Department of EducationRegion 1

Division of La UnionRegional Science High School for Region 1

Bangar, La Union

Antimicrobial Property of Nut Grass Rhizome

(Cyperus rotundus) against Escherichia

coli, Staphylococcus aureus

and Salmonella spp.

An Entry to the Intel Philippines Science FairRegional Level

Candon National High SchoolCandon City, Ilocos SurNovember 28-29, 2006

Proponents

Lyra Erika Liclican

Glen Bernard Asto

Mr. Rogelio Valdez

Research Adviser

Acknowledgement

Embarking on with this Investigatory Project was a long and onerous yet

challenging and rewarding task. But with the help and contributions of many persons and

entities, our study was realized.

To our Almighty God, for helping us hurdle all the obstacles in undertaking this

study.

To Dr. Amerfina Nelmida, School Principal for her guidance and valuable

suggestions.

To Mr. Rogelio Valdez, our adviser, who had endowed valuable time and

commitment in editing this piece of work. We could never repay him rational judgment

in giving profound ideas and suggestions for the betterment of this study.

To the judges for their favorable criticisms and inestimable suggestions in the

enrichment of this study.

To the Department of Science and Technology for their technical assistance in the

conduct of the Antimcrobial assay and Phytochemical Test of our study.

To Mrs.Ma. Luisa Allada for assisting us in the reflux method in the extraction of

our specimen.

Mrs. Marie Munar and Miss Herma Dingle for providing the necessary materials

needed; Mrs. Aurelia Garcia for lending us books; to Mrs. Edwina Manalang for assisting

us in the statistical tools involved; to Mr. Carlo Sojo for his patience in helping us in

collecting the Nut Grass ( Barsanga); to Mrs. Elma Gacutan and Mr. Kyle Mupas for

lending us their laptop and USB respectively.

To our parents, who gave us the chance to dive into the wide sea of education to

fish for knowledge.

And to the persons who, in one way or another had helped and inspired us .

We thank you all…

Lyra Erika Liclican

Glen Bernard Asto

Abstract

This study aimed to determine the Antimicrobial Property of Nut Grass (Cyperus

rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp.and it was

conducted on May to September 2005 at the Department of Science and Technology

Taguig City, Metro Manila..

Four hundred (400)g of Nut Grass rhizome was subjected to experimentation.

Antimicrobial assay test were subjected using the control Chloramphenicol and the

experimental variable –Nut Grass rhizome extract. Phytochemical Test was done to

determine the active principles present. It showed that Saponins and Glycosides were

abundant in all tissues of the rhizome while Triterpenes and Tannins were detectable

only.

There were two treatments used namely T0: the controlled antibiotic-

Chloramphenicol and T1: the Nut Grass rhizome extract. T- test showed that the Nut Grass

rhizome extract had a similar performance with the Chloramphenicol as an antibiotic.

Likewise, there is no significant difference of using the Antimicrobial Property of Nut

Grass rhizome extract and the Chloramphenicol in terms of zone of inhibition that

resulted to the acceptation of the Null Hypothesis.

Furthermore, it is highly recommended that it can be used as a medicinal plant.

Pure extracts of Nut Grass rhizome should be conducted, processed and be made as an

antibiotic. Wide dissemination of the latest technology must commence.

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Table of Contents

Approval Sheet………………………………………………………………..

Acknowledgement…………………………………………………………….

Abstract……………………………………………………………………….

CHAPTER 1…………………………………………………………………..

Introduction……………………………………………………………

Background of the Study……………………………………………..

Statement of the Problem…………………………………………….

Hypotheses……………………………………………………………

Significance of the Study……………………………………………..

Scope and delimitation……………………………………………..

Review of related Literature………………………………………..

Description of the Animal Specimen………………………

Histochemical Components………………………………..

Test Microorganisms………………………………………

CHAPTER II

Methodology

Experimental Design……………………………………….

Materials…………………………………………………..

Reagents…………………………………………………….

Collection of Animal Specimen……………………………

Preparation of the Extract…………………………………

Antimicrobial Assay Test…………………………………

Histochemical Test………………………………………….

Flowchart…………………………………………………….

CHAPTER III

RESULTS AND FINDINGS………………………………………

CHAPTER IV

COCLUSIONS AND RECOMMENDATIONS……………………

Conclusions………………………………………………….

Recommendations…………………………………………..

Bibliography…………………………………………………………………

Appendices…………………………………………………………………..

List of Plates

Plate 1. Collecting the plant specimen……………………………………………………

Plate 2. The plant specimen – Nut Grass

(Cyperus rotundus)…………………………………………………………………………

Plate 3. The Nut Grass Rhizome…………………………………………………………

Plate 4. Materials used for the Reflux

Method (Extraction)………………………………………………………………………

Plate 5. Researcher extracting the Nut Grass

rhizome using the Reflux Method………………………………………………………….

Plate 6.Filtering the extracted Nut Grass rhizome

using the filter paper………………………………………………………………………

Plate 7. Materials for the Antimicrobial Test……………………………………………

Plate 8. Aliquots of the bacterial and yeast suspensions

were transferred into pre-poured Nutrient Agar (NA) and

Glucose Yeast Peptone (GYP) Agar, respectively………………………………………..

Plate 9. The medium, melted and cooled to 450C,

was poured onto the agar plates…………………………………………………………….

Plate 10. Swirling the agar plate to distribute the inoculums

evenly in the agar surface…………………………………………………………………..

Plate 11. Make three (3) holes using a cork borer…………………………………………

Plate 12. The extracts were placed in each hole…………………………………………..

Plate 13.Incubating the agar plates at room temperature………………………………….

Plate 14.The treatments – To ( Chloramphenicol) and

T1 ( Nut Grass rhizome extract)…………………………………………………………

List of Figures

Figure 1.The Flowchart of the Experimental Design………………………………………

Figure 2. Mean of the Zone of Inhibition of E.coli,

S. aureus, C. albicans and A. niger treated by the

extract (5mL) from the Nut Grass rhizome…………………………………………………

Figure 3. Mean of the Zone of Inhibition of E. coli,

S. aureus C. albicans and A. niger treated with

5mL of Chloramphenicol syrup……………………………………………………………

Figure 4. Comparison of the Mean on the

Zone of Inhibition of E. coli, S. aureus,

C. albicans and A. niger when treated by

the Nut Grass rhizome extract and to

5mL of Chloramphenicol syrup…………………………………………………………..

List of Tables

Table 1. The inhibitory effect of Escherichia coli

growth when treated with 5mL of

Chloramphenicol (commercial antibiotic)

and the extract (5mL) from the Nut Grass rhizome…………………………………..

Table 2.The Inhibitory effect on the Staphyloccoccus aureus

growth when treated with 5mL of Chloramphenicol

(commercial antibiotic) and the extract (5 mL) from

the Nut Grass rhizome…………………………………………………………………

Table 3.The Inhibitory effect on the Candida albicans

growth when treated with 5mL of Chloramphenicol

(commercial antibiotic) and the extract (5 mL) from

the Nut Grass rhizome………………………………………………………………

Table 4.The Inhibitory effect on the Aspergillus niger

growth when treated with 5mL of Chloramphenicol

(commercial antibiotic) and the extract (5 mL)

from the Nut Grass rhizome………………………………………………………….

Table 5.Summary table of the Inhibitory effect

on the E. coli, S. aureus, C. albicans and A. niger

growth when treated with 5mL of Chloramphenicol

(commercial antibiotic) and the extract (5mL) from

the Nut Grass rhizome……………………………………………………………………..

Table 6 A Summary table of the Active Principles

of the Nut Grass rhizome………………………………………………………………….

Chapter IINTRODUCTION

A. Background of the Study

Many medicines and drugs are derived from plants. Pharmaceuticals and

synthetics are based on natural compounds first found in plants, bacteria and fungi.

Although modern medicines are predominantly composed of scientifically developed

synthetic drugs, still about 40% of all prescribed drugs are derived from natural

substances such as plant resources.

Due to the present economic crisis, people living in remote areas found it difficult

to buy medicines for their ailments. They usually resort to nature in the form of herbal

medicines which they found to be effective in many cases. Many plants in our country

have been reported to have medicinal uses.

Cyperus rotundus, commonly known as Nut Grass, is a pan tropic species of

sedge that has spread-out to become a worldwide introduced weed. It has been called “the

world’s worst weed” as it is known as s pest in over 90 countries, and infests over 50

crops worldwide. Its existence in a field significantly reduces crop yield, both because it

is a tough competitor for ground resources, and because its dead subterranean tissue

releases substances harmful to other plants. Similarly, it also has a bad effect on

ornamental gardening.

Philippines, being a tropical country, provide a name for countless species of

plants and animals, from the simplest to the most complex ones. Among these plants is

the Nut Grass (Cyperus rotundus) and can be a very good source of antibiotics.

With these information, it provoke the researcher’s mind that what if Cyperus

rotundus has an antibacterial property and then could be used as antibacterial agent to

inhibit the growth of harmful bacteria, thus the Antimicrobial Property of Nut Grass

(Cyperus rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp.

was conceived.

B. Statement of the Problem

This study aimed to determine the Antimicrobial Property of Nut Grass (Cyperus

rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp.

Specifically, this study sought to answer the following sub problems:

1) Can the Nut Grass rhizome extract inhibit the growth of harmful microorganisms?

2) What are the active components Nut grass rhizome using Phytochemical test?

3) Is there a significant difference of using the Antimicrobial Property of Nut grass

rhizome and the commercial antibiotic in terms of zone of inhibition?

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C. Hypotheses

This study anchors the following hypotheses:

1) The Nut Grass rhizome extract cannot inhibit the growth of harmful

microorganisms.

2) Triterpenes, Tannins, Saponins and Glycosides are not the active components of

the Nut Grass rhizome using the Phytochemical Test.

3) There is no significant difference of using the Antimicrobial Property of Nut

Grass rhizome and the commercial antibiotic in terms of zone of inhibition.

D. Significance of the Study

This study will engender information on the localization of the active principles or

constituents present in the plant tissue from which the plant extract was made. Moreover,

the presence of active principles in the plant would explain the antibacterial effect

exhibited by the plant extracts. The antibacterial susceptibility test will determine the

varying antibacterial effects of the plant extracts on the different test microorganisms.

This study will also generate information regarding the effect of the plant extracts

on Escherichia coli, Staphylococcus aureus, and. Salmonella spp. As a result, the

information would be useful for the preparation of drugs from plant extracts.

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E. Scope and Delimitation of the Study

This study is limited to the use of natural rhizome as source of plant extracts and

the use of three species of bacteria namely: Escherichia coli, Staphylococcus aureus,

and Salmonella spp.for antimicrobial assay test.

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F. Review of Related Literature

English Name: Nut Grass

Common Name: Barsanga

Other name: Curry Flower seed

Scientific Name: Cyperus rotundus

Kingdom: Plantae

Division: Magnoliophyta

Class: Liliopsida

Order: Poales

Family: Cyperaceae

Genus: Cyperus

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Description

· It is slender, erect, glabrous, and perennial glasslike plant. It can grow to10 to 40 cm

high. Its rhizomes or underground stems are wiry, bearing black, and hard; ovoid tubers

are about 1 cm in diameter. Its above ground stem is solitary, distinctly 3-angled.

· Its flowers are inflorescence umbel-type, simple or compound, 2 to 6 cm long, with

rather long rays or spikes. Their spikes with 3 to 8 spikelets are brown, flat, slender, 10 to

25 mm long with 10 to 25 florets per spikelet. Rachilla of the spikelet distinctly winged.

Glumes of the floret distichously arranged the first 2 empty, the third one bisexual.

Distribution

Found throughout the Philippines; a common weed in gardens, lawns and

wastelands.throughout the Philippines.

Part utilized

· Rhizome.

· Harvest from December to January.

· Wash and sun-dry or heat-dry in a clean frying pan.

· Scrape off the fibrous roots.

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General Description

Cyperus rotundus known also as Curry Flower Seed is a perennial plant that may

reach a height of up to 40 cm. The names "nut grass" and "nut sedge" are derived from its

tubers that somewhat resemble nuts, although botanically they have nothing to do with

nuts.

As in other Cyperaceae, the leaves of the Cyperus rotundus sprout in ranks of

three from the base of the plant. The flower stems have a triangular cross-section. The

flower is bisexual and has three stamina and a three-stigma carpel. The fruit is a three-

angled achene.

A young plant root system initially forms white, fleshy rhizomes. Some rhizomes

grow upward in the soil, and then form a bulb-like structure from which new shoots and

roots grow, and from the new roots – new rhizomes grow. Other rhizomes grow

horizontally or downward, and form dark reddish-brown tubers or tuber-chains.

Cyperus rotundus is one of the worst weeds mankind knows. Its existence in a

field significantly reduces crop yield, both because it is a tough competitor for ground

resources, and because its dead subterranean tissue releases substances harmful to other

plants. Similarly, it also has a bad effect on ornamental gardening. The difficulty to

control it is a result of its intensive system of underground tubers, and its resistance to

most herbicides. It is also one of the few weeds that cannot be stopped with plastic

mulch.

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Weed pulling in gardens usually results in breakage of roots, leaving tubers in the

ground from which new plants quickly emerge. ploughing distributes the tubers in the

field, worsening the infestation; even if the plough cuts up the tubers to pieces, new

plants can still grow from them.

Herbicides may kill the plant’s leaves, but most have no effect on the root system

and the tubers. In addition, the tubers can survive harsh conditions, further contributing to

the difficulty to eradicate the plant.

Phytochemical Components

Phytochemical studies on animals give basic information on the presence and

localization of the active constituents in the plants tissues. These tests include the test for

the presence of alkaloids, saponins, tannins, glycosides and sterols, flavonoids, formic

acid, tartaric acid, fats and oils and tipertenes.

Alkaloids

Alkaloids are new synthetic agents of greater potency and lesser toxicity. It is a

naturally occurring amine produced by a plant but amines produced by animals and fungi

are also called alkaloids. Many alkaloids have pharmacological effects on humans and

animals. Alkaloids are usually derivatives of amino acids. They are found as secondary

metabolites in plants, animals and fungi, and can be extracted from their sources by

treatment with acids.

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Alkaloids, group of mildly alkaline compounds, mostly of plant origin and of

moderate molecular complexity. Even in very small amounts, the alkaloids produce

strong physiological effects on the body. All contain nitrogen atoms that are structurally

related to those of ammonia.

Nearly 3000 alkaloids have been recorded; the first to be prepared synthetically

(1886) was one of the simplest, called coniine, or 2-propyl piperidine, C5H10NC3H7. It

is highly poisonous; less than 0.2 g (0.007 oz) is fatal. Coniine, obtained from seeds of

the hemlock, was the poison used in the execution of Socrates. Some 30 of the known

alkaloids are used in medicine. For example, atropine, obtained from belladonna, causes

dilation of the pupils; morphine is a painkiller; quinine is a specific remedy for malaria;

nicotine is a potent insecticide; and reserpine is a valuable tranquilizer.

Saponins

Saponins are subgroups of glycosides that have the characteristic ability to cause

foaming when shaken with water. They are sometimes used as emulsifying agents.

Saponins are believed to be useful in the human diet for controlling cholesterol, but some

are poisonous if swallowed and can cause urticaria. Any markedly toxic saponin is

known as a sapotoxin.

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Saponins, group of naturally occurring oily glycosides that foam freely when

shaken with water. They occur in a wide variety of plants, including acacia, soapwort,

soaproot, California pigweed, and many others. Saponins have been, and sometimes still

are, used as cleaning agents and as foam producers, notably in fire-extinguishing fluids.

They have a bitter taste and when ingested orally are practically nonpoisonous to warm-

blooded animals. When injected directly into the bloodstream, however, they are

dangerous and quickly dissolve red blood cells. Hydrolysis of a saponin, brought about

by acids or by enzymes, gives a sugar (often, but not necessarily, glucose) and a

sapogenin, the latter being either a triterpene or a steroid. Some of the sugars and

saponins are useful as raw materials for synthesis of steroid hormones.

Glycosides

Glycosides, class of complex chemical compounds in plants. They are broken

down by plant enzymes into sugars, among which glucose is generally included, and into

other substances. The term glucoside is often used synonymously with glycoside, but in

its more specific meaning it refers to glycosides that yield glucose.

Each glycoside in a plant is hydrolyzed (converted in a reaction with water) by an

enzyme, usually a specific enzyme found in the same plant. The enzyme emulsin,

however, causes hydrolysis of several glycosides. The enzymes and glycosides are stored

in separate plant cells until the reaction products of the glycosides are needed and the

enzymes are activated.

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Glycosides are believed to serve several purposes in the plant. Glycosides are

bitter tasting, and it is believed that they help keep birds and insects from eating seeds

and fruit before they are fully grown, by which time the glycosides have been converted

to sweet sugars. When a plant tissue is bruised, the enzymes hydrolyze the glycosides

into products, such as phenol compounds and acids that have an antiseptic action and

prevent decay of the damaged tissues.

Glycosides are soluble in water and are obtained from plants by water extraction.

They are mostly colorless crystalline solids with a bitter taste. Simple glycosides have

been synthesized in the laboratory, and several hundred glycosides have been extracted

from plants and used for many purposes. Among the important glycosides are indican,

used for dyeing; digitalin, used in medicine; and the saponins, foaming agents used

industrially and medicinally.

