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Lemon Peel (Citrus Limon) and Lagundi (Vitex Negundo) Extracts As Potential Mosquito Larvicide
Lemon Peel (Citrus Limon) and Lagundi (Vitex Negundo) Extracts As Potential Mosquito Larvicide
Lemon Peel (Citrus Limon) and Lagundi (Vitex Negundo) Extracts As Potential Mosquito Larvicide
An official entry
to the
Life Science
Team Category
Louis L. Uy
Researchers
Research Adviser
Co-Adviser
La Carlota City
I
Abstract
Mosquitos can transmit more diseases than any other group of anthropods and
affects millions of people throughout the world. WHO has declared the mosquitoes as
“public enemy number 1”. Mosquito borne diseases are prevalent in more than 100
countries across the world, infecting over 700,000,000 people every year globally. (World
Heatlh Organization, 2016). This study aims to determine the effect of Lemon peel (Citrus
limon) and Lagundi (Vitex negundo) extracts as potential mosquito larvicides. The Lemon
(C. limon) were bought at the Agora Public Market while the Lagundi (v. negundo) were
gathered from a farm located athda. Remedios, Brgy. Balabag. The Lemon (C. limon) and
Lagundi (V. negundo) were washed with running water then they were pounded using
mortar and pestle. There are 4 treatments in this study; Treatment A (Lemon peel extracts),
control). There are 4 mosquitoes in each replicate with the total of 12 mosquito larvae per
treatment. The One-Way Analysis Of Variance was used to determine the differences
among the 4 treatments respectively. Based on the results, Treatment A (lemon peel
extracts) were more effective than Treatment B (lagundi extracts) on the otherhand
Treatment C (positive control) were able to exterminate all mosquito larvae while
Treatment d (negatice control) did not record any number of mosquito larvae terminated.
Table of Contents
Pages
Abstract………………………………………………………………………………...I
Forms……………………………………………………………………………………
Research Plan…………………………………………………………………………i-v
Introduction……………………………………………………………………………1
Hypothesis………………………………………………………………………2
Methodology…………………………………………………………………………...12
Materials………………………………………………………………………..12
Equipments……………………………………………………………………..12
Collection………………………………………………………………………12
Preparation of Treatments……………………………...……………………...13
Preparation of Set-ups………………...…………………………………….....14
Application of Treatments………………...………...…………………………14
Disposal………………...……………………………………...………………14
Results………………...……………………………………...………………………16-18
Table 1.1………….……...……………….…………….……………………...16
Table 1.2……….………...………………….…………….…...………………17
Table 1.3………………...….…………….….…….…………...………………18
Discussion………………...……………………………………...……………………..19
Summary………………...……………………………………...………………. .....19-20
Conclusion………………...……………………………………...…………………...20
Recommendations………………...……………………………………..……..……20-21
Appendices………………...……………………………………...…………………..
Bibliography………………...……………………………………...…………………
Acknowledgement………………...……………………………………...……………
i
Research Plan
Mosquitoes can cause sickness and death through the diseases that they can carry.
Mosquitoes are mainly known for the disease they carry called as “Dengue”. Dengue is a
disease that debilitates a viral disease and viral fever and acute pains in the joints.
The Department of Health (DOH) has expressed alarm over the rising number of dengue cases
in Eastern Visayas (Region 8) this year, with 19 deaths and 4,809 confirmed cases. (Department of
Health, 2019) Mosquitoes can transmit many infectious diseases that are caused by a
B. Goals/Expected Outcome/Hypothesis:
friendly using the extracts of Lemon peel (Citrus limon) and Lagundi (Vitex negundo).
This study also aims to make an alternative for commercial products such as Spike
Larvicide.
ii
C. Methodology:
Sample Collection
Lemon (Citrus limon) will be bought Agora Public Market. Lagundi (Vitex
negundo) will be collected at Hda. Remedios, Brgy. Balabag. Mosquito larvae will be
collected at La Paz St., La Carlota City at the New Printing Press Beside the fishpond.
Spike larvicide and distilled water will be bought at Lopues La Carlota Branch.
