Cell Viability Studies and Anti-cancerous activity Evaluation of Pomegranate (Punica granatum L) extract
Rajasekhar Pinnamaneni
Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Green-fields, Vaddeswaram, Guntur, Andhra Pradesh-522 502, India
*Corresponding Author E-mail: pinnamaneniraj@yahoo.com
ABSTRACT:
Pomegranate, Punica granatum L belonging to the family Lythraceae is a widely used plant having medicinal properties. Pomegranate gained considerable recognition as a functional food in the modern era. In light of the importance of pomegranate extract as an anti-cancerous agent, an attempt was made to analyze its anti-cancerous activity by studying the toxicity studies on the cancerous cell lines by treating with pomegranate peel extract and studying the viability of cancerous cell lines. Dried pomegranate peel extract powder was obtained by rotary evaporation and freeze drying. The percentage viability of HCT 15 and T47D cancer cells carried out by using Trypan blue staining were 73.46%, 78.35% respectively which are most suitable to perform cytotoxicity studies. With the increase in the concentration of the pomegranate peel extract (20 to 120 µl/ml acetone, methanol and ethyl acetate pomegranate peel extracts); the percent viability of cells tends to decrease.
KEYWORDS: Pomegranate extract, Cell viability, Trypan blue staining, MTT assay.
INTRODUCTION:
Pomegranate and its constituents can efficiently affect multiple signaling pathways involved in inflammation, cellular transformation, hyperproliferation, angiogenesis, initiation of tumorigenesis, and eventually suppressing the final steps of tumorigenesis and metastasis7. The pomegranate constituents are shown to modulate transcription factors, pro-apoptotic proteins, anti-apoptotic proteins, cell cycle regulator molecules, protein kinases, cell adhesion molecules, pro-inflammatory mediators, and growth factors in various cancers8,9,10,11,12,13,14,15,16,17,18,19. In light of the anti-cancerous activity of Pomegranate extract, an attempt was made to analyze its anti-cancerous activity by studying the viability of cancerous cell lines and by studying the toxicity studies on the cancerous cell lines by treating with pomegranate peel extract.
MATERIALS AND METHODS:
Plant samples:
Ripe pomegranate fruits were collected in October, 2017 from pomegranate trees in Anantapuramu, Andhra Pradesh, India. Ripe pomegranate fruits were harvested from different trees of Jodhpur Red cultivar. The variety was authenticated by Department of Horticulture, Government of Andhra Pradesh. The English, scientific and family names of the plant under study are: Pomegranate, Punica granatum L and Lythraceae, respectively.
Preparations of crude pomegranate peel:
Peels were cut into small pieces (i.e. 3cm × 3cm), placed into plastic containers (50ml), and were kept in a freezer at -40°C for at least 16 hours. The frozen peels were freeze-dried (i.e. freeze drying started from -40°C and ended at 20oC) for 96 hours using Freeze Dryer. Dried peels were then stored in a desiccator for one week before being ground into powder20.
Preparation of extract:
Dried pomegranate peels (30g) were ground into powder using a grinder at 9676.8 g. Powders were kept in air tight plastic containers and stored at -40°C until used for extraction in Soxhlet apparatus using organic solvents such as Acetone, Methanol and Ethyl acetate. Three extracts of Acetone, Methanol and Ethyl acetate (100 mL) were added to 0.5g of dried sample in conical flasks and was stirred for 3h at room temperature (20°C). The extractions were performed for 48 h and concentrated by slow evaporation process21. The obtained extracts were kept in moisture free container and used for viability assays. To avoid light exposure the flasks were covered with aluminum foil. Mixture was then centrifuged for 30 minutes at 9676.8 g at 3°C (REMI-RM 1210, India). The supernatant was filtered using Whatman filter paper No 1,155mm. Extracts were then stored at 4°C until used for analysis22. Organic layer was separated and concentrated under reduced pressure to approximately 20mL of volume and dried using sodium sulphate23.
Sample preparation for biological studies:
The solvent was evaporated under vacuum using a rotary evaporator and the concentrated extracts were stored at -70°C until the use. From the dried pomegranate extract, the bioactive compounds were dissolved in DMSO and transferred to sterile vials of 2ml capacity; these samples were stored at cold temperature in freezer, protected from light.
