WO2023240100A1 - A method to encapsulate non-polar compounds to achieve extended thermal stability and attenuate/prevent thermal and redox isomerization - Google Patents

A method to encapsulate non-polar compounds to achieve extended thermal stability and attenuate/prevent thermal and redox isomerization Download PDF

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Publication number
WO2023240100A1
WO2023240100A1 PCT/US2023/068017 US2023068017W WO2023240100A1 WO 2023240100 A1 WO2023240100 A1 WO 2023240100A1 US 2023068017 W US2023068017 W US 2023068017W WO 2023240100 A1 WO2023240100 A1 WO 2023240100A1
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cyclodextrin
guest
molecules
encapsulating
encapsulating component
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PCT/US2023/068017
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French (fr)
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Clay Carew CLARKSON
Miracle Shaffon JACKSON
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Clarkson Clay Carew
Jackson Miracle Shaffon
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Publication of WO2023240100A1 publication Critical patent/WO2023240100A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A61K9/5057—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to compositions and methods for chemical entrapment of plant-based nutraceuticals and extracts for the fortification of food and beverage products. More particularly, the method provides for the encapsulation of polyphenols, terpenophenolics, and flavonoids for pre- and post-biotic systems.
  • the compositions and methods may be used to form nutraceutical supplements, pet feed, cannabis and hemp edibles, processed foods and beverages, and pharmaceuticals.
  • Microcapsules are polymeric layers that encapsulate active ingredients to mask flavors and smells, and to provide protection from heat, oxidation, and ultraviolet light.
  • a microcapsule consists of a polymeric outer shell and an oil phase core containing an active ingredient or compound of interest.
  • shell properties determine its stability and release mechanism. Microcapsules are employed by many industries for a variety of purposes.
  • microcapsules are often designed to target specific organs for controlled drug release.
  • Polymeric shells consisting of crosslinked polysaccharides and proteins are known in literature to exhibit acidic stability and swell under alkaline conditions.
  • These crosslinked microcapsules can be composed of the following polysaccharides and proteins: acacia gum, cellulose, chitosan, and pectin, and gelatin, soy proteins, whey proteins, and wheat proteins.
  • crosslinking of the proteins and polysaccharides often needs glutamine and lysine moieties to form isopeptide bonds via transglutaminase.
  • Tannic acid a natural polyphenol and antioxidant, is also capable of crosslinking microcapsules that are composed of proteins. Tannic acid can crosslink amine groups via Michael addition or Schiff base reaction, thiol groups via Michael addition or thiyl radical oxidation, and carboxyl groups via acylation.
  • Crosslinked polysaccharides-protein microcapsules are prepared following complex coacervation techniques. This technique is described as an electrostatic attraction of two or more biopolymers with opposite charges (e.g., proteins and polysaccharides) under varying pH conditions to induce changes in charges. Methods on microcapsule formation often employ high shear emulsification followed by addition of an oil phase containing the active ingredient.
  • microcapsules are readily used where a volatile compound of interest needs to be stabilized in a matrix. This includes but is not limited to encapsulation of flavors, fragrances, and nutrients. Microcapsules, however, are not ideal for bypassing first-pass metabolism effects as the size is generally larger than 500 nm in diameter.
  • Crosslinked gelatin acacia gum microcapsules are used in consumer products and have been used to deliver pre-biotics or good gut bacteria to the large intestine.
  • the crosslinking of the gelatin based microcapsules gives it acid stability or stability in the stomach and gastrointestinal tract.
  • the swelling or release of the microcapsule occurs at pH above 7, which is the condition of the large intestine.
  • Microcapsules described in published US patent application Publication No. 2017/021616 contain near fully hydrolyzed polyvinyl alcohols to better retain aromatic compounds and withstand the mechanical stress induced by washing machines.
  • This patent also makes the claim that scents or flavors can be released upon baking and chewing processes. The focus here was to obtain capsules with a wide fragrance release profile that releases under mechanical stress.
  • the ratio of capsule shell mass to core mass was described to be below 1. Where sheikcore mass ratio is defined by Equation 1.
  • Sheikcore (total mass polymeric shell / mass oil phase core). This characteristic gives rise to a capsule that does not exhibit extended thermal stability under traditional cooking / baking conditions. Where traditional cooking / baking temperatures are between 175 °C to 260 °C for 30 minutes to 60 minutes.
  • Increasing the amount of shell composition will increase the thermal stability of a microcapsule.
  • Shell composition also plays a role.
  • a method described in published PCT patent application Publication No. W02008085997 utilizes the advantage of multi-nuclear microcapsules. Where a multi-nuclear microcapsule is described to be a microcapsule containing smaller capsules or having internal chambers. Multi-nuclear constructs increase the ratio of shell to core but hinder the ability to effectively reduce particle size and hence bypass first-pass metabolism effects.
  • encapsulation vehicles for pharmaceutical or nutraceutical compounds should be able to bypass first-pass metabolism routes. Some medical and therapeutic compounds are metabolized in the liver. This results in reduced transport of active and desired compounds to the bloodstream. Therefore, to effectively control the delivery of oral based pharmaceuticals or nutraceuticals, sub-nano drug carriers that are released from fast digestible microcapsules are employed. The microcapsules should be formulated to be enzymatically released at the start of digestion (via buccal cavity).
  • the present invention provides a method and related compositions that encapsulate ingestible items of interest in a way that encapsulating material integrity is maintained through processes of interest that include elevated temperatures that would otherwise degrade the encapsulant.
  • the present invention employs a guest-host molecule derived from the class of cyclodextrins that is synthesized to enhance the complexation with, but not limited to; phytocannabinoids, steroids, flavonoids, polyphenols, and terpenophenolics. This is accomplished by dimerization and bridging of cyclic glucopyranose or cyclodextrins and all its derivatives, such as hydroxy-beta-cyclodextrin, into bis or nanosponge structures, occurs at its free -OH groups in the presence of dicarboxylic acids, like tartaric acid, following a Fischer esterification reaction. Dicarboxylic acids suitable for this reaction include, but are not limited to, tartaric acid. Excess amounts of dicarboxylic acids will push the reactions towards the formation of nanosponge or a polymer of cyclodextrins where bridging can occur at other -OH groups.
