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

Abstract

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.

Key words
antimicrobial; Antioxidant; Cyanobacteria; Cytotoxicity; GC-MS; Natural products

INTRODUCTION

The increase in the rate of infection by antibiotics- resistant 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 cyanobacteria-derived 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 anti-diabetic 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.

MATERIALS AND METHODS

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-²s^-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 10⁶ 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-1-picrylhydrazyl) 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:

$$\text{Free radical scavenging \%}=(Ac–As)/Ac\times 100$$

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:

$$(A_{570}\text{of treated samples}/A_{570}\text{of untreated sample})\times 100.$$

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. All extracts exhibited different degrees of antimicrobial activity unrelatedly to the tested pathogen. Generally, the methanolic extracts showed the highest antimicrobial activity followed by ethyl acetate and then hexane extracts. Methanolic extracts of A. variabilis showed broad activity with the highest inhibition zone recorded against B. subtilis STR-168 (19 mm) while all A. variabilis extracts showed no activity against the two tested fungal species. Similarly, methanolic extracts of N. muscorum recorded the highest inhibition zones against A. flavus GCA-3357 (17mm) and B. subtilis STR-168 (16mm). Regarding N. linckia methanolic extract, significant inhibition zones were recorded only for B. subtilis STR-168, K. pneumoniae HS-1286, C. albicans SC-5314 and A. flavus GCA-3357 (17, 10, 17 and 22 mm), respectively, with a noticeable antifungal activity. Hexane and ethyl acetate extracts of N. linckia showed comparable activities against the tested bacterial pathogens. The highest inhibition zones were recorded for A. flavus GCA-3357 followed by C. albicans SC-5314 hexane and ethyl acetate extracts of N. linckia, respectively. The majority of O. acuminata extracts exhibited considerable antifungal and antibacterial effects. Among which O. acuminata methanolic extracts recorded the highest inhibition zones against A. flavus GCA-3357 (26 mm), B. subtilis STR-168 (22 mm) and S. aureus STR-168 (20mm). In addition, the ethyl acetate of O. acuminata showed antifungal activity of 20 mm for A. flavus GCA-3357. Among all tested cyanobacterial extracts, the highest antifungal and antibacterial effects were recorded for O. ampigranulata different extracts (except for E. coli MG-1655 with the hexane extract). The highest inhibition zones were recorded by the methanolic extracts of O. ampigranulata as 31, 27 and 25mm against A. flavus GCA-3357, B. subtilis STR-168, S. aureus STR-168 and C. albicans SC-5314, respectively. Similarly, ethyl acetate and hexane extracts displayed significant antifungal inhibition zones of 27 and 25 mm, respectively against A. flavus GCA-3357 strain. For S. platensis, the methanolic extract presented promising antimicrobial inhibition zone diameters of 27, 24, 23, 21 and 19 mm against A. flavus GCA-3357, B. subtilis STR-168, S. aureus STR-168, C. albicans SC-5314 and K. pneumoniae HS-1286 pathogens, respectively (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 same conclusion was reported by (Soltani et al. 2005, Malathi et al. 2014, Abo-State et al. 2015). The recorded values were promising, although of significant difference than the used controls Ampicillin and Fluconazole (Table I). Evidently, methanolic extracts of O. amphigranulata, O. acuminata and S. platensis, respectively exhibited the best potent antimicrobial activities. Gram +ve B. subtilis STR-168, S. aureus STR-168 and A. flavus GCA-3357 fungus were the most affected pathogens followed by C. albicans SC-5314 (Table I).

Table I
Antimicrobial activity of cyanobacterial extracts using different solvents (mean± SD).

