Preparation of cosmetic carotenoid compositions

Abstract

The capsicum extract composition (600) comprises capsanthin in the range of 1-1.5%, zeaxanthin in the range of 0.5-1%, and cryptoxanthin in the range of 0.1-0.5%. The composition is produced, at least in part, from materials consumed from purifying carotenoids using countercurrent extraction, and the composition is for strengthening the skin, eyes, or hair.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 298,804, filed January 12, 2022, by Sevanti Mehta, entitled “Manufacture of Cosmetic Carotenoid Compositions.” [Technical field]

[0002] The capsicum annum extract composition includes capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 1%, and cryptoxanthin in the range of 0.1% to 0.5%. The composition is produced, at least in part, from spent materials from purifying carotenoids using countercurrent extraction, and the composition is intended to enhance the skin, eyes, or hair. Summary of the Invention [Means for solving the problem]

[0003] The method for producing the composition includes extracting carotenoids from spent material, the spent material being produced by purifying carotenoids from pepper fruits using countercurrent extraction with a solvent. The method further includes concentrating the extracted carotenoids using supercritical fluid extraction. The method further includes purifying the concentrated carotenoids using countercurrent extraction such that the composition strengthens the skin, eyes, or hair.

[0004] The method for producing the composition includes extracting carotenoids from spent material, the spent material being produced by purifying carotenoids from pepper fruits using countercurrent extraction with a solvent. The method further includes concentrating the extracted carotenoids using supercritical fluid extraction. The method further includes purifying the hydrolyzed carotenoids using countercurrent extraction, such that the composition comprises capsanthin in the range of 1%-1.5%, zeaxanthin in the range of 0.5%-15%, and cryptoxanthin in the range of 0.1%-0.5%, and the composition strengthens the skin, eye, or hair.

[0005] Accordingly, compositions and methods are provided according to one or more of the following examples. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 illustrates an exemplary method for making the composition. [Diagram 2] Illustrative bar graph showing reduction in intraocular pressure [Diagram 3] Illustrative diagram showing results of studies evaluating anti-obesity effects [Figure 4] Illustrative diagram showing results of studies evaluating anti-obesity effects [Diagram 5] Illustrative diagram showing results of studies evaluating anti-obesity effects [Figure 6] FIG. 1 illustrates an exemplary composition. [Figure 7] Exemplary Bar Graphs Showing Melanin Inhibition [Figure 8] Exemplary Bar Graphs Showing Tyrosinase Inhibition [Figure 9] Illustrative bar graph showing gene expression analysis of gene transcripts for fold increase DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] It should be understood, however, that the specific embodiments given in the drawings and their detailed description are not intended to limit the present disclosure, but rather provide a basis for those skilled in the art to recognize alternatives, equivalents, and modifications that are encompassed in the appended claims along with one or more of the given embodiments.

[0008] Certain terms are used throughout the following description and claims to refer to particular system components and configurations. As one skilled in the art will appreciate, companies may refer to components by different names. This specification does not intend to distinguish between components that differ in name but not function. In the following discussion and claims, the terms "including" and "comprising" are used in a non-limiting manner and therefore should be interpreted to mean "including, but not limited to."

[0009] A carotenoid is any of a variety of pigments, such as carotenes, found widely in plants and animals and chemically characterized by a long aliphatic polyene chain of eight isoprene units. Capsanthin is a carotenoid, as are beta-carotene, lycopene, lutein, and zeaxanthin. These pigments are found in red, orange, yellow, and green fruits and vegetables. Carotenoids are antioxidant tetraterpenoids that aid in the body's disease prevention mechanisms.

[0010] Capsicum is a widely consumed natural food ingredient used as a vegetable, spice and / or coloring, and paprika extract is an extract of the fruit of the Capsicum genus. The genus, native to Central and South America, belongs to the Solanaceae family and includes all peppers from the mild bell pepper to the spicy habanero. There are five domesticated species: annum, frutescens, chinense, pubescens and baccatum, with the most widespread being annum, frutescens and chinense. The first to be introduced in the world was annum, native to Mexico. Formerly, they were divided into two categories: sweet (or mild) peppers and hot (or chili) peppers, but modern plant breeding has eliminated that distinction. Currently, annum is the most widespread in terms of home consumption and industrial processing.

[0011] Annuum is a variety used to produce paprika extracts for food coloring. The colored extract has a lower capsaicin content compared to extracts used as spices. In red varieties, capsanthin and capsorubin are the main compounds responsible for the red color. Pure carotenoid crystals derived from Annuum fruits contain xanthophylls such as capsanthin, zeaxanthin and cryptoxanthin. The chemical structure of a carotenoid ultimately determines what potential biological function the pigment may have. The unique pattern of alternating single and double bonds in the polyene backbone of carotenoids allows them to absorb excess energy from other molecules, while the nature of certain end groups on carotenoids may affect their polarity. The carbonyl groups present in capsanthin and capsorubin make them unique when compared to other carotenoids such as lutein and zeaxanthin.

[0012] Age-related macular degeneration ("AMD") is a common medical eye disease and the leading cause of blindness among people over the age of 50. It involves damage to the macula, an oval-shaped pigmented area near the center of the retina used for sharp central vision. AMD can be of two types: dry (atrophic) or wet (exudative). In addition to aging, high blood pressure, oxidative stress, blue light exposure, obesity, etc. can also cause AMD. Carotenoids in the macula help filter blue wavelengths of sunlight and reduce free radicals near the retina, both of which are harmful to eye cells. Lutein and zeaxanthin are carotenoids present in the retina that protect eye cells from photooxidative damage. Specifically, chronic exposure to blue light, the main cause of AMD, causes a decrease in cone density and cone sensitivity, and zeaxanthin may prevent AMD by absorbing blue light.

[0013] Skin care is not only a cosmetic challenge but also a major health concern, especially considering the aging population that is significantly exposed to sunlight. Skin care products are therapeutic preparations intended to come into contact with various external sites of the human body, protecting against degenerative skin diseases. Synthetic chemicals used in various cosmetic formulations are associated with undesirable, sometimes irreversible, side effects and also cause skin allergies. Petroleum-based chemicals such as parabens and phthalates are perceived as health hazards, which is why consumers are seeking natural, herbal-based ingredients. Furthermore, synthetic fragrances have been linked to reproductive toxicity, cancer, and endocrine disruption. Therefore, cosmetics derived from natural products are preferred over synthetic chemicals, as natural products are free of undesirable side effects. The dermatological application of capsanthin as a cosmetic has been disclosed through in vitro experiments such as sun protection factor ("SPF"), melanin inhibitory activity, tyrosinase inhibitory activity, efficacy against acne and dandruff caused by microorganisms, and induction of gene expression of aquaporin 3 ("AQP-3") in human keratinocyte cell lines.

