Determination of Antibacterial Property of Guyabano

(Anona muricata Linn.) Leaves Extract

against Diarrhea

Fritz John M. Saloma

Submitted on partial fulfillment of the requirements

In Research II

Nabunturan National Comprehensive High School

Nabunturan, Compostela Valley

September, 2010

Mrs. Rowena Y. Pontillo

Research Adviser

ACKNOWLEDGEMENT

The author would like to express his heartfelt gratitude to the people who share their
knowledge, abilities and time for the success of this study.
He therefore extends his thanks to the people behind his success:
First and foremost to the almighty God, who provides guidance, courage, strength,
wisdom and instruments for the realization of this undertakings;
To Mr Norberto Casia for providing the laboratory equipments.
To all his research advisers, Maam Rowena Y. Pontillo and Sir Jose Banaag Catugal,
fortheir kind and careful criticisms which had motivated him to make his research
completeand correct.
To all the handsome and pretty faces inside the four corners of 1V- Charity, his
classmates who together with him endured all the hardships, helped and encouraged
each other in order for us all to finish our research.
A special acknowledgement is also extended to his loving parents for their love, patience,
advises and financial support.
Once again, thank you and God bless!

TABLE OF CONTENTS
Titles

Pages

Chapter 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1-3

Definition of terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3-5

Chapter 2
Review of Related Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6-34

CONCEPTUAL PARADIGM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35

Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

36

CHAPTER 3
METHODOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37-41

CHAPTER 1
INTRODUCTION
In this chapter, all about the problem will be discuss, like the background of the study.
This chapter also asks the questions is there anything about this study that we should
know And what are the things that we will be discussing and wont discuss.

Background of the study

The use of herbal medicine is an integral part of the Philippine culture .Within
this culture, various plants have been popularly studied to determine their
medicinal properties. In addition to this, other native plants are being investigated
to bring about other alternative methods which could be as effective as the
commercial products .This includes the investigation of Anona muricata Linn.
(GUYABANO). Guyabano is one of the healthiest fruits known to man
Anona muricata L. is reported to possess promising medicinal properties such as
cytotoxicity, antimicrobial and antiparasitic properties. Even though findings from
other countries have shown that Anona muricata has antimicrobial properties
against common pathogens, the Guyabano species in the Philippines could have a
different set of properties comparable to their foreign counterparts.
This study will then look at the possible antibacterial property of the
guyabano leaf.

-1-

Statement of the Problem

The general objective is to determine the antibacterial property of Anona muricata
L. leaf extract against Escherichia coli.
Specifically, this study aims:
1. To determine the potential of Guyabano (Anona muricata Linn.) leaves
extract to treat Diarrhea.
2. To determine if the extract will inhibit the growth of E.coli.

Hypothesis
1. Guyabano leaves extract is feasible to be a cure to diarrhea.
2. Guyabano leaves extract is better than the expensive commercial products

Scope and Delimitations

This study introduced the feasibility of guyabano leaves extract to cure Diarrhea.
It would also introduced the cheaper cost of the samples compared to the Commercial
medicine. This study uses leaves only and it doesnt include other parts of the plant.

-2-

Significance of the Study

The use of herbal medicine is an integral part of the Philippine culture. Within this
culture, various plants have been popularly studied to determine their medicinal
properties. In addition to this, other native plants are being investigated to bring about
other alternative methods which could be as effective as the commercial products .This
includes the investigation of Anona muricata Linn. (GUYABANO). Guyabano is one of
the healthiest fruits known to man Knowing these data, it would be concluded that
making a cheap antifungal natural cream shall be produced. Medical industry and people
attacked by bacterial infection are the ones to benefit from it, especially the poor people.

DEFINITION OF TERMS
Antibacterial property is the distinct property of an organism or a substance that
inhibits the growth of certain bacteria.

Bacteria

are microscopic organisms whose single cells have neither a membrane-

bounded nucleus nor other membrane-bounded organelles like mitochondria and

chloroplasts. Another group of microbes, the archaea, meet these criteria but are so
different from the bacteria in other ways that they must have had a long, independent
evolutionary history since close to the dawn of life. In fact, there is considerable evidence
that you are more closely related to the archaea than they are to the bacteria
-3Disc diffusion antibiotic sensitivity testing, is a test which uses antibiotic-impregnated

wafers to test whether particular bacteria are susceptible to specific antibiotics. A known
quantity of bacteria is 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).The size of
the zone and the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity
to that particular antibiotic. In general, larger zones correlate with smaller minimum
inhibitory concentration (MIC) of antibiotic for those bacteria.

