Anti-inflammatory activity of two varieties of pumpkin seed oil in an adjuvant arthritis model in rats

Abstract

The aim of the present research was to evaluate the anti-inflammatory activity of pumpkin seed oils (PSOs) of an Egyptian and European variety, in a rat model of adjuvant arthritis. Edema thickness, plasma tumor necrosis factor-α (TNF-α) and erythrocyte sedimentation rate (ESR) were determined as inflammatory biomarkers while malondialdehyde (MDA) and total antioxidant capacity (TAC) were assessed as indicative of oxidative stress. Chromosomal aberration, sperm shape abnormalities, and DNA fragmentations are cytogenetic parameters which aid in tracing inflammatory and oxidative activity. Phenolic contents and β-carotene were determined in PSOs. The results showed elevated ESR, plasma TNF-α, plasma MDA, liver cellular DNA fragmentation, bone marrow chromosomal aberration, sperm shape abnormalities with a reduction in plasma TAC and body weight gain in an adjuvant arthritis control compared to a healthy control. Administration of low and high doses of either Egyptian or European PSO improved all the aforementioned parameters with variable degrees.

Full text

GRASAS Y ACEITES 68 (1)
January–March 2017, e180
ISSN-L: 0017-3495
doi: http://dx.doi.org/10.3989/gya.0796161
Anti-inflammatory activity of two varieties of pumpkin
seedoilinan adjuvant arthritis model in rats
S.Y. Al-Okbia, D.A. Mohameda, E. Kandilb, M.A. Abo-Zeidc, S.E. Mohammeda and E.K. Ahmedb,*
aFood Sciences and Nutrition Department, National Research Centre, Dokki, Cairo, Egypt
bBiochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt
cGenetics and Cytology Department, National Research Centre, Dokki, Cairo, Egypt
*Corresponding author: emad.ahmed@sci.asu.edu.eg
Submitted: 13 July 2016; Accepted: 14 December 2016
SUMMARY: The aim of the present research was to evaluate the anti-inflammatory activity of pumpkin seed
oils (PSOs) of an Egyptian and European variety, in a rat model of adjuvant arthritis. Edema thickness, plasma
tumor necrosis factor-α (TNF-α) and erythrocyte sedimentation rate (ESR) were determined as inflammatory
biomarkers while malondialdehyde (MDA) and total antioxidant capacity (TAC) were assessed as indicative
of oxidative stress. Chromosomal aberration, sperm shape abnormalities, and DNA fragmentations are cyto-
genetic parameters which aid in tracing inflammatory and oxidative activity. Phenolic contents and β-carotene
were determined in PSOs. The results showed elevated ESR, plasma TNF-α, plasma MDA, liver cellular DNA
fragmentation, bone marrow chromosomal aberration, sperm shape abnormalities with a reduction in plasma
TAC and body weight gain in an adjuvant arthritis control compared to a healthy control. Administration of
low and high doses of either Egyptian or European PSO improved all the aforementioned parameters with vari-
able degrees.
KEYWORDS: Adjuvant arthritis; Anti-inflammatory; β-Carotene; Phenolic contents; Pumpkin seed oils
RESUMEN: Actividad antiinflamatoria de dos variedades de aceite de semillas de calabaza en un modelo de
artritis adyuvante en ratas. El objetivo de la presente investigación fue evaluar la actividad antiinflamatoria de
aceites de calabaza (PSOs) de variedades egipcia y europea, en un modelo de rata con artritis adyuvante. El
espesor del edema, el factor de necrosis tumoral (TNF-α) y la velocidad de sedimentación eritrocitaria (ESR)
se determinaron como biomarcadores inflamatorios, mientras que el malondialdehído (MDA) y la capacidad
antioxidante total (TAC) fueron evaluados como indicativos de estrés oxidativo. La aberración cromosómica,
las anomalías de la forma del esperma y las fragmentaciones del ADN son parámetros citogenéticos que ayudan
a localizar la actividad inflamatoria y oxidativa. Se determinaron contenidos fenólicos y β-caroteno en PSOs.
Los resultados mostraron elevado ESR, TNF-α plasmático, MDA plasmática, fragmentación del ADN del
hígado, aberración cromosómica de la médula ósea, anomalías de la forma espermática con una reducción del
TAC plasmático y un aumento del peso corporal en el control de la artritis adyuvante en comparación con el
control sano. La administración de dosis bajas y altas de PSO egipcia o europea mejoró todos los parámetros
mencionados en grados variables.
PALABRAS CLAVE: Aceites de semillas de calabaza; Antiinflamatorio; Artritis adyuvante; β-Caroteno; Contenido
fenólico
ORCID ID: Al-Okbi SY http://orcid.org/0000-0002-8114-2718, Mohamed DA http://orcid.org/0000-0003-0606-9378,
Kandil E http://orcid.org/0000-0003-4731-7315 Abo-Zeid MA http://orcid.org/0000-0003-1566-6684 Mohammed SE
http://orcid.org/0000-0003-3463-8945 Ahmed EK http://orcid.org/0000-0002-5511-1678
Citation/Cómo citar este artículo: Al-Okbi SY, Mohamed DA, Kandil E, Abo-Zeid MA, Mohammed SE, Ahmed EK.
2017. Anti-inflammatory activity of two varieties of pumpkin seed oil in an adjuvant arthritis model in rats. Grasas
Aceites 68, e180. http://dx.doi.org/10.3989/gya.0796161
Copyright: © 2017 CSIC. This is an open-access article distributed under the terms of the Creative Commons
Attribution (CC-by) Spain 3.0 License.

