Isolation, identification and antioxidant activity of bound phenolic compounds present in rice bran

Article abstract of DOI:10.1016/j.foodchem.2014.08.095

The bound phenolic compounds in rice bran were released and extracted with ethyl acetate based on alkaline digestion. An investigation of the chemical constituents of EtOAc extract has led to the isolation of a new compound, para-hydroxy methyl benzoate glucoside (8), together with nine known compounds, cycloeucalenol cis-ferulate (1), cycloeucalenol trans-ferulate (2), trans-ferulic acid (3), trans-ferulic acid methyl ester (4), cis-ferulic acid (5), cis-ferulic acid methyl ester (6), methyl caffeate (7), vanillic aldehyde (9) and para-hydroxy benzaldehyde (10). The structures of these compounds were determined using a combination of spectroscopic methods and chemical analysis. Among the compounds isolated, compound 3, 5 and 7 exhibited strong DPPH and ABTS⁺radical scavenging activities, followed by compounds 4 and 6. Compound 1 and 2 showed potent DPPH and ABTS⁺radical scavenging activities, compound 8 displayed moderate antioxidant activity against ABTS⁺radical, whereas compound 9 and 10 showed weak antioxidant activity.

Full text of DOI:10.1016/j.foodchem.2014.08.095

Accepted Manuscript
Isolation, identification and antioxidant activity of bound phenolic compounds
present in rice bran
Wei Wang, Jia Guo, Junnan Zhang, Jie Peng, Tianxing Liu, Zhihong Xin
PII:
S0308-8146(14)01320-X
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.08.095
FOCH 16318
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2014
18 August 2014
21 August 2014
Please cite this article as: Wang, W., Guo, J., Zhang, J., Peng, J., Liu, T., Xin, Z., Isolation, identification and
antioxidant activity of bound phenolic compounds present in rice bran, Food Chemistry (2014), doi: http://
dx.doi.org/10.1016/j.foodchem.2014.08.095
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Isolation, identification and antioxidant activity of bound phenolic compounds present in rice
bran
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Wei Wang, Jia Guo, Junnan Zhang, Jie Peng, Tianxing Liu, Zhihong Xin＊
Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing
Agricultural University, Nanjing 210095, PR China
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Abstract: The bound phenolic compounds in rice bran were released and extracted with ethyl acetate based
on alkaline digestion. An investigation of the chemical constituents of EtOAc extract has led to the isolation
of a new compound, para-hydroxy methyl benzoate glucoside (8), together with nine known compounds,
cycloeucalenol cis-ferulate (1), cycloeucalenol trans-ferulate (2), trans-ferulic acid (3), trans-ferulic acid
methyl ester (4), cis-ferulic acid (5), cis-ferulic acid methyl ester (6), methyl caffeate (7), vanillic aldehyde (9)
and para-hydroxy benzaldehyde (10). The structures of these compounds were determined using a
combination of spectroscopic methods and chemical analysis. Among the compounds isolated, compound 3, 5
and 7 exhibited strong DPPH and ABTS+ radical scavenging activities, followed by compounds 4 and 6.
Compound 1 and 2 showed potent DPPH and ABTS+ radical scavenging activities, compound 8 displayed
moderate antioxidant activity against ABTS+ radical, whereas compound 9 and 10 showed weak antioxidant
activity.
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Keywords: Rice bran; Alkaline hydrolysis; Bound penolic compounds; Structural identification;
Antioxidant activities;
1. Introduction
＊Corresponding author. Tel./fax: +86 25 8439 5618.
E-mail address: xzhfood@njau.edu.cn (ZH. Xin).
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Rice bran, produced as the most abundant and valuable byproducts in the rice milling process, is of
steadily growing interest in recent years due to its potential health benefits. It has been reported that rice bran
contained high levels of phytochemicals, such as γ-oryzanol (Xu & Godber, 1999), phytic acid (Canan et al.,
2011), tocopherols, tocotrienols (Xu, Hua & Godber, 2001), carotenoids (Stoggl, Huck, Wongyai, Scherz &
Bonn, 2005), γ-aminobutyric acid (Parrado, Miramontes, Jover, Gutierrez, Collantes De Terán & Bautista,
2006), octacosanol (Chen et al., 2007), squalene (Sugihara, Kanda, Nakano, Nakamura, Igusa & Hara, 2010),
unsaturated fatty acids (Xu & Godber, 1999), phytosterols and phenolic compounds (Liang et al., 2014).
Among them, phenolics have been extensively investigated because they exhibited a diverse range of
bioactivities such as antioxidative (Arab, Alemzadeh & Maghsoudi, 2011), antimicrobial (Kondo, Teongtip,
Srichana & Itharat, 2011), antiviral (Ray et al., 2013), anti-inflammatory properties (Akihisa et al., 2000) and
overall for the promotion of human health. These bioactive properties make these compounds play an
important role in the prevention of certain major chronic diseases, such as diabetes (Lai, Chen, Chen, Chang
& Cheng, 2012), chronic inflammation (Choi, Kim, Kang, Nam & Friedman, 2010), cardiovascular disease
(Qureshi, Bradlow, Salser & Brace, 1997) and certain kinds of cancer (Verschoyle, Greaves, Cai, Edwards,
Steward & Gescher, 2007), as strongly supported by animal studies and clinical trials.
Phenolics in rice bran are in soluble free, soluble conjugates and insoluble bound forms (Adom & Liu,
2002). Free forms present within the plant cell vacuoles (Pandey & Rizvi, 2009), whereas soluble esters or
conjugates are esterified to sugars and other low molecular mass components, and insoluble bound forms are
covalently linked to cell wall structural components, such as cellulose, hemicellulose, lignin, pectin and
rod-shaped structural proteins (Arranz, Silvan & Saura-Calixto, 2010; McKee & Latner, 2000). The last is the
major form in rice bran to enhance the mechanical strength of cell walls (Ryden, Sugimoto-Shirasu, Smith,
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Findlay, Reiter & McCann, 2003) as well as providing both physical and chemical barriers, protection against
pathogen invasion and in response to stress conditions such as infection, wounding and UV radiation, among
others (Rice-Evans, Miller & Paganga, 1997). An increasing number of research groups turned their attention
to these bound phenolics exist in fruits, vegetables and cereal grains, which have been given different names:
nonextractable, unextractable, insoluble, or bound phenolics (Perez-Jimenez & Torres, 2011). About 74% of
the total phenolics present in rice are in the insoluble bound forms, as also demonstrated by a comparison of
the antioxidant capacity of bound phenolics significantly higher than that of free or soluble conjugated forms,
with ferulic acid being the major phenolic compound present (Adom & Liu, 2002). In addition, some of these
phenolics such as ferulic acid and diferulates are predominantly found in grains but are not present in
significant quantities in fruits and vegetables (Adom & Liu, 2002).
Last decades, many studies have been reported the phenolic levels in rice bran using different
combinations of water and organic solvents to extract soluble phenolics, however, most of these studies
reported in the literature have largely ignored bound phenolics, hence underestimating the total content of
phenolics present (Arranz, Silvan & Saura-Calixto, 2010). Although a few dozen papers have focused on the
bound phenolics content and profiles, many of these studies were focused in the analysis of those known and
particular types of phenolics. Limitations of these studies are that the individual phenolic composition in the
sample usually must be known and the standards of some phenolics are commercially unavailable. Therefore,
the precise composition of bound phenolics is crucial for evaluating their physicochemical properties,
nutritional values, potential application, epidemiological and clinical studies addressing their potential health
effects, as well as for the lipid composition analysis and quality control of rice bran oils. However, to date,
there are no reports of the individual compositions of bound phenolics present in rice bran.
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In general, bound phenolics can be released by alkali, acid, or enzymatic treatment of samples prior to
extraction (Dai & Mumper, 2010). In most of cases, alkaline hydrolysis was the method mostly used for
extracting esterified or bound phenolics (Su et al., 2014), for example, in our preliminary studies, the content
of bound phenolics after alkaline hydrolysis of samples was significantly higher than after acid and enzymatic
hydrolysis. It was for this reason that alkaline hydrolysis was selected for releasing bound phenolics and
further chemical research.
