Optimization of Triterpene Saponins Mixture with Antiproliferative Activity

: In this study, three of the saponins present in leaves of Hedera helix L., α -hederin, hederagenin, and hederacoside C were studied for their antiproliferative activity. The three saponins were analyzed in di ﬀ erent concentrations by in vitro tests on normal ﬁbroblasts cells and cervix ephitelial tumor cells. Determination of cytotoxicity and antitumor e ﬀ ects was performed using the MTT method. From the tested saponins, α -hederin was biocompatible in normal ﬁbroblasts cells at concentrations between 2–10 µ g / mL. Its antiproliferative activity was exerted in the concentration range of 10–400 µ g / mL in cervix ephitelial tumor cells. Similarly, hederagenin presented antiproliferative activity at concentrations between 25–400 µ g / mL. In turn, hederacoside C was shown to be noncytotoxic in normal ﬁbroblasts and cervix ephitelial tumor cell culture at all the tested concentrations. The obtained experimental results were analyzed by “Mixture design”, a specialized form of the response surface method (RSM) provided by the Design Expert 11 software, and the optimal composition of obtained saponins mixture was selected and veriﬁed in vitro for antiproliferative activity. The results showed that an optimal saponins mixture has the potential to be used in pharmacological applications.

Structurally, saponins are amphiphilic compounds composed of one or more hydrophilic sugar parts and a lipophilic steroidal or triterpenic part (sapogenin). Saponins can be classified into monodesmosidic, bidesmosidic, or polydesmosidic saponins, having one, two, or more sugar chains. A large structural variety can be found in nature, due to the presence of different sugars, sugar branchings, and sapogenins [9][10][11].
The interesting properties of saponins have led us to investigate the monodesmosidic saponin α-hederin, which has shown activities towards cancer cells [12]. α-Hederin, or Kalopanax saponin

Cell Culture
The cell cultures used for in vitro testing were cultivated in MEM, supplemented with 10% FBS, 2 mM L-glutamine and a mixture of 100 U/mL penicillin, 100 µg/mL streptomycin, and 500 µg/mL neomycin.
Appl. Sci. 2019, 9, 5160 3 of 14 Fibroblasts were seeded at a density of 4 × 10 4 cells/mL for NCTC culture and 4 × 10 4 cells/ mL, while Hep-2 cells were seeded at a density of 6 × 10 4 cells/ mL in 96-well culture plates and incubated at 37 • C in a humid atmosphere with 5% CO 2 for 24 h using a Bioquell biology security cabinet.
Standard saponins were solubilized in a small amount of DMSO and diluted in the culture medium to obtain stock solutions which were filtered through 0.22 µm fillters (Millipore Merck, Burlington, MA, USA). They were added tothe culture medium at concentrations of 2, 5, 10, 25, 50, 100, 200, 300, and 400 µg/mL. The compounds tested in NCTC culture were diluted in MEM supplemented with 10% FSB, while for Hep-2 tumor cell culture, a parallel testing was performed in two variants: in the first one, the samples were diluted in MEM supplemented with 10% FBS, and in the other one, the samples were diluted in FBS-free of MEM [8]. In addition, mixtures of saponins determined by mathematical modeling were also tested in the same conditions. The tested samples were added in triplicate to the wells of the culture plates. The culture plates were incubated under standard conditions (37 • C, 5% CO 2 ) for 24 h. Untreated cultured cells were used as control.

In Vitro Cytotoxicity Testing by MTT Assay
Cell viability after cell cultivation with samples was determined using theMTT colorimetric method in which tetrazolium salt reacted with mitochondrial dehydrogenases from the metabolic active cells reducing, with the formation of blue-violet formazan crystals which were insoluble in the culture medium [29]. For the cytotoxicity assay, the culture medium was replaced with MTT solution at a concentration of 50 µg/mL, followed by culture plate incubation for 3 h. After incubation, isopropanol was added to solubilize the formazan crystals that formed in viable cells by shaking the plates for 15 min on an orbital shaker. Determination of cell viability was performed after 24 h of treatment by optical density (OD) measurement of the colored solution at a wavelength of 570 nm. The measured OD is directly proportional to the number of viable cells present in the cell culture. The results were calculated using the following formula: % cell viability = (OD sample/OD control) × 100% Control cells were considered to have 100% viability.

Mixture modeling using Design Expert 11 software
In a mixture experiment, the independent factors are proportions of different components of a blend, and the measured response is assumed to depend only on the relative proportions of the ingredients or components in the mixture [30].
The Response Surface Method (RSM) provided by Design Expert 11 software is a specialized statistical tool used to model the blending surface with some form of mathematical equation [28,31], so that:

1.
Predictions of the response for any mixture or combination of the ingredients can be made empirically or statistically.

