Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Antioxidant Properties of Water Extracts from Pigmented Rice
Grown in Southern Thailand
Asma Abru, Patcharin Pakdeechanun and Taewee T. Karrila*
Department of Food science and Nutrition, Faculty of Science and Technology.
Prince of Songkla University, Pattani Campus, Thailand
*Corresponding author: e-mail: [email protected], phone: +66-7331-2160
Abstract
Native pigmented rice in southern Thailand are under utilization although their colour and
antioxidant properties are very attractive. To promote pigmented rice its water extracts could be used as
beverage. The aim of this work was to determine properties of water extracts from 8 varieties of
pigmented rice from southern Thailand. The effect of extraction heating time on properties of water
extracts was also studied. De-husked pigment rice grains (8 varieties both waxy and non waxy) were
mixed with water (grain:water = 1:25) and heated at 1000C for 15 min. The water extracts obtained were
determined for colour parameters (L*, a* and b*), total phenol, anthocyanin and antioxidant activities
(DPPH., ABTS+ and SRSA). The results showed that total polyphenol and total anthocyanin contents in
water extracts from all varieties were in the range of 0.18-2.19 mg GAE/ml extracts and 0.11-0.99 mg
Cy-3-G/ml extracts, respectively. However, those from waxy rice had higher content for both polyphenol
and anthocyanin and hence performed higher antioxidant activities shown by DPPH., ABTS+ and SRSA
radical scavenging activity (p<0.05). The water extracts from waxy rice varieties gave lower L*, a* and
b* value indicated darker color.
Keywords: Pigmented rice, extraction, polyphenols, anthocyanins, antioxidant activity
Introduction
Thailand is well known as a rice production country. It has many thousand rice varieties spread
through out the country, including pigmented rice. Many different pigmented rice cultivars have been
collected at Pattani Rice Research Center (PRRC), Southern Thailand. Most of them are now rarely
available for consumption. Optical characteristics and antioxidant properties of 8 varieties of pigmented
rice grain grown in southern Thailand have been reported by Yodmanee (2010). Those rice varieties can
be grouped as dark purple color group and red brown color group. The first group has higher
polyphenolic content than the later group. The phenolic content in rice grain is greatly affected by
genotype and environment (Goffman and Bergman, 2004). Polyphenolic compounds are also known as
natural antioxidants (Hu et al., 2003), which recognized to have protective functions against oxidative
damage, and are associated with reduced risk of chronic diseases (Liu, 2007). They contain a group of
reddish purple water soluble substances called anthocyanins, and that are the primary pigments in the red
and black grains (Abdel-Aal et al., 2006). The major components of anthocyanin in colored rice are
cyanidin-3-glucoside and peonidin-3-glucoside (Yawadio et al., 2007). Those compounds are normally
extracted using methanol or inorganic solvent (Choi et al., 2007) prior to determine their content and
activity, but using water is rarely found. Extract of pigmented rice has very attractive color, and could be
used as food ingredients. Using water as extraction medium would be more interesting due to itsetability.
The aim of this research was to expand utilization of pigmented rice grown in southern, Thailand.
Therefore, the objective of this work was to determined antioxidant properties of water extracts from 8
varieties of pigmented rice available in southern Thailand.
Materails and Methods
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Rice samples
Pigmented rice samples used in this study were harvested in March 2009 and provided by Pattani
Rice Research center, Pattani, Thailand. Eight varieties of pigmented rice grains, consisted of 3 non waxy
rice Homkradunga, (HK), Kumyan (KN), Songyod (SY) and 5 varieties of waxy rice Red waxy rice96060 (RWR-96060), Kramrad (KR), Black waxy rice-96044 (BWR-96044), Black waxy rice-96025
(BWR-96025) and Chormaiphai (CMP). Rough rice grains were dehusked, and brown rice obtained was
stored at 4°C.
Rice water extracts preparation
Rice water extract of all rice varieties was prepared in the same manner as following steps.
Pigmented brown rice (10g) and water (250ml) were mixed, and heated at 100°C for 15 min. Water
extract was then filtered and followed by centrifuging at 1500 rpm for 10 min. Rice extracts were
determined for their properties as following.
