AND OR ITS ESTERS

Wolfberry - commercially called Goji berry - is the common name for the fruit of two very closely related species: Lycium barbarum and Lyci m chinense, two species of boxthorn. Since Lycium bar- barum L. is the only species approved for use in China the present invention is especially directed to the fruits of Lycium barbarum L., the "Goji berries".

Goji berries have the highest content of zeaxanthin of all known food sources. The berries have been cultivated in China for decades and are used as food and in Traditional Chinese Medicine. The content of carotenoids in Goji berries can range from 600 to 900 ppm, whereby the carotenoids are mainly located in the skin of such Goji berries. Thus, the content of carotenoids in the skin of such berries is even higher, and in the range of e.g. 2300 to 3300 ppm. The main carotenoid is zeaxanthin in its natural ester form.

Commercially available are Goji berries, mostly in dried form, as well as Goji juice obtained from Goji berries.

Goji juice can in general be obtained by the following procedure: Goji berries are harvested, washed with pure water, crushed, homogeneousness, centrifuged and filtered. Thereby the skin and most of the Goji berry seeds and pulp is separated from Goji juice. The separated material was until now regarded as waste and fed to animals.

The inventors of the present invention now discovered that this waste of the Goji juice production which consists mainly of the skin of the Goji berries as well as small amounts of pulp and seeds is a valuable starting material for the manufacture of extracts which are highly enriched in zeaxanthin and/or its esters. Despite the fact that the waste of the Goji juice production also contains pulp and seed, so it is more a "skin of the Goji Berries", it is called "skin of Goji berries" in the following. The inventors of the present invention have now succeeded in developing a process for the extraction of the dried skin of Goji berries with a lipophilic solvent and obtained in high yield an oily oleo- resin, rich in zeaxanthin (zeaxanthin content of ca. 4 to 15 weight-%). Further purification of the oleoresin could be achieved by crystallization from a solvent such as hot ethanol yielding an orange- red powder with a zeaxanthin dipalmitate content in the range of 36 to 72 weight-% corresponding to a zeaxanthin content in the range of 20 to 40 weight-% (yield in the range of 60 to 90%). In addi- tion the zeaxanthin dipalmitate containing oleoresin could be saponified with caustic ethanol to give a powder with a zeaxanthin content in the range of 30 to 95% (yield in the range of 30 to 95%).

The present invention is directed to a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin and/or its esters, comprising the following steps:

a) obtaining dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the dried skin of Goji berries or the size reduced pieces thereof obtained in step a) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and

c) removing the solvent from the extracts obtained in step b); and

d) optionally cleaving the esters in the dried extracts obtained in step c); and

e) optionally crystallizing the dried extracts obtained in step c) or d) in an aliphatic straight or branched C alcohol. Any person skilled in the art should understand, steps d) and e) are optional, so they may be per ^¬ formed or not in the process according to the present invention. Preferably the process according to the present invention consists only of these steps a) to e) and does not comprise any additional steps.

Thus, a preferred embodiment of the present invention is a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin and/or its esters, consisting of the following steps: a) obtaining dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the dried skin of Goji berries or the size reduced pieces thereof obtained in step a) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petro ^¬ leum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and

c) removing the solvent from the extracts obtained in step b); and d) optionally cleaving the esters in the dried extracts obtained in step c); and

e) optionally crystallizing the dried extracts obtained in step c) or d) in an aliphatic straight or branched C alcohol. Thus, one of these preferred embodiments is directed to a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin (di)esters consisting of the following steps:

a) obtaining dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the dried skin of Goji berries or the size reduced pieces thereof obtained in the preceding step with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and

c) removing the solvent from the extracts obtained in the preceding step; and

e) crystallizing the dried extracts obtained in the preceding step in an aliphatic straight or branched C alcohol.

The other preferred embodiment is directed to a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin consisting of the following steps:

a) obtaining dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the dried skin of Goji berries or the size reduced pieces thereof obtained in the preceding step with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and

c) removing the solvent from the extracts obtained in the preceding step; and

d) cleaving the esters in the dried extracts obtained in the preceding step; and

e) optionally crystallizing the dried extracts obtained in the preceding step in an aliphatic straight or branched Q _-4 alcohol.

Processes to obtain zeaxanthin are already known in the prior art, but have several disadvantages. Furthermore no prior art document deals with only the extraction of the skin of the Goji berries, but only with the extraction of the entire Goji berries. CN-A 1 158 263 e.g. uses an extraction solvent which contains water, so that also the sugars contained in the berries are extracted.

The process according to CN-A 1 086 730 focuses on the preservation of the natural nutritious com- ponents and special flavour of the Goji berries.

