About 3 kg of fresh oranges, Citrus Sinensis, of Navel Lane Late variation were purchased from a local store in Madrid (Spain). The oranges were squeezed, and the peels were dried in an oven at 313 K for 24 h, then, ground in a mill until a homogenous powder was obtained. The resultant powder, labeled as orange peel waste (OPW), was stored in a freezer, and maintained at 269 K until experiments were carried out. A schematic diagram of the preparation of the OPW can be observed in Fig. 1.

Fig. 1.

Representation of the orange peel waste (OPW) preparation.

The solvents used in this work were deionized water, ethanol absolute (CAS Number: 64-17-5; purity $\ge$99.5 wt.%), supplied by J. T. Baker (Gliwice, Upper Silesia, Poland), and ethyl acetate (CAS Number: 141-78-6; purity $\ge$99.5 wt.%), supplied by HoneyWell (Seelze, Lower Saxony, Germany). In all cases, solvents were used as received without any additional purification.

The Folin–Ciocalteu phenol reagent (FC) (2N) (CAS number: 12111-13-6) and sodium carbonate anhydrous (CAS Number: 497-19-8; purity $\ge$99.5%) were used to perform the Folin–Ciocalteu method following purchase from Sigma-Aldrich (St. Louis, MO, USA). To determine the flavonoid content in each essay, sodium nitrite (CAS Number: 7632-00-0; analysis purity) from Sigma-Aldrich, aluminum chloride (CAS Number: 7446-70-0; purity $>$98%) from EMD Millipore Corporation (Darmstadt, Hessen, Germany), and sodium hydroxide (CAS Number: 1310-73-2; for certificate analysis) from J.T. Baker were used to conduct the corresponding aluminum complexation reactions.

The chemicals used as standards for HPLC analysis were hesperidin (CAS Number: 520-26-3; purity $\ge$80 wt.%), naringin (CAS Number: 10236-47-2; purity $\ge$95 wt.%), (+)-catechin hydrate (CAS Number: 225937-10-0; purity $\ge$98 wt.%), rutin hydrate (CAS Number: 207671-50-9; purity $\ge$94 wt.%), quercetin (CAS Number: 117-39-5; purity $\ge$95 wt.%), gallic acid (CAS Number: 149-91-7; purity $\ge$97 wt.%), caffeic acid (CAS Number: 331-39-5; purity $\ge$98 wt.%), p-coumaric acid (CAS Number: 501-98-4; purity $\ge$98 wt.%), and trans-ferulic acid (CAS Number: 537-98-4; purity 99 wt.%), all of which were purchased from Sigma-Aldrich. For the HPLC mobile phase, acetonitrile (CAS Number: 75-05-08), and water (HPLC grade purity) were purchased from Honeywell, and acetic acid (CAS Number: 64-19-7; purity $\ge$99 wt.%) was bought from Labkem (Vilassar de Dalt, Barcelona, Spain).

All the extractions were carried out by adding 0.5 g of OPW to 5 mL of the solvent (i.e., biomass:solvent ratio = 1:10, in g/mL). In this work, deionized water (H${}_2$O), a solution of 80% (v/v) ethanol in water (80% EtOH), and ethyl acetate (EA) were used as solvents.

Extraction conditions for each technique (orbital shaker, ultrasound, and microwave) were evaluated in terms of extraction time and temperature. The selected values for each variable were based on preliminary assays and the operative limits of each instrument.

For orbital shaker extraction (OSE), an orbital shaker (Labnet VORTEMP 1550 orbital shaker) with controlled temperature and an accuracy of $\pm$ 0.5 K was used. The extractions were performed by varying the extraction temperature between 303 and 333 K, and the extraction time at 30, 60, and 90 min, under a fixed shaker rate of 900 rpm.

For ultrasound bath-assisted extraction (UBAE), a bath (Elmasonic S 15 H, Elma) was used with an ultrasound frequency of 37 kHz, power of 35 W, and a controlled temperature. In this case, the extractions were carried out at 333 K for 30 min.

Ultrasound probe-assisted extraction (UPAE) was evaluated using a portable ultrasonic homogenizer UP200Ht (200 W, 26 kHz) (Hielscher Ultrasound technology). The extraction process was carried out using a power of 10 W (based on preliminary tests) and various extraction times of 1.5, 3, and 6 min. Although temperature could not be controlled in this case, it was measured at the end of each experiment.

A microwave reactor (Monowave 400, Anton Paar) equipped with an infrared temperature sensor for temperature control was used in microwave-assisted extractions (MAEs). For the microwave extraction, a hermetically closed vessel was used, with a fixed stirring rate of 1000 rpm. Samples were heated at 333 and 373 K for 1.5, 3, or 6 min.

