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Antioxidant activity of fulvic acid: A living matter-derived bioactive compound



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 (O 2•-), hypochlorous acid (HOCl), hydrogen peroxide (H 2O 2), hydroxyl radical (OH•), peroxynitrite (ONOO -) and singlet oxygen ( 1O 2) scavenging capacity of the fulvic acid synthesized from a compost elaborated with poultry manure by spectrophotometric methods. The IC 50 (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 O 2•-, HOCl, H 2O 2, OH•, ONOO- and 1O 2, 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 O 2•-, HOCl, H 2O 2, OH•, ONOO - and 1O 2, 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.
Journal of Food, Agriculture & Environment, Vol.9 (3&4), July-October 2011 123
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
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.
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.
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
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
Element Contents (%)
Organic matter 58.13
Nitrogen (NH
) 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
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
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 OHradicals 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 OHradical 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
(mg/ml) Reference compound IC
3.87±0.19 NDGA 0.003 ± 0.0003 *
HOCl 14.93±0.47 Ascorbic acid 0.0016 ± 0.0009 *
49.79±7.86 Pyruvate 1.3 ± 0.0708 *
48.59±2.12 DMTU 0.166 ± 0.0634 *
57.2±5.78 Penicillamine 0.0017 ± 0.00013 *
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
ONOOscavenging capacity (%)
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
50 30 10 90 100 110 70
025 50 75 100
1O2 scavenging capacity (%)
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
OHscavenging capacity (%)
Figure 4. Fulvic acid scavenges OHin 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 (%)
0 0.27
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 (%)
0 10
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
40 70 90 100 80 50 30 60 20
O2•– scavenging capacity (%)
0.00664 40.700000
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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... Fulvic acids are natural compounds with phenolic, acidic and other groups, and they are of great interest to medicine. Despite the fact that this group of substances has a different composition, depending on the source of extraction (silt, soil, peat, lignins, and others), they have common antioxidant [1][2][3][4][5][6][7], antitumor [8][9][10][11], antiviral [12][13][14], anti-inflammatory [3,12,[15][16][17][18] properties. ...
... The amount of the drug was determined by using the calibration curve generated from known concentrations of FA to calculate the percentage of the drug released according to Equation (1). ...
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The formulation of the transdermal patch with fulvic acid (FA) on an emulsion basis using pluronic Kolliphor® p237 as a permeability enhancer was developed and studied for anti-inflammatory properties. FA was isolated from the peat in the Nizhny Novgorod region of Russia and characterized as a potential active pharmaceutical ingredient. In vitro studies of the release of FA from the transdermal patch, as well as the FA penetration through the acetyl cellulose membrane using the Franz diffusion cell, showed its high efficiency (56% and 90%, respectively, in 8 h). In the in vivo experiment, qualitative and quantitative features of the rat knee caused by complete Freund’s adjuvant-induced arthritis (morphological changes, the FA influence on the biochemical indexes) were studied. The inflammatory process that developed within 15 days was accompanied by the activation of antioxidant oxidoreductase enzymes (by 50–70%), the increase in the cross-sectional diameter of the cartilage, and the increase in the values of marker indicators of the process of rheumatoid arthritis. Within 7 days of treatment, under the influence of FA, the values of ESR, RF, leukocytes, C-reactive protein, as well as the biochemical parameters characterizing oxidative stress (SOD, catalase, glutathione reductase, LDH, G6PD) normalized, and the edema reduced. These results may be useful for arthritis treatment using the transdermal patch with FA.
... Humic (HA) and fulvic (FA) acids being the components of natural sources such as silt, soil, peat, lignin, coal, and others, are promising biologically active substances for pharmacy and medicine. This group of supramolecular or polymeric compounds of various composition and structure exhibits powerful antioxidant [1][2][3][4][5][6][7], antitumor [8][9][10][11], hypoglycemic [12][13][14], antiviral [15][16][17][18], and anti-inflammatory properties [3,15,[19][20][21][22]. ...
