The subject of the invention is a method of obtaining high-purity myxoxanthophyils from pigment extracts of cyanobacteria (cyanophyta). The obtained myxoxanthophyil fraction can be used in the pharmaceutical and cosmetic industries.

State of the art

Myxoxanthophyils are monocyclic carotenoids, derivatives of myxol ((2'S,3R)-3',4'- Didehydro-1 ',6'-seco-beta, beta-carotene-1 ',2',3-triol) glycosylated with 6-deoxy-D-glucose, L- fuco-a-pyranose (fucose), L-rhamnose, or other methyl-pentoses, and present in the cells of many species of cyanophyta (cyanobacteria) (Takaichi et al. 2001 ; Graham and Bryant, 2009; Mohamed et al., 2004; Takaichi et al. 2005, 2006). Despite early identification - in the 1930s (Heilbron and Lythgoe, 1936), both the physico-chemical properties and the biological function of these compounds have not been systematically studied so far.

The presence of a sugar residue (pentose or, more rarely, hexose) in the structure determines the high degree of amphiphilicity of the molecule compared to other xanthophylls, exhibiting predominantly hydrophobic properties. The presence of terpenoid aglycone having a conjugated system of 11 double bonds, similar to that present in other xanthophylls, such as zeaxanthin or lutein, suggests a high reactivity of these compounds with active oxygen species (Sandmann, 2019). Studies conducted in recent years by several independent research groups have proven that myxoxanthophyil is necessary for the formation of the correct structure of cell walls and the proper structural organization of thylakoids membranes, as well as that its accumulation is increased in response to biotic and abiotic stress factors, such as strong light, lowered or elevated temperature, destabilization of photosynthetic membranes or deficiency of certain minerals (Varkonyi et al. 2002; Mohammed and Vermaas, 2004, Mohammed etal. 2005; Schafer, 2005).

Given the wide use of carotenoids in the food industry (pigments of natural origin, dietary supplements), cosmetics (cosmetic ingredients with a protective/supportive effect) and pharmaceuticals (therapeutic substances), as well as the growing demand for new derivatives, an increased interest in myxoxanthophyils, and thus also the methods of obtaining them, can be anticipated.

The prior art knows methods for the preparation of pure carotenoid pigment derivatives on a preparative scale, which consist in their initial extraction from biological material with organic solvents (acetone, chloroform, methanol, ethanol and others, or with mixtures of these solvents or mixtures of these solvents with water). The obtained extracts, usually containing a mixture of many compounds (chlorophylls, carotenoids, lipids, etc.), are then purified by chromatographic, precipitation and/or partitioning techniques, resulting in pure preparations. In particular, the chromatographic methods include chromatography on adsorbents such as modified and neutralized silica gels (Britton & Young, 1991).

The literature describes a two-step method of purifying myxoxanthophyll from a pigment extract of Synechocystis PCC6803 cyanobacteria, where it is the main polar carotenoid. The pigment extract obtained by extraction with acetone/methanol in a volume ratio of 7:2 was applied to a silica gel 60 column (Merck, Germany), b-carotene was eluted with n-hexane, then chlorophyll a and nonpolar carotenoids were eluted with n-hexane/acetone in a volume ratio of 8:2 and 7:3. The fraction containing polar carotenoid was eluted with n- hexane/acetone in a volume ratio of 1:1 and applied to a DEAE-Toyopearl 650 M column (Tosoh, Japan), and myxoxanthophyll was eluted with n-hexane/acetone in a volume ratio of 1 :1 , leaving chlorophyll a and polar lipids bound to the column (Takaichi et at., 2001).

Another two-step method was used to purify myxoxanthophyll from cyanophyta Cylindrospermopsis raciborskii (Nagy et a!., 2009; Nagy et a/., 2018). The lyophilized cells underwent extraction by sonication in a mixture of methanol :acetone, in a volume ratio of 3:1 , and then, after separating the solid phase, the solvent was evaporated. The pigment- containing fraction was then separated in a two-phase system: hexane:methanol/water in a volume ratio of 85:15. The pigments dissolved in the polar phase were then purified on a silica gel column (Kieselgel 60, particle size 0.063-0.200 mm, Merck) modified by neutralization with sodium bicarbonate, using hexane:acetone mixtures in various proportions as eluent.

