POTENTIAL LARVICIDAL ACTIVITY OF PLEUROTUS FLORIDA (MONT.) SINGER METHANOLIC EXTRACT ON AEDES AEGYPTI (L.)

CHARLIZE MAIBETH GAREJO MARAMOT

CHRISTINE KAE NOCETE YBARRITA

Submitted to the

Department of Biology

College of Arts and Sciences

University of the Philippines - Manila

Padre Faura, Manila

In partial fulfillment of the requirements

for the degree of

Bachelor of Science in Biology

June 2019

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS …………………………………………………………. iv

LIST OF TABLES …………………………………………………………………… i

LIST OF FIGURES ………………………………………………………………….. ii

LIST OF APPENDICES …………………………………………………………….. iii

ABSTRACT …………………………………………………………………………. vi

CHAPTER I: INTRODUCTION ……………………………………………………. 1

Background of the Study……………………………………………………... 1

Statement of the Problem ……………………………………………………. 3

Objectives of the Study ……………………………………………………… 3

Significance of the Study ……………………………………………………. 4

Scope and Limitations ……………………………………………………….. 4

CHAPTER II: REVIEW OF RELATED LITERATURE ………………………….... 5

Dengue virus: a global public health threat …………………………………. 5

Aedes aegypti …………………………………………………………………... 7

Dengue management and vector control ……………………………………... 8

Larvicides: its advantages and challenges ……………………………………. 10

Pleurotus florida (Mont.) Singer ……………………………………………... 11

CHAPTER III: METHODOLOGY …………………………………………………... 13

Collection, Air-Drying, and Preparation of Methanolic

Extracts of Macrofungi Samples ……………………............................ 13

Qualitative Phytochemical Screening of Macrofungi Samples …………….... 14

Larvicidal Activity Assessment …………………………………………….... 18

Data Processing ………………………..……………………………………... 18

CHAPTER IV: RESULTS ……………………………………………………………. 19

CHAPTER V: DISCUSSION ……………………………………………………….... 22

CHAPTER VI: CONCLUSION AND RECOMMENDATIONS ……………………. 24

REFERENCES ……………………………………………………………………...... 25

APPENDICES ………………………………………………………………………... 54

CURRICULUM VITAE …………………………………………………………….... 58

i

LIST OF TABLES

Table Page

1 Qualitative phytochemical screening of the secondary metabolites

in crude methanol extracts from Pleurotus florida …………………………. 19

2 Mean ± SD Mortality of Aedes aegypti Larvae in varying

concentrations of crude methanolic extract of P. florida under

24 hours and 48 hourse exposure …………………………………………… 20

3 Number of dead larvae per concentration for Trial 1, Replicate 1 ………...... 54

4 Number of dead larvae per concentration for Trial 1, Replicate 2 ………….. 54

5 Number of dead larvae per concentration for Trial 1, Replicate 3 ………….. 55

ii

LIST OF FIGURES

Figure Page

1 Percent larval mortality of Aedes aegypti larvae in

varying concentrations of crude methanolic extract of

P. florida under 24 and 48 hours exposure …………………………………… 21

2 Pleurotus florida mushroom …………………………………………………. 56

3 Larvicidal activity assessment set-up ……………………………................... 57

4 Certificate of Registration from the Research Grants

Administration Office ……………………………………………………….. 58

iii

LIST OF APPENDICES

Page

Appendix A ………………………………………………………………………….. 54

Appendix B ………………………………………………………………………….. 56

Appendix C ………………………………………………………………………….. 57

Appendix D ………………………………………………………………………….. 58

ACKNOWLEDGEMENTS

We would like to extend our sincerest gratitude to our thesis adviser, Professor

Glenn Sia Su, who has been supportive of our endeavours. This scientific research would

not have been possible without his presence and guidance in the process of writing this

undergraduate thesis.

We thank our thesis panel member, Professor Myra S. Mistica, for imparting her

expertise and knowledge and for allowing us to use their laboratory for the completion of

our experiment protocol. We also thank Dr. Sharon Villanueva for her recommendations

toward the improvement of our thesis.

Our thanks also goes to the Department of Parasitology of the College of Public

Health for permitting us to use their Medical Entomology Laboratory for the duration of

our experiment proper.

We extend our gratitude to our parents and our beloved families for providing the

emotional, moral, and financial support that we need in order to duly comply with the

requirements of this thesis. Thank you for being our motivation and inspiration all

throughout this journey.

We thank Mr. Rodrigo Arellano Jr. for being accommodating and readily

providing us the mushroom sample.

Thank you to our dear batchmates and friends for extending their hands and

words of encouragement in times we mostly needed them.

Lastly, we would like to raise all our gratitude and praises to God. We surrender

this glory in His name. All these were possible because of Him who guided and

strengthened us.

iv

ABSTRACT

Mosquito borne diseases are responsible for causing numerous morbidities and

mortalities worldwide. The need to control these mosquitoes is a must to help curb the

problem brought about by these mosquitoes. Chemical insecticides and commercial

larvicides are used to control these mosquito populations, however, the use of these

chemicals bring about detrimental effects to other organisms and the environment.

