Research Article

Received: 15 November 2010 Revised: 27 December 2011 Accepted: 8 January 2012 Published online in Wiley Online Library: 13 February 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5615

Consumption of Hibiscus sabdariffa L. aqueous

extract and its impact on systemic antioxidant
potential in healthy subjects
Thomas Frank,a Gabriele Netzel,b Dietmar R Kammerer,c Reinhold Carle,c
Adolf Kler,d Erwin Kriesl,d Irmgard Bitsch,e Roland Bitschb
and Michael Netzelb∗

Abstract
BACKGROUND: To evaluate health benefits attributed to Hibiscus sabdariffa L. a randomized, open-label, two-way crossover
study was undertaken to compare the impact of an aqueous H. sabdariffa L. extract (HSE) on the systemic antioxidant potential
(AOP; assayed by ferric reducing antioxidant power (FRAP)) with a reference treatment (water) in eight healthy volunteers. The
biokinetic variables were the areas under the curve (AUC) of plasma FRAP, ascorbic acid and urate that are above the pre-dose
concentration, and the amounts excreted into urine within 24 h (Ae0 – 24 ) of antioxidants as assayed by FRAP, ascorbic acid,
uric acid, malondialdehyde (biomarker for oxidative stress), and hippuric acid (metabolite and potential biomarker for total
polyphenol intake).

RESULTS: HSE caused significantly higher plasma AUC of FRAP, an increase in Ae0 – 24 of FRAP, ascorbic acid and hippuric acid,
whereas malondialdehyde excretion was reduced. Furthermore, the main hibiscus anthocyanins as well as one glucuronide
conjugate could be quantified in the volunteers’ urine (0.02% of the administered dose).

CONCLUSION: The aqueous HSE investigated in this study enhanced the systemic AOP and reduced the oxidative stress in
humans. Furthermore, the increased urinary hippuric acid excretion after HSE consumption indicates a high biotransformation
of the ingested HSE polyphenols, most likely caused by the colonic microbiota.


c 2012 Society of Chemical Industry

Keywords: Hibiscus sabdariffa L.; polyphenols; antioxidant potential; malondialdehyde; hippuric acid; humans

INTRODUCTION acid, protocatechuic acid, 5-caffeoylquinic acid, 4-caffeoylquinic

Antioxidant properties elicited by plant species have a full range
of perspective applications in human healthcare. In recent years,
the prevention of cancer and cardiovascular diseases has been
associated with the ingestion of fresh fruits, vegetables or teas
rich in natural antioxidants, suggesting that a higher intake of
such compounds could lower the risk of mortality from these
diseases.1,2 Plant foods contain fibre, vitamins, phytosterols, sulfur
compounds, carotenoids, and organic acids, which contribute to
the health effects, but they also contain a variety of polyphenols,
which are increasingly regarded as effective protective agents.3
Therefore, in search for sources of novel antioxidants, in the last
few years some medicinal plants have been extensively studied
for their antioxidant potential and radical scavenging activity.4
Tropical roselle (Hibiscus sabdariffa L., Malvaceae) is an annual,
erect, bushy, herbaceous sub-shrub that grows to 8 ft (2.4 m)
in height and typically consists of a red calyx with five large
sepals.5 H. sabdariffa extracts are a rich source of polyphenols,
with anthocyanins as the major phenolic constituents. Peng
et al.6 could detect and characterize, in an H. sabdariffa extract,
anthocyanins (e.g. delphinidin-3-sambubioside and cyanidin-3-
sambubioside), kaempferol-3-glucoside, a quercetin derivative
acid, chlorogenic acid, feruloylquinic derivative, and caffeic acid.
Anthocyanins are responsible for the red, purple, and blue colours
of many fruits and vegetables. They also provide the food industry
with natural replacements for some synthetic food colorants.7
The most common naturally occurring anthocyanins are the 3-O-
glucosides or 3,5-di-O-glucosides of cyanidin, delphinidin (in order
of increasing hydroxylation), peonidin, petunidin and malvidin (in
order of increasing methoxylation).8,9 Numerous studies, mostly
in vitro and animal experiments, have demonstrated a broad range
∗
Correspondence to: Michael Netzel, CSIRO Food and Nutritional Sciences, PO
Box 745, Archerfield BC, QLD 4108, Australia. E-mail: Michael.Netzel@csiro.au
a Private Consultant, 65812 Bad Soden, Germany
b Institute of Nutrition, Friedrich Schiller University Jena, 07743 Jena, Germany
c Institute of Food Science and Biotechnology, University of Hohenheim, 70599
Stuttgart, Germany
d Plantextrakt GmbH & Co. KG, 91487 Vestenbergsgreuth, Germany
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as well as several phenolic acids and derivatives such as gallic e Institute of Nutrition, Justus Liebig University Giessen, 35392 Giessen, Germany