Flavonoid

The term flavonoid refers to a class of plant secondary metabolites based around

a phenylbenzopyrone structure. Flavonoids are most commonly known for their

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antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the

media – these terms are equivalent and interchangeable, since all flavonoids are

biological in origin.

The flavonoid synthetic pathway begins with a product of glycolysis,

phosphoenolpyruvate, entering into the Shikimate pathway to yield phenylalanine.

Phenylalanine is the starting material of the phenylpropanoid metabolic pathway, from

which 4-Coumaryl-CoA is produced. This can be combined with Malonyl-CoA to yield

the true backbone of flavonoids, a group of compounds called chalcones. Ring-closure of

these compounds results in the familiar form of flavonoids, a three-ringed phenolic

structure (polyphenols). The metabolic pathway continues through a series of enzymatic

modifications to yield flavanones → dihydroflavonols → anthocyanins. Along this

pathway many products can be formed, including the flavonols, flavan-3-ols,

proanthocyanidins (tannins) and a host of other polyphenolics.

Flavonoids are widely distributed in plants fulfilling many functions including

producing yellow or red/blue pigmentation in flowers and protection from attack by

microbes and insects. The widespread distribution of flavonoids, their variety and their

relatively low toxicity compared to other active plant compounds (for instance alkaloids)

mean that many animals, including humans, ingest significant quantities in their diet.

Flavonoids have been found in high concentrations in butterflies and moths sequestered

from dietary intake at the larval stage and then stored in adult tissues.

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Flavonoids have been referred to as "nature's biological response modifiers"

because of strong experimental evidence of their inherent ability to modify the body's

reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-

inflammatory [1] , anti-microbial and anti-cancer activity. In addition, flavonoids act as

powerful antioxidants, protecting against oxidative and free radical damage.

Consumers and food manufacturers have become interested in flavonoids for their

medicinal properties, especially their potential role in the prevention of cancers and

cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red

wine have been attributed to flavonoid compounds rather than to known nutrients and

vitamins

Tannins

These are also tannin acid, common name applied to a group of vegetable

products, both amorphous and crystalline, obtained from various plants, and important

commercially in the tanning of leather. Tannins have variable composition. Some, called

condensed tannins, are phenols of moderately complex structure, and others are esters of

glucose or some other sugar with one or more trihydroxybenzoic acids. The empirical

formula, C14H14O11, often given for common tannin, is only an average. Tannins occur in

many trees, and the best sources include oak galls and the bark of sumac. Extraction with

water, or water and alcohol, is the first step in the preparation of tannin. Settling,

followed by evaporation at a low temperature, yields the commercial product.

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Tannins have a yellow-white to brown color and a faint, characteristic odor.

Exposure to light deepens the color. They all taste bitter and are astringent. Water,

acetone, and alcohol dissolve tannins readily, but benzene, ether, and chloroform do not.

Heating to 210°C, (410° F) causes decomposition, accompanied by formation of

pyrogallol and carbon dioxide. The chemical property that provides the basis for most

uses of tannins is its ready formation of precipitates with albumin, with gelatin, and with

many alkaloidal and metallic salts. The ability of tannins to transform proteins into

insoluble products resistant to decomposition leads to their use as tanning agents. Ferric

salts react with tannins to give bluish-black products that are useful as inks. Tannins are

used as mordants for dyeing cloth, as sizes for paper or silk, and as coagulants for rubber.

The precipitating properties of tannins are used in clarifying, or cleaning, wines and beer.

Tannic acid is valuable as an external medicine because it is astringent and styptic.

Fats and Oils

They are group of naturally occurring organic compounds called triglycerides—

esters comprised of three molecules of fatty acids and one molecule of the alcohol

gylcerol. They are oily, greasy, or waxy substances that, in their pure state, are normally

tasteless, colorless, and odorless. Fats and oils are lighter than water and are insoluble in

it; they are slightly soluble in alcohol and are readily dissolved in ether and other organic

solvents. Fats are soft and greasy at ordinary temperatures, whereas fixed oils—as

distinct from essential oils and petroleum—are liquid. Some waxes, which are hard solids

at ordinary temperatures, are chemically similar to fats

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Formic Acid

They are the simplest of the organic acids, with the chemical formula HCOOH. A

colorless liquid with an irritating odor, it boils at 100.7° C (213° F) and freezes at 8.4° C

(47.1° F). It is prepared commercially by reacting sodium hydroxide and carbon

monoxide at high pressure and temperature. Formic acid is widely used in the chemical

industries and in dyeing and tanning. In nature, formic acid occurs in the poisons of

stinging ants and other insects and in stinging nettles.

Tartaric Acid

These are also dihydroxy-succinic acid, organic acid of formula C4H6O6, found

in many plants and known to the early Greeks and Romans as tartar, the acid potassium

salt derived as a deposit from fermented grape juice. The acid was first isolated in 1769

by the Swedish chemist Carl Wilhelm Scheele, who boiled tartar with chalk and

decomposed the product with sulfuric acid. Fermentation of the juices of grapes,

tamarinds, pineapples, and mulberries produces, on the inner surface of the container, a

white crust of potassium acid tartrate known as argol, or lees. Argol, boiled with dilute

hydrochloric acid, precipitates as calcium tartrate when calcium hydroxide is added.

Upon addition of dilute sulfuric acid, dextrotartaric acid is liberated, which rotates the

plane of polarized light to the right. Dextrotartaric acid has a melting point of 170° C

(338° F) and is extremely soluble in water and alcohol and insoluble in ether.

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Another variety, called levotartaric acid, is identical to dextrotartaric acid except

that it rotates the plane of polarized light to the left. This acid was first prepared from its

sodium ammonium salt by the French chemist Louis Pasteur. Tartaric acid synthesized in

the laboratory is a mixture of equal amounts of the dextro and levo acids, and this

mixture, called also racemic tartaric acid, does not affect the plane of polarized light. A

fourth variety, mesotartaric acid, also without effect on the plane of polarized light, is

said to be internally compensated.

Tartaric acid, in either the dextrorotary or racemic form, is used as a flavoring in

foods and beverages. It is used also in photography, in tanning, and as sodium potassium

tartrate, also known as Rochelle salt, as a mild laxative.

Sterols, or steroid alcohols are a subgroup of steroids with a hydroxyl group in the

3-position of the A-ring. They are amphipathic lipids synthetised from Acetyl coenzyme

A. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest

of the aliphatic chain is non-polar.

Sterols are important for the physiology of eukaryotic organisms. They form part

of the cellular membrane where they modulate their fluidity and function and participate

as secondary messengers in developmental signaling.

Different organisms utilize different sterols. The most important ones are

cholesterol, phytosterols, and some steroid hormones in animals, and campesterol,

sitosterol and stigmasterol in plants.

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Sterols are also known to block cholesterol absorption sites in the human gut thus helping

to reduce cholesterol in humans by up to 15%.

Sterols

Sterols may be found either as free sterols, acylated (sterol esters), alkylated (steryl alkyl

ethers), sulfated (cholesterol sulfate), or linked to a glycoside moiety (steryl glycosides)

which can be itself acylated (acylated sterol glycosides).

 Free Sterols

Sterols form an important group among the steroids. Unsaturated steroids with

most of the skeleton of cholestane containing a 3-hydroxyl group and an aliphatic side

chain of 8 or more carbon atoms attached to position 17 form the group of sterols. 

They are lipids resistant to saponification and are found in an appreciable quantity

in all animal and vegetal tissues. These unsaponifiable lipids may include one or more of

a variety of molecules belonging to 3-hydroxysteroids, they are C27-C30 crystalline

alcohols (in Greek, stereos, solid). These lipids can be classed as triterpenes as they

derive from squalene which gives directly by cyclization, unsaturation and 3-

hydroxylation, lanosterol in animals or cycloartenol in plants.

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In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol (Greek,

chole, bile), particularly abundant in adrenals (10%, w/w), nervous tissues (2%,w/w),

liver (0.2%,w/w) and gall stones, its fundamental carbon structure being a

cyclopentanoperhydrophenanthrene ring (also called sterane). It was the first isolated

sterol around 1770 by Poulletier de La Salle from gall stones. In 1815, it was isolated

from the unsaponifiable fraction of animal fats by M.E. Chevreul who named it

cholesterine (Greek, khole, bile and stereos, solid). The correct formula (C27H46O) was

proposed in 1888 by F. Reinitzer but structural studies from 1900 to 1932, mainly by

H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize

Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their

connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact steric

representation of cholesterol. In 1936, Callow and Young have designated steroids all

compounds chemically related to cholesterol.

While it became clear very early that cholesterol plays an important role in controlling

cell membrane permeability by reducing average fluidity, it appears now that it has a key

role in the lateral organization of membranes and free volume distribution. These two

parameters seem to be involved in controlling membrane protein activity and "raft"

formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1). 

The vertebrate brain is the most cholesterol-rich organ , containing roughly 25% of the

total free cholesterol present in the whole body.

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In late-step synthesis of cholesterol, discrete oxidoreductive and/or demethylation

reactions occur, which start with the common precursor lanosterol. It was also found as a

major constituent of the unsaponifiable portion of wool fat (lanoline). Animal tissues

contain in addition to cholesterol small amounts of 7-dehydrocholesterol which, on UV

irradiation, is converted to vitamin D3 (cholecalciferol).

Desmosterol (24-dehydrocholesterol), an intermediate between lanosterol and

cholesterol, has been implicated with myelination processes. While high desmosterol

levels could be detected in the brain of young animals (Paoletti R et al., J Am Oil Chem

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Soc 1965, 42, 400) no desmosterol was found in the brain of adult animals. It is

alsoknown as an abundant membrane component in some mammalian cells, such as

spermatozoa and astrocytes (Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J

Biol Chem 2004, 279, 48654). Inability to convert desmosterol to cholesterol leads to the

human disorder desmosterolosis (a severe developmental defect and cognitive

impairment) (Waterham HR et al., Am J Hum Genet 2001, 69, 685).

In higher plants, the first sterols were isolated by Hesse (1878) from the Calabar

beans (Phytostigma venenosum) which coined the term "phytosterine". This substance

was later named stigmasterol (Windaus and Hault, 1906) from the plant genus. The

denomination "phytosterol" was proposed in 1897 (Thoms H) for all sterols of vegetal

origin. Chemically, these sterols have the same basic structure as cholesterol but

differences arise from the lateral chain which is modified by the addition of one or two

supernumerary carbon atoms at C-24 with either or chirality. The 24-alkyl group is

characteristic of all phytosterols and is preserved during subsequent steroid metabolism

in both fungi and plants to give hormones that regulate growth and reproduction in a

manner similar to animals.

20

Most phytosterols are compounds having 28 to 30 carbon atoms and one or two

carbon-carbon double bonds, typically one in the sterol nucleus and sometimes a second

in the alkyl side chain. 

All phytosterols were shown to derive in plants from cycloartenol and in fungi from

lanosterol, both direct products of the cyclization of squalene.

More than 200 different types of phytosterols have been reported in plant species.

Representatives of these sterols are campesterol, stigmasterol (in soybean oil) and -

sitosterol which is present in all plant lipids and is used for steroid synthesis. An

important sterol from yeast and ergot is the C28 compound ergosterol (mycosterol). Upon

irradiation, this sterol gives rise to vitamin D2 (calciferol).

As ergosterol is a cell membrane component largely restricted to fungi, its amount in

environmental matrices may be used as an index molecule for these micro-organisms in a

living biomass (Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset

JY et al., Appl Environ Microbiol 2001, 67, 2051).

21

 

Considerable variability in the concentration of free sterols was observed among

different oils. While concentrations lower than 100 mg/100 g are found in oils from

coconut, palm, olive, and avocado, concentrations between 100 and 200 mg/100 g are

found in oils from peanut, safflower, soybean, borage, cottonseed, and sunflower, and

concentrations between 200 and 400 mg/100 g are found in oils from sesame, canola,

rapeseed, corn, and evening primrose (Phillips KM et al., J Food Comp Anal 2002, 15,

123).

22

Phytosterols produce a wide spectrum of biological activities in animals and

humans. They are considered efficient cholesterol-lowering agents. In addition, they

produce a wide spectrum of therapeutic effects including anti-tumor properties. Further

data on their metabolism and potential therapeutic action can be found in a review article

The European Commission authorized in 2004 the addition of phytosterols and

phytostanols in food products with conditions of labeling including their amount per 100

g and the statement that the human consumption of more than 3 g/day should be avoided.

Phytostanols are a fully-saturated subgroup of phytosterols (they contain no

double bonds). They occur in trace levels in many plant species but in high levels in

tissues of only in a few cereal species. They are in general produced by hydrogenation of

phytosterols.

Stanols often occur in dinoflagellates but are not common in other marine

microalgae. Hence, dinoflagellates are often the major direct source of 5(H)-stanols in

marine sediments (Robinson N et al., Nature 1984, 308, 439).

Fully saturated sterols are also found in animals but are of bacterial origin. Thus, the

5(H)-stanol coprostanol constitutes approximately 60% of the total sterols in human

faeces. 

23

While cholesterol was considered to be nearly absent in vegetal organisms, its

presence is now largely accepted in higher plants. It can be detected in vegetal oils in a

small proportion (up to 5% of the total sterols) but remains frequently present in trace

amounts. An unusual relatively high content of cholesterol was described in camelina oil

(about 200 mg per kg) (Shukla VKS et al., JAOCS 2002, 79, 965). However, several

studies have revealed the existence of cholesterol as a major component sterol in

chloroplasts, shoots and pollens. Furthermore, cholesterol has been detected as one of the

major sterols in the surface lipids of higher plant leaves (rape) where he may amount to

about 72% of the total sterols in that fraction (Noda M et al., Lipids 1988, 23, 439).

Although practical, the ancient distinction between zoosterols, mycosterols and

phytosterols is no more used, since the same sterol may have different sources, but the

appellation phytosterol is actually more frequently used.

Sterols are often isolated in the unsaponifiable fraction of any lipid extract and

determined by various chromatographic procedures (HPLC or GLC).

Avenasterol can be isolated from oat oil. This sterol was shown to protect

specifically frying oils from oxidation owing to its ethylidene group in the side chain

(White PJ et al., JAOCS 1986, 63, 525).

24

An extensive review on the diversity, analysis, and health-promoting uses of

phytosterols and phytostanols may be consulted with interest (Moreau RA et al., Prog

Lipid Res 2002, 41, 457).

Test Microorganisms

Escherichia coli

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

25

Genus: Escherichia

Species: Escherichia coli

E. coli, discovered by Theodor Escheric h , a German pediatrician and

bacteriologist, is one of the main species of bacteria that live in the lower intestines of

mammals. Specimens have also been located on the edge of hot springs. The bacteria are

necessary for the proper digestion of food and are part of the intestinal flora. Presence in

surface water is a common indicator of fecal contamination. It belongs among the

Enterobacteriaceae, and is commonly used as a model organism for bacteria in general.

One of the root words of the family's scientific name, "enteric", refers to the intestine,

hence "gastroenteritis" (from 'gastro-', stomach, 'entero-' intestine, '-itis', inflammation).

"Fecal" is the adjective pertaining to feces, so it is often used synonymously with

"enteric".

The number of individual E. coli bacteria in the feces that one human passes in

one day averages between 100 billion and 10 trillion. All the different kinds of fecal coli

bacteria, and all the very similar bacteria that live in the ground (in soil or decaying

plants, of which the most common is Enterobacter aerogenes), are grouped together

under the name coliform bacteria. Technically, the "coliform group" is defined to be all

the aerobic and facultative anaerobic, non-spore-forming, Gram-negative, rod-shaped

bacteria that ferment lactose with the production of gas within 48 hours at 35 °C (95 °F).

In the body, this gas is released as flatulence. E. coli cells are elongated, 1–2 µm in length

and 0.1–0.5 µm in diameter.

26

Escherichia coli, commonly known as E. coli, is a species of bacteria normally

present in human intestines. A recently recognized strain, E. coli 0157:H7, produces high

levels of toxins that can cause kidney damage, as well as septicemia, or blood poisoning.

Symptoms can include diarrhea, chills, headaches, and high fever and in some cases the

infection can lead to death, even with medical intervention.

Staphylococcus aureus

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Staphylococcaceae

Genus: Staphylococcus

Species: S. aureus

27

Staphylococcus aureus is a genus of spherical bacteria capable of producing a

heat stable toxin that cause illness in humans. The most common pathogen S. aureus, is

frequently responsible for carbuncles, boils, pneumonia, abscesses and osteomyelities It

exist in air, dust, sewage, water, humans, foods and animals.

Staphylococcus aureus (which is occasionally given the nickname golden staph) is

a bacterium, frequently living on the skin or in the nose of a healthy person, that can

cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulitis)

and abscesses, to life-threatening diseases such as pneumonia, meningitis, endocarditis,

Toxic shock syndrome (TSS), and septicemia. Each year some 500,000 patients in

American hospitals contract a staphylococcal infection. It is a spherical bacterium. It is

abbreviated to S. aureus or sometimes referred to as Staph aureus in medical literature,

and should not be confused with the somewhat similarly named streptococci which are

also medically important.

S. aureus is a Gram-positive coccus, which appears as g rape -like clusters when

viewed through a microscope and as large, round, golden-yellow colonies, often with β-

hemolysis, when grown on blood agar plates. The golden appearance is the etymological

root of the bacteria's name: aureus means "gold" in Latin.

