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In vitro cytotoxicity of cardamom oil, lemon oil, and jasmine oil on human skin, gastric, and brain cancer cell line


 Department of Pharmacology, H.K.E. Society's Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India

Correspondence Address:
Chetan Manjunath,
Assistant professor, Department of Pharmacology, H.K.E. Society's Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences, Sedam Road, Gulbarga - 585 105, Karnataka
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_915_17

 > Abstract 


Objective: The main objective of the study was to evaluate the cytotoxicity of selected essential oils on human skin, gastric, and brain cancer cell lines using microculture tetrazolium test.
Materials and Methods: Phytochemical analysis, as well as acute oral toxicity tests, was carried out in female albino mice with cardamom oil, lemon oil, and jasmine oil according to the Organization for Economic Co-operation and Development guidelines 425. Anticancer activities of the above test drugs were performed using human cancer cell lines. The studies were carried out at Skanda Life Sciences Pvt. Ltd., Bengaluru.
Results: Phytochemical analysis has shown the presence of carbohydrates and flavonoids in cardamom oil. While lemon oil has shown the presence of carbohydrates, flavonoids, steroids, terpenoids, and tannins, jasmine oil has shown the presence of carbohydrates, alkaloids, flavonoids, steroids, terpenoids, and glycosides. Toxicity studies showed that cardamom oil, lemon oil, and jasmine oil were all found to be safe up to 2000 mg/kg body weight. Results have shown that lemon oil exhibited the strongest cytotoxicity toward three human cancer cell lines, namely skin cancer (A431), gastric cancer (MKN-45), and brain cancer (U-87 MG) cell lines, with higher IC50 values of 62.82 μg/ml, 220.9 μg/ml, and 440.1 μg/ml compared to standard. Jasmine oil exhibited the strongest cytotoxicity toward skin cancer and brain cancer cell lines, whereas cardamom oil has shown stronger cytotoxicity only toward skin cancer cell line but did not show any level of inhibition of growth of brain and gastric cancer cells.
Conclusion: Our study reveals that lemon oil, jasmine oil, and cardamom oil possess potent antitumor activity compared to standard. At different concentrations, lemon oil has shown statistically significant (***P < 0.0001) anticancer activity toward all the three human cancer cell lines. While jasmine oil has shown statistically significant (***P < 0.0001) anticancer activity toward skin and brain cancer cell line, cardamom oil has also shown statistically significant (***P < 0.0001) anticancer activity but only toward skin cancer cell line.

Keywords: Cancer cell lines, cardamom oil, cytotoxicity, jasmine oil, lemon oil, 3-(4, 5 dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay



How to cite this URL:
Manjunath C, Mahurkar N. In vitro cytotoxicity of cardamom oil, lemon oil, and jasmine oil on human skin, gastric, and brain cancer cell line. J Can Res Ther [Epub ahead of print] [cited 2019 May 20]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=257731




 > Introduction Top


Cancer is a complex, multifaceted group of diseases characterized by uncontrolled cell growth, local tissue invasion, and metastasis. According to recent statistics, cancer accounts for about 23% of all disease-related deaths in the United States of America (USA). It is the second most common cause of death after heart disease, particularly in developing countries where it is estimated that the impact of cancer in the population corresponds to approximately 80% from the 20 million new cases estimated for 2025. In Brazil, the National Institute of Cancer estimates for the 2016–2017 period around 600,000 new cases of cancer.[1] Many synthetic anticancer agents are used in the treatment of cancer which in turn have aggravated the risk of side effects among the cancer patients. The use of essential oils of aromatic plants for dietary and therapeutic purposes has been a focus on research in health sciences since the secondary metabolites of plant have shown low side effects and toxicity.[2] The use of medicinal plants has a strong track record of contributing to drug development, and there is a broad consensus that the potential for new natural products from plants is not exhausted and still represents an important source for the lead in drug discovery.[3] The antitumor property of essential oils has been the source of investigation for the development of drugs to treat different types of cancer. Cardamom oil (Elettaria cardamomum) locally known as “elaichi” one of the world's most ancient spices, referred to as the Queen of all spices belonging to family Zingiberaceae. It is a perennial herb, indigenous to India, Pakistan, Burma, and Sri Lanka.[4] Cardamom is the most common ingredient of the Indian Ayurveda and Chinese traditional medicine, and it possesses an extensive array of bioactive compounds.[5] It is used in the treatment of bronchitis, hoarse voice, impotence, vomiting, blood pressure, vomiting, dry lips, arrhythmia diarrhea, toothache, and gum bleeding. Cardamom exhibits chemopreventive and anticancer qualities, which have been suggested to significantly reduce the diameter and weight of tumors and papillomas. In India, Kerala is the main producer of cardamom, responsible for 70% of total cultivation, followed by Karnataka (20%) and Tamil Nadu (10%).[6]

