Post on 25-Jan-2023
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
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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.
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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
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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