Flavonol glycosides of sea buckthorn (hippophae rhamnoides ssp. sinensis) and lingonberry (vaccinium vitis-idaea) are bioavailable in humans and monoglucuronidated for excretion

Article abstract of DOI:10.1021/jf9029942

Glucuronidation and excretion of sea buckthorn and lingonberry flavonols were investigated in a postprandial trial by analyzing the intact forms of flavonol glycosides as well as glucuronides in plasma, urine, and feces. Four study subjects consumed sea buckthorn (study day 1) and lingonberry (study day 2) breakfasts, and blood, urine, and fecal samples were collected for 8, 24, and 48 h, respectively. Both glycosides and glucuronides of the flavonol quercetin as well as kaempferol glucuronides were detected in urine and plasma samples after the consumption of lingonberries; 14% of flavonols in urine were glycosides, and 86% were glucuronidated forms (wt %). After the consumption of sea buckthorn, 5% of flavonols excreted in urine were detected intact, and 95% as the glucuronides (wt %). Solely glucuronides of flavonols isorhamnetin and quercetin were found in plasma after the consumption of sea buckthorn berries. Only glycosides were detected in the feces after each berry trial. Flavonol glycosides and glucuronides remained in blood and urine quite long, and the peak concentrations appeared slightly later than previously described. The berries seemed to serve as a good flavonol supply, providing steady flavonol input for the body for a relatively long time.

Full text of DOI:10.1021/jf9029942

620 J. Agric. Food Chem. 2010, 58, 620–627
DOI:10.1021/jf9029942
Flavonol Glycosides of Sea Buckthorn
€
(Hippophae rhamnoides ssp. sinensis) and Lingonberry
(Vaccinium vitis-idaea) Are Bioavailable in Humans and
Monoglucuronidated for Excretion
†
H
ENNA-MARIA
L
M
EHTONEN,*,† OUTI
‡,#
L
EHTINEN,† JUKKA-PEKKA
†
S
UOMELA
,
ATTI
V
IITANEN
,
AND
HEIKKI
K
ALLIO
†Department of Biochemistry and Food Chemistry, and ‡Department of Geriatrics, University of Turku,
FI-20014 Turku, Finland, and #Department of Geriatrics, Karolinska Institutet, Karolinska University
Hospital Huddinge, SE-17177 Stockholm, Sweden
Glucuronidation and excretion of sea buckthorn and lingonberry flavonols were investigated in a
postprandial trial by analyzing the intact forms of flavonol glycosides as well as glucuronides in
plasma, urine, and feces. Four study subjects consumed sea buckthorn (study day 1) and
lingonberry (study day 2) breakfasts, and blood, urine, and fecal samples were collected for 8,
24, and 48 h, respectively. Both glycosides and glucuronides of the flavonol quercetin as well as
kaempferol glucuronides were detected in urine and plasma samples after the consumption of
lingonberries; 14% of flavonols in urine were glycosides, and 86% were glucuronidated forms
(wt %). After the consumption of sea buckthorn, 5% of flavonols excreted in urine were detected
intact, and 95% as the glucuronides (wt %). Solely glucuronides of flavonols isorhamnetin and
quercetin were found in plasma after the consumption of sea buckthorn berries. Only glycosides
were detected in the feces after each berry trial. Flavonol glycosides and glucuronides remained in
blood and urine quite long, and the peak concentrations appeared slightly later than previously
described. The berries seemed to serve as a good flavonol supply, providing steady flavonol input
for the body for a relatively long time.
KEYWORDS: Glucuronidation; isorhamnetin; kaempferol; lingonberry; metabolism; postprandial;
quercetin; sea buckthorn
INTRODUCTION
picked wild berry in Scandinavia and Russia. Lingonberry has
been shown to possess both chemopreventive (13) and antimi-
crobial (1) properties in in vitro trials.
Sea buckthorn (Hippophae€ rhamnoides L.) is natively distri-
buted over a wide area of Eurasia, especially in China, where it
has traditionally been exploited for soil and water conservation
purposes in addition to being an important food and medicinal
plant. Recently, the health-related potential of sea buckthorn has
raised interest also among Western researchers. The berry has
been shown to possess antimicrobial (1, 2) and in vitro radio-
protective (3) properties as well as chemopreventive (4), anti-
neurotoxic (5), and antiatherogenic (6) properties in animal trials.
Sea buckthorn products may also have protective effect against
atopic dermatitis (7), castric ulcer (8), and coronary heart
disease (9) and inhibit platelet aggregation (10) as well as
nicotine-induced oxidative stress (11). In a recent double-blind
placebo-controlled trial in humans, a very modest amount of sea
buckthorn (28 g daily) reduced high-sensitivity CRP level in
plasma (12).
Sea buckthorn and lingonberry contain a wide range of
secondary metabolites produced via the shikimate and acetate
pathways. The phenolic compounds act as protecting agents, and
they are often condensed in the cuticular surface layers of the
plant. The phenolics include flavonoids (anthocyanins, flavonols,
and catechins), phenolic acids, and stilbenoids, as well as con-
densed and hydrolyzable tannins. In sea buckthorn, a wide range
of flavonol glycosides as well as oligomeric proanthocyanidins
have been identified (14-16). In lingonberry flavonols, antho-
cyanins, catechins and their glycosides, and different caffeoyl and
feruloyl conjugates have been identified (17-19). The basic
structures of flavonols and some flavonol compounds of interest
in this study are presented in Figure 1.
Phenolics of sea buckthorn have been shown to possess several
potential health effects. The flavonoid fraction of sea buckthorn
seeds has antihypertensive effect in high-sucrose fed rats (20); it
inhibits thrombosis in mouse femoral artery and in vitro platelet
aggregation (21) and enhances wound healing in rats (22). Iso-
rhamnetin, a predominant flavonol in sea buckthorn, has
been shown to prevent endothelial dysfunction and superoxide
Lingonberry (Vaccinium vitis-idaea L.) is a wild, semiwoody
chamephyte that keeps its leaves through winter and commonly
grows in the northern latitudes. Lingonberry is an abundantly
*Corresponding author (telephone þ358-40-709-9020; fax þ358-2-
3336860; e-mail henna-maria.lehtonen@utu.fi).
pubs.acs.org/JAFC
Published on Web 11/20/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 621
Stock solutions of reference compounds (0.5 mg/mL) were prepared in
methanol or methanol/dimethyl sulfoxide (9:1, v/v, for isorhamnetin-
3-glucoside and isorhamnetin-3-rutinoside) and stored at -70 °C.
