A Halophilic Marine Bacterium Shewanella Alga (A1): Isolation and Identification of New Bioactive Compound with Cytotoxicity Activity

Marine bacteria have been produced as a valuable source of bioactive substances, and marine natural products play an essential role in biomedical research and the pharmaceutical sector (1). Nature has been the source of medical remedies for thousands of years. Many modern medications are isolated from microbes, most of which are based on traditional medicine. In the preceding century, microorganisms became increasingly important in the production of antibiotics and other drugs (2). Terrestrial fungus and bacteria have been critical sources of valuable bioactive compounds for more than half a century (3). For nearly two centuries, people have been looking for fresh medicine goods from the sea. Prior research has demonstrated that while marine invertebrates are a huge source of new biochemical compounds, as seen by the enormous number of chemicals found in clinical studies, appropriate and consistent supplies of these compounds have proven infamously difficult to obtain from nature. As a result of these problems, researchers have well-documented the importance of marine microorganisms (4). As a result, the expected massive richness of sea organisms could have been overlooked at first glance for a variety of reasons, even though marine-derived microbes were not taxonomically defined. The foundation investigations revealed that the world's seas are rich in microbial diversity, making them potential frontiers for new drug discovery (5).  Salt tolerance microorganisms are known as halophiles. They can grow in a variety of high-salt environments (6). The salt concentration in the ocean is 3-5 per cent in a hypersaline environment. Halophiles were discovered in every domain of life, with archaea being the most common. Extreme habitats are defined by extreme environments that make higher life forms uninhabitable (7). Microorganisms are halophilic and produce important enzymes and bioactive chemicals. Secondary metabolites (extracellular polysaccharides, such as proteins, enzymes, amylase, cellulases, and amino acids) are also produced by halophilic bacteria (8). Salt-loving bacteria that live in hypersaline settings are known as halophilic microorganisms. Some microorganisms have evolved to diverse extreme environmental temperature conditions such as pH, salinity, radiation, and pressure in halophilic and halotolerant bacterial media that contain more than 5% salinity (9).  The objective of the work, to study therapeutic applications of halophilic bacteria. Marine habitats nevertheless have a vast diversity of bacteria with a wide range of bioactive compounds. A combination of culture and molecular methods will be used to describe halophilic bacteria from marine sediment samples (Ponnani shore in Kerala, India) and profile potential biotechnological applications.

MATERIALS:

Chemicals and reagents

Marine Zobell media (Himedia Pvt. Bombay, India), DMEM (Dulbecco's Modified Eagle's Medium), streptomycin, Penicillin-G, L-glutamine, phosphate-buffered saline (PBS), trypsin-EDTA, acridine orange, ethidium bromide, ethanol, ethyl acetate, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich Chemical (India). Hi media Laboratories Pvt. Ltd., India, supplied analytical grade hexane, chloroform, ethyl acetate, acetone, and benzene.

METHODOLOGY:

Sample collection and identification of antimicrobial agent producing bacteria

Grab sample collection method used to collect marine sediments from Ponnani shore in Kerala, India. The sediment sample was serially diluted in sterile water, and 1ml serially diluted sample from the dilutions was added to the marine Zobell agar plate. For seven days, the plate was incubated at 37°C. Bacterial colonies were isolated and purified using the streak plate method after being isolated using an inoculation loop.

Phenotypic identification

The strains' fundamental phenotypic analysis was carried out using the techniques described by Romanenko LA et al. (2003). The method described by Chao Y; Zhang T was used to conduct morphological studies on pure bacterial cultures using an electronic microscope (2011).

Genotypic identification

Bacterial DNA extraction was subject to normal PCR insulation conditions. The reaction was heated for 10 min to lyse the cells, and the DNA was centrifuged at 13,000 rpm for 3 min. The concentration of DNA in 1% of Agarose Gel Electrophoresis was measured.

PCR was performed using the 1492R (5?-TACGGCTACCTTGTTACG ACTT-3?) and 27F (5?-AGAGTT TGATCCTGCTCAG-3?) primers targeting the 16S rRNA domain Bacteria. The PCR product has been sequenced using primers. Sequencing reactions have been conducted using ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (Applied Biosystems).

The phylogeny analysis of the query sequence accompanied by multiple sequence alignment was conducted with the closely related sequence of blast data. For different alignments of sequences, the programme MUSCLE 3.7 was used. Phylogeny analysis PhyML 3.0 aLRT software was used (10).

