# Green and innovative extraction of polyphenols from Rhamnus alaternus using natural deep eutectic solvents and evaluation of their bioactivities

Amine Nekkaa, Akila Benaissa, Abd El Djalil Lalaouna, François Dupire, Arnaud Risler, Fabrice Mutelet, Laetitia Canabady-Rochelle

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Amine Nekkaa, Akila Benaissa, Abd El Djalil Lalaouna, François Dupire, Arnaud Risler, et al.. Green and innovative extraction of polyphenols from Rhamnus alaternus using natural deep eutectic solvents and evaluation of their bioactivities. Journal of Applied Research on Medicinal and Aromatic Plants, 2023, 35, pp.100503. 10.1016/j.jarmap.2023.100503. hal-04248451

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Green and Innovative Extraction of Polyphenols from
*Rhamnus alaternus* using Natural Deep Eutectic Solvents
and Evaluation of their Bioactivities

Amine Nekkaa, [a,b]* Akila Benaissa, [c] Abd El Djalil Lalaouna, [d] François Dupire, [e] Arnaud

Risler, [e] Fabrice Mutelet, [a] Laetitia Canabady-Rochelle [a]*

[a] Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France

[b] Process Engineering Laboratory for Sustainable Development and Health Products, National Polytechnic School of Constantine, Constantine 25000, Algeria

[c] Drug and Sustainable Development Laboratory (ReMeDD), Faculty of Process Engineering, University Salah Boubnider - Constantine 3, Constantine 25000, Algeria

[d] Laboratory of Analytical Chemistry, Faculty of Medicine, University Salah Boubnider, Constantine 3, Constantine 25000, Algeria

[e] Université de Lorraine, CNRS, L2CM, 54000 Nancy, France

* Corresponding authors

*Laetitia Canabady-Rochelle

E-mail: laetitia.canabady-rochelle@univ-lorraine.fr

Tel: +33(0) 3 72 74 38 86

*Amine Nekkaa

E-mail: amine.nekkaa25@gmail.com

Tel: +33(0) 6 12 49 67 91
25 Abstract

26 Phenolic compounds, naturally present in plants, are particularly relevant as nutraceutics for
27 their antioxidant activities. Here, choline chloride-based natural deep eutectic solvents
28 (NaDESs) were used for efficient extraction of total phenolic compounds (TPC) from leaves,
29 pods and roots of Rhamnus alaternus plant. In each extract, the polyphenols content and their
30 antioxidant activities were evaluated. More, their antibacterial activity was carried out against
31 Gram-positive and Gram-negative bacteria. The investigated extracts presented antioxidant and
32 antimicrobial activities. The leaves, pods and roots extracts obtained using the choline chloride-
33 glycerol (ChCl-Gly) mixture exhibited a high value of TPC comprised within 437.7 and 317.0 mg
34 GAE/100 g d.w. and showed the best antioxidant properties with ABTS radical scavenging
35 capacity (IC50: from 44.55 to 53.56 µg/mL), DPPH assay (IC50: from 26.36 to 118.23 µg/mL),
36 reducing activity (EC50: from 77.53 to 85.05 µg/mL) and iron chelation activity (EC50: from 91.73
37 to 108.7 µg/mL). In contrast, choline chloride-urea (ChCl-Ur) extracts showed a low antioxidant
38 capacity. Lastly, the best bioactive extracts were characterized using HPLC-MS/MS to determine
39 their bioactive compounds. The present study suggests that the DES-based method developed
40 was selective, efficient and sustainable for extraction of polyphenols.

41

42 **Keywords: Rhamnus alaternus**, natural deep eutectic solvents, green extraction, sustainable
43 process, bioactive compounds, antioxidant, antimicrobial activity.

44

45
46 **1. Introduction**

Recently, the green extraction techniques have become of utmost importance in research related to biomolecules production. Therefore, food, cosmetic and pharmaceutical industries operated a transition from conventional extraction to the green extraction processes, using alternative cost-effective and earth-friendly solvents (Nagarajan et al., 2019; Palos-Hernández et al., 2022). Considering environmental protection and collective awareness, consumers are more and more demanding for highly valued bio-based products (Bugge et al., 2016; Catone et al., 2021). Plants are an important source of various bioactive molecules notably polyphenolic compounds (Si et al., 2016), known for their pharmacological properties including antioxidant, antibacterial, anti-inflammatory and anticancer effects (Zhang et al., 2011).

Rhamnus alaternus, popularly called “Imlilesse”, belongs to the Rhamnaceae family and the Reynosia Genus. Widely distributed around the Mediterranean region, this plant has been widely consumed as infusion for thousands of years, notably for treating dermatological complications and for its hypotensive, laxative, purgative effect (Ben Ammar et al., 2007). Recent investigations have demonstrated the health benefits and high functional activities of bioactive compounds present in R. alaternus including antioxidant, antibacterial, antihyperlipidemic, antigenotoxic and antimutagenic activities (Bhouri et al., 2011; Tacherfiout et al., 2018). The main bioactive substances identified in various parts of R. alaternus are polyphenols, flavonoids, anthraquinones, coumarin and anthocyanins (Longo et al., 2005; Moussi et al., 2015). Polyphenols are the most active constituents in secondary metabolites of plants with high biological activities including antioxidant capacity (Quideau et al., 2011).
To date, the extraction of bioactive compounds from *R. alaternus* is largely carried out using conventional solid-liquid extraction methods involving organic solvents such as ethanol, acetone and ethyl acetate (Boussahel et al., 2015). However, the consumption of such solvents may contribute to environmental pollution due to their toxicity, volatility and flammability. In this regard, at the beginning of the 2000’s, Natural Deep Eutectic Solvents (NaDESs) emerged as alternative for green chemistry. NaDESs were simply prepared by mixing hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) (Trivedi et al., 2016). Characterized by their low toxicity, biodegradability, high solubility, biocompatibility, low vapor pressure and being eco-friendly, NaDESs are appropriate for substituting conventional solvents (Jeong et al., 2015). Furthermore, NaDESs were successfully applied for the extraction of bioactive compounds such as polyphenols, flavonoids and saponins from natural resources (Dai et al., 2013; Sillero et al., 2021).

To the author's knowledge, the extraction of bioactive compounds from *R. alaternus* has never been explored using Natural Deep Eutectic Solvents. Therefore, this study aimed to evaluate the efficiency of three NaDESs, in order to develop green and sustainable process for the extraction of high-value phenolic compounds, from various parts of *R. alaternus* (i.e., leaves, pods and roots) and to investigate the bioactive properties of collected extracts. To reach this aim, three NaDESs were investigated and constituted of choline chloride as HBA and of glycerol, ethylene glycol or urea, as HBD. Compared to conventional ethanol extraction, each extract was evaluated for its total polyphenol content (TPC) and its related bioactivities, notably its antioxidant and antimicrobial activities. Finally, the most promising NaDES extract in terms of TPC was characterized with LC-MS/MS analysis in order to identify its major constituents.
## 2. Material and methods

### 2.1. Plant materials, reagents, equipment and consumable

R. alaternus was collected from Beni Ourthilane region - Algeria (36°19′57″N,5°5′19″E) in July 2019. A voucher specimen (Ra-19-001) was kept in the laboratory of pharmacognosy, pharmacy department, University Salah Boubnider - Constantine 3, Algeria. Dried at room temperature under shadow, the leaves, pods and roots of R. alaternus were ground using a cutting mill (SM100 RETSCH, Retsch, Haan, Germany) to obtain a fine powder, stored in controlled atmosphere until use.

ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), DPPH (2,2-diphenyl-1-picrylhydrazyl) and Trolox® were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium phosphate dibasic dihydrate and sodium phosphate monobasic dihydrate were provided from VWR Chemicals (Leuven, Belgium). The ascorbic acid, potassium persulfate, ferrozine (3-(2-pycril)-5,6-diphenyl-1,2,4-triazine-4',4"-disulfonic acid sodium salt), iron (II) chloride, EDTA, Folin-Ciocalteu's phenol reagent (2N), gallic acid, potassium ferricyanide (III), iron (III) chloride, iron (III) ferrocyanide, trichloroacetic acid, sodium carbonate, ethylene glycol, choline chloride, urea and glycerol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and ethanol (Sigma-Aldrich) were of analytical grade. Water was purified by Arum® 611 water purification system (Sartorius, Göttingen, Germany). For the homogeneity of the unities, all concentrations in the manuscript were presented in mass/volume. CAS number and purity of each chemical used are given in the Supplementary data (Table S1). For antioxidant tests, all spectrophotometry experiments were carried out using Thermo Scientific Multiskan™ spectrophotometer and 96 wells-microplates (Thermo Scientific™, Roskild, Denmark).
111 **2.2. Solid-liquid extraction**

112 **2.2.1. Natural deep eutectic solvents preparation**

113 Three natural deep eutectic solvents (NaDESs) were selected for the extraction of phenolic
114 compounds from R. *alaternus's* leaves, pods and roots: choline chloride-glycerol (ChCl-Gly),
115 choline-chloride-ethylene glycol (ChCl-EG) and choline chloride-urea (ChCl-Ur). These NaDESs
116 were prepared as previously described (Tang & Row, 2020; Wan Mahmood et al., 2019). Briefly,
117 the hydrogen bond acceptor (HBA) i.e., choline chloride (ChCl) was mixed either with the
118 hydrogen bond donor (HBD) i.e., ethylene glycol (EG), glycerol (Gly) or with urea (Ur) at a molar
119 ratio 1:2. These solvents were prepared at 80 °C under stirring using a magnetic agitator (2mag
120 MIX 15; Merck, Darmstadt, Germany) until the formation of a transparent and homogenous
121 liquid (3 - 4 h).

122 The prepared NaDESs were mixed with water (8:2 v/v) to reduce their viscosity and change
123 their polarity in order to increase the extraction efficiency by improving the solubility of TPC in
124 the corresponding solvents (Chanioti & Tzia, 2018). Indeed, the high viscosity of NaDESs used
125 for extraction process can reduce the mass transport efficiency (Dai et al., 2013; Wei et al.,
126 2015). Yet, the water content mixed to NaDESs must not be too high since it could break the
127 hydrogen bonds within the NaDESs constituents (Vilková et al., 2020).

128 **2.2.2. Extraction process**

129 The phenolic compounds from various part of R. *alaternus* (i.e., leaves, pods and roots) were
130 separated by solid-liquid extraction (SLE). Briefly, 1 g of powder obtained from each part of the
131 plant was dissolved in 10 mL of solvent, constituted of one NaDES mixed with water in a 8:2
(v/v) ratio. Each SLE experiment was led for 24 h at 30 °C using a thermostated waterbath, and under stirring using a multipoint magnetic stirrer (2mag MIX 15, Merck, Darmstadt, Germany) set at 350 rpm. After extraction, each sample was centrifuged (Hettich universal 320, Sérézin du Rhône, France; 1008 g, 10 min) to remove any solid impurities. The collected supernatants were then filtered through 0.45 µm filter (Millex® Syringe Filter, PTFE, Merck KGaA, Darmstadt, Germany) in order to eliminate any remaining particles before further analysis. Each extract was named by its part of the plant (leaves, pods or roots) followed by the nature of the solvent used for SLE extraction (ChCl-Gly, ChCl-EG, ChCl-Ur, or Ethanol). After extraction, each extract was diluted by successive tenfold dilution in ultrapure water until the final dilution factor of 1:1000 (v/v) and was used as initial extract for further analysis (polyphenols identification, antioxidant and antimicrobial activity, and HPLC/MS/MS analysis).

## 2.3. Determination of the total phenolic content

For all investigated extracts, the total phenolic content (TPC) was determined by Folin-Ciocalteu method adapted to a microplate assay (Singleton & Rossi, 1965). Then, 220 µL of ultrapure water, 5 µL of each diluted extract, 15 µL of Folin-Ciocalteu reagent (10 % v/v) and 60 µL of aqueous sodium carbonate (7.5 % w/v) were mixed in each well. Finally, the microplate was stored in the dark for 60 min at ambient temperature, and analysed at 765 nm by spectrophotometry. The absorbance of each sample was measured five times.

The total phenolic compound was determined using a calibration curve performed with gallic acid as standard, in a concentration range varying from 0 to 35 µg/mL ($R^2$ = 0.998). The results were expressed as mg gallic acid equivalent (mg GAE) per 100 grams of plant powder (d.w.).
## 2.4. Determination of antioxidant capacity of the extracts

### 2.4.1. Radical scavenging activity

**ABTS radical scavenging activity.** To evaluate the ability of various extracts from *R. alaternus* to scavenge the ABTS⁺ radical, the method described by Re et al. (1999) was followed. Initially, 16.55 mg potassium persulfate was added to 25 mL of the ABTS solution (7 mM) prepared in ultrapure water, allowing the mixture to stand for 12 to 16 h in the dark, at ambient temperature. The concentrated ABTS⁺ solution was then diluted with 4 mM phosphate buffer pH 7.4 to reach an absorbance value of 0.7 ± 0.02 at 734 nm. Then, 150 µL of ABTS⁺ diluted solution was added to 150 µL of extracts, previously prepared at various concentrations (0 - 120 µg/mL) in the phosphate buffer. After 10 min of incubation, the absorbance was measured at 734 nm. Each concentration was repeated five times. The Trolox® was investigated as reference from 0 to 25 µg/mL in the 4 mM phosphate buffer. From the absorbance measurement, the radical scavenging activity (%) was calculated as follows (Equation 1) in order to plot a calibration curve (Radical scavenging activity (%) vs. Trolox® concentration):

$$ \text{Radical scavenging activity (\%)} = \frac{A_{734 (control)} - A_{734 (sample)}}{A_{734 (control)}} \times 100 \quad (1) $$

Where $A_{734 (control)}$ is the absorbance of the control (initial ABTS⁺ radical solution; the sample volume was replaced by ultrapure water), and $A_{734 (sample)}$ is the absorbance of the remaining radical in the presence of biological extracts or Trolox®.