Cyanobacteria species	Diameter of inhibition zones (mm)
	Solvent	E. coli MG-1655	P. aeruginosa AE-4091	K. pneumoniae HS-1286	S. aureus TC-8325	B. subtilis STR-168	C. albicans SC-5314	A. flavus GCA-3357
A. variabilis	Ethyl acetate Hexane Methanol	12aA± 0.311aA±0.02NA	12aA± 0.1NA14aB ± 0.04	10bA± 0.39bA ± 0.0510bA ± 0.03	12aA± 0.511aA ± 0.1NA	13cA± 0.312cA±0.219cC± 0.2	NA NA NA	NA NA NA
N. muscorum	Ethyl acetate Hexane Methanol	11aA±0.01 NA 12aA ± 0.2	NA 13aA ± 0.414bB ± 0.1	9bA ± 0.1 NA 10cA ± 0.4	NA NA 15dB ± 0.3	11aA±0.212bA±0.216dB±0.3	11aA±0.312bA± 0.213bA± 0.1	NA 15cB± 0.317eB ± 0.4
N. linckia	Ethyl acetate Hexane Methanol	11aA±0.0413aA±0.06 NA	12bA ± 0.03NANA	11aA ± 0.212bB± 0.0410aA ± 0.03	11aA ± 0.412bB± 0.2 NA	12bA±0.114cB± 0.6 17bB± 0.3	13cA±0.4 15dB±0.517bB± 0.5	15dB±0.217eB ±0.4 22cD ± 0.4
O. acuminata	Ethyl acetate Hexane Methanol	NA NA 14aB ± 0.3	13aA ± 0.0112aA ± 0.0117bB ± 0.02	13aA ± 0.0212aA ± 0.0312cA ± 0.04	15bB±0.0413bA±0.0420dC ± 0.1	17cB± 0.315cB±0.622eD±0.2	12dA± 0.1 NA 15fB ± 0.3	20eC ±0.318dC± 0.326gE ± 0.3
O. amphigranulata	Ethyl acetate Hexane Methanol	14aB±0.06NA18aC±0.05	17bC ± 0.116aC ± 0.221bC± 0.03	13cA ± 0.0315bB ± 0.0520bC ± 0.02	21dC±0.0518cC±0.0125cD±0.02	21dC±0.0319dC ±0.427dE±0.06	20dC±0.321eC±0.225cD±0.3	27eE±0.125fD± 0.431eF±0.2
S. platensis	Ethyl acetate Hexane Methanol	13aA± 0.112aA±0.01 16aB ± 0.3	12bA± 0.3NA17bB ± 0.2	13aA ± 0.210bA± 0.0119cC ± 0.3	15cB ± 0.316cB± 0. 123dD± 0.4	19dC ± 0.217dB ± 0.4 24eD ± 0.1	13aA± 0.1NA21fC±0.3	14eB ± 0.214eB ± 0.1 27gE ± 0.1
Ampicillin		26aE±0.3	29bE ± 0.1	28cE ± 0.2	29aE±0.4	30dF±0.3	--	--
Fluconazole		--	--		--	--	28aE±0.4	32bF± 0.6

• NA, no activity recorded. Difference in superscript letters in each row or column indicate significance (p ≤ 0.05).

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). For this, the most active extracts of O. amphigranulata, S. platensis and O. acuminata, in methanol were selected to test their antioxidant and cytotoxic activities on HepG-2, HCT-116 and MCF-7 cell lines. On the same direction, the MIC showed fluctuated values with methanolic extracts of different tested cyanobacteria. However, the values compared well as potent antifungal agents against C. albicans SC-5314 (1.17; 4.68 µg/ml) and A. flavus GCA-3357 (0.78 µg/ml; 1.56 µg/ml) for O. amphigranulata and S. platensis methanolic extracts, respectively. Lower MIC values were also recorded for O. amphigranulata extract against E. coli MG-1655 (4.68 µg/ml), P. aeruginosa AE-4091 (3.13 µg/ml), K. pneumoniae HS-1286 and B. subtilis STR-168 (2.34 µg/ml). 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).

Table II
The minimum inhibition concentration (MIC µg/ml) of cyanobacterial methanolic extracts against different tested pathogens.

