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Chapter 1

INTRODUCTION

Background

Euphorbia hirta, belongs to the family of Euphorbiaceae which is a large

family of dicotyledons, with about 300 genera and over 5,000 species. Here in

the Philippines, the Euphorbia hirta, is commonly referred to as Tawa-tawa or

Gatas-gatas in some provinces. It is also known as Asthma weed or Snake weed

in the United States. The plants of 3 different species share Phoretic variations,

these plants are: (1) Mutha (Cyperus rotundus), (2) Gatas-gatas (Euphorbia

hirta) and (3) Botoncillo (Gomphena globosa).

Tawa-tawa is usually very abundant in tropical regions such as the

Philippines. A simple weed scattered in sunny lawns, waste places and open

grasslands. It is pantropic in distribution.

The plant is an annual, hairy herb, usually branched from the base,

spreading up to 40 cm long. The stem is slender and often reddish and purplish

in color, covered with yellowish bristly hairs especially in younger parts. The

leaves are oppositely arranged, elliptical-oblong to oblong-lanceolate, 1 to 2.5 cm

long, toothed at the edge, and blotched with purple in the middle. In the axils

appear numerous involucres, purplish or greenish, dense, axillary, short stalk

clusters or crowded cymes, about 1 mm long. The capsules are broadly ovoid,

hairy, three-angled, about 1.5 cm. The small green flowers constitute the

inflourescence characteristics of the euphorbias. The stem and the leaves

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produce white or milky juice when cut (Lind and Tallantire, 1971;Anonymous

2005).

In some parts of Africa, extract of the plant are used in the treatment of

asthma and respiratory tract inflammations (Kokwaro, 1993). The plant contains

relatively abundant white latex. The white latex is capable of causing dermatitis

(Oliver, 1960). The plant shows antibiotic activity (Sofowora, 1993). Upon reading

some medical research studies on Euphorbia hirta revealed that the plant is very

popular amongst its specie; Euphorbia hirta as an herbal medicinal plant has

been presumed to possess therapeutic virtues, thus there is a need to determine

its antibacterial potentials.

Objectives

The study about Euphorbia hirta (Tawa-tawa) is intended to determine the

Minimum Inhibitory Concentration (MIC), anti-microbial properties using

Staphylococcus aureus, toxicity and platelet count.

Specifically, this study singled out to answer the following questions:

1. What is the Minimum Inhibitory Concentration (MIC) of crude extract

concoction of Tawa-Tawa roots?

2. Does Tawa-Tawa possess any anti-bacterial effect towards the test

organism- Staphylococcus aureus?

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3. Does the crude extract concoction of Tawa-Tawa roots possess any

toxic effect when administered orally to the test organism-albino mice?

4. Can Tawa-Tawa affect the platelet production of albino mice induced

with thrombocytopenic drugs such as aspirin?

Significance of the Study

Physician. This study would help medical practitioners especially doctors

of medicine particularly hematologist who specialize in the diagnosis and

treatment of Hemorrhagic Fever (H-Fever) or commonly known as Dengue

Fever. Pathologist is still searching and discovering for treatment for the disease.

Dengue Fever Patients. Dengue virus causes thrombocytopenia. This

study would help patients suffering from dengue fever lessen the burden of

having the disease.

Health Workers. Health workers play an important role in information

dissemination. Through the Health workers, information about the disease could

be avoided and minimized.

Community. The global burden of dengue has grown dramatically in

recent decades, and thus increased number of cases affects the community

especially the burden of the disease remains proportionately much greater in

Asian and Pacific countries including Philippines.

Future Researchers. This study would be of help to future researchers,

especially in the field of drugs and medicine to come up with a treatment of

Dengue fever. This would also serve as a guide to study other possible toxic, and

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anti-microbial properties of Tawa- Tawa. This work therefore, was undertaken to

authenticate the activities of Tawa- Tawa on toxicity, anti-microbial properties,

and amplification of platelet count.

Milieu

This study encompasses the potential pythochemistry of Euphorbia hirta

as well as the anti-microbial activities using Staphylococcus aureus, toxicity, and

animal inoculation assay.

This study was conducted from November 2005 to March 2008 in the City

of Bacolod, Negros Occidental.

This experimental study was performed in the Biochemistry Laboratory in

the University of Negros Occidental- Recoletos, under the supervision of Mr.

Ronnie Gicana.

The results of the research holds true only during the time the research

was done. Any modifications and further study maybe conducted to obtain more

accurate and precise results.

