Method for preparing plant extract composition

Abstract

The present invention discloses a method for preparing a caffeoylquinic acid composition from a plant source. The method may include chromatographically separating a biomass extract on an ion exchange stationary phase and obtaining an eluent containing the caffeoylquinic acid composition. The biomass may be, for example, stevia or yerba mate. The caffeoylquinic acid composition comprises one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts of the foregoing substances.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the following applications: U.S. Application No. 62 / 830,448, filed on April 6, 2019, entitled “Stevia Processing”; U.S. Application No. 62 / 832,273, filed on April 10, 2019, entitled “Methods For Making Botanical Extract Composition”; U.S. Application No. 16 / 373,206, filed on April 4, 2019, entitled “Steviol Glycoside Solubility Enhancers”, which was published as U.S. Patent Application Publication No. 2019 / 0223481 on July 25, 2019; International Application No. PCT / US2018 / 054691, filed on October 5, 2018, entitled “Steviol Glycoside Solubility Enhancers”; International Application No. PCT / US2018 / 054691, filed on October 6, 2017, entitled “Steviol Glycoside Solubility Enhancers” No. 62 / 569,279, entitled “Methods for Making Yerba Mate Extract Composition,” filed on May 25, 2018; No. 62 / 676,722, entitled “Methods for Making Yerba Mate Extract Composition,” filed on October 5, 2018; and No. 16 / 374,894, entitled “Methods for Making Yerba Mate Extract Composition,” filed on April 4, 2019, published as U.S. Patent Application Publication No. 2019 / 0231834 on August 1, 2019. The entirety of each of these applications is hereby incorporated by reference. Background Art

[0003] Compositions comprising monocaffeoylquinic acid (e.g., chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid) and dicaffeoylquinic acid (e.g., 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid) and their salts can be prepared from various plant sources. Compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts can be incorporated into edible materials to provide beneficial properties, including, for example, beneficial sensory properties. However, botanicals comprising compositions of monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts may contain compounds having off-flavors or other undesirable properties that limit the use of these botanicals. Summary of the invention

[0004] Due to the beneficial properties of compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts, methods for extracting these compounds from plant biomass, such as from, for example, yerba mate, stevia, and globe artichoke, are of interest. These compounds include, but are not limited to, monocaffeoylquinic acids (e.g., chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid) and dicaffeoylquinic acids (e.g., 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid), and their salts:

[0005]

[0006]

[0007]

[0008]

[0009] The compounds can each be isolated with high purity (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, and greater than 99%; or about 50% to about 99%, about 60% to about 90%, about 80% to about 95%, or about 70% to about 99% or more purity). In addition, the methods described herein provide the ability to separate compounds of interest such that a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof obtained by the methods described herein can comprise substantially the same amount by weight and / or substantially the same ratio by weight of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof relative to the plant biomass from which the compounds were isolated. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The drawings illustrate generally by way of example and not by way of limitation various embodiments described herein.

[0011] Figure 1 Flow chart of an example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0012] Figure 2 A flow chart of another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0013] Figure 3 Flow chart of an example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0014] Figure 4A A flow chart of another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0015] Figure 4B A flow chart of another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof from yerba mate biomass.

[0016] Figure 4C A flow chart of another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof from yerba mate biomass.

[0017] Figures 5 to 7 is a UHPLC-UV chromatogram of an initial yerba mate extract, which is a concentrate obtained after chromatography of the adjusted second initial extract on an ion exchange chromatography stationary phase; and after drying, following the process described in steps (a)-(h) described herein, wherein "DCQA" refers to "dicaffeoylquinic acid".

[0018] Figures 8 to 10 To show in table form Figures 5 to 7 Table of peak names, retention times and relative area percentage data for the UHPLC-UV chromatograms shown. Figure 8 The data table of the initial yerba mate extract is shown in Table 1. Based on the UV absorbance at 210 nm, the total purity of the target compounds was 49.7%. Fig. 9 The data table is a concentrate obtained after chromatography of the adjusted second initial extract on an ion exchange chromatography stationary phase. The total target compound is 87.1% pure according to the UV absorbance at 210 nm. Fig.10The data are after drying and following the process described in steps (a)-(h) described herein, wherein "DCQA" refers to "dicaffeoylquinic acid". The total of the target compounds was 93.2% pure based on UV absorbance at 210 nm.

[0019] Fig.11 The preparation of Stevia biomass is shown. The amounts of steviol glycosides and caffeoylquinic acid in the initial extract are shown in the loading flow-through, washing (first elution), second elution and column regeneration.

[0020] Repeated use of reference characters in the specification and drawings is intended to represent the same or analogous features or elements of the present disclosure, even when the numerals are increased by 100 between each figure (e.g., Figure 1 The drying operation 120 in Figure 2 4). It should be appreciated that numerous other modifications and examples can be devised by those skilled in the art, which fall within the scope and spirit of the principles of the present disclosure. DETAILED DESCRIPTION

[0021] Reference will now be made in detail to certain embodiments of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. Although the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0022] The present disclosure as a whole relates to methods for preparing compositions from plant biomass, the compositions comprising monocaffeoylquinic acid (e.g., chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid) and dicaffeoylquinic acid (e.g., 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid) and at least one of their salts. Since monocaffeoylquinic acid and dicaffeoylquinic acid can be considered as weak acids, they can each be present in at least one of their conjugate acid form, conjugate base form (e.g., in the form of their salts), and mixed conjugate acid-conjugate base form, wherein a portion (e.g., molar fraction) of the compound is present in the conjugate acid form and another portion is present in the conjugate base form. The fraction of the conjugate acid form to the conjugate base form of monocaffeoylquinic acid and dicaffeoylquinic acid will depend on a variety of factors, including the pKa of each compound and the pH of the composition.

[0023] Examples of salts of monocaffeoylquinic acid and dicaffeoylquinic acid include, but are not limited to, quaternary ammonium salts, sodium salts, potassium salts, lithium salts, magnesium salts, and calcium salts of caffeic acid, monocaffeoylquinic acid, dicaffeoylquinic acid, and the like.

[0024] Exemplary plant sources of compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof

[0025] Compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts can be prepared and / or isolated from plant sources, including but not limited to plants, such as leaves and stems of plants. Table 1 provides a variety of plant genera that are examples of plant sources that may contain monocaffeoylquinic acid, dicaffeoylquinic acid and their salts and can be used as biomass to prepare compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts.

[0026] Table 1 .

[0027]

[0028]

[0029]

[0030]

[0031] In some aspects, the composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts can be isolated from a plant source, such as those shown in Table 1. Examples of commercially available plant sources from which the composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts can be isolated include mate (Ilex paraguariensis), stevia, coffee, tea, chicory and globe artichoke. Some plant sources can produce a composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts, which is rich in one or more of monocaffeoylquinic acid and dicaffeoylquinic acid. For example, a composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts isolated from a mate plant can be rich in dicaffeoylquinic acid. In other aspects, the composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof isolated from a dicaffeoylquinic acid-rich yerba mate plant may comprise a combination of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more, 60% or more, 70% or more, or 80% or more, or 90% or more of one or more of 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, and salts thereof.

[0032] An example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0033] (a) contacting yerba mate biomass with an aqueous composition to obtain an initial extract;

[0034] (b) removing solids from the initial extract to obtain a second initial extract;

[0035] (c) adjusting the volume of the second initial extract with the aqueous composition to obtain an adjusted second initial extract;

[0036] (d) chromatographically separating the adjusted second initial extract on an ion exchange chromatography stationary phase;

[0037] (e) eluting the ion exchange chromatography stationary phase to obtain a first eluate comprising a solvent;

[0038] (f) removing the solvent to form a concentrate; and

[0039] (g) performing at least one of decolorization and desalination on the concentrate to obtain at least one of a filtrate and a retentate.

[0040] An example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0041] (a) contacting yerba mate biomass with an aqueous composition to obtain an initial extract;

[0042] (b) removing solids from the initial extract to obtain a second initial extract;

[0043] (c) adjusting the volume of the second initial extract with the aqueous composition to obtain an adjusted second initial extract;

[0044] (d) chromatographic separation of the adjusted initial extract on an ion exchange chromatography stationary phase;

[0045] (e) eluting the ion exchange stationary phase to obtain a first eluate comprising a solvent;

[0046] (f) removing the solvent to form a concentrate;

[0047] (g) performing at least one of decolorization and desalination on the concentrate to obtain at least one of a filtrate and a retentate; and

[0048] (h) drying at least one of the filtrate and the retentate to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof.

[0049] Step (a) of the method described herein involves contacting yerba mate biomass with an aqueous composition to obtain an initial extract comprising at least one of mono- and dicaffeoylquinic acids and salts thereof (e.g., quaternary ammonium, sodium, potassium, lithium, magnesium, and calcium salts).

[0050] The aqueous composition may contain water and does not contain any co-solvent, such as an organic solvent. However, in addition to water, the aqueous composition may also contain a co-solvent. Suitable co-solvents include organic solvents, such as (C1-C4)alkanols and mixtures of (C1-C4)alkanols. By "(C1-C4)alkanol" is meant an alcohol of the formula (C1-C4)alkyl-OH, wherein "alkyl" refers to a straight and branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl and tert-butyl, so that the resulting (C1-C4)alkanol is methanol, ethanol, n-propyl alcohol, n-butyl alcohol, isopropyl alcohol, isobutyl alcohol, sec-butyl alcohol and tert-butyl alcohol. The proportion of an organic solvent such as a (C1-C4)alkanol or a mixture of (C1-C4)alkanols can be any suitable proportion, such that the aqueous composition can comprise up to about 30%, up to about 40%, up to about 50%, or up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to 100% by volume of the organic solvent, with the remainder being water, except when the aqueous composition comprises 100% by volume of the organic solvent; or from about 30% to about 100%, from about 50% to about 100%, from about 60% to about 90%, from about 30% to about 60%, from about 40% to about 60%, from about 30% to about 50%, from about 40% to about 50%, or about 50% by volume of the organic solvent, with the remainder being water.

[0051] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0052] As used herein, the term "mate biomass" generally refers to any and all parts of a mate plant, such as Ilex paraguariensis, including leaves, stalks, stems, tips, roots, etc., of the mate plant. The mate biomass can be in any suitable form, including a comminuted form obtained by, for example, chopping the mate biomass before and / or during contact with the aqueous composition. For example, the mate biomass can be comminuted in a suitable container, and the aqueous composition can be added to the comminuted mate biomass, thereby "contacting" the mate biomass. The comminuted mate biomass can then optionally be further comminuted in a suitable container. Alternatively, the mate biomass can be placed in a suitable container, the aqueous composition can be added thereto, thereby "contacting" the mate biomass, and the resulting composition can be comminuted.

[0053] The yerba mate biomass may be stirred, sonicated, or otherwise agitated prior to and / or during contacting to maximize extraction of, among other things, the at least one of caffeic acid, monocaffeoylquinic acid, and dicaffeoylquinic acid, and salts thereof.

[0054] The initial extract may be applied as is to step (c), or the bulk solids and / or plant solids present may be removed in step (b) of the process described herein, such as ground yerba mate plant leaves, stalks, tops, roots, etc. When step (b) is performed, a second initial extract is obtained.

[0055] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that does not substantially retain at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0056] Step (c) of the method described herein involves adjusting the volume of the initial extract or the second initial extract with the first aqueous composition or the second aqueous composition, respectively, to obtain an adjusted initial extract or an adjusted second initial extract. The first aqueous composition and the second aqueous composition may be different or identical. The adjusted initial extract or the adjusted second initial extract may be filtered at this time, or it may be applied to step (d) as is. The initial extract or the second initial extract may be filtered using any suitable filtering method, including gravity filtration or vacuum filtration through any suitable filter, as long as the filter does not substantially retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, etc.

[0057] The volume of the initial extract or the second initial extract can be adjusted with a sufficient amount of an aqueous composition (e.g., water) to obtain an adjusted initial extract or an adjusted second initial extract to, among other things, increase the binding of the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to the ion exchange chromatography column used in step (d) of the method described herein relative to the unadjusted initial extract or the unadjusted second initial extract.

[0058] The volume of the initial extract or the second initial extract can be adjusted to adjust, among other things, the amount of organic solvent (when present) in the initial extract or the second initial extract. The volume of the initial extract or the second initial extract can be adjusted so that the adjusted initial extract or the adjusted second initial extract comprises less than about 60% by volume, less than about 50% by volume, less than about 40% by volume, less than about 30% by volume, less than about 20% by volume, less than about 10% by volume, less than about 5% by volume, less than about 1% by volume, or even about 0% by volume of organic solvent, the remainder being water; or about 0% by volume to about 40% by volume, about 0% by volume to about 30% by volume, about 10% by volume to about 40% by volume, about 10% by volume to about 30% by volume, about 20% by volume to about 40% by volume, about 30% by volume to about 40% by volume, or about 35% by volume of organic solvent, the remainder being water.

[0059] Step (d) of the methods described herein involves chromatographic separation of the adjusted initial extract or the second initial extract on an ion exchange stationary phase (eg, a weak anion exchange stationary phase). The chromatographic separation may be performed in any suitable manner, including in batch mode or using a chromatography column. Chromatographic separation can be performed using an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol) as an eluent (e.g., an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water), leaving at least one of caffeic acid, monocaffeoylquinic acid, and dicaffeoylquinic acid and salts thereof adsorbed on a weak ion exchange chromatography column while eluting other compounds including caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, and rutin (also known as rutin, quercetin-3-O-rutinoside, and sophora flavescens)

[0060]

[0061] and isomers thereof. Step (d) of the method described herein can reduce the concentration of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers to a concentration of less than 1 mass %, less than 0.5 mass %, less than 0.1 mass %, less than 0.05 mass %, less than 0.01 mass %, or less than 0.001 mass %. Therefore, the present disclosure contemplates a mate tea extract comprising less than 0.1% by mass of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers. The present disclosure also contemplates a yerba mate extract comprising less than 0.5% by mass of each of caffeine, quercetin, hyperoside, astragaloside, styloside, sophorafollicularis, rutin, and rutin isomers, and less than about 1% by mass of a combination of caffeine, quercetin, hyperoside, astragaloside, styloside, sophorafollicularis, rutin, and rutin isomers. The present disclosure also contemplates a yerba mate extract that is effectively free of at least one of caffeine, quercetin, hyperoside, astragaloside, styloside, sophorafollicularis, rutin, and rutin isomers (e.g., free of caffeine, free of quercetin, free of hyperoside, free of astragaloside, free of styloside, free of sophorafollicularis, free of rutin, free of rutin isomers, and / or free of caffeine, rutin, and rutin isomers).

[0062] The ion exchange stationary phase is non-limiting and can be any suitable ion exchange chromatography stationary phase. Examples of suitable ion exchange chromatography stationary phases include High flow resin, DEAE DEAE A25 resin, (FPA 53, FPA 55, CG-50I, IRC-50, IRC-50S and IRP-64), DIAION WA10 and CCR-3.

[0063] Prior to chromatographic separation of the conditioned initial extract or the conditioned second initial extract, the ion exchange chromatography stationary phase may optionally be preconditioned with an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol), such as an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water. For example, a weak ion exchange chromatography column may be preconditioned with about 2 or more bed volumes (BV) at a flow rate of about 2 BV / h.

[0064] The pH of the weak ion exchange chromatography column can optionally be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract. For example, the pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography with any suitable acid (e.g., hydrochloric acid) such that the pH of the weak ion exchange chromatography column (e.g., the pH of the resin / stationary phase) is a pH of less than about 10, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less; or a pH of about 2 to about 10, about 3 to about 8, about 5 to about 9, about 2 to about 6, about 3 to about 4, or about 3 to about 6. The pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract, before or after optionally preconditioning the column with an aqueous composition comprising (C1-C4).

[0065] After preconditioning and / or adjusting the pH of the weak ion exchange chromatography column, the adjusted initial extract or the adjusted second initial extract can be loaded onto the column at any suitable rate, such as at a rate greater than 2 BV / h (bed volume / hour). After loading the adjusted initial extract or the adjusted second initial extract, the column can be washed with any suitable volume of an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% to about 40% by volume, about 10% to about 30% by volume, about 20% to about 40% by volume, about 30% to about 40% by volume, or about 35% by volume of (C1-C4)alkanol, the remainder being water) at any suitable rate, such as at a rate greater than 2 BV / h. The volume of the aqueous composition comprising the (C1-C4)alkanol may be removed because it will contain, among other things, caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, rutin and rutin isomers.

[0066] Step (e) of the method described herein involves eluting the adsorbed at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from a weak ion exchange chromatography column to obtain a first eluent comprising the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. The elution is performed under any conditions suitable for eluting the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column.

[0067] Examples of suitable conditions for eluting at least one of the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column include eluting the column with any suitable volume of a solution comprising a salt (e.g., sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, etc.). Examples of solutions comprising a salt include solutions comprising at least one salt (e.g., about 5% by weight to about 25% by weight, about 15% by weight to about 20% by weight, or about 5% by weight to about 10% by weight of a salt) dissolved in an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% by volume to about 60% by volume, about 20% by volume to about 50% by volume, about 30% by volume to about 55% by volume, about 40% by volume to about 60% by volume, or about 50% by volume of the (C1-C4)alkanol).

[0068] Another example of suitable conditions for eluting at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column includes eluting the column with any suitable volume of a solution comprising an acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.). Examples of solutions comprising an acid include solutions comprising hydrochloric acid, etc. and optionally an acid solution comprising an aqueous composition comprising about 10% to about 60% by volume, about 20% to about 50% by volume, about 30% to about 55% by volume, about 40% to about 60% by volume, or about 50% by volume of (C1-C4)alkanol.

[0069] The first eluate collected from the elution step, comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, is collected and subsequently concentrated by removing the solvent in any suitable manner (e.g., to remove water and (C1-C4)alkanol) to provide a concentrate comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Solvent removal can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere). Without wishing to be bound by any particular theory, it is believed that solvent removal under an inert atmosphere can reduce the formation of highly colored polymeric substances naturally present in yerba mate biomass or formed at one or more of the steps described herein.

[0070] The first eluent containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts contains a solvent. The solvent may be removed to dryness in step (f), or may be removed to the extent that a certain volume of the aqueous composition containing (C1-C4)alkanol remains as solvent (e.g., about 50%, about 40%, about 30%, about 20%, about 10% or about 5% of the initial total volume of the eluent) to form a concentrate, but the ratio of the components constituting the aqueous composition containing (C1-C4)alkanol may be different from or may be equivalent to the ratio of the components constituting the aqueous composition containing (C1-C4)alkanol used to elute the adsorbed caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Alternatively, the solvent in the eluate comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be removed to an extent that a volume of an aqueous composition comprising (C1-C4)alkanol remains, wherein the aqueous composition comprising (C1-C4)alkanol comprises less than about 10% by volume, less than about 5% by volume, less than about 2% by volume or less than about 1% by volume of (C1-C4)alkanol.