Compound	E. coli MG-1655	P. aeruginosa AE-4091	K. pneumoniae HS-1286	S. aureus TC-8325	B. subtilis STR-168	C. albicans SC-5314	A. flavus GCA-3357
S. platensisO. acuminataO. amphigranulata Ampicillin Fluconazole	12.5aA±1.5 25.0aB±1.5 4.68aC±0.7 1.56aC±0.2 ----	6.25bA ±1.3 9.37bB±0.5 3.13aC±0.3 2.34bC±1.0 ----	9.37cA±0.5 6.25bB±1.3 2.34bC±1.0 1.17aC±0.2 ----	9.37cA±0.5 37.5cB±2.5 3.13aC±0.3 0.78cD±0.5 ----	12.5aA±1.5 18.75dB±1.1 2.34bC±1.0 1.17aC±0.2 ----	4.68bA±0.7 6.25bA±1.3 1.17cB±0.2 ---- 1.17aB±0.2	1.56dA±0.2 3.13eB±0.3 0.78dC±0.5 ---- 0.58bC±1.0

• Difference in superscript letters in each row or column indicate significance at p ≤ 0.05 level.

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).

Table III
Qualitative phytochemicals screening of cyanobacterial species methanolic extracts.

Phytochemical compounds/ Test	S. platensis	O. acuminata	O. amphigranulata
Phenolic compounds, ferric chloride test	+	++	+
Flavonoid, alkaline reagent test	+	+	+
Terpenoids, Salkowski’s test	+	___	+
Tannins, ferric chloride test	___	___	___
Saponins, Froth test	++	+	++
Alkaloids, Wagner’s test	++	++	++
Steroids, Liebermann-Burchard test	___	___	___
Glycosides, Keller-Kiliani’s test	___	+	___
Total Phenolics content (mgGA/g dry wt)	18.385±0.3	32.63±1.3	15.696±0.5

• * + = Presence of constituents; – = Absence of constituents.

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), it may reacted synergistically leading to the elevated antioxidant activity of this species. Many previous studies (Shanab et al. 2012, Rajishamol et al. 2016, Abd El Sadek et al. 2017) proposed synergetic action between secondary metabolites, especially phenolic compounds, flavonoids, polyunsaturated fatty acids, pigments and polysaccharides to be responsible for the antioxidant activity. Phenolic components are one of the most abundant classes of phytochemicals in algae and have potential antioxidant ability through scavenging singlet oxygen, superoxide and hydroxyl radicals, metal chelating activity, electron or hydrogen donation ability and also stabilizing lipid peroxidation. Furthermore, microalgae are famed with the ability to face oxidative stresses through stimulating their enzymatic and non-enzymatic antioxidants as a defense system. The contribution of these antioxidant products as chemo-preventive and tumors growth controlling agents were already established (Wang et al. 2010, Varshney & Singh 2013, Konickova et al. 2014, Ahmed et al. 2017). 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.

Table IV
The antioxidant activity of cyanobacterial species methanolic extracts.

Methanol extract	% inhibition of DPPH	% inhibition of ABTS+
Ascorbic acid	6.14A ± 0.62	49.30A ±1.2
S. platensis	2.41B ± 0.93	17.50B ±0.62
O. acuminata	6.58A ± 0.62	34.60C ±0.95
O. amphigranulata	5.48A ± 0.31	23.20B ±0.80

• Difference in superscript letters in each column indicate significance at p ≤ 0.05 level.

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). O. acuminata methanol extract recorded 90.7, 91.2 and 85.2 % with HepG2, HCT-116 and MCF-7, respectively at concentration of 100µg/ml. One the same trend, O. amphigranulata recorded cytotoxicity percentage of 81.7, 83.8 and 74.2%; while S. platensis revealed relative cell viability inhibition of 64.9, 73.6 and 67.3 % with the same cell lines, respectively. The results suggested that active compounds in the extracts interacted with cancer cell lines associated receptors or cancer cell special molecules and triggered some mechanisms that cause cancer cell death (Wang et al. 2010). In the present study, IC₅₀ value (the concentration that caused 50% loss of the cell monolayer) of O. acuminata extract was of insignificant difference at p ≤ 0.05 than that of Doxorubicin drug recording very strong cytotoxicity activity (8.91, 8.43 µg/ml) for HepG2, HCT-116, respectively; and strong toxic activity (11.16 µg/ml) for MCF-7 cell line (Table V). Similarly, O. amphigranulata methanol extract recorded strong IC₅₀ value with HepG2 (16.3 µg/ml), HCT-116 (13.6 µg/ml) cell lines and moderate IC₅₀ value with MCF-7 cell line (21.29 µg/ml). For S. platensis methanol extract, moderate IC₅₀ values (41.07, 26.96, 37.80 µg/ml) were noticed for liver, colon and breast cell lines, respectively. These data were in agreement with the findings of Konickova et al. (2014) who reported the role of S. platensis with promising potential for its use in the treatment of cancer. Oscillatoria sp. secondary metabolites extracted as fatty acids methyl esters (FAME) proved significant pharmaceutical potentials to control tumor activities (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 chemo-protective agents (Namvar et al. 2014).

