Accepted Manuscript

Phenolic acids, anthocyanins, proanthocyanidins, antioxidant activity, minerals

and their correlations in non-pigmented, red, and black rice

Yafang Shao, Zhanqiang Hu, Yonghong Yu, Renxiang Mou, Zhiwei Zhu, Trust
Beta

PII: S0308-8146(17)31145-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.07.009
Reference: FOCH 21393

To appear in: Food Chemistry

Received Date: 23 May 2017

Revised Date: 20 June 2017
Accepted Date: 2 July 2017

Please cite this article as: Shao, Y., Hu, Z., Yu, Y., Mou, R., Zhu, Z., Beta, T., Phenolic acids, anthocyanins,
proanthocyanidins, antioxidant activity, minerals and their correlations in non-pigmented, red, and black rice, Food
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.07.009

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Phenolic acids, anthocyanins, proanthocyanidins, antioxidant activity,

minerals and their correlations in non-pigmented, red, and black rice

Yafang Shao 1, Zhanqiang Hu 1, Yonghong Yu 1, Renxiang Mou 1, Zhiwei Zhu 1*,

Trust Beta 2*

1
China National Rice Research Institute, Hangzhou, 310006, China
2
University of Manitoba, Department of Food Science, Winnipeg R3T 2N2, Canada

*Corresponding authors. Addresses: University of Manitoba, Winnipeg, Manitoba,

Canada R3T 2N2. Tel.: +1-204-474-8214; fax: +1-204-474-7630 (T. Beta); China
National Rice Research Institute, Hangzhou, 310006, China (Z. Zhu). E-mail
addresses: zwzhu80@126.com (Z. Zhu), Trust.Beta@umanitoba.ca (T. Beta).

1
ABSTRACT
Soluble-free, soluble-conjugated, insoluble-bound phenolics and antioxidant activity,
flavonoid (TFC), proanthocyanidins (TPAC), anthocyanins and minerals of fifteen
whole rice grains with different colors were investigated. Soluble-free protocatechuic
and vanillic acids were only quantified in black rice, which had the most quantities.
Non-pigmented rice had no detectable conjugated protocatechuic and
2,5-dihydroxybenzoic acids both of which were found in black and red rice,
respectively. The main bound phenolic acids were ferulic and p-coumaric, as well as
2,5-dihydroxybenzoic in red rice and protocatechuic and vanillic acids in black rice.
Soluble-conjugated phenolics, TFC, and anthocyanins were negatively correlated with
L*, b*, C and H° values. TPAC was positively correlated with a* (P<0.01).
Protocatechuic, vanillic, syringic and ferulic acids were associated with TPC and
antioxidant activity in the soluble-conjugated fraction while protocatechuic and
ferulic acid were correlated with those in the insoluble-bound fraction. Principal
component analysis divided samples into non-pigmented, red and black rice groups.

Keywords: whole rice grain; phenolic acids; anthocyanins; proanthocyanidins;

antioxidant activity; minerals; principal component analysis

2
1. Introduction
Rice (Oryza sativa L.), as one of the most important staple crops, provides energy for
almost half of the world’s population. Because of the tradition of eating milled or
polished rice, the monotonous consumption of rice may lead to deficiencies of
essential vitamins, minerals and other nutritional and functional compounds (Bouis,
Chassy, & Ochanda, 2003). Most of the phytochemicals are present in bran layer and
embryo fraction (Shao, Xu, Sun, Bao, & Beta, 2014a). Whole rice grains are
hypothesized to contribute positively to human health due to their polyphenols,
minerals, fibre, vitamins and other phytochemicals (Deng, Xu, Guo, Xia, & Li, 2012;
Deng, Xu, Zhang, Li, Gan, & Li, 2013; Min, Mcclung, & Chen, 2011). These
compounds may influence biological functions individually or synergistically. Many
interventional and epidemiological studies show that the consumption of whole rice
grain is associated with the reduction in risk of developing chronic diseases such as
cardiovascular diseases, obesity, type II diabetes and some cancers (Mattei et al., 2015;
Shao & Bao, 2015).

Phenolics, as a large group of secondary metabolites in cereal grains, are present in

three forms, soluble-free, soluble-conjugated and insoluble-bound (Li, Shewry, &
Ward, 2008; Park et al., 2012). The soluble phenolic acids would become absorbed in
the stomach and small intestine for distribution to the whole body with concomitant
health benefits such as inhibition against oxidation of low-density lipoprotein
cholesterol and liposomes (Min, Gu, Mcclung, Bergman, & Chen, 2012; Shao & Bao,
2015), while the insoluble bound phenolic acids would be accessible in the intestine
after digestion by the enzymes and some are released in the colon by the colonic
microflora (Saura-Calixto, Serrano, & Goñi, 2007). Microelements (Zn, Mn, Cu and
Fe) and macroelements (P, K, Ca and Mg) can be found in every cell where they play
important roles in maintaining normal metabolic functions, proper fluid balance,
blood pressure regulation, nerve transmission, and immune system health (Speich,
Pineau, Ballereau, 2001).

3
Color is an important feature used by consumers for selection of food products. The
pigments of black and red rice samples are due to the accumulation of anthocyanins
and proanthocyanidins in rice bran, respectively (Min et al., 2011). The biosynthesis
pathway of phenolic acids, anthocyanins and proanthocyanidins begins with
L-phenylalanine via the phenylpropanoid pathway (Shao & Bao, 2015). Using
classical genetic analysis, two loci that are associated with red pericarp have been
identified (Sweeney, Thomson, Pfeil, & McCouch, 2006), and the regulation
mechanisms of anthocyanin biosynthesis have been revealed (Oikawa et al., 2015).
However, the biosynthesis regulatory relationships between phenolic acids and
anthocyanins in different colored rice grains are largely unknown. Although the
correlations among color parameters of L*, a*, b*, C, H°, total phenolic, flavonoid
content, and antioxidant activity were studied (Shen, Jin, Xiao, Lu, & Bao, 2009), the
exact phenolic compounds that contributed to the color differences, total phenolic
content, and antioxidant activity have not been identified.

In this study, we aimed to evaluate soluble-free, soluble-conjugated, and

insoluble-bound phenolics, antioxidant activity, total flavonoids, proanthocyanidins,
anthocyanins, and minerals of whole rice grains so as to determine their relationships
with grain color, as well as the relationships between the specific phenolic acids and
total phenolic content or antioxidant activity. The results of this study could provide
rice breeders or food industries new opportunities to promote the production of rice or
rice products with enhanced levels of the bioactive compounds.