28

S. aureus is catalase positive and thus able to convert hydrogen peroxide (H2O2)

to water and oxygen, which makes the catalase test useful to distinguish staphylococci

from enterococci and streptococci. S. aureus can be differentiated from most other

staphylococci by the coagulase test: S. aureus is coagulase-positive, while most other

Staphylococcus species are coagulase-negative.

The species has been subdivided into two subspecies: S. aureus aureus and S.

aureus anaerobius. The latter requires anaerobic conditions for growth, is an infrequent

cause of infection, and is rarely encountered in the clinical laboratory.

S. aureus may occur as a commensal on human skin (particularly the scalp,

armpits and groins); it also occurs in the nose (in about 25% of the population) and throat

and less commonly, may be found in the colon and in urine. The finding of Staph. aureus

under these circumstances does not always indicate infection and therefore does not

always require treatment (indeed, treatment may be ineffective and re-colonisation may

occur). It can survive on domesticated animals such as dogs, cats and horses, but has

never been found on food animals such as poultry or swine. It can survive for some hours

on dry environmental surfaces, but the importance of the environment in spread of Staph.

aureus is currently debated. It can host phages, such as the Panton-Valentine leukocidin,

that increase its virulence.

29

S. aureus can infect other tissues when normal barriers have been breached (e.g.

skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of

furuncles). In infants S. aureus infection can cause a severe disease Staphylococcal

scalded skin syndrome (SSSS).

S. aureus infections can be spread through contact with pus from an infected

wound, skin-to-skin contact with an infected person, and contact with objects such as

towels, sheets, clothing, or athletic equipment used by an infected person.

Deeply situated S. aureus infections can be very severe. Prosthetic joints put a

person at particular risk for septic arthritis, and staphylococcal endocarditis (infection of

the heart valves) and pneumonia may be rapidly fatal.

31

32

.

33

Definition of Terms

Antibiotics – are molecules that are produced by one microorganism that kill

(bacteriocidal) or inhibit (bacteriostatic) other microorganisms. They are one class of

antibacterial and antifungal antimicrobials that can potentially be used as medicinal

drugs to treat infections because of their low toxicity for humans or animals.

Antimicrobial agents – agents that kill or slow the growth of microbes like bacteria

(antibacterial activity), fungi (antifungal activity), viruses (antiviral activity), or parasites

(antiparasitic activity).

Chloramphenicol - is a bacteriostatic antibiotic originally derived from the bacterium

Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice

in 1949.It was the first antibiotic to be manufactured synthetically on a large scale.

Chloramphenicol is effective against a wide variety of microorganisms. In the West, the

main use of chloramphenicol is in eye drops or ointment for bacterial conjunctivitis.

34

Glucose Yeast Peptone – is recommended for the isolation of yeasts from soils

specimen. This is a highly nutritious medium, which may be used for microbial

examination.

Incubate – to give the best or optimum conditions (ex: temperature, moisture) for growth

and development.

Inoculate – to put microorganism or a substrate of organism on a medium.

Inoculation – a process of implanting infectious material into a culture medium.

Inoculum - population of a pure culture grown in a medium.

Inhibition zone - this is an area around a paper disk or colony of bacteria or mold where

no other organisms are growing.

Nutrient Agar – is used for the cultivation of bacteria and for the enumeration of

organisms in water, sewage, feces and other materials. It is used in the laboratory for the

cultivation and maintenance of nonfastidious species and used in microbiological

examination of a broad spectrum of materials. It is a simple medium composed of beef

extract, peptone, and agar. It has been one of the most generally used media in

bacteriological procedures. It is used for the ordinary routine examinations of water,

sewage, and food products, for the carrying of stock cultures, for the preliminary

cultivation of samples submitted for bacteriological examination, and for isolating

organisms in pure culture.

35

Chapter IIMETHODOLOGY

This chapter presents the Experimental Design Diagram, the materials,

procedures, methods of gathering data and the statistical tool used to interpret the data

gathered.

TITLE: Antimicrobial Property of Nut Grass rhizome against E. coli, Staphylococcus

aureus, and Salmonella spp.

HYPOTHESES:

1.) The Nut Grass rhizome extract cannot inhibit the growth of harmful microorganisms.

2.) Triterpenes, Tannins, Saponins and Glycosides are not the active components of the

Nut Grass rhizome using the Phytochemical Test.

4.) There is no significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotic in terms of zone of inhibition.

INDEPENDENT VARIABLE: Type of Antimicrobial agent

TREATMENTS

T0 (control)Chloramphenicol

T1

With Nut Grass rhizome extract

NUMBER OF TRIALS3 3

DEPENDENT VARIABLE: Zone of inhibition

CONSTANT: Amount of extract

36

Materials:

A. Extraction

Reflux apparatuses

condenser

rubber tubing

burner

iron ring

iron stand

Erlenmeyer flask

water

filter papers

container/ sterile vial

B. Antimicrobial Assay Test

Agar plates

Cork borer

Pipette

Nutrient Agar (NA)

Glucose Yeast Peptone (GYP) Agar

Culture Bacteria and Yeast- E.coli, S. aureus, and Salmonella spp.

Inoculating loop

Sterile cotton swab

Commercial antibiotic (Chloramphenicol)

37

Alcohol lamp

Beaker with 95% Ethanol

C. Phytochemical Test

acetic anhydride

sulfuric acid

1 ml 10% hydrochloric acid

1% hydrochloric acid

Mayer’s Reagent

ferric chloride

2 test tubes

anhydrous sodium carbonate

sodium hydroxide

potassium sodium tartrate

distilled water

GENERAL PROCEDURE

A. Reagents

All reagents (biochemical- equipment) were supplied by Regional Science High

School for Region 1 Laboratory and Department of Science and Technology Laboratory

(Main).

38

B. Collection of Plant Sample

400 grams of Nut grass rhizomes were collected in the Regional Science High

School for Region 1 campus.

C. Preparation of the Extract

Reflux Method was followed in extracting the Nut grass rhizome as indicated

below:

1) Put 400 grams of Nut Grass rhizome (sample) in the Erlenmeyer flask and add

some water enough for the sample.

2) Connect the Erlenmeyer flask containing samples into a condenser.

3) Subject the Erlenmeyer flask into heat to boil the water together with the sample.

Its temperature should not exceed to 1000C.

4) Wait for the water to completely evaporate. The extract will remain on the flask.

5) Then filter the sample left in the Erlenmeyer flask to remove unnecessary

samples.

D. Preparation of the Test Organisms

E.coli, S. aureus, and Salmonella spp.were obtained from the Department of

Science and Technology (Main) that were used as test organisms.

39

E. Antimicrobial Assay Test

Microbial suspensions were prepared from 24- hour cultures of the Escherichia

coli, Staphylococcus aureus and Salmonella spp. (bacteria) .The suspending medium

used was 0.1% peptone water.

One-tenth (0.1) mL aliquots of the bacterial and yeast suspensions were

transferred into pre-poured Nutrient Agar (NA) and Glucose Yeast Peptone (GYP) Agar,

respectively. Five (5) of the corresponding medium, melted and cooled to 450C, was

poured onto the agar plate and swirled to distribute the inoculums evenly on the agar

surface. Five (5)mL of the sample was placed in each hole.

The plates were incubated at room temperature. NA and GYP plates were

observed after 24-48 hours. The clearing zone was measured in millimeters and the

average diameter of the clearing zones was calculated. the antimicrobial index (AI) was

computed using the following formula:

AI = Diameter of clearing zone – Diameter of wellDiameter of well

40

F. Phytochemical analysis

Extract from the Nut Grass rhizome were use for Phytochemical analysis.

1) Test for Sterols and Tipertenes

Lieberman-Berchard Test

A small amount of the sample extract(Nut Grass Rhizome extract) in acetic

anhydride was dissolved. The soluble portion was decanted and to this, 1-2 drops of

concentrated sulfuric acid was added. Observe a green color, either immediately or solely

going into red and blue tones. A pink to red color is indicative of triterpenoids while a

blue color is indicative of steroids.

2) Test for Flavonoids

One (1) ml of sample extract was treated with 1 ml 10% hydrochloric acid and a

few magnesium turnings. Formation of red color is observed.

3) Test for Alkaloids

The sample extract was extracted with 1% HCL and drops of Mayer’s Reagent or

Wagner’s Rgt. was added to the filtered acid extract. A cream colored precipitate is

observed in the case of Mayer’s Rgt. while a reddish brown ppt. is observed in the case of

Wagner’s Rgt.

41

Formula of Mayer’s Rgt.:

1.358 g of mercuric chloride was dissolved in 60 ml distilled water and 5 g of

potassium iodide was dissolved in 10 ml distilled water. The two solutions were mixed

and diluted to 100 ml with distilled water.

Formula for Wagner’s Rgt.:

1.3 g iodine crystals and 2.0 g potassium iodide in sufficient amount of distilled

water to make a total volume of 100ml was dissolved.

4) Test for Tannins

The sample extract was extracted with hot water and the aqueous extract was

filtered. Upon addition of two drops of ferric chloride test solution, a dark color and

precipitate forms which may either be black, dark blue, blue black, green or blue green.

Ferric chloride TS: Dissolve 9g of ferric chloride in dist, water to make 100 ml.

42

5) Test for Saponins

The sample extract was dissolved in hot water. The aqueous extract when shaken

vigorously should become frontly. The froth, honeycomb in nature should persist for at

least 30 minutes.

6) Test for Glycosides

The sample extract was dissolved in hot water and filtered. The filtrate was used

for the test. Two test tubes were used. Two ml of sample was placed in each tube. One

ml of dilute hydrochloric acid was added to sample 1. Nothing is added to sample 2. The

2 test tubes were placed in a boiling water bath for 5 minutes. Then the test tubes were

cooled. The samples were both neutralized with anhydrous sodium carbonate until no

more effervescence is produced. Then add Fehling’ s B. One ml of Fehling’ s solution

was used. The 2 test tubes were heated in a water bath for 2 minutes. Observe the amount

of brick red precipitate that formed. An increase in the amount of brick red precipitate in

the hydrolyzed sample (the sample to which dilute acid was added) as compared to the

other sample indicates the presence of glycosides.

43

Fehling’s solution A:

Copper Sulfate (CuSO4. 5H2O)…34.66 grams

Distilled water, a sufficient quantity

To make…500 ml

Dissolve the copper sulfate in the distilled water and mixed.

Fehling’s Solution B:

Sodium Hydroxide…50 grams

Potassium Sodium Tartrate…173 grams

Distilled water, a sufficient quantity

To make …500 ml

The sodium hydroxide and the potassium sodium tartrate in the distilled

water and were dissolved and mixed.

Note: Mix fehling’s A and B in equal amount before using.

44

7.) Test for the presence of Organic acids

a.) Formic acid. Sections were placed in few drops of mercuric chloride

solutions, then heated on a water bath for an hour and washed with water. The

sections were transferred to a drop of 1% potassium hydroxide. Blackened cells

indicate the presence of formic acid.

b.) Tartaric acid. Sections were treated with 4% aqueous solution of any

ferrous salt and few drops of hydrogen peroxide or 10% potassium permanganate

with the addition of an excess of sodium hydroxide solution. Violet color reaction

indicates the presence of tartaric acid.

8.) Test for the presence of fats and Oils

Sections were treated or immersed in either Sudan III or IV for twenty

minutes, then washed with 50% alcohol, and transferred to glycerin for

observation. Deep red color reaction indicates the presence of fats and oils.

45

Figure 1. The Flowchart of Experimental Design

46

Chapter III

RESULTS AND DISCUSSONS

Reagents

Collection of the Plant Specimen

Preparation of the Culture Bacteria

Preparation of the Extract

Antimicrobial Assay Test

Phytochemical Analysis

This chapter dealt with the results and findings with their subsequent

discussions. The results were presented in tables and in graphs.

Table 1. Mean Zone of Inhibition Against E. coli in millimeters (mm).

The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome

extract) is 3.1mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is lesser than the Mean Zone of

Inhibition of T0. Likewise, results showed that there is no significant difference between

the treatments in terms of zone of inhibition.

47

Table2. Mean Zone of Inhibition Against S. aureus in millimeters (mm).

The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome

extract) is 3.5 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone

of Inhibition of T0. Likewise, results showed that there is no significant difference

between the treatments in terms of zone of inhibition.

48

Table3. Mean Zone of Inhibition Against Salmonella spp.in millimeters (mm).

The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome

extract) is 3.0 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is lesser than the Mean Zone of

Inhibition of T0. Likewise, results showed that there is no significant difference between

the treatments in terms of zone of inhibition.

49

Table 4. Mean Zone of Inhibition Test Microorganisms in millimeters (mm).

The table shows that the Mean Zone of Inhibition of T1 is 10.5mm and the

Mean Zone of Inhibition of T0 is 9.6 mm. This proves that the Mean Zone of Inhibition of

T1 is greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there

is no significant difference between the treatments in terms of zone of inhibition.

50

Table 5. Mean Zone of Inhibition Test Microorganisms in millimeters (mm).

The table shows that the Mean Zone of Inhibition of T1 is 10.5mm and the

Mean Zone of Inhibition of T0 is 9.6 mm. This proves that the Mean Zone of Inhibition of

T1 is greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there

is no significant difference between the treatments in terms of zone of inhibition.

51

Table 6. A Summary table of the Active Principles of the Dorsal Fin Venom

Active Principles Color reaction Nut Grass

rhizome

Alkaloids Reddish brown 3

Glycosides Red 1

Sterols Blue 3

Tannin black, dark blue,

blue black, green or

blue green

2

Saponin Yellow/ red 2

Formic acid Black 0

Tartaric acid Violet 0

Fats and oils Deep red 3

Flavonoids Red 3

Titerpenes pink to red 0

The table shows the Phytochemical Test result of the Nut Grass rhizome sample.

It shows that Alkaloids, Sterols, Flavonoids and Fats and Oils are very` abundant in all

tissues of the rhizome .Tannins and Saponins are observed abundant while Glycosides are

detectable only.This contributed much on the effectiveness of Nut Grass rhizome extract

an antibiotic agent as revealed on the table. Legends:

3 = Very Abundant (51-100%) 2 = Abundant (26-50%)

1= Detectable (1-25%) 0 = Absent

52

Figure 2. Mean of Zone of Inhibition of E.coli, S. aureus, C. albicans and A. niger

treated by the extract (5mL) from the Nut Grass rhizome.

3.3

3.35

3.4

3.45

3.5

3.55

3.6

Zo

ne

of

Inh

ibit

ion

of

Bac

teri

a in

Dia

met

ers

(mm

)

E.coli

S.aureus

C.albicans

A.niger

The figure shows the Mean of Zone of inhibition of E.coli is 3.6mm, the Mean

Zone of Inhibition of S. aureus is 3.5mm, the Mean Zone of inhibition of C. albicans is

3.4mm, whilethe Mean Zone of Inhibition of A. niger is 3.5mm.This proves that the Nut

Grass rhizome inhibit the growth of E.coli the most, compared to others.

53

Figure 3. Mean of the Zone of Inhibition of E. coli, S. aureus C. albicans and A.

niger treated with 5mL of Chloramphenicol syrup.

0

0.5

1

1.5

2

2.5

3

3.5

E.coli

S.aureus

C. albicans

A.niger

The figure shows the Mean Zone of Inhibition of E.coli, S.aureus and C.

albicans is 3.2 mm when treated with the control antibiotic

54

Figure 4. Comparison of the Mean on the Zone of Inhibition of Test

Microorganisms when treated by the Nut Grass rhizome extract and to 5mL of

Chloramphenicol syrup.

3

3.1

3.2

3.3

3.4

3.5

3.6

3.7

Co

mp

aris

on

of

Zo

ne

of

Inh

ibit

ion

of

Bac

teri

a in

Dia

met

ers

(mm

)

5mL ofChloramphenicol syrup

5mL of NutGrass rhizomeextract

The figure shows the Mean Zone of inhibition of the Test

Microorganisms when treated with the controlled antibiotic is 3.2mm. While the Mean

Zone of inhibition of the Test Microorganisms when treated with the Stonefish Dorsal

Fin Venom varies and it is greater than the Mean Zone of Inhibition of Test

Microorganisms when treated with the controlled antibiotic. Likewise, results showed

that there is no significant difference between the treatments in terms of zone of

inhibition

55

Chapter IVCONCLUSIONS AND RECOMMENDATIONS

Conclusions

Based from the findings, the following conclusions were drawn:

a) The Nut Grass rhizome extract can inhibit the growth of harmful microorganisms.

b) Phytochemical analysis showed the presence of active principles from the Nut

Grass rhizome tissues. It showed that Alkaloids, Sterols, Flavonoids and Fats and

Oils were very` abundant in all tissues of the rhizome .Tannins and Saponins were

observed abundant while Glycosides were detectable only. This contributed much

on the effectiveness of Nut Grass rhizome extract an antibiotic agent as revealed

on the table

c) There is no significant difference of using the Antimicrobial Property of Nut

Grass rhizome and the controlled antibiotic in terms of zone of inhibition.

