Mar 21, 1995 - CaIIuna (Ericaceae) which also contained ellagic acid, seem to be the most useful potential markers for the floral origin of heather honey.
Z Lebensm Unters Forsch (1996) 202:40-44
9 Springer-Verlag 1996
F e d e r i c o Ferreres 9 P a u l a Andrade 9 M a r i a I. Gil F r a n c i s c o A. Tomfis-Barberfin
Floral nectar phenolics as biochemical markers for the botanical origin of heather honey
Received: 21 March 1995
A b s t r a c t In order to find out biochemical markers for
the botanical origin of heather (Erica) honey, the phenolic metabolites present in heather floral nectar, collected from the honey-stomach of bees gathering nectar from these flowers, were analysed. The flavonoid fraction of nectar contained four main flavonoids. Two of them were quercetin and kaempferol 3-rhamnosides, and the other two were tentatively identified as myricetin Y-methyl ether and isorhamnetin 3-rhamnosides. Since the natural glycosides are hydrolysed by bee enzymes to render the corresponding aglycones, which are the metabolites detected in honey, acid hydrolysis of the nectar glycosides was achieved. The aglycones quercetin, myricetin 3'-methyl ether, kaempferol and isorhamnetin were identified, as well as the gallic acid derivative ellagic acid. The analysis of Portuguese heather honey samples showed that ellagic acid was present in all the samples in significant amounts ranging between 100 gg and 600 gg per 100 g honey. The other nectar-derived flavonoids were also present, although some of them in very variable amounts. Ellagic acid and myricetin 3'-methyl ether, which have not been detected in any of the monofloral honey samples investigated so far, with the only exception being a French honey sample of the botanically related CaIIuna (Ericaceae) which also contained ellagic acid, seem to be the most useful potential markers for the floral origin of heather honey. However, more detailed
F. Ferreres 9M.I. Gil. F.A. Tom~ts-Barbcrfin ( ~ ) Laboratorio de Fitoquimica, Departamento de Ciencia y Tecnologia de Alimentos, CEBAS (CSIC), P.O. Box 1495, E-30080 Murcia, Spain P. Andrade 1 Laboratorio de Farmacognosia, Faculdade Farmacia, Universidade Coimbra, P-3000 Coimbra, Portugal Present address:
1Laboratorio de Farmacognosia, Faculdade de Farmacia, Universidade do Porto, P-4000 Porto, Portugal
and extensive investigations are needed to prove the utility of these markers. Key words Heather honey- Nectar- Flavonoids 9 Ellagic acid" Biocl~emical marker
Introduction The botanical origin of honey is one of its main quality parameters, and its price is very often related to this floral origin. Some monofloral honeys are more appreciated than others due either to their flavour and aroma properties or to their pharmacological attributes, and these are generally more costly to buy than multifloral honeys [1]. The determination of the botanical origin of honey is usually evaluated by sensorial and pollen analysis [-2, 3]. However, some honey samples contain a very small amount of pollen (i.e. citrus honey), or the industrial treatments to which honey is submitted remove partially and sometimes totally the pollen grains. In these cases, other objective analytical techniques should be developed to help with floral origin determinations. Biochemical analysis of metabolites present in floral nectar seems to be a very valuable technique for this purpose [-4]. Recently, the use of hesperetin, a phenolic metabolite of citrus flower nectar, in the determination of the floral origin of citrus honey has been demonstrated [5, 6]. In a previous study on Portuguese heather honey, 22 flavonoids were detected, and a reduced number of substances seemed to be related to the floral origin I-7]. As a continuation of this previous study, the purpose of the present work was the identification of the phenolic compounds present in heather nectar collected with the help of honey bees, and the detection of these substances, or related metabolites, in honey samples from the same origin, in order to find out which phenolics would be suitable markers for the floral origin of heather honey.