Determinations of rice water extract properties
Total solid and light transmission
Total solid of water extract was determined using hot air oven method (AOAC, 2000). 5 ml of
water extracts was weighed and evaporated on water bath and then in hot air oven until it had stable
weight. Total solid content was calculated and expresses as % (w/w). Light transmission of rice water
extract was measured using spectrophotometer (Libra S22, Biochrom, England) at 760 nm (Palou et al.,
1999) and water was used as a blank.
Color
Color of water extracts was determined by HunterLab (ColorQuest XE, HunterLab, USA), color
measurements were expressed as tristimulus parameters, L*, a* and b*. L* indicates lightness (100=white
and 0=black) while a* and b* indicate redness-greenness and yellowness-blueness respectively (Bao et
al., 2005)
Total polyphenol content
Total polyphenols were assayed using the Folin–Ciocalteau method. Briefly, Folin-Ciocalteu
reagent was diluted with water 1:9 (v/v). To 1.25 ml of this reagent, 50 µl of sample was added. After 2
min incubation at room temperature, 1 ml sodium carbonate (75 g/L) was added. The mixture was
incubated for 15 min at 50°C and cooled quickly in an ice-water bath. The absorbance at 760 nm was read
within 15 min. The readings were compared to a standard curve using gallic acid (Aguilar-Garcia et al.,
2007)
Total anthocyanin content
Anthocyanins content was estimated by a pH differential method (Finocchiaro et al., 2010).
Absorbance (Abs) was measured at λmax nm (500nm) and at 700 nm for elimination of interference from
the background. Rice extract (400 µl) was diluted with buffer (2 ml) at pH 1.0 (0.025 mM KCl) and left at
rest for 15 min before measurement, whereas the samples diluted with the buffer pH 4.5 (0.4 mM
CH3COONa) were ready for measurement after 5 min. The corresponding pure buffer solution was used
as reference sample in the spectrophotometer. The absorbance shift was calculated as:
Δabs = (Abs500 nm - Abs700nm) pH 1.0 - (Abs500nm - Abs700nm) pH 4.5
The molar extinction coefficient of cyanidin-3-glucoside (29600 L/mol cm) was used to calculate
the concentration of total anthocyanins, expressed as cyanidin glucoside equivalents in mg/ml of the
extracts.
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Antioxidant capacity assays
Total antioxidant capacity (ABTS˙+, DPPH˙ of rice extracts (0.1 mg/ml) were determined as the
same method described by Choi et al. (2007). For 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)
radical or ABTS˙+ scavenging activity determination. ABTS˙+ solution (6 mL, absorbance of 1.4-1.5 at
414 nm) was added to 0.6 mL of the extracts and mixed thoroughly. The mixtures were kept at room
temperature for 1 hr before an absorbance at 414 nm was read, using a spectrophotometer (Libra S22,
Biochrom, England). The percentage of inhibition was expressed using the following equation:
ABTS˙+ radical scavenging activity (%) = [(Abst=0 – Abst=60 min)/Abst=0] x 100
For 1,1-diphenyl-2-picrylhydrazyl ( DPPH) radical scavenging activity determination, the extract
was first added to 3 mL of 200 µM DPPH solution in methanol. After incubation at room temperature for
30 min, the absorbance at 517 nm was measured using spectrophotometer (Libra S22, Biochrom,
England), and compared to that at 0 min. The percentage of inhibition was expressed using the following
equation:
DPPH radical scavenging activity (%) = [(Abst=0 – Abst=30 min)/Abst=0] x 100
Superoxide radical scavenging activity (SRSA) was also determined. The superoxide radical
which reacts with NBT generates a colored compound with absorbance at 560 nm (Rivero-Perez et al.,
2008). The reactive was made up with 50 µL NADH (77 µM), 50 µL NBT (50 µM), 5 µL of PMS (3.3
µM) and 10µL of the sample. The result was expressed as % inhibition in relation to a control test
(reactive mixtures without the sample) as:
Superoxide radical scavenging activity (%) = (T – T1) x 100/ (T – T2)
T = Abs of SRSA generation system, T1 = Abs of control, T2 = Abs test sample systems
Statistical analysis
All expressed values were mean + SD of triplicates. Data were statistically analyzed by analysis
of variance (ANOVA) test procedure and significant was statistically used to test the influence of rice
varieties, and Duncan’s Multiple Range test was used for comparisons among rice varieties at a
significant level of P<0.05.
