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EFFICACY OF TAWA-TAWA (Euphorbia hirta Linn.) LEAF CRUDE

EXTRACT AGAINST Microsporum canis IN-VITRO

Article · April 2013

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วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 161

RESEARCH ARTICLE
EFFICACY OF TAWA-TAWA (Euphorbia hirta Linn.)
LEAF CRUDE EXTRACT AGAINST
Microsporum canis IN-VITRO
Michael Galpa1, Dr. Ma. Asuncion G. Beltran1*

Abstract
Objective - This study was conducted to evaluate the antifungal activity of the tawa-tawa
leaf crude extract against Microsporum canis, in vitro, based on the zone of inhibition
at different concentrations of tawa-tawa leaf crude extract.
Materials and Methods - The different concentrations were 100 percent (T1), 75 percent (T2),
50 percent, 25 percent (T4), ketoconazole (T5) as the positive control and sample free disc as the
negative control.
Results and Conclusion - The result of the in vitro study revealed that the positive control had
the highest zone of inhibition of 41.02 mm (+++) with complete inhibitory activity and high
reactivity (4), and was significantly different from tawa tawa treated groups. However, all four
concentrations of tawa-tawa leaf crude extract showed no different effect on inhibition of
Microsporum canis; all treated groups produced complete inhibitory activity (+++) and severe
reactivity (4). Among four concentrations, 100 percent, 75 percent, 50 percent, and 25 percent,
the 100 percent tawa-tawa leaf crude extract produced the highest zone of inhibition of 26.20 mm
(+++) and 25.15 mm (+++), 23.50 mm (+++), 20.56 mm (+++) respectively but no significant
differences with each other.
The study showed that tawa-tawa leaf extract had highly significant biological influences in
inhibiting Microsporum canis, and this is based on the Universal Standard Procedure (USP)
of the Department of Science and Technology (DOST).

KKU Vet J. 2013;23(2):161-171. http://vmj.kku.ac.th

Keywords: Antifungal, Microsporum canis, Euphorbia hirta
1
Graduate and Faculty, Institute of Veterinary Medicine, Tarlac College of Agriculture, Camiling, Tarlac, Philippines.
*Corresponding author E-mail: marizonbeltran@yahoo.com
162 KKU Vet J Vol. 23 No. 2 July - December 2013

Introduction
Plants are invaluable sources of pharmaceutical products and use of ethno
pharmacological knowledge enhances the probability of success in new drug finding effort [1].
Screening active compounds from plants lead to discover of new medicinal drugs which have
efficient protection and treatment roles against various diseases. Euphorbia hirta Linn.,
(Euphorbiaceae) locally known as “tawa-tawa” and “maragatas”, a wild herbaceous plant is very
common in the Philippines. The plant has been widely acknowledged for the treatment of cough,
coryza, hay asthma, bronchial infections, bowel complaints, worm infestations, and kidney stones
in traditional medicine [2]. In recent publications, tawa-tawa extracts are used to treat dengue
fever by rural folks in the Philippines but no scientific studies have been made.
Ringworm is an infection by a fungus that most often affects the hair, nails and
superficial layers of the skin. The most commonly noted fungal types seen in cats and dogs are
Microsporum canis, Trichophyton metagrophytes and Microsporum gypseum [3]. Ringworm
fungi can be transmitted to humans, therefore owners of infected animals should considered
quarantining the pet indoors until the infection is cured. Precautions should be taken while treating
animals in order to prevent human infection and environmental contamination [4].
Tawa-tawa had been reported in other countries as antifungal agent [5,6]. It was
demonstrated that the plant extract has a strong antifungal activity but limited study has been
conducted in the Philippines yet. Hence, this study was conducted to determine the efficacy of
Tawa-tawa (Euphorbia hirta Linn.) crude extract against M. canis, in vitro.
This study was conducted to determine the antifungal efficacy of tawa-tawa
(Euphorbia hirta, Linn.) leaf extract against M. canis, in vitro. Specifically, the study aims to:
1. Determine the concentration of tawa-tawa leaf extract that inhibits growth of the fungi
M. canis
2. Determine the zone of inhibition at different concentrations of Euphorbia hirta Linn.
crude extract against M. canis, in vitro
Leaves of tawa-tawa (Euphorbia hirta Linn.) were collected in the fields of Tarlac
College of Agriculture, Camiling, Tarlac and processed and tested for efficacy at the Standard
and Testing Division/Industrial Technology Development Institute - Micro Laboratory Section
of the Department of Science and Technology DOST), General Santos Avenue, Bicutan, Taguig,
Metro Manila, from July, 2012 to September, 2012.
วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 163

