Chapter 1

The Problem and its Setting

The chapter presents the problem under study. It shows the nature, the scope and problem and its historical and theoretical background. It includes the background of the study, statement of the problem, hypotheses, significance of the study, review of related literature, operational definition of terms, and scope and limitation. 1.1 Background of the study

Imperata cylindrica, more commonly known as cogon grass, is a perennial

Rhizomatous grass native to east and Southeast Asia, India, Micronesia, Australia and eastern and southern Africa. It grows from 0.6-3 m (2-10 feet) tall. The leaves are about 2 cm wide near the base of the plant and narrow to a sharp point at the top; the margins are finely toothed and are embedded with sharp silica crystals. The main vein is a lighter colour than the rest of the leaf and tends to be nearer to one side of the leaf. The upper surface is hairy near the base of the plant while the underside is usually hairless. Roots are up to 1.2 meters deep, but 0.4 m is typical in sandy soil.

Eucalyptus deglupta is a tall tree, commonly known as the Rainbow Eucalyptus, the Mindanao Gum or the Bagras in the Philippines. It is the only Eucalyptus species found naturally in the Northern Hemisphere. Its natural distribution spans New Britain, New Guinea, Ceram, Sulawesi and Mindanao. Now, this tree is cultivated widely around the world, mainly for pulpwood used in making paper. It is the dominant species used 1

for pulpwood plantations in Philippines. This tree is also grown for ornamental purposes, due to the showy multi-coloured streaks that cover the trunk. Patches of outer bark are shed annually at different times, showing the bright-green inner bark. This then darkens and matures to give blue, purple, orange and then maroon tones.

The treatment of pain continues to be the subject of considerable pharmaceutical and clinical research. Microbial infections often produce pain and inflammation. Chemotherapeutic, analgesic, and anti-inflammatory drugs are prescribed

simultaneously in normal practice. The compound possessing in all three activities is not common. In view of these, it is the main goal of the study to determine the antibacterial and pharmacological properties through the following tests: Analgesic, Antipyretic and Antiinflammatory testings of the combined extract of cogon (Imperata cylindrica) and bagras (Eucalyptus deglupta). And also, to determine whether it is toxic or not with the use of guppies with different concentrations of the extract of cogon and bagras.

1.2

Statement of the problem This study aims to determine the in vitro synergistic pharmacological and

antibacterial properties of cogon (Imperata cylindrica) and bagras (Eucalyptus deglupta). Specifically this study was conducted to answer the following questions:

A. Does the combined extract of cogon (I. cylindrica) and bagra s (E. deglupta) have pharamcological properties?

B. Does the combined extract of cogon (I. cylindrica) and bagras (E. deglupta) have antibacterial property? C. What are phytochemical constituents present in the extracts of cogon (I. cylindrica) and bagras (E. deglupta)? D. What is the toxicity level of the individual extracts of cogon (I. cylindrica) and bagras (E. deglupta)?

1.3

General Objectives

This study aims to evaluate the synergistic pharmacological and antibacterial property of cogon (I. cylindrica) and bagras (E. deglupta). It also seeks to identify the phytochemical constituents of the two plants and its toxicity levels. The

pharmacological properties of the combined extracts of cogon (I. cylindrica) and bagras (E. deglupta) will be determined through the following tests: Anti-inflammatory Testing, Antipyretic Testing and Analgesic Testing. 1.4 Specific Objectives This study aims: A. To find out the pharmacological properties of the combined extracts of cogon (I. cylindrica) and bagras (E. deglupta) through analgesic, anti-inflammatory, and antipyretic tests. B. To assess the phytochemical constituents of cogon (I. cylindrica) and bagras (E. deglupta).

C. To discover if the combined extracts of cogon (I. cylindrica) and bagras (E. deglupta) have an antibacterial property. D. To evaluate the toxicity levels of the individual extracts of cogon (I. cylindrica) and bagras (E. deglupta).

1.5 Hypotheses ALTERNATIVE: There is a synergistic pharmacological and antibacterial property of cogon (I. cylindrica) and bagras (E. deglupta) extracts with the presence of secondary metabolites.

NULL: The combined extract of cogon (I. cylindrica) and bagras (E. deglupta) has no pharmacological and antibacterial properties with the presence of secondary metabolites.

1.6 Significance of the study It is a fact that nowadays, the prices of medicines are getting higher and higher, far above the affordable prices. So, we could confidently say that this study is significant in the discovery of other uses for the common plant, Imperata cylindrica and Eucalyptus deglupta. Through this experiment, we can discover more cures for those diseases that are caused by different kinds of bacteria. If the study is successful, a cheaper alternative to antibacterial, antipyretic, analgesic and anti-inflammatory agents may be found. This would help alleviate the concerns of rising medical cost. It would also add to the available alternative for

antibiotics. The need to conduct the study is to find an abundant natural resource of the Philippines. This study helps find other uses of the Ethanol extract of the Cogon and Bagras. Utilizing the available resources in the country and helping to benefit all members of the society are important factors of this study, with the application of different methods. Instead of becoming hazard, the methanol extract of the palm tree may just become a useful component to be studied.

1.7 Scope and Limitation This study is only limited to the study of the In vitro synergistic pharmacological and antibacterial interaction between bagras (E. deglupta) and cogon (I. cylindrica). The scope of this study is to prove whether the combined extracts of cogon (I. cylindrica) and bagras (E. deglupta) have pharmacological and antibacterial properties. The pharmacological property will be determined through the following tests: Antiinflammatory Testing, Antipyretic Testing and Analgesic Testing. Toxicity Testing was conducted to determine the toxicity level of the extracts with the use guppies with different concentrations. The researcher used four treatments. Treatment 1 is the positive control which is the reference drug, treatment 2 is the extract of bagras only, treatment 3 is the extract of cogon only and treatment 5 is the combined extract of cogon (I. cylindrica) and bagras (E. deglupta).Each treatment was replicated three times. These said experiments were conducted from August to September in the Science Laboratory of General Santos Hope Christian School.

Chapter 2 Review of Related Literature

This chapter presents the review of related literature and the operational definition of terms used in this research. Literatures were cited on the bases of their importance in the conduct of the study.

Agar Agar is prepared by boiling the algae in water, after which the filtered solution is cooled, purified, and dried. It is an amorphous, translucent material that is packaged in granules, flakes, bricks, or sheets. One of its chief uses is as a gelling agent in media for culturing microorganisms. It is also used in making confections, as an emulsifier in cosmetics and food products, as a sizing agent, as an inert carrier of drugs in medicine, and as a laxative. Agar is a gelatinous substance chiefly used as a culture medium for microbiological work. It is an unbranched polysaccharide obtained from the cell walls of some species of red algae or seaweed. It can be used as a laxative, a vegetarian gelatin substitute, a thickener for soups, in jellies, ice cream and Japanese desserts such as anmitsu, as a clarifying agent in brewing, and for paper sizing fabrics. The word agar comes from the Malay word agar-agar (meaning jelly). It is also known as kanten or agal-agal (Ceylon agar). Chemically, agar is a polymer made up of subunits of the sugar galactose. Agar polysaccharides serve as the primary structural support for the algae's cell walls.

(http://www.answers.com/topic/agar-1) 6

Alkaloids Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. The name derives from the word alkaline and was used to describe any nitrogen-containing base. Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants, and animals and are part of the group of natural products (also called secondary metabolites). Many alkaloids can be purified from crude extracts by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications and recreational drugs. Examples are the local anesthetic and stimulant cocaine, the stimulant caffeine, nicotine, the analgesic morphine, or the antimalarial drug quinine. Some alkaloids have a bitter taste. (http://en.wikipedia.org/wiki/Alkaloid) Amoxicillin Amoxicillin (INN), formerly amoxycillin (BAN), is a moderate-spectrum, bacteriolytic, -lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other -lactam antibiotics. Amoxicillin is susceptible to degradation by -lactamase-producing bacteria, and so may be given with clavulanic acid to decrease its susceptibility.

(http://en.wikipedia.org/wiki/Amoxicillin)

Analgesic

An analgesic (also known as a painkiller) is any member of the diverse group of drugs used to relieve pain (achieve analgesia). The word analgesic derives from Greek an- ("without") and algos ("pain"). Analgesic drugs act in various ways on the peripheral and central nervous systems; they include paracetamol (para-acetylaminophenol, also known in the US as acetaminophen), the non-steroidal anti-inflammatory drugs (NSAIDs) such as the salicylates, narcotic drugs such as morphine, synthetic drugs with narcotic properties such as tramadol, and various others.

In choosing analgesics, the severity and response to other medication determines the choice of agent; the WHO pain ladder, originally developed in cancer-related pain, is widely applied to find suitable drugs in a stepwise manner. The analgesic choice is also determined by the type of pain: for neuropathic pain, traditional analgesics are less effective, and there is often benefit from classes of drugs that are not normally considered analgesics, such as tricyclic antidepressants and anticonvulsants.

(http://en.wikipedia.org/wiki/Analgesic)

Antibacterial Anything that destroys bacteria or suppresses their growth or their ability to reproduce. Heat, chemicals such as chlorine and antibiotic drugs all have antibacterial properties. Many antibacterial products for cleaning and handwashing are sold today. Such products do not reduce the risk for symptoms of viral infectious diseases in

otherwise healthy persons. This does not preclude the potential contribution of antibacterial products to reducing symptoms of bacterial diseases in the home. (http://www.medterms.com/script/main/art.asp?articlekey=10215) Antibiotic Antibiotic: (adjective) capable of inhibiting or destroying life, especially of a substance produced b a microbe that affects other microbes. Antibiotic: (noun) a substance usually produced by a microbe that is used therapeutically to destroy or inhibit the growth of a pathogen. ( The New Lexicon Websters Encyclopedic Dictionary og the English Language, Deluxe Edition, 1991 )

Anti-inflammatory

Anti-inflammatory refers to the property of a substance or treatment that reduces inflammation. Anti-inflammatory drugs make up about half of analgesics, remedying pain by reducing inflammation as opposed to opioids which affect the brain.

(http://en.wikipedia.org/wiki/Anti-inflammatory)

Antipyretic

Antipyretics are drugs that reduce body temperature in situations such as fever. However, they will not affect the normal body temperature if one does not have fever.

Antipyretics cause the hypothalamus to override an interleukin-induced increase in temperature. The body will then work to lower the temperature and the result is a reduction in fever.

Most are also used for other purposes. For example, the most common antipyretics in the United States are aspirin and paracetamol (acetaminophen), which are used primarily as pain relievers. NSAIDs are antipyretic, anti-inflammatory, and pain relievers. There is some debate over the appropriate use of such medications: fever is part of the body's immune response to infection.

Herbal remedies with a fever-reducing effect are called febrifuges, and include catnip, chamomile, sage, wormwood and yarrow. However, the term febrifuge can also refer to a refrigerant, such as topical alcohol, which cools the body by physically removing heat rather than modifying the body's responses. This is not recommended currently, because alcohol can be transferred through the skin and affect the liver. In addition, alcohol slightly raises the body temperature before it brings it down, which, if the fever is already very high could cause permanent damage.

