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DEVELOPMENT OF TEST METHOD FOR ANTIAMOEBIC
ACTIVITY
Nurulhazwani Bt Mohd Rafiee
QL
368
AS
N974
2012
Bachelor of Science with Honours
(Biotechnology Resource)
2012
Pusat Khidmat Maklumat Akademik
l~lVERSm MALAYSIA SARAWAK
Development of Test Method for Antiamoebic Activity
P.KKtDMAT MAKLUMAT AKAC!MIK
111111111
111111111
1000235473
Nurulhazwani bt Mohd Rafiee
(24781)
A project submitted in fulfillment of requirements for degree in Bachelor of Science with Honours (Resource Biotechnology) Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
University Malaysia Sarawak
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ACKNOWLEDGMENT Thank you for all His Gracious that I am able to complete this project. It would not have
been possible to write this project without the help and support of the kind people around
me, to only some of whom it is possible to give particular mention here. Biggest gratitude
to my supervisor, Prof. Dr. Ismail b. Ahmad for his advice, expertise, and knowledge
towards completion of this project. This thesis would not have been possible without the
help, support and patience of my supervisor. Thank you to my co-supervisor Dr. Zainab
Ngaini for encouragement, insightful comments, teaching on chemistry part, and providing
chemical compound for this project.
I would like to express my appreciation to master's student of chemistry
department, Wan Sharifatun Handayani bt Wan Zullkiple for providing chemical
compound and sharing knowledge on chemical compound for this project. Not forget to aU
master' s student and laboratory assistants of Virology laboratory for their assistance.
Lastly, I would like to mention my appreciation to course mates for their
cooperation, assistance, and supports. To my family and friends, your moral supports are
really meaningful to me.
Thank you.
ii
Pusat Khidmat Maklumat Akademik
UNlVERSm MALAYSIA SARAWAK
Table of contents
ACKNOWLEDGEMENT
ii
CONTENT
iii
LIST OF ABBREVIATIONS
vi
LIST OF TABLES
vii
LIST OF FIGURES
viii
LIST OF APPENDICES
ix
ABSTRACT
x
ABSTRAK
x
1.0 INTRODUCTION
1
2.0 LITERATURE REVIEW
4
2.1 Amoebiasis
4
2.2 Amoeba
4
2.3 Free living amoeba
5
2.4 Non-pathogenic amoeba and non-parasitic amoeba
7
2.5 Anti-amoebic drugs
9
2.6 Thiourea and Amino acid-thiourea derivative
10
2.7 Amino acid-thiourea derivatives as anti amoebic
11
13
3.0 MATERIAL AND METHOD
12
3.1 Materials
3.1.1 Water samples
12
3.1.2 Media and Solutions
12
iii
3.1.3 Test Chemical compound
12 3.1.4 Heat inactivated E. coli
13 3.2 Media Preparation
15 3.2.1 Nutrient Agar (NA) and Nutrient broth (NB) preparation
15 3.2.2 NNA-E. coli and NA- E. coli preparation
15 3.3 Collection of Water samples
16 3.4 Isolation and Culturing of Free Living Amoeba (FLA)
3.4.1 Water Filtration Technique
3.4.2 Addition of Rice grains in Water Samples
3.5 Subculturing of free living amoebas (FLA)
3.5.1 Standard Method of subculturing FLA
3.5.2 Modified Method of Subculturing FLA
17 17 19 19 19 20 3.6 Detection of Free Living Amoeba (FLA)
21 3.7 Anti-amoebic screening test against isolated FLA
22 3.8 Statistical Analysis
23 4.0 RESULT
24 4.1 Collection water samples
24 4.2 Isolation and Culturing of Free Living Amoeba (FLA)
25 4.2.1 Heat inactivated E. coli
25 4.2.2 Water Filtration Techniques
26 4.2.3 Addition of Rice grains in Water Sample
27 4.2.4 Culturing of Free Living Amoeba (FLA)
28 iv
,
I
4.3 Subculturing of Free Living Amoeba (FLA)
28 4.4 Detection of Free Living Amoeba (FLA)
29 4.5 Antiamoebic Screening Test against Isolated Amoeba
31 32 4.6 Statistical Analysis
S.O DISCUSSION
33 5.1 Collection of Water Samples
33 5.2 Isolation and culturing of free living amoeba (FLA)
34 5.2.1 Heat inactivated E. coli
34 5.2.2 Water Filtration Technique
34 5.2.3 Addition of rice grains in Water sample
35 5.2.4 Culturing of Free Living Amoeba
35 5.3 Subculturing of Free Living Amoeba (FLA)
36 5.4 Detection of Free Living Amoeba (FLA)
36 5.5 Antiamoebic Screening Test against Isolated Amoeba
37 6.0 CONCLUSION
38 REFERENCES
39 APPENDICES
42 v
LIST OF ABBREVIATIONS
C
Control
CC
Cytotoxicity control
DMSO
Dimethyl sulfoxide
°C
Degree Ce1cius
FLA
Free Living Amoeba
FRST
Faculty of Resource Science and Technology
g
Gram
NaCI
Sodium chloride
NNA
Non-Nutrient Agar
MIC
Minimal Inhibitory Concentration
J.11
Micro liter
J.1m
Micro meter
mg
Milligram
ml
Milliliter
mm
Millimeter
PAS
Page's Modified Neffs Amoeba Saline
PBS
Phosphate Buffered Saline
%
Percent
TC
Thiourea control
TD
Amino acid-Thiourea Derivative
vi
I
I
.