Results and Discussion
Color, total solid and light transmission
Color parameters, total solid and light transmission of pigmented rice water extract are presented
in Table 1. The result shows that water extract from pigmented rice had lower L* value, but higher a* and
b* values than those of non waxy rice varieties. This would suggest that water extracts from waxy rice
had darker color. This result is agreed well with the previous study (Yodmanee, 2010). The dehusked
grains of non waxy and waxy rice varieties had been determined for their colors and it was found that
waxy pigmented rice varieties (BWR96025, BWR96044 and CMP) showed dark purple color while non
waxy rice showed red color (SY, KR, RWR96060, HK and KN) (Yodmanee, 2010). Color of water
extract from pigmented rice could be derived from anthocyanins contained in rice seed coat and is water
soluble (Furukawa et al., 2007).
Rice water extract, for all varieties had total solid in the range of 0.08-0.13%. This should be
related to transmission of water extract samples which affected light transmission through. Water extract
from BWR96044 variety showed the highest transmission and lowest total solid. Light transmission in the
range of 61.76-76.70 %, which were significantly different among those varieties. This should be related
to total solid of water extract samples which affected light transmission. Water extract from BWR96044
variety showed the highest transmission and lowest total solid.
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Total polyphenol and anthocyanin content
Total polyphenol in water extract was expressed as mg gallic acid equivalents (GAE) per ml of
the extracts. From Table 2 total polyphenol of all samples was in the range of 0.18-2.19 mgGAE/ml, and
ranked in descending order, as follows: BWR96025, BWR96044, CMP, RWR96060, KR, SY, KN and
HK. Yodmanee (2010) determined phenolic content of rice grain from those varieties, and found that the
first three highest phenolic compounds varieties were BWR96025, BWR96044 and CMP as found in this
study. Many studies have reported that polyphenol contents of the extracts from pigmented cereals,
including pigmented rice, are higher than colorless cereals (Finocchairo et al., 2010; Choi et al., 2007;
Chotimarkorn et al., 2008; Shen et al., 2009).
Table 1 Total solid, transmission and color parameters of pigmented rice extracts*
SY
HK
KN
KR
RWR 96060
CMP
BWR 96025
Total solid
(%)
0.10+0.01bc
0.13+0.01d
0.10+0.00bc
0.12+0.00d
0.09+0.00b
0.11+0.01c
0.09+0.01b
Transmission
(%)
64.86+0.41b
64.63+0.61b
76.30+0.60f
61.76+0.25a
72.03+0.60d
67.67+0.56c
74.10+0.36e
L*
94.18+0.00f
85.04+0.02e
85.57+0.00e
80.68+0.01d
86.15+0.01e
67.87+0.01c
43.66+0.01a
Color parameters
a*
3.30+0.01a
10.54+0.01b
12.04+0.11c
17.90+0.01e
16.96+0.01d
25.19+0.01f
44.95+0.01h
b*
10.49+0.01a
25.21+0.01b
26.02+0.01c
35.70+0.00e
38.38+0.02f
35.15+0.01d
45.58+0.03h
BWR 96044
0.08+0.00a
76.70+0.20f
52.63+0.01b
38.50+0.37g
41.52+0.02g
Rice varieties
Non-waxy
rice
Waxy-rice
*Rice grains: water ratio = 1:25, heating at 100C for 15 min
Mean value + standard deviation of triplicates.
Mean values with different letter in the same column are significantly different (p<0.05)
Total anthocyanin contents varied greatly in the range of 0.11-0.99 mg Cy-3-G/ml of the extracts
among rice varieties. It can be noticed that water extracts from waxy rice grain (KR, RWR96060, CMP,
BWR96025 and BWR96044) had higher anthocyanin content than those of non waxy rice grain. This is
related to their color parameter, especially a* and b*. The anthocyanins are composed of a group of
intensely colored pigments responsible for the purple color (Escribano-Bailon et al.,2004). The two color
parameters (a* and b*) are significantly correlated with phenolics, anthocyanin contents and antioxidant
capacity (Shen et al., 2009; Yodmanee, 2010)
Antioxidant activity
In this study the antioxidant activity of pigmented rice water extracts (rice grain: water = 1: 25,
heating at 100oC for 15 min) was determined by DPPH˙, ABTS˙+ and SRSA assays. The results are
presented in Figure 1. The water extract from waxy rice had DPPH radical inhibition activity in the range
of 52.38-67.82%, and were higher than those of non-waxy rice (31.31-39.59%). The first three highest
varieties were BWR96025, BWR96044 and CMP
ABTS+ method is employed for measuring the relative radical inhibition activity of hydrogen
donating and chain breaking antioxidants (Netzel et al., 2003). From this result, water extract of waxy
rice varieties had higher ABTS+ radical inhibition activity than those of non-waxy rice. The first three
higher values of rice extracts were from BWR96025, BWR96044 and CMP varieties, which were 84.02,
82.32 and 76.20% respectively.
