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


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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.
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January–March 2017, e180
ISSN-L: 0017-3495
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:
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
ORCID ID: Al-Okbi SY, Mohamed DA,
Kandil E Abo-Zeid MA Mohammed SE Ahmed EK
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.
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:
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.,
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.
2.1. 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:
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
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
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:
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. 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.
3.1. 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:
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
Inflammation thickness
(cm) (Mean± SE)
% Inhibition of
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
Total antioxidant (nmol/
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
Egyptian high dose 2.33b ± 0.33
(−52%) 23.72c ± 0.43
(−31%) 1.70abc ± 0.10
(26%) 2.70bc ± 0.22
Egyptian low dose 2.00bc ± 0.26
(−59%) 28.17b ± 0.79
(−18%) 1.55c ± 0.04
(15%) 2.65bc ± 0.23
European high dose 2.50b ± 0.43
(−48%) 24.07c ± 0.58
(−30%) 1.77ab ± 0.08
(31%) 3.07b ± 0.24
European low dose 2.50b ± 0.22
(−48%) 27.47b ± 0.78
(−20%) 1.58bc ± 0.06
(17%) 2.58bc ± 0.10
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:
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 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
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:
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). 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.
No. of
Abnormal sperms Different types of sperm shape abnormalities
No. Mean (%) ± SD Inhibition % Amorphous Triangular Banana shape
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:
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. 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.
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:
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,
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.)
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
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:
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:
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.
This work was completely financed by the
National Research Centre Cairo, Egypt.
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... Pumpkins are considered as healthy and functional food, and the consumption of pumpkins and pumpkin-based foods has been shown to confer several effects on human health, including hepatoprotective effects, antihyperglycaemic (antidiabetic) activity, anti-ulcer activity, anti-inflammatory activity, effects on prostatic hyperplasia (BPH) and urinary function, anti-microbial activity, and anticancer/antitumour effects [4][5][6][7][8][9]. The main factors that contribute to the nutritional and medicinal value of pumpkin fruits are their high total content of carotenoids and the presence of pectin and non-pectin polysaccharides, vitamins (A, C, E), dietary fibres, minerals (K, P, Mg, Fe, and Se), phenolic compounds (flavonoids, phenolic acids), and other compounds that possess health benefits [10][11][12][13][14][15]. ...
... The most consistent carotenoid detected was -carotene, found in all samples, albeit at different concentrations. Interestingly, zeaxanthin was detected in all pumpkins not originally from Serbia, except in two (23 and 384), whereas in Serbian pumpkins, this carotenoid was present in only four samples (2,3,4,212) out of the 13 tested. ...
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Pumpkin is considered a healthy and functional food. The consumption of pumpkins and pumpkin-based foods has been shown to confer several beneficial effects on human health due to their antioxidant capacity and terpenoid content. Consequently, this study aimed to characterize the in vitro antioxidant capacity (using FRAP and ABTS assays), terpenoid profile (using an untargeted lipidomics approach via high-resolution UHPLC-Orbitrap mass spectrometry), and carotenoid content (by HPLC-DAD) in pumpkin fruit pulp from accessions differing for species (11 Cucurbita maxima and 9 Cucurbita moschata), cultivar, and origin, belonging to a Serbian breeding collection. These accessions are candidates for inclusion within programs intended to improve pumpkin fruit quality. The results obtained in this work allowed us to highlight the best marker compounds, discriminating both the region of accession collection or breeding (“origin”) and the plant species. Furthermore, our findings have helped to identify the most suitable antioxidant-rich varieties to select for national breeding programs for improving human health. These findings provide valuable information to the overall current understanding of the potential health benefits of pumpkins and the discriminant triterpenoids underlying the C. maxima and C. moschata accessions investigated here, which include those of Serbian and non-Serbian origin.
... In addition to all of the aforementioned properties, pumpkin has been reported to play an important role in inflammatory diseases, such as arthritis due to its anti-inflammatory properties [139][140][141]. ...
