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Article

Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl.

by
José Osvaldo Bernal-Gallardo
1,
Hortencia Gabriela Mena-Violante
2,* and
Silvia Luna-Suárez
1,*
1
Instituto Politécnico Nacional, Centro de Investigación en Biotecnología Aplicada, Tepetitla 90700, Mexico
2
Instituto Politécnico Nacional, Department of Research, CIIDIR IPN Unidad Michoacán, Jiquilpan 59510, Mexico
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1091; https://doi.org/10.3390/horticulturae10101091
Submission received: 9 September 2024 / Revised: 6 October 2024 / Accepted: 10 October 2024 / Published: 12 October 2024
(This article belongs to the Special Issue Bioactivity of Horticultural Crops and Extracts)
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Abstract

:
Around 450 species of blueberries of the genus Vaccinium are known, of which some have gained preferential breeding, such as the ‘Biloxi’ variety. Some little studied species, such as Vaccinium leucanthum Schltdl. located in Mexico, could be a potential source of bioactive compounds. In this study, the phenolic compounds (chlorogenic acid content, hyperoside, phenols, flavonoids, tannins and total anthocyanins content) as well as the potential biological activity (antioxidant, antimicrobial, xanthine oxidase converting enzyme inhibition and angiotensin I inhibition) of Vaccinium leucanthum Schltdl. were studied, making a comparison with the Biloxi variety, which is the most widely cultivated one. The extract of V. leucanthum showed the highest content of flavonoids (4.853 ± 0.341 mg QE/g DW), total anthocyanins (0.303 ± 0.008 mg CGE/g DW), petunidin-3-glucoside (6.92 ± 0.12 mg PGE/g DW), malvidin-3-glucoside (11.80 ± 0.10 mg MGE/g DW) and hyperoside (5.137 ± 0.100 mg HE/g DW). It should be noted that V. leucanthum showed the same total tannin content and the same efficacy in the inhibition of Angiotensin I-converting enzyme as ‘Biloxi’, as well as the same antibacterial effect against the enterobacteria Salmonella choleraesuis ATCC 12022, Escherichia coli ATCC 12792and Shigella flexneri ATCC 10708. These findings demonstrate that V. leucanthum extracts could be an important source of preservatives as well as nutraceutical compounds for use in foods and medicines.

1. Introduction

The fruits of the genus Vaccinium in their final stage of ripening are red, blue or even black, depending on the species [1]. Blueberry is the most widely cultivated of the Vaccinium fruits, with production expected to increase by 20% by 2030 [2]. Around 90% of the world’s blueberry production takes place in the Americas [3], where Mexico in 2021 ranked fifth worldwide, with the state of Michoacán standing out as the largest producer and the ‘Biloxi’ variety as the most cultivated one [4]. Some varieties have been created from hybridizations of wild species, with Vaccinium corymbosum L. being one of the most widely used in plant breeding [5].
Vaccinium fruits, besides having a pleasant taste, have a high content of bioactive compounds that may contribute to the prevention of chronic diseases (e.g., cancer, diabetes, kidney damage, cardiovascular damage [6,7] and Alzheimer’s disease [8]) due to their antioxidant properties capable of protecting enzymatic activities in metabolic pathways, modulating second messenger systems and gene expression [6,9]. Among the secondary metabolites identified in Vaccinium fruits are flavonols mostly derived from quercetin, such as hyperoside; phenolic acids such as caffeic, p-coumaric, ferulic and chlorogenic acids; and anthocyanidins such as cyanidin, delphinidin, malvidin, petunidin and peonidin [10,11,12,13]. Chlorogenic acid and quercetin derivatives have also been reported in Vaccinium leaves, conferring beneficial effects in biological activities [14]. Chlorogenic acid is one of the main secondary metabolites found in Vaccinium fruits [15,16], being responsible for various biological activities such as anti-inflammatory [17,18], antihypertensive [19,20], antimicrobial, antioxidant and as a stimulator of the nervous system [21,22].
Extracts from Vaccinium fruits rich in anthocyanins have also been reported as good sources of antimicrobial compounds against pathogens such as Escherichia coli and Listeria monocytogenes [23]. Although many metabolites present in Vaccinium fruits have been found in other berries such as sweet cherry, better antioxidant and antimicrobial effect has been found in Vaccinium fruits [24]. Carbohydrates such as rhamnose, galactose and arabinose are also metabolites of interest in blueberries, since they are involved in the flavor and are part of the pectin polysaccharides of fruits, which have been reported to be effective inhibitors of Staphylococcus aureus and Escherichia coli [25].
Some wild Vaccinium species have been reported to have higher biological activity than cultivated species or cultivars due to their higher content of secondary metabolites [26,27,28]. In addition, the levels of secondary metabolites are influenced by genetic, biotic and abiotic factors [29], which in turn are related to the roles they play in plants. Phenolic compounds are important in the mechanisms of plant defense, fruit development and seed dispersal and accumulate to protect plants against various stresses such as exposure to ultraviolet light, drought, salinity, temperature and plant pathogenic microorganisms [30]. Additionally, the concentration of these compounds in the fruit is usually higher in the epidermis and in the outer layers of the hypodermis [28,31]. The stage of fruit collection is also important, as it has been reported in Vaccinium stenophyllum Steud. that there are differences in anthocyanin content according to ripening, with a higher content in the final stages of ripening, which is associated with a pleasant taste by the consumer [32].
Functional fruits with high levels of bioactive compounds are of interest to the food additive industry and plant breeders seeking to meet the demand for foods that improve consumer health. The genus Vaccinium has about 450 reported species [33,34], but only a small group has been studied. In Mexico, native Vaccinium species are distributed in the central and southeastern parts of the country [35]. In the state of Michoacán, Vaccinium leucanthum Schltd. has been identified, but little is known about its biological potential [36]. Therefore, the aim of the present work was to study the phenolic compounds and the biological activities of the fruits of V. leucanthum in comparison with the widely cultivated blueberry ‘Biloxi’.

