Patent Description:
Carotenoid compositions for use in the reduction of intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof and methods of manufacture of carotenoid compositions are disclosed herein. In the drawings:.

It should be understood, however, that the specific embodiments given in the drawings and detailed description thereto do not limit the disclosure. On the contrary, they provide the foundation for one of ordinary skill to discern the alternative forms, equivalents, and modifications that are encompassed together with one or more of the given embodiments in the scope of the appended claims.

Certain terms are used throughout the following description and claims to refer to particular system components and configurations. As one of ordinary skill will appreciate, companies may refer to a component by different names. This document does not intend to distinguish between components that differ in name but not function. In the following discussion and in the claims, the terms "including" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "including, but not limited to.

Carotenoids are any of various pigments such as carotenes found widely in plants and animals and characterized chemically by a long aliphatic polyene chain composed 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, which aid disease-prevention mechanisms of the body.

Age-related macular degeneration ("AMD") is a common medical eye condition and a leading cause of vision loss among people over the age of fifty. It includes damage to the macula, an oval-shaped pigmented area near the center of the retina that is used for sharp, central vision. AMD may be of two types: dry (atrophic) or wet (exudative). In addition to aging, high blood-pressure, oxidative stress, blue light exposure, obesity, and the like may also cause AMD. The 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 the carotenoids present in the retina, and they protect eye cells from photo oxidative damage. Specifically, chronic exposure to blue light, the major cause of AMD, causes reduction in cone density and cone sensitivity, and zeaxanthin may prevent AMD by absorbing blue light.

Capsicums are a widely consumed natural foodstuff used as a vegetable, spice, and/or color, and paprika extract is an extract of the fruits of the genus capsicum. The genus, which originates from Central and Southern America, belongs to the solanaceae family and includes all peppers, from the mild bell pepper to the spicy habanero. There are five domesticated species of capsicum: annum, frutescens, chinense, pubescens, and baccatum, and the most widely spread are annum, frutescens, and chinense. The first to be introduced worldwide was annum, originating from Mexico. It was previously divided into two categories: sweet (or mild) peppers and hot (or chili) peppers, though modern plant breeding removed that distinction. At present, annum is the most wide-spread in terms of household consumption and industrial processing.

Annum is the varietal used to manufacture paprika extract for food coloration. Color extracts have a low content of capsaicin compared with the extracts used as spice agents. In the red varietal, capsanthin and capsorubin are the main compounds responsible for the red color. Pure carotenoid crystals derived from the annum fruits include xanthophylls such as capsanthin, zeaxanthin, and cryptoxanthin. The chemical structure of the carotenoid ultimately determines what potential biological function(s) that pigment may have. The distinctive pattern of alternating single and double bonds in the polyene backbone of carotenoids is what allows them to absorb excess energy from other molecules, while the nature of the specific end groups on carotenoids may influence their polarity. The carbonyl group present in capsanthin and capsorubin makes them unique when compared to other carotenoids such as lutein and zeaxanthin.

<FIG> illustrates a method <NUM> of manufacturing a carotenoid composition for use in the reduction of intraocular pressure in accordance with some illustrated embodiments. Specifically, disclosed herein are methods for isolating and purifying carotenoids containing a specific composition of carotenoids such as trans-capsanthin, trans-zeaxanthin, and beta-cryptoxanthin from capsicum annum fruits leaving no trace of organic hazardous solvents. The method <NUM> includes selection of high colored composition of annum chili varieties, solvent extraction, super critical fluid extraction ("SCFE") enrichment, alkali hydrolysis of carotenoid esters with absolute alcohol, purification using counter current extractor, concentration, and drying.

At <NUM>, specific varieties of capsicum annum fruits are selected. In an embodiment, the capsicum annum fruit is selected from the Bydagi chili varieties, or other chilies high in color value, alone or in combination such as: Bydagi-Kaddi, Bydagi-Dyavnoor, Bydagi-Dabbi, KDL high color chili, <NUM> high color chili, and/or <NUM> high color chili. In an embodiment, the ratio of the combination is: (<NUM>) Bydagi-Kaddi : (<NUM>) Bydagi-Dyavnoor : (<NUM>) Bydagi-Dabbi (a ratio of <NUM>:<NUM>:<NUM>). In other embodiments, unexpected results have been found using the ratio of <NUM>:<NUM>:<NUM> and <NUM>:<NUM>:<NUM> of Bydagi-Kaddi : Bydagi-Dyavnoor : Bydagi-Dabbi. Additionally, unexpected results have been found using the ratio of KDL high color, <NUM> high color, and <NUM> high color in <NUM>:<NUM>:<NUM>, respectively. In various embodiments, the American Spice Trade Association ("ASTA") color value of the Bydagi chilies, or other high color chilies, are selected within the range from <NUM> to <NUM> units or a range from <NUM> to <NUM> units.

At <NUM>, carotenoids are extracted from the fruits with suitable solvents. In an embodiment, dried, deseeded, and flaked capsicum annum fruits undergo extraction using a solvent at a temperature ranging from <NUM> to <NUM>, preferably at <NUM>, for <NUM> hours to <NUM> hours, preferably <NUM> hours. The solvent extract is then concentrated under vacuum to produce capsicum oleoresin containing carotenoids esters. In various embodiments, the extraction solvent may be one or a combination of: methanol, ethanol, and/or isopropyl alcohol.

