Content uploaded by Hanaa abd El-Rahman
Author content
All content in this area was uploaded by Hanaa abd El-Rahman on Jan 06, 2016
Content may be subject to copyright.
Advances in Biochemistry
2015; 3(4): 40-50
Published online September 22, 2015 (http://www.sciencepublishinggroup.com/j/ab)
doi: 10.11648/j.ab.20150304.11
ISSN: 2329-0870 (Print); ISSN: 2329-0862 (Online)
Xanthine Oxidase Inhibitory Activity and Antigout of Celery
Leek Parsley and Molokhia
Hanaa S. M. Abd El-Rahman
1
, Nasra A. M. Abd–ELHak
2
1
Special Food and Nutrition Dept., Food Technology Research Institute, Agric. Res. Center, Giza, Egypt
2
Kitchen Experimental Unit, Food Technology Research Institute, Agric. Res. Center, Giza, Egypt
Email address:
Hanaasayed97@yahoo.com (H. S. M. A. El-Rahman), dr.nasraahmed@yahoo.com (N. A. M. Abd–ELHak)
To cite this article:
Hanaa S. M. Abd El-Rahman, Nasra A. M. Abd–ELHak. Xanthine Oxidase Inhibitory Activity and Antigout of Celery Leek Parsley and
Molokhia. Advances in Biochemistry. Vol. 3, No. 4, 2015, pp. 40-50. doi: 10.11648/j.ab.20150304.11
Abstract:
The present study was aimed at investigating in vitro xanthine oxidase inhibitory (XOI) and in vivo antigout
activity extracts of celery, leek, parsley, and molokhia. The degree of XO inhibitory activity was determined by measuring the
absorbance spectrophotometrically at 295 nm, which is associated with uric acid formation which is linked to gout. Our
preliminary screening study had employed the use of distilled water, and absolute ethanol to determine XOI from celery, leek,
parsley, and molokhia. In general, our study showed that the ethanolic extracts were found to be more active than the aqueous
extracts. Further in-vivo antigout was studied gout induced in rats by potassium oxonate. A total of 36 male albino rats were
randomly divided into 6 equal groups. Group 1 negative control given only standard diet, and group 2-6 given Potassium
oxonic acid (250 mg/kg, i.p.), Potassium oxonate an uricase inhibitor was used to induce gout. Oral administration (G3, G4,
and G5) of celery, leek, parsley (5 g/Kg), and (G6) molokhia (4.8 g/Kg) showed a significant decrease in uric acid, and
Creatinine levels in the gouty rats. All extracts (celery, leek, parsley, and molokhia) have shown significant decrease in level of
Malonaldehyde (MDA) and increase in activity of antioxidant enzyme level, comparable to positive rat (G2). No significant
changes between all extracts used and negative control in gain weights and organic phosphorus was noticed. The results
showed that increasing serum total calcium level with extracts of celery, leek, parsley, and molokhia in comparison to positive
control. The celery, leek, parsley, and molokhia extracts have some protective effects on the gout.
Keywords:
Celery, Leek, Parsley, Molokhia, Gout, Xanthine Oxidase Inhibitory
1. Introduction
Gout is a multi-factorial disease affecting the flexibility of
joints. It is usually characterized by re-current attacks of
acute inflammatory arthritis-a red, tender, hot, swollen joints
leading to bursitis (1). It is a serious disease that has been
growing in prevalence during the past several years in
Western civilizations (2). Gout is characterized by
abnormally high levels of uric acid in the body, resulting in
the formation and deposition of urate (as monosodium urate-
monohydrate) crystals, generally known as tophi crystals in
joints, tendons and surrounding tissues, characterized by
hyper-uricemia and in chronic stage, may lead to renal
failure(3).These crystals cause an acute inflammatory
response and can induce a permanent tissue damage which is
characterized by the appearance of ulceration of the joint
cartilage, marginal osteophytosis, geodic and erosive lesions
and chronic inflammation of synovial membrane (4 and
5).This is partly a reflection of changes in diet, increases in
longevity, hypertension, metabolic syndrome, and advanced
renal disease, and the broad use of diuretics in clinical
practice. Management of gout in the elderly, in organ
transplant recipients, and in patients with renal insufficiency
and allopurinol intolerance can be particularly challenging
(6).Uric acid is the end product of purine metabolism in
humans, and its overproduction by xanthine oxidase (XOD)
from purine compounds or under excretion can lead to
hyperuricemia as gout. Enzymatic degradation of
hypoxanthine and xanthine leads to the production of uric
acid (7 and 8). The major site of purine synthesis is in the
liver. The synthesis of uric acid occurs along two pathways,
referred to as the de novo and the salvage pathways.
Synthesis of the purine nucleotides begins with the formation
of phosphoribosyl pyrophosphate (PRPP) by PRPP
synthetase and leads to the first fully formed nucleotide,
inosine 5’-monophosphate (IMP). IMP is converted into
either adenosine 5’-monophosphate (AMP) or guanosine 5’-
41 Hanaa S. M. Abd El-Rahman and Nasra A. M. Abd–ELHak: Xanthine Oxidase Inhibitory Activity and Antigout of Celery, Leek,
Parsley, and Molokhia
monophosphate (GMP) through two distinct reaction
pathways. Catabolism of the purine nucleotides leads
ultimately to the production of uric acid (Figure 1) (9).
Abbreviations: ADA, adenosine deaminase; APRT, adenosine phosphoribosyl transferase; HPRT, hypoxanthine phosphoribosyl transferase; PNP, purine
nucleoside phosphorylase; XDH, xanthine dehydrogenase
Fig. (1). Purine metabolic pathway in humans.
XOD (EC 1.17.3.2) is a rate-limiting enzyme in the
biosynthesis of uric acid and catalyzes the oxidation of
hypoxanthine and xanthine to uric acid (8), which is
responsible for the medical condition leading to painful
inflammation called gout (10). XOD is distributed most
abundantly in the liver and intestine (11), situated at the end
of a catabolic sequence of the purine nucleotide metabolism
in humans and few other uric species (12). XOD also serves
as an important biological source of oxygen-derived free
radicals that contribute to oxidative damage to living tissues
involved in many pathological processes such as
inflammation, atherosclerosis, and cancer. In-vitro
bioassays are used to examine test material for XOD
inhibition, as inhibitors of XOD may be potentially useful
for the treatment of gout or other XOD induced diseases
(13). Therefore, XOD inhibitors can be potent therapeutic
agents for the prevention of hyperuricemia by inhibition of
uric acid biosynthesis (12). The treatment of gout entails
the use of the therapeutic agents such as xanthine oxidase
inhibitors (XOI) (14 and 12). XOI acts by blocking the
biosynthesis of uric acid from purine in the body (12) and it
is believed that either by increasing the excretion of uric
acid or reducing the uric acid production helps to reduce the
risk of gout (15).Current treatments to gout includes Non-
steroidal anti-inflammatory drugs (NSAIDS) such as
ibuprofen, naproxen, indomethacin, aspirin, etoricoxib
(cox-2 selective inhibitors); corticosteroids such as
prednisone; allopurinol, probencid, colchicines (to decrease
severity of episodes). Although these agents are generally
effective, they generates superoxide (16) and lead to several
side effects such as skin allergies, fever, rash and diarrhoea
progressively developing leukocytosis, eosinophilia,
vasculitis, aseptic meningitis, nephritis and renal
dysfunction, and hepatic dysfunction (17 and 18).