Figure 1
Cytotoxicity assay of cyanobacterial concentrations on different cell lines by MTT method.

Table V
Cytotoxicity (IC₅₀) of the tested cyanobacterial methanolic extracts on different cell lines*.

Methanolic extracts	In vitro Cytotoxicity IC50 (µg/ml)
	HePG2	HCT-116	MCF-7
Doxorubicin	4.50aA ± 0.2	5.23aA ±0.2	4.17aA ±0.2
S. platensis	41.07aC ± 3.0	26.96bC ±2.2	37.80cC ±2.5
O. acuminata	8.91aA ± 0.8	8.43aA ±0.6	11.16bD±1.2
O. amphigranulata	16.31aB ± 1.6	13.58bB ±1.4	21.29cB ±1.9

• *IC₅₀ (µg/ml): 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate). 51 – 100 (weak) and above 100 (non-cytotoxic). Difference in superscript letters in each row or column indicate significance at p ≤ 0.05 level.

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. According to the depicted peak area percentage, alcohols (1-Propanol, 1-Pentanol, 2-Methyl-1-propanol (isobutanol) and 1-Butanol, 2-methyl- (S)) constituted 18.179% of the total recorded peak areas in the chromatogram. The fatty acids content was noticeable (20.003%) of which 9-Hexadecenoic acid, methyl ester, (Z), Hexadecanoic acid, methyl ester, ç-Linolenic acid, methyl ester, 9, 12-Octadecadienoic acid (Z,Z)-, methyl ester and 9-Octadecenoic acid, methyl ester, (E). The GC-MS profile also contained valuable active compounds of 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (Phytol alcohol, 3.444%) and Diisooctyl phthalate (2.166%). In addition to betaine, guanidine, L-Isoleucine, N-benzoyl, Benzaldehyde, Oxime-, methoxy-phenyl and m-Xylene in small ratios.

