Beta-cryptoxanthin from plant source and a process for its preparation

Abstract

The present invention provides beta-cryptoxanthin crystals from plant source and a process for its preparation. The present invention particularly relates to a process for the preparation of high purity beta-cryptoxanthin crystals comprising at least about 10% by weight total xanthophylls, of which at least about 75% by weight is trans-beta-cryptoxanthin and the remaining including beta-carotene, and trace amounts of trans-capsanthin and other carotenoids derived from the plant source, including capsicum fruits. The production of beta-cryptoxanthin crystals with high content of trans-beta-cryptoxanthin makes it ideal and suitable for use as a provitamin A source material and also has potential effects on improving bone health and inhibiting bone resorption.

Claims

1. A process for the preparation of a trans-beta-cryptoxanthin enriched concentrate from plant material comprising about 10-80% by weight total xanthophylls, of which about 75-98% by weight is trans-beta-cryptoxanthin, the process comprising: (a) mixing an oleoresin of plant material comprising xanthophylls esters with an aliphatic alcoholic solvent; (b) saponifying the xanthophylls esters present in the oleoresin with an alkali at an elevated temperature; (c) removing the aliphatic alcoholic solvent followed by addition of water to obtain a diluted resultant mixture; (d) adding a diluted organic acid to the diluted resultant mixture to form a water layer and a precipitated xanthophylls mass; (e) removing the water layer and washing the precipitated xanthophylls mass with a polar solvent; (f) drying the precipitated xanthophylls mass to obtain a crude xanthophylls mass; (g) washing the crude xanthophylls mass with a non-polar solvent and concentrating the non-polar solvent washings to obtain a concentrated crude xanthophylls mass; (h) transferring the concentrated crude xanthophylls mass to a silica gel column and washing with a non-polar solvent; (i) eluting the column with a mixture of non-polar and polar solvent and concentrating the elutions to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; (j) admixing the trans-beta-cryptoxanthin-rich xanthophylls concentrate with an aliphatic alcohol and then cooling; and (k) filtering and drying the trans-beta-cryptoxanthin-rich xanthophylls concentrate to obtain the purified trans-beta-cryptoxanthin concentrate.

2. The process of claim 1, wherein the xanthophylls esters in the oleoresin of plant material in step (a) are present at about 6-8% by weight.

3. The process of claim 1, wherein the ratio of oleoresin to alcohol in step (a) ranges from about 1:0.25 to about 1:1 weight/volume.

4. The process of claim 1, wherein the ratio of oleoresin to alkali in step (b) ranges from about 1:0.25 to 1:0.5 weight/weight.

5. The process of claim 1, wherein the diluted organic acid of step (d) is acetic acid or phosphoric acid.

6. The process of claim 1, wherein the polar solvent of step (e) is water.

7. The process of claim 1, wherein a carotene concentrate is obtained by distilling the non-polar solvent washing of step (h).

8. The process of claim 1, wherein the non-polar solvent and polar solvent of step (i) are in a ratio of about 95:5 to about 98:2.

9. The process of claim 1, wherein the trans-beta-cryptoxanthin-rich xanthophylls concentrate of step (i) comprises at least about 10% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 75% by weight.

10. The process of claim 1, wherein the purified trans-beta-cryptoxanthin concentrate of step (k) comprises at least about 40% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 90% by weight.

11. The process of claim 1, further comprising a step (l): washing the purified trans-beta-cryptoxanthin concentrate with a mixture of non-polar and ester solvent and cooling for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.

12. The process of claim 11, wherein the high purity trans-beta-cryptoxanthin crystals of step (l) comprises at least about 80% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 98% by weight.

13. The process of claim 11, wherein the non-polar solvent and ester solvent of step (l) are in a ratio of about 80:20 to about 90:10.

14. The process of claim 11, wherein the cooling in step (l) is performed at temperature of about −10° C.

15. The process of claim 1, wherein the total xanthophylls comprise byproducts selected from zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids, and any combinations thereof.

16. The process of claim 1, wherein the plant material is selected from the group consisting of fruits, vegetables, and mixtures thereof.

17. The process of claim 16, wherein the plant material is from a capsicum.

18. The process of claim 1, wherein the aliphatic alcohol of step (a) or (j) is selected from the group consisting of ethanol, methanol, isopropyl alcohol, and mixtures thereof.

