Abstract
Melanin extracted from Auricularia auricula fruiting bodies (AAFB) was examined by element analyzer, amino acid analyzer, inductively coupled plasma-optical emission spectrometry. Elemental composition analysis revealed that main component of AAFB melanin was pheomelanin. Amino acid analysis showed that 16 amino acids were found in AAFB melanin and total amino acid content was 321. 63 mg/g. There were 13 detectable metal elements in AAFB melanin, which was rich in Ca, Fe, Cu and Zn. In addition, AAFB melanin exhibited stronger scavenging activities on 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical, superoxide radical and hydroxyl radical with IC50 values of 0.18, 0.59 and 0.34 mg/mL, respectively. These results indicated that AAFB melanin might be potentially used as a natural antioxidant.
Auricularia auricula ; melanin; chemical composition; radical scavenging activity
1 Introduction
It is well known that active free radicals are by-products of normal metabolism which
induce oxidative damage to biomacromolecules, including DNA, proteins, membrane
lipids and carbohydrates (Wiseman & Halliwell,
1996Wiseman, H., & Halliwell, B. (1996). Damage to DNA by reactive
oxygen and nitrogen species: role in inflammatory disease and progression to
cancer. The Biochemical Journal, 313(Pt 1), 17-29.
PMid:8546679.). In order to reduce the oxidative damage of active free radicals,
many synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated
hydroxyanisole (BHA) with a strong antioxidant capacity are widely used in food
industry. However, frequent discovery of potential harmful effects of synthetic
antioxidants on human health, such as liver damage and carcinogenesis (Sun & Kennedy, 2010Sun, Y. X., & Kennedy, J. F. (2010). Antioxidant activities of
different polysaccharide conjugates (CRPs) isolated from the fruiting bodies of
. Chroogomphis rutilus (Schaeff.: Fr.) O. K. MillerCarbohydrate
Polymers, 82(2), 510-514.
http://dx.doi.org/10.1016/j.carbpol.2010.05.010.
http://dx.doi.org/10.1016/j.carbpol.2010...
; Yuan et al., 2008Yuan, J. F., Zhang, Z. Q., Fan, Z. C., & Yang, J. X. (2008).
Antioxidant effects and cytotoxicity of three purified polysaccharides from .
Ligusticum chuanxiong HortCarbohydrate Polymers, 74(2),
822-827. http://dx.doi.org/10.1016/j.carbpol.2008.04.040.
http://dx.doi.org/10.1016/j.carbpol.2008...
), has led to public interest in natural
antioxidants as an alternative. Therefore, some natural and safe antioxidants,
including ascorbate, tocopherols, flavonoids and phenolic compounds from plants and
microorganisms, has been developed and used in food processing to improve body’s
antioxidant defenses and reduce the oxidative stress to human body.
Melanin is a dark-colored polyphenolic pigment produced from oxidative polymerization
of phenolic or indolic compounds by tyrosinase. These natural pigments are
synthesized by some fungi, plants, animals and several bacterial species (Dalfard et al., 2006Dalfard, A. B., Khajeh, K., Soudi, M. R., Naderi-Manesh, H.,
Ranjbar, B., & Sajedi, R. H. (2006). Isolation and biochemical
characterization of laccase and tyrosinase activities in a novel melanogenic
soil bacterium. Enzyme and Microbial Technology, 39(7), 1409-1416.
http://dx.doi.org/10.1016/j.enzmictec.2006.03.029.
http://dx.doi.org/10.1016/j.enzmictec.20...
). It is now well
documented that natural pigments contain both melanin (different kinds of monomer
units that are connected through carbon-carbon bonds) and melanoproteins which are
important in defining the assembly of melanins (Wakamatsu & Ito, 2002Wakamatsu, K., & Ito, S. (2002). Advanced chemical methods in
melanin determination. Pigment Cell Research, 15(3), 174-183.
http://dx.doi.org/10.1034/j.1600-0749.2002.02017.x.
