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Antioxidant and anti-inflammatory properties of C-phycocyanin from blue-green algae

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Phycocyanin is a pigment found in blue-green algae which contains open chain tetrapyrroles with possible scavenging properties. We have studied its antioxidant properties. Phycocyanin was evaluated as a putative antioxidant in vitro by using: a) luminol-enhanced chemiluminescence (LCL) generated by three different radical species (O2-, OH., RO.) and by zymosan activated human polymorphonuclear leukocytes (PMNLs), b) deoxyribose assay and c) inhibition of liver microsomal lipid peroxidation induced by Fe+2-ascorbic acid. The antioxidant activity was also assayed in vivo in glucose oxidase (GO)-induced inflammation in mouse paw. The results indicated that phycocyanin is able to scavenge OH. (IC50 = 0.91 mg/mL) and RO. (IC50 = 76 microg/mL) radicals, with activity equivalent to 0.125 mg/mL of dimethyl sulphoxide (DMSO) and 0.038 microg/mL of trolox, specific scavengers of those radicals respectively. In the deoxyribose assay the second-order rate constant was 3.56 x 10(11) M(-1) S(-1), similar to that obtained for some non-steroidal anti-inflammatory drugs. Phycocyanin also inhibits liver microsomal lipid peroxidation (IC50 = 12 mg/mL), the CL response of PMNLs (p < 0.05) as well as the edema index in GO-induced inflammation in mouse paw (p < 0.05). To our knowledge this is the first report of the antioxidant and anti-inflammatory properties of c-phycocyanin.
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Antioxidant and anti-inflammatory properties of C-phycocyanin
from blue-green algae
C. Romay
1
, J. Armesto
1
, D. Remirez
1
, R. Gonza
´
lez
1
, N. Ledon
1
and I. Garcı
´
a
2
1
Pharmacology Department, National Center for Scientific Research, CNIC, P.O. Box 6990, Havana, Cuba, Fax 53 7 330497,
e-mail: ricardo@quimica.cneuro.cu
2
Pharmacy and Foods Faculty, University of Havana, Ave. 23, No. 21425, La Coronela, Havana, Cuba
Received 21 April 1997; returned for revision 14 May 1997; accepted by M. J. Parnham 28 October 1997
Abstract. Objective: Phycocyanin is a pigment found in
blue-green algae which contains open chain tetrapyrroles
with possible scavenging properties. We have studied its
antioxidant properties.
Materials and methods: Phycocyanin was evaluated as a
putative antioxidant in vitro by using: a) luminol-enhanced
chemiluminescence (LCL) generated by three different
radical species (O
¹
2
,OH
,RO
) and by zymosan activated
human polymorphonuclear leukocytes (PMNLs), b) deoxy-
ribose assay and c) inhibition of liver microsomal lipid
peroxidation induced by Fe
þ2
-ascorbic acid. The antioxi-
dant activity was also assayed in vivo in glucose oxidase
(GO)-induced inflammation in mouse paw.
Results: The resultsindicatedthatphycocyanin is ableto scav-
enge OH
(IC
50
¼ 0:91mg/mL) and RO
(IC
50
¼ 76mg/mL)
radicals, with activity equivalent to 0.125mg/mL of
dimethyl sulphoxide (DMSO) and 0.038mg/mL of trolox,
specific scavengers of those radicals respectively. In the
deoxyribose assay the second-order rate constant was 3:56×
10
11
M
¹1
S
¹1
, similar to that obtained for some non-steroidal
anti-inflammatory drugs. Phycocyanin also inhibits liver
microsomal lipid peroxidation (IC
50
¼ 12 mg/mL), the CL
response of PMNLs (p<0.05) as well as the edema index in
GO-induced inflammation in mouse paw (p<0.05).
Conclusions: To our knowledge this is the first report
of the antioxidant and anti-inflammatory properties of
c-phycocyanin.
Key words: Antioxidant Chemiluminescence C-
Phycocyanin Blue-green algae Free radical scavenger
Introduction
C-phycocyanin is a protein-bound pigment found in blue-
green algae. Phycocyanin monomers are themselves made
up of two distinguishable protein subunits designated a and
b, which contain at least three covalently attached bilin
chromophores, open chain tetrapyrroles with no metal
complexes [1]. These prosthetic groups account for about
4% of the algae mass, indicating the presence of about
sixteen chromophoric groups per unit molecular weight [2].
It occurs in four different structural forms, monomeric,
trimeric, hexameric and decameric [3], and is the most
abundant pigment in blue-green algae, accounting for more
than 20% of algal dry weight [4].
The chemical structure of the bilin chromophores in
c-phycocyanin, (open chain tetrapyrroles) are very close to
that of bilirubin. Stocker et al. [5] reported that bilirubin is an
antioxidant of possible physiological importance because it
could scavenge peroxy radicals by donating a hydrogen atom
attached to the C-10 bridge of the tetrapyrrole molecule to
form a carbon-centered radical with resonance stabilization
extending over the entire bilirubin molecule.
It is well known that reactive oxygen species (ROS) are
involved in a diversity of important processes in medicine
including, among others: inflammation, atherosclerosis,
cancer, reperfusion injury [6]. One way by which a substance
can interfere with these processes is by acting as antioxidant
or free radical scavenger.
Taking these data into account, we postulated that
c-phycocyanin may be a putative antioxidant and decided to
evaluate it in some in vitro and in vivo experimental models.
Materials and methods
Reagents
Luminol and xanthine oxidase (XO 20U/mL) were obtained from
Boehringer Mannheim GmbH (Germany). Trolox and p-iodophenol
were from Aldrich Chemical Co. (Milwaukee, WI, USA). Superoxide
dismutase (SOD) (3333U/mL) from bovine erythrocytes and Hypox-
anthine (HX) were from Serva Feinbiochimica (Heidelberg, Germany).
