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Article

Evaluation of Polyphenol Profile from Citrus Peel Obtained by Natural Deep Eutectic Solvent/Ultrasound Extraction

by
Manuel Octavio Ramírez-Sucre
†,
Kevin Alejandro Avilés-Betanzos
†,
Anahí López-Martínez
and
Ingrid Mayanin Rodríguez-Buenfil
*
Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C., Subsede Sureste, Tablaje Catastral 31264, Km. 5.5 Carretera Sierra Papacal-Chuburná Puerto, Parque Científico Tecnológico de Yucatán, Mérida C.P. 97302, Yucatán, Mexico
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Processes 2024, 12(10), 2072; https://doi.org/10.3390/pr12102072
Submission received: 16 August 2024 / Revised: 6 September 2024 / Accepted: 22 September 2024 / Published: 25 September 2024
(This article belongs to the Special Issue Phytochemicals: Extraction, Optimization, Identification, Biological Activities, and Applications in the Food, Nutraceutical, and Pharmaceutical Industries)
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Abstract

:
Citrus fruits are widely consumed worldwide; however, one of their primary uses is juice production, resulting in over 40 million tons of agro-industrial waste. Citrus peel is the main agro-industrial by-product in citrus production. In recent years, secondary metabolites of interest, mainly polyphenols such as hesperidin, have been identified in citrus peels. Currently, green alternatives like natural deep eutectic solvents (NADES) based on choline chloride and glucose (Glu), combined with ultrasound-assisted extraction, are studied to obtain polyphenol-rich extracts with potential health applications. This study aims to evaluate the effect of: (1) molar ratios (MR) of 1:0.5, 1:1 or 1:2 mol/mol of choline chloride (ChCl):glucose (Glu); (2) the percentage of added water (WA: 50, 60 or 70%) to NADES; and (3) different citrus peels of Citrus aurantium (bitter orange), Citrus sinensis (sweet orange), and Citrus limon (lemon) used for extraction, on polyphenol profiles, total polyphenol content (TPC), and antioxidant capacity (Ax) of the extracts. The extracts were analyzed using ultra-performance liquid chromatography (UPLC) and evaluated using the Folin–Ciocalteu method for TPC and DPPH assay for quantifying AC. A factorial experimental design 33 was implemented. The extract obtained with an MR of 1:1 (ChCl:Glu) from Citrus aurantium peel exhibited the highest concentration of hesperidin (2003.37 ± 10.91 mg/100 g dry mass), whereas an MR of 1:2 (ChCl:Glu) exhibited the highest concentration of neohesperidin (1045.94 ± 1.27 mg/100 g dry mass), both using 60% WA. This extract also showed the highest antioxidant capacity, achieving 100% inhibition. On the other hand, the highest concentration of total phenolic content (TPC) (96.23 ± 0.83 mg GAE/100 g dry mass) was obtained using C. aurantium peel with an MR of 1:0.5 (ChCl:Glu) and 60% WA. The extracts also presented high concentrations of rutin and catechin. These findings highlight the potential of revalorizing citrus peels, particularly Citrus aurantium, and their extracts obtained with NADES for possible health applications.

1. Introduction

Citrus fruits, such as orange and lemon, are highly preferred and consumed worldwide. Their combined global production in 2023 reached 58.8 million tons, with Mexico ranking fourth. According to the Mexican Agri-Food and Fisheries Information Service (SIAP), this year, Mexico’s citrus fruit production was 8.1 million tons, representing 13.77% of the global production, following Brazil, China, and the European Union (USDA, 2024) [1].
According to Kumar et al. [2], the primary agricultural waste from citrus fruit production is the peel (CPe), which accounts for 30% of the fruit’s weight. Thus, in Mexico, this results in approximately 2.43 million tons of peel annually [3].
Although CPe is considered an agricultural waste, this plant matrix contains various components of interest, such as pectin (dietary fiber), carotenoids, essential oils, vitamin C (ascorbic acid), and polyphenols [4]; the latter have attracted particular interest due to their recognized antioxidant, anti-inflammatory, anticancer, antidiabetic, and other beneficial effects in humans [5]. High concentrations of these compounds have been reported in the peels of various citrus fruits. For example, Lagha-Benamrouche and Madani [6] determined the total polyphenol content (TPC) through maceration with methanol–water (4:1 v/v) in C. sinensis (25.60 ± 0.23 mg GAE/g dry mass) and C. aurantium (31.62 ± 0.88 mg GAE/g dry mass) peels. Similarly, Papoutsis et al. [7] registered the highest TPC (13.24 mg GAE/g dry mass) of C. limon peel by absolute methanol extraction.
While total polyphenols can be observed in citrus peel samples, the profile of individual polyphenols will not be the same for each type of citrus, even though they may share some characteristic compounds of the Citrus genus, such as hesperidin, naringenin, rutin, quercetin, and diosmin [8,9]. The characteristic phenolic compound of citrus fruits is hesperidin. This metabolite is classified as a flavonoid, and more specifically as a glycosylated flavanone, because its structure consists of the hesperetin molecule linked by a glycosidic bond to rutinose, a disaccharide formed by glucose and rhamnose [10].
Concentrations of this flavonoid have been reported in C. sinensis peel using various extraction methodologies, such as ultrasound (836 ± 29 mg/100 g dry mass), microwave (765 ± 12.13 mg/100 g dry mass), and ethanol (80%) maceration (551 ± 0.01 mg/100 g dry mass) [11]. For C. limon peel, extraction using the Soxhlet method, with absolute methanol at 85 °C for 12 h, has yielded concentrations of 331.50 ± 26.23 mg/100 g dry mass [9]. Finally, for C. aurantium, concentrations of 21.03 mg/100 g fresh mass have been reported. This metabolite has attracted significant interest in both the food and pharmaceutical industries, because of its various biological properties. It has notable cardioprotective and vasodilatory effects, which are harnessed in the creation of medications for circulatory system disorders [11]. Furthermore, it has been documented in relation to its capacity to enhance the body’s antioxidant enzymes, such as catalase and superoxide dismutase, its neuroprotective capabilities [12], and its preventive effects against SARS-CoV-2 [13,14].
Various methodologies have been reported for conducting flavonoid extraction, including traditional methods such as maceration or steam distillation, where organic solvents like ethanol, methanol, and/or acetone have been used [15,16]. However, in the search for new green extraction technologies aimed at obtain high phenolic content extracts with characteristic individual polyphenols, ultrasound-assisted extraction (UAE) and natural deep eutectic solvents (NADES) have recently been implemented in agro-industrial by-products of citrus. These studies are recent and there is limited information available, but the initial findings suggest significant potential for their use in the cosmetic, pharmaceutical, or food industries without posing a risk to handlers and consumers [17,18,19]. In this context, there is growing interest in combining both green technologies, UAE and NADES, to improve the extraction of bioactive compounds from citrus peels.
UAE, through ultrasonic waves, generates cavitation in the solvent medium, which enhances mass transfer and disrupts plant cell walls, leading to higher extraction yields and shorter processing times. UAE’s ability to improve solvent penetration and breakdown of plant structures makes it an ideal candidate for integration with other extraction methods to maximize efficiency [20], like NADES.
NADES have emerged as a promising alternative to conventional solvents, offering several favorable characteristics for the extraction of phenolic compounds. The effectiveness of NADES largely depends on the selection of their components, as these determine the solvent’s polarity. Specifically, the choice of a hydrogen bond donor (common donors: glucose, fructose, glycerol, malic acid, and citric acid, among others) and its combination in a defined molar ratio with a hydrogen bond acceptor (such as choline chloride, betaine, urea, proline, etc.) can optimize the extraction of a particular compound of interest [21].
Furthermore, the addition of a certain percentage of water to NADES results in a mixture with greater polarity and affinity to phenolic compounds. This adjustment not only reduces the solvent’s viscosity, thereby improving its diffusivity and mass transfer, but also promotes the formation of a hydrogen bond network between the solvent and the target metabolite. This network confers supramolecular stability on the mixture (through hydrogen bonds, van der Waals forces, and electrostatic interactions), preventing the degradation of bioactive compounds during the extraction process [22].
By integrating UAE with NADES, it is possible to harness the strengths of both technologies—UAE’s capacity for cell disruption and enhanced mass transfer, combined with NADES’ versatile properties and ability to stabilize bioactive compounds. This unified approach holds great potential for the efficient and sustainable extraction of bioactive compounds from citrus peels, contributing to the valorization of agricultural by-products and the advance of green chemistry, especially given the high production of citrus fruits and the substantial amount of peel generated as waste.
The innovative aspect of this study lies in evaluating different proportions of hydrogen bond donors, such as glucose, along with varying water content in NADES, to extract polyphenols from citrus peels, primarily the scarcely studied C. aurantium. There is very limited information available on the application of NADES and on the extracts obtained using these green solvents. Finally, the objective of this study is to evaluate the effect of the molar ratio and the percentage of added water in NADES on the polyphenol profile, highlighting the extraction combined with UAE from various citrus peels.