Materials and Methods

Preparation of Pure Culture of Microsporum canis
Cultured M. canis was purchased at the Microgenetics of the Department of Science and
Technology (DOST), Gen. Santos Avenue, Bicutan, Taguig City, Metro Manila, and were kept in
a refrigerator until use.
Preparation for Autoclaving the Materials
The glassware and the other laboratory materials to be used were washed first with
dishwashing soap and water. They were air dried and individually wrapped with manila
paper before autoclaving, together with the filter paper disc at 121 ํC (250 ํF) at a pressure of
103,421 Pascal (15 PSI) for 15 minutes.
Preparation of Tawa-Tawa Leaf Crude Extract
Fresh leaves of tawa-tawa were collected from the fields of Tarlac College of
Agriculture, Camiling, Tarlac. Only leaves without adhering dirt particles or associated insect or
insect bites were chosen and were placed in a plastic bag, labeled and transported to the Department
of Science and Technology (DOST), Gen. Santos Avenue, Bicutan, Taguig City, Metro Manila,
for plant crude extraction.
A 375 grams tawa-tawa leaves were macerated and soaked in a 2.4 liters of distilled
water for 48 hours with occasional agitation. The extract was filtered and concentrated under
vacuum at 60 ํC in rotary evaporator to a syrupy consistency. This was further dried in
water bath at the same temperature maintained at 60°C.
Maceration of 375 grams of the fresh tawa-tawa leaves with 2.4 liters of distilled
water, concentration of the filtrate in rotary evaporator under vacuum at 60 ํC produced 37.2 grams
crude extract. Percent yield of crude extract was 9.92 percent. The crude extract was
subjected to phytochemical analysis.
Preparation of Treatments
Five (5) empty small petri dishes were used in the study with an appropriate label
of the treatments. Series of dilution were followed to make each concentrations of tawa-tawa
crude extract using a micropipettor. The positive control used was antifungal drug (ketoconazole).
Each treatment was replicated three (3) times using filter paper disc.
The tawa-tawa crude extract treatments were as follows:
T1 = 100% tawa-tawa crude leaf extract
164 KKU Vet J Vol. 23 No. 2 July - December 2013

T2 = 75% tawa-tawa crude leaf extract

T3 = 50% tawa-tawa crude leaf
T4 = 25% tawa-tawa crude leaf extract
T5 = Ketoconazole as the positive control
T6 = Sample free disc as the negative control
Preparation of Filter Paper Discs
Using different sterile forceps that were designated for each prepared treatments,
three filter paper discs of standard sizes (10 mm) were placed and submerged in the following
treatment solution of tawa-tawa crude leaf extract: 100%, 75%, 50%, 25 % concentrations
and into ketoconazole as positive control. Sample free disc served as the negative control.
It was impregnated for about 30 minutes until the filter paper discs fully absorbed the treatment.
Preparation of Potato Dextrose Agar
In preparation of potato dextrose agar, 65 grams of the potato dextrose agar medium was
suspended in one litre of distilled water. It was heated with frequent agitation and boiled for one
minute until the medium completely dissolved. Then the prepared medium was autoclaved into
121°C for 15 minutes. After being autoclaved, the medium was thawed using water bath until the
temperature reached 44°C; the tolerable temperature for the microorganism that will be inoculated
for anti fungal sensitivity.
Disc Agar Diffusion Method
Inoculum of the prepared culture of M. canis was placed in a container bottle with
five grams of glass beads and 20 ml of distilled water. The mixture was manually stirred to have
an even distribution of M. canis. Using micropipettor, 0.6 ml of inoculum was placed into six
large petri dishes. Then after the prepared potato dextrose agar medium has reached 44 ํC, 45 ml
of potato dextrose agar was placed on six large petri dishes with the M. canis inoculum. It
was mixed by agitating the petri dishes in a clockwise motion until uniform distribution of
inoculums was observed. The inoculated plates were set aside and allowed the plate to dry for
five minutes. A small opening to the cover was placed so that excess moisture from the inoculums
was not absorbed by the agar. After the agar solidified, the plates were placed back into the
incubator for 45 minutes before planting the filter paper discs to acclimatize the fungi from
the environment of the plates with a temperature of 32°C.
The six large petri dishes were labeled properly according to what concentration of
วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 165