Extraction and isolation of secondary metabolites

The biologically active extract fraction 1 (F1) was used for its purity analysis. The purity of F1 was analysed by reversed-phase high-performance liquid chromatography (RP-HPLC- Shimadzu LC-10 system, Shimadzu Co., Kyoto, Japan) coupled with a PDA detector by Zhang et al. 2015. The C₁₈ column was used for separation (100×4.60mm 2.6 ?m, 100 Å) and the column temperature was maintained as 35^°C. The gradient elution was used for HPLC analysis. The mobile phase compositions were: A-water + 0.1% formic acid, B-methanol: acetonitrile: formic acid (80:20:0.1% (v/v). The gradient elution was started at 90:10 (A: B) and changed to 10:90 (A: B) with the flow rate of 1 mL/min for a total run time of 18 min. 10µl of injection volume was used, and the detecting wavelength was used as 280 nm.

Column Chromatography

The concentrated material was mixed with 80% ethanol, and the final extract was subjected to silica gel chromatography using methanol: dichloromethane (4: 1) ratio. The eluting solvent was collected and concentrated using a vacuum evaporator. The purity of the final single active compound was confirmed by TLC n-hexane: ethyl acetate (80:20: v/v) with a 0. 49 Rf value. The final eluent was recrystallised by ethanol (room temperature). The melting point of this compound is 71-73°C.

Determination of Compounds by TLC

The isolated compounds were assessed by TLC using silica gel-G (Merck grade) as the adsorbent. The solvent system (stationary, mobile phases) was indicated at appropriate places. The TLC plates are coated with silica and alumina. Silica gel is an essential material for the adsorption of water molecules.

Determination of functional group by FT-IR Spectrum

The functional group present in F1 was analysed using FTIR analysis. KBr powder was combined with the optimised extract to produce a 1% (w/v) slurry concentration, and the KBr pellet was prepared by pressing approximately 5.5 tons for 3 min. The measurements were then performed with a resolution of 4 cm?1 on a JASCO FT/IR-6300 instrument (JASCO Corporation, Tokyo, Japan), and the spectra were recorded over the IR spectrum of 400–4,000 cm^?1.

Determination of proton ¹HNMR and ¹³C NMR Studies

The identity of F1 was confirmed using Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker BioSpin, Rheinstetten, Germany). The ¹H NMR spectrometer was operated at 400 MHz, DMSO was used as the solvent of choice, and the spectra were recorded as follows. The ¹³C NMR spectrometer was performed at 100 MHz, DMSO was used as the solvent of choice, and the spectra were recorded as follows. Proton nuclear magnetic resonance spectra (1H NMR) and carbon nuclear magnetic resonance spectra (13C NMR) were recorded on a Bruker DPX400 spectrometer at 25 ºC with dimethyl sulfoxide (DMSO) and solvent signals allotted as internal standards.

Determination Mass Spectroscopy (LC-MS)

The molecular weight of F1 was analysed by LC-MS/MS analysis. Solvents used were 0.5% (v/v) acetic acid (A) and 100% methanol (B). The isocratic elution was as follows: (i) 55% of solvent A, from 0 to 10 min, (ii) 65%, from 11 to 20 min (iii) 35%, at 21-30 min of total run time. The PDA detector (UPLC LG 500 nm) was monitored at 340 nm, and the column temperature was maintained at 30 °C. The mass spectrometer (MS) was operated in the positive ionisation mode with the mass range of 150 m/z to 1000 m/z, the capillary voltage of 3.50 kV, cone voltage of 30 V, extractor voltage of 3V, the gas flow of 30 L/Hr and collision gas flow of 0.18 mL/Min. The mass spectrometry (MS) was determined for synthesised compounds using a Shimadzu QP0-2010 plus instrument. Direct injection detection of the ions was performed in an electrospray ionisation (ESI) quadrupole ion trap mass analyser.

Cytotoxic activity

SK-MEL-3 cells collected from National Centre for Cell Sciences (NCCS) in Pune, India. The cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10?tal Bovine Serum (FBS). To prevent bacterial contamination, streptomycin (100 g/ml) and penicillin (100 g/ml) was added to the medium. The cell culture medium was kept at 37°C in a humidified atmosphere with 5% CO₂.

Cell culture and cytotoxicity assay

In a 96-well plate, was used to assess the cytotoxicity of the ethyl acetate extract (F1). The cytotoxicity study carried out as described by Annamalai et al. (11) with modifications.

Statistical analysis

Statistical comparisons of cytotoxicity data were made using statistical product and service solutions (SPSS) version 12.0 for Windows, which included a one-way variance analysis (ANOVA) and a Duncan Multiple Range Test (DMRT). If the p-value is less than 0.05, results expressed as the mean of SD might be regarded statistically significant.

