ASEAN Food Journal 15 (1): 89-96 (2008)

Extraction and Determination of Oryzanol in Rice Bran of Mixed Herbarium UKMB 89

Extraction and Determination of Oryzanol in

Rice Bran of Mixed Herbarium UKMB; AZ 6807:
MR 185, AZ 6808: MR 211, AZ6809: MR 29
1
Azrina, A., 1*Maznah, I. and 2Azizah, A. H.
1
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2
Department of Food Science, Faculty of Food Science and Technology,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Abstract: The level of total lipid and oryzanol content, an important antioxidant compound in
locally produced bran was investigated. Total lipid in rice bran was extracted using 3:2
chloroform:methanol mixture yielding 16.4% fat. Oryzanol content was determined without
saponification using a reverse-phase HPLC. Four fractions of oryzanol were successfully separated
and quantitated. The 4 isomers were cycloartenyl ferulate, 24-methylene cycloartanyl ferulate,
campestryl ferulate and mixtures of β–sitosteryl ferulate and cycloartanyl ferulate. The oryzanol
content of local mixed varieties ranged from 23.7–43.0 mg g-1. The oryzanol concentration may
depend on factors such as plant varieties, processing methods employed, extracting solvent used
and ratio of extracting solvent to bran as well as extracting solvent temperatures. This study showed
the potential of oryzanol extract from rice bran as a source of antioxidant.

Keywords: Vitamin, oryzanol, rice bran, food analysis

INTRODUCTION 1991; Raghuram and Rukmini, 1995). The

oryzanol alone has been reported to constitute
Rice bran, a by-product of the rice milling around 20 - 30% in the unsaponifiable matter
process constitutes about 10 wt % of rough rice of the bran and has been shown to have many
grain. The bran layer contains 18 - 22% oil, pharmaceutical uses such as for growth
making it the richest oil source from a grain acceleration, regulation of estrous cycle and
by-product (Saunders, 1990). The an effective antioxidant compound
hypocholesterolemic effect of rice bran has (Seetharamaiah and Prabhakar, 1986).
been attributed to various fractions of the bran Oryzanol or γ–oryzanol is a mixture of
such as the neutral detergent fiber, sterol esters of ferulic acid. This antioxidant
hemicellulose, rice bran oil and its compounds was first isolated in 1955 by
unsaponifiable matter (Nicolosi et al., 1991; Kaneko and Tsuchita. Norton (1995) reported
Visser et al., 2000). Compared to other that the complete oryzanol group is unique
vegetable oils, rice bran oil contains to rice bran oil and the exact composition of
considerably high (4%) unsaponifiable matter or yzanol depends on the rice cultivars.
which includes phytosterols, triterpene, Gamma-oryzanol, a mixture of phytosteryl
alcohols, tocols and oryzanol (Nicolosi et al., ferulates comprises 3 major components;

*Corresponding author.
E-mail: maznah@medic.upm.edu.my or maznahis@putra.upm.edu.my