LIST OF TABLES
Title
Table No.
Page
Page's Modified Neffs Amoeba Saline (PAS) preparation
44
2
Result Heat Inactivation E. coli
45
3
Result for the absorbance value
45
4
The numbers of FLA in different samples and ponds
24
5
The numbers of FLA in different samples and day conditions
25
6
The numbers of FLA in culture plates by using different isolation
techniques
26
7
The numbers of FLA in samples with different amount of addition
rice grains
27
8
The numbers of FLA in culture plates by using different media
28
9
The numbers of free living amoeba by using different subcuituring
techniques
29
10
The numbers of viable cells after screening test
47
11
Summary table ANOV A F-test
49
12
Average mean for each treatment
50
13
Ascending order average mean
50
14
Mean comparison, mean difference of LSD
51
15
Fisher's LSD (Least Significant Difference)
32
vii
LIST OF FIGURES Figure No.
Title Page
1
Amino acid-Thiourea Derivative
13
2
Sampling sites of ponds at Sarawak Golf Club UNIMAS
16
3
Procedures of isolation and cultivation of FLA
18 4
Subculture ofFLA on NNA-E. coli plate
20 5
Method for counting FLA
21 6
FLA from NNA-E. coli culture under 200x magnification of
compound microscope 30 7
FLA from NNA-E. coli culture under 400x magnification
compound microscope 30 8
Antiamoebic activity comaprisons
treatment ofTD, TC, and CC 31 viii
between
individual
--
I
LIST OF APPENDICES
A
Heat Inactivation E. coli Procedures
B
Page's Modified Neff's Amoeba Saline (PAS) PAS Solution Preparation
C
Result for Heat Inactivation E. coli
D
Antiamoebic Activity Screening
E
Result for Antiamoebic Activity Screening Test
F
Statistical Analysis One-way ANOV A
G
Summary Statistical Analysis One-way ANOV A
H
Fisher' s LSD (Least Significant Difference)
I
Mean Comparison Fisher's LSD (Least Significant Difference)
ix
•
I
Development of Test Method for Anti-amoebic Activity
Nurulhazwani bt Mohd Rafiee
(24781)
Programme: Resource Biotechnology Department: Molecular Biology Department Faculty of Resource Science and Technology University Malaysia Sarawak ABSTRACT
A study was carried out to develop a preliminary method of antiamoebic screening test. Collection
water samples were conducted in less polluted pond at Sarawak Golf Club UNIMAS to isolate non­
pathogenic amoeba. Stepwise optimisation to initial amoeba isolation protocol resulted in a
protocol which was applied successful to isolate and culture amoeba. The antiamoebic activity was
conducted by treating free living amoebas with 50 ~g/ml concentration of Amino acid-Thiourea
derivative. The viability of cells was compared with the treatment Thiourea control and NaCl
cytotoxicity control with the same concentration. The antiamoebic activity was based on
comparison of percentage of viable cells from 50 first views of counted cells under 100x
magnification inverted microscope. Data was analysed using one-way ANOV A and significant data
was analysed with Fisher's LSD test. Results of treatment showed toxicity of Amino acid-thiourea
derivative on free living amoeba compared to NaCI was successfully developed. The prototype of
the model antiamoebic activity screening test was established preliminary method for further detail
study. The study suggests that antiamoebic activity of this compound may further be explored in
the future by using higher concentration of compound.
