In Vitro Cytotoxicity of Cardamom Oil and Carrot Seed Oil on Mouse Fibroblast and Urinary Bladder Cell Line

Introduction

Cancer is a major health problem worldwide, which involves uncontrolled growth of cells capable of invading neighbouring tissues and finally spreading to distant tissues of the body. In developed and developing countries cancer is one of the leading causes of death.¹ According to data collected by International Agency for Research in Cancer, about 14.1 million new cancer cases and 8.2 million deaths were reported in 2012.² Since plant-derived products induce lesser side effects when compared to synthetic drugs. Essential oils are a complex mixture of volatile, liquid, odorous and flavor substances synthesized by plants. Plant species producing EOs are classified as aromatic by their distinctive and characteristic aroma and odour.³ Cardamom oil (Elettaria cardamomum) is one of the world’s most ancient spices, known as the Queen of all spices and belongs to the family Zingiberaceae and locally known as “elaichi”. It is cultivated widely in the tropical region with Guatemala, India, and Sri Lanka as the main producer countries currently.^4,5 Cardamom is used to improve the circulation of the lungs and to treat asthma in Ayurvedic medicine.⁶ Cardamom seeds showed beneficial effect in anorexia, dyspepsia, asthma, bronchitis, haemorrhoids, halitosis, gastropathy and burning sensation⁷ . Carrot seed oil (Daucus carota L.) a biennial herbaceous species, belonging to Apiaceae family. Carrot is nutritionally important as an excellent source of provitamin a, carotenoids, vitamins C, D, E, K, B1, B6, biotin and minerals. Carrots are used in traditional herbal medicine due to its antilipemic, stomachic, hypotensive, carminative and diuretic properties.⁸

Materials and Methods

Essential oils : The essential oils cardamom oil and carrot seed oil were procured from Sigma- Aldrich (St.Louis, Mo, USA).

Standred drug : 5- Flurouracil (1g) was procured from Sigma-Aldrich (St.Louis, Mo, USA).

Animals : Adult Swiss Albino mice of either sex were used for the study. The mice weighing between 25-30 g were procured from central animal house of H.K.E.S Matoshree Taradevi Institute of Pharmaceutical Sciences, Kalaburagi. The animals were housed in polypropylene cages and maintained under standard conditions at 25±2^o C with relative humidity of 55-65% under 12 h light/dark cycle. Animals were fed with standard pellet diet with water ad libitum. The study was carried out in Research lab, Department of Pharmacology, H.K.E.’s Matoshree Taradevi HKECOP/IAEC/64/2014-19).

Preliminary phytochemical screening

The above mentioned essential oils were subjected to preliminary qualitative tests for various constituents using suitable chemical tests. The essential oils were solubilised in 70% ethanol and tests were carried out.⁹ The results are shown in Table 1.

Acute oral toxicity testing

The acute oral toxicity tests were conducted in mice using cardamom oil and carrot seed oil. Acute oral toxicity tests were performed according to OECD guidelines 425.¹⁰ Normal adult Swiss albino mice of either sex weighing between 20 - 25 g were used for the study. The food, but not water, was withheld for 4 h before the test drug was administered orally. The essential oils were given in doses of 2000 mg/kg orally and the number of mice per dose was three. The mice were observed for 24 h for behavioural, neurological and autonomic profiles and for any lethality or death over the next 48 h.

Chemicals

3-(4,5–dimethylthiazol–2–yl)–5–diphenyl tetrazolium bromide (MTT) Sigma Aldrich Co, St Louis, USA. Fetal Bovine serum (FBS), Phosphate Buffered Saline (PBS), Minimal Essential Media (DMEM) and Trypsin were were procured from Invitrogen. Ethylene diamine tetra acetic acid, Glucose and antibiotics were obtained from Hi-Media Laboratories Ltd., Mumbai. Dimethyl Sulfoxide (DMSO) and Propanol were obtained from E.Merck Ltd., Mumbai, India.

Cell lines and culture medium

L929 (mouse fibroblast) cell line and RT4 (urinary bladder) cell line was procured from ATCC, India. Stock cells were cultured in DMEM and McCoy′s 5a medium with 2mM Glutamine respectively. Both the media were supplemented with 10% inactivated Fetal Bovine Serum (FBS), 1% penicillin (100 IU/ml), and streptomycin (100 µg/ml) in an humidified atmosphere of 5% CO₂ at 37^o C until confluent. The cell was dissociated with TPVG solution (0.2 % trypsin, 0.02 % EDTA, 0.05 % glucose in PBS). The viability of the cells are checked using trypan blue and centrifuged. Further, 50,000 cells / well of L929 and RT4 were seeded in a 96 well plate and it was incubated for 24 h at 37^o C, 5 % CO₂ incubator.