Escherichia coli
Are a common type of bacteria that can get
into food, like beef and vegetables.
E. coli is short for the medical term Escherichia coli.
The strange thing about these
bacteria and lots of other bacteria is that they're
not always harmful to you. E. coli
normally lives inside your intestines, where it helps
your body break down and digest the
food you eat.
Unfortunately, certain types (called strains) of E. coli can get from the intestines
into the blood. This is a rare illness, but it can cause a very serious infection.

-4-

Nutrient agar
is a microbiological growth medium commonly used for the routine
cultivationof non-fastidious bacteria. It is useful because it remains solid even at
relatively high temperatures. Also, bacteria grown in nutrient agar grows on the surface,
and is clearly visible as small colonies. In nutrient broth, the bacteria grows in the liquid,
and is seen as a soupy substance, not as clearly distinguishable clumps. Nutrient agar
typically contains

0.5 % peptone

0.3 % beef extract/yeast extract

1.5 % agar

0.5% NaCl

distilled water

pH adjusted to neutral (6.8)at 25 C.

-5-

CHAPTER 2
REVIEW OF RELATED LITERATURE

This chapter contains the information about guyabano and diarrhea.

Guyabano
Scientific classification:
Kingdom: Plantae
(Unranked): Magnoliids
Family: Annonaceae
Species: A. muricata

(Unranked): Angiosperms
Order: Magnoliales
Genus: Annona
Binomial name Annona muricata

Belongs to the family of Annonaceae, (A. muricata L.). The flesh of the fruit
consist of a white edible pulp that is high in carbohydrates and considerable amounts of
Vitamin C, Vitamin B1, Vitamin B2, Potassium and dietary fiber. Guyabano is low in
cholesterol, saturated fat and sodium. No only is guyabano a good health food, it also
taste delicious. The tree and fruit is known in various names: Guyabano in Filipino,
Soursop in English, Graviola in Brazil, and Guanabana in Spanish.

-6-

About the Guyabano

The heart shaped / oblong guyabano fruit has a dark green, leathery and spike-like skin
that measures from 8 to 12 inches long and can weigh up to 2.5 kilos. The creamy and
delectable flesh contains from 60 to 100 black-brown seeds that are indigestible and nonedible.
The guyabano tree is relatively small. It usually grows from 8 to less than 20 feet high
and is sensitive to very cold temperatures. The guyabano tree requires a lot of water,
warmth and humidity and is usually grown in the tropics. It is cultivated commercially in
Central & South America, West Africa, Asia and South Florida in limited
numbers.Guyabano is one of the healthiest fruits known to man. The flesh of the fruit
consist of a white edible pulp that is high in carbohydrates (particularly fructose) and
considerable amounts of Vitamin C, Vitamin B1, Vitamin B2, potassium and dietary fiber.
Guyabano is low in cholesterol, saturated fat and sodium. Not only is guyabano a good
health food, it also tastes delicious.
Aside from being eaten raw, the guyabano fruit is processed into candies, tarts,
shakes,ice cream, and sherbets and other beverages. An assortment of punch and cocktail
drinks can be made by mixing the nectar with wine rum or cola drinks or buko (fresh
coconut) juice and ice.The fruit, seeds, and leaves have a number of herbal medicinal
uses in countries where the plant is common. The sap of the young leaves may be applied
directly on pimples to inducesuppuration. The sap is also considered parasitical. An
alcoholic extract of the leaves, when distilled with steam, yields a small amount of
essential oil.
-7-

The portion of alcoholic extract which is soluble in water contains a large amount of
potassium chloride together with dextrose tannins, amorphous products, and a small
amount of an alkaloid substance which could not be crystallized. The leaves and roots
also cure colic and convulsions.
Botany
Small tree, usually less than 7 meters high. Leaves are smooth, shiny oblong to oblongovate, pointed on both ends, with petioles about 5 mm long. Flowers are large, solitary,
yellow or greenish-yellow. Fruit is ovoid and large, covered with small spine-like
structures. Pulp is fleshy, soft, white and fibrous.

Distribution

Cultivated for its edible fruit.

Parts utilized

Leaves,flowers, fruiit.

Chemical constituents

Alcoholic extract of leaves yielded as essential oil.

The extract also yielded a dark-green resin containing: myricyl alcohol, sitosterol,
fatty
-8-

Acids (oleci, linolic, and stearic) and anonol.

Flesh of fruit contains saccharose 2.53%, dextrose 5.05 percent, and levulose
0.04%.

Bark contained an alkaloid found to be poisonous.

Seed contains a non-poisonoous alkaloid.

Recent studies isolated three acetogenins: annonacin, annonacin A and

annomuricin A.

