WO2000064922A1

WO2000064922A1 - Alkanol, methyl ethyl ketone water solvent system for the separation of sterols

Info

Publication number: WO2000064922A1
Application number: PCT/IB2000/000539
Authority: WO
Inventors: Antti Hamunen
Original assignee: Sterol Technologies Ltd.
Priority date: 1999-04-27
Filing date: 2000-04-27
Publication date: 2000-11-02
Also published as: AU3983900A

Abstract

A method for purifying 4-desmethylsterols from neutral substances containing the 4-desmethylsterols and undesired neutral components, the method comprising the step of treating the neutral substances with a solvent mixture comprising methyl ethyl ketone, a C1-C6 alkanol and water.

Description

ALKANOL, METHYL ETHYL KETONE WATER SOLVENT SYSTEM FOR THE SEPARATION OF STEROLS

This application claims priority under 35 USC § 119(e) of U.S. Provisional Application Serial No. 60/131,305, which is hereby incorporated by reference.

Field of the Invention

The present invention relates to a method for separation of commercially important sterols from neutral substances comprising those fractions of wood originated extractives which are soluble in organic solvents. Such neutral substances are included in e.g. tall oil and tall oil pitch.

Background of the Invention

Sterols useable in pharmaceuticals and foods need to be purified to quite a high degree to exclude undesired components. For example, in neutral substances originated at least partly from hardwoods like birch, such undesired components include a-sitosterol (citrostadienol), other methylsterols, dimethylsterols and triterpene alcohols e.g. betuline.

U.S. 5,670,669 discloses a method of separating tocopherols (vitamin E) from a mixture containing tocopherols and sterols, the mixture being essentially free of higher fatty acids. The tocopherols and sterols are separated by the crystallization of the sterols using a solvent blend comprised of a major amount of a low polarity organic solvent, a minor amount of a high polarity organic solvent, and a minor amount of water. The preferred low polarity organic solvents are the higher alkanes, mono-ketones such as acetone, 2- butanone or 2-octanone, mono-aldehydes, mono-esters and higher mono-hydric alcohols such as n-propanol, iso-propanol, n-butanol, sec-butanol, n-hexanol or 2-ethylhexanol. The preferred high polarity organic solvents are the low molecular weight, oxygenated hydrocarbons, preferably the lower alkanols. The solvent blend is preferably comprised of 90-99.5 % of the low polarity solvent and 0.5-10% of the high polarity solvent, with the water preferably being present in an amount essentially equal to the high polarity organic solvent. This general method is disclosed as being useful for crystallizing all types of sterols, however, there is no disclosure in U.S. '669 regarding the removal of desired sterols from undesired sterols/neutral components.

In U.S. 4,422,974, β-sitosterol is separated from an α-sitosterol- containing sterol mixture by crystallizing it from an organic solvent including one of 1 ,2-dichloroethylene, methyl ethyl ketone and ethyl acetate, or mixtures thereof, and also including the addition of water. The formed crystals are collected by filtration. The drawback with this method is that the filtration is difficult to perform, because the crystals are not structurally clear and therefore, the yield and the purity of the product is not acceptable.

In U.S. 4,420,427, sterols are separated from extracted and dried neutral substances using methanol or ethanol, or a mixture thereof, or a mixture of methyl ethyl ketone and water as a solvent. The neutral substances are mixed in the solvent, heated until dissolved, and the mixture is cooled to room temperature or below to precipitate the desired sterols, which are then filtered and washed with a suitable solvent. As washing solvents, acetone or a mixture of methanol and acetone are mentioned.

U.S. 2,843,610 discloses the use of a methanol-containing solvent medium composed of 9-100% by volume of methanol, 0-90% by volume of acetone or methyl ethyl ketone and 0-10% by volume of water, to crystallize sterols from sterol-containing materials such as deoderizer sludges, fatty acid distillation residues, tall oil still bottoms (i.e. , pitch) and soap stocks. It has been found that these starting materials cannot be used to crystallize sterols having a high purity. Summary of the Invention It has now surprisingly been shown that by using a mixture of methyl ethyl ketone (MEK), a lower (C_rC₆) alkanol and water as a solvent in any purification step in the production of desired sterols e.g. 4-desmethylsterols from neutral substances, a more purified product can be obtained. These purification steps may include crystallization, washing and/or recrystallization procedures.

Thus, in a first aspect of the present invention there is provided a method for the purification of 4-desmethylsterols from undesired neutral components comprising the treatment of neutral substances containing 4- desmethylsterols and undesired neutral components with a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water.

