Use of curcuminoid composition

Abstract

The present invention relates to methods and uses of compositions comprising curcumin; in particular, methods and uses relating to improving the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals.

Description

Technical Field

[0001] Technical Field The present invention relates to methods and uses of compositions comprising curcumin; in particular, to methods and uses relating to improving the bioaccessibility, bioavailability, biological effectiveness and / or biological activity of curcuminoids in mammals. Background Art

[0002] Background Art The listing or discussion of an apparently previously published document in this specification should not necessarily be construed as an admission that the document is part of the state of the art or is common general knowledge.

[0003] Turmeric, and compounds isolated from turmeric such as curcuminoids including curcumin, have long been used to treat a variety of diseases and conditions.

[0004] Curcuminoids are natural yellow-orange pigments and hydrophobic polyphenols derived from the rhizomes of the herb *Curcuma longa*. They are generally isolated from turmeric, a spice and food colouring agent.

[0005] Turmeric extract contains approximately 75-80% curcumin, 15-20% demethoxycurcumin (DMC), and 0-10% bisdemethoxycurcumin (BDMC). Curcuminoids have a unique conjugated structure, bis-α,β-unsaturated β-diketone (commonly called diferloylmethane), which exhibits keto-enol tautomerism, having a dominant keto form in acidic and neutral solutions and a stable enol form in alkaline media (see Figure 1) (Hoehle SI, Pfeiffer E, Solyom AM, Metzler M. Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver. J Agric Food Chem. 2006 Feb 8;54(3):756-64).

[0006] Curcumin is a highly pleiotropic molecule that was first shown to exhibit antimicrobial activity in 1949. Since then, this polyphenol has been shown to possess anti-inflammatory, hypoglycemic, antioxidant, wound-healing, and antimicrobial activities. Extensive preclinical and clinical research over the past 30 years has demonstrated the therapeutic potential of curcumin for a wide range of human diseases (Gupta SC, Sung B, Kim JH, Prasad S, Li S, Aggarwal BB. Multitargeting by turmeric, the golden spice: From kitchen to clinic. Mol Nutr Food Res. 2013 Sep;57(9):1510-28).

[0007] While curcumin has shown efficacy against many human disorders, it is also known to have limited bioavailability due to insufficient absorption, rapid metabolism, and rapid systemic elimination.

[0008] Low serum levels, limited tissue distribution, and rapid metabolism have been reported when curcumin is administered orally. For example, it has been demonstrated that serum levels of curcumin did not exceed low micromolar levels even after oral administration of up to 12 g. In one clinical study with 4–8 g of curcumin, the maximum serum concentration observed was 1.3 μg / mL, but several other clinical and animal studies have reported serum levels in the lower range of nanograms per milliliter (Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm. 2007 Nov-Dec;4(6):807-18).

[0009] Curcumin has been found to be poorly soluble in water. For example, the maximum solubility of curcumin in aqueous buffer (pH 5.0) has been reported to be as low as 11 ng / ml (Tonessen et al, 2002).

[0010] Curcumin is relatively stable at acidic pH, but rapidly decomposes at pH levels above neutral to form ferulic acid and ferleumethane, the latter of which yields vanillin (FAO 2004. CURCUMIN Chemical and Technical Assessment (CTA) First draft prepared by Ivan Stankovic. Chemical and Technical Assessment 61st JECFA).

[0011] Curcumin also exhibits extremely low intestinal absorption. The apparent permeability coefficient of curcumin was found to be extremely low in an in vitro human intestinal cell model (Papp value: 0.1 × 10⁻⁶ cm / s), which, according to the correlation between Papp values ​​measured in in vitro Caco-2 cells and in vivo human absorption, could predict low (0-20%) absorption in humans (Dempe JS, Scheerle RK, Pfeiffer E, Metzler M. Metabolism and permeability of curcumin in cultured Caco-2 cells. Mol Nutr Food Res. 2013 Sep;57(9):1543-9).

[0012] Once absorbed, curcumin undergoes both Phase I and Phase II metabolism (see Figure 2). In Phase I metabolism, curcumin and its two demethoxy congeners undergo sequential reduction to their dihydro-, tetrahydro-, hexahydro-, and octahydro- metabolites in the liver and intestinal mucosa. In Phase II metabolism, both curcumin and its reducing metabolites are conjugated with glucuronic acid and sulfate to form Phase II metabolites.

[0013] Reduction and conjugation appear to be common metabolic pathways for curcuminoids in the liver and intestinal tissues of rats and humans. Therefore, some authors suggest that the biological effects induced by curcumin in tissues other than the gastrointestinal tract are likely due to curcumin metabolites.

[0014] Curcumin has also been shown to be metabolized by intestinal microorganisms (see Figure 3). It has been found that the microbial metabolism of curcumin involves a two-step reduction process, in which curcumin is successively converted to dihydrocurcumin by NADPH-dependent curcumin / dihydrocurcumin reductase (CurA), and then to tetrahydrocurcumin (Hassaninasab A, Hashimoto Y, Tomita-Yokotani K, Kobayashi M. Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc Natl Acad Sci US A. 2011 Apr 19;108(16):6615-20).

[0015] Recent studies have reported that tetrahydroxycurcumin, rather than curcumin, can accumulate in rat tissues, suggesting that the microbiome can also be considered a potential actor in curcumin metabolism and bioavailability (Neyrinck AM, Alligier M, Memvanga PB, Nevraumont E, Larondelle Y, Preat V, Cani PD, Delzenne NM. Curcuma longa extract associated with white pepper lessens high fat diet-induced inflammation in subcutaneous adipose tissue. PLoS One. 2013 Nov 19;8(11):e81252).

[0016] Rapid metabolism, insufficient water solubility, instability at neutral pH, instability upon exposure to light and / or oxygen, and poor tissue uptake significantly limit the potential utility of curcuminoids, including curcumin, in the treatment of conditions such as cancer.

[0017] Due to its poor water solubility and absorption properties, organic solvents such as methyl sulfoxide (DMSO) are typically used to dissolve curcumin. However, while the use of such solvents helps to solubilize curcumin and improve its availability, the use of organic solvents as vehicles is controversial and undesirable, especially in an era of increasing consumer demand for natural ingredients.

[0018] Another possibility for aiding curcumin's water solubility is through emulsification. However, natural organic-grade emulsifiers are rare, and both synthetic emulsifiers such as polysorbate 80 and natural emulsifiers such as starch-based emulsifiers typically only allow for low levels (<7%) of curcumin to disperse in water.

[0019] Other strategies explored to address the aforementioned shortcomings and improve the therapeutic efficacy of curcumin include the implantation of curcumin into polymer nanoparticles, polymer micelles, and hydrophilic polymers.

[0020] The present invention aims to address the aforementioned problems associated with curcuminoids, such as the insufficient solubility of curcumin in water, and to improve the bioaccessibility, bioavailability, bioefficacy, and / or bioactivity of curcuminoids in mammals, by providing the following methods and uses. [Overview of the project]

[0021] Disclosure of the invention The inventors have surprisingly found that the bioaccessibility, bioavailability, bioefficacy, and / or bioactivity of curcuminoids in mammals can be improved by administering the curcuminoids in the form of a composition comprising (i) curcuminoids, (ii) quillaja, and (iii) modified starch and / or gum arabic.

[0022] Method and Use The present invention provides a method for improving the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals, the method comprising administering the curcuminoids in the form of a composition comprising (i) curcuminoids, (ii) an extract obtained or obtainable from quillaja, and (iii) modified starch and / or gum arabic. In particular, the present invention provides a method for improving the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals, the method comprising administering the curcuminoids in the form of a composition comprising (i) curcuminoids, (ii) an extract obtained or obtainable from quillaja, and (iii) modified starch or gum arabic. Such a method may hereafter be referred to as the "method of the present invention."

[0023] The compositions used in the methods of the present invention do not contain fenugreek; for example, some compositions of the present invention do not contain fenugreek fibers (i.e., fibers obtained or obtainable from fenugreek).

[0024] The compositions used in the methods of the present invention may contain small amounts of polyols and / or preferably low molecular weight sugars having one or two monosaccharide units, such as less than 5% by weight or less than 2.5% by weight of the composition. Alternatively, the compositions of the present invention may not contain low molecular weight sugars such as polyols and / or those having one or two monosaccharide units; that is, some compositions may not contain any low molecular weight sugars such as polyols and / or those having one or two monosaccharide units.

[0025] The present invention also provides the use of a composition comprising (i) curcuminoids, (ii) extracts obtained or obtainable from quillaja, and (iii) modified starch and / or gum arabic for improving the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals.

[0026] The compositions used in the methods / uses of the present invention described herein may be in the form of emulsions, or the compositions of the present invention may be in the form of solids, for example, powders.

[0027] As used herein, the term “emulsion” refers to a type of colloid formed by combining two liquids that are usually not mixed. Typically, one liquid contains a dispersion of the other liquid.

[0028] Sometimes the terms “colloid” and “emulsion” are used interchangeably, but as used herein, the term emulsion applies when both phases of a mixture are liquid. The particles in a colloid can be any phase of substance. Therefore, while an emulsion is a type of colloid, not all colloids are emulsions. A colloidal solution, sometimes identified as a colloidal suspension, is a mixture in which substances are regularly suspended in a fluid. Such use may hereafter be referred to as "use of the present invention."

[0029] In the methods or uses of the present invention, the curcuminoids in the composition may be obtained from any source. However, it is preferable that the curcuminoids be obtained from natural sources, i.e., that the curcuminoids are plant-based rather than synthetic.

[0030] In compositions used in the methods / uses described herein, the composition may include particles having an average diameter of about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm or 1500 nm to about 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm or 2000 nm, for example, about 1000 nm to about 6000 nm. The particles may also have an average diameter of about 200 nm to about 600 nm, or about 300 nm to about 500 nm or about 400 nm.

[0031] For example, in the compositions used in the methods / uses described herein, the compositions may contain particles having an average diameter of about 100 nm to about 700 nm, such as about 200 nm to about 600 nm, about 300 nm to about 500 nm, or about 400 nm.

[0032] For example, the composition may contain particles having an average diameter of approximately 550 nm to approximately 700 nm and particles having an average diameter of approximately 100 nm to approximately 250 nm, resulting in an average diameter of approximately 400 nm.

[0033] For example, if the composition is in the form of an emulsion, the composition may contain, for example, particles having an average diameter of about 550 nm to about 700 nm and particles having an average diameter of about 100 nm to about 250 nm, with an average diameter of about 400 nm. If the composition is in solid form, such as a powder, the composition may contain particles having an average diameter of approximately 1000 nm to approximately 6000 nm, for example, approximately 2000 nm to approximately 4000 nm.

[0034] The particles in the composition may be in the form of micelles. In the composition of the present invention, for example, if the composition is in solid form, the particles may be formed using techniques known in the art, such as spray drying. After the particles have formed (for example, after drying by spray drying), they may be ground and / or pulverized (e.g., by a ball mill) to provide a more uniform size.

[0035] The size and morphology of the loaded curcumin micelles were analyzed by dynamic light scattering (DLS), zeta potential (Z-potential), and scanning electron microscopy (SEM). For DLS and zeta potential analysis, a Zetasizer Nano ZS (NanoZS90, Malvern Instrument Ltd., UK) equipped with a He / Ne laser (λ=633nm) at a fixed scattering angle of 90° at a temperature of (25±0.1℃) was used. For example, particle size may be measured by the CQ-MO-304 method as defined in the example below.

[0036] In the composition, curcuminoids can be obtained from any source. However, it is preferable that the curcuminoids be obtained from natural sources; that is, the curcuminoids are plant-based rather than synthetic. The composition of the present invention may contain at least about 2.5% by weight of curcuminoids. Typically, in compositions used in the methods / uses described herein, curcuminoids may be present in amounts of about 2.5% to about 60% by weight, such as about 5% to about 50% by weight or about 10% to about 45% by weight, or about 15% to about 40% by weight.

[0037] Curcuminoids may also be provided by extraction and optional purification from the root (rhizome) of turmeric (Curcuma longa), oleoresin turmeric root, defatted oleoresin turmeric root and mixtures thereof, i.e., (i) the curcuminoids may be in the form of an extract or purified extract of turmeric containing about 30% to about 100% curcuminoids, such as about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% curcuminoids.

[0038] When curcuminoids are provided as an extract of turmeric, the turmeric may be extracted using an alcohol-based extraction solvent such as a water / alcohol mixture or alcohol. For example, the alcohol-based extraction solvent may be water / methanol (i.e., a mixture of water and methanol) or water / ethanol (i.e., a mixture of water and ethanol) or methanol or ethanol.

[0039] If the extraction solvent contains a water / alcohol mixture, the water-to-alcohol ratio may be approximately 25:75 to 1:99, such as approximately 20:80 to 5:95 or approximately 10:90. For example, the extraction solvent may be water / ethanol in a ratio of approximately 25:75 to 1:99, such as approximately 20:80 to 5:95 or approximately 10:90.

[0040] The turmeric extract may then be further purified to provide a curcuminoid extract containing approximately 30% to approximately 100% curcuminoids, such as approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% curcuminoids, based on the percentage of total curcuminoids in the extract.

[0041] The purification of the extract may be carried out using such techniques known in the art. Typically, the extract is purified using an alcohol-based solvent such as 100% methanol or 100% ethanol. The turmeric extract may be optionally dried to remove excess solvent.

[0042] If the curcuminoids are provided in the form of a turmeric extract as previously defined, the composition may contain about 2.5% to about 50% by weight of turmeric extract, such as about 5% to about 40% or about 35% by weight of the composition. For example, the composition may contain about 8% to about 40% by weight of turmeric extract, where the turmeric extract contains about 65% to about 95% by weight of curcuminoids in the turmeric extract, providing a composition containing about 6% to about 38% by weight of curcuminoids in the composition, or the composition may contain about 8% to about 15% by weight of turmeric extract, where the turmeric extract contains about 95% by weight of curcuminoids in the turmeric extract, providing a composition containing about 6% to about 13.5% of curcuminoids.

[0043] Curcuminoids may be provided as a liquid or powder, such as a powder. For example, powdered turmeric extract. As used herein, the term “curcuminoid” encompasses curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). For example, a turmeric extract may contain about 70% to about 85% curcumin (e.g., about 75% to about 80%), about 10% to about 25% DMC (e.g., about 15% to about 20%), and about 0% to about 10% BDMC.