[0071] Suitable conditions for removing solvent from an eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a concentrate comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include blowing an inert gas (e.g., nitrogen) on the surface of the eluent. The eluent can be heated while blowing nitrogen, or it can be heated at room temperature (e.g., 25°C). Other conditions for removing the solvent in the eluent include applying a vacuum to a container containing the eluent. A vacuum can be applied with the eluent at room temperature or while heating the container. Other conditions for removing the solvent in the eluent include passing the eluent through a wiped film evaporator or a stirred thin film evaporator.

[0072] At this time, the pH of the concentrate can be adjusted to obtain a concentrate adjusted by pH, but adjusting the pH at this time is optional. For example, the pH of the concentrate can be adjusted to a pH that protects at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from degradation. Suitable pH includes a pH of less than about 6, less than about 5, less than about 4, less than about 3 or less than about 2; such as a pH of about 2 to about 6, about 2 to about 5, about 2 to about 4, about 3 to about 5 or about 3.5. The pH of the concentrate can be adjusted using any suitable acid or base. When using acid, the acid can be hydrochloric acid, etc.

[0073] The concentrate or pH-adjusted concentrate may be used as is in the methods or removal step (f) described herein, or they may be filtered. The concentrate or pH-adjusted concentrate may be filtered using any suitable filter (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filter, polyethersulfone filter, glass fiber filter, diatomaceous earth pad, etc. In some cases, the pH-adjusted concentrate may be filtered through a polymer membrane such as a polyethersulfone (PES) filter having, for example, a pore size of 0.2 μm, or a pleated (flat membrane, vacuum filtration) or pleated PES membrane, depending on the volume of the concentrate or pH-adjusted concentrate.

[0074] The concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, whether pH adjusted, filtered, or both pH adjusted and filtered, can be used directly in the drying step (h), or can be used in step (g) to desalt / decolorize the highly colorable concentrate (in any order, including desalting, then decolorizing; decolorizing, then desalting; decolorizing, but not desalting; or desalting, but not decolorizing). Desalting / decolorizing can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere). Without wishing to be bound by any particular theory, it is believed that performing the one or more steps under an inert atmosphere can reduce the formation of highly colored polymeric species that are naturally present in the yerba mate biomass or formed at one or more of the steps described herein.

[0075] Whether the concentrate is pH adjusted, filtered, or both pH adjusted and filtered, it can be decolorized by any suitable means, including ultrafiltration (e.g., filtration through a molecular weight cutoff membrane, size exclusion chromatography, or gel permeation). The filtrate is obtained by decolorization. Ultrafiltration also achieves decolorization of a concentrate that can be highly colored, among other things. Without wishing to be bound by any particular theory, it is believed that ultrafiltration can remove highly colored polymeric substances that are naturally present in the yerba mate biomass or that are formed at one or more of the steps described herein.

[0076] The filtrate from decolorization can be used for drying step (h), or it can be desalted in step (g). Alternatively, no matter concentrate is pH-adjusted, filtered or both pH-adjusted and filtered, all can be desalted without first decolorizing. In any case, desalination can be achieved using nanofiltration membranes and hydrophobic resins. Those skilled in the art will recognize that when using nanofiltration membranes and hydrophobic resins, permeate is removed and retentate is retained. In one example, desalination can be achieved using hydrophobic resins (e.g., porous polydivinylbenzene / ethylvinylbenzene matrix, such as SEPABEADS TM SP70) is achieved, wherein a pH-adjusted concentrate containing less than about 20% by volume of (C1-C4)alkanol (e.g., an acidified concentrate having a pH of less than about 2) is loaded. The resin is then washed with a dilute alcohol (e.g., less than about 10% by volume of (C1-C4)alkanol, the remainder being water having a pH of less than about 2), and then eluted with an aqueous solution of an aqueous composition containing about 70% by volume of (C1-C4)alkanol to obtain a desalted second eluate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0077] If desalting is performed before decolorization in step (g), the solvent in the permeate from the desalting step can be removed to the extent that a certain volume of the aqueous composition comprising (C1-C4)alkanol remains as solvent (e.g., about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the initial total volume of the eluate) to form a first desalted concentrate. Alternatively, the solvent in the permeate from the desalting step can be removed until a certain volume of the aqueous composition comprising (C1-C4)alkanol remains to obtain a second desalted concentrate, wherein the aqueous composition comprising (C1-C4)alkanol comprises less than about 10% by volume, less than about 5% by volume, less than about 2% by volume, or less than about 1% by volume of (C1-C4)alkanol. The first desalination concentrate may also have the attributes of the second desalination concentrate such that the first desalination concentrate also has less than about 10 vol%, less than about 5 vol%, less than about 2 vol%, or less than about 1 vol% (C1-C4) alkanol.

[0078] Suitable conditions for removing solvent from a permeate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a first / second desalted concentrate comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include sparging an inert gas (e.g., nitrogen) on the surface of the eluent. The permeate may be heated while sparging nitrogen, or it may be heated at room temperature (e.g., 25° C.). Other conditions for removing the solvent in the eluent include applying a vacuum to a container containing the permeate. A vacuum may be applied to the permeate at room temperature or while heating the container. Other conditions for removing the solvent in the permeate include passing the permeate through a wiped film evaporator or an agitated thin film evaporator.

[0079] In another example, the concentrate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be filtered through filter paper to obtain a first filtrate, the first filtrate is ultrafiltered to obtain a second filtrate, and the second filtrate is nanofiltered using a nanofiltration membrane to obtain a first retentate, or the second filtrate is eluted through a hydrophobic resin to obtain a desalted second eluate. In another example, the concentrate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be filtered through filter paper to obtain a first filtrate, the first filtrate is nanofiltered using a nanofiltration membrane to obtain a third retentate, or the first filtrate is eluted through a hydrophobic resin to obtain a desalted second eluate, and the third retentate or the desalted second eluate is ultrafiltered to obtain a third filtrate.

[0080] As described herein, the eluate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts may be concentrated to dryness, or may be concentrated to a degree that a volume of an aqueous composition comprising a (C1-C4)alkanol is retained. If the eluate is concentrated to dryness, the dried material may be reconstituted using, for example, an aqueous composition comprising a (C1-C4)alkanol. The reconstituted material may then be filtered as described herein to perform at least one of desalting and decolorization, among other things.

[0081] The method described herein may include step (h) involving drying the first retentate, the desalted second eluate or the third filtrate to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof. The first retentate, the desalted second eluate or the third filtrate may be dried in any suitable manner, including by freeze drying or spray drying.

[0082] Figure 1 4 show methods using yerba mate and stevia as exemplary plant sources. Figure 1 Flow chart of method 100 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. In operation 102, mate biomass is contacted with an aqueous composition comprising 50% ethanol / water in a suitable container (e.g., a glass jar) for 1 hour (300 g of mate biomass is placed in 1.5 L of solvent) to obtain an initial extract. In operation 104, the initial extract is filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 54 low ash filter paper. In operation 106, the volume of the filtered initial extract is adjusted with an aqueous composition (in this case, water) to obtain an adjusted filtered initial extract containing a lower proportion of ethanol, in this case 35% by volume of ethanol. In operation 108, the adjusted filtered initial extract can be re-filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 44 low ash filter paper. In operation 110, the conditioned filtered initial extract is subjected to chromatographic separation on an ion exchange chromatography stationary phase. FPA 53 resin is filled in a glass column. The resin is preconditioned with 35% ethanol (2BV, 2BV / h). The adjusted filtered initial extract is loaded (2BV / h) onto the resin, and the loaded permeate is removed. The resin is washed with 35% ethanol (4BV, 2BV / h), and the washed permeate is removed. At least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts is eluted with 50% ethanol / water, 10% FCC sodium chloride (4BV, 0.5BV / h), and the permeate is retained. Optionally, water (4BV, 2BV / h) is used to regenerate the column / resin. In operation 112, the eluent / permeate is concentrated to form a concentrate. In this case, nitrogen is sparged on the top of the eluent / permeate for 2 days until the volume is about one-third of the initial volume of the eluent / permeate and / or ethanol is less than 1% of the eluent / permeate, thereby obtaining a concentrate. In operation 114, the concentrate is acidified to a pH of about 3.5 and then filtered through Whatman 44 filter paper on a Buchner funnel and then through a 0.2 μm polyethersulfone (PES) filter. In operation 116, the concentrate is filtered using a molecular weight cutoff membrane (MWCO; e.g., a MWCO membrane that removes materials with a molecular weight greater than 10 kDA, such as a 3 kDa NP010) to decolorize the filtered concentrate, among other things, to decolorize the filtered concentrate and obtain a permeate. In operation 118, the permeate is passed through a nanofiltration membrane (e.g., XN45 membrane), and then the retentate is dried in operation 120 to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof.

[0083] Figure 2Flow chart of method 200 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. In operation 202, mate biomass is contacted with an aqueous composition comprising 50% ethanol / water in a suitable container (e.g., a glass jar) for 1 hour (300 g of mate biomass is placed in 1.5 L of solvent) to obtain an initial extract. In operation 204, the initial extract is filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 54 low ash filter paper. In operation 206, the volume of the filtered initial extract is adjusted with an aqueous composition (in this case, water) to obtain an adjusted filtered initial extract containing a lower proportion of ethanol, in this case 35% by volume of ethanol. In operation 208, the adjusted filtered initial extract can be re-filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 44 low ash filter paper. In operation 210, the conditioned filtered initial extract is subjected to chromatographic separation on an ion exchange chromatography stationary phase. FPA 53 resin is filled in a glass column. The resin is preconditioned with 35% ethanol (2BV, 2BV / h). The adjusted filtered initial extract is loaded (2BV / h) onto the resin, and the loaded permeate is removed. The resin is washed with 35% ethanol (4BV, 2BV / h), and the washed permeate is removed. At least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts is eluted with 50% ethanol / water, 10% FCC sodium chloride (4BV, 0.5BV / h), and the permeate is retained. Optionally, water (4BV, 2BV / h) is used to regenerate the column / resin. In operation 212, the eluent / permeate is concentrated to form a concentrate, wherein the volume is about one-third of the initial volume of the eluent / permeate and / or ethanol is less than 1% of the eluent / permeate, thereby obtaining a concentrate. In operation 214, the concentrate is acidified to a pH of about 1 and then filtered through Whatman 44 filter paper on a Buchner funnel and then through a 0.2 μm polyethersulfone (PES) filter. In operation 218, a hydrophobic resin (e.g., a porous polydivinylbenzene / ethylvinylbenzene matrix such as SEPABEADS TM The concentrate is desalted at step SP70, and the solvent in the retentate is removed in operation 217. In operation 216, the retentate is desalted using a molecular weight cutoff membrane (MWCO; for example, a MWCO membrane that removes materials with a molecular weight greater than 10 kDA, such as 3 kDa NP010) decolorizes the desalted concentrate to, among other things, decolorize the filtered concentrate and obtain a permeate, which is then dried in operation 220 to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0084] Another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0085] (i) contacting yerba mate biomass with an aqueous composition to obtain an initial extract;

[0086] (ii) removing solids from the initial extract to obtain a second initial extract;

[0087] (iii) contacting the second initial extract with acidified ethyl acetate to obtain an acidic ethyl acetate extract;

[0088] (iv) neutralizing the acidic ethyl acetate extract to obtain a neutralized ethyl acetate and an aqueous extract;

[0089] (v) decolorizing the aqueous extract to obtain a decolorized aqueous extract; and

[0090] (vi) drying the decolorized aqueous extract to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0091] Steps (i), (ii) and (vi) are performed as described herein for steps (a), (b) and (h). Step (v) is similar to the filtration step (g), except that step (v) only involves a decolorization process, such as ultrafiltration, which includes filtration through a molecular weight cut-off membrane, size exclusion chromatography and gel permeation, as described herein. Therefore, the disclosure about steps (a), (b), (g) and (h) applies to steps (i), (ii), (v) and (vi).

[0092] Step (i) of the method described herein involves contacting yerba mate biomass with an aqueous composition to obtain an initial extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0093] The aqueous composition may include water and does not contain any cosolvent, such as an organic solvent. However, in addition to water, the aqueous composition may also include a cosolvent. Suitable cosolvents include organic solvents, such as (C1-C4) alkanols and mixtures of (C1-C4) alkanols. The ratio of an organic solvent such as (C1-C4) alkanols or mixtures of (C1-C4) alkanols may be any suitable ratio so that the aqueous composition may include up to about 30% by volume, up to about 40% by volume, up to about 50% by volume or up to about 60% by volume of an organic solvent, the remainder being water; or about 30% by volume to about 60% by volume, about 40% by volume to about 60% by volume, about 30% by volume to about 50% by volume, about 40% by volume to about 50% by volume or about 50% by volume of an organic solvent, the remainder being water.

[0094] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0095] The yerba mate biomass may be stirred, sonicated, or otherwise agitated prior to and / or during the contacting of step (i) to maximize extraction of, among other things, the at least one of caffeic acid, monocaffeoylquinic acid, and dicaffeoylquinic acid, and salts thereof.

[0096] The initial extract may be applied as is to step (iii), or the bulk solids and / or plant solids present may be removed in step (ii) of the process described herein, such as ground yerba mate plant leaves, stalks, tops, roots, etc. When step (ii) is performed, a second initial extract is obtained.

[0097] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that does not substantially retain at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0098] Prior to performing step (iii), the pH of the initial or second initial extract may optionally be adjusted to a pH between about 4 and about 7 with a suitable acid (e.g., hydrochloric acid, etc.) or a suitable base (e.g., sodium hydroxide). The pH-adjusted initial or second initial extract is then extracted with ethyl acetate that has not been pre-acidified as described herein. Without wishing to be bound by any particular theory, it is believed that when the pH of the initial or second initial extract is adjusted to between about 4 and about 7, it is possible to extract certain impurities into ethyl acetate while retaining the compounds of interest (e.g., caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts) in the aqueous layer.

[0099] Step (iii) of the method described herein involves contacting the first or second initial extract with acidified ethyl acetate to obtain an acidic ethyl acetate extract. Acidified ethyl acetate can be prepared in any suitable manner, including by adding any suitable acid, including hydrochloric acid, sulfuric acid and glacial acetic acid (e.g., 0.01%-1% volume / volume). The acidic ethyl acetate extract is washed with water (e.g., three times with 1:1 volume / volume of water). Under these conditions, at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid will be substantially in their conjugate acid form and will substantially reside in the acidic ethyl acetate layer formed when the acidic ethyl acetate extract is washed with water. The aqueous layer is removed, and the acidic ethyl acetate extract is continued to step (iv).

[0100] Step (iii) of the methods described herein can be performed in other suitable ways, including using ethyl acetate that is not pre-acidified as described herein (e.g., by pre-washing with glacial acetic acid), but rather pre-acidified by adjusting the pH of the initial or second initial extract with a suitable acid (e.g., hydrochloric acid, etc.), and then extracting the pH-adjusted initial or second initial extract with ethyl acetate that is not pre-acidified. Regardless of the acid used to adjust the pH of the initial extract or the second initial extract, the pH of the initial extract or the second initial extract is adjusted to about 4 or less, 3 or less, about 2 or less, or about 1 or less. The aqueous layer is removed, and the resulting acidic ethyl acetate extract is continued to step (iv).

[0101] Step (iv) of the method described herein involves neutralizing the acidic ethyl acetate extract to obtain a neutralized ethyl acetate and an aqueous extract. This is achieved in any suitable manner, including washing the acidic ethyl acetate extract with water (e.g., washing three times with 1:1 volume / volume water), the water comprising a suitable base, such as sodium hydroxide, potassium hydroxide, etc. and combinations thereof. Under these conditions, the at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid will be substantially in the form of their conjugate bases and will reside substantially in the aqueous layer formed when the acidic ethyl acetate extract is washed with water comprising a suitable base.

[0102] In an alternative optional step (iv-a) of step (iv), the acidic ethyl acetate extract obtained from step (iii) may be optionally removed, even to dryness. Any solids remaining may be reconstituted with pH neutral water (e.g., deionized water), the pH of the water may then be adjusted to about 3 to about 7; or the solids remaining may be reconstituted with water having a pH of about 3 to about 7.

[0103] The aqueous extract comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts (whether they are from step (iv) or from step (iv-a)) can then be used in step (v) to achieve, among other things, decolorization of the aqueous extract, which may be highly colored. Decolorization can be achieved by any suitable method, including ultrafiltration (e.g., by molecular weight cutoff membrane filtration, size exclusion chromatography or gel permeation). The filtrate is obtained by decolorization. Ultrafiltration also achieves, among other things, decolorization of a concentrate that may be highly colored. Without wishing to be bound by any particular theory, it is believed that ultrafiltration can remove highly colored polymeric substances that are naturally present in yerba mate biomass or formed at one or more steps in the steps described herein.

[0104] Another example of a modification of the method described herein comprising steps (i)-(vi) (including alternative optional step (iv-a)) comprises a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0105] contacting yerba mate biomass with an aqueous composition to obtain an initial extract;

[0106] removing solids from the initial extract to obtain a second initial extract;

[0107] adjusting the pH of the second initial extract to a pH of about 4 to about 7 to obtain a first pH adjusted second initial extract;

[0108] contacting the first pH adjusted second initial extract with ethyl acetate to obtain a first ethyl acetate extract and a second aqueous extract;

[0109] adjusting the pH of the second aqueous extract to a pH of less than 2 to obtain a pH-adjusted second aqueous extract;

[0110] contacting the pH adjusted second aqueous extract with ethyl acetate to obtain a second ethyl acetate extract;

[0111] removing ethyl acetate from the second ethyl acetate extract to obtain a purified composition;

[0112] reconstituting the crude composition with water to obtain a third aqueous extract; and

[0113] The third aqueous extract is decolorized to obtain a decolorized aqueous extract.

[0114] A "purified composition" will contain the compound of interest (e.g., at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts) and will be purified relative to at least the initial extract and the second initial extract in that the "purified composition" will be free of certain impurities in the initial extract and the second initial extract, but will contain highly colored polymeric substances that are naturally present in the yerba mate biomass or that are formed at one or more of the steps described herein and that are removed in the decolorization step.

[0115] Yet another example of a modification of the method described herein comprising steps (i)-(vi) (including alternative optional step (iv-a)) comprises a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0116] contacting yerba mate biomass with an aqueous composition to obtain an initial extract;

[0117] removing solids from the initial extract to obtain a second initial extract;

[0118] adjusting the pH of the second initial extract to a pH of less than about 2 to obtain a second pH-adjusted second initial extract;

[0119] contacting the second pH adjusted second initial extract with ethyl acetate to obtain a third ethyl acetate extract;

[0120] neutralizing the third ethyl acetate extract to obtain a first neutralized ethyl acetate extract and a third aqueous extract; and

[0121] The third aqueous extract is decolorized to obtain a decolorized aqueous extract.

