Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

S167

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

doi: 10.1016/S1995-7645(14)60225-X

Document heading

Larvicidal

activity of Persea americana Mill. against Aedes aegypti

1,2*

Rosalinda C. Torres

, Alicia G. Garbo1, Rikkamae Zinca Marie L. Walde2

Industrial Technology Development Institute, Department of Science and Technology, General Santos Ave., Bicutan, Taguig City 1631, Philippines

National Research Council of the Philippines, Department of Science and Technology, General Santos Ave., Bicutan, Taguig City 1631, Philippines

ARTICLE INFO

ABSTRACT

Article history:
Received 23 Jun 2014
Received in revised form 6 Jul 2014
Accepted 19 Jul 2014
Available online 20 Aug 2014

Objective: To evaluate the toxicity of the ethanol and hexane extracts of the different parts of
Persea americana Mill. (P. americana) toward third and fourth instars larvae of Aedes aegypti (Ae.
aegypti) and to characterize the ethanol extract by qualitative phytochemical analysis.
Methods: The seeds, peels and pulp of P. americana were processed for crude extraction using 95%
ethanol and n-hexane. Crude extracts were bio-assayed for larvicidal activity against Ae. aegypti
following the World Health Organization standard bioassay method. The mortality was observed
at 24 h and 48 h after treatment and data were subjected to probit analysis to determine lethal
concentrations (LC50 and LC90). The ethanol extract was characterized by phytochemical analysis.
Results: Both the hexane and ethanol extracts from the different parts of P. americana exhibited
evidence of larvicidal toxicity. The hexane extract from the seeds exhibited the highest toxicity
with LC50 and LC90 values of 9.82 mg/L and 22.19 mg/L, respectively, while the ethanol seed extract
exhibited LC50 of 16.48 mg/L and LC90 45.77 mg/L, respectively. This was closely followed by the
ethanol extract of the peels with an LC50 of 10.35 mg/L and LC90 of 26.29 mg/L. The pulp extracted
with ethanol also yielded great larvicidal toxicity with LC50 of 21.32 mg/L and LC90 of 59.45 mg/L.
Results of the phytochemical analysis of the ethanol seed extract indicated presence of alkaloids,
tannins, saponins, unsaturated steroids and triterpenoids, flavonoids (leucoanthocyanins), fats and
oils.
Conclusions: Both the hexane and ethanol extracts of P. americana showed promising potential
as an alternative source of a more sustainable, non-toxic and environmentally friendly solution
for the control of dengue vector, Ae. aegypti.

Keywords:

Persea americana
Dengue vector
Aedes aegypti
Larvicidal bioassay
Phytochemical analysis

1. Introduction
M osquito-borne diseases contribute significantly to
disease burden, death, poverty, and social debility all
over the world, particularly in tropical countries[1]. Among
these diseases, dengue remains the most serious vectorborne disease in the Philippines. Dengue is a severe, often
fatal, most rapidly emerging febrile disease transmitted
by female Aedes aegypti (Ae. aegypti) mosquitoes. It is an
acute infection that kills much faster than AIDS. In the
Philippines, dengue fever and dengue hemorrhagic fever
are widespread in all its regions according to World Health
Organization. Treatment is mainly supportive as there is no
specific therapy available to date. These are the reasons
*Corresponding author: Rosalinda C. Torres, Ph.D., Chemicals and Energy Division,
Industrial Technology Development Institute, Department of Science and Technology,
General Santos Ave., Bicutan, Taguig City, Metro Manila 1631, Philippines.
Tel: (632) 837-20-71 loc. 2226
E-mail: lindactorres@yahoo.com
Foundation Project: Supported by the National Research Council of the Philippines of
the Department of Science and Technology, Taguig City, Metro Manila 1631, Philippines.

