journal of functional foods 9 (2014) 78–88

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Antioxidant and α-glucosidase inhibitory

properties of soluble melanins from the fruits of
Vitex mollis Kunth, Randia echinocarpa Sessé et
Mociño and Crescentia alata Kunth

Esmeralda Cuevas-Juárez a,1, Korinthia Yamileth Yuriar-Arredondo a,1,

Juan Fernando Pío-León a,b,1, Julio Montes-Avila c,d,
Gabriela López-Angulo a,d, Sylvia Páz Díaz-Camacho c,d,
Francisco Delgado-Vargas a,d,*
a
Maestría en Ciencia y Tecnología de Alimentos, Facultad de Ciencias Químico Biológicas (FCQB), de la
Universidad Autónoma de Sinaloa (UAS). Ciudad Universitaria s/n, Culiacan, Sinaloa, Mexico
b
Centro de Investigaciones Biológicas del Noroeste, Mar Bermejo 195, Col. Playa Palo de Sta. Rita, P.O. Box 128,
La Paz, B.C.S. 23090, Mexico
c
Maestría en Ciencias Biomédicas, FCQB, UAS. Ciudad Universitaria s/n, Culiacan, Sinaloa, Mexico
d
Programa en Biotecnología, FCQB, UAS. Ciudad Universitaria s/n, Culiacan, Sinaloa, Mexico

A R T I C L E I N F O A B S T R A C T

Article history: Melanins have important pharmacological properties with limited applications due to their
Received 30 December 2013 poor solubility. Soluble melanins have been reported in different organisms, but not in plants.
Received in revised form 13 April In this study, aqueous soluble melanins, Impure (IMe) and Partially Purified (PMe), were pre-
2014 pared from the edible pulp of fruits native to Mexico (i.e. Vitex mollis, VM; Randia echinocarpa,
Accepted 14 April 2014 RE; and Crescentia alata, CA). IMe and PMe were tested for their phenolics content (TPC), an-
Available online tioxidant activities (AA), α-glucosidase inhibition (αGI) and spectroscopic features (UV-Vis
and IR). Melanins of VM showed the highest TPC (222.23 mg GAE/g) and AA (ABTS 2779.30 μmol
Keywords: TE/g; FRAP 1203.70 μmol TE/g). Purified melanins (PMe) showed better αGI than acarbose
Soluble melanins (IC50 = 8.38 mg/mL). The spectroscopic features of IMe and PMe corresponded to melanins
Vitex mollis and solubility may be by their complexation with carbohydrates. For the first time, plant-
Randia echinocarpa based soluble melanins and their remarkably high antioxidant and α-glucosidase inhibi-
Crescentia alata tory characteristics are presented.
Antioxidant activity © 2014 Elsevier Ltd. All rights reserved.
α-glucosidase inhibition