US4320050 describes a method of purifying a pool of glycosylated carotenoids (myxoxanthophyll and oscillaxanthin) from freeze-dried or dried Spirulina biomass, previously used to extract other pigments (phycocyanin, chlorophyll). The method consists in extracting carotenoid pigments from the biomass with acetone, and then binding them by mixing the extract with cellulose powder (1 kg/4 L of extract). The selective separation of the hydrophobic carotenoids fraction and the glycosylated carotenoids-containing fraction is achieved by sequential desorption first with petroleum ether and then with ethanol.

WO0224183 describes a method of obtaining myxoxanthophyll from cyanobacteria cells based on subjecting them to initial extraction with a 50% aqueous solution of acetone while grinding in a mortar with glass beads and then centrifuging the solids. Equal volumes of acetone, hexane and water with the addition of 10 g of NaCI are successively added to the extract obtained, and the whole is then shaken in a separatory funnel. As a result of the phase separation, myxoxanthophyll is accumulated in the hexane phase, which is subjected to separation and subsequent drying in a vacuum evaporator at 25°C for 30 min. The purity of the preparation obtained by using the above procedure is estimated to >90%. Optionally, the patent provides for fine purification of myxoxanthophyll with reverse phase chromatography. MD4360 describes a precipitation process for obtaining myxoxanthophylls from cyanophyta Arthrospira platensis, whereby the extraction of pigments from biomass with an aqueous-ethanol solution (70% - 96% ethanol) with separation of the biomass by centrifugation is repeated several times. Then, 40% potassium hydroxide is added to the combined volumes of the extracts in a 3:1 volume ratio to the initial volume of the extracted biomass. After 4-6 hours, the resulting solution is mixed with an equal volume of hexane. Then, after spontaneous phase separation and partitioning of the myxoxanthophyll-containing ethanol fraction, this fraction should be diluted with water to the ethyl alcohol concentration of 45% - 50%, which results in pigments precipitation, then centrifuged at 6000 rpm, and the obtained crystals are washed with ethanol solution (45% - 50%) and dried.

The above-described methods for obtaining myxoxanthophyll fractions are characterized by a multi-step procedure and usually require a combination of two or more partitioning, precipitation and/or chromatographic techniques. The multi-step methods for obtaining myxoxanthophylls are time-consuming and generate high costs. Additionally, depending on the reagents used, the myxoxanthophyll fractions obtained by partitioning and precipitation techniques may contain significant admixtures of other cyanobacterial metabolites (including chlorophylls, other carotenoids, lipids), detectable by chromatographic and/or spectroscopic methods.

The essence of the invention

Unexpectedly, the above-mentioned problems have been solved by the present invention.

The subject of the invention is a method of obtaining high-purity myxoxanthophylls from dye extracts of cyanobacteria, including: a) solvent extraction of biomass from cyanobacteria using sonication, b) centrifuging the cyanobacterial sediment from step a) and decanting the supernatant, c) obtaining an extract by evaporating the solvent or mixture of solvents from the supernatant obtained in step b), d) dissolving the extract obtained in step c) in a solvent or a mixture of solvents, e) introducing the extract solution from step d) onto a chromatographic column, f) purifying the dissolved extract on the chromatographic column, wherein steps a) - f) are carried out in the absence of light, characterized in that the purification with column chromatography is carried out in isocratic conditions using a polar adsorbent, with the elution in the first step being carried out in a constant flow of not more than 5 mL/min until the myxoxanthophylls adsorbed on the column are completely separated from the remaining dyes, while a solvent mixture of petroleum etheracetone, preferably having a volume ratio of 6:4, is used as the eluent, and in the second step the elution is carried out with a highly polar solvent or mixture of solvents until the myxoxanthophylls are completely washed out from the column.

Preferably, in the method of the invention, the solvent extraction is carried out in a highly polar solvent or mixture of solvents.

Preferably, in the method of the invention, the solvent extraction is carried out in a solvent mixture of acetone:methanol, preferably having a volume ratio of 7:2, or another solvent or mixture of solvents of analogous polarity

Preferably, in the method of the invention, the extraction, centrifugation and decanting steps are repeated until the extraction liquid does not change colour when the sediment is resuspended.