2. Materials and Methods

Methanol, chlorogenic acid, hydrochloric acid (HCl), benzenesulfonyl chloride (BSC), hippuryl-L-histidyl-L-leucine (HHL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), quinoline, angiotensin converting enzyme (ACE), hyperoside and Trolox were HPLC grade from Sigma-Aldrich® (St. Louis, MO, USA). Delphinidin-3-glucoside, cyanidin-3-glucoside, malvidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and pelargonidin-3-glucoside were purchased from Sigma-Aldrich® (St. Louis, MO, USA). The silica gel chromatographic plates 60 F254 (20 cm × 10 cm, Art. 1.05729.0001), used in the HPTLC technique, were supplied by Merck® (Darmstadst, Hesse, Germany). The solvents used for the mobile phases, ethyl acetate, acetic acid and formic acid were obtained from Sigma-Aldrich® (St. Louis, MO, USA). Nutrient broth and Mueller-Hinton agar were purchased from BD Bioxon (Ciudad de México, CDMX, México). The bacterial strains used in this study were the certified reference strains Salmonella choleraesuis ATCC 12022, Escherichia coli ATCC 12792 and Shigella flexneri ATCC 10708.
Mature fruits of V. leucanthum were harvested in the autumn season (September 2022) according to the color reported in the botanical literature [37]. Mature fruits of V. leucanthum Schltd. were collected in Morelia, Michoacán (19°35′54.7″ N 101°07′16.7″ W at 2258 m a.s.l.).
The plants of V. leucanthum were identified by M. en C. Ignacio García Ruíz (collection number 12569), who is a botanist dedicated to the identification of wild species. Specimens were deposited in the CIIDIR-MICHOACÁN Herbarium CIMI. Fruits of the ‘Biloxi’ were supplied by the company Driscoll’s, located in Chilchota, Michoacán, Mexico, in the autumn season (September 2022).

2.1. Fruit Quality

The maturity index of each fruit was determined by means of the soluble solids/titratable acidity ratio [38]. The evaluation was conducted in triplicate.

2.2. Extract Preparation

The preparation of the extracts was carried out as previously reported [32]. Lyophilized fruit samples (Lyophilizer, LABCON, Kansas City, MO, USA) were extracted with methanol in an ultrasonic bath (ULTRAsonik, DENSTPLY, NEYTECH, Yucaipa, CA, USA).

2.3. Total Phenol Quantification

Total phenols were quantified in a UV-Vis spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA) following the method previously described [39]. Absorbance readings were taken at a wavelength of 700 nm. For the construction of the calibration curve (A700 = 1.4722 [gallic acid] − 0.16, R2 = 0.9759), standard solutions of gallic acid were prepared in a range of concentrations from 0.2 to 2.0 mg/mL. Each determination was carried out in triplicate and the results obtained were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW).

2.4. Total Flavonoid Quantification

The total flavonoid content of the extracts was determined using a UV-Vis spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA) following previous reports [40]. Absorbance readings were taken at a wavelength of 425 nm. For the construction of the calibration curve (A425 = 1.0443 [quercetin] − 0.048, R2 = 0.9967), quercetin standard solutions were prepared in a range of concentrations from 0.2 to 2.0 mg/mL. Each determination was performed in triplicate and the results obtained were expressed as milligrams of quercetin equivalents per gram of dry weight (mg QE/g DW).

2.5. Total Anthocyanin Quantification

The total anthocyanin content of the extracts was determined using a UV-Vis spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA) following previous reports [41]. The absorbance of each extract was read at 535 nm using a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Vermont, VT, USA). The assay was performed in triplicate. Total anthocyanin content was expressed as cyanidin-3-glucoside equivalents per gram of dry weight (mg CGE/g DW) and was calculated using the following formula:
T o t a l   a n t h o c y a n i n s   c o n t e n t = ( A 535 n m 25,965 ) ( 449 ) ( 1 e x t r a c t   c o n c e n t r a t i o n ) ( 10 6 )

2.6. Total Tannins Quantification

Total tannin content of the extracts was determined using a UV-Vis spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA) following previous reports [42]. Absorbance readings were taken at a wavelength of 750 nm. For the construction of the calibration curve (A750 = 1.7142 [tannic acid] + 0.1896, R2 = 0.9418), tannic acid standard solutions were prepared in a range of concentrations from 0.2 to 1.2 mg/mL. Each determination was performed in triplicate and the results obtained were expressed as milligrams of tannic acid equivalents per gram of dry weight (mg TAE/g DW).

2.7. Detection and Quantification of Chlorogenic Acid, Hyperoside and Anthocyanins by HPTLC

Detection and quantification of reference compounds in methanolic extracts using High Performance Thin Layer Chromatography (HPTLC) was carried out as previously described, with some modifications [43]. Silica gel plates were activated at 120 °C for 5 min in a plate heater (TLC Plate Heater 3, CAMAG, Muttenz, Switzerland).
The reference solutions of chlorogenic acid, hyperoside, cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and pelargonidin-3-glucoside were prepared at 0.1 mg/mL in methanol. An automatic sampler (ATS 4, CAMAG, Muttenz, Switzerland) was used to apply the extracts and reference solutions to the plates. A 25 µL syringe was used to apply bands with a length of 6 mm on the different plate lanes. The distance between lanes was 7.3 mm, the distance from the bottom edge was 8.7 mm, and the distance from the left side was 9 mm. Five different volumes (0.5, 1.5, 3, 4.5 and 6 µL) of each mixture (hyperoside: chlorogenic acid, malvidin-3-glucoside: petunidin-3-glucoside, cyanidin-3-glucoside: delphinidin-3-glucoside and malvidin-3-glucoside: petunidin-3-glucoside) were applied to the plate. Additionally, 2.0 µL of the extracts were applied in triplicate at a constant application rate of 150 nL/s.
An automatic development chamber (ADC 2, CAMAG, Muttenz, Switzerland) was used for the chromatography run. The chamber was maintained at a relative humidity of 25 ± 2%, and the mobile phase was ethyl acetate (10): formic acid, (1.1): acetic acid and (1.1): water (2.3) (v/v/v/v). For standardized separation, the plate activity was controlled for 4 min using a saturated potassium acetate solution (257.6 g/100 g H2O) in the humidity control unit. The migration distance was 45 mm in 15 min, after which the plate was dried under a stream of air for 5 min.
After completion of the chromatography, the plate was heated at 120 °C for 5 min in a plate heater (TLC Plate Heater 3, CAMAG, Muttenz, Switzerland) and derivatized with natural products ethylene glycol reagent (NPEG). The derivatization was performed in the immersion device (Chromatogram Immersion Device, CAMAG, Muttenz, Switzerland) using a vertical speed of 5 cm/s. The immersion time was 1 s, and after derivatization the plate was dried under air flow for 3 min. The plates images were captured under visible light and UV light (254 nm and 366 nm) using a TLC Visualizer (CAMAG, Muttenz, Switzerland). The data were collected and analyzed using VisionCATS software (version 2.4, CAMAG, Muttenz, Switzerland). Table 1 shows the standard calibration curves used for phenolics quantification in the extracts.