Properties of fulvic acid (FA) extracted from the peat of the Nizhny Novgorod region of Russia by ultrasonic extraction with alkaline or ethanol solutions and further purification were studied in the paper. The purification of FA by the Lamar method was shown to possibly lead to the formation of FA polymorphs of the same composition but different structure (IR, solid-state 13С NMR, fluorescence, UV, energy-dispersive X-ray spectroscopy, SEM and optical microscopy, concentration of carboxyl and phenol groups). Aggregation of FA polymorphs in water led to the formation of nanoparticles with the average size of 8–10 nm and the zeta potential of –22 to –27 mV. The formation and properties of polymorphic supramolecular structures are greatly influenced by the drying mode. The solubility of the two FA polymorphs varied from freely soluble (3.3 mL of water per gram) after the FA solution freeze-drying to soluble (12 mL of water per gram) after the FA solution vacuum drying. The FA sample extracted by ethanol ultrasonic extraction from the peat, followed by convection drying, had a solubility of 666.7 mL of water per gram (Slightly soluble), a zeta potential of -6 mV, and a particle size of 15–20 nm.
... The main constituent of Andean Shilajit and BrainUp-10 ® is fulvic acid, a water-soluble compound endowed with potent antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects through the inhibition of tau protein aggregation [36][37][38]. According to our results, the fulvic acid present in both Andean Shilajit and the BrainUp-10 ® formula could be one of the bioactive compounds inducing neuritogenic and axogenic activity [22]. ...
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Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder without a cure, despite the enormous number of investigations and therapeutic approaches. AD is a consequence of microglial responses to “damage signals”, such as aggregated tau oligomers, which trigger a neuro-inflammatory reaction, promoting the misfolding of cytoskeleton structure. Since AD is the most prevalent cause of dementia in the elderly (>60 years old), new treatments are essential to improve the well-being of affected subjects. The pharmaceutical industry has not developed new drugs with efficacy for controlling AD. In this context, major attention has been given to nutraceuticals and novel bioactive compounds, such as molecules from the Andean Shilajit (AnSh), obtained from the Andes of Chile. Primary cultures of rat hippocampal neurons and mouse neuroblastoma cells were evaluated to examine the functional and neuroprotective role of different AnSh fractions. Our findings show that AnSh fractions increase the number and length of neuronal processes at a differential dose. All fractions were viable in neurons. The AnSh fractions inhibit tau self-aggregation after 10 days of treatment. Finally, we identified two candidate molecules in M3 fractions assayed by UPLC/MS. Our research points to a novel AnSh-derived fraction that is helpful in AD. Intensive work toward elucidation of the molecular mechanisms is being carried out. AnSh is an alternative for AD treatment or as a coadjuvant for an effective treatment
... The authors concluded that the rate of oxygen absorption was significantly reduced in the presence of HA, and at higher concentrations (10 g·L −1 ) the oxidation process was completely stopped. FAs show antioxidant activity, depending on their concentration, but are somewhat less effective than the mentioned reference compounds [40]. Radical-chain-initiated oxidation of 1,4-dioxane (I) under standard conditions was used to assess antioxidant activity by Avvakumova et al. [41]. ...
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Citation: Vašková, J.; Stupák, M.; Vidová Ugurbaş, M.; Žatko, D.; Vaško, L. Therapeutic Efficiency of Humic Acids in Intoxications. Life 2023, 13, 971. 10.3390/life13040971 Academic Editor: Ewa M. Urbańska Abstract: Humins, humic and fulvic acids represent molecules with complex structures. These compounds comprising humic substances (HS) exist naturally in soil, brown coal, peat, and water. They are formed during the decomposition and transformation of organic matter (animal and plant remains) and their formation explains several theories. Within their chemical structures, there are numerous phenolic and carboxyl groups and their derivatives that affect their different properties, such as their solubility in water or their absorption of cations or mycotoxins. The manifold chemical structure of HS alters their polyelectrolyte character and thus their chelating efficiency. For many years, HS have been studied due to their detoxification, anti-, and pro-inflammatory or anticancer and antiviral ability. This article summarizes the antioxidant and adsorption properties of humic acids, highlighting their usefulness in intoxications.
