Current Research in Microbiology and Biotechnology

Vol. 2, No. 3 (2014): 384-390

Research Article
Open Access

ISSN: 2320-2246

Effect of aqueous red cabbage extract on uricase

activity isolated from Pseudomonas aeruginosa
Mohannad S.AL-Fayyadh*
Biotechnology Dept. College of Science, University of Baghdad

* Corresponding author: Mohannad S.AL-Fayyadh; email: mohsal222@yahoo.com

Received: 06 May 2014

Accepted: 27 May 2014

Online: 05 June 2014

ABSTRACT
Separation of uricase enzyme from Pseudomonas aeruginosa was done using (70%) saturation ammonium
sulphate, and purification of this enzyme was done by ion exchange chromatography on DEAE- cellulose column
and eluted with linear NaCl (0-1M). Partial purified uricase gave an activity of (4.9 unit/ml), protein
concentration of (0.56 mg/ml), specific activity of (8.75 unit/mg) with purification folds (8.4) and a yield of
(48%). The maximum purified uricase activity was detected at 35c and pH 8.5. The crude extract was screened
for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, phenolic
compounds, flavonoids, saponins, tannins and anthraquinones using standard procedures. The results shown
that red cabbage extract(RCE) contain flavonoides which contain phenolic compounds and anthocyanines which
glycoslated with mono or dimolecules of saccharides, while test for alkaloids, steroids, saponins and tannins
were negative. Red cabbage extract (50mg/ml) have an inhibition activity against uricase compared with
negative and positive control using allopurinol. So red cabbage considered as a novel inhibition for uricase whiel
can used to treat gout disease in future.

Keywords: Pseudomonas aeruginosa; uricase; aqueous red cabbage; anthocyanins

INTRODUCTION
Cabbage is a popular cultivar of the species Brassica
oleraceaLinne (Capitata Group) of the Family
Brassicaceae and is vegetable of leafy green. It is a
herbaceous, biennial, dicotyledonous flowering
distinguished by stem upon which is crowded a mass of
leaves green but in some varieties red or purplish,
which while immature form a characteristic compact,
globular cluster (cabbagehead)[1]. Cabbage is an
excellent source of vitamin C. It also contains
significant amounts of glutamine, an amino acid that
has anti-inflammatory properties. Cabbage can also be
included in dieting programs, as it is a low calorie food
[2]. Cancer prevention all other of health research with
best to cabbage and its late benefits. More than 475
studies have examined the role of this cruciferous
vegetable in cancer prevention [3]. Pseudomonas
aeruginosa is aerobic rod shaped a Gram-negative,
bacterium with unipolar motility. An opportunistic
human pathogen, P. aeruginosa is also an opportunistic
pathogen of plants and animals [4]. It is found in soil,
skin flora, water, and most man-made environments

throughout the world. It development not only in

normal atmospheres, but also with little oxygen, and
has colonised many natural and artificial environments.
P. aeruginosa secretes a variety of pigments, including
pyocyanin (blue-green), fluorescein (yellow-green and
fluorescent), and pyorubin (red-brown) [5]. Uricase is
one of the oxidoreductases enzymes. This group of
enzymes is the first group in the rules of classification
according to the Enzyme Commission (EC.) in the
International Union of Biochemist (IUB). The
systematic name of this enzyme is (Urate: Oxygen
Oxidoreductase [EC 1.7.3.3] [6]. Uricase catalyzes the
oxidation of uric acid with molecular oxygen to
allantoin serving as an electron acceptor [7].

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384

MATERIALS AND METHODS

Sepharose-6B, DEAE-cellulose, Column were purchased
from Pharmacia Fine Chemicals. Coomassie Brilliant
Blue, Uric acid, Boric acid, Glucose, Ethanol absolute,
other chemicals were supplied by BDH Chemicals.

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Plant materials
Fresh Samples of red cabbage plant were purchased
from Iraqi local market in Baghdad. The samples were
washed with clean tap water to remove dirt on the
leaves. The dried plant material was manually
powdered and the powder kept in polyethylene bags
until used.

b) Liebermans test
A 0.5 g of the aqueous extract was dissolved in 2 ml of
acetic anhydride and cooled well in an ice-bath.
Concentrated sulfuric acid was then carefully added. A
colour change from purple to blue to green indicated
the presence of a steroid nucleus i.e. aglycone portion
of the cardiac glycosides [13].