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of biological properties for anthocyanins, including antioxidant,
anti-inflammatory, antimicrobial and anti-carcinogenic activities.9
Their daily intake has been estimated at between 12.5 mg in the
USA and 82 mg in Finland, which is considerably higher than
the intake estimated for many other polyphenols.9,10 Delphinidin-
3-sambubioside and cyanidin-3-sambubioside were identified as
the major hibiscus anthocyanins, with delphinidin-3-glucoside
and cyanidin-3-glucoside occurring in minor amounts.8
The pharmacological actions of calyx extracts include strong
in vitro and in vivo antioxidant activity.11 Induction of apoptosis
in human leukaemia cells and human gastric carcinoma cells
could be demonstrated with delphinidin-3-sambubioside and a
polyphenol-rich hibiscus extract, respectively.12,13 Furthermore,
dried flowers or extracts of H. sabdariffa are popular ingredients
in Asian herbal medicine and are used to treat hypertension,
pyrexia, liver disorders and inflammation.12 – 14 Recently, McKay
et al.15 reported that daily consumption of three servings of
H. sabdariffa (hibiscus) tea, an amount readily incorporated into
the diet, effectively lowered blood pressure in pre- and mildly
hypertensive adults.
There is no doubt that health benefits attributed to H. sabdariffa
need to be linked to the systemic exposure to the fraction
of ingested compounds that are available for antioxidant and
physiological activity, respectively. Only human intervention
studies using H. sabdariffa are able to determine its efficacy in
the prevention of chronic diseases and establishing science-based
dosing recommendations.
The present study has been carried out to determine the effect of
a single oral administration of an aqueous H. sabdariffa extract on
systemic, i.e. ex vivo antioxidant potential (AOP), evaluated by the
ferric reducing antioxidant power (FRAP) assay in young healthy
Caucasian male and female subjects. Plasma concentrations versus
time courses and their changes from baseline (i.e. pre-dose value)
as well as the AOP of urine were analysed. To characterize the
fraction of ingested phenolic compounds enhancing the AOP, the
area under the curve (AUC) above the baseline was derived from
individual plasma concentrations.
Furthermore, plasma and urine samples should be analysed for
hibiscus anthocyanins and metabolites as well as for ascorbic acid
and uric acid as important antioxidant compounds in biological
fluids.16,17 Hippuric acid, as a potential biomarker for total
polyphenols intake18 and malondialdehyde (MDA), a biomarker
for oxidative stress,19 were also quantified in the urine samples.
MATERIAL AND METHODS
Chemicals
Unless otherwise stated, all chemicals were purchased from Merck
(Darmstadt, Germany). Delphinidin-3-glucoside was purchased
from Polyphenols Laboratories AS (Sandnes, Norway), cyanidin-
3-glucoside was provided by Roth (Karlsruhe, Germany), and
cyanidin-3-sambubioside and delphinidin-3-sambubioside were
provided by the Institute of Food Chemistry of the Technische
Universität Braunschweig (Germany). Deionized water was used
throughout.
Subjects
Eight healthy non-smoking volunteers (four males and four
females) aged from 22 to 27 years (mean age 24 ± 1.9 years)
and a mean body mass index of 21.6 (±2.7 kg m−2 ) were
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included if they met the following inclusion criteria: 18–45 years
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T Frank et al.
of age and of good general clinical condition, as assessed by
clinical history. Subjects with known drug hypersensitivity, history
of clinically significant psychiatric or medical disease, aortic
stenosis, pregnancy (including presumed pregnancy), lactation,
pathological pattern of ECG, or history of drug or alcohol abuse
were excluded. The study was conducted in accordance with the
Declaration of Helsinki (Version October 2000) and amendments,
and international and local regulatory requirements. The trial
was approved by the Ethics Committee of the Friedrich Schiller
University Jena, Faculty of Medicine (code 1656-11/05). Subjects
gave their written informed consent prior to study enrolment.
Beverages
The following study beverages were administered:
• Experimental beverage: 10 g H. sabdariffa L. extract (pow-
der), dissolved immediately prior to drinking in 200 mL tap
water (referred to as HSE); manufacturer: Plantextrakt GmbH
& Co KG, D-91487 Vestenbergsgreuth, Germany; antioxidant
capacity: FRAP:20,21 3.24 mmol Fe2+ equivalents by anal-
ysis, total phenolic content by Folin–Ciocalteu assay:21,22
239.1 mg gallic acid equivalents (GAE) by analysis; total an-
thocyanins (sum of cyanidin-3-glucoside (0.15 mg), cyanidin-3-
sambubioside (65.56 mg), delphinidin-3-glucoside (2.42 mg),
and delphinidin-3-sambubioside (62.12 mg)): 130.25 mg by
high-performance liquid chromatography (HPLC);23 ascorbic
acid was not detectable.24,25 Details of the analytical methods
used are described in the following section.
• Reference beverage (control): 200 mL tap water.
Study design
This study was a randomized, open-label, two-way crossover
study. Subjects were randomized to receive a single dose of HSE
or water, and, after a washout period of 2 weeks, the dosing and
testing were repeated in a crossover fashion. The sample size was
fixed arbitrarily, i.e. without power and sample size calculation.
Participants adhered to their usual diet, but had to abstain from
food and beverages rich in polyphenols and ascorbic acid from 24 h
prior to treatment. They were instructed to refrain from alcohol
and medication, including over-the-counter drugs, throughout
the study. Subjects were free to withdraw from the study at any
time.
After an overnight fast, the treatments were taken together
with white bread rolls at 8 : 00 am on the study day. On these
days, in both periods, only the consumption of water (ad libitum)
and two standardized meals (two white wheat bread rolls with
60 g sliced cheese (Gouda) for lunch and dinner, respectively)
was allowed. Venous blood samples were drawn pre-dose (time
0) as well as 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8 and 10 h after
administration. Each blood sample (18 mL) was collected in an
ethylenediaminetetraacetic acid-coated tube. The blood samples
were centrifuged (10 min, 2000 × g, 8 ◦ C) within 10 min after
collection for plasma preparation.
In addition, voided urine of the subjects in periods 1 and 2
was collected during 24 h after treatment in dark laboratory urine
containers. During the collection period urine was stored at 4 ◦ C,
and no preservatives were added. For analysis of HSE anthocyanins
and metabolites, aliquots of plasma (2 mL) and urine (7 mL) were
stabilized with 0.44 mol L−1 trifluoroacetic acid (TFA; 0.4 mL) and
formic acid (2 mL), respectively, whereas for the analysis of ascorbic
acid and uric acid aliquots of plasma (0.6 mL) and urine (1 mL) were
stabilized with an equal volume of 10% (w/v) meta-phosphoric
acid. All samples were stored frozen at −80 ◦ C until analysed.