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Chapter 2

Review of Related Literature

This chapter presents related concepts on the risk factors and daily

practices lead to dengue fever. Important epidemiological measures,

environmental factors and prevention and control are presented to provide basis

that these factors contribute significantly to the occurrence of disease. The

effects of Tawa-Tawa were cited in the study on MIC, Agar Diffusion, and Animal

Inoculation Assays to identify their possible toxicity, anti-microbial property,

environmental management and amplification of platelets are also reviewed.

Medicinal Plants

Medicinal plants are a source of great economic value in the Philippine Island.

Nature has bestowed on us a very rich botanical wealth and a large number of

diverse types of plants grow in different parts of the country. Philippines is rich in all the

3 levels of biodiversity, namely; species, genetic diversity and habitat diversity. (Ahmad

I, et.al, Ethnopharmacol, 1998).

In Philippines, thousands of species are known to have medicinal value and the

use of different parts of several medicinal plants to cure specific ailments has been in

vogue since ancient times. Herbal medicine is still the mainstay of about 75-80% of the

whole population, mainly in developing countries, for primary health care because of

better cultural acceptability, better compatibility with the human body and fewer side

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effects. However, the last few years have seen a major increase in their use in the

developed world. (Rajkot and Jamnagar, 2004).

Nowadays multiple drug resistance has developed due to the indiscriminate use

of commercial antimicrobial drugs commonly used in the treatment of infectious

disease. In addition to this problem, antibiotics are sometimes associated with adverse

effects on the host including hypersensitivity, immune-suppression and allergic

reactions. This situation forced scientists to search for new antimicrobial substances.

Given the alarming incidence of antibiotic resistance in bacteria of medical importance,

there is a constant need for new and effective therapeutic agents. Therefore, there is a

need to develop alternative antimicrobial drugs for the treatment of infectious diseases

from medicinal plants and random screenin of active plants for active chemicals is

important. (Davis J, Science,1995)

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Euphorbia hirta or (Tawa- Tawa)

Description

Euphorbia hirta is a terrestrial, annual, erect herb, up to 60 cm tall. It is taproot

white or brown. The stem is round, solid, hairy, with abundant milk sap. Stipules are

present. The leaves are simple, not lobed or divided, opposite, sessile or stalked,

elliptic, less than 2 cm long/wide, hairy on both sides, denser pilosity along the veins in

the lower face, more scattered on the upper side; leaf base asymmetric, margin finely

dentate, apex acute, base acute, 3-veined not to the top. Flowers are unisexual, solitary

or grouped together in an axillary cyme, stalked, and petals are absent.

It is distributed in pan tropical, or partly sub-tropical areas and found in roadsides

and wastelands as cultivated areas. (Le Bourgeois T., et. al., 2001)

Medicinal Uses

Euphorbia hirta has traditionally been used in Asia to treat bronchitic asthma and

laryngeal spasm, though in modern herbalism it is more used in the treatment of

intestinal amoebic dysentery. It should not be used without expert guidance, however,

since large doses cause gastro-intestinal irritation, nausea and vomiting.

It is anti-asthmatic, antipruritic, sedative, antidote, diuretic, purgative and homostatic.

The aerial parts of the plant are harvested when in flower during the summer and

can be dried for later use. The stem, which is taken internally, is famed as a treatment

for asthma, bronchitis and various other lung complaints. The herb relaxes the

bronchioles but apparently depresses the heart and general respiration. It is usually

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used in combination with other anti-asthma herbs such as Grindelia camporum and

Lobelia inflata. It is also used to treat intestinal amoebic dysentery.

The whole plant is decocted and used in the treatment of athlete's foot, dysentery,

enteritis and skin conditions. It has been used in the treatment of syphilis.

The sap is applied to warts in order to destroy them. The treatment needs to be

repeated 2 - 3 times a day over a period of several weeks to be fully effective.

(http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta)

Cultivation details

Euphorbia hirta prefers a light well-drained moderately rich loam in an open

sunny position. It is not very tolerant of frost, though it can probably be grown

successfully in this country as a spring-sown annual. Hybridizes with other members of

this genus. The ripe seed is released explosively from the seed capsules. Members of

this genus are rarely if ever troubled by browsing deer or rabbits. This genus has been

singled out as a potential source of latex (for making rubber) for the temperate zone,

although no individual species has been singled out. (http://www.ibiblio.org/pfaf/cgi-

bin/arr_html?Euphorbia+hirta)

Propagation

Euphorbia hirta is propagated in seed - sow mid to late spring in situ.

