Conifer Bark Extracts as Modulators of Endothelial Function: Evidence from Abies alba and Cedrus brevifolia

TL;DR

This study shows that extracts from the bark of two conifer species can improve endothelial function by boosting nitric oxide and reducing oxidative stress.

Contribution

The novel contribution is the comparative evaluation of Abies alba and Cedrus brevifolia bark extracts for their endothelial benefits and vasorelaxant effects.

Findings

Abies alba extract significantly enhances nitric oxide production and reduces oxidative stress in endothelial cells.

Both conifer bark extracts demonstrate strong vasorelaxant activity, primarily through endothelial nitric oxide synthase.

Cedrus brevifolia extract shows greater vasorelaxant activity compared to Abies alba.

Abstract

Conifer bark extracts have attracted growing interest for their potential to protect and support endothelial function. The objective of this study was to evaluate the effects of Abies alba Mill. and Cedrus brevifolia (Hook. f.) Henry bark extracts on vascular endothelial function. The bark extracts were characterized by liquid chromatography coupled to high-resolution mass spectrometry. Bioactivity studies were first conducted in EA.hy926 endothelial cells to investigate the effects of bark extracts on cell viability and proliferation, nitric oxide production, oxidative stress, and angiogenesis. The vasorelaxant effects of bark extracts in rat aortic rings, as well as their impact on in vitro arginase activity, were further assessed. Abies alba bark extract was more effective in enhancing nitric oxide production (8.8-fold vs. 7.4-fold at 0.1 mg/mL), reducing oxidative stress (by 33% vs. 26% at 0.1 mg/mL), and inhibiting angiogenesis in EA.hy926 endothelial cells. It also exhibited stronger arginase inhibitory activity (IC50 = 68.30 µg/mL vs. 115.31 µg/mL). Both bark extracts showed marked vasorelaxant activity (EC50 < 15 µg/mL), mainly mediated by an endothelial nitric oxide synthase-related mechanism, with the Cedrus brevifolia bark extract being more active. Overall, our findings indicate that both bark extracts are promising candidates for supporting endothelial function.

Figures

Full text

1. Introduction

Conifer bark extracts exhibit a wide range of effects that contribute to the prevention and treatment of cardiovascular diseases. Numerous in vitro, in vivo, and clinical studies have highlighted their antioxidant capacity, anti-atherosclerotic, and cholesterol-lowering properties, as well as their abilities to protect the vascular endothelium function and to induce vasorelaxant and antihypertensive effects [1,2]. These beneficial activities are largely attributed to their high content of polyphenolic compounds, which play a key role in the modulation of vascular tone and endothelial homeostasis [3].

Several standardized conifer bark extracts, mainly derived from species of the genus Pinus, are currently commercially available in the form of capsules, tablets, and chewing gums. Enzogenol^®^, a water-soluble powdered extract obtained from the bark of Pinus radiata D. Don and standardized to contain more than 80% proanthocyanidins, was reported to reduce blood pressure following oral administration of 480 mg/day in a 12-week open label pilot study conducted on healthy people [4,5,6].

Oligopin^®^ is an aqueous extract obtained from the Pinus pinaster Aiton bark containing 65–75% oligomeric proanthocyanidins, together with catechin, taxifolin, and phenolic acids, including ferulic, gallic, and caffeic acids [7,8]. Due to its high content in soluble oligomers and low proportion of insoluble components, Oligopin^®^ is considered one of the most purified pine bark extracts on the market [7,9]. In a randomized, double-blind, placebo-controlled, crossover trial, daily supplementation with 150 mg Oligopin^®^ for five weeks led to improvements in cardiovascular risk factors, evidenced by reduced systolic blood pressure and low-density lipoprotein (LDL) oxidation, as well as increase in high-density lipoprotein (HDL) and apolipoprotein A1 levels in patients with stage 1 hypertension [10].

Pycnogenol^®^, another standardized and purified extract derived from Pinus pinaster bark, has been extensively investigated in both clinical and preclinical studies. This extract is standardized to contain 70 ± 5% procyanidins, mainly composed of catechin and epicatechin subunits [11]. Apart from its antioxidant capabilities, Pycnogenol^®^ has been shown to enhance constitutive endothelial nitric oxide synthase (eNOS) activity, thereby elevating nitric oxide (NO) bioavailability in isolated rat aortic rings [12,13]. Clinical studies in patients with diabetes mellitus have demonstrated that daily supplementation with 25 mg Pycnogenol^®^ for 12 weeks reduced LDL and maintained blood pressure within normal limits, allowing the use of lower doses of angiotensin-converting enzyme (ACE) inhibitors. At the same time, serum endothelin-1 (ET-1) levels, a vasoconstrictive peptide, exhibited a significant reduction [14]. The capacity of Pycnogenol^®^ to enhance endothelium-dependent vasodilation through the upregulation of NO production was also noted after two weeks of oral administration (180 mg/day) in a double-blind, randomized study [15]. It is well recognized that NO produced by endothelial cells regulates blood pressure, and its reduced levels are associated with hypertension and other cardiovascular disorders [2]. Additionally, following the administration of 200 mg Pycnogenol^®^ daily for eight weeks to patients with coronary artery disease, an improvement in endothelial function was observed. The vascular protective role of Pycnogenol^®^ was correlated with its antioxidant properties, as evidenced by a decrease in the oxidative stress biomarker 15-F2t-isoprostane [16].

Another commercially available extract obtained from Pinus pinaster bark is Flavangenol^®^, whose chemical composition differs from that of Pycnogenol^®^ and Oligopin^®^, owing to a distinct standardization process. Flavangenol^®^ is produced by hot-water extraction of the bark and contains 72.5% polyphenols, including procyanidin B1, catechin, and epicatechin [16,17]. This extract has been shown to activate the eNOS-NO-soluble guanylate cyclase (sGC) signaling pathway, resulting in pronounced vasorelaxant effects, associated with an upregulation of phosphorylated-eNOS (Ser1177) protein [18].

Beyond these commercially available Pinus species, antihypertensive effects have also been reported for bark extracts from other Pinus species. In a double-blind, randomized, placebo-controlled trial, daily administration of 1322 mg of Pinus massoniana Lamb. bark extract (equivalent to 432 mg of polyphenols) for 12 weeks resulted in a significant reduction in systolic blood pressure in healthy adults [19]. Similarly, administration of an aqueous bark extract from Pinus densiflora Sieb. et Zucc. (Korean red pine) to hypertensive rats, at doses of 50 and 150 mg/kg/day for seven weeks, significantly reduced blood pressure due to the downregulation of renin–angiotensin system components and malondialdehyde (MDA) levels [20,21].

