Bangladesh J Pharmacol. 2015; 10: 160-165

Available Online: 1 March 2015; DOI: 10.3329/bjp.v10i1.21462

Effect of crude saponins from Gaultheria trichophylla extract on growth inhibition in human colorectal cancer cells

¹Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan; ²Wolfson Cell Science Centre, Faculty of Health & Medical Sciences, University of Surrey, Guildford, UK.

Abstract

The genus Gaultheria also comprised of species with reported cytotoxic activities. Current research work was carried out to evaluate G. trichophylla crude extract and respective saponins fraction against human colorectal cancer cell line (Caco-2) based on cell viability assays. Caco-2 cells treated with the crude extract showed significant growth inhibition (p< 0.001) in a dose dependent manner with apparent IC₅₀ value of 200 μg/mL and 100 μg/mL in MTT and NRU assays respectively. The fractioned crude saponins showed an enhanced response and inhibited the growth of Caco-2 by 93.6 and 97.4% in MTT and NRU assays respectively, with compared to actinomycin-D (65%). The DAPI staining of cell treated with crude saponins observed under confocal microscope showed shrunken nuclei with apparent nuclear fragmentation and chromatin condensation indicating apoptosis mode of cell death. The study exhibited that the G. Trichophylla saponins induced apoptosis of Caco-2 cell lines. This study provides new evidences to further explore this plant for the novel targets in anticancer drug development.

Introduction

Colorectal cancer is the third most common malignant neoplasm worldwide and has become one of the major causes of cancer mortality (Wong, 2011).

It is well known that in the past some medicinally important plants like Catharanthus roseus, Podophyllum peltatum and Taxus brevifolia and others provided therapeutically active metabolites which were used to control the advance stages of malignancies (Huang, 2003). The flowers range in colour from red to pink; fruits are blue-colored berries; and small green leaves are approximately 3-7 mm in length. This plant is an inhabitant of cold and lofty locations of the mountains and like many plants of such locations, it is furnished with setae. The calyx is adherent to the lower part of the capsule, becomes succulent, and forms an edible fruit in the month of September (Liu et al., 2013; Zhang et al., 2011), antibacterial (Xiong et al., 2009). The plants of this genus including G. itoana Hayata and G. yunnanensis are reported to possess cytotoxic activities against the selected cancer cell lines (Chen et al., 2009; Li et al., 2010). Gaultherin a natural salicylate isolated from G. yunnanensis possess analgesic and anti-inflammatory activity (Alauddin et al., 1965; El-Basyouni et al., 1964; Ibrahim and Towers, 1960; Zhang et al., 1998).

Materials and Methods

Chemicals: The Caco-2 (human colon adenocarcinoma) cell line was obtained from the ECCC (European Collection of Cell Cultures) through Health Protection Agency, Salisbury, UK (Catalogue No. 86010202). Neutral red solution, fetal bovine serum (FBS), actinomycin-D, Dulbecco’s modified Eagle medium (DMEM) and 4,6-diamidineo-2-phenylinldole (DAPI) were obtained from Sigma Chemical Co. (St Lois, MO, USA).

Preparation of extract and crude saponins: The dried powdered plant material (200 g) was extracted with methanol using soxhlet extractor for 20 hours. It was filtered through a Whatman Grade-I filter paper. The filtrate was evaporated on a vacuum rotary evaporator under reduced pressure at 40°C. Extracted percent yield of the methanol fraction was 21.9%.

Extraction of saponins from powdered materials (200 g) of whole plant of G. trichophylla was done first with petroleum ether, followed by extraction with methanol in Soxhlet apparatus. The solvent was reduced on rotary evaporator under vacuum to obtain dry semi solid extracts. The methanol extract of the plant was further fractionated with n-butanol and water, in equal proportion. The n-butanol fraction was separated. The crude saponins were precipitated with petroleum ether, yield was approximately 4.5 g of crude saponin extract (Green and Kroemer, 2004). The MTT assay measures the mitochondrial function activity of mitochondrial dehydrogenases. MTT is a yellow colored dye [3-(4,5-dimethyl- 2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], which converts to a purple insoluble product formazan as a result of reduction reaction with concomitant oxidation of NADH (nicotinamide adenine dinucleotide-reduced) and NADPH (nicotinamide adenine dinucleotide phosphate-reduced) through the ability of the mitochondrial succinic dehydrogenase enzyme in living cells (Borenfreund et al., 1988) growth of cancer cells was quantified. Following a 24 hours exposure test period of drugs, cells were washed twice with phosphate buffer saline (PBS), and a 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well including the blanks, which contained medium only. The plates were returned to the incubator for 4 hours at 37°C. Subsequently, cells were washed twice with PBS, and 100 μL/well DMSO was added in each well as solvent to dissolve the insoluble crystalline formazan products. The effect of plant extracts on cancer cells was quantified as the percentage of control absorbance of reduced dye at 550 nm on microplate reader (LabtechLT-4000MS, Labtechm International Ltd., East Sussex, and UK). For each treatment, five replicate wells were examined, and each experiment was repeated three times (n=3). Standard error of mean (SEM) was calculated between three experiments. The results were calculated as percentage growth inhibition, untreated (control) cells versus treated cells according to the following formula (Qadir, 2014):

