Phytochemical Screening, Antibacterial and Anti-angiogenic Property of Water Spinach (Ipomoea aquatica) Through Choreoallantoic

Membrane Assay of Duck Egg

1

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

2

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.

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 Anti-

inflammatory 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?

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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).

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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

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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: Anti-

inflammatory 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.

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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 )

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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, β-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 β-lactam antibiotics.

Amoxicillin is susceptible to degradation by β-lactamase-producing bacteria, and

so may be given with clavulanic acid to decrease its susceptibility.

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

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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

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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 Webster’s Encyclopedic Dictionary og the English Language,

Deluxe Edition, 1991 )

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,

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

assays.

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

Bacteria

Extremely small—usually 0.3 to 2.0 micrometers in diameter—and relatively

simple microorganisms possessing the prokaryotic type of cell construction. Although

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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 )

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 )

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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)

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.

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Ethanol is a straight-chain alcohol, and its molecular formula is C2H5OH. Its

empirical formula is C2H6O. An alternative notation is CH3–CH2–OH, 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)

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

13

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

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

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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.

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 Gram’s Stain test in which they do not retain the

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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

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

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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 )

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 )

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Inhibit

It means to hold in check, restrain a natural impulse either consciously or

unconsciously; to obstruct, hinder or prohibit.

( The New Lexicon Webster’s Encyclopedic Dictionary of the English Language,

Deluxe Edition, 1991 )

Inoculation

It means the act of inoculating; an instance of this; mental preparation or

conditioning (noun).

( The New Lexicon Webster’s 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)

Disk diffusion testing

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is one of several phenotypic assays which can be utilised to determine the

antimicrobial resistance profile (antibiogramme) of an organism. Disk diffusion tests

estimate in vitro susceptibility. The principle of agar diffusion is simple: Agar plates are

inoculated with a standardised inoculum of the bacteria and an antimicrobial disk is

placed on the inoculated agar plate. The disks used for a disk diffusion assay contain a

standardised known amount of an antimicrobial agent, which diffuses into the agar

when in contact with the agar surface

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 )

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

19

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 )

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)

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

fermentable dietary fibers, meet significant scientific agreement to be allowed limited

health claims by the US Food and Drug Administration (FDA).[1]

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Phytochemicals have been used as drugs for millennia. For example,

Hippocrates may have prescribed willow tree leaves to abate fever. Salicin, having anti-

inflammatory 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

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.

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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 )

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

22

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

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.

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( 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 )

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

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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 HMG-

CoA 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

other animals, corticosteroids, such as cortisol act as signalling compounds in cellular

communication and general metabolism.

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

Synergism

25

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 )

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

26

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 )

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.

The size of the inhibition zone indicates the degree of resistance, and might also give important

information about the resistance mechanism and the resistance genes involved.

(http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm)

27

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.

28

Agar GSHCS

Alkaloids

Analgesic

Antibacterial Mice

Antibiotic Guinea Pigs

Anti-inflammatory Guppies

Antipyretic

Bacteria

Phytochemical Phytochemical Screening

Toxicity Kirby-Bauer Testing

Antipyretic Testing

Flick Test Method

Anti-inflammatory Testing

Moving Average Method

Bagras (Eucalyptus deglupta)

Cogon (Imperata cylindrica)

ANOVA

29

In Vitro Synergistic Pharmacological and Antibacterial Interaction and Toxicity Testing of Bagras (Eucalyptus deglupta) and Cogon

(Imperata cylindrica)

Description

Plants Investigated

Locale of the Study

Experimental Animals

Experimental Procedures

Statistical

Combined extract of Bagras and Cogon can be used as an alternative for commercial medicine available for fever, bacterial

infections, pain (reliever), and inflammation.

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.

3.4 Procedure Flow Chart

30

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

of the procedure is shown on this page.

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)

Soaking of extracts for 24 hours.

