Research Report 5 |1

Division of City Schools

CALOOCAN NATIONAL SCIENCE AND TECHNOLOGY HIGH SCHOOL
Caloocan City

VISION: We dream of Filipinos who passionately love their country and whose values and competencies enable
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Brine Shrimp Lethality Assay for the Screening of Ginger (Zingiber officinale), Grapes (Vitis
vinifera), and Mangosteen (Garcinia mangostana)

Collado, Sofia
Del Mundo, Marx Chryz
Tablanza, Krys Roger
Tolledo, Syber Blaise
Valentin, Ian Angel

11- Joule

Mr. Leogiver Maosca

Research Adviser
RATIONALE
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Cancer is widely known to be one of the causes of mortality worldwide and it is

characterized to be an abnormal growth of cells. Ginger (Zingiber officinale), grapes (Vitis
vinifera), and (Garcinia mangostana) are plants that are not proven cancer treatment but are
candidates to cure cancer. Brine shrimp lethality assay will be used instead of other assays
because it will be used to preliminary screen plants extracts to know if there is a possibility that
an antitumor compound is present on a specific extract (McLaughlin, 1991). The protocol made
by Apu, et al., 2010 will be used for the methanolic extraction and preparation of plants. The
plant methanolic extracts will be assessed with the following concentrations of 10, 100, and 1000
g/mL using brine shrimp lethality assay. Each concentration has three replicates and 10 brine
shrimps per trial, which results to 3 trials per concentration and 30 shrimps per dilution. A
control group containing methanol will also be prepared. The percent mortality will be calculated
by dividing the number of dead brine shrimps over the total number per vial, and then
multiplying it to 100%. If control death occurs, the results will be corrected using Abbots
formula. (Olowa & Nueza, 2013). The plant methanolic extracts will be used to assess if they
can cure cancers based on the LC50 values.

Materials and Methods

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Gathering of Supplies

The materials will be gathered from Laboratory supplies and from the market such as
chemicals and glasswares such as vials and pipettes. The plants needed namely mangosteen
(Garcinia mangostana), ginger (Zingiber officinale), and grapes (Vitis vinifera) will be bought
from the market. The brine shrimp eggs will be bought from a pet shop supplier online and the
hatching place will be created using a shallow dish placed with a plastic divider with 2mm holes
will be placed and the larger compartment will be covered while the small one will be
illuminated. The light will attract the hatched nauplii and this will separate them to the unhatched
eggs.

Brine Shrimp Lethality Assay

The brine shrimp eggs will be hatched in the hatchery created earlier with 100 ml distilled
water with 3.8g of salt. The protocol made by Apu et al., 2010 will be used for the methanolic
extraction of Ginger (Zingiber officinale), grapes (Vitis vinifera), and mangosteen (Garcinia
mangostana) and will be assessed with concentrations of 10, 100, and 1000 g/mL using brine
shrimp lethality assay (Guevara, 2005). Each concentration has three replicates and 10 brine
shrimps per trial, which results to 3 trials per concentration and 30 shrimps per dilution. A
control group containing methanol will also be prepared. When placing the brine shrimps on
each vial, a 10 inch pipette will be used. The pipette will be held against a well-lighted
background for easier counting. Each vial will be receiving a suspension of 3mg of yeast so it
can serve as food for the nauplii.

Preparation of Plant Extracts

The plant samples will be gathered and the fruits of mangosteen and grapes, and ginger
will be dried. The dried plants will be powderized and after that, methanol will be added to the
powderized plants. The mixture will be placed under rotary evaporator for 24 hours so that the
methanol will evaporate and the extracts will be purified. The extracts will be stored for the brine
shrimp lethality assay (Apu et al., 2010).

Data Collection Procedures

The number of dead brine shrimps will be counted after 6 and 24 hours of treatment with
the plant extracts. But the 24-hour count will be used in recording the number of deaths after the
application of the different concentrations of the three plant methanol extracts. The brine shrimps
on each vials will be counted using 3x magnifying glass. The percent mortality will be calculated
by dividing the number of dead brine shrimps over the total number per vial, and then
multiplying it to 100%. If control death occurs, the results will be corrected using Abbots
formula. (Olowa & Nueza, 2013)

Determination of Median Lethal Concentration

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The LC50s of the plant extracts and the 95% confidence intervals will be calculated using
the 24 hour counts and using probit analysis described by Finney. Plant extracts having LC 50 of
greater than 1000 g/mL will be considered as inactive, while plant extracts which have LC 50 of
less than 1000 g/mL will be considered as active. (Olowa & Nueza, 2013)

References:

Guevara, B. Q. (Ed.). (2005). A Guide to Plant Screening: Phytochemical and Biological. Manila: UST
Publishing House.
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Hamidi, %. R., Jovanova, B., & Panovska, . (2014). Toxiclogical evaluation of the plant products using
Brine Shrimp (Artemia salina L.) model. Macedonian pharmaceutical bulletin, 60(1), 9-18.

Micharl Dockey & Stephen Tonkins. "Brine shrimp ecology" (PDF). British Ecological Society.

Olowa, L. F., & Nueza, O. M. (2013). Brine Shrimp Lethality Assay of the Ethanolic Extracts of Three
Selected Species of Medicinal Plants from Iligan City, Philippines. International Research Journal of
Biological Sciences , 2(11), 74-77.

Pimentel Montanher, A., Pizzolatti, M., & Costa Brighente, I. (2002). An Application of the Brine Shrimp
Bioassay for General Screening of Brazilian Medicinal Plants. Acta Farm. Bonaerense , 21(5), 175-178.

Price KS, Waggy GT, Conway RA, Brine shrimp bioassay and seawater BOD of petrochemicals. J Water
Pollut Control Fed. 1974 Jan; 46(1):63-77.

McLaughlin, J.L. (1991) Methods Plant Biochem. 6: 1-32.

Vincent, K. (n.d.). Probit Analysis. Retrieved from

http://userwww.sfsu.edu/efc/classes/biol710/probit/ProbitAnalysis.pdf

Apu, A., Muhit, M., Tareq, S., Pathan, A., Jamaluddin, A., & Ahmed, M. (2010). Antimicrobial
Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica
Linn. Journal of Young Pharmacists,2(1), 50-53. doi:10.4103/0975-1483.62213