ASEAN Food Journal Vol. 15, 89-96

90 Azrina, A., Maznah, I. and Azizah, A. H.

cycloartenyl ferulates, 24-methylene- varieties (Herbarium UKMB; AZ 6807: MR

cycloartanyl ferulate and campesteryl ferulate. 185, AZ 6808: MR 211, AZ 6809: MR 29.
Ten fractions of γ –or yzanol isomers from Freshly milled rice bran was collected from mill
cr ude rice bran have been successfully break # 2, which was the first polisher
identified and isolated using reverse-phase immediately after the removal of hull. The
HPLC (Xu and Godber, 1999). There have sample was then transported immediately to
been many reports on the physiological Universiti Putra Malaysia (UPM) on dry ice in
properties of γ-oryzanol such as having the cold box containers. Upon arrival the sample
superoxide dismutase-like antioxidant activity was sieved using 600 mm sieve to obtain
and hypocholesterolemic effects in animal uniform particle size before subjecting to the
models (Hundermer, 1991; Kahlon et al., 1992, stabilization process.
1996; Rouanet et al., 1993) and human subjects Total lipid was extracted using a method
(Gerhardt and Gallo, 1998; Visser et al., 2000). described by Suzuki et al. (1996). Butylated
Besides the well-documented health benefits, Hydroxytoluene (BHT) (Sigma, UK),
oryzanol also has been reported as a potential Chloroform (BDH, England) and Methanol
additive in various food products, (BDH, England) were used to extract lipid. All
pharmaceuticals and cosmetics (Lloyd et al., reagents were of analytical grade. Oryzanol
2000). content was analysed using HPLC and the
Although numerous studies have been method developed by Rogers et al. (1993). The
conducted (Seetharamaiah and Prabhakar, solvent system consisted of HPLC-grade
1986; Shin and Godber, 1996; Hu et al., 1996) acetonitrile (Fisher Chemicals, UK), methanol
on the various stabilization methods and (Merck, Germany) and isopropanol (Merck,
solvent extraction systems to extract the Germany). Mobile phase was filtered and
maximum amount of this high-value degassed under vacuum immediately prior to
antioxidant from rice bran, only a single study use. The oryzanol standard was purchased
(Othman, 1987) has been done for the locally from Tokyo Kasei (Japan). Stabilization of rice
produced rice bran. Therefore the purpose of bran was carried out using Autoclave (Tomy
this study was to quantify and evaluate the SS-325, US) and Microwave (National
amount of γ–oryzanol in mixed local Malaysian Microwave/Convection Oven IEC-750W,
varieties. Japan) (Azrina et al., 2000).
In this study, the level of γ–oryzanol was
determined in rice bran from local mixed Standard and Sample Preparation
varieties following two different stabilization
methods. The stabilized rice bran samples were Standard: Stock solution of γ-oryzanol standard
stored at room temperature (26 o C) and was prepared at a concentration of 50 mg ml-1
analyzed at specific intervals for 48 weeks. In of mobile phase. A series of daily working
this study chloroform:methanol (3:2, v/v) was standards used were 2000 ppm, 1000 ppm, 500
used in a ratio of 1 part of rice bran to 5 parts ppm, 100 ppm and 50 ppm prepared from
of extracting solvent. diluting stock solution with mobile phase.

MATERIALS AND METHODS Sample: A known amount of lipid was diluted

in mobile phase at 20% concentration. The
Materials sample was vigorously vortexed (MS1
Rice bran sample was provided by a local Minishaker, Malaysia) for 5 min. The slight
milling company, Padiberas Nasional Berhad emulsion formed was broken by centrifugation
(BERNAS) at Sekinchan, Selangor, Malaysia. (Hettich, Germany) at 3000 rpm for 3 min.
The rice bran used was from mixed local Aliquots were then filtered (Whatman, USA;

ASEAN Food Journal Vol. 15, 89-96

 Extraction and Determination of Oryzanol in Rice Bran of Mixed Herbarium UKMB 91

0.2 um pore size, PTFE membrane) prior to

analysis.