Keyword: Amino acid-thiourea derivative, antiamoebic activity, free living amoeba, non­
pathogenic, preliminary method.
ABSTRAK
Satu kajian telah dijalankan untuk membangunkan satu kaedah awal ujian saringan antiamoebik.
Pengambilan sampel air dijalankan di Sarawak Golf Club UNIMAS untuk pemencilan amoeba
bukan patogenik. Pengoptimum setiap langkah terhadap protokol pemencilan amoeba telah
menghasUkan satu protokol yang dapat diaplikasikan untuk memencilkan dan seterusnya
mengkulturkan amoeba. Aktiviti antiamoebik telah dijalankan dengan cara menguji amoeba
dengan 50 pg/m/ kepekatan derivatif amino asid-Thiourea. Kemandirian sel adalah berdasarkan
perbandingan dengan rawatan Thiourea kawalan dan NaCI sitotoksiti kawalan dengan
menngunakan kepekatan yang sama. Aktiviti antiamoebik adalah berdasarkan perbandingan
peratusan kemandirian sel dari 50 permerhatian pertama sel-sel yang dikira di bawah mikroskop
, songsang dengan magnifikasi JOOx. Data dianalisis menggunakan ANOVA sehala dan data yang
signifikan telah dianalisis dengan ujian LSD Fisher. Keputusan rawatan menunjukkan ketoksikan
derivalifamino asid-thiourea pada amoeba berbanding kepada NaCl telah berjaya dibangunkan.
Model prototaip ujian saringan aktiviti antiamoebic yang telah ditubuhkan dengan kaedah awal
untuk kajian terperinci lanjut. Dapatan kajian ini mencadangkan bahawa aktiviti antiamoebik
untuk sebatian ini boleh dikaji pada masa hadapan dengan menggunakan kepekatan sebatian yang
lebih tinggi.
Keyword: derivatif Amino acid-thiourea, aktiviti antiamoebik, free living amoeba, bukan
patogenik, kaedah awal.
x
1.0 INTRODUCTION
Amoebiasis is responsible for 100, 000 deaths per year worldwide. It is second leading
cause of death due parasitic disease (Bharti et al., 2006). Infection by amoeba remains a
significant cause of morbidity or mortality (Moundipa et aI. , 2005) due to inadequate
sanitation, contaminated food and drinking water. Besides, free living amoebas are
ubiquitous organisms that have been isolated from various domestic water and may lead to
amoebic dysentery and liver abscess (Bharti et
at., 2006). The increase in world travel,
economic globalization, and the growing number of chronically immunosuppressed people
have also contributed to the rise in importance of intestinal protozoa (Sharma & Ahuja,
2003).
Amoebic infection can cause abdominal cramps, tenesmus, and blood and mucus in
the stool (Fernando et at., 2001). Treatment of amoebic infections is usually done by using
synthetic drug. Most anti-amoebic drugs have been shown to be relatively efficient for the
treatment of clinical cases. Presently, the most effective anti-amoebic medications are
nitromidazole drugs such as metronidazole [1 - (2 - hydroxyethyl) - 2 - methyl - 5 ­
nitroimidazole] . Nitroimidazole have been shown several important problems such as
mutagenic and toxic towards the host when they are used at high doses. Long term use of
medication produces undesirable side effects in patients. In addition, amoeba strains are
able to develop resistant towards these drugs (Barthi et at., 2006). As a result effectiveness
is reduced to the extent that it can eradicate only up to 50% of laminae infections and
ineffective against cysts (Moundipa et
at., 2005). Therefore, new a novel drug is needed
for effective treatment newly emerging resistant strains.