2. Materials and methods

2.1. Rice samples
Fifteen rice (Oryza sativa L.) genotypes, which consisted of 5 non-pigmented brown
rice (9311, Nipponbare, Tianyouhuazhan, II You 838, Yanfeng 47), 5 red rice
(Jinggangshanhongmi, Xiangwanxian 12, Qianxiuhong, Changhong No. 1, Hongmi 2),
and 5 black rice (D Youzinuo 161, Heimi No. 1, Heixiangnuo No. 3, Heinuomi, Heimi
2420) were selected for the study. Yanfeng 47 was planted in 2015 in the saline and
4
alkaline land of Jinan, Shangdong province, China. All the other genotypes were
grown in 2015 at a farm belonging to the China National Rice Research Institute
located in Hangzhou, China. After maturing, the grains were harvested, sun-dried to a
moisture content of about 13%, stored in an air-tight plastic bag at room temperature
for three months, and then stored at 4 oC in the dark before analysis. The rough rice
samples were de-hulled on a Satake Rice Machine (Satake, Tokyo, Japan) and milled
to pass through a 100-mesh sieve on a cyclone sample mill (Foss, Switzerland).

2.2. Chemicals
Folin-Ciocalteu reagent, DPPH (2,2-diphenyl-1-picrylhydrazyl), trolox
(6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid), ABTS
(2,20-azino-bis-(3-ethylbenzonthiazoline-6-sulfonic acid) diammonium salt) and
gallic acid, catechin, vanillin were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO, USA). Sodium hydroxide, sodium nitrite, aluminum chloride hexahydrate,
potassium preoxydisulfate, hydrochloric acid, sulfuric acid and sodium sulfate were
purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The HPLC
grade methanol and ethyl acetate, used in the extraction, purification and HPLC
analysis were purchased from Merck (Darmstadt, Germany) and Tedia (Fairfield, OH,
USA), respectively. The standards of gallic, protocatechuic, 2,5-dihydroxybenzoic,
p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, isoferulic,
o-coumaric acid, cyanindin-3-O-glucoside, peonidin-3-O-glucoside were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HPLC grade acetic acid
was purchased from Macklin (Shanghai, China). The certified mineral standards in
rice flour samples (GBW 10010, CRM Rice) were obtained from the National
Institute Center of Standards (Beijing, China).

2.3. Determination of color parameters

Color parameters of all samples were measured by the color difference meter (Konica
Minolta, Japan), and performed in triplicate. They were expressed as tristimulus
parameters, L*, a* and b*. L* indicates lightness (100 = white, 0 = black); a*
5
indicates redness-greenness (positive = red); b* indicates yellowness-blueness
(positive = yellow). In addition, the chroma (C) value which indicates color intensity
or saturation was calculated as C = (a*2 + b*2)1/2, and hue angle was calculated as H°
= tan-1 (b*/a*).

2.4. Extraction of soluble-free, soluble-conjugated and insoluble-bound phenolics

Soluble-free, soluble-conjugated, and insoluble-bound phenolics were extracted
according the method reported by Shao et al (2014a). Briefly, soluble phenolics of
rice flour were extracted with 80% methanol twice. The mixture was centrifuged
(Himac CR21G , Hitachi, Japan) at 10,000 ×g for 15 min at 4 oC. The soluble
phenolics were concentrated by a rotary evaporator (IKA RV10, German) at 37 oC to
get concentrated soluble phenolics. In order to get soluble-free phenolics, the
concentrated soluble phenolics were extracted by ethyl acetate three times, and then
dried by rotary evaporator, and dissolved in 5 mL of 50% methanol. To get
soluble-conjugated phenolics, the concentrated soluble phenolics were hydrolyzed
using 4 M NaOH for 2 h followed by adjusting pH to 1.5-2.0, extraction with ethyl
acetate, drying using a rotary evaporator, and then dissolving in 5 mL of 50%
methanol. After the extraction of soluble phenolics, the residues were used to extract
insoluble-bound phenolics. The protocols were similar to the extraction of
soluble-conjugated phenolics from concentrated soluble phenolics extracts by using 4
M NaOH and ethyl acetate. All extractions were performed in triplicate.

2.5. Determination of total phenilc content (TPC)

TPC was measured by Folin-Ciocalteu assay as reported by Shen et al. (2009). The
results were expressed as milligrams of gallic acid equivalent per 100 gram of dry rice
flour (mg GAE/ 100 g). Each extract was measured in duplicate.

2.6. Determination of ABTS radical scavenging activity (ABTS)

Total antioxidant activity of ABTS radical scavenging was assayed using
spectrophotometry (Shen et al., 2009). The results were expressed as micromoles of
6
trolox equivalent antioxidant activity per gram of dry rice flour (µM TE/ g). Each
extract was measured in duplicate.

2.7. Determination of DPPH radical scavenging activity (DPPH)

DPPH radical scavenging activity was carried out according to Beta, Nam, Dexter, &
Sapirstein (2005). Total antioxidant activity of DPPH radical scavenging was
expressed as micromoles of trolox equivalent antioxidant activity per gram of dry rice
flour (µM TE/ g). Each extract was measured in duplicate.

2.8. Determination of total flavanoid content (TFC)

TFC was assayed using a colorimetric method as reported by Shen et al. (2009). The
results were expressed as milligrams of catechin equivalent per 100 g of dry rice flour
(mg CE/ 100 g). Each extract was measured in duplicate.

2.9. Determination of total proanthocyanidin content (TPAC)

TPAC was determined using the vanillin assay (Sun, Ricardo-da-Silva, Spranger,
1998). The results were expressed as milligrams of catechin equivalent per 100 gram
of dry rice flour (mg CE/ 100 g). Each extract was measured in duplicate.

2.10. HPLC for analysis of phenolic acids

The phenolic acid content was analyzed by an HPLC system that consisted of a binary
pump (Waters 1525), an autosampler (Waters 2707), and a UV/Visible detector
(Waters 2489). A C18 column of dimensions 250 mm × 4.6 mm with 5 µm particles
(XBridge, Waters) was used for separation. The mobile phase consisted of A (0.1%
acetic acid in water) and B (0.1% acetic acid in methanol). The flow rate was 1
ml/min. A 75 min linear gradient was set according to the method of Shao et al.
(2014a). The injection volume was 10 µL. The column temperature was kept at 35 oC.
Soluble-free, soluble-conjugated, and insoluble-bound phenolic extracts were filtered
through 0.45 µm membrane filters before analysis. The phenolic acids were detected
at wavelengths of 280 and 320 nm, and quantified using the external calibration
7
curves according to the retention time of phenolic acid standards.

