Method for separating capsorubin and other carotenoids from capsicum oleoresin

Abstract

The present invention relates to a novel process for the isolation of capsorubin and several other carotenoids from capsicum oleoresin, which is a concentrated extract of capsicum fruit (Capsicum annuum). In another aspect, the present invention relates to the use of mild non-aqueous saponification processes and aqueous saponification processes for separating high purity capsorubin and other carotenoids from capsicum oleoresins at temperatures of about 20 DEG C to 50 DEG C. In another aspect, the present invention relates to a saponification process that produces a crystalline mixture of capsorubin, beta-carotene, beta-cryptoxanthin and zeaxanthin, which can be separated by solvent extraction and crystallization to provide high purity capsorubin as well as other carotenoids.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 424,514, filed on November 11, 2022, titled "PROCESS FOR ISOLATION OF CAPSANTHIN AND OTHER CAROTENOIDS FROM PAPRIKA OLEORESIN", the entire disclosure of which is incorporated herein by reference in its entirety.

[0003] Introduction

[0004] The present invention relates to a new method for isolating capsanthin and several other carotenoids from paprika oleoresin, which is a concentrated extract of chili pepper fruits (Capsicum annuum). In another aspect, the present invention relates to a mild saponification method for isolating high-purity capsanthin and other carotenoids from paprika oleoresin at a lower temperature, such as from ambient temperature to about 48 °C. In another aspect, the present invention relates to a method for obtaining capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin from paprika oleoresin by saponifying carotenoid esters and then crystallizing and separating these carotenoids. BACKGROUND OF THE INVENTION

[0005] The present invention relates to a new method for isolating capsanthin and several other carotenoids from paprika oleoresin, which is a concentrated extract of chili pepper fruits (Capsicum annuum). In another aspect, the present invention relates to a mild saponification method for isolating high-purity capsanthin and other carotenoids from paprika oleoresin at a lower temperature, such as from ambient temperature to about 48 °C. In another aspect, the present invention relates to a method for obtaining capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin from paprika oleoresin by saponifying carotenoid esters and then crystallizing and separating these carotenoids.

[0006] Capsanthin is the main carotenoid in paprika oleoresin, and the capsanthin is esterified with fatty acid esters. In addition to capsanthin, the other main carotenoids in paprika oleoresin are: β-carotene, β-cryptoxanthin, and zeaxanthin. Since capsanthin undergoes degradation in hot alkaline solutions, the conventional saponification of esterified carotenoids in paprika oleoresin at temperatures above 50 °C results in a significant loss of this thermally unstable carotenoid. Therefore, there has long been a need for a saponification method that does not cause a significant loss of these compounds.

[0007] Paprika oleoresin is an extract of capsicum fruits having about 5% major carotenoids and about 4% minor carotenoids; said major carotenoids consist mainly of trans- and cis-isomers of capsorubin (40%-48%), β-carotene (11%-14%), β-cryptoxanthin (9%-10%), zeaxanthin (10%-12%), cucurbitaxanthin (5%-7%) and capsanthone (5-6%). Except for β-carotene, these carotenoids are esterified with palmitic acid, myristic acid and lauric acid. Thus, the concentrated extract (paprika oleoresin) from capsicum fruits is saponified to convert the capsorubin fatty acid esters to the unesterified free capsorubin. Similarly, after saponification, zeaxanthin fatty acid esters and β-cryptoxanthin fatty acid esters are converted to their corresponding hydroxycarotenoids. The chemical structures of the major capsicum carotenoids are shown in Figure 1 In. The inventors have also identified a variety of minor carotenoids in paprika oleoresin; these are: zeaxanthin 3,6-epoxide (cucurbitaxanthin); capsanthone, zeaxanthin 5,8-furanoxide (mutatoxanthin); capsorubin 3,6-epoxide; capsorubin 5,8-furanoxide (capsochrome) and karpoxanthin.

[0008] In addition to the above carotenoids, capsicum fruits also contain capsanthin, cryptocapsin, karpoxanthin and their corresponding epoxides [J. Deli, P. Molnar, Current Organicchemistry. (2002), 6(13), 1197–1219]. Eleven minor apocarotenoids have also been isolated and identified in red capsicum fruits (peppers) [Maoka et al., J. Agric. Food Chem. (2001), 49, 1601–1606]. Due to the complex nature of the carotenoids in paprika oleoresin, the focus of the present invention is on the major carotenoids mentioned above.

[0009] In the past few decades, the health benefits of dietary carotenoids such as lutein, zeaxanthin, β-cryptoxanthin, and β-carotene have been well recognized. Similarly, the bioactivity of capsanthin in disease prevention is a subject of intense research. In 1998, Matsufuji et al. (J. of Agric. & Food Chem. 46(9), 3468 - 3472) reported the antioxidant activity of capsanthin and its fatty acid esters by measuring the free radical oxidation of methyl linoleate. The antioxidant, anti-nociceptive, and anti-inflammatory effects of carotenoids extracted from dried sweet peppers (Capsicum annuum L.) have also been documented by Hernandez-Ortega et al. [J. of Biomed. and Biotech. (2012), 524019, 10 pp]. In another study, Narisawa et al. [Proceedings of the Society for Experimental Biology and Medicine, 2000, 224(2), 116 - 122] reported the prevention of colon carcinogenesis in rats by capsanthin and capsanthin-rich pepper juice. Similarly, Maoka et al. [Cancer Letters. (2001), 172(2), 103 - 109] demonstrated the cancer chemopreventive activity of carotenoids in red pepper fruits (Capsicum). Capsanthin has also been shown to inhibit adipogenesis in 3T3-L1 preadipocyte obesity-induced inflammation and prevent weight gain in high-fat diet-induced obese mice [Jo, Sung Jun, Biomolecules & Therapeutics. (2017), 25(3), 329 - 336]. In addition, dietary capsanthin has been shown to have an HDL-cholesterol-elevating effect on plasma and an effect on liver gene expression in rats [Aizawa & Inakuma, British J. of Nutrition. (2009), 102(12), 1760 - 1766]. Another important health benefit of capsanthin is related to its ability to protect against non-alcoholic fatty liver disease in a mouse model [Joo et al., J. of Medicinal Food. (2021), 24(6), 635 - 644]. Capsanthin and capsicum carotenoids also protect human dermal fibroblasts from UVB-induced DNA damage [Fernandez-Garcia et al., Photochem. & Photobiologic. Sci. (2016), 15(9), 1204 - 1211].Finally, it has been reported that dietary capsicum carotenoids absorbed into the blood contribute to the endurance performance of athletes by reducing oxygen (VO2) and heart rate [Maeda, Hayato; Nishino, Azusa; Maoka, Takashi, Advances in Experimental Medicine & Biology. (2021), 1261, 285 - 293].

[0010] Despite the important health benefits of capsicum carotenoids, there are only a few patented methods for extracting and separating carotenoids from the fruits of capsicum. For example, Duanmu et al. (CN101906254 A, 2010 - 12 - 08) reported a method for extracting capsanthin oleoresin from dried capsicum using dimethyl ether as a solvent. However, this patent describes the extraction and preparation of a concentrated extract of red pepper powder containing capsanthin esters, but does not subject the extract to saponification and separation and purification of capsanthin.

[0011] Reilly et al. (U.S. Publication No. 20110282083A1, 2011 - 11 - 17) described a method for converting esterified lutein from capsicum to non - esterified lutein with a purity of 60% - 80%. However, Reilly et al. only focused on the separation of zeaxanthin from oleoresin capsicum. In addition, the saponification method described by Reilly et al. uses hexane, methanol, and an aqueous solution of KOH or NaOH (45% solution) to saponify and separate zeaxanthin at a temperature of 22 - 83°C. Since the hydrolysis of esters in an alkaline aqueous solution does not occur at ambient temperature, it is necessary to carry out saponification at an elevated temperature in this system. In some cases, Reilly et al. carried out the saponification of oleoresin capsicum in methanol - KOH at ambient temperature to separate zeaxanthin. However, this disclosure does not describe the separation of purified capsanthin, and one of ordinary skill in the art would understand that due to the use of an aqueous base, this method requires the separation of the organic phase and the aqueous phase. To remove the aqueous phase, Reilly et al. employed decantation of the upper phase in a centrifuge bottle and washed the precipitate with methanol to separate zeaxanthin. Employing decantation in a centrifuge bottle is not a method that can be scaled up to large - scale commercial production without the use of dedicated equipment.

[0012] Jacob et al. (WO2011135519 A1, 2011 - 11 - 03) described a supercritical fluid extraction method to obtain deodorized red pepper powder, which was then saponified to obtain a capsicum extract as a water - dispersible powder. However, this method focused on the separation of a capsanthin - rich carotenoid mixture from oleoresin capsicum and did not describe the separation or purification of individual carotenoids.

[0013] Umigai et al. (JP2015174858 A, October 5, 2015) described the preparation of a capsicum (red bell pepper) pigment extract containing β-cryptoxanthin (I) and capsanthin (II), where the content ratio of II to I ≤ 3 and does not turn feces red when ingested.

[0014] Sunilkumar et al. described in U.S. Patent No. 9,771,323 B2 on September 26, 2017, the isolation and purification of β-cryptoxanthin from a plant source and its preparation method. The plant source was oleoresin capsicum, but the authors used high temperatures (80 - 85 °C) for 3 - 5 h of saponification, followed by column chromatography for the purification of β-cryptoxanthin. Since capsanthin is sensitive to alkaline aqueous solutions at high temperatures, the method described in U.S. Patent No. 9,771,323 B2 was most clearly accompanied by a significant loss of this carotenoid. It should be noted that U.S. Patent No. 9,771,323 B2 did not describe a method for the isolation and purification of capsanthin. The details of the degradation of capsanthin in aqueous solution at high temperatures will be discussed in detail later in this application.

