Post on 19-Aug-2020
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Saint Mary’s University
Junior High School and Science High School
Bayombong, Nueva Vizcaya 3700
PAASCU Re-Accredited Level II
PHYTOCHEMICAL SCREENING, ANTIMICROBIAL AND
CYTOTOXICITY PROPERTIES OF PIKAW (Colocasia sp. cf. formosana
Hayata)
A Science Research Project
Samuel Levine L. Soliven
Ferylene C. Valentin
Felice Alexandria M. Sadueste
Student Researchers
Miss Elsa Cajucom, PhD
Research Adviser
October 2017
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TABLE OF CONTENTS
Title Page . . . . . . . . . . 1
Table of Contents . . . . . . . . . 2
Acknowledgement . . . . . . . . . 5
Acknowledgement . . . . . . . . . 6
Abstract . . . . . . . . . 7
INTRODUCTION
Background of the Study . . . . . . . 8
Statement of the Problem . . . . . . . 12
Statement of the Hypotheses . . . . . . . 12
Significance of the Study . . . . . . . 13
Scope and Limitations of the Study . . . . . . 13
Statistical Analyses . . . . . . . . 13
METHODOLOGY
Procedural Flowchart . . . . . . . . 16
Materials . . . . . . . . . 20
Methods/Procedures . . . . . . . . 20
Materials and Procedures (Documentation) . . . . . 27
Research Environment . . . . . . . 36
Experimental Designs . . . . . . . . 36
Treatment of Data . . . . . . . . 38
RESULTS AND DISCUSSIONS
DOST and CNS Lab Results on Phytochemical Screening of Pikaw
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Table 1: Phytochemical Components of Pikaw . . . 40
Antibacterial Properties of Pikaw
Table 2a: CNS Lab Results on Microbial Properties of Pikaw . 47
Table 2b: DOST Lab Results on Antibacterial Properties
of Pikaw . . . . . . . 47
Antifungal Properties of Pikaw
Table 3: DOST Lab Results on Antifungal
Properties of Pikaw Extract . . . . . 48
Table 4: Standard Zones of Inhibition of Selected Antifungal
Products on Candida albicans . . . . 48
Table 5: Comparison of Pikaw Ethanolic Extract Against
Selected Antifungal Products on Candida albicans . . 49
Cytotoxic Properties of Pikaw Ethanolic Extract
Table 6: CNS Lab Results on Cytotoxic Properties of Pikaw
Ethanolic Extract . . . . . . 52
CONCLUSIONS AND RECOMMENDATIONS . . . . . 54
BIBLIOGRAPHY . . . . . . . . . 55
APPENDICES
Appendix A. CNS Laboratory Results (Phytochemical) . . . . 58
Appendix B. CNS Laboratory Results (Bacteria) . . . . . 59
Appendix C. CNS Laboratory Results (Cytotoxicity) . . . . 60
Appendix D. DOST Laboratory Results (Phytochemical . . . . 61
Appendix E. DOST Laboratory Results (Bacteria and Fungus) . . . 62
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Appendix F. Laboratory Testing (Miconazole) . . . . . 64
Appendix G. Laboratory Testing (Fluconazole) . . . . . 65
Appendix H. Laboratory Testing (Clotrimazole) . . . . . 66
Appendix I. Laboratory Testing (Ketoconazole) . . . . . 67
Appendix J. Antifungal Properties of Miconazole, Fluconsazole,
Clotrimazole, Ketoconazole on Candida Albicans . . . 68
Appendix K. Letter to the Principal . . . . . . . 71
Appendix L. Letter to the Dean . . . . . . . 72
CURRICULUM VITAE . . . . . . . . 73
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ACKNOWLEDGEMENT
The young science researchers are grateful to the following:
Fr. Neil H. Sta. Ana, CICM, Dr. John Octavios S. Palina, Dr. Moises Alexander T. Asuncion and
Dr. Venica S. Acosta, the SMU Top Managers for making SMU a convenient place for scientific
investigation;
Mr. Melencio G. Bernardino Jr., SMU JHS/SHS Principal, Dr. Elsa L. Cajucom, Science
Research and Statistics 2 teacher and coordinator, Miss Khristine Ramirez, the adviser for the
inspiration and guidance given to the researchers;
Their parents Dr. and Mrs. Samuel R. Soliven, Mr. and Mrs. Federico S. Sadueste Jr., and Mr.
and Mrs. Ryan Valentin for the words of encouragement and assistance; and
Above all to God the Almighty for whom he owes everything.
The Researchers
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DEDICATION
We dedicate this project to our ever supportive parents Samuel and Anivel Soliven, Federico and
Ellen Sadueste, Ryan and Fema Valentin;
To our brothers;
To our friends, classmates, and teachers.
The Researchers
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ABSTRACT
The phytochemical, antibacterial, antifungal, and cytotoxic properties of the ethanol
extract of Pikaw (Colocasia sp. cf. formosana Hayata) were conducted in this study. These
were appropriately conducted in two accredited science laboratories, the Department of Science
and Technology in Tuguegarao City, Cagayan and Center for Natural Sciences, Saint Mary‘s
University in Bayombong, Nueva Vizcaya.
There are favorable results of the phytochemical screening, bacterial assay, fungal assay
and cytotoxicity assay of the Pikaw ethanolic extract. Pikaw has phythochemicals that include
flavonoids, tannins, saponins, essential oil, triterpenes, fatty acids, sugar, coumarins, anthrones,
phenols, alkaloids, steroids and anthraquinones.
In addition, pikaw ethanolic extract cannot inhibit bacteria S. aureus, E. coli and B.
subtilis but it has high ability to inhibit the fungus C. albicans. The range of the zones of
inhibition of Pikaw ethanolic extract on Candida albicans is 29-31. This range is comparable
with miconazole, cltrimazole and ketoconazole. Hence, the Pikaw ethanolic extract can be made
into products to serve as substitute of commercially available antifungal diseases caused by
Candida albicans.
Pikaw has also a cytotoxic property because after 18 hours the LC50 = 941.528 ppm,
after 21 hours the LC 50 = 743.894 ppm and after 24 hours the LC50=634.807 ppm.
Recommendations include the (1) preparation antifungal cream, ointment and other
antifungal products made out of pikaw ethanolic extract that resemble the commercial
preparations of miconazole, clotrimazole and ketoconazole; and (2) isolation the flavonoids since
this has a role on the cytotoxic property of the pikaw ethanolic extract then retest cytotoxicity.
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INTRODUCTION
Background of the Study
Nueva Vizcaya is blessed with flora from Diadi to Alfonso Castañeda. Its forest areas
have hidden water falls that contribute to its cool climate. Where perennial streams or springs
were located at the edge of forest, along roadsides is a morphologically distinct wild form or
species of taro (Colocasia sp. cf. formosana Hayata) growing on stream banks, and among rocks
next to small waterfalls. This wild taro is popularly known as the edible pikaw among Ilocano.
But there are many species of wild taro. Pikaw is just one of them. Matthews, et al.,
(2007) revealed that to understand the origins and history of a crop, it is necessary to consider
where wild populations of the same species were naturally distributed before people began
interacting with them and using them. Ethnographic observation of how people use wild
populations can also help by suggesting how wild plants were taken into cultivation, and how
they were domesticated. Domestication, or the genetic modification of a useful species, requires
selection from a variable population, and isolation of selected forms from the dominant genetic
influence of a wild gene pool. Their survey has not proven the existence of natural, indigenous
wild taro (C.esculenta) in the Philippines; wild taro is abundant in some areas, whether or not
breeding populations are present. Wild taro is economically significant, and is subject to loose
management as a community resource. Priorities for future research are on the natural and
cultural history of taro in the Philippines.
In the mountainous Ifugao region, in northern Luzon, wild taro did not appear abundant.
