Antiparasitic Activity of Methanolic and Ethyl acetate Extracts of Azadirachta indica against Haemonchus contortus

Abstract

Azadirachta indica (Neem) is an important medicinal plant containing diverse antimicrobial and antiparasitic activities. In the current scenario of emerging drug resistance, there is need of plant derived compounds having antiparasitic activity to counter infectious problem including helminths. Present research was conducted to evaluate the phytochemical composition, ovicidal and adulticidal activity of methanolic and ethyl acetate extracts of Azadirachta indica (Neem). The leaves of Neem were used for preparation of extracts and their composition was determined through high performance liquid chromatography (HPLC). Results showed that the methanolic extract had higher quercetin compounds than the ethyl acetate extract. The effect of these extracts on egg hatch and larval motility were tested in in vitro experiment. The results indicated that these were found effective to reduce the hatchability of eggs. Hatching percentage was 1.12% ± 1.01 in case of methanolic extract and 3.57% ± 0.97 in case of ethyl acetate extract. Ethyl acetate extract of A. indica killed all the worms at the concentration of 25 mg/mL at 6 hrs post exposure while methanolic extract showed 100 percent mortality at 10 hrs post exposure of same dose. Results were in dose-dependent manner. Ethyl acetate extract killed adult worms faster and at lower dose than methanolic extract. However, the methanolic extract of Neem had higher effectiveness than the ethyl acetate extract in preventing hatching of eggs. This research concludes that extraction solvent greatly affects the phytochemical composition and anthelmintic activities of Neem and this is effective to control the helminths.

Full text

199
Pakistan Veterinary Journal
ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE)
DOI: 10.29261/pakvetj/2023.014
Antiparasitic Activity of Methanolic and Ethyl acetate Extracts of Azadirachta indica against
Haemonchus contortus
Tauseef ur Rehman1*, Ahmed A. El-Mansi2,3, Sadeq K. Alhag4, Laila A. Al-Shuraym5, Zohaib Saeed6, Muhammad Arif7,
Muhammad Rashid1, Zeeshan Ahmad Bhutta8 and Muhammad Arfan Zaman9
.
1Department of Parasitology, The Islamia University of Bahawalpur, Pakistan; 2Biology Department, Faculty of Science,
King Khalid University, Abha, Saudi Arabia; 3Zoology Department, Faculty of Science, Mansoura University, Mansoura,
Egypt; 4Biology Department, College of Science and Arts, King Khalid University, Muhayl Asser, Saudi Arabia;
5Biology Department, Faculty of Science, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia;
6Department of Parasitology, University of Agriculture Faisalabad, 38040, Pakistan; 7Veterinary Officer, Livestock
production research institute, Bahadurnagar, Okara, Pakistan; 8Laboratory of Biochemistry and Immunology, College of
Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea; 9Department of
Parasitology, College of Veterinary and Animal Sciences, Jhang, Pakistan
*Corresponding author: drtauseef@iub.edu.pk
ARTICLE HISTORY (23-001)
A BS TR A C T
Received:
Revised:
Accepted:
Published online:
January 03, 2023
February 10, 2023
February 12, 2023
February 15, 2023
Azadirachta indica (Neem) is an important medicinal plant containing diverse
antimicrobial and antiparasitic activities. In the current scenario of emerging drug
resistance, there is need of plant derived compounds having antiparasitic activity to
counter infectious problem including helminths. Present research was conducted to
evaluate the phytochemical composition, ovicidal and adulticidal activity of
methanolic and ethyl acetate extracts of Azadirachta indica (Neem). The leaves of
Neem were used for preparation of extracts and their composition was determined
through high performance liquid chromatography (HPLC). Results showed that the
methanolic extract had higher quercetin compounds than the ethyl acetate extract.
