1. INTRODUCTION
In Latin America, traditional medicine constitutes an established and highly significant healthcare system. In Peru, medicinal plants play a fundamental role in therapeutic interventions [1]. Globally, approximately 422,000 plant species have been reported, with over 50,000 classified as medicinal; however, only a small fraction has been scientifically studied for therapeutic purposes [2]. Peru, characterized by its vast biodiversity and deep-rooted cultural tradition of medicinal plant use, is home to approximately 1,408 plant species utilized for medicinal purposes [3,4].
Today, it is crucial to identify and study new sources of bioactives with potential therapeutic activities, as well as to reveal their safe and effective use in order to establish the optimal consumption for positive outcomes [5]. Without scientific validation, traditional medicine would not persist, leading to the disappearance of traditional practices and, consequently, the loss of associated knowledge and culture [6]. Based on the sustainable use of biodiversity and the rational utilization of medicinal plants, phytotherapy can pave the way to improve the quality of life for the population and contribute to the economic and technological development of our country [7,8].
Polyphenolic compounds, including phenols, flavonoids, and anthocyanins, have been extensively studied in medicinal plants due to their diverse pharmacological properties, with antioxidant activity being the most prominent and well-researched [9]. It has been found that the majority of diseases are linked to oxidative stress and the accumulation of free radicals. These compounds have shown efficacy in neutralizing free radicals and halting cellular oxidative stress [10,11]. In recent years, interest has grown due to the crucial role of medicinal plants in addressing chronic diseases by counteracting oxidative stress [12,13].
Myrcianthes discolor (Kunth) McVaugh, commonly known as “lanche,” “mirto,” or “uñico,” is a shrub that belongs to the Myrtaceae family. It can reach up to 3 m in height, with brown-grayish stems and branches; the leaves are simple, entire, opposite, coriaceous, and aromatic. The flowers are simple cymes ranging from pink to red, and the fruit is smooth, blue–black, and an ovoid drupe [14–16].
The entire fresh plant is used, with the leaves commonly consumed as an infusion, while the fruits are edible, sweet-tasting, and consumed fresh [14,16]. The consumption of the leaves as an infusion after meals aids digestion due to their high concentration of phenolic compounds, flavonoids, essential oils, and tannins, which may help alleviate stomach discomfort by reducing the effects of fermented foods. Additionally, the bark is boiled and consumed to treat kidney conditions and inflammation [16,17].
Essential oils, such as E-caryophyllene, bicyclogermacrene, β-elemen, α-cubebene, δ-cadinene, α-humulene, and limonene, have been identified in M. discolor; however, a complete chemical profile of this species has yet to be reported [18].
Traditionally, M. discolor is used as an energizing food, memory enhancer, and for treating colds, inflammation, rheumatic pain, as well as for stomach and menstrual regulation [14–16]. Furthermore, studies have highlighted pharmacological and biological properties such as antibacterial, antioxidant, and anticholinesterase activities [18]. Given the limited information on this plant species, further research will expand scientific knowledge, and by thoroughly establishing these characteristics, it will be possible to formulate effective, safe, and high-quality phytopharmaceuticals as a natural therapeutic alternative. The aim is to validate traditional usage by incorporating it as a promising functional food with medicinal potential.
The growing interest in native plants as sources of bioactive compounds has revealed a scarce characterization of extraction conditions. In the case of M. discolor, we investigate how solvent polarity affects extraction efficiency and antioxidant activity. This research aims to systematically evaluate the effect of different solvent systems on the extraction of phenolic and flavonoid compounds, as well as on their antioxidant capacity, by establishing correlations between phytochemical contents and antioxidant activity. The aim is to contribute to the understanding of the potential of this species for nutraceutical or pharmaceutical applications.
This research aims to determine the influence of solvent ratio and its impact on antioxidant activity in the agitation-assisted extraction of total phenolic and flavonoid compounds from M. discolor leaves.
2. MATERIALS AND METHODS
2.1. Reagents and solvents
Ethanol 96° GL (CKF® ), distilled water (Dropaksa®), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Folin–Ciocalteu reagent (Sigma-Aldrich), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate, sodium bicarbonate, aluminum chloride, sodium acetate, and hydrochloric acid (Merck) were used. Gallic acid (Merck), quercetin (Sigma-Aldrich), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich) were used as standards.
