FI129376B

FI129376B - Purification of betulinol

Info

Publication number: FI129376B
Application number: FI20205819A
Authority: FI
Inventors: Suvi Pietarinen; Markus Ronkainen
Original assignee: Upm Kymmene Corp
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2022-01-14
Also published as: FI20205819A1

Abstract

Esitetään menetelmä betulinolin puhdistamiseksi. Menetelmä käsittää, että järjestetään betulinolia,joka on kiinteässä muodossa ja jonka puhtaus on 70 – 95 paino-%,ja altistetaan järjestetty betulinoli puhdistuskäsittelylle, jossa puhdistuskäsittely käsittää seuraavaa: (ia) suspendoidaan betulinoli, joka on kiinteässä muodossa, veteen siten, että läsnä on alkoholia ja ainakin yksi alkalinen komponentti, suspension muodostamiseksi; (iia) lämmitetään suspensiota lämpötilassa, joka on 140 – 180 oC, paineessa, joka on 5 – 25 bar, 1 – 5 tunnin ajan; ja (iiia) otetaan talteen betulinoli kohdasta (iia) saadusta suspensiosta kiinteässä muodossa. Lisäksi esitetään betulinoli ja sen käyttö.

Description

PURIFICATION OF BETULINOL

TECHNICAL FIELD The present disclosure relates to a method for purifying betulinol. The present disclosure further relates to betulinol obtainable by the method as disclosed in the current specification, to betulinol, and to the use thereof.

BACKGROUND Betulinol, also called betulin or betulinic alcohol, is an abundant, naturally occurring triter- pene. Chemically, betulinol is a triterpenoid of lu- pane structure. It has a pentacyclic ring structure, and hydroxyl groups in positions C3 and C28.

Betulinol is commonly isolated from the bark of birch trees. Betulinol can be produced by extrac- tion of birch outer bark by using a solvent, such as 2-propanol, ethanol, ethyl acetate, heptane, cyclopen- tyl methyl ether, or their mixtures. Betulinol may then be isolated from the solvent by a suitable pro- cess such as evaporative crystallization, total evapo- ration of solvents, or by precipitation by addition of a solvent, such as water. The resulting betulinol fraction is usually slightly yellow to brownish in — color depending as a result of including e.g. suberin O (yellow to beige in color when in ester form) and/or O lignin (brown) as impurities. Thus, the inventors have = recognized a need for a method to produce purified N 30 betulinol.

E Document CN 102321143A discloses a method for o preparing high-purity betulin by extracting crushed % silver birch bark by ethanol water solution, N collecting extract solution, concentrating, cooling, N 35 separating out crude product, and filtering to obtain betulin crude product.

SUMMARY A method for purifying betulinol is disclosed. The method may comprise providing betulinol being in solid form and having a purity of 70 - 95 weight-%3, and subjecting the provided betulinol to a purification treatment, wherein the purification treatment comprises: (ia) suspending betulinol, being in solid form, in water in the presence of an alcohol and at least one alkaline component to form a suspension; (iia) heating the suspension at a temperature of 140 — 180 *C under a pressure of 5 - 25 bar for 1 - 5 hours; and (iiia) recovering betulinol from the suspension received from (iia) in solid form.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawing, which 1s included to provide a further understanding of the embodiments and constitute a part of this specification, illustrates various embodiments. In the drawing: Fig. 1 discloses one embodiment of the method disclosed in the current specification.

= DETAILED DESCRIPTION N A method for purifying betulinol is S disclosed. The method may comprise providing betulinol S 30 being in solid form and having a purity of 70 - 95 Ek weight-%, and subjecting the provided betulinol to a + purification treatment, wherein the purification 2 treatment comprises: S (ia) suspending betulinol, being in solid S 35 form, in water in the presence of an alcohol and at least one alkaline component to form a suspension;

(iia) heating the suspension at a temperature of 140 — 180 °C under a pressure of 5 —- 25 bar for 1 - hours; and (iiia) recovering betulinol from the 5 suspension received from (iia) in solid form.

