FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
PREPARATION OF STEROLS AND TOCOPHEROLS
2. APPLICANT
(a) NAME: ' PRAJ Industries Limited
(b) NATIONALITY: Indian Company
(b) ADDRESS: PRAJ House, Bavdhan
Pune-411021, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention and the manner in which it is to be performed.

4. DESCRIPTION
FIELD OF INVENTION
The invention relates to a process for the preparation of sterols and tocopherols from edible vegetable oils. It particularly relates to the preparation of sterols and tocopherols from a deodorizer distillate from vegetable oils obtained from seed of soybean, sunflower, safflower, groundnut and other like plants.
BACKGROUND
Sterols [also known as phytosterols] and tocopherols of plant origin are widely used for different consumer and industrial applications. Sterols are used in medicinal formulations as such or in modified forms for the treatment of variety of ailments, especially, for the treatment of inflammatory, immune or chronic diseases. Tocopherols are used as vitamin supplements in various food and cosmetics products. These sterols and tocopherols are isolated from different plant materials like roots, stems and oil seeds. Phytosterols are also used in many neutraceutical and cosmetic products. Besides, they are used as raw materials for the preparation of several intermediate chemicals or derivatives of unique properties for the preparation of several medicinal molecules.
Soybean oil deodorizer distillate (SODD) is obtained as a by-product of the deodorization step in the refining of soybean oil. During the refining of soybean oil most bioactive compounds such as free phytosterols, fatty acid steryl esters, tocopherols and squalenes are concentrated in SODD. Furthermore, SODD also contains free fatty acids, polycyclic hydrocarbons and the bioactive compounds making it is a good natural source of phytosterols, tocopherols and squalenes. A few methods have been developed to recover and purify the sterols and tocopherols from the deodorizer distillates of vegetable oils.

The major issues with the recovery of sterols and tocopherols from materials like SODD are the yield and quality of the final compositions. They form raw materials for the preparation of many sterol and tocopherol derivatives used in pharmaceutical preparations. Thus, there is still a need for a process to produce sterols and tocopherols from plant materials using green, non-toxic and economic methods with higher quality of the final preparations for use in high-end applications like pharmaceutical and nutritional products.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a process for the recovery of sterols and tocopherols from plant origin materials like SODD using a benign method having higher process efficiencies and higher quality of the final preparations. The disclosed process further utilizes fewer amounts of organic solvents over known methods providing economic value and improved control over the waste by-product treatment. The present invention further provides a product comprising derivatives of sterols and tocopherols produced by a process of the invention disclosed herein, the said products being pharmaceutical, nutraceutical or cosmetic preparations.
In one embodiment of the present invention, disclosed is a process for isolation of sterols and tocopherols from a deodorizer distillate of oil comprising: providing a deodorizer distillate comprising free and bound sterols and tocopherols; saponifying said deodorizer distillate by contacting it with an inorganic base in water at an elevated temperature and pressure for a specific time period leading to formation of a soap composition; acidulating said soap composition with an inorganic acid at an elevated temperature for a specific time period leading to formation of an acidified composition; separating aqueous mass from said acidified composition by layer separation leading to formation of an organic composition; drying said organic composition by evaporation; esterifying said dried organic composition with an alcohol in the presence of an acid catalyst and allowing formation of crude sterol crystals in remaining liquid reaction mass; recovering said crude sterol crystals from said

