ISOLATION, IDENTIFICATION AND SCREENING FOR BIOACTIVE COMPOUNDS WITH ANTIMICROBIAL ACTIVITIES FROM SUB-AERIAL CYANOBACTERIA OF EASTERN REGION, ODISHA

ISOLATION, IDENTIFICATION AND SCREENING FOR BIOACTIVE COMPOUNDS WITH ANTIMICROBIAL ACTIVITIES FROM SUB-AERIAL CYANOBACTERIA OF EASTERN REGION, ODISHA

Sparsha Pattnaik ¹, Chandi Charan Rath ² and Lakshmi Singh ^{* 1}

Department of Botany ¹, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar - 751003, Odisha, India.

P.G. Department of Life Science ², Rama Devi Women’s University, Vidya Vihar, Bhubaneswar - 751022, Odisha, India.

ABSTRACT: Background and Objectives: Cyanobacteria are rich in various bioactive compounds. This study aims to screen the antimicrobial activity of sub-aerial Cyanobacteria isolated from sub-aerial habitats (Old monuments and building facades). Materials and Methods: Crude aqueous and organic extracts were screened for phytochemical constituents and tested for their antimicrobial efficacy against human pathogens. The crude extract that showed better antimicrobial activities were further fractionated by column chromatography and purified by TLC. The active bands were scraped out separately and concentrated in vacuo to isolate pure compounds. Each purified compounds dissolved in respective solvents, assayed for antimicrobial activities were further characterised through FTIR and LC-MS. Results: The results indicated the presence of many secondary metabolites. Of the isolated purified compounds, the compounds from three species, viz. Scytonema hyalinum, Tolypothrix rechingeri, and Fischerella species exhibited antimicrobial activities, whereas others showed antibacterial activity. The FTIR spectra revealed the peaks of carboxylic acid, alkaloids, and phenolic compounds. The chemical compounds were identified as 1,3-dihydroxycyclohexane [Lyngbya kuetzingiana], 4-(4-hydroxyphenyl) butanoic acid [Nostoc linckia], octahydro-1H-indole-2-carboxylic acid [Scytonema sp., S. pseudoguyanensis, S. pseudohofmani], ergost-5-en-3-ol [Scytonema hyalinum],7,11-dihydroxysolasodine [Scytonema ocellatum], 2, 4-Bis (2-methyl-2-propanyl)phenol-phosphorous acid [Tolypothrix rechingeri] and Pentaphenyl ferrocene carboxamide [Fischerella sp.] respectively. Conclusion: Though it is firsthand information regarding the isolation of sub-aerial Cyanobacteria, with a production of different bioactive chemical compounds, further investigation is required for use in pharmaceutical industries against costly harmful antibiotics and chemotherapeutics.

Sub-aerial Cyanobacteria, Antimicrobial activity, Bioactive compounds, FTIR, LCMS

INTRODUCTION: The development of multi-drug resistance (MDR) among pathogens is a global concern today. This necessitates the demand for the discovery of the new source of chemical compounds as alternative antibiotics.

In this regard, a large number of microbes are lagging behind which may produce potent and novel bioactive compound which can be used in the treatment of humans as well as animals. In this aspect, Cyanobacteria are considered to be one of the accessible organisms in the microbial world which are useful to humankind in several ways.

The use of Cyanobacteria to produce valuable chemicals and pharmaceutically applicable products is still little explored, and it seems a long way to go.

Modern research has focussed on a variety of bioactive compounds produced by Cyanobacteria from various biotypes, for example, marine ¹, freshwater ², terrestrial ³, hot spring ^4, etc. Some of the lead compounds with antibacterial activities are bacteriocin, scytophytin, didehydromirabazole, bas-tadin, muscoride, noscomin, bis-x-butyrolactone, nostocin A, and lipids ⁵. Butylated hydroxytoluene, hexadecanoic acid methyl ester, tjipanazole, ambi-guines, fischerllin A, nostofungicidin, phytoalexin, hassalldin, scytophycin, hapalindole, Y-lactone, calothrixins ⁶ have been reported with antifungal activity. Thus screening efforts aimed to identify antimicrobial agents in sub-aerial Cyanobacteria which might reveal pharmaceutically important compounds.

