MATERIALS AND METHOD

Time and Place of the Study

Pipinong-gubat (M. pendula) will be collected in Barangay Nonong Sr, San Luis, Aurora on

December, 2019 at 8 o’clock of the morning. Drying of plant sample will be done at the Interactive

Laboratory of Aurora National Science High School, Barangay Buhangin, Baler, Aurora. Plant

extraction will be executed at Isabela State University on San Fabian, Echague, Isabela. The Brine

Shrimp (A. salina) sample will be provided by the Interactive Laboratory of Aurora National Science

High School on the conduction of the study. The Laboratory Procedures will be conducted at the

Interactive Laboratory of Senior High School in Aurora National Science High School, Barangay

Buhangin, Baler, Aurora on December, 2019 to March 2020.

Collection of Plant Samples

A B
Figure 1. Photo Pipinong-gubat (Melothria pendula) taken from Barangay Nonong Sr., San Luis,
Aurora; A (Photo of leaves and stem) and B (Photo of ripe fruit)
 Ripe fruit of Pipinong-gubat (M. Pendula) were collected at Barangay Nonong Sr., San Luis,

Aurora. This area lies at Latitude 15º 38.8’ 8.6” N and Longitude 121º 28’ 6.49” E. The plant was

selected because of their availability (commonly in sandy roadsides and almost in any fence) in the area

aside from its medicinal potential. The samples were washed thoroughly with tap water and then

following with distilled water to remove contaminants. The ripe fruit were blotted. Clean ripe fruit were

placed in sealed plastic bag with label to transfer in the Interactive Laboratory of Aurora National

Science High School, Barangay Buhangin, Baler, Aurora.

Preparation of Plant Extracts

Clean ripe Pipinong-gubat is air-dried at room temperature about 2 weeks until constant weight

were achieved in the Interactive Laboratory of Aurora National Science High School, Barangay

Buhangin, Baler, Aurora. Then the air-dried fruit were pounded using blender and kept in closed plastic

container.

The extraction and Phytochemical Analysis of the prepared pounded fruit were then been

performed at the Isabela State University on San Fabian, Echague, Isabela.

Authentication and Classification

Authentication and classification for the plant species used in the study was recorded

encompasses the family name and scientific name. For fortifying, the researchers sought for an expert in

confirming the identified and classified species.

Source of the Test Organism

Brine Shrimp was obtained from Interactive Laboratory of Aurora National Science High

School, Baranagay Buhangin, Baler, Aurora.

Hatching and Growing of Brine Shrimp Lethality Assay

Using measuring cylinder one (1) liters of water were measured and pour into the rectangular jar.

About 30 g of table salt were weighed by a balance and add it into the jar containing water and mixing

the water with the aid of spatula. The tip of an airline were placed from an air pump into the bottom of

the jar for maintaining the proper aeration. About 2.5 g of brine shrimp eggs were added at the top water

level of the jar and mix with the water. Then, a light (60-100 Watt bulb) was placed a few inches away

from the jar to be followed by switching on the light. After 20-24 hours, the nauplii was then hatch. The

eggs and nauplii were observed. After the next 12 hours nauplii were collected. (Sarah, Anny and

Misbahuddin, 2019).

Hatched nauplii were then separated from the empty egg. It has been done by turning off the air

and switching off the lamp. The empty egg then had floated while the brine shrimp was concentrated in

the water column. Transfer 270 nauplii to a Pertri dish using a dropper. (Sarah, Anny and Misbahuddin,

2019)

When the shrimp larvae has been ready, 0.5 mL of the seawater was added to each Petri dish and

30 brine shrimps was then introduced into each Petri dish. Thus, there have been a total of 30 shrimps

per dilution. Then the volume was adjusted with seawater up to 1 mL per vial. The Petri dish was then

left uncovered under the lamp. (Olowa and Nuñeza, 2013)

Bioassay of A. Salina

For toxicity tests, ten A. salina nauplii (larva) was transferred into each sample vial using

dropper. The nauplii have been counted macroscopically. A drop of dry yeast suspension (Red star) (3

mg in 5 ml artificial sea water) will then be added as food to each vial. It was added in every second day

(Sarah, Anny and Misbahuddin, 2019), if the observation is not yet conducting. All the vials were

maintained under illumination.

Clean Petri dish have then be taken and labeled. Plant extract of 10000μg/ml had also been got

by a dropper. Three set of Petri dish were labeled as 1-3: Positive control, an aqueous solution of

Hydroxyurea, as the medine for anti-cancer, Negative Control were Artificial seawater, and the

Experimental Control, were Ethanolic Fruit Extract of Pipinong-gubat). Then 1 mL of prepared solution

had been taken into the respect test tubes containing 30 nauplii with 1 mL of seawater. The number of

dead nauplii had been counted after 24 hours. Moreover, three (3) replication of the study had been

executed.

Statistical Design and Analysis

Experimental Research Design methodology, specifically, the Randomized Complete Block

Design (RCBD) used in this study to evaluate its Phytochemical components present in Pipinong-gubat

(M. pendula) and it’s toxicological properties.

The data in mortality were calculated using Abbott’s formula as follows: % deaths = [(Test-

control)/control x 100]. The data were analyzed with the usage of IBM Statistical Package for the Social

Science (SPSS) statistical analysis software using Two-Way Analysis of Variance (Tw0-Way ANOVA).
The statistical analysis (with 95% confidence limits) were calculated which the differences among the

results were considered to be statistically significant when P value is < 0.05.