Sea buckthorn (H. rhamnoides var. Ljubitelskaja) berries were organi-
€
cally cultivated in Finland and purchased from Vinkkilan Luomutuote
(Vehmaa, Finland). Lingonberries (V. vitis-idaea L.) were of wild Finnish
origin and purchased frozen from Pakkasmarja Ltd. (Suonenjoki,
Finland).
Postprandial Study Design. To investigate the metabolism of sea
buckthorn and lingonberry flavonols in humans, a postprandial clinical
trial was designed. Four healthy, nonsmoking volunteers (two women and
two men) aged on average 26 years (22, 24, 28, and 29 years), BMI 20-25,
were recruited. The subjects followed a flavonoid-free diet for 30 h before
the study days as well as during the sample collection period of 48 h.
Between the study days, there was a wash-out period of 7 days. During the
flavonoid-free diet, white bread, white rice, and white pasta were allowed,
in addition to all foods of animal origin. After a 10 h fast, the subjects
consumed a sea buckthorn breakfast (study day 1, 300 g of frozen sea
buckthorn berries and some vanilla yogurt) or lingonberry breakfast
(study day 2, 300 g of lingonberries and some vanilla yogurt).
Before the breakfast, a basal sample of blood, urine, and, when
possible, feces was collected. Blood samples were drawn at 1, 2, 4, and 8
h after the consumption of berry breakfast, and urine and fecal samples
were collected for 24 (0-4, 4-8, 8-12, and 12-24 h) and 48 h (0-12,
12-36, and 36-48 h), respectively, after the consumption of the berry
breakfast. The plasma and urine samples collected on study day 1 (sea
buckthorn) were frozen and stored at -70 °C immediately. Samples
collected on study day 2 (lingonberry) were acidified with 0.1% TFA
[0.04:1, v(TFA-H2O)/v(sample)] and stored at -70 °C. Fecal samples were
lyophilized for 3-5 days, homogenized, and extracted with acidified
methanol (0.1% TFA, 20 mL for 5 g of sample). A 2 mL sample of
plasma, a 6 mL sample of urine, and a 5 g sample of lyophilized feces were
stored and used for analysis. The total amount of urine and feces excreted
was not recorded.
Figure 1. Basic structure of a flavonol aglycone, and exemplarily the
chemical structures of one flavonol aglycone, quercetin, and one flavonol
metabolite of that aglycone, quercetin-3-glucuronide. In the basic structure
R1 = OH and R2 = OH for quercetin, R1 = OH and R2 = H for kaempferol,
and R1 = OCH3, R2 = OH for isorhamnetin.
production in medically induced hypertensive rats’ aorta (23).
Isorhamnetin has also been shown to possess antiproliferative
and apoptotic effects in cancer cell cultures in vitro (24) as well as
to inhibit procarcinogen-activating CYP1 enzyme in vitro (25).
Although most in vitro research has been done with the aglycone
form, in the study of Tribolo et al. (26) isorhamnetin-3-glucur-
onide was shown to modulate the expression of vascular cell
adhesion molecule (VCAM). Isorhamnetin-3-glucoside has also
shown potential in decreasing the risk of diabetes and its further
complications such as cardiovascular diseases by lowering serum
glucose concentration and inhibiting aldose reductase action in
streptozotocin-induced diabetic rats (27). In some in vitro studies,
it has been shown that antioxidative activity may be decreased in
flavonoid metabolites found in vivo when compared with glyco-
sides or aglycone forms, even though other bioactive effects are
less affected by the intestinal and hepatic metabolism of
flavonols (28-30). Lingonberry flavonols have not been investi-
gated as such, although vast research concerning flavonol glyco-
sides known to exist in lingonberries has indicated potential
health effects (31).
The profiles of the phenolic compounds of sea buckthorn and
lingonberry have been already well characterized, but mecha-
nisms of absorption and metabolism of the compounds as
conjugates are largely unknown. Some research has been per-
formed bothin animals (32,33) and in human subjects (34-36) by
analyzing aglycone forms of flavonols present in sea buckthorn
and lingonberry. However, this approach enables only the detec-
tion of absorption, not providing information on metabolites in
various tissues. In most studies concerning flavonol metabolism,
urine or plasma, but not feces, has been investigated.
The subjects were provided with a standardized flavonoid-free lunch
and an early evening snack on the study day. A flavonoid-free late evening
snack was also permitted.
This study was conducted according to the guidelines laid down in the
Declaration of Helsinki (2000), and all procedures involving human
subjects were approved by the Ethics Committee of the Hospital District
of Southwest Finland. Written informed consent was obtained from all
subjects. Prior to the study, the subjects were informed about the
investigation and their right to discontinue at any time without an
explanation, and they had an opportunity to ask questions. The study
products used in the study were safe, traditional berry products.
Method Development. For sample pretreatment, a method pre-
viously reported by us for anthocyanin analysis in urine (37) was used.
Ultrahigh-performance liquid chromatographic (uHPLC) analysis of
flavonols was developed by testing solvents defined in the litera-
ture (38, 39) at different concentrations and combinations. The gra-
dient was optimized to enable a sufficient chromatographic separation
of analytes in about 5 min. For identification of the different isomers of
flavonol glucuronides, also another inlet method of a longer run time of
14.5 min was developed. Tandem mass spectrometric (MS/MS) analysis
was performed in the multiple reaction mode (MRM). Ionization
energies, collision energies, and daughter ions were carefully optimized
for reference compounds.
The aim of this study was to investigate the absorption and
excretion of sea buckthorn and lingonberry flavonols in a post-
prandial trial by analyzing flavonol glycosides and glucuroni-
dated flavonols in plasma, urine, and feces.
Method Validation. Detection and quantification limits of the
analytical method were determined by analyzing different concentrations
of reference compounds in TFA/H2O/MeOH (0.1:39.9:60, by volume). In
accordance with Bioanalytical Method Validation Guidance for Industry
(2003), the detection limit was considered to be the concentration that gave
a signal-to-noise ratio (S/N) > 3, and the quantification limit was
considered to be the concentration that gave S/N > 10. The lowest
concentration of reference compounds analyzed was 0.2 ng/mL.
