Research Article

Received: 22 October 2010 Revised: 7 January 2011 Accepted: 10 January 2011 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4333

Comparisons of protein, lipid, phenolics,

γ -oryzanol, vitamin E, and mineral contents
in bran layer of sodium azide-induced red rice
mutants
Toong Long Jeng,a Pei Tzu Ho,a Yi Ju Shih,a Chia Chi Lai,a Min Tze Wua
and Jih Min Sungb∗

Abstract
BACKGROUND: The bran part of red rice grain is concentrated with many phytochemicals, including proanthocyanidins,
oryzanol and vitamin E, that exert beneficial effects on human health, but it contains low levels of essential minerals such as Fe
and Zn. In the present study, the protein, lipid, phytochemicals and mineral contents in bran samples were compared among
red rice SA-586 and its NaN3 -induced mutants.

RESULTS: The plant heights of NaN3 -induced mutants were decreased. The contents of protein, lipid, total phenolics, total
flavonoids, total anthocyanins, total proanthocyanidins, total γ -oryzanol, total tocopherols and total tocotrienols also varied
among the tested mutants. The brans of mutants M-18, M-56 and M-50 contained more proanthocyanidins, γ -oryzanol,
vitamin E than that of SA-586, respectively. M-54 accumulated more Fe content (588.7 mg kg−1 bran dry weight) than SA-586
(100.1 mg kg−1 bran dry weight).

CONCLUSIONS: The brans of M-18, M-50 and M-56 are good sources of proanthocyanidins, vitamin E and γ -oryzanol, respectively,
while the bran of M-54 is rich in Fe. Thus these mutants could be used to produce high-value phytochemicals or Fe byproducts
from bran during rice grain milling or as genetic resources for rice improvement programs.

c 2011 Society of Chemical Industry

Keywords: anthocyanin; mutant; oryzanol; proanthocyanidins; red rice; tocotrienols

INTRODUCTION regions where red rice is consumed as a major carbohydrate

Rice (Oryza sativa L.) is consumed by about half of the world’s
population. Most rice varieties grown for human consumption are
white bran rice. However, some rice varieties produce grains with
a red, brown or purple bran layer. Cultivation and consumption
of red rice is limited in Western countries, but in some Asia
countries such as Bhutan, India, Sri Lanka and Philippines red rice
is cultivated and consumed as a traditional staple crop.1 Red rice
is also considered as a functional food in some growing areas
of China, Korea and Japan.2 The pigmentations in bran part are
mainly proanthocyanidins,3 which are colorless in vivo but are
oxidized into red colored pigments during grain desiccation. The
changes from white to red bran in rice grain occur through
spontaneous mutation, but it may also arise from chemical-
induced mutation.4 Red rice also contains considerable levels of
vitamin E and γ -oryzanol in its bran layer.5 These phytochemicals
are known to decrease oxidative stress in vivo and thus exert
beneficial effects on human health.6
Although red rice is concentrated with some phytochemicals
similar to those reported in common rice varieties,7 red rice
contains low levels of important mineral nutrients (e.g. Fe).8
Therefore increasing Fe content in red rice grain may help to
alleviate the deficiency incidences of these mineral elements in
source. A genetically modified approach is an effective strategy to
increase Fe content in rice grains,9 but genetically modified rice
may not be acceptable to all consumers. Conventional breeding
seems to offer a manageable and non-controversial solution.10
Therefore varieties with high grain Fe content are required as the
genetic source for a breeding program.
Chemical mutation has been used to create and increase genetic
variability in crop species and ultimately improves some plant
traits.11 The main advantage of chemical mutation is the possibility
of improving one or two specific characters without altering other
acceptable genotypes. SA-586 is a high yielding red rice line
mutated from a japonica type variety Tainung 67 by the Agricultural
Research Institute in Taiwan (Wufeng, Taiwan). However, the
∗ Correspondence to: Jih Min Sung, Department of Food Science and Technology,
Hungkuang University, Shalu, Taichung County, Taiwan, ROC.
E-mail: sungjm@sunrise.hk.edu.tw
a Biotechnology division, Taiwan Agricultural Research Institute, Wufeng,
Taichung County, Taiwan, ROC
b Department of Food Science and Technology, Hungkuang University, Shalu,
Taichung County, Taiwan, ROC

