Characterization of markers of botanical origin and other compounds extracted from unifloral honeys

Article abstract of DOI:10.1021/jf302798d

The possibility of tracing the botanical and geographical origin of products such as honey has become more important because of market globalization. As a consequence, numerous analytical methods have been applied to the determination of honey authenticity. The scope of the present work is to chromatographically purify and characterize 23 compounds from organic extracts of unifloral (chestnut, linden, orange, acacia, eucalyptus, honeydew) and polyfloral honeys. Of these compounds, 17 were identified as specific markers and were used for botanical discrimination in a previous study based on multivariate statistical analysis of proton nuclear magnetic resonance ( ¹H NMR) data. Together with the botanical markers, 6 other substances were isolated and characterized using NMR and mass spectrometry. These phytochemicals belong to several classes, that is, terpenes, organic acids, flavonoids, and others. For the first time, a diacylglyceryl ether and 5 other compounds present in different types of honey were identified and characterized.

Full text of DOI:10.1021/jf302798d

Article
pubs.acs.org/JAFC
Characterization of Markers of Botanical Origin and Other
Compounds Extracted from Unifloral Honeys
Elisabetta Schievano,* Elisa Morelato, Chiara Facchin, and Stefano Mammi
̀
Dipartimento di Scienze Chimiche, Universita di Padova, Via Marzolo 1, 35131 Padova, Italy
ABSTRACT: The possibility of tracing the botanical and geographical origin of products such as honey has become more
important because of market globalization. As a consequence, numerous analytical methods have been applied to the
determination of honey authenticity. The scope of the present work is to chromatographically purify and characterize 23
compounds from organic extracts of unifloral (chestnut, linden, orange, acacia, eucalyptus, honeydew) and polyfloral honeys. Of
these compounds, 17 were identified as specific markers and were used for botanical discrimination in a previous study based on
1
multivariate statistical analysis of proton nuclear magnetic resonance ( H NMR) data. Together with the botanical markers, 6
other substances were isolated and characterized using NMR and mass spectrometry. These phytochemicals belong to several
classes, that is, terpenes, organic acids, flavonoids, and others. For the first time, a diacylglyceryl ether and 5 other compounds
present in different types of honey were identified and characterized.
KEYWORDS: honey, botanical origin, marker compounds, NMR
INTRODUCTION
NMR has several advantages compared to other techniques:
sample preparation is simple and easy, which minimizes the
change in the chemical composition and the loss of metabolites
during sample preparation; also, many components, such as
flavonoids, sugars, terpenes, and organic acids, are detected
simultaneously. NMR is also a very powerful technique for the
determination of the chemical structure of organic molecules.
In the present context, NMR can provide the identity of reliable
marker compounds, a crucial step not only to characterize a
certain type of honey but also to expose possible adulterations.
In this paper, the isolation and identification of several
compounds present in chloroform extracts of honeys of
different floral origins are described. Among all of the
compounds that can be found in honey, most of the ones
considered in this work were selected because they are
responsible for statistical discrimination of different botanical
origins. In a previously proposed NMR-based metabolomic
■
Honey is the natural product obtained by honey bees (Apis
mellifera L.) from the nectar of flowers or from secretions of
other living parts of plants or excretions of sucker insects.
Honey is mainly a supersaturated sugar solution, with >95% of
its dry mass consisting of sugars, although different valuable
nutrients such as vitamins, minerals, enzymes, flavoring organic
compounds, free amino acids, and numerous volatile
1
compounds are present as minor components. The nature,
amount, and combination of the various components endow
each honey with individual organoleptic character and
nutraceutical properties. These compounds represent a finger-
print of a specific honey and therefore could be used to
differentiate honeys of different botanical and also geographic
origins. The main parameters of honey quality, which also
2
influence its price, derive from its botanical origin. The
11
approach that used O2PLS-DA multivariate data analysis, the
proton resonances of these compounds have allowed the
classification of different unifloral honeys, although some of
them were not characterized. Some compounds included in this
study are not specific markers, but they are nevertheless
intriguing. A few of these compounds were identified for the
first time in honey produced by A. mellifera.
knowledge of which minor components are present in different
unifloral honeys may also substantiate (or dispute) health
claims attached to some of these products.