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Hibiscus and systemic antioxidant potential
Procedures
FRAP assay
The ex vivo AOP in plasma and urine as well as the antioxidant
capacity of HSE were measured photometrically using the FRAP
assay according to Schlesier et al.21 and Benzie and Strain,20 with
minor modifications. 30 µL deionized water and 10 µL sample
solution (HSE, plasma or urine) were mixed with 200 µL FRAP
reagent consisting of ferric chloride and 2,4,6-tripyridyl-s-triazine
(TPTZ) in acetate buffer. The absorbance was measured after 8 min
at 595 nm with a microplate reader model Anthos ht2 (Anthos,
Krefeld, Germany). The AOP/antioxidant capacity was calculated
using the absorbance difference between sample and blank and a
further parallel Fe(II) standard solution, and results were expressed
as micromoles or millimoles of Fe2+ equivalents.
Total phenolic content
The total phenolic content of HSE was determined using the
Folin–Ciocalteu assay21,22 and was performed in an Uvidec-
610 spectrophotometer (Jasco, Gross-Umstadt, Germany). HSE
samples were diluted with deionized water (1 : 20), and were
directly assayed at 750 nm, with gallic acid serving as a standard.
The total phenolic content was expressed as milligrams of gallic
acid equivalents (GAE) per 200 mL HSE (administered dose).
HSE anthocyanins, anthocyanin metabolites and hippuric acid by
HPLC
HSE anthocyanins and metabolites in plasma and urine were
extracted with a solid-phase cartridge (Sep-Pak C18, Waters,
Milford, MA, USA), as described by Miyazawa et al.26 and Felgines
et al.,27 respectively, with slight modifications.
Plasma: The cartridge was washed with 10 mL methanol and
equilibrated with 10 mL of 1.5 mol L−1 formic acid. Subsequently,
2 mL sample plasma diluted with 0.4 mL of 0.44 mol L−1 TFA
solution was applied to the equilibrated cartridge. The water-
soluble components as well as polar and neutral lipids were
removed in succession by 10 mL formic acid (1.5 mol L−1 ), 10 mL
dichloromethane and 10 mL benzene. Anthocyanins were eluted
with 10 mL of 0.44 mol L−1 TFA in methanol. The methanol phase
was collected and solvent was removed under vacuum with a ro-
tary evaporator (Rotavap RE 121, Buchi, Flawil, Switzerland), with
the bath temperature maintained at 30 ◦ C. The extracts were redis-
solved in 300 µL of the HPLC mobile phase before analysis. Aliquots
of 15 µL (for identification of HSE anthocyanins and metabolites by
HPLC–photodiode array detection (PDA)–electrospray ionization
(ESI)–tandem mass spectrometry (MS/MS); system I) and of 50 µL
(for quantification by HPLC-PDA; system II), respectively, were used
for HPLC analysis.
Urine: Acidified urine samples were thawed and maintained for
60 min at room temperature before solid-phase extraction (SPE)
to obtain the maximal yield of the coloured flavylium cations. The
SPE cartridge was activated with 10 mL methanol and equilibrated
with 10 mL of 12 mmol L−1 aqueous HCl before use. Subsequently,
9 mL acidified urine was applied to the equilibrated cartridge. The
cartridge was then washed with 10 mL of 12 mmol L−1 aqueous
HCl, and anthocyanins were eluted with 15 mL of 12 mmol L−1
HCl in methanol. The methanolic extract was evaporated under
nitrogen to a volume of 1 mL at 35 ◦ C. Aliquots of 15 µL (for
identification of HSE anthocyanins and metabolites; system I) and
of 50 µL (for quantification; system II), respectively, were used for
HPLC analysis.
Hippuric acid: Untreated urine was centrifuged at 8400 × g
and diluted with deionized water (1 : 200, v/v) prior to HPLC
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analysis (system II). Sample aliquots for LC-MS analysis (system I)
were prepared using SPE as described for HSE anthocyanins and
metabolites.
The diluted HSE was centrifuged (5 min, 8400 ×g, 8 ◦ C) and
analysed for anthocyanins under the same conditions as described
below (system II).
Identification of HSE anthocyanins, anthocyanin metabolites,
and hippuric acid (system I): Characterization of HSE phenolics
was conducted using an Agilent HPLC series 1100 (Agilent,
Waldbronn, Germany), equipped with ChemStation software, a
G1379A degasser, a G1312A binary gradient pump, a G1313A
autosampler, a G1316A column oven, and a G1315B diode array
detector. The separation was performed with a Phenomenex
Aqua column (250 × 4.6 mm i.d.; 5 µm) with a C18 ODS guard
column (4.0 × 2.0 mm i.d.) operated at a temperature of 25 ◦ C.
The mobile phase (anthocyanins and anthocyanin metabolites)
consisted of water–formic acid–acetonitrile (87 : 10 : 3, v/v/v,
eluent A) and of water–formic acid–acetonitrile (40 : 10 : 50, v/v/v,
eluent B) using a gradient programme as follows: 10% B to 25% B
(10 min), 25% B to 31% B (5 min), 31% B to 40% B (5 min), 40% B to
50% B (10 min), 50% B to 100% B (10 min), 100% B to 10% B (5 min),
10% B isocratic (5 min). Monitoring was performed at 520 nm at a
flow rate of 0.8 mL min−1 . For the separation of non-anthocyanin
phenolics a solvent system of 2% (v/v) acetic acid in water (eluent
A) and of 0.5% acetic acid in water and acetonitrile (50 : 50, v/v;
eluent B) with the following gradient programme was used: 10%
B to 24% B (20 min), 24% B to 30% B (20 min), 30% B to 55%
B (20 min), 55% B to 100% B (15 min), 100% B isocratic (8 min),
100% B to 10% B (2 min), 10% B isocratic (5 min). Monitoring was
performed at 280 nm at a flow rate of 1.0 mL min−1 . The HPLC
system was coupled online to a Bruker (Bremen, Germany) model
Esquire 3000+ ion trap mass spectrometer using an ESI interface
and Esquire Control software. Positive (anthocyanins) and negative
(non-anthocyanin phenolics) ion mass spectra, respectively, were
recorded in the range m/z 50–1000. Nitrogen was used as drying
gas at a flow rate of 11 L min−1 and as nebulizing gas at a pressure
of 65 psi (anthocyanins); the parameters for the analysis of non-
anthocyanin phenolics were 12 L min−1 and 70 psi, respectively.
The nebulizer temperature was set at 365 ◦ C and a potential of
±4000 V was used on the capillary. Helium was used as collision
gas at a pressure of 4.0 × 10−6 mbar. Dissociation spectra were
obtained with a fragmentation amplitude of 1.0 V.
Quantification of HSE anthocyanins, metabolites, and hippuric acid
(system II): Quantification of HSE anthocyanins and metabolites
in plasma and urine was carried out following the method
of Kammerer et al.23 The HPLC system consisted of an L-
6200 pump, an AS-4000 auto-injector (Merck-Hitachi, Darmstadt,
Germany), and a 990 photodiode array detector (Waters, Eschborn,
Germany) equipped with a ProntoSIL Eurobond RP-18 column
(5 µm, 250 × 4.0 mm i.d., Bischoff, Leonberg, Germany) and a
LiChrospher 100 RP-18 guard column (5 µm, 4.0 × 4.0 mm i.d.,
Merck). Analytical HPLC was run at ambient temperature. The
composition of the mobile phase, gradient programme, detection
wavelength as well as the flow rate were the same as described
for system I (anthocyanins). Individual HSE anthocyanins and
metabolites were quantified using external calibration curves
of cyanidin-3-glucoside, cyanidin-3-sambubioside, delphinidin-3-
glucoside and delphinidin-3-sambubioside.
