WO2024107107A1 - Procede d'extraction de principes actifs naturels - Google Patents

Procede d'extraction de principes actifs naturels Download PDF

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WO2024107107A1
WO2024107107A1 PCT/SG2023/050754 SG2023050754W WO2024107107A1 WO 2024107107 A1 WO2024107107 A1 WO 2024107107A1 SG 2023050754 W SG2023050754 W SG 2023050754W WO 2024107107 A1 WO2024107107 A1 WO 2024107107A1
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nades
glycerol
water
waste
solvent
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PCT/SG2023/050754
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Xiao Hu
Sze Ying Lee
Yen Nan LIANG
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Nanyang Technological University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B63/00—Purification; Separation; Stabilisation; Use of additives

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  • the current invention relates to a method of extracting natural bioactive compounds with differing polarities from a solid plant waste, the resulting solid plant waste extract, a ternary solvent and use of the ternary solvent.
  • cruciferous vegetable such as kale (Brassica oleracea var. acephala), which is gaining popularity as a nutrient-dense “superfood”, contains exceptionally high levels of health-promoting phytochemicals like polyphenols and carotenoids that bring immense health benefits beyond basic nutrients.
  • Polyphenols are powerful antioxidants that can relieve oxidative stress by radical scavenging activity (RSA) and thus reduce the risk of chronic degenerative illnesses (Kaulmann, A. et al., Food Chemistry, 2014, 155, 240).
  • lipophilic pigments such as carotenoids and chlorophylls, which can be found in kale, are proven to exert positive effects on inflammatory behaviour, neuroprotection and reduction of coronary and eye disorders (Ma, L. & Lin, X. M., Journal of the Science of Food and Agriculture, 2010, 90, 2; Jubert, C. et al., Cancer prevention research, 2009, 2, 1015).
  • kale extract has been applied in the formulation of cosmetics, nutraceuticals (Danesi, F. et al., Electrophoresis, 2016, 37, 1805; Meinke, M.C. et al., European Journal of Pharmaceutics and Biopharmaceutics, 2013, 84, 365) and pharmaceutical products (Lemos, M. et al., Journal of Ethnopharmacology, 2011, 138, 503; Das, G. et al., International Journal of Nanomedicine, 2022, 17, 1125).
  • NADESs Natural deep eutectic solvents
  • VOCs Volts
  • Y. et al. Journal of Chromatography A, 2016, 1434, 50
  • They are liquids entirely composed of plant-based primary metabolites such as amino acids, sugars, sugar alcohols and organic acids (Choi, Y. H. et al., Plant Physiology, 2011 , 156, 1701).
  • NADESs feature a high- solubilising capacity for natural products, making them suitable extraction media.
  • the natural constituents of NADESs make possible the direct use of extracts in food, pharmaceutical and cosmetic applications and hence simplifying the product polishing step (da Silva, D.T. et al., Food Chemistry, 2021, 364, 130370).
  • hydrophobic NADESs are introduced and used in isolating ergosterol (Khare, L. et al., Food Chemistry, 2021 , 340, 127979) and carotenoids such as lutein (Fan, C. et al., Food Chemistry, 2022, 376, 131930), astaxanthin (Pitacco, W. et al., Food Chemistry, 2022, 379, 132156) and P-carotene (Stupar, A. et al., Ultrasonics Sonochemistry, 2021 , 76, 105638) from various matrices.
  • ergosterol Kele, L. et al., Food Chemistry, 2021 , 340, 127979
  • carotenoids such as lutein (Fan, C. et al., Food Chemistry, 2022, 376, 131930), astaxanthin (Pitacco, W. et al., Food Chemistry, 2022, 379, 132156)
  • NADESs may vary, depending on the nature of the target bioactive metabolites present in the source.
  • the use of NADESs to extract bioactive compounds from kale waste has not yet been adequately evaluated. Additionally, most of the studies often focus on a specific group of compounds (either hydrophilic or lipophilic), there is still limited information concerning the capacity of hydrophilic/hydrophobic NADESs to recover different classes of bioactive metabolites from kale waste. The development of sustainable recovery processes is needed to achieve efficient valorisation of kale waste for the production of natural antioxidants and pigments.
  • This invention provides a total and efficient extraction method of nutrients and active ingredients from vegetables, other plants and their waste streams using green and even foodsafe solvents in a single step.
  • the active ingredients that can be extracted from the materials include but are not limited to natural antioxidants and pigments for use in cosmetics, food supplements, pharmaceuticals and other uses. Minimal pre-treatment of the plants or plant waste is needed.
  • a method of extracting natural bioactive compounds with differing polarities from a solid plant waste comprising the steps of:
  • a ternary solvent mixture comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and ethyl acetate, or contacting the wet solid plant waste for suitable a period of time and at a suitable temperature with a binary solvent mixture comprising: a natural deep eutectic solvent (NADES) comprising glycerol; and ethyl acetate, wherein: the NADES is a hydrophilic NADES; and water is present in an amount of from 20 to 40 wt%, relative to: the total weight of the water and the NADES in the ternary solvent; or the total weight of water in the wet solid plant waste and the NADES in the binary solvent.