2 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
1. INTRODUCTION
Oxidative stress and inflammation are the main
causes of many chronic diseases including rheu-
matoid arthritis (RA). Oxidative stress plays an
important role in the initiation and progression
of joint diseases (Knight, 2000). Reactive oxygen
species (ROS) cause oxidative damage which accu-
mulates during the life cycle, the radical-related
damage affects DNA, proteins and lipids which
have been proposed to play a key role in the devel-
opment of arthritis.
RA is an autoimmune disease that causes
chronic inflammation of the joints and surround-
ing tissue with infiltration of macrophages and
activated T cells. The pathogenesis of this disease
is predominantly linked with the formation of
free radicals at the site of inflammation. The dis-
ease progression of RA is associated with chronic
soft tissue inflammation, which is often followed
by bone and cartilage destruction of inflamed
joints (Anderson et al., 2012). Oxidative injury
and inflammation is reflected in increased levels
of serum TNF-α that has been shown to play a
critical role in the onset and progression of RA.
The overproduction of TNF-α could stimulate
cartilage matrix degradation by inhibiting the pro-
duction of proteoglycans and type II collagen and
up-regulating the production of matrix-degrading
enzymes (Goldring, 2000). An excessive accumu-
lation of ROS may cause cellular oxidative dam-
age to nucleic acids, proteins and chromosomal
aberrations in the cells of several systems and that
could occur in RA as reported previously (Al-Okbi
et al., 2011).
The inverse association between fruit and
vegetable intake and the risk of chronic diseases
related to morbidity and mortality is often attrib-
uted to bioactive ingredients that may possess
anti-inflammatory and antioxidant activity such
as vitamins C and E, carotenoids and polyphe-
nols (Prakash and Gupta, 2009). Plant foods are
rich sources of such bioactive ingredients. The
pumpkin seed is among the plant foods that con-
tain high levels of bioactive ingredients, includ-
ing β-carotene, unsaturated fatty acids, phenolic
compounds, phytosterols and tocopherols espe-
cially in the oil compartment. These functional
food ingredients have been reported to possess
both anti-inflammatory and antioxidant activi-
ties in hyper-cholesterolemic rats (Al-Okbi et al.,
2014).
The aim of the present research was to assess
the functional food ingredients, β-carotene and
phenolic contents, in PSOs of the Egyptian and
European varieties. The main objective was to
evaluate the PSOs’ anti-inflammatory activity and
the related mechanism in a rat model of chronic
inflammation that simulates RA in humans.
2. MATERIALS AND METHODS
2.1. Plant materials
Egyptian pumpkin seeds (Cucurbita moschata,
L. Family Curcubitaceae) were purchased from
the local market, Cairo, Egypt. A European PSO
(Cucurbita pepo, L. Family Cucurbitaceae var.
styria) was obtained from Graz, Austria.
2.2. Animals
Male white albino rats of 100-110 g body weight
were used in the present study. The animals were kept
individually in stainless steel cages; water and food
were given ad-libitum. The animal experiment was
carried out adopting the Ethics Committee of the
National Research Centre, Cairo, Egypt, and fol-
lowed the recommendations of the National Institutes
of Health Guide for Care and Use of Laboratory
Animals (Publication No. 85-23, revised 1985).
2.3. Major chemicals
Colchicine and Folin-Ciocalteu reagent were
purchased from Sigma (USA) and Sigma-Aldrich,
Germany, respectively. Freund’s complete adju-
vant (FCA) was supplemented from DIFCO
LABORATORIES, Detroit, Michigan USA. All
chemicals and solvents used were of high quality
analytical grade.
2.4. Preparation of PSO
PSO was prepared from Egyptian pumpkin
seeds by extraction using petroleum ether (40–60ºC)
(Al-Okbi et al., 2014).
2.5. Determination of total phenolic content (TPC)
of the oils
TPC was extracted from PSOs and determined
colorimetrically using the Folin-Ciocalteu reagent
(Singleton and Rossi, 1965). Absorbance was mea-
sured at 765 nm using a UVPC spectrophotom-
eter. Gallic acid was used as a standard and results
were calculated as mg gallic acid equivalent (GAE)
pergm of oil. The reaction was conducted in tripli-
cate and results were averaged.
2.6. Assessment of β-carotene in Egyptian and
European PSO
β-Carotene was determined using the HPLC
method (Hart and Scott, 1995). The exact weights
of 2.5 grams from the Egyptian and European
PSOs were weighed in two separate glass screw
cap tubes. The oils were homogenized with 20 ml
ethanol (HPLC) by vortex mixing (5 min), then 10
ml of hexane (HPLC) were added to each tube,

Anti-inflammatory activity of two varieties of pumpkin seedoilinan adjuvant arthritis model in rats • 3
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
mixed for 2 min, centrifuged (5 min, 5000g) and
the clear upper layer was carefully transferred to
other glass screw cap tubes. The samples were
re-extracted twice with hexane. Then 7 ml of the
hexane extracts were dried to completion under
a nitrogen stream and re-suspended in 1 ml of
50% ethanol for analysis by the HPLC System.
HPLC conditions: Waters Melinnium 3.2 software
using a system equipped with a binary pump sys-
tem (Waters 515), an auto-injector (Waters 717
plus), a PDA detector (Waters 996), and a col-
umn heater (Spectra Physics SP8792). The com-
pound was separated on a 4.6 x 250 mm, 5µm,
YMC Carotenoid column (C-30 reverse-phase)
purchased from Waters (Milford, MA), which
was maintained at 35 °C. The following gradient
system was used: methanol/water/triethylamine
(90:10:0.1v/v/v) (A), and methanol/MTBE/trieth-
ylamine (6:90:0.1v/v/v) (B); gradient (min/%A)
0/99, 8/99, 45/0, 50/0, and 53/99. The column was
brought back to initial conditions, and allowed
to equilibrate for 10 minutes before injection. All
solvents were filtered and degassed before use.
The concentration of β-carotene in the samples
was obtained by comparing its peak area with the
peak area of standard β-carotene in relation to
concentration.
2.7. Preparation of dosage form
An emulsion of each oil was prepared using gum
acacia to easily manage the rats’ oral dose. The same
concentration of gum acacia was prepared in water
(the vehicle) to be given to control groups of rats.
2.8. Preparation of diet
A balanced diet was prepared containing 11.9
casein (10 protein), 10 sunflower oil, 45.73 corn
starch, 22.87 sucrose, 5 cellulose, 1 vitamin mixture
and 3.5 salt mixture as g./100g.
2.9. In-vivo study of the anti-inammatory activity
of PSO in rats
The potential anti-inflammatory effect of PSOs
was evaluated in an adjuvant arthritis model
(chronic inflammation model) of rats. Adjuvant
arthritis (AA) was induced by subcutaneous injec-
tion of 0.3 ml FCA/rat into the sub-planter region
of the right hind paw (Singh et al., 1992).
Thirty-six male albino rats were divided into
6groups, 6 rats each; all rats were fed a normal
balanced diet throughout the experiment. The
rats from the first group (control normal) received
no medication or injection and were given a daily
oral dose of the vehicle for 21 days. Rats of the
second group (adjuvant arthritis control) were
given a daily oral dose of the vehicle for 21 days;
the rats were treated with FCA 8 days from start-
ing vehicle dosing. The rats of group 3 were given
a daily oral dose of Egyptian PSO (40 mg/Kg rat
body weight) for 21 days along with treatment
by FCA 8 days later from starting the oral dose.
Rats of group 4 were given a daily oral dose of
Egyptian PSO (500 mg/Kg rat body weight) for
21 days while FCA was injected 8 days from start-
ing the oral dose. The rats of groups 5 and 6 were
treated like groups 3 and 4, respectively but with
applying the European PSO. The food intake and
body weight of rats were recorded once a week. At
the end of the experiment, total food intake, body
weight gain and food efficiency ratio were calcu-
lated. Paw thickness was measured immediately
before the induction of arthritis (zero time) and at
the end of the experiment using Vernier caliper to
assess the degree of inflammation. The increase
in the thickness of the injected paw (inflamma-
tion thickness) of the rats of all groups was cal-
culated. Also, at the end of the experiment, rats
were anesthetized and blood samples were with-
drawn from the eye vein orbital after an overnight
fast (16h). Blood samples were divided into two
parts. One was mixed with trisodium citrate (109
mmol/L) for the determination of the erythrocyte
sedimentation rate (Westergren, 1921), the second
part was mixed with heparin as anticoagulant,
followed by centrifugation at 3000 rpm for 15
min for separation of plasma and determination
of TNF-α (Stepaniak etal., 1995), MDA (Satoh,
1978) and total antioxidant capacity (Koracevic
et al., 2001). ESR and TNF-α are both consid-
ered as inflammatory biomarkers. Plasma MDA
and total antioxidant capacity were assessed for
evaluating the oxidative stress and/or antioxidant
status.
All rats were kept alive for an additional 24 hours
and treated by intraperitoneal injection of colchi-
cine at a concentration of 3 mg/kg body weight.
After 2 hours of injection, rats were anesthetized
with ether and sacrificed; the liver was separated,
washed with saline and received upon filter paper
to remove any excess blood then wrapped with foil
and kept at -20ºC until being analyzed for DNA
fragmentation assay. The reproductive tract was
exposed and the epididymus were excised. Both
epididymus of each rat were received in a tube
containing saline. The femurs bones of both legs
of each rat were also received on saline in another
tube. Both epididymus and the bone marrow of the
femur were taken immediately for cytogenetic and
chromosomal aberration tests.
For an extra cytogenetic study, two more groups
were run parallel to the previous groups; each of 6
rats; a group was given daily oral dose of Egyptian
PSO (500mg/Kg rat body weight) and the other
group was given the same dose from the European
variety for 21 days and fed on balanced diet.

