Optimización de la extracción hidroalcohólica de compuestos antioxidantes desde la pulpa de Syzygium cumini L.: efecto del etanol y tiempo J. Food Sci. Gastron. (July - December 2025) 3(2): 10-16 https://doi.org/10.5281/zenodo.16741108 ISSN 3073-1283 ORIGINAL ARTICLE Optimization of hydroalcoholic extraction of antioxidant compounds from Syzygium cumini L. pulp: effect of ethanol concentration and extraction time  Claudia E. Restrepo-Flórez claudia.restrepo@uniremington.edu.co Corporación Universitaria Remington, Medellín, Colombia. Received: 25 March 2025 / Accepted: 10 June 2025 / Published online: 31 July 2025 © The Author(s) 2025 Claudia E. Restrepo-Flórez 1 · Yenis E. Castillo 2 Abstract The hydroalcoholic extraction process of black cherry (Syzygium cumini) pulp was optimized, considering the yield of total polyphenols, anthocyanins, and antioxidant capacity as response variables. The highest yields of phe- nolic and anthocyanin compounds were achieved when pro- longed extraction times and an ethanol concentration close to 80% (v/v) were used, with statistically significant differences (p ≤ 0.05). These conditions did not show a relevant effect on antioxidant capacity from a practical point of view. The extract obtained under optimal conditions, with 83.23% eth- anol and 12 hours of extraction, presented yield values for polyphenols (25.19%), anthocyanins (23.24%), and antiox- idant activity (11886 mg/100 mL) that were lower than the values predicted by the optimization model. Visually, the ex- tract exhibited an intense purple hue, indicative of the pres- ence of anthocyanins, such as pelargonidin and delphinidin, as well as their glycosylated derivatives. Keywords Syzygium cumini, hydroalcoholic extraction, antioxidant compounds, total polyphenols, anthocyanins, ex- traction optimization. Resumen Se llevó a cabo la optimización del proceso de extracción hidroalcohólica de la pulpa de cerezo negro (Syzy- gium cumini), considerando como variables de respuesta el rendimiento de polifenoles totales, antocianinas y la capaci- dad antioxidante. Los mayores rendimientos de compuestos fenólicos y antociánicos se alcanzaron al emplear tiempos prolongados de extracción y una concentración de etanol cercana al 80 % (v/v), con diferencias estadísticamente sig- nificativas (p ≤ 0,05). Estas condiciones no mostraron un efecto relevante en la capacidad antioxidante desde el punto de vista práctico. El extracto obtenido bajo condiciones óp- timas, 83,23 % de etanol y 12 horas de extracción, presentó valores de rendimiento en polifenoles (25,19 %), antociani- nas (23,24 %) y actividad antioxidante (11886 mg/100 mL) por debajo de los valores predichos por el modelo de optimi- zación. Visualmente, el extracto mostró una tonalidad mora- da intensa, indicativo de la presencia de antocianinas como pelargonidina, delfinidina y sus derivados glicosilados. Palabras clave Syzygium cumini, hydroalcoholic extraction, antioxidant compounds, total polyphenols, anthocyanins, ex- traction optimization. How to cite Restrepo-Flórez, C. E., & Castillo, Y. E. (2025). Optimization of hydroalcoholic extraction of antioxidant compounds from Syzygium cumini L. pulp: effect of ethanol concentration and extraction time. Journal of Food Science and Gastronomy, 3(2), 10-16. https://doi.org/10.5281/zenodo.16741108 1 Corporación Universitaria Remington, Medellín, Colombia. 2 Instituto de Farmacia y Alimentos, Universidad de La Habana, Cuba.

