US 2014034 1934A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0341934 A1 van Spronsen et al. (43) Pub. Date: Nov. 20, 2014

(54) PROCESS FOR EXTRACTING MATERALS Publication Classification FROM BIOLOGICAL MATERAL (51) Int. Cl. (71) Applicant: Universiteit Leiden, Leiden (NL) C09K3/00 (2006.01) C07H 17/07 (2006.01) (72) Inventors: Jacob van Spronsen, Noordwijk (NL); C07D 3II/62 (2006.01) Geert-Jan Witkamp, Bergschenhoek (52) U.S. Cl. (NL); Frank Hollman, Den Haag (NL); CPC ...... C09K3/00 (2013.01); C07D 3II/62 Young Hae Choi, Leiden (NL); Robert (2013.01); C07H 17/07 (2013.01) Verpoorte, Leiden (NL) USPC ..... 424/184.1: 435/183: 530/425; 536/25.41: 536/123.1; 549/400: 536/18.1; 252/364 (21) Appl. No.: 14/451,845

(22) Filed: Aug. 5, 2014 (57) ABSTRACT Related U.S. Application Data (63) Continuation of application No. 13/702,633, filed on The invention is directed to a process for extracting materials Feb. 18, 2013, filed as application No. PCT/NL2011/ from biological material, which process is characterized in 050407 on Jun. 7, 2011. that the naturally occurring biological material is treated with an extractant consisting of a deep eutectic solvent of natural (30) Foreign Application Priority Data origin or a an ionic liquid of natural origin to produce a biological extract of natural origin dissolved in the said sol Jun. 7, 2010 (NL) ...... 2004.835 vent or ionic liquid. US 2014/034 1934 A1 Nov. 20, 2014

PROCESS FOR EXTRACTING MATERALS carboxylic acids. The deep eutectic phenomenon was first FROM BIOLOGICAL MATERAL described in 2003 for a mixture of choline chloride and urea 0001. The present invention is directed to a process for in a 1:2 mole ratio, respectively. Other deep eutectic solvents extracting materials from biological materials. of choline chloride are formed with phenol and glycerol. 0002 Drugs, flavors, fragrances, agrochemicals, dyes etc., Deep eutectic solvents are able to dissolve many metal salts both from synthetic and natural Sources are often poorly like lithium chloride and copper(II) oxide. Also, organic com soluble in water. Therefore extraction, purification, adminis pounds such as benzoic acid and cellulose have great Solubil tration requires the use of less polar solvents, such as alco ity in deep eutectic solvents. Compared to ordinary Solvents, hols, acetone, ethyl acetate, chloroform etc. Such solvents eutectic solvents have a very low volatility and are non present several problems such as: toxicity for the producer/ flammable. They share a lot of characteristics with ionic patient/consumer, environmental problems, explosions and liquids, but they are ionic mixtures and notionic compounds. the like. 0011 Instead, choline citrate is a real ionic liquid. This 0003 Ionic liquids can be environmentally benign and compound was formed by dissolving citric acid in water, safe replacements for the traditional Volatile organic solvents followed by addition of choline hydroxide (in the ratio 2:1) in various chemical processes. The reason that ionic liquids dissolved in methanol. The solvent (water and methanol) was are considered to be green solvents is their negligible vapor evaporated. The product choline citrate was a slightly yellow pressure. However, ionic liquids can have a hidden environ viscous liquid, and not a solid. This is probably the first mental cost because they are synthesized from petrochemical naturally occurring ionic liquid found. resources. In a lot of synthesis routes halogen atoms are involved. Halogen materials in ionic liquids are undesirable, because of the low hydrolysis stability, the high toxicity, the low biodegradability and the high disposal cost. For example, n-nu-O" fluorinated anions such as PF- and BF - are sensitive to water 1. O O and may release the corrosive and toxic hydrogen fluoride. Moreover, the alkyl halides used in the syntheses of many ionic liquids are greenhouse gases and OZone-depleting mate rials. choline citrate 0004. The reason that ionic