Journal of Oleo Science Copyright ©2010 by Japan Oil Chemists’ Society J. Oleo Sci. 59, (2) 65-70 (2010) Novel Fractionation Method for Squalene and Phytosterols Contained in the Deodorization Distillate of Rice Bran Oil Naoko Sugihara1, Ayato Kanda1, Teruyuki Nakano2, Takahiro Nakamura2, Hisao Igusa2 and Setsuko Hara 1＊ 1 Dept. of Materials and Life Science, Faculty of Science and Technology, Seikei University (3-1 Kichijoji-kitamachi 3, Musashino-shi, Tokyo 180-8633, JAPAN) 2 Technical Division, Boso Oil & Fat Co. Ltd. (17-1 Hinode 2, Funabashi-shi, Chiba Prefecture 273-0015, JAPAN)

Abstract: Since deodorization distillate, a by-product of rice bran oil production, contains squalene (ca. 8%) and phytosterols (ca. 4%) as unsaponifiable components, the concentration of those materials for their use in the cosmetics and food industries is desirable. In the present work, a novel fractionation method of concentrating squalene and phytosterols from deodorization distillate or the unsaponifiable components of the deodorization distillate without oxidative deterioration was examined. Supercritical fluid extraction (SFE) with supercritical carbon dioxide was investigated under the following conditions: temperature, 30℃; pressure, 100 kg/cm2; flow rate of carbon dioxide, 7 mL/min. Under these conditions, squalene was effectively concentrated to 25% with nearly quantitative recovery, and then a more highly concentrated squalene (ca. 50% purity) was obtained by using a supercritical fluid chromatography (SFC) with silica gel packed into the extraction vessel. In addition, squalene with ca. 68% purity could be obtained by repeating the SFC twice. After the saponification of the deodorization distillate, followed by solvent fractionation with hexane, highly purified phytosterols (97% purity) could be obtained, and highly purified squalene (81% and 100% purity) could be also obtained by using SFC combined with the solvent fractionation technique for the unsaponifiable materials. Therefore, it is considered that the present fractionation method combined with SFC and solvent fractionation is an effective means of concentrating squalene and phytosterols.

Key words: rice bran oil, squalene, phytosterol, supercritical fluid chromatography, unsaponifiable component, deodorization distillate

1 INTRODUCTION of plant origin, and the application of this type of squalene Squalene is widely found in marine animal oils as a trace to cosmetics, medicine and functional foods has been component, and has been used in cosmetics as a moisture- attempted. Although squalene originating from olive oil is retaining ingredient1,2). Since it has been well known that being produced in Europe, the production quantity is too the liver oil of some varieties of shark, especially those low to make up for the sagging production of squalene orig- inhabiting the deep sea, is rich in squalene, this substance inating from shark liver oil. Therefore, the possibility of has been fractionated from shark liver oil. extracting squalene from rice bran oil is being On the other hand, squalene obtained from shark liver oil investigated3). has not been fully utilized recently on humane grounds, The phytosterols contained in rice bran oil have choles- and due to the unstable supply of shark liver oil as an terol-reducing activity4-8) and are effective components for industrial material, the characteristic smell of fish oil, and utilization as functional foods called foods for specified the large variation of the constituents health use. Under these conditions, attention has shifted to squalene Although there are already some reports on methods of

＊ Correspondence to: Setsuko Hara, Dept. of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-1 Kichijoji-kitamachi 3, Musashino-shi,Tokyo 180-8633, JAPAN E-mail: [email protected] Accepted October 15, 2009 (received for review August 3, 2009) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/