  • the cyclodextrin reaction solutions are dried under vacuum freeze-drying conditions, rotoevaporation, or spray dried.
  • the resulting solid powder is then kneaded with the non-polar compounds of interest to form inclusion complexes.
  • the formed inclusion complexes are then mixed and emulsified with saccharides, oligosaccharides, polysaccharides, denatured proteins, or a formulated mixture to form micro- or nano-capsules around the inclusion complexes.
  • examples include, but not limited to; dextrins, arabic gum, inulin, hemicelluloses, etc.
  • the formulated emulsification mixture contains an equivalent or greater mass to cyclodextrin inclusion complexes.
  • the formulated microcapsule mixture should have a shell to core ratio that is equal or greater than 1. Where shell composition is composed of the emulsification mixture and the core is the cyclodextrin inclusion complexes.
  • the emulsification mixture is combined with the cyclodextrin inclusion complexes and is spray dried or freeze dried to form micro(nano)capsules around the cyclodextrin inclusion complexes.
  • cyclodextrin inclusion complexes can be omitted.
  • the oil phase is homogenized with the micro(nano)capsule formulation, and then spray dried or freeze dried to obtain a dry powder.
  • the micro(nano)capsule formulation follows a shell to core mass ratio of 1 or greater to achieve extended thermal stability.
  • Examples of this process is employed to encapsulate delta-9- tetrahydrocannabinol, cannabinol and other terpenoids and phytocannabinoids present in the extracts of Cannabis saliva L. and its close relatives into a thermally stable, tasteless, and odorless powder.
  • this method can be used to encapsulate other bioactive compounds of interest that are non-polar or slightly non-polar. This includes flavonoid polyphenols, like qucerterin and epicatechin from Theobroma cacao extracts.
  • the invention is a method for encapsulating an ingestible agent in an encapsulating component.
  • the method includes the steps of crosslinking together a plurality of guest-host molecules, wherein the guest-host molecule is a cyclic glucopyranose or a cyclodextrin containing free hydroxyl groups, to link together the plurality of guest-host molecules to form the encapsulating component, and complexating a plurality of molecules of the ingestible agent in the encapsulating component to form a complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent.
  • the step of crosslinking involves using a linker molecule to form ester bonds between the plurality of guesthost molecules through esterification.
  • the esterification may be a Fischer esterification and the linker molecule may be a diacid.
  • the diacid may be a dicarboxylic acid having at least two reactive carboxyl groups.
  • the dicarboxylic acid may be tartaric acid.
  • the cyclodextrin may be a hydroxypropyl-beta-cyclodextrin, a beta-cyclodextrin, or other cyclodextrin derivative with a free hydroxyl group.
  • the encapsulating component is a dimer structure.
  • three or more guest-host molecules are crosslinked together the encapsulating component is a bridged or polymerized structure.
  • the encapsulating component is a nanosponge.
  • an excess of di carboxylic acid is used in the crosslinking step to form the encapsulating component as the nanosponge.
  • the ingestible item is selected from the classes comprising phytocannabinoids, steroids, flavonoids, polyphenols, and terpenophenolics.
  • the method may further include the step of encapsulating the complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent in a polymeric shell with the combination forming a core substantially within the polymeric shell to enhance thermal stability of the core.
  • the polymeric shell is formed of either or both of proteins and carbohydrates.
  • the polymeric shell may be formed by crosslinking using either transglutaminase enzyme or tannic acid. A total mass of the polymeric shell is greater than a total mass of the core. BRIEF DESCRIPTION OF THE DRAWINGS.
  • FIG. 1 is a fluorescent microscope image of gelatin-acacia gum microcapsules. Average size of a microcapsule is 1 micron in diameter.
  • FIG. 2 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core ratio of 0.5.
  • the pre-heated sample contained a mixture of cannabinoids.
  • FIG. 3 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core mass ratio of 0.5. Sample was baked at 176.67°C for 30 minutes. Sample contained a mixture of cannabinoids.
  • FIG. 4 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core ratio of 0.5. Sample was baked at 204.44°C for 30 minutes. Sample contained a mixture of cannabinoids.
  • FIG. 5 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Pre-heated sample. Sample contained only THC.
  • FIG. 6 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Sample was baked at 176.67°C for 30 minutes. Sample contained only THC.
  • FIG. 7 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Sample was baked at 176.67°C for 90 minutes. Sample contained only THC.
  • FIG. 8 is a reaction mechanism to form a bis(X-cyclodextrin)s where there is only one linker molecule bridging two cyclodextrins.
  • X is either a, P, or y and directly relates to n, where n is 6, 7, or 8 , respectively.
  • R defines the cyclodextrin to be either in its parent form or a 2-hydroxypropyl form, most commonly found with P-cyclodextrins.
  • FIG. 9 is a representation of a result of bis(X-cyclodextrin)s where there are more than one linker molecules bridging two cyclodextrins. Where X is either a, , or y. The structure should have more structural rigidity compared to being linked by one linker molecule. The linker molecule is tartaric acid.
  • FIG 10 is a depiction of a cyclodextrin polymer or nanosponge. Crosslinking is occurring at the 6’ -OH and other free -OH groups along the cyclic glucopyranose structure. The linker molecule is tartaric acid.
  • FIG. 11 is a depiction of supramolecular cyclodextrin complexes encapsulated in polymeric shells.
  • the polymeric shell on the left depicts a micro-sized capsule at around 1 micron in diameter.
  • the polymeric shell on the right depicts a nano-sized capsule that is below 500 nm.
  • Cyclodextrins are mixed with dicarboxylic acids at a molar ratio where the number of free 6’ hydroxyls on the cyclic glucopyranose molecules is 2.25: 1 or greater.
  • 3-cyclodextrin contains seven 6’ hydroxyl groups, so for every mol of P-cyclodextrin there is 3.11 mols of tartaric acid in the system. Tartaric acid in the presence of strong acids will crosslink cyclodextrins into a bis(cyclodextrin) form where tartaric acid is used as the linker molecule.
  • An excess of dicarboxylic acids pushes beyond the formation of bis(cyclodextrin)s to form nanosponges. Where nanosponges are a polymer or crosslinked matrix of more than two cyclodextrin molecules. Crosslinking can also occur at other free hydroxyl groups on the glucopyranose subunits besides the 6’ carbon.
