Title of Invention

A PROCESS FOR THE PREPARATION OF ORYZANOL COMPOUND FROM THE RICE BRAN OIL

Abstract This invention is relates to a Process for preparation of Oryzanol compound from rice-bran oil comprising steps of preparation of Methyl ester by removing gummy matter of rice bran oil by reacting with ortho-phosphoric acid and citric acid, subjecting degummed oil thus prepared to a two stage conversion wherein first stage conversion comprises refluxing of free fatty acids present in the oil with methanol in the presence of mineral acid as a catalyst and second stage conversion comprises refluxing of reactant products using alkaline catalyst to complete the conversion of free fatty acids of rice bran oil to methyl ester wherein ratio of degummed oil to methanol by weight is from 1:1 to 1:2.4 depending upon the content of FFA in the oil, and mineral acid is selected from H2SO4) HCI, HNO3 and taken in quantity 0.1 to 3% by weight of oil and wherein alkaline catalyst is selected from KOH, NaoH and taken in quantity 0.1 to 0.2% by weight of oil depending upon the content of triglycerides in the oil; subjecting the reaction products obtained by step (a) to distillation at temperature of 65.0 to 70°C to distill out methanol followed by mixing residue with dilute mineral acid and washing with water, treating with aqueous salt solution taken in quantity of 0.1 to 0.2% by weight of rice-bran oil in case emulsion is formed, which is further followed by drying under vacuum of l-5torr; subjecting the dried methyl ester obtained by step (b) to vacuum distillation under vacuum of l-5torr at temperature of 185 to 200°C followed by fractional distillation to obtain methyl ester residue; subjecting methyl ester residue obtained by step (c) to column chromatography using 60-120 mesh silica gel, isolating different fractions, using recycled fractions hexane and diethyl ether as eluents, vacuum drying, weighing, analyzing the factions for their oryzonal content and isolating oryzonal rich fraction, wherein silica gel is taken in quantity of 60g for every lOg of residue obtained by step (c), purifying the oryzonal rich fraction by first crystallization with methanol taking said oryzonal concentrate and methanol in the ratio of 1:2 and then by second crystallisation with a mixture of methanol and acetone taken in weight ratio of 2:1, taking oryzonal obtained after first crystallization and the mixture of methanol and acetone in the ratio of 1:2 to obtain desired purified cryzonal compound.
Full Text FIELD OF INVENTION
This invention relates to a process for the preparation of oryzanol compound from rice bran oil. The oryzanol compound has advantageous applications in food, cosmetics and pharmaceutical industries.
PRIOR ART
The oryzanol compound is a very good antioxidant and has several beneficial health effects like cholesterol lowering property and anti-artheroscelerotic effect. As per the conventional process, oryzonal is extracted directly from rice bran oil or its derivatives. One of the conventional method is to isolate oryzonal from soap stock. Industrially soap stock is obtained as a by-product during chemical refining process of rice bran oil. During this step, free fatty acids present in the rice bran oil get converted to sodium salt. Another conventional process of obtaining oryzonol is splitting, washing and fractionation of soap stock containing unsaponifiable matter, which on extraction with diethyl ether gives oryzonal. The main disadvantage of the conventional processes is that not a single conventional process has been found useful on the commercial scale.
Another disadvantage of the known process is that the oryzonal is isolated or prepared from the soap stock or from the acid oil which are the by-products of the rice bran oil industry using conventional alkali refining. However, newer advanced methods of rice bran oil refining such

as physical refining is in practice which omits the chemical refining process responsible for the formation of soap stock and acid oil. Adoption of such advanced technologies would not have soap stock as by product, which would make conventional process as non-practicable due to nonavailability of soap-stock used in such process as a starting material.
According to another process known in the art as per US Patent No. 5869708, oryzanol is isolated from crude dark acid oil obtained by acidificaiton of soap stock obtained in the oil refining industries.
The above process has also the disadvantage that with new advanced refining technologies, alkali refining of rice-bran oil is out of practice due to which soap stock obtained as by-product would not be available and therefore the starting material for the above process would not be available
Yet another disadvantage associated with the conventional process is that the full amount of oryzanol present in the rice bran oil material is not isolated or recovered.
Still another disadvantage associated with the conventional process is that pure oryzonal compound is not isolated/prepared by using a conventional process.
OBJECTS OF PRESENT INVENTION

