Simultaneous Testing on Thin-Layer Chromatography Constituents, Antioxidant

Property, Efficacy from Selected Blaan Plant Extract as Antibacterial Agents: As

Basis for Ointment Formulation

Proponent:

Binolirao, Cesar Ryan D.

Gerolaga, Rio Marlo G.

Gutierrez, Devina Marie D.

Pasalinto, Psyche May C.

Tayoto, Meika Jin A.

In Fulfillment of the Requirements in Research II

General Santos City National High School

Calumpang, General Santos City

Pelmar B. Fernandez
Research II Adviser
 Iii

Approval Sheet
This research entitled Determination of Bioactive Compounds present in
selected Blaan Plants Extract prepared and submitted by Cesar Ryan D. Binolirao,
Rio Marlo M. Gerolaga. Devina Marie Gutierrez,Psyche Mae T. Pasalinto and Meika Jin
A. Tayoto has been examined and presented to the faculty and staff of General Santos
City National High School in fulfillment of the requirements in Research I.
PELMAR T. FERNANDEZ
Adviser

Accepted and Approved by the Examination Committee

IMELDA P. CASICAS
Chairman

HELEN I. PERPETUA DANILO U. CASICAS

Member Member

Accepted and Approved in fulfillment of the requirements in Research I

LALAINE BIBOSO
Coordinator
Science, Technology and Engineering Program

Date: ________________
 Iv

Acknowledgement

A lot of problems were encountered by the researchers as they went along their

study. From the formulation of this study down to the conduction of experiments, the

researchers would like to acknowledge the presence and help of everyone, including

their friends, classmates, acquaintances, teachers and parents. Their guidance, words

of wisdom and overwhelming support which contributed and gave a lot of help is one of

the reason why this study has been successful. The researchers would like to dedicate

their acknowledgement of gratitude towards the following entities for the completion of

this study.

To the researchers classmates and friends, who were very willing to lend their

hand in times of trouble, to ate Ludivie Dagmil, for giving us additional information in

completion of our study.

Sincerest gratitude to the following universities and medical clinic, Ateneo de

Davao University (ADDU), University of Immaculate Conception (UIC) and Davao

Analytical Incorporated for lending us their equipments and facilities in order to conduct

tests for the completion of the researchers research.

To Maam Lalaine Biboso, our curriculum coordinator, for her words of wisdom

and advices. And we also thank her for letting us borrow the laptop to encode and edit

our write-ups.

Sir Pelmar Fernandez, our research teacher and adviser, for all the knowledge

and wisdom that he shared to us, And for the guidance and effort in helping us to finish

the researchers study.

 To the researchers parents and relatives for their overwhelming support both

technical and financially, their sacrifices in order for the researchers sake, Mr. and Mrs.

Porceso Binolirao, Mr. and Mrs. Noel Tayoto, Mr. and Mrs. Dennis Gutierrez, Mr. and

Mrs. Henry Pasalinto and Mr. and Mrs. Mario Gerolaga.

Lastly, to our almighty God, who had been always there as our inspiration in

making this study. He gave us strength, and perseverance to overcome the hardships

and problems in making our study. He also gave us the knowledge and wisdom that

made this study successful. He gave us all we need to make this study possible and

successful.

- The Researchers
 ABSTRACT

Title: Simultaneous testing on TLC Constituents, Antioxidant Property, Efficacy from

Category
Selected Blaan Plant Extracts as Antibacterial Agents: as basis for Ointment
Formulation Pick one only-
Mark an X
Researchers: Cesar Ryan Binolirao, Devina Marie Gutierrez and Meika Jin Tayoto
in the box at
School: General Santos City National High School, JP Rizal St. Calumpang right

Plants has a great contribution to the innovations of the medical field. Blaans living Biochemistry
in the South Cotabato tend to use traditional herbal plants rather than synthetic drugs. Those Botany
herbal plants are Tangisang bayuak (Ficus variegata blume), Badyang (Alocasia
macrorrhiza) and Galinsoga (Galinsoga parviflora). For the completion of this study, the Chemistry
researchers conducted various tests such as bioassay, Thin-layer chromatography, antioxidant
Computers
assay, formulated an ointment and conducted a bioassay and heavy metals testing.
Earth and
The Bioassay results proved that Tangisang bayuak has a mean zone of inhibition of
Space Sciences
18mm which means it has an active inhibitory activity, so is with the Badyang with a mean
zone of inhibition of 17.7 mm which means that it has an active inhibitory activity and lastly Engineering
is the Galinsoga with a mean zone of inhibition of 19 mm which means it has an active
inhibitory activity against the reference strain, Staphylococcus aureus. Thin-layer Environmental
chromatography results showed that the pericarp extract of Tangisang bayuak detected the Science
presence of flavanoids, saponins and tannins. The leaves extract of Badyang and Galinsoga,
steroids, saponins and tannins; steroids, saponins, tannins and phenols respectively. Mathematics
Antioxidant results shows that Tangisang bayuak, Badyang and Galinsoga inhibits the
Microbiology
formation of DPPH at the percentage rate of 37.84 to 41.62, 29.62 to 33.56 and 41.75 to 48.71
respectively which confirms their potential as an antioxidant agent. Heavy metals testing of Physics
the formulated ointment results shows that the ointments contains no residual contamination
with pH value within the acceptable range. Zoology

Based on the results showed, it can be inferred that the commonly used medicinal Biochemistry
plants of the Blaan people in South Cotabato possesses medicinal values that can be used by
the Blaan people in the future.

1. As a part of this research project, the student directly handled, manipulated, of interacted
with (check ALL that apply):
Human subject Pathogenic agents Recombinant DNA
Non-Human vertebrae Controlled substances Human/Animal Tissues
Animal
2. Students independently performed all procedures as outlined in the subject?
Yes No
3. This project was conducted at a Registered Research Institute?
Yes No
 Table of Contents
Page

Cover Page I

Title Page Ii

Approval Sheet Iii

Acknowledgement Iv

Chapter I- The Problem and Its Settings

Background of the Study 1

Statement of the Problem . 3

Hypotheses . 4

Significance of the Study 5

Scope and Limitation 6

Operational Definition of Terms .. 7

Chapter II- Review of Related Literature

Tagisan bayauak 8

Galinsoga 8

Badyang 9

Antibacterial 10

Thin-Layer Chromatography . 10
 DPPH Antioxidant assay 11

Ointment 12

Heavy Metals 14

Lead .. 15

Cadmium .. 16

Saponins 16

Flavanoids 18

Phenols 19

Tannins .... 20

Steroids . 21

Staphylococcus aureus 21

Chapter III- Methodology

Research Site and Duration 23

Research Design 23

Procedure Flow Chart . 25

a. Bioassay .. 25

b. Thin-Layer Chromatography 26

c. Antioxidant assay 27
 Materials and Instruments 28

a. Pure Extract Extraction and Bioassay . 28

b. Pure Extract Extraction and Thin-Layer Chromatography .. 39

c. Pure Extract Extraction and Antioxidant assay . 30

Experimentation and General Procedure 31

a. Preparation of the selected Blaan plants extract . 31

b. Bioassay .. 31

c. Thin Layer Chromatography . 33

d. Antioxidant assay 33

Chapter IV- Presentation, Analysis and Interpretation of Data

Bioassay 35

Thin Layer Chromatography 44

Antioxidant assay ... 45

Physico-Chemical Testing 47
Heavy Metals Testing .. 48
Chapter V- Summary, Conclusion and Recommendation

Summary . 49

Conclusions . 50
 Recommendations . 51

Appendices .. 58

Bibliography . 53
 List of Tables

Table 1.1 Materials and Instruments of Crude extraction and TLC .. 21

Table 1.2 Materials and Instruments of Pure plants extract Extraction 22

Table 1.3 Materials and Instruments of Antioxidant assay . 23

Table 2.1 Results in Bioassay of Tangisan Bayauak Extract . 24

Table 2.2 Results in Bioassay of Badyang Extract .. 25

Table 2.3 Results in Bioassay of Galinsoga Extract 26

Table 3.1 Result in Antioxidant assay of Tangisan Bayauak Extract 27

Table 3.2 Result in Antioxidant assay of Badyang Extract 28

Table 3.3 Result in Antioxidant assay of Galinsoga Extract .. 29

 List of Figures

Figure 1 Research Design .. 20

Figure 2.1 Flowchart of Bioassay 21

Figure 2.2 Flowchart of TLC . 22

Figure 2.3 Flowchart of Antioxidant assay .. 23

 List of Appendices

Appendix A.. 48

Appendix B.. 49

Appendix C.. 50

Appendix D.. 51

Appendix E.. 52

Appendix F.. 53

Appendix G. 54

Appendix H. 55

Appendix I.. .. 56

Appendix J.. 57

Appendix K.. 58

Appendix L.. 59

Appendix M.. 60

Appendix N.. 61

Appendix O. 62

Appendix P. 63

Appendix Q.. .. 64
 Chapter I

The Problem and Its Settings

This chapter introduces the problem of the study. And explains the nature of the

study.

