Compositions and methods using nettle concentrate for cellular energy

Abstract

A composition comprising nettle concentrate is provided. The composition can be administered to an individual in need for (i) improvement of a physiological condition associated with metabolic fatigue in one or more cells, and / or (ii) increased mitochondrial energy and increased mitochondrial calcium uptake in one or more cells, and / or (iii) enhancement of mitochondrial function, and / or (iv) treatment or prevention of calcium deficiency / depletion disorders in the individual. Additionally or alternatively, the composition can be administered for the treatment or prevention of the above in an individual in need of or at risk of treatment or prevention of mitochondrial-related diseases or conditions associated with altered mitochondrial function.

Description

[Technical Field]

[0001] The present invention relates, in general, to compositions and methods for managing energy at the cellular level using nettle concentrate. In some embodiments, these compositions and methods can enhance mitochondrial function in middle-aged, elderly, or ICU patients, increase bioenergetics by activating mitochondrial calcium uniporters, and thereby promote cellular activation. [Background technology]

[0002] Population aging is a noteworthy phenomenon in terms of demographics. As life expectancy increases, the increase in the elderly population is outpacing the increase in the total population, and the proportion of the elderly to the rest of the population is increasing significantly, coupled with a declining birth rate. For example, in the 1950s, one in twelve people was over 60 years old, but by the end of the 2000s, this figure had risen to one in ten. It is predicted that by the end of the 2050s, one in five people worldwide will be over 60 years old.

[0003] In middle-aged and older adults, cognitive impairment, including a degree of decline in physical function and / or age-related cognitive decline, is common, and age-related changes in brain morphology and cerebrovascular function are generally observed. Cognitive decline is consistently reported in conjunction with aging in a wide range of cognitive areas, including processing speed, attention, episodic memory, spatial ability, and executive function. Brain imaging analyses have shown that these normal age-related cognitive declines are associated with a decrease in the volume of both gray and white matter in the brain, with the striatum frontalis system being the most severely damaged with age. This decrease in cortical volume may be due to many harmful cellular processes associated with normal aging, such as the time-dependent accumulation of damage from free radicals that lead to oxidative damage, chronic mild inflammation, homocysteine ​​accumulation (which, when increased, is a risk factor for cognitive impairment and dementia), and decreased mitochondrial function. In addition to direct cellular damage, the brain also suffers indirect damage from damage to the microvascular structure. It is clear that the pathology of aging, and even dementia, involves complex interactions between these interconnected factors. For example, mitochondrial dysfunction can result in increased oxidative stress, which can trigger inflammation and vascular damage.

[0004] Mitochondria are the primary source of aerobic energy production in mammalian cells and maintain a large Ca2+ gradient across the inner membrane, providing signaling potentials for this molecule. Furthermore, mitochondrial Ca2+ may play a role in regulating ATP production within mitochondria and contributing to the orchestration of cellular metabolic homeostasis (Glancy, B. et al. (2012). "Role of mitochondrial Ca2+ in the regulation of cellular energetics." Biochemistry 51(14):2959-2973). Changes in mitochondrial Ca2+ homeostasis have been associated with various pathological conditions and are important in the pathogenesis of several human diseases (Arduino et al. Journal Physiol. 2018 Jul;596(14):2717-2733).

[0005] In recent years, nutrition, education, exercise, and cognitive exercise have been demonstrated as effective interventions to prevent age-related declines in physical and cognitive function. Extensive clinical, epidemiological, and individualized evidence supports the reduction of dementia risk and age-related neurodegeneration by individual nutritional factors. However, the results of formal trials on nutritional interventions have been inconsistent (Schmitt et al., Nutrition Reviews 68:S2-S5 (2010)). Furthermore, while efforts have been made to utilize mitochondrial Ca2+ transport mechanisms for therapeutic interventions, there are currently no pharmacological compounds that induce or selectively regulate mitochondrial Ca2+ homeostasis.

[0006] Therefore, it is desirable to provide compounds that induce and selectively regulate mitochondrial Ca2+ homeostasis. It is also desirable that such compounds be natural. Furthermore, it is desirable that such compounds be suitable for vegetarian or vegan diets.

[0007] No reference to prior art documents in this specification should be considered to acknowledge that such prior art is well known or that it forms part of a common general understanding in the art.

[0008] [Overview of the prefecture] The present invention aims to improve the current state of the art, and in particular to overcome the problems of the prior art and to provide compositions, unit dosage forms and methods that address the above needs, or at least provide useful alternatives.

[0009] The inventors have surprisingly found that the object of the present invention can be achieved by the subject matter of the independent claim. The dependent claims further develop the idea of ​​the present invention.

[0010] In particular, considering the experimental data disclosed later herein, the nettle concentrate disclosed herein is thought to improve the energy production efficiency of mitochondria.

[0011] Accordingly, one embodiment of the present invention is a composition comprising nettle concentrate in an effective amount for use in (i) improving the physiological state associated with metabolic fatigue in one or more cells, and / or (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and / or (iii) enhancing mitochondrial function, and / or (iv) treating or preventing calcium deficiency / depletion disorders in an organism, Nettle concentrate, a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending nettle material suspensions to obtain nettle material slurry, c) A step of applying physical means to a nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate. We propose a composition that can be obtained or is obtained by a method including the following.

[0012] In certain embodiments, at least a portion of one or more cells is part of at least one body part selected from the group consisting of the liver, kidneys, brain, and skeletal muscle.

[0013] In further specific embodiments, the physiological conditions associated with metabolic fatigue include muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, particularly a lack of physical energy. In further specific embodiments, an effective amount of nettle concentrate is administered orally daily for at least one week.

[0014] In additional specific embodiments, the composition further comprises at least one compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

[0015] In further specific embodiments, the composition is selected from the group consisting of food compositions, dietary supplements, nutritional compositions, complete nutritional compositions, pharmaceuticals, oral nutritional supplements, medical foods, nutraceuticals, beverages, powdered nutritional products that are reconstituted with water or milk before ingestion, food additives, foods for specific medical purposes (FSMPs), medicinal products, pet foods, and combinations thereof.

[0016] In further specific embodiments, the composition may be in the form of a solid powder, powder stick, capsule, or solution.

[0017] In further specific embodiments, an effective amount of nettle concentrate is administered in a food product or beverage further comprising components selected from the group consisting of proteins, carbohydrates, fats, and mixtures thereof.

[0018] Another embodiment of the present invention is a composition for use in an effective amount for the treatment and / or reduction of mitochondrial-related diseases or conditions associated with altered mitochondrial function, in individuals who require or are at risk of such treatment and / or reduction, the treatment and / or reduction of the incidence and / or severity thereof, wherein the method comprises orally administering an effective amount of mitochondrial concentrate to an individual who requires or is at risk of such treatment and / or reduction. Nettle concentrate, a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending nettle material suspensions to obtain nettle material slurry, c) A step of applying physical means to a nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate. We propose a composition that can be obtained or is obtained by a method including the following.

[0019] In certain embodiments, the mitochondrial-related disease or condition is selected from the group consisting of stress, physiological aging, obesity, decreased metabolic rate, metabolic syndrome, type 2 diabetes, diabetic complications, hyperlipidemia, neurodegenerative diseases, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal death or dysfunction, musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, renal insufficiency, trauma, infections, cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

[0020] Another embodiment of the present invention is a composition comprising an ellagitannin concentrate for use in an effective amount to delay the onset of metabolic decline, maintain muscle mass and / or muscle function, maintain immune function, and / or maintain cognitive function in healthy middle-aged and elderly individuals, wherein the ellagitannin concentrate a) suspending an ellagitannin material in an aqueous liquid to form an ellagitannin material suspension; b) blending the ellagitannin material suspension to obtain an ellagitannin material slurry; c) applying physical means to the ellagitannin material slurry to separate and obtain the ellagitannin concentrate; d) optionally, drying the ellagitannin concentrate; and is obtainable or obtained by a method comprising the above steps.

[0021] Another embodiment of the present invention is a composition comprising an ellagitannin concentrate for use in an effective amount to enhance at least one of mental performance or muscle performance in an individual, wherein the ellagitannin concentrate a) suspending an ellagitannin material in an aqueous liquid to form an ellagitannin material suspension; b) blending the ellagitannin material suspension to obtain an ellagitannin material slurry; c) applying physical means to the ellagitannin material slurry to separate and obtain the ellagitannin concentrate; d) optionally, drying the ellagitannin concentrate; We propose a composition that can be obtained or is obtained by a method including the following.

[0022] Another embodiment of the present invention is a composition comprising nettle concentrate for use in an effective amount for improving or maintaining cognitive function in an individual, Nettle concentrate, a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending nettle material suspensions to obtain nettle material slurry, c) A step of applying physical means to a nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate. We propose a composition that can be obtained or is obtained by a method including the following.

[0023] In certain embodiments, cognitive functions are selected from the group consisting of perception, memory, attention, speech comprehension, speech production, reading comprehension, image creation, learning, reasoning, and combinations thereof.

[0024] In all of the aforementioned embodiments, in specific embodiments, an effective amount of nettle concentrate may be administered in a composition further containing calcium. In all of the aforementioned embodiments, in specific embodiments, the individual is a middle-aged, elderly, or ICU patient.

[0025] Another embodiment of the present invention is a unit dosage form comprising nettle concentrate for use in an effective amount for at least one of the following: (i) treatment of mitochondrial-related diseases or conditions associated with altered mitochondrial function, reduction of their incidence, or reduction of their severity, and / or (ii) improvement of physiological conditions associated with metabolic fatigue in one or more cells, and / or (iii) increase of mitochondrial energy and mitochondrial calcium uptake in one or more cells, and / or (iv) treatment or prevention of calcium deficiency / depletion disorders, and / or (v) increase of metabolic rate, and / or (vi) improvement or maintenance of cognitive function, and / or (viii) improvement or maintenance of mitochondrial function, Nettle concentrate, a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending nettle material suspensions to obtain nettle material slurry, c) A step of applying physical means to a nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate. We propose a unit dosage form that can be obtained or is obtained by a method including the following.

[0026] In certain embodiments, physiological conditions associated with metabolic fatigue include muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, and in particular, deficient physical energy.

