pubs.acs.org/JAFC Published on Web 03/15/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 4725–4730 4725 DOI:10.1021/jf904527n Studies on the Constituents of Yellow Cuban Propolis: GC-MS Determination of Triterpenoids and Flavonoids INGRID MA ´ RQUEZ HERNA ´ NDEZ, † OSMANY CUESTA-RUBIO, † MERCEDES CAMPO FERNA ´ NDEZ, † ARI ´ STIDES ROSADO PE ´ REZ, ‡ RODNY MONTES DE OCA PORTO, § ANNA LISA PICCINELLI, ) AND LUCA RASTRELLI* , ) † Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana. Avenida 23, 21425 La Lisa, Ciudad de La Habana, Cuba CP 13600, ‡ Estaci on Experimental Apı´cola. El Cano, Cuba, § Instituto de Medicina del Deporte, Laboratorio Antidoping, Calle 100 y Aldab o, CP 10800, Ciudad de La Habana, Cuba, and ) Dipartimento di Scienze Farmaceutiche, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy In this study, on the basis of the information supplied by NMR and HPLC-PDA data, we reported a quali-quantitative GC-MS study of 19 yellow Cuban propolis (YCP) samples collected in different regions of Cuba. The profiles of YCP samples allowed us to define two main types of YCP directly related to their secondary metabolite classes: type A, rich in triterpenic alcohols and with the presence of polymethoxylated flavonoids as minor constituents, and type B, containing acetyl triterpenes as the main constituents. For the first time, triterpenoids belonging to oleanane, lupane, ursane, and lanostane skeletons were reported as major compounds in propolis. Also, the presence of polymethoxylated flavones or flavanones was found for the first time in propolis. KEYWORDS: Cuban propolis; yellow variety; triterpenic alcohols; polymethoxylated flavonoids; acetyl triterpenes; 1D NMR spectroscopy; HPLC-PDA; GC-MS INTRODUCTION Propolis is a resinous substance collected by honeybees from various plant sources with a variable chemical composition and a multitude of pharmacological and nutritional applications (1 ). Because propolis is reputed to have antiseptic, antimycotic, bacteriostatic, astringent, choleretic, spasmolytic, anti-inflamma- tory, anesthetic, and antioxidant properties, the list of prepara- tions and uses is nearly endless (2 -4 ). The plant source of propolis depends on the specific flora at the site of collection. Thus, the constituents of propolis of different places may be different. In temperate zones, propolis originates from the bud exudates of Populus species and therefore has relatively constant qualitative composition (5 , 6 ). Propolis from tropical zones seems to show a major variety of organic compounds including pre- nylated p-coumaric acids, diterpenes, triterpenes, lignans, pre- nylated benzophenones, flavonoids, and so on (7 -11 ). In our previous studies, we reported for the first time the occurrence of prenylated benzophenones and flavonoids in two different varieties of Cuban propolis (12 -15 ). Subsequently, we developed a classification method of Cuban propolis employing a combination of NMR, HPLC-PDA, and HPLC-ESI/MS tech- niques, which allowed the definition of three main types of Cuban propolis directly related to their secondary metabolite classes: brown Cuban propolis (BCP), rich in polyisoprenylated benzo- phenones, red Cuban propolis (RCP), containing isoflavonoids as the main constituents, and yellow Cuban propolis (YCP) (16 ). The main chemical constituents of the last group were shown to be aliphatic compounds, but these remain unknown. Column chromatograhy, RP-HPLC-PDA, and RP-HPLC-ESI/ MS methods have been shown to be insufficient for isolation and characterization of the main constituents of YCP samples be- cause all of them seemed to be mixtures of very closely related aliphatic compounds. For these reasons, the suitability of gas chromatography-mass spectrometry (GC-MS) was evaluated in this study as a technique for the determination of aliphatic compounds in YCP. For this purpose, 19 propolis samples collected in western, central, and eastern regions of Cuba were analyzed by GC-MS to characterize their quali-quantitative chemical composition and to investigate possible differences and similarities between samples collected in the different regions. MATERIALS AND METHODS Chemicals. Methanol, hexane (Hex), ethyl acetate (EtOAc), dry pyridine (C 5 H 5 N), and acetic anhydride (Ac 2 O) were purchased from J. T. Baker (Baker Mallinckrodt, Phillipsburg, NJ). The derivatization reagent, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), was purchased from Supelco (Supelco Park, PA). R-Amyrin, β-amyrin, lupeol, cycloartenol, and cholesterol were from Sigma-Aldrich (Milan, Italy), lanosterol was purchased from Merck (Darmstadt, Germany), and ger- manicol, R-amyrone, β-amyrone, and 24-methylene-9,19-ciclolanostan- 3β-ol were obtained from Centro de Quı´mica Farmace´ utica (Havana, Cuba). Propolis Samples. Samples of Cuban propolis were collected between October and December 2008 in different provinces of Cuba (municipalities are included in parentheses): Granma, sample 20 (Bayamo); Habana, *To whom correspondence should be addressed. Phone: 0039 89969766. Fax: 0039 89 969602. E-mail: rastrelli@unisa.it.