Hippuric acid was analysed and quantified according to the
HPLC method reported by Kubota et al.,28 with slight modifica-
tions. The samples were eluted by a ProntoSIL Eurobond RP-18
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column under isocratic conditions with water–acetonitrile–acetic
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acid (78 : 20 : 2, v/v/v; pH 2.2), and monitored at 235 nm. Urinary
hippuric acid concentrations were calculated from an external
hippuric acid calibration curve.
Ascorbic acid and uric acid by HPLC
Plasma and urine (stabilized with meta-phosphoric acid) were
centrifuged (5 min, 8400 × g, 8 ◦ C) and the supernatants were
directly injected onto a ProntoSIL Eurobond RP-18 column (system
II) and eluted under isocratic conditions with water acidified with
sulfuric acid to pH 2.2.24,25 Detection was carried out at 245 nm
at a flow rate of 0.8 mL min−1 . Ascorbic acid and uric acid were
identified by comparing their retention times and characteristic
UV–visible spectra with those of synthetic L-ascorbic acid and
uric acid, respectively. The concentrations in the samples were
calculated from external L-ascorbic acid and uric acid calibration
curves, respectively.
The diluted HSE was centrifuged (5 min, 8400 × g, 8 ◦ C) and
analysed for ascorbic acid under the same conditions as described
above.
Malondialdehyde by HPLC
MDA concentrations in urine were determined by isocratic
reversed-phase (RP)-HPLC according to Volpi and Tarugi29 with
slight modifications. Urine was mixed with 2-thiobarbituric acid
(TBA) and butylated hydroxytoluene (BHT) and incubated at 95 ◦ C
for 45 min. After cooling on ice, the samples were centrifuged
(10 min, 8400 × g, 8 ◦ C) and the supernatants were diluted
(1 : 10) prior to HPLC analysis. The separation of the MDA–TBA
complex (system II) was performed using a ProntoSIL Eurobond
RP-18 column equipped with a LiChrospher 100 RP-18 guard
column and a mobile phase composed of 35% methanol and 65%
50 mmol L−1 disodium phosphate buffer, pH 7.0. The complex was
eluted at a flow rate of 0.8 mL min−1 and monitored by a Merck-
Hitachi F-1050 fluorescence detector (ex.: 515 nm; em.: 553 nm).
Urinary MDA concentrations were calculated from an external
MDA calibration curve.
Intra-day and inter-day variability proved to be below 10% for
all analytical methods.
Biokinetics and statistical evaluation
Non-compartmental biokinetic evaluation was performed accord-
ing to standard methods using model no. 220 of WinNonlin
software, version 5.0 (Pharsight Corporation, Mountain View, CA,
USA). The following biokinetic variables were determined from
the plasma concentration–time courses of FRAP, ascorbic acid
and uric acid: ‘baseline’, defined as the last non-missing plasma
concentration prior to ingestion of the study beverage, i.e. the
pre-dose value measured at t = 0 h, and the area under the curve
(AUC) above baseline. The AUC above baseline was calculated ac-
cording to the linear trapezoidal rule. The biokinetic analysis was
based upon assayed plasma concentrations and nominal sampling
times; the sampling schedule was followed exactly.
Furthermore, the amounts of antioxidants assayed by FRAP,
MDA, HSE anthocyanins and metabolites, hippuric acid, ascorbic
acid and uric acid excreted in 24 h urine (Ae0 – 24 ) were determined
by multiplying the concentration of the respective analyte in urine
by the volume of the 24 h urine sample.
The biokinetic variables in plasma and urine were summarized
using number of observations, arithmetic mean, standard devia-
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tion (SD), minimum, maximum and median for each treatment.
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Untransformed variables, baseline, AUC above baseline and
Ae0 – 24 were analysed with a linear mixed-effects model with fixed
terms for treatment, period, sequence and sex, and random term
for subject within sequence-by-sex:
Biokinetic variable = Sequence + Subject (Sequence × Sex)
+ Period + Treatment + Sex + Error
fitted by generalized least squares (GLS) with restricted maximum
likelihood (REML) estimates of variances and covariances, using
WinNonlin , version 5.2.1 (Pharsight Corporation). For baseline,
AUC above baseline and Ae0 – 24 , estimate and 90% confidence
interval (CI) for the ratio of treatment means (HSE/water) were
obtained by computing estimate and 90% CI for the contrast giving
the difference between treatment means within the linear mixed
effects model framework. The variable baseline was subjected to
analysis in order to check whether the assumption of no carryover
effects is viable. To check whether the assumption of normal
distribution is valid, QQ plots of the residuals were created. No
adjustment of the alpha-level was made for multiple analyses.
Pearson’s correlation coefficients were calculated to explore the
relationship between each ascorbic acid and uric acid versus FRAP
plasma concentrations, respectively.
The level of statistical significance was fixed at P < 0.05. All
statistical testing was purely exploratory.
RESULTS
Plasma
Concentration versus time curves of ascorbic acid, uric acid and FRAP
The absolute changes from baseline of ascorbic and uric acid
as well as FRAP plasma levels at the different sampling times
after administration of HSE or water are displayed in Fig. 1. On
average, there were only minor changes in plasma concentrations
of ascorbic and uric acid versus time upon comparison of the
two treatments. The highest average change in FRAP plasma
concentration was attained 1 h post-dose (Fig. 1). Beginning
from 2 h post-dose the average FRAP plasma level declined
continuously below the pre-dose level following administration
of HSE. Upon administration of water, there was an almost linear
decline in average FRAP plasma level below the pre-dose level
(Fig. 1).
Biokinetic variables
Data on the extent of change in antioxidant capacity in plasma as
characterized by AUC above baseline are summarized in Table 1.
The baseline values (defined as the pre-dose levels at t = 0 h)
are also given in Table 1. Data on urinary excretion are reported
in Table 2. Estimates of treatment ratios HSE (test) versus water
(reference) together with the 90% confidence limits are detailed
in Table 3.
FRAP AUC above baseline values ranged from 0.0 to 151 µmol h
L−1 , and averaged 67.0 ± 47.6 µmol h L−1 (mean ± SD) after
HSE treatment and 6.69 ± 10.2 µmol h L−1 after water. The
difference between the two treatments was statistically significant
(see Table 3). Figure 2 illustrates that a unique increase in AUC
above baseline FRAP level occurred after HSE treatment in all
subjects.
Ascorbic acid AUC above baseline values were 3.84 ± 5.45 mg h
L−1 after HSE treatment and 2.47 ± 2.89 mg h L−1 after water
(Table 1). The difference between the two treatments was not
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Table 1. Summary of biokinetic variables in plasma following administration of either a single oral dose of an aqueous Hibiscus sabdariffa L. extract
or water to young healthy subjects