[0027] These and other aspects, features and advantages of the present invention will become further apparent to those skilled in the art from the detailed description of embodiments of the invention in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0028] [Figure 1] The figures show the iron content (mg / kg) based on dry weight (DW) in the nettle material and the corresponding nettle concentrate obtained from the nettle material according to the method of Example 1. The values ​​represent the average between three independent samples, and the error bars represent the standard deviation. In Figure 1, "nettle material" is understood to be a combination of the leaves and stems of the dried nettle material that has been ground into a powder before further processing in the concentration method of Example 1. [Figure 2] Figure 2 shows the molar ratio (M / M) of iron and oxalic acid in the nettle material and the corresponding nettle concentrate obtained from the nettle material according to the method of Example 1. The values ​​represent the average between two consecutive measurements. In Figure 2, "nettle material" is understood to be a combination of the leaves and stems of the dried nettle material that has been ground into a powder before further processing in the concentration method of Example 1. [Figure 3]Figure 3 shows the molar ratio (M / M) of iron and phytic acid in the nettle material and the corresponding nettle concentrate obtained from the nettle material according to the method of Example 1. The values ​​represent the average between two independent samples, and the error bars represent the standard deviation. In Figure 3, "nettle material" is understood to be a combination of the leaves and stems of the dried nettle material that has been ground into a powder before further processing in the concentration method of Example 1. [Figure 4] The bioaccessibility of iron in nettle concentrates prepared with water (according to the method of Example 1) and nettle concentrates prepared with water in the presence of citric acid and hydrochloric acid (according to the method of Example 2) is shown. Values ​​represent the mean between two independent samples, and error bars represent the standard deviation. [Figure 5] Figure 5 shows the absolute amount of bioaccessible iron contained in the nettle material and in the corresponding nettle concentrate prepared with water according to the concentration method of Example 1, or prepared with water in the presence of citric acid or hydrochloric acid, respectively, according to the concentration method of Example 2. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. The value represents the average between two sets of independent samples, and the error bars represent the standard deviation. In Figure 5, "nettle material" is understood to be a combination of the leaves and stems of the dried nettle material that has been pulverized into a powder before further processing in the concentration method of Example 1 or Example 2. [Figure 6] This graph shows the time course of total mitochondrial Ca2+ uptake (expressed in relative luminescence units (RLU)) in C2C12-derived myotubes in the absence of nettle concentrate (no stimulation) or in the presence of nettle concentrate at different concentrations (1 / 10 to 1 / 80 dilution) prepared according to the method of Example 1. Arrows indicate the time points at which nettle concentrate at different concentrations (1 / 10 to 1 / 80 dilution) or standard aequorin buffer alone (no stimulation) was added. The results are representative of eight biological experiments. [Figure 7]This bar graph shows the total mitochondrial Ca2+ uptake in C2C12-derived myotubes after either no stimulation (i.e., only the addition of aequorin buffer) or stimulation with different concentrations of nettle concentrate (1 / 10 to 1 / 80 dilution) prepared according to the method of Example 1. Results are expressed as the mean ± SEM from n=8 biological replicates. * indicates a statistically significant difference between conditions with P<0.05 (one-way ANOVA test). [Modes for carrying out the invention]

[0029] When used herein, terms such as “comprise” and “comprising” should be interpreted not as exclusive or exhaustive, but as encompassing, i.e., “including, but not limited to.” Similarly, terms such as “include,” “including,” and “or” should all be interpreted as encompassing, unless such interpretation is clearly prevented by the context. However, compositions disclosed herein may not include elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term “comprising” include disclosures of embodiments that “essentially include / essentially consist of” the specified components, and embodiments that “consist of” the specified components.

[0030] When used herein, “compositions essentially comprising nettle concentrate” and “compositions essentially comprising calcium and nettle concentrate” do not include any additional compounds other than the nettle concentrate disclosed herein and any calcium that would affect mitochondrial calcium translocation. In certain non-limiting embodiments, the composition comprises an excipient, the nettle concentrate disclosed herein, and optionally calcium.

[0031] All numerical ranges should be understood to include all integers, whole numbers, or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims that cover any number or subset of a number within that range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, and 3.5 to 9.9.

[0032] All percentages given herein are by total weight of the composition unless otherwise stated. As used herein, “about,” “approximately,” and “substantially” are understood to mean a range of values, for example, within the range of -10% to +10% of the reference value, preferably within the range of -5% to +5% of the reference value, more preferably within the range of -1% to +1% of the reference value, and most preferably within the range of -0.1% to +0.1% of the reference value.

[0033] As used herein, the singular “one” (“a,” “an,” and “the”) includes multiple references unless otherwise indicated. Therefore, for example, a reference to “a bioactive compound” or “the bioactive compound” includes not only a single bioactive compound but also two or more bioactive compounds.

[0034] Unless otherwise stated, all percentages herein refer to weight percentages, where applicable.

[0035] Unless otherwise defined, all technical and scientific terms have the same meaning as those commonly understood by those skilled in the art in the field to which this invention pertains, and should be given the same meaning.

[0036] Where used herein, the term "and / or" in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "both X and Y". For example, "at least one of mental performance or muscular performance" means "mental performance", "muscular performance", or "both mental performance and muscular performance".

[0037] When used herein, the terms “example” and “such as” are merely illustrative and descriptive, and should not be considered exclusive or inclusive, especially when followed by a list of terms. However, disclosures of embodiments using the terms “example” and “such as” include disclosures of embodiments in which these terms are exclusive and / or inclusive.

[0038] As used herein, “associated with” and “linked with” mean that they occur simultaneously, preferably caused by the same underlying condition, and most preferably caused by one of the specified conditions.

[0039] As used herein, the terms “food,” “food product,” and “food composition” mean a product or composition intended for consumption by an individual, such as a human, and providing to such individual at least one nutrient. The compositions of this disclosure, including many embodiments described herein, may include, be composed of, or essentially include the elements disclosed herein, as well as any additional or optional raw materials, components, or elements described herein or not described herein that are useful in a diet.

[0040] As used herein, the terms “beverage” and “drink” generally refer to “food,” “food product,” or “food composition” that is consumed by drinking. In particular, the terms “beverage” and “drink” are used interchangeably.

[0041] As used herein, the terms “treatment” and “treatment” mean administering a composition disclosed herein to an object having a condition for the purpose of reducing, reducing or improving at least one symptom associated with that condition, and / or delaying, reducing or preventing the progression of that condition. The terms “treatment” and “treatment” include both deterrent or preventive treatment (treatment to prevent and / or delay the onset or progression of a targeted pathological condition or disorder) and curative, therapeutic, or disease-modifying treatment, such as therapeutic means for the cure, delay, reduction of symptoms, and / or cessation of progression of a diagnosed pathological condition or disorder, and treatment of patients at risk of or suspected of having the condition, and patients in poor health or diagnosed with a disease or medical condition. The terms “treatment” and “treatment” do not necessarily mean treating until the object is fully recovered. The terms “treatment / therapy” and “to treat / therapy” also refer to maintaining and / or promoting the health of an individual who is not suffering from a disease but is susceptible to unhealthy conditions. The terms “treatment / therapy” and “to treat / therapy” also aim to include the synergistic effect, or otherwise enhancement, of one or more primary preventive or therapeutic measures. In non-limiting examples, treatment / therapy may be performed by a patient, caregiver, physician, nurse, or other healthcare professional.

[0042] Therapies for both humans and animals are within the scope of this disclosure. Preferably, the nettle concentrates disclosed herein are administered in serving or unit dosage forms that provide a therapeutic or prophylactic effective dose.

[0043] As used herein, the terms “prevent” and “prevention” mean administering a composition disclosed herein to a subject who is not exhibiting any symptoms of the condition in order to suppress or prevent the onset of at least one symptom associated with the condition. Furthermore, “prevention” includes reducing the risk, incidence, and / or severity of the condition or disorder.

[0044] As used herein, “effective amount” means an amount that, in an individual, treats or prevents a deficiency, treats or prevents a disease or medical condition, or, more generally, reduces symptoms, controls the progression of a disease, or provides nutritional, physiological or medical benefit to the individual.

[0045] As used herein, relative terms such as “improved,” “enhance,” “increased,” “enhance,” “boosted,” and “enhance” refer to the effect of the compositions disclosed herein, i.e., compositions containing the nettle concentrate disclosed herein, compared to administration over the same period of time of a composition that lacks / does not contain the nettle concentrate disclosed herein but is otherwise identical.

[0046] As used herein, “administer” includes one individual providing the composition mentioned to another individual so that the individual can ingest the composition, as well as the individual’s own actions of ingesting the composition mentioned.

[0047] As used herein, the term “animal” includes, but is not limited to, mammals, including rodents; aquatic mammals; domestic animals, e.g., dogs, cats, and other pets; livestock, e.g., sheep, pigs, cattle, and horses; and humans. Where “animal,” “mammal,” or their plural forms are used, these terms also apply, depending on the context, to any animal from which the effects shown or intended may be demonstrated, for example, by the animal’s benefit from improved mitochondrial calcium transport. The terms “individual” or “subject” are often used herein to refer to humans, but this disclosure is not limited in that sense. Thus, the terms “individual” or “subject” refer to any animal, mammal, or human who may benefit from the methods and compositions disclosed herein.

[0048] As used herein, “subject” or “individual” means a mammal, preferably a human. In a human context, the term “elderly” means an age of at least 60 years, preferably over 63 years, more preferably over 65 years, and most preferably over 70 years. In a human context, the term “middle-aged and older” means an age of at least 45 years, preferably over 50 years, and more preferably over 55 years, and includes elderly. In a human context, the term “middle-aged and older” means an age of at least 45 years or older, preferably over 50 years, and more preferably over 55 years, and includes elderly.

[0049] As used herein, “frailty” is defined as a clinically identifiable condition in which increased vulnerability results from age-related declines in reserve capacity and function across multiple biological systems, impairing the ability to cope with everyday or acute stressors. Pre-frailty stages, in which one or two of these criteria are present, are identified as being at high risk of progressing to frailty.

[0050] As used herein, the terms “serving” or “unit dosage form” refer to physically discrete units that are interchangeable and suitable as unit doses for human and animal subjects, each unit containing a predetermined amount of a composition comprising a nettle concentrate disclosed herein in an amount sufficient to produce the desired effect, preferably together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the unit dosage form depend on the specific compounds used, the effect to be achieved, and the pharmacodynamics relating to each compound in the host body. In one embodiment, the unit dosage form may be a predetermined volume of liquid contained in a container such as a bottle.

[0051] As used herein, “oral nutritional supplements,” or “ONS,” are compositions comprising at least one major nutrient and / or at least one micronutrient, for example, in the form of a sterile liquid, semi-solid, or powder, and intended to supplement other nutritional intake, such as from food. Non-limiting examples of commercially available ONS products include MERITENE®, BOOST®, NUTREN®, and SUSTAGEN®. In some embodiments, ONS can be in the form of a liquid beverage, for example, with a liquid volume equal to one serving of the composition, and can be consumed without further liquid addition.

[0052] As used herein, "incomplete nutrition" preferably refers to a nutritional product in which the major nutrients (proteins, fats, and carbohydrates) or micronutrients contained are not in sufficient levels to serve as the sole source of nutrition for an animal administered the product. The term "complete nutrition" refers to a product that can serve as the sole source of nutrition for a given subject. An individual can obtain 100% of its nutritional requirements from a complete nutrition composition.

[0053] As used herein, “metabolic fatigue” means a decrease in mitochondrial function in one or more cells (e.g., one or more of the liver, kidneys, brain, and skeletal muscle) due to a lack of substrates within one or more cells and / or an accumulation of metabolites within muscle fibers, which inhibits either calcium release or the ability of calcium to stimulate mitochondrial function. Physiological conditions associated with metabolic fatigue may include muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, and in particular, a lack of physical energy.