The interest in honey has recently grown not only because of
the possible therapeutic effects that accompany its nutritional
value but also because legislations tend to protect its geographic
and botanical origin. A metabolomic approach has been widely
applied in food science to guarantee authenticity and is gaining
importance to determine the origin of unifloral honeys. Various
MATERIALS AND METHODS
Chemicals. Deuterated chloroform, CDCl (99.96% d, H O
<0.005%), was from Euriso-Top, Gif sur Yvette, France; acetonitrile
■
3
,4
5
methods of minor constituents analysis are employed,
3
2
including gas chromatography−mass spectrometry (GC-MS),
6
(CH CN), methanol (MeOH), and standard compounds (chrysin,
3
liquid chromatography−mass spectrometry (LC-MS), capil-
9
7% pure; pinocembrin, 95% pure; kynurenic acid, 98% pure;
lary electrophoresis−time-of-flight mass spectrometry (CE-
7
8
9
TOF-MS), MALDI-TOF MS, FTIR and nuclear magnetic
1
0,11
Special Issue: IX Italian Congress of Food Chemistry
resonance (NMR) spectroscopy.
In recent years, the use of
high magnetic fields and the greater sensitivity and spectro-
scopic resolution that they bring have stimulated interest in 1D
and 2D NMR spectroscopy as a routine method for the analysis
Received: June 29, 2012
Revised: January 15, 2013
Accepted: January 29, 2013
Published: January 29, 2013
12
10,11,13−15
of complex mixtures such as honey.
©
2013 American Chemical Society
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(
±)-abscisic acid, 99% pure; linalool, 97% pure; neurotensin mass spec
chromatography to afford and isolate the following compounds
standard, angiotensin mass spec standard, and bradykinin mass spec
standard) were purchased from Sigma-Aldrich (Milan, Italy).
(
Figure 1): 4-(1-hydroxy-1-methylethyl)cyclohexa-1,3-diene-
carboxylic acid methyl ester (1), 4-(1-hydroxy-1-methylethyl)-
cyclohexa-1,3-dienecarboxylic acid (2), 4-(1-methylethenyl)-
cyclohexa-1,3-dienecarboxylic acid (3), and 4-(1-hydroxy-1-
methylethyl)benzoic acid (4) from linden honey; deoxyvasici-
none (6), the γ-lactam derivative of 3-(2′-pyrrolidinyl)-
kynurenic acid, γ-LACT-3-PKA (7), 2-quinolone (8), and
Honey Samples. Honey samples of seven different floral origins
were obtained either from Rigoni S.p.A. or from trusted Veneto
apiaries: 75 acacia, 60 chestnut, 62 linden, 40 orange, 32 eucalyptus,
and 36 honeydew. The botanical origin of each sample was certified by
the provider and ascertained through sensorial analysis and, in several
cases, by melissopalinologic analysis. The NMR-based metabolomic
1
0,11
analysis previously described confirmed the declared origin.
Sample Preparation. A liquid/liquid extraction was carried out as
4
2
-quinolone (9) from chestnut honey; caffeine (14) and (E)-
,6-dimethylocta-2,7-diene-1,6-diol (8-hydroxylinalool) (15)
10
previously reported with slight modifications. The necessary amount
of honey (50−100 g) was obtained by combining different samples of
the same floral origin. Specifically, each honey sample was divided in
many Teflon tubes, each containing 12 g of honey, 10 mL of water,
and 10 mL of chloroform. After 10 min of mechanical stirring at room
temperature, the tubes were centrifuged at 10000 rpm for 15 min. The
lower chloroform phases were pooled, and the solvent was evaporated
under a gentle stream of nitrogen. The solid residues were purified
using the optimized procedure, described below.
