BAAKLINI GRACE (CH)
PICCIOLO FABIANO (CH)
RONCO CARLO (CH)
SCHAEFER CHRISTIAN (CH)
SCHLEGEL BERND (CH)
VERDURAND ELODIE (CH)
VOECKLER CHRISTA (CH)
WUESTENBERG BETTINA (CH)
FANTOU CÉLINE (CH)
| WO2014186683A1 | 2014-11-20 | |||
| WO2008079287A1 | 2008-07-03 | |||
| WO2014115037A2 | 2014-07-31 | |||
| WO2016174600A1 | 2016-11-03 | |||
| WO2008045405A2 | 2008-04-17 | |||
| WO2005110122A1 | 2005-11-24 | |||
| WO2008079287A1 | 2008-07-03 | |||
| WO2004037236A1 | 2004-05-06 | |||
| WO2016157157A1 | 2016-10-06 | |||
| WO2015089385A1 | 2015-06-18 | |||
| WO2014186683A1 | 2014-11-20 |
| US20090311761A1 | 2009-12-17 | |||
| US20090093015A1 | 2009-04-09 | |||
| US20090311761A1 | 2009-12-17 |
XIN WEN ET AL., JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 65, 2017, pages 6140 - 6151
D. E. LOEBER ET AL., J. CHEM. SOC. C, vol. 404, 1971
F. KHACHIK ET AL., SYNTHESIS, vol. 3, 2011, pages 509 - 516
R. KUHNC. GRUNDMANN, CHEMISCHE BERICHTE, 1933, pages 1746 - 1750
Y. J. JIANM. GUO, PROCESSING TECHNOLOGY FOR INFANT FORMULA
| Claims 1. A crystal form I of b-cryptoxanthin, wherein said crystal form I is defined by the following parameters: l-a) an X-Ray Powder Diffraction pattern comprising at least 3 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), preferably comprising at least 4 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10); more preferably comprising at least 5 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10), 25.192(4); most preferably comprising at least 6 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10), 19.270(16), 25.192(4). 2. The crystal form I of b-cryptoxanthin according to claim 1, further being defined by: l-b) a Differential Scanning Calorimeter scan at a heating rate of 10 K/min showing a phase transition at a temperature in the range of from 60 to 120°C, preferably at a temperature in the range of from 80 to 100°C, more preferably at a temperature in the range of from 80 to 90° C, most preferably at a temperature of 85 °C ± 1 °C, all at a heating rate of 10 K/min. 3. The crystal form I of b-cryptoxanthin according to claim 1 and/or 2, further being defined by: l-c) a Differential Scanning Calorimeter scan showing a melting peak at a temperature in the range of from 140°C to 200°C, preferably at a temperature in the range of from 150°C to 190°C, all at a heating rate of 10 K/min. 4. A process for the manufacture of the crystal form I of b-cryptoxanthin according to any one or more of claims 1 to 3, comprising the following steps: a) providing crude b-cryptoxanthin, whereby the crude b-cryptoxanthin is preferably obtained by the process according to claim 6 and/or 7; b) suspending the crude b-cryptoxanthin in an organic solvent and optionally heating the suspension; c) optionally cooling the suspension to a temperature of at most 30° C, preferably of at most 25 °C, more preferably of at most 20° C; d) optionally adding water to the cooled suspension; e) separating the crystal form I of b-cryptoxanthin; f) drying of the crystalline b-cryptoxanthin, preferably at a temperature in the range of from 40 to 60° C; whereby the organic solvent is selected from ketones R1-C(=0)-R2 with R1 and R2 being independently from each other straight-chain CM alkyl or branched C3-4 alkyl, cyclic ethers with a total amount of carbon atoms from 4 to 7, dialkyl ethers R3-C(=0)-R4 with R3 and R4 being independently from each other straight-chain CM alkyl or branched C3-4 alkyl, and dialkyl carbonates R50-C(=0)-0R6 with R5 and R6 being independently from each other straight-chain CM alkyl or branched C3-4 alkyl. 5. The process according to claim 4, whereby the organic solvent used in step b) is selected from acetone, a mixture of acetone and tetrahydrofuran with a volume ratio of 9:1, dimethyl carbonate, tert-butyl methyl ether and methyl ethyl ketone; preferably whereby the organic solvent is acetone. 6. A process for the manufacture of crude b-cryptoxanthin comprising the following steps: I) reacting 2,7,11 -trimethyl-13-(2, 6, 6-trimethylcyclohex-1-en-1 -yl)trideca- 2,4,6,8,10,12-hexaenal and 4-(5-(halotriaryl-5-phosphanyl)-3- methylpenta-1 ,3-dien-1 -yl)-3,5,5-trimethylcyclohex-3-en-1 -ol, preferably by reacting (2E, 4E, 6E, 8E, 10E,12E)-2, 7, 11 -trimethyl-13-(2, 6, 6- trimethylcyclohex-1-en-1 -yl)trideca-2,4,6,8,10,12-hexaenal and (/?)- ((2E,4E)-5-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3-methylpenta- 2,4-dien-1 -yl)triphenylphosphonium chloride, in the presence of a 30 weight-% methanolic solution of sodium methoxide; II) filtering the thus obtained b-cryptoxanthin to obtain crude b- cryptoxanthin. 7. The process according to claim 6, whereby the amount of sodium methoxide in step I) is in the range of from 1.00 to 1.50 equivalents, preferably in the range of from 1.00 to 1.20 equivalents, more preferably in the range of from 1.05 to 1.15 equivalents, based on the amount of 4-(5-(halotriaryl-5-phosphanyl)-3- methylpenta-1,3-dien-1-yl)-3,5,5-trimethylcyclohex-3-en-1-ol, preferably based on the amount of (/?)-((2E,4E)-5-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)- 3-methylpenta-2,4-dien-1 -yl)triphenylphosphonium chloride; and/or whereby step I) is performed in ethanol. 8. A liquid formulation comprising the crystal form I of b-cryptoxanthin according to any one or more of claims 1 to 3 and optionally another crystal form of b- cryptoxanthin as component i). 9. The liquid formulation according to claim 8, whereby the other crystal form of b-cryptoxanthin is crystal form II and/or crystal form IV, and their total amount together is at most 20 weight-%. 10. The liquid formulation according to claim 8 and/or 9 further comprising ii) at least an oil; and iii) optionally at least an antioxidant, preferably optionally at least a fat-soluble antioxidant. 11. The liquid formulation according to any one or more of claims 8 to 10, whereby the amount of component i) is in the range of from 0.1 to 30 weight-%/0.5 to 30 weight-%, preferably in the range of from 0.8 to 20 weight-%, more preferably in the range of from 1 to 17.0 weight-%, most preferably in the range of from 1 to 5 weight-% or from 10.0 to 17.0 weight-%, and/or the amount component ii) is in the range of from 70 to 99.9 weight-%/70 to 99.5 weight-%, preferably in the range of from 80 to 99.2 weight-%, more preferably in the range of from 83 to 99 weight-%, most preferably in the range of from 95 to 99 weight-% or from 83 to 90 weight-%, and/or the amount component iii) is in the range of from 0 to 10 weight-%, preferably in the range of from 0.2 to 2 weight-%, more preferably in the range of from 0.5 to 1.5 weight-%, all amounts based on the total weight of the liquid formulation and whereby the amounts of compounds i) to iii) sum up to 100%. 12. A process for the manufacture of the liquid formulation according to any one or more of claims 8 to 11 , comprising the following step: comminuting the crystal form I of b-cryptoxanthin according to any one or more of claims 1 to 3 and optionally the crystal form II of b-cryptoxanthin and optionally the crystal form IV of b-cryptoxanthin (component i)) in an oil (component ii)) and adding before, during or after comminuting optionally at least an antioxidant, preferably optionally at least a fat-soluble antioxidant (component iii)). 13. Dietary supplement comprising the liquid formulation according to any one or more of claims 8 to 11 , preferably being in form of soft capsules or in form of powdered products or in form of ready-to-drink products, more preferably being in form of soft gelatin capsules or in form of powdered infant formula or in form of ready-to-drink infant formula. |
Summary of the invention
The invention relates to a new crystal form of b-cryptoxanthin, processes for its manufacture, liquid formulations comprising such crystal form and products thereof as well as processes for their manufacture.
