ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITIES

OF AQUEOUS AND ETHANOL EXTRACTS OF COLA

NITIDA (STERCULIACEAE) IN CARRAGEENAN INDUCED
PAW EDEMA IN RATS

AYEBE EDWIGE K.1, YAPI HOUPHOUET F.1,*, TIANGA YAYA SORO3, YAPO ADOU F.1,
GOGAHY K.1, YEO DODEHE1, NGUESSAN JEAN D.1, DJAMAN ALLICO J.1,2
1
Pharmacodynamics Biochemical Laboratory, UFR Biosciences, Felix Houphouet Boigny University,
PO Box 582, Abidjan 22, Côte d’Ivoire
2
Laboratory of Basic and Clinical Biochemistry, Pasteur Institute of Cote d’Ivoire,
PO Box 01 BP 490, Abidjan 01, Côte d’Ivoire
3
Laboratory de physiologie animale, UFR Biosciences, Felix Houphouet Boigny University,
PO Box 582, Abidjan 22, Côte d’Ivoire
(Received 11 November, 2014)

This study aimed to evaluate the potential anti-inflammatory and antioxidant activities of Cola
nitida aqueous and ethanol extracts. Anti-inflammatory activities were investigated by carrageenan
induced paw edema and C reactive protein (CRP) concentration in rats. The effects of extracts on
lipid peroxidation (LPO) and reducing power assay were estimated. Extracts were injected
intraperitoneally into rats at doses of 100 mg/kg and 200 mg/kg body weight (bw). A significant anti-
inflammatory activity at 200 mg/kg bw for the ethanol extract was found, which was comparable to
Diclofenac (25 mg/kg) inhibition. The aqueous extract of Cola nitida was less effective than the
ethanol extract in the prevention of rat paw edema. This study showed an increased CRP
concentration (p <0.05) in rats treated with carrageenan, contrary to extracts and Diclofenac treated
groups, but no significant difference between CRP concentration of the extracts and Diclofenac
(p>0.05). Administration of Cola nitida at a dose of 200 mg/kg per day decreased the level of TBARS
and increased the reducing ability in serum. Our study showed that Cola nitida aqueous and ethanol
extracts have potential anti-inflammatory and antioxidant properties. However, the anti-inflammatory
activity was raised with the ethanol extract. Therefore, ethanol and aqueous extracts can be used for
therapeutic purposes.

Keywords: Cola nitida, inflammation, oxidative stress, rat, Cote d’Ivoire.

                                                            
*
Corresponding author (Email: felhouph@yahoo.fr; Pharmacodynamics Biochemical
Laboratory, UFR Biosciences, Felix Houphouet Boigny University, PO Box 582, Abidjan 22, Côte
d’Ivoire)

ROM. J. BIOCHEM., 52, 1, 19–29 (2015)

20 Ayebe Edwige K. et al. 2

INTRODUCTION

Utilization of natural products with therapeutic properties is as old as human

civilization. For a long period of time, minerals, plants and animal products were
the main sources of drugs with different properties (1). It is also estimated that less
than 10% of plant species have been studied for their biological activities. These
figures indicate that a thorough study of Cola nitida could lead to the discovery of
new therapeutic agents. Cola nitida plays an important social role in African
societies, where it symbolizes the sacred (2). Kola nut is used in community rituals
such as weddings, christenings, events friendships, funerals, rituals and sacrifices
(3, 4).
Indeed, the triphytochemical screening of Cola nitida extracts in our recent
work revealed the presence of polyphenols, leucoanthocyan, tannin catechin, gallic
tannin, flavonoids, alkaloids, saponins, sterols and terpenes (5).
Flavonoids are known to have anti-inflammatory activity (6). Santos and
Rao (7) reported an anti-inflammatory and antinociceptive activity of terpinoids.
Phenolic compounds are reported to possess anti-inflammatory and antioxidant
activities (8).
The present investigation was carried out to evaluate the anti-inflammatory
and antioxidant activities of Cola nitida aqueous and ethanol extracts, and to
determine which was the most active extract.