  • NADES natural deep eutectic solvent
  • water is present in an amount of from 20 to 40 wt%, relative to: the total weight of the water and the NADES in the ternary solvent; or the total weight of water in the wet solid plant waste and the NADES in the binary solvent.
  • NADES comprises glycerol and a further component selected from one or more of the group consisting of an amino acid, a sugar alcohol, a sugar, and urea.
  • NADES comprises glycerol and a further component is selected from one or more of the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.
  • volume to volume ratio of ethyl acetate to water and the NADES in the ternary solvent is from 1:5 to 5:1 , such as 1:3 to 3:1 , such as about 1 :1.
  • a solid to liquid ratio (g/mL) of the dried solid plant waste to the ternary solvent is from 1 :10 to 1 :40, such as from 1 :15 to 1 :35, such as from 1 :18 to 1 :35, such as about 1:20.
  • the suitable amount of time is from 10 minutes to 5 hours, such as from 15 minutes to 2 hours, such as from 20 minutes to 1 hour, such as about 30 minutes;
  • the suitable temperature is from 15 to 65 °C, such as from 20 to 45 °C, such as about 25 °C.
  • the solid plant waste is provided in a particulate form, optionally wherein the particles have an average diameter of from 0.1 to 10 mm, such as an average diameter of from 0.5 to 5 mm, such as an average diameter of about 2 mm.
  • the plant waste is a vegetable waste, optionally wherein the vegetable waste is a cruciferous vegetable waste, further optionally wherein the cruciferous vegetable waste is selected from one or more of spinach, broccoli, cauliflower and, more particularly, curly kale leaves (Brassica oleracea var. acephala).
  • the method further comprises separating and retaining a resulting ethyl acetate extract and a resulting NADES/water extract from the solid plant waste following step (b).
  • a solid plant waste extract comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and one or more polyphenol compounds.
  • NADES natural deep eutectic solvent
  • a ternary solvent comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and ethyl acetate, wherein: the NADES is a hydrophilic NADES; and water is present in an amount of from 20 to 40 wt%, relative to the total weight of the water and NADES in the ternary solvent.
  • NADES natural deep eutectic solvent
  • NADES comprises glycerol and a further component selected from one or more of the group consisting of an amino acid, a sugar alcohol, a sugar, and urea.
  • NADES comprises glycerol and a further component is selected from one or more of the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.
  • ternary solvent according to any one of Clauses 17 to 21, wherein water is present in an amount of from 25 to 35 wt%, such as about 30 wt% relative to the total weight of thewater and NADES in the ternary solvent.
  • FIG. 1 depicts the recovery yields of total phenolics, carotenoids (p-carotene, lutein) and chlorophylls (a and b) from kale waste using solvent (H 2 O - water, MeOH - methanol, 70% MeOH - 70% aqueous methanol, EtOH - ethanol, 70% EtOH - 70% aqueous ethanol, 70% Gly - 70% aqueous glycerol, 70% aqueous NADES based on Gly/Bet 2 - glycerol: betaine (2:1), Gly/Bet - glycerol: betaine (3:1), Gly/Sor 2 - glycerol: sorbitol (2:1), Gly/Sor - glycerol: sorbitol (3:1), Gly/Xyl - glycerol: xylose (3:1), Gly/Glu - glycerol: glucose (3:1), Gly/F
  • FIG. 2 depicts the chromatographic profiles acquired by reversed-phase high-performance liquid chromatographic (RP-HPLC) at 450 nm for kale waste extracts obtained using ethanol (EtOH) and ethyl acetate (EtOAc), underthe RP-HPLC operation described in Example 2, with the highlighted peaks of [3-carotene, lutein, chlorophyll a and b, and their chemical structures.
  • RP-HPLC reversed-phase high-performance liquid chromatographic
  • FIG. 4 depicts the recovery yields of total phenolics from kale waste using NADES based on glycerol: betaine (3:1), in the investigation of the effects of the (A) solid-liquid ratio (SLR), at a solvent concentration of 70% and at 25 °C for 30 min; and (B) solvent concentration (50, 60, 70 and 80%), extraction temperature (25, 45 and 65 °C) and time (30 and 60 min), and in comparison with the ultrasound-assisted extraction (that was operated at 37 kHz and 100% power), using a SLR of 1 :20.
  • SLR solid-liquid ratio
  • FIG. 4 depicts the recovery yields of total phenolics from kale waste using NADES based on glycerol: betaine (3:1), in the investigation of the effects of the (A) solid-liquid ratio (SLR), at a solvent concentration of 70% and at 25 °C for 30 min; and (B) solvent concentration (50, 60, 70 and 80%), extraction temperature (25, 45 and 65 °
  • FIG. 5 depicts the stability of kale waste extracts produced using solvent (H 2 O - water, 70% EtOH - 70% aqueous ethanol, 70% Gly - 70% aqueous glycerol and 70% Gly/Bet - 70% aqueous glycerol: betaine (3:1)), represented by the relative concentration of total phenolics over time after stored in the dark at (A) 25 °C and (B) 4 °C, respectively, for 30 days; and (C) the photographs of the extracts just after extraction and during the storage. The original extract prepared with water was used as the control for comparison.