The process of US 2007/0161826 needs a washing step with a polar solvent such as an ethanol- water or an acetone-water mixture (see page 2, paragraphs [0021] and [0024], as well as example 3). The process of US 2005/0038271 involves either an extraction step with tetrahydrofuran (see examples 2 and 3) which easily forms peroxides under the reaction conditions or a crystallization step in water (see example 4), which is much more difficult to remove than the aliphatic straight or branched Ci_4 alcohol used in step e) of the process of the present invention. According to example 3 of WO 97/23436 the berries were first put in water and then dried again after having removed the sugars.

US 6,191,293 also describes a process where Chinese wolfberries are pre-extracted with water to eliminate water-soluble gums (see example 4).

The aim of the process of US 2008/0124416 (see the examples) is to obtain an extract rich in polysaccharides and in carotenoids, so that an extraction step with water is mandatory, whereas the aim of the present invention is to obtain an extract highly enriched in zeaxanthin and preferably in the form of a powder and not a paste, since powders are much more easily manageable.

A further advantage of the present invention is that the process of the present invention is cost- efficient and can be used in industrial scale to obtain natural zeaxanthin and/or its esters, especially its dipalmttate esters, in a large amount (especially in an amount of several tons). The process of the present invention is now described in more detail. Preferably all steps a) to e) of the processes according to the present invention are performed under an inert gas atmosphere such as e.g. under nitrogen or argon or mixtures thereof.

The Goji berries which skins are used can either be of the species Lycium barbar m and/or of the species Lycium chinense, preferably they are of the species Lycium barbarum L. The skin is a valu _¬ able starting material being obtained as waste material by the production of Goji juice from the entire fresh Goji berries.

The term "zeaxanthin esters'" encompasses zeaxanthin monoesters, as well as zeaxanthin diesters. In the skin of Goji berries the zeaxanthin esters are found mainly in form of their dipalmitate diesters.

When only steps a), b) and c) of the processes according to the present invention are performed, a so-called "oleoresin" is obtained. The consistence of this oleoresin is pasty-like at room-temperature, and it mainly consists of lipophilic compounds such as fats. The content of zeaxanthin in form of its esters in this oleoresin is usually in the range of 7.2 to 27 weight-%, based on the total weight of the oleoresin.

When only steps a), b), c) and e) of the processes according to the present invention are performed, an extract is obtained, which has a zeaxanthin(di)ester content in the range of 36 to 72 weight-%, based on the total weight of the extract. This extract is pulverous which makes its handling very easy.

When steps a), b), c) and d) of the processes according to the present invention are performed, an extract is obtained, which has a zeaxanthin content in the range of 30 to 95 weight-% in form of free zeaxanthin, based on the total weight of the extract. This extract is pulverous which makes its han ^¬ dling also very easy.

The extracts obtained by the processes according to the present invention can be used as food sup ^¬ plements e.g. for eye health. Furthermore the extracts and food supplements containing them may be used as antioxidants and/or blue light absorber. The single steps of the processes of the present invention are now described in more detail below.

Step a): Obtaining the dried skin of Goii berries and optionally size reducing it The dried skin of Goji berries may be prepared from fresh skin of Goji berries which is commercially available after a simple dryness, or may be prepared from the fresh Goji berries with the known procedures in the art.

The fresh Goji berries may be crushed or squeezed to get a so-called "Goji juice". The Goji juice is then separated from the solids which consist of seeds and skins. In general this is done by means of a decanter centrifuge, which allows to separate also the seeds from the skins (preferred).

Usually such skin is not available in dried form, and has a water content in the range of 70 to 80 weight-%, based on the total weight of the skin of the Goji berries. Drying may be carried out by any method known to the person skilled in the art such as by sunshine drying, by oven drying or by freeze drying. Preferably, the skins get dried by freeze drying and can then be used for the extraction of zeaxanthin according to the present invention.

Preferably the dried skin of Goji berries according to the present invention has a water content of up to 20 weight- , more preferably of up to 15 weight-%, even more preferably it has a water content in the range of 3 to 10 weight-%, most preferably in the range of 5 to 7 weight-%, based on the total weight of the skin of the Goji berries.

The size reduction of the skin of the Goji berries is not compulsory, because the skin usually is in form of small pieces after crushing of the berries and the centrifugation process. However, it was found out that the extraction leads to extracts with higher enrichments in zeaxanthin if the size of the skin of the Goji berries was further reduced before the extraction.

Therefore in a preferred embodiment the size of skin of the Goji berries is further reduced. The size reduction can be done by any method known to the person skilled in the art. Preferred methods are mechanical reduction such as crushing, smashing, chopping and milling, whereby crushing and milling and their combination are preferred.