MATERIALS AND METHODS

COLLECTION AND AUTHENTICATION OF COLA NITIDA PLANT

The seeds of Cola nitida were collected from areas around the Anyama
forest in the district of Abidjan (Côte d’Ivoire, West Africa). Nuts were identified
and authenticated by Professor AKE ASSI at the Department of Botany, Felix
Houphouet Boigny University (Côte d’Ivoire, West Africa).

PREPARATION OF EXTRACT

Nuts were cut into pieces and dried out of the sun, then the dried seeds were
ground into powder (IKAMAG RCT®). One hundred (100) grams of cola nut
powder were extracted in 1000 mL of distilled water by maceration for 24 hours.
The homogenate was successively filtered twice on the cotton wool and once on
Whatman paper. The collected filtrate was then heated to 50°C. The resulted
powder represented the total aqueous extract which was used for the preparation of
extracts with different concentrations. This method was developed by Guede
et al. (9). Ethanol extracts were prepared similarly to the total aqueous extract.
3 Anti-inflammatory and antioxidant activities of Cola nitida extracts 21

EXPERIMENTAL ANIMALS TESTS FOR ANTI-INFLAMMATORY

AND ANTIOXIDANT ACTIVITIES

Carrageenan induced paw edema model

In the first set of experiments, Wistar albino rats weighing 150–200 g were
obtained from the animal center of Faculty of Pharmaceutical in Felix Houphouet
Boigny University. Rats were housed in standard cages at a constant temperature
of 22±1°C and relative humidity 55±5%, with 12 h light-dark cycle, for at least
1 week before the experiments. Each animal experiment was performed in six
groups:
– Group I: normal saline (0.9%) + carrageenan (0.2 mL of 1% in normal
saline);
– Group II: Diclofenac sodium (10 mg/kg, i.p.) + carrageenan (0.2 mL of 1%
w/v in saline solution);
– Group III: aqueous extract of Cola nitida (100 mg/kg, i.p.) + carrageenan
(0.2 mL of 1% w/v in saline solution);
– Group IV: aqueous extract of Cola nitida (200 mg/kg, i.p.) + carrageenan
(0.2 mL of 1% w/v in saline solution);
– Group V: ethanolic extract of Cola nitida (100 mg/kg, i.p.) + carrageenan
(0.2 mL of 1% w/v in saline solution);
– Group VI: ethanolic extract of Cola nitida (200 mg/kg, i.p.) + carrageenan
(0.2 mL of 1% w/v in saline solution).
In this experiment, all drugs (extracts) were administered intraperitoneally.
One hour after the treatment of all animals with extracts, 0.2 mL (10) of 1%
carrageenan solution were injected in the sub-plantar surface of the right hind paw
and the paw volume was measured by the caliper ruler at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h
and 24 h (11).

CRP and antioxidant status determination

In the second set of experiments, thirty rats were divided into 6 groups,
each containing six animals. Group I received normal saline 0.9% only (control).
Group II were given 0.2 mL carrageenan (with normal saline 0.9%). Groups III,
IV and V received 200 mg/kg b.w of aqueous and ethanol extracts and 25 mg/kg
b.w. Diclofenac respectively, with 0.2 mL carrageenan and normal saline 0.9%
after 1 h. After 5 hours of carrageenan administration, all animals were sacrificed,
blood samples were collected and serum was separated to be used later (11).
22 Ayebe Edwige K. et al. 4

Quantitative measurement of Rat C Reactive Protein in serum

Serum levels of C reactive protein (CRP) were measured using a commercially

available enzyme-linked immunosorbent assay (ELISA) kit (CRP, Rat ELISA Kit,
ab108827) supplied by ABCAM®, UK, as per the manufacturer’s instructions.

Essay procedure (12)

All materials and prepared reagents were equilibrated prior to use.

