ANTIMICROBIAL AND ANTIOXIDANT PROPERTY OF COMMONLY FOUND MICROALGAE SPIRULINA PLATENSIS, NOSTOC MUSCORUM AND CHLORELLA PYRENOIDOSA AGAINST SOME PATHOGENIC BACTERIA AND FUNGI

ANTIMICROBIAL AND ANTIOXIDANT PROPERTY OF COMMONLY FOUND MICROALGAE SPIRULINA PLATENSIS, NOSTOC MUSCORUM AND CHLORELLA PYRENOIDOSA AGAINST SOME PATHOGENIC BACTERIA AND FUNGI

Neelam Arun*, Shalini Gupta and D.P. Singh

Department of Environmental Science, Babasaheb Bhimrao Ambedkar (Central) University, Raibarelly Road, Lucknow-226025, Uttar Pradesh, India

ABSTRACT

In the present study, three algal species (Spirulina platensis, Chlorella pyrenoidosa and Nostoc muscorum (ATCC 2789) were used to investigate their antimicrobial and antioxidant properties against some human pathogenic bacteria and fungi (Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and fungus Aspergillus luchuensis, Aspergillus niger, Fusarium oxysporum). Four different solvents namely methanol, acetone, n-hexane and water were used for extraction.  The present investigation showed that methanolic extract was more effective against the pathogenic bacteria and fungi. Methanolic extract of C. pyrenoidosa and N. muscorum was found to be most effective against P. aeruginosa while methanolic extract of S. platensis showed maximum activity against S. aureus. S. platensis showed maximum antifungal activity in comparison to other algal extracts.  Antioxidant activity was determined by free radical scavenging activity (Nitric oxide radical scavenging activity) and it was found that methanolic extract of S. platensis showed maximum antioxidant activity.

Green algae, Antimicrobial activity, Antioxidant activity, Secondary metabolites

INTRODUCTION: Algae are photosynthetic organisms, which constitute a total of twenty-five to thirty thousand species, with a great diversity of forms and sizes ranging from unicellular microscopic organisms (microalgae) to multicellular organisms of great size (macroalgae). They can double every few hours during their exponential growth period ¹.

During the peak growth phase, some microalgae can double even every 3.5 hrs ². In fact, some algae are organisms that live in complex habitats submitted to extreme conditions as they can easily adapt to the new environmental conditions and are known to produce a great variety of secondary (biologically active) metabolites, which cannot be found in other organisms ³.

Algae can be a very interesting natural source of new compounds with biological activity that could be used as functional ingredients ⁴. Seaweeds represent a potential source of antimicrobial substances due to their diversity of secondary metabolites with antiviral, antibacterial and antifungal activities ^{5, 6}.

The search for similar antimicrobial activity in other algal species has gained importance in recent years due to growing worldwide concern about antibiotic-resistant micro-organisms. Efforts are being made to extract substances with antibacterial, antiviral, fungicidal, enzyme inhibitory, immunosuppressive and cytotoxic and algicidal property from the low cost algal biomass ^7-10.

Among algal species, Spirulina has been reported to prevent oxidative damage by scavenging free radicals and active oxygen, and hence can indirectly reduce cancer formation in human body ^{11, 12}. In this respect, the increased consumption of foods characterized by free radical scavenging activity, leads up to a doubling of protection against many common types of cancer formulation.  The antimicrobial activity and antioxidant status of microalgae as whole is considered a tool for evaluation of pharmaceutical potential of the algal extracts ^{13, 14}.

Bacterial infection causes high rate of mortality in human population and aquaculture organisms. For an example, Bacillus subtilis is responsible for causing food borne gastroenteritis ¹⁵. Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa cause diseases like mastitis, abortion, respiratory complications including some life threatening illness ¹⁶, while Salmonella sp. causes diarrhea and typhoid fever ^{17, 18}. Some fungi are opportunistic while others are pathogenic, causing disease if the immune system is not healthy ¹⁹. Preventing disease outbreaks or treating the diseases with drugs or chemicals alone may not be sufficient to tackles these problems as the microorganisms develop resistance against the applied chemical drugs ²⁰. It becomes a greater problem of giving treatment against resistant pathogenic bacteria ²¹.

Microalgae being a rich source of novel bioactive compounds, have recently found immense application in human and animal medicine. A promising strategy for the replacement of antibacterial and antifungal chemicals is to promote the natural biological control products obtained from microalgae. The present work is an effort to evaluate the pharmaceutical potential of some common fresh water microalgae against some pathogenic bacterial and fungal strains. In addition to this, antioxidant potential of these algal strains has also been evaluated.