Table VI
GC-MS analysis of Spirulina platensis methanolic extract**

Retention time	Compound Name/ Activity	Peak area %	Compound nature	Molecular formula
2.443	1-Propyne, 3-chloro-	3.524	Alkyne	C3H3Cl
2.483	2-Propanone, 1-(1-methylethoxy)	1.964	Ketone	C6H12O2
2.503	Betaine organic osmolyte, treatment of hypochlorhydria, gastrointestinal disturbances	1.596	Alkaloid	C5H11NO2
2.523	Guanidine Anti-inflammatory agents, antidiabetic, chemotherapy	1.589	Alkaloid	CH5N3
4.219	1-Propanol Antimicrobial	10.682	Alcohol	C3H8O
4.479	2-Methyl-1-propanol (isobutanol) Antimicrobial	4.063	Alcohol	C4H10O
5.195	L-Isoleucine, N-benzoyl Antitumor activity.	9.509	Amino acid derivative	C13H17NO3
5.270	1-Pentanol Antimicrobial	2.316	Alcohol	C5H12O
5.300	1-Butanol, 2-methyl- (S)- Antimicrobial	1.118	Alcohol	C5H12O
6.405	p-Xylene Antifungal, antioxidant, antimicrobial.	0.833	Aromatic hydrocarbon	C8H10
6.725	Butane, 1,1-diethoxy-	3.870	Alkane	C8H18O2
6.930	Benzaldehyde Antimicrobial, antioxidant, insecticidal	1.030	Aromatic aldehyde	C7H6O
7.305	m –Xylene Antifungal, antioxidant, antimicrobial	1.248	Aromatic hydrocarbon	C8H10
7.485	Oxime-, methoxy-phenyl- Antifungal, antibacterial, anticancer, antitumor	0.667	Ether	C8H9NO2
21.331	Eicosane Antifungal, antibacterial, antitumor, cytotoxic.	0.659	Alkane	C20H42
24.057	9-Hexadecenoic acid, methyl ester, (Z)- Antioxidant, hypochloesterolemic, haemolytic, antiandrogenic, 5-alpha reductase inhibitor	0.866	ω7fatty acid	C17H32O2
24.322	Hexadecanoic acid, methyl ester Antioxidant, anti-inflammatory, antifibrinolytic, cancer preventive, hemolytic, antialopecic, hepatoprotective, antihistaminic, antiacne,	7.195	Palmitic acid	C17H34O2
26.168	ç-Linolenic acid, methyl ester Antibacterial, anticandidal, anticancer, hypocholesterolemic, hepatoprotective, antiinflammatory, antihistaminic, antiarthritic, antieczemic, antiacne, antiandrogenic.	1.597	ω3fatty acid	C19H32O2
26.363	9,12-Octadecadienoic acid (Z,Z), methyl ester Anti-cancer, antiinflammatory, analgesic, hypocholesterolemic, ulcerogenic, antiarthritic, hepatoprotective, antihistaminic.	4.007	ω6 Linoleic acid	C19H34O2
26.443	9-Octadecenoic acid, methyl ester, (E)- Antibacterial, antitumor, antiinflammatory, 5-α reductase inhibitor, allergenic, anemiagenic, antialopecic, choleretic.	6.368	ω9 Oleic acid	C19H36O2
26.573	3,7,11,15-Tetramethyl-2-hexadecen-1-ol Vitamins E and K1 precursor, anticancer, antimicrobial, anti-inflammatory, chemopreventive, vaccine formulations.	3.444	Diterpene (Phytol)	C20H40O
31.680	Diisooctyl phthalate Antimicrobial, antifouling, plasticizer, melanogenesis inhibitor.	2.166	Ether	C24H38O4

• ** (Source: Dr. Duke’s Phytochemical and Ethnobotanical Databases).

Results listed in Table VII showed the GC-MS profile of O. acuminata methanolic extract. Alcohols of 1-Propanol and 2-Methyl-1-propanol (isobutanol) were present forming 2.821% of the total recorded peak areas in the chromatogram. Fatty acids and their esters were of pronounced ratios recording 72.763% of which 11-Octadecenoic acid, methyl ester (38.344%), 9,12-Octadecadienoic acid (Z,Z), methyl ester (22.771%) and Hexadecanoic acid, methyl ester were forming the bulk percentage. Other valuable components were 3-Aminobutanoic acid, á-Hydroxyisovaleric acid, L-Isoleucine, N-benzoyl, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (phytol alcohol), Diisooctyl phthalate, Octacosane and Heptacosane hydrocarbon. In addition to L-(+)-Ascorbic acid 2, 6-dihexadecanoate.

Table VII
GC-MS analysis of Oscilatoria acuminata methanolic extract**.