  • solvent H 2 O - water, 70% EtOH - 70% aqueous ethanol, 70% Gly - 70% aqueous glycerol and 70% Gly/Bet - 70% aqueous glycerol: betaine (3:1)
  • solvent H 2 O - water, 70% EtOH - 70% aqueous ethanol, 70% Gly - 70% aqueous glycerol and
  • FIG. 6 depicts the photographs of kale waste extracts (on a dark background) obtained from extraction using water (H 2 O), 70% aqueous ethanol (70% EtOH), 70% aqueous glycerol (70% Gly) and 70% Glycerol/Betaine with a molar ratio of 3:1 (70% Gly/Bet) during the storage under protection from light at 25 and 4 °C.
  • FIG. 7 depicts the recovery yields of total phenolics, carotenoids (P-carotene, lutein) and chlorophylls (a and b) from kale waste using sequential (Route 1 and 2) and integrated (Route 3) approaches designed in this work. Different letters in the same series indicate significant differences at p ⁇ 0.05 level.
  • FIG. 8 depicts the diagram of the integrative process for upcycling kale waste using green and cosmetic-/food-grade ternary solvent mixture (Gly/Bet - glycerol: betaine (3:1) + H 2 O - water + EtOAc - ethyl acetate) for the production of natural antioxidants and pigments with potential applications in the cosmetic and food industries. Dashed lines were not experimentally tested but were recurrently used in product polishing.
  • FIG. 9 depicts the recovery yields of total phenolics, carotenoids (p-carotene, lutein) and chlorophylls (a and b) from kale waste using the integrated approaches using glycerol/fructose (3:1) + H 2 O + ethyl acetate and glycerol/betaine (3:1) + H 2 O + ethyl acetate, respectively.
  • this invention provides a total and efficient extraction method of nutrients and active ingredients from vegetables, other plants and their waste streams using green and even food-safe solvents in a single step.
  • a method of extracting natural bioactive compounds with differing polarities from a solid plant waste comprising the steps of:
  • a ternary solvent mixture comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and ethyl acetate, or contacting the wet solid plant waste for suitable a period of time and at a suitable temperature with a binary solvent mixture comprising: a natural deep eutectic solvent (NADES) comprising glycerol; and ethyl acetate, wherein: the NADES is a hydrophilic NADES; and water is present in an amount of from 20 to 40 wt%, relative to: the total weight of the water and the NADES in the ternary solvent; or the total weight of water in the wet solid plant waste and the NADES in the binary solvent.
  • NADES natural deep eutectic solvent
  • water is present in an amount of from 20 to 40 wt%, relative to: the total weight of the water and the NADES in the ternary solvent; or the total weight of water in the wet solid plant waste and the NADES in the binary solvent.
  • the word “comprising” refers herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.
  • the phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present.
  • the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
  • the “solid plant waste” may be any suitable waste.
  • it may be the whole or part of a vegetable or other plant (e.g. a vegetable past its sell-by date) or a waste stream associated with the vegetable or other plant (e.g. the roots or the plant of the vegetable).
  • a single vegetable or plant may be use or combinations thereof may be used in the method.
  • the ternary solvent mixture in the method includes a first component, which is a natural deep eutectic solvent (NADES) comprising glycerol, water as a second component and ethyl acetate as a third component.
  • NADES natural deep eutectic solvent
  • the ternary solvent mixture in the method consists essentially of a first component, which is a natural deep eutectic solvent (NADES) comprising glycerol, water as a second component and ethyl acetate as a third component.
  • the binary solvent comprises (or consists of) a NADES and ethyl acetate, with water being supplied through the use of a wet plant waste.
  • the wet solid plant waste may comprise from 65 to 90 wt%, such as about 75 wt% water relative to the total weight of the wet solid plant waste. This may be measured by weighing a sample of the wet plant waste and then drying the sample and working out the percentage of water based on the weight loss from drying.
  • the water content of wet solid plant waste can be concentrated to a specific amount needed to maintain the same solid liquid ratio of the extraction process.
  • a Deep Eutectic Solvent is a solution of Lewis or Bronsted acids and bases which form a eutectic mixture.
  • a NADES is a bio-based deep eutectic solvent which is composed of two or more compounds that are generally plant based primary metabolites, i.e. organic acids, sugars, alcohols, amines and amino acids.
  • the NADES comprises glycerol and then at least one further suitable component to form the NADES.
  • Suitable further components include, but are not limited to amino acids, sugar alcohols, sugars, and urea, and combinations thereof.
  • the NADES may comprises (or consist of) glycerol and a further component that is selected from one or more of the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.
  • the NADES may be formed from:
  • the NADES may be formed from:
  • any suitable molar ratio between the glycerol and the further component(s) may be used.
  • suitable molar ratios include, but are not limited to one where the molar ratio of glycerol to the further component(s) is from 1:1 to 5:1 , such as from 2:1 to 3:1, such as about 3:1.