2.
Some measure of the influence on the response of each component and in combination with other components can be obtained.

3.
The optimization feature can be used to calculate the optimum composition for a mixture to maximize a certain effect.

Statistical Analysis
The results of cell culture experiments were expressed as mean value ± standard deviation (SD) of three independent samples (n = 3). Statistical analysis of the data was performed using one-tailed, paired Student's t-test (Excel, Office Excel 2007 software, Microsoft, Redmond, WA, USA) on each pair of interest. Differences were considered statistically significant at p ≤ 0.01.

Chemical Formula of Saponins
The structures of studied saponins are presented in Figure 1.

Cytotoxicity of Saponins in NCTC Cells
The results of the MTT assayfor the standard saponins in NCTC cells are presented in Figure 2. The values indicated that the compound α-hederin presented a high degree of biocompatibility in NCTC cells in the concentration range of 2-10 μg/mL (99-82% cell viability) at 24 h. It was slightly cytotoxic at 25 μg/mL (62% cell viability) and severely cytotoxic throughout the range of concentrations 50-400 μg/mL, when the cell viability decreased to 0.4%.
Hederagenin was biocompatible in NCTC cells from 2 to 50 μg/mL, had moderate cytotoxicity in the range of 100-300 μg/mL, with values of 73-56% cell viability, and was cytotoxic at 400 μg/mL, with only 49% cell viability at 24 h.

Cytotoxicity of Saponins in NCTC Cells
The results of the MTT assayfor the standard saponins in NCTC cells are presented in Figure 2. The values indicated that the compound α-hederin presented a high degree of biocompatibility in NCTC cells in the concentration range of 2-10 µg/mL (99-82% cell viability) at 24 h. It was slightly cytotoxic at 25 µg/mL (62% cell viability) and severely cytotoxic throughout the range of concentrations 50-400 µg/mL, when the cell viability decreased to 0.4%.
Hederagenin was biocompatible in NCTC cells from 2 to 50 µg/mL, had moderate cytotoxicity in the range of 100-300 µg/mL, with values of 73-56% cell viability, and was cytotoxic at 400 µg/mL, with only 49% cell viability at 24 h.
Comparing the cytotoxicity assay results of the three studied saponins in NCTC cells, it was concluded that α-hederin was biocompatible only up to10 µg/mL, while in the concentration range of 25-400µg/mL, it was cytotoxic; hederagenin had moderate cytotoxicity between 100-300µg/mL concentrations, and increased cytotoxicity at 400µg/mL, while hederacoside C was biocompatible in the concentration range of 2-400µg/mL. Comparing the cytotoxicity assay results of the three studied saponins in NCTC cells, it was concluded that α-hederin was biocompatible only up to10 μg/mL, while in the concentration range of 25-400μg/mL, it was cytotoxic; hederagenin had moderate cytotoxicity between 100-300μg/mL concentrations, and increased cytotoxicity at 400μg/mL, while hederacoside C was biocompatible in the concentration range of 2-400μg/mL.

Antiproliferative Activity of Saponins in Hep-2 Cells
The MTT testresults for the analyzed standard saponins in Hep-2 tumor cells indicated that α-hederin induced the most potent antitumoral effect (Figure 2). At a concentration of 2 μg/mL, α-hederin was biocompatible in NCTC cells (99% cell viability), while in Hep-2 tumor cells cultivated in medium, it was antiproliferative (51% viability) after 24 h of treatment.
The MTT assay results for hederagenin tested in Hep-2 cells cultivated in a FBS-free medium exerted a moderate cytotoxicity activity at 25-300 μg/mL and strong cytotoxicity at 400 μg/mL (52% cell viability). In cells cultivated in a medium with FBS, it was observed that hederagenin had no significant antitumor activity, with the viability of Hep-2 tumoral cells exceeding 80% within the entire range of concentrations (2-400 μg/mL).
Hederacoside C was biocompatible within the entire range of concentrations (2-400 μg/mL), with no significant antitumoral effect when cultivated in medium with or without FBS ( Figure 2).
Our study confirmed that saponins tested in the medium containing FBS presented very low antiproliferative activity. Hederagenin in the medium with FBS did not present any cytotoxicity in Hep-2 tumor cells, compared to the experiment in medium without FBS.
The antitumor activity of standard saponins was also calculated as the inhibitory concentration of compounds that killed 50% of cells (IC50, μg/mL). The results are presented in Table 1.

