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American Journal of  
Life Science and Innovation (AJLSI)

In VitroIn Vitro Antihelmintic Activity of  the Hydroethanolic Leaf  Extracts of  Avicennia marinaAvicennia marina 
(White Mangroves) on Eudrilus eugeniaeEudrilus eugeniae (African Nightcrawlers)

Malacad1, Magallanes1, Felizarte1, Fenete1, Guillermo1, Jaspe1, Jordan1, Legada1, Losañes1, Marañon1, Esteva1, Palmos1, Barrido1, 
Leong-on1*

Volume 4 Issue 2, Year 2025
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v4i2.5052
https://journals.e-palli.com/home/index.php/ajlsi

Article Information ABSTRACT

Received: April 12, 2025

Accepted: May 16, 2025

Published: June 25, 2025

Research is needed to determine the Avicennia marina hydroethanolic leaf  extract is effective 
against adult E. eugeniae, despite research showing that it can eliminate helminths such as 
Pheretima posthuma. The purpose of  this study was to ascertain the hydroethanolic leaf  
extracts of  A. marina antihelmintic effectiveness against adult E. eugeniae in vitro. Using E. 
eugenie adults (6–8 cm in length and 0.2–0.3 cm in breadth), the percentage vermicidal activity 
of  three concentrations of  dried crude hydroethanolic leaf  extract of  A. marina, 25, 50, and 
75 mg/mL of  distilled water, was assessed every three hours for 12 hours. Five replicates, 
three trials, distilled water as a negative control, and mebendazole as a positive control were 
all examined. According to the findings, the hydroethanolic leaf  extracts of  A. marina have 
shown antihelmintic efficacy against E. eugenie adults in vitro at doses of  50 mg/mL and 
75 mg/mL utilizing Kruskal-Wallis and LSD on the 3, 6, 9, and 12 hours, with a p-value 
of  less than .05. In both concentrations, 50 and 75mg/ml, until the 12-hour exposure to 
the extract, there was no discernible difference between them. In vitro antihelmintic activity 
was best demonstrated at 50 mg/mL for 3 hours (mean = 91.1; sd = 26.6), and the in vitro 
antihelminthic activity was significantly superior to that of  the positive control for the same 
duration (mean = 2.22; sd = 8.61). The results scientifically demonstrated the antihelmintic 
efficacy of  hydroethanolic leaf  extract of  A. marina against E. eugeniae and provided the 
foundation for testing against Ascaris, Trichuris trichiura, and hookworms. 

Keywords

Antihelmintic, Avicennia Marina, 
Eudrilus Eugeniae, Hydroethanolic 
Extract, Vermicidal Activity

1 Medical Laboratory Science, College of  Pharmacy and Medical Technology, University of  San Agustin, Iloilo City, Philippines
* Corresponding author’s e-mail: mleongon@usa.edu.ph

INTRODUCTION
According to estimates, 1.5 billion people, or 24% of  the 
world’s population, are affected by helminth infections, 
making it one of  the most common infections in the 
world (World Health Organization, 2023). It is especially 
common in third-world countries, where poor sanitation 
and hygiene create the perfect conditions for parasitic 
helminth infection and extensive transmission. Between 
33.8% to 75.9% of  Filipinos were afflicted with soil-
transmitted helminth (STH) infections in the Philippines, 
with schoolchildren being the most susceptible (Mationg 
et al., 2021). The roundworms Ascaris, Trichuris trichiura, 
and hookworms are the most frequent causes. Numerous 
health problems, such as stomach pain, diarrhea, loss 
of  blood and protein, rectal prolapse, and physical and 
cognitive growth retardation, can be caused by significant 
helminthic infections in humans (CDC, 2022).
Over the past few decades, numerous anthelmintic 
studies have been developed as a result of  the increased 
incidence and prevalence of  helminthic illnesses. Using 
roundworm species that are morphologically similar 
to parasitic species is one strategy. Ogen et al. (2017) 
proposed using the common soil earthworm Pheretima 
posthuma as test subjects because of  its morphological 
similarity to intestinal roundworms. In the Philippines, 
E. eugeniae (African nightcrawlers) are especially common 
and make excellent subjects for anthelmintic research. 
Additionally, they are helminths, which and intestinal 