The extracts were initially treated with an acetonitrile/water = 95:5
solution to precipitate waxes. Compound 23 also precipitates in these
conditions. The solutions were dried and purified through a 25 cm × 2
cm silica gel column, equilibrated in chloroform. For acacia honey, a
step gradient elution of petroleum ether/ethyl acetate from 0:5 to 5:0
from orange honey; and dehydrovomifoliol (16) and 3-oxo-
α-ionone (17) from eucalyptus honey. (E)-2,6-Dimethyl-3,7-
octadiene-2,6-diol (5) is a terpene that derives from linalool
and is present mainly in linden honey; however, the molecule
was also found in chestnut honey. This result can be explained
by the overlapping flowering periods of the two plants, which
can result in cross-contamination. Pinocembrin (10), chrysin
(
11), alpinone (12) and (Z,E)-abscisic acid (13) were found
mainly in acacia honey, but they are present in almost every
kind of honey. Also, other compounds are present in most
honeys because they derive from bee metabolism ((E)-2-
decenedioic acid (18), (E)-2-nonenedioic acid (19), decane-
dioic acid (20), nonanedioic acid (21)) or are ubiquitous in
plants (methyl syringate (22)). Compound 23 was isolated
from honeydew, where it is relatively more abundant, but it is
also present in honey.
Compounds 2 and 3 were identified directly in the organic
honey extract because of their strong and isolated signals,
whereas methyl syringate (22) was detected by spiking the
extract with the standard compound. The other molecules were
isolated in silica gel column fractions with the following
conditions: 5 (1% MeOH), 6 (3% MeOH), 7 (2.5% MeOH), 8
and 9 (0.5% MeOH), 14 (0.5% MeOH) 15 (1.25% MeOH),
and 16 and 17 (0.25% MeOH). From acacia honey, 10 (E:A =
(
E:A) was used. For the other honeys, a step gradient elution from 0
to 5% MeOH in CHCl was employed. The fractions were evaporated
3
under a stream of nitrogen to eliminate the mobile phase, redissolved
in 600 μL of CDCl , and placed in an NMR tube for the analysis.
3
NMR Measurements. A 600 MHz Bruker (Bruker, Rheinstetten,
Germany) Avance spectrometer was used for one- and two-
dimensional spectra. In a typical 1D experiment, 256 transients were
collected as 64K points with a spectral width of 14 ppm and a
relaxation time of 2 s, at 298 K, using a modified double-pulsed field
16
gradient spin echoes sequence when necessary. Two-dimensional
homo- and heteronuclear correlation NMR spectra (DQF-COSY,
TOCSY, NOESY, HMQC, and HMBC) were collected. The
experimental conditions were as follows: for TOCSY, spectral width
(
SW) = 10 ppm in both dimensions, data points (DP) in the F2
4
:1), both 11 and 12 (E:A = 3:2). After the last gradient
dimension = 2K, relaxation delay (RD) = 2 s, number of scans (NS) =
solution, all of the columns were always washed with
acetonitrile/water = 95:5, obtaining compound 13 from acacia
honey, compounds 1 and 4 from linden honey, and compounds
1
1
5
4
6, number of experiments (NE) = 400−512, spin-lock time = 100−
20 ms; for COSY, SW = 10 ppm, DP = 2K, RD = 2 s, NE = 400−
12; for NOESY, SW = 10 ppm, RD = 2 s, NS = 128−256, NE =
1
13
00−512, mixing time = 1.2 s; for H− C HMQC, SW = 10 ppm
1
8−21 from all of the botanical origins. Compound 23 was
1
13
1
13
(
H), SW = 220 ppm ( C), DP = 1K, NS = 128−256; for H− C
1 13
purified through a silica gel column with petroleum ether/ethyl
HMBC, SW = 10 ppm ( H), SW = 220 ppm ( C), DP = 1K, NS =
acetate = 3:1 as an isocratic eluent.
All of the compounds were identified and characterized by
6
00−800.
Mass Spectrometry. Molecular masses were determined on a
1
D and 2D NMR and by ESI-MS, using the procedure
Mariner (PerSeptive Biosystems, Foster City, CA, USA) quadrupole
mass spectrometer equipped with an electrospray ion source. The
instrument was operated in the positive ESI(+) ion mode at a probe
tip voltage of 4.3 kV. The samples were introduced into the mass
spectrometer ion source directly through a Rheodyne injector with a
described under Materials and Methods; the assignments of the
isolated molecules 1 and 4−9 are reported in Table 1; the
assignments of compounds 12, 13, 16−23 are reported in
Table 2, whereas the assignments of compounds 2, 3, 10, 11,
20 μL sample loop. The mobile phase flow (10 μL/min of 50:50 v/v
11
14, and 15 have been published previously.