56

Recommendations

Based on the results of the study, the following recommendations are given:

a) Being abundant, Nut Grass should be used as a medicinal plant. However, users

should follow the recommendations of the DOH, since it is more effective as an

antimicrobial.

b) Pure extracts of Nut Grass rhizome should be conducted, processed and be made

as tablets for medicine.

c) Wide dissemination of the latest technology must commence

57

Bibliography

Internet websites, Yahoo.com; Google .com; and MSN Network.

Microsoft Encarta Library Edition 2006.

Phillips KM et al., J Food Comp Anal 2002, 15, 123

Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051

Waterham HR et al., Am J Hum Genet 2001, 69, 685

Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654

Paoletti R et al., J Am Oil Chem Soc 1965, 42, 400

Wikipedia, free Dictionary

58

Appendices

I. Statistical test for the zone of inhibition of Escherichia coli treated with

Chloramphenicol (T0) and with the Nut Grass rhizome extract (T1).

A B

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 10.24 12.96

2 10.24 13.69

3 10.24 12.96

Total ( x1)

( x12)

30.72

( y2)

( y22) 39.61

N1=3 N2=3

H0: There is no significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

Ha: There is a significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

59

= 0.05

Df = N1 + N2- 2 Tcrit.= 2.776

= 6-2

= 4

Compute for t

A= 9.6/ 3 = 3.2 ; B = 10.9/ 3 = 3.63

S 1 - 2 =

=

=

60

=

=

= 0.0408

t cal = t =

= 3.2 – 3.63 / 0.0408

= -10.5392

Decision: Accept H0

The tcrit is greater than the t cal so the Null Hypothesis was accepted. Thus, there is no

significant difference of using the Antimicrobial Property of Nut Grass rhizome and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

61

II. Statistical test for the zone of inhibition of Staphylococcus aureus treated with

the commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract

(T1).

C D

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 10.24 12.25

2 10.24 12.25

3 10.24 12.25

Total ( x1)

( x12)

( y1)

( y12)

36.75

N1=3 N2=3

H0: There is no significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

Ha: There is a significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

62

= 0.05

Df = N1 + N2- 2 Tcrit.= 2.776

= 6-2

= 4

Compute for t

C= 3.2/ 3 = 3.2 ; D = 10.5/ 3 = 3.5

S 1 - 2 =

=

=

=

= 0

63

t cal = t =

= 3.2 – 3.5 / 0

=0

Decision: Accept H0

The tcrit is greater than the t cal so the Null Hypothesis was accepted. Thus, there is no

significant difference of using the Antimicrobial Property of Nut Grass rhizome and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

64

III. Statistical test for the zone of inhibition of Candida albicans treated with the

commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract (T1).

E F

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 11.56

2 11.56

3 10.89

Total 34.01

N1=3 N2=3

H0: There is no significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

Ha: There is a significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

65

= 0.05

Df = N1 + N2- 2 Tcrit.= 2.776

= 6-2

= 4

Compute for t

E = 9.6/ 3 = 3.2 ; F = 10.1/ 3 = 3.36

S 1 - 2 =

=

=

=

=

= 0.0408

66

t cal = t =

= 3.2 – 3.36 / 0.0408

=-3.9216

Decision: Accept H0

The tcrit is greater than the t cal so the Null Hypothesis was accepted. Thus, there is no

significant difference of using the Antimicrobial Property of Nut Grass rhizome and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

67

IV. Statistical test for the zone of inhibition of Aspergillus niger treated with the

commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract (T1).

G H

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 12.25

2 12.25

3 12.25

Total 36.75

N1=3 N2=3

H0: There is no significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

Ha: There is a significant difference of using the Antimicrobial Property of Nut Grass

rhizome and the commercial antibiotics in terms of zone of inhibition.

68

= 0.05

Df = N1 + N2- 2 Tcrit.= 2.776

= 6-2

= 4

Compute for t

G = 9.6 / 3 = 3.2 ; H = 10.5/ 3 = 3.5

S 1 - 2 =

=

=

=

= 0

69

t cal = t =

= 3.2 – 3.5 / 0

=0

Decision: Accept H0

The tcrit is greater than the t cal so the Null Hypothesis was accepted. Thus, there is no

significant difference of using the Antimicrobial Property of Nut Grass rhizome and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

70

PLATES

Plate 1. Researchers Collecting the Plant Specimen.

71

Plate 2. The Plant Specimen – Nut Grass (Cyperus rotundus).

72

Plate 3. The Nut Grass Rhizome.

73

Plate 4. Materials for the Reflux Method (Extraction)

74

Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method.

75

Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper.

76

Plate 7. Materials for the Antimicrobial Test.

77

Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to

Nutrient Agar and Glucose Yeast Peptone Agar.

78

Plate 9. Researcher Pouring the Medium onto the Agar Plates.

79

Plate 10. Researcher Swirling Agar Plates to distribute the inoculums evenly in the

Agar Surface.

80

Plate 11. Researcher making holes on the Agar Plates using the Cork Borer.

81

Plate 12. Researcher Placing the Extracts in each hole.

82

Plate 13. Researching Incubating the Plates at room temperature

83

Plate 14.The treatments – To ( Chloramphenicol) and T1 ( Nut Grass rhizome

extract).

84

CURRICULUM VITAE

Name: Lyra Erika Liclican

Age: 16years old

Address: Paratong, Sta.Cruz, Ilocos Sur

Birthday: September 1, 1990

Parents: Mr. Raul Liclican

Mrs. Lydia Liclican

Religion: Roman Catholic

Nationality: Filipino

Civil Status: Single

Educational Attainment:

Elementary

St. Joseph Institute

Candon City, Ilocos Sur

St. Augustine’s School

Tagudin, Ilocos Sur

Secondary

Regional Science High School for Region 1

Bangar, La Union

Name: Glen Bernard Asto

Age: 14years old

Address: Suyo, Luna, La Union

Birthday: September 8, 1992

Parents: Mr. Leonardo Asto

Mrs. Gloria Asto

Religion: Roman Catholic

Nationality: Filipino

Civil Status: Single

Educational Attainment:

Elementary

Suyo Elementary School

Suya, Luna, La Union

Secondary

Regional Science High School for Region 1

Bangar, La Union

DATA BOOK

SUCCESS CALENDAR

1. CHOOSING Planned Date Date Completed

A TOPIC May 3,2006 May 10,2006

This is the hardest thing to do when doing an Investigatory Project. A narrowed

down topic that would make sense and can be attained and give benefit to the majority.

And fortunately my title, “ Antimicrobial Property of Nut Grass rhizome against E.coli,

S.aureus, C. albicans and A,niger.”

2 COLLECTING Planned Date Date Completed

BACKGROUND May 11, 2006 May 15, 2006

INFORMATION

Collection of information regarding my study is not that easy. For two days, I went

to Department of Science and Technology, San Fernando City and Don Mariano Marcos

Memorial State University, Bacnotan La Union to research essential information from

books and manuscripts. Last May 13- 15, my adviser and I went to Manila. We went to

Adamson University, DOST Main, Natural Science Research Institute (NSRI) and

Marine Science Institute (MSI) of University of the Philippines to gather facts about my

study.

3. EXPERIMENTATION Planned Date Date Completed

AND GATHERING May 17, 2006 June 2, 2006

OF DATA

With the suggestions of UPD, DOST and ADU, I design for the procedures to

follow in extracting the Nut Grass rhizome. The extracted venom was tested for

Antimicrobial Assay and Phytochemical Analysis at DOST.

4 MAKING OF THE WRITE Planned Date Date Completed

UP (MANUSCRIPT) - July 28,2006 August 2, 2006

Background of the Study

5. MAKING THE STATEMENT Planned Date Date Completed

OF THE PROBLEM, August 4, 2006 August 14, 2006

HYPOTHESES, SIGNIFICANCE

OF THE STUDY UNTIL SCOPE

AND DELIMITATION

6.MAKING THE REVIEW OF Date Planned Date Completed

RELATED LITERATURE August 17,2006 August 26,2006

7. MAKING THE CHAPTER II Date Planned Date Completed

( Methodology) August 28,2006 September 12, 2006

8. MAKING THE CHAPTER III Date Planned Date Completed

(Results and discussions) September 13,2006 Sept. 17, 2006

9. MAKING THE CHAPTER IV Date Planned Date Completed

(Conclusions and Sept. 18, 2006 Sept. 20,2006

Recommendations)

10. MAKING THE Date Planned Date Completed

BIBLIOGRAPHY, Sept.22, 2006 Sept. 24, 2006

APPENDICES

10. FINALIZATION Date Planned Date Completed

Sept. 26, 2006 Sept. 28, 2006

11. PRINTING OF Date Planned Date Completed

THE MANUSCRIPT Sept. 29, 2006 Sept 30.2006

Department of EducationRegion 1

Division of La UnionRegional Science High School for Region 1

Bangar, La Union

Antibiotic Property from Stonefish Dorsal

Fin (Synanceia verrucosa) against

Escherichia

coli, Staphylococcus aureus, and

Candida albicans

By:

LYRA ERIKA T.LICLICAN

ROGELIO C. VALDEZ

Research Adviser

S.Y 2006-2007

Table of Contents

Abstract……………………………………………………………………….i

CHAPTER 1

Introduction……………………………………………………………

Background of the Study……………………………………………..

Statement of the Problem…………………………………………….

Hypotheses……………………………………………………………

Significance of the Study……………………………………………..

Scope and delimitation……………………………………………..

Review of related Literature………………………………………..

Description of the Animal Specimen………………………

Phytochemical Components………………………………..

Test Microorganisms………………………………………

CHAPTER II

Methodology

Experimental Design……………………………………….

Materials…………………………………………………..

Reagents…………………………………………………….

Collection of Animal Specimen……………………………

Preparation of the Extract…………………………………

Antimicrobial Assay Test…………………………………

Phytochemical Test………………………………………….

Flowchart…………………………………………………….

CHAPTER III

RESULTS AND FINDINGS………………………………………

CHAPTER IV

COCLUSIONS AND RECOMMENDATIONS……………………

Conclusions………………………………………………….

Recommendations…………………………………………..

Bibliography…………………………………………………………………

Appendices…………………………………………………………………..

Curriculum Vitae ……………………………………………………………

LIST OF TABLES

Table 1. Mean Zone of Inhibition against

E. coli in millimeters (mm)………………………………………………………….54

Table 2. Mean Zone of Inhibition against

S. aureus in millimeters (mm)…………………………………………………..…..55

Table 2 .Mean Zone of Inhibition against C. albicans in millimeters (mm)…………………………………………………….56

Table 4.Summary table of the Mean Zone

of Inhibition of Test Microorganisms in millimeters (mm)………………………..57

Table 5. A Summary table of the Active Principles………………………………..58

LIST OF FIGURES

Figure 1. The Flowchart of Experimental Design…………………………………52

Figure 2. Block presentation of the standard

protocol on fluid venom extraction of

Stonefish……………………………………………………………………………53.

Figure 3. Mean of Zone of Inhibition of

Test Microorganisms treated by the extract

(3mL) from Stonefish Dorsal Fin

Venom…………………………………………………………………… ………..59

Figure 4. Mean of Zone of Inhibition of

Test Microorganisms treated with 3mL

of Chloramphenicol syrup…………………………………………………………60

Figure 5. Comparison of the Mean of

Zone of Inhibition of Test Microorganisms

when treated by the Stonefish

Dorsal Fin venom and to 3 mL of

Chloramphenicol syrup……………………………………………………………61

LIST OF PLATES

Plate 1. The Stonefish Habitat………………………………………………….….74

Plate 2. The Animal Specimen…………………………………………………….75

Plate 3. Materials for the Extraction

and Dissection of Stonefish………………………………………………………..76

Plate 4. Researcher Dissecting the

Specimen……………………………………………………………………………77

Plate 5. Researcher Inserting the

Knife Above the Dorsal Fin………………………………………………………..78

Plate 6.Reseacher Pinning down

the tail end of the Dorsal Fin using

a Sterile Knife………………………………………………………………………79

Plate 7. Researcher Grasping the

Tail Fin and Pulling it way from

the Dorsal Fin…………………………………………………………………….80

Plate 8. Researcher Extracting the

Venom using a Sterile Syringe……………………………………………………..81

Plate 9: The Extracted

Venom………………………………………………………………………….…..82

Plate 10. Materials used for the

Antimicrobial Assay Test………………………………………………………….83.

Plate 11.Researcher transferring

Aliquots Bacterial and Yeast Suspension

o Nutrient Agar and Glucose Yeast Peptone

Agar………………………………………………………………………………..84

Plate 12. Researcher Pouring the

Medium onto the Agar Plates………………………………………………………85

Plate 13.Reseacher Swirling Agar Plates

to distribute the inoculums evenly in

the Agar Surface……………………………………………………………….… ..86

Plate 14.Reseacher making holes on the

Agar Plates using the Cork Borer………………………………………………… ..87

Plate 15. The bored Agra Plate Surface……………………………………………88

Plate 16.Researcher Placing the Extracts

in each hole………………………………………………………………………..89

Plate 17.Researching Incubating the

Plates at room temperature…………………………………………………………90

Plate 18. The treatments To (Chloramphenicol)

and T1 ( Stonefish Dorsal Fin Venom)……………………………………………..91

Abstract

This study aimed to determine the Antibiotic Property from Stonefish (Synanceia

verrucosa) Dorsal Fin Venom against Escherichia coli, Staphylococcus aureus and

Candida albican and it was conducted at the University of the Philippines

Fifteen Stonefish were subjected to experimentation. Antimicrobial assay test

were subjected using the control Chloramphenicol and the experimental variable –

Stonefish Dorsal Fin venom. Phytochemical Test was done to determine the active

principles present. It showed that Alkaloids, Saponin and Tannins were very abundant in

the Stonefish Dorsal Fin venom.

There were two treatments used namely T0: the controlled antibiotic-

Chloramphenicol and T1: the Stonefish Dorsal Fin Venom.T- test showed that the

Stonefish Dorsal Fin Venom had a similar performance with the Chloramphenicol as an

antibiotic. Likewise, there is no significant difference of using the Antibiotic

Property of Stonefish Dorsal Fin Venom and the Chloramphenicol in terms of zone of

inhibition that resulted to the acceptation of the Null Hypothesis.

Furthermore, it is highly recommended to the Pharmaceutical Industry, that this

will serve as a basis of making an antibiotic out of the Stonefish Dorsal Fin Venom.

Additional Brand of antibiotics shall be made available for comparing the effect of

Stonefish Dorsal Fin Venom to test further efficacy. Wide dissemination of the latest

technology must commence.

i

Chapter IINTRODUCTION

A. Background of the Study

According to one of the famous biologists, Barry Commoner, “Everything is

connected with everything else” and ”Everything goes somewhere” Plants- Animals,

Humans-Animals, Plants-Humans . . . .

In these present times, there is a need and exigency of the usage of antibiotics in

the field of medicine. The exposure of living creatures to a lot of flying particles may

bequeath danger to their physical state. Many living things accommodate to its

environment that defend them from the assault of other organisms or simply concealment

by blending in with the surroundings. In this case, the stonefishes an aquatic animal has

bulky, compact body shape with sloughing skin with variable color patterns and covered

with algae, providing a magnificent camouflage, simulating a stone or a mass of mud.

About 1000 known species of marine animals are venomous. And the venom of one, the

stonefish, can kill a man. It is armed with thirteen strong spines along its back carrying

enough venom to kill a human being. The evolution of a protective system is easily

understandable. The stonefish use their poisonous spines only in self-defense.

Previous years has been observed renascence of interest among students and

specialized researchers in seeking antimicrobial properties from chosen vertebrate and

invertebrate as well. This is accredited to the fact that artificial and presently available

medicines are ineffective, futile, and too expensive or tend to bring superb side effects.

Our tropical country is gifted with variety of flora and fauna. Among these is the

Stonefish (Synanceia verrucosa) which is considered undesirable organisms in the

environment however can be a very good source of antibiotic.

Inquisitiveness provoke the researcher’s mind that what if Synanceia verrucosa

has an antibiotic property and then could be used as antimicrobial agent to restrain the

growth of some bacteria, which motivated the researcher to conduct a study on the said

specie, thus the Antibiotic property from Stonefish Dorsal Fin Venom against Escherichia

coli, Staphylococcus aureus and Candida albicans was conceived.

B. Statement of the Problem

This study aimed to determine the Antibiotic Property from Stonefish Dorsal Fin

(Synaceia verrucosa) against Escherichia coli, Staphylococcus aureus and Candida

albicans was conceived.

Specifically, this study sought to answer the following sub problems:

1.) Can the Stonefish Dorsal Fin Venom inhibit the growth of harmful microorganisms?

2.) What are the active constituents of the Stonefish Dorsal Fin Venom using

Phytochemical test?

3.) Is there a significant difference of using the Stonefish Dorsal Fin and the commercial

antibiotic in terms of zone of inhibition?

2

C. Hypotheses

The study anchors the following hypotheses:

1.) The Stonefish Dorsal Fin Venom cannot inhibit the growth of harmful

microorganisms.

2.) Alkaloids, Saponin and Tannin are not the active constituents of the Stonefish Dorsal

Fin Venom using the Phytochemical Test.

3.) There is no significant difference of using the Antibiotic Property from Stonefish

Dorsal Fin and the commercial antibiotic in terms of zone of inhibition.