In contrast to the extensive literature on Pinus bark extracts, other members of the Pinaceae family remain comparatively underexplored. Abies alba Mill. (silver fir) and Cedrus brevifolia (Hook. f.) Henry (Cyprus cedar) are two such species whose bark has demonstrated promising bioactivities with potential pharmaceutical applications [22,23,24,25,26,27]. Cedrus brevifolia is a narrowly endemic conifer species native to the island of Cyprus [28]. Charalambous et al. identified several bioactive compounds in the methanolic bark extract (quercitrin hydrate, vanilloyl hexoside, taxifolin, dihydromyricetin, matairesinol), that contributed to the notable antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay (IC_50_ = 0.011 ± 0.001 mg/mL) [29]. On the other hand, Leone et al. showed that an ethyl-acetate soluble fraction of an aqueous bark extract of Abies alba (Abigenol^®^/AlbiPhenol^®^) reduced the production of reactive oxygen species (ROS) and glutathione oxidation in human umbilical vein endothelial cells (HUVEC) and hepatocytes (HepG2 cells), as well as superoxide dismutase (SOD) activity in H9c2 rat myoblasts [30]. This ethyl-acetate fraction, rich in phenolic acids, flavonoids and lignans, also reduced HDL and LDL oxidation in vitro, suggesting a potential protective effect against atheromatous plaque formation [1,30]. An additional noteworthy property of Abigenol^®^/AlbiPhenol^®^ was demonstrated in the H9c2 cardiomyocyte cell model, where the highest non-toxic concentration (700 µg/mL) significantly reduced ACE activity, exhibiting a stronger inhibitory effect than lisinopril. This outcome highlights its potential in modulating key factors associated with cardiovascular disorders, including vasorelaxant properties, likely mediated through endothelium-dependent mechanisms and NO production, which could be linked to a reduced risk of hypertension in vivo [30].

Given the high phenolic content of conifer bark extracts and the well-established involvement of phenolic compounds in endothelial regulation, we hypothesized that phenolic-rich, tannin-depleted extracts from Abies alba and Cedrus brevifolia bark may modulate endothelial function by affecting NO bioavailability, oxidative stress, angiogenesis-related processes, and vascular reactivity. To investigate this hypothesis, we performed phytochemical characterization of the extracts, followed by in vitro endothelial cell-based assays and ex vivo vascular reactivity studies.

2. Results

2.1. Phytochemical Analysis of Bark Extracts

Abies alba bark extract (AABE) and Cedrus brevifolia bark extract (CBBE) were obtained with yields of 7.55% and 4.34%, respectively. Their total phenolic, flavonoid, and proanthocyanidin contents are presented in Table 1. AABE exhibited a higher total phenolic content than CBBE (659.55 vs. 589.33 mg chlorogenic acid equivalents/g of extract), whereas CBBE had a higher total flavonoid content (246.26 vs. 167.92 mg catechin equivalents/g of extract). Regarding proanthocyanidin content, the values were comparable between the two extracts (98.2 mg vs. 101.6 mg cyanidin equivalents/g of extract).

Twenty-five compounds were tentatively identified in the two bark extracts by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) by comparing their mass spectral data and fragmentation with those found in the literature and KNApSacK database [31] (Figure 1, Table 2). A total of 21 compounds were detected in AABE, of which seven were simple phenolics, including glycosides of methoxy-dihydroxybenzoic acid (2), hydroxy-methoxybenzyl alcohol (3), hydroxybenzoic acid (4, 9), and methoxyhydroxy-benzaldehyde (17), as well as homovanillic acid (5). Among flavonoids, glycosides of kaempferol (12), isorhamnetin (13, 24), and taxifolin (21) were identified. Additionally, several flavan-3-ols such as epigallocatechin (10), gallocatechin (15), catechin (16), and epicatechin (19), were observed. Proanthocyanidin dimers were also observed, with peaks 8, 11, and 14, possibly corresponding to prodelphinidins and procyanidins. Lastly, two lignan glycosides, taxiresinol (20) and (iso)lariciresinol (22), were proposed. In contrast, CBBE displayed a slightly lower compound diversity, with many compounds also identified in AABE. Glycosides of vanillic acid (7), dimethoxybenzaldehyde (18), dihydro-isorhamnetin (23), and quercetin (25) were detected only in CBBE. Overall, both extracts shared a substantial number of common metabolites.

2.2. Effects of Bark Extracts on Proliferation of EA.hy926 Endothelial Cells

In order to evaluate the effects of bark extracts (0.01–0.20 mg/mL) on the proliferation of EA.hy926 cells, real-time experiments were performed to monitor cell growth for five days; untreated cells (0.00 mg/mL) were used as the control (Figure 2). The assay relies on the continuous recording of cellular impedance, with the generation of time- and concentration-dependent cellular profiles. These profiles reflect cellular proliferation, growth, and morphological changes, corresponding to alterations in cell physiology [56,57]. The addition of AABE at 0.01 mg/mL did not change the shape of the proliferation curve compared to the control during the first 48 h of treatment. A slight decrease in the cell index (CI) values of the treated cells was observed after 72–96 h of treatment, indicating a reduced proliferation rate. However, when treated with higher concentrations of AABE (0.05, 0.1, and 0.2 mg/mL), the cell response curves indicated time- and concentration-dependent alterations in cell physiology after 24 h of treatment. The proliferation curve of cells treated with AABE at 0.05 mg/mL suggested an antiproliferative effect, with CI values decreasing to approximately half of those recorded for the control. For the cells treated with AABE at 0.1 and 0.2 mg/mL, the CI values were significantly lower and the cellular profiles indicated a possible cell death process, which might occur after long-time incubation with AABE (Figure 2A). The proliferation curves of EA.hy926 cells treated with CBBE at all tested concentrations (0.01–0.2 mg/mL) had similar shapes to those of untreated cells. Treatment with increasing concentrations of CBBE appeared to slightly reduce the proliferation rate, as shown by decreasing CI values in a time-dependent manner (Figure 2B). However, the CI values for cells treated with either bark extracts at concentrations of 0.01, 0.05, and 0.1 mg/mL were similar to untreated cells, during the first 24 h of treatment. The results suggest no significant cytotoxicity of bark extracts at indicated concentrations in this time frame. Based on these findings, a 24 h treatment period was selected for all subsequent cell-based assays to ensure biological relevance while minimizing potential cytotoxic effects.

