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Cah. Biol. Mar. (2012) 53 : 45-51

Cah. Biol. Mar. (2012) 53 : 45-51 Antimycobacterialactivityofmarinecyanobacterialspecies against M yco b a cter iu m

Antimycobacterialactivityofmarinecyanobacterialspecies against M yco b a cter iu m s meg ma tis

Murugesan CHANDRASEKARAN 1 and Muthuraman SUNDARARAMAN 2 * (1) DepartmentofPlantScience,BharathidasanUniversity,Tiruchirrappalli-620024,Tamilnadu,India (2) DepartmentofMarineBiotechnology,BharathidasanUniversity,Tiruchirappalli-62024,Tamilnadu,India

*Correspondingauthor:Fax:+914312407084,E-mail:sundarbdu1@gmail.com

Abstract: The studies of antimycobacterial activity of extracts (ethanol, ethyl acetate and hexane) from 18 marine cyanobacterial strains have shown specific inhibitory activity against Mycobacterium smegmatis. All the strains were evaluated for antimycobacterial activity by disc diffusion method followed by spread plate, broth dilution and streak plate methods. It was found that among the 18 cyanobacterial species, Oscillatorialaetevirens BDU 141071 had the highest activity in all the three solvent systems. Among them three strains, Oscillatoriawillei BDU 130711, Oscillatoriawillei BDU 130791 and Phormidiumcorium BDU 60201 had no activity. The results revealed that most of the ethyl acetate extracts (13 cyanobacterial strains) were active against Mycobacterium smegmatis compared to ethanol (eight cyano- bacterial strains) and hexane (five cyanobacterial strains) extracts. The present study shows that some marine cyanobacteria harbor potential lead compounds having antimycobacterial activity.

Résumé:Activitéantimycobactériennedecyanobactériesmarinescontre Mycobacterium smegmatis. Les études d’activité antimycobactérienne d’extraits (ethanol, ethyl acetate and hexane) de 18 souches de cyanobactéries marines ont montré une activité inhibitrice spécifique contre Mycobacteriumsmegmatis. Toutes les souches ont été évaluées pour l’activité anti- mycobactérienne par la méthode de diffusion de disque suivie par la plaque de propagation, la dilution de bouillon et les méthodes de plaque latérale. Parmi les 18 espèces de cyanobactéries, Oscillatorialaetevirens BDU 141071 avait la plus forte activité dans les trois types de solvant. Trois souches, Oscillatoriawillei BDU 130711, Oscillatoriawillei BDU 130791 et Phormidiumcorium BDU 60201 n’ont montré aucune activité. Les résultats ont révélé que la plupart des extraits d’acétate d’éthyle (13 souches) étaient actifs contre Mycobacteriumsmegmatis, davantage qu’à l’éthanol (8 souches) et à l’hexane (5 souches). La présente étude montre que quelques cyanobactéries marines recèlent des composés potentielle- ment majeurs pour l’activité antimycobactérienne.

Keywords: Marine Cyanobacteria l Antimycobacterial compounds l Mycobacteriumsmegmatis l Blue green algae

Reçu le 24 septembre 2010 ; accepté après révision le 11 août 2011.

Received24September2010;acceptedinrevisedform11August2011.

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ANTIMyCOBACTERIALACTIVITy OF MARINE CyANOBACTERIA

Introduction

The demand of therapeutic drugs from natural resources is on the increase in the 21 st century (Borowitzka, 1995; Mundt et al., 2001; Ramamurthy & Raveendran, 2009; Al-

