ANTIMICROBIAL ACTIVITIES OF FRESHWATER CYANOBACTERIUM, NOSTOC SP. FROM TAMDIL WETLAND OF MIZORAM, INDIA: AN IDENTIFICATION OF BIOACTIVE COMPOUNDS BY GC-MS

ANTIMICROBIAL ACTIVITIES OF FRESHWATER CYANOBACTERIUM, NOSTOC SP. FROM TAMDIL WETLAND OF MIZORAM, INDIA: AN IDENTIFICATION OF BIOACTIVE COMPOUNDS BY GC-MS

Kshetrimayum Mirabai Devi and Surya Kant Mehta^*

Department of Botany, Mizoram University, Aizawl - 796004, India.

ABSTRACT: A freshwater microalga, Nostoc sp. isolated from Tamdil Wetland of Mizoram was tested for its algicidal, antibacterial and antifungal activity. Extracts were prepared with methanol, ethanol:water and dichloromethane:isopropanol and tested for their antimicrobial activity against nine microorganisms comprising of three chlorophycean algae (Chlorella vulgaris, Scenedesmus quadricauda and Selenastrum capricornatum), one cyanobacterium (Anabaena variabilis), three bacterial strains (Bacillus subtilis, Bacillus pumilus and Escherichia coli) and two fungal strains (Fusarium udum and Fusarium culmorum). Among the three extracts tested the methanol extract was found most effective on algal test organisms. The highest (60%) inhibitory effect was observed against A. variabilis by methanol extract followed by 31% inhibition by dichloromethane:isopropanol. When tested on bacterial strains, the highest inhibition zone (23.67±1.58mm) was observed in B. subtilis by dichloromethane:isopropanol extract followed by ethanol:water extract. In case of fungal activity, the highest inhibition zone (11.00±0.58mm) was observed in F. culmorum by dichloromethane:isopropanol extract and the rest of the extracts showed almost similar effect. MICs of each extract on all the organisms varied. The extracts were further analysed by UV-VIS Spectrophotometer, which reveals the presence of active compounds. Among the three extracts, methanol indicated the highest percentage inhibition and also obtained highest absorption spectra. So, this extract was analysed by GC-MS and we identified the presence of main components in the extract as 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, Phytol, n-Hexadecanoic acid, 9,12-Octadecadienoic acid, etc. Further study for purification of the potent compound will explain their usefulness in pharmaceutical and biotechnological industry

Antimicrobial, Algicidal, Antibacterial, Antifungal, Chlorophycean,   Cyanobacterium

INTRODUCTION: Cyanobacteria or blue-green algae are morphologically diverse group of prokaryotic, photosynthetic organisms that flourish in diverse type of habitats. The evolutionary history of this group of organisms dates back to the Proterozoic era ¹. Most species of cyanobacteria are free-living, freshwater, marine or terrestrial and symbionts in association with other plants and lichens.

Due to their occurrence in diverse habitats, these organisms are excellent materials for investigation by the ecologists, physiologists, biochemists, pharmacists and molecular biologists. Many metabolites with a diverse range of bioactivities have been reported in cyanobacteria ².

These metabolites probably originated in cyanobacterial mats and were presumably responsible for regulation of communities. Productions of these metabolites are highly species and even strain dependent ³. Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with diverse biological activities, such as antialgal, antibacterial and antiviral ⁴. Antimicrobial activity depends on both algal species and the solvents used for their extraction ^{5, 6}. Screening of cyanobacteria and algae for antibiotics and other pharmacologically active compounds have received considerable attention during the past few decades ⁷.

Nostoc has been shown as a good source of antifungal, algicide and cytotoxic metabolites ⁸. Many different bioactive substances mainly peptides, alkaloids and phenolics have been identified in Nostoc species ⁹. Cryptophycin-A and nostodione-A, which inhibit microtubule assembly and cyanovirin-N, which has anti-HIV property are detected in Nostoc strains ¹⁰. ‘Cyanobacterin’ LU-1 and LU-2 have also been reported from Nostoc linckia CALA 892 and 893, respectively. Cyanobacterial metabolite known as algicide acts as light reactions of photosynthesis ¹¹. Flores and Wolk identified one species, Nostoc species 31, as good source of an anticyanobacterial compound named nostocyclamide ¹². This compound inhibited the growth of several cyanobacteria and chlorophyceans. The application of bioactive compounds derived from algae will prove beneficial and is much more effective as compared with traditional treatment methods ¹³. Analytical methods play important roles in the discovery, development and manufacture of bioactive compounds ¹⁴.