4 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
2.10. Assessment of cytogenetic parameters
2.10.1. Chromosomal aberrations test
Bone marrow metaphases were prepared (Yosida
and Amano, 1965). Bone-marrow cells from both
femurs were collected in a 6-8 ml hypotonic solu-
tion of KCl (0.075 M). The cell suspension was
incubated for about 20 min. at 37 °C then centri-
fuged at 1000 rpm for 10 min. After discarding
the supernatant, cells were fixed by re-suspending
in freshly-prepared cold fixative methanol/ acetic
acid (3:1), and centrifuged again for 10 min. at
1000 rpm. The fixation step was repeated at least
three times. Finally, the cells were re-suspended in
the appropriate volume of fixative and were spread
by dropping the concentrated cell suspensions onto
slides that had just been removed from the freezer.
Five to seven slides were prepared for each rat.
The slides were stained with 7% Giemsa in phos-
phate buffer (pH 6.8). One hundred well-spread
Metaphases were analyzed per animal. Different
chromosome aberrations including gaps, breaks,
fragments and deletions were recorded.
2.10.2. Sperm shape abnormalities
The protocol recommended previously
(Wyrobek, 1978) was used. Both epididymus from
each rat were minced together with small scissors
in physiological saline, pipetted up and down and
then filtered into small test tubes to exclude large
tissue fragments to which the volume was made up
to 2 ml. Smears were prepared on clean dry slides.
The slides were stained in 1% Eosin Y (aqueous).
Six rats were taken for each treatment and at least
three slides were prepared for each rat to study
sperm abnormalities for microscopical examina-
tion. For each dose, at least 5000 sperms were
assessed for morphological abnormalities of the
sperm shape.
2.10.3. DNA fragmentation assay
The method of DNA fragmentation was car-
ried out (Perandones et al., 1993). Reagents
used were hypotonic lysis buffer pH 8.0, 10%
Trichloroaceticacid (TCA), 5% TCA and color
reagent. Hypotonic lysis buffer was made from
0.2%Triton X-100, 10mM Tris and 1mM EDTA
while the color reagent consisted of 0.088M
Diphenylamine (DPA), 98% v/v Glacial acetic
acid, 1.5% v/v Sulphuric acid and 0.5% v/v 1.6%
Acetaldehyde solution. The liver tissues (50 mg)
were mechanically dissociated in 400 µL hypotonic
lysis buffer to obtain cell lysate. The cell lysate was
centrifuged at 13.800 xg for 15 minutes. The super-
natant containing small DNA fragments was sepa-
rated immediately, the supernatant and the pellet
containing large pieces of DNA, were used for the
diphenylamine (DPA) assay.
2.10.3.1. Diphenylamine assay (DPA)
The colorimetric determination of DNA content
was assessed (Perandones et al., 1993). Both the
supernatant and the pellet were used for DPA assay
after acid extraction of DNA. The pellet contain-
ing large fragments of DNA and cell debris was re-
suspended in a 400 µl hypotonic lysis buffer. TCA
(10%) was added (400 μl) to both the supernatant
and the re-suspended pellet. The tubes were centri-
fuged at 2000 rpm for 10 min. The precipitate was
re-suspended in 400 µl of 5% TCA. The tubes were
incubated at 80 ºC for 30 min. The supernatant con-
taining the extracted DNA was left to cool at room
temperature. Two volumes of the color reagent were
added to one volume of extracted DNA. The sam-
ples were stored at 4 ºC for 48h till the blue color was
developed. The blue color was measured colorimet-
rically using a spectrophotometer at 578 nm. The
percentage of DNA fragmentation in each sample
was expressed by the formula: % DNA fragmenta-
tion = OD of supernatant x 100⁄OD of supernatant
+ OD of pellet, where OD is the optical density.
2.11. Statistical analysis
Results of total phenolic content were expressed
as mean ± SE with application of t-Students test. The
results of the animal experiments were presented as
the mean ± SE and were analyzed statistically using
one-way analysis of variance ANOVA followed by
LSD test. In all cases p < 0.05 was used as the cri-
terion for statistical significance. For the statistical
analysis of chromosomal aberrations in bone mar-
row cells, the Chi-square test (X2 contingency table)
was applied. One-way analysis of variance (ANOVA
test) was applied to the data of sperm shape abnor-
malities and DNA fragmentation in liver cells.
3. RESULTS
3.1. β-carotene and total phenolic contents of PSOs
In Table 1, the European oil showed a higher
β-carotene level compared with the Egyptian oil
(28.5 and 17.9 µg/100g oil, respectively). In addition,
Table 1. β-carotene and total phenolic content in PSOs
Parameters Egyptian PSO European PSO
β-carotene (μg/100 g oil) 17.9 28.5
Total phenolic† (mgGAE/g
oil) 1.71 ± 0.012 14 ± 0.15*
GAE: Gallic acid equivalent, †Total phenolic was calculated as
mean ± SE, *Phenolic contents of European oil was significantly
higher than that of the Egyptian oil,*P < 0.001.

