Antioxidant and Wound Healing Bioactive Potential of Extracts Obtained from Bark and Needles of Softwood Species

Interest in the extraction of phytochemical bioactive compounds, especially polyphenols from biomass, has recently increased due to their valuable biological potential as natural sources of antioxidants, which could be used in a wide range of applications, from foods and pharmaceuticals to green polymers and bio-based materials. The present research study aimed to provide a comprehensive chemical characterization of the phytochemical composition of forest biomass (bark and needles) of softwood species (Picea abies L., H. Karst., and Abies alba Mill.) and to investigate their in vitro antioxidant and antimicrobial activities to assess their potential in treating and healing infected chronic wounds. The DPPH radical-scavenging method and P–LD were used for a mechanistic explanation of the biomolecular effects of the investigated bioactive compounds. (+)-Catechin, epicatechin, rutin, myricetin, 4 hydroxybenzoic and p-cumaric acids, kaempherol, and apigenin were the main quantified polyphenols in coniferous biomass (in quantities around 100 µg/g). Also, numerous phenolic acids, flavonoids, stilbenes, terpenes, lignans, secoiridoids, and indanes with antioxidant, antimicrobial, anti-inflammatory, antihemolytic, and anti-carcinogenic potential were identified. The Abies alba needle extract was more toxic to microbial strains than the eukaryotic cells that provide its active wound healing principles. In this context, developing industrial upscaling strategies is imperative for the long-term success of biorefineries and incorporating them as part of a circular bio-economy.


Introduction
The promotion of renewable natural resources with potential applications in fields related to health, medicine, and nutrition is of great interest. The biomass of softwood species is rich in chemicals, with potential uses in various areas, from pharmaceutical and food industries to green polymers and bio-based materials [1,2]. The barks of softwood species contain high-level extractables, including a whole range of secondary metabolites such as valuable polyphenols, mostly condensed tannins, and hydrolysable tannins [3,4]. As natural antioxidants, polyphenols have important bioactive properties, which have been extensively described in the literature [5][6][7][8]. Several studies have confirmed that extractive substances from softwood bark show high antioxidant activity, revealing their potential for application in the pharmaceutical industry [9][10][11]. In addition, the use of polyphenolic altitude: 700 m) and Malaia (average altitude: 1130 m) localities of Southern Carpathians, Romania. The trees had diameters that ranged from 31 to 35.5 cm. We collected 13 of each spruce bark and needles and 4 of each fir bark and needles (from trees located at different altitudes). The raw plant materials were dried at 40 • C using an Biovita drier (Biovita, România), ground in a two-knife mill (Fritsch, Idar-Oberstein, Germany), and then ground into a fine powder by using a Retsch 200 mill (Haan, Germany). The bark was stored in sealed bags at +4 • C until they were required for use in the experiments.

Microwave-Assisted Extraction (MAE) of Bioactive Compounds from Bark and Needles
Microwave-assisted extraction was performed with a CEM Mars 6 closed microwave system (CEM Corp., Matthews, NC, USA)), with adjustable power settings ranging from 100 to 1600 W, closed vessel reactions (GreenChem, 100 mL), power and temperature sensors, and steering and cooling systems. To determine the best extraction solvent, different extractions were carried out at 300 W for 5 min with ethanol and methanol (30%, 50%, and 80%) solutions at 1:30 liquid/solid ratio (g/g). Low-power extraction (300 W) was used to better manage the reaction of solvents in a closed system [87]. The optimal extraction conditions that allowed us to obtain a high total polyphenol content (mg GAE/g) were as follows: 50% ethanol extraction solvent, 10 min ramp to 50 • C, followed by 5 min hold. Briefly, 1.0 g of bark/needle powder was weighed into extraction vessels, and then 30 mL of 50% ethanol was added. Teflon stir bars were inserted, and the vessels were closed and subjected to the MAE program. At the end of the extraction process, the extraction vessels were allowed to cool for 30 min at room temperature and then the extracts were centrifuged for 15 min at 4 • C and 10,000 rpm (Hettich Rotina 380 R Centrifuge, Tuttingen, Germany). After filtration through a 0.45 mm membrane, the aliquots of samples were preserved at −4 • C until they were required for analysis. Two replications were carried out for each treatment. The remaining extracts were evaporated to remove the ethanol in a gentle stream of nitrogen using a TurboVap LV (Biotage, Uppsala, Sweden) and then lyophilized using a Labconco freeze dryer (Biotage, Uppsala, Sweden). The obtained final masses were weighted to calculate the Total Extraction Yield, with the obtained values being 5.8% and 14.0% for fir and spruce bark, respectively, and 4.5% and 7.5% for fir and spruce needles, respectively.

Bioactive Polyphenolic Characterization of Bark and Needles Extracts 2.4.1. Bioactive Characteristics by UV-Vis Spectrophotometric Methods
The bioactive characteristics (total polyphenols (TP), total flavonoids (TF) and antioxidant activity (AA)) of the bark and needle extracts were determined by UV-Vis spectrophotometric measurements taken using an Specord 250 Plus UV-Vis spectrophotometer (Analytic Jena, Jena, Germany) equipped with 1 cm pathlength quartz cells. All analytical determinations were conducted in duplicate, and results were averaged.
The Total polyphenols (TP) were determined by using the Folin-Ciocalteau method according to the protocol described by Ciucure et al. [88]. In brief, 100 µL of 5-fold diluted extract was added to test tubes and mixed with 5 mL ultrapure water and 200 µL Folin-Ciocalteau reagent. After 5 min of reaction at room temperature and protected from light, 300 µL of 20% sodium carbonate solution was added to stop the reaction and to generate a characteristic blue color for 2 h. The resulting blue color had a maximum absorbance peak of 675 nm, with the absorbance being proportional to the amount of phenolic compounds. Water was used as blank. A calibration curve was built using gallic acid as a standard (0-1250 mg/L) to quantify the samples. The results were expressed as mg gallic acid equivalent (GAE) per gram of bark (mg GAE/g bark).
The Total Flavonoids (TF) were determined by using the AlCl 3 method as described by Geana et al. [89]. Briefly, 0.5 mL of extract were treated with 0.4 mL of 25 g/L AlCl3 solution, 0.5 mL of 100 g/L CH3COONa solution and 4 mL distilled water. After 15 min, the absorbance of the mixture was measured at 430 nm against water as blank. Total flavonoid content (TF) was quantified as mg quercetin equivalent (QE) per gram of bark using calibration curve obtained in the concentration range of 0-125 mg/L quercetin.
Antioxidant activity (AA) was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging method, as described by Geana et al. [89]. In this assay, after being left to stand for 20 min at room temperature, 6 mL of 0.09 mg/mL DPPH methanolic solution was mixed with 0.5 mL aliquots of 5-fold diluted extract, and the absorbance was measured at 517 nm against methanol as blank. Absorbance measurements were transformed to antioxidant activity using Trolox as standard, (in the concentration range of 50-1000 µmol/L), and the antioxidant activity was expressed as µmol/L Trolox equivalents per gram of bark/needles (dw).