Repeatability was tested by spiking reference compounds (a concentra-
tion of 100 ng/mL of each) into urine that did not contain flavonols and by
analyzing samples as several parallel samples within one day (five parallel
samples) and in three consecutive days (three parallel samples each day).
The yield of the extraction step was tested by spiking blank urine samples
before the extraction and the extract after the extraction at a concentration
MATERIALS AND METHODS
Reagents and Materials. Methanol and acetonitrile were of HPLC
grade and purchased from Sigma-Aldrich (Steinheim, Germany). Tri-
fluoroacectic acid (TFA, g98.0%) was obtained from Fluka
(Deisenhofen, Germany). Reference compounds (isorhamnetin-3-gluco-
side, isorhamnetin-3-rutinoside, kaempferol-3-glucoside, kaempferol-
3-rutinoside, quercetin-3-rhamnoside, quercetin-3-galactoside, quercetin-
3-glucoside, myricetin-3-rhamnoside, and syringetin-3-glucoside) were
purchased from Extrasynthese (Genay, France). Flavonol glucuronides
(isorhamnetin-3-glucuronide, quercetin-3-glucuronide, and quercetin-7-
glucuronide) were obtained from Dr. Paul Needs (Institute of Food
Research, Norwich Research Park, U.K.).

622 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Lehtonen et al.
of 100 ng/mL. Freeze-and-thaw stability was tested by analyzing one urine
sample before and after two freeze-and-thaw cycles.
All compounds were quantified by uHPLC-MS/MS analysis with one
internal standard, syringetin-3-glucoside (m/z 509.4/347.0). To eliminate
the error from differences in response, a standard curve was made by
spiking flavonol-free blank urine with 0, 40, 200, 1000, and 5000 ng/mL of
all the reference compounds used. For quantification of compounds for
which the reference compounds could not be obtained, a standard curve of
the most similar compound (another glycoside of the same aglycone or the
same glycoside part with different aglycone) was utilized. Glucuronides
were quantified as a sum because of coelution, and identification was
performed by comparing retention times obtained with another inlet
method.
The applicability of validation results for other matrices analyzed was
tested by comparing the matrix effects of all three matrices. Reference
compounds were spiked (a concentration of 100 ng/mL of each) into blank
plasma, urine, and fecal samples pretreated as described below
and analyzed by uHPLC-MS/MS to compare the retention times and
responses.
Quantification of Sea Buckthorn and Lingonberry Flavonols.
Half a gram of frozen berries was weighed, thawed, homogenized, and
extracted once with 4 mL of 0.1% TFA/H2O and twice with 1 mL of 0.1%
TFA/MeOH (1:1, v/v) by shaking the berries for 20 s, sonicating for 5 min,
and shaking in a vortex shaker for 20 s and subsequently centrifuging for 5
min at 3400g. Supernatants were combined and diluted with 8 mL of 0.1%
TFA/H2O. Extracts were applied in Supelco (C18, 500 mg) solid phase
extraction tubes preconditioned with 2 mL of methanol and 2 mL of 0.1%
TFA/H2O. Tubes were washed with 2 mL of 0.1% TFA/H2O, and
analytes were eluted with 1 mL of TFA/H2O/MeOH (0.1:39.9:60, by
volume). The samples were analyzed as such with the uHPLC-MS/MS
method described below and quantified with the QuanLynx program with
an internal standard method.
Glucuronide Synthesis. To tentatively identify the substitution posi-
tions of glucuronides detected in plasma and urine samples, quercetin,
kaempferol, and isorhamnetin aglycones were derivatized into corre-
sponding glucuronides by UGT1A1 enzyme. Briefly, 65 μL of UDP-
glucuronic acid, 6.25 μL of aglycone (10 mM in MeOH), 379 μL of
potassium phosphate (20 mM, pH 7.0), and 25 μL of UGT1A1 enzyme
(12 mg/mL) were mixed and incubated at 37 °C for 6 h. Reaction was
stopped by adding 1.5 mL of acetone. The reaction mixture was centri-
fuged for 10 min at 16000g, and acetone was evaporated from the
supernatant with nitrogen. The sample was analyzed by uHPLC-
MS/MS both with the 5.3 min method used for physiological samples
and with the other method developed to separate different isomers.
Physiological Sample Preparation. Sample pretreatment from a
method previously reported by us for anthocyanin analysis from urine (37)
was used for flavonol analysis. Briefly, acidified plasma, urine, or fecal
samples were purified with solid phase extraction with 96-well plates with
2 mg of Oasis packing material (Waters, Milford, MA). Samples were
injected directly from the collection plate wells into the uHPLC system.
uHPLC-MS/MS Method. All MS analyses were carried out on
uHPLC-MS/MS equipment consisting of an Acquity UPLC system with a
50 mm ꢀ 2.1 mm, 1.7 μm, Acquity UPLC BEH C18 column and a Quattro
Premier tandem quadrupole mass spectrometer (Waters). Elution of the
berry and physiological samples was performed using 1% acetic acid in
water as solvent A and acetonitrile as solvent B. The gradient was
optimized to achieve baseline separation of all the critical pairs of the
flavonol glycoside reference compounds. The flow rate was set at
0.45 mL/min. Initial solvent composition for 0.45 min was 90% A and
10% B. Subsequently, the compounds were eluted with a gradient from
0.45 to 2 min, resulting in 82.5% A and 17.5% B. After that, the
proportion of B was increased to 70% for column wash and the initial
conditions were stabilized at 4.8-5.3 min. The total run time was 5.3 min.
For the identification of the different isomers of flavonol glucuronides
synthesized, glucuronide reference compounds as well as some physiolo-
gical samples were analyzed by another uHPLC method. Elution was
performed using 0.33% formic acid in water as solvent A and methanol as
solvent B. The gradient was optimized to achieve baseline separation of
analytes. The flow rate was set at 0.3 mL/min. Initial solvent composition
was 85% A and 15% B. Subsequently, compounds were eluted with a
gradient from 0 to 12 min, resulting in 30% A and 70% B. After that, the
proportion of B was increased to 95% for column wash. The total run time
was 14.5 min.