Background of the invention b-Cryptoxanthin ^-carotene-3-ol) is a natural carotenoid pigment. It can be isolated from a variety of sources including the petals and flowers of plants in the genus Physalis (see WO 2014/115037, WO 2016/174600, and Xin Wen et al. , Journal of Agricultural and Food Chemistry 2017, 65, 6140-6151 ), be obtained by fermentation (see e.g. US 2009/0093015, WO 2008/045405), as well as be synthesized chemically (see e.g. D. E. Loeber et al., J. Chem. Soc. C 1971 , 404; F. Khachik et al., Synthesis 2011 , 3, 509-516).
In the human body, b-cryptoxanthin is converted to vitamin A (retinol) and is, therefore, considered a provitamin A. According to WO 2005/110122 it can be used to increase the protein formation in a human or an animal. Further health benefits of b-cryptoxanthin are e.g. disclosed in WO 2008/079287, WO 2004/037236, WO 2016/157157 and WO 2015/089385. b-Cryptoxanthin can be administered to humans or animals in the form of soft capsules. The term “soft capsules” encompasses “soft-gel capsules”, as well as non-gelatine soft capsules and further soft capsules based on other materials of non-animal origin such as e.g. starch. They may be manufactured according to processes known to the person skilled in the art.
In order to produce soft capsules b-cryptoxanthin has to be provided in a semi-solid form, in a liquid form or in a suspension, whereby the suspension can be oily. b-Cryptoxanthin, however, is sparingly soluble in water and only partially soluble in many common edible oils. Thus, b-cryptoxanthin is advantageously dispersed in oil in order to be processable into soft capsules. The dispersion in oil has the further advantage that higher concentrations of b-cryptoxanthin - compared to dispersions in water - may be reached.
Thus, there is an ongoing need for a process facilitating the preparation of liquid formulations of b-cryptoxanthin which are suitable for human consumption.
As b-cryptoxanthin is also very sensitive to oxidation and heat treatment such formulations furthermore need to be oxidation stable.
Preferably, such liquid formulations are manufactured without the use of an organic solvent, especially without the use of a halogenated hydrocarbon such as dichloromethane or chloroform.
Since the b-cryptoxanthin has to be comminuted and micronization techniques often require organic solvents in contrast to milling, the method of choice for comminuting is milling.
Thus, there was a need to improve the milling properties of solid b-cryptoxanthin.
Surprisingly it has now be found that a certain, novel crystal form of b- cryptoxanthin is easy to mill in oil and thus facilitates the preparation of the above-mentioned liquid formulations. A further advantage is that high concentrations of b-cryptoxanthin in the liquid formulation could be achieved.
The prior art is silent on specific crystal forms of b-cryptoxanthin and their potential utility for preparing (liquid) b-cryptoxanthin formulations. The inventors of the present patent application have found that it is of high importance to choose the right polymorph (= crystal-form) of b-cryptoxanthin and that said specific crystalline polymorph of b-cryptoxanthin can be more useful than others, especially for the preparation of an oily suspension. “Right” in this context means a polymorph of b-cryptoxanthin which is stable in the used oil and does not undergo polymorphism which could be accompanied by viscosity increase and thus, difficult handling of the oily suspension by high viscosity increase which makes high performance milling with a stirred media mill impossible.
Detailed description of the invention
Thus, this need is fulfilled by the present invention, which is directed to a new crystal form of b-cryptoxanthin: crystal form I of b-cryptoxanthin (“needles”; see Figure 5)
Crystal form IV of b-cryptoxanthin (“platelets”) is already known from the prior art.
Crystal form II of b-cryptoxanthin (“platelets”; see Figure 6) is also a new crystal form of b-cryptoxanthin.
Surprisingly, it has been found that crystal form I is easy to mill in oil, whereby a certain amount of another crystal form such as e.g. crystal form II and/or crystal form IV may be tolerated. This is surprising since the inventors expected crystal form II and crystal form IV to be milled more easily than crystal form I due to the higher surface of crystal form II and crystal form IV compared to crystal form I, whereas the opposite is the case.
The invention also relates to methods for preparing crystal form I. Interestingly, it was further found that the crystal form I can be prepared from crystal form II.
Additionally, the invention is directed to liquid formulations, preferably to oily suspensions, comprising crystal form I of b-cryptoxanthin (“needles”), whereby a certain amount of another crystal form of b-cryptoxanthin such as e.g. crystal form II of b-cryptoxanthin (“platelets”) and/or crystal form IV of b-cryptoxanthin (“platelets”) may be tolerated.
Finally, the invention is related to the use of such liquid formulations according to the invention in dietary supplements such as soft capsules.
Crystal forms I, and IV
In particular, the present invention relates to the new crystalline form I of b- cryptoxanthin, and any of its mixtures with crystal form II and crystal form IV.
Crystal form I of b-cryptoxanthin is anhydrous and ansolvated with needle-shaped crystals. Crystal form II is an ethanol solvate (ethanolate) which means that the ethanol is incorporated in the crystal lattice with the b-cryptoxanthin resulting in platelets crystals.
Crystal form IV of b-cryptoxanthin is obtained amongst others by drying crystal form II of b-cryptoxanthin by higher temperatures and are also platelets.
Many techniques are used to identify the polymorphic forms of a material and its relative temperature stability. These techniques include X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC).
Until now the prior art known to the person skilled in the art did not disclose the possibility of preparing specific crystal forms of b-cryptoxanthin clearly characterized by XRPD and DSC.
In most cases X-ray diffractometry is capable to reflect the differences in crystal structure. Ideally, X-ray diffraction on a single crystal (typical dimensions 100x100x100 pm3) yields a three-dimensional diffraction pattern of, normally, well-resolved peaks, which after phasing can be back-transformed into electron density. As a matter of fact, it is often difficult, if not impossible, to obtain single crystals of required quality and size from a given material. In powder diffraction experiment the sample consists of a huge number of crystallites with typical dimensions of 5x5x5 pm3. The powder is normally obtained by grinding or milling.
In case of crystal form I, II and IV, X-Ray Powder Diffraction (XRPD) patterns are obtained by collecting intensity data measured by a Rigaku Mim ' Flex 600 diffractometer. The system is equipped with a Cu anode and a monochromator providing Cu-Ka radiation (l = 1.54056 A). The measurements are carried out using step width 0.02 degree.
In case of crystal Form VIII, the XRPD pattern was measured using Bruker D8 (LynxEye in Reflection) diffractometer with a copper Cu-Ka radiation (l = 1.54056 A). The X-Ray analysis was performed with a step width 0.02 degree.
Thermal analysis methods are defined as those techniques in which a property of the analyte is determined as a function of an externally applied temperature. In many respects, DSC is an easy method to use routinely on a quantitative basis, and for this reason it has become a widely accepted method for identification and characterization.
In accordance with the present invention, a DSC1 from Mettler Toledo equipped with a sensor type FRS 5+ is used to investigate the thermodynamic relationship between different polymorphs. The DSC cell is calibrated with indium (melting temperature Tm = 156.6 °C, enthalpy of fusion AHfus=28.45J.g.-1 ). b-Crypto- xanthin (normally 3 - 6 mg) was heated up at 10 K/min rate, from 25 °C to 220 °C in Al-pans while being purged with nitrogen.
A two point calibration of the DSC with Indium and Zinc is performed. The tolerances for the calibration are: To investigate the different polymorphs, 3-5 mg samples of form I or II were heated from 20°C to 220°C at a heating rate of 10 K-min-1 , respectively, while being purged with nitrogen (10 ml-min-l ).
The morphology of crystal Form I and crystal Form II was examined by scanning electron microscopy from Schaefer-Tec-AG, the equipment type is Phenom ProX.