liquids are also considered to 0012. In addition to the ions, sugar-based liquids can be be safe solvents is because their lack of volatility greatly deep eutectic solvents. reduces any chance of exposure other than by direct physical 0013. According to the invention a process for extracting contact with skin or by ingestion. However, most conven materials from biological material is provided, which process tional ionic liquids are irritating and have a toxicity compa is characterized in that the naturally occurring biological rable to common organic solvents. From biological tests it material is treated with an extractant consisting of a deep appeared that the toxicity of ionic liquids is mainly deter eutectic solvent of natural origin oraan ionic liquid of natural mined by the type of cation and that ionic liquids with short origin to produce a biological extract of natural origin dis alkyl Substituents in the cation usually have a lower toxicity. Solved in the said solvent or ionic liquid. 0005. A solution to the problems mentioned above is the development of halogen-free ionic liquids, such as ionic liq 0014 Surprisingly it has been found that deep eutectic uids with the alkyl sulfate, the alkyl carbonate and the sul Solvents of natural origin, as defined herein, and natural ionic fonate anion. It was also found that some ionic liquids with liquids are suitable extractants for biological materials. These ester groups in their alkyl side chains are biodegradable. extractants are very efficient and selective, and as they are of However, these ionic liquids are still synthesized using pet natural origin, they are extremely efficient and suitable for rochemical resources. extracting components from biological materials, resulting in 0006. In WO2006/116126 a process is described for an efficient process, providing a good yield. The melting extracting biopolymers from biomass, using ionic liquids. points of the deep eutectic mixtures and ionic liquids is pref Generally the ionic liquids described therein are of petro erable below 25°C. The materials are thus preferably liquid at chemical nature. The biopolymers extracted are chitin, chito ambient temperatures. san, collagen and keratin. Polyhydroxyalkanoate is extracted 0015 Suitable deep eutectic solvents to be used in the from genetically engineered plants. present invention, i.e. mixtures of materials of natural origin, 0007 As indicated above, there is a need for an improved are based on mixtures of at least two compounds, Substan process for extracting organic compounds from natural tially without chemical or ionic bonding. The first component Sources, without the need for the use of organic solvents or of the solvents is preferably selected from at least one natu other synthetic materials. rally occurring organic acid or an inorganic compound. Such 0008 Further, there is a need for a process that can truly be as a salt. considered green, i.e. using only natural compounds. 0016. The second component is preferably selected from 0009. The invention is based on the surprising fact that at least one naturally occurring mono- or dimeric Sugar, Sugar Some specific naturally occurring materials can Suitably be alcohol, amino acid, di or trialkanol or choline derivatives, used for extracting materials from biological Sources. These Such as choline or phosphatidylcholine. materials are deep eutectic solvents (or mixtures) of natural 0017 Said sugar or sugar alcohol may be selected from the origin or ionic liquids of natural origin. group of Sucrose, glucose, fructose, lactose, maltose, cello 0010 Deep eutectic solvents are liquids having a melting biose, arabinose, ribose, ribulose, galactose, rhamnose, raffi point that is much lower than the melting points of the two nose, Xylose, Sucrose, mannose, trehalose, mannitol, Sorbitol, compounds that form the eutectic mixture. Generally, they are inositol, ribitol, galactitol, erythritol. Xyletol and adonitol, formed between a variety of quaternary ammonium salts and and, as well as their phosphates. US 2014/034 1934 A1 Nov. 20, 2014