[0122] The method described herein may include step (vi) involving drying the decolorized aqueous extract to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof. The first retentate or the second retentate or the third filtrate may be dried in any suitable manner, including by freeze drying or spray drying.

[0123] Figure 3Flow chart of a method 300 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. In operation 302, mate biomass is contacted with an aqueous composition comprising 50% ethanol / water in a suitable container (e.g., a glass jar) for 1 hour (300 g of mate biomass is placed in 1.5 L of solvent) to obtain an initial extract. In operation 304, the initial extract is filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 54 low ash filter paper to remove solids from, for example, mate biomass. The filtrate from operation 304 is extracted by acidified ethyl acetate extraction in operation 306. After the at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid is extracted into acidified ethyl acetate, the acidified ethyl acetate is washed with water containing a suitable base such as sodium hydroxide, potassium hydroxide, etc. in operation 308 to obtain neutralized ethyl acetate and an aqueous extract. Under these conditions, at least one of caffeic acid, monocaffeoylquinic acid, and dicaffeoylquinic acid will be substantially in the form of their conjugate bases and will reside substantially in the aqueous layer formed when the acidic ethyl acetate extract is washed with water containing a suitable base. In operation 310, the aqueous layer is filtered to obtain a filtrate. In operation 316, the filtrate is filtered using a 3 kDa molecular weight cutoff membrane ( NP010; six diafiltrations) decolorize the filtrate to decolorize the aqueous extract, among other things, to obtain a decolorized aqueous extract. In operation 320, the decolorized aqueous extract is dried to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0124] Figure 4AFlow chart of a method 400 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. In operation 402, mate biomass is contacted with an aqueous composition comprising 50% ethanol / water in a suitable container (e.g., a glass jar) for 1 hour (300 g of mate biomass is placed in 1.5 L of solvent) to obtain an initial extract. In operation 404, the initial extract is filtered into a 4 L glass side arm flask using, for example, a ceramic Buchner funnel with Whatman 54 low ash filter paper to remove solids from, for example, mate biomass. The pH of the filtrate from operation 404 is adjusted to about 4 to about 7, and the filtrate is extracted with ethyl acetate in operation 408, while the compound of interest is retained in the aqueous layer. In operation 406, the pH of the aqueous layer is adjusted to less than 2, and the aqueous layer is extracted with ethyl acetate. After extracting the at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid into ethyl acetate, the ethyl acetate is removed to dryness to obtain a solid in operation 407. The solid is reconstituted with water, and the pH of the water is adjusted to about 3 to about 7. Under these conditions, the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid will be substantially in the form of their conjugate bases and will dissolve in water. In operation 410, the aqueous layer is filtered to obtain a filtrate. In operation 416, the filtrate is filtered using a 3 kDa molecular weight cutoff membrane ( NP010; six diafiltrations) to decolorize the filtrate to decolorize the aqueous extract, among other things, to obtain a decolorized aqueous extract. In operation 420, the decolorized aqueous extract is dried to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0125] An example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0126] (AA) contacting the plant biomass with an aqueous composition to obtain an initial extract;

[0127] (BB) removing solids from the initial extract to obtain a second initial extract;

[0128] (CC) adjusting the volume of the second initial extract with the aqueous composition to obtain an adjusted second initial extract;

[0129] (DD) chromatographically separating the adjusted second initial extract on an ion exchange chromatography stationary phase;

[0130] (EE) eluting the ion exchange chromatography stationary phase to obtain a first eluent comprising a solvent;

[0131] (FF) removing the solvent to form a concentrate; and

[0132] (GG) performing at least one of decolorization and desalting on the concentrate to obtain at least one of a filtrate and a retentate.

[0133] An example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0134] (AA) contacting the plant biomass with an aqueous composition to obtain an initial extract;

[0135] (BB) removing solids from the initial extract to obtain a second initial extract;

[0136] (CC) adjusting the volume of the second initial extract with the aqueous composition to obtain an adjusted second initial extract;

[0137] (DD) chromatographically separating the adjusted initial extract on an ion exchange chromatography stationary phase;

[0138] (EE) eluting the ion exchange stationary phase to obtain a first eluent comprising a solvent;

[0139] (FF) removing the solvent to form a concentrate;

[0140] (GG) performing at least one of decolorizing and desalting the concentrate to obtain at least one of a filtrate and a retentate; and

[0141] (HH) drying at least one of the filtrate and the retentate to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0142] Step (AA) of the methods described herein involves contacting the plant biomass with an aqueous composition to obtain an initial extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts (e.g., quaternary ammonium, sodium, potassium, lithium, magnesium and calcium salts).

[0143] The aqueous composition may contain water and does not contain any co-solvent, such as an organic solvent. However, in addition to water, the aqueous composition may also contain a co-solvent. Suitable co-solvents include organic solvents, such as (C1-C4)alkanols and mixtures of (C1-C4)alkanols. By "(C1-C4)alkanol" is meant an alcohol of the formula (C1-C4)alkyl-OH, wherein "alkyl" refers to a straight and branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl and tert-butyl, so that the resulting (C1-C4)alkanol is methanol, ethanol, n-propyl alcohol, n-butyl alcohol, isopropyl alcohol, isobutyl alcohol, sec-butyl alcohol and tert-butyl alcohol. The proportion of an organic solvent such as a (C1-C4)alkanol or a mixture of (C1-C4)alkanols can be any suitable proportion, such that the aqueous composition can comprise up to about 30%, up to about 40%, up to about 50%, or up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to 100% by volume of the organic solvent, with the remainder being water, except when the aqueous composition comprises 100% by volume of the organic solvent; or from about 30% to about 100%, from about 50% to about 100%, from about 60% to about 90%, from about 30% to about 60%, from about 40% to about 60%, from about 30% to about 50%, from about 40% to about 50%, or about 50% by volume of the organic solvent, with the remainder being water.

[0144] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0145] As used herein, the term "plant biomass" generally refers to any and all parts of a plant origin comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts, including leaves, stalks, stems, tops, roots, etc. of plant origin. Plant biomass can be in any suitable form, including a pulverized form obtained by, for example, chopping plant biomass before and / or during contact with an aqueous composition. For example, plant biomass can be pulverized in a suitable container, and an aqueous composition can be added to the pulverized plant biomass, thereby "contacting" the plant biomass. The pulverized plant biomass can then be optionally further pulverized in a suitable container. Alternatively, the plant biomass can be placed in a suitable container, an aqueous composition can be added thereto, thereby "contacting" the plant biomass, and the resulting composition can be pulverized.

[0146] The plant biomass may be stirred, sonicated, or otherwise agitated prior to and / or during contacting to, among other things, maximize extraction of the at least one of mono- and dicaffeoylquinic acids and salts thereof.

[0147] The initial extract may be applied as such to step (CC), or the bulk solids and / or plant solids present may be removed in step (BB) of the process described herein, such as comminuted plant leaves, stalks, tops, roots, etc. When step (BB) is performed, a second initial extract is obtained.

[0148] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that substantially does not retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0149] Step (CC) of the method described herein involves adjusting the volume of the initial extract or the second initial extract with the first aqueous composition or the second aqueous composition, respectively, to obtain an adjusted initial extract or an adjusted second initial extract. The first aqueous composition and the second aqueous composition may be different or the same. The adjusted initial extract or the adjusted second initial extract may be filtered at this point, or it may be applied to step (DD) as is. The initial extract or the second initial extract may be filtered using any suitable filtering method, including gravity filtration or vacuum filtration through any suitable filter, as long as the filter does not substantially retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0150] The volume of the initial extract or the second initial extract can be adjusted with a sufficient amount of an aqueous composition (e.g., water) to obtain an adjusted initial extract or an adjusted second initial extract to, among other things, increase the binding of the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to the ion exchange chromatography column used in step (DD) of the method described herein relative to the unadjusted initial extract or the unadjusted second initial extract.

[0151] The volume of the initial extract or the second initial extract can be adjusted to adjust, among other things, the amount of organic solvent (when present) in the initial extract or the second initial extract. The volume of the initial extract or the second initial extract can be adjusted so that the adjusted initial extract or the adjusted second initial extract comprises less than about 60% by volume, less than about 50% by volume, less than about 40% by volume, less than about 30% by volume, less than about 20% by volume, less than about 10% by volume, less than about 5% by volume, less than about 1% by volume, or even about 0% by volume of organic solvent, the remainder being water; or about 0% by volume to about 40% by volume, about 0% by volume to about 30% by volume, about 10% by volume to about 40% by volume, about 10% by volume to about 30% by volume, about 20% by volume to about 40% by volume, about 30% by volume to about 40% by volume, or about 35% by volume of organic solvent, the remainder being water.

[0152] Step (DD) of the methods described herein involves chromatographic separation of the adjusted initial extract or the second initial extract on an ion exchange stationary phase (eg, a weak anion exchange stationary phase). The chromatographic separation may be performed in any suitable manner, including in batch mode or using a chromatography column. Chromatographic separation can be performed using an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol) as an eluent (e.g., an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water), leaving at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof adsorbed on a weak ion exchange chromatography column while eluting other compounds including caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, and rutin (also known as rutin, quercetin-3-O-rutinoside, and sophora flavescens)

[0153]

[0154] and isomers thereof. Step (DD) of the method described herein can reduce the concentration of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers to a concentration of less than 1 mass %, less than 0.5 mass %, less than 0.1 mass %, less than 0.05 mass %, less than 0.01 mass %, or less than 0.001 mass %. Therefore, the present disclosure contemplates a plant extract comprising less than 0.1% by mass of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers. The present disclosure also contemplates plant extracts comprising less than 0.5% by mass of each of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers, and less than about 1% by mass of a combination of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers. The present disclosure also contemplates plant extracts that are effectively free of at least one of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers (e.g., free of caffeine, free of quercetin, free of hyperoside, free of astragalus glycoside, free of succinoside, free of sophora glycoside, free of rutin, free of rutin isomers, and / or free of caffeine, rutin, and rutin isomers).

[0155] The ion exchange stationary phase is non-limiting and can be any suitable ion exchange chromatography stationary phase. Examples of suitable ion exchange chromatography stationary phases include High flow resin, DEAE DEAE A25 resin, (FPA 53, FPA 55, CG-50I, IRC-50, IRC-50S and IRP-64), DIAION WA10 and CCR-3.

[0156] Prior to chromatographic separation of the conditioned initial extract or the conditioned second initial extract, the ion exchange chromatography stationary phase may optionally be preconditioned with an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol), such as an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water. For example, a weak ion exchange chromatography column may be preconditioned with about 2 or more bed volumes (BV) at a flow rate of about 2 BV / h.

[0157] The pH of the weak ion exchange chromatography column can optionally be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract. For example, the pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography with any suitable acid (e.g., hydrochloric acid) such that the pH of the weak ion exchange chromatography column (e.g., the pH of the resin / stationary phase) is a pH of less than about 10, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less; or a pH of about 2 to about 10, about 3 to about 8, about 5 to about 9, about 2 to about 6, about 3 to about 4, or about 3 to about 6. The pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract, before or after optionally preconditioning the column with an aqueous composition comprising (C1-C4).

[0158] After preconditioning and / or adjusting the pH of the weak ion exchange chromatography column, the adjusted initial extract or the adjusted second initial extract can be loaded onto the column at any suitable rate, such as at a rate greater than 2 BV / h (bed volume / hour). After loading the adjusted initial extract or the adjusted second initial extract, the column can be washed with any suitable volume of an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% to about 40% by volume, about 10% to about 30% by volume, about 20% to about 40% by volume, about 30% to about 40% by volume, or about 35% by volume of (C1-C4)alkanol, the remainder being water) at any suitable rate, such as at a rate greater than 2 BV / h. The volume of the aqueous composition comprising the (C1-C4)alkanol may be removed because it will contain, among other things, caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, rutin and rutin isomers.

[0159] Step (EE) of the method described herein involves eluting the adsorbed at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from a weak ion exchange chromatography column to obtain a first eluent comprising the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. The elution is performed under any conditions suitable for eluting the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column.

[0160] Examples of suitable conditions for eluting at least one of the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column include eluting the column with any suitable volume of a solution comprising a salt (e.g., sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, etc.). Examples of solutions comprising a salt include solutions comprising at least one salt (e.g., about 5% by weight to about 25% by weight, about 15% by weight to about 20% by weight, or about 5% by weight to about 10% by weight of a salt) dissolved in an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% by volume to about 60% by volume, about 20% by volume to about 50% by volume, about 30% by volume to about 55% by volume, about 40% by volume to about 60% by volume, or about 50% by volume of the (C1-C4)alkanol).

[0161] Another example of suitable conditions for eluting at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column includes eluting the column with any suitable volume of a solution comprising an acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.). Examples of solutions comprising an acid include solutions comprising hydrochloric acid, etc. and optionally an acid solution comprising an aqueous composition comprising about 10% to about 60% by volume, about 20% to about 50% by volume, about 30% to about 55% by volume, about 40% to about 60% by volume, or about 50% by volume of (C1-C4)alkanol.

[0162] The first eluate collected from the elution step comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts is collected and subsequently concentrated by removing the solvent in any suitable manner (e.g., to remove water and (C1-C4)alkanol) to provide a concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Solvent removal can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere). Without wishing to be bound by any particular theory, it is believed that solvent removal under an inert atmosphere can reduce the formation of highly colored polymeric substances naturally present in plant biomass or formed at one or more steps in the steps described herein.

[0163] The first eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts comprises a solvent. The solvent may be removed to dryness in step (FF), or may be removed to the extent that a certain volume of the aqueous composition comprising (C1-C4)alkanol remains as solvent (e.g., about 50%, about 40%, about 30%, about 20%, about 10% or about 5% of the initial total volume of the eluent) to form a concentrate, but the ratio of the components constituting the aqueous composition comprising (C1-C4)alkanol may be different from or may be equivalent to the ratio of the components constituting the aqueous composition comprising (C1-C4)alkanol used to elute the adsorbed monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Alternatively, the solvent in the eluate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be removed to an extent that a certain volume of aqueous composition comprising (C1-C4)alkanol remains, wherein the aqueous composition comprising (C1-C4)alkanol comprises less than about 10% by volume, less than about 5% by volume, less than about 2% by volume or less than about 1% by volume of (C1-C4)alkanol.

[0164] Suitable conditions for removing solvent from an eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include blowing an inert gas (e.g., nitrogen) on the surface of the eluent. The eluent can be heated while blowing nitrogen, or it can be heated at room temperature (e.g., 25°C). Other conditions for removing the solvent in the eluent include applying a vacuum to a container containing the eluent. A vacuum can be applied with the eluent at room temperature or while heating the container. Other conditions for removing the solvent in the eluent include passing the eluent through a wiped film evaporator or a stirred thin film evaporator.

[0165] At this time, the pH of the concentrate can be adjusted to obtain a concentrate adjusted by pH, but adjusting the pH at this time is optional. For example, the pH of the concentrate can be adjusted to a pH that protects at least one of the monocaffeoylquinic acid and the dicaffeoylquinic acid and their salts from degradation. Suitable pH includes a pH of less than about 6, less than about 5, less than about 4, less than about 3, or less than about 2; such as a pH of about 2 to about 6, about 2 to about 5, about 2 to about 4, about 3 to about 5, or about 3.5. The pH of the concentrate can be adjusted by using any suitable acid or base. When using an acid, the acid can be hydrochloric acid, etc.

[0166] The concentrate or pH adjusted concentrate may be used as is in the methods or removal step (FF) described herein, or they may be filtered. The concentrate or pH adjusted concentrate may be filtered using any suitable filter (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filter, polyethersulfone filter, glass fiber filter, diatomaceous earth pad, etc. In some cases, the pH adjusted concentrate may be filtered through a polymer membrane such as a polyethersulfone (PES) filter having, for example, a pore size of 0.2 μm, or a pleated (flat membrane, vacuum filtration) or pleated PES membrane, depending on the volume of the concentrate or pH adjusted concentrate.

[0167] The concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, whether pH adjusted, filtered, or both pH adjusted and filtered, can be used directly in the drying step (HH), or can be used in step (GG) to desalt / decolorize a highly colorable concentrate (in any order, including desalting, then decolorizing; decolorizing, then desalting; decolorizing, but not desalting; or desalting, but not decolorizing). Desalting / decolorizing can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere). Without wishing to be bound by any particular theory, it is believed that performing the one or more steps under an inert atmosphere can reduce the formation of highly colored polymeric substances that are naturally present in plant biomass or formed at one or more of the steps described herein.

[0168] Whether the concentrate is pH adjusted, filtered, or both pH adjusted and filtered, it can be decolorized by any suitable means, including ultrafiltration (e.g., by filtration through a molecular weight cut-off membrane, size exclusion chromatography, or gel permeation). The filtrate is obtained by decolorization. Ultrafiltration also achieves decolorization of highly colorable concentrates, among other things. Without wishing to be bound by any particular theory, it is believed that ultrafiltration can remove highly colored polymeric substances that are naturally present in plant biomass or formed at one or more steps in the steps described herein.

[0169] The filtrate from the decolorization can be used for the drying step (HH), or it can be desalted in step (GG). Alternatively, whether the concentrate is pH adjusted, filtered, or both pH adjusted and filtered, it can be desalted without first decolorizing. In any case, desalination can be achieved using nanofiltration membranes and hydrophobic resins. Those skilled in the art will recognize that when using nanofiltration membranes and hydrophobic resins, the permeate is removed and the retentate is retained. In one example, desalination can be achieved using a hydrophobic resin (e.g., a porous polydivinylbenzene / ethylvinylbenzene matrix, such as SEPABEADS TM SP70) is achieved, wherein a pH-adjusted concentrate containing less than about 20% by volume of (C1-C4)alkanol (e.g., an acidified concentrate having a pH of less than about 2) is loaded. The resin is then washed with a dilute alcohol (e.g., less than about 10% by volume of (C1-C4)alkanol, the remainder being water having a pH of less than about 2), and then eluted with an aqueous solution of an aqueous composition containing about 70% by volume of (C1-C4)alkanol to obtain a desalted second eluate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0170] If desalting is performed before decolorization in step (GG), the solvent in the permeate from the desalting step can be removed to the extent that a certain volume of the aqueous composition comprising (C1-C4)alkanol remains as solvent (e.g., about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the initial total volume of the eluate) to form a first desalted concentrate. Alternatively, the solvent in the permeate from the desalting step can be removed until a certain volume of the aqueous composition comprising (C1-C4)alkanol remains to obtain a second desalted concentrate, wherein the aqueous composition comprising (C1-C4)alkanol comprises less than about 10% by volume, less than about 5% by volume, less than about 2% by volume, or less than about 1% by volume of (C1-C4)alkanol. The first desalination concentrate may also have the attributes of the second desalination concentrate such that the first desalination concentrate also has less than about 10 vol%, less than about 5 vol%, less than about 2 vol%, or less than about 1 vol% (C1-C4) alkanol.