Preparation of Treatment
Lagundi (Vitex negundo) will be pounded using a mortar and pestle the pounded
variables will be placed in a clean cloth and squeezed until extracts could be collected.
The extracts collected will be placed in a sterilized beaker. Lemon (Citrus limon) will be
peeled using a peeler and the lemon peel will be pounded using mortar and pestle the
pounded variables will be placed in a clean cloth and squeezed until the extracts could be
collected. The extracts collected will be placed in a sterilized beaker. The Lemon peel
(Citrus limon) extracts will be transferred in a 50 ml sterilized test tube and will be
ml sterilized test tube and will be labelled as B (treatment 2). The spike lavicide will also
be placed in a 50 ml sterilized test tube and will be labelled as C (treatment 3) the water
will also be in a 50 ml sterilized test tube and will be labelled as D (treatment 4).
iii
Preparation of Setup
Treatments R1 R2 R3
limon) extracts
(100%)
B. Lagundi (Vitex
negundo) extracts
(100%)
C. Spike larvicide
(100%)
D. Distilled Water
(100%)
Application of Treatment
peel (Citrus limon) extracts per replicates, Treatment B will be conducted once with 15 ml
of Lagundi (Vitex negundo) extracts per replicates. Treatment C with positive control will
be conducted once with 15 ml of Spike larvicide per replicates. Treatment D with negative
control conducted once with 15 ml of water per replicates. Each replicate will be containing
Waste disposal
The used petri dish, beaker, mortar and pestle will be washed using running water
and will be reused for future experimental uses. The left over extracts will be stored in
compatible containers with closed and fitted tops. The useless remains will be properly
The effectiveness of Lemon peel (Citrus limon) and Lagundi (Vitex negundo)
extracts based on the mosquito larva motality rate, survival and time of death were recorded
and compared among the four treatments having the same amount of extracts to which
The data obtained from the study will be subjected to the following
difference
Tukey test- to test which treatment is significantly effective than the other.
Bibliography
Gonazales, Eduardo. The disease that mosquitoes can cause, Manila Bulletin. Retrieved
news/5689-the-mosquito-menace-in-the-philippines
https://www.pna.gov.ph/articles/1074779
Report of the WHO Informal Consultationon the Evaluation on the Testing of insecticides,
Introduction
Mosquitoes can transmit more diseases than any other group of arthropods and
affect million of people throughout the world. WHO has declared the mosquitoes as “public
enemy number one”. Mosquito borne diseases are prevalent in more than 100 countries
across the world, infecting over 700,000,000 people every year globally and 40,000,000 of
the Indian population .they act as vector for most of the life threatening diseases like
malaria, yellow fever, filariasis, encephalitis, West Nile virus infection, etc. , in almost all
tropical and subtropical countries and many other parts of the world. (World Health
Organization, 2015).
In this study, the researchers used Lemon Peel (Citrus limon) and Lagundi (Vitex
Mosquitoes act as vector for most of the life-threatening diseases like malaria,
yellow fever, filariasis, encephalitis, West Nile virus infection, etc. The major tool in
and organophosphate compounds. due to lack of larvicide, high cost of synthetic larvicide,
concern for environmental sustainability, harmful effect on human health, and other non-
through ecosystems, and increasing insecticide resistance on a global scale (Anupam et.
al., 2012).
2
General
To determine the effects of Lemon Peel (Citrus limon) and Lagundi (Vitex
Specific objectives
1.2.1 To determine the effect of Lemon Peel (C. limon) extracts as potential mosquito
larvicide
1.2.2 To determine the effect of Lagundi (V. negundo) extracts as potential mosquito
larvicide
1.3 Hypothesis
There is a significant effect using Lemon Peel (C. limon) extracts as potential
mosquito larvicide
mosquito larvicide
does not need to cause high danger on the mortality rate on target organism in order to be
acceptable but should be eco-friendly in nature. Phytochemicals may serve as these are
relatively safe, inexpensive and readily available in many parts of the world
3
This study is limited only of using Lemon Peel (C. limon) and Lagundi (V.
