Journal of biological and health sciences http://biotecnia.unison.mx
Universidad de Sonora
ISSN: 1665-1456
Original Article
Recuperación de elagitaninos de Eucalyptus camaldulensis: Comparación de tecnologías híbridas convencionales y emergentes, su efecto antioxidante y antigiardia
1 Food Research Department, School of Chemistry, Autonomous University of Coahuila, Ing. José Cárdenas Valdés, República Ote, C.P. 25280, Saltillo, Coahuila, Mexico.
2 School of Professional Studies Huasteca Zone, Autonomous University of San Luis Potosí. Romualdo del Campo No 501, Rafael Curiel, C.P. 79060, Ciudad Valles, San Luis Potosí, Mexico.
Eucalyptus camaldulensis is a plant that offers phenolic com- pounds with antioxidant and antimicrobial properties. This study aimed to compare conventional and emerging extrac- tion techniques to recover a set of ellagitannins and evaluate their antioxidant and antiparasitic activities. Three extraction methods were employed under different conditions to de- termine the most effective method for obtaining phenolic compounds. The extraction yielding the highest number of phenolic compounds was subjected to purification, phenolic profile identification, antioxidant potential evaluation, and antiparasitic activity against Giardia lamblia. Results indica- ted that ultrasound-microwave assisted extraction method was the most effective, yielding seven compounds, predomi- nantly ellagitannins. In addition, it allowed to obtain an anti- oxidant activity on DPPH of an IC₅₀ of 371.13 mg/L, for lipid oxidation inhibition assay (LOI) IC₅₀ 173.09 mg/L, for the FRAP assay content of about 500 mq equivalents of Trolox/L, and ABTS IC₅₀ 25.28 mg/L. In addition, ellagitannins succeeded in inhibiting Giardia lamblia, reaching a maximum activity of around 80 % at 48 h. This suggests that hybrid extraction is effective for obtaining ellagitannins with antioxidant poten- tial and antiparasitic activity from Eucalyptus camaldulensis. Keywords: Extracts, Giardia lamblia, Maceration, Ultrasound- Microwave
Eucalyptus camaldulensis es una planta que ofrece com- puestos fenólicos con propiedades antioxidantes y antimi- crobianas. El objetivo de este estudio fué comparar técnicas de extracción convencionales y emergentes para recuperar un conjunto de elagitaninos y evaluar sus actividades an- tioxidante y antiparasitaria. Se emplearon tres métodos de extracción en diferentes condiciones para determinar el mé- todo más eficaz para la obtención de compuestos fenólicos. La extracción que produjo el mayor número de compuestos fenólicos se sometió a purificación, identificación del perfil fenólico, evaluación del potencial antioxidante y actividad
antiparasitaria contra Giardia lamblia. Los resultados indica- ron que el método de extracción asistido por ultrasonidos y microondas fue el más eficaz y produjo siete compuestos, predominantemente elagitaninos. Además, permitió obtener una actividad antioxidante sobre DPPH de un IC₅₀ de 371.13 mg/L, para el ensayo de inhibición de la oxidación lipídica (LOI) IC₅₀ 173.09 mg/L, para el ensayo FRAP un contenido de 500 mq equivalentes de Trolox/L, y ABTS IC₅₀ 25.28 mg/L. Además, los elagitaninos consiguieron inhibir Giardia lam- blia, alcanzando una actividad máxima de alrededor del 80 % a las 48 h. Esto sugiere que la extracción híbrida es eficaz para obtener elagitaninos con potencial antioxidante y actividad antiparasitaria a partir de Eucalyptus camaldulensis.
Palabras clave: Elagitaninos, Giardia lamblia, Maceración, Ultrasonido-Microondas
Free radicals are molecules that can exist on their own, and that have unpaired electrons in a molecular orbital (Mar- temucci et al., 2022); these molecules can be destructive to cells, cause degenerative illnesses, and other oxidative processes (Akbari et al., 2022). Some compounds, such as phenolic compounds are effective on free radicals (Sun and Shahrajabian, 2023), and are also known as antioxidants, and they work by neutralizing free radicals in biological cells (Munteanu and Apetrei, 2021).
Intestinal parasites are common infectious diseases that cause many health problems (Fauziah et al., 2022). Giardia lamblia is an intestinal parasite responsible for the disease called Giardiasis. It is the third biological agent that genera- tes diarrheal diseases in children and adults (Juarez-Saldivar et al., 2021; Morales-Luna et al., 2022). Phenolic compounds in addition to functioning as antioxidants, could be anti- parasitic (Sun and Shahrajabian, 2023), and they have been pursued in science and industry (Silva et al., 2023).
Ellagitannins are phenolic compounds considered hy- drolyzable tannins with a hexahydrodiphenyl group (HHDP) and a central glucose (Era et al., 2020); they have demons-
*Author for correspondence: Juan A. Ascacio-Valdés e-mail: alberto_ascaciovaldes@uadec.edu.mx Received: November 12, 2024
Accepted: April 7, 2025
Published: May 21, 2025
Volume XXVII
DOI: /10.18633/biotecnia.v27.2503
trated effectiveness as an antioxidant, and antimicrobial ac- tivities (Tolmie et al., 2023). Ellagitannins are found in berries, pomegranate, rambutan, and dried fruits such as nuts (Banc et al., 2023; Estrada-Gil et al., 2022). Eucalyptus camaldulensis is a plant characterized by terpenes and terpenoids (Aleksic Sabo and Knezevic, 2019) esters, flavonoids, ellagitannins, and phenolic acids (Nwabor et al., 2021; Singa et al., 2011) and due to its properties, it is important to recover their compounds.
Conventional extraction methods, such as maceration, and percolation, require large volumes of solvents and a large amount of manpower (Alara et al., 2021; Naviglio et al., 2019). Extraction by maceration is based on placing the plant material in a solvent of interest under specific conditions for a specific time which can be hours or days (Bitwell et al., 2023). In percolation, the sample is sprayed in a system, dropping the solvent on the sample from top to bottom, the system contains a filter that allows obtaining only the solvent with the extract, however, it requires more time, and there are problems in the solubility of the sample (Alara et al., 2021).
In contrast to conventional methods, non-conventional methods enable the retrieval of greater quantities of pro- ducts and the development of green extractions (Belwal et al., 2020). One of these, the ultrasound-assisted extraction process uses microbubbles to break down the cell walls of the plant material, allowing the release of these com- pounds in solvent (Machado-Carvalho et al., 2023). Likewise, microwave-assisted extraction, allows the extraction of compounds by using high temperatures to heat the solvents (González-González et al., 2022) and the plant material used using electromagnetic radiation (Castillo-Reyes et al., 2022) so that the solvent permeates in the plant matrix (Ordoñez- Torres et al., 2021).