This might be due to the much dissected nature of the terrain, which made access difficult, and
the lack of large open habitats that are not closely controlled and cultivated. It is also possible
that the cooler climate of this northern mountain region is not ideal for wild taro. On the dry
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slopes of Mt Arayat, further south, and in western Luzon, wild taro was completely absent. By
contrast, wild taro was very abundant in open, wet, and humid areas, and at middle altitudes on
Mt Banahaw and Mt Apo. Possible source populations may or may not be located at higher
altitudes on these mountains, in less-disturbed and more inaccessible forest areas. The most
common phenotype seen, from Ifugao to Mt Banahaw and Mt Apo, displayed green leaves, with
white lower parts, relatively small corms, and long stolons. In all obvious respects, this is like the
wild type taro present in northern Australia, Papua New Guinea, Indonesia, Myanmar, and
Vietnam—all areas where flowering and breeding populations are present (observations by
Matthews, and others).
As noted in the introduction, Brown (1920) did not mention wild taro in his list of wild
food plants of the Philippines. He did indicate that ‗personal judgment‘ was used in making the
list, and that: ‗Many people may consider some of those (plants) treated in the present list as
worthless, and some common species which are omitted as good‘, and that his list was ‗probably
very far from complete‘. In later publications, Brown (1941) and Monsalud et al. (1966)
mentioned the use of other wild aroids, including species that are also cultivated, but made no
mention of taro as a wild plant. From their own observations, and those of their local informants,
it is clear that wild taro is widely used and popular as a vegetable food, mainly for the leaves, but
also to some extent for the stolons, and as a source of leaves for pig fodder.
Cordero-Fernando (1992) stated that people in the Bikol region (southern Luzon) cook
and eat only the leaves of the gabi plant, and that most gabi grow wild. In the past, wild taro may
have been important as a source of starch in times of hunger, and as a free and nutritious leaf
vegetable at all times, especially among rural households.
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Taro has been focused on cultivars that are grown primarily for their corms. Corms may
have more nutritional or economic value, as a starchy food and for trade, in most social contexts.
Pardales and Villanueva (1984) did not mention leaf varieties in their report on field trials with
Philippine and Hawaiian cultivars at Baybay in Leyte. Lebot et al. (2004) surveyed the corm
qualities of 2,298 taro accessions collected in Asia (including the Philippines) and the Pacific,
and also did not comment on the use of leaves. In Vietnam, a national collection of 350 taro
accessions included approximately 25% with edible petioles or young leaves (Nguyen, 2000).
Wild and cultivated taros with edible leaves are likely to be under-represented in national
collections of the Philippines and most other countries. To encourage further research on edible-
leaf forms of taro in the Philippines, a collection of several distinct forms of wild taro in the
vicinity of Mt. Banahaw, and transferred them to a living taro collection maintained by the
College of Agriculture, University of Los Baños.
In the study of Adaoag, pikaw is under classification Araceae. It is a herbaceous aroid. It
has an underground fleshy stem or corm that extends to a large cylindrical stalk, and wide ovate
leaves. The leaves are cuneate-based and acute-apexed. Clusters of naked female flowers are in a
compact mass on the basal part of a fleshy stalk (spadix) give rise to berries. On the same stalk
are the male and neutral flowers protected by a hood, the spathe. The fruits are in triangular tube
containing the berries inside. Pikaw is documented in the highland municipalities of Abra like
Boliney, Sallapadan Lacub, Malibcong, Daguiman and Bucloc along river systems and streams
at higher altitudes. It anchors its roots in between and among rocks and well adapted to sandy
clay soil with good drainage and high organic matter.
The plant contains a sap that causes skin irritations or itchiness found concentrated in the
flowers and which gives a permanent black stain to clothes. Corms are not gathered by local
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folks as they are organs for plant horizontal growth and food reservoir for the plant. The
phytochemical analysis results show the plant contains gums, mucilages, glycosides,
carbohydrates, reducing sugars, tannins and derivatives, proteins and derivatives, flavonoids,
deoxysugars, unsaturated sterols and triterpenes, polyphenolic compounds Adaoag.
The young leaves are the edible portions leaving the corms for further growth. These
parts are hashed and bundled piled into the cooking pot. They are treated with fish sauce, onions,
garlic, vinegar, and ginger and sometimes with coconut milk, topped with meat, sardines, or
dried fish. It is best not to disturb/stir pot while cooking so the calcium oxalate crystals in the sap
will be thoroughly cooked.
Philippines has different epidemics that people suffer and that the researchers want to
help all the people that will be affected of diseases and after knowing about the plant Pikaw, the
researchers became curious about its chemical properties that can help not just Ilocanos but also
other people needed to be cured. This reason made the researchers to continue and aim for the
solutions that can help all people who are in need. The researchers aimed to know all the
properties of the plant that can treat diseases. It inspired the researchers to continue their journey
to know about the plant and how can it help people. Ilocanos kept on eating Pikaw and the
researchers want to tell them that the plant that they eat is one of the possible cures for their
sickness. All the possible effects of its chemical properties of Pikaw it can be cure of their
sickness. The researches always kept in mind that if they prove that Pikaw is an effective plant
they can help all the people who are suffering in all there sickness.
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Statement of the Problem
This research aimed to determine the phytochemical, antimicrobial and cytotoxic properties of
Pikaw (Colocasia sp. cf. formosana Hayata).
Specifically, it answered the following:
1. What are the phytochemical constituents of the Pikaw (Colocasia sp. cf. formosana
Hayata)?
2. What are the antimicrobial properties of Pikaw (Colocasia sp. cf. formosana Hayata)
when extracted using ethanol using gram-negative bacteria, gram-positive bacteria and
fungus?
3. Is there a cytotoxic property of Pikaw (Colocasia sp. cf. formosana Hayata)?
Statement of Null Hypothesis
The following null hypotheses were tested:
1. Is there a significant difference in the zones of inhibition when Pikaw (Colocasia sp. cf.
formosana Hayata) is:
applied as ethanol-based extract; or
applied with an antibiotic as control
Using gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus), gram-negative
bacteria (Escherichia coli), and fungus (Candida albicans)?
2. Is the mean percentage of death of brine shrimp (Artemia nauplii) per concentration of
Pikaw (Colocasia sp. cf. formosana Hayata) is not significantly different from 50%?
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Significance of the Study
This study would benefit the research community most especially the consumers of this
plant in the upland areas of Nueva Vizcaya. This study can help the people to understand the
properties of Pikaw and what can it do for their health. There are some chemical properties of the
plant that can treat some illnesses.
Scope and Limitations of the Study
The study will be confined on the phytochemical screening to know the chemical
properties of pikaw; antimicrobial to know if it is possible to kill bacteria and fungi and cytotoxic
properties to know if it is possible to kill cells. This study does not know if any attempt has been
made to assess the commercial value of wild taro.
Statistical Analysis
The following statistical procedures were used:
Descriptive statistics: Means, standard deviations and qualitative descriptions to describe the
data on zones of inhibitions. The qualitative descriptions of the means are as follows:
If the zone of inhibition is:
0 mm, there is no inhibitory effect
<10 mm, there is inhibitory effect but inactive
10-13 mm, there is inhibitory effect but partially active 14-19 mm, there is
inhibitory effect and is active
>19 mm, there is inhibitory effect and is very active
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Inferential statistics (anti-microbial assay): F-test for One-way Analysis of Variance (ANOVA)
was used to determine the antibacterial capabilities of pikaw using Gram-positive bacteria
(Bacillus subtilis, Staphylococcus aureus), Gram-negative bacteria (Escherichia coli) and fungus
(Candida albicans). Special values derived were the F-ratio and p-values. If the p-value > 0.05,
do not reject the null hypotheses and if the p-value <0.05, reject the null hypothesis.
Since the null hypothesis was rejected, the Scheffe method was used to determine which
pair of means was statistically different.
Special values derived are the MD (Mean Difference) and p-values. If the p-value >0.05,
the mean difference is not significant. This reveals that the mean zones of inhibition for the two
extracts against a certain bacterium are not significantly different. Meaning, the extracts have the
same inhibitory effects.