31

E. deglupta and I. cylindrica

were collected and soaked in

Ethanol for 24 hours. It was

then extracted using the rotary

evaporator.

Crude Extraction through Rotary Evaporator.

Collection of Eucalyptus deglupta and Imperata cylindrica

Kirby-Bauer Antibiotic Testing/Disc Diffusion Testing

32

19 grams of Mueller Hinton Agar weighed.

19 grams of Mueller Hinton

Agar was weighed and then

mixed with 500 mL of distilled

water

33

After mixing the Mueller Hinton

Agar with distilled water, it was

heated on the hot plate.

34

MHA heated on the hot plate.

Sterilization of materials in the Autoclave.

15 Petri Dishes, along with the

inoculating loop and the Mueller

Hinton Agar were sterilized in

the autoclave.

Petri Dishes with label.

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

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.

35

Bacteria being placed on the Petri Dish.

As the agar is still in its liquid

state, the proponent quickly

added the E. coli and S. aureus

bacteria on the petri dishes.

Treated paper discs placed on the Petri dish.

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

replications.

Petri dishes inside the Incubator.

The Petri dishes were then left

at 37°C for 24 hours.

Chapter 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF

DATA

This chapter presents the data gathered through actual observation and

experimentation of the tests.

e 4.3 Growth inhibition of Staphylococcus aureus

REPLICATIONTreatments I II III Sum Mean

Control 14.70mm 9.85mm 8.75mm 33.3mm 11.1mmCogon 0mm 0mm 0mm 0mm 0mmBagras 18.25mm 16.30mm 17.25mm 51.80mm 17.27mm

36

Measuring of zone of inhibition.

After 24 hours, the zone of

inhibition of each treatment was

then measured with the use of

the Vernier Caliper.

Combination 22.55mm 24.10mm 23.5mm 70.15mm 23.38mmTotal 55.50mm 50.25mm 49.50mm 155.25mm 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

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

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.

Table 4.4 SUMMARY ON Staphylococcus aureus with variance

SUMMARY        Groups Count Sum Average Variance

Amoxicillin 3 33.3 11.1 10.02Cogon 3 0 0 0Bagras 3 51.8 17.27 0.95Mixture  3 70.15 23.38 0.61

37

Treatments

Table 4.5 ANOVA

Source of Variation SS Df MS F P-value F crit

Between Groups 895.84 3.00 298.61 103.11 9.84E-07 4.07Within Groups 23.17 8.00 2.90                   

Total 919.0111.0

0        

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.

Table 4.6 Growth inhibition of Escherichia coli

REPLICATIONTreatments I II III Sum Mean

Control 11.80mm 13.30mm 15.60mm 40.70mm 13.57mmCogon 0mm 0mm 0mm 0mm 0mmBagras 16.35mm 15.20mm 18.40mm 49.95mm 16.65mm

38

Combination 22.80mm 20.55mm 22.15mm 65.50mm 21.83mmTotal 50.95mm 49.05mm 56.15mm 156.15mm 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

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 Count Sum Average VarianceAmoxicillin 3.00 40.70 13.57 3.66Cogon 3.00 0.00 0.00 0.00Bagras 3.00 49.95 16.65 2.63Mixture 3.00 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.

39

Treatments

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 SS Df MS F

P-value F crit

Between Groups 782.01 3.00 260.67 136.63 3E-07 4.07Within Groups 15.26 8.00 1.91                   Total 797.28 11        

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.

Chapter 5

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATION

40

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.

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

41

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 50-55°C. Then, it was

concluded that the extracts have an analgesic property.

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

42

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, anti-

inflammatory 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).

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

43

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.

Appendix

A. Pictorials

A. Soaking of Plants and extraction through Rotary Evaporator

44

B. Phytochemical Screening

45

Soaking of extracts for 24 hours. Crude Extraction through Rotary Evaporator.

Extracts of Bagras and Cogon evaporated.

