Nut Grass (Lyra Erika Liclican)

Department of Education Region 1 Division of La Union Regional 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 Fair Regional Level

Candon National High School Candon City, Ilocos Sur November 28-29, 2006
Proponents
Lyra Erika Liclican Glen Bernard Asto
Mr. Rogelio Valdez Research Adviser
Acknowledgement
Embarking on with this Investigatory Project was a long and onerous yet challenging and rewarding task. But with the help and contributions of many persons and entities, our study was realized.
To our Almighty God, for helping us hurdle all the obstacles in undertaking this study.
To Dr. Amerfina Nelmida, School Principal for her guidance and valuable suggestions.
To Mr. Rogelio Valdez, our adviser, who had endowed valuable time and commitment in editing this piece of work. We could never repay him rational judgment in giving profound ideas and suggestions for the betterment of this study.
To the judges for their favorable criticisms and inestimable suggestions in the enrichment of this study.
To the Department of Science and Technology for their technical assistance in the conduct of the Antimcrobial assay and Phytochemical Test of our study.
To Mrs.Ma. Luisa Allada for assisting us in the reflux method in the extraction of our specimen.
Mrs. Marie Munar and Miss Herma Dingle for providing the necessary materials needed; Mrs. Aurelia Garcia for lending us books; to Mrs. Edwina Manalang for assisting us in the statistical tools involved; to Mr. Carlo Sojo for his patience in helping us in collecting the Nut Grass ( Barsanga); to Mrs. Elma Gacutan and Mr. Kyle Mupas for lending us their laptop and USB respectively. To our parents, who gave us the chance to dive into the wide sea of education to fish for knowledge. And to the persons who, in one way or another had helped and inspired us .
We thank you all
Lyra Erika Liclican Glen Bernard Asto
Abstract
This study aimed to determine the Antimicrobial Property of Nut Grass (Cyperus rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp.and it was conducted on May to September 2005 at the Department of Science and Technology Taguig City, Metro Manila.. Four hundred (400)g of Nut Grass rhizome was subjected to experimentation. Antimicrobial assay test were subjected using the control Chloramphenicol and the experimental variable Nut Grass rhizome extract. Phytochemical Test was done to determine the active principles present. It showed that Saponins and Glycosides were abundant in all tissues of the rhizome while Triterpenes and Tannins were detectable only. There were two treatments used namely T0: the controlled antibioticChloramphenicol 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.
Table of Contents
Approval Sheet.. Acknowledgement. Abstract. CHAPTER 1.. Introduction Background of the Study.. Statement of the Problem. Hypotheses Significance of the Study.. Scope and delimitation.. Review of related Literature.. Description of the Animal Specimen Histochemical Components.. Test Microorganisms CHAPTER II Methodology Experimental Design. Materials.. Reagents. Collection of Animal Specimen Preparation of the Extract
Antimicrobial Assay Test Histochemical Test. Flowchart. CHAPTER III RESULTS AND FINDINGS
CHAPTER IV COCLUSIONS AND RECOMMENDATIONS Conclusions. Recommendations.. Bibliography Appendices..
List of Plates
Plate 1. Collecting the plant specimen
Plate 2. The plant specimen Nut Grass (Cyperus rotundus)
Plate 3. The Nut Grass Rhizome
Plate 4. Materials used for the Reflux Method (Extraction)
Plate 5. Researcher extracting the Nut Grass rhizome using the Reflux Method.
Plate 6.Filtering the extracted Nut Grass rhizome using the filter paper
Plate 7. Materials for the Antimicrobial Test
Plate 8. Aliquots of the bacterial and yeast suspensions were transferred into pre-poured Nutrient Agar (NA) and Glucose Yeast Peptone (GYP) Agar, respectively..
Plate 9. The medium, melted and cooled to 450C, was poured onto the agar plates.
Plate 10. Swirling the agar plate to distribute the inoculums evenly in the agar surface..
Plate 11. Make three (3) holes using a cork borer
Plate 12. The extracts were placed in each hole..
Plate 13.Incubating the agar plates at room temperature.
Plate 14.The treatments To ( Chloramphenicol) and T1 ( Nut Grass rhizome extract)
List of Figures
Figure 1.The Flowchart of the Experimental Design
Figure 2. Mean of the Zone of Inhibition of E.coli, S. aureus, C. albicans and A. niger treated by the extract (5mL) from the Nut Grass rhizome
Figure 3. Mean of the Zone of Inhibition of E. coli, S. aureus C. albicans and A. niger treated with 5mL of Chloramphenicol syrup
Figure 4. Comparison of the Mean on the Zone of Inhibition of E. coli, S. aureus, C. albicans and A. niger when treated by the Nut Grass rhizome extract and to 5mL of Chloramphenicol syrup..
List of Tables
Table 1. The inhibitory effect of Escherichia coli growth when treated with 5mL of Chloramphenicol (commercial antibiotic) and the extract (5mL) from the Nut Grass rhizome..
Table 2.The Inhibitory effect on the Staphyloccoccus aureus growth when treated with 5mL of Chloramphenicol (commercial antibiotic) and the extract (5 mL) from the Nut Grass rhizome
Table 3.The Inhibitory effect on the Candida albicans growth when treated with 5mL of Chloramphenicol (commercial antibiotic) and the extract (5 mL) from the Nut Grass rhizome
Table 4.The Inhibitory effect on the Aspergillus niger growth when treated with 5mL of Chloramphenicol (commercial antibiotic) and the extract (5 mL) from the Nut Grass rhizome.
Table 5.Summary table of the Inhibitory effect on the E. coli, S. aureus, C. albicans and A. niger growth when treated with 5mL of Chloramphenicol (commercial antibiotic) and the extract (5mL) from the Nut Grass rhizome..
Table 6 A Summary table of the Active Principles of the Nut Grass rhizome.
Chapter I INTRODUCTION
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 worlds worst weed as it is known as s pest in over 90 countries, and infests over 50 crops worldwide. Its existence in a field significantly reduces crop yield, both because it is a tough competitor for ground resources, and because its dead subterranean tissue releases substances harmful to other plants. Similarly, it also has a bad effect on ornamental gardening.
Philippines, being a tropical country, provide a name for countless species of plants and animals, from the simplest to the most complex ones. Among these plants is the Nut Grass (Cyperus rotundus) and can be a very good source of antibiotics.
With these information, it provoke the researchers mind that what if Cyperus rotundus has an antibacterial property and then could be used as antibacterial agent to inhibit the growth of harmful bacteria, thus the Antimicrobial Property of Nut Grass (Cyperus rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp. was conceived.
B. Statement of the Problem