Inferential statistics (cytotoxicity assay)
Probit analysis was used. According to Vincent (2013), probit analysis is a specialized
regression model of binomial response variable like death/no death of pests or brine shrimps in
toxicity studies. It is the preferred statistical model in understanding the death of brine shrimps
relative to solution concentration. It is the basis in determining the LC50 of the larvae and pupae
solution in this study. LC50 represents the concentration at which 50% of the brine shrimp would
die.
The probit regression model is a logarithmic function of the form Probit:
(p) = q(a + b log x)
Where a and b are characteristic constants depending on the solution and extract
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q - cumulative normal distribution function x is the solution concentration
Using statistical software, a table was generated to easily show the LC 50 based on the
probit regression model.
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METHODOLOGY
Procedural Flowchart
PREPATION OF THE CRUDE EXTRACT
Phytochemical
Screening
Antimicrobial
Assay Cytotoxicity
Assay
Gathering of Pikaw (Colocasia sp. cf.
formosana Hayata)
Air-drying
Pulverization
Extraction
Filtration
Water Bath
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PHYTOCHEMICAL SCREENING
Phytochemical Screening
Capillary Tube Spotting
Chromatography
REAGENTS
Naphthol-
sulfuric Acid
Antimony
(III) Chloride
Potassium Ferricyanide-
ferric Chloride
Magnesium
Acetate Ninhydrin
Dragendroff’s
Reagent
Methanolic
Potassium Hydroxide
Preliminary
Test
Vanillin
Sulfuric Acid
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ANTIMICROBIAL ASSAY
Antimicrobial Assay
Preparation of Culture
Media
Inoculation Procedure
Preparation of Culture
Media
Streak Plate Procedure Streaking Patterns
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CYTOTOXIC ASSAY
Cytotoxicity Assay
Preparing the artificial
sea water
Hatching of brine
shrimp
Preparation of test
samples
Brine shrimp counting
Observation and
measuring for 24
hours (3 hours interval)
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Materials
1 kilogram of fresh Pikaw leaves for air-drying
All standard materials for phytochemical screening, antimicrobial assay and cytotoxicity
study were obtained from a standard laboratory (DOST, SMU)
Methods/Procedures
Phytochemical screening: Phytochemical examinations were carried out for all the extracts as
per the standard methods.
Reagents used:
1. Preliminary Test. (Essential oils). Heat at 90°C; violet spot under UV 365 nm.
2. Vanillin Sulfuric Acid. (Higher alcohols, steroids, triterpenes, essential oils, phenols,
fatty acids). Heat at 90°C (char). Triterpenes and sterols appear mainly as blue violet
spots under long wave UV 365 nm. Essential oils from zones with wide range of colors
under long wave UV 365 nm. Phenols appear as brown spot under visible light. Fatty
acids as yellow spot under visible light.
3. Napthol-sulfuric Acid. (Sugars). Heat at 90°C (char). Blue dark spot under visible light.
4. Methanolic Potassium Hydroxide (KOH-MetOH). (Anthraquinones, coumarins,
anthrones). Anthraquinones give orange coloration under visible light. Coumarins react
to form blue colored zone under long wave UV 365 nm. Anthrones give yellow zones
long wave UV 365 nm.
5. Potassium Ferricyanide-ferric Chloride. (Tannins, flavonoids, phenols). Blue spots
under visible light.
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6. Dragendroff’s Reagent. (Alkaloids). Brown-orange visible spots immediately upon
immersing into test reagent; colors are not stable.
7. Antimony (III) Chloride. (Flavonoids, steroids). Intense yellow to orange upon
immersing for glycoside flavonoids; fluorescent colors under long wave UV 365 nm for
steroids.
8. Magnesium Acetate. (Anthraquinones). Heat at 90°C (char). Orange-violet color after
heating at 90°C.
9. Ninhydrin. (Amino Acids). Violet spot upon dipping.
Antimicrobial Assay
Preparation of Culture Media
Test tubes with 20 mm diameter were prepared. These were used for all pours: i.e.,
nutrient agar, Sabouraud‘s agar, EMB agar, etc. Pours were used for filling petri plates. The test
tubes were cleaned with warm water and detergent and using a test tube brush. These were rinsed
twice, first with tap water, and finally with distilled water to rid them of all traces of detergent.
These were placed in a test tube rack in an inverted position to allow draining.
Reagents needed were prepared as follows: 30 g agar/gulaman, 15 g peptone, 3 g
meat/beef extract, 1000 ml tap water. Dissolved the peptone, meat extract and agar in 1000 ml
tap water and heated to boiling by stirring constantly then transferred into tubes. Provided a
closure for each tube. Plastic (polypropylene) caps were used to close each tube. All caps that
slipped over the tube end have inside ridges that gripped the side of the tube and provided an air
gap to allow steam to escape during sterilization. The tubes were sterilized in the autoclave for
15 minutes at 121 C and 15 psi.
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If the tubes are to be converted to slants, it is necessary to lay the tubes down in a near-
horizontal manner as soon as they are removed from the autoclave. Solidification should occur in
about 30-60 minutes. If the tubes are to be converted into tubes of broth, agar deeps, nutrient
gelatin, etc., the tubes should be allowed to cool to room temperature after removal from the
autoclave. Once they have cooled down, place them in a refrigerator or cold-storage room. If
tubes of media are not to be used immediately, they should be stored in a cool place. When
stored for long periods of time at room temperature media tend to lose moisture. At refrigerated
temperatures, media will keep for months.
Dispensing Media in Agar Plates
The nutrient agar was liquefied, cooled to 50oC, and poured the medium into the bottom
of the plate. The tube was sterilized by flaming the neck of it. After pouring the medium into the
plate, the plate was gently rotated to evenly distribute it without any splash of the medium up
over the sides. There should be no moisture on the cover to prevent the drop of it on any colony
on the medium.
Inoculation Procedure
STREAK PLATE PROCEDURE. The culture tube was shaken from side to side to
suspend organisms. Do not moisten cap on tube. The loop and wire was heated to red-hot. The
handle was flamed slightly also. The cap was removed and the neck of the tube was flamed. Do
not place the cap down on the table. After allowing the loop to cool for at least 5 seconds, a
loopful of organisms was obtained. Avoid touching the sides of the tube. The mouth of the
culture tube was flamed again. Then, return the cap to the tube and placed the tube in a test-tube
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rack. The plate was streaked. Do not gouge into the medium with the loop. The loop was flamed
before placing it down.
STREAKING PATTERNS. One loopful of organisms was streaked back and forth over
area 1 near edge of the plate. The loop was applied lightly. Don‘t gouge into the medium. The
loop was flamed, cooled down in 5 seconds and made 5 to 6 streaks from Area 1 through Area 2
momentarily touching the loop to a sterile area of the medium before streaking insures a cool
loop. The loop was flamed again, cooled it, and made 6 or 7 streaks from area 2 through area 3.
The loop was flamed again and made as many streaks as possible from area 3 into area 4 using
up the remainder of the plate surface. The loop was flamed before putting it aside. Filter paper
discs of 6 mm diameter were immersed in the extract and in a positive control for 24 hours. The
discs were removed from the vial and aseptically put 3 of the paper discs immersed in the extract
and one of the paper discs immersed in the control equidistantly on the surface of the assay
plates. The plates were incubated at 35oC for bacteria-containing plates. Then the zones of
inhibition were measured by a digital caliper and interpreted data as follows:
<10 mm=inactive
10-13 mm =partially active
14-19 mm=active >19 mm =very active
In summary, the fresh extract of pikaw leaves and stalks was put in a blender and was
placed each on a beaker. 50 mL of the fresh mixture of pikaw leaves and stalks extract was
extracted using 10 mL ethanol. The extracts were covered using aluminum foil and left them for
one day. At the same time, the bacteria were cultured using an incubator at 35ºC and left them
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there for one day. Once extracted, the fresh, ethanol extract were filtered using cotton balls and
placed them in a beaker. Once filtered, around 12 paper discs were prepared and placed them on
a beaker including one for the control and waited for around 2 hours for it to absorb the extract.