Allopurinol is an XOD inhibitor used clinically for the
treatment of gout. However, it can have side effects, such as
hypersensitivity reaction, Stevens - Johnson syndrome,
renal toxicity, and even fatal liver necrosis (19). So, there is
a need of herbal extracts with antioxidant property to
nullify oxidative and inflammatory response produced by
xanthine oxidase. It is believed that XOD inhibitors from
natural sources can be used as alternatives to allopurinol
because of fewer potential adverse side effects (20). Some
tropical plants and their phytochemicals are worth to be
explored as potential XOI as they are already used as food
or food supplements for many years and found safe for
Advances in Biochemistry 2015; 3(4): 40-50 42
human bodies (21). Polyphenols, flavonoids, coumarins,
ellagic acid, and valoneic acid dilactone (VAD) have been
reported to be potent plant-based XOI (22; 23; 24; 25 and
12).
Celery (Apium graveolers dulce) is a biennial plant,
belongs to the Umbelliferae family. The celery plant
cultivated in the Mediterranean region and its Arabic name
is Karafs. Celery seeds contain several substances including
volatile oils; flavonoids, antioxidants that give plants their
colors and may protect cells from damage; coumarins,
chemicals that help thin the blood; and linoleic acid, an
omega-6 fatty acid. Celery seed is used for treating arthritis
and gout, and to help reduce muscle spasms, calm the
nerves, and reduce inflammation (26 and 27). Leeks
(Allium porrum or A. ampeloprasumvar. porrum),
sometimes called "the gourmet's onion". The thick leaf
bases and slightly developed bulb look like a giant green
onion, and are eaten as a fresh vegetable. Leeks contain
saponins and the major flavonoid in leeks is kaempferol,
with only a small amount of quercetin, carotenoids and
chlorophyll mainly in the green tops(28 and 29).Parsley
(Petroselinum crispum) is a member of Apiaceous family
that has been employed in the food, pharmaceutical,
perfume, and cosmetic industries (30). In folk medicine,
parsley is used to treat a wide variety of conditions (31).
Phytochemical screening of parsley has revealed the
presence of several classes of flavonoids (32). Flavonols
(kaempferol and quercetin) and flavones (apigenin and
luteolin), which occur as glycosidic form in nature, are
major flavonoids found in parsley (33).
Molokhia Corchorus olitorius (Tiliaceae) is an annual herb
whose leaves and roots are used as herbal medicine and eaten
as vegetable by local people in East Malaysia, India, Egypt,
and Philippines (34).Traditionally, its leaves are used in the
treatment of pain, fever, chronic cystitis and tumors (35).
Molokhia abundantly contains 5-caffeoylquinic acid, 3, 5-
dicaffeoylquinic acid, quercetin 3-galactoside, quercetin3-
glucoside, quercetin 3-(6-malonylglucoside), quercetin3-
(malonylgalactoside), ascorbic acid, α-tocopherol, and
chlorophyll, etc., and the content of quercetin glycosidesis
remarkable (36).
Therefore, the present study was carried out to evaluate the
xanthine oxidase inhibitory potential of some plants (celery,
leek, parsley, and molokhia) and in vivo anti-inflammatory in
experimental gout model in rats.
2. Materials and Methods
2.1. Reagents and Chemicals
Potassium oxonate, allopurinol, xanthine and xanthine
oxidase (buttermilk) were purchased from Sigma-Aldrich
Chemicals (St. Louis, MO, USA). Dimethylsulphoxide
(DMSO), hydrochloric acid (HCl), absolute ethanol, and
other reagents of analytical grade were obtained from Merck
(Darmstadt, FR, Germany). Potassium di-hydrogen
phosphate (KH
2
PO
4
) and dipotassium hydrogen phosphate
(K
2
HPO
4
) were of the highest purity. All other reagents were
purchased from Merck (Darmstadt, Germany). The reagents
used were from of analytical grades.
2.2. In-Vitro Xanthine Oxidase Inhibitory Activity
2.2.1. Preparation of Crude Extracts
The plants were washed and oven-dried for 72 h at 40°C.
The dried plant materials were grounded using domestic
blender to small particle size. All plant materials were
subjected to a standard procedure of solvent extraction
process (37). 1 g of each of the dried powdered plant material
was added into 10 ml of extraction solvent and all
experiments were conducted in triplicate. Two extraction
solvents were employed, namely, absolute ethanol and
distilled water. The mixture of the ground sample and solvent
were capped with aluminum foil, and placed in an incubator
shaker. The agitation speed of the incubator shaker was set at
100 rpm and ran for 6 h at 30°C. Each mixture of plant
material and extraction solvent was filtered using Whatman
No. 1 filter paper and the filtrate was collected, concentrated
by vacuum rotary evaporator and dissolved in Di-Methyl
Sulfoxide (DMSO) (100%). Then, it was subjected to XO
inhibitory activity assay spectrophotometrically at 295 nm to
determine the XOI properties.
2.2.2. Xanthine Oxidase Inhibitory Activity Assay
The crude extract was used for the analysis of XO inhibition
under in vitro assays. The inhibitory effect on XO was
measured spectrophotometrically at 295 nm under aerobic
condition, with some modifications, following the method
reported by Unno et al., (12) and Umamaheswari et al., (15).
A well-known XOI, allopurinol (100 µg/ml) was used as a
positive control for the inhibition test. The reaction mixture
consisted of 300 µl of 50 mM sodium phosphate buffer (pH
7.5), 100 µl of sample solution dissolved in DMSO 100 µl of
freshly prepared enzyme solution (0.2 units/ml of xanthine
oxidase in phosphate buffer) and 100 µl of distilled water. The
assay mixture was pre-incubated at 37°C for 15 min. Then,
200 µl of substrate solution (0.15 mM of xanthine) was added
into the mixture. The mixture was incubated at 37°C for 30
min. Next, the reaction was stopped with the addition of 200 µl
of 0.5 M HCl. The absorbance was measured using UV/VIS
spectrophotometer against a blank prepared in the same way
but the enzyme solution was replaced with the phosphate
buffer. Another reaction mixture was prepared (control) having
100 µl of DMSO instead of test compounds in order to have
maximum uric acid formation. The equation reported by
Naseem et al., (38) was used to evaluate the degree of XO
inhibitory activity. Thus, XOI activity was calculated using
Eq.1, in which α is the activity of XO without test extract and
β is the activity of XO with test extract. % XO inhibition = (1
– β/α) x 100 (1)
2.3. In-Vivo Antigout Activity
2.3.1. Preparation of Extract
The edible portions of fresh plant (celery, leek, parsley,
and molokhia) which were purchased from local markets in
43 Hanaa S. M. Abd El-Rahman and Nasra A. M. Abd–ELHak: Xanthine Oxidase Inhibitory Activity and Antigout of Celery, Leek,
Parsley, and Molokhia
Cairo, Egypt. Celery, Leeks, and Parsley, leaves were
carefully washed with water and left to dry at room
temperature. Then they were weighted and completely
blended in distilled water (1: 1 w/v) (39). Aqueous extract
of molokhia were washed with water and dried (solar
drying), they were weighted and completely blended in
distilled water and boiling for 10 min. The extract was
filtered to remove particulate matters, and then
administered orally to rats at dose of 4.8 mg/kg body
weight (40 and 41). All freshly prepared juicy samples were
administrated to the corresponding groups by oral gavage
once a day for 4 weeks.
2.3.2. Animals
A total of 36 male albino rats (body weights: 160-205 g)
were used in the present study. The rats were kept under
normal health laboratory conditions and fed on basal diet for
one week. Water and basal diet were provided ad libitum for
30 days. All the experimental procedures were carried out in
the Ophthalmology Research Institute, Giza, Egypt. The
animals were observed daily for any signs of toxicity. Body
weight was recorded at regular intervals throughout the
experimental period.
2.3.3. Animal Model of Gout in Rats
Experimentally-induced gout in rats (due to inhibition of
uricase with potassium oxonate) was used to study antigout
(42). Briefly, 250 mg/Kg, uricase inhibitor, potassium
oxonate (PO), dissolved in 0.9% saline solution was
administrated intraperitoneally to each animal except group 1
(group 2-6), 1 h before oral administration of test juicy
samples.
