VIETNAM Department: School of Biotechnology I. PROJECT

VIETNAM
NATIONAL UNIVERSITY
INTERNATIONAL UNIVERSITY School of Biotechnology

——  ? ? ? —–

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BSc THESIS PROJECT
PROPOSAL Student’s name: TRAN THI THU HIEN

ID: BTBTIU14328

Department: School of Biotechnology

 

 

 

I.  PROJECT GENERAL INFORMATION

 

1.   NAME OF STUDY:

 

       Biological activities of
methanol extract from the fungus Talaromyces
sp in Taxus wallichiana

 

2.  
FIELD OF STUDY:

 

       Pharmaceutical biotechnology

 

3.  
SUPPERVISORS:

 

Asso.Prof. Nguyen
Hoang Khue Tu

 

4.  
HOST
INSTITUTE

 

Vietnam National
University HCMC – International University

 

5.  
TIME
 (from …to…)

 

      March 2nd
2018 to July 2nd 2018

 

6.  
BUDGET ESTIMATION: 10 million VND

 

 

II.      PROJECT CONTENTS

 

1.   AIM

 

Obtain
the biological activities, antimicrobial activities, cytotoxicity, anti-biofilm
of the methanol extracts from the
fungus Talaromyces.

2.   BACKGROUND

 

2.1. LITERATURE REVIEW.

 

Talaromyces sp is an important fungal genus in the family Trichocomaceae widely found in rural areas in most South Eastern
Asian countries, including Thailand, Vietnam, Laos and southern China in soil,
indoor environments. It is described in 1955 by American mycologist Chester Ray
Benjamin, as a sexual state of Penicillium
that produces soft walled ascomata covered with interwoven hyphae.1 Some
of the species are heat resistant. Some of the species are famous because of
their enzymes applicable in the synthesis of saccharides, preparation of chiral
building blocks or biotransformations, and for its application in pest
biocontrol. Many of its species are used in food and agricultural production.
Interestingly, members of this genus have attracted the attention of chemists
and biologist on their secondary metabolites. 2

 

Scientists currently found that there are several strains of genus Talaromyces. Among of these, T. flavus is the commonest strain of the
genus Talaromyces. 3 T.flavus, an extremely variable species,
has been found in soils and on organic materials undergoing slow decomposition.
It also found in foods, probably as a contaminant rather as a spoilage fungus.
Some Talaromyces pathogens are known
such as T. marneffei generalized
papular skin eruptions among HIV-infected, 4 T. piceus caused both
fungaemia and rib osteomyelitis in an X-linked chronic granulomatous disease
(X-CGD) patient, the lungs and sputum of immunocompromised patients can be
infected by T. amestolkiae and T. stollii 5,6

 

Endophytic fungi have a great influence on plant health and growth, and
are an important source of bioactive natural compounds with a wide variety of
chemical structures and diverse biological activities. Among them, the fungi in
the genus Talaromyces have produced
various secondary metabolites with interesting biological activities and novel
structures including sesquiterpenes, macrolides, and cyclic peptides. Bioactive
azaphilone derivatives have been isolated from the the genus Talaromyces, exhibit a range of
biological activities, including antibacterial, antifungal, antioxidant,
cytotoxic, and anti-inflammatory 7. To understand clearly, several
studies have been conducted during recent years. In June 2014, Alexander Pretsch did the research on Talaromyces wortmannii. He saw that this
fungus extracts showed both antibacterial activity especially against P. acnes
and also anti-inflammatory properties byinhibiting TNF-a-induced ICAM-1
expression and P. acnes-induced IL-8 release 8. Next year, in
2015, Talaromyces Radicus, isolated
from Catharanthus Roseus, produces
vincristine and vinblastine, which induce apoptotic cell death 9.
Moreover, in 2016, another strain of Talaromyces,
Talaromyces flavus SP5 in the marine
sediment of Southern Coast of India also acted as a potent antimicrobial,
antifungal, and anticancer agent, providing basic information on the potency of
marine fungi towards biomedical applications and further investigation may lead
to the development of novel anticancer drugs 10. At the same year,
bioactive Azaphilone derivatives from the Talaromyces
aculeatus in the China by Jie Ren
exhibited stronger inhibitory activity against ?-glucosidase than the positive
control acarbose (IC50 = 101.5 ?M), with IC50 values of 78.6 and 22.9 ?M.
Nearly, in December 2016, three bioactive metabolites: the siderophore
ferrirubin, the platelet-aggregation inhibitor herquline B and the antibiotic 3-O-methylfunicone
obtained from Strain F36CF of Talaromyces pinophilus extracts isolated
from strawberry tree helped the development of 
new biopesticide formulations11.

