Phenolic profile and biological properties of the leaves of Ficus vasta Forssk. (Moraceae) growing in Egypt

F. vasta hydroalcoholic extract was subjected to paper chromatography (Whatman No.1) using three different solvent systems as n-butanol:acetic acid:water (BAW 4:1:5, upper layer), 15% acetic acid, and water. By comparison with standard compounds some flavonoids were identified. Then, each band was cut, and the compounds were dissolved in a mixture of MeOH/H₂O, purified over Sephadex LH-20 and identified by UV, ¹H-NMR and MS analyses [15, 16].

HPLC-PDA/ESI-MS analyses were performed on a Prominence LC system (Shimadzu, Milan, Italy) equipped with photo diode array (PDA) and mass spectrometry (MS) (LCMS-2020, Shimadzu) detection. Data acquisition was performed by Shimadzu LabSolution software ver. 5.53.

For chromatographic separations, an Ascentis Express C18 column (15 cm × 4.6 mm I.D.) packed with 2.7 μm partially porous particles, was employed (Supelco, Bellefonte, PA, USA). The injection volume was 5 μL, and the mobile phase consisted of water/acetic acid (0.1%) at pH = 3 (solvent A) and ACN/acetic acid (0.1%) (solvent B), respectively in the following linear gradient mode: 0 min, 0% B; 5 min, 5% B; 15 min, 10% B; 30 min, 20% B; 60 min, 50% B; 70 min, 100% B; 71 min, 0% B. The mobile phase flow rate was 1.0 mL/min, and it was splitted to 0.3 mL/min prior to MS detection. PDA wavelength range was 210–400 nm and the chromatograms were extracted at 280 and 350 nm.

The extract (10 mg) was dissolved in DMSO (1 mL) and filtered through a 0.45 μm membrane filters (Whatman, Clifton, USA).

Phenolics identification was carried out by the complementary information provided by chromatographic retention times, PDA and mass spectra, and further supported by comparison to existing literature data [13].

The quantitative determination of each compound was carried out by means of the external standard method using gallic acid (λ = 270), catechin (λ = 278), naringenin (λ = 283), chlorogenic acid (λ = 325), apigenin (λ = 330), rutin (λ = 355), kaempferol (λ = 365) and quercetin (λ = 370) as reference compounds in a concentration range of 1–100 ppm. With three different concentration levels. Triplicate injections were made for each level, and a linear regression was generated. The calibration curves with the external standards were obtained using concentration (mg/L) with respect to the area obtained from the integration of the PDA peaks at a wavelength of 270 nm for benzoic acid-like, 278 nm for flavan-3-ol-like, 283 nm for flavanone-like, 325 nm for cinnamic acid-like, 330 nm for flavone-glycoside-like, 354 nm for flavonol-glycoside-like and flavanone-glycoside-like, 365 nm for flavone-like and 370 nm for flavonol-like compounds. The results were obtained from the average of three determinations and are expressed as mg/g dried extract ± percent relative standard deviation (%RSD).

The free radical scavenging activity of F. vasta extract was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) test, according to the protocol previously reported [17]. An aliquot (0.5 mL) of 80% MeOH solution containing different amounts of the extract (0.0125–0.2 mg/mL) was added to 3 mL of daily prepared methanol DPPH solution (0.1 mM). The optical density change at 517 nm was measured, 20 min after the initial mixing, with a model UV-1601 spectrophotometer (Shimadzu). Butylated Hydroxytoluene (BHT) was used as reference. The scavenging activity was measured as the decrease in absorbance of the samples versus DPPH standard solution. The results were obtained from the average of three independent experiments, and are reported as mean radical scavenging activity percentage (%) ± SD. The results are also expressed as mean 50% Inhibitory Concentration (IC₅₀) ± standard deviation (SD), determined graphically by interpolation of the dose-response curve; lower IC₅₀ value indicates higher antioxidant activity.

The reducing power of F. vasta extract was evaluated by spectrophotometric detection of Fe³⁺-Fe²⁺ transformation method, as previously reported [18]. Different amounts of the extract (0.0125–0.2 mg/mL) in 1 mL solvent were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferrycyanide [K₃Fe(CN)₆]. The mixture was incubated at 50 °C for 20 min. The resulting solution was cooled rapidly, mixed with 2.5 mL of 10% trichloroacetic acid, and centrifuged at 3000 rpm for 10 min. The resulting supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% fresh ferric chloride (FeCl₃), and the absorbance was measured at 700_nm after 10 min; the increased absorbance of the reaction mixture indicates an increase in reducing power. As blank, an equal volume (1 mL) of water was mixed with a solution prepared as described above. Ascorbic acid and BHT were used as reference standards. The results were obtained from the average of three independent experiments, and are expressed as mean absorbance values ± SD. The reducing power was also expressed as ascorbic acid equivalent (ASE/mL); when the reducing power is 1 ASE/mL, the reducing power of 1 mL extract is equivalent to 1 μmol ascorbic acid.

