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In Vitro Evaluation of Anticancer Activity of Sorgamara Ilai Chooranam (Leaves of Simarouba Glauca) Against A549 Cell Line (Human Lung Cancer Cell)

Lung cancer is the leading cause of cancer-related deaths, both in women and among men. In India, Several population based cohort studies were conducted which confirm the significant burden of lung cancer in India, contributing significantly toward the cancer morbidity and mortality. In order to get rid of adverse effects from currently available treatment, scientists have focused on the development of safe anti-cancer agents from herbal sources. Sorgamara Ilai chooranam (Simarouba glauca) is one of the herb mentioned in Siddha text with anti-cancer potential. Hence this present study was performed to validate anti-cancer potential of Sorgamara Ilai Chooranam through MTT assay on A549 cell line at various concentrations. Result showed that the exposure of A549 cells to Sorgamara Ilai Chooranam at the various concentrations significantly reduced the cell viability in a concentration dependent manner. As the concentration increases the cell viability% significantly decreases. For Sorgamara Ilai Chooranam from 7.8μg/ml to 62.5 μg/ml concentration the cell viability % significantly decreased from 74.85 to 52.80. In conclusion, the present study shows that Sorgamara Ilai Chooranam obtained from Simarouba glauca exhibited cytotoxic and anti proliferative activities. The anticancer activities could be explained partly by the presence of high content of Quassinoids and phenolic compounds. However, pharmacological and toxicological studies, preclinical and clinical trials are needed to elucidate the utility of Sorgamara Ilai Chooranam in the treatment of lung cancer.


INTRODUCTION
Lung cancer is the leading cause of cancer-related deaths, both in women and among men. Tobacco use is responsible for nearly 90 percent of all lung cancer cases. Lung cancer affects approximately 7 million people each year globally in which more than 6 million deaths result from direct tobacco use and more than 890,000 deaths result from exposure to second-hand smoke (1,2) . The incidence and mortality of lung cancer have significantly increased worldwide (3) . It is the leading cause of cancer death in developed countries and is rising in alarming rates in developing countries (4) .
In India, Several population based cohort studies were conducted which confirm the significant burden of lung cancer in India, contributing significantly toward the cancer morbidity and mortality (5) . The overall estimated lung cancer mortality in India in 2012 was 63,759, making it the third most common cause of cancer-related mortality in India after breast and cervical cancer (6) .
Even though lung cancer can be treated with chemotherapy and radiotherapy, these treatments can have a negative effect on normal cells (7) . In order to get rid of these adverse effects, scientists have focused on the development of safe anti-cancer agents from herbal sources. Several researches have been done so far on herbs to extract anti-cancer agents with seemingly impressive results. For many years medicinal plants have been used in Siddha system of medicine for treating various diseases, including cancer. Many single, poly-herbal formulation are available in this system of medicine to treat cancer more effectively. These drugs kill cancer cells, support defense system and detoxify the carcinogens.
Among them Sorgamara ilai chooranam (Simarouba glauca) is one of the herb mentioned in Siddha text with anti-cancer potential (8) . It is also known as 'Laxmitaru' or 'paradise tree' has a long history of herbal medicine in many countries and belongs to the family Simaroubaceae. Because of its anticancer potential, it is known as tree of solace of cancer (9) . The main distribution hot spots are located at tropical areas of America, Africa, Madagascar and Australia, Cuba, Brazil, Mexico, Peru, India (10) . Hence this present study was performed to validate anti-cancer potential of Sorgamara Ilai Chooranam through MTT assay on A549 cell line (Human lung cancer cell).

Source of Collection
S. glauca leaves were collected from distinct region of Kolli hills. After collection the leaves were identified and authenticated by the Gunapadam experts in Government Siddha Medical College, Arumbakkam, Chennai -106. The specimen sample of the herb has been preserved in PG Gunapadam department for future reference.

Procedure
Fresh leaves were collected and dried at room temperature. The dried leaves were powdered by means of grinder and the powder was sieved by a cotton cloth and then bottled up. It was labeled as Sorgamara Ilai Chooranam (SMC).

In vitro evaluation of anticancer activity
Cell line and culture In Vitro assay for anti-cancer activity: (MTT assay) (11) Cells (1 × 105/well) were plated in 24-well plates and incubated in 370C with 5% CO2 condition.
After the cell reaches the confluence, the various concentrations of the samples were added and incubated for 24hrs. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) or DMEM without serum. 100μl/ well (5mg/ml) of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added and incubated for 4 hours. After incubation, 1ml of DMSO was added in all the wells. The absorbance at 540nm was measured with UV-Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50% inhibition (IC50) was determined graphically. The % cell viability was calculated using the following formula:

% Cell viability = A570 of treated cells / A570 of control cells × 100
Graphs are plotted using the % of Cell Viability at Y-axis and concentration of the sample in Xaxis. Cell control and sample control is included in each assay to compare the full cell viability assessments. www.ajptr.com

RESULTS AND DISCUSSION
Since the dawn of civilization, humans have been using plants, minerals and animal products as a medicine to fight against their diseases including cancer. Still, Siddha system of medicines treats many patients when other systems of medicine are no longer valid or too expensive. It should be noted that, active ingredients derived from plants always play a key role in the progress of many anticancer agents in modern medicine. Cancer for several decades has become a dominion reserved for modern medicine, thus depriving itself of all that can be traditional treatments especially herbal therapy.
In spite of recent advance in modern medicine in connection with anticancer treatments, some types of cancer remain incurable and are generally resistant to currently available treatments. In addition to that, cytotoxic agents lack specificity with respect to healthy cells in the body and can cause severe side effects. So, the search of safe, increase the specificity and effective anticancer agents still continues.
In order to contribute to the identification of new anticancer drug from Siddha system of medicine, Results were tabulated in Table.1 and graphically represented in Fig. 1.  Some chemical constituents present in Sorgamara Ilai Chooranam explain these results. It contains more than 300 active substances. The components that have been identified and extracted from Simarouba glauca include flavonoids, glycosides phenolic compounds, saponins and fixed oil (12) .
Simarouba glauca also has 11 medicinally important quassinoids, the active principles in the tree (13) .