Preferred embodiments of the present invention are also embodiments where several preferred em- bodiments of step a) as listed above are combined.

Step b): Extraction

Step b) is preferably performed several times, especially step b) is performed three times. Of course step b) could also be performed only two times, but with a longer extraction time.

Usually the extraction proceeds within 1 to 3 hours per extraction step. The most efficient time needed for the extraction may be found out easily by the person skilled in the art. Preferred extraction solvents used in step b) are ethyl acetate, hexane such as n-hexane, petroleum ether and ethanol. The most preferred extraction solvent is ethyl acetate.

Preferably the amount of solvent used in step b) is in the range of 1-10 liters, more preferably in the range of 1.5-6 liters, even more preferably in the range of 2-4 liters, most preferably in the range of 2-3 liters, per 1 kg of (optionally and preferably) dried skin of Goji berries obtained in step a).

Preferably step b) is performed at a temperature in the range of 20°C to 80°C, more preferably at a temperature in the range of 30°C to 70°C, even more preferably at a temperature in the range of 40°C to 70°C, most preferably at a temperature in the range of 50°C to 65°C.

Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step b) as listed above are combined.

Step c): Solvent removal

If the extract obtain in step b) contains non-soluble parts, they are removed, preferably by filtration. Then the solvent of the extract is evaporated under reduced pressure, preferably at a temperature in the range of 20°C to 78°C, more preferably at a temperature in the range of 30°C to 50°C, most preferably at a temperature in the range of 40°C to 45°C, whereby an "oleoresin" ("dried extract") is obtained.

The solvent may be removed batch wise, continuously or in a serial manner.

Preferred embodiments of the present invention are also embodiments where several preferred em- bodiments of step c) as listed above are combined.

Step d): Ester cleavage

In the oleoresin from the extraction of the skin of Goji berries most of the zeaxanthin present is es- terified and occurs mainly as its dipalmitate ester. In order to obtain free zeaxanthin the oleoresin has to be treated with a base, such as KOH and/or NaOH, to cleave the esters. If this is done with an aqueous base zeaxanthin is obtained together with fatty acid salts (saponification). If this is done with a base (for example with solid KOH and/or solid NaOH) in an aliphatic straight or branched d. 4 alcohol zeaxanthin is obtained together with fatty acid esters (trans-esterification).

Thus, the cleavage of the zeaxanthin esters in step d) can be performed by saponification or trans- esterification of the zeaxanthin esters of the dried extracts obtained in step c).

Preferably the cleavage of the zeaxanthin esters is performed by trans-esterification. More prefera- bly this trans-esterification in step d) is performed at a temperature in the range of 20°C to 120°C, preferably at a temperature in the range of 60°C to 100°C, more preferably at a temperature in the range of 70°C to 90°C.

The reaction can be performed at ambient pressure or at a higher pressure depending on the tem ^¬ perature chosen. Most preferably the trans-esterification is performed by reaction of the zeaxanthin esters with an aliphatic straight or branched Ci^ alcohol in the presence of a base.

Examples of the aliphatic straight or branched C, _-4 alcohol are methanol, ethanol, iso-propanol, n- propanol, n-butanol, iso-butanol and tert-butanol. Preferred alcohols are methanol, ethanol and any mixture thereof. The most preferred alcohol is ethanol.

Preferably the amount of said alcohol is in the range of 2 to 12 liter per kg of dried extract, more preferably in the range of 4 to 10 liter per kg of dried extract, most preferably in the range of 5 to 8 liter per kg of dried extract.

Examples of bases are alkali metal hydroxides such as KOH and NaOH and any mixtures thereof. Preferably the amount of said base is in the range of stoichiometrical or catalytic amounts, such as 25-40% weight- KOH based on the dried extract.

Preferred embodiments of the present invention are also embodiments where several preferred em ^¬ bodiments of step d) as listed above are combined.

Step e ): Crystallization

If a step d) is performed by trans-esterification, the aliphatic straight or branched C alcohol used in step e) is the same as used in step d). Here the same preferences for this alcohol apply as for step d). Thus, the most preferred solvent used in step e) is ethanol. When the crystallization is done in ethanol, preferably step e) is performed at a temperature in the range of room temperature to 78°C, more preferably at a temperature in the range of 40°C to 60°C, most preferably at a temperature around 50°C.

When step d) is not performed, the amount of ethanol used in step e) is preferably in the range of 2 to 12 liter, more preferably the amount of ethanol used in step e) is in the range of 4 to 10 liter, most preferably the amount of ethanol used in step e) is in the range of 5 to 8 liter, per 1 kg of dried extract obtained in step c).

