Group 6 - Del Mundo
The presence of terpenoids, alkaloids, amino acids, and tannins in the guyabano leaf ethanolic extract was tested via a qualitative phytochemical analysis as these metabolites are responsible for the reduction of copper ions into CuNPs. As indicated in Table 1, all of these compounds can be found in the extract, specifically in the case of tannins which are highly present in the extract. Therefore, the guyabano leaf ethanolic extract, with its metabolites, is capable of triggering the synthesis of nanoparticles. The results of the qualitative phytochemical analysis are aligned with the studies of Agu & Okolie (2017), Nguyen et al. (2020), and Mutakin et al. (2022). The highly present label of tannins in the extract can be proven by the researches of Gogola and Tango (2019) and Rafael et al. (2017) as they detected high tannin contents reaching 811.1 Gallic acid equivalence (GAE) g-1 and 25.33±0.00 mg GAE g-1, respectively.
PHYTOCHEMICAL
ANALYSIS
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ACARICIDAL ACTIVITY DATA ANALYSIS
Among the five concentrations of CuNPs, the 30% solution showed the most rapidly successful extermination of the ticks after 100 minutes of exposure to the CuNPs with a 100% mortality rate. Even so, the mortality rate of all the experimental groups and the positive control group was 100% after four hours. This may be attributed to copper’s mechanism of cellular damage through contact killing which has been a common phenomenon in the field since ancient times. This is when microorganisms such as yeast, bacteria, and viruses are rapidly killed after being in contact with a metallic copper surface. (Grass, 2011). In contrast, the mortality rate of the negative control group (water) was 0 and signifies that there was no intervention made between the treatment and the samples.
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Figure 4. Mortality rates of ticks per 10 minutes.
As seen in the figure, the positive control group was the fastest to eliminate the ticks, followed by the 30% concentration. The remaining concentrations exhibited a slower, however, still successful extermination after the third hour mark. The results do not align with the findings of Cadaoas et al. (2020) where the researchers based their concentrations of CuNPs. The 10% and 20% concentrations were the most efficient acaricides in their study. Zinc oxide nanoparticles (ZnONP) naturally have less antimicrobial effect on some bacteria such as E.coli (Asamoah, et al., 2020). ZnONPs also exhibit an approximately 80% mortality rate against close relative species of Rhipicephalus sanguineus which is the Rhipicephalus microplus (Kirti et al., l2011 as cited by Banumathi et al., 2015).
With this, the researchers suspect that, along with the difference between the binder and the metal used, the effectivity of the actual nanoparticles also played a part to what caused the mismatch between the results as sunflower seeds were used to synthesize zinc nanoparticles in the aforementioned literature. Since ZnONPs possessed less acaricidal property than CuNPs, the concentration that exhibited the fastest extermination was the one who had a higher sample concentration. Even so, this still fulfills the research hypothesis, which is to synthesize copper nanoparticles through guyabano leaf ethanolic extract that can result in high mortality rates of brown dog ticks in various concentrations over a short period of time when utilized as an acaricide.
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Table 3. Descriptive statistics of the mortality rate of brown dog ticks.
Table 3 shows the population of ticks per replicate, mean or the average number of exterminated ticks per 10 minutes, and standard deviation between the replicates. Lower SD values denote more precise and more consistent results and thus can be concluded from the concentrations of CuNPs. The SD of the negative control group is 0 since there was no change in the number of dead ticks from the start of the experimentation until the end.
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Table 4. One-way analysis of variance (ANOVA) for the results of the experimental group.
Table 4 shows the calculated p-value, which is 0.108, from the mortality rates of dog ticks in different experimental groups. This indicates the lack of evidence to reject the null hypothesis and thus suggests that there is no significant difference among the different concentrations of CuNPs. Moreover, this claim is further supported and strengthened by the F critical value of 3.478, which is less than the F value which is 2.517. These results show that the difference in concentrations did not have significant individual and distinct impacts on the mortality rates of the experimental units.
There is no significant difference among the concentrations of CuNPs.
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Both the negative control group and the positive control group had a significant difference from the experimental groups.
Table 5. T-test between the positive control group and the experimental group.
Table 6. T-test between the negative control group and the experimental group.
On the other hand, Tables 5 and 6 show the difference between the results of all of the experimental groups and the control groups. Both the negative and the positive control groups had a significant difference from the experimental groups. From this, it can be deduced that, although effective, the CuNPs exterminated the dog ticks in a much slower time interval than the positive control group. This suggests that all of the concentrations were capable of exterminating the brown dog ticks, but they were not as effective in doing so as the positive control group since the latter was still able to kill the ticks before any of the experimental groups. From this data, it can then be presumed that guyabano-mediated CuNPs are effective in exterminating Rhipicephalus sanguineus and can be further explored as an alternative organic acaricide.