Preparation of Red Cabbage extracts

Leaves were sliced into small pieces and oven-dried at
50 C. Dried plants (100 gm) were extracted. The uses of
dry plants can be effective to minimize enzymatic
degradation of phonetic compounds inside plant
tissues. After overnight maceration, the extract was
filtered through gauze and water was evaporated
under reduced pressure at 50C by using a rotary
evaporator. After evaporation, dried samples were
placed in a desiccator over calcium sulfate to remove
any remaining water. The resulting dried pigments
were then used for further studies. The extraction yield
for Red Cabbage extracts (RCE) was about 11% [8].

Test for phenolic compounds

a) 0.5 g of the aqueous extract was dissolved in 10 ml of
distilled water in a test tube. To 2 ml of filtered solution
of the extract, 3 drops of a freshly prepared mixture of
1 ml of 1% ferric chloride and 1 ml of potassium
ferrocyanide was added to detect phenolic compounds.
Formation of bluish-green color was taken as positive
[14].

Preliminary phytochemical screening

Standard screening test of the extract was carried out
for various plant constituents. The crude extract was
screened for the presence or absence of secondary
metabolites such as alkaloids, steroidal compounds,
phenolic compounds, flavonoids, saponins, tannins and
anthraquinones using standard procedures [9].

Test for flavonoids

a) Test for free flavonoids
Five milliliters of ethyl acetate was added to a solution
of 0.5 g of the extract in water. The mixture was
shaken, allowed to settle and inspected for the
production of yellow color in the organic layer which is
taken as positive for free flavonoids [15].

Test for alkaloids

a) Preliminary test
A 100 mg of an aqueous extract was dissolved in dilute
hydrochloric acid. Solution was clarified by filtration.
Filtrate was tested with Dragendr-offs and Mayers
reagents. The treated solutions were observed for any
precipitation [10].

b) Lead acetate test

To a solution of 0.5 g of the extract in water about 1 ml
of 10% lead acetate solution was added. Production of
yellow precipitate is considered as positive for
flavonoids [10].

b) Confirmatory test
Five grams of the aqueous extract was treated with
40% calcium hydroxide solution until the extract was
distinctly alkaline to litmus paper, and then extracted
twice with 10 ml portions of chloroform. Chloroform
extracts were combined and concentrated in vacuum to
about 5 ml. Chloroform extract was then spotted on
thin layer plates[10,11]. Solvent system (n-hexaneethyl acetate, 4:1) was used to develop chromatograms
and detected by spraying the chromatograms with
freshly prepared Dragendroffs spray reagent. An
orange or dark colored spots against a pale yellow
background was confirmatory evidence for presence of
alkaloids [11].

b) The dried aqueous extract (100 mg) was dissolved in

water. Few crystals of ferric sulfate were added to the
mixture. Formation of dark-violet color indicated the
presence of phenolic compounds [10].

c) Reaction with sodium hydroxide

Dilute sodium hydroxide solution was added to a
solution of 0.5 g of the extract in water. The mixture
was inspected for the production of yellow color which
was considered as a positive test for flavonoids [15].
Test for saponins
Froth test
0.5 g of the aqueous extract was dissolved in 10 ml of
distilled water in a test tube. The test tube was
stoppered and shaken vigorously for about 30 seconds.
The test tube was allowed to stand in a vertical position
and observed over a 30 minute period of time. If a
honey comb froth above the surface of liquid persists
after 30 min. the sample is suspected to contain
saponins [11].

Test for steroidal compounds

a) Salkowskis test
A 0.5 g of the aqueous extract was dissolved in 2 ml
chloroform in a test tube. Concentrated sulfuric acid
was carefully added on the wall of the test tube to form
a lower layer. A reddish brown colour at the interface
indicated the presence of a steroid ring (i.e. the
aglycone portion of the glycoside) [12].

Test for tannins

a) Ferric chloride test
A portion of the aqueous extract was dissolved in
water. The solution was clarified by filtration. 10%
ferric chloride solution was added to the clear filtrate.
This was observed for a change in color to bluish black
[15].