Background of the Study

Herbal medicines are the use of herbal plants to improve ones body or

wellbeing. According to a study of the ResearchGate.net, in the year 2014, almost 80%

of the countries across the world are using herbal plants as alternative for medical

drugs. The Philippines are one of those countries. Because of the rising prices of

medical drugs in the country, more than 100 million Filipinos are pushed into poverty

every year just because of pricey medical drugs. High-priced drugs are sometimes the

reason why bacterial diseases such as meningitis, ulcer, gastritis and pneumonia are

not treated and leads to death. Under the Republic Act (RA) 8423 or Traditional and

Alternative Medicine Act of 2007 by the former Sec. Juan M. Clavier states that the use

of plants as alternative medicine is encouraged by the Philippine Government. The

Philippines Institute of Traditional and Alternative Health Care (PITAHC) was created to

promote and advocate the use of traditional, alternative, preventive and curative health

care modalities that have been proven safe, effective, cost-effective and consistent with

government standards on medical practice.

There are over 40 ethnic groups present in the Philippines. And 18 of those tribes

are in Mindanao. They can be classified as Tribal groups and Indigenous groups.
The Blaan are the major indigenous groups in Mindanao. They believe in their God or

what they call Dawata. Blaan have rituals for everything- including their medicines.

They believe that all of the plants on earth are planted by Dawata and these plants will

help them to cure such illnesses that will make them healthier. But they are not aware of

what specific plant are they going to use and what are the good or bad effects to them.

Plant-based natural constituents can be derived from any part of the plant like

bark, leaves, flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain

active components. Tagisang bayauak (Ficus variegata blume) is a native plant which

commonly grows on primary forests and places with low and medium altitudes. It is a

tall, spreading plant with pale bark, broad leaves and fruits that grows or attached on its

bark where the fruit are on its twigs. The latex of its bark is commonly used in treating

external wounds together with its leaves. Native people in this area call by many names

according to their tribe. Badyang (Alocasia macorrhiza) are commonly found in Tboli,

South Cotabato, although this plant can be seen everywhere and is valued an

ornamental. This is sometimes referred to as Elephant ears and Giant taro because

of its upright and large leaves standing straight up pointing skyward. According to

folktales, the plants stems is edible and are eaten as vegetable during scarce times.

Galinsoga (Galinsoga parviflora) is a forb and an abundant seed-producing plant with

hairy leaves and stem. The stem of this plant is erect, branched, grows up to 60 cm

and is slightly hairy. The leaves of this plant is slightly hairy, grows up to 6cm long and

4m wide simple and ovate.

 The plants extract will be evaluated using tests such as Thin-Layer

Chromatography (TLC), determining its Antioxidant property, and using it as

antibacterial agents as basis for ointment formulation.

Statement of the Problem

This study aims to identify the Antibacterial activity of Tangisan bayuak (Ficus

variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga parviflora)

extracts and ointment, to identify the bioactive compounds using Thin-Layer

Chromatography, determine its antioxidant property and the heavy metals present in the

ointment. Specifically, the study will answer the following questions:

1. What is the level of antibacterial activity using the mean zone of inhibition of

Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia macorrhiza) and

Galinsoga (Galinsoga farviflora) plants extract against the reference strain S. aureus?

2. What is the level of antibacterial activity using the mean zone of inhibition of

Tagisang bayauak (Ficus variegata blume) ointment, Badyang (Alocasia

macrorrhiza) ointment and Galinsoga (Galinsoga farviflora) ointment against the

reference strain S. aureus?

3. What are the possible bioactive compounds present in the Tagisang bayauak

(Ficus variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga

farviflora) plants extract?

4. What are the percentage of the DPPH inhibited by the Tagisang bayauak (Ficus

variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga

farviflora) plants extract?

 6. What are the heavy metals present in each ointment?

Hypotheses

This study will be conducted to prove the following hypotheses:

Null

1. The zone of inhibition of Tagisang bayauak (Ficus variegata blume), Badyang

(Alocasia macrorrhiza) and Galinsoga (Galinsoga farviflora) plants extract are the same

with the positive control, Mupirocin.

2. The mean zone of inhibition of Tagisang bayauak (Ficus variegata blume) ointment,

Badyang (Alocasia macrorrhiza) ointment and Galinsoga (Galinsoga parviflora)

ointment is the same with the positive control, Mupirocin.

3. There is no bioactive compounds present in Tagisang bayauak (Ficus variegata

blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga farviflora).

4. The different plants extract did not inhibit the DPPH.

5. There are heavy metals present in each ointment.

Alternative

1. The zone of inhibition of Tagisang bayauak (Ficus variegata blume), Badyang

(Alocasia macrorrhiza) and Galinsoga (Galinsoga farviflora) plants extract are

greater than the positive control, Mupirocin.

2. The mean zone inhibition of the positive control is lesser than the mean zone of

inhibition of the Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia

macrorrhiza) and Galinsoga (Galinsoga parviflora) ointments.

3. There are bioactive compounds present in Tagisang bayauak (Ficus variegata

blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga farviflora.

4. The different plants extract inhibited the DPPH.

5. There are no heavy metals present in each ointment.

Significance of the Study

According to a study of Centers for Disease Control and Prevention, allmost 91%

of the deaths in the year 2013 are caused by bacterial diseases which were untreated

because of poverty. The use of medicinal plant extract for the treatment of human

diseases is ancient practice. Folktales and beliefs that herbal plants found in the

community which can be used as medicines are widespread in rural places and in tribes

like Blaan.

The results of this study is useful the Blaan people in understanding why certain

plants like Tangisang bayawak, Badyang and Galinsoga, which are present in their

community, can heal their illnesses. It gives them scientific explanations to support their

beliefs. It also creates a spot for the Blaan people to discover plants in their community

which can aid them in their illnesses.

This study created an option to those people who cant afford medicines

prescribed by their doctors for they can substitue these safe herbal plants as medicines.

This study can also create a breakthrough in medical field because this can give them

additional information about the bioactive compounds present in these plants and they

can use this study as a basis in creating alternative medicines.

 Lastly, this study is an addition to the list of the usage of natural resources, aiding

the government in lowering the high percentage of incurable bacterial diseases due to

poverty. The government can now provide medicines which the populace can afford.

Scope and Limitation

This study will focus on the effectiveness of Tagisang bayawak (Ficus variegata

blume), Badyang (Alocasia macorrhiza), Mani-manian (Arachis pintoi) in inhibiting the

growth of bacteria, on the testing of the bioactive compunds present in the plant and the

antioxidants present in the plants. This study will only test the effectivity of Tagisang

bayawak (Ficus variegata blume, Badyang (Alocasia macorrhiza), Mani-manian

(Arachis pintoi) to lessen the growth of Escherichia coli (ATCC 25922). This can be

obtained through the process of thin-layer chromatography (TLC), reducing property

test and DPPH. This study also focuses on the capacity of the Blaan plants extract in

the ointment formulation.

Operational Definition of Terms

The following terms are define to provide clarity and better understanding of the words

frequently used in the following pages.

Antibacterial- refers to the ability of the selected Blaan plants extract to suppress or

inhibit the growth of harmful bacteria through the measurement of the mean zone of

inhibitions.

Antioxidant- a substance that inhibits oxidation or reactions promoted by oxygen,

peroxides, or free radicals.

Ascorbic Acid- the standard used in the reducing property test to determine the

reducing property of the selected Blaan plants.

Badyang (Alocasia macrorrhiza)- one of the selected Blaan plants in South Cotabato

that are manipulated in the study.

Bioactive compounds refers to the constituents present in Tagisan bayauak (Ficus

variegata blume) leaves extract that may help or harm humans.

Bioassay- refers to the determination of the strength of a drug or biological product by

testing its effect on living organisms using aseptic techniques.

Galinsoga (Galinsoga farviflora)- one of the selected Blaan plants in South Cotabato

that are manipulated in the study.