2.3. Effects of Bark Extracts on Cell Viability and Cytotoxic Activity in Endothelial EA.hy926 Cells

To confirm the absence of significant cytotoxicity, cell viability and cytotoxic assays were performed in EA.hy926 cells following 24 h incubation with the bark extracts (0.01–0.20 mg/mL) (Figure 3). The viability of EA.hy926 cells, assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, was not significantly affected by treatment with bark extracts at 0.01 and 0.05 mg/mL. A slight reduction in cell viability was observed at 0.10 mg/mL, with cell viability decreasing to 92.83 ± 2.42% and 90.50 ± 2.93% following treatment with AABE and CBBE, respectively (p < 0.01). EA.hy926 cell viability decreased to 74.07 ± 3.70% (p < 0.001) in response to treatment with AABE at 0.2 mg/mL. The impact of CBBE at the same concentration was less pronounced, resulting in a cell viability of 82.00 ± 4.02% (p < 0.001). The cytotoxic potential of AABE and CBBE was evaluated by the lactate dehydrogenase (LDH) release assay. The latter confirmed the MTS assay results, showing no cytotoxicity for either bark extract, except for AABE at 0.2 mg/mL. At this concentration, cytotoxicity, evaluated as LDH release, was higher (17.91 ± 2.03%) compared to the control (13.73 ± 2.81%) (p < 0.05), likely explaining the reduction in cell viability below 80%. The results confirm the data provided by real-time experiments, indicating that EA.hy926 cells remain viable after treatment with bark extracts at 0.01–0.10 mg/mL for 24 h.

2.4. Effects of Bark Extracts on NO Production in Endothelial EA.hy926 Cells

The intracellular NO level in EA.hy926 cells, treated with bark extracts and untreated (control), was detected using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA), a non-fluorescent compound that reacts with NO to generate fluorescent triazolofluorescein [58]. The highest NO production was detected in EA.hy926 cells treated overnight (18 h) with bark extracts at 0.10 mg/mL and 1 μM DAF-FM DA. Under these conditions, NO levels in cells treated with AABE showed an approximately 8.8-fold increase compared to the control. Treatment with CBBE induced slightly lower NO production under identical conditions (7.4-fold increase compared to the control) (Figure 4A). The histograms support these findings (Figure 4B). Untreated EA.hy926 cells (control) showed a main peak (grey) corresponding to non-fluorescent cells (NO-negative cells) and a minor peak (green) representing fluorescent cells (NO-positive cells). Histograms of treated cells displayed well-defined peaks corresponding to fluorescent cells. The capacity of bark extracts to stimulate NO production in EA.hy926 cells was further confirmed by fluorescence microscopy. Under the same experimental conditions, AABE-treated cells exhibited a slightly higher proportion of NO-positive cells (fluorescent cells) compared with those exposed to CBBE (Figure 4C).

2.5. Effects of Bark Extracts on ROS Production in Endothelial EA.hy926 Cells

Treatment with bark extracts for 24 h dose-dependently reduced H_2_O_2_-induced ROS production in EA.hy926 cells. AABE was more effective in reducing ROS generation at all three tested concentrations. At 0.10 mg/mL, it reduced ROS level to 67%, whereas CBBE decreased ROS level to 74% (Figure 5A,B). The higher antioxidant potency of AABE was confirmed by fluorescence microscopy (Figure 5C).

2.6. Effects of Bark Extracts on Angiogenesis in Endothelial EA.hy926 Cells

The angiogenesis process involves several stages such as cell proliferation, migration, and sprouting, followed by the formation of tight cell–cell junctions. The final stage is characterized by the development of complex capillary structures forming tubular networks [59]. Both bark extracts dose-dependently reduced EA.hy926 cell migration compared to the control. Control cells nearly closed the wound within 24 h, whereas cells treated with bark extracts exhibited reduced wound closure, indicating impaired cell migration. AABE was more effective, causing wound area closure of 68% at 0.05 mg/mL and 46% at 0.10 mg/mL. At the same concentrations, CBBE was less active, inducing wound area closure of 77% and 61%, respectively (Figure 6).

The capacity of bark extracts to modulate the differentiation of EA.hy926 cells into capillary-like structures on Matrigel is illustrated in Figure 7. Morphological analysis revealed that both bark extracts inhibited the angiogenic organization of human epidermal growth factor (hEGF)-stimulated EA.hy926 cells in a dose-dependent manner. While the control group formed a dense, interconnected tubular lattice, treated cells exhibited a progressive breakdown of network connectivity. Specifically, treatment with AABE at 0.05 and 0.1 mg/mL resulted in a prevalence of shortened tube fragments, isolated cell aggregates, and numerous individual non-aligned cells, indicating a potent inhibition of endothelial differentiation. CBBE at 0.1 mg/mL also reduced the complexity of the tubular network compared to the control. However, the anti-angiogenic effect of CBBE was notably less pronounced than that of AABE at the same concentration, as evidenced by a lower frequency of isolated individual cells. Overall, the prevalence of isolated cell aggregates and fragmented tubular structures at high extract concentrations correlates with the anti-migratory effects observed in the wound healing assays.

2.7. Effects of Bark Extracts on Arginase Activity

As shown in Table 3, the initial screening showed that AABE and CBBE inhibited bovine arginase I by more than 45% at 100 µg/mL. After plotting the logarithm of bark extract concentration vs. the percentage of arginase inhibition (Figure 8), the calculated inhibitory concentration 50% (IC_50_) value was significantly lower for AABE (68.30 ± 3.92 µg/mL) compared with CBBE (115.31 ± 4.32 µg/mL). The area under the curve (AUC) across the tested concentration range indicated the superior potency of AABE. In the present study, S-(2-boronoethyl)-L-cysteine (BEC) served as the positive control, given its high potency as an arginase inhibitor, characterized by an IC_50_ value lower than 1 µg/mL and an AUC value of 205.9. Regarding the maximal effect (E_max_), all samples achieved >95% inhibition, indicating full inhibitory efficacy.

