Antioxidant and Antifungal Activities and Characterization of Phenolic Compounds Using HPLC- MS in Empetrum rubrum Vahl ex Willd.

1. Introduction

Plants, due to their biodiversity and the broad presence of secondary metabolites [1] in plant tissues, provide a source of antioxidants [2]. A nonenzymatic antioxidant [3] is defined as a molecule capable of stabilizing another that has a missing electron, by giving one [4]. This way prevents reactions with other molecules [5] and prevents oxidative stress [6]. Phenols and polyphenols are intrinsically antioxidants [7] and this group of secondary plant metabolites is present in all parts of plants [8] and possesses nonenzymatic antioxidant properties [9]. Furthermore, plant extracts with phenolic compounds exert different biological functions [10], such as antifungal [11,12], antibacterial [13,14], antiviral [15], and anti-inflammatory activities [16]. Another aspect to be considered is the benefit for human health, because phenolic compounds prevent coronary heart diseases [17], have cytotoxic effects [18], have been used in traditional medicine for the treatment of diabetes [19], possess inhibitory activities of enzymes related to Alzheimer`s disease [20] and anti-obesity activities [21]. Exposure of plants to abiotic stress affects plant secondary metabolism and influences the polyphenolic composition and therefore antioxidant properties [22]. Abiotic stresses are mainly extreme temperature, drought, salinity, and flooding [23]. Species of the genus Empetrum are keystone species, for maintaining mammals and birds, and dominate many tundra and heathland ecosystems excluding other plants through allelopathic toxins [24]. A wide range of biologically active compounds with pharmacological effects are present in the genus Empetrum, and Empetrum species from the northern hemisphere are used in traditional medicine [25]. Following a common practice and in order to avoid confusion, all Southern Hemisphere natural populations are assignable as E. rubrum Vahl ex Willd and all northern hemisphere natural populations as Empetrum nigrum L. [24]. Other species of the genus Empetrum present in the Northern Hemisphere are Empetrum atropurpureum and Empetrum eamesii [26]. There are no scientific reports about biological activities and chemical compounds such as polyphenols in aerial parts of this species E. rubrum Vahl ex Willd. This vascular plant lives in high-Andean zones [27] in the presence of living conditions that involve strong winds, high exposition to the sun during the summer months, and low temperatures down to -20 ºC. E. rubrum is a straight subshrub, that develops on sand of volcanic origin and survives after being covered by snow during the winter months [28]. The environmental conditions of E. rubrum play a role in the accumulation of polyphenolic compounds that have antioxidant properties. Empetrum nigrum L. var. andinum DC. and Empetrum maclovianum Gand. are synonymous with E. rubrum, it is native to Chile and is distributed across the Valparaiso Region to the Magallanes Region in Chile, and in adjacent areas of Argentina [29]. The distribution of Empetrum nigrum L. is predominantly circum-arctic-boreal [30] and there are scientific studies about antioxidant, anti-inflammatory, and α-glucosidase inhibitory effects of aerial parts extract [31], about the antioxidant effect of fruit extracts [32], about antifungal and antibacterial effects [33] and about cytotoxic activity against human cancer cells of a compound isolated from the leaves [34], of this species. The extraction, isolation, and structural elucidation of chemical compounds contained in Empetrum nigrum, have underpinned the uses in traditional and folk medicine for the treatment of tuberculosis [35], diarrhea [36], coronary heart disease, and alcoholic liver disease [37]. Considering that the antioxidant and antifungal activities of the southern hemisphere species of the genus Empetrum have not been investigated, the present study considers the evaluation of the above-mentioned biological activities in the methanolic extract of E. rubrum. With the aim of contributing to a preliminary knowledge of secondary metabolites contained in fruits, stems, and leaves of E. rubrum, we also carried out high-performance liquid chromatography (HPLC) and Mass Spectroscopic (MS) characterization of this species.

