Bioactive extraction method

Abstract

The present disclosure generally relates to decaffeinated caffeoylquinic acid plant extract compositions and extraction methods for removing bioactive compounds from plant material. In some embodiments, the method includes obtaining plant material, mixing the plant material with a natural deep eutectic solvent (NaDES), obtaining a crude extract solution containing the plant material, bioactive compounds, and the NaDES extract, loading the crude extract solution onto a macroporous resin, and recovering the plant extract from the macroporous resin, wherein the bioactive compounds and the NaDES have been removed from the plant extract. In some embodiments, the bioactive compound is caffeine. In some embodiments, the plant material is obtained from yerba mate.

Description

[Background technology]

[0001] Incorporation by reference to priority applications This application claims priority to U.S. Provisional Application No. 63 / 374,353, entitled "BIO ACTIVE EXTRACTION METHOD," filed September 1, 2022, which is incorporated by reference in its entirety. [Technical Field]

[0002] The present disclosure relates generally to the field of plant extraction processes. In particular, the present disclosure relates to a method for extracting bioactive compounds, such as caffeine, from plants, such as yerba mate. The method includes deep eutectic solvent extraction combined with macroporous adsorption resin chromatography.

[0003] Ilex paraguariensis (Yerba Mate) is a medicinal plant that contains numerous bioactive compounds, including caffeine, chlorogenic acid, saponins, and xanthine alkaloids. Traditional extraction of these compounds from Yerba Mate uses harsh processes and chemicals that are neither environmentally friendly nor efficient.

[0004] Natural deep eutectic solvents (NaDES) are universal compounds that solubilize a variety of metabolites and compounds, and they are environmentally friendly.

[0005] Macroporous adsorption resins (MARs) are synthetic porous cross-linked polymer beads used to separate and purify pharmaceuticals and natural products. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure generally relates to compositions of decaffeinated caffeoylquinic acid plant extracts and methods for removing caffeine from plants using a combination of natural deep eutectic solvents and microporous resin adsorption chromatography. [Means for solving the problem]

[0007] Some embodiments provided herein relate to methods for extracting bioactive compounds from plant material. In some embodiments, the method includes obtaining a plant material. In some embodiments, the method further includes mixing the plant material with a natural deep eutectic solvent (NaDES). In some embodiments, the method further includes obtaining a crude extract solution comprising an extract of the plant material, a bioactive compound, and a NaDES. In some embodiments, the method further includes loading the crude extract solution onto a macroporous resin. In some embodiments, the method further includes recovering the plant extract from the macroporous resin, wherein the bioactive compound and the NaDES have been removed from the plant extract. In some embodiments, the bioactive compound is caffeine. In some embodiments, the plant material is obtained from yerba mate.

[0008] In some embodiments, the NaDES includes choline chloride (ChCl) and glucose (GLU). In some embodiments, the choline chloride and glucose are present in a ratio of about 2:1. In some embodiments, the NaDES includes water in an amount ranging from 10% to 50% by weight. In some embodiments, the NaDES includes water present in an amount of about 30% by weight.

[0009] In some embodiments, the method also includes diluting the crude extract with water before applying the crude extract to the macroporous resin. In some embodiments, the dilution is about 10-fold.

[0010] In some embodiments, the recovered plant extract is rich in caffeoylquinic acids. In some embodiments, the recovered plant extract contains less than 0.05% caffeine by weight. In some embodiments, the recovered plant extract contains less than 0.01% caffeine by weight.

[0011] In some embodiments, the macroporous resin comprises a non-polar macroporous resin. In some embodiments, the macroporous resin comprises HDP200. In some embodiments, the macroporous resin is incorporated into a column.

[0012] In some embodiments, recovering the plant extract comprises eluting the plant extract from the macroporous resin. In some embodiments, the plant extract is eluted with a solution comprising ethanol in an amount ranging from 10% to 30% by volume. In some embodiments, the solution comprises about 20% by volume of ethanol.

[0013] In some embodiments, the method also includes recovering the NaDES from the macroporous resin. In some embodiments, recovering the NaDES from the macroporous resin includes washing the macroporous resin with water, capturing the eluate and water, and drying the captured eluate and water to recover the NaDES.

[0014] The above and other features of the present disclosure will become more fully apparent from the following description taken in conjunction with the accompanying drawings. It should be understood that these drawings illustrate only some embodiments according to the present disclosure and therefore should not be considered limiting of its scope. The present disclosure will be described with additional specificity and detail through the use of the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of an embodiment of a process for the recovery of natural deep eutectic solvent and the production of decaffeinated yerba mate extract. [Figure 2] 1 shows the biosynthesis of caffeine in tea. [Figure 3] The chemical structure and properties of caffeine are shown. [Figure 4] 1 shows exemplary HPLC chromatograms at 280 nm and 350 nm of yerba mate extracted with a 2:1 ratio of ChCl:CA (choline chloride:citric acid) solvent. [Figure 5] 1 shows a bar graph showing a comparison of extraction yields of yerba mate by different NaDES with water and ethanol, including, from left to right, water, CHCL:CA2:1, CHCL:CA1:1, CHCL:CA1:2, CHCL:CA1:3, CHCL:Glu2:1, Glu:CA1:1, and ethanol. [Figure 6A] The static adsorption / desorption capacity and desorption rate of bioactive substances in yerba mate on different macroporous resins are shown. [Figure 6B] The static adsorption / desorption capacity and desorption rate of bioactive substances in yerba mate on different macroporous resins are shown. [Figure 7A] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 7B] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 7C] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 7D] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 7E] Shown are exemplary HPLC chromatograms of samples obtained from the column chromatography process. Each sample contains two chromatograms: theobromine and caffeine at 280 nm, and all caffeoylquinic acids and rutin at 350 nm. [Figure 8A] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 8B] 1 shows an exemplary HPLC chromatogram of a sample obtained from the column chromatography process. [Figure 8C]Shown are exemplary HPLC chromatograms of samples obtained from the column chromatography process. Each sample contains two chromatograms: theobromine and caffeine at 280 nm, and all chlorogenic acids and rutin at 350 nm. [Figure 9] 1 is an exemplary line graph showing the results of a 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay of different fractions. [Figure 10] 1 shows the chemical structures of caffeoylquinic acid isomers. DETAILED DESCRIPTION OF THE INVENTION

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like elements unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects generally described in the present disclosure and illustrated in the figures can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0017] As summarized herein, provided herein are method embodiments for removing and extracting bioactive compounds from plant material using a combination of natural deep eutectic solvent (NaDES) and macroporous adsorption resin (MAR) chromatography.

[0018] It is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0019] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

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

[0021] As used herein, the terms "extract," "extraction," "extracting," or derivatives thereof, have their ordinary meaning as understood in light of this specification and refer to the process of removing a substance, compound, composition, or other component from a substance. The starting material extraction process can be carried out by a variety of means, including by physical or chemical extraction, such as pressing, grinding, heating, stirring, or other known methods for extracting components from starting materials.

[0022] The term "plant" as used herein has its ordinary meaning as understood in light of the present specification and refers to a whole plant or a part or derivative thereof, such as a plant cell, plant protoplast, plant cell tissue culture from which a plant can be regenerated, plant callus, embryo, pollen, ovule, fruit, flower, leaf, seed, root, root tip, etc. The term "botanical" is used interchangeably herein with plant and refers to a plant or any derived part thereof.

