Bioactivity Evaluation of Different Mango Cultivars Leaves Ethanolic Extract on

3rd - Early 4th Instar Aedes aegpyti Larvae

Hasinaah S. Candelario
hasinaahsadimcandelario@gmail.com
MIMAROPA Regional Science High School-Senior High School
Pag-asa, Bansud, Oriental Mindoro, MIMAROPA Region, Philippines, 5210

Abstract: Aedes aegypti is a main vector of diseases which causes morbidity. In

response to this, various low cost and easy to prepare plant-based larvicides have been
developed. In this study, the ethanolic extract of Indian Mango (Mangifera indica),
Carabao Mango (Mangifera rostrata), Pico Mango (Mangifera sylvatica), Paho Mango
(Mangifera altissima), and Apple Mango (Mangifera laurina) leaves were evaluated for
its bioactivity on 3rd – early 4th instar Aedes aegypti larvae. Sundried and grinded
mango cultivars leaves were macerated in ethanol and purified using water bath. The
ethanolic extracts were serially diluted into five different concentrations: 10,000 ppm,
15,000 ppm, 20,000ppm, 25,000ppm and 30,000ppm with separate treatment containing
50 ml crude extract. The mortality of the larvae was recorded and evaluated for its actual
morphology before and after exposure to the extracts. One-way ANOVA results showed
significant difference among the five treatments in terms of the mortality rate wherein
treatment 6 (crude extract), obtained the highest mean average of mortality rate and
treatment 7 (negative control) the lowest. This means that the extract is dose dependent
with lethal concentration to kill 50% and 90% of the larvae at 23,988.33ppm and
86,963.689ppm for M. indica, 20,090.93ppm and 50,118.72ppm for M. rostrata,
22,908.68ppm and 68,706.84ppm for M. sylvatica, 20,417.38ppm and 68,391.165ppm
for M. altissima, and 19,998.62 and 42,657.95ppm for M. laurina respectively. In
addition, there is no significant difference among the different mango cultivars when it
comes to effectivity. This means that any of the five mango cultivars can be used for its
bioactivity against Aedes aegypti larvae. The morphology of Aedes aegypti larvae
showed that there is serious aberration on the mid-gut and swelling after experimentation.
Moreover, coloration of the larvae is also observed. Therefore, the ethanolic extracts of
the five mango cultivars are potential bio-larvicide which could be utilized to control
Aedes aegypti which is the primary vector of the dengue virus.

KEYWORDS: Plant-based larvicide, Mangifera indica, Mangifera rostrata, Mangifera

sylvatica, Mangifera altissima, Mangifera laurina, Aedes aegypti

1
Introduction

The Philippines being a tropical country is subjected to various climates. During wet

climates, mosquitoes often see this instinctively as the beginning of mating season.

Akban and Nwaueze (2015) explained that with abundance of moist and water,

mosquitoes copulate and lay eggs due to careless waste disposal, irrigation and poor

drainage which proliferates the living conditions of the parasites. Interest in the control

of Aedes aegypti lies in the fact that they act as vectors of arboviruses such as dengue,

yellow fever and chikungunya. Meanwhile, as of July, 2019, the Department of Health

reported a total of 146,062 dengue cases; thus, the declaration of national dengue

epidemic. Aedes aegypti females has a habit of collecting blood with their stingrays

especially when they lay eggs. As such, they spread diseases to their victims. One of the

solutions to eradicate this problem in mosquito is to start mortality in its larval stage.

Over the years, synthetic insecticides were introduced but although these are effective,

the insect tends to develop resistance to such products (Jirakanjanakit et al., 2007). Aside

from being costly, the use of these repellents also generates problems such as

environmental pollution and has toxic side effect to humans (Sarwar, Ahmad & Toufiq,

2009). This imposes a need for other alternative methods which are efficient, economic

and environmentally safe. Currently, plant based bio-insecticides are intensively studied

worldwide, especially in tropical countries with a high diversity of plants. Particular

attention has been paid to the study on many plant extracts for determining their

larvicidal potential against Aedes aegypti.

2
 Mango, from the family Anacardiaceae, is a common domestic plant in the

Philippines. The tree often reach 15 to 18 m in height. The leaves in general are dark

green above and pale below, alternate, with no stipules, simple, leathery and varies in

shape. The roots are unbranched long taproots with a dense mass of superficial feeder

roots. The flowers are tiny, and yellowish or reddish. Native to Southern Asia, mango

has been cultivated, praised and even revered for its many uses. Mango contains

mangiferin (Ramirez et al, 2016); a unique phenolic compound known only to mango.