Hence, there is a need to find natural products that can act as an alternative in controlling

mosquito populations. This study aims to determine whether the crude methanolic extract

of Pleurotus florida (white oyster mushroom) is able to kill A. aegypti mosquito larvae

and to elucidate the presence of bioactive compounds through qualitative phytochemical

screening. The mushroom samples were collected, air-dried, and crude methanolic

extracted and screened for the presence of secondary metabolites namely flavonoids,

alkaloids, tannins, phytosterols, anthraquinone, glycosides, saponins, resins, reducing

sugars, carbohydrates, and proteins. Larvicidal activity was assessed using a modified

larvicide bioassay using three concentrations (v/v): 5%, 2%, and 1%. Results show that

only flavonoids, carbohydrates, alkaloids, glycosides, saponins, and phytosterols are

present. Complete larval mortality at 5% concentration v/v after 24 and 48 hours post-

exposure. Gradual larval mortality was observed in both the 1% and 2% concentrations

from 24 to 48 hours. All concentrations exhibited higher larval mortality relative to the

positive control (Piper nigrum), which had a 43.33% larval mortality. The P. florida

crude methanolic extract can be a potential larvicide against A. aegypti larvae.

Keywords: Pleurotus florida, Aedes aegypti, dengue virus, larvicide, phytochemical

CHAPTER I: INTRODUCTION

Background of the Study

Mosquito-borne diseases such as malaria, dengue fever, Japanese encephalitis,

filariasis, Chikungunya, and Zika virus affect millions of people (WHO, 2016). These

diseases affect people all around the world with more than a million cases of death each

year (CDC-NIOSH, 2016; WHO, 2016; Niang et al., 2018). Common mosquito genera

like Anopheles, Aedes, and Culex are recognized as important biological vectors of

protozoan, viral, and nematode pathogens (Rueda, 2007; Wilke & Marrelli, 2015). There

are approximately 500 million cases of malaria occurring every year with 2.7 million

deaths (WHO, 2013). A study by Brady et al. (2012) indicated that dengue virus brings

about harm to at least 128 countries and threatens the health of about 4 billion people.

These numbers do not represent the entire population who were affected and eventually

died as there are still cases that remain unreported and unrecorded, particularly in the

rural areas (Diaz-Quijano, 2015; de los Reyes & Escaner, 2018).

Dengue disease is predominantly a health concern in the South-East Asian region

and the Western Pacific region with approximately 75% of all globally-reported cases

occurring in the mentioned regions alone (Ferreira, 2012; de los Reyes & Escaner, 2018).

The Western Pacific region includes the Philippines, and its neighboring nations like,

Cambodia, Malaysia, etc. (WHO, 2014). Dengue is one of the most prevalent infectious

illnesses in the country (DOH, 2011). There are 59,139 cases of dengue with 237 deaths

in the Philippines as of April 1, 2019, which is higher than the recorded 32, 611 cases and

175 deaths during the same time last year based on the recent report by WHO (2019).

1

The Aedes aegypti mosquito is the primary vector for all five serotypes of the

dengue virus (Taylor-Robinsons, 2016). A. aegypti is also the common vector of other

diseases such as the Chikungunya virus, the Zika virus, and the yellow fever (Pialoux et

al., 2007; Vasconcelos et al., 2018).

Efforts to control these mosquitoes have been implemented through the years.

Control measures, which includes the use of chemical insecticides to kill the adult

mosquito and the use of commercial larvicides to prevent the larvae from developing into

adulthood, have helped in preventing the occurrence of mosquito-borne diseases (Pansit

et al., 2018). The use of synthetic pesticides, such as insecticides and larvicides, remains

successful in reducing the number of these disease vectors. However, overuse of these

substances bring about negative repercussions to the health and to the environment

(WHO, 2018), wherein there are possibilities that these substances may even enter the

food chain and affect the overall welfare of the general public (Oliveira et al., 2011).

Hence, there is a need to find better alternatives that can help control these vectors from

spreading more of these mosquito-borne diseases.

Currently, there are various researches (Rahuman et al., 2007; Arriaga, et al.,

2009; Hafeez et al., 2011; Ghosh et al., 2012; Kumar et al., 2014) on the potential use of

plant extracts as an alternative larvicide. There are also a few larvicidal studies that

evaluate mushroom extracts (Njogu et al., 2010; Thongwat et al., 2015; Carapeto, et al.,

2017; Chaiphongpachara et al., 2018) since there are also compounds found in some

mushrooms that are toxic to insects such as octenol, ibotenic acid, and muscimol

(Cárcamo et al., 2016; Chaiphongpachara et al., 2018). Xylaria nigripes, Chlorophyllum

2

spp., Steccherinum spp. and Thaeogyroporus porentosus are some of the mushrooms

which possess larvicidal activity against mosquitoes (Chaiphongpachara et al., 2018).