Germination usually takes place within 2 - 3 weeks at 20°c. It might be best to sow the

seed in a cool greenhouse in early March. When they are large enough to handle, prick

the seedlings out into individual pots and plant out the seedlings in late May. This will

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give the plants longer to grow and mature. (http://www.ibiblio.org/pfaf/cgi-

bin/arr_html?Euphorbia+hirta)

Biologic Activity

Minimum Inhibition Concentration.

In microbiology, is the lowest concentration of an antimicrobial that will inhibit the

visible growth of a microorganism after overnight incubation. Minimum inhibitory

concentrations are important in diagnostic laboratories to confirm resistance of

microorganisms to an antimicrobial agent and also to monitor the activity of new

antimicrobial agents.

MICs can be determined by agar or broth dilution methods. Commercial methods

are the E-test or Oxoid MICE valuator method using strips of a gradient of antibiotic

concentration. E-test strips create ellipses of microbial inhibition. The point at which the

MIC is taken within the ellipse of inhibition is the point where the bacterial growth

crosses the strip. Clinically, the minimum inhibitory concentrations are used not only to

determine the amount of antibiotic that the patient will receive but also the type of

antibiotic used, which in turn lowers the opportunity for microbial resistance to specific

antimicrobial agents. (www.wikipedia.com)

Toxicity.

The degree to which a substance can harm humans or animals. It can be acute,

sub-chronic, or chronic: Acute toxicity involves harmful effects in an organism through a

single or short-term exposure. Sub-chronic toxicity is the ability of a toxic substance to

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cause effects for more than one year but less than the lifetime of the exposed organism.

Chronic toxicity is the ability of a substance or mixture of substances to cause harmful

effects over an extended period, usually upon repeated or continuous exposure,

sometimes lasting for the entire life of exposed organism.

(http://www.medterms.com/script/main/art.asp?articlekey=34093)

Anti-microbial.

Clinical substance produced either by a microorganism or by a synthetic means

that is capable of killing or suppressing growth of microorganisms. Antimicrobial agents

play a central role in the control and management of infectious diseases, some

understanding of their mode of action and the mechanisms microorganisms deploy to

circumvent antimicrobial activity is important, especially because diagnostic laboratories

are expected to design and implement tests that measure a pathogen’s response to

antimicrobial activity. Several key steps must be completed for an antimicrobial agent to

successfully inhibit or kill the infecting microorganism. First, the agent must be in an

active form. This is ensured through the pharmacodynamic design of the drug, which

takes into account the route through which the patient will receive the agent (e.g. orally,

intramuscularly, intravenously). Second, the antibiotic must also be able to achieve

sufficient levels or concentrations at the site of infection so that it has a chance to exert

an antibacterial effect (i.e., be in anatomic approximation with the infecting bacteria).

The ability to achieve adequate levels depends on the pharmakokinetic properties of the

agent. (Bailey & Scott’s Diagnostic Microbiology, Eleventh Edition)

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Staphylococcus

Staphylococcus is group of bacteria that can cause a multitude of diseases as a

result of infection of various tissues of the body. Staphylococci that are associated with

infections in humans are colonizers of various skin and mucosal surfaces. Because the

carrier state is common among the human population, infections are frequently acquired

when the colonizing strain gains entrance to a normally sterile site as a result of trauma

or abrasion to the skin or mucosal surface.

Staphylococci are also transmitted from person to person. Upon transmission,

the organism may become established as part of recipient’s normal flora and later be

introduced to sterile sites by trauma or invasive procedures. Alternatively, a surgeon or

nurse may directly introduce the organism to normally sterile sites, such as during

surgery. Person-to-person spread of staphylococci, particularly those that have

acquired antimicrobial resistance, most notably occurs in hospitals.

Over 30 different types of Staphylococci can infect humans, but most infections

are caused by Staphylococcus aureus. Staphylococci can be found normally in the nose

and on the skin (and less commonly in other locations) of 20%-30% of healthy adults. In

the majority of cases, the bacteria do not cause disease. However, damage to the skin

or other injury may allow the bacteria to overcome the natural protective mechanisms of

the body, leading to infection. (Bailey & Scott’s Diagnostic Microbiology, Eleventh

Edition).