... In general, there are numerous advantages of using FA-Fe 3 O 4 NPs, which are as follows: the NPs subsume low cytotoxicity (no toxicity till 1000µg/mL), high level of cell protection, have the ability to pass the BBB, and inhibiting AD progression through overshadowing multifarious AD-related pathways simultaneously (in ammation, amyloid and tau aggregations, along with acidic pH), as the NPs can e ciently target the amyloid aggregations and hence prohibit the formation of tau aggregations (because that amyloid accumulation triggers tau deposition formation), furthermore, due to the alkaline nature of the FA-Fe 3 O 4 NPs (because in the nal step of synthesizing we raised the pH to 7.2), these NPs can also target the acidic pH of the brain, which is considered as one of the important factors that lead to amyloid aggregations [43][44][45][46][47]. Moreover, the magnetic nature of the iron oxide NPs facilitates their quick and easily evacuation through applying small magnetic eld, which itself lowers the level of Aβ plaques. ...
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Alzheimer’s Disease is a multi-factor malady, the main hallmarks of which are, extracellular amyloid-beta and intracellular tau protein aggregations, leading to a pathological cascade of events and ultimately neural death. With this in mind, most of the studies have been concentrated on eliminating the amyloid and tau aggregations. Fulvic acid is one of the polyphenolic compounds which exhibits strong anti-inflammation and anti-amyloidogenic activity. On the other hand, iron oxide nanoparticles exhibit anti-amyloid activity on their own, therefore, this study investigates the interactions between fulvic acid-coated iron oxide nanoparticles and the commonly used in-vitro model, lysozyme from chicken egg white, that forms the amyloid aggregation under acidic pH and appropriate heat. The average size of nanoparticles was 10.7±2.7nm. FESEM, XRD, and FTIR characterization confirmed that fulvic acid was coated onto the surface of the nanoparticles. The inhibitory effects of the fulvic acid coated iron oxide nanoparticles were verified by Thioflavin T assay, circular dichroism (CD), and FESEM analysis. Furthermore, the toxicity of the nanoparticles on the neuroblastoma SH-SY5Y human cell line was assessed through MTT assay. Our results indicate that fulvic acid-coated iron oxide nanoparticles can efficiently inhibit formation of amyloid aggregations while exhibiting no in-vitro toxicity. This data shed light on the anti-amyloid activity of combination of fulvic acid and iron oxide nanoparticles; paving the way for future drug development for treating Alzheimer’s Disease.
... In this study, FA increased the serum level of T-AOC and GSH-Px, suggesting FA may be able to protect laying hens against oxidation damage more effectively. It could be attributed to the antioxidant properties of FA (7). ...
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The purpose of this study was to appraise the effect of fulvic acid on production, biochemical indices, and gut microbiota of laying hens. A total of 252 Dawu Golden Phoenix laying hens (55-week-old) were allotted to two treatments randomly, each with six replicates and 21 hens per replicate, including the control group (CG) and fulvic acid (500 mg/kg) group (FA). The trial period was 8 weeks. Adding FA raised egg weight ( P = 0.03), shell-breaking strength ( P = 0.03), and reduced egg breaking rate ( P < 0.01), compared with CG. There was no difference in eggshell thickness and egg shape index between the two treatments; however, the FA group increased egg production by 1.45% and reduced the feed-to-egg ratio by 0.09. Moreover, dietary FA decreased the aspartate aminotransferase levels in serum ( P = 0.04), and glutathione peroxidase and total antioxidant capacity were increased ( P = 0.02 and 0.04, respectively). Despite this, the two groups had no differences in the alpha diversity indices (PD_whole trees, Shannon, Ace, Simpson, Chao1, and goods_coverage). Obviously, at the phylum level, the abundances of Firmicutes were improved ( P < 0.01), Actinobacteriota ( P < 0.01), and Proteobacteria ( P < 0.01) were reduced by dietary FA. Supplementation with FA could improve the abundances of Megamonas ( P < 0.01) and reduce Enterobacter ( P < 0.01) at the genus level. To sum up, this study showed the addition of 500 mg/kg FA may boost production and egg quality and modulate the cecal microflora abundance and serum biochemical indices of laying hens.
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The group of natural organic compounds referred to as humic substances are increasingly attracting attention both in Russia and globally. These compounds exhibit a fairly extensive range of biological properties, thus finding successful application in animal husbandry, agriculture and veterinary medicine. Fulvic acid represents one of the most prominent representatives of this group of substances. The chemical and biological properties of fulvic acid make it a promising candidate for application in traditional medicine and as a basis for the development of modern pharmacological preparations.