While waiting for it, the incubated bacteria were removed and streaked it on the ready-made
broth placed in a petri dish using an inoculating loop. The inoculating loop was sanitized using
the flame from an alcohol lamp. Once the streaking was done, placed 2 from the ethanol-extract
and 1 from the positive and negative controls in certain areas on each petri dish with
corresponding bacteria. Marked a certain part on the petri dish so that we may not forget which
paper disc is which. Did this thrice then leave the set-ups for a day. The same process was
followed for the pikaw extract. After a day, the zones of inhibition were measured using a digital
caliper.
Cytotoxicity Assay
Pikaw leaves were screened. These were processed for the brine shrimp bioassay test.
Cysts: For this experiment, resting Brine shrimp eggs were obtained from St. Mary‘s University,
Center for Natural Sciences (CNS) Laboratory. Pikaw leaves in desiccator were stored in a
refrigerator at 5 °C.
Culture Medium: The artificial seawater (ASW) was used in the study. Although natural
seawater (about 38 g of salts per liter) is the medium of choice, it has been shown the hatching
rate and the viability of the nauplii in natural seawater (NSW) are similar to those in the artificial
seawater (ASW).
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Hatching the shrimp. Brine shrimp eggs were hatched in a shallow rectangular dish
filled with artificial seawater. A plastic divider with several 2 mm holes was clamped in the dish
to make two unequal compartments. The eggs were sprinkled. After 48 hours, the phototropic
nauplii were collected by pipette from the lighted side, having been separated by the divider from
their shells. Lamp positioned above the uncovered side attracted hatched shrimp. It took 2 days
(48h) for the eggs to hatch and mature as nauplii. The lamp provided direct light and warmth
(about 25° C) throughout embryogenesis.
Preparation of test samples:
Samples were prepared by dissolving 20 mg of the ethanolic pikaw leaves extract in 2 mL
of a suitable solvent (Stock solution # 1). Dilution of this stock solution gave the series of
concentrations required for testing. Four concentrations (replicates) were obtained for each series
of tests.
Bioassay:
To each sample vial, a drop of DMSO solvent was added, ten shrimps were transferred
using a Pasteur pipette, and artificial seawater was added to make a total volume of 5 mL. The
nauplii were counted against a lighted background. Counting for the chronic LC50 began 24
hours after initiation of tests. Nauplii were considered dead if they were immobile at the bottom
of the vials, and the percentage of deaths at each dose was determined.
In summary, the brine shrimp eggs were hatch in artificial saline solution (3.8 g rock
salt/100 mL distilled water) placed in 2 petri dishes within 48 hours. While hatching the eggs, the
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fresh extract from pikaw leaves was prepared for blending. Once the eggs have been hatched, the
egg shells from the brine shrimp were separated using a pipette and placed the brine shrimp on a
separate petri dish. For the pikaw extract, a 6 mg extract/3 mL H2O mixture and a 6 mg extract/6
mL H2O mixture were created. 500 µL of the first mixture and 500 µL of the brine with the
hatched brine shrimp were measured using a micrometer to create a 1000 ppm concentration into
a micro-centrifuge tube (MCT). This was done 3 times. The MCT‘s according to concentration
was labeled, time to be measured (3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21
hours or 24 hours). The lid was kept open.
500 µL of the second mixture and 500 µL of the brine with the hatched brine shrimp
were measured using a micrometer to create a 500 ppm concentration into an MCT. This was
done 3 times. The MCT‘s according to concentration was labeled, time to be measured and the
extract. The lid was kept open. 250 µL of the second mixture and 750 µL of the brine with the
hatched brine shrimp was measured using a micrometer to create a 250 ppm concentration into
an MCT. This was done 3 times. The MCT‘s according to concentration was labeled, time to be
measured and type of extract. The lid was kept open. 125 µL of the second mixture and 875 µL
of the brine with the hatched brine shrimp was measured using a micrometer to create a 125 ppm
concentration into an MCT. This was done 3 times. The MCT‘s according to concentration was
labeled, time to be measured and type of extract. The lid was kept open. During the process, the
number of brine shrimp present was measured. After 6 hours, the number of brine shrimps
remaining of all of the MCT‘s labeled with 6 hours was counted. This was done also after 12
hours and 24 hours. The percentages of the dead per MCT were computed with the formula:
[(Original-Alive)/Alive]*100.
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Materials and Procedure (Documentation)
Materials
All standard materials for phytochemical screening, antimicrobial assay and cytotoxicity
study will be obtained from a standard laboratory (DOST, SMU).
Air-drying
1 kilogram of fresh Pikaw leaves and stalks for air-drying
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Pulverization
Extract Filtration
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Water Bath Pikaw Extract
Phytochemical Screening
Capillary Tube Spotting Drying
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Chromatography Testing Drying
Soaking Drying
Heating Ultra Violet Lighting
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Findings
Antimicrobial Assay
Preparation of Culture Media
Dispensing Media in Agar Plates
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Inoculation Procedure
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Findings
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Cytotoxic Assay
Shrimp Hatching and Culture Medium
Preparation of Test Samples
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Brine Shrimp Counting
Ready for Observation and Measuring
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Research Environment
The pikaw plant was collected in Amoccocan Falls, Bayombong, Nueva Vizcaya in
April-May 2017. The phytochemical screening, antimicrobial assay ang cytotoxicity assay were
conducted in May-July 2017 at the Center for Natural Sciences, Saint Mary‘s University,
Bayombong Nueva Vizcaya The validation of antimicrobial assay was done at the Department of
Science and Technology, Tuegegarao City, Cagayan last July 2017.
Experimental Designs
Antimicrobial Assay of Pikaw (Colocasia sp. cf. formosana Hayata)
Experimental Design 1
Title: The Effect of the Extraction Mechanism for Pikaw (Colocasia sp. cf. formosana Hayata)
on Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Gram-negative bacteria
(Escherichia coli) and fungus (Candida albicans)
Hypothesis:
There is no significant difference in the zones of inhibition when Pikaw (Colocasia sp.
cf. formosana Hayata),
applied as methanol-based extract
applied with an antibiotic as control
Using Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus), Gram-negative bacteria
(Escherichia coli), and fungus (Candida albicans)
IV: Extraction Mechanism for Pikaw (Colocasia sp. cf. formosana Hayata)
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DV: Zones of Inhibition (mm) with the use of: Gram-positive bacteria (Bacillus subtilis,
Staphylococcus aureus) Gram-negative bacteria (Escherichia coli) and fungus (Candida
albicans)
ANTIMICROBIAL ANALYSIS
Sample Description
ZONE OF INHIBITION
Staphylococcus aureus Escherichia coli Candida albicans
R1 R2 R3 MEAN R1 R2 R3 MEAN R1 R2 R3 MEAN
Pikaw Ethanolic extract
POSITIVE CONTROL:
Gentamicin Sulfate
NEGATIVE CONTROL:
Distilled Water
<10 mm=inactive
10-13 mm=partially active
14-19 mm=active
>19 mm=very active
Constants: materials and laboratory factors, No. of repeated trials: 3
Cytotoxicity Assay of Pikaw (Colocasia sp. cf. formosana Hayata)
Experimental Design 2
Title: The Effect of the Pikaw (Colocasia sp. cf. formosana Hayata) on the Artemia nauplii
brine shrimp
Hypothesis: The mean percentage of death of Artemia nauplii brine shrimp Pikaw (Colocasia
sp. cf. formosana Hayata) is not significantly different from 50%
IV: Pikaw (Colocasia sp. cf. formosana Hayata) DV: Death of Artemia nauplii brine shrimp
Mean Mortality Rate (Pikaw Extracts)
Concentration 3 hrs 6 hrs 9 hrs 12 hrs 15 hrs 18 hrs 21 hrs 24 hrs
1000 ppm
500 ppm
250 ppm
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125 ppm
62.5 ppm
Constants: materials and laboratory factors
Treatment of Data
The following statistical procedures were used.