However, in a hybrid approach, ultrasound and microwa- ves together allow an improvement in obtaining secondary metabolites from plants, interacting between the cavitation performed and the temperature (Estrada-Gil et al., 2022; Hernández-Hernández et al., 2020). The use of ultrasound- microwave assisted extraction to different polyphenols sour- ces has been reported (Cheng et al., 2023; Ramić et al., 2015; Yadav et al., 2023). This study aimed to compare maceration, percolation extraction and ultrasound-microwave assisted extraction for the recovery of ellagitannins from Eucalyptus camaldulensis and the evaluation of their antioxidant and antiparasitic activity.
Branches and leaves from Eucalyptus camaldulensis Dehnh
(22.23340° N, -100.86150° W, Record number 104039, Her-
barium ANSM) were provided by the School of Veterinary and Agronomy of the Autonomous University of San Luis Potosí, San Luis Potosi, Mexico in December 2022. For this study only leaves were used. Leaves from E. camaldulensis were dehydrated at 40-45°C for 48 h in an oven Binder Model BD 400 (Germany). Once dried, the plant material was stored
in plastic bags in the absence of light. Subsequently, it was ground a particle size of 0.8 mm in a grinder model Tecnal, TE-631/4 (Brazil).
The reagents gallic acid (No. G7384), catechin (No. C1251), 2,2-diphenyl-1-picrylhydrazyl radical (No. D9132), linoleic acid (No. L1376), 2,2-0-Azino-bis (3-ethylbenzothiazoline-
-6-sulfonic acid) (No. A1888) were of Sigma-Aldrich (St. Louis, MO, USA), acquired from Sigma Chemical Mexico ®, and all solvents for high-performance liquid chromatography analy- ses were HPLC grade.
To obtain polyphenol extracts, three extraction methods were evaluated using a fractional factorial design for each ex- traction method to evaluate mass/volume ratio and ethanol/ water ratio, with five levels and each performed in triplicate (Table 1). Each extraction method was systematically tested under varying conditions to assess its efficacy in extracting phenolic compounds from Eucalyptus camaldulensis. The polyphenol content was analyzed using the STATISTICA 7 software an ANOVA and Tukey test was used to compare mean treatments.
The Ultrasound-Microwave Assisted Extraction (EAU/M) was executed as reported by Hernández-Hernández et al. (2020), using a cooperative microwave/ultrasound work station (Nanjing ATPIO Instruments Manufacture Co., Ltd. company, Nanjing, China), with a microwave frequency of 2450 MHz, as well as 25 KHz to ultrasound. The sample was placed in a volume of 700 mL, according to the mass/volume ratio of Table 1, and subjected to ultrasound treatment for 20 min. After that, the extracts were recovered by filtrate (Whatman #2).
Table 1. Fractional factorial design used for the recovery of phenolic compounds.
Tabla 1. Diseño factorial fraccionado utilizado para la recuperación de compuestos fenólicos.
Run | Mass/volume ratio (g/mL) | Ethanol/water ratio (%) |
1 | 1:16 | 0 |
2 | 1:16 | 70 |
3 | 1:8 | 0 |
4 | 1:8 | 70 |
5 | 1:12 | 30 |
% Inhibition=(Abs control-Abs sample)/(Abs control) Eq. (1)
Where: Abs control is the difference of the absorbances at 0 and 24 h with respect to the control sample, and Abs sample is the difference of absorbances at 0 and 24 h with respect to each sample.
Donde: Abs control es la diferencia de las absorbancias a las 0 y 24 h con respecto a la muestra control, y Abs muestra es la diferencia de las absorbancias a las 0 y 24 h con respecto a cada muestra.
Maceration extraction was performed based according to the method reported by Hasni et al. (2021), using a shaker with a temperature of 50 °C, for 24 h with a shaking incubator (Mini Shaker, shel lab, Sheldon manufacturing inc, USA) at 200 rpm. After the extraction was completed, the liquid was filtrated (Whatman # 2),stored under refrigeration and kept away from light until evaluation.
Percolation was carried out following to information descri- bed by Tuane et al. (2021) and Cao-Ngoc et al. (2020) for the time of extraction with modifications in the temperature. The solvent was heated in an electric grill (Corning Model PC-220, USA) up to 50°C. The plant material was placed on a filter pa- per (Whatman #2) and a funnel, passing the solvent through, and completely covering the plant material and the extracts were stored away from light until evaluation.
Separation and determination of bound polyphenols Eucalyptus leaf residues after all extractions were collected after filtration of the extracts and were stored frozen to deter- mine the bound phenols in the plant material. The extraction of bound phenols was conducted following the method of Zhang et al. (2010) with modifications by Hernández- Hernández et al. (2020), using a digestion of 1.0 g of residue with 50 mL 2 M sodium hydroxide, at room temperature for 4
h. Subsequently, the mixture was acidified with hydrochloric acid pH 2.0. Later, sample was filtered using Whatman # 2 fil- ter paper. Lipids were removed with 30 mL of hexane and the mixture remains were extracted 3 times with 75 mL of ethyl acetate using a liquid-liquid separation. The obtained ethyl acetate fractions were evaporated to dryness in a conventio- nal oven and the bound phenolic compounds were diluted in 5 mL with the same extraction conditions, the obtained fractions were taken as the bound phenols and were quanti- fied according to phenolic compound determination.
The hydrolyzable tannin content was determined by the Folin-Ciocalteu technique, described by Gómez-Martínez et al. (2020). For that, 400 μL of sample were added in test tubes isolated from light, 400 μL of Folin Ciocalteu’s reagent were added, followed by stirring with rest for 5 min. After that, 400 μL of sodium carbonate (0.05 M) were added and left to stand for 1 min. Then, 2500 μL of water were added followed by a final stirring, the reaction was read in a spectrophotometer (Thermo spectronic Biomate3, USA) at 790 nm, and a gallic acid standard was used in the range of 0 to 500 ppm.
The extracts were evaluated as described by Estrada-Gil et al. (2022). A sample of 500 μL was placed in tubes of 8 mL capa- city, then 3 mL of Butanol-HCL solution (95:5 v/v) were added and mixed, after 100 μL of ferric reagent (2 %) were added.
The solutions were capped and heated in a water bath to 95° C for 1 h. Finally, the reactions were read in a spectrophoto- meter (Thermo spectronic Biomate3, USA) at 460 nm. Results were expressed based on of catechin standard of 0 to 1000 ppm.
All obtained extracts were evaluated according to their content of hydrolyzable and condensed polyphenols. The sum of both determinations was taken as the total phenolic content.
The extract with the highest content of total polyphenols was subjected to column liquid chromatography, using the amberlite XAD-16 chromatographic packing as the stationary phase and water and ethanol as the mobile phase. Only the ethanolic fraction containing the ellagitannin compounds of interest was obtained. The phenolic fraction was evaporated in an incubator NAPCO 1000, model 322 (USA) at 45 °C and recovered as a fine powder after complete drying for 48 h (Hernández-Hernández et al., 2020).