The aim of the present study was to determine the antimicrobial activity of Nostoc extracts against algae, bacteria and fungi. Furthermore, GC-MS autogram for Nostoc sp. extracts was employed for preliminary detection of active constituents.

MATERIALS AND METHODS:

Culture and growth condition:

The Nostoc sp. was collected from Tamdil Wetland of Mizoram, India. It was isolated on agar plates by using standard methods of isolation and purification ¹⁵ and was grown axenically in 250-ml Erlenmeyer flasks containing 100 ml Chu-10 medium ¹⁶ at pH 7.0±0.2 in an air-conditioned culture room under the photoperiod of 12 hrs. Cultures were hand-shaken thrice daily with an interval of 2-3 hrs. The pure culture of Nostoc, is deposited at the Microalgal Culture Collection of Mizoram University (Accession No. MZUCC029).

Determination of specific growth rate, protein, chlorophyll a and carotenoid:

The growth rate of Nostoc was determined by spectrophotometric method and the specific growth rate was calculated according to Guillard ¹⁷. 150 ml medium was inoculated to give final absorbance of 0.2 at 440 nm. The culture was kept under light for 16 days and at regular interval, 5 ml culture was removed and protein, chlorophyll a and carotenoids were determined. The protein content of the Nostoc was determined by Lowry’s method using the standard BSA ¹⁸. Quantitative estimation of chlorophyll a and carotenoid were performed by the method of Mackinney and Myers and Krantz ^{19, 20}. The cells were harvested by centrifugation and the pellet was suspended in 5 ml of 80% acetone. After overnight incubation in dark, the pellets were separated and the absorbance of the supernatant was measured at 665 and 480 nm using UV-VIS Spectrophotometer (Systronics, India, model: 117). The chlorophyll a and carotenoid were quantified as per the formula given below:

Where, α is the absorption coefficient (82.04 for Chlorophyll a and 200 for Carotenoid), D is the optical density, d is the inside path length of the spectrophotometer in cm (1cm) and C is the concentration of pigment in gl^-1.

Estimation of Lipid content:

0.06 g fresh weight of Nostoc was washed with 0.2% NaCl. The pellet was collected and suspended in 5 ml hot isopropanol. It was boiled for 3 min in order to inactivate the lipase activities. After cooling, the suspension was mixed with 5 ml chloroform and kept for 12 hrs at room temperature. 10 ml of water was added and the mixture was vortexed for 30 sec. The lower chloroform phase was collected. Chloroform was completely evaporated in a vacuum oven. 2 ml of acid dichromate reagent (stock) was added. After being heated for 30 min, 10 ml of water was added. Absorbance was recorded at 430 nm as previously described by Kalacheva et al., ²¹.

Estimation of Sugar content:

0.01g dry weight of Nostoc was mixed with 3 ml of 80% hot ethanol and kept at room temperature for 30 min. Subsequently, it was centrifuged at 5000 g for 10 min. Then, the supernatant was collected and used for estimation of soluble sugar contents as described by Gerhardt et al., ²².

Preparation of extracts:

The stationary phase culture was used for extraction of metabolites. After biomass was separated by centrifugation (4000 g for 10 min), the pellet was collected in a flask containing sterile Milli-Q water and kept at room temperature for 48 hrs to further stimulate the synthesis of secondary metabolites. Subsequently, the biomass was freeze dried at -20°C for 24 hrs ²³. 0.5 g freeze dried biomass was suspended in 10 ml of methanol and the cells were ruptured by ultrasonication (Sartorius, Germany; model: BBI 8535108) for 30 sec. The residues were separated by passing through Sephadex G column. The filtrates were used as methanol (100%) extract. Two other extracts, ethanol:water; 3:7 (v/v) and then dichloromethane:isopropanol (DCM:ISO) ; 1:1 (v/v) were also prepared from the remaining biomass.

Test Microorganisms

Three green algae (Chlorella vulgaris, Scenedesmus quadricauda and Selenastrum capricornatum), one cyanobacterium (Anabaena variabilis), two gram positive bacteria [Bacillus subtilis (strain ATCC 11774) and Bacillus pumilus (strain ATCC 14884)] and one gram negative bacteria [Escherichia coli (strain ATCC10536)] and two fungi (Fusarium udum and Fusarium culmorum) were used as target organisms for testing bioactivities. Algae were grown axenically in Chu-10 medium whereas bacterial strains were incubated in nutrient broth medium. Fungal strains were grown in potato dextrose agar (PDA) medium. The actively growing cultures of the organisms were used for various tests.

