Natural deep eutectic solvents characteristics determine their extracting and protective power on chlorogenic acids from sun�ower meal

Sun�ower meal is a residue of the edible oil extraction and a promising source for the extraction of phenolic compounds, especially the chlorogenic acid family. Their clean extraction and later conservation may be improved by the use of natural deep eutectic solvents (NaDES). This study evaluated the extraction and stability of phenolic compounds from sun�ower meal using different combinations of NaDES and ethanol, as control. The principal aim was �nding a clean extraction method for this rich bioactive compound by-product. The results showed that all solvents extracted similar phenolic compounds, but the most acidic NADES showed higher e�ciency. In terms of stability, this acidic NaDES showed better protection against heat treatment and storage, while ethanol exhibited better stability under light exposure. The �ndings suggest that the NaDES composition, pH and other characteristics may in�uence both, extraction e�ciency and stability, enabling the clean use and valorization of this residue from the agroindustry.


Introduction
Sun ower seed is the third most produced oilseed in the world, with an output of 57.27 million tons per year.Its largest producers are Ukraine (30.5%),Russia (27.2%) and the European Union (17.9%) [1].The foremost purpose of the seed is the production of edible oil.From this process, 30% of the seed remains as meal, an unavoidable co-product of the extraction of sun ower oil that is mainly used as animal feed, with a high protein content [2].Based on the world production, an estimate of circa 17.2 million tons of sun ower meal is globally generated every year.This co-product is rich in phenolic compounds, especially those belonging to the chlorogenic acid (CGA) family, which represent more than 70% of the phytochemicals in the sun ower meal [3,4].
In the extraction of "green" products, some of the desirable characteristics of the solvents are to be nontoxic, non-ammable and non-explosive, biodegradable, non-polluting, with the possibility of reuse or recycling, among others [5].With the growing demand for clean label products, natural deep eutectic solvents (NaDES) have emerged as an alternative to organic solvents.NaDES are formed by mixing at least two components, a hydrogen donor, and an acceptor that, when mixed under certain conditions, form a eutectic mixture, which is liquid at low temperature [6].These solvents show no ammability or volatility and little toxicity, unlike conventional organic solvents, characteristics that are among the main advantages of their use.In addition, NaDES have been used for the extraction of phenolic compounds due to some of their properties -viscosity and polarity, mainly -which, when optimized, show higher e ciency then the regularly used organic solvents or water [7].
The extraction of phenolic compounds has been studied using different techniques.For some raw materials, time consuming methods were used, such as extraction by mechanical agitation or using the Soxhlet apparatus, applying organic solvents such as ethanol and methanol [8,9].However, such methods can lead to hydrolysis and oxidation of phenolic compounds due to their susceptibility to thermal degradation [9].Ultrasound [10] or microwave [11] extraction methods have also been studied as alternatives for faster extractions, with better yield, quality, and purity, especially from fruits and their byproducts.
The storage stability of phenolic compounds depends on temperature and time, in addition to extraction and storage conditions [12].Different studies have evaluated the stability of phenolic compounds using NaDES as solvent e.g., for the extraction of Catharanthus roseus, an ornamental plant [13], turmeric [14] and sa ower [15], compared to conventional organic solvents or water.The extracts that used NaDES as solvent showed greater stability, which was attributed to the strong hydrogen bonds between the solutes and the solvent.The high viscosity of these solvents also seemed to contribute to the stabilization of these molecular interactions [15].
The aim of the present study was to investigate the extraction of phenolic compounds from sun ower meal using two different NaDES formulations, with the aid of ultrasound, and to assess the stability of the extract over time -after heat treatment, at different storage temperatures, and exposure to light.This work hypothesized that the use of NaDES would be more e cient to extract phenolic compounds from sun ower meal, in addition to improving stability, when compared to a hydroethanolic solution.This is, to the extent of our knowledge, the rst time this approach has been applied to the valorization of this residue and promising CGA source [4].

Vegetable raw material
The sun ower cake used in this work was provided by the Caramuru company, located in Itumbiara -GO, Brazil.The material was sent dry, degreased, desolventized and pelletized.