Anti-inflammatory activity of two varieties of pumpkin seedoilinan adjuvant arthritis model in rats • 5
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
the studied oils showed the presence of total pheno-
lic content at 1.71 and 14 mg GAE/g in Egyptian
and European oil, respectively. The total phenolic
content of the European oil was significantly higher
than that of the Egyptian variety.
3.2. In-vivo study of the anti-inammatory activity
of PSOs in rats
3.2.1. Assessments of Inammation
The increase in foot thickness (thickness of
inflammation) of the control arthritic rats at the end
of the experiment compared with the rats given dif-
ferent oil treatments is shown in Table 2. Different
doses of pumpkin oil produced a significant reduc-
tion in foot swelling compared to the arthritic con-
trol. Non-significant differences were observed
between arthritic rats given different oil doses.
Pumpkin oils exhibited a significant inhibition of
inflammation ranging from 31% to 50%.
3.2.2. Biochemical and nutritional parameters of
arthritic rats
The results of the biochemical changes in the dif-
ferent groups of rats in the adjuvant arthritis experi-
ment are shown in Table 3. Plasma TNF-α, MDA and
ESR were significantly higher in arthritic rats than
the normal control. The plasma level of total antioxi-
dants was significantly lower in the arthritic rats com-
pared to the normal control. Plasma TNF-α, MDA
and ESR showed significant reductions in arthritic
rats given different pumpkin oil doses compared to
the arthritic control. In addition; the plasma level
of total antioxidants was significantly higher in the
arthritic rats given different oil treatments compared
to the arthritic control. Both Egyptian and European
high pumpkin oil dose groups have comparable and
significant plasma TNF-α, ESR, MDA and total anti-
oxidant levels. It is noted that high pumpkin oil doses
have a much more potent effect rather than lower
doses on reducing plasma TNF-α. No significant
change was noticed in ESR and plasma MDA among
the different oil treatments and doses. Different oil
treatments normalized Plasma MDA levels except
for the high dose of the European PSO. The level of
ESR only normalized upon the administration of the
Egyptian low dose of PSO. The rats given a high dose
of Egyptian and European PSO showed no significant
change in plasma total antioxidants compared to the
normal control. However, the different oil treatments
and doses could not normalize the level of TNF-α.
The results of the nutritional parameters of the
different groups of rats in adjuvant arthritis experi-
ment are shown in Table 4. Final body weight, body
weight gain, total food intake, and Food efficiency
ratio decreased significantly in the control arthritic
rats compared to the normal control. Final body
Table 2. Inflammation thickness of the injected foot
(after 13 days of adjuvant arthritis induction) of arthritic
rats given different doses of PSO compared to the control
arthritic group
Groups
Inflammation thickness
(cm) (Mean± SE)
% Inhibition of
inflammation
Arthritic control 0.400a ± 0.013 -
European high dose 0.217b ± 0.017 46
European low dose 0.200b ± 0.026 50
Egyptian high dose 0.223b ± 0.009 44
Egyptian low dose 0.275b ± 0.017 31
In the same column different letters mean significant differences
at 0.01probability.
Table 3. Levels of ESR, TNF-α, total antioxidant, and malondialdehyde (MDA) in the different experimental groups
Parameters
ESR
(mm/h)
TNF-α
(pg/ml)
Total antioxidant (nmol/
ml)
MDA
(nmol/ml)
Normal healthy control 1.33c ± 0.21 20.22d ± 0.59 1.88a ± 0.07 2.12c ± 0.21
Arthritic control 4.83a ± 0.31
(263%) 34.52a ± 0.80
(71%) 1.35d ± 0.04
(−28%) 4.92a ± 0.29
(132%)
Egyptian high dose 2.33b ± 0.33
(−52%) 23.72c ± 0.43
(−31%) 1.70abc ± 0.10
(26%) 2.70bc ± 0.22
(−45%)
Egyptian low dose 2.00bc ± 0.26
(−59%) 28.17b ± 0.79
(−18%) 1.55c ± 0.04
(15%) 2.65bc ± 0.23
(−46%)
European high dose 2.50b ± 0.43
(−48%) 24.07c ± 0.58
(−30%) 1.77ab ± 0.08
(31%) 3.07b ± 0.24
(−38%)
European low dose 2.50b ± 0.22
(−48%) 27.47b ± 0.78
(−20%) 1.58bc ± 0.06
(17%) 2.58bc ± 0.10
(−47%)
Results are expressed as mean ± SE of 6 rats.
In each column the same letter means non-significant difference while different letters mean significant differences at 0.05probability.
% change in the row of arthritic control was calculated by comparing arthritic control with normal control. % change in the last 4 rows
was calculated by comparing the arthritic groups given PSO with the arthritic control.

6 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
weight and body weight gain increased significantly
upon the administration of different PSO types and
doses compared to the arthritic control. The final
body weight and body weight gain of these treatments
matched the normal control except for the high dose
of the European variety. Compared to the arthritic
control, the food efficiency ratio increased signifi-
cantly upon administration of different PSO types
and doses till it matched that of the normal control
rats. No significant difference in total food intake was
noticed between the groups given the European PSO
and the arthritic control. Compared to the arthritic
control, total food intake increased significantly upon
administration of either doses of the Egyptian PSO
variety till matching the normal control.
3.2.3. Cytogenetic parameters in adjuvant arthritic rats
3.2.3.1. Chromosomal aberrations
Rat bone marrow cells were collected from the
rats of normal control, adjuvant arthritic control
and from normal rats given high doses of either
European or Egyptian PSO. The results showed no
significant chromosomal aberrations after treat-
ing normal animals with high doses of Egyptian
or European PSO alone compared to the normal
control. Adjuvant arthritic rats showed an induced
genotoxicity in rat bone marrow cells represented
by chromosomal aberrations. It was noticed that
chromosomal aberrations including and exclud-
ing gaps increased significantly after treatment
withFCA (p < 0.05) compared to the normal rats
(Table 5). Different types of chromosomal aberra-
tions were observed (gaps, breaks, fragments and
deletion) after treating animals with FCA (Table 5
and Figure 1).
The Egyptian and European PSO significantly
reduced the percentages of chromosomal aberra-
tions produced by FCA in comparison to rats of
the adjuvant arthritic control group except for the
Egyptian PSO low dose. The low dose of Egyptian
PSO only reduced chromosomal aberrations includ-
ing and excluding gaps without any significance.
The percentages of inhibition after treating FCA
rats with Egyptian PSO reached 20.75% and 49.06%
Table 4. Nutritional parameters of different experimental groups
Parameters Initial body weight (g) Final body weight (g) Body weight gain (g) Total food intake (g) Food efficiency ratio
Normal healthy
control 107.3a ± 0.61 167.5a ± 2.39 60.2a ± 2.56 259.0a ± 7.32 0.234ab ± 0.01
Arthritic control 107.7a ± 2.27 142.7c ± 3.04 35.0c ± 3.29 217.2b ± 12.94 0.164c ± 0.01
Egyptian high dose 107.3a ± 2.72 167.8a ± 2.77 60.5a ± 1.43 261a ± 5.17 0.232ab ± 0.008
Egyptian low dose 107.7a ± 1.76 163.2a ± 3.88 55.5a ± 2.80 256.7a ± 7.48 0.216b ± 0.01
European high dose 107.3a ± 2.90 152.3b ± 3.37 45b ± 1.75 216.8b ± 2.16 0.208b ± 0.01
European low dose 107.3a ± 1.74 164.0a ± 3.05 56.7a ± 2.29 223b ± 6.50 0.254a ± 0.005
Results are expressed as mean ± SE of 6 rats.
In each column the same letter means non-significant difference while different letters mean significant differences at 0.05 probability.
Table 5. Number and mean percentages of chromosomal aberrations in rat bone-marrow cells of all experimental groups of the
adjuvant arthritis experiment
Treatments
Ch. Ab. Including Gaps Ch. Ab. Excluding Gaps Different types of Ch. Ab.
No. % Inhibition % No. % Inhibition % G. Br. F r. Del.
CN 32 6.4 - 19 3.8 - 13 11 3 5
N Egp HD 34 (ns) 6.8 - 22 (ns) 4.4 - 12 11 8 3
N Eur HD 36 (ns) 7.2 - 21 (ns) 4.2 - 15 10 9 2
Ad.Arth. C 53* 10.6 - 34* 6.8 - 19 14 11 9
Ad.Arth. + Egp LD 42 8.4 20.75 25 5 26.47 17 16 6 3
Ad.Arth. + Egp HD 27++ 5.4 49.06 17+3.4 50.00 10 7 8 2
Ad.Arth. + Eur LD 29++ 5.8 45.28 16+3.2 52.94 13 9 5 2
Ad.Arth. + Eur HD 25++ 5.0 52.83 14++ 2.8 58.82 11 10 3 1
Total number of scored metaphases 500 (100 metaphases/ animal). Ch. Ab.:Chromosomal aberration; No: Numbers; G.:Gap; Br.:
Break; Fr.: Fragment; Del.: Deletion. CN: control normal, Egp LD: Egyptian PSO low dose, N Egp HD: Normal rats treated with
Egyptian PSO high dose, Eur LD: European PSO low dose, N Eur HD: Normal rats treated with European PSO high dose, Ad.Arth.
C: Rats of control adjuvant arthritic group; Ad.Arth.: Rats with adjuvant arthritis
*: Significant compared to normal control (p < 0.05); ns: Insignificant compared to normal control; ++ & +: Significant in comparison to
adjuvant arthritis control (+p < 0.05; ++p < 0.01).