Tests for Bioactivities:

Algicidal assay:

Algal cultures of Chlorella vulgaris, Scenedesmus quadricauda, Selenastrum capricornatum and Anabaena variabilis were used to test potential bioactivities of Nostoc extracts. Different concentrations ranging from 25 µl to 100 µl of the extracts were added separately to 3 ml culture of C. vulgaris, S. quadricauda, S. capricornutum and A. variabilis (in triplicate each). After 4 days of incubation in light, the absorbance was recorded at 684 nm for C. vulgaris, 440 nm for S. quaricauda and S. capricornutum, and 417 nm for A. variabilis using spectrophotometer (Systronics, India, model: 117). Specific growth rate was calculated for treated and untreated cultures.

Anti-bacterial assay:

The antibacterial activities of different extracts were also tested using agar well diffusion assay as previously described ²⁴. Briefly, bacterial strains were inoculated in nutrient broth medium and then incubated at 35°C for 24 hrs. 0.5 ml of bacterial suspension was poured on sterilized nutrient agar plates and spread uniformly by using L-shape spreader. Plates were punched to make a well of 6 mm diameter with the help of sterile cork borer. Different concentrations (25 µl, 50 µl, 75 µl and 100 µl) of extracts were pipetted into the well and plates were incubated overnight at 35°C in an incubator. All the tests were done in triplicates. The plates were observed for the inhibition zone after 48-72 hrs.

Anti-fungal assay:

For antifungal assay, disc diffusion method was performed ²⁵. The fungal strains, (Fusarium udum and Fusarium culmorum) were inoculated on sterile petri dishes containing 10 ml of potato dextrose agar medium. The filter paper (8 mm diameter) were impregnated with 25 µl, 50 µl, 75 µl and 100 µl of the extracts and placed onto the agar plates previously inoculated with the fungal cultures by using a flamed forcep followed by gently pressing down to ensure contact ²⁶. The plates were incubated at 30^oC for 48 hrs, and diameters of the inhibition zones were measured.

Determination of Minimum Inhibitory Concentration (MIC):

Minimum inhibitory concentration (MIC) of different extracts was also evaluated using different concentrations (25 µl, 50 µl, 75 µl and 100 µl) of extracts against the selected microorganisms. These tests were done to determine the lowest concentration of algal extract that inhibits the growth of other organisms.

UV-Visible Spectrophotometric Analysis:

The absorption spectra of methanol, ethanol:water and dichloromethane:isopropanol extracts of Nostoc were determined using UV-Visible Spectrophotometer (HITACHI  U4100L). The wavelength ranged from 200 to 750 nm ²⁷.

GC-MS analysis of Nostoc extract:

Nostoc sp. crude methanol extract was dissolved in chloroform at the concentration of 10 mg/ml and was analysed by GC-MS -QP2010 Plus (Shimadzu, Japan). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used, we used helium as the carrier gas at a constant flow rate of 1.21 mL/min. The injector and MS transfer line temperature was set at 250^ᵒC and 260^ᵒC, respectively ²⁸. Compounds were identified based on the comparison of their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC-MS system.

Statistical Analysis: The data obtained from the study were analysed statistically using the Analysis of Variance (ANOVA). The test was performed using Statistical 5.0 software at p<0.05 significant level.

RESULTS:

Growth pattern and determination of protein, chlorophyll a, carotenoid, lipid and sugar contents: The growth pattern of Nostoc species was determined by recording the absorbance of culture at 440 nm ( Fig. 1). The growth of culture remained active upto 20 days, then entered into the stationary phase. The level of protein, chlorophyll a and carotenoid were found to be elevating till the stationary phase ( Fig. 2) and monitored during the course of 15 days growth period. The general biochemical profile including fresh- and dry-weights, lipid content, protein content, sugar content, and photosynthetic parameters (Fv/Fm, Y(II), qP, qL, NPQ)  are given in Table 1. The total protein, lipid and sugar contents were measured to be  0.148 mg ml^-1, 261.7 mg g^-1 dry weight, 176.00 mg g^-1 dry weight, respectively.

FIG. 1: GROWTH BEHAVIOR OF NOSTOC SPECIES. THE CYANOBACTERIUM WAS GROWN IN CHU-10 MEDIUM WITHOUT NITROGEN AND ABSORBANCE WAS RECORDED AT DIFFERENT DAYS. THE VALUES ARE MEAN OF THE THREE REPLICATES AND VERTICAL BARS INDICATES S.D.

