In vitro evaluation of antioxidant, anticancer, and antiviral activities of exopolysaccharide from Streptomyces hirsutus NRC2018

Sampling and isolation of streptomycetes

Samples were collected during summer 2016 from marine sediments from North Coast, Egypt. Samples were serially diluted according to Hayakawa and Nonomura (1987), and plated on starch nitrate agar medium containing (g/l): starch 10.0, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.5, NaCl 0.5, KNO₃ 2.0, CaCO₃ 2.0, FeSO₄.7H₂O 0.01, and agar 20.0, which was dissolved in 750 ml seawater then topped to 1 L with 250 ml distilled water, pH adjusted to 7.0 (Waksman, 1961). The streptomycete isolate was selected and picked up based on the morphological features of colonies.

Identification of streptomycete isolate

The isolate was identified according to morphological, physiological, and biochemical features (Goodfellow et al., 1987; Kutzner, 1976; Shirling and Gottlieb, 1966; Szabó et al., 1975; Tresner and Backus 1963). Also, molecular identification was performed by 16S rRNA gene sequencing. The bacterial genome was isolated and 16S rRNA gene was amplified using polymerase chain reaction technique using the following primers F(5′ GAGTTTGATCCTGGCTCAG 3) and R(5′GGTTACCTTGTTACGACTT 3′) based on Gardes and Bruns (1993) method. Sequencing was done using kit of Big Dye terminator cycle sequencing (Applied Biosystems, USA). Products of sequencing were analyzed on a model Applied Bio-systems 3730XL automated DNA sequencing system (Applied Biosystems, USA). The resulted 16S rRNA sequence was submitted to GenBank database and compared with the other sequences in https://www.ncbi.nlm.nih.gov/ using the BLAST program. The sequence of other bacterial strains with the most similarity to the 16S rRNA gene of our isolate was selected and aligned for making the suitable phylogenetic tree. Then, 16S rRNA sequence was deposited in the DDBJ/EMBL/GenBank databases with accession number MK050544.

EPS production

Streptomyces isolate was inoculated into production medium containing (g/l) Glucose 10.0, Tryptone 5.0, Yeast extract 5.0, K₂HPO₄ 3.0, NaCl 3.0, KH₂PO₄ 1.0, MgSO₄.7H₂O 0.5, CaCO₃ 0.5 dissolved in 750 ml seawater and topped to 1 L by distilled water and adjusted to pH 7 (Manivasagan et al., 2013) then incubated at 28°C for 5 days at 120 rpm. After incubation, the medium was centrifuged at 5,000 rpm for 30 minutes, the supernatant mixed with Trichloroacetic acid (TCA) (10%) and left overnight at 4 C. Then, centrifuged at 5,000 rpm for 20 minutes to remove the protein. The supernatant pH was neutralized to 7 with NaOH solution (Liu et al., 2010). The EPSs containing solution was collected and mixed with four volumes of ethanol (95%), left it overnight at 4°C. The precipitated EPSs were separated by centrifugation at 5,000 rpm for 20 minutes and redissolved in deionized water and dialyzed three times (1 L × 3) using dialysis tubing (MWCO 2000) for 48 hours. For purification and major fraction determination, absolute cold ethanol was added in 1, 2, 3, and 4 volumes gradually and the precipitated EPS was collected up to date. The major fraction obtained by one volume was dialyzed against distilled water for 72 hours, washed twice using acetone. Then, dehydrated by ether, dried at 40°C, and coded as EPSNC2 (Shene et al., 2008).

Homogeneity and molecular mass of EPSNC2

The mass average molar mass (Mw) of EPSNC2 was determined used high-performance gel permeation chromatography (HPGPC, Agilent 1100 Series System, Hewlett-Packard, Germany) with refractive index (RI) detection (You et al., 2013). The number average molar mass (Mn) was determined and polydispersity index (PI) was calculated from (Mw/Mn) ratio.

Monosaccharide composition analysis

EPSNC2 was subjected to complete acid hydrolysis with formic acid (88 %) at 100°C for 5 hours. High-performance liquid chromatography (HPLC, Agilent Pack, serics1, 200), equipped with Aminex carbohydrate HP-87C column (300 × 7.8 mm) was applied for monosaccharide composition and calculation of molar ratio (Randall et al., 1989).

Fourier-transform infrared spectroscopy (FTIR)

Dried EPSNC2 (2 mg) was mixed with 200 mg KBr powder then pressed to form a pellet of 1 mm. FTIR spectrum was measured in the range between 400 and 4,000 cm⁻¹ using FTIR-UNIT Bruker Vector 22 Spectrophotometer (Brock-Neely, 1957).

