Phenolic acids in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and their potential for floral authentication

Food Research International 38 (2005) 651–658 www.elsevier.com/locate/foodres

Phenolic acids in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and their potential for floral authentication Lihu Yaoa

b

a,c

, Yueming Jiang a,*, Riantong Singanusong b, Nivedita Datta c, Katherine Raymont d

a South China Botanic Garden, The Chinese Academy of Sciences, Guangzhou ReYiJu 510650, The PeopleÕs Republic of China Department of Agro-Industry, Faculty of Agriculture, Natural Resources and Environmental Sciences, Naresuan University, Muang, Phitsanulok 65000, Thailand c School of Land and Food Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia d School of Agriculture and Horticulture, The University of Queensland, Gatton, Queensland 4343, Australia

Received 4 November 2004; accepted 8 January 2005

Abstract Eight phenolic acids and two abscisic acid isomers in Australian honeys from five botanical species (Melaleuca, Guioa, Lophostemon, Banksia and Helianthus) have been analyzed in relation to their botanical origins. Total phenolic acids present in these honeys range from 2.13 mg/100 g sunflower (Helianthus annuus) honey to 12.11 mg/100 g tea tree (Melaleuca quinquenervia) honey, with amounts of individual acids being various. Tea tree honey shows a phenolic profile of gallic, ellagic, chlorogenic and coumaric acids, which is similar to the phenolic profile of an Australian Eucalyptus honey (bloodwood or Eucalyptus intermedia honey). The main difference between tea tree and bloodwood honeys is the contribution of chlorogenic acid to their total phenolic profiles. In Australian crow ash (Guioa semiglauca) honey, a characteristic phenolic profile mainly consisting of gallic acid and abscisic acid could be used as the floral marker. In brush box (Lophostemon conferta) honey, the phenolic profile, comprising mainly gallic acid and ellagic acid, could be used to differentiate this honey not only from the other Australian non-Eucalyptus honeys but also from a Eucalyptus honey (yellow box or Eucalyptus melliodora honey). However, this Eucalyptus honey could not be differentiated from brush box honey based only on their flavonoid profiles. Similarly, the phenolic profile of heath (Banksia ericifolia) honey, comprising mainly gallic acid, an unknown phenolic acid (Ph1) and coumaric acid, could also be used to differentiate this honey from tea tree and bloodwood honeys, which have similar flavonoid profiles. Coumaric acid is a principal phenolic acid in Australian sunflower honey and it could thus be used together with gallic acid for the authentication. These results show that the HPLC analysis of phenolic acids and abscisic acids in Australian floral honeys could assist the differentiation and authentication of the honeys.
2005 Elsevier Ltd. All rights reserved. Keywords: Phenolic acids; Honeys; Melaleuca; Guioa; Lophostemon; Banksia; Helianthus; Floral authentication

1. Introduction The phytochemical constituents of a honey play important roles in determining the quality of the honey, *

Corresponding author. Tel.: +86 203 725 2525; fax: +86 203 725 2831. E-mail address: [email protected] (Y. Jiang). 0963-9969/$ - see front matter
2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.01.002

and are closely associated with the botanical/floral origin of the honey (Cherchi, Spanedda, Tuberoso, & Cabras, 1994). These floral related constituents can be divided into two main groups of chemical compounds, volatiles and non-volatiles. The honey flavours hence the organoleptic quality are contributed by the volatile compounds, which include hydrocarbons (Bonaga, Giumanini, & Gliozzi, 1986), phenylalanine decomposition products