Preferably, in the method of the invention, the extract obtained after evaporating the solvent mixture from the supernatant is dissolved in a solvent mixture of petroleum etheracetone having a volume ratio of 6:4.

Preferably, in the method of the invention, the polar adsorbent is (meta)silicic acid, unmodified silica gel or alumina.

Preferably, in the method of the invention, the polar adsorbent is suspended in a solvent mixture of petroleum etheracetone having a volume ratio of 6:4 before it is introduced onto the column.

Preferably, in the method of the invention, the highly polar solvent or mixture of solvents used in the second elution step is methanol or ethanol or a solvent with analogous polarity.

In the context of the present invention, "polar adsorbent" is understood to mean a substance characterized by a non-zero electrical dipole moment or having functional groups characterized by a non-zero electrical dipole moment. Examples of polar adsorbents are ( eta)silicic acid, unmodified silica gel or alumina.

In the context of the present invention, "highly polar solvent" is understood to mean a substance other than water, characterized by a non-zero electrical dipole moment and a dielectric constant of not less than 20 under standard state conditions (i.e. pressure of 1000 hPa and temperature of 25°C). Examples of highly polar solvents are methanol , ethanol or any mixtures thereof.

In the context of the present invention, "isocratic conditions" are understood to mean elution conditions from the column in which an unchanging, constant composition of the mobile phase is ensured, resulting in a stable interaction between the stationary phase (adsorbent) and the mobile phase (eluent).

In the context of the present invention, "solvent with analogous polarity" is understood to mean solvents other than ethanol or methanol, or mixtures thereof, characterized by a dielectric constant of not less than 20 under standard state conditions. For a better understanding of the essence of the present invention, it is explained in the following examples and attached figures.

Fig. 1 presents chromatographic separation of a dye extract from Synechocystis PCC 6803 cyanophyta cells on a stationary phase ((meta)silicic acid) equilibrated with a petroleum ethenacetone solvent (6:4). (A) Initial separation phase - visible separation of dyes from the extract. (B) Final separation phase - myxoxanthophylls (carotenoid glycosides) visible at the top of the column.

Fig. 2 presents the analysis of the Synechocystis PCC 6803 myxoxanthophyll fraction. (A) HPLC/DAD chromatogram at a wavelength of 470 nm, retention times of the individual substances are indicated. (B) Absorption spectra corresponding to the separated substances.

Fig. 3 presents LC/MS analysis of the Synechocystis PCC 6803 myxoxanthophyll fraction. Chromatogram at 450 nm (A), mass spectrum (ESI, positive ions) for the peak with a retention time of 5.56 min - myxoxanthophyll (B) and a fragmentation spectrum (C) for this substance.

Fig. 4 presents analysis of Anabaena 7120 myxoxanthophyll fraction. (A) HPLC/DAD chromatogram at a wavelength of 470 nm, retention times of the individual substances are indicated. (B) Absorption spectra corresponding to the separated substances.

Fig. 5 presents LC/MS analysis of the Anabaena 7120 myxoxanthophyll fraction. Chromatogram at 450 nm (A) and mass spectra (ESI, positive ions) of peaks with a retention time of 10.32 min - ketomyxoxanthophyll (B) and 11.42 min - myxoxanthophyll (C) .

Fig. 6 presents analysis of Arthrospira platensis ( Spirulina ) myxoxanthophyll fraction. (A) HPLC/DAD chromatogram at a wavelength of 470 nm, retention times of the individual substances are indicated. (B) Absorption spectra corresponding to the separated substances.

Fig. 7 presents LC/MS analysis of Arthrospira platensis ( Spirulina ) carotenoid glycoside fraction. (A) Chromatogram at 450 nm (A) and mass spectra (ESI, positive ions) of peaks with a retention time of 7.22 min - hydromyxoxanthophyll (B) and 11.40 min - myxoxanthophyll (C).

Fig. 8 presents analysis of the Arthrospira platensis ( Spirulina ) carotenoid glycoside fraction isolated from a commercial preparation (A). HPLC/DAD chromatogram at a wavelength of 470 nm, retention times of the individual substances are indicated. (B) Absorption spectrum corresponding to the peak with a retention time of 2.18 min. (C) Absorption spectrum corresponding to the peak with a retention time of 2.37 min.