(http://en.wikipedia.org/wiki/Antipyretic) Assay

An assay is a procedure where a property or concentration of an analyte is measured.

There are numerous types of assays, such as an antigen capture assay, bioassay, competitive protein binding assay, crude oil assay, four-point assay, immunoassay, 10

microbiological assay, stem cell assay, and many others, including concentration assays.

(http://en.wikipedia.org/wiki/Assay) Bacteria Extremely smallusually 0.3 to 2.0 micrometers in diameterand relatively simple microorganisms possessing the prokaryotic type of cell construction. Although traditionally classified within the fungi as Schizomycetes, they show no phylogenetic affinities with the fungi, which are eukaryotic organisms. The only group that is clearly related to the bacteria are the blue-green algae. Bacteria are found almost everywhere, being abundant, for example, in soil, water, and the alimentary tracts of animals. Each kind of bacterium is fitted physiologically to survive in one of the innumerable habitats created by various combinations of space, food, moisture, light, air, temperature, inhibitory substances, and accompanying organisms. Dried but often still living bacteria can be carried into the air. Bacteria have a practical significance for humans. Some cause disease in humans and domestic animals, thereby affecting health and the economy. Some bacteria are useful in industry, while others, particularly in the food, petroleum, and textile industries, are harmful. Some bacteria improve soil fertility. As in higher forms of life, each bacterial cell arises either by division of a preexisting cell with similar characteristics or through a combination of elements from two such cells in a sexual process.

(http://www.answers.com/topic/bacteria)

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Culture Medium

Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties.

(http://lecturer.ukdw.ac.id/dhira/NutritionGrowth/culturemedia.html)

Escherichia coli

Escherichia coli (commonly known as E. coli) is a bacterium that is commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, or by preventing the establishment of pathogenic bacteria within the intestine.

E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology. E. coli was discovered by German pediatrician and bacteriologist Theodor Escherich in 1885, and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria.

(Guevera, Beatrice Q. et al. 2005)

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Ethanol

Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug, best known as the type of alcohol found in alcoholic beverages and in modern thermometers. Ethanol is one of the oldest recreational drugs. In common usage, it is often referred to simply as alcohol or spirits.

Ethanol is a straight-chain alcohol, and its molecular formula is C2H5OH. Its empirical formula is C2H6O. An alternative notation is CH3CH2OH, which indicates that the carbon of a methyl group (CH3) is attached to the carbon of a methylene group (CH2), which is attached to the oxygen of a hydroxyl group (OH). It is a constitutional isomer of dimethyl ether. Ethanol is often abbreviated as EtOH, using the common organic chemistry notation of representing the ethyl group (C2H5) with Et. The fermentation of sugar into ethanol is one of the earliest organic reactions employed by humanity. The intoxicating effects of ethanol consumption have been known since ancient times. In modern times, ethanol intended for industrial use is also produced from by-products of petroleum refining.

Ethanol has widespread use as a solvent of substances intended for human contact or consumption, including scents, flavorings, colorings, and medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of other products. It has a long history as a fuel for heat and light and also as a fuel for internal combustion engines. (http://en.wikipedia.org/wiki/Ethanol)

13

Eucalyptus deglupta

Eucalyptus deglupta is a tall tree, commonly known as the Rainbow Eucalyptus, the Mindanao Gum or Bagras in the Philippines, or the Rainbow Gum. It is the only Eucalyptus species found naturally in the Northern Hemisphere. Its natural distribution spans New Britain, New Guinea, Ceram, Sulawesi and Mindanao. Now, this tree is cultivated widely around the world, mainly for pulpwood used in making paper. It is the dominant species used for pulpwood plantations in Philippines.

This tree is also grown for ornamental purposes, due to the showy multi-coloured streaks that cover the trunk. Patches of outer bark are shed annually at different times, showing the bright-green inner bark. This then darkens and matures to give blue, purple, orange and then maroon tones.

(http://en.wikipedia.org/wiki/Eucalyptus_deglupta)

Flavonoids 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 referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to

14

allergens, viruses, and carcinogens. They show antiallergic, anti-inflammatory, antimicrobial and anticancer activity.

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.

(http://en.wikipedia.org/wiki/Flavonoid) Gram-negative Bacteria

Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol. Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution. In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on structural differences in their cell walls.

Many species of Gram-negative bacteria are pathogenic, meaning they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer.[1] In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation.

15

Inflammation is a common result of cytokine production, which can also produce host toxicity.

(http://en.wikipedia.org/wiki/Gram-negative_bacteria) Gram-Negative Bacteria (Pseudomonas Aeruginosa) are simply called so because of their detection by the Grams Stain test in which they do not retain the crystal violet color (dye) in their cell wall. The Gram-Negative bacteria cell-wall holds the pink or reddish dye once a counterstain chemical is used.

The outer layer of Gram Negative bacteria cell-wall is made up of Lypopolysaccharide and Protein (core and O-polysaccharide and Lipid A) and it covers a very few thinner layers of Peptidoglycan as compared to Gram Positive bacteria (Peptidoglycan forms the outer layer of the Gram Positive bacteria cell-wall) and does not contain lipoproteins. The outer layer of the cell wall contains porins (pore like structure for a specific type of molecule). Below the outer layer of Lypopolysaccharide, there exist layers of peri-plasmic space (space between two layers of peptidoglycan and internal cell membrane) and plasma membrane. Some Gram Negative bacteria also have flagella with four surrounding rings.

The cell wall of Gram Negative bacteria also home a component that helps in Endotoxic activity and it also has pyrogenic effects associated with the Gram Negative infections. The Gram-Negative bacteria side wall also has a part which is called side chain that is made up of Lipopolysaccharide (and has hexoses in various chemical compositions as part of its structures). These side chains carry bases of somatic

16

antigen. These side chains are very important in order to classify the Gram Negative bacteria based on their chemical composition.

(http://www.buzzle.com/articles/gram-negative-bacteria.html)

Gram-positive Bacteria

Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain (safranin) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.

(http://en.wikipedia.org/wiki/Gram-positive_bacteria)

Guinea pig

The guinea pig (Cavia porcellus), also commonly called the Cavy, is a species of rodent belonging to the family Caviidae and the genus Cavia. Despite their common name, these animals are not pigs, nor do they come from Guinea. They originated in the Andes, and studies based on biochemistry and hybridization suggest they are domesticated descendants of a closely related species of cavy such as Cavia aperea, C. fulgida, or C. tschudii, and therefore do not exist naturally in the wild. The guinea pig plays an important role in the folk culture of many Indigenous South American groups, especially as a food source, but also in folk medicine and in community religious

17

ceremonies. Since the 1960s, efforts have been made to increase consumption of the animal outside South America.

In Western societies, the guinea pig has enjoyed widespread popularity as a household pet since its introduction by European traders in the 16th century. Their docile nature, their responsiveness to handling and feeding, and the relative ease of caring for them, continue to make the guinea pig a popular pet. Organizations devoted to competitive breeding of guinea pigs have been formed worldwide, and many specialized breeds of guinea pig, with varying coat colors and compositions, are cultivated by breeders.

Biological experimentation on guinea pigs has been carried out since the 17th century. The animals were frequently used as a model organism in the 19th and 20th centuries, resulting in the metaphor "guinea pig" for a test subject, but have since been largely replaced by other rodents such as mice and rats. They are still used in research, primarily as models for human medical conditions such as juvenile diabetes, tuberculosis, scurvy, and pregnancy complications.

Because the guinea pig has a stout, compact body, the animal more easily tolerates excessive cold than excessive heat. Its normal body temperature is 101 104 F (38.540 C), and so its ideal ambient air temperature range is similar to the human's, about 6575 F (1824 C). Consistent ambient temperatures in excess of 90 F (32 C) have been linked to hyperthermia and death, especially among pregnant sows. Guinea pigs are not well suited to environments that feature wind or frequent

18

drafts, and respond poorly to extremes of humidity outside of the range of 3070%. (http://en.wikipedia.org/wiki/Guinea_pig)

Imperata cylindrica

Imperata cylindrica, also known as cogongrass or kunai grass, is a species of grass in the genus Imperata. It is placed in the subfamily Panicoideae, supertribe Andropogonodae, tribe Andropogoneae.

It is a perennial rhizomatous grass native to east and southeast Asia, India, Micronesia, Australia and eastern and southern Africa. It grows from 0.6-3 m (2-10 feet) tall. The leaves are about 2 cm wide near the base of the plant and narrow to a sharp point at the top; the margins are finely toothed and are embedded with sharp silica crystals. The main vein is a lighter colour than the rest of the leaf and tends to be nearer to one side of the leaf. The upper surface is hairy near the base of the plant while the underside is usually hairless. Roots are up to 1.2 meters deep, but 0.4 m is typical in sandy soil.

(http://en.wikipedia.org/wiki/Imperata_cylindrica)

Inhibit

It means to hold in check, restrain a natural impulse either consciously or unconsciously; to obstruct, hinder or prohibit. ( The New Lexicon Websters Encyclopedic Dictionary of the English Language, Deluxe Edition, 1991 ) 19

Inoculation It means the act of inoculating; an instance of this; mental preparation or conditioning (noun). ( The New Lexicon Websters Encyclopedic Dictionary of the English Language, Deluxe Edition, 1991 ) In Vitro In vitro (Latin: within the glass) refers to the technique of performing a given procedure in a controlled environment outside of a living organism, such as in a "test tube" or Petri dish. Many experiments in cellular biology are conducted outside of organisms or cells; because the test conditions may not correspond to the conditions inside of the organism, this may lead to results that do not correspond to the situation that arises in a living organism. Consequently, such experimental results are often annotated with in vitro, in contradistinction with in vivo. (http://en.wikipedia.org/wiki/In_vitro)

Kirby-Bauer Antibiotic Testing (Disc Diffusion Testing)

Kirby-Bauer Testing is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics. Known quantities of bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing, called a zone of inhibition.

(Burton, 2004) 20

Leucoanthocyanin

Proanthocyanidin (also known as procyanidin oligomeric proanthocyanidin (OPC), pycnogenol, leukocyanidin and leucoanthocyanin) is a class of flavanols. Proanthocyanidins are essentially polymer chains of flavonoids such as catechins.[1] One was discovered in 1936 by Professor Jacques Masquelier and called Vitamin P, although this name did not gain official category status and has since fallen out of usage. It was Masquelier who first developed techniques for the extraction of proanthocyanidins from certain plant species.

Proanthocyanidins have been sold as nutritional and therapeutic supplements in Europe since the 1980s, but their introduction to the United States market has been relatively recent.

(http://en.wikipedia.org/wiki/Proanthocyanidin)

Mouse

A mouse (plural mice) is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse (Mus musculus). It is also a popular pet. The American white-footed mouse (Peromyscus leucopus) and the deer mouse (Peromyscus maniculatus) also sometimes live in houses. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles. They are known to invade homes for food and occasionally shelter.

