Composition, structural characteristics, and antitumor properties of polysaccharides from the brown algae Dictyopteris polypodioides and Sargassum sp.

Article abstract of DOI:10.1007/s10600-011-9925-1

The polysaccharide compositions of the brown algae Dictyopteris polypodioides and Sargassum sp. from the Mediterranean Sea were determined. The principal polysaccharide of the studied algae (about 12% of the dry alga weight) was alginic acid. The content of water-soluble polysaccharides was low. The amount of fucoidan was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%. The monosaccharide compositions of fucoidans and neutral polysaccharides were investigated. Experiments on soft agar-agar models showed that fucoidans from D. polypodioides and Sargassum sp. exhibited antitumor activity against RPMI-7951 human melanoma cells.

Full text of DOI:10.1007/s10600-011-9925-1

Chemistry of Natural Compounds, Vol. 47, No. 3, July, 2011 [Russian original No. 3, May-June, 2011]
COMPOSITION, STRUCTURAL CHARACTERISTICS, AND
ANTITUMOR PROPERTIES OF POLYSACCHARIDES FROM
THE BROWN ALGAE Dictyopteris polypodioides AND Sargassum sp.
1*
1
2
R. V. Sokolova, S. P. Ermakova, S. M. Awada,
UDC 582.272.4;582.272.7
1
2
T. N. Zvyagintseva, and H. M. Kanaan
The polysaccharide compositions of the brown algae Dictyopteris polypodioides and Sargassum sp. from
the Mediterranean Sea were determined. The principal polysaccharide of the studied algae (about 12% of
the dry alga weight) was alginic acid. The content of water-soluble polysaccharides was low. The amount
of fucoidan was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%. The
monosaccharide compositions of fucoidans and neutral polysaccharides were investigated. Experiments on
soft agar-agar models showed that fucoidans from D. polypodioides and Sargassum sp. exhibited antitumor
activity against RPMI-7951 human melanoma cells.
Keywords: brown algae, polysaccharides, Dictyopteris, Sargassum, laminaran, fucoidan, alginic acid, biological
activity.
The search for biologically active compounds from natural sources is currently of great interest. Polysaccharide-type
compounds exhibit a broad spectrum of biological activity and low in vivo toxicity.
Brown algae contain compounds with structures that differ from those in red and green algae and terrestrial plants.
Mannitol and comparatively low-molecular-weight 1,3;1,6-ꢀ-D-glucans (laminarans) act as reserve material in them [1]. Salts
of alginic acids (alginates) [2] and complex sulfonated polysaccharides (fucoidans) [3, 4] occur in cell walls and intercellular
space. The latter exhibit a broad spectrum of biological activity. Structure–activity relationships of the polysaccharides are
rather difficult to establish because their structures are heterogeneous, irregular, branched, and highly sulfonated.
The goal of the present work was to study the composition, structural characteristics, and antitumor activity of
polysaccharides from the Mediterranean algae Dictyopteris polypodioides and Sargassum sp.
Alginic acids, neutral polysaccharides, and fucoidans were isolated from these brown algae.
Dried and ground algae were treated with EtOH (70%) to remove low-molecular-weight compounds and then extracted
with HCl solution. The extract containing water-soluble polysaccharides was concentrated, dialyzed, and placed on a column
with the hydrophobic sorbent Polychrom-1. Charged polysaccharides (Dp-F and S-F) were eluted by H O; neutral
2
polysaccharides, by aqueous EtOH (5 and 15%) to afford fractions Dp-5%, Dp-15%, S-5%, and S-15%. Remaining algae
were extracted by Na CO solution. The extracts were dialyzed. Alginic acid was precipitated by three times the volume of
2
3
EtOH (96%) to afford Dp-A and S-A. Fractions Dp-F and S-F contained fucoidans after Polychrom-1 and were separated over
a column of Macro-Prep DEAE to afford Dp-F1, Dp-F2, Dp-F3, and Dp-F4 and S-F1, S-F2, S-F3, and S-F4, which were
lyophilized to produce alginic acids, neutral polysaccharides, and fucoidans. Table 1 presents the yields of polysaccharides.
Table 1 shows that the principal polysaccharide from the studied algae was alginic acid (greater than 12% for both
algae), the content of which depended on the species, season, latitude, and ecological habitat [5]. According to the literature,
the content of alginic acids in algae of the genus Sargassum varies from 3 to 17% [6, 7]. The studied alga could be classified
as a Sargassum containing significant quantities of alginates.
