Green and highly extraction of phenolic compounds and antioxidant capacity from kinkeliba (Combretum micranthum G. Don) by natural deep eutectic solvents (NADESs) using maceration, ultrasound-assisted extraction and homogenate-assisted extraction

2.1 Plant material

The leaves of kinkeliba (Combretum micranthum G. Don) were collected from Abomey-Calavi/Benin Republic. The leaves sun-dried for seven days and packed in brown bottles with screw caps.

2.2 Chemicals and reagents

Distilled water purified by a Millipore-Q system (Millipore Billerica, Massachusetts, USA). methanol (HPLC grade), ethanol (HPLC grade), acetonitrile (HPLC grade), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, ≥99.0%), Folin-Ciocalteu reagent, Trolox (97%), sodium nitrite (99–100.5%), hydrochloric acid (36.5–38%), sodium carbonate (99.5–100.5%), choline chloride (≥98%), gallic acid (≥99.0) and other standards were bought from Sigma Aldrich Chemical Co. (St Louis, MO). Lactic acid (90%) was purchased from Isolab while aluminum chloride, iron (III) chloride and iron sulfate heptahydrate (≥99.5%) were brought from Merck. Sodium acetate anhydrous (≥99.0%), L(+) tartaric acid (>99%), glacial acetic acid (99.5%), potassium chloride (≥99.0%), and sodium hydroxide (≥97.0%) obtained from Carlo Erba.

2.3 Synthesis of NADESs

Three NADESs were synthesized using choline chloride as the hydrogen bond acceptor (HBA) and lactic acid, tartaric acid and acetic acid as hydrogen bond donors (HBD). The NADESs were obtained according to the preparation procedure described in Chanioti and Tzia (2018) with a slight modification. Briefly, choline chloride was mixed with each hydrogen bond donor at 1:2 M ratio, followed by the addition of 20% of distilled water. Then, the mixture was heated at 80 °C under constant stirring for 2 h. The NADESs obtained from the combination of choline chloride and lactic acid, tartaric acid and acetic acid were encoded as ChLa, ChTa and ChAa, respectively.

2.4 Extraction with NADES and conventional solvents

2.5 Total phenolic content (TPC)

The TPC was determined by the Folin-Ciocalteu method using the method of Singleton and Rossi (1965) with some modifications. Briefly, 150 µL of the appropriately diluted sample was mixed 750 µL of 10% Folin-Ciocalteu reagent and 600 μL of 7.5% (w/v) Na₂CO₃, respectively. The mixture was placed in the dark for 2 h and the absorbance was read at 760 nm using a UV- spectrophotometer (Thermo Spectronic). The TPC was expressed as mg gallic acid equivalent per g (mg GAE/g).

2.6 Total flavonoid content (TFC)

TFC was determined using the described method in Zannou and Koca (2020). Briefly, 1 mL of the appropriately diluted sample mixed with 300 µL of 5% NaNO₂ and 500 µL of 5% AlCl₃ and 500 µL of 1 M NaOH respectively. Afterwards, the mixture was placed in the dark for 10 min and the absorbance was read at 510 nm. The results were given as mg epicatechin equivalents per g (mg ECE/g).

2.7 DPPH radical scavenging activity assay (DPPH)

The DPPH assay was conducted according to the adopted method of Zannou et al. (2020). The DPPH solution was used as the control and the scavenging ratio was calculated as follows: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\left(\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\right)\times 100$$

The values of DPPH radical scavenging were determined with a calibration curve as mmol Trolox equivalent per g (mmol TE/g).

2.8 Ferric reducing antioxidant power assay (FRAP)

The FRAP assay was conducted according to the procedure indicated in Benzie and Strain (1996). The values of FRAP was calculated from a calibration curve of FeSO₄ and the results were given as mmol FeSO₄ equivalents per g (mmol ISE/g).

2.9 Phenolic compounds profile of C. micranthum extracts via HPLC-DAD

The individual phenolic compounds of sumac were determined using the previous method of Bosiljkov et al. (2017) with modifications. The phenolic compounds were identified using a high-pressure liquid chromatography (HPLC) system (Agilent 1260; Agilent Technologies) coupled with a diode array detector (DAD) at 520 nm wavelength for anthocyanins and 280 nm for other phenolic compounds. The phenolic compounds were separated in an Inertsil ODS-4 column (3 µm, 4,6 × 50 mm; GL Sciences Kat No: 5020–0404) at a 1 mL.min⁻¹ flow rate. The mobile phases were: (A) 94% 2 mM sodium acetate and 6% acetic acid (v/v); and (B) acetonitrile. The following elution gradient was used, according to solvent B: 0–20 min, 14–23%; 20–40 min, 23–35%; 40–50 min, 40%; 50–60 min, 60%; 60–65 min 95%. The column temperature was set at 30 °C. The individual phenolic compounds were identified by comparing their retention times with their respective standard. The identified phenolic compounds were quantified using a mixture of external standards which were prepared at different concentrations.

