Result of extraction

The extraction of the Dioscorea plant was carried out by the Soxhlet method. After determining the concentration of 667 mg/ml extract, the desired concentrations were prepared using DMEM extract and culture medium and by sequential dilution.

Evaluation of biological percentage of MCF-7 cell line under the influence of different concentrations of Dioscorea extract after 24, 48 and 72 h

After determining the optimal cell number, 20,000–30,000 cells were cultured in each well.

Figure 1 shows that the plant extract did not cause healthy cells to die in the HFF cell line. As the concentration of the extract increases, the survival rate of cancer cells decreases. This indicates that the plant extract can be used to kill cancer cells without harming healthy cells. This condition was also observed in the 48- and 72-h treatments. In the 24-h treatment of cancer cells with the plant extract, the IC50 was reached at a concentration of 1178.95 µg/ml.

Fig. 1

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 24 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

When treated with the plant extract for 48 h, it was found that the plant extract had no lethal effect on the HFF cell line. As the concentration of the extract increases, the survival rate of the cancer cell lines decreases. In the 48-h treatment with the plant extract, the IC50 was determined at a concentration of 568.25 µg/ml (Fig. 2).

Fig. 2

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 48 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

After 72 h of treatment with the plant extract, it was found that the plant extract had no lethal effect on the HFF cell line. During the 72-h treatment with the plant extract, the IC50 value was 438.35 µg/ml (Fig. 3).

Fig. 3

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 72 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

Table 3 compares the IC50 results obtained by treatment with the plant extract in the cancer cell line and the control. The results show that at the IC50 concentrations obtained for the cancer cell line, the control cells remained healthy, but the cancer cells died.

Table 3 IC50 value of MCF-7 cell line (right) and normal cell line (left)

The statistical studies on the 24-, 48-, and 72-h treatments with the plant extract with p value < 0.01 are shown in Tables 4 and 5. According to this table, the survival rate of MCF-7 cells is highest at a concentration of 31.25 µg/ml. The viability of MCF-7 cells decreases with increasing concentration.

Table 4 Mean bioavailability of MCF-7 cell line with different concentrations of Dioscorea extract in 24, 48 and 72 h by MTT assayTable 5 Mean bioavailability of normal cells with different concentrations of Dioscorea extract in 24, 48 and 72 h by MTT assayThe result of the quantified RNA extract

After the amount of RNA extracted from the cells of the cell line was evaluated using the Nanodrop device at an absorbance ratio of 260 to 280 nm, the RNA concentration was 480.5 ng/µl (Fig. 4).

Fig. 4

The results of quantitative analysis of RNA concentrations extracted from the MCF-7 breast cancer cell line using Nanodrop

Real-time PCR results

The melting curve shows that specific amplification of the desired gene fragments occurred well, and the absence of priming and nonamplification of nonspecific fragments for each gene indicates that there is no contamination in the samples. β-actin was considered as a control. After cDNA synthesis, a real-time PCR reaction was performed using primers for the Bax and Bcl-2 genes. Ct values were determined based on the amplification plot. Subsequently, gene expression changes were calculated using the Livak method (Table 6).

Table 6 Ct values and fold changes in genes expressionExpression profile of the studied genes in normal and cancer cells treated with Dioscorea extract

Using the Ct values from the real-time PCR reaction of the treated cells and the β-actin gene, gene expression analysis was performed using the Livak method. In Table 7 and Fig. 5, the fold change and the changes in the expression of Bax and Bcl-2 genes were calculated, respectively, compared with the reference gene and the control cell. The greatest decrease and increase in gene expression was seen at the 72-h treatment.

Table 7 Comparison of Bax and Bcl-2 gene expression in MCF-7 cancer cell lines treated with Dioscorea extract at 24, 48 and 72 hFig. 5

Fold change of Bax (right) and Bcl-2 (left) genes in 72-h treatment

The expression of Bcl-2 and Bax genes in 72 h after treatment with the extract has a significant decrease of 0.67 (p value < 0.05) and a significant increase of 2.72 (p value < 0.001), respectively.

The expression of Bax and Bcl-2 genes at 24, 48 and 72 h and the expression of Bax and Bcl-2 genes in MCF-7 cell lines treated with Dioscorea extract are shown in Table 7 and Fig. 6.

Fig. 6

Expression rate of Bax and Bcl-2 genes in 24-, 48- and 72-h treatment (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

Results of induced apoptosis at 24, 48 and 72 h

Based on the quadrant plots, it can be seen that the apoptosis induced by the studied plant extract increased in 48 h compared to the control group. After 48 h, the apoptosis induced by IC50 dilution of the plant extract was statistically significant in the cell line compared to the control group (p value < 0.001) (Table 8). The difference with the control group was not significant. After 72 h, more primary apoptosis was observed than delayed apoptosis and necrosis (Table 9).