This study aimed to determine the Antimicrobial Property of Nut Grass (Cyperus rotundus) against Escherichia coli, Staphylococcus aureus and Salmonella spp.
Specifically, this study sought to answer the following sub problems:
1) Can the Nut Grass rhizome extract inhibit the growth of harmful microorganisms? 2) What are the active components Nut grass rhizome using Phytochemical test? 3) Is there a significant difference of using the Antimicrobial Property of Nut grass rhizome and the commercial antibiotic in terms of zone of inhibition?
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.
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.
F. Review of Related Literature
English Name: Nut Grass Common Name: Barsanga Other name: Curry Flower seed Scientific Name: Cyperus rotundus Kingdom: Plantae Division: Magnoliophyta Class: Liliopsida Order: Poales Family: Cyperaceae Genus: Cyperus
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.
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 threeangled achene. A young plant root system initially forms white, fleshy rhizomes. Some rhizomes grow upward in the soil, and then form a bulb-like structure from which new shoots and roots grow, and from the new roots new rhizomes grow. Other rhizomes grow horizontally or downward, and form dark reddish-brown tubers or tuber-chains. Cyperus rotundus is one of the worst weeds mankind knows. Its existence in a field significantly reduces crop yield, both because it is a tough competitor for ground resources, and because its dead subterranean tissue releases substances harmful to other plants. Similarly, it also has a bad effect on ornamental gardening. The difficulty to control it is a result of its intensive system of underground tubers, and its resistance to most herbicides. It is also one of the few weeds that cannot be stopped with plastic mulch.
7 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 plants leaves, but most have no effect on the root system and the tubers. In addition, the tubers can survive harsh conditions, further contributing to the difficulty to eradicate the plant.
Phytochemical Components Phytochemical studies on animals give basic information on the presence and localization of the active constituents in the plants tissues. These tests include the test for the presence of alkaloids, saponins, tannins, glycosides and sterols, flavonoids, formic acid, tartaric acid, fats and oils and tipertenes.
Alkaloids Alkaloids are new synthetic agents of greater potency and lesser toxicity. It is a naturally occurring amine produced by a plant but amines produced by animals and fungi are also called alkaloids. Many alkaloids have pharmacological effects on humans and animals. Alkaloids are usually derivatives of amino acids. They are found as secondary metabolites in plants, animals and fungi, and can be extracted from their sources by treatment with acids.
8 Alkaloids, group of mildly alkaline compounds, mostly of plant origin and of moderate molecular complexity. Even in very small amounts, the alkaloids produce strong physiological effects on the body. All contain nitrogen atoms that are structurally related to those of ammonia.
Nearly 3000 alkaloids have been recorded; the first to be prepared synthetically (1886) was one of the simplest, called coniine, or 2-propyl piperidine, C5H10NC3H7. It is highly poisonous; less than 0.2 g (0.007 oz) is fatal. Coniine, obtained from seeds of the hemlock, was the poison used in the execution of Socrates. Some 30 of the known alkaloids are used in medicine. For example, atropine, obtained from belladonna, causes dilation of the pupils; morphine is a painkiller; quinine is a specific remedy for malaria; nicotine is a potent insecticide; and reserpine is a valuable tranquilizer.
Saponins
Saponins are subgroups of glycosides that have the characteristic ability to cause foaming when shaken with water. They are sometimes used as emulsifying agents. Saponins are believed to be useful in the human diet for controlling cholesterol, but some are poisonous if swallowed and can cause urticaria. Any markedly toxic saponin is known as a sapotoxin.
9 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 warmblooded animals. When injected directly into the bloodstream, however, they are dangerous and quickly dissolve red blood cells. Hydrolysis of a saponin, brought about by acids or by enzymes, gives a sugar (often, but not necessarily, glucose) and a sapogenin, the latter being either a triterpene or a steroid. Some of the sugars and saponins are useful as raw materials for synthesis of steroid hormones.
Glycosides Glycosides, class of complex chemical compounds in plants. They are broken down by plant enzymes into sugars, among which glucose is generally included, and into other substances. The term glucoside is often used synonymously with glycoside, but in its more specific meaning it refers to glycosides that yield glucose.
Each glycoside in a plant is hydrolyzed (converted in a reaction with water) by an enzyme, usually a specific enzyme found in the same plant. The enzyme emulsin, however, causes hydrolysis of several glycosides. The enzymes and glycosides are stored in separate plant cells until the reaction products of the glycosides are needed and the
enzymes are activated. 10 Glycosides are believed to serve several purposes in the plant. Glycosides are bitter tasting, and it is believed that they help keep birds and insects from eating seeds and fruit before they are fully grown, by which time the glycosides have been converted to sweet sugars. When a plant tissue is bruised, the enzymes hydrolyze the glycosides into products, such as phenol compounds and acids that have an antiseptic action and prevent decay of the damaged tissues.
Glycosides are soluble in water and are obtained from plants by water extraction. They are mostly colorless crystalline solids with a bitter taste. Simple glycosides have been synthesized in the laboratory, and several hundred glycosides have been extracted from plants and used for many purposes. Among the important glycosides are indican, used for dyeing; digitalin, used in medicine; and the saponins, foaming agents used industrially and medicinally. Flavonoid
The term flavonoid refers to a class of plant secondary metabolites based around a phenylbenzopyrone structure. Flavonoids are most commonly known for their 11 antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the media these terms are equivalent and interchangeable, since all flavonoids are biological in origin. The flavonoid synthetic pathway begins with a product of glycolysis, phosphoenolpyruvate, entering into the Shikimate pathway to yield phenylalanine. Phenylalanine is the starting material of the phenylpropanoid metabolic pathway, from which 4-Coumaryl-CoA is produced. This can be combined with Malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones. Ring-closure of these compounds results in the familiar form of flavonoids, a three-ringed phenolic structure (polyphenols). The metabolic pathway continues through a series of enzymatic modifications to yield flavanones dihydroflavonols anthocyanins. Along this pathway many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other polyphenolics. Flavonoids are widely distributed in plants fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet.
Flavonoids have been found in high concentrations in butterflies and moths sequestered from dietary intake at the larval stage and then stored in adult tissues. 12 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, antiinflammatory
[1]
, anti-microbial and anti-cancer activity. In addition, flavonoids act as
powerful antioxidants, protecting against oxidative and free radical damage. Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins Tannins
These are also tannin acid, common name applied to a group of vegetable products, both amorphous and crystalline, obtained from various plants, and important commercially in the tanning of leather. Tannins have variable composition. Some, called condensed tannins, are phenols of moderately complex structure, and others are esters of glucose or some other sugar with one or more trihydroxybenzoic acids. The empirical formula, C14H14O11, often given for common tannin, is only an average. Tannins occur in
many trees, and the best sources include oak galls and the bark of sumac. Extraction with water, or water and alcohol, is the first step in the preparation of tannin. Settling, followed by evaporation at a low temperature, yields the commercial product. 13 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 210C, (410 F) causes decomposition, accompanied by formation of pyrogallol and carbon dioxide. The chemical property that provides the basis for most uses of tannins is its ready formation of precipitates with albumin, with gelatin, and with many alkaloidal and metallic salts. The ability of tannins to transform proteins into insoluble products resistant to decomposition leads to their use as tanning agents. Ferric salts react with tannins to give bluish-black products that are useful as inks. Tannins are used as mordants for dyeing cloth, as sizes for paper or silk, and as coagulants for rubber. The precipitating properties of tannins are used in clarifying, or cleaning, wines and beer. Tannic acid is valuable as an external medicine because it is astringent and styptic.
Fats and Oils They are group of naturally occurring organic compounds called triglycerides esters comprised of three molecules of fatty acids and one molecule of the alcohol gylcerol. They are oily, greasy, or waxy substances that, in their pure state, are normally tasteless, colorless, and odorless. Fats and oils are lighter than water and are insoluble in it; they are slightly soluble in alcohol and are readily dissolved in ether and other organic
solvents. Fats are soft and greasy at ordinary temperatures, whereas fixed oilsas distinct from essential oils and petroleumare liquid. Some waxes, which are hard solids at ordinary temperatures, are chemically similar to fats 14 Formic Acid They are the simplest of the organic acids, with the chemical formula HCOOH. A colorless liquid with an irritating odor, it boils at 100.7 C (213 F) and freezes at 8.4 C (47.1 F). It is prepared commercially by reacting sodium hydroxide and carbon monoxide at high pressure and temperature. Formic acid is widely used in the chemical industries and in dyeing and tanning. In nature, formic acid occurs in the poisons of stinging ants and other insects and in stinging nettles.
Tartaric Acid
These are also dihydroxy-succinic acid, organic acid of formula C4H6O6, found in many plants and known to the early Greeks and Romans as tartar, the acid potassium salt derived as a deposit from fermented grape juice. The acid was first isolated in 1769 by the Swedish chemist Carl Wilhelm Scheele, who boiled tartar with chalk and decomposed the product with sulfuric acid. Fermentation of the juices of grapes, tamarinds, pineapples, and mulberries produces, on the inner surface of the container, a white crust of potassium acid tartrate known as argol, or lees. Argol, boiled with dilute hydrochloric acid, precipitates as calcium tartrate when calcium hydroxide is added. Upon addition of dilute sulfuric acid, dextrotartaric acid is liberated, which rotates the
plane of polarized light to the right. Dextrotartaric acid has a melting point of 170 C (338 F) and is extremely soluble in water and alcohol and insoluble in ether.
15 Another variety, called levotartaric acid, is identical to dextrotartaric acid except that it rotates the plane of polarized light to the left. This acid was first prepared from its sodium ammonium salt by the French chemist Louis Pasteur. Tartaric acid synthesized in the laboratory is a mixture of equal amounts of the dextro and levo acids, and this mixture, called also racemic tartaric acid, does not affect the plane of polarized light. A fourth variety, mesotartaric acid, also without effect on the plane of polarized light, is said to be internally compensated. Tartaric acid, in either the dextrorotary or racemic form, is used as a flavoring in foods and beverages. It is used also in photography, in tanning, and as sodium potassium tartrate, also known as Rochelle salt, as a mild laxative. Sterols, or steroid alcohols are a subgroup of steroids with a hydroxyl group in the 3-position of the A-ring. They are amphipathic lipids synthetised from Acetyl coenzyme A. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar. Sterols are important for the physiology of eukaryotic organisms. They form part of the cellular membrane where they modulate their fluidity and function and participate as secondary messengers in developmental signaling.
Different organisms utilize different sterols. The most important ones are cholesterol, phytosterols, and some steroid hormones in animals, and campesterol, sitosterol and stigmasterol in plants. 16 Sterols are also known to block cholesterol absorption sites in the human gut thus helping to reduce cholesterol in humans by up to 15%.
Sterols Sterols may be found either as free sterols, acylated (sterol esters), alkylated (steryl alkyl ethers), sulfated (cholesterol sulfate), or linked to a glycoside moiety (steryl glycosides) which can be itself acylated (acylated sterol glycosides). Free Sterols
Sterols form an important group among the steroids. Unsaturated steroids with most of the skeleton of cholestane containing a 3 hydroxyl group and an aliphatic side chain of 8 or more carbon atoms attached to position 17 form the group of sterols. They are lipids resistant to saponification and are found in an appreciable quantity in all animal and vegetal tissues. These unsaponifiable lipids may include one or more of a variety of molecules belonging to 3-hydroxysteroids, they are C27-C30 crystalline
alcohols (in Greek, stereos, solid). These lipids can be classed as triterpenes as they derive from squalene which gives directly by cyclization, unsaturation and 3hydroxylation, lanosterol in animals or cycloartenol in plants. 17 In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol (Greek, chole, bile), particularly abundant in adrenals (10%, w/w), nervous tissues (2%,w/w), liver (0.2%,w/w) and gall stones, its fundamental carbon structure being a cyclopentanoperhydrophenanthrene ring (also called sterane). It was the first isolated sterol around 1770 by Poulletier de La Salle from gall stones. In 1815, it was isolated from the unsaponifiable fraction of animal fats by M.E. Chevreul who named it cholesterine (Greek, khole, bile and stereos, solid). The correct formula (C27H46O) was proposed in 1888 by F. Reinitzer but structural studies from 1900 to 1932, mainly by H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact steric representation of cholesterol. In 1936, Callow and Young have designated steroids all compounds chemically related to cholesterol.
While it became clear very early that cholesterol plays an important role in controlling cell membrane permeability by reducing average fluidity, it appears now that it has a key role in the lateral organization of membranes and free volume distribution. These two parameters seem to be involved in controlling membrane protein activity and "raft" formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1).
The vertebrate brain is the most cholesterol-rich organ , containing roughly 25% of the total free cholesterol present in the whole body.
18
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 19 Soc 1965, 42, 400) no desmosterol was found in the brain of adult animals. It is alsoknown as an abundant membrane component in some mammalian cells, such as spermatozoa and astrocytes (Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654). Inability to convert desmosterol to cholesterol leads to the human disorder desmosterolosis (a severe developmental defect and cognitive impairment) (Waterham HR et al., Am J Hum Genet 2001, 69, 685).
In higher plants, the first sterols were isolated by Hesse (1878) from the Calabar beans (Phytostigma venenosum) which coined the term "phytosterine". This substance was later named stigmasterol (Windaus and Hault, 1906) from the plant genus. The denomination "phytosterol" was proposed in 1897 (Thoms H) for all sterols of vegetal origin. Chemically, these sterols have the same basic structure as cholesterol but differences arise from the lateral chain which is modified by the addition of one or two supernumerary carbon atoms at C-24 with either or chirality. The 24-alkyl group is characteristic of all phytosterols and is preserved during subsequent steroid metabolism in both fungi and plants to give hormones that regulate growth and reproduction in a manner similar to animals.
20 Most phytosterols are compounds having 28 to 30 carbon atoms and one or two carbon-carbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain.
All phytosterols were shown to derive in plants from cycloartenol and in fungi from lanosterol, both direct products of the cyclization of squalene.
More than 200 different types of phytosterols have been reported in plant species. Representatives of these sterols are campesterol, stigmasterol (in soybean oil) and sitosterol which is present in all plant lipids and is used for steroid synthesis. An important sterol from yeast and ergot is the C28 compound ergosterol (mycosterol). Upon irradiation, this sterol gives rise to vitamin D2 (calciferol).
As ergosterol is a cell membrane component largely restricted to fungi, its amount in environmental matrices may be used as an index molecule for these micro-organisms in a
living biomass (Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051). 21
Considerable variability in the concentration of free sterols was observed among different oils. While concentrations lower than 100 mg/100 g are found in oils from coconut, palm, olive, and avocado, concentrations between 100 and 200 mg/100 g are found in oils from peanut, safflower, soybean, borage, cottonseed, and sunflower, and concentrations between 200 and 400 mg/100 g are found in oils from sesame, canola,
rapeseed, corn, and evening primrose (Phillips KM et al., J Food Comp Anal 2002, 15, 123). 22
Phytosterols produce a wide spectrum of biological activities in animals and humans. They are considered efficient cholesterol-lowering agents. In addition, they produce a wide spectrum of therapeutic effects including anti-tumor properties. Further data on their metabolism and potential therapeutic action can be found in a review article The European Commission authorized in 2004 the addition of phytosterols and phytostanols in food products with conditions of labeling including their amount per 100 g and the statement that the human consumption of more than 3 g/day should be avoided.
Phytostanols are a fully-saturated subgroup of phytosterols (they contain no double bonds). They occur in trace levels in many plant species but in high levels in tissues of only in a few cereal species. They are in general produced by hydrogenation of phytosterols.
Stanols often occur in dinoflagellates but are not common in other marine microalgae. Hence, dinoflagellates are often the major direct source of 5(H)-stanols in marine sediments (Robinson N et al., Nature 1984, 308, 439).
Fully saturated sterols are also found in animals but are of bacterial origin. Thus, the
5(H)-stanol coprostanol constitutes approximately 60% of the total sterols in human faeces.
23
While cholesterol was considered to be nearly absent in vegetal organisms, its presence is now largely accepted in higher plants. It can be detected in vegetal oils in a small proportion (up to 5% of the total sterols) but remains frequently present in trace amounts. An unusual relatively high content of cholesterol was described in camelina oil (about 200 mg per kg) (Shukla VKS et al., JAOCS 2002, 79, 965). However, several studies have revealed the existence of cholesterol as a major component sterol in chloroplasts, shoots and pollens. Furthermore, cholesterol has been detected as one of the major sterols in the surface lipids of higher plant leaves (rape) where he may amount to about 72% of the total sterols in that fraction (Noda M et al., Lipids 1988, 23, 439).
Although practical, the ancient distinction between zoosterols, mycosterols and phytosterols is no more used, since the same sterol may have different sources, but the appellation phytosterol is actually more frequently used.
Sterols are often isolated in the unsaponifiable fraction of any lipid extract and determined by various chromatographic procedures (HPLC or GLC).
Avenasterol can be isolated from oat oil. This sterol was shown to protect
specifically frying oils from oxidation owing to its ethylidene group in the side chain (White PJ et al., JAOCS 1986, 63, 525).
24
An extensive review on the diversity, analysis, and health-promoting uses of phytosterols and phytostanols may be consulted with interest (Moreau RA et al., Prog Lipid Res 2002, 41, 457).
Test Microorganisms Escherichia coli
Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae 25 Genus: Escherichia Species: Escherichia coli E. coli, discovered by Theodor Escherich, 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, 12 m in length and 0.10.5 m in diameter. 26 Escherichia coli, commonly known as E. coli, is a species of bacteria normally present in human intestines. A recently recognized strain, E. coli 0157:H7, produces high levels of toxins that can cause kidney damage, as well as septicemia, or blood poisoning. Symptoms can include diarrhea, chills, headaches, and high fever and in some cases the infection can lead to death, even with medical intervention.
Staphylococcus aureus
Kingdom: Bacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae
Genus: Staphylococcus Species: S. aureus
27 Staphylococcus aureus is a genus of spherical bacteria capable of producing a heat stable toxin that cause illness in humans. The most common pathogen S. aureus, is frequently responsible for carbuncles, boils, pneumonia, abscesses and osteomyelities It exist in air, dust, sewage, water, humans, foods and animals.
Staphylococcus aureus (which is occasionally given the nickname golden staph) is a bacterium, frequently living on the skin or in the nose of a healthy person, that can cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulitis) and abscesses, to life-threatening diseases such as pneumonia, meningitis, endocarditis, Toxic shock syndrome (TSS), and septicemia. Each year some 500,000 patients in American hospitals contract a staphylococcal infection. It is a spherical bacterium. It is abbreviated to S. aureus or sometimes referred to as Staph aureus in medical literature, and should not be confused with the somewhat similarly named streptococci which are also medically important.
S. aureus is a Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and as large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacteria's name: aureus means "gold" in Latin.
28 S. aureus is catalase positive and thus able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci. S. aureus can be differentiated from most other staphylococci by the coagulase test: S. aureus is coagulase-positive, while most other Staphylococcus species are coagulase-negative. The species has been subdivided into two subspecies: S. aureus aureus and S. aureus anaerobius. The latter requires anaerobic conditions for growth, is an infrequent cause of infection, and is rarely encountered in the clinical laboratory. S. aureus may occur as a commensal on human skin (particularly the scalp, armpits and groins); it also occurs in the nose (in about 25% of the population) and throat and less commonly, may be found in the colon and in urine. The finding of Staph. aureus under these circumstances does not always indicate infection and therefore does not always require treatment (indeed, treatment may be ineffective and re-colonisation may occur). It can survive on domesticated animals such as dogs, cats and horses, but has never been found on food animals such as poultry or swine. It can survive for some hours
on dry environmental surfaces, but the importance of the environment in spread of Staph. aureus is currently debated. It can host phages, such as the Panton-Valentine leukocidin, that increase its virulence.
29 S. aureus can infect other tissues when normal barriers have been breached (e.g. skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of furuncles). In infants S. aureus infection can cause a severe disease Staphylococcal scalded skin syndrome (SSSS). S. aureus infections can be spread through contact with pus from an infected wound, skin-to-skin contact with an infected person, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person. Deeply situated S. aureus infections can be very severe. Prosthetic joints put a person at particular risk for septic arthritis, and staphylococcal endocarditis (infection of the heart valves) and pneumonia may be rapidly fatal.
31
32
33
Definition of Terms
Antibiotics are molecules that are produced by one microorganism that kill (bacteriocidal) or inhibit (bacteriostatic) other microorganisms. They are one class of antibacterial and antifungal antimicrobials that can potentially be used as medicinal drugs to treat infections because of their low toxicity for humans or animals.
Antimicrobial agents agents that kill or slow the growth of microbes like bacteria (antibacterial activity), fungi (antifungal activity), viruses (antiviral activity), or parasites (antiparasitic activity).
Chloramphenicol - is a bacteriostatic antibiotic originally derived from the bacterium Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice in 1949.It was the first antibiotic to be manufactured synthetically on a large scale. Chloramphenicol is effective against a wide variety of microorganisms. In the West, the main use of chloramphenicol is in eye drops or ointment for bacterial conjunctivitis.
34 Glucose Yeast Peptone is recommended for the isolation of yeasts from soils specimen. This is a highly nutritious medium, which may be used for microbial examination.
Incubate to give the best or optimum conditions (ex: temperature, moisture) for growth and development. Inoculate to put microorganism or a substrate of organism on a medium. Inoculation a process of implanting infectious material into a culture medium. Inoculum - population of a pure culture grown in a medium.
Inhibition zone - this is an area around a paper disk or colony of bacteria or mold where no other organisms are growing. Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in water, sewage, feces and other materials. It is used in the laboratory for the
cultivation and maintenance of nonfastidious species and used in microbiological examination of a broad spectrum of materials. It is a simple medium composed of beef extract, peptone, and agar. It has been one of the most generally used media in bacteriological procedures. It is used for the ordinary routine examinations of water, sewage, and food products, for the carrying of stock cultures, for the preliminary cultivation of samples submitted for bacteriological examination, and for isolating organisms in pure culture. 35
Chapter II METHODOLOGY
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 NUMBER OF TRIALS
T0 (control) Chloramphenicol 3
T1 With Nut Grass rhizome extract 3
DEPENDENT VARIABLE: Zone of inhibition CONSTANT: Amount of extract 36
Materials:
A. Extraction Reflux apparatuses condenser rubber tubing burner iron ring iron stand Erlenmeyer flask water filter papers container/ sterile vial
B. Antimicrobial Assay Test Agar plates
Cork borer Pipette Nutrient Agar (NA) Glucose Yeast Peptone (GYP) Agar Culture Bacteria and Yeast- E.coli, S. aureus, and Salmonella spp. Inoculating loop Sterile cotton swab Commercial antibiotic (Chloramphenicol) 37 Alcohol lamp Beaker with 95% Ethanol C. Phytochemical Test acetic anhydride sulfuric acid 1 ml 10% hydrochloric acid 1% hydrochloric acid Mayers Reagent ferric chloride 2 test tubes anhydrous sodium carbonate sodium hydroxide potassium sodium tartrate distilled water
GENERAL PROCEDURE
A. Reagents All reagents (biochemical- equipment) were supplied by Regional Science High School for Region 1 Laboratory and Department of Science and Technology Laboratory (Main).
38 B. Collection of Plant Sample 400 grams of Nut grass rhizomes were collected in the Regional Science High School for Region 1 campus.
C. Preparation of the Extract Reflux Method was followed in extracting the Nut grass rhizome as indicated below: 1) Put 400 grams of Nut Grass rhizome (sample) in the Erlenmeyer flask and add some water enough for the sample. 2) Connect the Erlenmeyer flask containing samples into a condenser. 3) Subject the Erlenmeyer flask into heat to boil the water together with the sample. Its temperature should not exceed to 1000C. 4) Wait for the water to completely evaporate. The extract will remain on the flask.
5) Then filter the sample left in the Erlenmeyer flask to remove unnecessary samples.
D. Preparation of the Test Organisms E.coli, S. aureus, and Salmonella spp.were obtained from the Department of Science and Technology (Main) that were used as test organisms.
39
E. Antimicrobial Assay Test Microbial suspensions were prepared from 24- hour cultures of the Escherichia coli, Staphylococcus aureus and Salmonella spp. (bacteria) .The suspending medium used was 0.1% peptone water.
One-tenth (0.1) mL aliquots of the bacterial and yeast suspensions were transferred into pre-poured Nutrient Agar (NA) and Glucose Yeast Peptone (GYP) Agar, respectively. Five (5) of the corresponding medium, melted and cooled to 450C, was poured onto the agar plate and swirled to distribute the inoculums evenly on the agar surface. Five (5)mL of the sample was placed in each hole.
The plates were incubated at room temperature. NA and GYP plates were observed after 24-48 hours. The clearing zone was measured in millimeters and the average diameter of the clearing zones was calculated. the antimicrobial index (AI) was computed using the following formula:
AI
Diameter of clearing zone Diameter of well Diameter of well
40 F. Phytochemical analysis Extract from the Nut Grass rhizome were use for Phytochemical analysis.
1) Test for Sterols and Tipertenes Lieberman-Berchard Test
A small amount of the sample extract(Nut Grass Rhizome extract) in acetic anhydride was dissolved. The soluble portion was decanted and to this, 1-2 drops of concentrated sulfuric acid was added. Observe a green color, either immediately or solely going into red and blue tones. A pink to red color is indicative of triterpenoids while a blue color is indicative of steroids.
2) Test for Flavonoids
One (1) ml of sample extract was treated with 1 ml 10% hydrochloric acid and a few magnesium turnings. Formation of red color is observed.
3) Test for Alkaloids The sample extract was extracted with 1% HCL and drops of Mayers Reagent or Wagners Rgt. was added to the filtered acid extract. A cream colored precipitate is observed in the case of Mayers Rgt. while a reddish brown ppt. is observed in the case of Wagners Rgt. 41 Formula of Mayers 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 Wagners Rgt.:
1.3 g iodine crystals and 2.0 g potassium iodide in sufficient amount of distilled water to make a total volume of 100ml was dissolved.
4) Test for Tannins
The sample extract was extracted with hot water and the aqueous extract was filtered. Upon addition of two drops of ferric chloride test solution, a dark color and precipitate forms which may either be black, dark blue, blue black, green or blue green.
Ferric chloride TS: Dissolve 9g of ferric chloride in dist, water to make 100 ml.
42 5) Test for Saponins
The sample extract was dissolved in hot water. The aqueous extract when shaken vigorously should become frontly. The froth, honeycomb in nature should persist for at least 30 minutes.
6) Test for Glycosides
The sample extract was dissolved in hot water and filtered. The filtrate was used for the test. Two test tubes were used. Two ml of sample was placed in each tube. One ml of dilute hydrochloric acid was added to sample 1. Nothing is added to sample 2. The
2 test tubes were placed in a boiling water bath for 5 minutes. Then the test tubes were cooled. The samples were both neutralized with anhydrous sodium carbonate until no more effervescence is produced. Then add Fehling s B. One ml of Fehling s solution was used. The 2 test tubes were heated in a water bath for 2 minutes. Observe the amount of brick red precipitate that formed. An increase in the amount of brick red precipitate in the hydrolyzed sample (the sample to which dilute acid was added) as compared to the other sample indicates the presence of glycosides.
43 Fehlings solution A:
Copper Sulfate (CuSO4. 5H2O)34.66 grams Distilled water, a sufficient quantity
To make500 ml
Dissolve the copper sulfate in the distilled water and mixed.
Fehlings Solution B: Sodium Hydroxide50 grams
Potassium Sodium Tartrate173 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 fehlings A and B in equal amount before using.
44 7.) Test for the presence of Organic acids a.) Formic acid. Sections were placed in few drops of mercuric chloride solutions, then heated on a water bath for an hour and washed with water. The sections were transferred to a drop of 1% potassium hydroxide. Blackened cells indicate the presence of formic acid.
b.) Tartaric acid. Sections were treated with 4% aqueous solution of any ferrous salt and few drops of hydrogen peroxide or 10% potassium permanganate with the addition of an excess of sodium hydroxide solution. Violet color reaction indicates the presence of tartaric acid.
8.) Test for the presence of fats and Oils
Sections were treated or immersed in either Sudan III or IV for twenty minutes, then washed with 50% alcohol, and transferred to glycerin for observation. Deep red color reaction indicates the presence of fats and oils.
45
Reagents Collection of the Plant Specimen Preparation of the Culture Bacteria Preparation of the Extract
Antimicrobial Assay Test
Phytochemical Analysis
Figure 1. The Flowchart of Experimental Design 46
Chapter III RESULTS AND DISCUSSONS