2. Materials and Methods

2.1. Collection of Wild Plant Material

The leaves, stems, and fruits of E. rubrum were collected during the summer from the locality of Callaqui, in the Region of Bio Bio, in Chile, particularly at 1550 meters above sea level on the west side of the Volcano Callaqui (37º54’57.2’’S 71º28’44.8’’O), on the Andes mountain range. The plant material was transported to the Laboratorio de Extractos Vegetales of the Universidad de Concepción, to be washed with water and dried at room temperature (25 °C) and stored for its later use. The plant material was authenticated at the Departamento de Botánica of the Universidad de Concepción.

2.2. Preparation of the Extracts

To obtain the MEE for the assays mentioned below in points 2.3 to 2.5 and in point 2.7, the dried and ground leaves and stems were treated in a Soxhlet apparatus (Glassco, 304918), carrying out 6 cycles at 65 ± 5 °C, and an overnight maceration of 14 hours between the cycles. Methanol was used as an extraction solvent for the obtention of the extract. The extract was then concentrated in a rotary evaporator (Heidolph, Laborota 4001 efficient) under reduced pressure, at 65 ± 5 °C. Subsequently, the extract was dried in a glass vessel at 35 °C in an oven, and stored at 4 °C until use. The percentage yield (Y(%)), expressed on the dry weight basis of plant material was calculated from the following equation (1):

Y(%) = W₁ x 100 / W₂

(1)

Where W₁ is the weight of the extract obtained after solvent removal and W₂ is the weight of plant material before the extraction procedure.

Three kinds of extracts from E. rubrum were prepared for the assay mentioned in point 2.7, about HPLC and MS analyses. The first (F1) was obtained by employing distilled water as an extraction solvent, from the leaves and stems. The extraction process was performed in a Soxhlet apparatus (Glassco, 304918) at 65 ± 5 °C, for two days, until the extract surrounding the leaves and stems was colorless. The drying process for calculating Y (%), and calculated by the equation (1), was done in the same way as the method for the obtention of MEE, as described above. The second extract (F2) was obtained with methanol, from the leaves and stems, which corresponds to the same method for obtaining MEE. For the obtention of the third kind of extract (F3) from the fruits, the lyophilized fruits were dissolved in methanol, and then the mixture was treated with ultrasound (Merck) for 30 seconds, followed by agitation for 16 hours. For separating the solvent from the remained fruits, the mixture was centrifuged (Merck Centrifuge) and was repeated until the supernatant methanol was colorless, adding pure methanol to the remained fruits after each centrifugation, carrying out three centrifugation cycles. The supernatant methanol was treated in a rotary evaporator (Heidolph, Laborota 4001 efficient) and finally was dried in a glass vessel at 35 °C in an oven, and stored at 4 °C until use.

2.3. Determination of Antioxidant Activity

2.3.1. Scavenging Activity against DPPH Radical

The method with modifications, proposed by Gaviria et al. [38] was used for the measurement of DPPH inhibition in the presence of MEE. The DPPH solution was prepared by dissolving 1.8 mg of DPPH (Sigma-Aldrich) in methanol, in a 50 mL volumetric flask, for obtaining the DPPH concentration of 36 µg mL^-1 and the DPPH solution was kept at 4 °C protected from light. Before the measurements, the DPPH solution was adjusted with methanol to obtain an absorbance of 0.90 ± 0.10 at a wavelength of 517nm. The homogenized mixtures composed of 2.0 mL of DPPH solution and 0.175 mL of different concentrations of MEE, were incubated for 1 minute (min), prior to the registration of absorbance in a spectrophotometer (Merck, Spectroquant Pharo 300). The MEE solutions employed in this assay were prepared in methanol, in a range of concentrations between 0.04 and 0.80 mg mL^-1. The assays were performed in triplicate, using 2.0 mL of DPPH solution and 0.175 mL of methanol as negative control. The inhibition percentage of absorbance (IA (%)) was calculated according to the equation (2) as indicated below:

IA(%) = (A₀ – A₁ / A₀ ) x 100

(2)