Example 1 - General procedure for myxoxanthophyll preparation from extract of cyanobacteria (cyanophyta)

All activities are carried out in the dark, or with the use of vessels shielded so that their contents do not come into contact with light. The collected cyanobacterial biomass is sonicated in a mixture of acetone:methanol in a volume ratio of 7:2 or in 100% acetone or another polar organic solvent (e.g. ethanol, dimethyl ether, etc.)· The cell pellet is separated from the supernatant by centrifugation (5 min, min. 5000 rpm) and decantation, then it is resuspended in the extraction mixture and shaken vigorously. Centrifugation and decantation are carried out again. The supernatant is collected each time. The procedure is repeated until the liquid does not change colour after the sediment is suspended. The dye extract thus prepared is concentrated by evaporation under reduced pressure on a rotary evaporator at a temperature of up to 50°C or with a stream of inert gas (e.g. nitrogen). After evaporation, the dyes are redissolved in a small volume of petroleum ether (boiling point 40-60°C):acetone mixture in a volume ratio of 6:4.

Unmodified chemically (meta)silicic acid (HaSiC , MM: 78.10), or another adsorbent with similar physicochemical properties (unmodified silica gel or alumina), suspended in a mixture of petroleum ethenacetone in a volume ratio of 6:4, should be prepared as a stationary phase and poured into a chromatographic column (20 x 120 mm). Once the column is stabilised, the column should be connected to a vacuum pump through a filter flask. Then, the previously prepared dye extract is introduced onto the column. By starting the pump, a sufficient vacuum must be created to generate a flow of 5 mL/min. The separation is carried out with isocratic elution under constant flow, replenishing the eluent (petroleum ethenacetone in a volume ratio of 6:4) over the column as necessary. The eluent may be another organic solvent or a mixture of solvents showing a dielectric constant of 9.5. The dyes are separated according to increasing polarity - the least polar substances, including carotenes, chlorophylls and xanthophylls other than myxoxanthophyll, leave the column first. Myxoxanthophyll(s) can be observed on the column as a distinctly orange band at the top of the column that migrates very slowly compared to the other dyes. The initial and final phase of separation, with the carotenoid glycoside fraction bound on the adsorbent are shown in Fig. 1. After removal of the other dyes, when only the myxoxanthophyll fraction remains on the column, the filter flask should be replaced and the elution continued with pure methanol (or another polar solvent with similar physicochemical properties, e.g. ethanol), which allows for the elution of myxoxanthophylls from the column. The elution should be carried out until the stationary phase is completely discoloured. The myxoxanthophyll solution collected in the filter flask should be concentrated by vacuum evaporation or evaporated with an inert gas (nitrogen) in the same way as for the pigment extract before the chromatographic separation.

Example 2 - Preparation of myxoxanthophyll from Synechocystis PCC6803 unicellular cyanophyta

Synechocystis sp. PCC 6803 (strain obtained from the Pasteur Culture Collection of Cyanobacteria (Institut Pasteur)) was cultured photoautotrophically in BG11 liquid medium (Rippka et at., 1979) with the addition of 5 mM HEPES-NaOH (pH 7.5), at a temperature of 23°C, under constant illumination with a flux density of 60 pmol photons m~ ² s~ ¹ , using fluorescent lamps (Philips TL-D 18 W/33-640). The suspension cultures (200 mL in Erlenmayer flasks with a total volume of 500 mL) were shaken on a rotary shaker at 150 rpm. Culture growth was monitored by measuring optical density OD750 with a Jasco-V650 spectrophotometer.

Cells cultured in a total volume of 3 L, in the stationary phase (usually 21 days after inoculation, OD ₇₅ o=0.5-0.8) were harvested by centrifugation (7000 rpm, 5 min). The supernatant was discarded and the cell pellet was sonicated (10 min, 70% power; Sonix, Vibracell VCX 750) in approx. 200 mL of acetone.methanol extraction mixture with a volume ratio of 7:2 added in 3 equal volumes, and then concentrated by evaporation using a vacuum evaporator at room temperature in the dark.

The dried pigment extract was dissolved in the adsorption chromatography solvent mixture of petroleum ethenacetone in a volume ratio of 6:4 and separated on a 300 mL bed volume column containing (meta)silicic acid equilibrated with this solvent mixture, as shown in Example 1. The column was eluted with 500 mL of methanol once the separation was completed.

