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Efficacy of the Combination of Arrowroot Rhizome (Maranta

Arundinacea L.), Neem Leaves (Azadirachta Indica A. Juss.), and

Malunggay Leaves (Moringa Oleifera Lam.) as an Herbal Larvipod

Against Aedes Aegypti Larvae

Pearleen Mae C. Liganad, Leslie D. Necesito, Eunice Pamela P. Montaño, Jarissa D. Gallaza, Apple Jane

D. Siroy, RPh

Pharmacy Department, St. Alexius College, Inc. Philippines.

DOI: https://dx.doi.org/10.51584/IJRIAS.2025.1009000103

Received: 10 September 2025; Accepted: 16 September 2025; Published: 25 October 2025

ABSTRACT

The Aedes aegypti is the primary vector that causes dengue worldwide. The goal of the study is to evaluate the

combination of three plant extracts for its larvicidal activity. Arrowroot also contains various bioactive

compounds typical of plants in the Marantaceae family, which may include phenolics and flavonoids,

contributing to its potential health benefits and larvicidal properties in herbal formulations. Azadirachta indica

A. Juss. (neem leaves) contained azadirachtin, a potent pesticidal compound, while Moringa oleifera Lam.

(malunggay leaves) had flavonoids that contributed to larvicidal activity. This study evaluated the larvicidal

effectiveness of combined extracts from these plants as an Herbal LarviPod at concentrations of 10%, 30%,

50%, and 70% over three varied time intervals. The study reported mortality rates of 56.66%, 60%, 70%, and

86.66% against I to IV instars of Aedes aegypti larvae after 48 hours. Larvicon achieved complete mortality,

while PVA (polyvinyl alcohol) with excipients showed no mortality. The 70% Herbal LarviPod showed

moderate cytotoxicity, resulting in 30 deaths among 30 brine shrimp after 24 hours. Complete dissolution of

the PVA pod occurred after 48 hours, indicating effective distribution of the active ingredients. Cold

storage (2-8°C) preserved Herbal LarviPod efficacy, while storage at 30-35°C caused degradation. In distilled

water, the PVA film dissolved within 24 hours, and a similar timeline was observed with oils or stagnant

water. Statistical analysis (two-way ANOVA and Tukey's HSD post-hoc test) showed no significant difference

in larvicidal effectiveness between the Herbal LarviPod and the commercial Larvicon larvicide. The F- value

was 0.658 with a p-value of 0.638, indicating that both performed similarly among treatments.

Keywords: Aedes aegypti, Larvicidal Pod, Azadirachta indica A. Juss. (neem leaves), Maranta arundinacea L.

(arrowroot rhizome), Moringa oleifera Lam. (malunggay leaves)

I. INTRODUCTION

The Aedes aegypti species brought on dengue. This particular viral infection was transmitted from mosquitoes

to humans. This type of mosquito was common in tropical and subtropical regions. Humans get the dengue

virus when bitten by female mosquitoes carrying the virus, specifically Aedes aegypti (World Health

Organization, 2024).

As stated by the World Health Organization (2024), dengue cases had emerged that year, as 7.6 million dengue

cases were reported, including 3.4 million confirmed cases, over 16,000 severe cases, and 3,000 deaths as of

April 30, 2024. Although there had been a significant rise in dengue cases reported worldwide over the past

five years, this increase had been especially noticeable in the Region of the Americas, where by the end of

April 2024, there were over seven million cases, exceeding the 4.6 million cases that were reported annually in

2023.

According to Montemayor (2024), the number of dengue cases registered in Manila between January 1 and

June 15 has climbed by 15% compared to last year. The number of cases had risen by about 10% from the

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previously reported level, from 5,547 cases recorded from May 5 to May 18, 2024, to 6,082 cases from May

19 to June 1. Based on the statistics, between the beginning of 2024 and June 29, 90,119

dengue cases and 233 fatalities were recorded. That year's case count is 19% greater than the 75,968 cases

recorded during the same period the previous year (Department of Health, 2024).

Dengue cases in Soccsksargen, composed of the provinces of South Cotabato, Cotabato, Sultan Kudarat,

Sarangani, and General Santos City, caused 79 deaths from January to October (Gabieta, 2024). The number of

dengue cases in South Cotabato has increased 19% compared to the previous year. The Provincial

Epidemiology and Surveillance Unit reported 2,930 cases in South Cotabato (Dati, 2024).

According to Barangay Kematu’s Rural Health Unit (RHU), the community had faced a notable dengue fever

burden over the past two years. In 2023, the area recorded 52 cases spanning an age range of 0 to 60 years,

with a gender distribution of 30 males and 22 females. The cases were dispersed across various puroks and

sitios, with Purok Centro experiencing the highest incidence (12 cases), followed by Purok Bato (7 cases).