A reversed-phase high-performance liquid chromatography analysis was carried out on a Varian HPLC system with an au- tosampler (Varian ProStar 410, USA), a Varian ProStar 230I ter- nary pump (USA) and a PDA detector (Varian ProStar 330, Al). Using a chromatography ion trap mass spectrometer (Varian 500-MS IT Mass Spectrometer, USA) coupled to electrospray ion source. Samples of 5 µL were injected onto a Denali C18 column (150 mm × 2.1 mm, 3 µm, Grace, USA) at a tempera- ture of 30 °C in the oven. Formic acid (0.2 %, v/v; solvent A) and acetonitrile as solvent B were used as eluents. The gra- dient was used as initial 3% B; 0-5 min, 9% linear B; 5-15 min, 16% linear B; 15-45 min, 50% linear B. The column was then washed. Subsequently, the column was washed and recondi- tioned. The flow rate remained at 0.2 mL/min and elution at 245, 280, 320 and 550 nm. All effluent (0.2 mL/min) was in- jected into the mass spectrometer source, without splitting. MS experiments were performed out in the negative [M-H]-1 mode, using nitrogen as the nebulizer gas and helium as the buffer gas. The ion source parameters: sputtering voltage of
5.0 kV, capillary voltage, and temperature of 90.0 V and 350
°C, respectively. Samples were initially analyzed using full- scan mass spectrometry (m/z 50-2000 range). The data were extracted and processed in the software MS Workstation (V 6.9) (Hernández-Hernández et al., 2020).
Determination of the 2,2-diphenyl-1-picrylhydrazyl radical scavenging was carried out in a microplate for the purified fraction of ellagitannins from E. camaldulensis, where 7 µL of sample were placed in concentrations of 7. 8 to 1000 mg/L. Then, 193 µL of DPPH reagent (60 µM concentration in methanol) were added, the samples were left to stand
for 30 min, at the end of the reaction, sample was taken to a microplate reader (EPOCH Biotek ELISA, USA) at 517 nm. Results were expressed as percentage inhibition and as the half-maximal inhibitory concentration (De La Rosa-Esteban et al., 2023).
Ten µL of sample were deposited in a 96-well microplate to testing concentrations of 7.8-1000 ppm. Afterward, 290 µL of FRAP reagent were added and incubated at 37 °C for 15 min. After the incubation was complete, the sample was analyzed in a microplate reader (EPOCH Biotek ELISA, USA) at 593 nm. A calibration curve was performed with TROLOX and distilled water as a control (Delgado-Garcia et al., 2023).
This assay was performed as reported by Estrada-Gil et al. (2022). The sample (50 µL) was mixed with 100 µL of a linoleic acid solution and 1.5 mL of acetate buffer adjusted to a pH of 4.0 were added to a screw capped tube, and ethanol 50% were used as control. Incubation was carried out for 1 min at 37 °C in an incubator NAPCO 1000, model 322 (USA). After the incubation, 750 µL of the FeCl₂-EDTA were added and in- cubated for 1 h. Then, 250 µL of the reaction were taken and placed in a test tube, later, 1 mL of 0.1M NaOH dissolved in 10% ethanol and 2.5 mL of 10% ethanol were added. Subse- quently, sample was mixed and read in a spectrophotometer (Thermo spectronic Biomate3, USA) at 232 nm. The reaction of the sample and linoleic acid was incubated for 24 h at 37 °C, at the end of this incubation time, again 250 µL were taken to which 1 mL NaOH w 2.5 mL of 10% ethanol were added, then read at 232 nm. Results were reported as the half-maximal inhibitory concentration and the percentage of antioxidant power was determined with the following formula.
ABTS radical was prepared out by mixing a solution of po- tassium persulfate in distilled water (2.45 mM) and an ABTS 2,2-0-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) solution at 7 mM diluted in distilled water. The mixture was stored in the dark for 12-16 h and at the end of the time the ABTS solution was conditioned by diluting in ethanol to an absorbance of 0.700 (measured at 734 nm in a spectropho- tometer). Once the required absorbance was reached, 10 µL of the sample was mixed with 1000 µL of the adjusted ABTS solution. Subsequently, the absorbance was measured at 734 nm in a spectrophotometer and the results were reported as percentage inhibition of the ABTS radical were reported as percentage inhibition of the ABTS radical (Carlos et al., 2020).
Giardia lamblia inhibition
The growth inhibitory activity in Giardia lamblia was con- ducted as reported by Vargas-Villanueva et al. (2023) with some modifications. Giardia lamblia (ATCC 50803) was cultured twice in TYI-S-33 medium with 10% bovine serum and 0.5 mg/mL bovine bile per week at 37 °C. To evaluate
the effect of ellagitannins from Eucalyptus camaldulensis on growth, an inoculum of 10,000 G. lamblia trophozoites was incubated for 24, 48 and 72 h at 37 °C with concentrations of
E. camaldulensis ellagitannins of 100, 150, 200, 250 and 300 µg/mL according to preliminary experiment (Non -reported data), with 0.1% dimethyl sulfoxide (DMSO) as diluent and albendazole 1.5 µg/mL as positive control, each concentra- tion was evaluated in triplicate. After the incubation time the trophozoites were separated from the wall of the tubes by cooling the tubes in an ice water bath for 30 min and counted by optical microscopy using a hemocytometer. The results were expressed as the percentage inhibition of trophozoite growth compared to the control tube. The results of Giardia lamblia growth inhibition were analyzed in GraphPad Prism 6 software using a Two way ANOVA test and as well as TUKEY post hoc analysis with a significant difference of p<0.05.
Different types of extraction (percolation, maceration, ultrasound-microwave) were tested to determine the hig- hest extraction content of phenolic compounds (Figure 1). The data presented are the mean values of three distinct extraction methods. Figure 1a shows that, for percolation extraction, the condition yielding the highest polyphenol content was the 1:16 mass/volume ratio with 0% ethanol (P1). This condition demonstrated a significant difference (p
< 0.05) compared to all other conditions according to Tukey’s test. In contrast to percolation extraction, the extraction by maceration with a relation mass/solvent 1:12, and 30% of ethanol (M5) presented a higher content of polyphenols (54.50 mg of polyphenols/g) (p < 0.05) than the other condi- tions used in this extraction method (Fig. 1b). Figure 1 shows that in both, maceration and percolation extractions, and ultrasound-microwave assisted extraction, the lowest phe- nolic compounds are obtained using a 1:8 mass-volume ratio and 0% ethanol. However, it is also observed that the highest content of phenolic compounds is obtained in the 1:12 con- dition with 30% ethanol, in the maceration and ultrasound- microwave extraction. For the hybrid extraction (ultrasound- microwave) UM5 allowed the recovery of a higher phenolic content (59.79 mg of polyphenols/g). Thus, water is a good solvent to extract polyphenols, combined with ethanol and the exposure time in maceration, and ultrasound-microwave extraction technology. However, these results show that a greater amount of plant material does not allow a greater extraction of phenolic compounds.
