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OP-29

The Antioxidant Activity and Characterization of the Proanthocyanidin from

the Bark of Xylia xylocarpa (Roxb.) Taub by HPLC-ESI-MS

1 2 1,3
Salfarina Ramli , Ken-ichi Harada , Nijsiri Ruangrungsi
1
College of Public Health Sciences, Chulalongkorn University, Pathumwan, 10330 Bangkok, Thailand.
2
Graduate School of Environmental and Human Sciences & Faculty of Pharmacy, Meijo University, Tempaku, 468-8503
Nagoya, Japan.
3
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thai-
land

Rationale: Recent studies on antioxidants have enhanced the study on local and indigenous plants as an
alternative source of antioxidant in Thailand. Although traditionally known as antipyretic, no scientific study
has been reported about the bark of Xylia xylocarpa. The species has been undervalued as only materials for
making furniture. Therefore, investigation on the phytochemicals of the extract was carried out to provide
evidence for the claimed beneficial effect and exploitation of the plant.
Objective: To evaluate the antioxidant activity and to characterise the phytochemicals from the extract of
X. xylocarpa using High performance liquid chromatography (HPLC) coupled with Electrospray ionisation Mass
spectrometry (ESI-MS).
Methodology: Petroleum ether, dichloromethane and ethanol were used to consecutively extract the bark
X. xylocarpa using sohxlet apparatus. Sequential extraction with different polarity simplifies the chemical
complexity in the extract thus could be regarded as technique of sample clean up. Percentage yield were
calculated whereas, the ethanol extract was evaluated for the antioxidant activity by DPPH assay. Two hundred
μl of extracts (1 mg / ml) were filtered using an Ultrafree-MC membrane centrifugation filtration unit and 5 μl
of filtered extract was injected into a TSK-gel ODS-80Ts column (5 μm, 2.0 x 150 mm, TOSOH, Tokyo, Japan) at
o
40 C. The mobile phase consisted of water (A) and acetonitrile (B) both containing 0.1 % formic acid. The
gradient condition was 5 % - 60 % B for 35 minutes, with the flow rate of 200 μl / min. The LC separation was
carried out using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA). For both LC / MS
and MS / MS, 150 to 1000 of scan range were applied. The standards employed were gallic acid, epicatechin,
ellagic acid and epigallocatechin (50 μg / ml).
Results: The extract showed an excellent antioxidant activity with IC50 0.006 ± 0.003 μg / ml. From the
HPLC-ESI-MS, condensed tannin was found to be major compound of the bark. Proanthocyanidin dimer and
trimer were found and successfully characterized from the [M-H]- and comparison with previous literatures.
Calculated from the percentage yield, the bark of X. xylocarpa was found to be a good source of proanthocyanidin.
Conclusion: From the results and previously study, it could be deduced that the excellent antioxidant
activity showed by the bark extract of X.xylocarpa was contributed by the proanthocyanidin compound
characterised in the extract. HPLC-ESI-MS has been an indispensible tool for the characterization of the

phytochemicals.