Numerous studies have been completed on the science of organic matter and humic substances (HS), including humic acids (HA) and fulvic acids (FA). This chapter discusses that issues related to the regulation of commercial HA and FA products. For soil microorganisms, HS would be utilized as a source of organic carbon and might serve as a functional group of the biosynthesis chains. HS had unique physical and chemical/biochemical properties that influenced living cell metabolism and enzyme activity. Product efficacy data from a range of literature have shown both scientifically and practically that HS, when applied at reasonable rates, are beneficial for soil, crops, and livestock. One of the most important applications of humic products is in the production of organic certified products for people. While the science of HS, HA, and FA is solid, and each has been proven effective for agricultural application, cautions and limitations must be taken into consideration.
Fulvic acids are highly beneficial molecules promoting several soil functions such as nutrient absorption, metal transportation and improving soil structure. These activities are assisted by their molecular structure upon interaction with soil medium. The present study describes the isolation of fulvic acids (FA) from mature marine fish waste and sugarcane bagasse co-compost using alkaline extraction method and studying its molecular and structural characteristics using spectroscopic and microscopic techniques. UV–visible spectroscopic analysis of fulvic acids revealed presence of absorption peak at 264 nm due to characteristics of double bonds of aromatic or unsaturated compounds. Fluorescence spectroscopic analysis of fulvic acids showed emission (455 nm) and excitation (395 nm) spectrum respectively. The photometric ratio (E4/E6) and fluorescence index (FI) confirmed high humification degree of FA. The low color co-ordinate values (L* and b*) measured by CIELAB ensured good maturity of the FAs present in the co-compost. Especially, Proton NMR spectroscopy detected resonance signals of aliphatic protons (0.82–2.0 ppm) and aromatic proton (7.5 ppm) in the molecular structure of FA. FTIR spectrum identified functional groups such as hydroxyl; aliphatic, amide I and polysaccharide and classified the FA as type II. Stereo microscopic examination visualized presence of light brown crystalline particles. Further, SEM analysis of the FA particles revealed presence of various shapes such as spherical; cubical; flakes and micro-aggregate structures. Therefore, it is concluded that spectroscopic and microscopic tools are reliable techniques to reveal the molecular and structural characteristics of FA isolated from co-compost for usage as an organic fertilizer.
The organic matter of soils, peats and waters consists of a mixture of plant and animal products in various stages of decomposition together with substances synthesized biologically and/or chemically from the breakdown products as well as microorganisms and small animals [3,50]. This organic matter is usually divided into two groups: (i) Nonhumic substances, and (ii) humic substances.
Composting enables a detailed evaluation of the humification process of various organic wastes within a short period of time (3 to 6 months). Humic substances (HS) constitute a large fraction of the organic matter (OM) in compost, and they are its most active fraction due to their effects on soil ecology, structure, fertility and metal complexes, and plant growth. The formation and properties of HS extracted from various composts such as municipal solid waste (MSW), grape marc (GM), composted separated cattle manure (CSM), sewage sludge (SS), wood compost (WC) and other organic wastes were studied. Degradative and non-degradative techniques (FTIR, DRIFT, 13C-NMR) were used to study the transformation of HS during composting of the various organic wastes. Time-course studies of composting some of these wastes showed an increase in the relative amount of humic acid (HA) (from 18% to 45% of OM in CSM compost and from 5% to 20% in MSW compost), whereas the formation rate of fulvic acid (FA) was inconsistent. A humification ratio (HR – the ratio of HA/FA) was used to evaluate compost maturity. Values of 0.9 to 3.4 and 3.0 to 9.2 were typical for immature and mature composts, respectively. Another humification index (HI) used to define compost maturity was calculated as the ratio between non-humified fraction (NHF) and the humified fraction (HA+FA). A HI decrease during composting represents the formation of HS. Elemental- and functional-group analyses indicated only minor differences between HA extracted from composts at various stages. Moreover, these values fell into a wide range, similar to that of soil HA. The 13C-NMR spectra of the HA exhibited strong bands representing aliphatic structures in various composts (50% of total C in CSM, 30% in MSW and 61% in GM) and a lower level of aromatic components (37% in CSM, 22% in MSW and 22% in GM). The FTIR spectra showed similar trends of strong aliphatic and carbohydrate components. Both techniques provided more qualitative information indicating that HA extracted from mature compost exhibits more aromatic structures and carboxyl groups and less carbohydrate components than that from immature compost. Studies on the effects of HS on plant growth showed stimulative effects. Typical response curves indicated enhanced growth with increasing HS concentration in solution, followed by decreases in growth at higher concentrations. In soils, the addition of composts was found to stimulate growth beyond that provided by mineral nutrients, presumably because of the effects of HS.
