**4. Discussion**

Some studies reported that crude extracts of natural products containing a variety of molecules that exhibit antitumoral activity are highly effective in cancer cell death [38–40].

However, some reports suggested that natural product mixtures often claim that they are more effective than purified compounds due to synergistic, additive, or antagonistic interactions [41]. Also, different species of the same genus may have different concentrations of the same compounds [42].

In our study, cytotoxicity of all extracts was determined by MTT assay. Accordingly, 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; and 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; and 63% for U1, 20% for U2, 21% for U3, 20% for U4, 23% for U5 in 72 h.

Several compounds derived from natural products, such as Genistein, Puerarin, Formononetin, Cyanidin 3-O-glucoside, Epigallocatechin gallate, Quercetin, Kaempferol, Hesperidin, Naringin, Apigenin 7-glucoside, Wogonin, Eugenol,

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

Anthocyanins, Curcumin, Emodin, Epifriedelanol, Mitomycin C, and Piperine, induce apoptosis in cervical cancers [6, 43]. Activation of apoptosis is used as an anticancer mechanism in cancer therapy research [6].

The determination of the AI is the method that helps to predict and diagnose patients by considering the tumor, its stage, the course of the disease, its consequences, the patient's strength of resistance, and the possibilities of helping the patient [44].

AI has been determined by DAPI respectively the fluorescence microscopies (**Figure 3**). DAPI staining was also conducted to show the apoptosis of HeLa cells. AI was calculated as follows (**Table 1**):

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

#### **Table 1.**

*AI calculation formula.*

AI 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.

Bungu et al. [23] applied the prepared extracts from the leaves and bulbs of *Tulbaghia violacea* on HeLa, HT29, MCF-7, and WHCO3 cells. After 24 h, a significant number of apoptotic cells were detected. If after 48 h, a further increase in the number of apoptotic cells was observed. The results of our study are consistent with this and other similar studies.

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 48 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 2.92% for control group, 7.89% for B1, 11.19% for B2, 11.76% for B3, 14.72% for B4, and 10.5% for B5 in the application of the *C. baytopiorum* extract; 2.82% for U1, 7.91% for U2, 15.22% for U3, 16.23% for U4, and 11% for U5 in the application of the *C. umbrosum* extract in 72 h.

In our experiments in B2, B3, B4, and B5 experimental groups applied *C. baytopiorum* extract and the U3, U4, and U5 experimental groups applied *C. umbrosum* extract was determined to have a significant cytotoxic effect according to control (p < 0.01) in 72 h.

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 the apoptosis such as blebbing of membrane and apoptotic body including cell organelles and chromatin parts [26, 45, 46] have been shown through phase-contrast, fluorescence, and light microscopies (**Figures 3** and **5**–**7**).

Most studies on apoptosis of plant extracts were confirmed by mechanisms such as caspase and *bcl-2* gene family [6]. A study showed that *Dendrobium chrysanthum*

ethanol extracts induct apoptosis by upregulated Bax, and p53 and downregulated Bcl-2 in HeLa cell lines at the density of 450 μg/ml for 24 h [47]. Another study reported that 0–100 μmol/L Wogonin induces apoptosis via elevating the Bax/Bcl-2 expression ratio, activating Caspase 3 and 9 in SiHa and CaSki cells [48].

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 experiment group of the HeLa cells that were treated with the 0.1 mg/ml concentration of the *C. baytopiorum* extract, an increase has seen in expressions of *mcl-1* and *bcl-x* genes compared to the control group, and some increase has been determined in the *bax*, *bak*, *bik,* and *bfl-1* genes expressions. On the other hand, no expression was observed in *bcl-2* gene (**Figure 8**).
