SCREENING OF PHYTOCHEMICALS & BIOACTIVE ANTIBACTERIAL ACTIVITY IN SPIROGYRA

Aeromonashydrophila ,. The GC-MS analysis of the extracts revealed the presence of several antimicrobial bioactive constituents in the extracts.


ISSN: 2320-5407
Int. J. Adv. Res. 5 (7), 1145-1154 1146 produced by these organisms may be potential bioactive compounds of interest in pharmaceutical industry. There are a number of reports on the evaluation of antioxidant activity in microalgae and cyanobacteria belonging to the genera Botryococcus (Rao et al. 2006), Chlorella (Wu et al. 2005; Goh et al.2010), Dunaliella (Herrero et al. 2006), Nostoc (Li et al.2007), Phaeodactylum (Guzman et al. 2001), Spirulina (Miranda et al. 1998;Jaime et al. 2005; Mendiola et al.2007), Haematococcus (Cerón et al. 2007) and Chaetoceros (Goh et al. 2010). These studies concluded that several microalgal genera contain potent antioxidants, both from lipophilic and hydrophilic nature. Material and method:-Sample preparation & extraction:-Samples were collected from Arasinamakki, near Shishila. Dakshinakannada, India .Algae samples were cleaned all epiphytes and necrotic parts were removed. Samples were rinsed with sterile water to remove any associated debris. Sample was kept under sunshade for 7 days. After drying the sample, it was ground thoroughly to powder form. The powdered samples were extracted with at room temperature with different solvents. The extraction is carried out for 10 days, after that extracts were filtered, the filtrate is dried using rot evaporator and concentration is determined& subjected to analysis. (Gonzalez del valet.al, 2001.)

Phytochemical Analysis:-
The extracts were subjected to phytochemical tests for presence of following biomolecules byUsing the standard qualitative procedures as described in literature [15]. 1. Test for Glycosides: 10 ml of 50% H2SO4 was added to the 1 ml of extract in a boiling tube. The mixture was heated in boiling water bath for 5 min. 10 ml of Fehling's solution (5 ml of each solution A and B) was added and boiled. A brick red precipitate indicated the presence of glycosides. 2. Test for Alkaloids: 1 ml of 1% HCl was added to the 3 ml of extract in a test tube and was treated with few drops of Meyer's reagent. A creamy white precipitate indicated the presence of alkaloids. 3. Test for saponins: 5 ml of extract was shaken vigorously to obtain a stable persistent froth. The frothing was then mixed with 3 drops of olive oil and observed for the formation of emulsion, which indicated the presence of saponins. 4. Test for flavonoids: A few drops of 1% NH3 solution was added to the 2 ml of extract in a test tube. A yellow coloration was observed for the presence of flavonoids. 5. Test for tannins: To 0.5 ml of extract solution, 1 ml of distilled water and 1-2 drops of ferric chloride solution were added and observed for brownish green ora blue black coloration. 6. Test for terpenoids: 5 ml of extract was mixed with 2 ml of CHCl3in a test tube. 3 ml of concentrated H2SO4 was carefully added along the wall of the test tube to form a layer. An interface with a reddish brown coloration was confirmed the presence of terpenoids. 7. Test for cardiac glycosides: 5 ml of extract was mixed with 2 ml of glacial acetic acid containing 1 drop of FeCl3.Theabove mixture was carefully added to the 1ml of concentrated H2SO4. Presence of cardiacglycosides was detected by the formation of a brown ring. 8. Test for phlobatannins: 10 ml of extract was boiled with 1% HCl in a boiling tube. Deposition of a red precipitate indicated the presence of phlobatannins 9. Test for Anthraquinones: Extract was mixed well with benzene, and then half of its own volume of 10% ammonia solution was added. Presence of a pink, red or violet coloration in the ammonial phase indicated the anthraquinones. 10. Test for Phenols: 3 mL of 10% lead acetate solution were added to 5mL of plant extract. A bulky white precipitates indicated the presence of phenols. 1147

Estimation of Phenolic Content:-
The amount of total phenolics [1] in methanol extract was determined with Folin-Ciocalteu reagent according to the method of Singleton and Rossi with Gallic acid as the standard [23]. Briefly standard stock solution of 10 mg/10 ml of gallic acid was prepared in distilled water. From this, various concentrations ranging from 200-1000 μg / ml were prepared. To this 1 ml Folin and Ciacalteau reagents (1:2 with water) was added and kept at room temperature for 5 min and then 1 ml of 7% sodium carbonate solution was added to the reaction mixture and incubated at room temperature for 90 minutes. The colour developed was read at 750 nm. A 100 μl of each extract of sample was mixed with the same reagents. Gallic acid was used as the reference standard and the results are expressed as milligram gallic acid equivalent (mg / g dry weight of Spirogyra sp).
Antibacterial Activity:-Pure cultures of Aeromonas hydrophila, was used as test micro-organisms. Different solvent extracts were checked for antibacterial activity against the lawn cultures by agar well diffusion method. In each respective solvent is chosen as in the form of control.
Gas chromatography and mass spectrometry Analysis:-Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an BR-5MS(5%Diphenyl/95% Dimethyl poly siloxane)capillary column (length 30 m × diameter 0.25 mm × film thickness 0.25 μm) with helium at 1 ml for 1 min as a carrier gas. The mass spectrometer was operated in the electron impact (El) mode at 70 eV in the scan range of 50-500 m/z. The split ratio was adjusted to 1:10, and the injected volume was 2 μl. The injector temperature was 280 °C, and the oven temperature was kept at 110 °C for 3.5 min, rose to 280 °C at 5 °C min −1 (total run time 37.50 min). Peak identification of crude spirogyra extracts were performed by comparison with retention times of standards, and the mass spectra obtained were compared with those available in NIST libraries (NIST 11 -Mass Spectral Library, 2011 version) with an acceptance criterion of a match above a critical factor of 80% according to Srinivasan et al 2016

Results and Discussion:-
Phytochemical Analysis:-Important phytochemicals, such as alkaloids, triterpenoids, steroids, tannin, saponin, coumarins, terpenoids, quinine, phytosteroids, phlobatannins and flavonoids were screened for their presence and presented in Table 1.   Table 3.The agar well diffusion method was used to evaluate the antibacterial activity by measuring the zone of inhibition. Among three extracts Spirogyra ethyl acetate extract was found to be superior controlling growth of all three pathogens.

Conclusion:-
Aeromonashydrophila have the potential of causing zoonotic disease, a disease spreads from animal to human being during accidental cases. Currently antibiotic treatment is preferred to resolve disease. But, use of antibiotics has 1151 potential problem of being include inadequate dosage, overdosing, drug resistance by bacteria. Hence in the present study natural antimicrobial substance as a substitute for synthetic antibiotics is analyzed. Spirogyra extracts are prepared in different solvents with increasing order of polarity. Extracts are subjected to phytochemical tests for Glycosides,alkaloids, saponins,flavonoids,tannins,phenols,cardiac glycosides, sterols, resins etc. Extracts showing positive for phenol, tannins, flavonoids were estimated for phenolic content & tested for antimicrobial activity against pure cultures of Aeromonashydrophila. Crude extracts subjected to GC -MS analysis reported many several bioactive compound showing antimicrobial, antioxidant & anti fungal property. The cold extraction procedure adopted helped in the accountability of lipidous& hydrocarbon molecule. Such natural antimicrobial substance showing broad spectrum activity can be used to replace synthetic antibiotics more effectively, less toxicity also can development of antibiotic resistant strains can be curtailed.