Where A₀ is the absorbance of the negative control and A₁ is the absorbance of the samples. The IC₅₀ value was calculated from the equation of the curve from the graph of MEE concentrations versus inhibition (%), and using the IC₅₀ value a kinetic assay was carried out. The kinetic assay consisted of absorbance measurements at 517 nm every 5 min for 3 hours, and it was carried out in triplicate. The antioxidant effect of MEE was also expressed as the gallic acid equivalents (GAE) in the unit of mg of gallic acid per 1000 mg (1 g) of MEE. The results of GAE were obtained from a calibration curve performed in duplicate, and it consisted of DPPH absorbance versus gallic acid concentrations, of 0.01, 0.02, 0.03, 0.04, and 0.05 mg mL^-1, and the regression equation of the calibration curve to express the results was y = 0.6883 – 0.0262 x + 0.0003 x² with R² = 0.9875. The GAE was calculated, interpolating the absorbance in the calibration curve, of a mixture composed of 2.0 mL of DPPH solution and 0.175 mL of MEE, incubated for one minute before the absorbance measurement at 517 nm. The concentration in 0.175 mL of MEE was 0.5 mg/mL, and this absorbance measurement was performed in triplicate.

2.3.2. Scavenging Activity against ABTS Radical

The method developed by Kuskoski et al. [39] was carried out with some modifications. The ABTS radical cation was obtained through the reaction of ABTS (7 mM) (Sigma-Aldrich) with potassium peroxodisulfate (2.45 mM) (Merck), in a 50 mL volumetric flask and methanol as the solvent The reaction was performed at room temperature, during 16 hours in darkness. Once the ABTS radical cation is formed, it acquired a dark blue color, and the solution was diluted with methanol to obtain an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm (Merck, Spectroquant Pharo 300). After adjusting the ABTS radical cation solution, a 2.0 mL aliquot of this solution was mixed with 0.175 mL of different concentrations of MEE. The mixture was incubated for 1 min for the determination, performed in triplicate, of the absorbance at a wavelength of 734 nm. The negative control was a mixture of 2.0 mL of ABTS radical cation solution with 0.175 mL of methanol. The inhibition percentage of the absorbance with concentrations of MEE between 0.01 and 0.20 mg mL^-1, was calculated using the equation (2), and with the value of IC_50, obtained from the graph of MEE concentrations versus inhibition (%), a kinetic assay was performed in triplicate. The kinetic assay was developed with the values obtained for absorbance at 734 nm every 5 min for 3 hours. The antioxidant effect was also expressed as Trolox equivalents (TEAC), by interpolating the absorbance of MEE in a calibration curve, according to the method described by dos Santos et al. [40] with some modifications, and the result was expressed as mg of Trolox per 1000 mg (1 g) of MEE. The absorbance of MEE was determined in triplicate at 734 nm, with a mixture composed of 2.0 mL of ABTS radical cation solution and 0,175 mL of MEE, after one minute of incubation. The concentration in 0.175 mL of MEE was 0.125 mg/mL. The calibration curve was performed in duplicate, with ABTS absorbance versus Trolox concentrations of 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, and 0.055 mg mL^-1 and the regression equation of the calibration curve to express the results was y = 0.5799 – 0.0143 x + 7.2967 x 10^-5 x² with R² = 0.9966.

2.4. Assay for Determination of TPC

The determination of TPC in MEE, was carried out using the Folin-Ciocalteu method [41], with modifications, and gallic acid was used as a reference substance, for expressing the TPC as gallic acid equivalents in the unit of mg of gallic per 1000 mg (1 g) of MEE. In a 25 mL graduated flask, 180 µL of MEE at a concentration of 1.0 mg mL^-1, 12.5 mL of distilled water, 1.25 mL of Folin-Ciocalteu reagent, 5 mL of sodium carbonate solution at 20 %, were added, and the volume of 25 mL was completed with distilled water. According to the dilution used, the concentration of MEE in the 25 mL graduated flask was 0.0072 mg mL^-1. The absorbance of the homogenized mixture described above was measured at a wavelength of 765 nm (Merck, Spectroquant Pharo 300), after thirty min of incubation at room temperature in darkness, and the assay was performed in triplicate. The phenol used as a reference for the calibration curve was gallic acid, at concentrations of 1, 2, 3, 5, and 6 µg mL^-1 in two series of five 25 mL graduated flasks because the calibration curve was performed in duplicate. The blank solutions were prepared with all the components, except gallic acid and MEE, that were not added to the 25 mL graduated flasks. The regression equation of the calibration curve to calculate the TPC was y = 0.041 + 0.1061 x + 0.0033 x² with R² = 0.9969.