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Pumpkin is a well-known multifunctional ingredient in diet, full of nutrients, has opened up new vistas for scientists in past years. Not just the fruit of pumpkin, but the flower, seed as well as peel are rich source of primary and secondary metabolites including proteins, carbohydrates, monounsaturated fatty acids, polyunsaturated fatty acids, carotenoids, tocopherols, tryptophan, delta-7-sterolsand many other phytochemicals. This climber is being used traditionally in many countries such as Austria, Hungary, Mexico, Slovenia, China, Spain, and several Asian as well as African countries as functional food and to promote health promising properties. Other benefits of pumpkin such as improving spermatogenesis, wound healing, anti-microbial, anti-inflammatory, anti-oxidative, anti-ulcerative properties and treatment of benign prostatic hyperplasia have also been confirmed by researchers. For better drug delivery, nanoemulsions and niosomes made by pumpkin seeds have also been reported as a health promising tool but still more researches need to be done in this field. This review mainly focuses on compiling and summarizing most relevant literature to highlight the nutritional value, phytochemical potential and therapeutic benefits of pumpkin.
... The antidepressant activity of the whole seed was observed by George and Nazn (2012). Many studies have reported the antioxidant and anti-inflammatory activities of PSO (Al-Okbi et al., 2017;Boujemaa et al., 2020). The bioactive components in PSO (ascorbic acid, tocopherols, b -carotene, unsaturated fatty acids and flavonoids) have antioxidant and anti-inflammatory effects (Kaur et al., 2019). ...
Purpose This study aims to investigate the potential of pumpkin seed oil (PSO) and zinc to attenuate oxidative stress and neuroinflammation caused by chronic mild stress (CMS) in the cerebral cortex of male rats. Design/methodology/approach The rats were submitted to stress for six weeks and then the behavior of the rats was tested by forced swimming test (FST) and novel cage test. The treated groups were given venlafaxine (20 mg/kg), pumpkin seed oil (40 mg/kg) and zinc (4 mg/kg). The cortex homogenate was used for the detection of the oxidative stress parameters, the concentration of neurotransmitters, tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), Na+/K+-ATPase activity, and the expression of histamine N-methyltransferase (Hnmt) and tyrosine hydroxylase (Th). Findings CMS causes a significant increase in immobility time in the FST and a significant decrease in the number of rearing in the novel cage test. CMS group showed a significant increase in alanine aminotransferase (ALT) activity, levels of cortisol, TNF-α, IL-1β, nitric oxide and malondialdehyde. CMS caused a significant decrease in the concentrations of serotonin, GABA, norepinephrine, and the activities of glutathione peroxidase, catalase, superoxide dismutase and Na ⁺ /K ⁺ -ATPase. CMS caused a marked reduction in the expression of Hnmt and Th in the cortex. PSO and zinc attenuated the Na ⁺ /K ⁺ -ATPase activity, oxidative parameters and neuroinflammation induced by the CMS, and this was reflected by the elevation of the concentration of neurotransmitters and reduction of cortisol and ALT, in addition to the behavior normalization. PSO and zinc attenuated the CMS by improving the antioxidant milieu and anti-inflammatory status of the cerebral cortex. Originality/value There are no studies on the effect of pumpkin seed oil on depression
... In a similar study conducted by Amin et al. (2020), the anti-inflammatory activity of indigenous and hybrid pumpkin seed oils was investigated and found that indigenous pumpkin seed oil had a higher effect with a rate of 89% at a concentration of 45 µg/mL compared to a hybrid one. In another in vivo study, the anti-inflammatory activity of pumpkin seed oils of an Egyptian and European variety was examined in a rat model of adjuvant arthritis and it was determined that PSO significantly reduced inflammation (Al-Okbi et al., 2017). Similarly, Arslanbaş et al. (2020) examined the anti-inflammatory activity of Turkey source pumpkin seed oil in rat oedema model and observed that a significant anti-inflammatory effect in the group to which a high dosage of Turkey-sourced PSO (100 mg/ kg) was administered. ...
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Abstract This study aimed to investigate the effect of press temperature on physicochemical properties, fatty acid, sterol, phenolic composition, in-vitro cytotoxicity assay, and anti-inflammatory activity of pumpkin seed oil. For this aim, the oils obtained at 100 °C (PSO2) and 150 °C (PSO3) press temperature were compared with the cold press oil (PSO1). The application of press temperature at 150 °C caused a significant decrease in the amount of sterol, while the press application at 100 °C did not cause a significant change in the sterol composition. Total phenolic content, antioxidant capacity values, and individual phenolic content of PSO2 and PSO3 samples were significantly lower than those of PSO1. 30 mg/mL of PSO1, PSO2, and PSO3 samples exhibited a cytotoxic effect on the cells with an inhibition ratio of 75%, 48%, and 39%, respectively, indicating that press temperature reduced the cytotoxic effect of pumpkin seed oil. PSO1 showed anti-inflammatory activity ranged from 79% to 59%, while at the same concentrations PSO2 and PSO3 exhibited approximately from 58% to 49%. This study indicated that the bioactive properties of the cold press oil could be negatively affected by higher press temperature.
