Plant Extraction

Abstract

A method for extracting a compound of interest from a plant material is disclosed. The disclosed method includes identifying an optimal natural deep eutectic solvent (NaDES), mixing the identified NaDES with a plant material, and isolating the compound of interest from the mixture. Additionally, a composition comprising the isolated compound of interest and the NaDES is described.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of plant extraction processes. Specifically, the present disclosure relates to a method for extracting compounds from plants, such as Matecha, and a composition comprising a plant extract and a natural deep eutectic solvent. The method of the present disclosure includes precisely determining and modeling a bioinspired natural deep eutectic solvent for improving the extraction of compounds from plants. [Background technology]

[0002] Ilex paraguariensis (Matecha) is a medicinal plant that contains numerous bioactive compounds, including chlorogenic acids, saponins, and xanthine alkaloids. Extraction of such compounds from Matecha has traditionally involved harsh, environmentally unfriendly and inefficient methods and harsh chemicals.

[0003] Natural deep eutectic solvents (NaDES) are common compounds that can dissolve a variety of metabolites and compounds and are environmentally friendly. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure generally relates to compositions comprising a plant extract and a natural deep eutectic solvent, and methods of purifying a plant extract using a natural deep eutectic solvent.

[0005] Some embodiments provided herein relate to a method of isolating a plant extract from a plant material. In some embodiments, the method includes obtaining a plant material. In some embodiments, the method includes processing the plant material. In some embodiments, the method includes mixing the processed plant material with a natural deep eutectic solvent (NaDES). In some embodiments, the method includes isolating a plant extract. In some embodiments, the plant material is obtained from Matecha. In some embodiments, the NaDES includes choline chloride, citric acid, glucose, sucrose, or a combination thereof. In some embodiments, the NaDES includes choline chloride and citric acid. In some embodiments, the choline chloride and the citric acid are in a ratio of 3:1. In some embodiments, the NaDES includes 10% to 80% water. In some embodiments, the plant extract is a xanthine alkaloid, a chlorogenic acid, or a matesaponin. In some embodiments, the xanthine alkaloid includes caffeine, theobromine, theophylline, or an analog or derivative thereof. In some embodiments, the chlorogenic acids include caffeic acid, tannin, or analogs or derivatives thereof. In some embodiments, the method further includes a heating step, a stirring step, a centrifugation step, and / or a filtering step after the mixing step. In some embodiments, the NaDES includes choline chloride and citric acid in a 3:1 ratio, and 50% water.

[0006] Some embodiments provided herein relate to a composition. In some embodiments, the composition comprises a plant isolate and a natural deep eutectic solvent (NaDES). In some embodiments, the plant isolate comprises an isolate from Matecha. In some embodiments, the plant isolate comprises xanthine alkaloids, chlorogenic acids, or matesaponins. In some embodiments, the plant isolate comprises caffeine, theobromine, theophylline, caffeic acid, tannins, or analogs or derivatives thereof. In some embodiments, the NaDES comprises choline chloride, citric acid, glucose, sucrose, or combinations thereof. In some embodiments, the NaDES comprises choline chloride and citric acid. In some embodiments, the choline chloride and the citric acid are in a 3:1 ratio. In some embodiments, the composition comprises caffeine, theobromine, theophylline, caffeic acid, tannins, or analogs or derivatives thereof, choline chloride and citric acid in a 3:1 ratio, and 50% water.

[0007] These features, and other features described further herein, are described in further detail in the following drawing description and detailed description. [Brief description of the drawings]

[0008] The foregoing and other features of the present disclosure will become better understood from the following description taken in conjunction with the accompanying drawings, in which: FIG. 1 illustrates only certain embodiments in accordance with the present disclosure, and is therefore not intended to limit the scope of the disclosure, and the present disclosure will be described with more specificity and detail by reference to the accompanying drawings, in which:

[0009] [Figure 1A-1B] Some of the major bioactive components identified in Matecha are shown below.

[0010] [Diagram 2]A bar graph showing the extraction yield of Matecha components using conventional extraction solvents. Typical extraction solvents used were (from left to right on the graph): water; 20% ethanol; 40% ethanol; 60% ethanol; 80% ethanol; and 100% ethanol.

[0011] [Diagram 3] 1 shows a line graph illustrating the results of antioxidant assay of Matecha extracts in various solvents using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.

[0012] [Figure 4] Figure 1 shows a bar graph illustrating the extraction yield of bioactive compounds from Matecha by various NaDES: (from left to right): CLCIT 1:1, 30% water; CLGLU 1:1, 30% water; CLSUC 1:1, 30% water; CITGLU 1:1, 30% water; CITSUC 1:1, 30% water; CLCITGLU 1:1:1, 30% water; CLCITSUC 1:1:1, 30% water; CLCIT 3:1, 30% water; CLCIT 2:1, 30% water; CLCIT 1:2, 30% water; CLCIT 1:3, 30% water; CLCIT 1:1, 50% water; CLCIT 1:1, 80% water; water; and buffered water. CL is choline chloride, CIT is citrate, GLU is glucose, and SUC is sucrose.

[0013] [Figure 5A-5C] Chromatograms of the mixed standard (Figures 5A and 5B) and one sample of Matecha extract (Figure 5C) are shown.

[0014] [Figures 6A-6D]Graphs of response surface methodology (RSM) analysis are shown. Figure 6A shows a graph of model predicted vs actual values ​​of total extraction yield of Matecha extract. Figure 6B shows response surface and contour plots showing the effect of extraction temperature (A), liquid / solid ratio (B), and extraction time (C) on the extraction yield of total extract from Matecha. Figure 6C shows a comparison of model predicted vs actual values ​​of chlorogenic acid. Figure 6D shows the results of extraction yield of chlorogenic acid from Matecha where the effects of extraction temperature, liquid / solid ratio, and extraction time were observed.

[0015] [Figure 7] 1 shows a bar graph illustrating the extraction yield of theobromine using various NaDES systems, from left to right: glucose:sucrose, glucose:fructose, citric acid:glucose, citric acid:sucrose, citric acid:fructose, choline chloride:citric acid, choline chloride:glucose, choline chloride:sucrose, erythritol:choline chloride, erythritol:citric acid, erythritol:glucose:fructose, methanol, and water, all in a 1:1 ratio (where applicable). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In the following detailed description, the present invention will be described with reference to the accompanying drawings, which form a part of this specification. Unless otherwise specified, similar symbols in the drawings generally indicate similar components. The embodiments described in the detailed description, drawings, and claims are for illustrative purposes only and are not intended to limit the present invention in any way. Other embodiments may be employed and other changes may be made without departing from the spirit or scope of the subject matter described herein. It is readily understood that the aspects of the present disclosure as outlined herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in various configurations, and all such aspects are expressly encompassed herein.

[0017] As outlined herein, provided herein are methods for isolating and extracting various compounds from plant material, as well as various embodiments of mixtures of components isolated by the disclosed methods with natural deep eutectic solvents (NaDES).

[0018] The present disclosure is not limited to the specific embodiments described herein, which may, of course, be modified in various ways. Furthermore, the terms used in the present specification are merely used for the purpose of describing the specific embodiments, and are not intended to limit the present invention in any way.

[0019] As will be apparent to those of skill in the art upon perusal of this disclosure, each embodiment depicted and illustrated herein has distinct elements and features which may be readily separated or combined with the features of any of several other embodiments without departing from the scope or spirit of the disclosure, and the methodology described herein may be carried out in the order of events described or in any other order which is logically possible.

[0020] Unless otherwise defined, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of this disclosure, the following terms are defined below.

[0021] In some embodiments, a method is provided for extracting compounds from plants using one or more NaDES.In some embodiments, the method of the present disclosure includes identifying a combination of NaDES, mixing the NaDES with plant material, processing the resulting mixture, and extracting compounds from the plant.The extracted compounds may be further isolated.

[0022] Further provided herein is a composition comprising a compound isolated from a plant and the NaDES used to isolate the compound.

[0023] As used herein, "plant" has its general meaning as understood in the context of the present specification and means a whole plant or a part or a part derived from a plant, such as a plant cell, a plant protoplast, a plant cell tissue culture from which a plant can be regenerated, a plant callus, an embryo, a pollen, an ovule, a fruit, a flower, a leaf, a seed, a root, a root tip, etc. As used herein, the term "botanical" is used interchangeably with "plant" to mean a plant or a part derived from a plant.

[0024] In some embodiments, the plant can be any plant that has components that are desirable to extract, purify, or obtain from the plant. For example, the plant may be Maca, Cashew, Alchornea castaneifolia, Sceletium Tortosum, Magnolia grandiflora, Ziziphi Spinosae, Fructus Cnidii, Corydalis yanhusuo, Cortex albiziae, Panax ginseng, Polygoni Multiflori, Poria cocos, Fructus corni, Rhizoma dioscoreae, Muira puama, Dendrobium, Glycyrrhizae Preparata, Cordyceps sinensis, Angelicae sinensis, Kratom (Mitragyna speciosa), Bacopa Monnieri, Catuaba, Ashwagandha, Peganum Harmala, Wheat, Alfalfa, Oats, Kamut, Echinacea, Chlorella, Amla, Nettle, Carob, Mesquite, Chuchuhuasi, Clavohuasca, Chanca Piedra, Guayusa, Rhodiola Rosea, Shilajit, Higenamine, Moringa oleifera), horny goat weed, astragalus, aloe vera, turmeric, pine, curcumin (turmeric compound), hops, xanthohumol (hop compound), passionflower, veitch's bean, tulsi, black pepper, bioperine (black pepper compound), Siberian ginseng, American ginseng, mate tea, lemon balm, astragalus, birch, schisandra chinensis, skullcap, valerian, poppy, epimedium, pau d'arco, ginkgo, lotus, lily, tea, coffee or cocoa, or parts of such plants, such as bark, leaves, stems, roots, flowers, fruits, pollen, seeds, etc. Plant ingredients include, but are not limited to, vegetable oils, such as linalool; beta-caryophyllene; beta-myrcene; d-limonene; humulene; alpha-pinene;Ylang jasmine (Cananga odorata);Yarrow (Achillea millefolium);Violet (Viola odorata);Vetiver (Vetiveria zizanoides);Vanilla (Vanilla plantifolia);Tuberose (Polianthes tuberosa);Thyme (Thymus vulgaris L.);Tea plant (Melaleuca alternifolia);Tangerine (Citrus reticulata);Spinus japonicus (Picea mariana);Spinus japonicus (Tsuga Canadensis);Spikenard (Nardostachys jatamansi);Spearmint (Mentha spicata);Sandalwood (Santalum spicatum);Rosewood (Aniba rosaeodora);Rosemary verbenone (Rosmarinus officinalis);Rosemary (Rosmarinus officinalis);Rose (Rosa damascena);Rose geranium (Pelargonium roseum;Ravensara aromatica;Zingiber cassumunar;Pine needle (Pinus sylvestris L.);Petitgrain (Citrus aurantium);Peppermint (Mentha piperita);Pepper (Piper nigrum L.);Patchouli (Pogostemon cablin);Palo santo (Bursera graveolens);Palmarosa (Cymbopogon martini);Osmanthus (Osmanthus fragrans);Oregano (Origanum vulgare);Orange (Citrus sinensis);Oakmoss (Evernia prunastri);Nutmeg (Myristica fragrans);Niaouli (Melaleuca viridifloria);Neroli (Citrus aurantium);Myrtle (Myrtus communis);Myrrh (Commiphora myrrha);Mimosa (Acacia decurrens);Melissa (Melissa officinalis L.);Marjoram (Origanum majorana);Manuka (Leptospermum scoparium);Mandarin (Citrus deliciosa);Mandarin (Citrus deliciosa);Lotus (Nelumbo nucifera);Lotus (Nelumbo nucifera);Lotus (Nelumbo nucifera);Lime (Citrus aurantifolia);Lily (Lilum aurantum);Lemongrass (Cymbopogon citratus);Lemon (Citrus limonum);Lavender (Lavandula angustifolium);Lavandula hybrida grosso);Kanuka (Kunzea ericoides);Juniper (Juniperus cummunis);Jasmine (Jasminum officinale);Jasmine (Jasminum sambac);Helichrysum (Helichrysum italicum);Grapefruit (Citrus xparadisi);Grapefruit (Citrus paradisi);Ginger (Zingiber officinalis);Geranium (Pelargonium graveolens);Geranium (Pelargonium graveolens 'Heit);Gardenia (Gardenia jasminoides);Galbanum (Ferula galbaniflua);Frankincense (Boswellia carterii);Frangipani (Plumeria alba);White fir (Abies alba);Siberian fir (Abies siberica);Canadian fir (Abies balsamea);Fennel (Foeniculum vulgare);Eucalyptus smitty, Eucalyptus radiata, Eucalyptus globulosus, Eucalyptus citriodora, Eucalyptus bluemary (Eucalyptus polybractea);Elemi (Canarium luzonicum);Dill (Anethum graveolens);Cypress (Cupressus Cumin (Cuminum cyminum); Coriander (Coriandum sativum);Cocoa (Theobroma cacao);Clove (Eugenia caryophylatta);Clary sage (Salvia sclarea);Cistus (Labdanum) (Cistus ladaniferus L.);Cinnamon (Cinnamomum zeylanicum);Chamomile (Anthemis nobilis);Chamomile (Matricaria chamomilla);Celery seed (Apium graveolins);Cedarwood (Thuja plicata);Cedarwood (Juniperus virginiana);Cedrus atlantica;Carrot seed (Daucus carota);Cardamom (Elettaria cardamomum);Caraway seed (Carum carvi);Cajeput (Melaleuca cajuputi);Cade (Juniperus oxycedrus);Bava (Betula alba);Bava (Betula lenta);Bergamot (Citrus bergamia);Bay laurel (Laurus nobilis); basil (Ocimum basilicum); basil (Ocimum sanctum); basil (Ocimum basilicum); balsam poplar (Populus balsamifera); balsam of Peru (Myroxylon balsamum); or angelica (Angelica archangelica L.);

[0025] In some embodiments, the plant is Matecha. As used herein, the term "Matecha" has its general meaning as understood in the context of the present specification and refers to the medicinal plant Ilex paraguariensis. Matecha leaves have been extracted using aqueous mixtures of methanol, ethanol and / or acetone in traditional solid / liquid extraction methods. These chemicals and traditional extraction methods involve harsh chemicals and harsh methods, which are economically and environmentally burdensome. Disclosed herein is a NaDES for use in the extraction of materials from plants such as Matecha, with the aim of developing an environmentally friendly process.