PMid:12028581
http://dx.doi.org/10.1034/j.1600-0749.20...
). In recent studies (Liu et al., 2005Liu, Y., Hong, L., Wakamatsu, K., Ito, S., Adhyaru, B., Cheng, C.
Y., Bowers, C. R., & Simon, J. D. (2005). Comparison of structural and
chemical properties of black and red human hair melanosomes. Photochemistry and
Photobiology, 81(1), 135-144. http://dx.doi.org/10.1562/2004-08-03-RA-259.1.
PMid:15504086
http://dx.doi.org/10.1562/2004-08-03-RA-...
; Tu et al.,
2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
), protein is considered to be an important component in melanins and
can affect the function of the assembled pigment. Melanins from different sources
possess a number of healthful functions, such as antioxidation (Tu et al., 2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
; Wu et al., 2008Wu, Y., Shan, L., Yang, S., & Ma, A. (2008). Identification and
antioxidant activity of melanin isolated from Hypoxylon archeri, a companion
fungus of Tremella fuciformis. Journal of Basic Microbiology, 48(3), 217-221.
http://dx.doi.org/10.1002/jobm.200700366. PMid:18506908
http://dx.doi.org/10.1002/jobm.200700366...
), anti-HIV activity (Manning et al., 2003Manning, J. T., Bundred, P. E., & Henzi, P. (2003). Melanin and
HIV in sub-Saharan Africa. Journal of Theoretical Biology, 223(1), 131-133.
http://dx.doi.org/10.1016/S0022-5193(03)00070-5. PMid:12782121
http://dx.doi.org/10.1016/S0022-5193(03)...
; Montefiori & Zhou, 1991Montefiori, D. C., & Zhou, J. Y. (1991). Selective antiviral
activity of synthetic soluble L-tyrosine and L-dopa melanins against human
immunodeficiency virus in vitro. Antiviral Research, 15(1), 11-25.
http://dx.doi.org/10.1016/0166-3542(91)90037-R. PMid:1709802
http://dx.doi.org/10.1016/0166-3542(91)9...
), and immunomodulatory activity (Sava et al., 2001Sava, V. M., Galkin, B. N., Hong, M. Y., Yang, P. C., & Huang,
G. S. (2001). A novel melanin-like pigment derived from black tea leaves with
immuno-stimulating activity. Food Research International, 34(4), 337-343.
http://dx.doi.org/10.1016/S0963-9969(00)00173-3.
http://dx.doi.org/10.1016/S0963-9969(00)...
). These functions promise
natural melanin with great development potential as a healthful food colorant.
Auricularia auricula (A. auricula)
is a precious macro-fungus distributed in the Northeast Provinces of China and has
been used as food and drug in China for a long time. Fruiting bodies of
A. auricula, a kind of edible black-brown
mushroom, are rich in nontoxic melanin and are increasingly popular as a “black
food” in China (Zou et al., 2010Zou, Y., Xie, C., Fang, G., Gu, Z., & Han, Y. (2010).
Optimization of ultrasound-assisted extraction of melanin from
Auricularia auricula fruit bodies. Innovative Food Science
& Emerging Technologies, 11(4), 611-615.
http://dx.doi.org/10.1016/j.ifset.2010.07.002.
http://dx.doi.org/10.1016/j.ifset.2010.0...
). Melanin
is considered to be one of the most important functional components in these “black
food”. However, most of this precious macro-fungus product is only used as cuisine
materials, and many of its functional components are not fully developed and
employed.
In previous research, one melanin had been successfully extracted from
A. auricula fruiting bodies (AAFB) using
ultrasound-assisted extraction technology (Zou et
al., 2010Zou, Y., Xie, C., Fang, G., Gu, Z., & Han, Y. (2010).