[0171] Suitable conditions for removing solvent from a permeate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a first / second desalted concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include sparging an inert gas (e.g., nitrogen) on the surface of the eluent. The permeate may be heated while sparging nitrogen, or it may be heated at room temperature (e.g., 25° C.). Other conditions for removing the solvent in the eluate include applying a vacuum to a container containing the permeate. A vacuum may be applied to the permeate at room temperature or while heating the container. Other conditions for removing the solvent in the permeate include passing the permeate through a wiped film evaporator or an agitated thin film evaporator.

[0172] In another example, the concentrate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be filtered through filter paper to obtain a first filtrate, the first filtrate is ultrafiltered to obtain a second filtrate, and the second filtrate is nanofiltered using a nanofiltration membrane to obtain a first retentate, or the second filtrate is eluted through a hydrophobic resin to obtain a desalted second eluate. In another example, the concentrate containing at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be filtered through filter paper to obtain a first filtrate, the first filtrate is nanofiltered using a nanofiltration membrane to obtain a third retentate, or the first filtrate is eluted through a hydrophobic resin to obtain a desalted second eluate, and the third retentate or the desalted second eluate is ultrafiltered to obtain a third filtrate.

[0173] As described herein, the eluate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts may be concentrated to dryness, or may be concentrated to a degree that a volume of an aqueous composition comprising a (C1-C4)alkanol is retained. If the eluate is concentrated to dryness, the dried material may be reconstituted using, for example, an aqueous composition comprising a (C1-C4)alkanol. The reconstituted material may then be filtered as described herein to perform at least one of desalting and decolorization, among other things.

[0174] The method described herein may include step (HH) involving drying the first retentate, the desalted second eluate or the third filtrate to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. The first retentate, the desalted second eluate or the third filtrate may be dried in any suitable manner, including by freeze drying or spray drying.

[0175] Another example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0176] (1) contacting plant biomass with an aqueous composition to obtain an initial extract;

[0177] (2) removing solids from the initial extract to obtain a second initial extract;

[0178] (3) contacting the second initial extract with acidified ethyl acetate to obtain an acidic ethyl acetate extract;

[0179] (4) neutralizing the acidic ethyl acetate extract to obtain a neutralized ethyl acetate and an aqueous extract;

[0180] (5) decolorizing the aqueous extract to obtain a decolorized aqueous extract; and

[0181] (6) Drying the decolorized aqueous extract to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0182] Steps (1), (2) and (6) are performed as described herein for steps (AA), (BB) and (HH). Step (5) is similar to the filtration step (GG), except that step (5) only involves a decolorization process, such as ultrafiltration, which includes filtration through a molecular weight cut-off membrane, size exclusion chromatography and gel permeation, as described herein. Therefore, the disclosure about steps (AA), (BB), (GG) and (HH) applies to steps (1), (2), (5) and (6).

[0183] Step (1) of the method described herein involves contacting the plant biomass with an aqueous composition to obtain an initial extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof.

[0184] The aqueous composition may include water and does not contain any cosolvent, such as an organic solvent. However, in addition to water, the aqueous composition may also include a cosolvent. Suitable cosolvents include organic solvents, such as (C1-C4) alkanols and mixtures of (C1-C4) alkanols. The ratio of an organic solvent such as (C1-C4) alkanols or mixtures of (C1-C4) alkanols may be any suitable ratio so that the aqueous composition may include up to about 30% by volume, up to about 40% by volume, up to about 50% by volume or up to about 60% by volume of an organic solvent, the remainder being water; or about 30% by volume to about 60% by volume, about 40% by volume to about 60% by volume, about 30% by volume to about 50% by volume, about 40% by volume to about 50% by volume or about 50% by volume of an organic solvent, the remainder being water.

[0185] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0186] The plant biomass may be stirred, sonicated, or otherwise agitated prior to and / or during the contacting of step (1) to, among other things, maximize the extraction of the at least one of mono- and dicaffeoylquinic acids and salts thereof.

[0187] The initial extract may be applied as is to step (3), or the bulk solids and / or plant solids present may be removed in step (2) of the process described herein, such as comminuted leaves, stalks, tops, roots, etc. When step (2) is performed, a second initial extract is obtained.

[0188] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that substantially does not retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0189] Prior to performing step (3), the pH of the initial or second initial extract may optionally be adjusted to a pH between about 4 and about 7 with a suitable acid (e.g., hydrochloric acid, etc.) or a suitable base (e.g., sodium hydroxide). The pH-adjusted initial or second initial extract is then extracted with ethyl acetate that has not been pre-acidified as described herein. Without wishing to be bound by any particular theory, it is believed that when the pH of the initial or second initial extract is adjusted to between about 4 and about 7, it is possible to extract certain impurities into ethyl acetate while retaining the compounds of interest (e.g., mono- and di-caffeoylquinic acids and their salts) in the aqueous layer.

[0190] Step (3) of the method described herein involves contacting the first or second initial extract with acidified ethyl acetate to obtain an acidic ethyl acetate extract. The acidified ethyl acetate can be prepared in any suitable manner, including by adding any suitable acid, including hydrochloric acid, sulfuric acid and glacial acetic acid (e.g., 0.01%-1% volume / volume). The acidic ethyl acetate extract is washed with water (e.g., three times with 1:1 volume / volume of water). Under these conditions, at least one of the monocaffeoylquinic acid and the dicaffeoylquinic acid will be substantially in their conjugate acid form and will substantially reside in the acidic ethyl acetate layer formed when the acidic ethyl acetate extract is washed with water. The aqueous layer is removed, and the acidic ethyl acetate extract is continued to step (4).

[0191] Step (3) of the methods described herein can be performed in other suitable ways, including using ethyl acetate that is not pre-acidified as described herein (e.g., by pre-washing with glacial acetic acid), but rather pre-acidified by adjusting the pH of the initial or second initial extract with a suitable acid (e.g., hydrochloric acid, etc.), and then extracting the pH-adjusted initial or second initial extract with ethyl acetate that is not pre-acidified. Regardless of the acid used to adjust the pH of the initial extract or the second initial extract, the pH of the initial extract or the second initial extract is adjusted to about 4 or less, 3 or less, about 2 or less, or about 1 or less. The aqueous layer is removed, and the resulting acidic ethyl acetate extract is continued with step (4).

[0192] Step (4) of the methods described herein involves neutralizing the acidic ethyl acetate extract to obtain a neutralized ethyl acetate and an aqueous extract. This is achieved in any suitable manner, including washing the acidic ethyl acetate extract with water (e.g., three times with 1:1 volume / volume water), the water comprising a suitable base, such as sodium hydroxide, potassium hydroxide, and the like, and combinations thereof. Under these conditions, the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid will be substantially in the form of their conjugate bases and will reside substantially in the aqueous layer formed when the acidic ethyl acetate extract is washed with water comprising a suitable base.

[0193] In an alternative optional step (4-a) of step (4), the acidic ethyl acetate extract obtained from step (3) may be optionally removed, even to dryness. Any solids remaining may be reconstituted with pH neutral water (e.g., deionized water), and the pH of the water may then be adjusted to about 3 to about 7; or the remaining solids may be reconstituted with water having a pH of about 3 to about 7.

[0194] Then the aqueous extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts (whether they are from step (4) or from step (4-a)) can be used in step (5) to achieve decolorization of the aqueous extract, which can be highly colored, among others. Decolorization can be achieved by any suitable method, including ultrafiltration (e.g., by molecular weight cut-off membrane filtration, size exclusion chromatography or gel permeation). The filtrate is obtained by decolorization. Among other things, ultrafiltration also achieves decolorization of a highly colored concentrate. Without wishing to be bound by any specific theory, it is believed that ultrafiltration can remove highly colored polymeric substances naturally present in plant biomass or formed at one or more steps in the steps described herein.

[0195] Another example of a modification of the method described herein comprising steps (1)-(6) (including alternative optional step (4-a)) includes a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0196] contacting the plant biomass with an aqueous composition to obtain an initial extract;

[0197] removing solids from the initial extract to obtain a second initial extract;

[0198] adjusting the pH of the second initial extract to a pH of about 4 to about 7 to obtain a first pH adjusted second initial extract;

[0199] contacting the first pH adjusted second initial extract with ethyl acetate to obtain a first ethyl acetate extract and a second aqueous extract;

[0200] adjusting the pH of the second aqueous extract to a pH of less than 2 to obtain a pH-adjusted second aqueous extract;

[0201] contacting the pH adjusted second aqueous extract with ethyl acetate to obtain a second ethyl acetate extract;

[0202] removing ethyl acetate from the second ethyl acetate extract to obtain a purified composition;

[0203] reconstituting the crude composition with water to obtain a third aqueous extract; and

[0204] The third aqueous extract is decolorized to obtain a decolorized aqueous extract.

[0205] A "purified composition" will contain the compound of interest (e.g., at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts) and will be purified relative to at least the initial extract and the second initial extract in that the "purified composition" will be free of certain impurities in the initial extract and the second initial extract, but will contain highly colored polymeric substances that are naturally present in the plant biomass or formed at one or more of the steps described herein and removed in the decolorization step.

[0206] Yet another example of a modification of the method described herein comprising steps (1)-(6) (including alternative optional step (4-a)) comprises a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0207] contacting the plant biomass with an aqueous composition to obtain an initial extract;

[0208] removing solids from the initial extract to obtain a second initial extract;

[0209] adjusting the pH of the second initial extract to a pH of less than about 2 to obtain a second pH-adjusted second initial extract;

[0210] contacting the second pH adjusted second initial extract with ethyl acetate to obtain a third ethyl acetate extract;

[0211] neutralizing the third ethyl acetate extract to obtain a first neutralized ethyl acetate extract and a third aqueous extract; and

[0212] The third aqueous extract is decolorized to obtain a decolorized aqueous extract.

[0213] The method described herein may include step (6) involving drying the decolorized aqueous extract to obtain a composition comprising at least one of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof. The first retentate or the second retentate or the third filtrate may be dried in any suitable manner, including by freeze drying or spray drying.

[0214] An example of a method for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the method comprising:

[0215] (A1) contacting plant biomass with an aqueous composition to obtain an initial extract;

[0216] (A2) adjusting the volume of the initial extract to obtain a second initial extract;

[0217] (A3) contacting the second initial extract with ethyl acetate to obtain an aqueous fraction;

[0218] (A4) acidifying the aqueous fraction and contacting it with ethyl acetate to obtain an ethyl acetate fraction;

[0219] (A5) drying and reconstituting the ethyl acetate fraction to obtain a decolorized fraction;

[0220] (A6) chromatographically separating the decolorized fraction on an ion exchange chromatography stationary phase;

[0221] (A7) eluting the ion exchange chromatography stationary phase to obtain a first eluate comprising a solvent;

[0222] (A8) removing the solvent to form a concentrate;

[0223] (A9) desalting the concentrate to form a desalted concentrate; and

[0224] (A10) drying the desalted concentrate to obtain a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof.

[0225] Step (A1) of the method described herein involves contacting the plant biomass with an aqueous composition to obtain an initial extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts (e.g., quaternary ammonium salts, sodium salts, potassium salts, lithium salts, magnesium salts and calcium salts).

[0226] The aqueous composition may contain water and does not contain any co-solvent, such as an organic solvent. However, in addition to water, the aqueous composition may also contain a co-solvent. Suitable co-solvents include organic solvents, such as (C1-C4)alkanols and mixtures of (C1-C4)alkanols. By "(C1-C4)alkanol" is meant an alcohol of the formula (C1-C4)alkyl-OH, wherein "alkyl" refers to a straight and branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl and tert-butyl, so that the resulting (C1-C4)alkanol is methanol, ethanol, n-propyl alcohol, n-butyl alcohol, isopropyl alcohol, isobutyl alcohol, sec-butyl alcohol and tert-butyl alcohol. The proportion of an organic solvent such as a (C1-C4)alkanol or a mixture of (C1-C4)alkanols can be any suitable proportion, such that the aqueous composition can comprise up to about 30%, up to about 40%, up to about 50%, or up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to 100% by volume of the organic solvent, with the remainder being water, except when the aqueous composition comprises 100% by volume of the organic solvent; or from about 30% to about 100%, from about 50% to about 100%, from about 60% to about 90%, from about 30% to about 60%, from about 40% to about 60%, from about 30% to about 50%, from about 40% to about 50%, or about 50% by volume of the organic solvent, with the remainder being water.

[0227] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0228] As used herein, the term "plant biomass" generally refers to any and all parts of a plant origin comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts, including leaves, stalks, stems, tops, roots, etc. of plant origin. Plant biomass can be in any suitable form, including a pulverized form obtained by, for example, chopping plant biomass before and / or during contact with an aqueous composition. For example, plant biomass can be pulverized in a suitable container, and an aqueous composition can be added to the pulverized plant biomass, thereby "contacting" the plant biomass. The pulverized plant biomass can then be optionally further pulverized in a suitable container. Alternatively, the plant biomass can be placed in a suitable container, an aqueous composition can be added thereto, thereby "contacting" the plant biomass, and the resulting composition can be pulverized.

[0229] The plant biomass may be stirred, sonicated, or otherwise agitated prior to and / or during contacting to, among other things, maximize extraction of the at least one of mono- and dicaffeoylquinic acids and salts thereof.

[0230] The initial extract may be applied to step (B1) as is, or bulk solids and / or plant solids present, such as comminuted plant leaves, stalks, tops, roots, etc., may be removed in step (A1) of the methods described herein.

[0231] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that substantially does not retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0232] Step (A2) of the method described herein involves adjusting the volume of the initial extract with the first aqueous composition to obtain a second initial extract. The second initial extract can be filtered at this point, or it can be applied to step (C3) as is. The second initial extract can be filtered using any suitable filtering method, including gravity filtration or vacuum filtration through any suitable filter, as long as the filter does not substantially retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, etc.

[0233] The volume of the initial extract can be adjusted to, among other things, adjust the amount of organic solvent (when present) in the second initial extract. The volume of the initial extract can be adjusted so that the second initial extract contains less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, or even about 0% by volume of organic solvent, the remainder being water; or about 0% to about 40%, about 0% to about 30%, about 10% to about 40%, about 10% to about 30%, about 20% to about 40%, about 30% to about 40%, or about 35% by volume of organic solvent, the remainder being water.

[0234] Step (A3) of the methods described herein involves contacting the second initial extract with ethyl acetate to obtain an aqueous fraction. The second initial extract is diluted with an equal volume of water, followed by an equal volume of ethyl acetate, shaken, and the resulting aqueous fraction retained.

[0235] Step (A4) of the method described herein involves acidifying the aqueous fraction and contacting with ethyl acetate to obtain an ethyl acetate fraction. The aqueous fraction is acidified, an equal volume of ethyl acetate is added, shaken, the ethyl acetate fraction is retained and the second aqueous fraction is removed. Optionally, the second aqueous fraction may also be retained and extracted again with ethyl acetate to obtain a second ethyl acetate fraction.

[0236] Step (A5) of the process described herein involves drying and reconstituting the ethyl acetate fraction to obtain a decolorized fraction. The retained ethyl acetate fraction (and the second ethyl acetate fraction) is dried to remove the solvent and then reconstituted with water to obtain a decolorized fraction.

[0237] Step (A6) of the method described herein relates to chromatographic separation of the decolorized fraction on an ion exchange stationary phase (e.g., a weak anion exchange stationary phase). The chromatographic separation can be carried out in any suitable manner, including batch mode or using a chromatographic column. The chromatographic separation can be carried out with an aqueous composition (e.g., an aqueous composition comprising (C1-C4) alkanol) as an eluent (e.g., an aqueous composition comprising about 0 volume % to about 40 volume %, about 0 volume % to about 30 volume %, about 10 volume % to about 40 volume %, about 10 volume % to about 30 volume %, about 20 volume % to about 40 volume %, about 30 volume % to about 40 volume %, or about 35 volume % of (C1-C4) alkanol and the remainder being water), leaving at least one of the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts adsorbed on a weak ion exchange chromatographic column, while eluting other compounds.

[0238] The ion exchange stationary phase is non-limiting and can be any suitable ion exchange chromatography stationary phase. Examples of suitable ion exchange chromatography stationary phases include High flow resin, DEAE DEAE A25 resin, (FPA 53, FPA 55, CG-50I, IRC-50, IRC-50S and IRP-64), DIAION WA10 and CCR-3.

[0239] Prior to chromatographic separation of the conditioned initial extract or the conditioned second initial extract, the ion exchange chromatography stationary phase may optionally be preconditioned with an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol), such as an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water. For example, a weak ion exchange chromatography column may be preconditioned with about 2 or more bed volumes (BV) at a flow rate of about 2 BV / h.

[0240] The pH of the weak ion exchange chromatography column can optionally be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract. For example, the pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography with any suitable acid (e.g., hydrochloric acid) such that the pH of the weak ion exchange chromatography column (e.g., the pH of the resin / stationary phase) is a pH of less than about 10, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less; or a pH of about 2 to about 10, about 3 to about 8, about 5 to about 9, about 2 to about 6, about 3 to about 4, or about 3 to about 6. The pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract, before or after optionally preconditioning the column with an aqueous composition comprising (C1-C4).

[0241] After preconditioning and / or adjusting the pH of the weak ion exchange chromatography column, the adjusted initial extract or the adjusted second initial extract can be loaded onto the column at any suitable rate, such as at a rate greater than 2 BV / h (bed volume / hour). After loading the adjusted initial extract or the adjusted second initial extract, the column can be washed with any suitable volume of an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% to about 40% by volume, about 10% to about 30% by volume, about 20% to about 40% by volume, about 30% to about 40% by volume, or about 35% by volume of (C1-C4)alkanol, the remainder being water) at any suitable rate, such as at a rate greater than 2 BV / h. The volume of the aqueous composition comprising the (C1-C4)alkanol may be removed because it will contain, among other things, caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, rutin and rutin isomers.

[0242] Step (A7) of the method described herein involves eluting the adsorbed at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from a weak ion exchange chromatography column to obtain a first eluent comprising the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. The elution is performed under any conditions suitable for eluting the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column.

[0243] Examples of suitable conditions for eluting at least one of the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column include eluting the column with any suitable volume of a solution comprising a salt (e.g., sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, etc.). Examples of solutions comprising a salt include solutions comprising at least one salt (e.g., about 5% by weight to about 25% by weight, about 15% by weight to about 20% by weight, or about 5% by weight to about 10% by weight of a salt) dissolved in an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% by volume to about 60% by volume, about 20% by volume to about 50% by volume, about 30% by volume to about 55% by volume, about 40% by volume to about 60% by volume, or about 50% by volume of the (C1-C4)alkanol).