Sitios like Butlehek, Maligaya, Lemblete, and Ipil-ipilan also reported multiple cases. The following year,

2024, saw a significant increase to 87 cases, affecting individuals aged 0 to 73. The male-to-female ratio

shifted slightly, with 49 males and 38 females affected. Purok Bato emerged as the area with the highest

number of cases (21), followed by Purok Maligaya (20) and Purok Ipil-ipil

(11). New locations such as Sitio Lamblobong, Domek, Blangas, and others also reported cases, indicating a

wider spread of the disease compared to the previous year.

This study aimed to develop an environmentally friendly larvicide using plant extracts of Arrowroot Rhizome

(Maranta arundinacea L.), Neem Leaves (Azadirachta indica A. Juss.), and Malunggay Leaves (Moringa

oleifera Lam.). The main method of controlling mosquitoes is through synthetic insecticides. By using

polyvinyl alcohol as the main packing material for our Herbal LarviPod, this study aimed to investigate and

focus on the latest advancements in larvicide. The success of the study would provide an ecofriendly

alternative to commercial larvicides to control the growth of mosquito larvae.

The combined Arrowroot Rhizome (Maranta arundinacea L.), Neem Leaves (Azadirachta indica A. Juss.), and

Malunggay Leaves (Moringa oleifera Lam.) that were being used in a structured formulation such as Pod had

not been well studied. Specifically, its effectiveness and user-centric elements such as odor, appearance, and

the dissolving dynamics of the polyvinyl film that is being

used.

Differences

phytochemical

composition resulting from different plant sources may affect the effectiveness. The thickness and other

physical properties of the pod may affect how quickly the plant extract is released, and no research has

determined the precise thickness needed to make the larvicide a pod. Furthermore, most existing research

emphasized the immediate larvicidal activity of plant extracts rather than formulation techniques or additives

that might extend the shelf life of larvicidal pods. This includes storage conditions such as the pods'

temperature.

MATERIALS AND METHODS

Research Design

This study employed a true experimental research design to investigate the larvicidal effect of Arrowroot

Rhizome (Maranta arundinacea L.), Neem Leaves (Azadirachta indica A. Juss.), and Malunggay Leaves

(Moringa oleifera Lam.) on the mortality of Aedes aegypti larvae. This design involved the manipulation of an

independent variable to determine its causal effect on one or more dependent variables. A control group was

also utilized to serve as a basis for comparison with the treatment groups, thus allowing accurate measurement

of the treatment effects (Sirisilla & Sirisilla, 2023). The use of this research design was appropriate for

observing and recording differences between experimental and control conditions.

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Research Locale

The research was conducted at the Pharmacy Laboratory 2 of St. Alexius College, located on Joaquin Street,

General Santos Drive, Koronadal City, South Cotabato, Philippines. This location was chosen due to the

availability of necessary tools, materials, and equipment for plant extraction procedures.

Data Gathering Procedure

Collection of Plant Sample

Arrowroot Rhizome (Maranta arundinacea L.)

Fifteen (15) kilograms of mature rhizomes were collected from Barangay Centrala, Municipality of Surallah,

South Cotabato.

Neem Leaves (Azadirachta indica A. Juss.)

Fifteen (15) kilograms of mature neem leaves were harvested from the Municipality of Tantangan, South

Cotabato.

Malunggay (Moringa oleifera Lam.)

Fifteen (15) kilograms of mature malunggay leaves were gathered from Barangay Triniville, Koronadal City,

South Cotabato.

Preparation of Plant Extract

Arrowroot Rhizome (Maranta arundinacea L.)

The samples were washed with distilled water, sliced into 2–3 cm sections, and oven- dried at 65°C for 5 hours

(Malki et al., 2023). One hundred (100) grams of dried rhizomes were macerated in 200 mL of 95% ethanol

for seven days, followed by filtration. The ethanol extract was evaporated at 50–65°C and 120 rpm to obtain

the crude extract (Jayakumar & Suganthi, 2017).

Neem Leaves (Azadirachta indica A. Juss.) Leaves were washed, air-dried for 24 hours, and subsequently

oven-dried at 45°C (Kalyabina et al., 2021). Fifty (50) grams of dried leaves were macerated in 800 mL

of 95% ethanol for seven days. The extract was filtered and concentrated using a rotary evaporator at 50–

55°C and 60 rpm (Kaewnang et al., 2015).

Malunggay Leaves (Moringa oleifera Lam.)

Cleaned malunggay leaves were patted dry, and oven-dried at 50°C for 4 hours (Younas et al., 2023). Fifty

(50) grams were macerated with 800 mL of 95% ethanol for seven days. The extract was filtered and

evaporated at 55°C and 60 rpm (Jayani et al., 2022; Marcus & Nwineewii, 2015).

Collection Process of Aedes aegypti Larvae

Larvae at instars I to IV were collected from Barangay Kematu, T'boli, South Cotabato, with assistance of four

trained professionals and one entomologist. Using pipettes, Petri dishes, and microscopes, larvae were

obtained from stagnant water in containers like tires, flower pots, and drains. A total of 170 larvae were

classified, and transported following World Health Organization (2020) guidelines.