Antiproliferative Activity of Saponins in Hep-2 Cells
The MTT testresults for the analyzed standard saponins in Hep-2 tumor cells indicated that α-hederin induced the most potent antitumoral effect (Figure 2). At a concentration of 2 µg/mL, α-hederin was biocompatible in NCTC cells (99% cell viability), while in Hep-2 tumor cells cultivated in medium, it was antiproliferative (51% viability) after 24 h of treatment.
The MTT assay results for hederagenin tested in Hep-2 cells cultivated in a FBS-free medium exerted a moderate cytotoxicity activity at 25-300 µg/mL and strong cytotoxicity at 400 µg/mL (52% cell viability). In cells cultivated in a medium with FBS, it was observed that hederagenin had no significant antitumor activity, with the viability of Hep-2 tumoral cells exceeding 80% within the entire range of concentrations (2-400 µg/mL).
Hederacoside C was biocompatible within the entire range of concentrations (2-400 µg/mL), with no significant antitumoral effect when cultivated in medium with or without FBS ( Figure 2).
Our study confirmed that saponins tested in the medium containing FBS presented very low antiproliferative activity. Hederagenin in the medium with FBS did not present any cytotoxicity in Hep-2 tumor cells, compared to the experiment in medium without FBS.
The antitumor activity of standard saponins was also calculated as the inhibitory concentration of compounds that killed 50% of cells (IC 50 , µg/mL). The results are presented in Table 1. α-Hederin presented the lowest value of IC 50 in Hep-2 cells in the medium without FBS (2 µg/mL) and with FBS (25 µg/mL), indicating the highest antiproliferative activity from the three tested saponins. In turn, hederagenin and hederacoside C presented low antiproliferative activity (IC 50 >400 µg/mL).
Previous in vitro studies suggested that the chemical bonds between saponins and proteins from fetal calf serum (FCS) reduced their antitumor effect [8]. In our saponin antitumoral activity evaluations, the growth factor added in the culture medium was FBS, known to have a similar composition to FCS and to be rich in proteins such as albumin, globulin, and gamma globulin [32]. FBS also diminishedthe antitumor activity of saponins by protein binding. Thus, in our in vitro study, saponin samples were processed (individually and mixtures variants) in a medium supplemented, or not, with FBS.
Similar studies showed that the most active compounds in mouse B16 tumor cells and 3T3 mouse normal fibroblasts αand β-hederins were cytotoxic at concentrations greater than or equal to 10 µg/mL [19]. Other in vitro experiments on B16 mouse melanoma and HeLa human tumor cells cultivated in serum-free media showed that α-hederin was toxic at concentrations less than 5 µg/mL after only 8 h of treatment [33]. α-Hederin induced the vacuolization of the cytoplasm and the alteration of the membranes that caused cell death. However, the cytotoxicity of the phytocompound was reduced in the presence of fetal calf serum (FCS) or bovine serum albumin (BSA) in the culture medium due to interactions between α-hederin and proteins.
In a study of hederagenin apoptosis effects and its possible mechanism of action in human colon cancer LoVo cells, the MTT assay showed significant inhibition of cell proliferation in a concentrationand time-dependent manner. The IC 50 was 1.39 µM at 4 h and 1.17 µM at 48 h of cultivation. The apoptosis percentage increased from 32% to 82% when the hederagenin concentration increased from 1 to 2 µM [34].

Mixture Modeling Using Design Expert 11 Software
In our study, the obtained cytotoxicity and antiproliferative experimental results were analyzed by RSM, provided by Design Expert 11, correlating the composition of the studied saponin mixtures with cytotoxic properties in order to optimize the saponin mixture with an antiproliferative effect.
The considered components and their selected concentration limits, estimated based on cytotoxicity assay results, are presented in the Table 2. The software generated 14 variants of mixture compositions for the tested saponins presented in Table 3.

In Vitro Cytotoxicity and Antiproliferative Activity of the Saponin Mixtures
Each of the 14 variants of saponin mixtures generated by Design Expert 11 software were tested in a similar way used for the cyototoxicity evaluation of standard saponins, with determination of cell viability in both normal and tumor cells. The obtained results are presented in Table 4. From the values obtained for cell viability at 24 h of Hep-2 response in medium without FBS, it can be seenthat for the variation limits were very large and it was used as a means of transformation: ln (Y).

Statistical Analysis
Using analysis of variance, the software proposed the models presented in the Tables 5 and 6. For each model, the F-and p-values were determined. These values showed that the models were significant. Also, the lack of fit of the F-and p-values implied that lack of fit was not significant relative to the pure error. The p-values were determined for each coefficient, with values greater than 0.1000 indicating the model terms were not significant ( Table 5). Using the models, we were able todescribe the response surface for each variant. The charts were ternary, and because there was a large difference between the α-hederin variation domains and the other two components, we had to present the larger diagrams in the so called concentration zone (Figure 3).