1
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•
Recently, a novel amino acid-thiourea derivatives have been synthesized at the
chemistry department of Faculty Resource of Science and Technology (FRST) UNIMAS
(Wan, 2011 ). Based on the study that showed cytotoxicity of some amino acid-thiourea
derivatives on pathogenic Acanthamoeba sp. (Maizatul et a!., 2011), the newly synthesized
amino acid-thiourea derivatives could have similar activity. To detennine the potential of
these compound chemical as anti amoebic drug, a preliminary experiment should be carried
out. In the literature testing of antiamoebic drug testing usually use pathogenic
Acanthamoeba sp. of free living amoeba. The use of these pathogenic amoebae is not
suitable for inadequately equipped laboratories as it poses risk of infections. Therefore, a
model of screening for antiamoebic drug using non-pathogenic amoeba species should be
developed to reduce risk of contamination in laboratories with less equipment.
Since, Sarawak is center of biodiversity, this study aims at utilizing free living
amoeba that are found in Sarawak that could be used as a model for anti amoebic drug
testing. For this purpose, isolation of Amoebas was carried out less polluted water which is
at Sarawak Golf Club ponds at UNIMAS. Once the amoebae are isolated, they will be used
for screening the novel thiourea for anti amoebic activity. Since this study was conducted
for the first time in this university, the study aims only to establish a prototype of the
model. Thus, the study of development of test method for anti-amoebic activity is
necessary to provide this prototype model was when applied for the preliminary screening
for anti-amoebic activity ofthe thiourea derivative compound.
2
Therefore the aims of study are:
1. To isolate non-pathogenic free living amoebas
2. To develop a model of preliminary screening for anti-amoebic activity of non­
pathogenic amoeba.
3. To determine presence of anti-amoebic activity m novel ammo acid-thiourea
derivative.
3
------~
- -- - --- - - ­
2.0 LITERATURE REVIEW
2.1 Amoebiasis
Amoebiasis is an infection caused by Entamoeba histoiytica, probably has existed very
long time ago and has world-wide distribution, and is one of the most prevalent intestinal
protozoa in developing countries (Sharma & Ahuja, 2003). Amoebic infections occur in
two major forms which are intestinal amoebiasis and extra-intestinal amoebiasis (Fernando
et ai., 2001). Intestinal amoebiasis is also known as amoebic dysentery and amoebic
colitis. Intestinal amoebiasis has mild symptoms or asymptomatic such as flatulence,
constipation, and loose stools (Okafor, 2011).
Dysentery development is characterized by abdominal cramps, tenesmus, and blood
and mucus in the stool (Fernando et ai., 2001). Extra intestinal occurs in small percentage
of patients. It is occur if the parasites spread through the body, most frequently ending up
in various organs including liver, lung and brain (Kuntz & Kuntz, 2006). A hepatic abscess
is the most common extra intestinal manifestation. The signs of a liver abscess include
fever, abdominal pain and weight loss (Kuntz & Kuntz, 2006). This invasive amoebiasis is
high in the areas of South East Asia (Thomas & Pak Leng, 1986).
2.2 Amoeba
Amoeba is a unicellular organism which is single-celled organism that can generate
pseudopods and is classified as a genus of Protozoa. Amoebae live mainly in water such as
freshwater, and seawater as well as live in damp soil and sediments on the seafloor. This
4
- --
----- - --
Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWA)( group of organisms exists as parasites on animals and plants while others are free-living
(Annstrong, 2001 ).
Amoeba has two types of cytoplasm, which are ectoplasm and endoplasm.
Ectoplasm is stiff but flexible and acts as a membrane that holds cell together. The other
type, endoplasm, is more watery consist of tiny structures that hold the cell's DNA and
perfonn other essential function. Ectoplasm can be extended into two a limb called
pseudopods which are used for feeding and can pull the amoeba along.
Amoeba usually feed on bacteria, simple algae, or other small single-celled
organism (Armstrong, 2001). Once amoeba have been isolated, it is very important to
establish optimum culture conditions immediately. They require constant care, feeding and
general sub-culturing. A species of free-living amoeba is grown at 25°C to 30°C (this
species tends to encyst at 37°C) (Greub & Raoult, 2004). Amoeba reproduce by process
called binary fission by splitting into two separate cells and is form of asexual
reproduction. Both of the new cells produced contain an exact copy of the DNA of the
original parent amoeba. The organism can reproduce at high rate (Bernabeo et al., 2000).