0018. The said organic acid may be selected from malic Deep eutectic mixture of malic acid with choline chloride acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric acid, Succinic acid, lactic acid, acetic acid, aconitic acid, OH O N C OH tartaric acid, malonic acid, ascorbic acid, glucuronic acid, O OH D-n- oxalic acid, neuraminic acid and sialic acids. 0019. In general it is preferred that the ionic liquid or deep Deep eutectic mixture of maleic acid with choline chloride eutectic solvent is free of chlorine/chloride. 0023 Suitable ionic liquids are based on naturally occur ring anions selected from the group of malic acid, maleic acid, 0020. In certain solvents additionally further components citric acid, lactic acid, tartaric acid glucosamine, glucuronic may be present, such as water, phenolics, etc. These addi acid, neuraminic acid and sialic acids. tional compounds are generally present in minor amounts, 0024. The said ionic liquid is further based on naturally such as below 5 wt.%. occurring cations selected from the group of choline, betaine, betanine, gamma-amino butyric acid, betalaine, acetylcho 0021 Suitable examples of inorganic compounds are the line, glucosamine, glutamine, glutamate, asparagine, aspartic phosphates, sulfates, sulfites and halogenides, such as acid, alanine, lysine, arginine, proline, threonine, putrescine, NaH2PO Na HPO, NaHSO, NaSO, CaCl, MgCl, cadaverine and choline derivatives. KC1, NaCl and KI. 0025. In a more preferred embodiment the said ionic liq 0022 Specific examples of deep eutectic solvents are uid is choline citrate. given in the table below, but also honey, maple syrup, and 0026. The ratio of the components of the deep eutectic nectar are examples of deep eutectic solvents that can be used Solvents and ionic liquids depends on the structure of the two or more constituents of the solvent or liquid. as extraction solvent (which are based on Sugar, and Small 0027. For deep eutectic solvents quite often the two com amounts of phenolics and amino acids). ponents are present in an equimolar ratio, although other ratio’s have also been observed. Generally however, the molar ratio can be expressed in whole numbers. These ratio’s C generally vary from 1:1 to 4:1. OH OH 0028 Ionic liquids are by definition salts anions and cat ions and accordingly the ratio is determined by the Valence of HO -- >~ the ions. 0029. In the following tables 1 and 2 the composition and properties of deep eutectic Solvent (des), as well as some solubility data have been given. TABLE 1. The composition and properties of deep eutectic solvent (des Water density viscosity Composition H2O w activity (40°C.) (40°C.) No. (molar ratio) % (40°C.) g/cm3 mm2/s Et (NR) T. C. T. C. MCH Ma:Ch:HO(1:1:2) 11.62%. O.195 246 445.9 44.81 2O1 -71.32 GlyCH Gly:Ch:HO(2:1:1) S.26% 0.126 1742 51.3 49.SS 187 -101.59 MAH Ma:f3-Ala:H2O (1:1:3) 19.48% 0.573 352 1746 48.05 164 -70.88 PMH Pro:Ma:HO(1:1:3) 17.81% 0.591 31.84 251 48.3 1S6 -61.29 CaGH8 CaCl2:Glc:HO(5/4:1:8) 31.11% 0.331 4904 720 54.56 137 -61.39 FCH Fru:Ch:HO (1:2.5:2.5) 7.84% 0.151 2O78 280.8 49.81 160 -84.58 XCH Xyl:Ch:HO (1:2:2) 7.74% 0.141 .2095 3O8.3 49.81 178 -81.8 SCH Suc:Ch:H2O (1:4:4) 7.40% 0.182 2269 581 49.72 >2OO -82.96 FGSH Fru:Gle:Suc:HO (1:1:1:11) 18.70% 0.662 3657 720 48.21 138 -50.77 GCH Glc:Ch:HO (1:2.5:2.5) 7.84% 0.162 197 3974 49.72 170 -83.86 PdCH 12Prop:Ch:H2O (1:1:1) 7.70% O.242 .0833 33.0 50.07 162 -109.SS LGH Lac:Glc:HO(5:1:2) 7.89% 0.496 2495 37.0 44.81 135 -77.06 SoCH So:Ch:HO(1:2.5:3) 11.17% 0.12 1853.85 138.4 49.98 >2OO -89.62 XoCH Xo:Ch:HO(1:2:3 11.17% 0.116 17841 86.1 49.72 >2OO -93.33 12.58% 0.218 .24729 49.13 -84.4 15.62% 0.213 18469 49.21 -72.96

Ala = alanine Ch = choline Fru = fructose Glc = glucose Gly= glycerol Lact—lactose Ma = malic acid 1,2Pro = 1,2-propanediol Pro = proline So = sorbitol Suc = sucrose Xo = xylitol Xyl = xylose US 2014/034 1934 A1 Nov. 20, 2014