[0026] Ilex paraguariensis (Matecha) is one of the most commercially available medicinal plants in South America, growing wild and cultivated in Argentina, Uruguay, Brazil and Paraguay. The constituent parts of Matecha are used to prepare tea-like beverages (Croge et al., 2020). In addition, Matecha has a long history of use worldwide. In Europe, it is used for weight loss, physical and mental fatigue, neurasthenia, rheumatic pain, and psychogenic and fatigue headaches. In Germany, it is widely used as an aid to weight loss. Matecha is included in the monographs of the German Pharmacopoeia. The German Pharmacopoeia states that it is approved for use against mental and physical fatigue, and that it has tonic, diuretic, positive inotropic, positive chronotropic, glycogenolytic, and lipolytic effects. In France, Mate tea is approved for the treatment of asthenia (weakness or fatigue), as an adjunct to weight loss programs, and to increase renal excretion. In addition, the British Pharmacopoeia (1996) lists applications for the treatment of fatigue, weight loss, and headaches. In the United States, Mate tea is recommended for use in arthritis, headaches, hemorrhoids, fluid retention, obesity, fatigue, stress, constipation, allergies, and hay fever, and is used to purify the blood, regulate the nervous system, slow aging, stimulate the mind, control appetite, and stimulate the production of cortisone, and is thought to enhance the healing powers of other herbs. Mate tea is also cultivated in India, where the Indian Ayurvedic Pharmacopoeia lists it for the treatment of psychogenic headaches, neurasthenia, fatigue, and rheumatic pain (Heck and De Mejia, 2007).

[0027] The bioactive chemical components contained in Matecha are mainly known to be xanthine alkaloids, chlorogenic acids, and matesaponins. Xanthine alkaloids include, for example, caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine), and theophylline (1,3-dimethylxanthine), as well as their analogs or derivatives. Chlorogenic acids include, for example, caffeic acid and tannins, as well as their analogs or derivatives (Oellig et al., 2018). Analysis of the components contained in Matecha leaves has been conducted, and it has been reported that Matecha leaves contain 0.7% to 2% by weight of caffeine, 0.3% to 0.9% by weight of theobromine, relatively small amounts of theophylline, saponins, and 10% of caffeic acid derivatives (chlorogenic acid, caffeic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid) (Matei et al., 2016). Other chemicals found in Matecha include α-amyrin, α-terpineol, arachidic acid, β-amyrin, butyric acid, 5-O-caffeoylquinic acid, calcium, carotene, choline, chlorophyll, chrysanthemum acid, cyanidin-3-O-xylosylglucoside, cyanidin-3-glucoside, essential oils, eugenol, geraniol, geranylacetone, guaiacan b, indole, inositol, ionone, isobutyric acid, isocaproic acid, isocyanic acid, and isocyanic acid. These include chlorogenic acid, isovaleric acid, kaempferol, lauric acid, levulose, linalool, linoleic acid, matesaponins, neochlorogenic acid, nerolidol, nicotinic acid, nudicaucin c, octan-1-ol, octanoic acid, oleic acid, palmitic acid, palmitoleic acid, pyridoxine, quercetin, raffinose, safrole, stearic acid, tannins, theobromine, theophylline, trigonelline, and ursolic acid.Additionally, Matecha leaf material has been reported to contain 7%-14% tannins (Bastos et al., 2007; Bojic et al., 2013; Burris et al., 2012; Chandrasekara and Shahidi, 2018; Chianese et al., 2019; Ferreira Cuelho et al., 2015; Frizon et al., 2018; Isolabella et al., 2010; Junior and Morand, 2016; Puangpraphant, 2012; Riachi et al., 2018; Souza et al., 2015). Figure 1A and Figure 1B show the main components of Matecha.

[0028] Theobromine is used as a vasodilator, diuretic, and cardiac inotropic agent. It is also known as an adenosine receptor antagonist and phosphodiesterase inhibitor and may be useful in the management of fatigue and orthostatic hypotension.

[0029] As used herein, the terms "extract", "extraction" or "extracting" or their derivatives have their ordinary meaning as understood in the context of this specification and refer to the process of removing a material, compound, composition or other component from a starting material. The extraction process can be carried out by various means, such as physical extraction by pressing, grinding, heating, stirring, etc., or chemical extraction, or other known methods for extracting components from a starting material.

[0030] As used herein, the terms "isolate", "isolated" or "isolating", or derivatives thereof, have their ordinary meaning as understood in the context of the present specification, and refer to a material that is substantially or essentially free of components that normally accompany or interact with it in its natural environment (e.g., components of a plant extract), or in a processed form of the material, e.g., processed Matecha material, from which it is substantially or essentially free of components that accompany or interact with it, e.g., materials from which substances used in the isolation process have been substantially or essentially removed. When the term "substantially purified" is used, it refers to a composition in which the desired component is the major or predominant component in the composition, e.g., a composition in which the desired component constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the total composition (e.g., on a weight / weight and / or weight / volume basis). As used herein, the term "substantially purified" refers to a compound that has been removed from its natural environment or in a processed form, or an isolated or separated compound, e.g., a compound derived from Matecha, that has been at least 60% free from other components with which it is associated in its natural environment, e.g., that has been 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% free from other components with which it is associated in its natural environment.

[0031] As used herein, the term "natural deep eutectic solvent (NaDES)" has its general meaning as understood in the context of this specification and refers to sugars, amino acids or organic acids that are normally solid at room temperature, but when mixed in certain mole fractions, have a significantly reduced melting point and become liquid at room temperature.

[0032] The discovery of deep eutectic solvents (DES) is a major breakthrough in the green chemistry industry. Deep eutectic solvents are often defined as binary or ternary mixtures composed of compounds that can associate primarily through hydrogen bonds. By mixing such compounds in specific molar ratios, a eutectic mixture is obtained. Eutectic solvents are composed of two or more inexpensive, non-toxic components, one of which can act as a hydrogen bond acceptor and the other has hydrogen bond donor properties. Eutectic solvents have melting points significantly lower than those of the individual components due to the formation of hydrogen bonds within the molecules and interactions through van der Waals forces. An example of a deep eutectic solvent is a mixture of hydroxyethyltrimethylammonium (choline chloride) (melting point Tm=302°C) and urea (Tm=133°C), which has a very low melting point (Tm=12°C). What is interesting about eutectic solvents is that they are not only liquid at ambient temperature, but also have tunable and highly soluble properties at ambient temperature (Abbott et al., 2004).

[0033] Natural deep eutectic solvents (NaDES) are a specific type of deep eutectic solvents (DES) prepared from biomolecules such as choline chloride and betaine as organic salts, urea as hydrogen bond donors, organic acids, amino acids, and sugars (Dai, 2006, 2007). The classification of natural deep eutectic solvents also includes DESs composed of primary metabolites such as organic acids, amino acids, sugars, polyols, and choline derivatives (Dai et al., 2013). In addition, water can also be part of the composition of NaDES. Remarkably, NaDES are ubiquitous in both intracellular and extracellular fluids of living organisms and may play a role in the synthesis and solubilization of poorly soluble metabolites such as flavonoids, as well as in enzyme reactivity and desiccation resistance. In this paradigm, NaDES constitute a third type of natural liquid distinct from water and lipids (Choi et al., 2011). A series of NaDESs have been prepared from a wide variety of biomolecules, including combinations of citric acid, malic acid, maleic acid, or ascorbic acid with choline chloride; combinations of proline and citric acid; combinations of malic acid and glucose; and sugar mixtures (e.g., fructose:glucose, fructose:sucrose, glucose:sucrose). NaDESs are considered as environmentally friendly alternative solvents for the extraction of biomolecules (Liu et al., 2018).

[0034] NaDES exhibits favorable properties as a solvent, including low vapor pressure, non-flammability, low or only slight toxicity, environmental friendliness, ease of preparation, and low cost. NaDES species have outstanding advantages as extraction media, due to their high dissolving ability for natural products. Among the components of NaDES, the most important functional groups are carboxylic acid, hydroxyl, and carbonyl groups. In the matrix of NaDES, these functional groups can form hydrogen bond networks through intermolecular interactions that affect the physicochemical environment. In general, the greater the intermolecular attraction, the greater the polarity. In other words, polarity usually means solubility. The unique intermolecular interactions or intermolecular arrangement of the NaDES matrix give NaDES its unique dissolving and stabilizing properties. For example, proline is almost insoluble in dimethyl sulfoxide (DMSO), but proline-based multicomponent NaDES can be completely miscible with DMSO.

[0035] NaDES are biologically active and can be designed to have specific biological activities. For example, if a solvent with antioxidant and / or antitumor activity is required, NaDES can be prepared using compounds with the desired biological activity. Previous reports have demonstrated that NaDES used for extraction purposes improved the antioxidant activity of the resulting plant extract, which can be explained by the fact that NaDES itself or its constituent compounds have scavenging activity against reactive oxygen species. The antioxidant activity of NaDES was not unexpected, since the constituent compounds of NaDES (malic acid, citric acid, proline, and betaine) have antioxidant activity. Furthermore, since the components contained in NaDES are abundant in nature and are part of our daily food and already on the market in dietary supplements (e.g., choline, citric acid, betaine, amino acids, etc.), it is expected that the extract obtained using NaDES can be used directly in food, pharmaceutical, cosmetic, and agrochemical products without the need for costly downstream purification steps. Furthermore, NaDES can also improve the biological activity of phenolic acids (Faggian et al., 2016), potentially allowing their direct use in food, cosmetic and pharmaceutical formulations.

[0036] NaDES are ubiquitous in both intracellular and extracellular fluids of living organisms and play a role in the synthesis and solubilization of poorly soluble metabolites such as flavonoids. NaDES also play a role in enzymatic reactivity and desiccation resistance. NaDES exhibit many favorable properties, such as low vapor pressure, non-flammability, low or only slight toxicity, no adverse environmental impact, low cost, and easy use. Furthermore, there are a huge number of structural combinations of NaDES, and the design of NaDES combinations for specific applications can be optimized. NaDES are also biocompatible and can improve biological activity. Thus, the design of compositions can be optimized using NaDES with specific biological activity. Extracts extracted by NaDES and containing NaDES in their composition may be used in foods, dietary supplements, pharmaceuticals, cosmetics, agricultural chemicals, and industrial applications.

[0037] The methods described herein relate to improved, efficient, and environmentally friendly processing of plant material, such as the whole plant, stems, leaves, or other parts of the plant (e.g., Matecha plant material). In some embodiments, the disclosed method includes the steps of providing Matecha plant material, screening for an optimal NaDES, mixing the Matecha plant material with the NaDES, and isolating one or more target compounds from the Matecha.

[0038] In some embodiments, the plant material is raw, unprocessed material, e.g., whole plant material as grown. In some embodiments, the plant material is processed material, e.g., dried, mixed, crushed, granulated, powdered, liquid, or other processed plant material. In some embodiments, the Matecha plant material is the whole plant, leaves, roots, stems, flowers, bark, or other parts.

[0039] In some embodiments, the NaDES comprises one or more NaDES including choline chloride, citric acid, malic acid, maleic acid, ascorbic acid, proline, glucose, sucrose, fructose, or any combination thereof. The combination of one or more NaDES can be provided in various ratios, for example, two NaDES can be combined in a ratio of 1:1, 1:2, 1:3, 1:4, 2:3, 3:2, 2:1, 3:1, or 4:1, or three or more NaDES can be combined in any of these ratios.

