Natural products from some soil cyanobacterial extracts with potent antimicrobial, antioxidant and cytotoxic activities

: The ethyl acetate, n hexane and methanol extracts of six cyanobacterial species isolated from paddy fields in Egypt were assessed for their antimicrobial activity, using disc diffusion method. Oscillatoria acuminata , Oscillatoria amphigranulata and Spirulina platensis methanolic extracts showed the highest inhibition zones. Minimum inhibitory concentration of O. amphigranulata extract recorded lower values using agar streak dilution method. O. acuminata methanolic extract exhibited the highest antioxidant activity (6.58 and 34.60 % using DPPH (2, 2- diphenyl-1- picrylhydrazyl) and ABTS + (2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) methods, respectively, followed by O. amphigranulata then S. platensis . Similarly, O. acuminata methanolic extract showed very strong cytotoxicity activity against HepG2 and HCT-116 cell lines and strong activity with MCF-7 cell lines. O. amphigranulata extract showed strong cytotoxicity for HepG2 and HCT-116 as well as moderate cytotoxicity for MCF-7 cell line. Whereas, S. platensis extract exhibited moderate cytotoxicity for all cell lines. Results of gas chromatography/mass spectroscopy analysis pointed out that the potential activity of these cyanobacterial extracts might be attributed to a synergistic effect between their pronounced contents of fatty acids, alkaloids, phytol, hydrocarbons, phenolics and phthalates, especially fatty acids. We recommend cyanobacteria as a rich source of natural products with potent pharmacological and medical applications .


INTRODUCTION
The increase in the rate of infection by antibioticsresistant microorganisms alarms for exploring of natural sources of antimicrobial compounds (Pandy 2015). The search for cyanobacteria with antimicrobial activity has gained importance in recent years due to their richness in natural metabolites with medicinal and pharmacological uses (Singh et al. 2011). The cyanobacteriaderived bioactive compounds have been reported to exhibit antibacterial (Heidari et al. 2012, Malathi et al. 2014), antifungal ( Soltani et al. 2005, Najdenski et al. 2013, antiviral activity, anticoagulant, antiinflammatory, antimalarial, antiprotozoal, antituberculosis and antitumor activities (Shanab et al. 2012, Varshney & Singh 2013, Pradhan et al. 2014, Abd El Sadek et al. 2017. Several active antimicrobial secondary metabolites were identified from cyanobacteria such as fatty acids (Gheda et al. 2013), acrylic acid, halogenated aliphatic compounds, terpenes, Sulphur containing heterocyclic compounds, carbohydrates and phenols (Plaza et al. 2010, Pandy 2015. In addition, cyanobacteria contained numerous biologically active molecules which have been known to exhibit antioxidant activity, such as polyunsatured fatty acids (PUFA), phycobiliproteins, β-carotene, pro-vitamins and phenolic compounds (Hajimahmoodi et al. 2010, Shanab et al. 2012 which may act in combination and induce antimicrobial as well as cytotoxic activities (Bharat et al. 2013). Nowadays, natural antioxidants are in greater demand than synthetic ones due to their non-carcinogenicity, high stability and better compatibility (Rajishamol et al. 2016). On the same direction, several studies have shown that extracts from Spirulina sp., Oscillatoria spp., Fischerella sp. and many other cyanobacterial species could prevent or inhibit cancer in humans and have immune-promoting effects. Natural compounds with antitumor activities against colon CT-26 and lung 3LL cell lines from different extracts of 24 cyanobacterial strains were reported (Silva-Stenico et al. 2013). Dietary supplementation of Spirulina platensis was helpful in the prevention and treatment of pancreatic cancer (Konickova et al. 2014). On their reviews, Singh et al. 2011, Vijayakumar & Menakha 2015 stated several compounds such as borophycin, cryptophycin 1, symplostatin, dolastatin, coibamide A, apratoxin A, curacin A, largazole and tolyporphin which were derived from different algal and cyanobacterial species that have valid mechanisms against variety of cancer cell lines. As reported by Ahmed et al. 2018 crude extracts of cyanobacterium Fischerella sp. BS1-EG had anti-cancer and antidiabetic activities.
Therefore, the development of natural, antimicrobial and antitumor products from cyanobacterial metabolites is a valuable trail. The wide distribution of cyanobacteria in the Egyptian environments such as in soil of the paddy fields supports this demand. In this study, six cyanobacterial species were isolated from paddy fields in Egypt. The antimicrobial and the antioxidant potentials of these cyanobacterial species were evaluated. The chemical composition of the extracts was identified by Gas Chromatography Mass Spectrometer (GC-MS) and their cytotoxicity effect against hepatocellular (HepG-2), mammary gland (MCF-7) and colorectal (HCT-116) cancer cell lines were investigated.

