isolasi senyawa aktif antikanker dari daun gaharu
DESCRIPTION
Rancangan Penelitian untuk Isolasi Senyawa Aktif dari Ekstrak Metanol Daun Muda Gaharu Aquilaria microcarpa Hasil InokulasiTRANSCRIPT
MAKKY JANUARI MUKTI
ISOLATION AND ANTICANCER ACTIVITY TESTOF ACTIVE COMPOUND FROM METHANOL
EXTRACTS OF INOCULATED GAHARUAquilaria microcarpa LEAVES
G44124017G44124017
Supervised by:Drs Dudi Tohir, MS
Dr Erdy Santoso, MS
Introduction
Aquilaria microcarpa
Natural
ingredients
Gaharu as therapeutic agent
Endangered species
Gaharu leaves
• Extract or pure isolate
• Promising agent as a major component in the synthesis of anticancer drugs
• Studies revealed that gaharu has remarkable anticancer activity (Huda et al. 2009). The benzene extracts of the plant have central nervous system antidepression activities (Khalil et al. 2013)
• Rise in demand for gaharu resulted in irrational cutting of the tree trunk (CITES 2004)
• Research on gaharu leaves should be developed
• Minimize the species extinction by using the leaves
In addition to induce the formation of gaharu, fungal infections also increases the plant metabolism so the secondary metabolites distributed to other parts of the tree, especially the leaves (Dewi 2013)
Study on comparison of toxicity among various gaharu leaves has been done
LC50 of crude extract of inoculated gaharu A. microcarpa young leaves is 26.47 μg/mL (Dewi 2013)
Crude extract is potential as an anticancer agent if its LC50 is under 30 µg/mL (Ariffin 2009)
This study aimed at an isolation of an anticancer active compound from the leaves extract and evaluation on its activity against human breast cancer cell T47D
Objective
Time and Place
Time
• March – August 2014Place•Organic Chemistry Laboratory, Chemistry Department, Faculty of Mathematics and Natural Science, Bogor•Research and Testing Laboratory, Academy of Analytical Chemistry, Bogor•Primate Research Center, Bogor
Materials and Methods
Instruments
• Analytical balance
• Oven• ELISA reader• Incubator• Preparative
HPLC• Column
chromatography
• TLC plate• UV lamp• 96-well plate• Micropipette• Rotary
evaporator• Glassware
Materials
• Young leaves of an inoculated gaharu A. microcarpa
• Methanol• n-Hexane• Chloroform• Ethyl Acetate• Ethanol• Shrimp larvae• Tween 80• H2SO4 2 M• Mg ribbon• Mayer’s reagent
• Wagner’s reagent• Dragendor reagent• Lieberman-
Buchard’s reagent• FeCl3 1%• NaOH 10%• n-Amyl alcohol• Human breast
cancer cell T47D• RPMI 1640 and SDS• Doxorubicin and
MTT
Workflow scheme
Young leaves of an inoculated gaharu A. microcarpa
• Dried, milled
Simplicia• Extracted with
methanol• Concentrated by rotary
evaporatorCrude extract• Fractionated by column
chromatography
Fractions
• Toxicity test (BSLT)
The most active fractions• Phytochemical test• Profiled by HPLC• Isolated by preparative
HPLCIsolates• Anticancer activity test• Purity test
IC50 of pure isolate
Sample Preparation
Methods
Young leaves of an inoculated gaharu A. microcarpa
Dried
3 days, 50 °C
Milled
Simplicia
Water Content Determination (ASTM D2216 – 10)
Methods
Dried
30 min, 105 °C
Cooled
Weighed
Constant weight
Dried
30 min, 105 °C
Cooled
Weighed
Constant weight
1 g
Extraction (Maceration)
Methods
60 g(1 part)
Methanol(13 parts)
3 x 24 hours
Filtered and concentrated in rotary evaporator
Crude extract
Optimum eluent determination
Methods
Eluted by single eluent:
n-hexane, chloroform,
ethyl acetate, methanol, and
waterλ = 254 and 366
nm
The optimum
eluent
Spotting of crude extract
Mixed and made
variations of composition ratio in case obtained two
optimum eluent
The optimum composition of
eluent
Fractionation by Column Chromatography
Methods
1 g ofcrude extract
The optimum eluent obtained from TLC test
Applied to TLC in every
5 min
Spots detected under UV light λ = 254 and 366 nm
Eluates in similar patterns and Rf value of TLC are combined as a single fraction
All fractions obtained are tested to determine the toxicity of the most active fractions
Brine Shrimp Lethality Test (BSLT)
Methods
±100 mg shrimp eggs in a
container filled with seawater fed air by an aerator
hatched
10 shrimp larvae
Fractions are dissolved in
seawater, 2 drops of Tween 80 are
added
Stock Solution
2000 ppm
Concentration series 0; 100; 200; 300; 400; 500 ppm
2 mL
24 hours
incubation
under the light
Number of
death is counted
LC5
0
Phytochemical AnalysisAlkaloid Test
Methods
0.25 g sample
2.5 mL chloroform-ammoniafiltered
filtrates
Few drops of H2S04 2 M
shaked
2 layers
Acid layer (colorless)
Acid layer (colorless)
Mayer
Wagner
Dragendorf
White precipitated
Brown precipitated
Red-orange precipitated
Methods
Phytochemical AnalysisTriterpenoid and Steroid Test
0.1 g sample
5 mL ethanol
50 °C
filtered
filtratesevaporat
ed to dry
ether
Ether layer
Ether layer
air-dried
ether
Lieberman-Buchard
triterpenoid
steroid
red
blue-green
Methods
Phytochemical AnalysisTriterpenoid and Steroid Test
0.1 g sample
10 mL aquadest
5 min
boiled filtere
d
Foam persisted in 10 min
shaked
Saponin
FeCl3 1%
Black-green or dark blue
Tanin
Methods
Phytochemical AnalysisPhenolic and Flavonoid Test
0.1 g sample
15 mL aquadest
2 min
boiled filtere
d
Red
NaOH
10%
0.1 g Mg ribbon
1 mL alcohol-chlorhydrate5 mL n-amyl alcohol
Red
Yellow
Orange
Flavonoid test
Phenolic test
Active Compound Isolation and Purification
Methods
10 μL of the most active
fractionProfiled by HPLC
Analytical column
100 μL of the most active
fractionIsolation by HPLC
Preparative column
Conditioning: ODS column, gradient mobile phase of water-acetonitrile, fraction collector, FDA detector
Anticancer Activity Test by MTT
Methods
100 μL T47D
cell (2 x 104
cell/well) 96-well
plate
CO2 incubat
or
10 hRPMI
100 μL isolates
(12.5; 25; 50; 100; 200;
400 ppm) are added
CO2 incubat
or
24 h
24 h100 μL
MTT are added
CO2 incubat
or
4 hBlue formaza
nSDS
CO2 incubat
or
10 h CO2 incubat
or
18 h Absorbance is measured by ELISA reader, λ = 570 nm
Methods
Anticancer Activity Test by MTT
Absorbance is measured by ELISA reader, λ = 570 nm
IC50 of pure isolate
Timetable
Bibliography
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