Cyanobacteria are adaptive to the exterior surface of monuments and building facades which belong to a group of organisms known as “Extremophiles” due to their ability to endure extreme conditions. Few studies have yet been identified for the antimicrobial potential of sub-aerial Cyanobacteria isolates from this habitat ^3, but no studies till date are adequate. In-state of Odisha, especially Bhubaneswar and Puri, known as temple city, rich in old cultural heritages including old/modern building is a home for diverse Cyanobacteria ⁷. So far, very little work has been done on the antimicrobial properties of extremophilic Cyano-bacteria. This prompted us to carry out this investigation to isolate and characterize different sub-aerial Cyanobacteria from the monument and building facades of Bhubaneswar and Puri and evaluate their antimicrobial activities against selected bacteria and fungi.

MATERIALS AND METHODS:

Isolation, Identification and Culture Condition: The biofilms/crusts collected from various sub-aerial sites (e.g., Stone monuments, caves, and building facades) were soaked in sterile distilled water and incubated under fluorescent light for 72 h and observed microscopically. Since the morpho-logically features needed for identification were not distinct even after prolonged soaking (up to 7 to 14days), a small amount of sample was transferred to BG-11 agar plates medium with and without nitrogen ⁸ (1.5% agar in the same medium) with the help of inoculation needle. Cultures were incubated at 28 ± 1°C under continuous fluorescent light at an intensity of 2000lux. After a period of 10 to 14 days of incubation, Cyanobacteria appeared in the culture were isolated using sub-culturing methods ⁹ and the axenic culture was cultured in a 500ml of BG-11 medium for biomass production, and were maintained under controlled conditions of room temperature (28 ± 1°C) and continuous illumination of 2000lux with 16:8 h light and dark regime in the laboratory. The cultures were identified through morphological variation studies following standard monographs ^{10, 11}.

Microbial Strains: Both bacterial [Bacillus subtilis (MTCC121), Pseudomonas aeruginosa (MTCC741), Staphylococcus aureus (MTCC902), Escherichia coli (MTCC723)] and fungal strains [Candida albi-cans (MTCC4748) and Epidermaphyton flocossum (MTCC7880)] were procured from Microbial Type Culture Collection (IMTECH, India) and were used in the study.

Experimental Organisms: Ten axenic sub-aerial Cyanobacteria species belonging to five genera were identified and used in the experiments.  These species are Lyngbya kuetzingiana (rock surface, Cave), Tolypothrix rechingeri (stone carving, Monument), Scytonema sp. (stone carving, Monument), S. pseudoguyanensis (stone surface, Temple), S. pseudohofmani (stone surface, Temple), S. hyalinum (limestone surface, Cave), Nostoc linckia and Fischerella sp. (cement wall surface, Building), and Scytonema ocellatum and S. crispum (lime-washed surface, Building wall), etc.

Preparation of Crude Extracts: After 32^th days of incubation, in BG-11 broth, isolates were harvested with Whatman No.1 filter paper and shade dried for 30 min, and grinded to a fine powder with the help of a glass homogenizer. One gram of Cyanobacterial powder was extracted separately in a soxhlet apparatus using aqueous and different solvents like hexane, benzene, chloroform, ethyl acetate, acetone, and methanol, etc. The extracts were collected in a pre-sterilized air-tight container and preserved at 4 ºC for further characterizations.

Phytochemical Screening of the Crude Extracts: Phytochemical screening of the crude extracts for the detection of alkaloids, phenolic, flavonoids, quinones, glycosides, terpenoids, tannins, steroids, saponin, and other secondary metabolites was carried out by following the standard procedure ¹².

Antimicrobial Efficacy and Screening of the Extracts: The crude extracts were further screened for antimicrobial activity through agar well diffusion technique ¹³ against the test pathogens. All the experiments were conducted in triplicates. Zone of inhibition was measured in mm, and the breakpoint for efficacy was taken as a zone of inhibition ≥ 10mm. Ciprofloxacin (10mg/ml) and Carbazine (10mg/ml) were used as controls.

