Insights on the Extraction Performance of Alkanediols and Glycerol: Using Juglans regia L. Leaves as a Source of Bioactive Compounds

Glycerol and alkanediols are being studied as alternative solvents to extract phytochemicals from plant material, often as hydrogen bond donors in deep eutectic solvents (DESs). Many of those alcohols are liquid at room temperature, yet studies of their use as extraction solvents are scarce. In this work, glycerol and a series of alkanediols (1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 1,2-pentanediol, 1,5-pentanediol, and 1,2-hexanediol) were studied for the extraction of phenolic compounds from Juglans regia L. leaves, a rich source of this class of bioactive compounds. The extraction yield was quantified, and the bioactivity of both extracts and pure solvents was evaluated by measuring the anti-inflammatory and cytotoxic activities. The solvents showing the best combined results were 1,2 and 1,3-propanediol, as their extracts presented a high amount of phenolic compounds, close to the results of ethanol, and similar cytotoxicity against cervical carcinoma cells, with no impact on non-tumor porcine liver cells in the studied concentration range. On the other hand, none of the extracts (and solvents) presented anti-inflammatory activity. Overall, the results obtained in this work contribute to the study of alternative solvents that could potentially be used also as formulation media, highlighting the importance of walnut leaves as a source of bioactive compounds.


Introduction
Plants are important sources of bioactive phytochemicals, with a wide range of applications in the food, pharmaceutical, and cosmetic sectors as natural ingredients, e.g., replacing synthetic additives or improving the quality of a final product [1]. In particular, the leaves of Juglans regia L. (walnut tree) have been reported as a rich source of phenolic compounds, especially hydroxycinnamic acids and flavonols [2,3], with potential application in cosmetics or pharmaceutics [4], and stand out for their bioactive properties (antioxidant, anti-inflammatory, antidiabetic, antiproliferative, and antibacterial, among others) [5,6].
In the case of systems containing glycerol, higher total phenolic contents were obtained, using small amounts of glycerol in water, from Olea europaea leaves (9.3%, w/v) [28], Hypericum perforatum dried aerial parts (10%, w/v) [26], and Oryza sativa bran (20%, v/v) [30]. On the other hand, an increasing concentration of glycerol (10-90%, w/v) leads to extracts of two Artemisia species with higher total phenolic content [27]. Philippi et al. [29] compared the extraction yields of aqueous solutions of glycerol or ethanol, obtaining similar total phenolic contents in the extracts of Solanum melongena. Regarding the use of propylene glycol, in some cases, higher extraction yields were obtained than by using water and/or ethanol. That was the case of the extracts obtained from Stevia rebaudiana leaves [22], Terminalia chebula fruits [23], Malus sylvestris wild fruit [18], and Pandanus amaryllifolius leaves [20]. On the other hand, the aqueous propylene glycol extract of Terminalia chebula fruits contained lower amounts of gallic and ellagic acids compared to the hydroethanolic extract [24]. Finally, using aqueous solutions of ethylene glycol, a higher total phenolic content was obtained from Morus alba L. when compared to acetone + (water or methanol) mixtures [31].
Besides the aqueous solutions of alcohols, other combinations were also studied. Binary mixtures of ethanol + propylene glycol or glycerol were more effective than ethanol for the extraction of rosmarinic acid, carvacrol, oleanolic acid, and ursolic acid from three Origanun species by heatassisted extractions (HAEs) [16], while higher amounts of isorhamnetin-3-O-rutinoside were extracted from Calendula officinalis L. flowers using an ethanol + propylene glycol + water equimolar mixture than using pure solvents or binary mixtures [17].
In this work, aqueous solutions of glycerol and alkanediols (1,2-ethanediol, 1,2-propanediol, 1,3propanediol, 1,3-butanediol, 1,2-pentanediol, 1,5-pentanediol, and 1,2-hexanediol) were studied to extract phenolic compounds from walnut leaves. These compounds were chosen not only to evaluate the effect of increasing the alkyl chain of the diols on the extraction yield, but also considering their potential application in different areas such as pharmaceuticals or cosmetics. Some of these compounds and their functions are included in a list of cosmetic ingredients (other than perfume and aromatic raw materials) [ [25] about the use of alternative solvents for food and cosmetic applications also includes 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2pentanediol, and glycerol, highlighting these ingredients as "bio-sourced natural compounds" and the possibility of obtaining them from renewable sources. Finally, 1,5-pentanediol was also studied as a preserving and humectant ingredient for dermatological care products [33][34][35], and 1,2hexanediol was proposed as a potential component for the preparation of topical formulations aiming to retard the percutaneous absorption of active pharmaceutical ingredients [36]. On an industrial scale, the use of alkanediols and glycerol could be also safer as they have lower vapor pressures compared to conventional organic solvents [37][38][39]. In addition, if the solvent could be part of the final formulation, the number of unit operations of the global process would be reduced by eliminating the need to separate the extracted compounds from the solvent [15,37]. Finally, to support the development of new applications, besides determining the extraction yield of the main phenolic compounds, the anti-inflammatory and cytotoxic activities of both extracts and pure solvents were also evaluated. UAE: ultrasound-assisted extraction; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; TBARS: thiobarbituric acid reactive substances; CRL-2522: human skin fibroblasts BJ; AAPH: 2,2′-Azobis(2-amidinopropane) dihydrochloride.

