Process for the isolation of oryzanols from crude dark acid oil (rice bran)

A process for the isolation of oryzanols from crude dark acid oil (rice bran) which comprises (a) distilling the free fatty acids from crude dark acid oil by conventional methods, (b) hydrolyzing the resultant residue by conventional methods, (c) dissolving the hydrolyzed product in water to form oryzanol containing micellar aggregates and adding dropwise aqueous solution of calcium chloride to form precipitate, (d) extracting the oryzanols from dried precipitate with polar organic solvent and if desired (e) purifying the oryzanols from the organic extract by column chromatography.

This invention relates to an improved process for the isolation of 
oryzanols from crude dark acid oil (Rice Bran). Oryzanols are mixtures 
containing ferulate (4-hydroxy-3-methoxy cinnamic acid) esters of 
triterpene alcohols and plant sterols. Recent investigations have 
unequivocally demonstrated the multitude of beneficial physiological 
effects associated with oryzanol intake. The hypocholesterolemic activity 
of rice bran oil has been shown to be due to its constituent oryzanol and 
to some other components of the unsaponifiable matter Seetharamaiah, G. 
S. and Chandrasekhara, N. Atherasclerosis: 78.219 (1989)!. Lipid 
peroxidation has been shown to be prevented in the retina by 
gamma-oryzanol because of its antioxidant property Heramitsu, Tadahisa 
and Armstrong Donald, Opthalmic Res: 23, 196 (1991)!. Pharmaceutical 
preparations containing oryzanols have been shown to successfully reduce 
wrinkles in aged women Sakai, Tatsu et al (Eisai Co. Ltd.); JP 05.30.526 
(1993)!. Melanin formation accelerating topical preparations containing 1 
weight % oryzanol have been shown to convert gray hair into natural black 
Sakai, Tatsu et al (Eisai Co. Ltd.); JP 05.225.037 (1993)!. Nail lacquers 
containing oryzanols prevent discoloration of nails Ito, Nobumasa (Pola 
Chemical Industries Inc.), JP 02.290.806 (1990)!. Deodorant formulations 
containing oryzanols are especially effective in controlling odor from 
perspiration and underarms Kumasaka, Sadao (Human Industry Corp.): JP 
633322 (1988)!. Oryzanol containing pharmaceutical formulations are used 
in preventing motion sickness Sakada, Hideharu; JP 82.32.229 (1982)! and 
in the treatment of nervous system disorder Sun. Zhide and Cong Yizi; CN 
87,101,519 (1988)!. A plethora or oryzanol containing transdermal 
pharmaceutical and moisturizing cosmetic preparations have been prepared 
for the treatment of skin disorders Courtin, Olivier (Clarins S. A.), FR 
2,688,137 (1993); Tokuda, Yasuaki et al (Nissei Marine Kogyo K.K.). JP 
01,290,613 (1989); Ichimaru Co. Ltd., Orizu Yuka Co. Ltd., JP 81,161,315 
(1981); Toyo Chemical Corp. JP 82,149,212 (1982); Zenyaku Kogyo Co. Ltd., 
JP 82,42,621 (1982); Nitto Electric Industrial Co. Ltd., JP 59,53,415 
(1984), JP 59,184,120 (1984)!. Oryzanol emulsions are used as antioxidants 
and preservatives for cosmetics & foods and such emulsions are also 
effective in preventing color changes in the products. Orita Yuka Co. 
Ltd., JP 58,45,728 (1983)!. Soft capsules containing oryzanols with or 
without riboflavin butyrate can be used to prevent arteriosclerosis 
Nisshin Kogaku K.K. JP 58,103,315 (1983)!. Bath preparations containing 
3-20% (by weight) oryzanols are used in treatment of atopic dermatitis and 
senile xeroderma Inoe, Toshio and Nunokawa Senzo (Otsuka Pharm Co. Ltd.; 
JP 05,279,272 (1993)!. Oryzanols have been shown to be highly effective 
against lipogenic liver cirrhosis in spontaneously hypertensive rats, an 
animal having natural abnormalism in lipid metabolism Ito, Masahiro et 
al; J. Clin Biochem. Nutr. 12,193 (1992)!. Investigations directed towards 
the safety assessment of oryzanols clearly indicate that oryzanols possess 
no genotoxic and carcinogenic initiation activities Tsushimoto. Gen et 
al, J. Toxicol. Sci. 16,191 (1991); Tamagawa, M et al. Food. Chem. 