[0015] In another patent method, Sunilkumar et al. (U.S. Patent No. 10,301,259 B2, May 8, 2019) described a composition and preparation method of β-cryptoxanthin from a plant source, which is used to improve lung health, physical performance, and heart and respiratory health and reduce oxidative stress markers. In this case, the plant source was oleoresin capsicum.

[0016] In another patent method, Deshpande et al. (U.S. Patent No. 10,568,846 B2, February 25, 2020) described a β-cryptoxanthin composition, its preparation method and uses, where the carotenoid was isolated from oleoresin capsicum according to the method of Sunilkumar et al. (U.S. Patent No. 9,771,323 B2, September 26, 2017). Deshpande et al. focused on using the β-cryptoxanthin composition to improve lung health, physical performance, and heart and respiratory health. The patent described administering an effective amount of a β-cryptoxanthin composition to an exercising individual, the β-cryptoxanthin composition containing an extract rich in trans-β-cryptoxanthin, where the extract contains about 75% to 100% by weight of trans-β-cryptoxanthin, and where the amount is effective to reduce oxidative stress markers and increase antioxidant muscle enzymes in the exercising individual compared to those of an exercising individual who has not been administered the β-cryptoxanthin composition.

[0017] Finally, a patent application recently published by Mehta (US2022 / 0009886 A1, January 18, 2022) describes the extraction of capsicum carotenoids by methanol and supercritical fluid extraction, followed by saponification with alcoholic KOH at a high temperature of 75 - 80 °C. The report also indicates the purification of carotenoids by ethyl acetate using countercurrent extraction. However, the present disclosure does not provide details or discussions related to the saponification and purification of carotenoids.

[0018] As described in detail above, the publicly available work to date focusing on the separation of β - cryptoxanthin and zeaxanthin from oleoresin capsicum has consistently disclosed the use of high temperatures during saponification, which necessarily leads to the degradation of these heat - labile target carotenoids such as capsanthin. There is an unmet need for a method that employs mild conditions for the saponification of carotenoid esters and the separation of capsanthin and other carotenoids from oleoresin capsicum.

[0019] For these and other reasons, the present invention is needed. SUMMARY OF THE INVENTION

[0020] The present invention generally relates to methods for separating and purifying capsanthin and several other major carotenoids from oleoresin capsicum. In certain embodiments, the present invention relates to methods for saponifying the major carotenoid esters of capsanthin, zeaxanthin, and β - cryptoxanthin from oleoresin capsicum, and then crystallizing and separating these carotenoids.

[0021] The inventors have surprisingly found new methods with conditions suitable for base - and heat - sensitive molecules, such as ambient temperature to low - temperature saponification methods, followed by the separation of carotenoids by crystallization without using column chromatography. These conditions address the technical challenges of conventional saponification methods that result in significant losses of these target carotenoids.

[0022] More specifically, the inventors have surprisingly found the use of a mixture of propylene glycol (PG) and ethanolic potassium hydroxide (KOH) solution for saponifying carotenoids in oleoresin capsicum. In at least one embodiment, the mixture comprises about 15% to about 20% ethanolic potassium hydroxide (KOH) solution for saponifying carotenoids in oleoresin capsicum. For example, in certain embodiments, the saponification of carotenoids in oleoresin capsicum is completed at ambient temperature using about 15% to about 20% ethanolic potassium hydroxide (KOH) solution in acetone, or using a 5% ethanolic solution without acetone.

[0023] The saponification according to the present invention provides a crystalline mixture of capsanthin, β - carotene, β - cryptoxanthin, and zeaxanthin separated by hexane extraction and crystallization to provide high - purity capsanthin as well as other carotenoids.

[0024] In certain embodiments, the saponification of oleoresin capsicum is completed in 3 - 4 h at 45 - 50 °C using a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH, 15% - 20%, w / w) and propylene glycol (PG). It should be noted that the oleoresin capsicum also contains β-carotene, which is a hydrocarbon carotenoid and does not require saponification. At the end of saponification, ethanol is distilled under reduced pressure at 40 - 50 °C, and the saponified oleoresin in propylene glycol (PG) is diluted with water and neutralized with an aqueous acetic acid solution to KOH. The oleoresin is filtered at 50 - 70 °C to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, and the capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin are washed with water at 50 - 70 °C to remove PG and water-soluble anthocyanins and flavonoids. After drying, the crystalline mixture of these carotenoids is subjected to solvent extraction and crystallization to separate capsanthin and other carotenoids.

[0025] In certain embodiments, a 40% - 60% aqueous potassium hydroxide (KOH) solution in ethanol is used to saponify the carotenoid esters in oleoresin capsicum at ambient temperature in about 24 h, such as in about 12 h, or alternatively in about 6 h, and in a preferred embodiment in about 3 h to 6 h. At the end of saponification, the product is neutralized with an aqueous acetic acid solution, and after distilling and recovering acetone and ethanol under reduced pressure at 40 - 50 °C, the saponified oleoresin is diluted with water. The aqueous oleoresin is filtered at 50 - 70 °C to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, and the capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin are washed with water at 50 - 70 °C to remove residual acetic acid and water-soluble anthocyanins and flavonoids. The crystalline carotenoids are dried overnight at 50 - 60 °C under high vacuum.

[0026] In certain embodiments, acetone is optionally used as a co-solvent together with a solution of KOH in ethanol (w / w) to facilitate stirring of the saponification mixture. However, in certain embodiments, for example, when saponifying the carotenoid esters in oleoresin capsicum with a 5% solution of KOH in ethanol (w / w), acetone as a co-solvent is not required.

[0027] In certain embodiments, the carotenoid esters in oleoresin capsicum are saponified with a 5% solution of KOH in ethanol at ambient temperature. At the end of saponification, KOH is neutralized with an aqueous acetic acid solution, and then ethanol is distilled and recovered under reduced pressure. Then the saponified oleoresin is treated with hot water (about 50 - 70 °C), and the crystalline carotenoids are collected by filtration. The crystalline carotenoids are washed with water (about 50 - 70 °C) to remove residual acetic acid, water-soluble anthocyanins, and flavonoids, and then dried at about 60 °C under high vacuum.

[0028] In certain embodiments, a mixture of hexane and ethanol is slowly added to an aqueous KOH solution (40%-62%, weight:weight) at ambient temperature to simultaneously saponify and separate the carotenoid esters in oleoresin capsicum for 4-5 h, and then stirred for about 24 hours. At the end of saponification, the KOH is neutralized with an aqueous acetic acid solution, and the saponified mixture is stirred at ambient temperature for 24 hours; this results in the crystallization of capsanthin and zeaxanthin, while β-carotene and β-cryptoxanthin remain in solution. The crystalline mixture of capsanthin and zeaxanthin is simply removed by filtration and washed successively with hot water (about 50-70 °C) and hexane to increase the purity of these carotenoids. Then the crystalline mixture of capsanthin and zeaxanthin is dried at about 50-60 °C under high vacuum. The filtrate from this crystallization contains β-carotene, β-cryptoxanthin and other minor capsicum carotenoids as well as their cis-isomers. The mixture of hexane and ethanol in the filtrate is recovered by azeotropic distillation of these solvents under reduced pressure. The recovered mixture of hexane and ethanol can be recycled without separation since the saponification of the carotenoid esters in oleoresin capsicum is carried out with this mixture of solvents. However, the ratio of these solvents needs to be adjusted.

[0029] In certain embodiments, the crystalline mixture of carotenoids from multiple saponification processes is subsequently extracted with hexane at about 25 °C to about 60 °C, which dissolves β-carotene and β-cryptoxanthin, while capsanthin and zeaxanthin remain insoluble in this solvent and are removed by filtration at ambient temperature. Since capsanthin and zeaxanthin exhibit slightly different solubility behaviors in an aqueous acetone solution, partial separation of these carotenoids is achieved by extraction. Thus, according to certain embodiments, a mixture of capsanthin (76%) and zeaxanthin (24%) is extracted with an aqueous solution of acetone, and then filtered to produce a crystalline mixture of capsanthin (85%) and zeaxanthin (15%) with a purity of 85%-90%.

[0030] Similarly, a mixture of β-carotene and β-cryptoxanthin is extracted with an alcohol such as ethanol, 1-propanol and 2-propanol to obtain an alcohol-soluble fraction consisting of β-cryptoxanthin, while β-carotene remains as an insoluble crystal. Each separated carotenoid is further purified by crystallization with a suitable solvent.

[0031] Detailed description of the drawings

[0032] Figure 1Shows the structures of all possible fatty acid esters of the main carotenoids in capsicum oleoresin: capsanthin, β - cryptoxanthin, zeaxanthin, and the minor carotenoids: cucurbitaxanthin and capsanthone; β - carotene is a hydrocarbon carotenoid and is thus unesterified.

[0033] Figure 2 Shows the degradation of capsanthin to β - citraurin by retro - aldol condensation as disclosed by Zechmeister and Cholnoky [Justus Liebigs Annalen der Chemie (1937), 530, 291 - 300].

[0034] Figure 3 Shows the degradation of cryptocapsin to β - apo - 8’ - carotenal by retro - aldol condensation as disclosed by L. Cholnoky and J. Szabolcs (Tetrahedron Letters, No. 19, 1257 - 1259, 1963).

[0035] Figure 4 Is a flow chart for saponifying the carotenoids in capsicum oleoresin with ethanol KOH in propylene glycol at 45 - 50 °C, and then separating capsanthin, zeaxanthin, β - carotene, and β - cryptoxanthin by solvent extraction and crystallization.

[0036] Figure 5 Is a flow chart for saponifying the carotenoids in capsicum oleoresin with ethanol KOH (15 - 20%, weight:weight) in acetone at ambient temperature, and then separating capsanthin, zeaxanthin, β - carotene, and β - cryptoxanthin by solvent extraction and crystallization.