The effect of these extracts on egg hatch and larval motility were tested in in vitro
experiment. The results indicated that these were found effective to reduce the
hatchability of eggs. Hatching percentage was 1.12% ± 1.01 in case of methanolic
extract and 3.57% ± 0.97 in case of ethyl acetate extract. Ethyl acetate extract of A.
indica killed all the worms at the concentration of 25 mg/mL at 6 hrs post exposure
while methanolic extract showed 100 percent mortality at 10 hrs post exposure of
same dose. Results were in dose-dependent manner. Ethyl acetate extract killed adult
worms faster and at lower dose than methanolic extract. However, the methanolic
extract of Neem had higher effectiveness than the ethyl acetate extract in preventing
hatching of eggs. This research concludes that extraction solvent greatly affects the
phytochemical composition and anthelmintic activities of Neem and this is effective
to control the helminths.
Key words:
Anthelmintic
Azadirachta indica
Ethyl acetate
Methanolic
Neem
To Cite This Article: Rehman TU, El-Mansi AA, Alhag SK, Al-Shuraym LA, Saeed Z, Arif M, Rashid M, Bhutta ZA
and Zaman MA, 2023. Antiparasitic activity of methanolic and ethyl acetate extracts of Azadirachta indica against
Haemonchus contortus. 43(1): 199-203. http://dx.doi.org/10.29261/pakvetj/2023.014
INTRODUCTION
Plants and plant compounds had been widely used for
the control of many diseases since ancient times (Chandran
et al., 2020; Hussain et al., 2021). Phytochemicals present
in plants enable them to control various diseases (Ghildiyal
et al., 2020). Parasites, especially helminths, are among the
major constraints in profitable livestock farming (Zaman et
al., 2014). They cause serious illness which may lead to
death (Rehman et al., 2016; Akram et al., 2019; Shaukat et
al., 2019; Strbac et al., 2020). Haemonchus is among
harmful parasites which are aggressive blood feeders of
ruminants (Cottingham et al., 2022). It causes anemia,
gastric problems and may even lead to death in ruminants
(Gareh et al., 2021). Development of resistance against
chemical drugs is compelling the researchers to find out
alternatives for the control of these parasites (Jimenez
Castro et al., 2019). Vaccines are being practiced but they
have limited prevention and unsure protection against the
helminths including Haemonchus (Ehsan et al., 2020). The
RESEARCH ARTICLE

Pak Vet J, 2023, 43(1): 199-203.
200
researchers are trying to find alternatives of these drugs.
Plants and plant derived compounds are attracting the
scientists due to high proportion of bioactive compounds,
safe mode of actions, and economic concern (Yasmin et al.,
2020; Jamil et al., 2022). Most of ancient and modern
medicinal compounds are still derived from the plants of
medical importance.
Azadirachta indica (Neem) is a well-known plant
having great medicinal and therapeutic effects (Mahmood
et al., 2018). Biologically active metabolites of Neem have
been proven to be effective in controlling multiple
infectious agents including bacteria, fungi, helminths, and
insects in various experiments (Tembe-Fokunang et al.,
2019). Multiple preparations of A. indica have been used
to control various diseases but the effective preparation is
still being researched (Srivastava et al., 2020). Various
extracts are being implemented in the research to
investigate a proper formulation but, still effective one
needs to be researched (Latif et al., 2020; Reddy and
Neelima, 2022).
Extracts are derived from the plants to obtain the
fractions which have high concentrations of bioactive
compounds (Lefebvre et al., 2021). Various solvents are
used for the extraction of phytochemicals from the plants,
so that the diversity of compounds may be achieved.
Research experiments suggested that the extraction
solvents have great effect on phytochemical composition of
the plants. Variation in solvents resulted in variety of
composition of the plants constituents extracted (Nobossé
et al., 2018; Nawaz et al., 2020).
This research trial was conducted to assess the
difference of phytochemical composition and in vitro
anthelmintic activity of the methanolic and ethyl acetate
extracts of the A. indica. In this experiment various
concentrations of the extracts were used to analyze the
effects of the A. indica on reduction in egg hatching and
percent worm motility of Haemonchus.