[0054] As used herein, the term “added organic solvent” refers to an organic solvent that is exogenous to the nettle material and is added to the nettle material in addition to the nettle material for the preparation of the nettle material concentrate. The term “added organic solvent” excludes organic solvents that are originally present in the nettle material of the nettle material concentrate.

[0055] As used herein, the term "vegan" refers to an edible composition that contains no animal products or animal-derived products whatsoever.

[0056] As used herein, the term "vegetarian" refers to an edible composition that does not contain meat, such as fish.

[0057] As used herein, the term "bioaccessibility" refers to the proportion of the total amount of a substance that is theoretically available for absorption.

[0058] As used herein, the terms “blend” and “mixture” may be used interchangeably.

[0059] How to obtain nettle concentrate The nettle concentrate provided herein is a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending and mixing nettle material suspensions to obtain nettle material slurry, c) A step of applying physical means to a nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate. It can be obtained or is obtained by a method that includes

[0060] In one embodiment, the ratio of nettle material to aqueous liquid in the nettle material suspension is 1:3 to 1:20, preferably 1:5 to 1:20, more preferably 1:10 to 1:20, and most preferably 1:12 to 1:18.

[0061] Nettle material contains nettle plant cells. Similarly, nettle material suspension contains nettle plant cells, which are inact nettle plant cells. The nettle plant cells in nettle material suspension are derived from nettle material.

[0062] The nettle material suspension is a product resulting from step a). The nettle material slurry is a product resulting from step b). The nettle material suspension and the nettle material slurry are different. Specifically, in the nettle material slurry, nettle plant cells are destroyed and intracellular substances of the nettle plant cells are released, but in the nettle material suspension, nettle plant cells are not destroyed, and as a result, intracellular substances of the nettle plant cells are not released.

[0063] The aqueous liquid, and therefore the nettle material suspension, does not contain any added organic solvents. For example, an aqueous liquid, and therefore a nettle material suspension, does not contain added organic solvents selected from the list consisting of acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 3-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (Glym, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, naphthalene, pentane, 1-propanol, 2-propanol, pyridine, toluene, triethylamine, tricine, tris, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0064] In a preferred embodiment, the aqueous liquid in step a) contains at least 80% by weight of water, more preferably 90% by weight of water, and even more preferably at least 95% by weight of water. Most preferably, the aqueous liquid in step a) is water.

[0065] In one embodiment, an osmotic regulator may be further added to the nettle material suspension before step b). The osmotic regulator may be selected from a list consisting of glucose, glycerol, sucrose, sorbitol, sodium chloride, potassium chloride, or a combination thereof. Preferably, the osmotic regulator is sucrose. The amount of osmotic regulator added to the nettle material suspension can be easily determined by those skilled in the art, depending on the type of osmotic regulator and the osmotic pressure of the suspension. The osmotic regulator may be used to adjust the osmotic pressure. Although not bound by theory, the osmotic regulator may contribute to preserving certain plant structures that store bioactive compounds and / or micronutrients, thus avoiding their dissolution due to osmosis. Although not bound by theory, this may make it possible to further improve the stability of bioactive compounds and / or micronutrients.

[0066] In a preferred embodiment, an acid may be further added to the nettle material suspension before step b).

[0067] In one embodiment, the acid is added to the nettle material suspension before step b) until the pH reaches 2 to 5.5, preferably 2.5 to 4.5, most preferably 3 to 4.

[0068] The acid may be selected from the group consisting of hydrochloric acid, citric acid, malic acid, ascorbic acid, acetic acid, lactic acid, propionic acid, fumaric acid, tartaric acid, phosphoric acid, adipic acid, succinic acid, gluconic acid, or mixtures thereof. Preferably, the acid is selected from the group consisting of malic acid, citric acid, hydrochloric acid, or mixtures thereof. In a more preferred embodiment, the acid is selected from the group consisting of citric acid, hydrochloric acid, or mixtures thereof. In the most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid. The acid may be provided as a pure acid solution, a diluted acid solution, or as an acid-containing food ingredient. Examples of acid-containing food ingredients include citrus juices, such as lemon juice, lime juice, orange juice, and tangerine juice.

[0069] The nettle material suspension may contain 0.01% to 5% by weight of acid.

[0070] When the acid is citric acid, the nettle material suspension may contain 0.01 to 3% by weight of citric acid, preferably 0.5 to 2.3% by weight of citric acid.

[0071] If the acid is hydrochloric acid, the nettle material suspension may contain 0.01% to 0.5% by weight of hydrochloric acid, preferably 0.02% to 0.54% by weight of hydrochloric acid.

[0072] When the acid is malic acid, the nettle material suspension may contain 0.01% to 3% by weight of malic acid, preferably 0.5% to 2.5% by weight of malic acid.

[0073] The use of acid has a dual effect. In particular, the acid lowers the pH and chelates micronutrients, especially iron. Lowering the pH and chelation contribute to improving the solubility of micronutrients, especially iron, and therefore contribute to improving the bioaccessibility of micronutrients, especially iron, in nettle concentrate.

[0074] In some embodiments, nettle refers to any plant of the genus Urtica, preferably any edible plant of the genus Urtica. Preferably, nettle refers to Urtica dioica.

[0075] The nettle material may include any part of the nettle plant, such as leaves, stems, flowers, buds, roots, etc. In one embodiment, the nettle material includes nettle leaves and / or stems. Preferably, the nettle material includes a substantial amount of leaves. The nettle material includes at least 80% by weight of nettle leaves, more preferably at least 90% by weight of nettle leaves, even more preferably at least 95% by weight of nettle leaves, and even more preferably at least 98% by weight of nettle leaves. The remainder of the nettle material may be any part of the nettle plant other than the nettle leaves disclosed herein. In one embodiment, the remainder of the green plant material consists only of nettle stems.

[0076] In the most preferred embodiment, the nettle material consists only of nettle leaves.

[0077] The leaves are generally edible and are preferable because they contain a high proportion of bioactive compounds and / or micronutrients. Therefore, the leaves are a good edible raw material for concentrating a considerable amount of bioactive compounds and / or micronutrients from nettle.

[0078] In one embodiment, the nettle material is dried nettle material and / or fresh nettle material. For example, the nettle material is dried nettle leaves and / or fresh leaves. Advantageously, the nettle material is dried nettle material. Dried nettle material has a longer shelf life than fresh nettle material, making it more convenient for handling on an industrial scale.

[0079] In one embodiment, if the nettle material includes or is dried nettle plant material, the dried nettle material may be pulverized into a powder before step a). The dried nettle material may be pulverized by dry grinding. Dry grinding can be achieved using any machine that provides shearing or includes a cutting device. For example, dry grinding can be performed using a hammer mill, stone mill, roller mill, ball mill, jet mill, colloid mill, agitated medium mill, bead mill, pin mill, roller grinder, roller refiner, impact mill, freeze grinder, rod mill, vibrating mill, cutting mill, disc mill, perforated disc mill, microcut mill, or extruder.

[0080] In one embodiment, blending step c) may be carried out by any type of shearing or mixing device. Examples of mixing devices include mixers, kitchen mixers, tumbler blenders, paddle mixers, agitators, flow impellers, planetary mixers, multi-axis mixers, Scanima mixers, or Stephan mixers. In one embodiment, blending may be carried out in step b) for at least 8 seconds, preferably 8 seconds to 5 minutes, more preferably 1 minute to 3 minutes. In one embodiment, blending may be carried out in step b) at a temperature of 4 to 80°C, preferably 4 to 25°C, more preferably 10 to 25°C. The preferred temperature range of 4 to 25°C is advantageous because it limits the oxidation / chemical decomposition of plant organelles that can occur at higher temperatures, e.g., 60°C to 100°C. For example, blending may be carried out at room temperature. This step disrupts plant cells and causes them to release their intracellular materials, including bioactive compounds and / or micronutrients. This contributes to improved bioaccessibility of bioactive compounds and / or micronutrients in the final concentrate when ingested by humans.

[0081] In one embodiment, step c) is carried out by filtration and / or centrifugation and / or decantation and / or heat treatment.

[0082] In one embodiment, the filtration in step c) may be carried out under the same conditions or characteristics as the filtration step c1) provided in the section "Filtration step c1)" below.

[0083] In one embodiment, the heat treatment in step c) may be carried out under the same conditions or characteristics as the heat treatment step c2) provided in the section "Heat Treatment Step c2)" below.

[0084] In one embodiment, the decantation or centrifugation in step c) may be carried out under the same conditions or characteristics as the decantation or centrifugation step c3) provided in the section “Decantation or centrifugation step c3)” below.

[0085] In one embodiment, step c) applying physical means is c1) A step of filtering the nettle material slurry to obtain a permeate, c2) Optionally, a step of heat-treating the permeate, c3) A step of obtaining nettle concentrate by centrifuging or decanting the permeate, It will be carried out by [company name].

[0086] Filtration process c1) As described above, in one embodiment, the method may include step c1) of filtering the nettle material slurry of step b) to obtain a permeate.

[0087] After filtration step c1), a retenate and a permeate are obtained. The substance that passes through the filter is called the "permeate," and the substance that does not pass through the filter and is recycled is called the "retenate." The retenate is removed after step c1), and the permeate is recovered after step c1 and further processed.

[0088] In a preferred embodiment, the filtration step c1) is carried out using a filter having a mesh size of 25 μm to 1000 μm, preferably 25 μm to 500 μm, and more preferably 100 μm to 200 μm. This mesh size contributes to improving purity by separating and concentrating the desired compounds, such as bioactive compounds and / or micronutrients, from nettle while discarding / reducing undesirable compounds, such as insoluble nettle compounds. The mesh size also reduces the particle size of the nettle concentrate to a level where the tendency for the concentrate to settle is reduced, especially when used in liquid form.

[0089] The filtration step c1) can be carried out in one or several steps. In one embodiment, the filtration step c1) can be carried out in 1 to 10 steps, preferably 1 to 5 steps. When the filtration step c1) is carried out in several steps, i.e., 2 to 10 steps, preferably 2 to 5 steps, the mesh size of the filter decreases with each successive filtration step. In other words, the mesh size of the filter used in a given filtration step (e.g., the first filtration step) is larger than the mesh size of the filter used in a successive downstream filtration step (e.g., the second filtration step), etc.

[0090] In a more preferred embodiment, the filtration step c1) is carried out in two steps, specifically, the nettle material slurry is first filtered through a filter having a mesh of 400-500 μm, preferably 500 μm, and then filtered through a filter having a mesh of 50-200 μm, preferably 180 μm.

[0091] Implementing multiple filtration processes, especially two, reduces the tendency for filters to clog.