Sulfate and uronic acid content determination

EPSNC2 was subjected to complete acid hydrolysis with formic acid (88%) at 100°C for 5 hours. The sulfate was estimated by the turbidity method using Barium Chloride-Tween 80 reagent according to Dodg=son and Price (1962). The content of uronic acid was determined using the m-hydroxybiphenyl colorimetric method at 525 nm (Filisetti-Cozzi and Carpita, 1991).

Evaluation of EPSNC2 antioxidant bioactivity

DPPH radical scavenging activity

The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined according to Brand-Williams et al. (1995). Different concentrations of EPSNC2 (100, 300, 500, 1,000, and 1,500 µg/ml) were added to 2 ml of DPPH solution. Then, left in dark for 10, 30, 60, and 90 minutes at 517 nm using UV-Visible spectrophotometer. The DPPH scavenging activity was calculated from the following equation:

Scavenging ability (%) = ( A 517 of control − A 517 of sample / A 517 of control ) × 100

Hydrogen peroxide scavenging activity

According to Ruch et al. (1989), different concentrations of EPSNC2 (200, 400, 600, 800, 1,000, and 1,500 µg/ml) were mixed with 40 mM hydrogen peroxide solution in 50 mM of phosphate buffer solution (pH 7.4). The results were measured at different periods (15, 30, 45, and 60 minutes) against phosphate buffer as a blank and at 230 nm using UV-Visible spectrophotometer. H₂O₂ scavenging activity was calculated as follows:

Scavenging ability (%) = ( A 230 of control − A 230 of sample / A 230 of control ) × 100

Reducing power (RP) activity

EPSNC2 concentrations (400, 600, 800, and 1,000 µg/ml) were added to 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml potassium ferricyanide (K₃Fe(CN)₆) 1% w/v and incubated at 50°C for 20 minutes then trichloroacetic acid (2.5 ml) 10% w/v was added. Then, mixture was centrifuged at 3,000 rpm for 10 minutes and the supernatant mixed with 2.5 ml H₂O and 0.5 mL of FeCl₃ (0.1% w/v). The absorbance of colored complex was measured at 700 nm (Oyaizu, 1986).

Metal chelating activity

The chelating activity was detected using Dinis et al. (1994) method by mixing 0.1 mL of EPSNC2 solution with concentrations (400, 600, 800, and 1,000 µg/ml) with 0.5 ml of 0.2 mM ferrous chloride; then 0.2 ml of ferrozine (5 mM) was added and the mixture incubated for 10 minutes at room temperature until red color complex was formed and then measured at 562 nm. The activity of metal chelating was calculated according to the following equation:

Metal-chelating activity (%) = ( A 562 of control − A 562 of sample / A 562 of control ) × 100

Assessment of antitumor activity

Cell line and culture

Human colorectal adenocarcinoma cell line (CaCo-2), human hepatocellular carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF-7), and human lung adenocarcinoma cell line (A-549) were obtained from Tissue Culture Unit, Holding Company for Biological Products and Vaccines (VACSERA) at Giza, Egypt. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% Glutamine, and 1% Penicillin/Streptomycin and incubated at 37°C in 5% CO₂ humidified incubator.

Cytotoxicity/Viability assay

The cytotoxicity of EPSNC2 was evaluated through the MTT viability assay (Mossman et al., 1983). The cancer cells incubated with different concentrations of EPSNC2 (7.8–500 µg/ml) in a 96-well micro-plate while, control was incubated with RPMI 1640 media without EPSNC2 for 24 hours at 37°C, with 5% CO₂. Cells were checked for any physical signs of toxicity, e.g. rounding, partial, or complete loss of the monolayer, cell granulation, or shrinkage. Then, 20 µ1 of MTT (BIO BASIC CANADA INC) solution (5 mg/ml dissolved in PBS) was added to the cells for 5 minutes then 200 µ1 of DMSO was added to dissolve formazan and then removed and the cells were washed. The absorbance was measured at 560 nm using a micro-plate ELISA reader. All tests were established in triplicate and averaged. The cell viability was calculated as follows:

[ 1 − ( OD test / OD control ) ] 1 × 100 (%)

Assessment of antiviral activity

Cell line and Cytotoxicity assay

African green monkey kidney epithelial (VERO) cell line was purchased from Tissue Culture Unit, VACSERA, Giza, Egypt. It was cultured in MEM-EARLE’s medium (VACSERA, Egypt) enriched with 2% fetal bovine serum and incubated with serially diluted concentrations of EPSNC2 (15.65, 31.25, 62.5, 125, 250, and 500 µg/ml) for up to 48 hours in 5% CO₂ at 37°C. Cytotoxicity was determined using the MTT viability assay (Mossman et al., 1983). The half-maximal cytotoxic concentration (IC₅₀) and the minimum non-toxic concentration were calculated.

