J Sci Food Agric 1998, 77, 506È510

Evaluation of Pollen as a Source of Kaempferol inRosemary HoneyF Ferreres,1 T Juan,2 C Pe� rez-Arquillue� ,3 A Herrera-Marteache,3 C Garc•�a-Viguera1and F A Toma� s-Barbera� n1*

1 Laboratorio de Fitoqu•�mica, Departamento de Ciencia y Tecnolog•� a de Alimentos, CEBAS (CSIC), POBox 4195, Murcia 30080, Spain2 Laboratorio de Bromatolog•� a Instituto Tecnolo� gico de Arago� n, Mar•� a de Luna 7, 50015, Zaragoza,Spain3 Departamento de Produccio� n Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidadde Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain

(Received 11 February 1997 ; revised version received 10 October 1997 ; accepted 12 December 1997)

Abstract : The content of the Ñavonoid kaempferol in di†erent experimental rose-mary honey samples produced in Arago� n (Spain) and in some commercial rose-mary honey samples produced in di†erent Spanish regions was evaluated byHPLC. The content ranged between 0É33 and 2É48 mg kg~1 of honey. Nectarhas been suggested recently as the origin of this Ñavonoid. Rosemary pollen con-tained kaempferol 3-diglucoside and 3-glucoside, and therefore could be an alter-native source for the kaempferol found in rosemary honey. However, there wasno correlation between the kaempferol present in honey and pollen content. Thissuggests that the contribution of pollen Ñavonoids to the pool of Ñavonoidsfound in honey is very small, and that the search for phenolic markers of thebotanical origin of honey should be addressed to the identiÐcation of nectarÑavonoids or other nectar constituents. 1998 SCI.(

J Sci Food Agric 77, 506È510 (1998)

Key words : honey ; kaempferol ; Ñavonoids ; botanical origin ; nectar ; pollen ;honey characterisation

INTRODUCTION

In the determination of the geographical and botanicalorigin of honey, the analysis of plant chemical constitu-ents has been considered as an alternative to the clas-sical pollen analysis. Thus, volatile compounds (Bonagaet al 1986), aromatic substances (Ha� usler and Montag1990 ; Tan et al 1990 ; Wilkins et al 1993), amino acids(Pe� rez-Arquillue� et al 1989) and degradation productsof amino acids (Speer and Montag 1987) have beenfound in honey and have been related to its Ñoralorigin. In addition, the possible use of Ñavonoids andother phenolic compound analyses as a complementarytechnique in the objective determination of the botan-

* To whom correspondence should be addressed.Contract/grant sponsor : CICYT.Contract/grant numbers : ALI 91-0486 and ALI 92-0151.

ical origin of honey, has been suggested (Amiot et al1989 ; Ferreres et al 1991, 1992). In fact, the Ñavonoidsdetected in Citrus nectar and honey (Ferreres et al 1993)and in heather nectar and honey (Ferreres et al 1994)support this original hypothesis.

In a recent paper, the Ñavonoid composition ofSpanish rosemary (Rosmarinus officinalis L) honey wasanalysed as well as that of rosemary Ñoral nectar col-lected with the help of bees (Gil et al 1995). Kaempferolglycosides were the main Ñavonoids found in rosemarynectar, and they were responsible for the fairly constantquantity of Kaempferol found in rosemary honeysamples, when compared with other honey phenolicswhich originate from propolis (a resinous material col-lected by bees from di†erent plants and which has dif-ferent roles in the hive). Another possible source forrosemary honey Ñavonoids could be the pollen grains

5061998 SCI. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

Pollen as a source of kaempferol in honey 507

which are also present in honey. It has been reportedthat pollen is a very rich source of Ñavonoid glycosides(Toma� s-Barbera� n et al 1989), and these compoundscould di†use from pollen to honey during honey matu-ration in the hive.

The purpose of the present work was to evaluate theÑavonoids in rosemary pollen and honey, and to studyif pollen could be an additional source for the kaemp-ferol found in honey.

MATERIALS AND METHODS

Honey and pollen samples

Experimental rosemary honeys were produced in di†er-ent areas of Arago� n (Spain). Geographical distributionof sampling locations was done according to the totalnumber of hives registered, and the speciÐc interest ofsome areas. Twenty one samples of unpasteurised andcentrifuged honeys were directly taken from the con-tainers used by the beekeepers for honey storage. Thesamples were stored in the laboratory at [20¡C untilanalysis. The characterisation of the samples as rose-mary honeys was achieved by physicochemical attrib-utes, pollen analysis and organoleptic analysis asdescribed in a previous work (Pe� rez-Arquillue� et al1994).

Commercial rosemary honey samples produced indi†erent Spanish regions (Table 1) were either pur-chased in the Spanish market, or provided by di†erentbeekeepers.

