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The anticancer activity of clove extract

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by

James Fong

on 25 April 2014

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Transcript of The anticancer activity of clove extract

The cervical cancer
Comparison of the Anti-cancer Activity of Clove Extract from Different Habitat
Group 12


Third most common cancer in women around the world.
In 2008, 529 000 new cases , over 85% occurred in developing countries.
Around 275 000 women died of this cancer, 88% of whom from developing countries.
cervical cancer around the world
Vaccine
effective but expensive
Anticancer drug and chemotherapy
expensive and side effect
common prevention and therapies
can not pay for expensive vaccine or therapies
Cervical cancer patient in developing countries
Clove
Eugenol a main aroma constituent of clove buds .

Main compound in clove extract
Anti proliferation activity against HeLa (cervical cancer), MCF-7 (ER + ve) and MDA-MB-231 (ER – ve) breast cancer, DU-145 prostate cancer and TE-13 esophageal cancer cell line in vitro has been proved.(Dwivedi and Shrivastava, 2011)
anticancer study of clove in India
The anticancer activity of clove in China
Use common and natural source Chinese Herbal Medicines
5,000 years of history
minimized the side effect
not expensive
popular in developing countries
(Asian and african countries)
solution
The chemical structure of eugenol
An indian spices
1000 years history in China
Different habitat
metabolism might change
anticancer activity may be different.

high morbidity and mortality rate compare to patient in developed countries
HeLa cell line

The oldest and most commonly used human cell line
Derived from cervical cancer cells
Adherent cell
Provide a in vitro model system for studying cervical cancer
Can be extracted by 70% ethanol .
Methodology
extraction of clove bud
clove bud
clove powder
milled
soxhelt extraction
(70% ethanol)
rotary evaporator
freeze dry
freeze dry product
Prepare different concentration of clove
extract
clove extract + 100% DMSO
Incubate overnight
Sonication for 10 minutes
syringe filtration
(0.2 µm)
Dilute to different concentration by serum free medium to target concentration(<1% DMSO)
MTT assay
Reflect cellular viability under different concentration of clove extract.

Measure the absorbance by a microplate reader at absorbance at wavelength 570nm
DNA fragmentation
after treatment
extraction the chromosome DNA in the cell
electrophoresis
check the DNA fragmentation
pattern under UV light source
One step RT-PCR
Use RT PCR to check the expression of apoptosis gene bax , anti-apoptosis gene bcl2 and internal control gene GAPDH.
extract total RNA of the cell
process one step RT-PCR
electrophoresis
check the band
under UV light source
Result
DNA fragmentation
cell viability
Apoptosis Analysis
what is apoptosis?
a process of programmed cell death that involves series of biochemical events.
Controlled by apoptosis gene bax , anti-apoptosis gene bcl2 and result in DNA fragmentation.

-VE:
negative control
sample 1:
clove grow in Yunnan
sample 2:
clove grow in Guangxi
cell morphology
Negative control with no clove extract
cell incubate with 2400µg/ml of clove extract for 24 hours
MTT assay result
One step RT-PCR
improvement
Extraction
use distilled water to prepare the aqueous extract
Prepare different concentration of clove extract
plot a standard curve about the
cell viability against DMSO concentration
MTT assay
Check the absorbance after the MTT formazan is completely dissolved
use rotating platform to help
dissolve the MTT formazan.
One step RT-PCR
check the RNA integrity before start the one step RT-PCR.
extract RNA at earlier time of incubation
Further improvement
High performance liquid hromatography
(HPLC) is needed to identify eugenol in
the extract.
In vivo experiment on testing animals, to further ensure the effectiveness of clove extract under metabolism of living animals.
expression of GAPDH and
Bcl-2 checking
conclusion
the anticancer ability of clove grow in Yunnan is more effective than clove grow in Guangxi
the anticancer mechanism is more likely to be necrosis rather than apoptosis
Full transcript