CHAPTER 1

THE PROBLEM

Tawa-Tawa (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 produce white or milky juice when cut.

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

and respiratory tract inflammations. The plant contains relatively abundant white latex.

The white latex is capable of causing dermatitis. The plant shows antibiotic activity.

1
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.

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 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.

2
 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.

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

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

developed world.

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

3
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.

Tawa-Tawa (Euphorbia hirta)

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.

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.

4
 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 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.

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.

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 give the plants longer to grow and mature.

5
 CHAPTER 3
RESEARCH METHODS

RESEARCH ENVIRONMENT

This study was conducted 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.

6
INSTRUMENTS

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

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

7
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.

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

species.

8
 CHAPTER 4
DATA ANALYSIS

This chapter presents the data collected using the research methodology, and

the analysis and interpretation of data using the prescribed statistical tool for the

experiment design.

The first experiment investigates the highest concentration of the extract that does

not produce significant reversion on the test organism- Staphylococcus aureus. Table

1: Shows the MIC of the Euphorbia hirta extract against Staphylococcus aureus.

Table 1: MIC of the Euphorbia hirta extract against Staphylococcus aureus

Concentration of Tawa-tawa extract g/ml Staphylococcus aureus growth
0% (+)
10% (+)
20% (+)
30% (+)
40% (+)
50% (+)
60% (+)
70% (+)
80% (-)
90% (-)
100% (-)

With 3 trials in every 10 serial dilutions used per trial for this experiment, in

triplicates, the minimum inhibitory concentration was produced against

Staphylococcus aureus with a concentration of 80%.

9
 The antibacterial screening of the extract was established using the cap

cylinder method with the test isolates- Staphylococcus aureus where 90% of the

concentration were used for the test control. The results show that the extract did not

inhibit growth of Staphylococcus aureus at 90% concentration used.

Streptomycin was used for the ( + ) control and showed zone of growth inhibition of 3.59 mm
in circumference as a total mean value of the 3 trials, in triplicates, was exhibited. Table 2:
shows the zones of inhibition for the 3 trials.

Table 2: Zones of inhibition for the 3 trials.

Sensitivity

First trial Streptomycin Distilled water T1 T2

A. 4.0 0 0 0

B. 3.8 0 0 0

C. 3.4 0 0 0

Total mean 3.73 0 0 0

Sensitivity
Second trial Streptomycin Distilled water T1 T2

A. 4.0 0 0 0

B. 3.0 0 0 0

C. 3.5 0 0 0

Total mean 3.50 0 0 0

Sensitivity
Third trial Streptomycin Distilled water T1 T2

A. 3.5 0 0 0

B. 3.7 0 0 0

C. 3.4 0 0 0

Total mean 3.73 0 0 0

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 The results of the platelet count analysis of the blood samples of mice

administered with different concentrations of the extract were shown in Table 3. In

general, although the values obtained for platelet count in determining the effects of

tawa-tawa on thrombocytopenia, differed. They were not significantly different

(α=0.05) from the values obtained from the control for this parameter.

For the values obtained from the mice before treatment of aspirin and extract,

there was no significant difference from those mice treated from control. But there was

significant difference from the value obtained from those mice treated with other

concentrations. This experiment yields the decrease in platelet count after

administering aspirin and the increase in platelet count after administering tawa-tawa

extract at different concentrations plus a control of which distilled water was used.

Figure 1: Platelet Count Curve for Albino Mice Administered to different

Concentrations of Extract, before taking Aspirin and tawa-tawa Extract, After Aspirin

and After Extract.

Table 3
Platelet Count Analysis

Concentration of Before Aspirin After Aspirin After Tawa-tawa

Extract and Tawa-tawa

Tail Blue 57 40 36

Left Ear Blue 51 50 48

Stomach Red 0% 181 35 12

Total mean 96 42 32

11
 Concentration of Before Aspirin After Aspirin After Tawa-tawa
Extract and Tawa-tawa

Right Ear Red 202 23 31

Left Foot Blue 48 21 87

Stomach Red 60% 44 35 50

Total mean 98 26 56

Concentration of Before Aspirin After Aspirin After Tawa-tawa

Extract and Tawa-tawa

Head Red 116 25 69

Right Hand Blue 74 29 33

Neck Blue 80% 50 28 86

Total mean 80 27 63

Concentration of Before Aspirin After Aspirin After Tawa-tawa

Extract and Tawa-tawa

Right Ear Blue 48 21 72

Right Ear Black 85 42 55

Right Foot Red 100% 255 24 30

Total mean 129 29 92

This experiment investigates the toxicity of the extract of different

concentrations to Albino mice as the test organisms. Three concentrations of extract

plus a control were used in order to know the test concentration lethal to the Albino

mice. This experiment yields the survival/ mortality curve for Albino mice, as well as

the test solutions median lethal concentration. Table 4: Shows the result of mortality

exhibited after 24 hours of exposure to the extract.