J Sci Food Agric (2011) www.soci.org

c 2011 Society of Chemical Industry
 www.soci.org
Figure 1. The dehulled grains of wild type SA-586 and its sodium azide-
induced mutants used in this study.
major drawback for commercializing SA-586 is its relatively
taller plant height (about 1.4 m), which makes it susceptible to
lodging. Therefore sodium azide (NaN3 ) is employed to induce
mutational changes in SA-586, and several mutants with reduced
plant height (average of 0.8 m) were identified and collected.
In the present study, an attempt was made to compare the
contents of several antioxidants, including phenolics, flavonoids,
anthocyanins, proanthocyanidins, γ -oryzanol, tocopherols and
tocotrienols, in bran parts of SA-586 and its selected NaN3 -
induced mutants with red bran. The composition of several
mineral elements was also determined and compared. Knowledge
of these variations should provide useful information concerning
the potential value of these mutants to breeding programs of red
rice.
EXPERIMENTAL
Plant materials
Grains of red rice line SA-586 and its eight NaN3 -induced red bran
mutants (M6 generation) (Fig. 1) with decreased plant height were
obtained from the Taiwan Agricultural Research Institute (Wufeng,
Taichung County, Taiwan, ROC). A mutant that changed from red
to white bran (M-30) was also selected to serve as a comparison. All
agronomic characteristics such as plant height, growth duration
and yield traits were recorded in the spring of 2009. The harvested
grains were stored at −4 ◦ C until used for research.
Prior to chemical analysis, all the grains were mechanically
dehulled using a Satake THU-35 dehusker (Satake Corp., Hiroshima,
Japan), and the resulting brown rice was milled using a Satake
Motor one-pass mill (Tokyo, Japan). The bran (including germ
parts) was sieved through a 0.84 mm sieve (EH Sargent & Co.,
Chicago, IL, USA), and stored at −20 ◦ C.
Chemicals
Gallic acid, quercetin, β-nicotinamide adenine dinucleotide re-
duced disodium salt hydrate (NADH) and lecithin, were purchased
from Sigma-Aldrich (St Louis, MO, USA). Folin–Ciocalteu phe-
nol reagent was purchased from Merck (Darmstad, Germany).
Purified delphinidin, cyanidin and petunidin for analysis by high-
performance liquid chromatography (HPLC) were purchased from
Extrasynthase (Genay, France). γ -Oryzanol standards were pur-
chased from Wako (Tokyo, Japan). Tocopherol and tocorienol
standards were purchased from Advanced Chemistry Develop-
ment, Inc. (Toronto, Canada). All other chemical reagents used
were of analytical grade.
Protein and lipid determinations
Protein content was determined using the Kjeldahl procedure. The
lipid content was determined gravimetrically with hexane for 12 h
in a Soxhlet extractor.
wileyonlinelibrary.com/jsfa

c 2011 Society of Chemical Industry
TL Jeng et al.
Total phenolics and total flavonoid determinations
Ground rice bran samples (1 g) were extracted with 20 mL
methanol (80 : 20, v/v in H2 O) for 15 min, and the filtrate volume
was refixed to 20 mL with extraction reagent. The content of total
phenolics in extracts was estimated using the Folin–Ciocalteu
assay.5 The absorption value was determined at 735 nm with
gallic acid used as standard.
Total flavonoid content was measured using the aluminium
chloride colorimetric assay12 with some modifications. Briefly,
an aliquot (0.5 mL) of extracts was mixed with 2.8 mL distilled
H2 O, 1.5 mL ethanol (95 : 5, v/v), 0.1 mL aluminium chloride
(1 : 10, w/v) and 0.1 mL of 1 mol L−1 potassium acetate. After
incubation at room temperature for 40 min, the absorbance of
the reaction mixture was measured at 415 nm with a Hitachi U-
2900 spectrophotometer (Tokyo, Japan); quercetin was used in the
construction of the standard curve. Total flavonoid content was
expressed as g quercetin equivalent (QE) kg−1 dry plant material.
Total proanthocyanidins and total anthocyanin determination
Ground rice brans (0.1 g) were extracted with 1.0 mL methanol
containing diluted HCl (1 : 99, v/v in methanol) at 4 ◦ C for
24 h. Part of the methanol extract was further acidified with
2.5 mL of 2.4 mol L−1 HCl at 100 ◦ C for 40 min to hydrolyze
anthocyanidins from proanthocyanidin polymers. Prior to analysis
all the samples were filtered through a 0.45 µmol L−1 membrane
filter and concentrated at 30 ◦ C in vacuo (model SPD111V,
Thermo Savant, USA). The concentrated extract was analyzed
using HPLC (model 2695, Waters, Milford, MA, USA) using an
Inertsil (Tokyo, Japan) ODS-3 column (4.6 × 250 mm, 5 µm; pre-
column: ODS-3, 4.6 × 33 mm, 5 µm). The flow rate was set at
1 mL min−1 using H2 O–acetic acid–methanol (69 : 10:21) with
monitoring at 530 nm, and the column temperature was set at
30 ◦ C. Anthocyanin contents were calculated by HPLC peak areas
and compared with external standard calibration curves. Linear
standard calibration curves were generated by injecting 0.05–1 µg
purified delphinidin, cyanidin and peonidin (Extrasynthase) in
20 µL of diluted HCl–methanol (1 : 100, v/v). Total anthocyanins
were expressed as the sum of delphinidin, cyanidin and petunidin.
Total proanthocyanidins were determined as the difference in
total anthocyanin content between acidified and non-acidified
samples.3
γ -Oryzanol, tocopherol and tocotrienol determinations
Ground rice brans (0.1 g) were dissolved in 1.0 mL methanol for
30 min and then filtered through a 0.45 µmol L−1 filter membrane.
The γ -oryzanol content in methanol extract was measured by
HPLC using a Waters model 2695 equipped with ODS column
(Inertsil ODS-3, 4.6×150 mm, 5 µm) and photodiode array detector
(325 nm) according to the method reported by Aguilar-Garcia
et al.5 with some modifications. Mobile phase was acetonitrile and
methanol at 60 : 40 (v/v) at a flow rate of 1 mL min−1 . The content
of γ -oryzanol was calculated from the peak area of standard
γ -oryzanol with various components (Wako, Osaka, Japan). The
same rice bran extract was also used for tocopherol and tocotrienol
determinations using HPLC under the same conditions, except that
a fluorescence detector (excitation 298 nm, emission 328 nm) was
applied. The chromatograph was calibrated using standards of
tocopherols and tocorienols with various components (Advanced
Chemistry Development, Inc., Toronto, Canada).
J Sci Food Agric (2011)
Nutrient composition in brans of red rice mutants www.soci.org