2.11. HPLC for analysis of anthocyanin compositions

The HPLC system and the column described above were also used for anthocyanin
analysis. The anthocyanins were extracted twice using acidified methanol (methanol:
1M HCl= 85:15, v/v). The clear supernatants contained anthocyanins. The mobile
phase consisted of A (0.5% formic acid in water) and B (0.5% formic acid in
methanol) at a flow rate of 1 mL/min. A 30 min linear gradient was set as follows: 0-2
min, 10% B; 2-6 min, 10-20% B; 6-10 min, 20-30% B; 10-15 min, 30-35% B; 15-20
min, 35-50% B; 20-24 min, 50-90% B; 24-29 min, 90-10% B; 29-30 min, 10% B. The
peaks of anthocyanins were detected at a wavelength of 520 nm. External calibration
curves were used to quantify the contents of anthocyanins.

2.12. Determination of some minerals

The minerals of rice (0.5 g) were extracted by ultrapure nitric acid (5 mL) on a
microwave digestion instrument (CEM Mars, Matthews, NC, USA). The digestion
procedure was set as follows: raising from room temperature to 120 °C, 0−5 min;
holding at 120 °C, 6−7 min; raising from 120 to 180 °C, 8−17 min; and holding at
180 °C, 18−32 min. After cooling to room temperature, the solution was transferred
into a 50 mL volumetric flask and diluted to 50 mL with deionized water. The
contents of K, Mg, Mn, Zn, Fe, Na, and Cu were measured by inductively coupled
plasma-atomic emission spectrometry (ICP-AES, Thermo Elemental, Franklin, MA)
at the wavelengths of 766.5, 280.3, 257.6, 213.9, 259.9, 589.0, and 324.8 nm,
respectively. The contents of minerals were calibrated using the certified standards in
rice flour samples. Results were expressed as milligrams per kilogram dry rice flour.
All the determinations were conducted in triplicate.

2.13. Statistical analysis

All the parameters were measured in triplicate, and the results were reported as means
± standard deviation (SD). Data analyses were performed with SAS version 8
8
software (SAS Institue, Cary, NC, U.S.A.). Differences among different rice varieties
were found by using ANOVA, followed by Duncan multiple comparison tests. The
correlation analysis among color parameters, phenolics, anthocyanins,
proanthocyanidins and minerals was performed with Pearson’s correlation test.
Statistical significance was defined at a level of P < 0.05. Principal component
analysis (PCA) was used to study the variation associated with different rice
genotypes.

3. Results and discussion

3.1. Color parameters
L* (lightness) ranged from 86.80 to 88.86, from 75.14 to 77.57, and from 63.56 to
76.41 for non-pigmented, red and black rice flours, respectively (Supplementary table
1). The a* values of red rice flour was significantly higher than that of non-pigmented
and black rice flours with the former higher than the latter. The b* and C values of
black rice flours were the lowest among three categories of flours. The non-pigmented
rice flours had higher H° values than red and black flours. The color parameters of
this study differed from the other report (Shen et al., 2009) due to the different forms
of samples in the measurement where rice flours and rice grains were used,
respectively. All the results of color parameters were in accordance with the previous
study reported by Shao, Tang, Xu, Wang & Bao (2013) as the same forms of samples
were used.

3.2. TPC, antioxidant activity, TFC, TPAC, anthocyanin composition and their
relations to color parameters
Soluble-free, soluble-conjugated and insoluble-bound TPC and antioxidant activity
are shown in Table 1.Soluble-free and soluble-conjugated TPC ranged from 0.79 to
51.86, and 4.52 to 40.83 mg GAE/ 100 g, respectively, with white rice showing the
lower values. The insoluble-bound TPC ranging from 33.17 to 62.55 mg GAE/ 100 g,
showed higher contents than soluble-free and soluble-conjugated TPC. Min et al.
(2012) reported that the soluble-free TPC in whole rice grain ranged from 44-697 mg
9
GAE/100 g, which was about 10 times higher than this study due to the differences in
methods used for extraction. The extractant of acetone/water/acetic acid might release
some of the soluble-conjugated and insoluble-bound phenolics (Choi, Jeong, & Lee,
2007) while some proteins, ascorbic acid and glucose could also be extracted as
interferences in Folin-Ciocalteu assay (Margraf, Karnopp, Rosso, & Granato, 2015).
The soluble-conjugated and insoluble-bound TPC were in accordance with the
previous reports (Min et al., 2012; Shao, Xu, Sun, Bao, & Beta, 2014b).

The antioxidant activity was measured by ABTS and DPPH assay (Table 1). In
generally, most of red and black rice had significantly higher soluble-free and
soluble-conjugated ABTS and DPPH than non-pigmented brown rice.
Insoluble-bound ABTS and DPPH did not differ much among non-pigmented and red
rice grains, respectively. As reported earlier on the high correlation between phenolic
contents and antioxidant activity (Shen et al., 2009; Zhou, Chen, Zhang, & Blanchard,
2014), ABTS and DPPH radical scavenging activity had similar tendency with TPC
among different rice samples.

Total flavonoid (TFC) and proanthocyanidin (TPAC) content, and anthocyanin

composition are shown in Table 3. The red and black rice grains had higher TFC
(162.86-415.10) than non-pigmented rice (63.06-114.49 mg CE/100 g). TPAC and
anthocyanins were only detected in red and black rice, respectively, and their contents
were dependent on the genotypes which differed from 58.13 to 254.80 mg CE/100 g,
and from 15.57 to 1417.12 mg/kg, respectively. In red rice, the majority of
proanthocyanidins are oligomers of 5-8 mers (40%), whereas the polymers (DP > 10)
accounted for 29% (Min et al., 2012). In black rice, the major anthocyanins were
cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G). Coloration of rice
is derived from the accumulation of anthocyanins or proanthocyanidins (Gunaratne et
al., 2013; Pereiracaro, Cros, Yokota, & Crozier, 2013). Thus, it could be expected that
red and black rice had higher TFC than the non-pigmented rice.

10
Pair-wise correlations between total phenolic content and antioxidant activity were
significantly positive (P < 0.01) among all the rice varieties either in soluble-free,
soluble-conjugated, or insoluble-bound forms (data not shown). The findings were
similar to many other reports (Choi et al., 2007; Masisi et al., 2016; Qiu, Liu, & Beta,
2010; Shen et al., 2009; Zhou et al., 2014). The correlations among color parameters,
TPC, ABTS and DPPH antioxidant activity, TFC, TPAC, and anthocyanin
composition are shown in Table 5. The color parameters of L*, b*, C, and H° of
whole rice flours had negative correlation with TPC, ABTS and DPPH antioxidant
activity either in soluble-free, soluble-conjugated or insoluble-bound fractions. The
negative correlations between the color parameters (L*, b*, C, and H°) and
soluble-conjugated TPC, ABTS or DPPH were significant (P < 0.01) suggesting that
pigmentation might be associated with the accumulation of soluble-conjugated
phenolics. There was partial agreement with a previous study that all the five grain
color parameters had negative correlations with the phenolics (soluble-conjugated
form), flavonoid contents and antioxidant activity (P < 0.001) (Shen et al., 2009),
although some differences still existed. Firstly, color parameters were measured by
different forms of samples as stated before. Secondly, different extraction methods
were performed.