2.3.4. Experimental Design
Animals were divided into 6 groups of six rats each.
Group 1 served as the normal control (negative control).
Group 2 served as gouty control (positive control). Group 3,
4, 5, (gouty rats) received celery, leek, and parsley at the
oral dose of 5 g/Kg extract (39).Group 6 (gouty rats)
received extract of molokhia at the oral dose of 4.8 g/Kg
(41).
2.3.5. Biochemical Evaluation
At the end of the treatment period, rats were weighed.
All animals were fasted for 12 h, and then blood samples
were collected from the animal’s eye plexus under diethyl
ether anesthesia. Serums were separated out by
centrifugation at 3000 rpm for 15 min. After the collections
of blood samples, animals were killed. Lipid peroxidation
in plasma was estimated by the method of Ledwozy et al.,
(43). Malonaldehyde (MDA) produced during peroxidation
of lipids served as an index of lipid peroxidation. MDA
reacts with TBA to generate a colour product, which
absorbs at 532 nm. Superoxide dismutase (SOD) activity
was determined by the method of Marklund and Marklund
(44)). The degree of inhibition of the auto-oxidation of
pyrogallol at an alkaline pH by SOD was used as a measure
of the enzyme activity. Catalase and Glutathione peroxidase
activities were estimated by the method of Sinha (45) and
Rotruck et al., (46). The activity of Catalase was expressed
as µg of H
2
0
2
consumed/mn/mg protein. Glutathione
peroxidase was expressed as µg of glutathione utilized
/minute/mg/ protein. The activity of acid phosphatase was
assayed by the method of King (47).Alkaline phosphatase
(ALP; EC 3.1.3.1) activity was measured at 405 nm by the
formation of para-nitrophenol from para
nitrophenylphosphate as a substrate using the method of
Varley et al., (48). Creatinine, and uric acid were
determined by using the methods described by Larsen (49)
and Caraway (50), total protein content was measured by
the method of Lowry et al., (51). Total calcium and organic
phosphourus was measured by the method of Ferguson et
al., (52) and Fiske and Subbarow (53).
2.4. Statistical Analysis
The resulted data were subjected to statistical analysis
using the standard analysis of variance (one-way ANOVA) as
outlined by Snedecor and Cochran (54)
.
and the differences
among means dose of all extracts effects were tested for the
least significance difference value (LSD) at 0.05 probabilities
by using Duncan’s multiple range tests by SPSS for
Windows statistical package, version 10.0.
3. Results
3.1. In-Vitro Xanthine Oxidase Inhibitory Activity
Fig. (2). XO inhibitory activity (%) of extracts of celery, leek, parsaly, and
molokhia using absolute ethanol and distilled water as the extraction
solvent.
The effectiveness of selected extraction solvents to extract
bioactive compounds responsible for XO inhibitory activity
was studied. The percentages of XO inhibitory activity of all
crude extracts obtained by using distilled water and absolute
ethanol were in figure 2. The comparison was also made
between the plant extracts in two extraction solvents and the
positive control (allopurinol), to determine the best extraction
solvent. In general, the ethanol extracts were found to be
more active than the aqueous extracts. The highest XOI
activity was shown by ethanol extract both of parsley and
celery with 82.5663% and 73.8867 %, respectively. While,
the lowest value were 15.3667% and 25.2767% for aqueous
extract of molokhia and leek, respectively. The results were
compared with the standard drug allopurinol, which showed
Advances in Biochemistry 2015; 3(4): 40-50 44
90.36% inhibition.
3.2. In-Vivo Anti-Gout Activity
The ability of extracts (celery, leek, parsley, and molokhia)
to inhibit uricase was investigated in this study. In the present
study, no significant changes had been observed in gain
weights (%) of the treated groups as compared with controls
(Table 1). Administration of uricase inhibitor, potassium
oxonate significantly affected various bio-chemical
parameters on rat blood. As shown in Table 1, an increase
was in the serum uric acid and creatinine levels in positive
control (G2) when compared to the negative control (G1).
Extracts of Leek (G4) and parsley (G5), significantly
(p<0.05) reduce serum uric acid levels of gouty rats to values
than that found in G2.
Administration of aqueous extract of molokhia at a dose of
4.8 g/kg was effectively reduced serum creatinine levels in
rat compared to the gouty rat groups (G3, G4, G5), though
still higher than the normal control level (G1).
As shown in Figure 3, significant decrease in serum total
calcium concentrations in G2. No significant changes were
observed in extracts of parsley (G5), molokhia (G6), and
negative control (G1).
Oral administration extracts of celery, leek, parsley, and
molokhia significantly reduced (p < 0.05) the serum organic
phosphorus levels of gouty rats but not positive control (G2).
The results also indicate that no significantly changes
between all extracts used and negative control (Figure 4).
Table 1. Effect of the orally administered of celery, leek, parsely, and molokhia on serum uric acid, creatinine, and gain weight in potassium oxonate -induced
rat.
Treatment Uric acid (mg/dl) Creatinine level (mg/dl) Gain weight (%)
Negative control (G1) 2.9286±0.2536
a
0.6855±0.0697
a
16.886±0.4640
a
Positive control (G2) 6.1402±0.5783
c
1.3966±0.04803
c
16.269±0.6496
a
Extract of Celery 5 g/Kg (G3) 4.2531±0.6665
b
1.2933±0.14629
bc
15.804±0.6692
a
Extract of Leek 5 g/Kg (G4) 3.4822±0.2362
ab
1.1816±0.0424
bc
17.216±1.1514
a
Extract of Parsley 5 g/Kg (G5) 3.5704±0.2149
ab
1.1520±0.06612
bc
16.736±1.1055
a
Extract of molokhia 4.8 g/Kg (G6) 4.1739±0.1836
b
1.046±0.0583
b
16.851±0.49
a
All the values are expressed as the mean ±standard error of the mean. The mean difference is significant (P < 0.05) when compared with the positive control
(gouty rat) group
Fig. (3). Total calcium (mg/dl) levels of aqueous extract of celery, leek,
parsaly, and molokhia in normal and potassium oxonate-induced gout rats.
Fig. (4). Organic Phosphorus (mg/dl) levels of aqueous extract of celery,
leek, parsaly, and molokhia in normal and potassium oxonate-induced gout
rats.
The present study also investigated the efficacy of orally
administered celery, leek, parsley, and molokhia extracts on
blood biomarkers of oxidative stress (Malonaldehyde
concentration) and lipid peroxidation levels in rats blood. In
gouty control rats (G2), the levels of serum MDA, as a
biomarker of lipid peroxidation, were statistically (p ≤ 0.05)
higher than normal rats (G1). Oral administration of celery,
leek, parsley, and molokhia to gouty rats induced a
significant reduction (p ≤ 0.05) in these elevated levels of
MDA, but could not yet reach these levels to the normal
value G1 (Table 2). Potassium oxinate treatment increased
MDA levels significantly at dose used (250 mg/Kg),
reflecting the increase in lipid peroxidation. In contrast to
these results, there was decrease in glutathione peroxidase
(GPx) enzyme activity compared with the controls (G1). GPx
enzyme activity levels were found to have increased in
parsley (G5) the value was 3.0597 U/mg protein).
The effect extracts of celery, leek, parsley, and molokhia
on the enzymatic antioxidant levels catalase (CAT) and
superoxide dismutase (SOD) in experimental rat were
tabulated in Table 2. CAT and SOD level was decreased
significantly in positive control (G2) when compared to
control group (G1). Administration of extracts to potassium
oxonate-induced rat altered the above changes by regulating
the CAT level to nearly that of normal levels. Molokhia and
parsley (G5, G6) also significantly inhibited the decreased
levels of MDA and increased the levels of SOD and CAT in
gouty rats (P < 0.05).
