Preparative Separation of Garcinia kola Heckel., Biflavonoids by Conventional and pH-Zone-Refining Counter-Current Chromatography
Garcinia kola (Family Clusiaceae) is a highly valued ingredient in African traditional medicine and is officially listed in the African Pharmacopoeias. A number of herbal products derived from G. kola seeds has been manufactured and marketed as dietary supplements. Several experimental studies have demonstrated that G. kola has significant pharmacological properties. These properties include antimicrobial, hepato-protective, anti-inflammatory and antidiabetic activities. The major bioactive principles are recognized to be biflavonones (BFs), prenylated benzophenones, xanthones and kolaviron. Thus, there is need for scale up isolation of the above-listed constituents for further studies. Two modes of type-J High-Speed Counter-Current Chromatography HSCCC were applied to scale-up the isolation of five BFs from G. kola seeds extracts. The conventional (HSCCC) was performed in an optimized two-phase solvent system composed of HEMW (3:5:3:5) with 10 – 500 mg of the methanol extract successfully separated during 10h. Using the new Spiral Tube Support (STS) , pH-Zone-refining CCC was performed with two-phase solvent systems composed of methyl-tert-butyl-ether-acetonitrile-water (4:1:5) and HEEW (7:10:7:10 v/v) where TFA (˜12mM) was added to the upper organic stationary phase as a retainer and NH3 (35 mM) and NaOH (10mM) to the aqueous mobile phases as eluters. The results revealed that pH-zone-refining technique yielded even more BFs that were separated at shorter elution time. From 5.0 g of methanol seed extract; five main BFs (GB-I-glucoside, GB-1a, GB-1, GB-2 and kolaflavonone were successfully separated with purities ranging from 95 – 98% as determined by HPLC. In comparison to conventional CCC, the results revealed that the new STS system yielded high partition efficiency with the best stationary phase retention and a short elution time. References:  Yang Y, Aisa HA, Ito Y (2009)J Chromatogr A, 1216(27): 5265 – 5271.