Although there are many various uses of mango, the leaves specifically have shown

different properties and activities such as antimicrobial (Bharti, 2013),

anti-hyperglycaemic (Aderibigbe, Emudianughe & Lawal, 1999) and anticancer

(Helen, Aswathy, Deepthi, Mol, Joseph & Sre, 2013). Furthermore, mango is one of

the most common and most cultivated fruits in the country of the Philippines. There

are diverse mango cultivars available such as Indian Mango (Mangifera indica),

Carabao Mango (Mangifera rostrata), Pico Mango (Mangifera sylvatica), Paho

Mango (Mangifera altissima), and Apple Mango (Mangifera laurina). Thus, in this

study, ethanolic extracts of different mango cultivars leaves were evaluated for its

efficacy as potential natural larvicide. Specifically, this work aims to determine the

mortality rate of the 3rd-early 4th Instar Aedes aegypti larvae after exposing to the

prepared treatments of M. indica, M. sylvatica, M. altissima, M. rostrata, M. laurina;

examine the morphology of the used Aedes aegypti larvae before and after treatment

exposures; and qualitatively determine the phytochemicals present in M. indica

ethanolic extract.

3
 This work promotes a simpler and cheaper natural larvicide which could help locals

to address problems on rapid increase of Aedes aegypti. In addition, the study will

contribute greatly to the community concerning to the need of immediate disposal of

mosquitoes in their early stage. Thus, the implementation of the practice simulated in the

study may prevent excessive growth of the mosquitoes’ population.

Methodology

Collection and Preparation of the Plant Sample

The leaves of mango cultivars (M. indica, M. sylvatica, M. altissima, M. rostrata, M.

laurina) were collected in Gloria, Oriental Mindoro. Harvested leaves were sun-dried for

three days. Using an electric blender, the dried plant samples were powdered and 500g

sample of mango leaves were placed into a container.

The leaf samples of mango cultivars were brought to the National Institute of

Biology at University of the Philippines - Diliman for proper identification and

authentication.

Figure 1. Collecting, sun-drying and powdering of different mango cultivar leaves.

4
Ethanol Plant Extraction

Five hundred (500) g of dried of mango cultivar leaves were placed in a container

containing 1000mL of 95% ethanol. It was left soaked for 72 hours. The soaked sample

were poured into clean cheesecloth that was prepared and squeezed into another

container through a filter paper and a funnel.

Subsequent to the process, the extract were purified through the process of water

bath for 1 hour. The extracts were placed into clean amber bottles and placed in the

refrigerator before laboratory use.

Figure 2. Maceration and water bath of the mango cultivars ethanolic extract

Phytochemical Analysis

The ethanol plant extract of the mango cultivars were subjected to qualitative

phytochemical screening to detect biological constituents (Daffodil, Lincy, & Mohan,

2014), as summarized in Table 1. Presence or absence of phytochemicals in the extracts

were determined by color reactions of the compounds with specific reagents/dyes.

5
 Table 1. Qualitative phytochemical screening procedures

Phytochemical Test Reagent and Positive Result

Chemicals Used
Alkaloid Mayer’s Test Hydrochloric acid, White precipitate
Mayer’s reagent
Saponin Foam Test Water Copious lather
formation
Tannin Lead Acetate Test Lead acetate White precipitate
Phenol Ferric Chloride Test Ferric chloride Blush green or red
color
Flavonoid Shindo’s Test Magnesium Red or red orange
turnings, color
Hydrochloric acid
Quinone Sulfuric Acid test Sulfuric acid Yellow precipitate

Figure 3. Phytochemical analysis of M. indica, M. rostrata, M. sylvatica, M. altissima, and M. laurina

Mosquito Acculturation

Larvicidal traps were used to collect A. aegypti eggs from open and closed habitats

and reared in a cup lined with filter paper strips and one third filled with tap water at the

Learning Resource Center of MIMAROPA Regional Science High School. A

homogenous population of late third to early fourth instars (5 days old and 4–5 mm in

length) were obtained five to seven days later.