MATERIALS AND METHODS
Plants and Preparation of extracts: Fresh leaves of A.
indica were collected and dried in shade. Leaves were then
ground into powder. This powder was mixed in sufficient
quantity of methanol and ethyl acetate. stirring was after
three days of suspension, solutions were filtered through
muslin cloth. This activity was repeated twice. Three
filtrates were then mixed and put into the rotary evaporator
for the preparation of methanolic and ethyl acetate extracts
of the leaves. Mondal et al. (2019) method was followed
for the preparation of extracts. The prepared extracts were
dried and stored at 4°C for experimental use.
Phytochemical analyses: Ethanolic and ethyl acetate
extracts were subjected to high performance liquid
chromatography (HPLC) for estimation of their
phytochemical constituents. CSW32-Chromatography
station was used, and the graphs were developed using the
Data Apex ® 2001 software. Shim-Pak CLC-ODS (C-18),
250mm x 46cm, 5um columns were used for the
chromatography. Flow rates were adjusted @ 1 mL/minute
in an ultraviolet-visible detector at a wavelength of 280nm.
Egg hatch assay: Fresh eggs were collected from female
worms, washed and strained through sieve. The filtered
eggs were counted and divided into 8 groups each having 3
replicates with 100 eggs in each replicate. The first 4
groups were administered the methanolic extracts of Neem
@ 25, 12.5, 6.25 and 3.125 mg/mL respectively while
groups 5-8 were subjected to ethyl acetate extract of Neem
@ 25, 12.5, 6.25 and 3.125 mg/mL respectively. Positive
control group contains Oxfendazole. The negative control
was given phosphate buffer saline. All the procedures were
done according to Coles et al. (1992).
Adult motility assay: Adult, living and motile female
worms of H. contortus were collected from abomasum of
the slaughtered sheep. They were washed and divided into
8 groups each having 3 replicates with 10 worms in each
replicate. The experimental design was the same as egg
hatch test. Motility of worms was observed and recorded
after 2hrs interval. The procedure was followed as Rehman
et al. (2021).
Statistical analyses: All the values were recorded, and
percent eggs hatched and percent reduction in worm
motility were calculated using Microsoft Excel 365®.
Means were compared through Tuckey test via Minitab®
26. The significance level was adjusted to 5% (P<0.05).
RESULTS
Phytochemical analysis: High-Performance Liquid
Chromatography (HPLC) method of the methanolic and
ethyl acetate extracts was performed in the similar
conditions. The results showed that the methanolic extract
had the higher Quercetins than the Ethyl acetate extract
(Fig. 1, 2; Table 1, 2).
Fig. 1: HPLC diagram of methanolic extract of Neem.
Fig. 2: HPLC diagram of ethyl extract of Neem.

Pak Vet J, 2023, 43(1): 199-203.
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Egg hatch assay: The effect of the various concentrations
of Neem was determined against the hatchability of eggs of
H. contortus. The results showed that both the extracts were
effective in dose dependent manner (Table 3). Highest dose
(25mg/mL) of methanolic and ethyl acetate extract allowed
hatching of only 1.12 and 3.57% of eggs. Methanolic
extract is more effective in inhibiting egg hatching as
compared to ethyl acetate extract.
Adult motility assay: Adult motility assays were
performed against the adult female H. contortus, and the
results are given in the Table 4 and Table 5. Ethyl acetate
extract with dose of 25 mg/mL killed/ inhibited motility of
all adult worms at 6hrs after administration of dose while
methanolic extract caused 100 percent mortality of adult
worms after 10hrs of administration of same dose. Ehtyl
acetate extract at dose of 3.125mg/mL killed 100 percent
worms 8hrs post-treatment. Ehtyl acetate extract proved to
be better in killing adult worms in comparison to
methanolic extract.
Table 1: Phytochemical composition of methanolic extract of Neem
Sr.
No
Retention
time
Area
(mV. s)
Compounds
Concentration
(ppm)
1.
2.853
994.969
Quercetin
52.73
2.