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(Speer & Montag, 1987), aromatic acrboxylic acids and their esters (Steeg & Montag, 1988) or organic acids (Cherchi et al., 1994), abscisic acid (Ferreres, Andrade, & Toma´s-Barbera´n, 1996a, Toma´s-Barbera´n, Martos, Ferreres, Radovic, & Anklam, 2001), degraded carotenoids (Tan, Wilkins, Holland, & McGhie, 1989), and aromatic aldehydes (Ha¨usler & Montag, 1990). In contrast, the taste, colour and other physical properties hence the overall quality of honey are contributed by the non-volatile compounds, which include sugars (Gomez-Barez et al., 2000; Mateo & Bosch-Reig, 1997; Serra-Bonvehi & Ventura-Coll, 1995), amino acids (Anklam, 1998; Davies, 1975, 1976; Davies & Harris, 1982), minerals (Anklam, 1998; Gonzalez-Paramas et al., 2000), and phenolic compounds (Amiot, Aubert, Gonnet, & Tacchini, 1989; Toma´s-Barbera´n et al., 2001). Interests in phenolic compounds have been increased in objective determination of the botanical (Amiot et al., 1989; Toma´s-Barbera´n, Toma´s-Lorente, Ferreres, & Garcı´a-Viguera, 1989; Toma´s-Barbera´n et al., 2001) and/or geographical (Martos, Ferreres, & Toma´s-Barbera´n, 2000a, 2000b; Toma´s-Barbera´n, Ferreres, Garcı´a-Viguera, & Toma´s-Lorente, 1993) origins of honeys in the past decades since the application of HPLC technology to honey analysis. Phenolic compounds occurring in honey have been classified into three groups: flavonoids, cinnamic acids and benzoic acids (Amiot et al., 1989). Some researchers classified phenolic compounds into two groups: phenolic acids including phenolic esters (Amiot et al., 1989; Andrade, Ferreres, & Amaral, 1997a; Andrade, Ferreres, Gil, & Toma´s-Barbera´n, 1997b; Ferreres, Andrade, Gil, & Toma´s-Barbera´n, 1996b; Sabatier, Amiot, Tacchini, & Aubert, 1992) and flavonoids (Ferreres, Andrade, & Toma´s-Barbera´n, 1994a; Ferreres, Garcı´a-Viguera, Toma´s-Lorente, & Toma´sBarbera´n, 1993; Ferreres, Giner, & Toma´s-Barbera´n, 1994b; Ferreres, Toma´s-Barbera´n, Gil, & Toma´sLorente, 1991; Ferreres et al., 1992; Martos et al., 2000a, 2000b). The contribution of these compositions to the generic phenolic profiles varies among honeys of different botanical origins (Amiot et al., 1989), which can be thus used in differentiating the floral sources hence the quality of a honey (Cherchi et al., 1994). Furthermore, the concentration of phenolics also varies among honeys of different origins (Vivar-Quintana, Baldi-Coronel, Sanchez-Sanchez, & Santos-Buelga, 1999), which can be used in assisting the differentiation. In the analysis and authentication of Australian (DÕArcy, Rintoul, Rowland, & Blackman, 1997; Rowland, Blackman, DÕArcy, & Rintoul, 1995) and New Zealand (Tan, Wilkins, Holland, & McGhie, 1990; Wilkins, Lu, & Tan, 1993) honeys, characteristic volatile compounds in the selected unifloral honeys have been identified and related to the botanical origins of honeys. Studies on flavonoids in Australian non-Eucalyptus honeys such as jelly bush (Leptospermum polygalifolium)

honey (Yao et al., 2003), tea tree (Melaleuca quinquenervia) and heath (Banksia ericifolia) honeys (Yao et al., 2004a) show that individual or grouped flavonoids are characteristic only to the honeys with similar floral/ botanical origins. However, some honeys with different botanical sources show similar flavonoid profiles thus could not be differentiated by their flavonoid profiles. In this case, phenolic acids and abscisic acid have been considered as the subsidiary phytochemical constituents to provide an authentication of botanical origins of these Australian honeys. The phenolic acids (Ferreres et al., 1996a; Joerg, 1996; Joerg & Sontag, 1993; Vivar-Quintana et al., 1999) and abscisic acid (Ferreres et al., 1996a) have been previously used as floral markers for honeys from other countries. The present work aims the analysis of the phenolic acids and abscisic acid in the selected non-Eucalyptus honeys from Australia, and the objective determination of the floral markers for these honeys using these compounds are also explored.