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Pathogenesis of S. aureus Infections

Staphylococcus aureus causes a variety of suppurative (pus-forming) infections

and toxinoses in humans. It causes superficial skin lesions such as boils, styes and

furunculosis; more serious infections such as pneumonia, mastitis, phlebitis, meningitis,

and urinary tract infections; and deep-seated infections, such as osteomyelitis and

endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of

surgical wounds and infections associated with indwelling medical devices. S. aureus

causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by

release of superantigens into the blood stream.

(http://www.medicinenet.com/staph_infection/index.htm).

Antibiotic Resistance

Methicillin-resistant staphylococcus aureus (MRSA), is a type of Staphylococcus

aureus that is resistant to the antibiotic methicillin and other drugs in the same class,

including penicillin, amoxicillin, and oxacillin. . Just as S. aureus can be carried on the

skin or in the nose without causing any disease, MRSA can be carried in this way also.

Stretomycin is an antibiotic drug, the first of a class of drugs called

aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It

is derived from the actinobacterium Streptomyces griseus. Streptomycin stops bacterial

growth by damaging cell membranes and inhibiting protein synthesis.

(http://www.medicinenet.com/staph_infection/index.htm).

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Laboratory Mice

Most of the mice used in laboratories are white albino house mice (Mus

musculus). The house mouse, a member of the rodent family, originated in ancient Asia

and later spread throughout Europe. In the 1500s, they traveled to North and South

America on English, French and Spanish ships. Today house mice appear both in the

wild and commensally (nesting within human dwellings) in nearly all parts of the United

States.

The mouse has been used in biomedical research since the early 20th century.

Today, more than 3,000 genetically defined strains of lab mice are used for research

purposes. The primary supply source of laboratory mice is commercial breeders.

Relatively few are captured from the wild or bred in research laboratories, although the

latter practice is becoming more common with some genetically engineered mice.

Several characteristics have led to the increased use of mice in research.

Scientists say that mice are genetically similar to humans (at least 80 percent of DNA in

mice is identical to that of humans). They are also used because of their small size,

short lifespan and reproductive cycle, low maintenance in captivity, and mild manner.

For these reasons, house mice constitute the majority of mammals used in research,

testing, and education. More than ten million mice are used each year in U.S.

laboratories alone, in tests of new procedures and drugs as well as in research involved

in the production of biological products such as vaccines.

(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

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Mouse Biology

There is no question that mice feel pain. In fact, mice are used in pain research

studies to help determine the role genes play in pain sensitivity. Pain may not be readily

noticeable in mice, as they instinctively attempt to hide illness and injury because

predators in the wild target weak or sick animals. However, mice in significant pain or

distress, or that are seriously sick, typically display a lack of activity, sunken eyes,

ruffled fur, and an arched back or huddled position.

Mouse Behavior

The natural habitat of mice is forests and grasslands, although nests may be

found in nearly any undisturbed place with a nearby food source. In the wild, mice live in

single, male-dominated groups called colonies. A typical colony consists of a dominant

male, several females, and their young. Occasionally, males may share territories.

Females are less aggressive than males but may establish their own hierarchy within a

territory.

Mice kept in a confined environment, such as a lab setting, are usually more

territorial than wild mice. There is rarely fighting among female mice groups but grouped

male mice can inflict serious injuries on each other. To show their dominance over other

mice in their territory, males may exhibit a behavior termed "barbering" in which they

chew distinct areas of fur, usually around the muzzle or whiskers, from subordinate

mice, leaving a bald patch.

(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

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Of Mice and Humans

Despite their bad reputation, not much about the average mouse's appearance

could be considered imposing or threatening. Adult mice generally weigh less than one

ounce and range in length from two-and-a-half to three-and-a-half inches, minus the tail.

A mouse's tail is usually only slightly shorter than the animal's body.

While the mild-mannered mouse is ill-suited for confrontation and nearly always

prefers flight to fighting, a single mouse can incite a near panic among people simply by

scurrying across a room. Their lightning speed most likely puts people off guard.

Albino house mice, the same kind used in laboratories, are the most commonly

kept pet mice. "Fancy mice," originally bred for show purposes, are also kept as pets.

Fancy mice are larger than other mice, have longer tails and larger ears, and occur with

a wide variety of colors, markings, and coats.

(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

Dangerous Medicine

Toxicity testing performed on rat’s exposes them to extremely painful

experiments such as single dose acute toxicity tests, which are used to determine the

lethal dose of a chemical. Besides the animal cruelty inherent in such tests, drugs found

safe through toxicity testing in rats in fact have caused severe, permanent side effects

and even death when taken by humans. (Daniel D Price, MD, 1998).