**DPPH radical scavenging activity.** The ability of various extracts from *R. alaternus* to scavenge the 2,2-diphenyl-1 picrylhydrazyl free radical was evaluated by the DPPH *in vitro* assay similarly to Brand-Williams et al. (1995), with slight modifications. After extraction, each extract was first diluted in ultrapure water. Briefly, 150 µL of each diluted extract after solid-liquid extraction
(final investigated concentration: 0 to 100 µg/mL) was added to 150 µL of the methanolic DPPH° solution (40 µg/mL) daily prepared. Each investigated concentration was repeated 5 times. The mixture was homogenized vigorously using a micropipette for 1 min and stored in the dark at ambient temperature for 30 min. Then, the sample absorbance was read at 517 nm by spectrophotometry. Ascorbic acid (150 µL) was investigated from 5 to 100 µg/mL as standard for plotting the calibration curve. The radical scavenging activity (%) of each extract or standard solution was calculated as follows (Equation 2):

$$182 \quad \text{Radical scavenging activity (\%)} = \frac{A_{517 (control)} - A_{517 (sample)}}{A_{517 (control)}} \times 100 \quad (2)$$

Where $A_{517 (control)}$ was the absorbance of the blank DPPH° reagent in the presence of methanol and $A_{517 (sample)}$ the absorbance of the DPPH° reagent solution mixed either with the ascorbic acid or the plant extracts.

The Inhibitory Concentration 50 (IC₅₀) – the concentration that scavenges 50 % of DPPH° free radicals (µg/mL) - was calculated for all R. alaternus extracts and compared with the one of ascorbic acid.

### 2.4.2. Reducing power

The reducing capacity of each biological extract was investigated by the method described by Oyaizu. (1986), with slight modifications. Briefly, 70 µL of various samples extracted and diluted in microplate from (0 to 250 µg/mL) were prepared in phosphate buffer (0.2 M, pH 6.6) and then added with 35 µL of potassium ferricyanide (K3FeCN6; 1 % w/v). After 20 min of incubation at 50 °C, 135 µL of ultrapure water was added in each well, followed by 33 µL of trichloroacetic acid (10 % w/v) and 27 µL of iron (III) chloride (FeCl₃; 0.1 % w/v). After 10 min of incubation at 25 °C, the absorbance was measured at 700 nm. Ascorbic acid (70 µL) was
investigated as positive control from 5 to 70 µg/mL. Each concentration was repeated 5 times.

The reducing capacity of the various extracts was evaluated as follows:

$$ Reducing capacity (\%) = 100 - \left( \frac{A_{700 (control)} - A_{700 (sample)}}{A_{700 (control)}} \right) \times 100 \quad (3) $$

Where: A<sub>700 (control)</sub> is the absorbance of a 66 µM-Prussian blue solution in the absence of ascorbic acid, which was experimentally determined at 0.83 absorbance value at 700 nm,

And A<sub>700 (sample)</sub> is the sample or ascorbic acid absorbance at 700 nm.

### 2.4.3. Iron (II) chelating activity

The ability of various extracts from R. alaternus to chelate Fe<sup>2+</sup> was evaluated using the method described by Canabady-Rochelle et al. (2015) adapted from Decker & Welch. (1990). Briefly, 7.5 µL of ferrous chloride solution (FeCl<sub>2</sub>, 253.4 µg/mL) was mixed with 277.5 µL of extract diluted in order to investigate various calculated concentrations (from 0 to 300 µg/mL) in ultrapure water. After 3 min of incubation at 25 °C, 15 µL of ferrozine solution (5 mM) was added in each well. The microplate was shaken using the spectrophotometer agitator, and then incubated for 10 min at 25 °C before absorbance measurement at 562 nm. EDTA was investigated as reference from 0 to 10 µg/mL. Each sample and standard were repeated (n = 5). The iron-chelating capacity (%) was calculated as follows (Equation 4):

$$ Iron - chelation capacity (\%) = \frac{A_{562 (control)} - A_{562 (sample)}}{A_{562 (control)}} \times 100 \quad (4) $$

Where the blank A<sub>562 (control)</sub> and A<sub>562 (sample)</sub> correspond to the absorbance of ultrapure water in the absence of sample and to the absorbance of sample, both mixed with all reagents.
216 **2.5. Antimicrobial activity of the plant extracts**

217 **2.5.1. Antimicrobial test**

218 To evaluate the antimicrobial activity of the samples and NaDESs, extracts of *R. alaternus* and
219 solvents alone were tested against two Gram+ bacteria (i.e., *Staphylococcus aureus* ATCC 29213
220 (*Sa*) and *Enterococcus faecalis* ATCC 29212 (*Ef*)) and three Gram- bacteria strains (i.e.,
221 *Pseudomonas aeruginosa* ATCC 27853 (*Pa*), *Escherichia coli* ATCC 25922 (*Ec*) and *Klebsiella*
222 pneumoniae ATCC 700603 (*Kp*)). All strains were obtained from the L2CM laboratory (UMR 7053
223 CNRS-UL, France). The minimum inhibitory concentrations (MIC) were determined (Section
224 2.5.3.).
225

225 **2.5.2. Preparation of the inoculum**

226 Bacteria cells were grown 18 ± 2 h at 35 °C in Mueller-Hinton broth, cation-adjusted (MHB-CA).
227 They were then centrifugated (3000 g, 5 min) and collected pellets were diluted in 10 mL NaCl
228 0.85 % (w/v). The absorbance was recorded at 540 nm with a microplate reader (Thermo
229 Multiskan FC). Bacteria cells were diluted in MHB-CA to 0.5 McFarland turbidity and further
230 diluted to 1:100 to reach a final concentration of 1 [0.4 - 1.6].10⁶ CFU/mL.

231 **2.5.3. MIC determination**

232 The MIC- defined as the lowest concentration of extract with no visible bacterial growth – was
233 determined using the microdilution broth method in 96-well microplates (Eloff, 1998) following
234 the standard (ISO 20776-1, 2019) guidelines with some adaptations.