Properties

Fruit is reported antiscorbutic and astrigent>

Flowers are pectoral.

Leaves are antispasmodic.

Seeds are emetic.

Infusion of leaves is sudorific.

Recent studies suggest a potential for antiviral, antiparasitic and anti-cancer

properties.

Toxicity

Presence of alkaloids anonaine and anoniine have been reported.The bark yields

muricine and muricinine and is high in hydrocyanic acid; small amounts are found in the
leaves and roots, and a trace in the fruit.

Caribbean study suggested a connection between consumption of soursop and

atypical forms of Parkinson's disease due to the very high concentration of
Annonacin

-9-

Uses
Nutrition

Juicy ripe fruit is eaten raw. Used in making beverages and jelly tarts.

Fruit is an excellent source of vitamins B and C.

Folkloric

Unripe fruit used for dysentery.

Ripe fruit is antiscorbutic.

Seeds and green fruit are astringent.

Flowers are antispasmodic.

Infusion of leaves used as sudorific, antispasmodic and emetic.

In some cultures, the fruits and leaves are used for tranquilizing and sedative
properties.

Juice of ripe fruit used as diuretic and for hematuria and urethritis.

Flesh of soursop used as poultice to draw out chiggers.

Decoction of leaves used of head lice and bedbugs.

Pulverized seeds and seed oil effective for head lice.

Fruit used as a bait in fish traps.

Decoction of leaves used as compresses for inflammation and swollen feet.

-10-

Others

Bark has been used in tanning.

The wood is a potential source of paper pulp.

For a good night's sleep, leaves are put in the pillow slip or strewn in bed.

Related Studies

Cytotoxicity / Antileishmanial: Cytotoxicity and antileishmanial activity of

Annona muricata pericarp: Extracts and fractionation led to the isolation of three
acetogeninsannonacin, annonacin A and annomuricin A

Diabetes: (1) Morphological Changes and Hypoglycemic Effects of Annona

Muricata Linn. (Annonaceae) Leaf Aqueous Extract on Pancreatic Cells of
Streptozotocin-Treated Diabetic Rats. (2) Histopathologic study showed
regeneration of -cells of pancreatic islets in A. muriatica- treated rats compared
to untreated diabetic group.

Anti-Herpes Simplex Virus: Study showed the extract of AM to inhibit the

cytopathic effect of HSV-1 on vero cells indicasting an anti-HSV1 potential.

Anticancer / Acetoginins: There are much publicized studies on the antitumor,

anti-cancer and selective toxicity of Annona muricata against several types of
cancer cells. One study showed that one of the acetoginins was selectively

cytotoxic to colon adenocarcinoma cells, with a potency 10,000 times that of

adriamycin.
-11-

Diarrhea

Diarrhea is an increase in the frequency of bowel movements or a decrease in the form

of stool (greater looseness of stool). Although changes in frequency of bowel
movementsand looseness of stools can vary independently of each other, changes often
occur in both. Diarrhea needs to be distinguished from four other conditions. Although
these conditions may accompany diarrhea, they often have different causes and different
treatments than diarrhea. These other conditions are:
1. Incontinence of stool, which is the inability to control (delay) bowel movement
until an appropriate time, for example, until one can get to the toilet
2. Rectal urgency, which is a sudden urge to have a bowel movement that is so
strong that if a toilet is not immediately available there will be incontinence
3. Incomplete evacuation, which is a sensation that another bowel movement is
necessary soon after a bowel movement, yet there is difficulty passing further
stool the second time
4. Bowel movements immediately after eating a meal

Diarrhea can be defined in absolute or relative terms based on either the frequency
of bowel movements or the consistency (looseness) of stools.

-12Consistency of stools. Absolute diarrhea is more difficult to define on the basis of the
Consistency of stool because the consistency of stool can vary considerably in healthy
individuals depending on their diets. Thus, individuals who eat large amounts
ofvegetables will have looser stools than individuals who eat few vegetables. Stools that
areliquid or watery are always abnormal and considered diarrhea. Relative diarrhea is
easierto define based on the consistency of stool.

Diarrhea develop
With diarrhea, stools usually are looser whether or not the frequency of
bowel movements is increased. This looseness of stool--which can vary all the way from
slightly soft to watery--is caused by increased water in the stool. During normal
digestion, food is kept liquid by the secretion of large amounts of water by the stomach,
upper small intestine, pancreas, and gallbladder. Food that is not digested reaches the
lower small intestine and colon in liquid form. The lower small intestine and particularly
the colon absorb the water, turning the undigested food into a more-or-less solid stool
with form. Increased amounts of water in stool can occur if the stomach and/or small
intestine secretes too much fluid, the distal small intestine and colon do not absorb
enough water, or the undigested, liquid food passes too quickly through the small
intestine and colon for enough water to be removed. Of course, more than one of these
abnormal processes may occur at the same time.

