Anti-proliferative Activity of Crotalaria pallida Aiton on MCF-7 Breast Cancer Cells

Crotalaria pallida Aiton ( C. pallida Aiton) is empirically used as dietary supplement to treat cancer by the people of North Sulawesi. However, its scientific pharmacology activity has not been explored yet. Therefore, this study was conducted to evaluate anti-proliferative activity of C. pallida Aiton on MCF-7 breast cancer cells. The extraction of leaves and seeds were performed using ethanol, ethyl acetate, n-hexane, and water. Phytochemical screening was then performed to identify secondary metabolites in this extract. Anti-proliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that ethyl acetate fraction of C. pallida Aiton has the lowest IC 50 (29,67). In conclusion, e thyl acetate fraction of C. pallida Aiton is potential to be developed as anti cancer agent.


Introduction
Breast cancer is the most common lifethreatening cancer in women and the leading cause of cancer death among women.[3] The toxicity of chemotherapeutic drugs sometimes creates a significant problem in the treatment of cancer using allopathy or established medicine.[6] Crotalaria pallida Aiton (C.pallida Aiton, Leguminoceae family, was empirically used by the people of North Sulawesi to treat cancer.However, its scientific pharmacology activity has not been explored yet.
Therefore, this study was conducted to evaluate anti-proliferative activity of Crotalaria pallida Aiton on MCF-7 breast cancer cells.Such information is essential to provide evidence on the anti-cancer activity of this medicinal plant from Indonesia.

Methods
The plant materials used in this study were 1195.45g and 2449.93 g of C. pallida Aiton leaves and seeds, respectively.The solvents used were ethyl acetate, n-hexane, distilled water, fetal bovine serum (FBS) (Gibco), phosphate buffer saline (PBS), water soluble tetrazolium (WST-8) (Dojindo) kit, and sodium dodecyl sulphate (SDS).The cell line used was MCF-7 breast cancer cells (Cell Culture Laboratory of Universitas Padjadjaran).
Cold extraction of leaves and seed of C. pallida Aiton was performed using ethanol.for 3 x 24 hours.These extracts were then fractionated using several solvents with different polarity.

Alkaloid Compound
The extract was moistened by adding a small amount of ammonia.Chloroform.was then added to the solution.The chloroform layer is then removed while being filtered.. Subsequently, hydrochloric acid was added.The mixture was shaken firmly to form several layers.The second layer was added by Mayer's reagent.The presence of white sediment indicated the presence of alkaloid group compounds.The third layer was then added with Dragendorff's reagent.The presence of yellow-orange solution indicated the presence of alkaloid compounds.

Flavonoid Compound
Magnesium powder and hydrochloric acid was added into the extract.The mixture was then heated over a water bath, then filtered.The obtained filtrate was then added with amyl alcohol.The presence of orange, red or yellow solution indicated the presence of flavonoids.

Polyphenol Compound
The extract was heated over a water bath and then filtered.The obtained filtrate was added with iron (III) chloride solution.The presence of the black-blue-green solution indicated the presence of polyphenol compounds.

Quinone Compound
The extract was heated over a water bath and then filtered.Potassium hydroxide solution.
(5%) was added into the solution.The presence of quinone compounds was shown by the formation of yellow to red solution.

Tanin Compound
The extract was heated over a water bath and then filtered.Gelatin solution (1%) was added into th extract.The presence of white sediment indicated the presence of tannin compound.

Monoterpenoid and Sesquiterpenoid
The extract was grinded and evaporated.Dried extract was then added with 10% vanillin solution in concentrated sulfuric acid solution.
The color changes indicated the presence of monoterpenoid and sesquiterpenoid.

Triterpenoid and Steroid
The extract was added with ether solution.Approximately 0.5 ml of anhydrous acetic acid, 0.5 ml of chloroform, and 1 ml of concentrated sulphuric acid were added into the solution.The formation of purplebrown ring on the second layer of the solution indicated the presence of steroid or triterpenoid.

Saponin Compound
The extract was heated over a water bath and then filtered.After the extract was cold, it was shaken rigorously for approximately 30 seconds.Hydrochloric acid.was then added into the foam to examine the presence of saponin compound.
Anti-proliferative test was performed using MTT assay.Various concentrations of extracts were put in a 96 well plate containing MCF-7 cells, and were then incubated for 24 hours.Ten μL of WST-8 solution was added to each well and incubated for 3 hours at 37 0 C. The reaction was discontinued with the addition of SDS reagent (100 μl).Cell proliferation rate was determined by reading the microtiter plate and measuring the absorbance at 450 nm.

Results and Discussion
Breast cancer is one of the most prevalent types of malignancy among woman in both developed and developing countries. 8Cancer The yields of concentrated extracts of C. pallida Aiton leaves and seeds were 57 g and 42 g, respectively.In this study, ethanol was used since it is a universal solvent which attract polar, semi polar and non polar secondary metabolites. 7The results of phytochemical screening can be found in Table 1.
The WST assay results showed that the extract and fraction of C. pallida Aiton had anti-proliferative activity.The lowest IC 50 was observed in ethyl acetate leaves fraction (29.67 μg/ml) and n-hexane seeds fraction (35.41 μg/ml) (Table 2).
The presence of several phytochemicals may contribute to its anti-cancer activities, such as alkaloid, flavonoid, and terpenoid.Previous studies showed that alkaloid might modulate microtubule activity.Microtubules contribute in the formation of mitotic spindle, making it becomes suitable target for anticancer drugs.10][11] Flavonoids might also induce antiproliferative activity of C. pallida extract.
Previous study showed that it might interferes with multiple mechanism, including signal transduction.Several flavonoid compounds could also activate apoptotic proteins and induce DNA damage in cancer cells. 12

Conclusion
Ethyl acetate fraction of C. pallida Aiton is potential to be developed as anti cancer agent.