Reagents and Cell Lines:
Human colorectal adenocarcinoma cancer cell line (HCT 15) and Human breast cancer cells (T47D) were procured from the National center for cell sciences (NCCS), Pune. Cells were maintained in Dulbecco’s modified Eagle medium (Hi-Media) supplemented with 10% fetal bovine serum and 100g/l penicillin/streptomycin and maintained in 5% CO2 (Thermo scientific) at 37°C. For experimental purpose, cells from exponentially growing culture were used. All experiments were replicated thrice.
Trypan blue dye exclusion technique:
Trypan Blue is an essential blue acidic dye containing two azo chromophores, used in estimating the number of viable cells present in a population24. A cell suspension was made with a fixed volume of cells (e.g. 1ml). Although an aseptic technique is not essential in all stages of this procedure, 50µl of cell suspension was taken and mixed with an equal volume of trypan blue solution with a pipette. Counting should commence in less than five minutes or else the cells will begin to take up the dye, after staining with trypan blue solution. The percentage viability of the HCT 15 and T47D cancer cells were calculated before the treatment with pomegranate extract in trypan blue staining. The percentage of viability of HCT 15 and T47D cancer cells were calculated after treating with the various concentrations of Acetone, Methanol, and Ethyl acetate pomegranate peel extracts (20, 40, 60, 80, 100 and 120 µg/ml) in trypan blue staining. It was transferred to a hemocytometer and then the live cells in clear form and dead cell in blue colour were counted. The hemocytometer was placed on the stage of an inverted microscope. Focus was adjusted until a single counting square fills the field.
The number of cells per ml, and the total number of cells were counted using the following formula:
% viability = (live cell count/total cell count) ×100
Microculture tetrazolium (MTT) assay:
This Colorimetric assay is based on the capacity of Mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble substrate 3- (4, 5-dimethyl thiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) into an insoluble, coloured formazan product which is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells25,26,27,28. Evaluation of anti-cancerous activity compound (Acetone, Methanol, Ethyl acetate extracts) of pomegranate peel was performed using HCT 15, T47D cancer cell line cultures. HCT 15 and T47D trypsinized cancer cells were seeded onto a 96-well plate at a density of 5000cells/well in 200µL of medium for 24 hours. Various concentrations of Acetone, Methanol, Ethyl acetate pomegranate extracts (20, 40, 60, 80, 100 and 120µg/ml), was added to the wells and were incubated for 24 h. A control well without extract was maintained to compare the percent cell viability. After the incubation period 100µl MTT (5 mg/ml in phosphate buffered saline) was added to each well and incubated for 3 hours in dark. Then MTT was discarded and 150 µl of DMSO was added to each well. The purple colour developed was measured at 570nm with microplate reader (Bio-Rad). Percent cell viability was calculated as follows.
Percentage of inhibition= 100-(Absorbance of test) / (Absorbance of control) × 100
Growth inhibition were expressed as mean ± SD value of the percentage of absorbance reading from treated cells versus untreated cells
RESULTS AND DISCUSSION:
Cancer cell line development and maintenance:
The cancer cell lines were maintained successfully in laboratory conditions and used for further studies, i.e., Trypan blue staining and MTT assay. Human colorectal adeno carcinoma Cancer cells (HCT 15), Human breast cancer cells (T47D) were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 4.5g/l glucose and 2mm 1- glutamine and 5% fetal bovine serum (FBS) (growth medium) at 37şC in 5% CO2 incubator.
Trypan blue staining:
Cell lines were free from bacterial and fungal contamination, percentage of cell viability of cell lines were carried out by using Trypan blue staining, The percentage of viability of the HCT 15 and T47D cancer cells before the treatment with pomegranate extract in trypan blue staining, showed viability of HCT 15 cell line and T47D cell line as 78.35% and 73.56% respectively, which are most suitable to perform cytotoxicity studies (Table.1; Fig.1). The percentage viability of HCT 15 and T47D cancer cells after treating with pomegranate extract (i.e., with acetone, methanol and ethyl acetate extracts) at different concentrations in trypan blue staining. With the increase in the concentration of the pomegranate peel extract (20 to 120 µl/ml), the percentage of viability of cells tends to be decreased.