2.3 Free living amoeba
Free living amoebae have been characterized as facultative parasites and opportunistic
pathogen. They can be isolated from soil, freshwater lakes, swimming pool, therapeutic
pools, tap water, natural thermal water and air all over the world. Furthermore, amoeba can
survive in severe condition by forming resistant cyst (Greub & Raoult, 2004). There are
four genera of free living amoeba, which are pathogenic and responsible for human
5
disease. These include the genera Acanthamoeba spp., Balamuthia mandifloris, Naegleria
fowleri and Sappinia diploidea (Zuhal et aI., 2010).
Acanthamoeba spp. and B. mandrillaris are common in soil and freshwater
habitats. They are known as oppurtinistic pathogens, causing infection of the central
nervous system, lungs, sinuses and skin, mostly in immunocompromised humans.
Balamunthia also associated with disease in immunocompetent children, according to
Zuhal et al., (2010). Acanthamoeba spp. also causes keratitis to soft contact lens users
with poor hygienic practices and also patients exposed to contaminated water. Naegleria
infection usually correlated with history of swimming lakes or brackish water, aspiration of
contaminated water, inhalation of contamination dust. Furthermore, immunosurpression is
a risk factor for infection from all free living amoeba (Couturier et al., 2010).
There are two types of developmental stages of free living amoebas, which are the
trophozoite and the cyst. The trophozoite stage is a vegetative feeding form and the
metabolically active stage, feeds on bacteria and multiplies by binary fission (Greub &
Raoult, 2004). The cyst is a resting form which is generally has two layers, the outer wall,
ectocyst and the inner wall, endocyst (Greub & Raoult, 2004). Some amoeba, such as
Naegleria spp. has an additional flagellate stage. Some species is presence a third layer,
mesocyst. Therefore, this structure cause cyst is resistant to biocides used for disinfecting
bronchoscopes and contact lenses. It is also resistant to chlorination and sterilization of
hospital water system. Cyst can be survived in condition of acidic pH of the stomach and
pass into intestine. Thus, adverse pH, osmotic pressure, and temperature condition cause
amoeba to encyst. Encystment also occur when food requirement are not fulfilled. As the
6
environmental condition become favorable, protozoa may excyst again (Greub & Raoult,
2004).
2.4 Non-pathogenic and non-parasitic amoeba
According to Lamps, (2009), non-pathogenic amoeba or amoeba of "low pathogenicity"
are referred as amoeba associated with low degree of pathogenicity. This group includes
Entamoeba dispar, Entamoeba hartmanni, Entamoeba eoli, Iodamoeba buetsehlii,
Endolimax nana, and Entamoeba poleeki. Entamoeba hartmanni is one of non-pathogenic
amoeba which is discovered by Von Prowazek in 1912. The species E. hartmanni shares
many morphological characteristics with E. histolytiea, except for their size. It has
geographical distribution similar to E. histolytiea. Previously, E. hartmanni is often called
"small histolytiea" (Ichhpujani & Bhatia, 2002). It is now considered as non-pathogenic
amoeba. Cyst of E. hartmanni measures usually 5-10 ).tm which is smaller than E.
histolytiea (10-20 ).tm). The trophozoite measures about 4-12 ).tm. The trophozoite of E.
hartmanni has ingested yeast, but not an erythrocyte. Besides, life cycle of E. hartmanni is
similar to life cycle of E. histolytiea (Ichhpujani & Bhatia, 2002).
The species E. dispar is non-invasive and non-pathogenic amoeba which was also
earlier considered as a non-pathogenic strain of E. histolytiea. Both species are
morphologically identical and the cysts of E. histolytiea and E. dispar could not be
differentiated microscopically (Bogitsh et al., 2013). Entamoeba eoli are worldwide
parasite which usually distributed in lumen of large intestine of man. It exists in three
stages, which are trophozoite, precyst and cyst. The life cycle is similar to E. histolytiea.
Trophozoites for this species have sluggish movement. The cytoplasm is not differentiated
7
into ectoplasm and endoplasm. They never contain red blood cells but bacteria and ceIrular
debris is present. The karyosome is eccentric and nuclear membrane is thick and is lined by
coarse chromatin granules. The cysts for Entamoeba coli are spherical in shape and the
size is 15 IJ.m to 20 J1m. The chromidial bars are filamentous (Bogitsh et al., 2013). Precyst
for this species resembles in shape with that of E. histolytica.