1) Pacific Institute of Bioorganic Chemistry, Far-East Division, RussianAcademy of Sciences, 690022, Vladivostok,
Prosp. 100-Letiya Vladivostoka, 159, fax: 8 (4232) 31 40 50, e-mail: Sokolova-R@yandex.ru; 2) Laboratory of Biotechnology
of Natural Substances and Health Products, Faculty of Pharmacy, Lebanese University, Beirut, Lebanon. Translated from
Khimiya Prirodnykh Soedinenii, No. 3, pp. 297–301, May–June, 2011. Original article submitted October 15, 2010.
©
0009-3130/11/4703-0329 2011 Springer Science+Business Media, Inc.
329

TABLE 1. Polysaccharide Content of Brown Algae Dictyopteris polypodioides and Sargassum sp.
Polysaccharide content, % of dry alga weight
neutral polysaccharides
Alga
alginic acid
fucoidan
EtOH elution
5%
15%
12.04
12.98
0.02
0.10
0.03
0.12
0.15
0.92
Dictyopteris polypodioides
sp.
Sargassum
TABLE 2. Monosaccharide Composition of Neutral Polysaccharides Obtained by Hydrophobic Chromatography over
Polychrom-1
Monosaccharide composition, mol/mol Fuc (Glc**)
Fraction
Yield, %*
Rha
Man
Fuc
Gal
Xyl
Glc
Dp-5%
Dp-15%
S-5%
0.02
0.03
0.10
0.15
0.3
0.1**
0.4
0.3
0.1**
0.2
1.0
0.1**
1.0
0.2
0.1**
0.4
0.1
0.1**
0.1
0.6
1.0**
0.6
S-15%
0.4
0.2
1.0
0.4
0.1
0.5
______
*% of dry alga weight; **monosaccharide composition, mol/mol Glc.
The ratio of mannuronic and glucuronic acid units (M/G) for the Dp-A and S-A fractions isolated by us was calculated
from the ratio of intensities of the corresponding resonances in the PMR spectrum. The quantity M/G for D. polypodioides
and Sargassum sp. was 2.0 and 1.5, respectively. According to the literature, the M/G ratio for many algae of the genus
Sargassum varies from 0.19 to 1.56. Alginates isolated from S. vulgare (Brazil) contained a large amount of mannuronic acid
[7], in contrast with alginates isolated from S. dentifolium, S. asperifolium, and S. latifolium (Egypt), which had high contents
of guluronic acid units [8]. Information on the content of alginic acids in algae of the genus Dictyopteris has not been
reported.
The algae studied by us, D. polypodioides and Sargassum sp., contained few water-soluble polysaccharides. The
amount of fucoidan in the studied algae was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%.
According to the literature, the content of water-soluble polysaccharides isolated from S. tenerrimum (India, Okha) was about
3% [6]; of polysaccharides isolated from S. stenophyllum (Brazilia, Bombinhas), about 0.4% calculated for dry alga weight
[9]. The differences in the polysaccharide compositions of algae of the genus Sargassum are probably related to the fact that
the specimens were collected at different sites at different times and were different species. The content of water-soluble
polysaccharides in algae of the genus Dictyopteris has not been reported.
Fractions Dp-5% and S-5%, which were eluted from Polychrom-1 by aqueous EtOH (5%), gave after hydrolysis
large amounts of fucose, mannose, rhamnose, and galactose and minor amounts of xylose (Table 2). The structures of these
13
compounds were not analyzed by C NMR and mass spectrometry because their monosaccharide compositions were highly
heterogeneous.
Fraction DP-15% gave a large amount of glucose and minor amounts of other monosaccharides upon hydrolysis. The
C NMR spectrum of isolated fraction Dp-15% (not shown) showed resonances with chemical shifts 103.21 (C-1), 73.98
13
(C-2), 84.86 (C-3), 68.81 (C-4), 76.31 (C-5), and 61.41 (C-6) ppm that were characteristic of 1,3-bound ꢀ-D-glucopyranose
13
[10]. The C NMR spectrum also showed a resonance with chemical shift 64.5 ppm that was characteristic of mannitol. In
addition to these strong resonances, there were additional weaker resonances with chemical shifts 96.58 and 92.77 ppm that
corresponded to C-1 of ꢁ- and ꢀ-Glcp and indicated that the compound was an oligosaccharide. This hypothesis was confirmed
by data obtained from MALDI-TOF mass spectrometry.
Fraction Dp-15% was a mixture of 1,3-ꢀ-D-glucooligo- and polysaccharides, the degree of polymerization of which
was in the range 7–30.