Viruses and evaluation of antiviral activity

The cytopathic effect of Herpes Simplex virus type 1 (HSV-1), Adenovirus, Hepatitis A virus (HAV), and Coxsackie B4 virus (Microbiology Department, Faculty of Medicine for girls, Al-Azhar University, Egypt) were tested on VERO cells and the half-maximal viral cytopathogenicity concentration was estimated using MTT method. Equal volume (1:1 v/v) of three dilutions of EPSNC2 (31.25, 62.5, and 125 µg/ml) were incubated with the half-maximal viral cytopathogenicity concentration of viruses for 1 hour. 100 µl of viral/sample suspension was added to 1 × 10⁵ cells which were cultured in 200 µl MEM media per well in a 96-well micro-plate then incubated at 37°C with 5% CO₂ humidity for 24 hours until either viral cytopathic effect or antiviral activity of EPSNC2 was obtained. According to MTT viability assay (Pauwels et al., 1988), optical density was measured at 560 nm and the background was subtracted at 620 nm. The experiment was carried out in triplicate. The concentration which inhibits 50% of viral cytopathic effect (EC₅₀) was determined.

Statistical analysis

Graph Pad InStat version 2 software was used for analyzed of all results. All graphs were plotted using the Graph Pad Prism version 8. Results were considered statistically significant with p values < 0.05.

Isolation and identification of EPS producing Streptomycetes strain

The streptomycete strain was isolated from the sediments of the Mediterranean Sea, North Coast, Egypt. The isolate was taxonomically identified morphologically, biochemically, and physiologically. Streptomycete strain was confirmed using 16S rRNA molecular identification. Table 1 shows the morphological, biochemical, and physiological features of the isolate. The isolate had a grayish color of spore mass with no melanin and diffusible pigment production. Also, it showed a variety in the utilization of different sugars as a carbon source. Whereas, the spore chain was spiral and spiny spore surface as obtained in Figure 1a and b. The partial analysis of NC2 isolate 16S rRNA gene and its comparison with the data included in GenBank proved that the isolate had 100% homology with S. hirsutus and gave an identification symbol NRC2018 with accession number MK050544.

The evolutionary history of the tree was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.00511422 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 10 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1,372 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

EPS production, purification, and chemical characterization

After 5 days of incubation in fermentation medium (Manivasagan et al., 2013), EPS 10 g/l of culture media was isolated and the major fraction was obtained by addition of one volume absolute ethanol and named EPSNC2. The novel EPSNC2 was subjected to chemical analysis which proved that it contains 72.73% uronic acid and no sulfate content. Furthermore, monosaccharide composition and molar ratio which were determined by HPLC showed that EPSNC2 consisted of glucuronic acid: galacturonic acid: glucose: mannose: arabinose with molar ratio 1.2: 0.6: 0.1: 0.2: 0.1, respectively, which meant that EPSNC2 is a highly acidic heterogeneous polysaccharide.

Table 1. Morphological, physiological, and biochemical characteristics of NC2 isolate. [Click here to view]

Figure 1. (a) Photomicrograph showing spiral aerial hyphae. (b) TEM micrograph showing spiny spore surface. [Click here to view]

Molecular weight determination

The mass average molar mass (Mw) and the number average molar mass (Mn) were 4.25 × 10⁵ g/mol and 2.71 × 10⁵ g/mol, respectively, as shown in Figure 2. While the PI which was calculated by Mw/Mn ratio was 1.57. Therefore, it is noted that EPSNC2 has a high molecular weight. These results are consistent with what has been published that Mw of EPSs between 10 and 6,000 kDa (Xie et al., 2010).