6
 Figure 4. Larvicidal traps used to culture eggs of Aedes aegypti

Preparation of Different Concentrations of Extract

Table 2. Different Concentrations Mango Cultivars Leaves Ethanolic Extract

Treatment Volume taken from 10% Volume of water

stock solution (ml)

T1 (10,000 ppm) 5 45
T2 (15,000 ppm) 7.5 42.5
T3 (20,000 ppm) 10 40
T4 (25,000 ppm) 12.5 37.5
T5 (30,000 ppm) 15 35
T6 (Crude Extract) 50 ml crude extract
T7 (Negative Control) Distilled water

Different concentrations of Mangifera rostrata (Carabao Mango), Mangifera

sylvatica (Pico Mango), Mangifera altissima (Paho Mango), and Mangifera laurina

(Apple Mango) ethanolic extracts were prepared.

7
Treatment Application

Batches of ten (10) third or fourth instar larvae were transferred by means of

droppers to small disposable test cups or vessels containing 50 ml of the different

treatments, crude extract and distilled water as negative control. Each cup was labeled

and then covered by a mosquito net. Small, unhealthy or damaged were removed and

replaced. After 48-hour exposure, larval mortality is recorded. Dead larvae are those that

cannot be induced to move when they are probed with a needle in the siphon or the

cervical region. The experiment was performed in five replicates.

Morphology Analysis

The morphology of the 3rd and early 4th instar larvae were observed before and

after the application of the plant extract using a HD-digital microscope. Swelling,

aberrations in the midgut and rear part of the larvae samples after death were also

observed.

Statistical Analysis

Data from all replicates were pooled for analysis. The lethal concentrations to kill

50% (LC50) and 90% (LC90) of larval population were calculated using linear

regression probit analysis.

One-Way Analysis of Variance (ANOVA) was used to determine the significant

difference in the number of dead mosquito larvae among the treatments. Meanwhile,

8
Two-Way Analysis of Variance (ANOVA) was used to determine the significant

difference among the effects of Mangifera rostrata (Carabao Mango), Mangifera

sylvatica (Pico Mango), Mangifera altissima (Paho Mango), and Mangifera laurina

(Apple Mango) ethanolic extracts.

Proper Disposal Procedures

After the experimentation, the treatments were thrown on one spot in which it was

poured with hot water. This method is according to the procedures given in using the

larvicide trap and following the Guidelines for Laboratory and Field Testing of Mosquito

Larvicides by World Health Organization. Chemical disposals were done according to

the proper clean up cited in the Safety Data Sheet. They were disposed on inert solids or

containerized before disposal.

Risk and Safety

According to the Safety Data Sheet of the Federal Register on rules and regulations

(2013), the use of chemicals demands personal precautions and emergency procedures in

case of accidental release measures. The use of protective equipment such as safety

glasses, gloves and protective clothing are basic standards. For safe handling, washing

possible exposed areas of the skin before the process and ventilation is required.

9
Results and Discussion

Phytochemical Screening of the Different Mango Cultivars

Table 3. Phytochemical Analysis of Mangifera indica Leaves Ethanolic Extract

Phyto- Test Result Result Result Result Result

chemical Mangifera Mangifera Mangifera Mangifera Mangifera
indica rostrata sylvatica altissima laurina
(Pico (Paho (Apple
(Indian (Carabao Mango) Mango) Mango)
Mango) Mango)
Alkaloid Mayer’s
Test
+ + + - +

Saponin Foam
+ + + + +
Test
Tannin Lead
+ + + + +
Acetate
Test
Phenol Ferric
+ + + + +
Chloride
Test
Flavonoid Shindo’s
+ + + + +
Test
Quinone Sulfuric
Acid Test
+ + + - +

Note: (+) present; (-) absent

The result of the phytochemical screening revealed the following metabolites as

present in the ethanolic extract of M. indica, M. rostrata, M. sylvatica, M. altissima and

M. laurina (Table 3).

All phytoconstituents have large roles in subjecting mortality to the Ae. Aegypti

larvae. Dietary phenolics such as flavonoids and phenolic compounds are used as

10
defense compounds and they have antioxidant properties that are used as protecting

agents against free radical-mediated disease processes. Alkaloids have antibacterial and

antifungal properties that ensure endurance against microorganisms, insects, etc. and

antimalarial activity (quinine). Saponin is antimicrobial, inhibits mold, and its

hypocholesterolaemic and anticarcinogenic properties have significantly affected the

growth, feed intake and reproduction in animals. Other than that, it is also an antioxidant,

it impairs the digestion of protein and the uptake of vitamins and minerals in the gut that

causes hypoglycaemia, and acts as antifungal and antiviral. Terpenoids have

anticarcinogenic, antimalarial activity, antibacterial and antifungal properties that inhibit

micro-organisms and reduce the risk of fungal infection. Tannins are astrigents and have

anti-inflammatory, antiseptic, antioxidant and haemostatic properties (Saxena, Nema,

Singh and Gupta, 2013).

