Green-based methods to obtain bioactive compounds from Ilex guayusa Loes. using polar solvent

Abstract Many biological activities have been reported for the Ilex genus. However, few studies in the literature have reported on guayusa. To address this gap in our knowledge, chemical analysis of guayusa leaves was made. Extracts were obtained by applying Soxhlet, maceration, supercritical CO2 and pressurised liquid extraction techniques, using water and ethanol as solvent/cosolvent. Extracts were evaluated for their phenolic content and antioxidant capacity. The chemical profile was obtained from HPLC. In raw guayusa leaves were identified caffeine (2.27 ± 0.05%), protein (15.31 ± 0.07%) and lipids (11.81 ± 0.14%). Extracts presented the highest phenolic content (156.56 ± 1.32 mg GAE g−1) and the best antioxidant activity (EC50= 61.85 ± 0.21 µg mL−1) when water was used as solvent/cosolvent. Through HPLC, three main substances were determined and quantified in the extracts: caffeine, theobromine and 5-caffeoylquinic acid. Based on these results, guayusa may be considered a natural source of compounds with potential application in the food and pharmaceutical industries. Graphical Abstract


Introduction
Guayusa (Ilex guayusa Loes.) belongs to the Ilex genus, Aquifoliaceae family. The Ilex genus is present worldwide with approximately 600 species (Pardau 2016). Some species from Ilex genus, such as yerba mate (Ilex paraguariensis), have been exploited because of stimulant and health-promoting properties associated with the presence of caffeine and other secondary metabolites (Garcia-Ruiz et al. 2017;Villac ıs-Chiriboga et al. 2018;Cadena-Carrera et al. 2019).
Guayusa has been used for centuries by native people of western Amazon region of three countries: southern Colombia, northern Peru but mainly Ecuador (Villac ıs- Chiriboga et al. 2018). Nowadays, guayusa is considered as an alternative to yerba mate when comparing its organoleptic and health-promoting properties, because of that, indigenous farmers in Ecuador produce guayusa in the agroforestry system and the export volume of this commodities has increased markedly. Between 2011 and 2019 guayusa exports showed an increasing trend reaching 294 tons in 2019. The US was the main importer of guayusa leaf, totalling the equivalent of 721 tons for this period. In addition to exporting in its raw form, this plant can be exploited to generate products with higher value added and more opportunities for use, contributing to sustainable development of producing regions and local farmers in the Andean rainforest (UN Comtrade 2021).
High-pressure extraction techniques such as supercritical fluid extraction SFE and pressurised fluid extraction PLE represent an alternative to traditional extraction techniques by using solvents that may be considered environmentally friendly. SFE with CO 2 as solvent (SC-CO 2 ) has many properties widely reported in the literature, but has non-polar solvent characteristics (Brunner 1994;Rosa et al. 2009). PLE has no limitation regarding polarity, as it uses high pressure allowing an increase in extraction rates, thus reducing the time of extraction and amount of solvent (Rosa et al. 2009). Up to now, however, few studies are available in the literature applying SFE (Londoño-Larrea et al. 2018;Cadena-Carrera et al. 2019) and none applying PLE. Therefore, the aims of this study were to evaluate the chemical composition of guayusa leaf and to compare low-pressure extraction techniques (Soxhlet and maceration) using a polar solvent (water) with high-pressure techniques (SC-CO 2 and PLE) using polar cosolvent/solvent (ethanol and water) regarding chemical profile by means of HPLC analysis and antioxidant capacity (total phenolic content TPC, Trolox equivalent antioxidant activity TEAC and DPPH radical (EC 50 )).

Results and discussion
The centesimal composition of guayusa leaves is presented in supplementary material, Table S1.
The global yield, phenolic content, antioxidant capacity (TEAC and EC 50 ) and the chemical profile quantified in guayusa extracts obtained by different extraction techniques is presented in supplementary material, Table S2.

