Method for isolating capsanthin and other carotenoids from paprika oleoresin

Abstract

The present invention relates to a novel method for isolating capsanthin and several other carotenoids from paprika oleoresin, a concentrated extract of paprika fruit (Capsicum annuum). In another aspect, the invention relates to the use of mild non-aqueous and aqueous saponification methods for isolating highly pure capsanthin and other carotenoids from paprika oleoresin at temperatures of about 20-50°C. In another aspect, the invention relates to a saponification method that results in a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, which can be separated by solvent extraction and crystallization to obtain highly pure capsanthin and other carotenoids.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 424,514, filed November 11, 2022, entitled "PROCESS FOR ISOLATION OF CAPSANTHIN AND OTHER CAROTENOIDS FROM PAPRIKA OLEORESIN," the entire disclosure of which is incorporated herein by reference.

[0002] Introduction The present invention relates to a novel method for isolating capsanthin and several other carotenoids from paprika oleoresin, a concentrated extract of paprika fruit (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 low temperatures, such as from ambient 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 the carotenoid esters, followed by crystallization and separation of these carotenoids.

[0003] Background of the Invention The present invention relates to a novel method for isolating capsanthin and several other carotenoids from paprika oleoresin, a concentrated extract of paprika fruit (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 low temperatures, such as from room 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 the carotenoid esters, followed by crystallization and separation of these carotenoids. [Background technology]

[0004] Capsanthin is the major carotenoid in paprika oleoresin, esterified with fatty acid esters. In addition to capsanthin, other major carotenoids in paprika oleoresin are β-carotene, β-cryptoxanthin, and zeaxanthin. Capsanthin is subject to decomposition in hot alkaline solutions, and conventional saponification of this esterified carotenoid in paprika oleoresin at temperatures above 50°C results in significant loss of this heat-labile carotenoid. Therefore, a saponification process that does not result in significant loss of these compounds has long been desired.

[0005] Paprika oleoresin is an extract of paprika fruit (Capsicum annuum) containing approximately 5% major carotenoids and approximately 4% minor carotenoids. The major carotenoids consist mainly of the trans- and cis-isomers of capsanthin (40-48%), β-carotene (11-14%), β-cryptoxanthin (9-10%), zeaxanthin (10-12%), cucurbitaxanthin (5-7%), and capsanthone (5-6%). With the exception of β-carotene, these carotenoids are esterified with palmitic, myristic, and lauric acids. Therefore, to convert capsanthin fatty acid esters to unesterified free capsanthin, the concentrated extract (paprika oleoresin) from paprika fruit is saponified. Similarly, saponification converts zeaxanthin fatty acid esters and β-cryptoxanthin fatty acid esters to their corresponding hydroxycarotenoids. The chemical structures of the major paprika carotenoids are shown in Figure 1. The inventors have also identified several minor carotenoids in paprika oleoresin, including zeaxanthin 3,6-epoxide (cucurbitaxanthin), capsanthone, zeaxanthin 5,8-furanoxide (mutatoxanthin), capsanthin 3,6-epoxide, capsanthin 5,8-furanoxide (capsochrome), and carpoxanthin.

[0006] In addition to the carotenoids mentioned above, paprika fruits also contain capsorubin, cryptocapsin, carpoxanthin, and their corresponding epoxides [J. Deli, P. Molnar, Current Organic Chemistry. (2002), 6(13), 1197-1219]. Eleven minor apocarotenoids have also been isolated and identified in red paprika (Capsicum annuum) fruits [Maoka et al. J. Agric. Food Chem. (2001), 49, 1601-1606]. Due to the complex nature of the carotenoids in paprika oleoresin, this invention focuses on the major carotenoids mentioned above.

[0007] Over the past few decades, the health benefits of dietary carotenoids, such as lutein, zeaxanthin, β-cryptoxanthin, and β-carotene, have been well established. Similarly, the biological activity of capsanthin in disease prevention has been the subject of intensive 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, antinociceptive, and anti-inflammatory effects of carotenoids extracted from dried chili peppers (Capsicum annuum L.) were also reported 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 paprika juice. Similarly, the cancer chemopreventive activity of carotenoids in red pepper (Capsicum annuum L.) fruit was demonstrated by Maoka et al. [Cancer Letters. (2001), 172(2), 103-109]. Capsanthin was also shown to suppress adipogenesis in obesity-induced inflammation of 3T3-L1 preadipocytes and prevent weight gain in high-fat diet-induced obese mice [Jo, Sung Jun, Biomolecules & Therapeutics. (2017), 25(3), 329-336]. Furthermore, dietary capsanthin has been shown to have an HDL cholesterol-raising effect on plasma and hepatic 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 protective ability against nonalcoholic fatty liver disease in mouse models [Joo et al. J. of Medicinal Food. (2021), 24(6), 635-644]. Capsanthin and paprika carotenoids also protect human skin fibroblasts from UVB-induced DNA damage [Fernandez-Garcia et al. Photochem. & Photobiologic. Sci. (2016), 15(9), 1204-1211]. Finally, dietary paprika carotenoids absorbed into the blood have been reported to contribute to endurance performance in athletes by reducing oxygen (VO2) and heart rate [Maeda, Hayato; Nishino, Azusa; Maoka, Takashi, Advances in Experimental Medicine & Biology. (2021), 1261, 285-293].

[0008] Despite the important health benefits of paprika carotenoids, there are only a few patented processes for the extraction and isolation of carotenoids from paprika fruits. For example, Duanmu et al. (Chinese Patent Application Publication No. 101906254(A), December 8, 2010) reported a method for extracting capsanthin ointment from dried paprika by using dimethyl ether as a solvent. However, this patent describes the extraction and preparation of a concentrated extract of paprika containing capsanthin esters, and does not subject the extract to saponification and isolation of purified capsanthin.

[0009] Reilly et al. (U.S. Patent Application Publication No. 20110282083(A1), November 17, 2011) described a process for converting esterified xanthophylls from Capsicum spp. to non-esterified xanthophylls with 60-80% purity. However, Reilly et al. focused only on the isolation of zeaxanthin from paprika oleoresin. Furthermore, the saponification process described by Reilly et al. used hexane, methanol, and aqueous KOH or NaOH (45% solution) for saponification and zeaxanthin isolation at temperatures ranging from 22 to 83°C. Because hydrolysis of esters in aqueous base solutions does not proceed at ambient temperature, saponification in this system had to be performed at elevated temperatures. In some cases, Reilly et al. performed saponification of paprika oleoresin in methanolic KOH at ambient temperature to isolate zeaxanthin. However, this disclosure does not describe the isolation of purified capsanthin, and one skilled in the art would understand that the use of aqueous base requires separation of the organic and aqueous phases in this process. Reilly et al. decanted the upper phase in a centrifuge bottle to remove the aqueous phase, and washed the precipitate with methanol to isolate zeaxanthin. Decanting in a centrifuge bottle is not a process that can be translated into large-scale commercial production without specialized equipment.

[0010] (WO 2011135519(A1), November 3, 2011) described a supercritical fluid extraction process to obtain deodorized paprika, followed by saponification to obtain a Capsicum annuum extract as a water-dispersible powder. However, this process focused on the isolation of a capsanthin-rich carotenoid mixture from Capsicum annuum oleoresin and did not describe the isolation or purification of individual carotenoids.

[0011] Umigai et al. (JP 2015-174858, October 5, 2015) described a formulation of Capsicum annuum (red pepper) pigment extract containing β-cryptoxanthin (I) and capsanthin (II) in a content ratio of II to I ≤ 3, which does not cause fecal redness upon ingestion.

[0012] Sunilkumar et al., in U.S. Patent No. 9,771,323(B2) (September 26, 2017), described the isolation and purification of β-cryptoxanthin from a plant source and a method for its preparation. The plant source was paprika oleoresin, and the authors performed saponification at high temperature (80-85°C) for 3-5 hours, followed by isolation of purified β-cryptoxanthin by column chromatography. Because capsanthin is sensitive to alkaline aqueous solutions at high temperatures, the process described in U.S. Patent No. 9,771,323(B2) almost certainly involves significant loss of this carotenoid. It should be noted that U.S. Patent No. 9,771,323(B2) does not describe a process for isolating purified capsanthin. Details of capsanthin decomposition in aqueous solutions at high temperatures are discussed in detail later in this application.