Purification of Crude Extracts through Column Chromatography and Thin Layer Chromato-graphy: The crude extracts that showed better antimicrobial efficacy during screening were further fractionated by column chromatography ¹⁴. The fraction obtained from respective isolates were collected and tested for antimicrobial properties. The selected antimicrobial fractions were further purified by preparatory TLC using ethyl acetate and hexane (1:1) as the mobile phase, visualized under UV, the bands were marked, and their R_f values were determined. The active fractions were scrapped out separately, vacuum evaporated, and the pure compound was obtained in the powdered form, dissolved in 3ml respective solvents, and their bioactivity was checked out using agar well diffusion methods against the test pathogens as described earlier. The antimicrobial activity index (AI) of a respective active fraction was calculated using the formula: inhibition zone of sample/inhibition zone of the standard × 100 ¹⁵. Further, the bioactivities of the active fractions were confirmed using the secondary screening technique (bioautography method) following method ¹⁶.

Minimum Inhibitory Concentration (MIC): Different active compounds observed from the previous study were further used to determine the minimum inhibitory concentration (MIC). The MIC values were determined by the microdilution method as described by National Committee for Clinical Laboratory Standards guidelines (NCCLS) ¹⁷. Compounds were serially diluted in order to obtain a range from 0.312mg/ml to 10mg/ml compounds in the respective solvent, in MHB and SDB for bacteria and fungi, respectively, in 96 well micro-titer plates. 20µl of overnight freshly grown pathogens were inoculated to each well. Wells, without extract, served as a negative control, and the antibacterial (Ciprofloxacin-10mg/ml) and anti-fungal (Carbazine-10mg/ml) agents were included in the assays as a positive control. The test plates were incubated at 37 ± 2°C and 28 ± 2°C for bacteria and fungi, respectively. After the incubation period, one loopful of the sample was sub-cultured onto NA and SDA plates for bacteria and fungi, respectively. A dilution where no growths of the pathogen were recorded is considered as the Minimum Inhibitory Concentration of the respective extract. The same procedure was followed for each extract and each organism separately.

Identification of Bioactive Compounds:

Fourier-Transform Infrared Spectroscopy: The antimicrobial compounds recorded with a highest antimicrobial index of different extracts were taken into consideration for identification of functional groups through FTIR analysis ¹⁸ using spectro-photometer (Nicolet 6700, USA and Perklin Elymer) in the range of wave number 500 to 4000 cm^-1.

Liquid Chromatography-Mass Spectroscopy: After FTIR analysis, the same fraction was further subjected to LCMS analysis for identification and characterization of bioactive compounds.

LC-MS analysis was carried out using High-Resolution Liquid Chromatography-Mass Spectro-meter (1260 Infinity Binary LC coupled with 6530B accurate mass Q-TOF, Agilent Techno-logies, USA).

Statistical Analysis: All the data obtained from the experiments were expressed as mean ± SD of three replicates using One-way analysis of variance (ANOVA) through SPSS version 17.0 statistical software. P-value ≤0.05 was considered significant.

RESULTS: During the study, ten sub-aerial Cyanobacteria species, viz. [Lyngbya kuetzingiana (1), Nostoc linckia  (1), six Scytonema of different species, Scytonema sp., S. pseudoguyanensis, S. pseudohofmani, S. hyalinum, S. ocellatum, S. crispum, Tolypothrix rechingeri (1) and Fischerella sp. (1)] inhabiting as microbial biofilms or crusts on different sub-aerial habitats of old and modern cultural heritages including building facades of the eastern region especially Puri and Bhubaneswar, in the state of Odisha were isolated and assayed for phytochemical screening, characterization, and identification of bioactive compound with anti-microbial potential.

Phytochemical Screening: Aqueous and organic crude solvent extracts of the ten sub-aerial Cyanobacterial species were prepared as described earlier and subjected to phytochemical screening. The presence of various phytochemical constituents in different extracts of the isolates is presented in Table 1. Phytochemical screening revealed the presence of major secondary metabolites like alkaloids, phenolic, flavonoids, glycosides, quinones, and steroids in the extracts. None of the compounds were observed in aqueous, and ethyl acetate extract of all the isolates studied.