roundworms have comparable anatomy (Pueblos et al., 
2015; Asiimwe et al., 2023; Hogade et al., 2014).
Commercial medications such as Albendazole, 
Mebendazole, Levamisole, and Pyrantel Pamoate were 
frequently used to treat helminth infections in humans 
(Moser et al., 2017). Despite their effectiveness, these 
medications have a number of  drawbacks when used over 
extended periods of  time. In their research, Kotze et al. 
(2019) observed that helminth parasites have a propensity 
to become resistant to some drugs following prolonged 
therapy. Agricultural research, which focused on natural 
chemicals with anthelmintic action, was therefore very 
interested in novel natural alternatives.
Other plants with comparable phytochemical 
components, including Leucaena leucocephala (Lead 
Tree), Carica papaya (Pawpaw), and Cayriata auriculata 
(ear-leafed Cayratia), have been the subject of  numerous 
studies and have been shown to be effective anthelmintic 
agents (Kancherla et al., 2019; Widaad et al., 2022). 
In particular, the synergistic anthelmintic effects of  
flavonoids and tannins mostly functioned by making 
the nematode cuticle stiffer, which inhibited growth and 
encouraged death (Greiffer et al., 2022).
The anthelmintic activity of  A. marina is one of  the 
plants that can be investigated. This kind of  mangrove 
can be found in rivers, streams, coastal regions, and the 
intertidal zones of  seabeds. For coastal barangays, it was 
a natural defense, particularly in remote, impoverished 



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areas. The effectiveness of  A. marina tree extracts against 
helminths, including the earthworm Pheretima posthuma, 
has been the subject of  a limited number of  studies 
(Kondepudi, 2018). Furthermore, previous research had 
shown that phytochemicals like phenolic compounds, 
alkaloids, flavonoids, sugars, carotenoids, aliphatic 
alcohols, amino acids, hydrocarbons, fatty acids, tannins, 
saponins, and terpenes were present (Wilfred et al., 2023). 
In support of  this, Mughal et al. (2016) also found that 
A. marina contains phytochemicals proteins, amino 
acids, flavonoids, phenols, carbohydrates, and alkaloids. 
Accordingly, the phytochemical substances saponins, 
flavonoids, and tannins were nematicidal chemicals 
according to research by Wilfred et al. (2023) and Mughal 
et al. (2016); D’Addabbo et al. (2022).
The use of  A. marina leaf  extract hydroethanolic against 
adult E. eugeniae has not been studied, despite the fact that 
it has been scientifically demonstrated to kill helminths 
like P. posthuma. The overall goal of  this work was to 
ascertain the hydroethanolic leaf  extracts of  A.marina’s 
antihelmintic effectiveness against E. eugeniae in vitro. The 
study’s specific goal was to ascertain the hydroethanolic 
leaf  extracts of  A.marina’s in vitro antihelmintic activity 
against adults of  E. eugeniae using varying concentrations 
(25, 50, and 75 mg/mL) and time (3, 6, 9, and 12 hours) in 
terms of  percent vermicidal activity (Va) in comparison 
to the negative control, distilled water and the positive 
control, Mebendazole (25 mg/mL). In terms of  percent 
vermicidal, the study identified the optimal concentrations 
(25, 50, and 75 mg/mL) and times (3, 6, 9, and 12 hours) 
that demonstrated the hydroethanolic leaf  extracts of  
A. marina’s in vitro anthelmintic activity against E. eugeniae 
adults. The study’s findings gave A. marina’s vermicidal 
action against E.eugeniae a scientific foundation.

MATERIALS AND METHODS
A quantitative post-test, fully randomized approach 
was employed in the investigation. The % vermicidal 
(mortality) effects of  A.marina at three different doses 
(25, 50, and 75 mg/mL) against E. eugeniae adults were 
evaluated. The study was reviewed by a private university’s 
recognized Ethics Review Committee. In addition, the 
study received biosafety and ethics clearance.
E. eugeniae were gathered from a vermicomposting region 
in Antique Province, A. marina were gathered from the 
riverbanks in Dumangas, Iloilo. The test was carried out 
in Antique between February and April of  2024.

Identification of  Samples
The pictures of  trees, fruits, and plant leaves (Figure 1) 
were sent to the Department of  Environment and Natural 
Resources for verification. This sample’s taxonomic 
classification is as follows:
Kingdom: Plantae
Phylum: Tracheophyta
Class: Magnoliopsia
Order: Lamiales
Family: Acanthaceae

Genus: Avicennia
Species name: marina
Common name: api-api or white/gray mangrove

Figure 1: Images of  the plant A. marina: A. yellowish 
leaf  abaxial surface; B. edge of  the leaf  (adaxial 
surface); C. arrangement of  leaves (opposite); D. roots 
(pneumatophores that resemble pencils); E. trunk of  a 
tree (lenticels and smooth bark); F. The crown of  a tree

Figure 2: Photograph of  A. marina whole plant

Collection of  plant material
A. marina leaves were gathered in Dumangas, Iloilo. 
Samples of  plants were gathered 100 meters from the 
road. The leaves were picked between 6:00 and 7:00 
a.m. Leaves that were not budding or decaying were 
used. Leaf  samples were free of  insect bites, trauma, and 
irregularities. Collection was avoided within 3 days after 
rainfall in the area.