19. The process of claim 1, wherein the alkali of step (b) is selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

(1) FIG. 1 depicts the chemical structures of alpha-cryptoxanthin, beta-cryptoxanthin and zeinoxanthin.

(2) FIG. 2 is a graph that depicts the Pyridinoline Crosslinks determination in the sham control, OVX control, and BCX-A, -B, and -C groups.

(3) FIG. 3 is a graph that depicts the bone density determination in the sham control, OVX control, and BCX-A, -B, and -C groups.

(4) FIG. 4 is a graph that depicts the failure load of bone in the sham control, OVX control, and BCX-A, -B, and -C groups.

DETAILED DESCRIPTION OF THE INVENTION

(5) The product and the process of the present invention is described herein below which is illustrative as shown in the examples and should not be construed to limit the scope of the present invention in any manner whatsoever.

Definitions

(6) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entireties for all purposes.

(7) As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.

(8) Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, that is, occurrences of the element or component. Therefore, “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

(9) The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

(10) As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term “about” includes, without limitation, ±10%.

(11) Units, prefixes, and symbols are denoted in their Systéme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

(12) Beta-Cryptoxanthin Concentrate

(13) The present invention provides a beta-cryptoxanthin concentrate, which contains about 10-80% by weight total xanthophylls, of which about 75-98% by weight is trans-beta-cryptoxanthin, the remaining including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids, derived from oleoresin or extract of plant material and which is useful for nutrition and health care.

(14) In certain embodiments, the concentrate comprises at least about 10% by weight total xanthophylls, of which at least about 75% by weight is trans-beta-cryptoxanthin.

(15) In certain embodiments, the concentrate comprises at least about 40% by weight total xanthophylls, of which at least about 90% by weight is trans-beta-cryptoxanthin.

(16) In certain embodiments, the concentrate comprises at least about 80% by weight total xanthophylls, of which at least about 98% by weight is trans-beta-cryptoxanthin.

(17) Natural Sources

(18) The plant material is derived from sources including, but not limited to, fruits and vegetables. In some embodiments of the invention, the plant material is derived from capsicums. Capsicum is a genus of flowering plants that includes several varieties of peppers, such as but not limited to red peppers, and the word “capsicum” is also used interchangeably in several parts of the world when referring to peppers. The capsicum oleoresin described herein also includes paprika oleoresin.

(19) Dosage and Administration

(20) The beta-cryptoxanthin enriched concentrates of the invention can be formulated in a dosage form including, but not limited to, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations. The beta-cryptoxanthin enriched concentrates of the invention can also be provided in a food or feed (including liquid or solid) composition. Thus, it is envisioned that suitable delivery methods include, but are not limited to, oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intracranial, or buccal administration.

(21) Compositions comprising the trans-beta-cryptoxanthin enriched concentrates of the invention include one or more suitable pharmaceutically acceptable ingredients or food grade ingredients such as, but not limited to, carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrators, solubilizers and isotonic agents.

(22) The compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of the trans-beta-cryptoxanthin enriched concentrates. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, for example in methods of treatment or pharmaceutical compositions for use in such methods. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic or preventive result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease or a condition requiring treatment is identified, the prophylactically effective amount will be less than the therapeutically effective amount.

(23) Dosage amounts useful for administering the trans-beta-cryptoxanthin enriched concentrates of the invention can range, e.g., from about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 1.0 mg/kg, and more usually from about 0.01 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 0.1 mg/kg, from about 0.01 mg/kg to about 0.05 mg/kg, from about 0.01 mg/kg to about 0.025 mg/kg, from about 0.01 mg/kg to about 0.2 mg/kg, or from about 0.05 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 1.0 mg/kg, or from about 0.05 mg/kg to about 0.1 mg/kg of the host body weight. For example dosages can be about 0.005 mg/kg body weight, about 0.01 mg/kg body weight, about 0.05 mg/kg body weight, about 0.1 mg/kg body weight, about 1.0 mg/kg body weight, or about 10 mg/kg body weight or within the range of about 0.001-1.0 mg/kg, preferably at least 0.005 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention (e.g., about 0.002 mg/kg, about 0.025 mg/kg, about 0.05 mg/kg, about 0.075 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 2 mg/kg, etc.). Subjects can take doses daily or be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months.