The correlations between color parameters and TPAC or anthocyanin composition

have not been well understood. As in this study, TPAC was positively correlated with
a* (P < 0.001), while negative correlations were found between anthocyanin
composition (C3G and P3G) and color parameters of L*, b*, C and H° (P < 0.05). The
findings suggested that color-related phytochemicals were associated with different
color parameters, which was dependent on the definitions of color parameters and the
levels of pigmentations.

3.3. Phenolic acids and their relation to color parameters, TFC, TPAC, anthocyanin
composition and antioxidant activity
Most phenolic acids are found in soluble-conjugated or insoluble-bound form as seen
11
in Table 2. For soluble-free phenolic acids, p-CA (p-coumaric acid), FA (ferulic acid),
SIA (sinapic acid) and IFA (isoferulic acid) were detectable in all the rice grains,
among which p-CA and FA were the most abundant. PA (protocatechuic acid) and VA
(vanillic acid) could be only quantified in black rice. The contents of all soluble-free
phenolic acids were in accordance with previous studies (Liu et al., 2015; Hu, Tang,
Liu, Zhu, & Shao, 2017). For soluble-conjugated phenolic acids, PA and 2,5-DHA
(2,5-dihydroxybenzoic acid) were detected in red and black rice. The black rice of
NF13-15 had higher PA content (27.75-175.43) than red rice (except NF9)
(5.95-24.66 µg/g), and red rice had higher 2,5-DHA content (21.56-93.76) than black
rice (4.94-22.60 µg/g). Black rice had higher VA and FA than red and non-pigmented
rice, as similarly reported before (Shao et al., 2014b). For insoluble-bound phenolic
acids, the main phenolic acids were FA and p-CA in non-pigmented rice, FA, p-CA,
and 2,5-DHA in red rice, and FA, VA, PA and p-CA in black rice. The findings were
partially in agreement with reports by Sumczynski, Kotásková, Družbíková, & Mlček
(2016) and Shao et al. (2014 a,b). It was notable that high amount of 2,5-DHA
(18.07-57.34 µg/g) was detected in red rice which was rarely reported before. Similar
to the findings by Shao et al. (2014 a,b), there were no detectable PA in
non-pigmented rice and similar contents of IFA in all samples except NF6 and NF15.
The variation of the soluble-free, soluble-conjugated, and insoluble-bound phenolics
among different rice grains might be associated with the inconsistent distribution of
phenolics at cellular and subcellular levels in the grains (Sumczynski et al., 2016).

To assess the contribution of the individual phenolic acid to color parameters, TPAC,
individual anthocyanins, TPC, antioxidant activity or minerals, as well as the
differentiations among three phenolic fractions, the relationships among those
parameters are shown in Table 5 and Supplementary table 2. For soluble-free phenolic
acids, PA and VA detected only in black rice (Table 2) were significantly negatively
correlated with L*, b*, C, H° (P < 0.05). C3G and P3G, the most abundant
anthocyanins in black rice (Pereiracaro et al., 2013) were significantly correlated with
color parameters, suggesting that soluble-free PA and VA might be the precursor or
12
the accelerant of the anthocyanins in the biosynthesis pathway. p-CA was positively
correlated with C3G and P3G likely because p-coumarate is needed in the
biosynthesis pathway of anthocyanins (Shao & Bao, 2015). C3G and P3G were
significantly correlated with PA (r > 0.96, P < 0.01). Anthocyanins in plants might
have an antioxidant role against reactive oxygen species caused by external stresses
such as ultraviolet, pathogens, and some microbes (Van & Dat, 2006). Moreover
anthocyanins could be metabolized into PA as a major metabolite (Wang, Wei, Yan,
Jin, & Ling, 2010). Compared with soluble-free and insoluble-bound phenolic acids,
there were more soluble-conjugated phenolic acids that correlated with the color
parameters, indicating that the coloration of rice grain might be more associated with
soluble-conjugated phenolics.

Among all the rice samples, significantly positive correlations were observed among
three parameters (TPC, ABTS and DPPH) and PA, VA, SYA, or FA in
soluble-conjugated fraction (P < 0.05) (Supplementary table 2). Sumczynski,
Kotásková, Orsavová, & Valášek (2017) reported that the main contributors to
soluble-conjugated antioxidant activity are FA and VA > SIA > SYA > CA in wild rice.
In bound phenolic fractions, PA, VA, FA and IFA were positively correlated with TPC,
and PA while p-HA, p-CA and FA were positively correlated with ABTS or DPPH
antioxidant activity (Supplementary table 2). Palafox-Carlos, Gil-Chávez,
Sotelo-Mundo, Namiesnik, Gorinstein, & González-Aguilar (2012) found that PA
exhibited the highest antioxidant activity probably due to its particular chemical
conformation and hydroxyl groups. They also reported that the interactions among
phenolic acids in a mixture can affect total DPPH antioxidant activity (Palafox-Carlos
et al., 2012). It could be speculated that individual phenolic acids present in
soluble-free, soluble-conjugated and insoluble-bound phenolic fractions exert their
antioxidant activity differently. They might act individually, synergistic or
antagonistically, and the molecular mechanisms need to be clarified with further
studies.

13
3.4. Minerals and the relationships among minerals and individual phenolic acids
Rice grain is a rich source of minerals, such as K, Mg, Mn, Zn, Fe, Na, Cu, and Se
which have critical functions in rice plants where they are required in varying
amounts among different tissues (Sperotto, Vasconcelos, Grusak, & Fett, 2012). The
mineral content of all rice samples are shown in Table 4. K and Mg were the most
abundant minerals in whole rice grains, accounting for about 60% and 30% of total
minerals, respectively. Mn and Zn showed higher content in black rice than in most of
non-pigmented and red rice samples. Interestingly, Na content was extremely high in
NF5. It might be correlated with a decrease in K+, Ca2+, and Fe2+ uptake and directly
correlated with increased Na+ in rice by maintaining Na+/K+ balance (Ahmad, Javed,
Javed, & Alvi, 2009). The contents of all the minerals were similar as the other whole
grains (Itani et al., 2002) and higher than the milled rice (Jiang, Wu, Feng, Yang, &
Shi, 2007; Yu et al., 2015).