2.2. Botanical material
The species used was M. discolor (Kunth) McVaugh, which was collected in the village of Santa Úrsula, Baños del Inca district, Cajamarca province, Cajamarca region, at geographic coordinates of latitude: 7.154531, and longitude: −78.40375, in UTM format (Zone: 17S East: 320951.65 meters, North: 790944.73 meters). A complete specimen was pressed and prepared according to the standard protocols of the Herbarium Truxillense and taxonomically identified with voucher number N° 65651.
2.3. Preparation and extraction
The M. discolor species acquired had its leaves selected as plant material, ensuring they were intact, free from inert material or decomposition. The plant material was dried in a Memmert oven at 40°C, followed by mechanical milling with an electric grinder to obtain fine particles. The pulverized M. discolor sample was extracted using six solvents: 96% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, water, and acetonitrile. A 5 g sample was used for each solvent, in a sample-to-solvent ratio of 1:20. The mixture was then placed on a heating plate with magnetic stirring (500 rpm) for 1 hour at a temperature of 100°C. The filtered extracts were stored at 6°C for further analysis [19].
2.4. Total phenols content
This was carried out using the Folin–Ciocalteu method with some modifications. In 10 ml volumetric flasks, 0.1 ml of extract was mixed with 2 ml of Folin–Ciocalteu reagent (1:10), 4.4 ml of a 7.5% sodium bicarbonate solution, and distilled water (to a final volume of 10 ml). A blank was prepared by omitting the sample. Total phenolic content (TPC) was estimated using an external standard calibration curve of gallic acid (1.26–10.08 μg/ml). After incubating the mixture for 60 minutes in the dark, the absorbance was measured at 765 nm using a UV-visible spectrophotometer (Peak Instruments C7000V). Each measurement was performed in triplicate. The total phenol concentration was expressed as gallic acid equivalents per gram of dry sample (mg GAE/g DS) [20,21].
2.5. Total flavonoid content
The total flavonoid content (TFC) was measured using the aluminum chloride colorimetric method with some modifications. In 10 ml volumetric flasks, 0.2 ml of extract was diluted with 1.5 ml of distilled water, and 0.4 ml of 10% (w/v) aluminum chloride, 0.4 ml of 1 M sodium acetate, and distilled water (to a final volume of 10 ml) were added to the mixture. TFC was estimated using an external standard calibration curve of quercetin (1–16 μg/ml). After incubating the mixture for 30 minutes in the dark, the absorbance was measured at 430 nm using a UV-visible spectrophotometer (Peak Instruments C7000V). Each measurement was performed in triplicate. The total flavonoid concentration was expressed as quercetin equivalents per gram of dry sample [22,23].
2.6. Antioxidant activity
2.6.1. 2,2-diphenyl-1-picrylhydrazyl
The free radical scavenging activity was measured using the DPPH method with some modifications. The stock solution was prepared at 0.1 mM in 96°GL ethanol. In 10 ml volumetric flasks, 300 μl of the extract was mixed with the 0.1 mM DPPH solution, gently shaken, and incubated for 30 minutes in the dark. Trolox was used as the standard for the calibration curve at concentrations ranging from 3 to 30 μM/ml. Finally, the absorbance was measured using a Peak Instruments C7000V spectrophotometer at 517 nm, with each measurement performed in triplicate. The results were expressed as Trolox equivalents (mg/100 g dry sample) [24,25].
2.6.2. 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid
The free radical scavenging activity was measured using the ABTS method with some modifications. The stock solution was prepared by mixing equal volumes of ABTS (7 mM) and K2S2O8 (2.45 mM) and then incubated in the dark for 16 hours, followed by dilution with 50% ethanol. In 10 ml volumetric flasks, 300 μl of the extract was mixed with the ABTS solution, gently shaken, and incubated for 30 minutes in the dark. Trolox was used as the standard for the calibration curve at concentrations ranging from 3 to 20 μM/ml. Absorbance was then measured using a Peak Instruments C7000V spectrophotometer at 734 nm, with each measurement performed in triplicate. The results were expressed as Trolox equivalents (mg/100 g dry sample) [19,23].