[0013] In another patented process, Sunilkumar et al. (U.S. Patent No. 10,301,259(B2), May 8, 2019) described a process and composition for the preparation of β-cryptoxanthin from a plant source that improves lung health, physical performance, and cardiopulmonary function and reduces oxidative stress markers. The plant source in this case was paprika oleoresin.

[0014] In another patented process, Deshpande et al. (U.S. Patent No. 10,568,846(B2), February 25, 2020) describes a β-cryptoxanthin composition, its manufacturing process, and its uses, in which the carotenoid is isolated from paprika oleoresin according to the process of Sunilkumar et al. (U.S. Patent No. 9,771,323(B2), September 26, 2017) mentioned above. Deshpande et al. focused on the use of the β-cryptoxanthin composition to improve lung health, physical performance, and cardiopulmonary function. The patent describes administering to an exercising subject an effective amount of a beta-cryptoxanthin composition comprising an extract enriched in trans-beta-cryptoxanthin, wherein the extract contains approximately 75-100% by weight of trans-beta-cryptoxanthin, in an amount effective to reduce oxidative stress markers and increase antioxidant muscle enzymes in the exercising subject compared to an exercising subject not administered the beta-cryptoxanthin composition.

[0015] Finally, a patent application was recently published by Mehta (U.S. Patent Application Publication No. 2022 / 0009886(A1), January 18, 2022) that describes the extraction of paprika carotenoids by extraction with methanol and supercritical fluids, followed by saponification with alcoholic KOH at elevated temperatures in the range of 75-80°C. This report also noted that the carotenoids were purified with ethyl acetate using countercurrent extraction. However, the disclosure did not provide any details or discussion regarding the saponification and purification of the carotenoids.

[0016] As detailed above, previous published studies focused on isolating β-cryptoxanthin and zeaxanthin from paprika oleoresin have consistently disclosed the use of high temperatures during the saponification process, which inevitably results in the degradation of these heat-labile target carotenoids, such as capsanthin. There is an unmet need for a process that uses the mild conditions required for the saponification of carotenoid esters and the isolation of capsanthin and other carotenoids from paprika oleoresin. Summary of the Invention

[0017] For these and other reasons, there is a need for the present invention. The present invention relates generally to a method for isolating and purifying capsanthin and several other major carotenoids from paprika oleoresin. In one particular embodiment, the present invention relates to a method for saponifying the major carotenoid esters of capsanthin, zeaxanthin, and β-cryptoxanthin from paprika oleoresin, followed by crystallization and separation of these carotenoids.

[0018] The inventors have surprisingly discovered a novel methodology, including an ambient to low temperature saponification process and subsequent separation of carotenoids by crystallization without column chromatography, under conditions suitable for alkaline and heat-labile molecules, which overcomes the technical challenges of conventional saponification processes that result in significant loss of these target carotenoids.

[0019] More specifically, the present inventors have surprisingly discovered that a mixture of propylene glycol (PG) and an ethanolic potassium hydroxide (KOH) solution can be used to saponify the carotenoids in paprika oleoresin. In at least one embodiment, the mixture comprises about 15% to about 20% ethanolic potassium hydroxide (KOH) solution for saponifying the carotenoids in paprika oleoresin. For example, in certain embodiments, saponification of the carotenoids in paprika oleoresin is achieved at ambient temperature using about 15% to about 20% ethanolic potassium hydroxide (KOH) solution in acetone or a 5% ethanolic solution without acetone.

[0020] Saponification according to the present invention yields a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin which can be separated by hexane extraction and crystallization to yield highly pure capsanthin as well as other carotenoids.

[0021] In one particular embodiment, saponification of paprika oleoresin was achieved with a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH, 15-20% (wt:wt)) and propylene glycol (PG) at 45-50°C within 3-4 hours. Note that paprika oleoresin also contains β-carotene, a hydrocarbon carotenoid that does not require saponification. After saponification was complete, the ethanol was removed by distillation under reduced pressure at 40-50°C, and the saponified oleoresin in propylene glycol (PG) was diluted with water and the KOH was neutralized with aqueous acetic acid. The oleoresin was filtered at 50-70°C to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, which was washed with water at 50-70°C to remove PG and water-soluble anthocyanins and flavonoids. After drying, these crystalline mixtures of carotenoids were subjected to solvent extraction and crystallization to isolate capsanthin and other carotenoids.

[0022] In certain embodiments, carotenoid esters in paprika oleoresin in acetone were saponified using a 40-60% aqueous solution of potassium hydroxide (KOH) in ethanol at ambient temperature within about 24 hours, e.g., within about 12 hours, or alternatively, within about 6 hours, and in a preferred embodiment, within about 3-6 hours. After saponification, the product was neutralized with aqueous acetic acid. The acetone and ethanol were recovered by distillation under reduced pressure at 40-50°C, and the saponified oleoresin was then diluted with water. This aqueous oleoresin was filtered at 50-70°C to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, which was washed with water at 50-70°C to remove residual acetic acid and water-soluble anthocyanins and flavonoids. The crystalline carotenoids were dried overnight under high vacuum at 50-60°C.

[0023] In certain embodiments, acetone is optionally used as a co-solvent with a KOH solution (wt:wt) in ethanol to facilitate stirring of the saponification mixture. However, in certain embodiments, for example, when saponification of carotenoid esters in paprika oleoresin was carried out with a 5% KOH solution (wt:wt) in ethanol, acetone as a co-solvent was not required.

[0024] In one particular embodiment, saponification of carotenoid esters in paprika oleoresin was carried out with a 5% solution of KOH in ethanol at ambient temperature. After saponification, the KOH was neutralized with aqueous acetic acid, followed by recovery of ethanol by distillation under reduced pressure. The saponified oleoresin was then treated with hot water (approximately 50-70°C), and the crystallized carotenoids were recovered by filtration. The crystalline carotenoids were washed with water (approximately 50-70°C) to remove residual acetic acid and water-soluble anthocyanins and flavonoids, and then dried under high vacuum at approximately 60°C.

[0025] In one particular embodiment, simultaneous saponification and isolation of carotenoid esters in paprika oleoresin was carried out using a mixture of hexane and ethanol by slowly adding aqueous KOH (40-62%, wt:wt) over 4-5 hours at ambient temperature, followed by stirring for approximately 24 hours. After saponification was complete, the KOH was neutralized with aqueous acetic acid, and the saponified mixture was stirred at ambient temperature for 24 hours, resulting in the crystallization of capsanthin and zeaxanthin, while β-carotene and β-cryptoxanthin remained in solution. The crystalline mixture of capsanthin and zeaxanthin was removed by simple filtration and washed sequentially with hot water (approximately 50-70°C) and hexane to enhance the purity of these carotenoids. The crystallized mixture of capsanthin and zeaxanthin was then dried under high vacuum at approximately 50-60°C. The filtrate from this crystallization contained β-carotene, β-cryptoxanthin, and trace amounts of other paprika carotenoids, as well as their cis-isomers. The mixture of hexane and ethanol in the filtrate was recovered by azeotropic distillation of these solvents under reduced pressure. Because the saponification of the carotenoid esters in paprika oleoresin was carried out using a mixture of these solvents, the recovered mixture of hexane and ethanol could be reused without separation. However, it was necessary to adjust the ratio of these solvents.

[0026] In certain embodiments, the crystalline mixture of carotenoids from various saponification processes was subsequently extracted with hexane at about 25 to about 60°C, which solubilized β-carotene and β-cryptoxanthin, while capsanthin and zeaxanthin remained insoluble in this solvent and were removed by filtration at room temperature. Because capsanthin and zeaxanthin exhibited slightly different solubility behavior in aqueous acetone, partial separation of these carotenoids was achieved by extraction. Thus, according to certain embodiments, extraction of a mixture of capsanthin (76%) and zeaxanthin (24%) with an aqueous solution of acetone, followed by filtration, resulted in a crystalline mixture of capsanthin (85%) and zeaxanthin (15%) at 85-90% purity.