  • the reaction mixture is stirred at 1000 RPM and heated to remove water formed by the formation of esters.
  • the reaction is pushed to completion by removal or boiling of water.
  • the cyclodextrin dimers are isolated by means of drying such as spray drying, freeze drying, or rotoevaporation.
  • the dried cyclodextrin dimer or polymer nanosponge powder is kneaded with the non-polar, slightly-nonpolar compounds or active ingredients to form inclusion complexes.
  • Cyclodextrin dimers and nanosponges have an enhanced complexation rate for active ingredients that are isolated compounds or within a plant extract, with non-polar or slightly polar properties including, but are not limited to, epicatechin, quercetin, terpenophenolics, phytocannabinoids, polyphenols, polyols, and phenolics. Cyclodextrin dimer complexes also give enhanced watersolubility properties compared to non-complexed non-polar compounds and single cyclodextrin inclusion complexes.
  • cyclodextrin inclusion complex will theoretically ensure the encapsulant oil phase will bypass the liver due to small particle size being on the sub-nano level (defined as below 10 nm in diameter).
  • the use of cyclodextrins is optional.
  • the formed complexes are then mixed with a homogenized emulsification mixture that contains more solid mass of emulsification reagents to the total mass of the cyclodextrin inclusion complexes.
  • a homogenized emulsification mixture that contains more solid mass of emulsification reagents to the total mass of the cyclodextrin inclusion complexes.
  • the shell : core mass ratio should be greater than 1, or the percent shell mass composition should be greater than 50% (Equation 2).
  • Percent shell mass composition (Total mass of polymeric shell / Total mass of polymeric shell and oil phase) X 100
  • the encapsulants are able to withstand elevated heat conditions (235 °C) for an extended period of time (60 min) with minimal volatilization, thermal degradation, or redox isomerization of compounds.
  • the mixture is homogenized at high shear speeds (8,000 + RPM) or ultrasonicated at a 20kHz frequency to achieve nano-sized emulsions or capsules. Once the mixture is homogenized it undergoes complex coacervation followed by crosslinking if employing two or more polymeric reagents or spray/freeze dried to form nano- or micro-capsules around the cyclodextrin dimer inclusion complexes.
  • Crosslinking of the nano- or micro-capsules may be employed, but no crosslinking results in a capsule that will release the core (cyclodextrin inclusion complexes) in the buccal cavities and stomach via enzymatic release and swelling caused by low pH of stomach acid.
  • Crosslinking of the nano- or micro-capsules occurs when a crosslinking agent, such as transglutaminase or tannic acid, is used to form isopeptide bonds between two amino acids or hydrogen bonding between the proteins and tannic acid.
  • a crosslinking agent such as transglutaminase or tannic acid
  • Non-crosslinked nano- or micro- capsules swell and release under acidic conditions over time.
  • crosslinked formulations swell and release their cargo under alkaline conditions; such as that of the large intestine where pH conditions go beyond 7.1. Swelling causes release of encapsulants at a rate that's directly linked to capsule wall thickness.
  • Encapsulation of the oil phase can omit the synthesized cyclodextrin supramolecular structures.
  • This formulation is ideal for a reduced cost of goods in consumer manufacturing. This is performed by formulating an emulsification mixture that contains more solid mass of emulsification reagents to the total mass of the oil phase by following Equation 1.
  • the emulsification mixture is homogenized at high shear speeds (8,000 + RPM) or ultrasonicated at a 20kHz frequency to achieve nano-sized emulsions or capsules.
  • Crosslinking of the nano- or micro-capsules may be employed, but no crosslinking results in a capsule that will release the oil phase core under acidic conditions or via enzymatic release.
  • crosslinked formulations swell and release their cargo under alkaline conditions; such as that of the large intestine where pH conditions go beyond 7.1. Swelling causes release of encapsulants at a rate that's directly linked to capsule wall thickness.
  • Example 1 Cyclodextrin Dimer Formation: Synthesis of bis (P-cyclodextrin)s with tartaric acid as the linker molecule.
  • Figure 8 depicts the structure with one linker.
  • Figure 9 depicts the structure with more than one linker molecule.
  • a solution of P-cyclodextrins and tartaric acid at a molar ratio of 1 :3.11 was prepared. The solution was gently heated to 50°C and stirred at 1000 RPM to fully dissolve. Hydrochloric acid was added as an acid catalyst to induce Fisher esterification at the hydroxyl moi eties on P-cyclodextrins. Maintain stirring and heat solution to 100°C to remove water from the system and push reaction to completion. Adding an excess of tartaric acid can push the reaction towards formation of nanosponge matrices. Where a nanosponge is defined as a polymer containing more than two cyclodextrin molecules; crosslinked in a matrix fashion. After several hours take the reaction and isolate formed bis(P-cyclodextrin)s and nanosponges by freeze drying, spray drying, or rotoevaporation.
  • Example 2 Cyclodextrin Dimer Inclusion Complexation: Inclusion of Cannabis sativa L. extract in bis (P-cyclodextrin)s.
  • Example 3 Gelatin Formulation: Micro(Nano)encapsulation of Cannabis sativa L. extract.
  • a surfactant stabilizer like tween 20 can be used with high shear RPM over 20,000 or an ultrasonic barbell probe at a 20kHz frequency.
  • a surfactant stabilizer like tween 20 can be used with high shear RPM over 20,000 or an ultrasonic barbell probe at a 20kHz frequency.
  • While homogenizing add MCT mixture dropwise or slowly. Stop homogenization after adding total volume of MCT mixture and maintain gentle stirring. Slowly add acacia gum solution to the homogenized mixture. Maintain gentle stirring and gentle heating. Dilute with water at a volume that's equivalent to the volume of the added acacia gum solution. Adjust pH to 4 using a food grade acid, such as acetic acid. The pH change will induce electrostatic attraction between acacia gum and gelatin molecules. Coacervation will begin and a soft gel will begin to grow as temperature is reduced.
  • the total mass of gelatin and acacia gum to oil phase mass was 1.2 (reference Equation 1).