NaDES
The components and the different proportions and combinations for preparing the solvents are shown in Table 1.
The components were weighed directly into hermetic asks and placed under magnetic stirring at 50ºC (Gehaka, model AA2050LED, São Paulo, Brazil), until a transparent and homogeneous liquid was formed.
Ultra-pure water was added to the solvents to reach 30 g.100g − 1 of NaDES, according to Radosevic et al (2016).

Extraction of phenolic compounds
Sun ower meal (4.50 g) was weighed and 30 mL of NaDES or hydroethanolic solution (40% ethanol -used as a control) were added.All samples were heated under agitation up to 45°C, followed by sonication by a probe ultrasound equipment (Ultronique, Desruptor, São Paulo, Brazil) for 1 minute, at maximum power (99%), according to Zardo et al (2017).After extraction, the extracts were ltered through polyester cloth, to eliminate solid particles, and stored at -80ºC in an ultra-freezer (INDREL, IULT 335 D, São Paulo, Brazil) until analysis.

Stability
The extracts in NaDES and the hydroethanolic extract, used as a control, were evaluated for stability of the CGAs to heat, exposure to light and storage for 30 days at room temperature, under refrigeration and freezing, according to Dai et al (2014).All stability tests were performed in triplicate and the results were evaluated by analysis of variance (two-way ANOVA), using the GraphPad Prism software (version 5.0) with a con dence level of 95%.
To evaluate the thermal stability, the extracts were placed in sealed cryogenic tubes and submitted to a metabolic bath (Marconi, MA093, São Paulo, Brazil) preheated at 40, 60 and 80°C.Samples were removed from the bath after 10, 30, 60 and 120 minutes, and immediately cooled in an ice bath to 25°C.
The effect of light incidence was evaluated on the extracts, in sealed cryogenic tubes, exposed to white light (LED 12 W OSRAM) or protected by aluminum foil (control), at room temperature.Samples were collected after 0, 3, 7 and 15 days for analysis.
During the stability assays quanti cation of CGAs, in NaDES and hydroethanolic extracts, was estimated by evaluating the total content of phenolic compounds by the method proposed by Wang et al. (2019), using CGA as a standard.Brie y, the samples were homogenized in vortex and 200 µL aliquots were taken and added to 200 µL of a Fe III solution (3.0 mM) -composed of Ferric Chloride (1.5 mM) and Potassium Ferricyanide (1.5 mM) -vortexed and reserved for 10 minutes.After that time, 200 µL were transferred to a 96-well microplate and the samples were read at 790 nm in a plate reader (PerkinElmer, VICTOR Nivo, Massachusetts, USA).The samples were analyzed in quadruplicate.

Extraction
As can be observed in Table 1, the solvent that extracted the highest concentration of CGA was the NaDES containing lactic acid and glycerol (LA:G), followed by the hydroethanolic solution (Et), with 26% less than LA:G and, lastly, the NaDES containing choline chloride and glucose (CC:GL), with a nal concentration of CGA, 54% lower than that of LA:G and 38% lower than that of the Et extract.
The chromatograms (Supplementary Fig. 1) show that the major compound extracted had an elution time of 8.5 min and maximum absorption at the wavelength of 320 nm.It was identi ed as CGA by comparison with the standard.It is also possible to verify that the 3 solvents extracted similar compound pro les, as observed by their absorbance of the 320 nm wavelength.

Heat stability
Figure 1 presents the results obtained for the heat stability tests of the 3 evaluated extracts.It is possible to observe that the heat treatment at 40ºC and 60ºC, regardless of their duration, caused a signi cant reduction in the content of phenolic compounds in both the hydroethanolic (Fig. 1A) and the CC:GL extracts (Fig. 1C).In the hydroalcoholic extract, these losses ranged from 30.6% (10'; 80°C) to 100% (60'; 40°C and 60°C), while in CC:GL the losses were 40.7% (10'; 80°C) to 100% (120'; 60°C).For treatments at these same temperatures, LA:G proved to be signi cantly more protective than the other extractors, showing no loss after treatment at 60°C (Fig. 1B).At 80ºC, this last extractor (LA:G) did not maintain its protective capacity, presenting a loss of 96.9% after 30 minutes of treatment, similar to those suffered in CC:GL at 80ºC, and hydroethanolic extract at 60°C.
However, in this extract (LA:G), at a temperature of 60ºC, there seems to have been an unexpected increase in the concentration of phenolic compounds in relation to the initial extract, regardless of treatment duration.In this same unforeseen way, treatment at 80ºC seems to have been less harmful to the phenolic compounds in the hydroethanolic extract than the lower temperatures tested.