2. Materials and Methods

2.1. Citrus Peel

The peels of bitter orange (Citrus aurantium), sweet orange (Citrus sinensis), and lemon (Citrus limon) were used. Approximately 1 kg of each citrus was obtained at a local supermarket (August 2023) in Mérida, Yucatán, Mexico. The citrus peels were carefully removed using a knife, ensuring that both structures, the flavedo and the albedo, were preserved. This process was carried out without squeezing the fruits or recovering the juice.

2.2. Drying, Grinding, and Sieving of Citrus Peel

The peels were placed in a tray oven and dried following the method described by Chel-Guerrero et al. [23] with slight modifications, drying at 32 °C for 24 h to achieve a moisture content below 15%.
The dried peels were ground using a MasterChef® coffee grinder until a homogeneous powder was obtained. The powder was sieved to obtain a particle size of ≤500 µm. The sieved powder was stored at room temperature in resealable bags lined with aluminum foil until further use.

2.3. Preparation of Natural Deep Eutectic Solvent Based on Choline Chloride

For the preparation of natural deep eutectic solvents (NADES), the methodology described by Avilés-Betanzos et al. [24] was followed. Choline chloride (ChCl, 139.62 g/mol) and glucose (Glu, 180.16 g/mol) were mixed in different molar ratios (ChCl:Glu, 1:0.5, 1:1, or 1:2 mol/mol), with ChCl serving as the hydrogen bond acceptor (HBA) and Glu as the hydrogen bond donor (HBD). The mixtures were heated to a temperature of 90 °C in a water bath with continuous stirring (90 min) until a clear liquid phase was obtained. To each prepared NADES, a percentage of water (50, 60, or 70%) was added according to the final weight of the preparation.

2.4. Polyphenol Extraction from Citrus Peels Using NADES by Ultrasound Bath

To evaluate the effect molar ratio (MR), percentage of added water (WA) and of citrus peel type (CPe), on the NADES extraction of phenolic compounds, a 33 factorial experimental design was established.
The levels of these three factors were: for MR, 1:0.5 (−1), 1:1 (0) or 1:2 (1) of ChCl:Glu, mol/mol; for WA, 50% (−1), 60% (0), and 70% (1); and for CPe, Citrus sinensis (−1), Citrus aurantium (0), and Citrus limon (1), the nomenclature is shown in Table 1. The response variables were: (a) Polyphenol profile (mg/100 g dry mass); (b) Total polyphenol content (TPC, mg GAE/100 g dry mass); and (c) Antioxidant capacity (Ax, % DPPH Inhibition).
The extraction of phenolic compounds was carried out following the procedure outlined by Avilés-Betanzos et al. [25], with some modifications. To a 1-g sample of citrus peel powder, 10 mL of NADES (MR and WA according to the experimental design) was added. The mixture was homogenized using a vortex mixer (Thermo Scientific®, Mexico City, Mexico, model Maxi Mix® II). The mixture was then sonicated in an ultrasonic bath for 30 min at 42 kHz (BRANSON®, model 351). Subsequently, the mixture was centrifuged (30 min, 4 °C, 4700 rpm) to recover the supernatant, which was then filtered through a Nylon filter (0.22 µm). The extract was stored in chromatographic vials under refrigeration (<18 °C) until analysis.