-13For example, some viruses, bacteria and parasites cause increased secretion of
fluid, either by invading and inflaming the lining of the small intestine (inflammation
stimulates the lining to secrete fluid) or by producing toxins (chemicals) that also
stimulate the lining to secrete fluid but without causing inflammation.
Inflammation of the small intestine and/or colon from bacteria or from
ileitis/colitis can increase the rapidity with which food passes through the intestines,
reducing the time that is available for absorbing water. Conditions of the colon such as
collagenous colitis can block the ability of the colon to absorb water.
Diarrhea generally is divided into two types, acute and chronic.

Acute diarrhea lasts from a few days up to a week.

Chronic diarrhea can be defined in several ways but almost always lasts more
than three weeks.

It is important to distinguish between acute and chronic diarrhea because they usually
have different causes, require different diagnostic tests, and require different treatment.

Common causes of acute diarrhea

The most common cause of acute diarrhea is infection--viral, bacterial, and parasitic.
Bacteria also can cause acute food poisoning. A third important cause of acute diarrhea is
starting a new medication.

-14-

Food poisoning
Food poisoning is a brief illness that is caused by toxins produced by bacteria. The toxins
cause abdominal pain (cramps) and vomiting and also cause the small intestine to secrete
large amounts of water that leads to diarrhea. The symptoms of food poisoning usually
last less than 24 hours. With some bacteria, the toxins are produced in the food before it
is eaten, while with other bacteria, the toxins are produced in the intestine after the food
is eaten.
Symptoms usually appear within several hours when food poisoning is caused by toxins
that are formed in the food before it is eaten. It takes longer for symptoms to develop
when the toxins are formed in the intestine (because it takes time for the bacteria to
produce the toxins). Therefore, in the latter case, symptoms usually appear after 7-15
hours. Staphylococcus aureus is an example of a bacterium that produces toxins in food
before it is eaten. Typically, food contaminated with Staphylococcus (such as salad, meat
or sandwiches with mayonnaise) is left un-refrigerated at room temperature overnight.
The Staphylococcal bacteria multiply in the food and produce toxins. Clostridium
perfringens is an example of a bacterium that multiplies in food (usually canned food),
and produces toxins in the small intestine after the contaminated food is eaten.

-15-

Traveler's diarrhea
There are many strains of E. coli bacteria. Most of the E. coli bacteria are normal
inhabitants of the small intestine and colon and are non-pathogenic, meaning they do not
cause disease in the intestines. Nevertheless, these non-pathogenic E. coli can cause
diseases if they spread outside of the intestines, for example, into the urinary tract (where
they cause bladder or kidney infections) or into the blood stream (sepsis).
Certain strains of E. coli, however, are pathogenic (meaning they can cause
disease in the small intestine and colon). These pathogenic strains of E. coli cause
diarrhea either by producing toxins (called enterotoxigenic E. coli or ETEC) or by
invading and inflaming the lining of the small intestine and the colon and causing
enterocolitis (called enteropathogenic E. coli or EPEC).
Traveler's diarrhea usually is caused by an ETEC strain of E. coli that produces a
diarrhea-inducing toxin.Tourists visiting foreign countries with warm climates and poor
sanitation (Mexico, parts of Africa, etc.) can acquire ETEC by eating contaminated foods
such as fruits, vegetables, seafood, raw meat, water, and ice cubes. Toxins produced by
ETEC cause the sudden onset of diarrhea, abdominal cramps, nausea, and sometimes
vomiting.
These symptoms usually occur 3-7 days after arrival in the foreign country and
generally subside within 3 days.

-16-

Bacterial enterocolitis
Disease-causing bacteria usually invade the small intestines and colon and cause
enterocolitis (inflammation of the small intestine and colon). Bacterial enterocolitis is
characterized by signs of inflammation (blood or pus in the stool, fever) and abdominal
pain and diarrhea. Campylobacter jejuni is the most common bacterium that causes acute
enterocolitis in the U.S. Other bacteria that cause enterocolitis include Shigella,
Salmonella, and EPEC. These bacteria usually are acquired by drinkingcontaminated
water or eating contaminated foods such as vegetables, poultry, and dairy products.
Enterocolitis caused by the bacterium Clostridium difficile is unusual because it
often is caused by antibiotic treatment. Clostridium difficile is also the most common
nosocomial

infection

(infection

acquired

while

in

the

hospital)

to

cause

diarrhea.Unfortunately, infection also is increasing among individuals who have neither

taken antibiotics nor been in the hospital.