Standards, controls and samples were prepared in duplicate. Reagents, working
standards and samples were prepared and equilibrated as instructed. The assay was
performed at room temperature (18-25°C). Excess microplate strips from the plate
frame were removed and returned immediately to the foil pouch with desiccant
inside. The pouch was resealed securely to minimize exposure to water vapor and
stored in vacuum desiccators. 50 µL of CRP standard or sample were added to the
wells, then covered with a sealing tape and incubated for two hours. The timer
started after the last sample addition. The plates were washed five times with
200 µL of the wash buffer manually, then inverted each time, and the contents were
decanted on absorbent paper towel to remove the liquid. 50 µL of 1X Biotinylated
CRP antibody were added to each well and the plates were incubated for one
hour. The microplates were washed as described above. 50 µL of 1X SP
Conjugate were added to each well and incubated for 30 min. The microplates
reader were turned on and the program was set up in advance. The
microplates were washed as described above. 50 µL of chromogen substrate
were added per well and incubated for about 20 min, until the optimal blue
colour density developed. 50 µL of stop solution were added to each well. The
colour should have changed from blue to yellow. The absorbance was read on a
microplate reader at a wavelength of 450 nm immediately.

Total Antioxidant Status Assay

The determination of total antioxidant capacity of serum using the method

of Benzie and Strain (13), based on the measurement of its ability to reduce Fe3+
to Fe2+ by the test of FRAP (Ferric Reducing Ability of Plasma). Thus, 10 µL of
serum were added to 300 µL of FRAP reagent (freshly prepared and preheated to
37°C) in a test tube. After 10 min of incubation at 37°C, the absorbance of the
blue complex was read against a blank (300 µL of FRAP reagent + 10 L of distilled
water) at 593 nm. The standards of Fe2+ in the concentration range from 100 to
1000 mmolxL-1 solutions were prepared from ferrous sulfate (FeSO4, 7H2O) in
5 Anti-inflammatory and antioxidant activities of Cola nitida extracts 23

water. Data were expressed as mmol of reduced to ferrous form per liter (FRAP
value) ferric ions.

Malondialdehyde assay

We used the thiobarbituric acid reactive substances (TBARS) method as

an index of lipid peroxidation for analyzing malondialdehyde (MDA) products
during an acid-heating reaction, as previously described by Satho (14). To
precipitate the serum proteins, 2.5 mL of 20% TCA (w/v) were added in 0.5 mL of
rabbit serum and centrifuged at 1500 g for 10 min. Then, 2.5 mL of sulfuric acid
and 2 mL TBA (0.2%) were added to the sediment. This reaction mixture was
shaken and incubated for 30 min in a boiling water bath. After adding 4 mL of n-
butanol, the solution was centrifuged, cooled and absorbance was read at 532 nm.
The calibration curve was obtained using different concentrations of 1, 1, 3,
3-tetramethoxypropane as standard to determine the TBA-MDA adduct
concentration in the sample.

Statistical analysis

The values expressed as mean ± SEM from 6 animals. The results were
subjected to statistical analysis by using one way ANOVA followed by Dunnett’s
test to verify the significant difference, if any, among the groups. P < 0.05*,
p < 0.01**and p < 0.001*** were considered significant.

RESULTS

CARRAGEENAN INDUCED PAW EDEMA

Edema was induced in the rat’s paw. The variation of edema was assessed
after administration of aqueous and ethanol extracts of Cola nitida in two doses
(100 and 200 mg/kg) intraperitoneally. The results were compared with those
provided by Diclofenac Sodium® and the physiological (normal saline 0.9%)
control. Administration of Diclofenac Sodium® at the dose of 25 mg/kg increased
significantly the rat paw diameter. Carrageenan treatment produced changes from
5.92±0.04% to 17.55%± 0.02. The results were significantly different from
controls. The ethanol extract of Cola nitida at the dose of 100 and 200 mg/kg
decreased significantly rat acute paw edema after 24 hours, while the aqueous
extract of Cola nitida was less effective. The ethanol extract at 100 and 200 mg/kg,
respectively, showed a percentage of inhibition similar to that of Diclofenac
(25 mg/kg).
24 Ayebe Edwige K. et al. 6

10
EA 100 + C

8 EE 100 + C
Diameter of oedema (mm)

EA 200 + C
6

EE 200 + C
4
D 25 + C

2
NaCl 
Nacl +carrageenan

0 Time (hours)
0 10 20 30

Fig. 1 – Effects of Cola nitida extracts and Diclofenac on the edema diameters after inflammation
induction by carrageenan. EA 100 + C : aqueous extract 100 mg/kg + carrageenan;
EE 100 + C : ethanol extract 100 mg/kg + carrageenan; EA 200 + C : aqueous extract
200 mg/kg + carrageenan; EE 200 + C : ethanol extract 200 mg/kg + carrageenan;
D25 + C : Dicofenac 25 mg/kg + carrageenan; NaCl + C : NaCl+ carrageenan.