2.8. Antioxidant Activity

2.8.1. Antioxidant Activity Using ABTS Assay

The antioxidant activity of the extracts was evaluated using the ABTS+• method, as previously reported [44]. The ABTS+• radical was generated via oxidizing with potassium persulfate incubating for 24 h. The ABTS+• radical was then adjusted with distilled water to an absorbance of 0.760 ± 0.001 using a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA). To 20 µL of methanolic extracts, 280 µL of ABTS solution were added and the mixture was kept in the dark for 6 min. The absorbance of the sample and blank were measured at 734 nm using a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Vermont, VT, USA). The antioxidant capacity of the extracts was expressed as μmoles Trolox equivalent per gram of dry weight (μmol ET/g DW) using a calibration curve (μmol ET = −0.9416[A734nm] + 0.828, R2 = 0.9981) with five different concentrations (0–0.60 μmol Trolox). The assay was performed in triplicate.

2.8.2. Antioxidant Activity Using DPPH Assay

The antioxidant activity of the methanolic extracts was quantified via DPPH assay in microplates, following an adaptation of the previously described method [45]. Mixtures of 20 μL extract and 200 μL DPPH● (150 mM) were incubated in darkness for 30 min. The decrease in absorbance at 515 nm was measured in a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA), quantified and related to Trolox concentration using a linear calibration curve (μM ET = −0.632 [A515nm] + 0.281, R2 = 0.9849). Each sample was analyzed in triplicate and the results were expressed as the antioxidant capacity in μM ET/g DW.

2.8.3. Antioxidant Activity Using HPTLC-DPPH●

The analysis of the antioxidant activity of the separated chromatographic zones was performed as previously reported [46]. Chromatography was performed according to Section 3.6 and derivatized with DPPH reagent in an immersion device (Chromatogram Immersion Device, CAMAG) at a vertical speed of 5 cm/s for 1 s. The plates were dried in the dark for 30 min. The plates’ images were captured under visible light and UV light (254 and 366 nm) using a TLC Visualizer and then analyzed with VisionCATS software.

2.9. Antimicrobial Activity

The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the extracts were determined against E. coli, Salmonella choleraesuis and Shigella flexneri using the reported broth microdilution method [47], with slight modifications. The methanolic extracts were concentrated on a rotary evaporator (Rotavapor r-200, BUCHI, Zurich, Switzerland) and adjusted with deionized water to 53.34, 26.67, 17.78, 8.89, 5.92, 2.96, 1.97 and 0.98 mg/mL. Aliquots of 100 μL of Mueller-Hinton broth, 50 μL of the extract dilution and 20 μL of the bacteria adjusted to a density of 1 × 107 CFU/mL were placed in sterile microplates and incubated at 37 °C for 19 h to allow bacterial growth. Then, 20 μL of MTT reagent was added and incubated for 45 min at 37 °C for MIC recording. Subsequently, 50 μL aliquots of the wells without bacterial growth were spread on plates with Mueller-Hinton agar medium, and the MBC was assessed after 24 h of incubation at 37 °C. The following controls were considered: positive control, broth + bacteria; negative control, broth only; and antibiotic control, broth + bacteria + antibiotic (35 μg ceftriaxone/mL). In the experiment, each dilution and control were applied in triplicate and repeated on three different days.

2.10. Xanthine Oxidase (XO) Inhibitory Assay

The xanthine oxidase inhibition assay was carried out as previously reported [48]. A total of 100 µL of methanol extracts with dimethyl sulfoxide (100 µg/mL) and 0.1 U/mL xanthine oxidase (100 µL) were added to 0.05 M potassium phosphate buffer (1.3 mL, pH 7.5). The mixtures were incubated at 25 °C for 10 min. The absorbance was measured at 295 nm using a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Winooski, VT, USA).

2.11. Angiotensin I-Converting Enzyme (ACE) Inhibition Assay

Angiotensin I-converting enzyme inhibition was performed based on previous reports [49]. First, 10 µL of sample was added to 30 µL of HHL, shaken and incubated for 5 min at 37 °C. Next, 20 µL ACE (0.1 U/mL) was added and kept at 37 °C for 1 h. Then, 70 µL of 1 M HCl was added. From this mixture, 30 µL was taken and added to 285 µL of quinoline and 75 µL of BSC and incubated for 30 min in the dark. Finally, 1.11 mL ethanol was added and shaken via inversion. Then, the absorbance was measured at 492 nm in a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, Vermont, VT, USA). For the blank, the same reagents were used, except the sample (10 µL of 0.1 M borate buffer); for the negative control, the same reagents were used, except 1 M HCl; captopril (50 µg/mL) was used as control inhibitor. The percentage inhibition of ACE was calculated as follows:
A C E   i n h i b i t o r y   a c t i v i t y % = A b l a n k − A s a m p l e A b l a n k − A c o n t r o l ( 100 )

2.12. Statistical Analysis

The results are presented as means ± standard deviations. To determine significant differences in each measured parameter, a t-Student test was performed with a significance level of 0.05, which was performed using R Studio software version 4.3.2.