Anti-inflammatory activity of two varieties of pumpkin seedoilinan adjuvant arthritis model in rats • 7
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
in including gaps and 26.47% and 50% in excluding
gaps for low and high doses, respectively (Table 5).
Both low and high doses of European PSO given
with FCA injection reduced chromosomal aberra-
tions including and excluding gaps significantly in
comparison to rats treated with FCA alone. The
percentages of inhibition after treating animals
with FCA and European PSO reached 45.28% and
52.83% in including gaps and 52.94% and 58.82%
in excluding gaps for low and high doses, respec-
tively (Table 5).
3.2.3.2. Sperm shape abnormalities
Sperm shape abnormalities are usually taken as
a characteristic criterion and as an applied test for
monitoring the mutagenic potential for many dis-
eases or chemicals. In this experimental study, rat
sperm cells were collected from normal healthy
rats, normal rats given high doses of Egyptian or
European PSO, rats treated with FCA alone or
in combination with high or low doses of either
Egyptian or European PSO to investigate their pro-
tective efficacy. The results showed that the treat-
ment of normal rats with Egyptian and European
PSO did not induce sperm shape abnormalities
in comparison to the non-treated normal control.
However, control adjuvant arthritic rats showed
significant sperm shape abnormalities in compari-
son to non-treated rats. The percentage of sperm
shape abnormalities (Table 6) was 2.8% (p < 0.001)
after treatment with FCA in comparison to con-
trol normal rats (0.48%). The main types of sperm
shape abnormalities observed were amorphous,
triangular, banana shape, without hook and small
head (Figure 2). Both Egyptian and European PSO
reduced the percentages of sperm shape abnor-
malities produced by FCA highly significantly
(p<0.001) in comparison to adjuvant arthritic con-
trol rats. The percentages of inhibition gradually
increased with increasing doses of PSO. The per-
centages of inhibition reached 32.14% and 35.36%
with low and high doses of Egyptian PSO, respec-
tively. The inhibition percentage was 37.5% and
Figure 1. Metaphases with different chromosome
aberrations in rat bone marrow cells after treatments with
arthritic adjuvant for 13 days, (a) normal metaphase,
(b)chromatid break, (c) fragment, (d) deletion. (X1250).
Table 6. Number and mean percentages of sperm shape abnormalities in rat sperms of the experimental groups of adjuvant
arthritis experiment.
Treatments
No. of
scored
sperms
Abnormal sperms Different types of sperm shape abnormalities
No. Mean (%) ± SD Inhibition % Amorphous Triangular Banana shape
Without
hook
Small
Head
CN 5024 24 0.48 ± 0.13 -10 1 4 7 2
N EgpHD 5029 29 0.58 ± 0.21 (ns) -10 0 0 16 3
N Eur HD 5028 28 0.56 ± 0.19 (ns) -11 1 2 13 1
Ad.Arth.C 5144 144 2.80 ± 0.14a- 58 15 15 48 8
Ad.Arth. + Egp LD 5097 97 1.90 ± 0.35b32.14 33 9 19 34 2
Ad.Arth. + Egp HD 5092 92 1.81 ± 0.29b35.36 25 13 22 32 0
Ad.Arth. + EurLD 5089 89 1.75 ± 0.25b37.50 24 11 24 28 2
Ad.Arth. + EurHD 5084 84 1.65 ± 0.13b41.07 18 5 28 31 2
No: Numbers, CN: control normal, Egp LD: Egyptian PSO low dose, N Egp HD: Normal rats treated with Egyptian PSO high dose,
Eur LD: European PSO low dose, N Eur HD: Normal rats treated with European PSO high dose, Ad.Arth. C: Rats of control adjuvant
arthritic group, Ad.Arth.: Rats with adjuvant arthritis.
At least 1000 sperms/animal were scored (6 rats/group).
a Significance in comparison to non-treated rats (control normal) (p < 0.001), (ns): Insignificant compared to normal control;
b Significance in comparison to adjuvant arthritis control (p < 0.001).

8 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
41.07% after treatment with low and high doses of
European PSO, respectively (Table 6). These results
indicated that European oil is slightly more effective
than Egyptian oil.
3.2.3.3. DNA fragmentation assay
The liver cells were collected from all experi-
mental animals and the percentages of DNA
fragmentation were recorded. Both Egyptian and
European oil did not induce DNA damage in the
liver cells of normal healthy rats in comparison
to non-treated animals (normal control). The per-
centages of DNA fragmentation were highly sig-
nificant (5.86%; p < 0.001) in animals treated with
FCA (adjuvant arthritic control) in comparison to
the normal healthy control (3.4%) (Table 7). Both
Egyptian and European pumpkin oils reduced the
DNA fragmentation induced by FCA. The reduc-
tion percentages were highly significant (p < 0.001)
in comparison to animals treated with FCA alone.
The percentages of inhibition were 30.92% and
37.52% after treatment with low and high doses
of Egyptian oil, respectively. The inhibition per-
centages of DNA fragmentation were 36.04% and
44.21% upon treatment with low and high doses of
European oil, respectively (Table 7) which indicated
a dose-dependent effect.
4. DISCUSSION
In the present research, the anti-inflammatory
activity of two varieties of PSO, an Egyptian and
a European, was studied in a chronic inflammatory
model of adjuvant arthritis in rats. These types of
oils were selected as they are expected to be rich
in bioactive ingredients which could have antioxi-
dant, free radical scavenging capacity and anti-
inflammatory effects.
In the present study, the induction of AA in rats
produced high oxidative stress reflected in elevated
plasma MDA and reduced total antioxidant capac-
ity. Also inflammatory biomarkers represented by
ESR and plasma TNF-α were significantly elevated
in AA rats with the induction of foot paw inflam-
mation. In support of the present work, arthritis is
described as being a condition that involves systemic
oxidative stress (Strosova et al., 1995). In previous
studies, it was shown that plasma oxidative stress
and inflammatory biomarkers increased in both AA
Figure 2. (a)- Normal sperm with a definite head and a marked hook from a control rat. (b–f) Different types of sperm shape
abnormalities in rats treated with Freund’s complete Adjuvant; (b) amorphous head; (c) triangular head; (d) without hook;
(e)banana shape; (f) small head. (X1250).

