# Patent

## Title
Process for extracting materials from biological material

## Abstract

The invention is directed to a process for extracting materials from biological material, which process is characterized in that the naturally occurring biological material is treated with an extractant consisting of a deep eutectic solvent of natural origin or a an ionic liquid of natural origin to produce a biological extract of natural origin dissolved in the said solvent or ionic liquid.

## Background
This application is the United States national stage of International Application No. PCT/NL2011/050407, filed Jun. 7, 2011, which was published under PCT Article 21 in English as International Publication No. WO 2011/155829, and which claims benefit of Netherlands Patent Application No. 2004835 filed Jun. 7, 2010 which is herein incorporated by reference.
The present invention is directed to a process for extracting materials from biological materials.
Drugs, flavors, fragrances, agrochemicals, dyes etc., both from synthetic and natural sources are often poorly soluble in water. Therefore extraction, purification, administration requires the use of less polar solvents, such as alcohols, acetone, ethyl acetate, chloroform etc. Such solvents present several problems such as: toxicity for the producer/patient/consumer, environmental problems, explosions and the like.
Ionic liquids can be environmentally benign and safe replacements for the traditional volatile organic solvents in various chemical processes. The reason that ionic liquids are considered to be ‘green’ solvents is their negligible vapor pressure. However, ionic liquids can have a hidden environmental cost because they are synthesized from petrochemical 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, fluorinated anions such as PF6− and BF4− are sensitive to water 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 materials.
The reason that ionic liquids are also considered to be safe solvents is because their lack of volatility greatly reduces any chance of exposure other than by direct physical contact with skin or by ingestion. However, most conventional ionic liquids are irritating and have a toxicity comparable to common organic solvents. From biological tests it appeared that the toxicity of ionic liquids is mainly determined by the type of cation and that ionic liquids with short alkyl substituents in the cation usually have a lower toxicity.
A solution to the problems mentioned above is the development of halogen-free ionic liquids, such as ionic liquids with the alkyl sulfate, the alkyl carbonate and the sulfonate anion. It was also found that some ionic liquids with ester groups in their alkyl side chains are biodegradable. However, these ionic liquids are still synthesized using petrochemical resources.
In WO2006/116126 a process is described for extracting biopolymers from biomass, using ionic liquids. Generally the ionic liquids described therein are of petrochemical nature. The biopolymers extracted are chitin, chitosan, collagen and keratin. Polyhydroxyalkanoate is extracted from genetically engineered plants.
As indicated above, there is a need for an improved process for extracting organic compounds from natural sources, without the need for the use of organic solvents or other synthetic materials.
Further, there is a need for a process that can truly be considered ‘green’, i.e. using only natural compounds.
The invention is based on the surprising fact that some specific naturally occurring materials can suitably be used for extracting materials from biological sources. These materials are deep eutectic solvents (or mixtures) of natural origin or ionic liquids of natural origin.
Deep eutectic solvents are liquids having a melting point that is much lower than the melting points of the two compounds that form the eutectic mixture. Generally, they are formed between a variety of quaternary ammonium salts and carboxylic acids. The deep eutectic phenomenon was first described in 2003 for a mixture of choline chloride and urea in a 1:2 mole ratio, respectively. Other deep eutectic solvents of choline chloride are formed with phenol and glycerol. Deep eutectic solvents are able to dissolve many metal salts like lithium chloride and copper(II)oxide. Also, organic compounds such as benzoic acid and cellulose have great solubility in deep eutectic solvents. Compared to ordinary solvents, eutectic solvents have a very low volatility and are non-flammable. They share a lot of characteristics with ionic liquids, but they are ionic mixtures and not ionic compounds.
Instead, choline citrate is a real ionic liquid. This compound was formed by dissolving citric acid in water, followed by addition of choline hydroxide (in the ratio 2:1) dissolved in methanol. The solvent (water and methanol) was evaporated. The product choline citrate was a slightly yellow viscous liquid, and not a solid. This is probably the first naturally occurring ionic liquid found.