(24) Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the trans-beta-cryptoxanthin enriched concentrates of the invention and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such trans-beta-cryptoxanthin enriched concentrates of the invention for treatment sensitivity in mammalian subjects. The individuals or mammalian subjects of the invention can include both human and animal subjects including domestic animals, pets, and farm-raised fish.

(25) Processes

(26) The present invention provides a process for the preparation of beta-cryptoxanthin enriched concentrate from plant material comprising about 10-80% by weight total xanthophylls, of which about 75-98% by weight is trans-beta-cryptoxanthin, the remaining including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids, suitable for human consumption as nutritional supplements, the process comprising: a) Mixing xanthophylls esters in an oleoresin with an aliphatic alcohol solvent; b) Saponifying the xanthophylls esters present in the oleoresin of plant material with an alkali at an elevated temperature; c) Removing the aliphatic alcoholic solvent followed by addition of water to get diluted resultant mixture; d) Adding a diluted organic acid to the diluted resultant mixture to form a water layer and a precipitated xanthophylls mass; e) Removing the water layer and washing the precipitated xanthophylls mass with a polar solvent; f) Drying the precipitated xanthophylls mass to obtain a crude xanthophylls mass; g) Washing the crude xanthophylls mass with a non-polar solvent and concentrating the non-polar solvent washings to get a concentrated crude xanthophylls mass; h) Transferring the concentrated crude xanthophylls mass to a silica gel column and washing with a non-polar solvent; i) Eluting the column with a mixture of non-polar and polar solvent and concentrating the elutions to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; j) Admixing the trans-beta-cryptoxanthin-rich concentrate with an aliphatic alcohol and then cooling; and k) Filtering and drying the trans-beta-cryptoxanthin-rich xanthophylls concentrate to obtain a purified trans-beta-cryptoxanthin concentrate.

(27) In certain embodiments, the xanthophylls esters in the oleoresin of plant material are present at about 2-12% by weight, about 4-10% by weight, or about 6-8% by weight.

(28) In certain embodiments, the aliphatic alcohol comprises a hydrocarbon fragment derived from a fatty, nonaromatic hydrocarbon and is selected from the group consisting of ethanol, methanol, isopropyl alcohol, and mixtures thereof. In some embodiments, the aliphatic alcohol is ethanol.

(29) In certain embodiments, the ratio of oleoresin to alcohol ranges from about 1:0.25 to about 1:1 weight/volume. In some embodiments, the ratio is about 1:1, about 1:0.75, or about 1:50.

(30) In certain embodiments, the alkali is a soluble hydroxide of the alkali metals, including lithium, sodium, potassium, rubidium, or cesium, and is selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the alkali is sodium hydroxide. In other embodiments, the alkali is potassium hydroxide.

(31) In certain embodiments, the ratio of oleoresin to alkali ranges from about 1:0.25 to about 1:0.5 weight/weight. In some embodiments, the ratio is about 1:0.25.

(32) In certain embodiments, the elevated temperature for saponification is above room temperature. In some embodiments, the elevated temperature ranges from about 65 to about 95° C., about 70 to about 90° C. about 75 to about 85° C., from about 75 to about 80° C., or from about 80 to about 85° C.

(33) In certain embodiments, the water added to form the diluted resultant mixture in step (c) above is about 2 to about 10 times, about 3 to about 9 times, about 4 to about 8 times, or about 5 to about 7 times that of the oleoresin (weight/weight). In some embodiments the water added is about 4 times, about 5 times, or about 6 times that of the oleoresin (weight/weight). In some embodiments, the water added is about 5 times that of the oleoresin (weight/weight).

(34) In certain embodiments, the diluted organic acid used in the process is acetic acid or phosphoric acid. In certain embodiments, the diluted organic acid is a solution of about 20% to about 50% of organic acid. In some embodiments, the diluted organic acid is about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 20% to about 40%, about 30% to about 50% of organic acid. In some embodiments, the diluted organic acid is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of organic acid.

(35) In certain embodiments, the polar solvent used to wash the precipitated xanthophylls mass is water.

(36) In certain embodiments, the non-polar solvent used in the process is selected from the group consisting of a hexane, a pentane, a heptane, and mixtures thereof.

(37) In certain embodiments, the crude xanthophylls mass and the non-polar solvent are in a ratio of about 1:5 to about 1:20 weight/volume, about 7.5 to about 17.5 weight/volume, or about 1:10 to about 1:15 weight/volume.