Optimization of ultrasound-assisted extraction of melanin from
Auricularia auricula fruit bodies. Innovative Food Science
& Emerging Technologies, 11(4), 611-615.
http://dx.doi.org/10.1016/j.ifset.2010.07.002.
http://dx.doi.org/10.1016/j.ifset.2010.0...
). However, up to now, no further investigation has been
conducted on physicochemical property and biological activity of AAFB melanin. In
this study, chemical composition of AAFB melanin was analysed. Meanwhile, scavenging
activity of AAFB melanin against radical was investigated for seeking new biological
functional principle used in food industry.
2 Materials and methods
2.1 Materials
Eighty grams dried fruiting bodies of A. auricula were purchased from a local market in Dongning City (Heilongjiang Province, China), pulverized and sifted through a 40-mesh sieve. The powder (moisture content 12-15% on dry basis) stored in dark bags to prevent from moisture and light. Synthetic melanin was purchased from Sigma Chemicals Co. (St. Louis, USA). All the other chemicals and reagents used in the experiment were of analytical grade.
2.2 Isolation and purification of AAFB melanin
The extraction process of melanin was carried out according to the method of
Zou et al. (2010)Zou, Y., Xie, C., Fang, G., Gu, Z., & Han, Y. (2010).
Optimization of ultrasound-assisted extraction of melanin from
Auricularia auricula fruit bodies. Innovative Food Science
& Emerging Technologies, 11(4), 611-615.
http://dx.doi.org/10.1016/j.ifset.2010.07.002.
http://dx.doi.org/10.1016/j.ifset.2010.0...
, fruiting bodies
powder of A. auricula was washed with running
water at a ratio of 30 mL/g (water/raw materials) for 5 min, followed by
centrifugation at 4000 rpm for 5 min. The precipitate was immersed into water at
a liquid-solid ratio 43 mL/g and the initial pH was adjusted to 12.0 with 1 M
NaOH. Then, the mixture was put into a conical flask and sonicated at 43 °C for
36 min by an ultrasound cleaning bath (Kunshan ultrasound instrument Co. Ltd.,
KQ250-DB, Kunshan, China) working at a frequency of 40 kHz and a ultrasound
intensity of 0.5 W/cm2 (the internal dimensions: 300 × 240 × 150 mm).
Afterward, the sample was centrifuged at 4000 rpm for 5 min and the supernatant
containing melanin was obtained.
Purification of melanin was performed as described by Wu et al. (2008)Wu, Y., Shan, L., Yang, S., & Ma, A. (2008). Identification and
antioxidant activity of melanin isolated from Hypoxylon archeri, a companion
fungus of Tremella fuciformis. Journal of Basic Microbiology, 48(3), 217-221.
http://dx.doi.org/10.1002/jobm.200700366. PMid:18506908
http://dx.doi.org/10.1002/jobm.200700366...
with proper modification. Melanin extract
was first adjusted to pH 2.0 with 3 M HCl to precipitate melanin, followed by
centrifugation at 10000 rpm for 20 min and the pellet was collected. The crude
melanin was hydrolyzed with 7 M HCl at 100 °C for 2 h. The non-hydrolysable
melanin collected by centrifugation (10000 rpm, 20 min) was washed with
chloroform, ethyl acetate and ethanol, and then dissolved in 1 M NaOH. The pH
value of water phase was adjusted to 2.0 with 3 M HCl. After centrifugation
(10000 rpm, 20 min), the pellet was washed with deionized water for three times.
Finally, the purified melanin was lyophilized and stored at -20 °C.
2.3 Chemical composition of AAFB Melanin
Elemental composition analysis
The amounts of C, N, H, O, and S in the AAFB melanin were determined with an element analyzer (Elementar analysen systeme, Vario EL III, Hanau, Germany) according to the national standard method of the People’s Republic of China (GB/T 19145-2003).
