Effect of Azima tetracantha Lam on Human Breast Cancer Cells MCF-7

 

B. Edwin Jose1, Dr. P. Muralidharan2

1Department of Pharmaceutical Chemistry, Sankaralingam Bhuvaneswari College of Pharmacy, Sivakasi.

2Department of Pharmacology, C. L. Baid Metha College of Pharmacy, Chennai.

*Corresponding Author E-mail: meeraharsa23@gmail.com

 

Abstract:

Current clinical trends involve the usage of plants as therapeutic agents in a wide range of applications. Present investigation is focused on the anticancer activity of the ethyl acetate extract and isolated compounds II, III of Azima tetracantha against MCF-7 cell line. The study was facilitated by collecting the plant sample and subjected to ethanol extraction using Soxhlet apparatus. The anticancer activity of the extracted sample against MCF-7 cell line was examined by MTT assay. The study confirms that the leaf extract of Azima tetracantha has pronounced anticancer potential against MCF-7 cell lines while compared to that of the stem extract. The plant investigated possesses remarkable anticancer activity and hence isolation of the compound contributing to the activity may lead to develop at a novel and natural phytomedicine for the disease.

 

KEY WORDS: Azima tetracantha, MCF-7 cell line, MTT assay, anti-cancer activity.

 

 

INTRODUCTION:

In recent years it has become clear that breast cancer does not represent a single disease but rather a number of molecularly-distinct tumors arising from the epithelial cells of the breast1. As for breast cancer, MCF-7 cells represent a very important candidate as they are used ubiquitously in research for estrogen receptor (ER)-positive breast cancer cell experiments and many sub-clones, which have been established and represent different classes of ER-positive tumors with varying nuclear receptor expression levels2.

 

MCF-7 is a commonly used breast cancer cell line that has been propagated for many years by multiple groups3. It proves to be a suitable model cell line for breast cancer investigations worldwide including those regarding anticancer drugs4. With time, MCF-7 has produced more data of practical knowledge for patient care than any other breast cancer cell line. MCF-7 is a poorly-aggressive and non invasive cell line5, normally being considered to have low metastatic potential.

 

Molecular Profile:

MCF-7 breast cancer cells are estrogen (E2)-sensitive cells and depend on E2 in order to proliferate8. They express high levels of ERα transcripts but low levels of ERβ9. MCF-7 cells increase expression of ER in the absence of estrogens. Short-term estrogen deprivation causes distinct responses of MCF-7 cells in comparison to long-term (over six months) estrogen deprivation. A reduced proliferation rate lasts for about a month after estrogen removal indicating that, during this period, MCF-7 has not found adaptive or compensatory growth mechanisms yet.

 

The growth of breast cancer cells is controlled not only by ER and PR but also by plasma membrane-associated growth factor receptors. Two particularly important members of this large family are the epidermal growth factor receptor (EGFR), which is activated by the epidermal growth factor (EGF), and the human epidermal growth factor receptor-2 (HER-2), both present in MCF-7 cells. Nevertheless, MCF-7 is considered to be moderate EGFR-expressing cell lines.

 

Azima tetracantha (Salvadoraceae) is a well known medicinal herb, termed Mulsangu in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant10. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea11,12. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol13,14. Azima tetracantha (lam) is reported to have antifungal15antitumour16, antidiabetic17, antidiarrhoeal18 and hepatoprotective activities.

 

Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.

 

Uses:

The plant is used in indigenous medicines for rheumatism, microbial infections, diahorrea, inflammatory conditions, reduce lipid and as hepato-protective.

 

MATERIALS AND METHODS:

Collection of plants:

The aerial part (leaves) of Azima tetracantha (lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccators and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis19

 

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.

 

Phytochemical Screening:

The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, tannins, sterols, and saponins in leaf extracts.

 

Experimental Design:

Evaluation of the cytotoxicity against human breast cancer cells MCF-7:

Preparation of plant extract:

The collected plant leaves were cleaned and shade dried for a week. 10 g of pulverized leaf material was mixed with 100 ml of methanol and kept in a rotary shaker at 100 rpm overnight and filtered with Whatman no. 1 paper and concentrated to dryness at 40C, lyophilized and stored at 4C until further use. Different concentrations of the ethyl acetate extract (0.4, 2, 10, 50 and 250 μg / ml) and isolated compound II and III were prepared in 0.5% DMSO for determining cytotoxicity.

