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by Bruce R D’Arcy
May 2005
RIRDC Publication No 05/040 RIRDC Project No UQ-102A
Executive summary
Polyphenols in foods are
thought to play important roles in human health such as cancer preventative
and anti-inflammatory, radical scavenging and antioxidative activities.
The most important classes of antioxidant polyphenols are the flavonoids
and phenolic acids. It is these substances in tea, wine, fruits and vegetables
that are most responsible for the antioxidant characteristics, and thus
the healthy image of these foods. However, little data exist on these components
in Australian floral honeys, hence the need for this study.
Multiple samples of the three floral types of Australian honey, yapunyah, leatherwood and Salvation Jane, and one sample of spotted gum honey were examined for their antioxidant polyphenol concentrations through an extraction and HPLC/LC-MS analysis of flavonoids and phenolics.
However, before this could occur, a chemical analysis method for the extraction of antioxidant flavonoids and phenolic acids from honey using Amberlite XAD-2 resin was optimised, and recovery studies were done. During the optimisation of the Amberlite XAD-2 extraction method, it was found that, contrary to previous studies (Yao, 2002), the phenolic acids, gallic acid and ellagic acid were not retained on the resin during the extraction under the acidic conditions ideal for such retention. This suggests that these two phenolic acids could not be extracted from honey using Amberlite XAD-2 resin as indicated by Yao (2002). In addition, the percentage recovery of the phenolic acids, caffeic acid, p-coumaric acid and ferulic acid, reported by Yao (2002) to be in yapunyah and other honey types, varied between 15.5 and 62%. However, this does not mean that other unknown (not identified) phenolic acids detected in this study were not recovered in higher yields. In contrast, the extraction efficiencies for flavonoid standards such as quercetin, hesperetin and chrysin were much better, with the latter two having recoveries of >83%, in agreement with previous literature studies.
Next, identification and quantification of honey flavonoids and other polyphenols were done using high performance liquid chromatography (HPLC) with diode array detection (DAD) at 290 nm (phenolic acids) and 340 nm (flavonoids), and using liquid chromatography (LC) - mass spectrometry (MS) with negative ionisation, including the use of the sensitive, selective ion recording (SIR) mode.
HPLC and LC-MS methodology was developed so that separation of flavonoids and phenolic acids was maximised, enabling accurate quantification.
After LC-MS analysis of extracts from five samples of yapunyah honey using the mass spectral, selected ion recording (SIR) mode with negative ionisation, it was found that the phenolic acids, gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ellagic acid were detected in negligible concentrations (<13.2 µg/ 100 g), which suggests that the previous reporting of these phenolic acids in two samples of yapunyah honey in much higher concentrations by Yao (2002) was in serious error.
This was because the study of Yao (2002) did not involve LC-MS analysis in the identification of these phenolic acids, only HPLC-DAD analysis. Such an approach is fraught with possible error.
However, a number of other phenolic acids were detected in the yapunyah honey samples during the study reported here, but could not be identified, even with the use of LC-MS.
Flavonoids identified and quantified in the five yapunyah honey samples were tricetin, pinobanksin, quercetin, luteolin, quercetin 3-methyl ether, and 8-methoxy kaempferol, in agreement with Yao (2002). These flavonoids have been reported before in Eucalypt honeys, with the three flavonoids, tricetin, quercetin and luteolin being characteristic of Eucalypt honeys. The concentration of flavonoids in yapunyah honey samples ranged from 568.98 to 990.37 µg/100 g honey (mean of 747.64 µg/100 g honey), while the concentrations of phenolic acids ranged from 407.15 to 625.17 µg/100 g honey (mean of 510.82 µg/100 g honey). These were lower than previously reported for two samples of yapunyah honey by Yao (2002). The reason for this difference in concentrations is not known at this time, but may be due to different samples being used.
The study of ten leatherwood honey samples quantified the phenolic acid, caffeic acid, and the flavonoids, tricetin, pinobanksin, luteolin, pinocembrin, and chrysin, as well as many unidentified phenolic acids. The concentration of flavonoids in the ten leatherwood honey samples ranged from 638.7 to 1579.1 µg/100 g honey (mean of 901.2 µg/100 g honey), while the concentrations of phenolic acids ranged from 1177.8 to 2718.8 µg/100 g honey (mean of 2066.6 µg/100 g honey). Leatherwood honey did not contain many flavonoids, but was rich in many phenolic acids. This is a very interesting result when considered in the light of the high concentrations of volatiles (some of which were phenolic compounds) previously found in leatherwood honey by D’Arcy et al. (2001) in an earlier RIRDC project.
For the six Salvation Jane honey samples, the flavonoids, pinobanksin, luteolin, kaempferol and pinocembrin, and the phenolic acids, 4-hydroxyphenyllactic acid and ?-cyano-4-hydroxycinnamic acid were identified and quantified. In addition, there were a significant number of other flavonoids and phenolic acids quantified, whose identity could not be determined even with the use of LC-MS.
The concentration of flavonoids in the seven Salvation Jane honey samples ranged from 245.11 to 484.54 µg/100 g honey (mean of 407.19 µg/100 g honey), while the concentrations of phenolic acids ranged from 344.27 to 954.75 µg/100 g honey (mean of 732.01 µg/100 g honey).
However, even with the use of LC-MS, many polyphenols could not be identified, although they were quantified against a standard polyphenol to give some indication of their relative concentrations. A future more detailed study is needed to identify these polyphenols.
Amongst the yapunyah, leatherwood and Salvation Jane honeys, leatherwood honey has approximately 2-3 times the concentration of total flavonoids/phenolic acids than the other two honeys. This was also the case for the volatile compound concentration, with leatherwood honey containing a larger number and range of volatiles and in much higher concentrations than other Australian floral honeys (D’Arcy et al., 2001).
Finally, the implications of this study are that since only four floral types were studied, a detailed comparison between floral types, to determine which Australian honey type has the highest concentrations of antioxidant flavonoids and phenolic acids, is not possible at this time.
In conclusion, the scientific data generated during this project on the identity and concentration of antioxidant flavonoids and phenolic acids in Australian honey will enable the further marketing of honey as a healthy and nutritious food to the Australian food industry and consumers, in addition to its use as a sweetener.
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