Target and Non-Target Polyphenolic Composition by HRMS Analysis
Chromatographic determinations were carried out using a high-resolution Q Exactive mass spectrometer Focus Hybrid Quadrupole-OrbiTrap (Thermo Fisher Scientific, Bremen, Germany) equipped with HESI and coupled to a high-performance liquid chromatograph UltiMate 3000 UHPLC (ThermoFisher Scientific, Bremen, Germany) with a DAD detector set at 280 nm. Separations were performed on Kinetex ® C18 column (100 × 2.1 mm, 1.7 µm particle diameter) at 30 • C. The mobile phase consisted of: solvent A-water with 0.1% formic acid and solvent B-acetonitrile with 0.1% formic acid. The UHPLC gradient for mass screening was: 0 min, 10% B, 0-10 min, 20% B; 10-20 min, 30% B; 20-30 min, 50% B; 30-33 min, 70% B; 33-43 min, 90% B; 43-45 min, 10%B and equilibration of the column for the next run. The flow rate was 0.3 mL/min, and the injection volume was 10 µL. All samples were filtered with 0.45 µm PTFE membrane filter prior to injection. The mass spectrometer was operated in negative mode in a range between 100 and 1000 m/z at a resolution of 70,000. HESI-source parameters were as follows: spray voltage-2.8 kV, capillary temperature-320 • C, auxiliary gas heater temperature-413 • C, sheath and auxiliary gas flow (N 2 )-35 and 10 (arbitrary units), respectively. Polyphenolic compounds from the samples were identified and quantified according to their spectral characteristics (mass spectra, accurate mass, and characteristic retention time) and compared against external standard solutions analyzed under the same conditions. In order to confirm the identified compounds, fragmentation studies were performed by employing a data-dependent scan with collision-induced dissociation (CID). The normalized collision energy of the CID cell was set at 25, 35, and 45 eV. Xcalibur software (Version 4.1) was used for instrument control, data acquisition, and data analysis. For quantification, in a concentration range between 0 and 2000 µg/L for each of the phenolic acids and flavonoids, calibration was performed via serial dilution with methanol of the standard mixture of concentration 10 mg/L. All stock and working solutions were stored in the dark at 4 • C. Calibration curves based on duplicate injections revealed good linearity, with correlation coefficients (R2) values exceeding 0.99 (peak areas versus concentration). For the non-target UHPLC-MS/MS screening, data processing, analysis, and interpretation were performed via Compound Discoverer v. 2 were obtained from the Microbial Strain Collection of Faculty of Biology, University of Bucharest, Romania, isolated from infected wounds, and confirmed via MALDI-TOF.

Qualitative Antimicrobial Activity
Antimicrobial activity was determined by employing an adapted version of the diffusion method described by Marinas et al. [90]. Briefly, a microbial inoculum with a density corresponding to 0.5 McFarland standard for bacterial strains and 1 McFarland for yeast strain was evenly swabbed on the agar surface, and thereafter 10 µL of the sample was spotted on the solid medium. The results were analyzed in triplicate and are expressed as the mean of diameter zone inhibition (DIZ) ± standard deviation (SD). In the same conditions, ethanol 50% was used as solvent control.

Quantitative Antimicrobial Activity
Quantitative analysis was performed by using a binary serial microdilution method in liquid medium (Brain Hart Infusion for bacteria and Sabouraud for yeast) in a 96-well plate. The concentration range for alcoholic extracts was from 7.81 to 500 µL/mL. Simultaneously, serial dilutions were made with 50% ethanol under the same working conditions in order to obtain a negative control. Each well was inoculated with 10 µL microbial suspension, adjusted to 1.5 × 10 8 CFU/mL from 18 to 24 h grown cultures. The MIC values were established both macroscopically, as the last concentration at which no microbial growth was observed, and spectrophotometrically. The absorbance of the microbial cultures was measured at 620 nm by using the FlexStation 3 UV-Vis (Molecular Devices, San Jose, CA, USA) Spectrophotometer.

Prevention of Biofilm Formation
Following the quantitative analysis of the antimicrobial activity, the adherence of biofilm biomass was assessed after fixation with methanol and crystal violet staining (0.1%) by using the slime method. The absorbance of the biological material resuspended in 33% acetic acid was measured at 490 nm.

MTT Assay
The MTT assay was used to assess cell viability and proliferation in the presence of extracts. This viability test facilitates the quantitative evaluation of live cells in culture. The compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] is permeable to living cell membranes and metabolized into soluble formazan crystals. The cells were incubated for 2 h with MTT at 37 • C, 95% humidity, 5% CO 2 . The formed formazan was dissolved in isopropanol, and the absorbance was measured at 595 nm on a FlexStation 3 plate reader (Molecular Devices, San Jose, CA, USA). The data obtained was analyzed using log (inhibitor) vs. response-Variable slope (four parameters) analysis function with the help of Prism GraphPad 9.0 software. The culture medium was collected after 24 h of incubation with the extracts, and LDH release was measured using the Cytotoxicity Detection Kit PLUS (Roche, NY, USA) according to the manufacturer's instructions. Volumes of 50 µL culture supernatants were mixed with 50 µL reaction mixture of catalyst and dye solution and incubated for 30 min in dark conditions. The reaction was stopped by adding 50 µL of stop solution, and the absorbance was measured at 490 nm using a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).

Hemolytic Index
The hemolytic index of the extracts was measured by using a spectrophotometric procedure as described elsewhere [91]. Nine milliliters of ram blood were collected in acid citrate dextrose (ACD) tubes and centrifuged for 10 min at 2000 rpm. The supernatant was removed, and the pellet was washed thrice with PBS (0.2 M, pH 7.4) before re-suspension in sterile saline solution (0.9%). Furthermore, 50 µL of the extracts (50-500 µL/mL in PBS, pH = 7.4) was dispensed into 200 µL of erythrocyte suspension (10%). The mixtures were incubated 1 h at 37 • C. The samples were centrifuged at 2000 rpm for 10 min, and the absorbance of the supernatant was measured at 540 nm. Relative hemolysis was assessed in comparison with the hemolysis in deionized water (A+), which was set as 100%. For the negative control, phosphate-buffered saline was used (A−). Each set of experiments was performed in triplicate, and inhibitory activity was calculated; expressed herein as percent inhibition of hemolysis. The hemolysis (%) was calculated as follows:

Anti-Hemolytic Activity
The anti-hemolytic potential of the extracts was measured via the use of a previously described spectrophotometric procedure [92] (with some modifications) [93,94]. Nine milliliters of ram blood were collected in acid citrate dextrose (ACD) tubes and centrifuged for 10 min at 2000 rpm. The supernatant was removed, and the pellet was washed thrice with PBS (0.2 M, pH 7.4) before re-suspension in saline solution (0.9%). Furthermore, 50 µL of the extracts (35-200 µL/mL in PBS, pH = 7.4) was dispensed into 200 µL of erythrocyte suspension (10%). The mixtures were incubated for 20 min at 37 • C, and 100 µL of AAPH (200 mM final concentration) was added to the reaction mixture to induce the oxidative degradation of the membrane lipids. The mixtures were incubated for 4 h at 37 • C. Subsequently, the samples were centrifuged at 2000 rpm for 10 min, and the absorbance of supernatant measured at 540 nm. The relative hemolysis was assessed in comparison with the hemolysis in the AAPH-treated negative control (A AAPH), which was set as 100%. For the positive control, phosphate-buffered saline instead of AAPH was used (A PBS). Each set of experiments was performed in triplicate, and inhibitory activity was calculated; expressed herein as percent inhibition of hemolysis. A stock solution of ascorbic acid (1 mg/mL) was used treated in a similar manner to the method employed for the samples in a range of concentrations between 35 and 200 µL/mL. The hemolysis (%) was calculated as follows:

Statistical Analysis
The data are expressed as means ± standard deviation (SD) (determined via triplicate analysis). The statistical analysis was conducted using GraphPad Prism 9v. The data were analyzed using ordinary two-way ANOVA with two-stage step-up for multiple comparisons (Benjamini, Kriefer, and Yekutieli) for biological assays. The level of significance was set to p < 0.05. Principal component analysis (PCA) and Heat Map Analysis (HMA) were carried out on the data matrix, including for the rows corresponding to the analyzed extracts, and variables derived from both the target and non-target HRMS analyses were analyzed by using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA) and XLSTAT Add in soft version 15.5.03.3707 (Addinsoft, New York, NY, USA).

