In silico solvent selection for the extraction of BB bioactives

The simultaneous extraction of both Bet and BAc from birch bark was evaluated in silico for the first time in this paper on the basis of their potential interactions estimated via COSMO-RS theory. Thus, the sigma profiles of both triterpenoids (Fig. 1) revealed that they are able to establish not only apolar interactions (–0.01 < σ < 0.01), but they are also capable of acting as hydrogen bond donors and acceptors, as shown by the peaks at σ < –0.01 and σ >  + 0.01, respectively. These common characteristics make their simultaneous extraction feasible under similar operating conditions.

Fig. 1

Chemical structures, sigma surfaces and sigma profiles of betulin and betulinic acid estimated by COSMO-RS

Taking into account the information provided by COSMO-RS, the use of a single h-NADES for the simultaneous recovery of Bet and BAc was considered. To select the HBD/HBA candidates with the best extraction performance, the individual solvent capacity of 25 terpenoids for these bioactives was evaluated. Monoterpenes such as limonene, which were previously described for the extraction of triterpenoic acids [10], as well as numerous terpenoids (e.g. menthol, thymol, camphor, etc.) reported as components of h-NADESs exhibiting improved extraction of plant and food bioactives [10, 21], were included in this study. Moreover, for comparison purposes, conventional organic solvents that were previously reported for the extraction of these compounds (e.g. methanol, ethanol and acetone) were also considered (Fig. 2).

Fig. 2

Infinite dilution solvent capacity (\(_^\)) of solvents under study estimated by COSMO-RS for Bet and BAc at 25 °C. Terpenoids and conventional organic solvents are shown as pale-colour and dark-colour bars, respectively

Thymol and carvacrol provided the highest \(_^\) for Bet (\(_}^\), 21.7 and 13.3, respectively); these values were also significantly higher than those provided by other terpenoids or conventional solvents. With respect to BAc, the \(_}^\) values were higher for acetone and isopulegol (23.1 and 12.5, respectively), with thymol (among other solvents) yielding intermediate results (\(_}^\)= 6.2). Considering the intended simultaneous extraction of both Bet and BAc, the sum of the solvent capacity for Bet and BAc (\(_+\text}^\)) was chosen as the criterion for selecting the optimal solvent. As shown in Fig. 2, thymol was the compound providing the highest \(_+\text}^\) value; therefore, it was selected as the first component of the h-NADES to be used in this study.

Because thymol mainly acts as a HBD, compounds such as alcohols and carboxylic acids were considered in the selection of the second component (HBA) of thymol-based h-NADESs. Eutectic mixtures of thymol and the different members of the homologous series of 1-alkanols and n-alkanoic acids were simulated to estimate the corresponding eutectic temperature (Teutectic). As shown in Fig. 3, the mixtures including alcohols had lower estimated melting temperatures than those obtained with acids did. Therefore, the former (specifically from ethanol to 1-hexadecanol) were chosen for additional assays to ensure that the h-NADESs to be prepared were liquid at room temperature and that no additional heating was needed. This energy savings would also positively contribute to the development of greener methods for BB bioactive extraction.

Fig. 3

Variation of estimated eutectic temperatures (Teutectic, °C) with the number of carbon atoms of n-alkanoic acids and 1-alkanols used as the second component of thymol-based h-NADES (1:1 molar ratio)

Fig. 4

Effect of the thymol:1-alkanol molar fraction (xthymol, from 0.2 to 0.8) and the chain length of the 1-alkanol on \(_+\text}^\) at 25 °C predicted by COSMO-RS

The last calculation step involved estimation of the \(_+\text}^\) values of the thymol:1-alkanol (from ethanol to 1-hexadecanol) mixtures with different HBD:HBA molar fraction (xthymol, from 0.2 to 0.8) at 25 °C. As shown in Fig. 4, a molar ratio of 4:1 (xthymol = 0.8) resulted in the highest solvent capacity for all the alcohols that were tested (\(_+\text}^\)= 20.3–21.7). With respect to the number of carbon atoms, the highest \(_+\text}^\) value was obtained for C7–C8 alcohols. Considering that low volatility and low hydrophilicity are desirable characteristics for obtaining green hydrophobic NADESs, the thymol:1-octanol (4:1) mixture was chosen as the optimal extractant of Bet and BAc (\(_+\text}^\) = 21.7).