Pleurotus spp. are one of the most studied mushrooms since it exhibits numerous

biological activities due to the beneficial bioactive molecules it contains (Jose et al.,

2004; Bellettini et al., 2017; Soni & Soni, 2017). There is that likelihood that such

bioactive molecules in the mushroom can bring about a larvicidal activity

(Chaiphongpachara & Laojun, 2018). Mosquito-related diseases are still rising despite the

efforts on mosquito control. Hence, this prompted the researchers to assess whether the

crude methanolic extract of Pleurotus florida may potentially be a larvicide. Results of

this study is vital as it can provide baseline information on the larvicidal activity of the

specified mushroom.

Statement of the Problem

Does the crude methanolic extract of Pleurotus florida exhibit a larvicidal

activity?

Objectives of the Study

The study aims to determine the potential larvicidal activity of the crude

methanolic extract of Pleurotus florida mushroom on the Aedes aegypti mosquito larvae.

Specifically, it aims to (1) assess whether the P. florida extracts will be able to

kill the A. aegypti mosquito larvae; and (2) determine the presence of certain bioactive

compounds through qualitative phytochemical screening.

3

Significance of the Study

This study will provide baseline information as to whether the mushroom extracts

of Pleurotus florida can be potentially used as a means to control the population of

mosquito larvae, particularly Aedes aegypti. It can likewise lead to the possible

development of alternative larvicides that can reduce health risks on the health of humans

and that of the environment.

Scope and Limitations of the Study

This study will only assess the mushroom Pleurotus florida (Mont.) Singer.

Samples will be acquired from a private mushroom cultivation farm in Brgy. Kaunlaran,

Cubao, Quezon City, Philippines. No sample will be obtained in the wild. Only

qualitative phytochemical screening and larvicidal activity will be determined and

assessed. The extract will only be applied on laboratory-reared third instar Aedes aegypti

larvae. All experiments will be administered in the Medical Entomology Laboratory of

the College of Public Health at the University of the Philippines Manila.

4

CHAPTER II: REVIEW OF RELATED LITERATURE

Dengue virus: a global public health threat

Dengue is the most prevalent arboviral disease affecting more than 50 million

people annually in the tropical and subtropical regions (WHO, 1999), with death rates

between 0.03% and 1.4% (WHO, 2005). Dengue virus (DENV) is a member of the

Flavivirus genus with a positive-sense single-stranded RNA (Costa et al., 2012) and is

usually transmitted by the female mosquitoes of Aedes aegypti (de los Reyes & Escaner,

2018).

Dengue virus is known to have four serotypes, DENV-1, DENV-2, DENV-3, and

DENV-4, wherein each serotype differs in terms of their clinical manifestations and

pathogenic properties (Balmaseda et al., 2006). These four were the serotypes known for

decades until it was reported on October 2013 that a fifth dengue serotype exists (Mustafa

et al., 2015). According to WHO (2017), being infected by one of these serotypes does

not guarantee immunity against the other serotypes. The transmission of the virus

involves a host-vector-host interaction through viremia at which the vector imbibes blood

from a viremic individual and transmits the virus to a secondary human host via the

mosquito’s saliva (Carrington & Simmons, 2014). Dengue diseases can vary based on

severity -- from the common dengue fever to severe dengue, which includes dengue

hemorrhagic fever or dengue shock syndrome (Wearing & Rohani, 2006). Symptoms

include high fever which lasts from 2 days to about a week (Hasan et al., 2016),

5

headache, retroocular pain, muscle and joint pain, nausea, vomiting, and rash

(Kalayanarooj et al., 1997).

The number of dengue fever and dengue haemorrhagic fever cases worldwide has

greatly increased over time (Messina et al., 2014). The recorded average of dengue cases

from 2000-2004 was 925,896, which is almost twice the recorded cases from 1990-1999

(Guzman et al., 2010). According to WHO (2019), 2016 was the year with massive

outbreak of dengue worldwide wherein 375,000 cases were reported in the Western

Pacific Region, with 176,411 cases from Philippines and 100,028 cases from Malaysia.

Dengue is one of the prevalent diseases in the Philippines (DOH, 2011) with a total of

585,324 cases with 3195 deaths reported from 2008 to 2012 (DOH, 2012).

According to the recent dengue update by WHO (2019), there are 43,065 cases

and 67 deaths reported in Malaysia as of April 27, 2019, which is twice more than the

recorded 19,348 cases and 36 deaths in 2018. There is also an increasing dengue activity

in Singapore, with 2,752 recorded cases as of week 17 of 2019 compared to the 790 cases

recorded in the same week in 2018. The recorded dengue cases throughout Southeast

Asia, America, and Western Pacific was over 1.2 million in 2008 and more than 3 million

in 2013, wherein 2.35 million cases were from the Americas alone (Sanyaolu et al.,

2017). Dengue is now a concern even in Europe, wherein the first dengue transmission

was reported in France and Croatia in 2010 (WHO, 2017). There has been a sudden surge

in dengue cases recorded in Madeira, a Portuguese autonomous region, with more than

2200 cases between October 2012 and January 2013 (Schaffner & Mathis, 2014).

