development of test method for antiamoebic activity

' \ I j 1 t' ·'

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

3.0 MATERIAL AND METHOD 13

3.1 Materials 12

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) 17

3.4.1 Water Filtration Technique 17

3.4.2 Addition of Rice grains in Water Samples 19

3.5 Subculturing of free living amoebas (FLA) 19

3.5.1 Standard Method of subculturing FLA 19

3.5.2 Modified Method of Subculturing FLA 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


4.2.2 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


4.5 Antiamoebic Screening Test against Isolated Amoeba 31

4.6 Statistical Analysis 32

S.O DISCUSSION 33

5.1 Collection of Water Samples 33

5.2 Isolation and culturing of free living amoeba (FLA) 34


5.2.2 Water Filtration Technique 34

5.2.3 Addition ofrice grains in Water sample 35

5.2.4 Culturing of Free Living Amoeba 35

5.3 Subculturing of Free Living Amoeba (FLA) 36


5.5 Antiamoebic Screening Test against Isolated Amoeba 37

6.0 CONCLUSION 38

REFERENCES 39

APPENDICES 42

v

C

CC

DMSO

°C

FLA

FRST

g

NaCI

NNA

MIC

J.11

J.1m

mg

ml

mm

PAS

PBS

%

TC

TD

LIST OF ABBREVIATIONS

Control

Cytotoxicity control

Dimethyl sulfoxide

Degree Ce1cius

Free Living Amoeba

Faculty of Resource Science and Technology

Gram

Sodium chloride

Non-Nutrient Agar

Minimal Inhibitory Concentration

Micro liter

Micro meter

Milligram

Milliliter

Millimeter

Page's Modified Neffs Amoeba Saline

Phosphate Buffered Saline

Percent

Thiourea control

Amino acid-Thiourea Derivative

vi

I I .

LIST OF TABLES

Table No. Title 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 26 techniques

7 The numbers of FLA in samples with different amount of addition 27 rice grains

8 The numbers of FLA in culture plates by using different media 28

9 The numbers of free living amoeba by using different subcuituring 29 techniques

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 30 compound microscope

7 FLA from NNA-E. coli culture under 400x magnification 30 compound microscope

8 Antiamoebic activity comaprisons between individual 31 treatment ofTD, TC, and CC

viii

-- 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 nonpathogenic 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, nonpathogenic, 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

, song sang 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

I •

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 ofE. 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 ofchoice 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

exhibited

have

com.).

synthesized and their anti-microbial properties were widely explored. Thiourea derivatives

a wide variety biological activity because this heterocyclic compounds

containing nitrogen and sulphur. In addition to antiamoebic activity, thiourea derivatives

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.

The preparation of this compound is performed by reaction between 3

10

2, 4-triozoles

the mycobacterial growth.

methanol as

substituted at

mycobacterial

daivatives had shown lower anti-mycobacterial activity.

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,

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

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

a solvent. The methyl substituent at position of triazole ring or the one

the terminal nitrogen of thiourea was reported to enhance the anti

activity of compound. The more bulky substituent in other thiourea

11

---~----------- - - -------- ----~-~-----------

Water samples

•1.2 Media and solutions

•

1210C before used.

~.iivaJtive.

3.0 MATERIAL AND METHOD

3.1 Materials

3.1.1 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 .

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,

Test Chemical compound

compound that was used as the test compound was synthesized Amino acid-Thiourea

This thiourea derivative with their molecular structure is shown in Figure 1.

:n_rea compound and NaCl compound used as control treatments.

12

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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development of test method for antiamoebic activity

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