Centesimal composition of guayusa leaves
The centesimal analysis of guayusa leaves found in this research was compared to data reported by Wise (2017) who analyzed and compared guayusa and yerba mate.
Differences in chemical composition may, for example, be attributed to species characteristics, geographical conditions, plant or leaf age, year or season (Pardau 2016;Villac ıs-Chiriboga et al. 2018).

Global yield (X 0 )
Global yield (X 0 ) was obtained using different extraction methods for guayusa leaves. For low-pressure techniques, the highest yield was obtained for the Soxhlet technique (39.35 ± 0.24%). Higher extraction temperatures applied in the Soxhlet technique allowed higher yields because higher temperature favors interactions between solute and matrix and also reduces the viscosity and surface tension of the solvent, allowing it to diffuse more easily in the matrix, the recycling of solvent also contributes to increased yield because, in each cycle, a fresh solvent is in contact with the matrix.
The global yield of SFE with water as cosolvent ranged between 27.13 ± 1.86% and 32.01 ± 2.41%. For this technique, no statistical difference was noted between the values of global yield for the different combinations of pressure and temperature applied. This could be attributed to the effect of cosolvent polarity in association with the attraction forces between solvent and solute , which was stronger on global yield when compared to either pressure or temperature. For the PLE technique, the global yield varied between 10.40 ± 0.56% and 24.58 ± 3.20%. Higher yields were obtained when water was used as solvent, rather than ethanol, suggesting that compounds present in guayusa leaves have intermediate to high polarity . When ethanol was used in the PLE technique, temperature had a positive effect on global yield (Figure (S1a) and Table S2); thus, higher yields were obtained with higher temperatures. For this technique, when water was used as the solvent (Figure (S1b) and Table S2), no statistical difference was observed in global yield when temperature increased from 60 to 73 C. This result occurred because the effect of solvent polarity was stronger than the effect of temperature, as demonstrated by a Pareto analysis (Table S3 and Figure S2). Although high extraction temperature was used (73 C), it was lower than the boiling point of water (100 C); consequently, solvent properties, such as dielectric constant, diffusivity, viscosity and density, were not influenced.

TPC, TEAC and DPPH (EC 50 )
TPC and antioxidant capacity (TEAC and EC 50 ) of extracts varied depending on solvent, pressure, temperature and/or technique applied. The highest value for TPC was obtained in the Soxhlet technique (63.13 ± 0.22 mg GAE g À1 ). Higher temperature, long extraction times and renewal of solvent applied in Soxhlet enhanced the obtention of phenolics, that is, owing to the positive effects of higher temperature on solvent properties that favours solute-solvent interactions, long periods of time that allows more interactions between solvent and compounds and finally, renewal of solvent that increases the difference of chemical potential, that promotes the transport of compounds to the unsaturated solvent (Rosa et al. 2009;Tramontin et al. 2019).
For high pressure techniques, SFE with water as cosolvent, values were low, ranging between 28.34 ± 0.47 mg GAE g À1 and 43.25 ± 1.20 mg GAE g À1 . For PLE when using ethanol as solvent, the lowest values of TPC were obtained, that is, 9.24 ± 0.53 mg GAE g À1 and 13.00 ± 1.10 mg GAE g À1 . TPC values were improved when using water (34.95 ± 2.79 mg GAE g À1 and 46.33 ± 5.01 mg GAE g À1 ) because of polar characteristics of phenolic compounds. However, TPC values were lower when compared with values obtained with Soxhlet in PLE, although fresh solvent was applied, that promotes the transport of compounds to the unsaturated solvent, the shorter periods of extraction (30 min), limited the time of contact between solvent and solute, as a result, less fresh solvent was in contact with the matrix.
TEAC varies from 11.93 ± 2.77 mmol TE g À1 to 156.56 ± 1.32 mmol TE g À1 , and the highest values were obtained for PLE when using water at 60 C and 73 C (154.36 ± 7.70 mmol TE g À1 and 156.56 ± 1.32 mmol TE g À1 , respectively). In this case, the value was higher than that obtained for Soxhlet (28.93 ± 1.56 mmol TE g À1 ), possibly because compounds responsible for antioxidant capacity detected by ABTS (EC 50 ) were extracted more efficiently in PLE by the use of high pressure, which enhanced the interactions between solvent and the matrix this allowed to overcome the matrixcompounds also when using PLE a swallowing effect occurs, this allows more interaction between solvent and compounds (Rosa et al. 2009).
Lower values of EC 50 , that is, better antioxidant activity, were obtained from the Soxhlet and maceration techniques (61.85 ± 0.21 and 98.50 ± 2.55 mg mL À1 ). Higher values of EC 50 , that is, less antioxidant activity, were obtained from SFE ranging from 109.25 ± 3.89 to 167.95 ± 3.32 mg mL À1 and from PLE when using water as a solvent for temperatures of 60 C and 73 C (131.23 ± 1.17 and 164.56 ± 0.66 mg mL À1 , respectively).