Anti-inflammatory activity of two varieties of pumpkin seedoilinan adjuvant arthritis model in rats • 9
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
rats and in patients with RA (Al-Okbi et al., 2000a;
2000b; 2011; Stepaniak et al., 1995).
TNF-α is a potent pro-inflammatory cytokine
that plays an important role in inflammation. It
binds to its cellular receptor TNF receptor 1, which
triggers signaling cascades that activate NF-κB
and activator protein 1 transcription factors. Pro-
inflammatory cytokines like IL-1β and TNF-α
could thus help to propagate the extension of a
local or systemic inflammatory process by activat-
ing NF-κB, forming a positive feedback mechanism
which exaggerates the inflammatory process (Sonis,
2002).
Overproduction of lipid peroxide oxidative stress
marker, MDA, was noticed in AA rats (Zhang etal.,
2014). AA was monitored by hind paw volume and
high level of MDA. During inflammatory joint dis-
eases, like adjuvant-induced arthritis, phagocytes
accumulate in the joints and produce superoxide
and hydroxyl radicals as well as hydrogen peroxide.
Such reactive oxygen species might contribute to the
elevated ESR, plasma MDA, and TNF-α observed
in the arthritic model of inflammation. An elevation
of the oxidative damage marker such as blood MDA
with reduction in total antioxidants in the present
study demonstrated a disturbance in the oxidant to
antioxidant balance in favor of lipid peroxidation,
which could lead to the tissue damage seen in the
disease. These results suggest the importance of
therapeutic co-administration of antioxidants with
conventional drugs in such condition.
Paw swelling, body weight loss and overproduc-
tion of serum TNF-α and MDA were reported as
the main pathological signs in FCA-induced arthri-
tis in rats (Zhang et al., 2014). Paw volume and ESR
were evaluated as arthritic markers (Banji et al.,
2014). During arthritis, TNF-α has been implicated
in the pathological mechanisms of synovial tis-
sue proliferation, joint destruction and apoptosis.
Hemshekhar et al. (2013) demonstrated a signifi-
cant reduction in arthritis-induced paw swelling and
body weight regain, establishing a close relationship
between inflammation and loss of body weight.
TNF-α played a role in the increased prevalence of
low body mass which has been reported in arthritic
individuals (Kaufmann et al., 2003). The decrease in
body weight gain and food efficiency ratio in the con-
trol AA rats in the present study is similar to that in
rheumatoid arthritis patients where the loss of lean
tissues could be due to tissue destruction. Elevated
TNF-α in arthritis could contribute in cartilage and
bone degeneration through the activation of extra-
cellular matrix degrading enzymes. The increase in
TNF-α in the present study could thus explain the
loss in weight gain. The enhanced degradation of
muscle protein and muscle wasting in experimental
arthritis might have a hand in body weight reduc-
tion. The decrease in total food intake in adjuvant
arthritis rats in the current study is similar to the
observed anorexia in RA patients and could partici-
pate in reducing body weight gain.
The administration of both PSOs to arthritic rats
in the present study produced significant improve-
ments in inflammatory and oxidative stress bio-
markers together with a reduction in inflammation
thickness. These effects are certainly attributed to
the presence of antioxidant and anti-inflammatory
constituents such as tocopherols, carotenoids, phy-
tosterols, and phenolic compounds in the POs. The
variation in degree of improvements may be related
to the different contents and concentration of bio-
active constituents and their synergistic effects.
The present study showed the presence of phenolic
compounds and beta- carotene in both varieties of
PSOs. Also, the two varieties of PSOs were reported
previously to contain phytosterols, unsaturated fatty
acids, and tocopherols (Al-Okbi et al., 2014). These
ingredients could collectively render the oils their
antioxidant and anti-inflammatory activity.
Egyptian and European PSOs significantly inhib-
ited the elevated plasma levels of TNF-α and MDA,
thereby reducing the severity of inflammation as
noticed from the reduced paw inflammation thick-
ness. Eventually, elevated ROS promotes liver dam-
age by elevating the levels of lipid peroxides. Lipid
peroxides, represented in the present study by MDA,
could behave as a secondary toxic trigger causing
further damage by modulating membrane fluid-
ity, permeability and transport in arthritic patients.
Thus the level of MDA may be a sensitive marker
Table 7. Mean percentage of DNA fragmentation
induced in rat liver cells injected with FCA with or without
daily oral doses of Egyptian or European pumpkin oil
(40and 500 mg/kg b. wt.)
Treatments
DNA
fragmentation
Mean (%) ± SD Inhibition %
CN 3.40 ± 0.26 -
N Egp HD 3.15 ± 0.51 -
N Eur HD 3.90 ± 0.69 -
Ad.Arth.C 5.86 ± 0.74a-
Ad.Arth. + Egp LD 4.04 ± 0.90b30.92
Ad.Arth. + Egp HD 3.66 ± 0.18b37.52
Ad.Arth. + Eur LD 3.74 ± 0.78b36.04
Ad.Arth. + Eur HD 3.27 ± 0.28b44.21
CN: normal control, Egp LD: Egyptian PSO low dose, N Egp
HD: Normal rats treated with Egyptian PSO high dose, Eur LD:
European PSO low dose, N Eur HD: Normal rats treated with
European PSO high dose, Ad.Arth. C: Rats of control adjuvant
arthritic group, Ad.Arth.: Rats with adjuvant arthritis (6 animals /
group were investigated).
a Significance in comparison to normal control (p < 0.001);
b Significance in comparison to adjuvant arthritis control
(p<0.001).