  • the total % shell composition in this example was 54% as defined by Equation 2.
  • Example 4 Inulin Formulation: Micro(Nano)encapsulation of Cannabis sativa L. extract.
  • HPLC samples of inulin formulated micro(nano)capsules are prepared by swelling the capsules in an acid medium with pH 2 and ultrasonicating or agitating for 5 minutes. Followinged by vacuum fdtration, concentrating, and re-suspending in methanol for HPLC analysis.
  • Gelatin-acaia gum micro(nano)capsules are digested with sodium hydroxide (0.1 M) and ethanol. With the aid of an ultrasonic bath at 50°C, the active ingredients are released from the micro(nano)capsules.
  • the solution is then vacuum fdtered through a 0.45 micron membrane fdter and concentrated via rotary evaporation. The residue is dissolved in methanol and prepared for HPLC analysis.
  • Encapsulated Cannabis sativa L. extract was obtained following the inulin formulation explained in Example 4 where the sheikcore ratio was 0.5 and the use of cyclodextrins was excluded. Samples of this powder were subjected to heating experiments followed by HPLC for qualitative and quantitative analysis.
  • Samples were made following the above examples. A set of samples was made following the inulin formulation to microencapsulate Cannabis sativa L. extract oil. The shelhcore ratio was 0.5 and the THC concentration was determined to be 11.3 mg/g via HPLC analysis. Samples of this powder were heated at 176.67°C for 30 minutes and another sample heated at 204.44°C for 30 minutes. The samples were then analyzed by HPLC to quantitate THC and determine the presence of degradants. The HPLC chromatograms are displayed in Figures 2-4. These chromatograms show that encapsulation vehicles with shell :core mass ratios of 0.5 produce detectable amounts of degradants. In these examples the degradant of THC is CBN.
  • the active ingredients or compounds of interest that can be encapsulated following the methods described herein include but are not limited to; epicatechin , quercetin, terpenophenolics, phytocannabinoids, polyphenols, polyols, phenolics, and any compound that has slightly non-polar properties, can be classified as a nutraceutical or pharmaceutical, and doesn’t exceed 2 nm in size dimensions. Active ingredients exceeding 2 nm in size dimensions can be complexed inside nanosponges and thermally stabilized in micro(nano)capsules.

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Abstract

A method for encapsulating non-polar compounds, such as steroids, terpenoids, and phytocannabinoids, at the micro, nano, and sub-nano scales. The invention involves a guest-host molecule formed of cyclodextrins crosslinked to form a dimerized, bridged, or polymerized encapsulating component. Crosslinking may be achieved using diacid linker molecules like succinic acid and tartaric acid. The resulting supramolecular encapsulating component exhibits improved complexation properties for a plurality of molecules of an ingestible item including, but not limited to, classes of polyphenols, terpenophenolics, phytocannabinoids, steroids, and flavonoids. These guest-host complexes may optionally be further enclosed within micro- and nano-capsules to enhance and prolong thermal stability. The stabilized active ingredients can be processed into a dry powder, which is highly suitable as a functional ingredient in pharmaceuticals, processed foods, culinary ingredients, and beverages.

Description

A method to encapsulate non-polar compounds to achieve extended thermal stability and attenuate/prevent thermal and redox isomerization
BACKGROUND OF THE INVENTION
1. Field of the Invention.
[0001] The present invention relates to compositions and methods for chemical entrapment of plant-based nutraceuticals and extracts for the fortification of food and beverage products. More particularly, the method provides for the encapsulation of polyphenols, terpenophenolics, and flavonoids for pre- and post-biotic systems. The compositions and methods may be used to form nutraceutical supplements, pet feed, cannabis and hemp edibles, processed foods and beverages, and pharmaceuticals.
2. Description of the Prior Art.
[0002] Microcapsules are polymeric layers that encapsulate active ingredients to mask flavors and smells, and to provide protection from heat, oxidation, and ultraviolet light. In essence a microcapsule consists of a polymeric outer shell and an oil phase core containing an active ingredient or compound of interest. Depending on the polymeric shell composition and degree of intermolecular and intramolecular bonds, shell properties determine its stability and release mechanism. Microcapsules are employed by many industries for a variety of purposes.
[0003] In the pharmaceutical industry, microcapsules are often designed to target specific organs for controlled drug release. Polymeric shells consisting of crosslinked polysaccharides and proteins are known in literature to exhibit acidic stability and swell under alkaline conditions. These crosslinked microcapsules can be composed of the following polysaccharides and proteins: acacia gum, cellulose, chitosan, and pectin, and gelatin, soy proteins, whey proteins, and wheat proteins. Though composition is not limited to the listed ingredients, crosslinking of the proteins and polysaccharides often needs glutamine and lysine moieties to form isopeptide bonds via transglutaminase. Tannic acid, a natural polyphenol and antioxidant, is also capable of crosslinking microcapsules that are composed of proteins. Tannic acid can crosslink amine groups via Michael addition or Schiff base reaction, thiol groups via Michael addition or thiyl radical oxidation, and carboxyl groups via acylation. [0004] Crosslinked polysaccharides-protein microcapsules are prepared following complex coacervation techniques. This technique is described as an electrostatic attraction of two or more biopolymers with opposite charges (e.g., proteins and polysaccharides) under varying pH conditions to induce changes in charges. Methods on microcapsule formation often employ high shear emulsification followed by addition of an oil phase containing the active ingredient.
[0005] In consumer markets, microcapsules are readily used where a volatile compound of interest needs to be stabilized in a matrix. This includes but is not limited to encapsulation of flavors, fragrances, and nutrients. Microcapsules, however, are not ideal for bypassing first-pass metabolism effects as the size is generally larger than 500 nm in diameter.
[0006] Crosslinked gelatin acacia gum microcapsules are used in consumer products and have been used to deliver pre-biotics or good gut bacteria to the large intestine. The crosslinking of the gelatin based microcapsules gives it acid stability or stability in the stomach and gastrointestinal tract. The swelling or release of the microcapsule occurs at pH above 7, which is the condition of the large intestine.