2.8. Vasorelaxant Activity of Bark Extracts in Rat Aortic Rings

As shown in Figure 9 and Table 4, AABE and CBBE induced concentration-dependent vasorelaxation of rat aortic rings precontracted with phenylephrine (PE) (10^−6^ M), with efficient concentration 50% (EC_50_) values of 13.88 ± 0.95 and 7.06 ± 0.77 µg/mL, respectively. The maximal relaxation response was obtained at 100 µg/mL for CBBE and 1000 µg/mL for AABE (90.08 ± 1.28% and 92.54 ± 0.96% respectively). Removal of the endothelium completely abolished the vasorelaxant effect of AABE at concentrations ranging from 0.01 to 100 µg/mL. At concentrations above 100 µg/mL, AABE was still able to relax the aortic rings, reaching a steady-state with a maximum relaxation of 90.15 ± 2.19% at 3000 µg/mL. Treatment with N(ω)-nitro-L-arginine methyl ester (L-NAME) suppressed the vasodilatory response to AABE, similar to the effect of endothelial removal. Notably, significant vasorelaxation was still observed at concentrations between 1000 and 3000 µg/mL, with a maximal relaxation of 91.53 ± 1.15% at 3000 µg/mL, closely matching that of endothelium-intact aortic rings. In addition, under endothelium-denuded condition, AABE-induced relaxation was slightly reduced by tetraethylammonium (TEA), a non-selective potassium channel blocker, but only at the concentration of 300 µg/mL. At all other concentrations, the concentration–response curves for endothelium-denuded rings and those treated with TEA were nearly superimposable, indicating that potassium channel activation contributes minimally to the endothelium-independent component of AABE-induced relaxation (Figure 9A). Similarly, endothelium removal and pretreatment of aortic rings with L-NAME markedly suppressed the vasorelaxant activity of CBBE, except at 1000 µg/mL, where a modest relaxation was still observed (35.16 ± 3.12% and 13.68 ± 1.64%, respectively). In contrast to AABE, preincubation with TEA in endothelium-denuded rings markedly reduced the vasorelaxant response to CBBE (1000 µg/mL) at 12.59 ± 1.84%. This partial inhibition indicates that activation of potassium channels contributes substantially to the vasorelaxant effect of CBBE, although additional mechanisms are likely involved (Figure 9B).

To understand the mechanisms involved in the endothelium-independent relaxation effect of AABE, especially at higher concentration, the contribution of extracellular calcium ion influx was investigated. In the presence of dimethyl sulfoxide (DMSO) 1% (control), aortic ring contraction induced by CaCl_2_ and KCl reached 46.99 ± 2.14%, whereas contraction induced by CaCl_2_ and PE attained 74.95 ± 2.94%. In contrast, AABE (1000 µg/mL) nearly abolished aortic ring contraction, lowering it to 0.58 ± 0.04% when induced by KCl (a stimulator of voltage-dependent calcium channels) and 0.55 ± 0.02% when induced by PE (a stimulator of receptor-operated calcium channels) (Figure 10).

3. Discussion

The present study provides the first experimental evidence of the vascular activity of two purified extracts obtained from the bark of Abies alba and Cedrus brevifolia (AABE and CBBE, respectively). By combining optimized extraction and purification procedures with complementary cellular and vascular assays, this work highlights the vasculoprotective potential of the two Pinaceae species.

AABE, obtained by ultrasound-assisted extraction using acetone–water–acetic acid and subsequently purified on polyamide, exhibited a high phenolic content (659.55 mg chlorogenic acid equivalents/g of dry extract), reflecting the efficiency of the extraction and purification protocols. By comparison, previous studies reported total phenolic contents of 298.6 mg gallic acid equivalents/g in the crude hydromethanolic extract [23] or 1520.12 ± 111.63 μg gallic acid equivalents/mL in the aqueous extract [60] of Abies alba bark. A similar enrichment was observed for CBBE, which reached 589.3 mg chlorogenic acid equivalents/g, whereas the crude hydromethanolic and aqueous Cedrus brevifolia bark extracts contained 248.9 mg gallic acid equivalents/g [24] and 38.4 mg gallic acid equivalents/g, respectively [29]. The phytochemical profiles of AABE and CBBE were consistent with previously reported compositions of Abies alba and Cedrus brevifolia bark extracts. Compounds tentatively identified in AABE such as catechin, epicatechin, proanthocyanidin dimers, homovanillic acid, derivatives of hydroxybenzoic acid, taxiresinol, lariciresinol, taxifolin, and isorhamnetin have been previously described in other Abies alba bark extracts [1,25,33,61,62]. Compounds similar to those identified in CBBE such as kaempferol, epicatechin, catechin, and taxifolin derivatives have been reported in other Cedrus brevifolia bark extracts [24,29]. The absence of higher-degree polymerized proanthocyanidins in the MS/MS spectra of both extracts is likely attributable to polyamide purification step, which preferentially retains low-molecular-weight flavan-3-ols, while limiting the recovery of high oligomeric and polymeric proanthocyanidins [63,64]. In addition, higher oligomers and polymers have poor ionization capacity, which impedes their detection by MS [65].

Functional bioassays demonstrated that AABE and CBBE exert vasodilatory, vasculoprotective, and anti-angiogenic effects, likely attributable to key phytochemicals, particularly phenolics. Phenolic compounds are well known to regulate endothelial NO production, oxidative stress, and angiogenesis, thereby playing a central role in vascular homeostasis [3].

NO is a key regulator of vascular tone and endothelial integrity. Under physiological conditions, eNOS converts L-arginine into NO, promoting vasodilation, inhibiting platelet aggregation, and protecting the vascular wall from inflammatory and oxidative injury. However, excessive arginase activity disrupts this balance by competing with eNOS for their common substrate, L-arginine, thereby reducing NO synthesis and promoting eNOS uncoupling. This results in decreased NO bioavailability and increased production of highly reactive species such as superoxide anion radicals and peroxynitrite anions. These species promote endothelial dysfunction and vascular inflammation, contributing to the pathogenesis of cardiovascular diseases [3,66]. In parallel, ornithine generated by arginase serves as a precursor for other metabolic pathways that exacerbate vascular remodeling. Through the action of ornithine decarboxylase (ODC), ornithine is converted into polyamines (putrescine, spermidine, and spermine), which promote vascular smooth muscle cell proliferation. Alternatively, ornithine aminotransferase (OAT) transforms ornithine into proline, a key amino acid for collagen synthesis and extracellular matrix deposition. Both pathways contribute to vascular hypertrophy, fibrosis, and structural remodeling [67].

In the present study, immortalized EA.hy926 endothelial cells were used due to important advantages over primary endothelial cells, such as high proliferative capacity, passage stability, ease of handling, and enhanced reproducibility [68]. Both AABE and CBBE increased NO production in EA.hy926 endothelial cells; however, AABE elicited a markedly stronger response, enhancing NO levels by more than eightfold compared to the control. This effect may be associated with the phenolic content of the extracts. For instance, taxifolin enhanced NO production in rat thoracic aorta in a concentration-dependent manner, inducing an almost threefold increase in NO levels and upregulating eNOS expression, with more pronounced effects in spontaneously hypertensive rats [69]. Similarly, epicatechin exhibited cardioprotective effects through eNOS-mediated NO production in endothelial cells [70]. In our study, cellular proliferation, MTS, and LDH assays indicated that AABE and CBBE are generally non-cytotoxic to EA.hy926 endothelial cells at concentrations up to 0.10 mg/mL after 24 h of treatment. Overall, both bark extracts maintained acceptable endothelial biocompatibility within the concentration range of 0.01–0.10 mg/mL, supporting their safe use in further studies.

