PROPOSAL
(1) PROJECT PROFILE
Project Title:Mosquito repellent spray made of Lemon grass (Cymbopogan citratus) and Tawa-
tawa (Euphorbia hirta) extract for repelling Mosquitoes (Aedis aegypti), a Vector of Dengue
Virus
Names of Project Proponent/s: Prince Jhayson Bartolome
Project Duration (number of months)
(2) CATEGORY OF RESEARCH (4) THEME
Physical Science Food Safety
Life Science Water Conservation
Robotics and Intelligent Renewable Energy
Machines Cyber Security
Mathematics and Computational Traffic / Road Congestion
Sciences Health
Disaster Mitigation
Agriculture and Environment
(3) Others (please specify)
_____ Individual ________________________
_____ Team
(5) INTRODUCTION
(5.1) RATIONALE/SIGNIFICANCE (not to exceed 300 words)
The mosquito, Aedes aegypti, the principal vector of dengue virus has caused an alarming
health issue worldwide. Commercial mosquito insecticides are readily available, but the
vectors develop resistance to these and may cause harm and pose negative effects on living
organisms and to the environment. This study evaluated the crude extracts of Lemon grass
(Cymbopogon citratus) and Tawa-tawa (Euphorbia hirta) leaves on the hatchability of
mosquito eggs, and the mortality, abdominal morphology and behavior of 4th instar A.
aegypti larvae. Mosquito eggs and larvae were exposed to 500 μL of each crude extract and
then observed after 24 hours. Mean level of hatchability and mean level of mortality were
recorded and the larvae were observed under a compound light microscope. Results showed
significant differences on the hatchability and mortality manifested in the treatment using
Tawa-tawa and Lemon grass crude extract. Qualitative analysis showed that the extracts from
Tawa-tawa and Lemon grass caused abnormalities on the abdominal morphology and
behavior of the 4th instar A. aegypti larvae when compared with the control. The results
showed a great potential for the production of organic insecticides from the plant extracts to
control A. aegypti, vector of the dengue virus.
(5.2) SCIENTIFIC BASIS/THEORETICAL FRAMEWORK/MATHEMATICAL THEORY
INVOLVED
Plant-based repellents have been used for generations in traditional practice as a personal
protection measure against host-seeking mosquitoes. Knowledge on traditional repellent
plants obtained through ethnobotanical studies is a valuable resource for the development of
new natural products. Recently, commercial repellent products containing plant-based
ingredients have gained increasing popularity among consumers, as these are commonly
perceived as “safe” in comparison to long-established synthetic repellents although this is
sometimes a misconception. To date insufficient studies have followed standard WHO
Pesticide Evaluation Scheme guidelines for repellent testing. There is a need for further
standardized studies in order to better evaluate repellent compounds and develop new
products that offer high repellency as well as good consumer safety. This paper presents a
summary of recent information on testing, efficacy and safety of plant-based repellents as
well as promising new developments in the field.Most plants contain compounds that they
use in preventing attack from phytophagous (plant eating) insects. These chemicals fall into
several categories, including repellents, feeding deterrents, toxins, and growth regulators.
Most can be grouped into five major chemical categories: (1) nitrogen compounds (primarily
alkaloids), (2) terpenoids, (3) phenolics, (4) proteinase inhibitors, and (5) growth regulators.
Although the primary functions of these compounds is defence against phytophagous insects,
many are also effective against mosquitoes and other biting Diptera, especially those volatile
components released as a consequence of herbivory [1]. The fact that several of these
compounds are repellent to haematophagous insects could be an evolutionary relict from a
plant-feeding ancestor, as many of these compounds evolved as repellents to phytophagous
insects [2], and this repellent response to potentially toxic compounds is well conserved in
the lineage of Diptera (True Flies). Insects detect odours when that volatile odour binds to
odorant receptor (OR) proteins displayed on ciliated dendrites of specialized odour receptor
neurons (ORNs) that are exposed to the external environment, often on the antennae and
maxillary palps of the insect, and some ORNs, such as OR83b that is important in olfaction
and blocked by the gold-standard synthetic repellent DEET (N, N-diethyl-3-
methylbenzamide) [3], are highly conserved across insect species [4, 5]. Plants commonly
produce volatile “green leaf volatiles” when leaves are damaged in order to deter herbivores
[6], and several authors have shown strong responses of mosquito odour receptors to this
class of volatiles including geranyl acetate and citronellal [7], 6-methyl-5- hepten-2-one and
geranylacetone [8]. Interestingly, the same odour receptors that respond to DEET also
respond to thujone eucalyptol and linalool in Culex quinquefasciatus[9]. In Anopheles
gambiae, the DEET receptor OR83b is stimulated by citronellal, but is also modulated by the
TRPA1 cation channel [10]. However, it is most likely that many plant volatiles are deterrent
or repellent because they have high vapour toxicity to insects.