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b) Formaldehyde test
To a solution of about 0.5 g of the extract in 5ml water,
3 drops of formaldehyde and 6 drops of dilute
hydrochloric acid were added. The resulting mixture
was heated to boiling for 1 minute and cooled. The
precipitate formed (if any) was washed with hot water,
warm alcohol and warm 5% potassium hydroxide
successively. A bulky precipitate, which leaves a
colored residue after washing, indicated the presence
of phlobatannins [11].
c) Test for Phlobatannins
Deposition of a red precipitate when an aqueous
extract of the plant part was boiled with 1% aqueous
hydrochloric acid was taken as evidence for the
presence of phlobatannins [15].
d) Modified iron complex test
To a solution of 0.5 g of the plant extract in five
milliliters of water a drop of 33% acetic acid and 1 g
sodium potassium tartarate was added. The mixture
was warmed and filtered to remove any precipitate. A
0.25% solution of ferric ammonium citrate was added
to the filtrate until no further intensification of colour is
obtained and then boiled. Purple or blackish
precipitates which is insoluble in hot water; alcohol or
dilute ammonia denotes pyrogallol tannin present [16].
Test for Anthraquinones
a) Test for free anthraquinones (Borntragers test)
The aqueous extract of the plant material (equivalent to
100 mg) was shaken vigorously with 10 ml of benzene,
filtered and 5 ml of 10% ammonia solution added to
the filtrate. The mixture was shaken and the presence
of a pink, red or violet color in the ammonia (lower)
phase indicated the presence of free anthraquinones
[15].
b) Test for O-anthraquinones glycosides (Modified
Borntragers test)
For combined anthraquinones, 5 g of the plant extract
was boiled with 10 ml 5% sulphuric acid for 1 hour and
filtered while hot. The filtrate was shaken with 5 ml
benzene; the benzene layer separated and half its own
volume of 10% ammonia solution added. The
formation of a pink, red or violet color in the ammonia
phase (lower layer) indicated the presence of
anthraquinones derivatives in the extract [11].
Enzyme extraction
Reagents:
0.1 M sodium borate buffer, pH 8.5. This buffer
consisted two kinds of solutions:
1- Solution A: prepared by dissolving 0.618 gm. of boric
acid in 25 ml of distilled water.
2- Solution B: prepared by dissolving 0.4 gm of sodium
hydroxide in 100 ml of distilled water. Then the pH
value of solution A was adjusted to 8.5 by using of
solution B and then diluted to a total volume of 100 ml
with distilled water [17].

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Microorganism
The bacterial strain used in this study was previously
isolated from poultry waste and was identified as
Pseudomonas aeruginosa by specific biochemical tests
[18]. The P. aeruginosa growth was optimized for mass
level uricase production by using basal media
containing several components such as 10.0g dextrose;
2.0g yeast extract; 3.0g uric acid and 5.0g NaCl. All
ingredients were dissolved in 1000ml distilled water
with 7.0 pH. The dextrose and yeast extract were used
as a carbon and nitrogen source. The organism was
allowed to grown at 35C temperature for 36 hours
incubation to obtain uricase for further analysis [17].
Enzyme assay
- Reagents
1. Sodium borate buffer: 0.1 M sodium borate buffer,
pH 8.5 was adjusted.
2. A 0.12 mM uric acid solution was prepared by
dissolve 0.002gm of uric acid in 100 ml borate buffer.
The principle of enzyme measurement was as follows:
Uricase could catalyze the oxidation of uric acid into
allantoin and H2O2, which was then measured by using
a reaction system containing 4-aminoantipyrine,
phenol and peroxidase as chromogen. In practical
analysis, 0.1ml enzyme solution was incubated with a
mixture of 0.6 ml sodium borate buffer (pH 8.5, 0.1M)
containing 2mM uric acid, 0.15 ml 4-aminoantipyrine
(30 mM), 0.1ml phenol (1.5%), 0.05ml peroxidase (15
unit/ml) at 37 C for 20 min.. The reaction was stopped
by addition of 1 ml ethanol, and the absorbance at 540
nm was read against the blank by a spectrophotometer.
One unit of enzyme was defined as the amount of
enzyme that produces 1ml of H2O2 per minute under
the standard assay conditions [17, 28].
Separation of uricase
The broth culture was centrifuged at 8000 xg for 30
minutes, at 4C. The pH of supernatant was adjusted to
8.5 with NaOH. Uricase activity and protein
concentration were estimated [17].
Precipitation step was by addition different
concentrations of solid ammonium sulfate to the crude
extract. Solid ammonium sulfate was slowly added
at 4 C with continue mixing for 30 minute, then
centrifuged at 8000 xg for 30 minutes at 4 C.
Precipitates were dissolved in a small volume of 0.01M
borate buffer (pH 8.5), then dialyzed against 1000 ml
of 0.01M borate buffer (pH 8.5) for 24 hours, and
concentrated by using sugar. Uricase activity and
protein concentration were determinate [19].
Purification of uricase by ion exchange
chromatography on DEAE-cellulose
A glass column (1.560 cm) was packed with DEAEcellulose. The concentrated and dialyzed cell free
supernatant was applied to a column, which previously
was equilibrated with 0.01M borate buffer (pH 8.5).
The column was washed with 3 times volumes of 0.01
M borate buffer, pH 8.5 at a flow rate of 40 ml/hour and
the bound proteins were eluted with a linear NaCl
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gradient (0-1 M) in the same buffer by using fraction