Handling Techniques

In handling laboratory mice, there are several things that must be kept in mind.

You must be firm but gentle and always handle the animal in the same way. A mouse is

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best handled by picking it up by the base of the tail, then gently grasping a pinch of lose

skin over the shoulder area between the thumb and forefinger (Figure 1).

Figure 1

The mouse is restrained in such a way can be easily manipulated. If the mice are

excitable and moving around in the cage so that grasping the base of the tail is difficult,

cup the hand over the top of the mouse and then grasp the tail gently at the base with a

thumb and forefinger. Care must be taken not to attempt to grab the tail other than close

to the base because this may result in slippage of the skin and subcutaneous tissue

from the bone leading to necrosis, infection, and sloughing of the tail area where the

skin has been pulled off. (http://www.fau.edu/research/ovs/VetData/mouse.php)

Husbandry

Mice should be kept in rooms with the temperature set at about 70F and humidity

at 50%. Lights should not be too bright since most white mice are albinos and too much

light hurts their eyes. They are diurnal which means they need about 12 hours of light

and 12 hours of darkness each day. Their bedding should not be wood shaving since

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some wood emits toxic fumes to mice. They should have fresh mouse or rat food and

water available at all times. Their bedding should be changed 2 or 3 times a week to

prevent the buildup of urea. (http://www.fau.edu/research/ovs/VetData/mouse.php)

Table 1. Physiological and General Data of Albino Mouse

COMMON NAME MOUSE

ORDER RODENTIA

FAMILY MURIDAE

GENUS MUS

SPECIES MUSCULUS

BLOOD PLATELETS 103/mm3 160-410

FEED DAILY gm/kg 15g/100g

WATER 15ml/100g

ROOM TEMPERATURE C 25

WEIGHT ADULT MALE gm 20-40

WEIGHT ADULT FEMALE gm 25-40

LIFE SPAN YEARS 1.5-3

ROOM TEMPERATURE °C 25

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Thrombocytopenia

A common clinical disorder and results from one or any combination of

mechanisms, including production, and splenic sequestration. Thrombocytopenia

diminishes the effectiveness of hemostasis, and bleeding may result. The severity of

bleeding is related to the degree of thrombocytopeniam. (Clinical Hematology,

Principles, Procedures, Correlations)

The causes of thrombocytopenia are often divided into three major causes of low

platelets: low production of platelets in the bone marrow, increased breakdown of

platelets in the bloodstream (intravascular), and increased breakdown of platelets in the

spleen or liver (extravascular).

The disorders that involve the breakdown of platelets include: immune

thrombocytopenic purpura (ITP), drug-induced immune thrombocytopenia, drug-induced

nonimmune thrombocytopenia, thrombotic thrombocytopenic purpura, disseminated

intravascular coagulation (DIC), hypersplenism.

Symptoms of thrombocytopenia include bruising, nosebleeds or bleeding in the

mouth and rashes. Other symptoms may be present as well, depending on the cause of

the condition. Mild thrombocytopenia can occur without symptoms.

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

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Dengue Fever

Overview

Dengue fever is an infectious disease carried by mosquitoes and caused by any

of four related dengue viruses. This disease used to be called "break-bone" fever

because it sometimes causes severe joint and muscle pain that feels like bones are

breaking, hence the name. Health experts have known about dengue fever for more

than 200 years.

Cause

Dengue fever can be caused by any one of four types of dengue virus: DEN-1,

DEN-2, DEN-3, and DEN-4. You can be infected by at least two if not all four types at

different times during your lifetime, but only once by the same type.

Transmission

You can get dengue virus infections from the bite of an infected Aedes mosquito.

Mosquitoes become infected when they bite infected humans, and later transmit

infection to other people they bite. Two main species of mosquito, Aedes aegypti and

Aedes albopictus, have been responsible for all cases of dengue transmitted in this

country. Dengue is not contagious from person to person.

Symptoms

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Symptoms of typical uncomplicated (classic) dengue usually start with fever

within 4 to 7 days after you have been bitten by an infected mosquito and include high

fever, up to 105ºF, severe headache, retro-orbital (behind the eye) pain, severe joint

and muscle pain, nausea and vomiting, and rashes.

Diagnosis

Your health care provider can diagnose dengue fever by doing two blood tests, 2

to 3 weeks apart. The tests can show whether a sample of your blood contains

antibodies to the virus. In epidemics, a health care provider often can diagnose dengue

by typical signs and symptom.