Haj et al., 2010; Villa & Gerwick, 2010) and the contribution of marine organisms is potentially remarkable (Sponga et al., 1999; Jha & Zi-rong, 2004; Bérdy, 2005; Berrue et al., 2007; Wei et al., 2007; Jain & Sonawane, 2008). Cyanobacteria (also known as blue–green algae) are

a group of unusually diverse Gram-negative photoau-

totrophic prokaryotes that originated 3.5 billion years ago. Various strains of cyanobacteria are known as producing intracellular and extracellular metabolites with diverse bio- logical activities such as antialgal, antibacterial, antifungal, antiviral, anthelmintic, and antiprotozoal activity (Issa, 1999; Burja et al., 2001; Tan, 2007; Mayer et al., 2007, 2009 & 2011; Patra et al., 2009). They have tremendous potential to be used as therapeutic agents for a variety of diseases (Shibib et al., 1993; Patterson et al., 1994, Romanos et al., 2002). Secondary metabolites from cyanobacteria have also exhibited toxic, hormonal, cyto- toxic and antineoplastic activities (Carmichael, 1992; Patterson et al., 1994; Harada et al., 2002; Tan, 2007; Mayer et al., 2007 & 2011). Tuberculosis (TB) is the leading cause of mortality all over the world due to infectious agent Mycobacterium

tuberculosis (Wallis, 1996; R ya n & R a y, 2004). WHO (2010) reported that more than 2 billion people equal to one-third of the world’s population are infected with Mycobacterium tuberculosis. The vast majority of TB deaths occur in the developing world and more than half of the deaths in Asia (WHO, 2010). There were 9.4 million new TB cases in 2008 (3.6 million of whom are women) including 1.4 million cases among them infected with HIV. During 2008, 1.8 million people have died due to TB and it indicates 4500 deaths a day due to Multidrug-Resistant TB (MDR-TB) (WHO, 2010). The resistance to second-line drugs developed on top of MDR-TB leads to extensively Drug-Resistant TB (XDR) (WHO, 2010). The worldwide problem caused by TB and the lack of new drugs in the market trigger an imperative search for novel lead molecules to fight efficiently against the rapid spread of

multidrug-resistant TB (Copp, 2003). In this context, there

is an urgent need for new anti-TB drugs with less toxic side

effects, improved pharmacokinetic properties, extensive and potent activity against resistant strains. Marine cyanobacteria have been recognized as an important renewable bioresource for novel bioactive compounds. We investigated the antimycobacterial activity of the organic solvent extracts from 18 marine cyanobacteria, as a pilot screening study by employing M. smegmatis which is a suitable surrogate screen for selecting

cyanobacterial strains with potential activities against multi-drug resistant M.tuberculosis.

Materialsandmethods

Bacterialstrainandgrowthcondition

Culture of Mycobacterium smegmatis was obtained from Tuberculosis Research Center (TRC), Chennai, Tamilnadu, India. This standard strain was confirmed with morphological and biochemical analysis. Culture suspensions were prepared from Middle Brook 7H9 broth and cultures were maintained on Middle Brook 7H10 agar medium containing 0.5% glycerol and 10% ADC enrichment, incubated at 37°C for 2 days.

Cyanobacterialstrainsandbiomass

Marine cyanobacterial strains (Table 1) were obtained from the Germplasm of National Facility for Marine Cyanobacteria (NFMC), Bharathidasan University, Tiruchirappalli, Tamilnadu, India. The cyanobacterial strains were grown in ASN-III (marine synthetic) medium (Rippka et al., 1979). The marine cyanobacterial cultures were inoculated into 250 mL Erlenmeyer flask containing 100 mLof sterileASN-III media and incubated under white fluorescent light 13.7 µE.m -2 .s -1 at 27 ± 2°C with 14/10 hr L/D cycle. After incubation, cultures were harvested at stationary phase (20 days) and centrifuged at 10,000 rpm for 20 min.

Table1. List of Marine cyanobacterial strains used for anti- mycobacterial studies. Tableau 1. Liste des souches cyanobactériennes marines utilisées pour les tests antimycobactériens.

SerialNo.

