidation, and inhibitory effects of some isolates on nitric oxide (NO) production by RAW 264.7 macrophages are described herein.
Compound 6 was obtained as gum. Its molecular formula, C 10 H 12 O 4 , was deduced from a [M] + peak at m/z 196.0733 in its HREIMS spectrum (calcd. 196.0736 for C 10 H 12 O 4 ). There are 10 carbon signals in the 13 C NMR spectrum, including one ester C=O at δ C 168.0, one methyl group at δ C 19.7, two methoxyls at δ C 51.7 and 55.9, and six aromatic carbon atoms with one deshielded signal at δ C 159.6. The 1 H and 13 C NMR spectra of 5 are quite similar to those of methyl-2,4-dihydroxy-6-methyl benzoate, which suggested that compound 5 has a benzenoid structure. Comparing the 1 H and 13 C NMR spectra of compound 5 with those of the similar analogue, methyl-2,4-dihydroxy-6-methyl benzoate, it was presumed that a hydroxyl group [δ H 11.60 (1H, s, OH-2); δ C 163.24 (C-2)] at C-2 of methyl-2,4-dihydroxy-6-methyl benzoate was substituted with a methoxy moiety in 8 from the signals at [δ H 3.72 (3H, s, OMe-2); δ C 55.9 (OMe-2)], which were confirmed by HMBC correlations. The HMBC correlations of OMe-2/C-2 and Me-6/C-6, 1 and 5 determined the positions of methoxyl and methyl groups, which was confirmed by the correlations between OMe-2/H-3 and Me-6/H-5 in the NOESY spectrum ( Figure 3). Finally, the NMR data were identical to those of methyl-4-hydroxy-2-methoxy-6-methylbenzoate, which has been obtained synthetically [15]. This is the first time 6 has been isolated from a nature source.

Biological Studies
Due to the small quantity of isolated compounds (5 and 6), we evaluated the inhibitory effects of saccharpiscinols A-C (1-3, respectively) and (2S)-5,7,3 ,4 -tetrahydroxy-6,8-dimethylflavanone (4) on the production of NO induced by LPS (Table 1). They showed potent inhibition with IC 50 values between 12.5 to 21.8 µM against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The high cell viability (>80%) indicated that the inhibitory activity of LPS-induced nitrite production by compounds 1 and 2 (IC 50 value: 12.5 µM and 18.0 µM) did not result from its cytotoxicity. Compounds 3 and 4 (IC 50 value: 21.8 µM and 20.0 µM) also showed inhibition of the NO production of macrophages, but the low cell viability (<80%) suggested the possibility of cytotoxicity. The RAW 264.7 cells were incubated with compounds in the presence of LPS (1 µg/mL). The medium was harvested 24 h later and assayed for nitrite production. NO release was measured by using the Griess reagent. The cell viability was evaluated with the MTT assay. The results are presented as a percentage of the control value obtained from non-treated cells. Values are expressed as mean ± SD of three individual experiments, performed in triplicate. * Statistically significant difference compared to LPS-activated cells (p < 0.05). # Statistically significant difference compared to untreated cells (p < 0.05).

Microorganism
Saccharomonospora piscinae (06168H-1 T ) was used throughout this study, and deposited at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI). This actinobacteria was identified by M. T.

Cultivation and Preparation of the Fungal Strain
The actinobacteria, Saccharomonospora piscinae, (06168H-1 T ), was isolated from dried fishpond sediment from the Kouhu township, in the south of Taiwan. The sample of fishpond sediment was dried at room temperature for 7 days. The sample suspension (100 µL) was plated on modified Sabouraud glucose agar (SGA; 7.5 g casamino acid, 10.0 g yeast extract, 20.0 g MgSO4·7H 2 O, 3.0 g trisodium citrate·2H 2 O, 2.0 g KCl, 34.0 g NaCl, 10.0 µg Fe 2+ , 18.0 g agar, 1.0 l distilled water, pH adjusted to 7.4) and incubated at 30 • C for 4 weeks. The spores or mycelia suspension were harvested with 20% (v/v) glycerol and stored at −20 • C.
The actinobacteria, Saccharomonospora piscinae, (06168H-1 T ), was maintained on Sabouraud glucose agar (SGA) and the strain was cultured on potato dextrose agar slants at 30 • C for 7 days, and then the spores were harvested by sterile water. The spores (5 × 10 5 ) were seeded into 500 mL shake flasks containing 50 mL CM+YM (Sabouraud glucose agar, SGA; 7.5 g casamino acid, 10.0 g yeast extract, 20.0 g MgSO4·7H 2 O, 3.0 g trisodium citrate·2H 2 O, 2.0 g KCl, 34.0 g NaCl, 10.0 µg Fe 2+ , 18.0 g agar, 1.0 L distilled water, pH adjusted to 7.4) and cultivated with shaking (150 rpm) at 30 • C for 5 days. After the mycelium enrichment step, an inoculum mixing 100 mL mycelium broth and 100 mL CM+YM medium was inoculated into plastic boxes (25 cm × 30 cm) containing 1.3 kg sterile rice and cultivated at 30 • C for producing rice, and the abovementioned CM+YM medium was added to maintain the growth. After 21 days of cultivation, the rice was harvested, and used as a sample for further extraction.

Isolation and Characterization of Secondary Metabolites
The rice fermentation was extracted five times with an equal volume of EtOAc. The combined EtOAc layers were evaporated to dryness under reduced pressure to give an EtOAc extract (12 g) which was subjected to silica gel column chromatography (CC) (petroleum ether-EtOAc v/v, gradient; from 10:1 to 0:1 to generate seven fractions (Frs. 1-7). Fr. 2 was isolated by CC on silica gel eluted with hexane-EtOAc (from 5:1 to 1:1) to afford four subfractions (Fr. atmosphere in a 5% CO 2 incubator. The cells were treated with 10, 25, and 50 µM of natural products in the presence of 1 µg/mL LPS (lipopolysaccharide, Sigma-Aldrich) for 20 h. The concentration of NO in culture supernatants was determined as nitrite, a major stable product of NO, by a Griess reagent assay [16], and cell viabilities were determined using the MTT assay as described previously [17].

Conclusions
Under the support of the Ministry of Economic Affairs, the Bioresource Collection and Research Center at the Food Industry Research and Development Institute has been dedicated to the research work on the collection, separation, and preservation of bioresource research in the past few years, and has constructed a complete indigenous strains resource bank in Taiwan. The applicant analyzed the active constituents from it, and obtained more than sixty active new compounds isolated from red yeast rice, endophytes, actinobacteria, and mushrooms, among which many new compounds have anti-cancer and anti-inflammatory effects [18][19][20][21][22][23].
Actinobacteria have the ability to produce a variety of physiologically active products, so they play a very important role in the food and pharmaceutical industries. Over the years, our team has also separated and collected actinomycetes resources from all over Taiwan and various environments. In addition to common Streptomyces, there are also many rare species of actinobacteria, and there are many new species. Based on the concept of "new species and new compounds", it is expected that special compounds can be found from these new strains. In recent years, studies have also found that these new species of actinobacteria can produce many active secondary metabolites. In order to further explore the efficacy of different strains of actinobacteria and expand the application range of actinomycetes, therefore, this project uses one new species of actinobacteria that has not been studied in the past. This species is cultured, extracted, purified and identified with high-level and highly active anti-inflammatory compounds with solid rice, in order to improve the research of actinobacteria at the country level, and develop health products related to actinobacteria. In summary, we have isolated and characterized three undescribed flavan derivatives, saccharpiscinols A-C, from an actinobacteria strain Saccharomonospora piscinae that was isolated from dried fishpond sediment from the Kouhu township, in the south of Taiwan. The absolute configurations of saccharpiscinols A-C were determined by comparing their optical activities with related derivatives. Saccharpiscinols A and B showed inhibitory activities against LPS-induced NO production. The discovery of flavan derivatives from actinobacteria pointed toward the potential use of endophytic or associated Saccharomonospora piscinae as alternative producers of flavan derivatives. The current results may encourage further investigations on the chemistry and bioactivity of flavan metabolites. These results also suggest that Saccharomonospora has distinct and diverse metabolites that arise under different fermentation conditions and soil-derived collections. It may therefore be possible to find more new bioactive natural products by searching Saccharomonospora species under special eco-environments. For the sake of better understanding the distribution of flavan acid analogs, the actinobacteria of the tilted research material and other special strains are worth examining for the presence of these secondary metabolites.