Absorbance of the media was subtracted both from control and treated cells.

Neutral red uptake assay: The neutral red assay is basically a metabolic impairment assay, which works simply on the principle that this dye accumulates in the lysosomes of viable cells by a combination of active endocytosis and pinocytosis until a stable equilibrium is reached. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes produced by toxic substances cause decreased uptake and binding of neutral red, making it possible to distinguish between viable, healthy and damaged or dead cells. Metabolically inactive cells lose their ability to accumulate and retain neutral red dye (Borenfreund and Puerner, 1985) by removal of the medium after dosing cells and 200 μL of neutral red solution (40 μg/mL) was added to each well (including the blanks, which contained medium only). After incubation for two and half hours, the neutral red was removed, cells were carefully rinsed with pre-warmed PBS, and 200 μL of ethanol/acetic acid (1% glacial acetic acid in 5% ethanol) was added to all wells.

The plates were covered in foil and placed on a plate shaker for 30 min to extract neutral red dye from the cells to form a homogeneous solution. Absorbance of the wells was measured at 540 nm in a microplate reader within 60 min. For each treatment, five replicates wells were examined and each experiment was repeated three times (n=3). The results were calculated as percentage growth inhibition, untreated (control) cells versus treated cells, according to the formula as described previously for MTT assay. Standard error means were calculated between three experiments.

Cytomorphological alterations (DAPI staining): DAPI (4', 6-diamidino-2-phenylindole) is a DNA-specific probe, which forms a fluorescent complex by attaching in the minor groove of A-T rich sequences of DNA. Binding of DAPI to DNA produces a ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove

Results

The present study was conducted to determine the ability of G. trichophylla crude extract and isolated saponins fraction against the proliferation of human colorectal cancer cells. Previous studies conducted showed that various isolated phytoconstituents provided a great potential for new strategies in exploring anticancer drugs.

Figure 1: Dose-dependent growth inhibitory activity of G. trichophylla methanol extract (G.Me) against Caco-2 human cancer cells. The graph also provides a comparison between MTT and NRU assay sensitivities

Figure 2: A comparison of G. trichophylla methanol and saponins extract and standard drug actinomycin D growth inhibitory activities against colorectal cancer cells (Caco-2). All the results are represented as mean ± SEM (n=3)

The DAPI staining of crude saponins treated cells when observed under confocal microscope. The nuclei were shrunken and signs of marked nuclear fragmentation and chromatin condensation were observed. It was very clear that the numbers of apoptotic body fragments were more as compared to untreated cells (control) where nuclei is intact and there was no apparent signs of DNA fragmentation. Based on these observations we may suggest that Caco-2 cells, after treating with saponins fraction followed a programmed cell death pathway, an indication of the apoptosis mode of cell death, that is more obvious than necrosis (Figure 3).

Figure 3: A. Control (untreated Caco-2 cells). B. Cells treated with G. trichophylla saponins (10 µg/mL, final concentration) for 24 hours and visualized under confocal microscope for DAPI stain

Discussion

The plants belonging to genus Gaultheria have also been reported to inhibit the human lung, prostate and liver cancer cell lines (Li et al., 2010). It is reported that saponins exert its cytotoxic activity through apoptosis through signalling pathways to prevent the tumors (Saha et al., 2013). In this study, saponins extract induced morphological changes in apoptotic cells which were observed in DAPI staining. This demonstrates that the treatment with saponins extract resulted in apoptotic body formation, chromatin condensation and nuclear fragmentation. It clearly indicates the potential of saponins extract to induce apoptosis against colon cancer cell lines i.e. Caco-2.

In conclusion, the present study shows that G. trichophylla methanol extract and its saponins fractions possess dose dependent growth inhibitory and potentially an apoptotic pathway against the human colon carcinoma Caco-2 cells.

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