NameoftheOrganism

1. Gleocapsacrepidium BDU 20121

2. Phormidiumcorium BDU 60201

3. Phormidiumvalderianum BDU 41001

4. Phormidiumvalderianum BDU 140041

5. Phormidiumvalderianum BDU 142552

6. Phormidiumvalderianum BDU 142022

7. Oscillatoriaboryana BDU 40261

8. Oscillatoriaformosa BDU 130511

9. Oscillatorialaetevirens BDU 100891

10. Oscillatorialaetevirens BDU 141071

11. Oscillatoriasalina BDU 92021

12. Oscillatoriawillei BDU 130711

13. Oscillatoriawillei BDU 130791

14. Lyngbya sp.

15. Lyngbya sp. BDU 30342

16. Lyngbya sp. BDU 140301

17. Spirulinasubsalsa BDU 141021

18. Nostoccalcicola BDU 40302

M. CHANDRASEKARAN, M. SUNDARARAMAN

47

Isolationofantimycobacterialmetabolites

To study the antimycobacterial activity, cyanobacterial extracts were derived (ethanol, ethyl acetate and hexane) by solvent extraction method (Shibib et al., 1993). Cyanobacterial biomass was ground with ethanol (1:2 w/v) and glass powder. The macerate was kept at 4ºC for 12 hrs. After incubation, the supernatant was separated. The deposit was re-subjected to grind with ethanol in the ratio of 1:1 (w/v), until it became pale in color; it was incubated at room temperature for 3 hrs. The pooled supernatant was concentrated with feed back vaccum concentrator (Savant, USA). The same procedure was used to prepare extracts from ethyl acetate and hexane solvents. The concentrated extracts were dissolved in DMSO (Dimethyl sulfoxide). The reconstituted (DMSO dissolved) extracts were examined for antimycobacterial activity against Mycobacteriumsmegmatis.

Determinationoftheantimycobacterialactivity

Disc diffusion assay. Antimycobacterial activity was evaluated by disc diffusion technique (Bauer et al., 1966). Sterile filter paper discs having 6 mm of diameter were prepared by pipetting 15 μL of extract to each disc and then the discs were placed on Middle Brook 7H10 agar plates. After incubation for 72 hours at 37°C, a clear zone around a disc was evidence of antimycobacterial activity. Diameter of the zone of inhibition was measured in millimeters. Each test was prepared in duplicate; discs loaded with the extracting agents were tested as controls.

Spread Plate Method. For the spread plate technique, the inoculum (100 µl of mycobacterial culture and 100 µL of ethyl acetate) was spread evenly over the entire surface of the Middle Brook 7H10 agar. The plates spread with DMSO (100 µL of mycobacterial culture and 100 µL of DMSO) and test organism (100 µL of mycobacterial culture and 100 µL of Middle Brook 7H9 broth) were treated as controls. The culture was incubated at 37°C for 72 hours. After incubation, the extracts added plates compared with controls.

Broth Dilution Method. The cyanobacterial extracts which showed inhibitory activity both in disc diffusion and spread plate method was further investigated using the broth dilution method. The experimental set up was made as follows (i) 1 mL of broth as control with 900 µL broth and 100 µL of Mycobacteriumsmegmatis (organism control); (ii) 800 µL broth with 100 µL culture, 100 µL DMSO (DMSO control) and (iii) 800 µLbroth with 100 µLculture, 100 µL ethyl acetate extracts (test). The culture was incubated for 72 hrs at 37°C. After incubation the results were observed for further confirmation.

Streak Plate Method. The confirmation of inhibitory

activity in broth dilution method was made by performing streak plate method.Aloopful of M.smegmatis culture was streaked uniformly over the Middle Brook 7H10 agar which contains 100 μLof extracts. In this technique, culture alone on the plate acted as organism control and DMSO as solvent control. The plates were incubated at 37°C for 72 hrs. All the experiments were conducted in triplicate. The results were observed and interpreted.