Isolation of cyanobacteria and culture conditions
Soil samples were collected from different regions of paddy fields in Nile Delta region, Gharbia Government, Tanta, Egypt. Samples were cultured on BG11 medium (Rippka et al. 1979). Axenic cyanobacterial cultures were identified using morphological and taxonomical approaches according to (Desikachary 1959, Prescott 1962 and further confirmed by AlgaeBase (http://www.algaebase.org). Six species were identified as Anabaena variabilis (Kutz.), Nostoc muscorum (Agardh), Nostoc linckia (Bornet), Oscillatoria acuminata (Gomont), Oscillatoria amphigranulata (Goor) and Spirulina platensis (Gomont). Working cultures were established on BG-11 medium for all species while Spirulina platensis was grown on its specific medium (Zarrouk 1966). The incubation temperature was 25 ±2 °C at 80 μEm-2 s -1 of illumination and 16hr light/8hr dark regime. The cultures were harvested at late exponential phase and the collected biomass was dried, weighted and used for further assays.

Collection of pathogenic microorganisms
Five gram +ve and gram -ve bacteria were used in this study as: Staphylococcus aureus TC-8325, Bacillus subtilis STR-168, Pseudomonas aeuroginosa AE-4091, Esherichia coli MG-1655 and Klebsiella pneumoniae HS-1286. The examined bacterial strains were identified by sequencing their 16S rRNA gene (SIGMA Scientific Service Company). The fungal strains were obtained from the Microbial Culture Collection (MCC), Faculty of Science, Assiut University, Egypt. These strains were identified as Candida albicans SC-5314 and Aspergillus flavus GCA-3357 using 18S rRNA PCR gene sequencing.

Preparation of cyanobacterial extracts
The extractions were performed by soaking the dried cyanobacterial material (0.1: 2 w/v) in (85%) methanol, n hexane or ethyl acetate solvents. The extract mixture was sonicated at 30% amplitude, interval 5 sec, pulse 5 sec for 15min. The extract was then shaken on a rotary shaker at 120 rpm at 28°C for 48 hrs; and filtered using Whatman No 4 filter paper. The solvent was evaporated under reduced pressure up to dryness and the obtained residue (crude extract) was stored at -20°C in airtight bottles until used ( Hajimahmoodi et al. 2010, Bharat et al. 2013.

Antimicrobial activity assay
The susceptibility of the tested pathogenic bacteria to various cyanobacterial extracts was assessed according to the Clinical and Laboratory Standards Institute (CLSI 2012) using the modified Kirby-Bauer disk diffusion method on Muller Hinton agar medium (Oxoid). Similarly, the anti-fungal activities were tested using Sabouraud dextrose agar medium (Oxoid). Each extract material was dissolved in DMSO solution (1 mg /ml). Previously sterilized filter paper discs soaked in the extract solutions were placed aseptically in the Petridishes containing agar media and previously seeded with the tested microorganisms (at a concentration of 10 6 cfu /ml). The Petridishes were incubated at 37 °C and the inhibition zones were recorded after 24 h and 48 h of incubation for bacteria and fungi, respectively. Each treatment was replicated three times. Ampicillin (100 µg/ml) and fluconazole (100 µg/ml) were used as common standard for antibacterial and antifungal activity while DMSO (1%) was used as a negative control using the same procedure as above. The minimum inhibitory concentration (MIC) of cyanobacterial extracts was determined by agar streak dilution method (Hawkey & Lewis 1994). A stock solution of the extracts (100 µg/ml) in DMSO was prepared and graded quantities were tested against different pathogenic microorganisms. The MIC value was considered to be the lowest concentration of test extract exhibiting no visible growth of bacteria or fungi on the plate.