* Corresponding author. Tel.: +52 667 7136615; fax: +52 667 7136615.
E-mail addresses: fdelgado@uas.edu.mx, fcodelgadovargas@gmail.com (F. Delgado-Vargas).
1
Equal contribution.
Abbreviations: AA, antioxidant activity; ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid; αGI, α-glucosidase inhibition; CA,
Crescentia alata; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalents; IMe, impure melanins, extracted at room (-R) or
boiling temperature (-B); PMe, purified melanins, obtained by ethanol precipitation (PMeEP) or by dialysis (PMeD) of melanins extracted
at room (-R) or boiling temperature (-B); RE, Randia echinocarpa; TE, Trolox equivalents; TPC, total phenolics content; VM, Vitex mollis
http://dx.doi.org/10.1016/j.jff.2014.04.016
1756-4646/© 2014 Elsevier Ltd. All rights reserved.
 journal of functional foods 9 (2014) 78–88
1. Introduction
Fruit and vegetable consumption has been associated with a
lower risk of developing cardiovascular diseases and in-
crease longevity. Phytochemicals (e.g. carotenoids and flavo-
noids) with diverse activities (e.g. antioxidant and α-glucosidase
inhibitory) have been related with those health beneficial prop-
erties (Gironés-Vilaplana et al., 2014).
The α-glucosidase inhibitors are important anti-diabetic
drugs that prevent postprandial hyperglycaemia by inhibit-
ing the hydrolysis of oligosaccharides in the gut (Kumar, Ghosh,
& Pal, 2013). Several efforts are currently underway to iden-
tify new natural sources of α-glucosidase inhibitors and an-
tioxidants that delay the damage caused by oxidative stress
in patients with type II diabetes mellitus (Ceriello, 2008; Coman,
Rugina, & Socaciu, 2012) or obesity (Gironés-Vilaplana et al.,
2014). Additionally, their potential to treat viral ills (e.g. hepa-
titis and dengue) (Norton et al., 2005; Zhong, Ma, & Shahidi,
2012) or cancer (Atsumi, Nosaka, Ochi, Iinuma, & Umezawa,
1993; Gamberucci et al., 2006; Pili et al., 1995) has been sug-
gested; these acting by inhibition of glycoprotein maturation.
Melanins are high-molecular-weight pigments that impart
light hues to black colours; they are found in animals, plants
and microorganisms and are important for human health due
to their functional properties (e.g. antioxidant and
immunostimulatory) (Huang et al., 2011; Pugh et al., 2005).
Studies about plant melanins are scarce compared to others
pigments or secondary metabolites. They have been isolated
from Osmanthus fragrans seeds (Wang, Pan, Tang, & Huang, 2006),
fruits and seeds of Nyctanthes arbor-tristis (Kannan & Ganjewala,
2009), Nigella sativa seeds (El-Obeid, Al-Harbi, Al-Jomah, & Hassib,
2006) and black tea (Sava, Yang, Hong, Yang, & Huang, 2001).
Melanins are commonly represented as covalent bonded
indoles (Bilinska, 1996), although their general structure is
unknown (Riley, 1997). It has been suggested that identical
melanin structures do not exist in nature (Kerestes, Kerestes,
& Vegner, 2001) and their chemical characterization is a com-
plicated task. These pigments have been scarcely studied due
to their low solubility in most of the common solvents (e.g.
water, methanol, ethanol, ethyl acetate, hexane) and harsh pH
extraction-conditions (i.e. ammonium hydroxide and hydro-
chloric acid) that give low yields. On the other hand, soluble
melanins have not been reported in plants, but in bacteria, fungi
and animals (Aghajanyan et al., 2005; Mazurkiewicz, 2006),
whose solubility has been associated with the formation of
complexes with carbohydrates or proteins (Aghajanyan et al.,
2011; Seniuk et al., 2010). Thus, the presence of soluble mela-
nins in plants is possible.
Vitex mollis Kunth, Randia echinocarpa Sessé et Mociño and
Crescentia alata Kunth, are endemic to Mexico and commonly
known as “uvalama”, “papache” and “ayale”, respectively. Their
edible pulps are dark, have therapeutic properties
(Delgado-Vargas et al., 2010; Santos-Cervantes, Ibarra-Zazueta,
Loarca-Piña, Paredes-López, & Delgado-Vargas, 2007; Vargas-Solis
& Perez-Gutierrez, 2002), and the spectroscopic characteris-
tics of their un-characterized pigments do not correspond to
those commonly found in other fruits (e.g. anthocyanins, ca-
rotenoids, chlorophylls, betalains), representing potential sources
of melanins. In fact, our research group has compared the
79
aqueous soluble (purified by dialysis) and insoluble (obtained
by alkali and acid extraction) melanins of V. mollis fruit; both
melanins show similar spectroscopical and biological proper-
ties, but the yield of the soluble is higher (Pío-León et al., 2014,
submitted for publication). Therefore, we hypothesized that pig-
ments of the black edible pulps of these fruits are soluble mela-
nins extractable by friendly environmental methods.
In this research, we partially purified aqueous soluble mela-
nins from the pulp of the fruits of V. mollis, R. echinocarpa and
C. alata, with high extraction yields, using two easy and en-
vironmentally friendly methods. Pigments were character-
ized by spectroscopic techniques and the effect of heating was
studied; their total phenolics content and in vitro antioxidant
and α-glucosidase inhibitory activities were also evaluated.
2. Material and methods
2.1. Plant material
Vitex mollis (VM), Randia echinocarpa (RE) and Crescentia alata (CA)
fruits were collected from the municipalities of Culiacan and
Elota (VM), and Badiraguato and Salvador Alvarado (RE and CA),
Sinaloa, Mexico. Taxonomic identification was corroborated by
Dr. Rito Vega Aviña of the Faculty of Agriculture of the Autono-
mous University of Sinaloa (UAS) and the assigned numbers
for the materials deposited in the herbarium of the Faculty of
Agriculture were VM (8258), RE (9035) and CA (9055). The fruits
were harvested at maturity as recommended by inhabitants
of the rural communities. Once collected, peel (except for VM)
and seeds were removed; recovered pulps were frozen (−40 °C),
freeze-dried (Model 5L Virtis Co., Gardiner, NY, USA) and milled
to obtain flours that passed through the sieve number 30.
Vitex mollis, R. echinocarpa and C. alata flours were stored for
no more than three months at −20 °C until use.
2.2. Preparation of impure melanin (IMe)
Impure melanins (IMe) were obtained by aqueous macera-
tion at room (IMe-R, 27 °C) and boiling (IMe-B) temperatures.
In an amber flask, 5 g of fruit flour and 100 mL of deionized
water were mixed and stirred for 30 min at 27 °C. The suspen-
sion was centrifuged (20000 g/ 15 min/ 20–25 °C), the super-
natant was recovered and then freeze-dried to obtain IMe-R.
IMe-B was obtained in the same way as IMe-R, but extraction
time (30 min) started after reaching the boiling temperature.
Both melanins were stored for no more than 3 months at −20 °C
until used.
2.3. Preparation of partially purified melanins (PMe)
2.3.1. Ethanol precipitation
The freeze-dried impure melanins (IMe-R and IMe-B) were re-
suspended in deionized water (50 mg/mL) and mixed with four
volumes of distilled anhydrous ethanol, stirred for 30 min and
centrifuged (20000 g/ 5 min/ 20–25 °C); the pellets were recov-
ered and freeze-dried to obtain partially purified melanins (PMe)
by ethanol precipitation (PMeEP) at room (PMeEP-R) and boiling
(PMeEP-B) temperature; and the yield was calculated. The PMe
80 journal of functional foods 9 (2014) 78–88
were stored for no more than 3 months at –20 °C until use. The
supernatants of the centrifugation processes were evaluated
for carbohydrates by using the Tollens’ reagent (Clugston &
Fleming, 2000).
2.3.2. Dialysis
The freeze-dried impure melanins (IMe-R or IMe-B) were
re-suspended in 10 mL of deionized water (50 mg/mL) and dia-
lyzed against 300 mL of deionized water using a 12 kDa pre-
hydrated cellulose membrane (Sigma-Aldrich, Seelze, Germany);
dialysis was carried out for four days under stirring and the
water was changed two times/day. Dialyzed retentates were
recovered, freeze-dried and stored at −20 °C until use; yields
were calculated. The fractions so obtained were partially pu-
rified melanins by dialysis of the aqueous extract at room tem-
perature (PMeD-R) and of the aqueous extract at boiling
temperature (PMeD-B).
2.4. Total phenolics content (TPC)
Total phenolics of the melanin samples (IMe and PMe) were
determined by the Folin-Ciocalteu reaction (Waterhouse, 2002).
Melanin dissolved in deionized water (20 μL) was mixed with
1.58 mL of deionized water and 100 μL of the Folin-Ciocalteau
reagent, gently stirred for 5 min and added with 300 μL of a
saturated solution of sodium carbonate. The mixture was allowed
to stand in darkness for 30 min at 40 °C and the absorbance
was measured at 765 nm (Spectronic Genesis 20, Spectronic In-
struments, Rochester, NY, USA). A calibration curve of gallic acid
in water-methanol (1:1, v/v) (0, 50, 100, 200, 400 and 500 μg/
mL) was prepared and TPC was calculated as milligrams of gallic
acid equivalents (GAE)/gram of melanin (mg GAE/g).
2.5. Antioxidant activity
Two methods were used: ABTS [2,2’-azino-bis(3-
ethylbenzothiazoline)-6-sulphonic acid] and FRAP (ferric re-
ducing antioxidant power).
2.5.1. ABTS assay
The radical 2,2’-azino-bis(3-ethylbenzothiazoline)-6-sulphonic
acid (ABTS•+) was produced by mixing 7 mM ABTS with 2.45 mM
potassium persulphate (5 mL of ABTS + 5 mL of potassium
persulphate 4.9 mM). The mixture was incubated in darkness
at room temperature for 16 h, diluted with 7 mM phosphate
buffer pH 7.4 to reach an absorbance between 1.0 and 1.2 at
734 nm. For the ABTS assay, 50 µL of melanin (IMe or PMe), or
dissolvent as control, were mixed with 1.95 mL of ABTS•+ so-
lution, incubated in darkness for 10 min at 37 °C and then the
absorbance was measured at 734 nm (Liu et al., 2009). A cali-
bration curve of Trolox in water-methanol (1:1, v/v) (400, 300,
200, 100 and 50 μg/mL) was prepared and the antioxidant ac-
tivity (AA) calculated as % AA = [(Ac – Am) / Ac] × 100; where, Ac
and Am are the absorbances for the control and melanin, re-
spectively. Results were reported as micromoles of Trolox
equivalents (TE)/gram of melanin (µmol TE/g).
2.5.2. FRAP assay
The FRAP reagent was prepared by mixing 2.5 mL of 10 mM
TPTZ (2,4,6-tris(2-pyridyl)-S-triazine) in 40 mM HCl, 2.5 mL of
20 mM FeCl3•6H2O and 25 mL of 0.3 mM acetate buffer (pH 3.6),
followed by incubation for 30 min at 37 °C. For the evalua-
tion, 900 μL of freshly prepared FRAP reagent, 90 μL of deion-
ized water and 10 μL of melanin sample (IMe or PMe, at known
concentrations) or appropriate reagent blank were mixed. The
mixture was incubated (30 min/37 °C) and the absorbance at
595 nm was read. Results were obtained by linear regression,
using a Trolox calibration curve (500, 400, 300, 200, 100 and 50 μg/
mL), and expressed as micromoles of TE/gram of melanin (µmol
TE/g) (Benzie & Strain, 1996).
2.6. α-Glucosidase inhibition (αGI)
In 96 microwell plates, 50 µL of melanin sample (IMe or PMe)
or acarbose (positive control) at different concentrations were
incubated for 10 min at 37 °C with 100 µL of α-glucosidase from
Saccharomyces cerevisiae (Sigma-Aldrich, Toluca, Estado de Mexico,
Mexico) (1 U/mL) in phosphate buffer (0.1 M, pH 6.9). Then, 50 µL
of 4-nitrophenyl β-D-glucopyranoside (5 mM in phosphate buffer
pH 6.9) were added and the mixture was incubated again for
10 min at 37 °C (Stat Fax-2200, Awareness Technology, Palm City,
FL, USA). For correction of sample interferences, a blank was
prepared by mixing 100 µL of α-glucosidase, 50 µL of melanin
sample and 50 µL of phosphate buffer. As control, 50 µL of
solvent was used instead of the melanin or acarbose. Absor-
bance at 405 nm was measured with a Multiskan Biochromatic
Microplate Reader (Fisher Scientific, Hudson, NH, USA) and the
inhibition was calculated as αGI (%) = [(Ac – Am) / Ac] × 100; where,
Ac and Am are the absorbance for the control and melanin, re-
spectively (Pinto et al., 2008). For each sample, the half maximal
inhibitory concentration (IC50) of the α-glucosidase activity was
determined with the Software GraphPadPrism Inc. version 5.03
(La Jolla, CA, USA).
2.7. Ultraviolet-visible spectroscopy (UV-Vis)
Melanin solutions were prepared at 0.5 mg/mL and the UV-
Vis spectra were obtained (200–800 nm) (SmartSpect™ 3000, Bio
Rad, Philadelphia, PA, USA).
2.8. Absorbance ratios (A300/A600)
The A300/A600 values of melanins were determined by measur-
ing the UV-Vis absorption spectra of different dilutions (at least
in triplicate). All dilutions followed the criteria 2.5>A300>0.25
(SmartSpect™ 3000, Bio Rad, Philadelphia, PA, USA); these limits
were established to ensure all spectra gave measurable A300 and
A600 values, and to reduce errors due to instrument satura-
tion at high absorbance measurements (Haywood, Lee, & Linge,
2006). Thus, the melanin concentrations (mg/mL) were as
follows: IMe-R-RE and IMe-B-RE (5.5); IMe-R-CA and IMe-B-
CA (6); IMe-R-VM (3); IMe-B-VM (1.75); all PMeEP (0.5), except
PMeEP-B-CA (2); all PMeD (0.4), except PMeD-R-RE (0.5) and
PMeD-B-CA (1.0). Absorbances of the dilutions at 300 nm were
around 2.0 (1.95≤A300 nm≤2.38).
2.9. Infrared spectroscopy (IR)
The IR spectra of melanin solid samples were obtained at room
temperature by attenuated total reflection with a Fourier
 journal of functional foods 9 (2014) 78–88 81

