isolation endophytic streptomyces from thai herb and screening

Isolation endophytic Streptomyces from Thai Herb and screening for anti-cancer activity from its metabolitesSaharat Jantaratrirat Department of Microbiology, Faculty of Science, Chulalongkorn University

Streptromyces sp.http://

• normal flora in soil

• Gram’s positive

• filamentous bacteria

• produce spores

• secrete a chemicals called “geosmens”

http://aspidisca.livejournal.com/10852.html

BIOACTIVE COMPOUND FROM Streptomyces sp.

• produce more than two-thirds of the clinically used natural antibiotics

• an important source of clinically important immunosupressants

• source of antitumor agents

http://www.popsci.com/science/article/2011-01/scientists-mine-dormant-bacterial-genes-new-antibiotics

Doxorubicin

• made by Streptomyces peucetius

• commonly used in the treatment of many different cancers.

http://www.procto.ro/doxorubicin/

Endophytic Streptomyces

http://aem.asm.org/content/69/7/4260/F1.expansion.html

cancer cells

http://www.unc.edu/depts/our/hhmi/hhmi-ft_learning_modules/cancermodule/pages/cancer.html

Liver cancer

Cancer in Thailand, Vol.V,2001-2003.2010

Leading cancer in Thailand (estimated), 2001-2003

ASR=Age-standardized incidence rates

Leading cancer in Thailand (estimated), 2001-2003

Cancer in Thailand, Vol.V,2001-2003.2010ASR=Age-standardized incidence rates

Thai herbs

Turmeric

Objectives

to screen Endophytic Streptomyces from turmeric

investigated the effect of Streptomyces ‘s metabolites on the growth of human hepatocarcinoma cell line

Outline

Isolation of Endophytic Streptomyces from turmeric

Verification isolated bacteria by 16S rDNA sequence analysis

test cytotoxic effects of metabolites on human hepatocarcinoma cell line(HepG2) by MTT assay

Isolation of Endophytic Streptomyces from turmeric

the turmeric were thoroughly washed in running water to remove all soil

surface sterilization

confirm the surface disinfection by roll plant tissues onto tryptic soy agar and incubate at 30oC

plate turmeric fragments on humic acid-vitamin B agar and incubate at 30oC

Verification isolated bacteria by 16S rDNA sequence analysis

extract genomic DNA(Li et al.,2007)

16S rRNA gene amplification by polymerase chain reaction

the PCR products were purified using QIAquick PCR Purification Kit

sequence 16s rDNA product

the sequence data were deposited in GenBank and compared with other Streptomycete gene sequences in GenBank

test cytotoxic effects of metabolites on human hepatocarcinoma cell line

extract metabolites by acetone and concentrate by a rotary vacuum evaporator

HepG2 cells are plated in 96-well plate incubate overnight

Treat with crude extracts at various concentrations for 4 days

Add MTT solution and incubate for 4 hours

Add 10%SDS and incubate overnight

the absorbance was measured using a microculture plate reader at 540 nm.

Isolation endophytic Streptomyces from Thai Herb and screening for anti-cancer activity from its metabolites

Saharat Jantaratrirat Department of Microbiology, Faculty of Science, Chulalongkorn University