Extraction Yields
Aqueous solutions of glycerol and several diols (1,2-ethanediol, 1,2-propanediol, 1,3propanediol, 1,3-butanediol, 1,2-pentanediol, 1,5-pentanediol, and 1,2-hexanediol) containing 20% water (w/w) were used to extract phenolic compounds from J. regia L. leaves. Water and ethanol + water (80:20, w/w) solvents were also studied for comparison purposes. To carry out this study, aqueous solutions of the solvents referred to were preferred as they have shown to improve the extraction yields by reducing the solvent viscosity, enhancing the mass-transfer from the plant to the solvent media and reducing energy consumption [40]. The optimum amount of water depends on several factors such as the plant matrix or the target molecules to be extracted, as summarized in Table 1. In this work, this amount was selected taking into account a previous study aimed at solvent screening for the extraction of phenolics from the same plant material [41].
The individual amounts of each phenolic compound obtained in this work, using conventional ethanol/water (80:20, w/w) mixtures, are generally in good agreement with the ones reported in the literature, as recently reviewed [42]. In previous studies, similar amounts of 3-O-caffeoylquinic acid were found using water as solvent, and also of quercetin 3-O-glucoside and quercetin O-pentoside using water and ethanol [43], or even eutectic mixtures based on choline chloride and carboxylic acids [41]. On the other hand, the values found in this work were higher than the ones presented by Zhao et al. [44], Amaral et al. [45], and Santos et al. [3], while Pereira et al. [46] reported higher amounts of 3-O-caffeoylquinic acid (12.06-14.82 mg/g dw), 3-p-coumaroylquinic acid (4.69-5.99 mg/g dw), and quercetin 3-O-galactoside (15.72-21.68 mg/g dw) in the aqueous (decoction) extract of different cultivars of walnut leaves. These differences arise from the combination of several factors, namely: (i) the type of solvent; (ii) extraction conditions; (iii) the plant (e.g., geographical location, time of collection, type of cultivar, etc.) [47,48]. In this work, the extraction conditions and the lot of the plant were the same, to allow the evaluation of the effect of changing the solvent, as discussed in the previous paragraph.

