Determination of LC50 Values of Extracts of Euphorbia hirta Linn and Euphorbia neriifolia Linn Using Brine Shrimp Lethality Assay

Sandeep B. Patil¹* and Chandrakant S. Magdum²

¹Appasaheb Birnale college of Pharmacy, South Shivaji nagar, Sangli 416 416.

²Rajarambapu College of Pharmacy, Kasegaon, Sangli

*Corresponding Author E-mail: sandeep_pharmacology@rediffmail.com

ABSTRACT:

Brine shrimp lethality assay is an important tool for preliminary assessment of cytotoxicity of herbal drugs. This method is used for the determination of LC₅₀ values of herbal extracts. In this study, extract of Euphorbia hirta Linn and Euphorbia nerifolia Linn, which are reported in traditional literature for their anti-tumour activity, had been selected to test for brine shrimp lethality activity. This experimentation shows that the LC₅₀ of ethyl acetate and acetone extract of Euphorbia hirta Linn and methanolic extract Euphorbia neriifolia Linn are found to be 71.15, 92.15 and 49.55ug/ml respectively.

KEYWORDS: Brine shrimp lethality assay, Euphorbia hirta Linn, Euphorbia neriifolia Linn

INTRODUCTION:

A general bioassay that appears capable of detecting a broad spectrum of bioactivity present in crude extracts is the brine shrimp lethality bioassay (BSLT). The technique is easily mastered, costs little, and utilizes small amount of test material. The aim of this method is to provide a front-line screen that can be backed up by more specific and more expensive bioassays once the active compounds have been isolated. It appears that BSLT is predictive of cytotoxicity and pesticidal activity¹, since its introduction in 1982 ².

Euphorbia hirta L. is a medicinal, rhizomatous herb distributed in Southern Western Ghats of India and Northern East Coast of Tamil Nadu³, found especially on roadsides and wasteland. In India it is used to treat worm infestations in children and for dysentery, gonorrhoea, jaundice, pimples, digestive problems and tumours⁴. The aerial parts of plant are well investigated for chemical information like quercitrin and quercitol, Euphorbins A, B, C, D, E, Gallic acid, myricitrin., This plant was reported in various pharmacological activities like Anti-inflammatory activity, Antibacterial activity, Anticancer activity, Diuretic activity etc⁵. Euphorbia neriifolia Linn is a shrubby, erect, branched, fleshy, cactuslike plant, 2 to 4 meters high, the trunk and older branches being grayish and cylindric.

The leaves arise from the sides of wings towards the end of the branches, are fleshy, oblong-obovate, 5 to 15 centimeters long, or in young plants somewhat longer, painted or blunt at the tip. The expressed juice of the leaves is reported as very effectual in relieving the paroxyms of spasmodic asthma. The leaves are considered diuretic ⁶.

MATERIALS AND METHODS:

Plants material and extract preparation:-

For the present study the plant was procured from the reliable source of Sangli region, and the identity of the drug was established by morphological study at the Department of Botany, Wellingdon College, Sangli. The whole plant of E. hirta Linn were shade dried at 37⁰C to 40^oC and coarsely powdered through mesh 20. The leaves of E. nerifolia Linn were shade dried and extracted with methanol by using soxhlet apparatus. The powdered plant E. hirta Linn was defatted with petroleum ether 60-80^o C successively extracted with chloroform, ethyl acetate, acetone, ethanol by soxhlet apparatus. The extract was used in the concentration of 20, 40, 80, 160 µg/kg.

Hatching the brine shrimp:

Brine shrimp eggs (Artemia salina, Sanders TM Great Salt Lake, Brine Shrimp Company L.C., U.S.A.) were hatched in artificial sea water prepared from commercial sea salt (Aqua Marine, Thailand) 40 g/l and supplemented with 6 mg/l dried yeast. The two unequal compartments plastic chamber with several holes on the divider was used for hatching.

Table 1: Percentage death of Brine shrimp in 24 hours

Sample ↓/Conc. (μg/ml) →	20	40	80	160
Methanol extract of Euphorbia neriifolia Linn	47%	44%	50%	100%
Ethyl acetate extract of Euphorbia hirta Linn	17%	54%	37%	100%
Acetone extract of Euphorbia hirta Linn	04%	37%	17%	100%

Table2: Brine shrimp larvicidal activity of euphorbiaceae plant extract

Treatment	LC50 value in μg/ml
Methanol extract of Euphorbia neriifolia Linn	49.55
Ethyl acetate extract of Euphorbia hirta Linn	71.15
Acetone extract of Euphorbia hirta Linn	92.18

Methanolic Extract of E. neriifolia Linn.

Ethyl Acetate Extract of E. hirta Linn.

Acetone Extract of E. hirta Linn.

The eggs were sprinkled into the larger compartment which was darkened, while the smaller compartment was illuminated. After 48 hours incubation at room temperature (25-29°C), nauplii (larvae) were collected by pipette from the illuminated side whereas their shells were left in another side.

BIOASSAY:

10 shrimp were transferred to each test tube and artificial sea water was added to make 5 ml. The nauplii can be counted macroscopically in capillary against a lighted background. The test tubes were maintained under illumination. Survivors were counted, with the aid of the 3 X magnifying glass, after 24 hours and the percentage death at each dose and control were determined the 24 hours count were more useful.

LETHAL CONCENTRATION (LC₅₀) DETERMINATION:

The lethal concentrations of plant extract resulting in 50% mortality of the brine shrimp (LC₅₀) was determined from the 24 h counts and the dose-response data were transformed into a straight line by means of a trendline fit linear regression analysis (MS Excel version 7); the LC₅₀ was derived from the best-fit line obtained..

RESULTS AND DISCUSSION:

Brine shrimp lethality activity of extracts of the plants of Euphorbiaceae family E. hirta Linn and E. neriifolia Linn were shown in Tables 1 and 2, respectively. Crude extracts resulting in LC₅₀ values of less than 250 μg/ml were considered significantly active and had the potential for further investigation⁷. They were Methanol extract of Euphorbia neriifolia Linn, Ethyl acetate extract of Euphorbia hirta Linn and Acetone extract of Euphorbia hirta Linn. Among the two Euphorbiaceae plants, the most active extract was the Methanol extract of Euphorbia neriifolia Linn. These extracts have a potential to be a candidate for the investigation of cytotoxic compounds.

REFERENCES:

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Received on 22.02.2011 Accepted on 16.03.2011

Asian J. Res. Pharm. Sci. 1(2): April-June 2011; Page 42-43