[0037] Figure 6 Is a flow chart for saponifying the carotenoids in capsicum oleoresin with ethanol KOH (5%, weight:weight) at ambient temperature, and then separating capsanthin, zeaxanthin, β - carotene, and β - cryptoxanthin by solvent extraction and crystallization.

[0038] Figure 7 Is a flow chart for simultaneously saponifying and separating the carotenoids in 200 g of capsicum oleoresin with an aqueous KOH solution (40% or 45%, or 62%, weight:weight) in hexane and ethanol at ambient temperature, and at the end of saponification, the main carotenoids: capsanthin and zeaxanthin crystallize in high purity. Detailed implementation

[0039] The present invention relates to a new method for saponifying carotenoid esters in capsanthin oleoresin under mild conditions to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, which has the advantage of avoiding degradation of carotenoids sensitive to bases and heat, such as capsanthin. Another aspect of the present invention relates to a new method for separating these carotenoids by solvent extraction and crystallization and isolating high-purity capsanthin.

[0040] Since conventional methods use alkaline solutions at high temperatures to drive the saponification of carotenoid esters in capsanthin oleoresin, the degradation of capsanthin under these conditions must be described. In 1937, Zechmeister and Cholnoky [Justus Liebigs Annalen der Chemie (1937), 530, 291 - 300] demonstrated that capsanthin subjected to an aqueous potassium hydroxide solution at 80 °C undergoes a retro-aldol condensation to β-citraurin as Figure 2 shown. Similarly, treatment of cryptocapsin, which is structurally similar to capsanthin, with hot aqueous potassium hydroxide solution was shown to result in cleavage of this ketocarotenoid to β-apo-8'-carotenal, as Figure 3 shown (L. Cholnoky and J. Szabolcs, Tetrahedron Letters, No. 19, 1257 - 1259, 1963). Thus, the present invention has developed ambient temperature and low-temperature saponification methods for hydrolyzing capsanthin esters to avoid degradation of this carotenoid. As used herein, "low temperature" refers to from about 20 °C to about 50 °C. For example, in certain embodiments, the saponification method is carried out at ambient temperature (about 20 - 25 °C). In alternative embodiments, the saponification method is carried out at a temperature of about 45 °C to about 50 °C. The resulting crystalline mixture of carotenoids obtained by these methods has been subjected to solvent extraction to separate capsanthin and other carotenoids.

[0041] According to certain embodiments, the present invention relates to a method for low-temperature saponification of carotenoid esters in capsanthin oleoresin to provide a mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, the method comprising:

[0042] treating capsanthin oleoresin with potassium hydroxide (KOH), or sodium hydroxide, or in alternative embodiments other known alkali metal hydroxides in a non-aqueous solution of ethanol (EtOH) or other C1 - C3 alcohols or mixtures and propylene glycol (PG) at low temperature, such as at about 45 - 50 °C, to obtain a saponified mixture;

[0043] heating the mixture to a temperature of, for example, about 40 °C to about 50 °C to saponify the carotenoid esters;

[0044] Treat the saponified paste with water and acetic acid (AcOH) or a 1:1 solution (v:v) in water of another weak organic acid such as propionic acid or butyric acid to neutralize the base;

[0045] Distill and recover ethanol at 45 - 50 °C under reduced pressure, such as about 200 - 120 Torr, to obtain the saponified paste;

[0046] Treat the saponified paste with water to obtain a suspension of carotenoids;

[0047] Filter the suspension and wash the crystals to obtain a crystalline mixture of trans - capsanthin, trans - β - carotene, trans - β - cryptoxanthin, and trans - zeaxanthin; and

[0048] Dry the crystalline mixture, for example, in some embodiments, dry the crystalline mixture at 40 - 60 °C under high vacuum, but in alternative embodiments, dry the mixture using other conventional drying techniques.

[0049] Figure 4The method for saponifying carotenoid esters in oleoresin capsici at low temperature using propylene glycol and KOH ethanol solution, and then separating capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin is shown. According to at least one embodiment, the saponification of carotenoid esters in oleoresin capsici is completed within 3 - 4 h at 45 - 50 °C using a non-aqueous solution of KOH (15% - 20%) in ethanol and propylene glycol (PG, 20% by weight of the oleoresin). In a preferred embodiment, the carotenoid esters in oleoresin capsici (50 g) are saponified with a 20% non-aqueous solution of KOH (10 g) in ethanol (40 g) and PG (10 g) at 45 - 50 °C within 3 - 4 h. After neutralizing the base with a 50% solution of acetic acid and water (23 mL, 1:1, v:v), ethanol is recovered by distillation under reduced pressure at 40 - 50 °C to prevent the loss of ethanol-soluble carotenoids, while propylene glycol (PG) remains in the saponified oleoresin due to its high boiling point (188.2 °C). Since carotenoids show poor solubility in PG, it is not necessary to remove the solvent before filtration. In addition, the use of PG aids in the filtration of saponified carotenoids. After evaporation of ethanol, the saponified mixture is diluted with water (50 g), and the mixture is heated at 50 - 70 °C to obtain a suspension. The crystallized carotenoids are filtered and washed with 50 g of water at 50 - 70 °C to remove propylene glycol, residual acetic acid, and water-soluble anthocyanins and flavonoids. The wet crystalline mixture of carotenoids is dried at 40 - 60 °C under high vacuum to obtain (4.80 g; 60% total carotenoids, 2.88 g) capsanthin (61.86%), β-carotene (12.71%), β-cryptoxanthin (8.62%) and zeaxanthin (16.81%), which are subjected to solvent extraction to separate individual carotenoids.

[0050] In another embodiment of the present invention, acetone is used as a co-solvent, and the carotenoid esters in oleoresin capsici are saponified with a non-aqueous solution of KOH in ethanol at ambient temperature, as Figure 5As shown in the flowchart. The weight ratio of acetone to oleoresin is from 2:1 to 4:1, and the concentration of KOH in ethanol is about 10% to 20%. In a preferred embodiment of the present invention, for example, the carotenoid esters in oleoresin capsici (50 g) are dissolved in acetone (100 g), and the resulting mixture is saponified with a 20% non-aqueous solution of KOH (10 g) in ethanol (40 g) at ambient temperature within 24 hours. Using acetone in the presence of KOH does not cause aldol condensation of acetone because the saponification is carried out at ambient temperature. Under these conditions, a large amount of soap and wax (23 g) is formed, which is dissolved by adding a 1:1 solution of acetic acid and water (20 - 24 mL). Acetone and ethanol are recovered by distillation under reduced pressure at 35 - 50 °C, yielding a dark red paste. It should be noted that ethanol (b.p. = 78.4 °C) and acetone (b.p. = 56.5 °C) do not form an azeotropic mixture, and due to their large boiling point difference, they can be easily separated and recovered by distillation. The saponified oleoresin is diluted with water (about 50 g). After heating the resulting mixture at 50 - 70 °C, a homogeneous suspension is obtained. The crystallized carotenoids are filtered and washed with 100 g of water at 50 - 70 °C to remove residual acetic acid and water-soluble anthocyanins and flavonoids. The wet crystalline mixture of carotenoids is dried under high vacuum at 40 - 60 °C to obtain 4.17 g (60% of total carotenoids, 2.50) of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, which are solvent-extracted to separate the individual carotenoids. The crystalline mixture is extracted with hexane to increase the purity of the mixture to 80% - 85%, and capsanthin and zeaxanthin are separated from β-carotene and β-cryptoxanthin.

[0051] In another embodiment of the present invention, after saponifying the carotenoid esters in oleoresin capsici with an aqueous KOH solution (40%) in acetone and ethanol at ambient temperature for 24 hours, a crystalline mixture with a purity of 85% of capsanthin (66.33%), zeaxanthin (20.63%), β-carotene (6.07%), and β-cryptoxanthin (6.97%) is obtained after post-treatment and purification. Similarly, saponifying the carotenoid esters in oleoresin capsici with a 45% aqueous KOH solution in acetone and ethanol, after post-treatment and purification, a crystalline mixture with a purity of 83% of capsanthin (66.11%), zeaxanthin (25.16%), β-carotene (2.81%), and β-cryptoxanthin (5.92%) is obtained.

[0052] In certain embodiments, when using a solution of KOH in ethanol (weight:weight) to saponify the carotenoid esters in oleoresin capsicum at ambient temperature, it is necessary to use acetone as a co-solvent to facilitate stirring of the saponification mixture. However, in alternative embodiments of the present invention, the carotenoid esters in oleoresin capsicum are saponified with a 5% solution of KOH in ethanol (weight:weight) without using acetone as a co-solvent, as shown in the flow chart of Figure 6 . The weight ratio of ethanol to oleoresin is from 3.80:1 to 5.70:1, and the weight ratio of oleoresin to KOH is from 3.3:1 to 5:1.

[0053] In certain embodiments, the carotenoid esters in oleoresin capsicum (100 g) are saponified with a 5% non-aqueous solution of KOH (20 g) in ethanol (380 g) at ambient temperature within 24 hours. The saponified oleoresin is then treated with an aqueous solution of acetic acid (1:1, v:v) to neutralize the base, and ethanol is recovered by distillation under reduced pressure. The saponified oleoresin is then treated with water (at about 50 - 70 °C), and the crystalline carotenoids are collected by filtration and washed with water (at about 50 - 70 °C) to remove residual acetic acid and water-soluble anthocyanins and flavonoids. After drying the crystals under high vacuum at 60 °C, the resulting crystalline mixture of carotenoids (9.00 g, 65% pure) is subjected to hexane extraction and crystallization to separate capsorubin and other carotenoids, as described above. Any drying method can be used according to certain embodiments.

[0054] Using normal-phase HPLC separation, the inventors have determined the detailed composition of the main capsicum carotenoids after saponification. Table 1 shows the relative composition of the main carotenoids in oleoresin capsicum after saponification at ambient temperature or low temperature according to the present invention.

