DOI: https://doi.org/10.21203/rs.3.rs-1272821/v1
Cyanobacteria contain a rich source of several bioprospective molecules with antibacterial, antifungal, antioxidant, and anticancer properties. The objective of the current study was determine the antioxidant, antimicrobial and anticancer potential of methanol and acetone extracts of two cyanobacterial species (Lyngbya majuscule and Lyngbya martensiana) isolated from Odisha coast. The free radical scavenging assay of the extracts was performed against DPPH and ABTS+. The reducing power assay of the extracts was also determined. The maximum DPPH and ABTS radical scavenging activity is recorded on L. majuscule and the IC50 values were 251.34±0.96 and 282.24±0.87 µg/ml respectively. Similarly, the reducing power assay was also highest in L. majuscule (0.762±0.0015). The total phenolic (69.18±0.79 µg gallic acid equivalents g−1) and total flavonoid (38.21±0.61 µg quersentin equivalents g−1) was found more in methanolic extract. The methanol extract of L. majuscule exhibited higher antimicrobial activity (23±0.74 mM) as compared to other test species. Further, methanol extract of L. majuscule, active fractions was analysed by gas chromatography-mass spectrometry (GC-MS), and bioactive compounds like 1-Monolinoleoylglycerol trimethylsilyl ether (31.182%) and n-Hexadecanoic acid(15.42%)were detected. Similarly the cytotoxicity results exhibited decreasing cell viability of HepG2 cells in methanol extracts L. majuscule in a dose-dependent manner without cytotoxicity on normal cells.These results concluded that extracts of studied cyanobacterial species exhibited appreciable antioxidant and antimicrobial activity and further the methanol extract of L. majuscule possess sufficient amount of bioactive compounds having antioxidant, antimicrobial and anticancer activity and thus could be a promising source for health products.
The distinct source of antibiotics is microorganisms and the major being the marine microalgae and cyanobacteria (Blunt et al. 2009).Cyanobacteria are reported as an important source of structurally and biologically active secondary bioactive metabolites (Tan 2007; Gademann and Portmann2008) and found to be an attractive sources of antiviral, antibacterial, antioxidant and anticancer molecules (Ruiz et al. 2010).At present there has been an increasing focus on cyanobacteria as a potential source for important drugs(Dvornyk and Nevo 2003).Marine cyanobacteria have been found to be one of the ample source for unique bioactive compounds (Tan 2007; Gerwick et al. 2008; Thajuddin and Subramanian 2005).Cyanobacteria present in marine system compete actively for light and nutrient resources. Cyanobacteria have produced a wide variety of antimicrobials and allelopathic secondary metabolites to achieve a competitive edge in complex and dynamic societies (Senhorinho et al. 2015; Burja et al. 2001).Cyanobacteria are versatile organisms, according to their morphological, biochemical and metabolic properties, they are considered as an attractive targets for identifying bioprospective compounds with functional utility (Falaise et al. 2016; Jena and Subudhi 2019; Barkia et al. 2019).Many Phytochemicals are present in cyanobacteria. They protect the cell constituents against destructive oxidative damage. Antioxidants present in many cyanobacteria are essential bioactive compounds that, through the defense of cells from oxidative destruction, play significant role against different diseases and anti-ageing processes. Against numerous illnesses (e.g. chronic inflammation, cancer and coronary disorders) and aging cycles, antioxidant compounds from cyanobacteria play an important role, explaining their immense market ability in medicine, food processing and the cosmetic industry. Several experiments have reported that cyanobacteria and microalgae are found to become a profound source of secondary metabolites of antimicrobials, antioxidants and anticancer activity (Mudimu et al. 2014; Iglesias et al. 2019). Thus the present study is focused with a purpose to examine the antimicrobial, antioxidant and anticancer activity of cyanobacterial species isolated across Odisha coast.
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.
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.
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.
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.
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.
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).
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.
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.
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 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%.
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.
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).