The sigma profiles and sigma potentials for thymol:1-octanol (4:1) h-NADES, betulin, and betulinic acid are shown in Fig. 5. The shape of the sigma potentials allows us to rationalize the expected ability of the selected h-NADES to easily coextract both bioactive compounds. There is a perfect match in the region of −0.01 < σ < 0.01, indicating favourable hydrophobic and electrostatic interactions. Moreover, this h-NADES has a good capacity to interact through donor (σ < −0.01) and acceptor (σ >  + 0.01) hydrogen bonds because it presents strong negative sigma potential values in both zones. Notably, the calculations performed adequately modulated the intensity of the interactions between the selected h-NADES and the two bioactives to be extracted, because the descending branches of the sigma potential in the hydrogen bond donor/acceptor zones of thymol:1-octanol (4:1) are between those of the two bioactives irrespective of the zone considered. This finding indicates a weighted interaction intensity for both compounds, which presumably can result in their good coextraction. On the basis of the excellent results obtained in this simulation study, thymol:1-octanol (4:1) h-NADES was used in the laboratory to develop a green and efficient method for the extraction of birch bark target bioactives.

Fig. 5

Sigma profiles (a) and sigma potentials (b) of betulin, betulinic acid and thymol:1-octanol (4:1) h-NADES estimated by COSMO-RS

Simultaneous extraction of BB bioactivesExperimental evaluation of thymol:1-octanol (4:1) h-NADES as an extractant

Prior to the use of thymol:1-octanol (4:1) as an extractant of BB bioactive compounds, the thermal behaviour of this h-NADES was evaluated by DSC. The DSC thermogram (Fig. S1) clearly shows a melting point depression, with an onset temperature (Tm, onset) of 23.1 °C, which is below the melting point of thymol (51.5 °C) and significantly lower than the estimated ideal eutectic mixture temperature (Tm, ideal = 40.7 °C). This thermal profile confirms the formation of a stable liquid phase at working temperatures, which fulfills the functional criteria for classification as a deep eutectic solvent [36] and supports its use as a BB bioactive extractant.

Moreover, this h-NADES was physicochemically characterized in terms of density and viscosity to confirm its feasibility of application. Compared with other high-density hydrophilic NADESs for the extraction of plant bioactives [17] and different terpenoid-based h-NADESs [37], thymol:1-octanol (4:1) was characterized by a noticeably lower density (0.94 g cm−3) and viscosity (12.4 mPa·s). From an application point of view, these properties not only favour the extraction process but also contribute to improving its sustainability because simpler facilities and a lower energy demand are required when low-density and low-viscosity solvents are used as extractants.

SLE vs. UAE

To evaluate the experimental performance in terms of the extraction yield (mg g−1 sample) of the h-NADES previously selected in “In silico solvent selection for the extraction of BB bioactives”, SLE assays that use thymol:1-octanol (4:1) as the extractant were carried out under different temperature conditions (25 and 55 °C). As shown in Table 1, the extraction yield of both bioactives significantly increased with temperature, with values that were 79% and 39% greater at 55 °C for Bet and BAc, respectively. In agreement with these results, an improved extraction yield of triterpenoid acids such as BAc has been reported when conventional extractions (maceration, stirring, etc.) with either organic solvents (ethanol, CH2Cl2, etc.) or h-NADESs (e.g. menthol:thymol combinations at different molar ratios) are carried out at temperatures above ambient temperature [10].

