Natural active ingredient extraction method

Abstract

The present invention relates to a method for extracting natural bioactive compounds having different polarities from solid plant waste, such as kale waste, by using a solid-liquid extraction-liquid-liquid extraction (SLE-LLE) integrated platform, using a ternary solvent mixture comprising a natural deep eutectic solvent (NADES) comprising glycerol, ethyl acetate and water, or a binary solvent mixture comprising NADES comprising glycerol and ethyl acetate is used. The invention also relates to a solid plant waste extract comprising NADES comprising glycerol, water and one or more polyphenol compounds.

Description

Technical Field

[0001] The present invention relates to a method for extracting natural bioactive compounds with different polarities from solid plant waste, the resulting solid plant waste extract, a ternary solvent, and the use of the ternary solvent. Background Art

[0002] The prior published documents listed or discussed in this specification should not be regarded as an admission that such documents are part of the prior art or common general knowledge.

[0003] Food loss and waste are serious global problems and are on the rise in many countries. For example, in 2021, Singapore wasted approximately 817,000 tons of food, a 23% increase from 665,000 tons in 2020, with nearly half of it being fruits and vegetables. Perishable agricultural products usually suffer significant losses during the post-harvest stage before reaching supermarket shelves. In addition to post-harvest spoilage, a large number of agricultural products with defects in size, shape, or color are rejected during strict aesthetic screening for sale in supermarkets. This loss has adverse effects on both the economy and the environment. For those rejected agricultural products that are discarded only because they fail to meet the requirements of consumers and retailers due to poor aesthetic appearance, they may have the same nutritional composition and food safety as marketable products. These wastes should be upgraded to higher-value products that can be fed back into the supply chain to achieve circularity and sustainability in agricultural production.

[0004] In particular, cruciferous vegetables, such as kale (Brassica oleracea var. acephala), are becoming increasingly popular as a nutrient-rich "superfood" that contains very high levels of health-promoting phytochemicals, such as polyphenols and carotenoids, and can bring significant health benefits in addition to basic nutrients.

[0005] Rejected kale leaves during cosmetic screening are an attractive source of phytochemicals and have potential applications in the cosmetic and food industries. In fact, kale is rich in phenolic compounds such as flavonoids (e.g., quercetin and kaempferol) and contains more antioxidants than other vegetables such as spinach, broccoli, and cauliflower. It is reported that the total phenols in freeze-dried kale are 14.03 mg g -1 , much higher than that in spinach (11.47 mg g -1 ) and broccoli (8.33 mg g -1)(Vargas, L. et al., LWT, 2022, 155, 112892). Polyphenols are powerful antioxidants that can mitigate oxidative stress through radical scavenging activity (RSA), thereby reducing the risk of chronic degenerative diseases (Kaulmann, A. et al., Food Chemistry, 2014, 155, 240). In addition, lipophilic pigments such as carotenoids and chlorophyll found in kale have been shown to have positive effects on inflammatory behavior, neuroprotection, and the reduction of coronary artery disease and ophthalmic diseases (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). Due to their health-promoting effects, kale extracts have been applied in the formulations 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 pharmaceuticals (Lemos, M. et al., Journal of Ethnopharmacology, 2011, 138, 503; Das, G. et al., International Journal of Nanomedicine, 2022, 17, 1125). Clinical studies have demonstrated that oral supplements containing curly kale extracts rich in carotenoids not only increase the RSA and lipids of the skin (daily dose of 4.45 mg total carotenoids) (Meinke, M. C. et al., European Journal of Pharmaceutics and Biopharmaceutics, 2013, 84, 365), but also avoid the degradation of type I collagen associated with aging in the dermis, even at a low daily dose of 1.65 mg carotenoids (Meinke, M. C. et al., Nutrients, 2017, 9, 775). Agricultural products rejected during the harvest process can be a valuable source of phytochemicals that have the potential to be used as natural antioxidants, pigments, and functional ingredients in cosmetic and food applications.

[0006] In addition to efficacy evaluation studies, the recovery process of kale extract is another important aspect, which directly affects the acceptance by regulatory agencies and consumers. In the past few decades, the problem of separating bioactive compounds from vegetable waste has been solved through conventional techniques involving volatile organic compounds (VOCs) derived from petroleum. Methanol, acetone, and their aqueous mixtures are commonly used to separate polyphenols from food waste (Nayak, B. et al., Food Chemistry, 2015, 187, 507; Meneses, N.G. et al., Separation and purification technology, 2013, 108, 152), while carotenoids are mainly extracted by hexane for food and pharmaceutical purposes. Volatile organic compounds above a certain concentration level are harmful to the environment and human health. The conventional organic solvents used for recovering these bioactive metabolites are harmful, so milder and harmless equivalents need to be sought. In addition to solution-based extraction methods, advanced techniques such as supercritical fluid extraction (Borja-Martínez, M. et al., Antioxidants, 2020, 9, 1195) and microwave-assisted extraction ( N. et al., Sustainable Chemistry and Pharmacy, 2022, 28, 100728) have also been studied. However, due to the use of high-cost specialized equipment in these extraction technologies, they may be economically unfeasible for industrial scale. Therefore, in order to extract nutrients, it is crucial to develop scalable, sustainable, and safe processes using harmless solvents.

[0007] Natural deep eutectic solvents (NADES) have been proposed as environmentally friendly alternatives to harmful volatile organic compounds (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50). They are liquids composed entirely 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). In addition to being non-volatile and biodegradable, NADES also have the characteristic of high solubilizing ability for natural agricultural products, making them suitable extraction media. Moreover, the natural components of NADES enable the direct use of the extracts in food, pharmaceutical, and cosmetic applications, thus simplifying the product purification steps (da Silva, D.T. et al., Food Chemistry, 2021, 364, 130370).

[0008] In addition to those polar-dominant NADES that have advantages in extracting polar metabolites, hydrophobic NADES have been introduced and used to isolate 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 β-carotene (Stupar, A. et al., Ultrasonics Sonochemistry, 2021, 76, 105638) from various matrices. The extraction ability of NADES may vary depending on the nature of the target bioactive metabolites present in the source. The extraction of bioactive compounds from kale waste using NADES has not been fully evaluated. In addition, most studies usually focus on a specific group of compounds (either hydrophilic or lipophilic), and the information on the ability of hydrophilic / hydrophobic NADES to recover different classes of bioactive metabolites from kale waste is still limited. Sustainable recovery processes need to be developed to effectively valorize kale waste for the production of natural antioxidants and pigments. Summary of the Invention

[0009] The present invention provides a comprehensive and effective extraction method for extracting nutrients and active ingredients from vegetables, other plants, and their waste streams using green and even food-safe solvents in a single step. The active ingredients that can be extracted from these materials include, but are not limited to, natural antioxidants and pigments for use in cosmetics, food supplements, pharmaceuticals, and other applications. Little pretreatment of the plant or plant waste is required.

[0010] Some aspects and embodiments of the present invention are described in the following numbered clauses.

[0011] 1. A method for extracting natural bioactive compounds with different polarities from solid plant waste, the method comprising the steps of:

[0012] (a) providing dry solid plant waste or wet solid plant waste; and

[0013] (b) contacting the dry solid plant waste with a ternary solvent mixture at a suitable temperature for a suitable period of time, the ternary solvent mixture comprising:

[0014] a natural deep eutectic solvent (NADES) containing glycerol,

[0015] water, and

[0016] Ethyl acetate; or

[0017] Contacting the wet solid plant waste with a binary solvent mixture at a suitable temperature for a suitable period of time, the binary solvent mixture comprising:

[0018] A natural deep eutectic solvent (NADES) containing glycerol, and

[0019] Ethyl acetate; wherein:

[0020] The NADES is a hydrophilic NADES; and

[0021] Relative to the total weight of water and NADES in the ternary solvent, or

[0022] Relative to the total weight of water in the wet solid plant waste and NADES in the binary solvent,

[0023] The water content is 20% to 40% by weight.

[0024] 2. The method according to clause 1, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of amino acids, sugar alcohols, sugars, and urea.

[0025] 3. The method according to clause 2, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.

[0026] 4. The method according to clause 3, wherein the NADES is formed from:

[0027] (ai) Glycerol and betaine;

[0028] (aii) Glycerol and sorbitol;

[0029] (aiii) Glycerol and glucose;

[0030] (aiv) Glycerol and fructose; or

[0031] (av) Glycerol and urea.

[0032] 5. The method according to clause 4, wherein the NADES is formed from:

[0033] (ai) Glycerol and betaine; or

[0034] (aiv) Glycerol and fructose.

[0035] 6. The method according to any one of clauses 2 to 5, wherein the molar ratio of glycerol to the other component is from 1:1 to 5:1, such as from 2:1 to 3:1, such as about 3:1.