  • the total of the molar values of the further components will be used to calculate the molar ratio.
  • water is present as a second component in the ternary solvent and it is present in an amount of from 20 to 40 wt%, relative to the total weight of the water and NADES in the ternary solvent. In further embodiments that may be mentioned herein, water may be present in an amount of from 25 to 35 wt%, such as about 30 wt% relative to the total weight of the water and NADES in the ternary solvent. It will be appreciated that when a wet solid plant waste is used that a similar proportion of water may be used in conjunction with the binary solvent (i.e.
  • the weight % of water may be calculated based on the weight of the solid plant waste versus the weight of water in the wet solid plant waste (calculated based on the weight % of water by mass in the wet solid plant waste) and the weight of the NADES in the binary solvent).
  • ethyl acetate is present as the third component of the ternary solvent (or second component in a binary solvent).
  • Ethyl acetate may be present in any suitable amount in the ternary solvent.
  • the volume to volume ratio of ethyl acetate to water and the NADES may be from 1 :5 to 5:1, such as 1 :3 to 3:1 , such as about 1:1.
  • Similar amounts of ethyl acetate may be used in the binary solvent, where the amount of ethyl acetate is based upon the calculated volume of water and NADES based on the NADES and expected volume of water in the wet solid plant waste.
  • the method disclosed herein requires the solid plant waste to be contacted with the ternary solvent in order to effect the extraction of the natural bioactive compounds with differing polarities. While any suitable solid to liquid ratio (g/mL) may be used, it has been found that a suitable ratio may be one in which the solid to liquid ratio (g/mL) of the solid plant waste to the ternary solvent may be from 1 :10 to 1:40, such as from 1 :15 to 1:35, such as from 1 :18 to 1:35, such as about 1:20. Any suitable solid to liquid ratio for the binary solvent and the wet plant waste may be used.
  • the solid (solid dry weight of plant waste) to liquid (binary solvent and water present in the wet plant waste) ratio may be from 1 :10 to 1 :40, such as from 1 :15 to 1 :35, such as from 1 :18 to 1 :35, such as about 1 :20.
  • any suitable period of time that allows for the effective extraction of the natural bioactive compounds with differing polarities from a solid plant waste may be used herein.
  • the suitable amount of time may be from 10 minutes to 5 hours, such as from 15 minutes to 2 hours, such as from 20 minutes to 1 hour, such as about 30 minutes.
  • Any suitable temperature that allows for the effective extraction of the natural bioactive compounds with differing polarities from a solid plant waste may be used herein.
  • the suitable temperature is from 15 to 65 °C, such as from 20 to 45 °C, such as about 25 °C.
  • the method may make use of temperatures around room temperature (i.e. about 25 °C) and a contact time of around 30 minutes to simple and effectively extract the desired materials.
  • the method may be conducted both expeditiously and without the need for heating or cooling of the ambient environment, thereby reducing energy expenditure.
  • These temperatures and times may be used with either the ternary or binary solvents mentioned herein, when used with dried or wet solid plant wastes, respectively.
  • the solid plant waste may be provided in any suitable form.
  • the solid plant waste may be provided as the whole plant or vegetable.
  • the solid plant waste may be provided in a particulate form, which may assist in the expeditious extraction of the desired materials.
  • Any suitable particle size may be used.
  • the particles may have an average diameter of from 0.1 to 10 mm, such as an average diameter of from 0.5 to 5 mm, such as an average diameter of about 2 mm. It will be appreciated that these particle sizes may apply to both dried and wet solid plant wastes.
  • the solid plant waste may be provided in a hydrated or a dried form.
  • a hydrated, or “wet”, form of the solid plant waste is used.
  • the wet form will be used with a binary solvent as discussed hereinbefore.
  • the solid plant waste may be provided in a dried form, which is used with a ternary solvent. More particularly, the solid plant waste may be provided in a dried form where substantially all of the water has been removed.
  • substantially all may mean that greater than or equal to 95%, such as greater than or equal to 96%, such as greater than or equal to 97%, such as greater than or equal to 98%, such as greater than or equal to 99%, such as greater than or equal to 99.5%, such as greater than or equal to 99.9% of the water has been removed from the solid plant waste.
  • the solid plant waste may be provided in a freeze-dried form.
  • the solid plant waste may be provided in a particulate form that has been (freeze) dried to remove water.
  • the solid plant waste may be a vegetable waste. While any suitable vegetable water may be used in the invention, the vegetable waste may be a cruciferous vegetable waste.
  • the cruciferous vegetable waste may be selected from one or more of spinach, broccoli, cauliflower and, more particularly, curly kale leaves (Brassica oleracea var. acephala).
  • the method may further comprise separating and retaining a resulting ethyl acetate extract and a resulting NADES/water extract from the solid plant waste following step (b) of the method. This may allow the separation of hydrophobic and hydrophilic natural bioactive compounds from one another, thereby allowing more effective use of the extracted compounds.
