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Article

Chemical Profiling and Vascular Effects of a Hydroalcoholic Extract of Calophyllum longifolium Willd: Role of Nitric Oxide and Potassium Channels

by
Diego Aravena
1,
Javier Palacios
1,*,
Cristián A. Véliz-Quezada
1,
Gabriela V. Aguirre-Martínez
1,
Julio Benites
2,
David J. Greensmith
3,*,
Adrián Paredes
4,5,
Fredi Cifuentes
5,6,
Mario J. Simirgiotis
7,
Frank R. León-Vargas
8,
Daniel Asunción-Alvarez
9,
Iván M. Quispe-Díaz
9 and
Roberto O. Ybañez-Julca
9
1
Química y Farmacia, Facultad de Ciencias de la Salud, Universidad Arturo Prat, Iquique 1110939, Chile
2
Laboratorio de Química Medicinal, Química y Farmacia, Facultad de Ciencias de la Salud, Universidad Arturo Prat, Casilla 121, Iquique 1100000, Chile
3
Biomedical Research and Innovation Centre, School of Science, Engineering and Environment, The University of Salford, Salford M5 4WT, UK
4
Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Antofagasta 1271155, Chile
5
Instituto Antofagasta (IA), Universidad de Antofagasta, Antofagasta 1271155, Chile
6
Departamento de Biomédico, Facultad Ciencias de la Salud, Universidad de Antofagasta, Antofagasta 1271155, Chile
7
Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia 5090000, Chile
8
Departamento de Ingeniería Química, Facultad de Ingeniería Química, Universidad Nacional de la Amazonia Peruana, Iquitos 16002, Peru
9
Laboratorio de Farmacología, Facultad de Farmacia y Bioquímica, Universidad Nacional de Trujillo, Trujillo 13011, Peru
*
Authors to whom correspondence should be addressed.
Plants 2026, 15(4), 594; https://doi.org/10.3390/plants15040594
Submission received: 24 December 2025 / Revised: 10 February 2026 / Accepted: 10 February 2026 / Published: 13 February 2026
(This article belongs to the Special Issue Phytochemical Characterization and Bioactivity of Medicinal Plants: From Analytical Techniques to Therapeutic Potential)
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Abstract

Calophyllum spp. infusions are used to treat varicose veins, hemorrhoids, and hypertension. However, the chemical composition and mechanisms of action are poorly understood. Accordingly, the aim of this study was to investigate the phytochemical composition and vascular effects of hydroalcoholic extracts of Calophyllum longifolium. Phytochemical profiling was performed using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Extract effects on rat aortic rings and aortic vascular smooth muscle cells (VSMCs) were evaluated using wire myography and photometric measurement of intracellular Ca2+, respectively. UHPLC-ESI-Q-TOF-MS revealed the presence of coumarins, xanthones, flavonoids, triterpenes, and phenolic acids. Coumarin–resveratrol hybrids, such as gut-70 derivatives, were also abundant. In aortic rings from normotensive rats, C. longifolium induced a biphasic vascular response whereby low concentrations (1 μg/mL) produced significant vascular relaxation, whereas high concentrations (100 μg/mL) produced contraction. Blockade of ATP-sensitive (KATP) or voltage-gated (KV) potassium channels attenuated these effects. Furthermore, effects were not observed in preparations preincubated with L-NG-Nitro-L-arginine methyl ester (L-NAME) or in endothelium-denuded rings. In aortic VSMCs, extracts (1 µg/mL) rapidly reduced sarcoplasmic reticulum (SR) Ca2+ content. This study provides the first UHPLC-ESI-Q-TOF-MS chemical profile of C. longifolium, revealing diverse bioactive metabolites. It is also the first to demonstrate that C. longifolium exerts an endothelium-dependent, nitric oxide- and Ca2+-mediated biphasic effect on vascular function. Taken together, these findings highlight C. longifolium as a potential novel source of vasculotropic phytopharmaceuticals.

1. Introduction

The genus Calophyllum includes 190 species of tropical tree. Among these, 179 species are distributed in Asia, Europe, and Africa, while only 10 species are found in America and Oceania [1]. Species within the Calophyllum genus have been chemically described and share several bioactive molecules, such as flavonoids, coumarins, chromanones, xanthones, and triterpenes [1]. Phenolic acids (protocatechuic acid and gallic acid) are common in the Calophyllum genus. However, coumarins are considered the chemotype of C. inophyllum, and chromanones and dipyranocoumarins that of C. brasiliense [2]. Given this, Calophyllum species may be pharmacologically useful. Indeed, certain species in the genus inhibit angiotensin-converting enzyme (ACE), which suggests that they may be useful for the modulation of blood pressure [3]. Furthermore, in traditional medicine, infusions of bark taken from Calophyllum species are used to treat varicose veins, hemorrhoids, and hypertension [3,4].
Here, we focus on C. longifolium, a light, stress-resistant tropical tree [5], typically reaching 6–30 m in height and with yellow latex in its bark. Its leaves are large, coriaceous, and glossy, with conspicuous venation. The species produces axillary inflorescences with few hermaphroditic flowers bearing numerous stamens and ovoid drupes with a detachable outer layer enclosing a hard stone. We are particularly interested in this species as it widespread and found in South America, including Bolivia (Santa Cruz), Brazil (Rondônia), Ecuador (Pastaza, Esmeraldas), Colombia (Amazonas, Choco, Vaupés, Valle del Cauca) and Peru (Jaén, Loreto) [6].
Originally, Vesque in 1893—the only author to revise the genus across both the Old and New World—proposed that C. longifolium be treated as a subspecies of C. brasiliense, naming it C. brasiliense ssp. longifolium (Willd.) Vesque [6]. The WFO Plant List still accepts Calophyllum brasiliense subsp. longifolium (Willd.) Vesque as a synonym of C. longifolium Willd [7], and both are sometimes referred to by the same common name, “Lagarto Caspi” [8]. However, D’Arcy and Keating (1979) [9] argue that C. longifolium is a different species given considerable anatomical differences from C. Brasiliense. These include the presence of transcurrent ducts, more frequent venation, midrib sclerenchyma forming an inverted V, and the presence of sclereids. It also exhibits smaller ducts and a slightly thicker lamina, cuticle, and mesophyll but a thinner hypodermis [10].
Given this, it may be that C. longifolium possesses discrete pharmacological potential, yet it remains chemically and biologically uncharacterized [1,3]. Furthermore, few studies report the vascular effects of the wider Calophyllum genus. Therefore, the present study sought to characterize the chemical composition and vascular effects of C. longifolium and then elucidate underlying mechanisms.

