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Flavonoid Patterns of French Honeys with Different Floral Origin C Soler, Mi Gil, C García-Viguera, Fa Tomás-Barberán

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Flavonoid Patterns of French Honeys with Different Floral Origin C Soler, Mi Gil, C García-Viguera, Fa Tomás-Barberán Flavonoid patterns of French honeys with different floral origin C Soler, Mi Gil, C García-Viguera, Fa Tomás-Barberán To cite this version: C Soler, Mi Gil, C García-Viguera, Fa Tomás-Barberán. Flavonoid patterns of French honeys with different floral origin. Apidologie, Springer Verlag, 1995, 26 (1), pp.53-60. hal-00891245 HAL Id: hal-00891245 https://hal.archives-ouvertes.fr/hal-00891245 Submitted on 1 Jan 1995 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original article Flavonoid patterns of French honeys with different floral origin C Soler MI Gil, C García-Viguera, FA Tomás-Barberán Laboratorio de Fitoquímica, Departamento de Ciencia y Technología de los Alimentos, CEBAS (CSIC), PO Box 4195, Murcia 30080, Spain (Received 9 August 1994; accepted 16 November 1994) Summary — The flavonoid profiles of 12 different unifloral French honey samples were analysed by HPLC to evaluate if these substances could be used as markers of the floral origin of honey. In this anal- ysis, the characteristic flavonoids from propolis and/or beeswax (chrysin, galangin, tectochrysin, pinocembrin and pinobanksin) were separated from those originating mainly from nectar and/or pollen (polyhydroxylated flavonoid aglycones), which would be related to their floral origin. All the analysed samples contained a common flavonoid profile consisting of polyhydroxylated flavonoid aglycones including 8-methoxykaempferol, kaempferol, quercetin, isorhamnetin, luteolin and apigenin, suggest- ing that flavonoid analysis does not generally prove differences between French monofloral honey samples. However, some individual honey samples showed potential floral markers. Thus, heather honey was characterized by the presence of myricetin, calluna honey by ellagic acid and citrus honey by the flavanone hesperetin. In other samples, the relative amount of 1 individual flavonoid could be related to the floral origin. Thus, sunflower honeys contained an important relative amount of quercetin, and in alder honey only 8-methoxykaempferol was detected. This preliminary study shows that flavonoid and phenolic compound analyses could be a very valuable complementary biochemical technique in the objective determination of the floral origin of some specific monofloral honey samples, but further studies with a larger number of samples is necessary to confirm the observed differences. honey / flavonoid / botanical origin / HPLC INTRODUCTION on expert ability and judgement. Alterna- tive methods that could be more widely and used for At present, the principal objective means accurately characterizing honeys of determining the geographical and floral have been sought for many years. Bonaga origin of honey is pollen analysis (Maurizio, and Giumanini (1986) have suggested that 1951; Louveaux et al, 1978). However, the the next step in this type of research will technique is tedious and very dependent be to correlate floral source with the pres- * Correspondence and reprints ence of certain compounds originating phenolic acid derivatives elute essentially either in the nectar or in some biochemical in a first fraction, while the polyhydroxylated modification of nectar compounds carried flavonoids originating mainly from by the bee. In fact, aromatic compounds nectar/pollen (although some have also and degraded carotenoid-like substances been detected in propolis) elute together in (Tan et al, 1988, 1989), amino acids (Davis, a second fraction (Ferreres et al, 1994a). 1975; Bosi and Battaglini, 1978), degrada- For the purposes of this study, only the tion products of phenylalanine (Speer and flavonoids present in the second fraction Montag, 1987), aromatic aldehydes and were collected and analysed by HPLC. heterocycles (Häusler and Montag, 1990) and aromatic acids and their esters (Speer and Montag, 1984; Steeg and Montag, MATERIALS AND METHODS 1988) have all proved quite useful for this purpose. In the same way, recent studies have Honey samples revealed that the analysis of flavonoids and other phenolic compounds constitute a very Honey samples and data on their floral and geo- promising technique to study the geo- graphical origin were provided by Dr Morlot graphical and floral origin of honey (Amiot et (Bernard Michaud, SA, Jurançon) via Dr Delgado, from INP-ENSC, Toulouse al, 1989; Ferreres et al, 1992; Ferreres et (France) (Delgado, 1993). al, 1993; Ferreres et al, 1994c). The flavonoids present in nectar and pollen are sometimes characteristic of the species, Flavonoid extraction from honey and can be used for the analysis of the flo- ral origin of honey. The utility of these sub- stances in the determination of the origin of Flavonoids were extracted from honey as citrus (Ferreres