Resultsanddiscussion

The antimycobacterial activity of crude extracts of 18 marine cyanobacterial strains were evaluated by disc diffusion method and the results were summarized in Table 2. Among 18 marine cyanobacterial strains subjected for primary screening process, all of them have shown activity against M. smegmatis, except three isolates (Oscillatoria willei BDU 130711, Oscillatoriawillei BDU 130791 and Phormidium corium BDU 60201). Hence, the cyanobacteria showed antimycobacterial activity were considered for further investigation. Among 15 marine cyanobacterial extracts, Oscillatoria formosa BDU 130511, Phormidium valderianum BDU 140041, Oscillatoriaboryana BDU 40261 showed activity only in ethyl acetate extract (Table 2 & Fig. 1). There was no activity observed in ethanol and hexane extracts. The zone of inhibition is less than 2 mm indicating minimum antimycobacterial activity in certain isolates. Ethanolic extracts of Oscillatoria salina BDU 92021 and Phormidium valderianum BDU 142552 exhibited reasonable activity, but there was no activity in ethyl acetate and hexane extracts. As the zone of inhibition was below 2 mm the compounds showed least activity. Nostoccalcicola BDU 40302, Spirulinasubsalsa BDU 141021, Gleocapsa crepidium BDU 20121, Lyngbya sp. BDU 30342 and Lyngbya sp. BDU 140301 have indicated higher activity (above 2 mm) only in ethanol and ethyl acetate extraction. Among them, Lyngbya sp. BDU 30342 exhibited good activity in both ethanol and ethyl acetate extraction than other cyanobacterial strains. Both ethyl actate and hexane extracts of Phormidium valderianum BDU 41001, Phormidiumvalderianum BDU 142022, Lyngbya sp. and Oscillatoria laetevirens BDU 100891 (Table 2) have exhibited antimycobacterial activity, but not in ethanolic extracts. Of all other cyanobacterial species tested, only Oscillatoria laetevirens BDU 141071 has shown activity against Mycobacteriumsmegmatis effectively (Table 2 & Fig. 1). In addition to that, it showed activity in all the three different solvent systems used in this study. The zone of inhibition was above 2 mm, indicating that some bioactive metabolites present in this organism showed antimyco- bacterial activity. In the previous studies, the variation of

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ANTIMyCOBACTERIALACTIVITy OF MARINE CyANOBACTERIA

Table2. Disc diffusion assay for the cyanobacterial extracts. Tableau2. Analyse de diffusion de disque pour les extraits cyanobactériaux.

SerialNo.

NameoftheOrganism

EthanolExtracts Ethylacetateextracts

Hexaneextracts

1.

Disc Control

--

-

2.

Solvent control

--

-

3.

DMSO control

--

-

4.

Gleocapsacrepidium BDU 20121

+

++

-

5.

Phormidiumcorium BDU 60201

--

-

6.

Phormidiumvalderianum BDU 41001

-

++

++

7.

Phormidiumvalderianum BDU 140041

-

++

-

8.

Phormidiumvalderianum BDU 142552

+-

-

9.

Phormidiumvalderianum BDU 142022

-

+

++

10.

Oscillatoriaboryana BDU 40261

-

++

-

11.

Oscillatoriaformosa BDU 130511

-

++

-

12.

Oscillatorialaetevirens BDU 100891

-

++

++

13.

O s cilla to r ia la etevir en s BDU141071

++

++

++

14.

OscillatoriasalinaBDU92021

+-

-

15.

Oscillatoriawillei BDU 130711

--

-

16.

Oscillatoriawillei BDU 130791

--

-

17.

Lyngbya sp.

-

++

++

18.

Lyngbya sp. BDU 30342

++

++

-

19.

Lyngbya sp. BDU 140301

+

++

-

20.

Spirulinasubsalsa BDU 141021

+

++

-

21.

Nostoccalcicola BDU 40302

+

++

-

- No activity; +≤ 4 mm (zone of inhibition); ++ ≥ 5 mm (zone of inhibition).

mm (zone of inhibition); ++ ≥ 5 mm (zone of inhibition). Figure 1. Disc Diffusion assay
mm (zone of inhibition); ++ ≥ 5 mm (zone of inhibition). Figure 1. Disc Diffusion assay

Figure 1. Disc Diffusion assay of three organic solvent extracts. Figure1. Analyse de diffusion de disque de trois extraits de

dissolvants organiques. Figure2. Spread plate method for Mycobacteriumsmegmatis against the selected ethyl acetate extracts from marine cyanobacteria S1-Nostoccalcicola BDU 40302, S12-Lyngbya sp.

