Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.


Introduction
Saponins contain a steroidal or a triterpenoid aglycone to which one or more sugar chains are attached [1]. These chemical structures determine their biological properties as natural nonionic detergents which have cytotoxic, hemolytic, molluscicidal, anti-inflammatory, antifungal, antiyeast, antibacterial, and antiviral activities [2][3][4]. Saponins are nonionic detergents belonging to the group of glycosides. They are used in the pharmaceutical industry because some forms are the starting point for the semisynthesis of steroidal drugs. Many have pharmacological properties and are used in phytotherapy and in the cosmetic industry. Saponins can be classified into two groups based on the nature of their aglycone skeleton: steroidal (consisting of a C27 spirostane skeleton comprising a six-ring structure) and triterpenoid (consisting of a C30 skeleton comprising a pentacyclic structure) [1]. The biological properties of saponins depend on the structure of the aglycone and/or the number of sugar units involved [5]. The therapeutic potential of saponins against eukaryotic cells is associated with their cell membrane-permeabilizing properties by complexing with cholesterol [6]. Our previous studies with a series of synthetic saponins showed their potential as anticancer drugs [7]. Saponins, having surface-active properties, might insert into the lipid bilayer, bind to cholesterol, form domains enriched with cholesterol-saponin complexes, and finally lyse cells [8]. On the other hand, the absence of cholesterol in the membrane structure does not inhibit pore formation by saponins [9,10]. In our previous study, we suggested that saponin might disturb the permeability of the bacterial outer membrane. About 90% of the surface of naturally cholesterol-free Gramnegative bacteria cell-wall outer membranes are covered by lipopolysaccharide (LPS). We concluded that saponin might interact with the lipid A part of Proteus LPSs and thereby increase the permeability of bacterial cell wall. Lipid A-saponin complexes might promote antibiotic (colistin, ampicillin) uptake to inherently resistant bacteria cells [11].
The potentially antibacterial effect of saponins from clinical point of view is associated with a comprehensive investigation of its antibacterial effect and minimal cytotoxic properties in eukaryotic cells. We previously showed that colistin or ampicillin in the presence of 15 μg/mL saponins reduced the numbers of cells of the laboratory strains P. mirabilis S1959 and R45 [11]. An important medical problem is the elimination of pathogenic Gram-positive and Gram-negative bacterial strains. Several types of actions are published, such as bacteriophage treatment [12], inhibition of bacterial adhesion [13], and increasing the permeability of bacterial cell walls [14].
The aims of the present study are (i) testing the saponin abilities to induce hemolytic and cytotoxic effects against eukaryotic cells, (ii) studies of saponin influences on growth of clinical E. coli strains, (iii) testing of saponin abilities to interfere with antibiotic action against E. coli cells.

MTT Test.
Saponin at final concentrations of 12 to 100 μg/mL was added to 500 μl of the CHO-K1 cell suspension (1.5 × 10 6 /mL of complete McCoy's 5A medium) onto plates. CHO-K1 cells were treated with saponin for 72 hours at 37 • C in 5% CO 2 . After treatment, the viability of cells was evaluated by the MTT assay. MTT reagent was added to each plate, and after 6 h of incubation in Hera Cell, 1 mL of SDS (10% in 0.01 N HCl) was added to dissolve the water-insoluble formazan salt. One hour later, the OD 650 nm-OD 570 nm difference was measured. Unexposed cells were regarded as 100% viable [15].

Apoptosis.
CHO-K1 cells were treated with saponin at different concentrations (6,12,24, and 50 μg/mL) for 72 hours. After treatment, the frequencies of early apoptotic, late apoptotic, and necrotic cells were evaluated with the Annexin V-FITC apoptosis detection Kit I (BD Pharmingen, USA) described elsewhere [16]. Briefly, the cells were washed two times with cold PBS and then resuspended in a 1 × binding buffer at a concentration of 1 × 106 cells/mL. An aliquot of 125 μL of the cell suspension was incubated with 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) at room temperature for 15 min in the dark. The cells were resuspended in 400 μL of 1 × binding buffer. The fluorescence was determined using a Becton Dickinson FACSscan flow cytometer. A computer system (CellQuest Pro, Becton Dickinson) was used for data acquisition and analysis. Data for 20000 events were stored. A cell gate containing CHO-K1 was established on the basis of forward and side light scatter. Four different populations of cells were detected with the Annexin V-FITC kit: normal cells that are Annexin negative and PI negative and express no fluorescence, early apoptotic cells that are Annexin positive and PI negative and express green fluorescence, late apoptotic/necrotic cells that are Annexin positive and PI positive and express green and orange fluorescence, and necrotic cells that are Annexin negative and PI positive and express orange fluorescence.