Table 1. Values of six agronomic traits from red rice wild type SA-586 and its nine sodium azide-induced mutants derived under field conditions

Accession Plant height (m) Growth duration (days) No. panicles per hill 1000-grain weight (g) Fertility (%) Grain yield (kg ha−1 )

SA-586 1.4 111 7.3 ± 1.0 30.4 ± 1.5 81.5 ± 1.8 5820 ± 600
M-18 0.8 115 9.8 ± 1.0 28.2 ± 1.0 78.8 ± 12.5 4520 ± 430
M-19 0.8 119 11.5 ± 1.3 26.3 ± 0.9 80.3 ± 2.3 5830 ± 840
M-20 0.8 119 10.3 ± 0.5 28.5 ± 0.7 78.3 ± 6.0 5140 ± 790
M-21 0.8 119 14.0 ± 4.1 26.9 ± 0.7 54.4 ± 12.5 4930 ± 1170
M-50 0.8 120 10.3 ± 2.2 27.3 ± 3.3 73.7 ± 3.6 4210 ± 820
M-54 0.7 105 12.5 ± 2.1 24.8 ± 0.6 65.0 ± 4.7 5460 ± 1250
M-55 0.7 105 12.5 ± 0.7 24.0 ± 0.7 70.7 ± 1.6 4530 ± 830
M-56 0.7 105 14.0 ± 1.4 23.8 ± 1.1 71.6 ± 3.5 4970 ± 390
M-30 0.8 115 12.6 ± 2.9 26.9 ± 2.1 73.4 ± 16.1 4810 ± 1040
LSD0.05 0.1 6 2.3 1.6 8.8 870

Values are means ± SD; n = 3.

Mineral element determination
Ground rice brans (1.0 g) were weighed and placed in a digestion
vessel to which 10 mL HClO4 –HNO3 (1 : 4) was added and kept
overnight for pre-digestion. Pre-digested samples were mixed well
by swirling and placed on a hotplate. Temperature was gradually
increased to 225 ◦ C for digestion. Digested samples were used for
the quantification of K, Ca, Na, Mg, Fe, Mn, Cu and Zn with an
inductively coupled plasma atomic emission spectrometer (JY50P,
Jobin-Yvon, Longjumeau, France).
bran dry weight (mutant M-20) to 141.3 g kg−1 bran dry weight
(mutant M-30), with extensive variation (P < 0.05) (Table 2). The
lipid content of rice bran is reported to be in the range of
133–198 g kg−1 bran dry weight.16 Results obtained in this study
showed the bran of SA-586 had a lipid content of 161.6 g kg−1
bran dry weight. The lipid content for the tested mutants ranged
from 145.5 (mutant M-20) to 187.8 g kg−1 bran dry weight (mutant
M-54), with significant differences (P < 0.05) existing among the
tested mutants (Table 2).

Statistical analysis Variations in total phenolic, total flavonoid, proanthocyanidin

Data were analyzed by analysis of variance using the Statistical
Package for Social Science (SPSS 10.0 for Windows, SPSS Inc.,
Chicago, IL, USA). Values were given as mean ± standard deviation
(SD) and means were separated using a least significant difference
(LSD) test.
RESULTS AND DISCUSSION
Variations in agronomic traits
Plant height in rice is an important trait for breeding high-yielding
cultivars, and numerous dwarf and semi-dwarf mutants have
been collected.13 In this study, all the NaN3 -induced rice mutants
exhibited reduced plant height (ranging from 0.7 to 0.8 m) in
comparison with their wild type SA-586 (1.4 m) (Table 1). Notable
variations were also observed for five other measured agronomic
traits. For example, growth duration (from planting to final harvest)
ranged from 105 to 120 days among the tested mutants. The
mutants M-21 and M-56 produced more panicles per hill (93%
more) than SA-586. Grain production of M-19, M-54 and M-20 were
statistically equal to SA-586 (Table 1). These results confirm that
the NaN3 -induced mutation can produce high levels of variation
for all measured agronomic traits such as plant height, 1000-grain
weight and grain yield.14
Variations in protein and lipid contents
Rice bran only constitutes about 10% of brown rice grain,15 but it
is a good source of protein, lipid and many antioxidants. Chemical
analysis16 shows the protein content of rice bran ranged from
100 to 130 g kg−1 dry weight. In the present study, the protein
content for SA-586 was 128 g kg−1 bran dry weight (Table 2). The
protein content for selected mutants ranged from 121.1 g kg−1
and anthocyanin contents
Red rice bran is concentrated with a considerable level of
phenolics.5 These phenolics bind and precipitate macromolecules
such as dietary protein, carhohydrate and digestive enzymes,
thereby reducing food digestibility.10 However, these phenolics
are also the most important antioxidative plant components.2,5
The results of total phenolics analysis for the bran samples of
SA-586 and its NaN3 -induced mutants are given in Table 2. Wild
type SA-586 had 50.3 g gallic acid kg−1 of total phenolics on
bran dry weight base. The levels of total phenolics for other eight
NaN3 -induced red bran mutants ranged from 29.8 (M-20) to 58.2
(M-54) g gallic acid kg−1 bran dry weight, which were greater
than in the red rice varieties reported by Goffman and Bergman.17
On the other hand, white bran mutant M-30 (Fig. 1) had a total
phenolics content of only 2.8 g gallic acid kg−1 bran dry weight.
This value was similar to the levels of total phenolics accumulated
in the bran of common rice varieties reported by Chotimarkorn
et al.18 However, only mutant M-54 contained significantly higher
total phenolics in its bran part than wild type SA-586.
Total flavonoids constitute the largest group of total phenolics
in plant seeds.14 It has been suggested that flavonoids regularly
introduced with the diet can act as mild pro-oxidants and
stimulate the endogenous antioxidant defenses, which prevents
disease development or reduces the impact of oxidative stress
when disease occurs.15 As shown in Table 2, the content of
total flavonoids in bran sample SA-586 was 0.7 g kg−1 bran dry
weight. The content of total flavonoids for eight red bran mutants
varied from 0.4 g kg−1 (mutant M-20) (35% lower than SA-586)
to 1.3 g kg−1 bran dry weight (mutant M-54) (91% higher than
SA-586). No flavonoids were detectable in white bran mutant
M-30 (Fig. 1 and Table 2). This result differed from the report of
Chotimarkorn et al.,18 in that a low level of flavonoids (ranging