-17-

Escherichia coli
Scientific classification:
Domain: Eubacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: E. coli

Binomial name Escherichia coli

What Is E. Coli:
Escherichia coli (E. coli) are a group of bacteria that can cause a variety of illnesses
in humans, including diarrhea, urinary tract infections, respiratory illnesses pneumonia
and other problems. There are also many types of E. coli bacteria that are harmless.
One type of E. coli that often causes illness and outbreaks in humans is known as E. Coli
O157. There are other strains of E. coli that cause illness as well, but O157 is the most
notorious, so we will focus on that strain in this article.

How Do You Get E. Coli O157?

E. coli lives in the intestinal tracts of humans and animals, such as cattle, goats, elk,
sheep and deer. Other animals, including pigs and birds, can pick up E. coli from the
environment and spread it. The most common cause of E. coli infection in humans is
cattle. E. coli is spread through contaminated fecal matter from animals that are carrying
the disease (these animals are typically not sick themselves) or from people who have an
infection but do not wash their hands properly after using the bathroom.

-18These microscopic amounts of fecal matter end up on food that we eat and then cause
infectionsOther sources of E. coli include raw (unpasteurized) milk, water that has not
been disinfected, unpasteurized apple cider, soft cheese made from unpasteurized milk,
swallowing lake water while swimming and touching animals in petting zoos or animal
exhibits.

Symptoms of E. Coli
Symptoms of an intestinal E. coli infection vary by person but can include:

Stomach cramps
Diarrhea (usually bloody)
Vomiting
Low-grade fever (typically not over 101)

These symptoms usually resolve on their own within 5 to 7 days. E. coli infections are
mild in most people, but they can be serious or even life threatening in others. Symptoms
of an E. coli infection typically start 3 to 5 days after exposure to the bacteria, but they
can occur as early as one day or as late as 10 days after exposure.

How Is E. Coli Treated:

For a typical E. coli infection, treatment mostly consists of staying hydrated. Drinking
water or electrolyte drinks, such as Gatorade or Pedialyte (for children), will help to
prevent dehydration. Eating when you have a stomach bug is not that important as long as
you are staying hydrated. For those people who are unable to keep any liquids down, IV
fluids may be needed.

-19Antibiotics are not effective against E. coli and should not be used, because they may
increase the risk of complications, such as HUS. Antidiarrheal medications, such as
Immodium, should be avoided, because they can also increase this risk. People who
develop HUS should be hospitalized to manage the illness. Various treatments may be
needed depending on the symptoms.

How Can E. Coli Infections be Prevented:

Nearly everyone is at risk for getting an E. Coli infection. Although infants, young
children, older adults and people with compromised immune systems are at the highest
risk for serious illness, even healthy adults can have complications. Some things you can
do to minimize your risk of infection include:

Good hand washing. Especially after using the restroom, changing diapers and
before preparing or eating food. Also wash your hands thoroughly after touching
any animals or their environments, even your own pets.

Cook meats thoroughly. Ground beef and meat that has been needle tenderized
should be cooked to an internal temperature of at least 160.

Avoid raw or unpasteurized milk, cheeses or other dairy products and

unpasteurized fruit juices or ciders.

Avoid swallowing water when swimming anywhere.

-20What is the natural habitat of E. coli:

The E. coli was first identified in the colon region of large intestine and so it was
given the name "coli" (found in colon) they are coliforms. It luxuriously grows in our
large intestine and it is an important normal microflora of human. Most strains won't do
any harm when present in the intestine. If they enter the blood or other sites of the body,
the

bacteria

can

cause

infections,

including

urinary

tract

infection.

E. coli is found in hugely varied environments and hence it is highly versatile in its
physiology. It responds in different ways to variances in pH, temperature, oxygen levels,
osmolarity, etc. As unstable-ga pointed out, it is a peritrichous flagellate bacillus(rodshaped bacteria with more than one flagellum or 'tail.')

Analogy
E. coli is described as Gram negative bacteria. (The term is derived from the fact
that the bacteria are not stained purple by a Gram stain.) The real difference is in the
composition of the bacteria's cell wall, which in Gram negative cells is thinner and
contains more lipopolysaccharide than Gram positive cell walls. Within the cell wall, the
bacteria's cell contents are very simple: from one to four identical molecules of DNA and
15,000 to 30,000 ribosomes. (Ribosomes are the site of protein-synthesis. Essentially,
E. coli grows by up taking nutrients directly from the environment through its cell wall.
Because it is found in so many different environments, it is able to use many different

chemicals as fuel.' After taking up these fuels, it converts them into energy and the
chemical building blocks it requires.