[0036] 7. The method according to any one of the preceding clauses, wherein the content of water is 25% to 35% by weight, such as about 30% by weight, relative to the total weight of water and NADES in the ternary solvent.

[0037] 8. The method according to any one of the preceding clauses, wherein the volume ratio of ethyl acetate to the volume of water and NADES in the ternary solvent is from 1:5 to 5:1, such as from 1:3 to 3:1, such as about 1:1.

[0038] 9. The method according to any one of the preceding clauses, wherein the solid-liquid ratio (g / mL) of the dry 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.

[0039] 10. The method according to any one of the preceding clauses, wherein one or both of the following apply:

[0040] (bi) A 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; and

[0041] (bii) A suitable temperature is from 15 °C to 65 °C, such as from 20 °C to 45 °C, such as about 25 °C.

[0042] 11. The method according to any one of the preceding clauses, wherein the solid plant waste is provided in particulate form, optionally wherein the average diameter of the particles is from 0.1 mm to 10 mm, such as an average diameter of from 0.5 mm to 5 mm, such as an average diameter of about 2 mm.

[0043] 12. The method according to any one of the preceding clauses, wherein the dry solid plant waste is provided in a form that has substantially removed all water, optionally wherein the dry solid plant waste is provided in a freeze-dried form.

[0044] 13. The method according to any one of the preceding clauses, wherein the plant waste is vegetable waste, optionally wherein the vegetable waste is cruciferous vegetable waste, and further optionally wherein the cruciferous vegetable waste is selected from one or more of spinach, broccoli, cauliflower, and more specifically curly kale (Brassica oleracea var. acephala) leaves.

[0045] 14. The method according to any one of the preceding clauses, wherein the method further comprises, after step (b), separating and retaining the resulting ethyl acetate extract and the resulting NADES / water extract from the solid plant waste.

[0046] 15. A solid plant waste extract, comprising:

[0047] A natural deep eutectic solvent (NADES) containing glycerol;

[0048] Water; and

[0049] One or more polyphenolic compounds.

[0050] 16. A ternary solvent, comprising:

[0051] A natural deep eutectic solvent (NADES) containing glycerol;

[0052] Water; and

[0053] Ethyl acetate, wherein:

[0054] The NADES is a hydrophilic NADES; and

[0055] Relative to the total weight of water and NADES in the ternary solvent, the content of water is 20% to 40% by weight.

[0056] 17. The ternary solvent according to clause 16, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of amino acids, sugar alcohols, sugars, and urea.

[0057] 18. The ternary solvent according to clause 17, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.

[0058] 19. The ternary solvent according to clause 18, wherein the NADES is formed from:

[0059] (ai) Glycerol and betaine;

[0060] (aii) Glycerol and sorbitol;

[0061] (aiii) Glycerol and glucose;

[0062] (aiv) Glycerol and fructose; or

[0063] (av) Glycerol and urea.

[0064] 20. The ternary solvent according to clause 19, wherein the NADES is formed from:

[0065] (ai) Glycerol and betaine; or

[0066] (aiv) Glycerol and fructose.

[0067] 21. The ternary solvent according to any one of clauses 17 to 20, wherein the molar ratio of glycerol to the other components is 1:1 to 5:1, such as 2:1 to 3:1, such as about 3:1.

[0068] 22. The ternary solvent according to any one of clauses 17 to 21, wherein the content of water is 25% by weight to 35% by weight, such as about 30% by weight, relative to the total weight of water and NADES in the ternary solvent.

[0069] 23. The ternary solvent according to any one of clauses 17 to 22, wherein the volume ratio of ethyl acetate to the volume of water and NADES is 1:5 to 5:1, such as 1:3 to 3:1, such as about 1:1.

[0070] 24. Use of the ternary solvent according to any one of clauses 1 to 8 or any one of clauses 16 to 23 for extracting natural bioactive compounds with different polarities from solid plant waste. Description of the Drawings

[0071] Figure 1 Shows the use of solvents (H2O - water, MeOH - methanol, 70% MeOH - 70% aqueous methanol solution, EtOH - ethanol, 70% EtOH - 70% aqueous ethanol solution, 70% Gly - 70% aqueous glycerol solution, respectively 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 / Fru - glycerol: fructose (3:1) and Gly / U - glycerol: urea (1:1) 70% aqueous NADES solution, Hex - hexane, EtOAC - ethyl acetate, Men / Thy - DL - menthol: thymol (1:1), Men / Thy 2 - DL - menthol: thymol (1:2), Men / Fen - DL - menthol: fenchyl alcohol (1:1), Thy / Fen - thymol: fenchyl alcohol (1:1)), the recovery rates of total phenols, carotenoids (β - carotene, lutein) and chlorophyll (a and b) recovered from kale waste after stirring at a solid - liquid ratio (SLR) of 1:40 and at 150 rpm and 25 °C for 30 minutes. ND indicates not determined. Different letters in the same series indicate significant differences at the p < 0.05 level.

[0072] Figure 2Shows the chromatogram obtained by RP-HPLC at a wavelength of 450 nm of the kale waste extract obtained using ethanol (EtOH) and ethyl acetate (EtOAc) under the reversed-phase high-performance liquid chromatography (RP-HPLC) operation described in Example 2, in which the peaks of β-carotene, lutein, chlorophyll a and b and their chemical structures are highlighted.

[0073] Figure 3 Shows (A) the antioxidant activity of the kale waste extract determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay method, which was obtained using solvents (H2O - water, MeOH - methanol, 70% MeOH - 70% aqueous methanol solution, EtOH - ethanol, 70% EtOH - 70% aqueous ethanol solution, 70% Gly - 70% aqueous glycerol solution, respectively 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 / Fru - glycerol:fructose (3:1) and Gly / U - glycerol:urea (1:1) in 70% NADES aqueous solution); and (B) the correlation (simple regression analysis) between the antioxidant activity and the total phenols in the extract. Different letters in the same series indicate significant differences at the p < 0.05 level.

[0074] Figure 4 Shows the recovery rate of total phenols recovered from kale waste using NADES based on glycerol:betaine (3:1), in which (A) the effect of the solid-liquid ratio (SLR) was studied under the conditions of a solvent concentration of 70%, 25 °C and a duration of 30 minutes, and (B) under the condition of using an SLR of 1:20, the effects of solvent concentration (50%, 60%, 70% and 80%), extraction temperature (25 °C, 45 °C and 65 °C) and time (30 minutes and 60 minutes) were studied and compared with ultrasonic-assisted extraction (operated at 37 kHz and 100% power). Different letters in the same series indicate significant differences at the p < 0.05 level.

[0075] Figure 5Shows the stability of kale waste extracts produced using solvents (H2O - water, 70% EtOH - 70% aqueous ethanol solution, 70% Gly - 70% aqueous glycerol solution, and 70% Gly / Bet - 70% glycerol:betaine (3:1) aqueous solution), represented as the change in the relative concentration of total phenols over time after storage in the dark at (A) 25 °C and (B) 4 °C for 30 days; and (C) photographs of the extracts just after extraction and during storage. The original extract prepared with water was used as a comparative control.

[0076] Figure 6 Shows the photographs of kale waste extracts (on a dark background) obtained using water (H2O),

[0077] 70% aqueous ethanol solution (70% EtOH), 70% aqueous glycerol solution (70% Gly), and 70% glycerol / betaine (70% Gly / Bet) with a molar ratio of 3:1 during storage in the dark at 25 °C and 4 °C.

[0078] Figure 7 Shows the recovery rates of total phenols, carotenoids (β - carotene, lutein), and chlorophylls (a and b) recovered from kale waste using the sequential methods (Routes 1 and 2) and the integrated method (Route 3) designed in this study. Different letters in the same series indicate significant differences at the p < 0.05 level.

[0079] Figure 8 Shows a diagram of an integrated process for the upgrading and recovery of kale waste using a green and cosmetic / food - grade ternary solvent mixture (Gly / Bet - glycerol:betaine (3:1) + H2O - water + EtOAc - ethyl acetate), which is used to produce natural antioxidants and pigments with potential applications in the cosmetic and food industries. The dashed lines have not been experimentally tested but are often used for product purification.