Preparation for the Different Concentrations of the Plant Extract

Crude extracts from arrowroot, neem, and malunggay were combined to prepare treatment solutions at 10%,

30%, 50%, and 70% concentration (Pam et al., 2021). Each solution was prepared in triplicate.10%

Concentration of Combined Plant Extracts Mixed 3.33 mL from each extract to yield 10 mL, with 3 mL

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glycerin, 0.5 mL acetic acid, and 1 g chitosan (Jamasri et al., 2023). The solution was exposed to 10 Aedes

aegypti larvae in 100 mL distilled water (Umar, 2015).

30% Concentration of Combined Plant Extracts

Mixed 10 mL from each extract to yield 30 mL, with 3 mL glycerin, 0.5 mL acetic acid, and 2.5 g chitosan

(Jamasri et al., 2023).

50% Concentration of Combined Plant Extracts

Mixed 16.66 mL from each extract to yield 50 mL, with 7 mL glycerin, 1 mL acetic acid, and 5.5 g chitosan

(Yusmaniar et al., 2023).

70% Concentration of Combined Plant Extracts

Mixed 23.33 mL from each extract to yield 70 mL, with 7 mL glycerin, 1 mL acetic acid, and 5.5 g chitosan

(Dhinakaran et al., 2019; Yusmaniar et al., 2023).

Formulation of Herbal LarviPod

In the Herbal LarviPod formulation, the three combined plant extracts were used as liquids in varying

quantities of 10%, 30%, 50%, and 70% concentration. The outer shell of the pods is made of polyvinyl alcohol

or a PVA derivative, which will dissolve in water to release the combined crude extract.

Formation: The commercially available polyvinyl alcohol (PVA) film were cut into 4

×5 cm rectangles and heat-sealed along three edges.

Filling: Approximately 5 mL of each

plant extract concentration was pipetted into the pods.

Sealing: The open edge was heat-sealed, with a small air volume retained to ensure buoyancy (Elie, 2023).

Packaging: The prepared LarviPods were stored in airtight glass containers under laboratory conditions until

use (Elie, 2023).

Statistical treatment

This study employed a two-way analysis of variance (ANOVA) to determine the effects of plant source

(Arrowroot rhizome, Neem leaves, and Malunggay leaves) and extract concentration (10%, 30%, 50%, and

70%) on the mortality rate and time of mortality of Aedes aegypti larvae. The two-way ANOVA allowed

the assessment of both main effects (the independent impact of each

factor) and interaction effects (the combined influence of plant source and concentration).

The Tukey post-hoc test was used to find the means that were significantly different between each treatment.

The combination of two-way ANOVA and the Tukey post-hoc test provided a comprehensive statistical

approach to analyze the efficacy of different plant extracts and concentrations against Aedes aegypti larvae

(Sakka et al., 2023). This methodology ensured accurate interpretation of teh results, particularly regarding

potential synergistic effects among natural plant extracts and commercial larvicides, thereby contributing to the

development of effective, plant-basedd mosquito control strategies.

RESULTS AND DISCUSSION

The data were gathered by determining the mortality rate of larvae by using the different concentrations of the

combination of Arrowroot Rhizome (Maranta arundinacea L.), Neem Leaves (Azadirachta indica A. Juss.),

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and Malunggay Leaves (Moringa oleifera Lam.) that consist of 10%, 30%, 50%, and 70% concentrations. The

identification of Aedes aegypti larvae was done using a microscope. The data on the different concentrations

as well as the result of the effectiveness of the study are presented in the table below.

TABLE 1.1. Mortality Rate of Different Plant Concentrations

Sum of

Squares

Mean

Square

p-value

Mortality

Rate

0.0

TWO-WAY ANOVA—Mortality Rate of Different Plant Concentrations

produce a meaningful difference in larval mortality rates.Similarly, the interaction between concentration and

mortality rate was also not significant (Sum of Squares = 265.04, df = 8, Mean Square = 33.13, F = 1.224, p =

0.392). This indicates that the combined effect of concentration levels and mortality rate did not contribute to

significant variation in larval outcomes.The residual variation accounted for a Sum of Squares of 361.06 with

72 degrees of freedom, reflecting the majority of unexplained variability in the dataset. Overall, the analysis

demonstrates that under the experimental conditions tested, neither concentration alone nor its interaction with

mortality rate significantly affected the time of mortality in Aedes aegypti larvae. These findings imply that

other factors beyond the tested concentration levels may play a more substantial role in determining larval

mortality patterns (Smith & Jones, 2019).

Table 1.2. Time and Mortality

TWO-WAY ANOVA - Time and Mortality

Sum of

Squares

Mean

Square

p-value

Mortality Rate

0.0

Time

91.0

45.49

3.468

0.090

Mortality

✻

Time

Rate

11.9

5.96

0.455

0.652

Residuals

91.8

13.12

n=30

Table 1.2 presents the results of the two-way ANOVA examining the effects of time and mortality rate on the

dependent variable. The main effect of time produced a Sum of Squares of 91.0 with 2 degrees of freedom,

yielding a Mean Square of 45.49. The

Concen

trati

ons

64.8

21.6

0.299

0.825

corresponding F-value was 3.468 with a p-value of

0.090,

indicating

that

while

the

effect

time

approached

Mortali

265.