Identification and Collection of  E. eugeniae 
A veterinarian identified the worm. A veterinarian 
verified the authenticity of  E. eugeniae worms that were 
gathered from vermicomposting facilities in the province 
of  Antique. According to the research of  Akande et al. 
(2018), the earthworms were measured to be 6–8 cm 
long and 0.2–0.3 cm wide. When the earthworms were 
collected, they were alive and moving. Following the 



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Am. J. Life Sci. Innov. 4(2) 1-5, 2025

confirmation of  the roundworm species, the E. eugeniae 
worms were cleaned of  dirt and debris using a standard 
saline solution.

Preparation of  A. marinaA. marina Extract
Following collecting, the leaves were carefully cleaned 
with tap water and then rinsed with distilled water. The 
leaves were allowed to air dry at room temperature for 
an hour without exposure to sunlight in a screen bag. 
The leaves were allowed to air dry before being ground 
into a fine powder in a blender. One gram of  powdered 
leaves was mixed with five milliliters of  95% ethanol for 
a duration of  seventy-two hours in order to homogenize 
the leaves. After that, a muslin cloth was used to filter 
the homogenized leaves. The ethanol was then extracted 
from the filtrate using a rotary evaporator set to 45 °C. 
The filtrate was dried in an oven set to 45 °C in order 
to recover either the powdered or sticky form.To be 
sure there was no ethanol in the extract, a flame test 
was conducted. An anthelmintic test was performed on 
the dried, crude hydroethanolic extract against adult E. 
eugeniae. Ibrahim et al. (2022) modified this process and 
used it in their studies.

Antihelmintic Assay Preparation
The Kondepudi (2018) method was used to perform the 
anthelmintic assay. The ethanolic leaf  extract was made 
into a stock solution at varying doses (25, 50, and 75 mg/
mL). A positive control solution containing 25 mg/mL 
Mebendazole and a negative control solution consisting 
of  distilled water were made. Each disposable plastic 
petri dish with a perforated lid and a 5.9-inch diameter 
was filled with three adult E. eugeniae. Each treatment’s 
5 mL solution is used to soak them. In accordance with 
the experiment carried out by Bestari et al. (2020), the 
percentage vermicidal of  the helminth was measured at 
3-hour intervals for 12 hours.

Validity and Reliability
A certified medical technologist verified the study’s 

findings. To ensure the study’s reliability, three 
concentrations—25 mg/mL, 50 mg/mL, and 75 mg/
mL—were used in five replicates with three trials. To 
guarantee the precision of  the tests conducted, the 
devices were also appropriately calibrated.

Waste Disposal
The adult E. eugeniae samples, both dead and alive, were 
disposed of  appropriately by filling the petri plates with a 
10% hypochlorite solution and securing the arrangement 
with tape. To guarantee the organisms’ total demise, the 
sample was submerged for a whole day. After being put 
in the biohazard bag, the petri plates were disposed of  in 
the proper garbage receptacle.

Statistical Analysis
The mean and SD of  the results were displayed. Using 
varying quantities of  A.marina hydroethanolic leaf  
extract, the study compared the mean results of  vermicidal 
counts of  E. eugeniae taken every three hours for 12 hours 
using the Kruskal-Wallis test. Statistical significance was 
defined as a p-value of  0.05. To determine whether there 
were any notable variations or differences between the 
treatments, a post-hoc Fisher’s least significant difference 
(LSD) test was also employed.

RESULTS AND DISCUSSIONS
Table 1 demonstrates that A. marina hydroethanolic leaf  
extracts at 50 and 75 mg/mL demonstrated antihelmintic 
efficacy against E. eugeniae. Vermicidal activity (Va) showed 
p< 0.05 for E. eugeniae adults on the 3, 6, 9, and 12 hours 
using Kruskal-Wallis and LSD. The antihelmintic effects 
of  the 50 mg/mL and 75 mg/5mL doses of  A. marina 
extract did not significantly change from the 3 to the 12 
hour. There was no antihelmintic effect from the 25 mg/
ml. 50 mg/mL was the optimal dose and duration that 
demonstrated in vitro antihelmintic action for the three 
hours of  exposure (mean = 91.1; sd = 26.6), and it was 
noticeably superior to the positive control during that 
period (mean = 2.22; sd = 8.61).