Heavy Metals- are traced metals that can be present on the selected Blaan plants

extract.

Staphylococcus aureus- common gram-positive bacteria which will be used to test the

antibacterial property of the extract.

Tagisang bayuak (Ficus variegata blume)- one of the selected Blaan plants extract

in South Cotabato that are manipulated in the study.

Thin-Layer Chromatography- the process to identify bioactive compounds present in

the plants leaves extract and testing the quantity of compounds.

Zone of inhibition- it is the area to be measured in order to know the potential of the

plant, Tagisan bayauak to inhibit the growth of a bacteria.

 Chapter II

Review of Related Literature

This chapter presents the literature taken from various works and researchers

which are significant in conducting this study.

Tagisang Bayauak
 Ficus variegate blume is a tall spreading tree with pale bark. It commonly grows

in primary forests and can also grow in countries in secondary forests. It is also widely

distributed in moist areas of low to medium elevations like forest, forest thickets,

farmlets, along gullies and rivebanks. The leaves of this plant are broad or elliptic-ovate,

that measure 10 to 17 centimeters long and are yellowish-brownish. Its receptacles are

about 1 cm in diameter and are clustered on long branches. The wood are known to be

used in temporary construction, mouldings, interior works and etc. It is known or

believed to treat external wounds using its boiled leaves extract. People sometimes use

this plant as decoration. Their young shoot-tips and young fruits are sometimes eaten

raw. (http://www.stuartxchange.org/Agi.html).

Galinsoga (Galinsoga parviflora)

Galinsoga parviflora is an herbaceous plant in the Asteraceae (daisy) family. It is

a herb and a leafy vegetable used in some cuisines in Colombia and on East African

countries. Galinsoga has medicinal benefits yet it still considered as noxious weed

(Cultural History of Plants, 2005). The leaf juices of the plant is used as astringent for

stings, cuts, wounds (Invasive Alien Plants: An Ecological Appraisal for the Indian
9
Subcontinent, 2012).

Galinsoga parviflora is a forb and an abundant seed-producing summer annual

with hairy leaves and stems. At seedling stage, it produces club-shaped cotyledons with

slightly indented tip. The stem below the cotyledon (hypocotyl) is very short, green,

becoming maroon with age. Young leaves are opposite, triangular with slightly toothed

margins, and covered with hairs. It has a shallow fibrous root system. The stem is erect,

branched, grows up to 60cm high and is slightly hairy. Leaves are opposite, up to 6cm
long and 4cm wide, simple, ovate and slightly hairy. Three distinct veins mark the leaf,

converging at the base and the margin is shallowly toothed. The inflorescence is flower

heads 5-8mm in diameter on stalks 12-25mm long. The leafy inflorescence is regularly

branched at the stem apex and from upper leaf axils. Flower heads consist of many

yellow tubular florets, and 4-5 white 3-lobed ray florets surrounded by membranous

bracts. Fruit is an achene and propagation is by seed. (http://agron-

www.agron.iastate.edu/~weeds/weedbiollibrary/517%20student%20pages/2000/Galinso

gad..html).

Badyang (Alocasia macrorrhiza)

Badyang (Alocasia macrorrhiza) or sometimes called Giant Taro is a large

evergreen plant with extremely large erect or spreading leaves; cultivated widely in

tropics for its edible rhizome and shoot and are used in wet warm regions as a stately

ornamental. Any plant of the genus Alocasia has large showy basal leaves and boat-

shaped spathe and reddish leaves. This plant is commonly used in rituals not only by

the Blaan people but also of the Folk-Healers of Siquijor. This plant is involved in the

minasa ritual where it is believed to further bestow potency to the brew. (Fuentes,

Evelyn & Mascunaa, Rolando V., 2004). 10

Anti-bacterial

Antibacterials are topical agents that eliminate the bacteria that causes epidermal

infections. The antibiotic neomycin is frequently used in ointments for this purpose.

(Allan, David M., Buchman, Michelle A. & Lockyer, Karen D., 2003).
 The basics of bacterial structure describing the basis for your understanding

mechanisms of antibacterial action. Antibacterial may refer to anything that destroys

bacteria or suppresses their growth or ability to reproduce. It covers the characteristic

features of bacterial pathogenicity, the genetic basis of resistance to anti-bacterial

drugs, the biochemical mechanisms of action of antibacterial drugs, how antibacterial

drugs reach their targets in gram-positive 3and gram-negative bacteria, and the wide

range of human immune responses against bacterial infection. Recent advances in

research and development of new classes of antibacterial drugs are examined.

(Khakdan, Fatemeh & Khosro, Piri, 2013).

Thin-layer Chromatography

Thin-Layer Chromatography (TLC) is a modern, reliable tool that complements

other chromatographic techniques. A chromatography technique used to separate non-

volatile mixtures. It is a simple, quick, and inexpensive procedure that gives a quick

answer as to how many components are in a mixture. Thin-layer chromatography is

performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer

of adsorbent material, usually silica gel, aluminum oxide, or cellulose. Thin-layer

chromatography can be used to monitor the progress of a reaction, identify compounds

present in a given mixture, and determine the purity of a substance (Wall, 2005). 11

Specific examples of this application include: analyzing ceramides and fatty

acids, detection of pesticides or insectisides in food and water, analyzing the dye

composition of fibers in forensics. TLC plates are usually commercially available, with

standard particle size ranges to improve reproducibility. They are prepared by mixing

the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate
(gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier

ship, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and

activated by heating in an oven for 30 minutes at 110 C. The thickness of the adsorbent

is typically around 0.5-2.0 mm for preparative TLC.

(http://bheem.hubpages.com/hub/tlc-thin-layer-chromatography-Principle-Procedure).

DPPH Antioxidant Assay

Scavenging of DPPH free radical is the basis of a common antioxidant assay.

2,2-diphenyl-1-picrylhydrazy (DPPH) free radical scavenging method offers the first

approach for evaluating the antioxidant potential of a compound or an extract. This is

the method wherein the prospective compound or extract is mixed with DPPH solution

and absorbance is recorded after a defined period. The primary feature distinguishing

antioxidant activity is the rate of reaction (less than 30 sec) is controlled by whether the

dominant antioxidant mechanism is an electron (very fast) or hydrogen atom (slow)

transfer and by impairment of steric accessibility to the DPPH radical site by bulking ring

adducts and multiple phenolic rings. (K.M Schaich & J. Xie, 2004).

This method was developed by Blois in the year 1958 with the viewpoint of

determining the antioxidant activity in a like many by using a stable free radical. The

assay is based on the measurement of the scavenging capacity of antioxidants towards

it. This assay is unique in carrying out the reaction of the sample with DPPH in

methanol/water, which facilitates the extraction of antioxidant compounds from the

sample. Antioxidant analysis by other methods may be limited to those compounds

soluble in selected solvents. The advantage of this method is that DPPH is allowed to
react with the whole sample and sufficient time given in the method allows DPPH to

react slowly even with weak antioxidants. It is why DPPH assay is considered a valid,

accurate, easy and economic method to evaluate radical scavenging activitY of

antioxidants. There are various methods for the determination of antioxidant potential of

different biological samples. Amongst all the available methods, DPPH method has

been widely applied for estimating antioxidant activity. (Kedare, Sugar B., Singh, R, P.

2011).

Ointment

For the past years, man has applied preparations to soothe and medicate the

skin. Creams, plasters and ointments were one of those. Ointments are thick liquid

substance that creates a soothing effect when applied to the skin. It is used especially

when a person has burns, insect bites and etc. Ointments are prepared in small

preparations and are made to fill the doctors prescribed formula. 13

The efficacy of different ointments have been proved through breakthroughs of

different studies. These ointments has different basis with different penetrations.

According to Ernest A. Strakoschs study, Penetration of Various Ointments Bases,

ointments were revealed to have different penetration with different bases. The relative

intensity of the penetration into the normal human skin of the different test substances

listed in the order from the best to the worst penetration, as revealed by this study is as

follows: the base consisting of: liquid petrolatum-peanut oil-steric acid-triethanolamine-

acetyl alcohol and water; "Hydrosorb" (Abbott), "Aquaphor" (Dule), lard, cold liver oil,

stearyl alcohol-mineral oil-water-petrolatum, lanolin, a base consisting of mannide

monooleate-ceresin wax-petrolatum-mineral oil-lanolin, lecithin ointment, petrolatum

and lanolin to equal parts, petrolatum plus five per cent cetyl alcohol, olive oil, rose

water ointment, vaseline (Chesebrough), and finally petrolatum as such. Because of

multi-drug resistance among many bacterial species has been reported to be on the

increase due to inappropriate or widespread use of antimicrobials. Resistance to

antimicrobials has been observed in various parts of the world. Because of this,

Alternative ointments were also introduced in the modern years. Ointments formulated

from herbal plants with bioactive compounds were proven safe and effective. Herbal

ointments were also introduced to the market.