J. Food Sci. Gastron. (July - December 2025) 3(2): 10-16 11 Introduction In recent years, there has been growing interest in repla- cing artificial colorants with natural alternatives that, in ad- dition to providing an attractive appearance, offer biological benefits and maintain stability during storage (Srinivasan & Rana, 2025). However, many natural pigments, especially anthocyanins, have low stability against factors such as pH, temperature, light, and oxygen, which limits their applica- tion in food products (Rodríguez, 2023; Molina et al., 2023; Xue et al., 2024). Anthocyanins are phenolic compounds of the flavonoid subgroup, widely present in fruits, flowers, and berries, and responsible for colors ranging from red to blue. These pig- ments have gained relevance not only for their sensory value but also for their antioxidant properties and potential protec- tive effects against cardiovascular disease, cancer, diabetes, and cognitive impairments (Kaur et al., 2024; Srinivasan & Rana, 2025). Therefore, in addition to their role as natural colorants, anthocyanins are of interest for developing va- lue-added functional ingredients. Recent studies have shown that the use of modern tech- niques, such as ultrasound-assisted extraction or the use of green solvents (aqueous glycerol), significantly improves the yield and stability of anthocyanins (De Sousa et al., 2021; Kaur & Qadri, 2024). However, there is still a need to opti- mize hydroalcoholic extraction processes from black cherry (Syzygium cumini L.) pulp, a fruit rich in anthocyanins that still requires validation of methods to maximize its yield and functionality under industrial conditions (Kaur et al., 2024). The objective of this work was to optimize the hydroalco- holic extraction of anthocyanins and total polyphenols from black cherry pulp, thereby maximizing their content and an- tioxidant capacity, using experimental design techniques. Methodology The fruits were harvested manually, selecting those with uniform characteristics of size, color, vegetative state, and no visible defects. From each fruit, the pulp and skin were separated using a No. 3 scalpel, and both parts were crushed and homogenized with an Ultra-Turrax IKA T25 (model T25 D S25). Design-Expert 8.0.6 software (Stat-Ease Inc., Minneapo- lis, USA) was used to design a factorial experiment with two factors: percentage of ethanol (A) and extraction time (B), evaluating the yield of total polyphenols, anthocyanins, and antioxidant activity as responses. Numerical optimiza- tion was applied using a response surface (optimal type IV), fitting second- and third-degree polynomial models for the response variables. Sixteen runs were defined, including four replicates, with factorial sets covering the levels of A and B according to the specified ranges. The experimental conditions evaluated were ethanol per- centages ranging from 30 to 70% and extraction times ran- ging from 30 to 60 minutes. Each combination was perfor- med in triplicate across 16 runs, resulting in a total of 64 determinations. Data were analyzed using the response sur- face model using Design-Expert software, with significance considered at p ≤ 0.05. For each run, dry matter was mixed with solvent in a 1:5 (m/v) ratio, using the established ethanol and time condi- tions. Extraction was performed on a sieve at 260 m -1 , at room temperature (25 °C). After extraction, the mixture was filtered to separate the extracts from the solid residues. Total polyphenols were determined by reaction with the Folin–Ciocalteu reagent (Slinkard & Singleton, 1977), ab- sorbance measurement at 765 nm, and expression of the content as mg gallic acid/100 mL of extract. The yield was calculated as a proportion of the total content in the original pulp. Total anthocyanins were determined using the differen- tial pH method (Lee et al., 2005), which involved measuring absorbance at 510 and 700 nm with buffers at pH 1.0 and 4.5, and calculating the content as cyanidin-3-glucoside equiva- lents based on the extinction coefficient. The yield was ex- pressed in the same way as for polyphenols. The antioxidant capacity was determined using the ABTS ●+ radical assay (Re et al., 1999), measured at 734 nm after a 10-min reaction with the extract at 25 °C; results are expressed as mg Trolox equivalent/100 mL of extract. Results and discussion Considering the presence of phenolic compounds and an- thocyanins in black cherry, it was decided to evaluate the ef- fectiveness of the extraction process based on the extraction yield of total polyphenols and anthocyanins, as well as the antioxidant capacity of the hydroalcoholic extracts for each of the conditions tested (Table 1). It is observed that runs 2 and 5, replicates of the extraction test, showed higher (p ≤ 0.05) yields of total polyphenols and anthocyanins, which may be because these runs correspond- ed to the longest extraction time and the use of ethanol at 80.1% (v/v), a concentration proposed by other works as the most appropriate for the extraction of phytochemical con- stituents, while runs 3 and 15 (replicates), with the shortest extraction time and a similar ethanol concentration, showed lower values for the response variables mentioned above. Table 2 shows the three statistical models used to analyze the effects of ethanol percentage (A) and extraction time (B) on three response variables: total polyphenols yield (TPY), antioxidant capacity (AC), and anthocyanin yield (AY).