235 First, solvents (Ethanol and NaDESs) or extracts were diluted in MHB-CA (final volume: 50 µL).

236 Then, 50 µL of bacterial inoculum were added to each well for a total volume of 100 µL per
237 microplate well (final bacterial concentration: 5 [2 – 8].10⁵ CFU/mL). Growth controls (i.e.,
238 bacteria + MHB-CA), sterility control (medium without bacteria), and product control (medium
239 without bacteria with solvents (i.e., Ethanol, ChCl-Gly, ChCl-EG, ChCl-Ur) or extracts) were
240 performed. Each test was repeated 8 times on the microplate. Plates were then incubated 18 ±
241 2 h at 35 °C. The MIC was expressed here as the % of extract (v/v) in the wells.
242

242 First, solvents were investigated alone at 25, 20, 15, 10, 5, 2.5, 1.25, 0.6 and 0.3 % to determine
243 the maximal concentration to use without affecting the cell growth. Then, the extracts were
244 studied from 1 % to 0.004 % (two-fold serial dilution) in a first set of experiments (n = 8). Last,
245 the remaining extracts were investigated at 10, 5, 1, 0.5 and 0.25 % (n = 2 only) in a second
246 series of experiments.
247

247 **2.6. Characterization of ChCl-Gly extracts by HPLC-MS/MS**

248 The *R. alaternus* leaves, pods and roots extracts using ChCl-Gly were analyzed by mass
249 spectrometry (Bruker Daltonics – micrOTOF – Q LCMS/MS, Bremen, Germany) system. The
250 separation was performed using Hypersil gold C18 column (100 mm × 2.1 mm, 3 µm) at 40 °C.
251 The mobile phase was composed of water containing 0.1% formic acid (solvent A) and
252 acetonitrile 100 % (solvent B). The elution program was set as follows: 95 % A/ 5 % B between 0
253 and 5 min, 1% A/ 99 % B between 5 and 40 min, stay at 99 % B between 40 and 45 min and 95
254 % A/ 5 % B between 45 and 55 min to reach the baseline. Before injection, all extracts were
255 filtered through 0.45 µm membrane filter (J.T. Baker®, Strasbourg, France). Then, 2 µL of each
256 one were injected at 200 µL/min flow rate during the whole experiment.
257
The MS analysis was performed with negative and positive ion modes, scanned m/z from 100 to 1000 for the determination of phenolic compounds. Each data was calibrated with Sodium formate at 1 mM in water/isopropanol 1/1. The ESI source conditions were as follows: drying gas at 5 L/min, the nebulizer pressure at 4 bar, the capillary voltage at 4500 V, and the capillary temperature was set at 190 °C. Data acquisitions were executed in autoMSMS mode at 20 eV collision energy.

## 2.7. Statistical analysis

Data were collected and expressed as the mean ± standard deviation of three independent experiments and analysed for statistical significance from control, using the Dunnett test SPSS 11.5 Statistics Software (SPSS, Chicago, IL, USA). The criterion for significance was set at $p < 0.05$. The $IC_{50}$ values, from the in vitro data, were calculated by regression analysis.

# 3. Results and discussion

## 3.1. Determination of total phenolic content

In this study, three different NaDESs (i.e., ChCl-Gly, ChCl-EG, and ChCl-Ur) were investigated for the extraction of TPC from leaves, pods and roots of *R. alaternus* plant and compared to ethanol as conventional solvent and used as reference (Fig. 1).

Fig. 1 shows the TPC for various *R. alaternus* extracts according to the nature of the NaDES used in comparison to ethanol. Whatever the plant part (i.e., leaves, pods and roots), the extracts obtained using NaDESs contains significantly more polyphenols (from 437.7 to 279.8 mg GAE/100 g d.w.) than those obtained using ethanol (from 250 to 222.6 mg GAE/100 g d.w.). Furthermore, the TPC are significantly more concentrated in leaves samples than in pods and
finally roots, whatever the solvents studied in this work. Such trend is the highest for ChCl-Gly and the lowest for ethanol. Comparing the solvents efficiency on extraction process, the ChCl-Gly is more efficient than other solvents studied in this work. In ChCl-Gly, the highest TPC values are determined at 437.7, 361.3 and 317.0 mg GAE/100 g d.w. for leaves, pods and roots, respectively. The ChCl-Gly is followed by ChCl-EG (TPC varying from 344.2 to 304.8 mg GAE/100 g d.w.) and ChCl-Ur (TPC varying from 346.2 to 279.8 mg GAE/100 g d.w.), for leaves, pods and roots, respectively. The lowest amounts of polyphenols determined for the ethanol extracts are of 250.0, 237.8 and 222.6 mg GAE/100 g d.w. for leaves, pods and roots, respectively. In general, this highest extraction efficiency of TPC with NaDESs comparing with ethanol is related to the H-bonding interactions between the polar compounds (polyphenols) and NaDESs as previously reported (Duan et al., 2016). Duan and co-workers extracted polyphenols from Chinese herbal using various deep eutectic solvents. Also, our results are in agreement with a recent study reported in the literature by Islamčević Razboršek et al. (2020) in which the extraction of phenolic compounds from *Aronia melanocarpa* plant using NaDESs is studied. This work found higher extraction yield of phenolic compounds with NaDESs than those observed with ethanol.

[Fig. 1]

## 3.2. Determination of antioxidant capacity of the extracts

### 3.2.1. Radical scavenging activity

**ABTS radical scavenging activity.** The capacity of extracts to scavenge ABTS free radicals was evaluated in this study to determine extracts with high radical scavenging capacity (lowest IC₅₀ values). As shown in Fig. 2A, the NaDESs are more efficient than the ethanol, since they show
300 the lowest IC₅₀ values. The highest radical scavenging activity is detected with ChCl-Gly extracts
301 followed by ChCl-EG extracts > ChCl-Ur extracts > Ethanol extracts, respectively. When solvents
302 are compared, extracts obtained using ChCl-Gly are more active with an IC₅₀ values ranging
303 from 44.55 to 53.56 µg/mL, followed by ChCl-EG with IC₅₀ values varying from 55.47 to 58.71
304 µg/mL, and finally ChCl-Ur extracts with IC₅₀ ranging from 60.22 to 63.72 µg/mL. In comparison
305 to NaDESs solvents, ethanol extracts present the lowest efficiency in terms of radical
306 scavenging activity with an IC₅₀ values comprised within 66.28 and 69.56 µg/mL. Whatever the
307 plant's part (i.e., leaves, pods or roots), the values of IC₅₀ and The Trolox Equivalent Antioxidant
308 Capacity (TEAC) values are close in the three parts (Fig. 2A, Table S2). These results confirm that
309 the phenolic compounds extracted from different parts of R. alaternus using NaDESs present an
310 excellent antioxidant activity.

311

[Fig 2.]

312 **DPPH radical scavenging activity.** The DPPH radical scavenging capacity was evaluated for the
313 different solvent extracts and for various parts of plant (leaves, pods and roots; Fig. 2B). The
314 IC₅₀ values range from 26.36 µg/mL for leaves extracted by ChCl-Gly (the highest activity) to
315 118.23 µg/mL for pods extracted using ChCl-Ur (the lowest activity) (Table S3). For ChCl-EG
316 extracts, the IC₅₀ values range from 51.36 to 68.81 µg/mL, with DPPH radical scavenging activity
317 rather similar to the ethanol extracts. On the whole, the IC₅₀ values increase in the following
318 order: ChCl-Gly extracts < ChCl-EG extracts < Ethanol extracts < ChCl-Ur extracts (Fig. 2B),
319 meaning the highest and the lowest DPPH radical scavenging activity are determined for the
320 ChCl-Gly and the ChCl-Ur extracts, respectively. All extracts obtained by using NaDESs present
321 stronger DPPH⁺ radical scavenging capacity than those obtained with ethanol, except ChCl-Ur.