Pearson correlation analyses were performed among phenolics and minerals. The
minerals were not significantly correlated with grain color (data not shown).
Significantly positive correlations were found for Zn vs soluble-free FA, and Mg vs
soluble-conjugated FA (Supplemental table 2). It might be due to the physiological
functions of Zn and Mg (Tucker 1999). Among minerals, Mg was positively
associated with K and negatively correlated with Fe. Significantly positive
correlations were also found between Zn and Na, or Cu content (Supplemental table
3). These results could probably reflect the fact that Mg regulates the uptake of the K
and Fe (Tucker 1999). These results were comparable with the results of previous
studies showing a positive relationship between Mg and K, Zn and Cu (Jiang et al.,
2007; Zeng, Shen, Wang, Liu, Pu, & Du, 2005). However, Fe content had a significant,
negative correlation with Mg content of the whole rice grain in the present study,
unlike in the report by Jiang et al. (2007). This was likely due to the interaction
between ions whose chemical properties were sufficiently similar and therefore
compete for the site of absorption, transport, and function in plant (Jiang et al., 2007).
Moreover, the mineral contents were determined in whole rice and milled rice,
14
respectively. The loss of minerals was quite different among the seven elements
during milling process (Tabekhia & Luh, 1979). The relationships among different
minerals could help to understand the co-transportation and absorption of minerals in
rice grain.

3.5. Principal component analysis

Principal component analysis was performed on 43 variables including TFC, TPAC,
C3G, P3G, soluble-free, soluble-conjugated, and insoluble-bound TPC, ABTS, DPPH,
and individual phenolic acids (Fig. 1). The results showed that the first three principal
components could explain 74.6% of total variance. The first principal component
(PC1) was the most important one, explaining 46.41% of total variance. PC1
represented the soluble-free PA and VA, soluble-conjugated TPC, antioxidant activity,
VA and PA, insoluble-bound VA, and anthocyanin components. The second principal
component (PC2) accounted for an additional 19.67% of the total variances, which
was mainly attributed to soluble-conjugated 2,5-DHA and p-HA, total
proanthocyanidin content, color parameter of a*, insoluble-bound 2,5-DHA, and
soluble-free TPC and antioxidant activity. The third principal component (PC3)
accounted for an additional 8.53% of the total variances. The variation was mainly
attributed to insoluble-bound IFA, p-CA, SIA and soluble-free p-CA. As seen in Fig. 1,
all of rice samples could be divided into three, as non-pigmented, red and black rice
groups. The non-pigmented rice was located in the negative part of PC1 and PC2. The
red rice had negative PC1 but positive PC2. The black rice had positive PC1 but
negative PC2. However, NF11 was in the negative part of PC1 and PC2. This was a
result of similar antioxidant activity of soluble-conjugated fractions contained in
non-pigmented rice and NF11, as well as the lower content of C3G and P3G in NF11
than in other black rice.

4. Conclusions
The color parameters, soluble-free, soluble-conjugated and insoluble-bound phenolics
and antioxidant activity, total flavonoid content, minerals differed within
15
non-pigmented, red and black rice groups. Correlation analyses confirmed that
phenolics significantly contributed to the antioxidant activity of soluble-free, soluble
conjugate and insoluble-bound fractions. The contributions of the individual
soluble-free, soluble-conjugated and insoluble-bound phenolic acids to the color
parameters, TFC, TPAC, anthocyanin components, TPC, antioxidant activity or
minerals were assessed. Soluble-conjugated phenolics, TFC, TPAC, C3G and P3G
were more relevant for the color parameters. Soluble-conjugated PA, VA, SYA, and
FA were associated with soluble-conjugated TPC and antioxidant activity while
insoluble-bound PA and FA were correlated with insoluble-bound TPC and
antioxidant activity. Principal component analysis also divided all the rice into three
subgroups as non-pigmented, red and black. The study contributed to further
understanding of phenolic acids of three fractions and minerals in different colored
whole rice grains. The findings may assist rice breeders or eventually commercial rice
producers with new opportunities to promote the production of rice with enhanced
levels of phytochemicals.

Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grant
No. 31500246), the special research funds for the Central Public Research Institute of
the China National Rice Research Institute (Grant No. 2014RG006-1), and the Special
Project of Agricultural Product Quality Safety Risk Assessment (Grant No.
GJFP201701502), Ministry of Agriculture, China.

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Figure caption
Fig. 1. Biplot from principal component analysis of soluble-free, soluble-conjugated
and insoluble-bound phenolics and antioxidant activity, TFC, TPAC, C3G and P3G
of all rice samples (NF1-NF15, shown in Table 1).

21
Table 1. TPC, ABTS and DPPH radical scavenging activity of soluble-free, soluble-conjugated and insoluble-bound phenolic fractions of all the
samples a
TPC (mg GAE/100 g) ABTS (µM TE/ g) DPPH (µM TE/ g)
Samplesb
Soluble F Soluble C Insoluble B Soluble F Soluble C Insoluble B Soluble F Soluble C Insoluble B
F EF EF H GH FGH H G
NF1 0.79±0.05 8.10±0.05 45.21±0.93 0.11±0.01 1.93±0.04 3.64±0.03 0.05±0.01 0.80±0.04 1.47±0.03FG
NF2 2.14±0.10EF 7.48±0.63EF 49.41±2.97CD 0.22±0.02H 1.87±0.12GH 3.76±0.07D-G 0.11±0.01H 0.71±0.05GH 1.50±0.04FG
NF3 1.21±0.05F 4.52±0.34G 33.17±0.88I 0.19±0.01H 1.47±0.02I 3.34±0.39GH 0.09±0.00H 0.58±0.01H 1.12±0.10H
NF4 1.17±0.10F 6.17±0.15FG 34.41±0.49I 1.79±0.01FG 1.70±0.03HI 3.38±0.10GH 0.62±0.06G 0.79±0.02G 1.28±0.02H
NF5 3.21±0.05EF 5.66±0.78FG 47.24±1.76DE 1.10±0.04GH 1.50±0.02I 3.87±0.11C-G 0.47±0.05GH 0.59±0.05H 1.46±0.04G
NF6 6.55±0.40E 21.39±1.22C 50.64±3.09C 0.24±0.01H 0.66±0.02J 4.31±0.02BCD 0.20±0.01H 0.37±0.01I 1.71±0.02CDE
NF7 7.21±0.54E 9.21±0.24E 43.31±3.32FG 3.42±0.16E 2.67±0.00F 4.16±0.16C-F 1.45±0.16E 1.31±0.03E 1.80±0.01C
NF8 22.93±4.15C 18.14±3.41D 38.38±0.24H 6.54±0.25C 6.35±0.05D 3.99±0.01C-F 3.88±0.50B 2.28±0.16D 1.63±0.07D-G
NF9 19.59±0.10CD 18.24±1.41D 43.86±0.29FG 6.54±0.49C 6.34±0.02D 4.26±0.04B-E 3.68±0.20BC 2.40±0.06D 1.79±0.11CD
NF10 51.86±2.54A 18.55±0.98D 35.17±1.37I 13.11±1.21A 6.61±0.10D 3.70±0.23E-H 5.69±0.28A 2.25±0.03D 1.50±0.03FG
NF11 4.31±0.15EF 6.41±0.29EFG 41.45±1.27GH 2.42±0.12F 2.14±0.03G 3.17±0.01HI 1.05±0.04F 1.04±0.02F 1.60±0.08EFG
NF12 15.93±0.20D 21.93±1.56C 38.55±0.29H 5.27±0.21D 4.93±0.49E 2.73±0.19I 2.32±0.05D 2.31±0.10D 1.27±0.09H
NF13 24.10±0.24C 29.72±0.59B 51.41±0.24C 8.82±0.27B 7.25±0.06C 4.41±0.08BC 3.30±0.07C 3.09±0.07C 2.01±0.03B
NF14 36.52±4.44B 40.83±1.37A 62.55±0.20A 8.86±0.72B 9.69±0.08B 5.77±0.67A 3.42±0.18C 4.09±0.16B 2.69±0.16A
NF15 23.72±4.88C 39.72±0.10A 54.83±2.15B 7.36±0.54C 11.13±0.26A 4.73±0.11B 3.38±0.24C 4.64±0.04A 1.64±0.05C-F
a
The results are present as mean ± SD (n=3), and values in each columns with different letters are significantly different (P < 0.05).
b
NF1: 9311; NF2: Nipponbare; NF3: Tianyouhuazhan; NF4: II You 838; NF5: Yanfeng 47; NF6: Jinggangshanhongmi; NF7: Xianwanxian 12;
NF8: Qianxiuhong; NF9: Changhong No. 2; NF10: Hongmi 2; NF11: D Youzinuo 161; NF12: Heimi No. 1; NF13: Heixiannuo N0. 3; NF14:
Heinuomi; NF15: Heimi 2420.