Statistical Analysis
Using analysis of variance, the software proposed the models presented in the Tables 5 and 6. For each model, the F-and p-values were determined. These values showed that the models were significant. Also, the lack of fit of the F-and p-values implied that lack of fit was not significant relative to the pure error. The p-values were determined for each coefficient, with values greater than 0.1000 indicating the model terms were not significant (Table 5). Using the models, we were able todescribe the response surface for each variant. The charts were ternary, and because there was a large difference between the α-hederin variation domains and the other two components, we had to present the larger diagrams in the so called concentration zone ( Figure 3). From the analysis of the results presented in Figure 4, it may be seen that the viability of Hep-2 cells was lower in the presence of MEM without FBS than with FBS. Areas with cell viability below 50% were determined in particular by the concentration of α-hederin for Hep-2 (MEM), and for From the analysis of the results presented in Figure 4, it may be seen that the viability of Hep-2 cells was lower in the presence of MEM without FBS than with FBS. Areas with cell viability below 50% were determined in particular by the concentration of α-hederin for Hep-2 (MEM), and for Hep-2 (MEM + FSB), the involved factors were more numerous and the area with viability below 50% was much more restricted (see also the coefficients of the model in Table 5).
Appl. Sci. 2019, 9, x FOR PEER REVIEW 11 of 15 Hep-2 (MEM + FSB), the involved factors were more numerous and the area with viability below 50% was much more restricted (see also the coefficients of the model in Table 5). For the combined optimization of response surfaces, we imposed the restrictions presented in Table 7. The solution for these restrictions is presented in Table 8 and the response area in Figure 5. For the combined optimization of response surfaces, we imposed the restrictions presented in Table 7. The solution for these restrictions is presented in Table 8 and the response area in Figure 5.    Figure 5 presents the response area forthe experimental desirability and also for the cell viability of NCTC and Hep-2 treated cells. It maybe seen that there was a fairly restricted area around the experimental point 2, where the two conditions of cell viability were fulfilled, respectively: >79% were met for NCTC cells and <50% for Hep-2 cells. This area had the following coordinates: α-hederin ranging between 3.44 and 3.96; hederagenin ranging from 100 to 116, and hederacoside C between 580 and 595.  Figure 5 presents the response area forthe experimental desirability and also for the cell viability of NCTC and Hep-2 treated cells. It maybe seen that there was a fairly restricted area around the experimental point 2, where the two conditions of cell viability were fulfilled, respectively: >79% were met for NCTC cells and <50% for Hep-2 cells. This area had the following coordinates: α-hederin ranging between 3.44 and 3.96; hederagenin ranging from 100 to 116, and hederacoside C between 580 and 595.
A statistical analysis of cell viability data was performed, showing that the finded solution with optimized ratio between the three saponins, α-hederin, hederagenin, and hederacoside C, was the same variant found by restriction solution, in which the 3.863:100.000:596.137 (w/w/w) variant had induced cell viability results of 80% in normal NCTC cells, 35% in Hep-2 tumor cells cultivated in MEM, and 76% in Hep-2 tumor cells in MEM with FBS (Table 8).
From the in vitro cytotoxicity experiments of the 14 variants obtained by mixture modeling using the Design Expert 11 software, the optimal variant of the saponin mixture, i.e., α-hederin, hederagenin, and hederacoside C, (no.9, Table 4), induced similar values: 81% in normal NCTC cells, 36% in Hep-2 tumor cells cultivated in MEM, and 77% in Hep-2 tumor cells in MEM with FBS (Table 4). This saponin combination was biocompatible in NCTC fibroblast cells and had a strong antiproliferative activity in Hep-2 cells in MEM. In addition, it had a mild cytotoxic effect on Hep-2 tumor cells in MEM with FBS. All these data correspond to the response optimization procedures using the desirability function.

Conclusions
α-Hederin, investigated in Hep-2 epithelial cervix tumor cells cultivated in medium without FBS, presented strong antiproliferative activity (0.5-51% viability) after 24 h of treatment within the entire range of concentrations, i.e., 2-400 µg/mL. Hederagenin exerted moderate antiproliferative activity between 25-400, decreasing the cell viability to 52%. In turn, hederacoside C did not present antiproliferative activity within the entire range of concentrations (2-400 µg/mL). The optimal ratio between the tested saponins was achieved by applying the optimization method with the Design Expert 11 software. The optimized ratio of 3.863:100.000:596.137 (w/w/w) between α-hederin, hederagenin, and hederacoside C was found to be biocompatible for normal NCTC cells (80% viability) and cytotoxic for Hep-2 epithelial cervix tumor cells cultivated in MEM without FBS (35% viability),as confirmed by in vitro experiments.
These results provide promising baseline information for the potential use of saponins and mixtures of saponins with antiproliferative effects in the treatment of cancer.

Conflicts of Interest:
The authors confirm that this article content has no conflicts of interest.