2. Results

2.1. UHPLC-ESI-Q-TOF-MS Spectrometry Analysis

Metabolomic analysis of the C. longifolium extract was carried out using ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS) and identified several metabolites, including specific coumarins previously reported in this species. The chromatograms are presented in Figure 1, the corresponding data are summarized in Table 1, and a detailed description of the metabolomic findings is provided below.

2.2. Phenolic Acids and Derivatives

Peak 2 was identified as gallic acid (C4H20O14) and peaks 13, 18 and 26 as grandidentatin isomers of formula C21H27O9. Peak 24 was identified as mulberrofuran X (C26H39O7), peak 34 as paucinone C (C37H49O9), and peaks 35 and 36 as isomers of benzoic acid derivatives.

2.3. Flavonoids

Peak 3 was identified as the flavonoid astilbin (C21H21O11) and peak 8 as 3-hydroxy-3’,4’,5’-trimethoxyflavone (C29H47O10).

2.4. Sugar Derivatives and Alcohols

Peak 5 was identified as a common sugar (C12H21O10), peak 7 as an oleate derivative (C27H45O10), peak 11 as 7-(2,4-dihydroxypentyl)-15-(ethoxymethyl)-2,5,8,11,14-pentaoxaicosane-17,19-diol, and peak 17 as a sugar derivative.

2.5. Fatty Acids

Peak 21 with an ion at m/z: 357.2464 was identified as a stearic acid derivative and peak 28 as a palmitoyl derivative (C16H19O6)

2.6. Terpenes

Peak 9 was identified as asiatic acid (Table 1), peak 26 as aoibaclyin [29], peak 10 as lepidolide (C34H47O6), peak 16 as tsugarioside B, peak 23 as cimiracemoside H (C37H57O11), peak 25 as charantoside Iii, peak 29 as the sesquiterpene paxidal, peak 30 as cotylenin F, peak 31 as caseagrewiifolin A, peak 33 as an epiceanothic acid derivative (C32H51O10), and, finally, peak 38 was identified as dryocrassin ABBA.

2.7. Xanthones

Peak 12 was identified as mangostanol, peak 20 as tajixanthone hydrate, and peak 37 as tetrahydrogambogic acid (C38H49O8).

2.8. Coumarins

Peaks 4, 14 and 15 were identified as important biomarkers. Peak 4 was identified as a coumarin calanolide, while peaks 14 and 15 were identified as gut-70 derivatives (Figure 2).

2.9. The Biphasic Contractile Effects of C. longifolium on Aortic Rings

To evaluate the vascular response of the extract, a first vascular reactivity experiment was performed. The experiment illustrated by Figure 3A shows that, in intact aortic rings precontracted with phenylephrine (PE), the hydroalcoholic extract of C. longifolium produced a biphasic effect.
On average (Figure 3B), concentrations of 1 and 10 μg/mL produced a significant vascular relaxation of 72 ± 8% (p = 0.001) and 76 ± 3% (p = 0.001), respectively, which preceded the onset of vasomotion (see Figure 3A inset).
We next evaluated whether the vascular effects of C. longifolium persisted under conditions of membrane depolarization induced by KCl (60 mM). As shown in Figure 4A, C. longifolium failed to elicit any relaxation in precontracted aortic rings, and the averaged data (Figure 4B) confirmed that its vascular effects were completely abolished under these conditions.

2.10. The Effect of C. longifolium Following Modulation of eNOS–NO–sGC Pathway

To further investigate the involvement of endothelial pathways, we assessed the contribution of nitric oxide (NO) to the responses shown in Figure 5. The same protocol was repeated following preincubation with either the nitric oxide synthase inhibitor L-NAME or the selective NO-sensitive guanylyl cyclase (sGC) inhibitor ODQ. As illustrated in the original traces (Figure 5A) and mean data (Figure 5B), preincubation with L-NAME or ODQ for 20 min completely abolished the vasorelaxant effect of C. longifolium observed in intact aortic rings. Consistently, AUC analysis (Figure 5C) revealed that control tissues (140.8 ± 12.5 a.u.) exhibited significantly greater responses compared with those preincubated with L-NAME (18.0 ± 1.3 a.u., p = 0.001) or ODQ (13.5 ± 1.0 a.u., p = 0.001). Collectively, these findings demonstrate that the endothelium-dependent vasorelaxation induced by C. longifolium is mediated through the participation of the eNOS–NO–sGC signaling pathway.
To evaluate whether C. longifolium extracts increased vascular endothelium NO, experiments were performed on rat aorta sections loaded with DAF-FM DA (a fluorescent NO probe, Figure 6A). The specimen record of Figure 6B shows that the extract (1 mg/mL) and acetylcholine (ACh, 10−5 M) produced an increase in relative fluorescence, which indicates the generation of NO.

2.11. Vascular Effects of C. longifolium Under Potassium Channel Blockade

To assess the contribution of potassium channels to the effects reported in Figure 3, the same experiment was repeated following preincubation (20 min) with various potassium channel blockers. Representative traces are shown in Figure 6A, where the preparation was preincubated with the voltage-gated potassium channel (KV) blocker 4-aminopyridine (4-AP, 1 mM). Furthermore, the figure shows that C. longifolium elicited vasomotion comparable to that induced by ACh (1), even when 4-AP-sensitive Kv channels were blocked (2). The same protocol was used on preparations preincubated with glibenclamide (10 μM, ATP-sensitive potassium channel [KATP] inhibitor), tetraethylammonium (TEA, 1 mM, a non-selective KCa channel blocker) and BaCl2 (10 μM, inward rectifier potassium channel [KIR] blocker).
As shown in the averaged data (Figure 7B), both 4-AP and glibenclamide abolished the relaxant effect of C. longifolium at 1 μg/mL. When the concentration was increased to 10 μg/mL, glibenclamide no longer influenced the effect of C. longifolium, whereas 4-AP reduced its effect by 57% (control: 76 ± 3%; 4-AP: 33 ± 3%; p = 0.001). The relative IC50 for 4-AP was 2.79 ± 0.39 μg/mL and for glibenclamide was 2.43 ± 0.23 μg/mL, values significantly different (p = 0.001) from the control of 0.25 ± 0.03 μg/mL.
Figure 7C shows a significant (p < 0.05 at 10 μg/mL extract) inhibition of the extract-dependent relaxation when the aortic rings were preincubated with TEA compared to the control. By contrast, BaCl2 did not influence the vasorelaxant effect of C. longifolium. The relative IC50 for BaCl2 was 0.24 ± 0.15 μg/mL, and for TEA, it was 0.22 ± 0.19 μg/mL, values similar to the control. Interestingly, in the presence of all blockers, the contractile effect induced by 100 µg/mL C. longifolium was comparable to that observed in the control rings. However, in the presence of glibenclamide, the contractile component of this response was attenuated, thereby enhancing the relaxant effect at this concentration (control: 36 ± 11%; glibenclamide: 63 ± 1%; p < 0.05).