[0080] Figure 9 Shows the recovery rates of total phenols, carotenoids (β - carotene, lutein), and chlorophylls (a and b) recovered from kale waste using the integrated method with glycerol / fructose (3:1) + H2O + ethyl acetate and glycerol /

[0081] betaine (3:1) + H2O + ethyl acetate, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0083] As described above, the present invention provides a comprehensive and effective extraction method for extracting nutrients and active ingredients from vegetables, other plants, and their waste streams using green and even food-safe solvents in a single step. Thus, in a first aspect of the present invention, there is provided a method for extracting natural bioactive compounds with different polarities from solid plant waste, the method comprising the following steps:

[0084] (a) providing dry solid plant waste or wet solid plant waste; and

[0085] (b) contacting the dry solid plant waste with a ternary solvent mixture at a suitable temperature for a suitable period of time, the ternary solvent mixture comprising:

[0086] a natural deep eutectic solvent (NADES) containing glycerol,

[0087] water, and

[0088] ethyl acetate; or

[0089] contacting the wet solid plant waste with a binary solvent mixture at a suitable temperature for a suitable period of time, the binary solvent mixture comprising:

[0090] a natural deep eutectic solvent (NADES) containing glycerol, and

[0091] ethyl acetate; wherein:

[0092] the NADES is a hydrophilic NADES; and

[0093] relative to the total weight of water and NADES in the ternary solvent, or

[0094] relative to the total weight of water in the wet solid plant waste and NADES in the binary solvent,

[0095] the water content is 20% to 40% by weight.

[0096] As used herein, the term "comprising" may be construed as requiring the stated features, but not precluding the presence of other features. Alternatively, the term "comprising" may also relate to cases where only the listed components / features are intended to be present (e.g., the term "comprising" may be replaced by the phrase "consisting of" or "consisting essentially of"). It is expressly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the term "comprising" and its synonyms may be replaced by the phrase "consisting of" or "consisting essentially of" or their synonyms, and vice versa.

[0097] The phrase "consisting essentially of" and variations thereof as used herein can be interpreted to mean a material in which there may be minor impurities. For example, the purity of the material can be greater than or equal to 90%, such as greater than 95%, such as greater than 97%, such as greater than 99%, such as greater than 99.9%, such as greater than 99.99%, such as greater than 99.999%, such as 100% purity.

[0098] As used herein, unless the context clearly dictates otherwise, the singular forms "a" and "the" include plural referents. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, and so forth.

[0099] As described above, "solid plant waste" can be any suitable waste. For example, it can be the whole or part of a vegetable or other plant (e.g., vegetables past their expiration date), or it can be a waste stream associated with a vegetable or other plant (e.g., the roots of a vegetable or plant). It is understood that a single vegetable or plant can be used in the method, or a combination thereof can be used.

[0100] As described above, the ternary solvent mixture in the method of the present invention comprises: a natural deep eutectic solvent (NADES) containing glycerol as a first component, water as a second component, and ethyl acetate as a third component. In some embodiments, the ternary solvent mixture in the method consists essentially of a natural deep eutectic solvent (NADES) containing glycerol as a first component, water as a second component, and ethyl acetate as a third component.

[0101] The binary solvent comprises NADES and ethyl acetate (or consists of NADES and ethyl acetate), and water is supplied by using wet plant waste. In such an embodiment, relative to the total weight of the wet solid plant waste, the wet solid plant waste can contain 65 wt% to 90 wt% (e.g., about 75 wt%) of water. This can be measured by weighing a sample of the wet plant waste, then drying the sample, and calculating the percentage of water based on the weight loss after drying. It is understood that the water content of the wet solid plant waste can be concentrated to a specific amount required to maintain the same solid-liquid ratio for the extraction process.

[0102] A deep eutectic solvent is a Lewis or Solutions of acids and bases. NADES are biobased deep eutectic solvents composed of two or more compounds, which are usually plant-based primary metabolites, namely organic acids, sugars, alcohols, amines, and amino acids. As mentioned above, NADES contains glycerol and additionally at least one other suitable component that forms the NADES. Suitable other components include, but are not limited to, amino acids, sugar alcohols, sugars, urea, and combinations thereof. In specific embodiments of the invention that may be mentioned herein, NADES may contain glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea (or consist of glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea).

[0103] In more specific embodiments of the invention that may be mentioned herein, NADES may be formed from:

[0104] (ai) glycerol and betaine;

[0105] (aii) glycerol and sorbitol;

[0106] (aiii) glycerol and glucose;

[0107] (aiv) glycerol and fructose; or

[0108] (av) glycerol and urea.

[0109] For example, NADES may be formed from:

[0110] (ai) glycerol and betaine; or

[0111] (aiv) glycerol and fructose.

[0112] In NADES, any suitable molar ratio may be adopted between glycerol and the other components. Examples of suitable molar ratios include, but are not limited to, a molar ratio of glycerol to the other components of 1:1 to 5:1, such as 2:1 to 3:1, such as approximately 3:1. It is understood that if there is more than one other component, the sum of the molar values of the other components will be used to calculate the molar ratio.

[0113] As described above, water is present as the second component in the ternary solvent, and the content of water is 20% to 40% by weight relative to the total weight of water and NADES in the ternary solvent. In further embodiments that may be mentioned herein, the content of water can be 25% to 35% by weight relative to the total weight of water and NADES in the ternary solvent, for example, about 30% by weight. It should be understood that when using wet solid plant waste, a similar proportion of water can be used in combination with the binary solvent (i.e., the weight percentage of water can be calculated based on the weight of the solid plant waste relative to the weight of water in the wet solid plant waste (calculated based on the mass percentage of water in the wet solid plant waste) and the weight of NADES in the binary solvent).

[0114] As described above, ethyl acetate is present as the third component of the ternary solvent (or the second component in the binary solvent). Ethyl acetate can be present in any suitable amount in the ternary solvent. For example, the volume ratio of ethyl acetate to the volume of water and NADES can be from 1:5 to 5:1, such as from 1:3 to 3:1, such as about 1:1. A similar amount of ethyl acetate can be used in the binary solvent, where the amount of ethyl acetate is based on the calculated volume of water and NADES (calculated based on the expected volume of water in NADES and wet solid plant waste).

[0115] It can be understood that the methods disclosed herein require the solid plant waste to be contacted with the ternary solvent to achieve the extraction of natural bioactive compounds of different polarities. Although any suitable solid-liquid ratio (g / mL) can be used, it has been found that a suitable ratio can be such that the solid-liquid ratio (g / mL) of the solid plant waste to the ternary solvent can 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-liquid ratio can be used for the binary solvent and the wet plant waste. For example, the ratio of the solid (dry weight of the solid in the plant waste) to the liquid (water present in the binary solvent and the wet plant waste) can 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.

[0116] This document can use any suitable time period that allows for the effective extraction of natural bioactive compounds with different polarities from solid plant waste. For example, a suitable time period can 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 approximately 30 minutes. This document can use any suitable temperature that allows for the effective extraction of natural bioactive compounds with different polarities from solid plant waste. For example, a suitable temperature is from 15 °C to 65 °C, such as from 20 °C to 45 °C, such as approximately 25 °C. Thus, the method can simply and effectively extract the desired substances using a temperature around room temperature (i.e., approximately 25 °C) and a contact time of approximately 30 minutes. Therefore, the method can be carried out quickly and effectively without the need to heat or cool the surrounding environment, thereby reducing energy consumption. These temperatures and times can be used with the ternary or binary solvents mentioned in this document when used with dry solid plant waste or wet solid plant waste, respectively.

[0117] The solid plant waste can be provided in any suitable form. For example, the solid plant waste can be provided as an entire plant or vegetable. In a more specific embodiment of the present invention, the solid plant waste can be provided in particulate form, which helps to quickly and effectively extract the desired substances. Any suitable particle size can be used. For example, the average diameter of the particles can be from 0.1 mm to 10 mm, such as an average diameter of from 0.5 mm to 5 mm, such as an average diameter of approximately 2 mm. It is understood that these particle sizes are applicable to both dry solid plant waste and wet solid plant waste.

[0118] The solid plant waste can be provided in a hydrated or dried form.

[0119] In some embodiments of the present invention, a hydrated or "wet" form of the solid plant waste is used. In such an embodiment, the wet form will be used with the binary solvent as described above.

[0120] In a preferred embodiment of the present invention, the solid plant waste can be provided in a dried form, which is used with the ternary solvent. More specifically, the solid plant waste can be provided in a dried form in which substantially all of the water has been removed. As used herein, "substantially all" can 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. In a specific embodiment of the present invention, the solid plant waste can be provided in a freeze-dried form. It is understood that the solid plant waste can be provided in particulate form that has been (freeze) dried to remove water.

[0121] In some embodiments of the present invention, the solid plant waste can be vegetable waste. While any suitable vegetable waste can be used in the present invention, the vegetable waste can be cruciferous vegetable waste. For example, the cruciferous vegetable waste can be selected from one or more of spinach, broccoli, cauliflower, and more specifically curly kale (Brassica oleracea var. acephala) leaves.

[0122] In a specific embodiment of the present invention, the method can further include, after step (b) of the method, separating and retaining the resulting ethyl acetate extract and the resulting NADES / water extract from the solid plant waste. This can separate the hydrophobic natural bioactive compounds and the hydrophilic natural bioactive compounds from each other, so that the extracted compounds can be utilized more effectively. It can be understood that although the binary solvent basically does not include water, at the end of the process, water has been extracted from the wet solid plant waste, so the resulting solution before separation will effectively be similar to a ternary solvent, and thus the separation of the mixture in step (b) of the above method will produce an ethyl acetate extract and a NADES / water extract, which are substantially the same as the extracts obtained when dry solid plant waste is used with a ternary solvent.