For example, <NUM> kilograms of deseeded, flaked capsicum annum fruits with ASTA color value from <NUM> to <NUM> units may be placed in a <NUM> liter capacity reactor with an agitator. A volume of methanol (<NUM>) may be added and the mixture may be stirred for <NUM> hours at <NUM>. The methanol layer may be filtered and collected. This methanol extraction may be repeated three times for efficiency purposes.

At <NUM>, the carotenoids are enriched using super critical fluid extraction. In an embodiment, carbon dioxide is used as a solvent, and the temperature for super critical fluid extraction ranges from <NUM> to <NUM>, preferably <NUM>. In an embodiment, the pressure employed for super critical fluid extraction ranges from <NUM> to <NUM> mPa, preferably 35mPa.

Continuing the above example, all the methanol layers may be combined and concentrated under vacuum. <NUM> of oleoresin may be obtained and formulated with approximately <NUM> of calcium carbonate in super critical fluid extractor bags. The extractor bags may be placed in an extraction chamber and extracted at <NUM> and 35mPa. The yield of enriched oleoresin may be approximately <NUM>.

At <NUM>, the carotenoid esters are hydrolyzed. In an embodiment, the enriched oleoresin is hydrolyzed with alcoholic potassium hydroxide to produce free carotenoids. In an embodiment, the base for hydroxylation is selected from a group consisting of potassium hydroxide (KOH), sodium hydroxide (NaOH), or a combination thereof. In an embodiment, the solvent media for hydrolysis is methanol, ethanol, isopropyl alcohol, or a combination thereof. In an embodiment, concentration of the hydrolysis agent ranges from <NUM>% to <NUM>%. In an embodiment, the temperature of hydrolysis ranges from <NUM>° to <NUM>, preferably <NUM>. In an embodiment, the time required for hydrolysis ranges from <NUM> hour to <NUM> hours, preferably <NUM> hours. As the carotenoids are hydrolyzed to free form, they become more bioavailable.

Continuing the above example, the enriched oleoresin may be placed in a <NUM> glass lined reactor. In a separate vessel, <NUM> of Potassium hydroxide may be added to <NUM> of ethyl alcohol while stirred. The alcoholic KOH may be added to the enriched oleoresin slowly while stirred at <NUM> for two hours.

At <NUM>, the hydrolyzed carotenoids are purified using counter current extractions. In an embodiment, the solvent used for counter current extraction is ethyl acetate, isopropyl acetate, or a combination thereof. In an embodiment, the immiscible aqueous phase is water or water acidified with hydrochloric acid (pH <NUM>-<NUM>), preferably acidified water.

Continuing the above example, after ensuring the degree of saponification to be more than <NUM>% by HPLC, <NUM> liters of demineralized hot water maintained at a temperature of <NUM>° C may be added to the reacted mass while stirred for <NUM> minutes. The diluted mass with carotenoid crystals may be pumped into a filter press to recover the crystals. Around <NUM> liters of additional hot water may be pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around <NUM>. After ensuring the neutralization, a positive pressure of nitrogen may be applied to the filter press to squeeze the crystals trapped inside the filter. The wet crystals with an approximate weight <NUM> may then be collected from the filter press, dissolved in <NUM> of ethyl acetate, and charged into a counter current extractor. <NUM> of water may be used, and the pH may be adjusted to <NUM> or <NUM> with dil-HCl. The acidified water may be bottom fed into the counter current extractor.

At <NUM>, the capsicum annum extract is blended with excipient(s). In various embodiments, the excipient is sunflower oil, safflower oil, soy lecithin, sunflower lecithin, phosphatidylcholine from sunflower or soy, starch, dextrin, lactose, dicalcium phosphate, colloidal silicon dioxide, and/or combinations thereof. In other embodiments, the excipient is a granulating agent, binding agent, lubricating agent, disintegrating agent, sweetening agent, glidant, anti-adherent, anti-static agent, surfactant, anti-oxidant, gum, coating agent, coloring agent, flavoring agent, coating agent, plasticizer, preservative, suspending agent, emulsifying agent, plant cellulosic material, spheronization agent, and/or combinations thereof. In an embodiment, the carotenoids are further isolated and/or purified by: addition of one or more solvents, addition of ionic resin, quenching, filtration, extraction, and/or ion exchange resin.

Continuing the example above, the ethyl acetate layer may be removed, dried over anhydrous sodium sulphate, and concentrated. The yield of composition may be about <NUM>. The carotenoid contents as measured by a spectrophotometer may be about <NUM>%, with all trans-capsanthin, all trans-zeaxanthin, and all the beta-cryptoxanthin by HPLC at <NUM>%, <NUM>% and <NUM>% respectively. The final product may contain a moisture content of about <NUM>% with no traces of residual methanol and ethyl acetate detected by gas chromatography analysis.