Descriptive statistics: Means, standard deviations and qualitative descriptions to describe the
data on zones of inhibitions. The qualitative descriptions of the means are as follows:
If the zone of inhibition is:
0 mm, there is no inhibitory effect
<10 mm, there is inhibitory effect but inactive
10-13 mm, there is inhibitory effect but partially active 14-19 mm, there is
inhibitory effect and is active
>19 mm, there is inhibitory effect and is very active
Inferential statistics (anti-bacterial assay)
F-test for One-way Analysis of Variance (ANOVA) was used for this null hypothesis.
There is no significant difference in the zones of inhibition of Pikaw (Colocasia sp. cf.
formosana Hayata)
applied as methanol-based extract; or
applied with an antibiotic as control
Using Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus), Gram-negative
bacteria (Escherichia coli) and fungus (Candida albicans)
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Special values derived were the F-ratio and p-values. If the p-value > 0.05, do not reject
the null hypotheses and if the p-value <0.05, reject the null hypothesis.
When the null hypothesis was rejected, the Scheffe method was used to determine which
pair of means were statistically different.
Special values derived are the MD (Mean Difference) and p-values.
If the p-value >0.05, the mean difference is not significant. This reveals that the mean
zones of inhibition for the two extracts against a certain bacterium are not significantly different.
Meaning, the extracts have the same inhibitory effects.
Inferential statistics (cytotoxicity assay)
Probit analysis was used. According to Vincent (2013), probit analysis is a specialized
regression model of binomial response variable like death/no death of pests or brine shrimps in
toxicity studies. It is the preferred statistical model in understanding the death of brine shrimps
relative to solution concentration. It is the basis in determining the LC50 of the larvae and pupae
solution in this study. LC50 represents the concentration at which 50% of the brine shrimp would
die.
The probit regression model is a logarithmic function of the form Probit:
(p) = q(a + b log x)
Where a and b are characteristic constants depending on the solution and extract
q - cumulative normal distribution function x is the solution concentration
Using statistical software, a table was generated to easily show the LC 50 based on the probit
regression model.
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RESULTS AND DISCUSSION
DOST and CNS Lab Results on Phytochemical Screening of Pikaw
Phytochemical analyses were conducted at the Saint Mary‘s University-Center for
Natural Sciences of Bayombong and DOST Laboratory Testing in Tuguegarao. This was done to
establish validity and reliability of results.
Table 1: Phytochemical Components of Pikaw
Phytochemical SMU Lab Result DOST Lab Result
Flavonoids Positive Positive
Tannins Positive Positive
Saponins Positive
Essential Oil Positive Not Tested
Triterpenes Positive Not Tested
Fatty Acids Positive Not Tested
Sugar Positive Not Tested
Coumarins Positive Not Tested
Anthrones Positive Not Tested
Phenols Positive Not Tested
Alkaloids Positive Not Tested
Steroids Positive Not Tested
Anthraquinones Positive Not Tested
At SMU-CNS the following phytochemicals were found: essential oils, triterpenes, fatty
acids, sugar, coumarins, athrones, tannins, flavonoids, phenols, alkaloids, steroids and
anthraquinones. At DOST Lab, the following phytochemicals were found: flavonoids, saponins
and tannins.
In both labs, the following were found: essential oils, triterpenes, fatty acids, sugar,
coumarins, athrones, tannins, flavonoids, phenols, alkaloids, steroids, anthraquinones and
saponins.
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Flavonoids
In both SMU and DOST Labs, they confirmed the presence of flavonoids in the Pikaw
extract.
According to Kozłowska A, Szostak-Wegierek D. (2014), flavonoids are a group of
bioactive compounds that are extensively found in foodstuffs of plant origin. Their regular
consumption is associated with reduced risk of a number of chronic diseases, including cancer,
cardiovascular disease (CVD) and neurodegenerative disorders. Their actions at the molecular
level include antioxidant effects, as well the ability to modulate several key enzymatic pathways.
The growing body of scientific evidence indicates that flavonoids play a beneficial role in
disease prevention. Among dietary sources of flavonoids there are fruits, vegetables, nuts, seeds
and spices. Consumption of these substances with diet appears to be safe. And this can include
Pikaw.
Tannins
In both SMU and DOST Labs, they confirmed the presence of tannins in the Pikaw
extract.
According to Chung KT, et al., (1998), tannins (commonly referred to as tannic acid) are
water-soluble polyphenols that are present in many plant foods. They have been reported to be
responsible for decreases in feed intake, growth rate, feed efficiency, net metabolizable energy,
and protein digestibility in experimental animals. Recent findings indicate that the major effect
of tannins was not due to their inhibition on food consumption or digestion but rather the
decreased efficiency in converting the absorbed nutrients to new body substances. Interestingly,
many reports indicated negative association between tea consumption and incidences of cancers.
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Tea polyphenols and many tannin components were suggested to be anticarcinogenic. Many
tannin molecules have also been shown to reduce the mutagenic activity of a number of
mutagens. Many carcinogens and/or mutagens produce oxygen-free radicals for interaction with
cellular macromolecules. The anticarcinogenic and antimutagenic potentials of tannins may be
related to their antioxidative property, which is important in protecting cellular oxidative
damage, including lipid peroxidation. The generation of superoxide radicals was reported to be
inhibited by tannins and related compounds. The antimicrobial activities of tannins are well
documented. The growth of many fungi, yeasts, bacteria, and viruses was inhibited by tannins.
The antimicrobial property of tannic acid can also be used in food processing to increase the
shelf-life of certain foods, such as catfish fillets. Tannins have also been reported to exert other
physiological effects, such as to accelerate blood clotting, reduce blood pressure, decrease the
serum lipid level, produce liver necrosis, and modulate immune responses.
Saponins
In the DOST Lab, it confirmed the presence of saponins in the Pikaw extract.
According to Man S., et al., (2010), saponins are a group of naturally occurring plant
glycosides, characterized by their strong foam-forming properties in aqueous solution. The
presence of saponins has been reported in more than 100 families of plants out of which at least
150 kinds of natural saponins have been found to possess significant anti-cancer properties. Due
to the great variability of their structures, saponins always display anti-tumorigenic effects
through varieties of antitumor pathways. Some special saponins with strong antitumor effects
have also been exhibited. Ginsenosides, belonging to dammaranes, have been found beneficial
targeted on inhibition of tumor angiogenesis by suppressing its inducer in the endothelial cells of
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blood vessels, and then on prevention of adhering, invasion, and metastasis of tumor cells.
Dioscin, one of the steroidal saponins, and its aglycone diosgenin also have been extensively
studied on its antitumor effect by cell cycle arrest and apoptosis.
Essential Oils
In the SMU Lab, it confirmed the presence of essential oils in the Pikaw extract.
According to Djilani A., Dicko A. (2012), essential oils (also called volatile or ethereal
oils, because they evaporate when exposed to heat in contrast to fixed oils) are odorous and
volatile compounds found only in 10% of the plant kingdom and are stored in plants in special
brittle secretory structures, such as glands, secretory hairs, secretory ducts, secretory cavities or
resin ducts. Essential oils have been used as perfumes, flavors for foods and beverages, or to heal
both body and mind for thousands of years.
Triterpenes
In the SMU Lab, it confirmed the presence of triterpenes in the Pikaw extract.
According to Nazaruk, J., Borzym-Kluczyk, M. (2014), triterpenes seem to demonstrate
adequate properties. Many experiments have shown that these compounds have several anti-
diabetic mechanisms. They can inhibit enzymes involved in glucose metabolism, prevent the
development of insulin resistance and normalize plasma glucose and insulin levels.
Triterpenes are also promising agents in the prevention of diabetic complications. They
have strong antioxidant activity and inhibit the formation of advanced glycation end products,
implicated in the pathogenesis of diabetic nephropathy, embryopathy, neuropathy or impaired
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wound healing. Until now very few clinical studies have been concerned with the application of
triterpenes in treating diabetes.
Fatty Acids
In the SMU Lab, it confirmed the presence of fatty acids in the Pikaw extract.