[0092] In one embodiment, the sequence of steps a), b), and c1) is repeated at least twice, preferably 2 to 5 times, before step c2), and in the second and subsequent sequences of steps a), b), and c1), the nettle material of step a) is replaced by the retaining liquid obtained in step c1) of the preceding sequence of steps a), b), and c1). For clarification, in the second and subsequent sequences of steps a), b), and c1), the retaining liquid from the preceding sequence of steps a), b), and c1) is suspended in place of the nettle material in the aqueous solution of step a) of the subsequent sequence of steps a), b), and c1). Therefore, in the second and subsequent sequences of steps a), b), and c1), the suspensions of steps a) and b) are retaining liquid suspensions, not nettle material suspensions, and the slurries of steps b) and c1) are retaining liquid slurry, not nettle material slurry. Furthermore, in the subsequent steps a), b), and c1), a permeate is still obtained in step c1). Furthermore, in the subsequent steps a), b), and c1), a retaining liquid is still obtained in step c1). The obtained retaining liquid can be further processed in a series of consecutive steps such as a), b), and c1).

[0093] Heat treatment process c2) As described above, in one embodiment, the method may optionally include step c2) of heat-treating the permeate obtained in step c1). This heat-treating step allows for an extension of the shelf life of the final nettle concentrate. In one embodiment, step c2) is not optional.

[0094] In one embodiment, the heat treatment step c2) is carried out at a temperature of at least 60°C for at least 2 seconds. Preferably, the heat treatment step c2) is carried out at a temperature of 60 to 125°C for 2 seconds to 30 minutes. More preferably, the heat treatment step c2) is carried out at a temperature of 70 to 85°C for 1 minute to 3 minutes.

[0095] decantation or centrifugation step c3) As described above, in one embodiment, the method may include step c3) of obtaining a nettle concentrate by centrifuging or decanting the permeate. Preferably, step c3) is a step of centrifuging the permeate. In one embodiment, the centrifugal separation step c3) is carried out with 500 to 10000 g, preferably 1000 g to 5000 g, more preferably 1000 g to 3000 g. In one embodiment, the centrifugal separation step c3) is carried out for 2 to 30 minutes, preferably 2 to 20 minutes, more preferably 5 to 15 minutes.

[0096] After centrifugation or decantation, a supernatant and a precipitate are obtained. The precipitate corresponds to a generally solid or semi-solid (especially paste-like) material that forms a deposit at the bottom of the centrifugation / decantation vessel, while the supernatant corresponds to a generally liquid material that floats or spreads over the precipitate. Discard the supernatant. Collect the precipitate. The precipitate obtained after step c3) corresponds to nettle concentrate.

[0097] In one embodiment, the centrifugation or decantation step c3) is performed only once. In other words, the precipitate obtained in step c3) is not subjected to further centrifugation or decantation.

[0098] After drying step d), the nettle concentrate is in powder form rather than semi-solid form (particularly paste form). For example, the drying step may be carried out by spray drying, roller drying, air drying, or freeze-drying. In one embodiment, drying step d) is not optional.

[0099] Instead of drying the nettle concentrate into a powder, the water activity of the nettle concentrate may be reduced to improve its microbiological stability over time. Therefore, in an alternative embodiment, the method may include a step d') of reducing the water activity of the iron nettle concentrate after step c) of applying physical means or step c3) of centrifugation or decantation. After the water activity reduction step d'), the nettle concentrate has a water activity of less than 0.85, preferably 0.5 to 0.85. After this water activity reduction step d'), the nettle concentrate is not in powder form. In practice, the nettle concentrate obtained after step d') is in the same form as the nettle concentrate obtained after step c) or c3), i.e., a semi-solid form (particularly a paste form). However, the nettle concentrate obtained after step d') has a lower water activity than the nettle concentrate obtained immediately after step c) or c3). The step d') to reduce the water activity can be carried out by evaporating the nettle concentrate, drying the nettle concentrate, or adding a water-retaining agent to the nettle concentrate. Preferably, the water-retaining agent is sucrose. The amount of water-retaining agent added to the nettle concentrate can be easily determined by those skilled in the art, depending on the type of water-retaining agent and the target water activity. Drying may be carried out by freeze-drying, spray-drying, air-drying, or roller-drying. The evaporation step may be carried out using an evaporator.

[0100] In some embodiments, the step d') of reducing the water activity of the nettle concentrate and the step d) of drying the nettle concentrate may be performed consecutively. In this embodiment, the step d') of reducing the water activity of the nettle concentrate is performed before the step d) of drying the nettle concentrate.

[0101] In some embodiments, particularly when step d) drying is applied, the method does not involve evaporation or drying before step d).

[0102] In some embodiments, particularly when step d) drying is applied, the method does not include any steps of evaporation or drying.

[0103] In some embodiments, if drying step d) is not applied, the method does not include any evaporation or drying steps.

[0104] In some embodiments, step c) is not carried out by evaporation, or does not involve any use of an evaporation apparatus, such as a rotary evaporator. Similarly, steps c1, c2, c3, and c4 are not carried out by evaporation, or do not involve any evaporation, or any use of an evaporation apparatus, such as a rotary evaporator.

[0105] It was observed that the iron concentration in ppm units in the final concentrate increased significantly in the method of the present invention compared to methods in which drying or evaporation is applied directly to the permeate as a concentration method, without additional physical separation, and especially without a centrifugation step.

[0106] In some embodiments, the method may include a step d) in which the nettle concentrate is heat-treated after step c) or step c3). This heat-treatment step d) may be performed before or after step d'). This heat-treatment step d) may be performed at a temperature of at least 60°C for at least 2 seconds. Preferably, the heat-treatment step d) is performed at a temperature of 60 to 125°C for 2 seconds to 30 minutes.

[0107] This method enables effective concentrations of bioactive compounds and / or micronutrients, including those that induce and selectively regulate mitochondrial Ca2+ homeostasis.

[0108] The resulting concentrate contains a substantial amount of bioactive compounds and / or micronutrients, such as iron. The concentrate induces and selectively regulates mitochondrial Ca2+ homeostasis through its bioactive compound and / or micronutrient composition. The concentrate is derived from plant material; therefore, it is from natural sources and suitable for vegetarian / vegan diets. Furthermore, the concentrate possesses good sensory properties and, when used in orally delivered / consumed products (e.g., beverages, food products), does not impart sensory defects, or imparts very limited sensory defects, particularly limited metallic off-flavors. Moreover, the concentrate is an important source of iron, and the iron in the concentrate has sufficient bioaccessibility properties. While not theoretically bound, the substantial amount of iron in nettle concentrate may confer immune effects, particularly contributing to the maintenance of immune function.

[0109] This method is substantially natural. Although it does not involve the use of added organic solvents, it still allows for the effective concentration of bioactive compounds and / or micronutrients, such as iron.

[0110] In a preferred embodiment, the method does not involve the use of added organic solvents. For example, this method does not involve the use of added organic solvents selected from a list consisting of acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 3-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (Glym, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, naphthalene, pentane, 1-propanol, 2-propanol, pyridine, toluene, triethylamine, tricine, tris, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0111] This method allows for the effective concentration of trace nutrients, such as iron, in nettle material. Advantageously, the weight percentage concentration of iron in the nettle concentrate obtained in step c) or c3) is at least twice as high, preferably 2 to 10 times higher, than in the nettle material in step a). For example, the iron concentrations in the nettle concentrate and nettle material can be measured according to the methods provided in the examples.

[0112] This method makes it possible to effectively reduce the proportion of anti-nutrient factors that are undesirable compared to micronutrients, such as iron, particularly anti-nutrient factors that may reduce or block the absorption of micronutrients, such as iron, in the body.

[0113] In particular, the molar ratio of iron to oxalic acid (M / M) is significantly increased by this method. Increasing the molar ratio of iron to oxalic acid is advantageous in order to reduce the effect of oxalic acid on iron. Indeed, oxalic acid can reduce or inhibit the absorption of iron in the body. Advantageously, the molar ratio of iron to oxalic acid in the nettle concentrate obtained in step c) or c3) is at least twice as high, preferably 2 to 10 times higher, than in the nettle material of step a). The molar ratio of iron to oxalic acid in the nettle concentrate is expressed by the dry weight of the nettle concentrate. The molar ratio of iron to oxalic acid in the nettle material is expressed by the dry weight of the nettle material. For example, the molar ratio of iron to oxalic acid in the nettle concentrate and nettle material can be measured according to the method provided in the examples.

[0114] In some embodiments, the iron-to-phytic acid molar ratio (M / M) is significantly increased by the method of the present invention. Increasing the iron-to-phytic acid molar ratio is advantageous to reduce the effect of phytic acid on iron. Indeed, phytic acid can reduce or inhibit the absorption of iron in the body. Advantageously, the iron-to-phytic acid molar ratio in the nettle concentrate obtained in step c) or c3) is at least twice as high, preferably at least three times higher, and more preferably three to ten times higher, than in the nettle material of step a). The iron-to-phytic acid molar ratio of the nettle concentrate is expressed by the dry weight of the nettle concentrate. The iron-to-phytic acid molar ratio of the nettle material is expressed by the dry weight of the nettle material. For example, the iron-to-phytic acid molar ratio in the nettle concentrate and nettle material can be measured according to the method provided in the examples.

[0115] In some embodiments, the method does not involve the addition of enzymes. For example, the method does not involve any addition of proteases, carbohydrateases, fiberases, oxalases, phytases, and / or phenolic enzymes.

[0116] Nettle concentrate In one embodiment, the nettle concentrate disclosed herein contains at least 500 ppm of iron by dry weight of the nettle concentrate. In a preferred embodiment, the nettle concentrate contains at least 1000 ppm of iron, more preferably at least 1500 ppm of iron by dry weight of the nettle concentrate. In one embodiment, the nettle concentrate contains up to 15000 ppm of iron, preferably up to 4000 ppm of iron, more preferably up to 2000 ppm of iron by dry weight of the nettle concentrate. For example, the iron concentration of the nettle concentrate can be measured according to the method provided in the examples.

[0117] In one embodiment, the nettle concentrate is derived from nettle material. In particular, the nettle concentrate includes nettle material. The nettle material may be the nettle material provided in the "Method for obtaining nettle concentrate" section above.

[0118] In some embodiments, the nettle concentrate disclosed herein has an iron bioaccessibility of at least 3%, preferably at least 5%, more preferably at least 7%, and even more preferably at least 8%. In some further embodiments, the nettle concentrate disclosed herein has an iron bioaccessibility of up to 50%, preferably up to 35%, more preferably up to 25%, and most preferably up to 9%. The iron bioaccessibility of the nettle concentrate can be measured as provided in the examples.

[0119] In one embodiment, the nettle concentrate disclosed herein contains an absolute amount of bioaccessible iron of at least 35 ppm, preferably at least 50 ppm, more preferably at least 100 ppm, and even more preferably at least 120 ppm. In some further embodiments, the nettle concentrate contains an absolute amount of bioaccessible iron of up to 1500 ppm, preferably at least 1000 ppm, more preferably at least 500 ppm, even more preferably at least 200 ppm, even more preferably at least 150 ppm, and even more preferably at least 50 ppm. For example, the absolute amount of bioaccessible iron in a nettle concentrate can be measured according to the method provided in the examples.