2.12. The Effects of C. longifolium on Sarcoplasmic Reticulum (SR) Ca2+ Content and Sarcolemmal Ca2+ Removal Pathways

Relative changes in VSMC sarcoplasmic reticulum (SR) Ca2+ content were estimated by measuring the amplitude of caffeine-evoked intracellular Ca2+ transients. The specimen records in Figure 8B show that preincubation with 1 μg/mL C. longifolium decreases this amplitude. On average (Figure 8C), C. longifolium reduced the amplitude of caffeine-evoked intracellular Ca2+ transients by 20% (control: 9.6 ± 0.4; C. longifolium: 7.6 ± 0.5; relative fluorescent units (F/F0); p = 0.023). Double exponential regression analysis was used to measure the rate constant of the fast and slow phases of the decay of caffeine-evoked Ca2+ transients, thus quantifying the activity of the sarcolemma Ca2+ removal pathways. On average (Figure 8D), C. longifolium increased the rate constant of the fast phase of decay by 27% (control: 0.192 ± 0.001; C. longifolium: 0.262 ± 0.005; s−1; p = 0.0001), while that of the slow phase was increased by 83% (control: 0.003 ± 0.001; C. longifolium: 0.018 ± 0.001; s−1; p = 0.0029).

2.13. Acute Toxicity Assessment of the Hydroalcoholic Extract of C. longifolium in Daphnia magna

To evaluate the potential toxic effects of C. longifolium, 24 h and 48 h exposure models were used to assess immobility in Daphnia magna. The Daphnia magna acute toxicity assay is a standardized 24 h to 48 h bioassay, according to OECD 202, that measures immobilization or mortality in neonates (less than 24 h old) to determine the EC50 (concentration that causes 50% of the effect) of chemicals, effluents, or environmental samples.
As shown in Figure 9A, under basal conditions (without extract), immobility was not affected after 24 h. Similarly, exposure to 100 μg/mL of C. longifolium did not significantly alter immobility (p = 0.5509). By contrast, higher concentrations of 150 μg/mL (40 ± 8.2%; p = 0.001) significantly increased immobility. After 48 h of exposure (Figure 9B), treatment with 100 μg/mL of the extract significantly increased immobility compared to the control (46.7 ± 2.4%; p = 0.0047). The highest concentrations, 150 μg/mL and 200 μg/mL, produced the greatest effects, with immobility values of 96.7 ± 2.4% (p = 0.001) and 100%, respectively. The 50% lethal concentration (LC50) of the hydroalcoholic extract of C. longifolium for D. magna was 115 µg/mL. These findings indicate that the hydroalcoholic extract of C. longifolium exerts a concentration- and time-dependent toxic effect in Daphnia magna, with marked lethality at concentrations above 150 μg/mL.

