INTRODUCTION:

The antimitotic activity was screened using Allium cepa root meristamatic cells which have been used extensively in screening of drug with antimitotic activity¹. Cells of this region undergo repeated divisions, known as meristamatic region, which is similar to cancer division in human. Hence, Allium cepa meristamatic cells can be used for preliminary screening of drug with anticancer activity^2,3. Yeast proteins and most of the proteins conserved between yeast and humans are involved in basic cellular processes such as small-molecule metabolism, protein synthesis, cell division, DNA synthesis and repair secretion, etc⁴. Hence, target identification in yeast has proven especially relevant for cancer, which at the simplest level is a disorder of proliferation control caused by accumulated mutations⁵.

Balanites aegyptiaca (L.) belongs to family Balanitaceae is a small tree reaching a height of around 10 m with compound leaves, greenish yellow flowers, and brown or pale brown fruits. It is widely used as good firewood and charcoal, fruit and seed has 30-40% of edible oil, young foliage and fruits are eaten by goats, sheep and camels. The other plant parts are used as soap substitutes because of high saponin contents, thorny branches used for fencing⁶. Plant bark shows antibacterial effects⁷, fruit mesocarp has larvicidal activity⁸ and root bark shows anthelmintic activity⁹.

The objective of the present study is to investigate the in vitro Anti-mitotic and Anti-proliferative activity of different extract of stem bark of Belanites aegyptiaca belonging to family Zygophyllaceae (Balanitaceae).

MATERIAL AND METHOD:

Plant materials and extraction

The plant materials (bark) were collected from the local area of Gondia district in Maharashtra, India during 2010 and confirmed by Dr. S. M. Bhiskute, Principal and Head, Department of Botany, Bhavbhuti Mahavidyalaya Amgaon, R.T.M. University, Nagpur. The plant parts after collection was shade dried and pulverized (40 mesh size) to get a coarse powder and subjected to the Soxhlet apparatus for defating with petroleum ether (60-80⁰) and followed by extraction with ethyl acetate, acetone and methanol.

In vitro antimitotic and antiproliferative Activity

The antimitotic and antiproliferative activity of different extract of bark of Belanites aegyptiaca were screened by using Allium cepa root meristamatic cells using methotrexate as a standard and yeast as a model system for screening of antiproliferative activity.

Antimitotic activity^10,11,12

The antimitotic activity was screened using Allium cepa root meristamatic cells which have been used extensively in screening of drug with antimitotic activity. Cells of this region undergo repeated divisions, known as meristamatic region, which is similar to cancer division in human. Hence Allium cepa meristamatic cells can be used for preliminary screening of drug with anticancer activity.

Procedure followed for carrying out antimitotic mentioned in following steps.

a) Roots development :

The experiment was planned as per the standard protocol. Onions were descaled and placed on glass cups which were filled with distilled water, kept in incubator at 24º for 72 hr then 2-3 cm roots were allow to germinate. These roots were used for further process.

b) Sample Preparation for Treatment:

Sample : Extracts of Belanites aegyptiaca (5 mg/ml and 10mg/ml),

Standard : Methotrexate (0.1mg/ml)

Control : Distilled water

c) Treatment : Developed roots were kept in different extracts for 3 hr at 18ºC .

d) Fixation :

Then root tips were cut and place in fixing solution caronys fixative for 24 hr at room temperature.

Composition of caronys fixative

Glacial acetic acid 25 ml and Ethanol 75 ml

e) Squash preparation:

a) Hydrolisation:-

These roots were hydrolised with 1N HCl, by keeping it with HCl in oven at 60º for 10 min.

b) Staining:-

Transferred root tips into 2% acetocarmine stain for 20 min.

c) Slide Preparation:-

Taken a stain root, cut it from the tip (which get dark stain) where meristematic cells were present. Then place cover slip on it and observed under microscope at 40 X objective and cells were counted.

Mitotic Indexwas determined using following formula

P + M + A + T

Mitotic index = ------------------------------ x 100

Total number of cells

Where, P = Prophase, M = Metaphase, A = Anaphase, T = Telophase

Antiproliferative activity^13,14,15

Yeast as a model system for anticancer study¹⁶

Most yeast proteins, and most of the proteins conserved between yeast and humans are involved in basic cellular processes such as small-molecule metabolism, protein synthesis, cell division, DNA synthesis and repair secretion, etc. Hence, target identification in yeast has proven especially relevant for cancer, which at the simplest level is a disorder of proliferation control caused by accumulated mutations. Many of the common mutations in human cancers (as well as genetic and physical interactions between the mutated genes/proteins) can be modelled in yeast, greatly simplifying and accelerating directed study The high degree of similarity and function in comparison with human has made this organisam as a model system for anticancer drug discovery.

Method of testing

1. Potatoes Dextrose Broth

2.5g broth was weighed and dissolved in 100 ml distilled water, sterilization of broth done by autoclaving at 121 lbs pressure for 15 min.