Treatment

Hibiscus extract Water

Analytical parameter AUC above baselinea Baselineb AUC above baseline Baseline

FRAPc N 8 8 8 8
Mean ± SD 67.0 ± 47.6 692 ± 85.9 6.69 ± 10.2 688 ± 113
Median (min., max.) 59.9 (14.3, 151) 698 (551, 795) 1.35 (0.00, 25.8) 683 (535, 856)
Ascorbic acidd N 8 8 8 8
Mean ± SD 3.84 ± 5.45 3.97 ± 1.15 2.47 ± 2.89 6.56 ± 2.41
Median (min., max.) 2.33 (0.00638, 16.5) 3.90 (2.79, 6.26) 1.94 (0.00, 8.48) 6.85 (2.68, 10.1)
Uric acidd N 8 8 8 8
Mean ± SD 7.82 ± 7.52 11.9 ± 3.26 14.4 ± 25.6 18.8 ± 5.62
Median (min., max.) 5.88 (0.00, 20.1) 11.5 (8.18, 17.9) 3.33 (0.00, 74.2) 18.7 (10.5, 27.7)
a AUC above baseline = area under the curve that is above the baseline, calculated according to linear trapezoidal rule.
b Baseline = the last non-missing plasma concentration prior to ingestion of the study beverage, i.e. pre-dose value at t = 0 h on study day.
c
Unit for baseline: µmol L−1 ; AUC: µmol h L−1 .
d
Unit for baseline: mg L−1 ; AUC: mg h L−1 .
Values are rounded to three significant figures.

Table 2. Summary of amount excreted within 24 h in urine (Ae0 – 24 ) following administration of either a single oral dose of an aqueous Hibiscus
sabdariffa L. extract or water to young healthy subjects

Treatment

Analytical parameter (unit) Hibiscus extract Water

FRAP (mmol) N 8 8
Mean ± SD 7.75 ± 1.80 6.14 ± 1.72
Median (min., max.) 7.24 (5.89, 10.7) 5.55 (4.66, 9.52)
Hippuric acid (mg) N 8 8
Mean ± SD 447 ± 78.1 331 ± 86.9
Median (min., max.) 452 (315, 541) 354 (180, 446)
MDA (µmol) N 8 8
Mean ± SD 1.60 ± 0.381 2.08 ± 0.694
Median (min., max.) 1.57 (1.18, 2.32) 1.87 (1.32, 3.18)
Ascorbic acid (mg) N 8 8
Mean ± SD 12.7 ± 5.66 8.48 ± 6.10
Median (min., max.) 10.5 (5.79, 24.2) 6.25 (1.75, 18.5)
Uric acid (mg) N 8 8
Mean ± SD 213 ± 81.3 249 ± 131
Median (min., max.) 198 (140, 388) 212 (134, 542)
Delphinidin-3-sambubioside (µg) N 8 8
Mean ± SD 6.54 ± 3.41 n.d.
Median (min., max.) 5.88 (2.47, 12.7) n.d.
Cyanidin-3-sambubioside (µg) N 8 8
Mean ± SD 18.7 ± 11.4 n.d.
Median (min., max.) 14.7 (9.39, 43.3) n.d.
Delphinidin monoglucuronide (µg) N 8 8
Mean ± SD 3.92 ± 1.60 n.d.
Median (min., max.) 4.11 (1.81, 6.77) n.d.
Total anthocyanins (µg) N 8 8
Mean ± SD 29.1 ± 12.6 n.d.
Median (min., max.) 26.3 (15.4, 53.3) n.d.

Values are rounded to three significant figures.

n.d., not detectable.
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Figure 1. Arithmetic means (±SD) of absolute changes in plasma FRAP, ascorbic acid and uric acid levels from pre-dose (baseline, t = 0 h) levels over
time following single oral administration of either hibiscus extract or water (n = 8 per treatment).

statistically significant (see Table 3). The AUC above baseline values
of uric acid ranged between 0.0 and 20.1 mg h L−1 (mean value
7.82 ± 7.52 mg h L−1 ) after HSE treatment, and between 0.0 and
74.2 mg h L−1 (mean value 14.4 ± 25.6 mg h L−1 ) after water (see
Table 1), with no significant difference between the two treatments
(see Table 3). Table 3 also shows almost identical baseline (pre-
dose) FRAP levels between treatments, whereas a statistically
significant lower ascorbic and uric acid baseline was proven upon
HSE administration. There were no significant periods, sequence
2212
Anthocyanins or common anthocyanin metabolites such as
methylated forms or glucuronide/sulfate conjugates were not
detected in plasma samples after HSE treatment.
Urine
FRAP Ae0 – 24 values ranged from 4.66 to 10.7 mmol, with a
mean value of 7.75 ± 1.80 mmol after HSE treatment and
6.14 ± 1.72 mmol after water. Hippuric acid (m/z 178/134) Ae0 – 24
values ranged from 180 to 541 mg, with a mean value of

or sex effects, either on baseline or on AUC above baseline. 447 ± 78.1 mg after HSE treatment and 331 ± 86.9 mg after water.

