Synergistic Effect of Lantana camara (Kantutay) and Tinospora boerl rumphii

(Makabuhay) as Plant-Based Mosquito Larvicide

Introduction
The Aedes aegypti mosquito is the main vector that transmits the viruses that cause
dengue. The viruses are passed on to humans through the bites of an infective female Aedes
mosquito, which mainly acquires the virus while feeding on the blood of an infected person
(World Health Organization, 2016). Additionally, Aedes aegypti is also the main mosquito
species that transmits Zika virus and several other serious diseases, and can breed practically
anywhere. These mosquitoes will lay eggs in the stagnant water collected in bird baths or old
tires, sure, but also in the few droplets gathered in a discarded candy wrapper, or on the damp
surface of a fallen leaf, or in a practically-empty soda can (Lafrance, 2016).
Most of its kind can be found in areas of great population, especially places with poor
sanitation, insufficient supply of water and improper waste disposal and management
(Tabachnick, 2013). It is suggested that Aedes aegypti evolved its domestic behavior in West
Africa and its widespread distribution and colonization in the tropics led to the highly efficient
inter-human transmission of viruses such as dengue (Weaver & Reisen, 2010). This domestic
behavior can provide protection against environmental conditions (as it rests indoors) and
numerous habitats suitable as oviposition sites, but can also result in increased sensitivity to
control measures used to eliminate them (Jansen & Beebe, 2010).
Dengue is a mosquito-borne viral disease that has rapidly spread in all regions of World
Health Organization in recent years. Dengue is widespread throughout the tropics, with local
variations in risk influenced by rainfall, temperature and unplanned rapid urbanization (World
Health Organization, 2016). This disease is brought about through vector transmission by the
specie Aedes aegypti.
Upon the acquisition of this illness, there will be a manifestation of symptoms such as
headache, fever, exhaustion, severe muscle and joint pain, swollen lymph nodes
(lymphadenopathy), and rash. The presence of fever, itchy rash, and headache (the "dengue
triad") is characteristic of dengue. Other signs of dengue fever include bleeding gums, severe
pain behind the eyes, and red palms and soles (Cunha, 2016).
Home treatment is not advised by most doctors for this illness. In addition, nonsteroidal
anti-inflammatory agents (NSAIDs) such as aspirin, ibuprofen, and other NSAIDs should be
avoided because of the tendency of the dengue viruses to cause hemorrhages. More severe
variations of dengue fever (hemorrhagic and shock syndrome) usually require additional
supportive treatments; these patients often require hospitalization. IV fluid hydration, blood
transfusions, platelet transfusions, blood pressure support, and other intensive-care measures
may need to be utilized in these patients. Consultation with infectious-disease and critical-care
specialists is often advised to optimize patient care (Davis, 2016).
In the Philippines alone, officials of the Department of Health–Epidemiology Bureau say
that the number of dengue fever cases in the country during the first 5 1/2 months of 2016 is up
41 percent compared to the same period in 2015. National data from the beginning of the year
through June 2011 shows that the Philippines has seen a total of 52,177 dengue cases. This
compares to 36,972 cases reported in 2015. Another disease that can be transmitted by an Aedes
aegypti mosquito is the Zika.
Zika is a virus that is spread mostly by mosquitoes. A pregnant mother can pass it to her
baby during pregnancy or around the time of birth. It can spread through sexual contact. There
have also been reports that the virus has spread through blood transfusions. There have been
outbreaks of Zika virus in the United States, Africa, Southeast Asia, the Pacific Islands, parts of
the Caribbean, and Central and South America (MedlinePlus, 2016).
According to the WHO, as of 10 August 2016, 69 countries and territories have reported
evidence of mosquito-borne Zika virus transmission since 2007 (66 of these countries and
territories have reported evidence of mosquito-borne Zika virus transmission since 2015). The
high rates of the number of cases are proportional to the rate of the illness’s proliferation. The
suggested treatments and medicines are rather expensive.
As stated by the Centers for Disease Control and Prevention (2012), the best way to
reduce mosquitoes is to eliminate the places where the mosquito lays her eggs, like artificial
containers that hold water in and around the home. The form elimination is by the use of
larvicides.
Larvicides target larvae in the breeding habitat before they can mature into adult
mosquitoes and disperse. Larvicide treatment of breeding habitats helps reduce the adult
mosquito population in nearby areas. Liquid larvicide products are applied directly to water
using backpack sprayers and truck or aircraft-mounted sprayers. Tablet, pellet, granular, and
briquet formulations of larvicides are also applied by mosquito controllers to breeding areas
(United States Environmental Protection Agency, 2016). Given the following information, the
researchers have decided to conceptualize a solution by tackling the dengue outrage by its roots,
through the modification of mosquito larvicides in the form a pellets, with the use of Lantara
camara (Kantutay) and Tinospora rumphii boearl (Makabuhay).
Materials and Methods
Preparation of the Extracts
For the collection of the raw materials, the researchers gathered a full sack of Lantana
camara at Diversion Road, Barangay Tatala, Binangonan, Rizal. The leaves were manually
separated from the stems and finished up with half a sack. Afterwards, the leaves were placed
under the sun with a temperature of 34 degrees Celsius and were dried for one day. Next, a half
sack of makabuhay stems were gathered from Sitio Caingin, Sta. Catalina Street, Barangay San
Juan, Morong, Rizal. After that, the dried leaves and fresh makabuhay were brought to
Department of Science and Technology-Industrial Technology Development Institute-Chemical
& Energy Division Pharmaceutical Section for Crude extraction.