et al, 1993; Ferreres et al, reported previously (Ferreres et al, 1994c). Honey (ca 200 g) was diluted with 5 parts acid and 1994b), rosemary (Ferreres et al, 1992) water (pH 2-3, adjusted with HCI) until com- heather (Ferreres et al, 1994a) honeys have pletely fluid and then filtered through cotton. The recently been reported. filtrate was passed through an Amberlite XAD-2 column (Sigma) and washed with acid water (100 In the present work, the flavonoid pro- ml) and distilled water (300 ml). The phenolic files of 12 different unifloral French honeys fraction was then eluted with methanol (300 ml). were analysed by HPLC to evaluate differ- This fraction was concentrated under reduced ences in their flavonoid patterns which could pressure and the flavonoids were further puri- be related to their botanical origin. It has fied by dissolving them in methanol and pass- been recently shown that honey flavonoids ing the solution through a Sephadex LH-20 col- umn (Pharmacia). The elution was followed under come either from propolis and/or beeswax or UV light at 360 nm to separate from nectar and/or and that have polyhydroxylated pollen they flavonoids, which were more retained in the col- different structural features depending on umn, from the flavanones and flavones with an their origin (Tomás-Barberán et al, 1993; unsubstituted ring B, characteristic of propolis Ferreres et al, 1994c). Using Sephadex LH- and/or beeswax, which eluted first (Tomás-Bar- 20 chromatography, it is possible to frac- berán et al, 1993). The polyhydroxylated tionate honey phenolic compounds, since flavonoid fraction had a dark-purple colour and was evaporated to under reduced the flavanones and dryness pres- pinobanksin pinocem- sure (40°C), redissolved in methanol (0.5 ml), the flavones and brin, chrysin tectochrysin, and 20 ml were injected for HPLC analysis (Fer- characteristic of propolis/beeswax, and the reres et al, 1994a). HPLC analysis of honey flavonoids RESULTS HPLC analysis was carried out on a reversed- The flavonoids present in the different honey phase column LiChrochart RP-18 (Merck, Darm- samples available were extracted and anal- x 0.4 5 stadt) (12.5 cm, μm particle size) using HPLC and the results are summa- water-formic acid and methanol ysed by (19:1) (solvent A) rized in table I. It is remarkable that 8- (solvent B) as solvents (Ferreres et al, 1994c). Detection was performed with a diode array detec- methoxykaempferol (P) (3,5,7,4’- tor, and chromatograms were recorded at 290 tetrahydroxy-8-methoxyflavone) was pre- and 340 nm. For the purposes of the present sent in 100% of the samples analysed, and work quantification of the individual flavonoids the flavonoids, kaempferol (K) (3,5,7,4’- was not necessary, and the presence of the dif- tetrahydroxyflavone), apigenin (A) (5,7,4’- ferent flavonoids was evaluated as the percentage isorhamnetin of the total absorbance of the whole chro- trihydroxyflavone), (I) (3,5,7,4’- matogram for each individual flavonoid. tetrahydroxy-3’-methoxyflavone), quercetin (Q) (3,5,7,3’,4’-pentahydroxyflavone) and luteolin (L) (5,7,3’,4’-tetrahydroxyflavone) Flavonoid identification were detected in the majority of the sam- ples analysed. In spite of the very different floral origin of the honey samples, they show The various flavonoids were identified in the chro- flavonoid patterns composed of only a by comparisons matograms cochromatographic reduced number of common with authentic markers previously isolated from compounds. On the other some were honey, and by their UV spectra recorded with an hand, compounds on-line diode array detector (Ferreres et al, 1993; detected in only 1 unifloral honey type, and Ferreres et al, 1994c). could be considered as potential floral mark- ers. Thus, ellagic acid (EA) (a dimer of gal- these honey samples are shown, and the lic acid) seems to be characteristic of cal- differences are clearly observed. The luna honey, myricetin (M) of heather honey flavonoids coming mainly from pollen and/or and the flavanone hesperetin (H) of citrus nectar (although they are also present in honey. In figure 1, the HPLC chro- propolis as minor constituents), which are matograms of the flavonoids present in related to the floral origin of honey, were marked with letters in the chromatograms, the sample of alder honey contained 8- and those coming exclusively from propolis methoxykaempferol as the only flavonoid and/or beeswax, and which do not have any and other unidentified phenolic acid deriva- relationship with the floral origin of honey, tives which were not present in the rest of were marked with numbers. Both flavonoid the samples. types were readily distinguished in the chro- To evaluate if these possible markers matograms. were present in the same proportion in dif- In other samples, it seems that the rela- ferent honey samples with the same floral tive amount of 1 individual flavonoid could be origin, but with different geographical ori- related to the floral origin of honey.
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