Phytochemicals as Markersof the Floral Origin of Honey
F.A. Tomás-Barberán, A. Allende, P. Truchado (CSIC) Murcia
L. Bortolotti, A.G. Sabatini (CRA) Bologna
J. Simuth, K. Bilikova (SAS) Bratislava
Honey quality (floral origin)
• Consumer appreciates different
origins
• Floral origin, different price
• Unifloral honeys
– Bioactive constituents
– Flavour constituents
• Certify the origin. The Objective
Phytochemicals used for
plant chemotaxonomy• Different secondary metabolites
– Polyphenols– Terpenoids
– Alkaloids– Sulfur-containing Compounds
• They are stable and can be analysed by chromatographic methods.
• They have proved useful for plant species classification
• They have been used for the determination of origin of processed fruit products
Hypothesis
• Use the plant-derived metabolites present in honey for honey plant-origin determination
– They are good taxonomical markers
– They can be analysed
– They are chemically stable
– Are they modified during honey maturation?
Sources for honey plant
secondary metabolites
• Floral Nectar (most representative) ******
– Water soluble compounds (glycosides)
• Propolis/beeswax (unrelated to floral) *
– Lipophilic compounds (resins)
• Pollen (differences in pollen content) ***
– Confounding factor.
Methods for honey
phytochemicals extraction
• Amberlite XAD resins + Ether extraction– Lipophilic compounds
– Propolis-derived
– Nectar phytochemicals hydrolysed by bee enzymes (aglycones)
• Solid Phase Extraction Cartridges (SEP-PACK)– Both polar and lipophilic compounds
– Need HPLC-MS-MS
Amberlite XAD Extraction of Honey Phenolics
HPLC-DAD Analysis
HPLC-DAD-MS Analysis
Fate of phytochemicals in honey
• Nectar phytochemicals
– Flavonoid glycosides
• Glucosides
– Hydrolyzed by bee saliva glucosidases
• Non-glucosides
– Rhamnosides, galactosides
– Esters
– Non hydrolyzed by rhamnosidases, galactosidases, esterases.
Fate of phytochemicals in honey
• Propolis/beeswax phytochemicals
– Flavonoid and other phenolic aglycones
• Flavonoids
– Characteristic flavones and flavanones
– Flavonol-methyl ethers
• Hydroxycinnamates
– Dimethyl-allyl caffeate
– Phenyl-ethyl caffeate
mA
U
0
500
1000
1500
2000
2500PROPOLIS290 nm
PROPOLIS 340 nm
RETENTION TIME
0 10 20 30 40 50 60
mA
U
0
500
1000
1500
2000
OOOO
OOOO
OOOOHHHHOOOOHHHH
OOOO
OOOOOOOOHHHH
HHHHOOOO
OOOO
OOOOOOOOHHHH
HHHHOOOO
Strategies to study Floral nectar phytochemicals
• Direct analysis of floral nectar
– Robinia, Citrus, Eucalyptus, Tilia
• Bee-honey sacs analysis
– Rosemary, Chestnut
• Freshly deposited honey in the comb
– Diplotaxis tenuifolia
Samples studied
• Nectar samples– Robinia nectar (Italy)
– Tilia nectar (Italy)
– Chestnut (Italy)
– Eucalyptus (Spain)
– Citrus (Spain)
– Diplotaxis (Argentina)
• Authentic honey samples– Robinia and Acacia (Italy and Slovakia)
– Tilia and Linden (Italy ans Slovakia)
– Chestnut (Italy and Spain)
– Eucalyptus (Spain)
– Citrus (Spain)
– Diplotaxis (Argentina)
• Experimental honey with sucrose (Italy and Slovakia)
Floral nectar
• Acacia
• Tilia and Linden
• Citrus
• Eucalyptus
Acacia Honey
Robinia nectar
Robinia pseudacacia
NECTAR ROBINIA340 nm
RETENTION TIME (min)
10 20 30 40 50
m A
U
0
20
40
60
80
HPLC-DAD Analysis of Robinia Nectar
O
OH
O
OOH
O
rhamn
glc-rhamn-glc
O
OH
OOH
O
OOOO
rhamn
glc-rhamn
O
OH
OOH
O
OOOOHHHH
rhamn-rhamn-glc-rhamn
O
OH
OOH
O
OOOOHHHH
rhamn
HPLC-MS analysis of Robinia nectar
F1
F2
739.3
901.4
937.2 983.2
-MS, 17.0min (#1096)
755.2
-MS2(901.4), 17.0min (#1097)
284.7
338.8
593.1
-MS3(901.4->755.2), 17.1min (#1099)
0.0
0.2
0.4
0.6
0.8
1.0
7x10
Intens.