[0023] In some embodiments, the plant is any plant that has components therein that are desirable to extract, purify, or obtain from the plant. For example, the plant may be or be derived from the following: maca, he shou wu, iporuru (Alchornea castaneifolia), canna (Sceletium Tortosum), honokiol (Magnolia grandiflora), jujube (Ziziphi Spinosae), cnidium (Fructus Cnidii), corydalis (Corydalis yanhusuo), albizia (Cortex albiziae), ginseng (Panax ginseng), pudding (Polygoni Multiflori), poria cocos, Chinese cherry (Fructus corni), dioscorea (Rhizoma dioscoreae), muira puama, Dendrobium sp., licorice root (Glycyrrhizae Preparata), Chinese ginseng root (Cordyceps sinensis, Angelicae sinensis, Kratom (Mitragyna speciosa), Bacopa monnieri, Catuaba, Ashwagandha, Peganum harmala, Wheat, Alfalfa, Oats, Kamut, Echinacea, Chlorella, Amla, Stinging Nettle, Carob, Mesquite, Chuchuhuasai, Clavo huasca, Chanca piedra, Guaiza, Rhodiola rosea, Shilajit, Higenamine, Moringa (Moringa oleifera), Horny goat weed (Epidmedium), Astragalus, Aloe vera vera, turmeric, pine, curcumin (turmeric compound), hops, xanthohumol (hop compound), passion flower, mucuna puriens, tusli, black pepperpepper), Bioperine (a black pepper compound), Siberian ginseng, American ginseng, yerba mate, lemon balm, astragulus, kava, schizandra, skullcap, valerian, California poppy, epidmedium, pau d'arco, ginkgo, lotus, lily, tea, coffee, or cacao, or any component of these plants, such as the bark, leaves, stems, roots, flowers, fruit, pollen, seeds, etc. Botanical ingredients may include, for example, plant oils such as linalool, b-caryophyllene, b-myrcene, d-limonene, humulene, a-pinene, ylang-ylang (Cananga odorata), yarrow (Achillea millefolium), violet (Viola odorata), vetiver (Vetiveria zizanoides), vanilla (Vanilla plantifolia), tuberose (Polianthes tuberosa), thyme (Thymus vulgaris L.), tea tree (Melaleuca alternifolia), tangerine (Citrus reticulata), spruce (Picea mariana), spruce (Tsuga Canadensis); pepper (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 (Ravensara aromatica); plai (Zingiber cassumunar); pine needles (Pinus sylvestrisL.); 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); lavandin (Lavandula hybrida grosso); kanuka (Kunzea ericoides); juniper (Juniperuscummunis); jasmine (Jasminum officinale); jasmine (Jasminum sambac); helichrysum (Helichrysum italicum); grapefruit (Citrus xparadisi); grapefruit (Citrus paradisi); ginger (Zingiber officinalis); Geranium (Pelargonium graveolens); Geranium (Pelargoniumgraveolens) (Herit); gardenia (Gardenia jasmine); galbanum (Ferula galbaniflua); frankincense (Boswellia carterii); frangipani (Plumeria alba); white fir needle (Abies alba); Siberian fir (Abies siberica); Canada fir needle (Abies balsamea); fennel (Foeniculum vulgare); eucalyptus smithie (Eucalyptus Smithii). Eucalyptus radiata (Eucalyptus Radiata), Eucalyptus Globulus (Eucalyptus Globulus), Eucalyptus Citriodora (Eucalyptus Citriodora), Eucalyptus Bluemary (Eucalyptus Polybractea); Elemi (Canarium Luzonicum); Dill (Anethum Gravelens); Cypress (Cupressus Sempervirens); 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 Gravelins); Cedarwood (Thuja) plicata); Cedarwood (Juniperus virginiana); Cedarwood Atlas (Cedrus atlantica); Carrot seeds (Daucus carota); Cardamom (Elettaria cardamomum); Caraway seeds (Carum carvi); Cajuput (Melaleuca cajuputi); Juniper (Juniperus oxycedrus); Birch (Betula alba); Birch (Betula lenta); Bergamot (Citrus bergamia), Laurel (Laurusnobilis), basil (Ocimum basilicum), basil (Ocimum sanctum), basil (Ocimum basilicum), balsam poplar (Populus balsamifera), Peru balsam (Myroxylon balsamum), or angelica (Angelica archangelica L.).

[0024] In some embodiments, the methods described herein include extracting yerba mate. As used herein, the term "yerba mate" has its ordinary meaning as understood herein and refers to the medicinal plant Ilex paraguariensis.

[0025] The dried leaves and stems of yerba mate, a perennial tree (Ilex paraguariensis St. Hilaire) (Luxner, 1995), are commonly used to prepare an infusion widely consumed in South America. Tea-like infusions are believed to have cleansing, diuretic, and stimulating properties. Yerba mate has been reported to help increase the organism's antioxidant defenses against free radicals. Yerba mate also plays an anti-glycation role, thereby helping to prevent chronic complications of diabetes. Consumption of yerba mate infusions is also important for nutritional purposes, as yerba mate is a rich source of minerals, including potassium, magnesium, and especially the trace element manganese. In addition to minerals, yerba mate infusions contain xanthines, such as caffeine, theobromine, and theophylline, saponins, and caffeoylquinic acids (a family of monoacylquinic and diacylquinic acids).

[0026] A typical 150 mL cup of yerba mate tea contains 10–78 mg of caffeine (Heck & Mejia, 2007), with levels that can vary depending on the amount of tea, the volume of boiling water used, the brewing temperature, and the length of brewing time. In the human body, the low levels of caffeine obtained from consuming such beverages act primarily as a mild central nervous system stimulant. Caffeine is a natural chemical with stimulating effects. It is found in coffee, tea, cola, cocoa, guarana, yerba mate, and over 60 other products (Heckman et al., 2010). Studies have linked caffeine consumption to a variety of human health benefits, including enhanced cognitive function (Foskett et al., 2009; Lorist & Tops, 2003), improved neuromuscular coordination (Glade, 2010; Samoggia & Rezzaghi, 2021; San Juan et al., 2019), elevated mood (Herz, 1999; Lieberman et al., 1987), reduced anxiety (Greden, 1974; Smith, 1988), and stimulation of the central nervous system and myocardium (Bolton & Null, 1981; Davis et al., 2003). Consequently, caffeine is used as an additive in soft and energy drinks. Caffeine has also been added to pharmaceuticals to improve analgesic effects (Derry et al., 2014; Ward et al., 1991; W.-Y. Zhang, 2001).

[0027] In yerba mate leaves, caffeine is the predominant xanthine (2-5% dry weight), with theophylline and theobromine accounting for only 0.2-0.4% and 0.02% dry weight, respectively. Caffeine is synthesized in tea leaves from adenosine (see Figure 2, which shows the biosynthesis of caffeine in tea), and adenosine is the major product of RNA metabolism throughout the life of the tea plant (Ashihara & Suzuki, 2004).

[0028] However, even small amounts of caffeine can cause gastrointestinal irritation, fatigue, and sleep deprivation in some humans (Marriott, 1994; Nawrot et al., 2003; Ranheim & Halvorsen, 2005; Wikoff et al., 2017; Wolde, 2014). Furthermore, when consumed in large amounts (>5 g), caffeine can cause severe toxicity with serious effects such as arrhythmia, tachycardia, vomiting, and convulsions, and can even cause coma and death (Andrade et al., 2018; Nojima et al., 2019). Some, but not most, observational studies have reported that consumption of caffeinated beverages is associated with bone loss and increased fracture risk.

[0029] Therefore, there is an increasing demand for decaffeinated versions of common beverages such as tea and coffee, as people wish to consume these beverages without the potentially harmful effects of caffeine. In response to this demand, decaffeinated coffee has become widely available (Ramalakshmi & Raghavan, 1999; Ogita et al., 2003; Reis et al., 2010). Similarly, there is a demand for decaffeinated tea, and therefore, interest in the methods used to decaffeine these teas (Banerjee & Chatterjee, 2015; Dong et al., 2011; Liang et al., 2007; Park et al., 2007; Ye et al., 2009). However, growing environmental awareness among consumers also raises the possibility that environmentally friendly and sustainable methods will become the most acceptable technology for decaffeinated tea ( Bermejo et al., 2013 ; Choung et al., 2014 ; Pietsch, 2017 ).

[0030] Many methods have been used to remove caffeine to produce decaffeinated tea. Organic solvents such as chloroform, methylene chloride, isopropanol, and ethyl acetate have been used effectively (Choung et al., 2014; Kanda et al., 2013; Villanueva-Bermejo et al., 2017). However, health concerns regarding the use of organic solvents in food processing have been raised among consumers, and alternative methods are being investigated. Supercritical fluid extraction with carbon dioxide has shown potential for removing caffeine from green tea (De Marco et al., 2017; do Espirito Santo et al., 2021; Lack & Seidlitz, 1993; Lee et al., 2007; (Park et al., 2007, 2012; Peker et al., 1992; Sun et al., 2010; Zabot, 2020; Zosel, 1978; Lack & Scidlitz, 1993). This process is fast, leaves no toxic residues, and results in less catechin degradation and flavor loss. However, it is costly to implement on an industrial scale. Microorganisms that can potentially reduce caffeine in tea extracts have been discovered (Dash & Gummadi, 2006; Gokulakrishnan et al., 2005; Mazzafera, 2002; Nanjundaiah et al., 2016; Vuong & Roach, 2014), but this biotechnological method requires strictly controlled conditions, increasing the difficulty and cost. The use of water as the sole solvent in the decaffeination process has also been investigated. This is because it lacks the health risks inherent in organic solvents (Katz, 1987; Liang et al., 2007). Steeping tea in boiling water for 3–10 minutes has been shown to work very well, and this method is relatively inexpensive and easy to scale up for industrial production. However, some of the other important bioactive components are also partially lost during the boiling process. Decaffeination using adsorbents such as activated carbon has also shown promise (Abebe et al., 2022; Lu et al., 2010; Oni et al., 2022; Roy, 1994; Saloko et al., 2020; Shiono et al., 2017; Quintero-Jaramillo et al., 2021). Adsorption methods include preparative paper chromatography, preparative column chromatography on silica gel and polyamide, medium-pressure liquid chromatography, and / or Sephadex LH-20 (size) column chromatography. These methods are inefficient, require more time and consume more solvent, and result in lower product recovery.

