Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe-emodine, emodine, rheine, chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae).
BACKGROUND
Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the flora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of different methods of extraction.


OBJECTIVE
After a partial validation of an HPLC method for the simultaneous determination of five anthraquinones, aloe-emodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three different methods of extraction.


METHODS
Anthraquinones were extracted from the bark of R. alpinus using different techniques (methanol maceration, ultrasonic and supercritical CO(2) extraction). Separation and quantification of anthraquinones were accomplished using a reversed-phase C(18) column with the mobile phase of H(2)O-methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm.


RESULTS
All calibration curves were linear over the concentration range tested (10-200 mM) with the determination coefficients >or=0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All five anthraquinones were found in the samples tested at concentrations reported in experimental data.


CONCLUSIONS
The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantification of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays.
Genovese
S
S
Dipartimento di Scienze del Farmaco, Università degli Studi G. D'Annunzio Chieti-Pescara, Via dei Vestini 31, 66100 Chieti (CH), Italy. s.genovese@unich.it
Tammaro
F
F
Menghini
L
L
Carlucci
G
G
Epifano
F
F
Locatelli
M
M
D000880:Anthraquinones / Q000032:analysis; D002245:Carbon Dioxide / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D025924:Chromatography, Supercritical Fluid / Q000379:methods; D004642:Emodin / Q000031:analogs & derivatives / Q000032:analysis; D000432:Methanol / Q000737:chemistry; D024301:Plant Bark / Q000737:chemistry*; D010936:Plant Extracts / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D031955:Rhamnus / Q000737:chemistry; D014465:Ultrasonics
D000880:Anthraquinones; D010936:Plant Extracts; D002245:Carbon Dioxide; C059635:aloe emodin anthrone; C008905:physcione; D004642:Emodin; C027113:chrysophanic acid; D000432:Methanol; C020491:rhein