Amino acid composition analysis
Melanin (100 μg) was heated in 0.4 mL of 6 M HCl at 120 °C for 24 h in an evacuated and sealed tube. The hydrolysate was evaporated to dryness in a vacuum and was dissolved in 800 μL of a pH 2.2 buffer for amino acid composition analysis using an amino acid analyzer (Hitachi Ltd., L-8800, Tokyo, Japan).
Metal composition analysis
Melanin (500 mg) was weighed into a beaker, digested in 7 mL of HNO3-H2O2 (5:2) for 10 min, and the mixture heated to near dryness. After cooling, the residue was treated with 0.1 M HNO3 and brought to 50 mL with bidistilled water. Metal composition was determined with an inductively coupled plasma-optical emission spectrometry (ICP-OES) (Teledyne Leeman Labs Ltd., Prodigy, Hudson, NH, USA).
2.4 Assessment of radical scavenging activity of AAFB melanin
Assay of scavenging activity on 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical
Assessment of the scavenging abilities of AAFB melanin and BHT on DPPH
radical was performed by the method previously described by Tu et al. (2009)Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
with a minor
modification. Melanin or BHT solutions (2 mL) in 95% ethanol at different
concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) were added to 2 mL 0.2 mM
solution of DPPH in 95% ethanol. The mixture was shaken vigorously and left
to stand for 30 min in the dark, and then the absorbance was measured at 517
nm against a blank (95% ethanol solution). The scavenging ability of DPPH
radical was calculated using the formula given below. IC50
(Inhibitory concentration) was the concentration of the sample required to
scavenge 50% of DPPH radicals (Equation 1).
where A0 was the absorbance of DPPH solution without sample (95% ethanol, instead of sample solution), A1 was the absorbance of the test sample mixed with DPPH solution and A2 was the absorbance of the sample without DPPH solution (95% ethanol, instead of DPPH solution).
Assay of scavenging activity on superoxide radical
The superoxide radical-scavenging activities of AAFB melanin and BHT were
evaluated according to the method detailed by Martinez et al. (2001)Martinez, C. A., Loureiro, M. E., Oliva, M. A., & Maestri, M.
(2001). Differential responses of superoxide dismutase in freezing resistant
Solanum curtilobum and freezing sensitive Solanum tuberosum subjected to
oxidative and water stress. Plant Science, 160(3), 505-515.
http://dx.doi.org/10.1016/S0168-9452(00)00418-0. PMid:11166438
http://dx.doi.org/10.1016/S0168-9452(00)...
Each 3 mL of reaction mixture
contained 50 mM of sodium phosphate buffer, pH 7.8, 13 mM of methionine, 2
μM of riboflavin, 100 μM of ethylene diamine tetraacetic acid (EDTA), 75 μM
of nitroblue tetrazolium (NBT), and 1 mL of melanin or BHT of different
concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL). The production of blue
formazan was then followed by monitoring the increase in absorbance at 560
nm after a 10 min illumination from a fluorescent lamp. The entire reaction
assembly was enclosed in a box lined with aluminum foil. Identical tubes
with reaction mixture were kept in the dark and served as a blank. The
scavenging activity of superoxide radical was calculated using the formula
given below. IC50 (Inhibitory concentration) was the
concentration of the sample required to scavenge 50% of superoxide radicals
(Equation 2).
where AC was the absorbance of the control (deionized water, instead of sample), and AS was the absorbance of the test sample mixed with reaction solution.
Assay of scavenging activity on hydroxyl radical
The scavenging ability of AAFB melanin on hydroxyl radical was determined by
the method previously reported by Sun &
Kennedy (2010)Sun, Y. X., & Kennedy, J. F. (2010). Antioxidant activities of
different polysaccharide conjugates (CRPs) isolated from the fruiting bodies of
. Chroogomphis rutilus (Schaeff.: Fr.) O. K. MillerCarbohydrate
Polymers, 82(2), 510-514.
http://dx.doi.org/10.1016/j.carbpol.2010.05.010.
http://dx.doi.org/10.1016/j.carbpol.2010...
