Background

Essential liver tumor is the fifth most normal threat on the planet, with a worldwide yearly occurrence of around one million new patients (Shah and Bhowmick 2006). The malady wins in parts of Asia and Africa, yet seems overflowing in numerous European nations lately. As need be, it is encouraged to investigate another approach for improvement of a viable treatment against this illness. In spite of the fact that chemotherapy is one of the compelling medication strategies against liver cancer, chemotherapeutic operator prompts serious unfavorable impacts while murdering tumor cells (Drake and Antonarakis 2010). At that point, it is extremely essential to discover novel bacteria hostile to tumor specialists with high natural exercises and low danger to host. Natural products have been the pillar of tumor chemotherapy for as far back as 30 years and are probably going to give a considerable lot of the lead structures, and these will be utilized as layouts for the development of novel compounds with upgraded organic properties (Wani et al. 1971). The marine condition is a rich wellspring of remarkable bioactive compounds, for example, exopolysaccharides (EPSs) from microorganisms with boundless chemical and functional varieties. Pretty much 30,000 regular items have been segregated from marine organisms, and a few of the medication competitors are right now in clinical trials. Marine microorganisms frequently deliver bioactive substances with novel capacities and structures in view of their uncommon living conditions (Fenical 1993). EPSs from marine microorganisms are important to new medication disclosure (Miranda et al. 2008; Xu et al. 2009; Casillo et al. 2018). EPSs have different biological activities with low poisonous quality and have pulled in broad considerations of researchers (Huang et al. 2012; Wang et al. 2014; Cai et al. 2017). As of late, various investigations on the mechanism of action of EPSs have exhibited that EPSs could repress the tumor development in vivo for their immunomodulatory activities. They apply hostility to tumor movement by boosting the host’s common insusceptible resistance. Along these lines in this investigation, different EPSs segregated from various marine bacteria were tried for their capacity on liver malignancy cell line (Yang et al. 2013; Peng et al. 2014; Yang et al. 2014). The present study aimed to isolate and identify new marine bacterial strains that produced EPSs. The cytotoxic effects of these crude EPSs were tested against hepatocellular carcinoma (HepG2) cells.

Methods

Sampling

Three samples were collected from marine sediment at different locations from (Mediterranean Sea) Sidi Bishr beach at Alexandria, (Red Sea) Marsa-Alam (rhizosphere around the mangrove tree) areas, and El-Ain El-Sokhna beach in September 2014.

Isolation of bacteria

The collected samples (10 g) were placed in 100 mL of sterile seawater and homogenized by shaking at 200 rpm for 15 min, and a serial dilution was performed (Hayakawa and Nonomura 1987). Finally, 50 μL of the supernatant of each dilution was inoculated on marine nutrient agar, and the plates were incubated at 37 °C for 24 h. The colonies that appeared per plate of each sample were subjected to purification. Isolated microorganisms showing mucous growth on these media were further screened for EPS production by inoculation into shake flasks.

Screening for production of EPSs

Isolates were screened for production of EPSs in a different liquid media which were composed of the following ingredients (g/L): peptone 4.0, yeast extract 2.0, and sucrose 20 (Jiang et al. 1999). The ingredients were dissolved in 750 mL seawater. After adjusting the pH, the final volume was completed up to 1 L with distilled water. After incubation at 37 °C for 3 days, the culture medium was centrifuged at 5000 rpm for 20 min (Sigma Laborzentrifugen, 2 K 15) to remove bacterial cells. Trichloroacetic acid (5%) was added and left overnight at 4 °C and centrifuged at 5000 rpm again. The pH of the clear solution was adjusted to 7.0 with NaOH solution and dialyzed three times against distilled water. The supernatant was completed to four volumes with absolute ethanol and left overnight at 4 °C. The precipitated polysaccharides were separated by centrifugation at 5000 rpm, washed twice with acetone, dehydrated by ether, and finally dried under vacuum at 40 °C (Shene et al. 2008).

Cytotoxic effects of isolated EPSs on HepG2 cells

Cell propagation and maintenance

Hepatocellular carcinoma HepG2 cells were purchased from ATCC (American Type Culture Collection) and maintained in the proper conditions. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Belgium) supplemented by 10% fetal bovine serum, 4 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate at 37 °C in a humidified incubator with 5% CO2. The cells were harvested after trypsinization (0.025% trypsin and 0.02% EDTA) and washed twice with Dulbecco’s phosphate-buffered saline (DPBS). When the cell density reached approximately 80%, cells were split for further culture. The experiments were made up when the cells were in the logarithmic growth phase.