(38) In certain embodiments, the concentrated crude xanthophylls mass and the non-polar solvent are in a ratio of about 1:2 to about 1:14 weight/volume, about 1:3 to about 1:12 weight/volume, about 1:4 to about 1:10 weight/volume, or about 1:5 to about 1:8 weight/volume.

(39) In certain embodiments, a carotene concentrate is obtained by distilling the non-polar solvent washing in step (h) above. In certain embodiments, the carotene concentrate is beta-carotene.

(40) In certain embodiments, the polar solvent used in the process is selected from the group consisting of a propanone, a pentanone, and mixtures thereof.

(41) In certain embodiments, the non-polar solvent and polar solvent in step (i) above are in a ratio of about 90:10 to about 99:1, about 92.5 to about 99:1, about 94:6 to about 98:2, or about 95:5 to about 98:2 (volume/volume).

(42) In certain embodiments, the temperature used to cool the trans-beta-cryptoxanthin-rich xanthophylls concentrate is from about 5° C. to about 15° C. or about 7.5° C. to about 12.5° C. In some embodiments, the temperature is about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., or about 12° C.

(43) In certain embodiments, the trans-beta-cryptoxanthin-rich xanthophylls concentrate comprises at least about 10% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 75% by weight.

(44) In certain embodiments, the purified trans-beta-cryptoxanthin concentrate comprises at least about 40% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 90% by weight.

(45) In certain embodiments, the process above further comprises a step (l): washing the purified trans-beta-cryptoxanthin concentrate with a mixture of non-polar and ester solvent and cooling for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.

(46) In certain embodiments, the high purity trans-beta-cryptoxanthin crystals of step (l) comprises at least about 80% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at least about 98% by weight.

(47) In certain embodiments, the ester solvent of step (l) is ethyl acetate and the non-polar solvent of step (l) is hexane.

(48) In certain embodiments, the non-polar solvent and ester solvent of step (l) are in a ratio of about 70:30 to about 90:10, about 80:20 to about 90:10, about 75:25 to about 95:5, or about 85:15 to about 95:5 (volume/volume).

(49) In certain embodiments, the temperature for cooling in step (l) above is from about −5° C. to about −15° C. or about −7.5° C. to about −12.5° C. In some embodiments, the temperature for cooling in step (l) above is about −7° C., about −8° C., about −9° C., about −10° C., about −11° C., about −12° C.

(50) In certain embodiments, the total xanthophylls comprise by-products selected from zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids, and any combinations thereof. In some embodiments, the trace amounts of other carotenoids include capsorubin or violaxanthin.

(51) In certain embodiments, the plant material is selected from the group consisting of fruits, vegetables, and mixtures thereof. In some embodiments, the plant material is from a capsicum.

(52) In certain embodiments, the solvent used in the process is removed by methods including, but not limited to, evaporation under vacuum. In certain embodiments, the saponification of the xanthophylls esters is carried out for at least 2 hours with agitation. In some embodiments, the saponification is carried out for about 2 to about 20 hours, about 2 to about 15 hours, about 2 to about 10 hours, about 3 to about 8 hours, about 3 to about 6 hours, or about 3 to 5 hours.

(53) In some embodiments, the non-polar hydrocarbon solvent used in the process is hexane or mixture of low boiling hydrocarbons, such as pentane or heptane. In some embodiments, the aliphatic alcohol selected for saponification is ethanol and the alkali used is selected from sodium or potassium hydroxide.

(54) In some embodiments, the silica gel column containing xanthophylls concentrate is eluted with non-polar solvent to remove the carotenes to obtain beta-cryptoxanthin concentrate.

(55) In some embodiments, the process comprises further washing the columns with non-polar:polar solvent and concentrating the washing results in beta-cryptoxanthin concentrate comprising about 10% by weight total xanthophylls out of which trans-beta-cryptoxanthin content is at least about 75% by weight and the remaining being beta-carotene, trans-capsanthin, zeaxanthin and traces of other carotenoids. The purified beta-cryptoxanthin concentrate comprises at least about 40% by weight of total xanthophylls out of which trans-beta-cryptoxanthin content is at least about 90% by weight and the high purity beta-cryptoxanthin concentrate obtained by washing the purified beta-cryptoxanthin concentrate with a mixture of non-polar:ester solvent and cooling for precipitation results in a concentrate which comprises at least about 80% by weight of total xanthophylls out of which trans-beta-cryptoxanthin content is at least about 98% by weight, the rest being beta-carotene, zeaxanthin, trans-capsanthin and traces of other carotenoids.