Table 1: Anthelmintic activity of  A. marina hydroethanolic leaf  extract against E. eugeniae taken at different hours using 
different concentrations in terms of  % vermicidal activity(Va)
Treatments Time (hours)

3 6 9 12
%Va  %Va %Va %Va
m sd M sd m sd m Sd

25 mg/mL a8.89 15.4 a20a 24.6 a33.3a 30.9 a46.7a 35.2

50 mg/mL b91.1a 26.6 b93.3a 25.8 c95.6a 17.2 c97.8a 8.61
75 mg/mL b91.1 a 26.6 b93.3 a 25.8 c93.3 a 25.8 c100 a 0.0
Negative-Control (Distilled Water) a0.0 a 0.0 a0.0 a 0.0 a0.0 a 0.0 a15.6b 30.5
Positive-Control Mebendazole) 25 
mg/mL

a2.22 a 8.61 a6.67 a 13.8 b51.1 ab 39.6 b84.4ab 27.8

Note: letters before the numbers mean comparison with the treatments; while letter after number means comparison among the hours



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Discussions
Significant anthelmintic activity was demonstrated by the 
hydroethanolic leaf  extract of  A. marina at concentrations 
of  50 and 75 mg/mL in terms of  percent vermicidal. 
Significant anthelmintic action was demonstrated by 
concentrations of  50 mg/mL and 75 mg/mL in terms 
of  percent vermicidal, with the peak activity occurring 
after 12 hours. Our results suggest that the phytochemical 
compounds present in A. marina, including alkaloids, 
flavonoids, sugars, carotenoids, aliphatic alcohols, amino 
acids, hydrocarbons, fatty acids, phenolic compounds, 
tannins, saponins, and terpenes, may have contributed 
to the anthelmintic activity (Wilfred et al., 2023; Mughal 
et al., 2016). Greiffer et al. (2022) found that flavonoids 
and tannins particularly inhibit helminth proliferation and 
cause helminth mortality.
In addition to A. marina, several plants were reported 
to possess these chemicals, including L. leucocephala 
(Widaad et al., 2022). Because of  its physical similarity to 
diseased helminths, E. eugeniae was used in the study as a 
reference for creating synthetic anthelmintic treatments.
Notably, the strongest anthelmintic activity against E. 
eugeniae was obtained at concentrations of  50 mg/mL 
and 75 mg/mL. The results of  Kondepudi’s (2018) 
investigation are consistent with the observed trend, 
which shows that higher concentrations exhibit stronger 
anthelmintic activity. The highest level of  anthelmintic 
activity of  A. marina was similarly seen at a concentration 
of  75 mg/mL to P. posthuma.
As demonstrated by the notable impacts on mortality 
employing varying doses of  hydroethanolic leaf  extracts 
of  A. marina, the results gave a scientific foundation for 
the vermicidal actions of  A.marina against E. eugeniae. For 
future research on helminths or organisms that share a 
biological morphology with African nightcrawler worms, 
such as hookworms, Trichuris trichiura, and roundworms 
(Ascaris suum), a concentration of  50 mg/mL may be 
utilized as a reference.
Given the persistently high incidence of  helminth 
infections in the Philippines, the experimental findings 
are positive about the best use of  readily available plants 
with anthelmintic qualities. The results show that natural 
products are reliable and strong enough to serve as a 
pointer to other possible sources of  helminth infection 
treatment. This is especially true for higher concentration 
therapies. Furthermore, by placing more significance 
and focus on the product’s source, this may also have 
positive economic effects. Last but not least, our research 
may help address the ongoing concern about the spread 
of  drug-resistant parasites (Fissiha & Kinde, 2021) by 
introducing a novel antidote that significantly slows the 
development of  undesired resistance.
Only the hydroethanolic leaf  extract of  A. marina at 
concentrations of  25 mg/mL, 50 mg/mL, and 75 mg/
mL and the anthelmintic assay method produced these 
results. Different results can be obtained by using a 
different extractant, concentration, technique, or part of  
the plant.

CONCLUSION
Comparing the extract concentrations at 50 mg/mL and 
75 mg/mL to the negative control (distilled water), there 
was notable antihelmintic activity based on the percentage 
of  vermicidal activity, from 3 to 12 hours. On the 3rd 
hour, the optimal concentration and duration of  in vitro 
anthelmintic action were 50 mg/mL. On the 3rd hour, 
the extract doses of  50 mg/mL and 75 mg/mL exhibited 
similar vermicidal effectiveness to the positive control, 
Mebendazole. The results scientifically demonstrated the 
antihelmintic efficacy of  hydroethanolic leaf  extract of  A. 
marina against E. eugeniae and provided the foundation for 
testing against Ascaris, Trichuris trichiura, and hookworms. 
Since the study demonstrated the plant’s effectiveness 
against E. eugenia in vitro, it is advised that a study on in 
vitro human helminths be carried out utilizing the crude 
ethanolic extract of  A. marina leaves. The phytochemical 
of  A. marina leaves should be used to identify or isolate 
the particular components that give it its anthelmintic 
effects. 

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