Further is disclosed betulinol obtainable by the method as disclosed in the current specification, wherein the betulinol has a purity of 95.5 — 99.5 weight-3. Further is disclosed betulinol having a purity of 95.5 — 99.5 weight-%3. In one embodiment, the betulinol has a purity of 96 - 99 weight-%, or 97 — 98 weight-3. The inventors thus surprisingly found out that by the method as disclosed in the current specification it may be possible to produce betulinol that has a purity as high as 95.5 - 99.5 weight, or 96 - 99 weight-%, or 97 — 98 weight-%.

The weight percentage (weight-%) purity of the betulinol may be determined by gas chromatography (GC), which calculates purity of a substance, such as betulinol, based on the amount of the main component peak area compared to the total peak areas.

Further, is disclosed the use of betulinol as disclosed in the current specification for the production of coatings, barriers, polymers such as polyesters and/or polyethers, cosmetics, pharmaceutical substances, pigments, hydrophobic surfaces and/or hydrophobic coatings.

N By the expression ”betulinol” should be un- N derstood in this specification, unless otherwise stat- S 30 ed, as referring to the provided betulinol or to betu- Q linol having already been subjected to steps (ia) - =E (iiia) and/or steps (ib) - (iiib). Therefore the term * ”betulinol” may include the betulinol that has not yet 2 been subjected to the purification treatment and the S 35 betulinol]l that has been subjected to steps (ia) - S (iiia) and/or steps (ib) — (iiib) one or more times.

The “purity” of betulinol thus changes during the method, when the purification treatment proceeds.

In one embodiment, the betulinol to be sub- jected to the purification treatment has a purity of 80 — 90 weight-%. I.e. the provided betulinol may have a purity of 70 — 95 weight-%, or 80 - 90 weight- By the expression "solid form” should be understood in this specification, unless otherwise stated, as referring to the betulinol having a dry matter content of 20 - 100 weight-%. In one embodiment, the betulinol in solid form has a dry matter content of 25 - 60 weight-%. The dry matter content may be determined by weighting a sample of “wet” solid form betulinol and then drying the sample in an oven at 105 °C until its weight does not essentially change. Then the weight of the oven-dried betulinol is divided by the weight of the “wet” solid form betulinol (i.e. the betulinol before being oven- dried).

The method for purifying betulinol comprises providing betulinol, so-called crude betulinol to be purified. Providing the betulinol may comprise extracting betulinol from birch bark, such as birch outer bark, by the use of at least one solvent. Examples of solvents to be used for extracting betulinol from birch bark are ethanol, 2-propanol, N ethyl acetate, heptane, cyclopentyl methyl ether, and N any mixture or combination thereof. Betulinol may be S 30 separated from the solvent by a suitable process such Q as evaporative crystallization, total evaporation of =E solvents or precipitation by addition of solvent such + as water. 2 However, so-called crude betulinol extracted S 35 from birch bark may not be sufficiently pure for use S in further applications. Crude betulinol may contain e.g. lignin, suberin and/or lupeol affecting the purity of the crude betulinol in an adverse manner in further applications. The inventors surprisingly found that betulinol with high purity may be formed by the method as disclosed in the current specification.

5 The betulinol produced e.g. by extracting from birch bark may have been subjected also to a pre- purification treatment before being subjected to the purification treatment as disclosed in the current specification. The provided betulinol being used directly after having been extracted from birch bark or after having been subjected to pre-purification treatment may have a purity of 70 - 95 weight-%.

In one embodiment, providing the betulinol comprises purchasing the betulinol.

In step (ia) betulinol, being in solid form, is suspended in water in the presence of an alcohol and at least one alkaline component to form a suspension.

In one embodiment, the at least one alkaline component comprises or consists of sodium hydroxide, sodium sulphite, or both sodium hydroxide and sodium sulphite. Thus, either sodium hydroxide or sodium sulphite or the combination of both sodium hydroxide and sodium sulphite may be used as the at least one alkaline component. The sodium sulphite may react with the impurities making them more soluble.