In a second aspect of the present invention there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising adding to neutral substances containing 4-desmethylsterols and undesired neutral components a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water to obtain a mixture, heating the mixture to dissolve the neutral substances, cooling the heated mixture to selectively crystallize 4-desmethylsterols , separating the obtained 4-desmethylsterol crystals, e.g. by centrifugation or filtration, and optionally subjecting the separated crystals to at least one of following additional treatment steps: washing and recrystallizing. In the third aspect of the present invention there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising providing a solution comprising neutral substances containing 4- desmethylsterols and undesired neutral components including undesired sterols dissolved in a solvent, crystallizing sterols from the solution, separating the obtained sterol crystals, e.g. by centrimgation or filtration, selectively washing the separated sterol crystals with a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water for removing undesired sterols, separating the desired 4-desmethylsterols, e.g. by filtration, and optionally recrystallizing the 4-desmethylsterols.

In the fourth aspect of the present invention there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising providing a solution comprising neutral substances containing 4- desmethylsterols and undesired neutral components including undesired sterols dissolved in a solvent, crystallizing sterols from the solution, separating the obtained sterol crystals, e.g. by centrifugation or filtration, optionally washing the obtained sterol crystals, selectively recrystallizing the sterols from a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water, to obtain desired 4- desmethylsterol crystals, and optionally washing the 4-desmethylsterol crystals.

Description of the Preferred Embodiments

The neutral substances, also known as unsaponifiables, can be obtained e.g. by extracting from soaps. Any soap of wood origin may be used. Neutral substances suitable as starting material in the method according to the invention may therefore be obtained from extracts of wood origin such as crude soap from the sulfate cellulose process or pitch soap obtained from tall oil. Any soap from the mentioned sources may be used as raw-material for producing the neutral substances used as starting material in the present invention. However, the method is particularly suitable for soaps obtained from the pulping of wood which contains hardwood.

For producing the neutral substances any known isolation method may be used, e.g. extraction or distillation. The extraction may preferably be performed using hydrocarbons alone or together with a ketone or an alcohol. The use of hydrocarbons alone is particularly suitable when performing the extraction at a temperature high enough to avoid emulsion formation.

In the solvent mixture, the weight ratio of methyl ethyl ketone : lower alkanol : water is preferably 50-75 : 15-40 : 5-15, and more preferably 60-70 : 20-35 : 5-10. Most preferably, the weight ratio can be about 70 : 20 : 10. The sum of the three components in the solvent mixture is preferably 100. The solvent components can be added one at a time or be mixed together before use in the purification.

The claimed treatment with the solvent mixture can comprise at least one of following treatment steps : crystallization, washing and recrystallization. In case the purification method of the present invention comprises two or more of these treatment steps, they can be carried out in optional order.

The obtained end product is practically free (e.g., contains less than 2 % , more preferably less than 1 %) of possible methyl and dimethyl sterols and triterpene alcohols (e.g. citrostadienol , cycloartenols , betulinol) which may be present in the wood raw material and also still in the neutral substances. The end product contains practically pure 4-desmethylsterols, including sitosterol and campesterol, and their corresponding saturated forms, sitostanol and campestanol.

By crystallizing and/or recrystallizing from and/or washing with a solvent mixture according to the present invention, structurally clearer and cleaner crystals are formed, and thereby the separation of the crystals e.g. by filtering or centrifuging is made easier, as compared to known methods.

Especially in a large scale process, this is of significant relevance.

By the expression "undesired neutral components" is meant all material included in the neutral substances other than the 4-desmethylsterols. The undesired neutral components include e.g. other sterols like 4-methyl- and 4,4'-dimethylsterols, fatty alcohols and terpene alcohols.

Preferred lower alkanols to be used in the invention are methanol and ethanol. Methanol is especially preferred, because of its higher specificity.

In the second aspect of the present invention, there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising

(a) adding to neutral substances containing 4-desmethylsterols and undesired neutral components a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water to obtain a mixture,

(b) heating the mixture to dissolve the neutral substances, (c) cooling the heated mixture to selectively crystallize 4- desmethylsterols,

(d) separating the obtained 4-desmethylsterol crystals, e.g. by centrifugation or filtration, and

(e) optionally subjecting the separated crystals to at least one of following additional treatment steps washing and recrystallizing.

