STUDY PROTOCOL
1.0 INTRODUCTION:
Brine Shrimp lethality bioassay is a rapid and comprehensive bioassay for the bioactive compounds of natural and synthetic origin. By this method, natural product extracts, fractions as well as pure compounds can be tested for their bioactivity. The method utilizes in vivo lethality in a simple zoological organism (Brine nauplii) as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products. Brine toxicity is closely correlated with 9KB (human nasopharyngeal carcinoma) cytotoxicity (p=0.036 and kappa = 0.56). ED50 values for cytotoxicities are generally about one-tenth of the LC50 values found in the Brine Shrimp test.
2.0 METHODOLOGY:
· Sea water will be prepared by dissolving 38 gm of sea salt (without iodine) in one liter of distilled water and filtered off to get a clear solution.
· Seawater will be taken in the small tank and shrimp eggs will be added to one side of the tank and covered.
· Allowed it for two days to hatch the shrimp and to be matured as nauplii.
· As hatched shrimps are attracted to the light (phototaxis) and so nauplii free from eggshells will be collected from the illuminated part of the tank.
· Constant oxygen supply will be carried out through the hatching time. The nauplii will be taken from the fish tank by a pipette and diluted in fresh clear sea water to increase visibility and 10 nauplii will be taken care of by micropipette.
· Vincristine sulfate will be used as the positive control group and different concentrations of vincristine will be prepared at 10, 5, 1, 0.5, 0.25, 0.125, and 0.06 μg/ml in seawater and makeup it to 5ml as the final solution.
· 50µl of DMSO will be added to each of three pre-marked test tubes containing 4.95 ml of simulated seawater and 10 shrimp nauplii will be used as control groups.
· Similarly, the different concentrations of the test sample will be prepared in seawater and makeup it to 5ml as the final solution.
· The test tube will be inspected using a magnifying glass against a black background and the number of survived nauplii in each tube will be counted.
· The percent (%) of the lethality of the brine shrimp nauplii will be calculated for each concentration.
· The mortality will be corrected using Abott’s formula
Pt= [(Po-Pc)/ (100-Pc)] × 100
· Where, Po= Observed mortality Pc= Control mortality.
· The effectiveness or the concentration-mortality relationship of plant product is usually expressed as a median lethal concentration (LC50). This represents the concentration of the chemical that produces death in half of the test subjects after a certain exposure time and is determined by the linear regression method by plotting % mortality against the correspondent log of concentration.
3.0 ENDPOINT PARAMETER(S):
· Total No. of nauplii survived/mortality
· LC50
· %Lethality
4.0 REFERENCE(S):
4.1 Isaac John Umaru, Fasihuddin A. Badruddin, Zaini B Assim and Hauwa A. Umaru. Cytotoxicity (Brine shrimp Lethality Bioassay) of Barringtoniaracemosa Leaves, Stem-Bark and Root Extract. Journal of Biotechnology and Bioengineering Volume 2, Issue 2, 2018, PP 45-50.
4.2 M. Obayed Ullah, Mahmuda Haque, Kaniz Fatima Urmi, Abu Hasanat Md. Zulfiker, Elichea Synthi Anita, Momtaj Begum, Kaiser Hamid. Anti-bacterial activity and brine shrimp lethality bioassay of methanolic extracts of fourteen different edible vegetables from Bangladesh. Asian Pacific Journal of Tropical Biomedicine (2013)1-7.
4.3 Mahmudul Hasan, Md. Mamunul Hoque Parvez, Jackie Barua, Fayejun Nesa, Md. Mohaiminul Islam, Ajoy Roy, Md. Jahid Hassan, Md. Mortuza Kamal, Mohammad Arafat Uddin, Md. Mahfuzur Rahman, Md. Sajjad Ul Hoque. Evaluation of Cytotoxic Activity by Brine Shrimp Lethality Bioassay of Ethanol Extract and Its Different Fractions of Anogeissus acuminata (Roxb.) Leave. Int J Pharm 2017; 7(4): 55-59.
4.4 Sandeep Waghulde, Mohan K. Kale and VijayR. Patil. Brine Shrimp Lethality Assay of the Aqueous and Ethanolic Extracts of the Selected Species of Medicinal Plants. Proceedings 2019, 41, 47; doi:10.3390/ecsoc-23-06703.
4.5 José Luis Carballo, Zaira L Hernández-Inda, Pilar Pérez and María D García-Grávalos. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnology 2002, 2.
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