Tert-butylhydroperoxide (t-BOOH) and butyl hydroxytoluene (BHT)
were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Sodium dodecylsulphate (SDS), hydrogen peroxide (H
2
O
2
), ascorbic
Inflamm. res. 47 (1998) 3641
q Birkha
¨
user Verlag, Basel, 1998
1023-3830/98/010036-06 $ 1.50+0.20/0
Inflammation Research
Correspondence to: C. Romay
m
acid, 2-deoxyribose and thiobarbituric acid (TBA), were from BDH Ltd
(Poole, UK). FeCl
3
and glucose oxidase (GO) (1.4U/mg) were from
Merck (Darmstadt, Germany). All other reagents were of analytical
grade.
Phycocyanin was obtained from Arthospira maxima species and
purified by the method of Neufeld and Riggs [7].
Phosphate-buffered saline solution (PBS) consisted of NaCl
0.14M, KCl 2.7mM, Na
2
HPO
4
12mM, KH
2
PO
4
1.5mM, CaCl
0.9mM and MgCl
2
0.49mM.
Animals
Male OF
1
mice weighing 2225g and male Sprague Dawley rats (220
250g) were purchased from the National Center for Laboratory
Animals Production (CENPALAB, Havana, Cuba). The animals were
housed under controlled temperature (t ¼ 25 8C) and air humidity
(60%) with a 12h light-dark cycle, and kept on a standard laboratory
diet and drinking water ad libitum.
Chemiluminescence measurements
The scavenging action of c-phycocyanin was determined against
different types of oxygen radicals, which were generated by specific
chemical reactions and detected by LCL.
A well known scavenger for each radical was used as control
for the paradigm and to compare its effect with that produced by
c-phycocyanin.
Chemiluminescence was measured in millivolts in an LKB Wallac
1250 luminometer coupled to an LKB 2210 two channel recorder.
Superoxide radical scavenging activity was determined as
described by Pascual et al. [8]. The reaction consisted of 800 mLofa
mixture containing 68mM glycine buffer pH 8.6, 10 mM luminol and
5mM p-iodophenol. Fifty mL of distilled water or c-phycocyanin
aqueous solutions were added. Then 2mL of xanthine oxidase (2U/mL)
were added and the reaction was initiated with 10mL of hypoxanthine
1mM. The intensity was registered immediately. SOD was used in the
system as a control.
Hydroxyl radicals were generated from the Fenton reaction. The
method used was described by Pascual and Romay [9]. In brief, to
800mL of a mixture containing: 50 mM K
2
HPO
4
-KH
2
PO
4
buffer
pH7.8, 2 mM EDTA and 0.1 mM luminol, 0.1mL of distilled water,
sample or specific scavenger (DMSO) was added. Then 5 mLof6mM
H
2
O
2
was also added and mixed. The reaction wasstarted with 10 mLof
20mM FeSO
4
and after rapid mixing, the CL signal was immediately
registered.
Determination of alkoxyl radical scavenging activity was per-
formed by measuring the inhibition of the CL produced by the reaction
of t-BOOH with ferrous ions in the presence of luminol, as previously
described [9]. The reaction mixture consisted of: 800 mLof50mM
glycine buffer pH8.6, 50mM SDS, 0.025 mM luminol. Ten mLof
distilled water was added and mixed prior to the addition and mixing of
5mL of 7.3mM t-BOOH.
The reaction was started with 10 mL of 0.4mM FeSO
4
and
immediately after rapid mixing the chemiluminescence signal (mV)
was recorded. Trolox, a water soluble analogue of vitamin E, was used
as specific scavenger of these radicals.
Double quenching experiments were done in each CL system in
order to determine whether the effect of the phycocyanin was due to the
scavenging of the desired oxygen free radical or the trapping of other
free radical species. These experiments were done by measuring the
luminous signal before (Io) and after (I) adding increasing concentra-
tions of c-phycocyanin in the absence and presence (sufficient to cause
50% inhibition) of the specific scavenger (SOD, DMSO or trolox). The
slope of the plot is equal to kt where t is the life time of the radical in
the absence of phycocyanin. When both phycocyanin and the specific
scavenger compete for the same radical, a decrease in the slope must be
expected.
Effect of phycocyanin on the CL response of isolated PMNLs
PMNL preparation
Human leukocytes were obtained as described previously [8] from
10mL heparinized (20 U/mL) venous blood from healthy volunteers,
who had not taken any drug during the week before blood sampling.
The blood was mixed with an equal volume of an ACD-dextran-glucose
mixture consisting of: 1.5 mL acid citrate dextrose (ACD) in 0.9%
NaCl (24.5g glucose/L, 22 g sodium citrate dihydrate/L and 7.3 g citric
acid/L), 5mL of 6% (w/v) Dextran T-500 in 0.9% of NaCl and 3.5mL
of 5% glucose in 0.9% NaCl.
After mixing well and allowing to stand for 4560min at room
temperature, the upper layer containing the leukocytes was removed by
aspiration and three times its volume of ammonium chloride 0.8%
added in order to hemolyze the remaining red cells. The cells were
centrifuged for 10min at 500 g at 48C. Then, 2 mL of 0.9% NaCl were
added, followed by 3mL of cold distilled water which were mixed and
allowed to stand for 2min, then mixed with 3.6% NaCl and centrifuged
for 10 min at 500g. Finally the cells were resuspended in approximately
5 ×10
6
cells per mL PBS.
Opsonisation of particles
Cells from a fresh culture of Saccharomyces cerevisiae were washed
and then put into a boiling water bath for 30 min. After washing in
saline they were resuspended at a concentration of 2 × 10
8
particles/mL
PBS. The opsonisation procedure was carried out by incubating a
mixture of 200mL human serum with 1.8mL of yeast suspension for
30min at 308C immediately prior to the experiment. A luminol stock
solution of 10
¹2
M in DMSO was prepared and was diluted to 10
¹4
M
in PBS prior to use.