Preferably the solvent mixture has the composition as defined above. In the crystallization step, the weight ratio of neutral substances : solvent mixture is preferably 1 : 0.5-15, and more preferably 1 : 1.5-8. The crystallization is performed preferably at about 0-40 °C, and more preferably at ambient temperature, i.e. about 20-25 °C. Typically, the yield is lower, but the purity is higher when higher crystallization temperatures are used, and vice versa. After separating the crystals in step (d) by any known method e.g. by filtration or centrifuging, the crystals may be washed with a suitable solvent e.g. with one or more solvents selected from the group consisting of hydrocarbons, more preferably short chain hydrocarbons containing 1-10 carbon atoms, preferably 5-8 carbon atoms, such as straight chain hydrocarbons (preferably pentane , hexane , heptane or octane) , branched chain hydrocarbons, hydrocarbons containing cyclic structures like cyclohexane or methylcyclohexane or hydrocarbon mixtures comprising any of these, lower alkanols such as methanol and/or ethanol and ketones such as methyl ethyl ketone. Water may also be added to the solvents. Preferably a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water may be used. More preferably the solvent mixture has the composition as defined above. Thus, a preferred solvent mixture comprises 50-75% by weight of MEK, 15-40% by weight of methanol and 5-15% by weight of water. The solvents can be added one at a time or be mixed together before use. A more preferred solvent mixture contains about 60-70% of MEK, about 20-35 % of MeOH and about 5-10% of water, and the most preferred about 70% of MEK, about 20% of MeOH and about 10% of water. Most preferably a mixture is used with the same composition as the one used in the crystallization step to avoid the need for many different solvent systems in the process as a whole. If the end product contains too much undesired material (with or without the use of washing steps as indicated above), it can be recrystallized. The recrystallization may be performed after a washing step or directly after the crystallization. The recrystallization may be performed by any known method, but preferably in the same manner as the first crystallization. In the recrystallization procedure the amount by weight of the solvent added to the sterol crystals is suitably somewhat higher than in the first crystallization, e.g. 5 to 15 times the amount of sterol crystals. Preferably a solvent mixture of the same composition as in the first crystallization may be used.

A third aspect of the present invention is to perform the crystallization in any prior known manner (e.g. as disclosed in U.S. 4,420,427, hereby incorporated by reference), and then perform the washing step using the solvent mixture according to the invention, i.e. a solvent comprising MEK, a lower alkanol and water.

Thus, in the third aspect of the present invention there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising

(a) providing a solution comprising neutral substances containing 4- desmethylsterols and undesired neutral components including undesired sterols dissolved in a solvent,

(b) crystallizing sterols from the solution,

(c) separating the obtained sterol crystals, e.g. by centrifugation or filtration, (d) selectively washing the separated sterol crystals with a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water for removing undesired sterols,

(e) separating the desired 4-desmethylsterols, e.g. by filtration, and

(f) optionally recrystallizing the 4-desmethylsterols. The solvent mixture used for washing preferably has the composition as defined above. Thus, a preferred solvent mixture comprises 50-75% by weight of MEK, 15-40% by weight of methanol and 5-15% by weight of water. The solvents can be added one at a time or be mixed together before use. A more preferred solvent mixture contains about 60-70% of MEK, about 20-35 % of MeOH and about 5-10% of water and the most preferred about 70% of MEK, about 20% of MeOH and about 10% of water. A recrystallization step, if needed, may also be included in the procedure after the washing step. In the recrystallization step, preferably a solvent mixture is used with the same composition as the one used in the washing step to avoid the need of many different solvent systems in the process.

A fourth aspect of the present invention is to recrystallize the desired sterols from the solvent mixture according to the present invention, i.e. a solvent comprising MEK, a lower alkanol and water, if the product still contains undesirable amounts of the undesired sterols.

Thus, in the fourth aspect of the present invention there is provided a method for selectively separating 4-desmethylsterols from undesired neutral components comprising (a) providing a solution comprising neutral substances containing 4- desmethylsterols and undesired neutral components including undesired sterols dissolved in a solvent,

(b) crystallizing sterols from the solution,

(c) separating the obtained sterol crystals, e.g. by centrifugation or filtration,

(d) optionally washing the obtained sterol crystals,

(e) selectively recrystallizing the sterols from a solvent mixture comprising methyl ethyl ketone, a lower alkanol and water, to obtain desired 4-desmethylsterol crystals, and (f) optionally washing the 4-desmethylsterol crystals.