88.3

1.224

0.392

significance, it did not meet the conventional

threshold

Rate

✻

Concentrati ons

Residuals 361.0 5 72.2

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n= 30

The results in Table 1.1 present the outcomes of a two-way ANOVA assessing the influence of concentration

on the mortality rate of Aedes aegypti larvae. The analysis revealed that the effect of concentration was not

statistically significant (Sum of Squares = 64.83, df = 21, Mean Square = 3.09, F = 0.299, p = 0.825). This

suggests that varying concentrations did not for statistical significance. This suggests a possible trend in

which time may influence the dependent variable, but the evidence is not strong enough to confirm a reliable

effect.The interaction effect between mortality rate and time yielded a Sum of Squares of 11.9 with 2 degrees

of freedom, a Mean Square of 5.96, an F-value of 0.455, and a p-value of 0.652. These results clearly

demonstrate that there is no significant interaction, meaning that the effect of time on the dependent variable

does not vary across different levels of mortality rate.The residual variation accounted for a Sum of Squares of

91.8 with 7 degrees of freedom, corresponding to a Mean Square of 13.12, representing the portion of

variance not explained by the model.Taken together, the findings indicate that neither mortality rate nor its

interaction with time significantly influenced the dependent variable. However, the near- significant p-value

for time suggests a potential trend that may warrant further investigation with a larger sample size or refined

experimental design (Smith & Lee, 2020)

TABLE 2.1.Mortality Rate of the Positive (Larvicon) and Negative Control (PVA Film with Excipients)

TWO-WAY ANOVA: Mortality and Positive Control

Sum of

Squares

Mean

Square

Mortality

0.222

0.111

0.400

0.694

Positive

Control

0.222

0.111

0.400

0.694

Residuals

1.111

0.278

n=30

Table 2.1 presents the results of the two-way ANOVA evaluating the effects of mortality and positive control

on the dependent variable. For mortality, the analysis produced a Sum of Squares of 0.222 with 2 degrees of

freedom, resulting in a Mean Square of 0.111. The corresponding F-value was 0.400 with a p-value of 0.694,

indicating no statistically significant effect.Similarly, the factor of positive control yielded identical values

(Sum of Squares = 0.222, df = 2, Mean Square = 0.111, F = 0.400, p = 0.694). This suggests that the inclusion

of a positive control did not contribute to significant variation in the outcome under the experimental

conditions.The residual variation accounted for a Sum of Squares of 1.111 with 4 degrees of freedom, resulting

in a Mean Square of 0.278. This relatively larger residual component highlights that the majority of the

variability in the dependent variable remains unexplained by the tested factors.Overall, the p-values for both

mortality and positive control (0.694) indicate that neither factor exerted a significant influence on the

dependent variable. In practical terms, this finding suggests that differences in mortality levels and the use of a

positive control did not meaningfully affect the measured outcome, and that other untested variables may play

a more critical role in shaping the results (Larvicon—Leads Environmental Health, 2021).

TABLE 2.2. Mortality Rate of the Positive (Larvicon) and NegativeControl (PVA Film with Excipients) Post

Hoc Comparisons—Time

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Note. Comparisons are based on estimated marginal means

Table 2.2 presents the results of Tukey’s post hoc comparisons, which were conducted to evaluate differences

in mean larval mortality rates across the three exposure times (4, 24, and 48 hours).The comparison between 4

and 24 hours showed a mean difference of –0.333 with a standard error of 0.385, a t-value of –0.866, and a p-

value of 0.679. This indicates that mortality rates did not significantly increase between these two time

intervals.The comparison between 4 and 48 hours yielded an almost negligible mean difference (–1.22 × 10 ¹

), with a t-value approaching zero (–3.17 × 10 ¹ ) and a p- value of 1.000. These results confirm that no

meaningful difference in larval mortality occurred between the shortest and longest exposure times.Similarly,

the comparison between 24 and 48 hours revealed a mean difference of 0.333, a t-value of 0.866, and a p-value

of 0.679, which was again not statistically significant.Taken together, the post hoc analysis demonstrates that

no significant differences in larval mortality were observed across the three exposure times. This suggests that

within the conditions of the experiment, the duration of exposure (up to 48 hours) did not exert a meaningful

effect on larval mortality. In practical terms, this finding implies that extending exposure time alone is

unlikely to enhance larvicidal effectiveness under the tested parameters (Larvicon—Leads Environmental

Health, 2021).