2.2 Lemon (Citrus limon) and Ginger (Zingiber officinale) extracts as potential
mosquito larvicide
(Graf, 1986) Medium-sized tree, open and spreading; almost thornless. 2 000 mg/kg
parasiticus after an incubation period of seven days. 300 mg/kg of oil in a glucose-yeast
extract medium were need to achieve the same result.Lemon uses natural and generally
non-toxic ingredients to repel or get rid of pests including ants, wasps, mites, moths, flies
and other insects. Citrus, like lemons, oranges, or grapefruit and harnesses the power of d-
limonene — the acidic oil found in the peels. This oil is toxic to ants (so it will kill them
on contact) and it messes up their trail, so live ones won’t be able to find the food source.
“Ants follow a pheromone trail when they find food, so if you can disrupt the trail, it will
confuse them and break them up,” says Scot Svenheim, an associate certified entomologist
Lemon has been celebrated for centuries for its amazing internal and external uses
that include antiseptic, cleansing agent, beauty aid and stain remover. Among lemon's
many home uses is as an insect repellent. Moth balls, ant hotels, flea collars and bug sprays
all contain toxic chemicals, so try using lemon as a cheap and natural way to get rid of
unwanted pests around the home.Many gardeners look for natural and cheap alternatives
to the sometimes expensive, chemical pesticides found in most garden centers. Often, you
5
don't need to go any farther than your pantry to find such alternatives. Lemon juice, for
example, can sometimes help control small infestations of garden pests, such as ants,
aphids and leaf beetles.Lemon juice acts as a natural herbicide in the garden and could
2.2.3 Citrus
Citrus crops, mainly sweet oranges, provide the main part of the world’s essential
oil production. Important quantities of essential oils are also extracted from other citrus
cultivars like tangerines, lemons, grapefruits, limes, and grapefruits. Citrus are damaged
by a broad range of pests and diseases and chemical phytosanitary products are commonly
applied in order to protect the crop. Essential oils are utilized in food, pharmaceutical and
cosmetic industries resulting in a very low tolerance for chemical residues. Not only the
legislations are becoming more stringent, but increasingly the industry, including citrus
producers, processors and end users are driving the demand for chemical free products. To
meet this demand non-chemical pest control strategies are required, that need to be both
Biological pest control, alone or as a part of an Integrated Pest Management (IPM) strategy,
represents such an alternative to chemical pesticides. As far as citrus essential oils are
concerned, recent studies demonstrate that essential oils produced without chemical
pesticides are not only residue free but also are of higher quality. The presentation will
describe the biological control and IPM principles and applications with special emphasis
on the citrus crop. Finally, Biobest’s program for the implementation of an IPM strategy
in citrus will be discussed.In a recent study by Di Bella et al. 2010, the presence of
6
originating from various countries (120 samples from Italy, and 70 samples from Brazil,
Argentine, South Africa and Spain) were investigated. According to the results, pesticides
from both categories were found in the majority of the samples examined. The largest
quantities of pesticides were found in essential oils from Brazil and Spain. The presence of
these pesticides in the citrus essential oils is probably associated with the insecticides used
2.2.4 D-limonene
oils. The spectrum of insecticidal activity of d-limonene was examined by using the
German cockroach, Blattella germanica (L.), house fly, Musca domestica L., rice weevil,
Sitophilus oryzae (L.), and western corn rootworm, Diabrotica virgifera virgifera leconte.
bioassays were performed to determine topical, fumigant, oral, repellent, residual, ovicidal,
and larvicidal activities. The material was slightly toxic topically to German cockroaches
and house flies and was synergized with piperonyl butoxide.High concentrations of vapors
caused mortality in German cockroaches and rice weevils. Oral administration did not
nymphs. Repellent activity against German cockroaches was noted at high concentrations.
No residual activity was observed on any of four surface types exposed to adult German
concentrations and showed moderate toxicity in soil against third-instar western corn
7
rootworm larvae. These findings indicate that the insecticidal properties of d-limonene are
limited.
pesticides may be found on the skin of many fruits, including citrus fruits, but there
are official regulatory efforts to keep the levels low in the American market and ways to
remove most of the chemicals before the fruit is consumed, said Lambert H. B. Kanga,
that essential oil from citrrus peels exhibited strong insecticidal activity against C.
maculatus, S. oryzae and T. confusum. ... The fumigant toxicity and repellent properties of
essential oils from citrus peels on agricultural pests and insects has also been
investigated.citrus peels are safe for use as insecticides. Many companies make flea
shampoos, medications and insecticides for gardens using the ingredients in citrus fruit.