This chapter dealt with the results and findings with their subsequent discussions. The results were presented in tables and in graphs.
Table 1. Mean Zone of Inhibition Against E. coli in millimeters (mm).
The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome extract) is 3.1mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is lesser than the Mean Zone of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
47
Table2. Mean Zone of Inhibition Against S. aureus in millimeters (mm).
The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome extract) is 3.5 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone
of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
48
Table3. Mean Zone of Inhibition Against Salmonella spp.in millimeters (mm).
The table shows that the Mean Zone of Inhibition of T1 (Nut Grass rhizome extract) is 3.0 mm and the Mean Zone of inhibition of T0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is lesser than the Mean Zone of
Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
49
Table 4. Mean Zone of Inhibition Test Microorganisms in millimeters (mm).
The table shows that the Mean Zone of Inhibition of T1 is 10.5mm and the Mean Zone of Inhibition of T0 is 9.6 mm. This proves that the Mean Zone of Inhibition of T1 is greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
50
Table 5. Mean Zone of Inhibition Test Microorganisms in millimeters (mm).
The table shows that the Mean Zone of Inhibition of T1 is 10.5mm and the Mean Zone of Inhibition of T0 is 9.6 mm. This proves that the Mean Zone of Inhibition of T1 is greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
51 Table 6. A Summary table of the Active Principles of the Dorsal Fin Venom Active Principles Alkaloids Glycosides Sterols Color reaction Reddish brown Red Blue Nut Grass rhizome 3 1 3
Tannin
black, dark blue, blue black, green or 2 2 0 0 3 3 0 blue green Yellow/ red Black Violet Deep red Red pink to red
Saponin Formic acid Tartaric acid Fats and oils Flavonoids Titerpenes
The table shows the Phytochemical Test result of the Nut Grass rhizome sample. It shows that Alkaloids, Sterols, Flavonoids and Fats and Oils are very` abundant in all tissues of the rhizome .Tannins and Saponins are observed abundant while Glycosides are detectable only.This contributed much on the effectiveness of Nut Grass rhizome extract an antibiotic agent as revealed on the table.
Legends: 3 = Very Abundant (51-100%) 2 = Abundant (26-50%) 1= Detectable (1-25%) 0 = Absent
52
Figure 2. Mean of Zone of Inhibition of E.coli, S. aureus, C. albicans and A. niger treated by the extract (5mL) from the Nut Grass rhizome.
3.6 Zone of Inhibition of Bacteria in Diameters (mm) 3.55 3.5 3.45 3.4 3.35 3.3 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.
Zone of Inhibit ion in Diame ters (mm)
3.5 3 2.5 2 1.5 1 0.5 0 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.
Comparison of Zone of Inhibition of Bacteria in Diameters (mm)
3.7 3.6 3.5 3.4 3.3 3.2 3.1 3 5mL of Chloramphenic ol syrup 5mL of Nut Grass rhizome extract
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 IV CONCLUSIONS AND RECOMMENDATIONS Conclusions
Based from the findings, the following conclusions were drawn:
a) The Nut Grass rhizome extract can inhibit the growth of harmful microorganisms.
b) Phytochemical analysis showed the presence of active principles from the Nut Grass rhizome tissues. It showed that Alkaloids, Sterols, Flavonoids and Fats and Oils were very` abundant in all tissues of the rhizome .Tannins and Saponins were observed abundant while Glycosides were detectable only. This contributed much on the effectiveness of Nut Grass rhizome extract an antibiotic agent as revealed on the table
c) There is no significant difference of using the Antimicrobial Property of Nut Grass rhizome and the controlled antibiotic in terms of zone of inhibition.
56
Recommendations
Based on the results of the study, the following recommendations are given:
a) Being abundant, Nut Grass should be used as a medicinal plant. However, users should follow the recommendations of the DOH, since it is more effective as an antimicrobial.
b) Pure extracts of Nut Grass rhizome should be conducted, processed and be made as tablets for medicine.
c) Wide dissemination of the latest technology must commence
57
Bibliography
Internet websites, Yahoo.com; Google .com; and MSN Network.
Microsoft Encarta Library Edition 2006.
Phillips KM et al., J Food Comp Anal 2002, 15, 123 Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051
Waterham HR et al., Am J Hum Genet 2001, 69, 685 Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654 Paoletti R et al., J Am Oil Chem Soc 1965, 42, 400 Wikipedia, free Dictionary
58
Appendices
I. Statistical test for the zone of inhibition of Escherichia coli treated with Chloramphenicol (T0) and with the Nut Grass rhizome extract (T1).
A
TRIALS 1 2 3 Total T0 (x) ( x1) N1=3 T0 (x2) 10.24 10.24 10.24 ( x12) 30.72 T1 (y) ( y2)
B
T1 (y2) 12.96 13.69 12.96 ( y22) 39.61 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 = 6-2 =4
Tcrit.= 2.776
Compute for t
= 9.6/ 3 = 3.2
= 10.9/ 3 = 3.63
SX 1- Y 2=
2 2 X 2 ( X 1 ) + Y 2 ( Y2 ) 2 1 N1 N2 N1 + N 2 2
1 + 1 N 1 N2
(9.6) 2 30 .72 3
(10 .9) 2 + 39 .61 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 39 .61 39 .6) (0.666 )

4
60 =
0.01 (0.666 ) 4
0.0 2 (0.6 6 ) 05 6
= 0.0408
t cal = t =
X 1 Y 2 0 S X 1 Y 2
= 3.2 3.63 / 0.0408
= -10.5392
Decision: Accept H0 The tcrit is greater than the t

cal
so the Null Hypothesis was accepted. Thus, there is no
significant difference of using the Antimicrobial Property of Nut Grass rhizome and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.
61
II. Statistical test for the zone of inhibition of Staphylococcus aureus treated with the commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract (T1).
C
TRIALS T0 T0 T1
D
T1
1 2 3 Total
(x) ( x1) N1=3
(x2) 10.24 10.24 10.24 ( x12)
(y) ( y1) N2=3
(y2) 12.25 12.25 12.25 ( y12) 36.75
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 = 6-2 =4 Tcrit.= 2.776
Compute for t
= 3.2/ 3 = 3.2
= 10.5/ 3 = 3.5
SX 1- Y 2=
2 2 X 2 ( X 1 ) + Y 2 ( Y2 ) 2 1 N1 N2 N1 + N 2 2
1 + 1 N 1 N2
(9.6) 2 (10 .5) 2 30 .72 + 36 .75 3 3 1 1 + 4 3 3
( 30 .72 30 .72 ) + ( 36 .75 36 .75 ) (0.666 )

4
0 (0.666 ) 4
=0
63 t cal = t = = 3.2 3.5 / 0

X 1 Y 2 0 S X 1 Y 2
=0
Decision: Accept H0
The tcrit is greater than the t
cal
64 III. Statistical test for the zone of inhibition of Candida albicans treated with the commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract (T1).
E
TRIALS 1 2 T0 (x) T0 (x2) T1 (y)
F
T1 (y2) 11.56 11.56
3 Total
N1=3
N2=3
10.89 34.01
65
= 0.05
Df = N1 + N2- 2 = 6-2 =4 Tcrit.= 2.776
Compute for t
= 9.6/ 3 = 3.2
= 10.1/ 3 = 3.36
SX 1- Y 2=
2 2 X 2 ( X 1 ) + Y 2 ( Y2 ) 2 1 N1 N2 N1 + N 2 2
1 + 1 N 1 N2
(9.6) 2 30 .72 3
(10 .1) 2 + 34 .01 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 34 .01 34 ) (0.666 )

4
0.01 (0.666 ) 4
= =
0 .0 5 ( 0 .6 6 ) 2 6
= 0.0408 66 t cal = t =
X 1 Y 2 0 S X 1 Y 2
= 3.2 3.36 / 0.0408 =-3.9216
Decision: Accept H0
The tcrit is greater than the t
cal
67 IV. Statistical test for the zone of inhibition of Aspergillus niger treated with the commercial antibiotic Chloramphenicol (To) and Nut Grass rhizome extract (T1).
G
TRIALS 1 2 T0 (x) T0 (x2) T1 (y)
H
T1 (y2) 12.25 12.25
3 Total
N1=3
N2=3
12.25 36.75
68
= 0.05
Df = N1 + N2- 2 = 6-2 =4 Tcrit.= 2.776
Compute for t
X
G
= 9.6 / 3 = 3.2
= 10.5/ 3 = 3.5
SX 1- Y 2=
2 2 X 2 ( X 1 ) + Y 2 ( Y2 ) 2 1 N1 N2 N1 + N 2 2
1 + 1 N 1 N2
(9.6) 2 30 .72 3
(10 .5) 2 + 36 .75 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 36 .75 36 .75 ) (0.666 )