-21Under favorable conditions, this can occur once every thirty minutes, growing
exponentially. However, a phenomenon called 'conjugation' occasionally occurs: it's the
single-celled organism's answer to sexual reproduction, a method of genetic shuffling. E.
coli may conjugate with bacteria of different species or genera, not just other E. coli

Cellular and Molecular Physiology of Escherichia coli

Upon exposure to oxygen, Escherichia coli increases the expression of

enzymes essential for aerobic respiration, such as components of the TCA cycle and
terminal oxidase complexes. This increase requires the elimination of repressionmediated
by the Arc regulatory system under anaerobic conditions. Coordinately, the synthesis of
enzymes that function in anaerobic processes such as fermentation decreases, partly due
to the inactivation of the transcription factor Fnr. E. coli is thus able to adjust the levels of
respiratory enzymes to fit its environmental circumstances, and in this case, reduces the
production of the less energy efficient fermentation enzymes in favor of the aerobic
pathways To alleviate the toxicity of these compounds, E. coli induces the synthesis of
protective enzymes, such as Mn-dependent superoxide dismutase (SodA) and catalase I
(HP I), and this induction is controlled by the regulatory proteins SoxRS, OxyR, and
ArcAB.

-22-

Thus, ArcAB, Fnr, SoxRS, and OxyR function in concert so that E. coli can optimize its
energy production and growth rate. Fnr and SoxRS are cytoplasmic, DNA-binding
proteins, and these regulatory systems utilize iron-sulfur clusters as cofactors which may
directly sense the redox environment. OxyR is also a cytoplasmic, DNA-binding protein,
and appears to respond to redox potential through the oxidation state of a specific
cysteine residue. In the ArcAB system (which belongs to the family of
During aerobic growth, the transphosphorylation of ArcA does not occur. In
this signal transduction mechanism, the ArcB C-terminal or receiver domain plays a
critical role; that is, it stimulates or abolishes the transphosphorylation depending on the
metabolic state of the cell, which in turn is influenced by the availability of oxygen. E.
coli thus employs at least four global regulatory systems which monitor the cellular
oxidative/metabolic conditions, and adjust the expression of more than 70 operons to give
the organism a better aerobic life.

Life Cycle of Escherichia coli

E. coli includes both asexual and sexual reproductions in its life cycle. Its
asexualreproduction is achieved by simple binary fission. The sexual reproduction of
E.coli takes place by conjugation which occurs only between genetically differentiated

strains. Some mating types of E. coli have a small extra-chromosomal DNA, particle
called sex factor or F, while other mating types may lack the sex factor.
-23The bacterial strains with sex factors are called F+ or Hfr (male or-donor) strains, while
bacterial strains lacking the sex factor are called F- or female acceptor strains. When a
chance collision of F+ and F- bacteria takes place, both types of cells attach with each
other and the walls between the two bacterial cells break open to form a small
cytoplasmic bridge between the two. Through this cytoplasmic bridge the transfer of sex
factor (DNA segment) of F+ cell to F- or female bacterial cell, takes place and thus
conjugation completes. The conjugating bacterial cells now separate and are called
exconjugants. The receptor F- cell thus, assumes a partial diploidy and is called
"merozygote". The F- merozygote reproduces by cell division and ultimately regains
monoploidy.

A. F+ or Hfr ('Male' or donor cell)

B. Closed bacterial chromosome

C. F-('Female' or acceptor cell)

D. Unclosed chromosome
F. DNA molecule

E. (Replication of chromosome Replica fail

to ring-close)

-24H. Conjugation and transfer of segment

G. Cytoplasmic bridge

of donor DNA

I. Fission

J. Partial diploid receptor or Merozygote

K. Recombination

L. Monoploid Cells

Genetic applications of Escherichia coli- E. coli has been the most favourite
experimental organism for the geneticists because it is non-pathogenic, easy to culture,
economical, small-sized, simply organized and has various biochemically and
physiologically detectable phenotypic traits. Further, it multiplies very rapidly.

When the doctor should be called for diarrhea:

Most episodes of diarrhea are mild and of short duration and do not need to be brought to
the attention of a doctor. The doctor should be consulted when there is:

High fever (temperature greater than 101 F

Moderate or severe abdominal pain or tenderness

Bloody diarrhea that suggests severe intestinal inflammation

Diarrhea in persons with serious underlying illness for whom dehydration may
have more serious consequences, for example, persons with diabetes,
heartdisease, and AIDS

Severe diarrhea that shows no improvement after 48 hours.