2.5. Qualitative Phytochemical Screening of Secondary Metabolites

With the aim to detect secondary metabolites, leaves and stems of E. rubrum , and also MEE, were subjected to a preliminary phytochemical screening, by utilizing standard procedures for detecting flavonoids [42], tannins [42], coumarins [43] , saponins [42], alkaloids [43]. The results of the qualitative assays were expressed as a marked presence of the metabolites (+++), a normal presence (++), a weak presence of the metabolites (+), and the absence of secondary metabolites was also informed (–).

2.6. HPLC and MS conditions for the analyses of phenolic compounds

A comparative study of the presence of phenolic compounds in aqueous and methanolic extracts of leaves and stems (F1 and F2 respectively) and fruits (F3).

Chromatographic analyses of phenolic compounds were conducted with a Shimadzu HPLC NEXERA system (Kyoto, Japan), equipped with a quaternary LC-30AD pump, a DGU-20A5R degasser unit, CTO-20AC column oven, a SIL-30AC autosampler, a CBM-20A controller system, and a UV–Vis diode array (DAD). An SPD-M20A detector coupled in tandem with a QTrap LC/MS/MS 3200 Applied Biosystems MDS Sciex detector (Foster City, CA, USA). Instrument control and data collection were done by using a CLASS-VP DAD Shimadzu Chromatography Data System and Analyst Software (version 1.5.2).

The anthocyanin analysis in F3 extract was carried out using a C18 column (Kromasil C18 250×4.6 mm, 5 μm) with a C-18 pre-column (Nova-Pak Waters, 22×3.9 mm, 4 μm) at 40 °C, using a mobile phase gradient constituted by water/acetonitrile/formic acid (87:3:10% v/v/v) (solvent A) and water/acetonitrile/formic acid (40:50:10% v/v/v) (solvent B). The flow rate was 0.8 mL/min, and the gradient program was from 6 to 30% of solvent B in 15 min, from 3 to 50% in 15 min, from 50 to 60% in 5 min, and from 60 to 6% in 6 min, followed by 9 min of stabilization at 94% [44].

The chromatographic analyses of flavonols and hydroxycinnamic acid derivatives (HCAD) to F1, F2, and F3 extracts were performed according to a method previously described by Ruiz et al. 2013b with some modifications. HPLC analyses were carried out on a Kinetex C18 column (core-shell 150 × 4.6 mm, 2.6 μm) with a pre-column (Phenomenex, Torrance, CA, USA) and a binary mobile phase of 0.1 % formic acid in water and acetonitrile at a flow rate of 0.5 mL/min with an injection volume of 10 μL. For flavonols, the mobile-phase gradient ranged from 15 to 25 % acetonitrile for 14 min, from 25 to 35 % for 11 min, from 35 to 100 % for 1 min, from 100 to 15 % for 1 min, and finally a stabilization period of 10 min. The column temperature was set at 40 °C. An additional clean-up step was performed on F3 fruit extract using solid-phase extraction previously described by Ruiz et al. [44], which was used to remove anthocyanins to improve flavonols and HCAD identification. Five mL of the fruit extract was diluted with 5 mL of hydrochloric acid 0.1 N. This solution was loaded on 500 mg Oasis MCX (Waters, USA) cartridges previously conditioned with 5.0 mL of methanol and 5.0 mL of water, followed by a rinsing step using 5.0 mL of hydrochloric acid 0.1 N and 5.0 mL of water. The fraction of interest that contains flavonols and hydroxycinnamic acid derivatives was eluted with three 5 mL volumes of methanol. Finally, the pooled solvents were evaporated and resuspended in 5 mL of the mobile phase.