Bark extracts significantly and dose-dependently reduced H_2_O_2_-induced ROS production in endothelial cells. Compared to CBBE, AABE was more effective across all tested concentrations, consistent with its higher total phenolic content. Catechin and epicatechin, present in both bark extracts, have been reported to protect endothelial cells from H_2_O_2_-induced oxidative injury through direct radical-scavenging activity [71].

Both AABE and CBBE also inhibited endothelial cell migration and their differentiation into tube-like structures, in a dose-dependent manner, indicating anti-angiogenic activity, with AABE exerting stronger effect. The anti-migratory activity is linked to phenolic compounds, particularly flavonoids, which downregulate pro-angiogenic signaling pathways. Kaempferol, quercetin, catechins, procyanidins, and taxifolin inhibit endothelial proliferation, migration, and tube formation by modulating vascular endothelial growth factor (VEGF)-mediated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), mitogen-activated protein kinase (MAPK), VEGF receptor-2 (VEGFR-2)/PI3K/Akt, and integrin signaling pathways [72,73,74,75]. Functionally, such anti-migrating effect is beneficial in pathological conditions characterized by excessive neovascularization within atherosclerotic plaques, or tumor-associated angiogenesis, while preserving endothelial integrity under oxidative stress conditions.

A particularly novel finding of this study is the demonstration that both extracts inhibit bovine arginase I in a concentration-dependent manner, with AABE being approximately twice as potent as CBBE. Anti-arginase activity represents a key mechanism for improving endothelial function and protecting against cardiovascular disorders, as it results in enhanced NO bioavailability [3,66]. To our knowledge, no previous studies have reported arginase inhibition by Abies alba or Cedrus brevifolia bark extracts. Phenolic compounds identified in AABE and CBBE, such as epicatechin, taxifolin, quercetin, and kaempferol, have been reported to inhibit bovine liver arginase I with IC_50_ values of 19.9, 23.2, 31.2, and 179.1 µM, respectively [76]. In another study, catechin and epicatechin displayed notable inhibitory activity against rat liver arginase (IC_50_ = 0.77 and 1.8 µM, respectively). Kinetic analyses demonstrated that catechin and epicatechin are competitive inhibitors of rat liver arginase, directly competing with L-arginine for the active site [77].

In isolated rat aortic rings, both bark extracts exhibited dose-dependent vasorelaxant effects, acting mainly through endothelium-dependent mechanisms at concentrations of 0.01–300 µg/mL. In the presence of L-NAME (an eNOS inhibitor), the vasorelaxation induced by each bark extract was blunted, suggesting the involvement of eNOS-dependent, downstream vasorelaxant signaling pathways [78,79]. The activation of potassium channels, an endothelium-independent mechanism, was found to contribute to the vasorelaxation induced by CBBE at 1000 µg/mL. On the other hand, at 1000 µg/mL, AABE induced marked vasorelaxation in endothelium-denuded aortic rings by inhibiting calcium ion influx through voltage-dependent and receptor-operated calcium ion channels. A dual, concentration-dependent mechanism of activity, similar to that observed for AABE and CBBE, has been previously described for gallic acid [80]. This highlights the multifaceted pharmacological nature of phenolic compounds, which can activate distinct molecular pathways depending on their local concentration and the cellular redox environment. Activation of eNOS has been reported for the phenolic compounds identified in AABE and CBBE. Epicatechin, taxifolin, kaempferol, and proanthocyanidins activated eNOS in various experimental models, including in vitro endothelial cell assays, ex vivo isolated rat aortic rings or porcine arteries, and in vivo in mice with high-fat-diet-induced endothelial dysfunction [69,70,81,82,83,84].

The consistently stronger effects observed with AABE compared to CBBE across multiple tests may be attributed to both quantitative and qualitative differences in their phenolic composition. AABE demonstrated superior activity in enhancing NO production, scavenging ROS, reducing angiogenesis, and stimulating arginase, consistent with its significantly higher total phenolic content. Beyond total phenolic levels, differences in the relative abundance of specific phenolic compounds may also contribute to the divergent biological profiles of the two bark extracts.

The effective concentrations determined in our assays fall within the µg/mL range. However, these values cannot be translated into expected human blood levels without pharmacokinetic studies. Following oral administration of conifer bark extracts, the native compounds attain very low plasma levels as a result of extensive gut and liver metabolism; their metabolites, often markedly different from the parent compounds, predominate in plasma. This limits direct extrapolation of effective experimental concentrations to in vivo conditions. For instance, after three weeks of oral supplementation with Pycnogenol^®^ (200 mg/day), catechin was detected in serum at mean concentrations of 48–53 ng/mL, while taxifolin was present at approximately 0.20 ng/mL. Ferulic and caffeic acid were detected at mean serum concentrations of 3.0 and 9–10 ng/mL, respectively [85]. Following a single 300 mg dose of Pycnogenol^®^, catechin reached plasma levels of 60 ng/mL at 0.5 h and 100 ng/mL at 4 h, remaining detectable throughout the 14 h sampling period. After repeated oral intake of Pycnogenol^®^ (200 mg/day for five days), catechin was detected in plasma at total concentrations of 48.6 ± 16.7 ng/mL [86]. The poor oral bioavailability of conifer bark-derived compounds does not necessarily limit their therapeutic application, since bioavailability can be improved through various strategies, including formulation-based approaches, co-administration with absorption enhancers, or alternative routes of administration [87,88]. In addition, in the case of many phenolic compounds (proanthocyanidins, flavonoids, and phenolic acids), reduced oral bioavailability does not necessarily translate into reduced bioactivity. This is attributable to their metabolites that exhibit similar or even higher biological effects compared with the parent compounds. The bioactive metabolites account for the health benefits of plant-derived products despite low oral bioavailability of their components [89].

Limitations of the study. The present study has several limitations that should be acknowledged. The endothelial protective effects were evaluated in vitro in EA.hy926 endothelial cells and the vasorelaxant effects were assessed ex vivo in isolated rat aortic rings. Arginase inhibition was demonstrated in vitro using the isolated enzyme. Although arginase inhibition may contribute to increased NO bioavailability and improved vasorelaxation, a direct causal relationship in intact vascular tissue would require additional ex vivo determinations, such as measurement of arginase activity or expression in isolated aortic rings. Therefore, the involvement of arginase is discussed as a plausible contributing mechanism rather than a definitively established mediator of the vasorelaxant response. Overall, the in vivo validation of our results in experimental models of endothelial dysfunction or cardiovascular disease, as well as pharmacokinetic studies are essential to confirm the therapeutic relevance of the observed effects. Despite these limitations, the integrative approach combining phytochemical characterization, endothelial cell assays, enzyme inhibition and vascular reactivity studies, provides a coherent and robust first evaluation of the endothelial and vasorelaxant effects of Abies alba and Cedrus brevifolia bark extracts and supports further in vivo investigations.