3. Discussion

The genus Calophyllum has attracted considerable attention due to its diverse pharmacological activities and traditional use in the treatment of hemorrhoids, varicose veins, and hypertension. Nevertheless, despite its wide distribution across South America, Calophyllum longifolium Willd. remains one of the least explored members of the genus, with no prior reports of systematic chemical characterization. In this context, the present work provides the first metabolomic profiling of C. longifolium using UHPLC-ESI-Q-TOF-MS, revealing the presence of multiple bioactive classes, including coumarins, xanthones, flavonoids, triterpenes, and phenolic acids. Furthermore, we demonstrate for the first time that C. longifolium induces a biphasic modulation of vascular tone in rat aortic rings, thereby adding a functional correlation to the phytochemical profile.
C. longifolium induced a clear biphasic vascular response, producing relaxation at low concentrations and contraction at higher concentrations. This phenomenon aligns with the concept of hormesis [34] and resembles the vascular actions reported for other phytomedicinal species such as Morus alba L. [35] and Acorus calamus [36]. An interesting case reported in the literature is the biphasic effect of Panax ginseng on blood pressure, characterized by an initial transient decrease followed by a sustained elevation [37]. This dual activity is attributed to the collective action of ginsenosides, rather than a single compound, which have been shown to stimulate α1-adrenergic receptors and enhance calcium influx in vascular smooth muscle [38].
Such evidence provides a useful framework for interpreting our findings with C. longifolium. Like ginseng, the biphasic vascular response observed with C. longifolium suggests that specific phytoconstituents within the extract may differentially modulate vascular tone in a concentration-dependent manner. In South America, several medicinal plants, such as Senecio nutans, Xenophyllum poposum, and Parastrephia quadrangularis, are traditionally used to alleviate altitude sickness, a condition often associated with elevated blood pressure.
Given its remarkable potency when compared with other South American medicinal plants, we further explored the mechanisms underlying the vasorelaxant activity of C. longifolium extract. The relaxing effects of the extract were evident in rat aorta precontracted with PE but not in preparations precontracted with KCl. This suggests that, like ACh, the extract is unable to counteract the strong membrane depolarization induced by extracellular K+. At low concentrations, the extract induced a characteristic relaxation accompanied by rhythmic oscillations in vascular tone, resembling the vasomotion typically triggered by acetylcholine. ACh produces relaxation in PE-precontracted aorta through endothelial NO-mediated hyperpolarization. Consistent with this pathway, our previous studies demonstrated that vasomotion depends on the endothelial NO signaling cascade [39], which suggests that this may be the mechanism by which C. longifolium extract produces relaxation at low concentrations. Supporting this, the vasorelaxant effect of the extract was completely abolished in endothelium-denuded aortas, as well as after preincubation with either the eNOS inhibitor L-NAME or the sGC inhibitor ODQ. Collectively, these observations confirm the involvement of the endothelial NO pathway in the vascular relaxation produced by C. longifolium. A putative model of this pathway is shown in Supplemental Material Figure S7.
Our results show that the inhibition of voltage-gated (Kᵥ) and ATP-sensitive (KATP) potassium channels abolished the relaxation induced by 1 µg/mL C. longifolium in rat aorta, which suggests that its vasorelaxant effect is at least partially dependent on these channels. Notably, 4-aminopyridine (4-AP), a Kᵥ channel blocker, significantly blunted the relaxation at 10 µg/mL, whereas glibenclamide, a KATP channel blocker, had no effect, which indicates that Kᵥ channels play a predominant role in mediating this response.
In support of this multifactorial action, our phytochemical analysis revealed that the hydroalcoholic extract of C. longifolium contains coumarins, xanthones, flavonoids, triterpenes, and phenolic acids. Compounds structurally related to coumarin–resveratrol hybrids, such as gut-70 derivatives (peak 15) identified in C. longifolium, have been associated with endothelium-dependent vasorelaxation through the stimulation of NO production and/or preservation of its bioavailability [40]. The mechanisms underlying this effect have been explored, with evidence pointing to the activation of sGC and the opening of K+ channels as key contributors [41]. Among the metabolites identified in the present study, asiatic acid has been shown to enhance vascular responses to ACh in the thoracic aorta and mesenteric vascular beds [42] through the restoration of eNOS protein expression and a subsequent increase in NO bioavailability [43]. Similarly, gallic acid induces relaxation of rat aortic rings via an endothelium-dependent mechanism involving eNOS phosphorylation and the subsequent opening of potassium channels, particularly KV channels [44]. Taken together, these findings suggest that C. longifolium-induced relaxation relies primarily on KV channel activation, although an additional contribution of the cAMP–PKA pathway cannot be excluded [45].
Notably, the contractile effect of C. longifolium observed at higher concentrations persisted despite the blockade of either Kᵥ or KATP channels, which indicates that this biphasic response is independent of potassium channel activity. Moreover, the myoendothelial hypothesis may partly account for the differential vasorelaxant responses of the extract under PE versus KCl stimulation. According to this hypothesis, the increase in intracellular Ca2+ in VSMC during PE-induced contraction can diffuse through myoendothelial gap junctions into the endothelium, where it triggers an anticontractile response via NO release [46]. Interestingly, tetrahydrogambogic acid (C38H49O8), a compound identified in C. longifolium in the present study, has been reported to inhibit gap junction communication [47], which could attenuate endothelial-mediated hyperpolarization and, thus, explain the contractile effect observed at high concentrations of the extract.
Preincubation of VSMCs with low concentrations of C. longifolium extract significantly decreased the amplitude of caffeine-evoked intracellular Ca2+ transients, indicating decreased SR Ca2+ content. Given the close relationship between SR Ca2+ content and cytosolic Ca2+ levels in VSMCs [48], this reduction likely contributes to the vasorelaxant effect of the extract. To elucidate the underlying mechanism, we measured the rate constant (RC) of decay of the caffeine-evoked Ca2+ transient, as this indicates the combined activities of the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA). In the presence of C. longifolium, the rate of Ca2+ decay was increased, indicating enhanced sarcolemma Ca2+ extrusion via these pathways and providing a mechanistic basis for the reduced SR Ca2+ content [49].
Finally, acute toxicity assays of the extract in Daphnia magna showed no evidence of general cytotoxicity at the lower concentrations (1–10 µg/mL) employed in our vascular experiments. This conclusion is supported by the fact that (1) these concentrations are well below the LC50 determined in the present study in D. magna (115 µg/mL) and the IC50 values reported by others (120 µg/mL) [50] and 2) the exposure times in our experiments were markedly shorter (20 min vs. 24–48 h), which would not be expected in a cytotoxic context. Importantly, however, we cannot exclude the possibility that the vascular contraction observed at 100 µg/mL may be attributable, at least in part, to cytotoxic effects [50].
We propose that the concentration of specific metabolites present in C. longifolium could underlie this hormetic effect. For instance, the dihydroflavonol derivative astilbin has been reported to promote endothelial cell proliferation and to significantly restore impaired vasculature in a zebrafish model of vascular insufficiency [51]. By contrast, gut-70, also identified in C. longifolium, has been shown to exert cytotoxic activity through G1 cell cycle arrest and the induction of apoptosis [10,52]. Moreover, the oligostilbene gnetin H has been described as a cytostatic agent via the inhibition of glycolysis, reduction of lactic acid synthesis, and suppression of thioredoxin-interacting protein expression [28]. Additionally, other compounds identified in C. longifolium, such as the clerodane-type diterpenoid caseagrewiifolin A (IC50 > 10 μM), the tigliane-type diterpene stelleracin A (IC50 = 4.7 μM), and the polyisoprenylated benzophenone derivative paucinone C (IC50 = 24.3 μM), have also been reported to exhibit cytotoxic effects [28,30,31].
Given the complexity of the signaling pathways involved and the different biological models employed, it is unlikely that a single mechanism fully accounts for the biphasic response [53], which warrants further investigation.

4. Conclusions

In summary, C. longifolium induces a biphasic vascular response. Relaxation produced by low concentrations is mediated by the vascular endothelial NO pathway, decreased SR Ca2+ content, and membrane repolarization through the activation of both Kᵥ and KATP channels. Higher concentrations produce contraction, and this may involve additional mechanisms that include cytotoxicity. Taken together, these findings suggest that extracts of C. longifolium are a promising source of vasoactive compounds.

5. Materials and Methods

5.1. Chemicals

Sephadex LH-20 was obtained from Pharmacia Fine Chemicals (Piscataway, NJ, USA); acetonitrile, methanol, hexane, and ethyl acetate from Merck (Santiago, Chile); and L-phenylephrine hydrochloride (PE), acetylcholine chloride (ACh), L-NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), tetraethylammonium (TEA), BaCl2, 4-aminopyridine (4-AP), and glibenclamide from Sigma-Aldrich (St. Louis, MO, USA).