[0044] If present, modified starch in the composition may be present in an amount of about 30% to 90% by weight of the composition, such as about 40% to 80% by weight of the composition. As used herein, the term “modified starch” includes starch that has been treated physically, enzymatically, or chemically, but does not include modified starch having a cyclic structure, such as cyclodextrin. Modified starch that may be used in the compositions described herein includes, but is not limited to, maltodextrin.

[0045] The gum arabic in the composition of the present invention may be present in an amount of about 40% to about 65% by weight of the composition, such as about 50% to about 60% by weight or about 58% by weight of the composition. The extract obtained or obtainable from quillaja in the composition of the present invention may be present in an amount of about 0.1% to about 5% by weight, such as about 0.5% to about 3% by weight of the composition.

[0046] In the composition, the extract obtained or obtainable from Quillaja may contain at least 50% by weight of saponins, such as at least 60% by weight of saponins or at least 65% by weight of saponins. For example, the Quillaja used in the composition of the present invention may contain about 50% to about 80% by weight or about 60% to about 75% by weight of saponins in the Quillaja extract.

[0047] The extract obtained or obtainable from quillaja used in the process of the present invention may be in any form, such as liquid or solid. For example, the quillaja extract may be used in a solid form, such as a powder.

[0048] The composition may optionally contain plant and / or vegetable oil. For example, the composition of the present invention may contain plant and / or vegetable oil selected from the group consisting of rapeseed oil (including coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, canola oil), safflower oil, sesame oil, soybean oil, sunflower oil, and mixtures thereof. The plant and / or vegetable oil present in the composition of the present invention may be present in an amount of about 1% to about 20% by weight, such as about 2.5% to about 10% by weight or about 5% by weight of the composition. Unless otherwise stated herein, the weight percentages indicated are based on the total weight of the resulting (dried) composition.

[0049] To avoid misunderstanding, preferences, choices, specific features, etc., to a given aspect, feature, or parameter of the present invention should be considered disclosed in conjunction with any and all other preferences, choices, specific features, etc., as shown with respect to the same or other aspects, features, and parameters of the present invention, unless the context otherwise indicates.

[0050] As used herein, the term “about” means, for example, a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or specifically ±0.1% relative to a specified amount when referring to a measurable value (such as the amount or weight of a specific component in a reaction mixture). For example, a variation of ±0.5% with respect to the percentage of components in the composition of the present invention means a variation of 0.5% relative to a given percentage, i.e., ±0.5% of 10% means a variation of 9.5% to 10.5%.

[0051] The compositions of the present invention may be provided in solid or liquid form, preferably in solid form such as powder. Solid form includes the possibility that the compound may be provided as an amorphous solid, or as a crystalline or partially crystalline solid.

[0052] The compositions of the present invention are typically highly water-soluble and / or stable at a pH of 4 or higher, such as pH about 4 to about 7. The term water solubility means that at least about 50% of a composition, such as at least about 60%, 70%, 80%, 90%, or 95%, will dissolve in water at room temperature, i.e., about 25°C.

[0053] In the methods or uses described herein, improvements in the bioaccessibility, bioavailability, bioefficacy, and / or bioactivity of curcuminoids in mammals may be due to improved gastrointestinal tolerance of curcuminoids and / or improved absorption of curcuminoids by intestinal cells and / or improved blood circulation. Therefore, the methods or uses described herein may be used to improve the gastrointestinal tolerance of curcuminoids, and / or to improve the absorption of curcuminoids by intestinal cells, and / or to improve blood circulation.

[0054] In the methods or uses described herein, improvements in the bioaccessibility, bioavailability, bioefficacy, and / or bioactivity of curcuminoids in mammals may be due to compositions that provide improved water solubility and / or improved stability at a pH of about 4 to about 7.

[0055] Therefore, the present invention provides a method for improving the water solubility and / or pH stability of curcuminoids, the method comprising administering the curcuminoids in the form of the composition of the present invention as defined above. The present invention also provides the use of the previously defined compositions to improve the water solubility and / or pH stability of curcuminoids.

[0056] In the methods or uses described herein, curcuminoids may be selected from the group consisting of curcumin and its phase I or phase II metabolites, bisdemethoxycurcumin and its phase I or phase II metabolites, and mixtures thereof. For example, phase I and / or phase II metabolites may be selected from the group consisting of curcumin glucuronide, curcumin sulfate, DMC glucuronide, DMC sulfate, BDMC glucuronide, BDMC sulfate, tetrahydrocurcumin (THC), THC glucuronide, THC sulfate, hexahydrocurcumin (HHC), HHC glucuronide, HHC sulfate, and mixtures thereof.

[0057] In the compositions, methods, or uses described herein, curcuminoids may be in their unmetabolized form (i.e., free form), for example, in a form that has not undergone the addition of glucuronide or sulfate, or curcumin, DMC, and BDMC. In the methods and uses described herein, the mammal may be a human.

[0058] As used herein, the term “bioavailability” can be defined as the fraction of ingested components available at the site of action for use in normal physiological function and is measured via in vivo assays (Guerra et al. 2012). Bioavailability is the result of three main steps: digestibility and solubility of elements in the gastrointestinal tract; absorption of elements by intestinal cells and transport into circulation; and integration into functional entities or targets (Wienk KJH, Marx JJM, Beynen AC (1999) The concept of iron bioavailability and its assessment. Eur J Nutr 38:51-75; Etcheverry P, Grusak MA, Fleige LE (2012) Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc, and vitamins B6, B12, D, and E. Front Physiol 3:1-21).

[0059] As used herein, the term “bioaccessibility” can be defined as the fraction of a compound released from its food matrix within the gastrointestinal tract and thus made available for intestinal absorption (typically established from in vitro procedures). It encompasses a range of events occurring during food digestion for conversion into potentially bioaccessible substances, but excludes absorption / anasimilation via epithelial tissue and presistemic metabolism (both intestine and liver). (Alegria A., Garcia-Llatas G., Cilla A. (2015) Static Digestion Models: General Introduction. In: Verhoeckx K. et al. (eds) The Impact of Food Bioactives on Health. Springer, Cham)

[0060] As used herein, the term “bioactive” can be defined as how a nutrient or bioactive compound is transported to target tissues, how it interacts with biomolecules, the metabolic or in vivo changes it may experience, and the generation of induced biomarkers and physiological responses (Alegria A., Garcia-Llatas G., Cilla A. (2015) Static Digestion Models: General Introduction. In: Verhoeckx K. et al. (eds) The Impact of Food Bioactives on Health. Springer, Cham).

[0061] In the methods / uses described herein, the composition may be administered or used to provide amounts of curcuminoids ranging from about 100 mg / day to about 2000 mg / day, or about 500 mg / day to about 1500 mg / day, or about 1000 mg / day, such as about 300 mg / day to about 1000 mg / day. For example, the composition may provide amounts of curcuminoids ranging from about 1 to about 10 mg / kg body weight, such as about 2.5 to about 7.5 mg / kg body weight or about 5 mg / kg.

[0062] Composition and administration According to the present invention, the compositions used in the methods and uses described herein may be provided in the form of nutritional supplements, dietary products or foods for human or animal use (functional food preparations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements, etc.), herbicides, nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary preparations, wine or cosmetic preparations, or form part of nutritional supplements, dietary products or foods for human or animal use (functional food preparations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements, etc.), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary preparations, wine or cosmetic preparations.

[0063] For example, the present invention provides a composition of the present invention that is essentially composed of (i.e., at least 90% w / w nutritional supplements, dietary products or foods for humans or animals (functional food formulations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements, etc.), nutritional supplements, fragrances or flavoring agents, pharmaceutical or veterinary formulations, wine or cosmetic formulations, etc., for example, at least 95%, 99%, or 99.5%), or a nutritional supplement, dietary products or foods for humans or animals (functional food formulations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements, etc.), nutritional supplements, fragrances or flavoring agents, pharmaceutical or veterinary formulations, wine or cosmetic formulations, etc.) comprising the composition of the present invention.

[0064] The present invention also provides for the use of the compositions of the present invention in nutritional supplements, dietary products or foods for human or animal use (functional food formulations, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplements, etc.), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary formulations, wine or cosmetic formulations.

[0065] If the composition of the present invention is a nutritional supplement, a dietary product or food for human or animal use (such as a functional food product, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a herbicide, a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary product, a wine or cosmetic product, or can form part of a nutritional supplement, a dietary product or food for human or animal use (such as a functional food product, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary product, a wine or cosmetic product, it may optionally further contain pharmaceutical / veterinary components such as excipients or carriers or (functional) food-acceptable components and mixtures thereof, as needed.

[0066] To avoid misunderstanding, when the terms “comprising” or “comprises” are used herein, it means that the described extract or composition must contain the listed components (singular or plural), but may optionally contain additional components. When the terms “consisting essentially of” or “consists essentially of” are used, it means that the described extract or composition must contain the listed components (singular or plural), and may contain small amounts of other components (for example, up to 5% by weight, or 1% by weight, or 0.1% by weight), but any additional components do not affect the essential properties of the extract or composition. When the terms “consisting of” or “consists of” are used, it means that the described extract or composition must contain only the listed components (singular or plural). Furthermore, it is also intended that, where necessary, the terms “comprise,” “comprises,” or “comprising” may be replaced throughout the application with “consist,” “consisting,” or “consisting essentially of.”

[0067] When used herein, references to pharmaceutically acceptable excipients may refer to pharmaceutically acceptable adjuvants, diluents and / or carriers as known to those skilled in the art. Food-acceptable ingredients include those known in the art (including those also referred to herein as pharmaceutically acceptable excipients), and may be natural or non-natural; that is, their structures may or may not occur in nature. In some cases, they may arise from natural compounds and may be modified before use (e.g., maltodextrin).

[0068] "Pharmacologically / nutritionally acceptable" means that any additional components of the composition are sterile and pyrogen-free. Such components must be "acceptable" in the sense that they are compatible with the composition of the present invention and not harmful to its recipient. Thus, "pharmaceutically acceptable" includes any compound used to form part of a formulation that is intended to act merely as an excipient, i.e., not intended to have biological activity of its own. Thus, pharmacovigilant excipients are generally safe, non-toxic, and not biologically or otherwise undesirable.

[0069] When the composition of the present invention forms part of a nutritional supplement, a dietary product or food for human or animal use (such as a functional food preparation, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary preparation, a wine or cosmetic preparation, the composition of the present invention is present in an amount of about 1 to about 99% by weight of the nutritional supplement, a dietary product or food for human or animal use (such as a functional food preparation, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary preparation, a wine or cosmetic preparation, Present in foods, beverages, feed or pet food or food, beverages, feed or pet food supplements, etc.), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary preparations, wine or cosmetic preparations, for example, in amounts of about 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, or 90% by weight ~ about 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, or 20% by weight, etc.

[0070] Administration Those skilled in the art will understand that, in the methods and / or uses described herein, the compositions of the present invention may be administered to a patient or subject (e.g., a human or animal patient or subject) by any preferred route, such as oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracapsular, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal) routes. This applies whether the compositions are in the form of a nutritional supplement, a dietary product or food for human or animal use (such as a functional food product, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary product, a wine or cosmetic product, or whether the nutritional supplement, a dietary product or food for human or animal use (such as a functional food product, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplement), a nutritional supplement, a fragrance or flavoring agent, a pharmaceutical or veterinary product, a wine or cosmetic product, or whether the composition comprises the compositions of the present invention.

[0071] In particular, in the methods and / or uses described herein, compositions, or compositions that are in the form of or form part of nutritional supplements, dietary products or foods for human or animal use (such as functional food formulations, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplements), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary formulations, wine or cosmetic formulations, may be administered orally. In such cases, pharmaceutical compositions according to the present invention may be specifically formulated for administration via the oral route.

[0072] Pharmaceutical formulations for oral administration include solid dosage forms such as hard or soft capsules, tablets, lozenges, sugar-coated tablets, pills, troches, powders, and granules. Where appropriate, they may be prepared with coatings such as enteric coatings, or may be formulated to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art. Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily / oil-based suspensions, syrups, and elixirs.

[0073] Nutritional supplements, dietary products or foods for human or animal use (such as functional food preparations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary preparations, wine or cosmetic preparations, and those intended for oral administration may be prepared by methods known to those skilled in the art, such as by mixing the components of the composition.

[0074] Such nutritional supplements, dietary products or foods for human or animal use (such as functional food preparations, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplements), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary preparations, wine or cosmetic preparations described herein may contain one or more additional ingredients selected from the group consisting of food ingredients such as sweeteners, flavorings, colorings and preservatives. Tablets may contain one or more active ingredients mixed with non-toxic, pharmaceutically acceptable excipients (or ingredients) suitable for the manufacture of tablets. These excipients (or ingredients) may, for example, include: inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, e.g., corn starch, maltodextrin or alginic acid; binders, e.g., starch, gelatin or acacia; and lubricants, e.g., magnesium stearate, stearic acid or talc. The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a long period. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate may be used.

[0075] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, gum arabic, modified starch, and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water. Furthermore, carriers or diluents may include any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, either alone or in combination with wax.

[0076] Depending on the disorder and subject being treated, as well as the route of administration, the compositions of the present invention can be administered in various doses (i.e., therapeutically effective doses administered to patients who need them). In this regard, those skilled in the art will understand that the dose administered to mammals, specifically humans, in the context of the present invention should be sufficient to influence the therapeutic response of the mammal over a reasonable time frame. Those skilled in the art will recognize that the selection of the exact dose and composition, as well as the most appropriate delivery regimen, is also influenced, among other things, by the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical and mental responsiveness of the recipient, as well as the efficacy of the particular compound, the age, condition, weight, sex and response of the patient receiving treatment, and the stage / severity of the disease.