Therefore, the main object of this invention is to propose a novel process for the preparation/isolation of oryzanol compound from the rice bran oil.
Another object of this invention is to propose a novel process by which complete yield present in the rice bran oil is isolated.
Yet another object of this invention is that pure oryzanol compound is isolated/prepared from the rice bran oil.
Still another object of this invention is to propose a novel process, which is capable of being used on an industrial scale for the preparation/ isolation of oryzonal compound from the rice bran oil.
STATEMENT OF INVENTION
According to this invention there is provided a process for preparation of oryzanol compound from rice bran oil comprising steps of:-
(a) preparation of methyl ester by subjecting rice-bran oil to a two stage conversion wherein first stage conversion comprises refluxing of free fatty acids present in rice bran oil with methanol in the presence of mineral acid as a catalyst and second stage conversion comprises refluxing of reactant products using alkaline catalyst to complete the conversion of free fatty acids of rice bran oil to methyl ester wherein ratio of rice bran oil to methanol by weight is from 1:1 to

1:2.4 depending upon the content of FFA in the oil, and mineral acid is selected from H2SO4, HCL, HNO3 and taken in quantity 0.1 to 3% by weight of rice-bran and wherein alkaline catalyst is selected from KOH, NaOH and taken in quantity 0.1 to 0.2% by weight of rice-bran depending the content of triglycerides in the rice-bran oil;
(b) subjecting the reaction products obtained by step (a) to
distillation at temperature of 65.0 to 70°C to distill out
methanol followed by mixing residue with dilute mineral acid
and washing with water, treating with aqueous salt solution
taken in quantity of 0.1 to 0.2% by weight of rice-bran oil in
case an emulsion is formed, which is further followed by
drying under vacuum of l-5torr;
(c) subjecting the dried methyl ester obtained by step (b) to
vacuum distillation under vacuum of 1-5 torr at temperature
of 185 to 200°C followed by fractional distillation to obtain
methyl ester residue;
(d) subjecting methyl ester residue to column chromatography
using 60-120 mesh silica gel isolating different fractions
using recycled fractions hexane and diethyl ether as eluents,
vacuum drying, weighing, analyzing the fractions for their
oryzonal content and isolating oryzonal rich fraction,
wherein silica gel is taken inquantity of 60g for every lOg of
residue obtained by step (c)

(e) purifying the oryzonal rich fraction by first crystallization
with methanol taking said oryzonal concentrate and
methanol in the ratio of 1:2 and then by second
crystallysation with a mixture of methanol and acetone
taken in weight ratio of 2:1 taking oryzonal obtained after first
crystallization and the mixture of methanol and acetone in the
ratio of 1:2 to obtain desired purified cryzonal compound.
DESCRIPTION OF INVENTION
In accordance with this invention, the process for preparation of oryzanol compound from rice-bran oil comprises following steps:
(a) Preparation of methyl ester
The preparation of methyl ester is a two-stage process. The first stage is methanolysis reaction involving conversion of free fatty acids (FFA) present in rice-bran oil to their respective methyl esters using methanol in the presence of mineral acid as a catalyst. The second stage of the process involves completion of methanolysis reaction by adding alkaline catalyst to the reaction products. The methanolysis reaction is carried out in a three-necked round bottom flask with middle neck of flask of the flask being attached to a reflux condenser, a thermometer being provided in another neck and a stopper provided in third neck of flask which is used for feeding the charge. For methanolysis reaction, methanol and degummed oil are taken in the ratio by weight of 1:1 to 1:2.4, depending