Crystal form I of b-cryptoxanthin is characterized by the following parameters: l-a) an X-Ray Powder Diffraction pattern comprising at least 3 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), preferably comprising at least 4 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10); more preferably comprising at least 5 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10), 25.192(4); most preferably comprising at least 6 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.209(7), 13.402(3), 16.056(5), 18.174(10), 19.270(16), 25.192(4).
The X-Ray Powder Diffraction pattern of crystal form I of b-cryptoxanthin shows also peaks at the following 2-theta positions (deg) (±0.2 deg):
4.821 (16), 6.64(3), 7.33(4), 8.17(2), 8.70(2), 9.268(15), 9.738(5), 11.11 (3), 11.784(5), 12.460(3), 13.031 (7), 13.886(4), 14.622(7), 15.342(8), 15.630(6), 16.508(10), 16.925(11 ), 17.558(8), 18.580(9), 19.586(15), 20.139(10), 20.532(12), 20.99(3), 21.712(12), 22.403048, 23.124(9), 24.557155, 25.999(6), 26.848(18), 27.412(10), 28.13(4), 28.91 (7), 30.47(3), 31.28(4).
The numbers in parentheses is the estimated standard deviation. “16” means “± 0.016”; “3” means “± 0.003” etc.
In a preferred embodiment the crystal form I of b-cryptoxanthin shows also l-b) a Differential Scanning Calorimeter scan showing a phase transition at a temperature in the range of 60 to 120°C, preferably at a temperature in the range of from 80 to 100°C, more preferably at a temperature in the range of from 80 to 90 °C, most preferably at a temperature of 85 °C ± 1 °C. In a further preferred embodiment the crystal form I of b-cryptoxanthin shows also
I-c) a Differential Scanning Calorimeter scan showing melting at a temperature in the range of from 140°C to 200°C, preferably at a temperature in the range of from 150°C to 190°C.
Crystal form II of b-cryptoxanthin is characterized by the following parameters:
II-a) an XRPD (X-Ray Powder Diffraction) pattern comprising 3 peaks at the following 2-theta positions (deg) (±0.2 deg): 13.646(3), 16.599(3), 21.203(5); preferably comprising 5 peaks at the following 2-theta positions (deg) (±0.2 deg): 13.646(3), 14.923(2), 16.599(3), 21.203(5), 24.331 (12); more preferably comprising 7 peaks at the following 2-theta positions (deg) (±0.2 deg): 13.266(2), 13.646(3), 14.923(2), 16.599(3), 18.892(7), 21.203(5), 24.331 (12); most preferably comprising 9 peaks at the following 2-theta positions (deg) (±0.2 deg): 13.266(2), 13.646(3), 14.562(4), 14.923(2), 15.846(3), 16.599(3), 18.892(7), 21.203(5), 24.331 (12).
The X-Ray Powder Diffraction pattern of crystal form II of b-cryptoxanthin shows also peaks at the following 2-theta positions (deg) (±0.2 deg):
3.60(2), 9.937 (10), 11.190(9), 12.31961 , 15.240(9), 16.194(3), 17.526(19), 17.845(11 ), 18.64(6), 19.99(4), 20.608(8), 22.035(11 ), 23.20(4), 25.008(13), 25.94(4), 26.873(9), 29.51 (3), 30.084(18), 31.51 (3).
The numbers in parentheses is the estimated standard deviation. “2” means “± 0.002”; “10” means “± 0.010” etc.
In a preferred embodiment the crystal form II of b-cryptoxanthin shows also ll-b) a Differential Scanning Calorimeter scan showing a de-solvation peak at a temperature in the range of 100 to 160°C, preferably at a temperature in the range of from 110° C to 150°C.
In a further preferred embodiment the crystal form II of b-cryptoxanthin shows also II-c) a Differential Scanning Calorimeter scan showing a melting peak at a temperature in the range of from 150°C to 195°C, preferably at a temperature in the range of from 155°C to 190°C.
Figure 1 shows the results, i.e. an X-Ray diffractogram of crystal form I and II of b- cryptoxanthin. The x-axis shows the 2-theta positions, the y-axis shows the counts.
The DSC analysis of crystal form I of b-cryptoxanthin (see Figure 2) shows a reversible endothermic peak in the range between 80° C and 100°C, followed by an intense endothermic peak between 150°C and 190°C corresponding to the melting peak.
The DSC analysis of crystal form II of b-cryptoxanthin (see Figure 3) shows a endothermic peak in the range between 110°C and 150°C corresponding to a de solvation peak, followed by an intense endothermic peak between 155°C and 190°C corresponding to the melting peak.
Crystal form IV of b-cryptoxanthin is characterized by the following parameters:
III-a) an X-Ray Powder Diffraction pattern comprising 3 peaks at the following 2- theta positions (deg) (±0.2 deg): 3.674(4), 16.8337(18), 22.093(2); preferably comprising 5 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.674(4), 16.8337(18), 17.568(5), 17.867(5), 22.093(2); more preferably comprising 7 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.674(4), 14.497(3), 16.8337(18), 17.568(5), 17.867(5), 22.093(2), 24.039(3); most preferably comprising 9 peaks at the following 2-theta positions (deg) (±0.2 deg): 3.674(4), 14.497(3), 16.0204(18), 16.8337(18), 17.568(5), 17.867(5), 18.995(8), 22.093(2), 24.039(3).
The X-Ray Powder Diffraction pattern of crystal form IV of b-cryptoxanthin shows also peaks at the following 2-theta positions (deg) (±0.2 deg):
5.52(4), 9.60(5), 10.150(16), 10.955(5), 11.749(8), 12.857(15), 13.180(19), 13.531 (11 ), 14.804(4), 15.15(2), 16.517(6), 19.271 (7), 20.283(9), 20.81 (3), 21.689(10), 22.495(5), 23.072(4), 23.606(4), 24.402(4), 24.889(7), 25.244(3), 25.982(12), 27.02(4), 27.715(7), 29.552(11 ) and 30.47(3).
The numbers in parentheses is the estimated standard deviation. “4” means “± 0.004”; “5” means “± 0.005” etc.
Crystal form IV of b-cryptoxanthin are platelets.
Figure 4 shows the X-Ray diffractogram of crystal form IV of b-cryptoxanthin. The x-axis shows the 2-theta positions, the y-axis shows the counts.
The inventors of the present invention thoroughly investigated the different crystal forms of b-cryptoxanthin and their stability. Crystal form I is the thermodynamically most stable form among them. Crystal forms II and IV of b- cryptoxanthin transform into crystal form I of b-cryptoxanthin in an edible oil such as corn oil, as well as in acetone.
The same may be expected with crystal form VIII of b-cryptoxanthin. Crystal form VIII of b-cryptoxanthin is an ethanol solvate that crystallizes in a polymorphic form which is different from crystal form II of b-cryptoxanthin. This can be concluded by comparing the XRPD patterns of crystal forms II and VIII of b-cryptoxanthin: The comparison reveals that the XRPD patterns are not similar.
Processes for the manufacture of the crystal forms and their conversion into each other; crystal form VIII
The process for the manufacture of b-cryptoxanthin is known to the person skilled in the art, see e.g. F. Khachik et al. , Synthesis 2011, 3, 509-516. Hereby crystal form VI of b-cryptoxanthin is obtained by dissolving the crude b-cryptoxanthin in methylene chloride, precipitating it with hexane and filtering it. The inventors have found that crystal form VI may also be obtained by digestion in ethyl acetate or diethyl ether. The manufacture of (3R)- and (3S)- b-cryptoxanthin is furthermore disclosed in examples 18 and 19 of US 2009/0311761 , whereby the crystallization is carried out with dichloromethane and hexane. Hereby crystal form VI of b-cryptoxanthin is obtained (see comparison example 3 of the present patent application).
The crystal form of b-cryptoxanthin obtained in R. Kuhn, C. Grundmann, Chemische Berichte 1933, pages 1746-1750 by crystallization in benzene-alcohol or benzene- methanol is described as butterfly- like connate, sharpened prisms or in regular prisms. It is thus different from any crystal form found by the present inventors.
