Bioactivity Evaluation of Melaleuca cajuputi (Myrtales: Myrtaceae) Crude Extracts against Aedes Mosquito - UPM
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Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)
TROPICAL AGRICULTURAL SCIENCE
Journal homepage: http://www.pertanika.upm.edu.my/
Bioactivity Evaluation of Melaleuca cajuputi (Myrtales:
Myrtaceae) Crude Extracts against Aedes Mosquito
Azlinda Abu Bakar1,2
1
Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan
Department of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan
2
Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur
ABSTRACT
Melaleuca cajuputi crude extract in four different solvents viz dichloromethane, ethyl
acetate, hexane, and methanol were evaluated for their insecticidal properties against
Aedes aegypti and Aedes albopictus mosquito. Bioassay against larva and adult mosquito
was done following World Health Organization’s guidelines. Late 3rd and/or early 4th
instar of Aedes larvae were assayed for different concentrations ranging from 10 to 120
mg/L of M. cajuputi crude extract. Larvicidal effects were observed in dichloromethane,
hexane, and methanol. Dichloromethane gave the highest of mean mortality, against Ae.
aegypti (12.6 ± 0.98) and Ae. albopictus (10.2 ± 0.37) with LC50 of 104.8 mg/L and 106
mg/L, respectively. The adulticidal bioassay was tested against 3 - 5 days old of female
mosquitoes with the range concentrations from 0.04 to 6.21 mg/cm2. Amongst solvents
used, extracts of dichloromethane and hexane showed effects against the adult mosquito.
Extract in hexane gave 100% mortality against both Aedes with LC50 of 0.015 mg/cm2
(Ae. aegypti) and 0.022 mg/cm2 (Ae. albopictus). In conclusion, the extract of M. cajuputi
could be exploited in the development of
potential plant-based products in controlling
dengue Aedes vectors, particularly in the
ARTICLE INFO
adult mosquito.
Article history:
Received: 4 February 2020 Keywords: Aedes sp., bioactivity, crude extracts,
Accepted: 27 April 2020
Published: 28 August 2020
Melaleuca cajuputi, solvents polarity
E-mail address:
Current affiliation
azlindaab@usm.my School of Biological Sciences, Universiti Sains
Malaysia, 11800 Gelugor, Pulau Pinang
ISSN: 1511-3701 © Universiti Putra Malaysia Press
e-ISSN 2231-8542Azlinda Abu Bakar
INTRODUCTION screening of various plant extracts in
In Asia, the mosquito Aedes aegypti is a Malaysia, had shown that Melaleuca was
primary vector for dengue and chikungunya the most effective when tested against
(Sam & Abu Bakar, 2006). Meanwhile, its Aedes spp. larva in the laboratory (Bakar
related taxon Aedes albopictus has been et al., 2018). Nevertheless, evaluation of
recognized as a secondary vector for dengue the essential oils of the same plant had also
and serves as a vector competence in the shown its potential insecticidal effects as
maintenance of dengue, and chikungunya well (Abu Bakar et al., 2012; Bakar et al.,
virus in areas where Ae. aegypti is less 2019). According to Lowe’s report (as cited
abundant or absent (Effler et al., 2005; in De Monte et al., 2014, p. 63), there are
Grard et al., 2014; Li et al., 2012; Wong et many different extractive techniques and
al., 2013). The global widespread of vector approaches working together with various
mosquitoes such as Aedes in certain tropical methodologies and solvents to improve
and sub-tropical areas causes a major the recovery and, the pharmacological
outbreak of dengue and other mosquito- profile of their extract products. However,
borne disease-related illness (Rezza, 2014). as a result of the differences among the
Utilization of insecticides in the strategy extractive processes and methods, there
for disease outbreak control is undeniably is a discrepancy in the qualitative and
effective, due to cost-effective, immediate quantitative composition of the extracts
action, and high efficiency against a obtained from the same plant.
broad range of vectors. Unfortunately, the It is known that various solvents of
effectiveness is threatened by negative and different polarities would extract different
harmful side effects on human, non-target phenolic compounds from plants with a high
animals, and the environment have become degree of accuracy (Wong & Kitts, 2006).
apparent. Nevertheless, the development Furthermore, previous studies have shown
of resistance among vector populations that solvents with a high polarity such as
(Hamid et al., 2018) is the biggest threat methanol displayed high effectiveness
to the program’s efficacy. Thus, the urge as antioxidants (Altemimi et al., 2017).
and interest in searching less hazardous The objective of this present study was to
alternatives of vector/pest control from plant evaluate the in vitro bioactivity of Melaleuca
resources are therefore being renewed and cajuputi plant extracts derived from four
continues today. different solvents polarity viz. hexane (non-
To date, many potential plant species polar), dichloromethane (moderately polar),
with known insecticidal properties and ethyl acetate (polar), and methanol (polar)
phytochemicals which are rich with against larvae and adult of dengue vector
biodegradable active compounds are being mosquitoes, Ae. aegypti and Ae. albopictus.
screened and evaluated (Sharma et al., 2006).