6

Dengvaxia is the first dengue vaccine to be developed and is currently licenced in

19 countries, including the Philippines (Fatima & Syed, 2018). This vaccine is the result

of years of search for possible immunization against dengue, but it still does not cover all

four dengue serotypes; thus, vector monitor and control measures are still the only means

to reduce dengue transmission (Qi et al., 2008; Guzmán & Kourí, 2001).

Aedes aegypti

Aedes aegypti is the primary vector of dengue virus, Chikungunya, and Zika virus

(Powell et al., 2018). This species belongs to the Order Diptera, alongside the true flies.

A. aegypti females prefer humans over other vertebrates as their food source (Ponlawat &

Harrington, 2005). They are capable of generating up to five sets of eggs in a lifetime

(Nelson, 1986) and would usually deposit their eggs in water tanks, flower vases,

buckets, or other water containers (Getachew et al., 2015). Their copulatory period lasts

for less than a minute, providing them the capacity to reproduce in a short span of time

(Oliva et al., 2013). They undergo complete metamorphosis, comprising the following

stages: an egg, larva, pupa, and adult stage (Goma, 1966). Moreover, they can live from

two weeks to a month depending on the existing environmental conditions (Muktar et al.,

2016).

A. aegypti mosquitoes are known to feed during the day (Ndenga et al., 2017) and

generally do not leave the place where they were laid. However, those that relocate can

only travel a maximum distance of 200 meters (Harrington et al., 2005).

7

Dengue management and vector control

It was only recently that the scientific community has developed and introduced

the first vaccine concocted for the dengue virus. This is considered as a breakthrough in

the global brawl against the deadly dengue virus. However, it still has limitations such

that it can only be effective once (WHO, 2017). It was also mentioned that an individual

who was once infected with any of the dengue serotypes and successfully treated with the

dengue vaccine will not have the same response once another serotype infects him/her.

The vaccine will be rendered inefficient and may just worsen the situation that may even

lead to death.

Rather et al. (2017) summarized dengue prevention and control strategies and

divided these into three categories, namely: physical control, biological control, and

chemical control. They included GIS mapping (Chang, et al., 2009; Eisen &

Lozano-Fuentes, 2009), effective monitoring and surveillance (Racloz et al., 2012),

determine behaviors and oviposition locations (Harrington et al., 2008; Ponnusamy, et al.,

2008; Wong et al., 2011), community-based programs (Gubler & Clark, 1994; Winch, et

al., 2002; Heintze et al., 2007), and educational strategies (Madeira et al., 2002; Khun &

Manderson, 2007; Jayawardene et al., 2011) for the physical control; use of Wolbachia

and recombinant bacteria (Federici, 2003; Moreira, et al., 2009; Hoffmann, et al., 2011;

Walker, et al., 2011), genetic modifications and paratransgenesis (Coutinho-Abreu et al.,

2010; Chavshin, et al., 2014; Wilke & Marrelli, 2015), usage of sterilized male

mosquitoes (Thomé et al., 2010; Valdez, et al., 2011; Benelli & Mehlhorn, 2016), and use

of larvivorous aquatic animals (Lardeux, 1992; Benelli et al., 2016; Pereira, et al., 2016;)

8

for biological control; and usage of insecticides, larvicides, etc. (Shaalan et al., 2005;

Deomena, et al., 2007; Pitasawat, et al., 2007; Ballenger-Browning & Elder, 2009; Ghosh

et al., 2012; Tennyson et al., 2012; Govindarajan, 2016), insect growth regulators

(Rodrigues & Wright, 1978; Yapabandara & Curtis, 2002; Vythilingam et al., 2005), and

pheromones and integrated pest management (Wynand Van Der Goes Van Naters &

Carlson, 2006; Rosell et al., 2008) for the chemical control.

The incidence of dengue cases has continuously increased in the past 50 years

despite the strategies implemented (Achee et al., 2015; Viennet et al., 2016). The actual

impact of dengue is perhaps greater than the reported cases since there are issues

regarding diagnosis and disease surveillance (Beatty et al., 2011; Gubler, 2011; Gubler,

2012). Researchers presume that the reported cases of dengue would further increase in

the future (Hales et al., 2002; Wilder-Smith & Gubler, 2008; Wilder-Smith et al., 2010;

Astrom et al., 2012). Problems affecting the effectiveness of the use of insecticides arise

due to improper usage of the insecticide equipment, unmonitored and inaccurate dosage

use, negative feedback and response from the public, and inadequacy to target vectors

(Chang et al., 2011). Moreover, pesticides are known to present negative impacts such as

contamination and degradation of the environment (Younes & Galal-Gorchev, 2000;

Turgut, 2003; Carvalho, 2017), pesticide intoxication and mutagenic effects in humans

(Bolognesi, 2003; Gomez-Arroyo et al., 2011; Sutris, et al., 2016), ecosystem imbalance

and food-web interference (Damalas & Eleftherohorinos, 2011), contamination of

agricultural produce (Schecter et al., 2010; Sun et al., 2016), soil and aquatic

microorganism population decline (Aktar et al., 2009; Kalia & Gosal, 2011). These

9

circumstances have raised efforts in reevaluating and improving existing strategies

(WHO, 2013) and discovering better alternatives (Ladner et al., 2017).