If crude b-cryptoxanthin is crystallized in a mixture of acetone and ethanol with a volume ratio of ~ 3:1 (similar conditions to the ones used in examples of WO 2014/186683) crystal form VIII of b-cryptoxanthin is obtained (see comparison example 4 of the present patent application).
Figure 10 shows the X-Ray diffractogram of crystal form VIII of b-cryptoxanthin.
The x-axis shows the 2-theta positions, the y-axis shows the counts.
Crystal form VIII of b-cryptoxanthin shows an X-Ray Powder Diffraction pattern comprising at least 3 peaks at the following 2-theta positions (deg) (±0.2 deg): 14.7695(13), 15.670(2), 16.2040(19), 18.252(4), 20.577(13), 22.513(7), 23.024(7), preferably comprising at least 5 peaks at the following 2-theta positions (deg) (±0.2 deg): 14.7695(13), 15.670(2), 16.2040(19), 18.252(4), 20.577(13), 22.513(7), 23.024(7); more preferably comprising 7 peaks at the following 2-theta positions (deg) (±0.2 deg): 14.7695(13), 15.670(2), 16.2040(19), 18.252(4), 20.577(13), 22.513(7), 23.024(7).
The X-Ray Powder Diffraction pattern of crystal form VIII of b-cryptoxanthin shows also peaks at the following 2-theta positions (deg) (±0.2 deg):
2.82559, 6.174611 , 8.415348, 9.6615(13), 11.0814(9), 11.226753, 12.260441 , 12.790(4), 13.237333, 13.684(8), 14.374(3), 15.19617, 17.243(19), 18.007536,18.827(12), 19.707(8), 21.602(16), 24.27288, 24.596(8), 25.486(7), 25.906691, 27.069(11), 28.193(15), 29.35(5), 29.821302, 30.75(3).
The numbers in parentheses is the estimated standard deviation. “13” means “± 0.013”; “9” means “± 0.009” etc.
The present invention is also directed to a process for the manufacture of crude b- cryptoxanthin (see Figure 7: compound of formula (1)) comprising the following steps:
I) reacting 2,7,11 -trimethyl-13-(2, 6, 6-trimethylcyclohex-1-en-1 -yl)trideca- 2,4,6,8,10,12-hexaenal (see Fig. 7: compound of formula (3)) and 4-(5- (halotriaryl-5-phosphanyl)-3-methylpenta-1,3-dien-1-yl)-3,5, 5- trimethylcyclohex-3-en-1 -ol (see Fig. 7: compound of formula (2)), in the presence of a 30 weight-% methanolic solution of sodium methoxide;
II) separating the thus obtained b-cryptoxanthin to obtain crude b- cryptoxanthin.
Step II) is preferably performed by filtration.
This reaction is shown in Fig. 7. The halogen X in the compound of formula (2) is Br or Cl, preferably it is Cl. Alternatively, for X also HSCV may be used. The substituent R is an aryl or a C1-4 alkyl substituted aryl or alkyl, preferably an aryl, more preferably phenyl. The preferred compound of formula (2) is (/?)-((2E,4E)-5- (4-hydroxy-2,6,6-trimethylcyclohex-1 -en-1 -yl)-3-methylpenta-2,4-dien-1 - yl)triphenylphosphonium halide/hydrogensulfate, especially the corresponding chloride.
Preferably the amount of sodium methoxide is in the range of from 1.00 to 1.50 equivalents, preferably in the range of from 1.00 to 1.20 equivalents, more preferably in the range of from 1.05 to 1.15 equivalents, based on the amount of 4- (5-(halotriaryl-5-phosphanyl)-3-methylpenta-1,3-dien-1 -yl)-3,5,5-trimethylcyclo- hex-3-en-1-ol, preferably based on the amount of (/?)-((2E,4E)-5-(4-hydroxy-2,6,6- trimethylcyclohex-1 -en-1 -yl)-3-methylpenta-2,4-dien-1 -yl)triphenylphosphonium halide/hydrogensulfate, especially the corresponding chloride.
In a preferred embodiment of this process the reaction I) is performed in ethanol.
Thus, an especially preferred process for the manufacture of crude b-cryptoxanthin (see Figure 7: compound of formula (1)) comprises the following steps:
I) reacting 2,7,11 -trimethyl-13-(2, 6, 6-trimethylcyclohex-1 -en-1 -yl)trideca- 2,4,6,8,10,12-hexaenal (compound of formula (3)) and (/?)-((2E,4E)-5-(4- hydroxy-2,6,6-trimethylcyclohex-1 -en-1 -yl)-3-methylpenta-2,4-dien-1 - yl)triphenylphosphonium chloride (compound of formula (2) with X being Cl and R being phenyl) to b-cryptoxanthin in ethanol in the presence of a 30 weight-% methanolic solution of sodium methoxide, whereby the amount of sodium methoxide is in the range of from 1.00 to 1.50 equivalents, preferably in the range of from 1.00 to 1.20 equivalents, more preferably in the range of from 1.05 to 1.15 equivalents, based on the amount of (/?)-((2E,4E)-5-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1- yl)-3-methylpenta-2,4-dien-1 -yl)triphenylphosphonium chloride;
II) separating the thus obtained b-cryptoxanthin to obtain crude b- cryptoxanthin.
Step II) is preferably performed by filtration.
To obtain crystal form I, such crude b-cryptoxanthin is subsequently treated as follows: b) suspending the crude b-cryptoxanthin in an organic solvent and optionally heating the suspension; c) optionally cooling the suspension to a temperature of at most 30 °C, preferably of at most 25 °C, more preferably of at most 20° C; d) optionally adding water to the cooled suspension; e) separating the crystal form I of b-cryptoxanthin; f) drying of the crystalline b-cryptoxanthin, preferably at a temperature in the range of from 40 to 60° C.
Step b)
Suitable organic solvents to be used in step b) are ketones R 1 -C(=0)-R 2 with R 1 and R 2 being independently from each other straight-chain CM alkyl or branched C3-4 alkyl, whereby preferably the total number of carbon atoms in the ketones is from 3 to 6, more preferably from 3 to 5, cyclic ethers with a total amount of carbon atoms from 4 to 7, preferably from 4 to 6, such as e.g. tetrahydrofuran, dialkyl ethers R 3 -C(=0)-R 4 with R 3 and R 4 being independently from each other straight- chain CM alkyl or branched C3-4 alkyl, whereby preferably the total number of carbon atoms in these dialkyl ethers is from 4 to 6, and dialkyl carbonates R 5 0- C(=0)-OR 6 with R 5 and R 6 being independently from each other straight-chain CM alkyl or branched C3-4 alkyl, whereby preferably the total number of carbon atoms in these dialkyl carbonates is from 3 to 6, more preferably from 3 to 4.
Most preferred organic solvents that are suitable for carrying out step b) are acetone, a mixture of acetone and tetrahydrofuran with a volume ratio of 9:1, dimethyl carbonate, tert-butyl methyl ether and methyl ethyl ketone.
In case the organic solvent is acetone (most preferred embodiment) the crude b- cryptoxanthin is suspended in it and the suspension heated to a temperature of at least 20° C, preferably to a temperature of at least 30° C, more preferably to a temperature of at least 40° C, even more preferably to a temperature of at least 45 °C, further preferably to a temperature of at least 50° C, most preferably at reflux. Afterwards the suspension is cooled to a temperature of at most 30° C, preferably of at most 25 °C, more preferably of at most 20° C (step c).
In case a mixture of acetone and tetrahydrofuran with a volume ratio of 9:1 or dimethyl carbonate or tert-butyl methyl ether or methyl ethyl ketone is used as organic solvent, the b-cryptoxanthin suspension is heated to reflux, whereby the transformation of b-cryptoxanthin platelets to b-cryptoxanthin needles took place during the heating up. Thus, if these organic solvents are used heating to a temperature of at least 10 K below the boiling temperature of the organic solvent may be necessary. Afterwards the suspension is cooled to a temperature of at most 30° C, preferably of at most 25 °C, more preferably of at most 20 °C (step c).
Thus, the present invention is directed to a process of obtaining crystal form I comprising the following steps: a) providing crude b-cryptoxanthin; b) to f) as described above.