Total Lipid Extraction

Twenty grams of stabilized rice bran sample
stored for a specific period of time was
homogenized with 100 ml chloroform:
methanol (3:2, v/v) mixture containing 0.05%
butylatedhydroxytoluene (BHT) on an orbital Figure 1: Molecular structure of ferulic acid
shaker (Protech, Malaysia) for 1 hour. The esterified with 24-methylene-cycloartanol
homogenate was centrifuged, and re- (Source: Lloyd et al., 2000)
homogenized twice with another 25 ml
mixture of chloroform: methanol. The preheated solvent extraction, a higher
extracts were combined and concentrated on percentage of crude oil can be extracted (24.9
a rotary evaporator (Butchi, Switzerland). The + 0.9%) (Hu et al., 1996).
lipid extract was kept in 20 ml chloroform: A typical HPLC chromatogram obtained
methanol solution (3:2, v/v) at temperature from crude rice bran oil extracted is shown in
below –20oC for further analysis. Figure 2. There were 4 major isomers detected
namely; cycloartenyl ferulate, 24-methylene
Determination Oryzanol Content cycloartanyl ferulate, campesteryl ferulate and
The oryzanol components of rice bran lipid mixtures of β –sitoster yl fer ulate and
were separated by reverse-phase HPLC. The cycloartanyl ferulate. The total γ–oryzanol
HPLC system consisted of a Hewlett-Packard concentration ranged from 23.7 - 43.0 mg g-
(Germany) Model G1311A High Performance 1
in the crude oil of the stabilized samples
Liquid Chromatography connected to ALS without saponification. Generally saponi-
Autoinjector Series 1100 (Hewlett Packard, fication has been employed in most lipid-
Germany). Or yzanol components were extracted samples to remove interfering
detected at 325 nm with a Hewlett-Packard triglycerides and other hydrolysable materials
Model 1100 Series Photodiode Array Detector and to aid the release of lipids from a sample
(PDA). Or yzanols were separated on a matrix (Diack and Saska, 1994). However, in
Hewlett-Packard 250 x 4 mm packed with 5- the case of oryzanol, the saponification process
mm ODS (C18) Hypersil silica. The mobile may hydrolyze the ester bond between
phase consisted of acetonitrile/methanol/ triterpenoids and ferulic acids.
isopropanol (50:45:5 by volume). The level of γ–oryzanol detected in crude
oil in this study was higher compared to the
RESULTS AND DISCUSSION earlier investigations such as 9.8 mg g-1 (Xu
and Godber, 1999), 12.8 - 13.9 mg g-1 (Hu et
Soxhlet extraction method was the most al., 1996), 14 mg g-1 (Zhao et al., 1987), 12.2
common technique used in the extraction of mg g-1 (Nicolosi et al., 1994) and only 2.4 - 3.1
lipid from plant and animal tissues. The mg g-1 (Shin and Godber, 1996) using different
extractability of rice bran lipid using this extracting solvents and mixture of solvents.
method has been reported in the range of 18 The higher extractability of γ–oryzanol in
- 20% (Saunders, 1986). In the present study, the present study could be due to the use of
approximately 3.3 g of crude oil was obtained 3:2 chloroform:methanol mixture as the
from 20 g of rice bran (16.4% yields). This extracting solvent, where oryzanol in both
result was supported by Xu and Godber non-polar (Xu and Godber, 1999) and polar
(1999), who reported that 14% of lipid was (Qureshi et al., 2000) lipid fractions were
extracted from 25 g rice bran. However using extracted. Earlier, Seetharamaiah and

ASEAN Food Journal Vol. 15, 89-96

92 Azrina, A., Maznah, I. and Azizah, A. H.

Figure 2: Ultraviolet detection of γ–oryzanol components.

1= cycloartenyl ferulate; 2= 24-methylene cycloartanyl ferulate; 3= campesteryl ferulate;
4=–β-sitosteryl ferulate and cycloartanyl ferulate.

Prabhakar (1986) have shown that a higher detected in raw samples was in week 2, while
concentration of oryzanol was extracted using in stabilized samples the highest concentration
chloroform:methanol mixture (2:1, v/v). was during week 12. There was steady
Besides the solvent type, other factors such as increment detected in the content of oryzanol
solvent to bran ratio and extraction from 0 - 5 and 0 - 12 weeks in raw and stabilized
temperatures may also influence the samples, respectively. The slow release of
extractability of rice lipid and its minor oryzanol in rice bran lipid observed could be
components (Hu et al., 1996). Diack and Saska due to the fact that fat-soluble antioxidants
(1994) found that when separating such as tocotrienol and tocotrienol-like
antioxidants of rice bran such as vitamin E and compounds (including oryzanol) are bound
oryzanol compounds, their concentrations to insoluble cellular components of the plant
also varied substantially according to the origin tissues (Qureshi et al., 2000). In this study, the
of the rice bran. effect of autoclave and microwave methods to
Figures 3 and 4 show the changes in stabilize rice bran was comparable as shown
oryzanol content over 48 weeks of storage by the observed similar trend.
following unstabilized (raw) and two different The reduced concentrations of oryzanol
stabilization methods (microwave and after the 5th week and 12th week for raw and
autoclave). Overall, the oryzanol levels in raw stabilized samples, respectively, indicated that
and stabilized samples reduced with storage the involvement of this compound in
time. The highest γ–oryzanol concentration combating non-enzymatic lipid oxidation