-25

Diarrhea that occurs during or immediately after completing a course of

antibiotics because the diarrhea may represent antibiotic-associated infection with
C. difficile that requires treatment

Diarrhea that develops in patients with chronic intestinal diseases such as colitis,
or Crohn's disease because the diarrhea may represent worsening of the
underlying disease or a complication of the disease, both requiring treatment

Acute diarrhea in an infant or young child in order to ensure the appropriate use
of oral liquids (type, amount, and rate), to prevent or treat dehydration, and to
prevent complications of inappropriate use of liquids such as seizures and
abnormal blood electrolytes

How is diarrhea treated:

Absorbents. Absorbents are compounds that absorb water. Absorbents that are taken
orally bind water in the small intestine and colon and make diarrheal stools less watery.
They also may bind toxic chemicals produced by bacteria that cause the small intestine to
secrete fluid; however, the importance of toxin binding in reducing diarrhea is unclear.
The two main absorbents are attapulgite and polycarbophil, and they are both available
without prescriptions.
Examples of products containing attapulgite are:

Donnagel,

Rheaban,
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Kaopectate Advanced Formula, Parepectolin, and Diasorb.

Examples of products containing polycarbophil are:

Equalactin,

Konsyl Fiber,
Mitrolan, and
Polycarb.
Equalactin is the antidiarrheal product containing attapulgite; however the

laxative, Konsyl, also contains attapulgite. Attapulgite and polycarbophil remain in the
intestine and, therefore, have no side effects outside of the gastrointestinal tract. They
may occasionally cause constipation and bloating. One concern is that absorbents also
can bind medications and interfere with their absorption into the body. For this reason, it
often is recommended that medications and absorbents be taken several hours apart so
that they are physically separated within the intestine.
Anti-motility medications. Anti-motility medications are drugs that relax the muscles of
the small intestine and/or the colon. Relaxation results in slower flow of intestinal
contents. Slower flow allows more time for water to be absorbed from the intestine and
colon and reduces the water content of stool. Cramps, due to spasm of the intestinal
muscles, also are relieved by the muscular relaxation.The two main anti-motility
medications are loperamide (Imodium), which is available without a prescription, and
diphenoxylate (Lomotil), which requires a prescription..

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Loperamide (Imodium), though

related to opiates, does

not cause addiction.

Loperamide and diphenoxylate are safe and well-tolerated. There are some
precautions, however, that should be observed.

Anti-motility medications should not be used without a doctor's guidance to treat

diarrhea caused by moderate or severe ulcerative colitis, C. difficile colitis, and
intestinal infections by bacteria that invade the intestine (for example, Shigella).
Their use can lead to more serious inflammation and prolong the infections.

Diphenoxylate can cause drowsiness or dizziness, and caution should be used if

driving or performing tasks that require alertness and coordination are required.

Most unimportant, acute diarrhea should improve within 72 hours. If symptoms

do not improve or if they worsen, a doctor should be consulted before continuing
treatment with anti-motility medications.

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When antibiotics should be used for diarrhea:

Most episodes of diarrhea are acute and of short duration and do not require
antibiotics. Antibiotics are not even necessary for the most common bacterial infections
that cause diarrhea. Antibiotics, however, often are used when (1) patients have more
severe and persistent diarrhea, (2) patients have additional debilitating diseases such as
heart failure, lung disease, and AIDS, (3) stool examination and testing discloses
parasites, more serious bacterial infections (for example, Shigella), or C. difficile, and 4)
traveler's diarrhea
RELATED STUDIES

Diarrhea
A number of different outcome measures have been proposed for use in
prospective studies of morbidity associated with childhood diarrhea. These include the
number of episodes experienced by each child over a defined period (a measure of
incidence) and the number of days of diarrhea divided by the total number of days of
observation for each child (a measure denoted longitudinal prevalence). The authors
examined data from Ghana to determine which of these measures is more strongly
associated with weight gain over a 4-month period and subsequent mortality.
-29-

Studies were sought in bibliographic databases of traditional biomedical as well as

complementary and alternative medicine literature published from 1966 to 2000. Search
terms were "competitive inhibition," "diarrhea," "gastroenteritis," "Lactobacillus,"
"probiotic," "rotavirus," and "yog(h)urt." We included studies that were adequately
randomized, blinded, controlled trials in which the treatment group received
Lactobacillus and the control group received an adequate placebo and that reported
clinical outcome measures of diarrhea intensity. These inclusion criteria were applied by
blind review and consensus. The original search yielded 26 studies, 9 of which met the
criteria. Multiple observers independently extracted study characteristics and clinical
outcomes.

