HPTLC fingerprint profile of antibacterial compound produced from forest soil Streptomyces SFA5

Ponnuswamy Shekar¹, Kannaiyan Sathish Kumar¹, Venugopal Gopikrishnan² and Manikkam Radhakrishnan²

¹Department of Chemical Engineering, SSN College of Engineering, Chennai 603 110, India; ²Centre for Drug Discovery and Development, Sathyabama University, Chennai 600 119, India.

Abstract

The present study investigates the presence of antimicrobial compounds from potent isolate Streptomyces sp. SFA5 from the Sabarimala forest ecosystem, Western Ghats, Kerala, India. Preliminary screening revealed Streptomyces sp. SFA5 isolate shows significant inhibition against the bacterial and fungal pathogens. The antibacterial compound from isolate SFA5 was produced by submerged fermentation using yeast extract malt extract broth and extracted using different solvents. Among the different solvent extracts tested, the ethyl acetate extract of SFA5 shows maximum zone of inhibition against Staphylococcus aureus (23 mm) and Bacillus cereus (24 mm). A simple, sensitive and accurate HPTLC method has been performed for the quantitative estimation of bioactive compounds from ethyl acetate extract of Streptomyces sp. SFA5. The optimized solvent system for mobile phase in HPTLC was toluene: ethyl acetate: methanol: acetic acid (5:3:1:0.5, v/v/v/v). Results of HPTLC finger printing of ethyl acetate extract of the strain SFA5 at UV 254 and 366 with corresponding Rf values substantiate the presence of bioactive compounds.

Introduction

High performance thin layer chromatography (HPTLC) is one of the modern sophisticated techniques that can be used for quick and easy determination of quality, authenticity and purity of the natural extracts, crude drugs and market formulations (Mamatha et al., 2011; Miyadoh et al., 1993). HPTLC provides valuable tool for reliable identification through chromatographic fingerprints that can be visualized and stored as electronic images.

The present study reports the HPTLC fingerprint profile and qualitative analysis of bioactive compounds produced by soil Streptomyces sp. SFA5 isolated from the Sabarimala forest ecosystem, Western Ghats, India.

Materials and Methods

Description of Streptomyces species SFA5

Streptomyces species (SFA5) was isolated from Sabari-mala Forest Ecosystem, Western Ghats, Kerala, India (Latitude: 09° 25′ 59″ N; Longitude: 77° 04′ 59″ E) using starch casein agar medium supplemented with filter sterilized nystatin (20 µg/mL) and nalidixic acid (100 µg/mL) by serial dilution plating method. Viability of strain SFA5 was maintained on ISP2 agar slants at 4°C (Shekar et al., 2011). Strain SFA5 produced soluble yellow pigment on ISP2 agar medium.

Production and extraction of bioactive compounds

For the preparation of inoculum, spores of Streptomyces sp. SFA5 were inoculated into 100 mL yeast extract malt extract (YEME) broth in 500 mL conical flask at 28°C in rotary shaker with 90 rpm for 7 days (Saravanan et al., 2012). After incubation the fermented broth which contains the bioactive compounds was centrifuged at 10,000 rpm for 30 min at 4°C. The cell free supernatant was tested for antimicrobial activity by agar well diffusion method (Chaudhary et al., 2013). To extract the antimicrobial compounds, cell free supernatant was extracted by liquid-liquid extraction method (Shekar et al., 2012) using equal volume of (1:1 v/v) different solvents such as ethyl acetate, methanol, chloroform, dichloromethane, and n-hexane and allowed to dry. The concentrated crude extract was impregnated in sterile 5 mm filter disc at 100 µg concentration placed over the test organism. Test organisms used for screening include standard pathogens such as Staphylococcus aureus NCIM 2079, Bacillus cereus NCIM 2106, Escherichia coli NCIM 2256, Pseudomonas aeruginosa NCIM 5031, Candida albicans ATCC 90028, Aspergillus fumigatus NCIM 6645 and Cryptococcus neoformans ATCC 66031 and clinical pathogens such as S. aureus, enteropathogenic E. coli, P. aeruginasa, K. pneumoniae and C. albicans. Zone of inhibition was measured after 24 hours of incubation at 37°C.

HPTLC profiling of ethyl acetate extract

The crude ethyl acetate extract of Streptomyces sp. SFA5 which showed good activity against test pathogens was subjected to HPTLC analysis. The ethyl acetate extract was dissolved in 200 μL of ethyl acetate from which one micro liter of crude extract was loaded as 6 mm band length in the 3 x 10 cm silica gel 60F254 coated TLC plate using hamilton syringe and camag linomat 5 instrument.

