isolation endophytic streptomyces from thai herb and screening
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StreptomycesTRANSCRIPT
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