TABLE 2

Summary of some solubility data in some ical Des at 40°C. (mg/ml) (n = 3 1,8-dihydroxy Des rutin quercetin cinnamic acid carthamin anthraquinone paclitaxel ginkgolide B

LGH 8.14 - 0.79 1.72 0.09 13.11 - 0.38 1240.21 O.20 OO1 5.39 O.S.S 1.93 - 0.31 GCH 121.63 - 1.45 2006 - 0.41 8.64 - 0.46 27.200.39 O.200.04 O.83 O16 585 0.42 PCH 352.90 31.19 205177.31 58.29 2.79 22.47 1.OO O.17 O.OO 11.71 - 0.68 78.42 1445 SOCH 149.21 2.61 145.84 2.82 4.50 - 0.21 14.05 - 0.66 O.O8 OO1 O.59 OO1 17O O.O1 HO O.O28 O.OO O.O3S O.OO3 O.S7 O.O1 1.43 O.O1 OOOOOO O.O1 OOO O.15 O.OO

0030 The present invention deals with extracting materi Solvent in enzymatic reactions. These reactions are then car als from biological products. In the most general scope, all ried out in the said solvent or liquid. An example is the laccase materials of biological origin may be used. Suitable examples reaction. are plants, insects, animals or micro-organisms. 0031. From these materials a great variety of products can 0035. The invention is now elucidated on the basis of the be isolated using the process of the present invention. More in following examples. particular the extracted or dissolved material is a flavonoid (e.g. rutin and quercetin), an anthocyanin, a colorant, an alka EXAMPLES loid, a terpenoid, a phenylpropanoid a glycoside, a phenolic compound, such as cinnamic acid, a ginkgolide, carthamin, 0036 First the solubility of natural products, which are not an anthraquinone, paclitaxel, taxoid, a lignan, a coumarin, a soluble in water, was evaluated in a few selected natural deep cinnamic acid derivative, azadirachtin, artimisinin, a hop bit eutectic solvents. Several flavonoids were chosen as the natu ter acid, a cannabinoid, Vanillin, a polyketide, a colorant, a ral water-insoluble products, because they are one of the most flavor, a fragrance, a dye, a biocide or a mixture of any of these abundant water-insoluble plant secondary metabolites. Up to compounds. Also proteins (enzymes), toxins, vaccins, DNA, now more than 500 flavonoids have been known. Most of RNA and polysaccharides may be extracted from suitable these flavonoids occur in their glycosides forms (bounded to SOUCCS, a sugar molecule) in plants. In spite of large abundance of 0032. In particular, the invention is directed to extracting flavonoids in plants, both the glycoside and the aglycone natural materials from natural Sources, i.e. not genetically engineered. In a further preferred embodiment, valuable (non-Sugar) part are not soluble in water. Thus, as a model materials are thus extracted or dissolved, such as non-poly research, the Solubility of typical flavonoids including quer meric compounds, as listed above. Non-polymeric com cetin (aglycone), quercitrin (quercetin-3-O-rhamnoside) and pounds are defined as those compounds that do not consist of rutin (quercetin-3-O-rhanmoglucoside), which have a very three or more repeating units of the same moiety (monomer) low water solubility, were tested in the naturally occurring or of the same type of monomers, such as amino acids or deep eutectic solvents. The structure of these flavonoids are Sugars. shown below. 0033. These non-polymeric materials are, for example, Suitable intermediates or products Suitable in food, pharma, cosmetics and agrochemicals. More in particular it is pre OH ferred to extract flavors and fragrance from plant, Vanillin from Vanilla, capsaicin from Capsicum, hop bitter acids from OH hops, cannabinoids from cannabis, azadirachtin from neem plant material, paclitaxel from Taxus plant material, artimisi HO O nin from Artemisia plant material, alkaloids from Catharan thus, morphine and codeine from Papaver plant material, atropine and hyoscyamine from Solanacea plant material, OH galanthamine from Amaryllidaceae plants, antixoxidants from plant material, antibiotics from microorganisms, colo rants from plants and microorganisms, flavonoids from plant materials, anthocyanins and carotenoids from flowers, an essential oil from a plant. 