3.

5 Biological Activity

3.5.1 Brine Shrimp (Artemia salina) Lethality Assay.

Brine shrimp lethality bioassay is carried out to investigation the cytotoxicity in

extract of Cassia alata. LC50 is Lethality Concentration in the chemical compound of Cassia
alata will be determined for this bioassay. This method is used because low cost, easily
mastered and requires small amount of test material. Lethality assay will be performed by
using the method establishes by Awal et al., (2004).

The eggs of brine shrimp, Artemia salina will be added in the tank that filled with
artificial sea water and covered it for hatching. The other tank is divided that contain the
same content in it which a light source is placed to attract the hatched shrimp (Awal et al.,
2004). After two days for hatching period of Artemia salina, this will produce a large
numbers of larvae known as (nauplii). Then, 3 mg of each extract is measured and dissolved
in 0.6 mL of solvent together. From the dilution of 0.5, 1, 2, 5, 10, 20 and 40 µL is placed in
seven different vials (Awal et al., 2012). After that, the solvent is evaporated under the
nitrogen and put it under the high vacuum about 30 minutes. The volatile solvent will
evaporate overnight. After two days, 5 ml of sea water and ten brine shrimp will be added to
each vials and left it for 24 hours of incubation. Then, the vials will be observed by using a
magnifying glass and the number of survivors in each vial is count and the mortality rate will
be determined. The resulting data were performed to probit analysis for determination of
lethal concentration (LC50) values for the extracts of Cassia alata.
3.5.2 Anti-Bacterial and Antifungal Assay

The evaluation of the antibacterial and antifungal activities on the extract of Cassia
alata is commonly used disc diffusion method. Four bacteria and one fungus are used as the
test organisms. Two Gram-positive bacteria such as Staphylococcus aureus and Bacillus
subtilis two Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa
and the fungi Candida albicans will be used in this study. According to disc diffusion
method, about 20 ml of sterile nutrient agar will be placed in a petri dish with 0.1 ml of 10 -2
dilution of bacterial culture of 24 hours. Filter paper discs will be placed on bacterial plate
and 30 µg of the extracts sample will be added. The plates have to be left for 30 minutes at
room temperature then will be incubated at 37°C for 24 hours for bacteria while for fungi is
72 hours. Diameter of zone of inhibition will be recorded and will be compared with a
standard antibiotic. The diameter will be measured in millimetres by using ruler.

3.5.3 Antioxidant Assay

Antioxidant activity will be determined used 2,2-diphenyl-1-picrylhydrazyl (DPPH)

solution. DPPH solution will be used as a stable radical. About 2 ml of the methanol extracts
will be mixed with 1 ml of 0.5 mM of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution and 2
ml of 0.1 M sodium acetate buffer (pH 5.5). Then, the mixture will be shaken and incubated
in the dark for 30 minutes for any reaction to occur and the absorbance will be measured at
517 nm. A solvent such as methanol can be used as negative control. The DPPH free radical
scavenging activity (%) is often expressed as percentage inhibition and will be calculated
using following equation:

% radical scavenging activity = (Ac – As) / Ac x 100

Ac= the absorbance of control solution (DPPH solution without test sample)

As= the absorbance of the sample solution (DPPH solution plus antioxidant)

The inhibitory concentration (IC50) value defined as the concentration of the test material
that leads to 50% reduction in the free radical concentration.
3.5.4 Anti-inflammatory Assay

5-Lipoxygenase Inhibition will be evaluated for anti-inflammatory activity. The rat

basophilic leukimia-1 (RBL-1) cells contains 5-Lipoxygenase enzyme. Referring to
Hiroyoshi et al. (2003) method, RBL-1 cells will be grown in RPMI-1640 medium
containing 10% heat-inactivated new born calf serum (NCS), penicillin 100 units/ml and
streptomycin 100 mg/ml. Cells will be cultured at room temperature in 5% CO 2 air. Then,
cells in the growth phase will be collected by centrifuging and will be suspended at a density
3x107 cells/ml in 50 mM phosphate buffer. The RBL-1 with 5-Lipoxygenase will be stored at
-80°C. After that, 50 mM of the phosphate buffer with varying concentration of the samples
in 1% DMSO, 2 mM CaCl2, 0.2 mg/ml of arachidonic acid (10 mg MeOH, 10 µl) and
ultrasonic pressed RBL-1 cells (1x107 cells/ml) in a final volume of 0.5 ml. Reaction of the
mixtures will be incubated at 37°C for 3 minutes and the reaction will be terminated by
addition of 0.5 ml methanol. The inhibitory effect of 5-Lipoxygenase activity (%) will be
calculated.
 2014 2015

Sept. Oct. Nov. Dec. Jan. Feb. Mac. Apr. May June

Proposal writing

and presentation
Data Collection

Bench work and

sample processing
Progress report

Data analysis

Data validation:

Statistical analysis
Progress report

Final report

writing and

presentation
Thesis writing