Antimicrobial Activity Studies: The organic crude extracts of the ten sub-aerial Cyanobacteria showed antimicrobial activities in terms of zone of inhibitions against the reference pathogenic bacterial and fungal strains during screening. Most of the antimicrobial activity was detected with the extract of relatively polar solvents. In two species, the antibacterial activity was observed in a non-polar solvent like hexane and benzene in Fischerella sp. and chloroform in Tolypothrix rechingeri. None of the sub-aerial Cyanobacteria showed bioactivity in aqueous as well as ethyl acetate extracts. The crude extract that showed better antimicrobial activities, further fractionated by column chromatography and purified by TLC. The active band on TLC were scrapped out separately, vacuum evaporated and pure compound is obtained in the powdered form, dissolved in 3ml respective solvents, and were checked for antimicrobial activities. The anti-microbial activities (zone of inhibition and respective activity index) of each ten isolates against pathogens were summarised in Table 2, Fig. 1-5. Results obtained from isolated compounds indicated that out of the ten isolates, three compounds (A₂, M₅ and A₂) respectively isolated separately from three sub-aerial Cyanobacteria species, Scytonema hyalinum, Tolypothrix rechingeri, and Fischerella species showed considerable inhibition zone respectively against target bacterial and fungal pathogens. It was noted that the extracts significantly inhibited gram-negative bacteria, whereas selectively inhibited gram-positive bacterial and fungal strains. Most activities were observed in the case of acetone dissolved compounds (Scytonema sp., S. hyalinum, S. ocellatum, Fischerella sp.), and methanol compounds (Lyngbya kuetzingiana, Nostoc linckia, Scytonema pseudoguyanensis, S. pseudohofmani, and Tolypothrix rechingeri) was found to be effective against E. coli and P. aeruginosa (accept) Fischerella sp. and Tolypothrix rechingeri Table 2; Fig. 1-5.

TABLE 1: PHYTOCHEMICAL SCREENING OF THE SUB-AERIAL CYANOBACTERIA CRUDE EXTRACTS

*Keys: +++ = indicate prominent; + = indicates slightly and - = indicates absence

Few isolated compounds (M₃, M_3, and M₂) from the methanol extracts of Lyngbya kuetzingiana, Scytonema pseudoguyanensis, and S. pseudo-hofmani respectively showed positive response against Bacillus subtilis and four compounds M₃ (Lyngbya kuetzingiana), M₂ (Nostoc linckia), A₂ (Scytonema hyalinum) and M₅ (Tolypothrix rechingeri) against Staphylococcus aureus respecti-vely. However, the compound (A₂) of acetone extracts of Fischerella species showed the highest inhibition zone (Complete inhibition) against E. coli. Concerning to fungal strains, out of isolated compounds from ten species, the compound (A₂) of acetone extract of Fischerella species was found to be active against C. albicans whereas, Scytonema hyalinum and Tolypothrix rechingeri also exhibited activity against both the fungal strains studied.

Each fraction had one active band obtained on TLC plates against target pathogenic strains. The bioautography was showed with one major active band separately obtained from hexane, benzene, chloroform, acetone, and methanol extract of ten isolates against respective pathogens with different R_f values Table 2.

The positive controls, ciprofloxacin and carbazine used in this study, showed better antimicrobial activities in comparison to the compound extracts studied Fig. 5. However, Fischerella species isolated compound showed better activities against E. coli, completely inhibiting the growth of the test organism in comparison to ciprofloxacin (28mm, zone of inhibition, Table 2; Fig. 4-5.

TABLE 2: ANTIMICROBIAL ACTIVITY OF ACTIVE FRACTIONS OF SUB-AERIAL CYANOBACTERIA AGAINST REFERENCE HUMAN PATHOGENIC STRAINS AS PRESENTED BY INHIBITION ZONE DIAMETER (in mm) WITH ANTIMICROBIAL INDEX (IN PARENTHESES)

*Results are the means of diameter values ±standard deviation of three replicates. *R_f = Retention factor; A = Acetone; M = Methanol; C= Chloroform; B= Benzene and H= Hexane.