Protein-Ligand Docking (P-LD)
A molecular docking study was carried out for several identified phytochemicals to evaluate the potential wound healing effect of the plant extracts (which is facilitated by modulating the PI3k/Akt signaling pathway and using 1E8W RCSB PDB entry for PI3Kγ due its better resolution; 2.50 Å) [73]. Two comparative P-LD runs were performed against the selected molecular target by using two different software types with two different docking algorithms, namely, (1) AutoDock Vina v.1.2.0 algorithm [95,96] with PyRx-Python Prescription v.0.9.7 software [97] as user interface (UI) control and (2) SwissDock web-service "http://www.swissdock.ch/ (30 May 2023)" [98] with EADock DSS algorithm [98,99]. A detailed description of the molecular docking technique (in silico technique) of each P-LD runs is provided in the Supplementary Materials. The co-crystallized ligand of PI3Kγ/1E8W (quercetin) was re-docked, serving as a reference or control molecule for both P-LD runs.

Bioactive Characteristics
Quantitative estimation of the bioactive characteristics (TP, TF, AA) of spruce and fir biomass (bark and needles) collected from mature trees distributed at different altitudes was performed in order to generate an overall estimation of the bioactive characteristics of each type of biomass. The obtained results are presented in a range of variation, mean, minimum, and maximum values, as shown in Figure 1 and Table S1.
The data are expressed as means ± standard deviation (SD) (determined via triplicate analysis). The statistical analysis was conducted using GraphPad Prism 9v. The data were analyzed using ordinary two-way ANOVA with two-stage step-up for multiple comparisons (Benjamini, Kriefer, and Yekutieli) for biological assays. The level of significance was set to p < 0.05. Principal component analysis (PCA) and Heat Map Analysis (HMA) were carried out on the data matrix, including for the rows corresponding to the analyzed extracts, and variables derived from both the target and non-target HRMS analyses were analyzed by using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA) and XLSTAT Add in soft version 15.5.03.3707 (Addinsoft, New York, NY, USA).

Protein-Ligand Docking (P-LD)
A molecular docking study was carried out for several identified phytochemicals to evaluate the potential wound healing effect of the plant extracts (which is facilitated by modulating the PI3k/Akt signaling pathway and using 1E8W RCSB PDB entry for PI3Kγ due its better resolution; 2.50 Å) [73]. Two comparative P-LD runs were performed against the selected molecular target by using two different software types with two different docking algorithms, namely, (1) AutoDock Vina v.1.2.0 algorithm [95,96] with PyRx-Python Prescription v.0.9.7 software [97] as user interface (UI) control and (2) SwissDock web-service "http://www.swissdock.ch/ (30 May 2023)" [98] with EADock DSS algorithm [98,99]. A detailed description of the molecular docking technique (in silico technique) of each P-LD runs is provided in the Supplementary Materials. The cocrystallized ligand of PI3Kγ/1E8W (quercetin) was re-docked, serving as a reference or control molecule for both P-LD runs.

Bioactive Characteristics
Quantitative estimation of the bioactive characteristics (TP, TF, AA) of spruce and fir biomass (bark and needles) collected from mature trees distributed at different altitudes was performed in order to generate an overall estimation of the bioactive characteristics of each type of biomass. The obtained results are presented in a range of variation, mean, minimum, and maximum values, as shown in Figure 1 and Table S1.  As highlighted in Figure 1, spruce bark and needles seemingly have more bioactive characteristics, with TP values (Figure 1a) [58]), higher values in Picea mariana bark (404.0 mg GAE/g [102], 426.0 mg GAE/g) [103], and lower values of TP in fir bark (14.32 mg GAE/g) [55]. No data were available in the literature for the TP content of spruce and fir needles, but the quanti-fied values were lower compared to those reported for pine needles (274.38 mg GAE/g) (see Table S2).
The amount of TF ( Figure 1b) ranged between 1.46 and 23.24 mg Q/g for bark and between 2.53 and 22.43 mg Q/g for needles, with average values of 9.91 mg Q/g for bark and 8.94 mg Q/g for needles. The amount of TF in fir bark and needles ranged between 1.24 and 2.81 mg Q/g for bark and between 2.76 and 3.67 mg Q/g for needles, with average values of 2.01 mg Q/g for bark and 3.17 mg Q/g for needles. The literature data (Table S2) reported the following values for TF (expressed as quercetin equivalents): 3.54-53.4 mg Q/g for spruce bark [58,[100][101][102], 7.46 mg Q/g for fir bark [100], 8.29-81.9 mg Q/g for pine bark [49,100], and 1.6 mg Q/g for pine needles [104].
Antioxidant activity of the studied coniferous biomass (Figure 1c) ranged between 84.77 and 348.51 µMoli Trolox/g, dw and from 220.26 to 348.76 µmol Trolox/g, dw for spruce bark and needles, respectively (  [103] and 404.12 µmol Trolox/g [100]) and fir (269.55 µmol Trolox/g [100]) barks (Table S1). It should be mentioned that, in the literature data, the bioactive properties were also reported as mg GAE/g extract for TP and mg Q or mg CE/g extract for TF and µg TE/mL extract (see Table S1).