In contrast with Dp-15%, fraction S-15%, which was eluted from Polychrom-1 by aqueous EtOH (15%), was a
13
heterogeneous polysaccharide. Table 2 presents the monosaccharide composition, which was also confirmed by C NMR
spectroscopy (spectrum not shown). It is known that many algae of the genus Sargassum contain practically no laminaran [6].
This agreed with our results.
330

TABLE 3. Characteristics of Fucoidans Obtained by Anion-Exchange Chromatography over Macro-Prep DEAE*
Content, %***
Monosaccharide composition, mol/mol Fuc
Fucoidan
Eluent [NaCl], M
Yield, %**
–
NaSO3
polyphenols
Rha
Man
Fuc
Gal
Xyl
Glc
Dp-F1
Dp-F2
Dp-F3
Dp-F4
S-F1
S-F2
S-F3
0–0.3
2.30
22.50
6.52
15.73
2.07
13.99
10.40
12.78
5.78
12.67
13.06
13.41
6.20
19.64
23.33
28.79
–
–
–
0.2
0.2
0.2
0.3
0.2
0.1
0.1
0.1
0.3
0.3
0.3
0.1
0.3
0.2
0.1
0.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.3
0.1
0.2
0.8
0.4
0.3
0.4
0.3
0.2
0.4
0.2
0.2
0.2
0.2
0.1
0.1
0.3
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.4–0.7
0.7–0.8
0.9–1.0
0.1–0.2
0.5–0.8
0.9–1.0
1.2–1.4
–
1.8
4.9
7.6
6.0
S-F4
______
*Protein-type compounds determined by the Bradford method [26] were absent in all fractions; **% of water-soluble fraction
content; ***% of weight.
a
b
OD490
2.0
OD490
1.2
C (NaCl), M
C (NaCl), M
Dp-F2
S-F2
2.0
2.0
1.5
1.0
Dp-F4
1.0
0.8
0.6
0.4
0.2
1.6
1.2
Dp-F3
1.5
1.0
0.5
0.0
S-F3
S-F4
Dp-F1
0.8
0.4
S-F1
0.5
0.0
0.0
0
0.0
0
50 100 150 200 250 300 350 400 450
V, mL
50 100 150 200 250 300 350 400 450
V, mL
–
Fig. 1. Ion-exchange chromatography over Macro-Prep DEAE (Cl -form, 8 ꢂ 2.5 cm) of fucoidans from Dictyopteris
polypodioides (a) and Sargassum sp. (b).
Fractions Dp-F and S-F contained after Polychrom-1 fucoidans that were separated over Macro-Prep DEAE by
elution with a linear gradient H O–NaCl (2 M). Figure 1 shows the elution profile for D. polypodioides and Sargassum sp.
2
Each alga afforded four fractions: Dp-F1, Dp-F2, Dp-F3, and Dp-F4 and S-F1, S-F2, S-F3, and S-F4. Table 3
presents their characteristics.
Table 3 presents the monosaccharide compositions of the isolated polysaccharides. Fractions obtained from anion-
exchange chromatography of polysaccharides Dp-F and S-F gave upon hydrolysis large quantities of fucose, galactose, rhamnose,
mannose, and xylose in addition to minor amounts of glucose. The contents of mannose and rhamnose decreased in both algae
on going from the first to the fourth fraction.
Fucoidans (S-F1, S-F2, S-F3, and S-F4) isolated from Sargassum sp. were sulfonated heterofucans, like analogous
polysaccharides isolated from algae of this genus (S. stenophyllum [9], S. tenerrimum [6], and S. hemiphyllum [10]).
Structural characteristics of fucoidans isolated from algae of the genus Dictyopteris (family Dictyotaceae) have not
been reported. However, it is known that other algae belonging to this family have afforded both practically pure sulfonated
fucans (Stoechospermum marginatum [11]) and heterogeneous polysaccharides (Spatoglossum schroederi [12], Lobophroa
variedata [13], Padina gymnospora [14], and Dictyota menstrualis [15]).
Fucoidans isolated from D. polypodioides did not contain polyphenols whereas the polyphenol content in fucoidans
isolated from Sargassum sp. varied from 1.8 to 7.6%. All isolated fucoidans contained no proteins. The degree of sulfonation
in the fucoidans increased with increasing eluent concentration. Thus, we isolated fucoidans with both low and high degrees
of sulfonation. Table 3 gives the values.