FTIR spectroscopy analysis

FT-IR spectrum showed patterns which are confirmatory for EPSNC2, as shown in Figure 3. A broad peak at 3,430.74 cm⁻¹ for OH stretching vibration (Kavita et al., 2014) and weak CH stretching bands were obtained at 2,961.16, 2,928.38, and 2857.99 cm⁻¹. The peak at 1,673.91cm^-1 was attributed to C=O group stretching vibration as a result of the presence of the carboxylate group of uronic acid (Ahluwalia and Goyal, 2005; Lillo et al., 2014). While, the ß-pyranose structure was proved by the existence of 837.91 cm⁻¹ band (Cheng et al., 2008) and 1,129.12 and 1,107.9 cm⁻¹ peaks which were referred to C-O-C and C-O groups (Vijayabaskar et al., 2011).

Evaluation of antioxidant activity

DPPH free radical scavenging activity

The ability of EPSNC2 to scavenge free radical was estimated using DPPH as a source of a free radical. The determination of antioxidant activity could be based on two methods: single electron transfer and hydrogen atom transfer (Gulcin, 2012; Tan and Lim, 2015). Polysaccharides have many functional groups such as hydroxyl groups which can quench free radical by hydrogen donation. EPSNC2 showed high powerful free radical scavenging activity in a dose-dependant manner with IC₅₀ value 158.5 µg/ml at 120 minutes, as shown in Figure 4. This antioxidant activity of EPSNC2 was due to presence of hydroxyl groups in addition to a high percentage of uronic acid (72.75%) containing carboxylic groups (Al-Sheraji et al., 2012; Wu et al., 2013). Ye et al., (2012) isolated and purified an acidic EPS which belonged to β-type heteropolysaccharide and has a pyran group from marine Pseudomonas PF-6. This EPS showed antioxidant activity against DPPH. El-Newary et al. (2017) noted that BAEPS an EPS has been isolated from Bacillus amyloliquefaciens 3MS 2017 could scavenge DPPH free radicals and the maximum activity was 99.39% at 1,000 µg/ml. Streptomyces carpaticus produced an EPS which exhibited DPPH antioxidant activity with EC₅₀ value 111 µg/ml (Selim et al., 2018).

Figure 2. Mass average molar mass (Mw) of EPSNC2. [Click here to view]

Hydrogen Peroxide scavenging activity

Although the EPSNC2 showed antioxidant activity against hydrogen peroxide, its activity did not exceed 75.6% at 1,500 µg/ml after 60 minutes. s illustrates the antioxidant activity of EPSNC2 and IC₅₀ value was estimated to be 501.2 µg/ml at 60 minutes. Besides the deleterious influences of H₂O₂, it is the source of hydroxyl free radical OH^●, the most potent and reactive free radical inside cells via Fenton and Haber-Weiss reactions (Fenton, 1894; Haber and Weiss, 1934). BAEPS which had been isolated by El-Newary et al. (2017) could scavenge H₂O₂ and the scavenging ability was found to be 61.72% at 100 µg/ml and was elevated progressively to 92.17% at 1,000 µg/ml.

Ferric ion reducing power and ferrous ion metal chelating activity

It is supposed that the reducing power antioxidant activity of polysaccharides is due to the existence of free terminal electrophilic aldehyde and ketone groups which could reduce the metal ion. The Fe⁺³ of potassium ferricyanide was reduced to Fe⁺² by EPSNC2 and Table 2 showed that RP increased in a dose-dependant manner. As observed by the metal chelating activity, 94.9% at 400 µg/ml and 98.5% at 1,000 µg/ml (Table 2). It is noteworthy that the hydroxyl and carboxyl groups found in EPSNC2 could interact with metal ions. Transition metal ions especially ferrous and cuprous ions are implicated in the formation of OH^● via Fenton reaction and electron transferring via electron transport chain. Streptomyces violaceus MM72 producing an EPS showed strong metal chelating activities and moderate reducing activities (Manivasagan et al., 2013).

In vitro anticancer activity against colon cancer cells

To assess the EPSNC2 cytotoxic effect on tumor cells, we observed the cancer cell lines after exposure to EPSNC2 for 24 hours. Interestingly, EPSNC2 showed a significant cytotoxic effect on CaCo-2 cell line without any effect on other cell lines (HepG2, MCF-7, and A-549) and the viability of CaCo-2 was remarkably decreased after EPSNC2 treatment in a dose-dependant manner (Fig. 6). Whereas there was no effect until 125 µg/ml, the cytotoxic effect increased to 44.8% and 75% at 250 and 500 µg/ml, respectively. IC₅₀ was estimated to be 295.1 µg/ml as shown in Figure 7, which also showed that a strong correlation between the EPSNC2 concentration and the anticancer effect on CaCo2 cells (R² = 0.9536, p = 0.0043).

