Phytochemical test:

Preliminary phytochemical analysis of the extract was performed by simple chemical tests.^7,8

Cancer Cell Cultures:

Hep G2, (liver cancer cell line) MDA MB 4355 (Human breast cancer) cell lines were purchased from NCCS, Pune, India. All cell lines were grown and maintained in suitable (RPMI-15-media and were grown and subculture in medium supplemented with 10% fetal bovine serum, 1% L-Glutamine.1% penicillin- streptomycin-amphotericine-B antibiotic solution. All cells were trypsinated using try sin-EDTA solution and seeded in96 well plates.

Ant proliferative assay of extract on MDA MB 4355 (Human breast cancer) ^9,10:-

The MDA MB 4355 (Human breast cancer) was maintained in L-15-media supplemented with 10 % fetal bovine serum. The cells were plated at a density of 1 × 10⁵ cells per well in a 96-well plate, and cultured for 24 h at 37 °C. The cells were subsequently exposed to 10, 50 µg/ml. The plates were incubated for 24 h, Test compounds were prepared in PBS and Controls were performed with medium alone. Ant proliferative activity was assessed by performing the Sulphorhodamine B (SRB) assay. Cells were fixed by adding 25 µL of ice-cold 50% trichloroacetic acid on top of the growth medium and the plates were incubated at 4 °C for 1 h, after which plates were washed to remove traces of medium, drug and serum. SRB stain (50 µL; 0.4% in 1% acetic acid) (Sigma) was added to each well and left in contact with the cells for 30 min after which they were washed with 1% acetic acid, rinsing 4 times until only dye adhering to the cells was left. The plates were then dried and 100 µL of 10 mM Tris buffer (Sigma) added to each well to solubilise the dye. The plates were shaken gently for 5 min and absorbance was read at 550 nm using a micro plate reader. Lastly percent cytotoxicity of the compounds was calculated by using following formula.

Percent Cytotoxicity =

Reading of control - Reading of treated cells X100

Reading of control

Antiproliferative assay of extract on Hep G2 (Liver cancer cell line) ^11,12

The Hep G2 cell line was maintained in MEM medium supplemented with 10 % fetal bovine serum. The cells were plated at a density of 1 × 10⁵ cells per well in a 96-well plate, and cultured for 24 h at 37°C. The cells were subsequently exposed to 10, 50 µg/ml. The plates were incubated for 48 h, and cell proliferation was measured by adding 10 µL of MTT (thiazolyl blue tetrazolium bromide) dye (5 mg ml^-1 in phosphate-buffered saline) per well. The plates were incubated for a further 4 h at 37°C in a humidified chamber containing 5% CO₂. Formazan crystals formed due to reduction of dye by viable cells in each well were dissolved in 200 µl DMSO, and absorbance was read at 490 nm. The results were compared with the standard drug inhibitors 5- flurouracil. (20µg/ml) percent inhibition was calculated as above formula.

RESULTS:

The preliminary Phytochemical analysis revealed the presence of alkaloids, flavonoids saponins and steroids

Table . 2: Antiproliferative activity of Successive solvent Extracts of Barleria gibsoni Dalz. against MDA MB 4355 (Human breast cancer) by SRB assay

Successive solvent Extracts of leaves	% Inhibition (10 µg/ml)	% Inhibition (50 µg/ml)
Pet ether	61.66	61.66
Chloroform	60.00	63.33
Acetone	< 50	31.66
Ethyl acetate	45.00	40.00
Ethanol	23.33	26.66
5- flurouracil (50 µg/ml)	55.00	64.33

As shown in Table. 2 the successive solvent extract displayed inhibition activities against breast human cancer cell lines. Notably, petroleum ether and chloroform extracts exhibited significant inhibitory activities against MDA MB 4355 cell lines compared to the positive control 5- fluorouracil. Remaining extracts showed moderate inhibitory activities against MDA MB 4355 cell lines.

Table . 3: Antiproliferative activity of Successive solvent Extracts of Barleria gibsoni Dalz. against Hep G2 cell line (Human liver cancer) by MTT assay

Successive solvent Extracts of leaves	% Inhibition (10 µg/ml)	% Inhibition (50 µg/ml)
Petroleum ether	21.07	21.53
Chloroform	>100	>100
Acetone	49.70	>100
Ethyl acetate	30.72	23.96
Ethanol	25.14	60.86
5- flurouracil (50 µg/ml)	64.33	80.43

As shown in Table.3, the successive solvent extract displayed inhibition activities against liver human cancer cell lines. Notably, ethanol extract of leaves exhibited moderate inhibitory activities against Hep G2 cell lines compared to the positive control 5- fluorouracil.

DISCUSSION:

From the present study it can be said that ethanol extract of leaves of Barleria gibsoni has antiproliferative activity against Hep G2 cell line by MTT assay method and pet ether and chloroform extract of leaves of Barleria gibsoni has antiproliferative activity against MDA MB 4355 breast cancer cell line by SRB assay method due to presence of phytoconstituents present in the plant. It is need to explore the isolation, characterization and development of drug which could be help to hopeful treatment in cancer disease.

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Received on 02.12.2015 Accepted on 20.12.2015

Asian J. Pharm. Res. 5(4): October- December, 2015; Page 183-185

DOI: 10.5958/2231-5691.2015.00028.3

Secondary Metabolites	Petroleum ether	Chloroform	Acetone	Ethyl acetate	Ethanol
Alkaloids	+	-	+	-	+
Flavonoids	-	+	+	+	+
Steroids	-	-	_	-	+
Saponins	-	-	+	+	+