[0014] SPF is a laboratory measurement of the effectiveness of protection against ultraviolet ("UV") radiation. Exposure to UV radiation causes sunburn and hyperpigmentation. In vitro SPF is measured by spectrophotometry; the higher the SPF, the greater the protection against UV radiation. Melanin pigment is produced by melanocytes and transferred to the surrounding keratinocytes. Melanocytes, specialized cells, are located in the stratum corneum of the epidermis. Melanin is primarily responsible for skin pigmentation and has a pivotal role in preventing UV radiation-induced skin damage. The enzymes tyrosinase, tyrosinase-related protein 1 ("TRP1"), and dopachrome tautomerase ("DCT") are responsible for the enzymatic conversion of tyrosine to melanin. The synthesis of melanin is promoted by various factors, including UV radiation and forskolin, a phytochemical derived from Coleus forskohlii that elevates cAMP. Abnormal biosynthesis of melanin can cause serious cosmetic problems such as melasma, freckles, hyperpigmentation, and age spots. Thus, the disclosed compositions and methods exhibit skin lightening properties by inhibiting melanin biosynthesis. Specifically, depigmentation is achieved by inhibiting melanin biosynthesis by reducing melanocyte proliferation or by inhibiting the copper-containing enzyme tyrosinase.

[0015] The lipophilic Malassezia furfur belongs to a monophyletic genus of fungi typically found on human and animal skin. More than 80% of the human skin fungal population comprises Malassezia furfur, and it is frequently isolated in both infected and healthy hosts. Malassezia is associated with several common skin diseases, including tinea versicolor, seborrheic dermatitis, and Malassezia folliculitis. Malassezia may also contribute to other skin diseases, such as atopic dermatitis and psoriasis. Skin conditions can be improved by reducing the number of Malassezia with antifungal agents. For example, dandruff is a skin disease characterized by a dry, itchy scalp and involves colonization of the skin by Malassezia. The most common and well-studied disease-causing Candida spp. is Candida albicans, which naturally colonizes the skin, intestinal mucosa, and genital tract of healthy humans. High morbidity and mortality have been reported due to severe infections, especially in hospitalized patients.

[0016] As disclosed herein, two capsanthin extracts obtained from red bell pepper fruit, also known as red paprika, provide skin protection against all the above conditions. The capsanthin extracts are characterized by high performance liquid chromatography ("HPLC"). Specifically, capsanthin obtained from red bell pepper fruit is effective in UV protection, melanin inhibition, skin whitening, tyrosinase inhibition, and induction of aquaporin 3 (AQP-3) gene expression in human keratinocyte cell line (HaCaT). Both capsanthin 50% w / w crystals ("CAP-50CR") and capsanthin 1.5% w / w soft extract ("CAP-1.5SE") exhibit these effects.

[0017] FIG. 1 shows a method 100 for producing two substances; specifically, carotenoid compositions according to some exemplary embodiments with the advantages described above. Also disclosed herein is a method for isolating and purifying carotenoids containing a specific composition of carotenoids such as trans-capsanthin, trans-zeaxanthin and β-cryptoxanthin from chili pepper fruits without traces of organic harmful solvents. The method 100 includes the selection of a highly colored composition of the Annuum chilli variety, solvent extraction, supercritical fluid extraction ("SCFE") concentration, alkaline hydrolysis of carotenoid esters with absolute alcohol, purification using a countercurrent extractor, concentration, and drying. The resulting crystals are suitable compositions for cosmetics, dietary supplements, food additives, and the like.

[0018] At 102, a particular variety of chili fruit is selected. In one embodiment, the chili fruit is selected from the Bydagi chili variety, or other high color value chili peppers, for example, the following, alone or in combination: Bydagi-Kaddi, Bydagi-Dyavnoor, Bydagi-Dabbi, KDL high color value chili pepper, 5531 high color value chili pepper, and / or 4431 high color value chili pepper. In one embodiment, the ratio of the combination is (1) Bydagi-Kaddi:(1) Bydagi-Dyavnoor:(1) Badagi-Dabbi (1:1:1 ratio). In other embodiments, unexpected results have been found using 1:2:2 and 1:1:2 ratios of Bydagi-Kaddi:Bydagi-Dyavnoor:Bydagi-Dabbi. Additionally, unexpected results were found using a 1:1:2 ratio of KDL High Color, 5531 High Color, and 4431 High Color, respectively. In various embodiments, the American Spice Trade Association ("ASTA") color value of the Byadagi pepper or other high color pepper is selected within the range of 2000-2600 units or within the range of 2000-2400 units.

[0019] At 104, carotenoids are extracted from the fruit using a suitable solvent. In one embodiment, dried, deseeded and flaked Capsicum fruits are extracted with a solvent at a temperature ranging from 40° C. to 90° C., preferably at 60° C., for 4 hours to 8 hours, preferably 6 hours. The solvent extract is then concentrated under vacuum to produce Capsicum oleoresin containing the carotenoid esters. In various embodiments, the extraction solvent may be one or a combination of methanol, ethanol and / or isopropyl alcohol.

[0020] For example, 250 kilograms of deseeded, flaked pepper fruits having an ASTA color value of 2000-2400 units can be placed in a 2000 liter capacity reactor equipped with an agitator. A volume of methanol (1000 L) can be added and the mixture stirred at 60°C for 6 hours. The methanol layer can be filtered and collected. This methanol extraction can be repeated three times for efficiency purposes. As another example, dried red bell pepper flakes (Capsicum annuum) can be extracted with n-hexane at 40°C-60°C for 6 hours, filtered, and concentrated under vacuum.

[0021] At 106, the carotenoids are concentrated using supercritical fluid extraction. In one embodiment, carbon dioxide is used as the solvent and the temperature of the supercritical fluid extraction ranges from 40° C. to 60° C., preferably 50° C. In one embodiment, the pressure used for the supercritical fluid extraction ranges from 25 to 50 mPa, preferably 35 mPa.

[0022] Following the above example, all the methanol layers can be combined and concentrated under vacuum. 22 kg of oleoresin can be obtained and combined with about 11 kg of calcium carbonate in a supercritical fluid extraction bag. The extraction bag can be placed in an extraction chamber and extracted at 50°C and 35 mPa. The yield of concentrated oleoresin can be about 11 kg.

[0023] At 108, the carotenoid esters are hydrolyzed. In one embodiment, the concentrated oleoresin is hydrolyzed with alcoholic potassium hydroxide to produce free carotenoids. In one embodiment, the base for hydroxylation is selected from the group consisting of potassium hydroxide (KOH), sodium hydroxide (NaOH), or a combination thereof. In one embodiment, the solvent medium for hydrolysis is methanol, ethanol, isopropyl alcohol, or a combination thereof. In one embodiment, the concentration of the hydrolyzing agent ranges from 10% to 30%. In one embodiment, the temperature of hydrolysis ranges from 70° C. to 85° C., preferably 80° C. In one embodiment, the time required for hydrolysis ranges from 1 hour to 3 hours, preferably 2 hours. Once the carotenoids are hydrolyzed to the free form, they become more bioavailable.

