6/15/2010

Evaluation of Toxicity and Mutagenicity of

Trichosetin, an Anti-MRSA Antibiotic Produced from
the Dual Culture of Catharanthus roseus Callus and
Trichoderma harzianum

EUFROCINIO C. MARFORI, Ph.D.

University Researcher, BIOTECH, UPLB

Presentation of Completed Researches

UPLB DA-BAR RDE Network Headquarters, UPLB, College, Laguna. March 30, 2010

OH

O
NH

O
O

Trichosetin

(Marfori et al., 2002. Z. Naturforsch. 57c: 465-470)

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Methanol Vancomycin

Trichosetin

Diwa and Peji (2005) established the antimicrobial activity

of trichosetin against local clinical MRSA isolates.

-minor skin infections

Methicillin-resistant Staphylococcus aureus

(MRSA)
-life-threatening diseases
such as pneumonia,
meningitis, endocarditis,
toxic shock syndrome and
septicemia.

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HO H2N HO
HO

O
CH2OH
O

O Cl

O O

HO
Cl
O
H
N O H
O H
N N N
H
O NH
HO O

NH2 O
O
O

OH
HO OH
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
Trichoderma harzianum

C. roseus callus dual culture

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Isolation of trichosetin
EtOAc fraction
Dual culture (10 kg)
open- column chromatography
soaked in 10 L MeOH overnight
34.5 cm x 1.6 cm column
MeOH extract (100.11 g) 30 g deactivated silica
resuspended in 500 ml dH2O eluted with solvents of
adjusted to pH 4.0; extracted increasing polarity
with 500 ml n-hexane (3x)
Fraction No. 1- Toluene
hexane Aqueous Fraction No. 2- Toluene: Acetone (9:1)
layer layer Fraction No. 3- Toluene: Acetone (8:2)
extracted with 500
ml EtOAc (3x) Fraction No. 4- Toluene: Acetone (7:3)
Fraction No. 5- Toluene: Acetone (6:4)
Aqueous EtOAC extract Fraction No. 6- Acetone
(2.82 g) Fraction No. 7- Methanol (145.2 mg)

rec Assay

rec Assay- used to determine DNA-damaging capacity of a compound

• involves the use of two strains of

Bacillus subtilis :
 recombination-proficient strain H17 Rec+
 recombination-deficient strain M45 Rec-

• M45 Rec- is much more sensitive to

agents 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 Prepared plates for the rec assay

Placing the paper disks containing

Streaking of the test strains
trichosetin

10 ppm 100 ppm 1000 ppm quinoline methanol

trichosetin trichosetin trichosetin (positive control) (negative control)

Length of inhibition, mm
Treatment
H17 Rec+ M45 Rec-

Trichosetin, 10 ppm
Trichosetin, 100 ppm
Trichosetin, 1000 ppm
Methanol
4-nitroquinoline oxide
0 0
0 0
0 0
0 0
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 Blood smearing

fetal calf serum

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Mean number of multinucleated polychromatic erythrocytes

(MPEs) scored per 1000 polychromatic erythrocytes (PCEs)

Treatment No. of MPEs

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

Note: A >2-fold increase in the frequency of micronuclei indicates that the

substance is mutagenic.

Toxicity Test:

Test animal: ICR-strain Swiss Webster mice

Route: Oral
Dose levels tested: 0, 0.16, 0.25, 0.40, 0.63, 0.80 g/Kg body wt.
Parameters taken:
1. weight of mice
2. mortality/morbidity
3. gross morphological
anatomy of major internal
organs

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male female

Mean body weight of male and female ICR-strain Swiss

Webster mice over the 14-d observation period

Lethal and non-lethal effects of trichosetin

Dose (g/Kg) % Mortality % Morbidity Observed Toxidromes

0 0 0 -
0.16 0 0 -
0.25 0 33 Ataxia, catalepsy
0.40 50 67 Ataxia, catalepsy,
analgesia, dyspnea,
pilomotor erection,
fine body tremors
0.63 75 100 Ataxia, catalepsy,
analgesia, dyspnea,
pilomotor erection,
coarse body tremors,
cyanosis, loss of
screen grip,
anesthesia, convulsion
0.80 100 -

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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
Dr. Ernesto Balolong, Jr., NIH, UP Manila
Danica Dimaya and Dr. Evangeline Amor
Institute of Chemistry, UP Diliman
Dr. Nelia Cortes-Maramba, Dr. Sid Sia and Prof. Susie Sio, Department
of Pharmacology, College of Medicine, UP Manila
Bureau of Animal Industry, Quezon City

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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)

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