Food Sci. Biotechnol. Vol. 13, No. 2, pp. 172 ~ 175 (2004)
+ The Korean Society of Food Science and Technology
Antioxidant Activity of Organic Solvent Extracts from Far InfraredTreated Rice Hulls
Seung-Cheol Lee, Jeong-Han Kim, Seok-Moon Jeong, Jung-Uk Ha, K. C. Nam1 and D. U. Ahn1
Division of Food and Biotechnology, Kyungnam University, Masan 631-701, Korea
1
Department of Animal Science, Iowa State University, Ames, IA 50011-3150, USA
Abstract Methanolic extracts of rice hulls with or without far infrared (FIR) irradiation were sequentially fractionated with
solvents (hexane, chloroform, ethyl acetate, butanol, and water), and antioxidant activities of the fractions were analyzed for total
phenol contents (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capability, reducing power, and antioxidant
potency. Yield of chloroform fraction increased significantly from 6.74 to 20.78% after FIR irradiation, while those of ethyl
acetate and butanol fractions slightly decreased. Antioxidant activity of ethyl acetate fraction increased significantly by FIR
radiation as TPC and DPPH radical-scavenging activity increased from 0.07 to 0.19 mM and 30.09 to 80.19%, respectively.
Lard induction time of ethyl acetate fraction increased from 1.15 to 1.49 hr by FIR radiation. GC-MS analysis indicated
amounts of phenolic compounds (3-vinyl-1-oxy benzene and benzaldehyde) in ethyl acetate fraction of FIR-irradiated rice hull
methanolic extract were greater than those of nonirradiated ones.
Keywords: rice hull extract, solvent fraction, far infrared, antioxidant
Introduction
Plants contain a diverse group of phenolic compounds
including simple phenolics, phenolic acids, anthocyanins,
hydrocinnamic acid derivatives, and flavonoids, among which
phenolic acids and flavonoids are the most active antioxidant
compounds (1). Phenolic compounds possess antioxidant
properties by hydrogen donation through the hydroxyl
group, and the subsequently formed radicals are stabilized
by resonance delocalization throughout the phenolic ring
structure (2). In addition, many phenolics contain acid or
ring groups that may participate in metal chelation (3).
Rice hull contains phenolic compounds such as isovitexin,
phytic acid, vanillic acid, syringic acid, and ferulic acid (47). Our pervious studies revealed that methanolic extracts
of rice hull contain several phenolic compounds including
cinnamic and benzoic acid derivatives and far infrared (FIR)
irradiation between 2 and 14 µm significantly increased
the antioxidant activities of rice hull extracts (8, 9), because
FIR irradiation on rice hull liberated and activated the
covalently bound phenolic compounds that have antioxidant
activities.
For concentration and isolation of useful components from
plant extracts, fractionation with several organic solvents
has been widely applied. In the present study, the antioxidant
activities of organic solvent fractions from rice hull extract
with or without FIR irradiation were determined. These
results will be important in developing an effective method
for the production of food-grade natural antioxidant from
rice hull extracts.
Materials and Methods
Materials Rice hulls from a Japonica-type rice cultivar
*Corresponding author: Tel: 82-55-249-2684. Fax: 82-55-249-2995
E-mail: sclee@kyungnam.ac.kr
Received December 8, 2003; accepted February 12, 2004
(Oriza Sativa L.) were purchased from a milling plant in
Kimcheon, Korea. They were ground in a mill and passed
through a 48-mesh sieve. 2-Thiobarbituric acid (TBA), butylated
hydroxytoluene (BHT), tannic acid, fish oil, 1,1-diphenyl2-picrylhydrazyl (DPPH), and lard were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteu
reagent was from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan).
FIR irradiation onto rice hulls Rice hulls (50 g) were put
in a wooden box (50 cm×40 cm×40 cm) and irradiated at
2-14 µm for 30 min with an FIR heater (35×10 cm; output
300 W; Hakko Electric Machine Works Co., LTD., Nagano,
Japan). The sample-holding tray in the middle of the treating
box was placed to parallelly face the FIR heater, and the
distance between sample and heater was 20 cm.
