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

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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

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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 analysis, 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 geographical 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 floral origin of honey. The utility of these substances 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 column (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 samples 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 markers. 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. Thus, gin, 3 sunflower honey samples produced in sunflower honey contains a considerable 3 French regions (Ariège, Aude and Loir- amount (around 30%) of quercetin, while et-Cher) were extracted and their flavonoids analysed. The results are shown in figure detected as the main flavonoid in sunflower 2. The 3 samples have an identical flavonoid pollen (Ferreres et al, 1992) and nectar profile, with slight differences in the relative (Tomás-Barberán, unpublished results). amounts of some components, and they are Quercetin was also present in heather, rape, characterized by the relative importance of calluna, lavender, citrus and lime tree honey, the flavonoid quercetin. but with smaller relative amounts than in the case of sunflower honey. DISCUSSION Although the profiles of flavonoid glyco- sides present in pollen can be used to dif- These results do not demonstrate that differentiate pollens with different botanical ori- ferences occur between the flavonoid pro- gin (Tomás-Barberán et al, 1989), they reflect files of different monofloral honeys, since glycosidic combinations of a relatively lim- they have a common flavonoid profile, as ited number of polyhydroxylated aglycones shown by a previous work on the flavonoids (quercetin, luteolin, 8-methoxykaempferol, of La Alcarria honey (Fererres et al, 1992). kaempferol, apigenin and isorhamnetin). However, in some cases, 1 individual These flavonoid aglycones also constitute flavonoid could be a potential marker for the the main flower- derived flavonoids in honey. floral origin of honey. Therefore, the pres- We conclude that the flavonoid profiles ence of hesperetin in the French citrus detected in the different French honey sam- honey agrees with previous reports on ples analysed are very similar, and it is not Spanish citrus honey (Ferreres et al, 1994b) easy to differentiate all honey samples by and its use as a marker of supports potential their flavonoid profiles. However, some indi- its The same origin (Ferreres et al, 1993). vidual compounds seem to be useful bio- to since it has applies myricetin, recently chemical markers of the floral origin of spe- been reported to be a possible marker of cific honey samples (calluna, citrus and the botanical origin in Portuguese heather heather), and the relative amount of other et and was found honey (Fererres al, 1994a) common flavonoids could help in the deter- in our French sample. mination of the floral origin of other honey In addition, the flavonoids that are com- samples (eg, quercetin in sunflower honey). mon to honeys with different floral origin These results are quite encouraging, but could be useful as an adjunct in the objective measurements in more monofloral honeys of determination of the plant origin of honey the same type should be conducted to con- when they are present as major compo- firm which flavonoids could be important for nents in the flavonoid profile. Therefore, the the characterization of a particular mono- importance of the relative amount of floral honey. quercetin observed in the 3 French samples of sunflower honey, agrees with previous work on Spanish (Ferreres et al, 1992) ACKNOWLEDGMENTS and French sunflower samples (Sabatier et al, 1992). This observation supports the The authors are grateful to the Spanish CICYT possible use of the determination of the rel- (Grants ALI91-0486 and ALI92-0151) for finan- ative amount of quercetin in honey as a cial support of this work, and to Drs Morlot and complementary analysis in determining the Delgado for providing honey samples. origin of sunflower honey. It is not surprising that quercetin is one of the main flavonoids in the chromatograms of sunflower honey, Résumé — Les flavonoïdes de miels since rutin (quercetin 3-rutinoside) has been français de diverses origines florales. Ces dernières années on s’est efforcé de plémentaires sur des échantillons d’origine mettre au point de nouvelles techniques géographique variée sont nécessaires pour analytiques objectives pour déterminer l’ori- confirmer l’utilisation de certains flavonoïdes gine des miels. Les flavonoïdes, les acides comme marqueurs de l’origine botanique phénoliques, les dérivés des caroténoïdes, des miels. les acides aminés et les composés aroma- tiques ont été utilisés dans ce but. Dans ce miel / flavonoïde / origine botanique / travail les flavonoïdes de 12 miels mono- chromatographie liquide haute pression floraux provenant de France ont été analy- sés par chromatographie liquide haute pression (HPLC) afin de voir s’il était possible Zusammenfassung — Das Flavonoid- de corréler le spectre des flavonoïdes et muster unterschiedlicher unifloraler Blü- l’origine botanique des miels. Les flavo- tenhonige