(5.3) OBJECTIVES
General: This study aims to test the effectiveness of repelency of the Tawa-tawa and Lemon
grass extracts to Aedis aegypti mosquito
Specific: Specifically, it seeks to find out the effectiveness of a mosquito repellent spray
made of Tawa-tawa and Lemon grass extract.
(6) REVIEW OF LITERATURE
Lemongrass (Cymbopogon citratus L.), which is a species of grass, is adapted to warm
climatic conditions. Upon steam distillation of dried leaves, a yellowish-colored, strongly
fragrant liquid called lemongrass oil is obtained, which has properties attributed to its strong
chemical composition. The active ingredients present in lemongrass essential oil are myrcene,
limonene, citral, geraniol, citronellol, geranyl acetate, neral, and nerol. Although myrcene and
limonene are aromatic compounds, citral and geraniol serve as an antimicrobial and
insecticidal, respectively. This oil counteracts fungi, bacteria, and also insects in general.
Additionally, lemongrass oil is a natural food preservative. Recent studies suggest that the
use of pure lemongrass essential oil is an innovative and useful tool as alternative to the use
of synthetic fungicides or other sanitation techniques in storage and packaging. Its use as an
alternative food preservative and the effectiveness of the essential oil depends on the target
pathogen. For example, the low pH of yogurt offers a selective environment for the growth of
acid-tolerant yeasts and molds. Therefore, it is not surprising that various investigators have
found that yeasts are the primary spoilage microorganisms for yogurt and that fruits, flavors,
and coloring agents are frequent contamination sources. The spoilage of yogurt by yeasts
generally has been characterized by yeasty off-flavors, loss of textural quality due to gas
production, and swelling and occasional rupturing of the product containers. As a result, there
is an apparent need for an effective preservation method to control acid-tolerant spoilage
yeasts and molds in yogurt. The study carried out by some researchers showed that
lemongrass essential oil was effective. It was observed that the addition of the appropriate
concentration of the essential oil (0.1%, w/v) improved the physicochemical properties as
well as sensory characteristics of yogurt, and this essential oil could be used for
decontamination of dairy products such as yogurt from mycotoxigenic fungi and prevent
mycotoxins formation, in addition to its beneficial properties as a functional
food.Cymbopogon citratus (DC. ex Nees) Stapf. (Fig. 18.1) is one of the best known species
of the genus Cymbopogon with the synonyms Andropogon citratus DC. ex Nees,
Andropogon ceriferus Hack., Andropogon nardus subsp. ceriferus L. (Hack.) Hack.,
Andropogon roxburghii Nees ex Steud., and Cymbopogon nardus subvar. citratus (L.) Rendle
(DC. ex Nees) Roberty. It is native to Asia (Indochina, Indonesia, and Malaysia), Africa, and
the Americas, but are widely cultivated in temperate and tropical regions of the world
(Jayasinha, 2001). C. citratus is known by numerous international common names, such as
West Indian lemon grass or lemon grass (English), hierba limon or zacate de limón (Spanish),
citronelle or verveine des indes (French) and xiang mao (Chinese) and locally identified with
over 28 vernacular names from different countries of the world (Jayasinha, 2001; Ross,
1999). Table 18.1 summarizes the geographical distribution of C. citratus around the
world.Myrcene, better known as the active sedating principle of hops and lemongrass, is also
found in basil, mangos, and its namesake, Myrcia sphaerocarpa, a medicinal shrub from
Brazil traditionally used to treat diabetes, diarrhea, dysentery, and hypertension (Ulbricht,
2011). In culinary and perfume use, myrcene’s aroma is earthy, fruity, and clove-like; it is
pungent in higher concentrations. Myrcene synergizes the activities of terpenes and other
compounds in a variety of ways. One mechanism that would be of particular note in cannabis
is its claimed effect on the permeability of cell membranes, particularly the blood–brain
barrier (BBB), increasing transport of cannabinoids into the brain; however, perusal of
claimed references in the popular literature shows a lack of hard data regarding brain
transport. Myrcene has been shown to enhance transdermal absorption (Schmitt et al., 2009).