The resulting myxoxanthophyll solution was concentrated by evaporation in a vacuum evaporator and dried at room temperature in the dark.

The composition of the obtained fraction was analyzed with HPLC/DAD method using a Jasco PU-4180 chromatograph with a Jasco MD-410 photodiode array detector and a Zorbax SB-C18 column (dimensions: 4.6 mm x 150 mm, porosity: 3.5 pm) (Agilent Technologies). The fraction was dissolved directly in the mobile phase of acetonitrile:methanol:water in a 72:8:1 volume ratio (Gillmore and Yamamoto, 1991). The chromatographic separation was performed as described in (Mysliwa-Kurdziel etal., 2012): flow rate of 0.7 mL/min increasing to 1.4 mL/min in the first 16.5 min; sample volume of 110 pi and detection wavelength of 470 nm.

One major peak (retention time 7.11 min; 67.83% of the total peak area) and 3 less intense peaks (retention times 8.26, 8.70 and 8.90 min; 3.37%, 12.66%, 16.15% of the total peak area, respectively) were identified in the HPLC/DAD chromatogram (Fig. 2A). The absorption spectra of the separated compounds are shown in Fig. 2B. They are very similar in the UV Vis range. In particular, substance 1 , with retention time RT=7.11 min, has the absorption maxima (A)max = 366, 452, 476, 506 nm; substance 2, with retention time RT=8.27 min, (A)max = 366, 448, 472, 504 nm; substance 3, with retention time RT=8.70 min, (A)max = 366, 446, 470, 502; substance 4, with retention time RT=8.90 nm; (A)max = 364, 446, 470, 500. The presented absorption spectra correspond to the absorption spectra of myxoxanthophyll known from the literature (Takaichi et at, 2001). The observed differences may correspond to the isomeric forms of the compound, in particular trans-myxoxanthophyll (substance 1 ) and cis-myxoxanthophyll (substances 2-4), characterized by marked absorption in the UV range (Melendez- artinez et al, 2013).

ESI LC/MS analysis was performed to further identify the identity of the isolated compounds (Figs. 3A-C). This analysis was able to confirm the presence of 4 substances in the studied fraction (Fig. 3A) with the mass spectrum of positive ions corresponding to myxoxanthophyll (Fig. 3B), characterized by the molecular weight of 758 u and the mass-to- charge ratio for the parent ion m/z=758 (Fig. 3C). This analysis confirms that there are 4 isomeric forms of myxoxanthophyll in the obtained fraction. For substances with retention times of 6.01; 6.30; 6.48 min, only the m/z value of the main ion is given. The m/z values for the remaining ions of these substances are identical to two decimal places as those reported for the substance with RT=5.56 min. The analysed fraction is characterized by a high degree of purity and homogeneity, due to the fact that the total area of the myxoxanthophylls peaks covers 91% of the total area under the chromatogram and there are no other accompanying substances.

The summary of the m/z values observed for ions of the identified substances is presented in Table 1a.

The m/z values observed for fragmentation ions of the identified substances are summarized in Table 1b. They allow for unambiguous identification of all components of the isolated fraction as myxoxanthophyll.

Table 1a - Summary of the retention times of the compounds present in the myxoxanthophyll fraction from Synechocystis based on the chromatogram with the corresponding m/z of selected ions determined by mass spectrometry along with the compound identification. Table 1b - List of fragmentation ions for the ion with m/z=781 (the major peak) for each of the compounds detected.

The concentration of myxoxanthophyll was quantified using the molar absorption coefficient e=1253 at a wavelength of 440 nm. Typically, depending on the specific culture conditions, from a 3-L culture of Synechocystis with OD75 ₀ of at least 0.5, approximately 200 micrograms of myxoxanthophyll with 91 % purity is obtained.

Example 3 - Myxoxanthophyll preparation from Anabaena 7120 colonial cyanophyta

Anabaena PCC 7120 (UTEX 2576 strain obtained from the UTEX collection of the University of Texas, Austin, also available as Nostoc sp. (ATCC® 27893™)) was cultured photoautotrophically in BG11 (-N) liquid medium (Rippka et ah, 1979) with the addition of 5 mM HEPES-NaOH (pH 7.5), at a temperature of 23°C, under constant illumination with a flux density of 60 pmol photons m~ ² s ^-1 , using fluorescent lamps (Philips TL-D 18 W/33-640). The suspension cultures (200 mL in Erlenmayer flasks with a total volume of 500 mL) were shaken on a rotary shaker at 150 rpm. Culture growth was monitored by measuring optical density OD ₇₅₀ with a Jasco-V650 spectrophotometer.