2. Materials and methods 2.1. Honey samples The honey samples analysed for this study were supplied by individual Australian apiarists, who sourced from the main honey production regions of New South Wales (NSW) and Queensland (Qld.), during the corresponding flowering season. The geographical locations, botanical origins and sourcing dates of these honeys are detailed in Table 1. All the samples were stored in a freezer at a temperature of 18 to 24 C prior to analysis. 2.2. Sample extraction (column chromatography) Extraction was carried out as described previously (Martos, Cossentini, Ferreres, & Toma´s-Barbera´n, 1997; Martos et al., 2000a, 2000b; Yao et al., 2003, 2004a). In brief, honey samples (100 g) were thoroughly mixed with five parts (500 ml) of distilled water, adjusted to pH 2 with concentrated HCl, until completely fluid. The fluid samples were then filtered through cotton wool to remove the solid particles. The filtrate was mixed with 150 g Amberlite XAD-2 (Supelco, Bellefonte, PA, USA, pore size 9 nm, particle size 0.3–1.2 mm) and stirred in a magnetic stirrer for 10 min, which was considered enough to absorb honey phenolics (including flavonoids, phenolic acids and abscisic acid) with a recovery rate more than 80% (Martos et al., 1997; Toma´s-Barbera´n, Blazquez, Garcı´a-Viguera, Ferreres, & Toma´s-Lorent, 1992; Yao et al., 2003, 2004a). The Amberlite particles were then packed in a glass column (42 · 3.2 cm) and the column was washed with acidified water (pH 2 with

L. Yaoa et al. / Food Research International 38 (2005) 651–658

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Table 1 Australian honey samples used in this experimenta Sample code

Common name

Floral origin

Year

Geographical origin

T/T LT1624 T/T LT0456 T/T A3940 C/Ash A0482 C/Ash Z4767 C/Ash A5144 C/Ash A0888 C/Ash A0629 C/Ash A0485 B/Box Z8258 B/Box Z7537 Heath Z9303 Heath A5433 Heath A3939a Heath A3939b S/F A5989

Tea tree Tea tree Tea tree Crow ash Crow ash Crow ash Crow ash Crow ash Crow ash Brush box Brush box Heath Heath Heath Heath Sunflower

Melaleuca quinquenervia Melaleuca quinquenervia Melaleuca quinquenervia Guioa semiglauca Guioa semiglauca Guioa semiglauca Guioa semiglauca Guioa semiglauca Guioa semiglauca Lophostemon conferta Lophostemon conferta Banksia ericifolia Banksia ericifolia Banksia ericifolia Banksia ericifolia Helianthus annuus

1999 1999 1998 1997 1994 1998 1998 1997 1997 1995 1995 1995 1998 1998 1998 1999

Curra County, Qld. Kempsey, NSW Woodburn, NSW North Coast, NSW Woodburn, NSW North Coast, NSW North Coast, NSW North Coast, NSW North Coast, NSW Gibralter Range, NSW Gibralter Range, NSW North Coast, NSW North Coast, NSW North Coast, NSW North Coast, NSW Allora, Qld.

a In this study, the aroma, taste and colour characteristics of the species-specific floral honey, together with information about season, hive location and available floral sources were utilised by supplying apiarists and a honey packer to accurately identify the floral source of the honey samples examined. This procedure is the standard honey-sourcing method utilised by the Australian honey industry (Yao, 2002).