[0123] The resulting ethyl acetate extract can be rich in carotenoids and chlorophyll, while the NADES / water extract can be rich in polyphenols.

[0124] In another aspect of the present invention, there is provided a solid plant waste extract comprising:

[0125] A natural deep eutectic solvent (NADES) containing glycerol;

[0126] Water; and

[0127] One or more polyphenol compounds.

[0128] It can be understood that this aspect of the present invention can be a product obtained after the step of further separating and retaining the resulting NADES / water extract from the solid plant waste in the method of the first aspect of the present invention and after step (b) of the method.

[0129] It should be understood that the solid plant waste and NADES are as described in the first aspect of the present invention. Therefore, to avoid repetition, these components will not be further discussed here.

[0130] It has also been surprisingly found that storing the polyphenols in the NADES / water extract can preserve the polyphenols for a longer time, both at ambient temperature (e.g., 25 °C) and at low temperature (e.g., in a refrigerator at 4 °C). For example, after storage at 4 °C and 25 °C for 30 days, the 70 wt% Gly / Bet NADES (30 wt% water) extract retained 91.7% and 88.6% of the original bioactive polyphenol content, respectively.

[0131] In another aspect of the present invention, there is provided a ternary solvent comprising:

[0132] a natural deep eutectic solvent (NADES) containing glycerol;

[0133] water; and

[0134] ethyl acetate, wherein:

[0135] the NADES is a hydrophilic NADES; and

[0136] relative to the total weight of water and NADES in the ternary solvent, the content of water is 20 wt% to 40 wt%.

[0137] Since the ternary solvent and its embodiments have been discussed in detail in the first aspect of the present invention, in order to avoid repetition, no further discussion will be made here.

[0138] In another aspect of the present invention, there is provided the use of the ternary solvent as described above in extracting natural bioactive compounds with different polarities from solid plant waste.

[0139] As disclosed herein, a ternary solvent comprising NADES, water, and ethyl acetate can be used to extract various bioactive metabolites from plant waste (such as kale waste), including polyphenols, carotenoids (such as β-carotene and lutein), and chlorophyll. This makes it possible to develop a sustainable process for producing natural antioxidants and pigments (such as chlorophyll).

[0140] Specifically, Gly / Bet (glycerol: betaine)-based NADES produced enhanced polyphenol-rich extracts under simple and mild conditions (25 °C, 30 minutes, solid-liquid ratio (SLR) of 1:20, solvent concentration of 70%). Additionally, it conferred good stability to the extracts, retaining >90% of polyphenols and transparency after storage at 4 °C for 30 days. Finally, to fully utilize kale waste, a combined method using an aqueous Gly / Bet solution and ethyl acetate (EtOAc) ternary mixture was employed to simultaneously recover and separate bioactive compounds with different polarities, dividing them into two fractions; one fraction was a ready-to-use polyphenol-rich extract, and the other was an extract rich in carotenoids and chlorophylls. The results disclosed herein demonstrate that the use of biobased solvents to extract multiple bioactive metabolites from kale waste under mild conditions and their application to a solid-liquid extraction - liquid-liquid extraction (SLE-LLE) integrated platform is a sustainable alternative for the efficient recovery of various valuable products from kale waste. It is understood that kale waste is used herein as an exemplary system, and other materials can also be used as the base waste material, and similar results are expected to be obtained.

[0141] Other aspects and embodiments of the present invention will be discussed in the following non-limiting examples. Examples

[0142] Material

[0143] Gallic acid (certified reference material), β-carotene (pharmaceutical secondary standard), lutein (pharmaceutical secondary standard), chlorophyll a from spinach (≥85%), chlorophyll b from spinach (≥90%), 6-hydroxy-2,5,7,8-tetramethylchroman-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%), anisyl alcohol (≥97%), triethylamine (≥99%), tetrahydrofuran (THF) (≥99%) and Folin-Ciocalteu phenol reagent were purchased from Sigma-Aldrich. Methanol (LCMS grade, ≥99.9%), acetonitrile (LCMS grade, ≥99.9%) and ethanol (HPLC grade, ≥99.8%) were supplied by Fisher Scientific. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (≥95%) was purchased from Alfa Aesar. All chemicals were used directly without further purification. Ultra-pure water was prepared using a Millipore Milli-Q water purification system.

[0144] The kale waste was provided by Sustenir Agriculture (Singapore). The curly kale leaves used in this study did not meet the commercial quality standards and were intended to be discarded as waste by the urban agriculture company. The kale waste was cleaned, freeze-dried, ground in a laboratory ball mill, and sieved to obtain a powder size of <0.2 mm, and stored in a sealed container at 4 °C until further use.

[0145] Data Analysis

[0146] The experiments were independently performed in triplicate for each case, and the results were expressed as mean ± standard deviation. One-way analysis of variance was used for statistical significance analysis, and post hoc Tukey's test was performed for all pairwise multiple comparisons. The statistical significance level was set at P < 0.05.

[0147] Example 1. Preparation and Characterization of Natural Deep Eutectic Solvents (NADES)

[0148] All selected NADES precursors are renewable, low-cost, and ingredients permitted for use in food and cosmetics.

[0149] Preparation of NADES

[0150] The NADES were prepared by mixing the respective precursors in a glass bottle at a certain molar ratio and continuously heating (maximum temperature of 80 °C) and stirring until a clear and homogeneous liquid was formed. They were kept at room temperature and no precipitation was observed in the liquid. All abbreviations and compositions of the NADES used in this study are shown in Table 1.

[0151] Viscosity and pH Measurement

[0152] The viscosities of all hydrophilic NADES containing 30% (w / w) water and pure hydrophobic NADES were measured using a modular compact rheometer (Anton Paar MCR 102, Germany) equipped with a cone-plate measuring geometry (CP50-1) with a diameter of 50 mm. The gap between the cone and the plate was set at 0.095 mm, and the temperature of the system was controlled by a Peltier temperature device (P-PTD200). All measurements were carried out at 25 °C with a constant shear rate of 10 s -1 , for 10 s. The final viscosity obtained was the average of the results. The pH values of all hydrophilic NADES aqueous solutions containing 30% (w / w) water were measured using a pH meter (Mettler

[0153] Toledo, Singapore) at 25 °C.

[0154] Table 1. List of NADES

[0155]

[0156] a The pH value of a 70% hydrophilic NADES aqueous solution.

[0157] b The viscosity of a 70% hydrophilic NADES aqueous solution.

[0158] c The viscosity of pure hydrophobic NADES.

[0159] Hydrophilic NADES was prepared by matching glycerol with betaine, sorbitol, xylose, glucose, fructose, and urea in appropriate molar ratios based on the literature (Jin, Y. et al., Applied Sciences, 2019, 9, 2581; Zheng, B. et al., LWT, 2022, 154, 112740). On the other hand, different combinations of terpenes including DL-menthol, thymol, and carvacrol formed the hydrophobic NADES disclosed in this example in appropriate molar ratios (Fan, C. et al., Food Chemistry, 2022, 376, 131930).

[0160] Example 2. Extraction of bioactive metabolites from kale waste and screening of solvents

[0161] Prepare NADES according to the protocol disclosed in Example 1.

[0162] First, the performance of hydrophilic and hydrophobic NADES in recovering bioactive metabolites (especially polyphenols, carotenoids (β-carotene and lutein), and chlorophylls (a and b)) from kale waste after stirring at 150 rpm and 25 °C for 30 minutes was screened. Since the high viscosity of hydrophilic NADES may hinder its extraction ability (Wojeicchowski, J. P. et al., Separation and Purification Technology, 2021, 258, 117975), they were used in a hydrated form, i.e., 30% water was added. For easy comparison, several reference solvents were tested under the same conditions, including water (H2O), methanol (MeOH), 70% methanol aqueous solution (70% MeOH), ethanol (EtOH), 70% ethanol aqueous solution (70% EtOH), 70% glycerol aqueous solution (70% Gly), n-hexane (Hex), and ethyl acetate (EtOAc).

[0163] Solid-liquid extraction (SLE)

[0164] Solid-liquid extraction (SLE) of freeze-dried kale waste powder was carried out for 30 minutes at 150 rpm and 25 °C with a solid-liquid ratio (SLR) of 1:40 using a series of solvents. All the hydrophilic NADES tested were in the hydrated form with 30 wt% water added, while pure hydrophobic NADES were used in the case of hydrophobic NADES. The mixture was vortexed at 3000 rpm for 30 s. All the studies were carried out under low light to prevent the degradation of photosensitive compounds. The mixture was then centrifuged at 12,000 g for 10 minutes, the supernatant was collected, filtered through a PTFE syringe filter (0.45 μm, 25 mm), and then subjected to quantitative analysis.