[0040] In some embodiments, the one or more NaDES comprises a first NaDES and water, and at least one hydrogen bond donor.In some embodiments, the hydrogen bond donor is an organic acid or a polyol.In some embodiments, the hydrogen bond donor is an organic acid, such as lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, citric acid, acetic acid, ursolic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, and lactone derivatives.Exemplary NaDES combinations are shown in Table 1 below. [Table 1]

[0041] In some embodiments, the compounds of interest isolated from Matecha are phenolic acids, chlorogenic acids, flavonoids, alkaloids, terpenes, flavors, antioxidants, or saponins, or any combination thereof.

[0042] In some embodiments, the step of mixing the plant and / or plant material with the NaDES comprises soaking the plant and / or plant material in a solution of the NaDES, spraying the plant and / or plant material with the solution of the NaDES, coating the plant and / or plant material with the solution of the NaDES, or contacting the plant and / or plant material with the solution of the NaDES. In some embodiments, the method of the present disclosure comprises heating the mixture, stirring the mixture, macerating the mixture, or impregnating the mixture, or any combination thereof. In some embodiments, the step of heating the mixture can be performed by heating the mixture to a temperature in the range of about 15° C. to about 80° C., for example, 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C., or a range of any two of these values.

[0043] In some embodiments, isolating the compound of interest comprises filtering the mixture, spray drying the mixture, or other process that isolates the compound of interest.

[0044] In some embodiments, NaDES is used as an extraction solvent for the treatment of plant material, such as leaves, for example, Matecha leaves. As described throughout this disclosure, NaDES is an ideal solvent for extracting bioactive components from plants and / or plant materials. In some embodiments, NaDES improves the solubility of phenolic acids.

[0045] NaDES are a greener alternative to organic solvents and, because their properties can be designed and tuned, can be further customized to extract desired components and for use in various sectors such as the nutraceutical, pharmaceutical, cosmetic and food industries.

[0046] Some embodiments provided herein relate to compositions comprising a bioactive compound purified, extracted or isolated from a plant and / or plant material using a NaDES using the methods described herein, and a NaDES. In some embodiments, the compositions of the present disclosure are formulated for use in pharmaceuticals, cosmetics or foods.

[0047] As used herein, the terms "composition" or "formulation" have their general meaning as understood in the context of this specification and refer to a combination of components, ingredients or compositions provided for a certain purpose.

[0048] The pharmaceutical compositions of the present disclosure may comprise one or more bioactive compounds isolated, purified or extracted from plants and / or plant materials, and an effective amount of NaDES, in combination with a pharma- ceutically acceptable carrier. The effective amount (and method of administration) is determined on an individual basis and is based on subject-specific considerations (length, age, general health), the severity of the condition being treated, the severity of the symptoms being treated, the desired outcome, the particular carrier or pharmaceutical formulation used, the route of administration, and other factors that are apparent to one of skill in the art. The effective amount can be determined by one of skill in the art using techniques known in the art. The therapeutically effective amounts of the compounds described herein and combinations thereof can be determined using in vitro tests, animal models, or other dose-response tests, as is well known in the art.

[0049] The pharmaceutical compositions of the present disclosure may be prepared, packaged, or sold in a formulation suitable for intradermal, intravenous, subcutaneous, oral, rectal, vaginal, parenteral, intraperitoneal, topical, pulmonary, intranasal, buccal, intraocular, intrathecal, epidural, or another route of administration. The compounds of the present disclosure may be administered by any convenient route, such as by infusion or bolus injection, or by absorption through epithelial or mucocutaneous surfaces (e.g., oral, rectal, intestinal, etc.), or may be administered together with other biologically active substances. Administration may be systemic or local. Furthermore, administration may be a single dose or multiple doses.

[0050] Therefore, the present disclosure further provides pharmaceutical compositions suitable for administration to a subject. The carrier may be liquid so that the composition of the present disclosure is suitable for parenteral administration, or solid so that the composition of the present disclosure is suitable for tablet or pill formulation for oral administration. Furthermore, the carrier may be in the form of a nebulizable liquid or solid so that the composition of the present disclosure is suitable for inhalation. For parenteral administration, the composition of the present disclosure should be pyrogen-free and is contained in an acceptable parenteral carrier. In another embodiment, the active compound can be formulated or encapsulated in liposomes using known methods. Other contemplated formulations include uneven nanoparticles and immunological formulations.

[0051] Liposomes are completely closed lipid bilayers that encapsulate water-soluble components. Liposomes can be unilamellar vesicles (vesicles made of a single membrane) or multilamellar vesicles (onion-like structures in which multiple membrane bilayers are separated from adjacent layers by aqueous layers). A lipid membrane bilayer is a stack of two lipid monolayers, each with a hydrophobic "tail" region and a hydrophilic "head" region. In a lipid membrane bilayer, the hydrophobic (non-polar) "tail" of the lipid monolayer faces inward from the bilayer, and the hydrophilic (polar) "head" faces toward the aqueous phase.

[0052] The compositions provided herein may further comprise suitable pharma- ceutically acceptable carriers, stabilizers, diluents or buffers; ingredients for use, preservation, bioavailability or solubility; or other ingredients for improving the efficacy, appearance or other properties of the compositions of the present disclosure.

[0053] A "pharmacologically acceptable" carrier is one that is not toxic to cells or mammals at the dosages and concentrations used therein. Pharmaceutically acceptable carriers include, but are not limited to, organic or inorganic solid or liquid additives that are suitable for the chosen mode of administration, such as topical, oral, or intravenous administration, and are administered in the form of conventional pharmaceutical formulations, such as solid formulations such as tablets, granules, powders, capsules, caplets, and liquid formulations such as solutions, sprays, emulsions, foams, suspensions, creams, lotions, ointments, liniments, gels, and the like. In many cases, physiologically acceptable carriers are aqueous pH buffers, such as phosphate buffers or citrate buffers. Additionally, physiologically acceptable carriers may include, for example, antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues in length) polypeptides; proteins such as serum albumin, gelatin, immunoglobulins, and the like; hydrophilic polymers such as polyvinylpyrrolidone; amino acids; sugars such as glucose, mannose, dextrin, and the like; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and Tween. TM , Polyethylene glycol (PEG), Pluronic TM and other nonionic surfactants such as: Auxiliaries, stabilizers, emulsifiers, lubricants, binders, pH adjusters, isotonicity adjusters, and other conventional additives may be added to these carriers.

[0054] As described herein, the composition of the present disclosure, which includes a plant and / or plant material derived bioactive compound and NaDES, can be formulated as an external preparation. The external preparation may further include, for example, a pharmaceutical base that does not interfere with the function or effectiveness of the NaDES and the bioactive compound. As described herein, "pharmaceutical base" refers to an inactive substance that is mixed into the preparation to facilitate measurement and administration of the external preparation.

[0055] In some embodiments, the active ingredient and mixtures thereof can be used in the form of an external preparation, for example, with a pharma- ceutically acceptable carrier, prepared for storage and subsequent administration. As used herein, "external" means administering or applying the preparation to the skin or various orifices of the body. Some embodiments include the use of the compositions described herein in combination with a pharma- ceutical acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), which is incorporated herein by reference in its entirety. Preservatives and stabilizers can be added to the external preparation. Preservatives can be used to prevent the degradation of nutritional ingredients for skin cells. As used herein, "carrier or diluent" can be a solid carrier or solid diluent for solid preparations, a liquid carrier or liquid diluent for liquid preparations, or a mixture thereof. Solid carriers / diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylic esters (e.g., methyl polyacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers for liquid formulations, such as topical and parenteral formulations, may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Examples of oils include mineral, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0056] Parenteral vehicles (for subcutaneous, intravenous, intraarterial or intramuscular injection) include sodium chloride solution, Ringer's glucose solution, glucose and sodium chloride solution, lactated Ringer's solution, and fixed oils. Vehicles for intravenous administration include fluids, nutrient replenishers, electrolyte replenishers (e.g., Ringer's glucose-based vehicles), and the like. Examples include sterile liquids such as water and oils, to which surfactants and other pharma-ceutically acceptable adjuvants may be added. In general, preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol and polyethylene glycol. Examples of oils include mineral, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0057] Additionally, the compositions of the present disclosure may contain additives such as binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers of various pH and ionic strength (e.g., Tris-HCI, acetates, phosphates), additives such as albumin and gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose), thickeners (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, paclitaxel, raven), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), glidants (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymeric coatings (e.g., poloxamers or poloxamines), coating and film formers (e.g., ethylcellulose, acrylates, polymethacrylates) and / or adjuvants.

[0058] The topical preparation can be formulated and used as a solution, spray, emulsion, foam, suspension, cream, lotion, ointment, liniment or gel for topical application. Suitable ingredients for topical preparations include, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, etc. If desired, an absorption enhancer (e.g., liposome) can be used. The topical preparation can further include, for example, one or more solvents and / or at least one skin softener.

[0059] As used herein, an "injectable composition" refers to a formulation prepared for administration by injection. Such injections can be administered by intravenous, subcutaneous, intradermal, intramuscular, intraarticular, or intrathecal routes.

[0060] In some embodiments, the pharmaceutical carrier is soybean, grapefruit, or almond oil; synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

[0061] In topical formulations, coconut oil, olive oil, sesame oil, peanut oil, and soybean oil can be used as a suspending agent or lubricant. In some embodiments, topical formulations may contain at least one thickening agent, at least one humectant, and / or at least one preservative. Thickening agents include, for example, triglycerides, palmitates, myristates, stearates, polyethylene glycols, plant-derived fatty alcohols, copolymers, cellulose gums, and xanthan gums. Humectants can be used for their moisturizing properties. Humectants include, but are not limited to, sodium PCA, nano lipid gel, glycerin, alpha-hydroxy acids, butylene glycol, propylene glycol, hexylene glycol, sorbitol, hyaluronic acid, urea, glyceryl triacetate, neoagarobiose, glycerol, xylitol, maltitol, polymeric polyols, polydextrose, quillaja, MP diol, seaweed extract, algae extract, and lactic acid. In some embodiments, the topical formulation further comprises at least one preservative.Preservatives include, but are not limited to, benzoin resin, jojoba, vitamin E, alcohol, phenoxyethanol, methylparaben, propylparaben, diazolidinyl urea, sorbic acid, and triclosan.In some embodiments, the at least one preservative is benzoin resin, jojoba, vitamin E, alcohol, phenoxyethanol, methylparaben, propylparaben, diazolidinyl urea, sorbic acid, and / or triclosan.

[0062] As used herein, the articles "a" and "an" are used to refer to one or to more than one (at least one) of the grammatical object of the article. For example, "an element" means one component or more than one component.

[0063] "About" means that a particular quantity, level, number, frequency, percentage, dimension, size, amount, weight or length may vary by 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, number, frequency, percentage, dimension, size, amount, weight or length.

[0064] Throughout this specification, unless otherwise stated, the terms "comprise" and "comprising" are meant to include the steps or components or steps or components described herein but not to exclude other steps or components or steps or components.

[0065] "Consisting of" means including only those listed before this term. Thus, the term "consisting of" indicates that the components listed before this term are necessary or essential, and other components may not be included. "Consisting essentially of" means including the components listed before this term, and including other components that do not interfere with or contribute to the activity or action described in connection with the disclosure of these components. Thus, the term "consisting essentially of" indicates that the components listed before this term are necessary or essential, but other components are optional and may or may not be included depending on whether they have a substantial effect on the activity or action of the components listed before this term.

[0066] In certain embodiments, the "purity" of a drug in a composition may be specifically defined. For example, a certain composition may contain a drug with a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (including decimals between each number) as measured by, for example, but not limited to, high pressure liquid chromatography (HPLC), a type of column chromatography known to be frequently used in biochemistry and analytical chemistry to separate, identify and quantify compounds.

[0067] Some embodiments relate to the embodiments listed below.

[0068] 1. A method for isolating a plant extract from a plant material, comprising: Obtaining plant material; treating the plant material; mixing the treated plant material with a natural deep eutectic solvent (NaDES); and Isolating the plant extract The method includes:

[0069] 2. The method of embodiment 1, wherein the plant material is obtained from Matecha.

[0070] 3. The method of embodiment 1 or 2, wherein the NaDES comprises a hydrogen bond donor molecule and a hydrogen bond acceptor molecule.

[0071] 4. The method of any one of embodiments 1 to 3, wherein the NaDES is a sugar-based NaDES, an organic acid-based NaDES, a choline chloride-based NaDES, or a polyol-based NaDES.

[0072] 5. The method of embodiment 3, wherein the sugar-based NaDES is glucose, sucrose, fructose, or a combination thereof.

[0073] 6. The method of embodiment 3, wherein the organic acid-based NaDES is citric acid in combination with glucose, sucrose, fructose, or a combination thereof.