CH CN/H O, 0.1% HCOOH) was delivered by a Harvard Apparatus
3
2
Deoxyvasicinone (6), for example, was identified by ESI-MS
P11 to the vaporization nozzle of the electrospray ion source (140
+
(
m/z 187 [M + H] ) and by 1D and 2D NMR spectra (Figure 2).
°
C), and nitrogen was employed as both a drying and nebulizing gas.
The spectrometer was calibrated using a mixture of neurotensin,
The TOCSY experiment shows a spin system that includes
protons H-5, H-6, H-,7 and H-8 and a second one that consists
of H-1′, H-2′, and H-3′. The carbonyl carbon (C-4) shows a cross
peak with H-5 in the HMBC experiment, which demonstrates
the connection between these parts of the molecules. Both H-1′
and H-2′ have a cross peak with C-2.
angiotensin, and bradykinin, at a concentration of 1 pmol/μL, as
external standard.
The ESI-MS analysis of the diacylglyceryl ether 23 was performed
by dissolving the compound in chloroform/methanol (1:1) containing
17
0.2 mM ammonium acetate.
Linden Honey. Compounds 1−4 are monoterpenoid acids
characterizing T. cordata honey. The structure of markers 2 and
3, which are the most abundant ones, was determined with the
RESULTS AND DISCUSSION
■
(
The chloroform extracts of the analyzed unifloral honeys
Citrus sinensis honey, Tilia cordata honey, Robinia acacia L.
18
aid of literature data. Compound 4 is possibly an oxidation
product of compound 2. The linalool derivative (E)-2,6-
dimethyl-3,7-octadiene-2,6-diol (5) is also characteristic of
honey, Castanea sativa honey, Eucalyptus spp. honey, and
honeydew honey) were subjected to silica gel column
1
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Figure 1. Structures of compounds 1−23.
linden honey, although it is present in smaller quantities than
the other markers.
honey because of their higher concentration with respect to
11
24
̈
other types of honeys. Aguero et al. demonstrated that
Chestnut Honey. Compounds 6−9 are typical molecules of
chestnut honey; their chemical shift values are reported in
Table 1. Deoxyvasicinone (6) is a quinazolinone fused with a
pyrrolidine ring: it had never been found in honey before, and
it has been demonstrated to have antimicrobial, anti-
pinocembrin (10) is moderately active against some types of
fungi. The assignment of compounds 10 and 11 has been
11
published. Another flavonoid was isolated from acacia honey
and identified as alpinone (12): the assignments are reported in
Table 2. This is the first time that this compound has been
19
25
inflammatory, and antidepressant activities. Compound 7
has been previously indicated as γ-LACT-3-PKA, the typical
found in honey: previously, its presence in propolis and in the
26
extracts of I. tenuifolia was reported. The plant hormone
abscisic acid (13) is important in seed maturation and germina-
tion and also in adaptation to abiotic environmental stresses; its
6
1
marker of chestnut honey. The H NMR spectra of
compounds 6 and 7, obtained from the isolated fractions of
chestnut honey, are reported in Figure 2. Compounds 8 and 9
are structural isomers with the molecular formula C H NO. In
27
content rises during exposure of a plant to drought. In the
plant, it is produced from the C carotenoid zeaxanthin through
9
7
40
28,29
the literature, compound 8 and its derivatives are endorsed with
the intermediate xanthoxin.
The assignment of abscisic acid
20
antibacterial, anticancer, antiviral, and antioxidative activities,
and compound 9 and its derivatives show antibacterial
is reported in Table 2.
Orange Honey. Compounds 14 and 15 are typical of
2
1
11
activity.
orange honey and have been previously assigned. A NOESY
Acacia Honey. Among the natural antioxidants present in
food, flavonoids represent one of the most important
cross peak between protons 1 and 3 confirms the E
configuration for the double bond C2−C3 in compound 15.
2
2,23
30
classes.
Compounds 10 and 11 (pinocembrin and chrysin)
This compound derives from ω-hydroxylation of linalool.
3
1
are flavonoids that can be regarded as characteristic of R. acacia L.
Polashock et al. reported antifungal and antimicrobial
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Figure 2. H NMR spectra of the isolated markers of chestnut honey deoxyvasicinone (6), γ-LACT-PKA (7), and 2-quinolone (8). The HMBC
(→) and TOCSY(--->) correlations for compound 6 are shown on the molecular structure.