[0244] Another example of suitable conditions for eluting at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the column includes eluting the column with any suitable volume of a solution comprising an acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.). Examples of solutions comprising an acid include solutions comprising hydrochloric acid, etc. and optionally an acid solution comprising an aqueous composition comprising about 10% to about 60% by volume, about 20% to about 50% by volume, about 30% to about 55% by volume, about 40% to about 60% by volume, or about 50% by volume of (C1-C4)alkanol.

[0245] The first eluate collected from the elution step comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts is collected and subsequently concentrated by removing the solvent in any suitable manner (e.g., to remove water and (C1-C4)alkanol) to provide a concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Solvent removal can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere). Without wishing to be bound by any particular theory, it is believed that solvent removal under an inert atmosphere can reduce the formation of highly colored polymeric substances naturally present in plant biomass or formed at one or more steps in the steps described herein.

[0246] The first eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts comprises a solvent. The solvent may be removed to dryness in step (A8), or it may be removed to the extent that a certain volume of the aqueous composition comprising (C1-C4)alkanol remains as solvent (e.g., about 50%, about 40%, about 30%, about 20%, about 10% or about 5% of the initial total volume of the eluent) to form a concentrate, but the ratio of the components constituting the aqueous composition comprising (C1-C4)alkanol may be different from or may be equivalent to the ratio of the components constituting the aqueous composition comprising (C1-C4)alkanol used to elute the adsorbed monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. Alternatively, the solvent in the eluate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts can be removed to an extent that a certain volume of aqueous composition comprising (C1-C4)alkanol remains, wherein the aqueous composition comprising (C1-C4)alkanol comprises less than about 10% by volume, less than about 5% by volume, less than about 2% by volume or less than about 1% by volume of (C1-C4)alkanol.

[0247] Suitable conditions for removing solvent from an eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include blowing an inert gas (e.g., nitrogen) on the surface of the eluent. The eluent can be heated while blowing nitrogen, or it can be heated at room temperature (e.g., 25°C). Other conditions for removing the solvent in the eluent include applying a vacuum to a container containing the eluent. A vacuum can be applied with the eluent at room temperature or while heating the container. Other conditions for removing the solvent in the eluent include passing the eluent through a wiped film evaporator or a stirred thin film evaporator.

[0248] The concentrate comprising the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof may be directly used in the drying step (A10), or may be used for desalting in the step (A9).

[0249] The filtrate from decolorization can be used for drying step (A10), or it can be desalinated in step (A9). Desalination can be achieved using a hydrophobic resin. In one example, desalination can be achieved using a hydrophobic resin (e.g., Diaion SP70), wherein concentrate is loaded onto the resin. The resin is then washed with dilute alcohol (e.g., less than about 10% by volume (C1-C4) alkanol, with the remaining being pH less than about 2 water), then eluted with an aqueous solution of an aqueous composition comprising about 70% by volume (C1-C4) alkanol, to obtain a desalination concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts.

[0250] The method described herein may include step (A10) involving drying the desalted concentrate to obtain a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. The desalted concentrate may be dried in any suitable manner, including by freeze drying or spray drying.

[0251] Single chromatographic separation step

[0252] In one aspect, if a method for preparing a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof results in an increased amount of at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof and a reduced preferred content level of other off-flavor compounds such as caffeine, rutin, rutin isomers, and other off-flavor compounds as described below (including, for example, compounds listed in Table 2 below), then the method is preferred. In one aspect, if a method for preparing a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof results in an increased amount of at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof and a reduced level of other off-flavor compounds such as caffeine, rutin, rutin isomers, and other off-flavor compounds as described in Table 2 below, then the method is preferred. In one aspect, if a method reduces the number of steps used to achieve an acceptably reduced content level of off-flavor compounds, then the method is preferred. In one aspect, a method is preferred if it reduces the number of steps used to achieve a composition of monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts having one or more off-flavor compounds as described in Table 2 below below that are below the corresponding acceptable cutoff values. For example, such a method that reduces the number of steps used to achieve an acceptably reduced content level of produced off-flavor compounds is commercially advantageous because it reduces the cost and / or time required to produce an acceptable product. In one aspect, an example of a method that reduces the number of steps used to achieve an acceptably reduced content level of produced off-flavor compounds includes a single chromatographic separation step. For example, a method that reduces the number of steps used to achieve an acceptably reduced content level of produced off-flavor compounds may include a single chromatographic separation step, and the decolorization and desalting steps may be eliminated while still achieving an acceptably reduced content level of produced off-flavor compounds. Examples of methods for preparing a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts while reducing the number of steps used to achieve an acceptably reduced content level of produced off-flavor compounds include

[0253] (B1) contacting the plant biomass with an aqueous composition to obtain an initial extract;

[0254] (B2) optionally filtering / removing solids from the initial extract;

[0255] (B3) chromatographically separating the initial extract on an ion exchange chromatography stationary phase;

[0256] (B4) eluting the stationary phase with a first elution composition to obtain a first eluate;

[0257] (B5) eluting the stationary phase with a second elution composition to obtain a second eluate; and

[0258] (B6) Solvent removal and / or drying.

[0259] Step (B1) of the method described herein involves contacting the plant biomass with an aqueous composition to obtain an initial extract comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts (e.g., quaternary ammonium salts, sodium salts, potassium salts, lithium salts, magnesium salts and calcium salts).

[0260] The aqueous composition may contain water and, in addition to water, may also contain a cosolvent. Suitable cosolvents include organic solvents such as (C1-C4)alkanols and mixtures of (C1-C4)alkanols. By "(C1-C4)alkanol" is meant an alcohol of the formula (C1-C4)alkyl-OH, wherein "alkyl" refers to straight and branched chain alkyl groups having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl and tert-butyl, such that the resulting (C1-C4)alkanol is methanol, ethanol, n-propyl alcohol, n-butyl alcohol, isopropyl alcohol, isobutyl alcohol, sec-butyl alcohol and tert-butyl alcohol. The proportion of an organic solvent such as a (C1-C4)alkanol or a mixture of (C1-C4)alkanols can be any suitable proportion, such that the aqueous composition can comprise up to about 30%, up to about 40%, up to about 50%, or up to about 60%, up to about 70%, up to about 80%, up to about 90%, or up to 100% by volume of the organic solvent, with the remainder being water, except when the aqueous composition comprises 100% by volume of the organic solvent; or from about 30% to about 100%, from about 50% to about 100%, from about 60% to about 90%, from about 30% to about 60%, from about 40% to about 60%, from about 30% to about 50%, from about 40% to about 50%, or about 50% by volume of the organic solvent, with the remainder being water.

[0261] In some cases, the aqueous composition can be buffered with any suitable buffer system, including but not limited to phosphate, citrate, ascorbate, lactate, acetate, etc. The buffer can be in the range of 1mM-1000mM of anions. Alternatively, water acidified to pH 5-6 with hydrochloric acid, sulfuric acid, nitric acid, etc. can be used in aqueous compositions with or without cosolvents. Alternatively, pure water adjusted to alkalinity to pH 7-11 with hydroxides (such as sodium hydroxide or potassium hydroxide) can be used in aqueous compositions with or without cosolvents. In other cases, it may be appropriate to add a suitable non-ionic solute that can help balance the osmotic potential of the aqueous composition.

[0262] As used herein, the term "plant biomass" generally refers to any and all parts of a plant source (e.g., a plant source as listed in Table 1) comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts, including leaves, stalks, stems, tops, roots, etc. of plant sources. Plant biomass can be in any suitable form, including a pulverized form obtained by, for example, chopping plant biomass before and / or during contact with an aqueous composition. For example, plant biomass can be pulverized in a suitable container, and an aqueous composition can be added to the pulverized plant biomass, thereby "contacting" the plant biomass. The pulverized plant biomass can then be optionally further pulverized in a suitable container. Alternatively, the plant biomass can be placed in a suitable container, an aqueous composition can be added thereto, thereby "contacting" the plant biomass, and the resulting composition can be pulverized.

[0263] The plant biomass may be stirred, sonicated, or otherwise agitated prior to and / or during contacting to, among other things, maximize extraction of the at least one of mono- and dicaffeoylquinic acids and salts thereof.

[0264] The initial extract may be applied as such to step (B3), or the bulk solids and / or plant solids present may be removed in step (B2) of the process described herein, such as pulverized plant leaves, stalks, tops, roots, etc. When step (B2) is performed, a second initial extract is obtained.

[0265] The bulk solids may be removed by any suitable method including centrifugation, skimming or filtration. For example, the initial extract may be filtered using any suitable filtration method including gravity filtration or vacuum filtration through any suitable filter that substantially does not retain at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts, including paper filters (e.g., low ash filter paper, such as Whatman 44 or 54 low ash filter paper), nylon filters, polyethersulfone filters, glass fiber filters, diatomaceous earth pads, and the like.

[0266] Step (B3) of the methods described herein involves chromatographic separation of the initial extract or the second initial extract on an ion exchange stationary phase (eg, a weak anion exchange stationary phase). The chromatographic separation can be performed in any suitable manner, including in batch mode or using a chromatography column. Chromatographic separation can be performed using an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol) as an eluent (e.g., an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water), leaving at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof adsorbed on a weak ion exchange chromatography column while eluting other compounds including caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, and rutin (also known as rutin, quercetin-3-O-rutinoside, and sophora flavescens) and its isomers.

[0267] Steps (B3-B5) of the method described herein can reduce the concentration of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers to a concentration of less than 1 mass%, less than 0.5 mass%, less than 0.1 mass%, less than 0.05 mass%, less than 0.01 mass%, or less than 0.001 mass%. Therefore, the present disclosure contemplates a plant extract comprising less than 0.1% by mass of at least one of caffeine, quercetin, hyperoside, astragaloside, scutellaria glycoside, sophora glycoside, rutin, and rutin isomers. The present disclosure also contemplates plant extracts comprising less than 0.5% by mass of each of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers, and less than about 1% by mass of a combination of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers. The present disclosure also contemplates plant extracts that are effectively free of at least one of caffeine, quercetin, hyperoside, astragalus glycoside, succinoside, sophora glycoside, rutin, and rutin isomers (e.g., free of caffeine, free of quercetin, free of hyperoside, free of astragalus glycoside, free of succinoside, free of sophora glycoside, free of rutin, free of rutin isomers, and / or free of caffeine, rutin, and rutin isomers).

[0268] Steps (B3-B5) of the methods described herein can achieve a composition of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof having one or more off-flavor compounds as described in Table 2 below that are below the corresponding acceptable cutoff values ​​for off-flavor compounds.

[0269] The ion exchange stationary phase is non-limiting and can be any suitable ion exchange chromatography stationary phase. Examples of suitable ion exchange chromatography stationary phases include High flow resin, DEAE DEAE A25 resin, (FPA 53, FPA 55, CG-50I, IRC-50, IRC-50S and IRP-64), DIAION WA10, Sunresin T5 and CCR-3.

[0270] Prior to chromatographic separation of the conditioned initial extract or the conditioned second initial extract, the ion exchange chromatography stationary phase may optionally be preconditioned with an aqueous composition (e.g., an aqueous composition comprising (C1-C4)alkanol), such as an aqueous composition comprising about 0 vol% to about 40 vol%, about 0 vol% to about 30 vol%, about 10 vol% to about 40 vol%, about 10 vol% to about 30 vol%, about 20 vol% to about 40 vol%, about 30 vol% to about 40 vol%, or about 35 vol% (C1-C4)alkanol and the remainder being water. For example, a weak ion exchange chromatography column may be preconditioned with about 2 or more bed volumes (BV) at a flow rate of about 2 BV / h.

[0271] The pH of the weak ion exchange chromatography column can optionally be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract. For example, the pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography with any suitable acid (e.g., hydrochloric acid) such that the pH of the weak ion exchange chromatography column (e.g., the pH of the resin / stationary phase) is a pH of less than about 10, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less; or a pH of about 2 to about 10, about 3 to about 8, about 5 to about 9, about 2 to about 6, about 3 to about 4, or about 3 to about 6. The pH of the weak ion exchange chromatography column can be adjusted prior to the chromatography of the adjusted initial extract or the adjusted second initial extract, before or after optionally preconditioning the column with an aqueous composition comprising (C1-C4).

[0272] After preconditioning and / or adjusting the pH of the weak ion exchange chromatography column, the adjusted initial extract or the adjusted second initial extract can be loaded onto the column at any suitable rate, such as at a rate greater than 2 BV / h (bed volume / hour). After loading the adjusted initial extract or the adjusted second initial extract, the column can be washed with any suitable volume of an aqueous composition comprising a (C1-C4)alkanol (e.g., at least about 2 BV, at least about 3 BV, or at least about 4 BV of an aqueous composition comprising about 10% to about 40% by volume, about 10% to about 30% by volume, about 20% to about 40% by volume, about 30% to about 40% by volume, or about 35% by volume of (C1-C4)alkanol, the remainder being water) at any suitable rate, such as at a rate greater than 2 BV / h. The volume of the aqueous composition comprising the (C1-C4)alkanol may be removed because it will contain, among other things, caffeine, quercetin, hyperoside, astragaloside, scutellarin, sophoroside, rutin and rutin isomers.

[0273] Step (B4) of the method described herein involves eluting the stationary phase with a first elution composition to obtain a first eluent. The first elution is performed under any conditions suitable for eluting other compounds bound to the ion exchange resin but not eluting the at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts adsorbed to the column.

[0274] Examples of suitable conditions for eluting these other compounds from the column include eluting the column with a first eluent composition of any suitable volume, the first eluent composition comprising about 10% by volume to about 50% by volume, about 20% by volume to about 50% by volume, or about 25% by volume of (C1-C4) alkanol (e.g., at least about 2BV, at least about 3BV, or at least about 4BV). Suitable first eluent compositions include an aqueous solution of 25% ethanol. The first eluent can be retained and can include the desired compound. For example, the stevia biomass processed by the method produces a first eluent comprising steviol glycosides.

[0275] The step (B5) of the method described herein relates to eluting the stationary phase with the second elution composition to obtain the second eluent. The second elution is carried out under any conditions suitable for eluting the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts adsorbed on the post. The example of the suitable conditions for eluting the monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from the post comprises a second eluent composition eluting column with any suitable volume, the second eluent composition comprising an acid (for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.) of a suitable concentration (0.1%-1.0%) and about 50% by volume to about 80% by volume, about 60% by volume to about 80% by volume, or about 70% by volume of (C1-C4) alkanol.

[0276] Step (B6) of the method described herein involves treating the second eluate to remove the solvent and / or drying the composition to obtain a composition having at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts. In one aspect, solvent removal can be achieved under an inert atmosphere (e.g., under a nitrogen atmosphere).

[0277] Suitable conditions for removing solvent from a second eluent comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts to form a concentrate comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts include blowing an inert gas (e.g., nitrogen) on the surface of the eluent. The eluent may be heated while blowing nitrogen, or it may be heated at room temperature (e.g., 25° C.). Other conditions for removing the solvent in the eluent include applying a vacuum to a container containing the eluent. A vacuum may be applied to the eluent at room temperature or while heating the container. Other conditions for removing the solvent in the eluent include passing the eluent through a wiped film evaporator or a stirred thin film evaporator. The methods described herein may include drying in any suitable manner, including by freeze drying or spray drying.

[0278] Figure 4B A flow chart of a method 500 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from yerba mate biomass using a single chromatographic separation step. In operation 502, the yerba mate biomass is contacted with deionized water heated to 70°C via a heat exchanger. The deionized water is heated to 70°C and applied to the bottom of a stainless steel column filled with yerba mate biomass at a flow rate of 2 BV per hour based on the volume of the yerba mate biomass. An initial extract is collected from the top of the stainless steel column. The volume of the initial extract is 10 times the volume of the yerba mate biomass. A 35 ml sample of the initial extract is collected for subsequent analysis. The initial extract is stored at 4°C.

[0279] In operation 510, the initial extract is treated with a single chromatographic separation step. The initial extract is applied to a weak anion exchange stationary phase (Sunresin T5 resin) at a rate of 2 BV per hour. The total amount of the initial extract loaded is about 40 g of caffeoylquinic acid and its salts per liter of resin. A 35 ml sample of the loaded flow-through is collected for analysis.

[0280] The stationary phase was then washed with 3 bed volumes of 25% ethanol in water at a rate of 2 BV per hour at 25° C. A 35 ml wash flow-through sample was collected for analysis.

[0281] The stationary phase was then eluted with 6 bed volumes of an elution composition comprising an aqueous solution of 0.75 wt % phosphoric acid and 70 % ethanol. The elution composition was heated to 50° C. via a heat exchanger before elution. The elution composition was applied at 1 BV per hour. The eluate was collected so that bed volumes 3, 4 and 5 were combined and further processed. Bed volumes 1, 2 and 6 were combined and could be reprocessed by the same method. 35 ml samples of the combined bed volumes 3, 4 and 5 were collected for analysis.

[0282] In operation 520, the salt content of the combined eluate is adjusted and processed into a dry powder. The content of caffeoylquinic acid and its salt is calculated, and then sodium hydroxide is added until about 70% of the combined eluate is present as caffeoylquinic acid salt. The salt-adjusted sample is then fed to an evaporator, heated to 40°C, and ethanol and water are stripped until a dissolved solids content of 5%-10% DS is reached. The composition is then dried to DS>95% and ground into a fine powder.

[0283] Figure 4C A flow chart of a method 600 for preparing a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts from stevia biomass using a single chromatographic separation step. Method 600 can also separate a steviol glycoside composition from stevia biomass. Method 600 can also separate a separate steviol glycoside composition and a separate caffeoylquinic acid composition from a single batch of stevia biomass. In operation 602, the stevia biomass is contacted with deionized water heated to 70°C via a heat exchanger. The deionized water is heated to 70°C and applied to the bottom of a stainless steel column filled with stevia biomass at a flow rate of 2BV per hour based on the volume of the stevia biomass. An initial extract is collected from the top of the stainless steel column. The volume of the initial extract is 10 times the volume of the yerba mate biomass. A 35mL initial extract sample is collected for subsequent analysis. The initial extract is stored at 4°C.

[0284] In operation 610, the initial extract is processed with a single chromatographic separation step. The initial extract is applied to a weak anion exchange stationary phase (Sunresin T5 resin) at a rate of 2 BV per hour. The total amount of the initial extract loaded is about 40 g of caffeoylquinic acid and its salt per liter of resin. Collect and merge the loaded flowthrough, which contains the steviol glycoside composition. Collect 35 mL of the loaded flowthrough sample for analysis.

[0285] The stationary phase was then eluted with 3 bed volumes of the first eluent composition (25% ethanol in water) at a rate of 2 BV per hour at 25°C. The first eluent, which contained steviol glycosides, was collected. A 35 mL sample of the first eluent was collected for analysis.

