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Brine Shrimp lethality bioassay for cytotoxic activity 5.2.1. Introduction: Brine shrimp lethality bioassay (Meyer et al.

,1982; Persoone, 1980) is a rapid general bioassay for the bioactive compound of the natural and synthetic origin. Bioactive compounds are almost always toxic at high dose. Pharmacology is simply toxicology at a lower dose or toxicology is simply pharmacology at a higher dose. Brine shrimp lethality bioassay is a bench top bioassay method for evaluating anticancer, anti-microbial and pharmacological activities of natural products and it is a recent development in the bioassay for the bioactive compounds. By this method, natural product extracts, fractions as well as the pure compounds can be tested for their biosphere-activity. Here, in vivo lethality in a simple zoological organism (Brine shrimp nauplii) is used as a convenient monitor for screening and in the discovery of new bioactive natural products. This bioassay is indicative cytotoxicity and a wide range of pharmacological activity of the compounds (Persoone,1980, McLaughlin,1992).

Figure 5.2.1: Artemia salina nauplli Brine shrimp lethality bioassay stands superior to other cytotoxicity testing procedures because it is a rapid method utilizing only 24 hours, inexpensive and requires no special equipment. Unlike

other methods, it does not require animal serum. Furthermore, it utilizes a large number of organisms for statistical validation and a relatively small amount of sample. Principle Brine Shrimp eggs are hatched in simulated seawater to get nauplii. Sample solutions are prepared by dissolving the test materials in pre-calculated amount of DMSO. Ten nauplii are taken in vials containing 5 ml of simulated seawater. The samples of different concentrations are added to the pre-marked vials with a micropipette. Survivors are counted after 24 hours. The median lethal concentration, LC 50 values of the test samples after 24 hours are obtained by a plot of percentage of dead Shrimps against the logarithm of the sample concentration using Microsoft Excel. Vincristine sulphate is usually used as the reference cytotoxic drug (Mazumder et al., 2009; Meyer et al 1982) Materials Artemia salina Leach (Brine Shrimp eggs), Sea salt (NaCl), PH meter Small tank with perforated dividing dam to hatch the Shrimp Lamp to attract Shrimps Pipettes (5, 25 ml) and Micropipette (5-50 l), (10-100 l).Pasteur pipette Glass vials Test samples of the plant under investigation Experimental procedure Preparation of seawater 38 g sea salt (pure NaCl) was weighed, dissolved in 1 litre of distilled water adjusted to pH 8.5 using 1N NaOH and was filtered off to get clear solution. Hatching of Brine Shrimps Artemia salina Leach (Brine Shrimp eggs) collected from pet shops was used as the test organism. Artificial seawater was taken in the small tank and Shrimp eggs were added to one side of the tank and then that side was covered. The tank was kept under constant aeration for 48 hrs to hatch the Shrimp and to be matured as nauplii. The hatched Shrimps were attracted

to the lamp through the perforated dam and with the help of a Pasteur pipette 10 living shrimps were added to each of the test tubes containing 5 ml of Brine solution.

Figure 5.2.2: Hatching of Artemia salina Preparation of test solutions Measured amount of each sample was dissolved in 60 l of DMSO. A series of solutions of lower concentrations were prepared by serial dilution with DMSO. From each of these test solutions 30 l were added to pre-marked glass vials/test tubes containing 5 ml of seawater and 10 Shrimp nauplii. So, the final concentration of samples in the vials/test tubes 400g/ml, 200g/ml, 100g/ml, 50g/ml,25g/ml,12.5g/ml, 6.25g/ml, 3.125g/ml, 1.5625g/ml, 0.78125g/ml. Preparation of Controls As for negative control, 30 l of DMSO was added to each of the pre-marked test tubes containing 5 ml of simulated seawater and 10 Shrimp nauplii. The test was considered invalid if the negative control showed a rapid mortality rate and therefore has to conduct again. The test tubes (containing nauplii) were then maintained at room temperature for 24 hrs under the light for observing the survival rate. Counting of nauplii and analysis of data After 24 hours, the test tubes were inspected using a magnifying glass and the number of survivors was counted. The percent (%) mortality was calculated for each dilution. The

concentration-mortality data were analyzed by using Microsoft Excel. The effectiveness or the concentration-mortality relationship of plant product is usually expressed as a median lethal concentration (LC 50) value. This represents the concentration of the chemical that produces death in half of the test subjects after a certain exposure period. However, LC90 values were also calculated in the similar way.

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