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Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011 123
www.world-food.net
Journal of Food, Agriculture & Environment Vol.9 (3&4): 123-127. 2011
WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net
Received 4 July 2011, accepted 10 September 2011.
Antioxidant activity of fulvic acid: A living matter-derived bioactive compound
Noemí Cárdenas Rodríguez 1*, Elvia Coballase Urrutia 1, Bernardino Huerta Gertrudis 1, José Pedraza
Chaverri 2 and Gerardo Barragán Mejía 1
1 Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, Insurgentes Sur 1300, Letra C, Col. Insurgentes-Cuicuilco, Del.
Coyoacán, 04530, Mexico D.F. 2 Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México.
*e-mail: noemicr2001@yahoo.com.mx
Abstract
Fulvic acid (FA) is a class of compound including humic substances together with humic acid and humin. It is formed through the degradation of
organic substances by chemical and biological process. FA consists of a mixture of closely related complex aromatic polymers with the presence of
aromatic rings, phenolic hydroxyl, ketone carbonyl, quinone carbonyl, carboxyl and alkoxyl groups. The possible application of coal-derived FA as
an antimicrobial and anti-inflammatory property has been reported. Actually, it is used as a soil supplement in agriculture and as a human nutritional
supplement. In this work, we examined, for the first time, the scavenging activity of biosynthesized fulvic acid in comparison with reference
compounds. It was evaluated the in vitro superoxide (O2•–), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), hydroxyl radical (OH•),
peroxynitrite (ONOO–) and singlet oxygen (1O2) scavenging capacity of the fulvic acid synthesized from a compost elaborated with poultry manure
by spectrophotometric methods. The IC50 (mg/ml) values were as follows: 4.97±0.02, 1.56±0.06, 1.39±0.03, 2.5±0.04, 5.73±0.05 and 3.54±0.03 for
O2•–, HOCl, H2O2, OH•, ONOO– and 1O2, respectively. FA displays a scavenging activity compared with the reference compounds although it was
less efficient than nordihydroguaiaretic acid (NDGA), ascorbic acid, pyruvate, dimethylthiourea (DMTU), penicillamine and glutathione (GSH) for
O2•–, HOCl, H2O2, OH•, ONOO– and 1O2, respectively. The antioxidant properties of the FA partially support the health beneficial properties of this
compound; and therefore, the FA is a good candidate to be used in pharmaceutical or food industries as an accessible source of natural antioxidants.
Key words: Fulvic acid, scavenging capacity, antioxidant properties.
Introduction
Fulvic acid (FA) is a class of compounds including humic
substances together with humic acid and humin. It is formed
through the degradation of such organic substances as dead
plants, microbes and animals by chemical and biological process.
FA is also found abundantly in peat, weathered coal and other
humified substances 1.
Chemical and spectroscopic analyses have revealed the
presence of aromatic rings and phenolic hydroxyls, ketone
carbonyl, quinone carbonyl, carboxyl and alkoxyl groups 1-3. Humic
substances are used in medicine and antimicrobial, anti-
inflammatory and antitumor agents, as liver stimulants, remedies
for gastric ulcer to stop bleeding and for the treatment for skin
burns 4.
FA has various useful effects due to its functional groups.
Studies on the physiological actions of FA exerted on the living
body are gradually being carried out. The possible application of
coal-derived FA as an antimicrobial and antioxidant substance
has been described and the inflammatory property of coal-derived
FA has been also reported 5-7. It has been used externally to treat
haematoma, phlebitis, desmorrhexis, myogelosis, arthrosis,
polyarthritis, osteoarthritis and osteochondrosis. Likewise, FA
has been taken orally as a therapy for gastritis, diarrhoea, stomach
ulcers, dysentery, colitis and diabetes mellitus 8. FA and humic
substances isolated from soil and water reservoirs have been
reported to stimulate neutrophil and lymphocyte immune function 1.
It is especially reactive with metals, forming strong complexes
with Fe3+, Al3+ and Cu2+ 9, 10. FA and its related compounds have
no toxic compounds 11. Humic and FA are commonly used as a soil
supplement in agriculture and as a human nutritional supplement.