behind the efforts of many institution and research bodies

to find a solution in controlling the vector of dengue, Ae.
aegypti. Chemical control is said to be an effective way
used generally in daily life[2], but alarms environmental
and human advocates since widespread use of synthetic
insecticides has led to many negative consequences[3].
This results in the increasing attention and consideration
to natural products [4]. G reen larvicides are now being
considered because plants consist of several bioactive
components that are of low toxicity and biodegradable.
Unlike the conventional insecticide which is based on
single active ingredient, green insecticides comprise
botanical blends of bioactive chemical compounds which
act concertedly on both behavioural and physiological
processes[5]. The decades of research about interactions
between plants and insects revealed the potential use of
plants in fundamental pest control programs[6].
Persea americana M ill. ( P. americana ) from the
L auraceae family is commonly known as avocado and
alligator pear. It is a much more branched, medium-sized
tree with shiny evergreen, elliptic leaves and delicious

Rosalinda C. Torres et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

fruit[7]. Originally, it was only native to humid tropical

areas of Mexico until it was later cultivated to other regions
of Latin America as well as to USA and Europe. Today, the
fruits are extensively cultivated worldwide on large scale
in various subtropical countries such as the Philippines[8].
O ur main focus is now on the research study of P.
americana as a potential vector control measure against Ae.
aegypti that is acceptable to the populace, cost effective
and more importantly, safe for the environment. The use
of natural alternatives such as plant extracts can provide
the best option as environmentally safe natural larvicides.
Therefore, the objectives of the study were to evaluate the
toxicity of the ethanol and hexane extracts of the different
parts of P. americana Mill. against third and fourth instars
larvae of Ae. aegypti and to characterize the ethanol extract
by qualitative phytochemical analysis.
2. Materials and methods
2.1. Botanical material
T he fruits of P. americana were collected in seven
different localities in the Philippines including Davao,
M indoro, B atangas, N egros O riental, I loilo, P alawan,
and Cavite. The seeds, peels and pulp of the plant were
processed for crude extraction and larvicidal bioassay.
Specimen labels were placed in each plant. Herbarium
specimens of the plants collected were prepared and sent
to the National Museum of the Philippines for necessary
identification and authentication.

2.2. Sample preparation

The seeds, peels and pulp of P. americana were separated
and processed individually. Both fresh and dried plant
materials were used in the study. These were ground with
either Waring blender or Wiley mill. About 300 g of plant
materials were macerated with 900 m L of ethanol and
n-hexane for 48 h. Then, the mixture was filtered with a

coarse filter paper and the filtrate was concentrated under

vacuum at 55 C to a syrupy consistency. This was further
evaporated in a water bath at 55 C until completely dried.
2.3. Rearing of Ae. aegypti larvae

The eggs of Ae. aegypti were reared in the Entomology

L aboratory of the I ndustrial T echnology D evelopment
Institute, Department of Science and Technology, Taguig
City, Metro Manila 1631, Philippines. The egg rafts were kept

in the basin containing tap water that served as the culture

medium at (282) C and a photoperiod of 12 h light followed
by 12 h dark (12L: 12D). Appropriate amount of brewers yeast
were added to enhance the growth of the larvae. The third
and fourth instars larvae were used in the study.
2.4. Larvicidal bioassay against third and fourth instars
larvae of Ae. aegypti
The larvicidal bioassay was conducted following the World

Health Organization method[9]. Batches of 20 third or fourth

instars larvae were transferred by means of droppers to small

disposable test vessels, each containing 100 mL of water. The
appropriate volume of dilution was added to 100 mL water
in the cups to obtain the desired target dosage, starting with
the lowest concentration. The test containers were held at
(282) C and a photoperiod of 12 h light followed by 12 h
dark (12L: 12D). Four or more replicates were set up for each
concentration and an equal number of controls were set up
simultaneously with tap water, to which 1 mL ethanol (or the
organic solvent used) was added. Each test was run three
times on different days. The results were recorded where the
LC50 and LC90 values and slope and heterogeneity analysis
were also noted.
2.5. Data analysis
Data from all replicates were pooled for analysis. LC50
and LC90 values were calculated from a log dosage-probit
mortality regression line using computer software programs.
S tandard deviation of the means of LC 50 values were
calculated and recorded. A test series is valid if the relative
standard deviation (or coefficient of variation) is less than
25% or if confidence limits of LC50 overlap (significant level
at P<0.05).