[0024] Continuing with the above example, the concentrated oleoresin may be placed in a 500 L glass lined reactor. In a separate vessel, 5 kg of potassium hydroxide may be added to 40 L of ethyl alcohol under stirring. The alcoholic KOH may be added slowly to the concentrated oleoresin under stirring at 80° C. for 2 hours.

[0025] At 110, the hydrolyzed carotenoids are purified using countercurrent extraction. In one embodiment, the solvent used for countercurrent extraction is ethyl acetate, isopropyl acetate, or a combination thereof. In one embodiment, the immiscible aqueous phase is water or water acidified with hydrochloric acid (pH 3-4), preferably acidified water.

[0026] Continuing with the above example, after ensuring that the degree of saponification is greater than 99% by HPLC, 40 liters of demineralized hot water maintained at a temperature of 70°C can be added to the reaction mass with stirring for 10 minutes. The dilute mass containing the carotenoid crystals can be fed to a filter press to recover the crystals. Approximately 250 liters of additional hot water can be fed through the filter press to wash out unwanted impurities and reduce the pH of the effluent to neutral, about 7.0. After ensuring neutralization, positive pressure of nitrogen can be applied to the filter press to squeeze out the crystals trapped within the filter. The wet crystals, having a weight of about 6.7 kg, can then be recovered from the filter press, dissolved in 60 L of ethyl acetate, and fed into a countercurrent extractor. 100 L of water can be used and the pH adjusted to 3 or 4 with dilute hydrochloric acid. The acidified water can be bottom fed to the countercurrent extractor.

[0027] As another example, the resulting oleoresin can be further purified by supercritical fluid extraction and saponified with 40% ethanolic caustic potash. The saponified extract can then be concentrated to remove ethanol and further purified with an ethyl acetate / water mixture.

[0028] At 112, the capsicum extract is blended with an excipient. In various embodiments, the excipient is sunflower oil, safflower oil, soy lecithin, sunflower lecithin, phosphatidylcholine from sunflower or soybean, starch, dextrin, lactose, dicalcium phosphate, colloidal silicon dioxide, and / or combinations thereof. In other embodiments, the excipient is a granulating agent, a binder, a lubricant, a disintegrant, a sweetener, a flow agent, an anti-adherent agent, an antistatic agent, a surfactant, an antioxidant, a gum, a coating agent, a colorant, a flavoring agent, a coating agent, a plasticizer, a preservative, a suspending agent, an emulsifier, a plant cellulosic material, a spheronizing agent, and / or combinations thereof. In one embodiment, the carotenoids are further isolated and / or purified by the addition of one or more solvents, the addition of an ionic resin, quenching, filtration, extraction, and / or ion exchange resin.

[0029] Following the above example, the ethyl acetate layer can be removed, dried over anhydrous sodium sulfate, and concentrated. The yield of the composition can be about 1.1 kg. The carotenoid content measured by spectrophotometer can be about 95.34%, and the total trans-capsanthin, all-trans-zeaxanthin, and total β-cryptoxanthin by HPLC are 98.71%, 8.31%, and 2.63%, respectively. The final product can contain a moisture content of about 0.2%, with no traces of residual methanol and ethyl acetate detected by gas chromatography analysis.

[0030] As another example, the ethyl acetate layer can be separated, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain 50% capsanthin crystals ("CAP-50CR"). The aqueous layer, usually an industrial waste, may contain a small amount of capsanthin. The pH of the aqueous layer may be adjusted to 5.5-7.0 with dilute hydrochloric acid, and ethyl acetate may be added and heated to 55-60°C. The separated ethyl acetate layer can be removed, dried over anhydrous sodium sulfate, and concentrated to obtain 1.5% capsanthin soft extract ("CAP-1.5SE").

[0031] FIG. 2 shows the results of a study using the disclosed compositions for the reduction of intraocular pressure ("IOP") in animals, and the following optional steps may be part of the method 100 in various embodiments. High intraocular pressure results from increased resistance to the drainage of aqueous humor through the trabecular meshwork. A persistent increase in aqueous humor may result from increased formation of aqueous humor, difficulty in draining aqueous humor, or increased pressure in the episcleral veins. In this study, IOP was induced by intravitreal injection. FIG. 2 shows a bar graph showing six groups (G1-G6) and the left and right eye IOP for each group. The first two groups (G1-G2) are control groups in which the IOP-lowering composition was not introduced. In G1, IOP was not induced, and in G2, IOP was induced. For the next groups (G3-G6), the left bar for each eye represents the IOP before introduction of the composition disclosed herein in one embodiment. The right bar for each eye represents the IOP after introduction of the composition. The results show that introduction of the composition reduced IOP to a level approximately equal to that of the group in which IOP was not induced (G1), and reduced IOP to a level significantly lower than that of the group in which the composition was not introduced (G2).

[0032] 3-5 show the results of a study to evaluate the anti-obesity effect of a composition disclosed herein, referred to in the figures as CapsiClear, and the following optional steps may be part of the method 100 in various embodiments. "HFD" refers to the high fat diet used to induce obesity. Six groups were used in this study: G1-G6. G1 refers to the group fed a normal diet, G2 refers to the group fed a high fat diet, G3 refers to the group fed a high fat diet with a low dose of the composition, G4 refers to the group fed a high fat diet with a medium dose of the composition, G5 refers to the group fed a high fat diet with a high dose of the composition, and G6 refers to the group fed a high fat diet with the reference drug orlistat.

[0033] In Figure 3, the y-axis represents body weight and the x-axis represents time in days. As can be seen, a significant decrease in body weight of 31% was observed at the high dose of 80 mg / kg.

[0034] FIG. 4 includes three charts 402, 404, 406 showing the results of different measurements in the study, any of the following steps may be part of the method 100 in various embodiments. At 402, liver mass was reduced in the group given the composition. At 404, adipose tissue weight was significantly reduced in the group given the high dose of the composition. At 406, epididymal fat mass was significantly reduced in the low and high dose groups of the composition.