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Table 3. Comparison of biokinetic variables obtained after administration of Hibiscus sabdariffa L. extract and water
Analytical Biokinetic Treatment ratio Hibiscus/water Significance
Matrix parameter variable (unit) (90% confidence interval)a (P-value)b

Plasma FRAP Baseline (µmol L−1 ) 100.5 (95.7, 105.4) 0.833

AUC above baseline (µmol h L−1 ) 1001.9 (504.2, 1499.7) 0.012
Ascorbic acid Baseline (mg L−1 ) 60.5 (40.2, 80.8) 0.009
AUC above baseline (mg h L−1 ) 155.2 (36.3, 274.3) 0.402
Uric acid Baseline (mg L−1 ) 63.3 (41.6, 85.0) 0.017
AUC above baseline (mg h L−1 ) 54.3 (−92.5, 201.1) 0.567
Urine FRAP Ae0 – 24 (mmol)c 126.3 (113.7, 138.8) 0.007
Hippuric acid Ae0 – 24 (mg) 135.1 (119.6, 150.5) 0.005
MDA Ae0 – 24 (µmol) 76.8 (61.0, 92.5) 0.028
Ascorbic acid Ae0 – 24 (mg) 149.2 (126.2, 172.2) 0.006
Uric acid Ae0 – 24 (mg) 85.8 (50.0, 121.6) 0.470
a
The least squares mean (LSM) of hibiscus as percentage of LSM of water.
b The probability (P-value) associated with F-statistics is given for the treatment term of the linear mixed effect model.
c Amount excreted into urine within 24 h.

Figure 2. Individual and arithmetic mean (±SD) FRAP AUC above baseline (pre-dose) by treatment.

MDA Ae0 – 24 values ranged from 1.18 to 3.18 µmol with a mean
value of 1.60±0.38 µmol after HSE treatment and 2.08±0.69 µmol
after water. Ae0 – 24 of ascorbic acid was 12.7 ± 5.66 mg after
HSE and 8.48 ± 6.10 mg after water. Ae0 – 24 of uric acid was
213 ± 81.3 mg after HSE and 249 ± 131 mg after water (Table 2).
Except for uric acid, the treatment difference was statistically
significant for each urinary analyte assayed (see Table 3). There
was a significant sequence effect on Ae0 – 24 of ascorbic acid, and a
significant sex effect on Ae0 – 24 of FRAP, hippuric acid and MDA.
The mean Ae0 – 24 of hibiscus anthocyanins and metabo-
lites (sum) was 29.1 ± 12.6 µg after HSE treatment. Cyanidin-
3-sambubioside was the major anthocyanin (18.7 ± 11.4 µg)
(Table 2). Representative mass spectra of collision-induced disso-
ciation experiments corroborating peak assignment are illustrated
in Fig. 3. At baseline and in the control treatment, none of the
or common anthocyanin metabolites (e.g. methylated and/or
glucuronidated anthocyanin derivatives).
The assumptions underlying the crossover design were met:
there were no statistically significant sequence or period effects
(with the aforementioned exceptions) that would have invalidated
the trial.
The correlation coefficients for plasma ascorbic acid versus
FRAP plasma levels were 0.17 (HSE, P > 0.05) and −0.12 (water,
P > 0.05), respectively. The correlation coefficients for plasma uric
acid versus plasma FRAP levels were 0.49 (HSE, P < 0.001) and 0.40
(water, P < 0.001), respectively (Fig. 4).
DISCUSSION
The objective of the present study was to evaluate the impact
of a single oral dose of an aqueous extract of H. sabdariffa L.
2213

urine samples contained any detectable amounts of anthocyanins (HSE) on the systemic AOP. The obtained data from eight healthy