Storage stability
The results obtained after the storage stability study are shown in Fig. 2. In the hydroethanolic extract (Fig. 2A), storage at -18°C showed the greatest loss, with a maximum of 42.5% (7d).The lowest loss in this extract was 10.4% after fteen days at 25°C, similarly to what was observed in the heat treatment, when higher temperatures unexpectedly resulted in higher nal concentrations of CGA in this extract.In CC:GL (Fig. 2C) a loss variation of 24.2 to 95.9%, in 1d at 25°C and 15d at 25°C, respectively, was found.The loss reached 100% in the 7d to 8°C.This extract also behaved similarly to what was observed in the heat treatment, with a constant reduction in the concentration of CGA over time, accelerated with the increase in the storage temperature.In LA:G (Fig. 2B), there was an increase in the concentration of total phenolic compounds at all storage temperatures, ranging from an addition of 21.5 (1d to 25°C) to 74.5% (15d to -18°C).This behavior was also similar to that observed during heat treatment, when the phenolic compounds content in this solvent was higher after treatment than before.It was observed that the increase in the concentration of phenolic content in the LA:G extract stabilized after day #1 at 8°C.In this solvent there was also a formation of granules (Supplementary Fig. 2) from day #7 in the samples stored at -18°C and 8°C and on day #30 in the extracts at 25°C.

Light stability
The results of stability to exposure to white light are presented in Fig. 3.In the hydroethanolic extract (Fig. 3A), the greatest loss was 55.1% after 15 days of direct exposure to white light.This was the only treatment after which the ethanol extract retained a higher CGA content than LA:G.In LA:G (Fig. 3B), there was a maximum loss of 79.2% after 15 days of light exposure.The protected samples of this extract showed a similar pattern of loss of phenolic content, but the maximum loss in these was 42.6% (15d).As in the storage stability study, CC:GL (Fig. 3C) showed total CGA loss after 15 days when exposed to or protected from arti cial white light.Also as with storage stability, there was an increase in phenolic content in the LA:G extracts after 1 day of exposure in all samples.However, after exposure to white light, this increase was also observed in hydroethanolic extracts and in CC:GL.A discoloration of the extracts was also observed, a little less pronounced in the samples with the re ective protection, except for LA:G, in which there seemed to be no difference between the two samples (Supplementary Fig. 3).