Average Mortality Rate of Aedes Aegypti

Table 4. Average Mortality Rate of Aedes aegypti Applied with Different Treatments
of Mangifera indica (Indian Mango)

R1 R2 R3 R4 R5 Mean
T1 (10,000ppm) 4 1 5 3 1 2.8
T2 (15,000ppm) 3 1 4 2 4 2.8
T3 (20,000ppm) 1 3 7 4 2 3.4
T4 (25,000ppm) 2 4 7 7 9 5.8
T5 (30,000ppm) 2 4 8 8 9 6.2
T6 (Crude extract) 10 10 10 10 10 10
T7 Negative Control 5 2 1 0 1 1.8

11
 Among the treatments of Mangifera indica, treatment six has the highest mortality

rate with mean of 10. This means that treatment six, or the crude extract of M. indica, has

the highest possibility of larval death, followed by treatment 5 with 30,000ppm,

treatment 4 with 25,000ppm, treatment 3 with 20,000ppm, treatment 2 with 15,000ppm

and treatment 1 with 10,000ppm concentrations respectively. The lowest mortality rate is

exhibited by treatment 7 with mean of 1.8, being the negative control treatment using

distilled water. The mortality of Ae. aegypti is directly proportional with the

concentrations of the ethanolic extract.

In the study of Yousaf and Zuharah (2015), M. indica extract is proven to have

strong larvicidal activity among the plants from the Anacardiaceae family tested, having

the the second lowest lethal concentration values. In addition to that, Mohammed and

Chadee cited that the toxic properties and efficacy of M. indica varies by the solvent

used or selected.

Table 5. Average Mortality Rate of Aedes aegypti Applied with Different Treatments of
Mangifera rostrata (Carabao Mango)

R1 R2 R3 R4 R5 Mean
T1 (10,000ppm) 2 2 3 1 2 2
T2 (15,000ppm) 3 3 4 2 4 3.2
T3 (20,000ppm) 2 3 5 6 4 4
T4 (25,000ppm) 5 5 5 6 8 5.8
T5 (30,000ppm) 7 8 8 9 8 8
T6 (Crude extract) 10 10 10 10 10 10
T7 Negative 2 1 0 2 1 1.2

12
 Among the treatments of Mangifera rostrata, treatment six has the highest mortality

rate with mean of 10. This means that treatment six, or the crude extract of M. rostrata,

has the highest possibility of larval death, followed by treatment 5 with 300,00ppm,

treatment 4 with 25,000ppm, treatment 3 with 20,000ppm, treatment 2 with 15,000ppm

and treatment 1 with 10,000ppm concentrations respectively. The lowest mortality rate is

exhibited by treatment 7 with mean of 1.2, being the negative control treatment using

distilled water. The mortality of Ae. aegypti is directly proportional with the

concentrations of the ethanolic extract.

Table 6. Average Mortality Rate of Aedes aegypti Applied with Different Treatments
of Mangifera sylvatica (Pico Mango)

R1 R2 R3 R4 R5 Mean
T1 (10,000ppm) 2 3 2 1 2 2
T2 (15,000ppm) 2 2 3 4 4 3
T3 (20,000ppm) 5 2 3 3 4 3.4
T4 (25,000ppm) 4 4 8 6 3 5
T5 (30,000ppm) 8 6 8 8 6 7.2
T6 (Crude extract) 10 9 10 10 9 9.6
T7 Negative Control 4 1 2 2 1 2

Among the treatments of Mangifera sylvatica, treatment six has the highest

mortality rate with mean of 9.6. This means that treatment six, or the crude extract of M.

sylvatica, has the highest possibility of larval death, followed by treatment 5 with

30,000ppm, treatment 4 with 25,000ppm, treatment 3 with 20,000ppm, treatment 2 with

13
15,000ppm and treatment 1 with 10,000ppm concentrations respectively. The lowest

mortality rate is exhibited by treatment 7 with mean of 2, being the negative control

treatment using distilled water. The mortality of Ae. aegypti is directly proportional with

the concentrations of the ethanolic extract.