[0074] 7. The method of embodiment 3, wherein the choline chloride-based NaDES is choline chloride in combination with citric acid, glucose, sucrose, fructose, or a combination thereof.

[0075] 8. The method of embodiment 3, wherein the polyol-based NaDES is erythritol in combination with choline chloride, citric acid, glucose, fructose, sucrose, or combinations thereof.

[0076] 9. The method of any one of embodiments 1 to 8, wherein the NaDES comprises choline chloride, citric acid, erythritol, glucose, sucrose, fructose, or a combination thereof.

[0077] 10. The method of any one of the preceding claims, wherein the NaDES comprises choline chloride:citric acid, choline chloride:glucose, choline chloride:sucrose, choline chloride:fructose, glucose:sucrose, glucose:fructose, citric acid:glucose, citric acid:sucrose, citric acid:fructose, erythritol:choline chloride, erythritol:citric acid, or erythritol:glucose:fructose.

[0078] 11. The method of any one of embodiments 1 to 10, wherein the NaDES comprises choline chloride and citric acid.

[0079] 12. The method of embodiment 11, wherein the choline chloride and the citric acid are in a ratio of 3:1.

[0080] 13. The method of any one of embodiments 1 to 12, wherein the NaDES comprises 10% to 80% water.

[0081] 14. The method of any one of embodiments 1 to 13, wherein the plant extract is a xanthine alkaloid, a chlorogenic acid, or a matesaponin.

[0082] 15. The method of embodiment 14, wherein the xanthine alkaloids comprise caffeine, theobromine, theophylline, or analogues or derivatives thereof.

[0083] 16. The method of embodiment 14, wherein the chlorogenic acids include caffeic acid, tannins, or analogues or derivatives thereof.

[0084] 17. The method of any one of the preceding embodiments, further comprising a heating step, a stirring step, a centrifugation step, and / or a filtration step after the mixing step.

[0085] 18. The method of any one of embodiments 1 to 16, wherein the NaDES comprises choline chloride and citric acid in a ratio of 3:1 and 50% water.

[0086] 19. The method of embodiment 10, wherein the NaDES is in a 1:1 molar ratio.

[0087] 20. The method of any one of embodiments 1 to 19, wherein the plant extract is theobromine extracted in an amount of about 5% to about 10% by weight.

[0088] 21. A plant isolate; Natural deep eutectic solvent (NaDES) and A composition comprising:

[0089] 22. The composition of embodiment 21, wherein the plant isolate comprises an isolate derived from Matecha.

[0090] 23. The composition of embodiment 21 or 22, wherein the plant isolate comprises xanthine alkaloids, chlorogenic acids, or matesaponins.

[0091] 24. The composition of any one of embodiments 21 to 23, wherein the plant isolate comprises caffeine, theobromine, theophylline, caffeic acid, tannins, or analogs or derivatives thereof.

[0092] 25. The composition of any one of embodiments 21 to 24, wherein the NaDES comprises choline chloride, citric acid, erythritol, glucose, sucrose, fructose, or a combination thereof.

[0093] 26. The composition of any one of embodiments 21 to 25, wherein the NaDES comprises choline chloride:citric acid, choline chloride:glucose, choline chloride:sucrose, choline chloride:fructose, glucose:sucrose, glucose:fructose, citric acid:glucose, citric acid:sucrose, citric acid:fructose, erythritol:choline chloride, erythritol:citric acid, or erythritol:glucose:fructose.

[0094] 27. The composition of any one of embodiments 21 to 26, wherein the NaDES is in a 1:1 molar ratio.

[0095] 28. The composition of any one of embodiments 21 to 27, wherein the NaDES comprises choline chloride and citric acid.

[0096] 29. The composition described in embodiment 28, wherein the choline chloride and the citric acid are in a ratio of 3:1.

[0097] 30. The composition of embodiment 21, comprising caffeine, theobromine, theophylline, caffeic acid, tannin, or an analogue or derivative thereof, choline chloride and citric acid in a 3:1 ratio, and 50% water. EXAMPLES

[0098] The following examples further disclose some of the aspects of the above-mentioned embodiments of the present invention, but are not intended to limit the scope of the present disclosure in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the present disclosure, as described herein and in the claims.

[0099] The examples provided herein relate to methods of extracting, purifying and isolating bioactive compounds from Matecha using NaDES, however, other plants and / or plant materials may also be used using the methods, techniques and assays described in the examples.

[0100] Materials and Methods The experiments were carried out with powdered organic Matecha leaves obtained from Triunfo. Chemicals, reagents and standards used in this study are listed in Table 2 below. [Table 2]

[0101] Preparation of eutectic solvent The various components of the eutectic mixture (choline chloride, glucose, sucrose and citric acid) were weighed and placed in an Erlenmeyer flask in a given order. Tap water, preferably demineralized or distilled water, was then added to a concentration (weight concentration) that maintained the physicochemical and microbiological integrity. The water content was 1-50%, preferably 30-50%, and desirably 50% by weight relative to the total weight of the mixture. The eutectic mixtures selected in this example were a mixture of citric acid and glucose (2mol:1mol, 1mol:1mol, or 1mol:2mol), a mixture of citric acid and sucrose (2mol:1mol, 1mol:1mol, or 1mol:2mol), a mixture of choline chloride and citric acid (1mol:1mol, 1mol:2mol, 1mol:3mol, 3mol:1mol, or 2mol:1mol), a mixture of choline chloride and glucose (1mol:1mol), and a mixture of choline chloride and sucrose (1mol:1mol), all with a water content of 30% by weight. Additionally, mixtures of choline chloride and citric acid (1 mol:1 mol) with moisture contents of 30%, 50% or 80% were also selected. However, it will be appreciated that other combinations of hydrogen bond donors and choline chloride as defined herein may be used. Each mixture was heated to 50°C ± 2°C and homogenized with magnetic stirring. Once the medium was completely dissolved and molten, it was allowed to stand at ambient temperature and then stored in a container until use.

[0102] Solid / liquid extraction by maceration 10 g of Matecha leaf powder was soaked in conventional solvents (water, water / citric acid, or aqueous ethanol) or eutectic solvents. Each mixture was heated in a 100 ml glass beaker with magnetic stirring. After extraction, the resulting liquid was transferred to a tube and centrifuged (3500 rpm for 10 min) and filtered through 5-7 μm cellulose filter paper to separate the solid and liquid fractions.

[0103] Experimental Design: The effects of three variables, namely extraction temperature (A), liquid / solid ratio (B) and extraction time (C), were investigated and optimized using a three-level Box-Behnken design (BBD). Thus, 15 experiments with the BBD design (including three replicates at the center point) were performed to fit regression equations and optimize the process factors affecting the extraction yield from Matecha leaves. The factors and their levels are shown in Table 3. For statistical calculations, each variable was transformed to +1, 0 and -1. These transformed variables indicate high, medium and low values, respectively. [Table 3]

[0104] These factors and levels were considered as important dependent variables in achieving optimal extraction conditions characterized by the content of total chlorogenic acids (CGAs) and caffeine and extraction yield. A second-order polynomial model was used for response surface analysis to predict the dependent variables and evaluate the effect of each extraction factor. This second-order polynomial model is described by Equation 1 below:

[0105]

number

[0106] where Y is the response variable, A0, Ai, Aii and Aij are the regression coefficients of the intercept, linear, quadratic and interaction terms, respectively, and Xi and Xj represent the independent variables (i ≠ j). This second-order polynomial model was used to assess the effect of each independent variable on the response.

[0107] The experimental data were analyzed using Design Expert version 13 to obtain the regression equation and determine the optimal parameter combination. The statistical significance of the model coefficients was determined by a combination of analysis of variance (ANOVA) and Fisher's F test, with a probability P value of 0.05. In addition, the coefficient of determination R as an index of the goodness of fit of the model was used. 2The accuracy of the model was investigated by the above method. Next, the fitted polynomial was plotted as a three-dimensional response surface and a two-dimensional contour plot to show the relationship between the response and the variables.

[0108] Quantitative determination of target compounds by high performance liquid chromatography HPLC analysis was performed using a Thermo Fisher Vanquish series system equipped with a diode array detector (DAD) and an Accucore C18 column (150 mm x 2.1 mm, particle size 2.6 μm). The mobile phase for separation was water / formic acid (99.9:0.1 (v / v)) (solvent A) and acetonitrile / formic acid (99.9:0.1 (v / v)) (solvent B). The column temperature was maintained at 30 °C and the autosampler was maintained at 4 °C. The injection volume was 1 μl and the flow rate was set at 0.5 ml / min. Before analysis, the samples were filtered through a 0.22 μm PTFE (polytetrafluoroethylene) filter and analyzed with the elution gradient shown in Table 4. [Table 4]

[0109] Retention times and spectral data of bioactive components from Matecha were compared with external standards. Caffeine and theobromine were identified at 280 nm, and chlorogenic acid (3-O-caffeoylquinic acid) (3-CQA), 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), 4,5-dicaffeoylquinic acid (4,5-diCQA) and rutin were identified at 350 nm.

[0110] Each standard solution was prepared in methanol at a concentration of 3 mg / ml and mixed in equal amounts to make a mixed solution with a concentration of 0.5 mg / ml, which was then serially diluted to 0.375 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.025 mg / ml and 0.005 mg / ml.

[0111] The concentration of each 3-CQA isomer was calculated as 3-CQA equivalent by the external standard method using a calibration curve. The total CGA content was calculated as the sum of the major CGA isomers 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA.

[0112] Testing physicochemical efficacy as an antioxidant 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a free radical used to evaluate the free radical scavenging properties of natural products. DPPH is characterized by the removal of the entire molecule's unpaired electron to become a stable free radical, and unlike most other free radicals, it does not form dimers. With the removal of the unpaired electron, DPPH turns dark purple in color. When a DPPH solution is mixed with a compound that can donate a hydrogen atom, it produces the reduced form diphenylpicrylhydrazine, and the purple color disappears. The free radical scavenging activity of all extracts was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to a previously reported method (Shen et al., 2010).

[0113] Thirty milligrams of dried extract of each sample was diluted with 10 ml of methanol to prepare a stock solution with a concentration of 3 mg / mL. From this stock solution, solutions with concentrations of 1.5 mg / mL, 0.75 mg / mL, 0.375 mg / mL, 0.1875 mg / mL, 0.09375 mg / mL, 0.046875 mg / mL, 0.0234275 mg / mL, or 0.01171825 mg / mL were prepared. Pure standards, ascorbic acid, rutin, and chlorogenic acid, were used as positive controls and were dissolved in methanol to prepare stock solutions with the same concentrations as the extracts.

[0114] DPPH (7.89 mg) was weighed out using an analytical balance with a minimum weighing value of 10 μg or less. It was then dissolved in HPLC-grade methanol and filled to volume in a 100 ml measuring flask (0.2 mM DPPH). This solution was further diluted to a 0.1 mM solution. It is experimentally known that the absorbance of the DPPH solution decreases over time for up to about 1 hour after preparation. For this reason, the solution was stored in the dark for 2 hours until the absorbance stabilized.

[0115] During the assay, the DPPH solution was stored in the dark at room temperature and used up on the day of preparation. 0.3 mL of each concentration of sample and positive control was added to 2.7 mL of 0.1 mM DPPH solution. The reaction was carried out in a tube covered with aluminum foil in a dark environment at 25 °C. The mixture was vigorously shaken and left at room temperature for 30 min. The absorbance was then measured at 517 nm using a microplate reader (Varioskan LUX, Thermo Fisher). The same amount of control samples were prepared without the extract and ascorbic acid standard. Methanol was used as a blank and the scavenging rate (%) of DPPH free radical was measured using the following formula:

[0116] DPPH scavenging effect (inhibition rate (%)) = {(Ac-As) / Ac) × 100}

[0117] In the formula, Ac represents the absorbance of DPPH alone, and As represents the absorbance of DPPH when various concentrations of extracts were added.

[0118] The measurements were performed at eight different concentrations. Each concentration of the analytical sample solution was measured three times.

[0119] IC of each analytical sample 50 The values ​​were calculated using the following procedure. The inhibition rates were plotted for all six sample concentrations, and each regression curve (y=ax+b) was obtained. The regression curve did not need to pass through the origin. In this process, it was confirmed that all measurement points were on the regression curve. Furthermore, it was confirmed that the two points near approximately 50% inhibition rate did not deviate from the regression curve. X (sample concentration) was calculated by setting Y in the regression equation to 50.

[0120] Example 1: Extraction of bioactive compounds from Matecha The following examples illustrate embodiments of methods for the extraction of bioactive compounds from Matecha.