BDU 30342, S14-Lyngbya sp. BDU 140301, S16-Oscillatoria laetevirens BDU 141071. Figure 2. Méthode de plaque de propagation pour Mycobacteriumsmegmatis contre les extraits d’acétate éthylique des cyanobactéries marines S1-Nostoc calcicola BDU 40302, S12-Lyngbya sp. BDU 30342, S14-Lyngbya sp. BDU 140301, S16-Oscillatorialaetevirens BDU 141071.

antibacterial activity of the organic solvent extracts was due to the presence of different antibacterial substances among these species (Schwartz et al., 1990; Patterson et al., 1994; Newman & Cragg, 2007; Rania & Hala, 2008; Mathivanan et al., 2010). Based on the experimental results, among 18 cyano- bacterial species, four organisms (Nostoc calcicola BDU

M. CHANDRASEKARAN, M. SUNDARARAMAN

49

Table3. Status of Mycobacteriumsmegmatis growth against the selected ethyl acetate extracts from marine cyanobacteria. Tableau3. Statut de croissance de Mycobacteriumsmegmatis contre les extraits choisis d’acétate éthylique du marine cyanobacteria.

Statusof M yco b a cter iu m s meg ma tis growth

NameoftheOrganism

Spreadplatemethod

Brothdilutionmethod

Streakplatemethod

Organism control

++

+

DMSO control

++

+

Nostoccalcicola BDU 40302 (S1)

--

-

Lyngbya sp. BDU 30342 (S12)

--

-

Lyngbya sp. BDU 140301(S14)

--

-

Oscillatorialaetevirens BDU 141071 (S16)

--

-

+ = Growth appeared; - = Not grown

141071 (S16) -- - + = Growth appeared; - = Not grown Figure 3. Broth dilution

Figure 3. Broth dilution method for Mycobacterium smegmatis against the selected ethyl acetate extracts from marine cyanobacteria T1-Broth Control, T2-Culture Control, T3-S1- Nostoccalcicola BDU 40302, T4-S12-Lyngbya sp. BDU 30342, T5-S14-Lyngbya sp. BDU 140301, T6-S16-Oscillatoria laete- virens BDU 141071, T7 - DMSO control. Figure 3. Méthode de dilution de bouillon pour Mycobacteriumsmegmatis contre les extraits d’acétate d’éthyle des cyanobactéries marines T1-Broth Control, T2-Culture témoin, T3-S1-Nostoccalcicola BDU 40302, T4-S12-Lyngbya sp. BDU 30342, T5-S14-Lyngbya sp. BDU 140301, T6-S16-Oscillatoria laetevirens BDU 141071, T7 - DMSO témoin.

40302, Lyngbya sp. BDU 30342, Lyngbya sp. BDU 140301, Oscillatoria laetivirens BDU 141071) showed higher activity. These organisms are subjected to further studies. In spread plate method, (Table 3 & Fig. 2) the growth was observed in organism control and DMSO control. Whereas in cyanobacterial extracts seeded plate, there was no growth, which may indicate that cyanobacterial compounds have had some antimycobacterial activity against Mycobacteriumsmegmatis. In broth dilution method (Table 3 & Fig. 3), complete growth inhibition was observed in extract added tubes. Whereas, the growth was observed in both organism and DMSO controls. Though extract added tubes showed some turbidity, which is not due to the growth of mycobacterium. This turbidity may be attributed to the reaction between compounds present in the extracts and broth. This was further confirmed through streak plate method (Table 3 & Fig. 4), in which organism and DMSO controls

(Table 3 & Fig. 4), in which organism and DMSO controls Figure4. Streak plate method for

Figure4. Streak plate method for Mycobacteriumsmegmatis against selected cyanobacterial extracts from marine cyanobacteria S1-Nostoccalcicola BDU 40302, S12-Lyngbya sp. BDU 30342, S14-Lyngbya sp. BDU 140301, S16-Oscillatoria laetevirens BDU 141071.