Table 7. Average Mortality Rate of Aedes aegypti Applied with Different Treatments
of Mangifera altissima (Paho Mango)

R1 R2 R3 R4 R5 Mean
T1 (10,000ppm) 4 3 2 2 3 2.8
T2 (15,000ppm) 5 2 3 4 3 3.4
T3 (20,000ppm) 4 2 4 4 5 3.8
T4 (25,000ppm) 6 5 7 5 8 6.2
T5 (30,000ppm) 7 8 7 6 8 7.2
T6 (Crude extract) 10 10 10 10 10 10
T7 Negative 5 2 1 0 2 2

Among the treatments of Mangifera altissima, treatment six has the highest

mortality rate with mean of 10. This means that treatment six, or the crude extract of M.

altissima, has the highest possibility of larval death, followed by treatment 5 with

30,000ppm, treatment 4 with 25,000ppm, treatment 3 with 20,000ppm, treatment 2 with

15,000ppm and treatment 1 with 10,000ppm concentrations respectively. The lowest

mortality rate is exhibited by treatment 7 with mean of 2, being the negative control

treatment using distilled water. The mortality of Ae. aegypti is directly proportional with

the concentrations of the ethanolic extract.

14
Table 8. Average Mortality Rate of Aedes aegypti Applied with Different Treatments
of Mangifera laurina (Apple Mango)

R1 R2 R3 R4 R5 Mean
T1 (10,000ppm) 1 0 1 3 2 1.4
T2 (15,000ppm) 3 3 2 2 4 2.8
T3 (20,000ppm) 3 6 4 5 6 4.8
T4 (25,000ppm) 6 5 6 5 7 5.8
T5 (30,000ppm) 8 7 8 9 9 8.2
T6 (Crude extract) 9 10 10 10 9 9.6
T7 Negative 0 2 1 1 0 0.8

Among the treatments of Mangifera laurina, treatment six has the highest mortality

rate with mean of 10. This means that treatment six, or the crude extract of M. laurina,

has the highest possibility of larval death, followed by treatment 5 with 30,000ppm,

treatment 4 with 25,000ppm, treatment 3 with 20,000ppm, treatment 2 with 15,000ppm

and treatment 1 with 10,000ppm concentrations respectively. The lowest mortality rate is

exhibited by treatment 7 with mean of 0.8, being the negative control treatment using

distilled water. The mortality of Ae. aegypti is directly proportional with the

concentrations of the ethanolic extract.

15
Difference on Mortality Rate of Aedes aegypti

Table 9. Difference on Mortality Rate of Aedes aegypti Applied with Different

Treatments of Mangifera indica (Indian Mango)

Source of
Variation SS df MS F P-value F crit
Between Groups 244.3429 6 40.72381 9.255411 1.282E-05 2.445259
Within Groups 123.2 28 4.4

For the results of mortality as shown in the table, there is significant difference

among the five mortality treatments on the mortality rate of the mosquito larvae at p

1.282E-05 < 0.05 level. These results suggest that high concentrations of the Mangifera

indica really have an effect on the mortality effect on the larvae of Ae. aegypti.

Table 10. Difference on Mortality Rate of Aedes aegypti Applied with Different
Treatments of Mangifera rostrata (Carabao Mango)

Source of
Variation SS df MS F P-value F crit
Between Groups 311.1429 6 51.85714 55 3.31E-14 2.445259

Within Groups 26.4 28 0.942857

For the results of mortality as shown in the table, there is significant difference

among the five mortality treatments on the mortality rate of the mosquito larvae at p

3.31E-14 < 0.05 level. These results suggest that high concentrations of the Mangifera

rostrata really have an effect on the mortality effect on the larvae of Ae. aegypti.

16
Table 11. Difference on Mortality Rate of Aedes aegypti Applied with Different
Treatments of Mangifera sylvatica (Pico Mango)

Source of
Variation SS df MS F P-value F crit
Between Groups 247.2 6 41.2 29.42857 7.3942E-11 2.445259
Within Groups 39.2 28 1.4

For the results of mortality as shown in the table, there is significant difference

among the five mortality treatments on the mortality rate of the mosquito larvae at p

7.3942E-11 < 0.05 level. These results suggest that high concentrations of the Mangifera

sylvatica really have an effect on the mortality effect on the larvae of Ae. aegypti.