It has a significant analgesic effect, which is blocked by the action of naloxone, an opioid
antagonist, suggesting a mechanism of action through the opioid receptor (Rao et al., 1990).
However, myrcene’s lack of affinity for opioid receptors points to α-2-adrenoceptor-
stimulated release of endogenous opiates. In contrast to morphine, no tolerance was observed
after repeated dosing in rats (Lorenzetti et al., 1991). At very high doses, myrcene in mice
was a sedative comparable to phenobarbital (Gurgel do Vale et al, 2002); the effect was
increased by simultaneous administration of citral, a mixture of other terpenes. Al-Omari
(2007) demonstrated that myrcene improved glucose tolerance in alloxan diabetic rats
comparable to metformin, without an effect on glucose levels in normal rats. Myrcene also
showed powerful antiinflammatory and anticatabolic effects in a human chondrocyte model
of osteoarthritis (Rufino et al., 2015). With inflammation underlying numerous diseases,
myrcene is the subject of a broad array of current research.C. citratus is a tall perennial fast
growing grass with tuft of lemon scented leaves from the annulate and sparingly branched
rhizomes. It grows to a height of 1 m and a width of 5–10 mm, and has distinct bluish-green
leaves which does not produce seed. However, it has many bulbous stems that increase the
clump size as the plant grows. The leaves are long, glabrous, glaucus green, linear, tapering
upward and along the margins, with very short ligule and tightly clasp sheaths at the base,
narrow and separating at distal end. The inflorescences are nodding with pairly racemes of
spirelets and subtended by spathes of about 1 m long with peduncles of 30–60 cm long
(Jayasinha, 2001; Ross, 1999).
Euphorbia hirta is an annual herb belongs to Euphorbiaceae family. It has been found at
warm and tropical regions mostly on roadsides and waste land throughout the world and used
as traditional medicines. Tawa tawa contains lycosidal substance, terpenoids, tannin, phorbic
acid, fatty acids and sterols, Flavonoids including quercitrol, quercetin, and its derivatives.
The extent or amount of these chemical constituents varies in different parts of the tawa tawa
plant and also depends on soil and climate condition. Recently published reports showed that
tea of tawa tawa plant is helpful as anti-malaria and anti-dengue. Euphorbia hirta has been
used for the remediation of respiratory disease, some female disease and also other such as
dysentery, jaundice, gonorrhoea, pimples, tumors, digestive problems and children infections.
Tawa-Tawa (Euphorbia hirta L.) is an annual plant which belongs to Euphorbiaceae family.