5.2. Plant Material

The leaves (150 g) of Calophyllum longifolium Willd. were collected in June 2023 in the town of Puerto Almendras, district of San Juan Bautista, province of Maynas, department of Loreto, Iquitos Region, Peru (−3.8383° S, −73.3803° W, at 98 m.a.s.l.).
The plant was identified by Dr. Juan Celidonio Ruiz Macedo from the University of the Amazonía Peruana, Peru (herbarium for collection; voucher # CONC 062-2023).

5.3. Extraction Preparation

Fresh leaves were dried at room temperature and then ground into powder. A 50% hydroalcoholic reflux system using ethanol and water in a 1:1 ratio was employed to extract compounds from powdered plant material. After 2 h of extraction, the solution was vacuum-filtered and concentrated using a rotary evaporator (Heidolph, Schwabach, Germany). The resulting concentrate was then dissolved in water, then stored at −80 °C in a Shell Freezer (Labconco, Kansas City, MO, USA) for subsequent lyophilization (Millrock, NY, USA). The lyophilized extract was stored at 4 °C until it was ready for use. The extraction yield was 34.7%.

5.4. LC Parameters and MS Parameters

The separation and identification of the compounds present in the C. longifolium extracts were performed with UHPLC-ESI-Q-TOF-MS using an Ultimate 3000 RS and Bruker maXis ESI-QTOF-MS with the software Data Analysis 4.0 (all Bruker Daltonik GmbH, Bremen, Germany). A total of 5 mg of dry extract was dissolved in 2 mL of methanol ≥99.9% and filtered with a polytetrafluoroethylene (PTFE) filter. Then, 10 µL was injected into the chromatographic equipment, which consisted of an autosampler, a quaternary pump, a thermostatted column compartment and a photodiode (PDA) array detector. Elution was performed with a gradient system with eluent (A) being 0.1% formic acid in the water and eluent (B) being 0.1% formic acid in the acetonitrile and the gradient: 12% B isocratic (0–1 min), 12–99% B (1–15 min), 99% B isocratic (15–18 min), 99–12% B (18–18.20 min), and 12% B (18.20–20 min). Separation was carried out with a Thermo 5 µm C18 80 Å column (150 mm × 4.6 mm) at a flow rate of 0.3 mL/min. UHPLC-ESI-Q-TOF-MS experiments were recorded in negative and positive ion mode, and the scan range was between 100 and 1250 m/z. Electrospray ionization (ESI) conditions included a capillary temperature of 200 °C, a capillary voltage of 2.0 kV, a dry gas flow rate of 8 L/min and a nebulizer pressure of 2 bar. The experiments were performed in MS/MS mode. The structural characterization of all metabolites was based on HR full MS, fragmentation patterns, and comparisons with the literature data. The scan range was set between m/z 100 and 1250 using the ultra-scan mode with a mass scanning range of 26.000 m/z per second. MS1 parameters were as follows: voltage of high-voltage (HV) capillary −3500 V, HV end plate offset −500 V, trap drive 64.0, octopole Rf amplitude 0.0 Vpp, lens 1 −200.0 V, capillary exit −200.0 V, target mass 500, max. accumulation time 200.000 μs, ion charge control (ICC) target 100,000, and an average of four spectra. With a collision energy of 5 eV, the cone voltage was 20 V. Results were evaluated with the software Data Analysis 4.0 (Bruker Daltonics, Bremen, Germany).

5.5. Animals

Experiments used male Wistar rats (6–8 weeks old; 170–200 g). The animals were randomly assigned and kept in an environment with a temperature range of 22–25 °C. They were exposed to a 12 h light/dark cycle, with lights turning on at 8:00 AM and off at 8:00 PM. The animals had unrestricted access to both water and food (Champion, Santiago). The Animal Research Committee at Antofagasta University approved the experimental protocol (CEIC #275/2020).

5.6. Vascular Reactivity Experiments

The animals were euthanized via cervical dislocation. Aortic rings (2–3 mm) were prepared as previously described and then placed in a wire myograph containing Krebs–Ringer bicarbonate (KRB) solution (in mM), 4.2 KCl, 1.19 KH2PO4, 120 NaCl, 25 NaHCO3, 1.2 MgSO4, 1.3 CaCl2, and 5 D-glucose (pH 7.4, 37 °C, 95% O2 and 5% CO2). After a 30 min equilibration period, the aortic rings were stabilized through three successive submaximal contractions using KCl (60 mM), with each contraction lasting 10 min. The integrity of the vascular endothelium was assessed by applying 10−5 M acetylcholine (ACh) in aortic rings precontracted with phenylephrine (10−6 M). Rings that exhibited 70–80% relaxation to ACh were considered intact aorta. The passive tension applied to the aorta was set at 1.0 g.
Vascular contractile response was assessed by application of phenylephrine (PE) in the presence or absence of vasoactive substances that included nitric oxide synthase inhibitor (L-NAME; 100 μM), soluble guanyl cyclase inhibitor (1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one; ODQ; 1 μM), and potassium channel blockers tetraethylammonium (TEA; 1 mM), BaCl2 (10 μM), 4-aminopyridine (4-AP; 1 mM), and glibenclamide (10 μM).
In the vascular reactivity experiments, the concentrations of the C. longifolium extract ranged from 0.1 to 100 µg/mL These concentrations were selected to permit harmonization with our previous experiments and those of others and the existing scientific literature on Calophyllum brasiliense extract [54]. Concentrations of 0.1–10 µg/mL of the extract are physiologically relevant in cells and vascular tissue, so low values would not have toxic effects.

5.7. Isolation and Culture of Aortic VSMCs

The aorta was removed and cleaned using sterile PBS. Next, 1–2 mm fragments were cut then placed in an Eppendorf tube containing 1 mL of DMEM medium with 0.4% collagenase and 0.2% bovine serum albumin (BSA), then incubated at 37 °C in 5% CO2 for 4 h. After the incubation, the contents of the Eppendorf tube were washed twice with DMEM medium containing 20% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). During each wash, samples were centrifuged for 7 min at 1000 rpm to eliminate excess collagenase and, thus, preserve the tissue. To eliminate fibroblasts, the remaining content was incubated on plates for 20 min. After this, the medium was replaced with fresh DMEM containing 20% FBS and 1% antibiotics, then incubated for 5 days at 37 °C in 5% CO2. Following this, the culture medium was replaced with DMEM containing 10% FBS and 1% antibiotics (penicillin and streptomycin).