MATERIAL AND METHODS:

Collection and culturing of Algae: Fresh water, unicellular, green algae Spirulina platensis, Chlorella pyrenoidosa and Nostoc muscorum (ATCC 2789) were used for the study.  Spirulina platensis culture was maintained in Zarrouk’s medium ²², while Chlorella pyrenoidosa and Nostoc muscorum was maintained in BG-11 medium ²³. Algal culturing was carried out in 1000 mL conical flask containing 600 mL respective medium, all the cultures were kept under fluorescent light (20 µmol m^{- 2}s^-1) with 16 h light period and at 25 ±2°C temperature.

Preparation of Algal Crude Extracts: The algal cultures were centrifuged at 2500 rpm for 10 minutes to harvest the algal biomass. After centrifugation, algal pastes were air dried in oven at 60°C. One gram of dried algal samples was extracted with 10 mL of the solvents (Methanol, acetone, n-Hexane, water).  The dried biomass was taken in sterile screw-capped bottles of 50 mL volume and was soaked in the solvents for 48 h. The mixture was then centrifuged at 2000 rpm for 10 min at 4°C.The supernatants were filtered through a sterile funnel and sterile Whatman filter paper No. 1. The filtrate was sterilized using 0.2 µm membrane filter. The extract obtained was used for screening of their antimicrobial potential ²⁴.

Test Microorganisms: Gram negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae  (K. pneumoniae), Gram positive bacterial strains Staphylococcus aureus  (S. aureus), Bacillus subtilis (B. subtilis),  Bacillus cereus (B. cereus), Micrococcus luteus  (M. luteus) and fungus Aspergillus luchuensis (A. luchuensis), Aspergillus niger (A. niger), Fusarium oxysporum (F. oxysporum) were used for the study. The parent cultures were obtained from NCIM, Pune and the subcultures were maintained in the nutrient media (Nutrient agar media (bacteria) and Czapek dox media (fungus)) once in 15 days.

Agar Well Diffusion Method: The antibacterial activity was performed by agar well diffusion method. The respective bacterial and fungal cultures were poured into the Muller Hinton agar plates and Czapax dox agar plates respectively, for uniform distribution of microorganisms. Using the sterile well puncture, 6 mm wide well was made on each agar plates.  0.1 ml of organic crude extracts   from  the stock were poured  into each well using a sterile micropipette ²⁵. Respective organic solvents were used as a negative control for each extract. Amoxicillin were used as standards. The plates were incubated for 24 hours at 37°C for bacteria and at 28°C for 3 days for fungus. At the end of incubation period, the zone of inhibition was measured.

Growth measurement of Fungus: Measuring the fungal growth was made according to the method described by Kane and Mullins ²⁶. The flasks of each triplicate were  filtered  off  using Whatman  filter  paper  after  the  end  of  the incubation period and the fungal mats were washed carefully and dried up to a constant dry weight at 70 - 80˚C for 24 h.

Qualitative analysis of Biochemicals in the Algal Extracts: Qualitative analysis of biochemicals in the methanolic extract of Chlorella pyrenoidosa,  Spirulina platensis and Nostoc muscorun was done.

1. Detection of Proteins and Amino Acids: The dried extract (100 mg) was dissolved in 10 mL of distilled water and filtered through Whatman no.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. To 2 mL of filtrate, few drops of Millon’s reagent were added and the observations were made for white precipitate (Millon’s test) ²⁷.
2. Detection of Carbohydrates: The extract (100 mg) is dissolved in 5 mL of water and filtered. To 2 mL of filtrate two drops of alcoholic solution of ά-naphthol were added, the mixture is shaken well and 1 mL of concentrated sulfuric acid was added slowly along the sides of the test tube and allowed to stand, then observed for the formation of violet ring (Molish’s test).
3. Detection of Phytosterols: The extract (50 mg) was dissolved in 2 mL of acetic anhydride. To this 1 or 2 drops of concentrated sulfuric acid was added slowly along the sides of the tube and observed for an array of colour ²⁸.
4. Detection of Phenolic Compounds: The extract (50 mg) was dissolved in 5 mL of distilled water. To this few drops of neutral ferric chloride solution was added and observed for a dark green coloration ²⁹.
5. Detection of Alkaloids: Preparation of filtrate solvent free extract (50 mg) is stirred with 2 mL of dilute hydrochloric acid and filtered. To 1 mL of filtrate, a drop of Mayer’s reagent was added by the side of tube and then observed for a white creamy precipitate ³⁰.
6. Detection of Flavonoids: To 5 mL of dilute ammonia solution a portion of the aqueous filtrate of each algal extract followed by addition of concentrated sulfuric acid. Then was observed for a yellow coloration. The yellow coloration disappears on standing ^{31, 32}.
7. Detection for Saponins: Two grams of the powdered sample was boiled in 20 mL of distilled water in a water bath and filtered. 10 mL of filtrate was mixed with 5 mL of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion ³³.
8. Test Glycosides: In the Keller-Killani test, 5 mL of each extract was treated with 2 mL of glacial acetic acid containing 1 drop of ferric chloride solution. This was underplayed with 1 mL of concentrated sulfuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring while in the acetic acid layer a greenish   ring   may   form just gradually throughout thin layer ³⁴.