Figure 4. Méthode de plaque latérale pour Mycobacterium smegmatis contre les extraits des cyanobactéries marines S1- Nostoccalcicola BDU 40302, S12-Lyngbya sp. BDU 30342, S14- Lyngbya sp. BDU 140301, S16-Oscillatoria laetevirens BDU

141071.

showed profuse growth, whereas complete growth inhibition was observed in test plates. The present work clearly indicated the potentials of marine cyanobacterial metabolites, which were effective against Mycobacterium smegmatis. For complete characterization of bioactive principles, the crude extracts should be purified further and analyzed for structure elucidation. Though antimycobacterial extracts analyzed might not be considered as new therapeutic agents, this preliminary study is found to be promising for future investigations. For the identification of the antimicrobial compounds, it is necessary to obtain it in pure form employing a series of purification process and different chemical analysis such as chromatography,

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ANTIMyCOBACTERIALACTIVITy OF MARINE CyANOBACTERIA

spectroscopy and other sophisticated techniques like GC- MS, NMR and so on. This study indicates that there is a vast scope for marine cyanobacteria in search for anti- mycobacterial compounds.

Acknowledgment

We thank National Facility for Marine Cyanobacteria (NFMC), Bharathidasan University, Tiruchirappalli-620 024, Tamilnadu, India, for providing facilities and support.

References

Al-Haj N.A., Mashan N.I., Shamsudin M.N., Mohamad H., VairappanC.S.&SekawiZ.2010. Antibacterial activity of marine source extracts against multidrug resistance organisms. AmericanJournalofPharmacologyandToxicology, 5: 95-102.

BauerA.W.,KirbyW.M.M.,SherrisJ.C.&TurokM.1966.

Antibiotic susceptibility testing by a standardized single disk method. AmericanJournalofClinicalPathology, 45: 493-496. BérdyJ.2005. Bioactive microbial metabolites. A personal view. JournalofAntibiotics,58: 1-27. BorowitzkaA.M.1995. Microalgae as source of pharmaceuticals and biologically active compounds. Journal of Applied Phycology,7: 3-15. BerrueF.,ThomasO.P.,LavilleR.,PradoS.,GolebiowskiJ., Fernandez R. & Amade P. 2007. The marine sponge Plakortiszygompha: a source of original bioactive polyketides. Tetrahedron,63: 2328-2334. BurjaM.A.,BanaigsB.,Abou-MansourE.,BurgessG.J.& WrightC.P.2001. Marine Cyanobacteria a prolific source of natural products. Tetrahedron, 57: 9347-9377. CarmichaelW.W.1992. Cyanobacteria secondary metabolites- the cyanotoxins. JournalofAppliedBacteriology,72: 445-459. Copp B.R. 2003. Antimycobacterial natural products. Natural ProductReports, 20: 535-557. Harada H., Yamashita U.H., Kurihara E., Fukushi J., KawabataJ.&KameiY.2002.Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. AnticancerResearch,22: 2587-2590. Issa A.A. 1999. Antibiotic production by the cyanobacteria Oscillatoria angustissima and Calothrix parietina. EnvironmentalToxicologyandPharmacology,8: 33-37. JainR.&SonawaneS.N.2008. Mandrekar, Marine organisms:

Potential source for drug discovery. CurrentScience, 94: 292. Jha R.K. & Zi-rong X. 2004. Biomedical Compounds from Marine organisms. MarineDrugs,2: 123-146. Mathivanan K., Ramamuthy V. & Rajaram R. 2010. Antimicrobial activity of Oscillatoria princeps and Lyngbya majuscula against pathogenic microbes. InternationalJournal ofCurrentResearch,5: 097-101. Mayer A.M.S., Rodriguez A.D., Berlinck R.G. & Hamann M.T. 2007. Marine pharmacology in 2003-4: marine compounds with anthelmintic antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affect-

ing the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology,145: 553-581. Mayer A.M.S., Rodriguez A.D., Berlinck R.G. & Hamann M.T. 2009. Marine pharmacology in 2005-6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, anti- tuberculosis, and antiviral activities; affecting the cardiovascu- lar, immune and nervous systems, and other miscellaneous mechanisms of action, BiochimicaetBiophysicaActa,1790:

283-308.