Chemical profile
The majoritarian compound detected in all guayusa extracts was the pseudoalkaloid caffeine ranging from 95.17 to 135.81 mg g À1 . The phenolic 5-caffeoylquinic acid was the second, ranging from 1.17 to 7.01 mg g À1 , and the lowest concentrations were for the pseudoalkaloid theobromine, ranging from 0.52 to 2.18 mg g À1 . Although rutin has been reported in guayusa leaf extracts (Garcia-Ruiz et al. 2017;Villac ıs-Chiriboga et al. 2018), it was not detected in the guayusa leaf extracts obtained in this research. The highest contents of caffeine and theobromine were observed for PLE with ethanol, which may have resulted from the intermediate polarity of ethanol and because the extraction temperature in PLE was close to the boiling point of ethanol, thus modifying dielectric constant value, that is, polarity, and capacity to extract methylxanthines . The highest content of 5-caffeoylquinic acid was for Soxhlet,maceration,and SFE at 45 C and 15 MPa,PLE with water at 60 C and 73 C and ethanol at 73 C, possibly owing to the polar characteristic of this phenolic acid, which is more soluble in polar solvents, such as water or ethanol. High yields of methylxanthines, mainly caffeine, when ethanol is used as solvent, have been reported (Brunner 1994;Cadena-Carrera et al. 2019).

Experimental
See supplemental material for standards and chemicals, SFE, PLE and low pressure extraction, antioxidant activity assays, and HPLC procedure.

Conclusions
Guayusa leaves appeared as a natural alternative source of caffeine, showing higher values when compared to Ilex paraguariensis. Extracts obtained from guayusa (Ilex guayusa Loes.) using polar solvents, water and ethanol showed good antioxidant activity when tested by the TPC, TEAC, and EC 50 methods. Water, as a cosolvent/solvent, was better than ethanol for obtaining extracts from guayusa leaves with high global yields and antioxidant capacity. The best extraction protocol depended on which response was aimed, regarding global yield, TPC, EC 50 the low pressure technique Soxhlet showed better results, and on the other hand, high-pressure technique PLE with ethanol or water showed better results regarding chemical profile. The compounds identified in the guayusa extracts were mainly caffeine, theobromine and phenolic 5-caffeoylquinic acid, but rutin was not detected. High caffeine content in guayusa extracts was obtained for PLE when using ethanol as solvent. High 5-caffeoylquinic acid content was obtained with Soxhlet, maceration, and SFE at 45 C and 15 MPa, PLE with water at 60 C and 73 C and ethanol at 73 C. Overall, guayusa represents a renewable source of high-value compounds, and further research is needed, applying other extraction techniques/solvents or analysis techniques to elucidate all the compounds present.
The present study developed an in vitro analysis of antioxidant capacity by means of ABTS and DPPH radicals, in vitro methods has some limitations, so it would be interesting to test the antioxidant capacity of extracts using in vivo methods in order to determine the total antioxidant capacity of the extracts that translates into tissues. Also the present study tested the properties of raw extracts; it would be interesting to isolate the compounds present in the raw extract and to evaluate the antioxidant and/or biological activities of isolates from extracts of guayusa.