The at least one alkaline component may N comprise one alkaline component, two alkaline N components, three alkaline components, etc. In one S 30 embodiment, the at least one alkaline component Q comprises or consists of one alkaline component. In Ek one embodiment, the at least one alkaline component * comprises or consists of two alkaline components. 2 The alcohol may be methanol, ethanol, 1- S 35 propanol, 2-propanol, butanol, or any mixture or S combination thereof. In one embodiment, the suspension comprises alcohol in an amount of 40 - 90 volume-%, or

50 — 80 volume-%, or 60 — 70 volume-%, based on the total volume of the suspension. The alcohol to be present in step (ia) may also come as a part of the betulinol extracted from birch bark. I.e. if the solvent used to extract betulinol from birch bark is an alcohol, such as ethanol and/or 2-propanol, then it may not be necessary to add any further alcohol in step (ia) but the alcohol that is needed to be present, is provided to step (ia) with the betulinol. If needed, the alcohol, the presence of which is needed in step (ia) may be added separately.

The alcohol used in step (ia) may be the same alcohol as used when extracting betulinol from the birch bark. Alternatively, the alcohol used in step (ia) may be a different alcohol than the one used when extracting betulinol from the birch bark.

In one embodiment, the purification treatment comprises before and/or after (ia) — (iiia): (ib) suspending betulinol, being in solid form, in water in the presence of at least one alkaline component to form a suspension; (iib) heating the suspension at a temperature of 140 — 180 °C under a pressure of 3 - 15 bar for 1 - 5 hours; and (1iiib) recovering betulinol from the suspension received from (iib) in solid form. N In one embodiment, the purification treatment N comprises before (ia) - (iiia): S 30 (ib) suspending betulinol, being in solid Q form, in water in the presence of at least one Ek alkaline component to form a suspension; * (iib) heating the suspension at a temperature 2 of 140 — 180 °C under a pressure of 3 - 15 bar for 1 - S 35 5 hours; and S (iiib) recovering betulinol from the suspension received from (iib) in solid form.

In one embodiment, the purification treatment comprises after (ia) - (iiia): (ib) suspending betulinol, being in solid form, in water in the presence of at least one alkaline component to form a suspension; (iib) heating the suspension at a temperature of 140 — 180 °C under a pressure of 3 - 15 bar for 1 - 5 hours; and (iiib) recovering betulinol from the suspension received from (iib) in solid form.

In one embodiment, steps (ia) - (iiia) are repeated at least once.

In one embodiment, steps (ia) — (iiia) are repeated once after steps (ia) - (iiia). In one embodiment, steps (ia) — (iiia) are repeated once after steps (ib) — (iiib). I.e. the purification treatment may comprise e.g.: - subjecting the betulinol to steps (ia) - (iiia); or - first subjecting betulinol to steps (ia) - (iiia) and then to steps (ib) - (iiib); or - first subjecting betulinol to steps (ia) - (iiia) and then again to steps (ia) - (iiia); or - first subjecting betulinol to steps (ia) - (iiia) and then again to steps (ia) - (iiia) and then further to steps (ib) — (iiib); or - first subjecting betulinol to steps (ia) - N (iiia) and then to steps (ib) -—- (iiib) and then N further to steps (ia) - (iiia); S 30 - first subjecting betulinol to steps (ib) - S (iiib) and then to steps (ia) — (iiia); or =E - first subjecting betulinol to steps (ib) - * (iiib) and then to steps (ia) - (iiia) and then 2 further to steps (ia) — (iiia); or S 35 - first subjecting betulinol to steps (ib) - S (iiib) and then to steps (ia) - (iiia) and then further to steps (ib) — (iiib).

By subjecting betulinol to the purification treatment comprising (ia) - (iiia), one may be able to remove or decrease the amount of betulinic acid and other triterpenes, as well as non-water soluble lignin and/or non-water soluble suberin from Dbetulinol to increase its purity.