Retention time	Compound Name	Peak area %	Compound nature	Molecular formula
2.453	3-Aminobutanoic acid Induces plant disease resistance, affect virus, fungal and nematodes plant pathogens.	0.382	Amino acid	C4H9NO2
2.513	3-Hydroxyisovaleric acid Dietary supplement in medical foods, increases lean body mass and muscle strength, inhibits breakdown of proteins in muscle tissue, promotes wound healing,	2.516	Organic acid	C5H10O3
4.139	1-Propanol*	1.818	Alcohol	C3H8O
4.404	2-Methyl-1-propanol (isobutanol)*	0.667	Alcohol	C4H10O
5.129	L-Isoleucine, N-benzoyl*	0.856	Amino acid	C13H17NO3
5.205	1-Pentanol*	0.336	Alcohol	C5H12O
23.912	7,10-Hexadecadienoic acid, methyl ester Antioxidant, antimicrobial, anti-inflammatory	0.685	Fatty acid	C17H30O2
24.067	9-Hexadecenoic acid, methyl ester, (Z)*	0.283	ω7 fatty acid	C17H32O2
24.362	Hexadecanoic acid, methyl ester*	7.104	Palmitic acid	C17H34O2
26.453	9,12-Octadecadienoic acid (Z,Z)-, methyl*	22.771	ω6 Linoleic acid	C19H34O2
26.578	11-Octadecenoic acid, methyl ester Anti carcinogenic, anemiagenic, hypocholesterolemic, dermatitigenic.	38.344	Vaccenic acid	C19H36O2
26.633	3,7,11,15-Tetramethyl-2-hexadecen-1-ol*	1.299	Diterpene (phytol)	C20H40O
26.783	Octadecenoic acid Cosmetic, lubricant, hypocholesterolemic, surfactant, softening, perfumery, flavor.	2.444	Stearic acid	C18H34O2
27.519	Hexadecanoic acid, ethyl ester*	0.301	Palmitic acid	C18H36O2
28.014	Heptacosane Antibacterial, antifungal, antioxidant, cytotoxic	0.616	Alkane	C27H56
28.494	6-Octadecenoic acid, methyl ester, (Z)- Cancer preventive, insectifuge.	0.501	Fatty acid	C19H36O2
28.759	Heptacosane*	0.871	Alkane	C27H56
28.989	Eicosanoic acid, methyl ester Alpha-glucosidase inhibitor activity, detergents, lubricants.	0.330	Arachidic acid	C21H42O2
29.069	Octacosane Antibacterial, antifungal, antioxidant, cytotoxic	0.544	Alkane	C28H58
29.709	Hexanedioic acid, bis (2-ethylhexyl) ester A plasticizer	0.826	Ester	C22H42O4
31.200	L-(+)-Ascorbic acid 2,6-dihexadecanoate Antioxidant, anti-inflammatory, antiscorbutic, cardio-protective, anticancer, flavor, wound healing property, antimutagenic, anti-infertility	0.412	Ester	C38H68O8
31.730	Diisooctyl phthalate*	2.638	Ester	C24H38O4

• **(Source: Dr. Duke’s Phytochemical and Ethnobotanical Databases).* Activity was mentioned in table VI.

Results in Table VIII listed the biologically active components of O. amphigranulata methanolic extract. Most of these compounds were of valuable medicinal activities. Alcohols of 1-Propanol, 2-Methyl-1-propanol (isobutanol) and 1-Butanol, 3-methyl- constituted 9.37% of the total recorded peak areas in the chromatogram in addition to 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (Phytol alcohol, 2.978%). Fatty acids and their methyl esters were obvious recording 35.382% of which Hexadecanoic acid, methyl ester, 9-Hexadecenoic acid, methyl ester, (Z)-, 11-Octadecenoic acid, methyl ester, Myristic acid and cis-10-Nonadecenoic acid were the dominant. Phenol compounds were represented by Cyclotrisiloxane, hexamethyl (1.040%) and Phenol, 2,4-bis(1,1-dimethylethyl)-(1.424%). The extract also contained other biologically active biomolecules as (ñ)-3-Hydroxybutyric acid (12.85%), D-2-Aminobutyric acid (0.768%), Benzoyl bromide (3.98%), p-Xylene (0.695%), Esculetin (0.692%), Heptadecane (0.724%) and Diisooctyl phthalate (2.866%).

Table VIII
GC-MS analysis of Oscilatoria amphigranulata in methanolic extract**.