Preferred embodiments of the present invention are also embodiments where several preferred em- bodiments of step e) as listed above are combined.

Further preferred embodiments of the present invention are embodiments where one or more of the preferred embodiments of step a) and/or step b) and/or step c) and/or step d) and/or step e) as listed above are combined.

The most preferred embodiment of the present invention is a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin, with a zeaxanthin content of at least 30-95 weight-%, based on the total weight of the extract, consisting of the following steps:

a) obtaining dried skin of Goji berries or the size reduced pieces thereof, with a water content of up to 20 weight-% based on the total weight of the dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the skin of Goji berries or the size reduced pieces thereof obtained in step a) with ethyl acetate at a temperature in the range of 45 to 65°C; and

c) removing the solvent at 40°C to 45°C from the extracts obtained in step b); and d) cleaving the esters in the dried extracts obtained in step c) by trans-esterification with ethanol in the presence of a base.

The other most preferred embodiment of the present invention is a process for the manufacture of an extract of the skin of Goji berries containing zeaxanthin (di)esters, with a zeaxanthin (di)esters con- tent of at least 36-72 weight-%, based on the total weight of the extract, consisting of the following steps:

a) obtaining dried skin of Goji berries or the size reduced pieces thereof, with a water content of up to 20 weight-% based on the total weight of the dried skin of Goji berries or the size reduced pieces thereof; and

b) extracting the skin of Goji berries or the size reduced pieces thereof obtained in step a) with ethyl acetate at a temperature in the range of 45 to 65 °C; and c) removing the solvent at 40°C to 45°C from the extracts obtained in step b); and e) crystallizing the dried extracts obtained in step c) in ethanol.

The invention will now further be illustrated by the following non-limiting examples. Examples

For analytical purposes samples of all products obtained were saponified and thus the zeaxanthin esters contained therein measured as free zeaxanthin. Thus, the zeaxanthin contents given below al- ways refer to free zeaxanthin and not to esterified zeaxanthin.

All examples were performed at an inert gas atmosphere, even if not explicitly mentioned.

Example 1 : Extraction of dried skin of Goji berries with ethyl acetate

The skin of Goji berries was purchased from Zhaokang Gojiberry Inc. (Ningxia province, China) and dried to less than 20 weight- % of water. 202.11 g of the dried skin of Goji berries was weighted into 2000 inL of flask. 400 mL of ethyl acetate was added and extracted for 2 h under stirring at 65°C. The mixture was cooled down to room temperature and then filtered. Repeated the above processes for another two times, combined all the extract, and evaporated off the solvent under high vacuum. 11.65 g of red Oleoresin was obtained. The zeaxanthin content was 48.6 g/kg with yield of 88.54%.

Example 2: Extraction of dried skin of Goji berries with n-hexane

The skin of Goji berries was purchased from Zhaokang Gojiberry Inc. (Ningxia province, China) and dried to less than 20 weight- % of water. 25 g of the dried skin of Goji berries was weighted into 500 mL of flask. 50 mL of n-hexane was added and extracted for 1 h under stirring at 45 ^° C. The mixture was cooled down to room temperature and filtered. Repeated the above processes for an- other two times, combined all the extract, and evaporated off the solvent under high vacuum. 0.8 g of red Oleoresin was obtained. The zeaxanthin content was 40.3 g/kg with yield of 30.17%. Example 3: Crystallization of the Oleoresin

1.01 g (48.6 g/kg) of Oleoresin obtained from the example 1 or 2 was distributed into 5 mL of etha- nol. The mixture was stirred at 50°C for 30 minutes, then cooled down to room temperature for 2 hours. The formed solid was filtered off, washed with cool ethanol (4°C, 5 mL, 3 times), and dried under high vacuum. 150 mg of zeaxanthin esters were obtained. The content of zeaxanthin is 229.6 g Kg analyzed by HPLC. The yield is 70.16%. Yield calculated formula is: yield= ((content x weight of product)/ (content x weight of oleoresin)) xlOO.

Example 4: Saponification of the Oleoresin

4.07 g of Oleoresin obtained from the example 1 or 2 was mixed with 41 mL of ethanol, and heated to reflux (oil bath 130 ^° C), then added into 40 mL of OH (1.3g) ethanol solution in 5 min, and re- fluxed for 20 min. Subsequently, the oil bath was removed, and acetic acid was added in dropwise over a period of 10 min. The mixture was then allowed to cool down to room temperature for 3 h. The formed solid was washed with ethanol (35 mL), ethanol : water=l : 1 (35 mL), water (70 mL) and ethanol (35 mL), then dried under high vacuum. 220 mg product was obtained. The content of free zeaxanthin is 837.2 g/Kg. The yield is 93.12 %.