After all extraction assays were performed, the obtained samples were centrifuged with Unicen 21 Centrifuge (Ortoalresa, Spain) for 10 min at 4200 rpm, to promote phase separation. The extracts were analyzed using different analytical techniques, which are described in the following section.

First, the compounds of interest were extracted from OPW and evaluated using the orbital shaker to study the effect of temperature on the process. The experiments were performed at 303 and 333 K, and the extraction time was maintained at 60 min. In this case, temperatures higher than 333 K were not tested to avoid possible thermal degradation of phenolic compounds since the extraction time used is quite prolonged. The results obtained from the TPC, TFC, and HPLC analyses of the extracts, for the three solvents evaluated, are shown in Fig. 2, and reported in Supplementary Table 1, available as Supporting Information.

Fig. 2.

Effect of temperature on the extraction from OPW using the orbital shaker. (A) Total phenolic content (TPC), total flavonoid content (TFC), and (B) high-performance liquid chromatography (HPLC) quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; t${}_{\text{extraction}}$ = 60 min; T = 303 and 333 K; stirring rate: 900 rpm.

As can be seen in Fig. 2A, in general, TPC and TFC increase alongside temperature, except for water, for which the TFC values are quite similar at both temperatures. These results suggest that a higher temperature enhances the extraction of phenolic compounds from OPW using the orbital shaker. The positive effect of the temperature on the extraction, specifically using 80% EtOH and ethyl acetate as solvents, could be explained due to the reduction in their surface tension and viscosity with temperature, which favors solubility and increases the diffusion coefficients of phenolic compounds [39].

Comparing Fig. 2A,B, it is also possible to observe that the TFC and HPLC analyses show a similar trend, which can be related to the fact that hesperidin (a flavonoid) was the major compound identified in the extracts by HPLC, as can also be seen in Supplementary Table 1, available as Supporting Information. Nevertheless, it is important to remark that only eight phenolic compounds were identified by HPLC analysis (quercetin was not found in any analysis), and therefore, any discrepancies between TFC and HPLC may be due to the extraction of other flavonoids not identified by HPLC.

The increase in TPC values by temperature for water, which does not agree with the values obtained by the TFC and HPLC analyses, could be attributed to the low selectivity of this solvent; in particular, water could be promoting the extraction of other compounds from OPW, such as sugar, short-chain carbohydrates, and/or ascorbic acid [40], which are known to interfere with the Folin–Ciocalteu method due to their reducing capacities [41, 42]. The low selectivity of water was confirmed by HPLC analysis since the presence of gallic acid, (+)-catechin hydrate, and trans-ferulic acid in water extract is remarkable, which does not happen for the other two solvents. The percentage of hesperidin in the aqueous extract represents about 84% of the phenolic compounds identified, which is lower than with 80% EtOH (about 99%) or in ethyl acetate, where only hesperidin was identified (see Supplementary Table 1).

Fig. 2 illustrates that the best extraction efficiency was achieved when using 80% EtOH at 333 K. Specifically, TPC increased from 3.9 to 6.8 mg GAE/g OPW, TFC increased from 8.8 to 12.5 mg QE/g OPW, and the total phenolic compounds quantified by HPLC increased from 5 to 29 mg PC/g OPW following the increases in temperature. The effectiveness of the ethanolic mixture agrees with the results published by Li et al. [36], who compared the ability of pure H${}_2$O and a solution of 80% EtOH to extract phenolic compounds from citrus wastes. The use of ethanol enhances the extraction capacity because the solubility of the phenolic compounds in this alcohol is higher than in water, while the addition of water to pure ethanol favors the desorption of the solute from the matrix [43].

In contrast, lower extraction values were obtained when using ethyl acetate, which also increased as the temperature increased (TPC increased from 0.4 to 1.1 mg GAE/g OPW, TFC increased from 2.5 to 4.1 mg QE/g OPW, and total phenolic compounds quantified with HPLC increased from 0.1 to 0.3 mg PC/g OPW). For this solvent, it is important to remark that only hesperidin was identified in the HPLC chromatograms, thereby revealing that the presence of flavonoids and phenolic acids in this extract is limited in comparison with the other studied solvents (8.7–9.4 and 5.3–27.8 mg PC/g OPW for water and 80% EtOH, respectively). These results agree with those published by Safdar et al. [43], who compared 80% EtOH and pure ethyl acetate in the extraction of phenolic compounds from Citrus reticulate L. (25 mg GAE/g with 80% EtOH and 9 mg GAE/g with ethyl acetate). This could be explained due to the hydrophobic character of ethyl acetate, whose capacity to be introduced into the cell wall is lower than the hydrophilic solvents.