[0077] Typically, the compositions or nutritional supplements of the present invention, dietary products or foods for human or animal use (such as functional food formulations, i.e., food, beverage, feed or pet food or food, beverage, feed or pet food supplements), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary formulations, wine or cosmetic formulations are administered to provide curcuminoids in amounts ranging from about 100 mg / day to about 2000 mg / day, or about 500 mg / day to about 1500 mg / day, or about 1000 mg / day, such as about 300 mg / day to about 1000 mg / day. For example, compositions or nutritional supplements, dietary products or foods for human or animal use (such as functional food formulations, i.e., food, beverages, feed or pet food or food, beverages, feed or pet food supplements), nutritional supplements, fragrances or flavorings, pharmaceutical or veterinary formulations, wine or cosmetic formulations may provide curcuminoids in amounts of about 1 to about 10 mg / kg body weight, such as about 2.5 to about 7.5 mg / kg body weight or about 5 mg / kg body weight.

[0078] In any case, physicians or other persons skilled in the art will be able to routinely determine the most suitable actual dosage for individual patients. The dosages described above are examples of average cases; of course, there may be individual cases where a higher or lower dosage range is beneficial, and such cases are within the scope of the present invention.

[0079] Preparation process of the composition of the present invention The present invention provides a process for preparing the composition of the present invention as defined above, the process comprising the following steps: (i) Prepare an aqueous solution of curcuminoids; (ii) Mix an aqueous solution from (i) with an aqueous solution of gum arabic, an extract obtained or obtainable from quillaja, and optionally a vegetable and / or plant oil to provide an emulsion; and optionally (iii)(ii) is dried to provide a composition containing particles having an average diameter of about 100 nm to about 10,000 nm, such as about 100 nm to about 700 nm or about 1,000 nm to about 6,000 nm. Includes.

[0080] For example, the present invention provides a process for preparing the composition of the present invention as defined above, the process comprising the following steps: (i) Prepare an aqueous solution of curcuminoids; (ii) Mix an aqueous solution from (i) with an aqueous solution of gum arabic, an extract obtained or obtainable from quillaja, and optionally a plant and / or vegetable oil to provide an emulsion; and (iii)(ii) is dried to provide a composition containing particles having an average diameter of about 100 nm to about 700 nm, such as about 100 nm to about 700 nm or about 1000 nm to about 6000 nm. Includes. Such a process will hereafter be referred to as the process of the present invention.

[0081] In the process of the present invention, the aqueous solution of gum arabic may be mixed with an aqueous solution of curcuminoids before being mixed with the extract obtained or obtainable from plant and / or vegetable oil and quillaja, i.e., the process of the present invention is as follows: (i) Prepare an aqueous solution of curcuminoids; (ii) Mix the aqueous solution from (i) with an aqueous gum arabic solution; (iii) Mix an extract obtained or obtainable from Quillaja, and optionally a plant and / or vegetable oil, with the product of (ii) to provide an emulsion; and optionally (iv)(iii) is dried (by spray drying, etc.) to provide a composition containing particles having an average diameter of about 100 nm to about 700 nm. It may include. After drying (e.g., spray drying), the particles may be ground and / or pulverized (e.g., using a ball mill) to provide a more uniform size.

[0082] In the process of the present invention, particles may have an average diameter of approximately 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm or 1500 nm to approximately 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm or 2000 nm, such as approximately 1000 nm to approximately 600 nm. Particles may also have an average diameter of approximately 200 nm to approximately 600 nm, or approximately 300 nm to approximately 500 nm or approximately 400 nm.

[0083] For example, the composition may include particles having an average diameter of about 550 nm to about 700 nm and particles having an average diameter of about 100 nm to about 250 nm, giving an average diameter of about 400 nm.

[0084] For example, if the composition of the present invention is in the form of an emulsion (i.e., before the drying step), the composition may contain particles having an average diameter of about 550 nm to about 700 nm and particles having an average diameter of about 100 nm to about 250 nm, with an average diameter of about 400 nm. If the composition of the present invention is in solid form (i.e., after the drying step), the composition may contain particles having an average diameter of about 1000 nm to about 6000 nm, such as about 2000 nm to about 4000 nm. The particles in the composition may be in the form of micelles.

[0085] In the process of the present invention, the curcuminoids present in the aqueous solution of curcuminoids may come from any of the sources previously defined with respect to the compositions of the present invention. Typically, in the process of the present invention, the curcuminoids may have a purity of about 5% to about 100% by weight of the curcuminoid source (based on total curcuminoids), i.e., the turmeric or curcuminoid extract may contain about 30% to about 100% curcuminoids, such as about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% based on the percentage of total curcuminoids in the extract, or about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% curcuminoids.

[0086] In the process of the present invention, curcuminoids can be mixed with water in a weight ratio of about 2:1 (curcuminoids:water) to about 4:1, such as about 3:1, to provide an aqueous solution of curcuminoids. Typically, in the process of the present invention, the weight concentration of curcuminoids in the aqueous solution can be about 1% to about 95%, such as about 5% to about 80% or about 7% to about 40%.

[0087] In the process of the present invention, an aqueous gum arabic solution can be prepared by mixing gum arabic with water in a gum arabic:water ratio of about 2:1 to about 4:1, such as about 3:1. Aqueous gum arabic solutions can have a weight concentration of gum arabic ranging from approximately 30% to 70%, such as approximately 40% to 60%. Typically, aqueous curcuminoid solutions and aqueous gum arabic solutions are mixed using stirring.

[0088] In the process of the present invention, the plant and / or vegetable oil may be from any plant and / or plant source. For example, the plant and / or vegetable oil may be sunflower oil. Typically, in the process of the present invention, plant and / or vegetable oil may be present in an amount of about 1% to about 10%, such as about 2.5% to about 7.5% or about 5%.

[0089] In the process of the present invention, the extract obtained or obtainable from quillaja may be as defined previously with respect to the composition of the present invention. The extract obtained or obtainable from quillaja used in the process of the present invention may be in any form, such as liquid or solid. For example, the quillaja extract may be used in a solid form, such as a powder. Typically, in the process of the present invention, quillaya may be present in an amount of about 0.5% to about 5%, such as about 1% to about 3%, or about 2%.

[0090] In the process of the present invention, the plant and / or vegetable oil and the quillaja extract may be mixed using stirring. An emulsion is provided by mixing an aqueous solution of curcuminoids, an aqueous solution of gum arabic, plant and / or vegetable oils, and quillaja as defined above.

[0091] The resulting emulsion may be dried using techniques known in the art to provide a composition containing micelles having an average diameter of about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm or 1500 nm to about 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm or 2000 nm, such as about 300 nm to about 500 nm or about 400 nm. Typically, the emulsion is spray-dried.

[0092] The process of the present invention may optionally include a step of removing additional solvents as needed to provide a substantially dry product, i.e., a product from which at least 90% of the present water has been removed, such as at least 95% or 99%. [Brief explanation of the drawing]

[0093] [Figure 1] Figure 1 - Chemical structure of curcuminoids from turmeric [Figure 2] Figure 2 - Phase I and Phase II metabolites of curcumin [Figure 3] Figure 3 - Bacterial metabolites of curcumin

[0094] [Figure 4] Figure 4- LDH activity measured in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation, data from standard curcumin extract. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB + colon 48h. [Figure 5] Figure 5- shows LDH activity measured in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation, with data normalized to TB+ colon 48h samples (100%) from standard curcumin extract. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB+ colon 48h.

[0095] [Figure 6] Figure 6-Data from turmeric phytosome preparations, measured in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB + colon 48h. [Figure 7]Figure 7- shows LDH activity measured in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation, with data normalized to TB+colon 48h samples (100%) from turmeric phytosome preparations. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB+colon 48h.

[0096] [Figure 8] Figure 8-Data from Quillaja base preparations, measured in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB + colon 48h. [Figure 9] Figure 9-Data from Quillaja base preparations with normalized data to TB+colon 48h samples (100%), measuring LDH activity in apical supernatant collected from Caco-2 cells treated with samples from different gastrointestinal compartments after 4h incubation. Bars represent mean ± SEM. PP: pure product; SI: small intestine; ST: stomach; TB: transport buffer. (*), (**), and (***) correspond to significance at p<0.05, p<0.01, and p<0.001, respectively, compared to TB+colon 48h.

[0097] [Figure 10] Figure 10 - Evolution of food intake over the adaptation and habituation periods in groups of mice sacrificed at the same time point. Average daily food intake (g) of different experimental groups during the adaptation (J0-J7) and habituation (J8-J14) periods. [Figure 11]Figure 11 - Evolution of body weight over the adaptation and habituation periods of groups of mice sacrificed at the same time point. Mean body weight (g) of different experimental groups during the same period. Data are expressed as mean ± SEM.

[0098] [Figure 12] Figure 12 shows the time course of total curcuminoid levels (sum of curcumin, DMC, BDMC and their related metabolites curcuminide and sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) in mouse plasma after a single oral administration of a turmeric phytosome preparation to a standard turmeric extract containing 300 mg / kg of curcuminoids. *, **, ***: Turmeric phytosome preparations significantly different from the standard turmeric extract at each time point (post-hoc t-test) (p<0.05, p<0.01, p<0.001, respectively). [Figure 13] Figure 13 shows the time course of total curcuminoid levels (sum of curcumin, DMC, BDMC and their related metabolites, curcumin glucuronide and sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) in mouse plasma after a single oral administration of the composition used in the method / use of the present invention to a standard turmeric extract containing 300 mg / kg of curcuminoids. *, **, ***: Compositions used in the method / use of the present invention that are significantly different from the standard turmeric extract at each time point (post-hoc t-test) (p<0.05, p<0.01, p<0.001, respectively).

[0099] [Figure 14]Figure 14: Total curcuminoid and metabolite concentrations (sum of curcumin, DMC, BDMC and their related metabolites, curcumin glucuronide and sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) as a function of time (h) after ingestion of different formulations (n=72 per formulation) (ppm). □, □□: Turmeric phytosomes significantly different from standard turmeric extract at each time point (p<0.05, p<0.01

[0100] [Figure 15] Figure 15 - Corresponding area AUC(0-8h) under the curves for different formulations. [Figure 16] Figure 16 - Corresponding area AUC(0-∞) under the curves for different formulations. [Figure 17] Figure 17 - Cmax (concentration at Tmax) of different formulations. [Figure 18] Figure 18 - Effect of pH on the color of the composition of the present invention dissolved in desalinated water (0.4%). [Figure 19] Figure 19 - DLS profile of the loaded composition of the present invention.

[0101] [Figure 20] Figure 20 - Z potentials of the present invention composition and individual components of the composition at different pH levels. [Figure 21] Figure 21 - Scanning electron microscope (SEM) image of the composition of the present invention at ×300. [Figure 22] Figure 22 - Scanning electron microscope (SEM) image of the composition of the present invention at ×1300. [Figure 23] Figure 23 - Scanning electron microscope (SEM) image of the composition of the present invention at ×9200. [Figure 24] Figure 24 - Graph showing AUC0-24h normalized by dosage for the ITT population. [Figure 25] Figure 25 - Graph showing AUC0-8h normalized by dosage for the ITT population.

[0102] [Figure 26] Figure 26 - Graph showing AUC0-∞ normalized by dosage for the ITT population. [Figure 27] Figure 27 - Graph showing AUC0-24h for the ITT population. [Figure 28] Figure 28 - Graph showing AUC0-8h for the ITT population. [Figure 29] Figure 29 - Graph showing AUC0-∞ for the ITT population. [Figure 30] Figure 30 - Graph showing the normalized Cmax for the ITT population.

[0103] [Figure 31] Figure 31 - Graph showing Cmax for the ITT group. [Figure 32] Figure 32 - Graph showing relative bioavailability from 0 to 24 hours for the ITT population. [Figure 33] Figure 33 - Graph showing relative bioavailability from 0 to 8 hours for the ITT population. [Figure 34] Figure 34 - Graph showing relative bioavailability from 0 to infinity for the ITT population. [Figure 35] Figure 35 - Graph showing the half-life for the ITT population. [Figure 36] Figure 36 - Graph showing the terminal elimination rate constant in the terminal phase for the ITT population. [Figure 37] Figure 37 - Graph showing Tmax for the ITT population.

[0104] example The present invention can be further described by reference to the following non-limiting examples. Example 1 - Testing the effect of the composition of the present invention on enhancing the bioavailability of curcuminoids in an in-vivo model of human gastrointestinal tract and intestinal absorptive cells. The human gastrointestinal tract (GIT) is one of the primary entry points into the human body. When food, beverages, or medicines are ingested orally, the intestines become the first point of contact between the ingested product and the host. To exert their biological activity, compounds must first pass through the stomach, where the acidic environment and the presence of digestive enzymes can lead to chemical or enzymatic modification. After leaving the stomach, ingested compounds reach the small intestine, where most of the host's metabolic enzymes are secreted, potentially causing further enzymatic modification. The compounds can then be absorbed and enter circulation, or pass further through the intestines, either in their original or modified form. Here, food compounds can acquire local biological activity by coming into contact with the complex microbial communities present in the terminal ileum (the last part of the small intestine) and colon (Alegria et al, 2015).

[0105] Human studies are certainly one of the most representative methods for studying different gut processes. However, they are highly labor-intensive, time-consuming, extremely costly, and do not allow for mechanical research. In humans, the gut can be considered a black box that allows for the quantification of inputs and outputs, but investigating underlying gut processes in different compartments is difficult due to sampling problems. Moreover, ethical constraints limit the general applicability of human trials.

[0106] Therefore, well-designed in vitro simulation techniques offer extremely useful alternatives to human and animal studies. Such models, representing specific processes, enable reproducible and thorough studies of those processes without ethical constraints. Easier setup and sampling allow for low-cost, medium-to-high-throughput studies. Nevertheless, the lack of a physiological host environment remains the most significant limitation of these models. However, the use of standardized in vitro cell cultures using human-derived cell lines provides a fast and reproducible method for studying the ultimate effects of compounds on the intestinal mucosa. Furthermore, extensive in vitro investigations allow for careful design of subsequent animal or human studies, thereby saving time and money.

[0107] In an in vitro approach, simulating a carefully designed GIT provides an excellent high-throughput screening setup for evaluating the putative metabolic fate of selected food components at different concentrations. Such components may be modified in the gut or alter the bacterial community, thus reaching the intestinal mucosa intact or in the form of modified by-products. Oral bioavailability of a dietary compound is defined as the fraction of doses that can be absorbed by intestinal cells and are available for use or storage.