FIG.2: PROTEIN, CHLOROPHYLL a AND CAROTENOID CONTENT OF NOSTOC CELLS IN mgl-1 DURING THE GROWTH PERIOD OF 15 DAYS. VERTICAL BARS INDICATE S.D. OF MEAN VALUE OF THREE REPLICATES.

TABLE 1 : SOME CHARACTERISTICS OF NOSTOC SPECIES

 Algicidal activity:

For antimicrobial activity assay by using three different extract solvents, methanol extract has maximum inhibitory effect against A. variabilis (60%). The inhibitory effect was noticed against both cyanobacterium and green algae tested in the present study. We also observed a dose dependent inhibitory response against the tested algae. The ethanol:water extract showed inhibitory effect against C. vulgaris, but surprisingly not against other two green algae tested. Instead, ethanol:water extract significantly stimulated the growth of S. quadricauda.  The dichloromethane:isopropanol extract also inhibited almost all the test organisms. A maximum of 31% inhibition was observed against A. variabilis. Out of three extracts tested in the present study, methanol and dichloromethane : isopropanol extracts have more growth inhibitory effect than ethanol:water extract on algal test organisms (Table 2; Fig. 3a, 3b and 3c).

FIG. 3: PERCENTAGE GROWTH OR INHIBITION OF C. VULGARIS, S. QUADRICAUDA, S. CAPRICORNUTUM AND A. VARIABILIS BY THE NOSTOC EXTRACTS PREPARED IN a) METHANOL b) ETHANOL:WATER AND c) DICHLOROMETHANE:ISOPROPANOL (DCM:ISO) IN VARIOUS CONCENTRATIONS.

TABLE 2: ANTIALGAL TEST OF EXTRACTS OF NOSTOC WITH FOUR DIFFERENT CONCENTRATIONS.

N.B. (-) and (+) indicate inhibition and stimulation in % growth rate, respectively. The values are corrected for solvent effect (data are not shown).

Anti-bacterial activity:

In antibacterial assay,  both ethanol:water and dichloromethane:isopropanol extracts inhibited the growth of B. subtilis, while  only dichloromethane:isopropanol extract inhibited the growth of B. pumilus. Methanol extract did not show any effect on all the bacterial strains. However, all the three extracts have no effect on E.

coli. The highest inhibition zone in B. subtilis by 100µl of dichloromethane:isopropanol extracts extract was 23.67±1.53 (Fig. 4a), followed by 14.00±1.00 (Fig. 4b) in B. pumilus (Table 3). As the concentration of the extracts increases, the zone of inhibition become higher.

TABLE 3: ANTIBACTERIAL ACTIVITIES OF NOSTOC EXTRACTS

(‘-’ sign indicates no effect, there was no effect of extracts on E. coli)

FIG. 4: INHIBITION ZONE DIAMETER OF a) B. SUBTILIS AND b) B. PUMILUS BY THE NOSTOC EXTRACT PREPARED IN METHANOL, ETHANOL:WATER AND DICHLOROMETHANE:ISOPROPANOL (DCM:ISO).

Anti-fungal activity: 

Table 4, Fig. 5a and 5b show the antifungal assay. In this case, the three extracts inhibited the growth of Fusarium culmorum, but ethanol:water extract had no effect against Fusarium udum. In F. culmorum, the dichloromethane:isopropanol extract showed higher inhibitory zone than the other extracts.

TABLE 4:  EFFECT OF NOSTOC EXTRACTS ON FUNGAL STRAINS.

FIG. 5: INHIBITION ZONE DIAMETER OF a) F. UDUM AND b) F. CULMORUM BY THE NOSTOC EXTRACTS PREPARED IN METHANOL, ETHANOL:WATER AND DICHLOROMETHANE:ISOPROPANOL (DCM:ISO).

Minimum inhibitory concentration

Minimum inhibitory concentrations of all the three extracts were varied (Table 5). 25 µl of all the extracts showed inhibitory effect against C. vulgaris. 100 µl of methanol and dichloromethane: isopropanol extracts inhibited the growth S. quadricauda. 75 µl of extract prepared in methanol has shown significant inhibition of growth of S. capricornutum and A. variabilis. 25 µl of dichloromethane:isopropanol extract also inhibited the growth of S. capricornutum, A. variabilis, B. subtilis and B. pumilus. The MIC of ethanol: water extract on A. variabilis and B. subtilis was 100 µl and 50 µl, respectively. All the extracts showed their MIC at 25 µl on the fungal strains tested.