J. Food Sci. Gastron. (July - December 2025) 3(2): 10-16 12 Table 1. Yield of total polyphenols, anthocyanins, and antioxidant capacity of hydroalcoholic extracts of black cherry Run Ethanol (%) Extraction time (h) Total polyphenol yield (%) Anthocyanin yield (%) Antioxidant capacity (mg/100 mL) 1 1 70.8 9.8 23.52 20.10 12000 2 80.1 12 29.60 24.83 19302.8 3 84.9 6 22.33 24.26 13708.3 4 78.9 9.46 28.95 24.34 11613.89 5 80.1 12 29.38 24.29 18488.9 6 70 6 27.57 24.50 14077.78 7 70 12 28.76 25.46 11705.56 8 90 12 25.38 21.33 15244.44 9 90 8.1 22.07 23.94 12658.33 10 77.6 6 28.03 24.43 14422.22 11 74 7.86 29.45 24.66 13977.78 12 78.9 9.46 27.32 24.66 15005.56 13 90 8.1 23.06 23.18 13186.1 14 86 10.11 29.38 23.86 13877.78 15 84.9 6 22.82 22.53 14786.11 16 78.9 9,462 25.81 23.18 16341.67 1 : Expressed as Trolox. Table 2. Analysis of variance for total polyphenols yield Fountain p-value TPY AC AY Model 0.0076 0.0371 0.0372 A 0.0562 0.0436 0.3296 B 0.0559 0.0295 0.1000 AB 0.0037 0.1419 0.1629 A 2 0.0013 0.0036 0.0142 B 2 0.1182 0.3816 0.0722 A 2 B 0.0298 0.1094 - AB 2 0.0077 0.0042 - A 3 0.0648 0.1119 - B 3 0.0368 0.0300 - R 2 0.9302 0.8752 0.6492 Lack of fit 0.1578 0.4131 0.5193 A: percentage of ethanol; B: extraction time; TPY: total polyphenols yield; AC: antioxidant capacity; AY: anthocy- anin yield. The cubic model applied to evaluate total polyphenol yield was statistically significant at the 95% confidence level, with a coefficient of determination (R 2 ) of 93.02%, indicating an adequate fit of the model to the experimental data. Although the main factors, percentage of ethanol and extraction time, did not show significant individual effects on the response, their interaction was significant at various levels (p ≤ 0.05). The analysis of anthocyanin yield revealed that the cubic model was adequate, accounting for 87.52% of the variabili- ty (R 2 ). In this case, the individual factors, the quadratic term (A 2 ), their interaction (AB 2 ), and the cubic term (B 3 ) were statistically significant (p ≤ 0.05). The quadratic model was significant (p ≤ 0.05) for antioxidant capacity, with an R² of 64.92%, reflecting a strong relationship between the study factors and the dependent variable. In this model, only the quadratic term for the percentage of ethanol (A 2 ) was statis- tically relevant. Together, these models demonstrate the complexity of bio- active compound behavior under different extraction condi- tions. The significance of interactions and nonlinear terms suggests the need for careful optimization to maximize both polyphenol and anthocyanin content and yield, similar to that reported by studies such as Gaibor et al. (2016) on black cherry and phenolic compound extraction, and Araújo et al. (2023) on anthocyanin optimization studies using response surface designs. The influence of these factors on the extraction yield of to- tal polyphenols and anthocyanins, as well as the antioxidant capacity, can be best observed in Figure 1. It was evident that, in none of the evaluated models, maximum antioxidant capacity values similar to those obtained experimentally in the runs with the best results were achieved. This discrepan- cy can be attributed to the absence of a direct linear relation- ship between antioxidant capacity and total polyphenol and anthocyanin concentrations. Antioxidant activity is influ- enced by multiple factors, including the compounds present and their structural characteristics, so a higher concentration of polyphenols or anthocyanins does not guarantee a high- er antioxidant capacity, as also pointed out by Prior et al.

J. Food Sci. Gastron. (July - December 2025) 3(2): 10-16 13 (2005) and de la Rosa et al. (2014). Figure 1. Influence of ethanol percentage and extraction time on a) total polyphenol extraction yield; b) anthocyanin extraction yield; and c) antioxidant capacity. On the other hand, it was observed that the highest yield values for total polyphenols were achieved with ethanol con- centrations of approximately 85% (v/v) and extraction times ranging from 6 to 10 hours. These results are consistent with those reported by Vergara-Salinas et al. (2012), who evaluat- ed the extraction of polyphenols in maqui (Aristotelia chilen- sis) fruits and highlighted that high ethanol concentrations favor the recovery of phenolic compounds. The significant differences (p ≤ 0.05) in yield between studies can be at- tributed to the physicochemical characteristics of the plant material used, including the plant matrix, the type and loca- tion of phenolic compounds, and genetic variability within the species. Table 3 presents the established ranges for the parameters evaluated during the optimization process of the hydroalco- holic extraction of bioactive compounds from black cherry pulp. The independent variables defined were the percent- age of ethanol (between 70 and 90%) and the extraction time (from 6 to 12 hours), both within a range considered ade- quate to guarantee process efficiency. The dependent vari- ables included the total polyphenol yield, the anthocyanin yield, and the antioxidant capacity of the extract, which were defined using the maximization criterion. Table 3. Restrictions for the optimization of the ex- traction process Parameter Lower limit Upper limit Criterion Ethanol (%) 70 90 In the interval Extraction time (h) 6 12 In the interval Total polyphenol yield (%) 22,0677 29,6007 Maximize Anthocyanin yield (%) 20,0965 25,4567 Maximize Antioxidant capacity (mg/100 mL) 11613.9 19302.8 Maximize Table 4 presents the three best solutions obtained during the optimization process of hydroalcoholic extraction of bio- active compounds from black cherry pulp, based on different combinations of ethanol and extraction time. Three respons- es were evaluated: total polyphenol yield, anthocyanin yield, and antioxidant capacity, in addition to the statistical conve- nience value, which indicates the suitability of each solution according to the established optimization criteria. Solution 1, with 83% ethanol and a 12-hour time, achieved the highest yields of total polyphenols (30.77%) and exhibit- ed good antioxidant capacity. This finding aligns with results from fruit extractions, such as S. cumini, where ethanol con- centrations of around 70–80% were found to favor the op- timal recovery of anthocyanins and polyphenols (De Sousa et al., 2021). Likewise, the maximum yield values reported for jaboticaba (Myrciaria cauliflora) with 74% ethanol sup- port the positive effect of high ethanol concentrations on ex- traction efficiency (Nunes et al., 2021).