[0120] In one embodiment, the nettle concentrate disclosed herein has an iron-to-oxalic acid molar ratio (M / M) of at least 0.3, preferably 0.3-3, more preferably 0.4-3, even more preferably 0.4-1.5, and most preferably 0.4-1.2. The iron-to-oxalic acid molar ratio is expressed by the dry weight of the nettle concentrate. For example, the iron-to-oxalic acid molar ratio of the nettle concentrate can be measured according to the method provided in the examples.

[0121] In some embodiments, the nettle concentrates disclosed herein contain less than 15,000 ppm, preferably less than 10,000 ppm, more preferably less than 5,000 ppm, and most preferably less than 3,000 ppm of oxalic acid by dry weight of the nettle concentrate. For example, the concentration of oxalic acid in the nettle concentrate may be measured according to the method provided in the examples.

[0122] In one embodiment, the nettle concentrate disclosed herein has an iron-to-phytic acid molar ratio (M / M) of at least 2, preferably at least 5, more preferably 5-80, more preferably 5-50, even more preferably 5-20, and most preferably 7-15. The iron-to-phytic acid molar ratio is expressed by the dry weight of the nettle concentrate. For example, the iron-to-phytic acid molar ratio of the nettle concentrate can be measured according to the method provided in the examples.

[0123] In some embodiments, the nettle concentrates disclosed herein contain less than 3000 ppm, preferably less than 2500 ppm, and more preferably less than 2200 ppm, of phytic acid by dry weight of the nettle concentrate. For example, the concentration of phytic acid in the nettle concentrate may be measured according to the method provided in the examples.

[0124] In one embodiment, the nettle concentrate disclosed herein does not contain added organic solvents, and in particular does not contain the added organic solvents listed in the section "Method for obtaining nettle concentrate" above.

[0125] In one embodiment, the nettle concentrate disclosed herein has a pH of 3 to 8.

[0126] In one embodiment, the nettle concentrate disclosed herein contains 0.01% to 5% by weight of an acid. The acid may be the acid provided in the section “Method for obtaining nettle concentrate” above. Preferably, the acid is selected from the group consisting of ascorbic acid, malic acid, citric acid, hydrochloric acid, or mixtures thereof. In a more preferred embodiment, the acid is selected from the group consisting of malic acid, citric acid, hydrochloric acid, or mixtures thereof. In an even more preferred embodiment, the acid is selected from the group consisting of citric acid, hydrochloric acid, or mixtures thereof. In the most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid.

[0127] The acid may be provided as a pure acid solution, a diluted acid solution, or as an acid-containing food ingredient. Examples of acid-containing food ingredients include citrus juices, such as lemon juice, lime juice, orange juice, and tangerine juice.

[0128] When the acid is citric acid, the nettle concentrate disclosed herein may contain 0.01 to 3% by weight, preferably 0.5 to 2.3% by weight, of citric acid.

[0129] When the acid is hydrochloric acid, the nettle concentrate disclosed herein may contain 0.01% to 0.5% by weight of hydrochloric acid, preferably 0.05% to 0.5% by weight of hydrochloric acid.

[0130] When the acid is malic acid, the nettle concentrate disclosed herein may contain 0.01% to 3% by weight of malic acid, preferably 0.5% to 2.5% by weight of malic acid.

[0131] In one embodiment, the nettle concentrate disclosed herein may include an osmotic regulator. The osmotic regulator may be the one provided in the section "Method for obtaining nettle concentrate" above.

[0132] In one embodiment, the nettle concentrate disclosed herein may include a water-retaining agent. The water-retaining agent may be the water-retaining agent provided in the section "Method for obtaining nettle concentrate" above.

[0133] In one embodiment, the nettle concentrate disclosed herein may have a total sucrose content of 1 to 50% by weight, preferably 5 to 20% by weight. The sucrose in the nettle concentrate may be used as a water-retaining agent and / or an osmotic pressure modifier. The total sucrose content range provided herein applies whether or not sucrose is used as a water-retaining agent and / or an osmotic pressure modifier.

[0134] The nettle concentrate disclosed herein contains substantial amounts of bioactive compounds and / or micronutrients such as iron. Through its bioactive compound and / or micronutrient composition, the nettle concentrate induces and selectively regulates mitochondrial Ca2+ homeostasis. The nettle concentrate is derived from plant material. Therefore, the nettle concentrate is derived from natural sources and is suitable for vegetarian / vegan diets.

[0135] Furthermore, nettle concentrate possesses good sensory properties and, when used in orally delivered / consumed products, does not cause sensory defects, or causes very limited sensory defects, particularly metallic off-flavors.

[0136] Furthermore, nettle concentrate is an important source of iron, and the iron in the concentrate is sufficiently bioaccessible. While not bound by theory, a substantial amount of iron in nettle concentrate may confer immune effects, and in particular, may contribute to maintaining immune function.

[0137] composition The composition may further contain one or more additional bioactive compounds. In particular, the composition may further contain at least one compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements, and / or vitamins. The compounds may be derived from natural sources. That is, the compounds may be derived from extracts of plants, animals, fish, fungi, algae, or microbial fermentations. Preferably, the compounds are derived from plants, fungi, or algae. Minerals are considered to be derived from natural sources. In preferred embodiments, the enzyme may be a protease, such as trypsin, or an enzyme extract, such as bromelain.

[0138] The effective dose of nettle concentrate varies depending on the specific composition, the age and condition of the user, and the specific disorder or disease being treated. However, in general embodiments, an individual may be administered 0.20 mg to 20 g / day, preferably 1 mg to 20 g / day, more preferably 1 mg to 10 mg / day, and more preferably 1 g to 10 g / day.

[0139] The composition may contain an effective amount of nettle concentrate. For example, a single serving or dose of the composition may contain an effective amount, and a package may contain one or more servings or doses. Optionally, the composition may further contain calcium.

[0140] In certain embodiments, nettle concentrate, particularly an effective amount of nettle concentrate, may be administered in a composition further containing calcium.

[0141] The composition may contain food additives selected from the group consisting of acidulants, thickeners, buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, osmotic pressure regulators, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, flavorings, vitamins, minerals, and combinations thereof.

[0142] Nettle concentrate, particularly an effective amount of nettle concentrate, can be administered in any composition suitable for human and / or animal intake. In preferred embodiments, such combinations are administered orally or enterally (e.g., via tube feeding) to the individual. For example, such combinations can be administered to the individual as beverages, food products, capsules, tablets, powders, or suspensions.

[0143] Non-limiting examples of suitable compositions include food compositions, dietary supplements (e.g., liquid ONS), nutritional compositions, complete nutritional compositions, beverages, pharmaceuticals, oral nutritional supplements, medical foods, nutraceuticals, foods for specific medical purposes (FSMPs), powdered nutritional products that are reconstituted with water or milk before ingestion, food additives, pharmaceuticals, pet food, and combinations thereof.

[0144] Examples of food products according to the present invention include dairy products, such as fermented dairy products like yogurt and buttermilk; ice cream; condensed milk; milk; dairy cream; flavored milk beverages; whey-based beverages; toppings; coffee creamer; plant-based dairy products; chocolate; cheese-based products; soups; sauces; purees; dressings; puddings; custards; infant formulas; nutritional formulas, such as complete nutritional formulas for infants, children, teenagers, adults, the elderly, or those with serious illnesses; cereals and cereal bars.

[0145] Beverages (or drinks) may include, for example, milk-based or yogurt-based beverages, fermented milk, protein drinks, coffee, tea, energy drinks, soy beverages, water-based beverages, dairy beverages, fruit and / or vegetable beverages, and fruit and / or vegetable juices.

[0146] Nettle concentrate, particularly an effective amount of nettle concentrate, can be administered in a food product or beverage further containing components selected from the group consisting of proteins, carbohydrates, fats, and mixtures thereof.

[0147] In one embodiment, the protein source is preferably purified protein (i.e., isolated from the natural food raw material from which the protein was produced). The protein content of the composition is preferably 20 to 99% by weight of the composition, for example, 20 to 90% by weight of the composition, for example, 30 to 80% by weight of the composition, for example, 40 to 80% by weight of the composition, for example, 50 to 80% by weight of the composition, for example, 40 to 70% by weight of the composition.

[0148] Non-limiting examples of proteins or sources suitable for use in the composition include hydrolyzed, partially hydrolyzed, or unhydrolyzed proteins or protein sources. These may be derived from any known or other suitable sources, e.g., milk (e.g., casein, whey), animals (e.g., meat, fish), grains (e.g., rice, corn), or vegetables (e.g., soybeans, peas). Multiple sources or combinations of multiple proteins may be used. Non-limiting examples of proteins or sources include raw pea protein, raw pea protein isolate, raw pea protein concentrate, milk protein isolate, milk protein concentrate, casein protein isolate, casein protein concentrate, whey protein concentrate, whey protein isolate, sodium caseinate or calcium caseinate, whole milk, partially or completely skimmed milk, yogurt, soy protein isolate, and soy protein concentrate, as well as combinations thereof. Multiple sources or combinations of multiple proteins may be used. Preferred proteins include pea protein, oat protein, broad bean protein, whey protein, soy protein, and casein. Casein protein may include, for example, sodium caseinate and calcium caseinate.

[0149] The protein source may be provided by individual amino acids, polypeptides containing amino acids, or mixtures thereof. In many treatments for muscle growth, muscle maintenance, and / or muscle enhancement, specific amino acids that are beneficial are, for example, L-arginine, L-glutamine, lysine, and branched-chain amino acids (i.e., leucine, isoleucine, and valine; leucine and isoleucine in particular). These specific amino acids may be provided as a protein source or added to the main source of protein. Thus, the protein source in the composition may include one or more branched-chain amino acids (leucine, isoleucine, and valine), one or both of L-arginine and L-glutamine, and lysine. In a preferred embodiment, the composition includes whey protein and / or casein protein together with one or more individual amino acids, for example, one or more (or all) of leucine, isoleucine, and L-arginine.

[0150] In one embodiment, the composition further comprises one or more medium-chain triglycerides, such as caproic acid, caprylic acid, capric acid, and lauric acid. In another embodiment, the composition further comprises phospholipids, such as phosphatidylcholine.

[0151] The composition may also contain a carbohydrate source and / or a fat source. Non-limiting examples of suitable fats include canola oil, corn oil, and high-oleic sunflower oil. Non-limiting examples of suitable carbohydrates include sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof. Further or alternatively, dietary fiber may be added. Dietary fiber passes through the small intestine undigested by enzymes and functions as a natural bulking laxative and purgative. The dietary fiber may be soluble or insoluble, and generally a blend of the two is preferred. Non-limiting examples of suitable dietary fiber include soy, pea, oat, pectin, guar gum, partially hydrolyzed guar gum, gum arabic, fructooligosaccharides, acid oligosaccharides, galactooligosaccharides, sialyl lactose, and oligosaccharides derived from animal milk. A preferred fiber blend is a mixture of inulin and relatively short-chain fructooligosaccharides. In one embodiment, the fiber content is 2 to 40 g / L of the composition, for example, 4 to 10 g / L.

