Brine Shrimp Lethality Bioassay for Cytotoxic Activity
-
Upload
radowan-ahmad -
Category
Documents
-
view
126 -
download
1
description
Transcript of Brine Shrimp Lethality Bioassay for Cytotoxic Activity
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.
3.3.2.1 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, LC50 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 µl).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 µl of DMSO. A series of solutions of
lower concentrations were prepared by serial dilution with DMSO. From each of these test
solutions 30 µl 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 400μg/ml,
200μg/ml, 100μg/ml, 50μg/ml,25μg/ml,12.5μg/ml, 6.25μg/ml, 3.125μg/ml, 1.5625μg/ml,
0.78125μg/ml.
Preparation of Controls
As for negative control, 30 µl 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 (LC50) 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.