By subjecting betulinol to the purification treatment comprising (ib) - (iiib), one may be able to remove or decrease the amount of water soluble lignin and/or water soluble suberin as well as other water soluble components from betulinol to increase its purity.

Suspending the betulinol may be conducted at a temperature of 20 - 80 °C, or 25 - 60 *C, or in room temperature.

I.e. first a suspension may be formed, which, after the different components have been mixed and suspended, is then subjected to the heating in a following step.

Step (ia) comprises suspending betulinol, being in solid form, in water in the presence of an alcohol and at least one alkaline component.

I.e. in step (ia) is formed a suspension comprising or consisting of betulinol, water, an alcohol, and at least one alkaline component.

Step (ib) comprises suspending betulinol, being in solid form, in water in the presence of at least one alkaline component.

I.e. in step (ib) is N formed a suspension comprising or consisting of N betulinol, water, and at least one alkaline component.

S 30 I.e. no additional alcohol may be present in the Q suspension formed in step (ib). However, some amount =E of alcohol may be provided into the suspension in step * (ib) as a part of the betulinol.

If the betulinol, 2 prior to step (ib), has been subjected to steps (ia) - S 35 (iiia), the Dbetulinol may comprise some residual S amount of alcohol that may then become a part in the suspension of (ib). The amount of alcohol that may remain in betulinol from step (iiia) may be 0.1 - 10 weight-3, or 1 -— 5 weight-%. In one embodiment, the heating is conducted at a temperature of 150 — 170 °C, or 145 - 155 °C, or 165 -— 175 °C.

In one embodiment, step (iia) of heating the suspension is carried out at a temperature of 145 - 155 °C.

In one embodiment, step (iib) of heating the suspension is carried out at a temperature of 165 - 175 °C.

In one embodiment, step (iia) of heating the suspension is carried out at a temperature of 145 - 155 °C and step (iib) of heating the suspension is carried out at a temperature of 165 — 175 °C.

In one embodiment, the heating is conducted in (iia) under a pressure of 6 - 15 bar, or 8 - 10 bar.

In one embodiment, the heating is conducted in (iib) under a pressure of 3 - 10 bar.

The heating of the suspension may be continued for 1 - 5 hours, e.g. for 2 - 4 hours, or for about 3 hours.

In one embodiment, the heating of the suspension is continued for 1 - 5 hours, or for 2 — 4 hours, or for about 3 hours.

In one embodiment, recovering of betulinol from the suspension is carried out by filtration, centrifugation, and/or by using a decanter.

The recovering of betulinol may be conducted while keeping the temperature of the suspension at 0 -— 120 °C , or N 10 - 105 °C, or at 20 — 50 °C, or at 20 — 40 °C.

The N recovering of betulinol] in step (iiia) may be S 30 conducted while keeping the temperature of the Q suspension at 0 - 50 °C, or 20 - 40 °C, or at about =E room temperature.

The recovering of betulinol in step * (iiib) may be conducted while keeping the temperature 2 of the suspension at 80 — 120 °C, or 95 -— 105 °C.

S 35 In one embodiment, the suspension is cooled S to a temperature of 0 - 50 °C, or 18 — 50 °C, or 25 - 50 °C before step (iiia) and/or (iiib). At these temperatures Dbetulinol is not soluble and may be recovered in solid form from the suspension. In one embodiment, the method comprises washing the recovered betulinol from (iiia) and/or (iiib) with water, alcohol, alkaline water, or any mixture or combination thereof. By washing the recovered betulinol one may be able to remove or decrease the amount of e.g. lupeol present. E.g. heptane may be used to wash lupeol from the recovered betulinol. In one embodiment, the method comprises crystallization of the recovered betulinol. In one embodiment, the recovered betulinol has a purity of 95.5 - 99.5 weight-%, or 96 - 99 weight-%, or 97 — 98 weight-%. The betulinol as recovered by the method as disclosed in the current specification may have a purity of 95.5 -— 99.5 weight- 2, or 96 - 99 weight-%, or 97 - 98 weight-%. I.e. the recovered betulinol may be considered as hbetulinol that has been subjected to the purification treatment. The method as disclosed in the current specification has the added utility of enabling to produce betulinol with a high purity of at least 95.5 weight-%, or at least 96 weight-%, or at least 97 weight-%3, or at least 98 weight-%, or at least 99 weight-3, or at least 99.5 weight-%. I.e. the method as disclosed in the current specification has the N added utility of efficiently removing or decreasing N the amount of e.g. suberin, lignin and/or lupeol from S 30 betulinol.