. Reaction mixtures in a final volume of 1.0 mL
contained deoxyribose (60 mM), phosphate buffer (pH 7.4, 20 mM), ferric
trichloride (100 μM), EDTA (100 μM), H2O2 (1 mM), and
different concentrations of melanin or BHT (0.2, 0.4, 0.6, 0.8, and 1.0
mg/mL). The reaction solution was incubated for 1 h at 37 °C, and then 1mL
of 1% thiobarbituric acid (TBA) and 1mL of 20% (v/v) HCl were added to the
mixture. The mixture was boiled for 15 min and cooled on ice. The absorbance
of the resulting mixture was measured at 532 nm. The scavenging activity of
hydroxyl radical was calculated according to the formula given below.
IC50 (Inhibitory concentration) was the concentration of the
sample required to scavenge 50% of hydroxyl radicals (Equation 3).
where AB was the absorbance of the control (deionized water, instead of sample), and AS was the absorbance of the test sample mixed with reaction solution.
2.5 Statistical analysis
The experimental results were expressed as means ± standard deviation (SD) of triplicates. Statistical analysis was performed using Fisher’s F-test and p<0.05 was regarded as significant.
3 Results and discussion
3.1 Element composition of AAFB melanin
Melanins present in pigmented tissues generally appear to be mixtures or
copolymers of eumelanins and pheomelanins (Chen
et al., 2008Chen, S. R., Jiang, B., Zheng, J. X., Xu, G. Y., Li, J. Y., &
Yang, N. (2008). Isolation and characterization of natural melanin derived from
silky fowl (. Gallus gallus domesticus Brisson)Food Chemistry,
111(3), 745-749.
http://dx.doi.org/10.1016/j.foodchem.2008.04.053.
http://dx.doi.org/10.1016/j.foodchem.200...
; Ito & Fujita,
1985Ito, S., & Fujita, K. (1985). Microanalysis of eumelanin and
pheomelanin in hair and melanomas by chemical degradation and liquid
chromatography. Analytical Biochemistry, 144(2), 527-536.
http://dx.doi.org/10.1016/0003-2697(85)90150-2. PMid:3993914
http://dx.doi.org/10.1016/0003-2697(85)9...
). Pheomelanin contains more sulfur than eumelanin. In this
study, the amounts of C, H, N, O, and S in AAFB melanin were quantified (Table 1). The experimental results showed
that the sulfur content of AAFB melanin was 5.44%. Thus, elemental composition
of AAFB melanin was very similar to that of pheomelanin. Fruiting bodies of
A. auricula grow on surfaces of withered
woods in natural environments. Therefore, withered woods might be source of
sulfur in AAFB melanin. The contents of sulfur and nitrogen in AAFB melanin were
lower than in pheomelanin. The contents of carbon and oxygen in AAFB melanin
were higher than in pheomelanin (Table
1). Thus, the structure of AAFB melanin might contain more aliphatic
groups or carboxylatecontaining monomers than pheomelanin.
3.2 Amino acid composition of AAFB melanin
The amino acid analysis showed that 16 amino acids were found in AAFB melanin and
total amino acid content was 321.63 mg/g, accounted for 32.16% of the mass of
the melanin sample (Figure 1). Previous
studies have shown that total amino acid contents of melanins extracted from
sepia (Chedekel et al., 1992Chedekel, M. R., Murr, B. L., & Zeise, L. (1992). Melanin
standard method: empirical formula. Pigment Cell Research, 5(3), 143-147.
http://dx.doi.org/10.1111/j.1600-0749.1992.tb00010.x.
PMid:1409451
http://dx.doi.org/10.1111/j.1600-0749.19...
),
black-bone silky fowl (Tu et al. 2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
),
black-hair, and red-hair (Liu et al.,
2005Liu, Y., Hong, L., Wakamatsu, K., Ito, S., Adhyaru, B., Cheng, C.