Chemiluminescence assay
Chemiluminescence was performed as described previously [8,10] with
minor modifications. Briefly, 200mL of opsonised yeast cells, 450 mL
of phosphate-buffered saline (PBS), 200 mL luminol 10
¹4
M and 50mL
of water or different phycocyanin concentrations, were incubated for
10min in the measuring cuvette. Just before the assay, 100mLofan
isolated leukocyte suspension were added and the light intensity was
measured every 3 min at 378C. In a system without cells there was
no interaction between luminol and phycocyanin. The viability of
PMNLs after being exposed to the higher c-phycocyanin concentration
(incubated at 37 8C water bath for 40min) was assessed by the Trypan
blue exclusion test. The viability obtained was 98%.
Inhibition of damage to 2-deoxyribose
Evaluation of the inhibition of damage to 2-deoxyribose, measured as
formation of thiobarbituric acid-reactive material [11], was carried out
as an alternative measure of the hydroxyl radical scavenger capacity of
c-phycocyanin. Mixtures contained, in a final volume of 1.2 mL:
deoxyribose 2.8mM, KH
2
PO
4
/KOH buffer 15 mM pH 7.4, FeCl
3
20mM, EDTA 100mM, H
2
O
2
2.8mM and ascorbic acid 100mM.
FeCl
3
and ascorbate solutions were made up in bidistilled water just
before use. FeCl
3
and EDTA were premixed prior to addition to the
reaction mixture. Ascorbic acid was added in order to start the reaction.
Reaction mixtures were incubated at 378C for 1 h. After addition of
1mL of TBA 1% (w/v) in 0.05mM NaOH and 1mL of TCA 2.8%
in water, the mixture was heated at 1008C for 20min. The pink
chromogen that progressively developed was then measured at 532nm
after cooling, against appropriate blanks. The second-order rate
constants k
s
were calculated using the data obtained in the presence
of EDTA from the slope of a plot of 1/A
532nm
against the test compound
concentration.
37Vol. 47, 1998 C-Phycocyanin as antioxidant
m
Inhibition of liver microsomal lipid peroxidation induced by
Fe
þ2
-ascorbic acid
Rat liver microsomes were prepared by differential ultracentrifugation
as previously described [12] and stored at ¹80 8C until use. Protein was
assayed by the Lowry method [13].
The procedure was carried out as described [14]. The microsomes
(final concentration 1.3 mg protein/ml) were incubated at 378C in Tris
buffer 50mM pH7.4 before induction of lipid peroxidation with 10mM
FeSO
4
and freshly prepared 0.2 mM ascorbic acid. The reaction was
stopped by adding 0.3mL of the incubation mixture to 2mL of ice-cold
TBA-TCA-HCl-BHT.Afterheatingfor15minat808Candcentrifugation
for15minat 2000g,theabsorbanceat535nmwasdetermined.The TBA-
TCA-HCl solution was prepared by dissolving 41.6mg TBA/10mL
TCA (16.8% w/v in 0.125N HCl). To 10mL TBA-TCA-HCL, 1mL of
BHT (1.5mg/mL ethanol) was added.
All the spectrophotometric measurements were done in a Spekol
220 from Carl Zeiss (Jena, Germany).
GO-induced inflammation in mouse paw
The animal model used was described by Spillert et al. [15].
Phycocyanin (50, 100 or 200mg/Kg in saline) or DMSO (1 g/Kg), as
positive control, were administered orally and i.p, respectively, to male
OF1 mice. One hour later, the mice were injected in the right hind foot
with 50mL of physiological saline and in the left foot with 50 mLof
100U/mL GO. The animals were killed at 1.5h post injection, both hind
feet amputated at the tibiotarsal joint and each paw weighed. The
38 C. Romay et al. Inflamm. res.
Fig. 1. Effect of c-phycocyanin on chemiluminescence intensity
produced by alkoxy radicals generated by the reaction of tert-
butylhydroperoxide with ferrous ion. (A) Decrease in Chemilumines-
cence signal (mV) with increasing c-phycocyanin (X) or trolox (W)
concentration. Each point represents the mean of three determinations.
Vertical bars represent 6 1 SD. (B) Double quenching experiment
carried out to determine the chemiluminescence signal before (Io) and
after (I) adding increasing concentrations of c-phycocyanin in the
absence (X) and presence (W) of trolox (46ng/mL). Each point
represents the mean Io/I value obtained from three determinations.
Fig. 2. Effect of c-phycocyanin as scavenger of hydroxyl radicals
produced by the Fenton reaction. (A) Decrease in chemiluminescence
signal (mV) with increasing c-phycocyanin (X) or DMSO (W)
concentrations. Each point represents the mean of three determinations.
Vertical bars represent 6 1 SD.(B) Io/I vs c-phycocyanin concentration
plot obtained as in Figure 2 in the absence (X) and presence (W)of
DMSO (149mg/mL). Each point represents the mean Io/I value
obtained from three determinations.
m
difference in weight of hind paws of each animal was called the edema
index (EI).
Statistical analysis
One-way ANOVA followed by Duncan’s multiple comparison test
were used to calculate the significance of the differences between the
means. The IC
50
was calculated using a GraphPad InPlot software
(GraphPad Software Inc., version 4.03, 1992).
Results
Chemiluminescent measurements
The CL produced by the reaction of t-BOOH with ferrous
ion was used for evaluation of alkoxyl radical scavenging
capacity of c-phycocyanin. The results show that c-phycocya-
nin inhibited the CL in this system (Fig. 1A). A comparison
with trolox, a water soluble analogue of vitamin E, indicates
that 0.038 mg/mL of trolox causes approximately the same
effect as 76 mg/mL of c-phycocyanin in terms of 50%
inhibition of the CL produced in this system.
C-phycocyanin was evaluated as a scavenger of hydroxyl
radicals by determining the inhibition of CL produced by the
Fenton reaction with luminol. As shown in Figure 2A, the
chemiluminescence signal (mV) was inhibited by increas-
ing phycocyanin concentrations. In this system, 0.91 mg/mL
of phycocyanin caused the same inhibition (50%) as
0.125mg/mL of DMSO, which was used as control.