Extraction
The main phenolic compounds of sun ower and its meal are the family of chlorogenic acids (CGAs), esters of quinic acid and trans-cinnamic acid, which may represent up to 70% of their total phenolic compounds [3,16,17].The extraction of CGAs usually applies methanol, ethanol, or acetone in association with water, which improves solubility.Among the above, ethanol may be considered a GRAS solvent, but it is still ammable, unlike NaDES.The concentration of organic solvent for extraction often depends on the food matrix and acidi cation may also be required [18].
The use of different extractors, in the present work, resulted in extracts with very similar characteristics when evaluated by HPLC, with a major chlorogenic acid peak and some minor peaks.These peaks, although not identi ed by comparison with standards, were quite similar in all extracts, regardless of the extractor used (Supplementary Fig. 1).This may indicate a similar a nity of the two NaDES and the hydroethanolic solution tested for the phenolic compounds present in the sun ower meal, despite the signi cant differences in pH, density, and viscosity among them.It could also be due to the uniform composition of the phenolic fraction in sun ower meal, as according to the work of Nascimento et al.
(2023), of the 10 most abundant secondary metabolites obtained from sun ower meal, six belonged to the CGA family.Another study that used different NaDES formulations to extract phenolic compounds and also obtained a similar peak pattern for different extractors was the work by Souza et al. (2022).In this study, Eugenia uni ora leaves extracted with different compositions of NaDES (malic acid:sorbitol, malic acid:glucose:fructose, lactic acid:sucrose, choline chloride:glucose and choline chloride:lactic acid) showed mainly gallic acid, acid ellagic acid and quercetin in all extracts.The same seems to have also been veri ed in the extraction of sun ower disks with NaDES (choline chloride:1,4-butanediol) and ethanol, where CGA was identi ed [19] and in the extraction of tomato co-product with NaDES (choline chloride:lactic acid, choline chloride:1,2-propanediol) and ethanol where gallic acid, caffeic acid and CGA were identi ed by HPLC [20].Thus, in the present study, the only difference between the extracts obtained seems to have been the concentration of CGA extracted from the initial raw material (AL:G > Et > CC:GL).
Regarding the relative extraction e ciency, the recent literature on the subject presents quite varied results when comparing similar NaDES formulations with ethanol.Barbieri et al. (2020) evaluated the extraction potential of four different NaDES formulated with choline chloride, compared to ethanol (100%).All NaDES showed better results than ethanol in the extraction of phenolic compounds from rosemary, except of the one containing glycerol (CC:GL), also used in the present study.Another study also obtained low concentrations of anthocyanin from blackberries using this same NaDES (CC:GL) formulation.In this case, however, the result obtained with NaDES was still superior to the ethanolic and aqueous extracts tested [21].In the extraction of phenolic compounds from orange peel, there was no signi cant difference between NaDES composed of lactic acid:glucose (LA:G), L-proline:malic acid and 50% ethanol [22].The extraction of chlorogenic acid from Artemisiae scopariae was studied by Yue et al. (2021), two formulations stood out when compared to ethanol and water, sorbitol:choline chloride and lactic acid:glucose (LA:G).
The performance of NaDES in the extraction of phenolic compounds may be in uenced by their viscosity.
Higher viscosities are due to a greater number of H bonds, which increases NaDES solubilization capacity.In contrast, excessively high viscosities hinder the diffusion of the desired compounds, decreasing the extraction [23,24].In the present study, the solvent with the highest viscosity was CC:GL, which was also the least e cient extractor of chlorogenic acid from sun ower meal.These results agree with the ndings of Alam et al. ( 2012), who stated that preparations with choline chloride tend to show higher viscosity and reduce the transfer of the target compound from the solid matrix.It is possible that the high viscosity of this NaDES has hindered the extraction of phenolic compounds from the raw material.If this was the case, it is also possible to infer that the small difference in viscosity between the tested NaDES (see Table 1) was already enough to affect their extraction ability.
Another factor that may in uence the extraction of CGAs is the pH of the solvent.In the present study, all solvents were acidic (LA:G > > Et > CC:GL -Table 1), and the most acidic NaDES showed higher extraction e ciency.Two other extraction studies also used NaDES composed of choline chloride:lactic acid as solvent and, in both, this composition was superior for the extraction of phenolic compounds, including CGAs, from tomato pomace and coffee pulp.In coffee pulp, extraction with betaine:glycerol (pH 8.78) was 60% lower when compared to choline chloride:lactic acid, and lower even than ethanolic extract [25].In tomato pomace [20] the NaDES preparation using choline chloride:propanediol (pH 2.2) extracted approximately 30% less when compared to choline chloride:lactic acid (pH 0.5).A different amount of water in the preparation of the acid NaDES in these two studies caused a pH value shift from 0.50 [20] to 0.78 [25].In the present study, the hydroethanolic solution (40% ethanol) showed the lowest viscosity and intermediary pH and achieved 1.6 times higher extraction of phenolic compounds than CC:GL, whereas LA:G resulted in concentrations of CGA1.3 higher than the Et extract.This NaDES showed the more acidic pH and intermediate viscosity.

Stability
In the present work, the protective capacity of three different extractors on phenolic compounds extracted from sun ower meal was evaluated when exposed to heat treatment, storage in freezer, under refrigeration and at room temperature, and exposure to light, with and without re ective protection.In general, it can be said that AL:G showed the greatest protection to the phenolic compounds against heat treatment, especially at temperatures up to 60ºC, and during storage for 30 days, regardless of the temperature.The ethanol extract granted the lowest decomposition of phenolic compounds after exposure to light for up to 7 days.