[0035] FIG. 5 includes four charts 502, 504, 506, 508 showing the results of different measurements in the study, and any of the following steps may be part of the method 100 in various embodiments. In 502, adiponectin was significantly increased in the groups given the medium and high doses of the composition. The increase in adiponectin causes insulin resistance and weight loss. In 504, 506, and 508, plasma leptin, plasma free fatty acids, and insulin were each significantly decreased in all groups given the composition. The decrease in insulin causes weight loss, the decrease in plasma free fatty acids causes improvement of mitochondrial function and whole body insulin sensitivity, and the decrease in plasma leptin causes restoration of hypothalamic leptin sensitivity, reduction in weight gain, and enhancement of insulin sensitivity. Therefore, based on all the above, the composition can be effectively used for weight loss purposes, along with or in combination with other benefits.

[0036] 6 shows a composition 600 of capsicum extract containing capsanthin, zeaxanthin and cryptoxanthin produced as described above in capsule embodiments for pharmaceutical, nutraceutical and / or cosmetology purposes. In other embodiments, the composition is embodied as a tablet, injection, cream, gel, ointment, lotion, solution, beverage, confectionery, emulsion, foam, troche, lozenge, aqueous suspension, oily suspension, patch, dentifrice, spray, drop, powder, granule, syrup, elixir, food, and / or combinations thereof. In a preferred embodiment, the composition comprises a tablet, soft gelatin capsule, hard gelatin capsule, cream, gel, lotion, and / or combinations thereof.

[0037] In one embodiment, the composition may be administered in various embodiments by topical administration, oral administration, intravenous administration, intraarticular administration, intramuscular administration, and / or combinations thereof.In a preferred embodiment, the mode of administration is oral, topical, intravenous intramuscular, and / or combinations thereof.In addition, materials such as flaxseed, flaxseed oil, vegetarian oil or vegetable oil and other oils can be combined with the composition to stabilize the active ingredient.Regardless of the amount of materials combined with the composition, the ratio of carotenoid to the total amount of carotenoid remains the same in at least some embodiments.

[0038] A 12-week randomized, placebo-controlled pilot clinical trial indicates that the composition may: increase macular pigment density ("MPOD"), which is associated with better visual performance; shorten recovery time after exposure to bright light; and improve reading under different lighting conditions (both white light and blue light), in addition to the benefits already described.

[0039] The composition may include capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 1%, and cryptoxanthin in the range of 0.1% to 0.5%. The composition may be made, at least in part, from materials consumed from purifying carotenoids using countercurrent extraction, the composition strengthening the skin, eyes, or hair. As a result of the above process, in one embodiment, the composition of carotenoids is in the following range: 50% to 80% capsanthin, 5% to 15% zeaxanthin, 1% to 5% cryptoxanthin, and trace amounts of other carotenoids. These percentages and all percentages herein refer to both actual percentages and percentages permitted by labeling regulations. In one embodiment, the composition of total carotenoids is in the range of 90% to 99%. In another embodiment, the composition may include total carotenoids in the range of 10% to 99%. In one embodiment, the carotenoids are trans-capsanthin: (3R,3'S,5'R)-3,3'-dihydroxy-β,κ-caroten-6'-one; trans-zeaxanthin: 3R,3'R-β,β-carotene-3,3'-diol; and beta-cryptoxanthin: (3R,6'R)-4',5'-didehydro-5',6'-dihydro-β,β-caroten-3-ol. Capsanthin may include trans-capsanthin (3R,3'S,5'R)-3,3'-dihydroxy-β,κ-caroten-6'-one, zeaxanthin may include trans-zeaxanthin (3R,3'R-β,β-carotene-3,3'-diol), and cryptoxanthin may include beta-cryptoxanthin (3R,6'R)-4',5'-didehydro-5',6'-dihydro-β,β-caroten-3-ol.

[0040] In one embodiment, the color value of the carotenoids ranges from 800,000 to 1,250,000. Thus, the composition is ideal for providing color and skin enhancing functions to cosmetics such as lipsticks, chapsticks, liquid glosses, lipstick pastes, blushes, lip liners, foundations, concealers, eye contours, eyeliners, mascaras, nail polishes, eye shadows, body make-up, and the like. WHOFAO, JECFA, and HPLC analysis can be performed to identify and quantify capsanthin in CAP-50CR and CAP-1.5SE using reference standards. In the HPLC chromatograms of the retention times of capsanthin in the reference standards, CAP-50CR and CAP-1.5SE are identical, which confirms the presence of capsanthin. In various embodiments, the method 100 includes the optional steps of:

[0041] Currently, the steady increase in solar ultraviolet radiation is responsible for non-melanoma skin cancers reported worldwide. UV radiation present in sunlight is implicated as the main cause. UV radiation is known to cause excess reactive oxygen species ("ROS"), thereby increasing oxidative stress and inducing skin damage and non-melanoma skin cancer. Therefore, reduction of ROS and oxidative stress is important for preventing skin from skin damage and cancer.

[0042] Carotenoids, a major class of polyene compounds in biology, and the long polyene bonds present in the molecule, scavenge free radicals by quenching excited molecules. Capsanthin is a very potent carotenoid due to the presence of 11 conjugated dienes and linked keto groups in its structure. The conjugated dienes are responsible for their radical scavenging and singlet oxygen quenching abilities. The skin-protecting, anti-cancer, anti-inflammatory, anti-obesity, anti-cholesterol, and anti-adipogenic effects exhibited by capsanthin are the result of its strong radical quenching ability.

[0043] The effectiveness of a sunscreen is determined by its Sun Protection Factor ("SPF"). An effective sunscreen must have a broad absorbance range (290 and 400 nm) to protect the skin from UV radiation and skin damage. According to the United States Food and Drug Administration (FDA), a sunscreen must have a minimum of 15 SPF. The SPF values ​​for the compositions disclosed herein, CAP-50CR and CAP-1.5SE, are 34.44 and 20.63, respectively. These values ​​qualify CAP-50CR and CAP-1.5SE as high and medium sunscreens, respectively. The following table shows the SPF tests and average values. For CAP-50CR, the photometric SPF values ​​were measured as 34.58, 33.62, and 35.13 in three tests, with an average value of 34.44. For CAP-1.5SE, photometric SPF values ​​were measured as 21.96, 19.96 and 19.97 in three tests, with an average value of 20.63. [Table 1]

[0044] In addition to SPF, the compositions also exhibit antifungal properties as shown in the table below. The antifungal activity of CAP-50CR and CAP-1.5SE against Malassezia furfur and Candida albicans is compared to the antifungal drug ketoconazole by the minimum inhibitory concentration ("MIC") method.

