ANTIMICROBIAL ACTIVITY OF NON EDIBLE SEEDS AGAINST IMPORTANT PATHOGENIC MICROORGANISMS PROJECT REFERENCE NO.: 38S _B_MSC_010 COLLGE BRANCH GUIDE STUDENTS : UNIVERSITY OF MYSORE, MYSORE : DEPARTMENT OF STUDIES IN MICROBIOLOGY : PROF. RAVISHANKER RAI V : MS. DIVYA P A MS. RANJITHA V R KEY WORDS: Non edible seeds, antimicrobial, bioactive Introduction Plants and their products are known for their high therapeutic value since ancient era due to their high therapeutic value and have been one of the major sources of bioactive compounds (Christaki et al., 2012). In recent years large numbers of scientific studies have been carried out by evaluating both edible and non edible seeds, as these seeds forms important component which develop into a whole plant which bear highly significant value (Ogrodowska et al., 2014). Owing to which there has been tremendous interest on seed research. But majority of scientific literatures on seed research is pertaining to edible seeds whereas non edible seeds are majorly reported to trade their applications in the production of bio-fuels and scanty reports are available on the complete evaluation of their secondary metabolites which can be promising area of interest for the scientific communities. These secondary metabolites have potential biological activities such antimicrobial activity when compared to the other plant based resources (Chhetri et al., 2008). Search for antimicrobial agents have been one of the major area of concern in recent year due to the increase in incidence of drug resistance in pathogenic microorganisms towards available antibiotics. Plant based antimicrobial metabolites have demonstrated significant activity against an array of pathogenic microorganisms and majority of antimicrobial drugs available in the market are of plant origin (Cowan, 1999). Bioactive compound in plants are produced as a secondary metabolites which elicit pharmacological or toxological effects in man and animals and several of them hold a prime importance function in living plants. For example, flavonoids can protect against free
radicals generated during photosynthesis. Terpenoids may attract pollinators or seed dispersers, or inhibit competing plants. Alkaloids usually ward off herbivore animals or insect attacks (phytoalexins) etc. Objectives of the study: 1. Selection of plant seeds for extraction of secondary metabolites 2. Screening for antibacterial activity of seed extracts 3. Characterization of bioactive compounds 4. Identification of bioactive compounds Material and Methods: Selection and processing of selected seeds Different seeds are selected based on the availability and its importance in production of bio-fuel from different regions of Karnataka. Selected seeds for the analysis were Garcinia indica and Chloroxylon swietenia. Seeds were cleaned, dried and fine powdered using mixer blender individually. Soxhlet extraction The fine powdered seed samples were extracted with methanol as solvents using Soxhlet extraction process. Dried seed sample of 40g was used for soxhlet extraction. Then, the obtained extracts were concentrated using a rotary evaporator (Hahn-Shin, Korea) at 50 ºC to obtain crude extract. The crude extract obtained was preserved at 4 ºC for further analysis. Antibacterial activity Bacterial strains About nine bacterial cultures were selected for the present study and all the cultures were procured from the Microbial Type Culture Collection Centre and Gene Bank, Chandigarh (MTCC). Three Gram positive bacteria, Staphylococcus aureus (MTCC-7443), Staphylococcus epidermidis (MTCC-435) and Bacillus subtilis (MTCC-121) and Gram negative Bacteria Pseudomonas aeruginosa (MTCC-7093), Enterobacter aerogenes (MTCC- 111), Escherichia coli (MTCC-40), Proteus mirabilis (MTCC-425), Shigella flexneri (MTCC-1457), Vibrio parahaemolyticus (MTCC-451) were used for the study. The cultures were maintained in nutrient broth (NB) with periodical sub-culturing before each assay at 37 ºC for 24 h Disc diffusion method
The disc diffusion method was followed as reported previously by kiran.et al (2008). Briefly, the bacterial culture were grown in NB media for 24 h at 37 ºC and culture inoculums approximately adjusted to 1.5X10 8 cfu/ml (using McFarnald Unit). Then, the bacterial cultures were swabbed over pre-solidified Muller-Hinton (MH) agar media and loaded with 30 µl of crude extract (1mg/disc). The disc with solvent (methanol) taken as negative control and chloramphenicol (30 µg/disc) as positive control. Then the plates were kept at 4 ºC for 10 min and incubated at 37 ºC for 24 h. After incubation, the plates were observed for the presence of inhibition zone around the sample disc and compared with positive and negative controls. Minimum Inhibitory Concentration (MIC) by Microtiter plate The minimum inhibitory concentrations (MIC) were followed as reported previously (Sarker et al., 2007). Briefly, the crude extract obtained from soxholation was loaded into the 96 well microtiter plates with 10 µl two fold micro dilution from 1mg/ml to 0.07 mg/ml. After that 170 µl of MH broth was added to all the wells and inoculated with 10 µl respective 24 h bacterial cultures (inoculums approximately adjusted to 1.5X10 8 cfu/ml using McFarnald Unit). The chloramphenicol (30 