RESULTS
Method Validation. The yield of the SPE extraction was
29-58% for different flavonol conjugates, and the repeatability
(CV) of the method was 4.4-28.4%. The variance explained
in a regression analysis, R2, of the standard curve of quercetin-
3 -galactoside was 0.9946. Detection and quantification limits for
the reference compounds were 0.75 and 1.5-3.1 ng/mL in
standard dilutions, respectively. Freeze-and-thaw stability was
75%, and freeze-and-thaw cycles were thus minimized in ana-
lyses. All samples were analyzed after a single freeze-and-thaw
cycle. Intensity differences of the peaks of reference compounds
as well as retention time shifts were modest in the matrices
analyzed; thus, validation with urine was applicable to all sample
matrices. The uHPLC-MS/MS run of 5.3 min utilizing small
solvent volumes together with small 96-well plate solid phase
extraction columns made the method economical and environ-
mentally responsible.
Flavonols in Berries. As presented in Table 1, isorhamnetin-
3-rutinoside was the major flavonol in sea buckthorn berries
[54% (wt %)] and quercetin-3-rhamnoside in lingonberries [33%
(wt %)]. Isorhamnetin-3-glucoside, isorhamnetin-3-galactoside,
and quercetin-3-glucoside were also detected, accounting for 22,
8, and 13% (wt %, respectively) of the total flavonols in sea
buckthorn. Quercetin-3-galactoside, quercetin-3-glucoside, and
quercetin-3-xyloside/-arabinoside were detected in lingonberries,
accounting for 29, 4, 6, 5, and 19% (wt %, respectively) of the
total flavonols. Isorhamnetin-3-glucoside, isorhamnetin-3-ruti-
noside, quercetin-3-glucoside, and quercetin-3-galactoside were
identified by comparing the retention times and the mother and
daughter ions in the uHPLC-MS/MS analysis with those of the
reference compounds, and isorhamnetin-3-galactoside and quer-
cetin-3-xyloside/-arabinoside were tentatively identified with the
help of literature data on m/z values and retention time. On
the basis of the berry analysis, the quantity of flavonols that
the human subjects consumed in the postprandial trial was
12.1 mg on day 1 (sea buckthorn, SB) and 18.1 mg on day
2 (lingonberry, LB).
Identification of Flavonol Glycosides and Their Metabolites in
Physiological Samples. Identification of flavonols in physiological
samples was performed by spiking blank urine with reference compounds,
when available, and comparing their retention times and parent and
product ions in MS/MS. Identification of some flavonol metabolites was
based on comparison of the retention time and respective m/z values of the
precursor and product ion information to that found in the litera-
ture (15, 18, 19, 36, 40), because all of the reference compounds could
not be obtained. As indicated in Table 1, isorhamnetin-3-rutinoside,
isorhamnetin-3-glucoside, quercetin-3-glucoside, quercetin-3-galactoside,
kaempferol-3-glucoside, kaempferol-3-rutinoside, quercetin-3-rhamno-
side, myricetin-3-rhamnoside, quercetin-3-glucuronide, quercetin-7-glu-
curonide, and isorhamnetin-7-glucuronide were used for identification.
Reference compounds were not obtained for kaempferol glucuronides,
quercetin-3-xyloside, and -arabinoside. Detection was carried out by using
electrospray ionization in positive ion mode with the desolvation gas flow
set to 598 L/h, capillary voltage to 3.2 kV, cone voltage to 13-15 V, and
collision energy to 10-22 eV (optimized separately for each reference
compound). MS/MS data were collected in the MRM mode by monitoring
the transition of precursor and product ions specific for each compound.
Identification of Flavonol Derivatives from Physiological Sam-
ples. Flavonol glycosides in their intact form prevailed in feces,
whereas glucuronides were the most abundant analytes in urine.
Both glycosidic and glucuronidic forms were detected in plasma
after the lingonberry meal, whereas after the sea buckthorn meal

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 623

624 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Lehtonen et al.
Figure 5. Total ion chromatograms of flavonol glucuronide analysis of
plasma (A) and urine (B) samples after the consumption of sea buckthorn.
Identification of peaks indicated is presented in Table 1.
Figure 2. Total ion chromatograms of the HPLC-MS/MS analysis of urine
(A) and feces (B) samples of one subject taken at 4-8 h and at 12-24 h
after the consumption of lingonberries, respectively. Identification of peaks
indicated is presented in Table 1.
taken at 4-8 h and at 12-24 h after the consumption of
lingonberries, respectively. Figure 3 shows an ion chromatogram
of flavonol glucuronide analysis of the urine sample of one subject
taken at 4-8 h after the consumption of sea buckthorn.
Isorhamnetin-3-rutinoside, isorhamnetin-3-glucoside, querce-
tin-3-glucoside, quercetin-3-galactoside, and quercetin-3-rham-
noside were identified by comparison with reference compounds
based on the retention times and detection of parent and product
ions of MS analysis as indicated in Table 1. Quercetin-3-xyloside
and -arabinoside were analyzed at m/z values of 435.00/303.00,
but reference compounds for these glycosides were not available.
Three peaks were detected in this mass window in the lingonberry
sample, whereas only one (Figure 2, peak 17) was present in
physiological samples, that is, in urine. On the basis of the
retention properties found in the literature (17, 19), this was
presumably quercetin-3-xyloside.
To define the site of glucuronidation, reference compounds of
isorhamnetin-3-glucuronide, quercetin-3-glucuronide, and quer-
cetin-7-glucuronide were obtained and other glucuronides were
synthesized. A total ion chromatogram of the synthesized glucur-
onides is shown in Figure 4. Synthesized glucuronides were
identified on the basis of the reference compounds obtained
and known retention order (41). In synthesized glucuronides,
peaks appearing at m/z values 493/317, 463/287, and 479/303
indicated the presence of isorhamnetin monoglucuronide,
kaempferol monoglucuronide, and quercetin monoglucuronide,
respectively, due to the cleavage of the characteristic fragments of
m/z 176. Because some glucuronides coeluted in our 5.3 min inlet
method, also another HPLC method was utilized to obtain
baseline separation of isomers.
Figure 3. Total ion chromatogram of flavonol glucuronide analysis of the
urine sample of one subject taken at 4-8 h after the consumption of sea
buckthorn. Identification of peaks indicated is presented in Table 1.