65 N. Sugihara, A. Kanda, T. Nakano et al.

concentrating squalene originating from plants, the ther- CH-201 (Scinics Corporation, Tokyo, Japan) and tempera- mal and oxidative deterioration of squalene poses signifi- tures ranging from 30 to 80℃ and pressures ranging from cant problems for its use in foods and medicines. In addi- 80 to 220 kg/cm2 were examined at an extraction time of 2 tion, solvent fractionation bears some difficulties related to h and a supercritical carbon dioxide flow rate of 7 mL/min. proper solvent selectivity during processing, the elimina- The squalene concentration in the extract was determined tion of the residual solvent from the squalene fraction, and by TLC-FID analysis with hexane as the primary develop- the disposal of the waste fluid. ing solvent and benzene/ethyl acetate (9:1, v/v) as the sec- In the present study, we examined a novel method of ondary developing solvent. concentrating the squalene and phytosterols contained in 2.2.2 Concentration of squalene by SFC the deodorization distillate of rice bran oil by combining The optimal conditions for squalene concentration by supercritical fluid extraction (SFE), supercritical fluid SFE were determined to be as follows: extraction tempera- chromatography (SFC) and solvent fractionation. Under ture, 30℃; supercritical carbon dioxide pressure, 100 the supercritical condition, gas acquires unique character- kg/cm2; flow rate, 7 mL/min. However, squalene was only istics: upon a slight pressure change, it becomes more concentrated to 25%, and the other components in the dense, less viscous and more soluble9). Therefore, super- extract had higher polarity, such as TG, DG and FA. There- critical fluid has the excellent ability to separate and fore, 1 to 3-fold higher quantities of activated silica gel as extract specific components from a mixture. Since the crit- compared with the deodorization distillate were placed in ical temperature and pressure of carbon dioxide are 31.1℃ the extraction vessel to adsorb the components with high- and 72.8 kg/cm2, respectively, there are many advantages er polarity, and then squalene was extracted and/or con- to using supercritical carbon dioxide for extraction at room centrated under the optimal conditions listed above by temperature: the extract undergoes no thermal and oxida- means of SFC as shown in Fig. 1. tive deterioration, no remnant gas is generated in the extract at atmospheric pressure, etc.10). 2.3 Concentration of phytosterols from the deodorization distillate 2.3.1 Concentration of phytosterols by SFC Since phytosterols were concentrated in the residue 2 EXPERIMENTAL after the extraction of squalene by SFC, further concentra- 2.1 Samples and reagents tion of the phytosterols contained in 6 g of residue was The deodorization distillate of rice bran oil used to con- attempted. centrate squalene and phytosterols was supplied from Boso In addition, the residue recovered as a result of SFE was Oil & Fat Co., Ltd., (Funabashi, Japan). The composition of also subjected to SFC to concentrate the phytosterols the deodorization distillate was as follows: squalene, 8.5%; adsorbed onto the silica gel. In this case, the concentration phytosterols, 3.5% (sitosterol: 2.05%, stigmasterol: 0.69%, ratio of phytosterols was compared with the amounts of campesterol: 0.76%); tocopherol (Toc), 1.6%; triglycerides residual phytosterols before and after the further extrac- (TG), 58.0%; diglycerides (DG) + fatty acids (FA), 26.5%. tion of squalene for 2 h, because phytosterols remained in Squalene standard of special grade and 2-propanol of HPLC the residue. grade were purchased from Wako Pure Chemical Indus- 2.3.2 Concentration of phytosterols by solvent fractiona- tries, Ltd. (Osaka, Japan), and all other reagents used were tion of special grade or first grade, as commercially available, The residue remaining in the vessel packed with silica and used without further purification, unless otherwise gel were extracted with ethanol and then saponified by specified. refluxing for 4 h with 3 mL of 25% potassium hydroxide aqueous solution and 40 mL each of ethanol and hexane. 2.2 Concentration of squalene from the deodorization dis- After the saponification, the reactant was separated into a tillate hexane layer and a hydrated ethanol layer. 2.2.1 Concentration of squalene by SFE 2.3.3 Concentration of the phytosterols from the To fractionate squalene from the deodorization distillate, unsaponifiable components of the deodorization distil- SFE with supercritical carbon dioxide was employed. SFE late makes it possible to extract the target compound by chang- Another means of concentrating phytosterols was also ing the density of supercritical carbon dioxide under cer- examined, as shown in Figs. 2: the phytosterols were con- tain conditions of temperature and/or pressure. In the pre- centrated after the saponification of the deodorization dis- sent experiment, SFE was carried out with an SFC appara- tillate. tus, model 880-81 (JASCO, Tokyo, Japan), equipped with a 2.3.4 Preparation of highly purified squalene column oven, model CP-80, and a 10- or 50-mL stainless To obtain more highly purified squalene, 6 g of the vessel. The column was kept in a thermostatic oven, model extract, which contained squalene having 81% purity and

66 J. Oleo Sci. 59, (2) 65-70 (2010) Fractionation of Squalene and Phytosterols in Deodorization Distillate of Rice Bran Oil

Fig. 1 Fractionation of Squalene and Phytosterols from the Deodorization Distillate.