331

108%
1200
91%
100%
91%
72%
*
59%
*
800
400
56%
*
0
0
C
50
100
200
50
100 200
C,
ꢀg/mL
Distyopteris polypodioides
Sargassum sp.
Fig. 2. Growth inhibition of RPMI-7951 human melanoma cell colonies by
fucoidans (Dp-F4, S-F4).
Fucoidans isolated from D. polypodioides and Sargassum sp., like many other natural algal fucoidans, have complicated
C NMR spectra (not shown) that are difficult to interpret fully. They all contained strong resonances in the anomeric region
13
(96–102 ppm) and at high frequencies (15–17 ppm) that were typical of ꢁ-fucopyranosides. Resonances at 19–21 ppm (CH )
3
and 173–176 (C=O) corresponded to acetyls in spectra of fractions Dp-F3, Dp-F4, S-F1, and S-F4.
Polysaccharides isolated from algae are known to exhibit various biological activities such as antitumor, antiviral,
anti-inflammatory, antiangiogenic, and immunostimulating. Thus, water-soluble polysaccharides and alginates isolated
from algae of the genus Sargassum exhibited antitumor properties [16–20]; sulfonated polysaccharides, antiviral properties
[6, 21–23].
According to the literature, sulfonated polysaccharides isolated from D. delicatula exhibited antioxidant and
anticoagulant activity [24]. Fucoidans isolated from algae of the family Dictyotaceae exhibited antiviral [11], antithrombotic
[12], and anticoagulant [13–15] activity. Therefore, it seemed interesting to determine the antitumor activity of the
polysaccharides isolated by us. Treatment of RPMI-751 cells with fucoidans from D. polypodioides and Sargassum sp.
(Dp-F4 and S-F4) at concentrations up to 200 ꢃg/mL did not inhibit their growth and were not associated with extensive cell
death. The growth inhibition of RPMI-7951 cell colonies treated with fucoidan from D. polypodioides at a dose of 200 ꢃg/mL
was 44%. Fucoidan from Sargassum sp. was a weaker inhibitor (28%) (Fig. 2).
The studied fucoidans had different structural characteristics with respect to both the monosaccharide composition
and degree of sulfonation. Apparently the differences in the antitumor activities among fucoidans from various sources are
due to these structural features.
EXPERIMENTAL
Reagents and Materials. EtOH, acetone, NaOH, and inorganic acids and salts were commercial products (Diaem,
Russia). Standards (mannose, rhamnose, maltose, glucose, galactose, xylose, bovine serum albumin) were obtained from
Sigma (USA). Sorbents for chromatography were Polychrom-1 (Reakhim, Russia) and Macro-Prep DEAE (Bio-Rad, USA).
Brown algae Dictyopteris polypodioides and Sargassum sp. were collected on the Mediterranean coast (Lebanon) in
July 2009 (Batroun: N 34° 15.069ꢄ, E 35° 39.3852 ; Barbara: N 34° 12.034ꢄ, E 35° 38.180ꢄ). Dried and ground algae were
treated with EtOH (70%, 1:1) for subsequent isolation of polysaccharides.
Carbohydrate contents were determined colorimetrically using the reaction with phenol and H SO [25]; protein
2
4
contents, by the Bradford method [26] using bovine serum albumin as a standard; polyphenol contents, colorimetrically by
the Folin–Ciocalteau method [27].
Extraction of Polysaccharides. Dried defatted algae Dictyopteris polypodioides and Sargassum sp. (50 g) were
extracted with HCl solution (0.1 M, 1:20 ratio, 2 ꢂ) for 2 h at 60°C. The extracts were neutralized and centrifuged. The
supernatant was concentrated in a rotary evaporator, dialyzed against distilled water, and evaporated to dryness to afford
fractions containing water-soluble polysaccharides. The algae remaining after the extraction were extracted with Na CO
2
3
solution (2%, 1:30 ratio) for 2 h at 55°C. The extracts were centrifuged. The supernatant was dialyzed against distilled water.
Alginic acid was precipitated from the extracts by three times the volume of EtOH (96%). The precipitates were washed
successively with EtOH (96%) and acetone and dried in air to afford Dp-A (6.9 g) and S-A (8.4 g).