[0286] The stationary phase was then eluted with 6 bed volumes of a second elution composition comprising an aqueous solution of 0.75 wt % phosphoric acid and 70 % ethanol. The second elution composition was heated to 40° C. via a heat exchanger prior to elution. The elution composition was applied at 1 BV per hour. The eluate was collected so that bed volumes 3, 4 and 5 were combined and further processed. Bed volumes 1, 2 and 6 were combined and could be reprocessed by the same method. 35 mL samples of the combined bed volumes 3, 4 and 5 were collected for analysis.

[0287] In operation 620, the salt content of the combined second eluate is adjusted and processed into a dry powder. The content of caffeoylquinic acid and its salt is calculated, and then sodium hydroxide is added until about 70% of the second combined eluate is present as caffeoylquinic acid salt. The salt-adjusted sample is then fed to an evaporator, heated to 40°C, and ethanol and water are stripped until a dissolved solids content of 5%-10% DS is reached. The composition is then dried to DS>95% and ground into a fine powder, and a composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts is obtained. Similarly, the flow-through and the first eluate can be dry-loaded to obtain a steviol glycoside composition.

[0288] Compositions of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts

[0289] The composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof prepared according to the methods described herein may comprise caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof in substantially the same amount by weight and / or substantially the same ratio by weight relative to mate biomass.

[0290] The composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and their salts prepared according to the methods described herein can have a ratio of total dicaffeoylquinic acid to total monocaffeoylquinic acid of about 1:1 to about 10:1 by mass (e.g., about 3:1 to about 10:1; about 3:2 to about 4:1; or about 3:1 to about 5:1). The composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts prepared according to the methods described herein can have a ratio of total dicaffeoylquinic acid to total monocaffeoylquinic acid of about 1:1 to about 0.01:1 by mass (e.g., about 0.5:1 to about 0.1:1).

[0291] The composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof prepared according to the method described herein may contain each of caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof in a ratio by mass of about 0.01 (e.g., about 0.005 to about 0.05) to about 1 (e.g., 0.5 to about 1.5) to about 1 (e.g., 0.5 to about 1.5), respectively.

[0292] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts as prepared according to the method described herein preferably does not contain odor compounds above the preferred content level. Table 2 shows the preferred content levels of odor compounds and corresponding odor compounds in the dry composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts. In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts does not contain one or more compounds or any combination thereof above the disclosed cut-off weight % as listed in Table 2 below. In one aspect, the composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts as prepared by the method comprising steps (B1-B6) does not contain one or more compounds or any combination thereof above the disclosed cut-off weight % as listed in Table 2 below.

[0293] Table 2 .

[0294]

[0295]

[0296]

[0297] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 3% by weight total of malonate, malonic acid, oxalate, oxalic acid, lactate, lactic acid, succinate, succinic acid, malate, malic acid, citrate, and citric acid in the composition when dried.

[0298] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.5% by weight total of tartrate, tartaric acid, pyruvate, pyruvic acid, fumarate, fumaric acid, ascorbic acid, sorbate, sorbic acid, acetate, and acetic acid in the composition when dried.

[0299] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total of sulfate, sulfuric acid, phosphate, phosphoric acid, nitrate, nitric acid, nitrite, nitrous acid, chloride, hydrochloric acid, ammonia, and ammonium in the composition when dried.

[0300] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 5% by weight total of flavones, isoflavones, and neoflavones (quercetin, kaempferol, myricetin, fisetin, galangin, isorhamnetin, horehound, rhamnetin, pyranoflavonols, furoflavonols, luteolin, apigenin, tangeretin, taxifolin (or dihydroquercetin), dihydrokaempferol, hesperetin, naringenin, eriodictyol, homoeriodictyol, genistein, daidzein, glycitein) in the composition when dried.

[0301] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 5% by weight total of hesperidin, naringin, rutin, quercetin, luteolin-glucoside, and quercetin-xyloside in the composition when dried.

[0302] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 5% by weight in total of cyanine, delphinium, malvaceae, pelargonium, peony, and petunias in the composition when dried.

[0303] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total of tannins and tannic acid in the composition when dried.

[0304] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.1% by weight total of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine in the composition when dried.

[0305] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total of monoglycerides, diglycerides, and triglycerides in the composition when dried.

[0306] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total of monosaccharides, disaccharides, polysaccharides, glucose, fructose, sucrose, galactose, ribose, trehalose, trehalulose, lactose, maltose, isomaltose, isomaltulose, mannose, tagatose, arabinose, rhamnose, xylose, dextrose, erythrose, threose, maltotriose, and panose in the composition when dried.

[0307] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total of sugar alcohols, glycerol, sorbitol, mannitol, xylitol, maltitol, lactitol, erythritol, isomalt, and inositol in the composition when dried.

[0308] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% dietary fiber, acacia (arabic) gum, agar, algin-alginate, arabinoxylan, β-glucan, β-mannan, carrageenan, carob or locust bean gum, fenugreek gum, galactomannan, gellan gum, glucomannan or konjac gum, guar gum, hemicellulose, inulin, karaya gum, pectin, polydextrose, psyllium husk mucilage, resistant starch, tara gum, tragacanth gum, xanthan gum, cellulose, chitin, and chitosan in total in the composition when dried.

[0309] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.1% by weight total of chlorophyll, furans, furan-containing chemicals, theobromine, theophylline, and trigonelline in the composition when dried.

[0310] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% total caffeine in the composition by weight when dried.

[0311] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 1% by weight total rutin in the composition when dried.

[0312] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.5 wt% glycosylated ursolic acid and glycosylated oleanolic acid in total in the composition when dried.

[0313] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.5% by weight total of volatile organic compounds, terpenes, eugenol, geraniol, geranial, α-ionone, β-ionone, epoxyionone, limonene, linalool, linalool oxide, nerol, and damascenone in the composition when dried.

[0314] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.5 wt% total of fatty acid oxidation products, decanone, decenal, nonenal, octenal, heptenal, hexenal, pentenals, pentenols, pentenone, hexenone, hydroxynonenal, and malondialdehyde in the composition when dried.

[0315] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.01 wt% total of polycyclic aromatic hydrocarbons (PAHs) (acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(ghi)perylene, benzo(k)fluoranthene, dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, naphthalene, phenanthrene, pyrene, etc.).

[0316] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein has less than 0.01 wt % of polycyclic aromatic hydrocarbons (PAH) (acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(ghi)perylene, benzo(k)fluoranthene, dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, naphthalene, phenanthrene, pyrene, etc.).

[0317] In one aspect, a composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein comprises less than 0.5 wt% total of fatty acid oxidation products, nonenal, hexenal, hydroxynonenal, and malondialdehyde in the composition when dried.

[0318] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein has color removed such that the % transmittance at 430 nm is > 80%.

[0319] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein has color removed such that the b value on the CIE L*a*b* color space is less than 4.

[0320] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof as prepared according to the methods described herein has had color removed such that the % transmittance at 430 nm is > 80% without the need for decolorization and / or desalting steps.

[0321] In one aspect, the composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof as prepared according to the methods described herein has had color removed such that the b value on the CIE L*a*b* color space is less than 4 without the need for a decolorization and / or desalting step.

[0322] Ingestible composition

[0323] The compositions comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof prepared according to the methods described herein can be incorporated into any ingestible composition, including beverages and food products.

[0324] For example, the ingestible composition can be an edible composition or an inedible composition. By "edible composition" is meant any composition that can be consumed by humans or animals as food, including solids, gels, pastes, foams, semisolids, liquids, or mixtures thereof. By "inedible composition" is meant any composition that is intended to be consumed or used by humans or animals rather than as food, including solids, gels, pastes, foams, semisolids, liquids, or mixtures thereof. Inedible compositions include, but are not limited to, medical compositions, which refer to inedible compositions intended to be used for therapeutic purposes by humans or animals. By "animal" is meant any non-human animal, such as farm animals and pets.

[0325] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0326] In this document, unless the context clearly dictates otherwise, the terms "a", "an" or "the" are used to include one or more than one. Unless otherwise indicated, the term "or" is used to refer to a non-exclusive "or". In addition, it should be understood that the words or terms used herein and not otherwise defined are for descriptive purposes only and not for limiting purposes. Any use of section headings is intended to assist in reading the document and should not be construed as limiting; information related to section headings may appear within or outside that particular section. In addition, all publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, just as if they were individually incorporated by reference. If there is a discrepancy in usage between this document and those documents so incorporated by reference, the usage in the incorporated references should be considered to supplement the usage of this document; for irreconcilable inconsistencies, the usage in this document shall prevail.

[0327] In the methods described herein, the steps may be performed in any order without departing from the principles of the invention, except when a temporal or operational order is explicitly enumerated. In addition, unless there is explicit declarative language stating that the specified steps are to be performed separately, they may be performed simultaneously. For example, a claimed step of performing X and a claimed step of performing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed process.

[0328] As used herein, the term "about" can allow for a degree of variability in values ​​or ranges, for example, within 10%, within 5%, or within 1% of a stated value or a stated limit of a range.

[0329] As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0330] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to "steviol glycoside" means one or more steviol glycosides.

[0331] Example

[0332] The following examples are provided to illustrate the present disclosure but are not intended to limit the scope thereof. Unless otherwise indicated, all parts and percentages are by weight.

[0333] Example 1 :

[0334] Materials and Methods

[0335] The yerba mate biomass that can be used is ECOTEAS TM Yerba Mate Unsmoked Leaf and Stem Traditional Cut is a commercially available product sold as yerba mate grown in the Misiones Province in northern Argentina. A crushed biomass was obtained. A portion (300 g) of the crushed yerba mate biomass was suspended in 50% ethanol / water (1.5 L) in a glass jar and shaken for 1 hour. After shaking, the resulting mixture was filtered into a 4L glass sidearm flask using a ceramic Buchner funnel with Whatman 54 low ash filter paper. The filtered material was diluted to 35% ethanol using water. Upon dilution, some unwanted substances precipitated because they were insoluble in 35% by volume ethanol. Therefore, the diluted material was refiltered into a 4L glass sidearm flask using a ceramic Buchner funnel with Whatman 44 low ash filter paper.

[0336] The glass column was prepared by treating the resin with aqueous hydrochloric acid to protonate the amines in the resin. FPA 53 resin is used for ion exchange chromatography. Chloride is then washed off with about 10 BV of water until pH is greater than 4. The resin is then preconditioned with an aqueous solution of 35% ethanol (2 BV, 2 BV / h) before loading. The material after refiltration is loaded onto the resin. The loaded permeate is removed. The resin is then washed with an aqueous solution of 35% ethanol (4 BV, 2 BV / h). The permeate is removed. The resin is then eluted with an aqueous solution of 50% ethanol containing 10% FCC sodium chloride (4 BV, 0.5 BV / h). The last permeate is used for the next step, wherein the solvent is slowly removed by sparging nitrogen on the top of the permeate for two days until the volume is about 1 / 3 of the initial volume and / or the ethanol concentration is <1% of the solution. The temperature is maintained at ambient temperature (about 25° C.) or lower, because high temperature, high oxygen content and / or high exposure can degrade the compound of interest. If not handled with caution, the compound will polymerize to form a highly colored hydrophobic polymer, some of the largest polymers of which are insoluble in water.

[0337] The concentrated material was filtered on a Buchner funnel through Whatman 44 filter paper and then through a 0.2 μm polyethersulfone filter. A 3 kDa molecular weight cutoff membrane was used. NP010 decolorizes the filtered material, retaining the permeate, but GE Osmonic Sepa CF TF (Thin Film) UF GK membranes may also be used. Decolorized materials will degrade / polymerize over time and may degrade due to oxidation processes, and this will reintroduce color into the system. Therefore, it is recommended to desalt and dry soon after decolorization (such as within one to two days). The decolorized material is then passed through XN45 nanofiltration membrane was used for desalination. TM FAST-FREEZE TM The desalted material was freeze dried in a 600 mL flask.

[0338] The freeze-dried material was characterized by UHPLC-UV analysis using a C18-based reverse phase column. Mobile phase A contained 0.025% TFA in water and mobile phase B was acetonitrile. After an initial hold at 5% B, the compounds were eluted at a flow rate of 0.4 mL / min from 1.2 minutes to 15 minutes using a gradient of 5% B to 25% B at elevated temperature. The column was then washed with 100% acetonitrile and re-equilibrated. The UV detector was set to record data at 210 nm and 324 nm.

[0339] Figures 5 to 7is a UHPLC-UV chromatogram of an initial yerba mate extract, which is a concentrate obtained after chromatography of the adjusted second initial extract on an ion exchange chromatography stationary phase; and after drying, following the process described in steps (a)-(h), wherein "DCQA" refers to "dicaffeoylquinic acid". Figure 5 It is shown that the initial yerba mate extract contains caffeine and rutin in addition to the compounds of interest, including chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, and various isomers of dicaffeoylquinic acid (including 3,4-DCQA and 3,5-DCQA). As described herein, and as Figure 6 As shown, the chromatographic separation removed a significant amount of caffeine and rutin present in the initial yerba mate extract. Figure 6 Does not exist Figure 5 The peak at a retention time of about 5.67 minutes corresponding to caffeine and the peak at a retention time of about 9.36 minutes corresponding to rutin are present in Figure 7 It is noteworthy that the relative intensities of the peaks of neochlorogenic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid and various isomers of dicaffeoylquinic acid are maintained, thus confirming the fact that the composition obtained using the method described herein comprises caffeic acid, monocaffeoylquinic acid and dicaffeoylquinic acid and their salts in substantially the same amount by weight or substantially the same ratio by weight relative to the mate biomass.

[0340] Figures 8 to 10 To show in table form Figures 5 to 7 Table of peak names, retention times and relative area percentage data for the UHPLC-UV chromatograms shown.

[0341] Example 2 :

[0342] Stevia Extract

[0343] A caffeoylquinic acid (CQA) composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts is prepared using stevia biomass. Stevia biomass is obtained in the form of dried stevia leaves. The dried stevia leaf biomass is extracted with 50% ethanol (v / v) in ultrapure water for a contact time of 1 hour to obtain an initial extract. The total mass of the initial extract is 10 times the mass of the stevia leaf biomass. The initial extract is filtered with a 20 μm filter, and the retained solids are washed with an amount of 50% ethanol extraction composition, the amount being about 5 times the amount of stevia leaf biomass.

[0344] The initial extract contains significant chlorophyll and other hydrophobic components. Ethyl acetate extraction is performed to reduce the level of chlorophyll and other hydrophobic components. The initial extract is transferred to a glass container, an equal volume of ultrapure water is added, and then the same volume of ethyl acetate is added. The glass container is shaken and the liquid phase is separated. Ethyl acetate is removed, and an additional volume of ethyl acetate is added. The glass container is shaken again and the liquid phase is separated. Ethyl acetate is removed, and the remaining aqueous fraction is retained. The remaining aqueous fraction is acidified with H2SO4 to convert caffeoylquinine from salt form to acid form. Another volume of ethyl acetate is added, the container is shaken, and the liquid phase is separated. Since caffeoylquinine is now present in the ethyl acetate fraction, the first ethyl acetate fraction is retained and further processed. Optionally, a second round of ethyl acetate extraction can increase the yield of CQA to obtain a second ethyl acetate fraction that can be combined with the first ethyl acetate fraction. The ethyl acetate fraction is dried under nitrogen for>24 hours, and the compound is reconstructed in water to obtain a reconstructed fraction.

[0345] Ion exchange chromatography

[0346] Weak anion exchange resin Dowex 66 is used as ion exchange stationary phase.The resin is filled into the column and regenerated in the following manner before use: the resin is suspended in water to form a slurry, and the slurry is loaded into the column until the bed volume reaches the desired amount. In order to remove the potential voids in the resin and prevent channeling, 2 times the bed volume of deionized water is made to pass through the column from the bottom to the top at a rate of 4 times the bed volume / hour. The preparation is equal to an aqueous solution (v / v) of 7% HCl and 50% ethanol of 4 times the bed volume, and is pumped into the column from the top to the bottom. The column is subsequently rinsed with deionized water at a rate of 4 times the bed volume / hour until the output of the column reaches pH>4.

[0347] The reconstituted fraction was loaded onto the prepared ion exchange column and the column was washed. The column was loaded at 25°C at a rate of 2 bed volumes per hour. The column was then washed with 3 bed volumes of 50% ethanol in water (v / v) at a temperature of 25°C and a rate of 2 bed volumes / hour.

[0348] The resin was eluted using a solvent solution containing a high concentration of salt. The resin was eluted using an elution composition containing 50% ethanol (v / v) and 10% (w / v) sodium chloride in water. The resin was eluted with 4 bed volumes of the elution composition solution at a rate of 2 bed volumes / hour. The eluate was collected. The resin was then regenerated using the above-mentioned HCl / ethanol method.

[0349] Evaporation and desalting resins

[0350] Eluent is evaporated with nitrogen to remove organic solvent.Fill hydrophobic resin Diaion SP70 and wash with 95% ethanol (v / v) before use.Then wash the hydrophobic resin with 4 bed volumes of water at a rate of 4 bed volumes / hour.The eluent is loaded onto the hydrophobic resin and washed with an aqueous solution (v / v) of 10% ethanol containing 0.1% HCl of 2 bed volumes at a flow rate of 2 bed volumes per hour.The hydrophobic resin is washed with 2 bed volumes of pure water at 2 bed volumes per hour.The hydrophobic resin is eluted with an aqueous solution (v / v) of 70% ethanol of 4 bed volumes, and the desalted eluent is dried under nitrogen to remove ethanol, thereby obtaining a desalted fraction.The desalted fraction is adjusted with sodium hydroxide until pH>3.0 but <4.0 (corresponding to a salt fraction of about 50%-80%), to obtain a caffeoylquinic acid (CQA) composition. The caffeoylquinic acid (CQA) composition was flash frozen in a dry ice / isopropanol bath and lyophilized to obtain the final powder.

[0351] Example 3 :

[0352] Yerba Mate Extract

[0353] A caffeoylquinic acid (CQA) composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and at least one of their salts is prepared using yerba mate biomass using a single chromatographic separation step. Yerba mate biomass in the form of dry yerba mate loose leaf tea is obtained. A stainless steel column is filled with yerba mate biomass. Deionized water is heated to 70° C. via a heat exchanger and applied to the bottom of the stainless steel column. Heated deionized water is applied at a flow rate of 2 BV per hour based on the volume of the yerba mate biomass. An initial extract is collected from the top orifice of the stainless steel column. The volume of the initial extract is 10 times the volume of the yerba mate biomass. A 35 ml initial extract sample is collected for subsequent analysis. The initial extract is stored at 4° C.