In the HX-XO chemiluminescence system (Fig. 3A),
phycocyanin inhibited the signal in a dose-dependent
manner. However, when the HX-XO reaction was used to
reduce nitroblue tetrazolium (NBT) dye, no inhibitory effect
was observed (data not shown). In the double quenching
experiments (Fig. 1B and 2B), the slope decreased when both
phycocyanin and the specific scavenger were added together,
indicating competition for the radical generated. This
behaviour was not observed in the O
¹
2
-generator system
(Fig. 3B) in which the slope is independent of the previous
addition of the specific scavenger (SOD) and is even
increased.
Effect of phycocyanin on the CL response of PMNLs
Human leukocytes stimulated with opsonized yeast cells
produced a typical time-dependent CL response with a
maximal intensity at approximately 16 min followed by a
decline. Table 1 shows the means of the areas under the
curve (AUC) obtained for each phycocyanin concentration
39Vol. 47, 1998 C-Phycocyanin as antioxidant
Fig. 3. Effect of various c-phycocyanin concentrations on chemilumi-
nescence produced in the luminol-XO-HX system. (A) Decrease in
chemiluminescence signal (mV) with increasing c-phycocyanin (X)or
SOD (W) concentrations. Each point represents the mean of three
determinations. Vertical bars represent 6 1 SD. (B) Io/I vs c-
phycocyanin concentration plot obtained as in Figure 2 in the absence
(X) and presence (W) of SOD (23ng/mL). Each point represents the
mean Io/I value from three determinations.
Table 1. Effect of phycocyanin on the chemiluminescence response of
human leukocytes.
AUC Inhibition %
X
¯
6 SD
Control 6662 6 221:3
a
Phycocyanin 1mg/mL 5800 6 246:5
b
12.9
2mg/mL 4647 6 227:0
c
30.2
3mg/mL 3841 6 296:5
d
42.3
Different letters: p<0.05 vs. each other, n ¼ 5.
Table 2. Effect of phycocyanin on GO-induced inflammation in the
mouse paw.
Treatment Edema index (g) Inhibition %
DMSO (1g/Kg) 0:045 6 0:007
a
64.05
Phycocyanin 50mg/Kg 0:110 6 0:010
b
4.43
100mg/Kg 0:080 6 0:007
c
35.46
200mg/Kg 0:061 6 0:009
d
51.05
Glucose oxidase 100 U/mL 0:125 6 0:008
b
Different letters: p<0.05 vs. each other, n ¼ 7 mice per group.
Phycocyanin was administered orally 1h before GO. The oedema was
measured 1.5 h after injection of GO into the paw. The oedema index
indicates the difference in weight between the hind paws.
m
and control. The CL signal was significantly reduced with
respect to control by increasing phycocyanin concentrations.
Significant statistical differences were also obtained among
phycocyanin concentrations.
Deoxyribose assay
In the deoxyribose assay, a more conventional method,
phycocyanin inhibited the deoxyribose damage in a
concentration-dependent fashion. The IC
50
calculated for
c-phycocyanin was 0.86mg/mL. In this system, a typical
competition profile was obtained (Fig. 4). This was suitable
for the calculation of a second-order rate constant of
3:56 ×10
11
M
¹1
S
¹1
, considering a molecular weight of
36700g/mol for c-phycocyanin monomer [16].
Microsomal lipid peroxidation inhibition
Addition of c-phycocyanin (8, 12, 20 mg/mL) to isolated
microsomes in the presence of Fe
þ2
-ascorbate, resulted in a
concentration dependent decrease in lipid peroxidation
(IC
50
¼ 12 mg/ml). Both the rate and the final extent of
lipid peroxidation were reduced by adding c-phycocyanin
(Fig. 5).
In the presence of iron alone, c-phycocyanin did not
cause lipid peroxidation, indicating that it does not have pro-
oxidant effect in this system. Furthermore, its inhibitory
effect was exerted during the incubation time, since it had
not effect when it was added with the TBA reagent.
GO-induced inflammation in mouse paw
Differences in weight between the hind paws of animals in
the various groups are shown in Table 2. Phycocyanin at
doses of 100 and 200mg/Kg orally was able to inhibit
significantly peroxide-induced inflammation in a dose
dependent fashion with a ED
50
of 170:3 6 1:62mg/Kg.
DMSO was used as a positive control and it also inhibited the
inflammatory response induced by GO in mouse paw.
Discussion
In this study, we have applied established in vitro and
in vivo assays in order to evaluate the antioxidant action of
c-phycocyanin. This natural product was able to scavenge
alkoxy and hydroxyl radicals. Two methods were used to
evaluate hydroxyl radical scavenging by c-phycocyanin
because hydroxyl radical is one of the most potent oxidizing
species and its extreme reactivity naturally poses problems
with regard to its detection [17]. In both methods, inhibition
was observed at relatively high concentrations of the
product.
Phycocyanin also quenched the CL signal generated in
the HX-XO system, but this effect can not be ascribed to O
¹
2
scavenging, as demonstrated by double-quenching and NBT
reduction assays. One possible explanation for this behaviour
is that phycocyanin quenches CL by binding to an inter-
mediate or co-oxidising species that may be involved in the
CL reaction [18].
The inhibitory effect observed on microsomal lipid
peroxidation most probably is due to a metal-binding
capacity of c-phycocyanin, since chain-breaking antioxi-
dants often introduce a lag period into the peroxidation
process, corresponding to the time taken for the antioxi-
dant to be consumed, whereas metal-binding antioxidants
will give a constant inhibition throughout the reaction
[19]. Another indication of such an action, is the ability of
c-phycocyanin to inhibit deoxyribose damage in a site-
specific manner (in the absence of EDTA) [19]. Further
experiments must be performed to obtain more evidence for
the ability of phycocyanin to chelate metal ions.
In the deoxyribose assay, a second-order rate constant
calculated for phycocyanin was similar to that obtained,
by the same method, for some non-steroidal anti-inflamma-
tory drugs, such as indomethacin and ibuprofen (1:8 ×10
10
M
¹1
S
¹1
) [20].