Cytotoxicity assay

Cell viability was measured by neutral red uptake assay (Repetto et al. 2008). The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. The cells were incubated with various concentrations of the test compounds (125, 250, 500, and 1000 μg/mL) for 48 h at a cell density of 104 cells/well of a 96-well plate. Using the relation between used concentrations and neutral red intensity value, IC50 of tested compounds was calculated (Repetto et al. 2008). Four parameter equation logistic curve was used (log concentration vs. percentage cell growth as compared to control cells). For the untreated cells (negative control), medium was added instead of the test compounds. A positive control doxorubicin (Mr = 579.9) was used as cytotoxic natural agent giving 100% inhibition. Dimethyl sulfoxide (DMSO) was the vehicle used for dissolution of tested compound, and its final concentration on the cells was less than 0.2%. All tests and analyses were done in triplicate, and the results were averaged.

Identification of promising isolates

The promising isolates which produced high amounts of EPS were identified based on biochemical, morphological, and physiological characteristics of the potential producer as determined by adopting standard methods (Cappucino and Sherman 2004). The strains were confirmed with 16S ribosomal RNA gene sequence and compared with other bacterial sequences by using NCBI BLAST. Taxonomic affiliation of the sequences was retrieved from classifier program of ribosomal database project (RDP-II) (Maidak et al. 1997; Tamura et al. 2007). RDP-II hierarchy is based on the new phylogenetically consistent higher order bacterial taxonomy.

Production, separation, and purification of EPSs

The EPSs were produced from the five strains Brevundimonas subvibrioides MSA1, Bacillus thuringiensis E4, Bacillus amyloliquefaciens MGA2, Pseudomonas fluorescens SGA3, and Advenella Kashmirensis NRC-7. Therefore, the crude EPS was precipitated with 1,2,3,4-volumes absolute ethanol, and supernatant was recovered by centrifugation. EPS fractions named MSA1, E4, MGA2, SGA3, and NRC7 were obtained from the supernatant mentioned above by precipitating with ethanol. The main fractions were collected, dialyzed, dried, and used for further analysis.

Chemical structure of EPSs

Analysis of chemical composition

The EPS samples (30 mg) were hydrolyzed with 88% HCOOH (5 mL) at 100 °C in a sealed tube for 5 h. Excess acid was removed by flash evaporation on a water bath at a temperature of 40 °C and co-distilled with water (1 mL × 3) (Sudhamani et al. 2004). Then, the hydrolyzed monosugars were extracted with absolute ethanol. The purified hydrolysis monosugars were analyzed by HPLC (Agilate Pack, serics1, 200), equipped with Aminex carbohydrate HP-87C column (300 × 7.8 mm). Deionized water was used as the mobile phase at flow rate 1 mL/min. Chromatographic peaks were identified by comparing the retention times with the respective retention times of known standard reference material. Retention time and peak area were used to calculate sugar concentration by the data analysis of Agilat Packard (Randall et al. 1989). Total sugar content was determined by the phenol-H2SO4 method using glucose as the standard (Dubois et al. 1956). Protein content was measured by the method of Bradford (1976). Sulfate ester content was measured using the turbidimetric method with sodium sulfate as standard (Therho and Hartiala 1971). Uronic acid content was determined by the carbazole-H2SO4 method using glucouronic acid as standard (Bitter and Muir 1962).

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared was obtained by grinding a mixture of EPS sample with dry KBr and pressing in a mold. An IR spectrum was recorded on a Fourier transform infrared spectrophotometer Brucker Scientific 500-IR (Ray 2006).

Average molecular weight determination

The average molecular weight (Mw) of the EPSs was determined by high-performance liquid chromatography (HPLC, Agilent 1100 Series System, Hewlett-Packard, Germany) with refractive index (RI) detection. The EPS (10 mg) was dissolved in 2 mL of solvent and filtrated by filter 0.45, then the EPS solution was put in a GPC device (Jun et al. 2009). The polydispersity index (PI) was calculated from the Mw/Mn ratio (You et al. 2013). Number average molecular weight (Mn) and weight average molecular weight (Mw) were directly calculated according to the definition of Mn and Mw using molecular weight and RI signal values at each elution volume (You et al. 2013).

Results

Screening for production of EPSs

Twenty isolates of marine bacteria were isolated from Marsa-Alam, El-Ain El-Sokhna, and Sidi Bishr. The bacterial isolates gathered from different marine sediments and displaying mucous morphology on marine supplement agar medium were inoculated into 50 mL of nutrient broth under shaking. Marine bacterial isolates were screened for their ability to deliver EPS (Table 1). The most elevated yield of EPS (7.5, 8.2, 8.1, 7.6, 8.2, 9.0, 8.0, 8.5, 9.2, 7.9, 7.3, 8.0, and 9.1 g/L) was acquired by isolates M1, M3, M6, M15, M18, M19, E2, E4, E10, E20, S5, S12, and S17, separately.