... Unlike many anti-inflammatory agents that have ulcerogenic effects, cold-pressed seed oils, such as those from coriander or black cumin are promising sources of anti-inflammatory agents that are highly desirable for treating inflammation (21). The anti-inflammatory activities of phenols, β-carotene, unsaturated fatty acids, and tocopherol in pumpkin seed oil have been demonstrated through their role in reducing the expression of inflammatory biomarkers in rat arthritis model (22). ...
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Fatty acid composition and antioxidant content are major determinants of vegetable oil quality. Antioxidants are important food components, and there is an increasing interest of replacing synthetic antioxidants with those from natural sources for food industry. The objective of this study was to evaluate fatty acid composition, total phenolic, carotenoid and chlorophyll contents, and antioxidant capacity of different varieties of two oilseed crops. Five niger seed and eight linseed varieties were used. For the analysis of fatty acid composition of the seed oil, gas chromatography method was used. Standard methods were used for total phenolic, carotenoid and chlorophyll contents, and antioxidant properties. In niger seed oil, linoleic acid (C18:2) was the dominant fatty acid, accounting for 73.3% (variety Esete) to 76.8% (variety Ginchi) of the total fatty acids. In linseed oil, linolenic acid (C18:3) was the dominant fatty acid accounting for 55.7 (variety Chilalo) to 60.1 (variety Belaye-96). The total phenolic content ranged from 22.4mg GAE/g (variety Esete) to 27.9mg GAE/g (variety Ginchi) in niger seed and from 20.5mg GAE/g (variety Belay-96) to 25.4mg GAE/g (variety Ci-1525) in linseed. In niger seed, variety Fogera had the highest values for FRAP and radical scavenging activity. The carotenoid content also showed significant variation among the varieties ranging from 2.57 (Esete) to 8.08 (Kuyu) μmol/g for niger and 4.13 (Tole) to 8.66 (Belay-96) μmol/g for linseed. The FRAP assay showed that variety Fogera of niger seed and variety Chilalo of linseed came on top among their respective varieties with values of 57.2 and 30.6, respectively. Both niger seed and linseed were shown to be rich in bioactive compounds. However, significant variation was observed among the varieties of each crop and among the two crops in their total phenolic and carotenoid contents as well as ferric reducing potential and radical scavenging capacity. Principal component analysis revealed the presence of more than one group in both niger seed and linseed. Hence, genetic variation among the varieties should be utilized for improving their desirable characteristics through breeding. Both oil crops can be used as the source of antioxidants for replacing synthetic compounds.
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Edible oils are one of the important products that have lately come to light for their beneficial and nutritional properties. As a result, scientists and the oil industry are always working to demonstrate the health-giving benefits of both fruit and vegetable seed oils. Fruits are popular for their fleshy parts. However, the seeds are often discarded since they are thought worthless. This research looked at the bioactive components found in Cucurbitaceae (Cucurbita spp., Cucumis melo L., Citrullus lanatus) seed oils extracted using various extraction procedures on Cucurbitaceae seeds from various species and geographical places throughout the globe. The outcomes of the study show that Cucurbitaceae seed oils are a good source of nutrients and may be classified as health-promoting compounds. The discoveries have also cleared the way for the use of these seed oil resources in the production of a broad variety of therapeutic products.