14
(http://jpet.aspetjournals.org/content/78/1/65.abstract).

Extracts from different plant extracts like Aloe vera, Ficus infectoria, Ficus

religiosa, and Piper betel were studied for antibacterial activity on resistant and sensitive

strains, isolated from skin and soft tissue infections. The combined extract was

formulated in different ointment bases such as polyethylene glycol, gelatin, sodium

alginate, carbopol, cream base and honey. These were then evaluated to find a suitable

base for preparation of an ointment. In vitro study of the release of antimicrobials and

kill-time studies of the herbal ointments was carried out against multi-drug resistant

isolate of Pseudomonas. The ointment showed bactericidal activity within 2 h against

the resistant strain of Pseudomonas spp.

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003181/).

Heavy Metals

Although, there is no clear definition of what a heavy metal is, density is in most

cases taken to be the defining factor. Heavy metals have been used in many different
areas for thousands of years. Heavy metals are naturally occurring elements that have

a high atomic weight and a density at least five times greater than that of water. Their

multiple industrial, domestic, agricultural, medical and technological applications have

led to their wide distribution in the environment; raising concerns over their potential

effects on the human health and the environment. Their toxicity depends on a lot of

factors like dose, route of exposure and chemical species. Because of their high degree

of toxicity, arsenic, cadmium, chromium, lead and mercury ranks among the priority

metals that are of public health significance. These metallic elements are considered

systemic toxicants that are known to induce multiple organ damage. (Patlolla, Anita K.,

Sutton, Dwayne J., Tchounwou, Paul B. & Yedjou, Clement G., 2014). 15

The term heavy metals refers to any metallic chemical element that has a

relatively high density and is toxic or poisonous at low concentrations. Examples of

heavy metals are Mercury (Hg), Cadmium (Cd), Arsenic (As), Chromium (Cr), Thallium

(Ti) and Lead (Pb). Heavy metals are trace elements. Some heavy metals like copper,

selenium and zinc helps our body and is essential to maintain the metabolism of the

human body. However at higher concentrations they can lead to poisoning. Heavy metal

poisoning could result, for instance, from drinking water contamination, high ambient air

concentrations near emission sources, or intake via food chain. Heavy metals are

dangerous because they tend to increase in the concentration of a chemical in a

biological organism over time. (www.lennetech.com/processes/heavy/heavy-

metals/heavy-metals .htm.).

Lead
 Lead poisoning, which is so severe as to cause evident illnesses.Lead is one of

the metals that has the most damaging effect on the human body. Lead is a poisonous

metal that affects the nervous and reproductive system of the human body. Also it

affects the behavior and the blood cells of a person. Even a small intake of lead can

cause severe damage to the human body which can damage the brain and other

important organs. Lead in the environment arises from both natural and anthropogenic

sources. Exposure can occur through drinking water, food, air, soil and dust from old

paint containing lead. Lead is the most recycled non-ferrous metals and its secondary

production has therefore grown steadily is spite of declining lead prices.

(http://www.lenntech.com).

The lead in our environment has been the use of leaded gasoline in automobile

engines. The use of leaded gasoline improves the performance of the engine. However,

the lead that has been released will be formed in the air, soil and water that maybe

mixed in food. Lead is more harmful if its released to water because water is the most

abundant in nature and it is the thing that man always need. If a man will take a

poisoned water it may affect the blood cells and may cause brain damage. Lead is

really a dangerous material that can affect the food chain. (Robblee & Brower, 2001).
16
Cadmium

Cadmium is used to manufacture pigments and batteries and in the metal-plating

and plastics industries.The main sources of cadmium in the air are the burning of fossil

fuels such as coal or oil and the incineration of municipal waste. Cadmium is an

important metal for many types of businesses and industrial processes. Cadmium is

most often used in the manufacturing sector but worker exposure can also occur in
other industry sectors including construction, wholesale trade, and transportation. The

acute short terms diseases that cadmium bring are mainly affecting the lungs such as

pulmonary irritation. While the long term effect causes kidney diseases. Cadmium and

its compounds are highly toxic and exposure to this metal is known to cause cancer and

targets the body's cardiovascular, renal, gastrointestinal, neurological, reproductive, and

respiratory systems. Requirements to protect workers from cadmium exposure are

addressed in specific OSHA cadmium standards covering general industry (1910.1027),

shipyards (1915.1027), construction (1926.1127) and agriculture (1928.1027).

(https://www.osha.gov/SLTC/cadmium/).

Saponins

Saponins are glucosides with foaming characteristics. Saponins consist of a

polycyclic aglycones attached to one or more sugar side chains. The foaming ability of

saponins is caused by the combination of a hydrophobic (fat-soluble) sapogenin and a

hydrophilic (water-soluble) sugar part. Saponins have a bitter taste. Some saponins are

toxic and are known as sapotoxin. Saponins are phytochemicals which can be found in

most vegetables, beans and herbs. The best known sources of saponins are peas,

soybeans, and some herbs with names indicating foaming properties such as soapwort,

saoproot, soapbark and soapberry. Commercial saponins are extracted mainly from

Yucca schidigera and Quillaja saponaria. Saponins have many health benefits. Studies

have illustrated the beneficial effects on blood cholesterol levels, cancer, bone health

and stimulation of the immune system. Most scientific studies investigate the effect of

saponins from specific plant sources and the results cannot be applied to other

saponins. Saponins have hemolytic, expectorative, anti-inflammatory and immune-

stimulating activity. Beyond that, saponins demonstrate antimicrobial properties

particularly against fungi and additionally against bacteria and protozoa.

(http://www.phytochemicals.info/phytochemicals/saponins.php).

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. There are more than 11 distinguished classes of

saponins including dammaranes, tirucallanes, lupanes, hopanes, oleananes,

taraxasteranes, ursanes, cycloartanes, lanostanes, cucurbitanes and steroids. Due to

the great variability of their structures, saponins always display anti-tumorigenic effects

through varieties of antitumor pathways. Saponins are common in a variety of higher

plants and usually found in roots, tubers, leaves, blooms or seeds. Based on the carbon

skeletons, saponins were classified into triterpenes and steroids. Their glycone parts

were mostly oligosaccharides, arranged either in a linear or branched fashion, attached

to hydroxyl groups through an acetal linkage. Modern research found that saponins

have antitumor effect on many cancer cells. Several saponins inhibit tumor cell growth

by cell cycle arrest and apoptosis with IC50 values up to 0.2 mM.

(http://www.sciencedirect.com/science/article/pii/S0367326X10001346, 2010).

18

Flavanoids

Flavonoids are one of the largest nutrient families known to scientists, and

include over 6,000 already-identified family members. They are water soluble

polyphenolic molecules containing 15 carbon atoms. Flavonoids belong to the

polyphenol family. This nutrient group is most famous for its antioxidant and anti-
inflammatory health benefits, as well as its contribution of vibrant color to the foods we

eat. As an especially delicate group of nutrients with respect to cooking heats,

flavonoids are often front and center in development of our cooking methods at

WHFoods, where we always look for cooking methods best able to preserve nutrients.

Flavonoids have antioxidant activity. Flavonoids are becoming very popular because

they have many health promoting effects. Some of the activities attributed to flavonoids

include: anti-allergic, anti-cancer, antioxidant, anti-inflammatory and anti-viral. The

flavonoids quercetin is known for its ability to relieve hay fever, eszema, sinusitis and

asthma. Epidemiological studies have illustrated that heart diseases are inversely

related to flavonoid intake. Studies have shown that flavonoids prevent the oxidation of

low-density lipoprotein thereby reducing the risk for the development of atherosclerosis.

(http://www.phytochemicals.info/phytochemicals/flavonoids.php).

One reason for this is because their concentration in the bloodstream is so much

lower. Another reason lies in the fact that many of the antioxidant functions of the

flavonoids are not performed by the flavonoids themselves, but by forms of the

flavonoids that have been altered by our metabolism. Even though we do not know all

the details about the way flavonoids function as antioxidants, however, studies have

documented better protection of certain cell typesfor example, red blood cells

following consumption of flavonoid-rich foods. Blueberries, for example, have been

repeatedly studied in this context for their flavonoid-related antioxidant benefits. In this

antioxidant context, it is also worth pointing out the potentially unique relationship

between flavonoids and vitamin C. Recent studies have shown the ability of flavonoids

to alter transport of vitamin C, as well as to alter function of an enzyme called ascorbate

oxidase, which converts vitamin C into a non-vitamin form (monodehydroascorbate).