Table 1 Production of EPS from isolates
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In vitro cytotoxicity on HepG2 cell line

The EPSs from 20 isolates have been examined for their anticancer activity against hepatocellular carcinoma HepG2 cell line. This was done using the ability of viable cells to incorporate and bind the survival dye neutral red in lysosomes. In an essential screening test, EPS (M1, M3, M6, M15, M19, E2, E4, E10, S5, S7, and S11) decreased the viability of HepG2 cell line in concentrations 125, 250, 500, and 1000 μg/mL (Fig. 1a–d and Table 2).

Fig. 1
figure 1

Effects of EPSs a S17, M19, M18, E14, and M13; b E16, M6, E10, S5, and S7; c M3, S11, E2, S12, and M1; and d E8, E4, M15, E20, and S9 on HepG2 cell line at 48 h

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Table 2 In vitro cytotoxicity on HepG2 cell line
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Identification of promising isolates

The promising isolates which give high yield of EPSs and cytotoxicity against HepG2 (M19, E4, M6, E2, S7) were distinguished in light of biochemical, morphological, and physiological characteristics (Table 3). The phylogenetic investigation of 16S rDNA exhibited that the bacteria had a place with the gamma subdivision of the Proteobacteria phylum and is firmly identified as Brevundimonas subvibrioides MSA1 (M19), Bacillus thuringiensis E4 (E4), Bacillus amyloliquefaciens MGA2 (M6), Pseudomonas fluorescens SGA3 (E2) and Advenella Kashmirensis NRC-7 (S7), with accession numbers KP064319, KP096416, KP064320, KP064321, and KM017000, separately.

Table 3 Morphology and biochemical characterization of promising isolates
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Chemical structure of EPSs

Chemical analysis of EPS fractions

The yields of the EPSs from Brevundimonas subvibrioides MSA1, Bacillus thuringiensis E4, Bacillus amyloliquefaciens MGA2, Pseudomonas fluorescens SGA3, and Advenella Kashmirensis NRC-7 were 7.5, 8.5, 8.2, 9.0, and 5.8 g/L broth media, individually. The main fractions MGA2, MSA1, E4, SGA3, and NRC7 were purified by fraction with ethanol precipitation from the crude EPSs. The fractions MGA2, MSA1, E4, SGA3, and NRC7 were gathered for further investigation of structure and activity. MGA2, MSA1, E4, SGA3, and NRC7 had a negative response to the Bradford test. This outcomes demonstrate the absence of protein and/or nucleic acid. The average molecular weights (Mw) and average molecular numbers (Mn) of MGA2, MSA1, E4, SGA3, and NRC7 were 2.51 × 104, 1.94 × 104, 1.18 × 105, 7.95 × 105, and 2.48 × 104 g/mol and 2.13 × 104, 1.51 × 104, 1.05 × 105, 7.53 × 105, and 2.32 × 104 g/mol, respectively (Table 4). Results from phenol-H2SO4 assay showed that MGA2, MSA1, E4, SGA3, and NRC7 contained 87.4, 90.2, 94.3, 97.5, and 97.1% carbohydrate; 1.9, 2.7, 8.6, 7.3, and 13.3% uronic acid; and 12.5, 9.6, 5.4, 2.1, and 2.8% sulfate, respectively (Table 5). These show that MGA2, MSA1, E4, SGA3, and NRC7 are acidic sulfated heteropolysaccharides. Concerning monosaccharide quantitative and qualitative determination, the use of HPLC is favored due to its sensitivity. MGA2, MSA1, E4, SGA3, and NRC7 are comprised of five distinct monosaccharides, including glucose, mannose, galactose, glucouronic acid, and mannouronic acid with molar ratios of 1.0:1.6:1.2:0.2:0.0; 2.3:1.0:0.0:0.2:0.0; 2.52:1.0:0.0:0.0:1.4; 3.6:0.0:1.0:0.5:0.4; and 1.0:2.5:0.0: 1.8:0.6, respectively (Table 5).

Table 4 Molecular weights and polydispersity of EPS
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Table 5 The uronic acid, sulfate percentage, and molar ratio of monosaccharides for MGA2, MSA1, E4, SGA3, and NRC7
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FTIR analysis of MGA2, MSA1, E4, SGA3, and NRC7

FTIR analysis of the EPS fractions MGA2, MSA1, E4, SGA3, and NRC7 has unmistakably demonstrated a number of peak characteristics of carbonyl compounds. The intensity of bands around 3431 cm−1 was assigned to υOH stretching frequency, and as expected, it was broad. The weak intensity of band attributed to C–H group stretching (~ 2933 cm−1) could also be noticed. Also, peaks at 1636 cm−1 and 1428 cm−1, for the asymmetric and symmetric stretching of carboxylate anionic groups, demonstrate the carboxyl groups in MGA2, MSA1, E4, SGA3, and NRC7. Absorption at 1429 cm−1 was possibly due to non-symmetric CH3 bending. The strong absorption at 1069 cm−1 was dominated by the glycosidic linkage υ (C–O–C) stretching vibration contribution (Fig. 2a–f).