J Sci Food Agric (2011)

c 2011 Society of Chemical Industry wileyonlinelibrary.com/jsfa
 www.soci.org TL Jeng et al.

Table 2. The contents of protein, lipid, total phenolics, total flavonoids, total proanthocyanidins and total anthocyanins in brans of SA-586 and its
sodium azide-induced mutants

Total phenolics (g gallic Total flavonoids (g quercetin Total proanthocyanidin Total anthocyanin
acid kg−1 bran equivalent kg−1 bran
Protein (g kg−1 Lipid (g kg−1
Accession bran dry weight) bran dry weight) dry weight) dry weight) (mg kg−1 bran dry weight)

SA-586 128.0 ± 0.9 161.6 ± 0.1 50.3 ± 0.6 0.7 ± 0.1 270.3 ± 23.5 56.6 ± 4.2
M-18 120.1 ± 0.6 156.0 ± 0.9 43.1 ± 0.2 0.8 ± 0.1 559.9 ± 18.8 31.8 ± 1.3
M-19 127.4 ± 0.3 166.7 ± 0.6 32.9 ± 0.3 0.7 ± 0.1 309.4 ± 40.0 27.8 ± 1.5
M-20 121.1 ± 0.3 145.5 ± 0.5 29.8 ± 0.2 0.4 ± 0.1 350.1 ± 14.6 30.6 ± 1.1
M-21 122.7 ± 0.3 152.7 ± 0.4 38.7 ± 0.5 0.7 ± 0.2 489.1 ± 28.6 30.7 ± 3.1
M-50 135.3 ± 0.3 181.8 ± 0.4 50.3 ± 1.5 0.8 ± 0.3 143.6 ± 23.9 38.4 ± 2.0
M-54 135.8 ± 0.6 187.8 ± 0.6 58.2 ± 0.4 1.3 ± 0.1 131.7 ± 19.5 33.7 ± 3.8
M-55 136.1 ± 0.6 164.8 ± 0.3 50.7 ± 1.9 1.2 ± 0.1 132.1 ± 16.9 36.2 ± 3.1
M-56 141.0 ± 0.3 171.5 ± 0.6 51.7 ± 3.0 0.9 ± 0.1 168.1 ± 23.4 43.1 ± 0.8
M-30 141.3 ± 0.3 185.7 ± 0.8 2.8 ± 0.1 – – –
LSD0.05 0.9 1.3 2.1 0.1 39.2 4.2

Values are means ± SD; n = 3.