Spot development

The crude extract loaded plate was kept in TLC twin trough developing chamber (after saturated with solvent vapour) with respective mobile phase and the plate was developed in the mobile phase up to 90 mm and it was then dried by hot air to evaporate solvents from the plate. Using UV light torch, the developed spots were marked, the distance travelled by each spot in baseline and relative R_f values were calculated (Baskar et al., 2010).

R_f value = Distance travelled by the substance / Distance travelled by the solvent

Photo documentation

The TLC plate was kept in photo documentation chamber and the images were taken at white light, UV 254 nm and UV 366 nm. The developed TLC plates were sprayed with anisaldehyde sulfuric acid reagent and dried at 100°C in hot air oven. After derivatization, the TLC plate was fixed in scanner stage (CAMAG TLC SCANNER 3) and scanning was done at 366 nm. The peak table and peak densitogram were also noted.

Results

Streptomyces sp SFA5 was showed good growth with ash white color aerial mycelium, brown color substrate mycelium and diffusible yellow pigment on YEME agar medium (Figure 1). The cell free supernatant of Streptomyces sp.SFA5 exhibited good activity over the standard as well as clinical pathogens in YEME broth (Radhakrishnan et al., 2013).

In total, the supernatant showed good activity over Gram positive bacteria and fungi than Gram negative bacteria. The supernatant of Streptomyces sp. SFA5 inhibited standard bacterial pathogen such as Staphylococcus aureus NCIM 2079 (22 mm) and Bacillus cereus NCIM 2106 (23mm) as well clinical pathogen such as Staphylococcus aureus (19 mm) with a maximum zone of inhibition (Table I). Further cell free supernatant of Streptomyces sp. SFA5 inhibited the standard fungal pathogen Candida albicans ATCC 90028 (18 mm) and clinical fungal pathogen Candida albicans (22 mm) with maximum zone of inhibition. Five different solvent tested for extraction, ethyl acetate extract of Streptomyces sp. SFA5 grown on YEME broth showed maximum zone of inhibition on antimicrobial activity, while other solvent extract such as dichloromethane, chloroform and n-hexane extract showed no zone of inhibition (Table II).

The HPTLC profiling of ethyl acetate extract of Streptomyces sp. SFA5 shows presence of antimicrobial compounds in the chromatograph as well as in UV after derivatization (Febina et al., 2013). The analysis was carried at volume of 10 µL Streptomyces sp. SFA5 extract at UV 254 nm and UV 366 nm (Figure 2). Yellow brown color spots were observed after derivatization confirmed the presence of antimicrobial compound and optimized solvent system for mobile phase involves toluene: ethyl acetate: methanol: acetic acid (5:3:1:0.5, v/v/v/v).

The Streptomyces sp. SFA5 shows nearly 14 peaks with good Rf and area% values for the peak 7 (0.4 and 29%) and peak 8 (0.5 and 18%) for finger print profile ethyl acetate extract indicates firmly the presence of antimicrobial compound at UV 254 nm and UV 366 nm (Figure 3). The Streptomyces sp. SFA5 shows nearly 6 peaks with good Rf and area percent values for the peak 2 (0.2 and 28%) and peak 3 (0.3 and 51%) for finger print profile of ethyl acetate extract indicates stoutly the presence of antimicrobial compound at UV 366 nm.

Figure 4 shows the profiling results of SFA5 ethyl acetate extract at UV 254 nm.

Discussion

Streptomyces species SFA5 from forest ecosystem exhibited good activity against Gram positive bacteria and fungi in agar well diffusion. Most of the metabolites reported from actinomycetes are found to be active against Gram positive bacteria when compared to Gram negative bacteria (Berdy, 2005; Radhakrishnan et al., 2010). The antimicrobial activity of ethyl acetate extract confirmed the extracellular nature of bioactive compounds. Most of the bioactive metabolites from Actionmycetes are extracellular in nature (Radhakrishnan et al., 2011). HPTLC is a reliable chromatographic method for analysing several samples of divergent nature and composition at the same time (Andola et al., 2010). HPTLC analysis of the ethyl acetate extract of SFA5 produced two yellow brown spots revealed the presence of two major antibacterial components at R_f value of 0.38 and 0.46 in the chosen solvent system.

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