collector (LKB Radi-Rac) and analyzed by UV
spectrophotometer at 280nm . Fractions containing
uricase enzyme were pooled and concentrated by using
sugar [20].
Determination of protein content
Total protein concentrations of cell free supernatant
and purified samples were assayed by the method of
Bradford [21] using a calibration curve established
with bovine serum albumin (BSA) as a standard and
Coomassie Brilliant Blue (CBB) G-250. Appropriate
volume (20l) was taken from the protein solution in
10 ml test tube. The volume in the test tube was
completed to 0.1 ml with distilled water (80 l) then
5ml of CBB dye were added to the tube and the
contents were well mixed. The absorbance of the
mixture was measured after 5 minutes at 595 nm
against reagent blank. The protein content calculated
from the standard curve.
Effect of different concentration of red cabbage
extract on uricase activity
The experiment was performed to investigate the effect
of different concentration or RCF on activity of uricase
(20 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, and 300
mg/ml) was used as follows:
Mix well and leave for 10 min. then stopping the
reaction. Calculate the uricase activity [29].

RESULTS AND DISCUSSION

Using aqueous solvent, yield of recovering red cabbage
extract was observed. High yield of crude aqueous
extract was produced from 100 g of dried whole plant
was 11.0 gm (11.0 %) with the violet dry material
similar to the optimum condition reported in the
literature to extract high yield of active compounds
without degradation were 70% ethanol as solvent for
extraction, 50C for temperature at overnight swirling.
The variations yields as seen between alcoholic and
aqueous extract depend on several factors, such as the
method of extraction and solvent polarity that was
used in the extraction process. Phenolic compound can
be extracted from plant samples. Usually before
extraction plant samples are treated by grinding and
homogenization, which may be preceded by freezedrying. Generally, freeze-drying retains higher levels of
Phenolic content in plant samples than air-drying.
However, drying processes, including freeze-drying,
can cause undesirable effects on the constituent
profiles of plant samples, therefore, caution when
prepare planning and analyzing research studies on the
medicinal properties of plants [3,8].