4. Materials and Methods

4.1. Chemicals and Reagents

Acetone, acetic acid, diethyl ether, dimethyl sulfoxide (DMSO), chlorogenic acid, (+)-catechin hydrate, quinic acid, p-hydroxybenzoic acid, epicatechin, phenylephrine (PE), N(ω)-nitro-L-arginine-methyl-ester (L-NAME), tetraethylammonium (TEA), and S-(2-boronoethyl)-L-cysteine (BEC) were acquired from Sigma-Aldrich (Steinheim, Germany). Polyamide CC6 (particle size 0.05–0.16 mm) was purchased from Macherey-Nagel GmbH (Düren, Germany). Purified bovine liver arginase I was obtained from MP Biomedicals (Illkirch-Graffenstaden, France). Folin–Ciocalteu’s phenol reagent was from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle medium (DMEM) was obtained from Capricorn Scientific (Ebsdorfergrund, Hessen, Germany). Fetal bovine serum (FBS) and 1% solution of penicillin-streptomycin were from Hyclone (Logan, UT, USA). CellTiter 96^®^ AQueous One Solution Cell Proliferation Assay kit (MTS test) and CytoTox 96^®^ Non-Radioactive Cytotoxicity Assay Kit (LDH release test) were procured from Promega Corporation (Madison, WI, USA). 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA), ProLong™ Gold Antifade, calcein AM, and hydrogen peroxide 35% wt.% solution in water, stabilized, were acquired from ThermoFisher Scientific (Waltham, MA, USA). CellROX Green reagent was from Invitrogen (Carlsbad, CA, USA). Corning^®^ Matrigel^®^ Basement Membrane Matrix was purchased from Sigma-Aldrich (St. Louis, MO, USA). Human epidermal growth factor (hEGF) was from Gibco (Billings, MT, USA). All other chemicals and reagents were of analytical grade.

4.2. Plant Material and Isolation of Bark Extracts

Details on the plant material, including source, drying, and storage conditions, have been described previously [22,23,24]. The taxonomic identity of the plant material was authenticated by Lecturer Dr. Cristina Lungu (Department of Pharmaceutical Botany, Grigore T. Popa University of Medicine and Pharmacy Iasi, Romania). The dried bark material was ground and sieved to obtain a powder with particle size below 250 µm (60 mesh). The bark powder (25 g) was extracted in an ultrasonic bath (3 × 20 min, 40 kHz, 80 W) with 3 × 60 mL solvent mixture (acetone/water/acetic acid (80/19.5/0.5, v/v/v); solid-to-liquid ratio 1:7.2, w/v). The combined extracts were delipidated with diethyl ether and freeze-dried, as previously described [35]. The resulting extracts were further purified by open column chromatography on polyamide 6. In brief, 1 g of each extract was suspended in 3 mL of methanol and applied to a polyamide 6 column (55 × 15 mm), followed by elution with water (200 mL), 50% methanol (200 mL), and methanol (200 mL). The eluates were combined, concentrated under reduced pressure at 40 °C, and freeze-dried to yield purified bark extracts which were subsequently subjected to chemical and biological assays. The extracts were stored at −20 °C until use.

4.3. Total Phenolic, Flavonoid, and Proanthocyanidin Contents

Total phenolic content was determined using the Folin–Ciocalteu method, with chlorogenic acid as the calibration standard. The calibration curve was linear over the tested concentration range (2–15 µg/mL) and described by the equation y = 0.0684x + 0.044, with a coefficient of determination R^2^ = 0.9992. Results were expressed as mg chlorogenic acid equivalents per g of extract [90,91]. Total flavonoid content was assessed using the aluminium chloride colorimetric assay, with catechin as the calibration standard. The calibration curve followed the equation y = 0.0217x − 0.0344, with R^2^ = 0.9997, and results were expressed as mg catechin equivalents per g of extract [90,91]. Total proanthocyanidin content was determined using the acid-butanol assay. The concentration of proanthocyanidins, expressed as mg cyanidin equivalents per g of extract, was estimated based on the molar absorptivity (ε = 17,360 L·mol^−1^·cm^−1^) and relative molecular mass (Mr = 287.24 g·mol^−1^) of cyanidin, using the equation M = A/(ε × l), where M is the molar concentration of the sample, A is the absorbance, and l is the cuvette path length (1 cm) [90,91].

4.4. Phenolic Profile Analysis by LC-HRMS/MS

The phenolic profile in bark extracts was analyzed by LC-HRMS/MS. The analysis was performed on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with auto-sampler (G1329B), degasser (G1379B), binary pump (G1312C), thermostat (G1316A), DAD detector (G1315D), Agilent ESI-Q-TOF mass spectrometer (G6530B), nitrogen generator (Parker Hannifin Corp., Cleveland, OH, USA), and compressed air generator (Jun-Air Oxymed, Łódź, Poland). Analyses were performed on a Phenomenex Gemini C18 column (2 × 100 mm, 3 μm) maintained at 20 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was as follows: 5% B at 0 min, increased linearly to 60% B over 45 min, then raised to 95% B between 45 and 55 min. The flow rate was set to 0.2 mL/min, and a 10 μL sample volume was injected. Mass spectrometric detection was performed in negative ion mode over an m/z range of 70–1000. Source conditions included a nitrogen gas temperature of 275 °C with a flow rate of 10 L/min, a nebulizer pressure of 35 psi, sheath gas at 325 °C and 12 L/min, a capillary voltage of 4000 V, nozzle voltage of 1000 V, skimmer voltage of 65 V, and fragmentor voltage of 140 V. Collision-induced dissociation was conducted at fixed energies of 10 and 30 V [60]. Data were processed using MassHunter Qualitative Analysis Navigator software version B.08.00 (Agilent Technologies, Santa Clara, CA, USA).

4.5. Cell Culture

Immortalized human endothelial cell line, EA.hy926 (CRL-2922™), was purchased from American Tissue Culture Collection (ATCC, Rockville, MD, USA). EA.hy926 cells were cultured in DMEM with high glucose content (4.5 g/L) and supplemented with 10% FBS and 1% solution of penicillin-streptomycin. The cells were grown in humidified atmosphere with 5% CO_2_ at 37 °C before use. Experiments were performed on cells within a passage range of 5–20.