Antioxidant Activity:

In vitro Nitric Oxide Radical (NO) Scavenging Assay: Nitric oxide radical scavenging activity was estimated on the basis of Griess Illosvey reaction using method followed by Govindarajan et al., ³⁵. Reaction mixture (3ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5ml, pH 7.4) and 0.5 ml of AVP (20- 120µg/mg) or standard ascorbic acid solution (0.5ml, 20-120µg/ml) was incubated at 25°C for 150 min.

After incubation, 0.5 ml of the reaction mixture mixed with 1 ml of sulfanilic acid reagent (0.33% in 29% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride (0.1% w/v) was added, mixed and allowed to stand for 30 min at 25°C. A pink coloured chromophore formed in diffused light was measured at 540 nm against the corresponding blank solutions.

RESULTS: In  the  present  study , antimicrobial  activity  of  algal  species  was  tested  against  the human  pathogenic  bacteria  and  plant  pathogenic  fungus  by  agar  well  diffusion method.  Single  concentration  was  used  for  the  study  which  was  0.1 g/ml.  The inhibition zones formed by the extracts at different concentrations against the specific test organisms were measured. The extract restricted the growth of pathogen on the media around the well.

Antimicrobial activity of Chlorella pyrenidosa extract: To check  the antimicrobial activity of C. pyrenoidosa  four different  solvents extracts  (methanol,  acetone, n-hexane  and water) were used  against  six different human  pathogenic  bacteria  and  three  different  plant  pathogenic  fungi.  It was observed that methanolic extract of C. pyrenoidosa was most effective against P. aeruginosa, with maximum zone of inhibition of (2.5 cm), while the minimum size of inhibition zone (0.2cm) was found in case of S. aureus. B. subtilis and, K. pneumoniae were insensitive.  The fungi A. niger and F. oxysporum were also found to be insensitive against methanolic extract of C. pyrenoidosa (Table 1). A .luchuensis showed zone of inhibition with a diameter of (1.8 cm).

In case of acetone extract zone of inhibition against B. subtilis was (1.5 cm) wide, while it was 1.0 cm in case of B. cereus and M. luteus.  P. aeruginosa showed complete resistance against acetone extract of C. pyrenoidosa.  In case of antifungal activity of  acetone  extract  of  C. pyrenoidosa,  the  A. luchuensis  showed maximum sensitivity  with  (2.5  cm)  wide  zone  of  inhibition.  A. niger was resistant against methanolic extract of C. pyrenoidosa.

Hexane extract was not very effective against selected pathogenic bacteria and fungi. Only  B. subtilis  among  the  tested  bacteria  showed  2.5  cm wide  zone  of inhibition, followed by K. pneumoniae.  Aqueous extract of C. pyrenoidosa was not effective against any selected pathogenic microbes (Table 1).

TABLE 1: ANTIMICROBIAL ACTIVITY AROUND THE WELLS OF DIFFERENT ORGANIC EXTRACTS OF C. PYRENOIDOSA. INHIBITION ZONE IN DIAMETER (CM). (DATA PRESENTED ARE THE MEAN OF TRIPLICATES ±STANDARD DEVIATION (SD))

 Antimicrobial activity of Spirulina platensis extract: Antimicrobial activity of extract of S. platensis against pathogenic bacteria and fungi revealed that S. aureus  was  relatively  more  sensitive  (1.5  cm  zone  of  inhibition)  to methanolic extract. While B.  subtilis, B. cereus, and M. luteus showed moderate sensitivity (1.0 cm for each) K. pneumoniae was found to be resistant against methanolic extract of S. platensis.

Among three fungi, only A. niger was  the most  sensitive, which  showed inhibition  zone  of  1.5  cm,  while  A. luchuensis and  F.  oxysporum were resistant against the methanolic extract.

In case of acetone extract of S. platensis;  B. subtilis,  K. pneumoniae  were found to be resistant, while P. aeruginosa showed the maximum zone of inhibition of 1.3 cm, followed by B. cereus and M. luteus exhibiting 1.2 cm wide zone ((Table 2).