3. Results and Discussion

3.1. Quality Attributes

The soluble solids content (Figure 1A) was 5% higher in V. leucanthum than that found in ‘Biloxi’. Previous studies have reported 11.78 ± 0.44% soluble solids in Vaccinium meridionale Swartz [50], which was 8% lower than that found in V. leucanthum. The soluble solids content generally increases sweetness, which is desirable for processing Vaccinium fruits [51]. Most of the total solids in the fruit are contributed by carbohydrates [52], with fructose, glucose and small amounts of sucrose being the most predominant in Vaccinium [53].
Titratable acidity, which is associated with the bitter taste of the fruit, was twice as high in V. leucanthum than in ‘Biloxi’ (Figure 1B). In Vaccinium angustifolium, quinic acid was reported to be the most abundant of all organic acids (65–85%), followed by citric and malic acids [54]. It is important to mention that both V. leucanthum and ‘Biloxi’ showed a different maturity index (Figure 1C), being higher in ‘Biloxi’. It should be noted that the maturity index is a characteristic value that can vary depending on the species or variety analyzed [55,56].

3.2. Total Phenols

The highest content of total phenolics (CTP) (Table 2) was observed in the fruits of ‘Biloxi’, which was 12% higher than that found in V. leucanthum. Previous research with ‘Biloxi’ reported a CTP of 2.87 ± 2.1 mg GAE/g DW [57], which is 85% lower than that found in the evaluated ‘Biloxi’ extracts. In addition, 11.7 ± 2.44 mg GAE/g DW was reported in fruits of ‘Nelson’ [57] and 9.44 ± 0.22 mg GAE/g DW in Vaccinium ashei [58], which were lower than those found in the evaluated ‘Biloxi’ extracts (39 and 51%, respectively). In Vaccinium myrtillus fruits [59], a CTP was reported to be 37% higher (30.5 ± 1.7 mg GAE/g DW) than that found in the evaluated ‘Biloxi’ extracts reported. Previous studies with ‘Elliott’, ‘Darrow’, ‘Duke’ and ‘Bluecrop’ fruits reported the highest CTP (5.05 ± mg GAE/g fresh weight) in ‘Elliott’, which correlated with the lowest MIC (450 mg/mL) and MBC (600 mg/mL) against Salmonella enteritidis [60], so it is, therefore, important to find fruits with a high CTP.

3.3. Total Flavonoids

The highest content of total flavonoids (CTF) was found in V. leucanthum (Table 2), which was 36% higher than that found in ‘Biloxi’. Previous studies have reported a CTF of 0.767 ± 0.026 mg QE/g DW in V. myrtillus fruits and 0.875 ± 0.083 mg QE/g DW in ‘Bluegold’ [61], which is lower than that in V. leucanthum (84 and 82%, respectively). Research has reported that the higher the CTF, the greater the antioxidant response [62,63], which could benefit the consumer.

3.4. Total Anthocyanins

The highest content of total anthocyanins (CTA) (Table 2) was obtained in V. leucanthum fruits (Table 2), which was 27% higher than that found in ‘Biloxi’. Previous studies with ‘Biloxi’ reported a CTA of 0.049 ± 0.002 mg CGE/g DW [64], 78% lower than that found in the evaluated ‘Biloxi’ extracts. Other studies have reported a CTA of 0.223 ± 0.003 mg CGE/g DW in ‘Misty’ [61] and 0.141 ± 0.004 mg CGE/g DW in V. stenophyllum [32], which were 26% and 53% lower than that found in V. leucanthum, respectively. In addition, 0.916 ± 0.001 mg CGE/g DW was reported in Vaccinium arctostaphylos, which was 67% higher than that found in V. leucanthum. Anthocyanins in blueberries are potent natural antioxidants, reduce inflammation, particularly in the gut, and protect against the development of cardiovascular disease via attenuating systemic microinflammation [65,66].
It is important to note that some cultivars have genetic characteristics derived from several Vaccinium species, such as ‘Biloxi’, which is derived from the species V. corymbosum, Vaccinium darrowii, Vaccinium virgatum, V. angustifolium and Vaccinium fuscatum [5]. However, this does not guarantee a higher content of these bioactive compounds since it depends on interactions with biotic and abiotic factors that modify their content [28,29,67], for example, the wavelength of UV radiation wavelengths absorbed by plants before harvesting significantly promotes anthocyanin biosynthesis [68].

3.5. Total Tannins

Tannins are the key factor that determines the degree of astringency of the fruit and appear in lower content in ripe fruit [69]. According to the total tannin content (CTT) (Table 2), no significant difference was found between ‘Biloxi’ and V. leucanthum. In Vaccinium vitis-idaea L., a CTT of 5.17 mg TAE/g DW was reported, which is 41% lower than that of V. leucanthum [70], while in Vaccinium macrocarpon fruit tannins accounted for 63% of the total phenolic compounds [71], giving the fruit antimicrobial power against Staphylococcus aureus VTT E-70045.
It is noteworthy that condensed tannins (proanthocyanidins) have been found in fruits of the genus Vaccinium and are formed via the polymerization of catechin and/or epicatechin units [72].