Toxicol. 30.49. (1992)!. All these remarkable beneficial effects of 
oryzanols on human health have generated, in recent years, an upsurge of 
global interest in developing commercially viable methods for the 
isolation of oryzanols from its various natural sources. 
Rice bran oil is perhaps the most readily accessible source for the 
isolation of oryzanol. The oryzanol content of rice bran oil varies within 
the range of 1.1 to 2.6%. The soap stock obtained by alkali refining of 
rice bran oil contains 1.3-3.1% oryzanol. Oryzanols were first isolated 
from rice bran oil Kaneko, R. and T. Tsuchiya; J. Chem Soc. Jpn. 
57,526(1954); Tsuchiya, T. et al; JP 4895 (1957)! was presumed to be a 
single component. Later is was determined to be a mixture containing 
ferulate (4-hydroxy-3-methoxy cinnamic acid) esters of triterpene alcohols 
and plant sterols. Individual components were identified as ferulate 
esters of cycloartenol, 24-methylene-cycloartanol, campesterol, 
beta-sitosterol and other sterols. Over the decades, the methods of 
oryzanol isolation from plant oils have been improved. Such methods 
include isolation of cycloartanol ferulate from plant oil using selective 
organic solvent for oryzanol extraction followed by chromatographic 
purification Kimura, Goro, Jap Pat 6314796 (1988) & Jap Pat 6314797 
(1988)!, isolation of oryzanols from rice bran dark oil by precipitating 
the stearins with aluminium sulfate followed by crystallization of 
oryzanols from the supernatant Beso oils & Fats Co. Ltd., Jap Pat 8295942 
(1982)!, highly concentrated separation of oryzanols from rice bran and 
rice germ oils by two-step alkali treatment Shimuzu, Hisashi; Jap Pat 
76123811 (1976)!, extraction of oryzanols from rice bran soapstock with 
diethylether at pH 9.5 followed by its chromatographic purification on a 
neutral alumina column G. S. Seetharamaiah and J. V. Prabhakar; J. Food 
Science Technology, 23,270 (1986)!, extraction of oryzanols from rice bran 
oil soap stock with ether after acidification of soap stock with HCl 
Tomotaro, Tsuchiya et al; Jap Pat 4895 (1957)!, isolation of oryzanols by 
transesterification of rice bran dark oil with methanol and sulfuric acid 
followed by column chromatography over pretreated Amberlite IRA-401 using 
mixed solvent methanol and ether as the eluent Tomaro, Tsuchiya and 
Osamu, Okubo; Jap Pat 13649 (1961)!, isolation of 98.3% pure oryzanols in 
overall about 35% yield by liquid-liquid extraction of hexane solution of 
20.2% concentrated solution of oryzanol using water-saturated furfural as 
the extractant Watanabe, Yasuo et al; Jap Pat 6812731 (1968)!, isolation 
of 85% pure oryzanols by passing carbon dioxide gas through a methanolic 
solution of alkaline oil cake of rice bran oil Nishihara, Masao and 
Shibuya, Yoshizane; Jap Pat 7812730 (1968)! and extraction of oryzanols 
from the raw oils of rice bran and ferment, maize & barley by distillation 
of these oils at comparatively low temperature followed by extracting the 
residue with hydroxol solvents Yamamoto, Takeshi, Ger Pat 1301002 (1969). 