[0108] The bioavailability of dietary compounds depends on many factors, namely, the individual nutritional and physiological state, the conjugation of compounds with other nutrients and / or bile salts, the enzymatic degradation of compounds by digestive enzymes, and the ability of gut-associated bacteria to metabolize them. In vitro gastrointestinal models offer the potential to screen large sets of molecules in a rapid and cost-effective manner in short-term experiments.

[0109] The following approach allows for rapid assessment of the intestinal fate of dietary compounds during digestion and colonic fermentation. This involves an in vitro cell model simulating human intestinal epithelium, enabling the investigation of the bioavailability of intact and modified compounds, thereby increasing both scientific output and commercial relevance.

[0110] First, a short-term screening assay was performed as a tool to evaluate the digestion fate of curcumin-based formulations with different solubility properties. Next, the results of these experiments were applied in vitro to Caco-2 cells to investigate the bioavailable fractions of different formulations compared to their unmodified / undigested forms. In addition, cytotoxicity was measured to compare the estimated cytotoxic effects of different digestion fractions.

[0111] Short-term screening assays consisted of sequential incubations (stomach, small intestine, colon) of representative doses (one or more) of selected lead compounds under simulated colonic conditions using representative bacterial inoculum. We used intestinal suspensions collected from the ascending colon compartment of the Human Gut Microbial Ecosystem Simulator (SHIME) (Van de Wiele et al, 2015). This inoculant consists of a stable microbial community adapted to the environmental conditions present in the proximal colon, both in terms of structure and activity.

[0112] The following curcumin-based preparations / compositions were evaluated: 1. Standard curcumin extract (Curcuma longa) - contains a mixture of three curcuminoids (curcumin - 75%, demethoxycurcumin (DMC) - 15-20%, and bidemethoxycurcumin (BDMC) - 5-10%). 2. Control formulation (Meriva®'s Turmeric Phytosome Thorne product, which contains 18-22% curcuminoids, with the present curcumin and soy lecithin blended in a 1:2 weight ratio (phytosome), followed by the addition of 2 parts microcrystalline cellulose to improve fluidity, resulting in an overall curcumin content of approximately 20% in the final product.) 3. A composition used in the method / use of the present invention (also referred to as Type I), comprising 8.6% turmeric extract (containing more than 6% curcuminoids), 15.9% sunflower oil, 2% quillaja extract, and 73.5% modified starch.

[0113] The short-term screening assay consisted of sequential incubation of three formulations under gastric, small intestinal, and colonic conditions. The formulations / compositions were tested to achieve a curcuminoid concentration of 0.5 g / L in the gastric compartment (the actual amount in mg was calculated based on the percentage of curcuminoids in each product - as shown in Table 1). Next, the different formulations / compositions were incubated in the presence of pepsin at 37°C and pH 2.0 for 1 hour (h). Next, the small intestine was simulated by adding pancreatic enzymes and bile salts, and the samples were incubated at 37°C for a total duration of 3 hours.

[0114] Finally, in the third incubation stage, the colon was simulated by adding representative fecal inoculation material and a nutrient-rich medium collected from SHIME. Colon incubation was carried out under anaerobic conditions with shaking at 37°C for a total of 48 hours. Each formulation / composition was tested three times to control for biological variability. It should be noted that these experiments were designed to respect the specific residence times of food components in the gastrointestinal tract. Considering the volume within each compartment, the concentrations of curcuminoids tested were 0.5 g / L in the stomach, 0.35 g / L in the small intestine, and 0.1 g / L in the colon. In the cell transport experiments, these samples were diluted more than 10-fold.

[0115] Table 1: Tested curcumin-based formulations / compositions and the percentage of each curcuminoid in the formulations / compositions. The percentage of curcuminoids was considered to calculate the amount of mg to be added to the gastric compartment. Natural forms are shown in bold. [Table 1]

[0116] Samples of each formulation / composition were collected at the following points in time. - Stomach: 30 minutes and 60 minutes - Small intestine: 60, 120, and 180 minutes - Colon: 2, 4, 6, 24 and 48 hours Next, the curcuminoid content (curcumin, DMC, and BDMC) of the samples was analyzed using high-pressure liquid chromatography (HPLC) combined with mass spectrometry.

[0117] Calibration curves were created for each of the three curcuminoids (Phytolab, Vestenbergsgreuth, Germany) in the range of 2–1000 ng / mL, and 54 ppb of curcumin-d6 (TLC pharmachem, Ontario, Canada) was added as an internal standard to ensure retention time stability and instrumental correction variability. Acetonitrile was used as the diluent for each solution. For free curcuminoid identification, exactly 450 μL of internal standard solution (60 ng / mL) was loaded into a Captiva 96-well plate (ND lipid from Agilent) via 50 μL of plasma sample. After mixing and filtration, the eluate was ready for injection into the LC / MS system. The Captiva ND lipid plate is designed to effectively remove phospholipids from plasma. To identify the total conjugated curcuminoid metabolites (glucuronides and sulfate metabolites), 100 μL of plasma sample was mixed with 100 μL of enzyme solution (either glucuronidase 1000 units / mL, Sigma#G7017; or sulfatase, Sigma#S9626, 100 units / mL) at 37°C for 2 hours. After this hydrolysis step, 50 μL of the solution was similarly mixed with 450 μL of acetonitrile on a Captiva 96-well plate. The sample procedure was the same as for free curcuminoids, with mixing and filtering before injection.

[0118] The LC / MS conditions were as follows: The autosampler (5°C) and LC system used were an Agilent Infinity 1290 integrated system. During the study, an Agilent 6420 triple quadrupole mass spectrometer was used with electrospray ionization. Metabolites were eluted from a BEH Shield RP 18 column (100 x 2.1, 1.7 μm; Waters) at a flow rate of 0.5 mL / min with mobile phases consisting of HPLC-grade 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Elution was a gradient from 40–80% B over 0–6 minutes. The injection volumes of standards and samples were 2 μL. For each reference compound, the relevant transition from precursor to product ion was detected using multiple reaction monitoring (MRM) mode. For each of the three analytes, the product ion was identified by MS1 full scan and then identified by MS / MS experiment. The MRM transitions for each analyte were optimized using direct injection and the Optimizer B.08.00 workstation software solution (Agilent Technologies, Santa Clara, CA, USA). See Table 2 for optimal selection criteria. The mass spectrometer parameters were set as follows: both negative and positive mode ESI sources; dry gas (N2) flow rate, 10 L / min; gas temperature, 350 °C; nebulizer, 40 psi; and capillary, 4.0 kV. The MS system was fully calibrated before execution according to the manufacturer's guidelines. Data analysis was performed using Agilent MassHunter quantitative / qualitative analysis B.07.00 (Agilent Technologies, Santa Clara, CA, USA).

[0119] Table 2: Retention times (Tr), multiple reaction monitoring (MRM) transitions, and optimized tandem mass spectrometry (MS / MS) detection parameters for three curcuminoids and an internal standard. [Table 2]

[0120] Table 3 shows the curcuminoid concentrations obtained between intestinal compartments during digestion. Surprisingly, the post-digestion concentrations of the composition used in the method / use of the present invention in the stomach at 60 minutes and in the small intestine at 120 and 180 minutes were superior to those obtained for the standard turmeric powder extract, demonstrating better digestibility resistance of the composition used in the method / use of the present invention compared to the standard extract. Furthermore, the post-digestion concentrations of the composition used in the method / use of the present invention in the stomach at 60 minutes and in the small intestine at 120 and 180 minutes were higher than those of the comparative turmeric phytosome preparation, demonstrating better digestibility resistance of the composition used in the method / use of the present invention compared to the turmeric phytosome comparison.

[0121] Table 4 shows the percentage of curcuminoids remaining in the intestinal compartment after digestion at initial concentrations (0.5 g / l or 500 mg / l of curcuminoids in the gastric compartment at the start of the experiment). Surprisingly, the results clearly indicate that curcuminoids from the compositions used in the method / use of the present invention were far more resistant to digestion in the stomach and small intestine compared to curcuminoids from standard turmeric extracts, that the compositions used in the method / use of the present invention were more protected from degradation during digestion after oral ingestion than curcuminoids from standard extracts or turmeric phytosome preparations, and that the amount of curcuminoids accessible for absorption in the small intestinal compartment after oral ingestion of the compositions used in the method / use of the present invention was far greater than that from oral ingestion of standard extracts or turmeric phytosome preparations.

[0122] [Table 3-1] [Table 3-2]

[0123] Table 4: Tolerance to gastrointestinal (GIT) digestion: Percentage of curcuminoids remaining in the intestinal compartment after digestion, based on initial concentration. [Table 4] The data show the mean ± SD percentage of curcuminoid concentration in different compartments and at different time points after digestion, compared to the initial concentration.

[0124] The following samples were also collected for transport experiments in Caco-2 cells. - Small intestine: 120 minutes and 180 minutes The pH of the sample applied to the cells was adjusted to 6.5 before use.

[0125] Caco-2 cells are widely used as a cellular model of intestinal function because they can spontaneously differentiate into intestinal cell-like cells during culture. When cultured on a semipermeable support, these cells develop into a functional, polarized monolayer similar to intestinal epithelium in the presence of apical brush-border enzymes and microvilli. Therefore, they are considered the “absolute standard” model for transport experiments because they acquire the morphological and functional characteristics of mature intestinal cells in culture (Sambuy et al, 2002).

[0126] Caco-2 cells (ATCC) are divided into 1 × 10⁻¹⁶ cells. 5 0.9 × 10 for cells / inserts 5 cells / cm 2Cells were seeded at a density into 12 transwell inserts (0.4 μm). Cells were differentiated until a functional monolayer was reached (21 days); apical (600 μL) and basal (1500 μL) medium were supplemented three times a week. Barrier function was assessed by measuring the transepithelial electrical resistance (TEER) of the monolayer on the day of the experiment. Cells were washed with HBSS to remove trace amounts of medium, and 2 mL of transport buffer (TB) was added to the basal side. Samples collected from short-term experiments were diluted with transport buffer at a ratio of 1:10 (v / v) and given to cells apically (600 μL). All products were also tested untreated, and powders were diluted with TB at a concentration of 0.025 mg / mL (the final theoretical concentrations of curcuminoids tested for all formulations can be seen in Table 2). These dilutions were prepared from stock solutions (250 mg / mL) prepared with HBSS, except for standard turmeric extract dissolved in DMSO due to its insufficient solubility. For control wells, colon samples diluted 1:10 (v / v) with TB (48h incubation) obtained by running a blank (without curcumin) during a short-term experiment were used. Transport buffer (TB) consisted of HBSS (pH 7.4) supplemented with 10 mM HEPES, 25 mM D-glucose, and 1× antibiotic-antifungal agent. Cells were incubated at 37°C for a total duration of 4 hours.

[0127] The following samples were collected: 1. Diluted sample (500 μL) used to stimulate the cells. Since it contains the diluted sample before being given to the cells, these correspond to the 0h time point, and the untreated formulation diluted in TB at a concentration of 0.025 mg / mL was also shipped. 2. Samples collected from the apical end after 2h and 4h incubation (250 μL each). 3. Samples collected from the side and bottom after incubation for 2 hours (800 μL) and 4 hours (1000 μL). 4. Samples from cells after 4 hours of incubation. These correspond to fractions taken into the cells. Briefly, ice-cold PBS1X was added to the cells to stop transport. The cells were then washed again with PBS1X to remove any uninternalized trace amounts of product, and the cells were permeabilized with a solution of PBS1X containing 20% ​​ethanol and 0.1% Tween-20 (600 μL); after 20 minutes, the cells were collected in a 1.5 mL tube in this solution and disintegrated and homogenized using a syringe and a 21 G needle. 5. The tube was centrifuged, and the supernatant was transferred to a new tube (450 μL). All samples were stored at -20°C until HPLC analysis.

[0128] To evaluate the cytotoxicity of different samples applied to Caco-2 cells, lactate dehydrogenase (LDH) released by apical Caco-2 cells (after 4 hours of incubation) was assessed using the LDH-Activity Kit. LDH is released into the supernatant by cells upon membrane damage and is therefore a marker of cell death. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc multiple comparison test. * ), ( ** ) and ( *** ) correspond to significance levels at p<0.05, p<0.01, and p<0.001, respectively.

[0129] As shown in Figure 4, the control well (TB + colon 48h) showed an LDH activity of approximately 1.0. As can be seen, for all products, both the undigested form and the stomach sample collected at 60 minutes showed the highest LDH activity compared to the control. In contrast, both the small intestine and colon samples showed levels comparable to or lower than the control. These results demonstrate that, with the exception of the stomach sample, all samples were not toxic to Caco-2 cells, and that the assays testing curcuminoid transport and bioavailability from Caco-2 cell samples can be utilized and judged as effective, since the results were obtained on viable cells.

[0130] Since Caco-2 cells are known to express UDP-glucuronosyltransferase and sulfotransferase ((Siissalo S, Zhang H, Stilgenbauer E, Kaukonen AM, Hirvonen J, Finel M), samples from the apical, basal, and intracellular compartments of Caco-2 cells incubated with either undigested products or samples from the small intestine (120 or 180 minutes) were further analyzed for their curcuminoid content (curcumin, DMC, and BDMC) and their associated metabolite content (curcumin sulfate, curcumin glucuronide, DMC sulfate and DMC glucuronide, BDMC sulfate and BDMC glucuronide). While the expression of most UDP-glucuronosyltransferases (UGTs) significantly increases during Caco-2 cell differentiation, UGT1A6 is highly expressed even in undifferentiated cells (Drug Metab Dispos. 2008). (Nov;36(11):2331-6), therefore, curcumin, DMC, and BDMC can be metabolized to their glucuronide or sulfate metabolites (Dempe JS, Scheerle RK, Pfeiffer E, Metzler M. Metabolism and permeability of curcumin in cultured Caco-2 cells. Mol Nutr Food Res. 2013 Sep;57(9):1543-9).

[0131] Apparent transmittance coefficient (P) of the transition from the apex to the base app) The value was calculated according to Artursson and Karlsson, using the following formula:

Math

[0132] Table 5 shows the P aap values at different time intervals after apical exposure of Caco-2 cells to a standard extract or two different formulations (turmeric phytosome or the composition used in the method / use of the present invention).