Lemon oil (Citrus limonum) locally known as “nimbu” belongs to family Rutaceae. The origin of lemon is unknown though lemons are thought to have grown in Assam, Northern Burma, and China.[4] Citrus fruit has an abundant amount of carotenoids, flavonoids, bitter limonoids, Vitamin C, and volatile compounds. Limonene (the major component of Citrus oils) is known for its medicinal and pharmacological actions such as antitumor, anti-inflammatory, digestive, and larvicidal activities. It eases constipation, relieves water retention, promotes circulation, increases absorption of Vitamin C to fight cold and flu, and also strengthens the skin.[7] Jasmine oil (Jasminum officinale L.) is commonly known as “mallige” belonging to family Oleaceae and native to tropical and subtropical regions of Asia, Africa, and Australia. The main constituents include methyl anthranilate, indole, linalool, ketone, benzyl acetate, cis-jasmone, and skatole.[8] J. officinale was used conventionally for the treatment of urinary tract infections, as central nervous system (CNS) depressant, sedative, mild anesthetic, and astringent. In addition, it was used in depression and stress-related conditions. It was also used for coughs, laryngitis, dysmenorrhea, labor pains, uterine disorders, skin problem such as dry, greasy, irritated, and sensitive skin, and muscular spasms and sprains. The buds of J. officinale L. were used as a folk remedy for the treatment of hepatitis and stomatitis in South China.[9]

In this study, we have investigated cytotoxic properties of selected essential oils on human cancer cell lines using microculture tetrazolium test.


 > Materials and Methods Top


Essential oils

The essential oils, namely cardamom oil, lemon oil, and jasmine oil, were all procured from Sigma-Aldrich (St. Louis, MO, USA).

Standard drug

Standard drug 5-fluorouracil (1 g) was procured from Sigma-Aldrich (St. Louis, MO, USA).

Animals

Adult Swiss albino mice of either sex are used for the study. The mice weighing between 25 and 30 g were procured from Central Animal House of M.R. Medical College, Kalaburagi. The animals were housed in polypropylene cages and maintained under standard conditions at 25°C ± 2°C with relative humidity of 55%–65% under 12-h light/dark cycle. Animals were fed with standard pellet diet with water ad libitum (IAEC NO-HKES/MTRIPS/IAEC/90/2017-19).

Preliminary phytochemical screening

The essential oils were subjected to preliminary qualitative tests for various constituents using suitable chemical tests. The essential oils were solubilized in 70% ethanol and the tests were carried out.[10] The results are shown in [Table 1].
Table 1: Preliminary phytochemical screening

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Acute oral toxicity testing

The acute oral toxicity tests were conducted in mice using cardamom oil, lemon oil, and jasmine oil. Acute oral toxicity tests were performed according to the Organization for Economic Co-operation and Development guidelines 425.[11] Normal adult Swiss albino mice of either sex weighing between 20 and 25 g were used for the study. The food, but not water, was withheld for 4 h before the extract 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 behavioral, neurological, and autonomic profiles and for any lethality or death over the next 48 h.