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Conceptual Paradigm

EXPERIMENTAL GROUP

Independent Variable

Dependent Variable

Guyabano extract

Zone of Inhibition

CONTROLLED GROUP
Compared Wide

Loperamide

Zone of Inhibition

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Experimental Design
Collection of Samples

Collection of Test Bacteria

Sterilization of Laboratory Materials

Extraction

Disc Diffusion Technique /Assay

Data Collection

Decontamination of Used Materials

Disposal of Agar Plates and Culture Medium

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CHAPTER 3
METHODOLOGY

Materials and Methods:

Research Design
This study utilized an Experimental Study Design.
Collection of Samples
Mature leaves of Guyabano (Anona muricata Linn.) were collected from
Nabunturan, Compostela Valley.
Collection of test bacteria
Culture of Escherichia coli were obtained from Department of Science and
Technology, Davao City.

Testing for Antibacterial Activity:

Sterilization of Laboratory Materials
All the materials used in the laboratory were disinfected or sterilized first,

using the pressure cooker or autoclave for 15minutes at 15psi before performing any of
the tests. Sterilized materials are entirely free from living organisms or contaminants,
which could affect the results of the tests and make it invalid.
-33The process of keeping the culture media and equipment clean or disinfected is called
Sterilization.
Petri dishes and flaks are sterilized by dry heat, such as that produce by
an oven at 170c for two hours. Cotton-stopper test tubes containing media are sterilized
in moist heat by placing them for 20 minutes in a large pressure cooker called autoclave
where they are subjected to live steam at 15lbs. of pressure at 121c. Broths and agar
should not be sterilized in an oven because they evaporate. Inoculating needles or loops
made of nichrome wire attached to metal handles are sterilized by direct flame of Bunsen
burner or an alcohol lamp. The end of the nichrome wire attached, shaped into loop,
provides an easy means which with to obtain bacterial growth from any surface.Desk tops
are ordinarily cleaned with disinfectants. Glass rods, slides, and alcohol cover glasses are
made sterile by dropping them in 20% alcohol lamp just before use.
Extraction
The leaves were air-dried for two weeks and were cut into small pieces.
Forty (40) grams of each type were then boiled in 250 ml of water for 15 minutes, cooled,
and then filtered using filtered paper. The filtrates were placed in water bath until 20 ml
of each of the filtrate was left. The concentrates were centrifuged at 2000rpm at 25C for

10 minutes. The supernatants from the mature leaves were filtered obtaining an amount
of 2 ml each. One ml of the filtered supernatant were labeled 50% M for mature leaves.
One ml of these solutions was then diluted with 1 ml each of sterilized water.

-34The resulting solutions were labeled 50% M for mature leaves.

Disc Diffusion Technique / Assay

When a filter disc impregnated with a chemical is placed on agar the
chemical or the substance exhibiting antimicrobial property will diffuse from the disc into
the agar. This diffusion will place the chemical in the agar only around the disc. The
solubility of the substance and its molecular size will determine the size of the area of
substance infiltration around the disc. If an organism placed on agar it will not grow in
the area around the disc if it is susceptible to the substance.
This area of no growth around the disc is known as a zone of inhibition.
Antiseptics, disinfectants and antibiotics are used in different ways to
combat microbial growth.
The discs prepared from filter paper had a diameter of 6.35 mm. Discs
were placed on top of an aluminum wire mesh. For the positive control, an aliquot of 0.02
ml of the antibiotic solution (ceftazidime for E. coli) of concentration were pipetted onto
the center of the disc. An aliquot of 0.02 ml of the A. muricata leaf extract of
concentrations (50%) was also pipetted onto the center of the discs. The discs were left to

air dry in a chamber that prevents contamination of the disc surfaces with foreign matter.

-35Application of Discs
For the susceptibility setup of the microorganisms (E. coli), each disc was
pressed down to ensure complete contact with the agar surface. The disc was distributed
evenly so that they are no closer than 24 mm from center to center (each disc was not
Relocated once it came in contact with the agar surface). The plates were incubated at
35 C for 16 to 18 hours before measuring the zones of inhibition.

Decontamination of Used Materials

After every use, the materials were decontaminated using the pressure cooker
or autoclave for 30 minutes at 15kpa after decontamination, cultures were properly
disposed in the bottles or containers to be thrown to the trash cans.

Disposal of Agar Plates and Culture Medium

Agar Plates, when finished were made safe before throwing away. The usual
method was used and is to place inside an autoclave bag and sterilized by autoclaving at
121c, 103 kPa (15psi) for 15 minutes. Plastic plates melted (hence the bag). After about
20 minutes the autoclave cooled down and the bag can safely thrown away. When all the

manipulations are done, the last step was to wash the thoroughly with antimicrobial soap
and hot water before leaving the laboratory.

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