FIG. 1: ANTIBACTERIAL ACTIVITY OF ACTIVE FRACTIONS OF METHANOL EXTRACT OF (A) LYNGBYA KUETZINGIANA AND (B) NOSTOC LINCKIA AGAINST TARGET PATHOGENIC STRAINS

FIG. 2: ANTIMICROBIAL ACTIVITY OF ACTIVE FRACTIONS OF ACETONE/OR AND METHANOL EXTRACT OF (A) SCYTONEMA SP., (B) S. PSEUDOGUYANENSIS AND (C) S. PSEUDOHOFMANI AGAINST TARGET PATHOGENIC STRAINS

FIG. 3: ANTIMICROBIAL ACTIVITY OF ACTIVE FRACTIONS OF ACETONE EXTRACTS OF (A) SCYTONEMA HYALINUM, (B) S. OCELLATUM AND (C) S. CRISPUM AGAINST TARGET PATHOGENIC STRAINS

FIG. 4: ANTIMICROBIAL ACTIVITY OF THE ACTIVE FRACTIONS OF HEXANE/BENZENE/CHLOROFORM/ ACETONE AND OR METHANOL EXTRACTS OF (A) TOLYPOTHRIX RECHINGERI AND (B) FISCHERELLA SP. AGAINST TARGET PATHOGENIC STRAINS

FIG. 5: ANTIMICROBIAL ACTIVITY OF THE STANDARD ANTIBIOTICS: CIPROFLOXACIN (10mg/ml) AND CARBAZINE (10mg/ml) AGAINST TARGET PATHOGENIC STRAINS

Minimum Inhibitory Concentration (MIC): The minimum inhibitory concentration (MIC) of the isolated compounds of different extracts exhibiting best antimicrobial activity of ten sub-aerial Cyano-bacteria were investigated to determine the effec-tiveness. The MICs value of different compounds isolated from sub-aerial Cyanobacteria exhibited antibacterial /or antifungal activity ranged between 0.312-5mg/ml, are shown in Table 3. It was noted that the lower MIC (0.312mg/ml) was obtained only against E. coli MTCC723 treated with isolated compound (A₂) of Fischerella species whereas, the other compound isolated from species such as Nostoc linckia, Scytonema ocellatum, S. hyalinum, and Tolypothrix rechingeri showed MIC similar to standard antibiotics (0.625mg/ml) respectively. The compounds of methanol and acetone extracts of four strains, Scytonema pseudoguyanensis, S. hyalinum, S. ocellatum and S. crispum exhibited with similar MIC value (0.625mg/ml) respectively against P. aeruginosa MTCC741. However, moderate MIC value ranged from 1.25-5mg/ml was observed with Lyngbya kuetzingiana, Nostoc linckia, Scytonema pseudoguyanensis, S. pseudohofmani, and S. ocellatum extracts (methanol and acetone) against E. coli and P. aeruginosa.

Compounds (A₂) isolated respectively from Fischerella sp. and Scytonema hyalinum showed MIC value (0.625 mg/ml, 1.25mg/ml) respectively against C. albicans followed by Tolypothrix rechingeri (M₅, 1.25 mg/ml). However, the isolated compounds of other species also showed moderate activity ranged between 0.625-5mg/ml against B. subtilis, E.coli, P. aeruginosa, S. aureus, C. albicans, and E. flocossum, respectively. MICs values of bioactive compounds and their zone of inhibition were significantly correlated, i.e., compounds with higher zones of inhibition showed lower MIC values with p ≤ 0.05.

TABLE 3: MINIMUM INHIBITORY CONCENTRATIONS (MIC) OF ACTIVE FRACTION OF SUB-AERIAL CYANOBACTERIA AGAINST TEST PATHOGENS

^*R_f = Retention factor; A = Acetone; M = Methanol; C= Chloroform; B= Benzene and H= Hexane; numerals with the extracts indicate the no of active fraction of the extract.

Characterization and Identification of Bioactive Compound: Based on highest activity index, the antimicrobial active purified compounds (acetone/ or methanol) of each extract of the isolates were further characterized by FTIR and LC-MS, and different functional groups identified in these compounds are presented in Table 4, Fig. 6(a-g).