2. Preparation of inoculums

Yeast was inoculated in conical flask containing 100 ml sterilized broth and incubated at 37⁰C for 24 h. This referred as seeded broth.

3. Preparation of test inoculums

1 ml seeded broth was taken and diluted it with sterilized distilled water up to 10 ml, it contain 25.4 x 10⁴ cells (average).

4. Preparation of test compounds

A solution of test extracts of concentration 20 mg/ml, 40 mg/ml, 60 mg/ml mg/ml, 80 mg/ml and 100 mg/ml were prepared. Test compounds include all four extracts.

5. Procedure

2.5 ml of sterilized broth were taken in test tubes, to this 0.5 ml of inoculum were added and then 0.1 ml of different concentration of extracts were added. This incubated for 24 h at 37⁰C. Control for petroleum ether soluble extract and methanol soluble extracts were prepared as above by means of adding 0.1ml petroleum ether and 0.1ml methanol instead of respective extracts.

6. Cell Count

Cell suspension were mixed with 0.1% methylene blue and examined under 40 X objective under microscope.

7. Protocol

1. 50µl of cell suspension was mixed with 50µl methylene blue.

2. About 20 µl cell suspension was placed on haemocytometer slide and cover slip was placed on it.

3. Leaved for 1 to 2 min.

4. Microscope under 40X objective was placed.

5. WBC chamber of hemocytometer was focused, viable cells (cells do not take stained) were counted which appeared transparent. Dead cells get stained. Viable cells in 16 chambers were counted and average taken.

Cells/ml = average number of cells in 1 chamber X 10⁴

RESULTS AND DISCUSSION:

Preliminary Phytochemical investigation

Preliminary phytochemical screening of Belanites aegyptiaca showed the presence of sterol, flavonoids, alkaloids, carbohydrates and glycosides.

In vitro Antimitotic and Antiproliferative activity

Maximum inhibition of growth of root shown by methanolic extract followed by acetone, ethyl acetate extract than other extract.

TABLE 1: Allium cepa root length attained after incubation with water, methotrexate, Belanites aegyptiaca extracts

Sr.No.	Sample	Root length (cm)
Sr.No.	Sample	0 hr	24 h	48 h	72 h
1.	Water	2.20±0.18 (n=8)	2.71±0.22	2.71±0.22	3.97±0.14
2.	Methotrexate	1.78±0.15 (n=6)	1.85±0.13	1.91±0.07	1.93±0.05
3.	Petroleum ether extract (5 mg/ml)	1.91±0.13 (n=10)	2.35±0.19	2.93±0.11	3.61±0.13
4.	Ethyl acetate extract (5 mg/ml)	1.96±0.12 (n=8)	2.16±0.2	2.31±0.18	2.45±0.21
5.	Acetone extract (5 mg/ml)	2.05±0.22 ⁽ⁿ⁼⁷⁾	2.40±0.24	2.80±0.18	3.12±0.17
6.	Methanolic extract (5 mg/ml)	1.98±0.30 ⁽ⁿ⁼¹¹⁾	2.40±0.21	2.82±0.14	3.24±0.17

n = Number of roots

TABLE 2: Cells count in different extract at increasing concentration

Name of extracts	Concentration in (mg/ml) and number of viable cells per ml
Name of extracts	20	40	60	80	100
Petroleum ether extract	242000	239000	234000	231000	224000
Ethyl acetate extract	194000	146000	113000	82000	57000
Acetone extract	235000	203000	176000	148000	123000
Methanolic extract	259000	235000	219000	198000	176000

Viable cells in control - Viable cells in test

% Inhibition of growth = ----------------------------------------------------- x 100

Control

TABLE 3 : Percentage growth inhibition of yeast cells

Name of extracts	Concentration in (mg/ml) and % Growth inhibition of viable cells					IC ₅₀ (mg/ml)
Name of extracts	20	40	60	80	100	IC ₅₀ (mg/ml)
Petroleum ether extract	2.41	3.62	5.64	6.85	9.67	517.06
Ethyl acetate extract	31.20	48.22	59.92	70.92	79.78	41.76
Acetone extract	16.66	28.14	37.58	47.51	56.38	84.19
Methanolic extract	8.15	16.66	22.34	29.78	37.58	133.04

Fig.1: Comparison of % inhibition of yeast cell growth between different extracts

Ethyl acetate extract have maximum growth inhibition of yeast cells and growth inhibition increases as concentration of extract increases

CONCLUSION:

The ethyl acetate extract and methanolic extract may be used as a potential anticancer agent by doing certain cell line studies and in future isolation of alkaloids and other potential agent by different separation technique such as preparative TLC, column chromatography method can lead to new potent compounds, from the extracts of these plants.

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Received on 26.04.2011 Modified on 19.06.2011

Research J. Pharm. and Tech. 5(4): April 2012; Page 474-477