[0027] Similarly, extraction of a mixture of β-carotene and β-cryptoxanthin with alcohols such as ethanol, 1-propanol, and 2-propanol resulted in an alcohol-soluble fraction consisting of β-cryptoxanthin, while β-carotene remained as insoluble crystals. Each isolated carotenoid was further purified by crystallization using an appropriate solvent. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the structures of all possible fatty acid esters of the major carotenoids in paprika oleoresin: capsanthin, β-cryptoxanthin, and zeaxanthin, as well as the minor carotenoids cucurbitaxanthin and capsanthone; β-carotene is not esterified, as it is a hydrocarbon carotenoid. [Figure 2] Figure 2 shows the decomposition of capsanthin to β-citraurin by retroaldol condensation, as published by Zechmeister and Cholnoky [Justus Liebigs Annalen der Chemie (1937), 530, 291-300]. [Figure 3] FIG. 3 shows the decomposition of cryptocapsin to β-apo-8′-carotenal by retroaldol condensation, as published by L. Cholnoky and J. Szabolcs (Tetrahedron Letters, No. 19, 1257-1259, 1963). [Figure 4] FIG. 4 is a flow chart for saponification of carotenoids in paprika oleoresin using ethanolic KOH in propylene glycol at 45-50°C and subsequent isolation of capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin by solvent extraction and crystallization. [Figure 5]Figure 5 is a flow chart for the saponification of carotenoids in paprika oleoresin at ambient temperature using ethanolic KOH (15-20% (wt:wt)) in acetone and subsequent isolation of capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin by solvent extraction and crystallization. [Figure 6] FIG. 6 is a flow chart for saponification of carotenoids in paprika oleoresin using ethanolic KOH (5% (wt:wt)) at ambient temperature and subsequent isolation of capsanthin, zeaxanthin, β-carotene, and β-cryptoxanthin by solvent extraction and crystallization. [Figure 7] FIG. 7 is a flow chart for simultaneous saponification and separation of carotenoids in 200 g of paprika oleoresin in hexane and ethanol at ambient temperature using aqueous KOH (40% or 45% or 62% (wt:wt)) and crystallization of the major carotenoids capsanthin and zeaxanthin in high purity after completion of saponification. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention relates to a novel process for saponifying carotenoid esters in paprika oleoresin under mild conditions to obtain a crystalline mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin, with the advantage of avoiding the degradation of alkaline and heat-labile carotenoids such as capsanthin. Another aspect of the invention relates to a novel process for the separation of these carotenoids by solvent extraction and crystallization and the isolation of capsanthin in high purity.

[0030] Because traditional methods use hot alkaline solutions to promote saponification of carotenoid esters in paprika oleoresin, it is inevitable to note the decomposition of capsanthin under these conditions. In 1937, Zechmeister and Cholnoky [Justus Liebigs Annalen der Chemie (1937), 530, 291-300] demonstrated that capsanthin subjected to aqueous potassium hydroxide at 80°C undergoes retroaldol condensation to β-citraurin, as shown in Figure 2. Similarly, treatment of cryptocapsin, which is structurally similar to capsanthin, with hot aqueous potassium hydroxide resulted in the cleavage of this ketocarotenoid to β-apo-8'-carotenal, as shown in Figure 3 (L. Cholnoky and J. Szabolcs, Tetrahedron Letters, No. 19, 1257-1259, 1963). Therefore, the present invention has developed ambient and low-temperature saponification methods for the hydrolysis of capsanthin esters that avoid degradation of this carotenoid. As used herein, "low temperature" means about 20 to about 50°C. For example, in certain embodiments, the saponification method is carried out at ambient temperature (about 20 to 25°C). In alternative embodiments, the saponification method is carried out at a temperature in the range of about 45 to about 50°C. The carotenoid crystalline mixtures obtained by these methods were subjected to solvent extraction to separate capsanthin and other carotenoids.

[0031] According to one particular embodiment, the present invention relates to a method for low temperature saponification of carotenoid esters in paprika oleoresin to obtain a mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin, the method comprising: treating paprika oleoresin with a non-aqueous solution of potassium hydroxide (KOH) or sodium hydroxide, or in alternative embodiments, other known alkali metal hydroxides, in ethanol (EtOH) or other C1-C3 alcohol or mixture, and propylene glycol (PG) at low temperature, for example, about 45-50°C, to obtain a saponified mixture; heating the mixture to a temperature, for example, a temperature in the range of about 40 to about 50°C, to saponify the carotenoid esters; Treating the saponified paste with water and a 1:1 (volume:volume) solution of acetic acid (AcOH) or other weak organic acid such as propanoic acid, butyric acid, etc. in water to neutralize the base; distilling off ethanol at 45-50°C under reduced pressure, e.g., in the range of about 200-120 Torr, to obtain a saponified paste; treating the saponified paste 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 crystal mixture, for example, in certain embodiments, the crystal mixture is dried under high vacuum at 40-60°C, although in alternative embodiments, the mixture is dried using other conventional drying techniques; Includes:

[0032] A process for low-temperature saponification of carotenoid esters in paprika oleoresin using propylene glycol and ethanolic KOH, and subsequent isolation of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin, is shown in Figure 4. According to at least one embodiment, saponification of carotenoid esters in paprika oleoresin was achieved using a non-aqueous solution of KOH (15-20%) in ethanol and propylene glycol (PG, 20% by weight of oleoresin) at 45-50°C within 3-4 hours. In one preferred embodiment, carotenoid esters in paprika oleoresin (50 g) were saponified using a 20% non-aqueous solution of KOH (10 g) in ethanol (40 g) and PG (10 g) at 45-50°C within 3-4 hours. After neutralizing the base with a 50% solution of acetic acid and water (23 mL, 1:1 (volume:volume)), ethanol was recovered by vacuum distillation at 40–50 °C to prevent loss of ethanol-soluble carotenoids during filtration, while propylene glycol (PG) remained in the saponified oleoresin due to its high boiling point (188.2 °C). Because carotenoids exhibit low solubility in PG, it was not necessary to remove this solvent before filtration. Additionally, the use of PG aided in the filtration of the saponified carotenoids. After ethanol evaporation, the saponified mixture was diluted with water (50 g), and the mixture was heated at 50–70 °C to obtain a suspension. The crystalline carotenoids were 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 was dried under high vacuum at 40-60°C to give 4.80 g (60% of total carotenoids, 2.88 g) of capsanthin (61.86%), β-carotene (12.71%), β-cryptoxanthin (8.62%), and zeaxanthin (16.81%), which was subjected to solvent extraction to separate the individual carotenoids.

[0033] In another embodiment of the present invention, carotenoid esters in paprika oleoresin were saponified at ambient temperature with a non-aqueous solution of KOH in ethanol using acetone as a cosolvent, as shown in the flow chart in Figure 5. The weight ratio of acetone to oleoresin was in the range of 2:1 to 4:1, and the concentration of KOH in ethanol was in the range of approximately 10-20%. In a preferred embodiment of the present invention, for example, carotenoid esters in paprika oleoresin (50 g) were solubilized in acetone (100 g), and the resulting mixture was saponified with a 20% non-aqueous solution of KOH (10 g) in ethanol (40 g) at ambient temperature within 24 hours. The use of acetone in the presence of KOH did not result in aldol condensation of acetone because the saponification was performed at ambient temperature. Under these conditions, significant amounts of soap and wax (23 g) were formed, which were solubilized by adding a 1:1 solution of acetic acid and water (20-24 mL). Acetone and ethanol were recovered by vacuum distillation at 35-50°C, yielding a dark red paste. Note that ethanol (boiling point = 78.4°C) and acetone (boiling point = 56.5°C) do not form an azeotrope; their significantly different boiling points allow for easy separation and recovery by distillation. The saponified oleoresin was diluted with water (approximately 50 g). After heating the resulting mixture at 50-70°C, a homogeneous suspension was obtained. The crystalline carotenoids were 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 carotenoid mixture was dried under high vacuum at 40-60°C to yield 4.17 g of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin (60% of total carotenoids, 2.5 g), which was then subjected to solvent extraction to separate the individual carotenoids. This crystalline mixture was extracted with hexane to increase the purity of the mixture to 80-85% and separate capsanthin and zeaxanthin from β-carotene and β-cryptoxanthin.