Fig. 2 Fractionation of Squalene and Phytosterols from the Unsaponifiable Components of the Deodorization Distillate.

recovered by SFC of the unsaponifiable components, were hexane as a primary developing solvent to detect squalene column-chromatographed with hexane/diethyl ether (95:5, and benzene/ethyl acetate at a ratio of 9:1 (v/v) as a sec- v/v) as an eluent in accordance with the procedure shown ondary developing solvent to detect the other components in Fig. 2. were used. The fatty acid composition of the lipids was analyzed by 2.4 Analysis GLC. The constituent fatty acids were methyl-esterified11), To confirm the composition of the deodorization distil- and a fused silica capillary column, CBP1-S25-050 (0.32 mm late of rice bran oil, TLC-FID analysis was carried out on a × 25 m; Sinwa-kagaku Co., Ltd.), was connected to a GLC Iatroscan, model MK-5 (Mitsubishi Kagaku Iatron Inc., (model GC-18A; Shimadzu Corp., Kyoto, Japan) for con- Tokyo, Japan) equipped with a Chromatopac C-R6A (Shi- stant-temperature GLC analysis. The column temperature madzu Corp., Kyoto, Japan).The deodorization distillate, was set at 200℃, helium was used as the carrier gas, and squalene, canola oil as TG, oleic acid as FA, phytosterols FID was used as the detector. and Toc as standard samples were loaded on silica gel sin- The Toc contents of the deodorization distillate and the tering quartz rods(Chromarod S-III, Mitsubishi Kagaku squalene standard were determined by HPLC analysis. A Iatron Inc., Tokyo, Japan).Developing solvents consisting of normal-phase column, Finepak SIL-5 (4.6 mm × 250 mm;

67 J. Oleo Sci. 59, (2) 65-70 (2010) N. Sugihara, A. Kanda, T. Nakano et al.

JASCO Corp.), was connected to an HPLC (model BIP-1; JASCO), and a mobile-phase solvent that consisted of hex- ane and 2-propanol at a ratio of 124:1 was allowed to flow through the column at a rate of 0.7 mL/min. A fluores- cence detector (model FP-2020 Plus, JASCO) with excita- tion at 295 nm and emission measured at 325 nm was used. For the quantitative analysis of Toc, 2,2,5,7,8-pentamethyl- 6-chromanol (PMC) was used as the internal standard, and the calibration curves prepared beforehand for the Toc iso- mers [for a-Toc, y=0.4508x-0.0927 (R2=0.9682); for b-Toc, y=0.7795x-0.3017 (R2=0.9488); for g-Toc, y=1.2532x-0.7692 (R2=0.9887); and for δ-Toc, y=1.2454x-0.9934 (R2=0.9752)] were used. Squalene content/recovery (%)

3 RESULTS 3.1 Concentration of squalene from the deodorization dis- tillate 3.1.1 Concentration of squalene by SFE Fig.4 As shown in Fig. 3, squalene was concentrated to an Changes in the Squalene Content and Recovery at average concentration of 25% under the following condi- Various Amounts of Activated silica Gel in SFC. tions: extraction temperature, 30℃; supercritical pressure, 100 kg/cm2; extraction time, 2 h; quantitative recovery. It on average, 50% squalene and 38% components with higher was confirmed that squalene could be concentrated to 3 polarity. Then, SFC was repeated after mixing fresh silica times the original content (8.5%) in the deodorization distil- gel and the extract obtained by the first SFC. As a result, it late with peroxide value (PV) of 3.0 meq/kg. was proved that squalene could be concentrated to 68% in 3.1.2 Concentration of squalene by SFC the extract with PV of 3.1 meq/kg by repeating SFC with As shown in Fig. 4, the higher squalene content was 3-fold more silica gel under the conditions stated earlier. obtained with more than 95% recovery when 3 times more Therefore, it was found that the present silica-gel SFC, silica gel was added to the deodorization distillate and the with the addition of silica gel as a stationary phase into the squalene was extracted with supercritical carbon dioxide supercritical vessel to create a chromatographic system, for 5 h. The extract obtained by silica-gel SFC contained, had a higher selectivity than mere SFE. The present silica- gel SFC is expected to became a very useful technique for concentrating squalene from the deodorization distillate of rice bran oil as shown in Fig. 1.

