Introduction

Brosimum alicastrum, commonly known as breadnut, ramon or Maya nut, is a tree distributed throughout the neotropics. Humans have found multiple uses for its vegetal elements. On the Yucatan Peninsula in southeast Mexico, the Maya use its leaves and bark in traditional medicine, and the seed of the fruit was a food resource for the ancient Maya (^{Moo-Huchin et al. 2019}, ^{Brechú-Franco et al. 2021}). Its leaves are used as fodder for sheep, pigs and rabbits (^{Sarmiento-Franco et al. 2022}). Some B. alicastrum products are marketed nationally and interationally, such as gluten-free flour made from the seed, roasted and ground seed as a coffee substitute, and tea from leaves. The fruit shell and seed skin are inedible by-products generated during processing which have no known use. The seeds are known to contain vitamins (A, C and B), minerals (Ca, K, Fe, etc.), fatty acids (linoleic acid and palmitic acid), nitrogen-free extract (starch and sugars) and essential amino acids (tryptophan, lysine, arginine and valine) (^{Carter and Northcutt 2023}).

Only limited research has been done on the phenolic compounds content and antioxidant activity of different B. alicastrum tissues using nonconventional extraction methods. In one study, phenolic compounds extraction of roasted and ground B. alicastrum seed with a Soxhlet extractor using an ethanol-distilled water mixture (80:20, v/v) at 80 °C for 30 min produced total phenolics content (TPC) (2467.0 ± 85.0 mg GAE 100 g⁻¹ extract) and DPP0048 radical scavenging activity (79.0 ± 2.7%) higher than in walnut, peanut and almond (^{Ozer 2017}). Another study compared extraction of unroasted B. alicastrum seed flour with an orbital shaker at 25 °C for 2 h using an ethanol-distilled water mixture (1:1, v/v) as solvent to other extraction solvents such as distilled water, methanol, ethanol, acetone, methanol-water (1:1, v/v) and acetone-water (1:1, v/v) (^{Moo-Huchin et al. 2019}); the ethanol-distilled water mixture produced the highest TPC content (1 230.12 ± 10. 61 mg GAE 100 g⁻¹ dry weight, dw) and DPPH antioxidant activity (872.75 ± 26.95 µM Trolox 100 g⁻¹ dw). One report found optimal conditions for ultrasound-assisted extraction (UAE) of phenolic compounds from B. alicastrum leaf powder to be 28 °C, 80% amplitude, 10 to 20 min UAE, and 80% aqueous methanol as extraction solvent; the resulting TPC was 45.18 mg GAE g⁻¹ and DPPH antioxidant activity was 67.27 µM Trolox g⁻¹ (^{Gullian-Klanian and Terrats-Preciat 2017}).

There is increasing interest in extracts rich in bioactive compounds with high antioxidant activity from B. alicastrum fruit and leaves. However, this requires a highly reproducible extraction method that provides high extraction yields in a short time, with low costs and easy development; an excellent option is UAE. The present study objective was to evaluate how the solvent-to-tissue ratio and the type of lyophilized tissue affects the efficiency of UAE in producing phenolic compounds from different tissues of B. alicastrum leaves and fruit.

Materials and methods

Results y discussion

Both factors, lyophilized tissue type (LT) and solvent-to-tissue ratio (STR), and their interaction (LT x STR) influenced (p ≤ 0.05) TPC and EY values (Table 1). The ethanol-water mixture (1:1 v/v) used here in the UAE of phenolic compounds was key to extraction results. Addition of distilled water in organic solvents (e.g., ethanol) generates a more polar medium, which facilitates extraction of phenolic compounds from plant matrices. In an ethanol- water system, ethanol breaks the hydrogen bonds and hydrophobic bonds existing between phenolic compounds and proteins, and phenolic compounds and cellulose (^{Kaderides et al. 2019}). A 50% ethanol concentration (in distilled water) produces the highest extraction yields from plant tissues, making it the most appropriate for phenolic compounds extraction (^{Drevelegka and Goula 2020}).

