Optimización del proceso de extracción hidroalcohólica del orégano ( Origanum vulgare L.) J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7https://doi.org/10.5281/zenodo.13996953ISSN: 3073-1283 ORIGINAL ARTICLE Optimization of the hydroalcoholic extraction process of oregano ( Origanum vulgare L.) Jaime O. Rojas jaime.rojas@utc.edu.ec Facultad de Ciencias Agropecuarias y Recursos Naturales, Universidad Técnica de Cotopaxi, Latacunga, Ecuador.Received: 4 March 2024 / Accepted: 16 June 2024 / Published online: 30 July 2024© The Author(s) 2024 Sixto A. Gavilanez · Jaime O. Rojas Abstract The present study aimed to optimize the hy-droalcoholic extraction process of raw material from orega-no plants. To achieve this, the plant was dehydrated in an oven at 40 °C for 4 hours, reaching a fnal moisture content of 12.09%, measured with a thermo-balance. A total of 19 experimental runs were established using the Design Expert 8.0.6 software, varying factors such as extraction time (6, 15, and 24 hours), temperature (30 and 60 °C), and mass/solvent ratio (1:10 and 1:20). Phytochemical analysis revealed that oregano contains favonoids, triterpenes, quinones, saponins, alkaloids, and phenolic compounds, with the latter being the most abundant. The hydroalcoholic extraction determined the total polyphenol content and antioxidant capacity of each run. The optimized extract showed an antioxidant capacity of 10,491.4 mg/L, higher than the total polyphenol content of 100.814 mg/L. Comparing these results with the numer-ical optimization, the antioxidant capacity aligned with the model, while the total polyphenol content was lower than the predicted value. Keywords antioxidant capacity, alkaloids, favonoids, op -timization, polyphenols. Resumen El presente estudio tuvo como objetivo opti-mizar el proceso de extracción hidroalcohólica de la droga cruda a partir de la planta de orégano. Para ello, la planta se deshidrató en una estufa a 40 °C durante 4 horas, obteniendo una humedad fnal del 12,09 %, medida con una termo-bal -anza. Se establecieron 19 corridas experimentales utilizando el software Design Expert 8.0.6, variando factores como el tiempo de extracción (6, 15 y 24 horas), la temperatura (30 y 60°C), y la relación masa/disolvente (1:10 y 1:20). Según el análisis ftoquímico, el orégano contiene favonoides, triterpenos, quininas, saponinas, alcaloides y compuestos fenólicos, siendo estos últimos predominantes. Al realizar la extracción hidroalcohólica, se determinó el contenido de po-lifenoles totales y la capacidad antioxidante de cada corrida. El extracto optimizado presentó una capacidad antioxidante de 10,491.4 mg/L, valor superior al contenido de polifenoles totales de 100.814 mg/L. Al comparar estos resultados con la optimización numérica, se encontró que la capacidad anti - oxidante se ajustaba al modelo, mientras que el contenido de polifenoles totales fue inferior al valor predicho. Palabras clave actividad antioxidante, alcaloides, favonoi -des, optimización, polifenoles. How to cite Gavilanez, S.A., & Rojas, J.O. (2024). Optimization of the hydroalcoholic extraction process of oregano ( Origanum vulgare L.). Journal of Food Science and Gastronomy , 2 (2), 1-7. https://doi.org/10.5281/zenodo.13996953
J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7 2 Introduction The food industry faces the challenge of developing high-quality foods that maintain nutritional characteristics and safety over prolonged storage periods. A promising al-ternative to achieve this is through plant extracts as natural preservatives. However, using these extracts can alter the organoleptic properties of foods, introducing favors and aromas that are not desirable compared to synthetic preser-vatives and antioxidants (Nieto, 2020).The hydroalcoholic extract of oregano ( Origanum vulgare L.) has gained attention due to its high total polyphenol con-tent and notable antioxidant capacity, making it a candidate for extending the shelf life of foods without compromising their physicochemical and organoleptic quality. Its potential as a natural preservative increases when technologies such as microencapsulation are employed, allowing for preserving its antioxidant and bioactive properties for food applications (Calderón-Oliver & Ponce-Alquicira, 2022). Antioxidants, both natural and synthetic, play a key role in reducing the adverse efects of reactive oxygen species, which can cause degenerative diseases. In this regard, inter -est has grown in utilizing natural antioxidants, such as poly-phenols, found in various medicinal plants (Ashok et al., 2022). This approach has led to an increase in the demand for food products containing natural additives, ofering a healthier alternative to chemical preservatives, whose long- term efects on consumer health are a concern. Oregano ( O. vulgare L.) is known for its richness in bio-active substances, including polyphenols with antioxidant properties, making it a valuable resource in the food industry as a natural preservative. These compounds can help replace synthetic additives, improving the safety