For instance, a study on the bioactivity
304 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)The Effect of M. cajuputi Extracts against Aedes
MATERIALS AND METHODS dichloromethane, and methanol with the
Collection of Plant Specimens ratio of 1g sample to 10 mL solvent in a 10L
plastic container. The samples were shaken
The leaves of M. cajuputi were collected
and mixed vigorously and left to sit for 72
from Port Dickson, Negeri Sembilan area,
hrs. The extracts were filtered through glass
2° 31′ 21.1440″ N, 101° 47′ 46.6620″ E in
funnel with filter paper Whatman No.1.
Malaysia. Port Dickson is located 120 km
The extract was concentrated using rotary
towards the south from Kuala Lumpur 3°
evaporator type EYELA (N-1001S-WD,
8’ 27.0708’’ N and 101° 41’ 35.5452’’ E.
Japan) at 45°C for eight hrs. The residue
The voucher specimen was sent to Forest
obtained was kept in an amber glass vial
Research Institute of Malaysia (FRIM) in
to be used for subsequent bioassay testing.
Kepong, Selangor for species confirmation
and specimen deposited at the herbarium.
Larval Bioassay
Preparation of Mosquito The larval bioassay was following the
standard guidelines (World Health
Laboratory strain mosquito was obtained
Organization [WHO], 2005). Five (5)
from Vector Control Research Unit, School
different concentrations of extracts were
of Biological Sciences, Universiti Sains
prepared at 10, 50, 80, 100, and 120 mg/L.
Malaysia, Pulau Pinang in the form of
A 10 mL stock solution was prepared at a
eggs. The mosquito colony was cultured
concentration of 100,000 mg/L (100,000
and reared in the laboratory under the
ppm) and kept in a refrigerator at 4 - 5 °C.
optimized condition: relative humidity (RH)
Five replicates of 20 late third instar larvae
80% ± 5% and room temperature 28.5 °C
were used in each bioassay. The numbers
± 2 °C. During the maintenance period,
of dead larvae were counted after 24 hrs.
mosquitoes’ larvae were provided with
of exposure. Positive and negative control
prepared powdered food which contains
solutions were prepared by mixing 1mL
cat’s biscuit, powder milk, grounded (dried)
solvent in 199mL of distilled water and
cow liver, yeast, and vitamin B complex.
2 mL of acetone in 198 mL of distilled
The mosquito colony was continuously
water respectively. During the observation,
maintained throughout the study period.
food was not supplied to the larvae. The
lethal concentrations (LC50 and LC90) were
Preparation of the Crude Extracts
calculated by probit analysis (Finney, 1971).
The freshly collected leaves of M. cajuputi
were dried at room temperature (29 - 31°C) Adulticidal Bioassay
for 5 - 7 days. The dried leaves were
Bioassay of adulticide was performed as
grounded mechanically using a household
described in the WHO (2016) guideline.
blender. The grounded leaves were extracted
Five (5) different concentrations of 2.0 mL
with solvents viz: hexane, ethyl acetate,
plant extracts of 0.04, 0.08, 0.12, 2.48, and
Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) 305Azlinda Abu Bakar
6.21 mg/cm2 were applied homogeneously ANOVA. Results with the value of p ≤ 0.05
at the filter papers Whatman No 1 (12 x 15 were reported to be statistically significant.
cm). The control paper was treated with All data were analyzed and calculated using
2.0 mL acetone. The impregnated papers SPSS statistics software.
were dried at room temperature for 24 hrs
and kept (4 - 5°C) in an aluminum foil. RESULTS
Four replicates of twenty-five female (3 - 5 The insecticidal bioefficacy of the M.