Heat stability
Phenolic compounds in general tend to be sensitive to heat and decompose at temperatures above 60°C [13,15,27].In the present study, at treatment temperatures of 40 and 60ºC, AL:G showed a protective capacity against the thermal degradation of the phenolic compounds in the extract, while Et and CC:GL did not.NaDES protection against thermal degradation of phenolic extracts has already been described by Yue et al. (2021).In this study, chlorogenic acid from Artemisiae scopariae was stable at 85°C, showing less than 5% loss, after 30 minutes, in both proline:malic acid and lactic acid:glucose (LA:G) formulations.In another study, which evaluated the heat stability of curcuminoids, the degradation at 50°C was similar in all extracts, but at 80°C, the thermal degradation in the ethanolic and methanolic extracts was higher when compared to NaDES of citric acid:glucose [23].Dai et al. (2016) observed that there was little degradation of anthocyanins in all extracts tested at 40°C, but that these compounds were more stable in lactic acid:glucose (LA:G) than in acidi ed ethanol, at 60°C.This same group [15] evaluated the heat stability of carthamin, which showed a half-life twice as high in xylitol:choline chloride than in water, at 60°C.In both studies, the greatest loss of phenolic compounds was recorded at 80°C, as well as in the present study.
The higher stability of phenolic compounds in DES media is associated with the binding between hydrogens of the target compound and the DES components, which is believed to decrease the oxidation and increase the solubility of phenolic compounds in these solvents [23].In the present work, although one NaDES promoted greater thermal protection than the organic solvent, the other formulation tested did not behave in the same way.It is possible that, despite the formation of H bonds in NaDES, the little acidic pH (6.35 -Table 1) of this formulation contributed to the degradation of the CGA.The stability of CGA was pH dependent in the study by Narita and Inouye (2013).Stability increased as the pH was reduced, and CGA showed signi cant degradation in aqueous solution with pH values greater than 5, at 37°C.Krungkri and Areekul (2019) also con rmed the low stability of phenolic compounds at pH 6.In their work, the stability decreased as the treatment temperature increased, in 95% ethanol, 60% acetone and water, reaching maximum degradation at 80°C, in an ethanolic solution at pH 6.However, the opposite was observed in the work by Zhou et al. (2022), where catechins were more stable to exposure at 80°C in NaDES composed of choline chloride:glycerol than in choline chloride:lactic acid.
Another unexpected behavior was also observed during the evaluation of the stability of the extracts to heat treatment and no similar results were found in the available literature.Both Et and LA:G showed higher concentrations of phenolic compounds after thermal treatment at 80ºC and 60ºC, respectively.Although all treatments were carried out in airtight tubes, several samples showed a reduction in volume after the treatment time, indicating a possible evaporation, either of the organic solvent or of the aqueous fraction (addition of 30% water) of the NaDES.In all cases, when a reduction in volume was veri ed, the sample was reconstituted with pure solvent, up to the initial volume.Even so, it is possible that the higher content of phenolic compounds detected in Et samples was a consequence of a partial concentration, by evaporation of the solvent during the thermal treatment at higher temperatures.When LA:G was heated to 60ºC, regardless of the treatment time, there was a signi cant increase in the content of phenolic compounds in the samples.This NaDES consisted of a liquid of high viscosity and density, which made it very di cult to separate the solid (sun ower meal) and liquid (NaDES) fractions after the extraction process.Thus, a residue of nely particulate solid material remained in suspension in the extract.It is possible that, with heating, changes occurred in the density, viscosity, and surface tension of the solvent, as described by Alam et al. ( 2021), thus promoting an improvement in the interaction with the phenolic compounds of the residues, generating an extraction of these compounds during the thermal treatment, which more than compensated for the thermal degradation that occurred over the treatment time.
Following the same reasoning, it is possible that this same extraction occurred at 80ºC, but the degradation that occurred at that temperature was greater than this additional extraction.The other formulation tested (CC:GL) was even more viscous and also contained suspended solid residue.However, apparently, the degradation of CGA that occurred in the little acidic pH of this solvent was greater than the possible additional extraction that may have occurred during the thermal treatment.Furthermore, this solvent was less e cient in extracting CGA under the initial extraction conditions, and may also have been less e cient during heating, when compared to LA:G.