Interestingly, a statistical comparison of means revea- led no significant difference between the phenolic content obtained from maceration (M5) and the hybrid extraction method (UM5) (p < 0.05) (See figure 2). However, it is notewor- thy that the hybrid extraction method (UM5) outperformed other conditions and extraction methods, showcasing its efficiency in recovering a greater quantity of polyphenols in a shorter duration of 20 min, been and an advantage of ultrasound-microwave assisted extraction (Ordoñez-Torres et
Figure 1. Total phenolic content from extraction by a) percolation, b) maceration, and c) ultrasound-microwave assisted extraction. Means with the same letter in the same column are not statistically different according to the Tukey test (p < 0.05).
Figura 1. Contenido fenólico total de la extracción por percolación a), maceración b) y extracción asistida por ultrasonidos-microondas c). Las medias con la misma letra en la misma columna no son estadísticamente diferentes según la prueba de Tukey (p < 0.05).
In the three different extraction methods (maceration, percolation, ultrasound-microwave), the most influential factor was the mass/solvent ratio. However, it´s impact was negative for percolation and ultrasound-microwave assisted extraction, in contrast to maceration. Notably, since this effect is quadratic, there is potential for optimization in fu- ture studies (Figure 3). Wong-Paz et al. (2015) reported that in E. camaldulensis leaves, the main factor interfering in the extraction of polyphenols was the ethanol/water ratio, where it was possible to obtain the highest content of 14 mg of hy- drolyzable tannins using 35% ethanol/water with a conven- tional heat reflux extraction system. Water is a great solvent for the obtention of polyphenols (Hernández-Hernández
Figure 2. Comparison between best extraction conditions (percolation, P1; maceration, M5; ultrasound/microwave, UM5). Different letters in the columns indicate significant differences between runs and equal letters indicate that there was no statistically significant difference (p < 0.05).
Figura 2. Comparación entre las mejores condiciones de extracción (percolación, P1; maceración, M5; ultrasonidos/microondas, UM5). Las letras distintas en las columnas indican diferencias significativas entre las series y letras iguales indican que no hubo diferencias estadísticamente significativas (p < 0.05).
al., 2021), unlike maceration method that require a long time (24 h to maceration). Polyphenol content of E. camaldulensis has been previously reported to be between 9.04 ± 0.26 mg/g of polyphenols (Nwabor et al., 2021), and 68 ±30 mg/g (Rosendal et al., 2020), which may vary according to growing conditions of the vegetal material and environmental factors.
et al., 2020), due to its capacity for solubilized natural com- pounds by their polarity, also the combination with ethanol allows to improve the extraction of phenolic compounds due to reducing the polarity to obtain more compounds (Lajoie et al., 2022). However, under the maceration and ultrasound- microwave assisted extraction methods used in this study, it was possible to obtain a more elevated content of phenolic compounds, using the same ethanol/water ratios. The factor that allowed obtaining a higher phenolic content could be due to the mass/volume ratio. The mass/solvent ratio in the extraction is an essential and strategic factor for obtaining phenolic compounds, as well as the polarity of the extractant solvents (Osorio-Tobón, 2020). A good mass/solvent ratio in
Figure 3. Pareto chart of variables effects in the extractions by a) percolation, b) maceration, and c) ultrasound-microwave assisted extraction.
Figura 3. Diagrama de Pareto de los efectos de las variables en las extracciones por percolación a), maceración b), y extracción asistida por ultrasonidos-microondas c).
te extracción of phenolic compounds allows that vegetal material have a good solubility in the solvent, improve the solubility of comnpounds, according to mass transfer princi- ples (Elboughdiri, 2018).
Bound phenolic compounds are covalently bound to other cell wall structures. Unlike free phenolic compounds that do not interact with other molecules and conventional solvents including organic solvents, bound phenolic compounds are not soluble because of the interaction that occurs with ma- cromolecules such as cellulose or proteins through covalent bonds in the plant cell wall (Rocchetti et al., 2022).
From the determination of the bound phenolic com- pounds, in the maceration extractions where a higher con- tent of bound phenols was found in the extraction with a 1:8 solvent mass ratio and 0% ethanol, this result shows that the use of these conditions is less favorable to obtain higher number of polyphenols in comparison with the other con- ditions. By the way, percolation and ultrasound-microwave assisted extractions can also observe a similar behavior, whe- re, using a 1:8 solvent mass ratio and 70% of ethanol retained the highest amount of bound polyphenols compared to the other conditions in their extraction method. In addition, the lowest percentage of bound polyphenols was found in the ultrasound-microwave-assisted extraction with 16 % bound polyphenols compared to soluble ones (Figure 4). This is due
to the ability of hybrid technology to break the cell through cavitation and joint temperature, thus allowing the solvent to penetrate the cell and obtain more polyphenols (Estrada- Gil et al., 2022). However, the bound phenolic content of E. camaldulensis has not been reported so far in other investiga- tions (Hernández-Hernández et al., 2020), that used the same extraction conditions and microwave ultrasound equipment, where they found that contrary to what was reported in our research, the extraction condition with the lowest number of bound phenols was 1:16 and 70% ethanol, and 1:8 with 70% ethanol.
The extract with the highest content of total polyphenols using ultrasound-microwave assisted extraction, mass/vo- lume ratio of 1:12, and 30 % ethanol, was partially purified using XAD-16 amberlite and then the purified fraction was dry. Phenolic compounds were identified via HPLC-ESI-MS, where six ellagitannins and one gallotannin, belonging to the group of hydrolyzable tannins, were found as the main chemical compounds in this fraction, being pedunculagin and tellimagrandin I, ellagic acid, some of those previously reported (Table 2).
Singa et al. (2011) reported several ellagitannins and other phenolic compounds in crude extracts of E. camaldu- lensis leaves, among which pedunculagin and tellimagrandin I and II were the most important, these reports have been
Figure 4. Comparative graph of soluble phenolic compounds vs. bound phenolic compounds obtained by a)percolation, b) maceration and c) ultrasound/microwave extractions. Different letters in the columns indicate significant differences between runs and equal letters indicate that there was no statistically significant difference (p < 0.05).
Figura 4. Gráfico comparativo de compuestos fenólicos solubles frente a compuestos fenólicos obtenidos mediante extracciones por percolación a), maceración b) y ultrasonidos/microondas c). Letras diferentes en las columnas indican diferencias significativas entre las series y letras iguales indican que no hubo diferencias estadísticamente significativas (p < 0.05).