tawa-tawa crude leaf extract treatment the filter paper discs were placed onto the prepared
media. Using the provided forceps on each treatment, each filter paper disc was gently
pressed onto the surface of the agar to ensure complete contact with the agar surface. The plates
were placed in the incubator at 25 ํC. The readings of results were recorded on the fifth and
eight days after treatment.

Reactivity = Total reaction diameter of disc (mm) - Diameter of the paper zone of inhibition (mm)

Statistical Design
The study was conducted using Completely Randomized Design (CRD). A total of six
plates were used for the six treatments and were replicated three times.
Analysis of data
The Analysis of Variance (ANOVA) of the Completely Randomized Design (CRD) was
used in the interpretation for all the data gathered. The different treatment means were compared
using the Duncan’s Multiple Range Test (DMRT).

Results and Discussion

Concentrations of tawa-tawa leaf extract that inhibit growth of M. canis.
Based on the results of initial measurements of inhibition zone at day 5 shown in
Table 1, the tawa-tawa leaf crude extract: 100%, 75%, 50% and 25% concentration produced
complete inhibitory activity (+++) with severe reactivity (4) against test organism M. canis.
Ketoconazole, which served as positive control, produced complete inhibitory activity (+++)
with severe reactivity (4) against the test organism. The sample free disc, which serveed as blank
control, had a negative activity (-) and no reactivity (0) against the organism.
166 KKU Vet J Vol. 23 No. 2 July - December 2013

Table 1. Activity and reactivity based on inhibition zone at day 5 on M. canis subjected to
different concentration of tawa-tawa leaf crude extract.

Microsporum canis
Test sample/Control Total Zone of Inhibition (mm) Activity Reactivity
R1 R2 R3 Mean
T1 = 100% Tawa-tawa 20.09 22.50 20.43 21.01b Complete Severe
leaf crude extract inhibition
T2 = 75%Tawa-tawa 26.46 18.26 17.80 20.84b Complete Severe
leaf crude extract inhibition
T3 = 50% Tawa-tawa 25.87 22.87 12.86 20.53b Complete Severe
leaf crude extract inhibition
T4 = 25% Tawa- tawa 10.00 23.42 19.07 17.50b Complete Severe
leaf crude extract inhibition
T5 = Ketoconazole 40.01 41.02 41.03 40.69a Complete Severe
(positive control) inhibition
T6 = Sample free disc 0
(negative control)
Note: Means followed by a common letter are not significantly different at the 5% level by DMRT.
Analysis of variance showed significant differences existed between the means. The
positive control (T5) has the highest mean of 40.68 mm, and significantly different with the treatment
groups which have comparable effects. The activity and reactivity of all treatment were similar,
with complete inhibition and severe reactivity of different treatment groups and the positive control.
Measurements of Zone of Inhibition at Day 8
The results of final measurements of zone of inhibition at day 8 was presented in Table 2,
ketoconazole, the positive control obtained a mean of 41.02 mm as the widest zone of inhibition
and produced severe reaction with complete inhibitory activity against M. canis. Analysis of
variance showed a highly significant difference between the different concentration of
tawa-tawa extract. However, all four concentrations of tawa-tawa leaf crude extract showed
วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 167

