ISOLATION AND ANTICANCER ACTIVITY TEST

OF ACTIVE COMPOUND FROM METHANOL

EXTRACTS OF INOCULATED GAHARU
Aquilaria microcarpa LEAVES

Supervised by:
Drs Dudi Tohir, MS
Dr Erdy Santoso, MS

G441240

MAKKY JANUARI MUKTI

Introduction

Aquilaria microcarpa

 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

Study on comparison of toxicity among various

Objective

Time and Place

Time
Place

Materials and Methods

Instrume
nts

Analytical
balance
Oven
ELISA reader
Incubator
Preparative
HPLC
Column
chromatograp
hy
TLC plate
UV lamp
96-well plate
Micropipette
Rotary

Material
s
Young leaves of an inoculated gaharu A.
microcarpa
Methanol
n-Hexane
Chloroform
Ethyl Acetate
Ethanol
Shrimp larvae
Tween 80
H2SO4 2 M
Mg ribbon
Mayers reagent
Wagners reagent
Dragendor reagent
Lieberman-Buchards reagent

Workflow scheme

Methods

Sample Preparation

Drie
d

Young leaves of
an inoculated
gaharu A.
microcarpa

3 days, 50
C

Mille
d

Simplic
ia

Methods

Water Content Determination (ASTM

min, 105
D2216 30
10)
C
Dried

1
g

Coole
d

Weighed

Constant
weight

30 min, 105
C
Dried

Coole
d

Weighed

Constant
weight

Methods

Extraction (Maceration)
Methanol
(13 parts)

60 g
(1
part)

3 x 24
hours

Crud
e
extra
ct
Filtered and
concentrated in rotary
evaporator

Methods

Optimum eluent determination

Spotting of crude
extract

The optimum
composition of
eluent

Eluted by
single eluent:
n-hexane,
chloroform,
ethyl acetate,
methanol, and
water
Mixed and
made
variations of
composition
ratio in case
obtained two

= 254 and 366

nm

The
optimum
eluent

Methods

Fractionation by Column
Chromatography
All fractions obtained are tested to

1 g of
crude
extract

The optimum
eluent
obtained from
TLC test

determine the toxicity of the most

active fractions

Spots
detected
under UV
light =
254 and
366 nm
Applied to
TLC in every

Eluates in similar
patterns and Rf
value of TLC are
combined as a
single fraction

Methods

Brine Shrimp Lethality Test (BSLT)

100 mg shrimp
eggs in a
container filled
with seawater fed
air by an aerator
hatch
ed

10
shrimp
larvae
Fractions are
dissolved in
seawater, 2 drops
of Tween 80 are
added

24
hours
incubati
on
under
the light

Number
of
death is
counted

2
mL

Stock
Solution
2000 ppm

Concentratio
n series 0;
100; 200;
300; 400;
500 ppm

LC5
0

Methods

Phytochemical Analysis
Alkaloid Test

0.25
g
samp
le

Acid
layer
(colorles
s)

2.5 mL
chlorofo
rmammoni
a
filtere
d

Few drops
of H2S04 2
M
filtrate
s

shake
d

Maye
r

White
precipitated

Wagne
r

Brown
precipitated

Dragendo
rf

Red-orange
precipitated

2
layers

Acid
layer
(colorles
s)

Methods

Phytochemical Analysis
Triterpenoid and Steroid Test

5 mL
ethanol
0.1 g
samp
le

Ethe
r
airlaye dried
r

50
C

ethe
r

filtere
d

Lieberm
anBuchard

filtrate
s
evaporat
ed to dry
ethe
r

Ethe
r
laye
r

triterpeno
id

red

steroi
d

bluegreen

Methods

Phytochemical Analysis
Triterpenoid and Steroid Test

0.1 g
samp
le

10 mL
aquades
t
boile
d5
min

shake Foam
persisted
d
in 10 min

Saponi
n

filtere
d
FeCl
1
%

Blackgreen
or dark
blue

Tanin

Methods

Phytochemical Analysis
Phenolic and Flavonoid Test

0.1 g
samp
le

15 mL
aquades
t
boile
d2
min

Phenoli
c test
NaOH
10%

filtere
d

Flavono
id test

Re
d
0.1 g Mg
ribbon

Re
d

1 mL
alcoholchlorhydra
5 mL nte
amyl
alcohol

Yello
w
Orang
e

Methods

Active Compound Isolation and

Purification
10 L of
the most
active
fraction

Profiled
by HPLC

Analytical
column

100 L of
the most
active
fraction

Isolation
by HPLC

Preparative
column

Conditioning: ODS column,

gradient mobile phase of
water-acetonitrile, fraction
collector, FDA detector

Methods

Anticancer Activity Test by MTT

100 L
T47D
cell (2 x
104
cell/well
)

RPM
I

Blue
formaza
n

10 h

96-well
plate
4h

CO2
incubat
or

SDS

CO2
incubat
or

10 h

CO2
incubat
or

CO2
incubat
or

100 L
MTT are
added
18 h

100 L
isolates
(12.5; 25; 50;
100; 200;
400 ppm) are
added

24 h

24
h

CO2
incubat
or

Absorbance is
measured by ELISA
reader, = 570 nm

Methods

Anticancer Activity Test by MTT

Absorbance is
measured by ELISA
reader, = 570 nm

IC50 of pure
isolate

Timetable

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THANK YOU