Cells cultured in a total volume of 3 L, in the stationary phase (usually 21 days after inoculation, OD _75C O.5-0.8) were harvested by centrifugation (7000 rpm, 5 min). The supernatant was discarded and the cell pellet was sonicated (10 min, 70% power; Sonix, Vibracell VCX 750) in approx. 200 mL of acetone:methanol extraction mixture with a volume ratio of 7:2 added in 3 equal volumes, and then concentrated by evaporation using a vacuum evaporator at room temperature in the dark.

The dried pigment extract was dissolved in the adsorption chromatography solvent mixture of petroleum ethenacetone in a volume ratio of 6:4 and separated on a 300 mL bed volume column containing (meta)silicic acid equilibrated with this solvent mixture, as shown in Example 1. The column was eluted with 500 mL of methanol once the separation was completed. The resulting myxoxanthophyll solution was concentrated by evaporation in a vacuum evaporator and dried at room temperature in the dark.

The composition of the obtained fraction was analyzed by HPLC/DAD followed by LC/MS as described in Example 2.

One major peak (retention time 5.05 min; 62.43% of the total peak area) and 4 less intense peaks (retention times 1.58, 5.71, 6.22 and 7.12 min; 2.67%, 13.24%, 12.75% and 3.50% of the total peak area, respectively) were identified in the HPLC/DAD chromatogram (Fig. 4A). The absorption spectra of the separated compounds are shown in Fig. 4B. They are very similar in the UV/Vis range. In particular, substance 1, with retention time RT=5.05 min, has the absorption maximum (A)max = 480 nm; substance 2, with retention time RT=1.58 min, (A)max = 450 nm; substance 3, with retention time RT=5.71 min, (A)max = 476 nm; substance 4, with retention time RT=6.22 min; (A)max = 475 nm; substance 5, with retention time RT=7.12 min; (A)max = 470 nm. The obtained absorption spectra correspond to the absorption spectra of keto-myxoxanthophyll and myxoxanthophyll known from the literature. Substances with retention times of 6.22 and 7.12 min may correspond to isomeric forms (cis-myxoxanthophyl!s).

ESI LC/MS analysis was performed to further identify the identity of the isolated compounds (Fig. 5A). This analysis was able to confirm the presence of a total of 9 substances in the studied fraction, including 4 substances with a positive ion mass spectrum corresponding to ketomyxoxanthophyll and 3 substances corresponding to myxoxanthophyll (Figs. 5B,C), characterized by a molecular weight of 744 and 730, respectively, and a mass-to-charge ratio for the parent ion m/z=744 and 730. This analysis confirms that there are at least 4 isomeric forms of ketomyxoxanthophyll and 3 isomers of myxoxanthophyll in the obtained fraction. The analysed fraction is characterized by a high degree of purity and homogeneity due to the fact that the total area of the myxoxanthophylls peaks covers 98% of the total area under the chromatogram and there are no other accompanying substances.

The summary of the most important parameters characterizing the identified substances is presented in Table 2.

Table 2 - Summary of the retention times of the compounds present in the myxoxanthophyll fraction from Anabaena 7120 based on the chromatogram with the corresponding selected ions determined by mass spectrometry along with the compound identification.

Typically, depending on the specific culture conditions, from a 3-L culture with OD750 of at least 0.5, approximately 300 micrograms of myxoxanthophyll are obtained with a purity of at least 95%.

Example 4 - Preparation of carotenoid glycoside fractions from non-toxic cyanophyta Arthrospira platensis (Spirulina) containing myxoxanthophylls

Arthrospira platensis cyanobacteria (strain SAG 85.79 available from the collection of University of Gottingen) (this strain is also annotated in UTEX (UTEX 2340) as "Spirulina platensis", the equivalent strain of Arthrospira platensis (Nordstedt) Gomont strain (ATCC® 29408™)) are cyanophyta, which carotenoid composition includes 2 types of glycosidic compounds: myxoxanthophyll and oscillaxanthin (1,1 ’-dihydroxy-2, 2’-di- -L-rhamnosyl-1, 2,1’, 2’-tetrahydro-3,4,3’,4’-tetradehydrolycopene) (Hertzberg and Liaaen-Jensen, 1969).