5.8. Nitric Oxide Measurements

Aortic ring sections (<1 mm) were preincubated with 5 μM 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) (Thermo Fisher Scientific, Waltham, MA, USA) in KRB for 10 min at 37 °C. Samples were then excited at 488 nm and emitted fluorescence measured at 515 nm.

5.9. Photometric Measurement of Intracellular Ca2+ and SR Ca2+ Content

Intracellular Ca2+ measurements were performed in cultured vascular smooth muscle cells (VSMCs) as previously described [53]. Briefly, cells were cultured on coverslips then pretreated with C. longifolium extract (1 μg/mL) or vehicle for 20 min after incubation with 10 µM Fluo-4 AM (Thermo Fisher Scientific) in Krebs–HEPES for 50 min at 37 °C. Krebs–HEPES included (in mM): 145 NaCl, 5 KCl, 2.6 CaCl2, 1 MgCl2, 10 HEPES, and 5.6 glucose. Cells were placed in a 1 mL chamber on the stage of a Carl Zeiss LSM-5 Pascal 5 Axiovert 200 microscope (Carl Zeiss® Göttingen, Germany). Cells were excited at 488 nm and emitted fluorescence measured at 527 nm. Images were collected every 500 ms and analyzed frame by frame using Carl Zeiss Zen 3.11 along with custom-written software. Relative changes in intracellular Ca2+ levels are expressed as F/F0, where F is point-in-time fluorescence and F0 is the initial fluorescence at 0 s.

5.10. Acute Toxicity Assessment

The juveniles of D. magna (cladocerans) were provided by the Toxicology Laboratory of Arturo Prat University. To evaluate acute toxicity, juveniles (n = 15) were exposed to control (no extract) or 50, 100, 150 or 200 µg/mL of the hydroalcoholic extract of C. longifolium leaves for a maximum period of 48 h. The bioassay was evaluated in triplicate following OECD 202 guidelines. Immobility and the median lethal concentration (LC50) were determined at 24 and 48 h.

5.11. Statistical Analysis

Statistical analysis was conducted with the average data expressed as mean values ± standard error of the mean. We utilized one-way ANOVA and two-way ANOVA, followed by Bonferroni’s post hoc test, for the statistical evaluations. Additionally, Student’s t test was employed. A p value of less than 0.01 was considered statistically significant. The software used was Graphpad Prism 8.0.2.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040594/s1, Full MS spectra of compounds identified in C. longifolium by UHPLC-ESI-Q-TOF-MS. Figure S1. Full MS spectra of peak 4. Figure S2. Full MS spectra of peak 6. Figure S3. Full MS spectra of peak 13. Figure S4. Full MS spectra of peak 14. Figure S5. Full MS spectra of peak 18. Figure S6. Full MS spectra of peak 38. Figure S7. Putative model of the biphasic effect of C. longifolium.