[0007] Microcapsules described in published US patent application Publication No. 2017/021616 contain near fully hydrolyzed polyvinyl alcohols to better retain aromatic compounds and withstand the mechanical stress induced by washing machines. This patent also makes the claim that scents or flavors can be released upon baking and chewing processes. The focus here was to obtain capsules with a wide fragrance release profile that releases under mechanical stress. The ratio of capsule shell mass to core mass was described to be below 1. Where sheikcore mass ratio is defined by Equation 1.
Equation 1. Sheikcore = (total mass polymeric shell / mass oil phase core). This characteristic gives rise to a capsule that does not exhibit extended thermal stability under traditional cooking / baking conditions. Where traditional cooking / baking temperatures are between 175 °C to 260 °C for 30 minutes to 60 minutes.
[0008] Increasing the amount of shell composition will increase the thermal stability of a microcapsule. Shell composition also plays a role. A method described in published PCT patent application Publication No. W02008085997 utilizes the advantage of multi-nuclear microcapsules. Where a multi-nuclear microcapsule is described to be a microcapsule containing smaller capsules or having internal chambers. Multi-nuclear constructs increase the ratio of shell to core but hinder the ability to effectively reduce particle size and hence bypass first-pass metabolism effects.
[0009] Ideally, encapsulation vehicles for pharmaceutical or nutraceutical compounds should be able to bypass first-pass metabolism routes. Some medical and therapeutic compounds are metabolized in the liver. This results in reduced transport of active and desired compounds to the bloodstream. Therefore, to effectively control the delivery of oral based pharmaceuticals or nutraceuticals, sub-nano drug carriers that are released from fast digestible microcapsules are employed. The microcapsules should be formulated to be enzymatically released at the start of digestion (via buccal cavity).
[0010] Published PCT patent application Publication No. W02008085997 describes a method for generating nanoparticles or nanoemulsions suspended in microcapsules. This method is ideal for overcoming the issue with first-pass metabolism; however, nanoemulsions are not stable under elevated temperatures (>200°C) and often are formulated using surfactants. Thus, to avoid the use of surfactants; the method described herein focuses on glucopyranose derived guest-host molecules or the class of cyclodextrins. The guest-host molecules are then further encapsulated in a polymeric shell where the shell :core mass ratio is greater than 1; to achieve extended thermal stability.
[0011] The need for this invention derives from the fact that phytocannabinoids, especially delta-9-tetrahydrocannabinol and cannabidiol, are subject to isomerization via reduction and oxidation reactions under room temperature and elevated heat temperatures (above 175°C). This poses issues to both the growing hemp and cannabis industries and an ultimate need to standardize how the extracts of the plants are processed and stored. Moreover, there is a desire to add volatile polyphenols to food products and culinary ingredients more broadly to stimulate good gut bacteria activity. Therefore, what is needed is a method and related compositions that encapsulate ingestible items of interest to ensure elevated processing temperatures do not degrade the encapsulating component in a way that adversely impacts the encapsulant.
SUMMARY OF THE INVENTION.
[0012] The present invention provides a method and related compositions that encapsulate ingestible items of interest in a way that encapsulating material integrity is maintained through processes of interest that include elevated temperatures that would otherwise degrade the encapsulant.
[0013] The present invention employs a guest-host molecule derived from the class of cyclodextrins that is synthesized to enhance the complexation with, but not limited to; phytocannabinoids, steroids, flavonoids, polyphenols, and terpenophenolics. This is accomplished by dimerization and bridging of cyclic glucopyranose or cyclodextrins and all its derivatives, such as hydroxy-beta-cyclodextrin, into bis or nanosponge structures, occurs at its free -OH groups in the presence of dicarboxylic acids, like tartaric acid, following a Fischer esterification reaction. Dicarboxylic acids suitable for this reaction include, but are not limited to, tartaric acid. Excess amounts of dicarboxylic acids will push the reactions towards the formation of nanosponge or a polymer of cyclodextrins where bridging can occur at other -OH groups.
[0014] The cyclodextrin reaction solutions are dried under vacuum freeze-drying conditions, rotoevaporation, or spray dried. The resulting solid powder is then kneaded with the non-polar compounds of interest to form inclusion complexes.
[0015] The formed inclusion complexes are then mixed and emulsified with saccharides, oligosaccharides, polysaccharides, denatured proteins, or a formulated mixture to form micro- or nano-capsules around the inclusion complexes. Examples include, but not limited to; dextrins, arabic gum, inulin, hemicelluloses, etc. The formulated emulsification mixture contains an equivalent or greater mass to cyclodextrin inclusion complexes. In other words, the formulated microcapsule mixture should have a shell to core ratio that is equal or greater than 1. Where shell composition is composed of the emulsification mixture and the core is the cyclodextrin inclusion complexes. The emulsification mixture is combined with the cyclodextrin inclusion complexes and is spray dried or freeze dried to form micro(nano)capsules around the cyclodextrin inclusion complexes.
[0016] Alternatively, cyclodextrin inclusion complexes can be omitted. In this case, the oil phase is homogenized with the micro(nano)capsule formulation, and then spray dried or freeze dried to obtain a dry powder. Again, the micro(nano)capsule formulation follows a shell to core mass ratio of 1 or greater to achieve extended thermal stability.
[0017] Examples of this process is employed to encapsulate delta-9- tetrahydrocannabinol, cannabinol and other terpenoids and phytocannabinoids present in the extracts of Cannabis saliva L. and its close relatives into a thermally stable, tasteless, and odorless powder. Though not limited to C. sativa L compounds; this method can be used to encapsulate other bioactive compounds of interest that are non-polar or slightly non-polar. This includes flavonoid polyphenols, like qucerterin and epicatechin from Theobroma cacao extracts.