The complexes formed between either Cd ²⁺ or Pb ²⁺ and fulvic acid extracted from sewage sludge mixtures were investigated by potentiometric titration of K/H fulvate with either Cd(ClO 4 ) 2 or Pb(ClO 4 ) 2 at pH 5.0 and 25°C, in an ionic medium of 0.1 M KClO 4 . Formation functions were calculated from the titration data and analyzed by the Scatchard plot method. The results were similar to those obtained previously for sludge‐fulvic acid complexes of Ca ²⁺ and Cu ²⁺ , in that two classes of complexes were found for both Cd ²⁺ and Pb ²⁺ . The common logarithms of the measured conditional stability constants for the two types of complexes with sludge‐derived fulvic acid were hypothesized to be linearly correlated with the Misono softness parameter, which is a quantitative measure of the tendency to covalent bonding on the part of a metal cation. The resulting linear correlation equations had r ² ‐values ≥ 0.99 and, therefore, were used to predict conditional stability constants for complexes of sludge‐derived fulvic acid with Mg ²⁺ , Mn ²⁺ , Fe ²⁺ , Ni ²⁺ , and Zn ²⁺ , which cannot be investigated by the method of potentiometric titration.
The antiinflammatory activity of topically applied coal-derived fulvic acids (called oxifulvic acid) at 4.5% and 9% was compared with that of diclofene sodium at 1% and betamethasone at 0.1% in a murine model of contact hypersensitivity. Mice were sensitised with dinitrofluorobenzene and challenged 6 days later by application to the dorsal surface of the right ear. The inflamed ears of the mice were then treated topically, and the thickness of the ears was measured daily. Oxifulvic acid at both concentrations compared favourably with both diclofene sodium and betamethasone in suppressing the cutaneous inflammatory response. Oxifulvic acid possesses antiinflammatory properties and may be of clinical benefit in the treatment of inflammatory skin conditions in humans. Drug Dev. Res. 53:29–32, 2001. © 2001 Wiley-Liss, Inc.
Fulvic acid, which is derived from the decay of plants and animals, is being studied for its role in the transport and toxicity of metal ions in soil and water. It is discussed in relation to the origin of humic substances and its interactions with metal ions. Techniques for investigating complexes of fulvic acid and metal ions are presented. They are separation and nonseparation analyses which are applied to speciation problems. The applicability, advantages, and disadvantages of both methods are presented. Separation of free and complexed metal ions can be done by chromatography, or with membranes that exclude the metal-ion complexes. Chromatographic techniques include liquid chromatography by size exclusion. Nonseparation techniques include voltametry and potentiometry, as well as fluorescence. A comparison of methods for calculation of the conditional stability constant K for complexes containing fulvic acid and copper (II) or cadmium (II) is presented.
The composition and toxicity of the aqueous oxidation fraction of coal were investigated, to gain information concerning process safety measures and the utilization of the oxidation products as antibacterial agents. Coal was oxidized with oxygen at 180°C under constant pressure (4 MPa). Fractions of the crude filtrate obtained during this process were sublimed, distilled and extracted with ether and ethanol. Almost 50 different acids were identified in these samples using g.c. and g.c.-m.s. analyses. The following groups of acids could be distinguished: (1) all possible unsubstituted acids containing up to four carbon atoms: (2) higher carboxylic acids containing even numbers of carbon atoms, agreeing with occurrence in nature; (3) oxygenated straight-chain or branched acids in the hydroxy or keto form containing up to six carbon atoms; (4) dicarboxylic aliphatic acids containing up to six carbon atoms; and (5) benzoic acid and its monohydroxy derivatives and phthalic acid. No highly toxic compounds could be identified, most of the compounds being common physiological metabolites. Primary acute toxicity studies were carried out on rats, using the crude aqueous solution and the drum-dried product of this solution. Apart from local irritation caused by their acidic nature, neither of these fractions exhibited significant acute toxicity in the test animals.