[0045] The inhibitory effect assay of CAP-50CR and CAP-1.5SE against Malassezia furfur and Candida albicans can be performed according to the culture microdilution standard method. Ketoconazole, an antifungal drug, can be used as a standard. Lyophilized Candida albicans (ATCC-10231) and Malassezia furfur (ATCC-14521) strains can be used as well. Loopful cultures of Malassezia furfur and Candida albicans can be streaked onto SDA (Sabouraud Dextrose Agar) and incubated at 35 ± 2 °C and 25 ± 2 °C, respectively, for 48 to 72 hours. Finally, the cells are suspended in RPMI 1640 medium and diluted to 1 × 10 6 Final concentrations of CFU / mL can be obtained. For the evaluation of inhibitory activity, the effects of CAP-50CR and CAP-1.5SE can be examined against strains of Malassezia furfur and Candida albicans cultured at different concentrations at 35 ± 2 °C and 25 ± 2 °C, respectively, in 96-well microplates for 48-72 h.

[0046] The antifungal standard ketoconazole showed a MIC value of 0.00048 mg / mL against both Malassezia furfur and Candida albicans. The MICs of CAP-50CR and CAP-1.5SE against Malassezia furfur were found to be 0.625 mg / mL and 5 mg / mL, respectively. CAP-50CR and CAP-1.5SE showed the same MIC value against Candida albicans, which was 2.5 mg / mL. Notably, the CAP-50CR extract showed moderate antifungal activity and CAP-1.5SE showed mild antifungal activity compared to the antifungal drug ketoconazole. Thus, the composition can help hair growth and the composition can also help remove and prevent dandruff. [Table 2]

[0047] Sterility and growth control may also be used. Yeast growth may be measured at 530 nm in an enzyme-linked immunosorbent assay microplate reader. The minimum inhibitory concentration (MIC50) may be defined as the lowest concentration of CAP-50CR and CAP-1.5SE that results in a 50% reduction in fungal growth compared to the control. Thus, the composition may further be an anti-yeast agent against Malassezia yeast.

[0048] The composition can aid in melanin inhibition as shown in Figure 7. Mouse skin melanoma cell line B16F10 can be cultured in DMEM-HG medium supplemented with 10% inactivated fetal bovine serum ("FBS"), penicillin (100 lU / mL), streptomycin (100 μg / mL), and amphotericin B (5 μg / mL) at 37°C in a humidified atmosphere of 5% CO2 until confluent. Cells can be dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). Stock cultures can be cultured in 25 cm 2 They can be grown in culture flasks and 96-well microtiter plates can be used.

[0049] Monolayer cell cultures may be trypsinized and the cells numbered at 1.0 × 10 5 The cells / mL may be adjusted. 0.1 mL of the diluted cell suspension was added to each well and maintained for 24 hours. After the monolayer was formed, the supernatant may be skipped, washed with medium, and 100 μl of test substances at different concentrations may be added and incubated for 48 hours at 37° C. in a 5% CO2 atmosphere. Microscopy may be performed and observations may be recorded every 24 hours.

[0050] Cell viability can be assessed by MTT reduction assay. After 72 hours of incubation, the drug solution can be discarded and 50 μl of MTT in PBS can be added to each well. The plate can be gently shaken and incubated for 3 hours at 37° C. in a 5% CO2 atmosphere. The supernatant can be removed and 100 μl of DMSO can be added. The plate can be gently shaken to solubilize the formazan formed. The absorbance can be measured at a wavelength of 540 nm using a microplate reader. The concentration of test substance required to inhibit cell proliferation by 50% (CTC50) can be calculated.

[0051] The cytotoxicity of CAP-50CR and CAP-1.5SE in the B16F10 cell line can be evaluated to determine CTC50 values. 24-hour cell cultures, which can be 70-80% confluent, can be used to determine melanin inhibition. Cells can be treated with non-toxic concentrations of test substances and forskolin (200 μM) for 48 hours.

[0052] At the end of the incubation period, the supernatant may be aspirated from the wells and the cultures may be washed twice with warm PBS. Cells may be harvested by trypsinization and the cell suspension may be centrifuged at 2500 rpm for 15 minutes. The cell pellet may be washed with PBS, centrifuged, and treated with 400 μL of 1 N NaOH containing 10% DMSO. Samples may be heated at 60° C. for 1 hour, cooled, and the absorbance of the cell lysate may be assessed by spectrophotometer at 450 nm.

[0053] The in vitro cytotoxicity of CAP-50CR and CAP-1.5SE can be evaluated in cutaneous melanoma cells (B16F10) by MTT assay. Different concentrations ranging from 1000 μg / mL to 7.8 μg / mL can be used to determine the proliferation inhibition percentage on B16F10 cells. CAP-50CR shows a CTC50 value of 98.44 ± 2.55 μg / mL on the B16F10 cell line, whereas CAP-1.5SE shows a CTC50 value of more than 1000 μg / mL. Furthermore, non-toxic concentrations can be evaluated for forskolin-induced melanin inhibition activity. The cytotoxicity properties of the substances are shown in Table 3 and the percentage of melanin inhibition is shown in Figure 7. For CAP-50CR, non-toxic concentrations of 7 and 3 μg / mL show melanin inhibition of 48.73 ± 0.75% and 42.29 ± 5.2%, respectively. Melanin inhibition for CAP-1.5SE is 55.84%±1.47 and 46.44%±1.05 for non-toxic concentrations of 50 and 25 μg / mL, respectively.

[0054] The compositions can inhibit tyrosinase as shown in Figure 8. CAP-50CR and CAP-1.5SE showed high tyrosinase inhibitory activity at non-toxic concentrations. Therefore, they are hypopigmenting agents for cosmetic and pharmaceutical applications. Specifically, overproduction of melanin leads to hyperpigmentation due to abnormal melanogenesis, and tyrosinase is an important marker for developing a cure for pigmentation disorders and skin whitening.

[0055] Tyrosinase inhibitory activity can be measured using the DOPA oxidase activity of mushroom tyrosinase measured by spectrophotometry. 10 μL of DMSO can be taken in a 96-well microplate and mixed with 60 μL of 50 mmol / L phosphate buffer (pH 6.8) on ice. Then, 20 μL of 0.9 mg / mL L-DOPA in phosphate buffer can be added. Finally, 10 μL of tyrosinase (500 U / mL in phosphate buffer) can be added to the mixture and incubated at 27°C for 10 minutes. After incubation, the amount of dopachrome produced in the reaction mixture can be measured by spectrophotometer at 450 nm (OD450) in a microplate reader. Kojic acid (5-100 μmol / L) dissolved in 50 mmol / L phosphate buffer can be used as a positive control. The 50% inhibitory concentration (IC50) can be determined.

[0056] CAP-50CR extract shows greater tyrosinase inhibitory activity than CAP-1.5SE. CAP-50CR extract shows 43.78% and 39.37% inhibition at concentrations of 7 and 3 μg / mL, respectively. CAP-1.5SE shows 34.6% and 22.9% inhibition at concentrations of 50 and 25 μg / mL, respectively. Therefore, CAP-50CR and CAP-1.5SE show whitening effect by inhibiting tyrosinase.