Retention time (RT)	Compound Name/ Activity	Peak area %	Compound Nature	Molecular formula
2.488	(ñ)-3-Hydroxybutyric acid An energy source for the brain when blood glucose is low, treatment of depression, anxiety, and cognitive impairment, polyesters, biodegradable plastics.	12.849	Organic acid	C4H8O3
2.724	D-2-Aminobutyric acid A substrate for D-amino acid oxidase, biosynthesis of non-ribosomal peptides.	0.768	Amino acid	C4H9NO2
3.944	1-Propanol*	6.319	Alcohol	C3H8O
4.239	2-Methyl-1-propanol (isobutanol)*	2.190	Alcohol	C4H10O
5.004	Benzoyl bromide Antibacterial, antifungal.	3.980	aromatic ketone	C7H5BrO
5.074	1-Butanol, 3-methyl Antimicrobial	0.861	Alcohol	C5H12O
5.450	Cyclotrisiloxane, hexamethyl- Antimicrobial, antioxidants.	1.040	Phenol	‎C6H18O3Si3
6.650	Butane, 1,1-diethoxy-	1.120	Alkane	C8H18O2
7.250	p-Xylene*	0.695	aromatic alkyl	C8H10
7.856	Esculetin Antitumor, anti-inflammatory, antimicrobial, antioxidant	0.692	Lactone	C9H6O4
18.55	Phenol, 2,4-bis(1,1-dimethylethyl)- Antioxidant, antimicrobial.	1.424	Phenol	C14H22O
21.34	Heptadecane antibacterial, antifungal and anti-inflammatory	0.724	Alkene	C17H36
21.506	Methyl myristoleate A cytotoxic component, induces apoptosis and necrosis in human prostate cancer LNCaP cells.	2.456	Ester	C15H28O2
21.686	Myristic acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant, cosmetic.	3.039	Fatty acid	C14H28O2
22.547	Pentadecanoic acid, methyl ester Antioxidant, antimicrobial	1.586	Fatty acid	C16H32O2
23.907	7,10-Hexadecadienoic acid, methyl ester*	0.687	Fatty acid	C17H30O2
24.072	9-Hexadecenoic acid, methyl ester, (Z)*	6.509	ω7 fatty acid	C17H32O2
24.192	9-Hexadecenoic acid, methyl ester, (Z)*	3.837	ω7 fatty acid	C17H32O2
24.342	Hexadecanoic acid, methyl ester*	9.582	Palmitic acid	C17H34O2
26.373	9,12-Octadecadienoic acid (Z,Z)-, methyl*	1.357	ω6 Linoleic acid	C19H34O2
26.448	9-Octadecenoic acid, methyl ester, (E)*	1.184	ω9 Oleic acid	C19H36O2
26.518	11-Octadecenoic acid, methyl ester*	5.145	Vaccenic acid	C19H36O2
26.583	3,7,11,15-Tetramethyl-2-hexadecen-1-ol *	2.978	Diterpene (Phytol)	C20H40O
26.798	cis-10-Nonadecenoic acid Potential antitumor activity, prevent LPS-induced tumor necrosis factor, inhibit p53 activity.	0.851	Fatty acid	C19H36O2
31.695	Diisooctyl phthalate *	2.866	Esters	C24H38O4

• **(Source: Dr. Duke’s Phytochemical and Ethnobotanical Databases). * Activity was mentioned in tables VI or VII.

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 and VIII). The saturated and unsaturated fatty acids were of concentrated ratios in O. acuminata extract while were more distributed with lower ratios in O. amphigranulata extract. The mechanism of fatty acids as antimicrobial agent may be through disruption of the microorganism cellular membrane, leakage and/or reduction of nutrient uptake and inhibiting cellular respiration. According to (Kumar et al. 2011), the fatty acids may diffuse into the peptidoglycan meshwork of the microbial cell wall causing disruption of the cellular membrane and its disintegration. As reported by (Desbois et al. 2009), fatty acids cause peroxidative stress to the microbial cells. Fatty acids longer than 10 carbon atoms in chain length, such as palmitoleic and oleic acids, may induce lysis of microbial cell protoplast. The polyunsaturated arachidonic acid exhibited a strong activity against methicillin resistant S. aureus (MRSA), E. coli, P. aeruginosa and S. epidermidis (Smith et al. 2010). Different volatile compounds from S. platensis extracts exhibited antibacterial activities (El-Sheekh et al. 2014). Furthermore, 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 chemo-preventive 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 compounds of antimicrobial and antioxidant activities. The results of O. acuminata extract recommended the potent anticancer activity of 90.7, 91.2 and 85.2% against HepG2, HCT-116 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.

ACKNOWLEGMENTS

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Publication Dates

• Publication in this collection
  07 Aug 2020
• Date of issue
  2020

History

• Received
  13 Aug 2019
• Accepted
  23 Oct 2019