In case of antifungal activity, only A. niger exhibited maximum sensitivity to acetone extract with zone of inhibition of 2 cm diameter, while A. niger and F. oxysporum were found to be resistant against acetone extract of S. platensis.  In case of hexane extract, only B. subtilis showed sensitivity with the zone of inhibition of 1 cm diameter. The other tested bacteria and fungi were found to be insensitive towards the aqueous extract (data not shown).

TABLE 2: ANTIMICROBIAL ACTIVITY AROUND THE WELLS OF DIFFERENT ORGANIC EXTRACTS OF S. PLATENSIS. Inhibition zone is in diameter (cm). (Data presented are the mean of triplicates ±standard deviation (sd))

 Antimicrobial activity of Nostoc muscorum (ATCC 2789): Antimicrobial activity of N. muscorum showed that among selected bacteria P. aeruginosa showed maximum zone of inhibition i.e. (2.0 cm), whereas B. cereus, M. luteus and S.aureus showed 0.8 cm wide zone of inhibition (0.8 cm). On the other hand, B. subtilis and K. pneumonia were found to  be  resistant  to  methanolic  extract  of  N. muscorum.  In antifungal assay, A. niger and A. luchuensis were resistant, while F. oxysporum showed the zone of inhibition of 1.2 cm in diameter. In case of acetone extract, P. aeruginosa showed maximum zone of inhibition with 1.8 cm diameter, followed by S. aureus and K. pneumoniae (1.6 and 1.0 cm, respectively). The bacterial strains B. subtilis and B. cereus were found to be resistant against acetone extract of N. muscorum.  In anti-fungal activity,  A.  niger showed  the  maximum  inhibition  zone  of  2.0  cm,  while  other  two  fungi  A. luchuensis and F. oxysporum showed resistance  against  the  acetone  extract. Extract with n-hexane  was  not  very  effective  in  inhibiting  the  growth  of  the  pathogens.  Only B. subtilis showed samall zone of inhibition of 1.4 cm diameter. In case of aqueous extract of N. muscorum, all the pathogenic microbes showed resistance (Table 3).

TABLE 3: ANTIMICROBIAL ACTIVITY AROUND THE WELLS OF DIFFERENT ORGANIC EXTRACTS OF N. MUSCORUM. Inhibition zone is in diameter (cm). (Data presented are the mean of triplicates ±standard deviation (SD))

 Antimicrobial Index of Algal Extracts: The antimicrobial efficacy of different algal extracts was calculated in terms of antimicrobial index by comparing the antimicrobial effect of all extracts with the effect known standard antibiotic amoxicillin;

Antimicrobial Index=

Inhibition zone of sample    X 100

Inhibition zone of standard

We found that methanolic extract of C. pyrenoidosa showed (Fig. 1) maximum percentage of inhibition (66.6%) against B. cereus, followed by S. platensis (55.5%) and N. muscorum (44.4%). The second most sensitive bacterial strain was S. aureus which showed maximum sensitivity to methanolic extract of S. platensis (35%), followed by C. pyrenoidosa (20%).  Whereas bacterial strains B. subtilis, P. aeruginosa and K. pneumoniae showed no sensitivity against methanolic extract of all the three algal strains selected for antimicrobial assay. From  the  foregoing  results,  it  was  deduced that gram-positive  bacteria  were  more sensitive  to methanolic extract of algal species than  gram-negative  bacteria.  B. cereus showed maximum percentage of inhibition, followed by S. aureus, M. luteus.

FIG. 1:  ANTIMICROBIAL INDEX OF THE METHANOLIC EXTRACTS OF ALGAE AGAINST SELECTED PATHOGENS. (DATA PRESENTED ARE THE MEAN OF TRIPLICATES WITH STANDARD ERROR (5%))

Antimicrobial index of acetone extract (Fig. 2) of all selected algae revealed that B. cereus was the most sensitive strain.  K. pneumoniae was more sensitive to extract of N. muscorum only (50% inhibition).  By observing the antimicrobial index we found that B. subtilis, P. aeruginosa were relatively more resistant against all the algal extract.

Based on the foregoing results, it may be concluded that the antibacterial efficiency of methanolic and acetone extracts was higher than the n-Hexane extract, particularly for gram-positive bacteria.