0034. In another embodiment, specific polymeric com pounds are extracted or dissolved, such as RNA, DNA, pro teinic materials such as enzymes, toxins, vaccines, but excluding keratin, elastin and collagen, or polysaccharides, excluding chitin and chitosan. Preferred polysaccharides to be extracted or dissolved are lentinan, heparin, hyaluronan, alginate, agar, starch and inuline. The extracted materials can Subsequently be isolated from the ionic liquid or deep eutectic solvent. It is also possible to use the solution as such for further processes. An example thereof of is the use of extracted enzymes, dissolved in the ionic liquid or eutectic US 2014/034 1934 A1 Nov. 20, 2014

-continued 19. The process according to claim 16, wherein said Sugar OH or Sugar alcohol is selected from the group consisting of Sucrose, glucose, fructose, lactose, maltose, cellobiose, ara binose, ribose, ribulose, galactose, rhamnose, raffinose, Xylose, Sucrose, mannose, trehalose, mannitol, Sorbitol, inositol. Xylitol, ribitol, galactitol, erythritol and adonitol, or a phosphate thereof. 20. The process according to claim 17, wherein said Sugar OH or Sugar alcohol is selected from the group consisting of Sucrose, glucose, fructose, lactose, maltose, cellobiose, ara binose, ribose, ribulose, galactose, rhamnose, raffinose, Xylose, Sucrose, mannose, trehalose, mannitol, Sorbitol, HO inositol. Xylitol, ribitol, galactitol, erythritol and adonitol, or HO a phosphate thereof. OH 21. The process according to claim 18, wherein said Sugar or Sugar alcohol is selected from the group consisting of Structures of quercetin, quercitrin and rutin (left to right) Sucrose, glucose, fructose, lactose, maltose, cellobiose, ara 0037. As shown in the table below the three flavonoids binose, ribose, ribulose, galactose, rhamnose, raffinose, were found to be well dissolved in the natural deep eutectic Xylose, Sucrose, mannose, trehalose, mannitol, Sorbitol, solvents, with solubilities that are 2 to 4 orders of magnitude inositol. Xylitol, ribitol, galactitol, erythritol and adonitol, or higher as compared to their solubilities in water. a phosphate thereof. 22. The process according to claim 16, wherein the said organic acid is selected from the group consisting of malic TABLE 3 acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric Solubility of flavonoids in several naturally occurring deep acid, Succinic acid, acetic acid, aconitic acid, tartaric acid, eutectic solvents ascorbic acid, malonic acid, oxalic acid, glucuronic acid, Solubility (mg/ml neuraminic acid and sialic acids. 23. The process according to claim 17, wherein the said Deep eutectic solvent Quercetin Quercitrin Rutin organic acid is selected from the group consisting of: malic Sucrose + Choline 15.63 O.S7 12.68 - 0.38 2.41 - 0.18 acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric chloride acid, Succinic acid, acetic acid, aconitic acid, tartaric acid, Glucose + Choline 21.56 0.94 7.81 - O20 4.78 O.84 ascorbic acid, malonic acid, oxalic acid, glucuronic acid, chloride neuraminic acid and sialic acids. Fructose + Choline 2334 2.54 11.25 + 0.64 10.94 1.70 chloride 24. The process according to claim 18, wherein the said Water O3OOOOO2 O.159, O.OO1 <0.001 organic acid is selected from the group consisting of malic acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric acid, Succinic acid, acetic acid, aconitic acid, tartaric acid, 0038. In order to confirm the solubility of flavonoids and ascorbic acid, malonic acid, oxalic acid, glucuronic acid, the related anthocyanins, the flowers of red rose were neuraminic acid and sialic acids. extracted in the naturally occurring ionic liquids. It was 25. The process according to claim 20, wherein the deep observed that the red color metabolites are localized in the eutectic solvent comprises water. epidermis cells. 