chemical extraction depends on the type of solvents,

extraction time and temperature, sample-to-solvent
ratio as well as on the chemical composition and
physical characteristics of the samples [26]. The
solubility of phenolic compounds is governed by the
chemical nature of the sample of plant, as well as the
polarity of the solvents used. Plant materials may
contain phenolics varying from simple e.g., phenolic
acids, anthocyanin). Moreover, phenolic may also be
associated with other plant components such as
proteins and carbohydrates. Therefore, there is no
union extraction procedure suitable for extraction of all
plant phenolic. Depending on the solvent system used
during exaction, a mixture of phenolic soluble in the
solvent will be extracted from plant materials. It may
also contain some non-phenolic material such as sugar,
organic acids and fats. As a result, additional steps may
be required to remove those undesirable components
[9, 11].
Anthocyanin are polar molecules with hydroxyl,
carboxyl, methoxyl and glycolyl groups bound to
aromatic rings. They are more soluble in water than in
non-polar solvents and this characteristic helps
extraction and separation processes, as described by
Corrales [22].
Hydrochloric acid diluted in methanol is generally used
to extract anthocyanins. Since methanol has toxic
characteristics, food scientists prefer other extraction
systems. Water: ethanol mixture of 80: 20 (v/v) is
commonly used as a solvent in the food industry, and it
is as good as methanol .In aqueous extractions, the
most used and efficient acids are acetic, citric, tartaric
and hydrochloric. Studies on anthocyanin extraction
using these solvents are found in literature .Although,
most of the literature concerns to the identification of
anthocyanins in several vegetable sources, studies are
carried out to develop the process and establish good
operational conditions.
Generally, temperatures higher than 70C cause rapid
degradation and discoloration of anthocyanins
.Opening of the pyrylium ring with a loss of the B-ring
and chalcone formation which further decompose to
coumarin glucoside derivatives was considered as the
first anthocyanin degradation step hydrolysis of the
glycosidic moiety and formation of aglycon even at
acidic pH as the initial degradation reaction has been
reported [23].

Solvent extirpation is the most commonly used

procedures to prepare extracts from plant materials
due to their ease of use, efficiency, and wide
applicability. It is generally known that the yield of

As anthocyanins are located inside the plant cell,

organic solvents are used to extract them from
homogenized plant material. In conventional extraction
procedures, acidified organic solvents like methanol,
ethanol or acetone are used. These solvents, along with
the acid can denature the cellular membrane and
release of intracellular anthocyanins into the solvent
and maintain the anthocyanins in their native form. Use
of excess acid may cause hydrolysis of the sugar
residues into furfural and hydroxymethyl furfural,
which have been shown to favor pigment decay [24].

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Phytochemical screening
Chemical tests
Phytochemical screening was done using color forming
and precipitating chemical reagents on the dried whole
leaves of red cabbage. Results obtained from the tests
were summarized in table (1).It was shown that red
cabbage extract (RCE) contain flavonoides which
contain phenolic compounds and anthocyanines which
glycoslated with mono
or
dimolecules
of

saccharides.The principle constituents of red cabbage

extract anthocyanins such as cyanidine 3-galactoside
,cyanidine 3-glucoside ,cyanidine 3-5 diglucoside and
acetylated anthocynins with sinapic acid ,coumaric
acid ,ferulic acid in addition to isothiocyanates (
glucosinolate), vitamins A, B, C and -carotenes .
Anthocyanins, a group of phenolic natural pigments
present in RCE, were found to have the strongest
antioxidizing power of 150 flavonoids.

Table 1. Results of phytochemical screening of aqueous RCE.