[0121] The extraction yield using NaDES was twice that using conventional solvents such as water and buffered water, indicating that NaDES has obvious advantages in the extraction of bioactive compounds from Matecha. Twenty types of NaDES were tested, and the extraction yield of the target compounds varied depending on the type of NaDES. Organic acid-based NaDES showed higher extraction yields than sugar-based NaDES. The presence of citric acid in NaDES had a significant effect, which was unexpected. For example, the extraction yield of 5-CQA increased by 10 times. The extraction yield was found to be closely related to some interaction between the solute and the solvent and the properties of the solvent. In other words, these results were due to the influence of hydrogen bonds formed between NaDES and the target components. In addition, among the NaDESs that contained citric acid, the one with a molar ratio of choline chloride to citric acid (CLCit) of 3:1 showed the highest extraction yield. Furthermore, the addition of water to reduce the viscosity of the solvent had a significant effect on the extraction yield. Based on these considerations, CLCit3:1 with a moisture content of 50% was selected as the favorable NaDES for extracting bioactive components from Matecha.

[0122] CLCit has favorable physicochemical properties for the extraction process, such as a polarity similar to that of water and polar organic solvents (ethanol and methanol) commonly used for the extraction of polyphenolic compounds, and a low pH value. Extraction with CLCit at 50% water content increased the extraction efficiency by reducing the viscosity of the NaDES upon dilution with water and improving the mass transfer from the plant matrix to the solution. Furthermore, it is interesting to note that the selected choline-based NaDES, which contain organic acids as hydrogen bond donors, are themselves biologically active compounds (choline and citric acid) that could possibly improve the biological activity of the plant extracts.

[0123] Choline is an essential component of the human diet, required for the synthesis of acetylcholine, cell membranes, and phospholipids of signaling molecules, and serves as an important methyl donor. Some studies have observed a clear association between dietary choline intake or plasma concentration and the risk of several types of cancer. The recommended daily requirement for choline is 550 mg / day, and the recommended daily requirement for choline for non-pregnant women is 425 mg / day. The upper daily intake for adults is 3,500 mg / day, which is probably the maximum intake that will not cause harm to the body. Citric acid also has a variety of interesting biological activities, including antioxidant, anti-inflammatory, and antitumor effects. Citric acid is found in large amounts in many fruits and vegetables, especially citrus fruits, and is a common food and beverage additive and is widely used in the food industry as a chemical oxidant, flavoring, or preservative. Citric acid is generally recognized as a natural health component, and the U.S. Food and Drug Administration (FDA) has not placed any restrictions on the amount of citric acid added to foods and beverages.

[0124] The disclosed extraction method utilizes heating and stirring and is based on dissolving the target analytes in the deep eutectic solvent under heating and stirring conditions. The dissolution of the target analytes in the deep eutectic solvent is optimized according to the type of target compound. Centrifugation and filtration steps can be used to separate the deep eutectic solvent phase from the sample solution. The synthesis and extraction of NaDES can be performed in a one-pot process, which is a simple method and is scalable.

[0125] The synergistic enhancing effect provided by the solvent is due to a natural physicochemical phenomenon called "eutectic formation" that occurs when the ratio of the mixture reaches a critical ratio that corresponds to the eutectic point, a point on a phase diagram located at the intersection of two liquidus curves, resulting in a composition that is liquid at the lowest liquidus temperature of the mixture.

[0126] Regarding the extract composition, the viscosity can be reduced to a threshold compatible with industrial applications by adding water in a proportion of 1-50% by weight, preferably 30-50% by weight.

[0127] The extraction method described herein uses a ternary mixture containing a hydrogen bond donor selected from polyols and organic acids, choline chloride, and water, and maintains the molar ratio of choline chloride to hydrogen bond donor (the typical ratio for binary mixtures (choline chloride:hydrogen bond donor)) or ternary mixtures (choline chloride with water:hydrogen bond donor) in the range of 1:3 to 3:1 to form a crucial eutectic. Surprisingly, by utilizing this extraction process, it was possible to significantly increase the active species contained in the extract. This extraction process can increase the yield of active substances by at least 6 times, for example, the yield can be increased by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 times.

[0128] The extraction methods described herein include, as exemplary steps, immersing crushed or uncrushed Matecha leaves in a eutectic solvent and macerating, impregnating, and / or saturating them for 1-5 hours at atmospheric pressure, e.g., at 40-70° C. with shaking. The extraction phase does not involve chemical conversion between compounds in the eutectic extraction solvent, which is completely inert to the biological and natural materials being extracted.

[0129] After optimization, it was found that a 3:1 ratio of choline chloride / citric acid showed high extraction efficiency for Matecha. Optimization of the extraction conditions was performed using response surface methodology (RSM). The optimal conditions were 50% water content in NaDES; 30% liquid / solid ratio; 55°C extraction temperature; and 180 min extraction time. The highest extraction yield was 78%. The contents of each bioactive compound in the optimized extract were caffeine 23mg / g; theobromine 16mg / g; rutin 7mg / g; total chlorogenic acids 241mg / g; chlorogenic acid 55mg / g; 4-CQA 63mg / g; 5-CQA 35mg / g; 3,4-diCQA 12mg / g; 3,5-diCQA 54mg / g; and 4,5-diCQA 21mg / g. The experimental values ​​were consistent with those predicted by the RSM model, indicating that the RSM model employed was suitable and that the extraction conditions were optimized by RSM. 50 The antioxidant properties evaluated from the results of the IC 2010 study showed that the antioxidant-containing NaDES solution had significantly higher antioxidant capacity than the aqueous form of the antioxidant. 50 = 64μg / mL) was observed in samples with a water content of 50% (v / v) H 2 The sample contained choline chloride:citric acid (molar ratio 3:1) of 0. The Matecha extract extracted with NaDES had an IC of 258 μg / ml of pure chlorogenic acid. 50 It has four times the antioxidant capacity.

[0130] Example 2: Improved extraction of bioactive components from Matecha The following examples illustrate embodiments of the improved extraction of bioactive components from Matecha.

[0131] The extraction of bioactive components from Matecha leaves was improved by using three variables: ratio of NaDES solution to Matecha leaves (5-30), extraction temperature (40-70°C) and extraction time (1-3 h). The Box-Behnken design principle was followed using Minitab software to obtain optimal yields, improve extraction efficiency, reduce energy consumption and decrease pollutant emissions.

[0132] The effects of three variables (extraction temperature, extraction time, and ratio of NaDES solution to charge) were investigated using a three-level Box-Behnken design (BBD). Thus, 15 experiments with BBD design (including three replicates at the center point) were performed to fit regression equations and optimize the process factors affecting the extraction yield and antioxidant properties of bioactive components. For statistical calculations, each variable was transformed to +1, 0, and -1, as shown in Table 5 below. These transformed variables represent high, medium, and low values, respectively. [Table 5]

[0133] The experimental data was analyzed using Minitab to obtain the regression equation and determine the optimal parameter combination. The statistical significance of the model coefficients was determined by a combination of analysis of variance (ANOVA) and Fisher's F-test, with a probability P-value of 0.05. In addition, the coefficient of determination R 2 The accuracy of the model was investigated by the above method. Next, the fitted polynomial was plotted as a three-dimensional response surface and a two-dimensional contour plot to show the relationship between the response and the variables.

[0134] The optimal values ​​of the independent parameters were obtained by solving the regression equations and analyzing the response surface and contour plots.Additional experiments were then carried out to verify the validity of this statistical experimental method.

[0135] The optimum conditions for the extraction process were aimed at obtaining the maximum extraction yield and high chlorogenic acids content. The optimum conditions were an extraction temperature of 55°C, an extraction time of 3 h, and a ratio of NaDES solvent to feed of 30. To achieve practical operation, three validation experiments were carried out to confirm the predictions under the improved optimum conditions. The result was an extraction yield of 78% and a total chlorogenic acids content of 24%. The results were comparable to the values ​​predicted by the regression model.

[0136] By performing multiple regression analysis of the experimental data, the extraction yield response can be predicted from the following second order polynomial: Extraction yield (%) = 0.621377 + -0.0110222 × A + 0.145756 × B + 0.0105304 × C + 0.0567341 × AB + 0.00778068 × AC + 0.126731 × BC + 0.0151577 × A 2 + -0.0655996 × B 2 + -0.0104967 × C 2

[0137] The significance of each coefficient was examined using F-tests and p-values. A large F-value and a small p-value indicate that the corresponding variable is significant. The F-value for this model was 3.64, suggesting that the model is not significant relative to noise. The probability that noise would produce an F-value of this magnitude is 15.80%. A P-value less than 0.0500 indicates that the model term is significant. In this case, the liquid / solid ratio is the significant model term. A P-value greater than 0.1000 indicates that the model term is not significant. As there are many model terms that are not significant (not including those required to support the hierarchy), further refinement was performed by linearly reducing the model. The reduced equation gave the following extraction yields: %=0.583877 + -0.0110222 × A + 0.145756 × B + 0.0105304 × C

[0138] The F-score of the model was 4.42, suggesting that the model was significant. A comparison of the model's predicted values ​​and actual values ​​is shown in Figure 6A. The probability of an F-score of this magnitude being caused by noise is only 3.60%. R 2 The value of 91.6% indicates that there is indeed a good correlation between the response and the independent variables in this model.

[0139] A three-dimensional response surface was constructed to characterize multiple nonlinear regression models to illustrate the interaction effects of the operating parameters. The method to visualize the relationship between the response of each variable parameter and the experimental value, as well as the type of interaction between two test variables, is shown. The results of the extraction yield as influenced by the extraction temperature, extraction time, and solvent / feed ratio are shown in Figure 6B. In the response surface and contour plots, the extraction yield is shown with two continuous variables, one of which is shown as a fixed constant with a zero level (the center value of the test range). It was clearly shown that the extraction yield responded sensitively to small changes in the test variables. With increasing the extraction temperature, the yield increased rapidly in the initial stage, but then slowed down, indicating that a higher extraction temperature was favorable for extraction to a certain extent. From the significance of the regression coefficients of the second-order polynomial model and the slope and contour plots of the three-dimensional response curves, it was found that the solvent / feed ratio had the greatest effect on the extraction yield, followed by the extraction temperature and extraction time. The highest extraction yield of 78% was obtained with the longest extraction time of 3 hours and the highest liquid / solid ratio of 30 at the intermediate extraction temperature of 55°C.

[0140] The disclosed extraction method was particularly beneficial in that it exerted a synergistic / enhancing effect on the Matecha extraction process. The biological and / or physicochemical activity of the compounds contained in the resulting extract was enhanced. The antioxidant activity induced by extraction with a choline chloride-based eutectic solvent was 2-3 times higher than that of the water-based extract.

[0141] Furthermore, since the eutectic solvent is edible and non-toxic, the extracts obtained by the extraction methods disclosed herein can be directly formulated into end products such as food and beverages (for humans and animals), as well as cosmetics, dietary supplements, cosmeceuticals, alcoholic beverages, flavors (spices and flavorings), and pharmaceuticals.

[0142] Example 3: Total Extraction Yield from Matecha The following examples show embodiments of extraction yield results of bioactive compounds from Matecha.

[0143] Figures 5A and 5B show the chromatograms of the mixed standard (Figure 5A and Figure 5B) and one sample of Matecha extract (Figure 5C). All major peaks were clearly separated and identified. Linearity was assessed by constructing external calibration curves in the concentration range of 5 to 500 ppm. The peak areas of the analyte responses were plotted against the six concentrations. The results showed that the coefficients of determination (R) were greater than 0.993% for all analytes. 2 ) was obtained, indicating that the peak area of ​​the response was linear and related to the concentration of the analyte, indicating that this method can be used for the quantification of bioactive components in the processing of Matecha.

[0144] The choice of solvent is crucial for the extraction process of a particular compound from plant material. It is generally believed that a solvent that dissolves a compound will be a good phase for its extraction. Therefore, a rough screening to select a good extraction solvent from thousands of possibilities is usually done by taking into account the following concepts: "Like dissolves like" is a general principle (two molecules or solvents with similar polarity are soluble or miscible). -The higher the temperature of the system, the higher the solubility. The larger the molecular weight of the solute, the less soluble it is in the solvent. Extraction is mainly determined by dissolution phenomena.

[0145] Matecha leaves were extracted with conventional aqueous ethanol. Extraction yield, purity of bioactive components tested by HPLC, and IC of antioxidants were determined. 50 The values ​​are shown in Table 6 and FIG. [Table 6]

[0146] Screening of phytochemicals from ethanol extracts of Matecha leaves showed the presence of alkaloids, phenols, and flavonoids. Although the concentrations of ethanol used for extraction were varied, the same types of chemical species could be extracted from Matecha leaves. Since ethanol is relatively safe (less toxic), the choice of ethanol as an extraction solvent was considered to have more advantages than other organic solvents. Ethanol can attract glycosides, polyacetylenes, sterols, polyphenols, tannins, flavonols, terpenoids, and alkaloids. Ethanol is a protic organic solvent with a polarity index value of 5.2 and a relative dielectric constant of 24.55. The polarity of the ethanol at six concentrations used in this example was influenced by the water content of the mixed solvent. The higher the water content, the higher the polarity compared to absolute alcohol. Solvents with high polarity can extract chemical species with a wide polarity range. These properties allow non-phenolic polar compounds such as sugars and proteins to be dissolved in the extraction process, which can increase the extraction yield. In this study, 40% ethanol was able to extract more metabolites than other ethanol solvents.