2.5. Determination of Polyphenol Profile of Citrus Peel Extracts

The analysis and quantification of polyphenols in extracts from citrus peels were conducted using an advanced Acquity UPLC H-Class system equipped with a diode array detector and a high-performance column (Acquity UPLC HSS C18), as adapted from methodologies outlined by Avilés-Betanzos et al. [26]. To accurately measure the polyphenol concentrations, a calibration curve incorporating 18 distinct standards was employed, ranging from 5 to 75 μg/mL, including gallic acid, protocatechuic acids, chlorogenic acid, coumaric acid, cinnamic acid, catechin, rutin, kaempferol, quercetin, luteolin, vanillin, ferulic acid, diosmin, hesperidin, neohesperidin, naringenin, apigenin, and diosmetin. This setup ensured precise control over the analytical conditions, with the column maintained at 45 °C and samples introduced in 2-μL injections. Detection was optimized at 280 nm using a solvent system of water with 0.2% acetic (phase A) and acetonitrile with 0.1% acetic acid (phase B). Each injection had a duration of 15 min, where the gradient for elution was 99% A to 70% A and 1% B to 30% B (0 min to 10 min), followed by a steady state of 70% A and 30% B (10 min to 12 min), and finally elution from 70% A back to 99% A and 30% B back to 1% B (3 min). Quercetin and luteolina were quantified collectively, due to their overlapping peaks during analysis. Figure S2 shows the chromatogram with the lowest concentration (2 µgmL) of the calibration curve of selected individual polyphenols, including gallic acid, protocatechuic acid, catechin, chlorogenic acid, p-coumaric acid, rutin, hesperidin, quercetin + luteolin, and kaempferol.

2.6. Evaluation of Total Polyphenol Content in Citrus Peel Extracts

The polyphenolic content of the citrus peel extracts (CPex) was determined using the Folin–Ciocalteu method with some modifications, following the procedure described by Singleton et al. [26]. Initially, a dilution (1:10) of the CPex was made with distilled water, then 25 μL of extract diluted was mixed with 25 μL of distilled water, followed by the addition of 3 mL of water and 250 μL of Folin reagent (Sigma-Aldrich®, St. Louis, MO, USA). After 5 min, 750 μL of 20% sodium carbonate (Na2CO3, Sigma-Aldrich) and 950 μL of distilled water were added, and the mixture was allowed to stand for 30 min. The samples were then analyzed at 765 nm using a UV–Vis spectrophotometer (Thermo scientific®, Mexico City, Mexico, model Genesys 140). Prior to the sample analysis, a calibration curve was generated (Figure S1) with gallic acid from 5 µg/mL to 75 µg /mL (R2 = 0.9965).

2.7. Antioxidant Capacity Assessment of Citrus Peel Extract

The assessment of antioxidant capacity (Ax) in CPex was performed using the DPPH assay, as outlined by Chel-Guerrero et al. [23]. Initially, 3.3 mg of DPPH was diluted to a total volume of 100 mL with methanol, and the solution subsequently was adjusted to an absorbance (Abs) of 0.700 ± 0.002 at 515 nm. The analysis was conducted using a Thermo Scientific® UV–vis spectrophotometer (Mexico City, Mexico, Genesys 140).
Following the standardization of the DPPH solution’s Abs, a 100-μL sample of the CPex was added to 3.9 mL of the adjusted DPPH solution. This mixture was mixed and incubated for 30 min. The Abs was then measured at 515 nm. The antioxidant capacity was quantified as the percentage of inhibition, calculated according to Equation (1):
% DPPH Inhibition = 100 − [(Abs of CPex × 100)/(Abs of adjusted DPPH solution)]

2.8. Statistical Analysis

The studies were carried out randomly. For each extract produced according to the experimental layout, measurements were taken in triplicate to ascertain both the total and specific polyphenols, along with the antioxidant capacity (Ax). The results are presented as mean values with standard deviations. The linear correlation analysis was conducted to examine the relationships between the total and specific polyphenols in by-products and their antioxidant capacities, as measured by DPPH assays. This involved calculating the Pearson correlation coefficient and conducting a principal component analysis (PCA). The statistical evaluations of the experimental design were performed using Statgraphics Centurion XVII.II-X64 (Statgraphics Technologies Inc.; Virgin, UT, USA), XLSTAT 2021.2.2 (Addison, Paris, France), and R software version 4.0.3 (The R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Polyphenol Profile from Citrus Peel Extracts

Among the 18 polyphenols examined in each extract from the factorial design, only 14 individual polyphenols were identified. Cinnamic acid, coumaric acid, ferulic acid, and diosmin were not detected (Table S1). Additionally, Figure S3 shows the chromatograms of three extracts from different citrus peels, obtained under the same conditions, highlighting the variability in the profiles of individual polyphenols. For a better perspective on the polyphenol profile results from the experimental design 33 factorial, the data are presented in Figure 1, considering the polyphenol profile of (a) C. aurantium, (b) C. sinensis, and (c) C. limon. and only de majoritarian individual polyphenols (catechin, rutin, quercetin + luteolin, hesperidin, and neohesperidin).
The profile of major polyphenols in extracts from the C. aurantium peel is shown in Figure 1a. Extract of C. aurantium showed the highest TPC (>2000 mg GAE/100 g dry mass) from all the CPex analyzed. The two characteristic polyphenols, hesperidin and neohesperidin, were identified. Under the extraction conditions of experiment #5 (MR = 1:1, WA = 60%), the highest concentration of hesperidin (2003.37 ± 10.91 mg/100 g DM) was obtained while experiment #6 (MR = 1:2, WA = 60%) resulted in the highest concentration of neohesperidin (1045.94 ± 1.27 mg/100 g DM). Furthermore, the presence of all major polyphenols was observed in the extracts obtained using NADES with an MR of 1:2 mol and 70% of added water.
Hesperidin and rutin were the characteristic individual polyphenols in the peel of C. sinensis (Figure 1b). High concentrations of hesperidin were obtained under NADES conditions with an MR of 1:2 mol and 50% added water, while the extract with the highest concentration of rutin was achieved using a NADES with an MR of 1:1 mol and a WA of 70%. Under these same conditions, the extract contained all the major polyphenols (catechin, rutin, quercetin + luteolin, hesperidin, and neohesperidin).
In the extracts obtained from the peel of C. limon, rutin was identified as the characteristic and predominant polyphenol. Under two conditions of NADES (p > 0.05), a high concentration of rutin was observed: 541.60 ± 0.81 mg/100 g DM with an MR of 1:2 mol and AW of 50% (#21), and 526.42 ± 2.30 mg/100 g DM with an MR of 1:0.5 mol and AW of 60% (#22). Unlike the extracts from C. aurantium and C. sinensis, all the major polyphenols analyzed were identified and quantified in all extracts from the peel of C. limon (Figure 1c).
Amidst the minor polyphenols found in the various extracts of the design (Table S1), gallic acid was identified and quantified only in two extracts (#16, #22). The highest concentration (7.45 ± 0.02 mg/100 g DM) was present in the extract obtained from C. sinensis peel with an MR of 1:0.5 and AW of 70%, while the lowest concentration (4.85 ± 0.00 mg/100 g DM) was found in the extract from C. limon (MR = 1:0.5, WA = 60%). Chlorogenic acid is another compound that was identified and quantified in few extracts (#6, #9, #11, #12, #16) of the experimental design, within concentrations of 3.73 ± 0.27 up to 31.62 ± 0.38 mg/100 g DM in extracts #11 (MR = 1:1, WA = 50%, C. sinensis) and #16, respectively.
Other minor metabolites were found in the extracts, including protocatechuic acid, kaempferol, vanillin, naringenin, apigenin, and diosmetin (Table S1). The highest concentrations of protocatechuic acid (36.25 ± 0.24 mg/100 g DM) and diosmetin (47.54 ± 1.28 mg/100 g DM) were identified in the extracts obtained under the conditions of experiment #12 (MR = 1:2, WA = 50%, CPe = C. sinensis). Conversely, high concentrations of kaempferol (27.77 ± 2.30 mg/100 g DM) and vanillin (15.33 ± 0.05 mg/100 g DM) were identified in experiment #5 (MR = 1:1, AW = 60%, CPe = C. aurantium).
The extract from experiment #20 (MR = 1:1, WA = 50%, CPe = C. limon) exhibited the highest concentration of naringenin (22.90 ± 0.05 mg/100 g DM), whereas the highest concentration of apigenin (10.50 ± 0.03 mg/100 g/DM) was observed in the extract obtained under the conditions of experiment #27 (MR = 1:2, WA = 70%, CPe = C. limon).
Table 2 shows the multifactorial ANOVA results on the polyphenol profile. These data indicate that concentrations of gallic acid (WA), protocatechuic acid (CPe), catechin (MR, CPe), rutin (MR, CPe), quercetin + luteolin (WA), kaempferol (WA), and diosmetin (WA, CPe) in citrus peel extracts are influenced (p < 0.05) by one or more primary factors, but not by their interactions (double or triple). Conversely, the concentrations of chlorogenic acid (WA*MR), hesperidin (WA*MR, MR*CPe, WA*MR*CPe), neohesperidin (WA*CPe), naringenin (WA*MR, WA*CPe), and apigenin (MR*CPe) are significantly affected (p < 0.05) by double or triple interactions.

