Anticancer Activity of Fruit and Leaf Extracts of Averrhoa Bilimbi on MCF-7 Human Breast Cancer Cell Lines: A Preliminary Study

Research Article

Austin J Pharmacol Ther. 2016; 4(2).1082.

Anticancer Activity of Fruit and Leaf Extracts of Averrhoa Bilimbi on MCF-7 Human Breast Cancer Cell Lines: A Preliminary Study

Maya S. Nair*, Kamala Soren, Virendra Singh and Bibari Boro

Department of Biotechnology, Indian Institute of Technology, India

*Corresponding author: Maya S Nair, Department of Biotechnology, Indian Institute of Technology, India

Received: May 04, 2016; Accepted: July 22, 2016; Published: July 27, 2016


The fruit and leaf extracts of Averrhoa bilimbi were analyzed for the presence of phytochemicals, flavonoid content and in vitro cytotoxic potential. The methanolic extract of the fruit showed the presence of various phytoconstituents viz flavonoids, saponins, tannins, terpenoids etc. on the basis of the qualitative chemical tests while the ethanolic leaf extract showed the presence of phenols, alkaloids and flavonoids. The total flavonoid content of the fruit extract was found as 358 ± 0.7 μg rutin /g plant extract while for leaf extract, it was 47 ± 1.2μg/g only. The methanolic fruit extract exhibited significant cytotoxic potential against MCF-7 human breast cancer cell lines with an IC50 value of 154.9 μg/ ml whereas an IC50 value of 668 μg/ml observed for ethanolic leaf extract. The present findings indicate that the methanolic fruit extract could be considered as a source of novel anticancer compounds.

Keywords: Averrhoa bilimbi; Flavonoid; Cytotoxicity; Rutin; MTT assay; MCF-7 cell lines


Though cancer has been considered as a group of diseases affecting the more developed countries, its incidence in many different forms is now rapidly increasing worldwide. It represents the largest cause of mortality in the world. Although many drugs are there in active development and many are in clinical trials, there is an urgent need to develop much more effective and less toxic drugs. It has been reported that about 50% of all drugs in clinical use are derived from natural products, many of which have the potential to induce apoptosis in various human cell lines [1].

Plants and their secondary metabolites have always been an exemplary source of medicine from time immemorial. A rapid progress has been made in the phytochemical analysis of the plant products. Natural products are formulated to develop different drugs having anticancer properties. Several plant extracts and their bioactive components are well recognized for their ability to exert anticancer effects [2]. Some of the most clinically useful chemotherapeutic agents developed from natural products include vincristine, podophyllotoxin, paclitaxel and camptothecin [3,4]. Flavonoids are polyphenolic compounds having free radical scavenging activity, inhibition of hydrolytic and oxidative enzymes, anti-inflammatory activity like properties and have been isolated from plants.

Averrhoa bilimbi plant belonging to Oxalidaceae family has been reported to have many pharmacological effects. Averrhoa bilimbi fruit and leaf have antibacterial, antiscourbutic, astringent, postpartum protective efficiency and has been used for the treatment of fever, mumps, inflammation of rectum, diabetes, rheumatism, whooping cough, hypertension etc in traditional medicine. The fruit juice is also helpful in removing stains and rust from clothes [5,6]. The hypoglycemic and hypolipidemic activity of the semi purified leaf extract of Averrhoa bilimbi on high fat diet streptozotocin induced diabetic rat was studied by Tan and group [7] and hypoglycemic activity by performing oral glucose tolerance test by Pushparaj et al. [8]. Their study also showed the hypotriglyceridemic, anti-lipid peroxidative, anti-atherogenic properties of the fruit extract in STZ diabetic rats. The fruit and its water extract are shown to have antihypercholesterolemic activity in rats through another study [9].

The ability of Averrhoa bilimbi fruit to heal gingival wound by formation of fibroblast was reported by Hartini [10]. The cytotoxic potential of the hydromethanolic fruit extract of Averrhoa bilimbi was demonstrated by Chowdhury et al. [11] using brine shrimp lethal bioassay with an IC50 value of 5.011μg/ml. However, to our knowledge investigating effect of the anticancer activity of Averrhoa bilimbi extract is lacking so far. The present study was carried out to estimate the total flavonoid content of the fruit and leaf extract of Averrhoa bilimbi (A. Bilimbi) and to demonstrate their anticancer potential against human breast cancer cell line.

Materials and Methods

Plant material and extraction

The fruits and leaves of A.Bilimbi were obtained from Kerala, India. The botanical identification was done by the local ayurvedic physician Dr. Ashwin Raj (Sreekumar Pharmacy, Thrickodithanam, Kerala).

Leaf extract

Shade dried leaves were crushed into powder and were extracted successively with petroleum ether, chloroform, ethyl acetate, 80% ethanol and water in a soxhlet apparatus.

Fruit extract: The fruits were cut into pieces and dried in a hot air oven at 40°C. The dried fruits were ground into fine powder. A methanolic extract was prepared by mixing 10g of the fruit powder with 80% methanol at a solid: liquid ratio of 1:10. The mixture taken in a conical flask was agitated at 50°C for 2 hours, filtered to obtain a clear solution and was used for further investigation.