To purify crystal form II, such crude b-cryptoxanthin is subsequently treated as follows:
B) suspending the crude b-cryptoxanthin in ethanol and heating the suspension to a temperature of at least 60° C, preferably of at least 65 °C, more preferably of at least 70° C, most preferably at reflux;
C) cooling the suspension to a temperature of at most 30° C, preferably of at most 25 °C, more preferably of at most 20° C;
D) separating b-cryptoxanthin;
E) drying the separated b-cryptoxanthin at a temperature in the range of from 30 to 50° C, preferably at a temperature in the range of from 35 to 45 °C, more preferably at a temperature around 40°C, to obtain the crystal form II of b-cryptoxanthin.
Thus, a process of obtaining crystal form II comprises the following steps:
A) providing crude b-cryptoxanthin;
B) to E) as described above.
When the crude b-cryptoxanthin as obtained in the process as disclosed above is dried at a temperature in the range of from 90 to 125°C, preferably at a temperature in the range of from 90 to 120°C, more preferably at a temperature in the range of from 90 to 110°C, even more preferably at a temperature in the range of from 95 to 105°C, most preferably at a temperature around 100°C, the crystal form IV of b-cryptoxanthin is obtained. Crystal form IV of b-cryptoxanthin may also be obtained by digestion of crude b- cryptoxanthin in methanol or in a mixture of methanol and toluene with a volume ratio of 4:1 or in a mixture of methanol and tetrahydrofuran with a volume ratio of 4:1 or in a mixture of methanol and 1 -propanol with a volume ratio of 1 : 1 or in a mixture of methanol and methyl ethyl ketone with a volume ratio of 1 : 1 or in a mixture of ethanol and water with a volume ratio of 1:1, followed by separation, preferably by filtration, and drying.
A process of obtaining crystal form IV comprises the following steps:
A) providing crude b-cryptoxanthin;
B) to E) as described above;
P) drying the separated b-cryptoxanthin at a temperature in the range of from 90 to 125°C, preferably at a temperature in the range of from 90 to 120°C, more preferably at a temperature in the range of from 90 to 110°C, even more preferably at a temperature in the range of from 95 to 105°C, most preferably at a temperature around 100°C, to obtain the crystal form IV of b-cryptoxanthin.
Step e) and Step D), respectively, may be performed by filtration.
An alternative process of obtaining crystal form IV of b-cryptoxanthin comprises the following steps:
A) providing crude b-cryptoxanthin;
B’) suspending the crude b-cryptoxanthin in an organic solvent and heating the suspension to reflux, whereby the organic solvent is selected from methanol, a mixture of methanol and toluene with a volume ratio of 4:1 , a mixture of methanol and tetrahydrofuran with a volume ratio of 4:1, a mixture of methanol and 1- propanol with a volume ratio of 1 :1, a mixture of methanol and methyl ethyl ketone with a volume ratio of 1 : 1 and a mixture of ethanol and water with a volume ratio of 1 : 1 ;
C) cooling the suspension to a temperature of at most 30° C, preferably of at most 25 °C, more preferably of at most 20° C;
D) separating b-cryptoxanthin;
E’) drying the separated b-cryptoxanthin. As starting material also crude b-cryptoxanthin obtained from other chemical syntheses or from fermentation or from extraction from natural sources may be used. Preferably, however, the crude b-cryptoxanthin is manufactured by the advantageous process as disclosed above.
As mentioned above, the invention also relates to a process for the transformation of crystal form II of b-cryptoxanthin into crystal form I of b-cryptoxanthin, which takes place in an organic solvent preferably selected from acetone, a mixture of acetone and tetrahydrofuran with a volume ratio of 9:1, dimethyl carbonate, tert- butyl methyl ether and methyl ethyl ketone, preferably by heating the suspension to reflux, and filtering after the transformation took place. The other organic solvents as mentioned above may also be used for that purpose.
A preferred embodiment of the invention is a process for the transformation of crystal form II of b-cryptoxanthin into crystal form I of b-cryptoxanthin, which takes place in acetone, preferably at a temperature in the range of from 20 to 60° C, more preferably at a temperature in the range of from 40 to 55 °C.
Hereby crystal form II of b-cryptoxanthin is suspended in acetone, optionally heated to a temperature in the range as disclose above, and filtered after the transformation took place.
Another embodiment of the invention is a liquid formulation comprising crystal form I of b-cryptoxanthin and optionally the crystal form II of b-cryptoxanthin, whereby preferably at most 20 weight-%, preferably at most 15 weight-%, more preferably at most 10 weight-%, of crystal form II of b-cryptoxanthin may be present, based on the total amount of crystal form I and II of b-cryptoxanthin in the liquid formulation, as well as the use thereof in the life science industry, especially as dietary supplement, more preferably in soft capsules. Instead of crystal form II of b-cryptoxanthin or additionally to crystal form II of b- cryptoxanthin, crystal form IV of b-cryptoxanthin or any other crystal form of b- cryptoxanthin may also be present. If one or more other crystal form(s) of b- cryptoxanthin such as crystal form II of b-cryptoxanthin and/or crystal form IV of b- cryptoxanthin are present, the total amount of the other crystal form(s) of b- cryptoxanthin together is preferably at most 20 weight-%, preferably at most 15 weight-%, more preferably at most 10 weight-%, based on the total amount of all crystal form(s) of b-cryptoxanthin in the liquid formulation.
For this purpose, the crystal form I of b-cryptoxanthin and optionally the crystal form II of b-cryptoxanthin, whereby preferably at most 20 weight-%, preferably at most 15 weight-%, more preferably at most 10 weight-%, of crystal form II of b- cryptoxanthin, based on the total amount of crystal form I and II of b- cryptoxanthin, may be present, may be milled in an edible oil to obtain a liquid formulation. Instead of or additionally to crystal form II of b-cryptoxanthin, crystal form IV of b-cryptoxanthin may also be used. If crystal form II of b-cryptoxanthin and crystal form IV of b-cryptoxanthin are present, the total amount of crystal form II and crystal form IV of b-cryptoxanthin together is preferably at most 20 weight-%, preferably at most 15 weight-%, more preferably at most 10 weight-%, based on the total amount of crystal form I, II and IV of b-cryptoxanthin in the liquid formulation.
The thus obtained liquid formulation may be directly incorporated in soft capsules or after dilution with an edible oil to the desired concentration incorporated in soft capsules. Therefore, the present invention is also directed to soft capsules comprising the liquid formulations of the present invention. The liquid formulation may further be incorporated in powdered products such as powdered infant formula. Therefore, the present invention is also directed to such powdered infant formula.
Liquid formulations according to the present invention
One formulation according to the present invention is a liquid formulation comprising i) crystal form I of b-cryptoxanthin, whereby optionally one or more other crystal form(s) of b-cryptoxanthin such as e.g crystal form II and/or crystal form IV of b-cryptoxanthin may be present; ii) at least an edible oil; and iii) optionally at least an antioxidant.
In case one or more crystal form(s) of b-cryptoxanthin such as e.g. crystal form II and/or crystal form IV of b-cryptoxanthin is/are present, its/their total amount together is preferably at most 20 weight-%, more preferably at most 15 weight-%, most preferably at most 10 weight-%, based on the total amount of all crystal forms of b-cryptoxanthin.
The single ingredients i) to iii) of said liquid formulation and their amounts are described in detail further below. The amounts of components i) to iii) are based on the total weight of the liquid formulation and sum up to 100%. i) b-Cryptoxanthin b-Cryptoxanthin (compound of formula I) can be obtained from a natural source, by fermentation or by chemical synthesis. A natural source might be mandarins. Chemical syntheses are e.g. described in D. E. Loeber et al. , J. Chem. Soc. C 1971, 404; F. Khachik et al., Synthesis 2011, 3, 509-516. The preferred chemical synthesis is disclosed above.