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Table 2 Total polyphenol and total anthocyanin contents of pigmented rice extracts*
Total polyphenol
(mg GAE/ml extracts)
0.28+0.00b
0.18+0.01a
0.26+0.02b
0.73+0.02c
0.81+0.02d
0.96+0.07e
2.19+0.02g
2.16+0.04f
Rice varieties
SY
HK
KN
KR
RWR 96060
CMP
BWR 96025
BWR 96044
Non-waxy rice
Waxy-rice
Total anthocyanin**
(mg Cy-3-G/ml extracts)
0.19+0.00b
0.11+0.00a
0.16+0.01b
0.45+0.00c
0.45+0.00c
0.53+0.01d
0.99+0.02e
0.96+0.03e
*Rice grains: water ratio = 1:25, heating at 100oC for 15 min
** λmax of anthocyanin was 500 nm.
Mean value + standard deviation of triplicates.
Mean values with different letter in the same column are significantly
(a)
DPPH radical scavenging activity (%)
80
70
60
50
40
30
20
10
0
A
different.
B
C
D
E
F
G
H
ABTS radical scavenging activity (%)
90
(b)
80
70
60
50
40
30
20
10
0
A
B
C
D
E
F
G
H
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
SRSA radical scavenging activity (%)
4.5
(c)
4
3.5
3
2.5
2
1.5
1
0.5
0
A
B
C
D
E
F
G
H
Figure 1 Radical inhibition (%) of water extract (0.1 mg/ml) from pigmented rice.
(a), DPPH˙, (b) ABTS+ and (c) and SRSA
A = SY; B,= HK; C= KN; D = KR; E= RWR96060; F= CMP; G= BWR96025;
H = BWR96044
The analysis of SRSA inhibition activity of determined oxygen free radicals could be considered
as a first approach of the potential healthy preventive effect of the moderate consumption of the extracts
(Zhang et al., 2006). The SRSA radical inhibition activity of the water extracts from waxy rice and non
waxy rice varieties were in the ranged of 1.34-3.90% and 0.83-1.23%, respectively. Similar to DPPH and
ABTS+ results, the three higher SRSA radical scavenging activity were waxy rice varieties, BWR96025,
BWR96044 and CMP. This result is agreed well with the previous study. Yodmanee (2010) has reported
for methanolic extracts of these 8 varieties pigmented rice on DPPH˙ and ABTS+ scavenging activity .The
result obtained also shows that BWR96044, BWR96025 and CMP varieties are higher than other
varieties. Choi et al. (2007) reported that, the DPPH radical scavenging activity of the extracts from red
sorghum and black rice showed relatively higher radical scavenging activity than non pigmented samples.
Conclusions
Eight varieties of pigmented rice grown in southern Thailand were determined for their water
extracts properties particularly color and antioxidant properties. Water extract of three varieties of waxy
pigmented rice BWR96025, BWR96044 and CMP had higher antioxidant, both content and activity, as
well as darker color than those of others. The results obtained provide very useful information for
selecting rice variety to expand pigmented rice utilization, especially for values added and healthy
products.
References
Abdel-Aal, E.M., Young, J.C, and Rabalski, I. 2006. Anthocyanin composition in black, blue, pink,
purple and red cereal grains. Journal of Agricultural and Food Chemistry. 54, 4696-4704.
Aguilar-Garcai, C., Gavino, G., Baragoño-Mosqueda, M., Hevia, P. and Gavino, V.C. 2007. Correlation
of tocopheral, tocotrienol, γ-oryzanol and total polyphenol content in rice bran with different
antioxidant capacity assays. Food Chemistry. 120, 1228-1232.
