EP4619125A1

EP4619125A1 - Method of natural active ingredient extraction

Info

Publication number: EP4619125A1
Application number: EP23892130.8A
Authority: EP
Inventors: Xiao Hu; Sze Ying Lee; Yen Nan LIANG
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2022-11-14
Filing date: 2023-11-14
Publication date: 2025-09-24
Also published as: CN120417979A; WO2024107107A1

Abstract

The invention relates to a method of extracting natural bioactive compounds with differing polarities from a solid plant waste such as kale waste using a ternary solvent mixture comprising a natural deep eutectic solvent (NADES) comprising glycerol, ethyl acetate and water, or a binary solvent mixture comprising a NADES comprising glycerol, and ethyl acetate by using solid-liquid extraction-liquid-liquid extraction (SLE-LLE) integrative platform. The invention further relates to a solid plant waste extract comprising a NADES comprising glycerol, water, and one or more polyphenol compounds.

Description

METHOD OF NATURAL ACTIVE INGREDIENT EXTRACTION

Field of Invention

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.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Food loss and waste have been serious global problems and are on an upward trend in many countries. For instance, around 817,000 tonnes of food were wasted in Singapore in 2021 , which was 23% higher than the 665,000 tonnes generated in 2020, almost half of which were fruit and vegetables. Perishable produce usually suffers from massive loss during the postharvest stage before reaching the supermarket shelves. Aside from post-harvest spoilage, large quantities of produce, with imperfections in size, shape or colour, are rejected when undergoing a strict process of aesthetic screening to be sold in supermarkets. This loss indicates adverse impacts on both the economy and the environment. For rejected produce that are discarded only due to their poor aesthetic appeal to consumers and retailers’ demands, they likely possess identical nutritional compositions and food safety as the saleable products. The waste should be upcycled into higher-value products that can contribute back to the supply chain, to achieve circularity and sustainability in agricultural production.

Particularly, 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.

Kale leaves rejected from cosmetic screening represent attractive sources of phytochemicals with potential applications in the cosmetic and food industries. Indeed, kale is rich in phenolic compounds like flavonoids (e.g., quercetin and kaempferol) and holds greater amount of antioxidants than other vegetables such as spinach, broccoli and cauliflower. It was reported that the total phenolics in freeze-dried kale were 14.03 mg g^_1, much higher than 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 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). In addition, 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). Attributed to its health-promoting effects, 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). Clinical studies proved that intake of an oral supplement of carotenoid-rich curly kale extract not only increased the skin’s RSA and lipids (with a daily dose of 4.45 mg total carotenoids) (Meinke, M.C. et al., European Journal of Pharmaceutics and Biopharmaceutics, 2013, 84, 365), but also avoided ageing-related collagen I degradation in the dermis, even with a daily dose as low as 1.65 mg carotenoids (Meinke, M.C. et al., Nutrients, 2017, 9, 775). The rejected produce from harvesting could be a valuable source of phytochemicals that could potentially serve as natural antioxidants, pigments and functional ingredients in cosmetic and food applications.

Apart from works on efficacy assessment, recovery processes of kale extract are another important aspect, which directly influence the level of acceptance by regulatory authorities and consumers. The isolation of bioactive compounds from vegetable waste has been addressed in past decades through conventional technologies that involve petroleum-derived volatile organic compounds (VOCs). Methanol, acetone, and their aqueous mixtures are often applied 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), whereas carotenoids are mostly extracted by hexane for food and pharmaceutical purposes. VOCs above certain concentration levels are detrimental to the environment and human health. Conventional organic solvents used for the recovery of these bioactive metabolites are hazardous, therefore more benign equivalents are sought. Besides solution-based extraction, advanced techniques like supercritical fluid extraction (Borja-Martinez, M. et al., Antioxidants, 2020, 9, 1195) and microwave-assisted extraction (Maravic, N. et al., Sustainable Chemistry and Pharmacy, 2022, 28, 100728) have been studied. However, they may not be economically viable on an industrial scale due to the use of high-cost specialised equipment. It would therefore be essential to develop scalable, sustainable, and safe processes with non-harmful solvents for the purpose of nutrient extraction.

Natural deep eutectic solvents (NADESs) have been proposed as environmentally benign substitutes for hazardous VOCs (Dai, 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). In addition to their non-volatility and biodegradability, NADESs feature a high- solubilising capacity for natural products, making them suitable extraction media. Moreover, 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).

Apart from the predominantly polar NADESs that are superior in extracting polar metabolites, 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. The extraction capacity of 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.

Summary of Invention

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.

Aspects and embodiments of the invention are described in the following numbered clauses. 1. A method of extracting natural bioactive compounds with differing polarities from a solid plant waste, the method comprising the steps of:

(a) providing a dried or a wet solid plant waste; and

(b) contacting the dried solid plant waste for suitable a period of time and at a suitable temperature with 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.