upon the content of FFA in oil. Degummed oil is. prepared by removing the gummy matters by reacting the oil with ortho phosphoric acid and citric acid, which brings down the phosphorus content to desirable limits. Mineral acid is selected from HaSO4, HCL and HNO3, however HaSO4 is preferred. Mineral acid is taken in weight percentage 1 to 2% by weight of oil. Mineral acid is added drop-wise to the above reaction mixture, with shaking in order to avoid charring of the oil. The reactants are allowed to reflux for a time period of 4 to 6 hours, preferably for 5 to 6 Hours at a temperature of 65 to 70°C. To the solution thus obtained, an alkaline catalyst selected from KOH, NaOH and sodium methoxide (NaOMe), preferably sodiummethoxide, taken in quantity 0.2 to 0.3% by weight of oil is added drop-wise and the reactants are again refluxed for 3 to 5 hours preferably for 4 to 5 hours at a temperature of 65 to 70°C. During refluxing, glass beads/porcelain chips are added to the flask to avoid bumping. After completion of the reaction, methanol is distilled out by distillation at temperature of 65 to 70°C. The product left over is washed with water. If an emulsion is formed, aqueous salt solution taken in quantity 0.1 to 0.2% by weight of oil is used to break the emulsion. The methyl ester is dried under vacuum of 1-5 torr.
(b) Vacuum Distillation of Methyl Ester
The dried methyl ester obtained as above is subjected to vacuum distillation in a three-necked round bottom flask. The distillation head is attached to a reflux condenser, which in turn is connected to a perkins

triangle. The vacuum pump is connected to the perkins triangle through CaC12 bottle. A manometer is provided to measure the pressure. The distilled methyl ester is collected in the round bottom flask fitted to the perkins triangle. The distilled methyl ester thus obtained is subjected to fractionation using a fractionation column in the middle neck of the flask.
(c) Isolation of oryzanol through Column Chromatography
Methyl ester residue obtained as above is subjected to column chromatography to isolate oryzonal therefrom. Chromatography is done on a column of silica gel of 60-120mesh taking silica gel in quantity of 60g per l0g of residue. Different fractions and volumes of hexane and diethyl ether are used as eluents. All the fractipns obtained by chromatography are distilled, vacuum dried, weighed and analysed for their oryzonal content. The identification and quantification of total oryzanol content is done by UV spectrophotometer. For this purpose, an accurately weighed quantity of the sample is dissolved in petroleum ether at 60-80°C and its absorbence is measured at 315 nm. Quantification is done by taking specific extinction co-efficient E1Cm% as 358.9. The third fractions obtained by chromatography using hexane and diethyl ether in 1:1 ratio contains orazynol concentration upto 53%.
(d) Purification of Oryzanol

The oryzonal concentrate obtained by the column chromatography method is washed with hexame and then subjected to first crystallization with methanol and then to second crystallisaiton with a mixture of methanol and acetone taken in the ratio of 2:1 by weight, to get 98% pure oryzanol. In the first crystatllisation, oryzonol concentrate and methanol are taken in the ratio of 1:2 and in second crystallization, prazynol obtained after first crystallization and the mixture of methanol and acetone are taken in the ratio of 1:2. the overall yield of oryzanol is about 60%.
WORKING EXAMPLE
About 1kg of degummed rice-bran oil having free fatty acid percentage of about 23% was taken in a three necked flask with about 980gm of methanol. 4.6 gm of HaSC4 was added to it and the mixture was refluxed for about 5 hours. After that 10.7 gm of sodium methoxide was added and it was again refluxed for 4 hours. Unreacted methanol was distilled upto 66°C and the methyl ester thus formed was washed with water and vacuum dried and distilled in vacuum at a temp upto 190°C. The residue obtained here was subjected to column chromatographic separation using 60-120 mesh silica gel. Hexane and diethyl ether were taken as eluting solvent. The third fraction containing 50:50 ratio of hexane and diethyl ether contained the oryzanol concentration upto 53.12%. the other fractions were recycled and after purification with methanol and acetone 98% pure oryzanol was obtained. The over all yield of oryzanol was found to be about 60%.