A subset of plasma and urine samples was analyzed by uHPLC-
MS/MS with the longer inlet method to obtain information on the
substitution sites of the glucuronides in the physiological samples.
Chromatograms of flavonol glucuronide analysis of plasma and
urine samples after the consumption of sea buckthorn are pre-
sented in Figure 5. The glucuronide isomers that could be identi-
fied (quercetin-3- and isorhamnetin-3-glucuronide, peaks 18 and 5,
respectively) or tentatively identified (isorhamnetin-7- and -40-
glucuronides, peaks 6 and 7, respectively) on the basis of reference
and synthesized glucuronides, respectively, are indicated.
Kaempferol glucuronides were tentatively identified on the
basis of the m/z values of precursor and parent ions, and
isomerism was assumed on the basis of the retention order of
different quercetin glucuronide isomers (41), because a reference
compound could not be obtained.
Figure 4. Total ion chromatograms of flavonol glucuronides synthesized
from quercetin (A), kaempferol (B), and isorhamnetin (C) aglycones.
Identification of synthesis products is based on the retention times and MS
data of reference compounds quercetin-3-glucuronide, quercetin-7-glucur-
onide, and isorhamnetin-3-glucuronide as well as retention order informa-
tion found in the literature. Identification of the peaks indicated is presented
in Table 1.
only glucuronides were detected. Figure 2 shows total ion chro-
matograms of a simultaneous flavonol glycoside and flavonol
glucuronide analysis of urine and fecal sample of one subject
After the consumption of sea buckthorn, isorhamnetin-
3-rutinoside, isorhamnetin-3-glucoside, quercetin-3-glucoside,

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 625
different proportions of intact and glucuronidated forms as 14 wt
% of flavonols analyzed from urine were glycosides and 86 wt %
were glucuronidated forms. Profiles of the derivatives in urine are
illustrated in Figure 6.
DISCUSSION
Quantities of flavonol glycosides in feces were about 100 times
higher than in urine. However, after the consumption of sea
buckthorn, the quantities of glucuronides in urine were of the
same magnitude as the quantities of glycosides in feces. Because
the total volume of urine or mass of feces was not measured, the
total excreted amounts of flavonols are not known. However, it
can be concluded on the basis of this study that glucuronidation
and excretion of isorhamnetin glycosides of sea buckthorn in
urine are approximately equally important excretion routes to the
excretion in feces without. It must be taken into account that
some of the isorhamnetin glucuronides detected might be methy-
lated and glucuronidated from quercetin glycosides also present
in sea buckthorn. In our study design, monoglucuronidation of
isorhamnetin and methylation and monoglucuronidation of
quecetin could not be distinguished.
Figure 6. Maximum concentrations of summed flavonol glucuronides and
summed flavonol glycosides in urine during the 24 h sample collection
period after the sea buckthorn (sb) or lingonberry (lb) breakfast.
Interindividual differences in both absorption extent and time
as well as relative proportions of different metabolites were high.
Figure 7 shows the concentrations of isorhamnetin-3-glucoside in
the urine of individual study subjects.
Also in previous studies, large interindividual differences
between study subjects in highest and lowest excreted concentra-
tions have been reported (42, 43). In a study by Bonetti et al.,
excreted concentrations of kaempferol was correlated with the
body mass index of the seven subjects participating in the
study (42). However, excretion rates of flavonols have been
remarkably similar in all study subjects (42, 43). The effect of
genotype on the ability to metabolize bioactive compounds such
as flavonols should be investigated in the future.
Figure 7. Concentrations of isorhamnetin-3-glucoside in urine samples of
the four study subjects after the ingestion of sea buckthorn.
The maximum concentration of flavonol derivatives in
plasma after the ingestion of lingonberries with yoghurt
appeared at 2-4 h time points. This is slightly later than
previously reported by Mullen et al. (44) after the ingestion
of onions. However, Moon et al. (45) found the peak concen-
tration of quercetin aglycone and its metabolites to appear
even later, at 3-4 h after the ingestion of quercetin aglycone.
Differences observed might be due to different food matrices,
resulting in potential flavonoid-protein interactions with milk
proteins in yogurt, or due to different sugar moieties attached
to flavonol aglycone. The highest concentrations of flavonols
in urine were found at 8-12 h of the sample collection period.
Bonetti et al. (42) have reported the peak concentration of
kaempferol in urine at 2-8 h after the consumption of beans.
Kaempferol, however, has been reported to be excreted more
efficiently than quercetin (46), indicating that the results might
not be comparable. After the ingestion of onions, Mullen
et al. (47) found the highest level of quercetin metabolites in
urine during the 0-4 h collection period. Onions in this study
were cooked, which might contribute to faster absorption than
in the present study, in which the study subjects consumed
uncooked berries. Berries thus seem to serve as foods or food
components providing a relatively slow and steady flavonol
supply to the body during several hours after ingestion.
Compared with the lingonberry meal, peak concentrations of
flavonol monoglucuronides in plasma and urine samples ana-
lyzed in the present study were higher after the consumption of
sea buckthorn, whereas the concentrations of flavonol glycosides
in feces were higher after the lingonberry meal. This indicates that
sea buckthorn and lingonberry flavonols are quite differently
and kaempferol-3-rutinoside were detected in feces, isorhamne-
tin-3-glucoside, quercetin-3-glucoside, isorhamnetin glucuro-
nides, and quercetin-3-glucuronide in urine, and isorhamnetin
glucuronides and kaempferol-7-glucuronide in plasma. After
consumption of lingonberries, quercetin-3-galactoside, querce-
tin-3-rhamnoside, and quercetin-3-xyloside were detected in
feces, quercetin-3-xyloside/-arabinoside, quercetin-3-glucuro-
nide, and quercetin-3-rhamnoside in urine, and quercetin-3-
rhamnoside, quercetin-3-glucuronide, and kaempferol-7-glucur-
onide in plasma. These compounds were not detectable before the
consumption of the berries, and the concentrations decreased in
plasma and urine almost to baseline values during the collection
period. For feces, the 48 h collectionperiod was rathershort as the
concentrations of the flavonol glycosides did not reach baseline
values at the 48 h time point. The flavonol-free diet of 32 h,
however, was sufficient, as only minor amounts of analytes were
detectable at baseline.