In the context of our ongoing search for new bioactive components from fruits, vegetables, cereal grains
and other natural products, a chemical investigation of the ethyl acetate (EtOAc) extract from an alkaline
treatment sample of rice bran, led to the isolation of one new and nine known compounds and evaluation of
their antioxidant activities. Herein, we describe the experimental details of the hydrolysis and isolation
process as well as providing information pertaining to the elucidation of the structures of these compounds
based on their spectroscopic properties and chemical reactivity.
2. Materials and methods
2.1. General methods
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All of the H and 13C NMR spectra were recorded on a Bruker Avance 300, 400 and 500 spectrometer
(Bruker BioSpin GmbH, Beijing, China), using tetramethylsilane (TMS) as an internal standard. The chemical
shifts in the NMR spectra were recorded as δ values. Two-dimensional NMR spectra include COSY
(Correlation Spectroscopy, COSY), HSQC (Heteronuclear Singular Quantum Correlation, HSQC) and HMBC
(Heteronuclear Multiple Bond Correlation, HMBC). Electrospray ionization mass spectrometry (ESI-MS)
analyses were measured on a Q-Tof Ultima Global GAA076 LC mass spectrometer (Waters Asia, Ltd,
Singapore). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254
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(10-40 µm), and over the silica gel (300-400 mesh, Qingdao Marine Chemical Factory, Qingdao, China), and
reversed phase C18 (Octadecylsilyl, ODS) silica gel (Silicycle, 50 µm, Parc-Technologique Blvd, Canada)
and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA), respectively. Columns used for silica gel
chromatography separation were 2.4cm in internal diameter and 30cm in length, and columns used for
Sephadex LH-20 were 3.0cm in internal diameter and 100cm in length.
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2.2. Materials and chemicals
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Rice bran was purchased from WeiGang trade market (Nanjing, China). Di(phenyl)-(2,4,6-trinitrophenyl)
iminoazanium (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), L-Cysteine methyl
ester hydrochloride, and N-trimethylsilylimidazole were purchased from Sigma Aldrich (St. Louis, MO, USA).
Dimethyl sulfoxide-d6 (DMSO-d6) and CDCl3 were obtained from Merck (Darmstadt, Germany). All of the
other chemicals and solvents used in the current study were purchased as the analytical grade.
2.3. Alkaline hydrolysis of rice bran and extraction of bound phenolic compounds
The alkaline hydrolysis of rice bran was performed according to the method described by Adom et al.
(2002) with minor modifications. Briefly, rice bran (1.2 kg) was grinded and homogenized in a Polytron
homogenizer before being extracted three times with 12 L 80% acetone (1:10, w/v) at ambient temperature for
24 h. The supernatant was discarded after each extraction. The residues were then digested with 2 M 10L
sodium hydroxide at ambient temperature for 2 h with shaking under nitrogen gas. The mixture was
neutralized with an appropriate amount of hydrochloric acid and extracted with hexane to remove lipids. The
final solution was extracted five times with EtOAc, and the combined EtOAc extracts were concentrated
under vacuum at 40 °C to give the crude extract (8.2 g).
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2.4. Isolation bound phenolic compounds
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The crude extract (8.2 g) was separated into four fractions (Fr. 1-4) using normal-phase silica gel CC (20
g silica gel, 300-400 mesh) using a step-wise gradient elution of petroleum ether: acetone: MeOH
(100:0:0−0:100:0−0:0:100, v/v/v).
Fr. 1 (2.5 g) was separated into 2 sub-fractions Fr. 1-1 and Fr. 1-2 on a silica gel CC using cyclohexane
eluent. Fr. 1-1 was subsequently purified by CC over silica gel with a choloroform eluent to yield Fr. 1-1-1,
which was further purified over a silica gel CC using petroleum ether: acetone eluent (100:1, v/v) to yield 1
(31.4 mg) and 2 (10.0 mg).
Fr. 2 (1.8 g) was purified by CC over silica gel with a petroleum ether: acetone eluent (30:1, v/v) to yield
Fr. 2-1, which was then passed through a Sephadex LH-20 column with a CHCl3: MeOH eluent (1:1, v/v) to
give 3 (153 mg) and 4 (16.2 mg).
Fr. 3 (2.0 g) was also purified on a normal-phase silica gel CC using a cyclohexane: ethyl acetate eluent
(20:1, v/v) to yield Fr. 3-1, which was further purified by a reversed-phase C18 ODS CC using a step-wise
gradient elution of MeOH: H2O (0:100-100:0) to give 5 (12.8 mg) and 6 (14.5 mg).
Fr. 4 (1.9 g) was subjected to CC over silica gel using a chloroform: acetone eluent (30:1, v/v) to give Fr.
4-1, Fr. 4-2 and Fr. 4-3. Fr. 4-1 was purified by CC over silica gel with a petroleum ether: acetone eluent (20:1,
v/v) to give 7 (6.8 mg). Fr. 4-2 was separated through a Sephadex LH-20 column with a CHCl3: MeOH eluent
(1:1, v/v) to give 8 (4.6 mg). Fr. 4-3 was purified on a silica gel CC using cyclohexane: ethyl acetate eluent
(30:1, v/v) to yield 9 (11.7 mg) and 10 (9.8 mg). Fig. 1 showed the extraction and separation processes
associated with EtOAc extract of rice bran.
2.5. Assay of DPPH and ABTS+ radical-scavenging
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DPPH radical scavenging assay was performed according to a previously reported protocol
(Rivero-Perez, Muniz & Gonzalez-Sanjose, 2007). Radical scavenging activity against ABTS+ was performed
as described in the literature (Zheleva-Dimitrova, Nedialkov & Kitanov, 2010). DPPH radical scavenging
activity (%)=[1-(A517 nm of sample/A517 nm of control)]×100, where A517 nm of sample is the absorbance of DPPH radical
solution mixed with the test compound/Vitamin E, A517 nm of control is the absorbance of DPPH radical solution
mixed with methanol solution. ABTS+ radical scavenging activity (%)=[1-(A734 nm of sample/A734 nm of control)]×100,
where A734 nm of sample is the absorbance of ABTS+ radical solution mixed with the test compound/Vitamin E,
A734 nm of control is the absorbance of ABTS+ radical solution mixed with methanol solution. The antioxidant
activities of the test compounds were expressed as IC50, with the IC50 being defined as the concentration of
test compound required to inhibit the formation of radicals by 50%. Vitamin E was employed as a positive
control.
2.6. Acid Hydrolysis of the Glucoside and Determination of the Absolute Configuration of the
Monosaccharides
The assay used to determine the absolute configuration of the monosaccharide was performed as
described in the literature (Liang, Hao, Zhang, Zhang, Chen & Yu, 2011).
2.7. NMR and ESI-MS Spectroscopic Data
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Cis-ferulates cycloeucalenol (1) and trans-ferulates cycloeucalenol (2): White amorphous powder; H
NMR and 13C NMR data, see Table 1; ESIMS m/z 601.1 [M - H]-.
Trans-ferulic acid (3): Colorless needle; 1H NMR (300 MHz, DMSO-d6, TMS): δ 7.50 (d, J = 15.87 Hz,
1H, H-7), 7.27 (d, J = 2.16 Hz, 1H, H-2), 7.08 (dd, J = 8.07, 1.89 Hz, 1H, H-6), 6.79 (d, J = 8.13 Hz, 1H, H-5),
6.38 (d, J = 15.87 Hz, 1H, H-8), 3.80 (s, 3H, 3-OCH3); 13C NMR: δ 126.5 (C-1), 110.3 (C-2), 149.1 (C-3),
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147.8 (C-4), 115.1 (C-5), 123.0 (C-6), 145.2 (C-7), 114.9 (C-8), 167.7 (C-9), 55.4 (3-OCH3); ESIMS m/z
193.0 [M - H]-.