reaction mass leading to formation of a mother liquor; purifying said crude sterol crystals by recrystallization; subjecting said mother liquor to a molecular distillation to get a first crude tocopherol composition; purifying said first crude tocopherol composition with solvent extraction to get a second crude tocopherol composition; and subjecting said second crude tocopherol composition to a resin purification to get a final tocopherol composition. These steps lead to the formation of a final sterol preparation of higher purity and yield compared to known methods.
DESCRIPTION OF DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:
Figure 1 depicts a process flow diagram for the preparation of sterols and tocopherols from a deodorizer distillate. Different elements of the process are identified and directional movements of different streams and components formed are shown to describe the features of one embodiment of the present invention.
Figure 2 [A] shows a graphical representation [gas chromatographic detection] of naturally occurring derivatives of sterols in a final preparation obtained as per a process of the invention disclosed: - campesterol RT: 33.7 min; stigmasterol RT: 34.4 min and β-sitisterol RT: 35.8 min; [B] shows a graphical representation [RP-HPLC detection] of naturally occurring derivatives of tocopherols in a final preparation obtained as per a process of the invention disclosed: - tocopherol - alpha RT: 18.2 min; tocopherol - delta RT: 17.1 min and tocopherol - gamma RT: 17.7 min.
DETAILED DESCRIPTION OF THE INVENTION
Sterols and tocopherols are an important group of organic molecules widely used in pharmaceutical, neutraceutical and cosmetic products. They are also

main components of several traditional medicinal preparations like ayurvedic products. Many vegetative plant materials like roots, stems, leaves or seeds produce and store the sterols and tocopherols.
Besides natural sterols many synthetic derivatives of the natural sterols have been prepared. These synthetic sterols have many properties not available in their natural derivatives like target specificity, potency, safety or solubility. These synthetic sterols are prepared from natural sterol derivatives isolated from plants or animals sources by regio- or stereo specific chemical reactions. Today most of the modern medicinal preparations used these synthetic sterols. To achieve the higher purity and yield of synthetic sterol derivatives in chemical syntheses, the quality of reagent sterols used is of crucial importance. In turn this quality of starting sterols mostly depends on the type of processes used for the preparation of sterols from natural sources like plant materials mentioned herein.
The natural tocopherols have been widely used in the preparations like cosmetic products, animal and human healthcare products. Of the natural tocopherols, alpha and gamma derivatives are more important as active ingredients in the above mentioned products.
Phytosterols and tocopherols are present in large amounts in oil seeds of several types. During extraction and refining of oils, sterols and tocopherols containing fractions are separated and further processed to isolate the sterols and tocopherols as by-products of higher values. Several methods have been known that separate these from other components. However, the quality of the final sterol and tocopherol preparations largely depends on the physical and chemical processes used to isolate it.
This invention discloses a process for the preparation of sterols and tocopherols from a deodorizer distillate as value added by-products of oil refining industry. The process having several advantages over the known methods like:

1. The saponification reaction in performed in water without used of any solvent or alcohol [aqueous saponification] leading to significant savings of solvents;
2. The process is also effective in splitting of glycerides and sterol esters at benign and economical conditions;
3. The recovery of sterols is improved as sterol esters are efficiently split into free sterols leading to an increase in the free sterols to be isolated;
4. Tocopherols of higher purity are recovered by molecular distillation operating at economical conditions;
5. The cost of solvents like alcohol is removed along with associated solvent recovery/ recycle units and related expenses; and
6. The quality of the final sterol and tocopherols preparations obtained by this invention is also superior for the preparation of pharmaceutical or cosmetic products of higher quality.
In one embodiment of the present invention, efficient recoveries of the sterols and tocopherols from a deodorizer distillate are obtained using a process comprising four steps namely: 1] aqueous saponification; 2] esterificatjon and crystallization; 3] recrystallization; and 4] isolation of tocopherols. Each step has one or more elements for performing specific or optional functions as required for achieving selective isolation of sterols from the raw materials. A person skilled in the art may appreciate different variations and/ or combinations of these elements that may be used to perform the objects of the invention disclosed herein.
In another embodiment of the invention, efficient recovery of sterols and tocopherols from SODD is obtained by treating it with an alkali like sodium hydroxide at an elevated temperature in a pressure vessel for about 2 hours. Then the reaction mass is subjected to acidulation with a mineral acid like sulphuric acid leading to the formation of saponified and acidulated material