Table 1 Concentration (average and standard deviation in brackets for n = 3) of betulin (Bet) and betulinic acid (BAc) recovered from sample BB1 by solid-liquid extraction (SLE) or ultrasound-assisted extraction (UAE) under different temperature conditions

The effect of the extraction technique was further evaluated by a comparison of SLE and UAE under identical extraction conditions (55 °C for 30 min). Compared with SLE, the use of an additional source of energy (US) in UAE improved the extraction yields of Bet (31%) and BAc (28%) (Table 1). These results are consistent with those of previous studies, which revealed that UAE, combined with organic solvents, outperforms (in terms of efficiency and speed) conventional techniques such as maceration for the extraction of birch triterpenoids [31, 32].

Development and analytical characterization of a green and efficient h-NADES UAE method for the simultaneous extraction of BB bioactives

A face-centred central composite experimental design was considered for the in-depth optimization of UAE operating conditions (time and temperature) in the ranges previously described in Table S1. In general, UAE experiments at low temperature (35 °C) or for short extraction times (up to 17.5 min) resulted in the lowest extraction of both bioactive compounds (Bet: 14.87–19.08 mg g−1; BAc: 0.61–0.78 mg g−1). Although an increased recovery of these two lupane-type triterpenoids was found when higher extraction temperatures were used (≥ 55 °C) (up to 26.78 mg g−1 Bet and 1.00 mg g−1 BAc), a significant degradation of both bioactives was detected at 75 °C, particularly when the extraction was extended for long times (down to 19.99 mg g−1 Bet and 0.89 mg g−1 BAc, respectively).

Response surface methodology was further used to calculate the regression coefficients of the models for the Bet and BAc responses (RBet and RBAc, respectively) and their statistical significance, as well as to estimate the prediction errors (standard error (SE) and mean absolute error (MAE)). The experimental conditions that individually maximized RBet and RBAc were also obtained (Table S2, Fig. S2). As indicated by the Pareto diagrams (Fig. S3), time (t) was the only significant factor at the 95% confidence level (p < 0.05) for both responses, and similar extraction temperature and time conditions were shown to be optimal for the individual extraction of either Bet or BAc (59 °C for 26 min and 62 °C for 22 min, respectively). With respect to the SE and MAE errors, the variability explained by the corresponding models was high enough to ensure the accuracy of the prediction. Moreover, when a multiple response (RD = 0.88) that simultaneously maximized RBet and RBAc was considered, 61 °C and 24 min were selected as the optimal UAE operating parameters to provide BB extracts rich in bioactives (25.17 mg g−1 Bet and 1.06 mg g−1 BAc, respectively). Under these conditions, a good match (relative error < 2%) between the experimental and predicted amounts of Bet and BAc recovered by UAE was obtained.

The number of extraction cycles of the optimized h-NADES UAE method was subsequently evaluated. As the percentage of both bioactives decreased similarly in cycles C1–C3 (87 > 9 > 4% for Bet and 85 > 10 > 5% for BAc), a single cycle was considered a trade-off to provide a high-throughput extraction method with enough recovery of both bioactive triterpenoids.

With respect to the development of not only an efficient UAE method based on the use of green solvents but also a sustainable approach reducing the energy demand to a minimum, the possibility of shortening the overall extraction process by considering the simultaneous preparation of the h-NADES thymol:1-octanol (4:1) and the UAE extraction of BB bioactives was considered under the UAE conditions previously optimized. However, the experimental recoveries of Bet and BAc via this joint approach were lower (15 and 13%, respectively) than those of the conventional two-step procedure. Therefore, this fully integrated method was discarded for applications in which maximization of the recovery of BB bioactives is intended, like in the present research.

The analytical characterization of the h-NADES UAE approach developed in this study was carried out by the LC-MS method previously optimized and validated for the analysis of Bet and BAc in biosolvent extracts, including the h-NADES thymol:1-octanol used here as an extractant [11].