[0055] Table 1. Relative composition of main carotenoids in oleoresin capsicum after saponification under various conditions before crystallization as determined by HPLC. a,b

[0056]

[0057] a The saponified mixture also contains about 6% - 8% of cucurbitaxanthin and capsanthone as minor carotenoids, the composition of which is not shown in Table 1; b Abbreviations: PG, propylene glycol; EtOH, ethanol; ACET, acetone;

[0058] KOH, potassium hydroxide; aq c : 40 wt% KOH in water; d Solvent-free.

[0059] It must be noted that oleoresin capsicum also contains 21%-23% minor carotenoids, and about 10%-12% of these carotenoids are identified as cucurbitaxanthin and capsanthone.

[0060] However, due to the complexity presented, the work described herein focuses on the isolation, separation, and purification of the major carotenoids in oleoresin capsicum using the new method described herein. The major carotenoids in saponified oleoresin capsicum are also accompanied by a large amount of their cis isomers; this is particularly evident in the case of capsorubin, as shown in Table 1. Therefore, the present inventors focused on the isolation of the major carotenoids in oleoresin capsicum, which consist of the trans isomers of capsorubin, β-carotene, β-cryptoxanthin, and zeaxanthin. This is because it is recognized that the cis isomers of carotenoids do not crystallize well due to their increased solubility in almost all organic solvents. At ambient temperature and low temperature, the saponification of carotenoid esters in oleoresin capsicum clearly shows that trans-capsorubin accounts for about 39%-48% of the total carotenoids, and the total carotenoids are accompanied by about 12%-23% of cis-capsorubin that is not expected to crystallize. Therefore, the saponification of carotenoid esters in oleoresin capsicum at high temperature can increase the composition of the less desirable cis carotenoids relative to their trans counterparts, resulting in a low recovery of crystalline carotenoids. In addition, saponification at high temperature (60-80 °C) can also cause the base-sensitive capsorubin to degrade to β-citraurin. These drawbacks prompted the inventors to search for alternative methods, and the criteria included determining a method for the low-temperature saponification of carotenoid esters in oleoresin capsicum.

[0061] As shown in Table 1, the relative composition of the major carotenoids in oleoresin capsicum saponified at ambient temperature or low temperature (45-48 °C) is consistent, and trans-capsorubin accounts for 39%-48% of the total carotenoids. As previously noted, the trans carotenoids in saponified oleoresin capsicum are accompanied by a large amount of their cis isomers. At the end of saponification, the trans isomers of the major carotenoids crystallize, while their cis isomers and minor carotenoids are removed by filtration. The relative composition of the major crystalline trans carotenoids determined by HPLC after crystallization is shown in Table 2. Hexane extraction of these crystalline mixtures is then carried out, followed by crystallization to increase the purity of the mixture to 80%-85% and to separate capsorubin and zeaxanthin from β-carotene and β-cryptoxanthin.

[0062] Table 2. Relative composition of the crystalline mixture of trans carotenoids as determined by HPLC after saponification of oleoresin capsicum and removal of cis isomers and other minor carotenoids, and before hexane extraction. a

[0063]

[0064] Abbreviations: PG, propylene glycol; EtOH, ethanol; ACET, acetone; KOH, potassium hydroxide;

[0065] aq b : 40 wt% KOH in water; c no solvent.

[0066] In addition, the inventors surprisingly observed that the saponification of carotenoid esters in oleoresin capsicum can be carried out with an aqueous KOH solution in hexane and ethanol at ambient temperature. Thus, in certain embodiments, the carotenoid esters in oleoresin capsicum (100 g and 200 g) were saponified for 24 hours at ambient temperature in hexane and ethanol by adding a 40%-62% (weight:weight) aqueous KOH solution. Although the ambient temperature saponification of esters in aqueous solution is a reversible reaction and does not proceed at ambient temperature, the carotenoid esters in oleoresin capsicum were successfully saponified by using a high concentration of aqueous KOH solution. This is because once the carotenoid esters are hydrolyzed in the presence of hexane, the unesterified carotenoids gradually crystallize and shift the reaction equilibrium forward. Also in these experiments, the aqueous KOH solution was added dropwise to the solution of oleoresin in hexane and ethanol over a period of 4 - 5 h to prevent the degradation of capsanthin. In this way, a low concentration of KOH was maintained during saponification, which is crucial for maintaining the integrity of capsanthin. The relative composition of the saponified carotenoids in the crude oleoresin determined by HPLC is shown in Table 3.

[0067] Table 3. Relative composition of major carotenoids determined by HPLC after 24 h of saponification of carotenoid esters in oleoresin capsicum with an aqueous KOH solution in hexane and ethanol at 20 - 25 °C and before crystallization. a

[0068]

[0069] a Abbreviations: EtOH, ethanol; KOH, potassium hydroxide.

[0070] The work-up of the saponification involves neutralizing the base with a 50% solution of acetic acid in water (v:v) and stirring the product for a further 24 h at ambient temperature. This allows for the crystallization of trans-capsanthin and trans-cryptoxanthin and other minor trans-carotenoids, which are simply removed as crystals by filtration and further purified by washing with water and hexane. The method is shown to be ideal for the simultaneous saponification and crystallization of trans-capsanthin and trans-cryptoxanthin, which are separated from other carotenoids by filtration and subsequently further purified with hexane. After removal of the cis-isomers of capsanthin and minor carotenoids, the relative composition of the crystallized carotenoids is shown in Table 4.

[0071] Table 4. Relative composition of crystallized trans-carotenoids determined by HPLC after saponification of carotenoid esters in oleoresin capsicum with aqueous KOH in hexane and ethanol at 20 - 25 °C for 24 h and after removal of cis-isomers and other minor carotenoids by extraction with hexane. a

[0072]

[0073] a Abbreviations: EtOH, ethanol; KOH, potassium hydroxide.

[0074] For the saponification of carotenoid esters in Experiments 19 - 23, 100 g of oleoresin was used, with a saturated solution of KOH in water (62%) and only 30 g of ethanol. After crystallization, a mixture of major and minor trans - carotenoids with a purity of 80% - 85% was obtained. In Experiments 19 and 20, equal weights of hexane and oleoresin (100 g / 100 g) were used, which led to the complete removal of β - carotene from the crystals into the filtrate and an increase in the capsorubin composition in the mixture. Thus, the crystallized carotenoids from Experiments 19 and 20 contained 72% and 76% of trans - capsorubin, respectively. In Experiments 21 - 23, the weight ratio of hexane / oleoresin was reduced to 75 g / 100 g and 50 g / 100 g (Table 3), and thus the crystallized carotenoids contained 2% - 7% of β - carotene and had a lower weight ratio of trans - capsorubin (64% - 69%). The weight ratio of trans - zeaxanthin in the crystallized product was not affected by the amount of hexane used in the saponification because this carotenoid is completely insoluble in hexane. Since the objective of the present invention is to obtain a crystallized product with the highest relative composition of trans - capsorubin, a scale - up saponification experiment (Experiments 24 - 29) was carried out with 200 g of oleoresin and an equal weight of hexane. When the saponification of carotenoid esters in oleoresin was carried out on a 200 g scale with 62% KOH (Experiments 24 - 26), 45% KOH (Experiments 27 and 28), and 40% KOH (Experiment 29), the relative composition of the crystallized carotenoids was reproducible and consistent. These saponification experiments yielded 8.70 - 10.40 g of a mixture of major and minor carotenoids with a purity of 80% - 85%, which contained 70% - 73% of trans - capsorubin, 16% - 18% of trans - zeaxanthin, 2.47% - 6.10% of β - cryptoxanthin, 2.40% - 4.60% of cucurbitaxanthin, and 4.15% - 5.44% of capsanthone. The detailed flow chart of the method for saponifying carotenoid esters in 200 g of oleoresin is shown in Figure 7 is shown in

[0075] In the first step, oleoresin capsicum is dissolved in hexane and ethanol, and aqueous KOH solutions of various concentrations (62%, 45% or 40%) are added over a period of 4 - 5 h, and the mixture is stirred at 20 - 25 °C for about 24 h. In the second step, the base is neutralized with a 1:1 solution (v:v) of acetic acid - water, and in the third step, the mixture is stirred at ambient temperature to promote the crystallization of capsorubin and zeaxanthin. In step four of the method, the crystallized carotenoids are filtered and washed with water at 70 °C (200 g / 200 g oleoresin) and dried on the funnel for 4 h. To increase the purity of the carotenoids, the solid is subsequently washed with an appropriate amount of hexane in step five of the method. The crystallized carotenoids are then dried under high vacuum at 50 °C to give 8.0 g of a mixture of capsorubin (71%), zeaxanthin (18%), β - cryptoxanthin (3%), cucurbitaxanthin (3%) and capsanthone (6%) with a purity of 80% - 85%. This crystalline mixture is extracted with an aqueous acetone solution to give 7.0 g of a crystalline mixture of capsorubin (85%) and zeaxanthin (15%) with a purity of 90%. In an alternative embodiment, after washing the impure crystallized carotenoids with water at 70 °C and drying, the solid can be stirred with hexane at ambient temperature for several hours and filtered to increase the purity of the crystallized carotenoids to 80% - 85%. The filtrate containing hexane and ethanol from the method is evaporated under reduced pressure, and these solvents are recycled without separation. Hexane (85%), ethanol (12%) and water (3%) form an azeotropic mixture and can be recycled without separation. The presence of a small amount of water carried over by azeotropic distillation with ethanol does not pose a problem. This is because the saponification of carotenoid esters in oleoresin capsicum is carried out with a mixture of these solvents in an aqueous KOH solution. However, the hexane / ethanol ratio needs to be adjusted according to the saponification protocol. The filtrate from saponification also contains excess acetic acid which does not form an azeotrope with hexane or ethanol.