2.6.3. Ferric reducing power
The antioxidant ferric reducing power (FRAP) was measured with some modifications. The FRAP reagent was freshly prepared before each measurement by mixing acetate buffer (300 mM), TPTZ [10 mM in HCl (40 mM)], and FeCl3 (20 mM) in a ratio of 10:1:1 (v/v/v), and incubated at 37°C for 10 minutes before use. In 10 ml volumetric flasks, 300 μl of the extract was mixed with the FRAP solution, gently shaken, and incubated at 37°C for 30 minutes in the dark. Trolox was used as the standard for the calibration curve at concentrations ranging from 5 to 30 μM/ml. Absorbance was then measured using a Peak Instruments C7000V spectrophotometer at 593 nm, with each measurement performed in triplicate. The results were expressed as Trolox equivalents (mg/100 g dry sample) [26,27].
2.7. Statistical analysis
The TPC, TFC, and antioxidant activity (using different assays) were determined in three replicates. The results, presented as mean ± SD, were subjected to analysis of variance (followed by Tukey’s test). The differences were considered statistically significant at p < 0.05. Principal component analysis (PCA) was conducted to correlate the TPC, TFC, DPPH, ABTS, and FRAP results using Jamovi v.17.05 software [28].
3. RESULTS AND DISCUSSION
The results of this study show that the type of solvent used has a significant influence on the extraction of phenolic and flavonoid compounds, as well as on the antioxidant capacity of M. discolor leaf extracts (Table 1).
Table 1. TPC, TFC, and antioxidant activity by different methods for the leaf extract of M. discolor extracted by agitation using different solvents.
| Solvents | TPC1* | TFC2* | DPPH assay3* | ABTS assay3* | FRAP assay3* |
|---|---|---|---|---|---|
| Acetonitrile | 18.99 ± 0.0d | 3.33 ± 0.02e | 0.18 ± 0.02e | 0.25 ± 0.01f | 0.14 ± 0.02f |
| Water | 48.63 ± 0.0b | 3.67 ± 0.02d | 0.49 ± 0.06c | 0.65 ± 0.17d | 0.55 ± 0.03d |
| 30% ethanolic | 58.79 ± 0.07a | 3.96 ± 0.02d | 0.71 ± 0.21a | 0.74 ± 0.24b | 0.82 ± 0.21a |
| 50% ethanolic | 59.31 ± 0.01a | 4.30 ± 0.02c | 0.68 ± 0.16a | 0.77 ± 0.27a | 0.74 ± 0.09b |
| 70% ethanolic | 54.53 ± 0.03a | 4.83 ± 0.03b | 0.58 ± 0.10b | 0.71 ± 0.23c | 0.69 ± 0.07c |
| 96% ethanolic | 35.40 ± 0.01c | 5.58 ± 0.02a | 0.34 ± 0.01d | 0.46 ± 0.05e | 0.43 ± 0.07e |
*X ± DE (n = 3), 1mg of gallic acid equivalent (GAE) g−1, 2mg of quercetin equivalent (QCE) g−1, 3mg of Trolox equivalent (TE) g−1. Statistical analysis: ANOVA followed by Tukey’s test. Different letters within the same column indicate statistically significant differences among treatments according to Tukey’s test (p < 0.05).
Several studies have reported differences in polyphenol and flavonoid content depending on the polarity and composition of the extracting solvent. Lalremruati et al. [29] found methanol to be the most efficient solvent, while El Oihabi et al. [30] reported that acetone yielded the highest TPC and TFC values. Purba and Paengkoum [31] also found that ethanol was effective in extracting phytoconstituents from plant matrices.
Specifically with regard to ethanol, it has been described that higher concentrations enhance flavonoid extraction [32,33]. However, moderate ethanol-water mixtures may improve the solubility of both hydrophilic and moderately lipophilic compounds. For example, a study using a pressurized liquid extraction system with a 1:1 water:ethanol ratio revealed a greater variety of extracted compounds [34]. Similarly, TPC, TFC, and antioxidant activities have been reported to increase when ethanol concentrations range from 60% to 100% [35]. In contrast, Linhares et al. [36] found that a mixture of ethanol and water in a ratio of 2:8 (v/v) performed best overall in the extraction of bioactives. This supports the idea that adding water to organic solvents creates a more polar medium that enhances the desorption of polyphenols from the plant matrix [37]. Furthermore, the solubility of phenolic compounds in alcohols such as methanol and ethanol is increased due to their lack of sugar moieties and relatively low molecular weight.
As shown in Table 1, the substantial variation in the concentration of bioactive compounds between different solvents is strongly linked to the chemical nature and polarity of the extraction solvent. Polar-protic alcoholic solvents such as ethanol and methanol facilitate the solubilization of low-molecular-weight phenolic compounds, including glycosylated and aglycone forms, due to their ability to act as hydrogen bond donors [38].