TABLE 4: FT-IR ANALYSIS OF ACTIVE FRACTIONS OF THE TWO DIFFERENT ORGANIC EXTRACTS OF POTENTIAL SUB-AERIAL CYANOBACTERIA SPECIES

The isolated compound (M₃) from methanol extract of Lyngbya kuetzingiana showed peak at 3317.3cm^-1, 2140.3cm^-1, 1635.10cm^-1, 1371.1cm^-1 due to the presence of O-H, -C=C, C=O, -CH₃ as functional properties Fig. 6a whereas compound (M₂) of methanol extract of Nostoc linckia showed peaks at 3350.78cm^-1, 2976cm^-1, 2896cm^-1, 1508.33cm^-1, 1384cm^-1, 1044cm^-1, 532.5cm^-1 contains O-H, =C-H, C-H, C=C, C-O group Fig. 6b. In addition to these, the isolated compounds (A₁, M_3, and M₂) from acetone/or methanol extract of three species, Scytonema sp., S. pseudoguyanensis, and S. pseudohofmani, respectively, from different sites showed similar peaks at 3366.5cm^-1, 1704.7cm^-1, 1384cm^-1, 1229.6cm^-1, 532.5cm^-1 indicated the presence of -N-H, OH group, C=O, C-H, C-O and alkyl halide group Fig. 6c, but the compound (A₂) of acetone extract of Scytonema hyalinum exhibited broad absorption bands at 2958.5cm^-1, 2924.1cm^-1 and 2859.7cm^-1, at 1459.2cm^-1, 1378.9cm^-1, 1037.7cm^-1 and at 724.5cm^-1 revealed the presence the functional properties like CH₂, C-N or C=C, C-O, –C=H functional groups Fig. 6d. The compound (A₂) isolated separately from two species, Scytonema ocellatum and S. crispum showed similarity in peaks at 3366cm^-1, 2945cm^-1, 2832cm^-1, 1704cm^-1, 1420cm^-1, 1364cm^-1, 1229cm^-1, 1093cm^-1, 1022cm^-1 and  532cm^-1 confirmed the presence of O-H, N-H, CH₂, C=O, C-O , C=C, –CH₃, P=O and alkyl halides Fig. 6e. However, compound (M₅) of methanol extract of Tolypothrix rechingeri showed peaks at 3314.3cm^-1,  2944.3cm^-1 and 2832.6cm^-1 in the presence of  O-H, N-H, and at 1449.2cm^-1, 1113.7cm^-1, 1022.1cm^-1 indicated the presence of -CH₃, C-O, C-O-C groups and at 616.7cm^-1 of alkyl halides Fig. 6f. The compound (A₂) of acetone extract of Fischerella sp. showed peaks at 3534cm^-1, 3003.8cm^-1, 1707.8cm^-1, 1420.3cm^-1, and 1358.8cm^-1 due to the presence of NH₂, OH, NH, CH₂, C=O, C-O, N-O group and at 1220.5cm^-1, 1092.1cm^-1, 903cm^-1, 529cm^-1 and at 420cm^-1 due to the presence of phosphodiester, N-H, alkyl halides and Fe group Fig. 6g.

FIG. 6: FT-IR SPECTRUM OF THE FUNCTIONAL GROUPS OF ACTIVE FRACTIONS OF THE TEN SUB-AERIAL CYANOBACTERIA. A= ACETONE EXTRACT AND M=METHANOL EXTRACT

LC-MS analyses were performed with purified compound of the crude extracts (acetone and methanol) of the target species. Chemical components of the analyzed active compounds are presented in Table 5. In our data, for each sub-aerial Cyanobacteria species, purified compounds chemical components was recorded such on 1,3-dihydroxycyclohexane, C₆H₁₂O₆: Mw-116g/mol [(compound-M₃), Lyngbya kuetzingiana]; 4-(4-hydroxylphenyl) butanoic acid, C₁₀H₁₂O₃:Mw-178g/mol [(compound-M₂), Nostoc linckia];  Octa-hydro-1H-indole-2-carboxylic acid, C₉H₁₅NO₂: Mw-169g/mol [(compound-A₁, M₃, M₂), Scytonema sp., S. pseudoguyanensis and S. pseudohofmani]; Ergost-5-en-3-ol, C₂₈H₃₈O₄: Mw- 704g/mol [(compound-A₂), Scytonema hyalinum]; 7, 11-dihydroxysolasodine, C₂₇H₄₃NO₄: Mw-413g/mol [(compound-A₂), Scytonema ocellatum and S. crispum]; 2, 4-Bis (2-methyl-2-propanyl) phenol - phosphorous acid, C₄₂H₆₉O₆P: Mw-700g/mol [(compound-M₅), Tolypothrix rechingeri] and Pentaphenyl ferrocene carboxamide, C₄₁H₃₁FeNO: Mw-610g/mol [(compound-A₂), Fischerella sp.]. The resultant major peaks of the active fractions of extracts were surveyed by the use of the available database PubChem, provided by the National Centre for Biotechnology Information (NCBI) at http://pubchem.ncbi. Nlm.nih.gov. Classification, biomedical features, and biological assay activity was obtained for some of the resultant fractions, and data are represented in Table 5.