Rosemary pollen was collected from mature stamenanthers, and also was collected from bees (bee-pollen).The nature and purity of pollen was tested by classicalanalytical techniques (Louveaux et al 1978).

Analysis of the sediment of honey samples

The method followed for sediment of honey sampleswas described by Louveaux et al (1978). BrieÑy, a sub-sample of 10 g of honey was dissolved in 20 ml warmdistilled water (around 40¡C) and centrifuged twice(2000] g) for 10 min. The dry sediment was mountedon a slide with glycerine/gelatine slightly stained withan alcoholic solution of fuchsine. Slides were micro-scopically observed and compared with the reference foridentiÐcation. All the samples contained more than 20%of rosemary pollen. The absolute number of plant ele-ments in honey was evaluated according to MaurizioÏsclassiÐcation that divides honey into Ðve groups(Louveaux et al 1978). The results of the analyses areshown in Table 2.

TABLE 1Kaempferol content of commercial rosemary honey samples

produced in Spain

Sample Kaempferol content Region of(mg kg~1 honey) production

S-457 0É52 La ManchaS-305 0É72 La ManchaS-434 0É44 La ManchaS-462 0É44 La ManchaS-465 0É70 La ManchaS-459 1É39 La ManchaS-435 0É76 La ManchaM-86 0É60 AndaluciaM-85 0É33 AndaluciaM-84 0É34 AndaluciaM-83 0É68 LevanteM-82 1É21 LevanteM-81 0É86 LevanteM-80 0É88 LevanteM-79 0É78 LevanteM-78 0É75 LevanteM-77 1É90 LevanteM-42 2É17 LevanteM-41 2É48 LevanteM-40 1É64 LevanteM-3 1É46 LevanteM-1 1É67 Levante

Pollen Ñavonoid analysis

Rosemary pollen was extracted with methanol/water(7 : 3 ; v/v) at room temperature overnight. The extractwas Ðltered through Whatman no 1 paper, concentratedto dryness under reduced pressure (40¡C), redissolved inmethanol and Ðltered through 0É45 lm before HPLCanalysis.

The extracts (20 lL) were injected in a Li ChrochartRP-18 column (Merck, Darmstadt, Germany)(12É5 ] 0É4 cm, 5 lm particle size), using as mobilephase water/formic acid (19 : 1 ; v/v) (solvent A) andmethanol (solvent B). Elution was isocratic with 35% Band 65% A with a solvent Ñow rate of 1 ml min~1 andchromatograms recorded at 350 nm. UV spectra of thedi†erent peaks were recorded with a diode array detec-tor.

Pollen Ñavonoids identiÐcation

The Ñavonoids present in the extract were chromato-graphed on Whatman no 1 paper with n-

(4 : 1 : 5, v/v, upper phase) and twoBuOH/HOAc/H2OÑavonoid fractions were detected under UV light(360 nm) and eluted with methanol. The purity of the

508 F Ferreres et al

TABLE 2Kaempferol and pollen content in rosemary honeys from

Arago� n

Sample Maurizio % Rosemary KaempferolPollen pollen (mg kg~1 honey)groupa

16T I 22 0É6245M I 33 0É6611M I 27 0É6828M I 20 0É7750T I 31 0É8434M I 20 0É8719H I 20 0É8735M I 20 1É0547M I 28 1É104M I 41 1É13

39M I 31 1É1437M I 29 1É1823M II 29 0É8424M II 23 0É9049T II 35 0É9558T II 31 0É994H II 25 1É02

48T II 26 1É0642M II 36 1É1848M III 21 0É8658M III 20 2É07

a I, \2000 grains g~1 ; II, 2000È10 000 grains g~1 ;III, 10 000È50 000 grains g~1 of honey.

compounds was tested by HPLC with the abovedescribed conditions. The two Ñavonoids were identiÐedby their UV spectra in methanol and after addition ofthe classical shift reagents (Mabry et al 1970) and by theidentiÐcation of the acid hydrolysis products (Ñavonoidaglycones and sugars). The aglycones were identiÐed bythe UV spectra and HPLC analysis against authenticmarkers, and the sugars by paper chromatography onWhatman no 1 paper using phenol/water 4 : 1 (w/v).

Extraction and analysis of Ñavonoids from honey

The Ñavonoids present in honey were extracted as pre-viously described (Ferreres et al 1991 ; Gil et al 1995)using an Amberlite XAD-2 column to remove polarcompounds and adsorb phenolics which were theneluted with methanol. The methanol extract containingthe phenolics was concentrated to dryness underreduced pressure (40¡C), extracted with ethyl ether and,after solvent removal, redissolved in methanol (0É5 ml).This was Ðltered through 0É45 lm and analysed byHPLC under the conditions previously reported (Gil etal 1995). Kaempferol was identiÐed and quantiÐed by

chromatographic comparison with an authentic marker,using diode-array detection.