Quantification of Polyphenolic Compounds in Picea abies L., H. Karst., and Abies alba Mill. Barks and Needles
A number of 24 compounds, including 11 phenolic acids and derivatives, 12 flavonoids, and stilbene t-resveratrol, were unambiguously identified and quantified in the coniferous biomass based on a comparison with available reference standards (retention time, highresolution accurate mass, and fragmentation pattern) ( Table S3). The quantitative results of the individual phenolic compounds in coniferous biomass (bark and needles of Picea abies L., H. Karst., and Abies alba Mill.) are presented in Table 1.
Flavonoids represent the most dominant bioactive compounds in spruce and fir biomass (bark and needles), and (+)-catechin is the representative compound of coniferous biomass, with values ranging between 108.7 and 1529.4 µg/g in spruce needles, 48.5-1420.4 µg/g in spruce bark, 94.0-894.5 µg/g in fir needles, and 53.8-186.6 µg/g in fir bark. The obtained data were comparable with data reported in the literature for fir (1499.61 µg/g) and pine (1502.3 µg/g) needles [100]. It should be noted that flavonoids such as (−)-epicatechin, myricetin, quercetin, isorhamnetin, and apigenin were also quantified in important amounts in both spruce and fir bark and needles; generally, higher amounts corresponded to spruce bark and needles rather than fir bark and needles. Higher amounts of kaempferol and rutin were quantified in spruce and fir needles, while lower amounts correspond to spruce and fir bark. Pinocembrin, chrysin, and galangin were quantified in higher amounts in fir biomass (bark and needles) compared with spruce biomass. The values of (−)-epicatechin, myricetin, quercetin, isorhamnetin, and apigenin quantified in spruce and fir biomass were lower compared with the data reported in the literature for spruce, fir, and pinus needle biomass [100,105].
Among the quantified phenolic acids, important amounts of 3,4-dihydroxybenzoic, 4-hydroxybenzoic, ferulic, syringic, and p-coumaric acids were quantified in spruce and fir biomass. Higher amounts of 3,4-dihydroxybenzoic, 4-hydroxybenzoic, chlorogenic, and pcoumaric acids correspond to needle biomass, while higher amounts of ferulic and syringic acids and CAPE correspond to bark biomass. The amounts of the analyzed individual phenolic acids were lower compared with those reported in the literature for spruce, fir, and pinus needle biomass [100,105]. Stilbene t-resveratrol was quantified in higher amounts in spruce biomass compared with fir biomass, with the obtained values being comparable with those reported in the literature for the Pinus sp. needles [105], but much lower than those reported for spruce needles [100] (see Table S4). As previously mentioned, catechin, epicatechin, 4-hydroxybenzoic acid, rutin, myricetin, p-cumaric acid, kaempherol, and apigenin were the main polyphenols found in the selected samples (in quantities around 100 µg/g). These substances can impart the following bioactive properties to the extracts: antioxidant, cytoprotective, anti-inflammatory, anticarcinogenic, antidiabetic, antiasthmatic, antiallergic, hypocholesterolemic, neuroprotective, or cardioprotective activity. Catechins possess significant antioxidant effects and strong activity against several pathogens, including bacteria, viruses, parasites, and fungi [106]. Modulation of the metabolism of nitric oxide and reactive nitrogen species, which results in the preservation and improvement of the endothelial function of arterial vessels, was demonstrated for epicatechin [107]. Rutin display neuroprotective activity on brain ischemia, sedative and anticonvulsant effects, and suppress the activity of pro-inflammatory cytokines by decreasing the production of TNF-α and IL-1β in microglia, an effect that seems to be useful in the treatment of Alzheimer's disease, by preventing the cytotoxicity of β-amyloid oligomers [108].
Ferulic acid demonstrated antioxidant, anti-inflammatory, antiviral, antiallergic, antimicrobial, antithrombotic, anticarcinogenic, and hepatoprotective activity; vasodilatory activity; modulation of the activity of some enzymes; and the activation of transcriptional and gene expression factors [109]. It can prevent oxidative damage and amyloid pathology in Alzheimer's disease by degrading the amyloid structures [110]. The displayed antidiabetic effect may be related to the reduction in oxidative stress, which could help the β-cells of the pancreas to secrete more insulin [109].
Myricetin displayed anti-proliferative activity against acute leukemia HL-60 cells [111] as well as protective activity against skin cancer by strongly inhibiting tumor and neoplastic cell transformation induced by the promoter by restricting the activity of MEK kinases, JAK1, AKT, and MKK4 [112]. The hypotensive action and the potential to modulate the immune system was also demonstrated by in vivo studies [113]. Moreover, myricetin showed a strong analgesic effect by reducing the ionas current in calcium channels [114] and presents several benefits related to the central nervous system, including protection against Parkinson's and Alzheimer's diseases [111].
Besides showing a significant antioxidant effect, 4-hydroxybenzoic acid exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria [115]. P-coumaric acid has a higher bioavailability compared to other phenolic acids and can be absorbed in all sectors of the gastrointestinal tract. p-Coumaric acid shows antidiabetic activity by reducing the intestinal absorption of dietary carbohydrates and also has a strong inhibitory effect on the enzymes involved in glucose metabolism (amylase and glucosidase) while stimulating insulin secretion [116].
The reported biological properties of apigenin include anti-oxidant, anti-mutagenic, anticarcinogenic, anti-inflammatory, anti-proliferative, and anti-progressive activity. Apigenyn is an effective anti-inflammatory agent and the strongest inhibitor of transcriptional activation regarding both inducible cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) in cells activated with lipopolysaccharide (LPS), reducing nitric oxide production and inhibiting the production of pro-inflammatory cytokines in lipopolysaccharidestimulated human monocytes [117]. Also, apigenyn is reportedly able to inhibit several viruses, including enterovirus 71 (EV71), herpes simplex virus HSV-1 and HSV-2, hepatitis C virus, and influenza virus [118].
Pearson correlation analysis further elucidated the bioactive characteristics (TP, TF, and AA), sum of the individual phenolic acids (Σ Phenolic acids), and sum of the individual flavonoids (Σ Flavonoids) ( Figure S1), showing positive and negative correlations. Strong positive correlations (coefficients higher than 0.5) were observed between the bioactive characteristics and Σ Flavonoids, demonstrating the major contribution of the flavonoids to the general bioactive characteristics (TP, TF, and AA) of the studied coniferous biomass.
A Total Ion Current (TIC) chromatogram of the spruce bark extract in the negative ion mode, covering a scan range between 75 and 1000 m/z, is shown in Figure 2, while the TIC chromatograms of the main bioactive compounds identified in spruce needles, fir bark, and fir needles extracts are presented in Figure S2, Figure S3, and Figure S4, respectively. The major identified compounds in spruce bark extracts ( Figure 2) were quinic acid, (+)-catechin, astringin, taxifolin, and isorhapontin, while quinic, shikimic, and toluic acids and (+)-catechin were the major identified compounds in the spruce needle extracts ( Figure S2). In the case of the studied fir bark and needles, quinic and shikimic acids, (+)-catechin, 7-deoxyloganic acid glucopyranosyl ester, lucidumoside A, diosbulbinoside F, combrestatatin A5, pinoresinol, carnosol, azelaic, and etienic acids were identified in the fir bark extracts, while quinic acid, epigallocatechin, (+)-catechin, syringin, sacranoside A, and denipride were identified in the fir needle extracts.
Quinic and shikimic acids show antioxidant, antimicrobial, anti-inflammatory, and neuroprotective effects; hence, they are used as industrial precursors for the synthesis of oseltamivir phosphate, known as the antiinfluenza drug Tamiflu, which is used to treat influenza A and B [125,126]. Taxifolin, azelaic, and etienic acids exhibit strong antioxidant, anti-inflammatory, antiviral, antitumor, antibacterial, and enzyme-inhibiting activities [124,134]; lucidumoside A was shown to have antioxidant activity against the hemolysis of red blood cells induced by free radicals and antiviral activity [135]; carnosol; and antioxidative, anti-inflammatory, and antiproliferative activity, while carnosol and carnosic acid shows antioxidant and antiproliferative activities [136], and pinoresinol is a plant lignan with anticancer, hypoglycemic, and antifungal activities [137]. Diosbulbinoside F is a compound identified in the rhizome of D. bulbifera that has been used in folk and traditional Chinese medicine for the treatment of thyroid diseases and cancer [138]. Syringin has significant anti-inflammatory activity [139], epigallocatechin is an effective radical scavenger that protects against nerve cell damage [140], sacranoside A has anti-HCV activity and represents a potential drug candidate for antiviral activity against HCV "https://pubchem.ncbi.nlm.nih.gov/ (accessed on 7 June 2023)" , and denipride has a positive impact on the central nervous system and has been studied for its potential use in treating neurological disorders such as Parkinson's disease "https://pubchem.ncbi.nlm.nih.gov/ (accessed on 7 June 2023)". Quinic and shikimic acids show antioxidant, antimicrobial, anti-inflammatory, and neuroprotective effects; hence, they are used as industrial precursors for the synthesis of oseltamivir phosphate, known as the antiinfluenza drug Tamiflu, which is used to treat influenza A and B [125,126]. Taxifolin, azelaic, and etienic acids exhibit strong antioxidant, anti-inflammatory, antiviral, antitumor, antibacterial, and enzyme-inhibiting activities [124,134]; lucidumoside A was shown to have antioxidant activity against the hemolysis of red blood cells induced by free radicals and antiviral activity [135]; carnosol; and antioxidative, anti-inflammatory, and antiproliferative activity, while carnosol and carnosic acid shows antioxidant and antiproliferative activities [136], and pinoresinol is a plant lignan with anticancer, hypoglycemic, and antifungal activities [137]. Diosbulbinoside F is a compound identified in the rhizome of D. bulbifera that has been used in folk and traditional Chinese medicine for the treatment of thyroid diseases and cancer [138]. Syringin has significant anti-inflammatory activity [139], epigallocatechin is an effective radical scavenger that protects against nerve cell damage [140], sacranoside A has anti-HCV activity and represents a potential drug candidate for antiviral activity against HCV "https://pubchem.ncbi.nlm.nih.gov/ (accessed on 7 June 2023)" , and denipride has a positive impact on the central nervous system and has been studied for its potential use in treating neurological disorders such as Parkinson s disease "https://pubchem.ncbi.nlm.nih.gov/ (accessed on 7 June 2023)".