Different parts of Annona muricata L., Annonaceae, are widely used for their biological activities. The fruit of this plant, called “graviola,” is recognized for its nutritional value, but its seeds are usually discarded, despite their potential as a source of substances with anti-inflammatory properties. This study investigated the effect of graviola seed oil in models of cutaneous inflammation after topical administration. Graviola seed oil was characterized regarding its fatty acid composition and evaluated in vitro for cytotoxicity in L929 fibroblasts. Acute cutaneous inflammation was induced by topical administration of 12-O-tetradecanoilphorbol-13-acetate, phenol, or capsaicin in the right ears of Swiss mice. Concomitantly, ears were treated with graviola seed oil (0.3, 1, and 3 mg/ear), oleic acid (0.3, 0.1, and 3 mg/ear), or dexamethasone (control). After the induction, inflammatory parameters were analyzed. The main fatty acids found in graviola seed oil were oleic (43.37%), linoleic (29.86%), palmitic (21.94%), and stearic acids (4.82%). Seed oil (10–150 μg/ml) did not alter L929 fibroblasts’ viability after 24 h of exposure. Treatment with seed oil, or dexamethasone reduced ear edema, myeloperoxidase activity, histological alterations, lipid peroxidation, and interleukin-1β and 6 levels induced by 12-O-tetradecanoilphorbol-13-acetate. Treatment with oleic acid reduced ear edema and lipid peroxidation, but not myeloperoxidase activity, in 12-O-tetradecanoilphorbol-13-acetate model. Treatment with seed oil and oleic acid reduced edema formation, but only treatment with seed oil reduced myeloperoxidase activity in phenol-induced ear inflammation. Administration of seed oil and oleic acid did not affect capsaicin-induced ear edema. These findings demonstrate the anti-inflammatory activity of graviola seed oil after skin injury, which is partially related to the presence of oleic acid.
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A large amount of wastes and by-products are generated during the vegetables and fruits production and food industry. These wastes create increasing disposal and severe environmental problems or discarded with a loss of valuable biomass and nutrients. However, these wastes contain bioactive compounds of great potential and value-added compounds. These wastes or by-products can be incorporated as food additives and/or used as nutraceuticals. Therefore, the valorization of agro wastes or by-products from the food industry significantly contributing to a sustainable food chain from an environmental and economic point of view. Pumpkin is a gourd-like fruit of the genus Cucurbita (family Cucurbitaceae), indigenous to the tropical and sub-tropical countries. Worldwide, three common pumpkin species are grown, namely Cucurbita pepo, Cucurbita maxima, and Cucurbita moschata, which economically represent the most important species. Globally, China, India, Ukraine, Egypt, and the United States are the major pumpkin-producing countries. Pumpkins are a rich source of important natural bioactive compounds such as carotenoids, tocopherols, phytosterols, phenolics, antidiabetic polysaccharides, minerals, vitamins, antifungal proteins, essential and nonessential amino acids, pectin, and fibers. Besides, the pumpkin seed oil is rich in unsaturated fatty acids (omega-6 and omega-9). The bioactive compounds found in pumpkin exhibit a wide range of biological activities such as antioxidant, anti-inflammatory, cardio protective, antiaging, antimicrobial anticancer, and prebiotic activities. The wastes from pumpkin fruits and biomass from seed oil production retained great amounts of these bioactive compounds, representing a potential for their use as a nutraceutical or dietary supplement. The present chapter describes the economic values, chemical composition, bioactive compounds, health benefits, and pumpkin fruits’ biological activity. In addition, the current status of the use, recovery, food, and non-food applications of pumpkin processing by-products, including peels, pulp, and seeds. The technologies employed to obtain and isolate the highly value-added components from these by-products will also be discussed.Keywords Cucurbita SeedSeed cakePeelPulpNutritional valueBio-wastesValorization
The use of cucurbit seed oil for the treatment of several diseases has been reported by many investigators and the consensus is that it has beneficial antimicrobial and allelopathic effects. The prevalence in literature is that some of the claims are based on folklore, preliminary antimicrobial, nutritional, or physicochemical data generated by investigators. In many cases, cucurbits seed oils showed high values for nutrients, so it is suitable for human health needs. There appears to be an empirical data generated from clinical trials in humans, which supports the beneficial inflammatory and antibacterial effects proposed in several disease conditions. This chapter gives an overview of the inflammatory, antimicrobial, and allelopathic activities of different cucurbit seed oils.
The photoprotective skincare products are in high demand to meet the consumer market with concern on skin health. Seed oils are commonly used as ingredients in many cosmetic products due to their natural antioxidants and now being increasingly recognized for their effects on skin health and photoprotection. This article briefly reviews the application of seed oils in sunscreen development focusing on the antioxidants that contribute to photoprotection, thus preventing UV‐induced erythema and photoaging. The addition of seed oils that contain specific natural bioactive compounds was discussed in the review. Besides that, seed oils acting in molecular pathways that benefit photoprotection were also summarized. Seed oils (pomegranate seed oil, castor oil, cocoa butter, jojoba oil, rosehip oil, grapeseed oil, kenaf seed oil, and pumpkin seed oil) utilization have high potential to act as natural UV filters and at the same time help in skin repairing. The seed oils contributed beneficial properties to the sunscreen formulation due to their synergistic effect with antioxidants, antiaging properties, anti‐inflammatory effect, and potential hormetic effect. The finding of specific bioactive compound from seed oils provides a better understanding of the contribution of seed oils in sunscreen formulation.