Antioxidant radical scavenging activities were evaluated and presented in (Table 1). The result of this assay was presented as IC50values which range from 251.34±0.96 to 309.11±0.92µg/ml (DPPH) and 282.24±0.87 to 353.03±0.81µg/ml (ABTS+). Among all the test species, methanolic extract of L. majuscule noted higher DPPH and ABTS+ radical scavenging activity (IC50: 251.34± 0.96 µg/ml and 282.24±0.87 µg/ml). In contrary, lower scavenging activity was noted in acetone extract of L. martensiana (IC50:339.11±0.92µg/ml and 353.03±0.81µg/ml).
| cyanobacterial species | Methanol | Acetone |
|---|---|---|
| DPPH radical scavenging activity (IC50 µg/ml) | ||
| L. majuscule | 251.34±0.96 | 306.04±0.94 |
| L. martensiana | 257.00±0.93 | 339.11±0.92 |
| ABTS +radical scavenging activity (IC50 µg/ml) | ||
| L. majuscule | 282.24±0.87 | 310.61±0.83 |
| L. martensiana | 326.69±0.84 | 353.03±0.81 |
The experimental result on reducing power assay was shown in (Fig. 1A, B). Higher absorbance value suggesting higher reducing power of the sample. Methanol extract of L. majuscule showed highest reducing power i.e., 0.762±0.0015 µg/ml. On the other hand L. martensiana exhibited minimum reducing power (0.679±0.0009 µg/ml).
The total phenolic and flavonoid content of solvent extracts (methanol and acetone) of two species of cyanobacteria was estimated in terms of Galic acid and Quercentin equivalent (µg/ml).The highest phenolic content was found in methanol (69.18±0.79 µg/ml) followed by acetone (57.14±0.75 µg/ml) extracts respectively in L. majuscule, whereas L. martensiana revealed lowest phenolic content i.e., 54.81±0.74and 48.27±0.73µg/ml in methanol and acetone extract respectively. Maximum flavonoid content was observed in methanol extract of L. majuscule (38.21±0.61 µg/ml) and minimum (22.53±0.64µg/ml) was recorded in acetone extract of L. martensiana (Table 2).
| Cyanobacterial species | Methanol | Acetone |
|---|---|---|
| TPC (µg/ ml) | ||
| L. majuscule | 69.18±0.79 | 57.14±0.75 |
| L. martensiana | 54.81±0.74 | 48.27±0.73 |
| TFC (µg/ml) | ||
| L. majuscule | 38.21±0.61 | 30.34±0.62 |
| L. martensiana | 29.39±0.66 | 22.53±0.64 |
The antimicrobial activity of methanol and acetone extracts of two marine cyanobacteria L. majuscule and L. martensiana showed differential zone of inhibition against bacterial and fungal pathogens. The zone of inhibition is clearly dependents on the type of solvent used for extract and test bacterial and fungal pathogens. The methanol extracts of L. majuscule showed higher inhibition zone (23±0.74 mM) against V. cholerae(Fig. 2A). However minimum zone of inhibition was observed in acetone extract of L. martensiana (10±0.77 mM) against E. coli(Fig. 2B). Similarly maximum antifungal activity was noted in methanol extract of L. majuscule (21±0.8 mM) (Fig. 3A), while acetone of L. Martensiana exhibits minimal zone of inhibition (10±0.72 mM) against C. albicans (Fig. 3B).
The efficacy of antimicrobial activity against the pathogens was determined by Minimum inhibitory concentration (Table 3). The MIC for the various concentrations was within the range of 62.5 to 500µg/ml against the tested bacterial/fungal pathogens. The highest activity was recorded in methanol extract of L. majuscule against V. cholerae, E. coli and C. albicans with MIC value (62.5 µg/ml) whereas least activity was shown in acetone extract of L. martensiana against all the test pathogens used. The MIC value was better against gram -ve bacteria than gram +ve bacteria.
| Pathogens | Minimum inhibitory concentration (µg/ml) | |||
|---|---|---|---|---|
| L. majuscule | L. martensiana | |||
| Methanol | Acetone | Methanol | Acetone | |
| S. aureus | 125 | 250 | 500 | 500 |
| B. subtilis | 125 | 500 | 500 | 500 |
| V. cholerae | 62.5 | 125 | 250 | 500 |
| E. coli | 62.5 | 250 | 125 | 250 |
| A. niger | 125 | 250 | 250 | 500 |
| C. albicans | 62.5 | 125 | 250 | 500 |
| P. verrucosun | 250 | 500 | 125 | 250 |
| F. oxysporum | 125 | 250 | 500 | 500 |
Data on the cytotoxic effects of L. majuscule methanol extract using liver cancer cell line In vitro are showed in figure 3&4. The results of this study showed that the methanolic extract from L. majusculecan inhibit the proliferation of liver cancer cells (HepG2). Morphological changes were noted in the treated cells included the cell shrinkage resulting the cells of various shape and size as compared to normal cells and (Fig. 4). The Cytotoxic effects of various concentrations (15.62, 31.25, 62.5, 125, 250, 500, 1000µg/ml) of L. majuscule crude extract was investigated using MTT assay (Fig. 5).The highest percentage (88.2%) of cell viability was observed with the treatment using 15.62 µg/ml while the lowest cell viability percentage was calculated as 17.6% with the treatment using 1000µg/ml.