41
Materials and methods Heather honey samples. Heather honey samples were produced in the Serra da Lousa (Coimbra) and were provided and guaranteed by the Direccao da Circunscricao Ftorestal de Coimbra. Samples were stored at 0 ~ until analysis, a process which has been published in more detail previously [7]. Collection of heather nectar. Due to the small size of heather flowers, and to the relatively small amount of nectar that they produce, the collection of heather nectar was achieved by extraction from the honey-stomach of bees gathering heather nectar in geographical areas where heather was the only flower available for nectar collection. Bees were trapped in liquid nitrogen and stored at - 2 0 ~ until analysis. The bees were thawed and the honey-stomach was dissected out with a scalpel and forceps. The contents of the different honey-stomachs were placed into Eppendorf tubes and stored at 20 ~ until needed. -
Flavonoid extraction from nectar. Of the heather nectar, 5 ml was diluted with 100 ml distilled water and filtered through cotton to remove solid particles. The filtrate was mixed with enough Amberlite XAD-2 non-ionic polymeric resin (Fluka, pore size 9 rim, particle size 0.3 1.2 ram) necessary to fill in a column of 19 cm x 3 cm, and was stirred with a magnetic stirrer for 1 h. The Amberlite XAD-2 with the phenolic compounds adsorbed was packed into the column, and washed with 1 1 distilled water. The phenolic metabolites were then eluted with methanol (200 ml), and concentrated in a rotary evaporator (40 ~ HPLC analysis of phenolics from nectar. The phenolics present in heather nectar were analysed on a reverse-phase column LiCrochart RP-18 (Merck, Darmstadt) maintained at a temperature of 40 ~ (12.5 cm x 0.4 cm, 5 gm particle size), using water:formic acid (19: 1) and methanol as solvents. Elution was performed at a solvent flow rate of 1 ml/min, starting with 15% methanol to reach 55% methanol at 30 min. The aglycones obtained after acid hydrolysis of the nectar flavonoid fraction were anlaysed with the same column conditions and solvents, but elution was performed starting with 5% methanol to reach 15% at 10 min, 30% at 15 rain, 35% at 25 min, 50% at 35 rain and 80% methanol at 40 min.
Sephadex LH-20 chromatography. This was achieved as described previously [7]. The methanol extract that eluted from the Amberlite XAD-2 column was concentrated and passed through a column (10 cm x 0.5 cm) of Sephadex LH-20 (Pharmacia, Upsala, Sweden). Elution was performed with methanol and the flavonoid fraction was visualized under UV light (360 nm) as described previously [8]. Acid hydrolysis. An aliquot of the flavonoid fraction purified by Sephadex LH-20 was dehydrated and redissolved in 2 ml of 2 N HC1 and heated at 90 ~ in a stoppered tube for 40 min. The aglycones were then extracted by liquid/liquid extraction with ethylic ether (2 ml x 3), and the extracts joined, dehydrated, redissolved in methanol and analysed by HPLC.
Phenolic compound identification and quantitation. The different phenolics were identified by chromatographic comparisons with authentic markers (commercial or previously isolated and identified from heather honey) [7], and by their UV spectra recorded with the diode array detector. Phenolics were quantified by the absorbance of their corresponding peaks in the chromatograms, the flavonols as quercetin detected at 340 nm and ellagic acid as an authentic marker (Sigma) at 256 nm.
Capillary electrophoresis analysis of nectar phenolics. This was achieved on a fused silica column (50 cm to detect 50 gm i.d.) at
30~ using 25 mM sodium borate as a buffer, pH 9.5, plus 20% methanol, at 20kV (average current of 38 hA). Detection was achieved with a diode array detector which allowed the recording of the UV spectra relating to the different flavonoids.
Analysis of honey flavonoids. This was achieved as described previously [9].