As previously reported, no interference arising from the coelution of h-NADES components with target bioactives was detected by LC-MS analysis of these BB extracts (Fig. S4). The linearity of this LC-MS method in terms of the concentration ranges encompassing the concentrations of Bet and BAc in h-NADES UAE extracts and well above the detection limits of this separation method (23 and 29 ng mL−1 for Bet and BAc, respectively) was also determined (Bet: y = 2.68·108x − 573,712, R2 = 0.992; BAc: y = 2.39·108x + 27,502, R2 = 0.997).

The figures of merit regarding the reproducibility and recovery of this new extraction method are shown in Table S3. On the basis of intraday and interday precision data (RSD ≤ 1.60% for Bet and RSD ≤ 2.27% for BAc) and recoveries estimated after the BB1 sample was spiked at three concentrations (≥ 96.22% and 79.88% for Bet and BAc, respectively), a precise and high enough accurate extraction of both BB bioactives was achieved via the h-NADES UAE methodology developed in the present study.

The quantitative sustainability assessment of this method was further evaluated by the AGREEprep metric [34]. Table S4 lists the assigned weights and individual scores estimated for each of the GSP principles, whereas the overall greenness score obtained by this method is presented in the pictogram of Fig. 6. The use of nonhazardous materials such as h-NADESs and the low volume of waste that is generated (factors 2 and 4), the minimal amount of a forestry byproduct rich in bioactives (factor 5) and operator safety (factor 10) were the main positive contributions to the AGREEprep overall score (0.76). The inevitable ex situ sample preparation placement was, in contrast, the most negative contribution.

Fig. 6

AGREEprep pictogram with the overall greenness score obtained in the sustainability assessment of the optimised h-NADES UAE method

Application of the optimized h-NADES UAE method to different BB samples

Table 2 summarizes the results obtained after the application of the h-NADES UAE method previously optimized for the simultaneous extraction of Bet and BAc from samples BB1–BB6. A wide variability in the concentrations of BB bioactive compounds was observed for the samples considered in this study. Although the UAE extract of BB5 was richer in both bioactives (32.17 mg Bet g−1 and 1.43 mg BAc g−1), the content of these lupane-type triterpenoids was not fully dependent on the species considered, because the concentrations of Bet and BAc were significantly lower in the other B. pendula extracts.

Table 2 Concentration of betulin (Bet) and betulinic acid (BAc) (average and standard deviation in brackets for n = 3) in the h-NADES UAE extracts obtained under optimal conditions (thymol:1-octanol (4:1), 61 °C for 24 min, 1 cycle) from birch bark samples under study

As previously described [12, 15, 27, 29], the concentration of bioactive triterpenoids in BB extracts is affected not only by the extraction procedure and conditions that were employed but also by many other factors, including Betula species, provenance and growth conditions, age of the tree and tissue considered, etc. Thus, contents of BAc (2.8 mg g−1 sample, Betula species not detailed) and Bet (in the range 20–30 mg g−1 depending on the type of birch bark) similar to those of the present study have been reported in the application of other advanced extraction techniques, such as subcritical water extraction (SWE, 27 min, 184 °C) [26] or ethanolic PLE (15 min, 120 °C) [24]. Although in both cases the extraction time was similar to that of the h-NADES UAE method here developed (24 min), the use of a noticeably lower extraction temperature (61 °C) in the present work would support its higher sustainability.

Considering the limited number of studies that address the use of h-NADESs for the extraction of bioactive triterpenoids, a lower extraction yield of BAc (0.41 mg g−1) and a much longer extraction time (4 h) have been reported in the SLE of Eucalyptus globulus biomass at 90 °C when menthol:thymol (molar ratio of 1:2) was used as the extractant [10]. These results support the previously mentioned requirement of careful selection of the most appropriate biomass and the optimal extraction approach when extracts rich in bioactive triterpenoids are intended.