[0034] In another embodiment of the present invention, carotenoid esters in paprika oleoresin in acetone and ethanol were saponified with aqueous KOH (40%) at ambient temperature for 24 hours to give, after work-up and purification, a crystalline mixture of capsanthin (66.33%), zeaxanthin (20.63%), β-carotene (6.07%), and β-cryptoxanthin (6.97%) in 85% purity. Similarly, carotenoid esters in paprika oleoresin in acetone and ethanol were saponified with aqueous KOH (45%) to give, after work-up and purification, a crystalline mixture of capsanthin (66.11%), zeaxanthin (25.16%), β-carotene (2.81%), and β-cryptoxanthin (5.92%) in 83% purity. In one particular embodiment, when a solution of KOH in ethanol (wt:wt) was used to saponify carotenoid esters in paprika oleoresin at ambient temperature, it was necessary to use acetone as a co-solvent to facilitate stirring of the saponification mixture. However, in an alternative embodiment of the present invention, the carotenoid esters in paprika oleoresin were saponified with a 5% solution of KOH in ethanol (wt:wt) without using acetone as a co-solvent, as shown in the flow chart of Figure 6. The weight ratio of ethanol:oleoresin was in the range of 3.80:1 to 5.70:1, and the weight ratio of oleoresin to KOH was in the range of 3.3:1 to 5:1.

[0035] In one particular embodiment, carotenoid esters in paprika oleoresin (100 g) were saponified with a 5% non-aqueous solution of KOH (20 g) in ethanol (380 g) at ambient temperature for 24 hours or less. The saponified oleoresin was then treated with aqueous acetic acid (1:1 (volume:volume)) to neutralize the base, and the ethanol was recovered by vacuum distillation. The saponified oleoresin was then treated with water (approximately 50-70°C), and the crystallized carotenoids were recovered by filtration and washed with water (approximately 50-70°C) to remove residual acetic acid and water-soluble anthocyanins and flavonoids. The crystals were dried under high vacuum at 60°C, and the resulting carotenoid crystal mixture (9.00 g, 65% purity) was then subjected to hexane extraction and crystallization to separate capsanthin and other carotenoids, as described above. According to one particular embodiment, any drying method may be used.

[0036] The inventors have used normal phase HPLC separation to identify the detailed composition of the major paprika carotenoids after saponification. The relative composition of the major carotenoids in paprika oleoresin after ambient or low temperature saponification according to the present invention is shown in Table 1.

[0037] [Table 1] It should be pointed out that paprika oleoresin also contains 21-23% minor carotenoids, and approximately 10-12% of these carotenoids have been identified as cucurbitaxanthin and capsanthone.

[0038] However, due to the presented complexities, the work described herein focused on the isolation, separation, and purification of the major carotenoids in paprika oleoresin using the novel method described herein. The major carotenoids in saponified paprika oleoresin were also accompanied by significant amounts of their cis-isomers, which was particularly evident in the case of capsanthin, as shown in Table 1. Therefore, the inventors focused on isolating the major carotenoids in paprika oleoresin, which consisted of the trans-isomers of capsanthin, β-carotene, β-cryptoxanthin, and zeaxanthin. This was because it was well established that the cis-isomers of carotenoids do not crystallize well due to their enhanced solubility in almost all organic solvents. Saponification of carotenoid esters in paprika oleoresin at ambient and low temperatures showed that trans-capsanthin accounted for approximately 39-48% of the total carotenoids, accompanied by approximately 12-23% of cis-capsanthin, which did not appear to crystallize. As a result, saponification of carotenoid esters in paprika oleoresin at high temperatures may increase the composition of less desirable cis-carotenoids compared to the corresponding trans-carotenoids, resulting in a low recovery rate of crystallized carotenoids. In addition, saponification at high temperatures (60-80°C) may result in the decomposition of base-sensitive capsanthin to β-citraurin. These drawbacks led the inventors to explore alternative methods, including the identification of a method for low-temperature saponification of carotenoid esters in paprika oleoresin.

[0039] As shown in Table 1, the relative composition of the major carotenoids in paprika oleoresin saponified at ambient or low temperatures (45–48°C) remained constant, with trans-capsanthin accounting for 39–48% of the total carotenoids. As previously noted, the trans-carotenoids in saponified paprika oleoresin were accompanied by significant amounts of their cis-isomers. After saponification, the trans-isomers of the major carotenoids crystallized, while their cis-isomers, as well as trace carotenoids, were removed by filtration. The relative composition of the major crystalline trans-carotenoids, as determined by HPLC after crystallization, is shown in Table 2. These crystalline mixtures were subjected to hexane extraction followed by crystallization to increase the purity of the mixture to 80–85% and separate capsanthin and zeaxanthin from β-carotene and β-cryptoxanthin.

[0040] [Table 2] Furthermore, the present inventors have surprisingly observed that saponification of carotenoid esters in paprika oleoresin can be carried out at ambient temperature with aqueous KOH in hexane and ethanol. Thus, in certain embodiments, carotenoid esters in paprika oleoresin (100 g and 200 g) were saponified by adding 40-62% (wt:wt) aqueous KOH in hexane and ethanol for 24 hours at ambient temperature. Although saponification of esters in aqueous solution at ambient temperature is a reversible reaction that does not proceed at ambient temperature, carotenoid esters in paprika oleoresin were successfully saponified using a highly concentrated aqueous KOH solution. This was because hydrolysis of carotenoid esters in the presence of hexane gradually crystallized unesterified carotenoids, shifting the reaction equilibrium in a forward direction. In these experiments, to prevent the degradation of capsanthin, aqueous KOH was added dropwise to the oleoresin solution in hexane and ethanol over a period of 4-5 hours. This approach maintained a low concentration of KOH during saponification, which is essential to preserve the integrity of capsanthin. The relative composition of saponified carotenoids in crude oleoresin as determined by HPLC is shown in Table 3.

[0041] [Table 3] The saponification workup involved neutralizing the base with 50% aqueous acetic acid (volume:volume) and stirring the product for an additional 24 hours at ambient temperature. This resulted in the crystallization of trans-capsanthin, trans-zeaxanthin, and other minor trans-carotenoids, which were isolated as crystals by simple filtration and further purified by washing with water and hexane. This method proved ideal for simultaneously saponifying and crystallizing trans-capsanthin and trans-zeaxanthin, and their separation from other carotenoids was achieved by simple filtration followed by further purification with hexane. The relative composition of the crystallized carotenoids after removal of the cis-isomer of capsanthin and minor carotenoids is shown in Table 4.

[0042] [Table 4] In Experiments 19–23, saponification of carotenoid esters was performed with 100 g of paprika oleoresin using a saturated aqueous solution of KOH (62%) and only 30 g of ethanol. After crystallization, a mixture of major and minor trans-carotenoids was obtained with a purity of 80–85%. In Experiments 19 and 20, equal weights of hexane and oleoresin (100 g / 100 g) were used. As a result, β-carotene was completely removed from the crystallization into the filtrate, and the composition of capsanthin in the mixture increased. As a result, the crystallized carotenoids from Experiments 19 and 20 contained 72% and 76% trans-capsanthin, respectively. In Experiments 21-23, the hexane / oleoresin weight ratio was reduced to 75 g / 100 g and 50 g / 100 g (Table 3), resulting in crystallized carotenoids containing 2-7% β-carotene and a reduced weight ratio of trans-capsanthin (64-69%). The weight ratio of trans-zeaxanthin in the crystallized product was not affected by the amount of hexane used for saponification, because this carotenoid is completely insoluble in hexane. Since the objective of this invention was to obtain a crystallized product with the highest relative composition of trans-capsanthin, scale-up saponification experiments were performed using 200 g of oleoresin and an equal weight of hexane (Experiments 24-29). When saponification of carotenoid esters in paprika oleoresin was performed at a 200 g scale using 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 80–85% purity, containing 70–73% trans-capsanthin, 16–18% trans-zeaxanthin, 2.47–6.10% β-cryptoxanthin, 2.40–4.60% cucurbitaxanthin, and 4.15–5.44% capsanthone. A detailed flowchart of this method for saponification of carotenoid esters in 200 g of paprika oleoresin is shown in Figure 7.