RESULTS AND DISCUSSION:

Phenotypic identification

Extracellular secretion and halophilic bacteria strain-A-1. With the culture growth in zobell media and studied biochemical characteristics. Shewanella alga shows the indole, voges proskauers, citrate utilisation, glucose, adonitol, arabinose, lactose sorbitol, mannitol, rhamnose and sucrose positive.  Bacterial cell morphology was detected using an electronic scanning microscope (Figure 1).

Molecular identification of the A1 through genomic analysis (16s rRNA)

Fermentation was carried out in aerobic conditions with a marine Zobell medium, and bacterial growth was observed on the second day of fermentation (Figure 5). Centrifuged and separated cell mass from fermented A1 (Shewanella alga) culture. The supernatant was collected for secondary metabolite screening.

TLC and HPLC

The purity of the final single active compound was confirmed by TLC (n-hexane: ethyl acetate (80:20: v/v).) it gave a discrete profiling pattern. Concentrated metabolite extract was run in a silica gel plate, and a spot was visualised with a retention factor (Rf) value of 0. 49 (Figure 3). The final eluent was recrystallised by Ethanol (Room Temperature). HPLC analysis (Figure 4) with RT 2.611d confirms the purity of the active compound. The final eluent was recrystallised by ethanol.

       
           
       
  Figure 3: TLC Single compound 1-(1H-indol-3-yl)-2-phenylpentan-3-one

The melting point of the isolated compound was found to be between 71-73°C.

FTIR spectrophotometry

The FTIR spectrum of the secondary metabolite scanned in the range 400 cm^-1 to 4000 cm^-1 showed significant peaks at 1889. The peaks at Ketone (C=O) (1889 cm^-1).

¹H NMR-

The spectrum of chemical shift (d) for proton NMR was between 0 and 14 ppm. The chemical sample shift was compared with the Tetramethyl silane (TMS) protons at 0 ppm. ¹H NMR (400 MHz, DMSO-d₆, ppm): ¹H NMR (CDCl­₃): ? 1.03 (3H, t, J = 7.4 Hz), 2.3 (2H, d, J = 7.4 Hz) 3.1(2H, d, J = 4.6 Hz), 4.2 (1H, t, J = 4.6 Hz), 6.29-6.46 (5H,m), 6.51-6.53 (5H,m), 10.34 (1H, s).

It is a modern technique with a very low natural abundance of up to 1.1%. Therefore, this further decreases the absorption intensity with the use of internal TMS comparison. The range of chemical shift (d) from 0-180 ppm is possible. An additional benefit of this methodology is that one can directly analyse the functional, group-containing carbon atom. ¹³C NMR (100 MHz DMSO-d₆): 210,137,136,128,128, 128,127,127,123,121,118,117,110,54,35,32,7.

The substance isolated from the K1 ethyl acetate extract was subjected to the spectral studies of Mass. The mass spectrum of the isolated compound suggested its molecular weight has been confirmed MS (ESI) m/z (% of relative abundance) calculated for C₁₉H₁₉NO: 277.36, Found 278.57 [M+1].

IR (?max, cm^-1, KBr): 1889 (C=O), Streching. ¹H NMR (400 MHz, DMSO): ? 1.03 (3H, t, J = 7.4 Hz), 2.3 (2H, d, J = 7.4 Hz) 3.1(2H, d, J = 4.6 Hz), 4.2 (1H, t, J = 4.6 Hz), 6.29-6.46 (5H,m), 6.51-6.53 (5H,m), 10.34 (1H, s). ¹³CNMR (400 MHz, DMSO): 210,137,136,128,128, 128,127,127,123,121,118,117,110,54,35,32,7. LC-MS (ESI) m/z (% of relative abundance) calculated for C₁₉H₁₉NO: 277.36, Found 278.57 [M+1]. The purity of the isolated compound was confirmed by HPLC with RT 2.61.

Anticancer activity was assessed for ethyl acetate extract SK-MEL-3 cells with different concentrations ranging from 10?g/mL, 20?g/mL, 30?g/mL, 40?g/mL, 50?g/ml, 60?g/ml and 70?g/ml. The cell viability analysis was calculated after 48hrs. Figure 6 shows the altered morphology of SK-MEL-3 cells after dose-dependent treatment with A1 ethyl acetate extract. Ethyl acetate extract (60?g) substantially decreased the spread of SK-MEL-3 cells relative to control cell viability.

SK-MEL-3 cells appear as the regular green nucleus, early apoptotic condensed or fragmented form of the yellow nucleus with chromatin, late apoptotic chromatin condensation or fragmentation of orange-stained nucleus and necrotic cells.