2. The method according to Clause 1, wherein the 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.

3. The method according to Clause 2, wherein the 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.

4. The method according to clause 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 clause 4, wherein the NADES is formed from:

(ai) glycerol and betaine; or

(aiv) glycerol and fructose. 6. The method according to any one of Clauses 2 to 5, wherein the molar ratio of glycerol to the further component is from 1:1 to 5: 1 , such as from 2:1 to 3: 1 , such as about 3: 1.

7. The method according to any one of the preceding clauses, wherein water is 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.

8. The method according to any one of the preceding clauses, wherein the 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.

9. The method according to any one of the preceding clauses, wherein 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.

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

(bi) 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; and

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

11. The method according to any one of the preceding clauses, wherein 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.

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

13. The method according to any one of the preceding clauses, wherein 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). 14. The method according to any one of the preceding clauses, wherein 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).

15. A solid plant waste extract comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and one or more polyphenol compounds.

16. 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.

17. The ternary solvent according to Clause 16, wherein the 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.

18. The ternary solvent according to Clause 17, wherein the 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.

19. The ternary solvent according to clause 18, 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.

20. The ternary solvent according to clause 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 Clauses 17 to 20, wherein the molar ratio of glycerol to the further 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 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.

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

24. Use of a ternary solvent as described in any one of Clauses 1 to 8 or any one of Clauses 16 to 23 in extracting natural bioactive compounds with differing polarities from a solid plant waste.

Drawings

FIG. 1 depicts the recovery yields of total phenolics, carotenoids (p-carotene, lutein) and chlorophylls (a and b) from kale waste using solvent (H₂O - 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/Bet² - glycerol: betaine (2:1), Gly/Bet - glycerol: betaine (3:1), Gly/Sor² - 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), Hex - hexane, EtOAc - ethyl acetate, Men/Thy - DL-menthol: thymol (1 :1), Men/Thy² - DL-menthol: thymol (1 :2), Men/Fen - DL- menthol: fenchyl alcohol (1 :1) and Thy/Fen - thymol: fenchyl alcohol (1:1)) at a solid-liquid ratio (SLR) of 1 :40 and after 30 min stirring at 150 rpm and 25 °C. ND indicates not determined. Different letters in the same series indicate significant differences at p < 0.05 level.

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. FIG. 3 depicts (A) the antioxidant activity measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay method for kale waste extracts obtained using solvent (H₂O - 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/Bet² - glycerol: betaine (2:1), Gly/Bet - glycerol: betaine (3:1), Gly/Sor² - 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)); and (B) the correlation (simple regression analysis) between antioxidant activity and total phenolics in the extracts. Different letters in the same series indicate significant differences at p < 0.05 level.

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. Different letters in the same series indicate significant differences at p < 0.05 level.

FIG. 5 depicts the stability of kale waste extracts produced using solvent (H₂O - 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.

FIG. 6 depicts the photographs of kale waste extracts (on a dark background) obtained from extraction using water (H₂O), 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) + H₂O - 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) + H₂O + ethyl acetate and glycerol/betaine (3:1) + H₂O + ethyl acetate, respectively.

Description

As noted above, 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. Thus, in a first aspect of the invention, there is provided a method of extracting natural bioactive compounds with differing polarities from a solid plant waste, the method comprising the steps of:

(a) providing a dried or a wet solid plant waste; and

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. For example, 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.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.

As noted above, the “solid plant waste” may be any suitable waste. For example, 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). As will be appreciated, a single vegetable or plant may be use or combinations thereof may be used in the method.

As noted above, 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. In certain embodiments, 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. In such embodiments, 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. As will be appreciated, 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. As noted hereinbefore, 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. In particular embodiments of the invention that may be mentioned herein, 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.

In more particular embodiments of the invention that may be mentioned herein, the NADES may be formed from:

(ai) glycerol and betaine;

(aii) glycerol and sorbitol;

(aiii) glycerol and glucose;

(aiv) glycerol and fructose; or

(av) glycerol and urea.

For example, the NADES may be formed from:

(ai) glycerol and betaine; or (aiv) glycerol and fructose.

In the NADES any suitable molar ratio between the glycerol and the further component(s) may be used. Examples of 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. As will be appreciated, where there is more than one further component then the total of the molar values of the further components will be used to calculate the molar ratio.

As noted above, 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).

As noted above, 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. For example, 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.

As will be appreciated, 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. For example, 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. For example, 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. For example, the suitable temperature is from 15 to 65 °C, such as from 20 to 45 °C, such as about 25 °C. As such, 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. As such, 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. For example, the solid plant waste may be provided as the whole plant or vegetable. In more particular embodiments of the invention, 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. For example, 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.

In embodiments of the invention where a hydrated, or “wet”, form of the solid plant waste is used. In such embodiments, the wet form will be used with a binary solvent as discussed hereinbefore.