N z EXAMPLES * Reference will now be made in detail to 2 various embodiments. S 35 The description below discloses some S embodiments in such a detail that a person skilled in the art is able to utilize the embodiments based on the disclosure. Not all steps or features of the embodiments are discussed in detail, as many of the steps or features will be obvious for the person skilled in the art based on this specification. The enclosed Fig. 1 discloses an example of an embodiment of the method for purifying betulinol. Fig. 1 discloses an embodiment comprising the general steps of the method. The method comprises providing betulinol, wherein the betulinol is in solid form and has a purity of 70 — 95 weight-3. The provided betu- linol is then subjected to a purification treatment. The purification treatment as disclosed in Fig. 1 com- prises: (ia) suspending betulinol, being in solid form, in water in the presence of an alcohol and at least one alkaline component to form a suspension; (iia) heating the suspension at a temperature of 140 — 180 *C under a pressure of 5 - 25 bar for 1 - 5 hours; and (iiia) recovering betulinol from the suspension received from (iia) in solid form. In addition as disclosed in Fig. 1, the purification treatment may comprise either before or after, or both before and after, the following: (ib) suspending betulinol, being in solid form, in water in the presence of at least one alkaline component to form a suspension; N (iib) heating the suspension at a temperature N of 140 — 180 °C under a pressure of 3 - 15 bar for 1 - S 30 5 hours; Q (1iiib) recovering betulinol from the =E suspension received from (iib) in solid form. > Further, as disclosed in Fig. 1, the method 2 may also comprise repeating steps (ia) - (iiia) and/or S 35 (ib) — (iiib) at least one time.

N Example 1 - Purifying betulinol

200 g of betulinol in solid form (purity 89 % GC (gas chromatography)) was suspended in 1.5-2 1 of 70 % (v/v) ethanol/H;O mixture with 40 g of NaOH and 20 g NaS03 mixed in 2 1 of the solvent. NaOH and Na,S03 were premixed with water before the 70 % solu- tion was prepared by addition of ethanol. Ethanol ad- dition forced some of the salt out of the solution. This suspension was mixed and used as such in the next step.

The suspension was heated at 150 °C for 3 h before cooled to room temperature, filtered, and washed with 2 1 of 70 % (v/v) ethanol.

The above procedure was repeated once, and the solids were “then washed with 2 1 of warm HO to remove excess ethanol.

The solids were suspended in 1.5-2 1 of HO with 40 g NaOH and 20 g Na»S03 mixed therein and then stirred at 170 °C for 3 h before cooled to room tem- perature and washed with alkaline water 4 1 (0.5 M, NaOH) to produce white betulinol (purity 96.5 weight- 3, 1.8 weight-% lupeol). Lupeol was removed by heptane wash.

The above described steps are summarized in the below table.

L KT Na2S03 H2O 96->97 0-1 170 3 Example 2 — Purifying betulinol 150 g of betulinol in solid form (purity 88.7 weight-% GC (gas chromatography)) was suspended in a water mixture with 60 g of NaOH and 30 g of Na,S0O3 dissolved in the solvent.

The suspension was heated at 170 °C for 3 h before cooled to room temperature.

The suspension was diluted in alkaline water (0.5 M, NaOH, 1.5 1). The suspension was vacuum fil- tered and the solids were washed with 2 | of alkaline water (0.5 M NaOH) and then dried in an oven at 105 °C overnight.