days old, blood starved) mosquitoes were cajuputi crude extracts of dichloromethane,
aspirated from the mosquito cage into a ethyl acetate, hexane, and methanol were
plastic holding tube. The mosquitoes were tested at 10 mg/L, 50 mg/L, 80 mg/L, 100
allowed to acclimatize in the tube for 1 mg/L, and 120 mg/L against dengue vectors,
hr. and later were exposed to the treated Ae. aegypti and Ae. albopictus. Table 1
impregnated filter paper for 1 hr. At the end summarizes the bioactivity against larvae
of the 1 hr. exposure period, the mosquitoes and adults’ stage of Aedes vectors. From the
were transferred back to the holding tube results obtained, dichloromethane, hexane,
and kept for mortality observation for 24 hrs. and methanol showed some larvicidal
A pad of cotton wool soaked in 10% sugar effects when tested against Aedes larvae.
water was placed on the mesh-screen. The Meanwhile, adulticidal effects were only
number of moribund and dead mosquitoes observed in dichloromethane and methanol.
was recorded at intervals of 1, 5, 10, 15, 20, The bioassay test against larvae and adults
25, 30, 45-, and 60-minutes post-exposure. Aedes mosquito showed a significant
Any knocked down mosquitoes, were increase in the mortality percentage (%)
considered moribund and counted as dead. A with the increase of concentration.
mosquito was classified as dead or knocked In Table 2, the paired t-test and the one-
down if it is immobile or unable to stand or way analysis of variance (ANOVA) were
take off. analyzed in mean mortality of Aedes sp.
larvae, and solvents used. Statistical analysis
Statistical Analysis of one-way ANOVA revealed no significant
Percentage mortality that lies between 5% difference between and within groups
to 20% will be corrected using Abbott’s among solvents and Aedes sp. Meanwhile
formula (Abbot, 1925). Larvicidal and paired t-test between solvents and Aedes sp.
adulticidal effects were reported in median showed a significant difference (p ≤ 0.05) in
lethal concentration (LC 50) with a 95% hexane (p = 0.04) and methanol (p=0.003)
confidence interval subjected to a log probit solvent respectively. The highest larvicidal
analysis test. The comparative effectiveness activity was observed in dichloromethane
of crude extracts among different types of against Aedes sp. with the LC50 values of
solvents and Aedes mean mortality were 104.8 mg/L and 106.0 mg/L for Ae. aegypti
analyzed using paired t-test and one-way and Ae. albopictus at 24 hrs. respectively
306 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)The Effect of M. cajuputi Extracts against Aedes
Table 1
Bioactivity of Melaleuca cajuputi crude extracts against Ae. aegypti and Ae. albopictus
Solvents Larvicidal Adulticidal
Dichloromethane √ √
Ethyl acetate - -
Hexane √ √
Methanol √ -
Note. √ toxic effects
Table 2
Mean mortality (± SE) of M. cajuputi crude extracts against larvae of Ae. aegypti and Ae. albopictus
Aedes sp. Dose *Mean mortality ± SE
(mg/L) Dichloromethane 1
Hexane2 Methanol3
a
Ae. aegypti 10 1.20 ± 0.20 0 1.40 ± 0.25
50 3.40 ± 0.25 1.20 ± 0.20 1.60 ± 0.25
80 6.80 ± 0.37 4.20 ± 0.37 2.40 ± 0.40
100 9.80 ± 0.66 5.40 ± 0.51 2.80 ± 0.20
120 12.6 ± 0.98 6.60 ± 0.51 3.00 ± 0.32
Control** 0 0 0
b
Ae. albopictus 10 0 0 0
50 0 0 0
80 7.40 ± 0.25 0.60 ± 0.25 0
100 10.0 ± 0.32 1.00 ± 0.32 1.60 ± 0.25
120 10.2 ± 0.37 2.00 ± 0.32 2.00 ± 0.00
Control** 0 0 0
Note. *Mean value of five replicates Control** = acetone 0.1%
1, a,b
No significant difference 1, 2, 3, a
No significant difference
Significant difference p < 0.05
2, a, b 1, 2, 3, b
No significant difference
(p = 0.04)
(Table 3). Figures 1 and 2 show the highest albopictus are presented in Table 4. There
percentage mortality values of 63% in Ae. was a significant difference between hexane
aegypti and 51% in Ae. albopictus at 120 and dichloromethane in mean mortality of
mg/L of dichloromethane extract in M. Aedes spp. with the p-values of 0.007 for
cajuputi. Ae. aegypti and 0.003 for Ae. albopictus.