321
Our results are similar to the study of Oliveira and co-workers (Oliveira et al., 2021) on the radical scavenging capacity of *Curcuma longa* L. plant. Former authors showed that *Curcuma longa* L. leaves extracts were better hydroxyl radical scavengers after deep eutectic solvents extraction notably for choline chloride / lactic acid (1:2) or choline chloride / acetic acid (1:2) in comparison to ethanol, used as reference solvent.

### 3.2.2. Reducing power

The reducing power of a biological extract indicates its ability to reduce the Fe³⁺/ferricyanide complex to the Fe²⁺ form, which is checked by measuring the formation of Perl's Prussian blue at 700 nm (Pan et al., 2010). The various NaDESs extracts from *R. alaternus* (i.e., leaves, pods and roots) were evaluated to determine the lowest EC₅₀ values (Fig. 3, panel A). The nature of solvent and the part of the plant also influence significantly the reducing capacity. The EC₅₀ values of the investigated extracts increases which mean the decreases of reducing power in the following order: ChCl-Gly extracts > Ethanol extracts > ChCl-EG extracts > ChCl-Ur extracts. Compared to other solvent extracts, the ChCl-Gly extracts give the highest reducing power, corresponding to the lowest EC₅₀ concentrations ranging from 77.53 to 85.05 µg/mL. The lowest reducing power was obtained in ChCl-Ur extracts, with EC₅₀ values ranging from 395.97 to 542.71 µg/mL (Table S4). This analysis suggests that ChCl-Gly extracts are the most efficient ones in terms of reducing power. While, according to the study of Ozturk and co-authors were studied the extraction of polyphenols from orange peel waste using various DESs, this may be related to a more efficient extraction of polyphenol compounds with this former NaDES (Ozturk et al., 2018). Indeed, the reducing power of *R. alaternus* may be due to their TPC presence,
343 which may contribute to the antioxidant capacity by different reaction mechanisms (Duh et al.,
344 2001).

345 [Fig. 3]

346 **3.2.3. Iron (II) chelating activity**

347 Plant extracts enriched in polyphenols and other bioactive compounds may also present metal
348 chelation properties including iron chelating capacity; this latter activity plays a central role in
349 antioxidative mechanisms while inhibiting the formation of free radicals (Halliwell, 2007;
350 Yoshino & Murakami, 1998).

351 The capacity of R. *alaternus* extracts to chelate Fe²⁺ enabled the calculation of EC₅₀ value, which
352 is the maximal effective concentration of extracts to obtain 50 % of iron chelating activity (Table
353 S5). The decrease of EC₅₀ values meaning an increase the iron chelating activity. Hence, the
354 iron-chelation properties of the various extracts (Fig. 3, panel B) evolve in the following order:
355 ChCl-Gly extracts > Ethanol extracts > ChCl-EG extracts > ChCl-Ur extracts. The ChCl-Gly extracts
356 have the highest iron-chelating activity (smallest EC₅₀) with values ranging from 91.73 to 108.7
357 µg/mL. Besides, the ChCl-Ur exhibits the lowest iron-chelating properties with EC₅₀ values
358 varying from 138.20 to 215.70 µg/mL. Furthermore, the Ethanol extracts chelate Fe²⁺ more
359 efficiently than ChCl-EG and ChCl-Ur ones. Hence, the ability of R. *alaternus* extracts to complex
360 iron is attributed to the presence of polyphenols, as previously reported in other plant study
361 (Oliveira et al., 2021). Indeed, extracts from various parts of *Curcuma longa* L. (i.e., leaves,
362 rhizome and flowers) present chelating properties after solid-liquid extraction carried out using
363 DESs choline chloride-based solvents or ethanol as reference. In accordance with ABTS⁺, DPPH
364 radical scavenging capacity and reducing power tests, ChCl-Ur is the less efficient solvent for
365 producing extracts endowed with the radical scavenging capacity and reducing capacity.

366 **3.3. Antimicrobial activity**

367 First, the antimicrobial activity of the solvents itself (Ethanol or NaDESs) was evaluated alone
368 against five bacterial strains of clinical interest. Although few MICs were obtained, in most
369 cases, the bacterial growth was affected by the solvents, even without reaching a MIC (Table
370 S6). Then, the microplates wells were gently homogenized and the OD540 was recorded. We
371 arbitrarily defined that a bacterial growth showing an optical density OD ≥ 75 % of the OD of
372 the growth control indicates few or no effect of the solvent. In some case, 1.3 % of the solvent
373 must be used as maximum value to avoid a negative effect on the bacterial growth (Table S7).
374 Our results also show the solvent negative effect on the bacterial growth in agreement with the
375 literature (Percevault et al., 2021). Former authors demonstrated that DES (choline chloride /
376 ethylene glycol (1:2) and betaine / citric acid (2:3)) are appropriate for the storage of bioactive
377 compounds to avoid microbial contamination, which could be promising for food and
378 pharmaceutical applications.

379 Therefore, the MIC of the extracts was determined against the bacteria with at most 1 % of
380 extract, in order to avoid any interference with the solvent activity. Only the extract of Roots-
381 ChCl-Gly showed a MIC at 0.5 % against E. faecalis. All other combinations showed no visible
382 effect. Hence, in a second series of experiments, higher concentrations of extracts (10, 5, 1, 0.5,
383 0.25 %) were investigated with care of the solvent potential effect (Table 1).
Among the Gram- bacteria, only the Leaves-ChCl-Ur extract has an effect on *E. coli* (MIC reached at 10 % without solvent interference). In three cases (i.e., Roots-ChCl-Gly vs. *P. aeruginosa*, Leaves-ChCl-Ur and Pods-ChCl-Ur against *K. pneumoniae*), a MIC value of 10 % is obtained, but at such concentration, the solvent fragilizes the cells (< 75 % of the growth control determined for the solvent alone) and is thus, a result of impaired bacteria.

On the other hand, the two Gram+ bacteria are more sensitive to the extracts. The Leaves-ChCl-Ur and Pods-ChCl-Gly extracts vs. *S. aureus*, and the Pods-Ethanol and Pods-ChCl-Gly extracts vs. *E. faecalis* show a MIC at 10 %, yet with solvent interference. Only the extracts Leaves-Ethanol, Leaves-ChCl-Gly and Leaves-ChCl-EG show no effect on *E. faecalis*. While, on all other combinations, MICs are defined between 0.5 and 10 %, without solvent interference on this strain.