TABLE 5: MINIMUM INHIBITORY CONCENTRATIONS OF EXTRACTS OF NOSTOC ON TEST ORGASNISMS.

UV-Visible spectrophotometric analysis:

From this analysis, several peaks were observed in different wavelengths, which indicate the presence of active compounds in the extracts of Nostoc. Among the extracts of Nostoc, the highest numbers of peaks were found in methanol extract which was followed by dichloromethane: isopropanol extract and lastly ethanol: water extract (Fig. 6).

FIG. 6: UV-VISIBLE SPECTRA OF METHANOL [A], ETHANOL: WATER (3:7) [B] AND DICHLOROMETHANE: ISOPROPANOL (1:1) [C] EXTRACTS OF NOSTOC SPECIES. THE MAJOR PEAKS DETECTED ARE SHOWN INSIDE THE FIGURE FOR RESPECTIVE EXTRACTS.

GC-MS analysis of Nostoc sp. extract:

GC-MS analysis of the Nostoc extract prepared in methanol has identified 32 active compounds. Some of the few compounds are 2,4,4-Trimethyl-1-hexene, Octadecane, 2,6,10-Ttrimethyl-14-Ethylene-14-Pentadecane, n-Hexadecanoic acid, 9,12-Octadecadienoic acid, 9,12-Octadecadienoyl chloride, Phytol, 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester and Vitamin E (Table 6). The chromatogram of the GC-MS is presented in Fig. 7.

TABLE 6: SELECTED LIST OF COMPOUNDS IDENTIFIED BY GC-MS IN METHANOL EXTRACT OF NOSTOC SPECIES

FIG. 7: GAS CHROMATOGRAPGIC PROFILE OF THE MAJOR CONSTITUENTS OF NOSTOC SPECIES

DISCUSSION: The present study has clearly shown the potential antimicrobial activity of Nostoc sp. Though the extracts prepared in different solvents showed variation in their inhibition properties in different test organisms of algae, bacteria and fungi, we have observed the potential pharmacological properties. Maximum growth inhibition was shown by the extract prepared in methanol (46.71% - 60.41%) followed by dichloromethane:isopropanol extract (11.33% - 31.16%) against A. variabilis. The growth inhibition of A. variabilis by the extracts of Nostoc suggests the possible presence of algicidal metabolites in Nostoc. In a similar study, it inhibited the growth of Nannochloris, Ankistrodesmus and Scenedesmus ²⁹. Nostoc possesses algicidal compounds like Crytophycin-A and nestodione-A, hexapeptides, Cyanobacterin LU-1 and LU-2 ³⁰. Compounds isolated from cyanobacteria act on the photosynthetic electron transport chain (ETC), proton coupling, pigment synthesis and photo-oxidative effects and synthesis of amino acids ³¹. It also reacts with cell membrane molecules thereby inhibiting the growth ³². Another very interesting observation was that the extract prepared in ethanol:water stimulated the growth of S. quadricauda (10.35% - 33.45%) and S. capricornutum (4.48% - 9.35%). Phytol and Vitamin E identified by GC-MS might have played key role in growth stimulation. Our observation is in corroboration with an earlier report on stimulation of growth of few phytoplanktons treated with extracts prepared from cyanobacteria ³³. Phytohormones like auxins, gibberellins and cytokinins are present in the extract, and may act on the different metabolic pathways. The target algae have ability to evolve pathways to effectively metabolize the compounds like allelochemical-like compounds ³⁴. We are yet unable to answer to this stimulatory mechanism. The bioactive compounds are associated with positive nutritional and adaptation mechanisms of target organism ³⁵. Our findings of algicidal assays using different solvents suggest that stimulation or inhibition of growth is not solvent specific, but a target (organism) specific.

The antibacterial activity was done in three widely used bacterial strains, such as B. subtilis, B. pumilus and E. coli. Out of the three extracts, two extracts prepared in ethanol:water and dichloromethane:isopropanol showed inhibition of growth of B. subtilis (7.33-23.67 mm). Nostoc extract prepared in dichloromethane:isopropanol inhibited the growth of B. pumilus (10.67-14.00 mm). 2, 6, 10 - Trimethyl, 14 – ethylene - 14 -pentadecane and 1,2-Benzenedicarboxylic acid were identified by GC-MS. They are well known for antibacterial activity ³⁶. Asthana et al., identified naphthalene compound from Nostoc CCC 537 which has antibacterial property ³⁷.