In addition to the ions, sugar-based liquids can be deep eutectic solvents.
According to the invention a process for extracting materials from biological material is provided, which process is characterized in that the naturally occurring biological material is treated with an extractant consisting of a deep eutectic solvent of natural origin or a an ionic liquid of natural origin to produce a biological extract of natural origin dissolved in the said solvent or ionic liquid.
Surprisingly it has been found that deep eutectic solvents of natural origin, as defined herein, and natural ionic liquids are suitable extractants for biological materials. These extractants are very efficient and selective, and as they are of natural origin, they are extremely efficient and suitable for extracting components from biological materials, resulting in an efficient process, providing a good yield. The melting points of the deep eutectic mixtures and ionic liquids is preferable below 25° C. The materials are thus preferably liquid at ambient temperatures.
Suitable deep eutectic solvents to be used in the present invention, i.e. mixtures of materials of natural origin, are based on mixtures of at least two compounds, substantially without chemical or ionic bonding. The first component of the solvents is preferably selected from at least one naturally occurring organic acid or an inorganic compound, such as a salt.
The second component is preferably selected from at least one naturally occurring mono- or dimeric sugar, sugar alcohol, amino acid, di or tri alkanol or choline derivatives, such as choline or phosphatidyl choline.
Said sugar or sugar alcohol may be selected from the group of sucrose, glucose, fructose, lactose, maltose, cellobiose, arabinose, ribose, ribulose, galactose, rhamnose, raffinose, xylose, sucrose, mannose, trehalose, mannitol, sorbitol, inositol, ribitol, galactitol, erythritol, xyletol and adonitol, and, as well as their phosphates.
The said organic acid may be selected from malic acid, maleic acid, citric acid, lactic acid, pyruvic acid, fumaric acid, succinic acid, lactic acid, acetic acid, aconitic acid, tartaric acid, malonic acid, ascorbic acid, glucuronic acid, oxalic acid, neuraminic acid and sialic acids.
In general it is preferred that the ionic liquid or deep eutectic solvent is free of chlorine/chloride.
In certain solvents additionally further components may be present, such as water, phenolics, etc. These additional compounds are generally present in minor amounts, such as below 5 wt. %.
Suitable examples of inorganic compounds are the phosphates, sulfates, sulfites and halogenides, such as NaH2PO4, Na2HPO4, NaHSO3, Na2SO4, CaCl2, MgCl2, KCl, NaCl and KI.
Specific examples of deep eutectic solvents are given in the table below, but also honey, maple syrup, and nectar are examples of deep eutectic solvents that can be used as extraction solvent (which are based on sugar, and small amounts of phenolics and amino acids).




Deep eutectic mixture of malic acid with choline chloride





Deep eutectic mixture of maleic acid with choline chloride

Suitable ionic liquids are based on naturally occurring anions selected from the group of malic acid, maleic acid, citric acid, lactic acid, tartaric acid glucosamine, glucuronic acid, neuraminic acid and sialic acids.
The said ionic liquid is further based on naturally occurring cations selected from the group of choline, betaine, betanine, gamma-amino butyric acid, betalaine, acetylcholine, glucosamine, glutamine, glutamate, asparagine, aspartic acid, alanine, lysine, arginine, proline, threonine, putrescine, cadaverine and choline derivatives.
In a more preferred embodiment the said ionic liquid is choline citrate.
The ratio of the components of the deep eutectic solvents and ionic liquids depends on the structure of the two or more constituents of the solvent or liquid.
For deep eutectic solvents quite often the two components 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 generally vary from 1:1 to 4:1.
Ionic liquids are by definition salts anions and cations and accordingly the ratio is determined by the valence of the ions.
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

activity
(40° C.)
(40° C.)


No.
(molar ratio)
H2O w %
(40° C.)
g/cm3
mm2/s
ET (NR)
Tdecom/° C.
Tg/° C.


