Preparation of solvent fractionation extracts Rice hull
samples (300 g), with or without 30-min FIR irradiation, were
extracted in 1.5 L methanol overnight at room temperature.
The extract was filtered through a Whatman nylon membrane
filter (0.2 µm), and the filtrate was evaporated to dryness
under reduced pressure on a rotary evaporator at 40oC. The
dried extract was dissolved in 300 mL of 10% methanol,
and 300 mL of hexane was added. The mixture was then
partitioned into hexane and aqueous layers. After separation
of the hexane layer, 300 mL of chloroform was added to
the aqueous layer and partitioned, and the chloroform layer
was separated. Using the same procedure, ethyl acetate, nbutanol, and final aqueous layers were separated (10). The
separated layers were evaporated to dryness under reduced
pressure and weighed to determine the yields. Each solvent
fraction was redissolved in methanol (1 g/100 mL) and
used for further analyses.
Identification of ethyl acetate fraction of rice hull extract
Dried ethyl acetate fraction of the rice hull methanol
extract was dissolved in ethanol (200 mg/mL) and centrifuged
172
173
Antioxidant Activity of Far Infrared-Treated Rice Hull
at 13,400 × g for 5 min to precipitate undissolved materials.
The supernatant was mixed with 4 volumes of BSA [N,Obis(trimethylsilyl)acetamide] and derivatized in a 70oC water
bath for 15 min. The compounds in ethyl acetate fractions
were identified through gas chromatography/mass spectrometry
(GC6890/MS5973, Hewlett-Packard Co., Wilmington, DE,
USA). A split inlet (100:1) was used to inject samples (5 µL)
into a combined column, an HP-5 column (30 m, 0.25 mm
i.d., 0.25-µm film thickness; Hewlett-Packard Co.) connected
to an HP-35 column (7.5 m, 0.25 mm i.d., 0.25-µm film
thickness; Hewlett-Packard Co.), using a zero-volume
connector. A ramped oven temperature was used (100oC
for 2 min, increased to 270oC at 10oC/min, and held for 6
min). The inlet temperature was 250oC, and the carrier gas
was He at a constant flow of 1.5 mL/min. Ionization potential
of mass selective detector and scan range were 70 eV and
19.1-400 m/z, respectively. Identification of compounds
detected was achieved by comparing mass spectral data of
samples with those of the Wiley library (Hewlett-Packard
Co.).
Total phenolic contents (TPC) TPC of each rice hull extract
fraction was determined using the Folin-Ciocalteu reagent
with tannic acid as a standard (11). One milliliter of each
fraction was mixed with 1 mL of 50% Folin-Ciocalteu reagent
and 1 mL of 2% Na2CO3, and centrifuged at 13,400 × g for
5 min. The prepared samples were stored at room temperature
for 30 min, and the optical density of each sample solution
was measured at 750 nm using a spectrophotometer
(Shimadzu UV-1601, Tokyo, Japan). TPCs were expressed
as mM tannic acid equivalents.
DPPH radical scavenging activity Antioxidant activity
was determined based on the radical scavenging activity of
the sample (12). After mixing 1 mL of 0.041 mM DPPH in
ethanol with 0.2 mL of rice hull extracts for 10 min, the
optical density (OD) was measured at 517 nm. Results were
expressed as a percentage DPPH-radical scavenging activity
of the sample and were calculated according to the following
equation:
% DPPH-radical scavenging activity
= [(control OD − sample OD)/(control OD)] × 100
Statistical analysis All measurements except GC/MS
analysis were done in triplicates, and Students t-test was
used to determine the difference between mean values (p <
0.05) of FIR irradiated and nonirradiated samples (15).
Results and Discussion
Extract yield Methanol extracts of intact rice hull (IRH)
and FIR-treated rice hull (FRH) were partitioned sequentially
into hexane, chloroform, ethyl acetate, and butanol fractions.