(56) In one embodiment, the present invention provides for the preparation of a beta-cryptoxanthin enriched concentrate from plant material comprising at least about 80% by weight total xanthophylls, of which at least about 98% by weight is trans-beta-cryptoxanthin, the process comprising: (a) mixing an oleoresin of plant material comprising xanthophylls esters with ethanol, wherein the ratio of oleoresin to ethanol is about 1:1 weight/volume; (b) saponifying the xanthophylls esters present in the oleoresin with potassium hydroxide without addition of water, wherein the ratio of oleoresin to potassium hydroxide is about 1:0.25 weight/weight; (c) applying heat to the oleoresin to elevate the temperature up to reflux at about 80-85° C.; (d) agitating the oleoresin for about 3 to 5 hours at about 80-85° C.; (e) evaporating the ethanol under vacuum followed by addition of water at about 5 times that of the oleoresin (weight/weight) to obtain a diluted resultant mixture and agitating for about 1 hour; (f) neutralizing the diluted resultant mixture with about 25% acetic acid to form a water layer and a precipitated xanthophylls mass; (g) separating the water layer from the precipitated xanthophylls mass and washing the mass with water to remove soaps and other polar soluble materials; (h) drying the precipitated xanthophylls mass under vacuum to obtain a crude xanthophylls mass; (i) washing the crude xanthophylls mass with about 1:10 hexane (weight/volume) and concentrating the hexane washings to obtain a concentrated crude xanthophylls mass; (j) transferring the concentrated crude xanthophylls mass to a silica gel column at a ratio of about 1:5 (weight/weight) and eluting with hexane to obtain a carotene fraction; (k) washing the column with about 98:2 hexane to acetone and concentrating the washings to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; (l) admixing the trans-beta-cryptoxanthin-rich xanthophylls concentrate with about 1:2 ethanol under stirring and then cooling at about 10° C. for about 8 hours; (m) filtering and drying the trans-beta-cryptoxanthin-rich xanthophylls concentrate under vacuum to obtain a purified trans-beta-cryptoxanthin concentrate; and (n) washing the purified trans-beta-cryptoxanthin concentrate with about 80:20 hexane:ethylacetate and cooling to about −10° C. for about 18 hours for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.

(57) The process by-products include beta-carotene, trans-capsanthin, zeaxanthin or mixtures thereof.

(58) A novel feature of the present process is the preparation of high purity trans-beta-cryptoxanthin concentrate crystals from a natural source such as capsicum extract, which has not been reported in the art.

(59) The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLES

Example 1

(60) A weighed quantity of 100 g of Paprika oleoresin containing 7.72% total xanthophylls and a color value of 1,23,515 units (HPLC profile of the oleoresin: beta-15.36% carotene; 10% trans-beta-cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85° C. with stirring. This saponification process was maintained for 3-5 hours at 80-85° C. with gentle agitation. The reaction mixture was cooled, and then ethanol was distilled out from the mass. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was separated, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponified mass concentrate obtained was 124 g with a total xanthophylls content of 3.73% by weight (HPLC profile of the saponified mass concentrate: 22.53% beta-carotene; 12.32% trans-beta-cryptoxanthin; 11% zeaxanthin; and 29.3% trans-capsanthin).

(61) The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to obtain a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 72 g with a total xanthophylls content of 3.2% (HPLC profile of the concentrated crude xanthophylls mass: 39.01% beta-carotene; 21.78% trans-beta-cryptoxanthin; 5.70% zeaxanthin; and 9.86% trans-capsanthin).

(62) The residue (saponified xanthophylls) remaining after hexane wash was 22 g, which on analysis showed a total xanthophylls content of 10% (HPLC profile of the residue: 0.7% beta-carotene; 3.43% trans-beta-cryptoxanthin; 15.32% zeaxanthin; and 52.84% trans-capsanthin).