[0133] Table 5: Papp values (10 -7 expressed as cm / s) of total curcuminoids and their metabolites calculated at different time intervals after apical exposure of Caco-2 cells to a standard extract or two different formulations

Table 5

[0134] As shown in Table 5, the apparent permeability coefficient (P aap ) value for apical-to-basolateral transport is surprisingly higher for the composition used in the method / use of the present invention than for the standard turmeric extract, and the P appThe values ​​doubled by 19.3 and 20.3 times. Apparent transmission coefficient (P) of transition to the lateral base aap The P values ​​were also higher in the turmeric phytosome formulation used as a positive control for the enhanced bioavailability formulation, compared to standard turmeric extract at 2h and 4h, respectively. app The values ​​doubled by 4.7 and 4.8. However, the results indicate that curcuminoid absorption by Caco-2 absorptive cells is greater with the composition used in the method / use of the present invention compared to the comparative turmeric phytosome formulation (4.1 times and 4.2 times better Papp values ​​at 2h and 4h, respectively).

[0135] Table 6 shows the P250 after apical exposure of Caco-2 cells to small intestinal digestion samples (120 or 180 minutes) of a standard extract or two different formulations (turmeric phytosomes or compositions used in the method / use of the present invention), with the quantified concentration of curcuminoids in the small intestinal compartment at 120 or 180 minutes used as Capi. app The values ​​are shown. This reflects the absorption capacity of curcuminoids from the digested preparation by cells. When a standard extract or preparation is digested in the gastric and small intestinal compartments, and therefore a sample from the small intestine (120 min or 180 min) is used to measure curcuminoid absorption by Caco-2 cells, the absorption of curcuminoids by Caco-2 absorptive cells was greater with the composition used in the method / use of the present invention compared with the turmeric phytosome preparation (P for small intestinal samples at 120 min or 180 min, respectively). app The values ​​were demonstrated to be 1.8 times and 16.4 times higher.

[0136] Table 7 shows the P values ​​after apical exposure of Caco-2 cells to small intestinal digestion samples (120 or 180 minutes) of standard extracts or two different formulations (turmeric phytosomes or compositions used in the method / use), with the theoretical concentration of curcuminoids in the small intestinal compartment at 120 or 180 minutes used as Capi to take into account not only the cellular absorption capacity but also tolerance to the digestive process. app The values ​​are shown. From these data, the level of curcuminoids that can reach the gastrointestinal compartment (mimicking blood circulation) after absorption and digestion by Caco-2 cells is higher in the composition used in the method / use of the present invention compared to standard turmeric extract (P app The values ​​were 2.0 times and 1.8 times higher for small intestinal samples at 120 minutes and 180 minutes, respectively, and compared to phytosomal preparations (P app The values ​​were significantly higher (3.4 times and 7.1 times higher, respectively, for small intestine samples at 120 minutes and 180 minutes).

[0137] Table 6: Total curcuminoids and their metabolites after apical exposure (120 or 180 minutes) of Caco-2 cells to small intestinal digestion samples of standard extracts or two different formulations. app value [Table 6] The data shows mean ± SD. P compared to turmeric phytosomes. app The doubling is shown in parentheses (). app The values ​​are calculated using the quantified concentration of curcuminoids in the small intestinal compartment at 120 or 180 minutes as Capi.

[0138] Table 7: Total curcuminoids and their metabolites after apical exposure (120 or 180 minutes) of Caco-2 cells to small intestinal digestion samples of standard extracts or two different formulations. app value [Table 7] The data shows mean ± SD. P compared to the standard extract. app The doubling is shown in parentheses (). P compared to turmeric phytosome formulations app The doubling is shown in square brackets []. app The values ​​are calculated using the theoretical curcuminoid concentration in the small intestinal compartment at 120 or 180 minutes as Capi.

[0139] Example 2 - Effects of the composition of the present invention on enhancing the bioavailability of curcuminoids through in-vivo comparative pharmacokinetic studies in mice In view of results obtained in an in vitro model that showed the composition used in the method / use of the present invention exhibits better tolerance to gastrointestinal digestion and better absorption through intestinal cells, it is hypothesized that the composition used in the method / use of the present invention (i.e., a composition comprising 8.6% turmeric extract (containing more than 6% curcuminoids), 15.9% sunflower oil, 2% quillaja extract, and 73.5% modified starch) would improve the bioavailability of curcuminoids in mice compared to standard turmeric extract. Therefore, comparative pharmacokinetic studies were conducted in mice.

[0140] Adult male C57Bl / 6J Rj mice from Janvier Labs (St-Berthevin, France) were 5 weeks old upon receipt and housed together in standard plastic cages (n=4 / cage). All animals had free access to water and standard pellet food (Pellet AO4; SAFE, Villemoisson-sur-Orge, France) and were maintained in a temperature-controlled (24.0-26.0°C) and humidity-controlled (40.0-50.0%) room with a 12-h light (07:00 AM-07:00 PM) / 12-h dark cycle.

[0141] All animals were allowed to acclimate to their new environment for one week after receipt. Total food intake and animal body weight were assessed twice a week to ensure both proper acclimatization and a standard growth curve. Following this acclimatization period, mice were acclimatized to daily oral administration of the vehicle (1% (w / v) carboxymethylcellulose sodium salt dissolved in distilled water at room temperature, CMC; Ref# C4888, batch number: SLBB5612V, SIGMA ALDRICH, St Quentin Fallavier, France) for six days prior to treatment. Total food intake and animal body weight were measured daily during this acclimatization period. These measures allowed for optimal acclimatization of the animals to both the injection procedure and manipulation by the experimenter.

[0142] During the adaptation and acclimatization period, mice were allowed free access to a pre-weighed amount of fresh food pellets (pellet AO4; SAFE, Villemoisson-sur-Orge, France). The remaining food was weighed the day after the measurement. Using a precision scale (THB-600G, PMC Millot; accuracy ±0.01g), the total food intake per cage (08:40-09:20 AM) was determined by subtracting the remaining food from the amount of food weighed beforehand. The average daily food intake was obtained by dividing this value by the number of days separating the two methods and the number of animals in each cage. At each weight measurement, the weight of the mice was measured in the morning (08:40-09:20 AM).

[0143] On the day before the treatment (the last day of the acclimatization period), 120 mice were fasted in the evening (17:40-18:20 PM). The following day, the mice (n=40 per formulation) were subjected to acute treatment in the morning (08:00-09:50 AM) by forced oral administration (30 ml / kg) of a standard turmeric powder extract, a turmeric phytosome formulation as a comparison, or a composition used in the method / use of the present invention (i.e., a composition containing 8.6% turmeric extract (containing more than 6% curcuminoids), 15.9% sunflower oil, 2% quillaja extract, and 73.5% modified starch).

[0144] An appropriate volume of vehicle (1% CMC dissolved in distilled water) was added to a suitable recipient under stirring, and the pH was adjusted to 5.5. An appropriate amount of the pre-weighed formulation was gradually added to the vehicle under constant stirring. Once homogenized, the pH of the resulting suspension was measured and adjusted to 5.5 if necessary. To avoid curcuminoid degradation, the final suspension was administered systematically 1 hour after preparation. The dose to animals receiving 300 mg / kg of total curcuminoids was calculated (see Table 8). This dose in mice was calculated using the formula from (United States Department of Health and Human Services, U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. 2005): Human equivalent dose (mg / kg) = Animal dose mg / kg × (Animal body weight kg / Human body weight kg) 0.33 Therefore, this corresponds to 21.37 mg / kg in humans, and 1282 mg assuming a 60 kg person. The dosage was adjusted to body weight measured immediately before fasting. To avoid the impact of feeding and drinking behavior on intestinal absorption after forced oral administration of the formulation, water and food were not given for the first 12 hours after the procedure.

[0145] Table 8: Curcuminoid content of standard turmeric extract and two formulations and the respective concentrations of the suspensions used to administer the products to mice with 300 mg / kg body weight of curcuminoids in the first in vivo study. [Table 8]

[0146] Blood was sampled from anesthetized mice by cardiac puncture at 0.5, 1h, 2h, 4h, 6h, 8h, 12h, or 24h after administration (n=5 / time point / product). Anesthesia was induced by intraperitoneal injection of a ketamine / xylazine mixture (100 mg / kg and 15 mg / kg, respectively). For cardiac puncture, a 26G syringe was inserted at a 45° angle to the longitudinal axis formed by the animal's body between the 8th and 10th sternal ribs to directly penetrate the left ventricle. Blood was then gently drawn to obtain a final volume of 0.6–1 ml. For the benefit of bioanalysis, the blood was then transferred to an Eppendorf tube, mixed with heparin sulfate (200 U.I / ml blood), and gently agitated. All samples were centrifuged at 3000 g and 4°C for 15 minutes within 30 minutes of blood collection to separate the plasma. Plasma (supernatant) was dispensed into a fresh 0.5 ml Eppendorf glass. Aliquots of plasma were centrifuged and frozen at -80°C within 1 hour.

[0147] Plasma doses of parent curcuminoids (curcumin, DMC, or BDMC) and their associated metabolites (curcumin glucuronide and sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) were determined by LC-MS-MS. Calibration curves were created for each of the five curcuminoids (Phytolab, Vestenbergsgreuth, Germany) in the range of 2–1000 ng / mL, with 54 ppb of curcumin-d6 (TLC pharmachem, Ontario, Canada) added as an internal standard to ensure retention time stability and instrumental correction variability. Acetonitrile was used as a diluent for each solution. For the identification of free curcuminoids, exactly 450 μL of internal standard solution (60 ng / mL) was loaded into a Captiva 96-well plate (ND lipids from Agilent) via 50 μL of plasma sample. After mixing and filtration, the eluate is ready for injection into the LC / MS system. Captiva ND lipid plates are designed to effectively remove phospholipids from plasma. For the identification of total conjugated curcuminoid metabolites (glucuronides and sulfate metabolites), 100 μL of plasma sample was mixed with 100 μL of enzyme solution (either glucuronidase 1000 units / mL, Sigma#G7017 or sulfatase, Sigma#S9626, 100 units / mL) at 37°C for 2 hours. After this hydrolysis step, 50 μL of the solution was also mixed with 450 μL of acetonitrile on a Captiva 96-well plate. The sample procedure is the same as for free curcuminoids, mixing and filtration before injection.

[0148] The LC / MS conditions were as follows: The autosampler (5°C) and LC system used were an Agilent Infinity 1290 integrated system. An Agilent 6420 triple quadrupole mass spectrometer was used with electrospray ionization during the study. Metabolites were eluted from a BEH Shield RP 18 column (100 × 2.1, 1.7 μm; Waters) at a flow rate of 0.5 mL / min with mobile phases consisting of HPLC-grade 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Elution was a gradient from 40–80% B over 0–6 minutes. The injection volumes of standards and samples were 2 μL. For each reference compound, the relevant transition from precursor to product ion was detected using multiple reaction monitoring (MRM) mode. For each of the five analytes, the product ion was identified by MS1 full scan and then identified by MS / MS experiment. The MRM transitions for each analyte were optimized using direct injection and the Optimizer B.08.00 workstation software solution (Agilent Technologies, Santa Clara, CA, USA). See Table 9 for optimal selection criteria. The mass spectrometer parameters were set as follows: both negative and positive mode ESI sources; dry gas (N2) flow rate, 10 L / min; gas temperature, 350 °C; nebulizer, 40 psi; and capillary, 4.0 kV. The MS system was fully calibrated before execution according to the manufacturer's guidelines. Data analysis was performed using Agilent MassHunter quantitative / qualitative analysis B.07.00 (Agilent Technologies, Santa Clara, CA, USA).

[0149] For the three formulations / compositions tested, the kinetics of plasma concentrations of each curcuminoid compound were determined 0.5–12 hours post-treatment by calculating the mean ± SEM plasma concentration at each time point of blood collection. Pharmacokinetic parameters T1 / 2 (half-life), Cmax, Tmax, AUC(0–12h), and AUC(0–∞) were determined from the 0–12h kinetics by non-compartmental analysis using PKSolver. PKSolver is a menu-based add-in program for Microsoft Excel written in Visual Basic for Applications (VBA) to solve pharmacokinetic problems (Zhang et al., 2010). All data are expressed as mean ± SEM. Statistical analysis was performed using Statview 5.0.1 (Statview software, Cary, NC, USA) and Excel 2013 programs. Data were analyzed by Student's t-test at each time point. Risk α was fixed at 0.05.

[0150] Table 9: Retention times (Tr), multiple reaction monitoring (MRM) transitions, and optimized tandem mass spectrometry (MS / MS) detection parameters for curcuminoids, tetrahydrocurcumin, and hexahydrocurcumin, and internal standards. [Table 9]

[0151] During two consecutive periods, adaptation (J1-J7) and habituation (J8-J14), 24-hour food intake and mouse body weight were regularly measured to ensure both correct adaptation and a standard growth curve before treatment. For this study, mice were housed in cages of four and treated with one of three formulations, with each used for blood sampling at the same time point. As a result, food intake and body weight data were first analyzed for each group of 15 mice sacrificed at the same time point (8 groups; 5 mice / time point / formulation). Figures 10 and 11 show the food intake and body weight of mice during the adaptation and habituation periods before treatment administration, respectively; different groups showed classical body weight curves and food intake before treatment. Note that the significant weight loss observed in all groups at J15 (Figure 11) resulted from overnight fasting conducted the night before treatment. These results confirmed that all mice used in this experiment behaved similarly and could be compared as expected.

[0152] Figure 12 shows the pharmacokinetic profiles of total curcuminoids (sum of curcumin, DMC, BDMC and their related metabolites, curcumin glucuronide sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) obtained at each time point in mice after oral administration (300 mg / kgbw curcuminoids) of turmeric phytosome formulations and standard extract formulations. The turmeric phytosome formulation enabled plasma concentrations of total curcuminoids to reach 41.5 ppm (μg / ml) after 30 minutes, which was significantly better than those obtained with the standard at each time point except 24 hours. Plasma concentrations of total curcuminoids from the standard turmeric extract reached a maximum of 12.9 ppm at 1 hour and were less than 10 ppm at all other time points. Turmeric phytosomes demonstrated a 3.2-fold increase in total curcuminoid Cmax and a 3.9-fold increase in AUC compared to standard turmeric extract. These results confirm the use of turmeric phytosome formulations as a positive control for enhancing curcuminoid bioavailability and validate the in vivo model for testing the ability of different formulations to enhance curcuminoid bioavailability compared to standard turmeric extract.