ROS contribute to the development of various diseases such
as atherosclerosis, diabetes, cancer, neurodegenerative diseases,
liver cirrhosis and ageing process 12. To prevent the damage caused
by ROS, tissues had developed an antioxidant defence system
that includes nonezymatic antioxidants (e.g., glutathione, uric acid,
bilirubin and vitamins C and E) and enzymatic activities such as
superoxide dismutase (SOD), catalase (CAT) and glutathione
peroxidase (GPx) 13. A second level of prevention against ROS-
induced damage is constituted by scavenging compounds, which
are able to reduce the incidence of free radical-mediated
diseases 13.
The use of antioxidants, both natural and synthetic, in the
prevention and cure of various diseases is expanding. There is a
considerable interest in the antioxidant activities of molecules
such as vitamins E and C and plant polyphenolic and carotenoid
components 14. In this sense, FA displays activity against
superoxide and hydroxyl radicals 15.
Despite the broad spectrum use of FA for a variety of medical
conditions, far less is known regarding the mechanisms of action
124 Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011
of FA. Therefore, the objective of this work was to evaluate, for
the first time, the in vitro superoxide (O2•–), hypochlorous acid
(HOCl), hydrogen peroxide (H2O2), hydroxyl radical (OH•),
peroxynitrite (ONOO–) and singlet oxygen (1O2) scavenging
capacity of the FA derived from a compost elaborated with poultry
manure (Miyamonte, Mexico) by established spectrophotometric
methods.
Materials and Methods
Chemicals: Sodium pyruvate, dimethyl thiourea (DMTU),
nordihydroguaiaretic acid (NDGA), ascorbic acid, histidine, xylenol
orange, butylated hydroxytoluene, ammonium iron (II), sulphate
hexahydrate, 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid
(ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), bovine serum
albumin (BSA), dimethyl sulphoxide (DMSO), NN-dimethyl-4-
nitrosoaniline (DMNA), catalase, xanthine, xanthine oxidase,
nitroblue tetrazolium (NBT), potassium nitrite (KNO2), manganese
dioxide (MnO2), diethylene triamine pentaacetic acid (DTPA),
butylated hydroxytoluene (BHT) and DL-penicillamine were
purchased to Sigma Aldrich (St. Louis, MO, USA), and 2,7-
dichlorodihydrofluorescein diacetate (DCF) and dihydrorhodamine
123 (DHR-123) were obtained from Cayman Chemical (Ann Arbor,
MI, USA). Potassium persulphate and sodium carbonate (Na2CO3)
were from Mallinckrodt (Paris, KY, USA). Absolute ethanol,
hydrogen peroxide (H2O2), sulphuric acid (H2SO4), methanol,
ethylenediamine-tetraacetic acid disodium salt (EDTA), sodium
hypochlorite (NaOCl) and sodium nitrite (NaNO2) were purchased
to JT Baker (Mexico City, Mexico). All other chemicals were reagent
grade and commercially available.
Preparation of fulvic acid: FA was obtained from a compost
elaborated with poultry manure, its characteristics are described
in Table 1. It was prepared by Miyamonte México S.A. de C.V.
FA was extracted from the compost following this process in
brief: a suspension was elaborated with 1 M NaOH, followed a
precipitation with 10% H2SO4. The obtained solution was
centrifuged to precipitate impurities and eliminate them. The
solution was placed on NH4OH (pH 2 to 7) in a roto-evaporator to
solidify the FA (greyish powder). Finally, it was dried in an oven
at 60ºC 16.
Determination of O2•– scavenging capacity: Xanthine-xanthine
oxidase system was used to determine the O2•– scavenging
capacity of the FA. O2•– in the assay system and xanthine oxidase
activity were measured as NBT reduction using a DU-64O series
Beckman spectrophotometer. This system is useful to test the
O2•– scavenging capacity only when the samples used do not
interfere with the xanthine oxidase activity. A compound with O2•–
scavenging capacity should decrease NBT reduction without
interfering with the xanthine oxidase activity measured as uric
acid production. Eight hundred µl of the following reaction mixture:
90 µM xanthine, 16 mM Na2CO3, 22.8 µM NBT and 18 mM
phosphate buffer (pH 7.0) were mixed with 100 µl of different
concentrations of FA. The reaction was started by the addition of
100 µl of xanthine oxidase (168 U/litre). Optical density was
registered both at 295 nm (for uric acid production) and 560 nm
(for O2•– in the assay system) 17. Scavenging percent was obtained
from the optical densities at 560 nm. NDGA was used as standard
for O2•– scavenging in this assay.