 

According Talaromyces extracts
research, Talaromyces endophytic
fungi can live in specific host plant to give the good metabolite product,
including Taxus tree. Taxus are one type of the host plants that are
distribution commonly in Lam Dong province in Viet Nam. The symbiotic
relationship between Taxus and Talaromyces
give researchers many interesting, especially in their extract and biological
activities. In the present study, biological activities of methanol extract
from Talaromyces are demonstrated.

 

Methanol, also known as methyl alcohol among others, is a chemical with
the formula CH?OH. It contains a methyl group linked to a hydroxyl group and its
polarity is too high compare with another substance like hexane or
toluene.  In this project, it is used as
a solvent in Talaromyces extract to
get a compound which is dissolvent in methanol without dissolving in other
solvent, later derived to test biological activities of this compound.

 

 

2.2. WHY THIS PROJECT NEED TO BE CARRIED
OUT

 

In recent years, the use of natural products has been the single most
successful strategy in the discovery of novel medicines. Therefore, more and
more researches are carried out based on the interaction between plant and
fungi to find out specific product as anti-cancer drugs through biochemical
pathway of fungi in plant. To be specific, the symbiotic relationship between
Taxus and Talaromyces are also as
promise for useful extracted compound that need to research more in the future.
Until now, although the researches on Talaromyces
extract with specific biological activities also have, they are too little. Therefore,
in this study, the methanol extract from fungus Talaromyces in Taxus with expressing biological activities need to
focus on.

                                                

 

2.3. THE CONTRIBUTION OF THIS PROJECT
INTO SCIENCE

 

The
discovery of novel medicines is very important in prevent and treatment
many diseases, so the methanol extract from fungus Talaromyces will produce at least one hydrophilic compound
(dissolve in methanol or water). This leads to further drug formulation.

 

    III.CONTENTS

 

3.1. HYPOTHESIS

 

Methanol
extract from the fungus Talaromyces
can give biological activity such as antimicrobial activities, cytotoxicity and
anti-biofilm, etc.

 

 

3.2. PROCESS OF PROJECT

 

(Flowchart
to show the process of the project is
required)

 

 

 

Cytotoxicity of purified
substance by Sulforhodamine
B assay

 

Antimicrobial test of the
purified substance by agar plate
diffusion

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.2. DETAIL CONTENTS:

 

3.2.1. Part I: Cultivation and optimization the
fungus Talaromyces

Material:

Malt extract

D-glucose

Yeast extract

Peptone

The spore
stock solution of Talaromyces was
kept at -80oC

Methods:

Talaromyces spore stock solution is inoculated into duran (1000mL) or Erlenmeyer
flask (1000mL) that contain the M1 broth medium. The fungus is cultured in 7-14
days at room temperature (25oC) under gently shaking.

3.2.2. Part II: Antimicrobial test of culture
collected after culturing

Material:

Peptone

Yeast extract

Sodium Chloride

Agar

Autoclave Machine

Bio Safety Cabinet

Micropipettes and tips

Test tubes

Incubator

Duran 500ml

Distilled water

Gram stain

Methods:

 Preparation
of bacterial strain:

 

The pathogens are used in this step, they are,
Salmonella typhi, Pseudomonas aeruginosa,
Staphylococcus sciuri, Serratia marcescens, Staphylococcus aureus, Vibrio
Harveyi, Micrococcus Luteis, Vibrio paraheamolyticus,.

Pathogens are grown in 5ml of LB broth medium
at 370C in 24 hours under aerobic conditions. Perform gram staining to check the formation and
contamination of the bacteria.

 Antimicrobial test the culture collected with
these strains of bacteria:

The cultured broths of Talaromyces are transferred into 50ml falcons. The supernatants are
harvested by centrifuging the culture at 10000 rpm for 10 minutes at 25oC.
Then, the supernatant are collected for antimicrobial test.

The antimicrobial test will be performed by agar
diffusion method. The M1 medium is used as the negative control. Amount of
pathogen cultures is spread onto the plate, using the sterile swab. Applying
aseptic technique, use a small sterile metal stick to punch into the LB agar
plate to form the wells. The supernatant is pipetted into the wells. All the
plates are incubated at room temperature. After 10-12 hours, measure the
inhibition zones around the wells.

 

3.2.3. Part III: Prepare methanol extract and
its activities:

 

Material:

Methanol

Chloroform

Falcon
50ml

Peptone

Yeast extract

Sodium Chloride

Agar

Autoclave Machine

Bio Safety Cabinet

Micropipettes and tips

Test tubes

Incubator

Duran 500ml

Distilled water

Gram stain

Silica gel

Column

TLC plate

Methods:

·        
Prepare
methanol extract:

The supernatants extract from Talaromyces sp cultures are concreted and
dissolved with methanol, then concreted again.