Can also be used as homemade spray, cleaner or compost to rid pets, homes and gardens
of pests without using harsh or dangerous chemicals. Citrus fruit repels ants, fleas, fungus,
analgesic, alterant, depurative, rejuvenating, stomachic. Its Roots are considered tonic,
febrifuge, anti-rheumatic, diuretic and expectorant. The Leaves and seeds are considered
vulnerary. The Leaves are also considered aromatic, bitter, anti-inflammatory, bronchial
While its Fruit is considered nervine, cephalic, aphrodisiac, emmenagogue and vermifuge.
Vitex negundo Linn. has been reported to have various advantageous uses, and
therefore, this has pushed scientists and researchers to test and further explore the
capabilities of this plant. Anti-cancer potential was tested by Diaz et al. (2003), on the
cytotoxicity of flavones isolated from the chloroform extract of Vitex negundo leaves.
Vitexicarpin, a flavone was investigated for its cytotoxic action in human cancer cell line.
Antimicrobial capabilities were also evaluated. Rideout et al. (1999) reported antibacterial
& antifungal activity of Vitexilactone & Casticin from the chloroform extract of Vitex
albicans and Aspergillus niger using agar plate method. Alagarsamy et al. (1999) reported
activity of chloroform, methanol and water extracts were evaluated by Agar cup plate
reference standards for said screeningsAmancharla et al. (1999) also tested mosquito
repellent activity of aqueous extract of Vitex negundo leaves. A new chemical ‘rotundial’
was tested for the said activity. The chloroform fraction of the aqueous extract of the fresh
9
leaves of Vitex negundo by bioactivity guided isolation yielded a pure compound rotundial
smaller slender tree with quadrangular, densely whitish tomentose branchlets up to 4.5-5.5
meters in height. The plant is used in a variety of means throughout its distribution points
bronchitis, asthma, and the enlargement of the spleen. Its roots are primarily used as a tonic,
in cephalalgia, otalgia, colic, uropathy, wound and ulcers. The bark is also useful in
odontalgia, verminosis and opthalmopathy. The flowers are mainly used as an astringent,
antihelmintic and vermifuge. The leaves (leaf oil) are also reported to have repellent action
against stored product pests (Tandon, 2005). Vitex negundo Linn. is a large aromatic shrub
or small slender tree of about 3 meters in height with quadrangular branches. It is mostly
found in moist areas, scattered all throughout Mediterranean countries and Central Asia. It
is a plant of the Verbenaceae family and is commonly known as five leaved chaste tree.
10
The plant is found throughout India, Ceylon- Afghanistan, tropical Africa, Madagascar,
China and Philippines (Kirtikar and Basu, 2008). The plant occurs in Bengal, Southern
India and Burma also (Nadkarni, 2002). It is common in waste places around villages, river
banks, moist localities and in the deciduous forests (Sharma et al., 2005)
Lagundi, Vitex negundo L., is one of the ten medicinal plants being promoted by the
member of the family Verbenaceae, alongside other economically and medically important
plants such as alagaw (Premna odorata), lemon verbena (Lippia citriodorata) and common
vervain or “mosquito plant” (Verbena officinalis). It is an erect, branched shrub with five-
altitudes and even in thickets and waste places. The leaves, bark, roots, and seeds of V.
negundo have long been used in traditional medicine to heal wounds, cleanseulcers,
prevent insect bites, treat snake bites, and relieve rheumatism. Its oil has also been used to
relieve sores and sinuses. Recent studies have proven its antiseptic, antitussive, as well as
its anti-inflammatory properties. It has been found to have antibacterial effects against
Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Other studies have also
shown its efficacy in reducing coughing and relaxing the bronchial smooth muscles
(Galvez Tan 2010). These properties make it an effective drug for coughs and
Methodology
I. Materials
Mosquito Larvae
Spike Larvicide
Distilled Water
II. Equipments
Peeler
Petri Dishes
Beaker
Test Tubes
Collection
Lemon (Citrus limon) were purchased at Agora Public Market. While Lagundi
(Vitex negundo) were gathered from a farm located at Hda. Remedios, Bryg. Balabag.