Identity assignment was carried out considering the retention times and by analysis of DAD and ESI-MS/MS spectra. In positive ionization mode for anthocyanins and negative ionization mode for flavonols and HCAD, under the following parameters: 5 V collision energy, 4000 V ionization voltage, capillary temperature at 450 °C, nebulizer gas 40 psi and auxiliary gas 50 psi.

2.7. Evaluation of Antifungal Activity of MEE

The antifungal activity of MEE was assayed against the pathogenic fungus Rhizoctonia solani Kühn (LBH-Rs-12), obtained from the Laboratorio de Biotecnología de Hongos of the Universidad de Concepción, Campus Los Angeles, and the fungal strain was conserved in potato dextrose agar at 4 degrees Celsius.

2.7.1. Evaluation of the Inhibitory Activity in Relation to the Fungal Growth

The antifungal activity of MEE, based on the growth inhibition of the mycelium was carried out according to Elgorban et al. [45] with some modifications. In sterile Petri dishes 19 mL of potato dextrose agar (PDA) and 1 mL of MEE concentrations of 100, 200, 300, and 400 mg mL^-1, were mixed to obtain the final MEE concentrations of 5, 10, 15, and 20 mg mL^-1. Plates with 19 mL of PDA and 1 mL of sterile water were used as the negative control. Mycelial disks of 0.91 cm² obtained from 8-day-old cultures of R. solani using a sterile cork borer, were inoculated at the center of the plates and the incubation temperature was 24 degrees Celsius. The growing area of R. solani was measured every 12 hours for 3.5 days, using the ImageJ2x software program, and all assays were performed in triplicate. The percentage of inhibitory activity in relation to the area of mycelial growth (IMG (%)) was calculated according to the following equation (3):

IMG (%) = (AC – AT / AC ) x 100

(3)

Where AC is the area of fungal colony without MEE (negative control) and AT is the area of fungal colony treated with different MEE concentrations.

2.7.2. Evaluation of the Inhibitory Activity in Relation to the Mycelial Weight

Additionally, in order to verify the inhibitory activity of MEE in relation to the fungal growth, the mycelial biomass was determined, by measuring the dry weight. After the 3.5 days of incubation as described above, the mycelial dry weight was determined according to Pereira et al. [46] with some modifications. After 4 days of incubation, the plates used as the negative control and the plates with different MEE concentrations were autoclaved at 121 degrees Celsius and 1 atmosphere for 15 min. After autoclaving, plates containing mycelium were filtered through filter paper, and the filter paper containing the mycelium was dried at 60 degrees Celsius to a constant weight. Finally, the filter paper containing the dry biomass was weighed, and the dry weight of mycelium was determined by means of the difference in weight. The assays conducted for determining the percentage inhibition of the dry mycelial weight (IMW (%)) were performed in triplicate, and the following equation was used (4):

IMW (%) = (MN – MM / MN) x 100

(4)

Where MN is the dry mycelial weight without MEE (negative control) and MM is the dry mycelial weight treated with different MEE concentrations.

2.8. Statistical Analysis

The results from the absorbance measurements for the DPPH, ABTS, and TPC assays, as well as for the antifungal effect were obtained using an analysis of variance (ANOVA) and multiple comparisons using the test of Tukey (p<0.05) with the statistical software InfoStat.

3. Results and Discussion

3.1. Extraction Yield

The total mass of dry leaves and stems was 97.989 g and the total mass of MEE was 27.739 g. The yield of extraction with methanol was 28.308 %, making three extractions in all, in the Soxhlet apparatus. In each extraction cycle, the extraction was carried out until the solvent remained transparent and colorless. The total mass for F1 was 18.254 g, extracted with water from 92.334 g of leaves and stems, obtaining a 19.769 % yield of extraction, after five extractions. According to Dias et al. [47] the Soxhlet apparatus was chosen to carry out a conventional technique to determine the total phenolic content and antioxidant capacity, making this technique an effective method for obtaining bioactive compounds.