1
Figure 3. H NMR spectra of the isolated markers of orange honey: caffeine (14) and (E)-2,6-dimethylocta-2,7-diene-1,6-diol (15).
activities of 15, which was found to accumulate in ripening
fruits. See Figure 3 for the H NMR spectra of compounds 14
and 15 isolated from orange honey.
Eucalyptus Honey. Norisoprenoids 16 and 17 are typical of
eucalyptus honey and derive from cleavage of the carotenoid
chain that can occur at different double bonds. Dehydrovomifoliol
3
2
1
1
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16) probably arises from the degradation of abscisic acid. On the
Article
(
other hand, 3-oxo-α-ionone (17) is generated by cleavage of the
C −C double bond of C carotenoids and has already been
9
10
40
33
found in sulla honey. See Table 2 for all of its assignments.
Compounds Isolated in All of the Honeys. Honeybee
larvae are fed by “nurse” bees with a jelly-like secretion (royal
34
jelly) of the mandibular and the hypopharyngeal glands.
Compounds 18−21 are characteristic of larval food of the
honeybee: although they mainly derive from royal jelly, some
traces can be found also in honey (Table 2). These compounds
were identified as free fatty diacids, because of the absence of
Figure 4. Compound 23 with all of the detected HMBC correlations
1
ester signals in the H NMR spectrum and of carbons of
(H → C).
carboxylic esters in the HMBC spectrum. The ESI-MS
spectrum confirmed the presence of all four of these
compounds in the same fraction.
are monounsaturated. This conclusion is corroborated by the
+
following peaks in the mass spectrum: m/z 873.8 [M + H] ,
+
These diacids are of special interest because they have long
been recognized as part of the pheromone system of the
m/z 890.9 [M + NH ] , and m/z 591.5. The latter peak results
4
from the loss of one of the fatty acids in position 3 or 2,
35−37
honeybee A. mellifera.
Queen and worker honeybees
yielding a monoacylglyceryl ether ion. The fact that a unique
+
produce functionalized 8-, 9-, and 10-carbon fatty acids in their
mandibular glands: these compounds regulate the queen’s and
worker’s activities in the hive. The honeybee queen produces
pheromones such as 9-hydroxy-(E)-2-decenoic acid (9-HDA),
[
M − RCO ] ion was found demonstrates that the same fatty
2
acid, an oleic acid chain, is present in these positions. This
finding and the value of the molecular weight derived from the
mass spectrum led us to conclude that a saturated C18 chain
occupies position 1.
9
-keto-(E)-2-decenoic acid (ODA), and other acids function-
38
alized at the penultimate (ω-1) position. Worker bees secrete
acids functionalized at the last (ω) position, such as 10-
hydroxy-(E)-2-decenoic acid (10-HDA), its saturated counter-
part, and the corresponding diacids (C10:1 DA and C10:0
To confirm the proposed structure, a transmethylation
reaction was performed. To a solution of the pure molecule
in 500 μL of CH OH was added 20 μL of H SO . The mixture
3
2
4
obtained was placed in a water bath at 80 °C for about 60 min,
and then 800 μL of n-hexane was added. The two phases were
separated. The n-hexane fraction was dried under a nitrogen
stream and characterized by 1D NMR and GC-MS. The results
3
9
DA). The worker’s mandibular gland secretions are believed
to function primarily as a preservative additive to the larval
4
0
food.
It has been previously shown that whereas 10-HDA is the
main acid in royal jelly, C10:1 DA represents the principal
(
not shown) indicate the presence of only methyl oleate.
The presence of this molecule in honey has never been
41
aliphatic acid in honey. As expected, we found only diacids
functionalized at the ω-position in honey, because worker bees
play the main role in the production of honey. Diacids with 9
carbon atoms were also found, confirming the work by Tan
reported in the literature. Therefore, a biosynthetic pathway
that might explain its formation is not yet available.
Interestingly, this type of molecule has been found only in
the muscles and liver of a giant squid and certain species of
sharks. Diacylglyceryl ethers are fundamental molecules for
these species as a short-term energy resource, and they may
3
5
et al., who found compound 21, even if only in traces, and by
3
6
Lercker et al., who found 19 and 21 in an amount comparable
to diacids with 10 carbon atoms.