A. platensis was cultured photoautotrophically in a liquid medium according to Zarrouk (Vonshak, 1997), with the addition of NaOH (final pH >9.0), at a temperature of 22°C, under illumination with a flux density of 70 pmol photons nr ² s ^~1 and photoperiod 16/8h, using fluorescent lamps (Philips TL-D 18 W/33-640). The suspension cultures (3000 mL in Erlenmayer flasks with a total volume of 5000 mL) were mixed at least once every 48 hours. Culture growth was monitored by measuring optical density OD ₇₅₀ with a Jasco-V650 spectrophotometer.

Cells cultured in a total volume of 3 L, in the stationary phase (usually 21 days after inoculation, OD ₇₅ o=0.5-0.8) were harvested by centrifugation (7000 rpm, 5 min). The supernatant was discarded and the cell pellet was sonicated (10 min, 70% power; Sonix, Vibracell VCX 750) in approx. 200 m!_ of acetonermethanol extraction mixture with a volume ratio of 7:2 added in 3 equal volumes, and then concentrated by evaporation using a vacuum evaporator at room temperature in the dark.

The dried pigment extract was dissolved in the adsorption chromatography solvent mixture of petroleum ethenacetone in a volume ratio of 6:4 and separated on a 300 mL bed volume column containing (meta)silicic acid equilibrated with this solvent mixture, as shown in Example 1. The column was eluted with 500 mL of methanol once the separation was completed.

The resulting myxoxanthophyll solution was concentrated by evaporation in a vacuum evaporator and dried at room temperature in the dark.

The composition of the obtained fraction was analyzed with HPLC/DAD method using a Jasco PU-4180 chromatograph with a Jasco MD-410 photodiode array detector and a Zorbax SB-C18 column (dimensions: 4.6 mmx 150 mm, porosity: 3.5 pm) (Agilent Technologies). The fraction was dissolved directly in the mobile phase of acetonitrile:methanol:water in a 72:8:1 volume ratio (Gillmore and Yamamoto, 1991). The chromatographic separation was performed as described (Mysliwa-Kurdzief et al., 2012): flow rate of 0.7 mL/min increasing to 1.4 mL/min in the first 16.5 min; sample volume of 110 pi and detection wavelength of 470 nm.

One major peak (retention time 5.76 min; 50.74%of the total peak area) and 3 less intense peaks (retention times 3.45, 4.66 and 5.27 min; 9.47%, 19.04%, 4.81% of the total peak area, respectively) were identified in the HPLC/DAD chromatogram (Fig. 6A). The absorption spectra of the separated compounds are shown in Fig. 6B. They show distinctive differences in both the position of the maxima and the structure of the spectrum in the UV/Vis range. In particular, the substance 1 , with retention time RT=5.76 min, has the absorption maximum (A)max = 474 nm. The absorption spectrum of this substance corresponds to the absorption spectra of keto-myxoxanthophyll and myxoxanthophyll known from the literature. Substance 2, with retention time RT=3.45 min, has the absorption maximum (A)max = 414 nm; and substance 3, with retention time RT=4.66 min, (A)max = 416 nm. The absorption spectra of these substances may correspond to carotenoid derivatives with hydrophilic properties (e.g. oscillaxanthin). The 4 ^th maximum, with retention time RT=5.27 min, is characterized by the lack of a distinct spectrum structure and probably corresponds to an unseparated mixture of substances.

ESI HPLC/MS analysis was performed to further identify the identity of the isolated compounds (Fig. 7A). This analysis confirmed the presence of a total of 17 substances in the studied fraction, including 1 substance with a positive ion mass spectrum corresponding to hydromyxoxanthophyll and 4 substances corresponding to myxoxanthophyll (Figs. 7B,C), as well as 3 substances corresponding to oscillaxanthin, characterised by a molecular weight of 730, 746 and 893, respectively and a mass-to-charge ratio for the parent ion m/z=730, 746 and 893. This analysis confirms that there are at least 3 isomeric forms of myxoxanthophyll (including hydromyxoxanthophyll) and at least 2 forms of oscillaxanthin in the obtained fraction. There is also a noticeable proportion of the unresolved mixture of presumably aggregated and/or ionized forms of carotenoid glycosides (shown in the chromatogram, Fig. 6A) and the presence of oleic acid as the impurity (around 5%). The found presence of deoxymethylpentose indicates that the remaining components of the fraction present in smaller amounts may be degradation products of glycosylated carotenoids, including mainly oscillaxanthin (Aakermann etal., 1992).