HOCl scavenging assay: The catalase assay involves a spectral
analysis of the enzyme. A spectrum (200-500 nm) of catalase,
catalase treated with HOCl and catalase containing varied mixtures
of HOCl treated with increasing concentrations of FA or the
reference compound was obtained. The HOCl scavenging
capacity of FA or the reference antioxidant was evident by the
inability of HOCl to eliminate/decrease the peak in a concentration-
dependent way. Experiments were carried out essentially as
described before 18. A solution of 49.8 µM bovine liver catalase
(16.6 µM, final concentration) was mixed with 18 mM HOCl (6 mM,
final concentration) in the presence of increasing concentrations
of FA or the reference compound.
Spectra (370-450 nm) of catalase alone, catalase plus HOCl,
catalase plus HOCl and the FA or the reference compound were
registered and the optical densities (OD) at 404 nm were obtained.
The value of the OD of catalase alone minus the OD of catalase
plus HOCl was considered as 100% of degradation of catalase (or
0% of scavenging activity), and the difference of the catalase
alone minus the OD of the catalase plus HOCl in presence of
either FA or reference compound was compared against this
value18. The ability of FA to scavenge HOCl was compared with
that of ascorbic acid.
Determination of H2O2 by the ferrous ion oxidation-xylenol
orange (FOX) assay: A solution of 75 mM H2O2 was mixed (1:1 v/
v) with water (0% scavenging tube) or with different concentrations
of FA and incubated for 30 min at room temperature. After this,
H2O2 was measured by the following method: briefly, 9 volumes of
4.4 mM BHT in HPLC-grade methanol were mixed with one volume
of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulphate
in 0.25 M H2SO4 to give the working FOX reagent.Of the extract
solutions 45 µl and 45 µl of 75 µM H2O2 were dispensed in 1.5 ml
Eppendorf tubes and mixed with 10 µl of HPLC-grade methanol
immediately followed by the addition of 0.9 ml of FOX reagent.
Solution was mixed on a Vortex mixer for 5 s and incubated at room
temperature for 10 min. The tubes were centrifuged for 15,000×g
for 10 min and absorbance at 560 nm was read against a methanol
blank. The concentration of H2O2 was calculated from a standard
curve prepared with increasing H2O2 concentrations. Pyruvate
was used as standard for H2O2 scavenging activity 18.
Table 1. Characteristics of a compost elaborated with
poultry manure by Miyamonte México S.A. de
C.V.
Element Contents (%)
Organic matter 58.13
Nitrogen (NH
3
) 23.57
Amoniacal nitrogen 0.2836
Nitric oxide 0.062
Phosphates 2.66
Calcium 8.08
Potassium 2.61
Magnesium 0.59
Zinc 0.024
Iron 0.36
Aluminium 0.059
pH 6.5-7.5
Humidity 15-20
Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011 125
Determination of OH• scavenging capacity: The ability of FA to
scavenge OH• was conducted in the Fe3+-EDTA-H2O2 -deoxyribose
system 19. The reaction mixture containing deoxyribose (0.056 mM),
H2O2 (1 mM), potassium phosphate buffer (10 mM, pH 7.4), FeCl3
(0.2 mM), EDTA (0.2 mM) and ascorbic acid (0.2 mM) was
incubated in a water bath at 37±0.5°C for 1h. The extent of the
deoxyribose degradation by the OH• formed was measured directly
in the aqueous FA phase by the thiobarbituric acid test at 532 nm.
The ability of FA to scavenge OH• was compared with that of
DMTU.