In preferred embodiments of the invention, 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. When used herein “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. In particular embodiments of the invention, the solid plant waste may be provided in a freeze-dried form. As will be appreciated, the solid plant waste may be provided in a particulate form that has been (freeze) dried to remove water.

In certain embodiments of the invention, 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. For example, 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).

In particular embodiments of the invention, 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. As will be appreciated, while the binary solvent will not substantially include water, at the end of the process water will have been extracted from the wet solid plant waste and so the resulting solution before separation will effectively be similar to the ternary solvent and so 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.

In a further aspect of the invention, there is provided a solid plant waste extract comprising: a natural deep eutectic solvent (NADES) comprising glycerol; water; and one or more polyphenol compounds.

As will be appreciated, 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. The resulting ethyl

It will be appreciated that the 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.

It has also been surprisingly found that 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). For example, 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.

In a further aspect of the invention, there is provided 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. As the ternary solvent and its embodiments has been discussed at length in the first aspect of the invention, it is not discussed further here in order to avoid repetition.

In a further aspect of the invention, there is provided a use of a ternary solvent as described hereinbefore in extracting natural bioactive compounds with differing polarities from a solid plant waste.

As disclosed herein, 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). This enables the development of a sustainable process for the production of natural antioxidants and pigments (e.g. chlorophylls).

Particularly, NADES based on Gly/Bet (glycerol: betaine) 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. Lastly, to address the full exploitation of kale waste, a combined approach using ternary mixtures of aqueous Gly/Bet and ethyl acetate (EtOAc) was applied to simultaneously recover and separate bioactive compounds with different polarities into two fractions; wherein one was a ready-to-use polyphenol-rich extract and another was an extract rich in carotenoids and chlorophylls. The results disclosed herein show using bio-based solvents in extracting multiple bioactive metabolites from kale waste under mild conditions, in addition to their use in the solid-liquid extraction-liquid-liquid extraction (SLE-LLE) integrative platform is a sustainable alternative for efficient recovery of various valuable products from kale waste. As will be appreciated, kale waste is used herein as an example system and other materials may be used as the based waste material and it is expected that similar results would be obtained.

Further aspects and embodiments of the invention will be discussed in the following non-liming examples.

Examples

Materials

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 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 acquired from Alfa Aesar. All the chemicals were used directly without further purification. Ultra-pure water was prepared by the Millipore Milli-Q water purification system.

Kale waste was provided by Sustenir 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.

Data Analysis

Triplicate independent runs were performed for each condition, and the results were expressed as average ± standard deviation. Statistical significance analysis was conducted using one-way ANOVA analysis and accompanied by a post-hoc Tukey method for all pair- wise multiple comparisons. The level of statistically significance was set at P < 0.05.

Example 1. Preparation and Characterisation of Natural Deep Eutectic Solvents (NADESs)

All precursors of NADESs selected are renewable and low-cost ingredients that are allowed for food and cosmetic use.

Preparation of NADESs

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.

Table 1 . List of NADESs

Type Component Component Molar Acronyms pH^a Viscosity at

1 2 ratio 25 °C (mPa s)

Hydrophilic Glycerol Betaine 2:1 Gly/Bet² 7.57 26.92 ± 0.52^b

Glycerol Betaine 3:1 Gly/Bet 7.31 19.33 ± 0.35^b

Glycerol D-sorbitol 2:1 Gly/Sor² 4.54 37.54 ± 0.45^b

Glycerol D-sorbitol 3:1 Gly/Sor 4.15 33.82 ± 0.53^b

Glycerol Xylose 3:1 Gly/Xyl 4.11 26.29 ± 0.37^b

Glycerol Glucose 3:1 Gly/Glu 4.71 32.41 ± 0.39^b

Glycerol Fructose 3:1 Gly/Fru 4.43 34.65 ± 0.87^b

Glycerol Urea 1 :1 Gly/U 9.76 9.98 ± 0.28^b

Hydrophobic DL-menthol Thymol 1 :1 Men/Thy - 36.07 ± 0.26^c

DL-menthol Thymol 1 :2 Men/Thy² - 44.26 ± 0.17^c

DL-menthol Fenchyl 1 :1 Men/Fen - 54.96 ± 0.36^c alcohol

Thymol Fenchyl 1 :1 Thy/Fen - 32.55 ± 0.23^c alcohol ^a pH of 70% hydrophilic NADES in aqueous solution. ^b viscosity of 70% hydrophilic NADES in aqueous solution. ^c viscosity of neat hydrophobic NADES.

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). On the other hand, 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

The NADESs were prepared by following the protocol disclosed in Example 1.

The performance of 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. To facilitate the comparison, several reference solvents, including water (H₂O), methanol (MeOH), 70% aqueous methanol (70% MeOH), ethanol (EtOH), 70% aqueous ethanol (70% EtOH), 70% aqueous glycerol (70% Gly), n-hexane (Hex) and ethyl acetate (EtOAc), were tested under the same conditions.