Mosquito larvae were collected at La Paz St., La Carlota City at the New Printing Press
Beside the fishpond. Spike larvicide was bought at Lopues La Carlota Branch. The
Preparation of Treatments
There were four (4) treatments in this study each having different variables. Each
The bought Lemon (Citrus limon) were washed thoroughly with clean water. The
Lemon (C. limon) were peeled using a peeler. The collected lemon peel was cut into smaller
pieces so that pounding it would be easier. The lemon peel was then pounded using mortar
and pestle the pounded peelings were placed in a clean cloth and squeezed until the extracts
could be collected. The extracts collected will be placed in a labeled and sterilized beaker.
The collected Lagundi (V. negundo) were washed thoroughly with clean water.
Then the leaves were separated from its stem. After Separating the stem healthy leaves
were chosen while the damaged one were disposed. The leaves were pounded using a
mortar and pestle the pounded, the pounded leaves were placed in a clean cloth and
squeezed until extracts could be collected. The collected extracts were placed in a sterilized
Preparation of Set-ups
contains 50ml of Lagundi (Vitex negundo) extracts. Treatment C the positive control
Application of Treatment
Lemon peel (Citrus limon) extracts per replicate. Treatment B with 15ml of Lagundi (Vitex
negundo) extracts per replicate. Treatment C the positive control was applied with 15ml of
Spike larvicide per replicate. Treatment D the negative control was applied with 15ml of
Disposal
The used container, beaker, graduated cylinder, mortar and pestle will be washed
using running water and will be reused for some experimental purposes. The left-over
extracts will be stored in compatible containers with closed and fitted tops. The useless
remains will be properly disposed in the garbage dump. All glasswares were autoclaved,
The data obtained from the study will be subjected to the following
difference
Tukey test- to test which treatment is significantly effective than the other.
16
Results
Table 1.1.
Replicates
Treatment Total Mean
1 2 3
Lemon peel (Citrus limon)
4 4 4 12 4.0
Extracts
Lagundi (Vitex negundo)
4 3 4 11 3.66
Leaves Extracts
Spike Larvicide 4 4 4 12 4.0
Distilled Water 0 0 0 0 0.0
The table 1.1. shows that the number of mosquito larvae exterminated was total of
12 in treatment A which is Lemon peel (Citrus limon) extracts. The total number of
mosquito larvae exterminated in treatment B which the Lagundi (Vitex negundo) extracts
was 11. The number of mosquito larvae killed the treatment C which is the spike larvicide
(positive control) was a total of 12. The treatment D which is the Distilled water (negative
Table 1.2.
One-way Analysis of Variance (ANOVA) for the mortality rate of the Mosquito Larvae
Degree of
Sum of Squares Mean Square F Sig.
Freedom
Between Groups 34.250 3 11.417 137.000 .000
Total 34.917 11
The table 1.2. shows that there is a significant difference in the mean of the
With p-value of <0.05 giving the researchers the reason to reject Null hypothesis and
using Lemon peel (Citrus limon) extracts and Lagundi (Vitex negundo) extracts in
exterminating mosquito larvae. As showed by the table above, it is certain that the
treatments have statistically significant effect against mosquito larvae at 0.05 or 5% level
of significance
18
Table 1.3.
The table 1.3. shows that Lemon peel (C. limon) extracts is significantly effective
than the Lagundi (V. negundo) and the negative control which is the Distilled water, and
equally significant with the positive control which is the spike larvicide. Lagundi (V.
negundo) is less effective than the the positive control which is the spike larvicide but the
spike larvicide is just slightly more effective than Lagundi (V. negundo)
19
Discussion
larvae in replicate 2. Treatment C, which is the positive control, killed 4 mosquito larvae
with a total of 12 and Treatment D which is the negative control put none of the larvae to
death.