  • step (b) of the process above the separation of the mixture in step (b) of the process above will result in an ethyl acetate extract and a NADES/water extract that is essentially identical to that obtained if a dried solid plant waste had been used with a ternary solvent .
  • the resulting ethyl acetate extract may be rich in carotenoids and chlorophyll, while the NADES/water extract may be rich in polyphenols.
  • a solid plant waste extract comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and one or more polyphenol compounds.
  • NADES natural deep eutectic solvent
  • this aspect of the invention may be the product obtained after the process of the first aspect of the invention and the further step of separating and retaining the resulting NADES/water extract from the solid plant waste following step (b) of the method.
  • solid plant waste and the NADES are as described in the first aspect of the invention. As such, these components will not be discussed further here in order to avoid repetition.
  • the storage of the polyphenols in the NADES/water extract may result in the preservation of the polyphenols for an extended period of time - both at ambient temperature (e.g. 25 °C) and reduced temperatures (e.g. in a fridge at 4 °C).
  • ambient temperature e.g. 25 °C
  • reduced temperatures e.g. in a fridge at 4 °C.
  • a 70 wt% Gly/Bet NADES (30 wt% water) extract retained 91.7 and 88.6% of the original bioactive polyphenols content after 30 days of storage at 4 and 25 °C, respectively.
  • a ternary solvent comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and ethyl acetate, wherein: the NADES is a hydrophilic NADES; and water is present in an amount of from 20 to 40 wt%, relative to the total weight of the water and NADES in the ternary solvent.
  • NADES natural deep eutectic solvent
  • water is present in an amount of from 20 to 40 wt%, relative to the total weight of the water and NADES in the ternary solvent.
  • a ternary solvent comprising NADES, water and ethyl acetate can be used to extract various bioactive metabolites, including polyphenols, carotenoids like 0- carotene and lutein, and chlorophylls from a plant waste (e.g. kale waste).
  • a plant waste e.g. kale waste
  • This enables the development of a sustainable process for the production of natural antioxidants and pigments (e.g. chlorophylls).
  • NADES based on Gly/Bet produced enhanced polyphenol-rich extract under simple and mild conditions (25 °C, 30 min, solid-liquid ratio (SLR) of 1 :20 and solvent concentration of 70%). Moreover, it rendered good stability of extract, retaining > 90% of polyphenols and clarity within it after storage at 4 °C for 30 days.
  • Gallic acid (certified reference material), 0-carotene (pharmaceutical secondary standard), lutein (pharmaceutical secondary standard), chlorophyll a from spinach (> 85%), chlorophyll b from spinach (> 90%), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (> 97%), glycerol (> 99%), betaine (> 99%), D-sorbitol (> 98%), D-(+)-xylose (> 99%), D-(+)- glucose (> 99.5%), D-(-)-fructose (> 99%), urea (> 99%), DL-menthol (> 95%), thymol (> 98.5%), fenchyl alcohol (> 97%), triethylamine (> 99%), tetrahydrofuran (THF) (> 99%) and Folin & Ciocalteu’s phenol reagent were purchased
  • Kale waste was provided by Suspar Agriculture (Singapore). Curly kale leaves used in this work did not meet commercial quality standards and were intended to be discarded as waste by the urban farming company.
  • the kale waste was cleaned, freeze-dried, ground in a laboratory ball mill, sieved to obtain a powder size of ⁇ 0.2 mm and stored in a sealed container at 4 °C until further use.
  • NADESs All precursors of NADESs selected are renewable and low-cost ingredients that are allowed for food and cosmetic use.
  • NADESs were prepared by mixing the respective precursors at certain molar ratios in glass vials with constant heating (maximum at 80 °C) and stirring until a clear homogeneous liquid was formed. They were kept at room temperature and observed to have no precipitate formed in the liquid. All abbreviations of NADESs and their compositions used in this work are detailed in Table 1.
  • Viscosity and pH Measurements Viscosities of all hydrophilic NADESs containing 30% (w/w) water and neat hydrophobic NADESs, respectively, were determined using a modular compact rheometer (Anton Paar MCR 102, Germany) fitted with a cone and plate measuring geometry with 50 mm of diameter (CP50-1). The gap between the cone and plate was set as 0.095 mm and the temperature of the system was controlled by a Peltier temperature device (P-PTD200). All measurements were performed at 25 °C and a constant shear rate of 10 s 1 for 10 s. Final viscosity was obtained as the average of the results. All aqueous solutions of hydrophilic NADESs containing 30% (w/w) water were measured for their pH using a pH meter (Mettler Toledo, Singapore) at 25 °C.
  • the hydrophilic NADESs were prepared by pairing glycerol with betaine, sorbitol, xylose, glucose, fructose and urea at proper molar ratios, based on the literature (Jin, Y. et al., Applied Sciences, 2019, 9, 2581 ; Zheng, B. et al., LWT, 2022, 154, 112740).
  • different combinations of terpenes including DL-menthol, thymol and fenchyl alcohol at appropriate molar ratios formed the hydrophobic NADESs disclosed in this example (Fan, C. et al., Food Chemistry, 2022, 376, 131930).
