Collection, isolation and culture conditions
Two species of Cyanobacteria (Lyngbya majuscule and Lyngbya martensiana) were collected from coastal regions of Odisha. The samples were cleaned to remove the necrotic parts and cultured in ASN III medium. The unialgal cultures were maintained under fluorescent light at an intensity of 2000-3000 lux with a photoperiod of 16hrs light and 8hrs dark at a temperature of 25 ± 10C.
Extracts preparation
The dried samples were grounded using mortar and pestle to a uniform powder. Two gram dried powder sample was dissolved in 10ml of an appropriate solvent (methanol and acetone) at room temperature for 72 hrs and the extracts were filtered with the help of Whatman No.1 filter paper. Under reduced pressure, the filtrate was evaporated and the extracts were then investigated against antioxidant, antimicrobial and anticancer activity.
Antioxidant activities
DPPH/ ABTS + radical scavenging activity
The radical scavenging activity of cyanobacterial extracts is assayed by 1,1-diphenyl-2-picryl-hydrazil (DPPH) (Shah et al. 2013). One ml of DPPH (0.1 mM) was mixed with different concentration of the extracts (100, 250, 500, 750 and 1000 µg/ml) and stored for 30 minutes in dark. The absorbance was recorded using UV-visible spectrophotometer (Systronics-2202) at 517nm and ascorbic acid is used as standard. The spectrophotometric analysis of ABTS+ [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt] radical scavenging activity was determined according to Re et al. (1999). ABTS+ was prepared by 5ml of ABTS (14mM) solution and K2S2O8(4.9mM) solution and stored in dark for 14-16 hrs. The absorbance is maintained 0.70±0.020 by diluting the ABTS+ solution in ethanol at 734nm. The antioxidant activity was compared using ascorbic acid as a standard.
The inhibition percentage of DPPH/ABTS+ was obtained by means of the formula: [(A0-A1)/A0]×100,where, A0- control absorption, A1- sample absorption. The 50 percent DPPH/ABTS+ scavenging effect (IC50) sample concentration was estimated from the DPPH/ABTS+ scavenging effect percentage of graph against various concentration of sample. Lower the IC50 value indicated stronger free radical scavenging activity.
Ferric reducing antioxidant power assay (FRAP)
The reducing power of the cyanobacterial extracts was determined according to the method of Oyaizu (1986). Various concentration of cyanobacterial extracts (100-1000µg/ml) was dissolved in 2.5 ml of K3Fe(CN)6 (1%) and PO4 buffer (0.2 M, pH 6.6) and stand for 25 min at 50°C followed by the addition of trichloroacetic acid (10%, 2.5ml) and centrifuged. Subsequently the supernatant (2.5ml) was added with same volume of double distilled water and FeCl3 (0.1%, 0.5 ml). At 700 nm, the absorbance was measured. Butylated hydroxyl toluene was taken as standard.
Total Phenolic Content (TPC)
According to Slinkard et al. (1977) the total phenolic content in cyanobacterial extracts was measured by using Folin’s reagent. The extract (100µl) was mixed with Na2CO3 (2%, 2ml) and allowed to stand for 2 minutes. Then Folin’s reagent (500 µl) was mixed and stands for 20 min in dark. The absorbance was reordered at 650 nm. The gallic acid equivalent (µg GAE/ g extract) was used to determine the total phenolic content in the extracts.
Total flavonoid contents (TFC)
The total flavonoid content was estimated by zhishen et al.(1999). One ml of cyanobacterial extract was added to NaNO2 (5%, 500 µl) and AlCl3.H2O (10%, 300 µl). After 10 minutes, one ml of NaOH (1M) was mixed and distilled water was added up to 5ml make up and placed at room temperature for 30 minutes. The absorbance was measured at 510 nm. The total flavonoid in the extract was determined in the form of quercentin equivalents (µg QE /g extract).