Multivariate Statistical Analysis for the Identification of the Specific Biomarkers of Picea abies L., H. Karst., and Abies alba Mill. Bark and Needles Extracts
Unsupervised multivariate methods, including Principal Component Analysis (PCA) and heat map analysis (HMA), were used in order to differentiate between different coniferous biomass (barks and needles of Picea abies L., H. Karst., and Abies alba Mill.) based on both the target and non-target HRMS profiling of the bioactive compounds. First, PCA was performed to facilitate an exploratory analysis of the data related to the quantitative data of some phenolic acids, flavonoids, and t-resveratrol (data presented in Table 1) and also for semiquantitative data (the area corresponding to the main repre-

Multivariate Statistical Analysis for the Identification of the Specific Biomarkers of Picea abies L., H. Karst., and Abies alba Mill. Bark and Needles Extracts
Unsupervised multivariate methods, including Principal Component Analysis (PCA) and heat map analysis (HMA), were used in order to differentiate between different coniferous biomass (barks and needles of Picea abies L., H. Karst., and Abies alba Mill.) based on both the target and non-target HRMS profiling of the bioactive compounds. First, PCA was performed to facilitate an exploratory analysis of the data related to the quantitative data of some phenolic acids, flavonoids, and t-resveratrol (data presented in Table 1) and also for semiquantitative data (the area corresponding to the main representative signals in the HRMS spectra) obtained from non-target HRMS analysis (data presented in Table 2) in order to identify specific biomarkers for each type of coniferous extract. The distribution of the studied extracts in the PC1-PC2 scores plot is presented in Figure 3. The first two components of the PCA model accounted for 47.46% of the variance pertaining to the targeted analysis ( Figure 3a) and 55.79% of the variance for the non-targeted screening analysis (Figure 3b), with a higher contribution being offered by PC1 compared to PC2 (in both cases).
From the HMA, it can be observed that the obtained results are in agreement with those obtained via PCA. Thus, as can be seen in Figure 4a, based on targeted data, the investigated coniferous extracts were clustered into two main clusters corresponding to needles (C1) and bark (C2) biomass, while the quantified variables were grouped into two main groups, with the first (G1) corresponding to the bark phytochemical compounds and the second (G2) being composed by the main tree subclusters that grouped phytochemical compounds representative of both needles and bark biomass. The heat map profiles based on the non-targeted HRMS data of the investigated coniferous extracts (Figure 4b) indicate two main clusters corresponding to bark (C1) and needles (C2) biomass, respectively, while also tentatively indicating subclusters for bark biomass corresponding to Picea abies L., H. Karst., and Abies alba Mill. (Figure 4b). The non-targeted identified variables were grouped into three main clusters corresponding to specific needle phytochemical biomarkers (G1), bark phytochemical biomarkers (G2), and both bark and needle phytochemical biomarkers (G3). targeted data (which refers to the greater number of the phytochemical bioactive compounds identified in the needles and bark extracts of Picea abies L., H. Karst., and Abies alba Mill.) were used to obtain the heat map profiles (Figure 4). From the HMA, it can be observed that the obtained results are in agreement with those obtained via PCA. Thus, as can be seen in Figure 4a, based on targeted data, the investigated coniferous extracts were clustered into two main clusters corresponding to needles (C1) and bark (C2) biomass, while the quantified variables were grouped into two main groups, with the first (G1) corresponding to the bark phytochemical compounds and the second (G2) being composed by the main tree subclusters that grouped phytochemical compounds representative of both needles and bark biomass. The heat map profiles based on the non-targeted HRMS data of the investigated coniferous extracts (Figure 4b) indicate two main clusters corresponding to bark (C1) and needles (C2)