[0147] The antioxidant activity test in this study was performed using DPPH radical. This method is considered to be relatively inexpensive, effective and efficient, and shows good sensitivity using UV-Vis spectrophotometry. DPPH is a dark blue free radical. When antioxidants are present, they donate a hydrogen atom to the DPPH radical (thus becoming DPPH-H). This reaction is characterized by a decrease in absorbance as DPPH loses its reactivity. The antioxidant activity in this study was measured by the IC of the samples, as shown in Figure 3. 50 The IC 50 The IC value indicated the concentration of sample required to inhibit 50% of the DPPH radical. This value was obtained as a result of calculation using linear regression analysis. The IC value of the antioxidant 50The smaller the value, the greater the ability to inhibit free radicals. From the results summarized in Table 6, it was found that the extract of Matecha leaves extracted with 20% ethanol had an IC 50 The extract of Matecha leaves extracted with 20% ethanol was shown to have strong DPPH radical scavenging activity. The antioxidant properties of the extracts were highly correlated with the amount of total chlorogenic acids with or without caffeine (total CQAs). As expected, caffeine does not contribute to the antioxidant properties of the extracts.

[0148] One of the important properties of an aqueous extraction medium is pH. Extracts are usually acidic or basic compounds, and the solubility of acidic or basic leachates (or extracts) depends on pH, so the pH of the extraction solvent is a crucial extraction design parameter. In this experiment, sodium citrate was used to adjust the pH of the aqueous solution, and the effect of pH on extraction efficiency in the pH range of 2-5 was investigated. The results are shown in Table 7 below. [Table 7]

[0149] The total extraction yield increased significantly with lower pH. Under strongly acidic conditions, the effect of pH on the extraction yield of Mtecha leaves was greater than that of H + This is thought to be due to the action of the fermentation agent, and the change in the structure of the Matecha leaves appears to have led to the release of more solids. + It was believed that the extraction opened up some of the leaf structure that is normally inaccessible, releasing more water-soluble components from the leaves. Meanwhile, some of the insoluble substances, such as polysaccharides, pectins and / or proteins, may have been hydrolyzed during the extraction process under strongly acidic conditions. The maximum amount of solids extracted from Matecha with hot water was about 200 g / kg, whereas the amount of solids extracted from dried leaves at pH 2 was about 600 g / kg. These results suggest that H +It was suggested that the action of the compound released approximately 400 g of solid matter per kg of matecha.

[0150] Compared to extraction with water, lowering the pH increases the recovery of bioactive components. The recovery of chlorogenic acid with water was 62.29%, but by lowering the pH to 4.56, the recovery increased to 91.12%. As mentioned earlier, the antioxidant properties of DPPH are directly related to the content of chlorogenic acids. By lowering the pH to 4-5, the IC 50 The value was also reduced, and the antioxidants could be improved. Water / citric acid is a preferred extraction solvent for Matecha leaves compared to aqueous ethanol.

[0151] The components of NaDES have a significant effect on its physicochemical properties, such as polarity, viscosity, and dissolving power. NaDES directly affect the extraction efficiency of target compounds. To select suitable NaDES, 20 NaDES were prepared from four primary metabolites, and the extraction yields of bioactive components from Matecha leaves by these NaDES were tested by HPLC. The extraction temperature was 50°C and the extraction time was 120 min. NaDES with 0% (w / w), 30% (w / w), 50% (w / w), or 80% (w / w) water was used as the extraction solvent because the viscosity was high, which may have reduced the mass transfer efficiency. Water or water / citric acid (buffered water) was used as a control for comparison. The results are shown in Table 8 and Figure 4. [Table 8]

[0152] As shown in Table 8 and Figure 4, the extraction yield with NaDES was three times that of water, indicating that NaDES had a surprising and unexpected advantage in the extraction of Matecha. Twenty different NaDES were tested, and the extraction yield of the target compounds varied depending on the type of NaDES. The organic acid-based NaDES showed a higher extraction yield than the sugar-based NaDES, indicating that the inclusion of organic acids provides an advantage in increasing the extraction yield. As expected, the extraction yield is closely related to some interaction between the solute and the solvent and the properties of the solvent. The high extraction yield was likely due to the effect of hydrogen bonds formed between NaDES and the target components.

[0153] These data also revealed that the ratio of various NaDESs affected the extraction yield. Furthermore, the above results showed that the extraction yield of chlorogenic acids was improved by changing the ratio of choline chloride (Cl) / citric acid (Cit) from 1:3 to 3:1. This was probably because the number of hydrogen bonds changed, which contributed to the destruction of plant cell walls and the dissolution of target components. Therefore, a combination of choline chloride / citric acid with a molar ratio of 3:1 was used in the following examples.

[0154] Similar to the molar ratio of the solvent components, various water contents of NaDES also affected the viscosity of the solvent, resulting in differences in extraction yield. Therefore, the extraction yields of NaDES with water contents ranging from 0% to 80% were investigated. As a result, the extraction process was improved by increasing the water content of NaDES from 0% to 80%. This is because the addition of water significantly reduced the viscosity and surface tension of the solvent, facilitating the mass transfer process from the material to the solvent. Water plays an important role in the properties and structure of NaDES. It can reduce the density and viscosity and increase the polarity. According to the previously mentioned studies, the supramolecular complex of NaDES can be protected by adding less than 50% water. Above this ratio, the addition of excess water may gradually weaken the H-bonds between the hydrogen bond acceptor and hydrogen bond donor, resulting in a mixture consisting only of dissociated NaDES compounds. Therefore, the water content was set to 50% in the subsequent studies.

[0155] Taking into account the eco-friendly extraction principle with NaDES, choline chloride (Cl) / citric acid (Cit) was selected as the optimized NaDES for the extraction of Matecha leaves. ClCit has favorable physicochemical properties for the extraction process, such as a polarity similar to that of water and polar organic solvents (ethanol and methanol) commonly used for the extraction of polyphenolic compounds, and a low pH value. Extraction with ClCit at 50% water content increased the extraction efficiency by decreasing the viscosity of the NaDES upon dilution with water and improving the mass transfer from the plant matrix to the solution. Moreover, it is interesting that the selected choline-based NaDES, which contains an organic acid as a hydrogen bond donor, is itself biologically active in forming compounds (choline and citric acid), possibly improving the biological activity of the plant extract.

[0156] Example 4: Bioactive Compounds in Matecha The following examples provide extraction parameters for various bioactive compounds from Matecha.

[0157] Box-Behnken design (BBD) coupled with response surface methodology (RSM) was used to optimize the extraction conditions for total chlorogenic acids (total CQAs), chlorogenic acid, 4-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), 4,5-dicaffeoylquinic acid (4,5-diCQA), rutin, theobromine, and caffeine.

[0158] Total CQAs The yield of total chlorogenic acids (Y1) was a function of the following variables: extraction temperature (A), liquid / solid ratio (B) and extraction time (C) were the independent variables. By applying multiple regression analysis to the experimental data, the following second order polynomial was obtained, which shows the extraction yield of total chlorogenic acids: Y1=209.381 + -37.8462 × A + 31.3421 × B + 9.12204 × C + 10.9688 × AB + -9.64654 × AC + 52.7593 × BC + -24.9719 × A 2 + -47.9325 × B 2 + -12.8844 × C 2

[0159] The model F value for total chlorogenic acids was 31.21 with a corresponding P value less than 0.05, indicating that the regression model was significant. There is only a 0.82% chance that an F value of this magnitude would be caused by noise. A P value less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, A 2 , B 2 is a significant model term. R 2 (0.9894) and adjusted R 2The high correlation coefficient (0.9577) indicates that the model indeed has a good correlation between the response and the independent variables. At the same time, the low coefficient of variation (CV) (7.38) clearly indicates that the experimental values ​​are highly accurate and reliable. A comparison of the model's predicted values ​​with the actual values ​​is shown in Figure 6C.

[0160] The regression was illustrated by three-dimensional (3D) response surface and contour plots, showing how to visualize the relationship between the response of each variable parameter and the experimental value, and the type of interaction between two test variables. Figure 6D shows the results of the extraction yield, where the effects of extraction temperature, liquid / solid ratio, and extraction time were observed. Table 9 shows the coefficients calculated from the regression model by analysis of variance (ANOVA). [Table 9]

[0161] The response surface and contour plots show the extraction yield of total chlorogenic acids along with two continuous variables, one of which is shown as a fixed constant with zero level (the center of the tested range). The large slope of the response surface indicated that the interaction between the variables was significant. When the liquid / solid ratio was maintained at the optimum level, the extraction yield of total chlorogenic acids increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased due to the decomposition of active compounds caused by high temperature. It was also shown that the active compounds may be decomposed by long extraction time. The liquid / solid ratio significantly affected the extraction yield. Increasing the solid / liquid ratio from 5 to 30 increased the yield from 76 mg / g to 241 mg / g. The maximum amount of total chlorogenic acids of 241 mg / g was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55°C, and the extraction time to 180 min.

[0162] Chlorogenic acid By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which shows the extraction yield of chlorogenic acid (Y2). Y2=47.0091 + -8.19396 × A + 7.72104 × B + 2.22975 × C + 2.71997 × AB + -2.21339 × AC + 11.771 × BC + -4.26374 × A 2 + -10.0948 × B 2 + -2.68677 × C 2

[0163] The model F value for chlorogenic acid was 26.57 with a corresponding P value of less than 0.05, indicating that the regression model was significant. There is only a 1.04% chance that an F value of this magnitude would be caused by noise. A P value of less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, and B 2 is a significant model term. R 2 (0.9876) and adjusted R 2 The high correlation coefficient (0.9504) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (7.71) clearly indicates the high precision and reliability of the experimental values.

[0164] The regression was illustrated by three-dimensional (3D) response surface and contour plots, showing how to visualize the relationship between the response of each variable parameter and the experimental value, and the type of interaction between the two test variables. In the response surface and contour plots, the extraction yield of chlorogenic acid is shown along with two continuous variables, one of which is shown as a fixed constant with a zero level (the center value of the test range).

[0165] The large slope of the response surface indicated that the interaction between the variables was significant. When the liquid / solid ratio was maintained at the optimum level, the extraction yield of chlorogenic acids increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the decomposition of chlorogenic acids caused by high temperature. Lower extraction temperatures showed that the extraction yield increased with increasing liquid / solid ratio. Both extraction time and liquid / solid ratio significantly affected the extraction yield. With an increase in solid / liquid ratio from 5 to 30, the yield increased from 17 mg / g to 55 mg / g. The maximum amount of total chlorogenic acids of 55 mg / g was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55 °C, and the extraction time to 180 min.

[0166] 4-Caffeoylquinic Acid (4-CQA) By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which shows the extraction yield of 4-CQA (Y3): Y3=56.1268 + -10.8746 × A + 8.03485 × B + 1.54769 × C + 2.90067 × AB + -3.13905 × AC + 14.025 × BC + -7.03086 × A 2 + -12.4312 × B 2 + -3.87252 × C 2

[0167] The model F-score for 4-CQA was 28.96 with a corresponding P-value of 0.0092, indicating that the regression model was significant. Noise would have caused an F-score of this magnitude only 0.92% of the time. A P-value less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, A 2 , B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9886) and adjusted R 2The high correlation coefficient (0.9545) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (7.73) clearly indicates the high precision and reliability of the experimental values.

[0168] The regression was illustrated by three-dimensional (3D) response surface and contour plots. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, as well as the type of interaction between the two test variables. The extraction yield of 4-CQA is shown in the response surface and contour plots along with two continuous variables, one of which is shown as a fixed constant with zero level (the center value of the test range). It was also observed that the interaction between the variables was significant, as shown by the large slope of the response surface. When the liquid / solid ratio was maintained at an optimal level, the extraction yield of 4-CQA increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the decomposition of 4-CQA due to high temperature. It was also shown that 4-caffeoylquinic acid (4-CQA) could be decomposed even with long extraction times at high temperatures. The higher the liquid / solid ratio, the longer the extraction time needed to be. The maximum amount of 4-CQA was obtained with 62.97 mg / g when the liquid / solid ratio was adjusted to 30 g / g, the temperature was adjusted to 55° C., and the extraction time was adjusted to 180 min.

[0169] 5-O-Caffeoylquinic Acid (5-CQA) By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which describes the extraction yield of 5-CQA: Y5=29.6757 + -5.48635 × A + 4.33813 × B + 1.65555 × C + 0.913624 × AB + -1.1249 × AC + 7.59666 × BC + -3.18214 × A 2 + -6.99323 × B 2 + -0.975141 × C 2

[0170] The model F-score for 5-CQA was 15.38 with a corresponding P-value of 0.0229, indicating that the regression model was significant. Noise would have caused an F-score of this magnitude only 2.29% of the time. A P-value less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, and B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9788) and adjusted R 2 The high value of the correlation coefficient (0.9151) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (10.39) clearly indicates the high precision and reliability of the experimental values.