In a similar study, Madhumati et al., showed growth inhibition of B. subtilis by the extract of cyanobacteria Oscillatoria latevirens (2.2 cm) and Lyngbya martensiana (2.5 cm) ³⁸. Methanol extract of Nostoc showed the growth inhibition of B. subtilis (17%) ²⁸. Our findings suggest the presence of bioactive compounds which interfere with the cellular and metabolic activity. Diterpenoid, isolated from Nostoc commune, was identified to have an antibacterial activity ³⁹. Another compound, Nostocyclamide M, also showed allelopathic effect ⁴⁰. However, all the three extracts showed no effect against E. coli. In another study, the Nostoc commune extract prepared in methanol inhibited the growth of Staphylococcus, B. subtilis, B. cereus, B. pumilus and E. coli ⁴¹. There is also report for antimicrobial properties of ethanol extract against E. coli ⁴². This discrepancy is explained by the genetic and physiological variations within the particular cyanobacterial strains.

Our present study has clearly shown antifungal activity of Nostoc. All the extracts of Nostoc inhibited the growth of F. culmorum, whereas methanol and dichloromethane:isopropanol extracts inhibited F. udum only. But, antifungal activity was found with the methanol extract on the two species as observed by Arun et al., ⁴³.  Nostofungicidine, a lipopeptide compound, isolated from Nostoc commune has antifungal activity ⁴⁴. Growth of fungus, Aspergillus niger was inhibited by Nostoc extract ²⁸. A polyunsaturated fatty acid hexadecatrienoic acid n4 has an antibacterial activity ⁴⁵. Antimicrobial effects are related with the compounds of hydrogen peroxide, terpenoid, bromoether and volatile fatty acids compounds isolated from algae ⁴⁶.  Phenols, plasticizer compound, phytol (acyclic diterpene alcohol) and flavonoid are abundant in methanol extract, while plasticizer compound, phytol, alkenes and ester are abundant in the acetone extract of Nostoc ²⁸. We are also looking for the bioactive compounds identified by GC-MS in different extracts, which is under validation phase.

The present findings on the overall antimicrobial activities clearly revealed that among the three different extracts of Nostoc, the methanol and dichloromethane:isopropanol extracts were more effective than the ethanol:water extract. When the extracts were analysed in UV-Visible spectra, methanol extract showed highest number of peaks which represents the presence of more active compounds in the extract that are responsible for antimicrobial activity.

CONCLUSION: From this study, we conclude that Nostoc has antimicrobial activities against both prokaryotic and eukaryotic target organisms (algae, bacteria and fungi). Further identification of the compounds in Nostoc sp. extracts followed by their phytochemical studies can elucidate the groups of compounds associated with such antimicrobial activity. Use of modern technology in breeding the culture will also increase the quality and quantity of algal production. Nostoc sp. has potential pharmacologic applications. 

ACKNOWLEDGEMENT: KMD gratefully acknowledges the research fellowship (MZU-UGC) availed by MZU. We are thankful to Dr. Bhim Pratap Singh, Dept. of Biotechnology, MZU, for providing us fungal cultures and Dr. Tekcham Dinesh Singh, Centre for Genomics, Jiwaji University, Gwalior, for helping us in GC-MS analysis.

REFERENCES:

1. Al-Wathnani H, Ara I, Tahmaz RR, Al-Dayel and Bakir MA: Bioactivity of natural compounds isolated from cyanobacteria and green algae against human pathogenic bacteria and yeast. Journal of Medicinal Plants Research 2012; 6:3425-3433.
2. Namikoshi M and Rinehart KL: Bioactive compounds produced by cyanobacteria. Journal of Indian Microbiology 1996; 17:373-384.
3. Le-Flaive J and Ten-Hage L: Algal and cyanobacterial secondary metabolites in freshwaters: a comparision of allelopathic compounds and toxins. Freshwater Biology 2007; 52:199-214.
4. Katricioglu H, Beyati Y, Aslim B, Yuksekdag Z and Atici T: Screening of antimicrobial agent production of some microalgae in freshwater. Internet Journal of Microbiology 2006; 2:1-9.
5. Prakash JW, Johnson M and Solomon J: Antimicrobial activity of certain freshwater microalgae from Thamirabarani. Asian Pacific Journal of Tropical Biomedicine 2011; 1:170-173.
6. Radhika D, Veerabahu C and Priya R: Antibacterial activity of some selected seaweeds from Gulf of Mannar Coast, South India. Asian Journal of Pharmaceutical and Clinical Research 2012; 5:89-90.
7. Rimsha R, Richa J, Sheela K, Shrivastava PN and Manju J: Bioactive substances of cyanobacteria (Nostos muscorum): a review. International Journal of Pharma Sciences and Research 2014; 5:320-322.
8. Hemscheidt T, Puglisi MP, Larsen LK, Patterson GML, Moore RE, Rios JL and Clardy J: Structure and biosynthesis of borophycin, a new boeseken complex of boric acid from a marine strain of the blue-green alga Nostoc linckia. Journal of Organic Chemistry 1994; 59:3467-3471.
9. Skulberg OM: Microalgae as a source of bioactive molecules–experience from cyanophyte research. Journal of Applied Phycology 2000; 12:341-348.
10. Esser MT, Mori T, Mondor I, Sattentau QJ, Dey B, Berger EA, Boyd MR and Lifson JD: Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120. Journal of Virology 1999; 73:4360-4371.
11. Smith GD and Doan NT: Cyanobacterial metabolites with bioactivity against photosynthesis in cyanobacteria, algae and higher plants. Journal of Applied Phycology 1999; 11:337-344.
12. Flores E and Wolk CP: Production, by filamentous, nitrogen-fixing cyanobacteria, of a bacteriacin and of other antibiotics that kill related strains. Archives of Microbiology 1986; 145:215-219.
13. Sanmukh S, Bruno B, Ramakrishnan U, Khairnar K and Swaminathan S: Bioactive compounds derived from microalgae showing antimicrobial activities. Journal of Aquaculture research and Development 2014; 5:224.
14. Mariswamy Y, Gnaraj WE and Johnson M: Chromatographic fingerprint analysis of steroids in Aerva lanata L by HPTLC technique. Asian Pacific Journal of Tropical Biomedicine 2011; 1:428-433.
15. Andersen RA: Algal Culturing Techniques. Phycological Society of America, Elsevier Academic Press. 2005; pp 83-98.
16. Gerloff GC, Fitzgerald GP and Skoog F: The mineral nutrition of Coccochlori peniocysis. American Journal of Botany 1950; 37:835-840.
17. Guillard RRL: Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press, Cambridge, Stein, J. R. (ed) 1973; pp 289-311.
18. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the folin-phenol reagent. Journal of Biological Chemistry 1951; 193:265-275.
19. Mackinney G: Absorption of light by chlorophyll solutions. Journal of Biological Chemistry 1941; 140:315-322.
20. Myers J and Krantz WA: Relationship between pigment content and photosynthetic characteristics in blue green alga. The Journal of General Physiology 1955; 39:11-12.
21. Kalacheva GS, Zhila NO, Volova TG and Gladyshev MI: The effect of temperature on the lipid composition of the green alga Botryococcus. Microbiology 2002; 71:286-293.
22. Gerhardt P, Murray RGE, Wood WA and Krieg NR: Methods General Molecular Bacteriology. ASM Washington DC (ISBN 1-55581-048-9) 1994; p-518.
23. Ördög V, Strik WA, Lenobel R, Bancířová M, Strnad M, Van Staden J, Szigeti J and Németh L: Screening of microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites. Journal of Applied Phycology 2004; 16:309-314.
24. Perez C, Pauli M and Bazerque P: An antibiotic assay by agar-well diffusion method. Acta Biol Med Exp 1990; 15:113-115.
25. de Mule M, de Caire G, de Cano M and Halperin D: Bioactive compound from Nostoc muscorum (Cyanobacterium). Cytobios 1991; 66:169-172.
26. NCCLS – National Committee for Clinical Laboratory Standards 2001, Performance standards for antimicrobial susceptibility testing: eleventh information supplement; Document M100-SII. National Committee for Clinical Laboratory Standards, Wayne, PA, USA.