Trans-ferulic acid methyl ester (4): Colorless needle; 1H NMR (500 MHz, CDCl3, TMS): δ 7.66 (d, J =
15.92 Hz, 1H, H-7), 7.10 (dd, J = 8.20, 1.84 Hz, 1H, H-6), 7.04 (d, J = 1.84 Hz, 1H, H-2), 6.95 (d, J = 8.16
Hz, 1H, H-5), 6.33 (d, J = 15.92 Hz, 1H, H-8), 5.98 (s, 1H, 4-OH), 3.94 (s, 3H, 3-OCH3), 3.82 (s, 3H,
9-OCH3); 13C NMR: δ 126.9 (C-1), 109.3 (C-2), 148.0 (C-3), 146.8 (C-4), 115.1 (C-5), 123.0 (C-6), 145.0
(C-7), 114.7 (C-8), 167.8 (C-9), 55.9 (3-OCH3), 51.6 (9-OCH3); ESIMS m/z 207.0 [M - H]-, 231.0 [M + Na]+.
Cis-ferulic acid (5): Colorless needle; 1H NMR (300 MHz, DMSO-d6, TMS): δ 7.66 (d, J = 1.83 Hz, 1H,
H-2), 7.13 (dd, J = 8.25, 1.86 Hz, 1H, H-6), 6.75 (d, J = 8.19 Hz, 1H, H-5), 6.64 (d, J = 12.96 Hz, 1H, H-8),
5.75 (d, J = 12.96 Hz, 1H, H-7), 3.74 (s, 3H, 3-OCH3), ESIMS m/z 193.0 [M - H]-.
Cis-ferulic acid methyl ester (6): 1H NMR (500 MHz, CDCl3, TMS): δ 7.82 (d, J = 2.15 Hz, 1H, H-2),
7.13 (dd, J = 8.25, 1.86 Hz, 1H, H-6), 6.91 (d, J = 8.35 Hz, 1H, H-5), 6.84 (d, J = 12.90 Hz, 1H, H-8), 5.82 (d,
J = 12.90 Hz, 1H, H-7), 3.95 (s, 3H, 3-OCH3), 3.75 (s, 3H, 9-OCH3); ESIMS m/z 207.0 [M - H]-.
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Methyl caffeate (7): White amorphous powder; H NMR (500 MHz, Acetone, TMS): δ (1H, d, J =
15.92 Hz, H-7), 7.17 (1H, d, J = 15.92 Hz, H-8), 7.06(1H, dd, J = 1.84, 8.16 Hz, H-6), 6.89 (1H, d, J = 8.20
Hz, H-8), 6.31(1H, d, J = 15.92 Hz, H-7), 3.72 (s, 3H, 9-OCH3); 13C NMR: δ 127.5 (C-1), 115.1 (C-2), 148.7
(C-3), 146.3 (C-4), 115.3 (C-5), 122.5 (C-6), 145.7 (C-7), 116.3 (C-8), 167.9 (C-9), 51.5 (9-OCH3); ESIMS
m/z 217.1 [M + Na]+, 193.1 [M - H]-.
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Para-hydroxy methyl benzoate glucoside (8): White amorphous powder; H NMR and 13C NMR data,
see Table 1; HRESIMS [M + Na]+ at m/z 337.0906 (calcd 337.0899).
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Vanillic aldehyde (9): Colorless needle; H NMR (400 MHz, CDCl3, TMS): δ 9.86 (s, 1H, CHO), 7.47
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(2H, d, J = 5.84 Hz, H-2, H-6), 7.09 (1H, d, J = 8.48 Hz, H-5), 4.00 (3H, s, 3-OCH3); 13C NMR: δ 127.6 (C-1),
129.8 (C-2), 147.2 (C-3), 151.8 (C-4), 114.4 (C-5), 108.7 (C-6), 191.0 (C-7), 56.1 (s, 3H, 3-OCH3); ESIMS
m/z 151.0 [M - H]-.
Para-hydroxy benzaldehyde (10): Colorless amorphous powder; 1H NMR (400 MHz, DMSO-d6, TMS):
δ 9.79 (s, 1H, H-7), 7.77 (2H, d, J = 8.48 Hz, H-2, H-6), 6.95 (2H, d, J = 8.48 Hz, H-3, H-5), ESIMS m/z
121.0 [M - H]-.
2.8. Statistical analysis
All the data were analyzed according to Duncan’s multiple comparison test (p < 0.05), using version 8.1
of the SAS software package. The resulting data were presented as mean SD.
3. Results and discussion
3.1. Structural elucidation of the isolated compounds
Compound 1 was isolated as a white amorphous powder. Its molecular formula was determined to be
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C40H58O4 based on its H and 13C NMR, and ESI-MS data. ESI-MS analysis of the material revealed an m/z
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value of 601.1, corresponding to [M - H]-. The H and 13C NMR spectra were typical of a feruloyl ester of
triterpene alcohols. The 1D NMR spectrum showed the presence of one olefinic proton at δ 5.10 (t, J = 6.75
Hz, 1H, H-24), one oxygenated methine proton at δ 4.71 (s, 1H, H-3), seven methyl protons at δ 0.90 (s, 3H,
H-18), 0.90 (s, 3H, H-21), 1.69 (s, 3H, H-26), 1.61 (s, 3H, H-27), 0.86 (s, 3H, H-28), 0.90 (s, 3H, H-29) and
0.90 (s, 3H, H-30), and two methylene protons of a cyclopropyl group at δ 0.35 (d, J = 3.95 Hz, 1H, H-19),
0.58 (d, J = 3.90 Hz, 1H, H-19), as well as two typical sp2 olefinic carbon signals at δ 125.2 (C-24) and 130.9
(C-25). Taken together, these data were corresponding to a typical cycloartenol moiety. The 13C NMR of 1
displayed the presence of 40 carbon signals, with 30 of these signals being assigned to the cycloartenol and 10
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to the feruloyl ester moieties being connected to C-3 position of cycloartenol (Table. 1), which was confirmed
by the key correlation from H-3 to C-1′ from the HMBC experiment. The double bond of feruloyl ester
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moieties was determined to be a cis-configuration by its JH-2′
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coupling constant of 12.60 Hz. Further
H-3′
analysis of the 2D NMR data, including the COSY, NOESY, HSQC and HMBC spectra, allowed for the
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complete assignment of the H and 13C NMR spectral data (Fig. 2A), and compound 1 was consequently
identified as cis-ferulates cycloeucalenol (Fig. 2). Although this compound has previously been isolated from
rice ran (Akihisa et al., 2000), the comprehensive NMR spectral data were not yet reported.
Compound 2 was isolated as a white amorphous powder. Its molecular formula was determined to be
C40H58O4 on the basis of its 1H and 13C NMR, and ESI-MS spectra, with the latter of these analyses giving an
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m/z value of 601.1 [M - H]-. The H and 13C NMR spectra of 1 and 2 were very similar but not identical. A
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direct comparison of the H and 13C NMR data of compound 2 with those of 1 revealed that they share the
same planar structure with the sole difference of the presence of a trans-configuration double bond at δ H 6.29
(d, J = 15.90 Hz, 1H) and 7.59 (d, J = 15.85 Hz, 1H) in 2 instead of the cis-configuration at δ H 5.84 (d, J =
12.60 Hz, 1H) and 6.79 (d, J = 12.85 Hz, 1H) in 1 (Table. 1). The structure of 2 was further confirmed by the
COSY and HMBC correlations and by comparing the NMR data with those of trans-ferulates cycloeucalenol
in the literature (Cho et al., 2012). Compound 2 was consequently identified as trans-ferulates cycloeucalenol
(Fig. 2).