Solid-liquid Extraction (SLE)

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.

Total Phenolic Content Assay

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. After 5 min, an aliquot of 25 pL of 10% (w/v) sodium carbonate was added, and the reaction mixture was shaken gently and incubated in the dark at 25 °C for 60 min. The total phenolics in the extract were determined calorimetrically by measuring the absorbance at 765 nm with a microplate reader (Tecan Infinite M200 Pro, Singapore) based on the calibration curve of gallic acid. To eliminate the effect of the solvents, a blank control system for each sample was prepared under the same conditions and used. Results were documented as mg gallic acid equivalent per g dry weight of kale waste (mg GAE g^-1 DW). It should be noted that for hydrophobic solvents studied, the extract collected was subjected to liquid-liquid extraction (LLE) by adding an equivalent volume of water. The mixture was shaken vigorously, and the aqueous layer collected after phase separation by centrifugation was assayed for total phenolics. The total phenolics in the extracts of Men/Thy, Men/Thy² and Thy/Fen were not determined due to the interference with the assay’s reagents.

Carotenoid (fi-carotene and lutein) and Chlorophyll (a and b) Quantification

A reversed-phase high-performance liquid chromatographic (RP-HPLC) protocol was used to identify and quantify p-carotene, lutein, and chlorophyll a and b in the extract. Separation and detection of these compounds were accomplished with an Agilent 1260 Infinity II LC system (Agilent Technologies, Singapore) equipped with a diode-array UV-Vis detector, set at 450 nm, on an Agilent ZORBAX Eclipse Plus C18 (3.0 mm x150 mm, 3.5 pm) column with a 15 min isocratic elution method at 1.0 mL min'¹. 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.

Identification of each compound was verified based on the retention time of commercial compounds. 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. Lutein and chlorophyll standard stock solutions were prepared using ethanol and methanol, respectively. The exact concentration of standard stock solutions was assessed spectrophotometrically (Shimadzu UV-1800, Japan) and corrected by applying the reported extinction coefficient, E^, of each compound at respective wavelength: p-carotene ( E¹ cm 'ⁿ ethanol = 2529 cm'¹ (g/100 mL)'¹ at 450 nm); lutein ( E^ in ethanol = 2550 cm'¹ (g/100 mL)'¹ at 445 nm); chlorophyll a (s^ in methanol = 799.5 cm'¹ (g/100 mL)'¹ at 665 nm) and chlorophyll b (s^ in methanol = 424.8 cm ¹ (g/100 mL)'¹ at 652 nm) (Craft, N. E. & Soares, J. H., Journal of agricultural and food chemistry, 1992, 40, 431 ; Porra, R. et al., Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1989, 975, 384). Results were expressed as mg per g dry weight of kale waste (mg g ¹ DW).

Results and Discussions

FIG. 1 presents the results of yields of target compounds using different solvents. In accordance with the mass transfer mechanism “like dissolve like”, all hydrophilic glycerol- based NADESs displayed selective extraction towards polyphenols, whereas lipophilic compounds were favourably extracted by hydrophobic terpene-based NADESs.

All aqueous solutions of glycerol-based NADESs, except for Gly/U, achieved higher yields of polyphenols compared to the reference solvents. This could be attributed to the high dissolution of polyphenol molecules in aqueous NADES caused by their strong interactions with NADES constituents which are good hydrogen bond donors and acceptors, as listed in Table 2. Table 2. Chemical structures of the precursors used in the preparation of NADESs, and their H-bond formation tendency.

Component Chemical structure No. of H-bond acceptor No. of H-bond donor

Glycerol

Betaine

2 1

D-sorbitol

6 6

D-(+)-xylose

D-(+)-glucose

5

D-(-)-fructose

5

Urea

DL-menthol Component Chemical structure No. of H-bond acceptor No. of H-bond donor

Thymol

Fenchyl alcohol

In the current invention, Gly/Bet gave the highest yield of polyphenols, which was around 2.2- and 1.3-fold higher than that of using MeOH and H₂O, respectively. Furthermore, glycerolbased NADESs coupled with sugar alcohols, namely sorbitol (Gly/Sor) and xylose (Gly/Xyl), demonstrated relatively good dissolution capacity towards polyphenols than the sugar counterparts, which were glucose (Gly/Glu) and fructose (Gly/Fru). Since the NADESs tested possess a common glycerol constituent, the variation of the yields among the solvents could be attributed to the different hydrogen bonding capacities of the second component. The extraction aptitude of glycerol-based NADESs for polyphenols from kale waste is in close agreement with the polarity of their constituent, as confirmed by the predicted logarithmic function of octanol-water partition coefficient (log Kow) obtained from ChemAxon: urea (log Kow = -1.364) < fructose (log Kow = -2.758) < glucose (log Kow = -2.933) < xylose (log Kow = -2.938) < sorbitol (log K_Ow = -3.730) < betaine (log K_Ow = -4.494). On the opposite, no linear relationship was observed between pH/viscosity and yield, based on the pH and viscosity data tabulated in Table 1. Nevertheless, it was noted that the best extractant, 70% Gly/Bet, possesses relatively low viscosity (19.33 mPa s) and almost neutral pH. In contrast, 70% Gly/U that has sufficiently low viscosity suffered from poor yield, probably due to its alkalinity. Chagnoleau et al. (Chagnoleau et al., Food Chemistry, 2022, 133992) suggested that the yield of phenolic compounds from kiwifruit seems to be controlled by the solvent pH when the solvent viscosity was below 20 mPa s, and the dissolution of phenolics was more favourable in acidic conditions.