Regarding total phenol content (Fig. 1A), values of 59.31, 58.79, and 54.53 mg gallic acid equivalents per gram of dry material (mg GAE/g DS) were obtained for the 50%, 30%, and 70% ethanolic extracts, respectively; the 50% ethanol extract was the most effective. These results are consistent with the idea that hydroalcoholic mixtures optimize the extraction of medium-polarity polyphenols.
 | Figure 1. (A) Total phenols content and (B) total flavonoid content in M. discolor extracts obtained by agitation using different solvents. DS = dry sample; GAE = gallic acid equivalent; QCE = quercetin equivalent. [Click here to view] |
In contrast, studies on M. pungens fruits revealed significantly higher TPC values. Seraglio et al. [39] reported values of 2,061.35 ± 51.26 and 1,739.28 ± 8.12 mg GAE/100 g for immature and mature fruits, respectively. In a comprehensive review, Peixoto et al. [40] reported a wide range of 28.41–59.34 mg GAE/g dry weight in Myrtaceae fruits. Similarly, Schulz et al. [41] found values between 1,739 and 4,613 mg GAE/100 g dry drug. These higher values are likely related to the use of different plant organs (fruits vs. leaves), extraction techniques, and environmental factors affecting phytochemical profiles.
Regarding TFC (see Fig. 1B), the values obtained for the 96%, 70%, and 50% ethanol extracts were 5.58 mg, 4.83 mg, and 4.30 mg quercetin equivalents per gram of dry material (mg QE/g DM), respectively. The superior performance of the 96% ethanol extract may be attributed to its effectiveness in extracting less polar flavonoid aglycones. By comparison, Andrade et al. [42] reported TFC values between 79.8 and 154 mg/100 g dry weight in Myrcianthes pungens fruit. Bombana et al. [32] also found very high concentrations in ethanolic M. pungens extracts obtained by ultrasound (10,544.04 and 1,621.78 mg QE/100 g). Spinelli et al. [43] further supported these findings, analyzing M. pungens fruit by LC-MS/MS and obtaining values of 6230 μg GAE/g and 91.61 mg QE/100 g.
Similarly, other Myrtaceae species exhibited diverse values. For instance, Eugenia myrcianthes exhibited values of 102.87 ± 1.80 μg GAE/g and 8.83 ± 0.08 μg QE/g in its extract [44], whereas the fruits of Myrciaria cauliflora and Myrciaria jaboticaba showed values of 1,443–3,160 mg and up to 6,000 mg GAE/100 g dry weight, respectively [41].
It is evident that agitation and moderate heating facilitate the release of secondary metabolites by disrupting plant cell walls, thereby enhancing compound diffusion into the solvent. Environmental factors such as temperature, rainfall, soil composition, plant maturity stage, and collection location can also contribute to phytochemical variability [45,46]. Additionally, the plant part used and the specific extraction methodology can markedly impact final yields [47,48].
Figure 2 shows the antioxidant activity of extracts prepared using different solvents. The extracts prepared using 30% and 50% ethanol exhibited the highest antioxidant capacities. Notably, the extract prepared with 30% ethanol yielded 0.82 mM TE (FRAP) and 0.71 mM TE (DPPH) per gram of dry material, whereas the extract prepared with 50% ethanol reached 0.77 mM TE in the ABTS assay. These values support the observation that intermediate ethanol concentrations favor the extraction of hydrophilic antioxidants.
 | Figure 2. Antioxidant activity in M. discolor extracts obtained by agitation using different solvents. DS = dry sample; mM TE = millimolar Trolox equivalent. [Click here to view] |
Romero et al. [18] reported markedly higher antioxidant capacities for M. discolor essential oil (144.93 and 3,599.6 μM TE for ABTS and DPPH, respectively). Antonelo et al. [49] evaluated three Myrtaceae species (M. gigantea, M. oblongata, and M. tenella), reporting values of 26.00–139.51 μM TE/g for DPPH and 29.35–88.08 μM TE/g for ABTS. However, other studies have reported even higher values, for example, in the peel of the fruit of M. cauliflora (6,834.5 ± 77.9 μM TE, ABTS; 316.2 ± 211.03 μM TE, FRAP) [50] and in the leaves of E. myrcianthes (1,052.32 ± 3.61 μM TE, DPPH; 6,132.94 ± 429.07 μM TE, ABTS) [44].