Chemicals

3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fetal bovine serum, phosphate buffered saline (PBS), minimal essential media, and trypsin were obtained from Sigma-Aldrich Co., St Louis, MO, USA. Ethylene diamine tetraacetic acid (EDTA), glucose, and antibiotics were obtained from Hi-Media Laboratories Ltd., Mumbai. Dimethyl sulfoxide and propanol were obtained from E. Merck Ltd., Mumbai, India.

Cell lines and culture medium

The U87-MG cell line was procured from the American Type Culture Collection (ATCC) with reference to the catalog number of the cell line, viz., ATCC® HTB-14™, and MKN-45 was procured from DSMZ with reference to the DSMZ number ACC 409. They were thawed and cultured in T25 tissue culture-treated flask at 37°C with 5% CO2. A431 cell line was procured from ATCC with reference to the catalog number A-431 (ATCC® CRL-1555™).

Stock cells were cultured in Dulbecco's minimal essential medium (DMEM) and Roswell park memorial institute medium supplemented with 10% inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and amphotericin B (5 μg/ml) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with trypsin phosphate versene glucose solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks, and all experiments were carried out in 96 microtiter plates (Tarsons India Pvt. Ltd., Kolkata, India). The results are shown in [Table 2], [Table 3], [Table 4] and [Figure 1], [Figure 2], [Figure 3], [Figure 4].
Table 2: Cytotoxic properties of test drugs against A431 cell line

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Table 3: Cytotoxic properties of test drugs against MKN-45 cell line

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Table 4: Cytotoxic properties of test drugs against U87 MG cell line

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Figure 1: Chemical reaction showing conversion of MTT to Formazan

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Figure 2: Cell line: A 431 American Type Culture Collection No: CRL-1555

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Figure 3: Cell line: MKN 45 American Type Culture Collection No: 40

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Figure 4: Cell line: U-87 MG American Type Culture Collection No: HTB-14

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Preparation of test solutions

For cytotoxicity studies, each test molecule was weighed and mixed to obtain the desired concentration and dissolved in distilled dimethyl sulfoxide, and the volume was made up with DMEM 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 test assay (3-(4,5 dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide)

Principle

The ability of the cells to survive a toxic assault has been the basis of most cytotoxicity assays.[12] 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 MTT into a blue-colored 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

The monolayer cell culture was trypsinized, and the cell count was adjusted to 1.0 × 105 cells/ml using minimal essential medium containing 10% fetal bovine serum. To each well of the 96-well microtiter plate, 0.1 ml of the diluted cell suspension (approximately 50,000 cells/well) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off and washed the monolayer once with medium, and 100 μl of different test concentrations of test drugs was added on to the partial monolayer in microtiter plates. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 μl of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 μl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula, and the 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



Statistical analysis

Data were expressed as mean ± standard error of the mean, and statistical analysis was carried out by one-way ANOVA followed by Dunnett's test using GraphPad Prism version 5.00 for Windows Vista™ BASIC, GraphPad Software, San Diego, CA, USA (www.graphpad.com). P < 0.05 was considered statistically significant


 > Results Top


Preliminary phytochemical screening

Phytochemical analysis has shown the presence of carbohydrates and flavonoids in cardamom oil. However, carbohydrates, flavonoids, steroids, terpenoids, and tannins were present in lemon oil. Analysis has also shown the presence of carbohydrates, alkaloids, flavonoids, steroids, terpenoids, and glycosides in jasmine oil.