transform infrared spectrometer (FTIR-ATR Cary 660; Agilent
Technologies, Santa Clara, CA, USA) in the range 4000–400 cm–1
(10 scannings, 1 cm−1 resolution).
2.10. Statistical analysis
Data from every measured property were analyzed by one way
analysis of variance and the means were compared by the
Fisher test (α = 0.05). Relationships between the measured
properties were analyzed by simple linear regression and the
Pearson correlation coefficient. All the analyses were
carried out with Statgraphics 5.1 (StatPoint Inc., Warrenton,
VA, USA). Property evaluations were done at least in
triplicate.
3. Results
3.1. Impure melanins (IMe)
3.1.1. Extraction yields
The extraction yields of IMe ranged from 69% dry weight (dw)
(IMe-B-VM) to 84% dw (IMe-R-RE) and, comparing for every fruit,
were not affected by the thermal treatment (Table 1).
3.1.3. α-Glucosidase inhibition (αGI)
The order of activities for αGI of impure melanins (IMe) were
VM>RE>CA. The IC50 values for VM (3.11 mg/mL and 3.79 mg/
mL) were better than that of acarbose (8.38 mg/mL). By con-
trast with TPC and AA, heating during extraction did not affect
the αGI of IMe from the same fruit (Table 1).
3.2. Purified melanins (PMe)
3.2.1. Extraction yield
The extraction yields (% of IMe) of PMe obtained by ethanol
precipitation (PMeEP) varied from 6.05 to 19.3%; whereas lower
values were obtained for melanins purified by dialysis (PMeD)
(3.63–13.55%). The decreasing order of extraction was VM>CA>RE
for both PMeEP and PMeD. Contrasting the effect of heating,
the extraction yields of PMeEP were not affected (PMeEP-R ≅
PMeEP-B), but increased for the PMeD of V. mollis and C. alata
(Table 2).
The supernatants obtained in the preparation of the PMeEP
gave a positive result for carbohydrates by the Tollens’ test
(Clugston & Fleming, 2000). In addition, the solubilities of
the PMe were lower than those of their corresponding IMe,
considering the absorption at the λ max and the ease of
dissolution.

3.1.2. Total phenolics content (TPC) and antioxidant
activity (AA)
The IMe of VM showed the highest TPC and AA; their TPC values
were at least 4.7 higher than those of RE and CA; whereas their
AA values were approximately 7 and 4 times higher than those
of IMe-B-RE and IMe-B-CA, respectively.
The AA values by ABTS were higher than those obtained
by FRAP (Table 1). Moreover, boiling extraction increased both
TPC and AA for VM and RE (IMe-B>IMe-R), but not for CA
(Table 1). In addition, the AA and TPC of IMe showed a posi-
tive correlation (ABTS R2 = 0.9732, p = 0.0003; FRAP R2 = 0.9124,
p = 0.0030).
3.2.2. Total phenolics content (TPC) and antioxidant
activity (AA)
The TPC values for melanins purified by ethanol precipita-
tion (PMeEP) were from 23.5 (PMeEP-B-CA) to 222.23 (PMeEP-
B-VM) mg GAE/g with a decreasing order of VM>RE>CA; in
contrast with the order VM>CA>RE for melanins purified by di-
alysis (PMeD), with values from 22.31 (PMeD-R-RE) to 217.85
(PMeD-B-VM) mg GAE/g (Table 2).
In general, the AA values (µmol TE/g) of PMeD (413.78 to
2779.3 by ABTS and 209.22 to 1203.7 µmol TE/g by FRAP) were
higher than those of PMeEP (262.73 to 1930.99 by ABTS and
171.43 to 869.18 by FRAP) (Table 2), and the AA of both PMe were

Table 1 – Extraction yields, total phenolics content, antioxidant activity and α-glucosidase inhibition (αGI) of the impure
melanins (IMe), obtained by aqueous extraction at room and boiling temperature, from the Vitex mollis, Randia
echinocarpa and Crescentia alata fruits.
Impure Extraction Total phenolics Antioxidant activity αGI (IC50)
melanin (IMe)a yield (% d.w.)b,f content (mg GAE/g)c,f (mg/mL)e,f
ABTS (μmolTE/g)d,f FRAP (μmolTE/g)d,f
IMe-R-VM 73.24 ± 1.31g,h,i 29.48 ± 0.46g 429.21 ± 28.53g 146.98 ± 18.81g 3.11 ± 0.47g
IMe-B-VM 69.45 ± 6.25g 40.15 ± 0.46h 531.14 ± 5.17h 271.17 ± 7.15h 3.79 ± 0.71g
IMe-R-RE 84.07 ± 1.98j 4.84 ± 0.05i 53.59 ± 1.12i 23.65 ± 0.13i 14.07 ± 4.49h
IMe-B-RE 80.52 ± 1.78i,j 6.28 ± 0.19j 76.06 ± 0.69j 37.90 ± 0.13i 10.22 ± 1.89h
IMe-R-CA 70.49 ± 6.26g,h 4.51 ± 0.37i 120.69 ± 0.45k 67.42 ± 9.71j 23.70 ± 0.22i
IMe-B-CA 77.25 ± 1.95h,i,j 4.71 ± 0.03i 124.86 ± 1.05k 64.97 ± 2.85j 26.77 ± 2.48i
a
Impure melanins obtained at room (IMe-R) and boiling temperature (IMe-B) from Vitex mollis (-VM), Randia echinocarpa (-RE) and Crescentia
alata (-CA) fruits.
b
Dry weight basis (d.w.).
c
Expressed as milligrams of gallic acid equivalents per gram of melanin (mg GAE/g).
d
Expressed as micromoles of Trolox equivalents per gram of melanin (μmol TE/g).
e
α-Glucosidase inhibition (αGI), expressed as inhibitory concentration of 50% of the enzyme activity (IC50), in milligrams of melanin per mil-
liliter (mg/mL) (IC50 for acarbose = 8.38 mg/mL).
f
Data shown are the mean of at least three independent experiments and different superscript letters (g–k) in the same column indicate sig-
nificant differences (P < 0.05).
82 journal of functional foods 9 (2014) 78–88