Yield of chloroform fraction increased significantly from
6.74 to 20.78% after FIR irradiation, while those of ethyl
acetate and butanol fractions decreased slightly (Fig. 1).
Results revealed yield and antioxidant activity of natural
extracts were strongly dependent upon the solvent used for
extraction (16) due to the difference in polarity of each
compound (17).
Total phenolic contents (TPC) All solvent fractions,
except for chloroform fraction, of the FIR-irradiated rice
hull extracts showed higher amounts of phenolic compounds
in comparison with those from the nonirradiated ones (Fig.
2), an indication that the antioxidant activities of most
fractions were increased by FIR irradiation. Ethyl acetate
fraction showed the highest TPC increase of 0.19 from
0.07 mM among all fractions, thus making ethyl acetate
the optimal solvent for the extraction of polyphenols from
rice hulls. Ethyl acetate was also used for the extraction of
low molecular weight phenols from oak wood (18).
Furthermore, polyphenols extracted with ethyl acetate from
natural materials were reported to have strong antioxidant
activity (17).
Radical scavenging activity DPPH-radical-scavenging
activities of solvent fractions from the FIR-irradiated rice
hull extract were higher than those of the nonirradiated
ones (Fig. 3). The overall patterns of radical-scavenging
activity of the solvent fractions were equal to those of total
phenolic contents, except for the chloroform fraction.
Ethyl acetate fraction also showed the highest increase in
radical-scavenging activity, 80.19%, from 30.09%. At equal
concentrations (1 mg/mL), chloroform, ethyl acetate, and
butanol fractions of FRH showed as strong radical-
Reducing power Reducing power of the solvent fractions
of rice hull extract was determined using the method of
Oyaizu (13). Extracts (1 mg/mL) in phosphate buffer (2.5
mL, 0.2 M, pH 6.6) were added to potassium ferricyanide
(2.5 mL, 10 mg/mL), and the mixture was incubated at 50oC
for 20 min. Trichloroacetic acid (2.5 mL, 100 mg/mL H2O)
was added to the mixture and centrifuged at 13,400 x g for
5 min. The supernatant (1 mL) was mixed with distilled water
(1 mL) and ferric chloride (0.1 mL, 10 mg/mL H2O), and
the absorbance was measured at 700 nm.
Rancimat method Induction periods of lard as affected by
the addition of antioxidant were determined using a Metrohm
793 Rancimat (Herisan, Switzerland) (14). Oxidation was
carried out at 100oC with an airflow rate of 20 L/hr. One
milliliter of each sample (10 mg/mL) was added to the lard
(2.5 g), and mixed vigorously with a vortex for 8 s
immediately before Rancimat measurement.
Fig. 1. Yield of different fractions of methanol extracts from intact
rice hull (− −) and FIR treated rice hull (− −). Each value means
percent for yield.
174
S.-C. Lee et al.
Table 1. Effect of solvent fractions of methanol extracts from
intact rice hull extract (IRH) and far infrared-treated rice hull
extract (FRH) on the induction time of lipid peroxidation
Solvent
fraction
Fig. 2. Total phenolic contents of different fractions of methanol
extracts from intact rice hull (IRH) and FIR treated rice hull (FRH).
Data represent the mean±SD of three independent measurements.
Control
Methanol
Hexane
Chloroform
Ethyl acetate
Butanol
Water
Induction time for lard (hr)
IRH
FRH
1.09±0.031)
1.08±0.01
1.09±0.02
1.09±0.12
1.20±0.03
1.25±0.10
1.54±0.14
1.41±0.06
1.49±0.05
1.15±0.04
1.18±0.13
1.17±0.10
0.79±0.17
0.85±0.02
1
Each value expresses the mean of triplicate measurements with standard derivation. All values within a column are significantly difference
at p<0.05.
Fig. 3. Radical scavenging activity of different fractions of methanol
extracts from IRH and FRH. Data represent the mean±SD of
three independent measurements.
Fig. 4. Reducing power of different fractions of methanol extracts
from IRH and FRH. Data represent the mean±SD of three
independent measurements.