having higher amounts of free sterols in the solution. During this treatment the amount of free sterols is increased by at least 20% of original by efficient splitting of sterols esters [bound sterols]. These steps are performed in aqueous medium without any alcohol or organic solvents. Next, the said saponified and acidulated material is subjected to esterification with an alcohol like methanol and allowed to form crude crystals of sterols in cooled reaction mixture for an extended time. The said crude crystals are recovered from the mother liquor and further subjected to recrystallization using an organic solvent like acetone to get the final sterol preparations of higher purity and yield useful for use in the preparation of pharmaceutical or cosmetic products. The said mother liquor separated from sterols crystals contain the most of tocopherols, which is subjected to the steps of molecular distillation following by solvent extraction and resin purification to get the final tocopherol preparation of higher yield and purity. It may be appreciate that the steps disclosed herein are equally applicable to a deodorizer distillate other than the SODD as primary components of these distillates remains similar.
Step 1: Aqueous Saponification
The aqueous saponification step is used for conversion of glycerides and sterol esters present in the materials like SODD to free fatty acids and free sterols, leading to an increase in the amount of free sterols in the stream at the end of saponification treatment. The said saponification is performed totally in water without use of any organic solvent like methanol or ethanol. During this step said deodorizer distillate is saponified at an elevated temperature in the presence of an alkali like sodium hydroxide in a pressure vessel under non-oxidizing atmosphere. Nitrogen may be used to maintain the non-oxidizing conditions during this step. Beside, an elevated pressure may be maintained so that the desired reaction takes place effectively. Then the reaction mass is subjected to acidulation by a mineral acid to neutralize said alkali leading to the formation of two liquid layers. The aqueous layer is removed and the organic layer is washed with hot water and concentrated [dried] by evaporation; then further subjected to the next step.

Step 2: Esterification and Crystallization
The stream obtained at the end of said saponification step is subjected to esterification in the presence of an acid catalyst with an alcohol like methanol or ethanol at a temperature of about 65°C for about 4 h. During this process the free fatty acids are converted into esters helping easy removal of said fatty acids from the crystallizing sterols during the process of crystallization by standing the reaction mixture at a temperature of about 27°C for about 18 h. Then the crude sterols crystals are removed by filtration from the mother liquor and subjected to the next step. The said mother liquor so obtained is washed with water to remove any acid, and further processed to recover tocopherols present in it in a subsequent step [step 4].
Step 3: Recrystallization of Sterols
Then said crude sterol crystals are again washed with water to remove any acid, and further purified for higher purity from an organic solvent like one or more of acetone, ethyl acetate, ethanol, hexane or heptane. This recrystallization step is performed at a temperature of about 5°C for about 18 h and the final crystals are dried. The quality of final sterol, preparations largely depends on the solvents used, the ratio of crude crystals to the solvents, the composition of the solvent system as well as treatment conditions as disclosed herein.
The amount of sterols and different components of the final sterol preparations were analyzed by gas chromatograph with flame ionization detector. An HP-50 [AGILENT J&W GC column] column of size 30 m X 0.32 mm OD X 0.25 mm ID was used. Helium was used as carrier gas with flow rate of 0.9 mL/min. Injector and detector temperatures were kept at 250°C and 325°C respectively. About 100 mg of a final sterol preparation was dissolved in 5 mlL of chloroform. To this 1 mL of 15 mg/mL in chloroform of 5-alpha-cholestane was added as an internal standard and the final volume was made to 10 mL with chloroform. About 100 mcL of this sample was taken and dried