[0153] Table 10 contains the mean ± SEM values ​​for each time point. The numbers in parentheses next to these values ​​indicate the number of samples that showed a positive value relative to the total number of samples. Statistical comparison results are also shown in the same table. Table 11 contains PK parameters obtained from non-compartmental analysis using PKSolver software. The percentage of variation between groups is also shown (%Var°). Data are expressed as mean ± SEM.

[0154] [Table 10] Tables 10 and 11 show the mean ± SEM and PK parameters obtained from non-compartmental analysis using PKSolver software at each time point shown in Figure 12.

[0155] Figure 13 shows the pharmacokinetic profiles of total curcuminoids (sum of curcumin, DMC, BDMC and their related metabolites, curcumin glucuronide sulfate, DMC glucuronide and sulfate, BDMC glucuronide and sulfate, THC, THC glucuronide and sulfate, HHC, HHC glucuronide and sulfate) obtained in mice at each time point after oral administration (300 mg / kgbw curcuminoids) of a mixture containing curcumin, quillaja oil and modified starch (Example 2 Type 1) and a standard extract formulation. The results showed that the compositions used in the method / use of the present invention could significantly increase the total curcuminoid concentration from 0.5h to 24h compared to the standard turmeric extract. The compositions used in the method / use of the present invention demonstrated a 1.8-fold increase in total curcuminoid Cmax and a 2.2-fold increase in AUC.

[0156] Table 12 shows the mean ± SEM values ​​for each time point. The numbers in parentheses next to these values ​​indicate the number of samples that showed a positive value relative to the total number of samples. Statistical comparison results are also shown in the same table. Table 13 shows the PK parameters obtained from non-compartmental analysis using PKSolver software. The percentage of variation between groups is also shown (%Var°). Data are expressed as mean ± SEM.

[0157] [Table 11] Tables 12 and 13: Show the mean ± SEM and PK parameters obtained from non-compartmental analysis using PKSolver software at each time point shown in Figure 13.

[0158] Table 14 shows the plasma concentrations of parental curcuminoids at each time point after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the methods / uses of the present invention. Specifically looking at the parental compounds (natural forms, i.e., unmetabolized curcumin, DMC, and BDMC), the compositions used in the methods / uses of the present invention were the only ones that could quantify a detectable amount of parental curcuminoids within the first 4 hours after administration, and therefore AUC(0-12h) and AUC(0-∞) could be calculated (Table 15). Compared with standard turmeric extract, a 10.9-fold increase in the Cmax of parental curcuminoids was obtained for the compositions used in the methods / uses of the present invention.

[0159] Table 16 shows the plasma concentrations of parental curcumin at each time point after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention. Specifically looking at the parent compound (natural form, i.e., unmetabolized curcumin), the compositions used in the method / use of the present invention were the only ones that could quantify a detectable amount of parental curcuminoids within the first 4 hours after administration, and therefore AUC(0-12h) and AUC(0-∞) could be calculated (Table 16). Compared with standard turmeric extract, the compositions used in the method / use of the present invention showed a 521.8-fold increase in the Cmax of parental curcumin. Furthermore, the compositions used in the method / use of the present invention induced higher plasma levels of parental curcumin than the turmeric phytosome formulation (a 1.8-fold increase in Cmax).

[0160] From this initial in vivo experiment, it can be concluded that the composition used in the method / use of the present invention, which contains more than 6% curcuminoids and is obtained from 8.6% turmeric extract, 15.9% sunflower oil, 2% quillaja extract, and 73.5% modified starch, and is prepared according to type 1, can enhance not only the bioavailability of total curcuminoids and their metabolites but also the bioavailability of the parent compound compared to standard turmeric extract. Furthermore, it can be concluded that the compositions used in the method / use of the present invention can improve the bioavailability of natural curcumin more effectively than turmeric phytosome formulations.

[0161] Therefore, the compositions used in the method / use of the present invention represent an attractive method for enhancing the bioavailability of parent curcumin without using soy-derived lecithin in the formulation, in contrast to turmeric phytosome formulations. Furthermore, curcumin is considered one of the most potent active substances of turmeric compared to DMC and BDMC and their associated reduced forms, glucuronides, or sulfate metabolites (reson C, Orr S, Jones DJ, Verschoyle R, Lim CK, Luo JL, Howells L, Plummer S, Jukes R, Williams M, Steward WP, ​​Gescher A. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production. Cancer Res. 2001 Feb 1;61(3):1058-64; Anand P, Thomas SG, Kunnumakkara AB, Sundaram C, Harikumar KB, Sung B, Tharakan ST, Misra K, Priyadarsini IK, Rajasekharan KN, Aggarwal BB. Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature. Biochem Pharmacol. 2008 Dec 1;76(11):1590-611; Pal A, Sung B, Bhanu Prasad BA, Schuber PT Jr, Prasad S, Aggarwal BB, Bornmann WG.Curcumin glucuronides: assessing the proliferative activity against human cell lines. Bioorg Med Chem. 2014 Jan 1;22(1):435-9), the composition used in the method / use of the present invention represents a good solution for improving the biological efficacy of curcumin against various health conditions such as joint health, inflammation, arthritis, atherosclerosis, fatty liver, hepatic fibrosis, diabetes, cognition, mild cognitive impairment, and irritable bowel syndrome.

[0162] Table 14: Concentrations of parent curcuminoids (sum of curcumin, DMC, and BDMC) at each time point after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in the first in vivo study. [Table 12] The numbers in parentheses next to these values ​​indicate the number of samples that showed a positive value relative to the total number of samples.

[0163] Table 15: PK parameters obtained from non-compartmental analysis using PKSolver software for parent curcuminoids after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in the first in vivo study. [Table 13]

[0164] Table 16: Concentration of parent curcumin at each time point after ingestion of 300 mg / kg curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in the first in vivo study. [Table 14] The numbers in parentheses next to these values ​​indicate the number of samples that showed a positive value relative to the total number of samples.

[0165] Table 17: PK parameters obtained from non-compartmental analysis using PKSolver software for curcumin after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in the first in vivo study. [Table 15]

[0166] Considering the results obtained in the first in vivo study in mice, which showed better bioavailability of total curcuminoids, parental curcuminoids, and curcumin, we decided to test in a second comparative pharmacokinetic study in mice the ability of a formulation optimized with a higher curcuminoid content (12% curcuminoids), prepared according to type 2 with 14.4% turmeric extract, 26.8% sunflower oil, 2% quillaja extract, and 56.8% modified starch, to improve the bioavailability of curcuminoids compared to its standard turmeric extract.

[0167] The same methodology (mouse rearing, acclimatization period, habituation period, and quantification of curcuminoids and their metabolites using LC / MS) was used in this example as previously described, but with a larger group and number of animals per time (n=12 / time point / formulation), and standard turmeric extracts (79.5, 15.0, and 3.0 g / 100g of curcumin, DMC, and BDMC, respectively, and a total of 97.5 g of curcuminoids / 100g), and turmeric phytosome preparations (18.6, 2.6, and 0.2 g / 100g of curcumin, respectively) were used. To clarify the kinetic profile of curcuminoids in the earliest phases after oral administration (300 mg / kg body weight) of curcuminoids from compositions used in the method / use of the present invention (9.8 g, 1.6 g, and 0.2 g / 100g of curcumin, DMC, and BDMC, and a total of 21.5 g of curcuminoids / 100g, respectively) or type 2 curcuminoids (9.8, 1.6, and 0.2 g / 100g of curcumin, DMC, and BDMC, and a total of 11.6 g of curcuminoids / 100g), blood was sampled at 0.25-, 0.5-, 0.75-, 1h-, 2h-, or 8h after administration.

[0168] Figure 14 shows the total curcuminoid and metabolite concentrations as a function of time after ingestion of different formulations (n=72 per formulation). The results clearly show a significant increase in curcuminoid and metabolite concentrations for turmeric phytosomes and the compositions used in the method / use of the present invention compared to standard turmeric extract. Total curcuminoid concentrations were higher at 0.5h and 0.75h after ingestion of the compositions used in the method / use of the present invention compared to the turmeric phytosome formulation, and surprisingly, the compositions used in the method / use of the present invention demonstrate better performance in terms of improved bioavailability of total curcuminoids and metabolites compared to the turmeric phytosome formulation.

[0169] This was confirmed while calculating the corresponding areas under the curves AUC(0-8h), AUC(0-∞), and Cmax (Figures 15, 16, and 17, respectively), which were 280%, 300%, and 337% higher, respectively, for the compositions used in the method / use of the present invention compared to the standard extract, and 6.5%, 30%, and 63% higher, respectively, for the compositions used in the method / use of the present invention compared to the turmeric phytosome formulation (Table 18). It was also shown that curcuminoids and metabolites from the compositions used in the method / use of the present invention were absorbed more rapidly, with a 1.5-fold reduction in Tmax compared to the standard extract (0.5h vs. 0.75h) and a 2-fold reduction in Tmax compared to the turmeric phytosome formulation (0.5h vs. 1h), respectively (Table 18). Surprisingly, the results also showed that curcuminoids and metabolites from the compositions used in the method / use of the present invention were not excreted more rapidly with a longer half-life (3.8h vs. 2.8h) compared to turmeric phytosome formulations.

[0170] As shown in Table 19, which gives the PK parameters of parent curcuminoids after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the methods / uses of the present invention, when specifically looking at the parent compounds (natural forms, i.e., unmetabolized curcumin, DMC, and BDMC), surprisingly, the compositions used in the methods / uses of the present invention were the only ones for which we were able to calculate the AUC(0-8h) for parent curcumin. Compared with standard turmeric extract, a 3.2-fold increase in the Cmax of parent curcuminoids was obtained for the compositions used in the methods / uses of the present invention. Since parent curcumin could not be found in plasma samples after ingestion of the turmeric phytosome preparations, the Cmax of curcumin could not be calculated.

[0171] From this second in vivo experiment, it can be concluded that the composition used in the method / use of the present invention, which has a higher curcuminoid content (12% curcuminoids) and is prepared according to type 2 with 14.4% turmeric extract, 26.8% sunflower oil, 2% quillaja extract, and 56.8% modified starch, can unexpectedly enhance the bioavailability of parent curcumin as well as total curcuminoids and their metabolites compared to standard turmeric extract and turmeric phytosome formulations. Therefore, the compositions used in the method / use of the present invention represent an attractive method for enhancing the bioavailability of curcuminoids without using soy-derived lecithin in the formulation, in contrast to turmeric phytosome formulations.

[0172] Table 18: PK parameters obtained from non-compartmental analysis using PKSolver software for total curcuminoids and metabolites after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in a second in vivo study. [Table 16] Doubling of AUC or Cmax compared to the standard extract is indicated in parentheses (). The doubling of AUC or Cmax compared to turmeric phytosome formulations is shown in square brackets [].

[0173] Table 19: PK parameters obtained from non-compartmental analysis using PKSolver software for parent curcuminoids after ingestion of 300 mg / kg of curcuminoids from standard turmeric extract, turmeric phytosomes, or compositions used in the method / use of the present invention in a second in vivo study. [Table 17]

[0174] Example 3 - Comparative pharmacokinetic studies in healthy volunteers to evaluate the ability of the compositions of the present invention to enhance the bioavailability of curcuminoids. This study had two objectives: 1. 1st purpose To evaluate the plasma concentration profile of total curcuminoids (curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and metabolites thereof) 24 hours after a single dose of 300 mg of the composition of the present invention (Turmipure GOLD TM curcuminoid formulation), in comparison with 1500 mg of standard turmeric powder extract comprising 95% curcuminoids.

[0175] 2. Secondary purpose To evaluate the plasma concentration profile of the following parameters after a single intake of five study products, each containing any one of 1425 mg (standard turmeric powder extract 95% curcuminoids, Curcumin C3 complex California Gold Nutrition), 200 mg (Curcuma Platinum MannaVital), 90 mg of the composition of the present invention (Turmipure GOLD TM 30% curcuminoids) or 60 mg of active substance (Curcumin Cell’Innov): · Total curcuminoids; · Parent compounds (curcumin, DMC, BDMC) and their metabolites: curcumin glucuronide and sulfate; DMC glucuronide and sulfate; BDMC glucuronide and sulfate; native tetrahydrocurcumin (THC), glucuronide and sulfate thereof; native hexahydrocurcumin (HHC), glucuronide and sulfate thereof. This study was a monocentric, randomized, crossover, and open-label clinical trial.

[0176] The study starts at the screening / inclusion visit (V0), followed by 5 experimental sessions (V1 to V5), during which the tested products are ingested by the subjects (one different product per session for each randomized subject). The V1 visit is conducted at a maximum of 3 weeks after V0, and may also constitute the randomization visit. Each experimental session (V1 to V5) was separated by a minimum of 1 week and a maximum of 2 weeks. During each experimental session, subjects underwent kinetic blood sampling over an 8-hour period. The last kinetic blood sample was collected 24 hours after the start of the kinetic study on the day following each experimental session. Urine collection was also performed during these visits for biobanking.

[0177] The first urination of the subject was collected on the morning of each experimental visit (the entire volume of this first urination), with additional collections on-site from 0 to 8 hours during kinetic blood sampling, and from 8 to 24 hours after returning home. The last urine collection was returned on the day after the experimental visit (when returning for the last blood sample of kinetic blood sampling, T24H). Standard meals were provided to volunteers for dinner before each experimental session and during all periods of each activity (breakfast, lunch, afternoon snack). The end of the study was the day after the last experimental session V5 (V5-24H).

