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Evaluation of Toxicity and Mutagenicity of
Trichosetin, an Anti-MRSA Antibiotic Produced from
the Dual Culture of Catharanthus roseus Callus and
Trichoderma harzianum
Presentation of Completed Researches
UPLB DA-BAR RDE Network Headquarters, UPLB, College, Laguna. March 30, 2010
EUFROCINIO C. MARFORI, Ph.D.
University Researcher, BIOTECH, UPLB
Trichosetin
NHO
O
O
OH
(Marfori et al., 2002. Z. Naturforsch. 57c: 465-470)
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Diwa and Peji (2005) established the antimicrobial activityof trichosetin against local clinical MRSA isolates.
Vancomycin
Trichosetin
Methanol
Methicillin-resistant Staphylococcus aureus
(MRSA)
-minor skin infections
-life-threatening diseases
such as pneumonia,
meningitis, endocarditis,
toxic shock syndrome and
septicemia.
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OO
O
O
OCH2OH
HOHO
H2NHO
Cl
NH
O
HN
O
NH2
O
O
NH
OHN
HN
HO
O
HO
OHHOOH
O
O
Cl
Vancomycin
Recent emergence of vancomycin-intermediate resistant S.
aureus (VISA) is now raising serious public health concerns.
•Any new compound proven to be effective against
certain microbial pathogens should first undergo
assessment of the potential risk it may cause to the
user, i.e. mutagenicity and toxicity studies.
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Objectives
General Objective:
To evaluate the potential risk of trichosetin as a therapeutic agent for
methicillin- resistant Staphylococcus aureus (MRSA) infection
Specific objectives:
a. To establish and maintain the dual culture of Catharanthus roseus callus
and Trichoderma harzianum
b. To determine the oral LD50 of trichosetin in mice
c. To determine the effect of trichosetin on vital organs
d. To determine the mutagenicity of trichosetin
Establishment of the dual culture of
Catharanthus roseus callus and Trichoderma harzianum
C. roseus plant
C. roseus callus dual culture
Trichoderma harzianum
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Dual culture (10 kg)
MeOH extract (100.11 g)
hexane
layerAqueous
layer
EtOAC extractAqueous
(2.82 g)
Isolation of trichosetin
soaked in 10 L MeOH overnight
resuspended in 500 ml dH2O
adjusted to pH 4.0; extracted
with 500 ml n-hexane (3x)
extracted with 500
ml EtOAc (3x)
open- column chromatography
34.5 cm x 1.6 cm column
30 g deactivated silica
eluted with solvents of
increasing polarity
EtOAc fraction
Fraction No. 7- Methanol (145.2 mg)
Fraction No. 1- Toluene
Fraction No. 2- Toluene: Acetone (9:1)
Fraction No. 3- Toluene: Acetone (8:2)
Fraction No. 4- Toluene: Acetone (7:3)
Fraction No. 5- Toluene: Acetone (6:4)
Fraction No. 6- Acetone
rec Assay
• involves the use of two strains of
Bacillus subtilis :
recombination-proficient strain H17 Rec+
recombination-deficient strain M45 Rec-
rec Assay- used to determine DNA-damaging capacity of a compound
• M45 Rec- is much more sensitive toagents which cause genetic alterations
• a test compound which inhibited the growth of M45 Rec- may be
considered as DNA-damaging and potential mutagen.
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Dilution of trichosetin
Streaking of the test strainsPlacing the paper disks containing
trichosetin
Prepared plates for the rec assay
10 ppm
trichosetin
100 ppm
trichosetin
1000 ppm
trichosetinquinoline
(positive control)
methanol
(negative control)
TreatmentLength of inhibition, mm
M45 Rec-H17 Rec+
Trichosetin, 10 ppm 0 0
Trichosetin, 100 ppm 0 0
Trichosetin, 1000 ppm 0 0
Methanol 0 0
4-nitroquinoline oxide 23.1 + 1.2 31.2 + 1.6
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Ames Test
- to determine the ability of a compound to induce frameshift and point
mutation
- being done using special tester strains of S.
typhimurium (TA 98 and TA 100), which are
his- and cannot synthesize histidine so that
they are unable to grow in its absence.
- tester strains spread on a culture plate that
lacks histidine
- a mutagen placed in the culture medium caused some of these his- bacteria
to revert to the his+ phenotype, as detected by their growth into visible
colonies after 18 h at 37oC
- mutagenicity of a compound scored based on the number of induced
revertant colonies
- a compound is mutagenic if the number of the induced revertant colonies is
more than double the revertant colonies of the negative control
Ames test of trichosetin using Salmonella typhimurium TA 98 and TA 100
Treatment Ave. Microbial Count (CFU mL-1)
TA 98 TA 100
No substance (inoculated with bacteria only)a 5.00 108.67
Trichosetin, 10000 mg.L-1 2.33 35.00
Trichosetin, 1000 mg.L-1 3.33 73.00
Trichosetin, 100 mg.L-1 7.67 42.67
Trichosetin, 10 mg.L-1 5.66 108.33
Methanol 2.33 23.67
Acridine orange, 1000 mg.L-1 32.66 546.00
aPlate for revertant. A >2-fold reversion rate indicates that the substance is mutagenic.