Chemiluminescence of PMNLs is the final result of
luminol oxidation by strong oxidants, such as oxygen
radicals and peroxides, emanating from enzymatic
reactions. For addition to the myeloperoxidase-H
2
O
2
-
halide system, the release of arachidonic acid by phospho-
lipase A
2
and of diacylglycerol and inositol trisphosphate by
phospholipase C, the metabolism of arachidonic acid by the
40 C. Romay et al. Inflamm. res.
Fig. 4. Plots of 1/A
532nm
against concentration of c-phycocyanin using
data obtained from deoxyribose oxidation in the presence (X)or
absence (W) of EDTA. Values for each point are means of three
experiments. Vertical bars represent 6 1 SD.
Fig. 5. Time course of microsomal non-enzymatic lipid peroxidation as
affected by various concentrations of c-phycocyanin: (X) no addition,
(W) 8mg/mL, (O) 12 mg/mL, (K) 20 mg/mL. Values for each point are
means of three determinations. Vertical bars represent 6 1 SD.
m
cyclooxygenase and lipooxygenase pathways, the activation
of membrane NADPH oxidase by diacylglycerol and
calcium mobilization by inositol trisphosphate are all able
to induce the CL reaction. Inhibition of any of these
mechanisms suppresses the CL response [21].
Phycocyanin was able to inhibit the LCL in a dose-
dependent fashion, most probably through its capacity to
scavengefreeradicals(OH
,H
2
O
2
,RO
)andperoxidesarising
during the respiratory burst of phagocytic cells. However, it is
also possible that phycocyanin could diminish CL signals in
other ways, e.g. by affecting enzymes involved in the
production of reactive oxygen species by activated phago-
cytes, NADPH oxidase and myeloperoxidase, or by interfer-
ing either with the binding of the stimulant or the arachidonic
acid metabolism pathway. In this regard, we have recent
evidence for inhibition by phycocyanin of LTB
4
release in an
animal model of inflammation (manuscript in preparation).
The peroxide-induced inflammatory response is a valu-
able in vivo model in order to test agents with potential
scavenging effect against H
2
O
2
and OH
. GO injected into
the mouse paw reacts with endogenous glucose and generates
H
2
O
2
which subsequently produces OH
radicals; both
together are responsible for tissue damage and for the
accompanying inflammatory changes [15]. Phycocyanin
reduced the edema produced by glucose oxidase in the
mouse paw. This anti-inflammatory effect must be due, at
least in part, to the scavenging of hydroxyl radicals, taking
into account the fact that DMSO, a well known scavenger of
OH
radicals, also inhibited the inflammatory response
induced by GO.
Currently, there is a consensus that much of the damage
induced by H
2
O
2
in vivo is due to its conversion to highly-
reactive oxidants, mainly OH
[19]. Therefore, the scaveng-
ing action of phycocyanin against OH is probably relevant to
its anti-inflammatory effects.
Very recently, research carried out in our laboratory has
confirmed the anti-inflammatory effects of phycocyanin in
other experimental models of inflammation such as cotton
pellet granuloma in the rat and TPA-induced inflammatory
response in the mouse ear (manuscript in preparation).
Finally, to our knowledge, this is the first time that both
antioxidant and anti-inflammatory properties have been
described for c-phycocyanin.
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41Vol. 47, 1998 C-Phycocyanin as antioxidant
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... It is well established that oxidative stress and inflammation are involved in the development of cardiovascular disease [7,29]. Phycocyanin, the main component of Spirulysat ® , has antioxidant activity as it is able to scavenge various radicals and inhibit lipid peroxidation [30,31] and has antiinflammatory properties [32,33]. We have shown that supplementation with Spirulysat ® , which contains a high amount of C-PC, significantly decreases the 24-h urinary isoprostanes concentration, suggesting a better redox balance. ...
... In the mice study, we observed a strong association between Spirulysat ® supplementation and antioxidant parameters, bile acid modification, impact on food intake and modulation of gene expression. Furthermore, numerous studies on human and animal models have reported the antioxidant effect of Arthrospira [22,35,36], and it was proved to be related essentially to C-PC [32,37]. It also appears that the antioxidant effect can also be linked to the presence of polysaccharides in the liquid extract isolated from Arthrospira platensis [38] and that these sulphated polysaccharides prevent many potential health risks, including cerebrovascular disease, cancer and chronic inflammation [39]. ...
... Using a higher dose of Spirulysat ® in hamsters for 12 weeks and in mice for 25 weeks, we did not observe deleterious effects on biological parameters or mortality [22,24]. No adverse effects are reported in previous studies using higher doses of phycocyanin, notably in mice [32,50] and in rats [51]. Thus, the safety profile of the products during the study can be considered as good. ...
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Lipid peroxidation is associated with the development of some pathologies, such as cardiovascular diseases. Reduction in oxidative stress by antioxidants, such as Arthrospira (formelySpirulina), helps improving this redox imbalance. The aim of the study was to evaluate the effect of the Arthrospira liquid extract “Spirulysat®” on oxidative markers—in particular, oxidized LDL (oxLDL)/total LDL cholesterol—and isoprostanes and to investigate its impact on lipid and glucose metabolism in the metabolic syndrome subject. A controlled, randomised, double-blind design was conducted in 40 subjects aged 18 to 65 years with metabolic syndrome after a daily intake of Spirulysat® or placebo for twelve weeks. Blood and urinary samples were collected at three visits (V1, V2, V3) in the two groups for parameters determination. Although the Spirulysat® group showed a decrease at all visits of the oxLDL/total cholesterol ratio, there was no significant difference compared to the placebo (p = 0.36). The urinary isoprostanes concentration in the Spirulysat® group was reduced (p = 0.014) at V3. Plasma triglycerides decreased at V3 (p = 0.003) and HDL-cholesterol increased (p = 0.031) at all visits with Spirulysat®. In conclusion, Spirulysat® did not change the oxidized LDL (oxLDL)/LDL ratio but decreased the urinary isoprostanes, plasma triglycerides and increased HDL cholesterol, suggesting a beneficial effect on metabolic syndrome.