[0076] Separation and purification of crystalline mixtures of carotenoids from saponified oleoresin capsici

[0077] As Figure 4As shown, the separation of the crystalline mixture of capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin obtained from the saponification of carotenoid esters in oleoresin capsicum is achieved by sequential extraction with a suitable solvent. In the first step, β-carotene and β-cryptoxanthin are dissolved and extracted from the mixture of carotenoids with a C5-C8 hydrocarbon, preferably hexane, while zeaxanthin and capsanthin remain insoluble in the hydrocarbon solvent. In a preferred embodiment of the present invention, at a temperature of 25 - 70 °C, a crystalline mixture (12.53 g) of capsanthin (61.86%), zeaxanthin (16.81%), β-carotene (12.71%), and β-cryptoxanthin (8.62%) is extracted with hexane (350 g) for 1 - 3 h. After stirring the mixture for 3 - 5 h at ambient temperature, the solid is filtered and washed with hexane (60 g). The crystalline solid (9.81 g) consists of capsanthin (76.36%) and zeaxanthin (23.64%). This crystalline mixture is extracted with an aqueous acetone solution and filtered. After drying the resulting solid, a 7.0 g mixture of capsanthin (85%) and zeaxanthin (15%) with a purity of 85% - 90% is obtained. The filtrate is evaporated to dryness to give a 2.72 g mixture of β-carotene (62.99%) and β-cryptoxanthin (37.01%).

[0078] In a preferred embodiment, the ratio of acetone to water is from 9:1 to 3:1 per gram of the mixture of capsanthin and zeaxanthin.

[0079] The following examples are provided to illustrate but not limit the invention. Thus, it should be understood that various formulation modifications as well as delivery method modifications can be made and still remain within the spirit of the invention.

[0080] Example 1

[0081] Saponification of carotenoid esters in oleoresin capsici using 20% KOH ethanol (EtOH) solution and propylene glycol (PG) as a co - solvent at low temperature of carotenoid esters in oleoresin capsici

[0082] Transfer the oleoresin capsicum (50 g) to a 250 mL round-bottom flask equipped with a magnetic stirrer and treat it with a solution of propylene glycol (PG, 10 g) and KOH (10 g) in ethanol (40 g) [20% weight:weight]. Place the flask in an oil bath at 55 °C and stir the mixture at 45 - 50 °C. Follow the saponification process by HPLC, which shows complete saponification after 3 h. Allow the saponification to proceed for an additional hour (4 h total) and remove the heating. Cool the mixture to ambient temperature and add a solution of acetic acid (AcOH) and water (23 mL, 1:1, v:v). Evaporate the ethanol on a rotary evaporator (water bath temperature: 55 °C) at 45 - 50 °C (solution temperature) under reduced pressure (100 Torr). Add hot water (70 °C, 50 g) and stir the mixture at 70 °C for 1 h until the paste dissolves and a suspension is obtained. Filter the resulting suspension while it is hot. Wash the purple solid with water (50 g) at 70 °C and dry the crystals in an oven at 60 °C under high vacuum for 24 h to obtain a crystalline mixture of carotenoids (4.80 g, 60% total carotenoids, 2.88 g). The relative composition of the carotenoids determined by HPLC was: capsanthin (61.86%), β-carotene (12.71%), β-cryptoxanthin (8.62%) and zeaxanthin (16.81%). Stir the crystalline mixture with hexane (15 g) at ambient temperature for 4 h and filter. Wash the crystals with hexane (15 g) and dry them in high vacuum at 40 - 60 °C for 24 h to obtain 3.05 g (84% total carotenoids, 2.56 g) of a mixture of capsanthin (76.20%), zeaxanthin (19.18%) and β-cryptoxanthin (4.62%).

[0083] Example 2

[0084] Saponification of carotenoid esters in oleoresin capsici using 20% KOH ethanol (EtOH) solution and propylene glycol (PG) as a co - solvent at low temperature of carotenoid esters in oleoresin capsici

[0085] Transfer oleoresin capsicum (100 g) into a 500 mL round-bottom flask equipped with a magnetic stirrer and treat it with a solution of propylene glycol (PG, 20 g) and KOH (20 g) in ethanol (80 g) [20% weight:weight]. Place the flask in an oil bath at 55 °C and stir the mixture at 45 - 50 °C. After 4 h, remove the heating and allow the mixture to cool to ambient temperature. Add a solution of acetic acid (AcOH) and water (45 mL, 1:1, v:v) and evaporate the ethanol on a rotary evaporator (water bath temperature: 55 °C) at 45 - 50 °C (solution temperature) under reduced pressure (100 Torr). Add hot water (70 °C, 100 g) and stir the mixture at 70 °C until the paste dissolves and a suspension is obtained. Filter the mixture while it is hot. Wash the purple solid with water at 70 °C (100 g) and dry the crystals in an oven at 60 °C under high vacuum for 24 h to obtain a crystalline mixture of carotenoids (8.30 g, 60% total carotenoids, 4.98 g). The relative composition of the carotenoids determined by HPLC was: capsorubin (63.65%), β-carotene (13.54%), β-cryptoxanthin (7.64%) and zeaxanthin (15.17%). Stir the crystalline mixture with hexane (30 g) at ambient temperature for 4 h and filter. Wash the crystals with hexane (30 g) and dry them in an oven at 40 - 60 °C under high vacuum for 24 h to obtain 5.15 g (85% total carotenoids, 4.37 g) of a mixture of capsorubin (74.5%), zeaxanthin (20.60%) and β-cryptoxanthin (4.90%).

[0086] Example 3

[0087] Saponification of carotenoid esters in oleoresin capsici using 20% KOH ethanol solution and acetone as a co - solvent at ambient temperature of carotenoid esters in oleoresin capsici

[0088] Transfer oleoresin capsicum (50 g) into a 250 mL conical flask equipped with a stir bar and dissolve in acetone (100 g). Treat the mixture with a solution of KOH (10 g) in ethanol (40 g) [20% w:w] at ambient temperature. This results in the formation of a large amount of soft wax and hard wax as well as potassium salts of fatty acids precipitating out of the solution. The saponification process was followed by HPLC, which showed complete saponification after 24 h. Add a solution of acetic acid (AcOH) and water (21 mL, 1:1, v:v) and stir the mixture at ambient temperature for 20 min. First, distill acetone at 35 - 40 °C under reduced pressure (300 Torr), then distill ethanol at 45 - 50 °C (100 Torr) to obtain oleoresin in water. Treat the oleoresin with hot water (70 °C, 50 g) and stir the mixture at 70 °C for 1 h until the paste dissolves and a homogeneous suspension is obtained. Filter the resulting suspension while it is hot. Wash the purple solid with water (100 g) at 70 °C and dry the crystals in an oven at 60 °C under high vacuum for 24 h to obtain a crystalline mixture of carotenoids (4.10 g, 62% total carotenoids, 2.54 g). The relative composition of carotenoids determined by HPLC was: capsanthin (67.14%), β-carotene (5.20%), β-cryptoxanthin (7.29%) and zeaxanthin (20.37%). Stir the crystalline mixture with hexane (15 g) at ambient temperature for 4 h and filter. Wash the crystals with hexane (15 g) and dry in high vacuum at 40 - 60 °C for 24 h to obtain 2.76 g (85% total carotenoids, 2.35 g) of a mixture of capsanthin (75.4%), zeaxanthin (21.30%) and β-cryptoxanthin (3.30%).

[0089] Example 4

[0090] Saponification of carotenoid esters in oleoresin capsici using 20% KOH ethanol solution and acetone as a co - solvent at ambient temperature of carotenoid esters in oleoresin capsici

[0091] Transfer oleoresin capsicum (100 g) to a 500 mL conical flask equipped with a stir bar and dissolve in acetone (300 g). Treat the mixture with a solution of KOH (20 g) in ethanol (80 g) [20% weight:weight] at ambient temperature. This results in the formation of a large amount of soft and hard waxes and the potassium salt of fatty acids precipitating out of the solution. The saponification process was followed by HPLC, which showed complete saponification after 24 h. Add a solution of acetic acid (AcOH) and water (42 mL, 1:1, v:v) and stir the mixture at ambient temperature for 20 min. First, distill acetone at 35 - 40 °C under reduced pressure (300 Torr), then distill ethanol at 45 - 50 °C (100 Torr) to obtain the oleoresin in water. Treat the oleoresin with hot water (70 °C, 100 g) and stir the mixture at 70 °C for 1 h until a homogeneous suspension is obtained. Filter the resulting suspension while hot. Wash the purple solid with water (200 g) at 70 °C and dry the crystals in an oven at 60 °C under high vacuum for 24 h to obtain a crystalline mixture of carotenoids (8.50 g, 61% total carotenoids, 5.19 g). The relative composition of the carotenoids determined by HPLC was: capsanthin (63.66%), β-carotene (7.92%), β-cryptoxanthin (8.38%) and zeaxanthin (20.04%). Stir the crystalline mixture with hexane (30 g) at ambient temperature for 4 h and filter. Wash the crystals with hexane (30 g) and dry in high vacuum at 40 - 60 °C for 24 h to obtain 5.67 g (83% total carotenoids, 4.71 g) of a mixture of capsanthin (75.8%), zeaxanthin (20.4%) and β-cryptoxanthin (3.8%).