Quantities of Flavonol Derivatives in Physiological Samples.
Flavonol glycosides ingested in this study as berry components
were excreted steadily and efficiently glucuronidated, although
large interindividual differences in the excretion rates and profiles
could be observed. All of the compounds detected in the physio-
logical samples taken before and during the 48 h sample collection
period after the sea buckthorn or lingonberry breakfasts, and
their quantities, are presented in Table 1.
After consumption of sea buckthorn, 5 wt % of flavonols
excreted in urine were detected intact and 95 wt % as the
glucuronidated form. The lingonberry meal resulted in somewhat

626 J. Agric. Food Chem., Vol. 58, No. 1, 2010
Lehtonen et al.
metabolized. Also, compared with isorhamnetin, quercetin was
excreted more in its intact glycosidic forms.
(8) Xing, J.; Yang, B.; Dong, Y.; Wang, B.; Wang, J.; Kallio, H. Effects
€
of sea buckthorn (Hippophae rhamnoides L.) seed and pulp oils on
experimental models of gastric ulcer in rats. Fitoterapia 2002, 73,
644–650.
The human ability to absorb isorhamnetin-3-rutinoside has
been indicated in an in vitro trial showing transportation through
the Caco-2 cell layer by MRP-transporter proteins (48). Human
studies concerning isorhamnetin metabolism, however, have not
been performed prior to the present study. Bioavailability of
isorhamnetin has been previously indicated (34, 35). After the
consumption of onions, both intact quercetin glycosides (49) and
glucuronidated and sulfonated forms have been detected (44,50).
Proportions of the intact and metabolized quercetin species in
plasma or urine are known to vary widely (51, 52), and in some
studies, only glucosides (49, 50) have been found. In this study,
quantifiable amounts of both flavonol glycosides and glucuro-
nides were detected after the ingestion of both berries in urine and
after lingonberry consumption in plasma. However, after the sea
buckthorn meal, glucuronides were present in urine at 10-50
times higher concentrations than glycosides, and in plasma
samples glycosides were below the detection limits, whereas after
the lingonberry meal, differences between glycoside and glucuro-
nide concentrations in urine and plasma were more modest. This
indicates that flavonols present in lingonberries are less prone to
monoglucuronidation than the main flavonol in sea buckthorn,
isorhamnetin, and are possibly directed toward other metabolic
routes such as sulfonation and methylation. According to the
present study, isorhamnetin, which exists as mono- and diglyco-
side derivatives in the sea buckthorn berries, is efficiently mono-
glucuronidated in vivo and is readily bioavailable.
In summary, this paper presents a rapid approach for flavonol
analysis from various physiological samples as well as from
berries and application of the method to the postprandial study
of sea buckthorn and lingonberry flavonols. Our results indicate
that isorhamnetin glucosides are absorbed and glucuronidated by
routes similar to those of other flavonol glycosides investigated to
date. Berries seem to serve as a good flavonol supply, providing a
steady flavonol input to the body for a relatively long time. By
consuming three berry meals daily, flavonol metabolite concen-
trations in blood would stay relatively high throughout the day,
which would potentially have positive effects on health status.
(9) Eccleston, C.; Yang, B.; Tahvonen, R.; Kallio, H.; Rimbach, G.;
Minihane, A. Effects of an antioxidant-rich juice (sea buckthorn) on
risk factors for coronary heart disease in humans. J. Nutr. Biochem.
2002, 13, 346–354.
(10) Johansson, A.; Korte, H.; Yang, B.; Stanley, J.; Kallio, H. Sea
buckthorn berry oil inhibits platelet aggregation. J. Nutr. Biochem.
2000, 11, 491–495.
(11) Suleyman, H; Gumustekin, K.; Taysi, S.; Keles, S.; Oztasan, N.;
Aktas, O.; et al. Beneficial effects of Hippophae rhamnoides L. on
nicotine induced oxidative stress in rat blood compared with vitamin
E. Biol. Pharm. Bull. 2002, 25, 1133–1136.
(12) Larmo, P.; Alin, J.; Salminen, E.; Kallio, H.; Tahvonen, R. Effects of
sea buckthorn berries on infections and inflammation: a double-
blind, randomized, placebo-controlled trial. Eur. J. Clin. Nutr. 2007,
62, 1123–1130.
(13) Wang, S. Y. Antioxidant activity in lingonberries (Vaccinium vitis-
idaea L.) and its inhibitory effect on activator protein-1, nuclear
factor-kappaB, and mitogen-activated protein kinases activation.
J. Agric. Food Chem. 2005, 53, 3156.
€
€
(14) Rosch, D.; Mugge, C.; Fogliano, V.; Kroh, L. W. Antioxidant
€
oligomeric proanthocyanidins from sea buckthorn (Hippophae
rhamnoides) pomace. J. Agric. Food Chem. 2004, 52, 6712–6718.
€
€
(15) Rosch, D.; Krumbein, A.; Mugge, C.; Kroh, L. W. Structural
investigations of flavonol glycosides from sea buckthorn
€
(Hippophae rhamnoides) pomace by NMR spectroscopy and
HPLC-ESI-MS(n). J. Agric. Food Chem. 2004, 52, 4039–4046.
(16) Yang, B.; Halttunen, T.; Raimo, O.; Price, K.; Kallio, H. Flavonol
glycosides in wild and cultivated berries of three major subspecies of
Hippophae€ rhamnoides and changes during harvesting period. Food
Chem. 2009, 115, 657–664.
€€ €
ꢀ
(17) Maatta-Riihinen, K. R.; Kamal-Eldin, A.; Mattila, P. H.; Gonzalez-
ꢀ
€ €
Paramas, A. M.; Torronen, A. R. Distribution and contents of
phenolic compounds in eighteen Scandinavian berry species.
J. Agric. Food Chem. 2004, 52, 4477–4486.
€
(18) Hakkinen, S.; Auriola, S. High-performance liquid chromatography
with electrospray ionization mass spectrometry and diode array
ultraviolet detection in the identification of flavonol aglycones and
glycosides in berries. J. Chromatogr., A 1998, 829, 91–100.
(19) Ek, S.; Kartimo, H.; Mattila, S.; Tolonen, A. Characterization of
phenolic compounds from lingonberry (Vaccinium vitis-idaea).