332

Hydrophobic Chromatography over Polychrom-1 (Polytetrafluoroethylene). The extract containing water-soluble
polysaccharides was placed on a column of Polychrom-1 (15 ꢂ 6.5 cm). Charged polysaccharides (fucoidans Dp-F, 0.16 g;
S-F, 1.52 g) were eluted with water (until the reaction of the eluent for total carbohydrates was negative; phenol–H SO
2
4
method). Neutral polysaccharides were eluted by aqueous EtOH (5 and 15%, until the reaction of the eluent for total hydrocarbons
was negative). The resulting fractions were evaporated in a rotary evaporator, frozen, and lyophilized to afford Dp-5%,
Dp-15%, S-5%, and S-15% (0.01, 0.02, 0.05, and 0.06 g, respectively).
Anion-Exchange Chromatography. Polysaccharide fractions (Dp-F and S-F) were dissolved in HCl (0.04 M),
placed on a column of Macro-Prep DEAE (8 ꢂ 2.5 cm) and eluted by a linear gradient of NaCl (2 M). The resulting fractions
Dp-F1, Dp-F2, Dp-F3, and Dp-F4 and S-F1, S-F2, S-F3, and S-F4 (0.01, 0.04, 0.01, 0.03, 0.03, 0.21, 0.16, 0.20 g, respectively)
were dialyzed against distilled water, evaporated in a rotary evaporator, and dried (lyophilized).
Acid Hydrolysis of Polysaccharides. A sample (5 mg) was dissolved in TFA (0.5 mL, 2 N). The hydrolysis was
performed at 100°C for 6 h. The sample was neutralized by aqueous ammonia and evaporated to dryness.
Monosaccharide compositions of the polysaccharides after acid hydrolysis were determined on a Biotronik
IC-5000 analyzer (Germany) using a Shim-pack ISA-07/S2504 column (0.4 ꢂ 25 cm) with elution by potassium borate buffer
at 0.6 mL/min. Detection used the bicinchonate method and integration by the Shimadzu C-R2 AX system. The standards
were maltose, rhamnose, mannose, fucose, galactose, xylose, and glucose.
Sulfonate contents in the polysaccharides were determined by turbidimetry [28].
13
PMR and C NMR spectra of D O solutions of the compounds were taken on a Bruker DPX-500 NMR spectrometer
2
at 303 K.
Cell Cultivation. RPMI-7951 human melanoma cells were cultivated in MEM growth medium with FBS (10%),
penicillin (100 U/L), and streptomycin (100 ꢃg/L) in an MCO-18AIC incubator (Sanyo, Japan) at 37°C with 5% CO .
2
4
Determination of Fucoidan Cytotoxicity. RPMI-7951 human melanoma cells (2 ꢂ 10 /mL) were seeded in a
96-well plate and cultivated in RPMI-1640 (10%, 200 ꢃL) in a CO -incubator at 37°C for 24 h. Then, cells were treated with
2
fucoidans at various concentrations (10, 50, 100, and 200 ꢃg/mL), incubated for 24 h, treated with 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT-reagent), and incubated for 4 h (37°C, 5% CO ). The optical density (OD) was
2
measured on a Bio-Tek Instruments spectrophotometer (USA) at 490/630 nm (A
).
490/630
Neoplastic Transformation of Cells (soft agar-agar model). The activity of the fucoidans from brown algae on the
formation and growth of colonies of RPMI-7951 human melanoma cells was determined by the soft agar-agar model.
3
Melanoma cells (8 ꢂ 10 cells) were treated with fucoidans from brown algae D. polypodioides and Sargassum sp. (50, 100,
and 200 ꢃg/mL) in BME agar-agar containing FBS (10%). Cells were cultivated at 37°C and 5% CO for 20 d. Colonies of
2
RPMI-7951 human melanoma cells were estimated using a Motic AE 20 inverted microscope (China) and the Motic Image
Plus program.
Statistical processing of results was carried out using Student’s t-criterion with 95% confidence probability (SigmaPlot
2000 program, Version 6, SPSS Inc., USA)
ACKNOWLEDGMENT
The work was supported by RFBR Grant No. 09-04-00761-a and the Molecular Cellular Biology Program. We thank
Prof. V. V. Isakov and Cand. Chem. Sci. S. D. Anastyuk (PIBOC, FEB, RAS) for recording spectra and assisting with their
interpretation.
REFERENCES
1.
2.
3.
4.
5.
6.
T. E. Nelson and B. A. Lewis, Carbohydr. Res., 33, 63 (1974).
A. I. Usov, Usp. Khim., 68, 1051 (1999).
T. Hu, D. Liu, Y. Chen, J. Wu, and S. Wang, Int. J. Biol. Macromol., 46, 193 (2010).
B. Kloareg and R. S. Quatrano, Oceanogr. Mar. Biol., 26, 259 (1988).
E. D. Obluchinskaya, G. M. Voskoboinikov, and V. A. Galynkin, Prikl. Biokhim. Mikrobiol., 38, 213 (2002).
S. Sinha, A. Astani, T. Ghosh, P. Schnitzler, and B. Ray, Phytochemistry, 71, 235 (2010).
333

7.