3.6. HPTLC

3.6.1. Detection and Quantification of Chlorogenic Acid and Hyperoside

According to the chromatogram (Figure 2), hyperoside (Rf = 0.57) and chlorogenic acid (Rf = 0.50) were detected and quantified in the fruit extracts. The chlorogenic acid (CCA) content (Table 3) of ‘Biloxi’ was 34% higher than that of V. leucanthum. Previous HPTLC studies [32] reported a CCA of 12.507 ± 0.289 mg CAE/g DW in V. stenophyllum, which was 26% lower than that found in ‘Biloxi’. The highest content of hyperoside (CH) (Table 3) was found in V. leucanthum, which was 31% higher than that of ‘Biloxi’.
Chlorogenic acid (5-O-caffeoylquinic acid) is produced from caffeic acid and quinic acid and is one of the most abundant phenolic compounds found in Vaccinium fruits [15,73]. Its isomers, such as cryptochlorogenic acid and neochlorogenic acid, are also found in this genus [22]. In general, chlorogenic acid and its isomers are responsible for scavenging reactive oxygen species and reducing cell membrane peroxidation, thus protecting plant cells from oxidative stress [74]. As a bioactive compound, chlorogenic acid modulates glucose and lipid homeostasis, protects the cardiovascular system, kidney and liver, facilitates recovery from neurological disorders including neurodegenerative diseases and inhibits tumor cell proliferation [75].
Hyperoside (quercetin-3-O-beta-D-galactopyranoside), a yellow solid flavonoid, is one of the predominant compounds of V. leucanthum that exhibits a broad spectrum of pharmacological properties, including cancer prevention and protection of the brain, neurons, heart and kidney of various vital organs via regulating various signaling pathways, metabolic processes, cytokines and kinases, making it a compound of great interest for the development of new therapies [76].

3.6.2. Detection and Quantification of Anthocyanins

Among the anthocyanins, delphinidin-3-glucoside (Rf = 0.23) and cyanidin-3-glucoside (Rf = 0.32) were more abundant in ‘Biloxi’ (Figure 3), whereas both petunidin-3-glucoside (Rf = 0.28) and malvidin-3-glucoside (Rf = 0.35) were detected only in V. leucanthum. Previous HPLC studies have reported glycosylated delphinidin and cyanidin as two of the most abundant anthocyanins in ‘Biloxi’ [77,78].
Table 4 shows the anthocyanin contents determined using HPTLC in the fruit extracts. Previous research [79] reported 3.44 mg MGE/g DW and 4.33 mg PGE/g DW in V. myrtillus quantified via HPLC, which were lower than those found in V. leucanthum (71% and 37%, respectively), and 10.47 mg DGE/g DW in V. myrtillus, which was 42% higher than that found in ‘Biloxi’; in contrast, 3.82 mg CGE/g DW was reported in V. myrtillus, which was 9% lower than that found in ‘Biloxi’.
The genus Vaccinium contains a wide variety of anthocyanins in high concentrations [80]. Subtle differences in anthocyanin profiles between different species or cultivars are not easily explained by genotypes alone, probably due to climatic factors interfering with anthocyanin biosynthesis [66].

3.7. Antioxidant Activity

3.7.1. DPPH● and ABTS●+ Assay

Using the DPPH● method for the analysis of lipophilic antioxidants [81], the highest antioxidant activity was found in ‘Biloxi’ (Table 5), which was 43% higher than that found in V. leucanthum. In V. corymbosum, 79.42 µmol TE/g DW was reported [82], which was 63% higher than that presented in ‘Biloxi’ but 17% lower than that reported in V. myrtillus (250 µmol ET/g DW) [59].
Regarding the ABTS●+ method, used to analyze both hydrophilic and lipophilic antioxidants [63], the highest antioxidant activity was found in ‘Biloxi’ (Table 5), which was 51% higher than that found in V. leucanthum. Previous studies have reported 192.32 µmol TE/g DW in V. corymbosum [82] and 72.76 ± 6.5 µmol TE/g DW in ‘Huron’ [83], which were lower than those presented in ‘Biloxi’ fruit (26 and 72%, respectively).
It has been suggested that consumption of antioxidant compounds reduces oxidative stress caused by oxidative free radical damage in humans, which is associated with diseases such as atherosclerosis, Alzheimer’s disease, cancer, eye diseases, diabetes, rheumatoid arthritis and neuronal diseases [84]. It has also been reported that consumption of a combination of antioxidant compounds is better than just a single one [85]. Blueberries have been identified as one of the fruits with the highest antioxidant activity, higher than strawberries, oranges, lemons, radishes and grapefruit [86]. Therefore, it is important to search for antioxidant compounds in fruits of the genus Vaccinium because of their high content and diversity.

3.7.2. HPTLC-DPPH● Antioxidant Assay

The HPTLC-DPPH● assay made it possible to note the chromatogram bands with antioxidant potential (Figure 4): the more intense the yellow of the band, the greater the antioxidant potential of the detected compound, marking the similarities and differences in the compounds involved in their antioxidant action. The main antioxidant bands in fruit extracts corresponded to chlorogenic acid (Rf = 0.50) and hyperoside (Rf = 0.57), in addition to some anthocyanins. In V. leucanthum, malvidin-3-glucoside (Rf = 0.43) and petunidin-3-glucoside (Rf = 0.35) were found to be involved in the antioxidant activity. In ‘Biloxi’, cyanidin-3-glucoside (Rf = 0.40) and delphinidin-3-glucoside (Rf = 0.23) were detected as other antioxidant compounds.
Phenolic acids are the most potent antioxidants among the polyphenolic compounds [87]. Chlorogenic acid, a representative phenolic acid, is a potent DPPH● radical-scavenger [21].
Within the group of flavonoids, anthocyanins are the ones with the highest antioxidant activity [87]. This is attributed to their unique chemical structure, as exemplified by cyanidin-3-glucoside, which contains an electron-deficient C-ring, hydroxyl (OH) substitutions, methylations on the B-ring, and sugar attachments, all of which contribute to the antioxidant activity of anthocyanins [88]. In addition, the antioxidant activity of the hyperoside may be related to the hydroxyl groups on the A and B rings and the glycosides attached to the C ring [76]. This suggests that the different antioxidant sites of these compounds give Vaccinium fruits their cardiovascular, anti-inflammatory, glycoregulatory and neuroprotective benefits [89].