While we do not yet know the full meaning of these relationships, it is clear that the

transport and cycling of vitamin C is flavonoid related. This association makes sense to

us, since so many foods high in vitamin C (such as our top five WHFoods for vitamin C

are papaya, bell peppers, broccoli, Brussels sprouts, and strawberries) are also high in

flavonoids. Flavonoids are phytochemical compounds that provide protection against

ultraviolet radiation, pathogens and herbivores to herbs. Flavonoids have effect on

human nutrition and health as antioxidant and chelating compounds.

(http://www.whfoods.com/genpage.php?tname=nutrient&dbid=119).

Phenols

Phenols are the largest category of phytochemicals and the most widely

distributed in the plant kingdom. Phenols are phytochemical compounds that function in

nutrient uptake, protein synthesis, enzyme activity, photosynthesis; structural

components and allelopathy in herbs. The phenolic compounds, have biological and

pharmacological properties especially their antimicrobial activity,antiviral, anti-

inflammatory and cytotoxic activity, antimutagenic and anticarcinogenic activities.

In Kisii region, southwest Kenya, amongst the herbs used as phytomedicines for

the treatment of diabetes, malaria and pneumonia are Carissa spinarum, Urtica dioica,

Warburgia ugandensis, Senna didymobotrya, Physalis Peruviana, Bidens pilosa,

Leonotis nepetifolia and Toddalia asiatica. A study was carried out on these herbs in the

year 2011 to 2012. The objective was to determine the phytochemical compounds of

flavonoids and phenolic acids present in the eight selected medicinal herbs by thin layer

chromatography (TLC) method. It has been recognized that phenolic compounds are a
class of antioxidant compounds which act as free radical terminators. (Gatebe, Erestus,

Gitu, Leonard, Maobe, Moses A. & Rotich, Henry, 2012).

Tannins

Tannin is a type of biomolecule, (as opposed to modern synthetic tannin), is an

astringent, bitter plant polyphenolic compound that either bonds and precipitates or

shrinks proteins and various other organic compounds including amino acids and

alkaloids (http://www.herbs2000.com/h/_menu/tannins.html).

Tannins can be present in the leaves, bark, and fruits, and are thought to protect

the plant against infection and herbivory (Phenolic Compound Biochemistry).

Tannin are used in leather manufacture and dyeing. Hey are also used in the

clarification of wine and beer, as a constituent to reduce viscosity of drilling mud for oil

wells, and in boiler water to prevent scale formation. Because of its styptic and

astringent properties, tannin has been used to treat tonsillitis, pharyngitis, hemorrhoids,

and skin eruptions; it has been administered internally to check diarrhea and intestinal

bleeding and as an antidote for metallic, alkaloidal, and glycosidic poisons, with which it

forms insoluble precipitates. Soluble in water, tannins form dark blue or dark green

solutions with iron salts, a property utilized in the manufacture of ink

(http://www.britanica.com/EBchecked/topic/582701/tannin).
21

Steroids

Steroids are used to treat a variety of conditions in which the bodys defense

system malfunctions and causes tissue damage. Steroids are used as the main

treatment for certain inflammatory conditions, such as systemic vasculitis (inflammation

of blood vessels) and myositis (inflammation of muscle). They may also be used

selectively to treat inflammatory conditions such as rheumatoid arthritis, lupus,

Sjgrens syndrome, or gout

(http://www.medicinenet.com/steroids_to_treat_arthritis/page2.html).

Steroids are used by prescription to treat certain types of medical disorders. They

are used to treat boys and men who dont produce enough male hormones for formal

development. For example, they are given to me who have testes removed because of

testicular cancer and in treatments for impaired growth. They are also used to treat

some forms of anemia (a condition in which a person has too few red blood cells),

certain types of cancers, autoimmune diseases, certain skin diseases, and osteoporosis

(the loss of bone, which often occurs as people age) (Steroids: High-risk Performance

drugs, 2009).

Staphylococcus Aureus

S. Aureus was first identified in 1880 by the surgeon Sir Alexander

Ogston in pus from a surgical abscess in a knee joint. It is estimated that 20% of the

human population are long-term carriers of S. aureus which can be found as part of the

normal skin flora and in anterior nares of the nasal passages. It is still one of the five
22
most common causes of nosocomial infections and is often the cause of postsurgical

wound infections. Many patients in the hospital are contracted to staphylococcal

infection. (http://en.wikipedia.org/wiki/Staphylococcus_aureus)

Staphylococcus aureus, the gold pigmented form, is a durable organism found on

the mucous membranes and skin of a considerable segment of the population. It

produces infection of the skin, but it is also noted for causing a variety of serious

infections, particularly in the debilitated. There is extensive information about the

products, metabolites, enzymes, extracts, structure and antigen of most cultivatable

microorganisms. The knowledge about these substances is used to identify the

microorganisms, to interpret their virulence, to explain their metabolic activities, and to

control the microorganisms. However, due to the presence of extrachromosomal

genetic units called plasmids, antibiotic resistance in S. aureus is created. Persistent

use of an antibiotic favors the continued presence of the specific antibiotic resistance

plasmid in the bacterial population. In staphylococcal treatment it has been

recommended that the use of tropical antibiotics be eliminated. The presence of

antibiotics at the carriage sites of man only increases the probability of selection of

resistant strains. Another involvement of staphylococci is in the most prevalent type of

food poisoning. Food preparers and handlers who have staphylococcal lessions of the

skin, especially on the hands are most likely to contaminate the food. (Boyd, Robert F.

& Hoerl, Ryan G., 2011).

 Chapter III

Methodology

Research Site and Duration

This study followed a simple research design. It assessed the selected Blaan

plants to show its properties through various tests; bioassay, thin layer chromatography

(TLC), antioxidant assay and heavy metals testing.

This experiment was conducted from February to July 2015 in three phases. The

1st and 2nd phase is the bioassay, TLC and antioxidant assay of the Badyang and

Galinsoga and the Tangisang Bayauak , respectively. While the third phase is the

formulation of ointments and heavy metals testing. First, was the bioassay, TLC and

antioxidant assay of Badyang and Galinsoga conducted on February March 2015 at

the University of the Immaculate Conception (UIC), Davao City. Then, the bioassay,

TLC and antioxidant assay of Tangisan Bayauak conducted on May 18 July 18, 2015

at the University of the Immaculate Conception (UIC), Davao City. Lastly, was the

formulation of ointments from the extract of the selected Blaan plants and heavy metals

testing of it, conducted on May 20 29, 2015 at the Davao Analytical Laboratories Inc.

Research Design

This study employed a descriptive-quantitative design for assessing the bioactive

compounds of Tagisan bayauak (Ficus variegate blume), Galinsoga (Galinsoga

parviflora) and Badyang (Alocasia macrorrhiza) leaves extract, its antioxidant property,

its efficacy against the growth of S. Aureus and the heavy metals present on each
 ointment. Quantitative determination of Blaan plants leaves extract activity and

secondary metabolites are measure by the following standard procedures.

Blaan plants extract

Bioassay Ointment

T1
S. Aureus

Zone of Inhibition

T2 R
2
Scavenging Activity
R3

R1
T3 R2 Legend:
R3 T Treatment

R1 R Replicate

T4 R2 T1 Positive Control (Mupirocin)

R3 T2 Negative Control (Water)

T3 50% concentration of Selected Blaan
R1
plants extract
T5
R2 T4 75% concentration of Selected Blaan
R3 plants extract
T5 100% concentration of Selected Blaan
plants extract

Thin Layer Chromatography

Antioxidant Assay

Heavy Metals
A. Procedure for Bioassay

Blaan Plants Extract

Antibacterial Screening

Reference strains
S. Aureus

Preparation of Inoculum

Preparation of Plate

Preparation of Extract disks

Reading of Zone inhibition

B. Procedure for Thin-Layer Chromatography

Preparation of Materials

Rotavaporization of the Selected

Blaan plants extract

Preparation of the Chemical

Solutions

Thin Sheets were prepared and a drop of the

chemical solution with the selected Blaan plants
extract were added, separately

Recording the Secondary

Metabolites present
C. Procedure for Antioxidant Assay

Preparation of the plants

extract

Dilluting the DPPH in

ethanol

Preparation of each
sample in triplicate

Treating each sample with the

ethanolic extracts

Reading the absorbance level of each

treatment through the spectrometer
Materials and Instruments
Table 1

List of Raw Materials and Laboratory Equipment for Pure Extract Extraction and
Bioassay.