TABLE 3 Brine Shrimp Lethality Assay

Trials

Trial 1

Trial 2

Trial 3

Total Deaths

10 % concentration

30 % concentration

50 % concentration

70 % concentration

n=30

Table 3 presents the outcomes of exposing brine shrimp to varying concentrations of Herbal Larvipod (10%,

30%, 50%, and 70%) over a 4-hour period, with three replicate trials conducted for each concentration (10

brine shrimp per trial, totaling 30 per concentration). As noted by N et al. (2020), standard bioassay

protocols typically use 10–15 larvae per test tube containing larval suspension.At the 10% concentration, 29

out of 30 brine shrimp died, demonstrating substantial toxicity even at the lowest tested dose. At both the 30%

and 70% concentrations, mortality reached 100%, with all 30 shrimp per group dying, indicating complete

lethality. The 50% concentration also showed near-total mortality, with 29 deaths, closely mirroring the

results of the 10% group.The LC value calculated at 24 hours indicated moderate cytotoxicity at higher

concentrations, consistent with previous findings in brine shrimp lethality bioassays (N et al., 2020).Taken

together, these results provide strong evidence that Herbal Larvipod possesses potent larvicidal and cytotoxic

properties, achieving near- complete mortality across all concentrations within a short 4-hour exposure period.

The minimal variation in mortality between low and high concentrations suggests that the extract’s bioactive

compounds are highly effective, even at reduced doses.

From an applied perspective, this points to the potential of Herbal Larvipod as a powerful biocontrol agent.

However, the high toxicity observed also highlights the need for further evaluation on target versus non-target

organisms, as well as assessments of its ecological safety and long-term environmental impact, before large-

scale application can be recommended.

Stability Test TABLE 4.1.

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Dissolution Time

Number of Hours Remarks

Harpaz et al. (2019), release mechanisms for embedded bioactive substances are often influenced by factors

such as temperature, pH, and ionic strength, all of which can affect both the rate and extent of dissolution.By

24 hours, the PVA pod was nearly dissolved, but the extract had not yet fully mixed with the water. This stage

reflects the transition from structural weakening to active release, implying that the dissolution process

proceeds gradually and that practical release of the extract occurred within approximately one day (POLYVA,

2019).At the 48-hour point, the PVA pod had completely dissolved, leaving no visible remnants. The water

turned green, confirming the successful release and uniform dispersion of the crude extract throughout the

medium. This final stage indicates the completion of both pod disintegration and extract distribution, with the

solution maintaining stability afterward.

Overall, the results demonstrate that the PVA pod served as an effective encapsulation and delivery system,

fully dissolving and releasing its contents within 48 hours. The staged dissolution rapid pod solubilization

followed by delayed extract release suggests that PVA pods can provide a controlled release mechanism,

which may be advantageous for applications requiring gradual dispersion of active compounds.

TABLE 4.2. Storage Duration and Temperature Effects

Temperature 1 Day 3 Days 7 Days

4 hours - The pod started sinking into the beaker. - Partial tearing of the pod is visible.

24 hours -The crude extract is partially mixed with water.

-The extract had not yet been fully distributed in the medium.

48 Hours -The PVA film dissolves completely in the container.

-The medium becomes green.

-No film is evident.

Table 4.1 describes the dissolution profile of polyvinyl alcohol (PVA) pod encapsulating a crude extract over

a 48-hour observation period.At the 4-hour mark, the PVA pod began to sink, and partial tearing of the film

was noted. However, the crude extract had not yet dispersed into the surrounding medium. This suggests that

while the PVA film exhibited early solubility in water, the release of the encapsulated extract was delayed,

Cold

Temperature (2°C - 8°C)

Room

Temperature (20°C - 25°C)

Hot

Temperature (30°C - 35°C)

Color: Dark Green to Brownish Texture: Deflated

Color: Dark Green to Brownish Texture: Creased

Color: Dark Green to Brownish Texture: Shrunk half its original size

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Color: Dark Green to Brownish Texture: Creased

Color: Brown to Black Texture: It' sa bit dry and has a jelly-like texture

Color: Dark Green to Brownish Texture: Creased

Color: Black

Texture: It's a bit dry and has a jelly-like texture Odor: Foul/Pung likely requiring additional pod

disintegration or more favorable environmental conditions. As highlighted by ent odor

Table 4.2 provides an analysis of the physical stability and preservation of PVA pods containing crude extract

when stored under three different temperature conditions—cold (2–8°C), room temperature (20–25°C), and

hot (30–35°C)—over a period of 1, 3, and 7 days. Under cold storage, the appearance of the extract was a dark

green to brownish liquid, and the pods showed only minor changes, such as air deflation that caused the

appearance to be creased on day 1. By day 3, the pods retained their appearance, indicating that the structural

integrity of the film was still largely intact. On day 7, the pods were notably deflated, but no changes in

appearance or color were observed across different concentrations, suggesting that cold storage is optimal for

preserving both the form and efficacy of the pods.

Moringa plant extract samples held at 4°C experienced the least amount of active content loss after four

months of storage when compared to ambient conditions (Sona et al. 2021). A 5%, 10%, and 20% neem leaf

extract at cold storage temperatures (2.8°C and 1.2°C, respectively) for three days of storage led to lower

levels of contamination, infection, and degradation compared to ambient conditions (Narayana C, 2018).