Table 2. Identification of obtained compounds from E. camaldulensis fraction (Ultrasound-microwave extraction, 1:12 mass/solvent, and 30 % ethanol). Tabla 2. Identificación de los compuestos obtenidos de la fracción de
Gallotannin
331, 169
46.02 483
glucose
Ellagitannin
Ellagitannin Ellagitannin
Ellagitannin Ellagitannin
Ellagitannin
Galloyl-HHDP- hexoside
Pedunculagin
Tellimagrandin I
Ellagic acid- hexoside
Ellagic acid
Methyl ellagic acid hexoside
Di-galloyl
463, 302, 301, 249
481, 375, 301
765, 633, 483
302, 301, 300
229, 185
315, 300
633
783
785
463
301
477
6.78
23.96
34.06
36.03
39.08
42.2
Group
Compound
MS2
RT [M-H]- (m/z)
E. camaldulensis (extracción por ultrasonidos y microondas, 1:12 masa/ disolvente y 30 % de etanol).
confirmed by Boulekbache-Makhlouf, Slimani and Madani (2013) in Eucalyptus leaves. This agrees with what was repor- ted in this study.
The antioxidant activity from of E. camaldulensis fraction ob- tain from hybrid extraction was determined through DPPH radical inhibition, FRAP, and lipid oxidation inhibition, and ABTS assay. Table 3 show the results of antioxidant assays, where the inhibition is presented as IC₅₀, are determined by the half-maximal inhibitory concentration.
To DPPH assay, has been reported in other research with extracts from E. camaldulensis, obtained by Nwabor et al. (2021) an activity of IC₅₀ 65.67 mg/L, in contrast with the results reported in our investigation, is a best activity (IC₅₀ 371.13 mg/L). Alghoraibi et al. (2020) described a DPPH sca- venging activity higher (IC₅₀ 90 mg/mL). Furthermore, these authors found the antioxidant activity of IC₅₀ 82.9 mg/mL to ABTS to be different from our results this activity is higher. However, these activities could be for other compounds in your extract, and in our study, only a fraction of ellagitannins were evaluated.
Another important antioxidant capacity is the ferric reducing antioxidant power (FRAP); in the results presented in Table 3 an antioxidant capacity higher than 500 mg eq. of Trolox/g. This result is an important finding, because there are reports of lower concentrations than those reported in a study realized by Gullón et al. (2019), who reported a content
Table 3. Antioxidant assays from Eucalyptus camaldulensis fraction.
Tabla 3. Ensayos antioxidantes de la fracción de Eucalyptus camaldulensis.
Antioxidant assay | Eucalyptus camaldulensis ellagitannins (IC₅₀ mg/mL) |
DPPH | IC₅₀ 371.13 mg/L |
ABTS | IC₅₀ 25.28 mg/L |
FRAP | 512 mg eq. of Trolox/g |
Lipid oxidation inhibition effect | IC₅₀ 173.09 mg/L |
of 112.4 mg equivalent of Trolox/g with Eucalyptus globulus, showing that in E. camaldulensis a higher activity on FRAP than E. globulus can be found.
On the other hand, no recent reports have been found on the inhibition of lipoperoxidation with compounds from Eucalyptus camaldulensis, nevertheless, Assaggaf et al. (2022) reported with another Eucalyptus species an IC50 0.17 μg/ mL, likewise. Harkat-Madouri et al. (2015) reported an IC50 of
6.75 mg/mL, both being higher than reported by our study.
Antioxidant capacity is an activity that has been related to anticancer and antitumor activities. Thus, some of the compounds found in isolation in this study show antioxidant activity. The activity of pedunculagin isolated from leaves of Eucalyptus spp. against liver tumor cells in-vitro (QGY-7703) has been reported with IC₅₀ 64.3 ± 6.1) μg/mL, causing holes in the membrane of tumor cells (Xiao et al., 2012). Furthermo- re, Al-Sayed and Esmat (2016) reported the antioxidant acti- vity of pedunculagin and tellimagrandin I in an in-vitro assay, demonstrating protection against carbon tetrachloride-in- duced hepatotoxicity in HepG2 cells. Pedunculagin increased superoxide dismutase levels by 76%, while tellimagrandin I elevated glutathione levels by 81%. Additionally, the isolated ellagic acid has shown activity on ABTS (4.59 ± 0.07 μg/mL) and DPPH (10.54 ± 0.07 μg/mL) (Yang et al., 2023).
Giardia lamblia inhibition
The results in Figure 5 show the inhibitory effect of ellagi- tannins from Eucalyptus camaldulensis at 24, 48 and 72 h. Inhibitory effects of 18 -52% were obtained at the first 24 h, from 150-300 µg/mL, reaching the maximum inhibitory
effect (83 ±6.03%), however, there is no difference between the control and the first two concentrations (100 and 150 µg/mL). The highest inhibitory effect took place at 48 h at the highest concentration (300 µg/mL), on the other hand, the effect of 150 µg/mL remained static with no difference p<0.05 compared to the control, reaching an IC₅₀ 183.6. At 72 hours the concentrations of 150-250 µg/mL decreased their effect to less than 40% inhibition. Unlike the 300 µg/mL concentration with an effect of 80% ±6.73. The use of ethanol extracts of pomegranate peel in the inhibition of Giardia lam- blia has been reported.
Garza-Ontiveros et al. (2024) observed that grape po- mace polyphenols had the greatest effect on G. lamblia at 48 h with 59% inhibition at a concentration of 200 µg/mL, and that grape pomace polyphenols induced an apoptosis-like process in Giardia lamblia trophozoites. It was also shown that all concentrations caused alterations in the distribution of α-tubulin in the ventral disc, flagella and midbody. They also showed a decrease in flagella size or loss of flagella as the concentration of polyphenols increased. In vitro effects could be due to alterations in cytoskeletal dynamics or the stress response culminating in apoptosis-like cell death in the parasite. The effect could be due to changes in cytoskeletal dynamics or the stress response culminating in apoptosis-like cell death in the parasite. Nevertheless, El-Kady et al. (2021) reported an inhibition of up to 98% in higher concentrations (50 mg/mL than those reported in this study (0.3 mg/mL) of pomegranate peel extracts, being that pomegranate peel possesses some ellagitannins such as pedunculagin, galloyl- HHDP-hexo side, ellagic acid-hexosid in addition to ellagic
Figure 5. Graphic effect of ellagitannins from E. camaldulensis on the inhibition of G. lamblia. The letters in the columns indicate significant differences between runs and equal letters indicate that there was no statistically significant difference (p < 0.05).