Average TPC values varied between the different B. alicastrum tissues (between 7.56 and 26.71 mg GAE g⁻¹ dw), seeds having the highest and seed skin the lowest: seed, 26.71 mg GAE g⁻¹; leaf, 21.40 mg GAE g⁻¹; shell, 10.51 mg GAE g⁻¹ and seed skin, 7.56 mg GAE g⁻¹. The seed TPC value was higher than the 11.61 ± 0.79 to 13.35 ± 0.80 mg GAE g⁻¹ reported elsewhere for B. alicastrum seeds (^{Losoya-Sifuentes et al. 2023}). Different TPC values have been reported for other B. alicastrum seed products. Dry extract of roasted seed has an average TPC content of 2 467 ± 85 mg GAE 100 g⁻¹ (^{Ozer 2017}), while seed flour has an average content of 1 230.12 ± 10.61 mg GAE 100 g⁻¹ (^{Moo-Huchin et al. 2019}). The present leaf TPC value (21.40 mg GAE g⁻¹) was higher than the 16.10 mg GAE g⁻¹ previously reported (^{Montes-Pérez et al. 2019}); a different study found a gallic acid content of 2.34 mg 100 g⁻¹ in lyophilized leaf extract (^{González-González et al. 2019}). Comparison of the present B. alicastrum fruit TPC values with those in the literature is challenging due to differences in tissue processing (roasting), extraction methods and results expression (dry extract weight, dry flour weight or extract volume).

Among the tissues analyzed here, the lyophilized shell had the highest EY (38.09%), while average EY for the leaf tissue was about two times higher than the seed and seed skin tissues (Table 1). This difference in EY values between tissues can be attributed to variation in morphology, structure and nutrient pool (glycoproteins and proteins) within B. alicastrum (^{Brechú-Franco et al. 2021}). In the leaf tissue, EY (19.94%) was higher than the 10% reported elsewhere (^{Dzib-Guerra et al. 2016}). Seed EY (11.92%) was comparable to the 11.0% previously reported by our team (^{Moo-Huchin et al. 2019}), but higher than the 1 to 6% reported for Ficus religiosa seeds (Moraceae) (^{Pinipay et al. 2022}).

The STR indicates the amount of solvent (mL) used in extraction of solute compounds (g). In the present results, the highest TPC value (17.89 mg GAE g⁻¹ dw) and lowest EY value (18.47%) were produced at the 60 mL g⁻¹ STR. Solvent viscosity and density are known to vary with increasing STR, which leads to higher EY values up to a certain point (maximum), after which they decrease as STR values increase (^{Kumar et al. 2021}). The high extraction efficiency observed in the present results is probably due to the fact that a higher STR reduces the density of the ethanol-water mixture, increasing ultrasound wave propagation velocity, consequently raising energy transfer and improving phenolic compound extraction efficiency from plant matrices (^{Dzah et al. 2020}). As solvent content increases in an extraction system, the effective ultrasonic intensity available for extraction reduces the number of extractables (^{Rao et al. 2021}); this explains the lower EY at 60 mL g⁻¹ (18.47%) relative to 40 mL g⁻¹ (21.02%). No difference in EY was observed between 20 mL g⁻¹ (20.52%) and 40 mL g⁻¹ (21.02%). This coincides with a study stating that EY does not vary between 20 and 40 mL g⁻¹ because certain compounds in the form of glycosides are highly soluble in a hydroalcoholic solution (^{Silva et al. 2007}). This would explain the absence of variability in EY at high STR values since the number of extractables would remain the same.

The present study is the first time that TPC content and EY have been reported for B. alicastrum fruit by-products (shell and seed skin). Use of the 60 mL g⁻¹ STR in UAE of the lyophilized B. alicastrum tissues (leaf, seed, shell and seed skin) produced the highest TPC content. Future research can focus on optimizing the UAE process to produce B. alicastrum extracts with high antioxidant activity.