[0092] Example 5

[0093] Saponification of carotenoid esters in oleoresin capsici using 40% KOH aqueous solution in acetone and ethanol at ambient temperature of carotenoid esters in oleoresin capsici

[0094] Transfer oleoresin capsicum (100 g) into a 500 mL conical flask equipped with a stir bar and dissolve it in acetone (150 g) and ethanol (30 g). Treat the mixture dropwise with a solution of KOH (20 g) in water (30 g) [40% w:w] over 4 h at ambient temperature. Stir the mixture at ambient temperature for 24 h. Add a solution of acetic acid (AcOH) and water (61 mL, 1:1, v:v) and stir the mixture at ambient temperature for 2 h. First, distill acetone at 35 - 40 °C under reduced pressure (300 Torr), then distill ethanol at 45 - 50 °C (100 Torr) to obtain oleoresin in water. Treat the oleoresin with hot water (70 °C, 100 g) and stir the mixture at 70 °C for 1 h until a homogeneous suspension is obtained. Filter the resulting suspension while it is hot. Wash the purple solid with water at 70 °C (200 g) and dry the crystals in an oven at 60 °C under high vacuum for 24 h to obtain a crystalline mixture of carotenoids (8.20 g, 62% total carotenoids, 5.08 g). The relative composition of carotenoids determined by HPLC was: capsorubin (66.33%), β-carotene (6.07%), β-cryptoxanthin (6.97%) and zeaxanthin (20.63%). Stir the crystalline mixture with hexane (30 g) at ambient temperature for 4 h and filter. Wash the crystals with hexane (30 g) and dry them in a high vacuum at 40 - 60 °C for 24 h to obtain 5.60 g (85% total carotenoids, 4.76 g) of a mixture of capsorubin (74.4%), zeaxanthin (20.8%) and β-cryptoxanthin (4.8%).

[0095] Example 6

[0096] Saponification of carotenoid esters in oleoresin capsici using 5% KOH ethanol solution at ambient temperature

[0097] Transfer the oleoresin capsicum (100 g) to a 1000 mL conical flask equipped with a stir bar and treat it at ambient temperature with a solution of KOH (20 g) in ethanol (380 g) [5% weight:weight]. The saponification process was followed by HPLC, which showed complete saponification after 24 h. The saponified oleoresin was treated with 42 mL of an aqueous solution of acetic acid - water (v:v) and the mixture was stirred at ambient temperature for 30 min. Ethanol was evaporated under reduced pressure at 45 - 50 °C (100 Torr) to give a red paste. The oleoresin was treated with hot water (70 °C, 100 g) and the mixture was stirred at 70 °C until the paste dissolved and a suspension was obtained. The resulting suspension was filtered while hot. The purple solid was washed with 70 °C water (200 g) and the crystals were dried in an oven at 60 °C under high vacuum for 24 h to give a crystalline mixture of carotenoids (9.00 g, 65% total carotenoids). The relative composition of the carotenoids determined by HPLC was: capsorubin (65.35%), β - carotene (11.07%), β - cryptoxanthin (7.68%) and zeaxanthin (15.90%).

[0098] Example 7

[0099] Saponification of carotenoid esters in oleoresin capsici using 5% KOH ethanol solution at ambient temperature

[0100] Transfer the oleoresin capsicum (100 g) to a 1000 mL conical flask equipped with a stir bar and treat it at ambient temperature with a solution of KOH (20 g) in ethanol (380 g) [5% weight:weight]. The saponification process was followed by HPLC, which showed complete saponification after 24 h. The saponified oleoresin was treated with 42 mL of an aqueous solution of acetic acid - water (v:v) and the mixture was stirred at ambient temperature for 30 min. Ethanol was evaporated under reduced pressure at 45 - 50 °C (100 Torr) to give a red paste. The oleoresin was treated with hot water (70 °C, 100 g) and the mixture was stirred at 70 °C until the paste dissolved and a suspension was obtained. The resulting suspension was filtered while hot. The purple solid was washed with 70 °C water (200 g) and the crystals were dried in an oven at 60 °C under high vacuum for 24 h to give a crystalline mixture of carotenoids (9.35 g, 63% total carotenoids). The relative composition of the carotenoids determined by HPLC was: capsorubin (64.79%), β - carotene (11.77%), β - cryptoxanthin (7.28%) and zeaxanthin (16.16%).

[0101] Example 8

[0102] Saponification of carotenoid esters in oleoresin capsici using 5% KOH ethanol solution at ambient temperature

[0103] Transfer the oleoresin capsicum (100 g) to a 1000 mL conical flask equipped with a stir bar and treat it with a solution of KOH (25 g) in ethanol (475 g) [5% w / w] at ambient temperature. The saponification process was followed by HPLC, which showed complete saponification after 24 h. The saponified oleoresin was treated with 51 mL of an aqueous solution of acetic acid - water (v:v), and the mixture was stirred at ambient temperature for 30 min. Ethanol was evaporated under reduced pressure at 45 - 50 °C (100 Torr) to give a red paste. The oleoresin was treated with hot water (70 °C, 100 g), and the mixture was stirred at 70 °C until the paste dissolved and a suspension was obtained. The resulting suspension was filtered while hot. The purple solid was washed with 70 °C water (200 g), and the crystals were dried in an oven at 60 °C under high vacuum for 24 h to give a crystalline mixture of carotenoids (9.72 g, 64% total carotenoids). The relative composition of the carotenoids determined by HPLC was: capsorubin (64.05%), β-carotene (10.73%), β-cryptoxanthin (8.11%) and zeaxanthin (17.11%).

[0104] Example 9

[0105] Saponification of carotenoid esters in oleoresin capsici using 5% KOH ethanol solution at ambient temperature

[0106] Transfer the oleoresin capsicum (100 g) to a 1000 mL conical flask equipped with a stir bar and treat it with a solution of KOH (30 g) in ethanol (570 g) [5% w / w] at ambient temperature. The saponification process was followed by HPLC, which showed complete saponification after 24 h. The saponified oleoresin was treated with 61 mL of an aqueous solution of acetic acid - water (v:v), and the mixture was stirred at ambient temperature for 30 min. Ethanol was evaporated under reduced pressure at 45 - 50 °C (100 Torr) to give a red paste. The oleoresin was treated with hot water (70 °C, 100 g), and the mixture was stirred at 70 °C until the paste dissolved and a suspension was obtained. The resulting suspension was filtered while hot. The purple solid was washed with 70 °C water (200 g), and the crystals were dried in an oven at 60 °C under high vacuum for 24 h to give a crystalline mixture of carotenoids (8.47 g, 60% total carotenoids). The relative composition of the carotenoids determined by HPLC was: capsorubin (65.32%), β-carotene (10.68%), β-cryptoxanthin (7.75%) and zeaxanthin (16.25%).

[0107] Example 10

[0108] Saponification of carotenoid esters in oleoresin capsici using 62% KOH aqueous solution in hexane and ethanol at ambient temperature of carotenoid esters in oleoresin capsici

[0109] Transfer oleoresin capsicum (200 g) to a 1000 mL conical flask equipped with a stir bar and stir in hexane (200 g) and ethanol (60 g) at ambient temperature until the oleoresin dissolves. Transfer a saturated solution of KOH (50 g) in water (30 g) [62% w:w] to an addition funnel and add dropwise over 4 h at 20 - 25 °C to the oleoresin in hexane and ethanol. Saponification generates heat and this slow addition allows the saponification to proceed at ambient temperature. After 24 h, add dropwise over a 1 h period a 1:1 solution of AcOH-H2O (152 mL, v:v); the slow addition of acid to base is crucial to keep the temperature of the mixture below 30 °C. The relative composition of carotenoids in the crude saponified mixture was: trans-capsanthin (44.63%), cis-capsanthin (11.64%), β-carotene (12.09%), β-cryptoxanthin (9.12%), zeaxanthin (10.19%), cucurbitaxanthin (7.12%) and capsanthone (5.21%). Stir the mixture at ambient temperature for 24 h to crystallize trans-capsanthin in hexane. Filter the saponified oleoresin and save the filtrate for solvent evaporation and recovery. Then wash the solid with hot water (70 °C, 200 g) and dry the crystals on the funnel for 4 h. Wash the crystals with hexane (60 g) and dry on the funnel for 1 h. Remove the crystals and dry in a vacuum oven at 50 °C for 24 h to obtain a mixture (8.41 g, 85% pure, 7.14 g) of trans-capsanthin (72.65%), zeaxanthin (17.29%), β-cryptoxanthin (2.47%), cucurbitaxanthin (2.81%) and capsanthone (4.78%). Combine the saved filtrate and hexane washings and evaporate and recover hexane and ethanol at 40 °C under reduced pressure (230 Torr) to 45 °C (100 Torr).

[0110] Example 11

[0111] Saponification of carotenoid esters in oleoresin capsici using 45% KOH aqueous solution in hexane and ethanol at ambient temperature of carotenoid esters in oleoresin capsici

[0112] Transfer oleoresin capsicum (200 g) to a 1000 mL conical flask equipped with a stir bar and stir in hexane (200 g) and ethanol (60 g) at ambient temperature until the oleoresin dissolves. Transfer a saturated solution of KOH (50 g) in water (61 g) [45% wt:wt] to an addition funnel and add dropwise over 4 h at 20 - 25 °C to the oleoresin in hexane and ethanol. Saponification generates heat and this slow addition allows the saponification to proceed at ambient temperature. After 24 h, add dropwise over a 1 h period a 1:1 solution of AcOH - H2O (152 mL, v:v); the slow addition of acid to base is crucial to keep the temperature of the mixture below 30 °C. The relative composition of carotenoids in the crude saponified mixture was: trans - capsorubin (48.25%), cis - capsorubin (11.42%), β - carotene (11.87%), β - cryptoxanthin (10.15%), zeaxanthin (11.92%), cucurbitaxanthin (5.93%) and capsanthone (5.56%). Stir the mixture at ambient temperature for 24 h to crystallize trans - capsorubin in hexane. Filter the saponified oleoresin and save the filtrate for solvent evaporation and recovery. Then wash the solid with hot water (70 °C, 200 g) and dry the crystals on the funnel for 4 h. Wash the crystals with hexane (60 g) and dry on the funnel for 1 h. Remove the crystals and dry in a vacuum oven at 50 °C for 24 h to obtain a mixture (9.28 g, 84% pure, 7.80 g) of trans - capsorubin (71.08%), zeaxanthin (18.27%), β - cryptoxanthin (2.90%), cucurbitaxanthin (2.81%) and capsanthone (4.94%). Combine the saved filtrate and hexane washings and evaporate and recover hexane and ethanol at 40 °C under reduced pressure (230 Torr) to 45 °C (100 Torr).