The yield was 146 g of white betulinol (pu- rity 92.6 weight-%, GC). The solids were then suspended in 1.5 1 of alkaline 70 % (v/v) EtOH with 30 g of NaOH and 15 g of Na,503 mixed therein.

The solution was prepared by first dissolving NaOH and Na»S03 into water followed by addition of EtOH.

The suspension was then heated and mixed at 150 °C for 2 h before cooled to about 50 °C.

The solids were then vacuum filtered and washed with 1 1 of 70 % (v/v) EtOH.

A white to greyish colored solid was formed (purity 97.3 weight-%, GC). This procedure was repeated once with the difference that the suspen- sion was cooled to 22 °C instead of 50 °C before being N vacuum filtered.

N The solids were then washed with 2 1 of alka- S line water (0.5 M NaOH) and then dried in an oven at a 105 °C overnight. 87 g of white betulinol with darker = 30 spots was received (purity 98.1 weight-%, GC). The > solids were then boiled in 1 1 of alkaline 30 3 (v/v) © ethanol (0.25 M NaOH), cooled to room temperature and S filtered to remove the darker spots/residual solvents.

The solids were then washed with 1 1 of water and dried in oven at 105 °C overnight and then boiled in 1.5 1 of n-heptane, then cooled to room temperature and filtered to remove lupeol. The solids were then dried in an oven at 90 °C overnight. 79 g of betulinol (purity 99.1 weight-%, 0.6 weight-% lupeol) was ob- tained. The above described steps are summarized in the below table. Purifi Pressur cation Purity |Purity e treatm|Alkaline change increase (bar) ent componen (weight-|(weight-|Temp Time steps |It(s) Solvent is) %) (oC) (h)

TE LL Na2S03 H2O 89->93 1-4 170 3 2 70 % NaOH, ETOH/H; Na2S03 O 93->98 3-5 150 2 3 70 % NaOH, ETOH/H; Na2S03 O 98->98 0-1 150 2 Examples 3 - Purifying betulinol 150 g of betulinol in solid form (purity 88.7 weight-%3 GC (gas chromatography)) was suspended in a water mixture with 60 g of NaOH dissolved in the N solvent. The suspension was heated at 170 °C for 3 h > 15 before cooled to room temperature. <Q The suspension was diluted in alkaline water N (0.5 M, NaOH, 1.5 1). The suspension was vacuum fil- E tered and the solids were washed with 2 | of alkaline o water (0.5 M NaOH) and then dried in an oven at 105 °C % 20 overnight. The yield was 146 g of white betulinol (pu- S rity 90.8 weight-%, GC). NN The solids were then suspended in 1.5 1 of alkaline 70 3 (v/v) EtOH with 30 g of NaOH mixed therein.

The solution was prepared by first dissolving NaOH into water followed by addition of EtOH.

The sus- pension was then heated and mixed at 150 °C for 2 h before cooled to about 50 °C.

The solids were then vacuum filtered and washed with 1 1 of 70 % (v/v) EtOH.

A white to greyish colored solid was formed (pu- rity 97.7 weight-%, GC). This procedure was repeated once with the difference that the suspension was cooled to 22 °C instead of 50 °C before being vacuum filtered.

The solids were then washed with 2 | of alka- line water (0.5 M NaOH) and then dried in an oven at 105 °C overnight.

As the yield was received 98 g of white betulinol (purity 98.3 weight-%, GC). The solids were then boiled in 1 1 of alkaline 30 % (v/v) ethanol (0.25 M NaOH), cooled to room temperature and filtered to remove residual solvents.

The solids were then washed with 1 1 of water and dried in oven at 105 °C overnight and then boiled in 1.5 1 of n-heptane, then cooled to room temperature and filtered to remove lupeol.

The solids were then dried in an oven at 90 °C overnight. 89 g of betulinol (purity 98.9 weight-%, 0.6 weight-% lupeol) was ob- tained.