AOAC. 2000. Official Method of Analysis. 16th ed. Verginia: The Associate Analysis Chemists.
Bao, J.S., Cai, Y., Sun, M., Wang, G.Y, and Corke, H. 2005. Anthocyanins, flavonols, and free radical
scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and
stability. Journal of Agricultural and Food Chemistry. 53, 2327-2332.
Cevallos-Casals, B.A. and Cisneros-Zevallos, L. 2004. Stability of anthocyanin-based aqueous extracts of
Andean purple corn and red-fleshed sweet potato compared to synthetic and natural colorants.
Food Chemistry. 86, 69-77.
53
Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Choi, Y., Jeong,H.S., and Lee, J. 2007. Antioxidant activity of methanolic extracts from some Grains
consumed in Korea. Food Chemistry. 103, 130-138.
Chotimakorn, C., Benjakul, S. and Silalai, N. 2008. Antioxidant components and properties of longgrained rice bran extracts from commercial available cultivars in Thailand. Food Chemistry.
111(3), 636-641.
Chung, H.S. and Shin, J.C. 2007. Characterization of antioxidant alkaloids and phenolic acids from
anthocyanin-pigmented rice (Oryza sativa cv. Heuginjubyeo). Food Chemistry. 104(4), 16701677.
Escribano-BailÓn, M.T., Santos-Buelga, C., and Rivas-Gonzalo, C. (2004). Anthocyanins in cereals.
Journal of Chromatography. 1054(1-2), 129-141.
Finocchiaro, F., Ferrari, B. and Gianinetti, A. 2010. A study of biodiversity of flavonoid content in the
rice caryopsis evidencing simultaneous accumulation of anthocyanins and proanthocyanidins in a
black-grained genotype. Journal of Cereal Science. 51, 28-34.
Furukawa, T., Maekawa, M., Oki, T., Suda, I., Lida, S., Shimada, H., Takamure, I. and Kadowaki, K.I.
2007. The Rc and Rd genes are involeved in proanthocyanidin synthesis in rice pericarp. Plant
Journal. 49, 91-102.
Goffman, F.D. and Bergman, C.J. 2004. Rice kernel phenolic content and its relationship with antiradical
efficiency. Journal of the Science of Food and Agriculture. 84, 1235-1240.
Hu, C., Zawistowski, J, Ling, W. 2003.Black rice (Oryza sativa L. indica) pigmented fraction suppresses
both reactive oxygen species and nitric oxide in chemical and biological model systems. Journal
of Agricultural and Food Chemistry. 51, 5271-5277.
Liu, R.H. 2007. Whole grain phytochemicals and health. Journal of Cereal Science. 46, 207-219.
Netzel, M., Strass, G., Bitsch, I., Konitz, R. Christmann, M. and Bitsch, R. 2003. Effect of grape
processing on selected antioxidant phenolics in red wine. Journal of Food Engineering. 56, 223228.
Palou, E., Lopez-malo, A., Barbosa-Canovas, G.V., Welti-Chanes, J. and Swanson, B.G. 1999.
Polyphenoloxidase activity and color of branched and high hydrostatic pressure treated banana
puree. Journal of Food Science. 64, 42-45.
Rivero-Perez, M.D., Muniz, P. and Gonzalez-Sanjose, M.L. 2008. Contribution of anthocyanin fraction to
the antioxidant properties of wine. Food and Chemical Toxicology. 46, 2815-2822.
Shen, Y., Jin, L., Xiao, P., Lu, Y. and Bao, J. (2009). Total phenolics, flavonoids, antioxidant capacity in
rice grain and their relations to grain color, size and weight. Journal of Cereal Science. 49(1),
106-111.
Yawadio, R., Tanimon, S. and Morita, N. 2007. Identification of phenolic compounds isolated from
pigmented rices and their aldose reductase inhibitory activities. Food Chemistry. 101(4), 16161625.
Yodmanee, S. 2010. Qualities of Southern Thailand Native Pigmented Rice. Master of Science Thesis in
Food Science and Nutrition, Prince of Songkla University.
Zhang, M.W., Guo, B.J., Zhang, R.F., Chi, J.W., Wei, Z.C., Xu, Z.H., Zhang, Y. and Tang, X.J. (2006).
Separation, purification and identification of antioxidant compositions in black rice. Agricultural
Science in China. 5(6), 431-440.
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