Measuring 5mL of Hydrochloric Acid.

Filtration of extract.

Evaporating of extract on hot plate and adding of Petroleum

ether.

46

Thorough mixing of substance.

Filtration of the solution.

The solutions used.

Testing for the presence of Leucoanthocyanin.

Magnesium strips added to the second test tube.

Measuring of 2mL extract.

Evaporation of extract.

C. Antibacterial Screening

47

Mixing thoroughly of extract.

Screening for Tannins.

19 grams of Mueller Hinton Agar weighed.

MHA heated on the hot plate.

Sterilization of materials in the Autoclave.

Petri Dishes with label.

D. Anti-inflammatory Screening

48

MHA being poured on the Petri dish.

Bacteria being placed on the Petri Dish.

Treated paper discs placed on the Petri dish.

Petri dishes inside the Incubator.

Measuring of zone of inhibition.

E. Antipyretic Testing

49

Carrageenan Extract.

Measuring of diameter.

Injecting of carrageenan extract.

Measuring of right hind paws.

Injecting of extract.

Measuring once again of right hind paws.

F. Analgesic Testing

50

Caged guinea pigs.

Measuring of Cochlear Temperature.

Inducing of fever.

Measuring of Cochleal Temperature once again.

Injecting of extract.

Measuring of cochlear temperature.

B. Analysis of Variance on Staphylococcus aureus

Source of Variation SS Df MS F P-value F crit

Between Groups 895.84 3.00 298.61 103.11 9.84E-07 4.07Within Groups 23.17 8.00 2.90                   Total 919.01 11.0        

51

Water boiled at 55°C.

Injection of extract.

Flick Test Method.

0

C. Analysis of Variance on Escherichia coli

Source of Variation SS Df MS F

P-value F crit

Between Groups 782.01 3.00 260.67 136.63 3E-07 4.07Within Groups 15.26 8.00 1.91                   Total 797.28 11        

D. Analysis of Variance on Anti-inflammatory Effect

Source of Variation SS df MS F P-value F crit

Between Groups 0.045 3.000 0.01528.14

1 0.00013 4.066Within Groups 0.004 8.000 0.001                   

Total 0.04911.00

0        

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

Source of Variation SS df MS F P-value F crit

Between Groups 7.310 3.000 2.4375.62

3 0.023 4.066Within Groups 3.467 8.000 0.433                   Total 10.777 11.000        

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

52

Source of Variation SS Df MS F P-value F crit

Between Groups 9.21 3.00 3.07 8.08 0.01 4.07Within Groups 3.04 8.00 0.38                   Total 12.25 11.00        

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

Source of Variation SS df MS F P-value F crit

Between Groups 8.97 3.00 2.99 7.01 0.01 4.07Within Groups 3.41 8.00 0.43                   Total 12.3825 11        

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

Source of Variation SS df MS F P-value F crit

Between Groups 88.33 3.00 29.44 0.82 0.52 4.07Within Groups 286.67 8.00 35.83                   Total 375 11        

I. Analysis of Variance on Analgesic Reaction One Hour and Thirty Minutes

After Injecting the Extract

Source of Variation SS df MS F P-value F crit

Between Groups 84.25 3 28.08 0.57 0.65 4.07

Within Groups394.6

7 8 49.33                   

Total478.9

2 11        

J. Analysis of Variance on Analgesic Reaction Two Hours and Thirty Minutes After Injecting the Extract

Source of Variation SS df MS F P-value F crit

53

Between Groups91.68

2 3 30.56 0.73 0.57 4.35Within Groups 294.5 7 42.07                   

Total386.1

8 10        

54

Results of Antibacterial Screening.

BibliographyBooks

55

Bagras Toxicity Test Setup.

Cogon Toxicity Test Setup

(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 Webster’s 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 )

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

56

(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 )

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

57

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

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

(http://www.newton.dep.anl.gov/askasci/mole00/mole00531.htm)

58