Another species of non-pathogenic amoeba is Iodamoeba butschilii distributed in
large intestine. This species is transmitted by a cyst that is very distinctive, facilitating
identification. Trophozoites are active in freshly evacuated unfonned stools and sluggish in
older stools. The ectoplasm is not well differentiated from endoplasm. Moisture and
wannth conditions are requirements for excystation. This species of amoeba is feeding on
bacteria and yeast (Bogitsh et al., 2013).
The first parasitic amoeba to be recognized is E. gingiva lis and found only in its
trophozoite form. This species is commensal in the gingival tissue around the teeth which
is described by Gros in 1849 in the soft tartar between the teeth. Also found in the diseased
tonsils and in the vaginal and cervical smears from women using intrauterine device. E.
gingivalis have pseudopodia that allow them to move quickly. Their spheroid nucleus is 2 ­
4 IJ.m in diameter and contains a small central endosome. There are numerous food
vacuoles and contain cellular debris, blood cells and bacteria (lnchpujani & Bhatia, 2002).
8
1.5 Anti-amoebic drugs
Antiamoebic drug is an agent used in the treatment of amoebic infections. Nitroimidazoles
such as metroniazole, tinidazole, secnidazole or ornidazole, is used to destroy amoeba that
have invaded tissues. This type of drug can be used to treat tissue amoebiasis for both
intestinal and extra intestinal. Besides, chloroquine can be used for extra intestinal
amoebiasis treatment only. Since most of the amoeba remains in the intestine when tissue
invasion occurs, some of anti amoebic drugs are developed to treat this luminal amoebiasis.
The anti-amoebic that can be used for the treatment are amide, diloxanidefuroate, 8­
hydroxy quinolones, Quinidochlor, antibiotics such as tetracycline.
Treatment with tissue amoebicide should always follow by luminal amoebicide to
eradicate source of infection. Metronidazole is prototype drug introduce in 1959 which is
act as bactericidal. As the metronidazole enters microorganism by diffusion, it will reduced
nitro group and causing DNA damaged. This drug has high selective anaerobic action. It is
also inhibits cell mediated immunity and induce mutagenesis. However, these drugs may
cause adverse drug reactions according to the level of doses and how often patient taking
this drug. If they take the drug frequently, the consequences are anorexia, nausea, metallic
taste and abdominal cramps. Less frequent taking will cause headache, glossitis, dry
mouth, dizziness, rashes, and transient neutropenia (Okpako, 1991).
According to Ordaz et al. (2005), anti-amoebic drugs have been classified
according to their site of action. Antiamoebic drugs that exert their site of action in the
large intestine are classified as luminal drugs. Antiamoebic drugs that exert their action in
the liver and other organs are classified as extra-luminal. Nitroimidazole is less effective in
9
the tissue than in gut lumen. In addition, it can eradicate only up to 50% of laminae
infections and has no action on cyst (Moundipa et aI., 2005).
However, nitroimidazole
derivatives such as metronidazole, tinidazole, and omidazole have been synthesized and
become the drugs of choice in invasive amoebiasis (Sharma & Ahuja, 2003).
1.6 Thiourea and Amino acid-thiourea derivative
Thiourea, SC(NH2h is an organic compound is potentially useful for development of new
drug. Thiourea is a white crystalline solid. Thiourea occurs in two tautomeric forms which
are thiourea and isothiourea. The compound has three functional groups that are essential
for structural modification. These functional groups are amino, imino and thiol. These
functional groups are required in order to synthesize new derivatives (Mohanta et ai.,
1999). Thiourea is soluble in water, soluble in polar protic and aprotic organic solvents,
and insoluble in non-polar solvents. Recently, many thiourea derivatives have been
synthesized and their anti-microbial properties were widely explored. Thiourea derivatives
exhibited a wide variety biological activity because this heterocyclic compounds
containing nitrogen and sulphur. In addition to antiamoebic activity, thiourea derivatives
have also been shown to possess antioxidant, anti-HIV and tuberculosis activities
(Maizatul et ai., 2011).