J. Agric. Food Chem. 2006, 54, 9834–9842.
LITERATURE CITED
(20) Pang, X.; Zhao, J.; Zhang, W.; Zhuang, X.; Wang, J.; Xu, R.; Xu, Z.;
Qu, W. Antihypertensive effect of total flavones extracted from seed
residues of Hippophae€ rhamnoides L. in sucrose-fed rats. J. Ethno-
pharmacol. 2008, 117, 325–331.
€
€
(1) Nohynek, L.; Alakomi, H.-L.; Kahkonen, M.; Heinonen, M.;
€
Helander, I.; Oksman-Caldentey, K.-M.; Puupponen-Pimia, R.
Berry phenolics: antimicrobial properties and mechanisms of action
against severe human pathogens. Nutr. Cancer 2006, 54, 18–32.
(21) Cheng, J.; Kondo, K.; Suzuki, Y.; Ikeda, Y.; Meng, X.; Umemura,
K. Inhibitory effects of total flavones of Hippophae€ Rhamnoides L.
on thrombosis in mouse femoral artery and in vitro platelet aggrega-
tion. Life Sci. 2003, 72, 2263–2271.
(22) Gupta, A.; Kumar, R.; Pal, K.; Singh, V.; Banerjee, P.; Sawhney, R.
Influence of sea buckthorn (Hippophae€ rhamnoides L.) flavone on
dermal wound healing in rats. Mol. Cell. Biochem. 2006, 290, 193–
198.
€
(2) Puupponen-Pimia, R.; Nohynek, L.; Hartmann-Schmidlin, S.;
€
€
€€ €
Kahkonen, M.; Heinonen, M.; Maatta-Riihinen, K.; Oksman-Cal-
dentey, K.-M. Berry phenolics selectively inhibit the growth of
intestinal pathogens. J. Appl. Microbiol. 2005, 98, 991–1000.
(3) Goel, H.; Kumar, P.; Samanta, N.; Rana, S. Induction of DNA-
protein cross-links by Hippophae€ rhamnoides: implications in radio-
protection and cytotoxicity. Mol. Cell. Biochem. 2003, 245, 57–67.
(4) Padmavathi, B.; Upreti, M.; Virendra, S.; Rameshi, R.; Rana, S.;
Pramod, R. Chemoprevention by Hippophae€ rhamnoides: effects on
tumorigenesis, phase II and antioxidant enzymes, and IRF-1 tran-
scription factor. Nutr. Cancer 2005, 51, 59–67.
(5) Xu, Y.; Li, G.; Han, C.; Sun, L.; Zhao, R.; Cui, S. Protective effects
of Hippophae€ rhamnoides L. juice on lead-induced neurotoxicity in
mice. Biol. Pharm. Bull. 2005, 28, 490–494.
(6) Basu, M.; Prasad, R.; Jatamurthy, P.; Pal, K.; Arumughan, C.;
Sawhney, R. Anti-atherogenic effects of sea buckthorn (Hippophae€
rhamnoides) seed oil. Phytomedicine 2007, 14, 770–777.
(23) Sanchez, M.; Lodi, F.; Vera, R.; Villar, I.; Cogolludo, A.; Jimenez,
R.; Moreno, L.; et al. Quercetin and isorhamnetin prevent endothe-
lial dysfunction, superoxide production, and overexpression of
p47phox induced by angiotensin II in rat aorta. J. Nutr. 2007, 137,
910–915.
(24) Ma, G.; Yang, C.; Qu, Y.; Wei, H.; Zhang, T.; Zhang, N. The
flavonoid component isorhamnetin in vitro inhibits proliferation
and induces apoptosis in Eca-109 cells. Chem.-Biol. Interact. 2007,
167, 153–160.
(25) Chang, T.; Chen, J.; Yeung, E. Effect of Ginkgo biloba extract on
procarcinogen-bioactivating human CYP1 enzymes: identification
of isorhamnetin, kaempferol, and quercetin as potent inhibitors of
CYP1B1. Toxicol. Appl. Pharmacol. 2006, 213, 18–26.
(7) Yang, B.; Kalimo, K.; Mattila, L.; Kallio, S.; Katajisto, J.; Peltola,
O.; Kallio, H. Effects of dietary supplementation with sea buckthorn
(Hippophae€ rhamnoides) seed and pulp oils on atopic dermatitis.
J. Nutr. Biochem. 1999, 10, 622–630.

Article
J. Agric. Food Chem., Vol. 58, No. 1, 2010 627
(26) Tribolo, S.; Lodi, F.; Connor, C.; Suri, S.; Wilson, V.; Taylor, M.;
Needs, P.; Kroon, P.; Hughes, D. Comparative effects of quercetin
and its predominant human metabolites on adhesion molecule
expression in activated human vascular endothelial cells. Athero-
sclerosis 2008, 197, 50–56.
(39) Zhao, Y.; Wang, Y.; Bao, Y.; Li, C. A sensitive method for the
detection and quantification of ginkgo flavonols from plasma. Rapid
Commun. Mass Spectrom. 2007, 21, 971–981.
€€ €
€ €
(40) Mikkonen, T.; Maatta, K.; Hukkanen, A.; Kokko, H.; Torronen,
€
R.; Karenlampi, S.; Karjalainen, R. Flavonol content varies among
(27) Lee, Y.; Lee, S.; Lee, H.; Kim, B.-K.; Ohuchi, K.; Shin, K. Inhibitory
black currant cultivars. J. Agric. Food Chem. 2001, 49, 3274–3277.
(41) Davis, B.; Brodbelt, J. Regioselectivity of human UDP-glucurono-
syl-transferase 1A1 in the synthesis of flavonoid glucuronides
determined by metal complexation and tandem mass spectrometry.
J. Am. Soc. Mass Spectrom. 2008, 19, 246–256.
effects of isorhamnetin-3-O-β-D-glucoside from Salicornia herbacea
on rat lens aldose reductase and sorbitol accumulation in strep-
tozotocin-induced diabetic rat tissues. Biol. Pharm. Bull. 2005, 28,
916–918.
(28) Janisch, K.; Williamson, G.; Needs, P.; Plub, G. Properties of
quercetin conjugates: modulation of LDL oxidation and binding
to human serum albumin. Free Radical Res. 2004, 38, 877–884.