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Although mice may live up to two and a half years in captivity, the average mouse in the wild lives only about four months, primarily owing to heavy predation. Cats, wild dogs, foxes, birds of prey, snakes and even certain kinds of arthropods have been known to prey heavily upon mice. Nevertheless, because of its remarkable adaptability to almost any environment, and its ability to live commensally with humans, the mouse is regarded to be the second most successful mammalian genus living on Earth today, after humans.

Mice can at times be harmful rodents, damaging and eating crops, causing structural damages and spreading diseases through their parasites and feces. In North America, breathing dust that has come in contact with mouse excrements has been linked to hantavirus, which may lead to Hantavirus Pulmonary Syndrome (HPS). Primarily nocturnal animals, mice compensate for their poor eyesight with a keen sense of hearing, and rely especially on their sense of smell to locate food and avoid predators. The original motivation for the domestication of cats is thought to have been for their predation of mice and their relatives, the rats.

(http://en.wikipedia.org/wiki/Mouse)

Mueller Hinton Agar

Mueller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. It is also used to isolate and maintain Neisseria and Moraxella species. (http://en.wikipedia.org/wiki/Mueller-Hinton_agar)

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Paracetamol /prsitml, prstml/)

Paracetamol

(INN)

(pronounced

or

acetaminophen (/sitmnfn/ (

listen)) (USAN) is a widely used over-the-counter

analgesic (pain reliever) and antipyretic (fever reducer). It is commonly used for the relief of fever, headaches, and other minor aches and pains, and is a major ingredient in numerous cold and flu remedies. In combination with non-steroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics, paracetamol is used also in the management of more severe pain (such as postoperative pain).

While generally safe for human use at recommended doses, acute overdoses (above 1000 mg per single dose and above 4000 mg per day for adults, above 2000 mg per day if drinking alcohol) of paracetamol can cause potentially fatal liver damage and, in rare individuals, a normal dose can do the same; the risk is heightened by alcohol consumption. Paracetamol toxicity is the foremost cause of acute liver failure in the Western world, and accounts for most drug overdoses in the United States, the United Kingdom, Australia and New Zealand.

Paracetamol is derived from coal tar, and is part of the class of drugs known as aniline analgesics; it is the only such drug still in use today. It is the active metabolite of phenacetin, once popular as an analgesic and antipyretic in its own right, but unlike phenacetin and its combinations, paracetamol is not considered to be carcinogenic at therapeutic doses. The words acetaminophen and paracetamol both come from chemical names for the compound: para-acetylaminophenol and para-

23

acetylaminophenol. In some contexts, it is simply abbreviated as APAP, for N-acetylpara-aminophenol.

Only

in

the

United

States

and

Canada

is

paracetamol

known

as

"acetaminophen".

(http://en.wikipedia.org/wiki/Paracetamol)

Pharmacology

It is the science of the effect of chemicals (except as food) on living organisms. It is applied to agriculture and horticulture, because chemicals are used to improve plants for economic purposes, to control plant disease, and to kill weeds.

(New Standard Encyclopedia, Standard Educational Corporation, Chicago)

Phytochemicals

Phytochemicals are plant-derived chemical compounds under scientific research for their potential health-promoting properties, but with unproved benefits.

"Phytonutrients" are plant-derived essential nutrients scientifically confirmed as important to human health.

There is evidence from laboratory studies that phytochemicals in fruits and vegetables may reduce the risk of cancer, possibly due to dietary fibers, polyphenol antioxidants and anti-inflammatory effects. Specific phytochemicals, such as

24

fermentable dietary fibers, meet significant scientific agreement to be allowed limited health claims by the US Food and Drug Administration (FDA).[1]

Phytochemicals have been used as drugs for millennia. For example, Hippocrates may have prescribed willow tree leaves to abate fever. Salicin, having antiinflammatory and pain-relieving properties, was originally extracted from the white willow tree and later synthetically produced to become the staple over-the-counter drug called Aspirin.

An important cancer drug, Taxol (paclitaxel), is a phytochemical initially extracted and purified from the Pacific yew tree.

Among edible plants with health promoting phytochemicals, diindolylmethane, from Brassica vegetables (broccoli, cauliflower, cabbage, kale, Brussels sprouts) may be useful for recurring respiratory papillomatosis tumors (caused by the human papilloma virus) is in Phase III clinical trials for cervical dysplasia (a precancerous condition caused by the human papilloma virus] and is in clinical trials sponsored by the National Cancer Institute of the United States for a variety of cancers (breast, prostate, lung, colon, and cervical). The compound is being studied for anti-viral, anti-bacterial and anti-cancer properties through a variety of pathways and has been shown to synergize with Taxol in its anti-cancer properties, making it a possible anti-cancer phytochemical as taxol resistance is a major problem for cancer patients.

Some phytochemicals with physiological properties may be elements rather than complex organic molecules. Abundant in many fruits and vegetables, selenium, for

25

example, is involved major metabolic pathways, including thyroid hormone metabolism and immune function. Particularly, it is an essential nutrient and cofactor for the enzymatic synthesis of glutathione, an endogenous antioxidant.

There are currently many phytochemicals possibly having medicinal properties in clinical trials for a variety of diseases. Lycopene, for example, from tomatoes has been tested in clinical trials for cardiovascular diseases and prostate cancer. These studies, however, did not attain sufficient scientific agreement to conclude an effect on any disease The FDA position reads:

"Very limited and preliminary scientific research suggests that eating one-half to one cup of tomatoes and/or tomato sauce a week may reduce the risk of prostate cancer. The FDA concludes that there is little scientific evidence supporting this claim."

Likewise, although lutein and zeaxanthin may affect visual performance and inhibit macular degeneration and cataracts, there was insufficient scientific evidence from clinical trials for such a specific effect or health claim.

Many phytochemicals have anti-inflammatory properties in vitro, including turmeric and chia. Inflammation is a factor in many diseases of aging including Alzheimer's and arthritis. Turmeric is also reported to be active against skin cancer (melanoma).

Clinical investigations continue to assess phytochemicals with medicinal properties.(http://en.wikipedia.org/wiki/Phytochemical)

26

Pour Plate Method

A technique for pure-culture isolation of bacteria; liquid, cooled agar in a test tube is inoculated with one loopful of bacterial suspension and mixed by rolling the tube between the hands; subsequent transfers are made from this to a second test tube, and from the second to a third; contents of each tube are poured into separate petri dishes; pure cultures can be isolated from isolated colonies appearing on the plates after incubation. (http://www.answers.com/topic/pour-plate-culture)

Saponins Saponins are a class of chemical compounds, one of very many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. Specifically, they are amphipathic glycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by their being composed of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative.

(Lippincott Williams and Wilkins, 2004)

Screening

Screening, in general, is the investigation of a great number of something (for instance, people) looking for those with a particular problem or feature. One example is at an airport, where many bags get x-rayed to try to detect any which may contain weapons or explosives. People are also screened going through a metal detector. Even 27

though the procedure aims at a large number of screens, it is always equivalent to sampling in statistics, because the complete population is almost always inaccessible for screening.

(http://en.wikipedia.org/wiki/Screening)

Secondary Metabolites

These are organic compounds that are not directly involved in the normal growth, development or reproduction of organisms. Unlike primary metabolites, absence of secondary metabolities results not in immediate death, but in long-term impairment of the organism's survivability/fecundity or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group. The function or importance of these compounds to the organism is usually of an ecological nature as they are used as defenses against predators, parasites and diseases, for interspecies competition, and to facilitate the reproductive processes (coloring agents, attractive smells, etc). Since these compounds are usually restricted to a much more limited group of organisms, they have long been of prime importance in taxonomic research. Secondary metabolites may be likely candidates for drug or other technological development directly, or as an inspiration for unnatural products. This will concern secondary metabolites in plants, bacteria, fungi and many marine organisms (sponges, tunicates, corals, snails). In some cases, higher organisms will host a microorganism which is the actual producer of the product in question, as part of a symbiotic relationship. (Burton, 2004)

28

Staphylococcus aureus Staphylococcus aureus is the most common cause of staph infections. It is a spherical bacterium, frequently found in the nose and skin of a person. About 20% of the population is long-term carriers of S. aureus. Staphylococcus aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo (may also be caused by Streptococcus pyogenes), boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis endocarditis, Toxic shock syndrome (TSS), and septicemia. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the four most common causes of nosocomial infections, often causing postsurgical wound infections. Abbreviated to S. aureus or Staph aureus in medical literature, S. aureus should not be confused with the similarly named (and also medically relevant) species of the genus Streptococcus.

(Guevera, Beatrice Q. et al. 2005)

Sterols Sterols are a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. They are amphipathic lipids synthesized from acetyl-coenzyme A via the HMGCoA reductase pathway. 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 and related compounds play essential roles in the physiology of eukaryotic organisms. For example, cholesterol forms part of the cellular membrane in animals, where it affects the cell membrane's fluidity and serves as secondary messenger in developmental signaling. In humans and

29

other animals, corticosteroids, such as cortisol act as signalling compounds in cellular communication and general metabolism.

(http://www.cyberlipid.org/sterols/ster0003.htm)

Synergism

Synergism, in general, may be defined as two or more agents working together to produce a result not obtainable by any of the agents independently. The word synergy or synergism comes from two Greek words: erg meaning "to work", and syn meaning "together"; hence, synergism is a "working together."

(http://en.wikipedia.org/wiki/Synergism)

Tannins

Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins. The astringency from the tannins is what causes the dry and puckery feeling in the mouth following the consumption of red wine, strong tea, or an unripened fruit. The term tannin refers to the use of tannins in tanning animal hides into leather; however, the term is widely applied to any large polyphenolic compound containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with proteins and other macromolecules. Tannins have molecular weights ranging from 500 to over 3,000. Tannins are incompatible with alkalis, gelatin, heavy metals, iron, lime water, metallic salts, strong oxidizing agents and zinc sulfate. (Burton, 2004)

30

Tannins are distributed all over the plant kingdom. They are commonly found in both gymnosperms as well as angiosperms. In terms of location of the tannins in a plant, they are mainly located in the vacuoles or surface wax of the plants. These sites are where tannins do not interfere with plant metabolism, and it is only after cell breakdown and death that the tannins are active in metabolic effects. Tannins are found in leaf tissues, bud tissues, seed tissues, root tissues and stem tissues. An example of the location of the tannins in the stem tissue is that they are often found in the growth areas of trees, such as the secondary phloem and xylem and the layer between the cortex and epidermis. Tannins may help regulate the growth of these tissues. They are also found in the heartwood of conifers and may play a role in inhibiting microbial activity, thus resulting in the natural durability of the wood. However, there may be a loss in the bioavailability of tannins in plants due to birds, pests, and other pathogens. [4]

The leaching of tannins from the decaying leaves of vegetation adjoining a stream may produce what is known as a blackwater river.

