Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line.

BACKGROUND: The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines.
MATERIALS AND METHODS: The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography.
RESULTS: The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 μg/mL and 75.0 μg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 μg/mL and 42.0 μg/mL, respectively.
CONCLUSION: The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction.

Cancer is a genetic disease that arises from an accumulation of mutations in critical genes. This allows a cell to escape the normal growth control and proliferate until it becomes a clinically evident tumor. The six essential alterations in cell physiology have been identified, which collectively dictate malignant growth; (i) Self-sufficiency in growth signals; (ii) insensitivity to growth-inhibitory (antigrowth) signals; (iii) evasion of programmed cell death (apoptosis); (iv) limitless replicative potential; (v) sustained angiogenesis; and (vi) tissue invasion and metastasis. These properties give the cancer cells a growth advantage over the general cell population.[1] Bacopa monnieri (Family-Scrophulariaceae)it is commonly known as Brahmi. It has been used in Ayurvedic and Unani systems of medicine for the treatment of various ailments and diseases. All the parts of the plant have been used for their therapeutic beneficiary effects from ancient times. It is used as memory enhancer,[2] antidepressant, adaptogenic agent,[3] analgesic and anti-inflammatory,[4] anti-epileptic agent,[5] nervine tonic,[6] anti-Parkinson's agent,[7] protective effect,[8] cardiotonic agent,[9] anti-Alzheimer's drug,[10] anti-amnesic agent,[11] anti-tumor agent,[12] anti-oxidant effects,[13] for the presence of bacoside A and B, brahmin, different types of cucurbitacins,[14] and betulinic acid.[15] Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells by signal transducer and activator of transcription-3 (STAT-3) independent but reactive oxygen species dependent mechanisms.[16] The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of an extract of dichloromethane (DCM) fraction of B. monnieri on human breast cancer (MCF-7 and MDA-MB 231) cell lines.

Materials and Methods

Chemicals

Roswell Park Memorial Institute (RPMI-1640), fetal calf serum (FCS), and phosphate buffer saline were procured from Gibco, USA. The trypsin-ethylenediaminetetraacetic acid, trypan blue, penicillin-streptomycin solution, dimethylsulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, USA. All other solvents and chemical used were of analytical grade and procured from Merck India Ltd.

Plant material

The drug samples were purchased from local market of Delhi, India and the specimen (Ref. NISCAIR/RHMD/Consult/-2010-11/1563/161/27/10-10) authenticated by botanist Dr H. B. Singh, Scientist F and Head Raw Material Herbarium and Museum, NISCAIR, New Delhi.

Preparation of ethanolic extract of Bacopa monnieri and its fractionations

The extraction was done by taking 500 g of powdered drug and extracting it using 70% alcohol as a solvent in soxhlet extractor on a water bath for 5 h. It was filled and evaporated to dryness under reduced pressure. Further, the ethanolic extract of B. monnieri, whole plant thus obtained was suspended in double distilled water and sonicated for 15 min at 45°C. Further, it was subjected to partitioning with different solvent viz. hexane, DCM, and acetone. The extract, left after partitioning, was evaporated to dryness and residue was sonicated with acetone, methanol and water for 20 min separately, to prepare different fractions. All the fractions were evaporated to dryness under reduced pressure, and the dried extract and fractions were further used for analysis and bioactivity.

Cytotoxicity assay of Bacopa monnieri extract and its fractions

Sample preparation for in vitro activity

It was prepared by dissolving 500 mg of each extract/fraction in DMSO and volume was made upto 10 mL in a volumetric flask, separately. These solutions were passed through 0.45 µ membrane filtered and stored at 4°C until used. The 50 mg/mL solution of every extract/fraction as prepared above was diluted 50 times using RPMI-1640 media (1–50 mL) to get concentration of 1,000 µg/mL of every extract/fraction. It was passed through 0.22 µ membrane filter before using for in vitro activity on two different cell lines. Similarly, DMSO control was also prepared and used in both the cell line.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The cells culture was trypsinated, and the cell count was adjusted to 1.0 × 105 cells/100 µL in RPMI-1640 containing 10% FCS. To each well of the 96-well microtiter plate, (approximately 100 µL media contain 10,000 cells) was added and kept it in the CO2 incubator overnight for adhering of cells on plate. Next day, the media was replaced with fresh complete media (100 µL). The drug (100 µL) was added in triplicate well (for one concentration). Then, serial dilution of the drug was done, again 100 µL of complete media was added to get the total volume of 200 µL (100 µL + 100 µL). After completing the treatment, the 96-well microtiter plate was kept in a CO2 incubator. After 24 h study, 20 µL of MTT was added and left for 3–4 h in a CO2 incubator and after that the media was discarded from the wells carefully. The vehicle (100 µL DMSO) was added for dissolving the formazan crystals formed after addition of MTT, and left for 30 min in the CO2 incubator. The plate was read spectrophotometrically done at 570 nm at 24 h, 48 h, and 72 h.

Percentage cytotoxicity of these extracts was calculated by using the formula:

([Ac – As]/Ac) × 100

Where, Ac is the absorbance of the control and As is the absorbance of the sample.

Results and Discussion

Cytotoxicity assay

The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.00 µg/mL and 75.00 µg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and the found 57.00 µg/mL and 42.00 µg/mL, respectively 24 and 48 h summarized in Table 1, show dose response curve in Figure 1 and microphotograph of MCF-7 and MDA-MB 231 cells after treatment with DCM fractions of B. monnieri Figure 2.

Table 1

Effect of ethanolic extract/fractions of Bacopa monnieri on MCF-7 and MDA-MB 231 cell lines

Figure 1

Dose response curve of Bacopa monnieri in MCF 7 cell line; (a): At 24 h; (b): At 48 h; (c): 72 h and in MDA-MB 231 cell line; (d): At 24 h; (e): At 48 h; (f): 72 h

Figure 2

Microphotograph MCF-7 cell (a) MDA-MB 231 cell (b) after treatment with DCM fractions of Bacopa monnieri

Conclusion

The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction.

Financial support and sponsorship

CCRUM, Department of AYUSH (Ministry of Health and family welfare).

Conflicts of interest

There are no conflicts of interest.