The lUPAC name of the isomer of b-cryptoxanthin occurring in nature is (1/?)-3,5,5- Trimethyl-4-[(3E,5E,7E,9E,11E,13E,15E)-3,7,12,16-tetramethyl -18-(2,6,6- trimethylcyclohex-1-en-1 -yl)octadeca-1,3,5,7,9,11,13,15,17-nonaen-1 -yl]cyclohex- 3-en-1 -ol. Another name is (3/?)^^-caroten-3-ol. Another isomer occurring in nature is (3R,6’R)-6,£-carotene-3-ol. b-Cryptoxanthin has two stereoisomers: the 3S-form and the 3/?-form depending on the steric configuration of the hydroxyl group at the 3-position of the ring structure present at one end of the molecule. b-Cryptoxanthin also has cis and trans geometrical isomers with respect to the conjugated double bond system of the polyene chain at the center of the molecule. Examples include the 9-cis isomer, the 13-cis isomer, 15-cis isomer and the all-E isomer. Thus, the term “b- cryptoxanthin” as used herein not only encompasses the (all-E)-isomer, but also any of its mono-, oligo- or poly-(Z)-isomers.
Amount of b-cryptoxanthin
The liquid formulation according to the present invention comprises preferably 0.1 to 30.0 weight-%/0.5 to 30 weight-%, more preferably 0.8 to 20.0 weight-%, even more preferably 1 to 17.0 weight-%, most preferably 1.0 to 5.0 weight-% or 10.0 to 17.0 weight-%, of b-cryptoxanthin, based on the total weight of the liquid formulation. ii) Edible Oil
The term “edible oil” in the context of the present invention refers to any oil suitable for human or animal consumption and encompasses e.g. any triglyceride such as vegetable oils or fats like corn oil, sunflower oil, soybean oil, safflower oil, rapeseed oil, peanut oil, palm oil, palm kernel oil, cotton seed oil, olive oil or coconut oil or MCT (middle chain triglycerides) as well as any mixture thereof.
The oils can be from any origin. They can be natural, modified or synthetic. If the oils are natural they can be plant or animal oils. The term “oil” in the context of the present invention thus also encompasses canola oil, sesame oil, hazelnut oil, almond oil, cashew oil, macadamia oil, mongongo nut oil, pracaxi oil, pecan oil, pine nut oil, pistachio oil, sacha Inchi (Plukenetia volubilis) oil or walnut oil.
Also, mixtures of oils may be present. Preferably, the oil is a plant oil or MCT or any mixture thereof. More preferably the oil is corn oil or safflower oil or any mixture thereof. Amount of edible oil
The liquid formulation according to the present invention comprises preferably 70 to 99.9/99.5 weight-%, more preferably 80 to 99.2 weight-%, even more preferably 83 to 99 weight-%, most preferably 95 to 99 weight-% or 83 to 90 weight-%, of edible oil, based on the total weight of the liquid formulation.
Antioxidant
Any food-grade antioxidant may be used. Such antioxidants are known to the person skilled in the art. Preferably the antioxidant is a fat-soluble antioxidant.
Fat-soluble antioxidant
Preferred fat-soluble antioxidants are selected from the group consisting of tocopherols, e.g. DL-a-tocopherol (i.e. synthetic tocopherol), D-a-tocopherol (i.e. natural tocopherol), b-, g- or d-tocopherol, or a mixture of two or more of these.
More preferably the fat-soluble antioxidant is DL-a-tocopherol or a mixture of tocopherols (“mixed tocopherols”). Most preferably the fat-soluble antioxidant is a mixture of tocopherols.
One non-limiting commercial example of mixed natural tocopherols (“MNT”) is “Tocomix 70 IP” from AOM (Buenos Aires, Argentina). Tocomix 70 IP comprises d-a- tocopherol, d- -tocopherol, d-y-tocopherol and d-6-tocopherol, whereby the total amount of tocopherols is at least 70.0 weight-% and the amount of non-a-toco- pherols is at least 56.0 weight-%.
Amount of antioxidant
Since the presence of the antioxidant is optional, its amount in the liquid formulation of the present invention is in the range of from 0 to 10 weight-%, based on the total weight of the liquid formulation.
If present, the total amount of the antioxidant(s) (especially DL-a-tocopherol or a mixture of tocopherols) in the liquid formulations according to the present invention is preferably in the range of from 0.1 to 10 weight-%, more preferably in the range of from 0.2 to 2 weight-%, most preferably in the range of from 0.5 to
1.5 weight-%, based on the total weight of the liquid formulation.
Since any of the ingredients i) to iii) may comprise water in a small amount as residue, a small amount of water may also be present in the liquid formulation according to the present invention. Preferably such amount of water is, however, < 5 weight-%, preferably < 3 weight-%, based on the total weight of the formulation.
Preferred embodiments of the liquid formulation
A preferred embodiment of the present invention is a formulation consisting essentially of the components i) to iii), whereby the amount of component i) is selected in the range from 0.1 to 30 weight-%/0.5 to 30 weight-%, preferably in the range from 0.8 to 20 weight-%, more preferably in the range from 1 to 17.0 weight- %, most preferably in the range from 1 to 5 weight-% or 10.0 to 17.0 weight-%, and/or the amount component ii) is selected in the range from 70 to 99.9/70 to
99.5 weight-%, preferably in the range from 80 to 99.2 weight-%, more preferably in the range from 83 to 99 weight-%, most preferably in the range from 95 to 99 weight-% or from 83 to 90 weight-%, and/or the amount component iii) is selected in the range from 0 to 10 weight-%, preferably in the range from 0.2 to 2 weight-%, more preferably in the range from 0.5 to 1.5 weight-%, all amounts based on the total weight of the liquid formulation.
The term “consisting essentially of” in the context of the present invention means that the amount of the listed ingredients sum up to 100 weight-%. However, it cannot be excluded that small amounts of impurities may be present such as e.g. in amounts of less than 5 weight-%, preferably less than weight-%, which are e.g. introduced via the respective raw materials or processes used.
Process for the manufacture of the liquid formulation
Preferably said liquid formulation is manufactured by comminuting, preferably by milling, a dispersion of crystal form I of b-cryptoxanthin in an oil, whereby optionally another crystal form of b-cryptoxanthin such as e.g. crystal form II of b- cryptoxanthin and/or crystal form IV of b-cryptoxanthin, and optionally at least an antioxidant may be present. If one or more other crystal form(s) such as e.g. crystal form II and/or crystal form IV of b-cryptoxanthin is/are present, their total amount is preferably at most 20 weight-%, more preferably at most 15 weight-%, most preferably at most 10 weight- %, based on the total weight of the b-cryptoxanthin. Thus, the present invention is also directed to a process for the manufacture of the liquid formulation according to the present invention comprising the following step: comminuting, preferably milling, the crystal form I of b-cryptoxanthin according to the present invention and optionally another crystal form of b- cryptoxanthin such as e.g. crystal form II of b-cryptoxanthin and/or crystal form IV of b-cryptoxanthin (component i)) in an edible oil (component ii)) and adding before, during or after comminuting optionally at least an antioxidant, preferably optionally at least a fat-soluble antioxidant (component iii)).
Preferably milling is performed until the particle size D (v, 0.9) of b-cryptoxanthin crystals is < 30 pm, preferably < 20 pm, more preferably < 15 pm, measured dispersed in Volasil® 344 by laser diffraction, Mastersizer3000 ex Malvern, Fraunhofer light scattering. Volasil® 344 is a clear colorless volatile blend of cyclic silicones.
The milling step is preferably carried out with a commercially available ball mill/stirred media mill. The desired mean particle size of the b-cryptoxanthin particles is achieved by adjusting the following parameters with respect to each other: rotor speed (peripheral speed), mean residence time in the mill, material and size of the milling beads and load of the mill. The milling step can either be performed in circulation or passage mode. Hereby a rotor speed, especially a peripheral speed, in the range of from 1 to 15 m/s, a short mean residence time in the mill (e.g. 1 to 10 min), and an average bead load of 65 to 90% may be applied. For such high performance bead milling/grinding stirred media mills using e.g. ZrO beads with an average size in the range of from 0.1 to 3 mm, preferably in the range of from 0.4 to 2 mm, most preferably in the range of from 0.6 to 1 mm may be used.