[0057] The composition can induce gene expression in human keratinocytes (HaCaT), as shown in FIG. 9. Keratinocytes, including the epidermis, produce loricrin, involucrin, and filaggrin to combine keratin filaments and form the keratinocyte envelope. In addition, hyaluronic acid present in keratinocytes acts as a moisture barrier in the skin. Water retention in the skin and other organs is regulated by aquaporins (AQPs), small hydrophobic protein molecules. Currently, nearly 13 types of AQPs have been identified in mammals. The function of AQP3 is thought to be important for the skin, as it is involved in the transport of glycerol and water. In response to skin damage, AQPs are downregulated. Thus, AQPs play an important role in the recovery of skin function and are involved in the biosynthesis of hyaluronic acid (HA). The reduction of HA in the skin leads to wrinkles, loss of elasticity, and premature aging. The effect of CAP-50CR and CAP-1.5SE on the expression of the gene encoding AQP3 in HaCaT cells can be examined using RT-PCR.

[0058] Stock cells can be cultured in DMEM supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU / mL), streptomycin (100 μg / mL), and amphotericin B (5 μg / mL) in a humidified atmosphere of 5% CO at 37 °C until confluent. Cells can be dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS).

[0059] Monolayer cell cultures may be trypsinized, cell numbers adjusted to 100,000 cells / mL with 10% FBS, and 0.1 mL of the diluted cell suspension may be harvested into a 96-well microtiter plate. After formation of the monolayer, the supernatant may be skipped, washed with medium, and 100 μl of test substances at different concentrations may be added and incubated for 72 hours at 37° C. in a 5% CO2 atmosphere. After 72 hours, the test solution may be discarded and 50 μl of MTT in PBS may be added to each well. The plate may be gently shaken and incubated for 3 hours at 37° C. in a 5% CO2 atmosphere. The supernatant may be removed and 100 μl of DMSO may be added to dissolve the formazan. The absorbance may be measured at a wavelength of 540 nm using a microplate reader. The percentage of growth inhibition and the concentration of test substance required to inhibit cell growth by 50% (CTC50) may be calculated.

[0060] HaCaT cells may be subjected to cell lysis by treatment with TriExtract reagent. Chloroform may be added and centrifuged to isolate total RNA from the sample. The upper layer may be collected, diluted with an equal volume of isopropanol, and incubated at -20°C for 10 minutes. After incubation, it may be centrifuged and resuspended by adding an appropriate volume of ethanol. The isolated RNA pellet may be air-dried or TAE buffer may be added. First strand cDNA may be synthesized from the RNA using oligo-dT primers followed by reverse transcriptase treatment. The cDNA may be subjected to PCR for amplification of AQP-3 and GAPDH.

[0061] The mRNA expression level of AQP-3 can be determined by using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. AQP-3 can be amplified using the following primers: for I strand synthesis: oligo dT primer; for II strand synthesis: forward: 5'-GCTGTCACTCTGGCATCCTG-3' and reverse: 5'-GCGTCTGTGCCAGGGTGTAG-3'. The amplification reaction can be carried out with the following scheme: 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing temperature for 30 s, and 72°C for 45 s, with a final extension at 72°C for 10 min.

[0062] CAP-50CR and CAP-1.5SE show CTC50 values ​​of 76.59 ± 3.25 μg / mL and 664.95 ± 2.88 μg / mL, respectively, on HaCaT cells. Furthermore, at non-toxic concentrations, CAP-50CR and CAP-1.5SE show increased levels of AQP-3 mRNA at lower and higher concentrations compared to the control in a semi-quantitative RT-PCR procedure. Figure 9 is a graph of the fold increase of the semi-quantitative gene expression analysis of gene transcripts, where the relative levels of AQP-3 gene expression are normalized to GAPDH. Values ​​are expressed as mean ± SEM (n = 2) and statistical significance is compared between the control group and the other treatment groups (CAP-50CR 7.5 μg / mL, CAP-50CR 15 μg / mL, CAP-1.5SE 125 μg / mL and CAP-1.5SE 250 μg / mL) ( *** p<0.0001, statistically significant). Both substances showed increased levels of AQP-3 mRNA at lower and higher concentrations compared to the control in a semi-quantitative RT-PCR procedure.

[0063] Based on the above, the composition can restore skin function and aid in skin moisturization by increasing the biosynthesis of hyaluronic acid. The composition can increase the level of AQP-3 mRNA. The composition can help protect the skin from ultraviolet radiation, UVA radiation, and UVB radiation. The composition can provide a sun protection factor (SPF) of at least 20. The composition can prevent and treat dry eye syndrome when administered orally. The composition can increase tear film breakup time and reduce tear osmolarity when administered orally. The composition can increase tear production when administered orally. The composition can reduce defects and abrasions in the corneal epithelium when administered orally. The composition can reduce damage to the ocular surface by reducing oxidative stress when administered orally. The composition can reduce degeneration and apoptotic cell death in the cornea by reducing serum MMP-2 and MMP-9 levels when administered orally. The composition can restore the mucin layer of the eye when administered orally. The composition may reduce expression of IL-2, IL-6 and MMP-9 genes.The composition may downregulate expression of TNF-α and IL-4 genes.

[0064] CAP-50CR and CAP-1.5SE exhibit skin protection properties from UV radiation and hyperpigmentation. They also exhibit antifungal, whitening, anti-wrinkle, and moisturizing properties. These results indicate that capsanthin from red bell pepper fruit can be used as a cosmetic active ingredient in skin guard formulations and as a potential treatment for various dermatological disorders.

[0065] Additional embodiments and features of the present disclosure will be apparent to those skilled in the art based on the description provided herein.The embodiments of the present specification and their various features and advantageous details are described with reference to non-limiting embodiments herein.The examples used herein are intended to merely promote understanding of the manner in which the embodiments of the present specification can be implemented, and further to enable those skilled in the art to implement the embodiments of the present specification.Therefore, the following examples should not be interpreted as limiting the scope of the embodiments of the present specification.

[0066] In some embodiments, compositions and methods are provided according to one or more of the following examples.

[0067] Example 1: A capsicum extract composition includes capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 1%, and cryptoxanthin in the range of 0.1% to 0.5%. The composition is made, at least in part, from spent materials from purifying carotenoids using countercurrent extraction, and the composition is intended to strengthen the skin, eyes, or hair.

[0068] Example 2: A method for producing a composition includes extracting carotenoids from spent material, the spent material being made by purifying carotenoids from pepper fruits using countercurrent extraction with a solvent. The method further includes concentrating the extracted carotenoids using supercritical fluid extraction. The method further includes purifying the concentrated carotenoids using countercurrent extraction, such that the composition strengthens the skin, eyes, or hair.