and quality of food products while minimizing the adverse efects of chemical preservatives. The research aimed to optimize the hydroal-coholic extraction process of oregano based on its total poly-phenol content and antioxidant capacity. Materials and methods The method for preparing the oregano extract was carried out as follows. First, the oregano stems and leaves were in-spected to ensure they were in good condition. Leaves were selected based on homogenous characteristics concerning the vegetative state, size, color, and absence of visible spots, cracks, morphological alterations, or infestations by fungi and parasites. The fresh leaves were dried at 40 °C in an oven without forced air circulation. Once dried, they were ground using a manual grinder and stored in Ziploc double-sealed bags, which were kept in a desiccator until further analysis. The extracts were obtained through maceration with occa-sional stirring; at the end of each extraction run, the resulting mixture was fltered, and the solid residue was discarded. Fi -nally, the oregano sample underwent various phytochemical analyses to determine its chemical composition.The determination of total polyphenol content was carried out in several stages. First, the pH was measured following the protocol established by Vázquez-Blanco et al. (2018). Then, the moisture content of the material was evaluated us-ing the method described by Tirado et al. (2015). Finally, the FRAP assay was conducted to determine the total polyphe- nols, allowing for the quantifcation of antioxidant capacity in the analyzed extracts.The experimental design and processing of hydroalcoholic oregano extracts were performed using the Design Expert 8.0.6 program to select the extract with the highest total polyphenol yield and antioxidant capacity. A numerical op-timization method was used through a response surface de- sign (IV Optimal), generating a mathematical model describ -ing the variations of variables in each extract. The factors evaluated were ethanol percentage (A), extraction time (B), mass/solvent ratio (C), and temperature (D), while the total polyphenol yield and antioxidant capacity were the response variables. The total number of combinations defned by the software was 19 runs, including 3 replicates.The experimental conditions used in the study included ethanol concentrations of 60, 75, and 90%, treated for 6, 15, and 24 hours, also expressed in numerical format. The temperature was maintained at nominal levels of 30 and 60 °C. Additionally, a mass/solvent ratio of 1:10 and 1:20 was established. Table 1 shows the experimental runs provided by the program. Results and discussion The raw material was subjected to chemical tests to detect bioactive components, including antioxidants, total polyphe-nols, alkaloids, and phenolic compounds, as detailed in Table 2.The table shows the presence of phytochemical com- pounds, highlighting that the ethanolic and aqueous extracts contain phenolic compounds, while the ether extract did not show their presence. Pereira et al. (2009) identifed the presence of quinones, benzoquinones, and free amino acids, indicating the presence of fatty compounds in an extract. The detection of these compounds was considered positive if red drops or a colored flm appeared in the liquid or on the walls of the test tube. Before conducting the analyses, the image displays the aqueous extracts and the presence of alkaloids and terpenes, as well as unidentifed metabolites during the phytochemical screening, which may be due to a possible reduction in their concentration during the drying of the plant. The Dragendorf test for the ether extract revealed
J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7 3 Table 1. Representation of experimental runs RunEthanol (%)Time (h)Temperature (ºC)Drug/solvent ratio 160153011601530116015301275156022751560227515602360243023602430236024302490630149063014906301560153015601530156015301660156026601560266015602775243017752430177524301875156028751560287515602975156019751560197515601109066021090660210906602119015302119015302119015302126063021260630212606302136024601136024601136024601149024301149024301149024301157515302157515302157515302169024602169024602169024602179015601179015601179015601187563021875630218756302197515601197515601197515601
J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7 4 residues of dry material (not precipitated) on the walls of the test tubes. In contrast, the ethanolic extract showed a distinct turbidity.The test with ferric chloride indicated that the phenolic compounds in the hydroalcoholic extract are derived from pyrocatechol, exhibiting a deep green coloration. Mean- while, the aqueous extract showed compounds derived from pyrogallol, characterized by an intense dark blue color. The phytochemical profle of the crude extract revealed that the leaves and stems are a rich source of antioxidants and total phenolics, particularly simple bioactive compounds widely distributed in the plant kingdom. The Baljet test showed that the ethanolic extract contained more triterpenes and steroids, while no