Figura 5. Gráfico del efecto de los elagitaninos de E. camaldulensis sobre la inhibición de G. lamblia. Las letras de las columnas indican diferencias significativas entre las corridas y las letras iguales indican que no hubo diferencias estadísticamente significativas (p < 0.05).
acid, which are found in our study (Hernández-Corroto et al., 2020). Other authors such as Palomo-Ligas et al. (2022) showed inhibition of Giardia lamblia with an extract of po- megranate peel with ellagitannins (punicalin, punicalagin and ellagic acid) at concentrations of 150, 175 and 200 µg/ mL, showing a maximum inhibition of 74% at 48 h. Surpri- singly, they also showed deformations of the trophozoites such as elongation, loss of the characteristic shape, protube- rances on the dorsal surface, irregularities on the periphery and, in addition, caused deformations of the flagella such as shortening or even loss, as well as perforations in the membrane with the treatments at 150, 175 and 200 µg/mL. Surprisingly, they demonstrated that this pomegranate peel extract interacts with the cytoskeleton of Giardia by altering tubulin, thus causing alterations to the microtubules of the ventral disc, flagella, causing deformation, decreasing their adhesion capacity and growth inhibition. This could be one of the inhibition factors of Giardia lamblia in our study, since ellagitannin compounds are similar to those reported for E. camaldulensis; however, in our research, more studies are needed to determine if this is the same behavior.
Hybrid extraction (ultrasound-microwave assisted extrac- tion) shows that it is a good method for obtaining ellagitan- nins from Eucalyptus camaldulensis compared to extraction methods such as maceration and percolation, and allows ob- taining compounds such as tellimagrandin I, pedunculagin. Therefore, E. camaldulensis is a plant source of ellagitannins, which possess antioxidant activity such as ferric-reducing antioxidant power, inhibition of lipid oxidation, DPPH radical inhibition, and ABTS radical inhibition. Furthermore, ellagi- tannins from E. camaldulensis possess antiparasitic activity against Giardia lamblia.
The authors declare that they have no conflicts of interest
Akbari, B., Baghaei-Yazdi, N., Bahmaie, M. and Mahdavi Abhari,
F. 2022. The role of plant-derived natural antioxidants in reduction of oxidative stress. BioFactors. 48(3): 611–633.
Al-Sayed, E. and Esmat, A. 2016. Hepatoprotective and antioxidant effect of ellagitannins and galloyl esters isolated from Melaleuca styphelioides on carbon tetrachloride- induced hepatotoxicity in HepG2 cells. Pharmaceutical Biology. 54(9): 1727–1735.
Alara, O.R., Abdurahman, N.H. and Ukaegbu, C.I. 2021. Extraction of phenolic compounds: A review. Current Research in Food Science. 4(December 2020): 200–214.
Aleksic Sabo, V. and Knezevic, P. 2019. Antimicrobial activity of Eucalyptus camaldulensis Dehn. plant extracts and essential oils: A review. Industrial Crops and Products. 132(March 2019): 413–429.
Alghoraibi, I., Soukkarieh, C., Zein, R., Alahmad, A., Walter, J.G. and Daghestani, M. 2020. Aqueous extract of Eucalyptus camaldulensis leaves as reducing and capping agent in
biosynthesis of silver nanoparticles. Inorganic and Nano- Metal Chemistry. 50(10): 895–902.
Assaggaf, H.M., Mrabti, H.N., Rajab, B.S., Attar, A.A., Hamed, M., Sheikh, R.A., El Omari, N., Menyiy, N.El, Belmehdi, O., Mahmud, S., Alshahrani, M.M., Park, M.N., Kim, B., Zengin,
G. and Bouyahya, A. 2022. Singular and combined effects of essential oil and honey of in vivo findings. Molecules. 27(5121): 1–17.
Banc, R., Rusu, M.E., Filip, L. and Popa, D.S. 2023. The impact of ellagitannins and their metabolites through gut microbiome on the gut health and brain wellness within the gut–brain axis. Foods. 12(2): 1–41.
Belwal, T., Chemat, F., Venskutonis, P.R., Cravotto, G., Jaiswal, D.K., Bhatt, I.D., Devkota, H.P. and Luo, Z. 2020. Recent advances in scaling-up of non-conventional extraction techniques: Learning from successes and failures. Trends in Analytical Chemistry. 127: 115895.
Bitwell, C., Indra, S. Sen, Luke, C. and Kakoma, M.K. 2023. A review of modern and conventional extraction techniques and their applications for extracting phytochemicals from plants. Scientific African. 19:1–19, e01585.
Boulekbache-Makhlouf, L., Slimani, S. and Madani, K. 2013. Total phenolic content, antioxidant and antibacterial activities of fruits of Eucalyptus globulus cultivated in Algeria. Industrial Crops and Products: 41(1): 85–89.
Cao-Ngoc, P., Leclercq, L., Rossi, J.C., Hertzog, J., Tixier, A.S.,
Chemat, F., Nasreddine, R., Banni, G.A.H.D., Nehme, R., Schmitt-Kopplin, P. and Cottet, H. 2020. Water-based extraction of bioactive principles from blackcurrant leaves and Chrysanthemum americanum: A comparative Study. Foods. 9(10): 1–26.
De León-Medina, J.C., Sepúlveda, L., Morlett-Chávez, J., Meléndez-Renteria, P., Zugasti-Cruz, A., Ascacio-Valdés,
J. and Aguilar, C.N. 2020. Solid-state fermentation with Aspergillus niger gh1 to enhance polyphenolic content and antioxidative activity of castilla rose (Purshia plicata). Plants. 9(11): 1–15.
Castillo-Reyes, F., De León-Juárez, E., Nery-Flores, S.D., Flores- Gallegos, A.C., Campos-Muzquiz, L.G., Ascacio-Valdés, J.A. and Rodríguez-Herrera, R. 2022. Polyphenols extraction from creosote bush, tarbush, and soursop using ultrasound- microwave and their effect against Alternaria alternata and Fusarium solani. Revista Mexicana de Fitopatología, Mexican Journal of Phytopathology. 40(3): 1–28.
Cheng, M., He, J., Wang, H., Li, C., Wu, G., Zhu, K., Chen, X., Zhang, Y. and Tan, L. 2023. Comparison of microwave, ultrasound and ultrasound-microwave assisted solvent extraction methods on phenolic profile and antioxidant activity of extracts from jackfruit (Artocarpus heterophyllus Lam.) pulp. Foods Science and Technology. 173(December 2022): 1–10, 114395.
De La Rosa-Esteban, K., Sepúlveda, L., Chávez-González, M.L., Torres-León, C., Estrada-Gil, L.E., Aguilar, C.N. and Ascacio- Valdés, J.A. 2023. Valorization of mexican rambutan peel through the recovery of ellagic acid via solid-state fermentation using a yeast. Fermentation. 9(8): 1–13.