3.2 Concentration of phytosterols from the deodorization distillate 3.2.1 Concentration of phytosterols by SFC The composition of the residue with PV of 3.0 meq/kg recovered under the conditions of 30℃ extraction temper- ature, 100 kg/cm2 supercritical pressure, 7 mL/min flow rate of supercritical carbon dioxide, and 5 h extraction

Squalene content (%) time by SFC with silica gel packed as a stationary phase was 10.4% phytosterols, 3.9% Toc, 48.6% TG, and 37.1% DG + FA. In addition, the residue recovered from the procedure described in 2.2.1 contained 17.3% phytosterols under the following conditions: extraction temperature 30℃; super- critical pressure 220 kg/cm2, flow rate of supercritical car- Fig. 3 Changes in Squalene Content under Various Condi- bon dioxide 7 mL/min, extraction time 7 h. From these tions of SFE. results, it was considered that SFC did not suit the separa- tion of phytosterols from a mixture of phytosterols, TG, DG

68 J. Oleo Sci. 59, (2) 65-70 (2010) Fractionation of Squalene and Phytosterols in Deodorization Distillate of Rice Bran Oil

and FA, which have nearly the same polarities, although were recovered. Then, hexane was added to the compo- SFC was suitable for the extraction of compounds with nents, and the crystalline phytosterols were recovered lower polarity, such as a squalene. Then, we examined sol- from the hexane-insoluble fraction under cooling. By a vent fractionation to concentrate the phytosterols from the series of processes, 9.16 g of hexane-soluble fraction and deodorization distillate. 1.29 g of hexane-insoluble fraction were obtained and ana- 3.2.2 Concentration of phytosterols by solvent fractiona- lyzed by TLC-FID. As a result, it was found that the phy- tion tosterols were concentrated to 97.2% in the hexane-insolu- The residual fraction shown in Fig. 1 contained 15.7% ble fraction as shown in Table 1. Therefore, it was con- phytosterols in 4.5 g of recovered residue. The 4.5 g of firmed that a combination of saponification and solvent residue remaining in the vessel packed with silica gel were fractionation of the deodorization distillate is an effective extracted with ethanol and then saponified by refluxing for means of concentrating phytosterols. Since the composi- 4 h with 3 mL of 25% potassium hydroxide aqueous solu- tion of the phytosterols fractionated by methods described tion and 40 mL each of ethanol and hexane. After the in 2.3 were analyzed by TLC-FID, changes of each molecu- saponification, the reactant was separated into a hexane lar species such as sitosterol, stigmasterol, and campes- layer and a hydrated ethanol layer. The unsaponifiable terol at fractionation have to be determined in the further components thus obtained in the hexane layer were then work. cooled to obtain 0.23 g of crystalline phytosterols with Since squalene was concentrated to 30.2% in the hexane- 97.3% purity. soluble fraction, this fraction were subjected to SFC with As described above, squalene was fractionated by SFC silica gel under the following conditions to obtain higher- with silica gel packed into the vessel, and phytosterols purity squalene: flow rate of supercritical carbon dioxide, 3 were highly concentrated from the residue by solvent frac- or 7 mL/min; extraction pressure, 80-140 kg/cm2. As a tionation. Therefore, it is considered that the combination result, it was found that higher squalene recovery tended of silica-gel SFC and solvent fractionation was a very effec- to be obtained at faster flow rates and higher pressures. tive means of obtaining both components with higher puri- Furthermore, the squalene content in the extract reached ty. This method, however, is rather time-consuming and 81.0%. costly, because SFC has to be repeated in order to concen- From these results, it is considered that these two func- trate the squalene, and the residue has to be extracted tional components contained in the deodorization distillate from the silica gel in the SFC column to concentrate the which are usually discarded as waste can be put to use eas- phytosterols. ily. In addition, the comparison of Fig. 1 with Fig. 2 indi- 3.2.3 Concentration of phytosterols from the unsaponifi- cates that the solvent fractionation of unsaponifiable com- able components of the deodorization distillate ponents of the deodorization distillate is a practicable and After the saponification of the deodorization distillate (80 convenient method of concentrating phytosterols and g) by refluxing for 4 h with 3 mL of 25% potassium hydrox- squalene. The combination of solvent fractionation and ide aqueous solution, 11.6 g of unsaponifiable components SFC developed in the present work is deemed to be an

Table 1 Composition and of the Hexane-soluble and Hexane-insoluble Fractions by Solvent Fractionation(%).