(63) The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200 mesh (wt/wt). The column was washed with hexane, and the separated band was collected and concentrated (yield 55 g with a total xanthophylls content of 2.3%, HPLC profile: 99.8% beta-carotene). The column was then eluted with 98:2 hexane:acetone (v/v), and the eluent collected and concentrated. This concentrate layer was enriched with beta-cryptoxanthin (yield 5.2 g with a total xanthophylls content of 10.26%, HPLC profile: 75.56% trans-beta-cryptoxanthin). Finally, the column was washed with acetone and the washings concentrated to obtain trans-capsanthin enriched residue.

Example 2

(64) A quantity of approximately 100 g of Paprika oleoresin containing 6.50% total xanthophylls and a color value of 1,05,457 units (HPLC profile of the oleoresin: 15.73% beta-carotene; 9.07% trans-beta-cryptoxanthin; 10.54% zeaxanthin and 31.38% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85° C. with stirring. This saponification process was maintained for 3-5 hours at 80-85° C. with gentle agitation. The reaction mixture was cooled, and then ethanol was distilled out from the mass. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 40% acetic acid solution. The water layer from the mass was separated, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponified mass concentrate obtained was 126 g with a total xanthophylls content of 3.73% by weight (HPLC profile of the saponified mass concentrate: 16.34% beta-carotene; 9.41% trans-beta-cryptoxanthin; 8.57% zeaxanthin; and 24.35% trans-capsanthin).

(65) The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to obtain a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 76.15 g with a total xanthophylls content of 3.26% (HPLC profile of the concentrated crude xanthophylls mass: 31.80% beta-carotene; 14.04% trans-beta-cryptoxanthin; 4.35% zeaxanthin; and 8.70% trans-capsanthin).

(66) The residue (saponified xanthophylls) remaining after hexane wash was 16 g, which on analysis showed a total xanthophylls content of 11% (HPLC analysis of the residue: 1.22% beta-carotene; 0.75% trans-beta-cryptoxanthin; 33.29% zeaxanthin; and 29.99% trans-capsanthin).

(67) The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200 mesh (wt/wt), eluted with hexane, and the first band separated was collected and concentrated (yield 54.72 g with a total xanthophylls content of 1.08%, HPLC profile: 85.88% beta-carotene). The column was then eluted with 98:2 hexane:acetone (v/v) collecting the eluent fraction and concentrated. This fraction was enriched with beta-cryptoxanthin, yielding 4.02 g with a total xanthophylls content of 9% (HPLC profile of the enriched beta-cryptoxanthin concentrate: 76.04% trans-beta-cryptoxanthin). Finally the column was washed with acetone.

(68) The 4.02 g fraction concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled for 8 hrs at 10° C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.42 g crystalline precipitate with a total xanthophylls content of 42.45%. The HPLC profile of the crystalline precipitate showed 98.3% trans-beta-cryptoxanthin.

Example 3

(69) A weighed quantity of Paprika oleoresin (100 g) containing 6-8% by weight total xanthophylls and a color value of 1,00,000 units (HPLC profile of the oleoresin: 15.36% beta-carotene; 10% trans-beta-cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85° C. with stirring. This saponification process was maintained for 3-5 hours at 80-85° C. with gentle agitation. The reaction mixture was cooled and then ethanol was distilled off from the mass under vacuum. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was removed, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponified mass concentrate obtained was 121.75 g with a total xanthophylls content of 4.92% by wt (HPLC profile of the saponified mass concentrate: 21.76% beta-carotene; 12.74% trans-beta-cryptoxanthin; 10.13% zeaxanthin; and 38.25% trans-capsanthin).

(70) The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to get a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 85.81 g with a total xanthophylls content of 3.21% by wt (HPLC profile of the concentrated crude xanthophylls mass: 35.28% beta-carotene; 19.65% trans-beta-cryptoxanthin; 3.99% zeaxanthin; and 13.88% trans-capsanthin).

(71) The residue (saponified xanthophylls) remaining after hexane wash was 25.65 g, which on analysis showed a total xanthophylls content of 10.42% by wt (HPLC analysis of the residue: 0.7% beta-carotene; 1.24% trans-beta-cryptoxanthin; 18.98% zeaxanthin; and 52.32% trans-capsanthin.