3.2. TPC and Antioxidant Capacity on Citrus Peel Extracts

The lowest and highest concentrations of total polyphenols in the experimental design were found in C. aurantium extracts (Table S2). The lowest concentration (1.36 ± 0.24 mg GAE/100 g DM) of total polyphenol content (TPC) was achieved with a NADES having an MR of 1:1 mol and a WA of 50%. Using an MR of 1:0.5 mol with a higher WA percentage (60%) resulted in an extract with the highest concentration (96.23 ± 0.83 mg GAE/100 g DM) of phenolic compounds (Figure 2); these values were also the lowest and highest in the entire experimental design.
On the other hand, only for C. sinensis extracts, the lowest concentration of TPC (18.03 ± 0.47 mg GAE/100 g DM) was obtained under the conditions of experiment #10 (MR = 1:0.5 mol, WAAW = 50%), while the highest concentration (71.73 ± 0.76 mg GAE/100 g DM) was observed in experiment #12 (MR = 1:2 mol, WA = 50%).
Figure 2 also displays the TPC concentrations for the C. limon extracts. Among the C. limon extracts obtained, the analysis revealed that the lowest (19.02 ± 0.43 mg GAE/100 g DM) concentration of phenolic compounds was achieved using a NADES with 1:1 (MR) and 70% added water (WA). In contrast, using 1:0.5 mol formulated with 60% WA resulted in an increased concentration (82.46 ± 1.74 mg GAE/100 g DM) of these metabolites of interest for C. limon extracts.
In a similar way, with TPC, the lowest and highest antioxidant capacity were found in C. aurantium peel extract. Under conditions of 11: (MR) and 70% of added water (WA), the lowest antioxidant capacity (12.82 ± 0.14% Inhibition) extract was obtained. For the highest antioxidant capacity extract, no difference (p > 0.05) was found between the conditions from experiments #6 (MR = 1:2 mol, WA = 60%, C. aurantium), #9 (MR = 1:2, WA = 70%, C. aurantium), both with 100 ± 0.00% de inhibition, and #20 (MR = 1:1 mol, WA = 50%, C. limon), with 98.58 ± 0.00% inhibition.
However, among extracts obtained from C. sinensis peel, the one with highest antioxidant capacity (83.05 ± 0.00% Inhibition) was obtained using an MR of 1:1 and 50% added water (Exp #11).
Figure 3a illustrates that any factor or interaction shows an effect (p > 0.05) on total polyphenol content in extracts obtained using choline chloride-based NADES. Conversely, the molar ratio was the only factor (main and interactions) to significantly affect (p < 0.05) the antioxidant capacity of extracts derived from a citrus by-product (Figure 3b).

3.3. Linear and Pearson Correlation

According to the linear correlation analysis results (Table 3), the antioxidant capacities of extracts obtained from C. limon peel were well correlated with diosmetin (r = 0.7863) and vanillin (r = 0.8487). On the other hand, neohesperidin (r = 0.7486) and vanillin (r = 0.8072) showed a strong correlation with the antioxidant capacity of C. sinensis extract.
Additionally, the antioxidant capacity of extract from C. aurantium presented a good correlation with several phenolic compounds such as quercetin + luteolin (r = 0.7716), kaempferol (r = 0.7110), neohesperidin (r = 0.7484), and the total polyphenol content (r = 0.7514).
There was no linear correlation between phenolic compounds and antioxidant capacity when analyzing the complete experimental design 33 (Table S3).
The data obtained from all the response variables of the 33 experimental design were analyzed using the Pearson correlation (Figure 4).
A positive correlation was observed between naringenin and rutin (r = 0.7547), kaempferol and Q + L (r = 0.7193), and neohesperidin and vanillin (r = 0.7193).
In Figure S4, the analysis of TPC and antioxidant capacity by citrus peel extract is shown. For example, in the extracts of C. aurantium (Figure S4a), a positive correlation was observed between the TPC and the individual polyphenols kaempferol (r = 0.8448), Q + L (r = 0.7552), vanillin (r = 0.7738), and neohesperidin (r = 0.7846). Meanwhile, Ax showed a correlation with TPC (r = 0.7614), neohesperidin (r = 0.7486), Q + L (r = 0.7706), and kaempferol (r = 0.7110). Almost all the phenolic compounds that showed a positive correlation with TPC also exhibited a similar correlation with Ax.
C. sinensis (Figure S4b) extracts showed a positive correlation between TPC and diosmetin (r = 0.8497). On the other hand, Ax was positively correlated with vanillin (r = 0.8072) and neohesperidin (r = 0.7129). A positive correlation was found between TPC and gallic acid (r = 0.8631), as well as between Ax with both, vanillin (r = 0.8487) and diosmetin (r = 0.7863) in C. limon (Figure S4c) extracts.