HCl, 250 ml) and subsequently rinsed with distilled water (300 ml) to remove all sugars and other polar constituents of the honey. The phenolics remain absorbed on the column (Ferreres et al., 1991) and could be eluted with methanol. The abscisic acid of the honey appears to have a similar chromatographic behaviour thus should remain in the phenolic fraction. The whole phenolic fraction was then eluted with methanol (400 ml). This extract was concentrated to dryness under reduced pressure in a rotary evaporator at 40 C. The residue was redissolved in distilled water (5 ml) and extracted with diethyl ether (5 ml · 3). The ether extracts were combined, and the diethyl ether was removed by flushing with nitrogen. The dried residue was then redissolved in 1 ml of methanol (HPLC grade) and filtered through a 0.45 lm membrane filter, ready for HPLC analysis. 2.3. HPLC Analysis Analyses of the extracts from the selected Australian honeys were carried out using a Shimadzu Class-VP HPLC system with a computer-controlled system containing upgraded Class-VP 5.03 software. Separations were carried out on a reversed phase column LiChroCART RP-18 (Merck, Darmstadt, Germany; 12.5 cm · 0.4 cm, particle size 5 lm), using a mobile phase of water–formic acid (19:1, v/v) (solvent A) and methanol (solvent B) at a constant solvent flow rate of 1 mL/min. The following gradient was used, according to the method of Martos et al. (1997, 2000a, 2000b): 30% methanol (B) flowed through the column isocratically with solvent A for 15 min; and then was increased to 40% methanol at 20 min, 45% methanol at 30 min, 60% methanol at 50 min, 80% methanol at 52 min, and 90% methanol at 60 min. Finally, isocratic elution with

90% methanol was done until 65 min. The honey extracts were injected with a SIL-10A XL Auto Injector and the phenolics were detected using a multichannel photodiode-array detector (SPD-M10A VP) to obtain the UV spectra of phenolics. In addition, the chromatograms were monitored at 290 and 340 nm, since the majority of the honey phenolics show their UV absorption maxima around these two wavelengths (Martos et al., 1997, 2000a, 2000b; Yao et al., 2003). The phenolics were identified and quantified according to the method reported previously (Andrade et al., 1997a, 1997b; Martos et al., 1997; Yao et al., 2003). Briefly, honey phenolics were identified by comparing their UV spectra and retention times against authentic compounds. When the authentic compounds for some compounds were unavailable, the stored UV spectra extracted from the same HPLC method for honey analysis (previous standard analytical data under same analytical conditions) and their corresponding retention times were utilised for the identification. In this study, the phenolic acids such as gallic, chlorogenic and coumaric acids were quantified against their standards at 290 nm. Ellagic acid was quantified against its standard at 340 nm. The abscisic acid, including both trans,trans- and cis,trans-isomers, were determined against the standard at 290 nm. The accuracy of the quantification for all the phenolic compounds is assured by closely following the similar work done on Australian and European honeys (Martos et al., 2000a; Martos et al., 2000b; Yao, 2002; Yao et al., 2003; Yao et al., 2004a; Yao, Jiang, Singanusong, Datta, & Raymont, 2004b). Two extractions were carried out for each sample and two HPLC analyses were done for each extraction. Only those compounds having a recovery more than 80%, calculated by on their response factors to their detection wavelength and retentions (Yao,

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2002), were analysed for their representative roles as honey phenolics for the analysis of potential floral authentication for Australian honey.

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3. Results and discussion

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3.1. Phenolic acids in Australian tea tree (M. quinquenervia) honeys

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The HPLC chromatogram of tea tree honey (Fig. 1A) shows that the main phenolic acids are gallic and coumaric acids. The content of total phenolic acids in this honey is 12.11 mg/100 g honey, with gallic acid being the most abundant (4.52 mg/100 g honey) (Table 2). Gallic acid represents 36.1%, whereas ellagic, chlorogenic and coumaric acids represent 14.0%, 14.0% and 10.8% of total phenolic acids, respectively (Table 2). Caffeic acid and an unknown phenolic acid (Ph1, described previously, Yao et al., 2003) are present in small amounts, with syringic and ferulic acids being present in minor amounts. This profile of phenolic acids in tea tree honey is very similar to that of bloodwood (Eucalyptus intermedia) honeys (Yao et al., 2004b). The main difference in their phenolic profiles between these honeys is the different percentages of chlorogenic acids to their to-

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Fig. 1. HPLC chromatograms of phenolic acids and abscisic acid in Australian tea tree (M. elaleuca quinquenervia) (A) and crow ash (Guioa semiglauca) (B) honeys (290 nm). Phenolic acids are: (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid, (4) p-coumaric acid, (5) ferulic acid, (6) o-coumaric acid, (7) ellagic acid and (8) trans,transabscisic acid (peaks of flavonoids are not numbered).