Antimicrobial Activity
The infectious process at the level of the skin involves, in the first stage, predominantly Gram-positive bacteria, such as S. aureus, S. epidermidis, E. faecalis, etc. Subsequently, Gramnegative bacteria such as P. aeruginosa, A. baumannii/haemolyticus, E. coli, P. mirabilis become involved when a chronic wound develops [141]. Yeast strains, e.g., species of Candida sp., can also be detected in infected wounds [142].
Among the four extracts studied, P. abies needles extract showed a significant inhibition zone for P. aeruginosa (p < 0.0001), E. coli (p < 0.0001), E. faecalis (p < 0.0001), and MRSA (p < 0.0.0001) strains, while A. alba bark extract showed a significant inhibition zone against P. aeruginosa (p < 0.0.0001), E. coli (p < 0.0.001), E. faecalis (p < 0.0.0001), and S. marcescens (p < 0.0.0001) (Figure 5a). The antimicrobial activity of the A. alba needles extract was evident only against S. aureus sc and E. faecalis strains, while P. abies bark extract showed activity against P. aeruginosa, E. faecalis, and S. marcescens strains. Regarding the diameters of inhibition zones (DIZs), the antibacterial activities of extracts ranged between 2.5 and 16.5 mm. Maximum DIZ was observed for the A. alba needles extract against S. aureus sc (16.5 ± 1.29 mm), and minimum zone of inhibition was given by the A. alba bark extract against a clinical strain of E. coli (2.5 ± 0.58 mm). Statistical analysis was performed by comparing the antimicrobial activities between the extracts and the solvent control. The antimicrobial effect of the P. abies needles extract was better than the A. alba needles extract, probably due to phenolic content, while the extract from the P. abies bark was more active than the A. alba bark extract. The variation of the chemical components significantly changes the spectrum of antimicrobial activity. species of Candida sp., can also be detected in infected wounds [142].
Among the four extracts studied, P. abies needles extract showed a significant inhibition zone for P. aeruginosa (p < 0.0001), E. coli (p < 0.0001), E. faecalis (p < 0.0001), and MRSA (p < 0.0.0001) strains, while A. alba bark extract showed a significant inhibition zone against P. aeruginosa (p < 0.0.0001), E. coli (p < 0.0.001), E. faecalis (p < 0.0.0001), and S. marcescens (p < 0.0.0001) (Figure 5a). The antimicrobial activity of the A. alba needles extract was evident only against S. aureus sc and E. faecalis strains, while P. abies bark extract showed activity against P. aeruginosa, E. faecalis, and S. marcescens strains. Regarding the diameters of inhibition zones (DIZs), the antibacterial activities of extracts ranged between 2.5 and 16.5 mm. Maximum DIZ was observed for the A. alba needles extract against S. aureus sc (16.5 ± 1.29 mm), and minimum zone of inhibition was given by the A. alba bark extract against a clinical strain of E. coli (2.5 ± 0.58 mm). Statistical analysis was performed by comparing the antimicrobial activities between the extracts and the solvent control. The antimicrobial effect of the P. abies needles extract was better than the A. alba needles extract, probably due to phenolic content, while the extract from the P. abies bark was more active than the A. alba bark extract. The variation of the chemical components significantly changes the spectrum of antimicrobial activity. The minimum inhibitory concentration (MIC) was used to accurately determine the plant extracts effectiveness. IC50 is a more precisely defined measure than MIC, and its standard error is readily calculable [143]. The concentration of plant extracts that inhibit the growth of half of a microbial inoculum (IC50) was estimated by calculating the percentage of viable cells with varying concentrations of plant extract or solvent control and fitting a probit inhibition curve to the results (Figure 5b). The minimum inhibitory concentration (MIC) was used to accurately determine the plant extracts' effectiveness. IC50 is a more precisely defined measure than MIC, and its standard error is readily calculable [143]. The concentration of plant extracts that inhibit the growth of half of a microbial inoculum (IC50) was estimated by calculating the percentage of viable cells with varying concentrations of plant extract or solvent control and fitting a probit inhibition curve to the results (Figure 5b).
The MIC values obtained from plant extracts exhibited antibacterial activity ranging between 15.625 and 250 µL/mL (Table S6). The highest antimicrobial activity was observed for the A. alba needles extract, followed by the P. abies needles extract. The P. abies needles extract was more active on Gram-negative bacteria strains than bark, but the last one showed broad-spectrum activity. The bark extracts had similar MICs. The A. alba needles gave a MIC value at 15.625 µL/mL against P. aeruginosa. In Table S6, the comparative antimicrobial activity between extracts and similar solvent concentrations is highlighted. The solvent had an antimicrobial activity between 62.5 and 250 µL/mL. P. aeruginosa and MRSA strains proved sensitive to all extracts compared to the solvent control. The S. aureus sc strain was sensitive to P. abies and A. alba needles extracts and P. abies bark extract. In the case of E. faecalis, it was observed that the antimicrobial activity was actually given by the solvent used, while the S. marcescens strain was only sensitive to P. abies needles extract. In the case of the C. albicans strain, for which no antimicrobial activity was observed following qualitative analysis, only the extracts from the needles showed activity.
The antimicrobial activity of these extracts may be attributable to a synergistic effect between all phenolic components [144]. In the case of the spruce bark extract, it was evident that the compounds like catechin, taxifolin, astringin, quinic acid, shikimic acid, and isorhapontin [145,146] have activity against E. faecalis, S. thermophilus, Prevotella intermedia, Fusobacterium nucleatum, Streptococcus spp. These compounds are also found in the other three extracts but in a smaller proportion. Probably due to the presence of compounds like procyanidine [147], sinapic acid [148], apigenin [149], lariciresinol [150], azelaic acid [151], and sinapic acid [129], the extracts of A. alba (needles and barks) demonstrated significantly increased antimicrobial activity compared to the extracts obtained from P. abies. Another main compound identified in spruce bark extract, isorhamnetin, is known for enhancing survival and reducing proinflammatory cytokine levels in the serum and lung tissue of E. coli-infected mice [152].
To facilitate a statistical analysis of the comparative antimicrobial activity between the extracts and the solvent control, the IC50 was evaluated. From Figure 5b, it can be seen that, in the case of P. aeruginosa and S. marcescens, the antimicrobial activity was not significantly different from that of the solvent (p > 0.05). Significant differences in antimicrobial activity were observed for P. abies bark and A. alba needles extracts (p < 0.0001) on S. aureus sc and MRSA strains. In the case of the E. faecalis strain, the P. abies bark extract demonstrated significant activity compared to the solvent used (p < 0.05), while the IC50 for C. albicans was significantly reduced by the extracts of needles (p < 0.01).
The association of microbial biofilms with the chronicity of infected wounds is an important topic. Microbial biofilms represent a physical barrier to wound healing, maintaining a constant inflammatory state, causing more lesions, and delaying tissue epithelization. Moreover, chronic inflammatory skin conditions are more susceptible to the development of pathogenic biofilms [153,154].
Infections associated with microbial biofilms are difficult to resolve [155]. The topical route allows the application of high concentrations of antibacterial agents directly to the site of infection, with low incidence of systemic side effects. The rise of antibiotic resistance and the emergence of multidrug-resistant (MDR) microorganisms are serious causes for concern, and the development of new tissue-biocompatible antimicrobial solutions has been slow [153]. Therefore, for these reasons, the anti-biofilm effect was studied by evaluating the reduction in microbial adhesion to the inert substrate.
From Table S6, it can be seen that the antibiofilm effect was manifested only for S. aureus sc, MRSA, and C. albicans strains. The antibiofilm activity against the S. aureus sc strain was given by the extracts obtained from needles and fir bark extract, while against MRSA, only A. alba bark extract showed activity. In the case of C. albicans, all extracts prevented microbial adhesion, with the most active being A. alba bark extract. It is likely that the fir bark extract contains other non-phenolic compounds that act on the formation of adhesins.
The extracts obtained from the barks had a similar anti-biofilm activity, but the extract from the fir needles was the most active.

Biocompatibility
The possible toxic effects of the extracts were evaluated via MTT assay and LDH leakage assay on HaCaT cells. A cell viability assay conducted after 24 h of incubation showed that cell viability was the lowest for A. alba needles extract, followed by P. abies bark extract. As shown in Figure 6a, the IC50 values obtained for spruce needles and fir bark extracts did not differ significantly.
LDH is an intracellular enzyme that is released from cells where membrane integrity has been affected. Therefore, the measurement of LDH leakage in cell culture is the best indicator for the in vitro cytotoxicity [156]. It can be seen in Figure 6b that the extract with the lowest IC50 value (the fir needles extract) exhibited the largest amount of LDH released. Moreover, an increase in LDH release from cells in a manner dependent on the extract concentration was also observed. Thus, an optimization of the extract concentration in the final formulation based on a compromise between the cell viability and therapeutic properties is required.
Lower LDH leakage (for 25 µL/mL extract, Figure 6b) values were obtained with increasing values for antioxidant activity (DPPH assay). Thus, the antioxidant compounds could protect the integrity of eukaryotic cells (Pearson correlation, R 2 = 0.9474, p < 0.05). LDH released. Moreover, an increase in LDH release from cells in a manner dependent on the extract concentration was also observed. Thus, an optimization of the extract concentration in the final formulation based on a compromise between the cell viability and therapeutic properties is required.
A relationship between cytotoxicity and antimicrobial activity was established through the Selectivity Index (SI) ( Table S7). The SI values were calculated by the ratio of the IC50 and the MIC values for each microorganism (SI = IC50/MIC). Values over 1 represent elevated selectivity against microorganisms, whereas values below 1 indicate elevated toxicity towards HaCaT cells [157].
The extract from the spruce bark has an SI < 1 for all strains, both against MIC and the minimal biofilm eradication concentration (MBEC), which highlights that this extract was more toxic to eukaryotic cells than to microbial ones. The other extracts proved to be more cytotoxic against the P. aeruginosa strain compared to the eukaryotic ones, which suggests that the MIC and MBEC values can be selective against P. aeruginosa infections without affecting the HaCaT cells. This effect was also observed for the extracts from the Figure 6. Biocompatibility of the extracts: (a) IC50 (µL/mL) and (b) LDH release (%) of HaCaT cells exposed to fir and spruce bark and needle extracts; significant differences (p < 0.05) in all comparisons are shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
A relationship between cytotoxicity and antimicrobial activity was established through the Selectivity Index (SI) ( Table S7). The SI values were calculated by the ratio of the IC50 and the MIC values for each microorganism (SI = IC50/MIC). Values over 1 represent elevated selectivity against microorganisms, whereas values below 1 indicate elevated toxicity towards HaCaT cells [157].
The extract from the spruce bark has an SI < 1 for all strains, both against MIC and the minimal biofilm eradication concentration (MBEC), which highlights that this extract was more toxic to eukaryotic cells than to microbial ones. The other extracts proved to be more cytotoxic against the P. aeruginosa strain compared to the eukaryotic ones, which suggests that the MIC and MBEC values can be selective against P. aeruginosa infections without affecting the HaCaT cells. This effect was also observed for the extracts from the needles in relation to the microbial adhesion of S. aureus sc pl cells. The selective antibiofilm effect was evident for the bark extract (MRSA and C. albicans) and spruce needles extract (S. marcescens and C. albicans). Therefore, we can conclude that the extracts could be used for the antimicrobial effect only in the case of monospecific P. aeruginosa infection, but spruce needles extract shows a broad-spectrum antibiofilm effect (P. aeruginosa, S. aureus sc pl, S. marcescens and C. albicans).