The results of the adulticidal activity However, no significant difference was
of hexane and dichloromethane extracts observed in mean mortality between Aedes
of M. cajuputi against Ae. aegypti and Ae. spp. in each solvent used, hexane and
Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) 307Azlinda Abu Bakar
Table 3
LC values of M. cajuputi crude extracts against Aedes sp. larvae
Ae. aegypti Ae. albopictus
Solvents LC50 95% df χ 2
LC50 95% df χ2
(mg/L) Confidence (mg/L) Confidence
Limit Limit
LCL UCL LCL UCL
Dichloromethane 104.8 94.9 117.8 2 0.43 106.0 N/A N/A 2 15.9
Hexane 164.4 132.1 260.3 2 0.71 429.0 N/A N/A 2 1.60
Methanol 349.5 N/A N/A 2 0.19 N/A N/A N/A N/A N/A
Note. LCL = lower confidence limit; UCL = upper confidence limit; df = degree of freedom
80
70 63
60
50
50
40 34
Mortality (%)
33
21 27
30
17
20 14 15
12
6 7 6 8
10
0
0
-10 10 50 80 100 120
-20
Hexane
Dose Concentration (mg/L) Dichloromethane
Methanol
Figure 1. Percentage mortality of M. cajuputi crude extracts against Ae. aegypti larvae
70
60 51
50
50
37
40
Mortality (%)
30
20
8 10 10
10 3 5
0 0 0 0 0 0 0
0
0 50 80 100 120
-10
-20
Hexane
Dose Concentration (mg/L) Dichloromethane
Methanol
Figure 2. Percentage mortality of M. cajuputi crude extracts against Ae. albopictus larvae
308 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)The Effect of M. cajuputi Extracts against Aedes
dichloromethane. The probit analysis of extract against Ae. albopictus showed
95% confidence limits LC50 (UCL-LCL) minimal larvicidal effects after 24 hr.
was also calculated and presented in Table 5. of exposure at various concentrations.
The chi-square values were not significantly Meanwhile, observation in adulticidal
different at p ≤ 0.05. Among the solvents assays using dichloromethane, ethyl
used, hexane gives lower LC50 of 0.015 mg/ acetate, hexane, and methanol showed
cm2 (0.005-0.025) and 0.022 mg/cm2 (0.009- nonconformity with the results of the
0.003) in Ae. aegypti and Ae. albopictus, larvicidal assays. Of these, dichloromethane
respectively. and hexane extracts of M. cajuputi showed
In this study, the results showed that adulticidal effects against Ae. aegypti and
bioactivity of M. cajuputi crude extracts Ae. albopictus. However, hexane extract was
against Aedes spp. was varied according the most effective against Ae. aegypti and
to the species, stage of life, and solvents Ae. albopictus adult’s mosquito. From Table
used (Tables 1, 2, and 4). The extract of 4, more than 50% mortality was observed in
M. cajuputi in dichloromethane possessed Ae. aegypti (74%) and Ae. albopictus (69%)
moderate effects against Ae. aegypti at lowest concentration of 0.04 mg/cm2 and
larvae. On the other hand, the sensitivity 100% mortality when exposed at higher
of dichloromethane, hexane, and methanol concentrations of 2.48 and 6.21 mg/cm2.
Table 4
Adulticidal effects of M. cajuputi crude extracts against Aedes aegypti and Aedes albopictus
Solvents Dose 1
Ae. aegypti 2
Ae. albopictus
Used (mg/cm2) Mortality *Mean mortality Mortality *Mean mortality
(%) ± SD (%) ± SD
a
Hexane 0.04 74 18.50 ± 1.29 69 17.25 ± 1.26
N=100 0.08 77 19.25 ± 0.96 71 17.75 ± 1.89
0.12 79 19.75 ± 0.96 89 22.25 ± 3.30
2.48 100 25.00 ± 0.00 100 25.00 ± 0.00
6.21 100 25.00 ± 0.00 100 25.00 ± 0.00
**Control 0 0 0 0
b
Dichloromethane 0.04 31 7.75 ± 1.15 54 13.50 ± 3.00
N=100 0.08 50 12.50 ± 3.00 56 14.00 ± 2.16
0.12 55 13.75 ± 0.96 57 16.75 ± 2.50
2.48 80 20.00 ± 1.83 80 20.00 ± 0.00
6.21 87 19.25 ± 6.29 81 20.20 ± 0.5
**Control 0 0 0 0
Note. *Mean value of four replicates **Control=acetone 0.1%
1, 2, a
No significant difference p > 0.05 1, a, b
Significant difference p ≤ 0.05 (p = 0.007)
1, 2, b
No significant difference p > 0.05 2, a, b
Significant difference p ≤ 0.05 (p = 0.003)
Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020) 309Azlinda Abu Bakar
Table 5
LC values of M. cajuputi crude extracts against Aedes sp. adults
Ae. aegypti Ae. albopictus
Solvents LC50 95% df χ 2
LC50 95% df χ2
(mg/L) Confidence (mg/L) Confidence
Limit Limit
LCL UCL LCL UCL
Dichloromethane 0.116 0.071 0.176 3 3.73 0.030 0.007 0.069 3 0.83
Hexane 0.015 0.005 0.025 3 4.57 0.022 0.009 0.033 3 5.86
Note. LCL = lower confidence limit; UCL = upper confidence limit; df = degree of freedom
DISCUSSION years ago (Walling, 1957). Most chemical
Many researchers have reported the potential reactions that are carried out in laboratories
of plant extracts for controlling mosquito- or the industry are in the form of solutions.
