Available online at www.buu.ac.th/BUUConference
ST-O-011
Burapha University International Conference 2015
“Moving Forward to a Prosperous and Sustainable Community”
Effect of metallic salt and ethanol concentration on the
extraction of γ-Oryzanol from rice bran acid oil
Piraporn Sombutsuwana, Kornkanok Aryusuka, *, Supathra Lilitchanb, Narumon
Jeyashokea, Kanit Krisnangkuraa
a
Biochemical Technology Division, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi,
Bangkok 10150, Thailand
b
Department of Nutrition, Faculty of Public Health, Mahidol University , Bangkok 10400,Thailand
Abstract
γ- Oryzanol is a natural antioxidant derived from rice bran oil. It has many health benefits, such as lowering low density
lipoprotein cholesterol (LDL-C), decreasing pallet aggregation and increasing high density lipoprotein cholesterol (HDLC). Commercial γ-oryzanol is normally prepared from soapstock or acid oil, byproducts from rice bran oil refinery. Removal
of free fatty acids (FFAs) and other impurities is a very difficult task for extraction and purification of γ-oryzanol from acid
oil. Extraction of γ-oryzanol from acid oil through the precipitation of metallic soap is a simple method for preparation of
γ-oryzanol to minimize the subsequent steps for purification. In this study, effects of metallic soap types and ethanol
concentration on the recovery and the purity of γ-oryzanol from rice bran acid oil were investigated. The soluble soap was
prepared by saponification of the acid oil with 1 M NaOH in aqueous 80% ethanol at 65 oC for 10 min. The soluble soap
was then converted to insoluble soap by inducing precipitation of different metallic soaps. Effects of ethanol concentrations
between 50% and 80% (v/v) on the extractions of γ-oryzanol were also compared. The optimum concentration for extraction
of γ-oryzanol was 80% ethanol. Recoveries of γ-oryzanol from the 80% ethanol were 70.36%, 64.40% and 49.89% for the
aluminium, magnesium and calcium soap, respectively. However, very low purity of γ-oryzanol (4.53%) was obtained due
to the metallic soap also co-dissolved in the ethanol. Thus, further step(s) for purification of γ-oryzanol is unavoidable and
need to be studied.
© 2015 Published by Burapha University
Keywords: γ-oryzanol; metallic salt; ethanol concentration; rice bran acid oil.
* Corresponding author. Tel.:+66-2470-7758
E-mail address: [email protected].
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1. Introduction
γ-Oryzanol, a natural antioxidant, which is a characteristic compound found only in rice bran oil. It has been
reported to decrease low density and increase high density lipoprotein cholesterol, to decrease pallet aggregation
and to induce muscle development in animal and human (Murase & Iishima, 1963; Nicolosi et al., 1991; Rong
et al., 1997; Seetharamaiah & Chandrasekhara, 1988) It is used as antioxidant in cosmetic, food and
pharmaceutical industries (Iqbal et al., 2005; Nanua et al., 2000).
Crude rice bran oil (CRBO) is a rich source of γ-oryzanol. Its content ranges from 1.5% to 2.9% in CRBO.
However, approximately 93-94% of γ-oryzanol is lost in the neutralization step of alkali refining (Gopala
Krishna et al., 2001). High amount of γ-oryzanol (14.2 mg/g, or 95.3% of total γ-oryzanol in CRBO) was found
in the soapstock (Pestana-Bauer et al., 2012). Therefore, the soapstock and/ or acid oil is the actual raw material
which is to be processed further to recover the -oryzanol.
The main components of acid oil are free fatty acids (FFAs), triglyceride (TG) and diglyceride (DG) with
small amount of monoglyceride (MG) and γ-oryzanol (Narayan et al., 2006). γ-Oryzanol is not soluble in water
but soluble in oil, FFAs and polar solvents such as ethanol, ethyl acetate and isopropanol (Lilitchan et al., 2008;
Xu & Godber, 2000). The major difficulties in isolating oryzanol from acid oil by extraction are time-consuming
and poor partitioning of γ-oryzanol into the extracting phase. The difference of boiling point between γ-oryzanol
and FFAs has been applied for the preparation of γ-oryzanol. The FFAs or its ester were removed from acid oil
by distillation. The γ-oryzanol was then extracted with an organic solvent, crystallized and purified by column
chromatography (Takeshi, 1969; Tsuchiya & Okubo, 1961; Yasuo et al., 1968). Disadvantages of this method
are high cost and high energy consumption for large scale application and numerous of unit operations are
required (Narayan et al., 2006).