45 Hanaa S. M. Abd El-Rahman and Nasra A. M. Abd–ELHak: Xanthine Oxidase Inhibitory Activity and Antigout of Celery, Leek,
Parsley, and Molokhia
Table 2. MDA levels and antioxidant enzyme activities of groups.
Treatment MDA (nmol/mL) Superoxide dismutase
(SOD) (U/mg protein)
Catalase (CAT) (U/mg
protein) GPX (U/mg protein)
Negative control (G1) 3.0619±0.1197
a
3.6201±0.2451
ab
10.134±0.7023
a
3.8180±0.1499
a
Positive control (G2) 5.2374±0.0808
c
1.9656±0.06397
c
5.9359±0.32497
e
1.0732±0.0885
e
Extract of Celery 5 g/Kg (G3) 4.1747±0.1528
b
3.1081±0.1924
b
7.2546±0.27389
d
2.0222±0.10558
d
Extract of Leek 5 g/Kg (G4) 4.0085±0.1939
b
3.3099±0.1937
ab
8.3224±0.05065
c
2.5289±0.10609
c
Extract of Parsley 5 g/Kg G5 3.839 ±0.4286
b
3.8174±0.1169
a
9.0268±0.100796
b
3.0597±0.08822
b
Extract of molokhia 4.8 g/Kg (G6) 3.5263±0.2626
ab
3.252±0.1913
ab
8.5267±0.39954
bc
1.9917±0.15365
d
All the values are expressed as the mean ±standard error of the mean. The mean difference is significant (P < 0.05) when compared with the positive control
(gouty rat) group
There was a significant (P<0.05) increase in acid
phosphatase (ACP) and alkaline phosphatase (ALP) enzyme
activities in rat treated with the uricase inhibitor, potassium
oxonate (G2) when compared to the normal control (G1).
These were found to be reverted back in extracts of celery,
leek, parsley, and molokhia treated animals. Treatment with
extract of parsley significantly (P<0.05) decreased the ACP
and ALP when compared to all extracts. On the other hand,
the activity produced by extract of parsley (G5) was almost
similar to that of the negative control (G1) (Table 3).
Table 3. Acid phosphatase and Alkaline phosphatase levels of groups.
Treatment acid phosphatase (ACP) Alkaline phosphatase (ALP)
Negative control (G1) 0.1108 ±0.1046
a
168.72±7.3169
a
Positive control (G2) 0.2778 ±0.0078
c
432.63±12.1662
d
Extract of Celery 5 g/Kg (G3) 0.16114 ±0.02044
b
292.48±5.2898
c
Extract of Leek 5 g/Kg (G4) 0.1915 ±0.00961
b
286.14±5.2285
c
Extract of Parsley 5 g/Kg (G5) 0.11384±0.01347
a
253.44±8.850
b
Extract of molokhia 4.8 g/Kg (G6) 0.1613±0.016634
b
286.24±4.2929
c
All the values are expressed as the mean ±standard error of the mean. The mean difference is significant (P < 0.05) when compared with the positive control
(gouty rat) group
4. Discussion
One of the most sensitive and dramatic indicator of gout is
neutrophil influx into the joint fluid. Neutrophils accumulate
in both the joint fluid and the synovial membrane, where a
small fraction of these cells actively phagocytose
monosodium urate crystals and release mediators, that are
chemotactic and amplify the inflammatory reaction (55). The
enzyme XO catalyzes the oxidation of hypoxanthine to
xanthine and then to uric acid, which plays a crucial role in
gout (56). XO is an important source of oxygen derived free
radicals. The enzyme catalyzes reduce oxygen (during
reperfusion phase), leading to the formation of superoxide
anion radicals and hydrogen peroxide, as well as hydroxyl
radicals (57). It has been proposed as a central mechanism of
oxidative injury in some situations like gout, ischemia, renal
damage, hypertension, diabetes, etc. (58, 16 and 59). Recent
findings show that the occurrence of gout is increasing
worldwide, possibly due to the changes in dietary habits like
intake of high-purine foods viz., organ meats, yeast, beer and
other alcoholic beverages (60 and 61). The main therapeutic
approach for gout is the use of XOI such as allopurinol,
which block the final step in the synthesis of uric acid from
purines (62 and 63). An alternative to allopurinol is the use
of medicinal plants which possess phytochemical
constituents. We thus began our program to look for xanthine
oxidase inhibitors of phytochemical origin from the extracts
of celery, leek, parsley, and molokhia. Phytochemical
screening of extracts revealed the presence of flavonoids,
phenolics, and saponins accounting for its antioxidant
property. Flavonoids are a group of polyphenolic compounds
which exhibit several biological effects such as anti-
inflammatory, anti-hepatotoxic, antiulcer activities, etc. Also,
the structure–activity relationship of different chemical
classes of flavonoids have been reported as potential
inhibitors of XOD (26, 27, 33, 28, 29, 22, 32and 36). Several
in-vitro studies confirmed the xanthine oxidase to Xanthine
dehydrogenase (XOD/XDH) inhibitory activity of some
flavonoids. These compounds are structurally similar to
XOD/XDH substrate and so can inhibit the enzyme activity
(64 and 65). Celery, leek, parsley, and molokhia have also
demonstrated the least XO inhibitory (XOI) activity probably
due to limited bioactive compounds present. The extent of
increasing in XOI activity elicited by allopurinol was much
higher than that observed with the celery, leek, parsley, and
molokhia in both extraction solvent. Similar results have
been reported by others (66 and 39).In the present study, we
noted Parsley and celery were the best source of raw material
for obtaining the XOI compound as each exhibits more than
70% inhibition of XOD under all two extraction solvents. In
fact, Parsley and celery under evaluation have shown
considerable activity for XOD inhibition, substantiate the
fact that secondary metabolites in the leaves contain diverse
classes of bioactive phenolic compounds such as
polyphenols, tocopherols and alkaloids (67, 68 and 32),
which may act as XOI.
It is well known that XOD is an inducible enzyme. Oxonic
acid and its salts are foreign substances that could interfere
with some other metabolic systems in mice. It was found that
oxonate was distributed to the intracellular sites of the small
Advances in Biochemistry 2015; 3(4): 40-50 46
intestine at a much higher concentration after oral
administration. In addition, it was converted mostly to
cyanuric acid in the gastrointestinal tract partly by XOD (69).
Serum urate level is partly regulated by the kidney in
rodents. Physiological and pharmacological studies have
suggested that urate produced in liver is transported
bidirectional in the proximal tubule. As oxonate is a
competitive uricase inhibitor, it is likely that oxonate
similarly competes with urate for binding at a specific site
within urate transporter and permit efflux of urate subsequent
to its intracellular production. Further, many studies
confirmed that oxonate specifically inhibited renal
electrogenic urate transport, and also blocks channel activity
of urate transporter in mammalian (70, 71, and 72).The levels
of uric acid, and creatinine were significantly elevated after
oxonate treatment in our study. In the goutyrat, serum uric
acid and creatinine levels reduced significantly after extracts
of celery, leek, parsley, and molokhia administration. Similar
results have been reported by others (20, 73, 74 and 75).
Although the elevated levels of uric acid in the circulation
could give rise to gout and possibly other pathological
conditions (76), the antioxidant action of uric acid,
particularly its ability to inhibit DNA damage, is also well
documented (76 and 19). Parsley (Petroselinum crispum) as a
dietary vegetable can be used safely long-term; this feature of
parsley makes it a possible alternative for allopurinol, or at
least in combination therapy to minimize the side-effects of
allopurinol (39).XOD was found to be significant activity in
liver. Uric acid synthesis appears to be mainly a hepatic
process. Therefore, the gout effect of celery, leek, parsley,
and molokhia could be explained, at least in part, by blocking
of liver of XOD activities. The present study was the first
account demonstrating the in vivo gout action of celery, leek,
parsley, and molokhiaas well as their ability to decrease
serum uric acid and creatinine levels in animals orally
administered with our extracts. Duke (77) and Balch et al.,
(78) recorded that the seeds and stalks of celery are known to
reduce uric acid levels, relieving symptoms of joint pain and
immobility. In one study, Apium graveolens has shown
significant reduction in serum uric acid level in rats (79).