from 0.03 to 0.10 g kg−1 bran dry weight) was detectable in the
bran part of common rice varieties. This could be due to the
different rice varieties used between the two studies.
Both proanthocyanidins and anthocyanins are concentrated in
the bran part of pigmented rice grain.19 These two phytochemicals
are reported to be beneficial to human health due to their
significant anti-oxidative capacities, which suppress the oxidative
changes in low-density lipoprotein, reduce the formation of nitric
oxide and prevent breaks supercoiled DNA strands.20 Oki et al.3
reported that proanthocyanidins are the major pigments in red
rice. In the present study, SA-586 had 270.3 mg kg−1 of total
proanthocyanidins on a bran dry weight base (Table 2). The levels
of total proanthocyanidins for eight red bran mutants varied from
a low of 131.6 to 559.9 mg kg−1 . The Three red bran mutants M-18,
M-20 and M-21 contained more total proanthocyanidins than wild
type SA-586, but no total proanthocyanidin was detectable in the
bran part of white bran mutant M-30 (Fig. 1 and Table 2).
Anthocyanins are reported to have high antioxidant activities as
well as anti-inflammatory properties.20 Kim et al.21 reported that
no anthocyanins were detectable in red rice. In this study, all the red
bran mutants and wild type SA-586 had measurable anthocyanins
(Table 2). The wild type SA-586 had 56.6 mg kg−1 of anthocyanins
on a bran dry weight basis. The levels of total anthocyanins for
eight red bran mutants, expressed on a bran dry weight basis,
varied from a low of 27.8 to 43.1 mg kg−1 . However, none of the
red bran mutants had an anthocyanin content higher than wild
type SA-586. In all the tested red bran mutants, the content of
total anthocyanins contained in the bran part was considerably
lower than total proanthocyanidins. These results reconfirm that
the major phytochemicals responsible for pigmentation in red
bran are proanthocyanidins.3
Variation in γ -oryzanol
The lipid antioxidant γ -oryzanol is a mixture of ferulic acid (4-
hydroxy-3-methoxycinnamic acid) esters of triterpene alcohol. It
is a powerful inhibitor of iron-driven hydroxyl radical formation.22
γ -Oryzanol has been reported to reduce plasma cholesterol and
decrease early atherosclerosis.23,24 It is also used in the treatment
of nerve imbalance and disorders of menopause.25 Rice bran is
a rich source of γ -oryzanol, but the content varied substantially
depending on the origin of the rice bran.6 With respect to the
total γ -oryzanol content accumulated in the bran part, mutant
M-56 recorded the highest content of 3.9 g kg−1 bran dry weight,
followed by mutant M-50 (3.4 g kg−1 bran dry weight) (Table 3).
These values were 35% and 17% higher than wild type SA-586
(2.9 g kg−1 bran dry weight), respectively (Table 3). The white
bran mutant M-30 (Fig. 1) exhibited 2.3 g kg−1 bran dry weight γ -
oryzanol, which was lower than SA-586 but higher than some of red
bran mutants (M-18, M-19 and M-20). However, all the detected
γ -oryzanol contents were higher than the data (0.5–0.8 g kg−1
bran dry weight) reported by Iqbal et al.26
The major components of γ -oryzanol founded in the tested
rice bran samples were 2,4-methylene cycloartenyl ferulate,
campesteryl ferulate, cycloartenyl ferulate and sitosteryl ferulate
(Table 3). Similar results were also reported by Parrado et al.14 As
for the levels of detected γ -oryzanol components, the highest level
was found for 2,4-methylene cycloartenyl ferulate (ranging from
30% to 50% of total γ -oryzanol), and then followed by campesteryl
ferulate, cycloartenyl ferulate or sitosteryl ferulate, depending on
the mutants examined.
Variations in tocopherols and tocotrienols contents
Both tocopherols and tocotrienols are reported to suppress tumor
cell proliferation, inhibit cholesterol synthesis and reduce serum
cholesterol level.27 However, tocotrienol is more powerful and
effective than tocopherol.28 Rice bran also contains a significant
amount of tocopherols and tocotrienols.29 In the present study,
both tocopherols and tocotrienols contents (Table 4) were
considerably lower than γ -oryzanol content (Table 3). These
results are in agreement with the report of Bergman and
Xu (2003).30 Mutant M-56 had the highest content of total
tocopherols (92.0 mg kg−1 bran dry weight), followed by M-50
(77.4 mg kg−1 bran dry weight) and M-19 (77.2 mg kg−1 bran dry
weight) (Table 4). The white bran mutant M-30 (Fig. 1) had more
total tocopherols (61.9 mg kg−1 bran dry weight) than SA-586
(54.6 mg kg−1 bran dry weight) (Table 4). Aguilar-Garcia et al.5
reported that only α-tocopherol (the major component) and
γ -tocopherol were detectable in bran layers of tested common
rice varieties. In this study, α-tocopherol, γ -tocopherol and
δ-tocopherol were obtained from all the tested red rice wild

wileyonlinelibrary.com/jsfa

c 2011 Society of Chemical Industry J Sci Food Agric (2011)
Nutrient composition in brans of red rice mutants www.soci.org

Table 3. Contents of various components of γ -oryzanol in brans of SA-586 and its sodium azide-induced mutants

Cycloartenyl ferulate 2,4-methylene cycloartenyl ferulate Campesteryl ferulate Sitosteryl ferulate Total γ -oryzanol

Accession (mg kg−1 bran dry weight)

SA-586 675.4 ± 70.3 1450.6 ± 80.1 241.2 ± 15.2 523.1 ± 18.9 2890.3 ± 179.1
M-18 369.3 ± 47.4 979.3 ± 67.5 429.1 ± 30.2 335.1 ± 40.0 2112.8 ± 175.9
M-19 262.9 ± 22.4 737.2 ± 30.2 279.7 ± 10.7 271.2 ± 13.5 1551.1 ± 37.9
M-20 383.1 ± 12.4 815.8 ± 30.2 371.0 ± 16.4 295.0 ± 31.3 1864.7 ± 70.5
M-21 435.0 ± 25.7 1080.3 ± 18.1 447.6 ± 16.6 365.8 ± 23.5 2328.7 ± 48.3
M-50 559.9 ± 47.3 1401.4 ± 99.6 725.1 ± 41.5 691.9 ± 80.7 3378.4 ± 282.4
M-54 538.2 ± 19.5 1275.4 ± 42.1 656.0 ± 26.6 697.0 ± 43.2 3166.5 ± 125.0
M-55 601.6 ± 49.1 1280.3 ± 80.3 584.2 ± 31.5 643.2 ± 44.5 3109.3 ± 204.8
M-56 742.5 ± 53.4 1612.4 ± 43.4 710.8 ± 34.2 823.7 ± 28.7 3889.4 ± 62.8
M-30 474.5 ± 21.8 1140.2 ± 13.9 201.8 ± 18.5 452.7 ± 15.8 2269.3 ± 46.0
LSD0.05 68.9 104.3 42.1 65.8 249.8

Values are means ± SD; n = 3.