Roots extracts (whatever the solvent) seems to have the best antimicrobial activity, with MICs on both Gram+ bacteria (i.e., *Staphylococcus aureus* and *Enterococcus faecalis*) without solvent interference. This antimicrobial activity towards Gram+ bacteria may be related to their absence of outer membrane, which could render them less resistant against extracts rich in bioactive molecules compared to Gram- bacteria (Brul & Coote, 1999). Our results are in agreement with Zhao and colleagues (Zhao et al., 2015), who found that extracts from *Sophora japonica* using DESs choline chloride-based (i.e., choline chloride / ethylene glycol (1:2), choline chloride / glycerol (1:2) and choline chloride / urea (1:2)) presented antimicrobial properties against gram+ bacteria (*Staphylococcus aureus*).
405

406

407

408

409

<table><caption>**Table 1.** Minimum inhibitory concentrations of the extracts against the five bacterial strains.</caption><thead><tr><th rowspan="2">Extract</th><th colspan="2">Gram+</th><th colspan="3">Gram-</th></tr><tr><th><i>Staphylococcus aureus</i> (Sa)</th><th><i>Enterococcus faecalis</i> (Ef)</th><th><i>Pseudomonas aeruginosa</i> (Pa)</th><th><i>Escherichia coli</i> (Ec)</th><th><i>Klebsiella pneumoniae</i> (Kp)</th></tr></thead><tbody><tr><td>Leaves-Ethanol</td><td>5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Leaves-ChCl-Gly</td><td>5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Leaves-ChCl-EG</td><td>5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Leaves-ChCl-Ur</td><td>10%<sup>++</sup></td><td>10%<sup>+</sup></td><td>≥ 10 %</td><td>10%<sup>+</sup></td><td>10%<sup>++</sup></td></tr><tr><td>Pods-Ethanol</td><td>10%<sup>+</sup></td><td>10%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Pods-ChCl-Gly</td><td>10%<sup>++</sup></td><td>10%<sup>++</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Pods-ChCl-EG</td><td>5%<sup>+</sup></td><td>10%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Pods-ChCl-Ur</td><td>5%<sup>+</sup></td><td>5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>10%<sup>++</sup></td></tr><tr><td>Roots-Ethanol</td><td>5%<sup>+</sup></td><td>5%<sup>+</sup></td><td>10%<sup>*</sup></td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Roots-ChCl-Gly</td><td>5%<sup>+</sup></td><td>0,5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Roots-ChCl-EG</td><td>5%<sup>+</sup></td><td>5%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr><tr><td>Roots-ChCl-Ur</td><td>5%<sup>+</sup></td><td>10%<sup>+</sup></td><td>≥ 10 %</td><td>≥ 10 %</td><td>≥ 10 %</td></tr></tbody></table>

410

(+): weak or no effect of the solvent; (++): combined effect of the solvent; (*): strong effect of the solvent; ≥ 10 %: no effect.

411

412

**3.4. Characterization of ChCl-Gly extracts by HPLC-MS/MS**

413

414

415

The qualitative determination of bioactive compounds extracted from three different extracts of *R. alaternus* (i.e., leaves, pods and roots) with ChCl-Gly - corresponding to the best extract in term of biological activities - were analyzed by HPLC-MS/MS (Table 2). Twenty-one compounds
416 were determined in the extracts. Among them, twelve molecules were newly identified in this
417 plant comparing to the literature (Nekkaa et al., 2021), including tyrosine (2.5 min),
418 dihydroquercetine pentoside glucoside (9.1 min), tryptophane (9.8 min), aloesin (11.3 min),
419 aloesol 7-glucoside (11.5 min), tectoridin (12.8 min), glucuronic acid (12.6/13.7 min), 7-hydroxy-
420 3-(2-hydroxypropyl)-5-methylisochromen-1-one (15.3 min), aloesol (15.4 min), piperonylidene
421 acetone (16.4 min), (R)-linalyl beta-vicianoside (18.1 min), neryl rhamnosyl-glucoside (18.3
422 min). Furthermore, six compounds i.e., aloesin (11.3 min), aloesol glucoside (11.6 min), aloesol
423 7-glucoside (11.5 min), 7-hydroxy-3-(2 hydroxypropyl)-5-methylisochromen-1-one (15.3 min),
424 aloesol (15.4 min) and emodin (25.1 min) were common to the three extracts collected from
425 the various part of plant. Based on Table 2, the leaves provide the richest part of R. *alaternus* in
426 term of number of bioactive compounds identified in this extraction comparing to other parts,
427 and is followed by pods than roots. On the other hand, these finding evidences that the R.
428 *alaternus* plant presents total phenolic compounds with high antioxidant effect (i.e.,
429 dihydroquercetine pentoside glucoside, kaempferol, dihydroquercetin 3-O-glucoside,
430 kaempferide 3-glucoside and tectoridin), and organic acids (i.e., malic acid, citric acid and
431 glucuronic acid), in addition to amino acids including tyrosine and tryptophan.

432 The ChCl-Gly (1:2) may have a better affinity towards bioactive compounds through hydrogen
433 bonding interactions, which would favour the antioxidant effects of extracts.

4. **Conclusion and Perspectives**

435 For the first time, three choline chloride-based NaDESs (choline chloride-glycerol (ChCl-Gly),
436 choline-chloride-ethylene glycol (ChCl-EG) and choline chloride-urea (ChCl-Ur)) were used for
437 the extraction of bioactive compounds from various parts of R. *alaternus* plant (i.e., leaves,
438 pods and roots). Among all the studied extracts, the ChCl-Gly extracts from R. *alaternus'* leaves
439 presents the best content of total phenolic compounds and provides the highest antioxidant
440 activity compared to other investigated solvents such as NaDESs and ethanol, used in
441 reference. All NaDESs extracts show interesting antimicrobial activity with significant inhibitory
442 potential against Gram+ strains. Furthermore, the leaves, roots and pods extract carried out
443 using ChCl-Gly were characterized and quantified by HPLC-MS/MS, which confirms that the
444 phenolic compounds and flavonoids are the main antioxidant compounds. The *in vitro*
445 biological analysis of extracts suggests that ChCl-Gly is an efficient solvent for the extraction of
446 secondary metabolite from leaves, pods and roots of R. *alaternus*. These bio-based sustainable
447 and green processes present a great potential for pharmaceutical, cosmetic and food
448 applications.