As opposed to the NADES studied, polar solvents such as MeOH, EtOH, 70% MeOH and 70% EtOH rendered much more complex extract contents encompassing polyphenols, carotenoids and chlorophylls. Alcoholic solvents showed good extraction capacity towards carotenoids and chlorophylls, however, relatively lower yields of polyphenols were acquired. Specifically, more polar MeOH (log Kow = -0.77) extracted more polar lutein and chlorophylls but lesser non-polar p-carotene compared to EtOH (log Kow = -0.31). The addition of water in polar solvent systems improved the ability of alcohols to dissolve polyphenols but diminish the solubilisation power for lipophilic compounds, especially [3-carotene that are non-polar, as exhibited by 70% MeOH and 70% EtOH. On the other hand, moderately polar EtOAc (log Kow = -0.31) presented a balanced ability to extract a wide range of lipophilic compounds from kale waste, from the most polar lutein to chlorophylls and non-polar [3-carotene (their chemical structures are provided in FIG. 2). 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. On the other hand, 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.

All hydrophobic terpene-based NADESs studied in this work (i.e., Men/Thy, Men/Thy², Men/Fen and Thy/Fen) exhibited comparable extractability for [3-carotene and lutein than Hex; but lower yields of lutein compared to that of obtained by EtOAc. It was observed that lutein and chlorophylls demonstrated similar extraction characteristics probably because of their similar polarity. Similar findings were also reported by Derrien et al. (Derrien, M. et al., LWT- Food Science and Technology, 2017, 79, 170) that lutein and chlorophylls can be extracted with the same level of process parameters such as solvent concentration and temperature. Although the yields of lutein and chlorophylls obtained using hydrophobic NADESs in this screening test were lower compared to reference solvents, it probably could be improved by optimising the process conditions.

Example 3. Antioxidant Potential of the Aqueous Extracts

After initial screening of solvents, the antioxidant activity of the extracts in Example 2 was analysed via DPPH (2,2-diphenyl-1-picrylhydrazyl ) procedure.

DPPH radical-scavenging activity assay

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_Sam_Pie 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).

Results and Discussions

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. Interestingly, the results of antioxidant capacity followed the identical trends of total phenolic contents in the extracts, as validated by a good relationship between them (R² = 0.9013, FIG. 3B), revealing that the antioxidant activity determined by the DPPH method was predominantly governed by polyphenols in the extracts. On the other hand, carotenoids and chlorophylls in methanolic and ethanolic extracts did not seem to exert a synergistic effect to significantly increase the antioxidant capacity of the respective extracts, although the antioxidant behaviours of carotenoids and chlorophylls are 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 were reported in several works involving extracts derived from fruit (Gil, M.l. et al., Journal of agricultural and food chemistry, 2002, 50, 4976) and vegetables (Biegariska-Marecik, R. et al., Food Chemistry, 2017, 230, 271; Armesto, J. et al., LWT-Food Science and Technology, 2017, 79, 267), and even microalgae (Goiris, K. et al., Journal of applied phycology, 2012, 24, 1477). Thaipong and co-workers (Thaipong, K. et al., Journal of Food Composition and Analysis, 2006, 19, 669) observed that the antioxidant capacity of methanolic guava extract was strongly correlated with total phenolics but had a negative relationship with total carotenoids. Likewise, Cervantes-Paz et al. (Cervantes-Paz et al., Food Chemistry, 2014, 146, 188) concluded that the antioxidant activity of the pepper extract was not governed by its carotenoid and chlorophyll contents in view of the poor relationship between the antioxidant activity and the pigment’s concentration (R² = 0.0024 - 0.1332). Polyphenols have been recognised as the dominant antioxidants of Brassica crops (Pods^dek, A., LWT - Food Science and Technology, 2007, 40, 1); they displayed higher antioxidant activity than vitamins and carotenoids, as proven by in vitro studies (Vinson, J. A. etal., 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 water-soluble antioxidants in Brassica vegetables contributed to > 89% of total antioxidant capacity, based on the data obtained using the oxygen radical absorbance capacity (ORAC) assay. Additionally, the antioxidant capacity of individual flavonoids isolated from kale has been validated in the work of Fiol et al. (Fiol, M. et al., Food Research International, 2012, 47, 80), and the results showed that the quercetin derivatives with catechol structure contributed to high antioxidant activity. Moreover, another work conducted by Lanfer-Marquez et al. (Lanfer-Marquez, U. M. et al., Food research international, 2005, 38, 885) stated that the chlorophylls extracted from fresh spinach leaves displayed extremely low DPPH RSA (<12%) when compared to Trolox scavenged around 80%. In the current invention, Gly/Bet appeared to be an excellent candidate to render a polyphenol-rich extract with remarkable antioxidant behaviour for applications in the cosmetic, food and pharmaceutical industries. In addition, its precursor like glycerol is frequently used as a humectant or moistener in cosmetics (Vaillant, L. et al., Journal of cosmetic dermatology, 2020, 19, 1399), besides being widely applied as an excipient in pharmaceutical formulations, such as providing lubrication and smoothness in many cough syrups and other drugs (EWccles, R. & Mallefet, P., Pharmacy, 2017, 5, 4). Furthermore, betaine is commonly 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 optimisation study.