(http://en.wikipedia.org/wiki/Tannin)

31

Zone of Inhibition This is an area around a paper disk or colony of bacteria or mold where no other organisms are growing. If you are testing antibiotic sensitivity for example, you can impregnate paper disks with antibiotic and then put them on an agar plate of growing bacteria. The antibiotic then diffuses into the agar away from the disk. If the bacteria are sensitive to the antibiotic, they will not grow near the disk. The size of the zone is proportional to how sensitive the organism is. If the organism is resistant to the antibiotic, it will grow right up to the disk. (http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm)

32

Chapter 3 Materials and Methods

3.1 Research Design This study will follow a simple research design. It attempts to show the importance and the medicinal effect of the combined extracts of Eucalyptus deglupta and Imperata cylindrica, specifically the pharmacological property, determination of its active secondary metabolites and the antibacterial property. The study will undergo various tests conducted at the Science Laboratory of General Santos Hope Christian School. The first part is where the proponent extracts the substances from the E. deglupta and I. cylindrica. The proponent then conducts the phytochemical screening which consists of six tests screening for alkaloids, screening for saponins, screening for unsaturated sterols, screening for flavonoids, screening for leucoanthocyanins, and screening for tannins. Then the proponent conducts the Kirby-Bauer testing to see if the extract is effective in inhibiting the growth of bacteria. The bacteria used are Escherichia coli and Staphylococcus aureus. Then, the antipyretic screening was conducted wherein boiled milk was used in inducing fever. Then, the proponent conducts the analgesic testing wherein the flick test was used. Also, the proponent conducts the anti-inflammatory testing with the use of the carrageenan extract to induce the inflammation.

33

In Vitro Synergistic Pharmacological and Antibacterial Interaction and Toxicity Testing of Bagras (Eucalyptus deglupta) and Cogon (Imperata cylindrica) Locale of the Study GSHCS Experimental Animals Mice Guinea Pigs Guppies Experimental Procedures Phytochemical Screening Kirby-Bauer Testing Antipyretic Testing Flick Test Method Anti-inflammatory Testing Plants Investigated Bagras (Eucalyptus deglupta) Cogon (Imperata cylindrica) Statistical Treatment ANOVA Moving Average Method

Description Agar Alkaloids Analgesic Antibacterial Antibiotic Anti-inflammatory Antipyretic Bacteria Phytochemical Toxicity

Combined extract of Bagras and Cogon can be used as an alternative for commercial medicine available for fever, bacterial infections, pain (reliever), and inflammation.

34

3.2

Population and Sampling The collection of the plants Bagras (E. deglupta) and Cogon (I. cylindrica) was

gathered in the school and at Katangawan. The said areas are abundant sources of the mentioned plants above. Extracting and testing of the substance were conducted at the Science Laboratory of General Santos Hope Christian School.

3.3 Materials and Equipments This sub-section presents the raw materials and laboratory equipments to be used in the experimental setup. These were categorized based on preparation of extract, preparation of paper disc, antibacterial susceptibility test, phytochemical screening and antipyretic testing.

35

Table 3.1 List of Raw Materials and Laboratory Equipments Antibacterial Susceptibility Test Raw Materials Ethanol extracts of E. deglupta and I. cylindrica Mueller Hinton Agar Pure Culture Baceria: Staphylococcus aureus Escherichia coli Amoxicillin Laboratory Equipments Autoclave Erlenmeyer Flask Petri Dishes Alcohol lamp Incubator Forceps Inoculating Loop Vernier Caliper Filter Paper Antiinflammatory Testing Raw Materials Ethanol extracts of E. deglupta and I. cylindrica Carageenan Solution Paracetamol

Phytochemical Screening Raw Materials Ethanol extracts of E. deglupta and I. cylindrica Distilled Water Laboratory Equipments Evaporating Dish Stirring Rod Hot Plate Filter Paper Funnel Erlenmeyer Flask Test Tubes Magnesium Strips Chemical Solvent 2M HCI Mayers Reagent Petroleum Ether Chloroform 70% Ethanol Sulfuric Acid NACl Solution Sodium Sulfate

Antipyretic Testing Raw Materials Ethanol extracts of E. deglupta and I. cylindrica Boiled Milk Paracetamol

Analgesic Testing Raw Materials Ethanol extracts of E. deglupta and I. cylindrica Paracetamol Boiled Water Laboratory Equipments

Toxicity Testing Raw Materials Ethanol extracts of E. deglupta and I. cylindrica

Laboratory Equipments

Laboratory Equipments Laboratory Equipments Syringe Micrometer Specimen Mice Graduated Cylinder Specimen Guppies

Beaker Beaker Thermometer Digital Syringe Thermometer Test Tube Syringe Test Tube Specimen Specimen Guinea Pigs Mice

36

3.4

Procedure Flow Chart This part shows the procedures undertaken for the study; a more detailed chart

of the procedure is shown on this page.

Collection of Eucalyptus deglupta and Imperata cylindrica

Soaking of plants in Ethanol for 24 hours

Crude Extraction through Rotary Evaporator

Combined Crude extract was used for the following tests:

Toxicity Testing

Phytochemical Screening

Antibacterial Screening

Pharmacological Tests

Phytochemical Constituents

Zone of Inhibition

Analgesic Testing

Antiinflammatory Testing

Antipyretic Screening

Pain Reliever

Fever Inflammation

37

3.5

Experimentation and General Procedure This section shows a step-by-step procedure on how the experiment was

thoroughly done. It tells us how the procedures were done and what equipments were used. Extracting the substances of Bagras (E. deglupta) and Cogon (I. cylindrica)

E. deglupta and I. cylindrica were collected and soaked in Ethanol for 24 hours. It was Soaking of extracts for 24 hours. then extracted using the rotary evaporator.

Crude Extraction through Rotary Evaporator.

38

Phytochemical Screening
Screening for Alkaloids 20mL of the extract was evaporated on an evaporating dish to a syrupy constituency over the hot plate. Extracts of Bagras and Cogon evaporated.

Then Hydrochloric added to

5mL acid the

2 (HCl)

M was

concentrated

extract. It was thoroughly mixed, then Measuring 5mL of Hydrochloric Acid. About powdered (NaCl) was 2.5 Sodium added grams of cooled to room

temperature.

Chloride to the

solution, was stirred and filtered. The filtrate was brought to a final volume of 5mL by adding more 2 M HCl. Filtration of extract.

39

Screening for Unsaturated Sterols

30mL of the Ethanol extract was evaporated to dryness on the hot plate. The residue was cooled at room temperature and added 15mL of Petroleum ether. It was then mixed and filtered. Evaporating of extract on hot plate and adding of Petroleum ether.

Additional volumes of petroleum ether were added until the last volume of petroleum ether was colorless. Thorough mixing of substance.

The

combined

ethereal

filtrates

were then evaporated to dryness and the residue was dissolved in 15mL chloroform. The chloroform solution was dried over anhydrous sodium sulfate, filtered. Filtration of the solution. 40

Screening for Leucoanthocyanins The ethereal filtrates from the experiment of screening for saturated sterols were combined and the defatted residue was dissolved with 30mL of 70% ethanol; filtered. 1-2mL of the filtrate was placed in The solutions used. each of the three test tubes.

To the first test tube, 0.5mL of concentrated HCl acid was added warmed on a steam bath for about five minutes and color changes were then observed. The development of a redviolet indicates the presence of

leucoanthocyanin. Testing for the presence of leucoanthocyanin.

To the second test tube, 0.5mL of concentrated HCl acid and 3-4 magnesium strips were added. The color changes were carefully observed for ten minutes. The third test tube served as the control.

Magnesium strips added to the second test tube. 41

Screening for Saponins

An equivalent of 2mL crude Ethanol extract was placed in a test tube. Each test tube was added with 10mL distilled water, stopped, and shook vigorously for 30 seconds. It was allowed to stand and was observed. The sample is presumed to contain saponins if honeycomb froth greater than 3cm persists the liquid surface. Measuring of 2mL extract.

Screening for Tannins One hundred (100) mL of the 95% ethanol extract was evaporated to dryness on a steam bath. The evaporating dish was removed and the residue was added with 25mL of distilled water. Evaporation of extract. The solution was mixed well with a stirring rod and was allowed to cool at room temperature. The cooled extract was allowed to stand for several minutes and the upper half was decanted from each tube used. Mixing thoroughly of extract.

42

Three to four drops of 10% sodium chloride solution was added to the decanted supernatant. Precipitation was observed as a result of the nontannin components. Positive test is confirmed by the addition of ferric chloride solution to the filtrate and should turn out blue, blue-black, green, or blue-green color and precipitate.

Screening for Tannins.

43

Kirby-Bauer Antibiotic Testing/Disc Diffusion Testing

19 grams of Mueller Hinton Agar was weighed and then mixed with 500 mL of distilled water 19 grams of Mueller Hinton Agar weighed.

After mixing the Mueller Hinton Agar with distilled water, it was heated on the hot plate.

MHA heated on the hot plate.

15 Petri Dishes, along with the inoculating loop and the Mueller Hinton Agar were sterilized in the autoclave.

Sterilization of materials in the Autoclave.

44

The

Petri

dishes

were

divided

into

five

treatments Bagras, Cogon, Combined extracts, Water, and Control. Five Petri dishes were labeled for Escherichia coli with Bagras, Cogon, Combined extracts, Water, and Control. Another five dishes were labeled for Staphylococcus Petri Dishes with label. aureus with Bagras, Cogon, Combined extracts, Water, and Control.

The Mueller Hinton Agar was poured on the five Petri dishes for E. coli, and the other five for S. aureus.

MHA being poured on the Petri dishes.

As the agar is still in its liquid state, the proponent quickly added the E. coli and S. aureus bacteria on the petri dishes.

Bacteria being placed on the Petri Dish.

45

Filter papers were soaked in the Bagras, Cogon, Combined extract, water and Control (Amoxicillin mixed with distilled water). When the agar

solidified, these treated paper discs were then placed on each of the three Treated paper discs placed on the Petri dish. replications.

The Petri dishes were then left at 37C for 24 hours.

Petri dishes inside the Incubator.

After 24 hours, the zone of inhibition of each treatment was then measured with the use of the Vernier Caliper.

Measuring of zone of inhibition.

46

Anti-inflammatory Testing

100mg of carrageenan was weighed then was sprinkled on 10mL sodium chloride. Then it was allowed to soak for one hour and was stirred so it will be suspended.

Carrageenan Extract.

The diameter of the right paws of the 12

mice, 18 days all male with the weight of 110-120 grams, were measured by the

use of the micrometer caliper.

Measuring of diameter.

The carrageenan extract was then injected to the right paws of the mice to induce

inflammation.

Injecting of carrageenan extract.

47

After 30 minutes, the diameters of the right hind paws of the mice were again measured to check inflammation.

Measuring of right hind paws.

After

the

inflammation

was

already induced, the mices

Analgesic Testing
right hind paws were treated with the extracts.

Injecting of extract.

The mices right hind paws were once again measured

prior to injection of extract.