Treatment

There is no specific treatment for classic dengue fever, and most people recover

within 2 weeks. To help with recovery, health care experts recommend getting plenty of

bed rest, drinking lots of fluids, and taking medicine to reduce fever.

Prevention

The best way to prevent dengue virus infection is to take special precautions to

avoid being bitten by mosquitoes. Several dengue vaccines are being developed, but

none is likely to be licensed by the Food and Drug Administration in the next few years.

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Since, Aedes mosquitoes usually bite during the day, be sure to take precautions,

especially during early morning hours before daybreak and in the late afternoon before

dark.

Complications

Most people who develop dengue fever recover completely within 2 weeks.

Some, especially adults, may be tired and/or depressed for several weeks to months

after being infected with the virus. The more clinically severe dengue hemorrhagic fever

and dengue shock syndromes can result in vascular (blood vessel) and liver damage,

and can be life threatening. (www.eMedicine.com)

Aspirin, or acetylsalicylic acid (ASA)

A salicylate drug often used as an analgesic (to relieve minor aches and pains),

antipyretic (to reduce fever), and as an anti-inflammatory. It also has an antiplatelet

("anti-clotting") effect and is used in long-term, low doses to prevent heart attacks and

blood clot formation in people at high risk for developing blood clots.

Low dose of aspirin (160 mg) may also be given immediately after an acute heart

attack; these doses may inhibit the synthesis of prothrombin and therefore produce a

second and different anticoagulant effect, although this is not well understood.

Aspirin was the first-discovered member of the class of drugs known as non-

steroidal anti-inflammatory drugs (NSAIDs), not all of which are salicylates, although

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they all have similar effects and most have some mechanism of action which involves

non-selective inhibition of the enzyme cyclooxygenase. (www.wikipedia.com)

Veterinary uses. Aspirin has been used to treat pain and arthritis in veterinary

medicine, primarily in cats and dogs, although it is not particularly recommended, as

there are better medications available. Also, dogs are particularly susceptible to the

gastrointestinal side effects associated with salicylates. Horses have also been given

aspirin for pain relief, although it is not commonly used due to its relatively short-lived

analgesic effects. Horses are also fairly sensitive to the gastrointestinal side effects.

Nevertheless, it has shown promise in its use as an anticoagulant, mostly in cases of

laminitis. Aspirin’s use in animals should only be done under the direct supervision of a

veterinarian. (www.wikipedia.com)

Resistance. For some people, aspirin does not have as strong an effect on platelets as

for others, an effect known as aspirin "resistance" or insensitivity. One study has

suggested that women are more likely to be resistant than men and a different,

aggregate study of 2,930 patients found 28% to be resistant. These results may be

contested, however, as there is currently no accepted method of determining who is and

who isn't resistant. (www.wikipedia.com)

Toxicity. The toxic dose of aspirin is generally considered greater than 150 mg per kg

of body mass. Moderate toxicity occurs at doses up to 300 mg/kg, severe toxicity occurs

between 300 to 500 mg/kg, and a potentially lethal dose is greater than 500 mg/kg. [60]

This is the equivalent of many dozens of the common 325 mg tablets, depending on

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body weight. However children cannot tolerate as much aspirin per unit body weight as

adults can. (www.wikipedia.com)

Table 2. Dosage of Aspirin

120-300 MG/KG PO OR
NONSTEROIDAL ANTI-
ACETHYLSALICYLICATE 100-150 MG/KG PO q4h
INFLAMMATORY,
ACID (ASPIRIN) (14) // 25 IP/20 SC/120 PO
ANALGESIC
MG/KG Q4H(2)

Chapter 3

MATERIAL AND METHODS

This chapter presents the research design, procedure, and materials that were

used in the study.

Research Design

The experimental design of the study was carried out under strict laboratory

conditions.

Equipment, facilities, and glasswares of the Biochemical Laboratory of the

University of Negros Occidental- Recoletos were utilized in the research with proper

permission.

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The University of Negros Occidental- Recoletos Biochemical Laboratory has

been put up to facilitate Unorians in the different laboratory course and to cater free

laboratory services.