Compound 3 was isolated as a colorless needle. The molecular formula of the material was determined to
be C10H10O4 on the basis of its 1H and 13C NMR, and ESI-MS data. ESI-MS analysis of the material revealed
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a peak at m/z 193.0, corresponding to [M - H]-, indicating that the molecular weight of 3 was 194. The H
NMR spectrum revealed the presence of two olefinic protons at δ 7.50 (d, J = 15.87 Hz, 1H, H-7) and 6.38 (d,
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J = 15.87 Hz, 1H, H-8), and three aromatic ring protons at δ 7.27 (d, J = 2.16 Hz, 1H, H-2), 7.08 (dd, J = 8.07,
1.89 Hz, 1H, H-6) and 6.79 (d, J = 8.13 Hz, 1H, H-5), and one methoxyl at δ 3.80 (s, 3H, 3-OCH3). The 13
C
NMR spectrum of 3 revealed two olefinic carbons at δ 145.2 (C-7), 114.9 (C-8), and six aromatic carbons at δ
126.5 (C-1), 110.3 (C-2), 149.1 (C-3), 147.8 (C-4), 115.1 (C-5) and 123.0 (C-6), and one methoxy carbon at δ
55.4, as well as a carboxyl carbon at δ 167.7 (C-9). The coupling constant of olefinic proton between C-7 and
C-8 was 15.87 Hz which allowed the determination of the double bond of 3 as a trans-configuration. Based on
a comparison of these data with data published in the literature (Yoshioka, Inokuchi, Fujioka & Kimura,
2004), compound 3 was identified as trans-ferulic acid (Fig. 2).
Compound 4 was isolated as a colorless needle, and its molecular formula was determined to be
C11H12O4 on the basis of its 1H and 13C NMR and ESI-MS spectra. ESI-MS analysis of the material gave m/z
values of 207.0 and 231.0, corresponding to [M - H]- and [M + Na]+, respectively. A careful comparison of the
1H and 13C NMR spectra of 4 with those of 3 revealed the existence of a close structural relationship between
the two compounds. Compared to the spectra of 3, an additional methoxyl signal at δ 3.94 (s, 3H, 9-OCH3)
instead of the 9-OH signal at δ 9.78 (1H, br s) was observed in 4. As expected, an additional methoxyl carbon
signal at δ 51.6 was observed in the 13C NMR spectrum of 4. By a comparison with data available in the
literature (Tanaka, Kato & Tsuchiya, 1971), compound 4 was determined to be trans-ferulic acid methyl ester
(Fig. 2).
Compound 5 and compound 3 share the same planar structure and the same molecular formula, C10H10O4,
1
established on the basis of the ESIMS ions detected at m/z 193.0, corresponding to [M - H]-. The H NMR
spectrum revealed the presence of three aromatic ring protons at δ 7.66 (d, J = 1.83 Hz, 1H, H-2), 7.08 (dd, J
= 8.25, 1.86 Hz, 1H, H-6) and 6.75 (d, J = 8.19 Hz, 1H, H-5), and one methoxyl at δ 3.74 (s, 3H, 3-OCH3), as
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well as two olefinic protons at δ 5.75 (d, J = 12.87 Hz, 1H, H-7) and 6.38 (d, J = 12.96 Hz, 1H, H-8) with a
lower coupling constant, corresponding to a cis-configuration double bond. Based on these data and a
comparison with those reported in the literature (Akihisa et al., 2000), compound 5 was identified as
cis-ferulic acid (Fig. 2).
Compound 6 and compound 4 share the same planar structure and the same molecular formula, C11H12O4,
1
determined on the basis of the ESIMS detected ions at m/z 207.0, corresponding to [M - H]-. The H NMR
spectra for 6 and 4 showed very similar signals, with the exception that a pair of trans-configuration olefinic
protons at δ 6.31 (d, J = 15.95 Hz, 1H, H-7) and 7.64 (d, J = 15.95 Hz, 1H, H-8) for 4 was replaced by a pair
of cis-configuration olefinic protons at δ 5.85 (d, J = 12.90 Hz, 1H, H-7) and 6.84 (d, J = 12.90 Hz, 1H, H-8)
for 6. Based on the comparison of the NMR and MS spectral data with those reported in the literature (Tanaka,
Kato & Tsuchiya, 1971), the structure of 6 was determined to be cis-ferulic acid methyl ester (Fig. 2).
Compound 7 was isolated as a white amorphous powder. The molecular formula was determined to be
1
C10H10O4 on the basis of its H NMR, 13C NMR and ESI-MS spectra. ESI-MS analysis gave m/z values of
1
217.1 and 193.1, corresponding to [M + Na]+ and [M - H]-, respectively. The H and 13C NMR spectra for 7
and 4 showed very similar signals, with the exception that the 3-OCH3 singlet at δ H 3.92 for 4 was replaced
by a new exchangeable proton at δ H 8.57 for 7, and the absence of one carbon signal at δ C 55.9
corresponding to 3-OCH3 carbon for 4. These data indicated that the methoxy at C-3 in 4 was substituted by a
hydroxy group in 7. Based on a comparison of these data with information reported in the literature
(Balachandran et al., 2012), compound 7 was identified as methyl caffeate (Fig. 2).
Compound 8 was isolated as a white amorphous powder. Its molecular formula was established as
C14H18O8 by High-resolution mass spectroscopy (HRMS) [M + Na]+ at m/z 337.0906 (calcd 337.0899),
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indicating six degrees of unsaturation. H NMR and 13C NMR spectrum of 8 revealed the presence of one
para-disubstituted benzene ring, one sugar unit, one carbonyl and one methoxyl. Analysis of the 1H-1H COSY,
HSQC and HMBC spectra of compound 8 allowed for the complete assignment of the H and 13C NMR
1
spectral data (Table. 2). Key correlations from H-2 to C-1, C-4 and C-6, from H-3 to C-1, C-2, C-5 and C-7,
from 7-OCH3 to C-7 were observed in the HMBC experiments. The sugar unit, which was identified as a
3
β-glucopyranosyl group on the basis of its JH-1′
,
coupling constant of 7.20 Hz, was placed at the C-1
H-2′
position of the benzene ring because of the HMBC correlation between the anomeric proton at δ H 5.00 and
the C-1 carbon resonance at δ C 161.5. The chemical shifts of all the individual protons and carbons of the
sugar unit from C-1′ to C-6′ were assigned on the basis of 1H-1H COSY and HSQC spectra analysis (Fig. 2B).
Acid hydrolysis of 8 with 1 N HCl liberated benzene acid and D-glucose, which were identified by gas
chromatography-mass spectrometry (GC-MS) analysis of the corresponding trimethylsilyl L-cysteine
derivative and a direct comparison with an authentic sample of the same material, prepared in the same
manner. This result was further confirmed through a comparison of the retention time of this derivative with
that of the authentic D-glucose derivative, with both samples providing the retention time of 7.92 min. Feng et
al. (2008) isolated a new compound that had the same planar structure as that of compound 8 and named
pseudolaroside C, however, the sugar moiety is not glucose but allose, therefore, they are a pair of isomers
with the different sugar unit. Thus, the structure of 8 was unambiguously identified as para-hydroxy methyl
benzoate glucoside, which is a new compound isolated and identified for the first time from a natural source
(Fig. 2).
Compound 9 was obtained as a colorless needle. The molecular formula of the material was determined
to be C8H8O3 based on its H and 13C NMR, and ESI-MS data. ESI-MS analysis revealed an m/z value of
1
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151.0, corresponding to [M - H]-. Analysis of its H NMR spectrum revealed the presence of a tri-substituted
aromatic ring with signals at δ 7.47 (2H, d, J = 5.84 Hz, H-2, H-6) and 7.09 (1H, d, J = 8.48 Hz, H-5), as well
as an aldehyde group signal at δ 9.86 (1H, br s, H-7), and three methoxy protons at δ 4.00 (3H, s, 3-OCH3).
Accordingly, the 13C NMR spectrum of 9 showed six aromatic ring carbon signals at δ 151.8 (C-4), 147.2
(C-3), 129.8 (C-2), 127.6 (C-1), 114.4 (C-5) and 108.7 (C-6), one aldehyde group carbon signal at δ 191.0
(C-7), one methoxy carbon signal at δ 56.1 (s, 3H, 3-OCH3). These data were the same as those reported for
vanillic aldehyde in the literature (Wang, Zhang, Zhao, Wang, Liu & Xin, 2013), and compound 9 was
consequently identified as vanillic aldehyde (Fig. 2).