[0165] Determination of total phenolic content

[0166] Total phenols were estimated as gallic acid equivalents (GAE) using a slightly modified 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). 25 μL of the diluted sample (supernatant after extraction) was mixed with 200 μL of water, and then 25 μL of 25% (v / v) Folin-Ciocalteu reagent was added. After 5 minutes, 25 μL aliquots of 10% (w / v) sodium carbonate were added, the reaction mixture was gently shaken, and incubated in the dark at 25 °C for 60 minutes. The total phenolic content in the extract was determined colorimetrically by measuring the absorbance at 765 nm using a microplate reader (Tecan Infinite M200 Pro, Singapore) based on a gallic acid calibration curve. To eliminate the influence of the solvent, blank control systems for each sample were prepared and used under the same conditions. The results were recorded as milligrams of gallic acid equivalents per gram of dry weight of kale waste (mg GAE g -1 DW). It should be noted that for the hydrophobic solvents studied, liquid-liquid extraction (LLE) was carried out on the collected extracts by adding an equal volume of water. The mixture was shaken vigorously, and the total phenol determination was carried out on the aqueous layer collected after phase separation by centrifugation. Men / Thy, Men / Thy 2 and the total phenols in the Thy / Fen extracts were not determined due to interference with the detection reagent.

[0167] Quantification of carotenoids (β-carotene and lutein) and chlorophylls (a and b)

[0168] β-carotene, lutein, and chlorophyll a and b in the extracts were identified and quantified using a reverse-phase high-performance liquid chromatography (RP-HPLC) protocol. Separation and detection of these compounds were accomplished on an Agilent ZORBAX Eclipse Plus C18 (3.0 mm × 150 mm, 3.5 μm) column with an Agilent 1260 Infinity II LC system (Agilent Technologies, Singapore), equipped with a diode array UV-Vis detector set at a wavelength of 450 nm, using an isocratic elution method for 15 minutes at a flow rate of 1.0 mL min -1 . The mobile phase was methanol and acetonitrile (9:1 v / v), with aliquots of 0.1% (v / v) triethylamine added to prevent non-specific adsorption and oxidation. The column temperature was maintained at 25 °C, and the sample injection volume was 5 μL.

[0169] Each identified compound was verified based on the retention time of commercial compounds. A β-carotene stock solution was prepared by dissolving β-carotene in a small amount of THF stabilized with butylated hydroxytoluene (BHT) (1% (v / v) of the total solvent volume), and then diluting with ethanol. Lutein standard stock solution and chlorophyll standard stock solution were prepared using ethanol and methanol, respectively. The exact concentration of the standard stock solutions was evaluated using a spectrophotometer (Shimadzu UV-1800, Japan), and was corrected by adopting the extinction coefficients of the respective compounds at their respective wavelengths : β-carotene (at a wavelength of 450 nm, in ethanol ); lutein (at a wavelength of 445 nm, in ethanol ); chlorophyll a (at a wavelength of 665 nm, in methanol ), chlorophyll b (at a wavelength of 652 nm, in methanol mL) -1 )(Craft, N.E. & Soares, J.H., Journal of agriculturaland food chemistry, 1992, 40, 431; Porra, R. et al., Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1989, 975, 384). The results were expressed as milligrams per gram of kale waste dry weight (mg g -1 DW).

[0170] Results and Discussion

[0171] Figure 1 The yield results of the target compound when using different solvents are shown. According to the mass transfer mechanism of "like dissolves like", all glycerol-based hydrophilic NADES showed selective extraction effects on polyphenols, while lipophilic compounds were favorably extracted by terpene-based hydrophobic NADES.

[0172] Compared with the reference solvent, all glycerol-based NADES aqueous solutions achieved higher polyphenol yields except for Gly / U. This may be due to the high solubility of polyphenol molecules in the NADES aqueous solution, which is caused by the strong interaction between polyphenol molecules and NADES components. The NADES components are good hydrogen bond donors and acceptors, as shown in Table 2.

[0173] Table 2. Chemical structures of the precursors used to prepare NADES and their hydrogen bond formation trends.

[0174]

[0175]

[0176] In the present invention, the highest polyphenol yield was obtained with Gly / Bet, which was approximately 2.2 times and 1.3 times that of using MeOH and H2O, respectively. In addition, among the glycerol-based NADES, those coupled with sugar alcohols (i.e., coupled with sorbitol (Gly / Sor), coupled with xylose (Gly / Xyl)) showed relatively better dissolution ability for polyphenols than the sugar counterparts (i.e., coupled with glucose (Gly / Glu), coupled with fructose (Gly / Fru)). Since the tested NADES have a common glycerol component, the variation in yields between these solvents can be attributed to the different hydrogen bond-forming abilities of the second components. The logarithmic function of the predicted octanol-water partition coefficient (log K OW ) obtained from ChemAxon confirmed that the extraction ability of glycerol-based NADES for polyphenols from kale waste was very consistent with the polarity of their components: urea (log K OW =-1.364) < fructose (log K OW =-2.758) < glucose (log K OW =-2.933) < xylose (log K OW =-2.938) < sorbitol (log K OW =-3.730) < betaine (log K OW= -4.494). In contrast, based on the pH and viscosity data listed in Table 1, no linear relationship was observed between pH / viscosity and yield. However, it is worth noting that the optimal extractant, 70% Gly / Bet, has a relatively low viscosity (19.33 mPa·s) and an almost neutral pH value. In contrast, the yield obtained with 70% Gly / U, which has a low enough viscosity, is low, which may be due to its alkalinity. Chagnoleau et al. (Chagnoleau et al., Food Chemistry, 2022, 133992) proposed that when the solvent viscosity is below 20 mPa·s, the yield of phenolic compounds in kiwifruit seems to be controlled by the solvent pH value, and the dissolution of phenolic compounds is more favorable under acidic conditions.

[0177] Contrary to the NADES studied, the extracts obtained with polar solvents (such as MeOH, EtOH, 70% MeOH, and 70% EtOH) are much more complex in composition, including polyphenols, carotenoids, and chlorophyll. Alcohol solvents show good extraction ability for carotenoids and chlorophyll, but the yield of polyphenols is relatively low. Specifically, compared with EtOH (log K OW = -0.31), the more polar MeOH (log K OW = -0.77) extracts more polar lutein and chlorophyll, but less non-polar β-carotene. As shown by 70% MeOH and 70% EtOH, adding water to the polar solvent system improves the ability of alcohol to dissolve polyphenols, but reduces the solubilization ability for lipophilic compounds (especially non-polar β-carotene). On the other hand, medium-polar EtOAc (log K OW = -0.31) shows a balanced ability to extract various lipophilic compounds from kale waste, covering from the most polar lutein to chlorophyll and then to non-polar β-carotene (their chemical structures are as Figure 2 shown). Lutein is a carotenol with a pair of hydroxylated groups on the terminal β-ring, making it more polar than the hydrocarbon β-carotene. Therefore, it is more easily extracted in polar solvents such as EtOH and MeOH, but most difficult to dissolve in non-polar hexane. On the other hand, the chlorophyll molecule consists of a hydrophilic porphyrin head and a long-chain lipophilic hydrocarbon tail, and its polarity is between lutein and β-carotene. Chlorophyll b has an aldehyde group at the 7-carbon position, and its polarity is slightly higher than that of chlorophyll a with a methyl group at the same position. It is worth noting that the elution order of pigments in RP-HPLC follows the order of decreasing polarity of each pigment, as shown in the chromatograms of several representative extracts shown in Figure 2 .

[0178] All terpene-based hydrophobic NADES studied in this work (i.e., Men / Thy, Men / Thy 2 , Men / Fen, and Thy / Fen) had comparable extraction capabilities for β-carotene and lutein to Hex; however, the yield of lutein was lower than that obtained using EtOAc. It was observed that lutein and chlorophyll exhibited similar extraction characteristics, which might be due to their similar polarities. Derrien et al. also reported similar findings (Derrien, M. et al., LWT - Food Science and Technology, 2017, 79, 170) that lutein and chlorophyll could be extracted with the same levels of process parameters (such as solvent concentration and temperature). Although the yields of lutein and chlorophyll obtained using hydrophobic NADES in this screening test were lower than those of the reference solvents, it was possible to improve the yields by optimizing the process conditions.

[0179] Example 3. Antioxidant potential of the aqueous extract

[0180] After preliminary screening of the solvents, the antioxidant activities of the extracts in Example 2 were analyzed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) procedure.