Storage stability
In the present study, with signi cant differences in pH were obtained, as shown in Table 1.AL:G, the most acidic of all, granted higher stability at different storage temperatures, while CC:GL, the closest to neutrality, did not.Et showed intermediate results, as well as pH value.According to Friedman and Jürgens (2000), the stability of phenolic compounds is associated with three main factors: pH, storage time and structure of phenolic compounds.In their study, alkaline media caused irreversible structural changes in caffeic, chlorogenic and gallic acids.Catechin, epigallocatechin and rutin were comparatively more stable, which was attributed to their greater number of aromatic structures, that provided higher stability at alkaline pH.
The protection of phenolic compounds in acidic NaDES was also observed by other authors.In the work by extracts in NaDES and ethanol for three months.In their study, the concentration of total phenolic compounds remained stable in NaDES, as opposed to acidi ed ethanol, which lost approximately 40% and 60% total phenols when stored at 4°C and 25°C, respectively.In Dai et al. (2014), carthamin was storagestable in all NaDES tested for 7 days at -20°C, however, in choline chloride:sucrose, the stability extended to the 15th day.Contrary to these studies, NaDES containing choline chloride, in the present study, was not as protective to sun ower meal extract after day #1, regardless of the temperature of storage.
As in the study of thermal stability, during storage there was also an increase in the concentration of CGAs in AL:G, which appears to have occurred earlier at higher temperatures and later at lower temperatures.This occurrence might also be attributed to continued extraction from residues of the sun ower meal that remained in suspension after ltration.Likewise, it can be assumed that the process was not repeated in the other extractor (CC:GL) due to its lower extracting capacity.

Light stability
CGAs were found to be sensitive to visible and ultraviolet light, undergoing trans to cis 5-caffeoylquinic acid isomerism.The degree of degradation varied according to exposure time, temperature, and wavelength [29][30][31].
In the present study, attention is drawn to the fact that there was little or no difference between the samples protected by aluminum foil and those exposed without protection to arti cial white light.To avoid the evaporation of solvents that occurred during the thermal treatment, these tests were conducted using cryogenic vials with screw caps.These asks are made of polypropylene, which allows the passage of light above 290 nm, but absorbs shorter wavelengths, especially below 250 nm [32], while the re ective covering should protect the sample from all wavelengths emitted by the lamp.The lack of signi cant difference between protected and unprotected samples indicates that exposing the extracts to wavelengths above 290 nm had the same effect as completely protecting the samples.It is possible that the degradation observed in the samples was mainly due to the period of exposure to a temperature slightly higher than the ambient temperature, an increase caused by the presence of the light bulb.However, the behavior of the extracts was quite different from the behavior observed for other tests involving storage or heat treatment.
In the light stability test, Et and CC:GL promoted better conservation of phenolic compounds than LA:G, at least for 7 days of exposure to the test conditions.It is also possible that the aluminum foil was ineffective in providing protection to the vials, as this was the only assay after which the samples showed marked visible discoloration, that may be observed in Supplementary Fig. 3.
The stability of phenolic compounds under white light is little studied and assays evaluating the role of NaDES on the stability of CGAs under white light or sunlight are rare.In the few works available, as a rule, the phenolic compounds showed greater stability in NaDES than in organic solvents.solvents, but NaDES were more protective than the organic solvent.In this same study, low-acidic NaDES (sucrose:choline chloride, glucose:choline chloride) and water ensured complete stability of carthamin under the test conditions.Blueberry pomace anthocyanins were extracted with acidi ed ethanol and acid NaDES (choline chloride:oxalic acid) and kept under arti cial light, sunlight or in the dark.The extracts behaved similarly in the absence of light, but exposure to sunlight showed the greatest loss of phenolic compounds, approximately 50% and 15% in the ethanolic extract and NaDES, respectively.Under arti cial light, the loss was 20% in ethanol and around 10% in NaDES [33].Ferreyra et al. (2023) recently evaluated the exposure to light of hydroethanolic (50%) extracts of grape co-products.After 2 weeks, a 43% reduction in total phenolic content was observed, similar to the obtained in the present study.Based on the results of the present test and the data available in the literature, apparently the acidic pH of the solvent negatively in uenced the stability of the phenolic compounds exposed to light, in opposition to what occurred during heat treatment and the storage protected from light.