2.6. Phytochemical analysis

The qualitative phytochemical analysis of the ethanol
extract of P. americana seeds was performed according to
the method of Guevara[10].

3. Results
Figures 1 to 4 show the lethal concentration expressed in
LC50 and LC90 values of crude ethanol and hexane extracts

of the seeds, peels and pulp of P. americana against third

and fourth instars larvae of Ae. aegypti. Figure 1 shows the
lethal concentration of the ethanol seed extracts collected
at different plant sites in the Philippines. Results showed
that the ethanol extract from Batangas yielded the most
potent toxicity effect with LC50 and LC90 values of 16.48 mg/
L and 45.77 mg/L, respectively. This was followed closely
by the ethanol extract from Davao with an LC50 of 18.21 mg/
L and LC90 of 41.38 mg/L, and the ethanol extract from Negros
Oriental with an LC50 value of 20.61 mg/L and LC90 of 44.39 mg/L,
respectively.
251

Lethal concentration (mg/L)

S168

201

151

101
51

Batangas
LC50

Davao Negros Oriental Mindoro

Sources
LC90

Palawan

Iloilo

Figure 1. Comparison of lethal concentration LC50 and LC90 of the ethanol

extracts of P. americana seeds from different sources.

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Rosalinda C. Torres et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

36
31
26
21
16
11
6
1

101
81
61
41
21
1

Davao
LC50

LC90

Sources

Palawan

Figure 4. Comparison of lethal concentration LC50 and LC90 of the hexane

extracts of P. americana peels from two sources.

LC50

LC90

Palawan
Sources

Cavite

Table 1 shows the results of the qualitative phytochemical

analysis of the ethanol extract of P. americana seeds.
T he presence of different phytochemicals in the seeds
of P. americana such as alkaloids, saponins, flavonoids
(leucoanthocyanins), unsaturated steroids and triterpenoids,
tannins, fats and oils may be attributed to its potent
toxicities against the dengue vector, Ae. aegypti.
Other parts of P. americana such as peels and pulp were
also extracted and tested for larvicidal activity. As shown
in Figure 3, the peels from Mindoro yielded the most potent
toxicity with LC50 of 10 .35 mg/L and LC90 of 26 .29 mg/L,
respectively.
101
81
61
41
21
1

LC50

Mindoro

LC90

Davao
Sources

Batangas

Figure 3. Comparison of lethal concentration LC50 and LC90 of the ethanol

extracts of P. americana peels from different sources.

As illustrated in Figure 4, the hexane extract of the P.

americana peels from Davao exhibited higher toxicity than
that from Palawan with an LC50 and LC90 values that ranged

As shown in Figure 5, the P. americana pulp from Davao

extracted with ethanol also had great larvicidal toxicity with
LC50 of 21.32 mg/L and LC90 of 59.45 mg/L. The pulp from
Mindoro extracted with hexane had an LC50 and LC90 values
that ranged from 32.77 mg/L to 46.09 mg/L (average=39.43
mg/L) and from 78.49 mg/L to 97.84 mg/L (average=88.17 mg/
L), respectively.
Lethal concentration (mg/L)

Davao

Figure 2. Comparison of lethal concentration LC50 and LC90 of the hexane

extracts of P. americana seeds from different sources.

Lethal concentration (mg/L)

from 19.87 mg/L to 22.80 mg/L (average=21.34 mg/L) and from

mg/L to 44.19 mg/L (average=43.02 mg/L), respectively.

41.84
Lethal concentration (mg/L)

Lethal concentration (mg/L)

A s shown in F igure 2 , the hexane extract from the

seeds of P. americana collected from the 3 sites, namely,
Davao, Palawan and Cavite exhibited very potent lethal
concentrations. The hexane extract from Davao yielded
the highest toxicity with LC50 and LC90 values of 9.82 mg/
L and 22.19 mg/L, respectively. This was followed by the
one collected in Palawan with LC50 of 13.39 mg/L and LC90 of
21.53 mg/L.