A novel amino-acid thiourea derivative which was synthesized recently is
[[3(carboxymethylcarbamothioylcarbamoyl) benzoyl] carbamothioylamino] acetic acid
(Wan, 2011). This amino-acid thiourea derivative was synthesized by chemistry research
group of University Malaysia Sarawak (Associate Professor Dr. Zainab Ngaini, pers.
com.). The preparation of this compound is performed by reaction between 3­
10
-
-- -- ------- --- -- -
acetylbenzoyl isothiocyanateand glycine. The compound was obtained as a yellowish solid
with yield of73% with the melting point at 226°C to 22TC.
2.7 Amino acid-thiourea derivatives as Anti-amoebic drug
Thiourea derivatives have been reported to have antibacterial activities. According to
Saeed et al. (2009), the l-aroyl-3-aryl thiourea with chlorine substituted synthesized by
reacting benzoyl isothiocyanate and aniline. This compound has shown just moderate
activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aueroginosa and
&chericia coli. The in vitro .evaluation of antibacterial activity against those four strains
was perfonned using Kirby-Bauer method. The presence of halo group in thiourea gave
enhancement of inhibitory activity.
Furthermore, Fernandez et al. (2005) have synthesized some 3-thioxo/alkylthio-l,
2, 4-triozoles with substituted thiourea moiety and are reported as excellent anti­
agent against M. tuberculosis in mono layers of mouse bone marrow
In . addition, I-methyl-3-[4-(4-methyl-5-thioxo-lH-I, 2, 4-triazol-3-yl)
phenyl] thiourea is the synthesized bis thiourea derivative that reported to inhibit 90% of
the mycobacterial growth. This compound is synthesized from reaction of methyl
thiocyanate and 3-(4-aminophenyl)-4-methyl-lH-l, 2, 4-triazole-5-thione in the present of
methanol as a solvent. The methyl substituent at position of triazole ring or the one
substituted at the terminal nitrogen of thiourea was reported to enhance the anti­
mycobacterial activity of compound. The more bulky substituent in other thiourea
daivatives had shown lower anti-mycobacterial activity.
11
---~ - ------ -- --
- - -- -- ---- - ---~-~-----------
3.0 MATERIAL AND METHOD
3.1 Materials
3.1.1 Water samples
Water samples were obtained from ponds with less polluted at Sarawak Golf Club
UNIMAS (Figure 2). Water samples were collected from different site of pond by using
250 ml Schott bottles. Water samples then were transferred immediately to the laboratory .
•1.2 Media and solutions
Media used for cultivation free living amoeba and E. coli in this study include Non­
nutrient agar (NNA), Nutrient agar (NA), and Nutrient broth (NB). The solutions were
required include Page's modified Neffs Amoeba Saline (PAS) solution, Phosphate Buffer
Saline (PBS) solution, and Dimethylsulphoxide (DMSO) solution (preparation of solutions
• Appendix B and Appendix D). All solution and media were sterilized at 15 lbs pressure,
1210C before used.
Test Chemical compound
compound that was used as the test compound was synthesized Amino acid-Thiourea
~.iivaJtive.
This thiourea derivative with their molecular structure is shown in Figure 1.
:n_rea compound and NaCl compound used as control treatments.
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Fipre I: Amino acid-thiourea derivative
2-[[3-(carboxymethylcarbamothioylcarbamoyl)benzoyIJ carbamothioylamino] acetic acid (reaction
with Glycine)
3.IA Heat Inactivated E. coli
Heat inactivated E. coli was used as food source for culturing amoeba in culture plate
(Zuhal et al., 2010). Heat inactivated E. coli was prepared by pipetting 50 III of E. coli
from glycerol stock and transferred into 40 ml nutrient broth in universal bottle, followed
by incubation at 37°C for 24 hours in incubator shaker. The next day, the E. coli colonies
growing on the nutrient broth were inoculated and streaked onto each of six nutrient agar
Single colony of E. coli growing on each the agar surface of these plates was then
iDoculated into each of six 3 ml nutrient broth in bijou bottles and incubated at 37°C for 24
hours in incubator shaker. Each two of these bijou bottles were labeled as 30 second, 1
.mil1lU1e, and 2 minute for heat inactivation process. After overnight incubation, all these E.
broth culture were heated in beaker containing boiling water (100°C) for 30 seconds, 1
~nute,
and 2 minutes according to their label. The heat-inactivated E. coli then was
ed for viability and integrity of the cells.
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