Galangoflavonoid Isolated from Rhizome of Alpinia galanga ( L ) Sw ( Zingiberaceae )

Purpose: The purpose of this investigation was to isolate novel flavonoids from Alpinia galanga rhizomes. Methods: The methanol extract of Alpinia galanga was subjected to column chromatography and eluted with ethyl acetate-methanol (9:1) to yield a compound, galangoflavonoside (AG 11). The structure of the compound was elucidated by various spectral techniques (UV, IR, 1 H NMR, 13 C NMR, and MS). Results: Chemical investigation of the extract furnished a new flavonoid galangoflavonoside (AG 11). Conclusion: The isolated compound, galangoflavonoside (AG 11,) could be a potential therapeutic compound as well as serve as a lead compound in the synthesis of other useful flavonoids. Isolation of the galangoflavonoside from Alpinia galanga rhizomes is being reported, to the best of our knowledge, for the first time.


EXPERIMENTAL Plant material
The dried rhizomes of Alpinia galanga (Zingiberaceae), collected in Pusad Province of India, were identified by Prof. Anjula Pandey, a taxonomist of the National Bureau of Plant Genetic Resources, Pusa, New Delhi.A voucher specimen, no.EP-542, was deposited in the herbarium of the Natural Medicines Research Center of S.N.Institute of Pharmacy, Pusad, Amravati University.

Extraction and isolation
Dried, ground rhizome of Alpinia galanga (3000 g) was defatted with 5.5 L of petroleum ether (60-80 °C), and successively extracted with 7.5 L of methanol using Soxhlet apparatus.The methanol extract was evaporated on a waterbath to yield a dark brown solid (35 g), which was subjected to Si-gel column chromatography (100-120 mesh) and eluted with ethyl acetate:methanol (9:1) to give a compound weighing 47 mg.In the Shinoda test [8], 95 % ethanol, HCl and magnesium turning were added to compound AG 11 and boiled for 1 -2 min.A positive test is indicated by magenta colour.In the lead acetate test [8], lead acetate was added to compound AG 11, and a positive is indicated by yellow colour.

Acid hydrolysis of compound AG11
Compound AG11 (20 mg) was dissolved in 5 ml of 2M HCl:methanol (1:1) and refluxed for 1 h.The solvent was evaporated completely under vacuum.The residue was dissolved in water (5 ml) extracted with petroleum ether to separate oleic acid, and extracted with ethyl acetate (3 X 5 ml) to remove flavones and aglycones.The sugars present in aqueous phase were detected by TLC with the aid of standard samples of D-glucose (Rf = 0.12) and L-arabinose (R f = 0.18) and with nbutanol:acetic acid: water (4:1:5) as the top layer.

Melting point and spectroscopic analysis of Isolate
Melting point was determined in an open capillary with Perfit melting point apparatus and was uncorrected.IR spectrum was recorded on Jasco FTIR-550 spectrophotometer using a thin film supported on KBR pellets, and values were reported as ν max (cm -1 ).I H NMR and 13 C NMR spectra were recorded on a Bruker DPX 300 (Bruker) system using DMSO as solvent.The chemical shift values were reported as values in ppm relative to tetramethyl silane (TMS, δ = 0) as internal standard.The mass spectrum was generated on FAB-JEOL-MS 303 system (JEOL) mass spectrometer operating at 70 eV, by fast atomic bombardment technique, using xenon as the carrier gas.The purity of the isolated compound AG 11 was determined (by observation of a single spot in a UV and iodine chamber) on aluminium TLC sheets coated with Silica gel 60 F254 (0.2 mm thick, Hi Media), using EtOAc-MeOH (9.5:0.5)solvent system.

RESULTS
Following column chromatography over silica gel using ethyl acetate:ethanol (9:1), a pale yellow crystalline mass of compound (AG 11) was obtained.The compound (AG 11) was positive to Shinoda and lead acetate tests for flavonoid glycosides.The compound (AG 11) was pure as indicated by a single spot with an R f value of 0.63 in EtOAc-MeOH (9.5:0.).

CONCLUSION
The present study has isolated and characterized a new flavone, galangoflavonoside, from Alpinia galanga rhizomes for the first time.Further pharmacological investigations are underway to investigate the biological activity of the isolated compound