**4. Screening of medicinal plants for biological activities**

From the collected data on the use of medicinal plants according to the traditional experience of the Tay ethnic people in Thai Nguyen province, the study on extracts of some herbal species *Ardisia gigantifolia* Stapf., *Excoecaria cochinchinensis* Lour.*,* and *Heliciopsis lobata* (Merr.) Sleum. was conducted ability to inhibit cancer cells.

MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to check the viability of certain cancer cell lines using the above-listed plant extract. It was shown that the ethanol extract from the leaves of *Ardisia gigantifolia* Stapf. was able to effectively inhibit the proliferation of all three AGS, MKN45, and MKN74 gastric cancer cell lines with IC50 values ranging from 50 to 140 μg/mL. Meanwhile, ethanol extract of *Excoecaria cochinchinensis* Lour. was also shown to inhibit the

*Medicinal Plants Used by the Tay Ethnic Group, Thai Nguyen Province, Vietnam DOI: http://dx.doi.org/10.5772/intechopen.108771*

**Figure 5.**

*Effect of Excoecaria cochinchinensis Lour. extracts on MKN45 cell proliferation. Cells were treated with extracts at different concentrations from 0.05 to 1 mg/mL. The control is extract - untreated cells (0 mg/mL): Cell image taken under an inverted microscope, 200X magnification. Scale: 50 μm.*

proliferation of gastric cancer cell line MKN45 with IC50 = 70 μg/mL (**Figure 5**, [36]). Also, by MTT screening on HepG2 liver cancer cells, it was shown that the methanol extract of *Heliciopsis lobata* (Merr.) Sleum. plant effectively inhibited cell proliferation after 48 h of treatment with the extract, with IC50 value = 84 μg/mL [37].

Research on the toxicological test of new triterpenoid saponins of *Ardisia gigantifolia* Stapf., results showed that the compound triterpenoid saponin from *Ardisia gigantifolia* Stapf. was able to inhibit the growth of liver cancer cell lines and Hela cells with IC50 values determined ranging from 1.9 to 4.8 μM [38]. A later study, also showed that triterpenoid saponin AG8, an analyzed compound from *Ardisia gigantifolia* Stapf., is capable of the growth mode of various cancer cell lines such as MDA-MB-231, BT-549, and MDA-MB-157 depending on the concentration in the culture medium [39].

The extract from *Excoecaria cochinchinensis* Lour. was determined to inhibit the growth of lung cancer cell lines A549, colorectal cancer cell line Col2, gastric cancer cell line SNU638 with IC50 in the range of 0.25–0.46 μg/mL [40]. The results of the study show that the ethanol extract from *Excoecaria agallocha* L. - a species of the same genus as *Excoecaria cochinchinensis* Lour. can inhibit the growth of breast cancer cell line MCF-7 with IC50 value is 56.5 μg/mL [41].

Invasion is the initiating form of cancer cells during metastasis. Cancer cells have a stronger ability to migrate than normal cells, giving them the ability to invade surrounding tissues and metastasize distantly in the body. Cell migration is indicated by the ability to modify the cell membrane, giving the cell the ability to shift positions. Currently developed anti-cancer drugs are mostly able to attack the ability of cells to migrate to minimize the possibility of spreading to surrounding tissues. One of the effects of herbs on cancer cells of particular interest is their ability to inhibit the spread of several different types of cancer. By analysis of cell migration, it was shown that ethanol extracts of some herbal species significantly reduced the invasion of cancer cells.

This study determined that the extract from the leaves of *Excoecaria cochinchinensis* Lour. was able to inhibit the translocation or migration activity of MKN45 gastric cancer cells (**Figure 6**, [36]). MKN45 cells were treated with the extract for 48 h at concentrations of 0.05 mg/mL; 0.2 mg/mL and 1 mg/mL for 48 h to evaluate its effect on cell migration. The analysis results (**Figure 6**) showed that the extract had a clear effect on cell migration. The ability of cells to migrate into the boundary region was reduced immediately after treatment with low concentrations (0.05 mg/mL), at this concentration, the migration level of cells was determined to be only 81.5 ± 5.3% compared to the control (100%). At higher concentrations (0.2 mg/mL), migration was only approximately 50% of the control (0 mg/mL). The cells observed were mostly dead and completely lost their ability to migrate.

Extracts from *Excoecaria cochinchinensis* Lour. contain many tepotinib compounds. Tepotinib has been shown to inhibit the migration of various gastric cancer cell lines (MKN45, Hs746T, SNU638) [42]. For the first time in this study, we have demonstrated that the extract from *Excoecaria cochinchinensis* Lour. can inhibit the migration of gastric cancer cell line MKN45.

Apoptosis is programmed cell death, and plays an important role in the regulation of the cell life cycle, the balance between living and dead cells, and the prevention of cancer development. Apoptosis offers an important implication in the development of anticancer therapies. The analysis of cell nucleus morphology by nuclear staining method with DAPI (4′,6-diamidino-2-phenylindole) dye showed that herbal extracts increased the number of cells with apoptosis karyotype. The percentage of apoptotic cells was also determined by Flow cytometry. This has shown a potential to kill cancer cells of extracts from some herbal species.