2.10 Statistical analyses

The experiments were carried out in triplicate. The results were given as mean ± standard deviation. The one-way analysis of variance (ANOVA) was performed for the statistical analyses using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Duncan's test was applied to determine the significance of the solvents' extractability results (p < 0.05). The independent t-test was performed to evaluate the significance of the extraction techniques (p < 0.05). Pearson's correlation test was used for the evaluation of the correlation between the antioxidant capacities of two independent tests (DPPH and FRAP) and total phenolic content, total flavonoid content and individual phenolic compounds. In addition, the principal component analysis (PCA) was performed (XL Stat software, Addinsoft, New York, NY, USA) to determine the correlations between the variables and properties of the extracts.

3.1 Effects of solvents and extraction methods on phytochemical properties of C. Micranthum

In the present study, the efficiency of the synthesized NADESs to recover phenolic compounds from C. micranthum leaves was tested using two representative phytochemical indices such as TPC and TFC. Distilled water, ethanol and methanol were chosen as control solvents since they are the conventional solvents usually used for the extraction of phenolic compounds from plant material. Furthermore, the extractions were carried out using ultrasound-assisted extraction (UAE), homogenate-assisted extraction (HAE) and maceration (ME) techniques to show out the appropriate extraction methods. Extraction is the first and essential step in the isolation and purification of bioactive components from plants. The maceration is a traditional extraction technique applied for the recovery of bioactive compounds such as phenolic compounds. In recent years, new extraction techniques have been experimented for the extraction of bioactive compounds including ultrasound-assisted extraction and homogenate-assisted extraction, (Khoddami et al., 2013; Chanioti and Tzia, 2018). Although the extraction technique is essential in the extraction process, the solvent and other extraction parameters such as time and temperature play a key role in the recovery of bioactive compounds.

The extractability of phenolic compounds with ChLa, ChTa and ChAa, water, methanol and ethanol was shown in Fig. 1. The recovery of phenolic compounds from C. micranthum leaves was significantly affected by the type of solvents (p < 0.05). ChLa exhibited the highest performance giving the TPC varying between 21.12 ± 0.13 mg GAE/g and 23.62 ± 0.58 mg GAE/g depending on the extraction methods. ChLa was followed by ChAc (15.49 ± 0.13–18.85 ± 0.39 mg GAE/g), water (17.08 ± 0.32–18.13 ± 0.13 mg GAE/g), ChTa (14.49 ± 0.26–17.44 ± 0.19 mg GAE/g), methanol (7.46 ± 0.45–11.64 ± 0.32 mg GAE/g) and ethanol (2.88 ± 0.39–4.60 ± 0.39 mg GAE/g), respectively. For TFC, ChLa (4.38 ± 0.09–5.01 ± 0.09 mg ECE/g) was the most prominent solvent extracting the flavonoid compounds, followed by ChAc (2.84 ± 0.04–5.01 ± 0.36 mg ECE/g), methanol (1.93 ± 053–4.85 ± 0.04 mg ECE/g), ethanol (1.49 ± 0.36–4.16 ± 0.04 mg ECE/g), ChTa (1.09 ± 0.04–3.22 ± 0.13 mg ECE/g) and water (1.15 ± 0.04–1.37 ± 0.44 mg ECE/g), respectively. The NADESs and particularly ChLa and ChAa exhibited the highest extraction efficiency compared to the conventional solvents. This finding is in close accordance with the results reported by others who found that carboxylic-based NADESs have a high ability to extract phenolics from plants (Chanioti and Tzia, 2018; Zannou et al.2020; Zannou and Koca, 2020; Alsaud et al., 2021). The addition of 20% of water to NADESs enhanced their hydrophilicity and decreased their viscosity. Thus, the phenolic compounds of C. micranthum leaves were more dissolved in NADESs. Moreover, the interaction between the phenolic compounds and hydrogen bonds of NADESs (OH and/or Cl-) facilitated their removal (Alsaud et al., 2021). Hao et al. (2020) have reported that the higher ability of ChLa and ChAc of extracting the flavonoids is linked with the strong multiple hydrogen-bonding networks that choline chloride and carboxylic acid-based NADESs form with flavonoids. ChLa has shown the best performance compared to ChAa and ChTa. Therefore, ChLa may have more affinity with the phenolic compounds of C. micranthum leaves since the NADES which form high hydrogen bonding can perform better than other NADESs (Dai et al., 2016; Alsaud et al., 2021).