TABLE 5: MASS SPECTRA COMPONENT IN ACTIVE FRACTIONS OF SUB-AERIAL CYANOBACTERIA SPECIES

DISCUSSION: The present investigation was carried out to evaluate the potential of ten sub-aerial Cyanobacteria with antimicrobial activities against pathogenic bacterial and fungal strains. During the study, secondary metabolites like alkaloids, phenols, flavonoids, glycosides, quinones, and steroids were observed to be present in the different crude extracts of the isolates studied. The presence of major secondary metabolites were observed in the acetone and methanol extracts as compared to the hexane, benzene, and chloroform extracts. Many reports indicated that natural alkaloids, phenolic compounds, and flavonoids are highly effective against a wide spectrum of pathogens. It was also reported that Cyanobacteria existing with secondary metabolites like alkaloids, phenols, flavonoids, quinones, glycosides, terpenoids, tannins, steroids, and saponin could be used in the development of antimicrobials, against resistant strains of bacteria, fungi, and mycobacterium, as base molecules use in drug development ¹⁹.

Further, while studying the antimicrobial activity of different extracts (and their compounds) of the ten sub-aerial Cyanobacteria the authors found that the purified compounds isolated from three species i.e. Fischerella sp., Scytonema hyalinum followed by Tolypothrix rechingeri showed significant antimicrobial activities against target reference human pathogenic strains. The compounds from other seven isolates, Lyngbya kuetzingiana, Nostoc linckia, Scytonema sp., S. pseudoguyanensis, S. pseudohofmani, S. ocellatum and S. crispum also exhibited antibacterial activities against Bacillus subtilis, E. coli, Pseudomonas aeruginosa and Staphylococcus aureus, but no activity was found against C. albicans and E. flocossum.

During the study we observed that the most purified compounds exhibited better antibacterial activities against E. coli and P. aeruginosa than Bacillus subtilis and Staphylococcus aureus.  In contrast to our findings it was reported that Gram-positive bacteria seemed to be more sensitive to the Cyanobacteria than Gram-negative bacteria. Among the isolates Fischerella sp. was observed to be more effective against the test pathogens ²⁰.

These finding somehow has an agreement with previous studies that indicated methanol extracts of Lyngbya kuetzingiana and Nostoc linckia had moderate antibacterial activities ^{21, 22}. While studying the MIC of different isolated, purified compounds of the isolates, through the broth dilution method, it was observed that the MIC values ranged between 0.312-5mg/ml Table 3.

Antimicrobial effects of the cyanobacteria extracts against pathogens were detected for the presence of various secondary metabolites ²³. The antimicrobial activity of the sub-aerial Cyanobacteria extract could be due to the presence of different chemicals that may include fatty acids, alkaloids, flavonoids, phenolic compounds, quinones, and steroids. These agreements are comparable with the present study's findings detected through direct bioautography with R_f value 0.72, 0.84, 0.86, and 0.96, which showed the presence of the phenolic compound, alkaloids, and steroids in the purified compounds of ten sub-aerial Cyanobacteria. In contrast to our observations, many studies have reported the presence of terpenoids, tannins, and saponin in the extracts of Cyanobacteria ²⁴.

The FTIR spectrum confirmed the presence of alcohol, alkanes, alkenes, amines, ester, aromatic, alkyl halides, aldehydes, carboxylic acids, and phenolic and nitro compounds in all tested purified compounds isolated from crude extracts (acetone/or methanol) of Cyanobacteria and were predicted through the presence of the functional groups like O-H, N-H, =C-H, C-N, C-C, C=O, C-O, N-O, >P=O stretching and iron group. The antimicrobial activity of the compounds could be due to the presence of these secondary metabolites ²⁵.