Das et al., (1998) developed method for extraction of γ-oryzanol by aqueous alkaline hydrolysis of the
acylglycerol in the acid oil after removed the FFAs by vacuum distillation. The calcium ions were then added
into the soluble soap to induce instant co-precipitation of the insoluble calcium soap and the aggregate associated
-oryzanol. The precipitated calcium soap was then air dried overnight and twice extracted with ethyl acetate.
The-oryzanol was purified from the evaporated ethyl acetate residue by silica gel column chromatography.
Although, satisfactory yield (96% purity with 76% recovery) of γ-oryzanol was obtained, however, several
extraction processes were used. In addition, the high energy consumption is required for removal of FFAs by
vacuum distillation.
Preparation of metallic soap by the addition of a metallic salt into the solution of soluble soap is a common
and simple method. Thus, extraction of γ-oryzanol from acid oil through the precipitation of calcium soap
suggested by Das et al., (1998) is interesting if the subsequent steps for extraction and purification are minimised
and simplified. γ-Oryzanol is soluble in ethanol. While, the metallic soap is slightly soluble or insoluble in
alcohol or ether (Harrison, 1924). Thus, in this study the different solubility between γ-oryzanol and metallic
soap in ethanol was adopted for extraction of γ-oryzanol from the acid oil. The γ-oryzanol was expected to
dissolve in the solution of ethanol during precipitation of the metallic soap and then recovery of γ-oryzanol
could be simplified.
2. Material and Method
2.1 Materials
Acid oil was a gift of Surin Rice Bran Oil Co., Ltd. (Surin Province, Thailand). Tri-, di- and mono-stearin
purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as standard tri-, di- and mono-acylglycerol,
respectively. A food grade γ-oryzanol (98.1% purity) was purchased froml Oryzan Oil & Fat Chemical Co.,
Ltd. (Ichinomiya, Japan). Stearic acid (C18:0) and its methyl ester were used as standard FFA and fatty acid
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methyl ester (FAME), respectively and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isooctane,
ethanol and toluene were HPLC grade and absolute ethanol was analytical grade obtained from RCI Labscan
Ltd. (Bangkok, Thailand). Sodium hydroxide, HCl and other chemicals were of analytical grade.
2.2 High performance size exclusion chromatography (HPSEC)
The HPLC system consisted of a pump model 515 (Waters Associates, Milford, MA, USA.), a Rheodyne
7125 six-port valve injector, a 20 µl loop and a Sedex 75 evaporative light scattering detector (ELSD; Sedere,
Alfortville, France). The ELSD drift tube was set at 30oC and N2 gas was 2 bars. Samples were prepared in
toluene and analyzed on a 100-Å Phenogel column (300 x 7.8 mm. ID., 5µm) (Phenomenex, Torrance, CA.)
protected with a Mightysil RP-18 Guard column (Kanto Chemical Co., Tokyo, Japan). The mobile phase was
toluene: isooctane: acetic 70:30:0.05 (v/v/v), its flow rate was set at 1.0 ml/min. Peaks were identified by
comparison with reference standards. The standard curves of tri-, di- and mono-stearin, stearic acid, γ-oryzanol
and methyl stearate were set up for quantifying the compositions of acid oil and samples.
2.3 Preparation of soluble soap
Two grams of acid oil was saponified with 20 ml of 1 M NaOH in 80% ethanol. The reaction temperature
was set at 65oC for 10 min. The reaction was stopped by addition of 4M HCl and the completeness of reaction
was monitored by HPSEC. The lipid composition of acid oil and saponified acid oil was monitored by HPSEC
and the lipid composition was calculated according to equation (1). The complete reaction was adjusted to pH
8 by 4 M HCl.
% Lipid composition (w⁄w) =
Amount of interested compound (g)
Total amount of sample compound (g)
×100
(1)
2.4 Effect of ethanol concentration and metallic insoluble soap types on extraction of γ-oryzanol
The soluble soap was prepared from 2 grams of acid oil as described in 2.3 was then converted to aluminium
soap by adding dropwise of 20 ml of 0.25 M of aluminium ammonium sulfate (AlNH4 (SO4)2 solution. The
absolute ethanol was added to adjust the ethanol concentration to be 50%, 60%, 70% or 80% (v/v). The reactions
were separated by Whatman filter paper No.1. The precipitates aluminium soap were further extracted with 20
ml ethanol. The ethanol soultion of each phase were acidified using 4 M HCl and extracted by ethyl acetate.
The lipid compositions of each phase were observed by HPSEC and its composition was calculated according
to equation (2). To study the effect of metallic soap, MgSO4 and CaCl2 was used instead of AlNH4 (SO4)2 and
the ethanol was fixed at 80% concentration.
