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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
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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.
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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
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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……………………………
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Preparation of the Extract…………………………………
Antimicrobial Assay Test…………………………………
Histochemical Test………………………………………….
Flowchart…………………………………………………….
CHAPTER III
RESULTS AND FINDINGS………………………………………
CHAPTER IV
COCLUSIONS AND RECOMMENDATIONS……………………
Conclusions………………………………………………….
Recommendations…………………………………………..
Bibliography…………………………………………………………………
Appendices…………………………………………………………………..
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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……………………………………………
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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)…………………………………………………………
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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…………………………………………………………..
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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………………………………………………………………
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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………………………………………………………………….
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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
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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?
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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
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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
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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
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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
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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.
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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
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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.
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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
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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.
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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
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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
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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
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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.
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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
Page 32
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.
Page 33
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
Page 34
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
Page 35
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
Page 36
(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
Page 37
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
Page 38
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
Page 39
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
Page 40
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
Page 41
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
Page 42
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
Page 45
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
Page 46
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
Page 47
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
Page 48
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
Page 49
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
Page 50
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
Page 51
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
Page 52
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.
Page 53
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.
Page 54
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.
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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.
Page 56
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
Page 57
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
Page 58
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).
Page 59
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).
Page 60
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).
Page 61
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.
Page 62
50
Table 5. Mean Zone of Inhibition Test Microorganisms in millimeters (mm).
Page 63
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
Page 64
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.
Page 65
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.
Page 66
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.
Page 67
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
Page 68
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:
Page 69
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.
Page 70
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
Page 71
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
Page 72
= 4
Compute for t
A= 9.6/ 3 = 3.2 ; B = 10.9/ 3 = 3.63
S 1 - 2 =
=
=
60
=
=
Page 73
= 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).
Page 74
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
Page 75
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
Page 76
=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).
Page 77
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
Page 78
= 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
Page 79
=-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).
Page 80
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
Page 81
= 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
Page 82
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
Page 83
Plate 1. Researchers Collecting the Plant Specimen.
71
Page 84
Plate 2. The Plant Specimen – Nut Grass (Cyperus rotundus).
72
Page 85
Plate 3. The Nut Grass Rhizome.
73
Page 86
Plate 4. Materials for the Reflux Method (Extraction)
74
Page 87
Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method.
75
Page 88
Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper.
76
Page 89
Plate 7. Materials for the Antimicrobial Test.
77
Page 90
Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to
Nutrient Agar and Glucose Yeast Peptone Agar.
78
Page 91
Plate 9. Researcher Pouring the Medium onto the Agar Plates.
79
Page 92
Plate 10. Researcher Swirling Agar Plates to distribute the inoculums evenly in the
Agar Surface.
80
Page 93
Plate 11. Researcher making holes on the Agar Plates using the Cork Borer.
81
Page 94
Plate 12. Researcher Placing the Extracts in each hole.
82
Page 95
Plate 13. Researching Incubating the Plates at room temperature
83
Page 96
Plate 14.The treatments – To ( Chloramphenicol) and T1 ( Nut Grass rhizome
extract).
84
Page 97
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
Page 98
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
Page 99
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
Page 100
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
Page 101
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
Page 102
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
Page 103
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…………………………………………………….
Page 104
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 ……………………………………………………………
Page 105
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
Page 106
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
Page 107
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
Page 108
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
Page 109
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
Page 110
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.
Page 111
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.
Page 112
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?
Page 113
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.
Page 114
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
Page 115
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
Page 116
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.
Page 117
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
Page 118
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.
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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.
Page 120
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.
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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
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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.
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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
Page 124
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
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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.
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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.
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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.
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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.
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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
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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
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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
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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
21
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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
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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
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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).
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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.
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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).
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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.
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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
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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
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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
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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.
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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
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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
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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
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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,
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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.
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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.
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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:
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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.
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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.
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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,
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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
Page 154
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)
Page 155
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
Page 156
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
Page 157
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.
Page 158
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:
Page 159
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.
Page 160
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.