Pure Leaves Extract Extraction Bioassay

Raw Materials Agar medium

Tagisan bayauak plant Nutrient agar
Galinsoga plant S. aureus
Mani-manian plant Laboratory equipment
Laboratory Equipment Autoclave
Weighing scale Petri dishes
Scissors Beaker
Petri dishes Erlenmeyer flasks
Graduated cylinder (500 mL) Forceps
Volumetric flasks Incubator
Beaker Whattman discs
Stirring rod Vernier caliper
Funnel Screw capped test tube
Spatula/Ladle Test tube stand
Erlenmeyer flasks Sterile cotton swabs
Extractor setup Vortex mixer
Rotary evaporator Micropipettor
Filter paper
Solvents
Distilled water
95% ethanol
Table 1.2
List of Raw Materials and Laboratory Equipment for Pure Extract
Extraction and Thin Layer Chromatography

Pure Leaves Extract Extraction Thin Layer Chromatography

Raw Materials Raw Materials
Tagisan bayauak plant
Tagisan bayauak plant
Galinsoga plant
Galinsoga plant
Badyang plant
Mani-manian plant
Laboratory Equipment
Laboratory Equipment
Weighing scale
Scissors
Beaker
Petri dishes
Stirring rod
Graduated cylinder (500 ml) Funnel
Volumetric flasks Cellulose
Beaker
Stirring rod Solvents
Funnel
Spatula/ladle Distilled water
Erlenmeyer Flasks 95% Ethanol

Extractor setup
Rotary evaporator
Filter paper
Solvents
Distilled water
95% Ethanol
Table 1.3
List of Raw Materials and Laboratory Equipment for Pure Extract Extraction and
Antioxidant assay

Pure Leaves Extract Extraction Antioxidant assay

Raw Materials Raw Materials

Tagisan bayauak plant

Galinsoga leaves extract
Galinsoga plant
Mani-manian plant Badyang leaves extract
Laboratory Equipment
Tagisan Bayauak pericarps extract
Weighing scale
Scissors Laboratory Equipment

Petri dishes
Beaker
Graduated cylinder (500 ml)
Volumetric flasks Test tube

Beaker
Stirring rod
Stirring rod
Funnel Pippete

Spatula/ladle
Dropper
Erlenmeyer Flasks
Extractor setup Spectrometer

Rotary evaporator
Solvents
Filter paper
Solvents Ethanol

Distilled water
Distilled water
95% Ethanol
Experimentation and General Procedure

A. Preparation of Plants Leaves Extract

Tagisang bayuak (Ficus variegata blume), Badyang (Alocasia macorrhiza) and

Galinsoga (Galinsoga Farviflora) plant was collected at South Cotabato. Five hundred

fifty grams (550 g) of freshly washed Tagisan bayauaks pericarp and Badyang and

Galinsoga leaves was completely soaked in 1.5 L of 95% ethyl alcohol for 48 hours and

was filtered. The crude ethanolic extract will then be concentrated by using a rotary

evaporator at temperature 60, resulting to a thick, viscous and syrupy extract which

was later used for the tests involved in the study.

B. Procedure for Bioassay: Antibacterial Activity of Tagisan bayauak (Ficus

variegate blume) Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga

Farviflora) Extract Against the Growth of Staphylococcus aureus

Preparation of Inoculum

Colonies of Staphylococcus aureus was inoculated onto 2 different 5 mL Mueller-

Hinton broth (MHB) and incubated at 35C for 2 hours to allow bacteria to grow. It will

be adjusted using 0.5 McFarland standard solution with sterile Normal Saline Solution

(NSS). These will serve as the standardized inoculum. (Guevara et al, 2005)

Preparation of Seeded Agar Plate

Twenty (20) mL of Mueller-Hinton Agar (MHA) was dispensed into sterile petri

dishes to a depth of 4 mm and allowed to cool and solidify. A sterile cotton swab was

dipped onto the standardized organism inoculums suspension, pressed and rotated
firmly against the inside wall of the tube just above the fluid to remove the excess liquid.

The entire surface of the solidified agar will be swabbed evenly to ensure even and

complete distribution of the inoculum. The plates will be allowed to dry for 15 minutes.

This preparation will be done in the biosafety cabinet. (Guevara et al., 2005)

Position of Paper Disks for Preliminary Screening of Antibacterial Activity

Twenty microliters (20 L) of each leaves extract was dispensed into sterile

paper disks with the use of a pipette. The impregnated paper disks was air-dried under

for two hours the biosafety hood. Using sterile forceps, the impregnated paper disks will

be placed in a clockwise order onto the seeded plate at equidistant positions.

Tetracycline disk and distilled water was used as positive and negative controls,

respectively. The drugs was allowed to diffuse through the agar for 60 minutes. The

plates was inverted and incubated for 24 hours at 350C. The diameter of the zone of

inhibition was measured in millimeter (mm) using a ruler. The assay will be done in

triplicate and the means will be computed.

Figure 8. Position of Paper Disks for Preliminary Screening as Antibacterial Agent

Legend:
1, 2, 3 - replicates
2

1 3
C. Procedure for Thin Layer Chromatography of Tagisan bayauak (Ficus
variegate blume), Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga
Farviflora) Extract

Preparation of the Plant Extract

The 0.50 g of Tagisan bayauak (Ficus variegate blume) leaves extract was

treated with ten (10) mL of a mixture of chloroform and acetic acid, CHCl 3:CH3COOH

(99:1V/V) and will be heated for five (5) to ten (10) minutes over a water bath. It was

filtered and the filtrate was labeled Solution A.

Another 0.50 grams leaves extract treated with ten (10) ml of mixture of

chloroform, methanol and acetic acid, CHCL3:CHCOH:CH3COOH (49.5:49.5:1) and was

heated for five (5) to ten (10) minutes over a water bath. It was filtered and the filtrate

labeled Solution B.

Finally the 0.50 grams leaves extract treated with ten (10) ml of mixture of

methanol and water, CH3OH:H20 (1:1) and was heated for five (5) to ten (10) minutes

over a water bath. It was filtered and the filtrate will be labeled Solution C.

D. Procedure for Antioxidant assay of Tagisan bayauak (Ficus variegate blume),

Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga Farviflora) Extract

Dissolved ethanolic extracts in absolute ethanol and water extract in distilled

water. Diluted each sample for at least 3 concentrations (two-fold dilutions). Prepared

6x10-5M of DPPH in absolute ethanol. Then, transferred 500 l of each sample solution

into an eppendorf tube with each concentration tested in triplicate. Transferred 500 l of

DPPH solution to mix with sample solution. Then shake and stand at the room
temperature for 30 minutes. Lastly, measured the absorbance at 520 nm, using a

mixture of 500 l sample solution and 500 l absolute ethanol as blank.

 Chapter IV

Presentation, Analysis and Interpretation of Data

This chapter presents and interprets the data gathered in textual, graphical and

tabular manner.

Average Zone of Inhibition of Tangisang bayuak (Ficus variegata

blume) plant extract against the growth of Staphylococcus aureus
REPLICATES

R1 R2 R3 MEAN
TREATMENTS

T1
(+) Control 24 24 24 24
(Mupirocin)
T2
(-) Control 6 6 6 6
(Sterilized Distilled Water)

T3
(50% Plant Extract) 17 13 11 13.7

T4
(75% Plant Extract) 16 17 15 16

T5
(100% Plant Extract) 19 17 18 18

The table above shows the activity of Tangisang bayuak (Ficus variegata blume)

against the growth of Staphylococcus aureus.

 Results revealed that the Positive control (T1 = Mupirocin) has the highest zone

of inhibition among the treatments with its mean zone of inhibition of 24 mm followed by

the T5, with a mean zone of inhibition of 18 mm. This means that the Tanguisang

bayuak pericarp extract has an active inhibitory activity against the reference strains,

Staphylococcus aureus.

Source of Degree of Sum of Mean of Computed Tabulated

Variation Freedom Squares Squares F F

Between 4 517.03 129.26

groups
58 3.48
Within 10 22.3 2.23
groups

The table above represents the statistical tool used for the activity of Tangisang

bayuak (Ficus variegata blume) plant extract against the growth of Staphylococcus

aureus.