Measuring once again of right hind paws. 48

Antipyretic Testing

These are the four cages with three guinea pigs the each three

representing

replications for each treatment in antipyretic testing.

Caged guinea pigs. Materials prepared. for Milk the as testing the were of

cause

disease (fever) was boiled in the hot plate and then put in a beaker. The guinea pigs cochlear temperatures were measured first. Measuring of Cochlear Temperature.

The

boiled

milk

was

then

injected in the lower limb of the guinea pigs.

Inducing of fever.

49

After 30 minutes, the cochlear temperature was once again measured after inducing fever.

Measuring of Cochlear Temperature once again.

Then, the Ethanol extract was injected to the lower limb of the guinea pig.

Injecting of extract.

Measuring

of

cochlear

temperature after injecting the extract.

Measuring cochlear temperature.

50

Analgesic Property

Water was boiled to 55C on the hot plate and was

maintained at 50-55C.

Water boiled at 55C.

The right hind legs were then injected with the extract.

Injection of extract. After 30 minutes of injection, tail flick method was conducted wherein 5cm of the mices tails were dipped to the 55C water and was observed whether how many seconds they would be able to resist the heat. Flick Test Method. 51

Toxicity Test: Moving Average Method The moving average method (Thompson and Weil, 1952; Dykstra and Robertson, 1983) gives a report and reasonable accurate estimate of this median effective dose (m) and the estimated standard deviation of its logarithms. Such methodology requires that the same number of animals be used per dosage level and that the spacing between successive dosage exposure levels be geometrically constant. Given this and access to a table for the computation of moving average. One can rapidly calculate the median effective dosage with the formula:

Log m = logD + d(K-1)/2 + df m = median effective dose or exposure D = the lowest dose tested d = the log of the ratio of successive dose/exposure f = a table value taken from Weil (1952) for the proper K ( the total number of level tested 1)

52

Chapter 4 PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents the data gathered through actual observation and experimentation of the tests. Table 4.1 Results of Phytochemical Screening of Cogon (Imperata cylindrica) Secondary Metabolites Saponins Alkaloids Flavonoids Leucoanthocyanins Tannins Unsaturated Sterols Results Negative Negative Positive Negative Positive Positive

The phytochemical screening showed that the Cogon extract is positive in Flavonoids. It also showed that Imperata cylindrica is positive in Tannins and Unsaturated Sterols. But the plants extract is negative for Saponins, Alkaloids and Leucoanthocyanins.

53

Table 4.2 Results of Phytochemical Screening of Bagras (Eucalyptus deglupta) Secondary Metabolites Saponins Alkaloids Flavonoids Leucoanthocyanins Tannins Unsaturated Sterols Results Positive Positive Negative Positive Positive Negative

The phytochemical screening showed that the Bagras extract is positive in Saponins. It also showed that Eucalyptus deglupta is positive in Alkaloids, Tannins and Leucoanthocyanins. But the plants extract is negative for Flavonoids and Unsaturated Sterols.

Results of the Antibacterial Screening

Table 4.3 Growth inhibition of Staphylococcus aureus

Treatments Control Cogon Bagras Combination Total

REPLICATION I II III Sum 14.70mm 9.85mm 8.75mm 33.3mm 0mm 0mm 0mm 0mm 18.25mm 16.30mm 17.25mm 51.80mm 22.55mm 24.10mm 23.5mm 70.15mm 55.50mm 50.25mm 49.50mm 155.25mm

Mean 11.1mm 0mm 17.27mm 23.38mm 17.25mm

Table 4.3 clearly showed that the mean of the different treatments did not have much difference to each other, having a mean difference of 12.94mm zone of inhibition

54

at most which is not so far apart. That is, the extract can be a substitute for the commercial medicine (amoxicillin) for there is a big zone of inhibition.

Graph 1 Growth of Inhibition of Staphylococcus aureus

30 25 20 Replication 1 15 10 5 0 Control Cogon Bagras Combination Replication 2 Replication 3

Zone of Inhibition (mm)

Treatments The graph above shows the summary of the data gathered for Staphylococcus aureus. The control has a result of 14.70mm, 9.85mm and 8.75mm respectively. The Cogon extract has a result of 0mm for all replications. The Bagras extract has a result of 18.25mm, 16.30mm and 17.25mm respectively. The combination of the two extracts has a result of 22.55mm, 24.10mm and 23.5mm respectively.

55

Table 4.4 SUMMARY ON Staphylococcus aureus with variance

SUMMARY Groups Amoxicillin Cogon Bagras Mixture

Count 3 3 3 3

Sum Average Variance 33.3 11.1 10.02 0 0 0 51.8 17.27 0.95 70.15 23.38 0.61

Table 4.5 ANOVA

Source of Variation Between Groups Within Groups Total

SS 895.84 23.17

Df 3.00 8.00

MS F 298.61 103.11 2.90

P-value 9.84E-07

F crit 4.07

919.01 11.00

Above table showed the analysis of variance on the individual effect of cogon, bagras, and the synergistic effect of the combined extract compared to the amoxicillin in inhibiting the growth of Staphylococcus aureus. Table showed that the F-computed of 103.11 is greater than the p-value of 9.84 x 10-7 with the critical value of 4.07 at 5% level of significance which means that the null hypothesis of no difference in inhibitory effect of the extract compared to the amoxicillin has to be rejected in favor of the alternative hypothesis. However, summary table revealed a higher mean zone of inhibition for bagras of 17.27 mm compared to the zone of inhibition showed by amoxicillin of 11.10mm which means that Bagras is effective in inhibiting the growth of Staphylococcus aureus. The negative response of cogon in inhibiting the growth of Staphylococcus aureus is the one responsible for rejecting the null hypothesis in the analysis of variance.

56

Table 4.6 Growth inhibition of Escherichia coli REPLICATION I II III Sum 11.80mm 13.30mm 15.60mm 40.70mm 0mm 0mm 0mm 0mm 16.35mm 15.20mm 18.40mm 49.95mm 22.80mm 20.55mm 22.15mm 65.50mm 50.95mm 49.05mm 56.15mm 156.15mm

Treatments Control Cogon Bagras Combination Total

Mean 13.57mm 0mm 16.65mm 21.83mm 17.35mm

Table 4.6 clearly showed that the mean of the different treatments were not so differentiated to each other having a mean difference of 17.35 mm zone of inhibition at most which are very far apart. That is, the extract can be a substitute for the commercial medicine (amoxicillin) with a zone of inhibition.

Graph 2 Growth of Inhibition of Escherichia coli

25

Zone of Inhibition (mm)

20

15 Replication 1 Replication2 10 Replication 3

0 Control Cogon Bagras Combined

Treatments

57

The graph above shows the summary of the data gathered for Escherichia coli. The control has a result of 11.80mm, 13.30mm and 15.60mm respectively. The Cogon extract has a result of 0mm for all replications. The Bagras extract has a result of 16.35mm, 15.20mm and 18.40mm respectively. The combination of the two extracts has a result of 22.80mm, 20.55mm and 22.15mm respectively.

Table 4.7 SUMMARY Table for the Growth Inhibition of Escherichia coli
Groups Amoxicillin Cogon Bagras Mixture Count 3.00 3.00 3.00 3.00 Sum Average Variance 40.70 13.57 3.66 0.00 0.00 0.00 49.95 16.65 2.63 65.50 21.83 1.34

Summary Table above showed that Bagras has a mean growth inhibition of 16.65 mm. while the mixture of cogon and Bagras has even higher mean of 21.83 mm. compared to the amoxicillin of 13.57 mm. Cogon showed no inhibitory effect on the growth of Escherichia coli.

Table 4.8 ANOVA for E. Coli

Source of Variation Between Groups Within Groups Total

SS 782.01 15.26 797.28

Df 3.00 8.00 11

MS 260.67 1.91

F 136.63

Pvalue 3E-07

F crit 4.07

58

Table above showed that the alternative hypothesis will be accepted having a higher F-value of 136.63 compared to the P-value of 3.0 x 10-7 at 5% level of

significance with 4.07 F-critical limit. This is again due to the negative response of cogon extract to the growth of E. coli. However, summary table showed that bagrass and has a comparable inhibitory effect with that of amoxicillin, which means that bagras is effective in treating patient under E. coli attack. Moreover, the positive synergistic effect of cogon and bagras extracts only showed the intense strength of bagras in inhibiting E. coli growth despite the negative inhibition of cogon against E. coli.

Results of Antibacterial Screening.

59

Results of the Anti-Inflammatory Test

Table 4.9 Normal Diameter of Mice Right Leg Paw (mm) Scheduled Treatment Paracetamol Cogon Bagras Cogon and Bagras Mixture R1 0.15 0.15 0.15 0.15 R2 0.15 0.15 0.15 0.15 R3 0.15 0.15 0.15 0.15 Total Mean 0.15 0.15 0.15 0.15

Table 4.10 Diameter of Mice Right Leg Paw (mm.) 30 minutes After Induction of Carageenan

Scheduled Treatment Paracetamol Cogon Bagras Mixed

R1 0.25 0.20 0.23 0.25

R2 0.25 0.25 0.23 0.25

R3 0.25 0.25 0.25 0.25

Total

Mean

Table 4.11 Diameter of Mice Right Leg Paw (mm.) 24 hours After Treatment Treatment Paracetamol Cogon Bagras Mixed R1 0.18 0.20 0.30 0.23 R2 0.13 0.15 0.30 0.23 R3 0.13 0.13 0.30 0.23 Total 0.44 0.48 0.30 0.23 Mean 0.15 0.16 0.30 0.23

Table 4.12 SUMMARY Anti-Inflammatory Effect

Groups Paracetamol Cogon Bagras Mixture

Count 3 3 3 3

Sum Average Variance 0.44 0.15 0.0008 0.48 0.16 0.0013 0.90 0.30 0 0.69 0.23 0

60

Table above showed the anti-inflammatory effect of extract on mice under study. Cogon extract revealed an excellent anti-inflammatory effect of 0.16 mm diameter comparable to Paracetamol of 0.15 mm. in diameter. Bagras has no anti-inflammatory property, it even destruct the anti-inflammatory property of cogon in synergy.

