Vasorelaxation endothelium-independent of the ethyl acetate phase from aerial parts of Solanum paludosum Moric. envolves channels-calcium L-type blockade

Previous results showed the ethyl acetate phase (SP-AcOEt), obtained from aerial parts of Solanum paludosum, relaxed the aorta isolated in the endothelium-dependent and -independent manner. The vasorelaxant effects of SPAcOEt was not characterized on aorta rings endothelium-denuded, thus this work aimed to elucidate the mechanisms endothelium-independent vasorelaxation on rat isolated aorta. The aorta was isolated from Wistar rats and mounted in glass baths containing 6 mL of normal Krebs physiological solution with pH at 7.4. The preparation was maintained at 37oC and bubbled continuously with a mixture of 95% O2 and 5% CO2. Aortic rings were maintained for 1 hour by a resting tension of 1g and next were contracted with phenylephrine after the sustained contraction ACh was added to access the integrity of the endothelium. SP-AcOEt relaxed pre-contracted aorta by KCl-30mM or -80mM in a similar manner, suggesting blockade CaV, but not channel-K participating. SP-AcOEt also inhibited the contraction induced by CaCl2 and relaxed pre-contracted aorta by (±)-BayK8644 (EC50 = 16.9±1.3 μg/mL), which confirms the involvement of L-type CaV blockade. SP-AcOEt presented vasorelaxation endothelium-independent that involves Ltype CaV blockade.


Introduction
The cardiovascular diseases have been the main causes of death, hospitalization and outpatient care in the world, including developing countries, as such Brazil (GBD, 2016). The medicinal plants are used, mainly, to treat hypertension (Zago et al., 2020). Thus, the researching for newer treatments is an ongoing process and plants may be a significant source of medicines.
Previous result our showed SP-AcOEt presents endothelium-dependent and independent vasorelaxation. Therefore, we investigated the endothelium-independent vasorelaxation mechanisms of the SP-AcOEt obtained from aerial parts of Solanum paludosum on rat aorta.

Methodology
This research is quantitative and experimental (Pereira et al., 2018), preclinical (Oliveira et al, 2019). All experiments are submitted and approved by Animal Experimentation Ethical Committee (CEPA) of Universidade Federal da Paraíba.

Plant material and extraction
The isolating was performed by Silva et al. (2002). Briefly, the aerial parts of the plant were collected in Brazil, State of Paraíba, municipality of João Pessoa, in summer of January 1999. Voucher specimens (M.F. Agra et al. 5257) are deposited at the Herbarium by Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraíba. The powdered aerial parts of Solanum paludosum were extracted with EtOH at room temperature. The extract was concentrated under vacuum. The crude residue was dissolved in H2O/AcOH (8:2) and extracted with benzene/ether (1:1). These solvents were removed, the residue was dissolved in MeOH/H2O (8:2) and extracted with hexane (3x) and then with AcOEt (3x), obtained the AcOEt phase (SP-AcOEt).

Preparation of rat aorta rings
Male Wistar rats (Rattus novergicus) weighting 200-350 g were euthanized by cerebral concussion. The aorta was cut into rings with width of about 3-5 mm in width and mounted in glass baths containing 6 mL of normal Krebs physiological solution with the following composition (mM): NaCl 118.0, KCl 4.6, CaCl2.2H2O 2.5, KH2PO4 1.1, MgSO4 5.7, glucose 11.0, NaHCO3 25.0. The pH was adjusted at 7.4. The nutritive solution was maintained at 37ºC and bubbled continuously with a mixture of 95% O2 and 5% CO2. Each ring had two metallic hooks inserted through the lumen; one was anchored to the glass bath and the other was attached to an isometric force transducer (FORT-10) connected to an amplifier (TBM-4M). Both equipaments are from World Precision Instruments (EUA) and are coupled to a data-acquisition system BioMed (BioData, Brazil) for recording isometric contractile response. Aortic rings were maintained for 1 hour by a resting tension of 1g and rinsed 4 times with Krebs solution. Followed, aortic rings were contracted with Phe (0.3 M) after the sustained contraction ACh (1 M) was added to access the integrity of the endothelium (Furchgot & Zawadki, 1980). The rings with more than 50% relaxation to ACh were considered to be endothelium-functional (Ajay et al., 2003).