Compound 10 was isolated as a colorless amorphous powder. The molecular formula of material was
1
determined to be C7H6O2 based on its H NMR and ESI-MS data. ESI-MS analysis of the material gave an
m/z peak of 121.0, corresponding to [M - H]-. The H NMR spectrum of 10 revealed the presence of an
1
aldehyde group proton at δ 9.79 (s, H-7), and four aromatic ring protons at δ 7.77 (d, J = 8.48 Hz, H-2, H-6)
and 6.95 (d, J = 8.32 Hz, H-3, H-5), corresponding to a para-disubstituted benzene ring. Based on these data
and a comparison with data available in the literature (Shengan, Rong, Wenhan & Hongquan, 2012),
compound 10 was determined to be para-hydroxy benzaldehyde (Fig. 2).
3.2. Antioxidant activities of the pure compounds isolated from rice bran
The antioxidant activities of the 10 pure compounds were tested according to their DPPH and ABTS+
radical scavenging assay. The results of these experiments have been shown in Fig. 3 and Fig. 4. DPPH
radical scavenging activity of compounds 1-10 (100µM) ranged from 15% to 97% (Figure 3A). Compound 3, 4,
5, 6 and 7 displayed a higher DPPH radical-scavenging activity than that of Vitamin E. Compound 3, 5 and 7
showed the highest antioxidant activity followed by compounds 4 and 6 and compounds 1, 2 and 8. Compound
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9 and 10 had the lowest DPPH radical-scavenging activity. There was no significant difference (p > 0.05) in
antioxidant activities among compound 3, 5 and 7, between 4 and 6, 1 and 2. Based on the data obtained,
compound 1-8 were selected to further determine whether their DPPH radical-scavenging capacity were
exhibited in a dose-dependent manner and the results were shown in Fig. 4A. The DPPH radical-scavenging
activities for compound 3, 5 and 7 increased sharply with increasing concentration in a dose-dependent
manner at the range of 0-40 µM, and increased up to a maximal value of 91.68 % DPPH inhibitory effect at
the concentration of 40 µM, then keep constant, and other samples also showed a dose-dependent manner in
DPPH radical-scavenging assay. The IC50 of compound 1-8 and Vitamin E were 87.06, 84.98, 14.59, 61.16,
16.29, 63.16, 15.26, >100 and 71.17 µM, respectively. These results indicated that the DPPH
radical-scavenging activities of trans-ferulic acid, cis-ferulic acid and methyl caffeate were nearly the same
and stronger than that of Vitamin E, followed by trans-ferulic acid methyl ester and cis-ferulic acid methyl
ester. The antioxidant activities of cycloeucalenol cis-ferulate and cycloeucalenol trans-ferulate were almost
identical and comparable with Vitamin E. However, para-hydroxy methyl benzoate glucoside was less active
than Vitamin E since its IC50 value was higher. Similarly, as shown in Fig. 4B, all samples showed a
dose-dependent ABTS+ scavenging-radical activity. IC50 values for compounds 1-8 and Vitamin E were 62.30,
60.60, 6.58, 35.35, 7.23, 37.63, 6.74, 65.52 and 57.91 µM, respectively.
It is well documented that ferulic acid and γ-oryzanol are the major antioxidants in rice bran, a
substantial of work has been carried out to investigate their antioxidant activity in vitro and vivo base on
different models. Hiramitsu et al. suggested that γ-oryzanol inhibited 61% reaction at a concentration of 10-4
M in a lipid peroxidation system induced by porcine retinal homogenates using ferric iron or UV light, while
α-tocopherol only 14% in the same conditions (Hiramitsu & Armstrong, 1991). Xu & Godber demonstrated
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that γ-oryzanol components (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl
ferulate) possessed significantly higher antioxidant activity than that of any of the four vitamin E components
(α-tocopherol, α-tocotrienol, γ-tocopherol, and γ-tocotrienol) in a cholesterol oxidation system accelerated by
2,2'-azobis(2-methylpropionamidine) dihydrochloride, of which 24-methylenecycloartanyl ferulate showed
the highest antioxidant activity (Xu, Hua, & Godber, 2001). The higher antioxidant activities of γ-oryzanol
components might because the structure of γ-oryzanol components is similar to that of cholesterol, an
important component in reducing oxidation stress and maintaining the functionality of cells, therefore, in
accordance with the structural relationship theory, the γ-oryzanol components may have greater ability to
associate with cholesterol in the small droplets of the emulsion and become more efficient in protecting
cholesterol against free radical attack. However, they also found that ferulic acid showed the highest
antioxidant activity, followed by vitamin E and γ-oryzanol at the three different ratios in a linoleic acid model
(Xu & Godber, 2001). The antioxidant properties of ferulic acid could be attributed to its aromatic phenolic
ring that stabilizes and delocalizes the unpaired electron within its aromatic ring, thereby acting as free-radical
scavengers (Srinivasan, Sudheer, & Menon, 2007). Whereas the triterpene portion of γ-oryzanol may affect its
antioxidant activity by lowering the mobility in the system due to its relatively larger molecular structure than
free ferulic acid (Xu & Godber, 2001). Wilson et al. reported that oryzanol has a greater effect on lowering
plasma lipid and lipoprotein cholesterol concentrations in hypercholesterolemic hamsters. However, they also
suggested that ferulic acid may have a greater antioxidant capacity via its ability to maintain serum vitamin E
levels compared to rice bran oil and oryzanol. Thus, both oryzanol and ferulic acid may exert similar
antiatherogenic properties, but through different mechanisms (Wilson, Nicolosi, Woolfrey, & Kritchevsky,
2007). To date there are various antioxidant activity assay models, all of them with strengths and limitations.
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In some case, it may have some controversy when using different model. There is not one method that can
provide unequivocal results and the best solution is to use various methods instead of a one-dimension
approach (Carocho & Ferreira, 2013).
In the current study, alkaline hydrolysis was used to release phenolic compounds present in the insoluble
bound form prior to extract with EtOAc. An investigation of the chemical constituents of EtOAc extract has
led to the isolation of a new compound, para-hydroxy methyl benzoate glucoside (8), together with nine
known compounds, cycloeucalenol cis-ferulate (1), cycloeucalenol trans-ferulate (2), trans-ferulic acid (3),
trans-ferulic acid methyl ester (4), cis-ferulic acid (5), cis-ferulic acid methyl ester (6), methyl caffeate (7),
vanillic aldehyde (9) and para-hydroxy benzaldehyde (10). The structures of these compounds were
elucidated using a combination of spectroscopic methods and chemical analysis. Among the compounds
isolated, compound 3, 5 and 7 exhibited strong DPPH hydroxy radical and ABTS+ radical scavenging
activities as compared with Vitamin E, followed by compounds 4 and 6, compound 1 and 2 showed potent
DPPH hydroxy radical and ABTS+ radical scavenging activities, compound 8 displayed moderate antioxidant
activity against ABTS+ radical, whereas compound 9 and 10 showed weak activities, and compound 3 was the
predominant antioxidant ingredient among the isolated bound phenolic compounds.
Compound 1 and 2 are a pair of isomers of γ-oryzanol naturally occurring in rice bran and rice germ.