The Fe²⁺ chelating activity of F. vasta extract was estimated by measuring the formation of the Fe²⁺-ferrozine complex, according to the method previously reported [18]. Briefly, different concentrations of the extract (0.0125–0.2 mg/mL) in 1 mL solvent were mixed with 0.5 mL of methanol and 0.05 mL of 2 mM FeCl₂. The reaction was initiated by the addition of 0.1 mL of 5 mM ferrozine. Then the mixture was shaken vigorously and left standing at room temperature for 10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm. The control contains FeCl₂ and ferrozine, complex formation molecules. Ethylenediaminetetraacetic acid (EDTA) was used as reference standard The results were obtained from the average of three independent experiments and are reported as mean inhibition of the Fe²⁺-ferrozine complex formation (%) ± SD and IC₅₀ ± SD.

The ability of F. vasta extract to protect bacterial growth and survival from the oxidative stress induced by hydrogen peroxide (H₂O₂) was evaluated according the protocol described by Smirnova et al. [19], with some modifications. Escherichia coli ATCC 25922 was obtained from the Department of Scienze Chimiche Biologiche Farmaceutiche ed Ambientali, University of Messina, in-house culture collection (Messina, Italy). Bacteria were grown overnight in LB medium. The overnight suspension was centrifuged (10 min at 3500 rpm), resuspended in LB fresh medium to obtain a final optical density at 600 nm (OD₆₀₀) = 0.1, and then grown aerobically at 37 °C with shaking at 150 rpm. In mid-log phase (OD₆₀₀ = 0.6) bacteria were centrifuged and the OD₆₀₀ adjusted to 0.2 value with fresh medium. The bacteria suspension was then aliquoted and F. vasta extract (1 mg/mL) and reference standard quercetin (0.2 mM) were added. Two control groups (Ctr), with and without H₂O₂ treatment, were included. After 30–40 min, when OD₆₀₀ reached a value equal to 0.4, in order to establish the ability of F. vasta extract to exert protection against E. coli growth inhibition induced from oxidative stress bacteria were treated with H₂O₂ (2 mM), and the growth was monitored every 20 min for 3 h.

For survival studies, the bacteria (OD₆₀₀ = 0.4) were exposed for 30 min to a higher concentration of H₂O₂ (10 mM), which caused bactericidal effect. Then an aliquot of each sample was diluted in 0.9% NaCl to obtain serial dilutions (1:10). Each sample was poured onto LB-agar plates and incubated at 37 °C; after 24 h the number of viable colonies was counted to estimate the cell survival. The percentage (%) of survival was calculated according to the formula: (colony forming units (CFU) of H₂O₂ treated culture/CFU of untreated Ctr) × 100 [20].

The results were obtained from the average of three independent experiments and are expressed as mean absorbance ± SD and surviving (%) ± SD for protective effect on E. coli growth and survival, respectively. Statistical comparisons of the data were performed by Student’s t-test for unpaired data. P-values lower than 0.05 were considered statistically significant.

The potential toxicity of F. vasta extract was investigated using brine shrimp (Artemia salina Leach) lethality bioassay, according to the method previously reported [21]. The extract was tested at different concentrations (10–1000 μg/mL). Ten brine shrimp larvae, taken 48 h after initiation of hatching in artificial seawater, were transferred to each sample vial, and artificial seawater was added to obtain a final volume of 5 mL. After 24 h of incubation at 25–28 °C, the vials were observed using a magnifying glass, and surviving larvae were counted. The assay was carried out in triplicate, and median lethal concentration (LC₅₀) values were determined using the probit analysis method. Extracts with LC₅₀ higher than 1000 μg/mL are considered non-toxic.

The following strains were used as indicators for the antimicrobial testing and were obtained from the Department of Scienze Chimiche Biologiche Farmaceutiche ed Ambientali, University of Messina (Italy), in-house culture collection: Bacillus subtilis ATCC 6633, Escherichia coli ATCC 10536, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 13932, Pseudomonas aeruginosa ATCC 15442, Salmonella typhimurium ATCC 13311, Salmonella enterica (Wild type), Staphylococcus aureus ATCC 29213, and Staphylococcus epidermidis ATCC 12228 were grown at 37 °C in MHB; the yeast Candida albicans ATCC 10231 was grown at 35 °C on SDA.