by passing nitrogen over it. To this was added about 60 mcL of BSTFA [a derivatization reagent] and about 240 mcL of pyridine solution. The reaction mixture was heated to about 70°C for about 30 min and cooled to room temperature and then diluted to 1.7 mL with heptane and injected for the GC analysis. Retention times of three components of the final sterol preparation, namely, campesterol, stigmasterol and sitosterol were observed at about 33, 34 and 35 minutes, respectively. Estimation of sterols content in the test samples was done using area under curve calculations relative to a standard. A representative graph of the amounts of different natural sterol derivatives in the final sterol preparation is shown in Figure 2.
Step 4: Isolation of Tocopherols
The mother liquor so obtained after recovery of sterol crystals is washed with water to remove any acid, dried under vacuum and further used for isolation of tocopherols. Said liquor is subjected to steps of molecular distillation wherein distillation conditions under vacuum and elevated temperature are maintained such that the tocopherols are fractionated from other components of the liquor and enriched to about 5-7 folds. In one embodiment, said about 200 gm of said liquor is subject to a temperature of about 180 °C under vacuum for about 3 h to remove high boiler components. The distillate obtained contained tocopherols; it is again subjected to another step of molecular distillation at 100 °C for about 3 h to remove low boiler components. The residue obtained here contained about 41% of tocopherols. Next, this intermediate is subjected to solvent extraction in an organic solvent like methanol, twice, to remove the methanol soluble fraction leading to formation enrichment of tocopherols to about 65%. To further purify this preparation a reverse phase adsorption chromatography technique is used, wherein a hydrophobic resin like polystyrene based resin without any functional group is employed. The samples are loaded to the column and washed with about 98% methanol followed by elution with 100% methanol. The tocopherol rich fractions are then collected leading to formation of a final tocopherol preparation with about 85% purity:

HPLC method details are: column: Waters Spherisorb (C18) 5 micron 4.6 X 250 mm; mobile phase; methanol (100%); flow 1.0 ml/min; column oven temp: 300°C; injection volume: 20 ul; run time: 30 min; and detector: UV @ 210 nm.
Representative features of the invention are illustrated in the DRAWINGS.
Figure 1 depicts a process flow for the preparation of sterols from a deodorizer distillate comprising using SODD as source of said sterols in accordance with one aspect of the present invention. In the first step [aqueous saponification] a deodorizer distillate comprising the sterol esters is saponified to break the sterol esters to free sterols, increasing the content of total free sterols. This treatment also splits other esters. Herein the saponification in performed in water only at elevated temperature in a pressure vessel under non-oxidizing atmosphere using an alkali. Then the organic layer is isolated after acidulation with a mineral acid like sulphuric acid to neutralize the effects of alkali, washed with water and subject to the next step. The second step [esterification and crystallization] is performed on the stream obtained from the first step, here the free fatty acids are esterified with an alcohol that helps in separation of sterols from the solution, in the crystallization step the sterols are allowed to form crystals on standing to remove other components from the sterols, then these crude crystals of sterols are collected by filtration. In the last step [recrystallization] said crude crystals of sterols are further purified by recrystallization from a solvent like acetone to improve purity of the final sterol preparations.
EXAMPLES
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples and tables, which demonstrate the advantageous and novel aspects of the process of performing saponification in water only at an

elevated temperature in a pressure vessel under non-oxidizing conditions for the efficient separation of sterols and tocopherols from a deodorizer distillate like SODD.
The characteristics of the suitable starting materials required for the above described process for producing the desired final sterol and tocopherol preparations are summarized in Table 1.
Table 1: Specifications of deodorizer distillates that may be used for the isolation of phytosterols and tocopherols

