Professional Documents
Culture Documents
INTRODUCTION
Background
family of dicotyledons, with about 300 genera and over 5,000 species. Here in
in the United States. The plants of 3 different species share Phoretic variations,
these plants are: (1) Mutha (Cyperus rotundus), (2) Gatas-gatas (Euphorbia
Philippines. A simple weed scattered in sunny lawns, waste places and open
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
long, toothed at the edge, and blotched with purple in the middle. In the axils
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
1
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
Objectives
2
3. Does the crude extract concoction of Tawa-Tawa roots possess any
Fever. Pathologist is still searching and discovering for treatment for the disease.
study would help patients suffering from dengue fever lessen the burden of
dissemination. Through the Health workers, information about the disease could
recent decades, and thus increased number of cases affects the community
Dengue fever. This would also serve as a guide to study other possible toxic, and
3
anti-microbial properties of Tawa- Tawa. This work therefore, was undertaken to
Milieu
This study was conducted from November 2005 to March 2008 in the City
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
4
Chapter 2
This chapter presents related concepts on the risk factors and daily
environmental factors and prevention and control are presented to provide basis
effects of Tawa-Tawa were cited in the study on MIC, Agar Diffusion, and Animal
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
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
5
effects. However, the last few years have seen a major increase in their use in the
Nowadays multiple drug resistance has developed due to the indiscriminate use
disease. In addition to this problem, antibiotics are sometimes associated with adverse
reactions. This situation forced scientists to search for new antimicrobial substances.
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
6
Euphorbia hirta or (Tawa- Tawa)
Description
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
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
intestinal amoebic dysentery. It should not be used without expert guidance, however,
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
7
used in combination with other anti-asthma herbs such as Grindelia camporum and
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
(http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta)
Cultivation details
sunny position. It is not very tolerant of frost, though it can probably be grown
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,
bin/arr_html?Euphorbia+hirta)
Propagation
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
8
give the plants longer to grow and mature. (http://www.ibiblio.org/pfaf/cgi-
bin/arr_html?Euphorbia+hirta)
Biologic Activity
antimicrobial agents.
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
Toxicity.
The degree to which a substance can harm humans or animals. It can be acute,
9
cause effects for more than one year but less than the lifetime of the exposed organism.
(http://www.medterms.com/script/main/art.asp?articlekey=34093)
Anti-microbial.
play a central role in the control and management of infectious diseases, some
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,
sufficient levels or concentrations at the site of infection so that it has a chance to exert
The ability to achieve adequate levels depends on the pharmakokinetic properties of the
10
Staphylococcus
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
the organism may become established as part of recipient’s normal flora and later be
nurse may directly introduce the organism to normally sterile sites, such as during
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).
11
Pathogenesis of S. aureus Infections
and toxinoses in humans. It causes superficial skin lesions such as boils, styes and
and urinary tract infections; and deep-seated infections, such as osteomyelitis and
surgical wounds and infections associated with indwelling medical devices. S. aureus
causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by
(http://www.medicinenet.com/staph_infection/index.htm).
Antibiotic Resistance
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.
aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It
(http://www.medicinenet.com/staph_infection/index.htm).
12
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
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.
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
(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).
13
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,
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
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
(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).
14
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
(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).
Dangerous Medicine
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
15
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
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
16
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
ORDER RODENTIA
FAMILY MURIDAE
GENUS MUS
SPECIES MUSCULUS
WATER 15ml/100g
ROOM TEMPERATURE C 25
ROOM TEMPERATURE °C 25
17
Thrombocytopenia
diminishes the effectiveness of hemostasis, and bleeding may result. The severity of
The causes of thrombocytopenia are often divided into three major causes of low
mouth and rashes. Other symptoms may be present as well, depending on the cause of
(http://en.wikipedia.org/wiki/Thrombocytopenia)
18
Dengue Fever
Overview
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
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
Symptoms
19
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
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
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.
20
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,
A salicylate drug often used as an analgesic (to relieve minor aches and pains),
("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
21
they all have similar effects and most have some mechanism of action which involves
Veterinary uses. Aspirin has been used to treat pain and arthritis in veterinary
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.
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
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
22
body weight. However children cannot tolerate as much aspirin per unit body weight as
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
This chapter presents the research design, procedure, and materials that were
Research Design
The experimental design of the study was carried out under strict laboratory
conditions.
University of Negros Occidental- Recoletos were utilized in the research with proper
permission.
23
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.
Fresh roots of Euhorbia hirta (tawa-tawa) were collected within the campus of the
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
Broth and agar cultures of the test organism (Staphylococcus aureus) were
prepared 24 hours before the procedure was commenced. The microorganism is from
24
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
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
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
26
Boil for 25-30 min until reduced
to 500ml distilled water
Store at ambient
temperature
27
Figure1: Schematic diagram showing the sample preparation and extraction procedure.
yeast, NaCl
Dispense in
test tubes. Get a loopful
Allowed to of culture
solidify.
28
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
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
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
Extract
Luria of tawa- Working
Broth tawa (2) culture
(1) (3)
31
32
Antibacteriocity
Luria agar and Luria broth. They were stored and incubated at 37˚ C or room
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
Table 2
33
Composition Base agar Composition Top agar
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
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
34
Sterilize at 115 psi for 15
minutes
Cool to 40° C
Cool at 40° C
Solidify
35
Dispense 100 цl of (+) and (-)
controls in each cylinder & the
other 2 for the aseptically test
substance
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
37