Fig. 5. A typical gas chromatography of ethyl acetate fraction
from (A) intact rice hull extract (IRH) and (B) FIR-irradiated
rice hull extract (FRH). The identities of peaks are: (A) 1, Silanol;
2, Hexanoic acid; 3, 7-Dioxa-2,8-disilanonane; 4, Benzoic acid; 5,
Tetradecanoic acid; 6, Galactose; 7, Mannitol; 8, Cinnamic acid; 9,
Hexadecanoic acid; 10, Indole acetic acid. (B) 1, Silanol; 2, Hexanoic
acid; 3, 7-Dioxa-2,8-disilanonane; 4, 3-Vinyl-1-oxy benzene; 5,
Benzaldehyde; 6, Benzoic acid; 7, Tetradecanoic acid; 8, Galactose;
9, Mannitol; 10, Cinnamic acid; 11, Hexadecanoic acid; 12, Indole
acetic acid.
scavenging activities as that of BHT, an indication that, in
rice hull extracts, antioxidant activities are closely related
to the total phenol contents.
significantly changed. The highest reducing power was
observed in the ethyl acetate fraction of FRH, also showing
that the reducing power of rice hull extracts is related to
the total phenol content.
Reducing power Reducing power of grape seed is
associated with its antioxidant activity (19). Duh (20) reported
that reducing properties are generally associated with the
presence of reductones. Figure 4 shows the reducing powers
of solvent fractions of FIR-irradiated and nonirradiated rice
hull extracts using the potassium ferricyanide reduction
method. The increased reducing powers of methanol, hexane,
chloroform, and ethyl acetate fractions by FIR irradiation
were 0.35, 0.50, 0.13, and 0.80 absorbance values, respectively,
whereas those of butanol and water fractions were not
Rancimat analysis Rancimat method is commonly used
to evaluate the antioxidant potency of various antioxidants
(21). The longer induction period of lard with the addition
of antioxidant compared to that of the control (pure lard)
increased the antioxidant activity of the antioxidant
compound. Table 1 shows the induction times of lard affected
by the addition of each solvent fraction. Chloroform fraction
showed the highest inhibition of lipid oxidation. However,
the lipid oxidation-retarding time of ethyl acetate fraction
of FIR-treated rice hull extract showed the greatest increase
Antioxidant Activity of Far Infrared-Treated Rice Hull
among the fractions, an increase from 1.15 to 1.49 hr.
GC analysis for ethyl acetate fraction of rice hull extracts
Phenolic compounds with antioxidant activity (benzoic,
cinnamic, and indole acetic acids) were detected in the ethyl
acetate fraction of nonirradiated rice hull methanolic extract
(Fig. 5A). However, the ethyl acetate fraction of FIR-irradiated
rice hull methanolic extract showed new phenolic compounds
such as 3-vinyl-1-oxy benzene and benzaldehyde (Fig. 5B).
Furthermore, the amount of cinnamic acid, a well-known
antioxidant phenolic compound, was higher than that from
nonirradiated rice hull. These results coincide with the
increase of TPC in ethyl acetate fraction of rice hull
extracts from 0.07 to 0.19 mM by FIR irradiation (Fig. 2).
In our previous study (8), FIR irradiation of rice hulls
liberated phenolic compounds, and increased the contents of
active compounds in the extracts. FIR irradiation activated
phenolic compounds in rice hull, thus increasing the
antioxidant activity of rice hull extract.
Acknowledgments
This study was supported by the Ministry of Science and
Technology (MOST) and the Korea Science and Engineering
Foundation (KOSEF) through the Coastal Resource and
Environmental Research Center (CRERC) at Kyungnam
University (R12-1999-025-08003-0), Korea, and State of
Iowa Funds. Jeong-Han Kim and Seok-Moon Jeong received
scholarships from the Brain Korea 21 Program of the
Korean Ministry of Education.
