**3. Results**

In our study, the cytotoxicity of all extracts was determined by MTT assay. Accordingly Cytotoxicity calculation method, it has been determined that the cytotoxicity and % vitality (calculated by assuming the control group as 100%) in HeLa cells, treated with the extracts from two species of Colchicum plant, are being increased in concentration compared to the control group (p < 0.05). Different concentrations of both extracts from the two Colchicum species have cytotoxic effects (p < 0.05) and HeLa cells are more sensitive to the species of *C. baytopiorum*. The meaningful cytotoxic effect of the 0.001 mg/ml concentration extracted from *C. umbrosum* has been occurred in only 72 h, compared to the control group (p < 0.01) (**Figures 1** and **2**).

Determined viability% values of HeLa cells treated with *C. baytopiorum* are respectively 70% for B1, 74% for B2, 58% for B3, 75% for B4, 77% for B5 in 24 h; 28% for B1, 23% for B2, 20% for B3, 26% for B4, 20% for B5 in 48 h; 15% for B1, 19% for B2, 27% for B3, 17% for B4, 14% for B5 in 72 h. Vitality % values of HeLa cells treated with *C. umbrosum* are respectively 101% for U1, 70% for U2, 82% for U3, 52% for U4, 77% for U5 in 24 h; 99% for U1, 28% for U2, 19% for U3, 33% for U4, 21% for U5 in 48 h; 63% for U1, 20% for U2, 21% for U3, 20% for U4, 23% for U5 in 72 h.

Apoptotic index is determined by fluorescence microscopy using DAPI staining (**Figure 3**). DAPI staining was also conducted to show the apoptosis of HeLa cells. The AI was calculated as follows:

#### **Figure 1.**

*MTT results of HeLa cells treated with C. baytopiorum and C. umbrosum (B1 and U1: 0.001, B2 and U2: 0.05, B3 and U3: 0.1, B4 and U4: 0.5, B5 and U5: 1 mg/ml) for 24, 48, and 72 h. \*p < 0.05, \*\*p < 0.01 (in comparison to control).*

*In Vitro Cytotoxic and Apoptosis Induction Potential of Two Plant Extracts on HeLa Cells DOI: http://dx.doi.org/10.5772/intechopen.105696*

#### **Figure 2.**

*Viability % values of HeLa cells treated with C. baytopiorum and C. umbrosum (1: 0.001, 2: 0.05, 3: 0.1, 4: 0.5, 5: 1 mg/ml) for 24, 48, and 72 h.*

#### **Figure 3.**

*Phase-contrast microscopy of control and B3 (0.1 mg/ml) concentration of the C. baytopiorum extract treatments groups on HeLa cells for 24, 48, and 72 h (×200).*

AI = (number of apoptotic cells/total number counted) × 100%.

Apoptotic index values of HeLa cells treated with *C. baytopiorum* and *C. umbrosum* for 24, 48, and 72 h are shown in **Figure 4**. AI values were determined as 2.3% for control group, 18.3% for B1, 16.4% for B2, 12.37% for B3, 25.12% for B4, 18.02% for B5 in the application of the *C. baytopiorum* extract; 8.66% for U1, 6.78% for U2, 14.49% for U3, 15.88% for U4, 18.91% for U5 in the application of the *C. umbrosum* extract in 24 h.

#### **Figure 4.**

*Determined apoptotic index (AI) values of HeLa cells treated with C. baytopiorum and C. umbrosum (B1 and U1: 0.001, B2 and U2: 0.05, B3 and U3: 0.1, B4 and U4: 0.5, B5 and U5: 1 mg/ml) for 24, 48, and 72 h. \*\*p < 0.01 (in comparison to control).*

In our experiments, in 24 h, it was determined significant cytotoxic effect according to the control (p < 0.01) in the experimental groups B1, B2, B4 and B5 applied the extract of *C. baytopiorum* and the experimental groups U3, U4 and U5 applied *C. umbrosum* extract.

AI values were determined as 2.84% for control group, 10.67% for B1, 35.91% for B2, 38.38% for B3, 22.04% for B4, 22.03% for B5 in the application of the C*. baytopiorum* extract; 8.9% for U1, 22.05% for U2, 30.19% for U3, 16.01% for U4, 23.58% for U5 in the application of the *C. umbrosum* extract in 48 h.

In our experiments in B2, B3, B4, and B5 experimental groups applied *C. baytopiorum* extract and U2, U3, U5 experimental groups applied *C. umbrosum*

#### **Figure 5.**

*Fluorescence microscopy of control and B3 (0.1 mg/ml) concentration of the C. baytopiorum extract treatments groups on HeLa cells for 24, 48, and 72 h (×1000; DAPI) ( : non-apoptotic cell, : apoptotic cell).*

### **Figure 6.**

*Light microscopy of control and B3 (0.1 mg/ml) concentration of the C. baytopiorum extract treatments groups on HeLa cells for 24, 48, and 72 h (×1000; Giemsa) ( : non-apoptotic cell, : apoptotic cell).*

*In Vitro Cytotoxic and Apoptosis Induction Potential of Two Plant Extracts on HeLa Cells DOI: http://dx.doi.org/10.5772/intechopen.105696*

extract were determined to have a significant cytotoxic effect according to control (p < 0.01) in 48 h.

AI values were determined as 2.92% for control group, 7.89% for B1, 11.19% for B2, 11.76% for B3, 14.72% for B4, 10.5% for B5 in the application of the *C. baytopiorum*

#### **Figure 7.**

*Light microscopy of control and B3 (0.1 mg/ml) concentration of the C. baytopiorum extract treatments groups on HeLa cells for 24, 48, and 72 h (×1000; Feulgen) ( : non-apoptotic cell, : apoptotic cell).*

#### **Figure 8.**

*RT-PCR analysis of bcl-2 members of control and B3 (0.1 mg/ml) concentration of the C. baytopiorum extract treatments groups on HeLa cells for 48 h (M: 1.5 kb marker, C: control, B3: 0.1 mg/ml).*

extract; 2.82% for U1, 7.91% for U2, 15.22% for U3, 16.23% for U4, 11% for U5 in the application of the *C. umbrosum* extract in 72 h.

In our experiments, in 72 h, it was determined significant cytotoxic effect according to the control (p < 0.01) in the experimental groups B1, B2, B4 and B5 applied the extract of *C. baytopiorum* and the experimental groups U3, U4 and U5 applied *C. umbrosum* extract.

The methanol extract of *C. baytopiorum* has been treated with the most effective concentration of 0.1 mg/ml in 48 h experiment group (after the evaluation of cytotoxicity and AI value).

The morphological changes belonging to apoptosis such as blebbing of membrane and apoptotic body including cell organelles and chromatin parts [32, 33] have been shown through phase-contrast, fluorescence, and light microscopies (**Figures 3** and **5**–**7**).

To determine the apoptosis at the molecular level, the expression rates of the genes of the *bax*, *bak*, *bik*, *mcl-1*, *bfl-1*, *bcl2*, and *bcl-x,* which are members of the *bcl-2* gene family, have been searched through the RT-PCR method. In the 48 h experimental group of HeLa cells which were treated with the concentration of 0.1 mg/ml of *C. baytopiorum* extract, an increase in the expression of the *mcl-1* and *bcl-x* genes by compared to the control group was observed, and an increase of the expression of *bax, bak, bik* and *bfl-1* genes was also determined . In contrast, any expression was observed in the *bcl-2* gene (**Figure 8**).
