Microbial inhibition
The EPSMSS1's antimicrobial properties protect against pathogenic fungi and bacteria. Data that was obtained indicated that EPSMSS1 exhibited a significant antibacterial effect versus S. aureus NRRLB-767 (70.25%), with a moderate activity against E. coli ATCC 25922 (55.25%). While, it no antibacterial activity against MRSA, Salmonella and P. aeruginosa ATCC 10145. On the other hand, EPSMSS1 displayed low antifungal activity against C. albicans ATCC 10231 and A. niger ATCC 10231. The MIC of the EPSMSS1 was measured and showed that MIC was 6.25, 12.5, 12.5 and 15.25 for E. coli ATCC 25922, S. aureus NRRLB-767, A. niger ATCC 10231, and C. albicans ATCC 10231 (Table 6).
Table 6: Antimicrobial activity and Minimum inhibitory concentration of EPS
Sta: Staphylococcus aureus NRRLB-767, Ps: Pseudomonas aeruginosa ATCC 10145, Sal: Salmonella typhi, Ech: Escherichia coli ATCC 25922, Can: Candida albicans ATCC 10231, Asper: Aspergillus niger ATCC 10231
Antibiofilm assay
The antibiofilm efficacy of EPSMSS1 was assessed using the MTT test against four harmful bacteria including (B. subtilis ATCC 6633, P. aeruginosa ATCC 10145, S. aureus NRRLB-767, and E. coli ATCC 25922). The results shown that EPSMSS1 exhibits a significant antibiofilm activity against S. aureus NRRLB-767 and E. coli ATCC 25922with biofilm inhibition ratio up to 66.35 and 50.23%, while weak biofilm inhibition activity against B. subtilis ATCC 6633 and P. aeruginosa ATCC 10145 (Fig. 11).
Figure 11. biofilm inhibitory activity of EPS (B. subtilis: Bacillus subtilis ATCC 6633, P. aeruginosa: Pseudomonas aeruginosa ATCC 10145, S. aureus: Staphylococcus aureus NRRLB-767, E. coli: Escherichia coli ATCC 25922)
Antioxidant activity of EPSMSS1
The antioxidant activity of EPSMSS1 was examined at various concentrations, the antioxidant activity was done by using different method as diphenylpicrylhydrazyl (DPPH), ROS scavenging capacity, NO scavenging capacity and Fe2+ chelation ability. The data provided indicate that EPSMSS1 demonstrated a promising ability to scavenge DPPH radicals, which was greatly enhanced as the EPSMSS1 concentration rose from 25 to 500 µg/ml (76.40 ± 1.09 and 96.81 ± 1.83%) Table (7a). while, it showed high activity by ROS scavenging capacity 92.838 ± 2.11% at 500 µg/ml Table (7b). Thus, the ability of EPSMSS1 to scavenge NO increased considerably from 14.821 ± 1.55% at a concentration of 25 µg/ml to 93.839 ± 2.29% at the concentration of 500 µg/ml. These values were lower than those of ascorbic acid, which were 49.402 ± 1.43% and 95.893 ± 2.32% at the same concentrations, as indicated in Table (7c). EPSMSS1 demonstrated the capacity to form a complex with ferrous ions in relation to ascorbic acid. EPSMSS1 hindered the development of the Fe2+-ferrozine complex by effectively capturing ferrous ions prior to the interaction with ferrozine, hence preventing complex formation. The chelating percentage of EPSMSS1 was 25.054 ± 1.42% at the lowest concentration and climbed to 95.206 ± 2.53% at the highest concentration, compared to vitamin C which had chelating percentages of 30.137 ± 1.89% and 97.893 ± 1.11% respectively, at the same concentrations Table (7d).
Table 7: Antioxidant activity of EPS as compared with reference standard Ascorbic acid
Antitumor Activity
After the experimental period (3 days), exopolysaccharide was collected and purified from protein, and then Ehrlich cells were treated with various EPSMSS1 concentrations from 20 µg to 40 µg/ml. The antitumor activity of polysaccharide has been evaluated. It was observed that tumor cell counts were significantly reduced with increasing in concentration of EPSMSS1 and the highest activity rate (96.67%) at 40 µg/ml exopolysaccharide concentration.