[0152] In addition to some form of calcium, one or more other minerals may be used in the composition. Non-limiting examples of suitable minerals include boron, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.

[0153] One or more other vitamins may be used in the composition. Non-limiting examples of suitable vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; generally, cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, folic acid, and biotin), as well as combinations thereof. "Vitamins" include compounds naturally obtained from plant and animal foods or produced synthetically, provitamins, derivatives thereof, and analogs thereof.

[0154] One or more food emulsifiers, such as diacetyl tartrate esters of monoglycerides and diglycerides, lecithin, and / or monoglycerides and diglycerides, may be incorporated into the composition. Suitable salts and stabilizers may also be included.

[0155] The compositions disclosed herein may be used in any of the various formulations for therapeutic administration. More specifically, the pharmaceutical compositions may include a suitable pharmaceutically acceptable carrier or diluent and may be formulated in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Accordingly, administration of the compositions can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active ingredient may be systemic after administration, or may be localized by topical administration, intramural administration, or by the use of implants that act to retain an effective dose at the implantation site.

[0156] In pharmaceutical dosage forms, nettle concentrate may also be used in appropriate association with other pharmacologically active compounds. The methods and excipients described below are merely illustrative and not limiting.

[0157] In oral formulations, nettle concentrate can be used alone or in combination with appropriate additives for manufacturing tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, cellulose functional derivatives, gum arabic, corn starch, or gelatin; with disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, if desired, in combination with diluents, buffers, humectants, preservatives, and flavorings.

[0158] In some embodiments, the composition is in the form of a solid powder, powder stick, capsule, or solution.

[0159] The composition, in particular nettle concentrate, more specifically an effective amount of nettle concentrate, can be administered for at least one day per week, preferably at least two days per week, more preferably at least three or four days per week (e.g., every other day), most preferably at least five days per week, six days per week, or seven days per week. The duration of administration may be at least one week, preferably at least one month, more preferably at least two months, most preferably at least three months, for example, at least four months. In one embodiment, administration is at least daily, for example, the subject may receive administration once or more times a day. In some embodiments, administration is continued for the remainder of the individual's life. In other embodiments, administration is continued until there are no detectable symptoms of the medical condition. In specific embodiments, administration is continued until there is a detectable improvement in at least one symptom, and in further cases, it is continued to maintain remission.

[0160] In some embodiments, the composition, in particular nettle concentrate, more specifically an effective amount of nettle concentrate, is administered orally daily for at least one week.

[0161] In one embodiment, the composition does not contain milk and / or soy.

[0162] In one embodiment, the composition is vegetarian. In another embodiment, the composition is vegan.

[0163] Treatment method One aspect of the present disclosure is a composition comprising nettle concentrate in an effective amount for use in (i) improving a physiological condition associated with metabolic fatigue in one or more cells, and / or (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and / or (iii) enhancing mitochondrial function, and / or (iv) treating or preventing calcium deficiency / depletion disorders in an organism.

[0164] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0165] In one embodiment, at least a portion of one or more cells is part of at least one body part selected from the group consisting of the liver, kidney, brain, and skeletal muscle. In a preferred embodiment, the cells are skeletal muscle-derived cells. Non-limiting examples of skeletal muscle-derived cells include myoblasts, myotubes, and combinations thereof. In a more preferred embodiment, the cells are myotubes.

[0166] In another embodiment, physiological conditions associated with metabolic fatigue include muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, and in particular, deficient physical energy.

[0167] In some embodiments, the method includes identifying individuals as having a condition or being at risk of developing a condition before administration.

[0168] A further aspect of this disclosure is a composition comprising nettle concentrate for use in an effective amount for the treatment or prevention (e.g., reduction of incidence and / or severity) of a mitochondrial-related disease or a condition associated with altered mitochondrial function in an individual who needs or is at risk of such treatment or prevention. The method comprises orally administering an effective amount of nettle concentrate to an individual who needs or is at risk of such treatment or prevention.

[0169] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0170] While not bound by theory, it is believed that various types of stress can lead to mitochondrial stress damage, reducing the ability of mitochondria to perform numerous roles essential to overall cellular function. The methods disclosed herein may be useful in treating conditions involving mitochondrial stress damage. This damage can manifest through one of many pathways, including but not limited to mitochondrial diseases.

[0171] Mitochondrial diseases result from either hereditary or spontaneous mutations in mitochondrial DNA or nuclear DNA, leading to alterations in the function of proteins or RNA molecules normally present within mitochondria. However, problems with mitochondrial function can affect only specific tissues due to factors that occur during development and growth and are not yet fully understood. Even considering tissue-specific isoforms of mitochondrial proteins, it remains difficult to explain the variable patterns of organ systems affected by mitochondrial disease syndromes observed clinically.

[0172] Mitochondrial diseases are caused by damage to mitochondria, specialized compartments present in all cells of the body except red blood cells. Mitochondria are involved in the production of over 90% of the energy the body needs to maintain life and support development. When mitochondria cease to function, the energy produced in cells decreases. Cells become damaged and eventually die. If this process is repeated throughout the body, the entire system begins to break down, and this breakdown severely threatens the person's life. Mitochondrial diseases mainly affect children, but cases in adults are also being observed.

[0173] Mitochondrial disorders appear to cause the most damage to cells in the brain, heart, liver, skeletal muscle, kidneys, and endocrine and respiratory systems.

[0174] Many symptoms in mitochondrial disorders are nonspecific. Symptoms may also present as a transient course with cyclical exacerbations. Review articles in mitochondrial medicine mention transient migraine episodes, as well as muscle pain, gastrointestinal symptoms, tinnitus, depression, chronic fatigue, and diabetes, among the various manifestations of mitochondrial disease. In patients with mitochondrial disorders, clinical symptoms typically arise when energy demands are high, associated with physiological stressors such as illness, hunger, excessive exercise, and extreme environmental temperatures. Furthermore, psychological stressors often trigger symptoms, possibly due to excessive brain energy demands that prevent the patient from producing sufficient ATP.

[0175] Depending on which cells are affected, symptoms may include loss of motor control, muscle weakness and pain, gastrointestinal disorders and dysphagia, stunted growth, heart disease, liver disease, development of diabetes, respiratory complications, seizures, visual / auditory problems, lactic acidosis, delayed response, and increased susceptibility to infections.

[0176] Mitochondrial diseases, though not limited to these, include Alpers syndrome; Barth syndrome; β-oxidation disorders; carnitine deficiency; carnitine-acyl-carnitine deficiency; chronic progressive extraocular muscle palsy syndrome; coenzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; CPT I deficiency; CPT II deficiency; creatine deficiency syndrome; cytochrome c oxidase deficiency; glutaric aciduria type 2; Kearns-Sayre syndrome; lactic acidosis; LCHAD (long-chain acyl-CoA dehydrogenase deficiency); Leber's hereditary optic neuropathy; Leigh syndrome; and lethal infantile cardiomyopathy. Cardiomyopathy; Luft's disease; MAD (medium-chain acyl-CoA dehydrogenase deficiency); mitochondrial cell dystrophy; mitochondrial DNA depletion; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms; mitochondrial encephalopathy; mitochondrial myopathy; mitochondrial recessive ataxia syndrome; muscular dystrophy, myoclonic epilepsy, and ragged-red fiber disease; myoneurogenic gastrointestinal encephalopathy; neuropathy, ataxia, retinitis pigmentosa, and ptosis; Pearson syndrome; POLG mutation; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; SCHAD (short-chain acyl-CoA dehydrogenase deficiency); and very long-chain acyl-CoA dehydrogenase deficiency.

[0177] In one embodiment, mitochondrial-related disorders or conditions are selected from the group consisting of stress (e.g., childhood stress and / or its effects), physiological aging, obesity, decreased metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative diseases, cognitive impairment, stress-induced or stress-related cognitive impairment, mood disorders (e.g., stress-induced or stress-related mood disorders), anxiety disorders (e.g., stress-induced or stress-related anxiety disorders), age-related neuronal death or dysfunction (e.g., age-related neuronal death or dysfunction not attributable to a specific neurodegenerative disease), musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, renal failure, trauma, infection (e.g., in the ICU), cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

[0178] Accordingly, one aspect of the present disclosure is a unit dosage form comprising nettle concentrate for use in an effective amount for the treatment or prevention of a condition selected from the group consisting of stress (e.g., childhood stress and / or its effects), physiological aging, obesity, decreased metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative diseases, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders (e.g., stress-induced or stress-related mood disorders), anxiety disorders (e.g., stress-induced or stress-related anxiety disorders), age-related neuronal death or dysfunction (e.g., age-related neuronal death or dysfunction not attributable to a specific neurodegenerative disease), musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, renal failure, trauma, infection (e.g., in an ICU), cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

[0179] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0180] Another aspect of the present disclosure is a method for treating in an individual having at least one condition selected from the group consisting of stress, obesity, decreased metabolic rate, metabolic syndrome, diabetes mellitus, cardiovascular disease, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive impairment, mood disorders (e.g., stress-induced or stress-related mood disorders), anxiety disorders (e.g., stress-induced or stress-related anxiety disorders), and age-related neuronal death or dysfunction (e.g., age-related neuronal death or dysfunction not attributable to a specific neurodegenerative disease), trauma, infection (e.g., in an ICU), or cancer, the method comprising administering to the individual having the at least one condition a composition comprising an effective amount of nettle concentrate.

[0181] In one embodiment, the hyperlipidemia to be treated or prevented includes hypertriglyceridemia. In one embodiment, the hyperlipidemia to be treated or prevented includes an increase in free fatty acids. In one embodiment, age-related neuronal cell death or dysfunction to be treated or prevented is by administration of the composition to middle-aged and elderly individuals, for example, the elderly.

[0182] The stress that can be treated or prevented may be early childhood stress, i.e., stress experienced during the period up to 5 years of age. Early childhood stress has been reported to have significant adverse effects on cognitive abilities, including an increased incidence of or susceptibility to psychological parameters such as depression, anxiety, and abnormal risk-taking behaviors. Individuals who have experienced early childhood stress have been reported to have a higher incidence of attention deficit / hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and major depressive disorder.

[0183] Another aspect of the present disclosure is a method for delaying the onset of metabolic decline, maintaining muscle mass, reducing oxidative stress, maintaining immune function, and / or maintaining cognitive function in healthy middle-aged and elderly persons, the method comprising administering a composition comprising an effective amount of nettle concentrate to the healthy middle-aged and elderly persons.

[0184] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0185] Another aspect of the present disclosure is a composition comprising nettle concentrate for use in healthy middle-aged and elderly individuals in an effective amount to delay the onset of metabolic decline and / or maintain muscle mass and / or maintain immune function and / or maintain cognitive function.

