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G4412401 7
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
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
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
Simplicia
Extracted with methanol Concentrated by rotary evaporator
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
Simplici a
Methods
Weighed
1g Dried
Constant weight
Constant weight
Methods
Extraction (Maceration)
Methanol (13 parts)
3 x 24 hours
60 g (1 part)
Crude extrac t
Methods
Mixed and made variations of composition ratio in case obtained two optimum eluent
Methods
1 g of crude extract
Eluates in similar patterns and Rf value of TLC are combined as a single fraction
Methods
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
LC5
0
Methods
Phytochemical Analysis Alkaloid Test
0.25 g sampl e
2 layers
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
red
steroi d
bluegreen
Methods
Phytochemical Analysis Triterpenoid and Steroid Test
Saponi n
filtered FeCl
31%
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
Flavonoi d test
Orang e
Methods
Isolation by HPLC
Preparative column
Conditioning: ODS column, gradient mobile phase of wateracetonitrile, fraction collector, FDA detector
Methods
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
CO2 incubator
SDS
10 h
CO2 incubator
CO2 incubator
18 h
Methods
Timetable
Bibliography
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