Preferably the temperature during milling is ranging from 20 to 90° C, more preferably from 25 °C to 80° C, even more preferably from 30 to 70° C, most preferably from 40 to 70° C.
The preferred milling parameters may differ depending on the ball mill used in the milling step, but can easily be adjusted by the person skilled in the art, as long as the amount of other crystal forms of b-cryptoxanthin than crystal form I such as the b-cryptoxanthin platelets (crystal form II and/or crystal form IV) does preferably not exceed an amount of 20 weight-%, based on the total amount of the b-cryptoxanthin.
Dietary supplements according to the present invention
The formulations according to the present invention are especially suitable for the manufacture of dietary supplements, especially in the form of soft capsules, with the preferences as given above.
A soft, soft gel or soft gelatin capsule is a solid capsule (outer shell) surrounding a liquid or semi-solid center (inner fill). An active ingredient can be incorporated into the outer shell, the inner fill, or both. They are oral dosage forms like capsules and tablets. Common soft gel shells are a combination of gelatin, water, opacifier and a plasticizer such as e.g. glycerin and/or sorbitol(s). Processes for their manufacture are well-known to the person skilled in the art (see e.g. Cornelia M. Keck and Rainer H. MCiller: “Moderne Pharmazeutische Technologie” 2009, Chapter 1.8 “Weichkapseln”).
Besides soft capsules, other dietary supplements also covered by the present invention are powdered products such as infant formula products, whereby the oily suspensions of the present invention may be incorporated by a wet mixing-spray drying process into baby milk powder. This process is known to the person skilled in the art and for example described by Y. J. Jian and M. Guo in their article “Processing technology for infant formula”. The infant formula products may be used as liquid such as ready-to-drink infant milks or as powder, i.e. dried e.g. spray-dried afterwards. The liquid formulation of the present invention may also added to other ready-to-drink beverages than ready-to-drink infant milks.
Infant formula products usually contain methylfolate, prebiotics, probiotics, vitamin E, calcium, iron, copper, zinc and magnesium.
Thus, the present invention is also directed to an infant formula product comprising methylfolate, prebiotics, probiotics, vitamin E, calcium, iron, copper, zinc, magnesium and a liquid b-cryptoxanthin formulation according to the present invention.
Thus, the present is directed to a dietary supplement comprising the liquid formulation according to the present invention, preferably being in form of soft capsules, more preferably being in form of soft gelatin capsules, or in form of powdered products, preferably in form of powdered infant formula.
The invention is now further illustrated in the following non-limiting examples. Examples
The following examples illustrate processes to manufacture crystal forms I, II and VIII of b-cryptoxanthin, as well as processes to manufacture liquid formulations comprising such b-cryptoxanthin crystal form I. The examples also illustrate that crystal form I is easy to mill compared to crystal form II.
Example 1 Synthesis of b-cryptoxanthin crystal form I (needles)
Under inert gas atmosphere C25-aldehyde (= (2E,4E,6E,8E,10E,12E)-2,7,11 - trimethyl-13-(2,6,6-trimethylcyclohex-1-en-1 -yl)trideca-2,4,6,8,10,12-hexaenal) (50 g, 135 mmol) and zeanyl salt such as e.g. zeanyl phosphonium chloride (= (/?)- ((2E,4E)-5-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3-m ethylpenta-2,4-dien- 1-yl)triphenylphosphonium chloride) (91 g, 155 mmol) are suspended in ethanol (432 ml). A solution of sodium methoxide (30% in methanol, 30.7 ml, 165 mmol) is added drop wise within 60 min, whereby the temperature is maintained between 22°C and 25°C. Afterwards the reaction mixture is stirred for 1 hour at 24°C before the temperature is raised to reflux. Stirring is continued for another 1.5 hours.
Then the reaction mixture is cooled to room temperature and filtered. The filter cake is rinsed subsequently with ethanol/water 9:1 and water and then dried in vacuum overnight (50°C/10 mbar). Afterwards the crude product is obtained as purple crystals in 93% yield.
Under inert gas atmosphere, the crude material is suspended in acetone (1000 ml) and heated to reflux. When the temperature reaches approximately 42 °C, a change of crystal form is observed. After 30 min at reflux, the reaction mixture is cooled to room temperature. Via addition funnel, water (200 ml) is added and stirring is continued for another 30 min. The red slurry is filtered, and the filter cake is rinsed with acetone/water 8:2. The crystals are dried in vacuum (60°C/10 mbar) for 13 hours. The product is obtained as red crystals (139.4 g, 97% yield).
Example 2: Synthesis of b-cryptoxanthin crystal form II (platelets)
Under inert gas atmosphere C25-aldehyde (= (2E,4E,6E,8E,10E,12E)-2,7,11 - trimethyl-13-(2,6,6-trimethylcyclohex-1-en-1 -yl)trideca-2,4,6,8,10,12-hexaenal) (100 g, 270 mmol) and zeanyl salt such as e.g. zeanyl phosphonium chloride (= (/?)- ((2E,4E)-5-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3-m ethylpenta-2,4-dien- 1-yl)triphenylphosphonium chloride) (182 g, 311 mmol) are suspended in ethanol (864 ml). A solution of sodium methoxide (30% in methanol, 61.7 ml, 331 mmol) is added drop wise and the reaction mixture is stirred for 1 hour at 24°C before the temperature is raised to reflux.
After 1.5 hours at reflux, the slurry becomes more liquid again and stirring is continued for another 1.5 hours. Then the reaction mixture is cooled to room temperature and filtered. The filter cake is rinsed subsequently with ethanol/water 9:1 and water and dried in vacuum overnight (50°C/10 mbar). After that the crude product is obtained as purple crystals (151.8 g, 93% yield).
The crude material is suspended in ethanol (2000 ml) and heated to reflux for 30 min. Then the oil-bath is removed, and the reaction mixture is cooled to room temperature.
The purple slurry is filtered, and the filter cake is rinsed with ethanol. The crystals are dried in vacuum (100°C/10 mbar) for 13 hours. The purified product is obtained as purple crystals (139.4 g, 98% yield).
Comparison Example 3: Synthesis of b-cryptoxanthin crystals with purification in the solvent system methylene chloride/n-hexane
Under inert gas atmosphere C25-aldehyde (9.29 g, 25 mmol) and zeanyl salt (15.41 g, 26.3 mmol) are suspended in ethanol (80 ml). A solution of sodium methoxide (30% in methanol, 5.22 ml, 28.0 mmol) is added drop wise within 60 min. After that the reaction mixture is stirred for 1 hour at 24 °C before the temperature is raised to reflux. After 1.5 hours at reflux, stirring is continued for another 1.5 hours. Then the reaction mixture is cooled to room temperature and filtered. The filter cake is rinsed subsequently with ethanol/water 9:1 and water and dried in vacuum overnight (40°C/10 mbar). After that the crude product is obtained as purple crystals (13.75 g).
Under inert gas atmosphere 5 g of the crude material are dissolved in methylene chloride (100 ml) with gentle heating. After cooling to room temperature, n-hexane (300 ml) is added dropwise over 30 min. After the first 100 ml of methylene chloride are added, crystal formation is observed. After complete addition, an orange-red suspension is obtained. Stirring is continued for 2 hours and after that, the suspension is filtered. The filter cake is rinsed with methylene chloride/hexanes 1 :3 and the crystals are dried under reduced pressure (16 hours at 60 °C/10 mbar). The product is obtained as light-red crystals (2.88 g of crystal form VI of b-cryptoxanthin) in 55% yield (yield of crude product: 91%, crystallization yield: 60%).
Comparison Example 4: Synthesis of b-cryptoxanthin crystals with purification in the solvent system acetone/ethanol (volume ratio ~ 3:1)
In this example the work-up conditions similar to the ones as disclosed in WO 2014/186683 have been used, b-cryptoxanthin is synthesized via Wittig reaction in ethanol as disclosed above.