Delgado-Garcia, M., Gómez-Secundino, O., Rodríguez, J.A., Mateos-Díaz, J.C., Muller-Santos, M., Aguilar, C.N. and Camacho-Ruiz, R.M. 2023. Identification, antioxidant capacity, and matrix metallopeptidase 9 (MMP-9) in silico inhibition of haloarchaeal carotenoids from Natronococcus sp. and halorubrum tebenquichense. Microorganisms. 11(9): 1–16.
Elboughdiri, N. 2018. Effect of time, solvent-solid ratio, ethanol concentration and temperature on extraction yield of phenolic compounds from olive leaves. Engineering, Technology & Applied Science Research. 8(2): 2805–2808.
El-Kady, A.M., Abdel-Rahman, I.A.M., Fouad, S.S., Allemailem, K.S., Istivan, T., Ahmed, S.F.M., Hasan, A.S., Osman, H.A. and Elshabrawy, H.A. 2021. Pomegranate peel extract is a potential alternative therapeutic for giardiasis. Antibiotics. 10(6): 1–15.
Era, M., Matsuo, Y., Saito, Y. and Tanaka, T. 2020. Production of ellagitannin hexahydroxydiphenoyl ester by spontaneous reduction of dehydrohexa-hydroxydiphenoyl ester. Molecules. 25(5): 1–14.
Estrada-Gil, L., Contreras-Esquivel, J.C., Flores-Gallegos, C., Zugasti-Cruz, A., Govea-Salas, M., Mata-Gómez, M.A., Rodríguez-Herrera, R. and Ascacio-Valdés, J.A. 2022. Recovery of bioactive ellagitannins by ultrasound/ microwave-assisted extraction from mexican rambutan peel (Nephelium lappaceum L.). Molecules. 27(5): 1–15.
Fauziah, N., Aviani, J.K., Agrianfanny, Y.N. and Fatimah, S.N. 2022. Intestinal parasitic infection and nutritional status in children under five years old: A systematic review. Tropical Medicine and Infectious Disease. 7(11): 1–26.
Garza-Ontiveros, M., Vargas-Villanueva, J.R., Gutiérrez-Gutiérrez, F., Nery-Flores, S.D., Ascacio-Valdés, J.A., Campos-Muzquiz, L.G., Rodriguez-Herrera, R. and Palomo-Ligas, L. 2024. In silico and in vitro antigiardiasic potential of grape pomace polyphenols extracted by hybrid microwave-ultrasound methodology. Revista Brasileira de Farmacognosia. 34(2): 313–327.
Gómez-Martínez, M., Ascacio-Valdés, J.A., Flores-Gallegos, A.C., González-Domínguez, J., Gómez-Martínez, S., Aguilar, C.N., Morlett-Chávez, J.A. and Rodríguez-Herrera, R. 2020. Location and tissue effects on phytochemical composition and in vitro antioxidant activity of Moringa oleifera. Industrial Crops and Products. 151(March 2020): 1–8, 112439.
González-González, G.M., Palomo-Ligas, L., Nery-Flores, S.D., Ascacio-Valdés, J.A., Sáenz-Galindo, A., Flores-Gallegos, A.C., Zakaria, Z.A., Aguilar, C.N. and Rodríguez-Herrera, R. 2022. Coffee pulp as a source for polyphenols extraction using ultrasound, microwave, and green solvents. Environmental Quality Management. June 2022: 1–11.
Gullón, B., Muñiz-Mouro, A., Lú-Chau, T.A., Moreira, M.T., Lema,
J.M. and Eibes, G. 2019. Green approaches for the extraction of antioxidants from Eucalyptus leaves. Industrial Crops and Products. 138(June 2019): 1–8, 111473.
Harkat-Madouri, L., Asma, B., Madani, K., Bey-Ould Si Said, Z., Rigou, P., Grenier, D., Allalou, H., Remini, H., Adjaoud, A. and Boulekbache-Makhlouf, L. 2015. Chemical composition, antibacterial and antioxidant activities of essential oil of Eucalyptus globulus from Algeria. Industrial Crops and Products. 78: 148–153.
Hasni, S., Rigane, G., Ghazghazi, H., Riguene, H., Bouallegue, A., Khedher, O., Oueslati, M.A. and Ben Salem, R. 2021. Optimum conditions and LC-ESI-MS analysis of phenolic rich extract from Eucalyptus marginata L. under maceration and ultrasound-assisted extraction methods using response surface methodology. Journal of Food Quality. 2021: 1–14.
Hernández-Corroto, E., Plaza, M., Marina, M.L. and García, M.C. 2020. Sustainable extraction of proteins and bioactive substances from pomegranate peel (Punica granatum
L.) using pressurized liquids and deep eutectic solvents. Innovative Food Science and Emerging Technologies. 60(December 2019): 1–11, 102314.
Hernández-Hernández, C., Aguilar, C.N., Flores-Gallegos, A.C., Sepúlveda, L., Rodríguez-Herrera, R., Morlett-Chávez, J., Govea-Salas, M. and Ascacio-Valdés, J. 2020. Preliminary testing of ultrasound/microwave-assisted extraction (U/M- AE) for the isolation of Geraniin from Nephelium lappaceum
L. (Mexican Variety) peel. Processes. 8(5): 1–10. Juárez‐Saldivar, A., Barbosa‐Cabrera, E., Lara‐Ramírez, E.E., Paz‐
González, A.D., Martínez‐Vázquez, A.V., Bocanegra‐García, V., Palos, I., Campillo, N.E. and Rivera, G. 2021. Virtual screening of fda‐approved drugs against triose phosphate isomerase from entamoeba histolytica and Giardia lamblia identifies inhibitors of their trophozoite growth phase. International Journal of Molecular Sciences. 22(11): 1–8.
Lajoie, L., Fabiano-Tixier, A.S. and Chemat, F. 2022. Water as green solvent: Methods of solubilisation and extraction of natural products—Past, present and future solutions. Pharmaceuticals. 15(12): 1–22.
Machado-Carvalho, L., Martins, T., Aires, A. and Marques, G. 2023. Optimization of phenolic compounds extraction and antioxidant activity from Inonotus hispidus using ultrasound- assisted extraction technology. Metabolites. 13(4): 1–15.
Martemucci, G., Costagliola, C., Mariano, M., D’andrea, L., Napolitano, P. and D’Alessandro, A. G. 2022. Free radical properties, source and targets, antioxidant consumption and health. Oxygen. 2(2): 48–78.
Morales-Luna, L., Hernández-Ochoa, B., Martínez-Rosas, V., Navarrete-Vázquez, G., Ortega-Cuellar, D., Rufino-González, Y., González-Valdez, A., Arreguin-Espinosa, R., Franco- Vásquez, A.M., Pérez de la Cruz, V., Enríquez-Flores, S., Martínez-Conde, C., Canseco-Ávila, L.M., Gómez-Chávez,
F. and Gómez-Manzo, S. 2022. Giardia lamblia G6PD::6PGL fused protein inhibitors decrease trophozoite viability: A new alternative against giardiasis. International Journal of Molecular Sciences: 23(22): 1–20.