4
0 (0.666 ) 4
=0 69 t cal = t =
X 1 Y 2 0 S X 1 Y 2
= 3.2 3.5 / 0 =0

cal
70
PLATES
Plate 1. Researchers Collecting the Plant Specimen.
71
Plate 2. The Plant Specimen Nut Grass (Cyperus rotundus).
72
Plate 3. The Nut Grass Rhizome.
73
Plate 4. Materials for the Reflux Method (Extraction)
74
Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method.
75
Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper.
76
Plate 7. Materials for the Antimicrobial Test.
77
Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient Agar and Glucose Yeast Peptone Agar.
78
Plate 9. Researcher Pouring the Medium onto the Agar Plates.
79
Plate 10. Researcher Swirling Agar Plates to distribute the inoculums evenly in the Agar Surface.
80
Plate 11. Researcher making holes on the Agar Plates using the Cork Borer.
81
Plate 12. Researcher Placing the Extracts in each hole.
82
Plate 13. Researching Incubating the Plates at room temperature
83
Plate 14.The treatments To ( Chloramphenicol) and T1 ( Nut Grass rhizome extract).
84
CURRICULUM VITAE
Name: Lyra Erika Liclican Age: 16years old Address: Paratong, Sta.Cruz, Ilocos Sur Birthday: September 1, 1990 Parents: Mr. Raul Liclican Mrs. Lydia Liclican Religion: Roman Catholic Nationality: Filipino Civil Status: Single Educational Attainment: Elementary St. Joseph Institute Candon City, Ilocos Sur St. Augustines School Tagudin, Ilocos Sur Secondary Regional Science High School for Region 1 Bangar, La Union
Name: Glen Bernard Asto Age: 14years old Address: Suyo, Luna, La Union Birthday: September 8, 1992 Parents: Mr. Leonardo Asto Mrs. Gloria Asto Religion: Roman Catholic Nationality: Filipino Civil Status: Single Educational Attainment: Elementary Suyo Elementary School Suya, Luna, La Union Secondary Regional Science High School for Region 1 Bangar, La Union
DATA BOOK
SUCCESS CALENDAR
1. CHOOSING A TOPIC
Planned Date May 3,2006
Date Completed 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 BACKGROUND INFORMATION
Planned Date May 11, 2006
Date Completed May 15, 2006
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 AND GATHERING OF DATA
Date Completed June 2, 2006
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 UP (MANUSCRIPT) Background of the Study
Planned Date July 28,2006
Date Completed August 2, 2006
5. MAKING THE STATEMENT OF THE PROBLEM, HYPOTHESES, SIGNIFICANCE OF THE STUDY UNTIL SCOPE AND DELIMITATION
Planned Date August 4, 2006
6.MAKING THE REVIEW OF RELATED LITERATURE
Date Planned August 17,2006
Date Completed August 26,2006
7. MAKING THE CHAPTER II ( Methodology)
Date Completed September 12, 2006
8. MAKING THE CHAPTER III (Results and discussions)
Date Planned September 13,2006
Date Completed Sept. 17, 2006
9. MAKING THE CHAPTER IV (Conclusions and Recommendations)
Date Planned Sept. 18, 2006
Date Completed Sept. 20,2006
10. MAKING THE BIBLIOGRAPHY, APPENDICES
Date Planned Sept.22, 2006
10. FINALIZATION
11. PRINTING OF THE MANUSCRIPT
Date Completed Sept 30.2006
Department of Education Region 1 Division of La Union Regional Science High School for Region 1 Bangar, La Union
Antibiotic Property from Stonefish Dorsal Fin (Synanceia verrucosa) against Escherichia coli, Staphylococcus aureus, and Candida albicans
By:
LYRA ERIKA T.LICLICAN
ROGELIO C. VALDEZ Research Adviser
S.Y 2006-2007
Table of Contents
Abstract.i CHAPTER 1 Introduction Background of the Study.. Statement of the Problem. Hypotheses Significance of the Study.. Scope and delimitation.. Review of related Literature.. Description of the Animal Specimen Phytochemical Components.. Test Microorganisms CHAPTER II Methodology Experimental Design. Materials.. Reagents. Collection of Animal Specimen Preparation of the Extract Antimicrobial Assay Test
Phytochemical Test. Flowchart. CHAPTER III RESULTS AND FINDINGS CHAPTER IV COCLUSIONS AND RECOMMENDATIONS Conclusions. Recommendations.. Bibliography Appendices.. Curriculum Vitae
LIST OF TABLES
Table 1. Mean Zone of Inhibition against E. coli in millimeters (mm).54
Table 2. Mean Zone of Inhibition against S. aureus in millimeters (mm)....55 Table 2 .Mean Zone of Inhibition against C. albicans in millimeters (mm).56
Table 4.Summary table of the Mean Zone of Inhibition of Test Microorganisms in millimeters (mm)..57
Table 5. A Summary table of the Active Principles..58
LIST OF FIGURES
Figure 1. The Flowchart of Experimental Design52
Figure 2. Block presentation of the standard protocol on fluid venom extraction of Stonefish53. 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 syrup60
Figure 5. Comparison of the Mean of Zone of Inhibition of Test Microorganisms when treated by the Stonefish Dorsal Fin venom and to 3 mL of Chloramphenicol syrup61
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 Specimen77
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 Knife79
Plate 7. Researcher Grasping the Tail Fin and Pulling it way from the Dorsal Fin.80
Plate 8. Researcher Extracting the Venom using a Sterile Syringe..81
Plate 9: The Extracted Venom...82
Plate 10. Materials used for the Antimicrobial Assay Test.83.
Plate 11.Researcher transferring Aliquots Bacterial and Yeast Suspension o Nutrient Agar and Glucose Yeast Peptone Agar..84
Plate 12. Researcher Pouring the Medium onto the Agar Plates85 Plate 13.Reseacher Swirling Agar Plates to distribute the inoculums evenly in the Agar Surface. ..86
Plate 14.Reseacher making holes on the Agar Plates using the Cork Borer ..87
Plate 15. The bored Agra Plate Surface88 Plate 16.Researcher Placing the Extracts in each hole..89
Plate 17.Researching Incubating the Plates at room temperature90
Plate 18. The treatments To (Chloramphenicol) and T1 ( Stonefish Dorsal Fin Venom)..91
Abstract
This study aimed to determine the Antibiotic Property from Stonefish (Synanceia verrucosa) Dorsal Fin Venom against Escherichia coli, Staphylococcus aureus and Candida albican and it was conducted at the University of the Philippines Fifteen Stonefish were subjected to experimentation. Antimicrobial assay test were subjected using the control Chloramphenicol and the experimental variable Stonefish Dorsal Fin venom. Phytochemical Test was done to determine the active principles present. It showed that Alkaloids, Saponin and Tannins were very abundant in the Stonefish Dorsal Fin venom. There were two treatments used namely T0: the controlled antibioticChloramphenicol and T1: the Stonefish Dorsal Fin Venom.T- test showed that the Stonefish Dorsal Fin Venom had a similar performance with the Chloramphenicol as an antibiotic. Likewise, there is no significant difference of using the Antibiotic
Property of Stonefish Dorsal Fin Venom and the Chloramphenicol in terms of zone of inhibition that resulted to the acceptation of the Null Hypothesis. Furthermore, it is highly recommended to the Pharmaceutical Industry, that this will serve as a basis of making an antibiotic out of the Stonefish Dorsal Fin Venom. Additional Brand of antibiotics shall be made available for comparing the effect of Stonefish Dorsal Fin Venom to test further efficacy. Wide dissemination of the latest technology must commence.
Chapter I INTRODUCTION
A. Background of the Study
According to one of the famous biologists, Barry Commoner, Everything is connected with everything else and Everything goes somewhere Plants- Animals, Humans-Animals, Plants-Humans . . . . In these present times, there is a need and exigency of the usage of antibiotics in the field of medicine. The exposure of living creatures to a lot of flying particles may bequeath danger to their physical state. Many living things accommodate to its environment that defend them from the assault of other organisms or simply concealment by blending in with the surroundings. In this case, the stonefishes an aquatic animal has bulky, compact body shape with sloughing skin with variable color patterns and covered with algae, providing a magnificent camouflage, simulating a stone or a mass of mud. About 1000 known species of marine animals are venomous. And the venom of one, the stonefish, can kill a man. It is armed with thirteen strong spines along its back carrying enough venom to kill a human being. The evolution of a protective system is easily understandable. The stonefish use their poisonous spines only in self-defense. Previous years has been observed renascence of interest among students and specialized researchers in seeking antimicrobial properties from chosen vertebrate and invertebrate as well. This is accredited to the fact that artificial and presently available medicines are ineffective, futile, and too expensive or tend to bring superb side effects.
Our tropical country is gifted with variety of flora and fauna. Among these is the Stonefish (Synanceia verrucosa) which is considered undesirable organisms in the environment however can be a very good source of antibiotic. Inquisitiveness provoke the researchers mind that what if Synanceia verrucosa has an antibiotic property and then could be used as antimicrobial agent to restrain the growth of some bacteria, which motivated the researcher to conduct a study on the said specie, thus the Antibiotic property from Stonefish Dorsal Fin Venom against Escherichia coli, Staphylococcus aureus and Candida albicans was conceived.
B. Statement of the Problem

This study aimed to determine the Antibiotic Property from Stonefish Dorsal Fin (Synaceia verrucosa) against Escherichia coli, Staphylococcus aureus and Candida albicans was conceived. Specifically, this study sought to answer the following sub problems: 1.) Can the Stonefish Dorsal Fin Venom inhibit the growth of harmful microorganisms? 2.) What are the active constituents of the Stonefish Dorsal Fin Venom using Phytochemical test? 3.) Is there a significant difference of using the Stonefish Dorsal Fin and the commercial antibiotic in terms of zone of inhibition?
C. Hypotheses
The study anchors the following hypotheses: 1.) The Stonefish Dorsal Fin Venom cannot inhibit the growth of harmful microorganisms. 2.) Alkaloids, Saponin and Tannin are not the active constituents of the Stonefish Dorsal Fin Venom using the Phytochemical Test. 3.) There is no significant difference of using the Antibiotic Property from Stonefish Dorsal Fin and the commercial antibiotic in terms of zone of inhibition.
D. Significance of the Study

Bacteria trigger many diseases in humans, including tetanus, diphtheria, plague, pneumonia, cholera, leprosy and meningitis. Massive times are spent in the effort to lessen the likelihood of these infections and in inhibiting the other destructive activities of bacteria. The viability of other organisms as sources of antibiotic property is inevitable and environmental friendly because of the notion of interdependent role. The complex integration of microorganisms in the web of nature is recognized and will be more completely understood as universal patterns of biodiversity are documented. The use of microorganisms in the production of antibiotics has saved many lives since the last century. Penicillin Antibiotic is known as the wonder drug.
While many kinds of microorganisms cause communicable diseases, others use by scientists to avert illnesses. It is for this reason that the researcher of the Antibiotic Property from Stonefish Dorsal Fin (Synaceia verrucosa) drove him to study the
chemical substance and its efficiency of fighting to harmful microorganisms
E.Scope and Limitations

