PHYTOCHEMICAL SCREENING,ANTIMICROBIAL AND CYTOTOXIC PROPERTIES

OF PIKAW (colocasia sp. cf. formosana Hayata)

CLARISSE JOY D. BINGA-AN

MA. ISABEL B. DONATO
DEXTER T. VILLALUNA

A Science Investigatory Project presented to the faculty of

President Manuel Roxas Memorial Integrated School SPED

Life Science

APRIL 2023
PHYTOCHEMICAL SCREENING, ANTIMICROBIAL AND CYTOTOXIC PROPERTIES
OF PIKAW (colocasia sp. cf. formosana Hayata)

RESEARCH PLAN

A. Questions or Problem being addressed.

Pikaw, or Colocasia sp. cf. formosana Hayata, is a plant commonly found

in Southeast Asia and has been used in traditional medicine for its potential

therapeutic properties. Previous studies have shown that various parts of the

plant, including its leaves, stems, and tubers, contain phytochemical compounds

that possess antimicrobial and cytotoxic activities.

Antimicrobial resistance has become a global health threat, and the

development of new antimicrobial agents is crucial to combat this problem.

Plants have been a rich source of natural compounds with antimicrobial

properties, and Pikaw could potentially provide a new source of such

compounds. Additionally, the cytotoxicity of natural compounds is important to

assess to ensure the safety of potential drugs.

Therefore, conducting a study on the phytochemical screening,

antimicrobial, and cytotoxic properties of Pikaw could provide valuable insights

into its potential medicinal properties. This could lead to the development of new

antimicrobial agents and contribute to the search for safer and more effective

treatments for various diseases. Furthermore, the study could also contribute to

the preservation of traditional knowledge and the sustainable use of natural

resources.

B. Goals/ Expected Outcomes/ Hypothesis

 This study will be conducted to determine the phytochemical, antimicrobial and

cytotoxic properties of pikaw (colocasia sp. cf. formosana Hayata) ethanolic leaves

extract.

Specifically, this will answer the following questions:

1. What are the phytochemical properties of pikaw (colocasia sp. cf. formosana

Hayata) ethanolic leaves extract?

2. What are the cytotoxic properties of pikaw (colocasia sp. cf. formosana Hayata)

ethanolic leaves extract?

3. What is the effect in using varying concentration of Pikaw (colocasia sp. cf.

formosana Hayata) ethanolic leaves extract to the zone of inhibition of

Staphylococcus aureus and Escherichia coli?

4. What is the effect in using varying concentration of Pikaw (colocasia sp. cf.

formosana Hayata) ethanolic leaves extract to the zone of inhibition of Bacillus

subtilis and Candida albicans?

5. Is there a significant difference in the zone of inhibition of Staphylococcus aureus

and Escherichia coli using varying concentration of Pikaw (colocasia sp. cf.

formosana Hayata) ethanolic leaves extract?

6. Is there a significant difference in the zone of inhibition of Bacillus subtilis and

Candida albicans using varying concentration of Pikaw (colocasia sp. cf.

formosana Hayata) ethanolic leaves extract?

Null hypothesis
 1. There is no significant difference in the zone of inhibition of Staphylococcus

aureus and Escherichia coli using varying concentration of Pikaw (colocasia sp.

cf. formosana Hayata) ethanolic leaves extract.

2. There is no significant difference in the zone of inhibition of Bacillus subtilis and

Candida albicans using varying concentration of Pikaw (colocasia sp. cf.

formosana Hayata) ethanolic leaves extract.

C. Procedure

Materials

1 kilogram of fresh pikaw leaves for air drying. All standard materials for

phytochemical screening, antimicrobial assay and cytotoxity study will be obtained from

a standard laboratory. For Phytochemical screening, phytochemical examination will be

carried out for all extracts as per the standard methods.

Experimental Design

This study will employ the Completely Randomized Design (CRD), with One

Factor experiment using four microorganisms, Staphylococcus aureus, Escherichia coli,

Bacillus Subtilis, and Candida Albicans; 100%, 75%, 50% Pikaw ethanolic leaves

extract with ciprofloxacin (for bacteria) and Flucinonide (for fungi) as the positive control

and distilled water for negative control in four replications through susceptibility testing.

Collection and preparation of plant Sample

The pikaw leaves will be collected from Bolo, Roxas City, Capiz.
Filtration of Extract

Pikaw extract will be filtered using a Grade I Filter paper collected in a 250 mL

Erlenmeyer flask. Cover with aluminum foil.

Phytochemical Analysis

Pikaw filtrate will be brought to the Central Laboratory of West Visayas State

University for phytochemical Analysis. The

The sample plant will be tested with the presence of saponins, tannins,

flavonoids, alkaloids protein and Carbohydrates.