[0031] Macroporous resins have attracted considerable attention due to their excellent performance in the separation and purification of pharmaceuticals and natural products (Du et al., 2012; Jin et al., 2015; A. Li et al., 2016; C. Li et al., 2011; H. Li et al., 2019; J. Li & Chase, 2010; Lin et al., 2012; Y. Liu et al., 2011; Z. Liu et al., 2013; Ma et al., 2009; Xiong et al., 2014; L. Zhang et al., 2018; Q.-W. Zhang et al., 2018; W. Zhang et al., 2016; Y. Zhang et al., 2008). As used herein, the term "macroporous adsorption resin" (MAR) has its ordinary meaning as understood in the context of this specification and refers to synthetic porous cross-linked polymer beads used to separate and purify pharmaceuticals and natural products. MARs can have different physical properties such as surface area, average pore size, particle diameter, polarity, and attached functional groups.

[0032] The principle of adsorption is based on electrostatic forces, hydrogen bonding interactions, and size sieving between the macroporous resin and different molecules in the solution. They have advantages such as good stability, high adsorption capacity and selectivity, fast adsorption and desorption, low operating costs, and ease of regeneration of the adsorbents, and therefore they are used in very different application fields (e.g., pharmaceutical, chemical, and food industries).

[0033] In some embodiments provided herein, MAR is used in the extraction process. The physical properties of six exemplary macroporous resins are shown in Table 1.

[0034] [Table 1]

[0035] In some embodiments, NaDES is used in the extraction process. As used herein, the term "natural deep eutectic solvent" (NaDES) has its ordinary meaning as understood in the context of this specification and typically refers to sugars, amino acids, or organic acids that are solid at room temperature but that, when mixed in certain mole fractions, exhibit a high melting point depression and become liquid at room temperature.

[0036] The discovery of deep eutectic solvents (DES) is a major advancement in the world of green chemistry. Deep eutectic solvents are often defined as binary or ternary mixtures of compounds that can associate primarily through hydrogen bonding. By combining these compounds in specific molar ratios, a eutectic mixture is obtained. These solvents consist of two or more inexpensive, non-toxic components, one of which has the ability to be a hydrogen bond acceptor and the other possesses hydrogen bond donor properties. Due to the formation of intramolecular hydrogen bonds and van der Waals interactions, these solvents have melting points much lower than those of the individual components. An example of a deep eutectic is hydroxyethyltrimethylammonium (choline chloride) (melting point T m =302℃) and urea (T m =133°C), and has a very low melting point (T m =12°C) cutoff temperature occurs. What is interesting about these solvents is that they are not only liquid at ambient temperature, but also have tunable and highly soluble properties (Abbott et al., 2004).

[0037] Natural deep eutectic solvents (NaDES) are a specific class of DESs prepared from biomolecules such as choline chloride and betaine as organic salts and urea, organic acids, amino acids, or sugars as hydrogen bond donors (Dai, 2006, 2007). This category includes DESs composed of primary metabolites such as organic acids, amino acids, sugars, polyols, and choline derivatives (Dai et al., 2013). Furthermore, water can also be part of the NaDES composition. Surprisingly, NaDESs are ubiquitous in living organisms, both in intracellular and extracellular media, and may be involved in the synthesis and solubilization of poorly soluble metabolites such as flavonoids, enzymatic reactions, and even drought tolerance. In this paradigm, NaDESs constitute a third type of natural liquid, separate from water and lipids (Choi et al., 2011). A series of NaDESs have been formed from abundant biomolecules, including combinations of choline chloride with citric acid, malic acid, maleic acid, and ascorbic acid, combinations of proline with citric acid, combinations of malic acid with 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).

[0038] NaDES exhibits favorable properties as a solvent, including low vapor pressure, nonflammability, low or negligible toxicity, environmental friendliness, ease of preparation, and low cost. NaDES species offer particular advantages as extraction media because they exhibit excellent solubilization capabilities for natural products. The most prominent functional groups of NaDES components are carboxylic acids, hydroxyl groups, and carbonyl groups. In NaDES matrices, these groups can form hydrogen-bonding networks through intermolecular interactions, thereby changing the physicochemical environment. Generally, the greater the intermolecular attraction, the greater the polarity. Therefore, polarity is generally a solubilizing property. The unique intermolecular interactions or configuration of the NaDES matrix result in its special solubilizing and stabilizing properties. For example, proline is only slightly soluble in dimethyl sulfoxide (DMSO), whereas proline-based multicomponent NaDESs are completely miscible with the same organic solvent.

[0039] NaDES possess biological activity and can be engineered to possess 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 studies have demonstrated that NaDES used for extraction purposes can improve the antioxidant activity of the resulting plant extract, which can be explained by the reactive oxygen species scavenging activity of NaDES itself or the NaDES-forming compounds. The antioxidant activity of these NaDES was not unexpected, as the forming compounds (malic acid, citric acid, proline, and betaine) also possess antioxidant activity. Furthermore, because the components of NaDES are abundant in nature and part of the daily diet, as well as food supplements already available on the market (e.g., choline, citric acid, betaine, amino acids, etc.), it is expected that extracts obtained by NaDES can be directly used in food, pharmaceutical, cosmetic, and agrochemical products without the need for expensive downstream purification procedures. NaDES also improves the biological activity of phenolic acids ( Faggian et al., 2016 ), and therefore can be directly used in food, cosmetic, and pharmaceutical formulations.

[0040] NaDES are ubiquitous in living organisms, both intracellularly and extracellularly, and are involved in the synthesis and solubilization of poorly soluble metabolites, such as flavonoids. NaDES are also involved in enzymatic reactivity and drought tolerance. NaDES exhibit numerous favorable properties, including low vapor pressure, non-flammability, low or negligible toxicity, no adverse environmental impact, low cost, and ease of use. Furthermore, the vast number of structural combinations of NaDES makes it possible to optimally design NaDES combinations for specific applications. NaDES are biocompatible and enhance biological activity. Therefore, it is possible to optimally design compositions containing NaDES with specific biological activity. Extracts obtained with NaDES and containing NaDES in the composition can be used in foods, dietary supplements, pharmaceuticals, cosmetics, pesticides, and industrial applications.

[0041] The extraction solvent is also an important factor in extraction efficiency. While various extraction techniques have been investigated, only a limited number of studies have reported the optimal extraction solvent for yerba mate extraction (da Silveira et al., 2017; Junior et al., 2019; Linares et al., 2010). However, there has been little research into environmentally friendly and safe solvents that can replace toxic organic solvents without compromising efficiency. Classic solvents often have drawbacks, such as high vapor pressure, which can pose hazards such as low flash points, high flammability, toxicity, corrosion to equipment, and the significant effort required for wastewater treatment. As public awareness of environmental issues grows and stricter environmental regulations are being implemented in an increasing number of countries, "green" solvents are gaining increasing attention. The search for such green solvents has led to the discovery of natural deep eutectic solvents (NaDES). NaDES can be synthesized by simply mixing two compounds (i.e., a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA)) within a specific molar ratio. These two components must be capable of forming a eutectic mixture. NaDES are typically mixtures of a salt and a hydrogen bond donor, exhibiting melting points significantly lower than those of the individual components. The interaction of the hydrogen bond donor molecule with the ion from the salt molecule, along with additional effects such as the symmetry of the cation molecule, causes a melting point depression of such mixtures (Choi & Verpoorte, 2019; Dai et al., 2013; Plotka-Wasylka et al., 2020; Vanda et al., 2018). NaDES offer many advantages, including ease of preparation, high purity, low toxicity, readily biodegradable properties, low melting points, high thermal stability, low volatility, nonflammability, and air stability. Their ease of synthesis and wide availability from relatively inexpensive components make them preferred in many applications compared to traditional organic solvents and ionic liquids (Choi & Verpoorte, 2019; Dai et al., 2013; Plotka-Wasylka et al., 2020; Vanda et al., 2018). Despite the vigorous testing of NaDES as alternatives to "traditional" solvents, little research has been done on their recovery and recycling, one of the main challenges for achieving cost-effective and environmentally friendly industrial applications. Without effective recovery, most NaDES-based options will likely never enter the commercial market.