The study was conducted at University of the Philippines Diliman, Quezon City from May to September 2006, S. Y. 2006-2007. The collection of the Stonefish Dorsal Fin Venom was done at Marine Science Institute Laboratory of UPD on the direct supervision of Dr. Lourdes Cruz in her protocol on fluid or venom extraction and proper handling of procedure. The extracted venom was brought to Natural Science Institute, UPD for antimicrobial assay test on the supervision of Mrs. Vina Argayosa and to Adamson University for the Phytochemical Test on the supervision of Mr. Ghel Balete This study is limited only to the use of the Stonefish Dorsal Fin Venom as source of extract and the use of three species of bacteria namely: Escherichia coli, Staphylococcus aureus and Candida albicans for antimicrobial tests.There were only two treatments namely T0 ( Chloramphenicol) and T1 (the Stone Dorsal Fin Venom).
F. Review of Related Literature
English Name: Stonefish Scientific Name: Synanceia verrucosa Kingdom: Animalia Phylum: Chordata Class: Actinoplerygii Order: Scorpaeniformes Family: Synanceiidae
Ecology
It lives along the external reef, often in sheltered bay or lagoon environments. Very well camouflaged, perfectly still for long times. It moves seldom, swimming heavily.
Association Often it is covered by algae and hydroids, contributing to camouflage.
Classification The stonefish comes from the fish family.
Habitat Some stonefish live in coral and sandy places. Stonefish live in warm water where it is clear. The stonefish lives primarily above the tropic of Capricorn. Its main habitat is on coral reefs, near and about rocks, or can be found dormant in the mud or sand. It feeds on small fish and shrimps. Appearance Some stonefish look like rocks and coral and are greenish brown color. Stonefish can grow up to 30cm in length. The Stone Fish is a mottled brown-greenish in color (which gives them camouflage) with many venomous spines along its back.
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Behavior Stonefish gobble small fish. They dont even give the small fish a chance
Description
Stonefish are indisputably ugly. In common with their relatives the scorpion-fish they have a bony ridged head and wavy fins but here ends the resemblance to any other living creature. The head and body of the stonefish are covered with lumps and fleshy growths and the eyes are deeply set in the bony hollows of the head. The large mouth is upturned and partly disguised by a notched fringe of skin. Their irregular shape and blotchy red-brown coloration afford stonefishes an extremely effective camouflage. This is enhanced by their ability to secrete a sticky fluid from the wart like growths on the skin, which covers the body and to which cling algae and mud. Even small invertebrates such as sea anemones and hydras colonize the apparently inanimate object. One might expect that the potential of the stonefish -- each spine carrying enough venom to kill a human being -- would be' exploited by it to obtain food. This is not the case, however, and its deadly arsenal is utilized only as a means of defense. Its diet composed of small fish and crustaceans such as shrimps, a stonefish uses its large front fins to scoop out a depression in the sand or mud where it lies motionless waiting for its prey to draw near. Deceived by the convincing camouflage, passing victims are swallowed completely as the stonefish makes an unexpectedly energetic lurch forwards.
Venom The sting causes excruciating pain and a great deal of swelling rapidly develops causing death to tissues. The severity of the symptoms depends on the depth of penetration and the number of spines penetrated. The symptoms of the venom are muscle weakness, temporary paralysis and shock, which may result in death if not treated. Venom Apparatus Among the representatives of the Scorpaenidae family, the stonefish has the most efficient and developed venom apparatus. Although three spines of the anal and two of the ventral fin contains venom glands, the 13 dorsal spines inflict most of the venomous stings. Each spine has two lateral grooves where a thick spindle-shaped is situated in the proximal part. A thin venom duct leads to the tip of the spine, and a thick integumentary sheath covers the spines. When the spine enters the body under pressure, its integument is ruptures and the glands are compressed injecting the venom through the venom ducts into the wound caused by the needle-sharp spines.
Venoms and Toxins Each venom gland of the dorsal spines contains about 0.03 ml liquid venom. It contains a mixture of high-molecular weight protein-toxins. In experimental animals, stonefish venom causes an attrio-ventricular blockade and ventricular fibrillation of the heart, a sudden hypertension and paralysis of skeletal muscles, which is either due to a massive of neuro-transmitter or to damage of nerve and muscle.
Scorpionfishes have a reddish to brownish color and are mottled. This enables them to disappear against the substrate.
Scorpaenopsis There are 4 very similar species of humpback scorpionfishes. Scorpaenopsis diabolus (devil scorpionfish - pectoral fin with orange, yellow and white) and Smacrochir (flasher scorpionfish - pectoral fin with orange and some black at the edge) can best be told apart by looking at their pectoral fins. The devil scorpionfish also has a more pronounced hump and is larger (up to 30cm) than the flasher scorpionfish (15cm). If the ridge above the eyes is serrated it is a bandtail scorpionfish (S. neglecta). Another
scorpionfish, Scorpaenopsis gibbosa (humpback scorpionfish), is only found in Africa and the Indian ocean.
9
The stonefish is extremely difficult to see because it usually buries most of its body under sand or rubble and only their widely separated eyes show. Often algae and hydroids grow on its back. It has been suggested, that stonefishes exude a white, milky substance over their bodies which encourages plant growth. Shrimps and other animals have been observed to climb over them. This is the worlds most venomous fish. Their near perfect camouflage and the venomous spines make them a hazard for swimmers, snorkelers and divers in shallow water. Wounds should be treated immediately with hot water or dry heat.
There is a scorpionfish that erroneously identified as stonefish, the humpbacked or devil scorpionfish. However there are two difference: First the shape of the mouth. Stonefishes have a mouth which is directed upwards like a upside-down "U". Second the stone fish curl their tail extremely to one side.
CHORDATA (VERTEBRATES) Scorpionfishes (Scorpaenidae) The family Scorpaenidae contains around 45 genera and 380 species.
Characteristics Scorpionfishes have large, heavily ridged and spined heads. Venomous spines on their back and fins with a groove and venom sack. Well camouflaged with tassels, warts and colored specks. Some scorpionfishes can change their color to better match their 10 surroundings. The stonefish is a master of disguise and deception, it looks like a piece of coral or sand covered rock. Thus, he can blend in with its surroundings and go unnoticed by its prey. Ecology and range Most scorpion fishes live on or near the bottom. They lie in crevices, in caves and under overhangs. Range: Red Sea , pacific ocean to Australia, Hawaii. A few scorpionfishes (no lionfishes or stonefishes) live in the Caribbean Behavior They feed on crustaceans, cephalopods and fishes employing a lie-in-wait strategy, remaining stationary and snapping prey that comes near. With their mouth they create a vacuum and suck prey in during a nearly imperceptible split-second movement (15 milliseconds). Some have algae and hydroid growth on their body surfaces( stonefish) and at least one species (Decoy scorpionfish Iracundus signifier) has a dorsal fin that looks like
a swimming fish, a behavior similar to that of the frogfish. Some species (for example the weed scorpionfish) sway their bodies from side to side so they look like a piece of debris. Scorpionfishes are not aggressive, but if threatened they will erect their dorsal spines. If danger continues they flee, usually very fast but only for a short distance and then quickly settle back and freeze. The stonefishes for example ususally bury themselves in sand or rubble using a shoveling motion of their pectoral fins. In a matter of less than 11 10 seconds only the dorsal portion of the head remains exposed, some sand is thrown on top to further enhancing concealment. Some species like the devilfish have very bright red and yellow colors on the inner surface of their pectoral fins. Those colors are not visible when resting but are flashed if threatened. Scorpion fishes produce a floating, gelatinous mass in which the eggs are embedded.
Phytochemical Components Phytochemical studies on animals give basic information on the presence and localization of the active constituents in the animal body. These tests include the test for The presence of alkaloids, saponins, tannins, glycosides, sterols, flavonoids and triterpenes.
Alkaloids
Alkaloids are new synthetic agents of greater potency and lesser toxicity. It is a naturally occurring amine produced by a plant but amines produced by animals and fungi are also called alkaloids. Many alkaloids have pharmacological effects on humans and animals. Alkaloids are usually derivatives of amino acids. They are found as secondary metabolites in plants, animals and fungi, and can be extracted from their sources by treatment with acids.
12 Alkaloids, group of mildly alkaline compounds, mostly of plant origin and of moderate molecular complexity. Even in very small amounts, the alkaloids produce strong physiological effects on the body. All contain nitrogen atoms that are structurally related to those of ammonia.
Nearly 3000 alkaloids have been recorded; the first to be prepared synthetically (1886) was one of the simplest, called coniine, or 2-propyl piperidine, C5H10NC3H7. It is highly poisonous; less than 0.2 g (0.007 oz) is fatal. Coniine, obtained from seeds of the hemlock, was the poison used in the execution of Socrates. Some 30 of the known alkaloids are used in medicine. For example, atropine, obtained from belladonna, causes dilation of the pupils; morphine is a painkiller; quinine is a specific remedy for malaria; nicotine is a potent insecticide; and reserpine is a valuable tranquilizer.
Saponins
Saponins are subgroups of glycosides that have the characteristic ability to cause foaming when shaken with water. They are sometimes used as emulsifying agents. Saponins are believed to be useful in the human diet for controlling cholesterol, but some are poisonous if swallowed and can cause urticaria. Any markedly toxic saponin is known as a sapotoxin.
13 Saponins, group of naturally occurring oily glycosides that foam freely when shaken with water. They occur in a wide variety of plants, including acacia, soapwort, soaproot, California pigweed, and many others. Saponins have been, and sometimes still are, used as cleaning agents and as foam producers, notably in fire-extinguishing fluids. They have a bitter taste and when ingested orally are practically nonpoisonous to warmblooded animals. When injected directly into the bloodstream, however, they are dangerous and quickly dissolve red blood cells. Hydrolysis of a saponin, brought about by acids or by enzymes, gives a sugar (often, but not necessarily, glucose) and a sapogenin, the latter being either a triterpene or a steroid. Some of the sugars and saponins are useful as raw materials for synthesis of steroid hormones.
Glycosides
Glycosides, class of complex chemical compounds in plants. They are broken down by plant enzymes into sugars, among which glucose is generally included, and into other substances. The term glucoside is often used synonymously with glycoside, but in its more specific meaning it refers to glycosides that yield glucose.
Each glycoside in a plant is hydrolyzed (converted in a reaction with water) by an enzyme, usually a specific enzyme found in the same plant. The enzyme emulsin, however, causes hydrolysis of several glycosides. The enzymes and glycosides are stored in separate plant cells until the reaction products of the glycosides are needed and the 14 enzymes are activated. Glycosides are believed to serve several purposes in the plant. Glycosides are bitter tasting, and it is believed that they help keep birds and insects from eating seeds and fruit before they are fully grown, by which time the glycosides have been converted to sweet sugars. When a plant tissue is bruised, the enzymes hydrolyze the glycosides into products, such as phenol compounds and acids that have an antiseptic action and prevent decay of the damaged tissues.
Glycosides are soluble in water and are obtained from plants by water extraction. They are mostly colorless crystalline solids with a bitter taste. Simple glycosides have been synthesized in the laboratory, and several hundred glycosides have been extracted from plants and used for many purposes. Among the important glycosides are indican,
used for dyeing; digitalin, used in medicine; and the saponins, foaming agents used industrially and medicinally.
Triterpene Triterpene refers to a particular type of molecules that has a four to five ring, planar base molecules containing 30 carbon atoms. It is synthesized from very simple compounds ( acetate units) that are found in all plants, but is mainly synthesized in higher plants by linking the acetate units head to tail. The triterpenes have an acidic quality, an acrid- bitter taste, and their function in plants remains unknown.
15 The triterpenes are subdivided into about 2o groups, depending on their particular structures. The base structure that is found in the largest variety of medicinal plants is the oleanane triterpene. This type of compound may be represented by four of the most frequently occurring forms: oleanolic acid, ursolic acid, and alpha and beta amyrin ( the latter 3 are sometimes put in the division of ursane triterpenes). Platycodin belongs to the very large class of aleanane triterpenes.
Tannins These are also tannin acid, common name applied to a group of vegetable products, both amorphous and crystalline, obtained from various plants, and important commercially in the tanning of leather. Tannins have variable composition. Some, called condensed tannins, are phenols of moderately complex structure, and others are esters of
glucose or some other sugar with one or more trihydroxybenzoic acids. The empirical formula, C14H14O11, often given for common tannin, is only an average. Tannins occur in many trees, and the best sources include oak galls and the bark of sumac. Extraction with water, or water and alcohol, is the first step in the preparation of tannin. Settling, followed by evaporation at a low temperature, yields the commercial product. Tannins have a yellow-white to brown color and a faint, characteristic odor. Exposure to light deepens the color. They all taste bitter and are astringent. Water, acetone, and alcohol dissolve tannins readily, but benzene, ether, and chloroform do not. Heating to 210C, (410 F) causes decomposition, accompanied by formation of pyrogallol and carbon dioxide. The chemical property that provides the basis for most 16 uses of tannins is its ready formation of precipitates with albumin, with gelatin, and with many alkaloidal and metallic salts. The ability of tannins to transform proteins into insoluble products resistant to decomposition leads to their use as tanning agents. Ferric salts react with tannins to give bluish-black products that are useful as inks. Tannins are used as mordants for dyeing cloth, as sizes for paper or silk, and as coagulants for rubber. The precipitating properties of tannins are used in clarifying, or cleaning, wines and beer. Tannic acid is valuable as an external medicine because it is astringent and styptic. Flavonoid
The term flavonoid refers to a class of plant secondary metabolites based around a phenylbenzopyrone structure. Flavonoids are most commonly known for their antioxidant activity. Flavonoids are also commonly referred to as bioflavonoids in the media these terms are equivalent and interchangeable, since all flavonoids are biological in origin. The flavonoid synthetic pathway begins with a product of glycolysis, phosphoenolpyruvate, entering into the Shikimate pathway to yield phenylalanine. Phenylalanine is the starting material of the phenylpropanoid metabolic pathway, from 17 which 4-Coumaryl-CoA is produced. This can be combined wih Malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones. Ring-closure of these compounds results in the familiar form of flavonoids, a three-ringed phenolic structure (polyphenols). The metabolic pathway continues through a series of enzymatic modifications to yield flavanones dihydroflavonols anthocyanins. Along this pathway many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other polyphenolics.
Flavonoids are widely distributed in plants fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Flavonoids have been found in high concentrations in butterflies and moths sequestered from dietary intake at the larval stage and then stored in adult tissues. Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, antiinflammatory
[1]
, anti-microbial and anti-cancer activity. In addition, flavonoids act as
powerful antioxidants, protecting against oxidative and free radical damage.
18 Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins. Sterols
Sterols may be found either as free sterols, acylated (sterol esters), alkylated (steryl alkyl ethers), sulfated (cholesterol sulfate), or linked to a glycoside moiety (steryl glycosides) which can be itself acylated (acylated sterol glycosides). Free Sterols Sterols form an important group among the steroids. Unsaturated steroids with most of the skeleton of cholestane containing a 3 hydroxyl group and an aliphatic side chain of 8 or more carbon atoms attached to position 17 form the group of sterols.