FIG. 2:  ANTIMICROBIAL INDEX OF THE ACETONE EXTRACTS OF ALGAE AGAINST SELECTED PATHOGENS. (DATA PRESENTED ARE THE MEAN OF TRIPLICATES WITH STANDARD ERROR (5%))

Effect of Algal Extract on Fungal Growth: To see the antifungal activity of algal extracts, the dry weight of fungal mat and protein content of the broth was determined. The methanolic extract of C. pyrenoidosa, S. platensis and N. muscorum was used to show the antifungal potential of the selected algae.

In case of Fusarium, the methanolic extract of Spirulina platensis showed the maximum reduction (57.44%) in the dry wt., followed by Nostoc muscorum (16.06%) and Chlorella pyrenoidosa (1.77%). The order of effectiveness was S. platensis (57.4%) > N. muscorum (16.06%) > C. pyrenoidosa (1.77%). In case of A. niger, the maximum growth inhibition was caused by the extract of Spirulina platensis (49.7%), followed by Nostoc muscorum and Chlorella pyrenoidos (Fig. 3).

31. luchuensis was most sensitive to Spirulina platensis extract (31.49%), followed by Chlorella pyrenoidosa (3.53%) and Nostoc muscorum (13.12%). On comparison of the antifungal results of different algal extracts, it was found that Spirulina platensis extract was more effective than the Nostoc muscorum and Chlorella pyrenoidosa extract.

FIG. 3: PERCENTAGE INHIBITION OF FUNGUS GROWTH IN PRESENCE OF METHANOLIC EXTRACT OF DIFFERENT ALGAE (DATA PRESENTED ARE THE MEAN OF TRIPLICATES WITH STANDARD ERROR (5%))

FIG. 4: PERCENTAGE INHIBITION IN PROTEIN CONTENT IN PRESENCE OF METHANOLIC EXTRACT OF DIFFERENT ALGAE (DATA PRESENTED ARE THE MEAN OF TRIPLICATES WITH STANDARD ERROR (5%))

Antioxidant activity: Antioxidant activity was determined, by free radical scavenging (Nitric oxide radical scavenging activity) in the methanolic extract C. pyrenoidosa, S. platensis and N. muscorum. It can be said that high percentage of NO scavenging activity was in S. platensis (40%) than the C. pyrenoidsa (18.85%) and N. muscorum (23.58%) (Figure 5). Qualitative analysis was done to check the presence of various biochemical constituents present in the algal extract obtained with different organic solvenst (methanol, acetone, hexane,). In case of methanolic and acetone extract, C. pyrenoidosa, S. platensis and N. muscorum showed the presence of glycosides, phenolic compounds, amino acid and protein.

FIG. 5: NITRIC OXIDE SCAVENGING ACTIVITY IN METHANOLIC EXTRACT OF ALGAE (DATA PRESENTED ARE THE MEAN OF TRIPLICATES WITH STANDARD ERROR (5%))

TABLE 4: QUALITATIVE ANALYSIS OF BIOCHEMICALS IN THE VARIOUS EXTRACTS OF S. PLATENSIS, C. PYRENOIDOSA AND N. MUSCORUM

+ Present, - Absent.

Phenolic compounds may affect growth and metabolism of bacteria. They could have an activating or inhibitory effect on microbial growth according to their constitution and concentration.

In case of hexane extract glycosides is absent only in N. muscorum, Saponins and flavonoids were absent in all the extracts. Glycosides, protein, amino acids, phenolic compounds were present in all the extract (Table 4).

Alkaloids are commonly found to have antimicrobial properties ³⁶ against both Gram-positive and Gram-negative bacteria ³⁷. Presence of alkaloids in all the extracts might be exerting a remarkable antibacterial activity against Gram-positive (S. aureus) and Gram-negative (K. pneumoniae) bacteria.

DISCUSSION: Marine organisms are a rich source of structurally novel and biologically active metabolites. Many chemically unique compounds of marine origin with various biological activities have been isolated to develop new pharmaceuticals ³⁸. The cell extracts and active constituents of various algae shown to have both antibacterial and antifungal activities ^{39, 40}. The extracts of C. pyrenidosa, S. platensis and N. muscorum possessed noticeable better activity against Gram positive and Gram negative bacteria when compared with amoxicillin.

The antimicrobial index for methanolic extract of C. pyrenoidosa showed maximum percentage of inhibition (66.6%) against B. cereus, followed by S. platensis (55.5%) and N. muscorum (44.4%). However, effect of acetone extract of all selected algae revealed that B. cereus was the most sensitive strain.

41. pneumoniae was relatively more sensitive to extract of N. muscorum only (50% inhibition). On comparison of the results on antifungal activity of different algal extracts showed that S. platensis extract was more effective than that of N. muscorum and C. pyrenoidosa extract. The n-hexane extract and aqueous extracts were least effective against microorganisms. The present results indicated that gram-positive bacteria are more sensitive to methanolic extract of algal species than gram-negative bacteria. Earlier many workers demonstrated that crude extracts of Indian seaweeds are more active against Gram-positive bacteria ⁴¹.