0.0
0.5
1.0
1.5
2.0
6x10
0.0
0.2
0.4
0.6
0.8
1.0
5x10
100 200 300 400 500 600 700 800 900 m/z
O
OH
O
OOH
O
rhamn
glc-rhamn-glc
O
OH
OOH
O
Ohex-rhamn-hex
O
OH
OOH
O
Ohex-rhamn
MS
MS2
MS3
ROBINIA NECTAR340 nm
m A
U
0
5
10
15
20
25
30
ROBINIA HONEY SEP-PAK340 nm
RETENTION TIME (min)
10 20 30 40 50 60
mA
U
0
50
100
150
ROBINIA HONEY ETER340 nm
mA
U
0
50
100
150
1*
F1
F2
O
OH
OOH
O
OOOO
rhamn
glc-rhamn
O
OH
O
OOH
O
rhamn
glc-rhamn-glcF1
F2
F3
F3
F3
F2
F2 O
OH
OOH
OOOO
HHHHOOOO
glc-rhamn
F3
Truchado et al., JAFC, 2008, 56, 8815
Conclusions Robinia Study
• The first time that flavonoid glycosides reported in honey
• This opens new possibilities of finding markers or methodologies to help in honey floral origin determinations
• Some flavonoids were degraded due to oxidation
Tilia nectar
Retention time (min)
0 10 20 30 40 50
mA
U
0
400
800
1200
mA
U
0
400
800
1200
1600
Detection of floral origin markers of
LINDEN and TILIA HONEY
Tilia honey
Floral markers: TERPENIC ACIDS
Linden honey
OOOOHHHHOOOO
HHHHOOOO
OOOO
HHHHOOOO
HHHHOOOO
HHHHOOOOOOOOHHHH
OOOO
OOOO
HHHHOOOOOOOOHHHH
HHHHOOOO
OOOOOOOO
OOOOHHHH
Cyclohexa-1-3-diene-
1-carboxilic acids
Cyclohexa-1-3-diene-1-
carboxilic acids gentiobioside
Retention time (min)
0 10 20 30 40 50 60
mA
U
0
100
200
300
400TILIA NECTAR
Citrus honey
10 20 30 40 50
0
20
40
mA
U
Retention time (min)
Detection of floral origin markers of ORANGE HONEY
HONEY
HESPERIDIN
Retention time (min)
10 20 30 40 50 60
mA
U
0
30
60
NECTAR
HESPERIDIN
HESPERIDIN
Floral markers: PHENOLIC COMPUNDS
(Flavanones )
Eucalyptus honey
Retention time (min)
0 10 20 30 40 50
mA
U
0
5
10
15
20
25
Detection of floral origin markers of EUCALIPTUS HONEY
NECTAR
MYRICETIN
TRICETIN
Floral markers: PHENOLIC COMPUNDS
(Aglicons in honey and Glucosides in nectar )
Retention time (min)
10 20 30 40 50
mA
U
0
20
40
60
80
LUTEOLIN
HONEY
TRICETIN SOPHOROSIDE
TRICETINLUTEOLIN
MYRICETIN
MYRICETIN SOPHOROSIDE
LEUTOLIN SOPHOROSIDE
Eucalyptus
Eucalyptus
E. melliodora (yellow box)
E. chamadulensis
E. pilligaensis (mallee)
E. tereticornis (red gum)
Honey bee sacs
• Chestnut
• Rosemary
• Heather
Chestnut honey
Detection of floral origin markers of CHESTNUT HONEY
nm260 280 300 320 340 360 3800
500
300
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700
nm260 280 300 320 340 360 3800
500
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0nm260 280 300 320 340 360 380
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0
nm260 280 300 320 340 360 3800
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nm260 280 300 320 340 360 3800
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nm260 280 300 320 340 360 380
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0nm260 280 300 320 340 360 380
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0
Time (min)
0 10 20 30 40 50 60
mA
U
0
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800
mA
U
0
200
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600
Time (min)
20 25 30 35
mA
U
0
50
100
150
L1
CH1
L2
F
CH1
CH2 CH5
Zoom
CH5
CH2
L2
CH1
L1
CH3
A
B
L1; L2
CH1
CH2
CH3
CH5
Ch
Pc M-Q
nm260 280 300 320 340 360 380
500
300
200
100
400
600
700