Sample Preparation and Extraction Procedure

Fresh roots of Euhorbia hirta (tawa-tawa) were collected within the campus of the

University of Negros Occidental- Recoletos. It was identified by Mr. Hermie Cordero, a

B.S. Agriculture major in Agronomy. The fresh roots were air-dried and cut finely into

pieces. One hundred grams (100g) of the finely cut roots were weighed into 1000ml of

distilled water in a beaker. This was boiled using Bunsen burner, and then allowed to

evaporate and reduced half of the volume of the distilled water (500ml), stirred from

time to time. The 500ml solution, which is the concentrated one, is cooled to 40˚C and

filtered using Whatman filter paper into 500ml volumetric flask. The filtrate was sterilized

using autoclave. Figure1 illustrates the schematic presentation of the sample

preparation and extraction procedure.

Inoculum Preparation Using Luria Broth and Luria Agar Slant

Broth and agar cultures of the test organism (Staphylococcus aureus) were

prepared 24 hours before the procedure was commenced. The microorganism is from

University of the Philippines, Los Baños.

Luria Agar Slant Culture

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Direct laboratory method was used to grow bacteria on Luria Agar Slant.

Peptone, yeast, NaCl, and agar are all the ingredients that were weighed and computed

by ratio and proportion to make a Luria Agar Slant. Table 1 shows the components of

Luria agar. In 1000 ml distilled water, all ingredients were mixed and diluted in

appropriate amount of distilled water in an Erlenmeyer flask with cotton plug, and then

placed the media into the water bath for heating until clear. The media is then sterilized

at 115 psi for 15 minutes, and allowed to cool to 40˚C before dispensing it to sterile test

tubes with cotton plugs and allowed to satisfy in a slant manner. The agar slant was

cultivated by using Staphylococcus aureus and was streaked into the agar slant

aseptically. The Luria agar with the culture was incubated for 18-24 hours at room

temperature and good for 72 hours consumption. Figure 2 illustrates schematic diagram

of the Luria agar inoculum preparation.

Luria Broth Culture

Direct laboratory method was used to grow Staphylococcus aureus on

Luria broth to used as working culture, same ingredients were used with the same ratio

and proportion as to the composition of Luria agar except for the agar. All ingredients

were weighed and computed as well, mixed and diluted in distilled water in an

Erlenmeyer flask with cotton plug. The solution was heated in a water bath until clear,

sterilized at 115 psi for 15minutes, allowed to cool to 40˚C a loopfull of inoculum from

the Luria agar slant was taken and was aseptically transferred to the Luria broth. The

Luria broth with the culture was incubated at room temperature for 18-24 hours good for

24 hours consumption.

25
Experimental methodology

The materials and methods for this study are divided into three experiments. The

first experiment is the establishment of MIC, which investigates the inhibition of

Staphylococcus aureus on the different concentrations of the crude extract of tawa-

tawa, while the second experiment is an investigation of the anti-bacterial property of

the tawa-tawa, against Staphylococcus aureus. The last experiment investigates the

effect of tawa-tawa to the platelet count of mice using albino mice as the model specie.

Euphorbia hirta
(Tawa-Tawa)
Roots 100g

Air dry, cut into fine pieces

Weigh into 1000ml-distilled


water in a beaker

26
Boil for 25-30 min until reduced
to 500ml distilled water

Cool for 40˚C, filter in


volumetric flask

Sterilized at 115 psi for 15 min

Store at ambient
temperature

27
Figure1: Schematic diagram showing the sample preparation and extraction procedure.

Luria agar Mixture of all ingredients computed Luria broth


By ratio and proportion

peptone and water

yeast, NaCl

Heat in water bath

Sterilize for 15min at


115ppm

Dispense in
test tubes. Get a loopful
Allowed to of culture
solidify.

28

Culture the Transfer to Luria


29
Figure2: Illustrates schematic diagram of the Luria agar inoculum preparation.
Minimum Inhibition Concentration

The first experiment of the research investigates the minimum inhibition

concentration that will give or show no observable growth after 24 hours of incubation.

Serial dilutions technique was used using 11 tubes assay. Luria broth is a

general media that does not cause microorganisms to grow in flocks or flogs and is

used as a diluting agent together with the crude extract of tawa-tawa to get the desired

concentrations. The tawa-tawa extract was dispensed in 11 test tubes aseptically from

0% to 100% concentrations. Table 1:shows the concentration of serial dilutions of

Tawa-Tawa root extract. Then from the working culture, dispensed 0.1 ml into the 11

test tubes. The tubes were incubated for 24 hours in 37˚ C or room temperature, and

then results were recorded. The experiment was done in three trials with triplicates in

every trial. Figure 3: Schematic diagram showing the methodology in conducting MIC.