10 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
of oxidative damage during arthritis and treatment
with PSO which reflects the potent antioxidant and
free radical scavenging activity of PSO.
PSOs have a significant inhibitory effect on foot
inflammation and this ability to reduce edema may
be related to its inhibitory action on TNF-α and
ESR which reflect their anti-inflammatory activity.
Also both Egyptian and European PSO doses sig-
nificantly elevate the antioxidant capacity and thus
they may play an important role in reducing the oxi-
dative stress associated with the arthritic condition,
and therefore has the potential to be used as an anti-
arthritic agent.
It was noticed that all treatments produced a sig-
nificant increase in body weight gain and food effi-
ciency ratio which is reflected in the improvement of
AA condition.
The presence of α, δ and γ-tocopherol in PSO
(Al-Okbi et al., 2014) could have a significant impact
on improving the changes in AA rats. The phenolic
content and the considerable concentration from
both tocopherol and β-carotene in PSO may explain
the antioxidant and anti-inflammatory effects. The
presence of a high phenolic content was reported
to play an anti-arthritic role through lowering ESR
and restoring body weight (Gupta et al., 2014).
Antioxidants play an important role in reliev-
ing arthritis in rats (Zhang et al., 2014). Chronic
administration of PSO in the present study causeda
remarkable elevation of serum antioxidant levels
and a marked inhibition of paw edema that could
beattributed to its high content of the aforemen-
tioned antioxidants.
PSO administration improved biochemical
parameters studied in the present research during
chronic arthritis which could be ascribed to the
high level of unsaturated fatty acids in PSO and
other antioxidants. Previously, extra-virgin olive
oil was reported to reduce inflammation and carti-
lage-matrix degradation in a mice model of arthri-
tis which was assigned to the adequate fatty acid
profile and the presence of high phenolic content
in the oil (Rosillo et al., 2016). Similarly, evening
primrose oil could reduce the pro-inflammatory
mediators in a model of fibromyalgia syndrome in
mice (Montserrat-de la Paz et al., 2013) and in RA
patients (Al-Okbi et al., 2000a) which might most
probably be due to the presence of polyunsaturated
fatty acids particularly gamma- linolenic acid. In
another study, functional foods containing either
fish oil or primrose oil showed beneficial effects in
a rat model of arthritis due to their high levels of
polyunsaturated fatty acids (Al-Okbi et al., 2012).
The present study indicated that AA has a highly
significant genotoxic effect on rat bone marrow cells
as well as sperm and liver DNA. Our results are in
agreement with a previous study which reported the
induction of structural and numerical aberrations
in the synovial fibroblasts in RA patients and other
inflammatory joint diseases (Wyllie et al., 1980).
The current work also indicated DNA fragmenta-
tion induced by AA in rat liver cells similar to the
work of Wyllie et al. (1980).
Oxidative metabolism is considerably enhanced in
the liver of AA rats. The livers of adjuvant-induced
arthritis rats show a pronounced high oxidative
stress which is the consequence of both a stimulated
pro-oxidant system and reduction in antioxidant
defense represented by the lowering in catalase and
glutathione peroxidase activities (Kelmer-Bracht
et al., 2003). This liver high oxidative stress could
be the cause of the observed DNA damage in the
AA rats in the present study. The mRNA levels of
mdr1a and mdr1b encoding P-glycoprotein (P-gp)
decreased significantly in the livers of the AA rats
(Kawase et al., 2014). In arthritic rats, it was observed
previously that there was a significant elevation
in DNA damage and genotoxicity (Al-Okbi et al.,
2011). Mutation rates might be increased due to the
elevated level of DNA damage in adjuvant arthri-
tis. The present study showed the protective role of
the tested PSOs by inhibiting the genotoxic effects
induced by adjuvant arthritis and the protection of
cells against oxidative DNA damage. The bioactiv-
ity of PO could be attributed to the presence of the
aforementioned bioactive constituents that could
impart antioxidant and anti-inflammatory activity.
It was reported that carotenoids reduced the induc-
tion of micronuclei in polychromatic erythrocytes in
mice bone-marrow cells (Rauscher et al., 1998). The
protective effect of nut oil was demonstrated against
free radical capacity due to the presence of tocopher-
ols. Tocopherol itself was shown previously to possess
protective effects against the induced genotoxicity
in mice bone-marrow cells (Arranz et al., 2008). So
tocopherols in combination with the different func-
tional ingredients of PSO in the present study could
have synergistic anti-genotoxicity. In the current
research, the mutagenic effect and DNA fragmenta-
tion in AA was minimized by the oral administration
of POs which significantly inhibited the sperm shape
abnormalities, DNA fragmentation and chromo-
somal aberrations of somatic and germ cells. Total
phenolics have been reported to have anti-mutagenic
effects (Batista etal., 2016), so the phenolic content
of PSO could have a similar effect.
The essential fatty acid, linoleic acid accounts for
nearly one-third of the total fatty acid in pumpkin
seeds while the contribution of α-linolenic acid was
considerably low (Glew et al., 2006; Tsaknis et al.,
1997). These fatty acids could participate in the bio-
activity of PSO.
Mini Paulista and Nova Caravela pumpkin vari-
eties showed high amounts of total phenolic com-
pounds in the lipid fractions and in the seeds. It was
also found that γ-tocopherol is the isomer that stood
out in the lipid fractions and in the seeds, mainly in
Menina Brasileira (Veronezi and Jorge, 2012).