During rice bran oil refining, a significant amount of rice bran oil gets 
trapped within the soap that is formed in the deacidification step. When 
the soap is removed by centrifugation, the entrapped oil also gets 
centrifuged with the soap. The centrifuged oil and soap is called 
soapstock. Soap industries use these soap stocks as their principal raw 
materials for manufacturing their bath preparations and toiletries. The 
soap stock is reacidified to prepare crude dark acid oil. The pure free 
fatty acids are distilled out from the crude dark acid oil under high 
vacuum. The entrapped oryzanols of soap stock is left in the residue, the 
so-called pitch, after the removal of free fatty acids from the crude dark 
acid oil. Thus it would be a plus to the arsenal of the oryzanol isolation 
methods if a commercially viable technology is developed for the isolation 
of oryzanol from the residue of crude dark acid oil after the distillation 
of free fatty acids under high vacuum. 
The main object of the present invention is therefore to provide a process 
for the isolation of oryzanols from the residue of crude dark acid oil 
after distilling off the free fatty acids under vacuum. Accordingly, the 
present invention provides an improved process for the isolation of 
oryzanol from the residue of crude dark acid oil which comprises (a) 
distilling the free fatty acids from crude dark acid oil by conventional 
methods, (b) hydrolyzing the resultant residue by conventional methods, 
(c) dissolving the hydrolyzed product mixture in predetermined amount of 
water to form oryzanol containing micellar aggregates, (d) adding dropwise 
aqueous solution of calcium chloride to form precipitate and extracting 
the oryzanol from the dried precipitate with polar organic solvent, and if 
desired (e) purifying the oryzanol from the organic extract by column 
chromatography. 
By the process of the present invention, a major part of the oryzanol that 
survives the vacuum distillation step for the free fatty acid removal, can 
be isolated in highly pure form. The byproducts are the calcium salts of 
various long chain fatty acids (mainly palmitic, stearic, oleic and 
linoleic acids) which can be utilized for various industrial applications. 
The invention accordingly provides a micellar chemistry based isolation of 
oryzanols from the crude dark acid oil. 
Micelles are roughly spherical aggregates of surfactant molecules in water. 
Such aqueous micellar aggregates can solubilize, in their hydrocarbon like 
interior, various hydrophobic molecules which are otherwise highly 
insoluble in water. Solubility of the ionic micelles in water can be 
drastically reduced by exchanging the counterions. In the present 
invention, the residue after distilling off the free fatty acids under 
high vacuum is hydrolyzed with aqueous sodium hydroxide solution at a 
temperature in the range of 60.degree. to 90.degree. C. for 0.5 to 4 hours 
and the hydrolyzed product mixture is dissolved in excess water to form 
oryzanol containing anionic micelles. For such anionic micelles of sodium 
salts of long chain fatty acids, addition of calcium ions induces 
immediate aggregation of the oryzanol containing micelles causing them to 
precipitate from water. The precipitate is then dried, extracted with 
polar organic solvent like ethyl acetate, chloroform and methanol. The 
organic extract is then washed with dilute aqueous alkali followed by 
water, dried over anhydrous sodium sulfate and the dried extract is 
evaporated. The oryzanol is finally purified from the residue by column 
chromatography using silica gel column and chloroform as eluent.

The details of the invention is further illustrated with the following 
examples which should not be however, be construed to limit the scope of 
the invention. 