Table 12. Difference on Mortality Rate of Aedes aegypti Applied with Different
Treatments of Mangifera altissima (Paho Mango)

Source of
Variation SS df MS F P-value F crit
Between Groups 245.4857 6 40.91429 31.47253 3.33E-11 2.445259
Within Groups 36.4 28 1.3

For the results of mortality as shown in the table, there is significant difference

among the five mortality treatments on the mortality rate of the mosquito larvae at p

3.33E-11 < 0.05 level. These results suggest that high concentrations of the Mangifera

altissima really have an effect on the mortality effect on the larvae of Ae. aegypti.

17
 Table 13. Difference on Mortality Rate of Aedes aegypti Applied with Different
Treatments of Mangifera laurina (Apple Mango)

Source of Variation SS df MS F P-value F crit

Between Groups 335.7714 6 55.9619 64.21858 4.52E-15 2.445259
Within Groups 24.4 28 0.871429

For the results of mortality as shown in the table, there is significant difference

among the five mortality treatments on the mortality rate of the mosquito larvae at p

4.52E-15 < 0.05 level. These results suggest that high concentrations of the Mangifera

laurina really have an effect on the mortality effect on the larvae of Ae. aegypti.

Table 14. Difference Among the Mango Cultivars Ethanolic Extract

Source of
Variation SS df MS F P-value F crit
Sample 4.457142857 4 1.114285714 0.105901521 0.980308959 2.431965056
Columns 17.2 4 4.3 0.408671253 0.802193113 2.431965056
Interaction 38.05714286 16 2.378571429 0.226059015 0.99928434 1.711346961
Within 1578.285714 150 10.52190476
Total 1638 174

The result of Two-Factor ANOVA, as shown in Table 14, shows no significant

difference among the mortality rates of Ae. Aegypti applied with the five mango cultivars

ethanolic extract. This means that any of the five mango cultivars can be used against Ae.

Aegypti for its larvicidal bioactivity.

18
Probit Analysis of Lethal Concentration of Mango Cultivars Ethanolic Extract

Table 15. Lethal Concentration LC50 and LC90

Mango Cultivars LC50 (ppm) LC90 (ppm)

M. indica (Indian Mango) 23,988.33 86,963.689
M. rostrata (Carabao Mango) 20,090.93 50,118.72
M. sylvatica (Pico Mango) 22,908.68 68,706.84
M. altissima (Paho Mango) 20,417.38 68,391.165
M. laurina (Apple Mango) 19,998.62 42,657.95

Using the regression equation, values of lethal concentration at 50% and 90% were

held at 23,988.33ppm and 86,963.689ppm for M. indica. This means that the guarantee

that half of the population was killed will be at concentration value of 23,988.33ppm and

potential at 90% will be 86,963.689ppm. For M. rostrata, values of lethal concentration

at 50% and 90% were 20,090.93ppm and 50,118.72ppm. For M. sylvatica, values of

lethal concentration at 50% and 90% were 22,908.68ppm and 68,706.84ppm. For M.

altissima, values of lethal concentration at 50% and 90% were 20,417.38ppm and

68,391.165ppm. For M. laurina, values of lethal concentration at 50% and 90% were

19,998.62ppm and 42,657.95ppm. Out of the five mango cultivars, M. laurina exhibited

the lowest 50% and 90% lethal concentration needed, while M. indica the highest.

According to the OPP classification criteria for acute toxicity, oral toxicity of the

extract is deemed to be Category IV while the dermal toxicity is classified as Category

III. Both have the signal word “caution”. This means that there is no hazard statement

19
required however as caution, the product may be harmful when swallowed and harmful

if in contact with skin.

Morphology of Aedes aegypti

Figure 5. Before and After Treatment of M. indica ethanolic extract

Figure 6. Before and After Treatment of M. rostrata ethanolic extract

Figure 7. Before and After Treatment of M. sylvatica ethanolic extract

Figure 8. Before and After Treatment of M. altissima ethanolic extract

20
 Figure 9. Before and After Treatment of M. laurina ethanolic extract

The figures above showed the digital microscopic photos of the larvae before and

after treatments of the mango cultivars ethanolic extracts. In Figure 5, the body was

hollow yet there the midgut is still present. There is also presence of coloration. This is

due to tannin which acts as a dye and astringent that produces the capability to preserve

tissues (Schrijvers et al., 1989) and as a result, has been used as preservatives for wood

products (Asante et al., 2017). In figure 6, the midgut of the larvae separates or rips into

two after application of the ethanolic extracts. In figure 7, swelling of the Ae. Aegypti

larvae is evident. In Figure 8, there is coloration and disintegration of the midgut. In

Figure 9, swelling of the Ae. Aegypti larvae is also evident.