Y., Bowers, C. R., & Simon, J. D. (2005). Comparison of structural and
chemical properties of black and red human hair melanosomes. Photochemistry and
Photobiology, 81(1), 135-144. http://dx.doi.org/10.1562/2004-08-03-RA-259.1.
PMid:15504086
http://dx.doi.org/10.1562/2004-08-03-RA-...
) were 6-8%, 25.4%, 14.6%, and 44%, respectively. This indicated
that protein was an important component in melanin. In previous reported
literatures, the term “melanin” was defined loosely and it might generally
contain a certain amount of protein. These proteins which spherically surround
monomer units of melanins could affect the function of melanins (Liu et al., 2005Liu, Y., Hong, L., Wakamatsu, K., Ito, S., Adhyaru, B., Cheng, C.
Y., Bowers, C. R., & Simon, J. D. (2005). Comparison of structural and
chemical properties of black and red human hair melanosomes. Photochemistry and
Photobiology, 81(1), 135-144. http://dx.doi.org/10.1562/2004-08-03-RA-259.1.
PMid:15504086
http://dx.doi.org/10.1562/2004-08-03-RA-...
; Tu et al., 2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
). The extensive proteolytic digestion
should be avoided because the intactness of melanin might be destroyed during
extraction process using proteases. The future work is required to delineate the
role of protein in the structure and property of AAFB melanin.
Amino acid composition of AAFB melanin. Values are means ± SD of three independent determinations.
3.3 Metal composition of AAFB melanin
There were 13 detectable metal elements in AAFB melanin, as found by ICP-OES
(Figure 2). The experimental results
showed that AAFB melanin was rich in Ca, Fe, Cu and Zn and their contents were
2.12, 1.43, 0.19, and 0.18 mg/g, respectively. The other investigator had
reported that melanin extracted from the muscles of black-bone silky fowl was
rich in Ca, Fe, Mg and Zn (Tu et al.
2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
), which was similar to the result obtained in this study. This
indicated that melanins from different sources could serve as a reservoir of
metal ions, such as Ca (II), Fe (II), Fe (III), Cu (II), Zn (II), and Mg (II).
Therefore, the ability of binding various metal ions was one of the most typical
characteristics of melanin.
Metal composition of AAFB melanin. Values are means ± SD of three independent determinations.
3.4 DPPH radical scavenging activity of AAFB melanin
Antioxidant properties, especially radical scavenging activities, were very
important because of the deleterious role of free radicals in foods and
biological systems. Excessive formation of free radicals accelerated the
oxidation of lipids in foods and induced severe damage to adjacent biomolecules
(Peksel et al., 2010Peksel, A., Arisan-Atac, I., & Yanardag, R. (2010). Evaluation
of antioxidant and antiacetylcholinesterase activities of the extracts of desf.
leaves. Pistacia atlanticaJournal of Food Biochemistry, 34(3),
451-476. http://dx.doi.org/10.1111/j.1745-4514.2009.00290.x.
http://dx.doi.org/10.1111/j.1745-4514.20...
). DPPH assay
was the simplest and most accurate method to evaluate the radical scavenging
ability of antioxidants. Under ethanol solution, antioxidants could interaction
with DPPH radical and transfer an electron or hydrogen atom to DPPH radical,
thus neutralizing its free radical character (Herchi et al., 2014Herchi, W., Kallel, H., & Boukhchina, S. (2014). Physicochemical
properties and antioxidant activity of Tunisian date palm (Phoenix
dactylifera L.) oil as affected by different extraction methods.
Food Science and Technology, 34(3), 464-470.
http://dx.doi.org/10.1590/1678-457x.6360.
http://dx.doi.org/10.1590/1678-457x.6360...
).