[0113] Example 12

[0114] Saponification of carotenoid esters in oleoresin capsici using 40% KOH aqueous solution in hexane and ethanol at ambient temperature of carotenoid esters in oleoresin capsici

[0115] Transfer oleoresin capsicum (200 g) to a 1000 mL conical flask equipped with a stir bar and stir in hexane (200 g) and ethanol (60 g) at ambient temperature until the oleoresin dissolves. Transfer a saturated solution of KOH (60 g) in water (90 g) [40% wt:wt] to an addition funnel and add dropwise over 4 h at 20 - 25 °C to the oleoresin in hexane and ethanol. The saponification generates heat and this slow addition allows the saponification to proceed at ambient temperature. After 24 h, add dropwise over a 1 h period a 1:1 solution of AcOH-H2O (152 mL, v:v); the slow addition of acid to base is crucial to keep the temperature of the mixture below 30 °C. The relative composition of carotenoids in the crude saponified mixture was: trans-capsanthin (43.40%), cis-capsanthin (12.22%), β-carotene (13.73%), β-cryptoxanthin (9.82%), zeaxanthin (10.73%), cucurbitaxanthin (5.23%) and capsanthone (4.87%). Stir the mixture at ambient temperature for 24 h to crystallize trans-capsanthin in hexane. Filter the saponified oleoresin and save the filtrate for solvent evaporation and recovery. Then wash the solid with hot water (70 °C, 200 g) and dry the crystals on the funnel for 4 h. Wash the crystals with hexane (60 g) and dry on the funnel for about 1 h. In certain embodiments, the drying can be carried out for one or more hours, such as about 1 h to 24 h. Remove the crystals and dry in a vacuum oven at 50 °C for 24 h to obtain a mixture (9.64 g, 83% pure, 7.99 g) of trans-capsanthin (70.23%), zeaxanthin (18.49%), β-cryptoxanthin (2.53%), cucurbitaxanthin (4.60%) and capsanthone (4.15%). Combine the saved filtrate and hexane washings and evaporate and recover hexane and ethanol at 40 °C under reduced pressure (230 Torr) to 45 °C (100 Torr).

[0116] It should be understood that minor modifications can be made to the reagent concentrations, saponification conditions and conditions for separating the carotenoids, their composition and the ranges expressed herein and still be within the scope and spirit of the present invention.

[0117] After describing the present invention by reference to specific compositions, theories of effectiveness, etc., it will be apparent to those skilled in the art that the present invention is not limited to this exemplary embodiment or mechanism and can be modified without departing from the scope or spirit of the present invention as defined by the appended claims. All such obvious modifications and variations are intended to be included within the scope of the present invention as defined by the appended claims. The claims are intended to cover the claimed components and steps as long as they effectively achieve their intended purpose in any order, unless the context clearly indicates the contrary.

[0118] It should also be understood that minor dosage and formulation modifications can be made to the compositions and ranges expressed herein and still be within the scope and spirit of the present invention.

[0119] It should further be understood that the formulations and methods described in the specification are merely exemplary embodiments of the inventive concepts defined in the appended claims. Accordingly, the specific conditions and other physical characteristics of the embodiments disclosed herein are not to be considered as limiting, unless the claims expressly state otherwise. When a numerical range is provided, each intermediate value (to the tenth of the unit of the lower limit) between the upper and lower limits of the range, as well as any other stated or intermediate value within the range, is covered within the scope of the present disclosure. The upper and lower limits of these smaller ranges can be independently incorporated into the smaller ranges and are also covered within the scope of the present disclosure, subject to the specific limitations excluded from the stated range. When the stated range includes one or both of the limitations, ranges excluding either or both of the included limitations are also included within the scope of the present disclosure. All ranges and parameters disclosed herein (including, but not limited to, percentages, parts, and ratios) are to be understood as covering any and all sub-ranges subsumed therein and all numerical values between the endpoints. For example, the range of "1 to 10" should be considered to include any and all sub-ranges starting with a minimum value of 1 or greater and ending with a maximum value of 10 or less (e.g., 1 to 6.1 or 2.3 to 9.4), as well as each integer (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) included within the range. Except where otherwise indicated in certain examples or expressly disclosed herein, all numerical values in this specification and the appended claims are understood to be modified by the word "about" when describing a numerical range.

[0120] In this specification and the appended claims, the English singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. All combinations of method steps or process steps used herein can be performed in any order, unless otherwise specified or the context of the recited combination clearly implies the contrary order.

[0121] With respect to the use of terms such as "comprising", "including", or "having" in the specification or claims, their meaning is similar to the meaning of the term "including" when used as a transitional word in a claim, and is intended to be inclusive. In addition, with respect to the use of the term "or" (e.g., A or B), it is intended to mean "A" or "B" or both "A" and "B". When the applicant intends to mean "only A or B but not both", the term "only A or B but not both" or a similar construction will be used. Thus, the use of the term "or" herein is inclusive and not exclusive. Additionally, with respect to the use of the terms "in" or "into" in the specification or claims, it is intended to also mean "on" or "upon". In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0122] The foregoing description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. It is contemplated that other alternative processes and methods that are obvious to those skilled in the art should also be considered to be included within the invention. This description is only an example of an embodiment. It is understood that any other modifications, substitutions, and / or additions can be made within the intended spirit and scope of the present disclosure. From the foregoing, it can be seen that the exemplary aspects of the present disclosure achieve at least all of the intended objectives.

[0123] The foregoing description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. It should be understood that in some exemplary embodiments, well-known processes, well-known methods, devices, and techniques have not been described in detail. One of ordinary skill in the art will understand that modifications and variations can be made to the disclosed embodiments within the scope of the invention so as to achieve substantially similar results.

[0124] References

[0125] 1.Deli J.,Molnar P.Paprika Carotenoids:Analysis,Isolation,StructureElucidation,Current Organic chemistry.2002,6(13),1197–1219.

[0126] 2. Maoka T., Fujiwara Y., Hashimoto K., Akimoto N. Isolation of a Series of Apocarotenoids from the Fruits of the Red Paprika (Capsicum annuum L.). J. Agric. Food Chem. 2001, 49, 1601–1606.

[0127] 3. Matsufuji H., Nakamura H., Chino M.; Takeda M. Antioxidant Activity of Capsanthin and the Fatty Acid Esters in Paprika (Capsicum annuum). J. Agric. Food Chem. 1998, 46(9), 3468–3472.

[0128] 4. Hernandez-Ortega M., Ortiz-Moreno A., Hernandez-Navarro M.D., Chamorro-Cevallos G., Dorantes-Alvarez L., Necoechea-Mondragon H. Antioxidant, antinociceptive, and anti-inflammatory effects of carotenoids extracted from dried pepper (Capsicum annuum L.). J. Biomed. and Biotech. 2012, 524019, 10 pp.

[0129] 5. Narisawa T. Fukaura Y, Hasebe M., Nomura S., Oshima S., Inakuma T. Prevention of N-methylnitrosourea-induced colon carcinogenesis in rats by oxygenated carotenoid capsanthin and capsanthin-rich paprika juice. Proceedings of the Society for Experimental Biology and Medicine. 2000, 224(2), 116-122.

[0130] 6. Maoka T., Mochida K., Kozuka M., Ito Y., Fujiwara Y., Hashimoto K., Enjo F., Ogata M., Nobukuni Y., Tokuda H. et al. Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L. Cancer Letters. 2001, 172(2), 103 - 109].

[0131] 7. Jo S.J., Kim J.W., Choi H.O., Kim J.H., Kim H.J., Woo S.H., Han B.H. Capsanthin inhibits both adipogenesis in 3T3 - L1 preadipocytes and weight gain in high - fat diet - induced obese mice. Biomolecules & Therapeutics. 2017, 25(3), 329 - 336.

[0132] 8. Aizawa K., Inakuma T. Dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), alters plasma high - density lipoprotein - cholesterol levels and hepatic gene expression in rats British J. of Nutrition. 2009, 102(12), 1760 - 1766.

[0133] 9. Joo H.K., Lee Y.R., Lee E.O., Kim S., Jin H., Kim S., Lim Y.P., An C.G., Jeon B.H. Protective Role of Dietary Capsanthin in a Mouse Model of Nonalcoholic Fatty Liver Disease. J. Medicinal Food. 2021, 24(6), 635 - 644.

[0134] 10. Fernandez-Garcia E., Carvajal-Lerida I., Perez-Galvez A. Carotenoids exclusively synthesized in red pepper (capsanthin and capsorubin) protect human dermal fibroblasts against UVB induced DNA damage. Photochem. & Photobiologic. Sci. 2016, 15(9), 1204 - 1211.

[0135] 11. Maeda H., Nishino A., Maoka T. Biological Activities of Paprika Carotenoids, Capsanthin and Capsorubin. Advances in Experimental Medicine & Biology. 2021, 1261, 285 - 293.

[0136] 12. Duanmu et al., China, CN101906254 A 2010 - 12 - 08, Method for extraction of capsanthin ointment from dry paprika by using dimethyl ether as solvent.

[0137] 13. Reilly et al., US20110282083 A1 2011 - 11 - 17. Process of converting esterified xanthophylls from Capsicum to non - esterified xanthophylls in high yields and purities.

[0138] 14. Jacob et al., WO2011135519 A1 2011 - 11 - 03, Extraction of capsanthin - rich carotenoid mixture from Capsicum annuum oleoresin.

[0139] 15. Umigai et al., JP2015174858 A 2015-10-05. Capsicum annuum (red pepper) pigment extract containing β-cryptoxanthin and capsanthin at controlled ratio.

