In Vitro Anti Cancer Activity of Leaf Extracts of Azima tetracantha Lam against Tryphan Blue Exclusion Method by using Dal Cell Lines

 

B. Edwin Jose¹, Dr. P. Muralidharan²

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

²Department of Pharmaceutical Chemistry, Bharath Institute of Higher Education and Research, Chennai

 

ABSTRACT:

Aim: Traditional medicine has a long history of serving people all over the world. In recent years, the use of traditional medicine information in cancer research received considerable interest. Ethanolic Extract of Azima tetracantha (lam) has been used in traditional and folklore medicine for the treatment of cancer. The aim of the present study was to evaluate the effect of Chloroform extract, Ethyl acetate extract, Ethanolic Extract and Aqueous extract of Azima tetracantha (lam) against trypan blue dye exclusion assay against Daltons ascites lymphoma cell lines. Materials and methods: The tumor cells were aspirated from the peritoneal cavity of tumor bearing mice were washed thrice normal saline and checked for viability using trypan blue dye exclusion method. The cell suspension (1x10⁶cells in 0.1ml) was added to tubes containing 10, 20, 50, 100 and 200 concentration of the test compounds and the volume was made up to 1ml using phosphate buffered saline (PBS). Control tube contained only cell suspension. These assay mixture were incubated for 3 hour at 37°C. Results: The number dead cells were found to increase with an increase in the concentration of drug used. The percentage toxicity was found to increase indicate that the given drug has potent cytotoxic activity in-vitro condition. For chloroform extract, the trypan exclusion method is evaluated as 2, 8, 14, 34 and 52% of dead cells. Ethyl acetate extract, the trypan exclusion method is evaluated as 0, 4, 11, 22 and 31% of dead cells. The ethanol extract, the trypan exclusion method is evaluated as 8, 14, 20, 40 and 60% of dead cells. For aqueous extract, the trypan exclusion method is evaluated as 4, 11, 22, 36 and 52% of dead cells. Conclusion: All these findings enable to conclude that the dose of Ethanolic Extract of Azima tetracantha (lam) possess a protective effect against DAL cell lines.

 

 

 

 

 

INTRODUCTION:

Cancer is a serious clinical problem that possesses significant social and economic challenges to the healthcare system. Despite improved imaging and molecular diagnostic techniques, cancer continues to affect millions of people globally¹. In many countries, cancer is the second leading cause of death after heart diseases². Lung, colorectal and stomach cancer are among the five most common cancers in the world for both men and women³. Over the past few decades, cancer has remained as the largest cause of mortality worldwide and the number of individuals living with cancer is steadily expanding. Hence, a major portion of the current pharmacological research is involved with the anticancer drug design customized to fit new molecular targets⁴. Due to the enormous propensity of plants, which synthesize a variety of structurally diverse bioactive compounds, the plant kingdom is a potential source of chemical constituents with antitumor and cytotoxic activities. Traditionally v7arious plants have long been used in the treatment of cancer^5,6,7,8. Plants have been a prime source of highly effective conventional drugs for the treatment of many forms of cancer.

 

Azima tetracantha (Salvadoraceae) is a wellknown 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 stimulant⁹. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea^10,11. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol^12,13. Azima tetracantha (lam) is reported to have antifungal¹⁴ antitumour¹⁵, antidiabetic¹⁶, antidiarrhoeal¹⁷ 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 desiccator and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis¹⁸

 

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80^oC) 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:

In-vitro cytotoxicity assay^19-22:

Trypan Blue:

Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye. Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.

 

Materials required:

DLA (Daltons lymphoma ascites) bearing mice. The standard drug 5-Flurouracil Injection. Phosphate buffered saline (PBS) contains Nacl- 4gm, Na₂HPO₄- 0.72gm, KH₂PO₄- 0.1gm, KCl- 0.1gm and Distilled water- 500ml. The dye used is Trypan blue and the cell is counted by using Haemocytometer.

 

Animals:

Adult male swiss albino mice weighing between 25-30 g were selected and acclimatized to the laboratory conditions for one week. They were fed with standard pellet diet obtained from Hindustan lever ltd. Bangalore, India, and water and ad libitum.

 

Induction of cancer:

The DAL cells were procured from Amala cancer Institute, Amala Nagar, Thrissur. The cells were introduced into the peritoneal cavity of the mice for tumour development. The cells were than aspirated aseptically from the tumour developed mice during log phase (on the 15th day after tumour transplantation) using 18gauge needle. The ascetic fluid was washed three times in PBS (Phosphate buffer Saline) or normal saline and the cell pellet was re suspended in PBS. The tumour cell count was done using tryphan blue dye exclusion method in a haemocytometer. The cell suspension was diluted to get 106 cells/ ml. Cancer was induced in the test mice by I.P inoculation of 106 cells per mouse.