γ-oryzanol is a mixture of steryl ferulates, which are formed by esterification of the hydroxyl group of sterols
(campesterol, stigmasterol, β-sitosterol) or triterpene alcohols (cycloartanol, cycloartenol, 2,4-methylene
cycloartanol, cyclobranol) with the carboxylic group of ferulic acid (Goufo & Trindade, 2014). Since
γ-oryzanol was first identified in rice bran oil in 1954 (Fang, Yu & Badger, 2003), at least 25 constituents of γ
-oryzanol have been found to date, with five of them accounting for about 95% of the total γ-oryzanol content,
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including 2,4-methylenecycloartanyl trans-ferulate (34-44%), cycloartenyl trans-ferulate (19-26%),
campesteryl trans-ferulate (15-23%), β-sitosteryl trans-ferulate (7-17%), and stigmasteryl trans-ferulate
(1-7%) (Goufo & Trindade, 2014). It has been reported that γ-oryzanol showed various important biological
profile such as antioxidant activities, lowering serum cholesterol levels, treating inflammatory diseases,
inhibiting tumor growth, decreasing platelet aggregation, promoting blood circulation, reducing blood
pressure and also promoting growth and development in humans and animals (Goufo & Trindade, 2014; Cho
et al., 2012). Besides the well-documented health benefits, γ-oryzanol also has been reported as a potential
additive in various food products, pharmaceuticals and cosmetics (Goufo & Trindade, 2014). Akihisa et al.
(2000) isolated and identified five pairs of trans- and cis-ferulate isomers include cycloeucalenol cis-ferulate
1
and cycloeucalenol trans-ferulate from rice bran by column chromatography, TLC, HPLC and H NMR.
However, other studies suggest that these cis-ferulates were most likely formed during the manufacture of rice
bran and the multistep separation process of individual γ-oryzanol components, because daylight and
long-wavelength UV radiation can induce cis-trans isomerization of feruloyl esters (Goufo & Trindade, 2014;
Fang, Yu & Badger, 2003). In the present study, it was found that the content of cycloeucalenol cis-ferulate
was increasing continuously following the decreasing of cycloeucalenol trans-ferulate by TLC detection
during purification, indicating that cycloeucalenol trans-ferulate was unstable and could convert to
cycloeucalenol cis-ferulate. This result therefore implied that trans-ferulates were not really natural products,
but artifact products.
Compound 3-6 are cis-trans isomers of ferulic acid and its methyl ester derivatives. Ferulic acid is
widely distributed in fruits, vegetables and cereal grains. In rice, ferulic acid is esterified to cell wall
components and thus form part of the dietary fiber and exist therefore in a insoluble bound form (Arranz,
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Silvan & Saura-Calixto, 2010). Ferulic acid is considered as one of the most important phenolic acids,
exhibiting a wide spectrum of therapeutic properties like anti-inflammatory, antiatherogenic, antidiabetic,
antiageing, neuroprotective, radioprotective and hepatoprotective effects (Adom & Liu, 2002). It also protects
against coronary disease, lowers cholesterol in serum and liver, and increases sperm viability (Adom, Sorrells
& Liu, 2003). Many of these activities can be attributed to its potent antioxidant capacity because its phenolic
nucleus and unsaturated side chain can readily form a resonance stabilized phenoxy radical delocalized across
the entire molecule (Rice-Evans, Miller & Paganga, 1997). On account of these properties it is receiving
increased attention with regard to applications in the food, health, cosmetic, and pharmaceutical industries.
Four forms of ferulic acid and its derivates were isolated as the major components of bound phenolics from
rice bran in this study, two cis-isomers are the converted products of the corresponding trans-isomers due to
cis-trans isomerization of the side chain of olefinic protons. Antioxidant experiments revealed that
trans-isomers possessed almost identical DPPH hydroxy radical and ABTS+ radical scavenging activities to
the corresponding cis-isomers and were the main antioxidant compounds among bound phenolics, and the
antioxidant activity of the ferulic acid methyl ester was slightly lower than the corresponding ferulic acid due
to a mehoxyl group instead of a hydroxyl group.
Compound 7, methyl caffeate, is a methyl esterification product of caffeic acid which is the major
representative of hydroxycinnamic acids widely present in almost every plant. Caffeic acid showed significant
antioxidant activity and might inhibit the formation of mutagenic and carcinogenic nitrosamine compounds
(Kuenzig et al., 1984). In vivo, when ingested with the diet, caffeic acid increase the plasma antioxidant
capacity and inhibit oxidation of low-density lipoprotein (Gulcin, 2006). Methyl caffeate showed strong
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antioxidant activities in DPPH and ABTS+ assay, indicating that it might be one of the important contributors
for antioxidant activity of bound phenolics.
Compound 8, para-hydroxy methyl benzoate glucoside, consists of a molecular of para-hydroxy benzoic
acid and glucose by esterification integrated to the aleurone, pericarp and embryo cell walls. Compound 8
exhibited moderate antioxidant activity against ABTS+ radical and might represent a new potential natural
antioxidant agent.
Compound 9, vanillic aldehyde, was isolated from the seedpods of Vanilla planifolia originally. This
compound has been shown to have a wide range of bioactivities such as antioxidant, anti-mutagenic,
anti-inflammatory, analgesic, hypolipidemic, and hepatoprotective activities (Wang, Zhang, Zhao, Wang, Liu
& Xin, 2013). It has been demonstrated that vanillic aldehyde can be conversed from ferulic acid due to
microbial metabolism (Venturi, Zennaro, Degrassi, Okeke & Bruschi, 1998). In general, vanillic aldehyde is
used as flavoring agents in foods, beverages, cosmetics and pharmaceuticals.
Compound 10, para-hydroxy benzaldehyde, is one of the three isomers of hydroxyl benzaldehyde. This
compound has been found in several plants such as the orchid Gastrodia elata, Galeola faberi and vanilla. It
has been reported that para-hydroxy benzaldehyde showed antioxidation and inhibition of
gamma-aminobutyric acid (GABA) transaminase associated with antiepileptic and anticonvulsive activity (Ha
et al., 2000).
It has been documented that cells in humans and other organisms are constantly exposed to a variety of
oxidizing agents, some of which are necessary for life (Wang, Zhang, Zhao, Wang, Liu & Xin, 2013).
Overproduction of oxidants can cause oxidative stress resulting in damage to DNA and proteins molecules, as
well as membrane lipid oxidation and an increased risk of degenerative diseases such as cancer,
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atherosclerosis, ageing, cardiovascular and inflammatory diseases (Adom & Liu, 2002). Therefore, the
consumption of sufficient amounts of antioxidants has been recommended to prevent or slow the oxidative
stress induced by free radicals. Phytochemicals derived from natural resources, particularly from fruits,
vegetables and cereal grains containing high levels of bound phenolics, have played an important role
associated with a decreased risk of cardiovascular disease (Sun, Chu, Wu & Liu, 2002). Moreover, it has been
reported that most of bound phenolics, which are strongly covalently linked to cell wall components thus act
as part of the dietary fiber that are resistant to digestion and absorption in the human stomach and small
intestine, are entrapped by dietary fibre and reach the colon intact, where they are released under the
fermentation of colonic microflore, and exert beneficial effects by scavenging the free radicals and
counteracting the effects of dietary pro-oxidants (Adom & Liu, 2002). This may partly explain the mechanism
of grain consumption in the prevention of colon cancer and other digestive cancers, which is supported by
epidemiological studies (Adom, Sorrells & Liu, 2003). The high levels of bound phenolic compounds with
significant antioxidant activity present in rice bran indicated that it is a valuable potential resource and worthy
of further utilizing and developing on industry scale. It is worth mentioning, however, that the bound phenolic
compounds are only a minor component of rice bran. This research therefore suggests that the antioxidant
activities of rice bran are derived from the combination of phytochemicals and not from a single compound.
This result is therefore consistent with similar findings in whole fruits and vegetables, where the additive and
synergistic effects of phytochemicals in whole foods have been reported to be responsible for their potent
antioxidant activities, with the potential health benefits being attributed to the complex mixtures of
phytochemicals present in whole foods (He & Liu 2008).
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In summary, this study has demonstrated that the individual bound phenolic compounds present in rice
bran can be released by alkaline hydrolysis and obtained by chromatographic purification. The majority of the
phenolic compound was ferulic acid with powerful antioxidant activity. Para-hydroxy methyl benzoate
glucoside, in particular, has been identified as a new compound and showed moderate antioxidant activity
against ABTS+ radical. The current study represents a useful addition to understand the potential application
of the bound phenolic compounds, lay the foundation for the clarification of mechanisms associated with
combating human diseases, and enhance better understanding of the relationship between bound phenolics
and human health.