[0181] Determination of DPPH radical scavenging activity

[0182] The antioxidant capacity of the extracts was determined based on the DPPH method. 25 μL of the diluted sample was mixed with 800 μL of 0.0072 mM DPPH methanol solution. The mixture was shaken vigorously and left to stand in the dark at 25 °C for 30 minutes. The RSA was estimated by measuring the decrease in DPPH radicals and expressed as the percentage of DPPH discoloration using Equation 1,

[0183]

[0184] where Abs 对照 and Abs 样品 are the absorbance values of the control and the sample at 517 nm, respectively. The influence of the solvent was eliminated by preparing a blank control system under the same conditions. The results were converted and recorded as milligrams of Trolox equivalents per gram of dry weight of kale waste (mg TE g -1 DW).

[0185] Results and Discussion

[0186] Antioxidant capacity is one of the most important biological properties of the extracts in their applications. The antioxidant capacities of all aqueous extracts were characterized based on the determination of their ability to scavenge the stable free radical DPPH˙, and the results are shown in Figure 3As shown in A. Interestingly, the results of antioxidant capacity followed the same trend as the total phenolic content in the extracts, which was verified by the good correlation between them (R 2 = 0.9013, Figure 3 B), indicating that the antioxidant activity determined by the DPPH method was mainly controlled by the polyphenols in the extracts. On the other hand, carotenoids and chlorophyll in the methanol and ethanol extracts did not seem to act synergistically to significantly improve the antioxidant capacity of their respective extracts, although the antioxidant behavior of carotenoids and chlorophyll has been well documented (Stahl, W. & Sies, H., Molecular Aspects of Medicine, 2003, 24, 345; Hsu, C.-Y. et al., Food and Nutrition Sciences, 2013, 4(8), 1, doi:10.4236 / fns.2013.48A001). Similar findings have been reported in several studies involving fruit extracts (Gil, M.I. et al., Journal of agricultural and food chemistry, 2002, 50, 4976), vegetable extracts (Biegańska-Marecik, R. et al., Food Chemistry, 2017, 230, 271; Armesto, J. et al., LWT-Food Science and Technology, 2017, 79, 267), and even microalgae extracts (Goiris, K. et al., Journal of applied phycology, 2012, 24, 1477). Thaipong and his colleagues (Thaipong, K. et al., Journal of Food Composition and Analysis, 2006, 19, 669) observed that the antioxidant capacity of guava methanol extract was closely related to the total phenols, but negatively correlated with the total carotenoids. Similarly, Cervantes-Paz et al. (Cervantes-Paz et al., Food Chemistry, 2014, 146, 188) concluded that the antioxidant activity of pepper extracts was not controlled by their carotenoid and chlorophyll contents, and the correlation between its antioxidant activity and pigment concentration was poor (R 2 = 0.0024 - 0.1332).

[0187] Polyphenols have been recognized as the main antioxidants in Brassica crops ( A., LWT – Food Science and Technology, 2007, 40, 1); As demonstrated in in vitro studies, they exhibit higher antioxidant activity than vitamins and carotenoids (Vinson, J. A. et al., Journal of Agricultural and Food Chemistry, 1995, 43, 2800; Re, R. et al., Free Radical Biology and Medicine, 1999, 26, 1231). Kurilich et al. (Kurilich, A. C. et al., Journal of Agricultural and Food Chemistry, 2002, 50, 5053) reported that based on data obtained using the oxygen radical absorbance capacity (ORAC) assay, water-soluble antioxidants in Brassica vegetables accounted for > 89% of the total antioxidant capacity. In addition, the antioxidant capacity of individual flavonoids isolated from kale was verified in the study by Fiol et al. (Fiol, M. et al., Food Research International, 2012, 47, 80), and the results showed that quercetin derivatives with a catechol structure contributed to the improvement of antioxidant activity. In addition, another study conducted by Lanfer-Marquez et al. (Lanfer-Marquez, U. M. et al., Food research international, 2005, 38, 885) showed that compared with the scavenging rate of about 80% of Trolox, chlorophyll extracted from fresh spinach leaves exhibited a very low DPPH RSA (< 12%). In the present invention, Gly / Bet appears to be an excellent candidate that can provide extracts rich in polyphenols and having significant antioxidant behavior, suitable for the cosmetic, food, and pharmaceutical industries. Additionally, its precursors such as glycerol are often used as humectants or moisturizers in cosmetics (Vaillant, L. et al., Journal of cosmetic dermatology, 2020, 19, 1399), and are also widely used as excipients in pharmaceutical formulations, for example, to provide lubrication and smoothness in many cough syrups and other medications (EWccles, R. & Mallefet, P., Pharmacy, 2017, 5, 4). In addition, betaine is usually consumed as a dietary supplement and is often used in personal care products (Di Gioacchino, M. et al., Journal of Molecular Liquids, 2020, 318, 114253). Therefore, Gly / Bet was selected for further process optimization studies.

[0188] Example 4. Extraction of polyphenols using glycerol: betaine

[0189] Next, the extraction conditions of the most promising solvents for polyphenols disclosed in Example 3 were evaluated.

[0190] Based on the selected NADES, single-factor experiments were conducted to evaluate the effects of each process parameter (i.e., SLR, solvent concentration, temperature, and time) on the total phenol yield. Achieving the maximum capacity of the extractant would be beneficial for improving productivity.

[0191] Optimization of polyphenol extraction process

[0192] After preliminary screening, the optimal solvent for polyphenol extraction was determined, and the process conditions were further studied by single-factor design. The effects of process parameters including SLR (1:10, 1:15, 1:20, 1:30, and 1:40), temperature (25 °C, 45 °C, and 65 °C), solvent concentration (50%, 60%, 70%, and 80%), and time (30 minutes and 60 minutes) were evaluated. For comparison, ultrasonic-assisted extraction was also carried out using an ultrasonic bath (Elma Elmasonic P, Germany) operating at a constant frequency of 37 kHz and an output power of 100%. The ultrasonic bath was at 25 °C at the start of the process and rose to 30 °C and 38 °C after 30 minutes and 60 minutes of operation, respectively. The supernatant was collected by centrifugation at 12000 g for 10 minutes and filtered for total phenol content determination.

[0193] Results and Discussion

[0194] First, the effect of SLR was examined, and the results are as Figure 4 shown in A. When using an SLR of 1:40 to 1:20, there was no significant difference in the yield. However, with a further increase in SLR, the yield of polyphenols decreased significantly. Considering using less solvent, an SLR of 1:20 was selected as the optimal value and used for subsequent studies.

[0195] Compared with traditional organic solvents, one of the main disadvantages of NADES is its high viscosity. The high viscosity of NADES reduces the diffusion of the target solute 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). Considering this constraint, appropriate dilution is very important for reducing the viscosity of the solvent while maintaining its eutectic properties. Therefore, in addition to extraction temperature and time, the effect of solvent concentration was also studied. Figure 4The results shown in B clearly indicate that the increase in temperature has an adverse effect on the total phenolic yield, and this effect is more obvious at low concentrations of Gly / Bet (i.e., 50%). In addition, when heat is provided during the extraction process, a long treatment time of 60 minutes results in a significant decrease in the yield. The results suggest that the polyphenols in kale are very susceptible to heat, which is very consistent with the research results of Lafarga et al. (Lafarga, T. et al., Innovative Food Science & Emerging Technologies, 2018, 47, 412), that is, heat treatments such as steaming (100 °C, 15 minutes) and vacuum low-temperature cooking (80 °C, 15 min) significantly reduce the total phenolic content of kale leaves. It is worth emphasizing that the adverse effects of high temperatures (i.e., 45 °C and 60 °C) are smaller at high solvent concentrations, indicating that Gly / Bet has a protective effect on polyphenols to prevent degradation. However, the protective effect of Gly / Bet disappears when its concentration is 50%, indicating that this may be the limit of the interruption of its intermolecular interactions. Dai et al. (Dai, Y. et al., Analytica chimica acta, 2013, 766, 61) also observed a similar phenomenon, that is, adding 50% (v / v) water to lactic acid - glucose causes a sharp change in structure and polarity, which may be due to the breakage of hydrogen bonds between the two components.

[0196] From Figure 4 From the results shown in B, the optimal conditions for isolating polyphenols from kale waste are to use 70% Gly / Bet at 25 °C for 30 minutes, and the yield is comparable to that obtained by ultrasonic-assisted extraction. The results indicate that when using the selected NADES as the extractant, there is no need to use ultrasonic treatment to enhance the process; the selected NADES can quickly dissolve the target solute. As observed in this study, especially when the process duration is extended to 60 minutes, ultrasonic treatment may be undesirable because local heat generation can damage heat-sensitive products. Dai et al. (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50) also observed that in their study on extracting anthocyanins from Catharanthus roseus using a lactic acid- and glucose-based NADES, simple mechanical stirring is much more effective than ultrasonic treatment.