26. The process according to claim 16, wherein the inor 0039 Extraction with the deep eutectic solvent fructose/ ganic compound is selected from the group consisting of glucose/malic acid (1:1:1 molar ratio) resulted in color phosphates, Sulfates, sulfites and halogenides, such as removal from the flowers into the deep eutectic solvent phase. NaH2PO Na HPO, NaHSO, NaSO, MgCl, CaCl, The structure of the flowers remained intact, with no break KC1, NaCl and KI. down of the natural structure. 27. The process according to claim 16, wherein the deep 1-15. (canceled) eutectic solvent has a melting point below 25°C. 16. A process for extracting materials from a biological 28. The process according to claim 16, wherein the material, which process is characterized in that the biological extracted material is recovered from said solvent. material is treated with an extractant consisting of a deep 29. The process according to claim 16, wherein said bio eutectic solvent to produce a biological extract dissolved in logical material is based on plants, insects, animals or micro said extractant, wherein the deep eutectic solvent is based on organisms. a combination of at least one organic acid or at least one 30. The process according to claim 29, wherein the inorganic compound and at least one mono- or dimeric Sugar, extracted material is a flavonoid (e.g. rutin and quercetin), an Sugar alcohol, amino acid, or di or trialkanol, or based on at anthocyanin, an antioxidant, an alkaloid, a terpenoid, a phe least two Sugars, wherein the molar ratio of the constituents of nylpropanoid a glycoside, a phenolic compound, Such as the deep eutectic solvent is between 1:1 and 5:1. cinnamic acid, a ginkgolide, carthamine, an anthraquinone, 17. The process according to claim 16, wherein said bio paclitaxel, a taxoid, a lignan, a coumarin, a cinnamic acid logical extract is a non-polymeric compound. derivative, azadirachtin, artimisinin, a hop bitter acid, a can 18. The process according to claim 16, wherein said bio nabinoid, Vanillin, a polyketide, a colorant, a flavor, a fra logical extract is a polymeric compound selected from the grance, a dye or a biocide or a mixture thereof. group consisting of RNA, DNA, proteins, enzymes, toxins, 31. An extractant comprising a deep eutectic solvent, vaccines and polysaccharides. wherein the deep eutectic solvent is based on a combination US 2014/034 1934 A1 Nov. 20, 2014 of at least one organic acid or at least one inorganic compound and at least one mono- or dimeric Sugar, Sugar alcohol, amino acid, or di or tri alkanol, or based on at least two Sugars, wherein the molar ratio of the constituents of the deep eutectic solvent is between 1:1 and 5:1. 32. The extractant according to claim 31, wherein said Sugar or Sugar alcohol is selected from the group consisting of Sucrose, glucose, fructose, lactose, maltose, cellobiose, arabinose, ribose, ribulose, galactose, rhamnose, raffinose, Xylose, Sucrose, mannose, trehalose, mannitol, Sorbitol, inositol. Xylitol, ribitol, galactitol, erythritol and adonitol, or a phosphate thereof. 33. The extractant according to claim 31, wherein the organic acid is selected from the group consisting of malic acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric acid, Succinic acid, lactic acid, acetic acid, aconitic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucu ronic acid, neuraminic acid and Sialic acids. 34. The extractant according to claim 31, wherein the inor ganic compound is selected from phosphates, Sulfates, sulfites and halogenides, such as NaH2PO Na HPO, NaHSO, NaSO, MgCl, CaCl2, KCl, NaCl and KI. 35. An extractant consisting essentially of a deep eutectic Solvent, wherein the deep eutectic Solvent is based on a com bination of at least one organic acid or at least one inorganic compound and at least one mono- or dimeric Sugar, Sugar alcohol, amino acid, or di or trialkanol, or based on at least two Sugars, wherein the molar ratio of the constituents of the deep eutectic solvent is between 1:1 and 5:1. k k k k k