44
also be important in imparting buoyancy regulation.
Methyl syringate (22) is a general aromatic constituent in
plants. It was previously found in many types of honeys and
In conclusion, the work presented here is a significant step
toward the characterization of the organic components of
unifloral honeys. The compounds described here were selected
on the basis of a previous multivariate statistical analysis of the
42
also in grapevines, leaf, and root. Jermnak et al. described its
43
potential as an aflatoxin production inhibitor.
1
0,11
Compound 23 is a diacylglyceryl ether that contains two
oleic acid chains in positions 2 and 3 and a saturated
hydrocarbon C18 chain in position 1. The chemical shifts,
the TOCSY connectivities, and the patterns of J-couplings of
protons H-1, H-2, H-3, and H-3′ are consistent with the
glycerol skeleton of a 2,3-diester (see Table 2). This is
confirmed by the correlations found in the HMBC spectrum
NMR signals of chloroform extracts.
The present results clearly show how the use of NMR in the
study of complex mixtures, in conjunction with appropriate
data analysis, can not only lead to the differentiation of food
products of different origin but can extend to the possibility of
elucidating the structure of single components in such mixtures.
In the case of unifloral honey, the identification of markers of
each botanical origin can lead to the formation of a database in
which each type of honey can be associated with one or more
specific molecule. The present work contributes in this
direction, although many unifloral honeys were not considered
here, and no consideration regarding concentration of analytes
was possible.
Much of this information is already present in the literature,
and it could all be put together in a database of markers of
botanical origin, which can be used to classify honeys of
different sources. Such a database could be used for different
purposes. In many cases, the markers identified may be
quantified directly using analytical techniques different from the
ones used here. This may provide an alternative method to
(
Figure 4) between H-3, H-3′, and carbonyl C-1(R3) (173.73
ppm) and between H-2 and carbonyl C-1(R2) (173.33 ppm).
Also, H-2(R2) correlates with both C-1(R2) and C-3(R2)
(
Figure 4). The presence of a correlation between H-1 and
C-1(R1) (71.4 ppm) is indicative of an ether oxygen in position 1.
This finding is further supported by the cross peaks in the NOESY
spectrum between H-1 and H-1(R1) that confirms an ether chain
−
OCH R linked in position 1.
In addition, the presence of a multiplet at 5.36 ppm for the
2
olefinic protons (H-9,10(R2,R3)), the TOCSY correlation with
the allylic protons resonating at 2.04 ppm (H-8,11(R2,R3)),
and the absence of signals from bisallylic protons in the
spectrum indicate that the fatty acid chains in positions 2 and 3
1
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1
melissopalinological analysis to control botanical claims and
protect consumers. Also, many of the various components are
endowed with specific nutraceutical properties and enhance the
value of the honey in which they are present. A marker database
could be used as a reference for health-related studies to derive
reliable information about the efficacy of honey in therapy and
prevention.
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(
experiments for quantifying minor components in complex mixtures:
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(
AUTHOR INFORMATION
■
Corresponding Author
2
(
*
Phone: 39-049-827-5742. Fax: 39-049-827-5239. E-mail:
elisabetta.schievano@unipd.it.
quantification of trace components in complex matrices by
Funding
bandselective excitation with adiabatic pulses. Magn. Reson. Chem.
2
(
009, 47, 868−872.
C.F. is the recipient of an “Annalaura Segre” Scholarship from
GIDRM (Gruppo Italiano Discussione Risonanze Magnetiche).
17) Endo, Y.; Tagiri-Endo, M.; Seo, H.-S.; Fujimoto, K.
Identification and quantification of molecular species of diacyl glyceryl
ether by reversed phase high-performance liquid chromatography with
refractive index detection and mass spectrometry. J. Chromatogr., A
Notes
The authors declare no competing financial interest.
2
001, 911, 39−45.
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flower to linden honey. Glycosidic precursors of cyclohexa-1,3-diene-
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19) Al-Shamma, A.; Drake, S.; Flynn, D. L.; Mitscher, L. A.; Park, Y.
ACKNOWLEDGMENTS
̀
■
The support of Barbara Biondi in the acquisition of mass
spectra is gratefully acknowledged.
1
(
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