CHAPTER III

MATERIALS AND METHODOLOGY

This section provides comprehensively all the materials and procedures

needed to successfully conduct the study.

Acquisition and Acclimatization of Zebrafish

The matured female and male zebrafish were obtained from Balagtas,

Bulacan, Philippines at 1:2 ratio, respectively. They were confined in an aquarium

containing untreated and clean tap water that continuously aerated using oxygen.

They were fed using dry flakes twice a day. After one (1) h, excess feeds were

removed out from the aquarium to maintain the good quality of water.

Acquisition of Botanical Specimen

The leaves of S. grandiflora were acquired from Santol Balagtas, Bulacan,

Philippines on January 2018. It was then brought to the National Museum of the

Philippines for verification and authentication. After which, fresh leaves were

collected again and washed, air-dried and prepared for hot water extraction.

Plant Hot Water Extraction

The 30 g pulverized plant sample was mixed in Erlenmeyer flask containing

300 mL distilled water (dH2O). After which, it was covered using foil and tied using

rubber bonds. It was then placed in a water bath for two hours at 80-90ºC for

extraction proper (Eguchi et al., 1999). After two hours, extracts was filtered and

filtrates was cooled. The filtrates were transferred in new sterilized Erlenmeyer flak,

covered using foil and refrigerated prior the assay.

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Preparation of Treatment Concentrations

The S. grandiflora leaves water extract was used to prepare the different

treatment concentrations. The extract was diluted in embryo medium (Thomas, 2000)

to formulate the following treatments: 10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05% and the

control (embryo medium).

Spawning of Zebrafish and Harvesting of Embryo

Twenty (20) male and 10 female zebrafish were localized in an aquarium

containing 30 L of dechlorinated tap water. Plastic mesh was used to prevent the

released eggs from cannibalism. To induce spawning, the aquarium was cover by a

black trash bag for 12 hours. Then, after 12 hours, the trash bag was uncovered and let

the eggs be exposed in light condition for another 12 hours to allow fertilization.

Fertilization occurs 30 minutes after the light was turned on. Embryos were siphoned

out using a hose and were transferred in a beaker. The collected embryos were rinsed

thrice using distilled water and were transferred into the petri dish then check the

uniformity and normal conditions of embryos using compound microscope. Deformed

and unfertilized eggs were discarded while uniformed eggs were used in the assay

(Jose et al., 2016)

Evaluation of Plant Extract Toxicity and Teratogenicity

Two mL of the prepared treatments were dispensed into each well of 24-well

ELISA Plate. Each treatment is consisted of three replicates. Four embryos of

zebrafish at segmentation period were distributed into each well. Afterwards, the plate

was placed at 26 ± 1oC (Reneses et al., 2016). Mortality was determined after 12, 24,

36 and 48 hours of exposure to extracts. Heartbeat and hatchability were also recorded

after 36 and 48 hours of exposure to the different extracts, respectively (Romagosa et

al., 2016). Morphological endpoint evaluation of zebrafish was based on parameters

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established by Nagel (2002): lethal (coagulation, tail not detached, no somites and no

heart-beat), teratogenic (malformation of head and tail, light pigmentation, scoliosis,

growth retardation, stunted tail, and limited movement) and normal.

Data Analysis

Treatments were laid out in a Complete Randomized Design (CRD) with three

replicates per treatment. Using SPSS Program (Version 17.0), data were gathered and

analyzed using One-Way ANOVA (Analysis of Variance) followed by a post-test,

Duncan’s Multiple Range Test (DMRT). Difference between means was considered

valid at 5% level of significance (P<0.05) (Bustillos et al., 2016).

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 Acquisition and Acclimatization
of D. rerio

Collection of Plant Leaves

Specimen

Verification and Authentication

of Botanical Specimen

Preparation of plant sample for

Water Bathing of hot water extraction Filtration of Hot
Plant Specimen water extract

Preparation of Test Samples

Preparation of Water Extract

Embryo Medium Spawning of Zebrafish Dilution

Collection of D. rerio Embryo

Checking of Egg Uniformity

Determination of Toxic and

Teratogenic Effects of Plant Extract

Statistical Analyses

Fig. 3.1. Framework of the Study

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