% Lipid composition (w⁄w) =
Amount of interested compound (g)
Total amount of sample compound ( sediment+ethanol phases) (g)
×100
(2)
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3
Results and discussion
3.1 Composition of acid oil and its soluble soap
Figure 1 shows the compositions of acid oil and its soluble soap prepared by alkali saponification determined
by HPSEC. The acid oil contained 4.27% γ-oryzanol. The main composition of acid oil was FFAs (66.05%),
TG (15.51%), DG (15.51%) and MG (1.36%). A slight decrease (around 0.8%) was observed in γ-oryzanol
content of saponified acid oil. Although, γ-oryzanol is classified as unsaponifiable constituents due to it cannot
saponified by alkalis (Gunstone, 2002). However, it was demonstrated that γ-oryzanol could be hydrolyzed to
triterpene and it was lost due to conversion to potassium salt during the preparation of unsaponificable matter
in rice bran oil by saponification (Afinisha Deepam & Arumughan, 2012).
Figure 1. Lipid compositions of acid oil and its soluble soap.
3.2 Effect of ethanol concentration on extraction of γ-oryzanol.
The metallic soaps are insoluble in water however, they can be dissolved in ethanol. In order to maximize
the γ-oryzanol and minimize the aluminium soap in the solution, ethanol concentration was varied between 50%
and 80% (v/v) during precipitation of the aluminium soap. The effect of ethanol concentrations on γ-oryzanol
extraction and purification are shown in Table 2.
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Table 2. The effect of ethanol concentration on extraction of γ-oryzanol.
% w/w
Ethanol
concentration
80%
70%
60%
50%
Phase
DG
γ-oryzanol
Aluminium
soap
Solution
0.76
2.87
70.85
Sediment
0.65
0.99
23.89
Solution
0.72
2.69
68.75
Sediment
0.69
1.15
26.00
Solution
0.71
1.63
46.51
Sediment
0.70
2.21
48.24
Solution
0.57
0.70
37.81
Sediment
0.57
2.43
57.92
% γ-oryzanol
recovery
74.36
70.00
42.51
22.36
The results showed that at higher ethanol concentration, higher content of γ-oryzanol in the solutions were
observed. On the other hand, γ-oryzanol was trapped or precipitated along with aluminum soap in the low
ethanol concentration. Thus, it may be concluded that solubility of γ-oryzanol and aluminium soap were affected
by the water content in the ethanol.
The similar trend in solubility characteristic of the aluminium soap and the γ-oryzanol in the ethanol solution
were observed. This solubility characteristic was differ from that Harrison (1924) who demonstrated that the
oleate, palmitate and stearate calcium soap were insoluble both in water and in the absolute ethanol at any
temperature.The ethanol concentration did not significantly effect on the amount of DG in both the solution
and the precipitated soap. The optimum concentration for extraction of γ-oryzanol was 80% (v/v) ethanol.
3.3 Effect of metallic insoluble soap types on the extraction of γ-oryzanol
Table 3 shows percentage of DG, γ-oryzanol and metallic soap in the precipitated soaps and ethanol
solutions of aluminium, magnesium and calcium soap prepared in 80% ethanol. Recoveries of γ-oryzanol from
the ethanol solutions were 74.36%, 64.40% and 49.89% for the aluminium, magnesium and calcium soap,
respectively. However, amounts of metallic soaps were also increased as increasing the recovery of γ-oryzanol
in the solutions. The dissolved metallic soaps were 70.85%, 60.09% and 34.65% for aluminium, magnesium
and calcium soap, respectively. The γ-oryzanol possibly prefer to aggregate precipitation with the metallic soap
in the ethanol solution like in water which was presented by Das (1998). These lead to difficulty in purification
of γ-oryzanol by this method. Solubility of aluminium soap in ethanol was greater than that of magnesium and
calcium soap. This probably was caused by the higher polary of aluminium soap than other soaps. Loncar et al.
(2003) reported that di- and/or mono- were the predominant form of the aluminium soap Thus, the more free
positive charges in this soap, made it more soluble than the other soaps. In cases of calcium stearate, it prefer
be in the form of di- calcium soap (Gönen et al., 2010). Comparing among metal salts, aluminium might be the
best choice for extracting γ-oryzanol by inducing precipitation of metallic soap.
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Table 3. The effect of insoluble soap types on γ-oryzanol extraction and purification.