Anti-Hemolytic Activity
In order to select the extract concentrations for the anti-hemolytic activity evaluation, the hemolysis index was determined and the extract concentrations below 200 µL/mL were selected. From Figure 7a, it is evident that at the concentration of 400 µL/mL, the spruce bark and needles extracts are slightly hemolytic, but with values below 5%. The differences are significant between the bark of P. abies and A. alba extracts (p < 0.0001), A. alba being non-hemolytic at all concentrations used. Therefore, extracts from A. alba could be used as active principles in open wounds while P. abies becomes hemocompatible at concentrations lower than 200 µL/mL. By comparison with the antimicrobial activity, it seems that the MIC and MBEC values are lower than the concentrations that induce hemolysis. Thus, these extracts can be used as active antimicrobial principles for infected wounds. Catechin possesses an accentuated hemolytic effect according to Veiko et al. [158], so due to the high content of catechin in spruce bark extract, the high hemolytic index compared to the other extracts could be explained. compatible at concentrations lower than 200 µL/mL. By comparison with the antimicrobial activity, it seems that the MIC and MBEC values are lower than the concentrations that induce hemolysis. Thus, these extracts can be used as active antimicrobial principles for infected wounds. Catechin possesses an accentuated hemolytic effect according to Veiko et al. [158], so due to the high content of catechin in spruce bark extract, the high hemolytic index compared to the other extracts could be explained. The antihemolytic activity of the fir and spruce extracts was evaluated by measuring the degree of hemolysis induced in ram erythrocytes by free radicals generated by AAPH. The pretreatment of RBCs with different doses (35-200 µL/mL) of the two needle extracts significantly attenuated the concentration of AAPH-induced hemolysis ( Figure  7b). The results clearly show that all four extracts were more effective than ascorbic acid in reducing AAPH-induced hemolysis. Indeed, the hemolysis inhibition percentages for the P. abies bark extract were almost 100% inhibition for all concentrations used, while the different concentrations of ascorbic acid (1 mg/mL was used as a standard) gave an inhibition percentage of 51.17 ± 1.53% to 27.61 ± 0.58% (Figure 7b). The inhibition of hemolysis by the extracts indicates that these extracts probably enhanced the antioxidant capacity of erythrocytes to quench free radicals and thereby attenuate oxidative hemolysis [94].
Basically, it was observed that when the erythrocytes were treated with AAPH and the four extracts, a significant reduction (p < 0.0001) in hemolysis was observed com- The antihemolytic activity of the fir and spruce extracts was evaluated by measuring the degree of hemolysis induced in ram erythrocytes by free radicals generated by AAPH. The pretreatment of RBCs with different doses (35-200 µL/mL) of the two needle extracts significantly attenuated the concentration of AAPH-induced hemolysis (Figure 7b). The results clearly show that all four extracts were more effective than ascorbic acid in reducing AAPH-induced hemolysis. Indeed, the hemolysis inhibition percentages for the P. abies bark extract were almost 100% inhibition for all concentrations used, while the different concentrations of ascorbic acid (1 mg/mL was used as a standard) gave an inhibition percentage of 51.17 ± 1.53% to 27.61 ± 0.58% (Figure 7b). The inhibition of hemolysis by the extracts indicates that these extracts probably enhanced the antioxidant capacity of erythrocytes to quench free radicals and thereby attenuate oxidative hemolysis [94].
Basically, it was observed that when the erythrocytes were treated with AAPH and the four extracts, a significant reduction (p < 0.0001) in hemolysis was observed compared to the cells treated only with phosphate-buffered saline (value set as 100% hemolysis) (p < 0.0001).
The antihemolytic activity was probably due to various phenolic compounds in the extracts, known as phenolic hydrogen atom donors. They help to stabilize free radicals through their antioxidant activity, increasing the resistance of erythrocytes to oxidative stress. Moreover, the phenolic components in the composition of the extracts could donate one or more electrons to neutralize the AAPH radical, leading to the inhibition of hemolysis. According to Balderrama-Carmona et al. [159], polyphenols show the ability to interact with the hydrophilic section of the lipid membrane, causing changes in the packing arrangement of the lipid polar ends.
The results of the antihemolytic effect were correlated with those obtained for the antioxidant activity achieved via the DPPH method. Thus, at a concentration of 35 µL/mL extract, it was observed that the greatest reduction in hemolysis was for the extract from spruce needles, followed by the extract from fir needles, spruce bark, and fir bark. Among these extracts, spruce needles had a higher content of flavonoids compared to the other extracts.
Flavonoids, as well as other polyphenols, protect erythrocyte cell membranes by interacting with membrane phospholipids, thus preventing lipid damage [160]. Flavonoids also promote Van der Waals interactions in the lipid bilayer and can be a source of membrane stabilization, protecting erythrocytes from damage generated by free radicals [161].

P-LD Runs against PI3Kγ
The superimposing of the structural files of the two RCSB PDB entries for PI3Kγ (1E8W with resolution = 2.50 Å and 1E90 with resolution = 2.70 Å) shows some folding differences, including in the region of the ATP binding pocket, where the co-crystalized ligands bind ( Figure 8); therefore, choosing structures with a higher resolution is a critical requirement for the accuracy of docking results.
teracting with membrane phospholipids, thus preventing lipid damage [160]. Flavonoids also promote Van der Waals interactions in the lipid bilayer and can be a source of membrane stabilization, protecting erythrocytes from damage generated by free radicals [161].