Larvicides: its advantages and challenges

The utilization of insecticides and larvicides is the primary vector control

measures implemented (Chang et al., 2011). The use of larvicides is a better and more

efficient strategy in targeting and controlling dengue vectors compared to other

insecticides, since mosquito larvae are typically deposited in large quantities and in small

areas (Becker et al, 2010).

Recent studies (Rodríguez et al., 2002; Braga et al., 2004; Seccacini et al., 2008;

Prophiro, et al., 2011) reported that vectors Aedes aegypti and Aedes albopictus have

developed resistance on common insecticide compounds such as organophosphate

temephos and pyrethroids. This is attributed to extensive use of vector control chemicals

and insufficient management of these methods thereafter (Morrison et al., 2008), which

has led to selection pressures inducing resistance (Paeporn, et al., 2003; Vontas, et al.,

2012).

Chemical pesticides with organochlorine, particularly DDT

(dichlorodiphenyltrichloroethane), HCHs (hexachlorocyclohexane isomers), aldrin,

dieldrin, and chlordanes are reported to have significantly accumulated in living

organisms, which are toxic to one’s overall health (Senthilkumar et al., 2001; Jayaraj et

al., 2016). According to Chelela et al. (2014), synthetic insecticides such as

organophosphorus, carbamates, pyrethroids, and pyrethrins are hazardous when overused,

10

since they may also be toxic to non-target organisms and the mosquitoes may develop

resistance against the compounds. Overexposure to these compounds may cause

endometriosis, neurotoxicity, immunotoxicity, and poisoning (Kleanthi et al., 2008;

Silberman & Taylor, 2018; Robb & Baker, 2019). There are also studies which suggest

that the constant application of Bacillus thuringiensis israelensis (Bti), a group of bacteria

used as a larvicide, for over 2-3 years to wetlands would result to a decrease in the overall

biodiversity (Siegel & Shadduck, 1990).

There have been researches (Row & Ho, 2009; Patil et al., 2010; Ghosh et al.,

2012) on the alternative larvicides using plant extracts due to the risks mentioned. In a

study by Pansit et al. (2018), Citrofortunella microcarpa and Carica papaya extracts

were used against Aedes sp. and results showed that they can be used as a larvicide,

surpassing the larvicidal activity of temephos, a commercial larvicide. They mentioned

that the aforementioned extracts’ larvicidal activity may be due to the biological

compounds present such as alkaloids, flavonoids, saponins, and tannins. There are studies

(Thongwat et al., 2015; Cárcamo et al., 2016; Chaiphongpachara et al., 2018) proving

that mushroom extracts can also be used as a larvicide, namely Lactarius densifolius,

Lactarius gymnocarpoides, Russula cellulata, Russula kivuensis, Amanita phalloides, and

Boletus species (Chelela et al., 2014).

Pleurotus florida (Mont.) Singer

The second most widely cultivated and produced mushrooms in the world are the

oyster mushrooms (Chang & Miles, 1991; Erkel, 1992; Ahmed et al., 2009; Royse,

11

2014). These mushrooms belong to the Pleurotus genus which is a part of the white-rot

basidiomycete fungi. According to Adebayo and Oloke (2017), this genus is famous for

its nutritional value and can readily grow on a wide cluster of forest and agricultural

wastes.

Pleurotus mushroom is the cheapest and easiest to grow among the cultivated

edible mushrooms (Alkoaik, 2015). Some examples include P. ostreatus, P. eryngii, P.

cystidiosis, P. flabellatus, P. cornucopie, P. sajor-caju, and P. florida. These species are

low maintenance mushrooms since they only require a few environmental requirements

for them to be cultivated and be maintained (Sanchez, 2010).

P. florida contains different biological compounds such as phenols, saponin,

flavonoids, steroids, terpenoids, and alkaloids (Menaga et al., 2012; Muthukumaran et al.,

2014). They also exhibit numerous biological activities, which includes antimutagenic,

anti-inflammatory, immunomodulatory (Yashvant et al., 2012; Ganeshpurkar & Rai,

2013), antioxidant (Prabu & Kumuthakalavalli, 2016; Khatun et al., 2014), and

antimicrobial (Menaga et al., 2012; Kumar et al., 2017) among many others. This

mushroom may be a potential larvicide considering the phytochemicals present and the

bioactivities it exhibits.

12

CHAPTER III: METHODOLOGY

I. Collection, Air-drying, and Preparation of Macrofungi Samples

The site of collection was a local macrofungi cultivation farm situated in Brgy.

