Evaluation of Cytotoxic Effect of Colutea persica on Human Gastric and Colon Cancer Cell Lines

Cancer is one of the most important issues in the world which affects public health as an important problem. Cancer is the third cause of death in Iran with an annual occurrence of 51,000 new cases [1]. Previous studies demonstrated the considerable increasing trends in the mortality of gastrointestinal cancer in Iran, especially for Gastric cancer and colorectal cancer [2]. In general, incidence rates of these two cancers are high in Eastern Europe, Eastern Asia, and South America and the lowest rates are in North America, and most parts of Africa [3]. The most critical factors that increases the risk of gastrointestinal cancer are aging, inappropriate diet, biological factors, and infectious diseases which contribute to the cancer occurrence [4]. Colon cancer is studied to be one of the most critical digestive diseases and is the second leading cause of cancer deaths [5]. Gastric cancer is important cancer in the world and the prevalence of gastric cancer is raised with aging in both men and women [6]. Colon cancer is a disorder in which malignant cells form in the large intestinal tissues [7]. On the other hand, in gastric cancer the malignant cells form in the lining layer of the stomach caused it. The risk of gastric cancer is high in Iran, and unlike in Western countries it is not under the control, thus it is on a dramatically increasing trend in Iran [8]. Secondary metabolites and phenolic compounds of plants can play an important role in reducing the side effects of chemotherapy drugs and have some positive effects on cancer cells, as well as in the expression of apoptotic pathway genes [9]. Colutea persica was used in traditional medicine as an anti-inflammatory agent in gastrointestinal problems [10]. The aim of this study was to investigate the cytotoxic effects of aqueous and hydroalcoholic extracts of Colutea persica on gastric cancer (AGS), colon cancer (HT-29), and normal fibroblast (SKM) cell lines.

Fresh Colutea persica leaves were collected from the Delfard region of Kerman province in 1399. Dried leaves of Colutea persica were ground into a fine powder by the electronic grinder. At the next step, the ground powder was extracted using ethanol by the maceration. Then, it was placed on a shaker at room temperature. After that, the collective extracted was filtered using a filter membrane. The solvent extracted was evaporated in the rotary evaporator. Finally, to completely remove the solvent, the extract was placed in an oven, and the hydroalcoholic extract was maintained at -20 °C until use.In order to measure the total flavonoid content (TFC) of the extracts the aluminum chloride complex-forming assay was applied. In this method, quercetin was applied as the standard which flavonoid content was distinguished based on the quercetin equivalent. Briefly, Colutea persica extract was added to the aluminum chloride hexahydrate and mixed with potassium acetate and distilled water. After 30 minutes, the absorbance of the reaction was checked using a UV-Vis spectrophotometer at 415 nm. The blank sample was made by replacing aluminum chloride with deionized water. Before measuring, all of the solutions were filtered by using Whatmann filter papers (number 41). Total flavonoid content in Colutea persica extract was measured using the standard calibration curve which is achieved from different concentrations of the standard reference.The total phenolic content (TPC) of organic crude extracts was calculated using the Folin- Ciocalteu reagent method. In this method, gallic acid was used as a reference standard (20-100 μg/mL) for plotting the calibration curve. 0.5 mL of the Colutea persica extract was added to 1.5 mL of Folin-Ciocalteu solution. Then, it was diluted (1:10) with deionized water and added to the sodium carbonate solution.The absorbance was recorded after one hour at 765 nm by using a UV-Vis spectrophotometer. Finally, the total phenolic content was calculated based on gallic acid equivalents (mgGAE/g). All the experiments were done in triplicate.Cytotoxic effects of 5, 10, 25, 50, 100 μg/ml concentrations of aqueous and hydroalcoholic extracts were evaluated on the cells for 24 hours using MTT assay. The apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. Cells were seeded and after 24 h they were incubated with a 1.25 mg/ml concentration of the extract and for each treated cell line, one control cell was considered. The annexin V/PI assay was performed to confirm the cytotoxic activity of the plant extracts against AGS and HT-29 cell lines. Total RNA was isolated from cells using RNX-plus TM Reagent before and after treatment. Total RNA was reverse transcribed to cDNA using M-MuLV-RT and random hexamers. The cDNA was assayed by real-time PCR using primers for BAX and BCL-2 genes. Data were analyzed using SPSS software, one way ANOVA, Tukey test at p ≤0.05...

The MTT assay revealed the cytotoxicity against both two cell lines (HT29 and AGS) in comparison to the HT29 and AGS normal cell line (SKM). The expression level of BAX gene increased and BCL2 gene decreased in AGS cell line after treatment by plant extract. (p<0.05). The total phenolic content was expressed in terms of milligram gallic acid equivalent per gram dry weight of plant extract. The total phenolic content of hydroalcoholic and aqueous extracts of Colutea persica were 5.02±.05 and 5.85.±0.08 mgGAE/g respectively.

In conclusion, our findings show that Colutea. persica has anti-cancer effects in vitro against AGS and HT-29 cancer cell lines. Induction of apoptosis by using plant extract was achieved by down-regulation of BCL2 and up-regulation of BAX. Finally, the current study suggests that Colutea. persica may have cancer-fighting properties and could be a promising new candidate in this field. Besides, the molecular target of Colutea. persica and its mechanism are unknown, and the author intends to use animal models and bioinformatics methods to discover the active ingredients of Colutea. persica.