Table 2 Content of phenolic acids of Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys Sample

Content of phenolic acids (mg/100 g honey)a,b GA

CA

Ph1

Caf

Cou

Fer

Ell

Syr

Total

T/T LT1624 T/T LT456 T/T A3940 Mean S.D. C/Ash A0482 C/Ash Z4767 C/Ash A5144 C/Ash A0888 C/Ash A0629 C/Ash A0485 Mean S.D. B/Box Z8258 B/Box Z7537 Mean S.D. Heath Z9303 Heath A5433 Heath A3939a Heath A3939b Mean S.D. S/F A5989

7.45(40.5) 1.94(32.9) 4.18(34.8) 4.52(36.1) 2.77(3.9) 3.29(38.4) 4.59(48.6) 5.45(45.3) 6.36(53.5) 1.69(25.7) 3.26(45.7) 4.11(42.8) 1.69(9.7) 1.17(34.9) 1.61(28.2) 1.39(31.5) 0.31(4.7) 1.03(31.1) 6.09(56.2) 2.21(35.6) 5.20(59.0) 3.63(45.5) 2.40(14.2) 0.54(25.2)

1.24(6.7) 0.70(11.9) 2.81(23.4) 1.58(14.0) 1.10(8.5) 0.73(8.5) 0.65(6.9) 0.84(7.0) 2.18(18.3) 0.78(11.8) 0.40(5.6) 0.93(9.7) 0.63(4.7) 0.16(4.8) 0.34(5.9) 0.25(5.3) 0.12(0.8)

2.82(15.3) 0.80(13.6)

1.32(7.2) 0.66(11.2) 1.24(10.4) 1.08(9.6) 0.36(2.1) 0.59(6.9) 0.79(8.4) 1.29(10.8) 0.41(3.5) 0.55(8.4) 0.72(10.1) 0.73(8.0) 0.31(2.6) 0.08(2.2) 0.39(6.8) 0.23(4.5) 0.22(3.2) 0.19(5.7) 0.83(7.7) 0.64(10.2) 0.77(8.8) 0.61(8.1) 0.29(1.9) 0.15(7.2)

2.37(12.9) 0.73(12.4) 0.85(7.1) 1.32(10.8) 0.91(3.2) 0.88(10.2) 1.25(13.3) 2.00(16.6) 0.71(5.9) 0.54(8.2) 0.95(13.3) 1.05(11.3) 0.52(3.9) 0.28(8.2) 0.73(12.7) 0.50(10.5) 0.32(3.2) 0.56(16.8) 1.06(9.8) 1.19(19.2) 1.01(11.4) 0.95(14.3) 0.27(4.4) 0.70(32.9)

0.68(3.7) 0.24(4.1) 0.22(1.8) 0.38(3.2) 0.26(1.2) 0.28(3.3) 0.50(5.3) 0.31(2.6) 0.21(1.8) 0.81(12.4) 0.36(5.1) 0.41(5.1) 0.22(3.8) 0.48(14.2) 0.21(3.7) 0.34(8.9) 0.19(7.4) 0.53(16.0) 0.11(1.0) 0.26(4.2) 0.15(1.7) 0.26(5.7) 0.19(7.0) 0.13(6.2)

1.56(8.5) 0.83(14.0) 2.36(19.7) 1.58(14.0) 0.77(5.6) 1.58(18.4) 0.90(9.5) 0.76(6.3) 0.58(4.9) 0.72(10.9) 0.67(9.5) 0.87(9.9) 0.36(4.7) 0.82(24.3) 1.55(27.2) 1.19(25.7) 0.52(2.0) 0.41(12.2) 0.45(4.2) 0.40(6.5) 0.31(3.5) 0.39(6.6) 0.06(4.0) 0.18(8.2)

0.98(5.3)

18.43 5.90 12.01

a

0.66(6.1) 0.39(6.3) 0.43(4.9) 0.49(4.3) 0.15(2.9) 0.20(9.5)