Acute toxicity studies

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

Cytotoxic activity toward cancer cells

In the present study, we have chosen cardamom oil, lemon oil, and jasmine oil against human skin, gastric, and brain cancer cell lines. Different concentrations of these essential oils were evaluated for their cytotoxic effect against above-mentioned cell lines. Cell viability was determined by MTT assay. As shown in [Table 2], [Table 3], [Table 4], lemon oil and jasmine oil showed different cytotoxic activities toward the three human cancer cell lines. However, cardamom oil showed cytotoxicity only toward A431 cell line but did not show any level of inhibition of growth of U87-MG and MKN-45 cells. A dose-dependent decrease in the growth of cells was observed with all the three cancer cell lines. The results suggest that lemon oil exhibited the strongest cytotoxicity toward three human cancer cells, namely skin cancer, gastric cancer, and brain cancer cell line, with IC50 values of 62.82 μg/ml, 220.9 μg/ml, and 440.1 μg/ml, respectively, when compared to standard. Jasmine oil exhibited the strongest cytotoxicity toward skin cancer and brain cancer cell line with IC50 values of 99.86 μg/ml and 336.2 μg/ml compared to standard. Jasmine oil also showed slight cytotoxicity toward gastric cancer cell line when compared to standard. Lemon oil has shown statistically significant (***P < 0.0001) anticancer activity toward A431, MKN-45, and U87-MG cell line at a concentration of 1000, 500, and 250 μg/ml when compared to standard. Cardamom oil has shown statistically significant (***P < 0.0001) cytotoxicity toward A431 cell line with an IC50 value of 166 μg/ml. The cytotoxicity was observed at concentration of 1000, 500, 125, and 62.5 μg/ml when compared to standard. Jasmine oil has shown statistically significant (***P < 0.0001) anticancer activity toward A431 and U87-MG cell line at a concentration of 1000 and 250 μg/ml compared to standard. Even at a concentration of 125 μg/ml, jasmine oil has shown statistically significant (***P < 0.01) anticancer activity toward MKN-45 cell line compared to standard.[13],[14]


 > Discussion Top


Skin cancer is one of the most prevalent cancers in the US and other regions of the world. Skin malignancy is manifested in many forms, such as melanoma, squamous cell carcinoma, and basal cell carcinoma. Common treatment protocols of skin cancers include surgical excision or radiation therapy. In addition, chemotherapy might be a better alternative in some situations where surgery is contraindicated.[15] In the US, the incidence of skin cancer has increased every year, resulting in a doubling of the total number of patients over the past 20 years.[16] Squamous cell carcinoma is the second most common skin cancer, with an incidence of 20%.[17] Gastric cancer is the most common malignant cancer of the gastrointestinal tract in the world. Gastric cancer is the second leading cause of mortality in the world after lung cancer. It is estimated that >750,000 new cases of gastric cancer are diagnosed every year throughout the globe.[18]

Gastrointestinal cancers are still the most important causes of morbidity and mortality among all cancer types. In 2016, 26,370 new cases and 10,730 deaths from gastric cancer were reported in the USA and even 133,999 new cases and 48,500 deaths in Japan. Mostly, gastric adenocarcinomas are diagnosed among gastric cancer patients.[19]

Gastric cancer has been found to be more prevalent in people aged 60 years or above. In 2005, the incidence rate of gastric cancer (3 million deaths and 4 million new cases) ranked third among the most common cancers in China.[20] Gliomas arise from glial cells and account for almost 80% of primary malignant brain tumors, which are refractive to all treatment modalities including surgical resection, chemotherapy, and radiotherapy. It is not considered to be the most frequent form of cancer; glioma results in more years of life with an incidence of 2–3 new cases per 100,000 persons annually.[21] Glioblastoma, one of several kinds of gliomas, is a cancer of astrocytes in the brain and the spinal cord and is the most common and malignant form of cancer of the CNS.[22]

Glioblastoma is among the most aggressive and lethal cancers to treat due to their rapid growth, highly invasiveness, and enhanced angiogenesis.[23] Natural products are a good source of compounds with novel chemical structures that are effective and less toxic. These natural compounds, in general, show multi-targeted effects and can modulate several oncogenic transcription factors that block the tumor microenvironment targets that usually sustain tumor growth.[24]