[0043] In the first step, paprika oleoresin was solubilized in hexane and ethanol, and various concentrations of KOH (62%, 45%, or 40%) were added over 4–5 hours. The mixture was stirred at 20–25°C for approximately 24 hours. In the second step, the base was neutralized with a 1:1 acetic acid-water (volume:volume) solution. In the third step, the mixture was stirred at ambient temperature to promote the crystallization of capsanthin and zeaxanthin. In the fourth step of this process, the crystallized carotenoids were filtered, washed with water at 70°C (200 g / 200 g oleoresin), and dried on the funnel for 4 hours. To increase the purity of the carotenoids, the solid was then washed with an appropriate amount of hexane in the fifth step of this process. The crystallized carotenoids were then dried under high vacuum at 50°C to yield 8.0 g of a mixture of capsanthin (71%), zeaxanthin (18%), β-cryptoxanthin (3%), cucurbitaxanthin (3%), and capsanthone (6%) with a purity of 80-85%. This crystalline mixture was subjected to extraction with aqueous acetone to yield 7.0 g of a crystalline mixture of capsanthin (85%) and zeaxanthin (15%) with a purity of 90%. In an alternative embodiment, the crude crystalline carotenoids were washed with water at 70°C and dried. The solid was then stirred with hexane at ambient temperature for several hours and filtered, increasing the purity of the crystallized carotenoids to 80-85%. The filtrate from this process, containing hexane and ethanol, was evaporated under reduced pressure and reused without separating these solvents. Hexane (85%), ethanol (12%), and water (3%) form an azeotropic mixture and can be reused without separation. The presence of a small amount of water carried over due to azeotropic distillation with ethanol is not a problem because the saponification of carotenoid esters in paprika oleoresin is carried out in aqueous KOH with a mixture of these solvents. However, the hexane / ethanol ratio needs to be adjusted according to the saponification protocol. The filtrate from the saponification also contains excess acetic acid, which does not form an azeotrope with either hexane or ethanol.

[0044] Isolation and purification of crystalline carotenoid mixtures from saponified paprika oleoresin. As shown in Figure 4, separation of the crystalline mixture of capsanthin, zeaxanthin, β-carotene, and cryptoxanthin obtained from the saponification of carotenoid esters in paprika oleoresin was achieved by sequential extraction with appropriate solvents. In the first step, β-carotene and β-cryptoxanthin were solubilized and extracted from the carotenoid mixture using a C5-C8 hydrocarbon, preferably hexane, while zeaxanthin and capsanthin remained insoluble in the hydrocarbon solvent. In a preferred embodiment of the present invention, a crystalline mixture (12.53 g) of capsanthin (61.86%), zeaxanthin (16.81%), β-carotene (12.71%), and β-cryptoxanthin (8.62%) was extracted with hexane (350 g) at temperatures ranging from 25 to 70 °C for 1 to 3 hours. After stirring the mixture at ambient temperature for 3-5 hours, the solid was filtered and washed with hexane (60 g). The crystallized solid (9.81 g) consisted of capsanthin (76.36%) and zeaxanthin (23.64%). This crystalline mixture was extracted with aqueous acetone and filtered. After drying the resulting solid, 7.0 g of a mixture of capsanthin (85%) and zeaxanthin (15%) was obtained with a purity of 85-90%. The filtrate was evaporated to dryness to yield 2.72 g of a mixture of β-carotene (62.99%) and β-cryptoxanthin (37.01%).

[0045] In a preferred embodiment, the ratio of acetone to water is in the range of 9:1 to 3:1 per gram of the capsanthin and zeaxanthin mixture. The following examples are offered to illustrate, not limit, the present invention, and as such, are presented with the understanding that various formulation modifications, as well as modifications to delivery methods, may be made and still fall within the spirit of the present invention.

[0046] Example 1 Low temperature saponification of carotenoid esters in paprika oleoresin using 20% ​​KOH in ethanol (EtOH) and propylene glycol (PG) as a cosolvent Paprika oleoresin (50 g) was transferred to a 250 mL round-bottom flask equipped with a stir bar and treated with a 20% (wt:wt) solution of KOH (10 g) in propylene glycol (PG, 10 g) and ethanol (40 g). The flask was placed in a 55 °C oil bath, and the mixture was stirred at 45–50 °C. The progress of saponification was monitored by HPLC, which showed complete saponification after 3 h. The saponification proceeded for an additional 1 h (4 h total), after which the heat was removed. The mixture was cooled to ambient temperature, and a solution of acetic acid (AcOH) and water (23 mL, 1:1 (v:v)) was added. The ethanol was evaporated under reduced pressure (100 torr) on a rotary evaporator (bath temperature: 55 °C) at 45–50 °C (solution temperature). Hot water (70 °C, 50 g) was added, and the mixture was stirred at 70 °C for 1 h until the paste dissolved and a suspension was obtained. The resulting suspension was filtered while hot. The purple solid was washed with water (50 g) at 70 °C, and the crystals were dried in an oven at 60 °C under high vacuum for 24 hours to yield a crystalline mixture of carotenoids (4.80 g, 60% total carotenoids, 2.88 g). The relative composition of carotenoids determined by HPLC was capsanthin (61.86%), β-carotene (12.71%), β-cryptoxanthin (8.62%), and zeaxanthin (16.81%). The crystalline mixture was stirred with hexane (15 g) at ambient temperature for 4 hours and filtered. The crystals were washed with hexane (15 g) and dried under high vacuum at 40–60°C for 24 h to obtain 3.05 g of a mixture of capsanthin (76.20%), zeaxanthin (19.18%), and β-cryptoxanthin (4.62%) (84% of total carotenoids, 2.56 g).

[0047] Example 2 Low temperature saponification of carotenoid esters in paprika oleoresin using 20% ​​KOH in ethanol (EtOH) and propylene glycol (PG) as a cosolvent Paprika oleoresin (100 g) was transferred to a 500 mL round-bottom flask equipped with a stir bar and treated with a solution of propylene glycol (PG, 20 g) and KOH (20 g) in ethanol (80 g) [20% (wt:wt)]. The flask was placed in a 55 °C oil bath and the mixture was stirred at 45–50 °C. After 4 h, the heat was removed and the mixture was cooled to ambient temperature. A solution of acetic acid (AcOH) and water (45 mL, 1:1 (v:v)) was added, and the ethanol was evaporated under reduced pressure (100 torr) on a rotary evaporator (bath temperature: 55 °C) at 45–50 °C (solution temperature). Hot water (70 °C, 100 g) was added, and the mixture was stirred at 70 °C until the paste dissolved and a suspension was obtained. The mixture was filtered while hot. The purple solid was washed with water (100 g) at 70 °C, and the crystals were dried in an oven at 60 °C under high vacuum for 24 hours to yield a crystalline mixture of carotenoids (8.30 g, 60% total carotenoids, 4.98 g). The relative composition of carotenoids determined by HPLC was capsanthin (63.65%), β-carotene (13.54%), β-cryptoxanthin (7.64%), and zeaxanthin (15.17%). The crystalline mixture was stirred with hexane (30 g) at ambient temperature for 4 hours and filtered. The crystals were washed with hexane (30 g) and dried under high vacuum at 40–60°C for 24 h to obtain 5.15 g of a mixture of capsanthin (74.5%), zeaxanthin (20.60%), and β-cryptoxanthin (4.90%) (85% of total carotenoids, 4.37 g).