Table 4.13 ANOVA for Anti-inflammatory Effect

Source of Variation Between Groups Within Groups Total

SS 0.045 0.004

df 3.000 8.000

MS F 0.015 28.141 0.001

P-value 0.00013

F crit 4.066

0.049 11.000

Table above showed the analysis of variance for the anti-inflammatory property of the plant extracts, its synergy and Paracetamol on mice. Table showed that alternative hypothesis has to be accepted having F-computed of 28.14 which is higher than the P-value of 0.00013 at 5% level of significance with Fcritical limit of 4.066. The acceptance of the alternative hypothesis is consonant with the negative inflammatory property of Bagras. Moreover, cogon showed comparable effectiveness in healing inflammation congruent to the effect of Paracetamol. Comparative Diameter of Mice before and after the Treatment a. Paracetamol (Table 4.14) After Treated Before injecting with extract Replications inducement carrageenan after 24 solution hours RI 0.15mm 0.25mm 0.18mm RII 0.15mm 0.25mm 0.13mm RIII 0.15mm 0.25mm 0.13mm

Sum 0.58mm 0.53mm 0.53mm

Mean 0.19mm 0.18mm 0.18mm 61

Graph 3 Increase and decrease in diameter of right hind paws (Paracetamol)

Diameter of Right Hind Paw in mm 0.3

0.2 Replication 1 Replication 2 0.1 Replication 3

0 Before Inducement After Injecting Carrageenan Solution Treated with extract after 24 hours

b. Cogon (Table 4.15) After Treated Before injecting with extract Replications inducement carrageenan after 24 solution hours RI 0.15mm 0.20mm 0.20mm RII 0.15mm 0.25mm 0.15mm RIII 0.15mm 0.25mm 0.13mm

Sum 0.55mm 0.55mm 0.53mm

Mean 0.18mm 0.18mm 0.18mm

Graph 4 Increase and decrease in diameter of right hind paws (Cogon)

Diameter of Right Hind Paw in mm 0.3 0.2 Replication 1 0.1 0 Before inducement After injecting carrageenan solution Treated with extract after 24 hours Replication 2 Replication3

62

c. Bagras (Table 4.16) After Treated Before injecting with extract Replications inducement carrageenan after 24 solution hours RI 0.15mm 0.23mm 0.30mm RII 0.15mm 0.23mm 0.30mm RIII 0.15mm 0.25mm 0.33mm

Sum 0.68mm 0.68mm 0.73mm

Mean 0.23mm 0.23mm 0.24mm

Graph 5 Increase and decrease in diameter of right hind paws (Bagras)

Diameter of Right Hind Paw in mm

0.3

0.2

Replication 1 Replication 2 Replication 3

0.1

0 Before inducement After injecting carrageenan solution Treated with extract after 24 hours

63

d. Mixture of Cogon and Bagras (Table 4.17) After Treated Before injecting with extract Replications inducement carrageenan after 24 solution hours RI 0.15mm 0.25mm 0.23mm RII 0.15mm 0.25mm 0.23mm RIII 0.17mm 0.25mm 0.23mm

Sum 0.63mm 0.63mm 0.65mm

Mean 0.21mm 0.21mm 0.22mm

Graph 6 Increase and decrease in diameter of right hind paws (Combination)

Diameter of Right Hind Paw in mm

0.3

0.2 Replication1 0.1 Replication 3 0 Before inducement After injecting Treated with carrageenan extract after solution 24 hours Replication 2 Replication1 Replication 2 Replication 3

64

Results of Antipyretic Testing

Table 4.18 Normal Body Temperature of Guinea Pig (C)

Scheduled Treatment

Paracetamol Cogon Bagras Mixed Mean

R1 38.3 36.4 38.0 37.2

R2 38.1 36.7 37.6 37.2

R3 38.1 35.6 37.9 38.4

Total 114.50 108.70 113.50 112.80

Mean 38.17 36.23 37.83 37.60 37.46

Table above showed the mean body temperature of 37.460C of the experimental mice taken using the digital thermometer prior to the experimentation in order to take the baseline temperature for statistical comparison. This temperature is congruent to the established body temperature of experimental mice. (Beatrice Bautista)

Table 4.19 Body Temperature of Guinea Pig after Induction

Scheduled Treatment

Paracetamol Cogon Bagras Mixed Mean

R1 38.8 37.0 39.0 37.6

R2 39.0 37.0 38.0 37.7

R3 38.6 37.1 38.5 39.1

Total 116.40 111.10 115.50 114.40

Mean 38.80 37.03 38.50 38.13 38.12

Thirty minutes after the induction of fever using milk, the temperature was then obtained and recorded on the table shown above. It is evident that the body temperature of the experimental mice is 38.120C. There is an increase of 0.660C which showed the onset of fever in the experimental mice.

65

Table 4.20 Temperature of Guinea Pig after Two Hours Treatment

Treatment Paracetamol Cogon Bagras Mixture Mean

R1 38.3 35.6 37.1 37.5

R2 37.5 36.7 35.9 37.3

R3 38.3 35.5 37.9 37.8

Total 114.10 107.80 110.90 112.60

Mean 38.03 35.93 36.97 37.53 37.12

Table 4.21 SUMMARY WITH VARIANCE

Groups Count 3 3 3 3 Sum Average Variance 114.100 38.033 0.213 107.800 35.933 0.443 110.900 36.967 1.013 112.600 37.533 0.063

Paracetamol
Cogon Bagras Mixture

Table above showed the mean body temperature of the experimental mice two hours after the treatment was done using the different extract and Paracetamol. It is evident that cogon has the effective antipyretic property, and even a more cooling effects lowering the mice body temperature below the normal body temperature of 37.460C to 35.930C. Moreover, the synergistic effect of cogon and bagras is as effective as Paracetamol in lowering the mice body temperature after the experiment.

Table 4.22 ANOVA

Source of Variation Between Groups Within Groups Total

SS 7.310 3.467 10.777

df 3.000 8.000 11.000

MS F 2.437 5.623 0.433

P-value 0.023

F crit 4.066

66

Table above showed the analysis of variance for the antipyretic property of cogon extract, Bagras extract and its combined mixture and Paracetamol. Table showed that alternative hypothesis is accepted which means that there is a difference in the effectiveness of the different extract in the antipyretic property. Moreover, the difference is not very significant as revealed in the F-computed of 5.623 which is close to the Fcritical limit of 4.066 at 5% level of significance and p-value of 0.023.

Table 4.23 Temperature of Guinea Pig after Six Hours of Treatment

Treatment Paracetamol Cogon Bagras Mixed

R1 37.9 35.8 36.5 35.7

R2 38.3 36.9 35.7 36.0

R3 37.7 34.7 36.3 36.1

Total 113.90 107.40 108.50 107.80

Mean 37.97 35.80 36.17 35.93

Table 4.24 TABLE SUMMARY WITH VARIANCE

Groups Count 3.00 3.00 3.00 3.00 Sum Average Variance 113.90 37.97 0.09 107.40 35.80 1.21 108.50 36.17 0.17 107.80 35.93 0.04

Paracetamol
Cogon Bagras Mixture

Table above showed the mean body temperature of the experimental mice six hours after the treatment was done using the different extract and Paracetamol. It is evident that cogon still has the effective antipyretic property lowering the body temperature to 35.80C on the average while Bagras lowered the mice body temperature to 36.170C on the average. The synergistic effect of cogon and Bagras is still effective lowering the guinea pigs body temperature to 35.930C after the experiment. The 67

synergistic antipyretic property of the cogon and bagras extract may be brought by the effectiveness of cogon extract.

Table 4.25 ANOVA

Source of Variation Between Groups Within Groups Total

SS 9.21 3.04 12.25

Df 3.00 8.00 11.00

MS 3.07 0.38

F 8.08

P-value 0.01

F crit 4.07

Table above showed the analysis of variance for the antipyretic property of cogon extract, Bagras extract and its combined mixture and Paracetamol. Table showed that alternative hypothesis is accepted which means that there is a difference in the effectiveness of the different extract in the antipyretic property. Moreover, the difference is not very significant as revealed in the F-computed of 8.08 which is close to the Fcritical limit of 4.066 at 5% level of significance and p-value of 0.010. The rejection of the null hypothesis or hypothesis of no difference is brought about by the wide range on the body temperature recorded observed on the data of cogon and Paracetamol with a mean difference of 2.170C.

Table 4.26 Temperature of Guinea Pig after Ten Hours of Treatment

Treatment Paracetamol Cogon Bagras Mixed

R1 37.6 36.3 36.2 34.0

R2 35.3 35.3 36.3 34.3

R3 36.1 35.4 36.4 34.3

Total 109.00 107.00 108.90 102.60

Mean 36.33 35.67 36.30 34.20

68

Table 4.27 SUMMARY WITH VARIANCE

Groups Amoxicillin Cogon Bagras Mixture

Count 3 3 3 3

Sum Average Variance 109.00 36.33 1.36 107.00 35.67 0.30 108.90 36.30 0.01 102.60 34.20 0.03

Table above showed the mean body temperature of the experimental guinea pig ten hours after the treatment was done using the different extract and Paracetamol. It is evident that the synergistic effect of cogon and Bagras extract has the effective antipyretic property, lowering the body temperature to 34.2 0C on the average while cogon lowered the guinea pigs body temperature to 35.670C on the average and

Bagras lowered the body temperature to 36.30C after the experiment.

Table 4.28 ANOVA

Source of Variation Between Groups Within Groups Total

SS 8.97 3.41 12.3825

df 3.00 8.00 11

MS 2.99 0.43

F 7.01

P-value 0.01

F crit 4.07

Table above showed the analysis of variance for the antipyretic property of cogon extract, Bagras extract and its combined mixture and Paracetamol. Table showed that alternative hypothesis is accepted which means that there is a difference in the effectiveness of the different extract in the antipyretic property. Moreover, the difference is not very significant as revealed in the F-computed of 7.01 which is close to the Fcritical limit of 4.07 at 5% level of significance and p-value of 0.010. The rejection of the 69

null hypothesis or hypothesis of no difference is brought about by the wide range on the body temperature recorded observed on the data of cogon and Paracetamol with a mean difference of 2.130C.