[0171] The regression was illustrated by three-dimensional (3D) response surface and contour plots. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, and the type of interaction between the two test variables. In the response surface and contour plots, the extraction yield of 5-CQA is shown along with two continuous variables, one of which is shown as a fixed constant at zero level (the center value of the test range). When the liquid / solid ratio was maintained at an optimal level, the extraction yield of 5-CQA increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. It was also shown that 5-CQA may degrade with prolonged extraction. It was observed that the interaction between the variables was significant, as shown by the large slope of the response surface. The higher the liquid / solid ratio, the longer the extraction time was required. The maximum amount of 5-CQA of 35.3 mg / g was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55°C, and the extraction time to 180 min.

[0172] 3,4-Dicaffeoylquinic Acid (3,4-diCQA) By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which describes the extraction yield of 3,4-diCQA: Y6=10.6223 + -1.84482 × A + 1.54172 × B + 0.451434 × C + 0.593569 × AB + -0.513514 × AC + 2.64981 × BC + -1.5156 × A 2 + -2.4587 × B 2 + -0.787695 × C 2

[0173] The model F value for 3,4-CQA was 30.46 with a corresponding P value of 0.0085, indicating that the regression model was significant. Noise would have caused an F value of this magnitude in only 0.85% of cases. A P value less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, A 2 , B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9892) and adjusted R 2 The high correlation coefficient (0.9567) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (7.59) clearly indicates the high precision and reliability of the experimental values.

[0174] The regression was illustrated by three-dimensional (3D) response surface and contour plots. The relationship between the response of each variable parameter and the experimental value and the type of interaction between the two test variables are shown in the response surface and contour plots. The extraction yield of 3,4-diCQA is shown along with two continuous variables, one of which is shown as a fixed constant at zero level (the center value of the test range). When the liquid / solid ratio was maintained at an optimal level, the extraction yield of 3,4-diCQA increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the degradation of 3,4-diCQA due to the high temperature. It was also observed that the interaction between the variables was significant, as shown by the large slope of the response surface. The liquid / solid ratio significantly affected the extraction yield. The higher the liquid / solid ratio, the longer the extraction time was required. The maximum amount of 3,4-diCQA, 12 mg / g, was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55°C, and the extraction time to 180 min.

[0175] 3,5-Dicaffeoylquinic Acid (3,5-diCQA) By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which describes the extraction yield of 3,5-diCQA: Y7=47.4372 + -8.25459 × A + 7.0397 × B + 2.14047 × C + 2.6516 × AB + -1.99397 × AC + 11.9534 × BC + -6.87771 × A 2 + -11.3866 × B 2 + -3.5024 × C 2

[0176] The model F value for 3,5-diCQA was 41.20 with a corresponding P value of 0.0055, indicating that the regression model was significant. Noise would have only a 0.55% chance of producing an F value of this magnitude. A P value less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, A 2 , B 2is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9920) and adjusted R 2 The high value of the coefficient of variation (0.9679) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (6.67) clearly indicates the high precision and reliability of the experimental values.

[0177] The regression was illustrated by three-dimensional (3D) response surface and contour plots. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, as well as the type of interaction between the two test variables. In the response surface and contour plots, the extraction yield of 3,5-diCQA is shown along with two continuous variables, one of which is shown as a fixed constant at zero level (the center value of the test range). When the liquid / solid ratio was maintained at an optimal level, the extraction yield of 3,5-diCQA increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the degradation of 3,5-diCQA caused by the high temperature. It was also observed that the interaction between the variables was significant, as shown by the large slope of the response surface. The liquid / solid ratio significantly affected the extraction yield. The higher the liquid / solid ratio, the longer the extraction time was required. The maximum amount of 3,5-diCQA of 21.5 mg / g was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55°C, and the extraction time to 180 min.

[0178] 4,5-Dicaffeoylquinic Acid (4,5-diCQA) By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which describes the extraction yield of 4,5-CQA: Y8=18.5102 + -3.19188 × A + 2.66662 × B + 1.09715 × C + 1.18942 × AB + -0.661707 × AC + 4.76348 × BC + -2.10181 × A 2 + -4.56793 × B 2 + -1.05986 × C 2

[0179] The model F value for 4,5-diCQA was 59.61 with a corresponding P value of 0.0032, indicating that the regression model was significant. There is only a 0.32% chance that an F value of this magnitude would be caused by noise. A P value of less than 0.0500 indicates that the model terms are significant. In this case, A, B, C, AB, BC, A 2 , B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9944) and adjusted R 2 The high value of the coefficient of variation (0.9798) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (5.41) clearly indicates the high precision and reliability of the experimental values.

[0180] The regression was illustrated by three-dimensional (3D) response surface and contour plots. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, as well as the type of interaction between the two test variables. In the response surface and contour plots, the extraction yield of 4,5-diCQA is shown along with two continuous variables, one of which is shown as a fixed constant at zero level (the center value of the test range). When the liquid / solid ratio was maintained at an optimal level, the extraction yield of 4,5-diCQA increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the degradation of 4,5-diCQA caused by the high temperature. It was also observed that the interaction between the variables was significant, as shown by the large slope of the response surface. The liquid / solid ratio significantly affected the extraction yield. The higher the liquid / solid ratio, the longer the extraction time was required. The maximum amount of 4,5-diCQA, 54 mg / g, was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55° C., and the extraction time to 180 min.

[0181] Rutin By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which shows the extraction yield of rutin. Y9=8.59949 + -2.02475 × A + -0.432504 × B + 0.134262 × C + 0.45758 × AB + -0.516049 × AC + 2.16226 × BC + -1.87299 × A 2 + -3.01976 × B 2 + -0.610253 × C 2

[0182] The model F value for rutin was 33.40, with a corresponding P value of 0.0075, indicating that the regression model was significant. A P value less than 0.0500 indicates that the model terms are significant. In this case, A, BC, and A 2 , B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9901) and adjusted R 2 The high value of the correlation coefficient (0.9605) indicates that the model indeed has a good correlation between the response and the independent variables, while the low coefficient of variation (CV) (9.84) clearly indicates the high precision and reliability of the experimental values.

[0183] The regression was illustrated by three-dimensional (3D) response surface and contour plot. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, and the type of interaction between the two test variables. In the response surface and contour plot, the extraction yield of 4-CQA is shown along with two continuous variables, one of which is shown as a fixed constant with zero level (the center value of the test range). When the liquid / solid ratio was maintained at the optimum level, the extraction yield of rutin increased with increasing temperature. However, when the temperature exceeded a certain value, the extraction yield decreased. This was due to the decomposition of rutin caused by high temperature. The longer the extraction time, the more decomposed rutin was. It was also observed that the interaction between the variables was significant, as shown by the large slope of the response surface. The liquid / solid ratio significantly affected the extraction yield. The maximum amount of rutin, 8.6 mg / g, was obtained by adjusting the liquid / solid ratio to 17.5 g / g, the temperature to 55°C, and the extraction time to 120 min.

[0184] Theobromine By applying multiple regression analysis to the experimental data, the following second-order polynomial equation was obtained, which shows the extraction yield of theobromine: Y10=9.96722 + -3.03923 × A + 3.69081 × B + 0.996837 × C + -1.61736 × AB + 1.3461 × AC + 3.1009 × BC + -2.5968 × A 2 + -0.983486 × B 2 + -0.320714 × C 2

[0185] The model F value for theobromine was 4.90, suggesting that the model is not significant relative to the noise. The probability that noise would produce an F value of this magnitude is 10.91%. Thus, linear reduction of the model gave the following equation: Y10=7.56661 + -3.03923 × A + 3.69081 × B + 0.996837 × C

[0186] The F-score for this model is 6.27, suggesting that the model is significant. There is only a 1.39% chance that an F-score of this magnitude would be caused by noise. A P-value less than 0.0500 indicates that the model terms are significant. In this case, A and B are the significant model terms. R 2 (0.9363) and adjusted R 2 The high value of (0.7451) indicates that there is indeed a good correlation between the response and the independent variables in this model.

[0187] The regression was illustrated by three-dimensional (3D) response surface and contour plots. It shows how to visualize the relationship between the response of each variable parameter and the experimental value, and the type of interaction between two test variables. In the response surface and contour plots, the extraction yield of theobromine is shown along with two continuous variables, one of which is shown as a fixed constant with zero level (the center value of the test range). When the temperature was maintained at the optimal level, the extraction yield of theobromine increased with increasing liquid / solid ratio. The maximum amount of theobromine of 16.5 mg / g was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55°C, and the extraction time to 180 min.

[0188] Caffeine By applying multiple regression analysis to the experimental data, the following second order polynomial was obtained, which describes the extraction yield of caffeine: Y11=20.9522 + -3.31823 × A + 2.72458 × B + 1.05134 × C + 1.35799 × AB + -0.56039 × AC + 5.43106 × BC + -2.51197 × A 2 + -5.37211 × B 2 + -1.51228 × C 2

[0189] The model F value for caffeine was 24.58 with a corresponding P value of 0.0117, indicating that the regression model was significant. A P value of less than 0.0500 indicates that the model terms are significant. In this case, A, B, BC, and B 2 is a significant model term. A P value of less than 0.0500 indicates that a model term is significant. In this case, A, BC, A 2 , B 2 is a significant model term. A P value greater than 0.1000 indicates that the model term is not significant. R 2 (0.9866) and adjusted R 2 The high value of the correlation coefficient (0.9465) indicates that there is indeed a good correlation between the response and the independent variables in this model, while the low coefficient of variation (CV) (8.31) clearly indicates the high precision and reliability of the experimental values.

[0190] The regression was illustrated by three-dimensional (3D) response surface and contour plots. The relationship between the response of each variable parameter and the experimental value and the type of interaction between the two test variables are shown. In the response surface and contour plots, the extraction yield of caffeine is shown with two continuous variables, one of which is shown as a fixed constant with a zero level (the center value of the test range). Caffeine is relatively stable at high extraction temperatures, but the extraction yield of caffeine increases with increasing liquid / solid ratio, and when the temperature exceeds a certain value, the extraction yield decreases. This is due to the longer extraction time, which is thought to be due to the decomposition of caffeine. The large slope of the response surface indicated that the interaction between the variables was significant. The maximum amount of caffeine, 23 mg / g, was obtained by adjusting the liquid / solid ratio to 30 g / g, the temperature to 55 °C, and the extraction time to 180 min.

[0191] The optimum conditions of the extraction process were aimed at obtaining the maximum extraction yield and high total chlorogenic acids content. Based on the above findings, an optimization study was carried out, and the optimum conditions were an extraction temperature of 55°C, an extraction time of 180 min, and a liquid / solid ratio of 30. Three validation experiments were carried out to confirm the predictions. The results showed that the extraction yield was 78±2% and the total chlorogenic acids content was 240±3.2 mg / g, which were almost the same as the yield (77.8%) and total chlorogenic acids content (241 mg / g) predicted by the regression model. [Table 10]

[0192] The antioxidant capacity of the extracts of Matecha obtained under various conditions was investigated by DPPH radical scavenging assay. The DPPH assay was chosen because it is a promising method that can indicate the presence of antioxidant compounds. The DPPH assay is simple and low-cost because the radical compound DPPH is relatively stable and does not require preparation of this compound. Surprisingly, all the antioxidant-containing NaDES solutions tested showed higher antioxidant capacity than the aqueous form of antioxidants. The DPPH assay results showed that the highest antioxidant activity was obtained in aqueous choline chloride / citric acid, and this high antioxidant activity was highly correlated with the high content of chlorogenic acids. The strong antioxidant activity could be attributed to citric acid, one of the components of the NaDES system. Citric acid is well known to be a natural antioxidant due to its ability to scavenge free radical species (ROS). The addition of citric acid would have contributed to the increase in the antioxidant activity of the NaDES system consisting of choline chloride / citric acid as an antioxidant. This result is in line with our hypothesis regarding the role of NaDES. Another notable finding is that the antioxidant activity of NaDES systems consisting of choline chloride / citric acid with water content of 30 wt%, 50 wt% or 80 wt% performed better than that of pure choline chloride / citric acid. This phenomenon is consistent with the effect of viscosity on the solubility of antioxidant compounds. It was suggested that the physicochemical properties of NaDES have a significant impact on the antioxidant activity. From these findings, it was concluded that the NaDES system consisting of choline chloride / citric acid with water content of 50 wt% is superior in improving antioxidant activity, clearly demonstrating its promise in the food, functional food and pharmaceutical fields.

[0193] Example 5: Extraction of theobromine Extraction of theobromine using the methods and compositions described herein is illustrated in the following examples.