The above described steps are summarized in the below table. — Purific Purity Pressur O ation Purity |increas e O treatme Alkaline change |e (bar) = nt componen (weight|(weight [Temp Time - steps t(s) Solventi-%) -%) (eC) (h) E | 1 eon o — [s9-01] 1-2 | 270 | 8 | 3 > ETOH/H, oO S NaOH o 91->98 | 3-7 150 2 - | 5 leon fos esos] 0-1 | 150 | & | 2

ETOH/H:

O It is obvious to a person skilled in the art that with the advancement of technology, the basic idea may be implemented in various ways. The embodiments are thus not limited to the examples described above; instead they may vary within the scope of the claims. The embodiments described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodiment. A method, betulinol, or a use, disclosed herein, may comprise at least one of the embodiments described hereinbefore. It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages. It will further be understood that reference to 'an' item refers to one or more of those items. The term “comprising” is used in this specification to mean including the feature(s) or act (s) followed thereafter, without excluding the presence of one or more additional features or acts. — 25

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LO O N O N

Claims

1. A method for purifying betulinol, wherein the method comprises providing betulinol being in solid form and having a purity of 70 - 95 weight-%, and subjecting the provided betulinol to a purification treatment, wherein the purification treatment comprises: (ia) suspending betulinol, being in solid form, in water in the presence of an alcohol and at least one alkaline component to form a suspension; (iia) heating the suspension at a temperature of 140 — 180 *C under a pressure of 5 - 25 bar for 1 - 5 hours; and (iiia) recovering betulinol from the suspension received from (iia) in solid form.

2. The method of claim 1, wherein providing betulinol comprises extracting betulinol from birch bark by the use of at least one solvent.

3. The method of any one of the preceding claims, wherein the at least alkaline component comprises or consists of sodium hydroxide, sodium sulphite, or both sodium hydroxide and sodium sulphite.

4. The method of any one of the preceding claims, wherein the purification treatment comprises, before and/or after (ia) — (iiia): — (ib) suspending betulinol, being in solid N form, in water in the presence of at least one > alkaline component to form a suspension; = 30 (iib) heating the suspension at a temperature N of 140 — 180 °C under a pressure of 3 - 15 bar for 1 - E 5 hours; and o (1iiib) recovering betulinol from the % suspension received from (iib) in solid form.

N 35 5. The method of any one of the preceding N claims, wherein (ia) - (iiia) are repeated at least once.

6. The method of any one of the preceding claims, wherein the heating is conducted at a temperature of 150 - 170 °C, or 145 — 155 °C, or 165 - 175 °C.

7. The method of any one of the preceding claims, wherein the heating is conducted in (iia) under a pressure of 6 - 15 bar, or 8 - 10 bar.

8. The method of any one of the preceding claims, wherein the heating is conducted in (iib) under a pressure of 3 - 10 bar.

9. The method of any one of the preceding claims, wherein recovering of betulinol in solid form from the suspension is carried out by filtration, centrifugation, and/or by using a decanter.

10. The method of any one of the preceding claims, wherein recovering of betulinol is conducted in (iiib) while keeping the temperature of the suspension at 80 — 120 °C , or 95 - 105 °C.

11. The method of any one of the preceding claims, wherein recovering of betulinol is conducted in (iiia) while keeping the temperature of the suspension at 0 - 50 °C, or 20 - 40 °C, or at room temperature.

12. The method of any one of the preceding claims, wherein the method comprises washing the recovered betulinol from (iiia) or (iiib) with water, alcohol, alkaline water, or a mixture or combination N thereof.

N

13. The method of any one of the preceding S 30 claims, wherein the method comprises crystallization Q of the recovered betulinol.

Ek

14. The method of any one of the preceding * claims, wherein the recovered betulinol has a purity 2 of 95.5 -— 99.5 weight-%, or 96 - 99 weight-%, or 97 - S 35 98 weight-%.

N