101
81
61
41
21
1

LC50

Davao (ethanol extract)

LC90

Mindoro (hexane extract)

Figure 5. Comparison of lethal concentration LC50 and LC90 of the extracts of

P. americana pulp from two sources.

4. Discussion
P eople have used various plant parts, products and
secondary metabolites of plant origin in pest control
since early historical times[5]. That is why several studies
have been dedicated on testing natural products against
mosquitoes specifically, Ae. aegypti, which produced
varying results [11-13] . S everal P hilippine plants have
been found to have various medicinal properties such as
larvicidal properties[14].
In the present study, hexane and ethanol extracts of the
seeds, peels and pulp of P. americana were subjected

Table 1
Result of the qualitative phytochemical analysis of the ethanol extract of P. americana seeds.
Qualitative tests
Mayers test
Dragendorffs test
Dragendorffs and Mayers test
Froth test
Na2CO3 test
Keller Killani test
Bate-Smith &Metcalf test
Willstatter Cyanidin test
FeCl3 test
Liebermann-Burchard test
Borntragers test
Spot test

Sources

Results
Formation of slightly turbid solution (+)
Formation of slightly turbid solution (+)
Formation of slightly turbid solution (+)
Formation of honeycomb froth greater than 2 cm (+)
Non-formation of dense and stable froth (-)
Formation of reddish-brown colored interface (+)
Formation of strong red colored solution (+)
Formation of red color (+)
Formation of blue-black colored solution (+)
Formation of red colored solution (+)
Non-formation of red colored solution (-)
Formation of greasy/oily appearance (+)

Indication
Presence of alkaloids
Presence of alkaloids
Absence of quaternary bases and/or amineoxides
Presence of saponins
Absence of free fatty acids
Presence of 2-deoxysugars
Presence of leucoanthocyanins
Presence of -benzopyrone nucleus (flavonoids)
Presence of hydrolyzable tannins
Presence of unsaturated steroid/triterpenoids
Absence of anthraquinones
Presence of fats and oils

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Rosalinda C. Torres et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S167-S170

to larvicidal toxicity assay. All the plant parts that were

studied showed great toxicities against Ae. aegypti,
especially the hexane extract from the seeds collected in
Davao with LC50 of 9.82 mg/L. One literature reported that
the hexane extract of P. americana seeds showed an LC50
8.87 mg/L, similar to the lethal concentration (9.82 mg/L)
previously reported in the present study[15]. The extracts
were considered to be bioactive since they showed lethal
concentrations (LC50 and LC90) extremely lower than 1 000 mg/
L[11]. Based from the results, P. americana from different
sources yielded varied results which can be attributed to
type of soil, the climate of the source and the season in
which the plant was collected. The solvent used can also
contribute to the variation since it has been shown that the
extraction of active biochemical from plants depends upon
the polarity of the solvents used[5].
P hytochemical analysis of the ethanol extract of P.
americana seeds showed presence of alkaloids, tannins,
saponins, unsaturated steroids and triterpenoids,
2-deoxysugars, flavonoids (leucoanthocyanins), fats and
oils. It is said that several groups of phytochemicals such as
alkaloids, steroids, terpenoids, essential oils and phenolics
from different plants have been reported previously for
their insecticidal activities[16].
P lants comprise a vast untapped pool of bioactive
phytochemicals that may be widely used in pest control
programs instead of the conventional synthetic insectides.
The remarkable toxicity effects exhibited by the seeds,
peels and pulp of P. americana against the third and
fourth instars larvae of Ae. aegypti indicate its potential
use as green or natural larvicides for the control of dengue
vector. Further study on the plant should therefore be fully
explored. It is worthwhile to study extensively the larvicidal
property by isolating and identifying the active components
that cause larval mortality and then use them in field trials
in order to assess their full potential as an alternative to
chemical larvicides.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
The National Research Council of the Philippines of the
Department of Science and Technology, Taguig City, Metro
Manila 1631, Philippines is gratefully acknowledged for
providing the research grant, and the Industrial Technology
Development Institute of the Department of Science and
Technology for the unwavering support in the continuous

implementation of this project.

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