The solvent mixture used in the recrystallization preferably has the composition as defined above. Thus, a preferred solvent mixture comprises 50- 75 % by weight of MEK, 15-40% by weight of methanol and 5-15 % by weight of water. The solvents can be added one at a time or be mixed together before use. A more preferred solvent mixture contains about 60-70% of MEK, about 20-35% of MeOH and about 5-10% of water and the most preferred about 70% of MEK, about 20% of MeOH and about 10% of water. The crystallization of the desired sterols from the neutral substances may be performed in any known manner (e.g. as disclosed in U.S. 4,279,827 or U.S. 4,420,427, each hereby incorporated by reference) prior to the recrystallization. A washing step may also be included in the procedure either before or after the recrystallization. In the washing step, preferably a solvent mixture is used with the same composition as the one used in the recrystallization step to avoid the need of many different solvent systems in the process.

The most preferred application of the invention is to perform the crystallization of the 4-desmethylsterols from the neutral substances using a mixture of MEK , MeOH and water , an especially preferred solvent comprising 50-75% by weight of MEK, 15-40% by weight of MeOH and 5-15 % by weight of water. A more preferred solvent mixture contains about 60-70% of MEK, about 20-35 % of MeOH and about 5-10% of water and the most preferred about 70% of MEK, about 20% of MeOH and about 10% of water. An even more preferred application of the present invention is to also perform the washing step with a similar solvent. Optionally, the process may also include a recrystallization step, preferably using a solvent of the same composition.

The following examples illustrate the invention in more detail. All the % given in the examples are calculated as weight- % .

Example 1 hexane/acetone/water extraction (according to U.S. 3,965,085, hereby incorporated by reference) was dissolved by refluxing in 40 g of a MEK/MeOH/water 70 : 20 : 10 blend and heating. The 4-desmethylsterols content (sitosterol + sitostanol + campesterol + campestanol) of the unsaponifiables was 24.5 % and the citrostadienol content was 2.9% . The solution was allowed to cool to 25 °C with gentle agitation. The crystals formed were filtered in a Buchner funnel and dried. The purity of the crystals was 82% .

Reference example 1 10 g of the similar unsaponifiables as used in example 1 was dissolved in 40 g of a solvent consisting of MEK/ water 90 : 10. The crystallization was done in a similar way as in example 1 and the crystals formed were filtered in a Buchner funnel. Filtration of the crystals was difficult. The purity of the dried product was 79% .

Example 2

The same crystallization procedure as in example 1 was performed. Thereafter the filtered crystals were washed with 50 g of a solvent with the same composition as the solvent used in the crystallization step. Filtrability of the crystals was very good and easy. When dried the yield of white crystals was 1.79 g (sterol content 97.8 % , citrostadienol content 0.1 %).

Reference example 2

The same crystallization procedure as in reference example 1 was performed. The filtered crystals were washed with 80 g of methanol added in small amounts at a time. The obtained crystals were brownish and the yield was 1.82 g, but sterol purity only 88% (citrostadienol content 0.4%). Example 3

The raw material of this example was the unsaponifiables separated from tall oil pitch (100% pine origin). The pitch was first saponified at 170°C with NaOH, and then the unsaponifiables were extracted in a closed system using a hydrocarbon solvent at the saponification temperature and prevailing vapor pressure of the mixture. After distilling off the extraction solvent, the fraction of unsaponifiables (35 % sterols) was dissolved by refluxing into a solvent mixture consisting of 65% MEK, 30% MeOH and 5% water (10 g unsaponifiables/80 g solvent). Crystallization of the sterols took place when the mixture was allowed to cool to 25 °C for 1.5 hours by gentle mixing with a magnetic stirrer. The crystals were filtered and washed with a solvent similar to the crystallization solvent. 2.8 g of 96.5 % pure dry sterols was obtained.

Example 4 The raw material was the unsaponifiables of 100 % Pinus radiata tall oil soap. The unsaponifiables were extracted from the soap using hexane/acetone solvent extraction (according to U.S. 3,965,085, incorporated by reference). 10 g of unsaponifiables was dissolved into 80 g solvent (60% MEK, 35 % MeOH, 5 % water) as in previous examples and the mixture was cooled to 20°C for 2 hours. After filtration, washing with 80 g of a solvent similar to the crystallization solvent added in small portions at a time, and drying, 2.85 g of 95 % pure sterols was obtained.