3.4. Principal Analysis Component

The response variables (excluding gallic acid, protocatechuic acid, chlorogenic acid, apigenin, and diosmetin) and the experimental conditions were analyzed together using principal component analysis (PCA) with the aim of simplifying the number of variables and identifying patterns and/or data groupings. The first two components explain 72.65% of the data variability.
In Figure 5a, the PCA biplot of the data from the 33 experimental design is shown, where the lines indicate the response variables (TPC, Ax, and polyphenol profile), and the points represent the observations (experimental conditions). The K-means clustering of the experimental conditions and response variables is also represented in Figure 5b.
The results show that TPC (1) exhibits a small distance from vanillin (10) and neohesperidin (12), indicating correlation and a similar behavior of the variables. TPC is also near hesperidin (11) and kaempferol (9). Catechin (5), rutin (7), and naringenin (13) are located at an angle close to 90° relative to TPC, which can be interpreted as a very low or null correlation. On the other hand, Ax (2) is very close to quercetin + luteolin (8), as these polyphenols have shown higher concentrations in extracts with a high antioxidant capacity.
Additionally, four different clusters were identified. It was observed that the response variables TPC, Ax, and the individual polyphenols analyzed were primarily associated with C. aurantium and C. sinensis, as well as the extraction conditions situated in clusters α and β, which could potentially enhance the extraction of these bioactive compounds and their antioxidant capacity.
Finally, clusters γ and δ exhibit unique characteristics and conditions that are likely not optimal for obtaining extracts rich in polyphenols, antioxidant capacity, or high concentrations of individual polyphenols of interest.

4. Discussion

Each analyzed CPex showed differences in the polyphenol profile, which varied according to the molar concentration of the NADES and its added water percentage. The characteristic and predominant polyphenols in the extracts obtained in this study were hesperidin and neohesperidin, both identified in the peels of C. aurantium and C. sinensis. Conversely, catechin was observed in higher concentrations in the peel of C. sinensis, while rutin was the predominant phenolic compound in the peel extract of C. limon.
Hesperidin (Hp) is a flavonoid characteristic of citrus fruits, classified as a glycosylated flavanone derived from the structure of hesperitin. Hp has been previously reported in the peel of C. aurantium from Yucatán by Covarrubias et al. [27]. The extraction was performed using organic solvents, with the highest concentration of Hp obtained using distilled water (292.00 ± 0.00 mg/100 g dry mass), followed by 50% ethanol (107.00 ± 2.00 mg/100 g dry mass) and 96% ethanol (90.00 ± 0.00 mg/100 g dry mass); all the extractions were assisted by ultrasound. These concentrations were significantly lower than those reported in this study, observing a highest concentration of 2000.37 ± 10.91 mg/100 g dry mass under conditions of 1:1 mol/mol (ChCl:Glu) and 60% of added water from C. aurantium peel. This difference is primarily attributed to the use of NADES instead of traditional organic solvents.
According to Liu et al. [28], the extraction of Hp from the peel of C. aurantium shows better yields when using NADES (244.00 ± 24.00 mg/100 g dry mass), which can be based on choline chloride and feature hydrogen bond donors such as amines, alcohols, and even sugars like glucose, compared to organic solvents like methanol (216.00 ± 15.00 mg/100 g dry mass), 80% methanol (190.00 ± 17.00 mg/100 g dry mass), and water (210.00 ± 23.00 mg/100 g dry mass). This is due to various factors, such as low concentrations (40%) of water percentage that reduces the solvent’s viscosity, allowing for proper mass transfer and the extraction of phenolic compounds like flavonoids. However, if the percentage reaches 80% of added water, it can disrupt the structure of the NADES, weakening the hydrogen bonds and decreasing the Hp extraction efficiency.
This same increase in added water would promote a highly polar environment, which, according to Pyrzynska [10] and Xu et al. [18], decreases the efficiency of Hp extraction due to its lower polarity behavior. Thus, the principle of “like dissolves like” would not apply. This explains the low concentrations of Hp found in the extracts of various citrus fruits, in this study, where a 70% water addition was used in NADES, compared to extracts with lower added water (50%, 60%).
Another factor in the extraction of hesperidin from citrus peels such as C. sinensis, as reported by Xu et al. [18], is the use of different ternary NADES (choline chloride-based, HBA) with various molar ratios and 25% added water. This study found that a neutral or slightly acidic pH (pH: 5.91–7.72) of the eutectic solvent resulted in higher extraction efficiency of Hp extraction. Under these conditions, the amorphous form of this flavanone is stabilized, improving its solubility [29]. In contrast, at a pH near 9, degradation of this metabolite is observed [18]. All factors involved in the extraction yield for Hp can be considered for the extraction of neohesperidin (Nhp) from citrus peels, as these metabolites are considered isomers due to their chemical structure. Both consist of hesperetin linked to a disaccharide; for Hp it is rutinose (Rhamnose-α1-6-glucose) and for Nhp it is neohesperidoside (Rhamnose-α1-2-glucose). However, Nhp might exhibit lower structural flexibility, potentially negatively affecting its extraction [30,31]. This behavior was observed in the extracts of C. aurantium and C. sinensis, where some extracts show similar concentrations of Hp, but was different in Nhp, verifying the data obtained from the PCA, where both metabolites presented a similar vector.
Another major polyphenol identified was rutin (quercetin-3-rutinoside), mainly in extracts from C. limon peels; this metabolite is a glycosylated flavonoid, meaning it is a quercetin molecule linked to a rutinose (glucose-α1-6-rhamnose) via a β-glycosidic bond at the 3rd carbon of the C-ring or pyran ring [32]. This coincides with the findings reported by Xi et al. [9], which indicate that the extract peel (methanol 80%:Dimethyl Sulfoxide [DMSO], 1:1 v/v, 12 h maceration) of C. limon contains a higher concentration of rutin (6.05 mg/100 g fresh peel) compared to other parts of the fruit (pulp > juice > seed). Nonetheless, the concentration is lower compared to hesperidin (331.5 mg/100 g fresh peel), which is opposite to the findings of this study by using NADES.
The concentrations of rutin found in the lemon extract can be attributed to the high solubility of this flavonoid in choline chloride-based NADES. Zang et al. [33] reported the extraction of rutin from Sophora japonica using various NADES preparations and added water combined with ultrasound. They observed that the increase in added water of a choline chloride-based NADES (choline chloride, 1:1 mol/mol) was from 10% to 20%, and the increase in extraction yield was from approximately 145 mg/g dry mass to 291 mg/g dry mass.
Regarding the extraction of total polyphenols, the highest concentration was observed in C. aurantium extracts (96.23 ± 0.83 mg/100 g DM), compared to C. sinensis and C. limon. This behavior was different from what has already been reported by Lagha-Benamrouche & Madani [6], where a maceration (80% methanol, 22 h) of C. aurantium and C. sinensis citrus peels (various varieties) was performed. The result was a TPC concentration in C. aurantium (bittersweet orange) peel extracts of 3162 ± 88.00 mg GAE/100 g DM, compared to C. sinensis with 2560 ± 23.00 mg GAE/100 g DM. The significant difference in these results is due to the extended extraction time when using maceration, which allows for greater diffusion of phenolic compounds into the solvent. However, the use of organic solvents and the required volume make it unsuitable for the extracts to be used for human consumption or at least to be considered GRAS (Generally Recognized as Safe) [34].
On the other hand, using NADES for the extraction of TPC from C. aurantium peel, it was observed that when using maceration of choline chloride-based NADES with a hydrogen donor such as 1,4-butanediol (1:3 mol/mol) and a water content of 49.9%, a concentration of 785.2 mg GAE/100 g DM was achieved [35]. This concentration does not align with that obtained in this study, with the use of glucose as the hydrogen donor at a ratio of 0.5 mol per 1 mol of choline chloride, 60% added water, and ultrasound-assisted extraction. These results are attributed to the affinity that choline chloride-based NADES exhibit with phenolic compounds, which is due to their ability to generate molecular stability. They present a slightly acidic pH, which, when combined with an appropriate percentage of water, can enhance diffusivity and solubility. Additionally, the 1,4-butanediol molecule has a linear configuration with two hydroxyl groups at the ends, unlike glucose, which has a cyclic structure. This allows 1,4-butanediol to generate NADES with greater stability among the hydrogen bond acceptor (HBA), water, and phenolic compounds [36]. Additionally, the use of ultrasound can further improve mass transfer by breaking down the plant matrix through the cavitation of ultrasonic waves and the differences in the concentration of TPC from these matrices reported in this same order, C. aurantium > C. sinensis > C. limon [18,28,37,38].
The antioxidant capacity observed in various citrus peel extracts was quite comparable, even though these extracts, such as those from C. limon, had significantly lower levels of total phenolic content (TPC) and individual polyphenols. This may be attributed to: (a) a more diverse polyphenol profile, creating a synergistic effect on DPPH radical inhibition [39]; and (b) the presence of other bioactive compounds such as vitamin C, limonoids, and coumarins, which are well-documented in citrus peels and possess an antioxidant capacity [40,41].
In contrast, extracts of C. aurantium and C. sinensis exhibited a less varied polyphenol profile but with high concentrations of hesperidin and neohesperidin, both reported to have a significant antioxidant capacity. Notably, neohesperidin showed a correlation with DPPH inhibition in both peels. According to Di Majo et al. [41], the glycosylation of hesperidin and neohesperidin could diminish their antioxidant capacity; however, it is comparable with molecules with a high antioxidant capacity like hesperitin.
Finally, all the extracts demonstrated a high capacity to inhibit the DPPH radical and exhibited elevated TPC concentrations. Most importantly, they showed high concentrations of polyphenols such as hesperidin, neohesperidin, rutin, and catechin, which have been reported for the prevention and treatment of various pathologies, including cardiovascular diseases, diabetes, hypercholesterolemia, and hypertension, among others [12,42,43].