Acknowledgments
This work was financially supported by the Fundamental Research Funds for the Central Universities
(KYZ201118), the Project of National Key Technology Research and Development Program for the 12th
Five-year Plan (2012BAD33B10) and the project funded by special funds of agro-product quality safety risk
assessment of Ministry of Agriculture of the People's Republic of China (2014FP11).
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References:
Adom, K. K., Sorrells, M. E., & Liu, R. H. (2003). Phytochemical profiles and antioxidant activity of wheat
460
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474
475
476
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481
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494
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499
varieties. Journal of Agricultural and Food Chemistry, 51(26), 7825-7834.
Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and Food
Chemistry, 50(21), 6182-6187.
Akihisa, T., Yasukawa, K., Yamaura, M., Ukiya, M., Kimura, Y., Shimizu, N., & Arai, K. (2000). Triterpene
alcohol and sterol ferulates from rice bran and their anti-inflammatory effects. Journal of Agricultural and
Food Chemistry, 48(6), 2313-2319.
Arab, F., Alemzadeh, I., & Maghsoudi, V. (2011). Determination of antioxidant component and activity of
rice bran extract. Scientia Iranica, 18(6), 1402-1406.
Arranz, S., Silvan, J. M., & Saura-Calixto, F. (2010). Nonextractable polyphenols, usually ignored, are the
major part of dietary polyphenols: a study on the Spanish diet. Molecular Nutrition & Food Research, 54(11),
1646-1658.
Balachandran, C., Duraipandiyan, V., Al-Dhabi, N. A., Balakrishna, K., Kalia, N. P., Rajput, V. S., Khan, I.
A., & Ignacimuthu, S. (2012). Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated
from Solanum torvum Swartz. Fruit. Indian Journal of Microbiology, 52(4), 676-681.
Canan, C., Cruz, F. T. L., Delaroza, F., Casagrande, R., Sarmento, C. P. M., Shimokomaki, M., & Ida, E. I.
(2011). Studies on the extraction and purification of phytic acid from rice bran. Journal of Food Composition
and Analysis, 24(7), 1057-1063.
Chen, F., Wang, Z., Zhao, G., Liao, X., Cai, T., Guo, L., & Hu, X. (2007). Purification process of octacosanol
extracts from rice bran wax by molecular distillation. Journal of Food Engineering, 79(1), 63-68.
Cho, J., Lee, H. J., Kim, G. A., Kim, G. D., Lee, Y. S., Shin, S. C., Park, K., & Moon, J. (2012). Quantitative
analyses of individual γ-Oryzanol (Steryl Ferulates) in conventional and organic brown rice (Oryza sativa
L.). Journal of Cereal Science, 55(3), 337-343.
Choi, S. P., Kim, S. P., Kang, M. Y., Nam, S. H., & Friedman, M. (2010). Protective effects of black rice bran
against chemically-induced inflammation of mouse skin. Journal of Agricultural and Food Chemistry, 58(18),
10007-10015.
Carocho, M., & Ferreira, I. C. (2013). A review on antioxidants, prooxidants and related controversy: natural
and synthetic compounds, screening and analysis methodologies and future perspectives. Food and Chemical
Toxicology, 51, 15-25.
Dai, J., & Mumper, R. J. (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer
properties. Molecules, 15(10), 7313-7352.
Fang, N., Yu, S., & Badger, T. M. (2003). Characterization of triterpene alcohol and sterol ferulates in rice
bran using LC-MS/MS. Journal of Agricultural and Food Chemistry, 51(11), 3260-3267.
Feng, S., Guo, H., Liu, P., Li, H., & Guo, D. (2008). Chemical constituents in bark of Pseudolarix kaempferi.
Chinese Traditional and Herbal Drugs, 39, 10-12.
Goufo, P., & Trindade, H. (2014). Rice antioxidants: phenolic acids, flavonoids, anthocyanins,
proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Food Science & Nutrition, 2(2),
75-104.
Gulcin, I. (2006). Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology, 217(2-3),
213-220.
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514
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517
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526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
Hiramitsu, T., & Armstrong, D. (1991). Preventive effect of antioxidants on lipid peroxidation in the retina.
Ophthalmic Research, 23(4), 196-203.
Ha, J. H., Lee, D. U., Lee, J. T., Kim, J. S., Yong, C. S., Kim, J. A., Ha, J. S., & Huh, K. (2000).
4-Hydroxybenzaldehyde from Gastrodia elata B1. is active in the antioxidation and GABAergic
neuromodulation of the rat brain. Journal of Ethnopharmacology, 73(1-2), 329-333.
He, X., & Liu, R. H. (2008). Phytochemicals of apple peels: isolation, structure elucidation, and their
antiproliferative and antioxidant activities. Journal of Agricultural and Food Chemistry, 56(21), 9905-9910.
Kondo, S., Teongtip, R., Srichana, D., & Itharat, A. (2011). Antimicrobial activity of rice bran extracts for
diarrheal disease. Journal of the Medical Association of Thailand, 94 Suppl 7, S117-S121.
Kuenzig, W., Chau, J., Norkus, E., Holowaschenko, H., Newmark, H., Mergens, W., & Conney, A. H. (1984).
Caffeic and ferulic acid as blockers of nitrosamine formation. Carcinogenesis, 5(3), 309-313.
Lai, M. H., Chen, Y. T., Chen, Y. Y., Chang, J. H., & Cheng, H. H. (2012). Effects of rice bran oil on the
blood lipids profiles and insulin resistance in type 2 diabetes patients. Journal of Clinical Biochemistry and
Nutrition, 51(1), 15-18.
Liang, D., Hao, Z. Y., Zhang, G. J., Zhang, Q. J., Chen, R. Y., & Yu, D. Q. (2011). Cytotoxic triterpenoid
saponins from Lysimachia clethroides. Journal of Natural Products, 74(10), 2128-2136.
Liang, Y., Gao, Y., Lin, Q., Luo, F., Wu, W., Lu, Q., & Liu, Y. (2014). A review of the research progress on
the bioactive ingredients and physiological activities of rice bran oil. European Food Research and
Technology, 238(2), 169-176.
McKee, L. H., & Latner, T. A. (2000). Underutilized sources of dietary fiber: a review. Plant Foods for
Human Nutrition, 55(4), 285-304.
Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human health and disease.
Oxidative Medicine and Cellular Longevity, 2(5), 270-278.
Parrado, J., Miramontes, E., Jover, M., Gutierrez, J. F., Collantes De Terán, L., & Bautista, J. (2006).
Preparation of a rice bran enzymatic extract with potential use as functional food. Food Chemistry, 98(4),
742-748.
Perez-Jimenez, J., & Torres, J. L. (2011). Analysis of nonextractable phenolic compounds in foods: the
current state of the art. Journal of Agricultural and Food Chemistry, 59(24), 12713-12724.
Qureshi, A. A., Bradlow, B. A., Salser, W. A., & Brace, L. D. (1997). Novel tocotrienols of rice bran
modulate cardiovascular disease risk parameters of hypercholesterolemic humans. The Journal of Nutritional
Biochemistry, 8(5), 290-298.
Ray, B., Hutterer, C., Bandyopadhyay, S. S., Ghosh, K., Chatterjee, U. R., Ray, S., Zeittrager, I., Wagner, S.,
& Marschall, M. (2013). Chemically engineered sulfated glucans from rice bran exert strong antiviral activity
at the stage of viral entry. Journal of Natural Products, 76(12), 2180-2188.
Rice-Evans, C., Miller, N., & Paganga, G. (1997). Antioxidant properties of phenolic compounds. Trends in
Plant Science, 2(4), 152-159.
Rivero-Perez, M. D., Muniz, P., & Gonzalez-Sanjose, M. L. (2007). Antioxidant profile of red wines
evaluated by total antioxidant capacity, scavenger activity, and biomarkers of oxidative stress methodologies.
Journal of Agricultural and Food Chemistry, 55(14), 5476-5483.