Bombana et al. [32] found an ABTS scavenging capacity of 337.35 μM TE/g DS and FRAP values ranging from 28.4 to 42.6 μmol TE/g DW in M. pungens [51]. This data suggests that, although M. discolor exhibits moderate antioxidant capacity, its efficacy is notable given the simplicity of the extraction method and solvent range used.
Of the methods applied (ABTS, DPPH, and FRAP), the highest antioxidant values were observed in the FRAP and ABTS assays. Pacheco et al. [52] suggested that these differences could be due to the physicochemical nature of each assay and the solubility of the active constituents. The antioxidant mechanism is primarily driven by hydrogen atom transfer (HAT), single electron transfer (SET), or mixed-mode processes [53,54]. High values in SET-based assays (such as the FRAP assay) suggest that electron donation is dominant, whereas lower responses in HAT assays may imply limited proton-donating capacity or suboptimal solvent extraction [49].
The results of this study highlight the promising antioxidant potential of M. discolor and support its inclusion in nutraceuticals as a means of mitigating oxidative stress-related pathologies. Furthermore, Romero et al. [18] emphasized the role of sesquiterpenes in M. discolor essential oil in inhibiting acetylcholinesterase, thereby enhancing its pharmacological relevance.
Nevertheless, data on M. discolor remain scarce, indicating the need for further research into its chemical profile and biological activities. A better understanding could lead to applications in the food, pharmaceutical, and cosmetic industries.
Figure 3A shows the correlations and PCA of TPC, TFC, and antioxidant activity. Strong correlations were observed between TPC and the antioxidant assays: ABTS (R² = 0.998), FRAP (R² = 0.987), and DPPH (R² = 0.985). Similarly, FRAP showed a strong correlation with both DPPH (R² = 0.987) and ABTS (R² = 0.975) (Fig. 3B). However, no significant correlation was found with TFC, suggesting that phenolic compounds, rather than flavonoids, are the main contributors to antioxidant capacity.
 | Figure 3. Correlation and PCA of TPC, TFC, and antioxidant activity by different methods in M. discolor extracts obtained by agitation using different solvents. [Click here to view] |
PCA revealed that TPC and the antioxidant assays formed a principal cluster, while TFC was less associated (Fig 3C). The first two components explained 79.77% and 19.45% of the variance, respectively, totaling 99.22%. This aligns with prior studies on M. pungens, in which phenolic compounds were identified as the main contributors to antioxidant function [55], and with evidence from other Myrtaceae species, which link TPC more strongly than TFC to radical-scavenging potential [26,56,57].
This study is one of the few experimental assessments of M. discolor. However, it is limited by the absence of metabolite identification through advanced chromatographic or spectrometric methods. The solvent range was restricted to polar systems, which may have excluded bioactive lipophilic constituents. Future research should, therefore, incorporate broader solvent systems (e.g., acetone and ethyl acetate), in-depth metabolomic profiling (e.g., LC-MS and GC-MS), and bioactivity-guided fractionation, in order to better understand the full therapeutic potential of this species and promote its use in health-related applications.
4. CONCLUSION
The highest TPC was obtained with a 50% ethanol solvent, while the highest TFC was achieved with a 96% ethanol solvent. The extracts demonstrated excellent antioxidant activity, particularly in the 30% and 50% ethanolic extracts. Strong positive correlations (R² > 0.95) were found between the FRAP, ABTS, and DPPH assays and TPC, whereas TFC showed no correlation. However, the lack of correlation between TFC and these methods does not imply that TFC is unsuitable for measurement in M. discolor extracts. It is recommended to continue research on this species, as there is limited information available, which could enhance the potential for therapeutic applications.
5. ACKNOWLEDGMENTS
The authors would like to express their gratitude to the technical support staff from the Laboratory of Nutrition at the School of Nutrition at Universidad César Vallejo and the Laboratory of Pharmacognosy at Universidad Nacional de Trujillo for their valuable assistance.
6. AUTHORS CONTRIBUTION
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. FINANCIAL SUPPORT
This research was funded by the Vice-Rectorate for Research of Universidad César Vallejo through Resolution No. 194-2023-VI-UCV, with project code P-2023-198, entitled “Potential source of polyphenols and antioxidants from the species Myrcianthes discolor (Kunth) McVaugh.”
8. CONFLICT OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
This project was approved by the Ethics Committee of the School of Nutrition at César Vallejo University with registration code PI-CEI-NUTRICIÓN-002.
10. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
11. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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