4.6. Real-Time Cell Proliferation Assay

EA.hy926 cell proliferation was monitored using the Real-Time Cell Analysis (RTCA) system (Agilent Technologies, Santa Clara, CA, USA), following the method described by Albulescu et al. [57] and Ke et al. [92], except that 8000 of EA.hy926 cells were plated on each well and CI was recorded every 30 min for the mentioned period of time. Bark extracts were used at concentrations of 0.01–0.2 mg/mL. RTCA software version 2.0 (Agilent Technologies, Santa Clara, CA, USA) was used to generate and compare the growth curves of treated and control cells.

4.7. Cell Viability and Cytotoxicity Assays

EA.hy926 cells were seeded in 96-well plates at a density of 8000 cells per well and allowed to adhere overnight in an incubator. Cells were then exposed for 24 h to bark extracts (0.01–0.20 mg/mL) or to corresponding concentrations of vehicle (50% ethanol); untreated EA.hy926 cells served as the control.

4.7.1. Cell Viability Assay

The MTS assay was performed as previously described. The results were expressed as percentages by normalizing the optical density at 490 nm of the treated cells to that of the control (untreated cells) [57].

4.7.2. LDH Release Assay

After incubation, the supernatant was collected and LDH release was measured using the CytoTox 96^®^ Non-Radioactive Cytotoxicity Assay Kit, according to the manufacturer’s instructions. The amount of LDH released into the culture medium reflects cell membrane integrity. Maximum LDH release (positive control) was achieved by exposing untreated cells to a lysis solution provided in the assay kit. Absorbance was measured at 490 nm using a BIOBASE-EL10 microplate reader (BIOBASE Group, Jinan, China). Cytotoxicity was expressed as a percentage of LDH released from treated EA.hy926 cells relative to that in the lysed cell culture medium [57].

4.8. Evaluation of Intracellular NO Level

NO production in EA.hy926 cells was measured by fluorescent staining with DAF-FM DA, a cell-permeable NO sensitive indicator at 1 µM, using flow cytometry and fluorescence microscopy, according to previously published protocols [58], with slight modifications. The cells were serum-starved for 8 h prior to overnight incubation with bark extracts.

4.8.1. Flow Cytometry

A total of 2.5 × 10^5^ EA.hy926 cells were seeded in each well of a 12-well plate. In brief, after serum starvation, the culture medium was replaced with bark extracts diluted in complete medium at 0.1 mg/mL or with medium alone for control cells. Fluorescence intensity was measured using a MoFlo Astrios EQ cell sorter (Beckman-Coulter, Brea, CA, USA) equipped with a 488 nm blue laser for excitation. DAF-FM DA fluorescence from at least 20,000 cells was collected through a 530/40 nm band-pass filter (fluorescein isothiocyanate [FITC] channel), after excluding debris and doublets using forward scatter/side scatter (FSC/SSC) and pulse-width gating [93]. Instrument voltages and thresholds were set using unstained cells and cells not treated with DAF-FM DA. Data were acquired and analyzed using Summit software version 6.3.1 (Beckman-Coulter, Brea, CA, USA).

4.8.2. Fluorescence Microscopy

A total of 40,000 EA.hy926 cells were plated on 16 mm glass coverslips and incubated overnight to allow adherence. The next day, the cells were serum-starved for 8 h and then treated with bark extracts at 0.1 mg/mL for 18 h. The cells were further stained with DAF-FM DA and fixed in 4% paraformaldehyde solution for 30 min; the coverslips were mounted with ProLong™ Gold Antifade. The fluorescence images were acquired using an EVOS^®^ FL Auto Imaging System for Fluorescence (Thermo Fisher Scientific, Waltham, MA, USA) with excitation/emission wavelengths of 495/515 nm [58]. Green channel images were collected for at least 100 cells per experimental condition. The images were analyzed using ImageJ program (version 1.50i, National Institutes of Health, Bethesda, MD, USA).

4.9. Evaluation of Intracellular ROS Level

4.9.1. Cellular Antioxidant Activity Assay

The antioxidant activity of bark extracts was assessed using the OxiSelect™ Cellular Antioxidant Assay Kit (Cell Biolabs, San Diego, CA, USA), following a previously published protocol with some modifications [57]. Oxidative stress was induced by adding H_2_O_2_ to the culture media at a final concentration of 500 µM [94]. After stimulation, fluorescence intensity was measured using a Microplate Multimode Detector Zenyth 3100 (Anthos Labtec Instruments GmbH, Salzburg, Austria) at excitation and emission wavelengths of 485 and 535 nm, respectively [57,95]. For data normalization, the fluorescence values of cells pretreated with bark extracts but unexposed to H_2_O_2_ were subtracted from the fluorescence values of cells pretreated with bark extracts and exposed to H_2_O_2_. Results were expressed as ROS production (%) relative to control (untreated cells).

4.9.2. Fluorescence Microscopy

The intracellular ROS levels were also measured by fluorescence microscopy using the CellROX Green reagent, (Invitrogen, Carlsbad, CA, USA) following the protocol provided by manufacturer with slight modifications. In brief, a number of 40,000 EA.hy926 cells were seeded on 16 mm glass coverslips and allowed to adhere for 24 h. The cells were pre-treated with bark extracts at concentrations of 0.01, 0.05, and 0.1 mg/mL for 24 h, followed by exposure to 500 µM H_2_O_2_ for 1 h. The cells were then washed with 1× PBS and stained with 5 µM CellROX Green reagent for 45 min, followed by fixation in 4% paraformaldehyde for 30 min at room temperature. Quercetin (20 µM) was used as reference antioxidant to validate the sensitivity of the method [96]. The fluorescence images were acquired using an EVOS^®^ FL Auto Imaging System for Fluorescence (Thermo Fisher Scientific, Waltham, MA, USA) for at least 100 cells per experimental condition. The images were analyzed using ImageJ program (version 1.50i, National Institutes of Health, Bethesda, MD, USA).

4.10. Angiogenesis Assays

4.10.1. Scratch Migration Assay

To evaluate endothelial cell migration, a scratch migration assay was performed using double-well silicone culture inserts (Ibidi GmbH, Gräfelfing, Germany). Each insert was placed in a 24-well plate, and 3.5 × 10^4^ cells were seeded into both wells of each insert with 70 μL of medium containing 10% FBS. When the cells reached confluence, the silicone inserts were gently removed, and the cells were incubated either with 1% FBS medium (control, untreated cells) or with bark extracts diluted in the same low-serum medium. The use of low-serum conditions is essential to ensure that cell migration measurements are not confounded by cell proliferation. Each well was photographed immediately after insert removal (T0) and after 24 h (T24) using a 10× objective on an EVOS^®^ FL Auto Imaging System for transmitted light (Thermo Fisher Scientific, Waltham, MA, USA). The wound closure was quantified using Wound Healing Size Tool, an ImageJ/Fiji^®^ plugin (ImageJ software version 1.50i, National Institutes of Health, Bethesda, MD, USA), as described by Suarez-Arnedo et al. [97].