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The aim of the present research was to discover plant food extracts and probiotics that may have bioactivity towards chronic inflammation. Three plant food extract mixtures expected to be rich in phenolic compounds, carotenoids and tocopherols were prepared. The anti-inflammatory activity of the different mixtures as well as probiotic bacteria (Bifidobacterium bifidum) were evaluated in adjuvant arthritis in rats. The anti-inflammatory effect, mechanism of action and safety of the three mixtures and Bifidobacterium bifidum were studied by measuring the size of inflammation and the determination of inflammatory and oxidative stress biomarkers, colonic bacteria profile and specific cytogenetic parameters. The contents of tocopherols, beta-carotene and phenolic compounds in the mixtures were determined. The results show that the tested mixtures and Bifidobacterium bifidum possess promising anti-inflammatory effects. The mechanism of action seems to involve a reduction in oxidative stress and inflammatory biomarkers and an effect on colonic microflora. Genotoxicity and DNA fragmentation induced by adjuvant arthritis were prevented after supplementation with the tested mixtures.
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The objective of the present study was to evaluate the cardiovascular protective effect of Egyptian and European pumpkin seed oil (PSO) in hypercholesterolemic rats. Tocopherols, fatty acids (FAs) and unsaponifiable matter (UNSAP) were assessed in both oils. The results showed that a-tocopherol was 108 and 273, y-tocopherol was 3.95 and 0 and 8-tocopherol was 0 and 1.58 mg-100 g-1 oil of the Egyptian and European, respectively. GLC analysis of FAs revealed the presence of linoleic acid as the major fatty acid in both oils. Feeding a hypercholesterolemic diet produced a significant increase in plasma total cholesterol (T-Ch), triglycerides (TGs), low density lipoprotein cholesterol, T-Ch/HDL-Ch, TGs/HDL-Ch and malondialdehyde and a significant reduction in high density lipoprotein cholesterol (HDL-Ch), vitamin E, and adiponectin. Rats fed on hypercholesterolemic diet with either oil showed a significant improvement in all biochemical parameters.
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Saraca asoca has been traditionally used in Indian system for treatment of uterine, genital, and other reproductive disorders in women, fever, pain, and inflammation. The hypothesis of this study is that acetone extract of Saraca asoca seeds is an effective anti-inflammatory treatment for arthritis in animal experiments. The antiarthritic effect of its oral administration on Freund's adjuvant-induced arthritis has been studied in Wistar albino rats after acute and subacute toxicities. Phytochemical analysis revealed presence of high concentrations of phenolic compounds such as flavonoids and tannins, while no mortality or morbidity was observed up to 1000 mg/kg dose during acute and subacute toxicity assessments. Regular treatment up to 21 days of adjuvant-induced arthritic rats with Saraca asoca acetone extract (at 300 and 500 mg/kg doses) increases RBC and Hb, decreases WBC, ESR, and prostaglandin levels in blood, and restores body weight when compared with control (normal saline) and standard (Indomethacin) groups. Significant (P < 0.05) inhibitory effect was observed especially at higher dose on paw edema, ankle joint inflammation, and hydroxyproline and glucosamine concentrations in urine. Normal radiological images of joint and histopathological analysis of joint, liver, stomach, and kidney also confirmed its significant nontoxic, antiarthritic, and anti-inflammatory effect.