[0042] Hydrogen bond donors and acceptors can be selected based on the properties of the target compound. For example, yerba mate extracts contain phenolic acids and alkaloids. Phenolic acids are highly polar compounds, more soluble in water than in nonpolar solvents, and their chemical form and stability depend on the pH value. Chlorogenic acid has been reported to be stable against acidic pH (Friedman & Jurgens, 2000).

[0043] Figure 3 shows a diagram of the chemical structure of caffeine and lists some of its properties (Quintero-Jaramillo et al., 2021). According to the information, the dipole moment of caffeine is high and increases depending on the polarity of the medium in which it is held. This means that the positive charge of nitrogen in caffeine interacts electrostatically with any negatively polarized functional group. pKa and pH affect the chemical behavior, and when pH < pKa, the protonated form of caffeine is produced.

[0044] Shafie et al., 2019 synthesized deep eutectic solvents using choline chloride and citric acid monohydrate at different molar ratios (e.g., NaDES 3:1, 2:1, 1:1, 1:2, and 1:3) and reported their physicochemical properties. They reported that NaDES was stable as a clear and viscous liquid over time by POM imaging. Based on the FTIR spectrum, they concluded that the formation of hydrogen bond interactions occurred. During the formation of NaDES, the hydroxyl group (OH) of citric acid monohydrate was attracted to the chlorine anion (Cl - ) of choline chloride, so an OH-Cl bond occurred. The interaction was different from the variation in the molar ratio of choline chloride and citric acid monohydrate. This is because the presence of the quaternary ammonium salt and hydrogen bond donors forms hydrogen bond (especially with the hydroxyl functional group) interactions, which changes the strength of the OH bond. These results are summarized in Table 2.

[0045]

Table 2

[0046] Increasing the molar ratio of choline chloride to NaDES increased the melting point and the extraction yield. This is due to the increased chloride ions (Cl) in choline chloride. - This may be explained by the fact that the increased choline chloride molar ratio induced stronger hydrogen-bonding interactions within NaDES, resulting in more energy required to break the interactions and higher extraction yields. These results indicated a significant effect of decreasing the molar ratio of choline chloride and increasing the molar ratio of citric acid monohydrate. The extraction yield continuously increased with increasing citric acid monohydrate content, except for NaDES 1:1, which showed the lowest extraction yield. This suggests that a eutectic solvent may not form at a molar ratio of 1:1. Generally, eutectic points were found at molar ratios of 1:2 or 2:1. For example, studies conducted by Abbott et al. (2003) and Hayyan et al. (2013) reported that eutectic points for choline chloride-urea and choline chloride-glucose were observed at molar ratios of 1:2 and 2:1.

[0047] The viscosity of NaDES was useful for understanding the internal resistance of the fluid to shear stress and the nature of the intermolecular interactions with choline chloride and citric acid monohydrate. It was reported that NaDES 1:3 exhibited the highest viscosity (1742.67 Pa-s) among the synthesized NaDESs due to the presence of more hydroxyl groups in citric acid monohydrate. The viscosity of NaDES increased with increasing the molar ratio of citric acid monohydrate. The presence of more citric acid monohydrate increases hydrogen-bonding interactions within NaDES, which increases the attractive forces and reduces the free volume of NaDES. However, for efficient extraction, lower viscosity is advantageous for mass transfer and improved extraction yield.

[0048] Surface tension is important for interpreting the energy required to increase the surface area of ​​a liquid and the strength of the intermolecular forces between compounds in NaDES. The data in Table 2 show that DESs 1:2 and 1:3 exhibited the highest surface tension. As the molar ratio of citric acid monohydrate increased, the surface tension increased continuously. Temperature, the interaction between the quaternary ammonium salt and the hydrogen bond donor, the alkyl chain, viscosity, and molecular weight are the main factors affecting the surface tension of NaDES. The increase in hydrogen-bonding interactions within NaDES leads to the formation of cohesive forces. This creates a stronger mutual attraction between the surface molecules to resist external forces, and the surface molecules develop and resist breakage. This can be explained by the fact that increasing the molar ratio of citric acid monohydrate as a hydrogen bond donor in DES induces an increase in the hydrogen-bonding interactions formed in DES, as previously mentioned. However, as observed, a system with lower surface tension is beneficial for mass transfer in the extraction process.

[0049] The type and molar ratio of quaternary ammonium salt and hydrogen bond donor affected the density of NaDES. NaDES3:1 (2.64 g / ml) had the lowest density, followed by NaDES2:1, NaDES1:1, NaDES1:2, and finally NaDES 1:3 had the highest density. When the molar ratio of citric acid monohydrate was increased, the density of NaDES dramatically increased from 2.64 g / ml to 3.11 g / ml. The increase in the molar ratio of citric acid monohydrate contributes to a decrease in the free space between NaDES, thereby increasing the density. Furthermore, the density of NaDES decreased significantly with the increase in the molar ratio of choline chloride. This is due to the increase in the chloride anion (Cl) induced by the addition of the molar ratio of citric acid monohydrate. - This can be explained by the interaction of the hydroxyl groups in citric acid monohydrate complexed with NaDES. This also contributed to the size of the formed NaDES. The packing structure and density of NaDES were also affected.

[0050] Hayyan et al., 2013, prepared D-glucose-based NaDESs at different molar ratios with choline chloride and reported their physical properties. These results revealed that the studied NaDESs had high viscosity, density, and surface tension at room temperature, which makes it more advisable to heat these types of NaDESs before processing them industrially. However, the nearly neutral pH and low sensitivity to temperature changes of these NaDESs make them good candidates for chemical, environmental, and biological applications.

[0051] In conclusion, the physicochemical properties of NaDES have a significant impact on extraction yield. Low viscosity, low density, small surface area, and high mass transfer efficiency physically increase extraction yield. Higher melting points and stronger intermolecular forces and hydrogen bonds chemically increase extraction yield. A clear relationship was observed between lower solvent viscosity and increased extraction efficiency, suggesting that viscosity is the main property affecting the extraction efficiency of organic acid-based NaDES.

[0052] Some embodiments provided herein include a NaDES containing choline chloride with D-glucose in a molar ratio of 2:1, which has the advantage of pH stability, which is very important for downstream processes and process reproducibility.

[0053] Yerba mate extracts prepared with NaDES can be considered ready for use in the food and pharmaceutical industries without the need for expensive downstream purification steps (Ruesgas-Ramon et al., 2017). However, the final goal is to remove caffeine, and furthermore, the solvent needs to be recycled after the process. Volatile organic solvents used in extraction are usually recycled by distillation. Volatile organic solvents are a major source of industrial waste (Turnbull et al., 2004). On the other hand, NaDES have very low vapor pressures (Lomba et al., 2019; Ozel & Elibol, 2021), making it very difficult to easily isolate the target compounds by evaporation, which can be problematic for industrial applications. Current literature has proposed several possibilities for the recovery of target compounds and the recycling of NaDES. These include liquid-liquid extraction using another solvent, solid-liquid extraction, and the addition of an antisolvent (Gullon et al., 2020; Huang et al., 2017; Vanda et al., 2018).

[0054] Method for extracting bioactive compounds from plant material Some embodiments provided herein relate to methods for extracting bioactive compounds from plant material. Figure 1 shows a schematic flow chart of an exemplary process for producing decaffeinated yerba mate extract and recovering NaDES.

[0055] In some embodiments, the method includes obtaining a plant material. In some embodiments, the method further includes mixing the plant material with a natural deep eutectic solvent (NaDES). In some embodiments, the method further includes obtaining a crude extract solution comprising an extract of the plant material, a bioactive compound, and a NaDES. In some embodiments, the method further includes loading the crude extract solution onto a macroporous resin. In some embodiments, the method further includes recovering the plant extract from the macroporous resin, wherein the bioactive compound and the NaDES have been removed from the plant extract. In some embodiments, the bioactive compound is caffeine. In some embodiments, the plant material is obtained from yerba mate.

[0056] In some embodiments, the NaDES includes a metal salt and an organic salt, a metal salt hydrate and an organic salt, an organic salt and a hydrogen bond donor, or a metal salt hydrate and a hydrogen bond donor. In some embodiments, the NaDES includes acetamide, acetic acid, 1,4-butanediol, choline acetate, choline chloride, choline fluoride, choline nitrate, citric acid monohydrate, ethylene glycol, fructose, glucose, glycerol, glycine, imidazole, lactic acid, lactose, malonic acid, maltose, mannitol, 1-methylurea, oxalic acid, phenylacetic acid, propionic acid, raffinose, resorcinol, sorbitol, sucrose, 2,2,2-trifluoroacetamide, urea, and xylitol, or any combination thereof. In some embodiments, the NaDES includes choline chloride (ChCl) and glucose (GLU). In some embodiments, the glucose is D-glucose. In some embodiments, the NaDES comprises citric acid monohydrate.