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TUKEY HSD POST-HOC
​ The Tukey HSD Post-hoc test was not conducted as the experimental groups are not significantly different from each other. The researchers suspect that the main factor for this is the hectic schedule of the school laboratory that was utilized for most of the experimentation process. The schedule was always subject to changes, possibly altering the efficacy of the guyabano extract and CuNPs as deterioration may occur over time. As stated in the study by Patra and Baek (2014), storage time is among the factors that influence both physical and chemical properties of nanoparticles. The study by Baer (2011) supports this by disclosing that the NPs may increase or decrease in size, or aggregate together as a function of time, thereby affecting its potential for application efficiency. Further, since a smaller sample size could translate to exaggerated or understated conclusions about the population, the small sample size could also be a reason for the insignificant difference between the concentrations. There were only two ticks per Petri dish due to a fast-paced process of experimentation resulting in a limited time to collect ticks. Moreover, almost half of the ticks collected died due to unknown reasons.
ULTRAVIOLET-VISIBLE
(UV-VIS) SPECTROPHOTOMETRY
ANALYSIS
The light absorbed by the guyabano-synthesized CuNPs was determined by spectrophotometry using a UV-2600 instrument. Readings were measured in a range of 200 – 800 nm. As seen in Figure 5, numerous wavelengths have obtained the absorbance peak of 10 A, thus interpretations for a single wavelength cannot be made. Rather, it can be concluded that the sample analyzed under UV-Vis was overly concentrated, in accordance with Beer-Lambert's Law which states that the intensity of light absorption is directly proportional to concentration (Picollo et al., 2019). This excessiveness in the amount of substance may be attributed to the sample’s nature of being powder in form, and not having been diluted before the analysis.
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To still distinguish a certain wavelength for the biosynthesized CuNPs, color analysis of the sample was alternatively considered. In HEX code, it is identified as #364638 which then equates to a wavelength of 380 nm which is in close agreement with the findings of Ravindran et al. (2017) revealing that the target indicator is at 350 nm. This wavelength measurement confirms the presence of biosynthesized CuNPs in the sample. Moreover, the study of Kayalvizhi et al. (2019) wherein the independent variables are also green synthesized Copper nanoparticles, 327 nm was one of the exhibited peaks of the CuNPs, which are relatively close to this study.
FOURIER TRANSFORM
INFRARED (FTIR)
SPECTROMETRY
ANALYSIS
FTIR was utilized to spot the functional groups that could have triggered the synthesis of guyabano-mediated CuNPs due to their ability to reduce copper ions. Figure 6 shows the IR spectrum of the CuNPS, carried out within the 4,000 to 650 cm-1 range, which revealed bands at 862.74, 917.96, 1207.2, 1629.98, 1664.6, 2927, 3110.63, 3350.9 cm-1. The band at 862.74 cm-1 is attributed to a strong C-Cl stretching vibration of halo compounds. The band at 1207.2 cm-1 corresponds to a strong C-O stretching vibration of tertiary alcohol. The band at 1629.98 cm-1 correlates to a medium C=C stretching vibration of cis alkene. The band at 2927 cm-1 is related to a strong N-H stretching vibration of amine salt. This band, along with 3110.63 cm-1, can also be attributed to a weak O-H stretching vibration of intramolecular-bonded alcohol and medium C-H stretching vibration of alkane, and. Finally, the band at 3350.9 cm-1 corresponds to a strong, broad O-H stretching vibration of intermolecular-bonded alcohol (Sigmaaldrich, 2023). These results are supported by the study of Avinash et al. (2016), which revealed the presence of the aforementioned bands in their green-synthesized CuO/TiO2 nanoparticles using the same spectrophotometry. These peaks suggest the presence and diversity of such functional groups on the CuNPs’ surface.
SCANNING ELECTRON MICROSCOPY (SEM) ANALYSIS
SIZE
> 60 nm
TEXTURE
BETWEEN ROUGH AND SLIGHTLY SMOOTH
STRUCTURE
PLATE-LIKE AND IRREGULAR
The nanoparticles were subjected to SEM for imaging purposes and to determine their morphological characteristics such as surface area, topography, and approximate size (Ural, 2021). These factors are important in determining the particles’ effectiveness in cell attachment and surface area, which provides wider reactive interference. Figure 7 shows the morphology of the CuNPs at 15,000x magnification. It shows the plate-like structure of the nanoparticles, which is seen as irregular, rough, and varying throughout the sample. The texture of the nanoparticles vary between rough and slightly smooth, which may affect binding affinity. The approximate sizes of the particles were recorded to be as small as 60 nanometers, which was measured using the provided scale bar in the lower right corner of the image. This is in line with the guyabano-mediated CuNPs of Kushwaha & Prakash (2021), which are 100 nm in size. The small size of the green CuNPs may be attributed to why they yielded 100% mortality rates.