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Fig. 1

Recovery of phtychemical content from C. micranthum: (A) total phenolic content (TPC) and (B) total flavonoid content (TFC). a-l = Different lowercase letters indicate significant differences between solvents (P < 0.05). A-L = Different uppercase letters indicate significant differences between the extraction methods (P < 0.05).

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The extraction methods did not have significant effects on the TPC, however, the TFC was significantly affected by the extraction methods (p < 0.05). For TPC, UAE was found to be the best method with extraction efficiency higher than HAE and ME. While the ME showed the best performance for the recovery of TFC, followed by UAE and HAE, respectively. It is worth mentioning that the dissolution of the phenolic compounds of C. micranthum leaves in the solvents was slow with ME (12 h), whereas UAE and HAE achieved close results with a very short time (30 min). These results are in close agreement with the previous studies where the novel extraction methods such as microwave-assisted extraction and ultrasound-assisted extraction have been reported to reduce significantly the extraction time as compared to maceration (Yılmaz et al., 2020; Tambun et al., 2021; Frohlich et al., 2022). The high yield and short timeframe of UAE and HAE are due to the disruption of the cell walls thanks to the ultrasound and high rotation speed, respectively. Wu et al. (2020) have demonstrated that the NADES-based UAE remarkably showed higher TPC, TFC, DPPH, ABTS⁺, OH⁻ and FRAP values than maceration and stirring extraction. Also, Chanioti and Tzia (2018) have proved that NADES-based HAE was the best method compared to ultrasound-assisted extraction, high hydrostatic pressure-assisted extraction and microwave-assisted extraction for the extraction of phenolic compounds from olive pomace. Although ME requires a long extraction time, UAE, HAE and ME could be good choices to perform the extraction of phenolic compounds with NADES.

3.2 Antioxidant capacity of C. micranthum

The antioxidant capacity of C. micranthum leaves was measured with DPPH and FRAP assays which are the most common antioxidant capacity assays used in the literature. The DPPH assay displayed the antiradical capacity of the extract, while the FRAP assay measures the ability of the extract to reduce the ferric ions. The results of the antioxidant capacity of C. micranthum leaves were given in Table 1. As can be seen, both antiradical activity and FRAP of C. micranthum extracts were significantly affected by the types of solvents (p < 0.05). The total antiradical activity was ranged from 73.33 ± 19.69 mmol TE/g to 255.22 ± 3.94 mmol TE/g, while the FRAP changed from 43.60 ± 3.73 mmol ISE/g to 160.27 ± 0.39 mmol ISE/g. The extracts obtained from ChLa, ChAa and ChTa provided the highest antioxidant capacity compared to the conventional solvents. Several studies have mentioned the same tendencies, reporting the highest antioxidant capacity from NADES extracts obtained from acidic NADESs when compared to aqueous, methanolic and ethanolic extracts (Bakirtzi et al., 2016; Chanioti and Tzia, 2018; Barbieri et al., 2020; Alsaud et al., 2021). The highest antiradical activity was observed with ChLa, followed by ChAc and ChTa, whereas the best FRAP was achieved with ChTa followed by ethanol and ChAa. Since the NADESs with higher antiradical activity were found different from those giving higher FRAP, it can be concluded that the composition of NADES influences greatly the extraction of the targeted analytes. Many previous studies have been reported the same behavior of NADESs on the antioxidant capacity of various plant materials (Bakirtzi et al., 2016; Barbieri et al., 2020; Oliveira et al., 2021; Alsaud et al., 2021). Although the UAE proved to be the best method, there was no significant statistical difference was determined between UAE, HAE and ME (p < 0.05). Thus, these extraction methods are well-adapted to assist the acidic NADESs for the extraction of antioxidants from plants.