Phytochemical analysis of cyanobacterial extracts
The cyanobacterial extracts were analyzed for the presence of secondary metabolites such as tannins, phenolics, flavonoids, saponins, terpenoids and sterols according to the standard phytochemical methods (Edeoga et al. 2005). Total phenolic content was estimated as Gallic acid (GA) equivalent per gram extract dry weight (Taga et al. 1984).

Antioxidant activity of cyanobacterial extracts DPPH assay
Antioxidant activities of cyanobacterial extracts were assayed by the DPPH (2, 2-diphenyl-1picrylhydrazyl) radical scavenging method (Blois 1958). Cyanobacterial samples were dissolved in methanol and the methanolic DPPH served as controls. Ascorbic acid was used as a reference and the percentage of DPPH-decolorization was calculated as: Free radical scavenging % = (Ac -As) /Ac ×100 Where: Ac = Absorbance of control and As = Absorbance of sample.

ABTS + assay
ABTS + assay was performed by modified method of (Paixão et al. 2007). ABTS + solution (3ml) was added to 3, 15 and 30 μl of each cyanobacterial methanolic extract to prepare 1, 5 and 10 ppm final concentration, respectively. The absorbance was measured at 415 nm using ascorbic acid as a positive control and ABTS + solution as negative control. Percentage of inhibition was measured as (% inhibition) according to the previous formula.

Cytotoxicity (MTT) assay of cyanobacterial extracts
The cell lines hepatocellular carcinoma (HepG-2), mammary gland (MCF-7) and colorectal carcinoma (HCT-116) were used to determine the inhibitory effects of the cyanobacterial extracts using the MTT assay (Skehan et al. 1990). This colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in the viable cancer cells. Doxorubicin was used as a standard anticancer drug for comparison (Fadda et al. 2012). The relative percentage of cell viability was calculated as: (A 570 of treated samples/A 570 of untreated sample) X 100.
Where: A is the absorbance at 570.

Gas chromatography-Mass spectrometry analysis
GC-MS analysis of the active cyanobacterial extracts was carried out as per standard procedure using Perkin Elmer: Clarus 580/560 S model system. Identification of metabolites in the extracts was recognized by comparison of retention time and fragmentation pattern with mass spectra in the NIST spectral database library software. Relative area value of each constituent (as a percentage of total volatile composition) were directly obtained from total ion current (TIC) and expressed as peak area normalization.

Statistical analysis
The results were expressed as mean±standard deviation of three replicates. Data was statistically analyzed using ANOVA (SPSS version 19) software.

RESULTS AND DISCUSSION
Antimicrobial activity of the cyanobacterial extracts Antimicrobial activity of the cyanobacterial extracts against tested bacterial and fungal species were presented in Table I (Table I). These results indicated that the antimicrobial activity of the extracts depended mainly on the type of cyanobacterial species, the used solvent and the tested pathogen. The  (Table I).
Cyanobacteria has been famed with living in diverse types of environments and under fluctuated growth conditions they produce different categories of primary and secondary metabolites to adopt with these environments and as a defense system to enable their survival (Heidari et al. 2012, Pandy 2015, Abd El-Karim 2016. Several metabolites as pigments, carbohydrates, polyphenols, fatty acids, lipids, hydrocarbons and some other cellular compound were endorsed with antimicrobial activity (Abu-Ghannam & Rajauria 2013, Pradhan et al. 2014).
Cyanobacterial species such as Spirulina platensis, Nostoc commune, N. muscorum, N. piscinale, Scytonema hofmanni, Oscillatoria anguistisima and Calothrix parietina, Tolypothrix tenuis and Anabaena variabilis, among others, have been accredited as antimicrobial producing species against human pathogens in in vitro studies (Soltani et al. 2005, Plaza et al. 2010, Gheda et al. 2013, Abo-State et al. 2015. The resistance of G-ve bacteria to antibiotics was a common notice among many previous studies, and that was also found in our results (Table I). This may be due to the complex lipopolysaccharides present in the G-ve bacteria cell wall, which hinders active compounds penetration. However, variation in inhibition zones using methanol, ethyl acetate and hexane might be ascribed to the difference in the active metabolites composition dissolved in these extracts (Bharat et al. 2013, Najdenski et al. 2013, Rajishamol et al. 2016 These values were nonsignificant to those recorded for Ampicillin (except for S. aureus STR-168) and Fluconazole standard antibiotics implying feasible antibacterial and antifungal activities for this extract (Table II).