Earlier the terpenoids, quinones and phenols have been identified as biologically active compounds against the microorganisms ⁴². Earlier Ely et al., ³⁹ showed that the methanolic extract of Cladophora prolifera was very effective antimicrobial agent with moderate bactericidal activity against S.  aureus and  Vibrio  cholerea. Freile-Pelegrin and Morales ⁴³ found that the ethanolic extract obtained from different regions of Caulerpa spp thallus (apical, basal and stolon) exhibit antibacterial activity. However, extract obtained from stolon region exhibited the highest bactericidal activity. A logical reason for variation in the antibacterial activity of different algal extracts could be due to specific distribution of antimicrobial substances as suggested by Lustigman and Brown ⁴⁴. A higher antimicrobial activity in the methanolic extract algal strains than the acetone extract may be due to abundance of some lipophilic, but polar compounds.

Previous investigations also reported  that  the compounds  such as  1-Octadecene,  1-Heptadeceane  present  in  both algae  and  higher plants are responsible for their anticancer, antioxidant and antimicrobial activities ^{45, 46}. It has been suggested that the lipids and fatty acids present in the algal strains could also be responsible for antimicrobial property ^{47, 48}. Fatty acids isolated from microalgae have been known to exhibit antibacterial activity ⁴⁹.

Nitric oxide radical scavenging activities in the different algal extracts have been used as an indicator of antioxidant property of different algal extracts ⁵⁰. The present results revealed high percentage of NO scavenging activity in S. platensis (40%), followed by C. pyrenoidsa (18.85%).

The antioxidant potential in the extract of Spirulina and Nostoc strains might be due to the total phycocyanin, triterpenoids and carotenoids present in the algal extracts.  

The antioxidant activity of Spirulina has been very well documented by Abd El-Baky et al., ⁵¹; Khan et al., ⁵²;  Athukorela et al., ⁵³. Other macroalgae also synthesize antioxidant molecules such as ascorbate, glutathione (GSH) as well as many more stable molecules including carotenoids, mycosporine-like amino acids, catechins, phlorotannins ^{54, 55, 56}. Cellular presence of phenolic compounds has also been coupled with both antioxidant and antibacterial activities ⁵⁵.

In view of foregoing results, it is concluded that algal extracts have potential to work as antimicrobial agents, which may be exploited either by application of individual extracts or in combination with chemical antibiotics. The antioxidative attributes of algal extracts would always contribute positively in pharmaceutical of the algal extracts.

REFERENCES:

1. Metting FB: Biodiversity and application of microalgae. Journal of Industrial Microbiology & Biotechnology 1996; 17(5-6): 477-489.
2. Chisti Y: Biodiesel from microalgae. Biotechnology Advances 2007; 25(3): 294-306.
3. Carlucci MJ, Scolaro LA, Damonte EB: Inhibitory action of natural carrageenans on Herpes simplex virus infection of mouse astrocytes. Chemotherapy 1999; 45 (6): 429-36.
4. Siddhanta  AK,  Mody KH,  Ramavat BK,  Chauhan VD,  Garg HS,  Goel  AK,  Doss MJ,  Srivastava MN, Patnaik G,  Kamboj KVP : Bioactivity of marine organisms: Part VIII -Screening of some marine flora of western coast of India. Indian J Exp Biol 1997; 36: 638-643.
5. Caccamese  S,  Azzolina R,  Funari G,  Cormaci  M,  Grasso S:  Antimicrobial  and  antiviral  activities  of  extracts  from Mediterranean  algae. Bot Mar 1980; 23: 285-288.
6. Gonzalez  del  Val  A,  Platas  G,  Basilio  A:    Screening  of antimicrobial  activities  in  red,  green  and  brown  macroalgae  from  Gran Canaria (Canary Islands, Spain). Int Microbiol 2001; 4: 35-40.
7. Knubel G, Larsen LK, Moore RE:  Cytotoxic antiviral indolocarbobazoles from a blue-green alga belonging to Nostocales. J Antibiot 1990; 43: 1236-1239.
8. Mule  MCZ,  Caire  GZ,  Cano  MS,  Halperin  DR: Bioactive compounds  from  Nostoc  musorum  (cyanobacteria).  Cytobios 1991; 66: 169-172.
9. Gerwork  WH,  Roberts  MA,  Proteau  PJ,  Chen  JL: Screening  cultured  marine  microalgae  for  anticancer  type  activity.  J  Appl Phycol 1994; 6:143-149.
10. Jaki B, Orjala J, Sticher O: A novel extracellular diterpenoid with antibacterial activity from the cyanobacteria Nostoc commume. J Nat Prod 1999; 62: 502-503.
11. Halliwell B, Gutteridge JMC: Free Radicals in Biology and Medicine. 2^nd edn. Clarendon Press, Oxford 1989; 299-357.
12. Kok FJ, Van Poppel G, Melse J, Merheul E, Schouten EG, Kruyssen EH, Hofman A: Antioxidant and polyunsaturated acids have a combined association with coronary atherosclerosis. Atherosclerosis 1990; 86: 85-90.
13. Gonzalez  del  Val  A,  Platas  G,  Basilio  A:  Screening  of antimicrobial  activities  in  red,  green  and  brown  macroalgae  from  Gran Canaria (Canary Islands, Spain). Int Microbiol 2001; 4: 35-40.
14. Smit AJ: Medicinal and pharmaceutical uses of seaweed natural products:  A review. J Appl Phycol 2004; 16: 245-262.
15. Kramer  JM,  Turnbull PCB,  Munshi G, Gilbert RJ: Identification  and characterization  of  Bacillus cereus  and other  Bacillus  species  associated  with  foods  and  food  poisoning.  In:  Corry JEL et al.  (eds)  Isolation and identification methods for food poisoning organisms. Academic Press, London 1982; 261-286.
16. Boyd RC: Basic Medical Microbiology. Little Brown Company, Boston. Fifth edition 1995.
17. Leven MM: Escherichia coli that causes diarrhea: Enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent. J Infect Dis 1987; 155: 41-47.
18. Jawetz E,  Mellnick  JL,  Adelberg  EA:  Review  of  Medical Microbiology,  20th Edition. Applellation Lange Norwalk, Connecticut. 1995; 139-218.
19. Eddy DM: Probabilistic reasoning in clinical medicine: problems and opportunities. In: Kahneman D, Slovic P, Tverksy A, eds. Judgement under uncertainty: heuristics and biases. New York: Cambridge University Press 1982; 249–67.
20. Walsh C: Where will new antibiotics come from. Nature Reviews Microbiology 2003; 1:65-70.
21. Sieradzki K, Robert RB, Haber SW, Tomasz A: The development of vanomycin resistance in patient with methicillin resistant S. aureus. N Engl J Med. 1999; 340: 517-523.
22. Zarrouk C: Contribution altitude cyanophyceae. Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthe ` se de Spirulina maxima. Ph. D. Thesis, Universite ´ de Paris. 1966.
23. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G:  Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev.1971; 35: 171-205.
24. Bhakuni DS, Dhawan BN, Garg HS, Goel AK, Mehrotra BN, Srimal RC, Srivastava M N : Bioactivity of marine organisms Part VI Screening of some marine flora from Indian Coasts, Indian J Exptl Biol. 1992; 30: 512-517.
25. Srinivasan KN, Devarajan C, Mohanasundari V, Chinthambi Nandakumar N: Antibacterial, preliminary phytochemical and pharmocognostical screening on the leaves of Vicoa indica (L) DC. Iran J Pharmacol therepeutics. 2007; 6: 109-113.
26. Kane  BE,  Mullins  JT: Thermophilic  fungi  in  municipal  waste compost system. Mycologia. 1973; 65(5): 1087-1100.
27. Walsh C: Where will new antibiotics come from.  Nature Reviews Microbiology. 2003; 1:65-70.
28. Finar G: Plants of economic importance. Medicinal Plants and Medicine in Africa. Spectrum Books Ltd. Ibadan. 1986; 78: 150-153.
29. Mace ME: Histochemial localization of phenols in healthy and diseased banana roots.  Physiol Plant. 1963; 16:915-925.
30. Evans WC: Treaus and Evans Pharmacognosy. Fourth eel. W. L3. Saunders

Company limited, V. P. India, 1997; 340-408.