[0354] Weak anion exchange resin Sunresin T5 is used as stationary phase, is filled into post and regenerated before use.Resin is suspended in deionized water to form slurry, and then slurry is loaded into post, until bed volume reaches desired amount.In order to remove the potential void in resin and prevent channeling, make the deionized water of 2 times of bed volumes reach top from bottom through post with the speed of 4 times of bed volumes / hour.Preparation is equal to 46.7% (v / v) aqueous solution of 15%HCl (w / w) and 50% ethanol (v / v) of 4 times of bed volumes, and is applied to post at the top orifice of post.Flush post with deionized water with the speed of 4 times of bed volumes / hour subsequently, until the output of post reaches pH>4.Make other deionized water pass through post with the increase flow rate of 8 times of bed volumes per hour to further fill post.

[0355] The initial extract was processed with a single chromatographic separation step. The initial extract was applied to the packed column at a rate of 2 BV per hour. The total amount of the initial extract loaded was about 33 g of caffeoylquinic acid and its salt per liter of resin. A 35 mL sample of the loaded flow-through was collected for analysis.

[0356] The column was then washed with 3 bed volumes of 25% ethanol in water at a rate of 2 BV per hour at 25° C. A 35 mL wash flow-through sample was collected for analysis.

[0357] The column was then eluted with 6 bed volumes of an elution composition comprising 70% ethanol (v / v) and 0.88% (v / v) of an 85 wt % phosphoric acid aqueous solution. The elution composition was heated to 50° C. via a heat exchanger prior to elution. The elution composition was applied at 1 BV per hour. The eluent was collected so that bed volumes 3, 4 and 5 were combined and further processed. Bed volumes 1, 2 and 6 were combined and may be reprocessed. A 35 mL sample of the combined bed volumes 3, 4 and 5 was collected for analysis. The resin was then regenerated using the method described above.

[0358] pH adjustment and drying

[0359] The combined eluate is adjusted for salt content and processed to a dry powder. The content of caffeoylquinic acid and its salts is calculated and then sodium hydroxide is added until about 70% of the combined eluate is present as caffeoylquinic acid salt. The salt adjusted sample is then fed to an evaporator, heated to 40°C, and ethanol and water are stripped until a dissolved solids content of 5%-10% DS is achieved. The composition is then dried to DS>95% and ground to a fine powder.

[0360] Example 4 :

[0361] Stevia Extract

[0362] Using a single chromatographic separation step, a caffeoylquinic acid (CQA) composition comprising at least one of monocaffeoylquinic acid, dicaffeoylquinic acid and their salts was prepared using stevia biomass. Stevia biomass in the form of dried stevia leaves (Stevia One, Peru) was obtained. A stainless steel column was filled with stevia leaf biomass. Deionized water was heated to 70°C via a heat exchanger and applied to the bottom of the stainless steel column. Based on the volume of stevia leaf biomass, heated deionized water was applied at a flow rate of 2BV per hour. The initial extract was collected from the top orifice of the stainless steel column. The volume of the initial extract was 10 times the volume of the stevia leaf biomass. 35mL initial extract samples were collected for subsequent analysis. The initial extract was stored at 4°C.

[0363] Weak anion exchange resin Sunresin T5 is used as stationary phase, is filled into post and regenerated before use.Resin is suspended in deionized water to form slurry, and then slurry is loaded into post, until bed volume reaches desired amount.In order to remove the potential void in resin and prevent channeling, make the deionized water of 2 times of bed volumes reach top from bottom through post with the speed of 4 times of bed volumes / hour.Preparation is equal to 46.7% (v / v) aqueous solution of 15%HCl (w / w) and 50% ethanol (v / v) of 4 times of bed volumes, and is applied to post at the top orifice of post.Flush post with deionized water with the speed of 4 times of bed volumes / hour subsequently, until the output of post reaches pH>4.Make other deionized water pass through post with the increase flow rate of 8 times of bed volumes per hour to further fill post.

[0364] The initial extract was processed with a single chromatographic separation step. The initial extract was applied to the packed column at a rate of 2 BV per hour. The total amount of the initial extract loaded was about 33 g of caffeoylquinic acid and its salt per liter of resin. A 35 mL sample of the loaded flow-through was collected for analysis.

[0365] The column was then washed with 3 bed volumes of 25% ethanol in water at a rate of 2 BV per hour at 25° C. A 35 mL wash flow-through sample was collected for analysis.

[0366] The column was then eluted with 6 bed volumes of an elution composition comprising 70% ethanol (v / v) and 0.88% (v / v) of an 85 wt % phosphoric acid aqueous solution. The elution composition was heated to 40° C. via a heat exchanger prior to elution. The elution composition was applied at 1 BV per hour. The eluent was collected so that bed volumes 3, 4 and 5 were combined and further processed. Bed volumes 1, 2 and 6 were combined and may be reprocessed. A 35 mL sample of the combined bed volumes 3, 4 and 5 was collected for analysis. The resin was then regenerated using the method described above.

[0367] pH adjustment and drying

[0368] The combined eluate is adjusted for salt content and processed to a dry powder. The content of caffeoylquinic acid and its salts is calculated and then sodium hydroxide is added until about 70% of the combined eluate is present as caffeoylquinic acid salt. The salt adjusted sample is then fed to an evaporator, heated to 40°C, and ethanol and water are stripped until a dissolved solids content of 5%-10% DS is achieved. The composition is then dried to DS>95% and ground to a fine powder.

[0369] Samples collected during the treatment were used for UHPLC-UV analysis. A total of six caffeoylquinic acids (CQAs) and seven steviol glycosides (SGs) were quantified. The results showed that the CQIs could be separated from the SGs using this treatment method (see Fig.11). Most of the SG was contained in the loading flow-through and ethanol wash steps (87% and 11% of the total, respectively). This is in contrast to CQA, where the elution effluent contained 80% of the mono-substituted CQA (MCQA) and 89% of the di-substituted CQA (DCQA). The initial extract loaded onto the ion exchange column contained a 4:1 ratio of SG to CQA (21.5 g SG, 4.9 g CQA), while the elution effluent contained a 1:38 ratio of SG to CQA (0.1 g SG, 3.8 g CQA). These results demonstrate that a single chromatographic separation method can be used to provide a dual purification scheme for the simultaneous separation of CQA and SG from a single batch of Stevia leaf biomass.

[0370] Example 5 :

[0371] Compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts prepared by the above method were analyzed to assess the residual color in the composition. Four batches were prepared using yerba mate biomass and analyzed for color. Samples of the initial extract were also analyzed. Four batches were analyzed using a HunterLab Vista spectrophotometer to record the L a*b* values ​​for each batch. If this instrument is not available, a spectrophotometer can be used to measure the transmittance of the sample at 430 nm.

[0372] In brief, a 1% (w / w) glacial acetic acid solution with a pH of about 4 was prepared in ultrapure water. Each test sample was prepared in a 1% (w / w) glacial acetic acid solution using an aliquot from each batch. The test samples were prepared to a final concentration of 1.0 mg / ml of total monocaffeoylquinic acid, dicaffeoylquinic acid, and their salts. Control samples were prepared using only 1% (w / w) glacial acetic acid solution. The La*b* values ​​of each test sample were measured using a HunterLab Vista spectrophotometer. For b* values, an acceptable cutoff value was <3.5 (or transmittance at 430 nm> 85%).

[0373] Batches 1, 2 and 4 were prepared by ion exchange chromatography, desalted with a hydrophobic column and decolorized by membrane filtration. Batch 3 was prepared by the method described above in steps (B1-B6) and Example 3. The sample with the initial extract was used as a reference. The b* values ​​of the test samples and controls are shown in Table 3 below.

[0374] Table 3 .

[0375] batch b* value Transmittance at 430nm Batch #1 – Good Color Removal 1.93 95.4％ Batch #2 – Acceptable Color Removal 2.77 92.9％ Batch #3 – Acceptable Color Removal 2.87 93.1％ Batch #4 – Poor Color Removal 4.37 89.2％ Initial crude hot water extract of yerba mate 17.13 40.7％

[0376] Batches 1, 2 and 4 showed acceptable color removal, as evidenced by b* values ​​below the cutoff of 3.5. Batch 3 also showed acceptable color removal, as evidenced by a b* value of 2.87 (below the 3.5 cutoff). Thus, surprisingly, samples corresponding to the methods described above in steps (B1-B6) and Example 3 showed acceptable color removal, even though only a single chromatographic separation step was used and no separate desalting and decolorizing steps were used.

[0377] Example 6 :

[0378] The composition comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts prepared by the above method is analyzed to evaluate the presence of polycyclic aromatic hydrocarbons. The presence of polycyclic aromatic hydrocarbons is analyzed using liquid chromatography with fluorescence detection. The method is similar to the method reported in EPA method 610 for analyzing the PAH content in drinking water. The sample is extracted into acetonitrile, filtered through a 0.2 μm PTFE filter, and injected directly without further purification. The raw material and the final product are prepared so that the total CQA content between them is equal. Additional samples containing only acetonitrile diluent are prepared in the same manner. The results in Table 4 show that the fluorescent material present in the initial leaves is completely removed from the final product, making it comparable to the blank sample.

[0379] Table 4 .

[0380] sample Fluorescence signal (area LU*min) Comparison 0.0415 Dried leaf material 1.7897 Caffeoylquinic acid composition obtained from yerba mate 0.0554

[0381] Example 7 :

[0382] The compositions comprising monocaffeoylquinic acid, dicaffeoylquinic acid and their salts prepared by the above method were analyzed to evaluate the presence of saponins. Table 5 below shows the determined saponin compounds and their corresponding masses.

[0383] Table 5 .

[0384] Compound Molecular formula [MH]-, [M-H+COOH]2- Oleanolic acid or ursolic acid + 3Glc C48H78O18 941.5115、493.2549 Oleanolic acid or ursolic acid + 4Glc C54H88O23 1103.5644、574.2813 Oleanolic acid or ursolic acid + 5Glc C60H98O28 1265.6172、655.3077 Oleanolic acid or ursolic acid + 6Glc C66H108O33 1427.6700、736.3341 Oleanolic acid or ursolic acid + 7Glc C72H118O38 1589.7228、817.3605

[0385] Compositions were prepared from yerba mate biomass using the methods described above in steps (B1-B6) and Example 3. Aliquots were taken throughout the process. Samples as described in Table 6 below were prepared by diluting in 55% methanol / water. The samples were injected into a high-resolution Orbitrap mass spectrometer and full scan data were collected at a mass resolution of 70,000. The accurate mass of the saponins was extracted and the area counts are reported in Table 6.

[0386] Table 6 .

[0387]

[0388] The present invention provides the following embodiments, the numbering of which should not be construed as designating a level of importance: Implementation

[0389] 1. A method for preparing a caffeoylquinic acid composition, the method comprising:

[0390] contacting the biomass with a first aqueous composition to obtain an initial extract;

[0391] chromatographing the initial extract on an ion exchange stationary phase;

[0392] eluting the stationary phase with a first aqueous eluent composition to obtain a first eluate;

[0393] as well as

[0394] The stationary phase is eluted with a second aqueous eluent composition to obtain a second eluent, wherein the second eluent comprises one or more of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts thereof.

[0395] 2. The method according to embodiment 1, wherein the biomass is selected from the group consisting of yerba mate, stevia and globe artichoke.

[0396] 3. The method of embodiment 1, wherein the biomass is comminuted.

[0397] 4. The method of embodiment 1, further comprising removing solids from the initial extract prior to chromatographic separation.

[0398] 5. The method according to embodiment 1 further comprises filtering the initial extract before chromatographic separation.

[0399] 6. The method according to embodiment 1, wherein the monocaffeoylquinic acid comprises one or more of 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid.

[0400] 7. The method according to embodiment 1, wherein the dicaffeoylquinic acid comprises one or more of 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and salts thereof.

[0401] 8. A method according to embodiment 1, wherein the stationary phase is a weak anion exchange stationary phase.

[0402] 9. The method of embodiment 1, wherein the first aqueous composition is water.

[0403] 10. The method of embodiment 1, wherein the first aqueous composition is heated to 50°C to 70°C prior to contacting with the biomass.

[0404] 11. The method according to embodiment 1, wherein the initial extract is contacted with the stationary phase at 20°C-25°C.

[0405] 12. The method according to embodiment 1, wherein the initial extract is contacted with the stationary phase at 1-2 bed volumes per hour.

[0406] 13. The method according to embodiment 1, wherein the initial extract is contacted with the stationary phase at a ratio of at most 40 g of caffeoylquinic acid composition per liter of resin.

[0407] 14. The method according to embodiment 1, wherein the initial extract is contacted with the stationary phase, and the stationary phase is washed with the first aqueous composition to obtain a first wash composition.

[0408] 15. The method of embodiment 1, wherein the first aqueous eluent composition comprises 1% to 40% of a (C1-C4)alkanol.

[0409] 16. The method of embodiment 1, wherein the first aqueous eluent composition comprises 20%-30% of a (C1-C4)alkanol.

[0410] 17. The method of embodiment 1, wherein the (C1-C4)alkanol is ethanol.

[0411] 18. The method of embodiment 1, wherein the first aqueous eluent composition comprises 25% ethanol.

[0412] 19. The method of embodiment 1, wherein the stationary phase is eluted with at least 3 bed volumes of the first aqueous elution composition.

[0413] 20. The method according to embodiment 1, wherein the stationary phase is eluted with the first aqueous elution composition at 20°C-25°C.

[0414] 21. The method of embodiment 1, wherein the stationary phase is eluted with the first aqueous elution composition at a rate of 1-2 bed volumes per hour.

[0415] 22. The method of embodiment 1, wherein the second aqueous eluent composition comprises 50% to 80% of a (C1-C4)alkanol.

[0416] 23. The method of embodiment 1, wherein the second aqueous eluent composition comprises 50%-80% ethanol.

[0417] 24. The method of embodiment 1, wherein the second aqueous eluent composition comprises 70% ethanol.

[0418] 25. The method of embodiment 1, wherein the second aqueous eluent composition comprises 1% to 10% salt.

[0419] 26. The method of embodiment 1, wherein the second aqueous eluent composition comprises sodium chloride.

[0420] 27. The method of embodiment 1, wherein the second aqueous eluent composition is acidified.

[0421] 28. A method according to embodiment 1, wherein the second aqueous elution composition is acidified with phosphoric acid.

[0422] 29. The method of embodiment 1, wherein the second aqueous elution composition is acidified with 0.5% to 1.0% phosphoric acid.

[0423] 30. The method of embodiment 1, wherein the stationary phase is eluted with at least 2 bed volumes of the second aqueous elution composition.

[0424] 31. The method of embodiment 1, wherein the stationary phase is eluted with the first aqueous eluent composition at a rate of 1 bed volume per hour.

[0425] 32. The method of embodiment 1, wherein the second aqueous elution composition is heated to 40°C-50°C prior to eluting the stationary phase.

[0426] 33. The method of embodiment 1, wherein the ratio of monocaffeoylquinic acid and salts thereof to dicaffeoylquinic acid and salts thereof of the second eluent is from about 0.01 to about 1 to about 1 by mass.

[0427] 34. The method according to embodiment 1 further comprises a decolorization step.

[0428] 35. The method according to embodiment 1 further comprises a desalting step.

[0429] 36. The method of embodiment 1 further comprises chromatographing the second eluate using a hydrophobic resin stationary phase to desalt the second eluate.

[0430] 37. The method according to embodiment 1 further comprises drying the first eluent.

[0431] 38. The method according to embodiment 1 further comprises drying the second eluent.

[0432] 39. The method of any one of embodiments 1 to 38, wherein the biomass is yerba mate.

[0433] 40. A method according to embodiment 39, wherein the stationary phase is a weak anion exchange stationary phase.

[0434] 41. The method of embodiment 39, wherein the first aqueous eluent composition comprises 25% ethanol.

[0435] 42. A method according to embodiment 39, wherein the stationary phase is eluted with the first aqueous elution composition at 25°C.

[0436] 43. A method according to embodiment 39, wherein the stationary phase is eluted with the first aqueous elution composition at a rate of 2 bed volumes per hour.

[0437] 44. A method according to embodiment 39, wherein the second aqueous elution composition comprises 70% (v / v) ethanol and 0.75% (w / v) phosphoric acid.

[0438] 45. A method according to embodiment 39, wherein the stationary phase is eluted with the second aqueous eluent composition at a rate of 1 bed volume per hour.

[0439] 46. ​​The method of any one of embodiments 1 to 38, wherein the biomass is Stevia.

[0440] 47. A method according to embodiment 46, wherein the stationary phase is a weak anion exchange stationary phase.

[0441] 48. A method according to embodiment 46, wherein the first aqueous eluent composition comprises 25% ethanol.

[0442] 49. A method according to embodiment 46, wherein the stationary phase is eluted with the first aqueous elution composition at 25°C.

[0443] 50. A method according to embodiment 46, wherein the stationary phase is eluted with the first aqueous eluent composition at a rate of 2 bed volumes per hour.

[0444] 51. The method of embodiment 46, wherein the initial extract is contacted with the stationary phase, and the stationary phase is washed with the first aqueous composition to obtain a first wash composition comprising steviol glycosides.

[0445] 52. The method of embodiment 46, wherein the first eluate comprises steviol glycosides.

[0446] 53. A method according to embodiment 46, wherein the second aqueous eluent composition comprises 70% (v / v) ethanol and 0.88% (w / v) 85 wt% phosphoric acid.

[0447] 54. The method of embodiment 46, wherein the second aqueous eluent composition comprises 70% (v / v) ethanol and 0.75% (w / v) phosphoric acid.

[0448] 55. A method according to embodiment 47, wherein the stationary phase is eluted with the second aqueous eluent composition at a rate of 1 bed volume per hour.

[0449] 56. A method for isolating a caffeoylquinic acid composition from yerba mate biomass, the method comprising:

[0450] contacting yerba mate biomass with a first aqueous composition to obtain an initial extract;

[0451] chromatographing the initial extract on a weak anion exchange stationary phase;

[0452] washing the stationary phase with the first aqueous composition;

[0453] eluting the weak anion exchange stationary phase with an aqueous 25% ethanol composition to obtain a first eluate; and

[0454] The stationary phase is eluted with an aqueous acidified 70% ethanol composition to obtain a second eluate comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof.

[0455] 57. A method for separating a steviol glycoside composition and a caffeoylquinic acid composition from a stevia biomass, the method comprising:

[0456] contacting a stevia biomass with a first aqueous composition to obtain an initial stevia extract;

[0457] chromatographing the initial stevia extract on a weak anion exchange stationary phase;

[0458] washing the stationary phase with the first aqueous composition to obtain a wash solution comprising a steviol glycoside composition;

[0459] Eluting the weak anion exchange stationary phase with an aqueous 25% ethanol composition to obtain a first eluate containing a steviol glycoside composition; and

[0460] The stationary phase is eluted with an aqueous acidified 70% ethanol composition to obtain a second eluate comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof.

[0461] 58. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 3% by weight total of malonate, malonic acid, oxalate, oxalic acid, lactate, lactic acid, succinate, succinic acid, malate, malic acid, citrate, and citric acid in the composition when dried.