französischer Herkunft. In den noïdes ont été extraits et purifiés sur une letzten Jahren wurden einige Anstrengun- colonne Amberlite XAD-2, puis sur une gen unternommen, um die Herkunft von colonne Sephadex LH-20 et analysés en Honigen mit neuen, objektiven, analytischen HPLC. Les flavonoïdes provenant principa- Methoden beurteilen zu können. Hierzu lement du pollen et/ou du nectar (marqués wurden bisher Flavonoide, Phenolsäuren, d’une lettre) et ceux caractéristiques de la Karotinoid-Abkömmlinge, Aminosäuren und propolis et/ou de la cire d’abeille (marqués aromatische Inhaltsstoffe benutzt. In die- d’un chiffre) se différenciaient nettement sur ser Arbeit wurden die Flavonoidprofile von les chromatogrammes (fig 1). Les premiers 12 verschiedenen unifloralen Honigen fran- étaient principalement des composés poly- zösischer Abstammung durch HPLC ana- hydroxylés, les seconds des flavonoïdes lysiert. Mögliche Unterschiede des Fla- lipophyles avec un cycle B non substitué. vonoidmusters sollten zu der botanischen Tous les miels monofloraux analysés Herkunft des Honigs in Beziehung gesetzt avaient en commun un spectre constitué werden. Die Honigflavonoide wurden extra- d’un petit nombre de flavonoïdes (tableau hiert und mit einer Kombination von Amber- I). Néanmoins certains miels contenaient lite XAD-2 und Sephadex LH-20 Chroma- un composé phénolique spécifique, sus- tographie gereinigt, und durch ceptible d’être utilisé pour déterminer l’ori- Hochdruckflüssigchromatographie mit Pha- gine des miels. Ainsi le miel de bruyère ren- senumkehr analysiert. Die hauptsächlich fermait de la myricétine (M) celui de callune ; aus Pollen und/oder Nektar stammenden de l’acide ellagique (EA) celui d’oranger ; Flavonoide (mit einem Buchstaben gezeich- la flavanone hespérétine (H). D’autres miels net) waren in den HPLC-Chromatogram- se caractérisaient par l’accumulation d’un men klar von den für Propolis und/oder Bie- flavonoïde assez commun. Ainsi, le miel de nenwachs charakteristischen Flavonoiden tournesol contenait des quantités de quer- (mit einer Zahl gezeichnet) getrennt (Abb cetine nettement supérieures à celles des 1). Die Flavonoide aus Pollen und/oder autres échantillons. Des quantités voisines Nektar bestanden hauptsächlich aus ont été trouvées dans 3 miels de tournesol polyhydroxilierten Anteilen. Die aus Pro- produits dans différentes régions de France polis und/oder Bienenwachs stammenden (fig 2). Cette étude montre que l’analyse Flavonoiden waren lipophil mit einem des flavonoïdes et des composés phéno- unsubstituierten Ring B. Die verschiede- liques peut être une technique biochimique nen unifloralen Honige hatten alle ein übe- complémentaire de valeur pour déterminer reinstimmendes Flavonoidmuster (Tabelle l’origine botanique de miels monofloraux I). Einige der Honige enthielten allerdings spécifiques, mais que des mesures com- spezifische phenolische Komponenten, die zur Herkunftsbestimmung genutzt werden Ferreres F, García-Viguera C, Tomás-Lorente F, Tomás- könnten. So enthielt der Heidehonig Myri- Barberán FA (1993) Hesperetin, a marker of the flo- cetin der Besenheide ral origin of citrus honey. J Sci Food Agric 61, 121- (M), Honig (Calluna) 123 Ellagsäure (EA) und Zitrushonig das Fla- Ferreres F, Andrade P, Tomás-Barberán FA (1994a) vanon Andere Hesperetin (H) (Abb 1). Flavonoids from Portuguese heather honey. Honige wurden durch das gehäufte Vor- Z Lebensm Unters Forsch 1991, 32-37 kommen von eher Flavonoi- gewöhnlichen Ferreres F, Giner JM, Tomás-Barberán FA (1994b) A den charakterisiert. Beispielsweise enthielt comparative study of hesperetin and methyl anthrani- Sonnenblumenhonig anteilsmäßig mehr late as markers of the floral origin of citrus honey. Quercetin als der Rest der analysierten Pro- J Sci Food Agric 65, 371-372 ben. Die Höhe dieses Anteils war bei 3 Ferreres F, Tomás-Barberán FA, Soler C, García- verschiedenen Honigen aus unterschiedli- Viguera C, Ortiz A, Tomás-Lorente F (1994c) A sim- chen Regionen Frankreichs ähnlich (Abb ple extractive technique for honey flavonoid HPLC analysis. Apidologie 25, 21-30 2). Die Studie ergab damit, daß die Ana- Häusler A Minor flavour/aroma com- der Flavonoide und der H, Montag (1990) lyse phenolischen pounds in honey. III. Distribution of aromatic alde- eine sehr nützliche zusätzli- Komponenten hydes in floral types, and occurrence of heterocyclic che biochemische Technik zur objektiven N- and S-compounds. Dtsch Lebensm Rundsch 86, Bestimmungen einiger bestimmter mono- 171-174 floraler Honige darstellt. Allerdings sind wei- Louveaux J, Maurizio A, Vorwohl G (1978) Methods in tere Messungen an Proben aus verschie- melissopalynology. Bee World 59, 139-157 denen geographischen Regionen Maurizio A (1951) Pollen analysis of honey. Bee World erforderlich, um die Verwendbarkeit eini- 32, 1-5 ger Flavonoide als Marker für die Blüten- Sabatier S, Amiot MJ, Tacchini M, Aubert S (1992) Iden- herkunft von Honigen zu bestätigen. tification of flavonoids in sunflower honey. J Food Sci 57, 773-774 Honig / Flavonoide / Blütenherkunft / Steeg E, Montag A (1988) Minor components of honey with organoleptic significance. Part I. Aromatic car- Hochdruckflüssigchromatographie boxylic acids and their esters. Dtsch Lebensm Rund- sch 84, 103-108 Speer K, Montag A (1984) Occurrence of benzoic acid REFERENCES and phenylacetic acid in honey. 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