University of Santo Tomas

Faculty of Medicine and Surgery

Department of Clinical Epidemiology

The Effects of Aqueous Extracts of Euphorbia

hirta in Increasing Platelet Count of Mus
musculus with Induced Thrombocytopenia

GROUP 66
Noel, Emmanuel Benedict S.
Ocampo, Innah Marie F.
Olalia, Michael Benjamin R.
Olasiman, Stephanie Rae P.
Olfato, Alex Guido III B.
Introduction
 Dengue
• One of the major concerns of the Filipinos
• most important mosquito-borne viral disease
• August 28, 2010: 69,594 cases (DOH)
• Death Toll: 501 in 2010
• The most severe form: Dengue haemorrhagic fever (DHF)
 World Health Organization Criteria for DHF
diagnosis:

• High continuous fever for 2 to 7

days
• Most frequent manifestations
presenting as skin petechiae
• Thrombocytopenia (<100 × 109/L)
• Increased capillary permeability
 Platelets
• natural hemostatic mechanism
of the body
• Platelet alterations in dengue:
– Thrombocytopenia
 peripheral destruction
and platelet production
(hemopoietic
suppression)

– Platelet Dysfunction
 platelet factor 4 and β-
thromboglobulin
• Early diagnosis and prompt management reduce risk of
progression

• Virus is self – limiting

• Management: non-aspirin analgesics and intravenous

fluid replacement
 Euphorbia hirta
• tawa-tawa

• Aqueous extracts of the leaves of the

plant

• Helps platelet counts of pediatric

patients with thrombocytopenia =
risk of hemorrhagic shock

• Still not approved by the Department of

Health as an alternative medicine for
Dengue
Hypotheses of the Study

Null Hypothesis (H0)

There is no significant difference between the means of
the platelet counts of Mus musculus after administration
of different concentrations of Euphorbia hirta

Alternative Hypothesis (H1)

There is a significant difference between the means of
the platelet counts of Mus musculus after administration
of different concentrations of Euphorbia hirta
Objectives
General Objective

To determine the effects of aqueous extracts of

Euphorbia hirta on the platelet count of Mus musculus
Specific Objectives

1. To determine the effects of lowering the platelet count of Mus musculus with
heparin without administration of Euphorbia hirta orally for two weeks

2. To determine if administration of Euphorbia hirta per orem for two weeks can
increase platelet count of Mus musculus after lowering the platelet count with
heparin

3. To determine if there is a significant difference in the means of the platelet count

of Mus musculus after administration of three different concentrations(100mg/ml,
150mg/ml, 200mg/ml) of Euphorbia hirta for two weeks

4. Document observable behavioral changes in mice after administration of aqueous

extracts of Euphorbia hirta
Conceptual Framework
REVIEW OF RELATED
LITERATURE
 Morphology and Habitat of E. hirta

• Family Euphorbiaceae
• small annual herb common
to tropical countries
• erect, slender-stemmed;
spreading up to 80 cm tall
• annual broad-leaved herb
• hairy stem with many
branches (base to the top)
Leaves
• opposite and elliptical
• oblong or oblong- lancelate, w/ a faintly toothed margin
•darker on the upper surface
Flowers
• small and numerous
•crowded together in dense cymes
•about 1 cm in diameter
• Stem and leaves produce a white or milky juice when cut
• Frequently seen occupying open waste spaces, banks of
watercourses, grasslands, road sides, and pathways
Pharmacological Studies
• Traditional medicinal herb in all the tropical countries of
Africa, Asia, America and Australia (Khare, 2007)
• Other names:
– Euphorbia pilulifera a member of the family of
Euphorbiaceae
– Australian asthma weed, Pill-bearing spurge or
Queensland asthma weed
• Said to treat cough, hay asthma, bowel complaints, worm
infestation, kidney stones, and others

• Traditional medicine practitioners consume aqueous

extracts of various parts (Hore et al., 2006)
• Aqueous extracts show analgesic, anti-pyretic, anxiolytic,
sedative, anti-inflammatory activities and inhibits platelet
aggregation (Khare, 2007)

• Quercetin (Tona et al., 1999; Tona et al., 2004)

– one of the main phenolic constituents
– an antioxidant, anti-inflammatory, anti-bacterial, anti-
aggregatory, and anti-carcinogenic
India (Patil et al. 2009)
• Treat worm infestations in children
• Dysentery, gonorrhoea, jaundice, pimples, digestive
problems and tumors
• Fresh Milky Latex on wounds & warts
• Root of the Plant on sprains, inflammation, etc.
• Shows selective cytotoxicity against several cancer cell
lines
• Effective treatment of cancers (malignant melanomas
and squamous cell carcinomas)
• Anti-platelet aggregation property
• Reduced the release of prostaglandins I2, E2, and D2.
• Inhibitory effect on platelet aggregation and depressed
the formation of carrageenin induced rat paw edema
• Other properties include:
– immunomodulatory, antifungal, antihelminthic, and
antioxidant, molluscicidal, larvicidal activities of the
aqueous stem bark and leaf extracts of the plant
Omeje et al. (2008)
• Nigeria
• Aqueous and methanolic extracts were administered to
albino mice
• Platelet count, bleeding, and clotting times were
measured
• Aqueous extracts showed a greater significant difference
than the methanol extracts.
Mortega and Parrocha (2008)
• Philippines
• Aimed to confirm the positive effect of E. hirta on dengue
by means of platelet counts of mice
• Induced thrombocytopenia by administering Emthexate®
• Aqueous extracts (50% and 100%) of E. hirta were then given
• 50% extracts had a significant difference
Sudhakar et al. (2006)