Kaunlaran, Cubao, Quezon City, Metro Manila, Philippines. The samples were cut or

pulled out from their corresponding substrates in the facility.

The air-drying procedure were based on the study by Deka et al. (2017) and were

done in the laboratory at the University of the Philippines Manila. The freshly-collected

macrofungi samples were weighed, cleaned with a dry brush and paper towel, and cut

into pieces prior to air-drying. They were then placed into nets and hanged for air drying.

The air-dried mushrooms were weighed and homogenized using a blender until it reaches

its powdered form once the samples were fully dried. The samples were weighed once

again after homogenization and the powdered samples were temporarily stored in

separate containers preceding the preparation of macrofungi extracts.

The methanol extract was prepared using 100 g of the powdered sample soaked in

1000 ml of 95% methanol in an Erlenmeyer flask which was covered with parafilm. They

were placed in a dark area for three days with occasional stirring. The extract was then

filtered using Whatman No.1 filter paper, and the filtrate was submitted to the National

Institutes of Health - Institute of Pharmaceutical Sciences (NIH-IPS) for extraction and

qualitative phytochemical screening of the crude methanolic extract.

The percentage of extraction was calculated using the following formula,

Extraction (100%)% = W eight of extractW eight of powdered material

13

II. Qualitative Phytochemical Screening of Crude Methanolic Extract

The macrofungal extracts were screened for the following phytochemicals:

flavonoids, alkaloids, tannins, phytosterols, anthraquinone, glycosides, saponins, resins,

reducing sugars, carbohydrates, and proteins by standard procedures (Johansen, 1940;

Clarke, 1975; Harborne, 1984; Sofowora, 1993; Singh & Bag, 2013). Results were

qualitatively expressed as either present or absent.

Test for Alkaloids (Johansen, 1940)

Hager’s Test

A few drops of Hager’s reagent was added to 2 mg of the crude methanolic

extract in a test tube. The presence of a yellow precipitate or turbid solution confirms the

presence of alkaloids.

Mayer’s Test

Two milligrams of the crude methanolic extract was added with a few drops of

Mayer’s reagent. The formation of a white precipitate or turbid solution indicated a

positive result.

Wagner’s Test

Two milligrams of the crude methanolic extract was acidified by adding 1.5% v/v

hydrochloric acid. A few drops of Wagner’s reagent was then added and the formation of

a reddish brown or turbid solution was noted.

14

Test for Anthraquinone (Harborne, 1984)

Diluted sulfuric acid was added to 3 mL of crude methanolic extract. The solution

was then boiled and filtered. Equal volume of benzene was added after the solution was

cooled. The organic layer was separated after shaking the solution and an equal volume

of diluted ammonia solution was added to the organic layer. A positive result is indicated

by the change of the ammonia layer into a pink or red solution.

Test for Carbohydrates (Sofowora, 1993)

Molisch Test

Three milliliters of the extract was mixed with 2 mL of Molisch’s reagent. Two

milliliters of concentrated sulfuric acid was added carefully down the side of the test

tube. The emergence of a violet ring between the two layers indicated a positive result.

Test for Flavonoids (Singh & Bag, 2013)

Alkaline Reagent Test

A few drops of sodium hydroxide was added to the extract. The formation of a

yellow solution which disappears upon the addition of dilute acetic acid indicated a

positive result

Lead Acetate Test

The extract was added with a few drops of 10% lead acetate solution. The

formation of a yellow precipitate indicated the presence of flavonoids.

15

Test for Glycosides (Harborne, 1984)

Keller killani Test

One milliliter of glacial acetic acid, a drop of 5% ferric chloride, and concentrated

sulfuric acid were added to 2 mL of the crude methanolic extract. The formation of a

reddish brown ring at the junction of the layers indicated the presence of glycosides.

Test for Phytosterols (Clarke, 1975)

Liebermann-Burchard Test

Ten milliliters of anhydrous chloroform was added to 0.5 g of the crude

methanolic extract. One milliliter of acetic anhydride was added to the solution and was

followed by the addition of 1 mL of concentrated sulfuric acid down the side of the test

tube. The presence of a brown ring indicated the presence of phytosterols.

Test for Proteins (peptide bonds) (Harborne, 1984)

Biuret Test

One to two drops of Biuret reagent was added to a small quantity of the extract.

Formation of purple or violet precipitate indicated the presence of proteins.

Test for Reducing Sugars (Johansen, 1940)

Fehling’s Test

Two milligrams (2 mg) of the extract was shaken with 10 mL of water, filtered

and the filtrate was concentrated. One milliliter of the solution was added to equal parts

16

of Fehling’s solution A and B and boiled for few minutes. The presence of reducing sugar

was indicated by the formation of red or brick-red precipitate.

Test for Resins (Johansen, 1940)

One milliliter (1 mL) of the extract was dissolved in acetone and was then poured

in distilled water. The presence of resins was determined by the presence of turbidity.