µg/ml) was taken as positive control and MH broth without bacterial inoculums is taken as negative control.the plates were incubated at 37 ºC for 24 h. After incubation, 10 µl of 2, 3, 5-triphenyl-tetrazolium-chloride (TTC, Himedia, Mumbai) was loaded with the concentration of 10 mg/ml in sterile water and incubated for 30 minutes at 37 ºC. MIC is read as the lowest dilution that showed no evidence of without pink/red colour change. Isolation and characterization of secondary metabolites from crude extracts The crude extract which is showing significant antimicrobial activity was subjected to purification using bio-molecular techniques. Thin layer chromatography (TLC) Thin layer chromatography was followed as described previously (Cieśla and Waksmundzka-Hajnos, 2009) with modifications. Briefly, the crude extract was resuspended with ethyl alcohol and centrifuged (Eppendroff, Germany) at 14000 rpm for 10 min. The supernatant was evaporated to concentrate the crude compound and dissolved to 1 mg/ml of extract. The mobile phase was optimized to get well separated crude extract with different combinations of solvent mixtures. Then, the TLC plate (Merck, Bangalore) was loaded with 10 µg of crude extract containing 80% alcohol as mobile phase and Rf value was calculated for each compound.
Ultra violet-visible spectroscopy (UV-Visible) The crude sample was dissolved in ethanol of the concentration 1 mg/ml and fraction from TLC was also dissolved in ethanol to get 100 µg/ml. The spectrum of crude compound and each TLC fraction were obtained at UV-visible range of 200 to 800 nm. Ethanol was taken as control for the analysis Fourier Transform Infrared Spectroscopy (FTIR) An FTIR spectrum has been done for crude sample and TLC fractions to known the presence of functional groups. The sample dissolved in ethanol was loaded into FTIR and scanned to get well optimized spectra of each sample ranging from 600 to 4000 cm -1. Ethanol was taken as control for the analysis. Liquid Chromatography-Mass Spectroscopy (LC-MS) LC-MS analysis was followed as described previously (Liu and Rochfort, 2014; Moco et al., 2006; Sawada and Hirai, 2013) with modifications. The samples of crude extract and TLC fractions were loaded into HPLC system connected with Waters 2996 PDA detector and set to acquire data every second from 240 to 600 nm with a resolution of 4.8 nm, and subsequently to a Mass Spectrometer (Waters-Corporation, MS technologies). An ESI source set with positive or negative ion mode for all MS analysis. Before each sample analysis, the mass spectrometer was calibrated using phosphoric acid: acetonitrile: water (1:103:103, v/v) solution. Capillary voltage, collision energy, and desolvation temperature calibration between m/z 80 and 1,500. Resolution was set at 10,000 and during calibration the MS parameters were adjusted to achieve such a resolution. TOF-MS data were acquired during LC-MS analysis scan of 0.9 s and an interscan time of 0.1 s. For LC-MS measurements, 10 μl of sample was injected into the system and MS measurements were made with 0.40 s of scan duration and 0.10 s of interscan delay with increasing collision energies. Results The seed extracts of Garcinia indica and Chloroxylon sweitenia (seed and rim) was obtained as reddish brown in colour after soxhletion extraction and preserved at 4 ºC for further analysis. 3.1 Antimicrobial activity by Disc diffusion assay The antibacterial activity of the crude extaract is carried out by disc diffusion method against nine different pathogens. The methanol seed extract of Garcinia indica showed significant inhibition activity against Gram positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis and Gram negative Bacteria Pseudomonas
aeruginosa, Enterobacter aerogens, Escherichia coli, Proteus mirabilis, Shigella flexneri, Vibrio parahaemolyticus (Table 1). But methanolic extract of seed and rim of Chloroxylon sweitenia did not showed antibacterial activity against tested organisms. The results presented in Table 1 shows that the crude extract under investigation exhibited significant antibacterial activity, as evidenced by their zones of inhibition for Garcinia indica (Figure 1). Table 1. Antibacterial activity of seed extract of Garcinia indica and C. sweitenia (seed and rim) Microorganisms Garcinia indica Methanol extract Zone of inhibition (mm) Hexane extarct Chloroxylon Swietenia Seeds extract Rim extract (methanol) (methanol) Staphylococcus aureus 10 - - - Staphylococcus 8 - - - epidermidis Bacillus subtilis 12 - - - Pseudomonas aeruginosa 12 - - - Enterobacter aerogens 12 -. - - Escherichia coli 11 - - - Shigella flexneri 10 - - - Vibrio parahaemolyticus 10 - - - Proteus mirabilis 12 - - - Figure 2. Antibacterial activity of methanolic crude extract from G. indica. A. Proteus mirabilis, B. Shigella flexneri, C. Vibrio parahaemolyticus, D. Enterobacter aerogenes, E. Staphylococcus epidermidis, F. Escherichia coli. MIC by Micro broth dilution assay The MIC assay was done using 96 well microtiter plates and showed in Table 2. The crude extract obtained from Garcinia indica exhibited significant MIC value of 62.5µg/ml against Enteribacter aerogenes and Bacillus subtilis, along with MIC value of 250 µg/ml for Vibrio parahaemolyticus, Staphylococcus aureus.