[0178] Thirty participants were selected for this study according to the following main selection and exclusion criteria: ■ I1: 18 to 45 years old (inclusive); ■ I2: 19 to 25 kg / m 2 BMI (inclusive); ■ I3: Stable body weight, within ±3 kg in the past 3 months; ■ I4: Routine blood chemistry values within the normal range;

[0179] ■ I5: For women: Not postmenopausal, have been using the same reliable method of contraception for at least three cycles prior to the start of the study and agree to maintain it for the entire duration of the study (accepting condoms with spermicidal gel and estrogen / progestin combination contraceptives), or are postmenopausal, with or without hormone replacement therapy (excluding estrogen replacement therapy started less than three months prior); ■ I6: I agree to quit smoking or limit my tobacco intake to ≤5 cigarettes / day and not smoke during all experimental sessions (V1-V5); ■ I7: I agree not to consume any foods, beverages, or condiments containing curcumin or other curcuminoids (DMC, BDMC) throughout the entire study period; ■ I8: Good general and mental health as determined by the principal investigator: No clinically significant or relevant abnormalities in medical history or physical examination;

[0180] ■ E1: Having a metabolic or endocrine disorder such as diabetes mellitus, uncontrolled or controlled thyroid disorder, or other metabolic disorder; ■ E2: Participants have a severe chronic disease (e.g., cancer, HIV, renal failure, progressive liver or biliary tract disease, chronic inflammatory bowel disease, arthritis or other chronic respiratory disease) or a gastrointestinal disorder that is found to be inconsistent with the principal investigator's conduct of the study (e.g., celiac disease); ■ E3: Suffering from liver disease; ■ E4: Current medical conditions that contraindicate this medication in patients receiving dietary supplementation: chronic diarrhea, constipation or abdominal pain, inflammatory bowel disease (Crohn's disease or ulcerative colitis), cirrhosis, chronic laxative use...; ■ E5: Has irritable bowel syndrome (IBS) diagnosed by a physician and treated with chronic medication;

[0181] ■ E6: According to the principal investigator, the patient has a history of a current medical condition that could affect the study results or expose the patient to additional risks; ■ E7: Recent foodborne illness such as gastroenteritis or confirmed food poisoning (less than 1 month ago); ■ E8: People who have donated blood within three months of their V0 visit, or who intend to donate blood within three months. ■ E9: According to the principal investigator's opinion, the blood sample has low venous capital and cannot be used to perform kinetic analysis; ■ E10: Having a known or suspected food allergy, intolerance, or hypersensitivity (such as gluten intolerance or celiac disease) to any of the ingredients of the research product and / or a standard diet; ■ E11: Pregnant or breastfeeding women, or women who intend to become pregnant within the next three months;

[0182] ■ E12: Presence of alcohol or drug dependence; ■ E13: Any chronic drug treatments except oral and topical contraceptives (e.g., anticoagulants, antihypertensive treatments, thyroid treatments, asthma treatments, anxiolytics, antidepressants, lipid-lowering treatments, corticosteroids, phlebotonics, veinotononics, drugs affecting blood circulation, etc.). ■ E14: Currently (and in the past 3 months) taking any supplements from plants; ■ E15: Participants consumed a curcumin-containing food supplement (curcumin, turmeric, and curry) or food (curcumin, turmeric, E100, and curry) at least three times a week for two weeks prior to the study; ■ E27: According to the principal investigator, the control record (blood glucose, GGT, ASAT, ALAT, urea, creatinine, and complete blood count) showed clinically significant abnormalities.

[0183] As part of this study, five dietary supplements in capsule form were tested: 1. Standard turmeric powder extract 95% curcuminoids 1500mg (4 capsules; 375mg powder per capsule) (STE), taken as capsules. 2. Commercially available product: C3 complex (registered trademark) 95% curcuminoids (1500mg) + BioPerine (registered trademark) 95% piperine (15mg) (3 caps; 500mg C3 complex powder + 5mg BioPerine powder per capsule) (TEP) 3. Meriva® (1000mg) Curcuma Platinum Mannavital 20% Curcuminoids (2 caps; 500mg powder per capsule) (PHYT), consumed as a commercially available product. 4. Novasol® (1000mg) Curcumin Cell innov 6% Curcuminoid (2 caps; 500mg liquid per capsule) (NOV), which is consumed as a commercially available product. 5. A composition as defined herein (1 capsule; 300 mg of powder per capsule) (TURMIPURE GOLD) comprising turmeric extract, sunflower oil, quillaja extract, and gum arabic, to be taken as a capsule.

[0184] To ensure the health status of the subjects and confirm eligibility criteria, blood samples were collected during the V0 visit, and control records were analyzed and pregnancy tests were performed on non-menopausal women (βhCG dosage). Samples were collected after physical examination and verification of eligibility criteria. A maximum of 10 mL was collected. Blood pressure was measured at each visit during the physical examination using an electronic blood pressure monitor (Carescape Dinamap® V100). Heart rate (HR, bpm), systolic blood pressure (SBP, mmHg), and diastolic blood pressure (DBP, mmHg) were also evaluated. All participants were in a state of 12-hour fasting before joining the study. In preparation for visits V1-V5, a venous catheter was inserted into the elbow crease of the subject after clinical examination. This catheter allowed for blood collection for dynamic analysis without additional punctures.

[0185] The dynamic study lasted approximately 8 hours, and all subjects stayed at the clinical trial center. Ten blood collections were performed according to the following schedule: - T-10 (baseline), - T15, T30, T45, T60, T90, T120, T240, T360, T480, A margin of ±30 seconds for T15, ±1 minute for T30 and T45, ±2 minutes for T60 and T90, and ±5 minutes for T120 to T480 was allowed. The time point T0 corresponds to the intake of the study product.

[0186] Volunteers were allowed to have a standard lunch approximately 4 hours after intake of the study product (immediately after the T240 time point) and a standard afternoon meal approximately 8 hours after intake of the study product. Lunch was consumed within a maximum of 30 minutes. Water was not permitted 1 hour before and 1 hour after product administration. The catheter was removed after the final time point, T480.

[0187] Subsequently, volunteers were requested to return to the clinical trial facility in a 12-hour fasted state on the day after the visit for the final dynamic blood collection, T24H. Classic venous blood sampling was used (single puncture). The biological parameters evaluated in these samplings were analyzed in plasma, therefore only EDTA tubes were used (5 mL per sampling).

[0188] Analysis group ■ ITT population: all randomized subjects in the study who received at least one dose of the product (n=30) ■ PP population: subjects included in the ITT population who completed the study without major protocol deviations (n=30). The following subject was excluded from the PP population: ■ Subject SN01-040-V5 for all parameters ■ Safety population: all randomized subjects in the study who received at least one dose of the product (n=30)

Table 18

[0189] Software Environment ■ Statistical analysis was performed using Biofortis with SAS® software version 9.3 (SAS Institute Inc., Cary, NC, USA). ■ Significance level ■ In all statistical tests (two-tailed), a significance level of 0.05 was used to justify claims of statistically significant effects.

[0190] ■ How to process missing data in dynamic data ■ If the dynamics data contains more than 2 values ​​or two consecutive values, the AUC calculation cannot be performed, and the dynamics data is considered missing in the statistical analysis (missing data was not replaced). ■ If data is missing at T-10, AUC calculation cannot be performed, and the dynamics are considered missing in the statistical analysis (no replacement of missing data was performed); ■ If values ​​(excluding baseline and last-point values) were missing from the dynamics data, they were replaced with values ​​obtained using the CopyMean method developed by Genolini (Genolini, 2013). ■ If the value at the final point in time of the dynamics (T1440=T24h) is missing, the missing data was not replaced. ■ AUC cannot be calculated when data processing is incomplete and dynamics are not fully understood. →This method was applied to the ITT and PP populations.

[0191] Derived variables ■ Total curcuminoids = curcumin + DMC + BDMC + THC + HHC + curcumin glucuronide + DMC glucuronide + BDMC glucuronide + THC glucuronide + HHC glucuronide + curcumin sulfate + DMC sulfate + BDMC sulfate + THC sulfate + HHC sulfate If all 15 of these elements are missing, the total curcuminoids cannot be calculated. If at least one of these 15 elements is quantified, the total curcuminoids can be calculated. ■ Total parent compound = sum of curcumin + DMC + BDMC ■ Total parent compounds and their related sulfate and glucuronide metabolites = curcumin + curcumin glucuronide + curcumin sulfate + DMC + DMC glucuronide + DMC sulfate + BDMC + BDMC glucuronide + BDMC sulfate

[0192] ■ Curcumin and its related sulfates and glucuronide metabolites = Curcumin + Curcumin glucuronide + Curcumin sulfate ■ DMC and its related sulfate and glucuronide metabolites = DMC + DMC glucuronide + DMC sulfate ■ BDMC and its related sulfate and glucuronide metabolites = BDMC + BDMC glucuronide + BDMC sulfate ■ Curcumin and all its related metabolites = Curcumin + Curcumin glucuronide + Curcumin sulfate + THC + THC glucuronide + THC sulfate + HHC + HHC glucuronide + HHC sulfate

[0193] ■ Relative bioavailability from 0 to 24 hours = Ratio of dose-normalized AUC0-24h for different test formulations to the dose-normalized AUC0-24h obtained for the reference product (turmeric extract 95% curcuminoids). ■ Relative bioavailability from 0 to infinity = Ratio of dose-normalized AUC0-∞ for different test formulations to dose-normalized AUC0-∞ obtained for the reference product (turmeric extract 95% curcuminoids).

[0194] Data processing of values ​​below the limit of detection (LOD) ■ For curcumin (natural, glucuronide, sulfate), which is represented as 《0.62》 in the database, several values ​​below the limit of detection (LOD) were identified. →The number and percentage of values ​​below LOD were given for each parameter and visit.

[0195] Create a graph display ■ Quantitative variable of the observed mean: Box plot of the AUC parameter (example shown in the figure below) [Table 19]

[0196] Confirmation of assumptions in statistical testing ■ Assumptions of normality and equal variances were investigated by graphical representation of the residuals generated by the statistical model. In cases of strong deviations from normality and / or equal variances, a logarithmic transformation (log10) of the study endpoint was considered. Notes on creating the results: ■ STE = Standard Turmeric Powder Extract 95% Curcuminoids 1500mg ■ TEP=Curcumin C3 complex California Gold Nutrition(1500mgC3 complex(registered trademark)) ■ NOV=Curcumin Cell'Innov (1000 mg Novasol(registered trademark)) ■ PHYT = Curcuma Platinum MannaVital (1000mg Meriva (registered trademark)) ■ Turmipure GOLD TM=Turmipure Gold 30% curcuminoids300mg

[0197] statistical methodology ■ Primary endpoint: Dose-normalized AUC for 0-24 hours was analyzed using the following mixed model for repeated measures (SAS® PROCMIXED, statistical model n°1): Y = Product + Visit + Baseline + Target ランダム Along with the following: ■ Y: Dose-normalized AUC of analyte plasma concentration over 0-24 hours; ■ Product: Turmipure Gold TM STE, TEP, NOV, PHYT; ■ Visit: Visit V1~V5; ■ Baseline: Parameter values ​​at time T-10 (T0 in the case of AUC calculation); ■ Target ランダム : Random factor. ■ In the case of a significant visit effect (p<0.05): A second analysis is conducted during the first period (visit) to evaluate the product effect. ■ Comparison of products of interest → Turmipure Gold compared to STE TM

[0198] Additional analysis: Investigation of gender effects ■ In this study, the following mixed model (SAS® PROCMIXED, statistical model n°2) was used to investigate the gender effect for repeated measures: Y = Product + Visit + Gender + Product * Gender + Baseline + Target ランダム Along with the following: ■ Y: Endpoint; ■ Product: Turmipure Gold TM STE, TEP, NOV, PHYT; ■ Visit: Visit V1~V5; ■ Gender: Female or male; ■ Products * Gender: The interaction between products and gender; ■ Baseline: Parameter values ​​at time T-10 (T0 in the case of AUC calculation); ■ Target ランダム : Random factor. ■ Comparison of products of interest; ■ Turmipure Gold compared to STE TM

[0199] ■ TEP compared to STE; ■ NOV compared to STE; ■ PHYT compared to STE; ■ Turmipure Gold TM TEP compared to; ■ Turmipure Gold TM NOV compared to NOV; ■ Turmipure Gold TM PHYT compared to this. ■ In the case of a significant visit effect (p<0.05): A second analysis is conducted during the first period (visit) to evaluate the product effect. ■ Significant Products * In the case of gender interaction effects (p<0.05): Treatment effects investigated separately for men and women (with the creation of descriptive statistics and graphs). ■ In the case of a significant product-gender interaction effect (p<0.05): Treatment effect investigated overall (women and men together)

[0200] Statistical methodology for the second endpoint (excluding relative bioavailability) ■ The second endpoint was analyzed using the following mixed model (SAS® PROCMIXED, statistical model n°1) for repeated measures: Y = Product + Visit + Baseline + Target ランダム ■ In the case of a significant visit effect (p<0.05): A second analysis is conducted during the first period (visit) to evaluate the product effect. ■ Comparison of products of interest; ■ Turmipure Gold compared to STE TM ■ TEP compared to STE; ■ NOV compared to STE; ■ PHYT compared to STE; ■ Turmipure Gold TM TEP compared to; ■ Turmipure Gold TM NOV compared to NOV; ■ Turmipure Gold TM PHYT compared to this.

[0201] ■ Statistical methodology of relative bioavailability ■ The second endpoint was analyzed using the following mixed model (SAS® PROCMIXED, statistical model n°1) for repeated measures: Y = Product + Visit + Baseline + Target ランダム ■ In the case of a significant visit effect (p<0.05): A second analysis is conducted during the first period (visit) to evaluate the product effect. ■ Comparison of products of interest; ■ Turmipure Gold TM TEP compared to; ■ Turmipure Gold TM NOV compared to NOV; ■ Turmipure Gold TM PHYT compared to this.