Test for Saponins (Clarke, 1975)

Froth Test

Three millilitres (3 mL) of the extract were mixed to 10 mL of distilled water in a

test-tube. The test-tube was stoppered and shaken vigorously for about 5 minutes and was

allowed to stand for another 30 minutes. The presence of a honeycomb froth indicated the

presence of saponins.

Test for Tannins (Clarke, 1975)

Ferric Chloride Test

Two millilitres (2 mL) of the extract were added to a few drops of 10% FeCl

solution (light yellow). A blackish-blue color indicated the presence of gallic tannins,

while a green-blackish colour indicated presence of catechol tannins.

III. Larvicidal Activity Assessment

17

Larvicidal activity was assessed through a modified larvicidal bioassay. The crude

methanolic extract was directly used as the starting solution. Three extract concentrations

were used (v/v %): 5%, 2%, and 1%. The extract concentrations were then added to 75

mL distilled water in glasses. Ten third instar larvae were also added to each glass using a

loop. The control groups were: (a) Piper nigrum as the positive control; and (b) distilled

water as the negative control. The positive control is an organic-based larvicide crudely

prepared by DOST and used in ovitrapping. Distilled water was used as the negative

control to maintain the typical physiological condition of the mosquito larvae. The

number of dead larvae was counted and recorded after a period of 24 and 48 hours for

each concentration. A larva was considered dead if there is no apparent movement even

after gentle probing. All experiments for each treatment was done in triplicate.

IV. Data Processing and Statistical Analysis

The number of dead larvae was presented as mean ± standard deviation (S.D.).

Larval mortality was corrected and calculated should the control mortality be between

5% and 20% using the Abbott’s formula,

ortality (%) (100%) M = XX −Y

where X = percentage survival in the untreated control and Y = percentage survival in the

treated sample.

18

CHAPTER IV: RESULTS

The Pleurotus florida mushroom were acquired from a cultivation farm situated

in Brgy. Brgy. Kaunlaran, Cubao, Quezon City, Metro Manila, Philippines. A total of 5

kilograms of mushroom samples were cut into pieces, air-dried for a month, and

homogenized. The total dry weight used for extraction was 300 grams. The crude

methanolic extract of P. florida mushroom was assayed to determine the potential

metabolites present. Table 1 shows the result of the qualitative phytochemical of P.

florida crude methanolic extracts.

Table 1. Qualitative phytochemical screening of the secondary metabolites in crude methanol extracts from

Pleurotus florida

Secondary Metabolites Result (Positive or negative)

Alkaloids Positive

Carbohydrates Positive

Flavonoids Positive

Glycosides Positive

Phytosterols Positive

Saponins Positive

Anthraquinone Negative

Proteins (peptide bonds) Negative

Tannin Negative

Reducing Sugars Negative

Resins Negative

19

The crude methanolic extract yield after the rotary evaporation procedure was

35.57 grams. A total of 150 third instar laboratory-reared Aedes aegypti larvae were used

for the larvicidal bioassay under 24 and 48 hours post-exposure. Table 2 shows the mean

± SD mortality of A. aegypti larvae in varying concentrations of the crude methanolic

extract of P. florida at 24 hours of exposure. Piper nigrum is used as the positive control

because of its efficacy as a plant-based larvicide (Briones & Garbo, 2016) due to the

presence of isobutylamide alkaloids, particularly pellitorine, guineensine, pipercide, and

retrofractamide (Park et al., 2002).

Table 2. Mean ± SD Mortality of Aedes aegypti Larvae in varying concentrations of crude methanolic extract of P. florida under 24 hours and 48 hourse exposure

Concentration

24 hours 48 hours

Mean ± SD Mortality Percentage Mean ± SD

Mortality Percentage

5% 10.00 ± 0.00 100.00% 10.00 ± 0.00 100.00%

2% 9.67 ± 0.47 96.67% 10.00 ± 0.00 100.00%

1% 5.67 ± 3.09 56.67% 10.00 ± 0.00 100.00%

Positive control (Piper nigrum) 4.33 ± 1.70 43.33% 7.00 ± 0.82 70.00%

Negative control 0.00 ± 0.00 0.00% 0.00 ± 0.00 0.00%

All mosquito larvae placed in 5% concentration died after 24 hours (Table 2). The

recorded larval mortality at 5% and 2% concentrations are greater than the observed

death in the positive control treatments. All larvae placed in 5%, 2%, and 1%

concentrations died after 48 hours, while 70% of the larvae placed in the positive control

20

died. Aedes aegypti larval mortality is observed to be 100% for 5% concentration of the

crude methanolic extract of P. florida at both 24 to 48 hours. There is a gradual larval

mortality observed for the concentrations 2% and 1% crude methanolic extract of P.

florida from 24 to 48 hours exposure.

Figure 1. Percent larval mortality of Aedes aegypti larvae in varying concentrations of crude methanolic

extract of P. florida under 24 and 48 hours exposure.