[0197] Example 5. Stability of Polyphenols in the Extract over Time

[0198] The preservation ability of the solvent system is important for the processing of the extract to achieve productization. Therefore, the stability of the polyphenol-rich extract during storage was investigated.

[0199] Stability of the extract over time

[0200] The stability of polyphenol-rich extracts obtained with the optimal solvent and several reference solvents under optimal conditions was analyzed over 30 days in the dark at 25 °C and 4 °C. The total phenols remaining in the extract were determined at least once a week. The results were expressed as the relative concentration of total phenols compared to the original extract obtained with water, as shown in Equation 2.

[0201]

[0202] Results and Discussion

[0203] The post-extraction stability of polyphenol-rich extracts obtained with 70% Gly / Bet and several reference solvents was investigated by regularly measuring the residual phenol content in the polyphenol-rich extracts during storage for 30 days in the dark at 25 °C and 4 °C. The results were expressed as the relative concentration of total phenols in the extract ( Figure 5 A and Figure 5B). Comparing the degradation rates of these extracts at 25 °C and 4 °C clearly shows that, in all the solvents tested, polyphenols deform faster at 25 °C than at 4 °C. Rapid deterioration of polyphenols was found in water, with more than half and nearly 30% of the original content lost after 30 days of storage at 25 °C and 4 °C, respectively. On the other hand, 70% Gly / Bet confers the highest stability on bioactive polyphenols, retaining 91.7% and 88.6% of the original content after 30 days of storage at 4 °C and 25 °C, respectively. Similarly, 70% EtOH also shows similar performance, maintaining the stability of polyphenols over time. However, among the solvents studied, the original content of polyphenols extracted with 70% EtOH is the lowest. The stabilizing behavior of NADES has been described in several studies on bioactive compounds such as anthocyanins (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50) and catechins (Jeong, K. M. et al., Journal of Cleaner Production, 2017, 151, 87). Dai et al. (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50) attributed this to the extensive interactions between biomolecules and NADES components, which control the movement of biomolecules, thus reducing oxidative degradation. In addition, transmission electron microscopy (TEM) studies conducted by Ling et al. (Ling, J. K. U. et al., LWT, 2020, 133, 110096) elucidated that antioxidant molecules extracted from mango (Mangifera pajang) fruit waste aggregate at the center of choline chloride:ascorbic acid-based NADES, forming a nanoscale cluster structure that protects the biomolecule.

[0204] All extracts except 70% EtOH were orange, which may be due to the presence of polyphenols such as flavonoids, while a large amount of chlorophyll caused the 70% EtOH extract to be bright green, as Figure 5 shown in C. Consistent with the results of the stability test, the fastest destabilization was observed in the water extract. After 2 days of storage at 25 °C, it began to become turbid, accompanied by visible discoloration, and precipitates appeared over time during storage, but the degradation rate slowed down when the extract was kept at 4 °C. Similarly, the 70% Gly extract also quickly became turbid, and over time, the color gradually changed from orange to light yellow (at Figure 6can be clearly seen). It has often been reported that a high degree of degradation of the polyphenol structure in the product is accompanied by a change in the color of the product (Sant’Anna, V. et al., Dyes and pigments, 2013, 98, 601). In addition, it was noticed that the color of the 70% EtOH extract changed from bright green to brownish olive green, accompanied by the formation and precipitation of green particles, which may be due to the conversion of chlorophyll to pheophytin and pyropheophytin (Sant’Anna, V. et al., Dyes and pigments, 2013, 98, 601). Chlorophyll pigments are prone to degradation during storage, as confirmed by Amir Ahmadi et al. (Ahmadi, A. et al., Food and Chemical Toxicology, 2022, 163, 112980), who found that chlorophyll a and b extracted from alfalfa by enzymatic and ultrasonic methods gradually degraded during storage for 45 days at -18 °C, 4 °C, and 25 °C. In contrast, the 70% Gly / Bet extract stored at 4 °C remained clear throughout the storage period and showed the smallest color change, verifying the good preservation ability of this solvent system. Since the components of this solvent are active and compatible, stable polyphenol-rich extracts can be easily applied to cosmetic or pharmaceutical formulations.

[0205] Example 6. Sequential and integrated recovery of bioactive compounds

[0206] To maximize the value-added potential of kale waste, a two-step sequential extraction process and a one-step integrated method were implemented to recover multiple target compounds from kale waste.

[0207] Sequential extraction method

[0208] A sequential extraction process for recovering polyphenols, carotenoids, and chlorophyll from kale waste was designed. In the first pathway (Route 1), freeze-dried kale waste powder was treated with a selected NADES (i.e., Gly / Bet) under optimal conditions (25 °C, 30 min, SLR of 1:20, concentration of 70%) to recover polyphenols. The polyphenol-rich supernatant was collected by centrifugation at 12,000 g for 10 min, and the total phenolic content was measured. Next, the residual particles were subjected to a second SLE with ethyl acetate; and the supernatant obtained after centrifugation was quantified for carotenoid and chlorophyll contents. In contrast, for the second route (Route 2), kale waste was treated with ethyl acetate followed by subsequent extraction with Gly / Bet. To simplify the process, an integrated strategy (Route 3) was attempted by using a ternary mixture of Gly / Bet-water mixture (30:70 w / w) and ethyl acetate at a 1:1 (v / v) ratio under the same process conditions. After centrifugation, the top layer (ethyl acetate-rich layer) and the bottom layer (Gly / Bet-water-rich layer) were collected for quantification of the target compounds.

[0209] Integrated extraction method for ternary solvent system

[0210] The weight proportion of water added to the deep eutectic solvent can be in a wide range, such as 20% to 40%. The volume ratio of the hydrophilic solvent (i.e., the mixture of NADES and water) and the hydrophobic solvent (i.e., ethyl acetate) can be adjusted according to different plant wastes used, such as 1:1.

[0211] The proposed ternary solvent system can simultaneously extract natural bioactive compounds with different polarities, including hydrophilic and lipophilic metabolites, from plant (e.g., vegetable leaves, stems and roots, as well as peels, seeds and pulp) wastes in a single one-step mild and rapid extraction process (e.g., 25 °C, 30 min). Specifically, using the rejected curly kale leaves as a model of vegetable waste, this method can recover various bioactive metabolites, such as phenolic compounds, carotenoids (e.g., lutein and β-carotene), and chlorophyll (a and b) from the freeze-dried and ground kale waste matrix. The pre-treatment processing of plant waste before extraction can be minimal. After the extraction process, the NADES aqueous solution (polyphenol-rich extract) and ethyl acetate (carotenoid / chlorophyll-rich extract) can be easily separated into two liquid phases by centrifugation and used for different applications. Examples of the ternary solvent system are glycerol / fructose (molar ratio of 3:1) + H2O + ethyl acetate and glycerol / betaine (molar ratio of 3:1) + H2O + ethyl acetate.

[0212] Results and Discussion

[0213] To promote the sustainable and effective valorization of kale waste, it is desirable to recover multiple value-added products. As can be seen from Figure 1 the results in -1 , compared with other wastes such as pepper leaves containing 0.23 mg g -1 lutein (Kang, J.-H. et al., Food Chemistry, 2016, 205, 140), kale waste contains a large amount of carotenoids, such as β-carotene (up to 0.45 mg g -1 ). In addition, chlorophyll is a recognized natural and permitted food colorant (European Standard No. E140) (Viera, I. et al., Molecules, 2019, 24, 154). Therefore, to further valorize the waste, it is recommended to use a second separation device to recover carotenoids and chlorophyll after treatment with 70% Gly / Bet (designated as Route 1). In this sense, EtOAc is a bio-based solvent and is selected for the extraction of lipophilic pigments due to its high extraction capacity. Another method with the opposite sequence (Route 2) was also implemented. In addition, an integrated method using a ternary mixture of aqueous Gly / Bet and EtOAc was attempted, forming a liquid-liquid extraction (LLE) two-phase system consisting of a top (EtOAc) liquid phase and a bottom (aqueous Gly / Bet) liquid phase (Route 3).

[0214] As can be seen from Figure 7 the results of the three methods plotted in -1 DW) and chlorophyll (7.86 mg g -1 DW) yields, while reducing the β-carotene (0.41 mg g -1 DW) and polyphenol (16.83 mg g-1 DW) maintained a similar yield. In addition, it should be noted that the total yield of bioactive compounds obtained using this integrated process is superior to that obtained using conventional solvents such as methanol. The presence of polar and non-polar properties in the mixed solvent system allows different classes of biomolecules with different polarities to be dissolved simultaneously, thus facilitating the penetration of solvent molecules to contact the target solutes in the biomass matrix. Specifically, the improved polarity of the mixed solvent system contributes to the extraction ability of EtOAc for polar pigments such as lutein and chlorophyll.