 

·        
Methanol extract
activity:

a.      Biological activities test :

–         
Antimicrobial test:
perform the same with part II

–         
Cytotoxicity test : perform
by Sulforhodamine B assay with Hep
G2 cell line

The cytotoxic percentage is calculated
by formula:

I%=1-(OD
live cell/OD initial cell) x 100%

b.      Perform Thin Layer Chromatography
(TLC) :

The methanol
extract was used as sample for performing TLC with three different ratios of chloroform: methanol (1:1, 1:2, 2:1) ?
choose the good ratio of solvent that help further getting a purified
substances.

c.       Purification by using Column
Chromatography (CC)

The crude
methanol extracts are dissolved into silica gel with suitable ratio, then pour
this mixture into the column and start to run with the ratio of solvent that
choose before.

Next, the eluted
substances/ fractions are collected into eppendorf. All the fractions (from F1
to Fn) are checked with TLC until getting the purified compound/
ideal fraction.

The purified
compound is storage to further analysis such as determining the chemical
formula and characteristics as well.

 

 

 

 

3.3. REFERENCES

 

1.     N. Yilmaz, C.M. Visagie, J.
Houbraken, J.C. Frisvad, and R.A. Samson, Polyphasic taxonomy of the genus
Talaromyces, 2014, Studies on Microbiology, 78, 175–341

2.     Zhai MM, Li J, Jiang CX, Shi
YP, Di DL, Crews P, Wu QX, The Bioactive Secondary Metabolites from Talaromyces
species, Nat Prod Bioprospect., 2016 Feb;6(1):1-24

3.     Yi Zang, Gre?gory
Genta-Jouve, Alexandre E. Escargueil,Annette K. Larsen, Laura Guedon, Bastien
Nay, and Soizic Prado, Antimicrobial Oligophenalenone Dimers from the Soil
Fungus, Journal of Natural Products, 2016, 79 (12), pp 2991–2996

4.     Maniar JK, Chitale AR,
Miskeen A, Shah K, Maniar A, penicillium marneffei infection: An AIDS-defining
illness, Indian J Dermatol Venereol Leprol 2005, 71, 202:4

5.     Weisenborn JLF, Kirschner R,
C aceres O, et al. (2010). Talaromyces indigoticus, Takada & Udagawa, The
first record from Panama and the American Continent. Mycopathologia 170:
203–208

6.     Santos PE, Piontelli E, Shea
YR, et al. (2006). Penicillium piceum infection: diagnosis and successful
treatment in chronic granulomatous disease, Medical Mycology 44: 749–753

7.     Jie Ren, Shuang-Shuang Ding,
Ao Zhu, Fei Cao, and Hua-Jie Zhu, Bioactive Azaphilone Derivatives from the
Fungus Talaromyces aculeatus, J. Nat. Prod. 2017, 80, 2199?2203

8.     Pretsch A, Nagl M,
Schwendinger K, Kreiseder B, Wiederstein M, et al. (2014) Antimicrobial and
Anti-Inflammatory Activities of Endophytic Fungi Talaromyces wortmannii
Extracts against Acne-Inducing Bacteria. PLoS ONE 9(6): e97929.
doi:10.1371/journal.pone.0097929

9.     Palem PPC, Kuriakose GC,
Jayabaskaran C (2016) Correction: An Endophytic Fungus, Talaromyces radicus,
Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which
Induce Apoptotic Cell Death. PLOS ONE 11(4): e0153111.
https://doi.org/10.1371/journal.pone.0153111

10.   Bibin G. Anand, C. K. Navin
Thomas, S. Prakash, In vitro cytotoxicity and antimicrobial activity of
Talaromyces flavus SP5 inhabited in the marine sediment of Southern Coast of
India, Chinese Journal of Natural Medicines 2016, 14(12): 0913-0921

11.   F. Vinale, R. Nicoletti, F.
Lacatena, R. Marra, A. Sacco, N. Lombardi, G.d’Errico, M. C. Digilio, M. Lorito
& S. L. Woo (2017): Secondary metabolites from the endophyticfungus
Talaromyces pinophilus, Natural Product Research, 2017,
http://dx.doi.org/10.1080/14786419.2017.1290624

 

IV.
PROGRESS AND
EXPECTED RESULTS

 

Contents

 
Expected results

 
Time

Part I :
Cultivation and optimization the fungus Talaromyces

 

March 2nd
to March 15th

Part II: Antimicrobial test of culture collected after culturing

 Appear the zone around the well

 

Experiment 1: Preparation of bacterial strain

 all bacteria strains growth with OD=
0.08-0.1

March 15th
to March 16th

Experiment 2 : Antibiotics test

Appear the
zone around the well

March 17th
to March 18th

Part III: Prepare methanol
extract and its activities

 

March 19th
to June 29th

Experiment 1: Biological activities test

 show the antimicrobial activity

March 19th
 to April 20th

Experiment 2: Perform Thin
Layer Chromatography (TLC)

 

April 21st
to April 22nd

 Experiment 3: Purification by using Column Chromatography (CC)
 

 get purify compound

April 23th to
June 29th

 

 

V. SUPERVISOR’S 
APPROVAL

 

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