Author Contributions

Conceptualization: J.P., D.J.G. and D.A.-A.; Methodology: J.P., D.A., C.A.V.-Q., A.P., F.C., I.M.Q.-D. and F.R.L.-V.; Formal analysis: M.J.S., D.A., C.A.V.-Q. and G.V.A.-M.; Writing—original preparation: J.P., D.J.G. and D.A.-A.; Writing—review and editing: D.A.-A., J.B., G.V.A.-M., J.P., D.J.G., R.O.Y.-J. and I.M.Q.-D.; Resources: J.P., R.O.Y.-J. and M.J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Agencia Nacional de Investigación y Desarrollo (ANID): Fondecyt Chile 1200610 to JP. MS received funds from Fondecyt 1220075.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors thank José Leal and Samuel Hermosilla from Universidad Arturo Prat for their technical assistance in the experiments. We thank Fondequip EQM170172 and CONICYT–Fondequip EQM180120 for allowing us to use their equipment.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. UHPLC-ESI-Q-TOF-MS total ion chromatograms of C. longifolium. (A) Negative ionization mode and (B) positive ionization mode.
Figure 1. UHPLC-ESI-Q-TOF-MS total ion chromatograms of C. longifolium. (A) Negative ionization mode and (B) positive ionization mode.
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Figure 2. Structures of some specific coumarins identified in Calophyllum longifolium.
Figure 2. Structures of some specific coumarins identified in Calophyllum longifolium.
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Figure 3. The effects of C. longifolium on aortic rings. (A) Specimen original record showing the effects of 0.1–100 μg/mL C. longifolium on aortic rings precontracted with 10−6 M phenylephrine (PE). (B) Concentration-dependent relaxation (%) and (C) area under the concentration–response curve (AUC) to C. longifolium in intact (Endo; black) and endothelium-denuded (Denuded; blue) aortic rings. Vascular relaxation is expressed as a percentage of the maximum contraction induced by PE. Data expressed as mean ± SEM (n = 3–6 experiments). For panel B, a two-way ANOVA was conducted, followed by Bonferroni’s post hoc test. For panel (C), a t-Student test was performed. ** p < 0.01 vs. Endo.
Figure 3. The effects of C. longifolium on aortic rings. (A) Specimen original record showing the effects of 0.1–100 μg/mL C. longifolium on aortic rings precontracted with 10−6 M phenylephrine (PE). (B) Concentration-dependent relaxation (%) and (C) area under the concentration–response curve (AUC) to C. longifolium in intact (Endo; black) and endothelium-denuded (Denuded; blue) aortic rings. Vascular relaxation is expressed as a percentage of the maximum contraction induced by PE. Data expressed as mean ± SEM (n = 3–6 experiments). For panel B, a two-way ANOVA was conducted, followed by Bonferroni’s post hoc test. For panel (C), a t-Student test was performed. ** p < 0.01 vs. Endo.
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Figure 4. The effects of C. longifolium on depolarized aortic rings. (A) Original recording showing the effects of 0.1–10 μg/mL C. longifolium on aortic rings precontracted 60 mM KCl. (B) Mean concentration-dependent relaxation response to C. longifolium in intact (E+) aortic rings. The vascular relaxation of the extract was expressed as a percentage of the maximum contraction induced by KCl. Data expressed as mean ± SEM (n = 5 experiments).
Figure 4. The effects of C. longifolium on depolarized aortic rings. (A) Original recording showing the effects of 0.1–10 μg/mL C. longifolium on aortic rings precontracted 60 mM KCl. (B) Mean concentration-dependent relaxation response to C. longifolium in intact (E+) aortic rings. The vascular relaxation of the extract was expressed as a percentage of the maximum contraction induced by KCl. Data expressed as mean ± SEM (n = 5 experiments).
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Figure 5. Endothelium-dependent effects of C. longifolium on aortic rings. (A) Representative traces showing the relaxant effects of C. longifolium (0.1–100 μg/mL) on endothelium-intact aortic rings (black), compared with preparations preincubated with L-NAME (100 μM; red) or ODQ (1 μM; orange). (B) Mean concentration-dependent relaxation response and (C) area under the concentration–response curve (AUC) to C. longifolium in intact (Endo), L-NAME-treated, or ODQ-treated aortic rings. Data expressed as mean ± SEM (n = 3–6 experiments). ** p < 0.01 vs. Endo.
Figure 5. Endothelium-dependent effects of C. longifolium on aortic rings. (A) Representative traces showing the relaxant effects of C. longifolium (0.1–100 μg/mL) on endothelium-intact aortic rings (black), compared with preparations preincubated with L-NAME (100 μM; red) or ODQ (1 μM; orange). (B) Mean concentration-dependent relaxation response and (C) area under the concentration–response curve (AUC) to C. longifolium in intact (Endo), L-NAME-treated, or ODQ-treated aortic rings. Data expressed as mean ± SEM (n = 3–6 experiments). ** p < 0.01 vs. Endo.
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Figure 6. The effect of C. longifolium on NO generation. (A) Micrograph (20×) of rat aorta sections. (B) Mean relative DAF-FM DA fluorescence response produced by C. longifolium (1 mg/mL) and acetylcholine (ACh; 10−5 M), n = 4.
Figure 6. The effect of C. longifolium on NO generation. (A) Micrograph (20×) of rat aorta sections. (B) Mean relative DAF-FM DA fluorescence response produced by C. longifolium (1 mg/mL) and acetylcholine (ACh; 10−5 M), n = 4.
Plants 15 00594 g006
Plants 15 00594 g007
Figure 7. The effect of C. longifolium on aortic rings following potassium channel inhibition. (A) Representative traces showing the relaxant effects of C. longifolium (0.1–100 μg/mL) in aortic rings preincubated with the voltage-gated potassium channel (KV) blocker 4-aminopyridine (4-AP, 1 mM). Interestingly, C. longifolium elicited vasomotion comparable to that induced by ACh (1), even in the presence of 4-AP (2). (B) Concentration–response curves comparing C. longifolium-induced relaxation in endothelium-intact rings (Endo) and those treated with either 4-AP or glibenclamide (10 µM; a KATP channel blocker). (C) Concentration–response curves comparing relaxation in Endo rings and those treated with tetraethylammonium (TEA, 1 mM; a non-selective KCa channel blocker) or BaCl2 (10 µM; a KIR channel blocker). Data shows mean ± SEM values (n = 5 experiments). ** p < 0.01 control vs. a given blocker.
Figure 7. The effect of C. longifolium on aortic rings following potassium channel inhibition. (A) Representative traces showing the relaxant effects of C. longifolium (0.1–100 μg/mL) in aortic rings preincubated with the voltage-gated potassium channel (KV) blocker 4-aminopyridine (4-AP, 1 mM). Interestingly, C. longifolium elicited vasomotion comparable to that induced by ACh (1), even in the presence of 4-AP (2). (B) Concentration–response curves comparing C. longifolium-induced relaxation in endothelium-intact rings (Endo) and those treated with either 4-AP or glibenclamide (10 µM; a KATP channel blocker). (C) Concentration–response curves comparing relaxation in Endo rings and those treated with tetraethylammonium (TEA, 1 mM; a non-selective KCa channel blocker) or BaCl2 (10 µM; a KIR channel blocker). Data shows mean ± SEM values (n = 5 experiments). ** p < 0.01 control vs. a given blocker.
Plants 15 00594 g007
Plants 15 00594 g008
Figure 8. The effects of C. longifolium extract on SR Ca2+ content and Ca2+ removal pathways in rat aortic vascular smooth muscle cells (VSMCs). (A) Specimen caffeine-evoked Ca2+ transients in control (Krebs–HEPES buffer as vehicle, black) and (B) following incubation with 1 μg/mL C. longifolium (blue). (C) Mean caffeine-evoked Ca2+ transient amplitude. (D) Mean rate constant of decay of the fast and slow phases of caffeine-evoked Ca2+ decay. Data expressed as mean ± SEM. n = 15 cells; ** p < 0.01 versus control.
Figure 8. The effects of C. longifolium extract on SR Ca2+ content and Ca2+ removal pathways in rat aortic vascular smooth muscle cells (VSMCs). (A) Specimen caffeine-evoked Ca2+ transients in control (Krebs–HEPES buffer as vehicle, black) and (B) following incubation with 1 μg/mL C. longifolium (blue). (C) Mean caffeine-evoked Ca2+ transient amplitude. (D) Mean rate constant of decay of the fast and slow phases of caffeine-evoked Ca2+ decay. Data expressed as mean ± SEM. n = 15 cells; ** p < 0.01 versus control.
Plants 15 00594 g008
Plants 15 00594 g009
Figure 9. The acute toxic effects of C. longifolium in Daphnia magna. Mean concentration-dependent toxicity, expressed as percentage immobility after (A) 24 h and (B) 48 h of exposure. Data are expressed as mean ± SEM (n = 4). ** p < 0.01.
Figure 9. The acute toxic effects of C. longifolium in Daphnia magna. Mean concentration-dependent toxicity, expressed as percentage immobility after (A) 24 h and (B) 48 h of exposure. Data are expressed as mean ± SEM (n = 4). ** p < 0.01.
Plants 15 00594 g009
Table 1. Metabolite profiling of C. longifolium by high-resolution UHPLC-ESI-Q-TOF-MS.
Table 1. Metabolite profiling of C. longifolium by high-resolution UHPLC-ESI-Q-TOF-MS.
PeakTentative
Identification		[M − H]− or [M + H]+ Retention Time (min)Theoretical Mass (m/z)Measured Mass (m/z)Accuracy (ppm)MS/MS fragments (m/z)Reference
1Sodium formate (internal standard) COOHNaCOO-C4H2O40.1112.9829112.98563.1 -
2Gallic acidC7H6O50.5169.0592169.0574−13.39151.0496[2]
3AstilbinC21H21O113.02449.1532449.1537-347.1418, 301.0708,
283.2533, 195.0892,
425.0534		[2]
4Calanolide derivative FC21H23O66.6371.1500371.1341−12.6327.1066[2]
5Methyl β-D-mannopyranosyl-(1→4)-β-d-xylopyranosideC12H21O107.0325.1126325.1140−4.25359.0859, 279.1001N.D
6Gut-70 derivativeC24H25O67.1409.1732409.16564.07325.1128[9]
7UnknownC27H45O107.2577.3143577.2866−26299.0849-
83-Hydroxy-3’,4’,5’-trimethoxyflavoneC29H47O107.6327.0859327.0832−2.7481.2993,
293.2536		[11]
9Asiatic acidC30H49O57.7489.3512489.35857.2413.2407, 297.2847[12]
10LepidolideC34H47O67.8551.3378551.3317−11.06409.17072,[13]
117-(2,4-Dihydroxypentyl)-15-(ethoxymethyl)-2,5,8,11,14-pentaoxaicosane-17,19-diol C27H47O108.10483.3128483.3138−5.9439.1614, 255.2744[14]
12MangostanolC24H25O78.27425.1605425.1627−5.9 [15]
13Grandidentatin C21H27O98.31423.1660423.1638−5.33847.3080 (2M-H), 409.1703, 356.1271[16]
14Gut-70 derivative 2 C24H25O58.57393.1744393.1707−5.47787.3248, 323.1341, 257.1576[9]
15Gut-70 derivative 3C24H25O68.76409.1682409.16566.29325.1128[9]
16Tsugarioside BC37H60O78.82617.4309617.4270−6.3453.3790, 395.1898[17]
171-Hexanol arabinosylglucosideC17H30O108.9395.1902395.1864−9.7281.0855[18]
18Grandidentatin C21H27O99.0423.1660423.1615−5.33409.1703, 356.1271[18]
19Unknown C33H61O109.2633.4219633.4457−23.7587.4387-
20Tajixanthone hydrateC25H27O79.4439.1973439.1955−4.12381.1164, 901.3562 (2M-H)[19]
21Hydroxy acetoxystearic acidC20H37O59.5357.2464357.248046.7245.1964N.D
22HelleboreinC37H55O1810.0787.3394787.3232−43393.1744[20]
23Cimiracemoside HC37H57O1110.3677.3906677.4004−14.5415.1954[21]
24mulberrofuran X C29H33O510.5463.2527463.270137.6377.1801[22]
25Charantoside IiiC36H57O710.6601.4335602.4251−1.9437.3698[23]
26GrandidentatinC21H27O910.7423.1640423.1602−2.1345.1155[24]
27UnknownC43H47O510.8643.3491643.3429−43615.4585, 473.3549[25]
28Methyl 6-palmitoyl-beta-D-glucopyranoside C23H43O710.9431.2837431.3014−43343.2888, N.D
29PaxidalC24H25O410.7377.2580377.1793−9.1321.1180[26]
30Cotylenin FC33H55O1111.3627.3519627.355036.7423.3618[27]
31Caseagrewiifolin AC37H49O1012.4653.3440653.3320−20.5593.3146[28]
32UnknownC32H51O1012.5595.3492595.3488−0.7453.2463-
333-O-Vanilloylepiceanothic acidC32H51O1012.6637.3495637.373524.0559.3115,
184.0406		[29]
34Paucinone C C37H49O912.7637.3490637.3371−12.5559.3110[30]
35Stelleracin AC37H49O813.1621.3522621.3531−161241.6844[31]
36Stelleracin A isomerC37H49O813.4621.3537621.3531−18.21241.6844[31]
37Tetrahydrogambogic AcidC38H49O813.7635.3686635.3538−23.3561.3264[32]
38Dryocrassin ABBAC43H49O1613.8821.3593821.36823.1783.2931, 459.2211, 393.1740[33]
39Unknown C56H87O713.9871.6441871.64780.6593.3254, 533.2912-
40Unknown C55H82O814.9871.6323871.665829593.3129-
N.D: No data.
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Aravena, D.; Palacios, J.; Véliz-Quezada, C.A.; Aguirre-Martínez, G.V.; Benites, J.; Greensmith, D.J.; Paredes, A.; Cifuentes, F.; Simirgiotis, M.J.; León-Vargas, F.R.; et al. Chemical Profiling and Vascular Effects of a Hydroalcoholic Extract of Calophyllum longifolium Willd: Role of Nitric Oxide and Potassium Channels. Plants 2026, 15, 594. https://doi.org/10.3390/plants15040594