As a result of the above process, in an embodiment the composition of carotenoids ranges from capsanthin: <NUM>% to <NUM>%, zeaxanthin: <NUM>% to <NUM>%, cryptoxanthin: <NUM>% to <NUM>%, and trace amounts of other carotenoids. These percentages, and all percentages herein, refer both to actual percentages and percentages allowable by labeling regulations. In an embodiment, the composition of total carotenoids ranges from <NUM>% to <NUM>%. In an embodiment, the carotenoids are trans-capsanthin: (3R,<NUM>'S,<NUM>'R)-<NUM>,<NUM>'-dihydroxy-β,κ-caroten-<NUM>'-one; trans-zeaxanthin: 3R, <NUM>'R-β,β-carotene-<NUM>,<NUM>'-diol; and beta-cryptoxanthin: (3R,<NUM>'R)-<NUM>',<NUM>'-didehydro-<NUM>',<NUM>'-dihydro-β,β-caroten-<NUM>-ol. In an embodiment, the color value of carotenoids ranges from <NUM>,<NUM> to <NUM>,<NUM>,<NUM>.

The composition lowers the intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof in various embodiments as shown in <FIG>. Thus, the invention provides the compositions as described herein for use in the reduction of intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof.

<FIG> depicts the results of a study to evaluate lowering of intraocular pressure ("IOP") in animals. The high intraocular pressure originated from an increased resistance to drainage of aqueous humor through the trabecular meshwork. A sustained increase in aqueous humor may be due to an increase in the formation of aqueous humor, a difficulty in its exits, or a raised pressure in the episcleral vein. In this study, IOP was induced by intravitreal injection. <FIG> shows a bar chart depicting six groups (G1-G6), and the left eye and right eye IOP for each group. The first two groups (G1-G2) are control groups in which no IOP lowering composition was introduced. In G1, IOP was not induced, and in G2, IOP was induced. For the next for groups (G3-G6), the left bar for each eye represents IOP prior to introduction of the composition disclosed herein in one embodiment. The right bar for each eye represents IOP after introduction of the composition. The results show that introduction of the composition reduced IOP to a level almost equal to the group in which IOP was not induced (G1), and significantly lower than the group in which the composition was not introduced (G2).

<FIG> illustrates a composition <NUM> of the capsicum annum extract including capsanthin, zeaxanthin, and cryptoxanthin for use in the reduction of intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof manufactured as described above in a capsule embodiment. In other embodiments the composition is implemented as a tablet, injectable, cream, gel, ointment, lotion, solution, beverage, confection, emulsion, foam, troche, lozenge, aqueous suspension, oily suspension, patch, dentifrice, spray, drop, powder, granule, syrup, elixir, food stuff, and/or combinations thereof. In preferred embodiments, the composition includes a tablet, soft gelatin capsule, hard gelatin capsule, cream, gel, lotion, and/or combinations thereof.

The composition may be administered by topical administration, oral administration, intravenous administration, intra articular administration, intramuscular administration, and/or combinations thereof in various embodiments. In preferred embodiments, the mode of administration is oral, topical, intravenous intramuscular, and/or combinations thereof. In addition, materials such as flaxseed, flaxseed oil, vegetarian or vegetable oil, and other oils may be combined with the composition to stabilize the active ingredients. Regardless of the amount of materials combined with the composition, the percentages of carotenoids in relation to the total amount of carotenoids remains the same in at least some embodiments.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein and the various features and advantageous details thereof are explained with reference to the nonlimiting embodiments in the description. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

In some aspects, apparatuses, systems, and methods are provided according to one or more of the following examples:.

Example <NUM>: A capsicum annum extract composition for use in the reduction of intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof includes capsanthin in the range from <NUM>% to <NUM>%, zeaxanthin in the range from <NUM>% to <NUM>%, and cryptoxanthin in the range from <NUM>% to <NUM>%.

Example <NUM>: A method for manufacturing a composition for use in the reduction of intraocular pressure includes extracting carotenoids from capsicum annum fruits using a solvent or solvents, enriching the carotenoids using super critical fluid extraction, hydrolyzing the carotenoids, and purifying the carotenoids using counter current extractions.

Example <NUM>: A method for manufacturing a composition for use in the reduction of intraocular pressure includes extracting carotenoids from capsicum annum fruits using a solvent or solvents, enriching the carotenoids using super critical fluid extraction, hydrolyzing the carotenoids, and purifying the carotenoids using counter current extractions such that the composition includes capsanthin in the range from <NUM>% to <NUM>%, zeaxanthin in the range from <NUM>% to <NUM>%, and cryptoxanthin in the range from <NUM>% to <NUM>%.

The following features may be incorporated into the various embodiments described above, such features incorporated either individually in or conjunction with one or more of the other features:.

Claim 1:
A capsicum annum extract composition (<NUM>) for use in the reduction of intraocular pressure associated with normal tension glaucoma, primary open angle glaucoma, angle closure glaucoma, and/or combinations thereof, wherein the composition comprises:
capsanthin in the range from <NUM>% to <NUM>%;
zeaxanthin in the range from <NUM>% to <NUM>%; and
cryptoxanthin in the range from <NUM>% to <NUM>%.