Preparation of Treatments

Five (5) treatments will be followed, and each treatment will be replicated

four (4) times. Treatment A – 100% Pikaw ethanolic leaves extract; Treatment B – 75%

Pikaw ethanolic leaves extract + 25% Distilled water; Treatment C – 50% Pikaw

ethanolic leaves extract + 50% Distilled water; Treatment D- Positive Control

(Ciprofluxacin for bacteria and Flucinonide for fungi); and Treatment E – Negative

Control – Distilled water.

Impregnation of Paper Disks

Five (5) sterile bottles will be prepared and labeled Treatment A, B, C, D and D.

Eight (8) 9 mm susceptibility disks will be placed in each treatment. Using a sterile

medicine dropper, each disk will be impregnated with 0.5 mL of Pikaw ethanolic leaves

extract. It will be covered and let it stand to dry overnight.

Sub-culturing of Fungal and Bacterial Isolates

 Four culture tubes with cover will be prepared. 5 mL NSS will be added to each

culture tubes and will be covered. Staphylococcus aureus, Escheriachia coli, B. subtilis

and C. albicans will be prepared. Using a sterile cotton applicator, one to two colonies

will be picked and dip into the designated culture tubes; flame and cover.

Antimicrobial Assay

Preparation of Culture Media

500 mL Erlenmeyer flask will be filled with 500 mL distilled water. 16 grams of Mueller

Hinton Agar (MHA) will be weighed and transferred to the Erlenmeyer Flask; will the be

swirled and sterilized for 20 minutes.

Pour Plating

16 sterile Petri dish will be plated with 20 mL culture media; cool until it will

solidified.

Plate Inoculation

Counter-clockwise plate-streaked method, the plates will be inoculated with four

different microorganisms.

Using a sterile cotton applicator, four (4) sterile MHA plates will be inoculated

with S. aureus; four (4) sterile MHA plates with E. coli, four (4) sterile MHA plates with

B. subtilis and four (4) plates with C. albicans isolates. After inoculation, using a twissor,

each inoculated plates will be planted with paper disks to be impregnated with the

different treatments. Flame and cover.

Incubation
 Petri dishes will be incubated for 18 hours with an optimum temperature of 370°C

in an upside-down position.

Measuring Zone of Inhibition

Zone of inhibition will be measured in millimeter (mm) interpreted as susceptible,

intermediate, and resistant based on the Antibacterial Index. A large zone of inhibitions

indicates that the organisms are susceptible to the treatment, while small or no zone of

inhibition indicates resistance. Interpretations of intermediate will be given for zones

which fall between the accepted cutoffs for the other interpretations.

Interpretation Diameter of Zone of Inhibition (mm)

Resistant 10 or less
Intermediate 11-15
Susceptible 16 or more

Source: Retrieved June 12, 2016 from “Chemical Methods

ofControl”http://www.accessexellence.org/AE/AEC/CC/chance_activity.html

Waste Disposal

Used culture media will be sprayed with Lysol disinfectant spray; it will be

removed from the petri dish after 30 minutes using spatula. The used media will be

buried into a hole dug outside the laboratory.

Data Collection

The that will be data collected is on the average mean and mean differences of

the zones of inhibitions of the microorganisms in millimeter.

Cytotoxicity Assay
 Brine Shrimp Lethality Method will be used in determining the cytotoxic activity of

the plant.

Statistical Tool

The data collected will be analyzed statistically using the Mean and Mean

difference. Mean will be used to find out and describe the quantified data of the four

treatments and Analysis of Variance (ANOVA) if there are significant differences in the

zone of inhibition among the four treatments.

 PROCEDURAL DESIGN

Collection and Preparation of Materials

Plant Extraction

Filtration

Phytochemical Analysis of Plant

Extract

Preparation of Treatments

Treatment B Treatment C
Treatment D Treatment E
Treatment A 75% Pikaw 50% Pikaw
100% Pikaw Ethanolic Ethanolic Positive Negative
Ethanolic Leaves Leaves Extract Control Control
Leaves Extract + 25% + 50% Distilled
(Cipro- (Distilled
Extract Distilled Water
Water fluxacin) water)

Phytochemical Screening Antimicrobial Assay Cytotoxicity Assay

Measuring Zones of Inhibitions

Waste Disposal

Recording and Analysis of Data

Conclusions and Recommendations

References:

McKechnie, D. (2022, November 19). Antifungal Medications.

https://patient.info/doctor/antifungal-medications

Thapa, U. (2018, September 1). Compendium of Vegetables. ResearchGate.

https://www.researchgate.net/publication/344157198_Compendium_of_Vegetables

http://www.elarjc.com/documents/A
Flavonoids--food sources and health benefits. (2014). PubMed.
https://www.ncbi.nlm.nih.gov/pubmed/25272572