Apium graveolens is used to treat fungal infections and
tumors. Apiumgraveolens contains furocoumarins that are
typically prescribed for their stomachic, carminative, diuretic
and emmenagogue properties (80).
Our study indicates that by increasing serum total calcium
level with extracts of celery, leek, parsley, and molokhia in
comparison to positive control, symptoms of gouty arthritis
are reduced. While comparing of organic phosphorus levels
of Group 2 (positive control) with group 1 and gouty rat
groups, highly significant correlation was observed (P <
0.05). It was due to high organic phosphorus levels of Group
G2 (10.517 mg/dL) which is the highest of all these groups.
Molokhia are edible and are used as an ingredient for popular
food in Egypt. This plant is also rich in potassium, calcium,
phosphorous, iron, ascorbic acid, and carotene (36). Celery
provides an excellent source of vitamin B1, B2, B6, C and
fiber. It's a very good source of folic acid, potassium, and
calcium (81 and 82). Leeks are a good source of dietary fiber,
folic acid, calcium, potassium, and vitamin C. Calcium in
leeks is also used for the proper clotting of blood in the
human body (83 and 84).
In recent years, there is increasing interest in free radicals;
they have been shown to modify biological molecules, which
may result in various pathological conditions. Thus
additional natural products need to be evaluated for their
antioxidant potential. Considerable evidence suggests that
oxidative stress and reactive oxygen species (ROS) play
significant roles in several aspects of acute and chronic
inflammation (85). The increased lipid peroxide level noticed
in potassium oxonate induced rat in our study (Group 2), due
to its release from neutrophils and monocytes during
inflammation (86). The result of the present study indicate
that the antioxidant defense system is compromised in
potassium oxante induced rat as evidenced by increased lipid
peroxidation concentration and decreased activity of
antioxidant enzyme. Our results show that the activities of
Superoxide dismutase, Glutathione peroxidase and Catalase
decreasing in potassium oxonate-induced animals which may
be due to consequence of their increased consumption during
oxidative stress and cellular lysis. All extracts of celery, leek,
parsley, and molokhiahave shown significant decrease in
level of MDA and increase in activity of antioxidant enzyme
level, comparable to positive rat (G2).Thereby revealing that
serum uric acid through lipid peroxidation, might be working
towards the etiopathogenesis of oxidative stress diseases and
its serum level may be a deciding factor for progression of
the disease, and also our study showed that parsley
(Petroselinum crispum) has a higher potential than celery,
leek, and molokhia to increase glutathione peroxidase
activity. Several studies have identified the active
antioxidants within parsley (Petroselinum crispum) including
flavonoids (32), carotenoids (87), ascorbic acid (88),
tocopherol and coumarines (32). These phytochemicals
improve total antioxidant capacity, suppress destructive
oxygen free radicals and prevents oxidative stress damage
(75 and 89). This was in agreement with Haidari et al., (39).
Leeks are a good source of allyl sulfides and also rich in the
flavonoid especially kaempferol. Allyl derivatives of leek
oils stimulate the activity of GP
X
and inhibited the decreased
ratio of reduced to oxidized glutathione produced by 12-O-
tetradecanoylphorbol-13- acetate in epidermal cells.
Diallyldisulfide increase GP
X
activity in animal tissues with
increased the activity of glutathione reductase, and
superoxide dismutase (90, 84 and 91). A phenolic extract of
molokhia exhibited antioxidant activity through the radical
generator-initiated peroxidation of linoleic acid (36).Al
Batran et al., (92) showed that gastroprotective effect of an
ethanolic extract of molokhia against ethanol-induced gastric
ulcers in adult Sprague Dawley rats. SOD and MDA levels
were significantly increased (P ≤0.05) and reduced (P
≤0.05), respectively pretreatment with molokhia. Oyedeji and
Bolarinwa (93) found that aqueous extract of molokhia has
beneficial potentialities on the blood chemistry of male
albino rats. As in traditional medicine they were used for
47 Hanaa S. M. Abd El-Rahman and Nasra A. M. Abd–ELHak: Xanthine Oxidase Inhibitory Activity and Antigout of Celery, Leek,
Parsley, and Molokhia
management of gout it is thought that their anti-gout activity,
at least by part, was related to this xanthine oxidase
inhibitory activity. Gouty arthritis is metablic disorder that is
characterized by deposition of uric acid into joints (94). Now
there is new trend to use herbal medicine because of their
potential to cure and fewer or no side effects. Medicinal
plants having anti-inflammatory, uricosuric and xanthine
oxidase inhibitory activities are used in gouty arthritis.
Herbal medicines are comparably safe, and are useful in the
management of gout. Although allopurinol is commonly used
in the treatment of gouty arthritis but this has side effects
such as skin rashes, nauseas and vomiting. The possible
explanation can be drawn from number of studies showing
that superoxide dismutase during catalyzing dismutation of
O
͞2
to H
2
O
2
can form copper bound hydroxyl radical from
hydrogen peroxide H
2
O
2
(95). Hydroxyl radical when gets
bounded to SOD, then it can attack adjacent histidine residue
which is attached to copper resulting in inactivation of both
SOD1 and SOD3 (96).
Conclusion, this study had established the xanthine
inhibitory action of several plant extracts used in Egypt folk
medicine. The extracts of celery, leek, parsley, and molokhia
have been tested for xanthine oxidase inhibitory activity. The
efficacy of distilled water as the extraction solvents was
commendable because most of the plants used to treat gout
were administered as decoctions and infusions, so the
biologically active compounds were most likely water-
soluble. In addition, the possibility of any harmful residue
due to use of organic solvent was also avoided. The selection
of tropical plants used in medicine and screening of their
extracts for pharmacological activity may provide
identification of newer medicaments for the treatment of
various ailments especially gout. Further research on celery,
leek, parsley, and molokhia have significantly reduced the
serum uric acid, lipid peroxidation, and increase activity of
antioxidant enzyme levels in gouty rats. This may be due to
the inhibition of XO activity and the presence of
phytochemical constituents.
Acknowledgements
The authors are thankful to Prof. Dr. Hanaa Sidky Special
Food and Nutrition Dept., Food Technology Research
Institute, Agricultural Research Center, for valuable helping
in this study.
References
[1] Singh, H.; Krishna, G. and Baske, P.K.(2010). Plants used in
the treatment of joint diseases (rheumatism, arthritis, gout and
lumbago) in Mayurbhanj district of Odisha, India. Rep. Opin.
2: 22-26.
[2] Falasca, G.F. (2006). Metabolic diseases: gout. Clin.
Dermatol24:498-508.
[3] Haidari, F.; Rashidi, M. R.; Keshavarz, S. A.; Mahboob, S. A.;
Eshraghian, M. R. and Shahi, M. M. (2008).Effects of onion
on serum uricacid levels and hepatic xanthine
dehydrogenase/xanthineoxidase activities in hyperuricemic
rats. Pak J Biol Sci. 11: 1779-84.
[4] Dalbeth, N. and Haskard, D. O. (2005).Mechanisms of
inflammation in gout. Rheumatology 44: 1090-1096.
[5] Corrado, A.; D'Onofrio, F.; Santoro, N.; Melillo, N. and
Cantatore, F. P. (2006).Pathogenesis, clinical findings and
management of acute and chronic gout. Minerva Med. 97:
495-509.