[0069] Example 3: A method for producing a composition includes extracting carotenoids from spent material, the spent material being made by purifying carotenoids from pepper fruits using countercurrent extraction with a solvent. The method further includes concentrating the extracted carotenoids using supercritical fluid extraction. The method further includes purifying the hydrolyzed carotenoids using countercurrent extraction, such that the composition comprises capsanthin in the range of 1%-1.5%, zeaxanthin in the range of 0.5%-15%, and cryptoxanthin in the range of 0.1%-0.5%, and the composition strengthens the skin, eye, or hair.

[0070] The following features can be incorporated in the various embodiments described above, and such features can be incorporated individually with one or more of the other features or in conjunction with one or more of the other features.

[0071] Capsanthin may include trans-capsanthin (3R,3'S,5'R)-3,3'-dihydroxy-β,κ-caroten-6'-one, zeaxanthin may include trans-zeaxanthin (3R,3'R-β,β-carotene-3,3'-diol), and cryptoxanthin may include beta-cryptoxanthin (3R,6'R)-4',5'-didehydro-5',6'-dihydro-β,β-carotene-3-ol. The color value of the carotenoids may range from 800,000 to 1,250,000. The composition may help manage age-related macular degeneration symptoms, such as blurred vision, distorted vision, reduced central vision, and difficulty adopting low light levels. The composition may protect against blue light-induced retinopathy by reducing oxidation and endoplasmic reticulum stress. The composition can provide color to cosmetics such as lipstick, chapstick, liquid gloss, lipstick paste, blush, lip liner, foundation, concealer, eye contour, eyeliner, mascara, nail polish, eye shadow, or body make-up. The composition can provide functional and morphological preservation of photoreceptors against blue light damage. The composition can reduce intraocular pressure associated with tension glaucoma, primary open-angle glaucoma, and angle-closure glaucoma. The composition can reduce body weight. The composition can reduce insulin, thereby reducing body weight. The composition can reduce plasma free fatty acids, thereby improving mitochondrial function and whole-body insulin sensitivity. The composition can reduce plasma leptin, thereby restoring hypothalamic leptin sensitivity, reducing weight gain, and enhancing insulin sensitivity. The composition can increase adiponectin, thereby reducing insulin resistance and body weight. The composition can reduce adipose tissue weight and epididymal fat pads. The composition may be in the form of a capsule, tablet, injection, cream, gel, ointment, lotion, liquid, beverage, confectionery, emulsion, foam, troche, lozenge, aqueous suspension, oily suspension, patch, dentifrice, spray, drops, powder, granules, syrup, elixir, or food, or the like.The composition may comprise total carotenoids in the range of 10% to 99%. The pepper fruit may be KDL high color, 5531 high color, and 4431 high color in a ratio of 1:1:2, respectively. The ASTA color value of the pepper fruit may range from 2000 to 2600 units. The solvent may comprise ethanol, methanol, and isopropyl alcohol. Extracting the carotenoids may include extracting the carotenoids at a temperature ranging from 40° C. to 90° C. for a period ranging from 4 hours to 8 hours. The solvent medium for the supercritical fluid extraction may be carbon dioxide. Concentrating the carotenoids may include concentrating the carotenoids using supercritical fluid extraction at a temperature ranging from 40° C. to 60° C. Concentrating the carotenoids may include concentrating the carotenoids using supercritical fluid extraction at a pressure ranging from 25 mPa to 50 mPa. Hydrolyzing the carotenoids may include hydrolyzing the carotenoids using KOH and NaOH. Hydrolyzing the carotenoids may include hydrolyzing the carotenoids using methanol, ethanol, and isopropyl alcohol. Hydrolyzing the carotenoids may include hydrolyzing the carotenoids using a hydrolyzing agent in the range of 10% to 30%. Hydrolyzing the carotenoids may include hydrolyzing the carotenoids at a temperature in the range of 70°C to 85°C. Hydrolyzing the carotenoids may include hydrolyzing the carotenoids in the range of 1 hour to 3 hours. Purifying the carotenoids may include purifying the carotenoids using a solvent for countercurrent extraction that includes ethyl acetate and isopropyl acetate. The immiscible aqueous phase may be used for the countercurrent extraction water and water acidified with hydrochloric acid (pH 3-4). The method may further include blending the carotenoid with an excipient such as sunflower oil, safflower oil, soy lecithin, sunflower lecithin, phosphatidylcholine derived from sunflower or soybean, starch, dextrin, lactose, dicalcium phosphate, or colloidal silicon dioxide.The capsanthin may include trans-capsanthin (3R,3'S,5'R)-3,3'-dihydroxy-β,κ-caroten-6'-one, the zeaxanthin may include trans-zeaxanthin (3R,3'R-β,β-carotene-3,3'-diol), and the cryptoxanthin may include beta-cryptoxanthin (3R,6'R)-4',5'-didehydro-5',6'-dihydro-β,β-carotene-3-ol. The composition may aid in melanin inhibition. The composition may inhibit tyrosinase. The composition may aid in the elimination and prevention of dandruff. The composition may be an anti-fungal agent against Malassezia furfur and Candida albicans. The composition may be an anti-yeast agent against Malassezia yeast. The composition may aid in hair growth. The composition may induce gene expression in human keratinocytes (HaCaT). The composition can restore skin function and aid in skin moisturization by increasing the biosynthesis of hyaluronic acid. The composition can increase the level of AQP-3 mRNA. The composition can help protect the skin from ultraviolet radiation, UVA radiation, and UVB radiation. The composition can provide a sun protection factor (SPF) of at least 20. The composition can prevent and treat dry eye syndrome when administered orally. The composition can increase tear film breakup time and reduce tear osmolarity when administered orally. The composition can increase tear production when administered orally. The composition can reduce defects and abrasions in the corneal epithelium when administered orally. The composition can reduce damage to the ocular surface by reducing oxidative stress when administered orally. The composition can reduce degeneration and apoptotic cell death in the cornea by reducing serum MMP-2 and MMP-9 levels when administered orally. The composition can restore the mucin layer of the eye when administered orally. The composition may reduce expression of IL-2, IL-6 and MMP-9 genes.The composition may downregulate expression of TNF-α and IL-4 genes.Extracting the carotenoids may include extracting the carotenoids at a temperature ranging from 40° C. to 90° C. for a period ranging from 4 hours to 8 hours, concentrating the carotenoids may include concentrating the carotenoids using supercritical fluid extraction at a temperature ranging from 40° C. to 60° C., concentrating the carotenoids may include concentrating the carotenoids using supercritical fluid extraction at a pressure ranging from 25 mPa to 50 mPa, and purifying the carotenoids may include purifying the carotenoids using a countercurrent extraction solvent comprising ethyl acetate or isopropyl acetate. Purifying the concentrated carotenoids may include removing an organic layer containing at least 50% capsanthin and adjusting the pH of the remaining aqueous layer via acid, and purifying the carotenoids using a solvent may include purifying the carotenoids using a solvent for countercurrent extraction containing ethyl acetate or isopropyl acetate such that the extract contains capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 15%, and cryptoxanthin in the range of 0.1% to 0.5%. The composition may aid in the removal and prevention of dandruff and may be an antifungal agent against Malassezia furfur and Candida albicans. The composition may aid in hair growth and induce gene expression in human keratinocytes (HaCaT). The composition may aid in skin moisturization and increase levels of AQP-3 mRNA. The composition may prevent and treat dry eye syndrome when administered orally. The composition may increase tear film breakup time and decrease tear osmolality when administered orally. The composition, when administered orally, can increase tear production. The composition, when administered orally, can reduce defects and abrasions in the corneal epithelium. The composition, when administered orally, can reduce damage to the ocular surface by reducing oxidative stress. The composition, when administered orally, can reduce degeneration and apoptotic cell death in the cornea by reducing serum MMP-2 and MMP-9 levels. The composition, when administered orally, can restore the mucin layer of the eye. The composition can reduce the expression of IL-2, IL-6 and MMP-9 genes. The composition can downregulate the expression of TNF-α and IL-4 genes.

