ACh (acetylcholine), PE (phenyl ephedrine), DMSO (dimethyl sulfoxide), and DPPH (2,2-diphenyl-1-picrylhydrazyl) were acquired from Sigma–Aldrich (Dorset, UK); DAF-FM (4-amino-5-methylamino-2’,7’-difluorofluorescein di-acetate) was acquired from Molecular Probes (New York, NY, USA). Deionized-Ultrapure H2O was utilized as solvent except for DPPH and natural metabolites, where DMSO (conc. not exceeding 0.1%) was utilized.

Seven-week-old male Wistar rats (180–200 g) were used (King Fahd Medical Research Center, KAU, KSA). The animals were housed with access to standard rodent pellets and purified water in clear polypropylene cages (4 rats each). Constant housing conditions were applied, including alternating 12 h light and dark, 22 ± 3 °C temperature, sufficient ventilation, and 50–60% relative humidity. The research ethics committee of King Abdulaziz University approved the study (approval number 126-1439). The study was carried out according to the Saudi Arabia Research Bioethics Guidelines and Regulations, which are in accordance with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines for research involving animals [15]. The animals were executed by decapitation using a rodent guillotine administered by qualified personnel in the animal housing. The method is acceptable to induce a rapid loss of consciousness, according to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition (section M3.7) [16], and the descending thoracic aorta was precisely removed and washed from connective tissues and fats.

Vasodilating capacities were assessed using the isolated artery method, as formerly reported [17,18]. Briefly, the aorta was removed, cleansed of any connective tissue and fats, and sliced into rings (3 mm). Each ring was hung in Krebs Henseleit buffer channels (4.8 mM KCl, 118 mM NaCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 11.1 mM glucose, and 25 mM NaHCO3) at 37 °C, with continuous aeration with gas (5% CO2 and 95% O2). Every 30 min, the channel buffer solution was exchanged. Quantification of the aortic tension was accomplished using an isometric force transducer, and the results were presented through a PowerLab data interface module linked to a PC running Chart software v8 (ADI Instruments).

The aortic rings were set aside for 30 min for equilibration at a 1500 mg ± 50 resting tension. Initial aorta contraction and relaxation were then carried out by the addition of PE, followed by ACh (both at 10 μM). After the tension was reverted to the rest state, accumulative concentrations of 1–10 μg/mL and 1–10 μM for the chloroform fraction or the pure metabolites, respectively, were added to the organ bath precontracted (PE, 10 μM)-isolated aortae. Tension reduction was estimated as a measurement of vasodilating actions. In other sets of experiments for investigating the role of the endothelium in the vasodilating effect, it was mechanically made bare. Additionally, L-NAME (100 μM) was added in the organ bath 15 min before adding the isolated metabolites or chloroform fraction to explore the role of nitric oxide in the vasodilating influence on different sets of experiments.

The separated aorta intracellular induction of NO production by the tested metabolites and the chloroform fraction was examined employing the DAF-FM fluorescence probe, as similarly outlined in our former work [19]. Similar to the earlier technique, the thoracic aorta was removed, the fats were washed off, and it was sliced into approximately 6 mm pieces. Every piece was put in a 96-well black plate separate well, maintained in dim light, having made a 2.5 µM DAF-FM/KHB mixture (37 °C) immediately before starting the procedure; 100 µL were accurately drawn and transported to the wells’ neighboring column; after that, ACh (10 µM) was included in one of the aortic segments, and the chloroform fraction (10 µg/mL) or separated metabolites (10 μM) was put in the other wells after 3 min. Again, after 3 min, 100 µL were taken from the wells with the aortae and transmitted to the nearby well columns. For the blank, a row without an aorta was retained for each ACh concentration, which was handled identically. The withdrawn volumes’ fluorescence intensity (and not the column including the aortae) was then assessed at λem = 515 nm and λex = 485 nm using a SpectraMax® M3 Monochromator plate reader.

ROS scavenging potential was assessed, as formerly stated in previous work from our laboratories [20]. In a 96-well clear plate, the pure metabolites (1–10 µM) or Fraction I (1–10 µg/mL) in MeOH was added to a DPPH (240 µM) solution in MeOH/tris (1:1 v/v). For the control (C), MeOH was utilized instead of the fraction or metabolites. DPPH was directly prepared before adding to the plate. The absorbance was estimated every minute at 520 nm for 10 min using a SpectraMax® M3-Monochromator plate reader.

Experimental values are depicted as the mean ± standard error of the mean (SEM). For statistical analysis, one- or two-way ANOVA (analysis of variance) was applied, as designated in the figure legends, succeeded by Dunnett’s posthoc test utilizing GraphPad Instat software version 5. If p < 0.05, the differences were recognized as significant.

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