449

450

451
<table><caption>**Table 2.** Identification of bioactive compounds present in leaves, pods and roots of *R. alaternus* extracted using NaDES (ChCl-Gly) analysed by LC-MS/MS.</caption><thead><tr><th rowspan="2">N°</th><th rowspan="2">Compound name</th><th rowspan="2">Molecular formula</th><th rowspan="2">tr (min)</th><th colspan="3">[M + H]<sup>-</sup></th><th colspan="3">[M + H]<sup>+</sup></th><th rowspan="2">plant part</th></tr><tr><th>Measured m/z (Da) [M + H]<sup>-</sup></th><th>Error (ppm)</th><th>MS/MS Fragmentation</th><th>Measured m/z (Da) [M + H]<sup>+</sup></th><th>Error (ppm)</th><th>MS/MS Fragmentation</th></tr></thead><tbody><tr><td>1</td><td>Dihydroquercetin pentoside glucoside</td><td>C<sub>26</sub>H<sub>30</sub>O<sub>16</sub></td><td>9.1</td><td>597.151</td><td>8.5</td><td>417.085 [M-hexose-H]<sup>-</sup><br>285.038 [M-hexose - pentosyl - H]<sup>-</sup></td><td>599.162</td><td>1.6</td><td>n.d</td><td>leaves</td></tr><tr><td>2</td><td>Kaempferol</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>6</sub></td><td>19.3</td><td>285.039</td><td>3.5</td><td>239.032 [M-HCOOH-H]<sup>-</sup><br>229.049 [M-CO-CO-H]<sup>-</sup><br>185.058 [M-CO-CO-CO<sub>2</sub>+H]<sup>+</sup></td><td>287.056</td><td>3.5</td><td>285.052 [M-CHO+H]<sup>+</sup><br>241.051 [M-HCOOH+H]<sup>+</sup><br>231.072 [M-CO-CO+H]<sup>+</sup><br>213.050 [M-CO-CO-H<sub>2</sub>O+H]<sup>+</sup><br>185.058 [M-CO-CO-H<sub>2</sub>O-CO+H]<sup>+</sup></td><td>Pods</td></tr><tr><td>3</td><td>Dihydroquercetin 3-O-glucoside</td><td>C<sub>21</sub>H<sub>22</sub>O<sub>12</sub></td><td>9.5</td><td>465.108</td><td>8.5</td><td>285.041 [M-hexose-2H]<sup>-</sup></td><td>467.116</td><td>8.5</td><td>305.065 [M-sucrose+H]<sup>+</sup><br>181.014 [M-sucrose-C<sub>6</sub>H<sub>4</sub>CH]<sup>+</sup><br>181.014 [C<sub>8</sub>H<sub>5</sub>O<sub>5</sub>]<sup>+</sup></td><td>leaves</td></tr><tr><td>4</td><td>Kaempferide 3-glucoside</td><td>C<sub>22</sub>H<sub>22</sub>O<sub>11</sub></td><td>14.6</td><td>n.d</td><td>n.d</td><td>n.d</td><td>463.125</td><td>4.3</td><td>301.069 [M-hexosyl+H]<sup>+</sup></td><td>Leaves, pods</td></tr><tr><td>5</td><td>Tectoridin</td><td>C<sub>22</sub>H<sub>22</sub>O<sub>11</sub></td><td>12.8</td><td>461.118</td><td>19.5</td><td>n.d</td><td>463.125</td><td>4.3</td><td>283.057 [M-hexose]<sup>+</sup><br>255.063 [M-hexose-CO<sub>2</sub>]<sup>+</sup></td><td>Leaves, pods</td></tr><tr><td>6</td><td>Emodin</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>5</sub></td><td>25.1</td><td>269.045</td><td>3.7</td><td>241.047 [M-CO-H]<sup>-</sup><br>255.053 [M-CO<sub>2</sub>-H]<sup>-</sup><br>210.029 [M-CO<sub>2</sub>-CH<sub>3</sub>-H]<sup>-</sup></td><td>271.058</td><td>7.3</td><td>229.046 [M-CH<sub>2</sub>CO+H]<sup>+</sup><br>225.051 [M-H<sub>2</sub>O-CO+H]<sup>+</sup><br>1978.059 [M-H<sub>2</sub>O-2CO+H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr><tr><td>7</td><td>Malic acid</td><td>C<sub>4</sub>H<sub>6</sub>O<sub>5</sub></td><td>1.8</td><td>133.015</td><td>7.5</td><td>114.004 [M-H<sub>2</sub>O-H]<sup>-</sup><br>71.021 [M-H<sub>2</sub>O-CO<sub>2</sub>-H]<sup>-</sup></td><td>n.d</td><td>n.d</td><td>n.d</td><td>Leaves, pods</td></tr><tr><td>8</td><td>Citric acid</td><td>C<sub>6</sub>H<sub>8</sub>O<sub>7</sub></td><td>2.1</td><td>191.015</td><td>10.5</td><td>129.014 [M-H<sub>2</sub>O-CO<sub>2</sub>-H]<sup>+</sup><br>111.007 [M-2H<sub>2</sub>O-CO<sub>2</sub>-H]<sup>-</sup><br>87.011 [M-H<sub>2</sub>O-CO<sub>2</sub>-CH<sub>2</sub>CO-H]<sup>-</sup></td><td>193.033</td><td>5.2</td><td>n.d</td><td>Leaves, roots</td></tr><tr><td>9</td><td>Tyrosine</td><td>C<sub>9</sub>H<sub>11</sub>NO<sub>3</sub></td><td>2.5</td><td>n.d</td><td>n.d</td><td>n.d</td><td>182.080</td><td>5.5</td><td>165.083 [M-OH+H]<sup>+</sup><br>135.077 [M-HCOOH+H]<sup>+</sup><br>119.057 [M-HCOOH-NH<sub>3</sub>+H]<sup>+</sup></td><td>Pods</td></tr><tr><td>10</td><td>Tryptophan</td><td>C<sub>11</sub>H<sub>12</sub>N<sub>2</sub>O<sub>2</sub></td><td>9.8</td><td>203.078</td><td>2.4</td><td>142.062 [M-CO<sub>2</sub>-NH<sub>3</sub>-H]<sup>-</sup><br>116.050 [M-CO<sub>2</sub>-NH<sub>3</sub>-CN-H]<sup>-</sup></td><td>205.095</td><td>9.7</td><td>188.078 [M-NH<sub>3</sub>+H]<sup>+</sup><br>144.080 [M-NH<sub>3</sub>-CO<sub>2</sub>+H]<sup>+</sup><br>118.068 [M-NH<sub>3</sub>-CO<sub>2</sub>-CN+H]<sup>+</sup></td><td>Leaves, pods</td></tr><tr><td>11</td><td>Aloesol 7-glucoside</td><td>C<sub>19</sub>H<sub>24</sub>O<sub>9</sub></td><td>11.5</td><td>395.135</td><td>0.0</td><td>351.103 [M-C<sub>2</sub>H<sub>4</sub>O-H]<sup>-</sup><br>231.063 [M-glycosyl-H]<sup>-</sup><br>203.064 [M-glycosyl-CO-H]<sup>-</sup></td><td>397.149</td><td>0.0</td><td>233.077 [M-glycosyl+H]<sup>+</sup><br>215.066 [M-glycosyl-H<sub>2</sub>O + H]<sup>+</sup><br>203.066 [M-glycosyl-CH<sub>2</sub>O + H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr></tbody></table>