The results of karyotype analysis (**Figure 7A**, [37]) showed that the leaf extract of *Heliciopsis lobata* (Merr.) Sleum. enhanced the appearance of cells with a typical karyotype of apoptosis. The cells after treatment with the extract were analyzed by Flow cytometry to determine the percentage of cells with apoptosis (**Figure 7B**, [37]). At low concentrations (0.1 mg/mL), the extract produced no significant difference in the rate of apoptosis compared with the control in either HepG2 cell line. However, at concentrations of 0.5 mg/mL or more, the increase in the percentage of apoptosis

#### **Figure 6.**

*Effect of Excoecaria cochinchinensis Lour. extracts on MKN45 cell migration. Cells were treated with extracts at different concentrations from 0.05 to 1 mg/mL. The control was cells that were not treated with the extract (0 mg/ mL). The image was taken under an inverted microscope, at a magnification of 200X.*

*Medicinal Plants Used by the Tay Ethnic Group, Thai Nguyen Province, Vietnam DOI: http://dx.doi.org/10.5772/intechopen.108771*

#### **Figure 7.**

*Effect of Heliciopsis lobata (Merr.) Sleum. extracts on karyotype (A) and apoptosis (B) of HepG2 liver cancer cells. (A) Cells were treated with extracts at different concentrations for 48 h, followed by cell nuclei stained with DAPI dye, observed at 200X magnification, scale: 50 μm. (B) Cells after treatment with the extract were analyzed by Flow cytometry to determine the percentage of apoptosis cells.*

cells was different from that of the control. In the HepG2 cell line, the rate of apoptosis induced when treated with the extract at a concentration of 0.5 mg/mL was 22.7 ± 2.7%, compared with the control was 2.5 ± 1.8%. Notably, at a high concentration (2 mg/mL), the methanol extract from the leaves of the *Heliciopsis lobata* (Merr.) Sleum. plant markedly induced apoptosis in the HepG2 cell line (50.5 ± 3.1%).

The composition of *Heliciopsis lobata* (Merr.) Sleum. has been identified to include many compounds, including myricetin, which has a significant ability to inhibit the growth of many different cancer cell lines, including cancer liver through its potentiating effect of apoptosis [43]. The research determined the inhibitory effect on HepG2 liver cancer cell proliferation of extracts from *Heliciopsis lobata* (Merr.) Sleum. through enhanced apoptosis [44]. Another study determined that an ethanol extract from *Excoecaria agallocha* L. was able to inhibit the growth of the breast cancer cell line MCF-7 by promoting the cancer cells to enter the apoptosis pathway [41].

Regulation of the cell division cycle is the way cells ensure division and maintenance of growth and development in the body, and it is strictly controlled by a series of different genes. In cancer cells, changes in genes caused by mutations can lead to changes in the cell cycle and are strongly associated with excessive proliferation compared to normal cells. Therefore, stopping the cell cycle is the anti-cancer approach of many current chemotherapy therapies. Typically, paclitaxel is an anti-cancer drug used in the treatment of cancer that currently inhibits cell division, stopping the cell cycle in the division phase (G2/M) through the mechanism of action on the control point of the mitotic cycle. Stopping the division cycle at this phase will prevent centromeric division between sister chromatids and lead to inhibition of the splitting of the mother cell into two daughter cells.

#### **Figure 8.**

*Extract of leaves of Excoecaria cochinchinensis Lour. affects the division cycle of gastric cancer cells MKN45. Cells were treated with extracts at different concentrations. The control was cells that were not treated with the extract (0 mg/mL); \* p < 0.05; n = 3.*

Cell cycle arrest at the G2/M division phase is closely related to genes encoding the CDK1-cyclin B1, CDC25 and CHEK1 proteins, and PLK1, which control the phase transition during the mitotic cycle. Analysis of the effect of the extract of *Excoecaria cochinchinensis* Lour. on the division cycle of gastric cancer MKN45 cells (**Figure 8**, [36]) showed that at concentrations of 0.2 mg/mL and 1 mg/mL affected the mitotic phases of gastric cancer MKN45 cells, stopping the cell division cycle at the G2/M phase. Specifically, the percentage of cells in the G2/M phase increased to 43%–49%, compared with 33% of the control. Meanwhile, the percentage of cells in the G0/G1 phase in the treated sample was reduced to 33%–38% compared to 47% in the control.

Similar results were also shown in breast cancer cell line MCF-7 when subjected to ethanol extract from *Excoecaria agallocha* L., according to which, there was a significant increase in the proportion of cancer cells at the stage G2/M after being treated with the extract [41]. The inhibitory effect on cancer cell growth through

*Medicinal Plants Used by the Tay Ethnic Group, Thai Nguyen Province, Vietnam DOI: http://dx.doi.org/10.5772/intechopen.108771*

the cessation of the cell division cycle in the G2/M phase has also been reported in various plant species. The extract of *Tamarix aucheriana* (Decne. ex Walp.) B.R. Baum inhibits the growth of colon cancer cells by stopping the cell cycle in the G2/M phase, thereby causing the cells to switch to apoptosis [45]. Another study showed the extract of *Calotropis procera* (Aiton) R. Br. enhanced apoptosis in skin melanoma cells through cyclization in the G2/M phase [46].

Experimental results have contributed to showing that the indigenous experiences of the Tay ethnic group in Thai Nguyen province are consistent with the scientific basis for the ability to inhibit some cell lines of stomach cancer and liver cancer. Explaining the scientific basis of indigenous experiences in the use of medicinal plants by experiments will strengthen and promote the conservation of folk knowledge as well as the conservation of ethnobotanical plants.
