Introduction

Cancer is a leading cause of death in the world, and the number of such cases and deaths is expected to grow rapidly. The stomach is one of the organs prone to malignancy (Rawla & Barsouk, 2019). Most patients diagnosed with gastric cancer need chemotherapy, surgery or radiotherapy. Each of these modes has different side effects which hamper the quality of the life of the individual. Numerous chemotherapeutic agents have been used in such cases with variable rates of success. The side effects of drugs include leukopenia, anemia, tiredness, mouth ulcers, nausea, vomiting, constipation or diarrhoea (Nurgali, Jagoe, & Abalo, 2018). To circumvent the above problem, a few herbal drugs or chemical extracts from plants have come to the fore in different malignant settings. Veratrum californicum, Scutellaria baicalensis and Wedelia Chinensis are some of the described herbal plants with therapeutic potential against cancer (Yin, Wei, Jian, & Yang, 2013). Citrus medica Linn., commonly known as a Citron in English, naarthai in Tamil and bijapura in Ayurvedic literature is a shrub. Extracts of leaves of these plants have been proved to have anti-ulcer potential (Li, Cai, Liu, Sun, & Luo, 2019). Hence, we wanted to investigate the possible role of extracts of this plant in combating gastric cancer through cell line studies. The study aimed to extract the phytoconstituents from the leaves of the above-said plant and study their effects as an antioxidant, their apoptotic and cytotoxic potential in AGS gastric cancer cell lines.

Materials and Methods

The fresh leaves of Citrus medica Linn. were collected in November 2018 from the local areas of Kumbakonam, Tamilnadu, India. The plant was identified and authenticated by Department of Botany, Government Arts and science college (Autonomous), Kumbakonam. The collected leaves of Citrus medica Linn. were washed and shade dried for two to three weeks. The dried samples were powdered using an electrical blender.

Then according to classical described techniques, ethanolic, petroleum ether and aqueous extracts of the same were prepared. Phytochemical screening was done to detect alkaloids, flavonoids, saponin and steroids. The antioxidant activity was analyzed with Ferric iron-reducing antioxidant power assay (FRAP). The radical scavenging activity was done by 2,2DPPH (2, 2-Diphenyl-picryl hydrazyl) scavenging assay (Parthasarathy, 2020). The human gastric cancer AGS cell line was purchased from National Centre for Cell Science (NCCS), Pune, India. The cell count was done, and the cell viability was tested by trypan blue dye exclusion test using a hemocytometer. The cell lines were treated with different concentrations of the plant extract ranging from 25 to 400 µg for varying periods (6 hours, 12 hours,18 hours,24 hours, 36 hours and 48 hours) and the dose and time of treatment was optimized in each cell line using MTT (4, 4-dimethyl-2-tetrazolyl)-2, 5-diphenyl-2, 4-tetrazolium salt) assay. The following table Table 1 describes the group with a concentration of citrus leaves extracts.

The extent of cytotoxicity was done with sulphorhodamine B (SRB) assay (Parthasarathy, 2020). The cell cycle analyses and the detection of apoptosis were done with flow cytometry according to described protocols. All tests were repeated in triplicate to confirm the findings and establish our results.

Results and Discussion

The phytoconstituents in different types of extracts is tabled below in Table 2. It can be inferred from the table that the ethanolic extract of the leaves had more phytoconstituents than the other ones. The effect of Citrus medica leaves on radical scavenging

activity (DPPH) was examined at different concentrations of various extracts (aqueous, and ethanol). Figure 1 represents the scavenging activity of Citrus medica leaf extracts in percentage. The aqueous extract showed 76.15% of scavenging effect, whereas the ethanolic extract showed 85.33% scavenging activity. Ascorbic acid was used as standard. The scavenging effect was more with ethanolic extract, which coincides with the constituents. The FRAP assay Figure 2 revealed the ethanolic extract (88.68%) of C. medica showed higher reducing activity compared to aqueous extract (83.34%). This measures the antioxidant power of the extracts. IC 50 was found to be 200 micgm Figure 3. Figure 4 represents the result of cytotoxicity effect of C. medica extract on the AGS cell line. It was observed that the percentage of cells was reduced to 50% in the treatment group when compared to the control group. It indicates that the ethanolic leaf extract of C. medica showed a cytotoxic effect in the AGS cell line. The untreated cell used as the control. Concerning the apoptotic detection of AGS cell line at 24 hours exposure of 175µg/ml dosage of C. medica leaf extract, the cells treated with ethanolic leaf extract of C. medica were arrested in G_0-G_1. In the early apoptosis, the percentage of cell death was slightly increased in the treated group when compared to the control group.

In late apoptosis, the percentage of cell death was significantly increased when compared to the control group. The results indicated that the ethanolic leaf extract of C. medica induces apoptosis in AGS cell line. The distribution of apoptotic cells with a comparison of both treated and untreated groups is tabled in Table 3. (Panara, Joshi, & Nishteswar, 2012) reported that the ethanolic leaf extract of Citrus medica contains flavonoids, alkaloids, steroids, glycosides, and phenolic compounds. Our findings go along with their results. Many authors have described the antioxidant properties regarding the antioxidant properties of the plant in general. The specific extracts of the leaves of citrus medica linn have been done only by a very few. (Cirmi et al., 2017) have described the various constituents and extracts of citrus juices and demonstrated the anti-cancer potential. Still, the leaf extracts were not noted in their study. As the leaves (naarthai leaves) have been traditionally used for gastric ulcer problems by this part of the continent, we chose the leaf extract and stomach cancer cell lines. The leaf extracts have been found to have antihelminthic and estrogenic (Bairagi, Kabra, & Mandade, 2011) properties. A demonstrable anti peptic ulcer activity and thereby influencing the malignant transformation of a peptic ulcer has so far not been published. Even though the citrus peels and other fruit part extracts have demonstrable medicinal values (Song, Shrestha, Gyawali, Lee, & Cho, 2015), this study is the first of its kind to demonstrate the citrus medica linn leaves in gastric cancer AGS cell lines. Even though the leaf extract induces early apoptosis, it's not much significant. Late apoptosis is very significant in our study. As apoptosis (Wong, 2011) assumes significance in the discovery of novel drugs countering cancer, our study may throw light on the leaf extracts of citrus medica linn which may play a role in the fight against gastric cancer.

Conclusions

The results of the present study demonstrated the presence of alkaloids, flavonoids, steroids, glycosides, and phenolic compounds in the ethanolic extract of Citrus medica (naarthai) leaves. Antioxidant activity of ethanolic stem extract of Citrus medica leaves was confirmed by DPPH and FRAP assay. The antioxidant potential is due to the phytochemical constituents present in the leafy aerial parts of the plant. Cytoprotection was measured by cleavage of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by surviving AGS cells following exposure to ethanolic extract of C. medica. The IC₅₀ value was found to be 200µg/ml by MTT assay. Flow cytometry analysis concludes that citrus medica mediated apoptosis and induced G₀-G₁ phase cell cycle arrest only in cancer cells.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.