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Claims

1. A method for low-temperature saponification of carotenoid esters in oleoresin capsici to provide a mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, the method comprising: Treating oleoresin capsici with potassium hydroxide (KOH), sodium hydroxide, or other alkali metal hydroxides in a non-aqueous solution of ethanol (EtOH) or other C1-C3 alcohols and propylene glycol (PG) at a low temperature of about 45°C to 50°C to obtain a saponified mixture; Heating the mixture to a temperature of about 40°C to about 50°C to saponify the carotenoid esters; Treating the saponified paste with water and acetic acid (AcOH) or other weak organic acids such as a 1:1 solution (v:v) of propionic acid or butyric acid in water to neutralize the base; Distilling and recovering ethanol at about 45°C to 50°C under reduced pressure, such as about 200 - 120 torr, to obtain a saponified oleoresin; Treating the saponified oleoresin with water to obtain a suspension of carotenoids; Filtering the suspension and washing the crystals to obtain a crystalline mixture of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin; and Drying the crystalline mixture.

2. The method according to claim 1, wherein the weight ratio of oleoresin capsici:PG:EtOH:KOH is 5:1:4:1 to 5:2:8:

2.

3. The method according to claim 2, wherein the concentration of KOH in EtOH is about 10% to 30% by weight.

4. The method according to claim 1, wherein the composition of the crystalline mixture of carotenoids is about 50% - 70% trans-capsanthin by weight, about 10% - 15% trans-β-carotene by weight, about 10% - 15% trans-β-cryptoxanthin by weight, and about 10% - 25% trans-zeaxanthin by weight.

5. The method according to claim 1, wherein the crystalline mixture of trans-capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin is separated by extraction with a C5-C7 hydrocarbon, preferably hexane, to obtain a hydrocarbon-soluble fraction and insoluble crystals.

6. The method according to claim 5, wherein the hydrocarbon-soluble fraction consists of β-carotene and β-cryptoxanthin, and the crystals consist of trans-capsanthin and trans-zeaxanthin.

7. The method according to claim 6, wherein the composition ratio of crystalline trans-capsanthin to trans-zeaxanthin is 95%:5% to 60%:40%.

8. The method according to claim 6, wherein the hydrocarbon-soluble fraction is evaporated to obtain a crystalline mixture of trans-β-carotene and trans-β-cryptoxanthin.

9. The method according to claim 8, wherein the relative composition of crystalline trans-β-carotene:trans-β-cryptoxanthin is 60%:40% to 20%:80%.

10. The method according to claim 7, wherein trans-capsanthin and trans-zeaxanthin are separated from an aqueous solution of acetone, wherein trans-capsanthin is soluble and trans-zeaxanthin remains as crystals and is removed by filtration.

11. The method according to claim 10, wherein trans-capsanthin is crystallized from water by distillation of acetone.

12. The method according to claim 11, wherein the crystalline trans-capsanthin is dried under high vacuum and produced with a purity of about 60% to 90%.

13. The method according to claim 8, wherein the trans-β-carotene and trans-β-cryptoxanthin are separated by extraction with a C1-C3 alcohol, preferably ethanol, to obtain an alcohol-soluble fraction and insoluble crystals.

14. The method according to claim 13, wherein the alcohol-insoluble fraction consists of trans-β-carotene, and the trans-β-carotene is removed by filtration.

15. The method according to claim 13, wherein the alcohol-soluble fraction consists of trans-β-cryptoxanthin, and the trans-β-cryptoxanthin is obtained by evaporating the alcohol and is crystals with a purity of 60 - 75%.

16. A method for ambient temperature saponification of carotenoid esters in oleoresin capsicum to provide a mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, the method comprising: dissolving the oleoresin capsicum in acetone, and treating it with a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH) or an aqueous KOH solution (40 - 45%) at ambient temperature with stirring to saponify the carotenoid esters and obtain a saponified mixture; treating the saponified mixture with a 1 / 1 solution (v:v) of acetic acid (AcOH) in water to neutralize the base to obtain a suspension of carotenoids; distilling and recovering acetone and ethanol from the filtrate to obtain the saponified oleoresin; adding water and filtering the suspension, and washing the crystals with water to obtain a crystalline mixture of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin; and drying the crystalline mixture of the above carotenoids.

17. The method according to claim 16, wherein the weight ratio of oleoresin capsicum:acetone:EtOH:KOH is 5:15:4:1 to 5:20:9:

1.

18. The method according to claim 17, wherein the concentration of KOH in EtOH is about 10% to 30%.

19. The method according to claim 16, wherein the weight ratio of oleoresin capsicum:acetone:EtOH:aqueous KOH solution is 4:6:1:1 to 5:8:2:

1.

20. The method according to claim 19, wherein the concentration of KOH in water is about 40% to 45% by weight.

21. The method according to claim 16, wherein the composition of the crystalline mixture of carotenoids is about 50% - 70% trans-capsanthin by weight, about 10% - 15% trans-β-carotene by weight, about 5% - 10% trans-β-cryptoxanthin by weight, and about 10% - 20% trans-zeaxanthin by weight.

22. The method according to claim 16, wherein the crystalline mixture of the trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin is separated individually to obtain a purity of about 50% - 90%.

23. A method for ambient temperature saponification of carotenoid esters in oleoresin capsicum to provide a mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, the method comprising: Treat oleoresin capsicum with a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH) at ambient temperature to obtain a saponification mixture; Stir the saponification mixture at ambient temperature for about 6 to 24 hours to saponify the carotenoid esters; Treat the saponified oleoresin with a 1:1 solution (v:v) of acetic acid (AcOH)-water; Distill and recover ethanol from the mixture at 45 - 50 °C under reduced pressure to obtain a saponified paste; Treat the paste with hot water; Filter the suspension and wash the crystals to obtain a crystalline mixture of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin; and Dry the crystalline mixture.

24. The method according to claim 21, wherein the weight ratio of ethanol:oleoresin capsicum is 3:1 to 6:1, and the weight ratio of oleoresin to KOH is 3.3:1 to 5:

1.

25. The method according to claim 22, wherein the concentration of KOH in EtOH is about 5% - 10%.

26. The method according to claim 21, wherein the composition of the crystalline mixture of carotenoids comprises trans-capsanthin in an amount of about 50% to 70% by weight, trans-β-carotene in an amount of about 10% to 15% by weight, trans-β-cryptoxanthin in an amount of about 5% to 10% by weight, and trans-zeaxanthin in an amount of about 10% to 20% by weight.

27. The method according to claim 21, wherein according to the method described in claims 5 - 15, the crystalline mixtures of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin are each separated in high purity.

28. A method for ambient temperature saponification of carotenoid esters in oleoresin capsicum to provide a mixture of capsanthin, β-carotene, β-cryptoxanthin, zeaxanthin, cucurbitaxanthin, and capsanthone, the method comprising: Treat a solution of oleoresin capsicum in hexane and ethanol with an aqueous solution of potassium hydroxide (KOH) at ambient temperature for a time of about 4 to 5 hours to obtain a saponification mixture; Stir the saponification mixture for at least 24 hours to saponify the carotenoid esters; Treat the saponified mixture with a 1:1 solution (v:v) of acetic acid (AcOH):water and stir the mixture at ambient temperature for 24 hours to crystallize trans-capsanthin and trans-zeaxanthin as the main carotenoids and β-carotene, β-cryptoxanthin, cucurbitaxanthin, and capsanthone as the minor carotenoids; Filter the crystallized carotenoids and wash the crystals with hot water at a temperature of about 50 - 70 °C; And dry the crystals for more than one hour; Wash the crystals with a hydrocarbon solvent, preferably hexane, to increase the purity of the carotenoids; and Dry the crystals under high vacuum at about 45 °C to 50 °C to obtain a crystalline mixture with a purity of 80% or higher of trans - capsanthin and trans - zeaxanthin as the main carotenoids and β - carotene, β - cryptoxanthin, cucurbitaxanthin, and capsanthone as the minor carotenoids.

29. The method according to claim 28, wherein the weight ratio of oleoresin:hexane:ethanol:KOH is 3.3:2:1:1 to 4:4:1.2:

1.

30. The method according to claim 28, wherein the concentration of KOH in water is about 40% to 62% by weight.

31. The method according to claim 28, wherein the main carotenoid capsanthin in the oleoresin crystallizes during the saponification process at ambient temperature and is protected from degradation by KOH in the alcohol.

32. The method according to claim 28, wherein the composition of the crystalline mixture of carotenoids contains trans - capsanthin in an amount of about 60% to 80% by weight, trans - zeaxanthin in an amount of about 10% to 20% by weight, β - carotene in an amount of 0 to about 4% by weight, β - cryptoxanthin in an amount of about 2% to 10% by weight, cucurbitaxanthin in an amount of about 2% to 5% by weight, and capsanthone in an amount of about 1% to 5% by weight, and the carotenoids in the crystalline mixture have a purity of 80% or higher.

33. The method according to claim 28, wherein the filtrate from the saponification is rich in β - carotene and β - cryptoxanthin.

34. The method according to claim 28, wherein the main carotenoids capsanthin and zeaxanthin are separated and further purified from the minor carotenoids β - carotene, β - cryptoxanthin, cucurbitaxanthin, and capsanthone.

35. The method according to claim 28, wherein the crystalline mixture contains trans - capsanthin in an amount of about 60% to 80% by weight, trans - zeaxanthin in an amount of about 10% to 20% by weight, β - carotene in an amount of about 0 to 4% by weight, β - cryptoxanthin in an amount of about 2% to 10% by weight, cucurbitaxanthin in an amount of about 2% to 5% by weight, and capsanthone in an amount of about 1% to 5% by weight. The crystalline mixture is extracted with an aqueous acetone solution to obtain a crystalline mixture with a purity of 80% or higher of about 85% trans - capsanthin and about 15% zeaxanthin.

36. The method according to claim 35, wherein the ratio of acetone to water is 9:1 to 3:1 per gram of the capsanthin and zeaxanthin mixture.

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