Comparative Temperature of Guinea Pig in a Prescribed Time Interval

a. Paracetamol (Table 4.29) Before Replications Inducement Inducement of Fever of Fever RI 38.3C 38.8C RII 38.1C 39.0C RIII 38.1C 38.6C After After 6 10 Sum Mean hours hours 37.9C 37.6C 190.9C 38.18C 38.3C 35.3C 188.2C 37.64C 37.7C 36.1C 188.8C 37.76C

After 2 hours 38.3C 37.5C 38.3C

Graph 7 Cochlear Temperatures of Guinea Pigs (Control)

40

Temperature in C

39 38 37 36 35 34 33 Replication 1 Replication 2 Replication 3

Before Inducemetnt of Fever Inducement of Fever After 2 hours prior to injecting the extract After 6 hours prior to injecting the extract After 10 hours prior to injecting the extract

70

b. Cogon (Table 4.30) Before Replications Inducement Inducement of Fever of Fever RI 36.4C 37.0C RII 36.7C 37.0C RIII 35.6C 37.1C After After 6 10 Sum Mean hours hours 35.8C 36.3C 181.1C 36.22C 36.9C 35.3C 182.6C 36.52C 34.7C 35.4C 178.3C 35.66C

After 2 hours 35.6C 36.7C 35.5C

Graph 8 Cochlear Temperatures of Guinea Pigs for Cogon treatment

37.5 37 Before Inducemetnt of Fever Inducement of Fever After 2 hours prior to injecting the extract After 6 hours prior to injecting the extract Replication 1 Replication 2 Replication 3 After 10 hours prior to injecting the extract

Temperature in C

36.5 36 35.5 35 34.5 34 33.5

c. Bagras (Table 4.31) Before Replications Inducement Inducement of Fever of Fever RI 38.0C 39.0C RII 37.6C 38.0C RIII 37.9C 38.5C After After 6 10 Sum Mean hours hours 36.5C 36.2C 186.6C 37.36C 35.7C 36.3C 183.5C 36.70C 36.3C 36.4C 187.0C 37.40C

After 2 hours 37.1C 35.9C 37.9C

71

Graph 9 Cochlear Temperatures of Guinea Pigs (Bagras)

Temperature in C

40 39 38 37 36 35 34 Replication 1 Replication 2 Replication 3 After 2 hours prior to injecting the extract Inducement of Fever Before Inducemetnt of Fever

d. Mixture of Cogon and Bagras (Table 4.32) Before Replications Inducement Inducement of Fever of Fever RI 37.2C 37.6C RII 37.2C 37.7C RIII 38.4C 39.1C After After 6 10 Sum Mean hours hours 35.7C 34.0C 182.0C 36.40C 36.0C 34.3C 182.5C 36.50C 36.1C 34.3C 185.7C 37.14C

After 2 hours 37.5C 37.3C 37.8C

Graph 10 Cochlear Temperatures of Guinea Pigs (Combined Extracts of Cogon and Bagras)
40

Temperature in C

39 38 37 36 35 34 33 32 31 Replication 1 Replication 2 Replication 3

Before Inducemetnt of Fever Inducement of Fever After 2 hours prior to injecting the extract After 6 hours prior to injecting the extract After 10 hours prior to injecting the extract

72

Results of Analgesic Testing

Table 4.33 Analgesic Reaction Thirty Minutes After Injecting the Extract

Treatment Paracetamol Cogon Bagras Mixed R1 23 5 13 3

Replication R2 3 6 4 4

R3 6 5 12 6

Total 32.00 16.00 29.00 13.00

Mean 10.67 5.33 9.67 4.33

Table 4.34 SUMMARY WITH VARIANCE

Groups Paracetamol Cogon Bagras Mixture Count 3 3 3 3 Sum Average Variance 32.00 10.67 116.33 16.00 5.33 0.33 29.00 9.67 24.33 13.00 4.33 2.33

Table above showed the mean analgesic response of the experimental mice using the tail flick method. Table above showed that Paracetamol has the highest analgesic response of 10.67 second on 500C water. This was followed by Bagras extract with 9.67 second heat resistance on water with 500C temperature.

Table 4.35 ANOVA

Source of Variation Between Groups Within Groups Total

SS 88.33 286.67 375

df 3.00 8.00 11

MS 29.44 35.83

F 0.82

P-value 0.52

F crit 4.07

73

Table above showed the analysis of variance for the analgesic property of cogon extract, Bagras extract and its combined mixture and Paracetamol. Table showed that alternative hypothesis is accepted which means that there is a difference in the effectiveness of the different extract in the analgesic property. Moreover, the difference is not very significant as revealed in the F-computed of 0.82 which is close to the Fcritical limit of 4.07 at 5% level of significance and p-value of 0.52. The rejection of the null hypothesis or hypothesis of no difference is brought about by the wide range differential on the resistance of mice treated with amoxicillin to the mice treated with the mixture of cogon and Bagras extract of 6.34 seconds. However, the resistivity to heat shown by mice treated with Paracetamol is very close to the resistivity to heat shown by mice treated with Bagras with 1.0 second time differential.

Table 4.36 Analgesic Reaction One Hour and Thirty minutes after Injecting the Extract

Treatment Paracetamol Cogon Bagras Mixed

R1 26 11 13 20

R2 12 8 8 15

R3 15 26 18 25

Total 53.00 45.00 39.00 60.00

Mean 17.67 15.00 13.00 20.00

Table 4.37 SUMMARY WITH VARIANCE

Groups Paracetamol Cogon Bagras Mixture

Count 3 3 3 3

Sum Average 53.00 17.67 45.00 15.00 39.00 13.00 60.00 20.00

Variance 54.33 93.00 25.00 25.00

74

Table above showed the mean analgesic response of the experimental mice using the tail flick method after one hour and thirty minutes of treatment. Table above showed that bagras and cogon extract has the highest synergistic analgesic response of 20.00 second on 500C water. This was followed by Paracetamol with 17.67 second heat resistance on water with 500C temperature.

Table 4.38 ANOVA

Source of Variation Between Groups Within Groups Total

SS 84.25 394.67 478.92

df 3 8 11

MS 28.08 49.33

F 0.57

P-value 0.65

F crit 4.07

Table above showed the analysis of variance for the analgesic property of cogon extract, bagras extract and its combined mixture and Paracetamol. Table showed that null hypothesis is accepted which means that there is no difference in the effectiveness of the different extract in the analgesic property with that of Paracetamol. Moreover, the difference is not very significant as revealed in the F-computed of 0.57 which is close to the p-value of 0.65 at 5% level of significance and F-critical limit of 4.07.

Table 4.39 Analgesic Reaction Two Hours and Thirty minutes after Injecting the Extract

Treatment Paracetamol Cogon Bagras Mixed

R1 28 12 12 19

R2 8 6 20 18

R3 11 13 15 17

Total 47.00 31.00 47.00 37.00

Mean 15.67 10.33 15.67 18.50 75

Table 4.40 SUMMARY WITH VARIANCE

Groups Paracetamol Cogon Bagras Mixture

Count 3 3 3 2

Sum Average 47.00 15.67 31.00 10.33 47.00 15.67 37.00 18.50

Variance 116.33 14.33 16.33 0.50

Table above showed the mean analgesic response of the experimental mice using the tail flick method after two hour and thirty minutes of treatment. Table above showed that bagras and cogon extract has the highest synergistic analgesic response of 18.50 second on the average on 500C water. This was followed by Paracetamol and bagras extract with 15.67 second heat resistance on water with 50 0C temperature.

Table 4.41 ANOVA

Source of Variation Between Groups Within Groups Total

SS 91.682 294.5 386.18

df 3 7 10

MS 30.56 42.07

F 0.73

P-value 0.57

F crit 4.35

Table above showed the analysis of variance for the analgesic property of cogon extract, bagras extract and its combined mixture and Paracetamol. Table showed that alternative hypothesis is accepted which means that there is a difference in the effectiveness of the different extract in the analgesic property with that of amoxicillin. Moreover, the difference is not very significant as revealed in the F-computed of 0.73 which is close to the p-value of 0.57 at 5% level of significance and F-critical limit of 4.35 76

Comparative Analgesic Reaction of Mice in a prescribed time interval according to treatments.

A. Paracetamol (Table 4.42)

Replications

RI RII RIII

30 minutes after injecting extract 23 s 3s 6s

1 hour and 30 minutes after injecting extract 26 s 12 s 15 s

2 hours and 30 minutes after injecting extract 28 s 8s 11 s

Sum

Mean

77 s 23 s 32 s

25.7 s 4.6 s 6.4 s

Graph 11 Increase in resistance of mice in seconds (Control)

30 25

Time in seconds

20 15 10 5 0 Replication 1 Replication 2 Replication 3

30 minutes after injecting extract 1 hour and 30 minutes after injecting extract 2 hours and 30 minutes after injecting extract

B. Cogon (Table 4.43)

Replications

RI RII RIII

30 minutes after injecting extract 5s 6s 5s

1 hour and 30 minutes after injecting extract 11 s 8s 26 s

2 hours and 30 minutes after injecting extract 12 s 6s 13 s

Sum

Mean

28 s 20 s 44 s

9.3 s 6.7 s 14.7 s 77

Graph 12 Increase in resistance of mice in seconds (Cogon)

30

Time in seconds

25 20 15 10 5 0 Replication 1 Replication 2 Replication 3 30 minutes after injecting extract 1 hour and 30 minutes after injecting extract 2 hours and 30 minutes after injecting extract

C. Bagras (Table 4.44)

Replications

RI RII RIII

30 minutes after injecting extract 13 s 4s 12 s

1 hour and 30 minutes after injecting extract 13 s 8s 18 s

2 hours and 30 minutes after injecting extract 12 s 20 s 15 s

Sum

Mean

38 s 32 s 45 s

12.7 s 10.7 s 15.0 s

Graph 13 Increase in resistance of mice in seconds (Bagras)

25

Time in seconds

20 15 10 5 0 Replication 1 Replication 2 Replication 3 30 minutes after injecting extract 1 hour and 30 minutes after injecting extract 2 hours and 30 minutes after injecting extract

78

D. Mixture: (Table 4.45)

Replications

RI RII RIII

30 minutes after injecting extract 3s 4s 6s

1 hour and 30 minutes after injecting extract 20 s 15 s 25 s

2 hours and 30 minutes after injecting extract 19 s 18 s 17 s

Sum

Mean

42 s 37 s 48 s

14.0 s 12.3 s 16.0 s

Graph 14 Increase in resistance of mice in seconds (Combined Extracts of cogon and Bagras)

30

Time in seconds

25 20 15 10 5 0 Replication 1 Replication 2 Replication 3 30 minutes after injecting extract 1 hour and 30 minutes after injecting extract 2 hours and 30 minutes after injecting extract

79

Toxicity Test Result Table 4.46 Guppies Lethality Bioassay (Moving Average Method) Mortality after 24 hours at 4 levels of sample concentration 10mg 20mg 40mg 80mg 0 1 10 10 0 0 0 0 0 1 5 5

Test Sample Bagras Cogon Bagras + Cogon

LC50 46 0 74

Table 4.46 above showed that bagras is lethal to aquatic life at LC50 of 46 ppm with 95% confidence interval with +/- 2.179, which will give a range of 28 ppm to 75 ppm, that is, bagras extract can cause fish poisoning if fishes ingest an amount equivalent to 20 mg of its body weight. This means that when an organism is exposed to 28 ppm of the extract, it will start to die, and 50% of the population will die when the concentration of the extract reaches 46 ppm and at 75 ppm, all organisms will die. Aside from aquatic poisoning, bagras extract can cause water pollution to the body of water where it was submerged or decomposed. Moreover, cogon has no lethal effect to guppies or any aquatic organism as per table above showed LC50 of zero (0) for cogon. Furthermore, the combined extract of cogon and bagras showed an LC50 of 74 ppm with 95% confidence interval and a range of 35 ppm to 105 ppm, that is, when the organism is exposed to 35 ppm of the extract, fish will start to die and 50% of the population will die when the concentration of the combined extract reaches 74 ppm and at 105 ppm, all fishes will die. It only showed that bagras has poisonous substance, in this case, alkaloids. It might be that the alkaloid present in the extract per phytochemical analysis is the poisonous alkaloid. It is also congruent to the phytochemical analysis that cogon is negative with alkaloid, hence not toxic to aquatic life.

80

Chapter 5 SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATION

This chapter presents the summary of findings and conclusions made from the study and recommendations given by the researcher.