MCH
Ma:Ch:H2O (1:1:2)
11.62%
0.195
1.246
445.9
44.81
201
−71.32


GlyCH
Gly:Ch:H2O (2:1:1)
5.26%
0.126
1.1742
51.3
49.55
187
−101.59


MAH
Ma:β-Ala:H2O (1:1:3)
19.48%
0.573
1.352
174.6
48.05
164
−70.88


PMH
Pro:Ma:H2O (1:1:3)
17.81%
0.591
1.3184
251
48.3
156
−61.29


CaGH8
CaCl2:Glc:H2O (5/4:1:8)
31.11%
0.331
1.4904
720
54.56
137
−61.39


FCH
Fru:Ch:H2O (1:2.5:2.5)
7.84%
0.151
1.2078
280.8
49.81
160
−84.58


XCH
Xyl:Ch:H2O (1:2:2)
7.74%
0.141
1.2095
308.3
49.81
178
−81.8


SCH
Suc:Ch:H2O (1:4:4)
7.40%
0.182
1.2269
581
49.72
>200
−82.96


FGSH
Fru:Glc:Suc:H2O (1:1:1:11)
18.70%
0.662
1.3657
720
48.21
138
−50.77


GCH
Glc:Ch:H2O (1:2.5:2.5)
7.84%
0.162
1.197
397.4
49.72
170
−83.86


PdCH
1,2Prop:Ch:H20 (1:1:1)
7.70%
0.242
1.0833
33.0
50.07
162
−109.55


LGH
Lac:Glc:H2O (5:1:2)
7.89%
0.496
1.2495
37.0
44.81
135
−77.06


SoCH
So:Ch:H2O (1:2.5;3)
11.17%
0.12
1.185385
138.4
49.98
>200
−89.62


XoCH
Xo:Ch:H2O (1:2:3
11.17%
0.116
1.17841
86.1
49.72
>200
−93.33


M2
Ma:Pro:Xo:Ch:H2O (1:1:1:2.5:6)
12.58%
0.218
1.24729

49.13

−84.4


M3
Ma:Pro:Ch:H2O (1:1:1:4)
15.62%
0.213
1.18469

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













TABLE 2







Summary of some solubility data in some typical Des at 40° C. (mg/ml) (n = 3)



















1,8-




Des
rutin
quercetin
cinnamic acid
carthamin
dihydroxyanthraquinone
paclitaxel
ginkgolide B





LGH
 8.14 ± 0.79
1.72 ± 0.09
13.11 ± 0.38 
 1.24 ± 0.21
0.20 ± 0.01
5.39 ± 0.55
1.93 ± 0.31


GCH
121.63 ± 1.45 
20.06 ± 0.41 
8.64 ± 0.46
27.20 ± 0.39
0.20 ± 0.04
0.83 ± 0.16
5.85 ± 0.42


PdCH
352.90 ± 31.19
205.17 ± 7.31 
58.29 ± 2.79 
22.47 ± 1.00
0.17 ± 0.00
11.71 ± 0.68 
78.42 ± 14.45


SoCH
149.21 ± 2.61 
145.84 ± 2.82 
4.50 ± 0.21
14.05 ± 0.66
0.08 ± 0.01
0.59 ± 0.01
1.70 ± 0.01


H2O
0.028 ± 0.00
0.035 ± 0.003
0.57 ± 0.01
 1.43 ± 0.01
0.00 ± 0.00
0.01 ± 0.00
0.15 ± 0.00









The present invention deals with extracting materials from biological products. In the most general scope, all materials of biological origin may be used. Suitable examples are plants, insects, animals or micro-organisms.
From these materials a great variety of products can be isolated using the process of the present invention. More in particular the extracted or dissolved material is a flavonoid (e.g. rutin and quercetin), an anthocyanin, a colorant, an alkaloid, a terpenoid, a phenylpropanoid a glycoside, a phenolic compound, such as cinnamic acid, a ginkgolide, carthamin, an anthraquinone, paclitaxel, taxoid, a lignan, a coumarin, a cinnamic acid derivative, azadirachtin, artimisinin, a hop bitter acid, a cannabinoid, vanillin, a polyketide, a colorant, a flavor, a fragrance, a dye, a biocide or a mixture of any of these compounds. Also proteins (enzymes), toxins, vaccins, DNA, RNA and polysaccharides may be extracted from suitable sources.
In particular, the invention is directed to extracting natural materials from natural sources, i.e. not genetically engineered. In a further preferred embodiment, valuable materials are thus extracted or dissolved, such as non-polymeric compounds, as listed above. Non-polymeric compounds are defined as those compounds that do not consist of three or more repeating units of the same moiety (monomer) or of the same type of monomers, such as amino acids or sugars.
These non-polymeric materials are, for example, suitable intermediates or products suitable in food, pharma, cosmetics and agrochemicals. More in particular it is preferred to extract flavors and fragrance from plant, vanillin from vanilla, capsaicin from Capsicum, hop bitter acids from hops, cannabinoids from cannabis, azadirachtin from neem plant material, paclitaxel from Taxus plant material, artimisinin from Artemisia plant material, alkaloids from Catharanthus, morphine and codeine from Papaver plant material, atropine and hyoscyamine from Solanacea plant material, galanthamine from Amaryllidaceae plants, antioxidants from plant material, antibiotics from microorganisms, colorants from plants and microorganisms, flavonoids from plant materials, anthocyanins and carotenoids from flowers, an essential oil from a plant.
In another embodiment, specific polymeric compounds are extracted or dissolved, such as RNA, DNA, proteinic 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 solvent in enzymatic reactions. These reactions are then carried out in the said solvent or liquid. An example is the laccase reaction.
The invention is now elucidated on the basis of the following examples.