Example 4. Extraction of polyphenols using Glycerol: Betaine

Next, the extraction conditions of the most promising solvent for polyphenols as disclosed in Example 3 were evaluated.

Based on the selected NADES, single-factor experiments were conducted to evaluate the effect of each process parameter (i.e. , SLR, solvent concentration, temperature and time) on the yield of total phenolics. It would be beneficial for productivity if the maximum capacity of extractant can be realized.

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. For comparison purposes, 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.

Results and Discussions

The influence of the SLR was first examined, and the results are presented in FIG. 4A. There was no significant difference in the yields when the SLRs between 1 :40 to 1 :20 were used. However, the yields of polyphenols significantly decreased with the further increase of the SLR. For consideration of less solvent use, a SLR of 1 :20 was chosen as the optimal and used for subsequent studies.

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). Given this constraint, an appropriate dilution is important to reduce the solvent’s viscosity and at the same time retaining their eutectic properties. Hence, the effect of the solvent concentration was studied, in addition to the extraction temperature and time. The results depicted in FIG. 4B clearly showed that an increase in the temperature has a deleterious effect on the yields of total phenolics, and the impact was more pronounced at low concentrations of Gly/Bet, i.e., 50%. Additionally, when the heat was supplied during extraction, a prolonged processing time of 60 min led to markedly reduced yields. The results implies that the polyphenols in kale are very vulnerable to heat, which is in good agreement with the findings reported by Lafarga et al. (Lafarga, T. et al., Innovative Food Science & Emerging Technologies, 2018, 47, 412) that thermal processing such as steaming (100 °C, 15 min) and sous-vide (80 °C, 15 min) drastically reduced the total phenolics of the kale leaves. It is noteworthy to highlight that the detrimental impact of high temperature, i.e., at 45 and 60 °C, was lesser at high solvent concentrations, revealing that the Gly/Bet exerted a protective effect on polyphenols from degradation. However, the protective effect of Gly/Bet vanished at its concentration of 50%, indicating that this is probably the limit whereby its intermolecular interactions get interrupted. A similar phenomenon was also observed by Dai et al. (Dai, Y. et al., Analytica chimica acta, 2013, 766, 61) that the addition of 50% (v/v) water to lactic acid-glucose provoked a dramatic change in structure and polarity, likely due to the rupture of hydrogen bonding between two constituents.

From the results presented in FIG. 4B, the optimal conditions to separate polyphenols from kale waste were at 25 °C for 30 min using 70% Gly/Bet, with yields comparable to that obtained with ultrasound-assisted extraction. The results revealed that, with the chosen NADES as an extractant, the process intensification using sonication is not necessary; the NADES selected are able to rapidly dissolve target solutes. Sonication might be undesirable due to the generation of localised heating that might destroy thermo-sensitive products, as observed in this work particularly when the process duration was extended to 60 min. Dai et al. (Dai, Y. et al., Journal of Chromatography A, 2016, 1434, 50) also observed that simple mechanical stirring was much more efficient than sonication in their study of extracting anthocyanins from Catharanthus roseus using NADES based on lactic acid and glucose.

Example 5. Stability of Polyphenols in Extract over Time

The preservation capability of the solvent system is important for the handling of the extract for productisation. Thus the stability of polyphenol-rich extract during storage was examined.

Stability of the Extract over Time

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.