[6] Bieber, J. D. and Terkeltaub, R. A. (2004).Gout on the Brink
of Novel Therapeutic Options for an Ancient Disease.Arthritis
& rheumatism 50 (8): 2400–2414.
[7] González, A. G.; Bazzocchi, I. L.; Moujir, L.; Ravelo, A. G.;
Correa, M. D. and Gupta, M. P. (1995). Xanthine oxidase
inhibitory activity of some Panamanian plants from
celastraceae and lamiaceae. The Journal of
Ethnopharmacology 46 (1): 25-29.
[8] Ramallo, I. A., Zacchino, S. A. and Furlan, R. L. E. (2006).A
rapid TLC autographic method for the detection of xanthine
oxidase inhibitors and superoxide scavengers. Phytochemical
Analysis 17 (1): 15-19.
[9] Moriwaki, Y. (2014).Effects on Uric Acid Metabolism of the
Drugs except the Antihy peruricemics. J. Bioequiv Availab. 6
(1):10-17.
[10] Nile, S. H. and Khobragade, C. N. (2011).In-vitro anti-
inflammatory and xanthine oxidase inhibitory activity of
Tephrosia purpurea L. shoot extract. Nat. Product Comm. 6:
1437-1440.
[11] Battelli, M. G.; Corte, E. D. and Stirpe, F. (1972). Xanthine
oxidase type D (dehydrogenase) in the intestine and other
organs of the rat. Biochemical Journal 126 (3):747-749.
[12] Unno, T.; Sugimoto, A. and Kakuda, T. (2004).Xanthine
oxidase inhibitors from the leaves of Lagerstroemia speciosa
(L.) Pers. J Ethnopharmacol 93:391-5.
[13] Sweeney, A.P.; Wyllie, S.G.; Shalliker, R.A. and Markham,
J.L.(2001).Xanthine oxidase inhibitory activity of selected
Australian native plants.J. Ethnopharmacol. 75: 273-277.
[14] Kong, L. D.; Abliz, Z.; Zhou, C. X.; Li, L. J.; Cheng, C. H. K.
and Tan, R. X. (2001).Glycosides and xanthine
oxidaseinhibitors from Conyza bonariensis. Phytochemistry
58 (4): 645-651.
[15] Umamaheswari, M.; Asok Kumar, K.; Somasundaram, A.;
Sivashanmugam, T.; Subhadradevi, V. and Ravi, T. K.
(2007).Xanthine oxidase inhibitory activity of someIndian
medicinal plants. Journal of Ethnopharmacology 109 (3):
547-551.
[16] Berry, C. E. and Hare, J. M. (2004).Xanthine oxidoreductase
and cardiovascular disease: Molecular mechanisms and
pathophysiological implications. J. Physiol. 555: 589-606.
[17] Nguyen, M. T. T.; Awale, S.; Tezuka, Y.; Tran, Q.L.;
Watanabe, H. and Kadota, S. (2004)Xnthine oxidase
inhibitory activity of Vietnames medicinal plants. Biol.
Pharm. Bull. 27: 1414-1421.
[18] Strazzullo, P. and Puig, J.G. (2007).Uric acid and oxidative
stress: Relative impact on cardiovascular risk.
Nutr.Metab.Cardiovasc. Dis. 17: 409-414.
Advances in Biochemistry 2015; 3(4): 40-50 48
[19] Wang, Y.; Zhu, J. X.; Kong, L. D. Yang, C.; Cheng, C. H. and
Zhang, X. (2004).Administration of procyanidins from grape
seeds reduces serumuric acid levels and decreases hepatic
xanthine dehydrogenase/oxidase activities in oxonate-treated
mice. Basic Clin Pharmacol Toxicol. 94: 232-7.
[20] Kong, L. D.; Yang, C.; Ge, F.; Wang, H. D. and Song, G.
Y.(2004). A Chinese herbal medicine Ermiao wan reduces
serum uric acid level and inhibits liver xanthine
dehydrogenase and xanthine oxidase in mice. J.
Ethnopharmacol. 93: 325–330.
[21] Abd Aziz, S. M.; Low, C. N.; Chai, L. C.; Abd Razak, S. S.
N.; Selamat, J.; Son, R.; Sarker, M. Z. I. and Khatib, A.
(2011).Screening of selected Malaysian plants against several
food borne pathogen bacteria. International Food Research
Journal 18(3)1195-1201.
[22] Costantino, L.; Albasini, A.; Rastelli, G. and Benvenuti, S.
(1992). Activity of polyphenolic crude extracts as scavengers
of superoxide radicals and inhibitors of xanthine oxidase.
Planta Medica 58: 342-344.
[23] Chang, W. S.; Lee, Y. J.; Lu, F. J. and Chiang, H. C.
(1993).Inhibitory effects of flavonoids on xanthine
oxidase.Anticancer Research 13: 2165-2170.
[24] Selloum, L.; Reichl, S.; Muller, M.; Sebihi, L. and Arnhold, J.
(2001).Effects of flavonols on the generation of superoxide
anion radicals by xanthine oxidase and stimulated neutrophils.
Archives of Biochemistry and Biophysics 395 (1): 49-56.
[25] Chang, W. S. and Chiang, H. C. (1995).Structure activity of
coumarins in xanthine oxidase inhibition. Anticancer
Research 15: 1969-1974.
[26] Cheung, M.C.; Lin, L. Y.; Yu, T. H. and Peng, R. Y.
(2008).Hypolipidemic and antioxidant activity of mountian
celery seed essential oils. J Agric Food Chem. 56 (11): 3997-
4003.
[27] Tsi, D.; Das, N.P. and Tan, B. K. (1995). Effects of aqueous
celery (Apium graveolens) extract on lipid parameters of rats
fed a high fat diet. Planta Med. 61: 18-21.
[28] Onyeagba, R. A.; Ugbogu, O. C.; Okeke, C. U AndIroakasi, O.
(2004). Studies On TheAntimicrobial Effects Of Garlic (Allium
SativumLinn), Ginger (Zingiber Officinale Roscoe) AndLime
(Citrus Aurantifolia Linn). Afr.J.Biotechnol. 3:552-554.
[29] Garth, L. N. And Rita, E. (2006). LipidReplacement And
Antioxidant NutritionalTherapy For Restoring Mitochondrial
FunctionAnd Reducing Fatigue In Chronic Fatigue Syndrome
And Other Fatiguing Illnesses. Journal Of Chronic Fatigue
Syndrome 13(1): 57-68.
[30] Lopez, M. G.; Sanchez-Mendoza, I. R. and Ochoa-Alejo,
N.(1999). Compartive study of volatile components and fatty
acids of plants and in-vitro cultures of parsley Petroselinum
crispum (Mill) nym ex hill. J. Agric. Food Chem. 47: 3292–
3296.
[31] Ozsoy-Sacan O, Yanardag R, Orak H, Ozgey Y, Yarat A and
Tunali T.(2006).Effects of parsley (Petroselinum crispum)
extract versus glibornuride on the liver of streptozotocin-
induced diabetic rats. J. Ethnopharmacol. 104: 175-181.
[32] Fejes, S. Z.; Blazovics, A.; Lemberkovics, E.; Petri, G.;
Szoke, E. and Kery, A.(2000).Free radical scavenging and
membrane protective effects of methanol extracts from
Anthriscus cerefolium (Hoffm) L. and Petroselinum
crispum(Mill) Nym. Ex A. W. Hill. Phytother. Res. 14: 362–
365.
[33] Peterson, S.; Lampe, J.W.; Bammler, T. K.; Gross-Steinmeyer,
K. and Eaton, D. L. (2006).Apiaceous vegetable constituents
inhibit human cytochrome P-450 1A2 (hCYP1A2) activity
and hCYP1A2-mediated mutagenicity of aflatoxin B
1
. Food
Chem. Toxicol. 44: 1474–1484.
[34] Zeghichi, S.; Kallithkara, S. and Simopoulus, A. P.