The dried 140.0 g lantana leaves were pulverized using Wiley mill and soaked in 1.4 L of
95% ethyl alcohol for 48 hours. Then, the mixture was filtered and the filtrate obtained was
concentrated using rotary evaporator at 60⁰C under vacuum for 2 hours. Next, the concentrated
extract was further evaporated using water bath at 60⁰C to obtain a semi-solid extract. The crude
extraction of dried 140.0g lantana leaves produced 1.2 L ethanolic extract and the concentration
of the filtrate yielded 30.0g of semi-solid extract.
On the other hand, the fresh 500g makabuhay stem were blended/osterized and soaked in
2.0L of 95% ethyl alcohol for 48 hours. The mixture was filtered and he filtered obtained was
concentrated using rotary evaporator at 60⁰C under vacuum for 2 hours. The concentrated
extract was further evaporated using water bath at 60⁰C to obtain a semi-solid extract. The crude
extraction of fresh 500.0g makabuhay stem produced 1.8L ethanolic extract and the
concentration of the filtrate yielded 45.0g of semi-solid extract.

Preparation of the pellet product

The other materials needed for this study were bought locally by the researchers because
aside from the plant itself, binders were needed in order to pelletize it. These binders are flour,
distilled water and CMC or caboxylmethylcellulose.
The 20g of the semi-solid extract of Lantana plant were set for mixing. Mixing was done
with the help of the binders in order for the mixture to stick together. The binders used for this
experiment are flour, distilled water and CMC or the carboxylmethyl cellulose, commonly
known as the cellulose gum. The first set or the Lantana Pellet 50 contains 12g of semi-solid
Lantana extract 5g of flour, 2mL of distilled water and 0.5g of CMC. On the other hand, the
second set or the Lantana Pellet 90 contains 8g semi-solid Lantana extract, 5g of flour, 2mL of
water and 0.5 g of CMC.
The 20g of the semi-solid extract of Makabuhay plant were set for mixing. Mixing was
done with the help of the binders in order for the mixture to stick together. Same binders were
used for the Makabuhay Pellets. The first set or the Makabuhay Pellet 50 contains 12g of semi-
solid Makabuhay extract , 5g of flour, 2mL of distilled water and 0.5g of CMC. On the other
hand, the second set or the Makabuhay Pellet 90 contains 8g semi-solid Lantana extract, 5g of
flour, 2mL of water and 0.5g of CMC.
The 5.6g of the semi-solid extract of Lantana and 5 g of the semi-solid extract of
Makabuhay were set for mixing with 70% Lantana+30% Makabuhay concentration. Mixing was
done with the help of the binders in order for the mixture to stick together. Same binders were
used for the Makabuhay Pellets. The first set or the Makalantana Pellet 50 contains 7g of semi-
solid lantana and 3g of semi-solid makabuhay extract , 2.4g of flour, 1mL of distilled water and
0.25 of CMC. On the other hand, the second set or the Makalantana Pellet 90 contains 2.8g of
semi-solid lantana and 4g of semi-solid makabuhay extract , 2.4g of flour, 1mL of distilled
water and 0.25g of CMC.
For the individual mixing of the 6 pellet concentration, the researchers made use of a
stand mixer with low to medium speed for about 5-10 minutes each depending on how fast the
mixture turns into a clay-like form.
Afterwards, the researchers made use of a pellet mill, to pelletize the clay-like mixture of
each concentration. By this time, the clay-like mixtures was turned into some small pencil like
strips. Next, it was placed in a dryer machine with a temperature of 60-80 degrees Celsius
overnight. The next day, it was cut into tiny pieces.