Table 2 – Extraction yields, total phenolics content, antioxidant activity and α-glucosidase inhibition (αGI) of the partially
purified melanins (PMe), obtained by ethanol precipitation (PMeEP) or by dialysis (PMeD), of the Vitex mollis (VM), Randia
echinocarpa (RE) and Crescentia alata (CA) fruits.
Partially purified Extraction Total phenolics Antioxidant activity αGI (IC50)
melanin (PMe)a yield (% d.w.)b,f content (mg GAE/g)c,f d,f d,f (mg/mL)e,f
ABTS (μmolTE/g) FRAP (μmolTE/g)
PMeEP-R-VM 18.08 ± 1.56g 174.17 ± 23.05g 1695.40 ± 59.60g 603.62 ± 42.32g 0.307 ± 0.029g
PMeEP-B-VM 19.03 ± 0.56f 222.23 ± 2.71h 1930.99 ± 71.42h 869.18 ± 25.55h 0.247 ± 0.012g
PMeEP-R-RE 6.05 ± 0.82h,i,j 42.88 ± 7.71i,j 436.16 ± 14.55i,j 206.50 ± 12.09i 1.94 ± 0.30h,i
PMeEP-B-RE 6.41 ± 0.35i,j 46.33 ± 2.17j 457.73 ± 3.93i,j 308.24 ± 4.57j 1.38 ± 0.14j,k
PMeEP-R-CA 8.99 ± 0.65k 26.23 ± 0.00k 317.42 ± 1.93k 200.88 ± 7.12i,k 4.97 ± 0.41l
PMeEP-B-CA 7.38 ± 0.58j,k 23.50 ± 1.15k 262.73 ± 2.81l 171.43 ± 0.69k 7.41 ± 0.74m
PMeD-R-VM 11.47 ± 0.23l 185.99 ± 1.31g 2682.9 ± 8.56m 1173.3 ± 13.88l 0.192 ± 0.014g
PMeD-B-VM 13.55 ± 0.55m 217.85 ± 2.63l 2779.3 ± 4.17n 1203.7 ± 35.73m 0.345 ± 0.069g
PMeD-R-RE 4.33 ± 0.45h,n 22.31 ± 1.12k 482.37 ± 12.35i 209.22 ± 7.00i 1.17 ± 0.069j,k
PMeD-B-RE 5.01 ± 0.25h,i,n 29.50 ± 0.98k 719.51 ± 2.21o 351.77 ± 6.16n 1.00 ± 0.010j
PMeD-R-CA 3.63 ± 0.95n 65.24 ± 0.51m 684.82 ± 3.73o 493.95 ± 26.45o 1.63 ± 0.307h,k
PMeD-B-CA 6.63 ± 1.88i,j 32.67 ± 0.59i,k 413.78 ± 3.56j 297.58 ± 3.87j 2.35 ± 0.222i
a
Partially purified melanins obtained by ethanol precipitation (PMeEP) or by dialysis (PMeD), extracted at room (-R) and boiling temperature
(-B) from Vitex mollis (-VM), Randia echinocarpa (-RE) and Crescentia alata (-CA) fruits.
b
The yield is with respect to the impure melanin in dry weight basis (d.w.).
c
Expressed as milligrams of gallic acid equivalents per gram of melanin (mg GAE/g).
d
Expressed as micromoles of Trolox equivalents per gram of melanin (μmol TE/g).
e
α-Glucosidase inhibition (αGI), expressed as inhibitory concentration of 50% of the enzyme activity (IC50), in milligrams of melanin per mil-
liliter (mg/mL) (IC50 for acarbose = 8.38 mg/mL).
f
Data shown are the mean of at least three independent experiments and different superscript letters (g–o) in the same column indicate sig-
nificant differences (P < 0.05).

higher than those of any impure melanin (IMe) (Table 1). The
PMe of V. mollis showed the highest AA; PMeD-B-VM was the
most active, being at least 3.9 (ABTS) and 4.1 (FRAP) times higher
than the corresponding activity values of the PMe of
R. echinocarpa and C. alata. Moreover, the AA ABTS-values for
PMe were higher than those obtained by FRAP and boiling ex-
traction only increased AA and TPC of the PMe of V. mollis and
R. echinocarpa (Table 2), as it was observed for the IMe (Table 1).
The AA values of PMe also showed a positive correlation
with their TPC content (ABTS R2 = 0.6505, p = 0.0015; FRAP
R2 = 0.6006, p = 0.0031) as it happened for IMe (see Section 3.1.2).
3.2.3. α-Glucosidase inhibition (αGI)
All of the partially purified melanins (PMe) showed better αGI
than any of the impure melanins (IMe) and acarbose, with that
of VM being the most active, followed by those of RE and CA
(Table 2). Contrasting the αGI values of PMe with those of IMe,
ethanol precipitated (PMeEP) were from 3.6 to 15.3 times better
whereas for the obtained by dialysis (PMeD) the range was from
10.2 to 16.2. In general, αGI values of PMe were only different
for CA (PMeD<PMeEP) and boiling extraction showed a nega-
tive effect only on αGI for the PMe of CA (Table 2).
3.3. Spectroscopical analyses of impure (IMe) and
partially purified (PMe) melanins
3.3.1. UV-Vis spectroscopy
Figure 1 shows the UV-Vis absorption spectra of the mela-
nins obtained at boiling temperature and partially purified by
dialysis (PMeD-B) from VM, RE and CA. They exhibit absorp-
tion peaks in the region from 353 to 296 nm and low or null
absorption in the visible region. The spectral behavior of IMe
and PMeEP (–R and –B) was very similar to that shown in Fig. 1,
with strong absorbances in the range from 289 to 304 nm. All
PMe showed higher absorption units (hyperchromic effect), at
the same concentration, than their corresponding IMe; also,
the λmax were higher for the PMe (bathochromic effect), except
for PMeEP-B-CA, which also showed the lowest increment in
the absorption.
3.3.2. Absorbance ratios (A300/A600)
Partially purified melanins (PMe) showed lower A300/A600 values
than their corresponding impure melanins (IMe), except for the
PMeEP-R-RE sample, which was slightly higher. Moreover, con-
trasting the same fruit and thermal treatment of extraction,
ethanol precipitated melanins (PMeEP) showed higher A300/
A600 ratios than those obtained by dialysis (PMeD) (Table 3). For
VM and RE, boiling during extraction decreased the A300/A600
ratios, but an opposite effect was registered for the CA mela-
nins (Table 3).
Analyzing the A 300 /A 600 ratios and the other measure-
ments, we found negative correlations for: VM with antioxi-
dant activity (AA) (ABTS p = 0.0283, R2 = 73.83; FRAP p = 0.0144,
R2 = 0.8105); CA with the total phenolics content (TPC) (p = 0.0440,
R2 = 0.6780) and AA (ABTS p = 0.0517, R2 = 0.6530; FRAP p = 0.0579,
R2 = 0.6344); whereas correlations were not found in the con-
trast with the properties of RE melanins.
3.3.3. IR spectroscopy
The infrared (IR) spectra of the impure melanins (IMe-R
and IMe-B) from the three fruits were characterized by bands
within the following wave numbers: 3400–3200, 2940–2920, 1630–
1590, 1440–1340 and 1250–990 cm−1 (Fig. 2), and heating during
extraction did not change the melanin spectra. The other evalu-
ated melanins showed similar IR spectra to those of Fig. 2; nev-
ertheless the IR spectra of partially purified melanins (PMe)
 journal of functional foods 9 (2014) 78–88 83

Fig. 1 – UV-Vis absorption spectra of partially purified melanins by dialysis and extracted at boiling temperature of: (a) Vitex
mollis (VM), (b) Randia echinocarpa (RE) and (c) Crescentia alata (CA). Data in the right side of each spectrum correspond to the
rest of the studied samples: impure melanins (IMe) and partially purified melanins by ethanol precipitation (PMeEP) or
dialysis (PMeD); extracted at room (R) and boiling (B) temperature.