Conclusion
The results obtained in the present work indicate that the pH was the determining factor in the ability of LA:G to extract and protect the sun ower meal CGAs, promoting greater extraction and greater protection against heating and storage over time when compared to CC:GL, a less acidic and more viscous NaDES formulation.On the other hand, these conditions were reversed when the extracts were exposed to light.When compared to the organic solvent used as a control (40% ethanol), the acidic NaDES formulation was more e cient in extracting CGAs and the less acidic NaDES formulation was less e cient, although the extracted compound pro le was the same for all extractors.
This study succeeded in the endeavor to shed light on the behavior of important phenolic compounds in natural deep eutectic solvents, at the same time that it indicates an alternative to add value to sun ower meal and avoid the waste of tons of bioactive phytochemicals annually.

Declarations
Author A study by Friedman; Jürgens (2000) evaluated the stability of CGAs and showed that pH values close to 7 tended to cause irreversible structural alterations.On the other hand, in the work of Mellinas et al. (2020), for the extraction of phenolic compounds from cocoa shells, alkaline extractors formed pores enabling the extraction, while acidic or neutral solvents did not show the same e ciency.When the use of hydroethanolic solutions was considered, the work of Zardo et al. (2017) evaluated ultrasound amplitude, temperature, and ethanol concentration.Ethanol concentrations higher than 45% lead to a decrease in the CGA extraction.The effect of ethanol concentration, temperature and pH was evaluated on the extraction of phenolic compounds from defatted sun ower seed husks.The best results were obtained at neutral pH, 50°C and at ethanol concentrations below 60% [26].

Gómez-
Urios et al. (2022) the phenolic compounds from orange by-products in formulations containing lactic acid showed better upkeep at lower temperatures (4ºC > 25ºC).Phenolic compounds extracted from co-products of olive, onion, tomato and pear seeds with lactic acid:glucose (AL:G) remained stable for two months at -18°C and 4°C, but there was a loss of 90% of apigenin and quercetin in the aqueous extract in the same conditions[28].The authors attributed the greater conservation in NaDES to the lower mobility of the compounds in these media, that might reduce contact with oxygen and delay oxidative degradation.The study by Aslan et al. (2022) evaluated the stability of anthocyanins from purple carrots in choline chloride:citric acid, at 4, 25 and 37°C, protected from light, for 90 days.Highest upkeep was achieved at 4°C, with a retention of 70% total phenols.The results of the present study, as well as those form the literature mentioned above, point to the acidic pH as an extremely important factor in the storage of phenolic extracts.However, other studies obtained better upkeep in NaDES (acidic or not) than in acidi ed ethanol, indicating a possible joint or synergistic effect between the H bonds of NaDES and their pH.This may be demonstrated by the following examples: Dai et al. (2016) evaluated the storage of anthocyanin Jeliński et al. (2019) tested the stability of curcumin in choline chloride based NaDES after exposure to arti cial light for 2 hours.Only 5% of curcumin remained after treatment in methanolic solution and 19% in the turmeric powder.In NaDES composed of choline chloride:glycerol (CC:GL), curcumin remained stable throughout the period of exposure to light.In the present work, CC:GL also granted stability of the CGAs in solution for up to 7 days.Dai et al. (2014) tested the stability of carthamin under white light, in acidic NaDES (lactic acid:glucose, proline:malic acid) and in 40% ethanol.Light accelerated the degradation process in all

Figure 1 Heat
Figure 1

Figure 2 Storage
Figure 2

Table 1
Composition and characteristics of solvents and concentration of total phenolic compounds in extracts.