Synthesis of ONOO–: ONOO– was synthesized as previously
described 17. Five ml of an acidic solution (0.6 M HCl) of H2O2 (0.7
M) was mixed with 5 ml of 0.6 M KNO2 on an ice bath for 1 s and
the reaction was quenched with 5 ml of ice-cold 1.2 M NaOH.
Residual H2O2 was removed using prewashed granular MnO2 and
the reaction mixture was then left overnight at -20°C. The resulting
yellow liquid layer on the top of the frozen mixture was collected
for the experiment.
ONOO– scavenging assay: The ONOO– mediated oxidation of
DHR-123 was performed as described before 18. A 50 mM solution
of DHR123 was prepared from a 28 mM stock solution in DMSO.
The solution was maintained protected from light at 4°C during
the assay. All reaction mixtures contained 5 mM DHR-123, 0.1 mM
DTPA, different concentrations of extracts and 25 mM ONOO–.
Optical density was registered at 500 nm, the optical density of a
mixture without sample was considered as 100% and the optical
densities of the mixtures containing the FA were compared against
it. The ability of the tested FA to scavenge ONOO– was compared
with that of penicillamine.
1O2 assay: The production of 1O2 by NaOCl and H2O2 was
determined using DMNA as selective acceptor of 1O2 as reported
elsewhere with minor modifications 18. The bleaching of DMNA
was monitored spectrophotometrically at 440 nm. The assay
mixture contained 45 mM Na-phosphate buffer (pH 7.1), 10 mM
histidine, 10 mM NaOCl, 10 mM H2O2, 50 µM DMNA and 0.1 ml of
FA. The total volume of reaction (2.0 ml) was incubated at 30°C for
40 min. The extent of 1O2 production was determined by measuring
the decrease in the absorbance of DMNA at 440 nm. The relative
scavenging efficiency (percentage of inhibition of 1O2 production)
of FA was estimated from the difference in absorbance of DMNA
with and without the addition of FA, being tested or reference
compound. Glutathione was used as standard for 1O2 scavenging.
Statistical analysis: Data are expressed as mean±SD. The data
were compared against the blank tube without FA or the reference
compounds using student t test. (GraphPad Prism 4.0 Software,
San Diego, CA, USA). P<0.05 was considered statistically
significant. The scavenging capacity was expressed as the 50%
inhibitory concentration value (IC50), which denotes the
concentration of FA or the reference compounds required to give
a 50% reduction in oxidating effect relative to the blank tube.
Results and Discussion
This is the first time that IC50 values of FA for HOCl, ONOO–, H2O2,
1O2 and OH• are described. The FA, as well as the reference
compounds, scavenged O2•–, HOCl, H2O2, ONOO–, 1O2, and OH•
in a concentration-dependent way (Figs. 1-6). The IC50 values,
calculated from the linear portion of the dose-response curve, are
shown in Table 2. The analysis of the IC50 values indicated that
FA displays a scavenging activity compared to the reference
compounds; although it was less efficient than NDGA, ascorbic
acid, pyruvate, DMTU, penicillamine and GSH for O2•–, HOCl, H2O2,
OH•, ONOO– and 1O2, respectively (P<0.0001).
FA is a class of compounds consisting of complex polymeric
aromatic structures. It is formed through environmental
degradation of animal, plant, fungal and bacterial biopolymers20-
22. The FA act as an antioxidant like other high molecular weight
plant phenolics such as tannins 7. In the present paper, we have
shown that FA in vitro scavenged of O2•–, HOCl, H2O2, OH•, ONOO–
and 1O2 in a concentration-dependent way. These specific
scavenging properties of FA contribute to explain their antioxidant
properties 7. The ability of FA to scavenge the above mentioned
reactive species was compared with reference compounds with
the purpose to know the relative efficacy of FA to scavenge these
species.
To our knowledge this is the first time that IC50 values of FA for
O2•–, HOCl, H2O2, OH•, ONOO– and 1O2 are described. Based on
these comparisons, FA is less effective than the reference
compounds to cope with all species studied. In this context, it
has been demonstrated the scavenging activity of four FA (named
XWCS-1, XWCS-4, XWCS-8 and XWCFA), obtained by
ozonolysis of humic acid extracted from Xinjiang (China) weathered
coal, for O2•– and OH• radicals investigated with an electron spin
resonance (ESR)-spin trapping method 7 and quenching 1O2
generated through visible light irradiation of Rose Bengal 23. FA
also reduced OH• radical formation, rate and time dependent, in
aqueous iron-hydrogen peroxide reaction 24.