similar effects on their activity and reactivity as to the commercial ketoconazole which also
produced complete inhibitory activity (+++) and severe reactivity (4), which means the zone
of inhibition extends greater than 10mm beyond the filter paper disc. Results showed (Table 2)
that the positive control (T5) has the highest zone of inhibition of 41.017 mm which has
a highly significant difference on the different treatment means. Treatment 1 with the 100%
concentration has a 26.20 mm zone of inhibition and was significantly different with treatments
2 and 3, with 25.15 mm and 25.50 mm respectively, while T4 has 20.56 mm. However,
all different concentrations gave complete inhibition and severe reactivity.
The different dilutions and pure concentration of tawa-tawa crude extract against
M. canis, in vitro, revealed a significant biological influences in inhibiting M. canis, and was based
on the Universal Standard Procedure (USP) of the Department of Science and Technology (DOST),
as a result of this study.
Table 2. Activity and reactivity based on inhibition zone at day 8 on M. canis subjected to
different concentration of tawa-tawa leaf crude extract.
Microsporum canis
Test sample/Control Total Zone of Inhibition (mm) Activity Reactivity
R1 R2 R3 Mean
T1 = 100% Tawa-tawa 24.51 28.42 25.64 26.20b Complete Severe
leaf crude extract inhibition
T2 = 75%Tawa-tawa 27.40 26.13 21.92 25.15bc Complete Severe
leaf crude extract inhibition
T3 = 50% Tawa-tawa 25.33 23.36 21.80 23.50bc Complete Severe
leaf crude extract inhibition
T4 = 25% Tawa- tawa 25.33 23.36 21.80 20.56c Complete Severe
leaf crude extract inhibition
T5 = Ketoconazole 40.01 41.02 41.03 41.02a Complete Severe
(positive control) inhibition
T6 = Sample free disc 0
(negative control)
Note: Means followed by a common letter are not significantly different at the 5% level by DMRT.
168 KKU Vet J Vol. 23 No. 2 July - December 2013

Phytochemical test shown in Table 3, showed that the tawa-tawa leaves contain fats
and oils, hydrolyzable tannins, flavonoids (leucoanthocyanins, and y-benzopyrone nucleus),
2-deoxysugar, unsaturated steroids and triterpenes (saponins).
Tannins are water-soluble polyphenols that are commonly found in herbaceous and
woody plants. The astringent property of the tannins may induce complexation with the enzymes
or substrates and also compensate the metal ions that produced tannin toxicity in the
microorganism. These bioactive compounds which are synthesized as secondary metabolites
as the plant grows, also serve to protect the plant against microbial attacts and predation
by animals [7].
Flavonoids collectively known as Vitamin P and citrin. Flavonoids are naturally
occurring phenols which possess numerous biological activities including anti-inflammatory,
anti-allergic, anti-thrombic and vasoprotective effects. It also inhibits DNA and RNA synthesis
in other microorganism and flavonoids also inhibit energy metabolism and cytoplasmic
membrane function of microorganism. Triterpenes (saponins) occur as glycosides that have
the effect of reducing surface tension which gives the membranes a soap-like quality promoting
a wound healing, anti-scarring property. They also have expectorant, diuretic and anti-inflammatory
properties [7]. With these components that tawa-tawa leaves possess, it demonstrates that the
tawa-tawa crude extract have an essential antifungal activity against M. canis, based on the
results of the phytochemical analysis of the aqueous extract of tawa-tawa leaves from the
Department of Science and Technology, Philippines.
วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 169

Table 3. Results of the phytochemical analysis of the aqueous extract of tawa- tawa leaves.
QUALITATIVE TEST RESULT INDICATION
Filter Paper Test Greasy appearance (+) Presence of fats and oils
Dragendorff’s Test No reaction (-) Absence of alkaloids
Mayer’s Test No reaction (-) Absence of alkaloids
Ferric Chloride Test Presence of blue-black Presence of hydrolysable tannins
coloration (+)
Froth Test Froth formed less than 2cm (-) Absence of saponins
Sodium Carbonate No stable and dense froth (-) Absence of free fatty acids
Keller-Kiliani Test Formation of reddish-brown Presence of 2-deoxysugar
color at the interface which turn
purple (+)
Liebermann-Burchard Test Formation of red color (+) Presence of unsaturated steroids
and triterpenes
Bate-Smith & Metcalf Test Formation of red color (+) Presence of leucoanthocyanins
Wilstatter “Cyanidin” Test Formation of red color (+) Presence of y-benzopyrene nucleus