(mostly PMeD) showed spectral lines more defined that those

of IMe (Fig. 3). The PMeD IR spectra of VM and CA showed a 4. Discussion
higher number of spectral lines in the range of wavelengths
1500–1000 cm−1. 4.1. Extraction yields of the impure (IMe) and purified
The IR spectra of CA melanins (IMe and PMe) showed a more (PMe) soluble melanins
intense spectral line around 1700 cm−1 (Figs. 2 and 3), and the
spectrum of PMeD-B-CA, compared with those of PMeD-B of The extraction yields obtained for the IMe (69.45–84.07% dw)
VM and RE, showed a less extended peak in 3347 cm−1. (Table 1) were higher than that reported for impure soluble

Table 3 – Absorbance ratios (A300/A600) and concentration of impure melanins (IMe) and partially purified melanins
(PMe)a.
Source A300/A600 of melanin (melanin concentration, mg/mL)b
Impure melanins Partially purified melanins (PMe)
(IMe)
by Ethanol by Dialysis (PMeD)
Precipitation (PMeEP)
R B R B R B
c,v c,w c,v c,x c,y
Vitex mollis (VM) 11.97 10.33 11.42 8.93 2.92 2.91c,y
(3.0) (1.75) (0.5) (0.5) (0.4) (0.4)
Randia echinocarpa (RE) 10.30d,v 9.83d,v 11.12c,w 8.06d,x,y 8.24d,x 7.66d,y
(5.5) (5.5) (0.5) (0.5) (0.5) (0.4)
Crescentia alata (CA) 7.26e,v 7.46e,v 3.52d,w 4.19e,x 3.21c,y 3.89e,z
(0.4) (0.4) (0.5) (0.4) (0.4) (1.0)
R, room temperature; B, boiling temperature. A300 and A600 stand for absorbances of the melanin solutions at 300 nm and 600 nm, respectively.
a
Superscript letters in the same column (c–e) or row (v–z) indicate significant differences (p < 0.05).
b
Concentrations were adjusted to obtain absorbance values at 300 nm around 2 (1.95 ≤ A300 nm ≤ 2.38).
84 journal of functional foods 9 (2014) 78–88
Fig. 2 – Infrared spectra of impure melanins (IMe) obtained at room (IMe-R) (a, b, c) and boiling temperature (IMe-B) (d, e, f)
of Vitex mollis (a, d), Randia echinocharpa (b, e) and Crescentia alata (c, f). Wave number values (cm−1) of the most
representative peaks are shown.
melanins from the fungus Inonotus obliquus (20%) (Mazurkiewicz,
2006). In the case of PMe, the extraction yields in this work (3.63–
19.03% dw) were higher than those reported for pure mela-
nins of black tea (1.36%) (Sava et al., 2001) and fruits and seeds
of Nyctanthes arbor-tristis (0.05%) (Kannan & Ganjewala, 2009),
obtained by alkaline extraction. Because we used water for ex-
traction, our methodology is easier, cheaper and more envi-
ronmentally friendly.
The improved water solubility of melanins from different
sources may be caused by the formation of complexes with
carbohydrates or proteins (Aghajanyan et al., 2011; Seniuk et al.,
2010). Results of this work suggested that plant soluble mela-
nins IMe and PMe showed high water solubility by the forma-
tion of complexes with carbohydrates; i.e., fruits are rich in
carbohydrates; high extraction yields for the obtained soluble
melanins (IMe and PMe) (Tables 1 and 2); positive Tollens’ test
for the supernatants obtained during the preparation of the
PMeEP; purified melanins (PMe) showed lower water solubili-
ties than impure melanins (IMe); and spectroscopic evidence
(see Sections 3.3. and 4.4.).
4.2. Total phenolics contents (TPC) and antioxidant
activities (AA) of the impure (IMe) and purified (PMe)
soluble melanins
The TPC of the V. mollis impure melanin IMe-B-VM (40.15 mg
GAE/g) (Table 1) was higher than the TPC reported for water-
ethanol extracts from skin of grapes (Vitis vinifera cv.
“Tempranillo”) (<1.6 mg catechin/ g) (López de Lerma, Peinado,
& Peinado, 2013). The IMe-B-VM also showed the highest an-
tioxidant activities (Table 1); their values were similar to those
reported for aqueous extracts of roasted and non-roasted coffee
beans (ABTS, 450–630 μmol TE/g) (Liu & Kitts, 2011) and higher
than those reported for the methanol-water extracts of several
red cabbage (Brassica oleracea L. var. capitata L. f. rubra) vari-
eties (ABTS, 87–169 μmol TE/g) (Wiczkowski, Topolska, & Honke,
2014). The high antioxidant activity of melanins was ex-
pected because the protection against UV-radiation and free
radicals generation are within their main functions (Huang
et al., 2011). The edible portion of V. mollis fruits is more exposed
to solar radiation and weather changes than those of R.
echinocarpa and C. alata, which are protected by a hard-
opaque pericarp. The highest TPC and AA of V. mollis could be
associated with this characteristic.
In vegetables, the total phenolics content (TPC) and the an-
tioxidant activity (AA) tend to decrease after cooking (Kaur &
Kapoor, 2001), contrasting with the increased values for the IMe
of VM and RE (Table 1). Melanins are very stable, probably not
so much affected by heating and considering that tempera-
ture of extraction did not affect the extraction yield (Table 1),
thus, the improved TPC of the mentioned fruits could be as-
sociated with differences in the profiles of the impure mela-
nins obtained at room (IMe-R) and boiling (IMe-B) temperatures.
Vitex mollis is traditionally consumed after cooking (Flores-Islas,
1999; Montiel-Herrera, Camacho-Hernández, Ríos-Morgan, &
Delgado-Vargas, 2004), and by contrast with raw fruit, boiled
 journal of functional foods 9 (2014) 78–88 85

Fig. 3 – Infrared spectra of partially purified melanins (PMe) by ethanol precipitation (PMeEP) (a, b, c) and dialysis (PMeD) (d,
e, f) of Vitex mollis (VM) (a, d), Randia echinocharpa (RE) (b, e) and Crescentia alata (CA) (c, f), obtained from impure melanins
extracted at boiling temperature (IMe-B). Wave number values (cm−1) of the most representative peaks are shown.