M. R. Torres, A. P. Sousa, E. A. Silva Filho, D. F. Melo, J. P. Feitosa, R. C. de Paula, and M. G. Lima, Carbohydr.
Res., 342, 2067 (2007).
8.
9.
10.
11.
B. Larsen, D. M. Salem, M. A. Sallam, M. M. Mishrikey, and A. I. Beltagy, Carbohydr. Res., 338, 2325 (2003).
M. E. R. Duarte, M. A. Cardoso, M. D. Noseda, and A. S. Cerezo, Carbohydr. Res., 333, 281 (2001).
T. N. Zvyagintseva, N. I. Shirokova, and L. A. Elyakova, Bioorg. Khim., 20, 1349 (1994).
U. Adhikari, C. G. Mateu, K. Chattopadhyay, C. A. Pujol, E. B. Damonte, and B. Ray, Phytochemistry, 67,
2474 (2006).
12.
13.
14.
15.
H. A. Rocha, L. C. Bezerra, I. R. de Albuquerque, L. S. Costa, C. M. Guerra, L. D. de Abreu, H. B. Nader, and
E. L. Leite, Planta Med., 71, 379 (2005).
V. P. Medeiros, K. C. Queiroz, M. L. Cardoso, G. R. Monteiro, F. W. Oliveira, S. F. Chavante, L. A. Guimaraes,
H. A. Rocha, and E. L. Leite, Biochemistry, 73, 1018 (2008).
T. M. Silva, L. G. Alves, K. C. de Queiroz, M. G. Santos, C. T. Marques, S. F. Chavante, H. A. Rocha, and
E. L. Leite, Braz. J. Med. Biol. Res., 38, 523 (2005).
I. R. Albuquerque, K. C. Queiroz, L. G. Alves, E. A. Santos, E. L. Leite, and H. A. Rocha, Braz. J. Med. Biol. Res.,
37, 167 (2004).
16.
17.
18.
A. M. Gamal-Eldeen, E. F. Ahmed, and M. A. Abo-Zeid, Food Chem. Toxicol., 47, 1378 (2009).
Q. Y. Liu and Q. Y. Meng, Nanfang Yike Daxue Xuebao, 28, 1378 (2008).
P. F. Dias, J. M. Siqueira, Jr., L. F. Vendruscolo, T. de Jesus Neiva, A. R. Gagliardi, M. Maraschin, and
R. M. Ribeiro-do-Valle, Cancer Chemother. Pharmacol., 56, 436 (2005).
19.
20.
A. de Paula Alves Sousa, P. S. Barbosa, M. R .Torres, A. M. Martins, R. D. Martins, R. de Sousa Alves,
D. F. de Sousa, C. D. Alves, L. V. Costa-Lotufo, and H. S. Monteiro, J. Appl. Toxicol., 28, 364 (2008).
O. S. Vishchuk, S. P. Ermakova, Fam Dyuk Tin, N. M. Shevchenko, Bui Ming Li, and T. N. Zvyagintseva,
Tikhookeanskii Med. Zh., No. 3, 92 (2009).
21.
22.
23.
24.
W. Zhu, L. C. Chiu, V. E. Ooi, P. K. Chan, and P. O. Ang, Jr., Phytomedicine, 13, 695 (2006).
S. Preeprame, K. Hayashi, J. B. Lee, U. Sankawa, and T. Hayashi, Chem. Pharm. Bull., 49, 484 (2001).
T. Hoshino, T. Hayashi, K. Hayashi, J. Hamada, J. B. Lee, and U. Sankawa, Biol. Pharm. Bull., 21, 730 (1998).
G. P. Fidelis, S. L. Cordeiro, R. M. Oliveira, D. A. Sabry, R. B. Camara, and L. T. Nobre, Biomed. Pharmacother.,
64, 21 (2010).
25.
26.
27.
28.
M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, Anal. Chem., 28, 350 (1956).
M. M. Bradford, Anal. Biochem., 72, 248 (1976).
V. L. Singleton and J. A. Rossi, Am. J. Enol. Vitic., 16, 144 (1965).
K. S. Dodgson and R. G. Price, Biochem. J., 84, 106 (1962).
334