MayerA.M.S.,RodriguezA.D.,BerlinckR.G.&FusetaniN. 2011. Marine pharmacology in 2007-8: Marine compounds with antibacterial, anticoagulant, antifungal, anti- inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 153: 191-222 Mundt S., Kreitlow S., Nowotny A. & Effmert U. 2001. Biochemical and pharmacological investigations of selected cyanobacteria. International Journal of Hygiene and EnvironmentalHealth, 203: 327-334. NewmanD.J.&CraggG.M.2007. Natural products as sources of new drugs over the last 25 years. Journal of Natural Products,70: 461-77. PatraJ.K.,PatraA.P.,MahapatraN.K.,ThatoiH.N.,DasS., Sahu R.K. & Swain G.C. 2009. Antimicrobial activity of organic solvent extracts of three marine macroalgae from Chilika Lake, Orissa, India. Malaysian Journal of Microbiology, 5: 128-131. PattersonG.M.L.,ParkerD.L.&BolisC.M.1994. Fungal cell wall polyaccaride elicit an antifungal secondary metabolite (phytoalexin) in the cyanobacterium Scytonema ocellatona. JournalofPhycology,33: 54-60. RamamurthyV.&RaveendranS.2009. Antibacterial and anti- fungal activity of Spirulinaplatensis and Lyngbyamajuscule. Journal of Ecobiology,24: 47-52. RaniaA.M.A.&HalaT.M.2008. Antibacterial and antifungal activity of Cyanobacteria and Green Microalgae. Evaluation of medium components by Placket-Burman Design for anti- microbial activity of Spirulina Platensis. Global Journal of Biotechnology&Biochemistry,3: 22-31. Rippka R., Deruelles J., Waterbury J.B., Herdman M. & StainerR.Y.1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of GeneralMicrobiology,111: 1-61. Romanos M.T., Andrada-Serpa M.J., Mourao P.A., Yoneshigue-ValentinY.,CostaS.S.,PereiraM.S.,Miranda M.M.,GoncalvesJ.L.&WiggM.D.2002. A sulphated fucan from the Laminaria abyssalis inhibits the human T cell lymphotropic virus type 1-induced syncytium formation in HeLa cells. AntiviralChemistry & Chemotherapy,13: 219-221. Ryan K.J. & Ray C.G. (editors) 2004. Sherris Medical Microbiology: an Introduction to infectious disease (4 th edition.). McGraw Hill Publishers Inc. pp. 442-456. SchwartzR.E.,HirschC.F.&SesinD.F.1990. Pharmaceuticals

M. CHANDRASEKARAN, M. SUNDARARAMAN

51

from cultured algae. Journal of Industrial Microbiology, 5:

113-124.

Shibib B.A., Khan L.A. & Rahman R. 1993. Hypoglycemic activity of Coccinia indica and Momordica charantia in diabetic rats: Depression of the hepatic gluconeogenic enzymes glucose-6 -phosphatase and fructose-1,6-bi- sphosphatase and elevation of both liver and red-cell shunt enzyme glucose-6 - phosphate dehydrogenase. Biochemical Journal,292: 267-70. SpongaF.,CavalettiL.,LazzariniA.,BorghiA.,CiciliatoI., Losi D. & Marinelli F. 1999. Biodiversity of potentials of marine-derived microorganisms. JournalofBiotechnology,70:

65-69.

Tan L.T. 2007. Bioactive natural products from marine

cyanobacteria for drug discovery. Phytochemistry, 68: 954-979. Villa F.A. & Gerwick L. 2010. Marine natural product drug discovery: Leads for treatment of inflammation, cancer, infections, and neurological disorders. Immunopharmacology andImmunotoxicology,32: 228-37. Wei X., RodriguezA.D., WangY. & Franzblau S.G. 2007. Novel ring B abeo-sterols as growth inhibitors of Mycobacterium tuberculosis isolated from a Caribbean Sea sponge, Svenzeazeai. TetrahedronLetters, 48: 8851-8854. WallisR.S.1996. New approaches to identification of antigens of Mycobacteriumtuberculosis. Infectiousagentsanddisease, 5:

119-25.

WHO2010. reports on TB-website: http://www.who.int/tb/publi- cations/en/