Preparation of test solutions

For cytotoxicity studies, 10mg of test drug was separately dissolved in media containing 1% Tween 80 and volume was made up with minimal essential medium supplemented with 2% inactivated Fetal Bovine Serum to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies.

Determination of cell viability by microculture tetrazolium assay (MTT)11

Principle: The ability of the cells to survive a toxic assault has been the basis of most cytotoxicity assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved is the cleavage of tetrazolium salt 3-(4,5 dimethyl thiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) into a blue coloured product (formazan) by mitochondrial enzyme Succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the cells used.

Procedure: Test samples were placed in each well of the 96 well microtiter culture plate. The L929 monolayer cells were subjected for trypsinization and the cell count was adjusted to 5.0 x 105 cells/ml using DMEM containing 10% FBS. The RT4 monolayer cells were subjected to trypsinization and the cell count was adjusted to 5.0 x 10⁵ cells/ml using McCoy′s 5a containing 10% FBS. To each well of the 96 well microtiter plate, 100 µl of the diluted cell suspension (50,000 cells/well) was seeded on each scaffold and cells seeded on cell culture plate. The plates were then incubated at 37^O C for 1 day in 5% CO₂ atmosphere. After 24 h, the test solutions in the wells were discarded and 100 µl of MTT (5 mg/10 ml of MTT in PBS) was added to each well. The plates were gently shaken and incubated for 4 h at 37^o C in 5% CO₂ atmosphere. The mitochondrial dehydrogenase enzymes of viable cells cleave the tetrazolium ring to an insoluble purple formazan. The supernatant was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the intracellular formed formazan and the absorbance was measured using a microplate reader at a wavelength of 590 nm.

The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC₅₀) values is generated from the dose-response curves for each cell line.

Calculating percentage growth inhibition:

% inhibition = 100 - (Mean OD of individual test group/Mean OD of control group) X 100

Statistical analysis:

The results are expressed as Mean ± S.E.M using one-way ANOVA followed by Dunnett’s test with help of Graph Pad Prism version 5.00 for Windows Vista TM BASIC. P value < 0.05 was considered as significant.

The results were expressed as Mean ± SEM using one-way ANOVA followed by Dunnett’s test of significance. ** indicates P < 0.001, *** indicates p < 0.0001 compared to standard. The P values were compared with control ***P < 0.0001.

The results were expressed as Mean ± SEM out using one-way ANOVA followed by Dunnett’s test of significance. * indicates P < 0.001, *** indicates p < 0.0001 compared to standard. The values were compared with control ***P < 0.0001.

Results

Preliminary phytochemical screening

Phytochemical analysis has shown the presence of carbohydrates and flavonoids in cardamom oil. In carrot seed oil, it has shown the presence of carbohydrates, alkaloids, flavonoids, steroids and glycosides 

Acute toxicity studies

In acute oral toxicity studies, no changes in the behavior and autonomic profiles as well no mortality were observed in all treated mice up to the dose of 2000 mg/kg. 

Cytotoxic activity towards cancer cells

In the present study we have chosen cardamom oil and carrot seed oil against mouse fibroblast and urinary bladder cell line. To investigate the cytotoxic activities, human cancer cell lines L929 (mouse fibroblast) and RT4 (urinary bladder cell line) were exposed to increasing concentrations of essential oils. Cell viability was determined by the MTT assay. The results showed that cardamom oil and carrot seed oil exhibited strongest cytotoxicity in L929 cell line with IC₅₀ values of 322.5 µg/ml and 171.9 µg/ml compared to standard. Both the essential oils at a concentration of 1000, 500, 250 & 125µg/ml have shown statistically significant (***p < 0.0001) anticancer activity towards L929 cell line as reported in Table 2. These essential oils too showed strongest cytotoxicity towards RT4 cell line with IC₅₀ values of 205.5 µg/ml and 224.5 µg/ml compared to standard. At higher concentrations (1000 µg/ml) cardamom oil and carrot seed oil have shown statistically significant (***P < 0.0001 & * P < 0.001) anticancer activity in RT4 cell line and even at a concentration of 250 µg/ml cardamom oil showed statistically significant (*P < 0.001) anticancer activity towards RT4 cell line as reported in Table 3.