• Exhibited a broad spectrum of antimicrobial activity

against Escherichia coli, Proteus vulgaris, Pseudomonas
aeruginosa and Staphylococcus aureus.
Youssouf et al. (2007)

• Antianaphylactic effect of E. hirta was demonstrated

• Ethanolic extract was observed on the release of TNF-
alpha and IL6 from anti-DNP-HSA activated rat
peritoneal mast cells.
More properties of the medicinal plants
validated
– Antiamoebic (Tona et al., 2004) &
antispasmodic activities (Tona et al., 2000)
MATERIALS AND METHODS
Research Design
• Randomized Control Trial
• Mus musculus will be used as subjects
• Heparin will be used to lower platelet count
• Control group: given water for two weeks
• Experimental Groups: 100mg/ml, 150mg/ml, and 200mg/ml
of E. hirta extracts
• Determination of platelet counts
 Before induction of decreased platelet count. This is
to determine the baseline data for comparison and
analysis.
 After administration of heparin
 After one, three, seven, ten, and fourteen days of
taking the aqueous extracts of E. hirta and water
respectively.
R
Sample Population

• White male mice of the species Mus musculus

• Sample size will be computed after pilot study
• Tentatively eighty (80) male mice 90-100g each
• four groups consisting of 20 mice per group
Experimental Intervention

a.) Collection and Authentication of E. hirta

• collected from various areas in Metro Manila
• identified and verified by a taxonomist from the UST
Research Center for Natural Sciences or Philippine
National Museum Herbarium
b.) Preparation of the Extracts
c.) Administration of Heparin

• Heparin tablets will be dissolved

to obtain 2.5 mg/ml solution
• Dosage will be calculated based
on the weight
• Given heparin after one hour
from gathering platelet count
• Use of a gavage
• After 1 to 2 hours, the platelet
count would be checked again
• Platelet count repeated every
hour until there is substantial
decrease
d.) Administration of E. hirta Extracts

• 5 ml of the three concentrations will be given to the three

test groups every two hours
• Water will be given to the control group
e.) Platelet Count
• Use of microhematocrit tubes or capillary
tubes from the tail***
• Normal platelet counts in mice: 900-1600 x
106/ml
• Platelet count will be checked before and
after administration of heparin, and after 1, 3,
7, 10, and 14 days
*** We can also use a new method of mouse bleeding

Use of the vascular bundle located at the rear of the Jaw

Bone w/c provides a convenient and consistent source of
blood (http://www.medipoint.com)
Outcome Measurement
• Outcome to be measured is the platelet number
• Done either manually w/ the use of a hemacytometer or
through automated hematology analyzers
• Discrepancies are due to:
 Some hematology analyzers are only capable of
measuring larger murine platelets
 Mouse platelets often become activated and form
clumps in vitro
 Clumps are counted as eosinophils
• To diminish such discrepancies, mouse peripheral blood
smears should first be evaluated for platelet clump
• Normal platelet counts in mice range between 900-1600
x 106/ml
• Some studies have gotten to as low a range as 207-385
x 106/ml (Mortega & Parrocha, 2008)
Statistical Analysis
• Determined by ANOVA (SPSS 16.0 Computer Software
or Open Epi)
• Compare more than two groups
• Probability value of 0.05 or less
Pilot Study
• Pre-testing on 10 mice per group only
• Factors that are possible to be changed are the
following:
 the amount or concentration of extract to be given to
the mice
 the amount or concentration of the platelet reducing
drug used for inducing the reduction of platelet
 the method for administering the drug and the tawa-
tawa extract
 the time in between induction of platelet reduction and
administration of tawa-tawa extract
 the time in between induction of platelet reduction and
administration of tawa-tawa extract
 the environment wherein the subjects would be stored upon
induction of the platelet reducing agent and the time
administering of the tawa-tawa extract up to increase of
platelet
 the way of obtaining the platelet count of the subjects
prior to and after the experiment
 percentages or the changes of the platelet count in
the induction of reducing it, in the waiting phase
before administration of the tawa-tawa extract, and
after administration of tawa-tawa extract
 other such variables that can be encountered within
the pilot testing phase
Ethical Concerns

• Protocol will be submitted to the University of Santo

Tomas-Institutional Animal Care and Use Committee
(UST-IACUC) for evaluation and approval
Budget
Items # of Units Unit Price Total
mice at 100g each 80 500 40,000
cages (1 cage for 10 mice) 8 300 2,400
water dispensers (2 for each cage) 16 100 1,600
mice food pellets (packs) 10 200 2,000
food containers (2 for each cage) 16 100 1,600
gavages (4 for each group) 16 150 2400
blades (boxes) 5 100 500
Gatorade/Sola bottles (replacement 50 15 750
for conical flask)
Distilled Water (1 gallon) 5 300 1,500
Whatman filter paper (sheets) 10 100 1000
microscope slides (box) 5 50 250
Transportation (500/person) 500 2500
Token/ Payment for taxonomist 1,000 1,000
Payment for medical technologist 2,500 2,500
Bond paper 500 500
Printing 1,000 1,000

TOTAL P59,000.00
Gannt Chart
Thank you!