Page 161
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
Page 162
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
Page 163
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
Page 164
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.
Page 165
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
Page 166
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.
Page 167
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.
Page 168
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.
Page 169
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
Page 170
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
Page 171
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.
Page 172
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
Page 173
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
Page 174
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
Page 175
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
Page 176
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
Page 177
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
Page 178
= 4
Compute for t
A= 9.6 /3= 3.2 ; B = 10.4/ 3 = 3.5
S 1 - 2 =
=
=
=
66
=
= 0.0408
Page 179
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).
Page 180
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
Page 181
= 6-2
= 4
Compute for t
C= 9.6/ 3 = 3.2 ; D = 10.1/ 3 = 3.37
S 1 - 2 =
=
=
= =
69
=
Page 182
= 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).
Page 183
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
Page 184
= 4
Compute for t
E= 9.6/ 3 = 3.2 ; F = 8.3/ 3 = 2.8
S 1 - 2 =
=
=
=
72
=
= 0.0408
Page 185
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
Page 186
Plate 1. The Stonefish Habitat.
74
Page 187
Plate 2. The Animal Specimen – Stonefish ( Synaceia verrucosa).
75
Page 188
Plate 3. Materials for the Extraction and Dissection of Stonefish.
76
Page 189
Plate 4. Researcher Dissecting the Specimen.
77
Page 190
Plate 5. Researcher Inserting the Knife Above the Dorsal Fin.
78
Page 191
Plate 6.Reseacher Pinning down the tail end of the Dorsal Fin using a Sterile Knife.
79
Page 192
Plate 7. Researcher Grasping the Tail Fin and Pulling it way from the Dorsal Fin.
80
Page 193
Plate 8. Researcher Extracting the Venom using a Sterile Syringe.
81
Page 194
Plate 9: The Extracted Venom.
82
Page 195
Plate 10. Materials used for the Antimicrobial Assay Test
83
Page 196
Plate 11.Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient
Agar and Glucose Yeast Peptone Agar.
84
Page 197
Plate 12. Researcher Pouring the Medium onto the Agar Plates.
85
Page 198
Plate 13.Reseacher Swirling Agar Plates to distribute the inoculums evenly in the Agar
Surface.
86
Page 199
Plate 14.Reseacher making holes on the Agar Plates using the Cork Borer.
87
Page 200
Plate 15. The bored Agar Plate Surface.
88
Page 201
Plate 16.Researcher Placing the Extracts in each hole.
89
Page 202
Plate 17.Researching Incubating the Plates at room temperature.
90
Page 203
Plate 18. The treatments To (Chloramphenicol) and T1 ( Stonefish Dorsal Fin Venom).
Page 204
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:
Page 205
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.
Page 206
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.
Page 207
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
Page 208
(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
Page 209
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
Page 210
Appendices………………………………………………………………………....65-73
Page 211
Table of Contents
Abstract…………………………………………………………………………………
…………………………………………………………………………………….i
CHAPTER 1
Introduction……………………………………………………………………………
…………………………………………………………………….1
Page 212
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
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Phytochemical
Components……………………………………………………………………………
…………………………8-25
Test
Microorganisms…………………………………………………………………………
……………………………………...25-33
Definition of
Terms……………………………………………………………………………………
………………………………34-35
CHAPTER II
Methodology
Experimental
Design……………………………………………………………………………………
…………………………….36
Materials…………………………………………………………………………………
………………………………………………...37-38
Reagents…………………………………………………………………………………
………………………………………………….38
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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
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CHAPTER IV
COCLUSIONS AND RECOMMENDATIONS
Conclusions………………………………………………………………………………
……………………………………56
Recommendations……………………………………………………………………
……………………………………57
Bibliography……………………………………………………………………………
…………………………………………………………………….58
Appendices………………………………………………………………………………
……………………………………………………………………59-70
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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
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Plate 14.The treatments – To ( Chloramphenicol) and T1 ( Nut Grass rhizome
extract).
List of Plates
Page 218
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
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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
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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
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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
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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
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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