[0018] The invention is a method for encapsulating an ingestible agent in an encapsulating component. The method includes the steps of crosslinking together a plurality of guest-host molecules, wherein the guest-host molecule is a cyclic glucopyranose or a cyclodextrin containing free hydroxyl groups, to link together the plurality of guest-host molecules to form the encapsulating component, and complexating a plurality of molecules of the ingestible agent in the encapsulating component to form a complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent. The step of crosslinking involves using a linker molecule to form ester bonds between the plurality of guesthost molecules through esterification. The esterification may be a Fischer esterification and the linker molecule may be a diacid. The diacid may be a dicarboxylic acid having at least two reactive carboxyl groups. The dicarboxylic acid may be tartaric acid. The cyclodextrin may be a hydroxypropyl-beta-cyclodextrin, a beta-cyclodextrin, or other cyclodextrin derivative with a free hydroxyl group. When two guest-host molecules are crosslinked together, the encapsulating component is a dimer structure. When three or more guest-host molecules are crosslinked together the encapsulating component is a bridged or polymerized structure. When more than three guest-host molecules are crosslinked together the encapsulating component is a nanosponge. For the nanosponge formation, an excess of di carboxylic acid is used in the crosslinking step to form the encapsulating component as the nanosponge. The ingestible item is selected from the classes comprising phytocannabinoids, steroids, flavonoids, polyphenols, and terpenophenolics. The method may further include the step of encapsulating the complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent in a polymeric shell with the combination forming a core substantially within the polymeric shell to enhance thermal stability of the core. The polymeric shell is formed of either or both of proteins and carbohydrates. The polymeric shell may be formed by crosslinking using either transglutaminase enzyme or tannic acid. A total mass of the polymeric shell is greater than a total mass of the core. BRIEF DESCRIPTION OF THE DRAWINGS.
[0019] FIG. 1 is a fluorescent microscope image of gelatin-acacia gum microcapsules. Average size of a microcapsule is 1 micron in diameter.
[0020] FIG. 2 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core ratio of 0.5. The pre-heated sample contained a mixture of cannabinoids.
[0021] FIG. 3 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core mass ratio of 0.5. Sample was baked at 176.67°C for 30 minutes. Sample contained a mixture of cannabinoids.
[0022] FIG. 4 is a HPLC chromatogram on inulin formulation following Example 4 to microencapsulate Cannabis sativa L. extracts at a shell: core ratio of 0.5. Sample was baked at 204.44°C for 30 minutes. Sample contained a mixture of cannabinoids.
[0023] FIG. 5 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Pre-heated sample. Sample contained only THC.
[0024] FIG. 6 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Sample was baked at 176.67°C for 30 minutes. Sample contained only THC.
[0025] FIG. 7 is a HPLC chromatogram on a crosslinked gelatin-acacia gum formulation following Example 3 to microencapsulate Cannabis sativa L. extracts at a sheikcore ratio of 1.2. Sample was baked at 176.67°C for 90 minutes. Sample contained only THC.
[0026] FIG. 8 is a reaction mechanism to form a bis(X-cyclodextrin)s where there is only one linker molecule bridging two cyclodextrins. Where X is either a, P, or y and directly relates to n, where n is 6, 7, or 8 , respectively. R defines the cyclodextrin to be either in its parent form or a 2-hydroxypropyl form, most commonly found with P-cyclodextrins.
[0027] FIG. 9 is a representation of a result of bis(X-cyclodextrin)s where there are more than one linker molecules bridging two cyclodextrins. Where X is either a, , or y. The structure should have more structural rigidity compared to being linked by one linker molecule. The linker molecule is tartaric acid. [0028] FIG 10 is a depiction of a cyclodextrin polymer or nanosponge. Crosslinking is occurring at the 6’ -OH and other free -OH groups along the cyclic glucopyranose structure. The linker molecule is tartaric acid.
[0029] FIG. 11 is a depiction of supramolecular cyclodextrin complexes encapsulated in polymeric shells. The polymeric shell on the left depicts a micro-sized capsule at around 1 micron in diameter. The polymeric shell on the right depicts a nano-sized capsule that is below 500 nm.
DETAILED DESCRIPTION OF INVENTION.
[0030] The present invention is described herein with respect to particular method steps and resultant compositions that provide for the effective encapsulation of materials of interest capable of being processed at elevated temperatures.
[0031] Cyclodextrin Dimer and Nanosponge Synthesis.
[0032] Cyclodextrins are mixed with dicarboxylic acids at a molar ratio where the number of free 6’ hydroxyls on the cyclic glucopyranose molecules is 2.25: 1 or greater. For example; |3-cyclodextrin contains seven 6’ hydroxyl groups, so for every mol of P-cyclodextrin there is 3.11 mols of tartaric acid in the system. Tartaric acid in the presence of strong acids will crosslink cyclodextrins into a bis(cyclodextrin) form where tartaric acid is used as the linker molecule. An excess of dicarboxylic acids pushes beyond the formation of bis(cyclodextrin)s to form nanosponges. Where nanosponges are a polymer or crosslinked matrix of more than two cyclodextrin molecules. Crosslinking can also occur at other free hydroxyl groups on the glucopyranose subunits besides the 6’ carbon.
[0033] The reaction mixture is stirred at 1000 RPM and heated to remove water formed by the formation of esters. The reaction is pushed to completion by removal or boiling of water. Once the reaction is complete, the cyclodextrin dimers are isolated by means of drying such as spray drying, freeze drying, or rotoevaporation.
[0034] Cyclodextrin Inclusion Complexation.
[0035] The dried cyclodextrin dimer or polymer nanosponge powder is kneaded with the non-polar, slightly-nonpolar compounds or active ingredients to form inclusion complexes. Cyclodextrin dimers and nanosponges have an enhanced complexation rate for active ingredients that are isolated compounds or within a plant extract, with non-polar or slightly polar properties including, but are not limited to, epicatechin, quercetin, terpenophenolics, phytocannabinoids, polyphenols, polyols, and phenolics. Cyclodextrin dimer complexes also give enhanced watersolubility properties compared to non-complexed non-polar compounds and single cyclodextrin inclusion complexes.
This is done by adding small amounts of water to the dried cyclodextrin dimer powder to form a paste. The paste is then kneaded with the oil phase or non-polar compounds.
[0036] Micro- and Nano-encapsulation of the Cyclodextrin Inclusion Complex.
[0037] The use of cyclodextrin inclusion complex will theoretically ensure the encapsulant oil phase will bypass the liver due to small particle size being on the sub-nano level (defined as below 10 nm in diameter). The use of cyclodextrins is optional.
[0038] The formed complexes are then mixed with a homogenized emulsification mixture that contains more solid mass of emulsification reagents to the total mass of the cyclodextrin inclusion complexes. To achieve extended thermal stability the shell : core mass ratio should be greater than 1, or the percent shell mass composition should be greater than 50% (Equation 2).