In the current investigation, the strong antimicrobial activity of the different purified compounds of acetone/or methanol extracts of each species was subjected for identification by LC-MS. The mass spectra of the compound were investigated with those similar in the PubChem database and some of our chemical components are reported to have a known importance in pharmaceutical fields Table 5. In this study, purified compound-M₃ of methanol extract of Lyngbya kuetzingiana contained 1, 3-dihydroxycyclohexane as alkyl compound and fatty alcohols, whereas other two compounds as carboxylic acids, 4-(4-hydroxylphenyl) butanoic acid [compound-M₂ of methanol extract of Nostoc linckia] and Octahydro-1H-indole-2-carboxylic acid [acetone/or methanol compounds-A₁, M₃, M₂ of three different species, Scytonema sp., S. pseudoguyanensis, S. pseudohofmani]. In the present study, fatty alcohol and carboxylic acid like compounds produced from Lyngbya kuetzingiana, Nostoc linckia, Scytonema sp., S. Pseudo-guyanensis, and S. pseudohofmani showed antibacterial activity with higher and MIC values against E. coli and P. aeruginosa Table 2-3.

Beside this, aromatic hydrocarbon like 2, 4-Bis (2-methyl-2-propanyl) phenol - phosphorous acid detected in the compound-M₅ of methanol extracts of Tolypothrix rechingeri also revealed better antimicrobial activities. This connection revealed that phenolic compound has broad application as antibacterial, antifungal, and antioxidant properties ²⁶. In present study, Tolypothrix rechingeri produced a compound having the highest antimicrobial action against P. aeruginosa, S. aureus, and C. albicans. In addition to these, two compounds, Ergost-5-en-3-ol from Scytonema hyalinum and 7,11-dihydroxysolasodine produced by two different species of Scytonema ocellatum and Scytonema crispum known as steroidal alkaloids, found to be present in the compound (A₂) of acetone extract respectively. In our study, steroids like compound ergost-5-en-3-ol and 7,11-dihydroxysolasodine found in two different Scytonema sp. showed good antibacterial activities against E. coli and P. aeruginosa. The heterocyclic alkaloids compound, amino (cyclopenta-2,4-dien-1-ylidene) methanolate also known as pentaphenyl ferrocene carboxamide was identified in the compound-A₂ isolated from acetone extract of  Fischerella sp. showed excellent antimicrobial activity against E. coli and C. albicans with MIC value (0.312mg/ml and 0.625mg/mol). It is pertinent to mention that the observation of the antimicrobial activity of these compounds isolated from sub-aerial Cyanobacteria against human pathogens is of its kind.

CONCLUSION: Sub-aerial Cyanobacteria that occupy extreme environments also known as extremophiles, are substantial resources for natural bioactive substances which could be exploited in the pharmacological industry. In this present study, we investigated the antimicrobial activity of some purified compounds of the crude extracts of sub-aerial Cyanobacteria, which showed significant results against human reference pathogens. The present investigation indicated that sub-aerial Cyanobacteria remain an interesting alternative source for new antimicrobial metabolites with better inhibition activity and also suggest that Scytonema hyalinum, Tolypothrix rechingeri, Fischerella sp. and could be potential sources for secondary metabolites with strong antibacterial and antifungal activities.  The findings recorded in this investigation are of their kind. Though it is firsthand information regarding the isolation of extremophilic sub-aerial Cyanobacteria  (from old monuments, temples, caves, building facades, etc. in Odisha, more specifically in the cities of Bhubaneswar and Puri), with the production of different antibacterial and antifungal compounds, further, studies are desired to find its way for use in pharmaceutical industries, for development of newer antimicrobials against costly harmful antibiotics and chemotherapeutics, in order to enjoy the benefits and/or the fruits of this investigation.

ACKNOWLEDGEMENT: Authors are thankful to the Science and Technology Department, the Govt of Odisha for their financial support, and also we thank the Director of College of Basic Science and Humanities, OUAT, for providing lab facilities throughout the study duration.

CONFLICTS OF INTEREST: The authors report no conflict of interest.

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    How to cite this article:

    Pattnaik S, Rath CC and Singh L: Isolation, identification and screening for bioactive compounds with antimicrobial activities from sub-aerial cyanobacteria of eastern region, Odisha. Int J Pharm Sci & Res 2021; 12(7): 3716-30. doi: 10.13040/IJPSR.0975-8232.12(7).3716-30.

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