3.2. Antioxidant Activity of MEE

The results of scavenging activity against DPPH and ABTS radicals reflect the antioxidant activity in all concentrations of MEE, observing a decrease of IA (%), which is directly proportional to MEE concentration. The assay performed with DPPH resulted in a decrease of 88,83 ± 0.62% and 4.98 ± 0.82% with a MEE concentration of 0.80 mg mL^-1 and 0.04 mg mL^-1, respectively. The decreases observed in the ABTS assay consisted of 92.63 ± 0.83% and 4.52 ± 0.29% with a MEE concentration of 0.20 mg mL^-1 and 0.01 mg mL^-1, respectively. The antioxidant activity also reflected in the IC₅₀ values of DPPH and ABTS, and in the GAE and TEAC values, is shown in Table 1. In the DPPH assay, gallic acid is commonly used to compare the antioxidant activity of a given substance [48]. On the other hand, in the ABTS assay as an antioxidant measuring method, Trolox is commonly used as a reference in measurements of antioxidant activity [49].

The antioxidant effect of aerial parts extracted from Korean crowberry (Empetrum nigrum var. japonicum) has been studied and a lower value of IC50 measured by the DPPH method was reported [50]. However, the method employed to obtain this lower value of 77.99 µg mL-1, was different, using a longer reaction time of 30 min between DPPH and the aqueous fraction. The aqueous fraction was obtained from a methanolic extract, and all stages of the extraction process were carried out at room temperature. The method employed by Gao et al. [51] , using ultrasound-assisted enzymatic extraction, yielded IC50 values of 212.919 µg mL-1 and 182.242 µg mL-1, measured by the DPPH method and the ABTS method, respectively. The authors noted above performed the extraction from Empetrum nigrum aerial parts. Gallic acid was used as a reference substance, and as a DPPH scavenger, owing to its effects on health, such as the strong antioxidant and free radical scavenging activities. Oxidation processes are of significant scientific interest, due to their involvement in the progressive genesis of diseases such as cancer, myocardial infarction, Alzheimer’s dementia, diabetes mellitus, and obesity [52]. On the other hand, Trolox has been used for ABTS assay, as a reference substance for measuring the antioxidant capacities of plant extracts [53]. TEAC of MEE could be compared with a lower TEAC value of 25.42 ± 1.98 for methanolic extract obtained from Nassauvia dentata, and it was collected at the same time as E. rubrum, at 1550 meters above sea level on the west side of the Volcano Callaqui [54]. When assessing the antioxidant effect of MEE at IC₅₀ concentration (0.414 mg mL^-1) during a period of 3 hours, it was observed a diminution of the percentage of absorbance for DPPH. Just 5 min after that the antioxidant effect began, a decrease of 73.13 % in DPPH absorbance was observed, and subsequently a recuperation of the free radical was observed (Figure 1). The recuperation of DPPH was reflected in a gradual decline of the antioxidant effect, reaching 44.62 % after 180 min. For the assay with ABTS using IC₅₀ concentration (0.108 mg mL^-1) of MEE, to determine the antioxidant effect during 3 hours, it was observed a decline of 68.17 %, of absorbance for ABTS, in the first 5 min. The decrease of absorbance for ABTS showed an increase until min 100, reaching a decrease of 91.11 %. Subsequently, the antioxidant effect of MEE remained practically constant, with slight variations of 1-2% (Figure 1).

3.3. Determination of TPC

According to the measurements carried out in triplicate, and based on the regression equation of the calibration curve, 0.007 mg mL^-1 of MEE is equivalent to 1.683 ± 0.024 µg mL^-1 of gallic acid. Consequently, the TPC expressed as gallic acid equivalents are 240.428 ± 3.428 mg of gallic per 1000 mg (1 g) of MEE. This result for MEE, in comparison with the total polyphenols from Empetrum nigrum aerial parts, extracted with ethanol at concentrations of 50 to 70 % and made under different extraction conditions [55], revealed a higher content of phenols in MEE.