The minimum inhibitory concentration (MIC) and minimum bactericidal and fungicidal concentration (MBC and MFC) values of F. vasta extract were determined using the in broth microdilution method according to the protocols recommended by the Clinical and Laboratory Standards Institute [22, 23].

Cultures of bacterial strains and C. albicans were prepared overnight in MHB and RPMI 1640, respectively; microorganism suspensions were therefore adjusted with sterile medium to give 1 × 10⁶ for bacteria and 1 × 10⁴ CFU/mL for C. albicans. The extract was dissolved in dimethyl sulfoxide (DMSO) (1%) and MHB to obtain a final concentration of 1 mg/mL. Two-fold serial dilutions were prepared in a 96-well plate. The tested concentrations ranged from 500 to 0.49 μg/mL. The MIC was defined as the lowest concentration (μg/mL) of extract which completely inhibit the visible growth of microorganisms in broth after 24 h of incubation for bacteria and 48 h for C. albicans. All experiments were performed in triplicate on three independent days. Positive and negative controls were also included.

Chromatographic separation of F. vasta extract allowed the identification of some flavonoid compounds, namely luteolin, quercetin, vitexin, quercetin-3-O-β-galactoside and rutin. Their structures were elucidated on the basis of, UV, ¹H-NMR, and MS analyses. The spectral information is summarized in Table 1.

The quali-quantitative characterization of the phenolic compounds present in the F. vasta leaves extract was accomplished by HPLC-PDA/ESI-MS. Baseline compound separation was achieved on the employed fused-core C18 stationary phase; as far as detection is concerned on-line coupling to PDA and MS detection provided complementary information for reliable identification purposes. The analysis revealed the presence of 12 compounds, 2 out of them belonging to the group of phenolic acids (77.09 mg/g extract) and 10 to flavonoids (135.98 mg/g extract). The flavonol quercetin-3-galactoside was found to be the main phenolic compound detected in the extract (81.5 mg/g ± 0.88% RSD), followed by gallic acid (76.36 mg/g ± 2.70% RSD) and isoquercitrin (22.5 mg/g ± 2.02% RSD) (Fig. 1, Table 2).

The results of DPPH assay are shown in Fig. 2a. F. vasta extract displayed strong radical scavenging effect, dose-dependent, which reached about 90% inhibition at the concentration of 0.15 mg/mL. The activity of the extract was higher than that of the standard BHT, as indicated also by the IC₅₀ values (0.0672 ± 0.0038 mg/mL and 0.0821 ± 0.0009 mg/mL, respectively).

F. vasta extract exhibited reducing power, that increased in a dose-dependent manner; the activity resulted lower than that of BHT, as confirmed by ASE/mL values (3.65 ± 0.48 ASE/mL and 1.97 ± 0.08 ASE/mL) (Fig. 2b).

In the Fe²⁺ chelating activity assay F. vasta extract showed mild, dose-dependent, effect (data not shown). As confirmed by the IC₅₀ values, the chelating ability of the extract resulted much lower than that of the standard EDTA (0.801 ± 0.007 mg/mL and 0.0067 ± 3.98 E-05 mg/mL, respectively).

In a preliminary experiment we established that F. vasta extract does not inhibit the growth of E. coli at the dose of 1 mg/mL under the experimental conditions utilized in this protocol, thus we tested the extract at the concentration of 1 mg/mL to evaluate its protective ability against the bacteriostatic and bactericidal effects of H₂O₂. As shown in Fig. 3, F. vasta extract displayed noticeable protective effect on E. coli growth under oxidative stress. Addition of 2 mM H₂O₂ resulted in a 60-min growth arrest of E. coli into the Ctr group. In the culture pretreated with quercetin (0.2 mM), addition of H₂O₂ did not inhibit bacterial growth. The pretreatment with F. vasta extract (1 mg/mL) provoked a strong protection against H₂O₂-induced damage, statistically significant at all time points compared to Ctr group treated with H₂O₂ (P < 0.001 and P < 0.0001). Further, the cell growth of the culture treated with F. vasta extract notably exceeded that of quercetin group at 20, 40 and 60 min.

The results of protective effect on E. coli survival are shown in Fig. 4. After 30 min, an elevated loss of viability in the Ctr culture treated with 10 mM H₂O₂ (approximately 68% survival) compared to untreated Ctr was observed. In the culture pretreated with F. vasta extract, high survival (approximately 110%) was maintained in the presence of 10 mM H₂O₂, statistically significant compared to Ctr culture treated with H₂O₂ (P < 0.0001); even in this case the observed effect was higher than that of quercetin.

F. vasta extract did not display any toxicity against brine shrimp larvae (LC₅₀ > 1000 μg/mL).