[0048] Example 3 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 20% ​​KOH in ethanol and acetone as cosolvent Paprika oleoresin (50 g) was transferred to a 250 mL Erlenmeyer flask equipped with a stir bar and dissolved in acetone (100 g). This mixture was treated with a solution of KOH (10 g) in ethanol (40 g) [20% (wt:wt)] at ambient temperature. This resulted in the formation of significant amounts of soft and hard waxes, as well as potassium salts of fatty acids that precipitated from the solution. The progress of saponification was monitored by HPLC, which showed complete saponification after 24 h. A solution of acetic acid (AcOH) and water (21 mL, 1:1 (v:v)) was added, and the mixture was stirred at ambient temperature for 20 min. Acetone was first distilled under reduced pressure (300 torr) at 35-40°C, followed by distillation of ethanol at 45-50°C (100 torr) to obtain the oleoresin in water. The oleoresin was treated with hot water (70°C, 50 g), and the mixture was stirred at 70°C for 1 h until the paste dissolved and a uniform suspension was obtained. The resulting suspension was filtered while still hot. The purple solid was washed with 70°C water (100 g), and the crystals were dried in an oven at 60°C under high vacuum for 24 hours to yield a crystalline mixture of carotenoids (4.10 g, 62% total carotenoids, 2.54 g). The relative composition of the carotenoids, as determined by HPLC, was capsanthin (67.14%), β-carotene (5.20%), β-cryptoxanthin (7.29%), and zeaxanthin (20.37%). The crystalline mixture was stirred with hexane (15 g) at ambient temperature for 4 hours and filtered. The crystals were washed with hexane (15 g) and dried under high vacuum at 40–60°C for 24 h to obtain 2.76 g of a mixture of capsanthin (75.4%), zeaxanthin (21.30%), and β-cryptoxanthin (3.30%) (85% of total carotenoids, 2.35 g).

[0049] Example 4 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 20% ​​KOH in ethanol and acetone as cosolvent Paprika oleoresin (100 g) was transferred to a 500 mL Erlenmeyer flask equipped with a stir bar and dissolved in acetone (300 g). This mixture was treated with a solution of KOH (20 g) in ethanol (80 g) [20% (wt:wt)] at ambient temperature. This resulted in the formation of significant amounts of soft and hard waxes, as well as potassium salts of fatty acids that precipitated from the solution. The progress of saponification was monitored by HPLC, which showed complete saponification after 24 h. A solution of acetic acid (AcOH) and water (42 mL, 1:1 (v:v)) was added, and the mixture was stirred at ambient temperature for 20 min. Acetone was first distilled under reduced pressure (300 torr) at 35-40°C, followed by distillation of ethanol at 45-50°C (100 torr) to obtain the oleoresin in water. The oleoresin was treated with hot water (70°C, 100 g), and the mixture was stirred at 70°C for 1 h until a homogeneous suspension was obtained. The resulting suspension was filtered while still 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 hours to yield a crystalline mixture of carotenoids (8.50 g, 61% total carotenoids, 5.19 g). The relative composition of the carotenoids, as determined by HPLC, was capsanthin (63.66%), β-carotene (7.92%), β-cryptoxanthin (8.38%), and zeaxanthin (20.04%). The crystalline mixture was stirred with hexane (30 g) at ambient temperature for 4 hours and filtered. The crystals were washed with hexane (30 g) and dried under high vacuum at 40–60°C for 24 h to obtain 5.67 g of a mixture of capsanthin (75.8%), zeaxanthin (20.4%), and β-cryptoxanthin (3.8%) (83% of total carotenoids, 4.71 g).

[0050] Example 5 Saponification of carotenoid esters in paprika oleoresin in acetone and ethanol using 40% aqueous KOH at ambient temperature Paprika oleoresin (100 g) was transferred to a 500 mL Erlenmeyer flask equipped with a stir bar and dissolved in acetone (150 g) and ethanol (30 g). This mixture was treated dropwise with a solution of KOH (20 g) in water (30 g) [40% (wt:wt)] for 4 hours at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. A solution of acetic acid (AcOH) and water (61 mL, 1:1 (v:v)] was added, and the mixture was stirred at ambient temperature for 2 hours. The acetone was first distilled under reduced pressure (300 torr) at 35-40°C, followed by distillation of ethanol at 45-50°C (100 torr) to obtain the oleoresin in water. The oleoresin was treated with hot water (70°C, 100 g), and the mixture was stirred at 70°C for 1 hour until a homogeneous suspension was obtained. The resulting suspension was filtered while still hot. The purple solid was washed with water (200 g) at 70 °C, and the crystals were dried in an oven at 60 °C under high vacuum for 24 hours to yield a crystalline mixture of carotenoids (8.20 g, 62% total carotenoids, 5.08 g). The relative composition of the carotenoids determined by HPLC was capsanthin (66.33%), β-carotene (6.07%), β-cryptoxanthin (6.97%), and zeaxanthin (20.63%). This crystalline mixture was stirred with hexane (30 g) at ambient temperature for 4 hours and filtered. The crystals were washed with hexane (30 g) and dried under high vacuum at 40–60 °C for 24 hours to yield 5.60 g (85% total carotenoids, 4.76 g) of a mixture of capsanthin (74.4%), zeaxanthin (20.8%), and β-cryptoxanthin (4.8%).

[0051] Example 6 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 5% KOH in ethanol Paprika oleoresin (100 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and treated with a solution of KOH (20 g) [5% (wt:wt)] in ethanol (380 g) at ambient temperature. The progress of saponification 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 and water (vol:vol), and the mixture was stirred at ambient temperature for 30 min. The 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 still hot. The purple solid was washed with water (200 g) at 70°C, and the crystals were dried in an oven at 60°C under high vacuum for 24 hours to give a crystalline mixture of carotenoids (9.00 g, 65% total carotenoids). The relative composition of the carotenoids determined by HPLC was capsanthin (65.35%), β-carotene (11.07%), β-cryptoxanthin (7.68%), and zeaxanthin (15.90%).

[0052] Example 7 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 5% KOH in ethanol Paprika oleoresin (100 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and treated with a solution of KOH (20 g) [5% (wt:wt)] in ethanol (380 g) at ambient temperature. The progress of saponification 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 and water (vol:vol), and the mixture was stirred at ambient temperature for 30 min. The 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 still hot. The purple solid was washed with water (200 g) at 70°C, and the crystals were dried in an oven at 60°C under high vacuum for 24 hours to give a crystalline mixture of carotenoids (9.35 g, 63% total carotenoids). The relative composition of the carotenoids determined by HPLC was capsanthin (64.79%), β-carotene (11.77%), β-cryptoxanthin (7.28%), and zeaxanthin (16.16%).

[0053] Example 8 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 5% KOH in ethanol Paprika oleoresin (100 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and treated with a solution of KOH (25 g) in ethanol (475 g) [5% (wt:wt)] at ambient temperature. The progress of saponification 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 and water (vol:vol), and the mixture was stirred at ambient temperature for 30 min. The 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 still hot. The purple solid was washed with water (200 g) at 70°C, and the crystals were dried in an oven at 60°C under high vacuum for 24 hours to give a crystalline mixture of carotenoids (9.72 g, 64% total carotenoids). The relative composition of the carotenoids determined by HPLC was capsanthin (64.05%), β-carotene (10.73%), β-cryptoxanthin (8.11%), and zeaxanthin (17.11%).

[0054] Example 9 Ambient temperature saponification of carotenoid esters in paprika oleoresin using 5% KOH in ethanol Paprika oleoresin (100 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and treated with a solution of KOH (30 g) [5% (wt:wt)] in ethanol (570 g) at ambient temperature. The progress of saponification 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 and water (vol:vol), and the mixture was stirred at ambient temperature for 30 min. The 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 still hot. The purple solid was washed with water (200 g) at 70°C, and the crystals were dried in an oven at 60°C under high vacuum for 24 hours to give a crystalline mixture of carotenoids (8.47 g, 60% total carotenoids). The relative composition of the carotenoids determined by HPLC was capsanthin (65.32%), β-carotene (10.68%), β-cryptoxanthin (7.75%), and zeaxanthin (16.25%).

