Group 6 - Del Mundo
RESEARCH
DESIGN
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The researchers utilized the Completely Randomized Design (CRD) to test the efficacy of guyabano-mediated copper nanoparticles as an acaricide against brown dog ticks (Rhipicephalus sanguineus) through their mortality rates since they are homogeneous and received different treatments. This design ensured that the treatment was assigned at random, wherein all of the subjects had the same chance of receiving any of the treatments, and that they received each treatment independently. The following treatments that were used for the in vitro testing of the acaricide, which are percentages of the amount of CuNPs mixed with ethanol, are as follows: (A) 5% CuNP solution, (B) 10% CuNP solution, (C) 20% CuNP solution, (D) 30% CuNP solution, (E) 40% CuNP solution, (F) commercially available acaricidal product (Amitraz) for positive control, and (G) distilled water for negative control. This setup is derived from the methodology of Cadaoas et al. (2020) which was recommended by experts from the Department of Science and Technology Entomology Laboratory. There were three (3) replicates per concentration to increase accuracy and avoid biased results. Each replicate received 1.75 mL of the treatment and an equal number of ticks. These replicates were checked every 10 minutes to see if the ticks were still alive in a span of four (4) hours. If no movement was observed from the test subjects after poking them with a needle, then they were assumed dead. The independent variable of this study is the guyabano-mediated CuNPs, while the dependent variable is the mortality rate of each test subject.
MATERIALS AND METHODS
Six hundred twenty-five (625) guyabano leaves were personally collected by the researchers in Calauan, Laguna, and were verified via physical authentication at the Bureau of Plant Industry in Malate, Manila.
COLLECTION AND VERIFICATION OF GUYABANO
The extraction process utilized is based on the maceration
method of Shyma et al. (2014), the methodology of the ethanolic extract acaricide
study of Banumathi et al. (2015), and the extraction process of Amer and Awwad (2020) with minor
adjustments for improved practicality and time efficiency. After verification, the guyabano leaves were surface
cleaned to remove dirt and other contaminants. These leaves then underwent shade drying and were powdered after 10 days using a bullet blender. One liter (1L) of ethanol was added with 145 grams of the powdered leaves for
extraction. The beaker was then tightly sealed and stored at room temperature. After two (2) days,
the mixture was filtered using a strainer and squeezed by hand
to obtain the supernatant ethanolic extract.
EXTRACTION OF GUYABANO LEAF ETHANOLIC EXTRACT
The guyabano leaf ethanolic extract was subjected
to Phytochemical Screening at the Makati Science High School to
determine its bioactive constituents and verify the plant extract’s components.
Specifically, the researchers utilized the Liebermann-Burchard test for terpenoids, the
Mg+ Turning test for flavonoids, the Biuret test for amino acids, and the Ferric Chloride test for tannins.
PHYTOCHEMICAL ANALYSIS OF GUYABANO LEAF ETHANOLIC EXTRACT
In line with the biosynthesis procedure of
Amer and Awwad (2020), 200 mL of the guyabano leaf ethanolic
extract was combined with eight grams (8g) of copper sulfate pentahydrate (CuSO4∙5H2O)
in a magnetic stirrer for four (4) hours at room temperature (27°C). The blue color of CuSO4∙5H2O
changed in a few minutes to green, suggesting the occurrence of a chemical reaction. Cu2+ ions were reduced
into Cu metals which resulted in the synthesis of copper nanoparticles (CuNPs). After the color change, the mixture was then centrifuged for 10 minutes under the maximum rpm of the centrifuge at the Makati Science High School at room temperature to separate the excess solvent from the nanoparticles. The supernatant from the centrifugation
was removed and the CuNPs were stored in an amber vial to prevent degradation
resulting from their light sensitivity. It was then sent to UP Diliman
where it was subjected to a drying oven at
80-90°C overnight.