Under inert gas atmosphere, a suspension of b-cryptoxanthin (10 g, 16.53 mmol) in ethyl acetate (500 ml) is warmed in an oil-bath to 70° C. When the crystalline material is completely dissolved, the solution is allowed to cool to room temperature and the solvent is removed. To the resulting orange-colored solid are added acetone (300 ml) and ethanol (90 ml), and the mixture is heated to reflux for 30 minutes. Then, it is cooled to 5°C and stirring is continued for another 30 minutes. After that the suspension was filtered (G3 sinter) and the filter cake is rinsed with 2x 50 ml of acetone/ethanol (3:1). The crystalline material is dried overnight in the oven under vacuum (50°C / 10 mbar). The product b-cryptoxanthin is obtained as dark-red crystals (5.63 g of crystal form VIII of b-cryptoxanthin,
52.2% yield, 84.7% purity, 7.3% ethanol, 1.5% acetone).
Examples 5-11
Examples 5-7 and 10-11: Preparation of oily suspensions, i.e. liquid formulations according to the present invention
Examples 8 and 9: Comparison examples
The content of b-cryptoxanthin in the prepared liquid formulations has been determined by HPLC. Furthermore, the following particles sizes: D(v, 0.1), D(v, 0.5) and D(v, 0.9) have been measured dispersed in Volasil® 344 (clear colourless volatile blend of cyclic silicones) by laser diffraction, Mastersizer3000 ex Malvern, Fraunhofer light scattering. The per-se stability of the liquid formulations has also been investigated.
Therefore, samples have been stored in amber glass or aluminum bottles at 25 °C and 40° C, and the b-cryptoxanthin retention measured by HPLC [%].
All results are summarized in Table 1 below.
Example 5: Preparation of an oily suspension with b-cryptoxanthin needles (crystal form I) in safflower oil
16 weight-% of b-cryptoxanthin needles (crystal form I) are suspended in 83 weight- % of safflower oil and 1 weight-% of DL-a-tocopherol, all amounts based on the total weight, with a rotor/stator device starting at 3500 rpm at room temperature and increasing to 6000 rpm and 55 °C within 7 min. Low-viscous crude suspension (see Figure 8) is transferred to a laboratory stirred media mill. Milling is done in passage mode with grinding media size of 1 mm (6 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The outlet temperature of the suspension during milling is adjusted to max. 55 °C by water cooling. Decrease of b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage. No transformation of the needle-shaped b-cryptoxanthin crystals into another crystal shape is observed under optical microscope with polarized light.
Example 6: Preparation of an oily suspension with b-cryptoxanthin needles (crystal form I) in corn oil
11 weight-% of b-cryptoxanthin needles (crystal form I) are suspended in 89 weight- % of corn oil, both amounts based on the total weight, and stirred with a rotor/stator device starting at 4000 rpm at room temperature and increasing to 6000 rpm and 46°C within 6 min. The low-viscous crude suspension is transferred to a laboratory stirred media mill. Milling is done in passage mode with grinding media size of 1 mm (6 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The outlet temperature of the suspension during milling is adjusted to max. 55°C by water cooling. Decrease of the b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage. No transformation of the needle-shaped b-cryptoxanthin crystals into another crystal shape is observed under optical microscope with polarized light.
The final suspension is split in two halves. One half is not further processed. To the second half DL-a-tocopherol is added and the suspension is mixed to end up with 1 weight-% DL-a-tocopherol.
Example 7: Preparation of an oily suspension with b-cryptoxanthin needles (crystal form I) in corn oil
16 weight-% of b-cryptoxanthin needles (crystal form I) are suspended in 84 weight- % of corn oil, both amounts based on the total weight, with a laboratory dissolver at room temperature for 20 min, starting at 400 rpm and increasing to 1200 rpm. Afterwards a rotor/stator device is used at 4000 rpm starting at room temperature and increasing to 50° C within 4 min. The low-viscous crude suspension is transferred to a laboratory stirred media mill. Milling is done in passage mode with grinding media size of 1 mm (6 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The outlet temperature of the suspension during milling is adjusted to max. 65 °C by water cooling. Decrease of the b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage. No transformation of the needle-shaped b-cryptoxanthin crystals into another crystal shape is observed under optical microscope with polarized light.
Comparison-Example 8: Attempt to prepare an oily suspension with crystal form IV of b-cryptoxanthin (= b-cryptoxanthin platelets)
16 weight-% of b-cryptoxanthin platelets (crystal form IV) are suspended in 83 weight% safflower oil and 1 weight-% DL-a-tocopherol, all amounts based on the total weight, with a rotor/stator device starting at 3500 rpm at room temperature and increasing to 4500 rpm and 36°C within 3 min. While suspending the b- cryptoxanthin platelets in oil, surprisingly the oily suspension turns into a very viscous paste. Due to very high viscosity of the crude suspension, milling of the crude suspension with a laboratory stirred media mill is not possible at this concentration. Suspension with that high viscosity cannot be transferred into the milling chamber. Thus, milling of b-cryptoxanthin platelets in oil is not possible.
Comparison example 9: Attempt to prepare an oily suspension with crystal form II of b-cryptoxanthin (= b-cryptoxanthin platelets)
Comparison example 7 is repeated with corn oil instead of safflower oil and crystal form II of b-cryptoxanthin instead of crystal form IV of b-cryptoxanthin. The result is the same: Milling of b-cryptoxanthin platelets in oil is not possible.
Example 10: Preparation of an oily suspension with a mixture of b-cryptoxanthin needles (crystal form I) and b-cryptoxanthin platelets (crystal form II) in a weight ratio of 95:5
11 weight-% of b-cryptoxanthin (whereof 95% are crystal form I and 5% are crystal form II) are suspended in 89 weight% of corn oil, both amounts based on the total weight, with a laboratory dissolver at room temperature at 1200 rpm for 20 min. Further suspending is carried out with a rotor/stator device starting at 5000 rpm at 27°C and increasing to 6000 rpm and 48°C within 5 min. The low-viscous crude suspension is transferred to a laboratory stirred media mill (Dispermat SL-12C1, VMA-Getzmann Gmbh). Milling is done in passage mode with grinding media size of 0.65 mm (8 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The temperature is adjusted during milling to max. 63 °C by water cooling. Decrease of b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage.
Example 11: Preparation of an oily suspension with a mixture of b-cryptoxanthin needles and b-cryptoxanthin platelets in a weight ratio of 95:5
16 weight-% of b-cryptoxanthin (whereof 95% are crystal form I and 5% are crystal form II) are suspended in 84 weight-% of corn oil, both amounts based on the total weight, with a laboratory dissolver at room temperature at 500 rpm for 20 min. Further suspending is carried out with a rotor/stator device starting at 3000 rpm at room temperature and increasing to 7000 rpm and 53 °C within 4 min. Low-viscous crude suspension is transferred to a laboratory stirred media mill (Dispermat SL- 12C1 , VMA-Getzmann Gmbh). Milling is done in passage mode with grinding media size of 1 mm (6 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The temperature is adjusted during milling to max. 62°C by water cooling. Decrease of b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage.
Example 12: Preparation of an oily suspension with b-cryptoxanthin needles and mixed tocopherols as antioxidant
16 weight-% of b-cryptoxanthin needles (crystal form I) are suspended in 83 weight- % of corn oil and 1 weight-% of mixed tocopherol, all amounts based on the total weight, with a laboratory dissolver at room temperature at 1000 rpm for 20 min. Further suspending is carried out with a rotor/stator device starting at 3000 rpm at room temperature and increasing to 7000 rpm and 53 °C within 4 min. Low-viscous crude suspension is transferred to a laboratory stirred media mill. Milling is done in passage mode with grinding media size of 1 mm (6 passages), a bead filling ratio of 80% and a rotor speed of 11 m/s. The outlet temperature of the suspension during milling is adjusted to max. 58° C by water cooling. Decrease of b-cryptoxanthin crystal particle size is measured by laser diffraction analysis after each milling passage. No transformation of the needle-shaped b-cryptoxanthin crystals into another crystal shape is observed under optical microscope with polarized light.
Table 1 (FS = fluid suspension): The amounts of the ingredients of the compositions are given in weight-%, based on the total weight of the composition