D. Significance of the Study

Bacteria trigger many diseases in humans, including tetanus, diphtheria, plague,

pneumonia, cholera, leprosy and meningitis. Massive times are spent in the effort to

lessen the likelihood of these infections and in inhibiting the other destructive activities

of bacteria.

The viability of other organisms as sources of antibiotic property is inevitable and

environmental friendly because of the notion of interdependent role.

The complex integration of microorganisms in the web of nature is recognized

and will be more completely understood as universal patterns of biodiversity are

documented.

The use of microorganisms in the production of antibiotics has saved many lives

since the last century. Penicillin Antibiotic is known as the wonder drug.

3

While many kinds of microorganisms cause communicable diseases, others use

by scientists to avert illnesses. It is for this reason that the researcher of the Antibiotic

Property from Stonefish Dorsal Fin (Synaceia verrucosa) drove him to study the

chemical substance and its efficiency of fighting to harmful microorganisms

E.Scope and Limitations

The study was conducted at University of the Philippines Diliman, Quezon City

from May to September 2006, S. Y. 2006-2007. The collection of the Stonefish Dorsal

Fin Venom was done at Marine Science Institute Laboratory of UPD on the direct

supervision of Dr. Lourdes Cruz in her protocol on fluid or venom extraction and proper

handling of procedure. The extracted venom was brought to Natural Science Institute,

UPD for antimicrobial assay test on the supervision of Mrs. Vina Argayosa and to

Adamson University for the Phytochemical Test on the supervision of Mr. Ghel Balete

This study is limited only to the use of the Stonefish Dorsal Fin Venom as source of

extract and the use of three species of bacteria namely: Escherichia coli, Staphylococcus

aureus and Candida albicans for antimicrobial tests.There were only two treatments

namely T0 ( Chloramphenicol) and T1 (the Stone Dorsal Fin Venom).

4

F. Review of Related Literature

English Name: Stonefish

Scientific Name: Synanceia verrucosa

Kingdom: Animalia

Phylum: Chordata

Class: Actinoplerygii

Order: Scorpaeniformes

Family: Synanceiidae

5

Ecology

It lives along the external reef, often in sheltered bay or lagoon environments.

Very well camouflaged, perfectly still for long times. It moves seldom, swimming

heavily.

Association

Often it is covered by algae and hydroids, contributing to camouflage.

Classification

The stonefish comes from the fish family.  

Habitat

Some stonefish live in coral and sandy places. Stonefish live in warm water where

it is clear. The stonefish lives primarily above the tropic of Capricorn. Its main habitat is

on coral reefs, near and about rocks, or can be found dormant in the mud or sand. 

It feeds on small fish and shrimps.

Appearance

Some stonefish look like rocks and coral and are greenish brown color.

Stonefish can grow up to 30cm in  length. The Stone Fish is a mottled brown-greenish in

color (which gives them camouflage) with many venomous spines along its back.

6

Behavior

Stonefish gobble small fish. They don’t even give the small fish a chance

Description

Stonefish are indisputably ugly. In common with their relatives the scorpion-fish they

have a bony ridged head and wavy fins but here ends the resemblance to any other living creature.

The head and body of the stonefish are covered with lumps and fleshy growths and the eyes are

deeply set in the bony hollows of the head. The large mouth is upturned and partly disguised by a

notched fringe of skin. Their irregular shape and blotchy red-brown coloration afford stonefishes

an extremely effective camouflage. This is enhanced by their ability to secrete a sticky fluid from

the wart like growths on the skin, which covers the body and to which cling algae and mud. Even

small invertebrates such as sea anemones and hydras colonize the apparently inanimate object.

 One might expect that the potential of the stonefish -- each spine carrying enough venom to kill a

human being -- would be' exploited by it to obtain food. This is not the case, however, and its

deadly arsenal is utilized only as a means of defense. Its diet composed of small fish and

crustaceans such as shrimps, a stonefish uses its large front fins to scoop out a depression in the

sand or mud where it lies motionless waiting for its prey to draw near. Deceived by the

convincing camouflage, passing victims are swallowed completely as the stonefish makes an

unexpectedly energetic lurch forwards.

7

Venom

The sting causes excruciating pain and a great deal of swelling rapidly develops

causing death to tissues. The severity of the symptoms depends on the depth of

penetration and the number of spines penetrated. The symptoms of the venom are muscle

weakness, temporary paralysis and shock, which may result in death if not treated.

Venom Apparatus

Among the representatives of the Scorpaenidae family, the stonefish has the most

efficient and developed venom apparatus. Although three spines of the anal and two of

the ventral fin contains venom glands, the 13 dorsal spines inflict most of the venomous

stings. Each spine has two lateral grooves where a thick spindle-shaped is situated in the

proximal part. A thin venom duct leads to the tip of the spine, and a thick integumentary

sheath covers the spines. When the spine enters the body under pressure, its integument is

ruptures and the glands are compressed injecting the venom through the venom ducts into

the wound caused by the needle-sharp spines.

Venoms and Toxins

Each venom gland of the dorsal spines contains about 0.03 ml liquid venom. It

contains a mixture of high-molecular weight protein-toxins. In experimental animals,

stonefish venom causes an attrio-ventricular blockade and ventricular fibrillation of the

heart, a sudden hypertension and paralysis of skeletal muscles, which is either due to a

massive of neuro-transmitter or to damage of nerve and muscle.

8

Scorpionfishes have a reddish to brownish color and are mottled. This enables

them to disappear against the substrate.

Scorpaenopsis

There are 4 very similar species of humpback scorpionfishes. Scorpaenopsis

diabolus (devil scorpionfish - pectoral fin with orange, yellow and white) and Smacrochir

(flasher scorpionfish - pectoral fin with orange and some black at the edge) can best be

told apart by looking at their pectoral fins. The devil scorpionfish also has a more

pronounced hump and is larger (up to 30cm) than the flasher scorpionfish (15cm). If the

ridge above the eyes is serrated it is a bandtail scorpionfish (S. neglecta). Another

scorpionfish, Scorpaenopsis gibbosa (humpback scorpionfish), is only found in Africa

and the Indian ocean.

9

The stonefish is extremely difficult to see because it usually buries most of its

body under sand or rubble and only their widely separated eyes show. Often algae and

hydroids grow on its back. It has been suggested, that stonefishes exude a white, milky

substance over their bodies which encourages plant growth. Shrimps and other animals

have been observed to climb over them. This is the worlds most venomous fish. Their

near perfect camouflage and the venomous spines make them a hazard for swimmers,

snorkelers and divers in shallow water. Wounds should be treated immediately with hot

water or dry heat.

There is a scorpionfish that erroneously identified as stonefish, the humpbacked

or devil scorpionfish. However there are two difference: First the shape of the mouth.

Stonefishes have a mouth which is directed upwards like a upside-down "U". Second the

stone fish curl their tail extremely to one side.

CHORDATA (VERTEBRATES)

Scorpionfishes (Scorpaenidae)

The family Scorpaenidae contains around 45 genera and 380 species.

Characteristics

Scorpionfishes have large, heavily ridged and spined heads. Venomous spines on

their back and fins with a groove and venom sack. Well camouflaged with tassels, warts

and colored specks. Some scorpionfishes can change their color to better match their

10

surroundings. The stonefish is a master of disguise and deception, it looks like a piece of

coral or sand covered rock. Thus, he can blend in with its surroundings and go unnoticed

by its prey.

Ecology and range

Most scorpion fishes live on or near the bottom. They lie in crevices, in caves and

under overhangs. Range: Red Sea , pacific ocean to Australia, Hawaii. A few

scorpionfishes (no lionfishes or stonefishes) live in the Caribbean

Behavior

They feed on crustaceans, cephalopods and fishes employing a lie-in-wait

strategy, remaining stationary and snapping prey that comes near. With their mouth they

create a vacuum and suck prey in during a nearly imperceptible split-second movement

(15 milliseconds).

Some have algae and hydroid growth on their body surfaces( stonefish) and at

least one species (Decoy scorpionfish Iracundus signifier) has a dorsal fin that looks like

a swimming fish, a behavior similar to that of the frogfish. Some species (for example the

weed scorpionfish) sway their bodies from side to side so they look like a piece of debris.

Scorpionfishes are not aggressive, but if threatened they will erect their dorsal

spines. If danger continues they flee, usually very fast but only for a short distance and

then quickly settle back and freeze. The stonefishes for example ususally bury themselves

in sand or rubble using a shoveling motion of their pectoral fins. In a matter of less than

11

10 seconds only the dorsal portion of the head remains exposed, some sand is thrown on

top to further enhancing concealment. Some species like the devilfish have very bright

red and yellow colors on the inner surface of their pectoral fins. Those colors are not

visible when resting but are flashed if threatened.

Scorpion fishes produce a floating, gelatinous mass in which the eggs are embedded.

Phytochemical Components

Phytochemical studies on animals give basic information on the presence and

localization of the active constituents in the animal body. These tests include the test for

The presence of alkaloids, saponins, tannins, glycosides, sterols, flavonoids and

triterpenes.

Alkaloids

Alkaloids are new synthetic agents of greater potency and lesser toxicity. It is a

naturally occurring amine produced by a plant but amines produced by animals and fungi

are also called alkaloids. Many alkaloids have pharmacological effects on humans and

animals. Alkaloids are usually derivatives of amino acids. They are found as secondary

metabolites in plants, animals and fungi, and can be extracted from their sources by

treatment with acids.

12

Alkaloids, group of mildly alkaline compounds, mostly of plant origin and of

moderate molecular complexity. Even in very small amounts, the alkaloids produce

strong physiological effects on the body. All contain nitrogen atoms that are structurally

related to those of ammonia.

Nearly 3000 alkaloids have been recorded; the first to be prepared synthetically

(1886) was one of the simplest, called coniine, or 2-propyl piperidine, C5H10NC3H7. It

is highly poisonous; less than 0.2 g (0.007 oz) is fatal. Coniine, obtained from seeds of

the hemlock, was the poison used in the execution of Socrates. Some 30 of the known

alkaloids are used in medicine. For example, atropine, obtained from belladonna, causes

dilation of the pupils; morphine is a painkiller; quinine is a specific remedy for malaria;

nicotine is a potent insecticide; and reserpine is a valuable tranquilizer.

Saponins

Saponins are subgroups of glycosides that have the characteristic ability to cause

foaming when shaken with water. They are sometimes used as emulsifying agents.

Saponins are believed to be useful in the human diet for controlling cholesterol, but some

are poisonous if swallowed and can cause urticaria. Any markedly toxic saponin is

known as a sapotoxin.

13

Saponins, group of naturally occurring oily glycosides that foam freely when

shaken with water. They occur in a wide variety of plants, including acacia, soapwort,

soaproot, California pigweed, and many others. Saponins have been, and sometimes still

are, used as cleaning agents and as foam producers, notably in fire-extinguishing fluids.

They have a bitter taste and when ingested orally are practically nonpoisonous to warm-

blooded animals. When injected directly into the bloodstream, however, they are

dangerous and quickly dissolve red blood cells. Hydrolysis of a saponin, brought about

by acids or by enzymes, gives a sugar (often, but not necessarily, glucose) and a

sapogenin, the latter being either a triterpene or a steroid. Some of the sugars and

saponins are useful as raw materials for synthesis of steroid hormones.

Glycosides

Glycosides, class of complex chemical compounds in plants. They are broken

down by plant enzymes into sugars, among which glucose is generally included, and into

other substances. The term glucoside is often used synonymously with glycoside, but in

its more specific meaning it refers to glycosides that yield glucose.

Each glycoside in a plant is hydrolyzed (converted in a reaction with water) by an

enzyme, usually a specific enzyme found in the same plant. The enzyme emulsin,

however, causes hydrolysis of several glycosides. The enzymes and glycosides are stored

in separate plant cells until the reaction products of the glycosides are needed and the

14

enzymes are activated.

Glycosides are believed to serve several purposes in the plant. Glycosides are

bitter tasting, and it is believed that they help keep birds and insects from eating seeds

and fruit before they are fully grown, by which time the glycosides have been converted

to sweet sugars. When a plant tissue is bruised, the enzymes hydrolyze the glycosides

into products, such as phenol compounds and acids that have an antiseptic action and

prevent decay of the damaged tissues.

Glycosides are soluble in water and are obtained from plants by water extraction.

They are mostly colorless crystalline solids with a bitter taste. Simple glycosides have

been synthesized in the laboratory, and several hundred glycosides have been extracted

from plants and used for many purposes. Among the important glycosides are indican,

used for dyeing; digitalin, used in medicine; and the saponins, foaming agents used

industrially and medicinally.

Triterpene

Triterpene refers to a particular type of molecules that has a four to five ring,

planar base molecules containing 30 carbon atoms. It is synthesized from very simple

compounds ( acetate units) that are found in all plants, but is mainly synthesized in higher

plants by linking the acetate units “ head to tail”. The triterpenes have an acidic quality,

an acrid- bitter taste, and their function in plants remains unknown.

15

The triterpenes are subdivided into about 2o groups, depending on their particular

structures. The base structure that is found in the largest variety of medicinal plants is the

oleanane triterpene. This type of compound may be represented by four of the most

frequently occurring forms: oleanolic acid, ursolic acid, and alpha and beta amyrin ( the

latter 3 are sometimes put in the division of ursane triterpenes). Platycodin belongs to the

very large class of aleanane triterpenes.

Tannins

These are also tannin acid, common name applied to a group of vegetable

products, both amorphous and crystalline, obtained from various plants, and important

commercially in the tanning of leather. Tannins have variable composition. Some, called

condensed tannins, are phenols of moderately complex structure, and others are esters of

glucose or some other sugar with one or more trihydroxybenzoic acids. The empirical

formula, C14H14O11, often given for common tannin, is only an average. Tannins occur in

many trees, and the best sources include oak galls and the bark of sumac. Extraction with

water, or water and alcohol, is the first step in the preparation of tannin. Settling,

followed by evaporation at a low temperature, yields the commercial product.

Tannins have a yellow-white to brown color and a faint, characteristic odor.

Exposure to light deepens the color. They all taste bitter and are astringent. Water,

acetone, and alcohol dissolve tannins readily, but benzene, ether, and chloroform do not.

Heating to 210°C, (410° F) causes decomposition, accompanied by formation of

pyrogallol and carbon dioxide. The chemical property that provides the basis for most

16

uses of tannins is its ready formation of precipitates with albumin, with gelatin, and with

many alkaloidal and metallic salts. The ability of tannins to transform proteins into

insoluble products resistant to decomposition leads to their use as tanning agents. Ferric

salts react with tannins to give bluish-black products that are useful as inks. Tannins are

used as mordants for dyeing cloth, as sizes for paper or silk, and as coagulants for rubber.

The precipitating properties of tannins are used in clarifying, or cleaning, wines and beer.

Tannic acid is valuable as an external medicine because it is astringent and styptic.

Flavonoid

The term flavonoid refers to a class of plant secondary metabolites based around

a phenylbenzopyrone structure. Flavonoids are most commonly known for their

antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the

media – these terms are equivalent and interchangeable, since all flavonoids are

biological in origin.

The flavonoid synthetic pathway begins with a product of glycolysis,

phosphoenolpyruvate, entering into the Shikimate pathway to yield phenylalanine.

Phenylalanine is the starting material of the phenylpropanoid metabolic pathway, from

17

which 4-Coumaryl-CoA is produced. This can be combined wih Malonyl-CoA to yield

the true backbone of flavonoids, a group of compounds called chalcones. Ring-closure of

these compounds results in the familiar form of flavonoids, a three-ringed phenolic

structure (polyphenols). The metabolic pathway continues through a series of enzymatic

modifications to yield flavanones → dihydroflavonols → anthocyanins. Along this

pathway many products can be formed, including the flavonols, flavan-3-ols,

proanthocyanidins (tannins) and a host of other polyphenolics.

Flavonoids are widely distributed in plants fulfilling many functions including

producing yellow or red/blue pigmentation in flowers and protection from attack by

microbes and insects. The widespread distribution of flavonoids, their variety and their

relatively low toxicity compared to other active plant compounds (for instance alkaloids)

mean that many animals, including humans, ingest significant quantities in their diet.

Flavonoids have been found in high concentrations in butterflies and moths sequestered

from dietary intake at the larval stage and then stored in adult tissues.

Flavonoids have been referred to as "nature's biological response modifiers"

because of strong experimental evidence of their inherent ability to modify the body's

reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-

inflammatory [1] , anti-microbial and anti-cancer activity. In addition, flavonoids act as

powerful antioxidants, protecting against oxidative and free radical damage.

18

Consumers and food manufacturers have become interested in flavonoids for their

medicinal properties, especially their potential role in the prevention of cancers and

cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red

wine have been attributed to flavonoid compounds rather than to known nutrients and

vitamins.

Sterols

Sterols may be found either as free sterols, acylated (sterol esters),

alkylated (steryl alkyl ethers), sulfated (cholesterol sulfate), or linked to a glycoside

moiety (steryl glycosides) which can be itself acylated (acylated sterol glycosides).

 Free Sterols

Sterols form an important group among the steroids. Unsaturated steroids with

most of the skeleton of cholestane containing a 3-hydroxyl group and an aliphatic side

chain of 8 or more carbon atoms attached to position 17 form the group of sterols. 