Table 4. Contents of various components of tocopherol and tocotrienol in brans of SA-586 and its sodium azide-induced mutants

α-Tocopherol γ -Tocopherol δ-Tocopherol Total tocopherol α-Tocotrienol γ -Tocotrienol δ-Tocotrienol Total tocotrienol

Accession (mg kg−1 bran dry weight)

SA-586 15.8 ± 1.2 34.7 ± 3.2 4.0 ± 0.2 54.6 ± 3.6 6.7 ± 0.3 129.8 ± 8.0 14.5 ± 0.4 151.0 ± 8.5
M-18 20.8 ± 1.2 26.4 ± 2.4 2.1 ± 0.3 49.1 ± 3.2 6.6 ± 0.1 175.9 ± 14.8 6.4 ± 0.3 188.9 ± 15.0
M-19 29.4 ± 1.2 43.8 ± 2.1 4.3 ± 0.2 77.2 ± 3.2 5.7 ± 0.2 149.3 ± 3.1 6.2 ± 0.2 161.2 ± 3.6
M-20 29.5 ± 2.5 36.8 ± 2.5 2.1 ± 0.3 68.3 ± 4.9 6.1 ± 0.2 157.5 ± 4.2 6.9 ± 0.3 170.2 ± 4.5
M-21 24.4 ± 1.3 31.9 ± 6.4 2.0 ± 0.3 58.4 ± 5.2 7.2 ± 0.2 194.5 ± 5.7 11.2 ± 0.1 212.9 ± 5.7
M-50 33.7 ± 2.5 41.7 ± 3.6 1.9 ± 0.1 77.4 ± 6.2 14.3 ± 1.1 230.6 ± 23.6 16.8 ± 1.9 261.6 ± 26.5
M-54 23.0 ± 1.2 28.6 ± 1.2 2.2 ± 0.1 53.5 ± 2.5 12.2 ± 0.8 213.6 ± 9.7 14.6 ± 0.9 240.4 ± 11.2
M-55 15.8 ± 2.6 16.7 ± 3.6 2.0 ± 0.1 34.3 ± 5.3 11.7 ± 0.8 176.8 ± 8.4 11.5 ± 0.6 199.9 ± 9.6
M-56 53.9 ± 2.3 36.2 ± 2.4 1.9 ± 0.1 92.0 ± 4.4 12.9 ± 0.5 184.4 ± 6.4 11.5 ± 0.5 208.9 ± 7.3
M-30 25.1 ± 1.2 34.7 ± 1.2 2.1 ± 0.1 62.0 ± 2.5 4.3 ± 0.2 109.4 ± 1.7 2.8 ± 0.1 116.4 ± 2.0
LSD0.05 3.2 5.4 0.4 7.3 0.9 17.9 1.3 19.7

Values are means ± SD; n = 3.

type SA-586 and mutants as well as the white bran mutant M-30
(Table 4). In almost in all cases (except mutant M-56), the highest
content was found for γ -tocopherol, followed by α-tocopherol
and δ-tocopherol. Except for mutant M-56, α-tocopherol showed
the highest level (Table 4). These results suggest that the NaN3 -
induced mutation also affects the synthetic pathways of individual
tocopherols in red rice bran.
All the red bran mutants and wild type SA-586 accumulated
more tocotrienols than tocopherols (Table 4). The mutant M-
50 (261.6 mg kg−1 bran dry weight) had the highest level of
tocotrienols and followed by mutant M-56 (208.8 mg kg−1 bran dry
weight), whereas white bran mutant M-30 (Fig. 1) (116.4 mg kg−1
bran dry weight) was the lowest (Table 4). As for the tocotrienol
profile, the highest content was found for γ -tocotrienol, which
represented almost 85–90% of total tocotrienol accumulated in
the tested rice brans (Table 4). A similar tocotrienol profile was
detected in several common varieties.5
Variations in minerals contents
Rice bran is also an important dietary source of many elements
such as K, Ca, Mg, Mn, Fe and Zn.31 The contents of most
elements examined in the bran part varied considerably among
the tested mutants (Table 5). However, the most striking variation
was found for Fe content. The wild type SA-586 had Fe content
of 100.1 mg kg−1 bran dry weight. The highest Fe content
(588.7 mg kg−1 bran dry weight) was obtained from mutant M-54,
which was 488% higher than SA-586. This value is also considerably
higher than the bran data measured from red rice varieties (ranging
from 14.4 to 84.6 mg kg−1 bran dry weight) reported by Kaneda
et al.31 The lowest Fe content (66.8 mg kg−1 bran dry weight) was
obtained from mutant M-20 (33% less than SA-586).
The wild type SA-586 had a Zn content of 99.02 µg g−1 bran dry
weight (Table 5). The content of Zn measured for nine mutants
ranged from 62.5 to 112.8 mg kg−1 bran dry weight (Table 5). The
Zn content measured from the tested SA-586 and mutants were
higher than most of the red rice varieties (30.7–61.2 mg kg-1 bran
dry weights) listed in the study by Kaneda et al.31
CONCLUSION
The results obtained indicate the great influence of NaN3 -induced
mutation on plant height and other agronomic traits of tested
rice mutants. Large variations in total phenolics, anthocyanins,
proanthocyanidins, γ -oryzanol, total tocopherols and tocotrienol

J Sci Food Agric (2011)

c 2011 Society of Chemical Industry wileyonlinelibrary.com/jsfa
 www.soci.org TL Jeng et al.