Cytotoxic and Anti-Inflammatory Activities
To assist the identification of potential applications for these extracts, cytotoxicity assays were applied not only to the extracts, but also to the solvents. The cytotoxicity was evaluated using a human tumor cell line (HeLa) and the non-tumor porcine live primary cell culture (PLP2). The HeLa cell line represents a suitable in vitro model to study cytotoxicity that has already been applied to new solvents [49] such as DES [50,51] and ionic liquids prepared from biomaterials [52]. On the other hand, PLP2 cells can be used to evaluate one specific type of cytotoxicity-hepatotoxicity [53]. Therefore, the potential toxicity occurring during/after metabolization is here represented by the porcine liver primary cells [54]. The results of the cytotoxicity assays are summarized in Table 3. Table 3. Bioactive properties of J. regia leaves. Cytotoxicity in human tumor cell lines HeLa and nontumor liver primary cells PLP2 (GI50). Quantification for extracts and solvents (mean ± SD). It is important to mention that none of the extracts were as active as the positive control (GI50 ellipticine = 1.0 ± 0.1 and 2.3 ± 0.2 µg/mL for HeLa and PLP2, respectively). As can be seen, the extracts obtained using standard solvents (water and ethanol) have no toxicological impact in PLP2 cells, allowing normal cell proliferation under concentrations up to 500 µg/mL. The same was observed for the aqueous extract applied to the HeLa cell culture; however, for the ethanolic extract, a GI50 of 245 ± 14 µg/mL was obtained. Regarding the extracts showing cytotoxic potential only against the tumor cell line but not the non-tumor one (1,2-and 1,3-propanediol, and 1,3-butanediol), 1,3-propanediol was the most effective solvent (GI50 = 216 ± 10 µg/mL and > 500 µg/mL, respectively, for the HeLa and PLP2 cell cultures). Interestingly, the extracts obtained using 1,2-ethanediol and glycerol showed toxicity against the selected control cell culture (PLP2), with GI50 values of 142 ± 5 µg/mL and 143 ± 5 µg/mL, respectively. Nevertheless, the pure solvents (without extracts) were absent of cytotoxicity up to concentrations of 4%. Besides the cytotoxicity of 1,2-ethanediol and glycerol extracts against the PLP2 cells, the GI50 values for the HeLa cells were lower (GI50 = 97 ± 10 µg/mL and 88 ± 4 µg/mL, respectively); thus, the extracts were more active against the cervical human tumor cell line. Finally, the most cytotoxic extract was obtained with 1,2-hexanediol, the concentrations of extract being significantly lower than those obtained with the remaining solvents (GI50, HeLa = 37 ± 2 µg/mL). The same was observed for the pure solvent, constituting a strong cytotoxic agent even when highly diluted (GI50, PLP2 = 0.285 ± 0.005% and GI50, HeLa = 0.36 ± 0.02%). This was also observed for both pentanediol isomers, which presented activity for concentrations lower than 1%. The 1,5-pentanediol extract showed significantly higher GI50 values against the HeLa cell line (GI50 = 212 ± 4 µg/mL) than glycerol and 1,2-ethanediol extracts, but the effective GI50 for the PLP2 cells was lower (141 ± 3 µg/mL), which is not desirable as ideally, GI50, PLP2 > GI50, HeLa. To complete this discussion, it is important to clarify the contribution of the solvent to the final toxicity of the extract. For that reason, the toxicity of the solvent was evaluated in a concentration range that includes the concentration of the solvent in the liquid extracts studied in the bioactivity studies. When both extracts and solvents presented cytotoxicity (1,2-pentanediol, 1,5-pentanediol, and 1,2-hexanediol), it was found that the concentrations providing cytotoxicity for the pure solvents are similar to those presented by the extracts (HeLa = 0.41%, 0.57%, and 0.10%; PLP2 = 0.63%, 0.38%, and 0.13%, respectively). Thus, the toxicity found is probably due to the presence of the solvent. On the other hand, for the remaining extracts, the bioactivity could be attributed to the bioactive substances in the extract since the solvents do not show toxicity against the studied cell lines up to a 4% concentration, which is higher than the amount of solvent present in the analyzed liquid extracts.

Solvent HeLa PLP2 Extract (µg/mL) Solvent (%,v/v) Extract (µg/mL) Solvent (%,v/v)
The cytotoxicity of walnut leaf extracts was previously studied by Santos et al. [3] for several cell lines, including HeLa and PLP2 cell cultures. The authors reported the absence of toxicity of extracts obtained by decoction (GI50 > 400 µg/mL), a specific type of aqueous extract, which is in good agreement with our results. In contrast, the methanolic extract of the same plant material, as well as those obtained from green and yellow leaves using ethanol + water [42], inhibited the proliferation of HeLa but not PLP2 cells, which also agrees with the results obtained in the present study using conventional solvents.
The evaluation of the cytotoxicity of the liquid extracts obtained with alkanediols and glycerol has seldom been carried out (Table 1). A comparative study of the cytotoxic potential of Stevia rebaudiana leaf extracts and the selected solvents (water, ethanol, and aqueous propylene glycol) was performed by Gaweł-Bȩben et al. [22] using skin fibroblast cells as a model to evaluate the extracts' potential as food and cosmetic ingredients. Generally, the extracts show higher cytotoxic potential than the solvents for the same solvent concentration. Furthermore, the aqueous ethanolic and propylene glycol extracts (between 2% and 5%) promoted a significant decrease in the skin fibroblasts' viability. Thus, the authors concluded that the potential of those extracts for cosmetic or food formulations are dependent on further studies to establish an appropriate dose, adapted to the desired product.
Finally, in a previous study, the extracts of green leaves of J. regia showed anti-inflammatory activity [42]; thus, this assay was also applied here. However, neither the liquid extracts nor solvents showed anti-inflammatory potential at the studied concentration range (EC50 > 500 µg/mL or 4% solvent). To the best of our knowledge, this is the first time that this cell-based assay was applied to the selected solvents and extracts.