Fig. 2
figure 2

Infrared of EPSs a MGA1, b MGA2, c E4, d SGA3, and f NRC7

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Discussion

Recently, EPSs have great potential as antitumor drug, as recent treatment strategies showed limitations of severe side effects and multidrug resistance occurred (Romanenko et al. 2008). Marine microbes have turned out to be well known and novel sources of EPSs, albeit numerous known marine bacteria can produce EPSs. Few of them are of biotechnological significance, so the search of EPSs that may have imaginative applications is still of potential intrigue (Casillo et al. 2018). While trying to get the marine bacterial strains, we separated from various areas and assessed based on the productivity of the EPSs. From the 20 marine EPSs, the promising IC50 of 11 EPSs (M1, M3, M6, M15, M19, E2, E4, E10, S5, S7, and S11) were 524.0, 549.0, 562.0, 501.0, 309.0, 537.0, 630.9, 549.0, 436.0, 389.0, and 407.0 μg/mL, individually. However, the remaining EPSs showed moderate anti-proliferative activities (Table 2). A few polysaccharides significantly inhibit the development of malignancy cells as illustrated by Huang (2013), while other polysaccharides demonstrated different functions. For instance, the polysaccharides from Ganoderma lucidum fundamentally decreased tumor cells in vitro (Zhao et al. 2010); on the other hand, polysaccharide from Pleurotus sp. mycelium demonstrated its antioxidant activities in vitro (Liu et al. 2010). Marine bacterial strain GWS-BW-H8hM was accounted for to hinder development of gastric cell line (HM02), liver cell line (HepG2), and bosom cell line (MCF7) (Bitzer et al. 2006). EPSs and sulfated polysaccharides from Halomonas stenophila sp. repressing hypersaline condition have likewise been accounted for their pro-apoptotic effects for T-leukemia cells (Ruiz-Ruiz et al. 2011; Xu et al. 2014). These show that MGA2, MSA1, E4, SGA3, and NRC7 are acidic sulfated heteropolysaccharides. MGA2, MSA1, E4, SGA3, and NRC7 are comprised of different monosaccharides including glucose, mannose, galactose, glucouronic acid, and mannouronic acid. A lot of marine bacteria are encompassed by EPSs, which may help bacterial groups to endure extremes of saltiness, temperature, and nutrient availability (Casillo et al. 2018). In view of the chemical and rheological properties of the EPSs generated by these bacteria, examinations were performed to test their potential applications in biotechnology and ecological protection (Nichols et al. 2005; Guezennec 2002). The item and nature of bacterial EPSs are exceptionally affected by the biological and dietary status (Kumar et al. 2007). Most EPSs created by marine microbes are heteropolysaccharides containing diverse unit of monosugars coordinated in a range of around 10 to compose repeating units (Decho 1990). For the most part, EPSs are straight, with molecular weight from 1 to 3 × 105 Da (Sutherland 1977). Numerous EPSs are neutral molecules, but the greater parts are polyanionic for the SO3−, PO4−, and uronic acid presence. The physical properties of EPSs are incredibly influenced by the method for the monosugars arranged and the accumulation of the one polymer chain (Vanhooren and Vandamme 1998). Biological activities of EPSs are depending on its chemical structure and the molecular weight. The presence of rate sugars as ribose and arabinose besides the uronic acid in the order of polygalacturonic, glucouronic, galacturonic acid, and polymers of low molecular weight are important indicators reflecting the antioxidant activity of the EPSs (Roca et al. 2015). Furthermore, in the FTIR spectrum of MGA2, MSA1, E4, SGA3, and NRC7, the characteristic band at 929 cm−1 belonged to the β-anomeric configuration in MGA2, SGA3, and NRC7 (Synytsya et al. 2003; Zhbankov et al. 1997), and the band at 840 cm−1 was ascribed to α-pyranoses in the MSA1 and E4 (Park 1971). Besides, absorption at 1271 cm−1 was assigned to the stretching vibration of S=O indicating MGA2, MSA1, E4, SGA3, and NRC7 were sulfated polysaccharides (Percival and Wold 1963).

Conclusion

EPSs produced from marine bacteria are very promising for treating the hepatocellular carcinoma HepG2 cells.