borne diseases (Ghosh et al., 2012; Kamaraj Hence the proper and appropriate solvent
et al., 2010; Rehimi et al., 2011). Up to date, selection as one of the reaction parameters is
there are now more than 2,000 potential important for a good and successful reaction
plant species that have been evaluated for (Reichardt, 2005). According to Rawani et
their insecticidal properties worldwide al. (2010), phytochemicals found in plants
(Maiza et al., 2013; Roark, 1947; Shaalan may play an important role (bioactivities) in
et al., 2005; Sukumar et al., 1991). In some vector control if applied appropriately. The
developing countries, pesticidal plants phytochemicals in plants can be obtained
offer unique and challenging opportunities from the whole plant or specific parts of
for the exploration and development of the plant with different solvents such as
their botanical sources. Furthermore, one petroleum ether, benzene, chlorophyll,
of the most important factors affecting the methanol, and acetone.
benefits and efficiency of bioactivity from Ghosh et al. (2012) showed that the
plant materials and their consequent health extraction of active biochemical from plants
is the extraction solvents used (Ngo et al., depended upon the polarity of the solvents.
2017). Thus, the selection of solvents used Polar solvents will extract polar molecules
depends on the purpose either as of choice and non-polar solvents extract non-polar
for yielding high content or for specific molecules. In this study, hexane (polarity
extraction of phytochemical compounds index of 0.1), dichloromethane (polarity
that would be useful for its medicinal and/ index of 3.1), ethyl acetate (polarity index
or insecticidal properties. of 4.4), and methanol (polarity index
The polarity effect depends on the of 5.1) (Corradini et al., 1998; Harris,
reactivity and selectivity of radical 2015) had been used to investigate the
chemistry that has been identified over 50 insecticidal properties of M. cajuputi extract
310 Pertanika J. Trop. Agric. Sci. 43 (3): 303 - 313 (2020)The Effect of M. cajuputi Extracts against Aedes
against Ae. aegypti and Ae. albopictus. in different solvents used can be carried out
From the results obtained, hexane and to characterize the insecticidal properties in
dichloromethane solvents that had lower different research settings. It can be used as a
to moderate polarity index were observed solution of variants efficacy in a situation of
to give moderate effects against Aedes chemical instability of whole or unprocessed
mosquito. These findings agree with the plant products.
previous study by Ghosh et al. (2012), which
described the efficacy of solvents polarity ACKNOWLEDGEMENTS
in the bioassays. Biochemical extracted The author is thankful to the Universiti Sains
using moderate polarity index solvents Malaysia and Ministry of Higher Education
showed good results in a few bioassays. for the scholarship program. The gratitude
This study revealed the bioactivity variance is also extended to Dr. Sallehudin Sulaiman
of M. cajuputi crude extracts when tested and Dr. Rasadah Mat Ali for their guidance
against larvae and adults of Aedes sp. Even and advice during the research, and for
though larvicidal effects were observed in providing the laboratory facility at the Forest
dichloromethane, hexane, and methanol Research Institute Malaysia. This research
extracts of M. cajuputi, the effectiveness did not receive any specific grant from
was slightly weak. However, the adulticidal funding agencies in the public, commercial,
activity showed good effects against Ae. or not-for-profit sectors.
aegypti and Ae. albopictus.
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