(29) Saito, A.; Sugisawa, A.; Umegaki, K.; Sunagawa, H. Protective
effects of quercetin and its metabolites on H2O2-induced chromo-
somal damage to WIL2-NS cells. Biosci., Biotechnol., Biochem. 2004,
68, 271–276.
(42) Bonetti, A.; Marotti, I.; Dinelli, G. Urinary excretion of kaempferol
from common beans (Phaseolus vulgaris L.) in humans. Int. J. Food
Sci. Nutr. 2007, 58, 261–269.
(43) DuPont, M.; Day, A.; Bennett, R.; Mellon, F.; Kroon, P. Absorp-
tion of kaempferol from endive, a source of kaempferol-3-glucur-
onide, in humans. Eur. J. Clin. Nutr. 2004, 58, 947–954.
(44) Mullen, W.; Boitier, A.; Stewart, A. J.; Crozier, A. Flavonoid
metabolites in human plasma and urine after the consumption of
red onions: analysis by liquid chromatography with photodiode
array and full scan tandem mass spectrometric detection. J. Chro-
maogr., A 2004, 1058, 163–168.
(45) Moon, Y.; Wang, L.; DiCenzo, R.; Morris, M. Quercetin pharma-
cokinetics in humans. Biopharm. Drug Dispos. 2008, 29, 205–217.
(46) Wang, F.; Yao, T.; Zeng, S. Determination of quercetin and
kaempferol in human urine after orally administrated tablet of
Ginkgo biloba extract by HPLC. J. Pharm. Biomed. Anal. 2003, 33,
317–321.
ꢀ
ꢀ
(30) Perez-Vizcaıno, F.; Ibarra, M.; Cogolludo, A.; Duarte, J.; Zaragoza-
´
ꢀ
ꢀ
ꢀ
Arnaez, F.; Moreno, L.; Lopez-Lopez, G.; Tamargo, J. Endothe-
lium-independent vasodilator effects of the flavonoid quercetin and
its methylated metabolites in rat conductance and resistance arteries.
J. Pharmacol. Exp. Ther. 2002, 302, 66–72.
(31) Williamson, G.; Manach, C. Bioavailability and bioefficacy of
polyphenols in humans. II. Review of 93 intervention studies. Am.
J. Clin. Nutr. 2005, 81, 230S–242S.
(32) Lan, K.; Jiang, X.; He, J. Quantitative determination of isorhamne-
tin, quercetin and kaempferol in rat plasma by liquid chromatogra-
phy with electrospray ionization tandem mass spectrometry and its
application to the pharmacokinetic study of isorhamnetin. Rapid
Commun. Mass Spectrom. 2007, 21, 112–120.
(47) Mullen, W.; Edwards, C.; Crozier, A. Absorption, excretion and
metabolite profiling of methyl-, glucuronyl-, glucosyl- and sulpho-
conjugates of quercetin in human plasma and urine after ingestion of
onions. Br. J. Nutr. 2006, 96, 107–116.
(33) Morrice, P.; Wood, S.; Duthie, G. High-performance liquid chro-
matographic determination of quercetin and isorhamnetin in rat
tissues using β-glucuronidase and acid hydrolysis. J. Chromatogr., B
2000, 738, 413–417.
(48) Tian, X.; Yang, X.; Wang, K.; Yang, X. The efflux of flavonoids
morin, isorhamnetin-3-O-rutinoside and diosmetin-7-O-β-
xylopyranosyl-(1-6)-β- -glucopyranoside in the human intestinal
cell line caco-2. Pharm. Res. 2006, 23, 1721–1728.
D-
D
(34) Suomela, J.-P.; Ahotupa, M.; Yang, B.; Vasankari, T; Kallio, H.
(49) Mauri, P.; Iemoli, L.; Gardana, C.; Riso, P.; Simonetti, P.; Porrini,
M.; Pietta, P. Liquid chromatography/electrospray ionization mass
spectrometric characterization of flavonol glycosides in tomato
extracts and human plasma. Rapid Commun. Mass Spectrom.
1999, 13, 924–931.
(50) Day, A.; Williamson, G. Biomarkers for exposure to dietary flavo-
noids: a review of the current evidence for identification of quercetin
glycosides in plasma. Br. J. Nutr. 2001, 86, S105–S110.
(51) Wittig, J.; Herderich, M.; Graefe, E.; Veit, M. Identification of
quercetin glucuronides in human plasma by high performance liquid
chromatography-tandem mass spectrometry. J. Chromatogr., B
2001, 735, 237–243.
€
Absorption of flavonols derived from sea buckthorn (Hippophae
rhamnoides L.) and their effect on emerging risk factors for cardio-
vascular disease in humans. J. Agric. Food Chem. 2006, 54, 7364–
7369.
(35) Larmo, P.; Yang, Y.; Hurme, S.; Alin, J.; Kallio, H.; Salminen, E.;
Tahvonen, R. Effect of a low dose of sea buckthorn berries on
circulating concentrations of cholesterol, triacylglycerols, and fla-
vonols in healthy adults. Eur. J. Nutr. 2009, 48, 277-282.
(36) Erlund, I.; Marniemi, J.; Hakala, P.; Alfthan, G.; Meririnne, E.; Aro,
A. Consumption of black currants, lingonberries and bilberries
increases serum quercetin concentrations. Eur. J. Clin. Nutr. 2003,
57, 37–42.
(52) Fang, T.; Wang, Y.; Ma, Y.; Su, W.; Bai, Y.; Zhao, P. A rapid LC-
MS/MS quantification assay for naringin and its two metabolites in
rats plasma. J. Pharm. Biomed. Anal. 2006, 40, 454–459.
(37) Lehtonen, H. M.; Rantala, M.; Suomela, J. P.; Viitanen, M.; Kallio,
H. Urinary excretion of the main anthocyanin of lingonberry
(Vaccinium vitis-ideae), cyanidin-3-O-galactoside, and its meta-
bolites. J. Agric. Food Chem. 2009, 57, 4447–4451.
(38) Chen, H.; Zuo, Y. Identification of flavonol glycosides in American
cranberry fruit. Food Chem. 2007, 101, 1357–1364.
Received for review August 27, 2009. Revised manuscript received
November 6, 2009. Accepted November 09, 2009.