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 www.soci.org
(A)
∆ AU
(b)
(a)
(d)
(c)
[A1] [A2]
Time
Figure 3a. Representative HPLC-PDA chromatograms and mass spec-
tra. (A) Human urine (subject 3) collected after the ingestion of
200 mL tap water (control; A1) and 200 mL HSE (A2), respectively.
Detection was performed at 520 nm. Peak assignment: (a) delphinidin-
3-sambubioside, (b) cyanidin-3-sambubioside, (c) cyanidin monoglu-
curonide and (d) delphinidin monoglucuronide. (B) Representative mass
spectra of collision-induced dissociation experiments corroborating peak
assignment. Anthocyanins were detected in the positive ion mode.
(a) Delphinidin-3-sambubioside; m/z 597 (b) cyanidin-3-sambubioside;
m/z 581 (c) cyanidin monoglucuronide; m/z 463 (d) delphinidin monoglu-
curonide; m/z 479.
male and female volunteers indicate that HSE consumption led
to an enhanced AOP as compared to water used as a reference
treatment. The enhanced AOP as assayed by FRAP was of short-
term nature with an average peak time around 1 h after dosing.
This result is in line with findings reported by others: increased
AOP of plasma assayed by FRAP has been observed following the
consumption of red wine and whisky,30 spinach and strawberries,31
black tea and green tea,32 as well as wheat bran.33
As shown in Table 2, there was an increased amount of
antioxidants assayed by FRAP excreted in the volunteers’ 24 h
urine after consumption of HSE as compared to water. A similar
result was found in a recently published study. Price et al.33
reported a significant increase in urinary total polyphenols and
antioxidant potential (assayed by FRAP) in 18 healthy subjects after
consumption of polyphenol-rich wheat bran. It should be noted
that Tepel et al.34 stated in a review about antioxidative therapies
and vascular diseases that antioxidants can prevent the acute
2214
decrease of renal function caused by ischaemia, contrast media
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c 2012 Society of Chemical Industry
T Frank et al.
or drugs. Therefore, the enhancement of urinary concentration
in antioxidants may have physiological relevance regarding the
protection of functional renal tissue.
Ascorbic acid and uric acid are important antioxidants in
biological fluids like blood and urine and contribute substantially to
the in vivo antioxidant capacity.16,17 Since the FRAP assay measures
a wide range of plasma and urine antioxidants including uric acid
and ascorbic acid,20,35,36 both compounds were determined to
exclude the possibility that the increase in AOP observed after the
ingestion of HSE was due to increased levels of these antioxidants.
Furthermore, previous studies demonstrated the correlation of
fructose consumption and increased uric acid concentrations in
human plasma and subsequently increased plasma antioxidant
capacity.37,38 Since ascorbic acid and urate levels did not
significantly change from the baseline after HSE consumption,
which did not contain measurable amounts of fructose or other
sugars, we conclude that the enhanced plasma AOP was most likely
caused by HSE phenolics and their metabolites. This is supported
by the fact that we found only a weak correlation between uric acid
and FRAP, but no correlation between ascorbic acid and FRAP. As
demonstrated for plasma, our data of urine samples also showed
that consumption of HSE did not result in statistically significant
higher excretion of uric acid within 24 h. In contrast to plasma AUC
above baseline, there was a markedly increased treatment ratio
for Ae0 – 24 of ascorbic acid exceeding 20% of reference (water).
This result is surprising since the tested HSE was virtually devoid
of ascorbic acid. Thus, it cannot be excluded that the increased
AOP in urine is confounded by increased ascorbic acid excretion
after HSE intake (12.7 versus 8.48 mg within 24 h). However, as
mean uric acid excretion within 24 h after HSE consumption was
∼ 36 mg lower (tendency; P > 0.05) compared to the control
(213 versus 249 mg), it is unlikely that the small increase (absolute
value) of ascorbic acid has a significant effect on the urinary AOP
after HSE ingestion.
We minimized the impact of the small number of recruited
subjects by using the crossover study design. We found statistically
significant elevated plasma ascorbic acid and urate baseline (pre-
dose) levels in our study at control treatment. These baseline
measurements were period dependent and might indicate that the
washout period was not sufficient to ensure that these baselines
are not influenced by previous treatment; that is, they were
affected by carryover. It is also possible that confounders such
as seasonal changes in temperature, environmental exposure to
allergens, pollution and pathogens may have influenced ascorbic
and uric acid status in subjects. However, this observation did not
invalidate the results of this study since the AUC calculation is
based on changes referred to the baseline. In addition, the impact
of any baseline differences in a crossover design was minimized
because each subject acted as their own control.
Urinary MDA was significantly reduced from 2.08±0.69 to 1.60±
0.38 µmol (−23% versus control) following HSE consumption and
values are in the same range as reported by Piche et al.39 for
human adults (2.05 µmol). Urinary MDA has been widely used
in animal models and in humans as a non-invasive biomarker of
lipid peroxidation induced by oxidative stress.19 The reduced
MDA excretion suggests that the renal generation of MDA
and/or the systemic production of MDA were reduced after HSE
ingestion. MDA has been found to be elevated in various diseases
purportedly related to free radical damage.40 Recently, Gorelik
et al.41 demonstrated significantly decreased urinary excretion
rates of MDA 6 h after the consumption of turkey cutlets with
red wine compared with a control (in a randomized crossover
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(B)
Intens.
a Delphinidin-3-sambubioside: +MS2 (597)
4000 302.8

3000

2000

1000
579.2

0
100 200 300 400 500 600 700 800 900 m/z

Intens.
b Cyanidin-3-sambubioside: +MS2 (581)
286.7
3000

2000

1000 562.9

0
100 200 300 400 500 600 700 800 900 m/z

Intens.
x104 c 462.7 Cyanidin-glucuronide: +MS2 (463)

6
286.6

0
100 200 300 400 500 600 700 800 900 m/z

Intens.
d Delphinidin-glucuronide: +MS2 (479)
302.6
2500

2000 478.7

1500

1000

500

0
100 200 300 400 500 600 700 800 900 m/z

Figure 3b. (Continued).

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Figure 4. Correlation between ascorbic acid and FRAP in plasma after hibiscus extract (r = 0.17, P > 0.05) and water (r = −0.12, P > 0.05), and
correlation between uric acid and FRAP in plasma after hibiscus extract (r = 0.49, P < 0.001) and water (r = 0.40, P < 0.001). Grey line, locally weighted
scatter-plot smoothing (LOESS curve).

study with 10 healthy subjects). Based on their results, they
suggested that red wine polyphenols exert a beneficial effect
by a novel function: inhibition of absorption of MDA due to
the formation of an adduct between red wine polyphenols and
aldehydes in the gastrointestinal tract. In another double-blind,
randomized, crossover study with 12 healthy men, Weinbrenner
et al.42 demonstrated a significant decrease in urinary MDA (24 h
urine) following a 4-day intervention with olive oils containing
different amounts of phenolic compounds. The observed decrease
in urinary MDA following HSE ingestion in the present study
suggests that the antioxidant compounds of hibiscus, either in
their intact forms or as metabolites, may have a reducing effect
on oxidative stress in vivo and could therefore provide protective
benefits for human health.
The consumption of HSE resulted in the appearance of native
HSE anthocyanins (cyanidin-3-sambubioside and delphinidin-3-
sambubioside) and at least of two identified anthocyanin metabo-
lites (cyanidin monoglucuronide and delphinidin monoglu-
curonide) in the volunteers’ urine (Table 2 and Fig. 3). It must
be noted that the excretion rates for cyanidin monoglucuronide
were not determined as urinary concentrations were below the
2216
ery of intact anthocyanins and metabolites was 29.1 ± 12.6 µg
within 24 h, corresponding to 0.022 ± 0.010% of the admin-
istered anthocyanin dose. These data are within the range as
reported in the literature for urinary excretion rates of antho-
cyanins and metabolites after ingestion of anthocyanin-rich food
(reported range: 0.004–5%).3,9,43 Furthermore, the detection of
intact anthocyanins and metabolites is in agreement with re-
sults reported by others: several bioavailability studies have
found that the glycosidic forms of anthocyanins are absorbed
intact and appear in biological fluids like plasma and urine,
whereas other studies have found anthocyanins to be present
as metabolites (key data of 30 published human studies dealing
with anthocyanin bioavailability were summarized by Mazza and
Kay).9
No native HSE anthocyanins or common anthocyanin metabo-
lites such as methylated derivatives or glucuronide/sulfate conju-
gates were found in plasma after HSE treatment. We minimized
the risk of anthocyanin degradation during sample preparation
and storage due to the fact that we used evaluated methods and
stored the samples under appropriate conditions (−80◦ C and ≤
pH 2).44,45 However, despite these precautions we cannot exclude

lower limit of quantification (1.3 ng mL−1 ). The urinary recov- minor losses during sampling and the extraction procedure.