No. Composition [%] SODD Type-1 SODD Type-2 SODD Type-3
1 Free fatty acids 40.83 30.00 53.92
2 Glycerides 25.32 30.80 12.84
3 Free sterols 5.01 5.06 8.82
4 Total sterols 7.49 7.44 10.38
5 Free tocopherols 4.62 4.29 9.71
Example 1
About 1000 gm of SODD [containing about 5.0% free sterols, about 7.49 % " total sterols and about 41% free fatty acids] was saponified using 120 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 10% solution of sulphuric acid at 80°C for about 1 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 7.94% free sterols and about 69% free fatty acids. The amount of free sterols in the dried material was enriched to about 48% over that of the starting material. The sterol esters splitting efficiency of about 98% was achieved by the above steps. Then about 100 gm of the above dried material was esterified with about 47 gm of methanol in the presence of about 2.72 gm of p-toluene sulphonic acid at

about 65°C for about 4 h. After the reaction about 14 gm of water [i.e. 10% of the total esterified mass of fatty acids] was added and reaction mixture was allowed to cool at ambient temperature and kept for about 18 h. Next, the crude sterol crystals settled in the solution [mother liquor] were separated by filtration and washed with hot water and then dried under vacuum. These crystals were then dissolved in acetone five times the weight of crystals at about 55°C for about 30 min, allowed to cool and recrystallized at about 5°C for about 18 h. Purified crystals were recovered by filtration and dried. These steps afforded about 6.5 gm of purified sterols crystals having a purity of about 88% by weight by gas chromatographic [GC] analysis. The recovery of total sterols from SODD was about 72%.
Example 2
About 1000 gm of SODD [containing about 5.0% free sterols, about 7.44% total sterols and about 29% free fatty acids] was saponified using 120 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 10% solution of sulphuric acid at 80oC for about 1 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 6.74% free sterols and about 69% free fatty acids. The amount of free sterols in the dried material was enriched to about 25% over that of the starting material, The sterol esters splitting efficiency of about 52.2% was achieved by the above steps. Then about 350 gm of the above dried material was esterified with about 164 gm of methanol in the presence of about 9.66 gm of p-toluene sulphonic acid at about 65°C for about 4 h. After the reaction water about 10% of the total esterified mass of fatty acids was added and reaction mixture was allowed to cool at ambient temperature and kept for about 18 h. Next, the crude sterol crystals settled in the solution [mother liquor] were separated by filtration and washed once with hot water and then dried under vacuum. These crystals were then dissolved in acetone five times the weight of crystals at about 55°C

for about 30 min, allowed to cool and recrystallized at about 5°C for about 18 h. Purified crystals were recovered by filtration and dried. These steps afforded about 18.88 gm of purified sterols crystals having a purity of about 91.5% by weight by GC analysis. The recovery of total sterols from SODD was about 61.33%.
Example 3
About 1000 gm of SODD [containing about 8.82% free sterols, about 10.38% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.68% free sterols and about 68% free fatty acids. The amount of free sterols in the dried material was enriched to about 16% over that of the starting material. The sterol esters splitting efficiency of about 92.6% was achieved by the above steps. Then about-300 gm of the above dried material was esterified with about 139 gm of methanol in the presence of about 8.16 gm of p-toluene sulphonic acid at about 65°C for about 4 h. After the reaction water about 10% of the total esterified mass of fatty acids was added and reaction mixture was allowed to cool at ambient temperature and kept for about 18 h. Next, the crude sterol crystals settled in the solution were separated by filtration and washed once with hot water and then dried under vacuum. These crystals were then dissolved in acetone five times the weight of crystals at about 55°C for about 30 min, allowed to cool and recrystallized at about 5°C for about 18 h. Purified crystals were recovered by filtration and dried. These steps afforded about 27.37 gm of purified sterols crystals having a purity of about 85% by weight by GC analysis. The recovery of total sterols from SODD was about 73%.