According to Rustan, A. and Drevon, C. (2001), fatty acids, both free and as part of
complex lipids, play a number of key roles in metabolism – major metabolic fuel (storage and
transport of energy), as essential components of all membranes, and as gene regulators. In
addition, dietary lipids provide polyunsaturated fatty acids (PUFAs) that are precursors of
powerful locally acting metabolites, i.e. the eicosanoids. As part of complex lipids, fatty acids
are also important for thermal and electrical insulation, and for mechanical protection. Moreover,
free fatty acids and their salts may function as detergents and soaps owing to their amphipathic
properties and the formation of micelles.
Sugar
In the SMU Lab, it confirmed the presence of sugar in the Pikaw extract.
According to Eveland, A. and Jackason, D. (2012), carbohydrates or sugars are essential
to the fundamental processes required for plant growth. Therefore, carbohydrate production,
metabolism, and use must be carefully coordinated with photosynthate availability,
environmental cues, and timing of key developmental programmes. Sugars in general can act as
signalling molecules and/or as global regulators of gene expression, for example acting like
hormones and translating nutrient status to regulation of growth and the floral transition.
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Coumarins
In the SMU Lab, it confirmed the presence of coumarins in the Pikaw extract.
According to Trenor, S., Shultz, A., Love, B., Long, T. (2004), the uses of coumarins are
as diverse as the structures of the 800 different derivatives in the coumarin family. Coumarins
are used in the fields of biology, medicine, and polymer science. They are also present or used in
perfumes and cosmetics, cigarettes, alcoholic beverages, and laser dyes. In addition, coumarins
are well documented as therapeutic agents and have been used as medicines.
Anthrones
In the SMU Lab, they confirmed the presence of anthrones in the Pikaw extract.
According to Clegg, W. et al., anthrone is a tricyclic aromatic ketone. It is used for a
popular cellulose assay and in the colorometric determination of carbohydrates. The anthrones
are used in pharmacy as laxative. They stimulate the motion of the colon and are responsible for
less water reabsorption. They may only be used for a short amount of time, because long time
use may lead to loss of electrolytes.
Phenols
In the SMU Lab, it confirmed the presence of phenols in the Pikaw extract.
According to Hollman, P. (2001), plant phenols are mostly products of the
phenylpropanoid pathway and comprise a large variety of compounds: cinnamic acids, benzoic
acids, flavonoids, proanthocyanidins, stilbenes, coumarins, lignans and lignins. They are strong
anti-oxidants and might prevent oxidative damage to biomolecules such as DNA, lipids and
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proteins may interfere with all stages of the cancer process, potentially resulting in a reduction oo
cancer risk.
Alkaloids
In the SMU Lab, it confirmed the presence of alkaloids in the Pikaw extract.
According to Marciano, M., alkaloids are a very mixed group of plant constituents that
contain a nitrogen-bearing molecule that makes them particularly pharmacologically
active. Despite this chemical similarity, the structures and functions vary so widely it would be
very silly to link all ―alkaloids‖ together.
Steroids
In the SMU Lab, it confirmed the presence of steroids in the Pikaw extract.
According to Zorumski, C., Mennerick, S., Isenberg, K., Covey, D. (2000), neuroactive
steroids that alter the function of glutamate receptors could be useful for treating
neurodegenerative disorders, and as cognitive enhancers. Recent progress in developing water-
soluble steroids and steroids with enhances oral efficacy foster optimism that certain neuroactive
steroids will be developed for clinical use.
Anthraquinones
In the SMU Lab, it confirmed the presence of anthraquinones in the Pikaw extract.
According to Dave, H. and Ledwani, D., anthraquinones are organic compounds found in
some plants. Chemically they come in the form of simple anthrones or bianthrones.
Anthraquinones are used for dyes, pigments as well as for medicinal purposes.
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Antibacterial Properties of Pikaw
The tables 2a and 2b below present the zones of inhibition of Pikaw extract using
bacteria.
Table 2a: CNS Lab Results on Microbial Properties of Pikaw
Staphylococcus aureus Escherichia coli Bacillus subtilis
R1 R2 R3 MEAN R1 R2 R3 MEAN R1 R2 R3 MEAN
Pikaw Ethanolic extract 6 6 6 6 6 6 6 6 6 6 6 6
POSITIVE CONTROL:
Streptomycin 32.2 34.0 35.3 33.8 32.7 31.7 33.3 32.6 27.6 28.3 27.8 27.9
NEGATIVE CONTROL:
Ethyl Alcohol 6 6 6 6 6 6 6 6 6 6 6 6
Through the CNS lab, the table reveals that the positive control has high zones of
inhibition on the S. aureus, E. coli and B. subtilis but not the Pikaw extract. The table further
reveals that the negative control has no zones of inhibition on the bacteria. Meaning, distilled
water cannot kill bacteria.
For validity, the said experiment was also conducted at the DOST lab. The result is
shown on the Table 2b below.
Table 2b: DOST Lab Results on Antibacterial Properties of Pikaw
Staphylococcus aureus Escherichia coli
R1 R2 R3 MEAN R1 R2 R3 MEAN
Pikaw Ethanolic extract 6 6 6 6 6 6 6 6
POSITIVE CONTROL: Gentamicin
Sulfate 35 22 37 31 26 29 25 27
NEGATIVE CONTROL: Distilled
Water 6 6 6 6 6 6 6 6
Comparing DOST and CNS results, they validated that Pikaw Extract does not have
antibacterial properties for these identified bacteria.
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Antifungal Properties of Pikaw
The table below presents the zones of inhibition of Pikaw extract using Candida albicans.
Table 3: DOST Lab Results on Antifungal Properties of Pikaw Extract
Candida albicans
R1 R2 R3 MEAN
Pikaw Ethanolic extract 29 31 31 30
POSITIVE CONTROL - - -
NEGATIVE CONTROL: Distilled Water 6 6 6 6
The table reveals that the Pikaw extract has high zones of inhibition on Candida albicans.
Meaning, this can kill this kind of fungus. The table further reveals that the negative control has
no zones of inhibition on the fungus. Meaning, distilled water cannot kill fungus.
The positive control is usually in the form of commercially available antifungal products
with generic names like miconazole, fluconazole, and clotrimazole. The table below shows the
standard zones of inhibition when these antifungal preparations are applied to Candida albicans.
Table 4: Standard Zones of Inhibition of Selected Antifungal Products on Candida albicans
Drug Concentration in
(mcg/disc)
Zone of inhibition in mm
Candida albicans
Miconazole
(24-48 hrs.) 50 26-32
Fluconazole
(24-48 hrs.) 25 28-39
Clotrimazole
(24-48 hrs.) 10 18-32
Ketoconazole
(24-48 hrs) 10 20-32
Pikaw ethanolic extract 29-31
It can be gleaned from the table that the range of the zones of inhibition of Pikaw
ethanolic extract is comparable with those with miconazole, fluconazole, and clotrimazole.
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Probably, this Pikaw ethanolic extract is as effective as miconazole, fluconazole and
clotrimazole.
To further investigate this, analysis of variance was used. The table below shows the
results.