[0057] The combination of ingredients can be present in a ratio ranging from about 10:1 to about 1:10, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, or any ratio within a range defined by any two of the foregoing values. In some embodiments, choline chloride and glucose are present in a ratio of about 2:1. In some embodiments, choline chloride and glucose are present in a ratio of about 3:1. In some embodiments, choline chloride and glucose are present in a ratio of about 1:1. In some embodiments, choline chloride and glucose are present in a ratio of about 1:2. In some embodiments, choline chloride and glucose are present in a ratio of about 1:3.

[0058] In some embodiments, the NaDES comprises water present in an amount ranging from 10% to 50%, e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by weight, or an amount within a range defined by any two of the foregoing values. In some embodiments, the NaDES comprises water present in an amount of about 30% by weight.

[0059] In some embodiments, the method also includes diluting the crude extract with water before applying the crude extract to the macroporous resin. In some embodiments, the dilution is about a 10-fold dilution. In some embodiments, the dilution is about a 9-fold dilution. In some embodiments, the dilution is about an 8-fold dilution. In some embodiments, the dilution is about a 7-fold dilution. In some embodiments, the dilution is about a 6-fold dilution. In some embodiments, the dilution is about a 5-fold dilution. In some embodiments, the dilution is about a 4-fold dilution. In some embodiments, the dilution is about a 3-fold dilution. In some embodiments, the dilution is about a 2-fold dilution. In some embodiments, the dilution is about an 11-fold dilution. In some embodiments, the dilution is about a 12-fold dilution. In some embodiments, the dilution is about a 13-fold dilution. In some embodiments, the dilution is about a 14-fold dilution. In some embodiments, the dilution is about a 15-fold dilution. In some embodiments, the dilution is about a 16-fold dilution. In some embodiments, the dilution is about a 17-fold dilution. In some embodiments, the dilution is about an 18-fold dilution. In some embodiments, the dilution is about a 19-fold dilution. In some embodiments, the dilution is about a 20-fold dilution. In some embodiments, the dilution is an amount within a range defined by any two of the foregoing values.

[0060] In some embodiments, the recovered plant extract is rich in caffeoylquinic acids. In some embodiments, the recovered plant extract contains less than 0.05% by weight of caffeine, such as 0.04%, 0.03%, 0.02%, or 0.01% by weight of caffeine. In some embodiments, the recovered plant extract contains less than 0.01% by weight of caffeine.

[0061] In some embodiments, the macroporous resin comprises a non-polar macroporous resin. In some embodiments, the macroporous resin comprises a polar macroporous resin. In some embodiments, the macroporous resin comprises a weakly polar macroporous resin. In some embodiments, the macroporous resin comprises a moderately polar macroporous resin. In some embodiments, the macroporous resin comprises an H-bond macroporous resin. In some embodiments, the macroporous resin is or comprises HDP200. In some embodiments, the macroporous resin is or comprises HDP722. In some embodiments, the macroporous resin is or comprises HDP400. In some embodiments, the macroporous resin is or comprises HDP750. In some embodiments, the macroporous resin is or comprises HDP600. In some embodiments, the macroporous resin is or comprises HDP826. In some embodiments, the macroporous resin is present in a column.

[0062] In some embodiments, recovering the plant extract comprises eluting the plant extract from the macroporous resin. In some embodiments, the plant extract is eluted with a solution comprising ethanol in an amount ranging from 10% to 30%, e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by volume, or an amount within a range defined by any two of the foregoing values. In some embodiments, the solution comprises about 20% ethanol by volume.

[0063] In some embodiments, the method also includes recovering the NaDES from the macroporous resin. In some embodiments, recovering the NaDES from the macroporous resin includes washing the macroporous resin with water, capturing the eluate and water, and drying the captured eluate and water to recover the NaDES.

[0064] Therefore, some embodiments are described in the alternative embodiments listed below.

[0065] 1. A method for extracting bioactive compounds from plant material, comprising obtaining the plant material, mixing the plant material with a natural deep eutectic solvent (NaDES), obtaining a crude extract solution containing an extract of the plant material, the bioactive compounds, and the NaDES, loading the crude extract solution onto a macroporous resin, and recovering the plant extract from the macroporous resin, wherein the bioactive compounds and the NaDES have been removed from the plant extract.

[0066] 2. The method of alternative embodiment 1, wherein the biologically active compound is caffeine.

[0067] 3. The method of alternative embodiment 1 or 2, wherein the plant material is obtained from yerba mate.

[0068] 4. The method of any one of alternative embodiments 1-3, wherein the NaDES comprises choline chloride (ChCl) and glucose (GLU).

[0069] 5. The method of alternative embodiment 4, wherein the choline chloride and glucose are present in a ratio of about 2:1.

[0070] 6. The method of any one of alternative embodiments 1-5, wherein the NaDES comprises water present in an amount ranging from 10% to 50% by weight.

[0071] 7. The method of any one of alternative embodiments 1-6, wherein the NaDES comprises water present in an amount of about 30% by weight.

[0072] 8. The method of any one of alternative embodiments 1-7, further comprising diluting the crude extract with water before applying the crude extract to the macroporous resin.

[0073] 9. The method of alternative embodiment 8, wherein the dilution is about a 10-fold dilution.

[0074] 10. The method of any one of alternative embodiments 1-9, wherein the recovered plant extract is enriched in caffeoylquinic acids.

[0075] 11. The method of any one of alternative embodiments 1-10, wherein the recovered plant extract contains less than 0.05% caffeine by weight.

[0076] 12. The method of any one of alternative embodiments 1-11, wherein the recovered plant extract contains less than 0.01% caffeine by weight.

[0077] 13. The method of any one of alternative embodiments 1-12, wherein the macroporous resin comprises a non-polar macroporous resin.

[0078] 14. The method of any one of alternative embodiments 1-13, wherein the macroporous resin comprises HDP200.

[0079] 15. The method of any one of alternative embodiments 1-14, wherein the macroporous resin is in a column.

[0080] 16. The method of any one of alternative embodiments 1-15, wherein recovering the plant extract comprises eluting the plant extract from a macroporous resin.

[0081] 17. The method of alternative embodiment 16, wherein the plant extract is eluted with a solution comprising ethanol in an amount ranging from 10% to 30% by volume.

[0082] 18. The method of alternative embodiment 17, wherein the solution comprises about 20% ethanol by volume.

[0083] 19. The method of any one of alternative embodiments 1-18, further comprising recovering the NaDES from the macroporous resin.

[0084] 20. The method of alternative embodiment 19, wherein recovering the NaDES from the macroporous resin comprises washing the macroporous resin with water, capturing the eluate and water, and drying the captured eluate and water to recover the NaDES.

[0085] Example Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the present invention, as described hereinabove and in the claims.

[0086] Materials and Methods Materials and Chemicals: Yerba mate leaves were sourced from Triunfo and used as received. Citric acid, glucose, and choline chloride were purchased from TCI Chemicals. HPLC-grade water, methanol, acetonitrile, and LC-grade formic acid were purchased from Fisher Chemicals. Reference standards chlorogenic acid, caffeine, theobromine, rutin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were purchased from Sigma Chemicals. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), a product of Caymon Chemicals, was purchased from DNA Biotechnology, SG.

[0087] Macroporous resins: Six types of macroporous resins (HDP200, HDP400, HDP600, HDP722, HDP750, and HDP826) were purchased from Bonchem Co. Ltd. (Guanghou, China). Their physical properties are listed in Table 1. Briefly, the resins were pretreated by soaking in ethanol for 24 hours. They were then thoroughly re-washed with fresh ethanol, distilled until no oligomers, porous materials, or other ethanol-soluble substances remained, and dried in a drying oven at 70 °C. Before use, the resins were soaked in ethanol and thoroughly washed with distilled water.

[0088] Preparation of NaDES: NaDES were prepared by mixing choline chloride (ChCl), which acts as a hydrogen bond acceptor (HBA), citric acid (CA), or glucose (GLU), which acts as an HBD. Choline chloride (ChCl) was dried in a vacuum oven at 60 °C for 24 h before use. ChCl and citric acid (CA) in molar ratios of 3:1, 2:1, 1:1, 1:2, or 1:3, and glucose (GLU) in a molar ratio of 2:1 with 30% water by weight were placed in a glass beaker and stirred using a magnetic stirrer (Multichannel Magnetic Stirrer, HS-8, Yooning, Hangzhou, China) at 350 rpm. The glass beaker was covered with parafilm and heated to 50 °C for 2 h until a homogeneous, colorless, and transparent liquid was formed.