Average Zone of Inhibition of Tangisang bayuak (Ficus variegata

blume) ointment against the growth of Staphylococcus aureus
REPLICATES
TREATMENTS
R1 R2 R3 MEAN
T1
(+) Control 24 24 24 24
 (Mupirocin)

T2
(-) Control 6 6 6 6
(Sterilized Distilled Water)

T3 (50%) 15 13 11 13

T4 (75%)
16 13 11 13.3

T5 (100%)
15 14 10 13

Results shows that the T1 is the most effective treatment that can inhibit the

growth of bacteria against the other treatments. (One-Way ANOVA Fcomp= 58 > Ftab =

3.48 = 0.05)

The table above shows the activity of Tangisang bayuak (Ficus variegata blume)

ointment against the growth of Staphylococcus aureus.

Results revealed that the Positive control (T1 = Mupirocin) has the highest zone

of inhibition among the treatments with its mean zone of inhibition of 24 mm followed by

the T5, with a mean zone of inhibition of 13.3 mm. This means that the Tanguisang

bayuak pericarp extract ointment has an active inhibitory activity against the reference

strains, Staphylococcus aureus.

Source of Degree of Sum of Squares Mean of Computed Tabulated

Variation Freedom Squares F F
Between 4 499 124.75
groups 19.0 3.48
 Within 10 66.7 6.57
groups

The table above represents the result of the statistical tool used for the activity of

Tangisang bayuak (Ficus variegata blume) ointment against the growth of

Staphylococcus aureus.

Results shows that the T1 is the most effective treatment that can inhibit the

growth of bacteria against the other treatments. (One-Way ANOVA Fcomp= 19.0 > Ftab =

3.48 = 0.05)

Average Zone of Inhibition of Badyang (Alocasia macrorrhiza) plant

extract against the growth of Staphylococcus aureus
TREATMENTS REPLICATES
R1 R2 R3 MEAN
T1
(+) Control 24 24 24 24
(Mupirocin)

T2
(-) Control 6 6 6 6
 (Sterilized Distilled Water)

T3
(50% Plant Extract) 12 13 15 13.3

T4
(75% Plant Extract) 15 14 14 14.3

T5
(100% Plant Extract) 18 17 18 17.7

The table above shows the activity of Badyang (Alocasia macrorrhiza) against

the growth of Staphylococcus aureus.

Results revealed that the Positive control (T1 = Mupirocin) has the highest zone

of inhibition among the treatments with a mean zone of inhibition of 24 mm followed by

the Treatment 5 (100% Plant Extract) with a mean zone of inhibition of 17.7 mm. This

means that the A. macrorrhiza has an active inhibitory effect towards the reference

strains Staphylococcus aureus.

Source of Degree of Sum of Squares Mean of Computed Tabulated

Variation Freedom Squares F F

Between 4 657.28 131.46

groups
27.73 3.48
Within 10 56.85 4.74
groups

The table above represents the result of the statistical used for the activity of

Badyang (Alocasia macrorrhiza) against the growth of Staphylococcus aureus.

 Results shows that the T1 is the most effective treatment that can inhibit the

growth of bacteria against the other treatments. (One-Way ANOVA Fcomp= 58 > Ftab =

3.48 = 0.05)

Average Zone of Inhibition of Badyang (Alocasia macrorrhiza)

ointment against the growth of Staphylococcus aureus
TREATMENTS REPLICATES
R1 R2 R3 MEAN
T1
(+) Control 24 24 24 24
(Mupirocin)

T2
(-) Control 6 6 6 6
(Sterilized Distilled Water)

T3
(50% Plant Extract) 15 11 10 12

T4
(75% Plant Extract) 16 13 11 13.3

T5
(100% Plant Extract) 15 13 12 13.3

The table above shows the activity of Badyang (Alocasia macrorrhiza) ointment

against the growth of Staphylococcus aureus.

Results revealed that the Positive control (T5 = Mupirocin) has the highest zone

of inhibition among the treatments with a mean zone of inhibition of 24 mm followed by

the Treatment 1 and 2 (100% and 75 % plant ointment respectively) with a mean zone
of inhibition of 13.3 mm. This means that the A. macrorrhiza ointment has an active

inhibitory effect towards the reference strains Staphylococcus aureus.

Source of Degree of Sum of Squares Mean of Computed Tabulated

Variation Freedom Squares F F

Between 4 505.6 126.4

groups
40.3 3.48
Within 10 31.33 3.13
groups

The table above represents the statistical used for the activity of Badyang

(Alocasia macrorrhiza) ointment against the growth of Staphylococcus aureus.

Results shows that the T1 has the most effective inhibitory activity among the

other treatments. (One-Way ANOVA Fcomp= 40.3 > Ftab = 3.48 = 0.05)

Average Zone of Inhibition of Galinsoga (Galinsoga parviflora)

against the growth of Staphylococcus aureus
TREATMENTS REPLICATES
R1 R2 R3 MEAN
T1
(+) Control 24 24 24 24
(Mupirocin)

T2
(-) Control 6 6 6 6
(Sterilized Distilled Water)
 T3
(50% Plant Extract) 12 13 12 12.3

T4
(75% Plant Extract) 15 17 16 16

T5
(100% Plant Extract) 19 18 20 19

The table above shows the activity of Galinsoga (Galinsoga parviflora) extract

against the growth of Staphylococcus aureus.

Results shows that the Positive Control (T1 = Mupirocin) has the highest zone of

inhibition of 24 mm followed by the Treatment 5 (100% G. parviflora Plant Extract) with

a mean zone of inhibition of 19 mm. This means that G. parviflora has an active

inhibitory effect against the reference strain, Staphylococcus aureus.

Source of Degree of Sum of Squares Mean of Computed Tabulated

Variation Freedom Squares F F

Between 4 417.72 104.43

groups
173.76 3.48
Within 10 6.01 6.01
groups

The table below represents the statistical tool used for the activity of Galinsoga

(Galinsoga parviflora) against the growth of Staphylococcus aureus.

Average Zone of Inhibition of Galinsoga (Galinsoga parviflora) ointment
against the growth of Staphylococcus aureus

Results shows that the T1 is the most effective treatment that can inhibit the

growth of bacteria against the other treatments. (One-Way ANOVA Fcomp= 173.76 > Ftab

= 3.48 = 0.05)
 REPLICATES
TREATMENTS
R1 R2 R3 MEAN
T1 24 24 24 24
(+) Control
(Mupirocin)
T2 6 6 6 6
(-) Control
(Sterilized Distilled Water)
T3 15 11 10 12
(50%)
T4 15 13 11 13
(75%)
T5 14 13 11 12.7
(100%)

The above shows the activity of Galinsoga (Galinsoga parviflora) ointment

against the growth of Staphylococcus aureus.

Results shows that the Positive Control (T1 = Mupirocin) has the highest zone of

inhibition of 24 mm followed by the Treatment 4 (75% G. parviflora plant ointment) with

a mean zone of inhibition of 13 mm. This means that G. parviflora ointment has a

partially active inhibitory effect against the reference strain, Staphylococcus aureus.

Source of Degree of Sum of Mean of Computed F Tabulated F

Variation Freedom Squares Squares

Between 4 73.75 18.40 30.62 3.48

groups
Within 10 6.01 0.601
groups
 The table below represents the statistical tool used for the activity of Galinsoga

(Galinsoga parviflora) ointment against the growth of Staphylococcus aureus.

The results shows that the T1 is the most effective treatment that can inhibit the

growth of the bacteria among the other treatments. One-Way ANOVA Fcomp= 54.53 >

Ftab = 3.48 = 0.05)

Thin-Layer Chromatography

Presented are the results of the Thin-Layer chromatography of Tangisang

bayuak (Ficus variegata blume), Badyang (Alocasia macrorrhiza) and

GalinsogaGalinsoga parviflora).

DISTANCE TRAVELLED
TEST Solvent Solute Hrf RESULTS
100 76 0.76
100 69 0.69
Tangisang bayuak Flavanoids,
(Ficus variegata blume) 100 49 0.49 Saponins,
Tannins
100 70 0.70
Badyang 100 60 0.60 Steroids,
(Alocasia macrorrhiza) Tannins,
100 50 0.50 Saponins
120 60 0.50
120 88 0.73
Galinsoga 120 75 0.63 Steroids,
(Galinsoga parviflora) Tannins,
120 90 0.75 Saponins,
Phenols

Thin layer chromatography results of Badyang (Alocasia macrorrhiza) detected

the presence of the bioactive compounds of Steroids, Tannins, Saponins. The distance

travelled by the solvent (from the tip) is 100mm and the distance travelled by the solute

(from the tip) is 70mm, 60mm, 50mm with the hrf value as follows, 0.70, 0.60, 0.50.