Euphorbiaceae is one of the large families of angiosperms that comprises of 300 genera and
about 7500 species. Almost all members of this family comprise of herbs some are trees and
shrubs. Certain species of these genera are xerophytes. Euphorbiaceae Family is widely
dispersed in both hemispheres with variety of morphological arrangement, from trees to
largest desert lush herbs. The most varied genera in plant kingdom are Euphorbia. Plants of
Euphorbia may also be perennial herbs, possess trees or woody shrubs with corrosive and
poisonous latex. The roots of tawa tawa are thick and some other plants have fine, tuberous or
fleshy. Numerous species are relatively thorny, succulent or unarmed. In luscious species the
leaves are frequently short- lived and small. Euphorbia hirta L. is a remedial, rhizomatous
plant scattered in south west Ghats of Asian country India and north east Coastline of Tamil
Nadu. Euphorbia hirta L. is a recognized as a therapeutic herb with numerous
pharmacological outlines. Tawa-tawa is small, annual (yearly) plant have hairy stems and
fruits of yellow colour [1]. Euphorbia hirta is known by various names which are given in
different countries of the world, according to their language. In English is typically called
snake weed. In India and Pakistan, it is called Dudhi or Dudhani. Other vernacular names of
Euphorbia hirta are Sheer jiyah, Dhudi Kalan (Unani), Raktavinduchada. Euphorbia hirta also
known as E. pilulifera L. have positive effect on diverse female disorders and in the cure of
respiratory illnesses including bronchitis and asthma moreover it has character for cumulative
milk flow because of having its milky latex. In India this plant is used for the treatment of
worm infections in kids and also for gonorrhea, dysentery, jaundice, pimples, tumors and
digestive problems. On the warts and wounds, the fresh milky latex obtained from tawa tawa
plant is used. Roots are used in inflammation, sprains, miscarriage, epilepsy and irregular
growth of teeth. Dengue fever is initiated by the arbovirus known as dengue virus, transferred
by the mosquito named as Aedes aegypti. It is a big approach to treat this fetal fever using
traditional or herbal medicines. Euphorbia hirta in the Philippines, natively known as “tawa
tawa” has applications in herbal medicine to treat dengue fever in rural areas. The exact
mechanism is yet unknown but it has considerable significance for the treatment of the
dengue fever. Euphorbia hirta contains tannin, lycosidal substance, fatty acids, Jambulol
melissic acid, phorbic acid, eciphosterol, sterols, and small amount of alkaloids and sugars.
Chemical composition varies with different season [5]. Aerial parts of Euphorbia hirta
contain terpenoids. Study of leaves of this plant shows the presence of phytochemicals such
as flavonoids, alkaloids, tannins, steroids, carbohydrates and glycosides. But fats, saponins
and protein are absent
(8) METHODOLOGY
1. Gather the ingredients. You’ll need half a cup of distilled water, half a cup of witch
hazel, rubbing alcohol, or vinegar, and 30 to 40 drops of essential oil of your choice.
(Rubbing alcohol may be irritating if sprayed on the skin, and witch hazel is the gentlest
option for sprays.)
2. Create a custom scent. Combine essential oils to create a custom scent. Your
homemade mosquito repellent spray can also serve as a natural bug repellent, depending
on the combination of oils. For example, cedarwood oil also deters ticks, and lavender
essential oils drive away gnats, flies, and ticks.
3. Pour the ingredients into a spray bottle. Shake to combine and spray as needed. You
should shake the bottle before every use because the oils and liquid will separate over
time.
(9) EXPECTED OUTPUTS AND POTENTIAL IMPACTS
Mosquitoes are common vectors of health hazards, a legitimate cause of health concerns
in humans such as malaria, dengue, and encephalitis, they are a nocturnal blood-feeding and
considered as pest with public health importance in human environments.
We aimed to test the effectiveness of the repellency of the Tawa-tawa and Lemon grass
extract on Mosquitoes.
In recent years, a large number of insect repellents have been developed with the idea of
consumer benefits. In addition to already known advantageous application of insect
repellents, there is increasing concern about the potential toxicity in environment leading to
health caused by random use of these compounds. An increasing number of evidence
suggests that insect repellents may trigger undesirable hazardous interactions with biological
systems with a potential to generate harmful effects including intermediate metabolites.
Biotransformation followed by bioaccumulation (vice e versa) may be an important
phenomenon for toxic response of this chemicals. In this review, we have summarized the
current state of knowledge on the insect repellent toxicity, including biochemical pathway
alteration under in vitro and in vivo conditions considering different classes of organisms,
from lower to higher vertebrate.
(10) WORK PLAN AND TARGET DELIVERABLES
Date Task Target Actual Accomplishment
Day 1
Day 1
Day 1
Day 2
Day 3
Day 4
Day 5
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extracts for their antibacterial and antifungal
activity and brine shrimp lethality. Molecules.
15(9): 6008-6018.
S.B. Patil, C.S. Magdum. (2011). Determination of
LC50 values of extracts of Euphorbia hirta Linn
and Euphorbia neriifolia Linn using brine shrimp
lethality assay. Asian Journal of Research in
Pharmaceutical Science. 1(2): 42-43
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