5. Conclusions

The use of natural deep eutectic solvents (NADES) based on choline chloride and glucose, as a hydrogen bond donor, demonstrated high effectiveness in extracting phenolic compounds from the peels of various citrus fruits. These NADES showed a particular affinity for compounds such as hesperidin and neohesperidin in the peels of C. aurantium and C. sinensis, as well as rutin and catechin in the peels of C. limon, all of which are metabolites of interest for the pharmaceutical industry.
The addition of water to the eutectic solvents had a notable effect on the antioxidant capacity of the extracts. Furthermore, key factors, such as interactions among components (both double and triple), influenced the extraction of certain individual polyphenols, demonstrating that NADES can be modified to target the extraction of specific desired compounds.
Finally, extracts from C. aurantium and C. sinensis have great potential for use in the formulation of functional foods, nutraceuticals, or pharmaceuticals for the treatment of cardiovascular diseases. For this reason, it is recommended that extraction conditions be optimized to maximize their concentration.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr12102072/s1, Figure S1: Calibration curve of gallic acid for the determination of total polyphenols from citrus peel; Figure S2: Chromatogram of individual standards polyphenols mix. Numbering: 1 = gallic acid (RT = 0.6305 min, R2 = 0.9940); 2 = protocatechuic acid (RT = 1.3252, R2 = 0.9998 min); 3 = catechin (RT = 3.7071 min, R2 = 0.9993); 4 = chlorogenic acid (RT = 3.8682 min, R2 = 0.9993); 5 = coumaric acid (RT = 4.8438 min, R2 = 0.9995); 6 = rutin (RT = 6.2894 min, R2 = 0.9993); 7 = hesperidin (RT = 7.6707 min, R2 = 0.9996); 8 = quercetin + luteolin (RT = 8.5638 min, R2 = 0.9998); 9 = kaempferol (RT = 9.7681 min, R2 = 0.9991); Figure S3: Chromatogram of selected individual polyphenols analyzed in citrus peel extracts of (a) C. aurantium, (b) C. sinensis and (c) C. limon. Numbering: 1 = gallic acid; 2 = protocatechuic acid; 3 = catechin; 4 = chlorogenic acid; 5 = coumaric acid; 6 = rutin; 7 = hesperidin; 8 = quercetin + luteolin; 9 = kaempferol; Figure S4: Pearson Correlation Heatmap of the 33 Experimental Design for the Evaluation of Polyphenol Extraction Using Different NADES from (a) C. aurantium (b) C. sinensis and (c) C. limon. TPC = Total polyphenol content; Ax = Antioxidant capacity; GAc = Gallic acid; PAc = Protocatechuic acid; Chn = Catechin; ChAc = Chlorogenic acid; Rt = Rutin; Q + L = Quercetin + Luteolin; Kp = Kaempferol; Vn = Vanillin; Hp = Hesperidin; NeHe = Neohesperidin; Ng = Naringenin; Apg = Apigenin; Dt = Diosmetin. Table S1: Citric peel extracts polyphenol profile obtained using choline chloride-based NADES by ultrasound-assisted extraction. Table S2: Citric peel extracts TPC and antioxidant capacity obtained using choline chloride NADES based by ultrasound-assisted extraction.