1.81(9.6) 1.43(8.4) 0.69(8.0) 0.76(8.1) 1.37(11.4) 1.22(10.3) 0.68(10.3) 0.77(10.8) 0.92(9.8) 0.30(1.4) 0.27(8.0) 0.62(10.9) 0.44(9.4) 0.25(2.0) 0.61(18.2) 1.63(15.0) 1.12(18.0) 0.95(10.8) 1.08(15.5) 0.43(3.5) 0.23(10.8)

0.35(2.9) 0.66(4.1) 0.45(1.7) 0.54(6.3)

0.22(1.9) 0.82(12.4) 0.53(6.9) 0.30(5.3) 0.12(3.5) 0.27(4.8) 0.20(4.1) 0.11(0.9)

8.58 9.46 12.03 11.89 6.59 7.13

3.37 5.72

3.33 10.83 6.21 8.82

2.13

GA, gallic acid; CA, chlorogenic acid; Caf, caffeic acid; Cou, coumaric acid; Fer, ferulic acid; Ell, ellagic acid; Syr, syringic acid; Ph1, unknown phenolic acid. b Values in parentheses are % of each individual phenolic acid in the total phenolic acids detected in the samples.

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tal phenolic acids, which is 14.0% in tea tree honey and 29.6% in bloodwood honey. In addition, the level of total phenolic acids detected in Australian tea tree honey (12.11 mg/100 g honey) is the highest among the Australian honeys analysed so far, which, together with the phenolic profile or constituents, could be related to the floral origin of tea tree honey.

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3.2. Phenolic acids in Australian crow ash (Guioa semiglauca) honeys

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3.3. Phenolic acids in Australian brush box (Lophostemon conferta) honey In Australian brush box honey, the content of total phenolic acids is 4.54 mg/100 g honey, with gallic and ellagic acids as the main contributors (1.39 and 1.19 mg/ 100 g honey, respectively) (Table 2). The HPLC chromatogram of brush box honey (Fig. 2A) shows that gallic, coumaric, ferulic and ellagic acids are the main components of the phenolic acid profile. Gallic and ellagic acids represent 31.5% and 25.7% of total phenolic acids, respectively, with coumaric acid, an unknown phenolic acid (Ph1) and ferulic acid present in much smaller amounts (Table 2). This phenolic profile (Fig. 2A) for brush box honey, comprising mainly gallic and

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In Australian crow ash (G. semiglauca) honey, the HPLC chromatogram (Fig. 1B) shows a characteristic profile comprised of gallic acid and trans,trans-abscisic acid. The content of total phenolic acids is 9.28 mg/ 100 g honey, with gallic acid dominating the profile and representing 42.8% of the total phenolic acids (Table 2). These results demonstrate that crow ash honey can be readily differentiated from other Australian honeys, including Eucalyptus honeys (Yao et al., 2004b), based on its profile of phenolic acids, or the occurrence of abscisic acid as a main component (Table 3). The level of total phenolic acids detected in crow ash honey (9.28 mg/100 g honey) is the third highest amount in the Australian honeys analysed so far, only lower than tea tree honey the first highest (Table 2) and then bloodwood honey (Yao et al., 2004b) the second highest. Thus, this phenolic profile and the constituents could be used as the floral markers for crow ash honey.

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Fig. 2. HPLC chromatograms of phenolic acids and abscisic acid in Australian brush box (Lophostemon conferta) (A) and heath (Banksia ericifolia) (B) honeys (290 nm). Phenolic acids are: (1) gallic acid, (2) chlorogenic acid, (3) cafeic acid, (4) p-coumaric acid, (5) ferulic acid, (7) ellagic acid and (8) trans,trans-abscisic acid (peaks of flavonoids are not numbered).