Hemolysis
Assay. Blood was obtained from healthy young male donors. The red blood cells (RBCs) were washed three times and resuspended in sterile PBS to give about 15 × 10 6 cells per mL and further processed [17]. The erythrocytes were incubated with saponin in a range of 0.5-50 μg/mL for 1 h at 37 • C. After centrifugation of the nonhemolysed erythrocytes (1400 rpm, 15 min), the absorbance of the released hemoglobin in the wavelength range of 400-600 nm was measured. The percentage of hemolysis was determined by comparing the absorbance of hemoglobin at 435, 540, and 575 nm released from the RBCs in the presence of saponin. The positive control (100% hemolysis) was determined by the amount of hemoglobin released from 15 × 10 6 RBCs after 10 min of incubation with water.    were found as assessed by the Snedecor-Fisher test, the differences were compared by the ANOVA test.

The Effect of Saponin on Eukaryotic Cells
3.1.1. Drug Resistance. Figure 1 shows the percent viability of the CHO-K1 cells after 4-day treatment with saponin at concentrations ranging from 12 to 100 μg/mL measured by the MTT assay. It was found that saponin at concentrations 25 and 50 μg/mL caused 70% and 50% cell killing, respectively. Table 1 shows the level of apoptosis measured by flow cytometer with the Annexin V-FITC kit and propidium iodide staining according to [16]. Saponin dosedepended lyses of CHO-K1 cells were observed, as in MTT test ( Figure 1). The number of early and late apoptotic cells was detected after incubation with saponin in a concentration range of 12-50 μg/mL. The flow cytometric analyses of the CHO-K1 cells population showed that saponins at concentrations of 12-50 μg/mL significantly increased the percentage of early apoptotic cells up to 30.88%. It was in a saponin dose-dependent manner compared with the untreated control. The increase in the number (6.21%) of late apoptosis was notice at the dose of 12 μg/mL of saponin. The necrosis of cells varied and did not excite 2.52% at saponin dose 50 μg/mL.

Hemolysis Assay.
In addition to CHO-K1 cell line, the human red blood cells (RBCs) were used as hemolysis marker. Figure 2 shows the amount of hemoglobin released from RBCs that was determined by the absorbance at 435, 540, and 575 nm in the presence of saponin at concentrations ranging from 3 to 100 μg/mL. We observed a statistically significant, dose-depended increase in hemolysis at concentrations from 25 up to 100 μg/mL. The proportion of pick at 435, 540, and 575 nm was similar. This may suggest that porfirine rings and iron molecules are not released from hemoglobin, and observed hemolysis is the effect of RBCs wall destruction. The saponin in range 3-12 μg/mL does not induce hemolysis. This indicates that critical amount of saponin molecules 25 μg/mL is required to destabilize cell wall of erythrocytes. Figure 3(a) shows the effect of saponin at concentrations ranging from 1 to 12 μg/mL on the growth of the six clinical E. coli strains incubated for 18 h at 37 • C in LB medium. The presence of saponin does not inhibite E.coli cells growth. It is worth to notice that we observed statistically significant increases in CFU of the multidrug resistant-(MDR-) E. coli strains in the presence of saponin at a concentration of 6 and 12 μg/mL in LB medium, in contrast to growth of bacterial strains in M9 medium which was influenced by saponin presence (Figure  3(b)).

The Effect of Saponin and Antibiotics on Bacterial
Growth. In next experiment, dose of 12 μg/mL of saponin in LB medium was used for testing antibiotic action against E. coli cells. Figure 4 shows the effect of the presence of saponin and ampicillin, streptomycin, or ciprofloxacin at concentrations ranging from 25 to 400 μg/mL on the growth of the clinical E. coli strains incubated for 18 h at 37 • C in LB medium. All six strains were sensitive to ciprofloxacin and resistant to ampicillin and streptomycin. The addition of saponin caused in strains 3 and 6 that ciprofloxacin was less effective (

Discussion
Medically desired situation is the concentration of saponins with antibacterial properties that has minimal cytotoxic activity against eukaryotic cells. Saponins possess detergent-like properties and might increase the permeability of bacterial cell membranes without destroying them [19]. In theory, this activity might facilitate antibiotic influx through the bacterial cell wall membrane. In the presented studies, we compared saponins' activities against prokaryotic and eukaryotic cells. We used commercial saponin from Quillaja saponaria, which is one of the major sources of industrial triterpenoid saponins. Saponins extracts from Quillaja saponaria have been in practical use as foaming agents in beverages or emulsifiers in foods [20]. In our investigations, we analyzed the activity of saponin from Quillaja saponaria against CHO-K1 cell line. The saponin extracts increase the number of early apoptotic cells in a dose-dependent manner. Saponins do not induce significantly necrosis and late apoptosis. Effect of saponin on membrane receptors for apoptosis was shown also by others [5]. In our studies, we also have shown that saponin at concentrations higher than 12 μg/mL has significantly cytotoxic effect on CHO-K1 cells.    saponin at a concentration of 12 μg/mL confirmed that saponins enhance bacteria growth, even in antibiotic presence.
In conclusion, we presented varied potencies of saponins against eukaryotic and prokaryotic cells. That is probably due to differences on cell membranes of two cell types. The biological properties of chemical substances potentially useful in clinical use should be characterized by their defined activity in correlation with the optimal concentration.