4.10.2. Tube Formation Assay

A previously reported method [98] with minor modifications was used to investigate the effect of bark extracts on the differentiation of EA.hy926 cells into tubular structures on a Matrigel matrix. hEGF was used as positive angiogenic stimulus [99]. A volume of 50 µL of Corning^®^ Matrigel^®^ Basement Membrane Matrix (4.5 mg/mL) was added to each well of a 96-well plate and incubated for 1 h at 37 °C to allow polymerization. Suspensions of EA.hy926 cells (30,000 cells/50 µL) in complete medium supplemented with 10 ng/mL hEGF were seeded into each coated well. Solutions of 2 × concentrated bark extracts, prepared in the same medium, were added in a volume of 50 µL to each test well to reach final concentrations of 0.01, 0.05, and 0.1 mg/mL. Untreated cells served as control. Cells were stained with 1 µM calcein AM prior to observation under a fluorescence microscope. Calcein AM-labeled cells were visualized and photographed using the EVOS^®^ FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA), with excitation at 485 nm and emission at 520 nm [100]. Qualitative morphological analysis was performed to evaluate the degree of network complexity ranging from isolated cells to a fully developed, continuous tubular lattice [101]. Images were analyzed using ImageJ software (version 1.50i, National Institutes of Health, Bethesda, MD, USA).

4.11. Arginase Inhibition Assay

The experimental protocol was performed as previously described in our publications [35,102], with minor changes. For the calculation of the IC_50_ values, stock solutions of 14 mg/mL were used for both bark extracts. The stock solutions were successively diluted with ultrapure water to obtain final well concentrations of 2000, 600, 200, 60, 20, 6, 2, 0.6, and 0.2 µg/mL. For CBBE, the concentration range was extended up to 6000 µg/mL in order to reach the maximal inhibitory plateau. For the reference arginase inhibitor (BEC), the screening was performed at concentrations of 10 and 100 µM. IC_50_ values were determined by nonlinear regression analysis using sigmoidal dose–response curves.

4.12. Vascular Reactivity Studies

Experiments were performed in isolated aorta from male Sprague Dawley rats (8–9-week-old, obtained from Janvier Labs, Le Genest Saint Isle, France). We used experimental protocols (no. 2015/001-CD/5) detailed in our previous publications [35,102] that were approved by the local Ethics Committees and adhered to the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines (ethical approval: CEBEA no. 58/18 June 2017. Rats were sedated with sodium pentobarbital (60 mg/kg, i.p.), and euthanasia was performed via exsanguination of the abdominal aorta. Then, the thoracic aorta was excised, cleaned of connective tissue, and cut into rings of approximately 2 mm in length, yielding up to 10 aortic rings per animal, which were randomly allocated to the different experimental groups. In total, vascular reactivity experiments were performed using 8 animals. In some rings, the endothelium was mechanically removed. Aortic ring viability was first assessed by vasoconstriction with 100 mM KCl in Krebs solution. Endothelial integrity was confirmed by more than 80% relaxation induced by the endothelium-dependent agonist acetylcholine (10^−6^ M) in aortic rings precontracted with α_1_-adrenergic agonist PE (10^−6^ M). Less than 10% relaxation response to acetylcholine indicated the absence of endothelium in denuded rings [35,102].

To investigate the vasodilatory effects, endothelium-intact and endothelium-denuded rings were precontracted with PE (10^−6^ M, a concentration inducing submaximal contraction) until a contractile plateau was reached. The rings were then exposed to cumulative concentrations of the bark extracts to achieve concentrations of 10^−2^–3·10^3^ µg/mL for AABE and 10^−2^–10^3^ µg/mL for CBBE. Cumulative concentration–response curves were generated by the stepwise addition of increasing concentrations of the extract to the same ring, without intermediate washout or subsequent exposure to any other extract or treatment. Concentrations were applied at intervals of 3–5 min, depending on the time required to reach a stable relaxation plateau before the addition of the next concentration [35,102].

To evaluate the contribution of the NO pathway to extract-induced vasorelaxation, some experiments were conducted in the presence of the eNOS inhibitor L-NAME (10^−4^ M). The involvement of vascular smooth muscle cell potassium channels in vasorelaxation was assessed using TEA (10^−3^ M) as non-selective potassium channel blocker, applied prior to PE induced-contraction in endothelium-denuded aortic rings. For AABE at 1000 µg/mL, the effects on extracellular calcium ion influx were evaluated in endothelium-denuded aortic rings previously depleted of calcium ions. Calcium influx was induced by CaCl_2_ (10^−2^ M) and assessed via receptor-operated or voltage-dependent calcium channels in the presence of PE (10^−6^ M) or KCl (60 mM), respectively [35,102].

EC_50_ values in vascular reactivity studies were calculated from cumulative concentration–response curves and represent the concentration of extract inducing 50% of the maximal vasorelaxant effect (E_max_). Curve fitting and parameter estimation were performed by nonlinear regression analysis using GraphPad Prism [35,102].

4.13. Data Analysis

All experiments were performed with a minimum of three independent biological replicates, and each sample was measured in technical triplicate. Data are shown as mean ± standard error of the mean (SEM). The statistical differences in the phytochemical contents of the extracts were assessed using an unpaired Student’s t-test. Statistical differences between vasorelaxation curves were analyzed using two-way ANOVA for repeated measures. In arginase inhibition assay, differences between two groups were analyzed using Student’s t-test or the Mann–Whitney test when data were not normally distributed. For comparisons among multiple groups, one-way ANOVA was used, followed by Tukey’s post hoc test. Statistical significance was accepted at least at p < 0.05. When applicable, different levels of statistical significance are indicated in the figures. All statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA).

5. Conclusions

In conclusion, our study provides the first evidence that two purified extracts obtained from the bark of Abies alba and Cedrus brevifolia (AABE and CBBE, respectively) exhibit beneficial vascular activity, which is associated with increased NO production in endothelial cells, protection of endothelial cells from oxidative stress, suppression of angiogenesis, inhibition of arginase, and vasorelaxation mediated predominantly by the eNOS pathway. Together, these findings support the relevance of Abies alba and Cedrus brevifolia bark extracts in supporting endothelial function. Mechanistic investigations and in vivo validation are essential to harness the potential of these bark extracts.