They are lipids resistant to saponification and are found in an appreciable quantity in all animal and vegetal tissues. These unsaponifiable lipids may include one or more of a variety of molecules belonging to 3-hydroxysteroids, they are C27-C30 crystalline alcohols (in Greek, stereos, solid). These lipids can be classed as triterpenes as they derive from squalene which gives directly by cyclization, unsaturation and 3hydroxylation, lanosterol in animals 19 or cycloartenol in plants.
In the tissues of vertebrates, the main sterol is the C27 alcohol cholesterol (Greek, chole, bile), particularly abundant in adrenals (10%, w/w), nervous tissues (2%,w/w), liver (0.2%,w/w) and gall stones, its fundamental carbon structure being a cyclopentanoperhydrophenanthrene ring (also called sterane). It was the first isolated sterol around 1770 by Poulletier de La Salle from gall stones. In 1815, it was isolated
from the unsaponifiable fraction of animal fats by M.E. Chevreul who named it cholesterine (Greek, khole, bile and stereos, solid). The correct formula (C27H46O) was proposed in 1888 by F. Reinitzer but structural studies from 1900 to 1932, mainly by H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact steric representation of cholesterol. In 1936, Callow and Young have designated steroids all compounds chemically related to cholesterol.
While it became clear very early that cholesterol plays an important role in controlling cell membrane permeability by reducing average fluidity, it appears now that it has a key role in the lateral organization of membranes and free volume distribution. These two parameters seem to be involved in controlling membrane protein activity and "raft" formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1).
The vertebrate brain is the most cholesterol-rich organ , containing roughly 25% of the total free cholesterol present in the whole body.
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In late-step synthesis of cholesterol, discrete oxidoreductive and/or demethylation reactions occur, which start with the common precursor lanosterol. It was also found as a major constituent of the unsaponifiable portion of wool fat (lanoline). Animal tissues contain in addition to cholesterol small amounts of 7-dehydrocholesterol which, on UV irradiation, Desmosterol is converted to an vitamin intermediate D3 between (cholecalciferol). lanosterol and
(24-dehydrocholesterol),
cholesterol, has been implicated with myelination processes. While high desmosterol levels could be detected in the brain of young animals (Paoletti R et al., J Am Oil Chem 21
Soc 1965, 42, 400) no desmosterol was found in the brain of adult animals. It is also known as an abundant membrane component in some mammalian cells, such as spermatozoa and astrocytes (Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654). Inability to convert desmosterol to cholesterol leads to the human disorder desmosterolosis (a severe developmental defect and cognitive impairment) (Waterham HR et al., Am J Hum Genet 2001, 69, 685).
In higher plants, the first sterols were isolated by Hesse (1878) from the Calabar beans (Phytostigma venenosum) which coined the term "phytosterine". This substance was later named stigmasterol (Windaus and Hault, 1906) from the plant genus. The denomination "phytosterol" was proposed in 1897 (Thoms H) for all sterols of vegetal origin. Chemically, these sterols have the same basic structure as cholesterol but differences arise from the lateral chain which is modified by the addition of one or two supernumerary carbon atoms at C-24 with either or chirality. The 24-alkyl group is characteristic of all phytosterols and is preserved during subsequent steroid metabolism in both fungi and plants to give hormones that regulate growth and reproduction in a manner similar to animals. Most phytosterols are compounds having 28 to 30 carbon atoms and one or two carbon-carbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain.
All phytosterols were shown to derive in plants from cycloartenol and in fungi from lanosterol, both direct products of the cyclization of squalene. 22
More than 200 different types of phytosterols have been reported in plant species. Representatives of these sterols are campesterol, stigmasterol (in soybean oil) and sitosterol which is present in all plant lipids and is used for steroid synthesis. An important sterol from yeast and ergot is the C28 compound ergosterol (mycosterol). Upon irradiation, this sterol gives rise to vitamin D2 (calciferol).
As ergosterol is a cell membrane component largely restricted to fungi, its amount in environmental matrices may be used as an index molecule for these micro-organisms in a living biomass (Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051).
23
Considerable variability in the concentration of free sterols was observed among different oils. While concentrations lower than 100 mg/100 g are found in oils from coconut, palm, olive, and avocado, concentrations between 100 and 200 mg/100 g are found in oils from peanut, safflower, soybean, borage, cottonseed, and sunflower, and concentrations between 200 and 400 mg/100 g are found in oils from sesame, canola, rapeseed, corn, and evening primrose (Phillips KM et al., J Food Comp Anal 2002, 15, 123). 24
Phytosterols produce a wide spectrum of biological activities in animals and
humans. They are considered efficient cholesterol-lowering agents. In addition, they produce a wide spectrum of therapeutic effects including anti-tumor properties. Further data on their metabolism and potential therapeutic action can be found in a review article (Ling WH et al., Life Sci 1995, 57, 195).
The European Commission authorized in 2004 the addition of phytosterols and phytostanols in food products with conditions of labeling including their amount per 100 g and the statement that the human consumption of more than 3 g/day should be avoided.
Phytostanols are a fully-saturated subgroup of phytosterols (they contain no double bonds). They occur in trace levels in many plant species but in high levels in tissues of only in a few cereal species. They are in general produced by hydrogenation of phytosterols.
Stanols often occur in dinoflagellates but are not common in other marine microalgae. Hence, dinoflagellates are often the major direct source of 5(H)-stanols in marine sediments (Robinson N et al., Nature 1984, 308, 439).
Fully saturated sterols are also found in animals but are of bacterial origin. Thus, the 5(H)-stanol coprostanol constitutes approximately 60% of the total sterols in human faeces.
25
While cholesterol was considered to be nearly absent in vegetal organisms, its presence is now largely accepted in higher plants. It can be detected in vegetal oils in a small proportion (up to 5% of the total sterols) but remains frequently present in trace amounts. An unusual relatively high content of cholesterol was described in camelina oil (about 200 mg per kg) (Shukla VKS et al., JAOCS 2002, 79, 965). However, several studies have revealed the existence of cholesterol as a major component sterol in chloroplasts, shoots and pollens. Furthermore, cholesterol has been detected as one of the major sterols in the surface lipids of higher plant leaves (rape) where he may amount to about 72% of the total sterols in that fraction (Noda M et al., Lipids 1988, 23, 439).
Although practical, the ancient distinction between zoosterols, mycosterols and phytosterols is no more used, since the same sterol may have different sources, but the appellation phytosterol is actually more frequently used.
Sterols are often isolated in the unsaponifiable fraction of any lipid extract and determined by various chromatographic procedures (HPLC or GLC).
Avenasterol can be isolated from oat oil. This sterol was shown to protect specifically frying oils from oxidation owing to its ethylidene group in the side chain (White PJ et al., JAOCS 1986, 63, 525).
26
An extensive review on the diversity, analysis, and health-promoting uses of phytosterols and phytostanols may be consulted with interest (Moreau RA et al., Prog Lipid Res 2002, 41, 457).
Sterols, or steroid alcohols are a subgroup of steroids with a hydroxyl group in the 3-position of the A-ring. They are amphipathic lipids synthesized from Acetyl coenzyme A. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar.
Sterols are important for the physiology of eukaryotic organisms. They form part of the cellular membrane where they modulate their fluidity and function and participate as secondary messengers in developmental signaling. 27
Different organisms utilize different sterols. The most important ones are cholesterol, phytosterols, and some steroid hormones in animals, and campesterol, sitosterol and stigmasterol in plants. Sterols are also known to block cholesterol absorption sites in the human gut thus helping to reduce cholesterol in humans by up to 15%. Test Microorganisms Escherichia coli
Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Escherichia Species: Escherichia coli 28
Escherichia coli, commonly known as E. coli, is a species of bacteria normally present in human intestines. A recently recognized strain, E. coli 0157:H7, produces high levels of toxins that can cause kidney damage, as well as septicemia, or blood poisoning. Symptoms can include diarrhea, chills, headaches, and high fever and in some cases the infection can lead to death, even with medical intervention. E. coli, discovered by Theodor Escherich, a German pediatrician and bacteriologist, is one of the main species of bacteria that live in the lower intestines of mammals. Specimens have also been located on the edge of hot springs. The bacteria are necessary for the proper digestion of food and are part of the intestinal flora. Presence in surface water is a common indicator of fecal contamination. It belongs among the Enterobacteriaceae, and is commonly used as a model organism for bacteria in general. One of the root words of the family's scientific name, "enteric", refers to the intestine, hence "gastroenteritis" (from 'gastro-', stomach, 'entero-' intestine, '-itis', inflammation). "Fecal" is the adjective pertaining to feces, so it is often used synonymously with "enteric". The number of individual E. coli bacteria in the feces that one human passes in one day averages between 100 billion and 10 trillion. All the different kinds of fecal coli bacteria, and all the very similar bacteria that live in the ground (in soil or decaying plants, of which the most common is Enterobacter aerogenes), are grouped together under the name coliform bacteria. Technically, the "coliform group" is defined to be all 29
the aerobic and facultative anaerobic, non-spore-forming, Gram-negative, rodshapedbacteria 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, 12 m in length and 0.10.5 m in diameter. Staphylococcus aureus
Kingdom: Bacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: S. aureus 30
Staphylococcus aureus is a genus of spherical bacteria capable of producing a heat stable toxin that cause illness in humans. The most common pathogen S. aureus, is frequently responsible for carbuncles, boils, pneumonia, abscesses and osteomyelities It exist in air, dust, sewage, water, humans, foods and animals. Staphylococcus aureus (which is occasionally given the nickname golden staph) is a bacterium, frequently living on the skin or in the nose of a healthy person, that can cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulitis) and abscesses, to life-threatening diseases such as pneumonia, meningitis, endocarditis, Toxic shock syndrome (TSS), and septicemia. Each year some 500,000 patients in American hospitals contract a staphylococcal infection. It is a spherical bacterium. It is abbreviated to S. aureus or sometimes referred to as Staph aureus in medical literature, and should not be confused with the somewhat similarly named streptococci which are also medically important. S. aureus is a Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and as large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacteria's name: aureus means "gold" in Latin. S. aureus is catalase positive and thus able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci. S. aureus can be differentiated from most other 31
staphylococci by the coagulase test: S. aureus is coagulase-positive, while most other Staphylococcus species are coagulase-negative. The species has been subdivided into two subspecies: S. aureus aureus and S. aureus anaerobius. The latter requires anaerobic conditions for growth, is an infrequent cause of infection, and is rarely encountered in the clinical laboratory. S. aureus may occur as a commensal on human skin (particularly the scalp, armpits and groins); it also occurs in the nose (in about 25% of the population) and throat and less commonly, may be found in the colon and in urine. The finding of Staph. aureus under these circumstances does not always indicate infection and therefore does not always require treatment (indeed, treatment may be ineffective and re-colonisation may occur). It can survive on domesticated animals such as dogs, cats and horses, but has never been found on food animals such as poultry or swine. It can survive for some hours on dry environmental surfaces, but the importance of the environment in spread of Staph. aureus is currently debated. It can host phages, such as the Panton-Valentine leukocidin, that increase its virulence. S. aureus can infect other tissues when normal barriers have been breached (e.g. skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of furuncles). In infants S. aureus infection can cause a severe disease Staphylococcal scalded skin syndrome (SSSS).[8] 32
S. aureus infections can be spread through contact with pus from an infected wound, skin-to-skin contact with an infected person, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person. Deeply situated S. aureus infections can be very severe. Prosthetic joints put a person at particular risk for septic arthritis, and staphylococcal endocarditis (infection of the heart valves) and pneumonia may be rapidly fatal
Candida albicans
Kingdom: Fungi Phylum: Ascomycota Subphylum: Saccharomycotina Class: Saccharomycetes Order: Saccharomycetales Family: Saccharomycetaceae Genus: Candida Species: C. albicans 33
Fungi belong to the genus Candida, especially Candida albicans, can infect both internal organs and mucous membranes of the mouth, throat and genital tract. In people with impaired immunity, this organism can cause a chronic infection.
Candida albicans is a diploid sexual fungus (a form of yeast), and a causal agent of opportunistic oral and vaginal infections in humans. Systemic fungal infections (fungemias) have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). In addition, hospital-related infections in patients not previously considered at risk (e.g. patients on an intensive care unit) have become a cause of major health concern. C. albicans is among the many organisms that live in the human mouth and gastrointestinal tract. Under normal circumstances, C. albicans lives in 80% of the human population with no harmful effects, although overgrowth results in candidiasis. Candidiasis is often observed in immunocompromised individuals such as HIV-positive patient. Candidiasis also may occur in the blood and in the genital tract. Candidiasis is commonly known as "thrush", and is a common condition that is usually easily cured in people who are not immunocompromised. To infect host tissue, the usual unicellular yeast-like form of Candida albicans reacts to environmental cues and switches into an invasive, multicellular filamentous form. 34
Materials used
A Soxhlet extractor is a type of laboratory glassware invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of lipid from a solid test material, but can be used whenever it is difficult to extract any compound from a solid. Typically, dry test material is placed inside a "thimble" made from filter paper, which is loaded into the Soxhlet extractor. The extractor is attached to a flask containing a solvent (commonly diethyl ether or petroleum ether) and a condenser. The solvent is heated, causing it to evaporate. The hot solvent vapor travels up to the condenser, where it cools and drips down onto the test material. The chamber containing the test material slowly fills with warm solvent until, when it is almost full, it is emptied by siphon action, back down to the flask. This cycle may be allowed to repeat many times. During each cycle, a portion of the lipid dissolves in the solvent. However, once the lipid reaches the solvent heating flask, it stays there. It does not participate in the extraction 35
cycle any further. This is the key advantage of this type of extraction; only clean warm solvent is used to extract the solid in the thimble. This increases the efficiency of the extraction when compared with simply heating up the solid in a flask with the solvent.
Liebig condenser The Liebig condenser is a piece of laboratory equipment where a straight glass pipe goes through a water jacket (a glass cylinder through which water constantly flows). It is used in distillation and reflux to condense vapours into liquid. History Though named after the German chemist Justus Baron von Liebig, he cannot be given credit for having invented it because it was already in use for some time before him. However, it is believed that the apparatus was made popular by him. The true inventors, all of them making the discovery independently, and the year of the invention were the German chemist Christian Ehrenfried Weigel in 1771, French scientist, P. J. Poisonnier, in 1779 and the Finnish chemist Johan Gadolin in 1791. Liebig himself incorrectly attributed the design to the German pharmacist Johann Gttling who had made improvements to the Weigel design in 1794 [1]. 36 Efficiency