The hydrophobicity of the antioxidants plays a role in the
efficacy of inhibition. The presence of structural units O-
functionalized, including aromatic domains in FA, could explain
their tendency to form molecular aggregates (hydrogen bridges,
metal bridges and hydrophobic interactions) in solution 2, 3, 7.
Moreover, it has been known that phenolic hydroxyl group is the
main active group which scavenges OH• (this effect can be
primarily attributed to the hydrogen donation and electron transfer
capacities of OH group) and favours the encapsulation of the pro-
oxidant iron species, which generates OH• through the Fenton
reaction 25-27. This suggests that the phenolic hydroxyl group and
metal-chelating ability by FA could explain the ROS scavenging
activity observed.
Data are presented as mean ± SD of six independent assays.
*P<0.0001 vs FA.
Fulvic acid
Species IC
50
(mg/ml) Reference compound IC
50
(mg/ml)
O
2
Ɣ-
3.87±0.19 NDGA 0.003 ± 0.0003 *
HOCl 14.93±0.47 Ascorbic acid 0.0016 ± 0.0009 *
H
2
O
2
49.79±7.86 Pyruvate 1.3 ± 0.0708 *
OH
x
48.59±2.12 DMTU 0.166 ± 0.0634 *
ONOO
-
57.2±5.78 Penicillamine 0.0017 ± 0.00013 *
1
O
2
32.56±2.22 GSH 0.75 ± 0.0732 *
Table 2. Scavenging capacity of FA and reference compounds.
126 Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011
ONOO– scavenging capacity (%)
mg/ml
0
25
50
75
100
0
Figure 5. Fulvic acid scavenges ONOO– in a concentration-dependent
way; () reference compound: penicillamine and () FA. Data are
mean±SD, n = 6 assays. P<0.0001 vs reference compound.
40 60 80 20
010 20 30 40 50 60 70 80 90 100 110
0
5
0
5
0
50 30 10 90 100 110 70
025 50 75 100
0
5
0
5
0
1O2 scavenging capacity (%)
mg/ml
0
25
50
75
100
0
Figure 6. Fulvic acid scavenges 1O2 in a concentration-dependent way;
() reference compound: GSH and () FA. Data are mean±SD, n = 6
assays. P<0.0001 vs reference compound.
50 75 100 25
020 40 60 80
0
5
0
5
0
OH• scavenging capacity (%)
mg/ml
0
25
50
75
100
0
Figure 4. Fulvic acid scavenges OH• in a concentration-dependent
way; () reference compound: DMTU and () FA. Data are mean±SD,
n = 6 assays. P<0.0001 vs reference compound.
40 60 80 20
H2O2 scavenging capacity (%)
mg/ml
0 0.27
25
50
75
100
0
Figure 3. Fulvic acid scavenges H2O2 in a concentration-dependent
way; () reference compound: pyruvate and () FA. Data are mean±SD,
n = 6 assays. P<0.0001 vs reference compound.
2.2 20.37 86.0 40.70 9.5 1.1 10.8 0.55 5.5
HOCl scavenging capacity (%)
mg/ml
0 10
25
50
75
100
0
Figure 2. Fulvic acid scavenges HOCl in a concentration-dependent
way; () reference compound: ascorbic acid and () FA. Data are
mean±SD, n = 6 assays. P<0.0001 vs reference compound.
010 20 30 40 060 080 90 100
0
5
0
5
0
40 70 90 100 80 50 30 60 20
0
5
0
5
0
O2•– scavenging capacity (%)
mg/mL
0.00664 40.700000
25
50
75
100
0
Figure 1. Fulvic acid scavenges O2•– in a concentration-dependent
way; () reference compound: NDGA and () FA. Data are mean±SD,
n = 3 assays. P<0.001 vs 0 g/ml.
Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011 127
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