 

They are lipids resistant to saponification and are found in an appreciable quantity

in all animal and vegetal tissues. These unsaponifiable lipids may include one or more of

a variety of molecules belonging to 3-hydroxysteroids, they are C27-C30 crystalline

alcohols (in Greek, stereos, solid). These lipids can be classed as triterpenes as they

derive from squalene which gives directly by cyclization, unsaturation and 3-

hydroxylation, lanosterol in animals or cycloartenol in plants.

19

In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol (Greek,

chole, bile), particularly abundant in adrenals (10%, w/w), nervous tissues (2%,w/w),

liver (0.2%,w/w) and gall stones, its fundamental carbon structure being a

cyclopentanoperhydrophenanthrene ring (also called sterane). It was the first isolated

sterol around 1770 by Poulletier de La Salle from gall stones. In 1815, it was isolated

from the unsaponifiable fraction of animal fats by M.E. Chevreul who named it

cholesterine (Greek, khole, bile and stereos, solid). The correct formula (C27H46O) was

proposed in 1888 by F. Reinitzer but structural studies from 1900 to 1932, mainly by

H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize

Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their

connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact steric

representation of cholesterol. In 1936, Callow and Young have designated steroids all

compounds chemically related to cholesterol.

While it became clear very early that cholesterol plays an important role in controlling

cell membrane permeability by reducing average fluidity, it appears now that it has a key

role in the lateral organization of membranes and free volume distribution. These two

parameters seem to be involved in controlling membrane protein activity and "raft"

formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1). 

The vertebrate brain is the most cholesterol-rich organ , containing roughly 25% of the

total free cholesterol present in the whole body.

20

 

In late-step synthesis of cholesterol, discrete oxidoreductive and/or demethylation

reactions occur, which start with the common precursor lanosterol. It was also found as a

major constituent of the unsaponifiable portion of wool fat (lanoline). Animal tissues

contain in addition to cholesterol small amounts of 7-dehydrocholesterol which, on UV

irradiation, is converted to vitamin D3 (cholecalciferol).

Desmosterol (24-dehydrocholesterol), an intermediate between lanosterol and

cholesterol, has been implicated with myelination processes. While high desmosterol

levels could be detected in the brain of young animals (Paoletti R et al., J Am Oil Chem

21

Soc 1965, 42, 400) no desmosterol was found in the brain of adult animals. It is also

known as an abundant membrane component in some mammalian cells, such as

spermatozoa and astrocytes (Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J

Biol Chem 2004, 279, 48654). Inability to convert desmosterol to cholesterol leads to the

human disorder desmosterolosis (a severe developmental defect and cognitive

impairment) (Waterham HR et al., Am J Hum Genet 2001, 69, 685).

In higher plants, the first sterols were isolated by Hesse (1878) from the Calabar

beans (Phytostigma venenosum) which coined the term "phytosterine". This substance

was later named stigmasterol (Windaus and Hault, 1906) from the plant genus. The

denomination "phytosterol" was proposed in 1897 (Thoms H) for all sterols of vegetal

origin. Chemically, these sterols have the same basic structure as cholesterol but

differences arise from the lateral chain which is modified by the addition of one or two

supernumerary carbon atoms at C-24 with either or chirality. The 24-alkyl group is

characteristic of all phytosterols and is preserved during subsequent steroid metabolism

in both fungi and plants to give hormones that regulate growth and reproduction in a

manner similar to animals. Most phytosterols are compounds having 28 to 30 carbon

atoms and one or two carbon-carbon double bonds, typically one in the sterol nucleus and

sometimes a second in the alkyl side chain. 

All phytosterols were shown to derive in plants from cycloartenol and in fungi from

lanosterol, both direct products of the cyclization of squalene.

22

More than 200 different types of phytosterols have been reported in plant species.

Representatives of these sterols are campesterol, stigmasterol (in soybean oil) and -

sitosterol which is present in all plant lipids and is used for steroid synthesis. An

important sterol from yeast and ergot is the C28 compound ergosterol (mycosterol). Upon

irradiation, this sterol gives rise to vitamin D2 (calciferol).

As ergosterol is a cell membrane component largely restricted to fungi, its amount in

environmental matrices may be used as an index molecule for these micro-organisms in a

living biomass (Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset

JY et al., Appl Environ Microbiol 2001, 67, 2051).

23

 

Considerable variability in the concentration of free sterols was observed among

different oils. While concentrations lower than 100 mg/100 g are found in oils from

coconut, palm, olive, and avocado, concentrations between 100 and 200 mg/100 g are

found in oils from peanut, safflower, soybean, borage, cottonseed, and sunflower, and

concentrations between 200 and 400 mg/100 g are found in oils from sesame, canola,

rapeseed, corn, and evening primrose (Phillips KM et al., J Food Comp Anal 2002, 15,

123).

24

Phytosterols produce a wide spectrum of biological activities in animals and

humans. They are considered efficient cholesterol-lowering agents. In addition, they

produce a wide spectrum of therapeutic effects including anti-tumor properties. Further

data on their metabolism and potential therapeutic action can be found in a review article

(Ling WH et al., Life Sci 1995, 57, 195).

The European Commission authorized in 2004 the addition of phytosterols and

phytostanols in food products with conditions of labeling including their amount per 100

g and the statement that the human consumption of more than 3 g/day should be avoided.

Phytostanols are a fully-saturated subgroup of phytosterols (they contain no

double bonds). They occur in trace levels in many plant species but in high levels in

tissues of only in a few cereal species. They are in general produced by hydrogenation of

phytosterols.

Stanols often occur in dinoflagellates but are not common in other marine

microalgae. Hence, dinoflagellates are often the major direct source of 5(H)-stanols in

marine sediments (Robinson N et al., Nature 1984, 308, 439).

Fully saturated sterols are also found in animals but are of bacterial origin. Thus, the

5(H)-stanol coprostanol constitutes approximately 60% of the total sterols in human

faeces. 

25

While cholesterol was considered to be nearly absent in vegetal organisms, its

presence is now largely accepted in higher plants. It can be detected in vegetal oils in a

small proportion (up to 5% of the total sterols) but remains frequently present in trace

amounts. An unusual relatively high content of cholesterol was described in camelina oil

(about 200 mg per kg) (Shukla VKS et al., JAOCS 2002, 79, 965). However, several

studies have revealed the existence of cholesterol as a major component sterol in

chloroplasts, shoots and pollens. Furthermore, cholesterol has been detected as one of the

major sterols in the surface lipids of higher plant leaves (rape) where he may amount to

about 72% of the total sterols in that fraction (Noda M et al., Lipids 1988, 23, 439).

Although practical, the ancient distinction between zoosterols, mycosterols and

phytosterols is no more used, since the same sterol may have different sources, but the

appellation phytosterol is actually more frequently used.

Sterols are often isolated in the unsaponifiable fraction of any lipid extract and

determined by various chromatographic procedures (HPLC or GLC).

Avenasterol can be isolated from oat oil. This sterol was shown to protect

specifically frying oils from oxidation owing to its ethylidene group in the side chain

(White PJ et al., JAOCS 1986, 63, 525).

26

An extensive review on the diversity, analysis, and health-promoting uses of

phytosterols and phytostanols may be consulted with interest (Moreau RA et al., Prog

Lipid Res 2002, 41, 457). 

Sterols, or steroid alcohols are a subgroup of steroids with a hydroxyl group in the

3-position of the A-ring. They are amphipathic lipids synthesized from Acetyl coenzyme

A. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest

of the aliphatic chain is non-polar.

Sterols are important for the physiology of eukaryotic organisms. They form part

of the cellular membrane where they modulate their fluidity and function and participate

as secondary messengers in developmental signaling.

27

Different organisms utilize different sterols. The most important ones are

cholesterol, phytosterols, and some steroid hormones in animals, and campesterol,

sitosterol and stigmasterol in plants.

Sterols are also known to block cholesterol absorption sites in the human gut thus

helping to reduce cholesterol in humans by up to 15%.

Test Microorganisms

Escherichia coli

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: Escherichia coli

28

Escherichia coli, commonly known as E. coli, is a species of bacteria normally

present in human intestines. A recently recognized strain, E. coli 0157:H7, produces high

levels of toxins that can cause kidney damage, as well as septicemia, or blood poisoning.

Symptoms can include diarrhea, chills, headaches, and high fever and in some cases the

infection can lead to death, even with medical intervention.

E. coli, discovered by Theodor Escheric h , a German pediatrician and

bacteriologist, is one of the main species of bacteria that live in the lower intestines of

mammals. Specimens have also been located on the edge of hot springs. The bacteria are

necessary for the proper digestion of food and are part of the intestinal flora. Presence in

surface water is a common indicator of fecal contamination. It belongs among the

Enterobacteriaceae, and is commonly used as a model organism for bacteria in general.

One of the root words of the family's scientific name, "enteric", refers to the intestine,

hence "gastroenteritis" (from 'gastro-', stomach, 'entero-' intestine, '-itis', inflammation).

"Fecal" is the adjective pertaining to feces, so it is often used synonymously with

"enteric".

The number of individual E. coli bacteria in the feces that one human passes in

one day averages between 100 billion and 10 trillion. All the different kinds of fecal coli

bacteria, and all the very similar bacteria that live in the ground (in soil or decaying

plants, of which the most common is Enterobacter aerogenes), are grouped together

under the name coliform bacteria. Technically, the "coliform group" is defined to be all

29

the aerobic and facultative anaerobic, non-spore-forming, Gram-negative, rod-

shapedbacteria that ferment lactose with the production of gas within 48 hours at 35 °C

(95 °F). In the body, this gas is released as flatulence. E. coli cells are elongated, 1–2 µm

in length and 0.1–0.5 µm in diameter.

Staphylococcus aureus

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Staphylococcaceae

Genus: Staphylococcus

Species: S. aureus

30

Staphylococcus aureus is a genus of spherical bacteria capable of producing a

heat stable toxin that cause illness in humans. The most common pathogen S. aureus, is

frequently responsible for carbuncles, boils, pneumonia, abscesses and osteomyelities It

exist in air, dust, sewage, water, humans, foods and animals.

Staphylococcus aureus (which is occasionally given the nickname golden staph) is

a bacterium, frequently living on the skin or in the nose of a healthy person, that can

cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulitis)

and abscesses, to life-threatening diseases such as pneumonia, meningitis, endocarditis,

Toxic shock syndrome (TSS), and septicemia. Each year some 500,000 patients in

American hospitals contract a staphylococcal infection. It is a spherical bacterium. It is

abbreviated to S. aureus or sometimes referred to as Staph aureus in medical literature,

and should not be confused with the somewhat similarly named streptococci which are

also medically important.

S. aureus is a Gram-positive coccus, which appears as g rape -like clusters when

viewed through a microscope and as large, round, golden-yellow colonies, often with β-

hemolysis, when grown on blood agar plates. The golden appearance is the etymological

root of the bacteria's name: aureus means "gold" in Latin.

S. aureus is catalase positive and thus able to convert hydrogen peroxide (H2O2)

to water and oxygen, which makes the catalase test useful to distinguish staphylococci

from enterococci and streptococci. S. aureus can be differentiated from most other

31

staphylococci by the coagulase test: S. aureus is coagulase-positive, while most other

Staphylococcus species are coagulase-negative.

The species has been subdivided into two subspecies: S. aureus aureus and S.

aureus anaerobius. The latter requires anaerobic conditions for growth, is an infrequent

cause of infection, and is rarely encountered in the clinical laboratory.

S. aureus may occur as a commensal on human skin (particularly the scalp,

armpits and groins); it also occurs in the nose (in about 25% of the population) and throat

and less commonly, may be found in the colon and in urine. The finding of Staph. aureus

under these circumstances does not always indicate infection and therefore does not

always require treatment (indeed, treatment may be ineffective and re-colonisation may

occur). It can survive on domesticated animals such as dogs, cats and horses, but has

never been found on food animals such as poultry or swine. It can survive for some hours

on dry environmental surfaces, but the importance of the environment in spread of Staph.

aureus is currently debated. It can host phages, such as the Panton-Valentine leukocidin,

that increase its virulence.

S. aureus can infect other tissues when normal barriers have been breached (e.g.

skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of

furuncles). In infants S. aureus infection can cause a severe disease Staphylococcal

scalded skin syndrome (SSSS).[8]

32

S. aureus infections can be spread through contact with pus from an infected

wound, skin-to-skin contact with an infected person, and contact with objects such as

towels, sheets, clothing, or athletic equipment used by an infected person.

Deeply situated S. aureus infections can be very severe. Prosthetic joints put a

person at particular risk for septic arthritis, and staphylococcal endocarditis (infection of

the heart valves) and pneumonia may be rapidly fatal

Candida albicans

Kingdom: Fungi

Phylum: Ascomycota

Subphylum: Saccharomycotina

Class: Saccharomycetes

Order: Saccharomycetales

Family: Saccharomycetaceae

Genus: Candida

Species: C. albicans

33

Fungi belong to the genus Candida, especially Candida albicans, can infect both

internal organs and mucous membranes of the mouth, throat and genital tract. In people

with impaired immunity, this organism can cause a chronic infection.

Candida albicans is a diploid sexual fungus (a form of yeast), and a causal agent

of opportunistic oral and vaginal infections in humans. Systemic fungal infections

(fungemias) have emerged as important causes of morbidity and mortality in

immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow

transplantation). In addition, hospital-related infections in patients not previously

considered at risk (e.g. patients on an intensive care unit) have become a cause of major

health concern.

C. albicans is among the many organisms that live in the human mouth and

gastrointestinal tract. Under normal circumstances, C. albicans lives in 80% of the human

population with no harmful effects, although overgrowth results in candidiasis.

Candidiasis is often observed in immunocompromised individuals such as HIV-positive

patient. Candidiasis also may occur in the blood and in the genital tract. Candidiasis is

commonly known as "thrush", and is a common condition that is usually easily cured in

people who are not immunocompromised. To infect host tissue, the usual unicellular

yeast-like form of Candida albicans reacts to environmental cues and switches into an

invasive, multicellular filamentous form.

34

Materials used

A Soxhlet extractor is a type of laboratory glassware invented in 1879 by Franz

von Soxhlet. It was originally designed for the extraction of lipid from a solid test

material, but can be used whenever it is difficult to extract any compound from a solid.

Typically, dry test material is placed inside a "thimble" made from filter paper,

which is loaded into the Soxhlet extractor. The extractor is attached to a flask containing

a solvent (commonly diethyl ether or petroleum ether) and a condenser. The solvent is

heated, causing it to evaporate. The hot solvent vapor travels up to the condenser, where

it cools and drips down onto the test material. The chamber containing the test material

slowly fills with warm solvent until, when it is almost full, it is emptied by siphon action,

back down to the flask. This cycle may be allowed to repeat many times. During each

cycle, a portion of the lipid dissolves in the solvent. However, once the lipid reaches the

solvent heating flask, it stays there. It does not participate in the extraction

35

cycle any further. This is the key advantage of this type of extraction; only clean warm

solvent is used to extract the solid in the thimble. This increases the efficiency of the

extraction when compared with simply heating up the solid in a flask with the solvent.

Liebig condenser

The Liebig condenser is a piece of laboratory equipment where a straight glass pipe goes

through a water jacket (a glass cylinder through which water constantly flows). It is used

in distillation and reflux to condense vapours into liquid.

History

Though named after the German chemist Justus Baron von Liebig, he cannot be given

credit for having invented it because it was already in use for some time before him.

However, it is believed that the apparatus was made popular by him.

The true inventors, all of them making the discovery independently, and the year of the

invention were the German chemist Christian Ehrenfried Weigel in 1771, French

scientist, P. J. Poisonnier, in 1779 and the Finnish chemist Johan Gadolin in 1791.

Liebig himself incorrectly attributed the design to the German pharmacist Johann

Göttling who had made improvements to the Weigel design in 1794 [1].

36

Efficiency

The Liebig condenser is much more efficient than a simple retort due to its use of liquid

cooling. Water can absorb much more heat than the same volume of air, and its constant

circulation through the water jacket keeps the condenser's temperature constant.

Therefore a Liebig condenser can condense a much greater flow of incoming vapour than

an air condenser or retort.

At the end of an extraction, the excess solvent may be removed using a rotary

evaporator, leaving behind only the extracted lipid.

Petroleum ether

Petroleum ether, also known as benzine or X4, is a group of various volatile,

highly flammable, liquid hydrocarbon mixtures used chiefly as nonpolar solvents.

Petroleum ether is obtained from petroleum refineries as the portion of the

distillate which is intermediate between the lighter naphtha and the heavier kerosene. It

has a specific gravity of between 0.6 and 0.8 depending on its composition.

Benzine should not be confused with benzene. Benzine is a mixture of alkanes, e.g.,

37

pentane, hexane, and heptane, whereas benzene is a cyclic, aromatic hydrocarbon, C6H6.

Likewise, petroleum ether should not be confused with the class of organic compounds

called ethers, which contain the -O- functional group.

Anhydrous

An ionic crystal is said to be anhydrous if it contains no water.

An example of anhydration can be seen in copper (II) sulfate. If the water of

crystallization is removed from blue crystals of copper (II) sulfate, a white powder

(anhydrous copper sulfate) is formed.

