Chromatographic approaches for the characterization of stem bark extracts from phyllanthus muellerianus (Kuntze) excel.

INTRODUCTION [The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat kidney and urinary bladder disturbance, intestinal infections, diabetes and hepatitis B [1]. Phyllanthus muellerianus (Kuntze) Excell is a medicinal plant widespread in the tropical region of West Africa and the Baka pygmies from Cameroon prepare a water decoction of Phyllanthus muellerianus stem bark which is used as a remedy for tetanus and wound infections [2,3].] In our previous work [4] the antimicrobial properties of P. muellerianus stem bark methanol extract have been demonstrated against different micro-organisms such as Clostridium sporogenes, Streptococcus mutans and Streptococcus pyogenes. Bioassay-guided isolation together with preliminary phytochemical investigations suggested the presence of a basic fraction responsible for the biological activity. MATERIALS AND METHODS [Amberlite IR120-H resin was used for a preliminary purification process. HPLC method based on RP-18 analytical column with a binary mobile phase water/acetonitrile (TFA 0,05%) under gradient elution. A second analytical method based on a cation-exchange column with a binary mixture of 80% ammonium acetate buffer (20 mM) and 20% acetonitrile was developed. The separation procedure was carried out under isocratic elution at a flow rate of 0.7 ml/min. UV detection was fixed at 254 nm for both methods.] RESULTS AND DISCUSSION [The basic fraction has been preliminary purified by means of a 'catch and release' process: the crude methanol extract was dissolved in methanol and Amberlite IR120-H resin was added. The purification process was followed by HPLC on RP-18. The chromatographic analysis of the basic fraction, after releasing from the resin by treatment with ammonia solution, highlighted the success of the purification procedure. On the other hands, ion-exchange interactions between the basic analytes and the residual acidic silanol on the surface of the stationary phase provoked poor separation and massive peak tailing. Thus, a different analytical method was developed , based on a cation-exchange analytical column, as described in a previous section, in order to improve the separation.] CONCLUSIONS [Based on our experiments, advantages and limitations of the employed analytical methods will be discussed.