21

CHAPTER V: DISCUSSION

Qualitative Phytochemical Screening of the Methanolic Extract

The qualitative phytochemical screening results indicate the presence of

flavonoids, carbohydrates, alkaloids, glycosides, saponins, and phytosterols in the crude

methanolic extract of P. florida mushroom. In a study by Prabu and Kumuthakalavalli

(2014), it was reported that the qualitative phytochemical analysis of the methanolic

extracts of P. florida confirmed the presence of secondary metabolites namely,

flavonoids, saponins, and sterols. The presence of carbohydrates and glycosides was also

reported (Menaga et al., 2012). Our results corroborate with other studies (Prabu &

Kumuthakalavalli, 2014; Menaga et al., 2012; Muthukumaran et al., 2014) done

indicating the presence of the substances in the crude methanolic extract of P. florida

mushroom.

Plants are known to possess secondary metabolites such as alkaloids, phenolics,

flavonoids, steroids, tannins, saponins, and essential oil (Crozier et al., 2006), which aid

in protecting the plant against herbivores and microbial infections (Danga et al., 2014).

Nicotine, ryanodine, and anabasine are the alkaloids which are often used as pesticides

(Mann & Kaufman, 2012). Alkaloids have varying mode of action on insect vectors

depending on their molecular structure, but a lot of them have an effect on

acetylcholinesterase and sodium channels (Flattum & Shankland, 1971; Amar et al.,

1991). Saponin also has an effect on the larvicidal activity because as it comes in contact

with the cuticle membrane of the larvae, the membrane may be disorganized, resulting to

the death of the larva (Pansit et al., 2018). Some of the flavonoids such as

22

karanjachromene, pongamol, and pongatorene were reported to restrain mosquito larval

cholinesterase (Permalsamy et al., 2015).

Larvicidal Activity Assessment

Mushrooms under the Basidiomycota division are known to have exhibited

larvicidal activity on different mosquito larvae, alongside other biological activities such

as antimicrobial and nematicidal (Sivanandhan et al., 2017). Extracellular mushroom

compounds, 4-(2-hydroxyethyl)phenol, tyrosol, 2-hydroxy-4-(4-hydroxychroman-7yl)

But-3-enal, and 3-methoxy-5-methyl-1,2-benzenediol, were recently isolated from a

basidiomycete culture (Basidiomycete JO5289), successfully showing potential larvicidal

activity (Kipngeno, 2010; Kipngeno et al., 2013).

Our results have showed high mortality upon the exposure of the crude

methanolic extract of P. florida to the A. aegypti larvae at 24 to 48 hours. Studies

(Chelela et al., 2014; Cárcamo et al., 2016; Thongwat et al., 2015) in the past have

indicated that mushrooms have larvicidal activities. A study by Chaiphongpachara et al.

(2018) have reported that P. pulmonarius ethanolic extract did not kill A. aegypti larvae,

but killed Culex sitiens larvae with mean ± SD of 3.00±1.73, 4.00±2.65, 4.50±2.00,

4.00±1.00, and 4.50±0.50 at 0.012 mg/L, 0.12 mg/L, 1.2 mg/L, 12 mg/L, and 120 mg/L

concentrations respectively. They may belong to the same genus, but their activity may

be different depending on the solvent used for extraction which can affect the percentage

extraction and the secondary metabolites present (Prabu & Kumuthakalavalli, 2014).

23

CHAPTER V: CONCLUSION

In conclusion, the crude methanolic extract of Pleurotus florida can be a potential

larvicide for the Aedes aegypti mosquito. The P. florida crude methanolic extract showed

qualitatively the presence of the following secondary metabolites namely, flavonoids,

carbohydrates, alkaloids, glycosides, saponins, and phytosterols. The crude methanolic

extract of P. florida killed the A. aegypti larvae completely at 5% concentration in both

24 to 48 hours exposure. Gradual mortality of the A. aegypti larvae was observed for both

the 2% and 1% concentrations of the crude methanolic extract of P. florida was seen

from 24 to 48 hours.

24

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APPENDIX A

Table 3. Number of dead larvae per concentration for Trial 1, Replicate 1.

Concentration Duration

After 24 hours After 48 hours

5% 10 10

2% 10 10

1% 3 10

Positive control 6 7

Negative control 0 0

Table 4. Number of dead larvae per concentration for Trial 1, Replicate 2.

Concentration Duration

After 24 hours After 48 hours

5% 10 10

2% 9 10

1% 10 10

Positive control 5 6

Negative control 0 0

54

Table 5. Number of dead larvae per concentration for Trial 1, Replicate 3.

Concentration Duration

After 24 hours After 48 hours

5% 10 10

2% 10 10

1% 4 10

Positive control 2 8

Negative control 0 0

55

APPENDIX B

Figure 2. Pleurotus florida mushroom

56

APPENDIX C

Figure 3. Larvicidal activity assessment set-up

57

APPENDIX D

Figure 4. Certificate of Registration from the Research Grants Administration Office

58