Discussion

Bladder cancer is a very common cancer in the United States (with an estimated 68,810 new cases and 14,100 deaths per year) and a significant cause of morbidity and mortality throughout the world.¹² Urothelial cell carcinoma of the bladder is the second most common malignancy of the genito-urinary tract. In Japan, the incidence of bladder cancer was estimated to be 12.4 per 100 000 in men and 3.5 per 100 000 in women. More than 90% of case of UBC occur in people older than 55 and 50% of cases occur in people older than 73. UBC has high world wide incidence for cancer. It stands as seventh most common malignancy in men and seventeenth in women.^13,14 An estimated 386,300 new bladder cases and 150,200 deaths from bladder cancer were diagnosed worldwide in 2008.¹⁵

RT4: Species: human (Homo sapiens), Cell Type: Epithelial, Tissue of Origin: Bladder,

Derived from explants of a recurring papillary tumour of the bladder. L929: Biological source: Adipose from mouse, Tissue: subcutaneous connective tissue; areolar and adipose, Morphology: fibroblast.

Fibroblasts are widely distributed in many types of tissues, such as tendon, ligament and skin. Fibroblasts are the cells that produce collagens and are considered to be the primary source of most extracellular matrix components. They play a critical role in regulating the turnover of extracellular matrix and play an important part in wound healing.¹⁶ L929 cell line is usually used as tool in many standard testing. It is a cell line which is popular in many experimental aspects such as material biocompatibility testing,¹⁷ drug cytotoxicity testing,18 and cell biology studies,¹⁹ etc.

Previous studies reported that Limonene, one of the active ingredients present in cardamom, has been shown to exert a chemopreventive activity against cancers of the mammary liver, lung, stomach and skin in rodents. Limonene has been found to inhibit various cancer cell growths without significant toxicity including prostate cancer cells.²⁰ Previous studies also revealed that Daucus carota oil extract exhibited potent cytotoxicity against colon (Caco-2, HT-29), breast (MCF-7, MDA-MB-23)21 and human acute myeloid leukemia cells.²² It also inhibited motility of MDA-MB 231 and SF-268 cells and reduced invasion of B16F-10 cells.²³ Researchers also reported the anti-carcinogenic effect of carrot juice extracts on myeloid and lymphoid leukemia cell lines. Investigators considered that β-carotene and falcarinol present in the carrot juice extract may have been responsible for this beneficial effect of “kill” leukemia cells and inhibit their progression.²⁴ β-carotene and several polyacetylenes are shown to induce cell cycle arrest and apoptosis in human tumor cell lines.

In the present study, it has been observed that both carrot seed oil & cardamom oil have shown significant cytotoxic activity towards L929 cell line. The cytotoxic activity of cardamom oil is due to the presence of limonene,²⁰ whereas cytotoxic activity of carrot seed oil is due to the presence of β-carotene and falcarinol.²⁴ The carrot seed oil & cardamom oil at higher concentration shows significant reduction in the mouse fibroblast cell line, when compared to control. The cardamom oil at higher concentrations also showed significant cytotoxic activity towards RT4 cell line and even carrot oil too showed moderate cytotoxic activity towards RT4 cell line compared to control. The results are shown in Table 1 & Table 2 and Conc. vs % inhibition can be studied from Graphical representation 1 & 2.

Conclusion

The present study indicates that both cardamom oil and carrot seed oil possess significant (***P < 0.0001) anticancer activity towards L929 cell line which might be due to the presence of phytoconstituents such as limonene in cardamom oil, β-carotene and falcarinol (flavonoid) in carrot seed oil which reduced the number of cancer cells. In L929 cell line, among the above essential oils, carrot seed oil showed highest cytotoxicity when compared to cardamom oil (Carrot seed oil> Cardamom oil). Hence from above results we can say that carrot seed oil has greater cytotoxicity on L929 cell line. In RT4 cell line, among the above essential oils cardamom oil showed higher cytotoxicity when compared to carrot seed oil (Cardamom oil> Carrot seed oil). Hence from above results we can say that cardamom oil has greater cytotoxicity on RT4 cell line. 

Conflict of Interest

The authors declare no conflict of interest.