Phytochemical analysis of cyanobacterial extracts
The results shown in Table III represented the qualitative phytochemical composition of the active methanolic extracts for the three tested cyanobacteria species. Obviously, the extracts were containing active secondary metabolites for which antimicrobial activity have been already established in many studies ( Shanab et al. 2012, Silva-Stenico et al. 2013, Abd El-Karim 2016. O. acuminata extract was rich in the phenols content (32.63±1.3 mg GA/g dry wt.) and in the presence of glycosides while flavonoids, alkaloids and saponins were apparent in all the tested extracts. As reported by (Mujeeb et al. 2014), these bioactive components exert diverse mechanisms for their antimicrobial action. Flavonoids, as a natural phenol, may interact with soluble and extracellular proteins of microbial cells. They are synthesized by plants in response to microbial infection and have been proved as an effective antimicrobial agent. Terpenoids also can cause dissolution of microorganism cell walls through weakening the membranes. Saponins can cause proteins and enzymes outflow from the cells. Alkaloids inhibit nucleic acid synthesis and attenuate microbial cells pathogenicity and virulence gene mechanisms (Cushnie et al. 2014). The antimicrobial action of phenols is due to alteration of microbial cell membrane permeability, loss of internal macromolecules, cellular integrity and eventual cell death (Abu-Ghannam & Rajauria 2013, Namvar et al. 2014, Rajishamol et al. 2016). Glycosides also have been reported to effect on S. aureus and C. albicans pathogens (Bilková et al. 2015).

Antioxidant activity of cyanobacterial extracts
As shown in Table IV, O. acuminata methanol extract exhibited the highest antioxidant activity (6.58 and 34.60 %) with both methods, respectively, followed by O. amphigranulata and then S. platensis. Since, O. acuminata contained phenolic compounds (32.63 mg Gallic acid/g dry wt.) as well as flavonoids, saponins, alkaloids and  glycosides (Table III) However, the phenomenon of synergistic effects of biological extracts was frequently crucial since sometimes this activity was lost when purified fractions were made. The advantage remains that bacterial resistance to synergistic drug formulations, like those of crude extracts, is often slower than for a single drug component (Cos et al. 2006). This justifies the feasibility of exploring crude extracts from cyanobacterial source for more biological activities than using isolated compounds.

Viability (Cytotoxicity) test of cyanobacterial extracts
The cytotoxicity assay was used to examine the cyanobacterial extracts as antitumor agent (Fadda et al. 2012). The crude extracts of all tested cyanobacteria showed significant increased inhibition of cell viability with increasing concentration (Fig. 1) (Sutharsana et al. 2016). In addition, phenolic compounds found in algae, have been reported to exert several biological effects including anti-apoptosis, anti-aging and anticarcinogenic properties and as chemoprotective agents (Namvar et al. 2014).

GC-MS analysis of cyanobacteria methanol extracts
As shown in Table VI, S. platensis methanolic extract contained some valuable biomolecules of antimicrobial and antitumor potent activity.