Conclusion

Based on the results, the study showed that Indian Mango (Mangifera indica),

Carabao Mango (Mangifera rostrata), Pico Mango (Mangifera sylvatica), Paho Mango

(Mangifera altissima), and Apple Mango (Mangifera laurina) leaves ethanolic extracts

are effective against mosquito larvae. This is initially supported by the phytoconstituents

such as tannin, alkaloids, flavonoids, terpenoids and phenolic compounds present in the

extract. In addition, it was found that there is significant difference among the prepared
21
treatments of the different mango cultivars ethanolic extracts wherein treatment 6 (crude

extract) exhibited highest mortality rate for Ae. aegypti larvae. This means that the five

mango cultivars ethanolic extract is dose dependent. The results of the study also showed

no significant difference among the five mango cultivars ethanolic extracts. It means that

any of the mango cultivars aforementioned can be used for its larvicidal potential. The

morphology of Ae. aegypti shows that there is serious aberration on the mid-gut and

swelling after experimentation. This may be associated to the presence of

phytoconstituents flavonoids, terpenoids, alkaloids, phenolic compounds and saponin.

Recommendations

For plant extraction, it is best if the ratio of the sample and the solvent is 1:3. This is

to ensure reserves in case complications occur in mosquito rearing. It is also best to use

rotary evaporator to purify the extracts properly. Make sure the cap of the bottle of the

plant extraction is sealed and keep the bottle refrigerated at all times.

Acknowledgment. The researcher would like to express sincerest gratitude to Ms.

Mischelle F. Puntod for providing great supervision during the experimentation period.

References

Abdalla A. (2017), Damage caused by Castor seed oil and its Ethanolic Extract on
Anopheles arabienses larvae. Geriza State, Sudan. Faculty of Science, University of
Hail, KSA, Saudi Arabia

22
Akaban, I.O.M. & Nwabueze, E. (2015). A Survey of Mosquito Larval Habitats and
Species Distribution in Rivers State University of Science and Technology
Nkpolu Porthacourt, Nigeria. International Journal of Science and Research
(IJSR), ISN: 2319- 7064.

Almiron, G., et al. (2008). Larvicide and oviposition deterrent effects of fruit and leaf
extracts from Melia azedarach L. on Aedes aegypti (L.) (Diptera: Culicidae).
Bioresource Technology, 99 (2008): 3066–3070.

Andrew J., and Ananya B. (2013). Morphonology and Morphometry of

Aedes aegypti Adult Mosquito. India. www.sciencedomain.org

Asante, B., Haapala, B., Li, P., Liimatainen, H., Sirviö, J.H. (2017). Adsorption Of
Tannin Onto Functionalized Nanocellulose.

Bharti, R. P. (2013). Studies on Antimicrobial Activity and Phytochemical Profile of

Mangifera Indica Leaf Extract. IOSR Journal Of Environmental Science,
Toxicology And Food Technology (IOSR-JESTFT), 7(3). e-ISSN: 2319-2402, p-
ISSN: 2319-2399.

Christophers, S.R. (1960) Aedes aegypti (L.). The Yellow Fever Mosquito, its Life
History, Bionomics and Structure. Cambridge Univ. Press, London.

Derese, S., et. al. Medicinal Spices and Vegetables from Africa: Mangifera
indica L. Anacardiaceae. http://dx.doi.org/10.1016/B978-0-12-809286-6.00021-2

Doughari, J.H. (2012). Phytochemicals: Extraction Methods, Basic Structures and

Mode of Action as Potential Chemotherapeutic Agents. Phytochemicals - A
Global Perspective of Their Role in Nutrition and Health, (Ed.), InTech, DOI:
10.5772/26052.

Finney, D. J.(1952). Probit Analysis. (2nd Ed), Journal of the Institute of Actuaries.
78 (3) :388-390.