ellagic acids, could be used to differentiate this type of honey from Australian yellow box (Eucalyptus melliodora) honey since they could not be differentiated based on their flavonoid profiles (Yao et al., 2004a). For this differentiation, gallic acid represents 72.7% of the total phenolic acids present in yellow box honey, but only 31.5% of total phenolic acids in brush box honey. It is noteworthy that the maximum absorption of the ellagic acid in the UV range is about 325 nm. Thus, it cannot be visualised as a main peak in Fig. 2A as those for the other phenolic acids. 3.4. Phenolic acids in Australian heath (B. ericifolia) honeys In Australian heath honey, the HPLC chromatogram contains gallic and coumaric acids as the main components of the phenolic profile (Fig. 2B). The content of total phenolic acids is 7.30 mg/100 g honey, with gallic

Table 3 Abscisic acid (ABA) in various Australian honeys Common name

Botanical name

Content of abscisic acid (mg/100 g honey) trans,trans-ABA

cis,trans-ABA

Total

Tea tree Crow ash Brush box Heath Sunflower

Melaleuca quinquenervia Guioa semiglauca Lophostemon conferta Banksia ericifolia Helianthus annuus

2.09 3.98 0.63 1.91 8.70

1.21 1.77 0.89 0.71 0.65

3.30 5.75 1.52 2.62 9.35

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acid as the main component (3.63 mg/100 g honey) (Table 2). Gallic acid represents 45.5% of total phenolic acids, whereas an unknown phenolic acid (Ph1) and coumaric acid represent 15.5% and 14.3%, respectively (Table 2). This profile of phenolic acids, comprising mainly gallic acid and secondly an unknown phenolic acid (Ph1) and coumaric acid, is characteristic to Australian heath honey only and could be used as its floral marker. This finding can assist the differentitation of heath honey from tea tree honey and bloodwood honey, which could not be differentitated by their flavonoid profiles (Yao et al., 2004a). 3.5. Phenolic acids in Australian sunflower (Helianthus annuus) honey The content of total phenolic acids in Australian sunflower (H. annuus) honey is only 2.13 mg/100 g honey, of which coumaric acid contributes 32.9% and gallic acid contributes 25.2% (Table 2). The profile of phenolic acids mainly comprising coumaric and gallic acids is characteristic only to this honey and could be used to authenticate its floral origin. However, since there was only one available sample analysed in this experiment, further analysis on a larger number of Australian sunflower honeys is necessary to confirm this profile of phenolic acids as floral markers for this honey. 3.6. Comparison of phenolic acids in Australian Melaleuca, Guioa, Lophostemon, Banksia, and Helianthus honeys with those found in honeys from other countries The levels of total phenolic acids present in Australian non-eucalyptus honeys range from 2.13 mg/100 g sunflower honey to 12.11 mg/100 g tea tree honey (Table 2), showing variable levels in various honeys of different floral origins. The levels of phenolic acids in these Australian honeys are lower than the level of New Zealand manuka honey (13.99 mg/100 g honey) analysed in an earlier study (Yao et al., 2003). However, these levels are higher than those found in honeys produced in other countries, of which Joerg and Sontag (1992) showed an average total phenolic acids in various honeys being at a level of 3.5 mg/100 g honey. For individual phenolic acid, ellagic acid was detected in heather honey at levels of 0.3–1.1 mg/100 g honey (Toma´s-Barbera´n et al., 2001). In the strawberry tree honey, Cabras et al. (1999) found that a phenolic acid (homogentistic acid) varied 19.7–54.0 mg/100 g honey, with an average of 37.8 mg/100 g honey. This is the highest level of single phenolic acid reported in honey so far, and this level is higher than the level of total phenolic acids found for any floral type of honey examined in this study. Homogentistic acid has thus been suggested as a floral marker for strawberry tree (Arbutus unedo) honey (Cabras