1. Harborne JB: Phytochemical methods. Chapman and Hall Ltd., London, 1973; 49-188.
2. Sofowora A: Medicinal plants and Traditional medicine in Africa.  Spectrum Books Ltd, Ibadan, Nigeria. 1993; 289.
3. Kokate A: Phytochemical Methods. Phytotherapy, 2^nd Edn. 1999; 78: 126-129.
4. Evans WC:  Trease and Evans Pharmacognosy, 15th Ed., W.B. Sanders, London, 2002; 183-184 and 191-393.
5. Govidarajan R, Rastogi S, Vijayakumar M, Shirwaikar A et al.: Studies on the Antioxidant Activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin. 2003; 26, 1424.
6. Omulokoli E, Khan B, Chhabra S: Antiplasmodial activity of four Kenyan medicinal plants. J Ethnopharmacol. 1997; 56(2):133–137.
7. Cowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12:564-582.
8. Freitas RA: The future of nanofabrication and molecular scale devices in nanomedicine. Stud. Health Technol Inform. 2002; 80: 45–59.
9. Ely  R,  Supriya  T,  Naik  CG:  Antimicrobial  activity  of  marine organisms collected off  the coastof South East  India. J Exp Marine Biol Ecol. 2004; 309(1): 121-127.
10. Schwartz RE, Hirsch CF, Sesin DF, Flor JE, Chartrain M, Fromtling RE et al.: Pharmaceuticals from cultured algae. J Ind Microbiol. 1990; 5:113–23.
11. Rao P, Parekh KS: Antibacterial activity of Indian seaweeds. Phykos. 1981; 23: 216-221.
12. Vairappan CS, Daitoh M, Suzuki M, Abe T, Masuda M: Antibacterial halogenated metabolites from the Malaysian Laurenciaspecies. Phytochemistry. 2001; 58: 291-297.
13. Freile-Pelegrin Y, Morales JL:  Antibacterial activity in marine algae from the coast of Yucatan, Mexico. Bot Mar. 2004; 47: 140-146.
14. Lustigman B, Brown C:  Antibiotic production by marine algae isolated from the New York/New Jersey coast. Bull. Environ. Contam.  Toxicol. 1991; 46: 329-335.
15. Lee  SH,  Chang  KS,  Su MS,  Huang  YS,  Jang  HD:  Effects  of some Chinese medicinal  plant  extracts  on  five  different  fungi. Food Control. 2007; 18: 1547-1554.
16. Mishra PM, Sree A: Antibacterial Activity and GCMS Analysis of the Extract of Leaves of Finlaysonia obovata (A Mangrove Plant). Asi J Pl Sci. 2007; 6: 168-172.
17. Demule MCZ, Decaire GZ, Decano MS: Bioactive substances from Spirulina platensis. Int J Exp Biol. 1996; 58: 93-96.
18. Lampe MF, Ballweber LM, Isaacs CE, Patton DL, Stamm WE:  Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk.  Antimicro Agents Chemo. 1998; 45: 1239-1244.
19. Kellam SJ, Walker JM: Results of a large scale screening programme to detect antifungal activity from marine and freshwater microalgae in laboratory culture. J Br Phycol. 1989; 23: 45-47.
20. Rojas A, Hernandez L, Pereda MR, Mata R: Screening for antimicrobial activity of crude drug extracts and pure natural products from Mexican medicinal plants. J Ethnopharmacol. 1992; 35: 275-283.
21. Abd El-Baky HH, El Baz FK, El-Baroty GS:  Over-production of Lipid Rich  in  y-Linolenic Acid  by  Blue Green Alga Spirulina maxima and  its  Inhibitory  Effect  on Carcinoma Cells. Ad Food Sci. 2006; 4: 206-212.
22. Khan  M,  Shobha  CJ,  Rao  UM,  Sundaram  CM,  Singh  S,  Mohan  J.I, Kuppusamy  P  and  Kutala  KV: Protective  effect  of  Spirulina against doxorubicin-induced cardiotoxicity. Phytother. Res. 2005; 19, 1030–1037.
23. Athukorala Y, Nam K, Jeon Y:  Antiproliferative  and  antioxidant properties  of  an  enzymatic  hydrolysate  from  brown  alga,  Ecklonia cava. Food Chem. Toxicol. 2006; 44: 1065-1074.
24. Zubia M, Payri C, Deslandes E: Alginate, mannitol, phenolic compounds and biological activities of two range-extending brown algae, Sargassum mangarevense and Turbinaria ornata (Phaeophyta: Fucales), from Tahiti (French Polynesia). J. Appl. Phycol. 2008; 20, 1033-1043.
25. Yao YQ, Ding X, Jia YC, Huang CX, Wang YZ, Xu YH: Anti-tumor effect of β-elemene in glioblastoma cells depends on p38 MAPK activation. Cancer Lett. 2008; 264, 127-134.
26. Liang H, He J, Ma AG, Zhang PH, Bi SL, Shi D: Effect of ethanol extract of alga Laurencia supplementation on DNA oxidation and alkylation damage in mice. Asia Pac J Clin Nutr. 2007; 16(1), 164-168.How to cite this article: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. Int J Pharm Sci Res. 3(10); 4866-4875.
    

    ---