22
Table 2. Soluble-free, soluble-conjugated and insoluble-bound phenolic acids of all rice samples a
Samples PA 2,5-DHA p-HA VA SYA p-CA FA SIA IFA
Soluble-free phenolic acids (µg/g)
NF1 nd nd nd nd nd 1.46±0.04FG 2.15±0.11EE 0.43±0.03B 0.52±0.05D
NF2 nd nd nd nd nd 0.76±0.07I 1.60±0.01F 0.12±0.02D 0.51±0.05D
NF3 nd nd nd nd nd 1.45±0.04FG 2.21±0.05E tr 0.39±0.00E
NF4 nd nd nd nd nd 2.06±0.04D 3.60±0.00B 0.26±0.03C 0.61±0.05C
NF5 nd nd nd nd nd 2.76±0.00B 1.53±0.02F 0.21±0.02C 0.27±0.02F
NF6 tr nd nd nd tr 1.70±0.11E 1.32±0.10F tr 0.82±0.06B
NF7 nd nd nd nd nd 1.66±0.09EF 2.59±0.25D tr 0.36±0.00E
NF8 tr tr tr nd nd 1.76±0.04E 2.01±0.05E tr 0.32±0.01EF
NF9 tr tr tr nd nd 1.68±0.02E 1.32±0.02F tr 0.27±0.00F
NF10 tr tr tr nd nd 1.56±0.03EF 1.62±0.04F 0.28±0.03C 0.52±0.04D
NF11 tr nd nd tr nd 1.11±0.05H 2.27±0.13DE 0.47±0.05B 0.61±0.05C
NF12 34.72±0.06C tr tr 15.28±0.24C tr 1.28±0.03GH 4.28±0.19A 0.49±0.05B tr
NF13 40.53±0.38B tr tr 18.63±0.77B tr 2.04±0.08D 3.18±0.10C 0.22±0.02C 0.50±0.03D
C
NF14 37.16±0.03 tr tr 12.46±0.92D nd 2.50±0.01C 2.14±0.17E tr 0.99±0.02A
NF15 71.46±4.44A tr tr 24.50±1.27A tr 3.63±0.31A 4.40±0.44A 1.81±0.11A 0.58±0.06CD
Soluble-conjugated phenolic acids (µg/g)
NF1 nd nd 2.62±0.09F 2.41±0.14F 1.02±0.02GH 4.73±0.29G 23.28±0.39E 21.90±2.10D 4.80±0.25E
NF2 nd nd 6.52±0.09E 1.94±0.05F tr 4.60±0.07G 21.92±0.04E 19.40±0.12D 4.75±0.04E
NF3 nd nd 2.45±0.12F 1.68±0.10F 1.88±0.06D 4.30±0.19G 18.42±0.98F 20.29±0.31D 5.84±0.27C
NF4 nd nd 5.28±0.27EF 1.84±0.13F 1.07±0.00FG 4.58±0.49G 22.50±1.42E 26.35±0.38C 7.64±0.21A
NF5 nd nd 3.86±0.24EF 1.50±0.01F 0.84±0.05H 9.76±1.04A 22.26±1.85E 9.37±0.60EF 6.37±0.69B
G
NF6 5.95±0.52 21.56±0.62E 3.27±0.32EF 0.90±0.09F 2.35±0.02C 5.69±0.40F 10.79±0.10G tr 3.99±0.19F
G
NF7 6.37±0.15 36.69±1.47D 19.5±0.25C 1.33±0.09F 1.23±0.06EF 8.84±0.18B 22.34±0.32E 11.88±0.51E 4.73±0.08E
NF8 8.38±0.56G 61.72±0.09C 19.59±1.67C 1.78±0.15F 1.33±0.05E 6.85±0.02DE 18.67±0.05F 21.97±1.04D 4.15±0.08F