NaCl+ C
8 EA 200 + C
EE 200 + C
Concentration of CRP mg/L

CEA + C
6
CEE + C
D25+ C
4 * * * * * *
NaCl

0
Fig. 2 – Effect of Cola nitida extracts on CRP concentration 5 hours after injection of carrageenan.
Drugs are injected by intraperitoneal one hour before oedema induction and simultaneously for
competitions. Values are means ± SEM (standard error of the mean), with n = 6; *P < 0.05
by compared to NaCl + carrageenan; AE 200 + C: aqueous extract 200 mg/kg + carrageenan; EE 200+ C:
ethanol extract 200 mg/kg + carrageenan; CEA + C: competition aqueous extract + carrageenan; CEE + C:
competition ethanol extract + carrageenan; D25 + C: Diclofenac 25 mg/kg + carrageenan; NaCl + C:
NaCl + carrageenan.
7 Anti-inflammatory and antioxidant activities of Cola nitida extracts 25

The CRP concentration of group II (NaCl + carrageenan) was significantly

higher (p <0.05) than that of other groups (extracts, Diclofenac and NaCl only).
However, CRP concentrations of extracts, Diclofenac and NaCl only did not differ
significantly (p <0.05).
Extract effects on the concentration of substances reacting with
thiobarbituric acid (TBARS) are shown in Fig. 3. In the groups treated with
carrageenan, the concentration of TBARS (25.68 ± 0.32 mmol/L) increased
significantly (p <0.001) compared to the control group (7.69 ± 0.63 mmol/L).
The preventive treatment by ethanolic extracts of Cola nitida and vitamin C
decreased the level of TBARS relative to controls. On the other hand, in the groups
treated with the aqueous extract and competition aqueous extract, the concentration
of TBARS was significantly reduced (p <0.05) compared to the treated groups.

30
NaCl
Concentrati on of TBARS (µmol /L)

* ** NaCl + C
EA 200+C
20
EE 200+C
CEE+C
CEA+ C
10 ***
*** VC 100 mg/kg+ C
***

Fig. 3 – Effect of Cola nitida extracts and vitamin C on the TBARS concentration after
induction of oxidative stress by carrageenan at 5th time. Values are means ± SEM (standard error of
the mean), with n=6; *P <0.05, **P<0.01, ***P <0.0001 compared with NaCl + carrageenan;
EA 200 + C: aqueous extract 200 mg/kg + carrageenan; EE 200+ C: ethanol extract
200 mg/kg + carrageenan; CEA + C: competition aqueous extract + carrageenan; CEE + C:
competition ethanol extract + carrageenan; VC 100 mg/kg + C: vitamin C 100 mg/kg + carrageenan;
NaCl + C: NaCl+ carrageenan.

The total antioxidant power (TAP) of the serum of rats subjected to different
treatments is shown in Fig. 4. The results highlight that the preventive treatment
with ethanol extracts and vitamin C increased the reducing ability of serum
comparatively with treated and control rats. The total power antioxidant decreased
significantly (p < 0.01) in the groups treated with aqueous extract and competition
aqueous extract.
26 Ayebe Edwige K. et al. 8

Total Power anti oxi dant (µmol Fer II /L) 15

*** *** *** NaCl

10 NaCl + C
VC 100+C
** ** EA 200+C
5 EE200 +C
CEA+C
CEE+ C
0

Fig. 4 – Total power antioxidant of extracts and vitamin C 5 hours after administration
of carrageenan. Values are means ± SEM (standard error of the mean), with n=6; *P <0.05,
**P<0.01, ***P <0.0001 compared with NaCl + carrageenan; AE 200 + C: aqueous extract
200 mg/ kg + carrageenan; EE 200 + C: ethanol extract 200 mg/ kg + carrageenan; CAE + C:
competition aqueous extract + carrageenan; CEE + C: competition ethanol extract + carrageenan;
VC 100 mg/ kg + C: vitamin C 100 mg/ kg + carrageenan; NaCl + C: NaCl + carrageenan.

DISCUSSION

The anti-inflammatory activity was performed by the carrageenan test and

the determination of CRP concentration (15). The rat paw edema induced by this
phlogogenic agent (carrageenan) was considered as a sign of inflammation (16).
This technique was selected due to its simplicity of implementation, rapid
induction of the characteristic symptoms of inflammation (development of edema
in the first hour after injection, with a maximum effect after 5 hours) and
reproducibility. Increasing the diameter of edema following carrageenan injection
was divided into two phases. The first was attributed to the release of
inflammatory mediators such as histamine and serotonin, and the other, to a
release of pro-inflammatory mediators like prostaglandin (17). The significant
inhibitory activity represented by the ethanol extract (200 mg/kg) during 4 hours
in the inflammation induced by carrageenan was similar to that of the group
treated with Diclofenac Sodium®. Previous studies of other plants, such as
Solanum trilobatum (18), Plumeria acuminata (19) and Thesium chinense (20),
showed the same effect. Our results highlight that ethanol extract injection
inhibited the edema from the first time and during all phases of inflammation.
The inhibition of the chemical mediators of inflammation was most likely to
occur. Also, CRP reacts in the serum of patients with acute inflammation (21).
9 Anti-inflammatory and antioxidant activities of Cola nitida extracts 27

Our results showed that the administration of extracts and the reference
molecule (Diclofenac Sodium®) reduced the concentration of CRP when
carrageenan was given. Indeed, carrageenan (carrageenan + NaCl) injection
increased CRP concentration. These results confirm the anti-inflammatory activity
of Cola nitida extracts. The antioxidant activity in vivo of Cola nitida after
inflammation induction in rats was assessed from serum TBARS and the
concentration of iron ions III reduced iron ions II.
Our results indicate that vitamin C and ethanol extracts can reduce the level
of TBARS and increase the ability to reduce serum. The activity of the ethanol
extract was higher than that of the aqueous extract. These results are in agreement
with those of Aouacheri et al. (22) that highlighted the protective effect of vitamin
C against oxidative stress. Preliminary phytochemical screening showed the strong
presence of polyphenols, flavonoids and tannins. Tannins are known for their
binding capacity to proteins, with a tendency to the impermeability and protection
(23). The anti-inflammatory effect is due to inhibition of prostaglandin synthesis
flavonoids and their antioxidant power (24). However, many studies have shown
that polyphenols and their metabolites were also acting as modulators of signaling
pathways of inflammation, with many molecular targets in the center of the
signaling pathways of inflammation (25). Various studies on the protective effects
of polyphenols in these pathological contexts have reported that they decreased
inflammation markers. Studies in healthy humans have shown that following a diet
rich in fruits and vegetables is inversely correlated with increased markers of
inflammation (CRP, IL-6) in plasma (26). Also, anthocyanin consumption was
associated with reduced levels of cytokines (IL-8, IL-13 and IFN-α) in circulation
(27). Similarly, increased plasma antioxidant consumption due to concentrated fruit
juice was associated with a decrease in the breakage of DNA strands (28). In view
of these results of inhibition, we note that the reduction caused by the ethanol
extract was higher than that of the aqueous extract. But the ethanol extract is
identical to the reference molecule. So, the average value of reduction was more
than 2.6% with regard to the reference molecule.

CONCLUSION

The results obtained from the present study have clearly demonstrated that
the extracts of Cola nitida had anti-inflammatory and antioxidant activities. This
supports their traditional utilization in Africa, particularly in Côte d’Ivoire. The
ethanol extract seemed more active than the aqueous extract and this activity is
comparable to that of the reference molecule.
28 Ayebe Edwige K. et al. 10

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