Example 5

The crystallization feed was the distillate fraction obtained from thin film evaporation of dried saponified pitch (according to U.S. 4,076,700, hereby incorporated by reference). The distillate contained 25 % sterols, and about 30% free fatty and resin acids. 1 part (100 g) was mixed with 3 parts of solvent mixture (70% MEK, 20% MeOH, 10% water) and heated until the mixture was a homogeneous solution. The solution was allowed to cool to 25 °C and was filtered and washed with a solvent similar to the crystallization solvent. 18.6 g of dry sterol blend (purity 95 %) was obtained.

Example 6

The starting material and the solvent were the same as in example 5 as well as the crystallization and washing procedure, but for the ratio of solids/ solvent, which was 1 : 1.5. The yield of 96% pure sterols was 18.1 g from 100 g of starting material.

Example 7

The crystallization was done in the same manner as in example 4. After filtration the crystals were washed with heptane. The sterol yield was 2.7 g of a 94.8 % sterol blend.

Example 8

The procedure was as in the previous example but washing was performed with methanol containing 15 % MEK. The sterol yield was 2.8 g and the purity was 93.9% .

Example 9 Pitch soap (Pinus taeda based, unsaponifiables 29.6% , sterols 41 % of the unsaponifiables) was extracted with a commercial hydrocarbon mixture (LIAV 110, Neste Oy) in a two-stage mixer-settler extraction equipment. The feed ratios of dry soap : water : solvent were 1 : 2.4 : 4.7, and the total feed rate was 730 kg/hour. The temperature in the settler vessels was 182°C and 184°C, and the pressures were 20.7 and 20 bars, respectively. The sterol content of the unsaponifiables included in the hydrocarbon phase was 35 % .

The hydrocarbon phase was cooled to 20 °C and the precipitated sterols were separated by filtration (purity 80%). The crystals were recrystallized from a solvent mixture consisting of 70% MEK, 20% MeOH and 10% water, the amount of crude sterols : solvent being 1 : 9 by weight. The yield of dry crystals was 75% , and the purity was 96% .

Reference example 9

The same procedure as in example 9 was performed, but for the recrystallizing step, which was realized using a solvent mixture consisting of MEK and water (90 : 10). The filtration was very difficult to accomplish. The purity of the sterol product was 85 % .

Example 10

The same procedure was performed as in example 9, however instead of the recrystallizing step, the crystals were washed by slurry ing them in a solvent mixture of the same composition as used in the crystallization step.

The amount of crystals to solvent was 1 : 9 by weight. After filtration, the purity of the sterols was 87% .

Claims

What is claimed is:

1. A method for purifying 4-desmethylsterols from neutral substances containing the 4-desmethylsterols and undesired neutral components, the method comprising the step of treating the neutral substances with a solvent mixture comprising methyl ethyl ketone, a C_rC₆ alkanol and water.

2. The method of claim 1 , wherein the methyl ethyl ketone, the C_rC₆ alkanol and the water are present in the solvent mixture in a weight ratio of 50- 75 : 15-40 : 5-15.

3. The method of claim 2, wherein the weight ratio is 60-70 : 20-35 : 5- 10.

4. The method of claim 2, wherein the weight ratio is about 70 : 20 : 10.

5. The method of claim 1 , wherein the C_rC₆ alkanol is methanol.

6. The method of claim 1 , wherein said treating step comprises at least one of the following steps: crystallization, washing and recrystallization.

7. A method for separating 4-desmethylsterols from neutral substances containing the 4-desmethylsterols and undesired neutral components, the method comprising

(a) providing a mixture comprising the neutral substances and a solvent mixture comprising methyl ethyl ketone, a C_rC₆ alkanol and water, and heating the mixture to dissolve the neutral substances in the solvent mixture,

(b) cooling the heated mixture to crystallize the 4-desmethylsterols; (c) separating the 4-desmethylsterol crystals from the mixture; and

(d) optionally subjecting the separated crystals to at least one of following additional treatment steps: washing and recrystallizing.

8. The method of claim 7, wherein the methyl ethyl ketone, the C_rC₆ alkanol and the water are present in the solvent mixture in a weight ratio of 50-

75 : 15-40 : 5-15.

9. The method of claim 8, wherein the weight ratio is 60-70 : 20-35 : 5- 10.

10. The method of claim 8, wherein the weight ratio is about 70 : 20 : 10.

11. The method of claim 7, wherein the C_rC₆ alkanol is methanol.

12. The method of claim 7, wherein the neutral substances and the solvent mixture are present in the mixture in a weight ratio of 1 : 0.5-15.