References
1. Aruoma OI. Extracts as antioxidant prophylactic agents. Int.
News Fats, Oils Relat. Mater. 8: 1236-1246 (1997)
2. Shahidi F, Wanasundara PK. Phenolic antioxidants. Crit. Rev.
Food Sci. Nutr. 32: 67-103 (1992)
3. Laughton MJ, Evans PJ, Moroney MA, Hoult JR, Halliwell B.
Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase
by flavonoids and phenolic dietary additives. Relationship to
antioxidant activity and to iron ion-reducing ability. Biochem.
Pharmacol. 42: 1673-1681 (1991)
4. Asamarai AM, Addis PB, Epley RJ, Krick TP. Wild rice hull
175
antioxidants. J. Agric. Food Chem. 44: 126-130 (1996).
5. Osawa T, Narasimhan R, Kawakishi S, Namiki M, Tashiro T.
Antioxidant defense system in rice hull against damage caused
by oxygen radicals. Agric. Biol. Chem. 10: 3085-3087 (1985)
6. Ramarathnam N, Osawa T, Namiki M, Kawakishi S. Chemical
studies on novel rice hull antioxidants. 2. Identification of isovitexin,
a c-glycosyl flavonoid. J. Agric. Food Chem. 37: 316-319 (1989)
7. Wu K, Zhang W, Addis PB, Epley RJ, Salih AM, Lehrfeld J.
Antioxidant properties of wild rice. J. Agric. Food Chem. 42:
34-37 (1994)
8. Lee SC, Kim JH, Jeong SM, Kim DR, Ha JU, Nam KC, Ahn
DU. Effect of Far-Infrared Radiation on the Antioxidant Activity
of Rice Hulls. J. Agric. Food Chem. 51: 4400-4403 (2003)
9. Lee SC, Kim JH, Nam KC, Ahn DU. Antioxidant properties of
far infrared-treated rice hull extract in irradiated raw and coked
turkey breast. J. Food Sci. 68: 1904-1909 (2003)
10. Roh JS, Sun WS, Oh SU, Lee JI, Oh WT, Kim JH. In vitro
antioxidant activity of safflower (Carthamus tinctorius L.) seeds.
Food Sci. Biotechnol. 8: 88-92 (1999)
11. Gutfinger T. Polyphenols in olive oils. J. Am. Oil Chem. Soc.
58: 966-968 (1981)
12. Blois MS. Antioxidant determination by the use of a stable free
radical. Nature 181: 1199-1200 (1958)
13. Oyaizu M. Studies on products of browning reaction: antioxidative
activities of products of browning reaction prepared from
glucosamine. Jpn. J. Nutr. 44: 307-315 (1986)
14. Chen JH, Ho CT. Antioxidant activities of caffeic acid and its
related hydroxycinnamic acid compounds. J. Agric. Food
Chem. 45: 2374-2378 (1997)
15. Walepole RE, Myers RH. Probability and Statistics for
Engineers and Scientists. Macmillan Publishing Co.: New York,
NY, USA. (1978)
16. Min JG, Son KT, Kim JH, Kim TJ, Park JH. Physiological and
functional properties of Salicornia herbacea (Tungtungmadi)
leaf extracts. Nutraceut. Food 7: 261-264 (2002)
17. Marinova EM, Yanishlieva NV. Antioxidative activity of extracts
from selected species of the family Lamiaceae in sunflower oil.
Food Chem. 58: 245-248 (1997)
18. Simon BF, Cadahia E, Garcia-Vallejo MC. Low molecular
weight phenolic compounds in Spanish oak woods. J. Agric.
Food Chem. 44: 1507-1511 (1996)
19. Jayaprakasha GK, Singh RP, Sakariah KK. Antioxidant activity
of grape seed (Vitis vinifera) extracts on peroxidation models in
vitro. Food Chem. 73: 285-290 (2001)
20. Duh PD. Antioxidant activity of budrock (Arctium lappa L.): Its
scavenging effect on free radical and active oxygen. J. Am. Oil
Chem. 75: 455-461 (1998)
21. Chen JH, Ho CT. Antioxidant properties of polyphenols extracts
from green and black teas. J. Food Lipids 2: 35-46 (1995)