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Micronucleus Test
Micronucleus test- for determination of the chromosome-damaging property of
a test compound.
DNA damage by a mutagen causes the formation of acentric chromatids and
chromosome fragments.
After mitosis, acentric chromatids and
chromosome fragments are included in the
daughter cells but a considerable
proportion is transformed into one or
several secondary nuclei which are smaller
than the principal nucleus and are called
micronuclei.
Intraperitoneal administration of
trichosetin Cervical dislocation of mouse
Flushing the bone marrow with
fetal calf serumBlood smearing
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Trichosetin, 10 mg/kg 3.53 + 1.93
Trichosetin, 50 mg/kg 3.87 + 1.02
Trichosetin, 100 mg/kg 3.00 + 1.15
Methanol (negative control) 2.07 + 1.62
Tetracycline (positive control) 7.07 + 2.62
Treatment No. of MPEs
Note: A >2-fold increase in the frequency of micronuclei indicates that the
substance is mutagenic.
Mean number of multinucleated polychromatic erythrocytes
(MPEs) scored per 1000 polychromatic erythrocytes (PCEs)
Toxicity Test:
Route: OralDose levels tested: 0, 0.16, 0.25, 0.40, 0.63, 0.80 g/Kg body wt.
Test animal: ICR-strain Swiss Webster mice
Parameters taken:
1. weight of mice
2. mortality/morbidity
3. gross morphological
anatomy of major internal
organs
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Mean body weight of male and female ICR-strain Swiss
Webster mice over the 14-d observation period
male female
Dose (g/Kg)
0 0 0 -
0.16 0 0 -
0.25 0 33 Ataxia, catalepsy
0.40 50 67
0.63 75 100
0.80 100 -
% Mortality Observed Toxidromes % Morbidity
Ataxia, catalepsy,
analgesia, dyspnea,
pilomotor erection,
fine body tremors
Ataxia, catalepsy,
analgesia, dyspnea,
pilomotor erection,
coarse body tremors,
cyanosis, loss of
screen grip,
anesthesia, convulsion
Lethal and non-lethal effects of trichosetin
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Gross anatomy of major internal organs from male
ICR-strain Swiss Webster mice
0 0.16 0.25 0.40 0.63
g /Kg
Gross anatomy of major internal organs from
female ICR-strain Swiss Webster mice
0 0.16 0.25 0.40 0.63
g /Kg
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Conclusion
Rec assay, Ames test and Micronucleus test showed that
trichosetin is not mutagenic.
Toxicity assay showed a dose-related increase in the
magnitude of biological response observed in the
toxidromes. Increasing doses results to decreasing body
weights; however, no dose-related changes were
observed in the gross morphological anatomy of major
internal organs.
LD 50 in mice= 0.40 g/ Kg; NOAEL= 0.16 g/Kg
Based on therapeutic index, it was found that
trichosetin has a wide margin of safety as an antibiotic
against MRSA.
Acknowledgements
UPLB Basic Research Program
Tonton Alad, Angeli Cocos, Bia Catibog, Lian Caluag and
Prof. Marilen Parungao Department of Biology, CAS, UP Manila
Danica Dimaya and Dr. Evangeline Amor
Institute of Chemistry, UP Diliman
Bureau of Animal Industry, Quezon City
Dr. Nelia Cortes-Maramba, Dr. Sid Sia and Prof. Susie Sio, Department
of Pharmacology, College of Medicine, UP Manila
Dr. Ernesto Balolong, Jr., NIH, UP Manila
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Related Publications:
1. Marfori, E.C., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2002. Trichosetin, a novel
tetramic acid antibiotic produced in the dual culture of Trichoderma harzianum and
Catharanthus roseus callus. Zeitscrift fur Naturforschung 57c: 465-470.
2. Marfori, E.C., Bamba, T., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2002.
Biosynthetic studies of the tetramic acid antibiotic trichosetin. Tetrahedron 58: 6655-
6658.
3. Marfori, E.C., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2003. Phytotoxicity of the
tetramic acid metabolite trichosetin. Phytochemistry 62: 715-721.
4. Marfori, E.C. and Malasa, A.B. 2007. Efficacy, mutagenicity and toxicity of trichosetin
as a new antibiotic for poultry. Phillipp. Agric. Scientist 90(2): 91-95.
5. Alad, P.O., Cocos, A.S., Balolong, E.C., Parungao, M.M. and Marfori, E.C. 2009.
Mutagenicity potential of the novel drug trichosetin estimated by using the rec assay
and micronucleus test. Philipp. J. Sci.138(2): 119-124.
6. Caluag, M.B., Catibog, I.D., Balolong, E.C., Parungao, M.M. , Cortes-Maramba, N.P.,
Sia, I.C., Sio, S.O. and Marfori, E.C. 2010. Toxicity of the novel drug trichosetin
(In Preparation)