The scavenging activities of AAFB melanin and BHT of various concentrations on DPPH radical are given in Figure 3. The scavenging activities of AAFB melanin and BHT increased with a concentration increase. Of all the concentrations tested (0.2-1.0 mg/mL), AAFB melanin showed significantly higher (p<0.05) activities than BHT. Meanwhile, IC50 values of AAFB melanin was significant lower (p<0.05) than BHT (Table 2). The experimental results indicated that AAFB melanin had a stronger scavenging activity on DPPH radical than BHT at an identical concentration.
Scavenging effect of AAFB melanin on DPPH radical. Values are means ± SD of three independent determinations.
3.5 Superoxide radical scavenging activity of AAFB melanin
Although the superoxide was a relatively weak oxidant, its combination with other
reactive species, such as nitric oxide and hydroxyl, might yield stronger
reactive species,which possessed greater oxidative ability than the precursor to
initiate lipids peroxidation (Miguel et al.,
2014Miguel, M. G., Nunes, S., Dandlen, S. A., Cavaco, A. M., &
Antunes, M. D. (2014). Phenols, flavonoids and antioxidant activity of aqueous
and methanolic extracts of propolis (Apis mellifera L.) from
Algarve, South Portugal. Food Science and Technology, 34(1), 16-23.
http://dx.doi.org/10.1590/S0101-20612014000100002.
http://dx.doi.org/10.1590/S0101-20612014...
; Peksel et al., 2010Peksel, A., Arisan-Atac, I., & Yanardag, R. (2010). Evaluation
of antioxidant and antiacetylcholinesterase activities of the extracts of desf.
leaves. Pistacia atlanticaJournal of Food Biochemistry, 34(3),
451-476. http://dx.doi.org/10.1111/j.1745-4514.2009.00290.x.
http://dx.doi.org/10.1111/j.1745-4514.20...
).
Furthermore, superoxide was also known to indirectly induce lipid peroxidation
as a result of H2O2 formation, creating precursors of
hydroxyl radical (Sun & Kennedy,
2010Sun, Y. X., & Kennedy, J. F. (2010). Antioxidant activities of
different polysaccharide conjugates (CRPs) isolated from the fruiting bodies of
. Chroogomphis rutilus (Schaeff.: Fr.) O. K. MillerCarbohydrate
Polymers, 82(2), 510-514.
http://dx.doi.org/10.1016/j.carbpol.2010.05.010.
http://dx.doi.org/10.1016/j.carbpol.2010...
). The hydrogen-donating ability is a primary index of
antioxidants that donate hydrogen to superoxide radicals, resulting in
detoxification and inhibition of the propagation phase of lipid oxidation.
The results of the scavenging superoxide radical of AAFB melanin and BHT are
shown in Figure 4. AAFB melanin possessed
higher (p<0.05) superoxide radical scavenging activity than
BHT in a concentration-dependent manner. As was shown in Table 2, IC50 values of AAFB melanin with
superoxide radical scavenging activity were significant lower
(p<0.05) than BHT. At an identical concentration,
scavenge activity of AAFB melanin on superoxide radical was stronger to that of
melanin from black-bone silky fowl and synthetic melanin (Tu et al., 2009Tu, Y., Sun, Y., Tian, Y., Xie, M., & Chen, J. (2009).
Physicochemical characterisation and antioxidant activity of melanin from the
muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus
Brisson). Food Chemistry, 114(4), 1345-1350.
http://dx.doi.org/10.1016/j.foodchem.2008.11.015.
http://dx.doi.org/10.1016/j.foodchem.200...
).
Scavenging effect of AAFB melanin on superoxide radical. Values are means ± SD of three independent determinations.