Table 5: Comparison of Pikaw Ethanolic Extract Against Selected Antifungal Products on
Candida albicans
Mean SD Mean Diff. P
value
Decision* % better
Zone C. albicans
(30.33)
Pikaw
ethanolic
extract (p)
Miconazole
(m)
(26-32)
26 .00000 4.33 .00000 p>m
71.4%
27 .00000 3.33 .00000 p>m
28 .00000 2.33 .00000 p>m
29 .00000 1.33 .00000 p>m
30 .00000 .33 .00000 p>m
31 .00000 -.67 .00000 p<m
32 .00000 -1.67 .00000 p<m
Fluconazole
(f)
(28-39)
28 .00000 2.33 .00000 p>f
25%
29 .00000 1.33 .00000 p>f
30 .00000 .33 .00000 p>f
31 .00000 -.67 .00000 p<f
32 .00000 -1.67 .00000 p<f
33 .00000 -2.67 .00000 p<f
34 .00000 -3.67 .00000 p<f
35 .00000 -4.67 .00000 p<f
36 .00000 -5.67 .00000 p<f
37 .00000 -6.67 .00000 p<f
38 .00000 -7.67 .00000 p<f
39 .00000 -8.67 .00000 p<f
Clotrimazole
(c )
(18-32)
18 .00000 12.33 .00000 p>c
86.7%
19 .00000 11.33 .00000 p>c
20 .00000 10.33 .00000 p>c
21 .00000 9.33 .00000 p>c
22 .00000 8.33 .00000 p>c
23 .00000 7.33 .00000 p>c
24 .00000 6.33 .00000 p>c
25 .00000 5.33 .00000 p>c
26 .00000 4.33 .00000 p>c
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27 .00000 3.33 .00000 p>c
28 .00000 2.33 .00000 p>c
29 .00000 1.33 .00000 p>c
30 .00000 .33 .00000 p>c
31 .00000 -.67 .00000 p<c
32 .00000 -1.67 .00000 p<c
Ketoconazole
(k)
20-32
20 .00000 10.33 .00000 p>k
84.62%
21 .00000 9.33 .00000 p>k
22 .00000 8.33 .00000 p>k
23 .00000 7.33 .00000 p>k
24 .00000 6.33 .00000 p>k
25 .00000 5.33 .00000 p>k
26 .00000 4.33 .00000 p>k
27 .00000 3.33 .00000 p>k
28 .00000 2.33 .00000 p>k
29 .00000 1.33 .00000 p>k
30 .00000 .33 .00000 p>k
31 .00000 -.67 .00000 p<k
32 .00000 -1.67 .00000 p<k
Note:
p>m Pikaw ethanolic extract is significantly better than miconazole
p<m Pikaw ethanolic extract is NOT significantly better than miconazole
p>f Pikaw ethanolic extract is significantly better than fluconazole
p<f Pikaw ethanolic extract is NOT significantly better than fluconazole
p>c Pikaw ethanolic extract is significantly better than clotrimazole
p<c Pikaw ethanolic extract is NOT significantly better than clotrimazole
p>k Pikaw ethanolic extract is significantly better than ketoconazole
p<k Pikaw ethanolic extract is NOT significantly better than ketoconazole
The table above reveals that pikaw ethanolic extract is better than ketoconazole,
clotrimazole and miconazole since the range of values in the zones of inhibition of pikaw
ethanolic extract is wider than these three anti C. albicans products except fluconazole.
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Pikaw Ethanolic Extract is better than Miconazole
Miconazole is used to treat skin infections such as athlete's foot, jock itch, ringworm, and
other fungal skin infections (candidiasis). This medication is also used to treat a skin condition
known as pityriasis (tinea versicolor), a fungal infection that causes a lightening or darkening of
the skin of the neck, chest, arms, or legs. Miconazole is an azole antifungal that works by
preventing the growth of fungus.
Thus, Pikaw ethanolic extract can be used for the same purpose as miconazole.
Pikaw Ethanolic Extract is inferior to Fluconazole
Fluconazole is used to prevent and treat a variety of fungal and yeast infections. It
belongs to a class of drugs called azole antifungals. It works by stopping the growth of certain
types of fungus.
Thus, Pikaw ethanolic extract cannot be used as substitute for fluconazole
Pikaw Ethanolic Extract is better than Clotrimazole
Clotrimazole is used to treat yeast infections of the vagina, mouth, and skin such as
athlete's foot, jock itch, and body ringworm. It can also be used to prevent oral thrush in certain
patients. This medication is used to treat vaginal yeast infections. Clotrimazole reduces vaginal
burning, itching, and discharge that may occur with this condition. This medication is an azole
antifungal. It works by stopping the growth of yeast (fungus) that causes the infection. The
vaginal product comes in 2 forms (a vaginal cream or tablet). Some products come with a skin
cream to be applied to the area around the outside of the vagina.
Thus, Pikaw ethanolic extract can be used for the same purpose as clotrimazole.
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Pikaw Ethanolic Extract is better than Ketoconazole
Ketoconazole is both an over-the-counter and a prescription medication. The over-the-
counter form is used to treat dandruff. The prescription form is used to treat serious fungal
infections that can spread to different parts of the body. Ketoconazole belongs to a class of drugs
called antifungals, which work by slowing the growth of fungi that cause infection. This
medication comes in tablet form and is taken once daily, with or without food. It is also available
as a topical gel, cream, and foam and as a shampoo. A form of the shampoo is available without
a prescription.
Thus, Pikaw ethanolic extract can be used for the same purpose as ketoconazole.
Cytotoxic Properties of Pikaw Ethanolic Extract
The table below shows the result of Probit Analysis.
Table 6: CNS Lab Results on Cytotoxic Properties of Pikaw Ethanolic Extract
Concentration 3 hrs 6 hrs 9 hrs 12 hrs 15 hrs 18 hrs 21 hrs 24 hrs
1000 ppm 0 0 2 2.67 3.67 5.67 7.33 9
500 ppm 0 0 0 0.33 0.67 1 1.67 1.67
250 ppm 0 0 0 0 0 0.33 0.67 0.67
125 ppm 0 0 0 0 0 0 0 0
62.5 ppm 0 0 0 0 0 0 0 0
0 0 1745.945 1463.334 1220.509 941.528 743.894 634.807
Qualitative
Interpretation
Non-
cytotoxic
Non-
cytotoxic
Non-
cytotoxic cytotoxic cytotoxic cytotoxic
According to Meyer et al., crude plant extract is toxic (active) if it has an LC50 value of
less than 1000 µg/mL (ppm) while non-toxic (inactive) if it is greater than 1000 µg/mL (ppm).
Using the standard, it can be inferred that after 18 hours the LC50 = 941.528 ppm, after 21 hours
the LC 50 = 743.894 ppm and after 24 hours the LC50=634.807 ppm.
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Therefore, the pikaw ethanolic extract can kill shrimp nauplii which means that it can kill
cells. Thus this extract has cytotoxic properties.
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CONCLUSIONS AND RECOMMENDATIONS
There are favorable results of the phytochemical screening, bacterial assay, fungal assay
and cytotoxicity assay of the Pikaw ethanolic extract. Pikaw has phythochemicals that include:
Flavonoids, Tannins, Saponins, Essential Oil, Triterpenes, Fatty Acids, Sugar, Coumarins,
Anthrones, Phenols, Alkaloids, Steroids and Anthraquinones
Pikaw cannot inhibit these bacteria, namely: S. aureus, E. coli and B. subtilis but it has
high ability to inhibit the fungus named C. albicans.
The range of the zones of inhibition of Pikaw ethanolic extract on Candida albicans is
29-31. This range is comparable with miconazole, cltrimazole and ketoconazole but not with
fluconazole. Hence, the Pikaw ethanolic extract can be made into products to serve as substitute
of commercially available antifungal diseases caused by Candida albicans.
Pikaw has also a cytotoxic property because after 18 hours the LC50 = 941.528 ppm,
after 21 hours the LC 50 = 743.894 ppm and after 24 hours the LC50=634.807 ppm.
The researchers recommend to (1) prepare antifungal cream, ointment and other
antifungal products made out of pikaw ethanolic extract that resemble the commercial
preparations of miconazole, clotrimazole and ketoconazole; and (2) isolate the flavonoids since
this has a role on the cytotoxic property of the pikaw ethanolic extract then retest cytotoxicity.