Example 4
About 1000 gm of SODD [containing about 7% free sterols, about 10% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [10% solution, with 20% excess of NaOH] at about 140°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 9.99% free sterols and about 70% free fatty acids. The amount of free sterols in the dried material was enriched to about 13% over that of the starting material. The sterol esters splitting efficiency of about 78% was achieved by the above steps.
Example 5
About 1000 gm of SODD [containing about 8% free sterols, about 10.38% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 140°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.4% free sterols and about 69% free fatty acids. The amount of free sterols in the dried material was enriched to about 18% over that of the starting material. The sterol esters splitting efficiency of about 85% was achieved by the above steps.
Example 6
About 1000 gm of SODD [containing about 8.82% free sterols, about 10.3% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 110°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After

acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 9% free sterols and about 69% free fatty acids. The amount of free sterols in the dried material was enriched to about 0.64% over that of the starting material. The sterol esters splitting efficiency of about 2.5% was achieved by the above steps.
Example 7
About 1000 gm of SODD [containing about 8% free sterols, about 10.3% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 15% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.16% free sterols and about 68% free fatty acids. The amount of free sterols in the dried material was enriched to about 18% over that of the starting material. The sterol esters splitting efficiency of about-75% was achieved by the. above steps.
Example 8
About 1000 gm of SODD [containing about 8.8% free sterols, about 10.3% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [10% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.7% free sterols and about 68% free fatty acids. The amount of free sterols in the dried material was enriched to about 24% over that of the starting material. The sterol esters splitting efficiency of about 99% was achieved by the above steps.

Example 9
About 1000 gm of SODD [containing about 8% free sterols, about 10.4% total sterols and about 53.92% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 1 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.6% free sterols and about 66% free fatty acids. The amount of free sterols in the drfed material was enriched to about 22% over that of the starting material. The sterol esters splitting efficiency of about 88% was achieved by the above steps.
Example 10
About 1000 gm of SODD [containing about 8.3% free sterols, about 10.3% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 1 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 90°C for about 1 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.6% free sterols and about 67% free fatty acids. The amount of free sterols in the dried material was enriched to about 24% over that of the starting material. The sterol esters splitting efficiency of above 98% was achieved by the above steps.
Example 11
About 1000 gm of SODD [containing about 8.3% free sterols, about 10.38% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and

acidulated using 30% solution of sulphuric acid at 90°C for about 1 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.6% free sterols and about 68% free fatty acids. The amount of free sterols in the dried material was enriched to about 23% over that of the starting material. The sterol esters splitting efficiency of about 97% was achieved by the above steps.
Example 12
About 1000 gm of SODD [containing about 8.3% free sterols, about 10.3% total sterols and about 54% free fatty acids] was saponified using 100 gm of NaOH [48% solution, with 20% excess of NaOH] at about 150°C and about 5 bar pressure for about 2 h. Reaction mass was cooled to about 70°C and acidulated using 30% solution of sulphuric acid at 80°C for about 2 h. After acidulation organic layer containing free sterols, free fatty acids, etc was isolated from aqueous layer, washed once with water and dried under vacuum. This dried material contained about 10.7% free sterols and about 69% free fatty acids. The amount of free sterols in the dried material was enriched to about 21% over-that of the starting material. The sterol esters splitting efficiency of about 96% was achieved by the above steps.
Example 13
To identify the optimized conditions for the recrystallization of crude sterol crystals different solvents were examined as enlisted in Table 13-1. Further to identify the optimized conditions for the recrystallization of crude sterol crystals different ratios of crude crystals to solvent were examined as enlisted in Table 13-2. The percent purity and the percent yield of the recrystallized sterol preparations were used as a measure of suitability of solvents for recrystallization.
Example 14