Anti-inflammatory activity of two varieties of pumpkin seedoilinan adjuvant arthritis model in rats • 11
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
5. CONCLUSIONS
The present study showed that both the Egyptian
and European varieties of PSO improved the bio-
chemical and nutritional changes in an adjuvant
arthritis model and reduced the genotoxicity and
paw inflammation which nominate PSO as being a
protective agent towards RA or complementary to
therapy used for this disease. The bioactivity of PSO
might be ascribed to its contents of total phenolics
and beta-carotene determined in the present study,
in addition to phytosterols, unsaturated fatty acids
and tocopherols, as reported previously.
ACKNOWLEDGMENTS
This work was completely financed by the
National Research Centre Cairo, Egypt.
REFERENCES
Al-Okbi SY, Ammar NM, Soroor KhA, Mohammed DA. 2000a.
Impact of natural oils supplements on disease activity and
antioxidant state of Egyptian patients with rheumatiod
arthritis. Med. J. Islamic Acad. Sci. 13, 161–171.
Al-Okbi SY, Sorour Kh, Abdel-Samed AM, Rashed MM,
Mohamed DA. 2000b. Nutritional status and blood anti-
oxidant levels of Egyptian rheumatoid arthritis patients.
Kasr El Aini Med. J. 6, 43–64.
Al-Okbi SY, Mohamed DA, Donya SM, Abd El Khalek
AB. 2011. Role of Bifidobacterium bifidum and Plant
Food Extracts in Improving Microflora and Biochemical
and Cytogenetic Parameters in Adjuvant Arthritis.
Grasas Aceites 62, 308–320. http://dx.doi.org/10.3989/
gya.089810
Al-Okbi SY, Mohamed DA. 2012. Preparation and evaluation
of functional foods in adjuvant arthritis. Grasas Aceites
63,394–402. http://dx.doi.org/10.3989/gya.130811
Al-Okbi SY, Mohamed DA, Kandil E, Ahmed EK, Mohammed
SE. 2014. Functional ingredients and cardiovascular pro-
tective effect of pumpkin seed oils. Grasas Aceites 65, e007.
http://dx.doi.org/10.3989/gya.062813
Anderson J, Caplan L, Yazdany J, Robbins ML, Neogi
T, Michaud K, Saag KG, O’Dell JR, Kazi S. 2012.
Rheumatoid arthritis disease activity measures: American
College of Rheumatology recommendations for use in
clinical practice. Arthritis Care Res. 64, 640–647. http://
dx.doi.org/10.1002/acr.21649
Arranz S, Cert R, Pérez-Jiménez J, Cert A, Saura-Calixto F. 2008.
Comparison between free radical scavenging capacity and
oxidative stability of nut oils. Food Chem. 15, 985–990.
http://dx.doi.org/10.1016/j.foodchem.2008.03.021
Banji OJ, Banji D, Soumya N, Chilipi KK, Kalpana CH, Kranthi
Kumar CH, Annamalai AR. 2014. Combination of car-
vacrol with methotrexate suppresses Complete Freund’s
Adjuvant induced synovial inflammation with reduced
hepatotoxicity in rats. European J. Pharmacol. 15, 91–98.
http://dx.doi.org/10.1016/j.ejphar.2013.12.009
Batista AG, Ferrari AS, da Cunha DC, da Silva JK, Cazarin CB,
Correa LC, Prado MA, Carvalho-Silva LB, Esteves EA,
Marostica Junior MR. 2016. Polyphenols, antioxidants,
and antimutagenic effects of Copaifera langsdorffii fruit.
Food Chem. 15, 1153–1159. http://dx.doi.org/10.1016/j.
foodchem.2015.11.093
Glew RH, Glew RS, Chuang LT, Huang YS, Millson M,
Constans D, Vanderjagt DJ. 2006. Amino acid, mineral
and fatty acid content of pumpkin seeds (Cucurbita spp)
and Cyperus esculentus nuts in the Republic of Niger. Plant
Foods Human Nutrit. 61, 51–56. http://dx.doi.org/10.1007/
s11130-006-0010-z
Goldring SR. 2000. The final pathogenetic steps in focal bone
erosions in rheumatoid arthritis. Annals Rheumatic Diseases
59, 72–74.
Gupta M, Sasmal S, Mukherjee A. 2014. Therapeutic Effects
of Acetone Extract of Saraca asoca Seeds on Rats with
Adjuvant-Induced Arthritis via Attenuating Inflammatory
Responses. ISRN Rheumatology. http://dx.doi.
org/10.1155/2014/959687
Hart DJ, Scott KJ.1995. Development and evaluation of an
HPLC method for the analysis of carotenoids in foods, and
the measurement of the carotenoid content of vegetables
and fruits commonly consumed in the UK. Food Chem. 54,
101–111. http://dx.doi.org/10.1016/0308-8146(95)92669-b
Hemshekhar M, Thushara RM, Jnaneshwari S, Devaraja S,
Kemparaju K, Girish KS. 2013. Attenuation of adjuvant-
induced arthritis by dietary sesamol via modulation of
inflammatory mediators, extracellular matrix degrading
enzymes and antioxidant status. European J. Nutrit. 52,
1787–1799. http://dx.doi.org/10.1007/s00394-012-0482-6
Kaufmann J, Kielstein V, Kilian S, Stein G, Hein G. 2003.
Relation between body mass index and radiological pro-
gression in patients with rheumatoid arthritis. J. Rheumatol.
30, 2350–2355
Kawase A, Norikane S, Okada A, Adachi M, Kato Y, Iwaki M.
2014. Distinct alterations in ATP-binding cassette trans-
porter expression in liver, kidney, small intestine, and brain
in adjuvant-induced arthritic rats. J. Pharmaceutical Sci.
103, 2556–2564. http://dx.doi.org/10.1002/jps.24043
Kelmer-Bracht AM, Santos CP, Ishii-Iwamoto EL, Broetto-
Biazon AC, Bracht A. 2003. Kinetic properties of the
glucose 6-phosphatase of the liver from arthritic rats.
Biochim. Biophys. Acta 20, 50–56. http://dx.doi.org/10.1016/
s0925-4439(03)00041-3
Knight JA. 2000. Review: Free radicals, antioxidants, and the
immune system. Annals Clinical Lab. Sci. 30, 145–158.
Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V.
2001. Method for the measurement of antioxidant activ-
ity in human fluids. J. Clinic. Pathol. 54, 356–361. http://
dx.doi.org/10.1136/jcp.54.5.356
Montserrat-de la Paz S, García-Giménez MD, Ángel-Martín M,
Marín-Aguilar F, Fernández-Arche A. 2013. Dietary sup-
plementation evening primrose oil improve symptoms of
fibromyalgia syndrome. J. Functional Foods 5, 1279–1287.
http://dx.doi.org/10.1016/j.jff.2013.04.012
Perandones CE, Illera VA, Peckham D, Stunz LL, Ashman RF.
1993. Regulation of apoptosis in vitro in mature murine spleen
T cells. J. Immunol. (Baltimore, Md :1950) 1, 3521–3529
Prakash D, Gupta KR. 2009. Antioxidant phytochemicals of
Nutraceutical Importance. Open Nutraceutic. J. 2, 20–35.
http://dx.doi.org/10.2174/1876396000902010020
Rauscher R, Edenharder R, Platt KL. 1998. In vitro antimuta-
genic and in vivo anticlastogenic effects of carotenoids
and solvent extracts from fruits and vegetables rich in
carotenoids. Mutation Res. 16, 129–142. http://dx.doi.
org/10.1016/s1383-5718(98)00017-5
Rosillo MA, Sánchez-Hidalgo M, Sánchez-Fidalgo S,
Aparicio-Soto M, Villegas I, Alarcon-de-la-Lastra C.
2016. Dietary extra-virgin olive oil prevents inflamma-
tory response and cartilage matrix degradation in murine
collagen-induced arthritis. Eur. J. Nutr. 55, 315–325. http://
dx.doi.org/10.1007/s00394-015-0850-0
Satoh K. 1978. Serum lipid peroxide in cerebrovascular disorders
determined by a new colorimetric method. Clin. Chim. Acta
15, 37–43. http://dx.doi.org/10.1016/0009-8981(78)90081-5
Singh GB, Singh S, Bani S, Gupta BD, Banerjee SK. 1992.
Anti-inflammatory activity of oleanolic acid in rats and
mice. J. Pharmacy Pharmacol. 44, 456–458. http://dx.doi.
org/10.1111/j.2042-7158.1992.tb03646.x
Singleton VL, Rossi JA. 1965. Colorimetry of total phenolics
with phosphomolybdic-phosphotungstic acid reagents.
Am. J. Enol. Vitic. 16, 144–158.
Sonis ST. 2002. The biologic role for nuclear factor-
kappaB in disease and its potential involvement in
mucosal injury associated with anti-neoplastic therapy.
Crit. Rev. Oral Biol. M. 13, 380–389. http://dx.doi.
org/10.1177/154411130201300502

12 • S.Y. Al-Okbi, D.A. Mohamed, E. Kandil, M.A. Abo-Zeid, S.E. Mohammed and E.K. Ahmed
Grasas Aceites 68 (1), January–March 2017, e180. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.0796161
Stepaniak JA, Gould KE, Sun D, Swanborg RH. 1995. A com-
parative study of experimental autoimmune encephalomy-
elitis in Lewis and DA rats. J. Immunol. (Baltimore, Md
:1950) 1, 2762–2769
Tsaknis J, Lalas S, Lazos ES. 1997. Characterization of
crude and purified pumpkin seed oil. Grasas Aceites
48, 267–272. http://dx.doi.org/10.3989/gya.1997.v48.
i5.802
Veronezi CM, Jorge N. 2012. Bioactive compounds in lipid frac-
tionsof pumpkin (Cucurbita sp) seeds for use in food. J. Food
Sci. 77, 653–657. http://dx.doi.org/10.1111/j.1750-3841.2012.
02736.x
Westergren A. 1921. Studies of the suspension stability of the
blood in pulmonary tuberculosis. Acta Medica Scandin.
54, 247–245. http://dx.doi.org/10.1111/j.0954-6820.1921.
tb15179.x
Wyllie AH, Kerr JF, Currie AR. 1980. Cell death: the signifi-
cance of apoptosis. Int. Rev. Cytol. 68, 251–306. http://
dx.doi.org/10.1016/s0074-7696(08)62312-8
Wyrobek AJ, Bruce WR. in Hallaender A, De Serres FJ (eds.).
1978. The Induction of Sperm-shape Abnormalities in
Mice and Humans, in Chemical Mutagens: Principles and
Methods for Their Detection 5, 257–285.
Yosida TH, Amano K. 1965. Autosomal polymorphism in
laboratory bred and wild Norway rats, Rattus norvegicus,
found in Misima. Chromosoma 28, 658–667. http://dx.doi.
org/10.1007/bf00285115
Zhang CF, Zhang SL, He X, Yang XL, Wu HT, Lin BQ, Jiang
CP, Wang J, Yu CH, Yang ZL, et al., 2014. Antioxidant
effects of Genkwa flos flavonoids on Freunds adjuvant-
induced rheumatoid arthritis in rats. J. Ethnopharmacol.
14, 793–800. http://dx.doi.org/10.1016/j.jep.2014.03.046