27. El-Sheekh MM, Osman MEH, Dyab MA and Amer MS: Production and characterization of antimicrobial active substance from the cyanobacterium Nostoc muscorum. Environmental Toxicology and Pharmacology 2006; 21:42-50.
28. Salem OMA, Hoballah EM, Ghazi SM and Hanna SN: Antimicrobial activity of microalgal extracts with special emphasize on Nostoc Life Science Journal 2014; 11:752-758.
29. Volk RB: Screening of microalgal culture media for the presence of algicidal compounds and isolation and identification of two bioactive metabolites, excreted by the cyanobacteria Nostoc insulare and Nodularia harveyana, respectively. Journal of Applied Phycology 2005; 17:339-347.
30. Gromov BV, Vepritski AA, Titova NN, Mamkayeva KA and Alexandrova OV: Production of the antibiotic cyanobacterin LU-1 by Nostoc linckia CALU 892 (cyanobacterium). Journal of Applied Phycology 1991; 3:55-59.
31. Böger P and Sandmann G: Pigment biosynthesis and herbicide interaction. Photosynthetica 1993; 28:481-493.
32. Spassova MI, Mellor R, Petrov AG, Beattie KA, Codd GA, Vais H and Usherwood PNR: Pores formed in lipid bilayers and in native membranes by nodularin, a cyanobacterial toxin. European Biophysics Journal 1995; 24:69-76.
33. Lopes VR and Vasconcelo VM: Bioactivity of benthic and picoplanktonic estuarine cyanobacteria on growth of photoautotrophs: inhibition versus stimulation. Marine Drugs 2011; 9:790-802.
34. Windust AJ, Quilliam MA, Wright JLC and McLachlan JL: Comparative toxicity of the diarrhetic shellfish poisons, okadaic acid diol-ester and dinophysistoxin-4, to the diatom Thalassiosira weissflogii. Toxicon 1997; 35:1591-1603.
35. Kearns KD and Hunter MD: Green algal extracellular products regulate antialgal toxin production in cyanobacterium. Environmental Microbiology 2000; 2:29-297.
36. Singh R, Dar SA and Sharma P: Antibacterial activity and toxicological evaluation of semipurified hexane extract of Urtica dioica Research Journal on Medicinal Plants 2012;6:123-135.
37. Asthana RK, Deepali A Tripathi MK, Srivartava A, Singh AK, Singh SP, Nath G, Srivastava R and Srivastava BS: Isolation and identification of a new antibacterial entity from the Antractic cyanobacterium Nostoc CCC 537. Journal of Applied Phycology 2009; 21:81-88.
38. Madhumathi V, Deepa P, Jeyachandran S, Manoharan C and Vijayakumar S: Antimicrobial activity of cyanobacteria isolated from freshwater lake. International Journal of Microbiology Research 2011; 2:213-216.
39. Jaki BB, Orjul JJ and Sticher O: A novel extracellular diterpenoid with antibacterial activity from the cyanobacteria Nostoc commune. Journal of Natural Products 1999; 62:502-503.
40. Jüttner F, Todorova AK, Walch N and von-Phillipsborn W: Nostocyclamides M: a cyanobacterial cyclic peptides with allelopatic activity from Nostoc Phytochemistry 2001; 57:613-619.
41. Kaushik P, Chauhan A, Chauhan G and Goyal P: Evaluation of Nostoc commune for potential antibacterial activity and UV-HPLC analysis of methanol extract. Internet Journal of Microbiology 2008; 5:1-5.
42. Amaro HM, Guedes AC and Malcata FX: Antimicrobial activities of microalgae; an invited review. Science against microbial pathogens: communicating current research and technological advances 2011; 3:1272-1280.
43. Arun N, Gupta S and Singh DP: Antimicrobial and antioxidant property of commonly found microalgae Spirulina platensis, Nostoc muscorum and Chlorella pyrenoidosa against some pathogenic bacteria and fungi. International Journal of Pharmaceutical Science and Research 2012; 3:4866-4875.
44. Kajiyama SI, Kanzaki H, Kawazu K and Kabayashi A: Nostofungicidine, an antifungal lipopeptide from the field-grown terrestrial blue-green alga Nostoc commune. Tetrahedron Letters 1998; 39:3737-3740.
45. Desbois A, Spragg AM and Smith VJ: A fatty acid from the diatom Phaeodactylum tricornutum is antibacterial against diverse bacteria including multiresistant Staphylococcus aureus (MRSA). Marine Biotechnology 2009; 11:45-52.
46. Tuney I, Cadirci BH, Unal D and Sukatar A: Antimicrobial activities of the extracts of marine algae from the coast of Urla (Izmir, Turkey). Turkish Journal of Biology 2006; 30:171-175
    

    ---

How to cite this article:

Devi KM and Mehta SK: Antimicrobial Activities of Freshwater Cyanobacterium, Nostoc Sp. From Tamdil Wetland of Mizoram, India: An Identification of Bioactive Compounds by GC-MS. Int J Pharm Sci Res 2016; 7(3): 1179-91.doi: 10.13040/IJPSR.0975-8232.7(3).1179-91.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.