EXAMPLE 1 
About 61 g of commercial dark acid oil containing 50-60% free fatty acids 
was distilled at 250.degree. C.-260.degree. C. under high vacuum (1 mm Hg) 
for removing most of its free fatty acids. The weight of the residue after 
distilling off the free fatty acids was about 34 g and its free fatty acid 
content was determined to be about 10%. About 11 g of this residue was 
hydrolyzed with aqueous sodium hydroxide (1.8 g NaOH in 25 ml water) at 
80.degree.-90.degree. C. for 3 hr. The hydrolyzed product mixture turned 
to a dark brown and somewhat viscous homogeneous solution. About 200 ml 
distilled water was added to this hydrolyzed product mixture, stirred at 
room temperature for about 30 min and to the solution, while being 
stirred, an aqueous solution of calcium chloride (2.5 g CaCl.sub.2 
dissolved in 30 ml. distilled water) was added dropwise. A huge 
precipitate appeared and the mixture was kept under stirring condition for 
another 15 minutes. The precipitate was then filtered and air dried, the 
dried precipitate was extracted with 100 ml. ethyl acetate by stirring at 
50.degree. C. for 30 min. centrifuged (5000 rpm) at 15.degree. C. and the 
supernatant was saved. This extraction protocol was repeated two times 
with the centrifuged precipitate. The combined ethylacetate extracts was 
then washed with 5% aqueous alkali (3.times.100 ml) and then with water 
(3.times.100 ml) and dried over anhydrous sodium sulfate. Ethyl acetate 
was evaporated off using a rotatory evaporator and finally the oryzanols 
were purified from the residue by silica gel column chromatography using a 
3.2.times.25 cm column and pure chloroform as the eluent. The oryzanol 
containing fractions (the volume of each collected fraction was about 10 
ml) were combined and chloroform was evaporated. At this state, after 
removal of chloroform, the residue (almost white in appearance) was 
further purified by activated charcoal (&lt;1%) treatment in hot methanol 
solution. The filtrate, on overnight cooling, gave perfect white oryzanols 
crystals (320 mg). The recovery of oryzanols based on the maximum 
literature value of total oryzanol content (3%) G. S. Seetharamaiah and 
J. V. Prabhakar; J. Food Science Technology, 23,270 (1986)! of the 
starting crude dark acid oil was about 54%. The UV spectra of the isolated 
oryzanol in chloroform was found to be practically superimposable with the 
published spectra (Observed .lambda. max values: 319 mn, 294.4 nm & 243.4 
nm) E. J. Rogers et al, J. Am. Oil Chem. Soc. 70,301 (1993)! and 
quantitative UV spectrophometric analysis indicated the purity level of 
the isolated oryzanol to be about 85%. The melting point of the isolated 
oryzanol sample was determined to be within the range of 
120.degree.-125.degree. C. The Reverse phase C-18 (Water's millipore 
column) HPLC of the purified oryzanols showed the presence of all the four 
components (with the same retention times) those are present in a 
commercially available oryzanol samples. The observed retention times are 
13.08 min. 14.42 min. 16.23 min. and 18.69 mins. The mobile phase 
consisted of acetonitrile/methanol/isopropanol (50:45:5 vol/vol/vol). The 
LC system used was of Shimadzu with 20 .mu.l sample loop. Oryzanol 
components were detected at 325 nm following a published protocol (E. J. 
Rogers et al. J. Am. Oil Chem. Soc. 70,301 (1993)!. 
EXAMPLE 2 
About 10.2 g of the residue, obtained after removal of the free fatty acid 
from crude dark acid oil, was hydrolyzed with aqueous sodium hydroxide 
(1.7 g NaOH in 25 ml water) at 80.degree.-90.degree. C. for 1/2 hr. The 
hydrolyzed product mixture turned to a dark brown and somewhat viscous 
homogeneous solution. About 200 ml distilled water was added to this 
hydrolyzed product mixture, stirred at room temperature for about 30 min 
and to the solution, while being stirred, an aqueous solution of calcium 
chloride (2.3 g CaCl.sub.2 dissolved in 30 ml. distilled water) was added 
dropwise. A huge precipitate appeared and the mixture was kept under 
stirring condition for another 15 minutes. The precipitate was then 
filtered and air dried, the dried precipitate was extracted with 100 ml. 