„ . . . . . . Total phenolics in the extract

Relative concentration = Tota —l phenoli —cs i _:n the ori 7gi —na :l - extract o :b —tai : —ned : — wi nth - water X 100% (Eq. 2)

Results and Discussions

The stability of polyphenol-rich extracts obtained with 70% Gly/Bet and several reference solvents after extraction was investigated by periodically measuring their residual phenolic contents during storage in the dark at 25 and 4 °C for 30 days. The results were presented in terms of the relative concentration of total phenolics in the extracts (FIG. 5A and FIG. 5B). Comparing the degradation rates of the extracts at 25 and 4 °C, it was clearly shown that polyphenols deformed faster at 25 °C than at 4 °C in all solvents tested. Rapid deterioration of polyphenols was found in water, in which more than half and close to 30% of the original contents were lost after being stored at 25 and 4 °C for 30 days, respectively. On the other hand, 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. Likewise, 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. et al., Journal of Chromatography A, 2016, 1434, 50) correlated it with the presence of extensive interactions between the biomolecules and NADES constituents that regulates the movement of biomolecules and consequently reduces oxidative degradation. Additionally, a transmission electron microscopy (TEM) study conducted by Ling et al. (Ling, J.K.U. et al., LWT, 2020, 133, 110096) elucidated that the antioxidant molecules from fruit waste of Mangifera pajang agglomerated in the centre of NADES based on choline chloride: ascorbic acid, forming a nano-scale cluster structure that protects the biomolecules.

All extracts, except for 70% EtOH, appeared orange probably due to the presence of polyphenols like flavonoids, while an abundance of chlorophylls led to a bright green colour of 70% EtOH extract, as depicted in FIG. 5C. Consistent with the results of the stability test, the fastest destabilisation was observed in the aqueous extract. It started to become turbid accompanied by visible colour fading after storing at 25 °C for 2 days, and precipitates were developed over time during storage, though the degradation slowed down when the extract was kept at 4 °C. Similarly, 70% Gly extract turned cloudy rapidly too, with a gradual colour change from orange to pale yellow over time (can be seen clearly in FIG. 6). It is frequently reported that a higher degradation of polyphenolic structure in the product was accompanied by an alteration of the product’s colour (Sant’Anna, V. et al., Dyes and Pigments, 2013, 98, 601). Besides, colour change from bright green to brownish olive green was perceived for 70% EtOH extract, with a concomitant formation and sedimentation of green particles, probably due to the conversion of chlorophylls to pheophytins and pyropheophytins (Sant’Anna, V. et al., Dyes and Pigments, 2013, 98, 601). Chlorophyll pigments are vulnerable to degradation during storage, as demonstrated by Amir Ahmadi et al. (Ahmadi, A. et al., Food and Chemical Toxicology, 2022, 163, 112980) that the chlorophylls a and b extracted from alfalfa by enzymatic and ultrasound methods had a gradual deterioration during storage at -18, 4 and 25 °C for 45 days. In contrast, 70% Gly/Bet extract that was stored at 4 °C remained clear and had the least colour change throughout the storage period, validating the good preservation capability of the solvent system. The stable polyphenol-rich extract could be readily applicable to cosmetic or pharmaceutical formulations since the solvent constituents are active and compatible ingredients.

Example 6. Sequential and Integrated Recovery of Bioactive Compounds

Aiming at maximising the valorisation potential of kale waste, two-step sequential processes as well as single-step integrated approach were conducted to recover multiple compounds of interest from kale waste.

Sequential Extraction Approaches

Sequential processes were designed to recover polyphenols, carotenoids and chlorophylls from kale waste. In the first pathway (Route 1), 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. Next, 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. Inversely, for the second route (Route 2), kale waste was treated with ethyl acetate and followed by subsequent extraction using Gly/Bet. To simplify the process, an integrated strategy (Route 3) was attempted by employing ternary mixtures of 1 :1 (v/v) of Gly/Bet-water mixture (30:70 w/w) and ethyl acetate at the same process conditions. After centrifugation, the top (ethyl acetate-rich) and bottom (aqueous Gly/Bet-rich) layers were collected for the quantification of target compounds.

Integrated Extraction Approach via Ternary Solvent System

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). Particularly, with the rejected curly kale leaves as the model of vegetable waste, 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. After the extraction process, the aqueous NADES (polyphenol-rich extract) and ethyl acetate (carotenoid/chlorophyll-rich extract) could be separated into two liquid phases easily by centrifugation, and used for different applications. 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.

Results and Discussions

To promote sustainable and efficient valorisation of kale waste, the recovery of multiple value- added products is desirable. From the results in FIG. 1, considerable amounts of carotenoids like p-carotene (up to 0.45 mg g^-1) and lutein (up to 0.59 mg g ¹) are found in kale waste, compared to other waste such as paprika leaves containing 0.23 mg g^-1 lutein (Kang, J.-H. et al., Food Chemistry, 2016, 205, 140). Furthermore, chlorophyll is a well-recognised natural and permitted food colourant (European standard no. E140) (Viera, I. et al., Molecules, 2019, 24, 154). Therefore, to further valorise the waste, a second separation unit was suggested to recover carotenoids and chlorophylls after the treatment with 70% Gly/Bet (designated as Route 1). In this sense, EtOAc, a bio-based solvent, was selected to extract lipophilic pigments owing to its high extraction aptitude. Another approach with an inverse sequence was also conducted (Route 2). Moreover, an integrated method using ternary mixtures of aqueous Gly/Bet and EtOAc was attempted, forming a liquid-liquid extraction (LLE) biphasic regime composed of the top (EtOAc) and bottom (aqueous Gly/Bet) liquid phases (Route 3).