Table 8 Comparison of induced apoptosis with necrosis by plant extract in 24-, 48- and 72-h treatment on MCF-7 cell lineTable 9 Evaluation of apoptosis induced by plant extract in 24-, 48- and 72-h treatment on MCF-7 and HFF cell lines

Flow cytometry results in Fig. 7 show the rate of induced apoptosis in HFF cells. The results show that 99.8% of the control cells survived because they did not become apoptotic as a result of treatment with the plant extract. The number of cells showing necrosis during the collection and processing phase is very low, 0.032%. The percentage of cells with primary apoptosis is 0.074% and the percentage of cells with delayed apoptosis is 0.064% (Fig. 7). The results of induction of apoptosis with three replicates in MCF-7 cells treated with IC50 dose for 24 h are shown in Fig. 7. The results show that at the IC50 concentration of the plant extract (1178.95 µg/ml), 92.9% of the cancer cells remained healthy. In this study, we used two different assays on separate instruments to measure two different parameters. It is important to note that it is very unlikely to get identical results, especially when comparing the percentage of cell populations to the relative absorbance.

Fig. 7

Results from the study of induction of apoptosis in control cells (left side) and in MCF-7 cells treated with IC50 concentration of Dioscorea extract in 24 h (right side)

The MTT assay was used to assess the metabolic activity of the cells, whereas the annexin V/PI (FACS) assay was used to measure the cell populations stained with a DNA antibody, which allowed the assessment of apoptosis induction and progression. The results do not have to match exactly; rather, it is critical that a similar trend be observed.

From the results of the MTT and FACS cytotoxicity assays, it can be concluded that the cytotoxicity of the extract increases with time. The tendency to cytotoxicity depends on duration, as the degree of cytotoxicity increases with time.

The results of induction of apoptosis with three replicates in MCF-7 cells treated with IC50 dose for 48 h are shown in Fig. 8. The results show that at IC50 concentration of the plant extract (568.25 µg/ml), 84.6% of cancer cells survived. The percentage of cells affected by necrosis in the collection and preparation phase was 1.12%, the percentage of cells with primary apoptosis was 4.51%, and the percentage of cells with delayed apoptosis was 9.77%. The results of apoptosis induction upon 72-h treatment with the plant extract are shown in Fig. 8. MCF-7 Cells were treated with an IC50 concentration of 438.35 µg/ml for 72 h. The results show that 77.5% of the cancer cells survived. Comparison of the three treatments shows that the 72-h treatment has the greatest effect on cancer cell death. The percentage of cells affected by necrosis was 1.94%, and the percentage of cells with primary and delayed apoptosis was 11.2% and 9.29%, respectively.

Fig. 8

Results of apoptosis induction in MCF-7 cells treated with IC50 concentration of Dioscorea extract in 48 h (right side) and 72 h (left side)

The amount of primary and delayed apoptosis in the 24-h treatment compared to the control group was 6.63% and the rate of necrosis was 0.477%, which is statistically significant with a p value < 0.001. In the 48-h treatment with the plant extract, the amount of primary and delayed apoptosis compared to the control group was 14.28% and the rate of necrosis was 1.12%, which is statistically significant (p value < 0.001). In the 72-h treatment with the plant extract, the percentage of primary and late apoptosis compared to the control group is 20.49% and the percentage of necrosis is 1.94%, which is statistically significant (p value < 0.001) (Table 8). Therefore, it can be concluded that 72 h treatment with a plant extract in IC50 concentration has the greatest effect on killing MCF-7 cancer cells. The rate of primary apoptosis, delayed apoptosis, and necrosis for all three treatments can be seen in Fig. 9 and Table 9.

Fig. 9

Comparison of delayed apoptosis, primary apoptosis and necrosis in 24-, 48- and 72-h treatment with plant extract (*p value < 0.05, **p value < 0.01***, p value < 0.001)

As shown in Table 9, the amount of primary apoptosis and necrosis was not significant at 24- and 48-h treatment. Delayed apoptosis was significant at 24- and 48-h treatment with the plant extract (p value < 0.001). In 72-h treatment with the plant extract, the amount of primary and delayed apoptosis was significant with p value < 0.001, while the amount of necrosis was not statistically significant. Therefore, it can be said that the rate of necrosis is not statistically significant in any of the three treatments. Among the three treatments of 24, 48 and 72 h with the plant extract, the 72-h treatment with the plant extract at a concentration of (IC50) 438.35 µg/ml has the best effect on inducing apoptosis in MCF-7 cell line cells.