  • Example 2 Extraction of Bioactive Metabolites from Kale Waste and Screening of Solvents
  • NADESs were prepared by following the protocol disclosed in Example 1.
  • hydrophilic and hydrophobic NADESs for the recovery of bioactive metabolites, particularly polyphenols, carotenoids (P-carotene and lutein) and chlorophylls (a and b), from kale waste after 30 min of stirring at 150 rpm and 25 °C was first screened. Because of the high viscosity of hydrophilic NADESs that might hinder their extraction ability (Wojeicchowski, J. P. et al., Separation and Purification Technology, 2021, 258, 117975), they were used in hydrated forms, i.e., with 30% water added.
  • the lyophilised kale waste powder was subjected to solid-liquid extraction (SLE) using a series of solvents at a solid-liquid ratio (SLR) of 1 :40 at 150 rpm and 25 °C for 30 min. All hydrophilic NADESs tested were in hydrated form with added 30 wt% water, while neat hydrophobic NADESs were used. The mixture was vortexed at 3000 rpm for 30 s. All studies were performed under reduced lighting to prevent the degradation of light-sensitive compounds. The mixture was then centrifuged at 12000 g for 10 min, the supernatants were collected and filtered with PTFE syringe filters (0.45 pm, 25 mm) before quantitative analysis.
  • SLE solid-liquid extraction
  • SLR solid-liquid ratio
  • Total phenolics were estimated as gallic acid equivalents (GAE) using the Folin-Ciocalteu protocol described by Singleton et al. (Singleton, V. L. et al., [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent, in Methods in enzymology. 1999, Elsevier, p. 152), with slight modifications. 25 pL of diluted sample (supernatant after extraction) was mixed with 200 pL of water and followed by the addition of 25 pL of 25% (v/v) Folin-Ciocalteu reagent.
  • RP-HPLC reversed-phase high-performance liquid chromatographic
  • the mobile phase was methanol and acetonitrile (9:1 v/v), with an aliquot of 0.1% (v/v) of triethylamine added to prevent both nonspecific adsorption and oxidation.
  • the column temperature was maintained at 25 °C, while the sample injection volume was 5 pL.
  • the stock solution of p-carotene was prepared by dissolving it in a small amount of THF stabilized with butylated hydroxytoluene (BHT) (1 % (v/v) of total solvent volume) before diluting it with ethanol.
  • BHT butylated hydroxytoluene
  • Lutein and chlorophyll standard stock solutions were prepared using ethanol and methanol, respectively.
  • FIG. 1 presents the results of yields of target compounds using different solvents.
  • all hydrophilic glycerol- based NADESs displayed selective extraction towards polyphenols, whereas lipophilic compounds were favourably extracted by hydrophobic terpene-based NADESs.
  • Gly/Bet gave the highest yield of polyphenols, which was around 2.2- and 1.3-fold higher than that of using MeOH and H 2 O, respectively.
  • glycerolbased NADESs coupled with sugar alcohols namely sorbitol (Gly/Sor) and xylose (Gly/Xyl)
  • polar solvents such as MeOH, EtOH, 70% MeOH and 70% EtOH rendered much more complex extract contents encompassing polyphenols, carotenoids and chlorophylls.
  • Lutein is a xanthophyll having a pair of hydroxylations in the terminal [3 rings, making it more polar than hydrocarbon [3-carotene, and thus it was more extractable in polar solvents like EtOH and MeOH but least soluble in nonpolar Hex.
  • chlorophyll molecule consists of a hydrophilic porphyrin head and a long lipophilic hydrocarbon tail, and it has an intermediate polarity between lutein and [3-carotene.
  • Chlorophyll b which has an aldehyde group at position 7-carbon, is slightly more polar than chlorophyll a that has a methyl group at the same position. It was noted that the pigment elution sequence in RP-HPLC followed the order of decreasing polarity of each pigment, as presented in the chromatograms of several representative sets of extracts depicted in FIG. 2.
  • the antioxidant capacity of the extract was assayed based on the DPPH method. 25 pL of diluted sample was mixed with 800 pL of 0.0072 mM methanolic DPPH solution. The mixture was shaken vigorously and left to stand in the dark at 25 °C for 30 min. RSA was estimated by measuring the reduction of DPPH radicals, which was expressed in a percentage of DPPH discolouration using Eq. 1, AbScontrol where AbScontroi and Abs S am P ie were the absorbance values of the control and sample, respectively, at 517 nm. The influence of the solvents was eliminated by preparing a blank control system under the same conditions. Results were converted and recorded in mg of Trolox equivalent per g dry weight of kale waste (mg TE g _1 DW).
  • Antioxidant capacity is one of the most important biological characteristics of an extract concerning its applications.
  • the antioxidant capacity of all aqueous extract was characterised based on the measurement of its ability to scavenge stable radical DPPH', and the results are presented in FIG. 3A.
  • Process optimisation for the extraction of polyphenols After preliminary screening, the best solvent to extract polyphenols was identified and the process conditions were further investigated by one-factor designs. The effects of process parameters including SLR (1:10, 1 :15, 1 :20, 1 :30 and 1:40), temperature (25, 45 and 65 °C), solvent concentration (50, 60, 70 and 80%) and time (30 and 60 min) were assessed.