Taking all these results into account, the best temperature for stabilization was 333 K and then the effect of extraction time at this temperature was also evaluated using three different times (30, 60, and 90 min). The results for TPC, TFC, and HPLC analysis are shown in Fig. 3 and Supplementary Table 1 (available as Supporting Information).

Fig. 3.

Effect of time on the extraction from OPW using the orbital shaker. (A) Total phenolic content (TPC) and total flavonoids content (TFC), and (B) HPLC quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; T = 333 K; t${}_{\text{extraction}}$ = 30, 60, and 90 min; stirring rate: 900 rpm.

The obtained results show little or no influence of the stirring time on the extraction of phenolic compounds, suggesting that 30 min should be sufficient to achieve equilibrium when these solvents are used. A similar conclusion was also obtained by Vergara-Salinas et al. [44], who found no influence by extraction time, between 5 and 30 min when extracting phenolic compounds from thyme samples using pressured hot water as the solvent. Thus, the choice of a longer time can lead to no significant effect on the extraction efficiency, as suggested by Silva et al. [45]. The decreasing extraction efficiency for phenolic compounds at 90 min using water and 80% EtOH could be attributed to the oxidation of some phenolic compounds by extended exposure to oxidizing factors, such as oxygen and light [46, 47, 48].

The results obtained in this part of the study agree with those trends plotted in Fig. 2, confirming that 80% EtOH is the solvent with the highest extraction capacity (27.8–35.2 mg PC/g OPW) followed by deionized water (8.8–9.5 mg PC/g OPW), and ethyl acetate (0.3–0.4 mg PC/g OPW) from the HPLC results. Then, since longer extraction times do not promote further recovery of phenolic compounds from OPW by OSE, it could be concluded that the best operating conditions for this technique are 333 K and 30 min, thereby avoiding the possible degradation of the phenolic compounds and saving costs by reducing extraction time. Since the operational power of the orbital shaker is 515 W, the energy consumption for the optimal conditions (333 K and 30 min) is 927 kJ.

Extractions by an ultrasonic bath (USBE) were performed using the best conditions established for OSE (333 K and 30 min). The obtained results are reported in Supplementary Table 2, available as Supporting Information, and a comparison between the USBE and OSE results is also plotted in Fig. 4. The best extraction efficiency was also achieved using the 80% EtOH mixture as the solvent but the use of this ultrasonic bath did not improve the extraction process. In fact, the TPC and TFC values were lower for USBE than OSE, while HPLC analysis revealed a small increase in phenolic compounds in water (9.3 to 10.7 mg PC/g OPW) and ethyl acetate (3 to 4 mg PC/g OPW) extracts, although also a significant decrease in phenolic compounds in the 80% EtOH extract (35.2 to 17.4 mg PC/g OPW). In all cases, a higher percentage of hesperidin compared to the other seven phenolic compounds was detected.

Fig. 4.

Extraction from OPW by orbital shaker (OSE) and an ultrasonic bath (USBE). (A) Total phenolic content (TPC), total flavonoid content (TFC), and (B) HPLC quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; t${}_{\text{extraction}}$ = 30 min; T = 333 K.

Although ultrasound energy is commonly applied to intensify extraction by increasing the number of cavitation bubbles, which causes the cell walls to rupture, thereby reducing particle size and improving mass transfer [7, 49], the ultrasonic bath used in this work did not improve the extraction results. This could be due to the fact that this bath only allowed the temperature to be controlled, and no other parameters, such as frequency, power, or distance of the sample to the irradiation surface, known to affect the intensification process [24]. Moreover, the performance of the equipment could also be influenced by the orientation of the sample, sonication volume, and purity of the water used in the bath [26, 50]. It is worth noting that, although there was no significant improvement in the extraction efficiency using USBE, there was a reduction in energy consumption from 927 kJ, when using OSE, to 63 kJ with UBAE.

Since extraction using indirect ultrasound (ultrasonic bath) was not as effective as expected, an ultrasound probe was tested as an alternative for performing the ultrasound-assisted extraction (USPE). After some preliminary assays, the power was set at 10 W since higher power values resulted in solvent evaporation due to higher final temperatures, while the recipient was open. In this case, the extraction time effect was evaluated at 1.5, 3, and 6 min, reaching a maximum final temperature of 333 K. The results of the TPC, TFC, and HPLC analysis are shown in Fig. 5 and Supplementary Table 3 available as Supporting Information.

Fig. 5.