Table 1. Percentage of cell viability of cell lines before Trypan blue staining
|
Cell line |
% viability |
Live cell count |
Total cell count |
pH |
|
HCT 15 |
73.56 |
1.48×105 |
2.71×105 |
6.7 |
|
T47D |
78.35 |
1.65×105 |
2.38×105 |
7.6 |
Fig.1: Trypan blue staining (A: Live cells, B: Dead cells)
MTT Assay with pomegranate peel extracts:
In our present study the impact of pomegranate peel extracts of Acetone, Methanol and Ethyl acetate on the growth of HCT 15 and T47D cancerous cell line was examined by performing MTT assay. After the treatment of cell lines with various concentrations of extracts, the results from MTT assay shows that there is an exponential increase in the growth inhibition as the concentration is increased. With the increase in the concentration of the pomegranate peel extract from 20 to 120µl/ml, the percent inhibition for HCT 15 cancer cell line increased from 27.01 to 68.34% in Acetone pomegranate extract, 30.34% to 75.31% in Methanolic pomegranate peel extract and 35.34 to 84.56% in Ethyl acetate pomegranate peel extract, which means they induced cell arrest to inhibit the growth of the HCT 15 cancer cells. In the same way, in case of T47D, as the concentration of Acetone, Methanol and Ethyl acetate pomegranate peel extracts increased from 20 to 120 µl/ml, the percent inhibition of T47D cancer cell growth increased from 22.56 to 63.13% in Acetone pomegranate peel extract, 25.82 to 64.37% in Methanol pomegranate peel extract and 28.34 to 68.37% in Ethyl acetate pomegranate peel extract (Fig.2 & 3).
Fig. 2: Percentage of HCT 15 cell inhibition at various concentrations of Pomegranate peel extract
Fig. 3: Percentage of T47D cell inhibition at various concentrations of Pomegranate peel extract
DISCUSSION:
Natural plant extracts and phytoconstituents have excellent bioactivity in vitro but less in vivo due to their poor lipid solubility or multiple ring large molecules or destruction in gut. Novel drug delivery system phytosomes were prepared by complexing polyphenolic phyto-constituents with phospholipid mainly phosphatidylcholine which bind components to each other on a molecular level. Bioavailability is enhanced due to their capacity to cross the lipid rich bio-membranes and to protect the valuable components of the herbal extract from destruction by digestive secretions and gut bacteria29. Pomegranate (Punica granatum L) gained considerable recognition as a functional food in the modern era. Phytochemical characterization has been carried out on the peel extract using ethyl acetate. The phytochemical analysis showed the presence of phenols, flavonoids, quinones, saponins, cardinolites, steroids, tanins and terpenoids in various extracts30. My findings were in correlation with the works of others carried out on natural sources. The leaf extract of Azima tetracantha has shown anticancer potential against MCF-7 cell lines while compared to that of the stem extract. The plant possesses remarkable anticancer activity and hence isolation of the compound contributing to the activity may lead to develop at a novel and natural phytomedicine for the disease31. The in vitro studies performed by MTT assay using human breast cancer cell line MCF-7 displayed that the various extracts of sweet cherry, Prunus avium (ethanolic and ethyl acetoacetate) possessed a very good anticancer activity and all were exhibiting the potential capability to inhibit the cancer cell when compared with standard drug doxorubicin and the cell growth inhibition was found to be the highest 92.90% growth inhibition at 10 µg (IC50 = 2.4 µg/ml) 32.
CONCLUSION:
The percentage of viability of HCT 15 and T47D cancer cells free from bacterial and fungal contamination carried out by using Trypan blue staining were 73.56%, 78.35% respectively which were most suitable to perform cytotoxicity studies. With the increase in the concentration of the pomegranate peel extract (20 to 120 µl/ml acetone, methanol and ethyl acetate extracts), the percentage of viability of cells tends to be decreased. By subjecting the cell lines to MTT assay, it was concluded that ethyl acetate pomegranate peel extract showed maximum inhibition of cancerous cell growth.
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Received on 06.07.2019 Modified on 16.08.2019
Accepted on 21.09.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(1): 303-307.
DOI: 10.5958/0974-360X.2020.00061.X