[0215] As Figure 8 shown, given the synergistic effect of the integrated platform on the recovery rate and related advantages such as time savings and energy conservation, this integrated platform appears to facilitate the upgrading and recycling of kale waste. Using a customized biphasic system composed of a ternary mixture of natural deep eutectic solvents (NADES), water, and ethyl acetate, the complete extraction of natural bioactive compounds with different polarities from plant waste in one step was achieved.

[0216] The results are as Figure 9 shown. The performance of the solvent system can be finely tuned by carefully selecting the NADES components and by changing the pH and viscosity (e.g., by adding water to the system).

[0217] This method uses "green" or even food-grade solvents and is thus sustainable. NADES components such as glycerol are often used as humectants or moisturizers in cosmetics and are widely used as excipients in pharmaceutical formulations, for example, to provide lubrication and smoothness in many cough syrups and other medications. Another example of a NADES component is betaine, which is a trimethylated form of glycine and was originally found in beets. Due to its moisturizing and emollient properties, it is a common active ingredient in cosmetics, food, and pharmaceuticals. In addition, fructose is a keto monosaccharide obtained from sugarcane, beets, and corn and is commonly added to foods and beverages to improve palatability and taste and to brown baked products. Considering that the NADES components themselves are food / cosmetic-compatible components, a stable aqueous solution of NADES rich in polyphenol extracts (natural antioxidants) can be easily applied to cosmetic or pharmaceutical formulations. In addition, according to the application requirements, the ethyl acetate portion of the extract can be subjected to subsequent solvent evaporation to obtain a dry solid extract rich in carotenoids / chlorophyll (natural pigments).

[0218] Considering the different applications of these two extracts, the liquid extract rich in polyphenols (with strong antioxidant capacity) can be easily incorporated into cosmetics and foods, while the extract rich in pigments requires further solvent evaporation according to the application requirements to obtain a dry solid extract.

[0219] Example 7. Extraction using wet kale waste

[0220] The applicability of the integrated extraction method to wet kale waste was also investigated. The kale waste was cleaned and cryogenically ground (SPEX 6875 Freezer / Mill, UK) to form a wet slurry containing 89.6% water. The wet slurry was further concentrated to a water content of approximately 75% to maintain the SLR of the extraction process. Considering using the water in the kale waste slurry to replace the water in the solvent system, the integrated extraction method was changed to use only Gly 3 :Bet and ethyl acetate.

[0221] The extraction was carried out under the same process conditions as the freeze-dried powder.

[0222] Thus, by simplifying the biomass pretreatment, the recycling of kale waste can be further improved, as freeze-drying is an energy-intensive process and is best avoided in downstream processing. Therefore, the feasibility of applying the integrated method to wet kale waste slurry was evaluated. Since the water contained in the kale waste slurry can be used as a diluent in the solvent system, no external water was added in the extraction process. The results in Table 3 show that compared with the freeze-dried powder, using the wet kale waste slurry significantly increased the recovery of all bioactive compounds except polyphenols. Considering that the extraction method has the same efficiency for these two biomasses, the lower recovery of the freeze-dried kale waste powder may be due to the degradation of phytochemicals during the freeze-drying and grinding process. On the other hand, the yield of polyphenols in the wet biomass did not increase significantly, which may be due to some losses during the process of removing excess water from the wet slurry.

[0223] Table 3. Recovery rates of total phenols, carotenoids (β-carotene, lutein), and chlorophylls (a and b) from different forms of kale waste using the integrated method.

[0224]

[0225] a The reported total phenols are in units of mg GAE g -1 DW.

Claims

1. A method for extracting natural bioactive compounds with different polarities from solid plant waste, the method comprising the following steps: (a) Providing dry solid plant waste or wet solid plant waste; and (b) Contacting the dry solid plant waste with a ternary solvent mixture at a suitable temperature for a suitable period of time, the ternary solvent mixture comprising: A natural deep eutectic solvent (NADES) containing glycerol, Water, and Ethyl acetate; or Contacting the wet solid plant waste with a binary solvent mixture at a suitable temperature for a suitable period of time, the binary solvent mixture comprising: A natural deep eutectic solvent (NADES) containing glycerol, and Ethyl acetate; wherein: The NADES is a hydrophilic NADES; and Relative to the total weight of water and NADES in the ternary solvent, or Relative to the total weight of water in the wet solid plant waste and NADES in the binary solvent, The water content is 20% to 40% by weight.

2. The method according to claim 1, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of amino acids, sugar alcohols, sugars, and urea.

3. The method according to claim 2, wherein the NADES comprises glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.

4. The method according to claim 3, wherein the NADES is formed from: (ai) Glycerol and betaine; (aii) Glycerol and sorbitol; (aiii) Glycerol and glucose; (aiv) Glycerol and fructose; or (av) Glycerol and urea.

5. The method according to claim 4, wherein the NADES is formed from: (ai) Glycerol and betaine; or (aiv) Glycerol and fructose.

6. The method according to any one of claims 2 to 5, wherein the molar ratio of glycerol to the other components is 1:1 to 5:1, such as 2:1 to 3:1, such as about 3:

1.

7. The method according to any one of the preceding claims, wherein relative to the total weight of water and NADES in the ternary solvent, the water content is 25% to 35% by weight, such as about 30% by weight.

8. The method according to any one of the preceding claims, wherein the volume ratio of ethyl acetate in the ternary solvent to the volume of water and NADES is 1:5 to 5:1, such as 1:3 to 3:1, such as about 1:

1.

9. The method according to any one of the preceding claims, wherein the solid-liquid ratio (g / mL) of the dry solid plant waste to the ternary solvent is 1:10 to 1:40, such as 1:15 to 1:35, such as 1:18 to 1:35, such as about 1:

20.

10. The method according to any one of the preceding claims, wherein one or both of the following apply: (bi) The suitable amount of time is 10 minutes to 5 hours, such as 15 minutes to 2 hours, such as 20 minutes to 1 hour, such as about 30 minutes; and (bii) A suitable temperature is from 15°C to 65°C, such as from 20°C to 45°C, such as about 25°C.

11. The method according to any one of the preceding claims, wherein the solid plant waste is provided in particulate form, optionally wherein the average diameter of the particles is from 0.1 mm to 10 mm, such as an average diameter of from 0.5 mm to 5 mm, such as an average diameter of about 2 mm.

12. The method according to any one of the preceding claims, wherein the dry solid plant waste is provided in a form substantially free of all water, optionally wherein the dry solid plant waste is provided in a lyophilized form.

13. The method according to any one of the preceding claims, wherein the plant waste is vegetable waste, optionally wherein the vegetable waste is cruciferous vegetable waste, further optionally wherein the cruciferous vegetable waste is selected from one or more of spinach, broccoli, cauliflower, and more specifically curly kale (Brassica oleracea var. acephala) leaves.

14. The method according to any one of the preceding claims, wherein the method further comprises, after step (b), separating and retaining the resulting ethyl acetate extract and the resulting NADES / water extract from the solid plant waste.

15. A solid plant waste extract comprising: A natural deep eutectic solvent (NADES) containing glycerol; Water; and One or more polyphenolic compounds.

16. A ternary solvent comprising: A natural deep eutectic solvent (NADES) containing glycerol; Water; and Ethyl acetate, wherein: The NADES is a hydrophilic NADES; and Relative to the total weight of water and NADES in the ternary solvent, the content of water is 20% by weight to 4%d by weight.

17. The ternary solvent according to claim 16, wherein the NADES contains glycerol and one or more other components selected from the group consisting of amino acids, sugar alcohols, sugars, and urea.

18. The ternary solvent according to claim 17, wherein the NADES contains glycerol and one or more other components selected from the group consisting of betaine, sorbitol, xylitol, glucose, fructose, and urea.

19. The ternary solvent according to claim 18, wherein the NADES is formed from: (ai) Glycerol and betaine; (aii) Glycerol and sorbitol; [[ID=]](aiii) Glycerol and glucose; (aiv) Glycerol and fructose; or (av) Glycerol and urea.

20. The ternary solvent according to claim 19, wherein the NADES is formed from: (ai) Glycerol and betaine; or (aiv) Glycerol and fructose.

21. The ternary solvent according to any one of claims 17 to 20, wherein the molar ratio of glycerol to the other component is from 1:1 to 5:1, such as from 2:1 to 3:1, such as about 3:

1.

22. The ternary solvent according to any one of claims 17 to 21, wherein the content of water is 25% to 35% by weight, such as about 30% by weight, based on the total weight of water and NADES in the ternary solvent.

23. The ternary solvent according to any one of claims 17 to 22, wherein the volume ratio of ethyl acetate to the volume of water and NADES is 1:5 to 5:1, such as 1:3 to 3:1, such as about 1:

1.

24. Use of the ternary solvent according to any one of claims 1 to 8 or any one of claims 16 to 23 for extracting natural bioactive compounds with different polarities from solid plant waste.

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