(72) The hexane concentrate was dissolved in minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 concentrate to Silica gel 100-200 mesh (wt/wt), eluted with 5-8 volumes of hexane, and the first band separated was eluted and concentrated (yield 55 g with a total xanthophylls content of 2.29% wt, HPLC profile: 99% beta-carotene). The column was then eluted with 98:2 hexane:acetone (vol/vol) collecting the eluent fraction and concentrated. This concentrate was enriched with beta-cryptoxanthin, yielding 9.06 g with a total xanthophylls content of 6.12% by wt (HPLC profile of the enriched beta-cryptoxanthin concentrate: 71.80% trans-beta-cryptoxanthin). Finally the column was eluted with acetone.

(73) The 9.06 g beta-cryptoxanthin concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled for 8 hours at 10° C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.5 g with a total xanthophylls content of 42.35% by wt. The HPLC profile of the crystal showed 98.3% trans-beta-cryptoxanthin content.

(74) The 0.5 g beta-cryptoxanthin precipitate was dissolved in a minimum amount of 80:20 hexane:ethylacetate (vol/vol) and chilled for 18 hrs at −10° C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.03 g with a total xanthophylls content of 80% and HPLC profile for trans-beta-cryptoxanthin of 98.50%.

Example 4

Anti-Resorptive Property of β-Cryptoxanthin and its Effect on Bone Mechanical Strength

(75) Description of Test Materials:

(76) TABLE-US-00001 Total Xanthophyll Samples content Source BCX-A 80.3% Obtained through Marigold oleoresin BCX-B 0.793%  Obtained through Orange fruits BCX-C   10%* Obtained through Paprika oleoresin *For experimental purpose the sample was diluted to 1%.

(77) Test System:

(78) Wistar rats of age between 8 to 10 weeks and weighing between 180-230 gm.

(79) Housing of Animals:

(80) Animals were divided into 5 groups of 6 animals in each group. Each cage was labeled with the name of group, protocol number, species/strain and sex of animal. The total number of cages used was 10 for 30 animals. Each cage housed three animals at the temperature (25° C.±2) and 50-70% relative humidity with 12 h light/dark cycle. All the animals had free access to water. Rice husk was used as bedding in the cages. The cages were cleaned on daily basis.

(81) Bilateral Ovariectomy Procedure:

(82) All surgical instruments were sterilized before use. The dorsal skin of rat was shaved and disinfected using Povidone iodine solution. Ovariectomy was performed by two dorso-lateral incisions, approximately 1 cm long above the ovaries. With the use of a sharp dissecting scissors, skin cut was made almost together with the dorsal muscles to access peritoneal cavity. The ovary was found surrounded by a variable amount of fat. Blood vessels were ligated to prevent blood loss. The connection between the fallopian tube and the uterine horn was cut and the ovary was moved out and three single catgut stitches were placed on the skin.

(83) Grouping:

(84) TABLE-US-00002 No. of GROUP TREATMENT Dose Animals Group-1 Sham Control (Corn oil) 0.5 ml/100 g 6 Group-2 Ovariectomized control (OVX control) — 6 Group-3 OVX + β Cryptoxanthin (BCX-A)  20 μg/100 g 6 Group-4 OVX + β Cryptoxanthin (BCX-B)  20 μg/100 g 6 Group-5 OVX + β Cryptoxanthin (BCX-C)  20 μg/100 g 6

(85) Study Procedure:

(86) Rats from Group 1 were operated for sham surgery under Ketamine (70 mg/kg)+Xylazine (10 mg/kg) (intra peritoneal) anesthesia. Rats from Groups 2, 3, 4 and 5 were operated for bilateral ovariectomy (OVX) under Ketamine+Xylazine (i.p.) anesthesia. The OVX-operated animals were fed with standard commercial laboratory chow amounts matched with the Sham operated group. The operated animals were housed individually and were allowed to recover for 2 weeks.

(87) Test compounds were dissolved in corn oil. Concentrations of 20 μg/100 g of body weights were administered orally to rats in respective groups through oral gavage needle once daily for 3 weeks. Control rats received corn oil (0.5 ml/100 g of body weight) orally.

(88) On last day of treatment, urine samples were collected by micturation induced by manual pressure from overnight fasted animals and preserved at −20° C. till further analysis.

(89) Statistical Analysis:

(90) All results were analyzed using One Way ANOVA followed by Dunnett's multiple comparison test. Considering confidence interval of P<0.05.

(91) Estimation of Bone Collagen Metabolite (Pyridinoline Crosslinks) in Urine:

(92) Pyridinoline Crosslinks are the bone collagen metabolites which appear in urine when the bone resorption process is accelerated and is considered as an early important marker of osteoporosis.