[0072] The foregoing description of specific embodiments fully reveals the generic nature of the embodiments herein, so that others, by applying current knowledge, can easily modify and / or adapt such specific embodiments for various applications without departing from the generic concept, and therefore such adaptations and modifications should and are intended to be understood within the meaning and equivalents of the disclosed embodiments. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. Thus, although the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be implemented with modifications within the spirit and scope of the embodiments as described herein. Once the above disclosure is fully appreciated, numerous other modifications, equivalents, and alternatives will become apparent to those skilled in the art. It is intended that the following claims be construed to embrace all such modifications, equivalents, and alternatives, where applicable.

Claims

1. A capsicum extract composition (600), comprising: Capsanthin in the range of 1% to 1.5%, Zeaxanthin in the range of 0.5% to 1%, and Cryptoxanthin in the range of 0.1% to 0.5% Including, the composition is produced, at least in part, from spent materials from purifying carotenoids using countercurrent extraction; The composition strengthens the skin, eyes, or hair. A composition comprising:

2. The composition according to claim 1, characterized in that the capsanthin comprises trans-capsanthin (3R,3'S,5'R)-3,3'-dihydroxy-β,κ-caroten-6'-one), the zeaxanthin comprises trans-zeaxanthin (3R,3'R-β,β-carotene-3,3'-diol), and the cryptoxanthin comprises beta-cryptoxanthin (3R,6'R)-4',5'-didehydro-5',6'-dihydro-β,β-carotene-3-ol.

3. 10. The composition of claim 1, wherein the composition aids in inhibiting melanin by inhibiting tyrosinase.

4. 2. The composition according to claim 1, characterized in that it is an antifungal agent against Malassezia furfur and Candida albicans and an antiyeast agent against Malassezia yeast, thereby helping to eliminate and prevent dandruff.

5. The composition of claim 1, characterized in that the composition promotes hair growth by inducing gene expression in human keratinocytes (HaCaT).

6. The composition of claim 1, wherein the composition increases the level of AQP-3 mRNA.

7. The composition of claim 1, wherein the composition increases tear film breakup time, increases tear production, and decreases tear osmolality when orally administered.

8. The composition of claim 1, wherein the composition, when orally administered, reduces defects and abrasions in the corneal epithelium and reduces damage to the ocular surface by reducing oxidative stress.

9. The composition of claim 1, wherein the composition, when orally administered, reduces degeneration and apoptotic cell death in the cornea by reducing serum MMP-2 and MMP-9 levels.

10. The composition of claim 1, characterized in that the composition reduces the expression of IL-2, IL-6, and MMP-9 genes and downregulates the expression of TNF-α and IL-4 genes.

11. A method (100) for producing a composition, comprising the steps of: extracting carotenoids from spent material (102), the spent material being produced from a process for purifying carotenoids from pepper fruits using countercurrent extraction with a solvent; concentrating the extracted carotenoids using supercritical fluid extraction (106); and (11) purifying the concentrated carotenoids using countercurrent extraction so that the composition strengthens the skin, eyes, or hair. A method comprising:

12. 12. The method of claim 11, wherein the step of extracting the carotenoids comprises extracting the carotenoids at a temperature ranging from 40°C to 90°C for a period ranging from 4 hours to 8 hours, the step of concentrating the carotenoids comprises concentrating the carotenoids using supercritical fluid extraction at a temperature ranging from 40°C to 60°C, the step of concentrating the carotenoids comprises concentrating the carotenoids using supercritical fluid extraction at a pressure ranging from 25 mPa to 50 mPa, and the step of purifying the carotenoids comprises purifying the carotenoids using a countercurrent extraction solvent comprising ethyl acetate or isopropyl acetate.

13. Purifying the concentrated carotenoids comprises removing an organic layer containing at least 50% of the capsanthin, and adjusting the pH of the remaining aqueous layer via an acid; The step of purifying the carotenoids using a solvent includes purifying the carotenoids using a countercurrent extraction solvent comprising ethyl acetate or isopropyl acetate such that the extract contains capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 15%, and cryptoxanthin in the range of 0.1% to 0.5%.

12. The method according to claim 11 .

14. 12. The method of claim 11, wherein the composition increases tear breakup time and decreases tear osmolarity when orally administered.

15. 12. The method of claim 11, wherein the composition increases tear production when administered orally.

16. 12. The method of claim 11, wherein the composition, when administered orally, reduces defects and abrasions in the corneal epithelium.

17. 12. The method of claim 11, wherein the composition, when orally administered, reduces damage to the ocular surface by reducing oxidative stress.

18. 12. The method of claim 11, wherein the composition, when orally administered, reduces degeneration and apoptotic cell death in the cornea by reducing serum MMP-2 and MMP-9 levels.

19. The method of claim 11, wherein the composition reduces expression of IL-2, IL-6 and MMP-9 genes and downregulates expression of TNF-α and IL-4 genes.

20. A method (100) for producing a composition, comprising the steps of: extracting carotenoids from spent material (104), the spent material being produced from a process for purifying carotenoids from pepper fruits using countercurrent extraction with a solvent; concentrating the extracted carotenoids using supercritical fluid extraction (106); and purifying the hydrolyzed carotenoids using countercurrent extraction (110), such that the composition comprises capsanthin in the range of 1% to 1.5%, zeaxanthin in the range of 0.5% to 15%, and cryptoxanthin in the range of 0.1% to 0.5%, and the composition strengthens the skin, eyes, or hair. A method comprising:

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