Results in
The GC-MS profiles revealed that methanolic extracts of S. platensis, O. acuminata and O. amphigranulata were represented consistent sources of bioactive compounds. The ingredient ratios were different between these species. However, there were basic compounds such as fatty acids, alcohols including phytol, phthalate and hydrocarbons which were commonly abundant and could be responsible for the recorded biological activity of these species (Tables VI, VII Chauhan et al. (1992) reported that ether extract of Oscillatoria sp. demonstrated antibiotic activity which may be due to the isolated and identified saturated fatty acids (C14:0, C16:0 and C18:0). In the present study, the G +ve bacteria were more susceptible than the G -ve bacteria. Similar results were obtained with other studies (Bharat et al. 2013, Abo-State et al. 2015. These differences may be due to the hydrophobicity of the G -ve bacteria outer membrane, of lipopolysaccharide composition, which acts as effective barrier against permeability of long chain fatty acids as antimicrobial substances. However, lauric, palmitic, linolenic, linoleic, oleic, stearic and myristic acids were known to have potential antibacterial and antifungal and to inhibit G-ve bacteria like E. coli with neither bacterial resistance to free fatty acids nor resistance phenotype has been developed (Desbois et al. 2009). Furthermore, antimicrobial activity of lipids may be attributed to the constituents of fatty acids and their ratios in a certain extract which could disrupt the oxidative phosphorylation, the electron transport chain and the energy production of microbial cells. Similarly, fatty acids may cause inhibition of enzyme activity, prompting peroxidation and auto-oxidation and degradation of microbial cells products (Desbois & Smith 2010). In the same context, the activity of antifungal compounds may be due to altering vital components of the fungal cell causing membrane permeability impairment, inhibition of spore germination and/ or inhibition of B-(1,3)-D-glucan synthesis. Furthermore, antifungal compounds may prevent lipid synthesis in the targeted fungal species by decreasing the ratio of unsaturated to saturated fatty acids or inhibiting the biosynthesis of ergosterols (Gupta et al. 2013).
The methanolic extracts of the tested cyanobacterial species proved potent chemopreventive or chemotherapeutic agents. The cytotoxicity percentages were proportional to the increase in the extracts concentrations and dependent on the extract ingredients of each species, especially the fatty acids content. O. acuminata extract was rich in fatty acids methyl esters content (72.76%) of 11-Octadecenoic acid, 9,12-Octadecadienoic acid (Z,Z) and Hexadecanoic acid; in addition to the other biologically active compounds in the extract (Table VII), interacted together to manifest the highest antioxidant and anti-proliferative activity of the tested extracts. Although of more diversity in composition, the fatty acids profile of O. amphigranulata extract exhibited lower antioxidant and cytotoxic activity than O. acuminata, and so applied for S. platensis extract. This may be due to the lower relative ratios of these fatty acids and their esters as 35.4% and 20% for O. amphigranulata and S. platensis, respectively. However, it should be noted that most of the principal bioactive components were common in the three chromatograms yet with different ratios. This implied the role of synergistic action between the ingredients in each extract to show its activity. According to (Kim et al. 2009), fatty acids as α-linolenic, linoleic and their derivatives exhibited pro-apoptotic and growth inhibitory activities on breast cancer-oestrogen positive cells. In their study using diethyl ether extracts of (10) cyanobacterial species, (Bharat et al. 2013) stated that saturated fatty acids of tetradecanoic, eicosanoic, docosanoic and heptadecanoic acids as well as PUFAs of α-Linolenic and Linoleic were abundant and responsible for the antimicrobial and cytotoxicity effects of these extracts. The mechanism(s) of action by which fatty acids trigger tumor cell death is still controversial. As described by (Dai et al. 2013), the tumor killing activity of PUFAs may be due to: (a) increased ROS generation (b) caspase enzymes activation (c) accumulation of lipid peroxidation toxic products leading to cell apoptosis (d) activation of peroxisome proliferator-activated receptors (PPARs) (e) modifying the expression of gene/ anti-oncogene, and (f) chromosomal damage stimulation of the cancer cells. Recently, Ahmed et al. (2017) recorded that Plectonema and Cyanothece sp. exhibited a pronounced cytotoxicity against hepatocellular carcinoma by inhibition of cell proliferation, stimulation of apoptosis and cell cycle arrest at diverse phases. Moreover, Ahmed et al. (2018) recorded that Fischerella BS1-EG crude extract has a pronounced influence on liver cancer (HepG-2), lung cancer (A549), colon cancer (HCT-116) and breast cancer (MCF-7).

CONCLUSION
In this study, different organic extracts of some soil cyanobacterial species showed a substantial antimicrobial activity. The methanolic extracts of S. platensis, O. acuminata and O. amphigranulata exhibited a significant antibacterial and antifungal action especially against B. subtilis and A. flavus. The phytochemical analysis revealed that these extracts contained many

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2.488 C 4 H 9 NO 2 Amino acid 0.768 D-2-Aminobutyric acid A substrate for D-amino acid oxidase, biosynthesis of non-ribosomal peptides.    and MCF-7 cell lines, respectively. According to the results of GC-MS analysis, fatty acids and their esters were responsible for the antimicrobial and the antitumor activities of these extracts. In addition, considerable ratios of biologically active compounds like organic alcohols including phytol, benzene derivatives, hydrocarbons, phenolics and phthalates were also detected which contributed synergistically to manifest the recorded activities. However, further studies should be conducted to visualize the crude extracts from cyanobacteria as a safe, natural and low-cost source for medicinal uses and drug industry after compass clinical trials.