Arrowroot rhizome extract should be stored at a cold temperature, ideally between 4°C (40°F) and -20°C (-

4°F), to preserve its active constituents.

In contrast, storage at room temperature resulted in moderate stability. The extract remained intact and

appeared as a dark green to brown liquid or gel-like substance, and PVA was partially creased on day 1. There

were no significant physical or chemical changes observed throughout the 7-day period. The pod maintained

its form and appearance, indicating that room temperature is acceptable for short- to mid-term storage. The

active substance in arrowroot is primarily a heat-stable polysaccharide. Research shows that extracts prepared

at room temperature (25°C) do not inhibit biological activity (Malki et al., 2023). Extraction studies show

that moderate compounds at 30 to 35°C led to the degradation of the plant extract. Also, the neem extract that

has been exposed to high temperatures, such as 30°C, will reduce its effectiveness. According to Boursier et

al. (2020), their study stated that increased temperature for neem extract, specifically its active compound,

azadirachtin, decreased its effectiveness by 30% between the third and seventh day of their storage. Arrowroot

extract could withstand higher temperatures. As stated by the study of Nogueira et al. (2018), even at 60°C, the

arrowroot indicated good functionality. The significant degradation of arrowroot occurs between 330°C -

410°C.

However, hot storage conditions (30- 35°C) caused notable degradation. By day 1, the pod’s appearance

changed, and it began shrinking to half of its original size. Over the next two days, the film developed

wrinkles, and the extract appeared dry and took on a jelly-like texture with slight discoloration. By day 7, a

foul odor developed, the texture became tacky, and the appearance deteriorated. These signs indicate that high

temperatures negatively affect the pod's integrity and lead to the death of active constituents, making this

condition unsuitable for storage. Overall, the data suggests that cold storage is most effective in maintaining

pod stability and bioactivity, while elevated temperatures accelerate deterioration. Since it slows down

hydrolytic and thermal degradation processes, preserving both the physical structure of the film and the

bioactivity of encapsulated ingredients (Orapan Romruen et al., 2024).

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TABLE 4.3. Solubility

Solvent

4 Hours

24 Hours

48 Hours

temperatures (25–40°C) do not significantly reduce total phenolic content, but higher temperatures may cause

degradation of the neem extract (Wirwis & Sadowski, 2023). The phenolic compounds in moringa are stable at

room temperature (28.5-33.5°C) for up to six months, with only minor decreases in content and activity

WATER (distilled water)

OILS

The PVA film started to shrink at 1-3 hours of exposure. The PVA film- The PVA film completely dissolved

in water.

- No fragments of the PVA remain.

- The PVA film

-The appearanc e of the medium remained the same.

-The over time (Jayani et al., 2022).

(canal water, started to shrink completely medium As stated by Castro-López et al. (2020), the increasing

storage temperature of Malunggay stagnant water) at 1-3 hours of exposure. dissolved in water.

- No fragments of the PVA film are visible becomes murky due to the

original appearance of the water used.

Table 4.3 examines the solubility of the PVA film in two different solvents—distilled water and oil-

contaminated water (such as canal or stagnant water)—over 4, 24, and 48 hours. In distilled water, the PVA

film began to shrink within the first 1–3 hours. By 24 hours, the PVA film had completely dissolved, with no

visible fragments of PVA film present. After 48 hours, the appearance remained the same. This suggests that

distilled water is an effective solvent for dissolving PVA films.

According to the research (Mangaraj et al., 2018), the PVA film showed high solubility in distilled water due

to the effect of the hydroxyl group in the water absorption mechanism. The water solubility and water

absorption of the PVA film were directly proportional, which resulted in the normal behavior of the increase of

water absorption of the PVA film, which in turn made the PVA film easier to dissolve in distilled water.

In contrast, when exposed to oils or stagnant water, the PVA film followed a similar initial disintegration

timeline, beginning within 1–3 hours. By 24 hours, the films were completely dissolved, and no visible

remnants of film were visible. At the 48-hour mark, the solution appeared cloudier due to the initial

appearance of the water used. PVA film has poor resistance to water due to its molecular structure that

includes hydrophilic hydroxyl groups; exposure to a wet environment causes them to swell or dissolve (Liu et

al., 2022).

TABLE 5.1. Effectiveness of Herbal LarviPod at Different Concentration

TWO-WAY ANOVA - Effectiveness of Herbal LarviPod at Different Concentration

The table summarizes the results of a two- way ANOVA used to assess the efficiency of the Herbal LarviPod

at various concentrations. For the concentration component, the sum of squares was

16.3 with three degrees of freedom, yielding a mean square of 5.44. The F-value for concentration was 0.358,

and the associated p- value was 0.786. This implies that concentration had no statistically significant effect on

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Page 1053

the efficiency of the Herbal LarviPod. In contrast, the time factor has a sum of squares of 158.2 with 2 degrees

of freedom, resulting in an average square of 79.08. The F-value for time was 5.205, with a p-value of 0.049,

slightly below the 0.05 significance level.