GC-MS Chromatogram in methanol extract of L. majuscule along with molecular formula, Retention time and peak area were depicted in (Table 4). Eleven compounds were identified in methanolic extract of L. majuscule. The 1-Monolinoleoylglycerol trimethylsilyl ether with percentage composition of (31.182%) was the major compound followed by n-Hexadecanoic acid (15.42%), Heptadecane (13.3%), Phytol, acetate (12.47%), 3,7,11,15-Tetramethyl- 2-hexadecen-1-ol (10.37%), Hexadecanoic acid, ethylester (5.02%)
| Sl.No | Name of the compound | Molecular Formulae | Retention time | Peak area (%) |
|---|---|---|---|---|
| 1 | 2-Mercaptopropanoic acid | C3H6O2S | 4.3 | 0.39 |
| 2 | Benzoic acid | C7H6O2 | 5.6 | 0.78 |
| 3 | Undecanoic acid | C11H22O2 | 9.5 | 1.73 |
| 4 | 3,7,11,15-Tetramethyl- 2-hexadecen-1-ol | C20H40O | 14 | 10.37 |
| 5 | Phytol, acetate | C22H42O2 | 14 | 12.47 |
| 6 | n-Hexadecanoic acid | C16H32O2 | 16 | 15.42 |
| 7 | Hexadecanoicacid, ethylester | C18H36O2 | 16 | 5.02 |
| 8 | Heptadecane | C17H36 | 16.75 | 13.3 |
| 9 | Phytol | C20H40O | 18 | 1.58 |
| 10 | Erucic acid | C22H42O2 | 25 | 0.87 |
| 11 | 1-Monolinoleoylglycerol trimethylsilyl ether | C27H54O4Si | 29 | 31.7 |
Marine cyanobacteria are excellent producers of bioactive secondary metabolites that provide antioxidant and antimicrobial compounds (Pahlich et al. 1983).Compounds of cytotoxic, antifungal, antibacterial, antiviral and antioxidant functions have been reported in studies throughout the past decades. Previously hydrophilic and lipophilic extracts of cyanobacterial strains isolated from fresh, brackish, and marine water were tested for antibiotic efficacy against microbes (Thacker et al. 1997). In current study, four marine cyanobacteria isolated from the coastal region of Odisha were tested against various human pathogens. The findings imply that selected cyanobacterial species are potential source for antimicrobial and antioxidant compounds. Hossain et al. (2016) exhibited the antioxidant properties of four cyanobacterial species such as (Spirulina sp.; Oscillatoria sp.; Lyngbya sp and Microcystis sp.) of which the higher DPPH radical scavenging activity was expressed in Oscillatoria sp.and Lyngbyasp. which is inclined to our work. However, the DPPH assay on Lyngbya sp. was studied by Hossain et al.(2015) showed marginally lower scavenging activity which contradict to our findings as the test species was from a freshwater environment. Likewise, Babic et al. (2016) also reported that the Phormidium sp. exhibited maximum antioxidant activity followed by Nostoc sp. This differential response might be of varied strains and different habitats of their occurrence. The capacity to reduce indicates that the antioxidant components are electron donors, and that the lipid peroxidation mechanism will reduce the oxidised intermediates, allowing them to function as primary and secondary antioxidants (Kokilam and Vasuki 2014).Overall, for the extraction of antioxidant compounds from cyanobacteria, methanol was the most effective solvent, this might be because methanol has a greater dielectric constant than acetone. The present study revealed that methanol extracts of Lyngbya majuscule marked high contents of both total phenols and total flavonoids which agree to the results reported by Sahin (2019); Patil and Kaliwal (2019) which states that higher total phenolic contents in Scenedesmus sp. Several workers have concluded that cyanobacterial phenolic compounds (along with flavonoids) substantially refer as their antioxidant potential (Hajimahmoodi et al. 2010; Shetty and Sibi 2015; Morowvat et al. 2019).These compounds show a broad variety of biological activities in addition to their antioxidant function, explaining their commercial applications in food and pharmaceutical industries (Machu et al. 2015; Ali and Doumandji 2017; Galasso et al. 2019).All the cyanobacteria extracts inhibited the test pathogens and were more potent against Gram-negative bacteria, for which is consistent with the findings ofCaires et al. (2018) and Pramanik et al. (2011). The test species Lyngbya majuscule expressed the maximum inhibitory activity against both bacteria and fungi. Similar observation was reported by Sundaramanickam et al. (2015), that Lyngbya sp. has a significant inhibitory impact on Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).Similarly, Sethubathi and Prabu (2010) demonstrated that solvent extracts of Oscillatoria sp., Phormidium sp., and Lyngbya majuscule have antimicrobial activity against human pathogens such as S. mutants, S. aureus, P. aeruginosa, B. subtilis and K. pneumonia which supplement to our result. Further, MIC assay result was found to be coherence to the results of earlier author showing lowest MIC against V. cholerae and E. coli among the test cyanobacterial extracts. The antibacterial activity of the cyanobacterial extracts was found to be more promising than the antifungal activity in the current investigation, which is consistent with the findings of Martins et al. (2008). GC-MS assessment of present study revealed higher Hexadecanoic acid in L.majuscule, similar to the Plaza 2010 results, which showed superior antimicrobial activity in the presence of different fatty acids and volatile compounds such as fucosterol, phytol, neophytadiene, palmitic, palmitoleic and oleic acids in Synechocystis sp. Similarly Geetha (2010) investigated the presence of diisooctyl ester, 1,2-Benzenedicarboxylic acid, 9, 12,15-Octadecatrienoic acid, Octadecanoic acid, n-Hexadecanoic acid, 9,12-ctadecadienoic acid, n-Hexanoic acid, Decanoic acid, Oleic acid, and other compounds in chlorella extracts. Inoue et al. 2005 demonstrated that the growth of Staphylococcus aureusis inhibited by phytol. The anticancer activity as determined in L.majuscule using HepG2 cells was encouraging depicting lower cell viability at higher concentration of extract which was supported the work of Yusof et al. 2010, reported anticancer screening of the microalga Chlorella vulgaris against cancerous cell lines HepG2 and found to prevent cell proliferation and to promote apoptosis.
It can be summarized that two extracts (methanol and acetone) from two test species of cyanobacteria demonstrated various levels of antioxidant and antimicrobial activity. We also observed that the phenolic content of cyanobacteria plays a significant role in antioxidants and antimicrobial activities. Eleven compounds with various biological activities were proposed in the GC/MS study to extend our understanding of this species as a producer of biologically active substances. Our analysis indicates that the methanol extract of Lyngbya majuscule has substantial biological activity (antioxidant and antimicrobial), rendering this species an important topic for further studies in the food, nutraceutical and pharmaceutical sectors.Further, we found that Lyngbya majusculeextract, affect cell viability by inducing apoptosis in Human liver cancer cell lines in vitro indicating it as an anti-cancer agent. This study suggests that further work will be helpful in pharmaceutical sciences for determining cyanobacteria as potential source for therapeutic purposes.
DPPH- 1,1-diphenyl-2-picryl-hydrazil, ABTS-2, 2-azinobis-(3-ethylbenzothiazoline-6)- sulfonic acid, PUFA- Poly unsaturated fatty acid, ASN III- Artificial sea nutrient, Abs- Absorbance, FRAP -Ferric reducing antioxidant power assay, BHT- Butylated hydroxyl toluene, TPC - Total phenolic content, GAE- Gallic acid equivalent, TFC- Total flavonoid contents, QE- Quercentin equivalents, MTCC- Microbial type culture collection and gene bank, MHA-Mueller-hinton agar, PDA- Potato dextrose agar, CFU- Colony forming unit, DMSO- Dimethyl sulfoxide, MIC- Minimum inhibitory concentration, MHB-Mueller hinton broth, GC-MS- Gas Chromatography-Mass Spectrometry, SD- Standard deviation, ANOVA- Analysis of variance, P. molle- Phormidium molle , P. angustissium – Phormidium angustissium, L. majuscule-Lyngbya majuscule, L. martensiana- Lyngbya martensiana, S. aureus - Staphylococcus aureus, B. subtilis -Bacillus subtilis, V. cholerae - Vibrio cholerae, E. coli- Eschercichia coli, C. albicans-Candida albicans , A. niger-Aspergillus niger, P.verrucosun- Penicillium verrucosun, F. oxysporum- Fusarium Oxysporum
Acknowledgments
The authors would like to convey their heartfelt thanks to Head, Department of Biotechnology, MSCBD University, for facilitating laboratory for smooth conduct of experiments.
Financial support
No financial support received for the work
Conflict of Interest
The authors declare that there is no conflict of interest.