(2003).Nutritional composition of Molokhia (Corchorus
olitorius) and Stammagathi (Cichorium spinosum) in plants,
in human health and nutritional policy. Simopoulus, A. P and
C. Gopalan (Eds.) Karger, Basel, pp 1-21.
[35] Abu- Hadid, A. F.; El-Shinawy, M. Z.; El-Bethagy, A. S.;
Gaafer, S. A.; Medany, M. (1994).Studies on the production of
off-season Jew’s mallow (Molokhia) in Egypt. Egypt J. Hort.
21:187-193.
[36] Azuma, K.; Nakayama, M.; Koshioka, M.; Ippoushi, K.;
Yamaguchi, Y.; Kohara, K.; Yamauchi, Y.; Ito, H. and
Higashio, H.(1999).Phenolic antioxidants from the leaves of
Corchorus olitorius L. Journal of Agricultural and Food
Chemistry 47, 3963–3966.
[37] Harborne, J. B. (1998). Phytochemical methods: A guide to
modern technique of plant analysis, 3rd edn. London:
Chapmann & Hall.
[38] Naseem, S. A.; Muhammad, F.; Muzammil, H. N.; Kouser, B.
M. and Aurangzeb, H. (2006). Activity of polyphenolic plant
extracts as scavengers of free radicals and inhibitors of xanthine
oxidase. The Journal of Basic and Applied Sciences 2 (1).
[39] Haidari, F.; Keshavarz, S. A.; Shahi, M. M.; Mahboob, S.
A.and Rashidi, M. (2011).Effects of Parsley (Petroselinum
crispum) and its Flavonol Constituents, Kaempferol and
Quercetin, on Serum Uric Acid Levels, Biomarkers of
Oxidative Stress and Liver Xanthine Oxidoreductase
Aactivity in Oxonate-Induced Hyperuricemic Rats.Iranian
Journal of Pharmaceutical Research 10 (4): 811-819.
[40] Das, A. K.; Bag, S.; Sahu, R.; Dua, T. K.; Sinha, M. K.;
Gangopadhyay, M.; Zaman, K. and Dewanjee, S. (2010).
Protective effect of Corchorus olitorius leaves on sodium
arsenite-induced toxicity in experimental rats. Food and
Chemical Toxicology 48:326–335.
[41] Mahgoub, S., El-Sharkawi, F. & Badary, O. (2007) Effect of
Molokhia soup on blood sugar, hepatic antioxidant status &
plasma lipid profile in diabetic rats. The Egyptian journal of
biochemistry & molecular biology (EJBMB), 25, 305-325.
[42] Hall, I. H.; Scoville, J. P.; Reynolds, D. J.; Simlot, R. and
Duncan, P. (1990).Substituted cyclic imides as potential anti-
gout agents.Life Sci. 46: 1923-1927.
[43] Ledwozyw A, Michalak J,.Stepien A, Kadziolka A.(1986).
The relationship between plasma triglycerides,cholesterol,
total lipids and lipid peroxidationproducts during human
atherosclerosis. Clin. Chim. Acta 155: 275-284.
[44] Marklund, S.L. and Marklund, G. (1974) Involvement
ofsuperoxide anion radical in the autoxidation ofpyrogallol
and a convenient assay for superoxidedismutase. Eur. J.
Biochem,; 47: 469–474.
[45] Sinha, A. K. (1972) Colorimetric assay of Catalase. Anal.
Biochem 47: 389–394.
49 Hanaa S. M. Abd El-Rahman and Nasra A. M. Abd–ELHak: Xanthine Oxidase Inhibitory Activity and Antigout of Celery, Leek,
Parsley, and Molokhia
[46] Rotruk, J. T.; Pope, A. L.; Ganther, H. E.; Swanson, A. B.;
Hafeman, D. G. and Hekstra, W. G.(1973). Selenium,
biochemical role as a component of glutathioneperoxidase
purification and assay. Science 179: 588–590.
[47] King, J. (1965). The hydrolases-acid and alkalinephosphatases,
In: Van D (ed), Practical clinicalenzymology, Nostrand
Company Limited, London, pp, 191-208.
[48] Varley, H.; Gewenlock, A. and Bell, M. (1980).Practical
Clinical Biochemistry. Vol. 1. 5th edn, pp. 741, 897. London:
William Heinemen Medical. Books, Ltd.
[49] Larsen, K. (1972).Creatinine assay by a reaction kinetic
principle.Clin.Chem. Acta. 41: 209-213.
[50] Caraway, W. T. (1955).Determination of uric acid in serum by
a carbonate method Amer. J. of Clin. Pathology2: 840-845.
[51] Lowry, O. H.; Rosebrough, N. J.; Farr, A. I and Randall, R.
J.(1951).Protein measurement with the folin phenolreagent. J.
Biol. Chem 193: 265-275.
[52] Ferguson, E.; Vaughan, A. and Swale, J.(1976). A method for
the estimation of total calcium in serum or heparinized plasma.
Clinica Chimica Acta 67(3): 281–286.
[53] Fiske C. H. and Subbarow Y. (1925).The colorimetric
determination of phosphorus. J Biol Chem 66:375-400.
[54] Snedecor, G. W. and Cochran, W.G. (1980).Statistical
methods Book, p420, 7th Ed Iowa Stat Univ. Press, Ames,
Iowa, USA.
[55] Landis, R. C. and Haskard, D. O. (2001). Pathogenesis of
crystalinduced inflammation. Curr Rheumatol Rep. 3:36-41.
[56] Bowman, W. C. and Rand, M. J.(1980).Text book of
pharmacology, 2nd ed. Blackwell, Sydney 243-244.
[57] Halliwell, B.and Gutteridge, J. M. C. (2001). Free radicals in
biology and medicine, 3rd ed. Oxford University press,
Oxford 28-30.
[58] Mazzali, M.; Hughes, J.; Kim, Y.; Jefferson, J.A.; Kang, D.
and Gordon, K.L. (2001). Elevated uric acid increases blood
pressure in the rat by a novel crystal-independent mechanism.
Hypertension. 38, 1101-1106.
[59] Nakagawa, T; Mazzali, M.; Kang, D.; Sanchez-Lozada, L. G.;
Herrera-Acosta, J. and Johnson, R.J. (2006).Uric acid – a
uremic toxin? Blood. Purif. 24, 67-70.
[60] Choi, J. W. and Ford,E. S. (2008).Sugar Sweetened Soft
Drinks, Diet Soft Drinks, and Serum Uric Acid Level: The
Third National Health and Nutrition Examination Survey,” Ar
- thritis & Rheumatism 59 (1): 109-116.
[61] Mayes, P. A. (1993).Intermediary Metabolism of Fructose,”
Ame- rican Journal of Clinical Nutrition 58 (5): 754S-765S.
[62] Kumar, A.; Edward, N.; White, M. I.; Johnston, P.W. and
Catto, G. R.(1996).Allopurinol, erythema multiforme and
renal insufficiency. BMJ. 312, 173-174.
[63] Wallach, S.L. (1998).The side effects of allopurinol. Hosp.
Pract. 33, 22.
[64] Mo, S. F.; Zhou, F.; Lv, Y. Z.; Hu, Q. H.; Zhang, D. M. and
Kong, L. D. (2007). Hypouricemic action of selected
flavonoids in mice: structure–activity relationships. Biol.
Pharm. Bull. 30: 1551-1556.
[65] Lin, C. M.; Chen, C. S.;Chen, C. T.; Liang, Y. C. and Lin, J.
K. (2002).Molecular modeling of flavonoids that inhibits XO.
Biochem. Biophys. Res. Commun. 294: 167–172.
[66] Azmi, S. M. N.; Jamal, P. and Amid, A. (2012).Xanthine
oxidase inhibitory activity from potential Malaysian medicinal
plant as remedies for gout.International Food Research
Journal 19(1): 159-165.