Ryden, P., Sugimoto-Shirasu, K., Smith, A. C., Findlay, K., Reiter, W. D., & McCann, M. C. (2003). Tensile
properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and
rhamnogalacturonan II-borate complexes. Plant Physiology, 132(2), 1033-1040.
24

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574
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577
578
579
580
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Shengan, T., Rong, X., Wenhan, L., & Hongquan, D. (2012). Jaspiferin A and B: Two New Secondary
Metabolites from the South China Sea Sponge Jaspis stellifera. Records of Natural Products, 6, 398-401.
Stoggl, W., Huck, C., Wongyai, S., Scherz, H., & Bonn, G. (2005). Simultaneous determination of
carotenoids, tocopherols, and gamma-oryzanol in crude rice bran oil by liquid chromatography coupled to
diode array and mass spectrometric detection employing silica C30 stationary phases. Journal of Separation
Science, 28(14), 1712-1718.
Su, D., Zhang, R., Hou, F., Zhang, M., Guo, J., Huang, F., Deng, Y., & Wei, Z. (2014). Comparison of the
free and bound phenolic profiles and cellular antioxidant activities of litchi pulp extracts from different
solvents. BMC Complementary and Alternative Medicine, 14(9), 1-10.
Sugihara, N., Kanda, A., Nakano, T., Nakamura, T., Igusa, H., & Hara, S. (2010). Novel fractionation method
for squalene and phytosterols contained in the deodorization distillate of rice bran oil. Journal of Separation
Science, 59(2), 65-70.
Srinivasan, M., Sudheer, A. R., & Menon, V. P. (2007). Ferulic acid: therapeutic potential through its
antioxidant property. Journal of Clinical Biochemistry and Nutrition, 40(2), 92.
Sun, J., Chu, Y. F., Wu, X., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of common fruits.
Journal of Agricultural and Food Chemistry, 50(25), 7449-7454.
Tanaka, A., Kato, A., & Tsuchiya, T. (1971). Isolation of methyl ferulate from rice bran oil. Journal of the
American Oil Chemists’ Society, 48(3), 95-97.
Venturi, V., Zennaro, F., Degrassi, G., Okeke, B. C., & Bruschi, C. V. (1998). Genetics of ferulic acid
bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358. Microbiology,
144 ( Pt 4), 965-973.
Verschoyle, R. D., Greaves, P., Cai, H., Edwards, R. E., Steward, W. P., & Gescher, A. J. (2007). Evaluation
of the cancer chemopreventive efficacy of rice bran in genetic mouse models of breast, prostate and intestinal
carcinogenesis. British Journal of Cancer, 96(2), 248-254.
Wilson, T. A., Nicolosi, R. J., Woolfrey, B., & Kritchevsky, D. (2007). Rice bran oil and oryzanol reduce
plasma lipid and lipoprotein cholesterol concentrations and aortic cholesterol ester accumulation to a greater
extent than ferulic acid in hypercholesterolemic hamsters. The Journal of Nutritional Biochemistry, 18(2),
105-112.
Wang, X., Zhang, M., Zhao, Y., Wang, H., Liu, T., & Xin, Z. (2013). Pentadecyl ferulate, a potent antioxidant
and antiproliferative agent from the halophyte Salicornia herbacea. Food Chemistry, 141(3), 2066-2074.
Xu, Z., & Godber, J. S. (1999). Purification and identification of components of gamma-oryzanol in rice bran
Oil. Journal of Agricultural and Food Chemistry, 47(7), 2724-2728.
Xu, Z., Hua, N., & Godber, J. S. (2001). Antioxidant activity of tocopherols, tocotrienols, and
gamma-oryzanol components from rice bran against cholesterol oxidation accelerated by
2,2'-azobis(2-methylpropionamidine) dihydrochloride. Journal of Agricultural and Food Chemistry, 49(4),
2077-2081.
Xu, Z., & Godber, J. S. (2001). Antioxidant activities of major components of γ-oryzanol from rice bran using
a linoleic acid model. Journal of the American Oil Chemists' Society, 78(6), 645-649.
Yoshioka, T., Inokuchi, T., Fujioka, S., & Kimura, Y. (2004). Phenolic compounds and flavonoids as plant
growth regulators from fruit and leaf of Vitex rotundifolia. Zeitschrift für Naturforschung. C, Journal of
Biosciences, 59(7-8), 509-514.
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Zheleva-Dimitrova, D., Nedialkov, P., & Kitanov, G. (2010). Radical scavenging and antioxidant activities of
methanolic extracts from Hypericum species growing in Bulgaria. Pharmacognosy Magazine, 6(22), 74-78.
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Figure Captions
Fig. 1 Extraction and separation process for the bound phenolics of rice bran
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Fig. 2 Bound phenolic compounds isolated from rice bran and Key COSY and HMBC correlations for
compound 1 (A) and 8 (B)
Fig. 3 DPPH hydroxy radical-scavenging activity (A), ABTS radical-scavenging activity (B) of the isolated
compounds at the concentration of 100 µM. cycloeucalenol cis-ferulate (1), cycloeucalenol trans-ferulate (2),
trans-ferulic acid (3), trans-ferulic acid methyl ester (4), cis-ferulic acid (5), cis-ferulic acid methyl ester (6),
methyl caffeate (7), para-hydroxy methyl benzoate glucoside (8), vanillic aldehyde (9) and para-hydroxy
benzaldehyde (10). Each value has been presented as the mean SD of three experiments. Note: a-e, results
with a different letter differ significantly (p < 0.05).
Fig. 4 DPPH hydroxy radical-scavenging activity (A) and ABTS radical-scavenging activity (B) of
compounds 1-8 under different concentrations.
Table 1. NMR spectral data for compound 1 and 2 in CDCl3 at 400 (1H) and 100 MHz (13C)
Table 2. NMR spectral data for compound 8 in DMSO-d6 at 400 (1H) and 100 MHz (13C)
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Figure. 1
rice bran (1.2kg)
extracted with 80% acetone
discard the supernatant
residues
digested with 2 M NaOH
the mixture extracted with ethyl acetate
ethyl acetate extract (8.2g)
column chromatography on silica gel
(petroleum ether/acetone/methanol 100:0:0-0:100:0-0:0:100)
Fr.2(1.8g)
silica gel column
Fr.3(2.0g)
silica gel column
Fr.4(1.9g)
Fr.1 (2.5g)
silica gel column
cyclohexane
silica gel column
chloroform:acetone 30:1
petroleum ether:
acetone 30:1
cyclohexane:
ethyl acetate 20:1
Fr.3-1
reversed phase
silica gel column
methanol: H
Fr.2-1
Sephadex LH-20
chloroform:methanol 1:1
2O
Fr.1-2
silica gel column
Fr.1-1
(0:100-100:0)
Fr.4-2
silica gel column
petroleum ether:
acetone 20:1
Fr.4-3
silica gel column
cyclohexane:
Fr.4-1
Sephadex LH-20
chloroform:methanol
1:1
chloroform
ethyl acetate 30:1
Fr.1-1-1
5(12.8mg)
6(14.5mg)
4(16.2mg)
3(153mg)
8(4.6mg)
7(6.8mg)
silica gel column
petroleum ether:
acetone 100:1
10(9.8mg)
9(11.7mg)
1(31.4mg)
2(10.0mg)
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Figure. 2
21
18
22
20
26
12
25
11
9
27
19
10
17
1
14
O
1'
H3CO
HO
O
O
15
8
3'
9'
3
H3CO
HO
30
4
7
O
5
O
2'
7'
28
5'
29
1
2
6'
O
OR
7
9
1
5
OCH3
OR
O
8
HO
HO
HO
O
3
O
OH
R
H
CH3
6
R
3
H
CH3
7
5
4
O
O
H
HO
6'
O
5'
2
H
OH
OH
O
1
3
OH
4
1'
3'
H3CO
7
O
OH
O
OH
10
8
9
HO
O
OH
O
OH
OH
H3CO
HO
O
H3CO
O
O
B
A
HMBC
COSY
656
657
658
659
660
661
29