As it was shown in the tables Treatment A has better potential than Treatment B in
terms of Mosquito Larvae exterminated. Treatment A killed all of the larvae in the
replicates while Treatment B nearly killed all the larvae but there was 1 left. The Lagundi
but only lesser capabilities than the positive control (spike larvicide) and the treatment A
Summary
This research was conducted to determine the effect of Lemon Peel (C. limon) and
Lagundi (Vitex negundo) Extracts as potential mosquito larvicide. This was conducted
from July 2019 to August 2019 at Dona Hortencia Salas Benedicto National High School,
Negros Occidental.
There are four mosquitoes in each replicate with the total of twelve mosquitoes
every treatment. There are four treatments: Treatment A which is Lemon peel (Citrus
20
limon) extracts, Treatment B which the Lagundi (Vitex negundo), Treatment C which is the
spike larvicide (positive control) and Treatment D which is the Distilled water (negative
control)
In Treatment A, four mosquito larvae were killed in each replicate with the total of
12 mosquito larvae exterminated while in Treatment B, only 11 mosquito larvae were slain,
In Treatment C, four mosquito larvae were put to death in every replicate totaling to 12
mosquito larvae deaths. Treatment A and Treatment C killed the mosquito larvae in 30
Conclusion
Therefore we conclude that Treatment A, which is Lemon Peel (C. limon) Extracts
is more effective than Treatment B, which is Lagundi (V. negundo) and D which is the
negative control (Distilled Water). There is no significant relationship between the positive
and negative control. The results show that the study achieved its objectives.
Recommendations
Based on the results of the research, the following recommendation are presented
The use of Lemon Peel (C. limon) and Lagundi (Vitex negundo) extracts should
also be considered
Use the oil of the Lemon Peel (C. limon) Extracts rather than just the extracts
Gonazales, Eduardo. The disease that mosquitoes can cause, Manila Bulletin. Retrieved
https://www.pna.gov.ph/articles/1074779
Report of the WHO Informal Consultationon the Evaluation on the Testing of insecticides,
Anupam et. al. Plants extracts as potential mosquito larvicide, Retrieved on August 14,
1154f18f91de155
Creative Home making. Pesticidal effects of Lemon. Retrieved on August 14, 2019.
a-pesticide/
Hunker. How to use Lemon as insect repellent. Retrieved on August 14, 2019. Retrieved
from https://www.hunker.com/13421047/how-to-use-lemon-as-insect-repellent
Pesticide Science Society of Japan. Properties of D-limonene Retrieved on August 14,
Tandon, V. R & Gupta, R.K. (2011). Properties of Lagundi. Retrieved on August 14,2019
Ganhinhin C.M. & A.P. Kintanar(2013). Studies on Lagundi. Retrieved on August 8, 2019
603aa94b949ad79c.pd
Ganhinhin C.M. & A.P. Kintanar. Larvicidal Activity Of Vitex negundo (Lagundi) Against
https://pdfs.semanticscholar.org/0abe/2ecdfce98006cb6b1c7c603aa94b949ad79c
Bautista, N., Cubos A.F. and Lim, M.C. 2015. Genetic Diversity of Vitex negundo L.
11%20Bautista-fullpaper
Acknowledgement
First of all this study will not be successful without the guidance and protection of
our Father God Almighty that blessed us and gave us strength, knowledge and wisdom to
finish and complete this task. We would like to thank him for his faithfulness and we offer
our success to him. To him we bring back the honor and glory.
Secondly, we would like to thank our research teacher Mrs. Adaiah A. Opeña who
has been our guide in finishing this project step by step and the one who gave us the golden
opportunity to do this meaningful project on the topic Lemon peel (Citrus limon) and
We would like to thank our ever supportive and loving families, To our friends and
classmates who gave their full support and motivation helped us a lot in finalizing this
Furthermore we would also like to acknowledge with much appreciation the crucial
role of Mrs.Mavil C. Moncada , who gave the permission to borrow all required equipment
and the necessary materias for us to successfully conduct the experiment Last but not least,
many thanks go to our project adviser Mr. Christian Floyd I. Erad who have invested his
full effort in guiding and leading us to the right path in having the best possible outcome