Identification and isolation of methyl gallate as a polar ... - Mardi

Polar chemical marker for Labisia pumila
and verification purposes. Based on
European Medicines Agency (EMEA),
standardization of herbal product is defined
as herbal preparation containing a substance
or group of substances with known
therapeutic activity which can be used to
standardise a biological effect or used as
marker compounds (Zhari and Jamshed
2010). Therefore, identification of an active
chemical marker from L. pumila extract is
essential for standardization of L. pumila
based products.
The objective of this study was
to isolate a potential chemical marker
for L. pumila which can be used for
standardization and verification of the herbs.
In this study, methyl gallate was isolated
from L. pumila leaves and its spectroscopy
data were analysed to prove the identity of
the compound. In a previous study, methyl
gallate was demonstrated to have high
antioxidant activity and cytotoxic towards
cancer cell lines (Yong et al. 2005). Various
types of bioactivities were also reported
such as antimicrobial, sucrase inhibitor,
collagenase inhibitor and used as treatment
for enteritis (Chaubal et al. 2005).
Materials and methods
Sample preparation
Three kg of fresh L. pumila leaves were
collected from MARDI Kluang station and
ground to 2.0 mm particle size.
Preparation of methanol extract
The fresh L. pumila leaves were ground to
2.0 mm particle size and soaked in 100%
methanol (MeOH, which is close to aqueous
extract) for 4 days at room temperature
after which the extract was decanted (Mohd
Nazrul Hisham et al. 2010). The plant
material was replenished with fresh MeOH
and the same extraction procedure repeated
twice. The extracts collected from each
soaking were pooled and concentrated to
dryness using a rotary evaporator to obtain
the crude extract.
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Isolation of methyl gallate
The crude methanol extract of L. pumila
was then resuspended in distilled water
and solvent partitioned with hexane,
dichloromethane (CH 2 Cl 2 ), ethyl acetate
(EtOAc) and butanol (BuOH) (Yong et al.
2005). Ethyl acetate fraction (10 g) was
first subjected to normal phase column
chromatography (CC). Elution started with
100% chloroform (CHCl 3 ) followed by
mixtures of CHCl 3 :MeOH of increasing
polarity to yield a total of ten fractions.
Based on similar TLC patterns,
fractions 3 to 5 (4 g) were recombined and
subjected to normal phase open column
chromatography using 100% chloroform
(CHCl 3 ) as mobile phase, followed by
mixtures of CHCl 3 :MeOH of increasing
polarity. Ten fractions were collected, and
based on similar TLC patterns, fractions
7 to 10 (2.5g) were recombined and
subjected to open mini column normal phase
chromatography using 100% chloroform
(CHCl 3 ) as mobile phase, followed by
mixtures of CHCl 3 :MeOH of increasing
polarity to obtain 20 mg of white crystals.
Mass analysis
Mass spectra were recorded on a Shimadzu
GC-17A gas chromatography mass
spectrometer (GCMS). The GCMS was
equipped with a direct injection probe
(DI-probe) to analyse pure samples without
passing through the capillary column. The
pure isolated compound (in white crystal
form) was subjected to mass spectroscopy
analysis and the mass spectrum elucidated to
support the NMR analysis for determination
of the chemical structure.
Nuclear Magnetic Resonance (NMR)
analysis
The pure white crystals were placed on
top of the probe and subjected to 1 H, 13 C
NMR and two dimensional analyses such
as Heteronuclear Multiple Bond Correlation
(HMBC). The different spectrum of the
isolated compound were recorded on a
Varian Unity Inova (500 MHz) equipped