J. Food Sci. Gastron. (July - December 2025) 3(2): 10-16 14 Table 4. Optimized solutions that meet the constraints Parameter Solution 1 2 3 Ethanol (%) 83.23 79.23 82.70 Extraction time (h) 12 6.34 9.45 Total polyphenol yield (%) 30,7684 29,6007 30,3069 Anthocyanin yield (%) 25,0582 26,1989 25,4567 Antioxidant capacity (mg/100 mL) 17715 15206.6 14885.8 Statistical convenience 0.902 0.776 0.752 Solution 2, with a shorter extraction time (6.34 h) and 79.23% ethanol, yielded the highest anthocyanin content (26.20%), although with a lower antioxidant capacity. Stud- ies using ultrasound-assisted extraction in jaboticaba have also shown that moderately short times can better preserve anthocyanins, reducing their thermal degradation and gener- ating greater selectivity (Sharma & Dash, 2022). In all solutions, the antioxidant capacity was lower than the theoretical maximum values, which coincides with ob- servations in jaboticaba, where higher anthocyanin contents did not always correspond to higher antioxidant capacities, according to the ABTS assay, due to antagonistic effects or lower solubility of antioxidant compounds in high-concen- tration ethanol. Solution 1 presented the highest statistical convenience (0.902), suggesting an adequate combination of factors to maximize multiple responses. This is consistent with the principles of response surface design, where the balance be- tween variables allows for robust and replicable results, as demonstrated by numerous optimization studies on anthocy- anin extractions (Miranda-Medina et al., 2018; Khan et al., 2020). The results obtained in this study reflect trends observed in the scientific literature: ethanol concentrations around 80% combined with moderate to long times favor high yields of polyphenols and anthocyanins. However, higher concentra- tions or excessive times may not significantly improve an- tioxidant capacity, due to possible limitations in the solubi- lization or stability of specific compounds. In this context, solution 1 represents the best alternative analyzed, reconcil- ing high total yield with considerable functional activity and statistical robustness. Several biological and environmental factors contribute to the variability observed among different studies on phenolic compounds, including cultivar, ripening stage, and climatic conditions, among others (Tomás-Barberán & Espín, 2001; Bouaziz et al., 2004). Furthermore, the absence of a stan- dardized method for measuring phenolics complicates the comparison between results, given that the Folin–Ciocalteu assay, although widely used, is influenced by parameters such as temperature, sodium carbonate concentration, and the type of standard used (Singleton et al., 1999). Slinkard and Singleton (1977) proposed modifications to the classical method, improving its specificity by standard- izing the ratio of reagent to alkaline substance, reaction time and temperature, absorbance wavelength (765 nm), and the use of gallic acid as a reference (Slinkard & Singleton, 1977). However, numerous published works fail to strictly follow these recommendations strictly, adopting instead al- ternative standards such as catechin, chlorogenic acid, caffe- ic acid, or tannic acid, which introduces comparative biases between studies. The lack of standardization in analytical methods can lead to significant differences in the results obtained. For exam- ple, in the determination of total phenols in blueberries, val- ues ranging from 22 to 4180 mg/100 g of fresh weight are reported, mainly due to variations in test conditions (Lee, 2005). All of this limits the comparability of results with oth- er sources. When analyzing the visible spectrum (Figure 2), a well-de- fined peak was observed between 520 and 560 nm, consis- tent with the UV-Vis spectrum of anthocyanins in general, as reported by Giusti and Wrolstad (2001). Figure 2. Spectrum of absorption of the extract hydroal- coholic optimized of cherry tree black. The shape of the UV-Vis spectrum of anthocyanins (490- 550 nm) can provide information about their concentration and the type of anthocyanin present. The anthocyanins pelar- gonidin (520 nm) and delphinidin (546 nm), as well as their glycosidic variants (10 to 15 nm lower), could be found in the optimized extract. Conclusions The highest yields of total polyphenols and anthocyanins were obtained using 80% (v/v) ethanol and a prolonged

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