0nm260 280 300 320 340 360 380
500
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0
500
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700
0
OH
nm260 280 300 320 340 360 3800
500
300
200
100
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600
700
nm260 280 300 320 340 360 3800
500
300
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nm260 280 300 320 340 360 380
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0nm260 280 300 320 340 360 380
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0
nm260 280 300 320 340 360 3800
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700
nm260 280 300 320 340 360 3800
500
300
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700
nm260 280 300 320 340 360 380
500
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700
0nm260 280 300 320 340 360 380
500
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0
Time (min)
0 10 20 30 40 50 60
mA
U
0
200
400
600
800
mA
U
0
200
400
600
Time (min)
20 25 30 35
mA
U
0
50
100
150
L1
CH1
L2
F
CH1
CH2 CH5
Zoom
CH5
CH2
L2
CH1
L1
CH3
A
B
L1; L2
CH1
CH2
CH3
CH5
Ch
Pc M-Q
nm260 280 300 320 340 360 380
500
300
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700
0nm260 280 300 320 340 360 380
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0
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0
OH
NECTAR (Extracted from the honeybee gut)
HONEY
UV spectrum of floral markers
ALKALOIDS
4-hydroxyquinaldic acid
(kynurenic acid)
4-hydroxy-quinaldinium
cation
NNNN
OOOOHHHH
CCCCOOOOOOOOHHHH
(((( CCCCHHHH1111))))
NNNN
OOOOHHHH
CCCCOOOOOOOOHHHH
(((( CCCCHHHH1111))))
NNNN
OOOOHHHH
CCCCOOOOOOOOHHHHHHHH
(((( CCCCHHHH2222))))
++++NNNN
OOOOHHHH
CCCCOOOOOOOOHHHHHHHH
(((( CCCCHHHH2222))))
++++CH3 and CH5: Unidentified coumpounds
L1: Cyclohexa-1-3-diene-1-carboxilic acids gentiobioside
L2: Cyclohexa-1-3-diene-1-carboxilic acids
Freshly-deposited honey
• Diplotaxis
• ?
Diplotaxis tenuifolia
• Rapessed
Nectar of Diplotaxis tenuifolia, UV 330nm
0
5
10
15
20
25
Intens.
[mAU]
15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 Time [min]
1+2+3
4+5
6
7
8
1011
12
13
9
Diplotaxis tenuifolia. ‘Nectar’ Freshly Collected from the comb.
chrysin
pinocembrin
pinostrobin
Km, qu, isor, glucosides
HPLC Analysis of Diplotaxis nectar
0
20
40
60
80
[mAU]
15 20 25 30 35 40 Time [min]
Diplotaxis tenuifolia, UV 330
Honey 1
Diplotaxis tenuifolia, UV 330Diplotaxis tenuifolia, UV 330
7 10
14
12
15
13
HPLC Analysis of Diplotaxis Honey
Different compounds derived from propolis, pollen and
nectar found in different monofloral honeys
Retention time (min)
0 10 20 30 40 50 60
mA
U
0
20
40
60
80
100
120
140
Retention time (min)
0 10 20 30 40 50 60
mA
U
0
20
40
60
80
100
120
140Taraxacum honey
Rhododendro honey
PropolisPropolis
Nectar+Pollen
Nectar+Pollen
Truchado et al., J. Chromatogr. 2009
Conclusions
• Different strategies can be used to identify floral-origin markers
• The presence of flavonoid glycosides in honey enlarges the potential markers
• These phytochemicals are bioactive
• All analyses have to be confirmed with the analysis of a sufficient number of unifloral honey samples from different origins