3.8. MIC and MBC of Methanolic Extracts of Fruits of V. leucanthum Schltdl. and ‘Biloxi’

Each fruit extract showed the same MIC (8.89 mg/mL) and MBC (17.78 mg/mL) for the bacteria studied (Table 6). This phenomenon has been previously reported in extracts of Prunus spinosa L., with an MIC of 250 μg/mL for E. coli ATCC 25922 and S. sonnei ATCC 25931 [90]. There are other studies in which MICs and MBCs for two or more bacteria have a difference of less than 50% of their concentrations, for example, extracts of V. corymbosum showed MICs with a difference of 1.58 mg/mL between E. coli (3.74 mg/mL) and Salmonella enteritidis (2.16 mg/mL) [91].
According to the previous report [92], MICs between 100 and 500 μg dry extract/mL (μg DE/mL) were considered active; MICs between 500 and 1000 μg DE/mL were considered moderately active; MICs between 1000 and 2000 μg DE/mL were considered to have low activity; and MICs > 2000 μg DE/mL were considered inactive. In this study, the MICs of the extracts were expressed in mg dry sample/mL. Therefore, the 8.89 mg dry sample/mL found in the extracts was equivalent to 382.27 μg DE/mL, so it can be considered that the extracts have active compounds against the studied enterobacteria. Considering the above, ceftriaxone with 96% less concentration than the fruits achieved the MIC; however, it should be noted that in the dry extract of the fruits, there are compounds that may not be participating in the microbial inhibition.
In particular Escherichia, Salmonella and Shigella are foodborne opportunistic pathogens of the Enterobacteriaceae family that primarily affect the gastrointestinal tract and cause diarrhea [93]. These bacteria are considered to be a major public health problem worldwide due to the emergence of antibiotic resistance in recent years [94].
The antimicrobial activity of phenolic compounds is not clearly related to a class of polyphenols [95]. The mechanism that has been suggested is the toxicity of phenolic compounds to microorganisms, including enzymatic inhibition by oxidized compounds, possibly due to reactions with sulfhydryl groups or nonspecific contacts with enzymes leading up to their inactivation [96].
One of the most abundant compounds detected in fruit extracts in the present study was chlorogenic acid. Previous studies [97] have reported that chlorogenic acid has inhibitory activity against Bacillus subtilis without directly altering the membrane, but induces a significant decrease in the intracellular concentration of adenosine triphosphate (ATP). In addition, metabolomic results indicated that chlorogenic acid had a bacteriostatic effect via inducing an intracellular metabolic imbalance of the tricarboxylic acid (TCA) cycle and glycolysis, leading to metabolic derangement and death of Bacillus subtilis.

3.9. Xanthine Oxidase Inhibitory Assay

The best xanthine oxidase inhibition (Table 7) occurred in ‘Biloxi’, which was 11% superior to V. leucanthum. In V. corymbosum L., a 50% inhibition of xanthine oxidase was reported at a concentration of 73.4 ± 2.5 μg DW/mL [98], which is 56% lower than that used in the present study.
Elevated uric acid concentrations are associated with high xanthine oxidase activity, which is characteristic of many vascular diseases such as gout [99]. Xanthine oxidase inhibitors work via blocking uric acid biosynthesis from purines in the body and are thought to increase uric acid excretion [99,100].
Previous studies [101] have reported that chlorogenic acid produces noncompetitive inhibition of xanthine oxidase, although the binding site has not yet been identified. In addition, it has been reported [102] that anthocyanins are mixed-type inhibitors of xanthine oxidase, which could insert into the hydrophobic active region and induce changes in the secondary structure.

3.10. Angiotensin I-Converting Enzyme Inhibitory Activity

Inhibition of angiotensin I-converting enzyme (ACE) activity is of great importance, because its involvement in the pathogenesis of hypertension is focused on the production and vasoconstrictive effects of angiotensin II along with the generation of free radicals [103]. According to the ACE inhibition assay (Table 8), the extracts were shown to inhibit the enzyme and did not differ in the percentage of inhibition. Notably, no ACE inhibition was found in a study with V. ashei [104]. Both quercetin (a component of hyperoside) and chlorogenic acid have been reported to be ACE inhibitors [105] and are components of the fruits in the present study that may be involved in the ACE inhibitory effect.
The degree of inhibition of ACE activity by phenolic acids depends on the nature of interactions between the phenolic acids and the residual fractions (Zn2+ and the disulfide bridge) in the active site of ACE [84].
Finally, it is well known that the consumption of the phenolic compounds that have been identified in V. leucanthum extracts can prevent the development of chronic degenerative diseases (e.g., diabetes, obesity, Parkinson’s, Alzheimer’s) due to their antioxidant, antimicrobial and therapeutic activity. In addition, the extracts can also be used as a source of preservatives, natural pigments or prebiotics for food manufacturing, showing interesting potential uses in innovative applications in the food, pharmaceutical and chemical industries [106,107].

4. Conclusions

In the continuous search for new sources of bioactive compounds and the generation of scientific knowledge, this work is the first report of the biological activities (antioxidant, antimicrobial, ACE and XO inhibition) of V. leucanthum extracts. Additionally, this is the first report that promotes and describes the use of HPTLC-DPPH to analyze the antioxidant activity of the studied fruits.
Antioxidant compounds identified in the fruit extracts included chlorogenic acid, hyperoside and anthocyanins, known for their potent antioxidant properties. These extracts also showed a significant antimicrobial effect against Escherichia coli ATCC 12792, Shigella flexneri ATCC 10708 and Salmonella choleraesuis ATCC 12022, which are bacteria of public health concern. In addition, the extracts showed an inhibitory effect on xanthine oxidase (XO) and angiotensin converting enzyme (ACE), which could be attributed to the presence of phenolic compounds.
This study expands the knowledge on the biological activity of the fruits of Vaccinium leucanthum Schltdl. and opens new avenues for their application in the food, pharmaceutical and chemical industries.