Hexane soluble Hexane insoluble

Less polar components 11.9 0

Squalene 30.2 0

Phytosterol 14.1 97.2

Triglyceride(TG) 17.5 2.8

Diglyceride(DG) 26.3 0 Fatty acid(FA)

Peroxide value(PV) 3.5 3.8 (meq/kg)

69 J. Oleo Sci. 59, (2) 65-70 (2010) N. Sugihara, A. Kanda, T. Nakano et al.

effective and safe means of fractionating the two functional References components squalene and phytosterols, which can then be 1. Abstract for the 3rd Health Food Exposition under the used as additives in cosmetics and functional foods. Auspices of Tsuno Food Industrial Co., Ltd and 3.3.4 Preparation of highly purified squalene Shokuhin Kagaku Shinbunsha. The 3.50 g of extract containing 81.0% squalene obtained 2. Hirota, H. Keshouhinyo Yushi no Kagaku, p.1, Fra- from the SFC were further purified by column chromatog- grance Journal Co., Ltd., (2001). raphy with hexane/diethyl ether (95:5, v/v). As a result, 3. Tsuno Food Industrial Co., Ltd., Technical Report of 2.55 g of squalene with 100% purity and PV of 4.0 meq/kg Shokuhin Sangyo Center, 24, (1998). could be obtained with 500 mL of eluate. 4. Chen, P.R.; Tsai, C.E. Various high monounsaturated edible oils might affect plasma lipids differently in man. Nutr. Res. 15, 615-621 (1995). 5. Raghuram, T.C.; Brahmaji Rao, U.; Rukmini, C. Srudies 4 DISCUSSION on hypolipidemic effects of dietary rice bran oil in In the present work, a novel method of fractionating human subjects. Nutr. Rep. Int. 39, 889-895 (1989). squalene and phytosterols contained in the deodorization 6. Sharma, R.D.; Rukmini, C. Hypocholesterolemic activi- distillate of rice bran oil without any oxidative rancidity ty of unsaponifiable matter of rice bran oil. Indian J. was established by the combination of solvent fractionation Med. Res. 85, 278-281 (1987). and SFC after saponification of the deodorization distillate. 7. Sharma, R.D.; Rukmini, C. Rice bran oil and hypoc- Although there are some industrial production meth- holesterolemia in rats, Lipids 21, 715-717 (1986). ods12-16) of squalene or squalane from the deodorization dis- 8. Nicolosi, R.J.; Ausman, L.M.; Hegsted, D.M. Rice bran tillate of rice bran oil, those methods have to perform many oil lowers serum total and low density lipoprotein processes such as saponification, solvent fractionation, dis- cholesterol and apo B levels in non-human primates. tillation, hydrogenation, and final molecular distillation to Atherosclerosis 88, 133-142 (1991). avoid the oxidative rancidity of squalene, or another is a 9. Xiao-Wen, W. Leading technology in the 21st century cultivation method with yeast extracts for 6 days at 30℃. “Supercritical fluid extraction”. Shokuhin to Kaihatsu A Japan patent17) for the production for phytosterols are 40, 68-69 (2005). released from Kao Corporation, in which phytosterols are 10. Xiao-Wen, W. Supercritical fluid extraction method. p.14- concentrated to 90-94% purity from crude phytosterols 20, Health Business Magazine Co., Ltd. (2005). (purity: ca. 80%) with hydrocarbon solvents. Commercial 11. Jham, G.N.; Teles, F.F.F.; Campos, L.G. Use of aqueous squalenes obtained from shark liver oil, olive oil, and rice HCl/MeOH as esterification reagent for analysis of bran oil are now on sale as 1,000-1,500 yen/kg, 2,500 fatty acids derived from soybean lipids. J. Am. Oil yen/kg, and 15,000 yen/kg, respectively. The market prices Chem. Soc. 59, 132-133 (1982). of phytosterols are 3,500-15,000 yen/kg based on their puri- 12. Ando, Y.; Watanabe, Y.; Nakazato, M. Japan Pat. ties. 306387 (1994). Therefore, the present method has some merits such as 13. Ando, Y.; Watanabe, Y.; Nakazato, M. Japan Pat. a fewer operation process, time-saving, no oxidative ran- 306388 (1994). cidity and continuous production of the two functional 14. Tsujiwaki, Y.; Yamamoto, H.; Minami, K. Japan Pat. components. In addition, there is a strong possibility of 327687 (1995). lower prices production than those of existent methods, 15. Ikuta, Y.; Maeda, K.; Yamaguchi, E.; Shimura, M.; since carbon dioxide used as a supercritical gas is costly Ishii, K.; Ohta, M.; Shirato, Y. Japan Pat. 309785 but recyclable. (1995). It was found that the present method very safely and 16. Hirata, Y.; Ohta, Y. Japan Pat. 176057 (1997). effectively fractionates the functional components con- 17. Kohno, J. Japan Pat. 316996 (2002). tained in deodorization distillate, which is usually regarded as waste.

70 J. Oleo Sci. 59, (2) 65-70 (2010)