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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)

59 | P a g e

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)

63 | P a g e

64 | P a g e

Appendix F. Laboratory Testing (Miconazole)

Source: http://himedialabs.com/TD/SD272.pdf

C. albicans (90028)* 26-32

65 | P a g e

Appendix G. Laboratory Testing (Fluconazole)

Source: http://himedialabs.com/TD/SD232.pdf

C. albicans (90028)* 28-39

66 | P a g e

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

68 | P a g e

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

73 | P a g e

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