3.4. General Qualitative Analysis of Secondary Metabolites

According to the phytochemical screening (Table 2), leaves, stems and MEE contain a marked presence of flavonoids and condensed tannins, and these classes of secondary metabolites are directly related to the observed antioxidant effects of MEE [56].

3.5. HPLC and MS analyses of phenolic compounds

The anthocyanin chromatographic profile in E. rubrum fruit extract F3 shows (Figure 2, Table 3) that the main anthocyanins identified are delphinidin-3-glucoside and malvidin-3-glucoside, followed by cyanidin and petunidin-3-glucosides. In a lesser proportion, some pentoside conjugates can be observed, but their identity was not confirmed with commercial standards, as the glucoside derivatives were. Other authors [57] identified the anthocyanin profile in other Empetrum berries (Black Crowberry) with the main identified anthocyanins were delphinidin, cyanidin, peonidin, and malvidin, but conjugated with galactose instead of glucose.

The phenolic compounds chromatographic analysis of the F1 (leaves and steams aqueous extract), F2 (leaves and steams methanolic extract), and the cleaned F3 fruit extract showed that the main compounds were HCAD and conjugated flavonols (Figure 3). There was no significant presence of flavanol or larger molecular weight procyanidins. The principal flavonols found in the samples were quercetin-3-glucoside and a laricitin hexoside, which can be either galactoside or glucoside in the steam and leaves samples. The other significant signals were tentatively identified as a caffeoylquinic acid isomer, and a coumaric acid and caffeic acid derivate (Table 4), also more prevalent in steam and leaves samples. In the F3 sample, which was previously treated to remove the anthocyanins, maintained the same proportion of flavonols and HCAD, but in much lesser quantities. Laaksonen et al. [58] analyzed flavonol and HCAD in Empetrum nigrum berries and found that the main flavonols identified were similar in profile with galactosides of laricitin and quercetin, and coumaric acid derivates.

3.6. Antifungal Activity of MEE

The observed antifungal activity of MEE against R. solani, consisted of a growth inhibition of mycelium and a diminution of the mass of fungus. The reduction of both parameters mentioned above was directly related to the MEE concentration. In fact, as shown in Figure 4, the maximum concentration of 20 mg mL^-1 inhibited the mycelial growth in a percentage of 97.70 ± 0.72 %, after 84 hours. With respect to the inhibition of the mass of fungus, and as seen in Figure 5 the same concentration, of 20 mg mL^-1, mentioned above leads to a decrease of 98.10 ± 1.34 % after 84 hours.

Similarly, the research of an algal methanolic extract performed by Al-Nazwani et al. [59] demonstrated that there is a correlation between the increase of the extract concentration tested, and the inhibition of growth and the biomass decline of R. solani. The authors mentioned above also concluded that phenol in comparison with other tested compounds, exerted the greatest antifungal effect, reflected in the mycelial growth of R. solani. Other research demonstrated the association of antifungal and antioxidant activities of methanolic plant extracts, with their phenols, tannins, and flavonoids contents [60]. According to the observed inhibition of mycelial growth of R. solani, Sclerotium rolfsii, and another fungus, a higher content of total phenols and flavonoids in a 70% ethanolic extract in comparison with an aqueous extract, is directly related to the growth inhibition of these microorganisms [61]. The above-mentioned research is in agreement with the higher levels of TPC detected in MEE, in comparison with TPC detected in the methanolic extract obtained from Nassauvia dentata [54], because MEE has a higher antifungal effect than the methanolic extract from Nassauvia dentata, against R. solani. Another modality of antifungal activity consists of biocontrol agents against the fungus Botrytis cinerea, using microorganisms inhabiting the leaf surface of E. rubrum [62].

4. Conclusions

The results of our research revealed a considerable content of phenols, extracted from the aerial parts of E. rubrum, using a polar solvent such as methanol, and these phenol compounds exhibited antioxidant activity. Furthermore, and based on our results, the phenol compounds in E. rubrum could have an antifungal effect against R. solani. Therefore, the methanol extract obtained from E. rubrum may be a natural antifungal agent in the future.