[0089] Preparation of NaDES extract: The NaDES extraction process was carried out using a water bath shaker (WS-10, Yooning, Hangzhou, China). 3 g of yerba mate leaves were added to a 50 ml centrifuge tube and mixed with 30 g of solvent containing both NaDES and other organic solvents. The extraction was carried out at 60 °C for 3 h. The mixture was centrifuged at 4000 rpm for 8 min (LC-8S, Joan Lab equipment, Huzhou, China) to separate the NaDES liquid extract from the biomass. The filtrate was collected and stored in a refrigerator until further analysis.

[0090] Static adsorption / desorption properties of macroporous resins. To select a suitable macroporous resin for caffeine removal and recovery of other bioactive substances and NaDES, a static adsorption / desorption test was performed. The pretreated hydrated resin (3.0 g dry weight) was placed in a 250 ml Erlenmeyer flask, and 40 ml of sample solution was then added. The flask was then tightly sealed with a stopcock and continuously shaken on an orbital shaker at 120 rpm for 12 hours at 25°C. The contents of bioactive substances in the solutions before and after adsorption were analyzed by HPLC.

[0091] After reaching adsorption equilibrium, the adsorbate-containing resin was first filtered, thoroughly washed three times with distilled water, and then desorbed with 50 ml of 70% (v / v) ethanol. The flask was continuously shaken at 120 rpm at 25°C for 12 hours. The content of bioactive substances in the desorption solution was analyzed by HPLC.

[0092] Adsorption capacity (Q e ), desorption capacity (Q d The following equations were used to calculate the desorption rate (D):

[0093] JPEG2025527440000004.jpg4599

[0094] In the formula, Q c is the adsorption capacity at adsorption equilibrium (mg / g, dry resin); Q d is the desorption capacity after adsorption equilibrium (mg / g, dry resin); C and C e are the initial concentration in solution and the adsorption equilibrium concentration (mg / ml); C d is the concentration in the desorption solution (mg / ml); V i and V d is the volume of the initial sample and desorption solution (ml); D is the desorption rate (%); W is the weight of the dry resin (g).

[0095] Caffeine removal, chlorogenic acid recovery, and NaDES recycling: Caffeine removal, chlorogenic acid recovery, and NaDES recycling were achieved by adsorption onto a macroporous resin in a wet-packed glass column (2.5 cm x 24.5 cm) containing 80 g of HDP200 (BV = 120 ml). The resin was pretreated with 2 BV of 96% ethanol and then with 3 BV of deionized water. 15 ml of extract prepared with ChClGlu 2:1 was diluted 10-fold with 135 ml of water. 150 ml of the extract solution was loaded onto the column at a flow rate of 1.14 BV / h. ChClGlu was eluted from the column with 1.6 BV of deionized water; decaffeinated chlorogenic acid-rich yerba mate extract was eluted with 1.6 BV of 20% v / v ethanol; and other bioactive compounds in the yerba mate extract were eluted with 1.6 BV of 70% v / v ethanol. The aqueous fraction was evaporated under vacuum to recover ChClGlu. The 20% ethanol and 70% ethanol fractions were analyzed by HPLC, and the yield was calculated.

[0096] High-Performance Liquid Chromatography (HPLC) Analysis: Quantitative analysis of bioactive compounds was performed using a Thermo Fisher Vanquish series HPLC system equipped with Chromeleon software, a VC-P10-A-01 pump, and a VC-D11A-01 diode array detector (DAD). Liquid chromatographic separation was performed on a reversed-phase Accucore C18 column (150 mm x 2.1 mm, 2.6 μm particle size, Thermo Fisher) at an oven temperature of 30°C. The detection wavelengths were 280 nm for caffeine and theobromine, and 350 nm for rutin and all other chlorogenic acids. The flow rate was 0.4 ml / min, and the injection volume was 10 μl. The mobile phase consisted of 0.1% formic acid / water (A) and 0.1% formic acid / acetonitrile (B). The mobile phase gradient elution was as follows: 5–20% B from 0–10 min, 20% B from 10–12 min, 20–5% B from 12–15 min, 5–95% B from 15–15.1 min, 95% B from 15.1–16.5 min, 95–5% B from 16.5–16.6 min, and 5% B from 16.6–19 min. The tested samples and standards were dissolved in HPLC-grade methanol and filtered through a 0.22 μm PTFE (polytetrafluoroethylene) filter before use.

[0097] Chromatographic peaks of all bioactive compounds were identified by retention time. The retention times of theobromine, neochlorogenic acid, chlorogenic acid, caffeine, cryptochlorogenic acid, rutin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were 1.54, 2.48, 4.337, 4.673, 5.300, 9.937, 11.450, 11.737, and 12.747 min, respectively. Standard solutions were prepared at 1 mg / ml in methanol and mixed in equal volumes to produce a 1 mg / ml mixture, which was then serially diluted to 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 mg / ml. Calibration was performed for each standard in the range of 2.23 μg / ml to 142.86 μg / ml or 2.23 ppm to 142.86 ppm. Calibration curves were plotted using the six experimental points. The regression equations for theobromine, caffeine, chlorogenic acid, rutin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were Y = 0.0335X - 0.0619 (R 2 =0.9978), Y=0.0969X+0.0434(R 2 =0.9999), Y=0.0498X-0.0459(R 2 =0.9992), Y=0.0648X+0.0345(R 2 =1.000), Y=0.0632X-0.0358(R 2 =0.9992), Y=0.0812X+0.0332(R 2 =0.9999) and Y=0.0819X-0.056(R 2 =0.9997), where Y is the peak area and X is the concentration (μg / ml).

[0098] DPPH radical scavenging ability assay: The DPPH free radical scavenging ability of the plant extracts was evaluated by using the method described by Takao et al. (Takao et al., 1994).

[0099] A stock solution of the plant extract was prepared in methanol to a concentration of 2000 μg / ml. Further 2-fold dilutions were made to obtain concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.62 μg / ml. Each diluted extract solution (2 ml) was mixed with 2 ml of DPPH methanol solution (80 μg / ml). After 30 minutes at room temperature in the dark, the absorbance was read at 517 nm using a spectrophotometer. Control samples included 2 ml of DPPH solution plus 2 ml of methanol. Caffeoylquinic acid and ascorbic acid were used as positive controls. Experiments were performed in triplicate. The scavenging activity is expressed as the percentage of inhibition calculated using the following formula: scavenging activity (%) = 100 × [(A control -A sample ) / A control )], where A control is the absorbance of the control, and A sample is the absorbance of the extract.

[0100] I C 50 The IC value is the effective concentration at which 50% of the DPPH radicals are removed. This was obtained from a graph of scavenging activity (%) against the concentration of the sample. A low IC 50 The values ​​indicate that the extract has a strong ability to act as a DPPH scavenger.

[0101] Experimental setup for process scale-up: 20 g of yerba mate leaves were extracted using 200 g of ChClGLU 2:1 and 30 wt% water. Extraction was performed at 60°C for 3 hours. To reduce viscosity and facilitate subsequent processing, 80 g of water was added to achieve a concentration of approximately 50%. The mixture was filtered. The filtrate was retained for further processing. Decaffeination, chlorogenic acid recovery, bioactive substance recovery, and NaDES recovery were achieved using a glass column (6 cm x 33.5 cm) wet-packed with approximately 670 g of HDP200 (bed volume (BY) = 1 L). The resin was pretreated with 2 BV of 95% ethanol and then with 3 BV of deionized water. The extract prepared above was further diluted to 90% with water and loaded onto the column at a flow rate of 1.14 BV / h. ChClGLU was eluted from the column with 2 BV of deionized water. The decaffeinated caffeoylquinic acid-enriched fraction was desorbed with 2 BV of 20% v / v aqueous ethanol, and the caffeine-enriched fraction was desorbed with 2 BV of 70% v / v aqueous ethanol. The eluate and aqueous fraction were evaporated under vacuum to recover ChClGLU. The EtOH fraction was analyzed by HPLC, and the yield was calculated. The recycled NaDES was made up to 30 wt% with water and used again for extraction.

[0102] Example 1 HPLC Results: The HPLC test method was successfully applied to test for phenolic acids and caffeine in yerba mate leaf extracts obtained under various conditions. As shown in Figure 4, theobromine (Rt = 1.45 min) and caffeine (Rt = 4.55 min) were detected at 280 nm; neochlorogenic acid (Rt = 2.327 min), chlorogenic acid (Rt = 4.257 min), cryptochlorogenic acid (Rt = 5.037 min), rutin (Rt = 9.837 min), 3,4-dicaffeoylquinic acid (Rt = 11.357 min), 3.5-dicaffeoylquinic acid (Rt = 11.597 min), and 4.5-dicaffeoylquinic acid (Rt = 12.643 min) were detected at 350 nm. UV detection was used at 280 nm.