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APPENDICES
Appendix A. CNS Laboratory Results (Phytochemical)
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Appendix B. CNS Laboratory Results (Bacteria)
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Appendix C. CNS Laboratory Results (Cytotoxicity)
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Appendix D. DOST Laboratory Results (Phytochemical)
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Appendix E. DOST Laboratory Results (Bacteria and Fungus)
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64 | P a g e
Appendix F. Laboratory Testing (Miconazole)
Source: http://himedialabs.com/TD/SD272.pdf
C. albicans (90028)* 26-32
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Appendix G. Laboratory Testing (Fluconazole)
Source: http://himedialabs.com/TD/SD232.pdf
C. albicans (90028)* 28-39
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Appendix H. Laboratory Testing (Clotrimazole)
Source: http://himedialabs.com/TD/SD115.pdf
C. albicans (90028)* 18-32
67 | P a g e
Appendix I. Laboratory Testing (Ketoconazole)
Source: http://himedialabs.com/TD/SD224.pdf
C. albicans (90028)* 20-32
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Appendix J. Antifungal Properties of Miconazole, Fluconsazole, Clotrimazole,
Ketoconazole on Candida Albicans
Antifungal Diseases (Internal) Description Antifungal Medicine
Candidal Vulvovaginitis
This is a yeast infection of the
lower female reproductive
tract.
Imidazole drugs (clotrimazole,
econazole, fenticonazole, and
miconazole) are effective in
the treatment of vulvovaginal
candidiasis.
Oral treatment with
fluconazole or itraconazole is
also effective.
Oral candidiasis
Candida spp. are yeast-like
fungi which can form true
hyphae and pseudohyphae.
They may be part of the
normal body flora, or may
become an invasive pathogen.
Candidal infection varies from
a benign local mucosal
membrane infection to
disseminated disease; it can
involve any organ. Severe
disease is associated with an
immunodeficiency - eg,
malignancy, HIV infection or
immunosuppressive therapy.
First-line therapy is with
topical treatment with
miconazole gel.
For extensive or severe
candidiasis, prescribe oral
fluconazole 50 mg a day for
one week. If the infection has
not resolved after seven days,
offer treatment for a further
week.
Nail infections
Different fungal organisms
may infect the nails, with
different patterns of
presentation, affecting any
part of the nail from the nail
bed to the nail matrix and
plate. The most common
result is a poor cosmetic
appearance of the affected
nail(s); however, the condition
may also cause pain,
disfigurement and functional
impairment.
Oral itraconazole is an
alternative. (Terbinafine is
most effective against
dermatophyte nail infections.
It has fungistatic activity
against Candida albicans.
Itraconazole is highly active
against Candida spp. but much
less so against
dermatophytes.)
Skin infections
Candida spp. are yeast-like
fungi which can form true
hyphae and pseudohyphae.
They may be part of the
Topical antifungals should be
prescribed in most cases. The
imidazoles (clotrimazole,
econazole, and miconazole)
69 | P a g e
normal body flora, or may become an invasive pathogen.
Candidal infection varies from
a benign local mucosal
membrane infection to
disseminated disease; it can
involve any organ. Severe
disease is associated with an
immunodeficiency - eg,
malignancy, HIV infection or
immunosuppressive therapy.
Candida spp. are yeast-like
fungi which can form true
hyphae and pseudohyphae.
They may be part of the
normal body flora, or may
become an invasive pathogen.
Candidal infection varies from
a benign local mucosal
membrane infection to
disseminated disease; it can
involve any organ. Severe
disease is associated with an
immunodeficiency - eg,
malignancy, HIV infection or
immunosuppressive therapy.
Pityriasis versicolor is a
common skin complaint in
which flaky discoloured
patches appear mainly on the
chest and back. It is
sometimes called tinea
versicolor, although the term
'tinea' should strictly refer to
infection with a dermatophyte
fungus.
Dermatophytosis (tinea)
infections are fungal
infections caused by
dermatophytes - a group of
fungi that invade and grow in
dead keratin. Several species
commonly invade human
are all effective.
70 | P a g e
keratin and these belong to the Epidermophyton,
Microsporum and
Trichophyton genera. They
tend to grow outwards on skin,
producing a ring-like pattern -
hence the term 'ringworm'.
They are very common and
affect different parts of the
body. They can usually be
successfully treated but
success depends on the site of
infection and on compliance
with treatment.
Immunocompromised Patients
Immunocompromised patients
are at increased risk of fungal
infections and may need
prophylactic antifungal drugs.
Management is a challenge,
and a specialist field, and
guidelines differ.
Oral triazole antifungals are
the drugs of choice for
prophylaxis. Fluconazole is
more reliably absorbed than
itraconazole but is not
effective against Aspergillus
spp. Therefore, itraconazole is
preferred in patients at risk of
invasive aspergillosis.
(Voriconazole is the treatment
of choice for established
aspergillosis.)
Posaconazole can be used for
prophylaxis in patients who
are undergoing haematopoietic
stem cell transplantation or
receiving chemotherapy for
acute myeloid leukaemia and
myelodysplastic syndrome, if
they are intolerant of
fluconazole or itraconazole.
Micafungin can be used when
fluconazole, itraconazole or
posaconazole cannot be used.
71 | P a g e
Appendix K. Letter to the Principal
72 | P a g e
Appendix L. Letter to the Dean
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CURRICULUM VITAE
I. Personal Data
Name: Samuel Levine L. Soliven
Date of Birth: February 4, 2002
Place of Birth: Bayombong, Nueva Vizcaya
Age: 15
Name of Father: Dr. Samuel R. Soliven
Occupation: Educator
Name of Mother: Mrs. Anivel L. Soliven
Occupation: Librarian
Sibling/s: Samuel Riemann L. Soliven
Samuel Heinrich L. Soliven
II. Educational Attainment
Elementary Level School Attended Rewards/Honors
Grade 1 SMUGS 5th
Honors
Grade 2 SMUGS 5th
Honors
Grade 3 SMUGS 5th
Honors
Grade 4 SMUGS 5th
Honors
Grade 5 SMUGS 5th
Honors
Grade 6 SMUGS 5th
Honors
Secondary Level School Attended Rewards/Honors
Grade 7 SMU/SHS With Academic Distinction
Grade 8 SMU/SHS With Academic Distinction
Grade 9 SMU/JSHS With Academic Distinction
Grade 10 SMU/JSHS Candidate for Moving Up
74 | P a g e
I. Personal Data
Name: Felice Alexandria M. Sadueste
Date of Birth: March 14, 2002
Place of Birth: Bambang, Nueva Vizcaya
Age: 15
Name of Father: Mr. Federico S. Sadueste Jr.
Occupation: Self-employed
Name of Mother: Mrs. Ellen M. Sadueste
Occupation: Self-employed
Sibling/s: None
II. Educational Attainment
Elementary Level School Attended Rewards/Honors
Grade 1 Bambang East Elementary School 2nd
Honors
Grade 2 Bambang East Elementary School 1st Honors
Grade 3 Bambang East Elementary School 1st Honors
Grade 4 Bambang East Elementary School 2nd
Honors
Grade 5 Bambang East Elementary School 2nd
Honors
Grade 6 Bambang East Elementary School Valedictorian
Secondary Level School Attended Rewards/Honors
Grade 7 SMU/SHS 3rd
Honors
Grade 8 SMU/SHS 2nd
Honors
Grade 9 SMU/JSHS With Honors
Grade 10 SMU/JSHS Candidate for Moving Up
75 | P a g e
I. Personal Data
Name: Ferylene C. Valentin
Date of Birth: December 9, 2001
Place of Birth: Bayombong, Nueva Vizcaya
Age: 15
Name of Father: Engr. Ryan T. Valentin
Occupation: Engineer
Name of Mother: Mrs. Fe Ma C. Valentin
Occupation: Teacher
Sibling/s: Benjamin Ryan C. Valentin
II. Educational Attainment
Elementary Level School Attended Rewards/Honors
Grade 1 La Torre Elementary School 4th
Honors
Grade 2 La Torre Elementary School 4th
Honors
Grade 3 La Torre Elementary School 4th
Honors
Grade 4 La Torre Elementary School 4th
Honors
Grade 5 La Torre Elementary School 4th
Honors
Grade 6 La Torre Elementary School 2nd
Honorable Mention
Secondary Level School Attended Rewards/Honors
Grade 7 SMU/SHS
Grade 8 SMU/SHS
Grade 9 SMU/JSHS
Grade 10 SMU/JSHS Candidate for Moving Up