Author Contributions

Conceptualization, H.G.M.-V., S.L.-S. and J.O.B.-G.; methodology, H.G.M.-V., S.L.-S. and J.O.B.-G.; validation, J.O.B.-G.; formal analysis, H.G.M.-V. and S.L.-S.; investigation, J.O.B.-G.; resources, H.G.M.-V. and S.L.-S.; data curation, H.G.M.-V., S.L.-S. and J.O.B.-G.; writing—original draft preparation, J.O.B.-G.; writing—review and editing, H.G.M.-V. and S.L.-S.; visualization, H.G.M.-V. and S.L.-S.; supervision, H.G.M.-V. and S.L.-S.; project administration, H.G.M.-V. and S.L.-S.; funding acquisition, H.G.M.-V. and S.L.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Secretaría de Investigación y Posgrado-IPN, grant numbers 20230837 and 20240556.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Acknowledgments

The first author thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT) in México for the economic support through national scholarship.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 10 01091 g001
Figure 1. Fruit quality of Vaccinium leucanthum Schltd. and ‘Biloxi’. (A) Soluble solids, (B) titratable acidity, and (C) maturity index. The mean ± standard deviation is shown, different letters (a and b) indicate a significant statistical difference in the determination using a t-Student test (p < 0.05).
Figure 1. Fruit quality of Vaccinium leucanthum Schltd. and ‘Biloxi’. (A) Soluble solids, (B) titratable acidity, and (C) maturity index. The mean ± standard deviation is shown, different letters (a and b) indicate a significant statistical difference in the determination using a t-Student test (p < 0.05).
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Horticulturae 10 01091 g002
Figure 2. Detection and quantification of chlorogenic acid and hyperoside from extracts (Track 1: ‘Biloxi’; Track 2: V. leucanthum). Mobile phase: ethyl acetate, (10): formic acid, (1,1): acetic acid, (1,1): acetic acid and (1,1): water (2,3) (v/v/v/v/v/v). The image of the derivatized plate was recorded at 366 nm. A mixture (1:1) of chlorogenic acid and hyperoside was applied in 4 different volumes (1.5, 3, 4.5 and 6 µL, corresponding to 0.15, 0.3, 0.45 and 0.6 µg). A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate.
Figure 2. Detection and quantification of chlorogenic acid and hyperoside from extracts (Track 1: ‘Biloxi’; Track 2: V. leucanthum). Mobile phase: ethyl acetate, (10): formic acid, (1,1): acetic acid, (1,1): acetic acid and (1,1): water (2,3) (v/v/v/v/v/v). The image of the derivatized plate was recorded at 366 nm. A mixture (1:1) of chlorogenic acid and hyperoside was applied in 4 different volumes (1.5, 3, 4.5 and 6 µL, corresponding to 0.15, 0.3, 0.45 and 0.6 µg). A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate.
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Horticulturae 10 01091 g003
Figure 3. HPTLC detection of anthocyanins in fruit extracts. Track 1: ‘Biloxi’; Track 2: V. leucanthum; Track D: delphinidin-3-glucoside; Track Pt: petunidin-3-glucoside; Track C: cyanidin-3-glucoside; Track M: malvidin-3-glucoside; Track Po: peonidin-3-glucoside; Track Pl: pelargonidin-3-glucoside. The image was captured under white light after derivatization with a NP reagent. A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate.
Figure 3. HPTLC detection of anthocyanins in fruit extracts. Track 1: ‘Biloxi’; Track 2: V. leucanthum; Track D: delphinidin-3-glucoside; Track Pt: petunidin-3-glucoside; Track C: cyanidin-3-glucoside; Track M: malvidin-3-glucoside; Track Po: peonidin-3-glucoside; Track Pl: pelargonidin-3-glucoside. The image was captured under white light after derivatization with a NP reagent. A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate.
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Horticulturae 10 01091 g004
Figure 4. Detection of HPTLC-DPPH• antioxidant compounds in fruit extracts under white light illumination after derivatization with DPPH reagent. Fruit extracts were applied in triplicate, volume = 2 µL (20 mg DW/mL, w/v). Track 1: delphinidin-3-glucoside (Rf = 0.23) and cyanidin-3-glucoside (Rf = 0.32); Track 2: petunidin-3-glucoside (Rf = 0.28) and peonidin-3-glucoside (Rf = 0.38); Track 3: malvidin-3-glucoside (Rf = 0.35) and pelargonidin-3-glucoside (Rf = 0.41); Track 4: hyperoside (Rf: 0.57) and chlorogenic acid (Rf = 0.50); Track 5: ‘Biloxi’; Track 6: V. leucanthum. A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate. Of each anthocyanin, 2 µL (1 mg/mL) of chlorogenic acid and hyperoside were applied in triplicate.
Figure 4. Detection of HPTLC-DPPH• antioxidant compounds in fruit extracts under white light illumination after derivatization with DPPH reagent. Fruit extracts were applied in triplicate, volume = 2 µL (20 mg DW/mL, w/v). Track 1: delphinidin-3-glucoside (Rf = 0.23) and cyanidin-3-glucoside (Rf = 0.32); Track 2: petunidin-3-glucoside (Rf = 0.28) and peonidin-3-glucoside (Rf = 0.38); Track 3: malvidin-3-glucoside (Rf = 0.35) and pelargonidin-3-glucoside (Rf = 0.41); Track 4: hyperoside (Rf: 0.57) and chlorogenic acid (Rf = 0.50); Track 5: ‘Biloxi’; Track 6: V. leucanthum. A volume of 2 µL (20 mg DW/mL) of each extract was applied in triplicate. Of each anthocyanin, 2 µL (1 mg/mL) of chlorogenic acid and hyperoside were applied in triplicate.
Horticulturae 10 01091 g004
Table 1. Standards quantified using HPTLC in the extract phenolics of V. leucanthum Schltdl. and ‘Biloxi’.
Table 1. Standards quantified using HPTLC in the extract phenolics of V. leucanthum Schltdl. and ‘Biloxi’.
StandardExpressionCalibration Curve
chlorogenic acidmilligrams chlorogenic acid equivalent per gram of dry weight (mg CAE/g DW)y = 0.9723x − 0.0823, R2 = 0.9821
hyperosidemilligrams of hyperoside equivalent per gram of dry weight (mg HE/g DW)y = 0.239x + 0.0182, R2 = 0.9878