Thin layer chromatography results of Galinsoga (Galinsoga parviflora) detected

the presence of the bioactive compounds of Steroids, Tannins, Saponins and Phenols.

The distance travelled by the solvent (from the tip) is 120mm and the distance travelled
by the solute (from the tip) is as follows 60mm, 88mm, 75mm, 90mm with the hrf values

accordingly 050, 0.73, 0.63, 0.75.

Antioxidant Assay

Presented is the result of the Antioxidant Assay of Tangisang bayuak (Ficus

variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga

parviflora).

TEST TREATMENT ABSORBANCE SCAVENGING ACTIVITY

LEVEL (%)
T1 .230 37.84%
Tangisang bayuak T2 .216 41.62%
(Ficus variegata blume)
T3 .219 40.81%

T1 .2604 29.62%
Badyang T2 .256 30.78%
(Alocasia macrorrhiza) T3 .2458 33.56%
T1 .2155 41.75%
Galinsoga T2 .2086 43.62%
(Galinsoga parviflora) T3 .1898 48.71%
T4 (Blank) .370

The findings indicated that Tangisang bayauak (Ficus variegata blume) pericarp

extract have inhibit the formation of DPPH at the percentage rate of 37.84% to 40.81%

which confirms its potential as antioxidant agent.

 The results indicated that Badyang (Alocasia macrorrhiza) plant extract have

inhibit the formation of DPPH at the percentage rate of 29.62% to 33.56% which

confirms its potential as antioxidant agent.

The results indicated that Galinsoga (Galinsoga parviflora) plant extract have

inhibit the formation of DPPH at the percentage rate of 41.75% to 48.71% which

confirms its potential as antioxidant agent.

Physico-Chemical Test

Presented are the results of the Physico-Chemical Test of Tangisang bayuak

(Ficus variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga

parviflora) ointments.

TEST TREATMENTS COLOR APPERANCE pH

T1 Greenish Uniform coloration with no 4.9

liquid formation
Tangisang bayuak
(Ficus variegata T2 Greenish Uniform coloration with no 4.8
blume) liquid formation

T3 Greenish Uniform coloration with no 5.0

liquid formation

T1 Greenish Uniform coloration with no 4.8

liquid formation
Badyang
(Alocasia macrorrhiza) T2 Greenish Uniform coloration with no 4.5
liquid formation

T3 Greenish Uniform coloration with no 4.3

liquid formation

T1 Greenish Uniform coloration with no 5.5

liquid formation
Galinsoga
(Galinsoga parviflora) T2 Greenish Uniform coloration with no 5.2
liquid formation

T3 Greenish Uniform coloration with no 5.4

liquid formation
Based on the results, the formulated ointment of Tangisang bayuak (Ficus

variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga parviflora)

has no residual contamination with pH value within the acceptable range.

Heavy Metals

Presented are the results of the Heavy Metals Test of Tangisang bayuak (Ficus

variegata blume), Badyang (Alocasia macrorrhiza) and Galinsoga (Galinsoga parviflora)

ointments.

OINTMENT PARAMETERS RESULTS

Cadmium, ppm
Tangisang bayuak (Ficus
variegata blume Lead, ppm Not Detected

Cadmium, ppm
Badyang (Alocasia
macrorrhiza) Lead, ppm Not Detected

Cadmium, ppm
Galinsoga (Galinsoga
Lead, ppm Not Detected
parviflora)
 Chapter V

Summary of Findings, Conclusions and Recommendations

This chapter presents the summary of findings, conclusion made from the study

and the recommendations given by researchers.

Summary of Findings

The study investigated the components of commonly used medicinal plants by

Blaan of South Cotabato. In the procedure, the plants extract and the ointment was

assess by antibacterial activity, thin layer chromatography, antioxidant and heavy

metals. The researchers followed standard procedures in testing the plant extract and

the ointment.

Bioassay proved that Galinsoga (Galinsoga parviflora) plant extract has a

moderate (mean, ZI=15.89 mm) inhibition to Staphylococcus aureus. The Badyang(

Alocasia macrorrhiza) plant extract has a moderate inhibition of Staphylococcus aureus

(mean, ZI=15.11 mm).

Thin layer chromatography detected the presence of the phytochemicals:

steroids, tannins, saponins and phenols for Galinsoga plant extract; phytochemicals:

steroids, tannins and saponins for Badyang plant extract; and lastly

flavonoids,tannins,and saponins for Tanguisang Bayawak plant extract. This means that

the composition of the leaves extract of commonly used medicinal plants by Blaan of

South Cotabato can be elucidated to a certain extent.

 The antioxidant property test result showed that the Tagisang bayauak (Ficus

variegata blume), Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga parviflora)

forms the inhibition of DPPH which confirms its potential as antioxidant agent.

The heavy metals test for the Tagisang bayauak (Ficus variegata blume)

ointment, Badyang (Alocasia macorrhiza) ointment and Galinsoga (Galinsoga parviflora)

ointment did not detect the heavy metals (cadmium and lead) from the ointment.

The Tagisang bayauak (Ficus variegata blume) ointment, Badyang (Alocasia

macorrhiza) ointment and eGalinsoga (Galinsoga parviflora) have the appearance of

uniform coloration with no liquid formation and its greeninsh color. It shows that the

formulated ointment has no residual contamination with pH value within the acceptable

range.

Conclusions:

1. Bioassay proved that the Galinsoga (Galinsoga parviflora) plants extract has a

moderate (mean, ZI: 15.89 mm) for Staphylococcus Aureus. The Badyang(

Alocasia macrorrhiza) plant extract has a moderate inhibition of Staphylococcus

aureus (mean, ZI=15.11 mm).

2. The Tangisang Bayauak (Ficus variegeta bume) ointment has a partially active

inhibitory effect against the Staphylococcus Aureus (mean, ZI=13.3). The

Galinsoga (Galinsoga parviflora) has a partially active inhibitory effect against the

Staphylococcus Aureus (mean, ZI=13). The Badyang (Alocasia macrorrhiza) has

a partially active inhibitory effect against the Staphylococcus Aureus (mean,

ZI=13.3)
 3. There are bioactive compounds present in Tagisang bayauak (Ficus variegata

blume), Badyang (Alocasia macorrhiza)- steroids, stanins and saponins and

Galinsoga (Galinsoga parviflora)-steriods, stanins, saponins and phenol plants

extract.

4. The antioxidant property shows that the scavenging activity of the Tagisang

bayauak (Ficus variegata blume), Badyang (Alocasia macorrhiza) and Galinsoga

(Galinsoga parviflora) is greater than the absorbance level of 1.5 which shows its

potential to antioxidant activity.

5. There are no detected heavy metals (cadmium and lead) present in the ointment

formulation of Tagisang bayauak (Ficus variegata blume), Badyang (Alocasia

macorrhiza) and Galinsoga (Galinsoga parviflora).

6. The Tangisang bayauak (Ficus variegeta blume), Badyang (Alocasia

macrorrhiza), Galinsoga (Galinsoga parviflora) has no residual contamination.

Recommendations:

1. Conduct a bioassay for a longer time other than observing for 24 hours.

2. Formulate another medicinal pills out of the Tagisang bayauak (Ficus variegata

blume),Badyang (Alocasia macorrhiza) and Galinsoga (Galinsoga parviflora)

plant extract.

3. Conduct more test the Tagisang bayauak (Ficus variegata blume) ointment,

Badyang (Alocasia macorrhiza) ointment and Galinsoga (Galinsoga parviflora)

ointment for more accurate result and to assess its safeness.

4. Investigate on Tagisang bayauak (Ficus variegata blume),Badyang (Alocasia

macorrhiza) and Galinsoga (Galinsoga parviflora) plant extract mutagenic

activities.

5. Conduct a test to identify the bioactive chemical constituents present in the

Tagisang bayauak (Ficus variegata blume),Badyang (Alocasia macorrhiza) and

Galinsoga (Galinsoga parviflora) plant extract that can be isolated for medicinal

uses.

6. Conduct a test of Tagisang bayauak (Ficus variegata blume),Badyang (Alocasia

macorrhiza) and Galinsoga (Galinsoga parviflora) for anti-fungal ativities.

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