The summary of the most important parameters characterizing the identified substances is presented in Table 3.

Table 3 - Summary of the retention times of the compounds present in the myxoxanthophyll fraction from Arthrospira platensis based on the chromatogram with the corresponding selected ions determined by mass spectrometry along with the compound identification.

Typically, depending on the specific culture conditions, from a 3-L culture with OD750 of at least 0.5-0.8, approximately 800 micrograms of glycosylated carotenoids are obtained with a purity of about >80%. In addition to carotenoid glycosides, the only organic substance of biological origin that can be identified by mass spectrometry is oleic acid.

Example 5 - Preparation of myxoxanthophyll from dry matter (commercial formulation) of Arthrospira platensis ( Spirulina )

1 g of Arthrospira platensis dry matter (commercially available preparation - tablets) Witpak Spirulina Tablets 250 g; dietary supplement; country of origin: China, manufacturer WITPAK sp. z 0.0. sp. k. ul. Chatubinskiego 13, 25-619 Kielce; batch number: 061119-1 extracted with 250 mL of acetone:methanol extraction mixture with a volume ratio of 7:2, and then concentrated by evaporation using a vacuum evaporator at room temperature in the dark.

The dried pigment extract was dissolved in the adsorption chromatography solvent mixture of petroleum ethenacetone in a volume ratio of 6:4 and separated on a 300 L bed volume column containing (meta)silicic acid equilibrated with this solvent mixture, as shown in Example 1. The column was eluted with 500 mL of methanol once the separation was completed.

The resulting myxoxanthophyll solution was concentrated by evaporation in a vacuum evaporator and dried at room temperature in the dark.

The composition of the obtained fraction was analyzed with HPLC/DAD method using a Jasco PU-4180 chromatograph with a Jasco MD-410 photodiode array detector and a Zorbax SB-C18 column (dimensions: 4.6 mm x 150 mm, porosity: 3.5 pm) (Agilent Technologies). The fraction was dissolved directly in the mobile phase of acetonitrile:methanol:water in a 72:8:1 volume ratio (Gillmore and Yamamoto, 1991). The chromatographic separation was performed as described (Mysliwa-Kurdziel et a!., 2012): flow rate of 0.7 mL/min increasing to 1.4 mL/min in the first 16.5 min; sample volume of 110 pi and detection wavelength of 470 nm.

One major peak (retention time 2.18 min) and a second peak having a retention time of 2.37 min were identified in the HPLC/DAD chromatogram (Fig. 8A). The structure of the absorption spectrum of the main peak (Fig. 8B), in particular the presence of many peaks characteristic for myxoxanthophyll and/or oscillaxanthin isomers, as well as an increased background level and a relatively short retention time, suggest that different forms of these compounds do not separate under the conditions of chromatographic separation, possibly constituting multi-molecular aggregates. The complete absence of structure in the absorption spectrum of the second peak (retention time 2.37 min) indicates aggregated material with low molecular weights (Fig. 8C) which is a contamination of the glycosylated carotenoid fraction.

This result shows that the technological processes that the biomass is subjected to, including multi-stage mechanical dewatering and spray drying at elevated temperature, have a significant impact on the purity and quality of carotenoid glycosides obtained by the proposed method. The observed contamination may also come from additional, unknown admixtures added to Arthrospira platensis biomass in the process of preparing a commercial preparation, e.g. preservatives, anti-caking agents, pharmaceutical vehicle, etc.

The resulting myxoxanthophyll solution was concentrated by evaporation in a vacuum evaporator and dried at room temperature in the dark.

Typically, 1 g of the commercial preparation produces about 15 mg of glycosylated carotenoids. The fraction has an estimated purity of > 50%, with the absence of other organic substances of biological origin, identifiable by mass spectrometry.