... SP contains several vital antioxidant and anti-inflammatory compounds as mentioned above, such as chlorophyll, phycocyanin, and carotenoids (β-carotene). The antioxidant and anti-inflammatory properties of phycocyanin have been determined in numerous studies [28, [83][84][85][86][90][91][92][93][94][95][96][97]. Phycocyanin is responsible for reducing oxidative stress and NADPH oxidase [28]. ...
... It scavenges free radicals, such as alkoxy, hydroxyl, and peroxyl radicals, and decreases nitrite production and inducible nitric oxide synthase (iNOS) expression. Phycocyanin also inhibits liver microsomal lipid peroxidation [28, [83][84][85][86][90][91][92][93][94][95][96][97]. ...
... NNRTIs are associated with life-threatening skin reactions and toxic hepatitis [114], these conditions may be ameliorated by SP. Phycocyanin from SP can inhibit liver microsomal lipid peroxidation [28, [83][84][85][86][90][91][92][93][94][95][96][97], and hence reducing toxic hepatitis. Moreso, SP incorporated into skin creams showed promising results as an anti-inflammatory and a wound-healing agent [89]; this can be beneficial in the mitigation of NNRTI-induced skin reactions. ...
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The human immunodeficiency virus (HIV) is one of the most prevalent diseases globally. It is estimated that 37.7 million people are infected with HIV globally, and 8.2 million persons are infected with the virus in South Africa. The highly active antiretroviral therapy (HAART) involves combining various types of antiretroviral drugs that are dependent on the infected person's viral load. HAART helps regulate the viral load and prevents its associated symptoms from progressing into acquired immune deficiency syndrome (AIDS). Despite its success in prolonging HIV-infected patients' lifespans, the use of HAART promotes metabolic syndrome (MetS) through an inflammatory pathway, excess production of reactive oxygen species (ROS), and mitochondrial dysfunction. Interestingly, Spirulina platensis (SP), a blue-green microalgae commonly used as a traditional food by Mexican and African people, has been demonstrated to mitigate MetS by regulating oxidative and inflammatory pathways. SP is also a potent antioxidant that has been shown to exhibit immunological, anticancer, anti-inflammatory, anti-aging, antidiabetic, antibacterial, and antiviral properties. This review is aimed at highlighting the biochemical mechanism of SP with a focus on studies linking SP to the inhibition of HIV, inflammation, and oxidative stress. Further, we propose SP as a potential supplement for HIV-infected persons on lifelong HAART.
... NADPH oxidase (NOX) is the primary source of ROS because the overactivation of NOX causes the accumulation of ROS [39]. PCB can decrease diabetic vascular complications by degrading NOX and normalising urinary and renal oxidative stress markers [15,24,40]. In the treatment of acute liver failure (ALF), PCB decreases injuries to the liver structure under CCl 4 and protects the proliferation of liver cells with antioxidants [41]. ...
... Parkinson's disease (PD) results primarily from the death of dopaminergic neurons in the substantia nigra. Clinical characteristics include tremors at rest, Scavenge ROS and radicals [15,16] Anti-inflammation Phycocyanobilin Inhibit the expression of inflammation-related genes Downregulate the quantity level of proinflammatory mediator [15,17,18] Anticancer Phycocyanobilin Antiproliferative effect on cancer cells [19,20] Phycocyanin Induce cell apoptosis and cell cycle arrest Prevent cell migration and colony formation [21,22] Other Journal of Immunology Research rigidity, slowness, lack of voluntary movement, and postural instability freezing, which significantly interfere with patients' daily lives [46]. When the antioxidant ability of cells decreases, free radicals can cause severe damage and even death to dopamine-produced cells. ...
... Parkinson's disease (PD) results primarily from the death of dopaminergic neurons in the substantia nigra. Clinical characteristics include tremors at rest, Scavenge ROS and radicals [15,16] Anti-inflammation Phycocyanobilin Inhibit the expression of inflammation-related genes Downregulate the quantity level of proinflammatory mediator [15,17,18] Anticancer Phycocyanobilin Antiproliferative effect on cancer cells [19,20] Phycocyanin Induce cell apoptosis and cell cycle arrest Prevent cell migration and colony formation [21,22] Other Journal of Immunology Research rigidity, slowness, lack of voluntary movement, and postural instability freezing, which significantly interfere with patients' daily lives [46]. When the antioxidant ability of cells decreases, free radicals can cause severe damage and even death to dopamine-produced cells. ...
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Phycocyanobilin (PCB) is a linear open-chain tetrapyrrole chromophore that captures and senses light and a variety of biological activities, such as anti-oxidation, anti-cancer, and anti-inflammatory. In this paper, the biological activities of PCB are reviewed, and the related mechanism of PCB and its latest application in disease treatment are introduced. PCB can resist oxidation by scavenging free radicals, inhibiting the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and delaying the activity of antioxidant enzymes. In addition, PCB can also be used as an excellent anti-inflammatory agent to reduce the proinflammatory factors IL-6 and IFN-γ and to up-regulate the production of anti-inflammatory cytokine IL-10 by inhibiting the inflammatory signal pathways NF-κB and mitogen-activated protein kinase (MAPK). Due to the above biological activities of phycocyanobilin PCB, it is expected to become a new effective drug for treating various diseases, such as COVID-19 complications, atherosclerosis, multiple sclerosis (MS), and ischaemic stroke (IS).
... The antioxidant activity of PBP was firstly demonstrated by in vitro and in vivo assays by Romay et al. [63]. C-PC from Arthospira maxima was able to scavenge alkoxy radicals (RO•, IC50 = 76 mg/mL) and hydroxyl radicals (OH•, IC50 = 0:91 mg/mL). ...
... The relative antioxidant ratio and IC50 value indicated that PC is a more efficient ONOO-scavenger than PCB [68]. PC and PCB derived from Aphanizomenon flos-aquae was shown to have similar activities against peroxyl radicals, The antioxidant activity of PBP was firstly demonstrated by in vitro and in vivo assays by Romay et al. [63]. C-PC from Arthospira maxima was able to scavenge alkoxy radicals (RO•, IC 50 = 76 mg/mL) and hydroxyl radicals (OH•, IC 50 = 0:91 mg/mL). ...