 

Cell line and culture condition:

MCF-7 (breast cancer cell line) was used for the in vitro cytotoxicity studies. The cells were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100 U / ml), and streptomycin (100 g / ml) in a humidified atmosphere of 50 g/ml CO2 at 37C.

 

In- vitro cytotoxic activity assay (MTT assay):

The Cytotoxic activity of sample on MCF-7 (breast cancer cell line) was determined by the MTT assay20-23. Cells (1 106 / well) were plated in 1ml of medium/well in 24-well plates. After 48 hours incubation the cell reaches the confluence. Then cells were incubated in the presence of various concentrations (0.4 g/ ml, 2 g/ml, 10 g/ml, 50 g/ml and 250 g/ml) of the ethyl acetate extract and isolated compound II and III of Azima tetracantha LAM in 0.5 % DMSO for 48 h at 37C. After removal of the sample solution and washing with phosphate-buffered saline (pH 7.4), 200 l / well (5 mg / ml) of 0.5 % 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl--tetrazolium bromide cells (MTT) phosphate buffered saline solution was added. After 4 h incubation, 0.04 M HCl / isopropanol were added. The absorbance at 570 nm was measured with a UV- Spectrophotometer using wells without sample containing cells as blanks. MTT assay is a quantitative colorimetric assay for measuring cellular growth, cell survival and cell proliferation based on the ability of living cells. The assay was carried out using (3-(4, 5-dimethyl thiazol-2yl) - 2, 5-diphenyl tetrazolium bromide (MTT). MTT is cleaved by mitochondrial enzyme dehydrogenase of viable cells, yielding a measurable purple product formazan. This formazan production is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity24-27. Triplicate analysis of in-vitro cytotoxicity of ethyl acetate extract and isolated compound II and III of Azima tetracantha LAM was carried out with various concentrations.

 

The effects of the samples on the proliferation of cell lines were expressed as the % cell viability, using the following formula:

% cell viability= A570 treated cells/A 570 control cells x100

 

Statistical analysis:

The data are expressed as mean standard deviation (SD) for at least three independent determinations in triplicate for each experimental point. The percentages of cell growth were used to obtain the full dose response curves and to determine the IC50 values (Lethal concentration inhibiting 50% of the cell growth compared with control). The data was analysed using Probit Analysis of SPSS package28.

 

RESULTS AND DISCUSSION:

In the present study, in-vitro cytotoxicity effects of ethyl acetate extract and isolated compound II and III of Azima tetracantha LAM was carried out with various concentrations for cancer cell line MCF-7 (breast cancer cell line). The leaf of the plant was collected and shade dried powdered and extracted with ethyl acetate solvent and also the compounds II and III were isolated by column chromatography. Five different concentrations (0.4, 2, 10, 50 and 250 g/ml) of ethyl acetate extract and isolated compounds II and III were used to study the cytotoxicity potential of the plant. The ctytotoxicity potential of various concentrations of ethyl acetate extract and isolated compounds II and III with IC50 values of Azima tetracantha LAM were displayed in Table 1. The results revealed that the cytotoxicity rate was increased when the concentrations of leaf extract increases. MTT assay measured the cell viability based on the reduction of yellow tetrazolium MTT to a purple formazan dye mitochondrial dehydrogenase enzyme. So, the amount of formazan produced reflected the number of metabolically active viable cells. The IC50 values observed in Cell line of ethyl acetate extract and isolated compounds II and III were 49.98g/ml and 51.34g/ml and 52.28 g/ml.

 

CONCLUSION:

Azima tetracantha LAM is one of the potent medicinal plants used in Traditional medicinal systems of India. All the parts of the plants have the potent medicinal property and used to cure many diseases. The current research also added one more potent activity of the leaf of the plant. The further research is necessary to design the drugs for cancer diseases in pharmaceutical industries.

 

Table 1: Cytotoxic activity of A.tetracantha

Sample Description

Concentrations g/mL

0.4

2

10

50

250

IC50

Ethyl acetate extract

17.9 2.67

29.6 1.1

43.87 1.03

78.5 2.03

87.0 1.98

49.98

Isolated compound II

18.2 2.46

31.1 1.8

45.56 1.11

79.7 2.08

89.2 1.95

51.34

Isolated compound II

18.6 2.72

31.8 1.2

45.96 1.05

80.0 2.12

89.8 2.06

52.28

 

 

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Received on 18.12.2018 Modified on 18.02.2019

Accepted on 04.04.2019 A&V Publications All right reserved

Research J. Science and Tech. 2019; 11(2):109-112.

DOI: 10.5958/2349-2988.2019.00017.2