GC-MS Determination of Isoflavonoids in Seven Red Cuban Propolis Samples MERCEDES CAMPO FERN ´ ANDEZ, † OSMANY CUESTA-RUBIO, † AR ´ ISTIDES ROSADO PEREZ, ‡ RODNY MONTES DE OCA PORTO, § INGRID M´ ARQUEZ HERN ´ ANDEZ, † ANNA LISA PICCINELLI, | AND LUCA RASTRELLI* ,| Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana, Avenida 23, 21425 La Lisa, CP 13600, Ciudad de La Habana, Cuba; Estacio´n Experimental Apı ´cola, El Cano, Cuba; Laboratorio Antidoping, Instituto de Medicina del Deporte, Calle 100 y Aldabo´, CP 10800, Ciudad de La Habana, Cuba; and Dipartimento di Scienze Farmaceutiche, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy In the present study, the phenolic composition analysis of seven red varieties of propolis, collected in different regions of Cuba, was evaluated by gas chromatography/mass spectrometry (GC-MS). Seventeen compounds were identified in all samples by the interpretation of their mass spectra. This appears to be the first report on the GC-MS analysis of isoflavonoids in the propolis. The results confirmed the presence of the main isoflavonoids isolated previously and suggested the general structure for the other five isoflavonoids. Vestitol, 7-O-methylvestitol, and medicarpin were present in high amounts in all propolis samples analyzed. This result indicates that propolis samples rich in isoflavonoids are not exclusively found in Pinar del Rı ´o province and proves that GC-MS technique is a useful and alternative tool for the chemical analysis of tropical red propolis. KEYWORDS: Red Cuban propolis; isoflavonoids; pterocarpans; GC-MS INTRODUCTION Isoflavonoids constitute a distinct group of plant natural products derived from the phenylpropanoid pathway. They are abundant in soybeans and other leguminous plants. These compounds have diverse biological activities and play numerous roles in the interaction between plants and the environment. In man, dietary intake of isoflavonoids has been associated with lower incidences of hormonally dependent cancers (1, 2), relief from symptoms of postmenopausal problems (3), and a reduction in the risk of osteoporosis (4) and cardiovascular disease (5). Thus, there is considerable interest in this class of natural compounds as health-promoting agents. Propolis contains sticky plant substances mixed with beeswax and other bee secretions, and its chemical composition is qualitatively and quantitatively variable, depending on the vegetation in the area from which it was collected (6). Because of the geographical differences, propolis samples from Europe, South America, and Asia have different chemical compositions. Propolis from Europe and China contains many kinds of flavonoids and phenolic acid esters (7). In contrast, the major components in propolis of Brazilian origin are terpenoids and prenylated derivatives of p-coumaric acids (8 -10). In our previous studies, we reported for the first time the occurrence of isoflavonoids and pterocarpans in a red variety Cuban propolis sample, suggesting new biological potentialities for this natural product (11). Subsequently, we demonstrated that the coupling of liquid chromatography (LC) with ultraviolet (UV) and/or electrospray ionization (ESI) mass spectrometry was a powerful tool for the identification and quantification of isoflavonoids and pterocarpans in red Cuban propolis (12). GC- MS determination has appeared to be a useful method to assay for phytoestrogens (13) and this approach has been used successfully in the detection of many isoflavonoids and their related glucoside in red and white clover (14) and in plant- derived foods (15). GC-MS was also reported for the analysis of propolis (16) and the determination of volatile constituents of propolis from various regions of Greece (17, 18). In this study, the suitability of gas chromatography/mass spectrometry (GC-MS) was evaluated as technique for deter- mination of isoflavonoids in red Cuban propolis. Seven red- type propolis samples (A-G) collected in different regions of Cuba were analyzed to investigate possible differences in the chemical composition of red propolis samples and to suggest similarities and differences between samples collected in different regions. MATERIALS AND METHODS Chemicals. Phenolics standards of isoliquiritigenin (1), (3S)-vestitol (5), and (6aS,11aS)-medicarpin (8) used for this study were isolated from a Cuban red propolis sample as previously described (11). Methanol was purchased from J. T. Baker (Baker Mallinckrodt, * Author to whom correspondence should be addressed. Phone: 0039 89 969766. Fax: 0039 89 969602. E-mail: rastrelli@unisa.it. † Universidad de La Habana. ‡ Estacio´n Experimental Apı ´cola. § Instituto de Medicina del Deporte. | University of Salerno. J. Agric. Food Chem. 2008, 56, 9927–9932 9927 10.1021/jf801870f CCC: $40.75  2008 American Chemical Society Published on Web 10/14/2008