[0103] Theobromine and caffeine, as well as caffeoylquinic acids and other bioactive substances, were tested using a UV detector at 350 nm. All compounds were well resolved. A peak analysis table is shown in Table 3. These results indicate that the nine biomarkers were successfully separated by the chromatographic method applied to the analysis of yerba mate extract. The resolution value indicates that the compound peaks are separated by a baseline gap, whereas 1.2 is the minimum value required for complete separation of two peak compounds (Ahuja, 2002).

[0104] [Table 3]

[0105] Example 2 To reduce viscosity and improve mass transfer between the solid and liquid phases, NaDES was prepared in 30 wt% water. These results were compared with ethanol, the most suitable and environmentally friendly conventional organic solvent. The concentration of chlorogenic acid in the ethanol extract was only 61 μg / ml, whereas in ChCl:Glu 2:1 it increased 20-fold to 1221 μg / ml. The extraction yield of total caffeoylquinic acids (total CQA) increased 10-fold, from 528 μg / ml in ethanol to 6346 μg / ml in ChCl:Glu 2:1. The results demonstrated that NaDES significantly outperformed organic solvents.

[0106] All citric acid-based NaDES were polar, with a polarity similar to that of water, and pH values ​​ranged from 0.16 to 1.62. These results also revealed that the extraction yields obtained with NaDES were similar to those obtained with water, but that changing the molar ratio significantly affected the extraction efficiency. For the extraction of bioactive substances in yerba mate, the highest extraction efficiency was obtained by using a 2:1 molar ratio of choline chloride / citric acid or a 2:1 molar ratio of choline chloride / glucose. The lowest extraction efficiency was obtained by using a 1:1 molar ratio of choline chloride / citric acid or glucose / citric acid. The ability of NaDES to extract bioactive substances from natural product matrices correlated well with physical and chemical properties, such as hydrogen bonding, polarity, acidity, and viscosity.

[0107] The contents of bioactive substances in the NaDES extract are shown in Table 4 and Figure 5. Total CQA and total caffeoylquinic acids were calculated by combining monocaffeoylquinic acid and dicaffeoylquinic acid.

[0108] [Table 4]

[0109] Example 3 Selection of Macroporous Resins: Commercially available HDP series resins with different polarities and physical properties were selected for this study. These resins were prepared by post-crosslinking chloromethylated gel-type styrene-DVB copolymers via the Friedel-Crafts reaction, followed by treatment of the residual chloromethyl groups with dimethylamine. Polarity was adjusted by adding functional groups. The adsorption / desorption properties of macroporous resins are closely related to their chemical structure and polarity, as well as their physical properties, such as surface area and pore size. Because caffeine and caffeoylquinic acid both contain nonpolar benzene rings, nonpolar, polar, or H-bond resins were selected based on the similarity of resin adsorption selectivity and the principle of mutual miscibility to optimize the process. The results of static adsorption and desorption experiments are shown in Table 5 and Figures 6A-6B.

[0110] [Table 5A]

[0111] [Table 5B]

[0112] [Table 5C]

[0113] Table 5A and Figure 6A show the adsorption capacities of the six studied resins, ranging from nonpolar, polar, to hydrogen-bonded. For caffeoylquinic acid, HDP200, HDP400, HDP600, HDP722, and HDP750 had higher adsorption capacities than HDP826, indicating that the five resins have better adsorption selectivity for caffeoylquinic acid and rutin. These resins exhibited a high adsorption selectivity between 500 and 700 m 2 Due to its large surface area of ​​1000 s / g and multiple benzene rings, van der Waals forces and π-π conjugation may contribute to its strong affinity. HDP826 showed the lowest adsorption capacity; this suggests that hydrogen bonding interactions are not important in the synergistic effect.

[0114] Table 5B and Figure 6B show the desorption capacities. In Table 5C, HDP200 exhibited the highest desorption rate, suggesting that HDP200 also possesses excellent desorption capacity. HDP826 resin exhibited the lowest desorption rate, approximately 50% lower than HDP200 resin, suggesting that both caffeine and chlorogenic acid were difficult to dissociate from HDP826 resin. These results indicate a significant waste of bioactive substances by HDP826 resin. Therefore, HDP826 was not selected for further studies.

[0115] Since caffeine was the impurity to be removed, the adsorption capacities of these resins for caffeine were investigated. Among the resins with high adsorption capacities for phenolic acids, the non-polar HDP200 resin showed the highest adsorption and desorption capacities for caffeine.

[0116] Therefore, HDP200 was selected as the optimized medium for recovering bioactive substances and removing caffeine.

[0117] Example 4 HDP200-based column separation for caffeine removal and NaDES recovery: Dynamic desorption experiments were carried out on a 120 ml column using HDP200 as the separation medium. The results are listed in Table 6.

[0118] Table 6A

[0119] [Table 6B]

[0120] The results showed that the total extraction yield of all bioactive substances in the crude extract of yerba mate using NaDES was 5.99%, which was the total yield of all bioactive substances contained in yerba mate leaves. Due to the presence of a large amount of NaDES solvent in the crude extract, the purity of the bioactive substances was less than 1%. Therefore, concentrating the bioactive substances to an appropriate concentration by removing and recovering NaDES may be an important step for practical applications.

[0121] 150 g of crude extract solution (Sample 10) containing 11.2 g of solids was loaded onto the column. The solid content included bioactive substances, impurities, and NaDES components. The loading solution had a concentration of 0.75%, and the large amount of water disrupted hydrogen bonds, so NaDES was not present. However, the individual components, choline chloride, and glucose were present in the solution. The HPLC chromatogram of Sample 10 is shown in Figure 7A. All bioactive substances were present in this sample.

[0122] First, Sample 10 was loaded onto the column. During the loading process, 5.4 g of solids (48% of the loading solution) were collected in the eluate. The HPLC chromatogram of the eluate Sample 11 is shown in Figure 7B. Only a small amount of bioactive substance was detected. After concentration, the fraction was sticky, indicating the presence of NaDES components. This fraction was recycled.

[0123] The packed column was washed sequentially with water, 20% v / v ethanol, and 70% v / v ethanol, and the following fractions were collected for mass balance and chemical testing: Sample 12 (water fraction), Sample 13 (20% ethanol fraction), and Sample 14 (70% ethanol fraction). The HPLC chromatogram of Sample 12 is shown in Figure 7C.

[0124] A total of 3.3 g of solids, representing 30% of the solids in the crude extract, was recovered in the aqueous fraction, sample 12. HPLC chromatograms indicated that a total of approximately 0.4 mg (0.3% of the crude extract) of bioactive material was collected in this fraction. After concentration, this fraction was also sticky, indicating the presence of NaDES components. This fraction was also recycled.

[0125] The 20% ethanol fraction, sample 13, recovered 38% of the bioactive compounds in the crude extract. Surprisingly, caffeine was completely removed, but theobromine was present and its purity increased from 0.06% in the crude extract to 2.44%; all three chlorogenic acid isomers were significantly enriched: chlorogenic acid was enriched from 0.15% to 5.24%; neochlorogenic acid was enriched from 0.22% to 7.64%; and cryptochlorogenic acid was enriched from 0.09% to 3.14%. Dicaffeoylquinic acid was also present at higher concentrations than in the crude extract. The process yield of this fraction was 10% based on the raw material. The HPLC chromatogram of this sample 13 is shown in Figure 7D.

[0126] Caffeine and dicaffeoylquinic acids in the crude extract were eluted with 70% ethanol in Sample 14. Compared to caffeoylquinic acids, caffeine is nonpolar and elutes in less polar solvents; dicaffeoylquinic acids are less polar than caffeoylquinic acids due to spatial hindrance. The HPLC chromatogram of Sample 14 is shown in Figure 7E.

[0127] Example 5 Based on the above results, the decaffeination and NaDES recycling processes were also carried out at a 1 L pilot scale. These results are shown in Table 7 and Figures 8A-8C.

[0128] [Table 7A]

[0129] [Table 7B]

[0130] This process was successfully scaled up to a 1 L column, which is 10 times larger than the small column. Scale-up showed reproducibility and similar trends in results, as follows:

[0131] The 1500 ml crude extract solution (Sample 15) contained 113 g of solids, including 98 g of NaDES and 0.95 g of measurable bioactive substances. The remainder was impurities extracted from the yerba mate leaves. The crude extract (Sample 15) was loaded onto the column, and 61.5 g of solids and 33.7 g of solids were collected in the eluate (Sample 16) and water wash solution (Sample 17). NaDES was recovered from these fractions and recycled.