Results and discussion The heather floral nectar, obtained from the honeystomach of bees, was diluted with distilled water and the phenolic compounds adsorbed onto particles of the non-ionic polymeric resin Amberlite XAD-2. The resin particles with the adsorbed phenolic metabolites were packed into a column and then washed with enough water to elute sugars and other polar compounds, and the phenolic fraction was then eluted with methanol. HPLC analysis of the methanol fraction indicated that the main constituents were phenolic acid derivatives, and that flavonoids were also present as trace elements. This agrees with previous studies on heather honey, which showed that flavonoids were in a minority and that in order to analyse them a purification by Sephadex LH-20 chromatography was necessary to prepare a flavonoid fraction which was suitable for HPLC analysis. Therefore, to concentrate the flavonoid fraction of heather nectar, the methanol extracts were subjected to chromatography on a Sephadex LH-20 column with methanol, and the elution of flavonoids was followed under UV light (360 rim). The last eluting fraction (a dark brown fraction under UV light) was constituted mainly by flavonoids. Two other fractions eluting previously were also collected and analysed by HPLC, and no flavonoids were detected. The HPLC analysis of the last fraction showed that four main flavonoids were present (compounds 1-4). Compounds 1 (retention time, tr 14.7 rain, 261, 348 nm) and 2 (tr 15.0 min, 261, 348 nm) were not clearly separated under the HPLC conditions used, and these two flavonoids accounted for 83 % of the total absorbance of the chromatogram at 340 nm. Compounds 3 (tr 18.4 min, 264, 347 nm) and 4 (tr 19.8 min, 261,348 nm) were minor constituents with 5% and 12% of the total absorbance of the chromatogram respectively. As separation of these substances by HPLC was not possible, these nectar flavonoids were then analysed by capillary zone electrophoresis (CZE), a technique which allows separation with a higher resolution than HPLC [10] and in this case a much better separation was obtained (Fig. 1). After CZE analysis, the same relative amounts for the individual flavonoids as compared to HPLC analysis were observed. As nectar metabolites are hydrolysed by the enzymes present in honey to render the corresponding aglycones [-5, 6], the nectar flavonoid fraction was hydrolysed with acid to obtain the metabolites which should be the constituents detected in honey. After acid hydrolysis,
42 Fig. 1 Capillary zone electrophoresis electropherogram of the naturally occurring heather nectar falvonoid glycosides. Detection at 340 rim. Compound 1, quercetin 3-rhamnoside; compound 2, myricetin 3'-methyl ether 3-rhamnoside; compound 3, kaempferol 3-rhamnoside; compound 4, Isorhamnetin 3.rhamnoside
mAbs
5-
4 3
\ O, 10
mV b 200
100
0
10
20
30
rain
Fig. 2 HPLC chromatogram of heather nectar phenolics after acid hydrolysis, compound a, ellagic acid; compound b, quercetin; compound c, myricetin 3'-methyl ether; compound d, kaempferol; compound e, isorhamnetin. Detection at 340 nm
the aglycones obtained were analysed using HPLC and the chromatogram shown in Fig. 2 was obtained. Five different compounds were detected, and identified as ellagic acid (compound a), quercetin (compound b), myricetin 3'-methyl ether (compound c), kaempferol (compound d) and isorhamnetin (compound e) by their UV spectra recorded with the diode array detector, and chromatographic comparisons with authentic markers. The natural nectar glycosides were studied by paper electrophoresis (pH 4.4) to discard the presence of glucuronic acid derivatives and the presence of acylations with dicarboxylic acids [-11], and by HPLC and TLC comparisons with some available flavonol glycoside markers. Their chromatographic behaviour suggested that they were flavonol 3-monoglycosides. The main glycoside (compound 1) was identified as quercetin 3-rhamnoside (compared with a marker from Fluka), and the minor glycoside (compound 3) coincided with kaempferol 3-rhamnoside, which is an impurity of the above-mentioned marker. This impurity was isolated by semipreparative HPLC (Spherisorb ODS-2 column
20
3uOrain