signifcance was observed in the ether and aqueous extracts.The evaluation of the ether, ethanolic, and aqueous extracts did not show the presence of resins. Similar colorations were observed in the extracts, except in the third test, which ex-hibited a more intense coloration. According to the phytochemical assays, when the difer - ent extracts were subjected to ferric chloride (III) reagent, a higher presence of phenolic compounds was observed in the ethanolic and aqueous extracts, while no such compounds were detected in the ether extract. The identifcation of these secondary metabolites was performed using the Shidona test (Zn/HCl), where the mag- nesium reaction in an acidic medium reduces favonoids, generating a color that ranges from reddish-orange to a dark violet hue. In this case, the ethanolic and aqueous extracts showed greater signifcance, while the ether extract did not present any favonoids, although a slight precipitation was observed in each sample. The alcoholic degree has a signifcant efect on the ex -traction of compounds present in oregano ( O. vulgare L.). Subjecting the crude drug to a hydroalcoholic solution al-lows for determining the presence of these compounds, as well as their antioxidant capacity and total phenolic con- tent in diferent ethanol concentrations (60, 75, and 90%) for each sample (Alvis, 2012). Considering the presence of compounds in both the aqueous and alcoholic extracts, the efectiveness of the process was evaluated based on total phenolics and antioxidant capacity (Rodríguez et al., 2022).The analysis of variance performed on the coefcients of response variables related to total phenolic content revealed that the quadratic model was signifcant at a 95% confdence level. This suggests a statistically signifcant relationship between mass/solvent and extraction time. Additionally, the coefcient of determination (R²) indicated that the model ex -plained 96.04% of the variability in total phenolic content (Table 3). In the analysis of variance for antioxidant capacity, the model was also signifcant with a 95% confdence level, in - dicating a statistically signifcant relationship between the interaction of the factors and the dependent variable of the model. In this case, the R² showed a signifcance of 98.91% of the variability in the antioxidant capacity present in the plant (Table 3). Table 2. Phytochemical profle of oregano MetaboliteTestEther extractEthanolic extractAqueous extract Fatty compoundsSudan+++Alkaloids Dragendorf -++-Lactonic grouping Baljet -+++Triterpenes/steroids Lieberman-Burchard +++-CatechinsCatechins+++ResinsResins-Reducing sugarsFehling++++++SaponinsFoam-+-Phenolic compounds Ferric chloride (III) ++++++Free amino acids/aminesNinhydrin+++ Quinones/benzoquinonesBromothymol blue +++FlavonoidsShinoda++++++Cardiotonic glycosidesKedde-AnthocyaninsAnthocyanidins+-MucilagesMucilages+- Bitter principlesBitter principles +++ +: Presence, ±: Regular, -: Absence.
J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7 5 According to the results, the mass/solvent ratio and ex- traction time, along with their homologous quadratic terms, showed signifcant diferences in their interactions. Studen -tized residuals are used as indicators of normality in the dis-tribution of errors in a regression model. They are obtained by dividing the residuals (diferences between observed and predicted values) by an estimate of their standard deviation. Upon examining the distribution of the studentized residu- als, it was observed that the data ft a normal distribution, indicating that the models for the total polyphenol content and antioxidant capacity were adequate, and the inferences drawn from them are valid. Figure 1 illustrates the relation-ship between extraction time and ethanol concentration, demonstrating a total polyphenol content higher than the op-timized value. The optimal temperature for extraction is 60 °C, with a mass/solvent ratio of 1:10. Table 3. Analysis of variance for the total phenolic content and antioxidant capacity of the hydroalcoholic extract of oregano VariableSource p -value Total phenolic contentModel 0.003 A 7.84 B 24.34 AB 4.7913A 2 1.107 B 2 34.33R 2 0.9604Antioxidant capacityModel0.017 A 893.2638 B 358.8194 AB 741.5972R 2 0.9891 Figure 1. Infuence of extraction time and ethanol concentration on total polyphenols: a) 30 °C, b) 60 °C.Martínez-Flores et al. (2021) noted that the efects of tem - perature and ethanol concentration were similar. In Figure 2, it can be observed that the optimal points for the extraction of total polyphenols varied, allowing for the determination of the optimal polyphenol value based on the mass/solvent ratio with the percentage of ethanol diluted in 1 gram of crude oregano.Figure 2 shows that the antioxidant capacity reached its highest level of 6325.46 mg/L with an ethanol concentra-tion of 75% and an extraction time of 15 hours. This value was obtained at a temperature of 30 °C and a crude drug/sol-vent ratio of 1:10. At 60 °C, an antioxidant capacity value of 6325.46 mg/L was obtained, which is higher than predicted.