Novel natural products offer opportunities for innovation in drug discovery. Plant essential oil, its components, and secondary metabolites have many applications in folk medicine. Essential oils are lipophilic compounds containing volatile aroma compounds. The constituents of the oils are mainly monoterpenes and sesquiterpenes. Many authors have emphasized that the active components of dietary phytochemicals are potent biological agents with interesting anticancer properties that appear to be protective against cancer.[25]

Major constituents of cardamom essential oil 1,8-cineole and limonene block the activities of cyclooxygenase-2 and cytochrome P450 and downregulate several signal transduction molecules.[6] Limonene has been found to inhibit various cancer cell growths without significant toxicity including prostate cancer cells.[26] Administration of limonene resulted in partial response in a patient with breast cancer and stabilized the disease for >6 months in patients with colorectal cancer.[27] Linalool has been shown to have antitumor activity against several human tumor cell lines including leukemia cells.[28]

Authors have reported that the anticancer properties of Citrus flavonoids focus primarily on the anticarcinogenic properties of the flavone and flavanone glycosides and on the antitumor properties of the polymethoxylated flavones (PMFs). The PMFs in Citrus are distinctive for their high antiproliferative activities against a number of human cancer cell lines.[29] It has been reported that oral administration of extracts from C. limonum has wound-healing effects on the skin of diabetic rats. Oral administration of limonoids present in Citrus fruits inhibits intestinal tumorigenesis using Apc-mutant Min mice.[30]

Jasmine oil was evaluated in vitro for their antiproliferative activity against Hep-2, MCF-7, and Vero cell lines. Jasmine oil exhibited significant antiproliferative activity against one or more of the cell lines.[31]

In the present study, it has been observed that lemon oil has shown significant cytotoxic activity toward all the three cell lines A431, MKN-45, and U87 MG.

In A431 cell line, among the above essential oils, jasmine oil showed the highest cytotoxicity when compared to cardamom oil which was in turn higher than lemon oil (jasmine oil > cardamom oil > lemon oil). Hence, from the above results, we can say that jasmine oil has greater cytotoxicity on A431 cell line.

In MKN-45 cell line, among the above essential oils, lemon oil showed the highest cytotoxicity when compared to jasmine oil which was in turn higher than cardamom oil (lemon oil > jasmine oil > cardamom oil). Hence, from the above results, we can say that lemon oil has greater cytotoxicity on MKN-45 cell line.

In U87-MG cell line, among the above essential oils, lemon oil showed the highest cytotoxicity when compared to jasmine oil which was in turn higher than cardamom oil (lemon oil > jasmine oil > cardamom oil). The cytotoxic activity of lemon oil is mainly due to presence of flavonoid and the phytochemical tests have shown positivity for flavonoid. Hesperidin is a major flavonoid present in lemon and considerable amount of research has been carried out on the anticancer activities of hesperidin and its aglycone hesperetin. Encouraging results of carcinogenesis inhibition were observed by using a hesperidin/diosmin combination on male mouse urinary bladder.[32] High concentrations of hesperidin, eriocitrin, and diosmin are known to inhibit human hepatocellular carcinoma. Further, evidence for the anticancer property of Citrus flavonoids, such as flavanones and PMFs, has been provided by numerous in vitro and in vivo studies.[33]


 > Conclusion Top


The present study indicates that lemon oil has shown statistically significant (***P < 0.0001) anticancer activity toward all the three human cancer cell lines, jasmine oil has shown statistically significant (***P < 0.0001) anticancer activity toward skin and brain cancer cell line, whereas cardamom oil has shown statistically significant (***P < 0.0001) anticancer activity only toward A431 cell line. Anticancer activity of all above-mentioned oils might be due to the presence of certain phytoconstituents which reduce the number of cancer cells.

Acknowledgment

Authors are thankful to the authorities of H.K.E. Society's MTRIPS for the facilities provided to carry out the work.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare that they have no actual or potential conflicts of interest.



 
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[PUBMED]  [Full text]  
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

 
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