[67] Zhou, K. and Yu, L.(2006).Total phenolic contents and
antioxidant properties of commonly consumed vegetables
grown in Colorado.Food Sci. Technol. 39, 1155–1162.
[68] Halliwell, B.; Gutteridge, J. M. C.; FreeSreeramulu, D. and
Raghunath, M. (2010). Antioxidant activity and phenolic
content of roots, tubers and vegetables commonly consumed
in India. Food Res 43, 1017–1020.
[69] Yoshisue, K.; Masuda, H.; Matsushima, E.; Ikeda, K.;
Nagayama, S. and Kawaguchi, Y. (2000). Tissue distribution
and biotransformation of potassium oxonate after oral
administration of a novel antitumor agent (drug combination of
tegafur, 5-chloro-2, 4-dihydroxypyridine, and potassium
oxonate) to rats. Drug Metabolism and Disposition 28, 1162–
1167.
[70] Leal-Pinto, E.; Cohen, B. E.; Lipkowitz, M. S. and Abramson,
R.G. (2002).Functional analysis and molecular model of the
human urate transporter/ channel, hUAT. The American
Journal of Physiology—Renal Physiology 283, F150–F163.
[71] Hosoyamada, M.; Ichida, K.; Enomoto, A.; Hosoya, T. and
Endou, H.(2004).Function and localization of urate transporter
1 in mouse kidney.Journalof the American Society of
Nephrology 15, 261–268.
[72] Lipkowitz, M. S.; Leal-Pinto, E.; Cohen, B. E. and Abramson,
R. G.(2004). Galectin9 is the sugar-regulated urate
transporter/channel UAT. Glycoconjugate Journal 19, 491–
498.
[73] Zhao, X.; Zhu, J.X.; Mo, S.F.; Pan, Y. and Kong, L.D. (2006).
Effects of cassia oil on serum and hepatic uric acid levels in
oxonate-induced mice and xanthine dehydrogenase and
xanthine oxidase activities in mouse liver. J. Ethnopharmacol.
103: 357–365.
[74] Zhu, J.X.; Wang, Y.; Kong, L.D.; Yang, C. and Zhang, X.
(2004).Effects of Biota orientalis extract an dits flavonoid
constituents, quercetin and rutin on serum uric acid levels in
oxonate-induced mice and xanthine dehydrogenase and
xanthine oxidase activities in mouse liver. J. Ethnopharmacol.
93: 133-140.
[75] Neguyen, M. T. T.; Awale, S.; Tezuka, Y.; Shi, L.; Zaidi, S. F.
H. and Ueda, J. (2005). Hypouricemic effects of acacetin and
4, 5-O-dicaffeoylquinic acid methyl ester on serum uric acid
levels in potassium oxonate-pretreated rats. Biol. Pharm. Bull.
28: 2231-2234.
[76] Nuki G.(2006). Gout. Medicine 34: 417-423.
[77] Duke, J. A. (1997).The Green Pharmacy. Rodale Press,
Pennsylvania.
[78] Balch, C. N. C.; James, F. and Phyllis, A. (1997).Prescription
for Nutritional Healing. Avery Publishing Group, New York.
[79] Doha, S. (2008). Evaluation of anti-gout activity of some
plant food extracts. Polish J. of Food and Nutr. Sci.,58: 389-
395.
Advances in Biochemistry 2015; 3(4): 40-50 50
[80] Nehal, M. A. (2011). Hepatoprotective effect of feeding celery
leaves mixed with Chicory leaves and barley grains to
hypercholesterolemic rats. Pharmacog.Magaz.,7: 151-156.
[81] Ang, J. F. and Miller, W.B. (1991).Multiple functions of
powdered cellulose as a food ingredient.Cereal Foods World
36, 558-564.
[82] Thebaudin, J. Y.; Lefobvre, A. C.; Harrington, M. and
Bourgeois, C. M.(1997). Dietary fibers: Nutritional and
technological interest. Trends in Food Science and Technology
8, 41-48.
[83] Dorant, E.; Van Den, P. A. and Goldbohm, R. A. (1996). A
Prospective Cohort Study On The Relationship Between
Onion And Leek Consumption, Garlic Supplement Use And
The Risk Of Colorectal Carcinoma In The Netherlands.
Carcinogenesis Mar 17(3):477-84.
[84] Riley, D. M.; Bianchini, F. and Vainio, H.(2001). Allium
Vegetables And Organosulfur Compounds: Do They Help
Prevent Cancer. Environ Health Perspect. Sep 109(9):893-
902.
[85] Ramprasad, V. R.; Shanthi, P. and Sachdanandam, P. (2005).
Evaluation of antioxidant effect of Semecarpus anacardium
Linn.nut extract on the components of immune system in
adjuvant arthritis. Vasc. Pharmacol. 42: 179-186.
[86] Sabina, E. P.; Chandel, S. and Rasool, M. Kh. (2008).
Inhibition of Monosodium Urate Crystal-Induced
Inflammation by Withaferin A. J Pharm Pharmaceut Sci. 11
(4): 46-55.
[87] Francis, G. W. and Isaksen, M. (1989).Droplet counter current
chromatography of the carotenoids of parsley Petroselinum
crispum. Chromatographia 27: 549–551.
[88] Davey, M. W.; Bauw, G. and Montagu, M. V.(1996).Analysis
of ascorbate in plant tissues by high performance capillary
zone electrophoresis. Anal. Biochem. 239: 8–19.
[89] Vijayalakshmi, B. and Chandrasekhar, M. (2008). Oxidative
stress and their resultant products as the target molecules in
clinical diagnosis of age related disease. Curr. Trends
Biotechnol. Pharm. 2: 239-250.
[90] Jin, L. and Baillie, T.A. (1997). Metabolism Of The
Chemoprotective Agent Diallyl Sulfide To Glutathione
Conjugates In Rats. Chem Res Toxicol 10:318–327.
[91] Al Hamedan, W. A and Anfenan, M. L. Kh.
(2011).Antioxidant Activity of Leek towards Free Radical
Resulting from Consumption of Carbonated Meat in Rats.Life
Science Journal 8 (1): 169-176.
[92] Al Batran, R.; Al-Bayaty, F.; Abdulla, M. A.; Al-Obaidi, M.
M. J.; Hajrezaei, M.; Hassandarvish, P.; Fouad, M.;
Golbabapour, S. and Talaee, S. (2013). Gastroprotective
effects of Corchorus olitorius leaf extract against ethanol-
induced gastric mucosal hemorrhagic lesions in rats. Journal
of Gastroenterology and Hepatology 28: 1321–1329
[93] Oyedeji, K. O. and Bolarinwa, A. F. (2013).Effect of
Corchorus Olitorius Extract On Haematological and Plasma
Biochemical Parameters in Male Albino Rats.Journal of
Dental and Medical Sciences 3(5):68-71.
[94] Terkeltaub, R. (2010). Gout: Novel therapies for treatment of
gout and hyperuricemia, Arthritis Research andTher.,11: 234-
236.
[95] Hink, H. U.; Santanam, N.; Dikalov, S.; Mc Cann, L.;
Nguyen, A. D.; Parthasarathy, S.; Harrison, D. G. and Fukai,T.
(2002).Peroxidase Properties of Extracellular Superoxide Dis-
mutase. Role of Uric Acid in Modulating in Vivo Activity,”
Arteriosclerosis, Thrombosis, and Vascular Biology
22(9):1506-1508.
[96] Jewett,S. L.; Rocklin, A. M.; Ghanevati, M.; Abel, J. M. and
Marach, J. A.(1999).A New Look at a Time-Worn System:
Oxidation of CuZn-SOD by H
2
O
2
. Free Radical Biology &
Medicine 26 (7-8): 905-918.