Hemingway, R.W. & Laks, P.E.(2012). Plant Polyphenols: Synthesis, Properties,

Significance. Springer Science & Business Media. 625.

23
Jirakanjanakit, N., Rongnoparut, P., Saengtharatip, S., Chareonviriyaphap, T., Duchon,
S., Bellec, C. & Yoksan, S. (2007). Insecticide susceptible/resistance status in Aedes
(Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in
Thailand during 2003-2005. Journal of Economic Entomology (in press).

Laboratory Biosafety Manual (2004), World Health Organization. Retrieved from

https://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Mamta S., et. Al (2013), Jornal of Pharmacology and Phytochemistry. Retrieved from
phytojournal.com

Mary Helen, P.A., Aswathy, M.R., Deepthi, K.G., Rathi Mol, R., Jaison Joseph, J.
and Jaya Sree, S. (2013). Phytochemical Analysis and Anti-cancer Activity of
Leaf Extract of Mangiferin indica (Kotutukonam Varika).

Maslbo, M. and He, Q.(2008). Major Mango Polyphenols and Their Potential
Significance to Human Health. Comprehensive Reviews in Food Science and
Food Safety, 7, 309 - 319.

Noratto, G.D. et al. (2010). Anticarcinogenic Effects of Polyphenolics from Mango

(Mangifera indica) Varieties. Journal of Agricultural and Food Chemistry, 58,
4104 – 4112. DOI:10.1021/jf903161g

Ramirez et al. (2016). Extraction of Mangiferin and Chemical Characterization and

Sensorial Analysis of Teas from Mangifera indica L. Leaves of the Ubá Variety.
Beverages.2, 33.

Rey, D., David, J.P., Besnard, G., Jullien, J.L., Lagneau, C. and Meyran, J.C.(2001),
Comparative sensitivity of larval mosquitoes to vegetable polyphenols versus
conventional insecticides. Entomologia Experimentalis et Applicata, 98:
361–367. DOI:10.1046/j.15707458.2001.00793.x

Saxena, M., Saxena, J., Nema, R., Singh, D. and Gupta, A. (2013). Phytochemistry
of Medicinal Plants. Journal of Pharmacognosy and Phytochemistry, 1(6):
168-182.

24
Schrijvers, A. H., Frederik, P.M., Stuart M.C., van der Vusse, G.J., Reneman, R.S.
(1989). Dual effect of tannic acid on the preservation and ultrastructure of
phosphatidyl choline vesicles. Mol Cell Biochem, 88(1-2):91-6.

Stuart, G. (2016). Mangifera indica. Philippine Alternative Medicine. Retrieved June

13, 2017 from http://www.stuartxchange.com/Mango.html

Yousaf, A. & Zuharah, W.F. (2015). Lethal response of the dengue vectors to the plant
extracts from family Anacardiaceae. Asian Pacific Journal of Tropical
Biomedicine, 5(10): 812-818. https://doi.org/10.1016/j.apjtb.2015.05.016

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 Appendix

Flow Chart

Preparation and Extraction of Plant Sample

Collection of Mango Washing of Mango Drying of leaves Powdering of dried leaves

cultivars leaves cultivars leaves

Extract filtration Squeezing of macerated Maceration Weighing of powdered

leaves leaves

Water Bathe The extracts were poured in

a bottle

26

Preparation of Chemicals and
Laboratory Equipment to be
used
Test of Flavonoid: A few Mg
turnings and a few drops of
hydrochloric acid were added
to the extract and were boiled
for five minutes
Test of Quinone: The extract was mixed with
few drops of aqueous sodium hydroxide
solution
PRESENT: Carabao, Piko and Apple Mango
Phytochemical Testing
Putting a small amount of
extract
Test of Phenol: A few drops
of ferric chloride solution
were added in the extract
PRESENT: All cultivars
Test of Tannin: Lead Acetate
was added to the extract
PRESENT: All cultivars
Test of Saponin: Extract was
shaken with 2 ml of water.
PRESENT: All cultivars
Test of Alkaloids: Extract
was mixed with little amount
of dilute hydrochloric acid
and Mayer’s reagent
PRESENT: Carabao, Piko
and Apple Mango
27
Treatment Applications (Mangifera indica)

28
Treatment Applications (Mangifera rostrata)

29
Treatment Applications (Mangifera sylvatica)

30
Treatment Applications (Mangifera altissima)

31
Treatment Applications (Mangifera laurina)

32
33