[0194] Matecha leaves were obtained and theobromine was extracted using NaDES. Specifically, theobromine was extracted using various types of NaDES as shown in Table 11 below. [Table 11]

[0195] As shown in Figure 7, the extraction yield of theobromine from Matecha leaves using citric acid:sucrose was 6.50%, and the extraction yield of theobromine from Matecha leaves using citric acid:fructose was 8.39% (fourth and fifth bars from the left in Figure 7), which were significantly higher than the extraction yields from other NaDES systems and water (rightmost bar in Figure 7). The extraction yield of theobromine using water was 0.08%-0.75%. The combination of fructose or sucrose (glucose and fructose combined in a 1:1 ratio) with citric acid increased the extraction yield of theobromine by 10-20 times compared to extraction with water, which was most likely due to the improved ability of theobromine to form hydrogen bonds with these NaDES. However, such observations were not observed with the combination of citric acid:glucose.

[0196] Jelinski et al. have investigated the solubility of pure theobromine in various solvents and NaDES (Jelinski et al., 2021). Jelinski et al. report that the solubility of theobromine in NaDES containing choline chloride and glycerol is almost 32 times higher than in water, and that the solubility of theobromine in NaDES containing choline chloride and glucose is almost the same as that in NaDES containing choline chloride and fructose. Interestingly, there was no correlation between the solubility of theobromine in NaDES reported in this literature and the extractability of theobromine from Matecha leaves by the NaDES described herein, suggesting the matrix effect of the present disclosure in the extraction. These results were therefore unexpected and surprising.

[0197] As shown in this example, the extraction yield of theobromine from Matecha leaves reaches approximately 8-9% by weight, which has not been achieved before. The tests in this study were performed in three replicates, with reproducible observations. The specific combination of fructose or fructose-containing sugars (e.g., sucrose) and citric acid shows remarkable selectivity for theobromine. Furthermore, this particular NaDES system is also beneficial for the extraction of other plants rich in theobromine, such as cocoa.

[0198] All headings are provided for ease of reading and should not be construed as limiting the meaning of the text following the heading, unless specifically stated otherwise.

[0199] All patents and publications mentioned herein are indicative of the level of knowledge of those skilled in the art to which this invention pertains. All patents and publications mentioned herein are hereby incorporated by reference for any disclosure specifically cited herein to the same extent as if each individual publication was specifically set forth for the purpose of being incorporated by reference.

[0200] Various aspects and embodiments have been disclosed herein, and other aspects and embodiments will be readily apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative of the invention and are not intended to limit the invention in any way, the scope and spirit of the invention being indicated by the claims.

[0201] References The following references are incorporated herein by reference in their entirety for all purposes: Abbott, A. P., Boothby, D., Capper, G., Davies, D. L., & Rasheed, R. K. (2004). Deep Eutectic Solvents Formed between Choline Chloride and Carboxylic Acids: Versatile Alternatives to Ionic Liquids. Journal of the American Chemical Society, 126(29), 9142-9147. Abbott, A. P., Capper, G., Davies, D. L., Rasheed, R. K., & Tambyrajah, V. (2003). Novel solvent properties of choline chloride / urea mixtures. Chemical Communications, 1, 70-71. Bastos, D. H. M., De Oliveira, D. M., Matsumoto, R. L. T., Carvalho, P. de O., & Ribeiro, M. L. (2007). Yerba mate: pharmacological properties, research and biotechnology. Med Aromat Plant Sci Biotechnol, 1(1), 37-46. Bojic, M., Simon Haas, V., Saric, D., & Males, Z. (2013). Determination of flavonoids, phenolic acids, and xanthines in mate tea (Ilex paraguariensis St.-Hil.). Journal of Analytical Methods in Chemistry, 2013. Burris, K. P., Harte, F. M., Davidson, P. M., Stewart Jr, C. N., & Zivanovic, S. (2012). Composition and bioactive properties of yerba mate (Ilex paraguariensis A. St.-Hil.): a review. Chilean Journal of Agricultural Research, 72(2), 268. Chandrasekara, A., & Shahidi, F. (2018). Herbal beverages: Bioactive compounds and their role in disease risk reduction-A review. Journal of Traditional and Complementary Medicine, 8(4), 451-458. Chianese, G., Golin-Pacheco, S. D., Taglialatela-Scafati, O., Collado, J. A., Munoz, E., Appendino, G., & Pollastro, F. (2019). Bioactive triterpenoids from the caffeine-rich plants guayusa and mate. Food Research International, 115, 504-510. Choi, Y. H., van Spronsen, J., Dai, Y., Verberne, M., Hollmann, F., Arends, I. W. C. E., Witkamp, G.-J., & Verpoorte, R. (2011). Are Natural Deep Eutectic Solvents the Missing Link in Understanding Cellular Metabolism and Physiology? Plant Physiology, 156(4), 1701-1705. Croge, C. P., Cuquel, F. L., & Pintro, P. T. M. (2020). Yerba mate: cultivation systems, processing and chemical composition. A review. Scientia Agricola, 78(5). Dai, Y. (2006). Natural deep eutectic solvents and their application in natural product research and. Carbohydr. Polym, 66, 546-550. Dai, Y. (2007). Natural deep eutectic solvents and their application in natural product research and. Plant J, 50, 967-981. Dai, Y., Witkamp, G.-J., Verpoorte, R., & Choi, Y. H. (2013). Natural Deep Eutectic Solvents as a New Extraction Media for Phenolic Metabolites in Carthamus tinctorius L. Analytical Chemistry, 85(13), 6272-6278. Ferreira Cuelho, C. H., de Franca Bonilha, I., Scotti do Canto, G., & Palermo Manfron, M. (2015). Recent advances in the bioactive properties of yerba mate. Revista Cubana de Farmacia, 49(2), 375-383. Frizon, C. N. T., Perussello, C. A., Sturion, J. A., & Hoffmann-Ribani, R. (2018). Novel Beverages of Yerba-Mate and Soy: Bioactive Compounds and Functional Properties. Beverages, 4(1), 21. Heck, C. I., & De Mejia, E. G. (2007). Yerba Mate Tea (Ilex paraguariensis): a comprehensive review on chemistry, health implications, and technological considerations. Journal of Food Science, 72(9), R138-R151. Isolabella, S., Cogoi, L., Lopez, P., Anesini, C., Ferraro, G., & Filip, R. (2010). Study of the bioactive compounds variation during yerba mate (Ilex paraguariensis) processing. Food Chemistry, 122(3), 695-699. Jelinski, T. et al. (2021) ‘Experimental and Theoretical Study on Theobromine Solubility Enhancement in Binary Aqueous Solutions and Ternary Designed Solvents.’, Pharmaceutics, 13(8). doi: 10.3390 / pharmaceutics13081118. Junior, E. L. C., & Morand, C. (2016). Interest of mate (Ilex paraguariensis A. St.-Hil.) as a new natural functional food to preserve human cardiovascular health-A review. Journal of Functional Foods, 21, 440-454. Liu, Y., Friesen, J. B., McAlpine, J. B., Lankin, D. C., Chen, S. N., & Pauli, G. F. (2018). Natural Deep Eutectic Solvents: Properties, Applications, and Perspectives. Journal of Natural Products, 81(3), 679-690. Matei, M. F., Jaiswal, R., Patras, M. A., & Kuhnert, N. (2016). LC-MSn study of the chemical transformations of hydroxycinnamates during yerba mate (Ilex paraguariensis) tea brewing. Food Research International, 90, 307-312. Oellig, C., Schunck, J., & Schwack, W. (2018). Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography. Journal of Chromatography A, 1533, 208-212. Puangpraphant, S. (2012). Anti-inflammatory and anti-colon cancer potential of yerba mate (Ilex paraguariensis St. Hilaire) bioactive constituents. University of Illinois at Urbana-Champaign. Riachi, L. G., Simas, D. L. R., Coelho, G. C., Marcellini, P. S., da Silva, A. J. R., & de Maria, C. A. B. (2018). Effect of light intensity and processing conditions on bioactive compounds in mate extracted from yerba mate (Ilex paraguariensis A. St.-Hil.). Food Chemistry, 266, 317-322. Shen, Q., Zhang, B., Xu, R., Wang, Y., Ding, X., & Li, P. (2010). Antioxidant activity in vitro of the selenium-contained protein from the Se-enriched Bifidobacterium animalis 01. Anaerobe, 16(4), 380-386. Souza, A. H. P., Correa, R. C. G., Barros, L., Calhelha, R. C., Santos-Buelga, C., Peralta, R. M., Bracht, A., Matsushita, M., & Ferreira, I. C. F. R. (2015). Phytochemicals and bioactive properties of Ilex paraguariensis: An in-vitro comparative study between the whole plant, leaves and stems. Food Research International, 78, 286-294.

Claims

1. 1. A method for isolating a plant extract from a plant material comprising the steps of: Obtaining plant material; treating the plant material; mixing the treated plant material with a natural deep eutectic solvent (NaDES); and Isolating the plant extract The method includes:

2. The method of claim 1 , wherein the plant material is obtained from Matecha.

3. The method of claim 1 , wherein the NaDES comprises a hydrogen bond donor molecule and a hydrogen bond acceptor molecule.

4. 2. The method of claim 1, wherein the NaDES is a sugar-based NaDES, an organic acid-based NaDES, a choline chloride-based NaDES, or a polyol-based NaDES.

5. The method of claim 3 , wherein the sugar-based NaDES is glucose, sucrose, fructose, or a combination thereof.

6. 4. The method of claim 3, wherein the organic acid based NaDES is citric acid in combination with glucose, sucrose, fructose or a combination thereof.

7. 4. The method of claim 3, wherein the choline chloride-based NaDES is choline chloride in combination with citric acid, glucose, sucrose, fructose, or combinations thereof.

8. 4. The method of claim 3, wherein the polyol-based NaDES is erythritol in combination with choline chloride, citric acid, glucose, fructose, sucrose, or combinations thereof.

9. 2. The method of claim 1, wherein the NaDES comprises choline chloride, citric acid, erythritol, glucose, sucrose, fructose, or a combination thereof.

10. 2. The method of claim 1, wherein the NaDES comprises choline chloride:citric acid, choline chloride:glucose, choline chloride:sucrose, choline chloride:fructose, glucose:sucrose, glucose:fructose, citric acid:glucose, citric acid:sucrose, citric acid:fructose, erythritol:choline chloride, erythritol:citric acid, or erythritol:glucose:fructose.

11. The method of claim 1 , wherein the NaDES comprises choline chloride and citric acid.

12. 12. The method of claim 11, wherein the choline chloride and the citric acid are in a ratio of 3:

1.

13. The method of claim 1 , wherein the NaDES comprises 10% to 80% water.

14. The method of claim 1 , wherein the plant extract is a xanthine alkaloid, a chlorogenic acid, or a matesaponin.

15. 15. The method of claim 14, wherein the xanthine alkaloids comprise caffeine, theobromine, theophylline, or analogs or derivatives thereof.

16. 15. The method of claim 14, wherein the chlorogenic acids include caffeic acid, tannins, or analogs or derivatives thereof.

17. The method of claim 1 further comprising the steps of heating, stirring, centrifuging, and / or filtering after the mixing step.

18. 2. The method of claim 1, wherein the NaDES comprises choline chloride and citric acid in a ratio of 3:1 and 50% water.

19. The method of claim 10, wherein the NaDES is in a 1:1 molar ratio.

20. 2. The method of claim 1, wherein the plant extract is theobromine extracted in an amount of about 5% to about 10% by weight.

21. A plant isolate; Natural deep eutectic solvents (NaDES) and A composition comprising:

22. 22. The composition of claim 21, wherein the plant isolate comprises an isolate from Matecha.

23. 22. The composition of claim 21, wherein the plant isolate comprises xanthine alkaloids, chlorogenic acids, or matesaponins.

24. 22. The composition of claim 21, wherein the plant isolate comprises caffeine, theobromine, theophylline, caffeic acid, tannins, or analogs or derivatives thereof.

25. 22. The composition of claim 21, wherein the NaDES comprises choline chloride, citric acid, erythritol, glucose, sucrose, fructose, or a combination thereof.

26. 22. The composition of claim 21, wherein the NaDES comprises choline chloride:citric acid, choline chloride:glucose, choline chloride:sucrose, choline chloride:fructose, glucose:sucrose, glucose:fructose, citric acid:glucose, citric acid:sucrose, citric acid:fructose, erythritol:choline chloride, erythritol:citric acid, or erythritol:glucose:fructose.

27. 22. The composition of claim 21, wherein the NaDES is in a 1:1 molar ratio.

28. 22. The composition of claim 21, wherein the NaDES comprises choline chloride and citric acid.

29. 29. The composition of claim 28, wherein the choline chloride and the citric acid are in a ratio of 3:

1.

30. 22. The composition of claim 21 comprising caffeine, theobromine, theophylline, caffeic acid, tannins, or analogs or derivatives thereof, choline chloride and citric acid in a ratio of 3:1, and 50% water.

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