Many researchers found that polysaccharides had antitumor activity and their mode of action followed these mechanisms: i) stimulation and enhancement of immune system; ii) induction of apoptosis; iii) inhibition of angiogenesis; and iv) cell cycle arrest (Meng et al., 2016; Xu et al., 2016). EPS isolated from Lactobacillus acidophilus showed anticancer activity against two types of colon cancer cell lines HCT15 and CaCo-2 (Deepak et al., 2015). Zhou et al. (2017) reported that EPS116 from Lactobacillus plantarum NCU116 inhibits the proliferation and survival of mouse colorectal carcinoma CT26 cells through induction of apoptosis via c-Jun, Fas/FasL pathways. Furthermore, the anticancer activity of EPS116 may be toll-like receptor (TLR)-2 dependent.

Figure 3. FT-IR spectrum of EPSNC2. [Click here to view]

Figure 4. DPPH free radical scavenging activity of EPSNC2. [Click here to view]

Figure 5. H2O2 scavenging activity of EPSNC2. [Click here to view]

Table 2. RP and metal chelating activity of EPSNC2. [Click here to view]

EPSNC2 has high molecular weight with monosaccharide composition diversity so it is believed to exhibit more interactions with many different cell surface receptors especially on macrophages and dendritic cells such as TLRs, mannose-binding lectins, and C-type lectins which have carbohydrate recognition domain enhancing innate immune system and complement system against tumor (Brown and Gordon, 2005; Willment et al., 2005). High molecular weight polysaccharides cannot enter the cells but, instead they bind with cancer cell receptors controlling cell signaling and cellular transduction.

Antiviral activity and EC50 determination

Before assessment of antiviral activity, cytotoxicity of EPSNC2 was evaluated against African green monkey normal kidney epithelial cell line (VERO). The half-maximal cytotoxic concentration (CC₅₀) was 387.19 µg/ml, while the maximum non-toxic concentration was 125 µg/ml. The antiviral activity of EPSNC2 was 84.9%, 20.3%, and 45.4% against HSV-1, HAV, and Coxsackie B4, respectively, at the non-toxic concentration 125 µg/ml. However, there was no antiviral activity against adenovirus at used EPSNC2 concentrations as shown in Figure 8. Figure 9 shows that the relationship between the logarithm of EPSNC2 concentration and its antiviral activity and the concentration which prevented the half-maximal viral cytopathic effect (EC₅₀) of HSV-1 was estimated to be 32.4 µg/ml. It has been reported that the antiviral activity of polysaccharides follows one or more of these mechanisms: i) Direct Virucidal Action, ii) Inhibition of Viral Adsorption, iii) Inhibition of Virus Internalization and Uncoating, iv) Inhibition of Virus Transcription and Replication, and v) Improvement of Host Antiviral Immune Responses. Carlucci et al. (1997) reported that the sulfate-containing λ-carrageenan could bind with HSV virion inhibiting its replication and change the structure of the glycoproteins of HSV (Carlucci et al., 2002). Also, they demonstrated that λ-carrageenan and cyclized μ/ι-carrageenan could interact with HSV-1 and HSV-2 cell surface receptors preventing their adsorption and entry (Carlucci et al., 1997). Without any doubt, polysaccharides have potent antiviral activity against enveloped viruses rather than non-enveloped and EPSNC2 consistent with what was published previously (Ahmadi et al., 2015). Obviously, EPSNC2 contains a high content of glucuronic and galacturonic so; it can block the viral cell surface receptor preventing adsorption or interaction with HSV gC and/or gB glycoproteins preventing HSV gC and/or gB-heparan sulfate interaction via mimicking heparan sulfate proteoglycans.

Figure 6. Anticancer effect against Caco-2 cell line. (a) Control; Caco-2 treated with EPSNC2 (b) 500 µg/ml; (c) 250 µg/ml; (d) 125 µg/ml; (e) 62.5 µg/ml. Partial or complete loss of the confluent monolayer, rounding, shrinkage, and cell granulation were observed. [Click here to view]

Figure 7. (A) Cytotoxicity (%) of EPSNC2 against CaCo-2 colon cancer cell line, (B) Correlation of Anticancer Activity. Significance: p value = 0.0076**. [Click here to view]

Figure 8. (A) Viral Cytopathic effect. (B) Antiviral activity of EPSNC2 against HAV, COXB4, HSV1, and ADENO viruses. Significance: p value = 0.0048**. [Click here to view]

Figure 9. EC50 of EPSNC2 on HSV-1. [Click here to view]