The original formula for crystalline copper (II) sulfate is CuSO4·5H2O. The

formula for anhydration is as follows:

CuSO4·5H2O + heat → CuSO4 + 5H2O

Another example is in the heating of magnesium sulfate heptahydrate,

MgSO4·7H2O. On heating, it undergoes the following reaction:

MgSO4·7H2O + heat → MgSO4 + 7H2O

38

Analytical balances are accurate and precise instruments used to measure masses.

They require a draft-free location on a solid bench that is free of vibrations. Some modern

balances have built-in calibration masses to maintain accuracy. Older balances should be

calibrated periodically with a standard mass.

39

DEFINITION OF TERMS

Antibiotics – are molecules that are produced by one microorganism that kill

(bacteriocidal) or inhibit (bacteriostatic) other microorganisms. They are one class of

antibacterial and antifungal antimicrobials that can potentially be used as medicinal

drugs to treat infections because of their low toxicity for humans or animals.

Antimicrobial agents – agents that kill or slow the growth of microbes like bacteria

(antibacterial activity), fungi (antifungal activity), viruses (antiviral activity), or parasites

(antiparasitic activity).

Chloramphenicol - is a bacteriostatic antibiotic originally derived from the bacterium

Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice

in 1949.It was the first antibiotic to be manufactured synthetically on a large scale.

Chloramphenicol is effective against a wide variety of microorganisms.In the West, the

main use of chloramphenicol is in eye drops or ointment for bacterial conjunctivitis.

Glucose Yeast Peptone – is recommended for the isolation of yeasts from soils

specimen. This is a highly nutritious medium, which may be used for microbial

examination.

40

Incubate – to give the best or optimum conditions (ex: temperature, moisture) for growth

and development.

Inoculate – to put microorganism or a substrate of organism on a medium.

Inoculation – a process of implanting infectious material into a culture medium.

Inoculum - population of a pure culture grown in a medium.

Inhibition zone - this is an area around a paper disk or colony of bacteria or mold where

no other organisms are growing.

Nutrient Agar – is used for the cultivation of bacteria and for the enumeration of

organisms in water, sewage, feces and other materials. It is used in the laboratory for the

cultivation and maintenance of nonfastidious species and used in microbiological

examination of a broad spectrum of materials. It is a simple medium composed of beef

extract, peptone, and agar. It has been one of the most generally used media in

bacteriological procedures. It is used for the ordinary routine examinations of water,

sewage, and food products, for the carrying of stock cultures, for the preliminary

cultivation of samples submitted for bacteriological examination, and for isolating

organisms in pure culture.

41

Chapter IIMETHODOLOGY

This chapter presents Experimental Design Diagram, the materials, procedures,

methods of gathering data and the statistical tool used to interpret the data gathered.

TITLE: Antibiotic Property from Stonefish dorsal Fin Venom (Synaceia verrucosa)

against Escherichia coli, Staphylococcus aureus and Candida albicans

HYPOTHESES: 1.) The Stonefish Dorsal Fin Venom cannot inhibit the growth of harmful

microorganisms.

2.) Alkaloids, Saponin and Tannins are not the active constituents of the Stonefish Dorsal

Fin Venom using the Phytochemical Test.

3.) There is no significant difference of using the Antibiotic Property from Stonefish

Dorsal Fin and the commercial antibiotics in terms of zone of inhibition.

INDEPENDENT VARIABLE: Stonefish Dorsal Fin Venom Extract

TREATMENTST0 (control)

ChloramphenicolT1

With Stonefish Dorsal Fin

Venom extract

NUMBER OF TRIALS 3 3

DEPENDENT VARIABLE: Zone of inhibition

CONSTANTS: Amount of extract

Materials:

A. Extractionsterile syringe

sterile cotton

sterile knife

sterile blade

sterile pair of scissor

test tubes

gloves

ice cubes

basin

sterile forceps

Solvent extraction unit, fitted with Liebig Condenser

Thimbles

Heat source

Analytical balance (capable of weighing up to 0.1 mg)

Desiccators

Distilling flasks (125-250 mL)

Grinder or mortar and pestle

Reagents ( Petroleum Ether, Anhydrous, Analytical Grade)

B. Antimicrobial Assay Test

Agar plates

Cork borer

43

Nutrient Agar (NA)

Glucose Yeast Peptone (GYP) Agar

Culture Bacteria and Yeast- E.coli, S. aureus, C. albicans

Inoculating loop

Sterile cotton swab

Commercial antibiotic (Chloramphenicol)

Alcohol lamp

Beaker with 95% Ethanol

Pippette

C. Phytochemical Test

acetic anhydride

sulfuric acid

1 ml 10% hydrochloric acid

1% hydrochloric acid

Mayer’s Reagent

ferric chloride

2 test tubes

anhydrous sodium carbonate

sodium hydroxide

potassium sodium tartrate

distilled water

44

GENERAL PROCEDURE

A. Reagents

All reagents (biochemical- equipment) were supplied by Regional Science High

School for Region 1 Laboratory, Adamson University, Marine Science Institute and

Natural Science of Research institute of University of the Philippines.

B. Collection of Animal Specimen

Fifteen Stonefish (Synanceia verrucosa) were gathered in the coastal area of

Paraoir, Balaoan, La Union. They were subjected to observation for 2 days with food and

seawater in a wide aquarium, the said specimen were in good physical condition and

ready for dorsal fin venom extraction.

C. Preparation of the Extract

Each Stonefish was applied with sterile cotton having 90% isopropyl alcohol

before inserting a sterile knife on the point where the head meets the body and cutting

through the joint. Most of extracted liquid was found in the specimen’s dorsal fin venom.

There were about 10mL of liquid extract obtained and placed in a test tube covered with

cotton plug then refrigerated at 150C for 24 hours and was used for the Antimicrobial

assay.

This was conducted under the direct supervision of Dr. Lourdes Cruz and Mr.

Chris Mendoza in their technical assistance of proper handling procedures of Stonefish.

45

The needed extract for the Phytochemical Test was prepared using the soxhlet

method as stated below:

1) The sample (Stonefish Dorsal Fin venom) was placed into the extraction thimble.

2) The thimble was placed in the soxhlet extraction fitted with a Liebig Condenser.

3) Thirty five (35) mL of pure petroleum ether was transferred to dry and the

distilling flask with 125-250 mL capacity was connected to the extraction unit.

4) Turn the heat source on.

5) The extraction proceed to 6 to 8 hours.

6) Turn off the heat source after, and then allow the extraction tube to drain for 15

minutes.

7) The thimble with the sample was removed.

8) Turn the heat source to recover the ether.

9) After the ether has been completely evaporated and collected into the extraction

tube, turn off the heat source.

10) The distilling flask was removed and was put it in the preheated (1050C) oven for

1 hour.

11) It was cooled in a desiccator and weighed.

12) The flask was again heated in the oven for 1 hour.

13) The procedure # 11 and 12 was repeated until the weight of the flask after two

consecutive weighing agrees to within 0.5mg.

46

D. Preparation of the Test Organisms

Escherichia coli, Staphylococcus aureus and Candida albicans were obtained from

Natural Science Research Institute (NSRI) at UP Diliman that were used as test

organisms.

E. Antimicrobial Assay Test

Microbial suspensions were prepared from 24- hour cultures of the Escherichia

coli, Staphylococcus aureus (bacteria) and Candida albicans (yeast). The suspending

medium used was 0.1% peptone water.

One-tenth (0.1) mL aliquots of the bacterial and yeast suspensions were

transferred into pre-poured Nutrient Agar (NA) and Glucose Yeast Peptone (GYP) Agar,

respectively. Five (5) of the corresponding medium, melted and cooled to 450C, was

poured onto the agar plate and swirled to distribute the inoculum evenly on the agar

surface. Three (3)mL of the sample was placed in each hole.

The plates were incubated at room temperature. NA and GYP plates were

observed after 24-48 hours. The clearing zone was measured in millimeters and the

average diameter of the clearing zones was calculated. The antimicrobial index (AI) was

computed using the following formula:

AI = Diameter of clearing zone – Diameter of wellDiameter of well

47

F. Phytochemical Test

Dorsal Fin Venom extract from fresh specimens of Synanceia verrucosa were

used for Phytochmical tests.

7) Test for Sterols and Tipertenes

Lieberman-Berchard Test

A small amount of the sample extract(Stonefish Dorsal Fin Venom) in acetic

anhydride was dissolved. The soluble portion was decanted and to this, 1-2 drops of

concentrated sulfuric acid was added. Observe a green color, either immediately or solely

going into red and blue tones. A pink to red color is indicative of triterpenoids while a

blue color is indicative of steroids.

8) Test for Flavonoids

One (1) ml of sample extract was treated with 1 ml 10% hydrochloric acid and a

few magnesium turnings. Formation of red color is observed.

9) Test for Alkaloids

The sample extract was extracted with 1% HCL and drops of Mayer’s Reagent or

Wagner’s Rgt. was added to the filtered acid extract. A cream colored precipitate is

observed in the case of Mayer’s Rgt. while a reddish brown ppt. is observed in the case of

Wagner’s Rgt.

48

Formula of Mayer’s Rgt.:

1.358 g of mercuric chloride was dissolved in 60 ml distilled water and 5 g of

potassium iodide was dissolved in 10 ml distilled water. The two solutions were mixed

and diluted to 100 ml with distilled water.

Formula for Wagner’s Rgt.:

1.3 g iodine crystals and 2.0 g potassium iodide in sufficient amount of distilled

water to make a total volume of 100ml was dissolved.

10) Test for Tannins

The sample extract was extracted with hot water and the aqueous extract was

filtered. Upon addition of two drops of ferric chloride test solution, a dark color and

precipitate forms which may either be black, dark blue, blue black, green or blue green.

Ferric chloride TS: Dissolve 9g of ferric chloride in dist, water to make 100 ml.

49

11) Test for Saponins

The sample extract was dissolved in hot water. The aqueous extract when shaken

vigorously should become frontly. The froth, honeycomb in nature should persist for at

least 30 minutes.

12) Test for Glycosides

The sample extract was dissolved in hot water and filtered. The filtrate was used

for the test. Two test tubes were used. Two ml of sample was placed in each tube. One

ml of dilute hydrochloric acid was added to sample 1. Nothing is added to sample 2. The

2 test tubes were placed in a boiling water bath for 5 minutes. Then the test tubes were

cooled. The samples were both neutralized with anhydrous sodium carbonate until no

more effervescence is produced. Then add Fehling’ s B. One ml of Fehling’ s solution

was used. The 2 test tubes were heated in a water bath for 2 minutes. Observe the amount

of brick red precipitate that formed. An increase in the amount of brick red precipitate in

the hydrolyzed sample (the sample to which dilute acid was added) as compared to the

other sample indicates the presence of glycosides.

50

Fehling’s solution A:

Copper Sulfate (CuSO4. 5H2O)…34.66 grams

Distilled water, a sufficient quantity

To make…500 ml

Dissolve the copper sulfate in the distilled water and mixed.

Fehling’s Solution B:

Sodium Hydroxide…50 grams

Potassium Sodium Tartrate…173 grams

Distilled water, a sufficient quantity

To make …500 ml

The sodium hydroxide and the potassium sodium tartrate in the distilled

water and were dissolved and mixed.

Note: Mix Fehling’s A and B in equal amount before using.

51

Reagents Collection of Animal Specimen

Preparation of the Test MicroorganismsPreparation of the

Extract

Antimicrobial Assay Test

Phytochemical Test

Figure 1. The Flowchart of Experimental Design

52

Preparing the necessary laboratory materials

Sterilizing laboratory materialsPositioning the live Stonefish under the supervision of an animal supervisor

Insertion of the knife at the point where the head meets the body and cutting through the joint

Laying the fish with its head towards you and inserting and cutting the knife above the dorsal fin

Cutting along the other side of the dorsal fin as before

Pinning down the tail end of the dorsal fin with the heel of the sterile blade. Grasping the tail fin and

pulling it way from the dorsal fin.

Preserving the fluid venom extract in a sterilized bottle and refrigerate.

Using a sterile syringe, eject it on the thin venom duct that leads to the tip of the spine.

Figure 2. Block presentation of the standard protocol on fluid venom extraction of

Stonefish

53

Chapter III

RESULTS AND DISCUSSONS

This chapter presents the presentation, analysis and interpretation of data

regarding significant inhibitory effect on the Antibiotic Property from Stonefish Dorsal

Fin Venom.

The observation and findings of the study were presented in tables and

graphics, which are analyzed and discussed thoroughly.

Table 1. Mean Zone of Inhibition against E. coli in millimeters (mm).

Trials T0 (mm) T1(mm)

1 3.2 3.52 3.2 3.53 3.2 3.4

Total (mm) 9.6 10.4Mean (mm) 3.2 3.5

The table shows that the Mean Zone of Inhibition of T1 (Stonefish Dorsal Fin

Venom) is 3.5 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone

of Inhibition of T0. Likewise, results showed that there is no significant difference

between the treatments in terms of zone of inhibition.

Table 2. Mean Zone of Inhibition against S. aureus in millimeters (mm)

Trials T0 (mm) T1(mm)

1 3.2 3.4

2 3.2 3.4

3 3.2 3.3

Total (mm) 9.6 10.1

Mean(mm) 3.2 3.4

The table shows that the Mean Zone of Inhibition of T1 (Stonefish Dorsal Fin

Venom) is 3.4 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone

of Inhibition of T0. Likewise, results showed that there is no significant difference

between the treatments in terms of zone of inhibition.

55

Table 2 .Mean Zone of Inhibition against C. albicans in millimeters (mm)

Trials T0(mm) T1(mm)

1 3.2 2.8

2 3.2 2.8

3 3.2 2.7

Total(mm) 9.6 8.3

Mean(mm) 3.2 2.8

The table shows that the Mean Zone of Inhibition of T1 (Stonefish Dorsal Fin

Venom) is 2.8 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm.

This proves that the Mean Zone of Inhibition of T1 is less than the Mean Zone of

Inhibition of T0. Likewise, results showed that there is a significant difference between

the treatments in terms of zone of inhibition.

56

Table 4.Summary table of the Mean Zone of Inhibition of Test Microorganisms in

millimeters (mm)..

Bacteria/ Yeast T0(mm) T1(mm)

Escherichia coli 9.6 10.4

Staphylococcus aureus 9.6 10.1

Candida albicans 9.6 8.3

Total(mm) 28.8 28.8

Mean(mm) 9.6 9.6

The table shows that the Mean Zone of Inhibition of T1 is 9.6 mm and the

Mean Zone of Inhibition of T0 is 9.6 mm. This proves that the Mean Zone of Inhibition of

T1 is similar to the Mean Zone of Inhibition of T0. Likewise, results showed that there is

no significant difference between the treatments in terms of zone of inhibition.

57

Table 5. A Summary table of the Active Principles of the Dorsal Fin Venom.

Active

Principles/Analytes

Color reaction RESULT

Alkaloids Reddish brown 3

Glycosides Red 0

Tannins black, dark blue,

blue black, green or

blue green

0

Saponin Yellow/. red 3

Flavonoids Red 0

Tipertenes pink to red 0

Sterols blue 3

The table shows the Phytochemical Test result of the Stonefish Dorsal Fin Venom

sample. It shows that alkaloids, Tannins and Saponin are very abundant. This contributed

much on the effectiveness of Stonefish Dorsal Fin Venom as an antibiotic agent as

revealed on the table

Legends:

3 = Very Abundant (51-100%) 2 = Abundant (26-50%)

1 = Detectable (1-25%) 0 = Absent

58

Figure 3. Mean of Zone of Inhibition of E.coli, S. aureus and C. albicans treated by

the extract (3mL) from Stonefish Dorsal Fin Venom.

0

0.5

1

1.5

2

2.5

3

3.5

Zo

ne

of

Inh

ibit

ion

of

Bac

teri

a in

D

iam

eter

s (m

m)

E.coli

S.aureus

C.albicans

The figure shows the Mean of Zone of inhibition of E.coli is 3.5 mm, the

Mean Zone of Inhibition of S. aureus is 3.4mm while the Mean Zone of inhibition of C.

albicans is 2.8mm.This proves that the Stonefish Dorsal Fin Venom inhibit the growth of

E.coli the most compared to others.

59

Figure 4. Mean of Zone of Inhibition of E. coli, S. aureus and C. albicans treated

with 3mL of Chloramphenicol syrup.

The figure shows the Mean Zone of Inhibition of E.coli, S.aureus and C.

albicans is 3.2 mm when treated with the control antibiotic.

60

Figure 5. Comparison of Mean of Zone of Inhibition the Test Microorganisms when

treated by the Stonefish Dorsal Fin venom and to 3 mL of Chloramphenicol syrup.

0

0.5

1

1.5

2

2.5

3

3.5Zo

ne o

f Inh

ibiti

on o

f B

acte

ria

in D

iam

eter

s

E.coli

S.aureus

C.albicans

0

0.5

1

1.5

2

2.5

3

3.5

4

Co

mp

aris

on

of

Zo

ne

of

Inh

ibit

ion

of

Bac

teri

a in

Dia

met

ers

(mm

)

3mL ofChloramphenicol syrup

3mL ofStonefishDorsal Finvenom

The figure shows the Mean Zone of inhibition of the Test Microorganisms

when treated with the controlled antibiotic is 3.2mm. While the Mean Zone of inhibition

of the Test Microorganisms when treated with the Stonefish Dorsal Fin Venom varies.

