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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

G4412401 7

MAKKY JANUARI MUKTI

Introduction
Gaharu: resin, non-timber forest product, highly valuable in terms of perfumery, incense, and pharmaceutical industry Gaharu formed as an impact of defence mechanism of plants against diseases caused by fungal infections: phytoalexin production Main components: sesquiterpenes and chromones (Burfield 2005) Inoculation technology helps planning and speeding up the formation of gaharu through the induction of fungus on gaharu trees The best Indonesian gaharu trees: Aquilaria microcarpa, Aquilaria malaccensis, Gyrinops versteegii
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)

Objective
This study aimed at an Crude isolation of an extract is anticancer potential as active LC50 of crude an compound extract of anticancer from the inoculated agent if its leaves extract gaharu A. LC50 is microcarpa under 30 and young leaves g/mL evaluation on is 26.47 (Ariffin 2009) its activity g/mL (Dewi against 2013) human breast cancer cell

Study on comparison of toxicity among various gaharu leaves has

Time and Place


Time March August 2014 Place
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


Instrumen ts
Analytical balance Oven ELISA reader Incubator Preparative HPLC Column chromatography TLC plate UV lamp 96-well plate Micropipette Rotary evaporator

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 Mayers reagent Wagners reagent Dragendor reagent Lieberman-Buchards 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

The most active fractions

Simplicia
Extracted with methanol Concentrated by rotary evaporator

Phytochemical test Profiled by HPLC Isolated by preparative HPLC

Isolates
Anticancer activity test Purity test
IC50 of pure isolate

Crude extract
Fractionated by column chromatography

Fractions
Toxicity test (BSLT)

Methods

Sample Preparation

Drie d

3 days, 50 C

Milled

Young leaves of an inoculated gaharu A. microcarpa

Simplici a

Methods

Water Content Determination (ASTM D2216 30 min, 105 10) C


Dried
Coole d

Weighed

1g Dried

30 min, 105 C Coole d Weighed

Constant weight

Constant weight

Methods

Extraction (Maceration)
Methanol (13 parts)

3 x 24 hours

60 g (1 part)

Crude extrac t

Filtered and concentrated in rotary evaporator

Methods

Optimum eluent determination


Eluted by single eluent: n-hexane, chloroform, ethyl acetate, methanol, and water

Spotting of crude extract

= 254 and 366 nm

The optimum composition of eluent

Mixed and made variations of composition ratio in case obtained two optimum eluent

The optimum eluent

Methods

Fractionation by Column Chromatography


All fractions obtained are tested to determine the toxicity of the most active fractions

1 g of crude extract

The optimum eluent obtained from TLC test

Spots detected under UV light = 254 and 366 nm

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

Applied to TLC in every 5 min

Methods

Brine Shrimp Lethality Test (BSLT)

100 mg shrimp eggs in a container filled with seawater fed air by an aerator
hatche d

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

24 hours incubatio n under the light


2 mL

Number of death is counted

LC5
0

Stock Solution 2000 ppm

Concentration series 0; 100; 200; 300; 400; 500 ppm

Methods
Phytochemical Analysis Alkaloid Test

2.5 mL chlorofor mammonia filtere d Few drops of H2S04 2 M filtrates shake d

0.25 g sampl e

2 layers

Acid layer (colorless )

Mayer
Acid layer (colorless ) Wagner Dragendorf

White precipitated

Brown precipitated
Red-orange precipitated

Methods
Phytochemical Analysis Triterpenoid and Steroid Test

5 mL ethanol 0.1 g sampl e filtrates 50 C filtere d evaporate d to dry ether triterpenoid Lieberma nBuchard ether

Ether layer

Ethe r air-dried layer

red

steroi d

bluegreen

Methods
Phytochemical Analysis Triterpenoid and Steroid Test

10 mL aquadest 0.1 g sampl e boile d 5 min

shaked Foam persisted in 10 min

Saponi n

filtered FeCl
31%

Blackgreen or dark blue

Tanin

Methods
Phytochemical Analysis Phenolic and Flavonoid Test

15 mL aquadest 0.1 g sampl e boile d 2 min Phenolic test NaOH 10% filtered

Re d 0.1 g Mg ribbon 1 mL alcoholchlorhydrat 5 mL ne amyl alcohol Red Yellow

Flavonoi d test

Orang e

Methods

Active Compound Isolation and Purification


10 L of the most active fraction 100 L of the most active fraction Profiled by HPLC Analytical column

Isolation by HPLC

Preparative column

Conditioning: ODS column, gradient mobile phase of wateracetonitrile, fraction collector, FDA detector

Methods

Anticancer Activity Test by MTT


RPM I 10 h

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

CO2 incubato r

24 h

96-well plate

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

Blue formaza n

4h

CO2 incubator

100 L MTT are added

CO2 incubator

SDS
10 h

CO2 incubator

CO2 incubator

18 h

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

Bibliography
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