Previous studies have indicated that the main fractions of humic substances (HS), gray humic acid (GHA), brown humic acid (BHA), and fulvic acid (FA), present different molecular patterns in water solution that are probably associated with specific structural features. However, the techniques used in these previous studies did not permit clarification of the principal qualitative characteristics of these structures. To study more in depth this subject several GHA, BHA, and FA have been analyzed through the complementary use of UV-visible and FTIR spectroscopy, (13)C NMR, thermogravimetry, and pyrolysis GC-MS. The results indicate that the studied humic fractions have different and distinctive structural features. Thus, large and nonpolar structural units (paraffins, olefins, terpenes) and aliphatic structures seem to accumulate in the gray fraction, whereas the smallest and more polar (furfural, phenols) and simpler structural units (sugar- and amino acid-related structures) are present in the fulvic one. BHA has a higher content in polycyclic aromatic moieties, S-containing compounds and aromatic structures, thus suggesting the presence of more condensed aromatic rings. Likewise, differences in both the presence of polar groups and the apparent molecular size explain the pattern of solubility as a function of pH and ionic strength (I) that defines each HS fraction. These results also indicate that the structural differences among the HS fractions are not only quantitative (the presence of the same type of structures differing in size and the concentration of functional groups) but also qualitative, because each fraction presented different and distinctive structural domains. These structural domains explain the molecular patterns associated with each HS fraction. Thus, the presence of smaller and more O-functionalized structural units including aromatic domains in FA explain their tendency to form molecular aggregates (hydrogen bridges, metal bridges, and hydrophobic interactions) in solution. This fact could also explain the presence of molecular aggregates in BHA, although to a lesser extent than in FA. Finally, the dominant aliphatic and less functionalized character of GHA may justify its lower tendency to form aggregates in solution at neutral and alkaline pH. Likewise, the results also indicate that the different structural domains associated with these fractions may be the consequence of diverse biosynthetic pathways involving different precursors.
Singlet oxygen (1O2) is a reactive oxygen species produced by dissolved organic matter (DOM) in sunlit waters. While the production of 1O2 by DOM has been studied, little is known on interactions between 1O2 and DOM. The central objective of this work was to quantify the rate constants of reaction and quenching of 1O2 with Suwannee River and Pony Lake fulvic acids, the terrestrial and microbial end-member reference aquatic humic substances of the International Humic Substance Society. Fulvic acids were reacted with 1O2 generated through visible light irradiation of Rose Bengal. Uptake of 1O2 by the fulvic acids was followed through changes in dissolved oxygen concentrations via membrane inlet mass spectrometry (MIMS). Results from multiple diagnostic tests for 1O2-processes in solution suggested that 64-70% of the observed uptake of oxygen by the fulvic acid solutions was due to reaction with 1O2; the remaining O2 uptake was likely due to non-1O2 processes initiated by the excited-state sensitizer. The rate constants of reaction (krxn) and physical quenching (kphys) with 1O2 were determined to be 2.6 x 10(5) M-C(-1) s(-1) and 2.7 x 10(5) M-C(1-) s(-1) (krxn) and 1.5 x 10(5) M-C(1-) s(-1) and 1.3 x 10(6) M-C(1-) s(-1) (kphys) for Suwannee River and Pony Lake fulvic acids, respectively. Results from this study demonstrated that1O2 reacts with microbially and terrestrially derived DOM at rate constants comparable to phenols, naphthols, or aromatic amines, on a per carbon basis.
Shilajit has been used traditionally in folk medicine for the treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding the physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, extracts of commercial Shilajit were fractionated using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are the products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, a strong correlation was found between the complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts.
Toxic free radicals can be produced by many reactions required for the maintenance of normal metabolism and the production of energy in the cell. The reactivity of both primary and secondary radicals with biomolecules and in whole tissue systems is of interest, not only because of their importance in radiobiology but also because of the role these species play in toxicity and various disorders. Oxidant stress is known to increase the production of free radicals. In the presence of metals, especially iron, these radicals are converted into more damaging species. Trace elements play an important role in many systems that have evolved to deal with free radicals. The dietary status of the cell can affect the preventative antioxidant constituents of the cell. The chain-breaking antioxidant status can clearly be influenced by the dietary content of substances such as vitamins E and C.