 

Statistics:

% Cytotoxicity = (Number of dead cell/number of life cell + number of dead cell) 100

 

RESULTS:

The number dead cells were found to increase with an increase in the concentration of drug used. The percentage toxicity was found to increase indicate that the given drug has potent cytotoxic activity in-vitro condition. The tumor cells were aspirated from the peritoneal cavity of tumor bearing mice were washed thrice normal saline and checked for viability using trypan blue dye exclusion method. The cell suspension (1x10⁶cellsin 0.1ml) was added to tubes containing 10, 20, 50, 100 and 200 concentration of the test compounds and the volume was made up to 1ml using phosphate buffered saline (PBS). Control tube contained only cell suspension. These assay mixture were incubated for 3 hour at 37°C.  For chloroform extract, the trypan exclusion method is evaluated as 2, 8, 14, 34 and 52% of dead cells (Table 1). Ethyl acetate extract, the trypan exclusion method is evaluated as 0, 4, 11, 22 and 31% of dead cells (Table 2). The ethanol extract, the trypan exclusion method is evaluated as 8, 14, 20, 40 and 60% of dead cells (Table 3). For aqueous extract, the trypan exclusion method is evaluated as 4, 11, 22, 36 and 52% of dead cells (Table 4).

 

DISCUSSION:

Cancer is uncontrolled proliferation of tumor cells. The present study was carried out to investigate the antitumor potential of Azima tetracantha Lam leaf all extracts against DAL bearing rats. DAL is a very rapidly growing carcinoma with very aggressive behavior²³. It is able to grow in almost all strains of rats. The ascitic fluid is essential for tumor growth, since it constitutes a direct nutritional source for tumor cells²⁴.

 

Now a days researcher are focusing their research towards the development of an eco friendly anti caner drug from plant sources, which resulted in newer chemotherapeutic agents such as paclitaxel, vincristine, podophyllotoxin and camptothecin existing in clinical trials. In the present study a common plant Azima tetracantha. Lam was selected based on Literature review its chloroform, ethyl acetate, ethanolic extract and aqueous was screened for its anticancer potential against DAL cell lines employing In-vitro methods²⁵.

 

The trypan blue assay based on the assumption that the dead cells will take the dye and viable cells do not²⁶. From the study, it was observed that extracts showed moderate cytotoxic against both cancer and normal cell lines. The cytotoxicity of the extract was found to be in a dose dependent.

 

Table-1: Chloroform Extract

S. No	Volume of drug(ml)	Con.of drug (µg)	Volume of PBS(µl)	Volume of cell (1x106)	Volume of trypan blue	No. of viable cell	No. of dead cell	Average	% toxicity
1	Con. I	-	900	-	-	99	1	1	1
	Con. II	-	900	-	-	99	1	1	1
2	50	10	950	0.1ml	0.1ml	97	2	2	2
3	50	20	950	0.1ml	0.1ml	91	8	8	8
4	100	50	900	0.1ml	0.1ml	85	14	14	14
5	200	100	800	0.1ml	0.1ml	65	34	34	34
6	400	200	600	0.1ml	0,1ml	47	52	52	52

 

Table-2: Ethyl Acetate Extract

S. No	Volume of drug(ml)	Con.of drug (µg)	Volume of PBS(µl)	Volume of cell (1x106)	Volume of trypan blue	No. of viable cell	No. of dead cell	Average	% toxicity
1	Con. I	-	900	-	-	99	1	1	1
	Con. II	-	900	-	-	99	1	1	1
2	50	10	950	0.1ml	0.1ml	99	0	0	0
3	50	20	950	0.1ml	0.1ml	95	4	4	4
4	100	50	900	0.1ml	0.1ml	88	11	11	11
5	200	100	800	0.1ml	0.1ml	77	22	22	22
6	400	200	600	0.1ml	0,1ml	68	31	31	31

 

 

 

Table-3: Ethanol Extract

S. No	Volume of drug (ml)	Con.of drug (µg)	Volume of PBS (µl)	Volume of cell (1x106)	Volume of trypan blue	No. of viable cell	No. of dead cell	Average	% toxicity
1	Con. I	-	900	-	-	99	1	1	1
	Con. II	-	900	-	-	99	1	1	1
2	50	10	950	0.1ml	0.1ml	91	8	8	8
3	50	20	950	0.1ml	0.1ml	85	14	14	14
4	100	50	900	0.1ml	0.1ml	79	20	20	20
5	200	100	800	0.1ml	0.1ml	59	40	40	40
6	400	200	600	0.1ml	0,1ml	39	60	60	60

 

 

Table-4: Aqueous Extract

S. No	Volume of drug(ml)	Con.of drug (µg)	Volume of PBS(µl)	Volume of cell (1x106)	Volume of trypan blue	No. of viable cell	No. of dead cell	Average	% toxicity
1	Con. I	-	900	-	-	99	1	1	1
	Con. II	-	900	-	-	99	1	1	1
2	50	10	950	0.1ml	0.1ml	95	4	4	4
3	50	20	950	0.1ml	0.1ml	88	11	11	11
4	100	50	900	0.1ml	0.1ml	77	22	22	22
5	200	100	800	0.1ml	0.1ml	63	36	36	36
6	400	200	600	0.1ml	0,1ml	47	52	52	52

 

 

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Received on 19.12.2018       Modified on 29.12.2018

Accepted on 13.01.2019       ©A&V Publications All right reserved