From the results of the three approaches plotted in FIG. 7, it was observed that the yield of polyphenols obtained using 70% Gly/Bet decreased significantly when the kale waste was first treated with EtOAc compared to the untreated biomass, likely due to the poor wettability of residual biomass. Apparently, subsequent extraction with EtOAc after the first treatment with 70% Gly/Bet improved the yields of lutein and chlorophylls but negatively impacted the recovery of p-carotene. It can be deduced that the prewetting of biomass with polar solvents facilitates the liberation of polar solutes like lutein and chlorophylls from their protein complexes, which in turn favours their recovery. A similar observation was reported by Mussagy et al. (Mussagy, C.U. et al., Green Chemistry, 2020, 22, 8478) in their work on recovering carotenoids and fatty acids from yeast. Interestingly, the integrated strategy achieved increased yields of lutein (0.50 mg g^-1 DW) and chlorophylls (7.86 mg g ¹ DW) while maintaining comparable yields of -carotene (0.41 mg g^-1 DW) and polyphenols (16.83 mg g ¹ DW) than the sequential SLE processes. Besides, it should be noted that the total yields of bioactive compounds obtained using this integrative process were superior to those obtained using a conventional solvent such as methanol. 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. Specifically, the improved polarity of the mixed solvent system aided in the extractability of polar pigments like lutein and chlorophylls by EtOAc.

In view of its synergistic effect on recovery yield and associated advantages such as time- and energy-saving, 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.

The results are presented in FIG. 9. The performance of the solvent system can be fine-tuned by the judicious selection of NADES constituents, in addition to the alteration of pH and viscosity (e.g., by the addition of water in the system).

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. Besides, fructose, a ketonic simple sugar derived from sugar cane, sugar beets and maize, is often supplemented into food and beverages for palatability and taste enhancement, as well as for browning of bakery products. Considering the NADES constituents themselves are food-/cosmetic-compatible ingredients, the stable aqueous NADES rich in polyphenol extracted (natural antioxidants) could be readily applicable to cosmetic or pharmaceutical formulations. Additionally, 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.

Considering different applications envisioned for the two kinds of extracts, 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. Considering the water in the kale waste paste as the substitute for water in the solvent system, the integrated extraction approach was modified with the use of only Gly³: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. Hence, 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.

Table 3. Recovery yields of total phenolics, carotenoids (P-carotene, lutein) and chlorophylls (a and b) from different forms of kale waste using the integrative method.

Recovery yield (mg g ¹ DW)

Kale waste Extraction method At Bottom

At Top Phase

Phase

Water Carotenoids Chlorophylls

Solvent SLR Total

Type content p- system (gmL¹) phenolics³ Lutein a b

(%) carotene

Gly³:Bet + 0.50 6.04 1.82

Lyophilised 16.83 ± 0.41 ±

None EtOAc + 1:20 ± ± ± powder 0.30 0.02

H₂O 0.02 0.03 0.02

0.57 7.83 2.35

Gly³:Bet + 16.10 ± 0.48 ±

Wet paste 74.34% 1:20 ± ± ±

EtOAc 0.42 0.04

0.05 0.04 0.02 ^aThe total phenolics reported were in mg GAE g^_1 DW.

Claims

1. A method of extracting natural bioactive compounds with differing polarities from a solid plant waste, the method comprising the steps of:

(a) providing a dried or a wet solid plant waste; and

(b) contacting the dried solid plant waste for suitable a period of time and at a suitable temperature with 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. .

2. The method according to Claim 1 , wherein the 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.

3. The method according to Claim 2, wherein the 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.

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 further component is from 1:1 to 5: 1 , such as from 2:1 to 3: 1 , such as about 3: 1.

7. The method according to any one of the preceding claims, wherein water is 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.

8. The method according to any one of the preceding claims, wherein the 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.

9. The method according to any one of the preceding claims, wherein 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.

10. The method according to any one of the preceding claims, wherein one or both of the following apply:

11. The method according to any one of the preceding claims, wherein 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.

12. The method according to any one of the preceding claims, wherein the dried solid plant waste is provided in a form where substantially all water has been removed, optionally wherein the dried solid plant waste is provided in a freeze-dried form.

13. The method according to any one of the preceding claims, wherein 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).

14. The method according to any one of the preceding claims, wherein 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).

17. The ternary solvent according to Claim 16, wherein the 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.

18. The ternary solvent according to Claim 17, wherein the 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.

19. The ternary solvent according to claim 18, 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.

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 further 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 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.

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

24. Use of a ternary solvent as described in any one of Claims 1 to 8 or any one of Claims 16 to 23 in extracting natural bioactive compounds with differing polarities from a solid plant waste.