4.880
118.196
Gallic Acid
4.255
3.
15.920
2.791
Chlorogenic acid
0.217
4.
20.253
4.656
M-Coumaric Acid
0.05
5.
24.580
411/707
Cinnamic Acid
45.19
Table 2: Phytochemical composition of ethyl acetate extract of Neem
Sr.
No
Retention
time
Area
(mV. s)
Compounds
Concentration
(ppm)
1.
2.660
196.754
Quercetin
10.42
2.
4.213
90.158
Gallic Acid
3.24
3.
13.200
5.961
Vanillic Acid
0.36
4.
14.373
3.181
Benzoic Acid
0.33
5.
15.280
78.562
Chlorogenic acid
0.30
6.
17.200
109..434
P-Coumaric Acid
0.12
Table 3: Effect of various concentrations of methanolic and
ethyl acetate extract of Neem on percent egg hatchability of H.
contortus
Concentrations
Percent (%) Eggs Hatched
Methanolic extract
Ethyl Acetate extract
25 mg/mL
1.12±1.01d
3.57±0.97de
12.5 mg/mL
8.25±3.8cd
12.11±4.73d
6.25mg/mL
22.09±5.47c
31.02±5.03c
3.125mg/mL
39.47±10.12b
55.06±4.39b
Positive Control
0.49±0.49d
0.49±0.49e
Negative control
96.17±2.47a
96.17±2.47a
Difference among means with different superscript is statistically
significant (P<0.05).
DISCUSSION
In this study, the effect of extraction solution on
phytochemical properties and in vitro anthelmintic activity
is evaluated. The result of this study showed that there is
great variation in the composition of methanolic and ethyl
acetate extracts of Neem. The methanolic extract of Neem
had a high proportion of active constituents and higher
Quercetin components. Ethyl acetate extract had the less
amount of active phenolics and flavonoids in it. The
methanolic extract of Neem had higher efficiencies in both
in vitro trials. Both the extracts were significantly effective
to control the egg hatchability and worm motility at 25
mg/mL concentration. The results were in dose dependent
manner. Multiple researchers have conducted similar
studies using plant extracts for in vitro efficacy against H.
contortus (Elandalousi et al., 2013; Politi et al., 2018;
Orengo et al., 2022).
The results of our study are in line with the results of
Alowanou et al., (2019) who mentioned that the herbal
extract of Bridelia ferruginea, Mitragyna inermis and
Combretum glutinosum plants were effective to reduce
worm motility and egg hatchability of the H. contortus in
in vitro environment. Similar results have been obtained by
Rehman et al. (2021), who conducted a research
experiment to control the worm motility and egg
hatchability of the H. contortus in in vitro environment
using an extract of Citrullus colocynthis. Kuiseu et al.
(2022) conducted similar research using the herbal extract
of Adansonia digitata and Anogiessus leiocarpus. The
results of their study suggested that the herbal extracts of
Adansonia digitata and Anogiessus leiocarpus were
effective against helminths. Many other studies are also in
line with the results of current study (Sisay et al., 2021;
Sebai et al., 2021).
The anthelmintic efficacy of herbals has been proven
and it is linked to phenolics, and flavonoids present in the
plants (Lima et al., 2021). The phenolics like Quercetin,
Gallic acid, and Benzoic acids etc. have known biomedical
activities (Salem et al., 2021). They are capable of inducing
apoptosis and cell death in the helminths (Mahmoudvand
et al., 2022). They can cause cell-cycle arrest (Liu et al.,
2022) i.e., lead to disturbing growth and differentiation of
the worms. They are also supposed to be involved in the
energy uptake mechanisms of the worm which leads to
their death (Adak and Kumar, 2022). Although exact
mechanisms of action are under investigation, methanolic
and ethyl acetate extracts of Neem contained high fractions
of Quercetin, and phenolic acids, these may be responsible
for the anthelmintic activities of Neem. Although,
methanolic extract had the higher proportion of compounds
but the ethyl acetate had a rich fraction of these compounds.