ASEAN Food Journal Vol. 15, 89-96

 Extraction and Determination of Oryzanol in Rice Bran of Mixed Herbarium UKMB 93

Figure 3: Total γ-oryzanol in unstabilized and autoclaved rice

bran samples stored for up to 48 weeks.
AC- Autoclaved, Uns- Unstabilized samples, PE- Polyethlene bags,
PEF- Polyethlene bags covered with foil

Figure 4: Total γ-oryzanol in unstabilized and microwave-heated rice

bran samples stored for up to 48 weeks.
MW- Microwave-heated, UnS- Unstabilized samples, PE- Polyethlene bags,
PEF- Polyethlene bags covered with foil

(Sowbhagya and Bhattacharya, 1976). The The quantity of plant-sterol in the lipid fraction
oxidation that occurred could have been of rice bran is influenced by plant genetics,
catalyzed by the presence of the naturally growing and harvesting conditions, the state
present metal ions in the rice bran or of maturity at har vest and processing
introduced by contamination from the techniques employed (Houghton and Raman,
shelling equipment during polishing 1998). The unsaponifiable fraction of crude
(Champagne, 1994). rice bran oil contains a unique complex of

ASEAN Food Journal Vol. 15, 89-96

94 Azrina, A., Maznah, I. and Azizah, A. H.

naturally occurring antioxidant compounds In a study that compares the concentration of

such as oryzanol and vitamin E. However in oryzanol in 5 different brands of commercial
this study, the vitamin E content was not oils, Roger et al. (1993) showed that the range
determined. Simultaneous assessment of oryzanol concentration was 0.115 - 0.787 mg
(identification and quantification) of oryzanol g-1 of oil. Besides the effect of the specific oil
and vitamin E in rice bran oil has been processing step, rice varieties used also may
developed by Rogers et al. (1993) using reverse- influence its’ final concentration of oryzanol
phase HPLC. Through the HPLC methods, it in commercial cooking oil.
has been clearly established that γ–oryzanol is
a mixture of several components (Diack and CONCLUSION
Saska, 1994; Norton, 1995). However,
depending on the chromatographic approach, In conclusion, factors such as rice varieties,
different numbers of individual components procedures used using brown rice and
have been identified. In this study, only 4 major extraction parameters, influenced the level of
components of oryzanol were successfully oryzanol in rice lipid. The level of oryzanol in
identified following the method and mixed Herbarium UKMB was higher than
experimental conditions of Rogers et al. some earlier reports and contained four major
(1993). isomers (cycloartenyl ferulate, 24-methylene
Oryzanol, is a unique compound that cycloartanyl ferulate, campesteryl ferulate and
imparts various physiological functions such β–sitosteryl ferulate). This finding is important
as decreasing plasma cholesterol level in as or yzanol is a potent and high value
animals (Nicolosi et al., 1991; Seetharamaiah antioxidant compound and locally produced
and Chandrasekhara, 1989; Sunitha et al., bran could be the source of this compound.
1997), decreased platelet aggregation
(Seetharamaiah et al., 1990) and showed ACKNOWLEDGEMENTS
antioxidant functionality (Duve and White,
1991). In plant tissues, γ-oryzanol is present at This study was supported by BERNAS
a very high concentration such as 43 000 ppm DOMINALS SDN. BHD and IRPA grant
in these mixed local varieties (Herbarium number 06-02-04-0216 from the Ministry of
UKMB; AZ 6807: MR 185, AZ 6808: MR 211, Science, Technology and Innovation, Malaysia.
AZ6809: MR 29). The high concentration of The author would also like to thank the
oryzanol is important as part of the plant Malaysian Heart Foundation for the Research
defense system. Fellowship Award.
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