To identify the optimized conditions for the recrystallization of crude sterol crystals using acetone as solvent, different amounts of the solvent were examined as enlisted in Table 14-1. The percent purity and the percent yield, of the recrystailized sterol preparations were used as a measure of suitability of the amount of the solvent required for recrystallization.
Example 15
The said mother liquor obtained in the step for recovery of sterols crystals contained the most of tocopherols present in the feedstock. About 200 gm of said mother liquor [comprising about 10% tocopherols] was subjected to first part of molecular distillation at about 100 °C at a vacuum of about 0.1 torr for about 3.5 hours. The low boiler components [FAME] present in the distillate were removed and the residue so obtained was again subjected to second part of molecular distillation at about 180 °C at vacuum of about 0.1 torr for about 3.5 hours. After this step the distillate comprise about 50% of tocopherols leading to about 5 fold enrichment of tocopherols over the said mother liquor. Next, the said tocopherol rich distillate was subjected to solvent extraction step using methanol as extracting solvent. About 10 gm of said distillate was extracted with 100 mL 95% methanol at-about 25 °C for 15 minutes. After layer separation methanol layer was separated and the remaining residue was again subject to said methanol extraction. These methanol extracts were collected and methanol was evaporated leading to formation of a tocopherol composition with about 62% tocopherols. To further purify this tocopherol composition it was subjected to reverse phase chromatographic purification using a polystyrene based adsorption resin. On a 95 mL bed volume column [1.0 x 120 cm] packed with a styrene based non-ionic hydrophobic copolymer was loaded with about 3.3 gm of said solvent extracted residue comprising about 68% tocopherols. Then the column was washed with about 8 column volumes of 98% methanol in water and eluted with about 3 column volumes 100% methanol. The tocopherol rich fractions were collected and methanol distilled out to obtain about 85% pure

tocopherols with a process yield of about 83%. The final preparation of tocopherols was analyzed with RP-HPLC.
Example 16
About 100 mg of finally purified sterols crystals were dissolved in 5 mL of chloroform and subject to a standart GC analysis method. Representative amounts of natural sterol derivatives in the final sterol preparations obtained as per Examples 1, 2 and 3 are listed in Table 16-1. See FIGURE 2[A].
Example 17
About 100 mg of finally purified tocopherols were subject to a standard RP-HPLC analysis method. Representative amounts of natural tocopherol derivatives in the final tocopherol preparations obtained as per Example 15 are listed in Table 17-1. See FIGURE 2[B].
While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made therein which are within the spirit of the invention and within the scope of the claims.

Table 13-1:

No. Crude Free Solvent Crystal to Crystalli- Crystalli- Purified Sterol purity Recovery of
sterol sterols solvent zation temp. zation sterol (%) free sterols on
crystal
(gm) (%) ratio (w/w) (°C) time (h) crystals (gm) basis of crude crystals (%)
1 3 70.42 Acetone 1:5 5 18 2.2 85.80 89.35
2 3 70.42 Ethyl acetate 1:5 5 18 1.66 90.81 71.36
3 3 70.42 Ethanol 1:5 5 18 2 89.05 84.30
4 3 69.82 Hexane 1:5 5 18 2.2 85.49 87.18
5 3 69.82 Heptane 1:5 5 18 2.2 84.87 86.54

Table 13-2:

No. Crude Free Solvent Crystal to Crystalli- Crystalli- Purified Sterol purity Recovery of
sterol sterols solvent zation temp. zation sterol (%) free sterols on
crystal (%) ratio
(w/w) (°C) time (h) crystals
(gm) basis of crude crystals (%)
1 3 68.24 Acetone 1:5 5 18 2.10 85.41 87.20
2 3 68.24 Acetone 1:10 5 18 1.86 87.16 79.19
3 3 68.24 Acetone 1:15 5 18 1.71 88.40 73.84
4 3 68.24 Acetone 1:20 5 18 1.58 87.38 67.44

Table 14-1:

No. Crude Free Solvent Crystals Crystallization Crystallization Purified Sterol Recovery of
sterol sterols to solvent temp. (°C) time (h) sterol purity free sterols
crystal (gm) (%) ratio (w/w) crystals
(gm) (%) on basis of
crude crystals (%)
1 3 68.24 Acetone 1:05 5 18 2.09 85.41 87.20
2 3 68.24 Acetone 1:10 5 18 1.86 87.16 79.19
3 3 68.24 Acetone 1:15 5 18 1.71 88.40 73.84
4 3 68.24 Acetone 1:20 5 18 1.58 87.38 67.44