  • ultrasound-assisted extraction was also conducted using an ultrasonic bath (Elma Elmasonic P, Germany) operating at a constant frequency of 37 kHz and 100% output power. The ultrasonic bath was at 25 °C at the beginning of the process and increased to 30 and 38 °C after 30- and 60-min operation, respectively. The supernatants were collected by centrifugation at 12000g for 10 min and filtered before assaying for total phenolics.
  • NADESs One of the main drawbacks of NADESs is their high viscosity compared to traditional organic solvents.
  • the high viscosity of NADESs reduces the diffusion of solute of interest and slows down the mass transfer rate, which in turn leads to slower recovery performance (Cao, J. et al., Journal of Molecular Liquids, 2020, 318, 113997).
  • an appropriate dilution is important to reduce the solvent’s viscosity and at the same time retaining their eutectic properties.
  • the effect of the solvent concentration was studied, in addition to the extraction temperature and time. The results depicted in FIG.
  • the preservation capability of the solvent system is important for the handling of the extract for productisation.
  • the stability of polyphenol-rich extract during storage was examined.
  • the polyphenol-rich extracts obtained with the best solvent and several reference solvents at the optimal conditions were analysed for the stability over time at 25 °C and 4 °C under protection from light for 30 days.
  • the residual total phenolics in the extracts were determined at least once a week.
  • the results are presented in the relative concentration of total phenolics compared to the original extract obtained with water, as described in Eq. 2.
  • 70% Gly/Bet rendered the greatest stability of bioactive polyphenols by retaining 91.7 and 88.6% of the original contents after 30 days of storage at 4 and 25 °C, respectively.
  • 70% EtOH displayed a comparable performance maintaining the stability of polyphenols overtime, however, the original contents of polyphenols extracted with 70% EtOH were the lowest amongst the solvents studied.
  • the stabilising behaviour of NADESs has been described in several works for bioactive compounds such as cyaniding (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50) and catechin (Jeong, K.M. et al., Journal of Cleaner Production, 2017, 151, 87). Dai et al. (Dai, Y.
  • Sequential processes were designed to recover polyphenols, carotenoids and chlorophylls from kale waste.
  • the lyophilised kale waste powder was treated with the selected NADES, i.e., Gly/Bet, at the optimal conditions (25 °C, 30 min, SLR of 1:20 and concentration of 70%) to recover polyphenols.
  • the polyphenol-rich supernatant was collected by centrifugation at 12000 g for 10 min and measured for total phenolics.
  • the residual pellet was subjected to the second SLE using ethyl acetate; and the supernatant obtained after centrifugation was quantified for carotenoid and chlorophyll contents.
  • the proportion by weight of water added to the eutectic solvent can be from a wide range, e.g., 20 to 40%.
  • the volume ratio of hydrophilic solvent (i.e., the mixture of NADES and water) and hydrophobic solvent (i.e., ethyl acetate) can be adjusted based on the different plant wastes used, e.g., 1 :1.
  • the proposed ternary solvent systems can simultaneously extract natural bioactive compounds with different polarities, including hydrophilic and lipophilic metabolites, from the plant (e.g., vegetable-leaves, stems and roots, and fruit-peels, seeds and pulps) waste in one single-step, gentle and rapid extraction process (e.g., 25 °C, 30 min).
  • the method can recover a wide variety of bioactive metabolites such as phenolic compounds, carotenoids (e.g., lutein and 0- carotene) and chlorophylls (a and b) from the lyophilized and ground kale waste matrix.
  • the pretreatment processing of plant waste before the extraction could be minimal.
  • the aqueous NADES polyphenol-rich extract
  • ethyl acetate carotenoid/chlorophyll-rich extract
  • the examples of the ternary solvent systems are glycerol/fructose (molar ratio of 3:1) + H2O + ethyl acetate and glycerol/betaine (molar ratio of 3:1) + H2O + ethyl acetate.
  • the presence of polar and non-polar properties in the mixed solvents system allowed concurrent solubilisation of different classes of biomolecules with different polarities and thus facilitates the penetration of solvent molecules to access target solutes in the biomass matrix.
  • the improved polarity of the mixed solvent system aided in the extractability of polar pigments like lutein and chlorophylls by EtOAc.
  • the integrated platform seems viable to promote the upcycling of kale waste, as depicted in FIG. 8.
  • Single-step total extractions of natural bioactive compounds with different polarities from plant waste were performed with the use of a tailored-made biphasic system that is composed of a ternary mixture of natural deep eutectic solvent (NADES), water and ethyl acetate.
  • NADES natural deep eutectic solvent
  • the method is sustainable with the use of “green” and even food grade solvents.
  • the NADES constituent such as glycerol is frequently used as a humectant or moistener in cosmetics, besides being widely applied as an excipient in pharmaceutical formulations, such as providing lubrication and smoothness in many cough syrups and other drugs.