Article
Full-text available
Phycobiliproteins (PBPs) are colored and water-soluble biliproteins found in cyanobacteria, rhodophytes, cryptomonads and cyanelles. They are divided into three main types: allophycocyanin, phycocyanin and phycoerythrin, according to their spectral properties. There are two methods for PBPs preparation. One is the extraction and purification of native PBPs from Cyanobacteria, Cryptophyta and Rhodophyta, and the other way is the production of recombinant PBPs by heterologous hosts. Apart from their function as light-harvesting antenna in photosynthesis, PBPs can be used as food colorants, nutraceuticals and fluorescent probes in immunofluorescence analysis. An increasing number of reports have revealed their pharmaceutical potentials such as antioxidant, anti-tumor, anti-inflammatory and antidiabetic effects. The advances in PBP biogenesis make it feasible to construct novel PBPs with various activities and produce recombinant PBPs by heterologous hosts at low cost. In this review, we present a critical overview on the productions, characterization and pharmaceutical potentials of PBPs, and discuss the key issues and future perspectives on the exploration of these valuable proteins.
... There is evidence that spirulina has an impact on the immune system by stimulating phagocytosis, modulating production of antibodies and cytokines. The photosynthetic pigment C-phycocyanin may modulate inflammatory response in a dose-dependent manner through its inhibitory effects on cyclooxygenase-2 activity and suppression of production of proinflammatory substances such as prostaglandin E2 and tumor necrosis factor alpha.284 Spirulina's protective and symptomatic relief role through inhibition of histamine release from mast cells in patients with allergic rhinitis has been confirmed in animal and human studies.283,285 It has also been noticed that spirulina may reduce inflammation in patients with rheumatoid arthritis through stimulation of secretion of interleukin-2.281 ...
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According to the new classification, periodontitis is defined as a chronic multifactorial inflammatory disease associated with dysbiotic biofilms and characterized by progressive destruction of the tooth‐supporting apparatus. This definition, based on the current scientific evidence, clearly indicates and emphasizes, beside the microbial component dental biofilm, the importance of the inflammatory reaction in the progressive destruction of periodontal tissues. The idea to modulate this inflammatory reaction in order to decrease or even cease the progressive destruction was, therefore, a logical consequence. Attempts to achieve this goal involve various kinds of anti‐inflammatory drugs or medications. However, there is also an increasing effort in using food supplements or so‐called natural food ingredients to modulate patients’ immune responses and maybe even improve the healing of periodontal tissues. The aim of this chapter of Periodontology 2000 is to review the evidence of various food supplements and ingredients regarding their possible effects on periodontal inflammation and wound healing. This review may help researchers and clinicians to evaluate the current evidence and to stimulate further research in this area.
... One of the most important natural antioxidant compounds present in cyanobacteria is its pigment, a tetrapyrrole linear derivative, along with a rich source of vitamins and minerals; however, their primary biological role is to do photosynthesis, thus contributing to the production of a major amount of biomass in the ecosystem (Romay et al., 1998(Romay et al., , 2003Romay and Gonzalez, 2000;Remirez et al., 2002;Eriksen, 2008;Thangam et al., 2013;Zhang et al., 2022). Among the functional components identified in cyanobacteria, natural pigments such as chlorophylls, carotenoids, and phycobilins have received attention due to their linear tetrapyrrolic structure. ...
Article
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Cyanobacteria have attracted the attention of researchers because of their promising role as primary and secondary metabolites in functional food and drug design. Due to an ever-increasing awareness of health and the use of natural products to avoid the onset of many chronic and lifestyle metabolic diseases, the global demand for the use of natural drugs and food additives has increased in the last few decades. There are several reports about the highly valuable cyanobacterial products such as carotenoids, vitamins, minerals, polysaccharides, and phycobiliproteins showing antioxidant, anti-cancerous, anti-inflammatory, hypoglycemic, and antimicrobial properties. Recently, it has been shown that allophycocyanin increases longevity and reduces the paralysis effect at least in Caenorhabditis elegans. Additionally, other pigments such as phycoerythrin and phycocyanin show antioxidative properties. Because of their high solubility in water and zero side effects, some of the cyanobacterial tetrapyrrole derivatives, i.e., pigments, facilitate an innovative and alternative way for the beverage and food industries in place of synthetic coloring agents at the commercial level. Thus, not only are the tetrapyrrole derivatives essential constituents for the synthesis of most of the basic physiological biomolecules, such as hemoglobin, chlorophyll, and cobalamin, but also have the potential to be used for the synthesis of synthetic compounds used in the pharmaceutical and nutraceutical industries. In the present review, we focused on the different aspects of tetrapyrrole rings in the drug design and food industries and addressed its remaining limitations to be used as natural nutrient supplements and therapeutic agents.
... The effect of AgNPs was more pronounced than that of the Spirulina treated groups. In vivo experimental models, mouse arthritis [42], mouse-ear edema [43], mouse-paw edema [44], and rat colitis [45] support our findings; hence, phycocyanin, a biliprotein obtained from the microalgae Spirulina (Arthospira) maxima, exerts anti-inflammatory activity by reducing LTB4 levels, TNF-α, NO, and arachidonic acid metabolites involved in the inflammatory response [46]. But others reported the opposite direction when LPS-activated macrophages and monocytes were treated with Spirulina [47]. ...