Metallic soap
Al3+
Mg2+
Ca2+
% w/w
DG
γ-Oryzanol
Metallic soap
% γ-Oryzanol
recovery
74.36
Phase
Ethanol solution
0.76
2.87
70.85
Precipitated soap
0.65
0.99
23.89
Ethanol solution
0.70
2.47
60.09
Precipitated soap
0.92
1.37
34.45
Ethanol solution
0.00
2.06
34.65
Precipitated soap
1.29
1.79
60.21
64.40
49.89
4. Conclusion
High yield recovery of γ-oryzanol from the ethanol solution during induced precipitation of insoluble
metallic soap could be obtained when AlNH4 (SO4)2 was used as metallic salt. The optimum ethanol
concentration was 80% (v/v). However, very low purity (4.53%) was obtained due to the metallic soap also codissolved in the ethanol. Thus, further step(s) for purification of γ-oryzanol is unavoidable and need to be
studied.
Acknowledgment
This work was supported by the Agricultural Research Development Agency, Public Organization (ARDA)
and the Higher Education Research Promotion and National Research University Project of Thailand, Office of
the Higher Education Commission.
References
Afinisha Deepam, L.S., Arumughan, C., 2012. Effect of saponification on composition of unsaponifiable matter
in rice bran oil, J Oleo Sci, 61(5),p. 241-7.
Gönen, M., Öztürk, S., Balköse, D., Okur, S., Ülkü, S., 2010. Preparation and Characterization of Calcium
Stearate Powders and Films Prepared by Precipitation and Langmuir−Blodgett Techniques, Industrial &
Engineering Chemistry Research, 49(4),p. 1732-1736.
Gopala Krishna, A., Khatoon, S., Shiela, P., Sarmandal, C., Indira, T., Mishra, A., 2001. Effect of refining of
crude rice bran oil on the retention of oryzanol in the refined oil, Journal of the American Oil Chemists'
Society, 78(2),p. 127-131.
Gunstone, F.D. 2002. Vegetable Oils in Food Technology: Composition, Properties, and Uses
Harrison, G.A., 1924. A Note on the Solubilities of Calcium Soaps, Biochemical Journal, 18(6),p. 1222-1223.
Iqbal, S., Bhanger, M.I., Anwar, F., 2005. Antioxidant properties and components of some commercially
available varieties of rice bran in Pakistan, Food Chemistry, 93(2),p. 265-272.
Lilitchan, S., Tangprawat, C., Aryusuk, K., Krisnangkura, S., Chokmoh, S., Krisnangkura, K., 2008. Partial
extraction method for the rapid analysis of total lipids and -oryzanol contents in rice bran, Food Chemistry,
106(2),p. 752-759.
Loncar, E., Lomic, G., Malbasa, R., Kolarov, L., 2003. Preparation and characterization of aluminum stearate,
Acta Periodica Technologica(34),p. 55-60.
Murase, Y., Iishima, H., 1963. Clinical studies oral administration of gamma-oryzanol on climacteric
complaints and its syndrome, 44,p. 135-143.
553
Nanua, J.N., McGregor, J.U., Godber, J.S., 2000. Influence of high-oryzanol rice bran oil on the oxidative
stability of whole milk powder, Journal of dairy science, 83(11),p. 2426-31.
Narayan, A.V., Barhate, R.S., Raghavarao, K.S.M.S., 2006. Extraction and purification of oryzanol from rice
bran oil and rice bran oil soapstock, Journal of the American Oil Chemists' Society, 83(8),p. 663-670.
Nicolosi, R.J., Ausman, L.M., Hegsted., D.M., 1991. Rice bran oil lowers serum total and low density
lipoprotein cholesterol and apo B levels in nonhuman primates, Atherosclerosis 88,p. 133-142.
Pestana-Bauer, V.R., Zambiazi, R.C., Mendonca, C.R.B., Beneito-Cambra, M., Ramis-Ramos, G., 2012. Oryzanol and tocopherol contents in residues of rice bran oil refining, Food Chemistry, 134(3),p. 14791483.
Rong, N., Ausman, L., Nicolosi, R., 1997. Oryzanol decreases cholesterol absorption and aortic fatty streaks in
hamsters., Lipids, 32,p. 303-309.
Seetharamaiah, G.S., Chandrasekhara, N., 1988. Hypocholesterolemic activity of oryzanol in rats, Nutrition
reports international, 38(5),p. 927-935.
Takeshi, Y. 1969. -Oryzanol. in: [Cited in Chemical Abstract 69:128704r]. Germany.
Tsuchiya, T., Okubo, O. 1961. Oryzanol. in: Japanese Patent 13,649 (1961) [Cited in Ibid. 56:8867a]. Japan.
Xu, Z., Godber, J., 2000. Comparison of supercritical fluid and solvent extraction methods in extracting γoryzanol from rice bran, Journal of the American Oil Chemists' Society, 77(5),p. 547-551.
Yasuo, W., Tsukasa, A., Tomisei, I. 1968. Oryzanol. in: Japanese Patent 68 12,731 (1968) [Cited inChemical
abstract. 69:107772e].
554