ethyl acetate by stirring at 50.degree. C. for 30 min, centrifuged (5000 
rpm) at 15.degree. C. and the supernatant was saved. This extraction 
protocol was repeated two times with the centrifuged precipitate. The 
combined ethyl acetate extracts was then washed with 5% aqueous alkali 
(3.times.100 ml) and then with water (3.times.100 ml) and dried over 
anhydrous sodium sulfate. Ethyl acetate was evaporated off using a 
rotatory evaporator and finally the oryzanols were purified from the 
residue by silica gel column chromatography using a 3.2.times.25 cm column 
and pure chloroform as the eluent. The oryzanol containing fractions (the 
volume of each collected fraction was about 10 ml) were combined and 
chloroform was evaporated. At this state, after removal of chloroform, the 
residue (almost white in appearance) was further purified by activated 
charcoal (&lt;1%) treatment in hot methanol solution. The filtrate, on 
overnight cooling, gave perfect white oryzanols crystals (362 mg). The 
recovery of oryzanols based on the maximum literature value of total 
oryzanol content (3%) G. S. Seetharamaiah and J. V. Prabhakar; J. Food 
Science Technology, 23,270 (1986)! of the starting crude dark acid oil was 
66%. The quantitative UV spectrophometric analysis indicated the purity 
level of the isolated oryzanol to be about 85%. The melting point of the 
isolated oryzanol sample was determined to be within the range of 
120.degree.-125.degree. C. 
EXAMPLE 3 
About 11 g of the residue, obtained after removal of the free fatty acid 
from crude dark acid oil, was hydrolyzed with aqueous sodium hydroxide 
(1.8 g NaOH in 25 ml water) at 80.degree.-90.degree. C. for 1 hr. The 
hydrolyzed product mixture turned to a dark brown and somewhat viscous 
homogeneous solution. About 200 ml distilled water was added to this 
hydrolyzed product mixture, stirred at room temperature for about 30 min 
and to the solution, while being stirred, an aqueous solution of calcium 
chloride (2.5 g CaCl.sub.2 dissolved in 30 ml. distilled water) was added 
dropwise. A huge precipitate appeared and the mixture was kept under 
stirring condition for another 15 minutes. The precipitate was then 
filtered and air dried, the dried precipitate was extracted with 100 ml. 
ethyl acetate by stirring at 50.degree. C. for 30 min. centrigured (5000 
rpm) at 15.degree. C. and the supernatant was saved. This extraction 
protocol was repeated two times with the centrifuged precipitate. The 
combined ethyl acetate extracts was then washed with 5% aqueous alkali 
(3.times.100 ml) and then with water (3.times.100 ml) and dried over 
anhydrous sodium sulfate. Ethyl acetate was evaporated off using a 
rotatory evaporator and finally the oryzanols were purified from the 
residue by silica gel column chromatography using: 3.2.times.25 cm column 
and pure chloroform as the eluent. The oryzanol containing fractions (the 
volume of each collected fraction was about 10 ml) were combined and 
chloroform was evaporated. At this state, after removal of chloroform, the 
residue (almost white in appearance) was further purified by activated 
charcoal (&lt;1%) treatment in hot methanol solution. The filtrate, on 
overnight cooling, gave perfect white oryzanols crystals (510 mg). The 
recovery of oryzanols based on the maximum literature value of total 
oryzanol content (3%) G. S. Seetharamaiah and J. V. Prabhakar; J. Food 
Science Technology, 23,270 (1986)! of the starting crude dark acid oil was 
about 86%. Quantitative UV spectrophomeric analysis indicated the purity 
level of the isolated oryzanol to be about 85%. The melting point of the 
isolated oryzanol sample was determined to be within the range of 
120.degree.-125.degree. C. 
Advantages of the present invention: 
1. The process can recover highly pure oryzanols from dense dark brown 
pitchlike byproducts of rice bran oil processing industries. 
2. Recovery of oryzanol is quite high. 
3. The byproducts calcium soaps (mainly calcium oleate, calcium palmitate 
and calcium sterate) can be utilized by industries manufacturing water 
proofing fabrics, cements, releasing agents for plastic molding powder, 
stabilizers for polyvinyl chloride resins, lubricants, conditioning agents 
in pharmaceutical products etc.