The Liebig condenser is much more efficient than a simple retort due to its use of liquid cooling. Water can absorb much more heat than the same volume of air, and its constant circulation through the water jacket keeps the condenser's temperature constant. Therefore a Liebig condenser can condense a much greater flow of incoming vapour than an air condenser or retort.
At the end of an extraction, the excess solvent may be removed using a rotary evaporator, leaving behind only the extracted lipid. Petroleum ether Petroleum ether, also known as benzine or X4, is a group of various volatile, highly flammable, liquid hydrocarbon mixtures used chiefly as nonpolar solvents.
Petroleum ether is obtained from petroleum refineries as the portion of the distillate which is intermediate between the lighter naphtha and the heavier kerosene. It has a specific gravity of between 0.6 and 0.8 depending on its composition. Benzine should not be confused with benzene. Benzine is a mixture of alkanes, e.g., 37
pentane, hexane, and heptane, whereas benzene is a cyclic, aromatic hydrocarbon, C6H6. Likewise, petroleum ether should not be confused with the class of organic compounds called ethers, which contain the -O- functional group.
Anhydrous An ionic crystal is said to be anhydrous if it contains no water. An example of anhydration can be seen in copper (II) sulfate. If the water of crystallization is removed from blue crystals of copper (II) sulfate, a white powder (anhydrous copper sulfate) is formed.
The original formula for crystalline copper (II) sulfate is CuSO45H2O. The formula for anhydration is as follows: CuSO45H2O + heat CuSO4 + 5H2O
Another example is in the heating of magnesium sulfate heptahydrate, MgSO47H2O. On heating, it undergoes the following reaction: MgSO47H2O + heat MgSO4 + 7H2O 38
Analytical balances are accurate and precise instruments used to measure masses. They require a draft-free location on a solid bench that is free of vibrations. Some modern balances have built-in calibration masses to maintain accuracy. Older balances should be calibrated periodically with a standard mass.
39
DEFINITION OF TERMS
Antibiotics are molecules that are produced by one microorganism that kill (bacteriocidal) or inhibit (bacteriostatic) other microorganisms. They are one class of antibacterial and antifungal antimicrobials that can potentially be used as medicinal drugs to treat infections because of their low toxicity for humans or animals. Antimicrobial agents agents that kill or slow the growth of microbes like bacteria (antibacterial activity), fungi (antifungal activity), viruses (antiviral activity), or parasites (antiparasitic activity). Chloramphenicol - is a bacteriostatic antibiotic originally derived from the bacterium Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice in 1949.It was the first antibiotic to be manufactured synthetically on a large scale. Chloramphenicol is effective against a wide variety of microorganisms.In the West, the main use of chloramphenicol is in eye drops or ointment for bacterial conjunctivitis. Glucose Yeast Peptone is recommended for the isolation of yeasts from soils specimen. This is a highly nutritious medium, which may be used for microbial examination.
40
Incubate to give the best or optimum conditions (ex: temperature, moisture) for growth and development.
Inoculate to put microorganism or a substrate of organism on a medium.
Inoculation a process of implanting infectious material into a culture medium.
Inoculum - population of a pure culture grown in a medium.
Inhibition zone - this is an area around a paper disk or colony of bacteria or mold where no other organisms are growing.
Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in water, sewage, feces and other materials. It is used in the laboratory for the cultivation and maintenance of nonfastidious species and used in microbiological examination of a broad spectrum of materials. It is a simple medium composed of beef extract, peptone, and agar. It has been one of the most generally used media in bacteriological procedures. It is used for the ordinary routine examinations of water, sewage, and food products, for the carrying of stock cultures, for the preliminary cultivation of samples submitted for bacteriological examination, and for isolating organisms in pure culture. 41
Chapter II METHODOLOGY
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 TREATMENTS T0 (control) Chloramphenicol 3 T1 With Stonefish Dorsal Fin Venom extract 3
NUMBER OF TRIALS
DEPENDENT VARIABLE: Zone of inhibition CONSTANTS: Amount of extract
Materials:
A. Extraction sterile syringe sterile cotton sterile knife sterile blade sterile pair of scissor test tubes gloves ice cubes basin sterile forceps Solvent extraction unit, fitted with Liebig Condenser Thimbles Heat source Analytical balance (capable of weighing up to 0.1 mg) Desiccators Distilling flasks (125-250 mL) Grinder or mortar and pestle Reagents ( Petroleum Ether, Anhydrous, Analytical Grade) B. Antimicrobial Assay Test Agar plates Cork borer 43
Nutrient Agar (NA) Glucose Yeast Peptone (GYP) Agar Culture Bacteria and Yeast- E.coli, S. aureus, C. albicans Inoculating loop Sterile cotton swab Commercial antibiotic (Chloramphenicol) Alcohol lamp Beaker with 95% Ethanol Pippette C. Phytochemical Test acetic anhydride sulfuric acid 1 ml 10% hydrochloric acid 1% hydrochloric acid Mayers Reagent ferric chloride 2 test tubes anhydrous sodium carbonate sodium hydroxide potassium sodium tartrate distilled water
44
GENERAL PROCEDURE
A. Reagents All reagents (biochemical- equipment) were supplied by Regional Science High School for Region 1 Laboratory, Adamson University, Marine Science Institute and Natural Science of Research institute of University of the Philippines.
B. Collection of Animal Specimen Fifteen Stonefish (Synanceia verrucosa) were gathered in the coastal area of Paraoir, Balaoan, La Union. They were subjected to observation for 2 days with food and seawater in a wide aquarium, the said specimen were in good physical condition and ready for dorsal fin venom extraction.
C. Preparation of the Extract Each Stonefish was applied with sterile cotton having 90% isopropyl alcohol before inserting a sterile knife on the point where the head meets the body and cutting through the joint. Most of extracted liquid was found in the specimens dorsal fin venom. There were about 10mL of liquid extract obtained and placed in a test tube covered with cotton plug then refrigerated at 150C for 24 hours and was used for the Antimicrobial assay. This was conducted under the direct supervision of Dr. Lourdes Cruz and Mr. Chris Mendoza in their technical assistance of proper handling procedures of Stonefish.
45
The needed extract for the Phytochemical Test was prepared using the soxhlet method as stated below: 1) The sample (Stonefish Dorsal Fin venom) was placed into the extraction thimble. 2) The thimble was placed in the soxhlet extraction fitted with a Liebig Condenser. 3) Thirty five (35) mL of pure petroleum ether was transferred to dry and the distilling flask with 125-250 mL capacity was connected to the extraction unit. 4) Turn the heat source on. 5) The extraction proceed to 6 to 8 hours. 6) Turn off the heat source after, and then allow the extraction tube to drain for 15 minutes. 7) The thimble with the sample was removed. 8) Turn the heat source to recover the ether. 9) After the ether has been completely evaporated and collected into the extraction tube, turn off the heat source. 10) The distilling flask was removed and was put it in the preheated (1050C) oven for 1 hour. 11) It was cooled in a desiccator and weighed. 12) The flask was again heated in the oven for 1 hour. 13) The procedure # 11 and 12 was repeated until the weight of the flask after two consecutive weighing agrees to within 0.5mg.
46
D. Preparation of the Test Organisms Escherichia coli, Staphylococcus aureus and Candida albicans were obtained from Natural Science Research Institute (NSRI) at UP Diliman that were used as test organisms. E. Antimicrobial Assay Test Microbial suspensions were prepared from 24- hour cultures of the Escherichia coli, Staphylococcus aureus (bacteria) and Candida albicans (yeast). The suspending medium used was 0.1% peptone water. One-tenth (0.1) mL aliquots of the bacterial and yeast suspensions were transferred into pre-poured Nutrient Agar (NA) and Glucose Yeast Peptone (GYP) Agar, respectively. Five (5) of the corresponding medium, melted and cooled to 450C, was poured onto the agar plate and swirled to distribute the inoculum evenly on the agar surface. Three (3)mL of the sample was placed in each hole. The plates were incubated at room temperature. NA and GYP plates were observed after 24-48 hours. The clearing zone was measured in millimeters and the average diameter of the clearing zones was calculated. The antimicrobial index (AI) was computed using the following formula:
AI
Diameter of clearing zone Diameter of well Diameter of well
47
F. Phytochemical Test Dorsal Fin Venom extract from fresh specimens of Synanceia verrucosa were used for Phytochmical tests.
7) Test for Sterols and Tipertenes Lieberman-Berchard Test
A small amount of the sample extract(Stonefish Dorsal Fin Venom) in acetic anhydride was dissolved. The soluble portion was decanted and to this, 1-2 drops of concentrated sulfuric acid was added. Observe a green color, either immediately or solely going into red and blue tones. A pink to red color is indicative of triterpenoids while a blue color is indicative of steroids.
8) Test for Flavonoids One (1) ml of sample extract was treated with 1 ml 10% hydrochloric acid and a few magnesium turnings. Formation of red color is observed.
9) Test for Alkaloids The sample extract was extracted with 1% HCL and drops of Mayers Reagent or Wagners Rgt. was added to the filtered acid extract. A cream colored precipitate is observed in the case of Mayers Rgt. while a reddish brown ppt. is observed in the case of Wagners Rgt. 48
Formula of Mayers 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 Wagners Rgt.:
1.3 g iodine crystals and 2.0 g potassium iodide in sufficient amount of distilled water to make a total volume of 100ml was dissolved.
10) Test for Tannins
The sample extract was extracted with hot water and the aqueous extract was filtered. Upon addition of two drops of ferric chloride test solution, a dark color and precipitate forms which may either be black, dark blue, blue black, green or blue green.
Ferric chloride TS: Dissolve 9g of ferric chloride in dist, water to make 100 ml.
49
11) Test for Saponins
The sample extract was dissolved in hot water. The aqueous extract when shaken vigorously should become frontly. The froth, honeycomb in nature should persist for at least 30 minutes.
12) Test for Glycosides
The sample extract was dissolved in hot water and filtered. The filtrate was used for the test. Two test tubes were used. Two ml of sample was placed in each tube. One ml of dilute hydrochloric acid was added to sample 1. Nothing is added to sample 2. The 2 test tubes were placed in a boiling water bath for 5 minutes. Then the test tubes were cooled. The samples were both neutralized with anhydrous sodium carbonate until no more effervescence is produced. Then add Fehling s B. One ml of Fehling s solution was used. The 2 test tubes were heated in a water bath for 2 minutes. Observe the amount of brick red precipitate that formed. An increase in the amount of brick red precipitate in the hydrolyzed sample (the sample to which dilute acid was added) as compared to the other sample indicates the presence of glycosides.
50
Fehlings solution A:
Copper Sulfate (CuSO4. 5H2O)34.66 grams Distilled water, a sufficient quantity
To make500 ml
Dissolve the copper sulfate in the distilled water and mixed.
Fehlings Solution B: Sodium Hydroxide50 grams Potassium Sodium Tartrate173 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 Fehlings A and B in equal amount before using.
51
Reagents
Collection of Animal Specimen
Preparation of the Extract
Preparation of the Test Microorganisms
Antimicrobial Assay Test
Figure 1. The
Phytochemical Test
Flowchart of Experimental Design
52
Preparing the necessary laboratory materials
PositioningSterilizing laboratory materials 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.
Using a sterile syringe, eject it on the thin venom duct that leads to the tip of the spine.
Preserving the fluid venom extract in a sterilized bottle and refrigerate. 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 1 2 3 Total (mm) Mean (mm)
T0 (mm) 3.2 3.2 3.2 9.6 3.2
T1(mm) 3.5 3.5 3.4 10.4 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 T 0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
Table 2. Mean Zone of Inhibition against S. aureus in millimeters (mm)
Trials
T0 (mm)
T1(mm)
1 2 3 Total (mm) Mean(mm)
3.2 3.2 3.2 9.6 3.2
3.4 3.4 3.3 10.1 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 T 0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is slightly greater than the Mean Zone of Inhibition of T0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
55
Table 2 .Mean Zone of Inhibition against C. albicans in millimeters (mm)
Trials 1 2 3 Total(mm) Mean(mm)
T0(mm) 3.2 3.2 3.2 9.6 3.2
T1(mm) 2.8 2.8 2.7 8.3 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 T 0 (Chloramphenicol) is 3.2mm. This proves that the Mean Zone of Inhibition of T1 is less than the Mean Zone of Inhibition of T0. Likewise, results showed that there is a significant difference between the treatments in terms of zone of inhibition.
56
Table 4.Summary table of the Mean Zone of Inhibition of Test Microorganisms in millimeters (mm)..
Bacteria/ Yeast
Escherichia coli Staphylococcus aureus Candida albicans
T0(mm) 9.6 9.6 9.6 28.8 9.6
T1(mm) 10.4 10.1 8.3 28.8 9.6
Total(mm) Mean(mm)
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 T 0. Likewise, results showed that there is no significant difference between the treatments in terms of zone of inhibition.
57 Table 5. A Summary table of the Active Principles of the Dorsal Fin Venom.
Active Principles/Analytes Alkaloids
Color reaction Reddish brown
RESULT 3
Glycosides Tannins
Red black, dark blue, blue black, green or
0 0 3 0 0 3
Saponin Flavonoids Tipertenes Sterols
blue green Yellow/. red Red pink to red blue
The table shows the Phytochemical Test result of the Stonefish Dorsal Fin Venom sample. It shows that alkaloids, Tannins and Saponin are very abundant. This contributed much on the effectiveness of Stonefish Dorsal Fin Venom as an antibiotic agent as revealed on the table
Legends:
3 = Very Abundant (51-100%) 2 = Abundant (26-50%) 1 = Detectable (1-25%) 0 = Absent
58
Figure 3. Mean of Zone of Inhibition of E.coli, S. aureus and C. albicans treated by the extract (3mL) from Stonefish Dorsal Fin Venom.
Zone of Inhibition of Bacteria in Diameters (mm)
3.5 3 2.5 2 1.5 1 0.5 0 E.coli S.aureus C.albicans
The figure shows the Mean of Zone of inhibition of E.coli is 3.5 mm, the Mean Zone of Inhibition of S. aureus is 3.4mm while the Mean Zone of inhibition of C. albicans is 2.8mm.This proves that the Stonefish Dorsal Fin Venom inhibit the growth of E.coli the most compared to others.
59 Figure 4. Mean of Zone of Inhibition of E. coli, S. aureus and C. albicans treated with 3mL of Chloramphenicol syrup.
3.5 3 Zone of Inhibition of Bacteria in Diameters 2.5 2 1.5 1 0.5 0 E .c oli S .aureus C.albic ans
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.
Comparison of Zone of Inhibition of Bacteria in Diameters (mm)
4 3.5 3 2.5 2 1.5 1 0.5 0 3mL of Chloramphenic ol syrup 3mL of Stonefish Dorsal Fin venom
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 IV CONCLUSIONS AND RECOMMENDATIONS Conclusions
Based from the findings, the following conclusions were drawn:
d) The Stonefish Dorsal Fin Venom can inhibit the growth of harmful microorganisms.
e) Phytochemical analysis showed the presence of active principles from the Stonefish Dorsal fin venom. Alkaloids, Tannins and Saponins were observed very abundant.
f) There is no significant difference of using the Antibiotic Property of Stonefish Dorsal Fin and the Controlled antibiotic in terms of zone of inhibition.
62
Recommendations
Based from the findings and conclusions the following are recommended:
1. This can be a basis for the Pharmaceutical industries in making an antibiotic out of the Stonefish Dorsal Fin Venom.
2. Additional Brand of antibiotics shall be made available for comparing the effect of Stonefish Dorsal Fin Venom to test further efficacy.
3. Wide dissemination of the latest technology must commence.
63
BIBLIOGRAPHY
Gwee, M.C., Gopalakrishnakone, P., Yuem,R.,Khoo,H.E.,Low,K.S.Y.,A review of stonefish venoms and toxins.Pharmac.Ther.64, 509(1994).
Internet websites, Yahoo.com; Google .com; and MSN Network.
Microsoft Encarta Library Edition 2006.
Sutherland,S.K.,Tibballs,J.,Australian animal toxins. Oxford Univ.Press, Melbourne (2001).
Phillips KM et al., J Food Comp Anal 2002, 15, 123 Barajas-Aceves M et al. J Microbiol Methods 2002, 50, 227; Charcosset JY et al., Appl Environ Microbiol 2001, 67, 2051 Waterham HR et al., Am J Hum Genet 2001, 69, 685 Lin DS et al., J Lipid Res 1993, 34, 491 - Mutka AL et al., J Biol Chem 2004, 279, 48654 Paoletti R et al., J Am Oil Chem Soc 1965, 42, 400 Wikipedia, free Dictionary 64
Appendices
I. Statistical test for the zone of inhibition of Escherichia coli treated with Chloramphenicol (T0) and with the Stonefish Dorsal Fin Venom (T1).
A
TRIALS 1 2 3 Total T0 (x) 3.2 3.2 3.2 9.6 N1=3 T0 (x2) 10.24 10.24 10.24 30.72 T1 (y) 3.5 3.5 3.4 10.4
B
T1 (y2) 12.25 12.25 11.56 36.06 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 = 6-2 =4 Tcrit.=- 2.776
Compute for t
X
A
= 9.6 /3= 3.2
= 10.4/ 3 = 3.5
SX 1- Y 2=
( X 1 ) 2 + Y 2 ( Y2 ) 2 2 X 2 1 N1 N2 1 1 N + N N1 + N 2 2 1 2
(9.6) 2 30 .72 3
(10 .4) 2 + 36 .06 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 36 .06 36 .05 ) (0.666 )