Table 16-1:

Natural sterol derivative [%] Example 1 Example 2 Example 3
Campesterol 23.27 25.13 24.28
Stigma sterol 23.92 28.03 26.23
β-Sitisterol 40.65 38.31 37.59
Total 88.04 91.47 88.15

Table 17-1:

Natural tocopherol derivative [%] Example 1 Example 2 Example 3
Alpha 7.03 8.1 1.0
Beta 29.38 35.1 27.45
Gamma



Delta 12.87 16.11 52.03
Total 49 59 80.45

5. CLAIMS
WE CLAIM:
1. A process for isolation of sterols and tocopherols from a deodorizer, distillate of oil comprising:
(a) providing a deodorizer distillate comprising free and bound sterols and tocopherols;
(b) saponifying said deodorizer distillate by contacting it with an inorganic base in water at an elevated temperature and pressure for a specific time period leading to formation of a soap composition;
(c) acidulating said soap composition with an inorganic acid at an elevated temperature for a specific time period leading to formation of an acidified composition;
(d) separating aqueous mass from said acidified composition by layer separation leading to formation of an organic composition;
(e) drying said organic composition by evaporation;
(f) esterifing said dried organic composition with an alcohol in the presence of an acid catalyst and allowing formation of crude sterol crystals in remaining liquid reaction mass;
(g) recovering said crude sterol crystals from said reaction mass leading to formation of a mother liquor;
(h) purifying said crude sterol crystals by recrystallization;
(i) subjecting said mother liquor to a molecular distillation to get
a first crude tocopherol composition; (j) purifying said first crude tocopherol composition with solvent
extraction to get a second crude tocopherol composition; and (k) subjecting said second crude tocopherol composition to a
resin purification to get a final tocopherol composition.

2. The process of claim 1, wherein:
(a) said inorganic base is sodium hydroxide or potassium
hydroxide; (b)said saponification reaction is performed at a temperature
between about 70°C to about 170°C and a pressure between
about 3 bars to about 7 bars;
(c) said saponification reaction is performed for a time period of between about 1 hour to about 4 hours;
(d) said acidulation reaction is performed using hydrochloric acid or sulphuric acid;
(e) said acidulation reaction is performed at a temperature between about 70°C to about 110°C for a time period of between about 30 minutes to about 200 minutes;
(f) said esterification reaction is performed using methanol, ethanol, butanol, propanol or a mixture thereof;
(g) said crude sterol crystals are recovered by decantation, filtration or centrifugation;
(h) said crude sterol crystals are further purified by
recrystallisation to a purity of at least 85% by weight; (i) said recrystallisation is performed using acetone, ethyl
acetate, methanol, ethanol, hexane, heptane or a mixture
thereof; (j) said deodorizer distillate comprises at least about 4% free
and bound sterols and at least about 3% tocopherols; (k) solvent extraction of said crude tocopherol composition is
performed using methanol, ethanol, butanol, propanol or a
mixture thereof; (I) said second crude tocopherol composition is further purified
by a reverse phase adsorption chromatography using a
mixture of water and a lower alcohol as a mobile eluting
phase;

(m)said final tocopherol composition has a purity of at least 30%
by weight; and (n) said deodorizer distillate is selected from one or more of
soybean oil, sunflower oil, rice bran oil, coconut oil and palm
kernel oil.
3. A sterol composition according to the process of claim 1 comprising campesterol, stigmasterol and β-sitosterol.
4. A tocopherol composition according to the process of claim 1 comprising alpha, beta, gamma and delta derivatives of tocopherol.
5. A sterol derivative prepared from sterols obtained according to the process of claim 1.
6. A tocopherol derivative prepared from tocopherols obtained according to the process of claim 1.
7. A pharmaceutical, neutraceutical or cosmetic product comprising a sterol or a tocopherol prepared according to the process of claim 1.