preparation may be characterized by high TPC and AA improv-
ing its beneficial properties.
Melanins are high molecular weight polymers and less con-
taminated samples must have higher TPC and AA values, as
it was registered, the PMe showed higher values than IMe
(Tables 1 and 2). Moreover, the highest AA of the dialysis pu-
rified melanins (PMeD) suggested their higher purities in con-
trast with the ethanol precipitated (PMeEP). Probably, ethanol
precipitation induced the co-precipitation of other compo-
nents non-bound to melanins (e.g. proteins and oligosaccha-
rides) and if they were characterized by low or null AA, the
observed high extraction yields and the low AA values for the
PMeEP may be explained.
In the ABTS assay, AA values of purified melanins from RE
and CA were similar to those reported for melanoidins (another
aqueous-soluble pigment) of roasted coffee beans (480-
730 μmol TE/g) (Liu & Kitts, 2011), but the AA for the V. mollis
PMe was superior (Table 2). Contrasting with the FRAP method,
the highest ABTS values (Tables 1 and 2) could be associated
with differences in sample composition and in the mecha-
nisms on which the assays are based on. The FRAP method
only detects ferric reducing compounds with potentials lower
than 0.7 V and is not sensitive to antioxidants acting by proton-
transfer reaction. Whereas, the ABTS method measures both
electron- and proton-transfer reactions, sensing lipophilic and
hydrophilic compounds (Re, Pellegrini, Proteggente, & Pannala,
1999). Consequently, synergic interactions among antioxi-
dants impact our results.
The positive correlation of the AA and TPC for the impure
(IMe) and the purified (PMe) soluble melanins could be due to
their structures; melanin (IMe and PMe) molecules have phe-
nolic groups (Harbone, 1984; Marcano & Hasegawa, 2002; Riley,
1997) and their quantification could estimate the pigment con-
centration in the sample.
Natural antioxidants (e.g. green tea extracts) are impor-
tant in the food industry to avoid oxidative reactions in diverse
products (e.g. meat, poultry, emulsions, beverages, snacks) and
to increase their shelf-life (Senanayake, 2013). Some desir-
able characteristics of water-soluble antioxidants are an easy
incorporation to diverse food matrixes, heat stability, low tox-
icity and cost; in particular, heat stability is important to use
such antioxidants in cooked foods. Soluble melanins re-
ported in this work have the aforementioned characteristics
but their lack of toxicity must be corroborated in order to rec-
ommend them as a food ingredient.
4.3. α-Glucosidase inhibition (αGI) of the impure (IMe)
and purified (PMe) soluble melanins
Soluble melanins (IMe and PMe) of the studied fruits were better
inhibitors than acarbose (Tables 1 and 2), a drug used to treat
diabetes mellitus type II (Clissold & Edwards, 1988). The αGI
86 journal of functional foods 9 (2014) 78–88
of extracts from different strawberry cultivars (Fragaria ×
ananassa Duch.) were evaluated by the same methodology used
in this work (IC50 ≈ 25 mg/mL) (Pinto et al., 2008); our values
for the IMe of VM were more than six times lower and those
for RE and CA were comparable. All the PMe were better in-
hibitors than IMe suggesting an association of melanin com-
ponents with the αGI. However, αGI values were neither
correlated with TPC nor with AA, although both of these pa-
rameters increased with purification (Tables 1 and 2); conse-
quently, sample composition was affecting differentially the
mentioned parameters.
The V. mollis melanin obtained at room temperature and by
dialysis (PMeD-R-VM) was one of the most potent α-glucosidase
inhibitors reported in the literature; it was 43 times better than
acarbose. Li et al. (2009) isolated different flavonoids (e.g. api-
genin, luteolin, vitexin, isovitexin) with such activity from
Crataeagu soxyacantha leaves, which were 9 to 17 times supe-
rior to acarbose. In another study, the α-GI by (+)-α-viniferin
isolated from Carex baccans, a plant used to treat diabetes, was
1.5 times higher than acarbose (Kumar, Gupta, Ghosh, Gaonkar,
& Pal, 2013).
Inhibitors of α-glucosidase, like acarbose, decrease the post-
prandial absorption of glucose after eating complex carbohy-
drates, helping with the prevention/ control of the diabetes
illness. In patients with diabetes, glucose oxidation increases
the oxidative stress and the risk of cardiovascular diseases
(Ceriello, 2008). The melanins evaluated here could have a thera-
peutic advantage over acarbose since they combine high αGI
and AA activities (Tables 1 and 2).
On the other hand, natural α-glucosidase inhibitors have
been suggested as antiviral and antitumor agents. Glycopro-
teins are involved in crucial steps of tumor progression and
viral reproductive cycle; thus, blocking protein glycosylation
is a strategy against these illnesses. In particular, the plant
alkaloid castanospermine is one of the best studied
phytochemicals showing antitumoural and antiviral activi-
ties through α-glucosidase inhibition (Atsumi et al., 1993; Pili
et al., 1995; Whitby et al., 2005); moreover, dietary green tea
phenolics inhibit the α-glucosidase II of rat liver microsomes
(Senanayake, 2013). However unlike castanospermine and green
tea phenolics, soluble melanins reported in this work are char-
acterized by high molecular weights, so their bioavailability
must be previously demonstrated.
Plants, fungi and microorganisms have been sources of
several compounds with α-glucosidase inhibitory activity (e.g.
flavonoids, alkaloids, organic acids, and peptides) (Coman et al.,
2012). However, we are reporting for the first time the exis-
tence of soluble melanins with α-glucosidase inhibitory ac-
tivity in different plant sources.
4.4. Spectroscopical analyses of impure (IMe) and
partially purified melanins (PMe)
The UV-Vis absorption spectra of the impure (IMe) and
purified (PMe) soluble melanins (Fig. 1) were similar to those
reported in the literature. Melanins obtained from black
tea, Nyctanthes arbor-tristis, Cinnamomum burmannii,
Osmanthus fragrans and marine Pseudomonas sp. showed λmax
values in the region from 200 to 300 nm and lower absorp-
tion at higher wavelengths (Huang et al., 2011; Kannan &
Ganjewala, 2009; Sava et al., 2001; Tarangini & Mishra, 2013).
This phenomenon is a characteristic of melanins and has been
associated with their complex structure, the presence of phe-
nolics groups as the main chromophores and the formation
of complexes with substances of low or no absorption in the
visible region (Aghajanyan et al., 2011; Seniuk et al., 2010).
As suggested in Section 4.1., water solubility of melanins
(IMe and PMe) may be caused by the formation of complexes
of melanin–carbohydrates. Supporting this proposal, the UV-
Vis absorption spectra of the evaluated melanins suggested that
proteins are not part of the soluble melanin complexes; they
lacked a well defined absorption-peak for aromatic amino
acids (320–380 nm), which should be evident when compar-
ing the spectra of impure (IMe) and partially purified (PMe)
melanins.
In relation with IMe, the UV-Vis absorption spectra of the
PMe showed hyperchromic and bathochromic effects (Fig. 1)
that could be explained by the elimination of components
during the purification process; purer melanins must induce
an additive effect of chromophores characterized by a higher
molar absorption (hyperchromic effect), as well as higher
interactions among these chromophores (bathochromic
effect).
The A300/A600 ratios offer us information about the oxida-
tion state and the range size of the melanin molecules. Melanin
oxidation induces lower absorbance values at 600 nm (A600), and
the A300/A600 absorbance ratio was proposed as a measure of
the oxidation extent, high values corresponded to more oxi-
dized melanin molecules. Also, it was argued that during the
oxidation of melanins, the phenolics are converted to
semiquinones or quinones, which produces more oxidized
(higher A300/A600 absorbance ratios) and smaller melanins (mo-
lecular weight < 1000 Da) (Haywood et al., 2006). Thus, impure
soluble melanins (IMe) showed higher A300/A600 ratios than pu-
rified ones (PMe); and as expected for PMe, the ratios for PMeEP
were higher than PMeD (Table 3). These data support that IMe
are a more complex mixture of melanin molecules than that
of PMe, with variability in the size and degree of oxidation; as
it happened with PMeEP compared with PMeD. Thus, the dif-
ferences in our A300/A600 values were associated with the pu-
rification process (IMe > PMe) and the range in the size of
melanins (PMeEP > PMeD), with PMeEP containing more oxi-
dized and smaller melanins; this last point may be partially
associated with the negative correlation of the A300/A600 ratio
with the antioxidant activity (AA) for VM and CA. Moreover,
soluble melanins of VM and RE obtained by boiling may be
better reduced and characterized by lower A300/A600 ratios
(Table 3). In melanins synthesized by oxidation of (D)L-3-(3,4-
dihydroxyphenyl)-alanine, free protons found in their inter-
molecular spaces mediate the equilibrium between
semiquinones, indolquinones, and hydroquinones forms
(Goncalves, Filho, & Graeff, 2006). The thermal treatment induces
the capture of H+ by the COO− groups present in the molecule
(according the IR spectra), which produces more reduced mela-
nins. This phenomenon could be associated with higher an-
tioxidant activities (AA) of VM and RE for boiled than room
temperature extracted soluble melanins (Tables 1–3). The pattern
found for purified melanins (PMe) of C. alata (CA) was oppo-
site. Data suggested that PMe of CA were more heat sensi-
tive; thus, boiling during extraction produced lower values for
 journal of functional foods 9 (2014) 78–88
TPC and AA (Table 2), associated with higher A300/A600 ratios
(negative correlation) and αGI (Table 3).
The IR bands of the soluble melanins obtained in this work
(Figs. 2 and 3) were similar to melanins of black tea (Sava et al.,
2001), fungi and humic substances (Bilinska, 1996) and
Nyctanthes arbor-tristis fruits (Kannan & Ganjewala, 2009). The
assigned groups for the infrared (IR) spectra of impure mela-
nins (IMe-R and IMe-B) from the three fruits (Fig. 2) were as
follows: 3400–3200 cm−1, associated with –OH and –NH2 groups;
2940–2920 cm−1 corresponding to stretching vibrations of C–H
(from aromatic rings) and C–H (from aliphatic compounds);
1630–1590 cm−1, caused by oscillation of coupled double bonds
of C=C and C=O types; 1440–1340 cm−1, attributed to methy-
lene vibration; 1250–990 cm−1, caused by oscillation of C–O
groups of carbohydrates, alcohols and phenols. Moreover, the
additional and more defined bands for the purified melanins
(PMe), and in particular of the PMeD of VM and CA, at 1500–
1000 cm−1 were possibly associated with alcohol groups (C–
OH from aromatic rings, CH2–OH, CH–OH) and with CH3–, CH2–,
or C–N groups. Peaks around 1100 cm−1 are associated with a
carbohydrate moiety in the water soluble chitin-melanin-
glucan complex (Veleshko et al., 2012). The IR band at 1700 cm−1
for CA melanins (IMe and PMe) (Figs. 2 and 3) was character-
istic of C=O group; whereas differences in the peak in 3347 cm−1
for the PMeD-B-CA corresponded to the presence of O–H groups
(Pretsch, Bèuhlmann, & Badertscher, 2009). This information
could suggest that the CA melanins were polymers of lower-
molecular-mass and less moisture; only during the partial pu-
rification by dialysis of CA melanins, colored pigments of lower
sizes (molecular mass ≤12 kDa) were eliminated, the molecu-
lar interactions were reduced and best defined spectral lines
were registered. However, A300/A600 ratios of the CA melanins
(Table 3) did not correspond with the suggested explanation.
Thus, more
information about the structure of these melanins must be
generated.
5. Conclusions
The soluble melanins from the fruits of V. mollis, R. echinocarpa
and C. alata were extracted and partially purified by facile and
environmentally friendly methods. The analyses suggested that
the aqueous solubility of the obtained melanins could be due
to their presence as melanin-carbohydrate complexes. Based
on the high antioxidant and α-glucosidase inhibitory activi-
ties of these soluble melanins, they could be used as
nutraceutical ingredients for the food and the pharmaceuti-
cal industries; e.g. their use could prevent the oxidative stress
and avoid the increase of postprandial glucose, helping with
the prevention/treatment of diabetes mellitus type II. This is
the first report contrasting the α-glucosidase inhibitory and an-
tioxidant activities attributed to soluble melanins obtained from
fruits.
Acknowledgements
Authors acknowledge the financial support of CONACYT,
PROFAPI –“Universidad Autónoma de Sinaloa” (UAS) and INAPI-
87
Sinaloa, the identification of plant material by Rito Vega Aviña
of the “Facultad de Agronomía” – UAS, as well as the critical
reading of the manuscript by José Ángel López-Valenzuela of
the “Facultad de Ciencias Químico Biológicas” – UAS.
REFERENCES
Aghajanyan, A. A., Asaturian, R. A., Hambardzumyan, A. A.,
Sargsyan, L. B., Hovsepyan, A. S., Vardanyan, A. H., & Saghyan,
A. S. (2011). Obtaining of water soluble microbial melanin and
study of its some properties. Applied Biochemistry and
Microbiology, 47, 500–506.
Aghajanyan, A. E., Hambardzumyan, A. A., Hovsepyan, A. S.,
Asaturian, R. A., Vardanyan, A. A., & Saghiyan, A. A. (2005).
Isolation, purification and physicochemical characterization
of water-soluble Bacillus thuringiensis melanin. Pigment Cell
Research, 18, 130–135.
Atsumi, S., Nosaka, C., Ochi, Y., Iinuma, H., & Umezawa, K. (1993).
Inhibition of experimental metastasis by an α-glucosidase
inhibitor, 1,6-epi-cyclophellitol. Cancer Research, 53,
4896–4899.
Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of
plasma (FRAP) as a measure of antioxidant power: The FRAP
assay. Analytical Biochemistry, 239, 70–76.
Bilinska, B. (1996). Progress of infrared investigations of melanin
structures. Spectrochemica Acta, 52, 1157–1162.
Ceriello, A. (2008). Cardiovascular effects of acute
hyperglycaemia: Pathophysiological underpinnings. Diabetes
and Vascular Disease Research, 5, 260–268.
Clissold, S. P., & Edwards, C. (1988). Acarbose. A preliminary
review of its pharmacodynamic and pharmacokinetic
properties, and therapeutic potential. Drugs, 35,
214–243.
Clugston, M., & Fleming, R. (2000). Advanced chemistry. Oxford, NY,
USA: Oxford University Press.
Coman, C., Rugina, O. D., & Socaciu, C. (2012). Plants and natural
compounds with antidiabetic action. Notulae Botanicae Horti
Agrobotanici Cluj-Napoca, 40, 314–325.
Delgado-Vargas, F., Félix-Favela, F., Pío-León, J. F., López-Angulo,
G., López-Valenzuela, J. A., Díaz-Camacho, S. P., & Uribe-
Beltrán, M. (2010). Antibacterial activity and qualitative
phytochemical analysis of Vitex mollis fruit. International
Journal of Green Pharmacy, 4, 288–291.
El-Obeid, A., Al-Harbi, S., Al-Jomah, N., & Hassib, A. (2006). Herbal
melanin modulates tumor necrosis factor alpha (TNF-α),
interleukin 6 (IL-6) and vascular endothelial growth factor
(VEGF) production. Phytomedicine: International Journal of
Phytotherapy and Phytopharmacology, 13, 324–333.
Flores-Islas, E. (1999). Flora silvestre de Sinaloa su fenología y relación
económica. Mazatlán, Sinaloa, México: Gobierno del estado de
Sinaloa.
Gamberucci, A., Konta, L., Colucci, A., Giunti, R., Magyar, J. E.,
Mandl, J., Banhegyi, G., Benedetti, A., & Csala, M. (2006). Green
tea flavonols inhibit glucosidase II. Biochemical Pharmacology,
72, 640–646.
Gironés-Vilaplana, A., Baenas, N., Villaño, D., Speisky, H., García-
Viguera, C., & Moreno, D. A. (2014). Evaluation of Latin-
American fruits rich in phytochemicals with biological
effects. Journal of Functional Foods, 7, 599–608.
Goncalves, P. J., Filho, O. B., & Graeff, C. F. O. (2006). Effects of
hydrogen on the electronic properties of synthetic melanin.
Journal of Applied Physics, 99, 104701.
Harbone, J. B. (1984). Phytochemical methods. New York, USA:
Chapman and Hall.
Haywood, R. M., Lee, M., & Linge, C. (2006). Synthetic melanin is a
model for soluble natural eumelanin in UVA-photosensitised
88 journal of functional foods 9 (2014) 78–88
superoxide production. Journal of Photochemistry and
Photobiology B, 82, 224–235.
Huang, S., Pan, Y., Gan, D., Ouyang, X., Tang, S., Ekunwe, S. I. N., &
Wang, H. (2011). Antioxidant activities and UV-protective
properties of melanin from the berry of Cinnamomum
burmannii and Osmanthus fragrans. Medicinal Chemistry
Research, 20, 475–481.
Kannan, P., & Ganjewala, D. (2009). Preliminary characterization
of melanin isolated from fruits and seeds of Nyctanthes arbor-
tristis. Journal of Scientific Research, 1, 655–661.
Kaur, C., & Kapoor, H. C. (2001). Antioxidants in fruits and
vegetables – The millennium’s health. International Journal of
Food Science and Technology, 36, 703–725.
Kerestes, J., Kerestes, J., & Vegner, L. A. (2001). Biologically active
fraction of vegetable melanin, process for its production and
its use. Application number EP19990935267. Patent number
EP1104446 A1. United States.
Kumar, D., Ghosh, R., & Pal, B. C. (2013). α-Glucosidase inhibitory
terpenoids from Potentilla fulgens and their quantitative
estimation by validated HPLC method. Journal of Functional
Foods, 5, 1135–1141.
Kumar, D., Gupta, N., Ghosh, R., Gaonkar, R. H., & Pal, B. C. (2013).
α-Glucosidase and α-amylase inhibitory constituent of Carex
baccans: Bio-assay guided isolation and quantification by
validated RP-HPLC–DAD. Journal of Functional Foods, 5, 211–218.
Li, H., Song, F., Xing, J., Tsao, R., Liu, Z., & Liu, S. (2009). Screening
and structural characterization of alpha-glucosidase
inhibitors from hawthorn leaf flavonoids extract by
ultrafiltration LC-DAD-MS(n) and SORI-CID FTICR MS. Journal
of the American Society for Mass Spectrometry, 20, 1496–1503.
Liu, L., Sun, Y., Laura, T., Liang, X., Ye, H., & Zeng, X. (2009).
Determination of polyphenolic content and antioxidant
activity of kudingcha made from Ilex kudingcha C.J. Tseng. Food
Chemistry, 112, 35–41.
Liu, Y., & Kitts, D. D. (2011). Confirmation that the Maillard
reaction is the principle contributor to the antioxidant
capacity of coffee brews. Food Research International, 44, 2418–
2424.
López de Lerma, N., Peinado, J., & Peinado, R. A. (2013). In vitro
and in vivo antioxidant activity of musts and skin extracts
from off-vine dried Vitis vinifera cv. “Tempranillo” grapes.
Journal of Functional Foods, 5, 914–922.
Marcano, D., & Hasegawa, M. (2002). Fitoquímica orgánica. Caracas,
Venezuela: Universidad Central de Venezuela. Consejo de
Desarrollo Científico y Humanistico.
Mazurkiewicz, W. (2006). Analysis of aqueous extract of Inonotus
obliquus. Acta Poloniae Pharmaceutica, 63, 497–501.
Montiel-Herrera, M., Camacho-Hernández, I. L., Ríos-Morgan, A.,
& Delgado-Vargas, F. (2004). Partial physicochemical and
nutritional characterization of the fruit of Vitex mollis
(Verbenaceae). Journal of Food Composition and Analysis, 17, 205–
215.
Norton, P. A., Conyers, B., Gong, Q., Steel, L. F., Block, T. M., &
Mehta, A. S. (2005). Assays for glucosidase inhibitors with
potential antiviral activities: Secreted alkaline phosphatase as
a surrogate marker. Journal of Virological Methods, 124, 167–172.
Pili, R., Chang, J., Partis, R. A., Mueller, R. A., Chrest, F. J., &
Passaniti, A. (1995). The α-glucosidase I inhibitor
castanospermine alters endothelial cell glycosylation,
prevents angiogenesis, and inhibits tumor growth. Cancer
Research, 55, 2920–2926.
Pinto, M. D. S., Kwon, Y. I., Apostolidis, E., Lajolo, F. M., Genovese,
M. I., & Shetty, K. (2008). Functionality of bioactive
compounds in Brazilian strawberry (Fragaria x ananassa
Duch.) cultivars: Evaluation of hyperglycemia and
hypertension potential using in vitro models. Journal of
Agricultural and Food Chemistry, 56, 4386–4392.
Pretsch, E., Bèuhlmann, P., & Badertscher, M. (2009). Structure
determination of organic compounds: Tables of spectral data. Berlin,
Germany: Springer-Verlag.
Pugh, N. D., Balachandran, P., Lata, H., Dayan, F. E., Joshi, V., Bedir,
E., Makino, T., Moraes, R., Khan, I., & Pasco, D. S. (2005).
Melanin: Dietary mucosal immune modulator from Echinacea
and other botanical supplements. International
Immunopharmacology, 5, 637–647.
Re, R., Pellegrini, N., Proteggente, A., & Pannala, A. (1999).
Antioxidant activity applying an improved ABTS radical
cation decolorization assay. Free Radical Biology and Medicine,
26, 1231–1237.
Riley, P. A. (1997). Melanin. The International Journal of Biochemistry
and Cell Biology, 29, 1235–1239.
Santos-Cervantes, M. E., Ibarra-Zazueta, M. E., Loarca-Piña, G.,
Paredes-López, O., & Delgado-Vargas, F. (2007). Antioxidant
and antimutagenic activities of Randia echinocarpa fruit. Plant
Foods for Human Nutrition, 62, 71–77.
Sava, V., Yang, S.-M., Hong, M.-Y., Yang, P.-C., & Huang, G. (2001).
Isolation and characterization of melanic pigments derived
from tea and tea polyphenols. Food Chemistry, 73, 177–184.
Senanayake, S. P. J. N. (2013). Green tea extract: Chemistry,
antioxidant properties and food applications – A review.
Journal of Functional Foods, 5, 1529–1541.
Seniuk, O. F., Gorovoj, L. F., Beketova, G. V., Savichuk, H. O., Rytik,
P. G., Kucherov, I. I., Prilutskay, A. B., & Prilutsky, A. I. (2010).
Anti-infective properties of the melanin-glucan complex
obtained from medicinal tinder bracket mushroom, Fomes
fomentarius (L.: Fr.) Fr. (Aphyllophoromycetideae). International
Journal of Medicinal Mushrooms, 13, 7–18.
Tarangini, K., & Mishra, S. (2013). Production, characterization
and analysis of melanin from isolated marine Pseudomonas sp.
using vegetable waste. Research Journal of Engineering Sciences,
2, 40–46.
Vargas-Solis, R., & Perez-Gutierrez, R. M. (2002). Diuretic and
urolithiatic activities of the aqueous extract of the fruit of
Randia echinocarpa on rats. Journal of Ethnopharmacology, 83,
145–147.
Veleshko, I. E., Veleshko, A. N., Rumyantseva, E. V., Budantseva,
N. A., Gal’braikh, L. S., & Sushinskaya, N. V. (2012). Co-
precipitation of radionuclides by water-soluble melanin from
the chitin-melanin glucan complex “Mikoton” in solutions.
Fibre Chemistry, 44, 83–89.
Wang, H., Pan, Y., Tang, X., & Huang, Z. (2006). Isolation and
characterization of melanin from Osmanthus fragrans’ seeds.
LWT – Food Science and Technology, 39, 496–502.
Waterhouse, A. L. (2002). Determination of total phenolics. In R.
E. Wrolstad, E. A. Decker, M. H. Penner, D. S. Reid, S. J.
Schwartz, C. F. Shoemaker, D. M. Smith, & P. Sporns (Eds.),
Current protocols in food analytical chemistry (p. I1.1, F1.1, F1.).
Hoboken, NJ, USA: John Wiley & Sons, Inc.
Whitby, K., Pierson, T. C., Geiss, B., Lane, K., Engle, M., Zhou, Y.,
Doms, R. W., & Diamond, M. S. (2005). Castanospermine, a
potent inhibitor of dengue virus infection in vitro and in vivo.
Journal of Virology, 79, 8698–8706.
Wiczkowski, W., Topolska, J., & Honke, J. (2014). Anthocyanins
profile and antioxidant capacity of red cabbages are
influenced by genotype and vegetation period. Journal of
Functional Foods, <http://dx.doi.org/10.1016/
j.jff.2014.1002.1011>.
Zhong, Y., Ma, C. M., & Shahidi, F. (2012). Antioxidant and
antiviral activities of lipophilic epigallocatechin gallate
(EGCG) derivatives. Journal of Functional Foods, 4, 87–93.