23
NF9 35.55±1.20C 93.76±2.04A 27.29±2.39B 1.82±0.17F 1.94±0.12D 10.30±0.39A 22.79±0.65E 6.66±0.29F 2.73±0.24G
E
NF10 24.66±0.38 85.03±0.71B 39.88±3.59A 2.11±0.14F 1.32±0.15E 6.05±0.24EF 23.25±0.98E 10.15±0.74E 0.30±0.03H
NF11 7.98±0.13G 4.94±0.27H 16.32±1.35D 20.54±0.00E 0.89±0.02GH 2.91±0.10H 21.78±0.34E 6.70±0.57F 2.72±0.09G
F
NF12 19.94±0.09 22.60±1.67E 28.11±0.22B 76.57±1.82C 1.27±0.00EF 6.26±0.07EF 42.31±0.25B 38.75±2.31A 4.12±0.03F
NF13 27.75±0.68D 12.67±0.92G 20.61±1.92C 125.88±3.99B 2.65±0.25B 8.12±0.01BC 61.33±0.84A 33.59±0.34B 6.58±0.08B
NF14 105.96±4.90B 11.34±0.83G 6.24±0.64E 59.41±3.71D 2.64±0.09B 6.22±0.17EF 40.22±0.16C 3.58±0.31G 5.14±0.16DE
NF15 175.43±0.63A 15.39±0.83F 15.14±0.03D 138.42±2.22A 2.90±0.09A 7.63±0.32CD 36.48±1.09D 32.64±3.47B 5.36±0.15CD
Insoluble-bound phenolic acids (µg/g)
NF1 nd nd 1.24±0.12FG 1.42±0.12EF 0.94±0.00F 66.13±0.09CD 181.57±0.50EF 28.85±0.17C 9.30±0.36DE
NF2 nd nd 2.36±0.11D 1.34±0.05EF 0.66±0.01F 60.76±0.36DE 206.43±0.25CD 27.59±0.10C 8.54±0.36EF
NF3 nd nd 1.01±0.12G 1.04±0.02EF 0.64±0.01F 68.88±0.49C 152.42±0.15H 22.61±0.36D 4.56±0.25I
NF4 nd nd 1.89±0.20E 0.81±0.04F 0.60±0.07F 56.66±0.68E 137.88±1.88IJ 22.87±1.00D 5.27±0.05HI
NF5 nd nd 2.23±0.11D 1.40±0.12EF 1.01±0.05F 71.42±1.77C 199.42±1.38D 32.38±1.53B 6.43±0.08GH
NF6 3.82±0.25F 18.07±1.64D tr tr tr 118.09±9.57A 147.68±11.6HI 6.19±0.68J 25.44±1.66A
NF7 3.42±0.22F 57.34±5.60A 1.68±0.12E 0.77±0.05F 1.55±0.17E 66.40±1.02CD 170.76±2.64FG 17.57±1.45F 8.70±0.02EF
NF8 3.54±0.17F 48.50±0.45B 1.34±0.05F 3.89±0.42E 1.71±0.03E 54.95±1.39E 135.00±0.27J 14.89±0.06G 6.58±0.27GH
F
NF9 3.05±0.35 49.09±3.32B 1.82±0.03E 1.26±0.08EF 4.15±0.39C 80.61±1.32B 169.32±5.36FG 19.97±0.85E 7.23±0.11FG
NF10 3.14±0.29F 23.45±1.37C 1.33±0.07F 1.23±0.08EF 4.72±0.16B 66.51±4.35CD 151.21±8.54H 19.05±0.10EF 7.59±0.58FG
NF11 35.74±3.18D 6.71±0.70FG 3.64±0.12B 36.87±1.70D 4.84±0.45B 19.92±0.37GF 185.11±0.81E 14.05±0.54G 12.66±0.78C
E
NF12 21.49±2.05 11.82±0.11EF 2.94±0.14C 60.40±0.57C 4.44±0.31BC 15.50±0.24G 165.93±4.53G 7.71±0.14IJ 7.23±0.14FG
NF13 95.65±9.15B 13.04±0.03DE 1.69±0.18E 73.52±1.25B 4.5±0.27BC 22.13±0.93F 220.50±8.57B 10.57±0.37H 10.55±0.64D
A
NF14 159.55±1.53 11.71±0.62EF 3.90±0.14A 61.93±3.76C 3.00±0.16D 22.28±1.08F 218.09±10.11BC 8.62±0.22I 12.05±0.39C
NF15 47.21±0.36C 4.10±0.35G 2.94±0.09C 98.27±1.95A 12.87±0.33A 25.21±0.26F 254.74±1.61A 52.16±1.12A 21.71±1.42B
a
The results are presented as mean ± SD (n=3), and values in each columns of soluble-free, soluble-conjugated and insoluble-bound phenolic
acids with different letters are significantly different (P < 0.05). PA: protocatechuic acid; 2,5-DHA: 2,5-dihydroxybenzoic acid; p-HA:
p-hydroxybenzoic acid; VA: vanillic acid; SYA: syringic acid; p-CA: p-coumaric acid; FA: ferulic acid; SIA: sinapic acid; IFA: isoferulic acid;
nd:not detected; tr: detected in trace amount. NF1-NF15: shown in Table 1.

24
Table 3. Total flavonoid, proanthocyanidin content and anthocyanin composition of
all rice samples a

Variety TFC (mg CE/100 g) TPAC (mg CE/100 g) C3G (mg/kg) P3G (mg/kg)
I
NF1 83.27±0.00 nd nd nd
H
NF2 114.49±0.87 nd nd nd
I
NF3 82.65±4.33 nd nd nd
NF4 65.10±2.02I nd nd nd
I
NF5 63.06±2.60 nd nd nd
G D
NF6 175.92±6.93 58.13±3.77 nd nd
G C
NF7 162.86±1.15 78.13±3.77 nd nd
E B
NF8 322.24±2.02 226.13±1.89 nd nd
BC A
NF9 383.67±2.89 254.8±14.14 nd nd
CD B
NF10 364.69±3.75 219.47±0.00 nd nd
F E
NF11 198.57±3.75 nd 12.03±0.02 3.54±0.35E
NF12 314.69±27.13E nd 82.32±1.66D 12.71±0.10D
NF13 361.84±15.87D nd 226.75±6.49C 84.28±1.87C
NF14 415.10±4.04A nd 683.74±24.81B 145.25±15.61B
NF15 393.27±22.22B nd 1106.00±13.29A 311.12±4.28A
a
The results are presented as mean ± SD (n=3), and values in each column with
different letters are significantly different (P < 0.05). TFC: total flavonoid content;
TPAC: total proanthocyanidin content; C3G: cyaniding-3-O-glucoside; P3G:
peonidin-3-O-glucoside; nd: not detected. NF1-NF15: shown in Table 1.

