Isolasi Senyawa Aktif Antikanker dari Daun Gaharu

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

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