(93) OVX control animals showed significant increase in Pyridinoline Crosslinks in urine which confirms the successful induction of osteoporosis after ovariectomy procedure. BCX-C treatment moderately reduced the urinary excretion of Pyridinoline Crosslinks to a considerable extent, as did BCX-A and BCX-B, as shown below in Table No. 1. This confirms the therapeutic utility of BCX treatment in estrogen deficiency related osteoporosis.

(94) TABLE-US-00003 TABLE NO. 1 Pyridinoline Crosslinks Determination Pyridinoline Crosslinks (nmol/mmol of Creatinine) Sham Control OVX control BCX-A BCX-B BCX-C 17.15 17.64 6.71 10.27 14.48 1.89 21.69 9.80 9.64 15.64 29.71 22.31 10.52 10.04 16.87 33.50 14.68 12.15 10.80 10.26 4.40 14.79 34.37 24.17 13.77 1.50 13.97 12.19 23.12 24.62 Mean = 14.69 17.51 14.29 14.68 15.94

(95) Determination of Bone (Femur) Density:

(96) Cessation of the ovarian function in humans leads to increase in bone turnover, a negative bone balance, and a net decrease in bone density; these changes are also evident in surgically ovariectomized rats.

(97) A significant OVX induced decrease in bone density was observed in Group 2. BCX-A, BCX-B and BCX-C treatment prevented OVX associated decrease in bone density. BCX-C showed better activity than BCX-A and BCX-B in preventing OVX induced loss of bone density as shown below in Table No. 2.

(98) TABLE-US-00004 TABLE NO. 2 Bone Density Determination Bone Density (g/cm3) Sham Control OVX control BCX-A BCX-B BCX-C 1.253 1.121 1.173 1.1858 1.1902 1.186 1.131 1.178 1.0669 1.2437 1.169 1.167 1.175 1.1785 1.2065 1.208 1.131 1.186 1.1893 1.1593 1.230 1.162 1.141 1.0782 1.1965 1.205 1.130 1.171 1.2490 1.1978 Mean ± SD = 1.140 ± 1.171 ± 1.157 ± 0.070 1.198 ± 0.027 1.209 ± 0.030 0.019 0.015

(99) The results obtained from the above experiments confirm that beta-cryptoxanthin obtained from Paprika source (BCX-C) shows better anti-resorptive property than beta-cryptoxanthin obtained from Marigold and Orange source.

(100) Determination of Ultimate Failure Load of Bones (Tibiae):

(101) Bone fragility can be defined broadly as the susceptibility to fracture. One function of bones is to carry loads. Fractures occur when loads exceed the bone strength, so weakened bones should be considered fragile. During traumatic loading, such as falling on the ground, fracture will occur if the energy from the fall exceeds the mechanical energy that the bone can absorb. Osteoporotic bones absorb very little energy before breaking (failure load) and are therefore more susceptible to fracture resulting from trauma. In this study, the failure load was measured using Three Point Bending test.

(102) In ovariectomized animals there was significant decrease in maximal load values for tibial mid shaft indicating the significant loss of cancellous bone. From the results given below in Table No. 3 it is evident that, BCX-C treatment significantly prevented the loss of mechanical strength of cancellous bone.

(103) TABLE-US-00005 TABLE NO. 3 Determination of Failure Load of Bone Failure load (N) Sham Control OVX control BCX-A BCX-B BCX-C 77.14 45.47 51.9  59.63 66.66 69.89 43.22 65.25 56.91 57.00 58.33 54.11 53.41 59.77 65.72 61.22 45.63 49.71 62.38 54.31 58.33 42   73.71 75.23 65.66 55.76 45.62 63.28 57.65 48.90 Mean ± SD = 46.00 ± 4.24 59.54 ± 9.38 61.92 ± 6.79 59.70 ± 7.39 63.445 ± 8.31

(104) The data represented in Tables No. 1, 2 and 3 are plotted in FIGS. 2, 3, and 4, respectively, as shown in the drawings accompanied with the specification.

CONCLUSION

(105) Trans-beta-cryptoxanthin possesses significant anti-osteoporotic activity in an OVX rat model. BCX-C (Paprika source) significantly improved mass and mechanical strength of bones in ovariectomized rats compared to BCX-A (Marigold source) & B (Orange source).

(106) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

(107) The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.