Table 5. Contents of various mineral elements in brans of SA-586 and its sodium azide-induced mutants

P K Ca Mg Fe Mn Cu Zn

Accession (g kg−1 bran dry weight) (mg kg−1 bran dry weight)

SA-586 26.4 ± 0.8 21.2 ± 0.6 0.7 ± 0.1 11.9 ± 0.4 100.1 ± 7.6 183.4 ± 5.7 12.4 ± 0.6 99.0 ± 4.6
M-18 20.9 ± 0.2 18.0 ± 0.3 0.5 ± 0.1 8.9 ± 0.1 72.0 ± 2.5 119.9 ± 0.6 5.0 ± 0.4 112.8 ± 2.6
M-19 21.0 ± 0.1 18.2 ± 0.2 0.6 ± 0.1 8.8 ± 0.1 76.0 ± 1.1 129.3 ± 1.4 7.6 ± 0.6 71.4 ± 1.2
M-20 19.8 ± 0.4 18.0 ± 0.4 0.5 ± 0.1 8.4 ± 0.1 66.8 ± 2.1 110.9 ± 2.2 6.5 ± 0.5 82.9 ± 2.7
M-21 22.2 ± 0.1 19.5 ± 0.1 0.5 ± 0.1 9.7 ± 0.1 79.5 ± 1.3 127.7 ± 1.5 8.5 ± 0.2 93.3 ± 0.9
M-50 25.8 ± 0.3 21.7 ± 0.4 0.6 ± 0.1 11.0 ± 0.2 229.4 ± 9.6 133.4 ± 2.9 11.2 ± 1.0 82.7 ± 1.7
M-54 25.9 ± 0.4 21.3 ± 0.2 0.6 ± 0.1 11.0 ± 0.1 588.7 ± 25.0 141.9 ± 4.0 11.4 ± 1.0 62.5 ± 3.8
M-55 26.9 ± 0.1 21.4 ± 0.1 0.7 ± 0.1 11.4 ± 0.1 151.4 ± 11. 149.7 ± 0.8 12.2 ± 0.1 69.1 ± 0.5
M-56 27.0 ± 0.3 21.4 ± 0.1 0.7 ± 0.1 11.7 ± 0.1 134.1 ± 2.8 148.1 ± 1.1 12.6 ± 1.6 80.1 ± 0.4
M-30 20.8 ± 0.26 17.4 ± 0.1 0.5 ± 0.1 8.6 ± 0.1 71.2 ± 0.8 127.4 ± 1.7 6.7 ± 0.3 96.0 ± 1.8
LSD0.05 0.1 0.1 0.1 0.1 11.6 6.2 1.7 5.1

Values are means ± SD; n = 3.