Bontrager’s Test No reaction (-) Absence of anthraquinones
The study was conducted to evaluate the antifungal activity of the tawa-tawa leaf crude
extract against M. canis, in vitro, based on the inhibition zone at different concentrations of
Euphorbia hirta Linn. leaf crude extract and to compare it to the commercial systemic antifungal
drug, ketoconazole.
The data gathered were statistically analyzed using the Analysis of Variance (ANOVA)
of the completely Randomized Design (CRD) and the treatment means were compared using the
Duncan’s Multiple Range Test (DMRT). The result of the in vitro study revealed that the positive
control had the highest zone of inhibition of 41.02 mm (+++) with complete inhibitory activity
and high reactivity (4), with a highly significant difference with the tawa tawa treatments. However,
all four concentrations of tawa-tawa leaf crude extract showed a significant antifungal effect on
the M. canis that produced complete inhibitory activity (+++) and high reactivity (4), thus zone of
inhibition extends greater than 10 mm beyond filter paper disc. The study showed that among four
170 KKU Vet J Vol. 23 No. 2 July - December 2013

concentrations: 100 percent, 75 percent, 50 percent, and 25 percent, the 100 percent tawa-tawa leaf
crude extract produced highest zone of inhibition of 26.20 mm (+++) and 25.15 mm (+++), 23.50
mm (+++), 20.56 mm (+++) respectively, with similar effects or no significant difference3s with
each other.
This data gathered showed that the different dilutions and pure concentration of tawa-tawa
crude extract against M. canis, in vitro in terms of zone of inhibition revealed that the study had
significant biological influences in inhibiting M. canis, and this is based on the Universal Standard
Procedure (USP) of the Department of Science and Technology (DOST).

Conclusion
By phytochemical tests, tawa-tawa leaves had been shown that they contain fats
and oils, hydrolyzable tannins, flavonoids (leucoanthocyanins, and y-benzopyrone nucleus),
2-deoxysugar, unsaturated steroids and triterpenes (saponins). With these components of
tawa-tawa leaves, it demonstrates that the tawa-tawa crude extract has an essential antifungal
activity against M. canis, based on the results of the phytochemical analysis of the aqueous extract
of tawa-tawa leaves from the Department of Science and Technology.
Based on the result of the study, the different concentrations of tawa-tawa leaf crude extract
at 100 percent, 75 percent, 50 percent and 25 percent had a significant antifungal effect having a
complete inhibitory activity (+++) and high reactivity (4) based on their inhibition zone against
M. canis. With similar effects, the positive control (ketoconazole), a commercial antifungal drug,
exhibited complete inhibitory activity and severe reactivity against M. canis.
The following recommendations were made: The use of ethanolic extraction and
other preparation of tawa-tawa leaves should be further explored; Crude extract preparation of
tawa-tawa leaves with other microorganisms should be evaluated; An in vivo study of
tawa-tawa leaves crude extract should be performed.

References
1. Akinpelu DA and TM Onakoya. 2006. Antimicrobial Activities of Medicinal Plants Used in Folklore
Remedies in South-Western Nigeria. Afr. J. Biotechnol. 5(11):1078-1081
2. Amutha S, Rajeh MAB, Zuraini Z, Sasidharan S and LY Latha. 2010. Assessment of
Euphorbia hirta L. Leaf, Flower, Stem and Root Extracts for Their Antibacterial and Antifungal Activity
and Brine Shrimp Lethality. Molecules. 2010; 15(9):6008-6018.
 วารสารสัตวแพทยศาสตร์ มข. ปีที่ 23 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2556 171

3. Descamps. 2002. Isolation of a Microsporum canis Gene Family Encoding Three Subtilisin-Like Proteases
Expressed in vivo. Journal of Investigative Dermatology 2002; 119: 830-835
4. Anonymous. 2012. Microsporum canis. Accessed on line. URL: http://www.doctorfungus.org/thefungi/
microsporum_canis.htm
5. El-mahmood AM. 2009. Antibacterial activity of crude extracts of Euphorbia hirta against
some bacteria associated with enteric infections. J. Med. Plant Res. 3(7): 498-505.
6. Patil SB, Naikwade, NS and CS Magdum. 2009. Review on phytochemistry and pharmacological aspects
of Euphorbia hirta linn. JPRHC Vol. 1 No.1 113-133.
7. Hulqvist M. 1999. Review on phytochemistry and pharmacological aspects of Euphrobia hirta Linn.
Phytomedicine 1999, 59-66.

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