[0462] 59. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.5% by weight total of tartrate, tartaric acid, pyruvate, pyruvic acid, fumarate, fumaric acid, ascorbic acid, sorbate, sorbic acid, acetate, and acetic acid in the composition when dried.

[0463] 60. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of sulfate, sulfuric acid, phosphate, phosphoric acid, nitrate, nitric acid, nitrite, nitrous acid, chloride, hydrochloric acid, ammonia, and ammonium in the composition when dried.

[0464] 61. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 5% by weight total of flavones, isoflavones, and neoflavones (quercetin, kaempferol, myricetin, fisetin, galangin, isorhamnetin, horehound, rhamnetin, pyranoflavonols, furoflavonols, luteolin, apigenin, tangeretin, taxifolin (or dihydroquercetin), dihydrokaempferol, hesperetin, naringenin, eriodictyol, homoeriodictyol, genistein, daidzein, glycitein) in the composition when dried.

[0465] 62. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 5% by weight total of hesperidin, naringin, rutin, quercetin, luteolin-glucoside, and quercetin-xyloside in the composition when dried.

[0466] 63. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 5% by weight total of cyanidin, delphinidin, malvidin, pelargonidin, peonyidin, and petuniidin in the composition when dried.

[0467] 64. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of tannins and tannic acid in the composition when dried.

[0468] 65. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.1% by weight total of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine in the composition when dried.

[0469] 66. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of monoglycerides, diglycerides, and triglycerides in the composition when dried.

[0470] 67. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of monosaccharides, disaccharides, polysaccharides, glucose, fructose, sucrose, galactose, ribose, trehalose, trehalulose, lactose, maltose, isomaltose, isomaltulose, mannose, tagatose, arabinose, rhamnose, xylose, dextrose, erythrose, threose, maltotriose, and panose in the composition when dried.

[0471] 68. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of sugar alcohols, glycerol, sorbitol, mannitol, xylitol, maltitol, lactitol, erythritol, isomalt, and inositol in the composition when dried.

[0472] 69. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total of dietary fiber, acacia (arabic) gum, agar, algin-alginate, arabinoxylan, β-glucan, β-mannan, carrageenan, carob or locust bean gum, fenugreek gum, galactomannan, gellan gum, glucomannan or konjac gum, guar gum, hemicellulose, inulin, karaya gum, pectin, polydextrose, psyllium husk mucilage, resistant starch, tara gum, tragacanth gum, xanthan gum, cellulose, chitin, and chitosan in the composition when dried.

[0473] 70. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.1% by weight combined of chlorophyll, furans, furan-containing chemicals, theobromine, theophylline, and trigonelline in the composition when dried.

[0474] 71. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total caffeine in the composition when dried.

[0475] 72. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 1% by weight total rutin in the composition when dried.

[0476] 73. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.5% by weight total of glycosylated ursolic acid and glycosylated oleanolic acid in the composition when dried.

[0477] 74. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.5% by weight total of volatile organic compounds, terpenes, eugenol, geraniol, geranial, α-ionone, β-ionone, epoxyionone, limonene, linalool, linalool oxide, nerol, and damascenone in the composition when dried.

[0478] 75. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.5% by weight total of fatty acid oxidation products, decanone, decenal, nonenal, octenal, heptenal, hexenal, pentenals, pentenols, pentenone, hexenone, hydroxynonenal, and malondialdehyde in the composition when dried.

[0479] 76. The method of any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.01% by weight in total of polycyclic aromatic hydrocarbons (PAHs) such as acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(ghi)perylene, benzo(k)fluoranthene, acenaphthene ...acenaphthene, anthracene, acenaphthene, anthracene, acenaphthene, anthracene, acenaphthene, anthracene, acenaphthene, anthracene dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, naphthalene, phenanthrene and pyrene.

[0480] 77. The method according to embodiment 1, wherein the polycyclic aromatic hydrocarbons (PAH) (acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(ghi)perylene, benzo(k)fluoranthene, Dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, naphthalene, phenanthrene, pyrene, etc.) were removed to less than 0.01 wt%.

[0481] 78. A method according to any one of embodiments 1 to 57, wherein the caffeoylquinic acid composition comprises less than 0.5% by weight in total of fatty acid oxidation products, decanone, decenal, nonenal, octenal, heptenal, hexenal, pentenals, pentenols, pentenone, hexenone, hydroxynonenal, and malondialdehyde in the composition when dried.

[0482] 79. The method of any one of embodiments 1 to 57, wherein color is removed such that the % transmittance at 430 nm is > 80%.

[0483] 80. A method according to any one of embodiments 1 to 57, wherein color is removed so that the b value on the CIE L*a*b* color space is less than 4.

[0484] 81. The method according to any one of embodiments 1 to 33 and 37 to 57, wherein color is removed such that the % transmittance at 430 nm is > 80% without the need for a decolorization and / or desalting step.

[0485] 82. The method of any one of embodiments 1 to 33 and 37 to 57, wherein color is removed such that the b-value on the CIEL*a*b* color space is less than 4 without requiring a decolorization and / or desalting step.

[0486] 83. A composition comprising at least one of monocaffeoylquinic acid and dicaffeoylquinic acid and salts thereof, the composition being prepared by the method according to any one of embodiments 1 to 82.

[0487] 84. An ingestible composition comprising the composition according to embodiment 83.

[0488] 85. The ingestible composition of embodiment 84, wherein the ingestible composition is a beverage or a food product.

Claims

1. A method for preparing a caffeoylquinic acid composition, the method comprising: i) contacting the biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on an ion exchange stationary phase; iii) eluting the stationary phase with a first aqueous eluent composition comprising 10% to 50% by volume of a (C1-C4)alkanol to obtain a first eluent; and iv) eluting the stationary phase with an acidified second aqueous eluent composition comprising 60% to 80% by volume of a (C1-C4)alkanol to obtain the caffeoylquinic acid composition, The caffeoylquinic acid composition comprises one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts of the foregoing substances, wherein the caffeoylquinic acid composition comprises less than 1% by weight of caffeine or rutin in the caffeoylquinic acid composition when dried, and wherein the caffeoylquinic acid composition has a transmittance % at 430 nm greater than 80% without the need for a decolorization or desalination step after step iv), The biomass is selected from yerba mate and stevia.

2. A method for preparing a caffeoylquinic acid composition, the method comprising: i) contacting the biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on an ion exchange stationary phase; iii) eluting the stationary phase with a first aqueous eluent composition comprising 10% to 50% by volume of a (C1-C4)alkanol to obtain a first eluent; and iv) eluting the stationary phase with an acidified second aqueous eluent composition comprising 60% to 80% by volume of a (C1-C4)alkanol to obtain the caffeoylquinic acid composition, The caffeoylquinic acid composition comprises one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts of the foregoing substances, wherein the caffeoylquinic acid composition comprises less than 1 wt% caffeine or rutin in the caffeoylquinic acid composition when dried, and wherein the caffeoylquinic acid composition has a transmittance % at 430 nm greater than 80% without requiring a decolorization or desalting step after step iv), wherein the method prepares the caffeoylquinic acid composition using a single chromatographic separation step, The biomass is selected from yerba mate and stevia.

3. The method according to claim 1 or 2, wherein the biomass is yerba mate.

4. The method of claim 1 or 2, further comprising removing solids from the initial extract prior to chromatographic separation.

5. The method according to claim 1 or 2, wherein the monocaffeoylquinic acid comprises one or more of 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid.

6. The method according to claim 1 or 2, wherein the dicaffeoylquinic acid comprises one or more of 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and salts thereof. The method according to claim 1 , wherein the stationary phase is a weak anion exchange stationary phase.

8. The method of claim 1 or 2, wherein the first aqueous composition is water.

9. The method of claim 1 or 2, wherein the first aqueous composition is heated to 50°C to 70°C prior to contacting with the biomass.

10. The method according to claim 1 or 2, wherein the initial extract is contacted with the stationary phase at 20°C-25°C.

11. The method according to claim 1 or 2, wherein the initial extract is contacted with the stationary phase at 1-2 bed volumes per hour.

12. The process of claim 1 or 2, wherein the initial extract is contacted with the stationary phase at a rate of at most 40 g of caffeoylquinic acid composition per liter of resin.

13. The method of claim 1 or 2, wherein the initial extract is contacted with the stationary phase, and the stationary phase is washed with the first aqueous composition to obtain a first wash composition.

14. The method of claim 1 or 2, wherein the first aqueous eluent composition comprises 20%-30% or 25% by volume of (C1-C4)alkanol.

15. The process of claim 14, wherein the (C1-C4)alkanol is ethanol.

16. The method of claim 1 or 2, wherein the stationary phase is eluted with at least 3 bed volumes of the first aqueous elution composition.

17. The method of claim 1 or 2, wherein the stationary phase is eluted with the first aqueous elution composition at 20°C-25°C.

18. The method of claim 1 or 2, wherein the stationary phase is eluted with the first aqueous elution composition at a rate of 1-2 bed volumes per hour.

19. The method of claim 3, wherein solids are removed from the initial extract by filtration.

20. The method of claim 1 or 2, wherein the second aqueous elution composition comprises 1%-10% of a salt selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, potassium sulfate, sodium phosphate, potassium phosphate.

21. The method of claim 1 or 2, wherein the second aqueous elution composition is acidified with 0.5% to 1.0% phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, or formic acid.

22. The method of claim 1 or 2, wherein the stationary phase is eluted with at least 2 bed volumes of the second aqueous elution composition.

23. The method of claim 1 or 2, wherein the second aqueous elution composition is heated to 40°C-50°C prior to eluting the stationary phase.

24. The method of claim 1 or 2, wherein the ratio of monocaffeoylquinic acid and salts thereof to dicaffeoylquinic acid and salts thereof of the caffeoylquinic acid composition is 0.01:1 to 1:1, 0.1:1 to 0.5:1, 1:1 to 10:1, 3:1 to 10:1, 3:1 to 5:1 or 3:2 to 4:1 by mass.

25. The method according to claim 1 or 2, further comprising a decolorization step.

26. The method according to claim 1 or 2, further comprising a desalting step.

27. The method of claim 1 or 2, further comprising chromatographically separating the caffeoylquinic acid composition using a hydrophobic resin stationary phase to desalt the caffeoylquinic acid composition.

28. The method of claim 1 or 2, further comprising drying the first eluate, drying the caffeoylquinic acid composition, or drying the first eluate and the caffeoylquinic acid composition.

29. The method of claim 1 or 2, wherein the biomass is Stevia.

30. The method of claim 3, wherein the stationary phase is a weak anion exchange stationary phase.

31. The method of claim 1 or 2, wherein the biomass is yerba mate and the second aqueous eluent composition comprises 70% (v / v) ethanol and 0.75% (w / v) phosphoric acid.

32. The method of claim 1 or 2, wherein the first eluate comprises steviol glycosides.

33. The method of claim 1 or 2, wherein the biomass is Stevia and the second aqueous elution composition comprises 70% (v / v) ethanol and 0.75% (w / v) phosphoric acid.

34. A method for isolating a caffeoylquinic acid composition from yerba mate biomass, the method comprising: i) contacting yerba mate biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluent; and iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60 to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof, wherein the caffeoylquinic acid composition comprises less than 1% by weight of caffeine or rutin in the caffeoylquinic acid composition when dried, and The transmittance % of the caffeoylquinic acid composition at 430 nm is greater than 80%, without the need for a decolorization or desalting step after step iv).

35. A method for isolating a caffeoylquinic acid composition from yerba mate biomass, the method comprising: i) contacting yerba mate biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluent; and iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60 to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof, wherein the caffeoylquinic acid composition comprises less than 1 wt% caffeine or rutin in the caffeoylquinic acid composition when dried, and wherein the caffeoylquinic acid composition has a transmittance % at 430 nm greater than 80% without requiring a decolorization or desalting step after step iv), wherein the method separates the caffeoylquinic acid composition using a single chromatographic separation step.

36. A method for separating a steviol glycoside composition and a caffeoylquinic acid composition from a stevia biomass, the method comprising: i) contacting a stevia biomass with a first aqueous composition to obtain an initial stevia extract; ii) chromatographically separating the initial stevia extract on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluate comprising a steviol glycoside composition; and iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60 to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof, wherein the caffeoylquinic acid composition comprises less than 1% by weight of caffeine or rutin in the caffeoylquinic acid composition when dried, and The transmittance % of the caffeoylquinic acid composition at 430 nm is greater than 80%, without the need for a decolorization or desalting step after step iv).

37. A method for separating a steviol glycoside composition and a caffeoylquinic acid composition from a stevia biomass, the method comprising: i) contacting a stevia biomass with a first aqueous composition to obtain an initial stevia extract; ii) chromatographically separating the initial stevia extract on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluate comprising a steviol glycoside composition; and iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60% to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts of the foregoing, wherein the caffeoylquinic acid composition when dried comprises less than 1% by weight of caffeine or rutin in the caffeoylquinic acid composition, and wherein the caffeoylquinic acid composition has a transmittance of greater than 80% at 430 nm, without a decolorization or desalting step after step iv), wherein the method separates the caffeoylquinic acid composition using a single chromatographic separation step.

38. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises one or more of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts of the foregoing.

39. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 3 wt% total of malonate, malonic acid, oxalate, oxalic acid, lactate, lactic acid, succinate, succinic acid, malate, malic acid, citrate, and citric acid based on the dry weight of the composition.

40. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.5 wt% total of tartrate, tartaric acid, pyruvate, pyruvic acid, fumarate, fumaric acid, ascorbic acid, sorbate, sorbic acid, acetate, and acetic acid based on the dry weight of the composition.

41. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt % total of sulfate, sulfuric acid, phosphate, phosphoric acid, nitrate, nitric acid, nitrite, nitrous acid, chloride, hydrochloric acid, ammonia, and ammonium, based on the dry weight of the composition.

42. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 5% by weight total of flavones, isoflavones, and neoflavones based on the dry weight of the composition.

43. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 5 wt% in total of hesperidin, naringin, rutin, quercetin, luteolin-glucoside, and quercetin-xyloside, based on the dry weight of the composition.

44. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 5% by weight in total of anthocyanin, delphinium, malvaceae, pelargonium, peony, and morning glory pigments, based on the dry weight of the composition.

45. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt% tannins and tannic acid combined, based on the dry weight of the composition.

46. ​​The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.1 wt% total of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, based on the dry weight of the composition.

47. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt% total of monoglycerides, diglycerides, and triglycerides based on the dry weight of the composition.

48. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt% in total of monosaccharides, disaccharides, polysaccharides, glucose, fructose, sucrose, galactose, ribose, trehalose, trehalulose, lactose, maltose, isomaltose, isomaltulose, mannose, tagatose, arabinose, rhamnose, xylose, dextrose, erythrose, threose, maltotriose, and panose, based on the dry weight of the composition.

49. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt % total of sugar alcohols, glycerol, sorbitol, mannitol, xylitol, maltitol, lactitol, erythritol, isomalt, and inositol, based on the dry weight of the composition.

50. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1 wt% dietary fiber, acacia gum, agar, algin-alginate, arabinoxylan, beta-glucan, beta-mannan, carrageenan, carob or locust bean gum, fenugreek gum, galactomannan, gellan gum, glucomannan or konjac gum, guar gum, hemicellulose, inulin, karaya gum, pectin, polydextrose, psyllium husk mucilage, resistant starch, tara gum, tragacanth gum, xanthan gum, cellulose, chitin, and chitosan in total, based on the dry weight of the composition.

51. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.1 wt% in total of chlorophyll, furans, furan-containing chemicals, theobromine, theophylline, and trigonelline, based on the dry weight of the composition.

52. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1% total caffeine by weight based on the dry weight of the composition.

53. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 1% by weight total rutin based on the dry weight of the composition.

54. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.5 wt% glycosylated ursolic acid and glycosylated oleanolic acid in total, based on the dry weight of the composition.

55. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.5 wt% in total of volatile organic compounds, terpenes, eugenol, geraniol, geranial, α-ionone, β-ionone, epoxyionone, limonene, linalool, linalool oxide, nerol, and damascenone, based on the dry weight of the composition.

56. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.5 wt% in total of fatty acid oxidation products, decanone, decenal, nonenal, octenal, heptenal, hexenal, pentenals, pentenols, pentenone, hexenone, hydroxynonenal, and malondialdehyde, based on the dry weight of the composition.

57. The method of any one of claims 1, 2, and 34-37, wherein the caffeoylquinic acid composition comprises less than 0.01 wt% in total of polycyclic aromatic hydrocarbons (PAHs), acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(ghi)perylene, benzo(k)fluoranthene, acenaphthene ...acenaphthene, anthracene, acenaphthene, anthracene, acenaphthene, anthracene, acenaphthene, anthracene, anthracene, dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, naphthalene, phenanthrene and pyrene.

58. The method of any one of claims 1, 2, and 34-37, wherein color is removed such that the % transmittance at 430 nm is > 80%.

59. The method of any one of claims 1, 2, and 34-37, wherein color is removed such that the b value on the CIEL*a*b* color space is less than 4.

60. The method of any one of claims 1, 2, and 34-37, wherein color is removed such that the % transmittance at 430 nm is > 80% without requiring a decolorization and / or desalting step.

61. The method of any one of claims 1, 2, and 34-37, wherein color is removed such that the b-value on the CIEL*a*b* color space is less than 4 without requiring a decolorization and / or desalting step.

62. The method of any one of claims 1, 2, and 34-37, wherein the first aqueous composition is free of organic solvents.

63. A method for isolating a caffeoylquinic acid composition from Stevia biomass, the method comprising: i) contacting Stevia biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluate; as well as iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60% to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof, wherein the caffeoylquinic acid composition comprises less than 1% by weight of caffeine or rutin in the caffeoylquinic acid composition when dried, and The transmittance % of the caffeoylquinic acid composition at 430 nm is greater than 80%, without the need for a decolorization or desalting step after step iv).

64. A method for isolating a caffeoylquinic acid composition from Stevia biomass, the method comprising: i) contacting Stevia biomass with a first aqueous composition to obtain an initial extract; ii) subjecting the initial extract to chromatographic separation on a weak anion exchange stationary phase; iii) eluting the weak anion exchange stationary phase with an aqueous ethanol composition comprising 10% to 50% by volume of ethanol to obtain a first eluate; as well as iv) eluting the stationary phase with an acidified aqueous ethanol composition comprising 60% to 80% by volume of ethanol to obtain a caffeoylquinic acid composition comprising one or more of monocaffeoylquinic acid, dicaffeoylquinic acid and salts thereof, wherein the caffeoylquinic acid composition comprises less than 1 wt% caffeine or rutin in the caffeoylquinic acid composition when dried, and wherein the caffeoylquinic acid composition has a transmittance % at 430 nm greater than 80% without requiring a decolorization or desalting step after step iv), wherein the method separates the caffeoylquinic acid composition using a single chromatographic separation step.

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