contents are also detectable among wild type SA-586 and NaN3 -
induced mutants. However, the increased proanthocyanidins
(mutant M-18), γ -oryznol (mutant M-56), total tocopherols (mutant
M-56) or tocotrienols (mutant M-50) were generally coupled with
concomitant declines in other phytochemicals. These results are
reasonable because all the measured phytochemicals, including
anthocyanins, proanthocyanidins, γ -oryzanol, total tocopherols
and tocotrienols, are derivatives of the pentose phosphate,
shikimate and phenylpropanoid pathways. Thus the mutation-
diverged substrate flows among these pathways might be
responsible for the observed changes in the formation of
these phytochemicals. NaN3 -induced mutation also affects the
contents of several important mineral elements. The mutant M-54
accumulated a considerably higher level of Fe than SA-586 and
other mutants. Thus some mutants that are rich in antioxidants
(M-18, M-56 and M-50) or Fe (M-54 and M-50) may be useful in
food and other applications. These mutants can also be used as
genetic resources for a rice nutrient improvement programs.
ACKNOWLEDGEMENT
The authors thank the National Science Council of Taiwan, ROC,
for supporting this work under grant NSC98-2324-B-241-001.
REFERENCES
1 Patindol J, Flowers A, Kuo M -I, Wang, Y -J and Gealy D, Comparison
of physicochemical properties and starch structure of red rice and
cultivated rice. J Agric Food Chem 54:2712–2718 (2006).
2 Finocchiaro F, Ferrari B, Gianinetti A, Dall’Asta A, Galaverna G,
Scazzina F, et al, Characterization of antioxidant compounds of
red and white rice and changes in total antioxidant capacity during
processing. Mol Nutr Food Res 51:1006–1019 (2007).
3 Oki T, Masuda M, Kobayashi M, Nishiba Y, Furuta S, Suda I, et al,
Polymeric procyanidins as radical-scavenging components in red-
hulled rice. J Agric Food Chem 50:7524–7529 (2002).
4 Brooks SA, Yan W, Jackson AK and Deren CW, A nutural mutation in rc
reverts white-rice-pericarp to red and results in a new, dominant,
wild-type allele: Rc-g. Theor Appl Genet 117:575–580 (2008).
5 Aguilar-Garcia C, Gavin G, Baragaño-Mosqueda M, Hevia P and
Gavino VC, Correlation of tocopherol, tocotrienol, γ -oryzanol and
total polyphenol content in rice bran with different antioxidant
capacity assays. Food Chem 102:1228–1232 (2007).
6 Yu S, Nehus ZT, Badger TM and Fang N, Quantification of vitamin E
and γ -oryzanol components in rice germ and bran. J Agric Food
Chem 55:7308–7313 (2007).
7 Welch RM and Graham RD, Breeding for micronutrients in staple food
crops from a human nutrition perspective. J Exp Bot 55:353–364
(2004).
8 Jiang AL, Wu JG, Thang NB, Feng Y, Yang XE and Shi CH, Genotypic
variation of mineral elements contents in rice (Oryza sativa L.). Eur
Food Res Technol 228:115–122 (2008).
9 Pintasen S, Prom-u-thai C, Jamjod S, Yimyam N and Rerkasem B,
Variation of grain iron content in a local upland rice germplasm
from the village of Huai Tee Cha in northern Thailand. Euphytica
158:27–34 (2007).
10 Gregorio GB, Progress in breeding for trace minerals in staple crops.
J Nutr 132:500S–502S (2002).
11 Ahloowalia BS and Maluszynski M, Induced mutations: a new
paradigm in plant breeding. Euphytica 118:167–173 (2001).
12 AbouZid SF and Elsherbeiny GM, Increase in flavonoids content in red
onion peel by mechanical shredding. J Med Plants Res 82:258–260
(2008).
13 Kurata N, Miyoshi K, Nonomura K, Yamazaki Y and Ito Y, Rice mutants
and genes related to organ development, morphogenesis and
physiological traits. Plant Cell Physiol 46:48–62 (2005).
14 Parrado J, Miramontes E, Jover M, Gutierrez JF, de Terán LC and
Bautista J, Preparation of rice bran enzymatic extract with potential
use as function food. Food Chem 98:742–748 (2006).
15 Al-Qurainy F and Khan S, Mutagenic effects of sodium azide and its
application in crop improvement. World Appl Sci J 6:1589–1601
(2009).
16 Amissah JGN, Ellis WO, Oduro I and Manful JT, Nutrient composition
of bran from new rice varieties under study in Ghana. Food Control
14:21–24 (2003).
17 Goffman FD and Bergman CJ, Rice kernel phenolic content and
its relationship with antiradical efficiency. J Sci Food Agric
84:1235–1240 (2004).
18 Chotimarkorn C, Benjakul S and Silalai N, Antioxidant compounds and
properties of five long-grain rice bran from commercial available
cultivars in Thailand. Food Chem 111:636–641 (2008).
19 Finocchiaro F, Ferrari1 B and Gianinetti1 A, A study of biodiversity of
flavonoid content in the rice caryopsis evidencing simultaneous
accumulation of anthocyanins and proanthocyanidins in a black-
grained genotype. J Cereal Sci 51:28–34 (2010).
20 Hu C, Zawistowski J, Ling W and Kitts DD, Black rice (Oryza sativa L.
indica) pigmented fraction suppresses both reactive oxygen species
and nitric oxide in chemical and biological model systems. J Agric
Food Chem 51:5271–5277 (2003).
21 Kim M, Kim H, Koh K, Kim H, Lee YS and Kim YH, Identification and
quantification of anthocyanin pigments in colored rice. Nutr Res
Prac 2:46–49 (2008).
22 Fardet A, Rock E and Rémésy C, Is the in vitro antioxidant potential
of whole-grain cereals and cereal products well reflected in vivo?
J Cereal Sci 48:258–276 (2008).

wileyonlinelibrary.com/jsfa

c 2011 Society of Chemical Industry J Sci Food Agric (2011)
Nutrient composition in brans of red rice mutants
23 Cicero AE and Derosa G, Rice bran and its main components: potential
role in the management of coronary risk factors. Curr Top Nutraceut
Res 3:29–46 (2005).
24 Chen M-H and Bergman CJ, A rapid procedure for analyzing rice bran
tocopherol, tocotrienol and γ -oryzanol contents. J Food Compos
Anal 18:319–331 (2005).
25 Sookwong P, Murata K, Nakagawa K, Shibata A, Kimura T, Yamaguchi
M, et al, Cross-fertilization for enhancing tocotrienol biosynthesis in
rice plants and QTL analysis of their F2 progenies. J Agric Food Chem
57:4620–4625 (2009).
26 Iqbal S, Bhanger MI and Anwar F, Antioxidant properties and
components of some commercially available varieties of rice bran
in Pakistan. Food Chem 93:265–277 (2005).
27 Lerma-Garcia MJ, Herrero-Martinez JM, Simo-Alfonso EF, Men-
donca CRB and Ramis-Ramos G, Composition, industrial processing
and applications of rice bran γ - oryzanol. Food Chem 115:389–404
(2009).
J Sci Food Agric (2011)

c 2011 Society of Chemical Industry
www.soci.org
28 Rogers EJ, Rice SM, Nicolosi RJ, Carpenter DR, McClelland CA and
Romanczyk LJ, Identification and quantitation of γ -oryzanol
components and simultaneous assessment of tocols in rice bran oil.
J Am Oil Chem Soc 70:301–307 (1993).
29 Butsat S and Siriamornpun S, Antioxidant capacities and phenolic
compounds of the husk, bran and endosperm of Thai rice. Food
Chem 119:606–613 (2010).
30 Bergman CJ and Xu Z, Genotype and environment effects on
tocopherols, tocotrienols and gamma-oryzanol contents of
Southern US rice. Cereal Chem 80:446–449 (2003).
31 Kaneda I, Kubo F and Sakurai H, Relationship between trace metal
concentration and antioxidative activity of ancient rice bran (red
and black rice) and present-day rice bran (Koshihikari). J Trace Elem
Med Biol 21:43–51 (2007).
wileyonlinelibrary.com/jsfa