<table><tr><td>12</td><td>7-hydroxy-3-(2-hydroxypropyl)-5-methylisochromen-1-one</td><td>C<sub>13</sub>H<sub>14</sub>O<sub>4</sub></td><td>15.3</td><td>233.078</td><td>17</td><td>189.051 [M-CH<sub>3</sub>COH-H]<sup>-</sup><br/>159.036 [M-CH<sub>3</sub>COH-CH<sub>2</sub>O-H]<sup>-</sup></td><td>235.095</td><td>4.2</td><td>217.077 [M-H<sub>2</sub>O+H]<sup>+</sup><br/>191.068 [M-CH<sub>3</sub>COH+H]<sup>+</sup><br/>176.046 [M-CH<sub>3</sub>COH-CH<sub>3</sub>+H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr><tr><td>13</td><td>Piperonylidene acetone</td><td>C<sub>11</sub>H<sub>10</sub>O<sub>3</sub></td><td>16.4</td><td>189.051</td><td>26</td><td>174.030 [M-CH<sub>3</sub>-H]<sup>-</sup><br/>159.042 [M-CH<sub>2</sub>COH-H]<sup>-</sup><br/>146.034 [M-CH<sub>2</sub>COH-CH<sub>3</sub>-H]<sup>-</sup></td><td>191.069</td><td>5.2</td><td>176.047 [M-CH<sub>3</sub>+H]<sup>+</sup><br/>151.037 [M-H<sub>2</sub>O-CH<sub>2</sub>CO+H]<sup>+</sup><br/>148.050 [M-CH<sub>3</sub>-CO+H]<sup>+</sup></td><td>Leaves, pods</td></tr><tr><td>14</td><td>(R)-Linalyl betavicianoside</td><td>C<sub>21</sub>H<sub>36</sub>O<sub>10</sub></td><td>18.1</td><td>449.233</td><td>9.1</td><td>447.231 [M-H]<sup>-</sup><br/>315.179 [M-pentosyl-H]<sup>-</sup><br/>161.042 [M-pentosyl-C<sub>10</sub>H<sub>17</sub>OH-H]<sup>-</sup></td><td>471.216</td><td>8.5</td><td>333.079 [M-C<sub>10</sub>H<sub>18</sub>+Na]<sup>+</sup></td><td>leaves</td></tr><tr><td>15</td><td>Neryl rhamnosyl-glucoside</td><td>C<sub>22</sub>H<sub>38</sub>O<sub>10</sub></td><td>18.3</td><td>507.251</td><td>16</td><td>461.246 [M-H]<sup>-</sup><br/>315.178 [M-Dehydrohexosyl-H]<sup>-</sup><br/>141.042 [M-Dehydrohexosyl-C<sub>10</sub>H<sub>17</sub>OH-H]<sup>-</sup></td><td>485.236</td><td>0.0</td><td>347.093 [M-C<sub>10</sub>H<sub>18</sub>+Na]<sup>+</sup><br/>204.040 [M-C<sub>10</sub>H<sub>18</sub>-dehydrohexosyl+Na]<sup>+</sup></td><td>leaves</td></tr><tr><td>16</td><td>Glucuronic acid</td><td>C<sub>16</sub>H<sub>20</sub>O<sub>10</sub></td><td>12.6/1<br/>3.7</td><td>371.098</td><td>0.2</td><td>249.062 [M-benzoic acid-H]<sup>-</sup><br/>175.019 [M-benzoic acid-glycerol-H]<sup>-</sup><br/>121.029 [benzoic acid-H]<sup>-</sup></td><td>373.111</td><td>5.3</td><td>n.d</td><td>leaves</td></tr><tr><td>17</td><td>Aloesol di glycoside</td><td>C<sub>25</sub>H<sub>34</sub>O<sub>14</sub></td><td>10.3</td><td>n.d</td><td>n.d</td><td>n.d</td><td>559.202</td><td>0.0</td><td>397.151 [M-Hexosyl+H]<sup>+</sup><br/>233.074 [M-2Hexosyl+H]<sup>+</sup></td><td>Leaves, pods</td></tr><tr><td>18</td><td>Aloesin-glycoside</td><td>C<sub>25</sub>H<sub>32</sub>O<sub>14</sub></td><td>10.1</td><td>555.180</td><td>13</td><td>n.d</td><td>557.189</td><td>3.6</td><td>395.139 [M-Hexosyl+H]<sup>+</sup><br/>233.078 [M-2Hexosyl+H]<sup>+</sup></td><td>Pods</td></tr><tr><td>19</td><td>Aloesin</td><td>C<sub>19</sub>H<sub>22</sub>O<sub>9</sub></td><td>11.3</td><td>393.121</td><td>5.1</td><td>273.079 [M-HOC<sub>6</sub>H<sub>3</sub>CO-H]<sup>-</sup><br/>231.061 [M-glycosyl-H]<sup>-</sup><br/>203.068 [M-glycosyl-CO-H]<sup>-</sup></td><td>395.135</td><td>3.5</td><td>233.077 [M-glycosyl + H]<sup>+</sup><br/>215.067 [M-glycosyl-H<sub>2</sub>O + H]<sup>+</sup><br/>203.065 [M-glycosyl-CH<sub>2</sub>O + H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr><tr><td>20</td><td>Aloesol-glucoside</td><td>C<sub>19</sub>H<sub>24</sub>O<sub>9</sub></td><td>11.6</td><td>395.135</td><td>2.5</td><td>275.089 [M-HOC<sub>6</sub>H<sub>3</sub>CO-H]<sup>-</sup><br/>231.066 [M-glycosyl-H]<sup>-</sup><br/>203.068 [M-glycosyl-CO-H]<sup>-</sup></td><td>397.151</td><td>6.0</td><td>233.077 [M-glycosyl + H]<sup>+</sup><br/>215.067 [M-glycosyl-H<sub>2</sub>O + H]<sup>+</sup><br/>203.066 [M-glycosyl-CH<sub>2</sub>O + H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr><tr><td>21</td><td>Aloesol</td><td>C<sub>13</sub>H<sub>14</sub>O<sub>4</sub></td><td>15.4</td><td>233.078</td><td>60</td><td>189.051 [M-C<sub>2</sub>H<sub>4</sub>O - H]<sup>-</sup></td><td>235.094</td><td>8.5</td><td>217.077 [M-H<sub>2</sub>O+H]<sup>+</sup><br/>191.068 [M-C<sub>2</sub>H<sub>4</sub>O + H]<sup>+</sup><br/>176.047 [M-C<sub>2</sub>H<sub>4</sub>O-CH<sub>3</sub> + H]<sup>+</sup></td><td>Leaves, pods, roots</td></tr></table>

n.d: not determined

454

455

456

457
458 Acknowledgements

459 This research work was supported by PHC-Tassili (Code 20MDU917). Besides, the authors

460 acknowledge the support of the "Laboratoire Réactions et Génie des Procédés" by the "Impact

461 Biomolecules" project of the "Lorraine Université d'Excellence" (Investissements d'avenir – ANR

462 project number 15-004).
463 References

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615 **Figure captions**

616 **Fig. 1.** Total phenolic content (TPC) of *R. alaternus's* leaves, pods and roots extracted with 617 natural deep eutectic solvents (NaDESs: ChCl-Gly, ChCl-EG, ChCl-Ur) and compared to Ethanol as 618 reference.

619 **Fig. 2.** Radical scavenging activity of different parts from *R. alaternus* extracted using natural 620 deep eutectic solvents (NaDESs): ChCl-Gly, ChCl-EG, ChCl-Ur in reference to Ethanol. (A) ABTS 621 test and (B) DPPH test.

622 **Fig. 3.** Reducing activity (A) and Iron chelating capacity (B) of different parts from *R. alaternus* 623 extracted using natural deep eutectic solvents (NaDESs); ChCl-Gly, ChCl-EG, ChCl-Ur and 624 Ethanol.

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