Author Contributions

Conceptualization, M.O.R.-S. and I.M.R.-B.; methodology, K.A.A.-B. and A.L.-M.; software, M.O.R.-S. and K.A.A.-B.; validation, I.M.R.-B. and M.O.R.-S.; formal analysis, K.A.A.-B.; investigation, K.A.A.-B. and I.M.R.-B.; resources, M.O.R.-S. and I.M.R.-B.; data curation, M.O.R.-S. and I.M.R.-B.; writing—original draft preparation, K.A.A.-B., M.O.R.-S. and I.M.R.-B.; writing—review and editing, M.O.R.-S., K.A.A.-B. and I.M.R.-B.; visualization, M.O.R.-S.; supervision, M.O.R.-S. and I.M.R.-B.; project administration, M.O.R.-S.; funding acquisition, M.O.R.-S. and I.M.R.-B. All authors have read and agreed to the published version of the manuscript.

Funding

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.; which financed the project UEVISCOEUTECTIC No 2004500186, and the scholarship 661099 for Kevin Alejandro Avilés-Betanzos, financed by CONAHCYT.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Major individual polyphenols in the peel of (a) C. aurantium, (b) C. sinensis, and (c) C. limon. Where Ct = catechin; Rt = rutin; Q + L = quercetin + luteolin; Hp = hesperidin; Nhp = Neohesperidin. Different letters with the same color indicate a statistically significant difference for a single individual polyphenol (LSD, p < 0.05). ** Molar ratio of glucose per 1 mol of Choline chloride. Values are means ± SD (n = 3).
Figure 1. Major individual polyphenols in the peel of (a) C. aurantium, (b) C. sinensis, and (c) C. limon. Where Ct = catechin; Rt = rutin; Q + L = quercetin + luteolin; Hp = hesperidin; Nhp = Neohesperidin. Different letters with the same color indicate a statistically significant difference for a single individual polyphenol (LSD, p < 0.05). ** Molar ratio of glucose per 1 mol of Choline chloride. Values are means ± SD (n = 3).
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Figure 2. Total polyphenol content (TPC) and antioxidant capacity (Ax) of citrus peel extracts obtained using a choline chloride-based NADES with ultrasound. Different lowercase letters indicate statistically significant differences in TPC, while uppercase letters denote differences in antioxidant capacity. Different letters of the same color indicate a statistically significant difference (LSD, p < 0.05) for the total polyphenol content (blue) and antioxidant capacity (red); * Au = Citrus aurantium; Si = Citrus sinensis; Li = Citrus Limon; ** Molar ratio of glucose per 1 mol of Choline chloride. Values are means ± SD (n = 3).
Figure 2. Total polyphenol content (TPC) and antioxidant capacity (Ax) of citrus peel extracts obtained using a choline chloride-based NADES with ultrasound. Different lowercase letters indicate statistically significant differences in TPC, while uppercase letters denote differences in antioxidant capacity. Different letters of the same color indicate a statistically significant difference (LSD, p < 0.05) for the total polyphenol content (blue) and antioxidant capacity (red); * Au = Citrus aurantium; Si = Citrus sinensis; Li = Citrus Limon; ** Molar ratio of glucose per 1 mol of Choline chloride. Values are means ± SD (n = 3).
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Figure 3. Pareto charts of (a) total polyphenol content (TPC) and (b) antioxidant capacity (Ax). Where MR = Molar Ratio; WA = Added Water; CPe = Citrus peel.
Figure 3. Pareto charts of (a) total polyphenol content (TPC) and (b) antioxidant capacity (Ax). Where MR = Molar Ratio; WA = Added Water; CPe = Citrus peel.
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Figure 4. Pearson correlation heatmap of the 33 experimental design for the evaluation of polyphenol extraction from citrus peel using different NADES. TPC = Total polyphenol content; Ax = Antioxidant activity; GAc = Gallic acid; PAc = Protocatechuic acid; Chn = Catechin; ChAc = Chlorogenic acid; Rt = Rutin; Q + L = Quercetin + Luteolin; Kp = Kaempferol; Vn = Vanillin; Hp = Hesperidin; NeHe = Neohesperidin; Ng = Naringenin; Apg = Apigenin; Dt = Diosmetin.
Figure 4. Pearson correlation heatmap of the 33 experimental design for the evaluation of polyphenol extraction from citrus peel using different NADES. TPC = Total polyphenol content; Ax = Antioxidant activity; GAc = Gallic acid; PAc = Protocatechuic acid; Chn = Catechin; ChAc = Chlorogenic acid; Rt = Rutin; Q + L = Quercetin + Luteolin; Kp = Kaempferol; Vn = Vanillin; Hp = Hesperidin; NeHe = Neohesperidin; Ng = Naringenin; Apg = Apigenin; Dt = Diosmetin.
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Figure 5. (a) Principal component analysis (PCA) and (b) k-means of the 33 experimental design for the evaluation of polyphenol extraction from citrus peel using different NADES. Where letters are conditions of experiments (Table 1): A = #1; B = #2; C = #3; D = #4; E = #5; F = #6; G = #7; H = #8; I = #9; J = #10; K = #11; L = #12; M = #13; N = #14; O = #15; p = #16; Q = #17; R = #18; S = #19; T = #20; U = #21; V = #22; W = #23; X = #24; Y = #25; Z = #26; Z.a = #27 and numbers are variables response that correspond to the order specified at Tables S1 and S2, 1 = Total polyphenol content; 2 = Antioxidant capacity; 5 = catechin; 7 = rutin; 8 = quercetin + luteolin; 9 = kaempferol; 10 = vanillin; 11 = hesperidin; 12 = neohesperidin; 13 = naringenin. Clusters are shown in Greek letters.
Figure 5. (a) Principal component analysis (PCA) and (b) k-means of the 33 experimental design for the evaluation of polyphenol extraction from citrus peel using different NADES. Where letters are conditions of experiments (Table 1): A = #1; B = #2; C = #3; D = #4; E = #5; F = #6; G = #7; H = #8; I = #9; J = #10; K = #11; L = #12; M = #13; N = #14; O = #15; p = #16; Q = #17; R = #18; S = #19; T = #20; U = #21; V = #22; W = #23; X = #24; Y = #25; Z = #26; Z.a = #27 and numbers are variables response that correspond to the order specified at Tables S1 and S2, 1 = Total polyphenol content; 2 = Antioxidant capacity; 5 = catechin; 7 = rutin; 8 = quercetin + luteolin; 9 = kaempferol; 10 = vanillin; 11 = hesperidin; 12 = neohesperidin; 13 = naringenin. Clusters are shown in Greek letters.
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Table 1. Experimental design 33 for the evaluation of polyphenol extraction form citrus peel using different NADES.
Table 1. Experimental design 33 for the evaluation of polyphenol extraction form citrus peel using different NADES.
#EXPEncoded ValuesActual ValuesResponse Variable **
X1X2X3MRWACPe *
1−1−1−11:0.550C. aurantiumY1
20−1−11:150C. aurantiumY2
31−1−11:250C. aurantiumY3
4−10−11:0.560C. aurantiumY4
500−11:160C. aurantiumY5
610−11:260C. aurantiumY6
7−11−11:0.570C. aurantiumY7
801−11:170C. aurantiumY8
911−11:270C. aurantiumY9
10−1−101:0.550C. sinensisY10
110−101:150C. sinensisY11
121−101:250C. sinensisY12
13−1001:0.560C. sinensisY13
140001:160C. sinensisY14
151001:260C. sinensisY15
16−1101:0.570C. sinensisY16
170101:170C. sinensisY17
181101:270C. sinensisY18
19−1−111:0.550C. limonY19
200−111:150C. limonY20
211−111:250C. limonY21
22−1011:0.560C. limonY22
230011:160C. limonY23
241011:260C. limonY24
25−1111:0.570C. limonY25
260111:170C. limonY26
271111:270C. limonY27
Note: EXP = Experiment number; MR = Molar Ratio of glucose per 1 mol of choline chloride; WA = Percentage of added water to NADES; CPe = Citrus Peels; TPC = Total Polyphenol Content; GAE = Gallic Acid Equivalent; DM = dry mass; * C. = Citrus; ** The response variables are: (a) Polyphenol profile (mg/100 g dry mass); (b) Total polyphenol content (TPC, mg GAE/100 g dry mass); and (c) Antioxidant capacity (Ax, % DPPH Inhibition).
Table 2. p-values of the main factors and their interactions on the polyphenol profile in citrus peel extracts.
Table 2. p-values of the main factors and their interactions on the polyphenol profile in citrus peel extracts.
Individual PolyphenolMain Factors and Interactions
ABCABACBCABC
Gallic acid0.01030.11220.29650.05330.20241.00001.0000
Protocatechuic acid0.54600.63030.00030.12880.54730.66430.3163
Chlorogenic acid0.46020.58580.33880.01880.24260.55440.4694
Catechin0.28200.0428<0.00010.48600.75610.4020.8375
Rutin0.11860.0495<0.00010.19300.95390.44210.5229
Quercetin + Luteolin0.04500.43000.12990.10400.12980.65070.7122
Kaempferol0.00270.46170.6230.24980.16550.8540.8114
Vanillin0.38140.45210.01240.39080.9980.53480.0877
Hesperidin0.08930.02240.05440.04580.07620.04620.0235
Neohesperidin0.02970.7984<0.00010.32420.04340.72980.5838
Naringenin0.34830.0651<0.0001<0.00010.00780.10650.1957
Apigenin0.00100.0913<0.00010.780.77840.03310.1322
Diosmetin0.03940.25560.00460.32140.36210.90920.5612
Note: A = Added Water (%): B = Molar Ratio (mol/mol); C = Citric Peel; bold letters indicate that there is an effect (p < 0.05) from the main factors and/or their interactions on the concentration of individual polyphenols observed in the extracts.
Table 3. Linear correlation of phenolic compounds with antioxidant capacity of extracts from different citrus peels.
Table 3. Linear correlation of phenolic compounds with antioxidant capacity of extracts from different citrus peels.
Phenolic CompoundAntioxidant Capacity (DPPH)
C. aurantiumC. sinensisC. limon
TPC0.55640.10510.2280
Gallic acid0.0000−0.2372−0.3056
Protocatechuic acid0.0000−0.0315−0.0460
Catechin0.33950.5492−0.1918
Chlorogenic acid−0.03940.12110.0000
Rutin0.44200.5211−0.3178
Q + L0.77060.57470.6023
Kaempferol0.71100.43120.3271
Vanillin0.44960.80720.8487
Hesperidin0.1803−0.24670.0456
Neohesperidin0.74860.71290.6004
Naringenin0.33530.60910.6722
Apigenin−0.35620.3604−0.4075
Diosmetin0.2755−0.29510.7863
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MDPI and ACS Style