Plant Material
The Juglans regia L. (walnut) dried leaves were purchased from Soria Natural, S.A., Soria, Spain. According to the distributor, the leaves were collected in Soria (Spain) during June 2014 and naturally dried in a room with controlled humidity. Before the extractions, the samples were milled (60 to 20 mesh) and stored in a desiccator protected from light for subsequent assays.
Extraction technique: the heat-assisted extractions (HAE) were performed using heating equipment with magnetic stirring (Carousel 12 Plus Reaction Station™, Radleys Tech, Essex, UK). The powdered samples (0.3 g) were extracted with 10 mL of each solvent for 120 min at 50 °C and 600 rpm. These conditions were set for solvent screening, taking into consideration previous results using, as solvents, mixtures of water and ethanol [43] and aqueous mixtures of choline chloride and carboxylic acids [41]. After extraction, the mixtures were filtered through a Whatman nº 4 paper type (Prat Dumas, Couze-St-Front, France) for further analysis.

Chromatographic Analysis of the Main Phenolic Compounds
Before chromatographic analysis, the samples of the solutions containing the extracts were diluted with water and filtered through 0.2 µm disposable liquid chromatography (LC) filter disks (30 mm, regenerated cellulose; Whatman, Maidstone, UK). The samples were analyzed using a Shimadzu 20A series UFLC (Ultra-Fast Liquid Chromatograph, Shimadzu Corporation, Kyoto, Japan) with a quaternary pump and a photodiode array detector (PDA) coupled to an LC solution software data-processing station. A Waters Spherisorb S3 ODS-2 C18, (3 µm, 4.6 mm × 150 mm; Waters Associates, PA, USA) column was used, operating at 35 °C. The chromatographic method was previously described by the authors [43]. A diode array detector (DAD) was used at 280 and 370 nm wavelengths. The target phenolic compounds were identified according to their UV spectra and retention time [55]. For the quantitative analysis, a baseline to valley integration with baseline projection mode was used to calculate peak areas, and external standards were used for quantification. The results were expressed in mg per g of dry plant (mg/g dry plant).

Cytotoxicity
J. regia leaf extracts (30 mg/mL) and solvents were studied regarding their inhibitory growth activity of the HeLa (cervical carcinoma) cell line and the non-tumor PLP2 cells (porcine liver primary cells) by using a sulforhodamine B assay. The extracts were diluted to obtain a stock solution of 10 mg/mL, and the concentration of the working solutions ranged from 500 to 7.8 µg/mL, while the concentration of the solvents ranged from 4% to 0.0156%. Ellipticine was used as positive control. The experimental protocol related to the cell cultures was previously described by Barros et al. [55] and Abreu et al. [53]. The concentration needed to reach 50% of the growth inhibition effect (GI50) was determined.

Anti-Inflammatory Activity
The walnut leaf extracts (30 mg/mL) and the pure solvents were tested with a lipopolysaccharide-induced nitric oxide (NO) production assay according to Corrêa et al. [56], using a mouse macrophage-like cell line (RAW264.7). The extracts were diluted to obtain a stock solution of 10 mg/mL, and the concentration of the working solutions ranged from 500 to 7.8 µg/mL. Again, the concentration of the solvents ranged from 4% to 0.0156%. Then, the anti-inflammatory activity was assessed by measuring the nitrite concentration in the cell culture medium, using the Griess Reagent System kit. Dexamethasone was used as a positive control. The concentration providing 50% of the inhibition of NO production (EC50) was determined.

Statistical Analysis
In the bioactivity assays, duplicates of each extract were assayed, and three repetitions of each methodology were performed, with the results expressed as mean values and standard deviations (SD). One-way ANOVA followed by Tukey's HSD test (p = 0.05) were used to analyze the results. Furthermore, significant differences between two samples were established by applying a student's t-test, with p = 0.05 using the IBM SPSS Statistics for Windows, Version 23.0. (IBM Corp., Armonk, New York, USA).

Conclusions
Aqueous solutions of several alkanediols were able to extract similar amounts of phenolic compounds to ethanol, a conventional volatile solvent for these target compounds, ethylene glycol being the best solvent. Among alkanediols, propanediols (1,2 and 1,3 isomers) stand out if both extraction yield and bioactivity studies are considered. Their extraction efficiency was close to ethanol, and their extracts also presented similar cytotoxicity, being active against the human cervical carcinoma cell line but not against the non-tumor porcine liver primary cell culture. None of the aqueous mixtures of solvents showed anti-inflammatory activity. Regarding the studied solvents (without extracts), those having up to four carbon atoms were not toxic for the selected cell cultures in the studied concentration range.
The results obtained suggest the use of the extracts prepared in 1,2 and 1,3-propanediol as a source of important phytochemicals with bioactive properties. Depending on the envisioned application, further tests should be carried out to ensure the safety of the solvents and extracts, while optimizing the extraction conditions.