This implies that the period of exposure had a statistically significant effect on the efficiency of the Herbal

LarviPod, implying that time is a critical factor in its larvicidal action (Nawarathne, M. P., & Dharmarathne,

C. 2024). The residuals, which indicate unexplained variability in the data, had a total of squares of 91.2, 6

degrees of freedom, and a mean square of 15.19. The results demonstrate that the concentration of the Herbal

LarviPod did not significantly influence its effectiveness; the time of exposure was a significant factor. Based

on the study of Araj et al. (2015), increased exposure time of larvae to larvicides enhances the larval mortality.

It also implies the importance of considering exposure duration when evaluating the larvicidal efficacy of this

herbal product.

TABLE 5.2.

Post Hoc Comparisons – Concentrations

Sum of Squares

Square

Concentrations

16.3

5.44

0.358

0.78

Time

158.2

79.08

5.205

0.04

Residuals

91.2

15.19

n=30

The post hoc comparisons for concentrations revealed no statistically significant differences in larval mortality

between any of the tested Herbal LarviPod concentrations (10%, 30%, 50%, and 70%). All p-values were well

above 0.05, indicating that none of the concentration pairs produced meaningful differences in mortality.

Even the largest mean difference, observed between 10% and 70% (-3.000), was not significant (p = 0.910).

These results clearly indicated that increasing the concentration of Herbal LarviPod did not lead to a

significant increase in larvicidal effectiveness, suggesting that concentration alone was not a determining factor

in larval mortality under the conditions of this study Piplani et al. (2019).

INTERNATIONAL JOURNAL OF RESEARCH AND INNOVATION IN APPLIED SCIENCE (IJRIAS)

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Page 1054

Table 6.

result implied that the duration of exposure may influence mortality rates to some extent. It's worth noting that

had the p-value been marginally lower, it would have suggested that larval mortality increased with time,

potentially due to the prolonged contact between the larvae and the active ingredients in the larvicides (Araj et

al., 2015).

Despite the lack of statistical significance, the practical differences observed in raw data were informative.

LarviCon consistently showed 100% mortality across all time intervals, while Herbal LarviPod's effectiveness

increased with concentration and exposure time (Larvicon—Leads Environmental Health 2021). For example,

the 70% Herbal LarviPod concentration resulted in notably higher mortality rates than lower concentrations,

especially at the 48- hour mark (Opoggen et al., 2019). These patterns indicate that while differences were not

yet statistically validated, they could be meaningful in real-world applications and might have become

significant with larger sample sizes or repeated trials.

The implications of these findings were important for public health and mosquito control strategies. While

Herbal LarviPod shows potential—particularly at higher concentrations and longer exposure durations—it

did not match the efficacy of LarviCon under the conditions tested. This suggests that Herbal LarviPod could

serve as a sustainable or natural larvicide alternative, but further development, optimization, and larger-scale

testing were necessary. Programs considering Herbal LarviPod would benefit from refining its formulation and

conducting more robust trials to support its effectiveness with statistical confidence.

Table 6.1.

The results of the two-way ANOVA examined the effects of treatment (LarviCon and various concentrations

of Herbal Concentrations of LarviPod) and time (4, 24, and 48 hours) on larval mortality and showed that

neither factor reached statistical significance at the conventional α = 0.05 level. For the treatment variable

comparing LarviCon and Herbal LarviPod concentrations, the F- value is 0.658 with a p-value of 0.638, which

was far greater than the significance threshold. This indicates that there was no statistically significant

difference in larvicidal effectiveness among the different treatments, including the commercially available

LarviCon and the various concentrations of Herbal LarviPod.

Regarding the time factor, the F-value is 4.12 with a p-value of 0.0588, which was slightly above α = 0.05,

suggesting a borderline effect of time on larval mortality. Although not statistically significant, this

Tukey’s HSD Post Hoc Test Comparing Treatment Means of

Larvicidal Effectiveness

Pairwise

Mean

HSD

Critical

Comparison Difference Value Decision

Not

Larvicon vs. 10%

4.33

8.79

Significant

Not

Larvicon vs. 30%

4.00

8.79

Significant

Not

Larvicon vs. 50%

3.00

8.79

Significant

Not

Larvicon vs. 70%

1.33

8.79

Significant

8.79

Not

10% vs. 30%

0.33

Significant

10% vs. 50%

1.33

8.79

Not

Significant

Not

10% vs. 70%

3.00

8.79

Significant

Not

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Page 1055

8.79 30% vs. 50% 1.00

Significant

0% vs. 70% 2.67 8.79

Not Significant Not

for environmentally conscious mosquito control programs. Given the comparable effectiveness and the

observed trend towards increased mortality50% vs. 70% 1.67 8.79

Significant

Significance level at α = 0.05

The results of Tukey's HSD post hoc test revealed that none of the pairwise comparisons between the

commercially available larvicide Larvicon and the different concentrations of Herbal LarviPod showed

statistically significant differences in larvicidal effectiveness. The mean differences between all treatment pairs

were below the critical HSD value of 8.79, leading to the conclusion that the observed differences in larval

mortality rates among these treatments are not statistically meaningful at the 0.05 significance level. This

indicates that Larvicon and Herbal LarviPod, regardless of concentration, have comparable effects on

mosquito larvae under the tested conditions.