3.3 Phenolic profile of C. micranthum leaves

The phenolic compounds extracted from C. micranthum using the acidic NADESs and conventional solvents combined with ME, UAE and HAE were shown in Tables 2, 3 and 4. Twelve phenolic compounds including gallic acid, catechin, hydroxybenzoic acid, chlorogenic acid, vanillic acid, epicatechin, syringic, ρ-coumaric acid, ferulic acid, sinapic acid and quercetin-3-glucoside were identified in all the extracts. According to the literature in the C. micranthum, gallic acid, catechin, epicatechin, caffeic acid, chlorogenic acid, ρ-coumaric acid, vanillic acid and syringic acid have been reported in various extracts C. micranthum (Touré et al., 2011; Kpemissi et al., 2019; Kpemissi et al., 2020; Zeitoun et al., 2020).

3.4 Correlation uisng Pearson's correlation test

3.5 Correlation and properties of differents extracts obtained with acidic NADESs and conventional solvents using ME, UAE and HAE

Principle component analysis (PCA) was performed to display the correlation of TPC, TFC, the antioxidant capacity and the phenolic compounds extracted from C. micranthum by using the green acidic NADESs combined with UAE, HAE and ME (Fig. 2). A total of sixteen variables (TPC, TFC, DPPH, FRAP and 12 individual phenolic compounds) were used for the PCA, contributing to a total of 79.27% of the total variation extraction experiments. The points on the loading plot assigned the contribution of a variable to the score, while the points on the score plot represented an investigated sample. The first principal component factor (PC1) contributed for 64.4% of the total variation of extraction experiments while the second principal component factor (PC2) described 14.83% of the variations. As can be seen in the PCA biplot, most of the investigated variables had positive effects on PC1, except FRAP, catechin and TFC which had a negative effect on PC1. Gallic acid, chlorogenic acid, quercetin-3-glucoside, vanillic acid, DPPH, ferulic acid, sinapic acid, TPC, ρ-coumaric acid and syringic acid positively correlated with each other. Furthermore, there are positive correlations between TPC, syringic acid, epicatechin, hydroxybenzoic acid, caffeic acid and gallic acid. Likewise, there is a positive correlation between FRAP and catechin.

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Fig. 2

PCA biplot of the extracts obtained from C. micranthum with NADESs and conventional solvents combined with ME, UAE and HAE.

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The coded values of the PCA score plot representing the extracts were shown in Table 6. According to the PCA score plot, 4 main groups of extracts were observed: (1) ME4, ME6, UAE4, UAE5, UAE6, HAE4, HAE5 and HAE 6; (2) ME1, UAE1 and HAE1; (3) ME2, UAE2 and HAE2; and (4) ME3, UAE3 and HAE3. The extracts of the group (1) confirmed that the best efficiencies were achieved with UAE, HAE and ME combined with ChLa ChAc and ChTa. These extracts conditions were particularly efficient for the recovery of TFC, chlorogenic acid, gallic acid, quercetin-3-glucoside, vanillic acid, DPPH, ferulic acid, sinapic acid, TPC, ρ-coumaric acid and syringic acid. The extraction time (12 h) applied during the maceration extraction was quite long for the solvents to penetrate and break the cell walls of the raw material, allowing the analytes to dissolve efficiently in the solvents. As can be acknowledged, the great performance of HAE at a short time (30 min) could be related to both processes of raw material-pulverizing with mechanical shear force and mixing of solid with solvent (Sun et al., 2017; Duan et al., 2018; Chanioti and Tzia, 2018). Likewise, the highest performances achieved by UAE at a short extraction time (30 min) could be attributed to the ultrasound waves that disrupted the plant cell walls, increased the solvent penetration and improved the mass transfer between the solvents and solute (Hossain et al., 2012; Altemimi et al., 2016).

The values of the group (2) revealed that the extracts obtained with water using ME, UAE and HAE showed similar characteristics of syringic acid, TPC, epicatechin, hydroxybenzoic acid caffeic acid and gallic acid. ME, UAE and HAE were found effective for the extraction of these phenolic compounds. The highest recovery of these phenolic compounds could be attributed to their high hydrophilic properties (Deng et al., 2012; Rodríguez-Roque et al., 2015). The samples of groups (3) and (4) indicated that the extraction of phenolic compounds from C. micranthum using ME, UAE and HAE combined with pure methanol and ethanol was less efficient. In addition, the lowest yields were determined with the ethanolic extracts. The previous studies have demonstrated that the extraction of antioxidants such as phenolic compounds with pure methanol or ethanol is less efficient and many authors have suggested the addition of 10–50% of water to pure methanol or ethanol in order to improve their efficiency in extracting the bioactive compounds (Odabaş and Koca, 2016; Kumar and Srinivasa Rao, 2020; Phuong et al., 2020; Saha et al., 2020).