Table 1

Concentration of Serial Dilutions of Tawa-Tawa Root Extract

30
Tubes Concentration Tawa-tawa Luria Broth Staphylococci

Extract Culture

1 0% 0 ml 10 ml 0.1 ml

2 10% 1 ml 9 ml 0.1 ml

3 20% 2 ml 8 ml 0.1 ml

4 30% 3 ml 7 ml 0.1 ml

5 40% 4 ml 6 ml 0.1 ml

6 50% 5 ml 5 ml 0.1 ml

7 60% 6 ml 4 ml 0.1 ml

8 70% 7 ml 3 ml 0.1 ml

9 80% 8 ml 2 ml 0.1 ml

10 90% 9 ml 1 ml 0.1 ml

11 100% 10 ml 0 ml 0.1 ml

Add aseptically Add aseptically Add aseptically

Extract
Luria of tawa- Working
Broth tawa (2) culture
(1) (3)

Dispense Dispense Dispense 0.1ml

31
32
Antibacteriocity

Staphylococcus aureus were reisolated and the pure cultures subcultured on

Luria agar and Luria broth. They were stored and incubated at 37˚ C or room

temperature for 24 hours.

The agar diffusion method was adopted for the study. Base and Top Agars

composition were computed by ratio and proportion. All ingredients for Mueller Hinton

Agar Media include beef extract, cassein, starch and agar powder and were diluted in

distilled water. Top agar composition includes peptone, agar, and Sodium Chloride

(NaCl) diluted in distilled water. Table 2: Shows the composition of Top and Base Agars

with their ratio and proportion.

Table 2

Components of Top and Base Agars in 1000ml Distilled Water

33
Composition Base agar Composition Top agar

Agar 6g Agar 70g

Starch 53g Peptone 30g

Beef Extract 4.5g NaCl 50g

Cassein 51g

All ingredients of base and top agars were computed by ratio and proportion,

sterilize at 115ppm for 15 minutes, allowed to cool to 40˚C. For base agar was

dispensed on a petridish aseptically and then allowed to solidify. Top agar was added

with 1.0 ml of 1x108 CFU/ml of bacteria, swirled gently to ensure uniform distribution of

the microorganism. Dispensed top agar on the petri dish with base agar, overlay and

solidify, within 2 hours, 4 cu cylinders were placed in the plates (about 5.0 mm dm)

using a sterile force and an equal volume of 1ml of the extract for the 2 test controls,

streptomycin ampule for the positive control and sterile distilled water for the negative

control were transferred into the holes using micropipettor. These plates were allowed

to stand for 1 hour for prediffusion of the extract to occur and were incubated at 37°C for

24 hour. Figure 4: Shows schematic diagram of Antibacteriocity and Sensitivity.

Three trials were done and three petri dish are made for every trial. At the end of

incubation, zones of inhibition that developed were measured and the average of zones

of inhibition was calculated.

Base Agar Top Agar


All ingredients mixed & All ingredients mixed &
computed by ratio and computed by ratio &
proportion proportion.

34
Sterilize at 115 psi for 15
minutes
Cool to 40° C

Cool at 40° C

Dispense to petridish aseptically

Add bacterial working culture


1ml of 1 x 108 CFU/ml

Solidify

Overlay on base agar & solidify

Put 4 glass cylinder within 2


hours

35
Dispense 100 цl of (+) and (-)
controls in each cylinder & the
other 2 for the aseptically test
substance

Incubate upright for 24-48


hours

Figure 4: Schematic diagram of Antibacteriocity and Sensitivity.

Toxicity

Initial LC50 studies carried out were used to determine the maximum

concentration that did not produce an increase in the platelet count in albino mice. Four

groups of albino mice each comprising 10mice, randomly selected. They were placed in

36
different cages. Based on the LC50, studies, concentrations of the crude extract of

tawa-tawa are 100%, 80%, 60%, 0% were orally administered into each group of the

mice respectively, the administration of 1ml of tawa-tawa extract was carried out on

daily basis for 2 days. The control group was orally administered with distilled water.

Food and water were provided. The albino mice were first drawn blood samples through

tail vein checked for their normal platelet count on the 2 nd day, they were induced with

thrombocytopenic drugs such as aspirin for a day and platelet count were checked. On

the third day, tawa-tawa extract were administered to them and blood samples were

analyzed for any effect on their platelet count.

37

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