CYTOTOXIC EFFECT OF CRUDE EXTRACTS OF ACACIA NILOTICA

CYTOTOXIC EFFECT OF CRUDE EXTRACTS OF ACACIA NILOTICA

Fahad Hussain ¹ and Mohammad Musarraf Hussain*²

Department of Pharmacy, Noakhali Science and Technology University ¹, Sonapur, Noakhali-3802, Bangladesh. Department of Pharmacy, Jagannath University ², Dhaka-1100, Bangladesh

The cytotoxic effect of crude extracts (n-hehane, ethyl acetate and chloroform soluble fractions) of root of Acacia nilotica was observed by brine shrimp lethality bioassay. The LC₅₀ values of n-hexane, ethyl acetate and chloroform soluble fraction found to be 1.956, 2.079 and 1.981 respectively indicating that the some of the compounds of the extract are biologically active. From this experiment, it was revelead that the test sample showed different response at different concentrations. The mortality rate of brine shrimp was found to be increased with the increased concentrations of sample, and a plot of log of concentration versus percent mortality on the graph produced an approximate linear correlation.

chloroform soluble fractions

INTRODUCTION:Acacia nilotica (Family: Fabaceae, Local name: babul) is a tree 5-20 m high with a dense spheric crown, stems and branches usually dark to black coloured, fissured bark, grey-pinkish slash, exuding a reddish low quality gum.

The tree has thin, straight, light, grey spines in axillary pairs, usually in 3 to 12 pairs, 5 to 7.5 cm long in young trees, mature trees commonly without thorns. The leaves are bipinnate, with 3-6 pairs of pinnulae and 10-30 pairs of leaflets each, tomentose, rachis with a gland at the bottom of the last pair of pinnulae.

Flowers in globulous heads 1.2-1.5 cm in diameter of a bright golden-yellow color set up either axillary or whorly on peduncles 2-3 cm long located at the end of the branches. Pods are strongly constricted, hairy, white-grey, thick and softly tomentose. Its seeds number approximately 8000/kg ^{1, 18}. Acacia nilotica is being used as a potencial medicinal agent of anticancer ¹⁴, antimutagenic ^{11, 14}, antibacterial ⁶, anti-inflammatory ⁶, kinase Inhibitor ², free radical scavenging ¹⁰, anti-helmintic ⁵, antioxidant ¹⁷, anti-quorum sensing ¹⁷, larvicidal activity ⁷,  cytotoxic effects ¹¹, dose related contractile activity ³, anti-spasmodic activity ⁹, antihypertensive ⁹, molluscidal ⁴.  A. nilotica  is a good sources of crude protein and mineral supplements to ruminants ¹⁶, a novel polyphenol, (+)-catechin-5-galloyl ester ¹²  and stimulate milk production and PRL release in the female rat ¹³.

METHODS AND MATERIALS:

Collection andidentification of the plant:  The plantsample (root) of Acacia nilotica was collected from Noakhali Science and Technology University campus in January, 2010.

Plant material preparation: The root of the plant was collected in fresh condition. It was sun-dried to make it suitable for grinding purpose. The coarse powder was then stored in air-tight container with marking for identification and kept in cool, dark and dry place for future use.

Extraction of plant material: The plant material in powdered form (800 g) was soaked in  methanol (2.30 L) in April 20, 2010 in a desecrator, kept on  occasional  stirring for 27days,  filtered through filter-cloth and the filtrate was kept to the open air for evaporation of solvent  to afford a crude extract  of biological investigation.

Preparation of Mother Solution: The methanolic extract of Acacia nilotica root  (5 g) was triturated with  ethanol (100 mL) containing distilled water (10 mL). The crude extract went to the solution completely. This is the mother solution, which was partitioned off successively by three solvents of different polarity.

Partitioning with n-hexane: The mother solution was taken in a separating funnel.  The solvent,  n-hexane (100 mL) was added to it, funnel was shaken and then kept undisturbed. The organic portion was collected and the process was repeated in three times.

The organic phases  (n-hexane fractions) were added together, solvent was evaporated in the air dry and the crud extract was found (0.60 gm).

Partitioning with Ethyl acetate: Distilled water (10 mL) was added with the mother solution left after washing with n-hexane. The mother solution was then taken into a separating funnel and extracted with ethyl acetate (3x100 mL). The ethyl acetate fractions were added together, solvent was evaporated in the dry air and extract was found (0.90 gm).

Partitioning with Chloroform:  Distilled water (20 mL)  was added with the mother solution that left after washing with n-hexane and ethyl acetate. The mother solution was then taken in a separating funnel and extracted with chloroform (3x100 mL). The chloroform soluble fractions were added together; solvent was evaporated in the air dry and extract was found (1.20 gm).

RESULT AND DISCUSSION: The brine shrimp test (BST) represents a rapid, inexpensive and simple bioassay for testing plant extract lethality which in most cases correlates reasonably well with cytotoxic and anti-tumor properties. Following the procedure of Meyer ¹⁵ the cytotoxic effect (lethality) of the crude extracts of Acacia nilotica, n-hexane, ethyl acetate and chloroform fractions were determined and the summary of the result are expressed in Table 1.

The LC₅₀ values of n-hexane, ethyl acetate and chloroform soluble fraction found to be 1.956, 2.079 and 1.981 respectively (Table 2 and Figure 2, 3, 4).  The LC₅₀ value for the positive control (vincristine sulphate) found to be -0.2001. From the results of the brine shrimp lethality bioassay, it can be well predicted that  the n-hexane, ethyl acetate and chloroform soluble fractions possess cytotoxic effect.

TABLE 1: RESULTS OF THE TEST SAMPLES OF ACACIA NILOTICA

TABLE 2: EFFECT OF n-HEXANE, ETHYL ACETATE AND CHLOROFORM SOLUBLE FRACTION ON SHRIMP NAUPLII

FIGURE 1: EFFECT OF VINCRISTINE SULPHATE ON BRINE SHRIMP NAUPLII

FIGURE 2: EFFECT OF n-HEXANE SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII

FIGURE 3: EFFECT OF ETHYL ACETATE SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII

FIGURE 4: EFFECT OF CHLOROFORM SOLUBLE FRACTION ON BRINE SHRIMP NAUPLII

CONCLUSION: The present study indicates that the crude extracts of Acacia nilotica has got intense cytotoxic effect and may have potential use in medicine. From the previous studies and our current investigation, it may be concluded  that further study can be carried out to investigate the individual bioactive principles.

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