Test Mosquito Larvae

The Aedes aegypti 3rd instar larvae used in the larvicidal study of Pelletized Lanatana,
Makabuhay and Makalantana was reared in the Insectary of Standards and Testing Division,
Industrial Technology Development Institute, Department of Science and Technology at a
laboratory condition of 27 ± 2 degrees Celsius temperature, 80 ± 10% relative humidity.
Homogeneous batches of third instar to early fourth instars larvae were used in the test.

Bioassay
In the preliminary test, the 3rd instar and early 4th instar mosquito larvae were exposed to
a range of test concentration from 0.3% to 5% and a control using dechlorinated tap water with
1% Ethanol to determine the effective doses. After determining the mortality of larvae in this
range of concentrations, another batch of larvae was exposed to concentrations from 0.3% to 3%.
Final test for lethal doses were re-evaluated at this range of concentrations.
The appropriate volume of extract from the stock solution used in each concentration is
added to 100mL de chlorinated tap volume starting with the lowest concentration as tabulated
below.
Final Test Amount of 10%
Concentrations plant extract
solution added to
100mL water
Lantana Pellet
0.3% or 3000ppm 3mL
0.6% or 6000ppm 6mL
0.9% or 9000ppm 9mL
1.2% or 12000ppm 12mL
1.5% or 15000ppm 15mL
Makabuhay pellet
1% 10mL
1.5% 15mL
2.0% 20mL
2.5% 25mL
3.0% 30mL
70% Lantana ± 30% Makabuhay
0.5% 5mL
1% 10mL
1.5% 15mL
2.0% 20mL
2.5% 25mL
Batches of 20 third instar and early fourth instar larvae were transferred by means of
Pasteur pipette to 250mL beakers containing the appropriate volume of solutions concentrations
under test. Three (3) replicates were set up to each concentration. Equal number of controls were
set up simultaneously. For negative control, dechlorinated tap water with 1% ethanol was used.
For positive Control Abate 1SG mosquito larvicide was used. The test containers were held at
25-28 degrees Celsius and relative humidity of 80± 10%.

After 48 hours exposure, larval mortality was recorded. Moribund larvae were counted
and added to dead larvae for calculating percentage mortality. Dead larvae are those that cannot
be induced to move when they are probed with a needle in the siphon or the cervical region.
Moribund larvae are those incapable of rising to the surface or not showing the characteristic
diving reaction when the water is disrupted.

Data Analysis
The mean percentage larval mortality at each concentration will be calculated including
the Mean Standard deviations. Lethal concentrations of Lantana camara at 50% and 90% or
LC50 and LC90, lethal concentrations of Makabuhay at 50% and 90% or LC50 and LC90, lethal
concentrations of MakaLantana at 50% and 90% or LC50 and LC90 will be determined by
Linear Regression –Probit Analysis.
If the control mortality is between 5% and 20%, the mortalities of treated groups should
be corrected by Abbott’s Formula:
% Mortality= X-Y (100)
X
where: X= percentage survival in untreated control and
Y= percentage survival in treated sample