Shigella flexneri, E. coli and Proteus mirabilis and least potential were seen in the case of Pseudomonas aerogenosa with MIC value of 500 µg/ml (Table 2). The results suggest that significance of antibacterial activity with irrespective of Gram positive or Gram negative bacteria. Table 2. Minimum Inhibitory Concentration for seed extract of Garcinia indica Microorganisms MIC (µg/ml) Staphylococcus aureus 250 Staphylococcus epidermidis 125 Bacillus subtilis 62.5 Pseudomonas aeruginosa 500 Enterobacter aerogens 62.5 Shigella flexneri 250 Vibrio parahaemolyticus 250 Proteus mirabilis 250 Escherichia coli 250 Thin layer chromatography The crude extract was loaded into TLC pre-equilibrated with mobile phase showed single prominent band under short UV range. The single band was separated from the silica and suspended in ethanol, used for further analysis. Ultra violet-visible spectroscopy (UV-Visible) The TLC separated compound was subjected to UV-visible spectra from 200 to 800 nm and showed a single band from TLC with maximum absorbance at 286 nm (Figure 3). 3.5 Fourier Transform Infrared Spectroscopy (FTIR) The FTIR analysis of crude sample as well as TLC fraction was correlated with presence of same functional groups. The spectra revealed that, the presence of Aromatic ring stretch (C=C-C, 1610-1580 cm -1 ), Tertiary amine (C-N, 1210-1150 cm -1 ), Six member lactone ring (1738 cm -1 ), Dimethyl or iso (doublet, -CH 3, 1370-1365 cm -1 ) and Methyl C-H asymmetry/symmetry stretch (2880-2860 cm -1 ) (Figure 4). Figure 3. UV-Visible spectra of TLC fractionated bioactive compound. Spectrum scanning range was selected from 200 to 800 nm and bioactive compound showed 286 nm of absorption maxima. Liquid Chromatography-Mass Spectroscopy (LC- MS) TLC purified was loaded into HPLC equipped with mass spectrometry. Then,
chromatogram showed the presence of major peak with some negligible impurities and under negative mode TOF-MS an ionization pattern was obtained based on m/z value for the major peak (Figure 5 and 6). Based splitting pattern, abundance of m/z value and following previous interpretations (Desai and Dodiya, 2014; Gali et al., 2014) the bioactive molecule was putatively predicted as Benzo-quinazoline derivative namely 1,3-bis(3-ethoxyphenyl)-9- methylbenzo-quinazoline with molecular weight 435 (Figure 7). Figure 4. FTIR spectrum of TLC fractionated bioactive compound from G. indica. Figure 5. HPLC chromatogram of TLC fractionated bioactive compound from G. indica Figure 6. Mass spectra showing splitting pattern, abundance and m/z value of bioactive compound from G. indica
Figure 7. Putatively identified bioactive compound an Benzo-quinazoline derivative (1,3- bis(3-ethoxyphenyl)-9-methylbenzo-quinazoline) from seed extract of G. indica. Conclusion Garcinol from Garcinia indica is well known for its medicinal property from fruit pulp and rind. The seeds of G. indica are non-edible and oil is used in Ayurvedic preparations. The study reports presence of Benzo-quinazoline derivative in methanolic seed extract with significant antibacterial activity against Gram negative and Gram positive bacteria. Furthermore, study reveals the importance of non-edible seeds from G. indica in industrial and medicinal uses.