[0055] Example 10 Saponification of carotenoid esters in paprika oleoresin in hexane and ethanol using 62% aqueous KOH at ambient temperature Paprika oleoresin (200 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and stirred at ambient temperature in hexane (200 g) and ethanol (60 g) until the oleoresin dissolved. A saturated solution of KOH (50 g) [62% (wt:wt)] in water (30 g) was transferred to a dropping funnel and added dropwise to the oleoresin in hexane and ethanol over 4 hours at 20-25 °C. Saponification generates heat, and this slow addition allowed the saponification to proceed at ambient temperature. After 24 hours, a 1:1 solution of AcOH-HO (152 mL (v:v)) was added dropwise over 1 hour; however, slow addition of the acid to the base was essential to maintain 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%). The mixture was stirred at ambient temperature for 24 hours to crystallize trans-capsanthin in hexane. The saponified oleoresin was filtered, and the filtrate was saved for solvent evaporation. The solid was then washed with hot water (70°C, 200 g), and the crystals were dried on the funnel for 4 hours. The crystals were washed with hexane (60 g) and dried on the funnel for 1 hour. The crystals were removed and dried in a vacuum oven at 50°C for 24 hours to yield a mixture of trans-capsanthin (72.65%), zeaxanthin (17.29%), β-cryptoxanthin (2.47%), cucurbitaxanthin (2.81%), and capsanthone (4.78%) (8.41 g, 85% purity, 7.14 g). The pooled filtrate and hexane washes were combined, and the hexane and ethanol were evaporated under reduced pressure (230 Torr) at 40°C to 45°C (100 Torr).

[0056] Example 11 Saponification of carotenoid esters in paprika oleoresin in hexane and ethanol using 45% aqueous KOH at ambient temperature Paprika oleoresin (200 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and stirred at ambient temperature in hexane (200 g) and ethanol (60 g) until the oleoresin dissolved. A saturated solution of KOH (50 g) in water (61 g) [45% (wt:wt)] was transferred to a dropping funnel and added dropwise to the oleoresin in hexane and ethanol over 4 hours at 20-25 °C. Saponification generates heat, and this slow addition allowed the saponification to proceed at ambient temperature. After 24 hours, a 1:1 solution of AcOH-HO (152 mL (v:v)) was added dropwise over 1 hour; however, it was essential to add the acid slowly to the base to maintain the temperature of the mixture below 30 °C. The relative composition of carotenoids in the crude saponified mixture was trans-capsanthin (48.25%), cis-capsanthin (11.42%), β-carotene (11.87%), β-cryptoxanthin (10.15%), zeaxanthin (11.92%), cucurbitaxanthin (5.93%), and capsanthone (5.56%). The mixture was stirred at ambient temperature for 24 hours to crystallize trans-capsanthin in hexane. The saponified oleoresin was filtered, and the filtrate was saved for solvent evaporation. The solid was then washed with hot water (70°C, 200 g), and the crystals were dried on the funnel for 4 hours. The crystals were washed with hexane (60 g) and dried on the funnel for 1 hour. The crystals were removed and dried in a vacuum oven at 50°C for 24 hours to yield a mixture of trans-capsanthin (71.08%), zeaxanthin (18.27%), β-cryptoxanthin (2.90%), cucurbitaxanthin (2.81%), and capsanthone (4.94%) (9.28 g, 84% purity, 7.80 g). The pooled filtrate and hexane washes were combined, and the hexane and ethanol were evaporated under reduced pressure (230 Torr) at 40°C to 45°C (100 Torr).

[0057] Example 12 Saponification of carotenoid esters in paprika oleoresin in hexane and ethanol using 40% aqueous KOH at ambient temperature Paprika oleoresin (200 g) was transferred to a 1000 mL Erlenmeyer flask equipped with a stir bar and stirred at ambient temperature in hexane (200 g) and ethanol (60 g) until the oleoresin dissolved. A saturated solution of KOH (60 g) [40% (wt:wt)] in water (90 g) was transferred to a dropping funnel and added dropwise to the oleoresin in hexane and ethanol over 4 hours at 20-25 °C. Saponification generates heat, and this slow addition allowed the saponification to proceed at ambient temperature. After 24 hours, a 1:1 solution of AcOH-HO (152 mL (v:v)) was added dropwise over 1 hour; however, slow addition of the acid to the base was essential to maintain 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%). The mixture was stirred at ambient temperature for 24 hours to crystallize trans-capsanthin in hexane. The saponified oleoresin was filtered, and the filtrate was saved for solvent recovery by evaporation. The solid was then washed with hot water (70°C, 200 g), and the crystals were dried on the funnel for 4 hours. The crystals were washed with hexane (60 g) and dried on the funnel for about 1 hour. In certain embodiments, drying may be carried out for more than 1 hour, for example, for about 1 to 24 hours. The crystals were removed and dried in a vacuum oven at 50°C for 24 hours to yield a mixture of trans-capsanthin (70.23%), zeaxanthin (18.49%), β-cryptoxanthin (2.53%), cucurbitaxanthin (4.60%), and capsanthone (4.15%) (9.64 g, 83% purity, 7.99 g). The pooled filtrate and hexane washes were combined, and the hexane and ethanol were evaporated under reduced pressure (230 Torr) at 40°C to 45°C (100 Torr).

[0058] It is understood that minor modifications may be made to the concentrations of reagents, saponification conditions, and carotenoid separation conditions, their compositions, and ranges expressed herein and still fall within the scope and spirit of the present invention.

[0059] While the present invention has been described with reference to particular compositions, theories of operation, and the like, it will be apparent to those skilled in the art that the invention is not intended to be limited to such exemplary embodiments or mechanisms, and that modifications may be made without departing from the scope or spirit of the invention as defined in the appended claims. All such obvious modifications and variations are intended to be included within the scope of the invention as defined in the appended claims. The claims are intended to encompass the claimed components and steps in any order that is effective to meet the intended purpose, unless the context clearly indicates otherwise.

[0060] It is further understood that minor dosage and formulation variations of the compositions and ranges expressed herein may be made and still fall within the scope and spirit of the invention. It should also be understood that the formulations and processes described herein are merely exemplary embodiments of the inventive concept defined in the appended claims. Therefore, specific conditions and other physical characteristics relating to the embodiments disclosed herein should not be considered limiting unless expressly stated otherwise in the claims. Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intermediate value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intermediate value within that stated range, is encompassed within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the disclosure, even if any limit in the stated range is specifically excluded. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the scope of the disclosure. All ranges and parameters disclosed herein, including but not limited to percentages, parts, and ratios, are understood to encompass every subrange contemplated and incorporated therein, and every number between the endpoints. For example, a range stated as "1 to 10" should be considered to include all subranges beginning at a minimum value of 1 or more and ending at a maximum value of 10 or less (e.g., 1 to 6.1, or 2.3 to 9.4), and each integer subsumed within that range (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Except in certain specific examples or as expressly disclosed herein, all numerical values ​​in this specification and the appended claims, when describing numerical range regions, are understood to be modified by the word "about."

[0061] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As used herein, all combinations of method or process steps may be performed in any order unless otherwise specified or unless the contrary is clearly implied by the context in which the combination is recited.

[0062] To the extent the term "includes," "including," "have," or "having" is used in this specification or the claims, it is intended to be inclusive, similar to the interpretation of the term "comprising" when used as a transitional phrase in the claims. Furthermore, to the extent the term "or" is used (e.g., A or B), it is intended to mean either "A" or "B," or both "A" and "B." If the applicant intends to indicate "A or B, but not both," the term "A or B, but not both" or similar construction would be used. Thus, the use of the term "or" herein is inclusive, not exclusive. Also, to the extent the term "in" or "into" is used in this specification or the claims, it is intended to also mean "on" or "onto." In this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0063] The foregoing description has been presented for purposes of illustration and description. It is not intended to be an exhaustive list or to limit the invention to the precise form disclosed. It is intended that other alternative processes and methods apparent to those skilled in the art be considered within the scope of the present invention. The description is merely an example of an embodiment. It is understood that any other modifications, substitutions, and / or additions may be made and are 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 their intended objectives.