RESULTS AND DISCUSSION

Analysis of rosemary pollen Ñavonoids

Pollen grains collected directly from rosemary maturestamens, and from rosemary bee-pollen (pollen collectedby bees), were extracted and the Ñavonoids analysed byHPLC. Both pollens presented an identical ÑavonoidproÐle, indicating that pollen collection, transport andstorage by the bee does not a†ect Ñavonoid composi-tion. These results are consistent with previouslypublished data on the Ñavonoid composition of di†er-ent bee-pollens (Toma� s-Barbera� n et al 1989). A typicalHPLC chromatogram of the Ñavonoids from rosemarypollen is shown in Fig 1. It is clear that a kaempferolglycoside is the main component of this pollen. Inter-estingly, the Ñavonoids present in rosemary nectarincluded a quercetin glycoside as a minor Ñavonoid,although a kaempferol derivative was the main phenoliccompound present (Gil et al 1995).

The Ñavonoids present in rosemary pollen (A and B)were isolated by paper chromatography, and identiÐedas kaempferol 3-glycosides by UV spectrophotometry inmethanol and after the addition of the classical shiftreagents (Mabry et al 1970) (which indicated akaempferol-type oxigenation with free hydroxyls in 5, 7and 4@ positions and that the hydroxyl in 3-position wasblocked). After acid hydrolysis the aglycone and sugarsreleased were identiÐed by chromatographic compari-sons with authentic markers. Compound A was tentati-vely identiÐed as kaempferol 3-diglucoside, whichco-chromatographed with a marker of this compoundpreviously identiÐed in almond pollen (Ferreres et al1989), and compound B as kaempferol 3-glucoside.

The previous data show that rosemary pollen Ñavo-noids could be a source of the kaempferol detected in

Fig 1. HPLC chromatogram of rosemary pollen Ñavonoids.A, Kaempferol 3-diglucoside ; B, kaempferol 3-glucoside.

Pollen as a source of kaempferol in honey 509

rosemary honey. Could the compounds present in thepollen exine be transferred to the honey and be hydro-lysed there by the bee enzymes, giving the aglycone? Inorder to answer this question, pollen and kaempferolcontent of selected honey samples was determined.

HPLC analysis of honey Ñavonoids

The kaempferol content of the experimental rosemaryhoney samples produced in Arago� n (Spain) rangedbetween 0É62 and 2É07 mg kg~1 honey (Table 2). Thesevalues were in the same range as those of di†erent com-mercial samples produced in di†erent Spanish regions(Table 1), in which the content of this constituentranged between 0É33 and 2É48 mg kg~1 honey.

The pollen analysis of the experimental rosemaryhoney samples (Table 2), indicated that the samplescould be allocated in the three Maurizio pollen contentgroups. Thus, twelve samples were placed in group I(\2000 pollen grains per gram of honey), seven ingroup II (between 2000 and 10 000 pollen grains g~1)and only two in group III (between 10 000 and 50 000pollen grains g~1). The percentage of rosemary pollenin these samples was between 20% and 40%, valueswhich fall in the range of rosemary monoÑoral honey(Pe� rez-Arquillue� et al 1988). If pollen is a source forhoney Ñavonoids, there should be signiÐcant di†erencesbetween the kaempferol content of honeys located indi†erent Maurizio groups. Figure 2 shows a representa-tion of the kaempferol content vs the three di†erentMaurizio groups, and it shows that there is no corre-lation between kaempferol and pollen content.However, the sample with the highest kaempferolcontent (58M) belongs to the group richer in pollen(group III), and the sample with the lowest kaempferolcontent (16T) belongs to group I which is the poorest inpollen.

Therefore, it can be concluded that although rose-mary pollen only contains kaempferol derivatives, these

Fig 2. Correlation between honey kaempferol content(mg kg~1 honey) and pollen content. Pollen content expressedas Maurizio groups. I, \2000 pollen grains g~1 of honey ; II,2000È10 000 grains g~1 ; III, 10 000È50 000 grains g~1 of

honey.

Ñavonoids do not contribute in a signiÐcant manner tothe kaempferol content of rosemary honey, and kaemp-ferol present in this rosemary honey should originatefrom nectar as a major source. In addition, these resultssuggest that the search for phenolic markers of thebotanical origin of honey should be addressed to theidentiÐcation of nectar Ñavonoids or other nectar con-stituents.

ACKNOWLEDGEMENTS

The authors are grateful to the Spanish CICYT forÐnancial support of this work (Grants ALI 91-0486 andALI 92-0151).

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