SYNTHESIS OF COPPER NANOPARTICLES
CHARACTERIZATION OF COPPER NANOPARTICLES
The biosynthesized CuNPs were characterized
using three analyses: Ultraviolet-Visible Spectrophotometry (UV-Vis),
Scanning Electron Microscopy (SEM), and Fourier Transform Infrared Spectrometry (FTIR). These
characterizations were done to ensure that nanoparticles were indeed synthesized during the process. UV-Vis
quantifies the optical characteristics of the nanoparticles, specifically, the light absorption which may give information about their size, shape, and concentration among others (Faraji et al., 2021). Similarly, SEM also illustrates its properties such as topography, morphology, composition, and crystallography, but it does so through high-quality imaging of the nanoparticle surface (Akthar, 2018). On the other hand, FTIR reveals the changes that have occurred in the total composition of the material by utilizing infrared light in detecting changes in their functional
groups (Eid, 2018). Samples from the guyabano-mediated CuNPs were sent to the De La Salle University (DLSU)
for UV-Vis and SEM, and the Department of Science and Technology Philippine Nuclear
Reaction Institute (DOST-PNRI) for FTIR. However, measurements
will vary due to the different requirements
per analysis.
Brown dog ticks were
harvested from infested cats and dogs in Metro Manila a week before the in vitro assay. This is the recommendation of the veterinarian who verified
the ticks. The removal of the aforementioned parasites will be done with care by following the TROCCAP:
Guidelines for the control of ectoparasites of dogs and cats in the tropics First Edition. Ticks will be collected from the heads, bodies, and feet of the dogs and will be placed in specimen cups to avoid leakage, wherein one cup was assigned per host. Then, the researchers consulted experts in the field of entomology, specifically the aforementioned veterinarian from a pet clinic in Taguig, to verify if the acquired parasites are R. sanguineus. Once presented with a valid species certification, the researchers proceeded and used the test subjects for experimentation. Amitraz was used on parasites assumed not to fit into the morphology of the desired species. Conversely, those successfully identified as R. sanguineus were placed back in the cups and subjected to the in vitro assay.
Identical in vitro setups were done for all the brown dog ticks setups. (Banumathi et al., 2015). The in vitro assay utilized in this study is derived from Cadaoas et al. (2020). The guyabano-mediated CuNPs was diluted with ethanol to get concentrations of 5%, 10%, 20%, 30%, and 40%. There were three (3) replicates per treatment, which included the different CuNP concentrations and the control groups. The researchers used 21 sterile Petri dishes, wherein each received 1.75 mL of the treatments. Subsequently, each Petri dish received two (2) ticks. The acaricidal activity of the guyabano-mediated CuNPs will be measured through the parasites' mortality rate from different treatment doses. The parasites will be recorded dead when no movement is observed after being poked with a needle. This will be determined every 10 minutes for four hours. Although the dependent variable in this study is the mortality rate, the survival rate, to be used in creating the survival curves, can be computed using the following equation:
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Survival Rate = (1 - number of dead ticks / number of ticks) * 100
COLLECTION AND VERIFICATION OF TICKS
IN VITRO TESTING OF ACARICIDE
PROCESS
DATA ANALYSIS
Kaplan-Meier survival curves will be used to analyze the number of dead ticks recorded from the experiment. Kaplan-Meier curves will compare the survivability of the subjects over time. In addition, the researchers will utilize the One-Way Analysis of Variance (ANOVA), the Tukey Honestly Significant Difference (HSD) post-hoc test, and a one sample t-test to analyze the data that will be collected for the hypotheses testing. One-way ANOVA is used to evaluate whether there are statistical differences between the means of the experimental and control groups (Lund Research Company, 2018). In line with the preceding study, the Tukey HSD post-hoc test is used to confirm if there is a significant difference between the computed values of the groups. Subsequently, a one sample t-test will be used to explore the differences within the various experimental groups. In particular, the mortality rate among different concentrations and the commercial acaricide (Amitraz). These tests will aid the researchers in deciding whether they will reject or fail to reject the null hypotheses in this study. If the p-value is determined to be less than or equal to 0.05, the null hypothesis will be rejected; otherwise, the null hypothesis will fail to be rejected. Moreover, the researchers will employ the Statistical Package for Social Sciences or the SPSS Statistics software for easier data interpretation.