It is to be understood that the process of present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes and adaptations are intended to be within the scope of the present invention, which is further set forth under the following claims:









I CALI1&
1. A process for preparation of oryzanol compound from rice-bran oil comprising steps of:-
(a) preparation of methyl ester by removing gummy matter of rice bran oil by reacting with ortho-phosphoric acid and citric acid, subjecting degummed oil thus prepared to a two stage conversion wherein first stage conversion comprises refluxing of free fatty acids present in the oil with methanol in the presence of mineral acid as a catalyst and second stage conversion comprises refluxing of reactant products using alkaline catalyst to complete the conversion of free fatty acids of rice bran oil to methyl ester wherein ratio of degummed oil to methanol by weight is from 1:1 to 1:2.4 depending upon the content of FFA in the oil, and mineral acid is selected from H2SO4, HC1, ^HN03 and taken in quantity 0.1 to 3% by weight of oil and wherein alkaline catalyst is selected from KOH, NaOH and taken in quantity 0.1 to 0,2% by weight of oil depending upon the content of triglycerides in the oil;
(b) subjecting the reaction products obtained by step (a) to distillation at temperature of 65.0 to 70°C to distill out methanol followed by mixing residue with dilute mineral acid and washing with water, treating with aqueous salt solution taken in quantity of 0.1 to 0.2% by weight of rice-bran oil in case an emulsion is formed, which is further followed by drying under vacuum of l-5torr;
c) subjecting the dried methyl ester obtained by step (b) to vacuum
distillation under vacuum of 1-5 torr at temperature of 185 to 200°C
followed by fractional distillation to obtain methyl ester residue;
(d) subjecting methyl ester residue obtained by step (c) to column chromatography using 60-120 mesh silica gel, isolating different fractions, using recycled fractions hexane and diethyl ether as eluents, vacuum drying, weighing, analyzing the fractions for their oryzonal content and isolating oryzonal rich fraction, wherein silica gel is taken in quantity of 60g for every lOg of residue obtained by step (c);
(e) purifying the oryzonal rich fraction by first crystallization with
methanol taking said oryzonal concentrate and methanol in the ratio of 1:2 and then by second crystallization with a mixture of methanol and acetone taken in weight ratio of 2:1, taking oryzonal obtained after first crystallization and the mixture of methanol and acetone in the ratio of 1:2 to obtain desired purified cryzonal compound.
2. A process as claimed in claim 1 wherein said mineral acid is H2SO4.
3. A process as claimed in claim 1 wherein said alkaline catalyst is
sodium methoxide (NaOMe).
4. A process for preparation of oryzonal compound from ricebran oil as herein described and illustrated.

Documents:

85-del-2003-abstract.pdf

85-del-2003-claim.pdf

85-del-2003-Claims-(22-12-2009).pdf

85-del-2003-correspondence others.pdf

85-del-2003-correspondence po.pdf

85-DEL-2003-Correspondence-Others-(09-09-2008).pdf

85-DEL-2003-Correspondence-Others-(13-08-2009).pdf

85-DEL-2003-Correspondence-Others-(16-04-2010).pdf

85-del-2003-Correspondence-Others-(22-12-2009).pdf

85-del-2003-description (complete).pdf

85-del-2003-description (provisional).pdf

85-del-2003-form-1.pdf

85-del-2003-form-18.pdf

85-del-2003-form-2.pdf

85-del-2003-form-3.pdf

85-del-2003-form-4.pdf

85-del-2003-form-5.pdf

85-del-2003-gpa.pdf

85-del-2003-petition-138.pdf


Patent Number 240729
Indian Patent Application Number 85/DEL/2003
PG Journal Number 23/2010
Publication Date 04-Jun-2010
Grant Date 26-May-2010
Date of Filing 05-Feb-2003
Name of Patentee R.P. SINGH
Applicant Address 4T/53 HBTI COLONY, CAMPUS, KANPUR-208002
Inventors:
# Inventor's Name Inventor's Address
1 R.P. SINGH 4T/53 HBTI COLONY, CAMPUS, KANPUR-208002
PCT International Classification Number A61K8/19
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA