Biological activities of new secondary metabolite produced by Streptomyces badius

Secondary metabolites obtained from Actinomycetales provide a potential source of many new antibacterial, antitumour, antifungal, antiviral, antiparasitic compounds and others. The majority of discovered substances are widely used as medicines for combating multidrug-resistant Gram-positive and Gram-negative bacterial strains, as well as in veterinary practice, agriculture and industry. Members of the Streptomyces genus are outstanding producers of already known secondary metabolites. In our study we isolated a new metabolite from Streptomyces badius fermentation broth. This compound, 2-acetamido-5-hydroxybenzoic acid (1) was already described in literature, however, not yet discovered as Streptomyces badius product. The compound was firstly isolated from fermentation broth and purified by chromatography methods (ion exchange resin DowexWX40, Solid Phase Extraction C18 Polar Plus; HPLC, dC18). Then, the chemical structure of metabolite was determined by 1H NMR, 13C NMR and 2D homo- and heteronuclear (1H-13C HSQC, 1H-13C HMBC) NMR. The molecular formula of (1), C9H9NO4 was identified by high resolution ESI-MS. The chemical structure was analyzed by the UV and IR methods, as well. Biological activities of 2-acetamido-5-hydroxybenzoic acid were evaluated. The compound shows moderate DD-peptidase 64-575 inhibitory activity as well as antioxidative properties (ABTS radical scavenging). Such chemical moiety may serve as model compound for further modern drug discovery and be a source of active substance in anti-ageing cosmetics.


Introduction
Search for bioactive compounds from nature play crucial role in fashioning new therapeutic agents.Especially secondary metabolites have major importance in drug discovery process.They are diverse and unusual in their chemical structures and may be used as scaffolds for further modifications.Actinomycetes are main producers of bioactive metabolites [1,2].
Actinomycetes are the most widely distributed groups of microorganisms in nature.They can be found in various environments such as soil and water.Their metabolites are active against bacteria, viruses, fungi, parasites and cancer cells [3].

Materials and Methods
Strain: Streptomyces badius ATCC 19888 was isolated from the soil in Kaukasus (Russia).Fermentation and purification: fermenter (Sartorius Biostat A, Germany), HPLC (Knauer).NMR analysis: The 1D 1 H and 13C NMR spectra as well as 2D homo-and heteronuclear spectra were collected on a 700MHz Bruker AVANCE III spectrometer, equipped with a QCI CryoProbe Experiments were performed at 25°C.Spectra were processed and prepared with TopSpin 3.0 Bruker Software.HR MS analysis: MaldiSYNAPT G2-S HDMS (Waters) coupled with ACQUITY UPLC I-Class System (Waters).

Biological assays:
The DD-peptidase 64-575 inhibition was measured spectrophotometrically according to the method previously described [4,5] with modifications.
The DPPH and ABTS radicals scavenging activity was assayed based on methods previously described [6].

Results: Fermentation
The Streptomyces badius ATCC 19888 was grown at 28°C for 10 days on yeast-malt agar medium.Then, S. badius spores were inoculated into a 500-ml Erlenmeyer flasks containing 35 ml of liquid medium M [7].
The inoculated flasks were incubated for 24h at 28°C in a rotary shaker at 220 rpm (Ecotron, Infors AG, Switzerland).Then, 280 ml of seed culture was transferred to 4.5 l of the same medium in fermenter.Fermentation process was carried out 144h at 28°C with minimal aeration 30% of air.After that the supernatant was collected for purification. The molecular formula of 1 was determined to be 2-acetamido-5hydroxybenzoic acid (C 9 H 9 NO 4 ).

References
 This compound shows DD-peptidase 64-575 inhibitory activity as well as prolonged antioxidative activity.
 Such chemical moiety may serve as model compound for further modern drug discovery and be a source of active substance in anti-ageing cosmetics.

Figure 1 .
Figure 1.The 1 H NMR spectrum of 1 recorded in D 2 O at 25°C.

Figure 2 .
Figure 2. The 13 C NMR spectrum of 1 recorded in D 2 O at 25°C.
The 13 C NMR spectrum of 1 contains two signals in the carbonyl region, eleven in the aromatic region and one in aliphatic region.We observed five carbon signals more than expected from ESI-MS, thus they must have come from the contamination of the sample.

Figure 3 .
Figure 3.The 2D COSY spectrum of 1 recorded in D 2 O at 25°C.
The molecular formula of 1 (C 9 H 9 NO 4 ) was determined by high resolution ESI-MS [ESI-MS spectra for the peak with the RT 0.92 min of the sample 1 ep.30negative ion mode (top) and positive ion mode (bottom)].The molecular formula prediction for the deprotonated molecule (m/z 194) in negative ion mode is shown in the table.


New metabolite 1 was isolated from S. badius fermentation broth and purified by chromatography methods.