AMA Style

Aravena D, Palacios J, Véliz-Quezada CA, Aguirre-Martínez GV, Benites J, Greensmith DJ, Paredes A, Cifuentes F, Simirgiotis MJ, León-Vargas FR, et al. Chemical Profiling and Vascular Effects of a Hydroalcoholic Extract of Calophyllum longifolium Willd: Role of Nitric Oxide and Potassium Channels. Plants. 2026; 15(4):594. https://doi.org/10.3390/plants15040594

Chicago/Turabian Style

Aravena, Diego, Javier Palacios, Cristián A. Véliz-Quezada, Gabriela V. Aguirre-Martínez, Julio Benites, David J. Greensmith, Adrián Paredes, Fredi Cifuentes, Mario J. Simirgiotis, Frank R. León-Vargas, and et al. 2026. "Chemical Profiling and Vascular Effects of a Hydroalcoholic Extract of Calophyllum longifolium Willd: Role of Nitric Oxide and Potassium Channels" Plants 15, no. 4: 594. https://doi.org/10.3390/plants15040594

APA Style

Aravena, D., Palacios, J., Véliz-Quezada, C. A., Aguirre-Martínez, G. V., Benites, J., Greensmith, D. J., Paredes, A., Cifuentes, F., Simirgiotis, M. J., León-Vargas, F. R., Asunción-Alvarez, D., Quispe-Díaz, I. M., & Ybañez-Julca, R. O. (2026). Chemical Profiling and Vascular Effects of a Hydroalcoholic Extract of Calophyllum longifolium Willd: Role of Nitric Oxide and Potassium Channels. Plants, 15(4), 594. https://doi.org/10.3390/plants15040594

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