It can be Justified that these compounds were the reason of
anthelmintic properties of Neem (Rahaman et al., 2022).
Differences in the activity of methanolic and ethyl
acetate extracts of Neem can be attributed to differences in
the amount and type of phenolics in the phytochemical
composition of both extracts. Multiple scientists have
suggested that the variation in the composition is a primary
factor in bioactivities (Ma et al., 2021). Due to these
variations, they have differences in their effect on egg hatch
and worm motility of H. contortus.
Table 4: Effect of various concentrations of methanolic extract of Neem on the percent reduction in motility of female worms
Treatment groups
Times (hours)
0
2
4
6
8
10
12
25 mg/mL
0.0±0.0l
6.66±5.77jk
26.66±5.77hi
50±17.32e
96.66±5.77b
100.0±0.0a
100.0±0.0a
12.5 mg/mL
0.0±0.0l
10.0±10.0j
30.0±10.0h
46.66±15.27f
73.33±11.54d
100.0±0.0a
100.0±0.0a
6.25 mg/mL
0.0±0.0l
3.33±5.77k
23.33±5.77i
36.66±5.77g
73.33±5.77d
100.0±0.0a
100.0±0.0a
3.125 mg/mL
0.0±0.0l
0.0±0.0l
6.66±5.77 jk
20.0±10.0i
46.66±15.27f
80.0±10.0c
100.0±0.0a
Positive Control
100.0±0.0a
100.0±0.0a
100.0±0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
Negative control
0.0±0.0l
0.0±0.0l
0.0±0.0l
0.0±0.0l
0.0±0.0l
0.0±0.0l
0.0±0.0l
Values carrying different superscript letters have statistically significant difference (P<0.05).

Pak Vet J, 2023, 43(1): 199-203.
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Table 5: Effect of various concentrations of ethyl acetate extract of Neem on the percent reduction in motility of female worms
Treatment groups
Times (hours)
0
2
4
6
8
10
12
25 mg/mL
0.0±0.0i
33.33±5.77f
70.0±5.77c
100.0±5.77a
100.0±0.0a
100.0±0.0a
100.0±0.0a
12.5 mg/mL
0.0±0.0i
23.33±5.77g
60.0±7.32d
90.0±10.0b
100.0±0.0a
100.0±0.0a
100.0±0.0a
6.25 mg/mL
0.0±0.0i
23.33±5.77g
43.33±15.27e
90.0±7.32b
100.0±0.0a
100.0±0.0a
100.0±0.0a
3.125 mg/mL
0.0±0.0i
13.33±5.77h
56.66±5.77d
90.0±10.0b
100.0±0.0a
100.0±0.0a
100.0±0.0a
Positive Control
100.0±0.0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
100.0±0.0a
Negative control
0.0±0.0i
0.0±0.0i
0.0±0.0i
0.0±0.0i
0.0±0.0i
0.0±0.0i
0.0±0.0i
Values carrying different superscript letters have statistically significant difference (P<0.05).
Conclusions: Present research concludes that Neem has a
variety of multiple bioactive compounds. The method of
extraction has a great impact on the type, amount, and
diversity of these compounds. Depending upon these
variations, the biomedical activity like anthelmintic
activity is varied. This research shows that the Neem has in
vitro anthelmintic activities and recommends that further
trials should be conducted to evaluate in vivo biomedical
activities.
Acknowledgements: Princess Nourah bint Abdulrahman
University Researchers Supporting Project number
(PNURSP2023R365), Princess Nourah bint Abdulrahman
University, Riyadh, Saudi Arabia. Also, authors extend
their appreciation to the Deanship of Scientific Research of
King Khalid University, Abha, Saudi Arabia for funding
this work with a grant number (R.G.P.1/29/43).
Authors contribution: TR, MA, AAEM, SKA, LAAS,
designed study plan; TR, ZS, MR performed in vitro tests;
ZAB, AAEM, SKA, LAAS, MA, MAZ performed
statistical analyses.
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