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 Table 4
Result of mortality exhibited after 24 hours of exposure to the extract

Number of Test Organisms Dead at

Concentration Number of Test
of Extract Organism 8 hour 16 hour 24 hour
0% 10 0 0 0
60% 10 0 1 1
80% 10 0 0 0
100% 10 0 0 1

Establishment of LC50 is very important for this experiment because it is from

this concentration that lethal effects of the extract on Albino mice survival after

prolonged exposure were determined.

With ten mice used per treatment for this experiment, in triplicates, the number

of survivors left after 24 hours of continuous exposure to extract, were counted.

13
 CHAPTER 5
SUMMARY OF FINDINGS

CONCLUSION

Using the statistical analysis of the data, these are the results of the study:

1. Minimum Inhibition Concentration of crude extract concoction of Tawa-

Tawa roots is 80%.

2. Crude extract concoction of Tawa- Tawa roots does not posses any anti-

bacterial effect towards Staphylococcus aureus.

3. Crude extract concoction of Tawa- Tawa roots, haematologically, is less

toxic, atleast to the mice, when administered orally with different

concentration.

4. Results show that there is no significant difference in the crude extract

concoction of Tawa- Tawa roots, thus does not affect the platelet count.

RECOMMENDATIONS

Based on the findings and conclusions of this study, the proponent

recommends the following studies should be made:

1. Further testing of the test substance should be made, this time utilizing

other strains of test organisms such as E. coli, P. aeruginosa, B.

subtilis and S. tyhi.

2. Mutagenicity should be done using Ames Test to determine any

mutagenic effects of the test substance.

14
 3. Conduct a research by using the other parts of Euphorbia hirta to be

extracted and analyzed.

4. Studies should be conducted on other disease that could be treatable

by Euphorbia hirta.

5. Investigate other serum biochemical parameters in mice subjected to

studies on Euphorbia hirta.

REFERENCES

- Forbes, B. A., et al. (2002). Anti microbial Principle. Staphylococcus. ELESEVIER

SCIENCE Publisher, Philippines.
- Lotspeich- Steininger, C. A., et al. (1992). Thrombocytopenia. Educational
Publishing House, Philippines.
- Lind and Tallantire. (1971). Description of E. hirta.
- Kokwaro. (1993). Treatment of asthma.
- Oliver. (1960). Latex cause dermatidis.
- Sofowora. (1993). Antibiotic activity.
- Ahmad I, et.al. (1998) Ethnopharmacol. Medicinal plants are a source of great
economic value.
- Rajkot and Jamnagar . (2004). Herbal medicine is still the mainstay of about 75-
80% of the whole population.
- Davis J, Science. (1995). Alternative antimicrobial drugs.
- Le Bourgeois T., et. al. (2001). Description
- http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta. Medicinal Uses,
Cultivation details, propagation.
- http://www.medterms.com/script/main/art.asp?articlekey=34093. Toxicity.
- www.wikipedia.com. MIC.
- http://www.medicinenet.com/staph_infection/index.htm. Pathogenesis of
S.aureus, and antibiotic resistance.
- http://www.hsus.org/animals_in_research/species_used_in_research/mouse.h
tml-laboratory. Mice.

15
- Daniel D Price, MD. (1998). Dangerous Medicine.
- http://www.fau.edu/research/ovs/VetData/mouse.php. Handling Techniques.
- http://www.fau.edu/research/ovs/VetData/mouse.php. Physiological and
General Data of Albino Mouse, Husbandry.
- Clinical Hematology, Principles, Procedures, Correlations. Thrombocytopenia.
- www.eMedicine.com. Dengue fever.
- Akujobi et al. (2004). Euphorbia hirta inhibits the growth of various
microorganisms.
- Chanda S., Turk J Biol. (2005). Crude extract of Euphorbia hirta was inactive
against the gram-positive bacteria.
- Braude. (1982). No growth exhibited by the extract against Staphylococcus
aureus.
- Singleton. (1999). Staphylococcus aureus produces the enzyme penicillinase.
- Igoli et al. (2005). Euphorbia hirta is commonly used traditionally in the
production of platelet count.

16