Article
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Prostate cancer (PCa) is the most diagnosed cancer in 112 countries and the second leading cause of death in men in 48 countries. We studied the outstanding agents silver nanoparticles (AgNPs) and Spirulina algae (Sp) for the management of PCa once as monotherapy or last as a combination. PCa in rats was induced using bicalutamide (Casodex®) and testosterone, followed by (7, 12-dimethylbenz[a]anthracene). Then, testosterone was injected s.c. for 3 months. Rats were divided into six groups, with 12 rats in each group. Group I was assigned as the control (co), group II as the PCa model, group III treated with AgNPs, group IV treated with Spirulina extract, group V treated with a combination of AgNPs plus Spirulina, and group VI treated with bicalutamide. The results show that AgNPs could normalize IL-6 levels and could overcome the hormonal disturbance induced in PCa rats along the hypothalamic–pituitary–testis axis. Spirulina revealed a significant reduction in the level of total and free prostatic specific antigen (PSA) to the same level as bicalutamide treatment, which was the same as the control group. Histopathological study revealed regression (75%) of the histological pattern of high-grade prostatic intraepithelial neoplasia (HGPIN) for Spirulina alone, and (50%) for bicalutamide. The best effect on IL-6 decline was reached with the AgNPs/Spirulina combination as well as bicalutamide treatment compared with the PCa group. Bicalutamide treatment significantly decreased the PSA concentration relative to the PCa group and reached the normal level. Adding Spirulina to AgNPs as a combination enhanced its effect on all mentioned drawbacks associated with PCa except hormonal imbalance that needs more adjustments.
Chapter
Seaweeds are considered one of the most potential yet renewable marine resources in the food, pharmacy, as well as cosmetic industries. A great deal of interest has been developed to extract bioproducts from seaweeds because of their numerous health-beneficial effects. Seaweed-derived bioproducts such as polysaccharides, fatty acids, natural pigments, proteins, and bioactive peptides exhibit many beneficial biological activities including antiviral, antioxidative, anticancer, brain development, neuroprotective, and immunomodulatory activities. However, in order to be used in pharmacological industries, green environmental-friendly technologies to refine bioproducts from seaweeds need to be developed. This contribution focuses on the development of bioproducts from seaweeds and elaborates the development of environmental-friendly technologies to extract bioproducts from seaweeds, their biological activities, health benefit effects, as well as potency in pharmacological industries.
Article
As the host defense response to various injuries and pathogens in the body, inflammation can remove damaged cells and pathogens in the host organism and protect the body. However, excessive inflammation may cause damage to normal tissue cells while removing pathogens, which in turn cause numerous inflammatory diseases and adversely affect the human health. Phycocyanin is an active substance extracted from Spirulina; it has outstanding antioxidant and anti-inflammatory activities, and can effectively inhibit various diseases caused by inflammation. This review systematically summarizes recent applications of phycocyanin against various inflammatory diseases in lung, liver, cardiovascular, and cerebrovascular systems. In addition, possible anti-inflammatory action pathways of phycocyanin are reviewed to canvass the anti-inflammatory mechanism. At last, based on the existing research, phycocyanobilin in phycocyanin is proposed as a bilirubin analog by inducing heme oxygenase 1 in vivo to suppress inflammation.
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Psoriasis is a chronic autoimmune disease caused by an abnormal proliferation of keratinocytes in the epidermis. The efficacy of C-Phycocyanin, a photosynthetic pigment isolated from Spirulina Maxima, was assessed via histological examinations, and anti-inflammatory properties were investigated in mouse models of imiquimod-induced psoriasis (BALB/C-nu and BALB/C). C-phycocyanin lowered epidermal thickening, as well as immune cell clustering in the dermis. Furthermore, C-phycocyanin modulated the levels of inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-6, cyclooxygenase-2, IL-1b) in the BALB/C-nu mouse model and psoriasis-related cytokines (IL-17a, interferon-gamma, calcitonin gene-related peptide) in BALB/c mice. We show that C-phycocyanin could be developed as a natural pharmaceutical against psoriasis.
Article
— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O2-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H2O2 could not be explored in this way, because superoxide dismutase is inactive under such conditions.
Article
Performing the deoxyribose (DR) assay for determination of the rate constants for reaction of non steroidal antiinflammatory drugs with hydroxyl radicals led to some unusual competition plots. The molecules from the arylpropionic family of drugs: ibuprofen, flurbiprofen, ketoprofen and naproxen produced the linear relationship. However, acemetacin, diclofenac Na, flufenamic acid, indometacin, niflumic acid, tolmetin Na and sulindac presented non linear competition plots manifesting at relatively low drug concentrations. This effect was corrected by increasing DR concentrations from 2.8 mM to 15 mM. The modification did not affect rate constants values for those derivatives which already presented a linear plot at 2.8 mM, but allowed to calculate rate constants for other compounds. It is suggested that the experimental conditions have to be adapted particularly for those derivatives with a relatively high. rate constant for reaction with the radical species. The oxicam derivatives (tenoxicam and piroxicam) presented another kind of deviation that revealed a prooxidant effect in this system: non linear plots were also observed at relatively low drug concentrations, but in the opposite direction than for the other molecules. This last effect was independent of DR concentration but could be corrected by increasing ascorbate concentration in the system.
Article
This chapter discusses the deoxyribose assay, and the practical application of the method. Iron ions might play a role in the deoxyribose assay via formation of other species, such as the ferryl or perferryl radical, whose properties are different from that of hydroxyl (•OH). However, no real evidence has been found that ferryl or the perferryl species exist in Fenton systems at physiological pH. Use of Fe(III)-EDTA in the presence of a reducing agent, such as ascorbate or the superoxide radical from the xanthine-hypoxanthine system mimics the conditions of ionizing radiation. The de-oxyribose assay has been adapted to assess the ability of food additives and nutrient components to act as pro-oxidants and hence mediate damage to the food matrix. The deoxyribose method remains an easy-to-use laboratory tool. When performed with an understanding that the inherent mechanisms involved are complex, the assay allows for (1) measuring rate constants for reactions with •OH and (2) assessing the iron-binding ability of the compound being tested.
1.1. Gel filtration measurements indicate that the minimum molecular weight of C-phycocyanin from Anacystis nidulans is about 30 000. Both the gel filtration and sedimentation data support the view that the molecule associates to form dimers, hexamers and dodecamers.2.2. The hexamer is the predominant component at low pH and high protein concentration. Dilution completely converts the hexamer to monomer at low pH and to dimer at high pH.3.3. The extinction coefficient of the protein at the absorption maximum is pH dependent; maximal absorption occurs at about pH 6.0. The absorption maximum shifts from 613 mμ at pH 3.5 to 622 mμ at pH 8.5.
Article
Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.