Equation 2: Percent shell mass composition = (Total mass of polymeric shell / Total mass of polymeric shell and oil phase) X 100
The encapsulants are able to withstand elevated heat conditions (235 °C) for an extended period of time (60 min) with minimal volatilization, thermal degradation, or redox isomerization of compounds.
[0039] The mixture is homogenized at high shear speeds (8,000 + RPM) or ultrasonicated at a 20kHz frequency to achieve nano-sized emulsions or capsules. Once the mixture is homogenized it undergoes complex coacervation followed by crosslinking if employing two or more polymeric reagents or spray/freeze dried to form nano- or micro-capsules around the cyclodextrin dimer inclusion complexes.
[0040] Crosslinking of the nano- or micro-capsules may be employed, but no crosslinking results in a capsule that will release the core (cyclodextrin inclusion complexes) in the buccal cavities and stomach via enzymatic release and swelling caused by low pH of stomach acid. Crosslinking of the nano- or micro-capsules occurs when a crosslinking agent, such as transglutaminase or tannic acid, is used to form isopeptide bonds between two amino acids or hydrogen bonding between the proteins and tannic acid. Non-crosslinked nano- or micro- capsules swell and release under acidic conditions over time. Whereas crosslinked formulations swell and release their cargo under alkaline conditions; such as that of the large intestine where pH conditions go beyond 7.1. Swelling causes release of encapsulants at a rate that's directly linked to capsule wall thickness.
[0041] Micro- and Nano-encapsulation without Cyclodextrin Supramolecular Structures.
[0042] Encapsulation of the oil phase can omit the synthesized cyclodextrin supramolecular structures. This formulation is ideal for a reduced cost of goods in consumer manufacturing. This is performed by formulating an emulsification mixture that contains more solid mass of emulsification reagents to the total mass of the oil phase by following Equation 1.
[0043] The emulsification mixture is homogenized at high shear speeds (8,000 + RPM) or ultrasonicated at a 20kHz frequency to achieve nano-sized emulsions or capsules. Once the mixture is homogenized it undergoes complex coacervation followed by crosslinking if employing two or more polymeric reagents or spray / freeze dried to form nano- or microcapsules. Crosslinking of the nano- or micro-capsules may be employed, but no crosslinking results in a capsule that will release the oil phase core under acidic conditions or via enzymatic release. Whereas crosslinked formulations swell and release their cargo under alkaline conditions; such as that of the large intestine where pH conditions go beyond 7.1. Swelling causes release of encapsulants at a rate that's directly linked to capsule wall thickness.
[0044] METHODOLOGY.
[0045] Example 1. Cyclodextrin Dimer Formation: Synthesis of bis (P-cyclodextrin)s with tartaric acid as the linker molecule. Figure 8 depicts the structure with one linker. Figure 9 depicts the structure with more than one linker molecule.
[0046] A solution of P-cyclodextrins and tartaric acid at a molar ratio of 1 :3.11 was prepared. The solution was gently heated to 50°C and stirred at 1000 RPM to fully dissolve. Hydrochloric acid was added as an acid catalyst to induce Fisher esterification at the hydroxyl moi eties on P-cyclodextrins. Maintain stirring and heat solution to 100°C to remove water from the system and push reaction to completion. Adding an excess of tartaric acid can push the reaction towards formation of nanosponge matrices. Where a nanosponge is defined as a polymer containing more than two cyclodextrin molecules; crosslinked in a matrix fashion. After several hours take the reaction and isolate formed bis(P-cyclodextrin)s and nanosponges by freeze drying, spray drying, or rotoevaporation.
[0047] Example 2. Cyclodextrin Dimer Inclusion Complexation: Inclusion of Cannabis sativa L. extract in bis (P-cyclodextrin)s.
[0048] Take the formed bis (P-cyclodextrin)s dry powder from Example 1 and mix with small amounts of water to make a paste. Knead the paste with Cannabis sativa L. extract at a molar ratio of 1 : 1. Assume Cannabis sativa L. extract is all delta-9-tetrahydrocannabinol.
[0049] Example 3. Gelatin Formulation: Micro(Nano)encapsulation of Cannabis sativa L. extract.
[0050] Prepare a 60 mL 8.33% gelatin solution in water and gently heat to dissolve. Prepare a separate 60 mL solution of 11% acacia gum in water and gently heat to dissolve. Mix Cannabis sativa L. extract with medium chain triglyceride oil (MCT) to decrease viscosity. Total amount of MCT mixture added was 9.6g. Alternatively use a bis (P-cyclodextrin)s inclusion complex of Cannabis sativa L. extract as explained in Examples 1 and 2. Homogenize gelatin solution at 8,000 RPM under gentle heat. To achieve nano-sized capsules (< 500 nm); a surfactant stabilizer like tween 20 can be used with high shear RPM over 20,000 or an ultrasonic barbell probe at a 20kHz frequency. While homogenizing, add MCT mixture dropwise or slowly. Stop homogenization after adding total volume of MCT mixture and maintain gentle stirring. Slowly add acacia gum solution to the homogenized mixture. Maintain gentle stirring and gentle heating. Dilute with water at a volume that's equivalent to the volume of the added acacia gum solution. Adjust pH to 4 using a food grade acid, such as acetic acid. The pH change will induce electrostatic attraction between acacia gum and gelatin molecules. Coacervation will begin and a soft gel will begin to grow as temperature is reduced. The temperature was reduced 1°C every minute to reach 4°C. Maintain gentle mixing throughout the process. Allow coacervation to continue for many several hours under cold temperature (4°C) while maintaining gentle stirring. Allow the micro(nano)capsule solution to reach room temperature. Slowly adjust pH to 6 with a food grade base, such as 10% sodium hydroxide. Add transglutaminase to crosslink the microcapsule solution. Let the solution stand for many several hours for crosslinking. Use a spray dryer or freeze dryer to powderize the micro(n no)capsule slurry. Figure 10 depicts a depiction of a cyclodextrin polymer or nanosponge. Figure 11 depicts supramolecular cyclodextrin complexes encapsulated in polymeric shells.