In vitro anticancer activity against human cell lines
The EPSMSS1 isolated polysaccharide was subjected to MTT assay using 3 different human cancer cell lines namely, human breast carcinoma (MCF-7 cell line), human colorectal carcinoma (HCT-116 cell line), and human pancreatic tumor cell line (Paca2). The initial screening concentration was 100 µg/ml. Each result is the average of three replicated samples, with values indicating the degree of growth suppression (GI) %. According to our results, EPSMSS1 polysaccharides possessed promising cytotoxic potentiality on the colon cell line with GI 63.8%. On the other hand, EPSMSS1 showed weak activity on the remaining breast and pancreatic cancerous cells with 22.3 and 3.8 GI % respectively. Followed by further assay of EPSMSS1 on 3 different concentrations to calculate their IC50 Fig. 12. According to our results, EPSMSS1 showed significant anticancer activity with IC50 20.1µg/ml. Afterward, we studied the effect of EPSMSS1 polysaccharide on normal cells BJ-1, and our results, EPSMSS1 was safe without causing any harm to the natural cells. These findings encourage us to further study the selective apoptotic effect of EPSMSS1 on different apoptotic parameters of the colon HCT-116 cell line.
Figure 12: IC50 of EPS on HCT-116, three different concentrations (100, 50, 25 µg/ml) were assayed their growth inhibition (GI) % values.
Effect of EPSMSS1 on the level of CYC, BCL2, BAX and BAX/BCL2 ratio in HCT-116
To investigate the apoptotic impact of EPSMSS1 on colon cells, we first examine the impact of EPSMSS1 on the structural integrity of the membrane of the mitochondria and the release of cytochrome C into the cytoplasm. Figure 13. Cytochrome C is a protein present in the mitochondrial electron transport chain of healthy cells. A finding of cytochrome C release in the cell's cytoplasm is considered an indicator of apoptosis. Figure 13 states that EPSMSS1 induce mitochondrial membrane permeability and resulted in cytochrome c release into the cytoplasm as compared using cells that have not been treated, with concentrations varying between 0.14 to 0.5 ng/ml. EPSMSS1 induce apoptotic protein BAX in the HCT-116 treated cells as compared to untreated cell from 73 to 292.2 pg/ml. unlike, the downregulation effect of EPSMSS1 on antiapoptotic protein Bcl2 7.9 to 3.3. likely, EPSMSS1 disrupt the BAX/ Bcl2 ratio of colon treated cells and compared to untreated from 0.2 to 1.7.
Figure 13: Cytochrome c (CYC) concentration ng/ml, BAX concentration pg/ml, BCL2 concentration ng/ml and BAX/ BCL2 ratio of HCT-116 treated with 20.1 ug/ml of EPS for 24 hr. compared with reference drug doxorubicin and untreated cells.
Cell cycle arrest
HCT-16 treated with 20.1 ug/ml of EPSMSS1 resulted in an increase in both early and late apoptosis compared with untreated cells, from 0.4 to 11.8% and 0.2 to 21.2%, respectively. Cell cycle examination using flow cytometry was done by exposure of HCT-116 to EPSMSS1 IC50 (20.1 ug/ml) for 24 hr. to examine the apoptotic molecular mode of action of EPSMSS1 on HCT-116. EPSMSS1 elevated the accumulation of cells at the G1 phase and S phase by 1.1 fold in comparison to untreated HCT-116 colon cells. induce an apoptotic cascade on colon HCT-116 through cell cycle arrest at the G1/S phase, as shown in Fig. 14.
Figure 14. Cellular mechanism of action of EPS (A, B), HCT-116 was treated with EPS 24 h and analyzed by annexin V/PI staining. The percentage of apoptotic cells is the sum of early apoptotic (annexin V+/PI−) cell percentage and late apoptotic (annexin V+/PI+) cell percentage. Cell cycle analysis of HCT-116 (C, D) after incubation with compound EPS for 24 hr. Untreated cells were used as a control.