Extraction from OPW by ultrasound probe (USPE). (A) Total phenolic content (TPC), total flavonoid content (TFC), and (B) HPLC quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; t${}_{\mathrm{\text{extraction}}}$ = 1.5, 3 and 6 min; p = 10 W; T${}_{\mathrm{\text{max}}}$ $\approx$ 333 K.

Comparing the Figs. 3,5, it is possible to observe that the TPC, TFC, and HPLC results follow a similar trend, yet those obtained by the ultrasonic probe are clearly better. Furthermore, they were obtained at shorter times (30–90 min using an orbital shaker vs. 1.5–6 min for the ultrasound probe). These facts confirm that ultrasound-assisted extraction using a probe instead of a bath improves the extraction of phenolic compounds from OPW since the probe transmits a higher energy to the medium and the mass transfer is improved due to the lack of barriers in the propagation path of ultrasound waves, hence, enabling shorter extraction times [26, 51]. Once again, the best extraction efficiency was achieved using 80% EtOH as solvent.

The HPLC analysis clearly shows a decrease in the extraction of phenolic compounds as time increases, which can be due to the heat generated during the sonication process or the sample oxidation since the vials are opened. This can lead to a change in the characteristics of the medium, resulting in the degradation of the phenolic compounds [51]. Such decreases were not observed in the TPC and TFC values because sonication could also be facilitating the extraction of other compounds that can interfere with the spectrophotometric measurements. Therefore, a time of 1.5 min was considered enough to ensure a high extraction yield and avoid possible degradation of the phenolic compounds. In addition, the energy consumed during USPE (18 kJ) is significantly lower than during the extraction using the bath (63 kJ). Therefore, the direct use of ultrasound as an assisted extraction technique is a better option in terms of extraction and energy efficiency. In brief, ultrasound-assisted extraction using the probe is more efficient than the orbital shaker and ultrasound bath-assisted extraction, as the extraction time can be shortened from 30 min to 1.5 min, achieving better extraction yields.

Subsequently, the best conditions established for USPE (1.5 min and 333 K) were used to test whether microwave-assisted extraction (MAE) could improve the obtained results. Moreover, the study temperature was extended to 373 K to evaluate the effect of temperature and time alongside this type of extraction technology. The obtained results are plotted in Figs. 6,7, and reported in Supplementary Table 4, available as Supporting Information.

Fig. 6.

Extraction from OPW by ultrasound probe (USPE) and microwaves. (A) Total phenolic content (TPC), total flavonoid content (TFC), and (B) HPLC quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; t${}_{\text{extraction}}$ = 1.5 min.

Fig. 7.

Effect of time on the extraction from OPW by microwave-assisted extraction (MAE). (A) Total phenolic content and total flavonoid content, and (B) HPLC quantification of the flavonoids and phenolic acids present in the extract. Extraction conditions: biomass:solvent ratio = 1:10; t${}_{\mathrm{\text{extraction}}}$ = 1.5, 3, 6 min; T = 373 K; stirring rate: 1000 rpm.

As can be seen in Fig. 6, microwave-assisted extraction at 333 K failed to improve the results obtained by USPE. A possible explanation is that the energy required to increase the temperature up to 333 K is very low; therefore, the effect of the microwaves is also very small. Nevertheless, a significant improvement in extraction efficiency, especially for hesperidin, can be observed from the HPLC analysis when the temperature was set at 373 K (11.4 to 22.8 mg PC/g OPW in water, 22.3 to 43 mg PC/g OPW in 80% EtOH, and 0.1 to 0.4 mg PC/g OPW in ethyl acetate). At 373 K, the required energy is higher and the effect of microwaves on the sample is more relevant, which facilitates the desorption of the compounds from the matrix and the solvent penetration on the matrix by reducing the surface tension and surface viscosity [31]. On the other hand, microwaves accelerated extraction by increasing the temperature and internal pressure in the plant cell walls, which leads to an enhancement in porosity, thereby allowing the solvent to better penetrate [10].

Considering the good results obtained at 373 K, this work was completed by evaluating the effect of time on the MAE (Fig. 7), concluding that MAE efficiency increases with time while achieving the best results using 80% EtOH at 6 min (58.6 mg PC/g OPW). This can be explained by the fact that ethanol is known to be a good microwave absorber, which increases the extraction efficiency [52].

The energy required when using MAE for 6 min with a maximum operating power of 850 W was 306 kJ. The extraction using microwave and 80% EtOH as a solvent, enhanced the efficiency from 35.2 mg PC/g OPW using OSE to 58.6 mg PC/g OPW with MAE; moreover, MAE was able to reduce the energy consumption from 927 kJ to 306 kJ. This demonstrates that MAE is a good alternative to conventional extraction techniques.