4
0.01 (0.666 ) 4
66 =
0.0 2 (0.6 6 ) 05 6
= 0.0408
t cal =
X 1 Y 2 0 S X 1 Y 2
= 3.2 3.5 / 0.0408
= -7.353

cal
significant difference of using the Antibiotic Property of Stonefish Dorsal Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.
67 II. Statistical test for the zone of inhibition of Staphylococcus aureus treated with the commercial antibiotic Chloramphenicol (To) and with the Stonefish Dorsal Fin venom ( T1).
C
TRIALS 1 2 3 T0 (x) 3.2 3.2 3.2 T0 (x2) 10.24 10.24 10.24 T1 (y) 3.4 3.4 3.3
D
T1 (y2) 11.56 11.56 10.89
Total
9.6 N1=3
30.72
10.1
34.01 N2=3
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 = 6-2 =4 Tcrit.= -2.776
Compute for t
= 9.6/ 3 = 3.2
= 10.1/ 3 = 3.37
SX 1- Y 2=
( X 1 ) 2 + Y 2 ( Y2 ) 2 2 X 2 1 N1 N2 1 1 N + N N1 + N 2 2 1 2
(9.6) 2 30 .72 3
(10 .1) 2 + 34 .01 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 34 .01 34 ) (0.666 )

4
==
0.01 (0.666 ) 4
69
0.0 2 (0.6 6 ) 05 6
= 0.0408
t cal = t =
X 1 Y 2 0 S X 1 Y 2
= 3.2 3.37 / 0.0408
= -4.1667

cal
so the null Hypothesis was accepted. Thus, there is no
significant difference of using the Antibiotic Property of Stonefish Dorsal Fin and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.
70
III. Statistical test for the zone of inhibition of Candida albicans treated with the commercial antibiotic Chloramphenicol (To) and with the Stonefish Dorsal Fin venom ( T1).
E
TRIALS 1 2 3 Total T0 (x) 3.2 3.2 3.2 9.6 T0 (x2) 10.24 10.24 10.24 30.72 T1 (y) 2.8 2.8 2.7 8.3
F
T1 (y2) 7.84 7.84 7.29 22.97
N1=3
N2=3
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 = 6-2 =4 Tcrit.= 2.776
Compute for t
= 9.6/ 3 = 3.2
= 8.3/ 3 = 2.8
SX 1- Y 2=
2 2 X 2 ( X 1 ) + Y 2 ( Y2 ) 2 1 N1 N2 1 1 + N N1 + N 2 2 N2 1
(9.6) 2 30.72 3
(8.3) 2 + 22.97 3 4
1 1 + 3 3
( 30 .72 30 .72 ) + ( 22 .97 22 .96 ) (0.666 )

4
0.01 (0.666 ) 4
72 =
0.0 2 (0.6 6 ) 05 6
= 0.0408
t cal = t =
X 1 Y 2 0 S X 1 Y 2
= 3.2 2.8/0.0408
=9.8039
Decision: Reject H0
The t
cal
is greater than the tcrit so the Null Hypothesis was rejected. Thus, there is a
significant difference of using the Antibiotic Property of Stonefish and the commercial antibiotic Chloramphenicol in terms of zone of inhibition.
73
PLATES
Plate 1. The Stonefish Habitat.
74
Plate 2. The Animal Specimen Stonefish ( Synaceia verrucosa).
75
Plate 3. Materials for the Extraction and Dissection of Stonefish.
76
Plate 4. Researcher Dissecting the Specimen.
77
Plate 5. Researcher Inserting the Knife Above the Dorsal Fin.
78
Plate 6.Reseacher Pinning down the tail end of the Dorsal Fin using a Sterile Knife.
79
Plate 7. Researcher Grasping the Tail Fin and Pulling it way from the Dorsal Fin.
80
Plate 8. Researcher Extracting the Venom using a Sterile Syringe.
81
Plate 9: The Extracted Venom.
82
Plate 10. Materials used for the Antimicrobial Assay Test
83
Plate 11.Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient Agar and Glucose Yeast Peptone Agar.
84
85
Plate 13.Reseacher Swirling Agar Plates to distribute the inoculums evenly in the Agar Surface.
86
Plate 14.Reseacher making holes on the Agar Plates using the Cork Borer.
87
Plate 15. The bored Agar Plate Surface.
88
Plate 16.Researcher Placing the Extracts in each hole.
89
Plate 17.Researching Incubating the Plates at room temperature.
90
Plate 18. The treatments To (Chloramphenicol) and T1 ( Stonefish Dorsal Fin Venom).
91
CURRICULUM VITAE
Name: Lyra Erika Liclican Age: 16years old Address: Paratong, Sta.Cruz, Ilocos Sur Birthday: September 1, 1990 Parents: Mr. Raul Liclican Mrs. Lydia Liclican Religion: Roman Catholic Nationality: Filipino
Civil Status: Single Educational Attainment: Elementary St. Joseph Institute Candon City, Ilocos Sur St. Augustines School Tagudin, Ilocos Sur Secondary Regional Science High School for Region 1 Bangar, La Union
DATA BOOK
SUCCESS CALENDAR
1. CHOOSING A TOPIC Planned Date May 3,2006 Date Completed May 10,2006
This is the hardest thing to do when doing an Investigatory Project. A narrowed down topic that would make sense and can be attained and give benefit to the majority. And fortunately my title, Antibiotic Property of Stonefish (Synanceia verrucosa) Dorsal Fin Venom against E,coli, C. albicans and S.aureus.
2 COLLECTING BACKGROUND INFORMATION
Date Completed May 15, 2006
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 AND GATHERING OF DATA
Date Completed June 2, 2006
With the suggestions of Dr. Lourdes Cruz of MSI at UPD, I design for the procedures to follow in dissecting and extracting the Stonefish Dorsal Fin Venom. Before going again go to UPD, Stonefish were collected at Paraoir, Balaon, La Union and were placed in an ice box. Dr. Cruz of MSI allowed me to conduct the dissection and extraction of my specimen with hier supervision. The extracted venom was tested at NSRI for Antimicrobial assay with Mrs.Vina Argayosa.
Then, soxhlet method was used for the extraction of my specimen for the Phytochemical Test at Adamson University.
4 MAKING OF THE WRITE UP (MANUSCRIPT) Background of the Study
Planned Date July 28,2006
5. MAKING THE STATEMENT OF THE PROBLEM, HYPOTHESES, SIGNIFICANCE OF THE STUDY UNTIL SCOPE AND DELIMITATION
Planned Date August 4, 2006
6.MAKING THE REVIEW OF RELATED LITERATURE
Date Completed August 26,2006
7. MAKING THE CHAPTER II ( Methodology)
Date Completed September 12, 2006
8. MAKING THE CHAPTER III (Results and discussions)
Date Planned September 13,2006
9. MAKING THE CHAPTER IV
Date Planned
Date Completed
(Conclusions and Recommendations)
Sept. 18, 2006
Sept. 20,2006
10. MAKING THE BIBLIOGRAPHY, APPENDICES
Date Planned Sept.22, 2006
10. FINALIZATION
11. PRINTING OF THE MANUSCRIPT
Date Completed Sept 30.2006
Table of Contents
Abstract.....i CHAPTER 1 Introduction..1 Background of the Study.....1-2 Statement of the Problem.........2 Hypotheses...3 Significance of the Study.3-4 Scope and delimitation.4 Review of related Literature
Description of the Animal Specimen..5-12 Phytochemical Components..12-28 Test Microorganisms...28-34 Materials used.....34-39 Definition of terms..40-41 CHAPTER II Methodology Experimental Design.42 Materials....43-44 Reagents...45 Collection of Animal Specimen45 Preparation of the Extract.45-46 Preparation of Test Organisms..47 Antimicrobial Assay Test.47 Phytochemical Test...48-51
CHAPTER III RESULTS AND FINDINGS54-61 CHAPTER IV COCLUSIONS AND RECOMMENDATIONS Conclusions...62 Recommendations.....63 Bibliography.64
Appendices....65-73
Table of Contents
Abstract .i CHAPTER 1
Introduction .1 Background of the Study ..1-2
Statement of the Problem ..2
Hypotheses ..3 Significance of the Study .3 Scope and delimitation ..4 Review of related Literature ...5 Description of the Animal Specimen 6-8 Phytochemical Components 8-25
Test Microorganisms ...25-33 Definition of Terms 34-35 CHAPTER II Methodology Experimental Design .36
Materials ...37-38
Reagents .38 Collection of Animal Specimen 39
Preparation of the Extract .39 Preparation of the Test Organisms ..39 Antimicrobial Assay Test ..40 Histochemical Test ...41-45
CHAPTER III RESULTS AND FINDINGS ...47-55
CHAPTER IV COCLUSIONS AND RECOMMENDATIONS
Conclusions 56
Recommendations 57 Bibliography .58 Appendices 59-70
Plate 1. Researchers Collecting the Plant Specimen.
Plate 2. The Plant Specimen Nut Grass (Cyperus rotundus). Plate 3. The Nut Grass Rhizome.
Plate 4. Materials for the Reflux Method (Extraction)
Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method.
Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper. Plate 7. Materials for the Antimicrobial Test.
Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient Agar and Glucose Yeast Peptone Agar
Plate 10. Researcher Swirling Agar Plates to distribute the inoculums evenly in the Agar Surface.
Plate 11. Researcher making holes on the Agar Plates using the Cork Borer.
Researcher Placing the Extracts in each hole Plate 13. Researching Incubating the Plates at room temperature Plate 14.The treatments To ( Chloramphenicol) and T1 ( Nut Grass rhizome extract).
List of Plates
Plate 1. Researchers Collecting
the Plant Specimen ..71
Plate 2. The Plant Specimen Nut Grass (Cyperus rotundus) ............................ .......................................72
Plate 3. The Nut Grass Rhizome ..73
Plate 4. Materials for the Reflux Method (Extraction) 74
Plate 5. Researchers extracting the Nut Grass Rhizome using the Reflux Method .75
Plate 6.Resechers Filtering the extracted Nut Grass Rhizome using the Filter paper......................................... ........................76 Plate 7. Materials for the Antimicrobial Test.. 77
Plate 8. .Researcher transferring Aliquots of Bacterial and Yeast Suspension to Nutrient Agar and Glucose Yeast Peptone Agar 78
Plate 9. Researcher Pouring the Medium onto the Agar Plates 79
Plate 10. Researcher Swirling Agar Plates to
distribute the inoculums evenly in the Agar Surface..80
Plate 11. Researcher making holes on the Agar Plates using the Cork Borer ..81
Plate 12.Researcher Placing the Extracts in each hole.82
Plate 13. Researching Incubating the Plates at room temperature ...83
Plate 14.The treatments To ( Chloramphenicol) and T1 (Nut Grass rhizome extract) .84
List of Tables
Table 1. Mean Zone of Inhibition Against E. coli in millimeters (mm). .47
Table 2. Mean Zone of Inhibition Against S. aureus in millimeters (mm). ..48
Table3. Mean Zone of Inhibition Against C.albicans in millimeters (mm) .49
Table 4. Mean Zone of Inhibition Against A. niger in millimeters (mm). ..50
Table 5. Mean Zone of Inhibition
Test Microorganisms in millimeters (mm) 51
Table 6 A Summary table of the Active Principles of the Nut Grass rhizome 52
List of Figures
Figure
1.
The
Flowchart
of
Experimental
Design 46
Figure 2. Mean of Zone of Inhibition of E.coli, S. aureus, C. albicans and A. niger treated by the extract (5mL) from the Nut Grass rhizome .53
Figure 3. Mean of the Zone of Inhibition of E. coli, S. aureus C. albicans and A. niger treated with 5mL of Chloramphenicol syrup 54
Figure 4. Comparison of the Mean on the Zone of Inhibition o f Test Microorganisms when treated by the Nut Grass rhizome extract and to 5mL of Chloramphenicol syrup ....55

Nut Grass (Lyra Erika Liclican)

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Department of Education Region 1 Division of La Union Regional Science High School for Region 1 Bangar, La Union

An Entry to the Intel Philippines Science Fair Regional Level

Lyra Erika Liclican Glen Bernard Asto

Mr. Rogelio Valdez Research Adviser

We thank you all

Lyra Erika Liclican Glen Bernard Asto

CHAPTER IV COCLUSIONS AND RECOMMENDATIONS Conclusions. Recommendations.. Bibliography Appendices..

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 7. Materials for the Antimicrobial Test

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)

Figure 1.The Flowchart of the Experimental Design

B. Statement of the Problem

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

D. Significance of the Study

E. Scope and Delimitation of the Study

F. Review of Related Literature

, anti-microbial and anti-cancer activity. In addition, flavonoids act as

Test Microorganisms Escherichia coli

Genus: Staphylococcus Species: S. aureus

TREATMENTS NUMBER OF TRIALS

T1 With Nut Grass rhizome extract 3

DEPENDENT VARIABLE: Zone of inhibition CONSTANT: Amount of extract 36

B. Antimicrobial Assay Test Agar plates

Diameter of clearing zone Diameter of well Diameter of well

1) Test for Sterols and Tipertenes Lieberman-Berchard Test

2) Test for Flavonoids

Formula for Wagners Rgt.:

4) Test for Tannins

42 5) Test for Saponins

6) Test for Glycosides

Copper Sulfate (CuSO4. 5H2O)34.66 grams Distilled water, a sufficient quantity

Dissolve the copper sulfate in the distilled water and mixed.

Fehlings Solution B: Sodium Hydroxide50 grams

Potassium Sodium Tartrate173 grams Distilled water, a sufficient quantity

Note: Mix fehlings A and B in equal amount before using.

8.) Test for the presence of fats and Oils

Antimicrobial Assay Test

Chapter III RESULTS AND DISCUSSONS

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

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

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

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

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

Zone of Inhibit ion in Diame ters (mm)

3.5 3 2.5 2 1.5 1 0.5 0 E.coli S.aureus C. albicans A.niger

Comparison of Zone of Inhibition of Bacteria in Diameters (mm)

Chapter IV CONCLUSIONS AND RECOMMENDATIONS Conclusions

Based from the findings, the following conclusions were drawn:

c) Wide dissemination of the latest technology must commence

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

Microsoft Encarta Library Edition 2006.

(10 .9) 2 + 39 .61 3 4

( 30 .72 30 .72 ) + ( 39 .61 39 .6) (0.666 )

= 3.2 3.63 / 0.0408

Decision: Accept H0 The tcrit is greater than the t

so the Null Hypothesis was accepted. Thus, there is no