25
Table 4. Contents of potassium (K), magnesium (Mg), manganese (Mn), zinc (Zn), iron (Fe), sodium (Na) and copper (Cu) of whole rice grain
(mg/kg) a
Variety K Mg Mn Zn Fe Na Cu
CD C E DE FG G
NF1 2427.50±1.06 1257.88±19.27 35.16±0.97 23.75±0.52 11.05±0.11 2.53±0.12 2.97±0.08F
NF2 1468.25±30.05I 755.63±20.68G 25.78±0.73F 50.40±0.33A 16.05±0.37C 4.26±0.19EF 7.10±0.16A
NF3 2153.38±59.22FG 1168.25±37.12DE 18.74±0.79G 20.19±0.05G 13.31±0.21DE 6.58±0.65BCD 1.33±0.09I
NF4 2142.63±12.90FG 1277.13±24.57C 21.13±0.22G 24.44±0.32D 14.15±0.43D 2.56±0.24G 2.56±0.06G
NF5 2084.13±25.99G 1069.75±27.22F 15.86±0.45H 14.95±0.06H 20.62±0.78A 42.78±2.25A 2.17±0.11H
NF6 2064.38±68.06G 1358.88±15.03B 38.38±0.41D 30.66±1.11B 13.67±0.35DE 5.20±0.22DE 4.03±0.03B
NF7 2262.75±33.94EF 1210.00±38.18CD 27.74±1.02F 21.02±0.11FG 12.20±0.02EFG 3.87±0.15EFG 2.11±0.11H
NF8 1882.50±29.70H 1096.75±30.05EF 50.93±1.87A 22.14±0.17EF 18.64±0.12B 3.32±0.06FG 3.53±0.14D
NF9 2530.38±34.83BC 1039.25±40.66F 33.77±1.12E 31.72±0.01B 15.87±0.02C 7.38±0.52BC 3.83±0.21C
NF10 2097.13±82.55FG 1222.00±68.59CD 33.57±1.78E 21.35±0.50FG 13.95±1.10D 6.17±0.06CD 2.28±0.11H
NF11 2219.00±23.33EFG 1204.63±36.95CD 38.83±1.13D 31.63±0.14B 10.73±0.34G 6.58±0.16BCD 2.23±0.15H
NF12 2673.75±101.47AB 1518.13±62.40A 49.89±2.60A 32.13±0.22B 12.60±0.29DEF 7.93±0.28B 3.92±0.15BC
NF13 2791.63±263.22A 1428.50±123.04B 44.50±4.76B 31.98±2.81B 12.45±2.14DEF 6.13±0.18CD 3.31±0.33E
NF14 2347.75±3.18DE 1272.00±16.26C 41.86±0.29C 32.08±0.38B 11.05±0.25FG 6.12±0.37CD 2.54±0.15G
NF15 2105.50±5.66FG 1238.63±9.02CD 34.43±0.66E 27.47±0.09C 12.23±0.54EFG 5.17±0.13DE 2.12±0.16H
a
The results are presented as mean ± SD, and values in each column with different letters are significantly different (P < 0.05). NF1-NF15: shown
in Table 1.

26
Table 5. Correlations among color parameters, TFC, TPAC, anthocyanin compositions and
soluble-free, soluble-conjugated and insoluble-bound phenolics of whole rice grain
L* a* b* C H TFC TPAC C3G P3G
Soluble-free
TPC -0.65** 0.49 -0.48 -0.32 -0.71** 0.85*** 0.65 0.63 0.55
ABTS -0.71** 0.45 -0.54* -0.38 -0.72** 0.88*** 0.74 0.56 0.55
DPPH -0.68** 0.57* -0.43 -0.26 -0.72** 0.90*** 0.88 0.71 0.70
PA -076*** -0.22 -0.91*** -0.90*** -0.72** 0.62** - 0.83 0.86
VA -0.76** -0.23 -0.91*** -0.91*** -0.68** 0.61* - 0.66 0.72
p-CA -0.40 -0.08 -0.44 -0.41 -0.51 0.31 -0.06 0.98** 0.99***
FA -0.46 -0.33 -0.61* -0.64** -0.32 0.20 -0.29 0.29 0.37
SIA -0.42 -0.20 -0.54* -0.52* -0.45 0.25 -0.32 0.64 0.72
IFA -0.06 -0.07 -0.29 -0.28 -0.24 0.07 -0.64 0.47 0.39
Soluble-conjugated
TPC -0.86*** 0.17 -0.85*** -0.75** -0.90*** 0.84*** 0.28 0.83 0.79
ABTS -0.80*** 0.21 -0.75** -0.64** -0.84*** 0.91*** 0.97** 0.92* 0.90*
DPPH -0.85*** 0.14 -0.82*** -0.73** -0.86*** 0.89*** 0.95* 0.92* 0.89*
PA -0.71** 0.00 -0.77*** -0.70** -0.80*** 0.66** 0.75 0.99*** 0.97**
2,5-DHA -0.31 0.88*** 0.14 0.33 -0.41 0.58* 0.94* 0.11 0.11
p-HA -0.54* 0.62* -0.21 -0.05 -0.47 0.66** 0.72 -0.56 -0.46
VA -0.73** -0.24 -0.91*** -0.90*** -0.65** 0.59* 0.90* 0.57 0.68
SYA -0.65** 0.20 -0.65** -0.57* -0.75** 0.62* -0.28 0.80 0.81
p-CA -0.31 0.36 -0.04 0.03 -0.34 0.36 0.29 0.51 0.57
FA -0.60* -0.31 -0.80*** -0.83*** -0.43 0.55* 0.59 0.01 0.07
SIA -0.16 -0.40 -0.33 -0.40 -0.01 0.08 0.46 0.03 0.14
IFA 0.27 -0.70** -0.06 -0.22 0.31 -0.41 -0.55 0.45 0.50
Insoluble-bound
TPC -0.44 -0.17 -0.59* -0.60* -0.49 0.36 -0.68 0.75 0.69
ABTS -0.13 -0.10 -0.18 -0.19 -0.13 0.29 0.47 0.35 0.25
DPPH -0.50 -0.06 -0.57* -0.56* -0.43 0.60* 0.59 0.19 0.10
PA -0.61* -0.22 -0.82*** -0.83*** -0.56* 0.58* -0.73 0.32 0.22
2,5-DHA -0.30 0.83*** 0.18 0.32 -0.36 0.40 0.20 -0.48 -0.53
p-HA -0.44 -0.39 -0.61* -0.66** -0.28 0.33 0.59 0.13 -0.02
VA -0.79*** -0.25 -0.96*** -0.96*** -0.68** 0.62* 0.62 0.81 0.87
SYA -0.73** 0.09 -0.72** -0.63* -0.74** 0.66** 0.79 0.71 0.80
p-CA 0.55* 0.41 0.75** 0.80*** 0.34 -0.44 -0.56 0.79 0.83
FA -0.41 -0.43 -0.68** -0.72** -0.39 0.31 -0.13 0.88 0.92*
SIA 0.17 -0.30 0.03 0.00 0.03 -0.17 0.67 0.78 0.86
IFA -0.49 0.14 -0.51 -0.44 -0.64* 0.29 -0.72 0.84 0.90*
Other phenolics
TFC -0.87** 0.46 -0.69** -0.55* -0.88*** - 0.98** 0.74 0.70
TPAC -0.12 0.86*** 0.34 0.51 -0.25 - - - -
C3G -0.65** -0.16 -0.80*** -0.77*** -0.73** - - - 0.98**
P3G -0.64* -0.16 -0.79*** -0.76** -0.72** - - - -
*, **, and *** indicate the significant levels at 0.05, 0.01, and 0.001, respectively.
27
Fig. 1.

28

Soluble-free, soluble-conjugated and insoluble-bound phenolic fractions were quantified in rice.

Soluble conjugated phenolics and anthocyanins were negatively correlated with color parameters.

Some soluble conjugated and insoluble bound phenolic acids had positive correlations with TPC and antioxidant activity.

The correlation ships among the seven mineral contents were exhibited.

Principal component analysis divided the rice samples into non-pigmented, red and black rice groups.

29