5.1

Findings The researcher started with the collection of the samples in the certain area. The

area is particularly in the field of General Santos Hope Christian School for the Cogon plant and in Katangawan for the Bagras plant. A suitable area was found having an abundant source of Imperata cylindrica and Eucalyptus deglupta. The collected samples were then soaked with Ethanol in Erlenmeyer flasks in the science laboratory of the said school. The phytochemical screening was conducted. Screenings for different chemicals were conducted in the laboratory. After each screening, results were again recorded despite the positive or negative outcomes. The results on some of the screening were positive and some are negative. Antibacterial screening was conducted after the phytochemical screening. A pure culture of a gram positive and a gram negative were produced in GSHCS science laboratory. Glass wares were then prepared for the antibacterial testing. The testing was then followed using aseptic technique. The Petri dishes used in the testing were then put in the incubator in 24 hours. Observations of the zone of inhibition were read on the next day and the results were recorded. The results showed that the combined extract of Imperata cylindrica and Eucalyptus deglupta has an antibacterial property.

81

After the antibacterial screening, the antipyretic testing was then done. The researcher prepared the materials as well as the specimens, which are the guinea pigs. Before the start of the testing, temperature of the specimens was measured as a guidance that it is in good condition. Boiled milk was used to cause fever in the specimens and was injected. Extracts of the samples individually and combined, were also injected to the specimens. Observations were pursued in the next day and the temperature of the specimens was recorded saying that the temperature was back to normal and was compared to the results of the positive Control which was the commercial product Paracetamol. Then, the Anti-inflammatory testing followed to determine if the extracts can cure inflammation. Inflammation of the right hind paws of the mice were induced by injecting the carrageenan extract. The mice were observed for 24 hours. This test concluded that the combined extract of Cogon and Bagras have an anti-inflammatory property and therefore, can reduce inflammation. Right after this, the analgesic testing wherein the proponent would determine whether the extracts can be a substitute to the commercial pain relievers that are available. The flick test method was used to the 12 mice with 3 mice representing the 3 replications for each treatment namely positive control, Cogon, Bagras and combined extracts. The analgesic property will be determined by recording the time the mice will be able to resist the heat of the boiled water maintained at 50-55C. Then, it was concluded that the extracts have an analgesic property.

82

After all the said tests, the toxicity test was conducted to determine the toxicity levels of the extracts of Imperata cylindrica and Eucalyptus deglupta with the use of guppies with different concentrations based on the method of Thomson and Weil. The test results reveal that the Bagras extract (Eucalyptus deglupta) is toxic with a range of 28 ppm to 75 ppm and an LC50 of 46 ppm. The cogon extract (Imperata cylindrica), on the other hand, has no lethal effect to guppies or any aquatic organism. The combined extract of cogon and bagras showed an LC50 of 74ppm with 95% confidence interval and a range of 35ppm to 105ppm. 5.2 Conclusions Based on the experimentation, results and data gathered, the researcher was able to formulate the following conclusions: 1. The individual Ethanol extracts of cogon (Imperata cylindrica) and bagras (Eucalyptus deglupta) have phytochemical properties that can cure diseases like fever. 2. Combined Ethanol extracts of cogon (Imperata cylindrica) and bagras (Eucalyptus deglupta) has antibacterial properties that it can inhibit the growth of bacteria. 3. Combined Ethanol extracts of cogon (Imperata cylindrica) and bagras (Eucalyptus deglupta) has pharmacological properties namely antipyretic, antiinflammatory and analgesic properties. 4. The extract of cogon (Imperata cylindrica) has no lethal effect to guppies or any aquatic organism as results showed an LC50 of zero (0).

83

5. The extract of bagras (Eucalyptus deglupta) is lethal to aquatic life at LC50 of 46 ppm, with a range of 28 ppm to 75 ppm. 6. The combined extract of cogon and bagras showed an LC50 of 74 ppm with 95% confidence interval and a range of 35 ppm to 105 ppm, that is, when the organism is exposed to 35ppm of the extract, fish will start to die and 50% of the population will die when the concentration of the combined extract reaches 74 ppm and at 105 ppm, all fishes will die.

5.3

Recommendations From the findings of the study, it can be observed that there were research gaps.

The following would serve as research agenda and methodological suggestions in future studies. 1. Further studies to be conducted with more bacterial cultures to prove the validity of the inhibitory effect. 2. Testing with other commercially available antibiotics besides the ones used in this study. 3. Comparison of the combined extracts of Imperata cylindrica and Eucalyptus deglupta to the commercially available antibiotics. 4. Further experimentation of the Bagras plant in terms of its anti-carcinogenic property.

84

Appendix
A. Pictorials A. Soaking of Plants and extraction through Rotary Evaporator

Soaking of extracts for 24 hours. B. Phytochemical Screening

Crude Extraction through Rotary Evaporator.

Extracts of Bagras and Cogon evaporated. Filtration of extract.

Measuring 5mL of Hydrochloric Acid. Evaporating of extract on hot plate and adding of Petroleum ether.

85

Thorough mixing of substance. Testing for the presence of Leucoanthocyanin.

Filtration of the solution.

Magnesium strips added to the second test tube.

The solutions used.

Measuring of 2mL extract.

Evaporation of extract.

86

Mixing thoroughly of extract.

Screening for Tannins. C. Antibacterial Screening

19 grams of Mueller Hinton Agar weighed.

Sterilization of materials in the Autoclave.

MHA heated on the hot plate. Petri Dishes with label.

87

MHA being poured on the Petri dish.

Petri dishes inside the Incubator.

Bacteria being placed on the Petri Dish.

Measuring of zone of inhibition.

Treated paper discs placed on the Petri dish.

88

D. Anti-inflammatory Screening

Carrageenan Extract.

Measuring of right hind paws.

Measuring of diameter.

Injecting of extract.

Injecting of carrageenan extract.

Measuring once again of right hind paws.

89

E. Antipyretic Testing

Caged guinea pigs.

Measuring of Cochleal Temperature once again.

Measuring of Cochlear Temperature.

Injecting of extract.

Inducing of fever.

Measuring of cochlear temperature. 90

F. Analgesic Testing

Water boiled at 55C.

Injection of extract.

Flick Test Method.

91

B. Analysis of Variance on Staphylococcus aureus

Source of Variation Between Groups Within Groups Total

SS 895.84 23.17

Df 3.00 8.00

MS F 298.61 103.11 2.90

P-value 9.84E-07

F crit 4.07

919.01 11.00

C. Analysis of Variance on Escherichia coli

Source of Variation Between Groups Within Groups Total Pvalue 3E-07

SS 782.01 15.26 797.28

Df 3.00 8.00 11

MS 260.67 1.91

F 136.63

F crit 4.07

D. Analysis of Variance on Anti-inflammatory Effect

Source of Variation Between Groups Within Groups Total

SS 0.045 0.004

df 3.000 8.000

MS F 0.015 28.141 0.001

P-value 0.00013

F crit 4.066

0.049 11.000

E. Analysis of Variance on Antipyretic Property (2 hours after treatment)

Source of Variation Between Groups Within Groups Total

SS 7.310 3.467 10.777

df 3.000 8.000 11.000

MS F 2.437 5.623 0.433

P-value 0.023

F crit 4.066

92

F. Analysis of Variance on Antipyretic Property (6 hours after treatment)

Source of Variation Between Groups Within Groups Total

SS 9.21 3.04 12.25

Df 3.00 8.00 11.00

MS 3.07 0.38

F 8.08

P-value 0.01

F crit 4.07

G. Analysis of Variance on Antipyretic Property (10 hours after treatment)

Source of Variation Between Groups Within Groups Total

SS 8.97 3.41 12.3825

df 3.00 8.00 11

MS 2.99 0.43

F 7.01

P-value 0.01

F crit 4.07

H. Analysis of Variance on Analgesic Reaction Thirty Minutes After Injecting the Extract

Source of Variation Between Groups Within Groups Total

SS 88.33 286.67 375

df 3.00 8.00 11

MS 29.44 35.83

F 0.82

P-value 0.52

F crit 4.07

I. Analysis of Variance on Analgesic Reaction One Hour and Thirty Minutes After Injecting the Extract
Source of Variation Between Groups Within Groups Total

SS 84.25 394.67 478.92

df 3 8 11

MS 28.08 49.33

F 0.57

P-value 0.65

F crit 4.07

93

J. Analysis of Variance on Analgesic Reaction Two Hours and Thirty Minutes After Injecting the Extract
Source of Variation Between Groups Within Groups Total

SS 91.682 294.5 386.18

df 3 7 10

MS 30.56 42.07

F 0.73

P-value 0.57

F crit 4.35

Results of Antibacterial Screening.

94

Bagras Toxicity Test Setup.

Cogon Toxicity Test Setup

95

Bibliography
Books (Guevera, Beatrice Q. et al. 2005)

(Burton, 2004)

(New Standard Encyclopedia, Standard Educational Corporation, Chicago)

(Lippincott Williams and Wilkins, 2004)

(Burton, 2004)

(Guevera, Beatrice Q. et al. 2005) ( The New Lexicon Websters Encyclopedic Dictionary of the English Language, Deluxe Edition, 1991 ) Internet

(http://www.answers.com/topic/agar-1) (http://en.wikipedia.org/wiki/Alkaloid) (http://en.wikipedia.org/wiki/Amoxicillin) (http://en.wikipedia.org/wiki/Analgesic) (http://www.medterms.com/script/main/art.asp?articlekey=10215) (http://en.wikipedia.org/wiki/Anti-inflammatory) (http://en.wikipedia.org/wiki/Antipyretic) (http://en.wikipedia.org/wiki/Assay) (http://www.answers.com/topic/bacteria) 96

(http://lecturer.ukdw.ac.id/dhira/NutritionGrowth/culturemedia.html)

(http://en.wikipedia.org/wiki/Ethanol)

(http://en.wikipedia.org/wiki/Eucalyptus_deglupta)

(http://en.wikipedia.org/wiki/Flavonoid) (http://en.wikipedia.org/wiki/Gram-negative_bacteria) (http://www.buzzle.com/articles/gram-negative-bacteria.html)

(http://en.wikipedia.org/wiki/Gram-positive_bacteria)

(http://en.wikipedia.org/wiki/Guinea_pig)

(http://en.wikipedia.org/wiki/Imperata_cylindrica)

(http://en.wikipedia.org/wiki/In_vitro)

(http://en.wikipedia.org/wiki/Mouse)

(http://en.wikipedia.org/wiki/Mueller-Hinton_agar)

(http://en.wikipedia.org/wiki/Paracetamol)

http://en.wikipedia.org/wiki/Proanthocyanidin)

(http://en.wikipedia.org/wiki/Phytochemical) (http://www.answers.com/topic/pour-plate-culture) (http://en.wikipedia.org/wiki/Screening)

97

(http://www.cyberlipid.org/sterols/ster0003.htm)

(http://en.wikipedia.org/wiki/Synergism)

(http://en.wikipedia.org/wiki/Tannin) (http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm)

98