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.

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1.5

2.0

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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

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800

1200

mA

U

0

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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

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Time (min)

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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

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OH

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CH1

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CH1

CH2 CH5

Zoom

CH5

CH2

L2

CH1

L1

CH3

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L1; L2

CH1

CH2

CH3

CH5

Ch

Pc M-Q

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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

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100

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140

Retention time (min)

0 10 20 30 40 50 60

mA

U

0

20

40

60

80

100

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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