13. The method of claim 12, wherein the weight ratio is 1 : 1.5-8.

14. The method of claim 7, wherein, after step (c), the separated crystals are subjected to one or more washing steps.

15. The method of claim 14, wherein said one or more washing steps are carried out by using a washing solvent selected from the group consisting of a hydrocarbon, a C_rC₆ alkanol and a ketone, or mixtures thereof, optionally together with water.

16. The method of claim 15, wherein the hydrocarbon is selected from the group consisting of pentane, hexane, heptane, octane, cyclohexane and methylcyclohexane, and mixtures thereof.

17. The method of claim 15 , wherein the C _rC₆ alkanol is selected from the group consisting of methanol and ethanol.

18. The method of claim 15, wherein the ketone comprises methyl ethyl ketone.

19. The method of claim 14, wherein said one or more washing steps are performed with a washing solvent comprising the solvent mixture of step (a).

20. The method of claim 7, wherein, after step (c), the separated crystals are subjected to one or more recrystallizing steps.

21. The method of claim 20, wherein said one or more recrystallizing steps are performed with a recrystallization solvent comprising the solvent mixture of step (a).

22. A method for separating 4-desmethylsterols from neutral substances containing the 4-desmethylsterols and undesired neutral components, wherein the undesired neutral components include undesired sterols, the method comprising

(a) providing a solution comprising the neutral substances dissolved in a solvent;

(b) crystallizing sterols from the solution, wherein the sterols comprise the 4-desmethylsterols and the undesired sterols; (c) separating the sterol crystals obtained in step (b) from the solution;

(d) washing the separated sterol crystals with a solvent mixture comprising methyl ethyl ketone, a C_rC₆ alkanol and water; (e) separating the 4-desmethylsterols from the undesired sterols ; and

(f) optionally recrystallizing the 4-desmethylsterols.

23. The method of claim 22, wherein the methyl ethyl ketone, the C_rC₆ alkanol and the water are present in the solvent mixture in a weight ratio of 50- 75 : 15-40 : 5-15.

24. The method of claim 23, wherein the weight ratio is 60-70 : 20-35 : 5- 10.

25. The method of claim 23 , wherein the weight ratio is about 70 : 20 : 10.

26. The method of claim 22, wherein the C_rC₆ alkanol is methanol.

27. A method for separating 4-desmethylsterols from neutral substances containing the 4-desmethylsterols and undesired neutral components, wherein the undesired neutral components include undesired sterols, the method comprising

(a) providing a solution comprising the neutral substances dissolved in a solvent; (b) crystallizing sterols from the solution, wherein the sterols comprise the 4-desmethylsterols and the undesired sterols;

(c) separating the sterol crystals obtained in step (b) from the solution; (d) optionally washing the separated sterol crystals;

(e) combining the sterol crystals with a solvent mixture comprising methyl ethyl ketone, a C_rC₆ alkanol and water, and recrystallizing 4- desmethylsterol crystals from the solvent mixture; and (f) optionally washing the 4-desmethylsterol crystals.

28. The method of claim 27, wherein the methyl ethyl ketone, the C_rC₆ alkanol and the water are present in the solvent mixture in a weight ratio of 50- 75 : 15-40 : 5-15.

29. The method of claim 28, wherein the weight ratio is 60-70 : 20-35 : 5- 10.

30. The method of claim 28, wherein the weight ratio is about 70 : 20 : 10.

31. The method of claim 27, wherein the C_rC₆ alkanol is methanol.

32. The method of claim 1 , wherein the neutral substances are derived from wood material.

33. The method of claim 7 , wherein the neutral substances are derived from wood material.

34. The method of claim 22, wherein the neutral substances are derived from wood material.

35. The method of claim 27, wherein the neutral substances are derived from wood material.

36. The method of claim 1 , wherein the 4-desmethylsterols comprise one or more sterols selected from the group consisting of sitosterol, campesterol, sitostanol and campestanol.

37. The method of claim 7, wherein the 4-desmethylsterols comprise one or more sterols selected from the group consisting of sitosterol, campesterol, sitostanol and campestanol.

38. The method of claim 22, wherein the 4-desmethylsterols comprise one or more sterols selected from the group consisting of sitosterol, campesterol, sitostanol and campestanol.

39. The method of claim 27, wherein the 4-desmethylsterols comprise one or more sterols selected from the group consisting of sitosterol, campesterol, sitostanol and campestanol.