Test

Reagent

Result

Test for alkaloids

Dragendroff's s , Mayer's

Negative

Test for steroids

Acetic anhydride , conc. Sulfuric acid ,

Chloroform and conc. sulfuric acid

Negative

Test for phenolic

Ferric chloride and potassium ferrocyanide

Test for flavonoids

Test for saponins
Test for tannins

10 % lead acetate sodium hydroxide ,ethyl

positive
positive

acetate
Negative

Froth s test
Ferric chloride aqueous hydrochloric acid

Negative

Formaldehyde , Modified iron complex

Test for

Test for free anthraquinones

anthraquinones

Test for O-anthraquinones

Test for glucosides

Test for reducing sugars

positive
positive

Anthocyanins are potent antioxidants in vitro, quench

free radicals and terminate the chain reaction that is
responsible for the oxidative damage, demonstrated
the antioxidant effects of anthocyanins in vitro using
human colon cancer cell line (CaCo2). Cyanidin and
Cyanidin-3-glucoside treatment reduced cell growth
and cell proliferation in a dose-dependent way,
decreased ROS level by any concentration of Cyanidin
and only at the lowest concentration, by Cyanidin-3glucoside and increased cell cycle/stress proteins
expression [25,27]. Both the compounds affected DNA
fragmentation, highlighting their antioxidant activity.
The authors concluded that anthocyanins exhibit
antioxidant properties and could therefore be useful in
the treatment of pathologies where free radical
Production plays a key role .The antioxidant activity of
anthocyanins is largely because of the presence of
hydroxyl groups in position 3 of ring C and also in the
3, 4 and 5 positions in ring B of the molecule.
Anthocyanidins have superior antioxidant activity
compared to their respective anthocyanins and the
activity decreases as the number of sugar moieties
increase. The antioxidant activity of many anthocyanins
was comparable to the commercial antioxidants such
as
tert-butylhydroquinone
(TBHQ),
butylated
hydroytoluene (BHT), butylated hydroxyanisole (BHA)
and vitamin E .By analyzing TLC chromatogram of
falvonoids as seen in figure 1- 1,we can note the
presence of several spots corresponding to compounds
with flavonid behavior( yellow ,yellow brown after
spraying with diphenylboriloxiethilamine) or de
hydroxycinnamic acid derivatives (blue or green-blue
fluorescensce, after spraying with the mentioned
reagent),.Among these, quercetin (Rf = 0.92) have been

identified in all the analyzed plant extract. The

consulted scientific data mentions flavonoids
glycosides of quercetin with xylose,galactose as
monosaccharides [22, 25, 26].

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388

Separation of uricase:
Separation of uricase from culture media of
Pseudomonas aeruginosa. Activity of enzyme and
protein concentration was determined. Results shown
in table (2).
Ammonium sulfate precipitation:
Ammonium sulfate was used at different concentration
to precipitate the enzyme. Results indicate that
precipitation of crude uricase was done with saturated
ammonium sulfate 70% .The activity and protein
concentration was determined, the amount of protein
was 0.96 mg/ml and activity 5.99U/ml, and specific
activity was estimated as 6.23 U/mg, purification fold
of 6 and yield of 67% as show in table(2).
Purification of uricase by ion exchange
chromatography:
Purification of uricase from P. aeruginosa was done use
ion exchange chromatography. During the experiment,
it was found that washing with borate buffer pH 8.5, no
peak was observed. As shown in figure (1). Addition of
300ml of borate buffer with NACL gradients (elution
step) allows one peak to be obtained and represented
by fraction 58-73 . Each fraction was tested for uricase
activity and protein concentration. The fraction in
elution step were showed uricase activity. Result also
indicates that uricase produced from P. aeruginosa
carried negative charges which attraction with DEAE-

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cellulose of positive charge therefore uricase was

eluted and goes to elution step. As described in table
(2) protein concentration of 0.56 mg/ml, activity of 4.9

U/ml, and specific activity of 8.75 U/mg, with

purification fold of 8.4 and yield of 49% were obtained.

Table 2. Purification step of uricase from Pseudomonas aeruginosa.

Yield%

Fold
of
purification

Total activity
(units)

Specific
activity (U/mg
protein)

Protein
(mg/ml)

Activity
(U/ml)

Volume ml

100

71.5

1.03

1.39

1.43

50

67

6.0

47.9

6.23

0.96

5.99

48

8.4

34.32

8.75

0.56

4.9

Steps

Crud extract
Pellet
after
70%
ammonium
sulphate and
Dialysis
Ion-exchange
DEAE
cellulose and sucrose

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Figure 1. Ion exchange chromatography for purification of

uricase from P. aeruginosa by using DEAE-cellulose column,
equilibrated with borate buffer (0.005 M, ph 8.0), eluted with
borate buffer with NaCl gradient (0.1-1 M).

Effect of different concentration of red cabbage

extract on uricase activity
The experiment was performed to investigate the effect
of different concentration from RCF on activity of
uricase (20 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml,
and 300 mg/ml). The results shown in figure (2),
indicated that uricase activity was decreased with
increased concentration red cabbage extract.

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Figure 2. Effect of different concentration of red cabbage

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A- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml D.W.
B- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml RCE (20 mg/ml).
C- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml RCE (50 mg/ml).
D- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml RCE (100 mg/ml).
E- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml RCE (200 mg/ml).
F- 0.5 ml of uricase + 1 ml uric acid + 0.5 ml RCE (300 mg/ml).

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