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Hibiscus and systemic antioxidant potential
In a previous study with H. sabdariffa L. extract, Frank
et al.44 determined maximum plasma concentrations of 2.2
and 1.3 ng mL−1 for cyanidin-3-sambubioside and delphinidin-
3-sambubioside, respectively, 1.5 h after intake. However, it has
to be noted that the ingested dose of total anthocyanins was
∼ 300 mg compared to the present study (∼ 130 mg). In a current
study by McKay et al.,15 the antihypertensive effects of hibiscus
tea consumption in pre- and mildly hypertensive humans were
investigated. A blood pressure lowering effect of hibiscus tea was
observed, but the authors failed to detect hibiscus anthocyanins
in blood plasma or urine of the subjects. Based on their results,
McKay and co-workers suggested that due to the low daily total
anthocyanin dose (three 7.04 mg servings) the absorbed and
excreted amounts of anthocyanins may have been below the
limit of detection in plasma and urine as measured by HPLC.
Another possible explanation for the failed detection of hibiscus
anthocyanins in the investigated body fluids could be an extensive
metabolism of the absorbed delphinidin and cyanidin glycosides.
Recently, Vitaglione et al.43 reported that protocatechuic acid,
which exhibits a marked antioxidant activity, was the major
metabolite of cyanidin glucosides in humans, accounting for
∼ 44% of the ingested pigments in 6 h post-consumption plasma.
They concluded that the ‘phenomenon’ reported in some clinical
trials, that the increased antioxidant capacity in plasma did not
correlate with the small or negligible amount of intact antho-
cyanins and/or their glucuronidated and methylated metabolites,
could be explained by the presence of protocatechuic acid.43
Metabolites like protocatechuic acid can be formed in the small
intestine, bloodstream and by the intestinal microbiota (colon),
respectively.43 The presence of protocatechuic acid and other not
yet identified anthocyanin metabolites with antioxidant activity
in the analysed plasma and urine samples after HSE consumption
cannot be excluded, since the analytical procedure was focused
on the detection and identification of native HSE anthocyanins
and their common glucuronidated/methylated derivatives. Fur-
thermore, the significant contribution of polyphenol metabolites
to the antioxidant activity in vivo was also reported by others.46 – 48
Therefore, it would be highly desirable in future in vivo studies with
animals and/or human subjects to utilize labelled anthocyanins for
the identification of all major metabolites, particularly metabolites
produced by the intestinal microbiota.
In this study, the mean urinary excretion of hippuric acid
increased significantly by 116 mg 24 h−1 after consumption of
200 mL HSE (∼ 1.4−fold increase versus control). Hippuric acid is
considered as a metabolite that results from microbial degradation
of polyphenols in the colon followed by hepatic conjugation
with glycine.18 Hippuric acid may be a biomarker of intake of
total polyphenols, having been reported as a urinary indicator
of polyphenol consumption from tea and other polyphenol-rich
food.18,49,50 Recently, Mulder et al.51 reported that black tea and
green tea consumption had comparable effects on urinary hippuric
acid excretion: the daily dose of polyphenols in the black tea solids
assayed by the Folin–Ciocalteu method was 9.08 mmol gallic acid
equivalents (GAE), and the corresponding increase in hippurate
excretion in urine was 1.86 mmol 24 h−1 (20%). For green tea,
the corresponding values were 13.3 mmol GAE consumed per
day, resulting in an increase in urinary hippurate excretion of
2.33 mmol 24 h−1 (18%). In the present study, a single oral dose
of 200 mL HSE (1.4 mmol GAE) resulted in an additional excretion
of 0.65 mmol hippuric acid 24 h−1 (46% of dose), indicating that
HSE polyphenols may have a higher biotransformation rate in
the colon compared to black and green tea polyphenols. The
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potential physiological significance of colonic metabolites of
polyphenols was reviewed and summarized by Williamson and
Clifford.52 The authors suggested that, owing to the fact that these
catabolites occur in vivo in significant higher concentrations than
their respective parent compounds, at least part of the biological
activities ascribed to plant polyphenols are due to their colonic
metabolites.
CONCLUSIONS
In the present study, we could demonstrate that a single oral dose
of HSE caused a significant increase in the antioxidant potential
of human plasma and urine as well as a significant decrease in
urinary MDA, which indicates the in vivo antioxidant potential of
hibiscus. Furthermore, a significantly increased urinary hippuric
acid excretion within 24 h after HSE consumption indicates a high
biotransformation of the ingested HSE polyphenols, most likely
caused by the colonic microbiota. To our knowledge, the present
study is the first one describing such physiological effects in healthy
humans after acute HSE consumption. Although based on a small
number of subjects, our results have established a basis for more
comprehensive investigations to evaluate potential benefits of
HSE consumption both acute and chronic, and to assess the mode
of action. Therefore, follow-up studies are warranted to identify
the complete spectra of in vivo metabolites in plasma and urine
and to investigate their interaction with chronic disease processes
by using appropriate cell based assays and animal models.
ACKNOWLEDGEMENTS
This study was funded by Plantextrakt GmbH & Co. KG,
Vestenbergsgreuth, Germany. The authors are indebted to
all volunteers who participated in this study. Furthermore,
the preparation of cyanidin-3-sambubioside and delphinidin-
3-sambubioside by Prof. Dr Peter Winterhalter, Technische
Universität Braunschweig (Germany) is gratefully acknowledged.
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