Munteanu, I.G. and Apetrei, C. 2021. Analytical methods used in determining antioxidant activity: A review. International Journal of Molecular Sciences. 22(7): 1-30.
Naviglio, D., Scarano, P., Ciaravolo, M. and Gallo, M. 2019. Rapid solid-liquid dynamic extraction (RSLDE): A powerful and greener alternative to the latest solid-liquid extraction techniques. Foods, 8(7): 1–22.
Nwabor, O.F., Singh, S., Syukri, D.M. and Voravuthikunchai,
S.P. 2021. Bioactive fractions of Eucalyptus camaldulensis inhibit important foodborne pathogens, reduce listeriolysin O-induced haemolysis, and ameliorate hydrogen peroxide- induced oxidative stress on human embryonic colon cells. Food Chemistry. 344: 1–31.
Ordoñez-Torres, A., Torres-León, C., Hernández-Almanza, A., Flores-Guía, T., Luque-Contreras, D., Aguilar, C.N. and Ascacio- Valdés, J. 2021. Ultrasound-microwave-assisted extraction of polyphenolic compounds from Mexican “Ataulfo” mango peels: Antioxidant potential and identification by HPLC/ESI/ MS. Phytochemical Analysis. 32(4): 495–502.
Osorio-Tobón, J.F. 2020. Recent advances and comparisons of conventional and alternative extraction techniques of phenolic compounds. Journal of Food Science and Technology. 57(12): 4299–4315.
Palomo-Ligas, L., Estrada-Camacho, J., Garza-Ontiveros, M., Vargas-Villanueva, J.R., Gutierrez-Gutierrez, F., Nery-Flores, S.D., Canas Montoya, J.A., Ascacio-Valdes, J., Campos- Muzquiz, L.G. and Rodriguez-Herrera, R. 2022. Polyphenolic extract from Punica granatum peel causes cytoskeleton- related damage on Giardia lamblia trophozoites in vitro. PeerJ. 10: 1–18.
Porter, L.J., Hrstich, L.N. and Chan, B.G. 1985. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry. 25(1): 223–230.
Ramić, M., Vidović, S., Zeković, Z., Vladić, J., Cvejin, A. and Pavlić,
B. 2015. Modeling and optimization of ultrasound-assisted extraction of polyphenolic compounds from Aronia melanocarpa by-products from filter-tea factory. Ultrasonics Sonochemistry. 23: 360–368.
Rocchetti, G., Gregorio, R.P., Lorenzo, J.M., Barba, F.J., Oliveira, P.G., Prieto, M.A., Simal-Gandara, J., Mosele, J.I., Motilva, M.J., Tomas, M., Patrone, V., Capanoglu, E. and Lucini, L. 2022. Functional implications of bound phenolic compounds and phenolics–food interaction: A review. Comprehensive Reviews in Food Science and Food Safety. 21(2): 811–842.
Rosendal, E., Ouédraogo, C.J.W., Dicko, C., Dey, E.S. and Bonzi-Coulibaly, Y.L. 2020. Geographical variation in total phenolics, flavonoids and antioxidant activities of Eucalyptus camaldulensis leaves in Burkina Faso. African Journal of Pure and Applied Chemistry. 14(3): 51–59.
Silva, A., Silva, V., Igrejas, G., Aires, A., Falco, V., Valentão, P. and Poeta, P. 2023. Phenolic compounds classification and their distribution in winemaking by-products. European Food Research and Technology. 249(2): 207–239.
Singa, A.N., Ayoub, N., Al-Sayed, E., Martiskainen, O., Sinkkonen,
J. and Pihlaja, K. 2011. Phenolic constituents of Eucalyptus camaldulensis Dehnh, with potential antioxidant and cytotoxic activities. Records of Natural Products. 5(4): 271–280.
Sun, W. and Shahrajabian, M.H. 2023. Therapeutic potential of phenolic compounds in medicinal plants—natural health products for human health. Molecules. 28(4): 1–43.
Tolmie, M., Bester, M.J., Serem, J.C., Nell, M. and Apostolides,
Z. 2023. The potential antidiabetic properties of green and purple tea [Camellia sinensis (L.) O Kuntze], purple tea ellagitannins, and urolithins. Journal of Ethnopharmacology. 309(February 2023): 1–13.
Tuane, A., Paz, S., Luiza, A., Negris, C., Marreto, R.N. and Cardoso,
E. 2021. Enhanced skin permeation of punicalagin after topical application of pluronic micelles or vesicles loaded with Lafoensia pacari extract. Planta med. March 2021.
Vargas-Villanueva, J.R., Gutiérrez-Gutiérrez, F., Garza-Ontiveros, M., Nery-Flores, S.D., Campos-Múzquiz, L.G., Vazquez- Obregón, D., Rodriguez-Herrera, R. and Palomo-Ligas, L. 2023. Tubulin as a potential molecular target for resveratrol in Giardia lamblia trophozoites, in vitro and in silico approaches. Acta Tropica, 248 (September 2023): 1–10.
Wong-Paz, J.E., Contreras-Esquivel, J.C., Rodríguez-Herrera, R., Carrillo-Inungaray, M.L., López, L.I., Nevárez-Moorillón,
G.V. and Aguilar, C.N. 2015. Total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid Mexican region. Asian Pacific Journal of Tropical Medicine. 8(2): 104–111.
Xiao, S.Y., Wen, B.Q., Zhuang, H.Y., Wang, J.L., Tang, J., Chen, H.Z., Chen, X.X. and Cao, Y. 2012. Extraction and antitumor activity of pedunculagin from Eucalyptus leaves. International Conference on Biomedical Engineering and Biotechnology. July 2012: 280–282.
Yadav, R., Mohapatra, D., Subeesh, A., Shabeer, T.P.A. and Giri,
S.K. 2023. Optimization of sequential ultrasound-microwave assisted extraction of polyphenols-rich concrete from tuberose flowers through modelling. Process Biochemistry. 134: 175–185.
Yang, Y., Liu, M., Liu, C., Tang, S., Gu, D., Tian, J., Huang, D. and He,
F. 2023. Ellagic acid from pomegranate peel: Consecutive countercurrent chromatographic separation and antioxidant effect. Biomedical Chromatography. 37(9): e5662.
Zhang, M.W., Zhang, R.F., Zhang, F.X. and Liu, R.H. 2010 . Phenolic profiles and antioxidant activity of black rice bran of different commercially available varieties. Journal of Agricultural and Food Chemistry. 58(13): 7580–7587.