et al., 1999), since it was not detected in any other monofloral honeys. 3.7. Abscisic acid in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys In an early study, it was found that the flavonoid profile of tea tree honey is very similar to that of Australian bloodwood honey (Yao et al., 2004a), and, thus, their differentiation based on their flavonoid profiles is not possible. Another approach has to consider a subsidiary tool such as using the content of abscisic and phenolic acids for such differentiation. In tea tree honey, the content of abscisic acid isomers is 2.90 mg/100 g honey (Table 3), lower than that found in bloodwood honey (4.99 mg/100 g honey). Thus, tea tree honey could be differentiated from bloodwood honey based on differences in the levels of abscisic acid. In other words, abscisic acid could be used together with the phenolic acids and/or flavonoids for honey floral authentication. In Australian crow ash (G. semiglauca) honey, the content of abscisic acid is 5.75 mg/100 g honey (Table 3). Abscisic acid is the characteristic component that may be useful in differentiating this type of honey from other honeys, as shown in the characteristic HPLC chromatogram (Fig. 1B). In comparsion to those in crow ash honey, the contents of abscisic acid in brush box (L. conferta) and heath (B. ericifolia) honeys are 1.52 and 2.62 mg/100 g honey, respectively (Table 3). They are the lowest levels of absicisic acids detected in the five Australian honeys of this study. However, abscisic acid contributes to the characteristic phenolic profiles to these two types of honeys (Fig. 2), which enables them to be differentiated from the other honey types. In contrast, abscisic acid in Australian sunflower (H. annuus) honey may be used as an important phytochemical marker for floral authentication due to its large concentration (9.35 mg/100 g honey; Table 3), which is more than twice that of the sum of its total phenolic acids (2.13 mg/100 g honey) (Table 2) and total flavonoids (1.79 mg/100 g honey) (Yao et al., 2004a). However, analysis of a larger number of samples is necessary to confirm this finding. The level of abscisic acid found in Australian honeys is variable, ranging 1.52 mg/100 g brush box honey to 9.35 mg/100 g sunflower honey, which is lower than the levels found in Australian jelly bush honey and New Zealand manuka honey (Yao et al., 2003). However, this range is close to the levels of abscisic acid in Portuguese heather honey (2.5–16.6 mg/100 g honey) (Ferreres et al., 1996a). Abscisic acid has been suggested as a floral marker for Portuguese heather honey (Ferreres et al., 1996a). Thus, the results of this study may also suggest that abscisic acid could be used as a complementary floral marker in association with the profiles of flavonoids and phenolic acids of various honeys.

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4. Conclusion In Australian crow ash honey, gallic acid is the dominant component in the profile of phenolic acids, which is characteristic to this honey, and, thus, could be used as a floral marker. The phenolic acid profile of Australian brush box honey comprises mainly gallic and ellagic acids as a characteristic profile and could be used to differentiate this type of honey from yellow box honey since the flavonoid profiles could not assist in their differentiation. The results of this study indicate that distribution of phenolic acids and abscsic acid in honeys is affected by the floral origins of honeys. Thus, honeys could be differentiated by the profiles of these phytochemical constituents. For honeys from similar floral origins, the profiles of phenolic acids or flavonoids cannot always assist in the differentiation. In these cases, contents of individual or total phenolic compounds may be used. A non-phenolic phytochemical constitute abscisic acid occurs in some honeys at high levels, and may also assist in the differentiation of species-specific floral types of honey. In conclusion, the results suggest that phenolic acids, together with abscisic acid and their profiles, could be used for floral authentication of Australian floral honeys. The HPLC analysis of these compounds should be a useful tool for objectively authenticating Australian species-specific floral honeys.

Acknowledgements We thank Mr. William G. Winner of Capilano Honey Ltd., Australia for supplying honey samples, Dr. Bruce DÕArcy of the University of Queensland for the discussion during the preliminary preparation of this work, and Drs. Brenda Mossel and Gavin Rintoul of the University of Queensland for their technical support and assistance with sample sourcing. Appreciation is also expressed to Department of Education, Science and Training (DEST, formerly DETYA, Australia) for providing an IPRS (formerly OPRS) fund support. Our gratitude is extended to Drs. Francisco A. Toma´s-Barbera´n, Federico Ferreres and Isabel Martos of Laboratorio de Fitoquı´mica, Department of Food Science and Technology, CEBAS (CSIC), Murcia, Spain for supplying some standards of honey phenolic acids and abscisic acid, and their discussion and technical support during this work.

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