  • Another example of NADES constituent, betaine, which is a trimethylated form of glycine first discovered in sugar beet is a common active ingredient in cosmetic, food and pharmaceutical products owing to its moisturising and emollient properties.
  • fructose a ketonic simple sugar derived from sugar cane, sugar beets and maize
  • a ketonic simple sugar derived from sugar cane is often supplemented into food and beverages for palatability and taste enhancement, as well as for browning of bakery products.
  • the stable aqueous NADES rich in polyphenol extracted could be readily applicable to cosmetic or pharmaceutical formulations.
  • ethyl acetate fraction from the extraction could undergo subsequent solvent evaporation to yield dry carotenoid/chlorophyll- rich solid extract (natural pigments) depending on the demands of the application.
  • polyphenol-rich liquid extract (with powerful antioxidant capacity) can be readily incorporated into cosmetics and food products, whereas the pigment-rich extract needs to undergo further solvent evaporation to obtain dry solid extract depending on the demands of the application.
  • Example 7 Extraction using Wet Kale Waste
  • the applicability of the integrated extraction approach for wet kale waste was also investigated.
  • the kale waste was clean and cryogenically ground (SPEX 6875 Freezer/Mill, U.K ), forming a wet paste containing 89.6% water.
  • the wet paste was further concentrated to around 75% water content to maintain the SLR of the extraction process.
  • the integrated extraction approach was modified with the use of only Gly 3 :Bet and ethyl acetate. The extraction was carried out under the same process conditions as the lyophilized powder.
  • Kale waste upcycling can be further improved by simplifying the biomass pretreatment as lyophilisation is an energy-intensive process and best avoided in downstream processing.
  • the feasibility of applying the integrated method on the wet kale waste paste was evaluated. Since the water held within the kale waste paste could work as a diluent in the solvent system, no external water was added to the extraction process.
  • the results in Table 3 show that the recovery yields of all bioactive compounds, except polyphenols, were greatly enhanced with the use of wet kale waste paste compared to lyophilized powder. Considering the identical efficiency of the extraction method for both types of biomass, the lower recovery yields with lyophilized kale waste powder could be due to the degradation of phytochemicals during lyophilisation-pulverization. On the other hand, there was no significant increase in the yield of polyphenols from wet biomass, possibly due to some loss during the removal process of excess water from wet paste.
  • Type content p- system (gmL 1 ) phenolics 3 Lutein a b

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Abstract

L'invention concerne un procédé d'extraction de composés bioactifs naturels présentant des polarités différentes à partir d'un déchet végétal solide tel que des déchets de kale à l'aide d'un mélange de solvant ternaire comprenant un solvant eutectique profond naturel (NADES) comprenant du glycérol, de l'acétate d'éthyle et de l'eau, ou un mélange de solvant binaire comprenant un NADES comprenant du glycérol, et de l'acétate d'éthyle à l'aide d'une plateforme intégrative d'extraction solide-liquide/extraction liquide-liquide (SLE-LLE). L'invention concerne en outre un extrait de déchets végétaux solides comprenant un NADES comprenant du glycérol, de l'eau et un ou plusieurs composés polyphénoliques.
PCT/SG2023/050754 2022-11-14 2023-11-14 Procede d'extraction de principes actifs naturels WO2024107107A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108992541A (zh) * 2018-07-23 2018-12-14 广西壮族自治区农业科学院农产品加工研究所 一种用于提取茶籽粕中茶多酚的低共熔溶剂及提取工艺
US10960042B2 (en) * 2015-04-10 2021-03-30 Universite D'avignon Et Des Pay De Vaucluse Eutectic extraction solvents, extraction methods by eutectigenesis using said solvents, and extracts derived from said extraction methods
CN113563393A (zh) * 2021-06-09 2021-10-29 华南农业大学 一种高效提取三叉苦药渣生物碱及联产生物甲烷的方法
WO2022101490A1 (fr) * 2020-11-13 2022-05-19 Universidade Do Minho Compositions eutectiques, procédés et utilisations associés
EP4011352A1 (fr) * 2020-12-09 2022-06-15 Beiersdorf AG Nouveaux solvants cosmétiques basé sur trois composants différents

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10960042B2 (en) * 2015-04-10 2021-03-30 Universite D'avignon Et Des Pay De Vaucluse Eutectic extraction solvents, extraction methods by eutectigenesis using said solvents, and extracts derived from said extraction methods
CN108992541A (zh) * 2018-07-23 2018-12-14 广西壮族自治区农业科学院农产品加工研究所 一种用于提取茶籽粕中茶多酚的低共熔溶剂及提取工艺
WO2022101490A1 (fr) * 2020-11-13 2022-05-19 Universidade Do Minho Compositions eutectiques, procédés et utilisations associés
EP4011352A1 (fr) * 2020-12-09 2022-06-15 Beiersdorf AG Nouveaux solvants cosmétiques basé sur trois composants différents
CN113563393A (zh) * 2021-06-09 2021-10-29 华南农业大学 一种高效提取三叉苦药渣生物碱及联产生物甲烷的方法

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