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Adjuvant arthritis is an animal model that closely resembles rheumatoid arthritis in humans. It is a successful working model used to study new anti-inflammatory agents. In previous studies (animal and clinical) we have shown that evening primrose oil, fish oil and the methanol extract of date fruits and fenugreek seeds have anti-inflammatory activity and that the methanol extract of dates has an antioxidant effect. Based on these studies, the aim of the present study was to prepare 7 functional foods containing such bioactive fractions separately or in combination and to evaluate them in adjuvant arthritis in rats, study the stability of bioactive ingredients and evaluate their sensory properties. The studied biochemical parameters were erythrocyte sedimentation rate, erythrocyte superoxide dismutase, glutathione peroxidase and plasma copper, zinc and interlukin 2. Nutritional parameters, including body weight gain, food intake and food efficiency ratio were monitored during the feeding of the functional foods. The bioactive ingredients assessed were total phenolic contents and fatty acids. The results showed improvement in the biochemical parameters, body weight gain and food efficiency ratio of arthritic rats fed on the functional foods with different degrees. All the prepared functional foods were sensory accepted. The active ingredients showed stability during storage. In conclusion, all the tested functional foods showed promising antiinflammatory activity and were determined to be acceptable through sensory evaluation which means that their potential beneficial use as dietary supplements in rheumatoid arthritis patients may be recommended. La artritis adyuvante es un modelo utilizado en animales y se caracteriza por ser muy parecida a la artritis reumatoide en humanos. Se trata de un modelo de trabajo utilizado con éxito para estudiar nuevos agentes anti-inflamatorios. En estudios previos (animales y clínica) hemos demostrado que el aceite de onagra, el aceite de pescado y los extractos metanólicos de semillas de fenogreco o alholva y de dátiles tienen actividad anti-inflamatoria y que el extracto metanólico de dátiles tiene efecto antioxidante. Basado en estos estudios, el objetivo del presente trabajo consistió en preparar alimentos funcionales con siete fracciones bioactivas por separado y conjuntamente y evaluarlas en artritis adyuvante en ratas, estudiar la estabilidad de los principios bioactivos y evaluar sus propiedades sensoriales. Los parámetros bioquímicas estudiados fueron la velocidad de sedimentación globular, la enzima superóxido dismutasa eritrocitaria, la glutatión peroxidasa, cobre plasmático, y la zinc interleucina-2. Los parámetros nutricionales, incluyendo ganancia de peso corporal, la ingesta de alimentos y la relación de eficiencia alimentaria se han seguido durante la ingesta de los alimentos funcionales. Los ingredientes bioactivos evaluados fueron el contenido de fenoles totales y ácidos grasos. Los resultados mostraron una mejoría de los parámetros bioquímicos, aumento de peso corporal y la relación de eficiencia alimentaria en las ratas artríticas que se alimentaron con los diferentes grados de alimentos funcionales. Todos los alimentos funcionales preparados fueron aceptables sensorialmente. Los ingredientes activos mostraron estabilidad durante el almacenamiento. Como conclusión, todos los alimentos funcionales probados mostraron una prometedora actividad anti-inflamatoria y fueron aceptables sensorialmente, siendo recomendable su uso como suplementos dietéticos por su potencial beneficio en pacientes con artritis reumatoide.
Evening primrose oil (EPO) is widely used as a dietary supplement from which beneficial effects have been reported in rheumatic and arthritic conditions, atopic dermatitis, premenstrual and menopausal syndrome, and atopic dermatitis. This study was designed to examine the effect of diets supplemented with EPO on generalized chronic pain, including fibromyalgia syndrome, induced by intermittent cold stress (ICS) in mice. After completing the in vivo test, we proceeded to isolation and culture of peritoneal macrophages in order to determine the effects on the inflammatory status. The results indicate that dietary-EPO is suitable to improve mechanical and thermal allodynia and hyperalgesia and it is also being able to improve behavioural disturbances, anxiety and depression. Besides, EPO reduced significantly the proinflammatory mediators as ON, PGE(2), TXB2 and IL-1 beta in LPS-macrophages stimulated from EPO fed animals. In conclusion, this study demonstrates that dietary-EPO can modify the nociceptive response and other symptoms associated with fibromyalgia syndrome, as well as to reduce the release of the inflammatory state.
Current experimental studies support a beneficial role of extra-virgin olive oil (EVOO) in several inflammatory diseases. The present study was designed to evaluate the effects of dietary EVOO on type II collagen-induced arthritis (CIA) in mice. DBA-1/J mice were randomized in four experimental groups (10 or 15 animals per group): (1) Sham sunflower diet (SO-Sham), (2) CIA sunflower diet (SO-CIA), (3) Sham EVOO diet (EVOO-Sham) and (4) CIA EVOO diet (EVOO-CIA) group. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrified 42 days after first immunization. In addition to macroscopic and histological analyses, serum levels of cartilage olimeric matrix protein (COMP), metalloproteinase-3 (MMP-3) and pro-inflammatory cytokines levels were evaluated by ELISA. The expressions of heme oxygenase-1 (HO-1), nuclear factor E2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), Janus kinase-signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. EVOO diet significantly reduced joint edema and cartilage destruction, preventing the arthritis development. Dietary EVOO significantly decreased serum COMP and MMP-3 levels, as well as, the pro-inflammatory cytokines levels (TNF-α, IL-1β and IL-17). Moreover, the activation of JAK/STAT, MAPKs and NF-κB pathways was drastically ameliorated. According to Nrf2 and HO-1, the protein expressions were up-regulated in those mice fed with EVOO. These results support the interest of EVOO as a beneficial functional food to prevent the development of the rheumatoid arthritis (RA).