[0051] The total mass of gelatin and acacia gum to oil phase mass was 1.2 (reference Equation 1). The total % shell composition in this example was 54% as defined by Equation 2.
[0052] Example 4. Inulin Formulation: Micro(Nano)encapsulation of Cannabis sativa L. extract.
[0053] Prepare a solution of inulin that contains a greater mass than the bis (0- cyclodextrin)s inclusion complex of Cannabis sativa L. extract. Follow Equation 1 and Equation 2 where the ratio is greater than 1 and shell mass composition is greater than 50% to achieve extended thermal stability. Alternatively, bis (0-cyclodextrin)s can be omitted and 0- cyclodextrins can be used. Homogenize the solution at high shear 8,000+ RPM for 6-7 minutes. To achieve nanocapsules or nanoemulsions add tween 20 as a stabilizer and perform homogenization at 20,000+ RPM or ultrasonicate at 20kHz. Powderize the homogenized slurry via drying methods such as freeze drying and spray drying.
[0054] Qualitative and Quantitative Analysis via high-pressure liquid chromatography (HPLC).
[0055] HPLC samples of inulin formulated micro(nano)capsules are prepared by swelling the capsules in an acid medium with pH 2 and ultrasonicating or agitating for 5 minutes. Followed by vacuum fdtration, concentrating, and re-suspending in methanol for HPLC analysis. Gelatin-acaia gum micro(nano)capsules are digested with sodium hydroxide (0.1 M) and ethanol. With the aid of an ultrasonic bath at 50°C, the active ingredients are released from the micro(nano)capsules. The solution is then vacuum fdtered through a 0.45 micron membrane fdter and concentrated via rotary evaporation. The residue is dissolved in methanol and prepared for HPLC analysis.
[0056] Extended Thermal Stability Study.
[0057] Encapsulated Cannabis sativa L. extract was obtained following the inulin formulation explained in Example 4 where the sheikcore ratio was 0.5 and the use of cyclodextrins was excluded. Samples of this powder were subjected to heating experiments followed by HPLC for qualitative and quantitative analysis.
[0058] Samples were made following the above examples. A set of samples was made following the inulin formulation to microencapsulate Cannabis sativa L. extract oil. The shelhcore ratio was 0.5 and the THC concentration was determined to be 11.3 mg/g via HPLC analysis. Samples of this powder were heated at 176.67°C for 30 minutes and another sample heated at 204.44°C for 30 minutes. The samples were then analyzed by HPLC to quantitate THC and determine the presence of degradants. The HPLC chromatograms are displayed in Figures 2-4. These chromatograms show that encapsulation vehicles with shell :core mass ratios of 0.5 produce detectable amounts of degradants. In these examples the degradant of THC is CBN.
[0059] Another sample was prepared following Example 4 where the shelhcore mass ratio was 1.2 and the use of cyclodextrins are excluded. These samples were subjected to a heating experiment at 176.67°C for 0, 30, and 90 minutes (Figures 5-7). Figure 1 depicts this microencapsulation. The HPLC analysis on these samples showed that the gelatin formulation with a shell: core mass ratio greater than 1 prevented the thermal degradation and isomerization of THC. Quantitative analysis also showed no declining trend in THC concentration over time during the heating experiment.
[0060] Active Ingredients.
[0061] The active ingredients or compounds of interest that can be encapsulated following the methods described herein include but are not limited to; epicatechin , quercetin, terpenophenolics, phytocannabinoids, polyphenols, polyols, phenolics, and any compound that has slightly non-polar properties, can be classified as a nutraceutical or pharmaceutical, and doesn’t exceed 2 nm in size dimensions. Active ingredients exceeding 2 nm in size dimensions can be complexed inside nanosponges and thermally stabilized in micro(nano)capsules.
[0062] While the present invention has been described with reference to certain specific examples, it is not intended to be limited thereto. Instead, the present invention is defined by the following claims and reasonable equivalents.

Claims

WHAT TS CLAIMED IS:
1. A method for encapsulating an ingestible agent in an encapsulating component, the method comprising the steps of: crosslinking together a plurality of guest-host molecules, wherein the guest-host molecule is a cyclic glucopyranose or a cyclodextrin containing free hydroxyl groups, to link together the plurality of guest-host molecules to form the encapsulating component; and complexating a plurality of molecules of the ingestible agent in the encapsulating component to form a complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent.
2. The method of Claim 1, wherein the step of crosslinking involves using a linker molecule to form ester bonds between the plurality of guest-host molecules through esterification.
3. The method of Claim 2, wherein the esterification is Fischer esterification and the linker molecule is a diacid.
4. The method of Claim 3, wherein the diacid is a dicarboxylic acid having at least two reactive carboxyl groups.
5. The method of Claim 4, wherein the dicarboxylic acid is tartaric acid.
6. The method of Claim 1, wherein the cyclodextrin is a hydroxypropyl-beta-cyclodextrin, a beta-cyclodextrin, or other cyclodextrin derivative with a free hydroxyl group.
7. The method of Claim 1, wherein two guest-host molecules are crosslinked together to form the encapsulating component is a dimer structure.
8. The method of Claim 1, wherein three or more guest-host molecules are crosslinked together to form the encapsulating component as a bridged or polymerized structure.
9. The method of Claim 8, wherein more than three guest-host molecules are crosslinked together to form the encapsulating component as a nanosponge.
10. The method of Claim 9, wherein an excess of dicarboxylic acid is used in the crosslinking step to form the encapsulating component as the nanosponge.
11. The method of Claim 1, wherein the ingestible item is selected from the classes comprising phytocannabinoids, steroids, flavonoids, polyphenols, and terpenophenolics.
12. The method of Claim 1, further comprising the step of encapsulating the complexated combination of the encapsulating component and the plurality of molecules of the ingestible agent in a polymeric shell with the combination forming a core substantially within the polymeric shell to enhance thermal stability of the core.
13. The method of Claim 12, wherein the polymeric shell is formed of either or both of proteins and carbohydrates.
14. The method of Claim 13, wherein the polymeric shell is formed by crosslinking using either transglutaminase enzyme or tannic acid.
15. The method of Claim 12, wherein a total mass of the polymeric shell is greater than a total mass of the core.
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Citations (2)

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