[0186] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0187] Another aspect of the present disclosure is a method for enhancing at least one of mental or muscular performance in an individual, comprising administering to the individual a composition comprising an effective amount of nettle concentrate.

[0188] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0189] Another aspect of the present disclosure is a composition comprising nettle concentrate for use in an effective amount to enhance at least one of mental or muscular performance in an individual.

[0190] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0191] Another aspect of the present disclosure is a method for improving or maintaining cognitive function in an individual, comprising administering to the individual a composition comprising an effective amount of nettle concentrate.

[0192] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0193] In one embodiment, cognitive functions are selected from the group consisting of perception, memory, attention, speech comprehension, speech production, reading comprehension, image creation, learning, reasoning, and combinations thereof.

[0194] Another aspect of this disclosure is a composition comprising nettle concentrate for use in an effective amount for improving or maintaining cognitive function in an individual.

[0195] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0196] In one embodiment, cognitive functions are selected from the group consisting of perception, memory, attention, speech comprehension, speech production, reading comprehension, image creation, learning, reasoning, and combinations thereof.

[0197] The compositions disclosed herein can also be used to treat any of a variety of additional diseases and conditions in which a defect or reduction in mitochondrial activity is involved in the pathophysiology of the disease or condition, or in which improvement of mitochondrial function results in a desired beneficial effect. Non-limiting examples of such conditions include male infertility associated with reduced sperm motility, macular degeneration, and other age-related and hereditary eye disorders and hearing loss (e.g., age-related hearing loss).

[0198] A further aspect of the present invention is a unit dosage form comprising an amount of nettle concentrate effective for at least one of the following: (i) treatment of mitochondrial-related diseases or conditions associated with altered mitochondrial function, reduction of their incidence, or reduction of their severity; and / or (ii) improvement of physiological conditions associated with metabolic fatigue in one or more cells; and / or (iii) increase of mitochondrial energy and mitochondrial calcium uptake in one or more cells; and (iv) treatment or prevention of calcium deficiency / depletion disorders; and / or (v) increase of metabolic rate; and / or (vi) improvement or maintenance of cognitive function; and / or (viii) improvement or maintenance of mitochondrial function.

[0199] Nettle concentrate is the nettle concentrate disclosed herein, which is obtainable or obtainable by the method disclosed herein.

[0200] In one embodiment, physiological conditions associated with metabolic fatigue include muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, and in particular, a lack of physical energy.

[0201] In one embodiment, mitochondrial-related disorders or conditions are selected from the group consisting of stress (e.g., childhood stress and / or its effects), physiological aging, obesity, decreased metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative diseases, cognitive impairment, stress-induced or stress-related cognitive impairment, mood disorders (e.g., stress-induced or stress-related mood disorders), anxiety disorders (e.g., stress-induced or stress-related anxiety disorders), age-related neuronal death or dysfunction (e.g., age-related neuronal death or dysfunction not attributable to a specific neurodegenerative disease), musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, renal failure, trauma, infection (e.g., in the ICU), cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

[0202] In one embodiment, cognitive functions are selected from the group consisting of perception, memory, attention, speech comprehension, speech production, reading comprehension, image creation, learning, reasoning, and combinations thereof.

[0203] In further embodiments, the unit dosage form essentially consists of a nettle concentrate.

[0204] In some embodiments of different aspects of this disclosure, the individual may be a middle-aged or elderly person, an elderly person, or an ICU patient.

[0205] The above administration examples do not require uninterrupted daily administration. Rather, administration may include some short-term interruptions, for example, 2 to 4 days during the administration period. The ideal duration of administration of the composition of the present invention can be determined by those skilled in the art.

[0206] In any preferred embodiment of the above-described aspects, the above-described benefits / effects (e.g., enhancement of at least one of mental performance or muscular performance, improvement or maintenance of cognitive function, etc.) are obtained by enhancing mitochondrial calcium uptake in cells, preferably skeletal muscle cells.

[0207] In any preferred embodiment of the above configuration, any effect / benefit on mitochondrial function (e.g., increased mitochondrial energy, increased mitochondrial calcium uptake, enhanced mitochondrial function) is preferably an effect / benefit on mitochondria in skeletal muscle cells.

[0208] Those skilled in the art will understand that all features of the present invention disclosed herein can be freely combined. Specifically, features described for the products / compositions of the present invention may be combined with the therapeutic methods of the present invention, and vice versa. Furthermore, features described for different embodiments or aspects of the present invention / disclosure may be combined.

[0209] Furthermore, where known equivalents exist for a particular feature, such equivalents are incorporated as specifically referred to herein. Further advantages and features of the present invention are evident from the drawings and non-limiting embodiments. [Examples]

[0210] In all of the following examples, the nettle material is Urtica dioica, and the nettle concentrate is a concentrate obtained by processing Urtica dioica in the manner described in the examples.

[0211] Example 1 - Method of the present invention for concentrating nettle from dried herbs without using acid Dried nettle (Urtica dioica) material, including leaves and stems, was ground into a powder. The powder and water were mixed in a ratio of 1:15 (w:v), and the powder was hydrated for 5 minutes to form a suspension. The suspension was then blended for 1 minute to obtain a slurry. The obtained slurry was filtered through a 500 μm mesh filter. The permeate was collected and subsequently filtered through a 180 μm mesh filter. The permeate was collected again, and the resulting permeate was heat-treated to a temperature of 71°C and held for 2 minutes. After cooling to 4°C, the permeate was centrifuged with 2500 g for 10 minutes. The precipitate was collected, and a nettle concentrate was formed. Optionally, the concentrate can be dried.

[0212] Example 2 - Method of the present invention for concentrating nettle from dried herbs in the presence of acid Dried nettle (Urtica dioica) material, including leaves and stems, was ground into a powder. A suspension was prepared by mixing the powder (50 g) with water (700 mL) in a ratio of 1:15 (w:v). Pure citric acid (15.8 g of anhydrous citric acid) or hydrochloric acid (15 mL of 6 M HCl solution) was added to the suspension to adjust the pH to 3.5. After adding the acid, the suspension was allowed to stand for 2 minutes to ensure proper hydration of the powder in the suspension. The suspension was then blended for 2 minutes to obtain a slurry. The obtained slurry was filtered through a 500 μm mesh filter. The permeate was collected and subsequently filtered through a 180 μm mesh filter. The permeate was collected again and the resulting permeate was heat-treated to a temperature of 71 °C and held for 2 minutes. After cooling to 4 °C, the permeate was centrifuged at 2500 g for 10 minutes. The precipitate was collected to form a nettle acid concentrate. Optionally, the concentrate can be dried.

[0213] Example 3 - Quantitative determination of iron Materials and methods The iron content of the nettle material (i.e., dried nettle powder obtained after grinding) and the nettle concentrate obtained in either Example 1 or 2 was measured by atomic emission spectroscopy using microwave plasma atomic emission spectroscopy (MP-AES) 4200 (Agilent, Switzerland). For MP-AES analysis, samples (approximately 100-400 mg) were mineralized in a double-stage process using an Xpress microwave bomb in a Microwave Digestion System (Mars 6, CEM, USA) with 4 mL of ultra-high purity 70% HNO3 (Sigma-Aldrich, St. Louis, MO, USA) and 1 mL of 30% H2O2 (Merck KGaA, Darmstadt, Germany). The mineral solution was then transferred to a 50 mL Falcon tube and adjusted to 20 mL with Milli-Q water. Iron content was measured using external calibration with multi-element standards at a wavelength of 371 nm. The accuracy of the analysis was confirmed by analyzing a standard reference material (SRM 3233, Typical Diet; NIST, MD, USA).

[0214] result Figure 1 shows the iron concentration (based on DW) of the nettle material (i.e., dried nettle powder obtained after grinding the dried nettle material) and the nettle concentrate obtained according to the method of Example 1.

[0215] The concentration method of the present invention significantly increased the iron concentration from 288 ppm in the nettle material to 1355 ppm in the nettle concentrate.

[0216] Example 4 - Quantification of anti-trophic factors Materials and methods: Oxalic acid was extracted from the sample using water under mechanical stirring. The oxalic acid was measured by mass spectrometry (SCIEX Triple Quad 5500 with Selexion) coupled to ion chromatography (Dionex ICS-5000 with Dionex Ion PAC AS16 REFIC Analytical column (250×2mm)).

[0217] Phytic acid was measured according to the "Phytic Acid (Phytate) / Total Phosphorus" Megazyme kit. This kit allows for the quantification of free and total phosphorus in a sample by colorimetric detection. Total phosphorus is defined as phosphorus derived from phytic acid and other sources, and is measured after the sample is first treated with phytase and then with alkaline phosphatase. Free phosphorus, on the other hand, is defined as phosphorus derived from non-phytic acid sources in the sample, and is measured from the kit without enzymatic treatment. Briefly, 1 g of the sample was mixed with 20 mL of HCl acid (0.66 M) and vigorously stirred for 3 hours. The extract (1 mL) was centrifuged at 13000 rpm for 10 minutes, and 0.5 mL of the resulting supernatant was neutralized with 0.5 mL of NaOH solution (0.75 M). A neutralized sample extract (0.05 mL) was mixed with distilled water (0.60 mL), the provided buffer I (0.20 mL), and a phytase suspension for total phosphorus quantification (0.02 mL). A control sample was prepared by mixing a sample extract (0.05 mL) with distilled water (0.62 mL) and the provided buffer I (0.20 mL), and free phosphorus was quantified. Both samples were vortexed and incubated at 40°C for 10 minutes. Distilled water (0.02 mL) and the provided buffer 3 (0.2 mL) were added to the control, and buffer 3 (0.20 mL) and suspension 4 (ADP, 0.02 mL) were added to the total phosphorus sample. The samples were vortexed and incubated at 40°C for 15 minutes. The reaction was stopped by adding 0.30 mL of trichloroacetic acid (50% w / v). The samples were centrifuged at 13000 rpm for 10 minutes. The supernatant (1 mL) was used for the colorimetric measurement of phosphorus. The sample was mixed with 0.5 mL of colorimetric reagent. The colorimetric reagent was prepared by mixing 1 part of ammonium molybdate solution (5% w / v) with 5 parts of ascorbic acid (10% w / v) / sulfuric acid (1 M) solution. After mixing the sample with the colorimetric reagent, it was incubated at 40°C for 1 hour, and then the absorbance at 655 nm was read.

[0218] The phosphorus concentration was calculated using the measured absorbance, and this was used to calculate the PA concentration. The concentrations of free phosphorus and phosphorus in the total phosphorus reaction were calculated using equation (1).