Ramírez-Sucre, M.O.; Avilés-Betanzos, K.A.; López-Martínez, A.; Rodríguez-Buenfil, I.M. Evaluation of Polyphenol Profile from Citrus Peel Obtained by Natural Deep Eutectic Solvent/Ultrasound Extraction. Processes 2024, 12, 2072. https://doi.org/10.3390/pr12102072

AMA Style

Ramírez-Sucre MO, Avilés-Betanzos KA, López-Martínez A, Rodríguez-Buenfil IM. Evaluation of Polyphenol Profile from Citrus Peel Obtained by Natural Deep Eutectic Solvent/Ultrasound Extraction. Processes. 2024; 12(10):2072. https://doi.org/10.3390/pr12102072

Chicago/Turabian Style

Ramírez-Sucre, Manuel Octavio, Kevin Alejandro Avilés-Betanzos, Anahí López-Martínez, and Ingrid Mayanin Rodríguez-Buenfil. 2024. "Evaluation of Polyphenol Profile from Citrus Peel Obtained by Natural Deep Eutectic Solvent/Ultrasound Extraction" Processes 12, no. 10: 2072. https://doi.org/10.3390/pr12102072

APA Style

Ramírez-Sucre, M. O., Avilés-Betanzos, K. A., López-Martínez, A., & Rodríguez-Buenfil, I. M. (2024). Evaluation of Polyphenol Profile from Citrus Peel Obtained by Natural Deep Eutectic Solvent/Ultrasound Extraction. Processes, 12(10), 2072. https://doi.org/10.3390/pr12102072

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