3.6 hydroxyl radical scavenging activity of AAFB melanin
Among all reactive oxygen radicals, hydroxyl radical was known as the most
powerful radical. It could induce severe damage to adjacent biomolecules in the
body, which result in cell damage that caused ageing, cancer and several other
diseases (Yang et al., 2014Yang, X., Yan, F., Huang, S., & Fu, C. (2014). Antioxidant
activities of fractions from longan pericarps. Food Science and Technology,
34(2), 341-345.
http://dx.doi.org/10.1590/S0101-20612014005000034.
http://dx.doi.org/10.1590/S0101-20612014...
). The
removal of hydroxyl radical was probably one of the most effective ways to
defense oxidative damage of human body. Therefore, hydroxyl radical scavenging
activity was considered to be one of the most important antioxidant
mechanisms.
The scavenging abilities of AAFB melanin and BHT on hydroxyl radical are shown in Figure 5. With the increase of concentration, the scavenging abilities of AAFB melanin and BHT on hydroxyl radical also increased. At the concentration range of 0.2-1.0 mg/mL, AAFB melanin showed significantly stronger (p<0.05) scavenging activities than BHT. As was shown in Table 2, IC50 values of AAFB melanin was significant lower (p<0.05) than BHT. These results suggested that AAFB melanin was better natural antioxidant than BHT in scavenging hydroxyl radical.
Scavenging effect of AAFB melanin on hydroxyl radical. Values are means ± SD of three independent determinations.
In recent studies, Chiarelli-Neto et al.
(2011)Chiarelli-Neto, O., Pavani, C., Ferreira, A. S., Uchoa, A. F.,
Severino, D., & Baptista, M. S. (2011). Generation and suppression of
singlet oxygen in hair by photosensitization of melanin. Free Radical Biology
& Medicine, 51(6), 1195-1202.
http://dx.doi.org/10.1016/j.freeradbiomed.2011.06.013.
PMid:21723388
http://dx.doi.org/10.1016/j.freeradbiome...
found that singlet oxygen induced hair damage was produced by
photosensitization of melanin. However, Brenner
& Hearing (2009)Brenner, M., & Hearing, V. J. (2008). The protective role of
melanin against UV damage in human skin. Photochemistry and Photobiology, 84(3),
539-549. http://dx.doi.org/10.1111/j.1751-1097.2007.00226.x.
PMid:18435612
http://dx.doi.org/10.1111/j.1751-1097.20...
and Geng et al.
(2008)Geng, J., Yu, S. B., Wan, X., Wang, X. J., Shen, P., Zhou, P., &
Chen, X. D. (2008). Protective action of bacterial melanin against DNA damage in
full UV spectrums by a sensitive plasmid-based noncellular system. Journal of
Biochemical and Biophysical Methods, 70(6), 1151-1155.
http://dx.doi.org/10.1016/j.jprot.2007.12.013. PMid:18272228
http://dx.doi.org/10.1016/j.jprot.2007.1...
reported that melanin was the most important photoprotective
factor and had antioxidant and radical scavenging properties. Therefore, the
role of melanin in photosensitization was still controversial. The future
research on photo-stability of melanin would be helpful to fully understand the
mechanism of photosensitization in organism and delineate the role of melanin in
the photoprotection.
4 Conclusions
Chemical composition and radical scavenging activity of AAFB melanin were examined and analysed. The experimental results indicated that AAFB melanin consisted mostly of pheomelanin. AAFB melanin contained 16 amino acids and 13 metal elements. It was rich in Ca, Fe, Cu and Zn. The antioxidant activities of AAFB melanin were evaluated by DPPH radical, superoxide radical, and hydroxyl radical scavenging assay. AAFB melanin exhibited stronger antioxidant activity compared to BHT. Results from this study indicated that AAFB melanin could be potentially used as a natural antioxidant.
Acknowledgements
This work was supported by the Doctor Research Fund of Dalian Nationalities University (No. 0701-110015).
-
Practical Application: A. auricula melanin can be potentially used as a natural antioxidant.
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Publication Dates
-
Publication in this collection
June 2015
History
-
Received
17 Sept 2014 -
Accepted
08 Mar 2015