P-LD Runs against PI3Kγ
The superimposing of the structural files of the two RCSB PDB entries for PI3Kγ (1E8W with resolution = 2.50 Å and 1E90 with resolution = 2.70 Å) shows some folding differences, including in the region of the ATP binding pocket, where the co-crystalized ligands bind ( Figure 8); therefore, choosing structures with a higher resolution is a critical requirement for the accuracy of docking results.  The results of the two comparative P-LD runs (against 53 compounds, quercetin-the reference compound-is included) are depicted briefly in Figures 9 and 10 for the best poses for each docking run. The P-LD runs carried-out with PyRx-Python Prescription v.0.9.7 software using Autodock Vina as a docking algorithm (PyRx w/Autodock Vina) were used as ranking criteria for the best pose of each ligand; the zero values of the RMSD terms (RMSD/lb and RMSD/ub) combined with the lowest value of the Binding Affinity (BA in kcal/mol). In the case of the P-LD run carried-out with SwissDock web-service using the EADock DSS algorithm (SwissDock w/EADock DSS), the best-ranked poses were selected using the FullFitness ranking system (kcal/mol) and Estimated ∆G (kcal/mol) values for the best binding affinity to the target. The full dataset used for the two P-LD runs is presented in the Supplementary Materials (Supplementary Table S8).
Overall, the two docking algorithms agreed regarding the docking results for the 36 analyzed ligands, representing 67.9% from all the investigated compounds (53 ligands). The seven strongest binders identified via consensus between the results of the PyRx w/Autodock Vina P-LD run and the SwissDock w/EADock DSS P-LD run and taxifolin (identified as strong binder only in the results of the PyRx w/Autodock Vina P-LD run, and also an analogs of quercetin) were analyzed further to determine their binding modes with PI3Kγ ( Figure 11).
Overall, the two docking algorithms agreed regarding the docking results for the 36 analyzed ligands, representing 67.9% from all the investigated compounds (53 ligands). The seven strongest binders identified via consensus between the results of the PyRx w/Autodock Vina P-LD run and the SwissDock w/EADock DSS P-LD run and taxifolin (identified as strong binder only in the results of the PyRx w/Autodock Vina P-LD run, and also an analogs of quercetin) were analyzed further to determine their binding modes with PI3Kγ ( Figure 11).  Figure 11 shows the two binding areas of ligands in the ATP binding pocket of PI3Kγ. The ligands with a greater molecular weight and a higher structural complexity (naringin, rutin, and enrasentan) bind in a different region than the ligands with a lower molecular weight (ellagic acid, quercetin, and analogs isorhamnetin, myricetin, and taxi-  Figure 11 shows the two binding areas of ligands in the ATP binding pocket of PI3Kγ. The ligands with a greater molecular weight and a higher structural complexity (naringin, rutin, and enrasentan) bind in a different region than the ligands with a lower molecular weight (ellagic acid, quercetin, and analogs isorhamnetin, myricetin, and taxifolin), which are flat-type molecules. Moreover, from Figure 11a it can be observed that the docking cavity where the five flat-type molecules were fitted is tighter than the wider docking site of naringin, rutin, and enrasentan and therefore inaccessible to them.
Disregarding their binding patterns in the ATP binding pocket of PI3Kγ, the eight strong binders (naringin, rutin, ellagic acid, quercetin, isorhamnetin, myricetin, and enrasentan) may act as inhibitors of PI3Kγ. Therefore, those strong binders can be involved in the downregulation of the effectors of the PI3K/AKT signaling pathway and can exert anti-inflammatory, antioxidant, and autophagy regulatory effects during the healing process for the treatment the (chronic) wounds. The results of the P-LD runs against PI3Kγ correlate with the results regarding antioxidant activity obtained via the DPPH method and antihemolytic tests from a biomolecular mechanistic point of view. Moreover, as the flat-type inhibitors of PI3Kγ are typically non-selective [66], it is reasonable to assume that ellagic acid and quercetin and its analogs may also bind with PI3Kα (a class IA PI3K).
Phenolic compounds, such as those evaluated using the DPPH method, often exhibit antioxidant activity. Antioxidants can influence cellular signaling pathways, including the PI3K/AKT signaling pathway, by modulating ROS levels or directly affecting the activity of signaling molecules (PI3Kγ). Therefore, phenols with antioxidant properties may indirectly impact the PI3K/AKT signaling pathway through their effects on oxidative stress and redox signaling.

Conclusions
The major identified compounds in Picea abies L., H. Karst. biomass were (+)-catechin, astringin, taxifolin, isorhapontin, quinic, shikimic, and toluic acids, while (+)-catechin, epigallocatechin, quinic and shikimic acids, lucidumoside A, diosbulbinoside F, combrestatatin A5, sacranoside A, denipride, pinoresinol, carnosol, azelaic, and etienic acids were representative of Abies alba Mill. biomass. The studied extracts showed antimicrobial activity, especially for those isolated from infected wounds, with the A. abies needles extract being the most active. A major reason for the persistence of chronic wounds is the development of biofilms by the opportunistic pathogenic species. Thus, the prevention of the microbial biofilm's formation and the antioxidant effect on ram erythrocytes and eukaryotic cell viability (IC50) encourages us to continue our research regarding the development of new products that use spruce needles extract as an active principle for the healing of the chronic wounds. The wet-lab assays were supported by the in silico technique of the molecular docking and revealed the mechanistic interactions of the selected phenolic compounds with PI3Kγ and their possible effects over the PI3K/AKT signaling pathway, as the PI3K/AKT signaling pathway and its interaction with ROS are linked to the healing process of (chronic) wounds [13,69]. Overall, the wet-lab assays combined with the in silico techniques intertwine in the study of bioactive natural compounds. The evaluation of antioxidant properties and the investigation of cellular signaling pathways like PI3K/AKT showcase the multifaceted nature of biochemistry and molecular biology research.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antiox12071383/s1, Figure S1: Pearson correlation between the bioactive characteristic and total phenolic acids and total flavonoids quantified by HRMS analysis in the coniferous extracts; Figure S2. The total ion current (TIC) chromatogram showing the identification of the main bioactive compounds in spruce needle extract using UHPLC-MS/MS detection in negative ionization mode; Figure S3. Total ion current (TIC) chromatogram showing the identification of the main bioactive compounds in fir bark extract using UHPLC-MS/MS detection in negative ionization mode; Figure S4. Total ion current (TIC) chromatogram showing the identification of the main bioactive compounds in fir needles extract using UHPLC-MS/MS detection in negative ionization mode; Table S1. The results of the quantitative estimation of the bioactive properties of the studied vegetal material, expressed in mg per g of dried weight vegetal material (range/mean ± SD); Table S2. Content of total polyphenols (TP), total flavonoids (TF), and antioxidant activity (AA) in coniferous biomass (bark and needles) according to data available in the literature; Table S3. Peak numbers, target compounds, expected retention times (tR), exact mass, accurate mass, mean mass accuracy (ppm), and resulted fragments (negative ionisation); Table S4. Phenolic compound content in coniferous biomass (bark and needles), according to data available in the literature; Table S5. Pharmacological activities of the bioactive compounds identified in spruce (Picea abies L., H. Karst) and fir (Abies alba Mill.) biomass (bark and needles) by non-target HRMS analysis; Table S6. The antimicrobial and antibiofilm activities of P. abies and A. alba alcoholic extracts vs. solvent control; Table S7. Selectivity index values of P. abies bark and needle extracts and A. abies bark and needle extracts related to MIC and MBEC values; Table S8. Docking data for the best-ranked poses of the ligands in the two P-LD runs against PI3Kγ [1E8W entry] [55][56][57][58][73][74][75][76][95][96][97][98][99][100][101][102][103][104][105][120][121][122]124,[126][127][128][129][130]134,135,138,139,145,.