For biological assay, the crude extracts obtained were lyophilized and redissolved in dimethyl sulphoxide to a concentration of 100 mg/ ml stock solution and was mixed with culture media to obtain the desired concentration.


Rutin hydrate was obtained from Sigma (St.Louis, USA). Minimal Essential Medium eagle (MEM), Foetal Bovine Serum (FBS), Trypsin, Phosphate Buffer Saline (PBS), Ethylthiazolyldiphenyl-Tetrazolium Bromide (MTT), Acridine Orange, Ethidium Bromide, Dimethyl Sulphoxide, Trypan blue dye were from Himedia (Mumbai, India). Aluminium chloride was from SD Fine (RFCL, Mumbai), Methanol and ethanol from Qualigens (GSK, NewDelhi). All chemicals used were of analytical grade.

Phytochemical screening

Phytochemical components of fruit and leaf extracts of A. blilimbi were identified by qualitative analysis. Freshly prepared methanolic fruit extract was qualitatively tested for the presence of tannin, phlobatannins, saponin, flavonoids, terpenoids cardiac glycosides and anthraquinones using the method described elsewhere [12]. The ethanolic leaf extract was tested for the presence of phenols, alkaloids and flavonoids using the protocol described by Kumar et al [13].

Total flavonoid content

The total flavonoid content of the ethanolic leaf extract and methanolic fruit extract of A. bilimbi was determined according to the protocol by Mervart et al. [14] 100 μl of fruit extract in 900 μl of methanol was taken and mixed with 1 ml of aluminium chloride (2% in methanol). The mixture was vigorously shaken and absorbance at 367 nm was read using a UV spectrophotometer (Cary Bio100, Varian, USA) after 10 minutes of incubation. Aqueous ethanol (80%) was used as reference for the leaf extract and aqueous methanol (80%) was used for fruit extract. The absorption of standard rutin solution (1 was measured under the same conditions. The flavonoid concentrations in both leaf and fruit extracts were calculated from the rut in calibration curve and expressed as rutin equivalent (RE) in mg of rutin /g of plant extract [15].

Cell lines and cell cultures

MCF-7 human breast cell line was obtained from National Center for Cell Science (NCCS), Pune, India. Cells were cultured in Minimum Essential Medium Eagle (MEM) medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic mix (100U/ml of pencillin and 100U/ml streptomycin). The cells were grown in tissue culture flask at 37°C in the presence of humidified 5% CO2.

Trypan blue dye exclusion assay

The dye exclusion test is used to determine the number of viable cells present in a cell suspension. The cells were treated with appropriate concentrations of crude ethanolic leaves extracts and incubated for 24 hrs at 37° C. After treatment the cells were collected and suspended in 1 ml Phosphate Buffer Saline (PBS). 0.4% trypan blue dye solution was added to this suspension and incubated for 1 minute at room temperature [16]. After incubation the cells were counted using haemocytometer (Superior Marienfield, Germany) and the percentage viability was determined using the formula as follows:

Cytotoxicity assay (MTT assay)

Cytotoxic effect of the leaf and fruit extracts was determined by MTT assay, as previously described [17]. The cells were seeded on 96-well plate at a density of 5× 104 cells per ml per well and treated with different concentrations of crude leaf extract ranging from 600μg/ml upto 1000μg/ml. The cells were then incubated for about 36 hours. After incubation the medium was changed and MTT solution (5mg/ml) was added and incubated for 4-8 hours in dark. MTT was removed without disturbing the formazan crystals and 100μl of DMSO was added into the wells. The contents of the plate were mixed for 5 minutes to dissolve the crystals properly and then absorbance was measured on ELISA reader (Molecular Devices, Spectromax M2e, USA) at 570 nm [18]. Viability and inhibition of cell growth by the plant extract was calculated relative to the absorbance (OD) of control treated with DMSO for 100% cell viability [19].

Experiments were performed in triplicate. The IC50 values for fruit and leaf extracts were obtained using nonlinear regression in Graphpad prism as shown in Figure 2. Doxorubicin and DMSO were used as positive and negative controls respectively.

Acridine Orange/Ethidium Bromide staining

Acridine Orange (AO) and ethidium bromide (EtBr) are fluorescent dyes capable of binding to DNA. Based on the fluorescence emitted by these compounds upon excitation, differentiation of healthy, early and late apoptotic and necrotic cells can be done. The cells were seeded in a 24 well plate and treated with different concentrations of methanolic fruit extract and ethanolic leaves extract for 24 h. After incubation the media was removed and the cells were washed with PBS. 4 μl of Acridine orange/Ethidium bromide dye mixture (100 μ AO and 100 μ EB) was added to cells for staining and was observed under fluorescent microscope (Axiovert, 25 Carl Zeiss) for differential staining of live and dead cells [20].


Phytochemical screening

Freshly prepared crude extracts from the fruit and leaves of A.bilimbi were tested for the presence of various chemical constituents qualitatively [12]. The results are as given in (Table 1). The fruit extract showed the presence of tannins, phlobatannins, saponins, flavonoids, terpenoids, cardiac glycosides and anthocyannins. The leaf extract contains flavonoid, alkaloids and phenols.