[0132] The caffeine content in the crude product (Sample 15) was 0.09%, and the total caffeoylquinic acid content (total CQAs), which includes caffeoylquinic acids and dicaffeoylquinic acids, was 0.68%. The HPLC chromatogram of Sample 15 is shown in Figure 8A. Due to the presence of large amounts of NaDES, the content of bioactive substances in the crude extract was low.

[0133] The 20% EtOH eluate (Sample 18) contained zero caffeine but 18.36% total CQA, 27-fold higher than that present in the crude extract. The HPLC chromatogram for Sample 18 is shown in Figure 8B. The process yield for this fraction was 10%. In this fraction, more than 90% of the total CQA was caffeolquinic acid.

[0134] The 70% EtOH eluate (Sample 19) contained 6.7% caffeine and 22.81% total CQAs. This fraction also contained over 90% dicaffeolquinic acid. The HPLC chromatogram of Sample 19 is shown in Figure 8C.

[0135] Example 6 To evaluate the antioxidant activity of the different fractions, an assay based on the reduction of DPPH was performed. Pure chlorogenic acid and ascorbic acid were used as positive controls. The results of the assay are shown in Figure 9 and the calculated IC 50 The values ​​are shown in Table 8.

[0136] [Table 8]

[0137] Surprisingly, decaffeinated yerba mate extracts Samples 13 and 18, which are rich in caffeolquinic acid, exhibited IC values ​​of 104–107 μg / ml, which is nearly equivalent to that of pure chlorogenic acid. 50 In this fraction, more than 90% of the total caffeoylquinic acid content was monoferroylquinic acid; interestingly, in the 70% ethanol elution fractions Sample 14 and Sample 19, the total caffeoylquinic acid content was high, but the IC 50 The values ​​ranged from 142 to 167 μg / ml. In this fraction, more than 90% of the total caffeoylquinic acid content was dicaffeoylquinic acid. These results suggest that dicaffeoylquinic acid has weaker antioxidant activity than the caffeoylquinic acid isomers and that caffeine does not contribute to the antioxidant activity.

[0138] Polyphenols perform diverse biological functions, many of which are responsible for their antioxidant activity (Pandey & Rizvi, 2009). Their structures are shown in Figure 10. In this study, caffeoylquinic acids (3-, 4-, and 5-) had superior antioxidant activity to dicaffeoylquinic acids (3,4-, 3,5-, and 4,5-). In general, antioxidant activity increased with an increase in the number of hydroxyl groups, enhancing the presence of ortho-hydroxyl groups attached to the aromatic ring. Dicaffeoylquinic acids have one more hydroxyl or ortho-hydroxyl group attached to the aromatic ring than caffeoylquinic acids, which is thought to be the primary factor contributing to their superior antioxidant activity. Surprisingly, however, the antioxidant activity of smaller caffeoylquinic acids against DPPH inhibition was higher than that of dicaffeoylquinic acids, indicating that the amount of hydroxyl groups is not the only factor important for protection against DPPH oxidation. This result suggests that the antioxidant activity of dicaffeoylquinic acids may be affected by the position of esterification of the quinic acid moiety. It can be predicted that the steric hindrance effect of dicaffeoylquinic acids, due to their longer bond length, may reduce the chemical bond energy compared to caffeoylquinic acids, stabilizing the molecule.

[0139] Taken together, these examples demonstrated that the decaffeinated caffeoylquinic acid isomer (3-, 4-, 5-) enriched fraction exhibited unexpectedly superior antioxidant activity comparable to that of pure caffeoylquinic acid and ascorbic acid. The caffeoylquinic acid-enriched fraction exhibited better DPPH inhibition than dicaffeoylquinic acid, even though dicaffeoylquinic acid has more hydroxyl or ortho-hydroxyl groups. This suggests that the amount of hydroxyl groups is not the only important factor in protecting against DPPH oxidation, and that the position of esterification of the quinic acid moiety in caffeoylquinic acid also has a significant effect. These results suggest that the steric hindrance effect of dicaffeoylquinic acid weakens its antioxidant activity.

[0140] Other Considerations Headings are included herein for reference purposes and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described therein. Such concepts may be applicable throughout the entire specification.

[0141] Each patent, patent application, publication, and document referenced herein is incorporated herein by reference in its entirety. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or dates of such publications or documents. Such citations do not indicate a search for relevant disclosures. All statements as to the date(s) or contents of documents are based on available information and do not constitute any admission as to their accuracy or correctness.

[0142] Although specific details are provided in the foregoing description to provide a thorough understanding of the embodiments, it will be understood by those skilled in the art that the embodiments may be practiced without these specific details.

[0143] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0144] The methods disclosed herein comprise one or more steps or acts for achieving the described method. Method steps and / or acts may be interchanged with one another without departing from the scope of the present disclosure. In other words, unless a specific order of steps or acts is required for the proper operation of the described method, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the present disclosure.

[0145] In at least some of the described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter defined by the appended claims.

[0146] With respect to the use of virtually any plural or singular term herein, one of ordinary skill in the art can translate from plural to singular or from singular to plural as appropriate to the context or application. For clarity, various singular / plural permutations may be expressly set forth herein.

[0147] The illustrative embodiments illustrated herein may be suitably practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions used are used as terms of description rather than limitation, and the use of such terms and expressions does not exclude any equivalents of the shown and described function or portions thereof, but various modifications are possible within the scope of the invention as defined in the claims. The terms "a" or "an" can refer to one or more of the elements they modify, unless the context clearly indicates that one or more elements of the elements are being described (e.g., "a reagent" can mean one or more "reagent" reagents). As used herein, the term "about" refers to a value within 10% (i.e., plus or minus 10%) of the underlying parameter, and when the word "about" is used at the beginning of a value string, the respective value is modified (i.e., "about 1, 2, and 3" refer to about 1, about 2, and about 3). For example, a weight of "about 100 grams" includes weights from 90 grams to 110 grams. Furthermore, when a list of values ​​is provided herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and subvalue values ​​therein (e.g., 54%, 85.4%). Therefore, while the technology of the present invention has been specifically disclosed by exemplary embodiments and optional features, it is understood that variations and modifications of the concepts disclosed herein may be referenced by those skilled in the art, and such variations and modifications are considered within the scope of the embodiments.

[0148] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will understand that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0149] As will be understood by those skilled in the art, for all purposes, including those relating to providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. It will be readily apparent that any recited range fully expresses, and can be readily recognized as, that same range can be divided into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," "less than," and the like, includes the recited numbers and refers to a range that can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual element. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items.

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Claims

1. 1. A method for extracting bioactive compounds from plant material, said method comprising: Obtaining plant material; mixing the plant material with natural deep eutectic solvent (NaDES); obtaining a crude extract solution comprising an extract of the plant material, the bioactive compound, and NaDES; loading the crude extract solution onto a macroporous resin; recovering the plant extract from the macroporous resin, wherein the bioactive compounds and NaDES have been removed from the plant extract.

2. 10. The method of claim 1, wherein the bioactive compound is caffeine.

3. 10. The method of claim 1, wherein said plant material is obtained from yerba mate.

4. 2. The method of claim 1, wherein the NaDES comprises choline chloride (ChCl) and glucose (GLU).

5. 5. The method of claim 4, wherein the choline chloride and glucose are present in a ratio of about 2:

1.

6. 10. The method of claim 1, wherein the NaDES comprises water present in an amount ranging from 10% to 50% by weight.

7. 10. The method of claim 1, wherein the NaDES comprises water present in an amount of about 30% by weight.

8. 10. The method of claim 1, further comprising diluting the crude extract with water before applying the crude extract to the macroporous resin.

9. 9. The method of claim 8, wherein the dilution is about a 10-fold dilution.

10. 10. The method of claim 1, wherein the recovered plant extract is enriched in caffeoylquinic acids.

11. 10. The method of claim 1, wherein the recovered plant extract contains less than 0.05% by weight of caffeine.

12. 10. The method of claim 1, wherein the recovered plant extract contains less than 0.01% by weight of caffeine.

13. The method of claim 1 , wherein the macroporous resin comprises a non-polar macroporous resin.

14. The method of claim 1 , wherein the macroporous resin comprises HDP200.

15. The method of claim 1 , wherein the macroporous resin is in a column.

16. 10. The method of claim 1, wherein recovering the plant extract comprises eluting the plant extract from the macroporous resin.

17. 17. The method of claim 16, wherein the plant extract is eluted with a solution comprising ethanol in an amount ranging from 10% to 30% by volume.

18. 18. The method of claim 17, wherein the solution comprises about 20% ethanol by volume.

19. 10. The method of claim 1, further comprising recovering NaDES from the macroporous resin.

20. 20. The method of claim 19, wherein recovering NaDES from the macroporous resin comprises washing the macroporous resin with water, capturing the eluate and water, and drying the captured eluate and water to recover NaDES.

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