Pathophysiological changes of infection or inflammation are associated with alterations in the production of numerous absorption, distribution, metabolism and excretion-related proteins. However, little information is available on the effects of inflammation on the expression levels and activities of ATP-binding cassette (ABC) transporters. We examined the effect of acute (on day 7) and chronic (on day 21) inflammation on the expression of ABC transporters in some major tissues in rat. Adjuvant-induced arthritis (AA) in rats was used as an animal model for inflammation. The mRNA levels of mdr1a and mdr1b encoding P-glycoprotein (P-gp) decreased significantly in livers of AA rats on day 21. Hepatic protein levels of P-gp, Mrp2, and Bcrp decreased significantly in membranes but not homogenates of AA rats after 7 days and after 21 days of treatment with adjuvant. Contrary to liver, protein levels of P-gp and Mrp2, but not Bcrp in kidney, increased significantly in membranes. The biliary excretion of rhodamine 123 was decreased in rats with chronic inflammation owing to decreases in efflux activities of P-gp. Our results showed that the expression of transporters in response to inflammation was organ dependent. In particular, hepatic and renal P-gp and Mrp2 exhibited opposite changes in membrane protein levels. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
Genkwa flos (Daphne genkwa Sieb. et Zucc.), a Chinese herbal medicine, has been traditionally used for over two thousand years in China for inflammation related symptoms, including joint pain. To evaluate the antioxidative effects of flavonoid aglycones (FA) isolated from Genkwa flos on adjuvant arthritis in rats and to identify the relationship between antioxidant potential and whole blood viscosity (WBV). FA compounds were identified using LC-MS and the content was assayed by HPLC. Arthritis was induced by an intradermal injection of Freund's complete adjuvant in the footpad. The effects of FA on paw volumes, secondary arthritis scores, histopathology of joints, and body and organ weights were measured. The antioxidant effects of FA and WBV were determined. LC-MS analysis showed that the FA contained four major compounds: luteolin, apigenin, hydroxygenkwanin and genkwanin. FA significantly decreased paw edema, arthritis scores, and weight loss. These observations were consistent with the reduction of oxidative stress and the improvement of the WBV. FA significantly decreased arthritis in a rat model through antioxidant and hemorheological modulatory mechanisms. The Genkwa flos flavonoids may have clinical potential for the treatment of rheumatoid arthritis.
The present study evaluated the therapeutic benefit of the combination of carvacrol, an isoprenoid having potential anti-inflammatory action, with methotrexate in suppressing Complete Freund's Adjuvant induced arthritis and attenuating methotrexate induced hepatic damage. Arthritis was induced in rats with Complete Freund's Adjuvant. Animals received methotrexate (2mg/kg) intraperitonealy once a week for 5 weeks alone and along with carvacrol orally (50 and 100mg/kg) respectively from the 10(th) to the 42(nd) day. Control and carvacrol alone group were also studied. Paw volume, hypernociception, and erythrocyte sedimentation rate were evaluated as arthritic markers. Hepatic marker enzymes in serum; myeloperoxidase, protein oxidation, and oxidative measures were determined in the liver homogenate. Liver histological assessments were also carried out. Methotrexate significantly controlled arthritis; however, liver damage was evident due to oxidative stress and rise in myeloperoxidase levels. Carvacrol suppressed the hyperalgesic response, significantly alleviated arthritis and reduced damage to the hepatocytes owing to a decline in the levels of myeloperoxidase and oxidative markers. High dose of the combination reduced the levels of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alkaline phosphatase by 24.74%, 30.2% and 28.14% compared with methotrexate treatment. Histological assessment also revealed that carvacrol minimizes methotrexate induced liver toxicity. In combination, carvacrol promoted the anti-arthritic action of methotrexate, reduced neutrophils infiltration and peroxidative damage to the liver. Therefore, carvacrol can serve as a useful adjuvant and promote the safe use of methotrexate in the management of arthritis.