Antimicrobial activity
Human Pathogens
Four bacterial pathogens (Staphylococcus aureusMTCC-96; Bacillus subtilisMTCC-441; Vibrio CholeraeMTCC-3906; Eschercichia coli MTCC-443) and four fungal pathogens (Candida albicans MTCC 183; Aspergillus niger MTCC-1344; Penicillium verrucosun MTCC-1758 and Fusarium oxysporum MTCC-284) were obtained from IMTECH Chandigarh and maintained in Department of Biotechnology, Maharaja Sriram Chandra Bhanja Deo University, Takatpur, Mayurbhanj, Odisha. These pathogens are used for the experimental purposed.
Agar well diffusion assay
Mueller Hinton and Potato dextrose agar medium was used for evaluation of antimicrobial activity by agar well diffusion method for Bacteria and fungi respectively (Abedin and Taha 2008). The test pathogens (20µl, 106 CFU/ml) were swabbed on all the plates. The extracts were dissolved in DMSO (1 mg/ml, 50 µl) and supplied into 6mm diameter wells and kept at 37ºC for 20 hours (bacteria) and 28ºC for 56 hours (fungi). The inhibited zone around the wells was measured in millimeter. Ampicillin and Clotrimazole (100g/ml) were employed as +ve controls for antibacterial and antifungal activities.
Determination of minimum inhibitory concentration (MIC)
A conventional 96-well microplate with Mueller hinton broth (MHB) was used to determine the MIC, MHB was used to developed various concentration of extracts (15.62, 31.25, 62.5, 125, 250, 500, 1000 µg/ml) using two-fold serial dilution. Fifty micro liter of pathogens (106 CFU/ml) was added. After 24hr (Bacteria) / 48hr (Fungus) of incubation microtiter plate reader (Bio-rad,iMark-11457) was to analyze the MIC. The minimum inhibition concentration was determined as the lowest concentration of the extracts that inhibit the visible growth of the test pathogens (Skov et al. 2019; Prasannabalaji et al. 2012).
Cytotoxicity assay
Cytotoxicity activity (In vitro bioassay) in human hepatocellular carcinoma cell line (HepG2) was conducted using methanol extract of Lyngbya majuscule by MTT assay (Mosmann1983).The cellular viability was evaluated by the reduction of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide(MTT). The cells were maintained in RPMI1640 medium. After 24h of adhesion, the media was supplemented with various concentrations of sample extracts (0.1% DMSO) and incubated for 48hours.After removal of the sample solution and washing with phosphate-buffered saline (pH 7.4) cells were exposed to 10 µL of MTT solution(0.5 mg /ml) for 4hours.MTT solution was removed after centrifugation at 2000 rpm for 10 min and the immiscible blue formazan crystals trapped in cells were dissolved and maintained in 100 µl of DMSO and the absorbance was measured at 550 nm in a microplate reader (Bio-Rad, model 3350), using wells without sample containing cells as blanks. All experiments were performed in triplicate and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of human Liver cancer cells was expressed as the % cell viability, using the following formula:
% cell viability = A570 of treated cells / A570 of control cells × 100%.
GC-MS Analysis
The chemical components of methanolic extracts of L. majuscule was identified by gas chromatography-mass spectrometry (GC-MS) (Swamy et al. 2017), Agilent Model 7890A-5975B [Column, DB 5ms, Agilent form (30m x 250 µm x 0.25 µm)] in the Unit of Analytical Chemistry, Triyat Scientific Co., Nagpur, Maharashtra, India. The compounds were tentatively identified by comparing their relative retention times and mass spectra to those of the NIST, WILLY library data of the GC/MS system. A relative percentage peak area was used to explore the quantification of all identified components.
Statistical analysis
For statistical purpose the experimental results were obtained in triplicate and expressed as the mean with standard deviations (±SD).The data were analyzed by one way ANOVA, and the means were compared by the least significant difference test (p=0.05).