J. Food Sci. Gastron . (July - December 2024) 2 (2): 1-7 6 For the numerical optimization of the extraction process, previously evaluated intervals of the mass/solvent ratio and extraction time were used. This allowed for higher fnal re -sults regarding total polyphenols and the antioxidant capaci-ty present in the crude oregano.The numerical optimization of the extraction process was carried out according to pre-established parameters. An in- terval for the mass/solvent ratio was defned between 1.0 and 2.0, and the extraction time was set in the range of 6 to 24 hours. To maximize total polyphenols, limits between 25.7625 and 124.88333 mg/L were considered. Additionally, the goal was to maximize antioxidant capacity within a range of 2099.4444 to 6325.4629 µmol Fe 2+ /mL. Together, these parameters provide a framework for optimizing the process, ensuring that they remain within the established limits and achieving optimal results in the total polyphenols extraction and antioxidant capacity.The results indicated the optimal parameters for the ex-traction of bioactive compounds. The mass/solvent ratio was established at 1:10, suggesting an appropriate proportion to maximize the extraction of the desired components. The ex- traction time was fxed at 6 hours, a period considered ef - cient for achieving a signifcant recovery of the metabolites. The results showed a total polyphenol content of 100.814 mg/L, refecting a considerable amount of these antioxidant compounds in the extracted sample.The antioxidant capacity reached a value of 10,491.4, indi-cating the strong antioxidant capacity of the extract. Finally, the statistical convenience (0.87015782) suggested that the model used to optimize these parameters is adequate and that the results obtained are reliable. These data provide a solid foundation for future research on the antioxidant potential of the analyzed crude drug.To obtain the optimized extract, the mass/solvent ratio was established using 1 g of crude drug with 10 ml of 60% ethanol diluted in distilled water, for 6 hours at 60 °C. The optimized extract presented a content of 0.111 mg/L of total polyphenols, a value lower than theoretical. This is attribut- ed to the diferent dilutions performed on the extract, which modifed the concentration of polyphenols in the hydroal -coholic extract of the crude oregano (de Torre et al., 2020).The Design Expert 8.0 program estimated an antioxidant capacity of 10,491.4 mg/L. When comparing this result using spectrophotometry, a value of 10,340.55 mg/L was obtained, demonstrating that the diference between the theoretical and practical value is minimal, aligning with the mathematical model. Conclusions The drying process of oregano leaves and stems was car-ried out for 48 hours at a temperature of 40 °C, resulting in leaves with a fnal moisture content of 12%, which matched the desired moisture indicator. During the extraction of the hydroalcoholic extract from the crude drug, several key compounds were identifed, such as total polyphenols and antioxidant capacity, in addition to detecting the presence of quinones, alkaloids, triterpenes, saponins, phenolic com - pounds, and favonoids through phytochemical analysis. The optimized hydroalcoholic extract showed a concentration of total polyphenols of approximately 100 mg/L, a value ob-tained through numerical optimization, while the antioxidant capacity was higher than expected, exceeding 10,000 mg/L in the laboratory. Finally, the phytochemical analysis using various assays such as Shidona, foam, and ferric chloride (III) allowed for determining the presence of metabolites in the three extracts analyzed: aqueous, ethereal, and ethanolic. Figure 2. Infuence of extraction time and ethanol concentration on antioxidant capacity: a) 30 °C, b) 60 °C.
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