The Mean zone of Inhibition of C.albicans treated with the Stonefish Dorsal Fin Venom

is lesser than the Mean Zone of Inhibition of C. albicans treated with the controlled

antibiotic. Likewise, Stonefish Dorsal fin Venom did not inhibit the growth of C.

albicans.

61

Chapter IVCONCLUSIONS AND RECOMMENDATIONS

Conclusions

Based from the findings, the following conclusions were drawn:

d) The Stonefish Dorsal Fin Venom can inhibit the growth of harmful

microorganisms.

e) Phytochemical analysis showed the presence of active principles from the

Stonefish Dorsal fin venom. Alkaloids, Tannins and Saponins were observed very

abundant.

f) There is no significant difference of using the Antibiotic Property of Stonefish

Dorsal Fin and the Controlled antibiotic in terms of zone of inhibition.

62

Recommendations

Based from the findings and conclusions the following are recommended:

1. This can be a basis for the Pharmaceutical industries in making an antibiotic

out of the Stonefish Dorsal Fin Venom.

2. Additional Brand of antibiotics shall be made available for comparing the

effect of Stonefish Dorsal Fin Venom to test further efficacy.

3. Wide dissemination of the latest technology must commence.

63

BIBLIOGRAPHY

Gwee, M.C., Gopalakrishnakone, P., Yuem,R.,Khoo,H.E.,Low,K.S.Y.,A review of

stonefish venoms and toxins.Pharmac.Ther.64, 509(1994).

Internet websites, Yahoo.com; Google .com; and MSN Network.

Microsoft Encarta Library Edition 2006.

Sutherland,S.K.,Tibballs,J.,Australian animal toxins. Oxford Univ.Press, Melbourne

(2001).

Phillips KM et al., J Food Comp Anal 2002, 15, 123

Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051

Waterham HR et al., Am J Hum Genet 2001, 69, 685

Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654

Paoletti R et al., J Am Oil Chem Soc 1965, 42, 400

Wikipedia, free Dictionary

64

Appendices

I. Statistical test for the zone of inhibition of Escherichia coli treated with

Chloramphenicol (T0) and with the Stonefish Dorsal Fin Venom (T1).

A B

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 3.2 10.24 3.5 12.25

2 3.2 10.24 3.5 12.25

3 3.2 10.24 3.4 11.56

Total 9.6 30.72 10.4 36.06

N1=3 N2=3

H0: There is no significant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

Ha: There is a significant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

65

= 0.05

Df = N1 + N2- 2 Tcrit.=- 2.776

= 6-2

= 4

Compute for t

A= 9.6 /3= 3.2 ; B = 10.4/ 3 = 3.5

S 1 - 2 =

=

=

=

66

=

= 0.0408

t cal =

= 3.2 – 3.5 / 0.0408

= -7.353

Decision: Accept H0

The tcrit is greater than the t cal so the Null Hypothesis was accepted. Thus, there is no

significant difference of using the Antibiotic Property of Stonefish Dorsal Fin and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

67

II. Statistical test for the zone of inhibition of Staphylococcus aureus treated with

the commercial antibiotic Chloramphenicol (To) and with the Stonefish Dorsal Fin

venom ( T1).

C D

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 3.2 10.24 3.4 11.56

2 3.2 10.24 3.4 11.56

3 3.2 10.24 3.3 10.89

Total 9.6 30.72 10.1 34.01

N1=3 N2=3

H0: There is no significant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

Ha: There is a significant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

68

= 0.05

Df = N1 + N2- 2 Tcrit.= -2.776

= 6-2

= 4

Compute for t

C= 9.6/ 3 = 3.2 ; D = 10.1/ 3 = 3.37

S 1 - 2 =

=

=

= =

69

=

= 0.0408

t cal = t =

= 3.2 – 3.37 / 0.0408

= -4.1667

Decision: Accept H0

The tcrit is greater than the t cal so the null Hypothesis was accepted. Thus, there is no

significant difference of using the Antibiotic Property of Stonefish Dorsal Fin and the

commercial antibiotic Chloramphenicol in terms of zone of inhibition.

70

III. Statistical test for the zone of inhibition of Candida albicans treated with the

commercial antibiotic Chloramphenicol (To) and with the Stonefish Dorsal Fin

venom ( T1).

E F

TRIALS T0

(x)

T0

(x2)

T1

(y)

T1

(y2)

1 3.2 10.24 2.8 7.84

2 3.2 10.24 2.8 7.84

3 3.2 10.24 2.7 7.29

Total 9.6 30.72 8.3 22.97

N1=3 N2=3

H0: There is no significant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

Ha: There is asignificant difference of using the Antibiotic Property of Stonefish Dorsal

Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.

71

= 0.05

Df = N1 + N2- 2 Tcrit.= 2.776

= 6-2

= 4

Compute for t

E= 9.6/ 3 = 3.2 ; F = 8.3/ 3 = 2.8

S 1 - 2 =

=

=

=

72

=

= 0.0408

t cal = t =

= 3.2 – 2.8/0.0408

=9.8039

Decision: Reject H0

The t cal is greater than the tcrit so the Null Hypothesis was rejected. Thus, there is a

significant difference of using the Antibiotic Property of Stonefish and the commercial

antibiotic Chloramphenicol in terms of zone of inhibition.

73

PLATES

Plate 1. The Stonefish Habitat.

74

Plate 2. The Animal Specimen – Stonefish ( Synaceia verrucosa).

75

Plate 3. Materials for the Extraction and Dissection of Stonefish.

76

Plate 4. Researcher Dissecting the Specimen.

77

Plate 5. Researcher Inserting the Knife Above the Dorsal Fin.

78

Plate 6.Reseacher Pinning down the tail end of the Dorsal Fin using a Sterile Knife.

79

Plate 7. Researcher Grasping the Tail Fin and Pulling it way from the Dorsal Fin.

80

Plate 8. Researcher Extracting the Venom using a Sterile Syringe.

81

Plate 9: The Extracted Venom.

82

Plate 10. Materials used for the Antimicrobial Assay Test

83

Plate 11.Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient

Agar and Glucose Yeast Peptone Agar.

84

Plate 12. Researcher Pouring the Medium onto the Agar Plates.

85

Plate 13.Reseacher Swirling Agar Plates to distribute the inoculums evenly in the Agar

Surface.

86

Plate 14.Reseacher making holes on the Agar Plates using the Cork Borer.

87

Plate 15. The bored Agar Plate Surface.

88

Plate 16.Researcher Placing the Extracts in each hole.

89

Plate 17.Researching Incubating the Plates at room temperature.

90

Plate 18. The treatments To (Chloramphenicol) and T1 ( Stonefish Dorsal Fin Venom).

91

CURRICULUM VITAE

Name: Lyra Erika Liclican

Age: 16years old

Address: Paratong, Sta.Cruz, Ilocos Sur

Birthday: September 1, 1990

Parents: Mr. Raul Liclican

Mrs. Lydia Liclican

Religion: Roman Catholic

Nationality: Filipino

Civil Status: Single

Educational Attainment:

Elementary

St. Joseph Institute

Candon City, Ilocos Sur

St. Augustine’s School

Tagudin, Ilocos Sur

Secondary

Regional Science High School for Region 1

Bangar, La Union

DATA BOOK

SUCCESS CALENDAR

1. CHOOSING Planned Date Date Completed

A TOPIC May 3,2006 May 10,2006

This is the hardest thing to do when doing an Investigatory Project. A narrowed

down topic that would make sense and can be attained and give benefit to the majority.

And fortunately my title, “ Antibiotic Property of Stonefish (Synanceia verrucosa) Dorsal

Fin Venom against E,coli, C. albicans and S.aureus.

2 COLLECTING Planned Date Date Completed

BACKGROUND May 11, 2006 May 15, 2006

INFORMATION

Collection of information regarding my study is not that easy. For two days, I went

to Department of Science and Technology, San Fernando City and Don Mariano Marcos

Memorial State University, Bacnotan La Union to research essential information from

books and manuscripts. Last May 13- 15, my adviser and I went to Manila. We went to

Adamson University, DOST Main, Natural Science Research Institute (NSRI) and

Marine Science Institute (MSI) of University of the Philippines to gather facts about my

study.

3. EXPERIMENTATION Planned Date Date Completed

AND GATHERING May 17, 2006 June 2, 2006

OF DATA

With the suggestions of Dr. Lourdes Cruz of MSI at UPD, I design for the

procedures to follow in dissecting and extracting the Stonefish Dorsal Fin Venom. Before

going again go to UPD, Stonefish were collected at Paraoir, Balaon, La Union and were

placed in an ice box. Dr. Cruz of MSI allowed me to conduct the dissection and

extraction of my specimen with hier supervision. The extracted venom was tested at

NSRI for Antimicrobial assay with Mrs.Vina Argayosa.

Then, soxhlet method was used for the extraction of my specimen for the

Phytochemical Test at Adamson University.

4 MAKING OF THE WRITE Planned Date Date Completed

UP (MANUSCRIPT) - July 28,2006 August 2, 2006

Background of the Study

5. MAKING THE STATEMENT Planned Date Date Completed

OF THE PROBLEM, August 4, 2006 August 14, 2006

HYPOTHESES, SIGNIFICANCE

OF THE STUDY UNTIL SCOPE

AND DELIMITATION

6.MAKING THE REVIEW OF Date Planned Date Completed

RELATED LITERATURE August 17,2006 August 26,2006

7. MAKING THE CHAPTER II Date Planned Date Completed

( Methodology) August 28,2006 September 12, 2006

8. MAKING THE CHAPTER III Date Planned Date Completed

(Results and discussions) September 13,2006 Sept. 17, 2006

9. MAKING THE CHAPTER IV Date Planned Date Completed

(Conclusions and Sept. 18, 2006 Sept. 20,2006

Recommendations)

10. MAKING THE Date Planned Date Completed

BIBLIOGRAPHY, Sept.22, 2006 Sept. 24, 2006

APPENDICES

10. FINALIZATION Date Planned Date Completed

Sept. 26, 2006 Sept. 28, 2006

11. PRINTING OF Date Planned Date Completed

THE MANUSCRIPT Sept. 29, 2006 Sept 30.2006

Table of Contents

Abstract…………………………………………………………………….…..…..i

CHAPTER 1

Introduction………………………………………………………….…….1

Background of the Study………………………………………….....……1-2

Statement of the Problem…………………………………………….........2

Hypotheses……………………………………………………………...…3

Significance of the Study………………………………………………….3-4

Scope and delimitation…………………………………………………….4

Review of related Literature

Description of the Animal Specimen……………………………..5-12

Phytochemical Components……………………………………..12-28

Test Microorganisms………………………………….…………..28-34

Materials used……………………………………………….…....34-39

Definition of terms………………………………………………..40-41

CHAPTER II

Methodology

Experimental Design……………………………………………….42

Materials…………………………………………………………....43-44

Reagents…………………………………………………………...45

Collection of Animal Specimen……………………………………45

Preparation of the Extract……………………………………….…45-46

Preparation of Test Organisms……………………………………..47

Antimicrobial Assay Test………………………………………….47

Phytochemical Test………………………………………………...48-51

CHAPTER III

RESULTS AND FINDINGS………………………………………………54-61

CHAPTER IV

COCLUSIONS AND RECOMMENDATIONS

Conclusions………………………………………………………...62

Recommendations……………………………………………..…...63

Bibliography…………………………………………………………………….…64

Appendices………………………………………………………………………....65-73

Table of Contents

Abstract…………………………………………………………………………………

…………………………………………………………………………………….i

CHAPTER 1

Introduction……………………………………………………………………………

…………………………………………………………………….1

Background of the

Study………………………………………………………………………………………

…………………………….……….1-2

Statement of the

Problem…………………………………………………………………………………

……………………………….……….2

Hypotheses………………………………………………………………………………

…………………………………………………………………..3

Significance of the

Study………………………………………………………………………………………

…………………………………….3

Scope and

delimitation………………………………………………………………………………

………………………………………………..4

Review of related

Literature…………………………………………………………………………………

…………………………………...5

Description of the Animal

Specimen…………………………………………………………………………………

………6-8

Phytochemical

Components……………………………………………………………………………

…………………………8-25

Test

Microorganisms…………………………………………………………………………

……………………………………...25-33

Definition of

Terms……………………………………………………………………………………

………………………………34-35

CHAPTER II

Methodology

Experimental

Design……………………………………………………………………………………

…………………………….36

Materials…………………………………………………………………………………

………………………………………………...37-38

Reagents…………………………………………………………………………………

………………………………………………….38

Collection of Animal

Specimen…………………………………………………………………………………

………………39

Preparation of the

Extract……………………………………………………………………………………

………………….39

Preparation of the Test

Organisms………………………………………………………………………………

….….39

Antimicrobial Assay

Test………………………………………………………………………………………

……………..40

Histochemical

Test………………………………………………………………………………………

……………………...41-45

CHAPTER III

RESULTS AND

FINDINGS…………………………………………………………………………………

…………………………………...47-55

CHAPTER IV

COCLUSIONS AND RECOMMENDATIONS

Conclusions………………………………………………………………………………

……………………………………56

Recommendations……………………………………………………………………

……………………………………57

Bibliography……………………………………………………………………………

…………………………………………………………………….58

Appendices………………………………………………………………………………

……………………………………………………………………59-70

Plate 1. Researchers Collecting the Plant Specimen.

Plate 2. The Plant Specimen – Nut Grass (Cyperus rotundus).

Plate 3. The Nut Grass Rhizome.

Plate 4. Materials for the Reflux Method (Extraction)

Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method.

Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper.

Plate 7. Materials for the Antimicrobial Test.

Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to

Nutrient Agar and Glucose Yeast Peptone Agar

Plate 9. Researcher Pouring the Medium onto the Agar Plates.

Plate 10. Researcher Swirling Agar Plates to distribute the inoculums evenly in the

Agar Surface.

Plate 11. Researcher making holes on the Agar Plates using the Cork Borer.

Researcher Placing the Extracts in each hole

Plate 13. Researching Incubating the Plates at room temperature

Plate 14.The treatments – To ( Chloramphenicol) and T1 ( Nut Grass rhizome

extract).

List of Plates

Plate 1. Researchers Collecting

the Plant

Specimen…………………………………………………………………………………

………………………………………………..71

Plate 2. The Plant Specimen – Nut Grass

(Cyperus rotundus)

………………………………………………………………………............................

.......................................72

Plate 3. The Nut Grass

Rhizome…………………………………………………………………………………

…………………….…….73

Plate 4. Materials for the Reflux Method

(Extraction)

………………………………………………………………………………………………

………………………………………………74

Plate 5. Researchers extracting the Nut Grass

Rhizome using the Reflux

Method……………………………………………………………………………………

…………………….75

Plate 6.Resechers Filtering the extracted Nut Grass

Rhizome using the Filter

paper…………………………………………………….........................................

........................76

Plate 7. Materials for the Antimicrobial

Test………………………………………………………………………………………..

77

Plate 8. .Researcher transferring Aliquots of

Bacterial and Yeast Suspension to Nutrient Agar

and Glucose Yeast Peptone

Agar………………………………………………………………………………………

…………………………78

Plate 9. Researcher Pouring the Medium onto the

Agar

Plates……………………………………………………………………………………

………………………………………………………………79

Plate 10. Researcher Swirling Agar Plates to

distribute the inoculums evenly in the Agar

Surface………………………………………………………………………………..80

Plate 11. Researcher making holes on the Agar Plates

using the Cork

Borer………………………………………………………………………………………

…………………………………………..81

Plate 12.Researcher Placing the Extracts in each

hole…………………………………………………………………………….82

Plate 13. Researching Incubating the Plates at

room

temperature……………………………………………………………………………

………………………………………………………...83

Plate 14.The treatments – To ( Chloramphenicol) and

T1 (Nut Grass rhizome extract)

………………………………………………………………………………………………

………………….84

List of Tables

Table 1. Mean Zone of Inhibition

Against E. coli in millimeters (mm).

………………………………………………………………………………………………

……………………….47

Table 2. Mean Zone of Inhibition

Against S. aureus in millimeters (mm).

………………………………………………………………………………………………

………………..48

Table3. Mean Zone of Inhibition

Against C.albicans in millimeters (mm)

………………………………………………………………………………………………

……………….49

Table 4. Mean Zone of Inhibition

Against A. niger in millimeters (mm).

………………………………………………………………………………………………

………………..50

Table 5. Mean Zone of Inhibition

Test Microorganisms in millimeters (mm)

………………………………………………………………………………………………

………51

Table 6 A Summary table of the Active Principles

of the Nut Grass

rhizome…………………………………………………………………………………

……………………………………………52

List of Figures

Figure 1. The Flowchart of Experimental

Design……………………………………………………………………………………

…………46

Figure 2. Mean of Zone of Inhibition of E.coli, S. aureus,

C. albicans and A. niger treated by the extract (5mL)

from the Nut Grass

rhizome…………………………………………………………………………………

………………………………………….53

Figure 3. Mean of the Zone of Inhibition of E. coli,

S. aureus C. albicans and A. niger treated with 5mL

of Chloramphenicol

syrup………………………………………………………………………………………

…………………………………………54

Figure 4. Comparison of the Mean on the Zone of Inhibition o

f Test Microorganisms when treated by the Nut Grass rhizome

extract and to 5mL of Chloramphenicol

syrup………………………………………………………………………………………

…....…55