[0202] Summary of results for the primary endpoint Tables 21 and 22: Initial analysis of ITT and PP populations [Table 20-1] [Table 20-2]

[0203] Tables 23 and 24: Additional analysis of ITT and PP groups (investigation of gender effects) [Table 21]

[0204] [Table 22]

[0205] Based on the above analysis, the following conclusions were drawn regarding the ITT group: Intergroup analysis (combining all genders) ■ No significant visits were identified → As a result, analysis was performed on all visits. ■ Significant product effect (p<0.0001): ■ Primary endpoint: Turmipure GOLD TM There is a statistically significant difference between and STE (adjusted p<0.0001; diff[(adjusted CI95%]=1.32[1.18;1.46]). ■ Other comparisons: ■ TEP vs STE (adjusted p=0.6948) ■ NOV vs STE (adjusted p<0.0001; diff[adjusted CI95%]=1.62[1.48;1.76]) → NOV > STE ■ PHYT vs STE (adjusted p < 0.0001; diff [adjusted CI 95%] = 0.48 [0.34; 0.62]) → PHYT > STE ■ TEPvsTurmipure GOLD TM (Adjusted p<0.0001; diff[adjusted CI95%]=-1.39[-1.53;-1.25])→TEP <Turmipure GOLD TM ■ NOVvsTurmipure GOLD TM (Adjusted p<0.0001; diff[adjusted CI95%]=0.29[0.15;0.43]) → NOV>Turmipure GOLD TM ■ PHYTvsTurmipure GOLD TM (Adjusted p<0.0001; diff[adjusted CI95%]=-0.84[-0.99;-0.70])→PHYT <Turmipure GOLD TM ■

[0206] ■ Additional analysis: Investigation of gender effects ■ Significant visits (p=0.2456) and products * No effect of gender interaction (p=0.3804): Analysis was performed for all genders together and for all visits. ■ Significant product effect (p<0.0001): ■ TEP vs STE (adjusted p=0.7091) ■ NOV vs STE (adjusted p<0.0001; diff[adjusted CI95%]=1.61[1.47;1.75]) → NOV > STE ■ PHYT vs STE (adjusted p < 0.0001; diff [adjusted CI 95%] = 0.48 [0.34; 0.62]) → PHYT > STE ■ Turmipure GOLD TM vsSTE (adjusted p<0.0001; diff[adjusted CI95%]=1.32[1.18;1.46]) → Turmipure GOLD TM >STE ■ TEPvsTurmipure GOLD TM (Adjusted p<0.0001; diff[adjusted CI95%]=-1.39[-1.52;-1.25])→TEP <Turmipure GOLD TM ■ NOVvsTurmipure GOLD TM (Adjusted p<0.0001; diff[adjusted CI95%]=0.29[0.15;0.43]) → NOV>Turmipure GOLD TM ■ PHYTvsTurmipure GOLD TM(Adjusted p<0.0001; diff[adjusted CI95%]=-0.84[-0.98;-0.70])→PHYT <Turmipure GOLD TM

[0207] About the PP group: ■ Intergroup analysis (combining all genders) ■ The results are similar to those observed in the ITT population. ■ Additional analysis: Investigation of gender effects ■ The results are similar to those observed in the ITT population.

[0208] Summary of results for the secondary endpoint The results for the secondary endpoints are shown in the tables below for both the ITT (Tables 26-42) and PP (Tables 43-59) populations. The results are illustrated in Figures 24-37. The following keys are applied to each table, and the statistical significance of each result is shown. [Table 23]

[0209] [Table 24]

[0210] [Table 25]

[0211] [Table 26]

[0212] [Table 27]

[0213] [Table 28]

[0214] Table 29

[0215] Table 30

[0216] Table 31

[0217] Table 32

[0218] Table 33

[0219] Table 34

[0220] Table 35

[0221] Table 36

[0222] Table 37

[0223] Table 38

[0224] Table 39

[0225] Table 40

[0226] Table 41

[0227] Table 42

[0228] Table 43

[0229] Table 44

[0230] Table 45

[0231] Table 46

[0232] Table 47

[0233] Table 48

[0234] Table 49

[0235] [Table 50]

[0236] [Table 51]

[0237] [Table 52]

[0238] [Table 53]

[0239] [Table 54]

[0240] [Table 55]

[0241] [Table 56]

[0242] [Table 57]

[0243] Serious adverse events : ■ Subject SN01-009: Neck pain / V0~V1 visit traffic accident (no research product) (with motor function / event unrelated to rheumatoid body tissue, disease history, moderate intensity, no action taken with the research product, event unrelated to the investigation and research product, event unrelated to corrective treatment, recovery without sequelae).

[0244] ■ Treatment of severe emergency AEs: ■ Target SN01-007: Lower back pain during V2~V3 visits (Turmipure GOLD) TM (Below the product) (Events unrelated to athletic ability / rheumatoid arthritis, medical history, severe intensity, no action taken on the research product, events unrelated to the investigation and research product, events unrelated to corrective treatment, recovery without sequelae).

[0245] ■ AE for research products: ■ Target SN01-008: Headache on the day of V3 visit (Turmipure GOLD TM (Under the product) (Events unrelated to neurological / psychiatric tissue, medical history, moderate intensity, no action taken on the research product, events unrelated to the investigation and research product, events unrelated to corrective treatment, recovery without sequelae). ■ Target SN01-030: Headache on the day of V3 visit (Turmipure GOLD TM (Under the product) (Events unrelated to neurological / psychiatric tissue, medical history, moderate intensity, no action taken on the research product, events potentially related to the investigation and research product, events unrelated to corrective treatment (paracetamol), recovery without sequelae). ■ Subject SN01-032: Headache on the day of V2 visit (under NOV product) (event unrelated to neurological / psychiatric tissue, medical history, moderate intensity, no action taken against the research product, event potentially related to the investigation and research product, event unrelated to corrective treatment, recovery without sequelae).

[0246] The results observed in the PP group were similar to those in the ITT group. The mean ± SD (ITT population) for total curcuminoids is shown below.

[0247] [Table 58]

[0248] conclusion The results demonstrate that there is little difference in the bioavailability of the compounds found in TEP and STE (only 5 differences). Compositions within the scope of the present invention (Turmipure GOLD) TM It has been found to offer better bioavailability of the compound than STE, TEP, and PHYT, and can provide similar bioavailability to NOV even when administered at low doses (300 mg compared to 1000 mg), and uses a natural source of curcuminoid compounds. More specifically, Novasol was used in 1000mg doses, but in this invention (Turmipure GOLD TM The composition of the product was 300 mg. Since Turmipure produces an effect of 6520 at 300 mg, when used at the same dose as Novasol (1000 mg), it produces an effect of 21733, which is much higher than the effect of Novasol (8539) at the same dose.

[0249] Example 4 - General preparation of compositions used in the methods / uses described herein. A mixture of curcuminoids in water was prepared using an organically purified curcuminoid extract in distilled water (at least 10%, but preferably 95% purity (total curcuminoids)) (3 volumes powder weight / water).

[0250] A 58% gum arabic mixture (substrate) was prepared using distilled water (3 volumes of powder weight / water). To 500 ml of aqueous gum arabic solution, 500 ml of the curcuminoid solution prepared in Example 1 was added under stirring (5000 rpm), and then 2% organic quillaja, standardized with 5% organic sunflower oil and saponins, was added. The resulting mixture was stirred at 5000 rpm for 10 minutes. The resulting emulsion was then spray-dried.

[0251] Example 5 - Characterization of the composition used in the method / use described herein. The size and morphology of the compositions of the present invention were analyzed by dynamic light scattering (DLS), zeta potential (Z-potential), and scanning electron microscopy (SEM). For DLS and zeta potential analysis, a Zetasizer Nano ZS (NanoZS90, Malvern Instrument Ltd., UK) equipped with a He / Ne laser (λ=633nm) at a fixed scattering angle of 90° at a temperature of (25±0.1℃) was used. The samples used were in liquid emulsion form (the final step before drying). The samples were suspended in demineralized water at a volume concentration of 0.4%, and sonication was applied for 1 minute. DLS analysis was performed immediately on these samples (measurement time = 60 seconds). Zeta potential analysis was performed over a wide pH range (2-11). The samples were prepared and analyzed at different pH levels using 0.1 M HCl and 0.1 M NaOH solutions, as follows. The resulting 10 samples (pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) were stored at room temperature (23°C) (Figure 4).

[0252] As shown in Figure 18, the color of the composition of the present invention in water is caused by pH. The keto form (yellow) is the dominant form present in the solution when the pH range changes from acidic to neutral (2-7). At pH 8 and 9, the colored solution turns orange, and at pH 10 and 11, a translucent reddish color becomes dominant. The color change is due to the successive deprotonation of the hydroxyl groups of the curcumin molecule, which is caused by the increase in pH that gives curcumin high solubility and instability. The DLS analysis results are shown in Figure 19. There are two groups of particle sizes. One group is concentrated at 616±160 nm (20.8% of all particles), and the most interesting group is concentrated at 188±42 nm (79.2% of all particles). The average hydrodynamic particle size of curcumin loaded into an aqueous solution (pH 5.4) was found to be 476.5 nm with a PDI (polydispersion index) of 0.337.

[0253] Figure 20 shows the Z potentials of the compositions of the present invention at different pH values ​​(2-11). The higher the Z potential, the more unstable the mixture. The compositions of the present invention have a negative Z potential between pH 2 and pH 11. The particles are negatively charged in the aqueous phase. At pH 2, the Z potential is close to 0 (isoelectric point: pH at which the potential is zero), and there is an unstable zone for the emulsion. Between pH 2 and pH 4, the Z potential is relatively low (<25mV), and above pH 4, the sample enters the stable zone. This stability is strongly confirmed from pH 5. A rapid shift in the isoelectric point was observed at pH 8.0, and the zeta potential of loaded curcumin was surprisingly high in the pH range of 2-7. In the aqueous phase at pH < 4.0, loaded curcumin is mostly in the lowest surface energy state. At pH 8.0, loaded curcumin faces its low-charge side toward gum arabic and exposes its high-charge side to interact more readily with water, which results in an elevated zeta potential.

[0254] Example 6 - Particle size distribution (PSD) of the composition of the present invention using CQ-MO-304 Materials and Reagents material -Mastersizer 3000 from Malvern Instruments, or equivalent - Hydro 2000SM sample dispersion unit, or equivalent (for liquid phase), - Malvern AERO S sample dispersion unit, or equivalent (for solid phase). reagent -water

[0255] procedure Analysis parameters - Background time: 10 seconds - Measurement time @ 10 seconds -Refractive index of distilled water: 1.33 -Calculation of results: General purpose - Pump / Agitation Speed: 1800 RPM -Liquid dispersant: Water -Solid dispersant: Environmental air Specific parameters -100705 (Refractive index: 1, Absorption: 1) -100019 (Refractive index: 1, Adsorption: 2) -3CAA0075 and 3CAA0076 (Compositions of the present invention) Samples of the composition of the present invention were mixed with distilled water, and the samples were tested using either a Hydro 2000SM unit or a Mastersizer 3000 (using a Scirocco 2000 unit).

[0256] result Several batches of the composition of the present invention obtained after drying and grinding were tested according to the method described above. The results are shown in Table 61 below.

[0257] Table 61. Particle size distribution of the compositions of the present invention (where (D90) corresponds to 90% of the particle size population, and (D4:3) corresponds to the average volume moment of the particle size population). [Table 59-1] [Table 59-2] [Table 59-3]

[0258] Example 7 - Form of the composition of the present invention (by scanning electron microscope, SEM) For SEM analysis, samples were prepared as follows: The composition of the present invention in powder form was deposited onto a sample holder by simply dusting. The platinum / palladium deposit was then metallized and observed and photographed with a scanning electron microscope equipped with an energy-dispersive X-ray detector. The SEM images shown in Figures 21, 22, and 23 provide visualization of the composition of the present invention.

[0259] The composition of the present invention exhibits conjugates that self-assemble into spherical micelles having a size of + / - 170 nm. The roughly spherical morphology observed in SEM analysis supported the sizing analysis performed by dynamic light scattering. SEM analysis reveals that the particles in the composition of the present invention clearly exhibit a chitosan outer layer, which was not present in the uncoated lecithin nanoparticles. Curcumin was found to be well dispersed within the lecithin core of the nanoparticles. SEM measurements also support evidence of a roughly spherical shape, and surface roughness indicates surface absorption. This suggests that the driving force for this type of adsorption is either direct electrostatic interaction or ion-ion interaction.

Claims

1. (i) Curcuminoids; (ii) Quillaja extract; (iii) Gum arabic; and (iv) Vegetable oil A composition for oral administration or ingestion, comprising, wherein gum arabic is present in an amount of about 40% to about 65% by weight of the composition.

2. The composition according to claim 1, further comprising modified starch.

3. The composition according to claim 1 or 2, wherein the quillaja extract is present in an amount of about 0.1% to about 5% by weight of the composition.

4. The composition according to any one of claims 1 to 3, wherein curcuminoids are present in an amount of about 2.5% to about 60% by weight of the composition.

5. (i) Curcuminoids; (ii) Quillaja extract; (iii) Gum arabic, and (iv) Vegetable oil A composition according to any one of claims 1 to 4, comprising:

6. A composition according to any one of claims 1 to 5 for improving the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals.

7. The composition according to claim 6, wherein the improvement in the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals is due to improved gastrointestinal tolerance of curcuminoids and / or improved absorption of curcuminoids by intestinal cells and / or improved blood circulation of curcuminoids.

8. The composition according to claim 6 or 7, wherein the curcuminoid is selected from the group consisting of curcumin, demethoxycurcumin, bisdemethoxycurcumin, and mixtures thereof.

9. The composition according to any one of claims 6 to 8, wherein the mammal is a human.

10. The composition according to any one of claims 1 to 9, wherein the curcuminoids are in a free form.

11. Use of compositions comprising (i) curcuminoids, (ii) quillaja extract, (iii) gum arabic, and (iv) vegetable oil in the manufacture of oral or ingestible formulations for improving the bioaccessibility, bioavailability, bioefficacy, and / or bioactivity of curcuminoids in mammals.

12. The use according to claim 11, wherein the improvement in the bioaccessibility, bioavailability, bioefficacy and / or bioactivity of curcuminoids in mammals is due to improved gastrointestinal tolerance of curcuminoids and / or improved absorption of curcuminoids by intestinal cells and / or improved blood circulation of curcuminoids.

13. The use according to claim 11 or 12, wherein the curcuminoid is selected from the group consisting of curcumin, demethoxycurcumin, bisdemethoxycurcumin, and mixtures thereof.

14. The use according to any one of claims 11 to 13, wherein the mammal is a human.

15. The use according to any one of claims 11 to 14, wherein the curcuminoid is in a free form.

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