EXAMPLES
First the solubility of natural products, which are not soluble in water, was evaluated in a few selected natural deep eutectic solvents. Several flavonoids were chosen as the natural water-insoluble products, because they are one of the most abundant water-insoluble plant secondary metabolites. Up to now more than 500 flavonoids have been known. Most of these flavonoids occur in their glycosides forms (bounded to a sugar molecule) in plants. In spite of large abundance of flavonoids in plants, both the glycoside and the aglycone (non-sugar) part are not soluble in water. Thus, as a model research, the solubility of typical flavonoids including quercetin (aglycone), quercitrin (quercetin-3-O-rhamnoside) and rutin (quercetin-3-O-rhanmoglucoside), which have a very low water solubility, were tested in the naturally occurring deep eutectic solvents. The structure of these flavonoids are shown below.




Structures of quercetin, quercitrin and rutin (left to right)

As shown in the table below the three flavonoids were found to be well dissolved in the natural deep eutectic solvents, with solubilities that are 2 to 4 orders of magnitude higher as compared to their solubilities in water.







TABLE 3







Solubility of flavonoids in several naturally occurring deep


eutectic solvents









Solubility (mg/ml)










Deep eutectic solvent
Quercetin
Quercitrin
Rutin





Sucrose + Choline
15.63 ± 0.57
12.68 ± 0.38 
2.41 ± 0.18


chloride


Glucose + Choline
21.56 ± 0.94
7.81 ± 0.20
4.78 ± 0.84


chloride


Fructose + Choline
23.34 ± 2.54
11.25 ± 0.64 
10.94 ± 1.70 


chloride


Water
 0.300 ± 0.002
0.159 ± 0.001
<0.001









In order to confirm the solubility of flavonoids and the related anthocyanins, the flowers of red rose were extracted in the naturally occurring ionic liquids. It was observed that the red color metabolites are localized in the epidermis cells.
Extraction with the deep eutectic solvent fructose/glucose/malic acid (1:1:1 molar ratio) resulted in color removal from the flowers into the deep eutectic solvent phase. The structure of the flowers remained intact, with no breakdown of the natural structure.

## Claims

1. An extractant comprising a deep eutectic solvent, wherein the deep eutectic solvent comprises a combination of lactose, glucose and water in a 5:1:2 molar ratio, and wherein the deep eutectic solvent has a lower melting temperature as compared to the melting temperature of the individual components.

2. An extractant comprising a deep eutectic solvent, wherein the deep eutectic solvent comprises a combination of malic acid, alanine and water in a 1:1:3 molar ratio, or a combination of malic acid, proline and water in a 1:1:3 molar ratio, and wherein the deep eutectic solvent has a lower melting temperature as compared to the melting temperature of the individual components.

3. An extractant comprising a deep eutectic solvent, wherein the deep eutectic solvent comprises a combination of CaCl2, glucose and water in a 5/4:1:8 molar ratio, and wherein the deep eutectic solvent has a lower melting temperature as compared to the melting temperature of the individual components.

4. The extractant according to claim 3, wherein the deep eutectic solvent comprises malic acid, alanine and water in a 1:1:3 molar ratio.

5. The extractant according to claim 3, wherein the deep eutectic solvent comprises malic acid, proline and water in a 1:1:3 molar ratio.