[0064] The foregoing description has been presented for purposes of illustration and description. It is not intended to be an exhaustive list or to limit the invention to the precise form disclosed. It should be understood that in some exemplary embodiments, well-known processes, methods, devices, and techniques have not been described in detail. Those skilled in the art will appreciate that modifications and variations of the disclosed embodiments can be made within the scope of the present invention to achieve substantially similar results.

[0065] References 1.Deli J., Molnar P. Paprika Carotenoids: Analysis, Isolation, Structure Elucidation, Current Organic chemistry. 2002, 6(13), 1197 - 1219. 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. 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. 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. 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. 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]. 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. 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. 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. 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. 11.Maeda H., Nishino A., Maoka T. Biological Activities of Paprika Carotenoids, Capsanthin and Capsorubin. Advances in Experimental Medicine & Biology. 2021, 1261, 285-293. 12. Duanmu et al. China, China Patent Application Publication No. 101906254(A) 2010-12-08, Method for extraction of capsanthin ointment from dry paprika by using dimethyl ether as solvent. 13. Reilly et al. U.S. Patent Application Publication No. 20110282083(A1) 2011-11-17. Process of converting esterified xanthophylls from Capsicum to non-esterified xanthophylls in high yields and purities. 14. Jacob et al. WO 2011135519(A1) 2011-11-03, Extraction of capsanthin-rich carotenoid mixture from Capsicum annuum oleoresin. 15.Umigai et al., JP2015-174858A 2015-10-05. Capsicum annuum (red pepper) pigment extract containing β-cryptoxanthin and capsanthin at controlled ratio. 16. Sunilkumar et al. U.S. Patent No. 9,771,323(B2), Sep. 26, 2017, Omni Active Health Technology Ltd. beta-Cryptoxanthin from plant source and process for its preparation. 17. Sunilkumar et al. U.S. Patent No. 10,301,259(B2), May 28, 2019, Omni Active Health Technology Ltd. beta-Cryptoxanthin from plant source and process for its preparation. 18. Deshpande et al. U.S. Patent No. 10,568,846(B2), February 25, 2020, beta-Cryptoxanthin compositions, processes for preparation and uses thereof. 19. Mehta S. U.S. Patent Application Publication No. 2022 / 0009886(A1), Jan. 18, 2022, Manufacture of carotenoid compositions 20.Zechmeister Cholnoky L. The studies on paprika dye; citraurin from capsanthin. Justus Liebigs Annalen der Chemie, 1937, 530, 291-300. 21.Cholnoky L., Szabolcs J. The structure of cryptocapsin, Tetrahedron Letters, 1963, No. 19, 1257 - 1259.

Claims

1. 1. A process for the saponification of carotenoid esters in paprika oleoresin at low temperature to obtain a mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin, comprising: treating paprika oleoresin with a non-aqueous solution of potassium hydroxide (KOH) or sodium hydroxide or other alkali metal hydroxide in ethanol (EtOH) or other C1-C3 alcohol and propylene glycol (PG) at low temperature of about 45-50°C to obtain a saponified mixture; heating the mixture to a temperature in the range of about 40 to about 50°C to saponify the carotenoid esters; treating the saponified paste with a 1:1 solution (volume:volume) of water and acetic acid (AcOH) or other weak organic acid such as propanoic acid, butyric acid, etc. in water to neutralize the base; distilling off the ethanol at about 45-50°C under reduced pressure, e.g., 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; A method comprising:

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

2.

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

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

5. The crystalline mixture of trans-capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin is C ５ ~C ７ 2. The method of claim 1, wherein the separation is carried out by extraction with a hydrocarbon, preferably hexane, to obtain a hydrocarbon-soluble fraction and insoluble crystals.

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

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

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

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

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

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

13. Trans-β-carotene and trans-β-cryptoxanthin are C １ ~C ３ 9. The method according to claim 8, wherein the separation is carried out by extraction with alcohol, preferably ethanol, to obtain an alcohol-soluble fraction and insoluble crystals.

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

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

16. 1. A process for the saponification of carotenoid esters in paprika oleoresin at ambient temperature to obtain a mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin, comprising: solubilizing paprika oleoresin in acetone and treating the solution with a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH) or an aqueous solution of KOH (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 of acetic acid (AcOH) in water (volume:volume) to neutralize the base and obtain a suspension of carotenoids; recovering acetone and ethanol from the filtrate by distillation to obtain a saponified oleoresin; adding water, 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 said crystalline mixture of said carotenoids; A method comprising:

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

1.

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

19. 17. The method of claim 16, wherein the weight ratio of paprika oleoresin:acetone:EtOH:aqueous KOH is in the range of 4:6:1:1 to 5:8:2:

1.

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

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

22. 17. The method of claim 16, wherein the crystalline mixture of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin, and trans-zeaxanthin is separated from each other to obtain a purity in the range of about 50-90%.

23. 1. A process for the saponification of carotenoid esters in paprika oleoresin at ambient temperature to obtain a mixture of capsanthin, β-carotene, β-cryptoxanthin and zeaxanthin, comprising: treating paprika oleoresin with a non-aqueous solution of potassium hydroxide (KOH) in ethanol (EtOH) at ambient temperature to obtain a saponified mixture; stirring the saponification mixture at ambient temperature for about 6 to 24 hours to saponify the carotenoid esters; treating the saponified oleoresin with a 1:1 solution (volume:volume) of acetic acid (AcOH)-water; distilling ethanol from the mixture at 45-50°C under reduced pressure to obtain a saponified paste; treating the paste with hot water; 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; A method comprising:

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

1.

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

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

27. 22. The method of claim 21, wherein the crystalline mixture of trans-capsanthin, trans-β-carotene, trans-β-cryptoxanthin and trans-zeaxanthin is separated from each other in high purity according to the method of any one of claims 5 to 15.

28. 1. A process for the saponification of carotenoid esters in paprika oleoresin at ambient temperature to obtain a mixture of capsanthin, β-carotene, β-cryptoxanthin, zeaxanthin, cucurbitaxanthin and capsanthone, comprising: treating a solution of paprika oleoresin in hexane and ethanol with aqueous potassium hydroxide (KOH) in water at ambient temperature for about 4-5 hours to obtain a saponified mixture; stirring the saponification mixture for at least 24 hours to saponify the carotenoid esters; treating the saponified mixture with a 1:1 solution (volume:volume) of acetic acid (AcOH):water and stirring the mixture at ambient temperature for 24 hours to crystallize trans-capsanthin and trans-zeaxanthin as major carotenoids and β-carotene, β-cryptoxanthin, cucurbitaxanthin, and capsanthone as minor carotenoids; filtering the crystallized carotenoid and washing the crystals with hot water at a temperature of about 50-70°C; drying the crystals for more than 1 hour; washing the crystals with a hydrocarbon solvent, preferably hexane, to increase the purity of the carotenoids; and drying the crystals under high vacuum at about 45-50°C to obtain a crystalline mixture of trans-capsanthin and trans-zeaxanthin as major carotenoids, and β-carotene, β-cryptoxanthin, cucurbitaxanthin, and capsanthone as minor carotenoids, with a purity of greater than 80%; A method comprising:

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

1.

30. 29. The method of claim 28, wherein the concentration of KOH in water is in the range of about 40 to 62 wt.%.

31. 29. The method of claim 28, wherein capsanthin, the major carotenoid in paprika oleoresin, is crystallized during ambient temperature saponification and protected from degradation by KOH in alcohol.

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

33. 29. The method of claim 28, wherein the filtrate from the saponification is enriched in β-carotene and β-cryptoxanthin.

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

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

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

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