[0219]

Number

[0222] ,

[0220] , sample free , , , , sample , , , , phosphorus , , , , , , PA= , , , sample tot , , , , standard0 , , , , ,

[0221] , , , , ,

[0223] = Concentration of phosphorus in the sample [g / 100g] mean M = Mean value of phosphorus standard [μg / ΔA standard , where M = μg of phosphorus in the standard solution (i.e., 0.5 - 7.5) divided by ΔA standard (i.e., A standardX - A standard0 ) vHCl = Volume of the original sample extraction [mL] F = Dilution factor (in this study, F = 1) ΔA sample = Absorbance difference of the sample (i.e., A sample tot - A sample free ) w = Weight of the original sample material [g] v = Volume of the sample used in the colorimetric measurement process (= 1 mL) 10000 = Conversion from μg / g to g / 100g

[0220] Equation (2) is used to convert the calculated concentration of bound phosphorus to the PA concentration. It is assumed that the measured amount of bound phosphorus is only from PA.

[0221]

Number

[0224] Figure 2 shows the molar ratios of iron and oxalic acid in the nettle material (i.e., dried nettle powder obtained after grinding the dried nettle material) and the nettle concentrate obtained according to the method of Example 1. The concentration method of the present invention increases the iron-to-oxalic acid ratio from 0.23 to 1.11, indicating that the concentration of iron that can be chelated by oxalic acid in the nettle concentrate is lower than in the nettle material, and therefore there is potentially more iron available for absorption.

[0225] Figure 3 shows the molar ratios of iron and phytic acid in the nettle material (i.e., dried nettle powder obtained after grinding the dried nettle material) and the nettle concentrate obtained according to the method of Example 1.

[0226] The concentration method of the present invention increased the iron-to-phytic acid ratio from 1.3 in the nettle material to 8.9 in the nettle concentrate.

[0227] These results indicate that the concentration of iron that can be chelated by phytic acid in nettle concentrate is lower than in nettle material, and therefore has potentially greater availability for absorption.

[0228] Example 5 - In vitro digestion for quantifying iron bioaccessibility Materials and methods: Briefly, 1 g of nettle material (i.e., dried nettle powder obtained after grinding) and nettle concentrate prepared according to the concentration methods of Examples 1 and 2 were mixed with 10 mL of KCl 5 mmol + NaCl 140 mmol pH 2. After adjusting the pH to 2, 0.5 mL of pepsin solution (prepared by dissolving 200 mg of pepsin in 10 mL of 0.1 M HCl) was added, and the sample was incubated at 37°C for 1 hour. After 1 hour, the pH was adjusted to 5.5 with 1 M NaHCO3. The volume of the sample was adjusted to 15 mL by adding 6.7 μL of KCl 5 mmol + NaCl 140 mmol. Pancreatin solution (2.5 mL, prepared by adding 87.5 mg of pancreatin and 525 mg of bile extract to 44 mL of 0.1 M NaHCO3) was added, and the sample was incubated at 37°C for 2 hours. An aliquot (2.5 g) of the complete digest was analyzed for iron content by MPAES. The remaining sample was centrifuged at 10,000 g for 30 minutes at 4°C, and 2.5 g of the supernatant was analyzed for iron content by MPAES. Iron bioaccessibility was defined as follows:

number

[0229] result: Figure 4 shows the bioaccessibility of iron in nettle concentrate prepared with water (according to Example 1) or with water in the presence of citric acid and hydrochloric acid (according to Example 2), respectively. Citric acid and hydrochloric acid had a positive effect on the bioaccessibility of iron in nettle concentrate.

[0230] Figure 5 shows the absolute amount of bioaccessible iron contained in nettle material and in nettle concentrates prepared with water according to the concentration method of Example 1, or prepared with water in the presence of citric acid and hydrochloric acid, respectively, according to the method of Example 2. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value.

[0231] Nettle concentrates prepared with water, citric acid, or hydrochloric acid have a clear advantage over raw nettle material because they contain a larger amount of bioavailable iron (39 ppm, 114 ppm, and 147 ppm in nettle concentrates prepared with water, citric acid, and hydrochloric acid, respectively, compared to 28 ppm in dried nettle).

[0232] Example 6 - Nettle concentrate increases mitochondrial activity through increased mitochondrial calcium. Materials and methods: To test the effect of nettle concentrate prepared according to Example 1 on mitochondrial activation in living cells, the inventors measured the elevation of mitochondrial calcium in myotubes differentiated from C2C12 cells.

[0233] C2C12 cells were purchased from ATCC. C2C12 cells were seeded at a density of 4500 cells / well in DMEM high glucose (Gibco) + 10% fetal bovine serum in 384-well plates. Myotubes were differentiated from C2C12 cells by growing the cells in DMEM containing 2% horse serum for 7 days.

[0234] Mitochondrial calcium levels were measured using myotubes infected with an adenovirus (Sirion biotech) expressing mitochondrial-modified aequorin, a calcium sensor targeted for luminescence in mitochondria (Montero et al., 2004). For aequorin reconstitution, 48 hours after infection, cells or myotubes were incubated at room temperature (22±°C) for 2 hours in aequorin standard buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM Hepes, pH 7.4) supplemented with 1 μM wild-type coelenterazine. Cells were washed with aequorin buffer before stimulation.

[0235] For sample preparation, nettle concentrates of different concentrations / dilutions were prepared by diluting the nettle concentrate with the standard aequorin buffer described above.

[0236] For treatment, nettle concentrate (at different concentrations / dilutions, as shown in Figure 6 or Figure 7) was directly injected into the myotube cultures during measurement. For the control (unstimulated), only standard aequorin buffer was directly injected into the myotube cultures during measurement.

[0237] Total mitochondrial calcium uptake during stimulation (= increase in mitochondrial calcium) was calculated as the area under the curve (AUC). Emissions were measured using a FLIPR cell imaging reader (molecular device). Calibration of emission data to calcium concentration was performed using the algorithm described above (Alvarez & Montero, 2002). Quantification was performed using custom module analysis based on Excel (Microsoft) and GraphPad Prism 7.02 (GraphPad) software.

[0238] result: The results are shown in Figures 6 and 7. As shown in Figures 6 and 7, nettle concentrate (at different concentrations) activates mitochondria in C2C12-derived myotubes via an increase in mitochondrial Ca2+.

[0239] In particular, Figure 6 is a graph showing the effect of nettle concentrate on mitochondrial Ca2+ uptake in C2C12-derived myotubes over time. Figure 6 shows the dose-dependent boost effect of nettle concentrate according to the present invention on mitochondrial activation (10 to 80-fold dilution of nettle concentrate). When only aequorin buffer was injected (no stimulation), no mitochondrial activation was observed.

[0240] Figure 7 confirms the dose-dependent boosting effect of the nettle concentrate according to the present invention on total mitochondrial calcium uptake in C2C12-derived myotubes.

Claims

1. A composition comprising nettle concentrate in an effective amount for use in (i) improving physiological conditions related to metabolic fatigue in one or more cells, and / or (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and / or (iii) enhancing mitochondrial function, and / or (iv) treating or preventing calcium deficiency / depletion disorders in an organism, The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A composition that can be obtained or is obtained by a method including the following.

2. The composition for use according to claim 1, wherein at least a portion of the one or more cells is part of at least one body part selected from the group consisting of the liver, kidney, brain, and skeletal muscle.

3. The composition for use according to any one of claims 1 or 2, wherein the physiological state associated with metabolic fatigue includes muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, in particular deficient physical energy.

4. The composition for use according to any one of claims 1 to 3, wherein the effective amount of nettle concentrate is administered orally daily for at least one week.

5. A composition for use according to any one of claims 1 to 4, further comprising at least one compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

6. A composition for use according to any one of claims 1 to 5, selected from the group consisting of food compositions, dietary supplements, nutritional compositions, complete nutritional compositions, pharmaceuticals, oral nutritional supplements, medical foods, nutraceuticals, beverages, powdered nutritional products that are reconstituted with water or milk before ingestion, food additives, foods for special medical purposes (FSMPs), pharmaceuticals, pet foods, and combinations thereof.

7. A composition for use according to any one of claims 1 to 6, which is in the form of a solid powder, powder stick, capsule, or solution.

8. The composition for use according to any one of claims 1 to 7, wherein the effective amount of nettle concentrate is administered in a food product or beverage further comprising components selected from the group consisting of proteins, carbohydrates, fats and mixtures thereof.

9. A composition comprising nettle concentrate for use in an effective amount for the treatment and / or reduction of mitochondrial-related diseases or conditions associated with altered mitochondrial function, in individuals requiring treatment and / or reduction of the incidence and / or reduction of the severity thereof, wherein the method comprises orally administering an effective amount of the nettle concentrate to the individual requiring or at risk thereof. The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A composition that can be obtained or is obtained by a method including the following.

10. The composition for use according to claim 9, wherein the mitochondrial-related disease or condition is selected from the group consisting of stress, physiological aging, obesity, decreased metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive impairment, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

11. A composition comprising nettle concentrate for use in healthy middle-aged and elderly individuals in an effective amount to delay the onset of metabolic decline, maintain muscle mass and / or muscle function, maintain immune function, and / or maintain cognitive function, The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A composition that can be obtained or is obtained by a method including the following.

12. A composition comprising nettle concentrate for use in an effective amount to enhance at least one of mental or muscular performance in an individual, The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A composition that can be obtained or is obtained by a method including the following.

13. A composition comprising nettle concentrate for use in an effective amount to improve or maintain cognitive function in an individual, The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A composition that can be obtained or is obtained by a method including the following.

14. The composition for use according to claim 13, wherein the cognitive function is selected from the group consisting of perception, memory, attention, speech comprehension, speech production, reading comprehension, image creation, learning, reasoning, and combinations thereof.

15. The composition for use according to any one of claims 1 to 14, wherein the effective amount of nettle concentrate is administered with a composition further comprising calcium.

16. The composition for use according to any one of claims 1 to 15, wherein the individual is a middle-aged or elderly person, an elderly person, or an ICU patient.

17. A unit dosage form comprising nettle concentrate for use in an effective amount for at least one of the following: (i) treatment of mitochondrial-related diseases or conditions associated with altered mitochondrial function, reduction of their incidence, or reduction of their severity, and / or (ii) improvement of physiological conditions associated with metabolic fatigue in one or more cells, and / or (iii) increase of mitochondrial energy and increase of mitochondrial calcium uptake in one or more cells, and / or (iv) treatment or prevention of calcium deficiency / depletion disorders, and / or (v) increase of metabolic rate, and / or (vi) improvement or maintenance of cognitive function, and / or (viiii) improvement or maintenance of mitochondrial function, The nettle concentrate mentioned above a) A step of suspending nettle material in an aqueous liquid to form a nettle material suspension, b) A step of blending the nettle material suspension to obtain a nettle material slurry, c) A step of applying physical means to the nettle material slurry to separate and obtain nettle concentrate, d) Optionally, a step of drying the nettle concentrate, A unit dosage form that can be obtained or is obtained by a method including the following.

18. The unit dosage form according to claim 17, wherein the physiological state associated with metabolic fatigue includes muscle fatigue, muscle weakness, lack of vitality, decreased or deficient energy, in particular deficient physical energy.

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