Analyzing the data more closely, although no significant differences were found, there was a clear numerical

trend where Larvicon consistently showed higher mean effectiveness compared to Herbal LarviPod

concentrations. Among the Herbal LarviPod treatments, increasing concentration corresponds to higher mean

mortality rates, with the 70% concentration being the most effective among the Herbal LarviPod groups. This

pattern suggested a dose-response relationship, where increased concentration of the natural larvicide

correlates with better larvicidal outcomes, even if these differences are not statistically significant in this

sample (Opoggen et al., 2019).

The lack of statistical significance might have been influenced by sample size or variability within the data. A

small sample size or high variability could reduce the power of the test, limiting the ability to detect real

differences that exist in the population. As stated by the World Health Organization (2025), large-scale trials

verify the larvicide's effectiveness in the chosen dosage(s) of the application against the target vector when

applied to expansive areas in natural breeding grounds. Therefore, while the current results do not confirm that

any treatment is superior, they do highlight the promising effectiveness of Herbal LarviPod at higher

concentrations, warranting further investigation with larger or more controlled studies.

The implications of this analysis suggested that Herbal LarviPod, a natural larvicide composed of varying

concentrations of botanical extracts, could be a viable alternative to commercial larvicides like Larvicon,

particularly with higher Herbal LarviPod concentrations, further research to optimize concentration and

application methods could help enhance its practical use. Additionally, expanding sample size and replication

will be critical to confirm these preliminary findings and establish Herbal LarviPod’s role in integrated vector

management strategies (Prates et al., 2024).

CONCLUSION

The study highlighted the potential combination of the extracts of Arrowroot Rhizome (Maranta arundinacea

L.), Neem Leaves (Azadirachta indica A. Juss.), and Malunggay Leaves (Moringa oleifera Lam.)—

encapsulated in a polyvinyl alcohol (PVA) film pod—as a potential larvicide against Aedes aegypti larvae,

through the following concentrations of 10%, 30%, 50%, and 70% that showed significant differences in the

mortality rate of the larvae. It sought to assess mortality across various concentrations and time intervals,

compare efficacy against controls, evaluate cytotoxicity via brine shrimp assay, and examine product stability

under diverse conditions.

The findings indicated a dose-dependent increase in larval mortality, with the 70% concentration producing the

highest mortality rate (86.66%) at 48 hours. Although the positive control (Larvicon) achieved consistent

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Page 1056

100% mortality in all trials and time intervals, statistical analyses (Two-way ANOVA and Tukey’s HSD Post

Hoc Test) revealed no significant differences (p > 0.05) between Larvicon and Herbal LarviPod

concentrations, suggesting that higher concentrations approximate the efficacy of commercial larvicide.

The brine shrimp assay supported the high cytotoxicity of Herbal LarviPod even at lower concentrations, with

near-complete lethality across all dose levels, indicating the extract’s strong bioactivity. Regarding stability,

the Herbal LarviPod showed favorable dissolution behavior, completing release by 48 hours, and maintained

structural and chemical integrity under cold and room temperature conditions for up to 7 days. However,

degradation occurred at elevated temperatures, reducing its potential efficacy under such storage conditions.

In Conclusion, The Study Demonstrates That The Herbal Larvipod Is A Promising,

Plant-Based

larvicidal agent, particularly effective at higher concentrations

(≥50%), with stable performance under

controlled conditions. While not statistically

superior to Larvicon, its

comparable

larvicidal

outcomes,

natural composition, and

low cytotoxic threshold point to its potential as an environmentally

sustainable

mosquito control method.

Further optimization and scaled-up testing are

recommended

to refine its application and validate its broader efficacy.

RECOMMENDATIONS

This study recommends Herbal LarviPod, a combination of arrowroot rhizome, neem leaves, and malunggay

leaves extracts encapsulated in a PVA film, as an eco-friendly, sustainable, and cost-effective larvicide against

Aedes aegypti. Further development is suggested for community use in dengue-prone areas, advocating

collaboration with local health authorities and integration into existing vector control programs. Large-scale

trials, formulation improvements (including slow-release mechanisms and olfactory component optimization),

and comprehensive safety evaluations are needed for regulatory approval and broader adoption. Future

research should investigate synergistic or antagonistic interactions between the plant extracts, optimize the

pod's physical form for consistent release and reduced loss, and conduct stability testing under controlled

conditions (humidity, temperature, pH) to determine shelf life accurately.

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