25 cm x 0.7 cm, 5 gm particle size with MeOH-H20), in which quercetin 3-rhamnoside eluted with MeOH: H20 (2 : 3), and the impurity eluted with M e O H - H 2 0 (1:1). After acid hydrolysis the impurity rendered kaempferol and rhamnose. The remaining glycosides were tentatively identified as the 3-rhamnosides of isorhamnetin (quercetin 3'-methyl ether) and myricetin 3'-methyl ether, by their close correlation between the chromatographic behaviour of the glycosides and that of the corresponding aglycones, which suggested that all compounds were conjugated with the same sugar (rhamnose). The detection of ellagic acid deserves a special comment. Ellagic acid is a dimeric derivative of gallic acid, which exists in plants combined with its precursor hexahydroxydiphenic acid or bound as ellagitannins esterified with glucose. It has been suggested that when consumed by animals, the glucose moieties of ellagitannins are probably removed by enzymatic activity, thus "freeing up" ellagic acid [12]. Recent evidence has shown the activity of ellagic acid to be antioxidant and that it is an anticarcinogenic metabolite [13, 14]. This compound was not detected in nectar, where it was most likely present as ellagitannin, but it was detected after acid hydrolysis of the flavonoid fraction purified by chromatography on a Sephadex LH-20 column. Therefore, this compound should be present in heather nectar as a constituent of ellagitannins, and is transformed to ellagic acid by the action of the bee enzymes. In order to test for the presence of heather nectar phenolic metabolites in honey, 20 heather honey samples collected in la Serra da Lousa in Portugal, were analysed for ftavonoids by HPLC as reported previously [-7]. These honey samples contained pollens from Erica erigena and E. arborea as the main pollen
43 Table 1 Content of heather flower nectar phenolic metabolites in honey
Honey samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Metabolite (gg/100 g honey) Ellagic acid
Quercetin
Myricetin 3-methyl ether
Kaempferol
Isorhamnetin
187 440 400 133 187 240 173 547 400 427 547 613 93 147 120 187 153 133 533 173
4.1 14.2 1.6 2.0 31.3 49.8 48.3 30.1 18.6 10.0 19.4 28.7 5.2 7.2 28.8 9.2 4.2 34.1 1.1 1.2
6.0 31.2 24.4 8.7 9.0 16.9 20.2 42.0 21.7 8.6 13.8 57.3 49.3 3.8 16.2 8.4 1.9 13.9 1.9 1.0
27.1 4.2 13.4 t 14.1 15.7 31.3 25.3 7.7 4.9 6.0 10.2 24.6 4.6 11.9 3.5 22.5 12.1 2.2 -
13.0 4.7 16.8 4.4 24.3 42.3 49.8 37.1 14.0 7.6 4.4 7.7 24.0 7.7 15.5 2.8 12.1 49.3 6.9 3.4
Flavonols were quantified at 340 nm, and ellagic acid at 256 nm
components. In addition to the flavonoids which are common to all the honey samples produced in temperate areas, since they come from propolis/beeswax origin (namely pinobanksin, pinocembrin, chrysin, galangin, techtochrysin and di- and tri-methyl ethers of quercetin) [15], some flavonoids which seemed to be related to the floral origin of heather nectar were identified in a previous study [7]. These were identified as myricetin, myricetin T-methyl ether and tricetin, as well as other more common flavonols such as quercetin, kaempferol and isorhamnetin. In Table 1, the content of floral-derived flavonoids of the different honey samples studied, as well as the content of ellagic acid, are shown. The main phenolic compound detected in all samples was ellagic acid, the content of which ranged from between 100 gg and 600 gg per 100 g honey. The amount of floral-nectar-derived flavonoids was much smaller and variable, as is evident from the results shown in Table 1. Nevertheless, the flavanoid myricetin 3'-methyl ether is present in all the samples analysed. With respect to phenolic markers for the floral origin of heather honey, myricetin 3'methyl ether and ellagic acid are the most interesting metabolites, Since they have not been detected in the majority of monofloral honey samples analysed so far (rosemary, sunflower, citrus, lavender, eucalyptus, almond, chesnut, acacia, thyme, rape, fir, alder, rhododendron, lime tree, etc) and seem to be very promising markers to be used as an adjunct in the objective determination of the floral
origin of heather honey. However, in a recent work on the flavonoids of different monofloral French honeys [16], ellagic acid was detected in one sample of CaIluna honey (Ericaceae), and a white heather honey sample (Erica sp.) contained myricetin and lacked ellagic acid. These results suggest that the presence or absence of ellagic acid in the nectar of different Erica species (and related genera such as Calluna), can be specific to species, and therefore distinction between different heather species would be possible. The finding of ellagic acid in Ericaceae species, and thus in the flower nectar, is not surprising since ellagitannins have been found to be present in many members of the Ericaceae family [17, 18]. However, the occurrence of ellagitannins in the plant kingdom is not widespread, and this is probably the reason why this metabolite has not been detected in any other honey samples from other floral origins studied so far. These results are very promising, but more detailed studies are necessary to prove that ellagic acid and myricetin 3'-methyl ether are useful floral markers for this purpose. Acknowledgements The authors are grateful to the Spanish CICYT (Grants AL191-0486 and AL192-0151) for financial support of this work, and to Alexandra Maria Amaral, Eng. Duarte Pessoa and Eng. M. Eduarda Campos da Delega~ao Florestal da Beira litoral for helping in nectar collection. P.A. is indebted to the Junta Nacional de Investigacao Cientifica e Tecnologica de Portugal for a fellowship.
44
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