cyanidin-3-glucosidemilligrams cyanidin-3-glucoside equivalent per gram of dry weight (mg CGE/g DW)y = 0.9138x + 0.0203, R2 = 0.9742
petunidin-3-glucosidemilligrams of petunidin-3-glucoside equivalent per gram of dry weight (mg PGE/g DW)y = 0.7244x − 0.0112, R2 = 0.9896
malvidin-3-glucosidemilligrams of malvidin-3-glucoside equivalent per gram of dry weight (mg MGE/g DW)y = 0.3203x + 0.0172, R2 = 0.9955
delphinidin-3-glucosidemilligrams of delphinidin-3-glucoside equivalents per gram of dry weight (mg DGE/g DW)y = 0.9331x + 0.0002, R2 = 0.9804
Table 2. Total phenolic, flavonoid and anthocyanin content of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 2. Total phenolic, flavonoid and anthocyanin content of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
ExtractsTotal Phenols
(mg GAE/g DW)		Total Flavonoids
(mg QE/g DW)		Total Anthocyanins
(mg CGE/g DW)		Total Tannins
(mg TAE/g DW)
‘Biloxi’19.23 ± 0.08 a3.12 ± 0.15 b0.221 ± 0.016 b9.32 ± 0.12 a
V. leucanthum16.75 ± 0.09 b4.85 ± 0.34 a0.303 ± 0.008 a8.81 ± 0.53 a
GAE = gallic acid equivalents; QE = quercetin equivalents; CGE = cyanidin-3-glucoside equivalents; TAE = tannic acid equivalents; DW = dry weight. Data are expressed as mean ± standard deviation. Statistical analysis using Student’s t-test (p < 0.05) revealed significant differences in the columns, indicated by different letters (a and b).
Table 3. Chlorogenic acid and hyperoside quantified using HPTLC in methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 3. Chlorogenic acid and hyperoside quantified using HPTLC in methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
ExtractsChlorogenic Acid
(mg CAE/g DW)		Hyperoside
(mg HE/g DW)
‘Biloxi’16.87 ± 0.33 a3.52 ± 0.36 b
V. leucanthum7.25 ± 0.11 b5.14 ± 0.10 a
CAE = Chlorogenic acid equivalents. HE = Hyperoside equivalents. Data are expressed as mean ± standard deviation. Statistical analysis using Student’s t-test (p < 0.05) revealed significant differences in the columns, indicated by different letters (a and b).
Table 4. Anthocyanins quantified via HPTLC in methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 4. Anthocyanins quantified via HPTLC in methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Extractsmg MGE/g DWmg PGE/g DWmg DGE/g DWmg CGE/g DW
‘Biloxi’n.d.n.d.6.06 ± 0.233.47 ± 0.30
V. leucanthum11.80 ± 0.106.92 ± 0.12n.d.n.d.
MGE = malvidin-3-glucoside equivalents; PGE = petunidin-3-glucoside equivalents; DGE = delphinidin-3-glucoside; CGE = cyanidin-3-glucoside equivalents; n.d. = not detectable. Data are expressed as mean ± standard deviation.
Table 5. Antioxidant capacity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 5. Antioxidant capacity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
ExtractsABTS●+
(µM TE/g DW)		DPPH●
(µM TE/g DW)
‘Biloxi’259.93 ± 15.2 a214.08 ± 15.9 a
V. leucanthum127.92 ± 4.9 b121.83 ± 5.9 b
TE = Trolox equivalents. Data are expressed as mean ± standard deviation. Statistical analysis using Student’s t-test (p < 0.05) revealed significant differences in the columns, indicated by different letters (a and b).
Table 6. Antimicrobial activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’ against E. coli, Shigella flexneri and Salmonella choleraesuis.
Table 6. Antimicrobial activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’ against E. coli, Shigella flexneri and Salmonella choleraesuis.
ExtractsE. coli
ATCC 12792		S. flexneri
ATCC 10708		S. choleraesuis
ATCC 12022
MICMBCMICMBCMICMBC
‘Biloxi’8.8917.788.8917.788.8917.78
V. leucanthum8.8917.788.8917.788.8917.78
Ceftriaxone0.0150.0310.0150.0310.0150.031
Mean values (mg dry weight/mL). MIC = minimum inhibitory concentration; MBC = minimum bactericidal concentration.
Table 7. Xanthine oxidase inhibitory activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 7. Xanthine oxidase inhibitory activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
ExtractsInhibition (%)
‘Biloxi’67.66 ± 1.194 a
V. leucanthum56.97 ± 0.782 b
Data are expressed as mean ± standard deviation. Statistical analysis using Student’s t-test (p < 0.05) revealed significant differences in the columns, indicated by different letters (a and b). Percent inhibition was measured at 165 μg DW/mL.
Table 8. Angiotensin I-converting enzyme inhibitory activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
Table 8. Angiotensin I-converting enzyme inhibitory activity of methanolic extracts of V. leucanthum Schltdl. and ‘Biloxi’.
ExtractsInhibition (%)
‘Biloxi’43.03 ± 4.0 a
V. leucanthum42.28 ± 2.4 a
Data are expressed as mean ± standard deviation. Statistical analysis using Student’s t-test (p < 0.05) revealed significant differences in the columns, as indicated by different letters. Percent inhibition was measured at 5 mg DW/mL.
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Bernal-Gallardo, J.O.; Mena-Violante, H.G.; Luna-Suárez, S. Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl. Horticulturae 2024, 10, 1091. https://doi.org/10.3390/horticulturae10101091

AMA Style

Bernal-Gallardo JO, Mena-Violante HG, Luna-Suárez S. Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl. Horticulturae. 2024; 10(10):1091. https://doi.org/10.3390/horticulturae10101091

Chicago/Turabian Style

Bernal-Gallardo, José Osvaldo, Hortencia Gabriela Mena-Violante, and Silvia Luna-Suárez. 2024. "Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl." Horticulturae 10, no. 10: 1091. https://doi.org/10.3390/horticulturae10101091

APA Style

Bernal-Gallardo, J. O., Mena-Violante, H. G., & Luna-Suárez, S. (2024). Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl. Horticulturae, 10(10), 1091. https://doi.org/10.3390/horticulturae10101091

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