Characterization of the endothelium-independent vasorelaxation mechanism
In order to investigate the endothelium-independent mechanism involving the vasorelaxation of SP-AcOEt, we used aorta rings without functional endothelium. To observe whether K + -channel or CaV-channels were involved in relaxant response, we accessed the effects of SP-AcOEt on contraction induced by high K + solution (KCl-30 or -80 mM).
Concentration-response curves were plotted as % relaxation against logarithmic concentration of plant products and the concentration producing a half-maximal response (EC50) values were calculated by non-linear regression. To confirm the involvement of CaV-channels, contractions induced by Ca +2 were stimulated gradually by adding CaCl2 (10 -7 -10 -1 M) to the high K + Krebs solution without Ca 2+ , as a control reference. After the aorta rings were washed three times, during 30 minutes, with high-K + Krebs solution without Ca 2+ , the addition of SP-AcOEt (9; 27 and 81 g/mL) to the glass bath followed 15 minutes the cumulative Ca 2+ -induced contractions were repeated. Once again, the effects of SP-AcOEt and flavonoids on ()-BayK8644-(0.3 M) induced contractions (on dimmer light) were observed (Schramm et al., 1983). The contraction rate was expressed as a percentage of the maximum tension obtained in the control reference.

Statistical analysis
All results were expressed as mean  SEM and were statistically analyzed using the T-test or ANOVA following Bonferroni's test. In all cases, statistical differences were considered significant only if the 'P' value was less than 0.05 (<0.05). The responses were computer-fitted to a sigmoid curve using non-linear regression (GraphPad Prism version 3.02, USA.) and EC50 were calculated.
The SP-AcOEt was five times more potent to relax KCl-80 than rings were pre-contracted with phenylephrine (data not shown). Those results match the ones reported by Sakata & Karaki (1991) in vascular smooth muscle, which demonstrated blockade of CaV-channels are more potent for relaxing response on high-K + than norephinephrine. Therefore, all these results indicate the vasorelaxant action of SP-AcOEt could be due to blockade Ca 2+ -influx through CaV-channels. Channels-Ca 2+ represent the main route for Ca 2+ translocation across the plasma membrane and support several functions, including muscle contraction (Fusi et al., 2017). Although various families channels-Ca 2+ are expressed in cardiovascular myocytes, CaV-channels constitute the dominant Ca 2+ -influx route (Catterall et al., 2020). The confirmation of CaV-channels blockade by SP-AcOEt (9; 27 or 81 g/mL) was observed in the inhibition curves induced by CaCl2 in high-K + and without Ca 2+ Krebs. The curves induced by CaCl2 were right shifted in a non-parallel manner and decreased the maximal effect (Emax = 78.75.5; 52.38.3; 22.65.6 %, respectively) ( Figure 2). The fraction of total flavonoids of Elsholtzia splendens inhibited also Ca 2+ -induced contraction in endothelium-denuded aortic rings (Wang et al., 2014). Another evidence was that SP-AcOEt (EC50 = 16.91.3 g/mL) relaxed pre-contracted rings by ()-BayK8644 (Fig.   3), an activator of L-type CaV-channels (Schramm et al., 1983), and the EC50 value did not show a significant difference compared to KCl-80mM. L-type CaV-channels have long been considered the primary route of Ca 2+ entry in vascular smooth muscle (Ghosh et al., 2017). Thus, all data strongly corroborate the involvement of L-type CaV-channels blockade in this endothelium-denuded vasorelaxant mechanism of SP-AcOEt. The chemical analyses from SP-AcOEt showed flavonoids and alkamide (Silva et al., 2002), maybe those flavonoids had induced blockade the CaV-channels. Because flavonoids and phenol compounds have been presented inhibiting contractions on smooth and cardiac muscles by CaV-channels blockade (Vourela et al., 1997;Penso et al., 2014;Fusi et al., 2017) and flavones inhibited contractions induced by Ca 2+ (Lin et al., 1997;De Rojas et al., 1999;Tew et al., 2020;Migkos et al., 2020). The calcium-blockers decrease Ca 2+ -level on vascular smooth muscle, resulting vascular tonus relaxation and decreasing blood pressure (Kochegarov, 2003), and are include medicines, frequently, prescribe for treatment of hypertension

Conclusion
The results of the present study make us to conclude the phase SP-AcOEt from aerial parts of Solanum paludosum presents vasorelaxation endothelium-independent that involves, mainly, blockade of L-type channels-CaV.
In the future, it is intended to determine whether isolated flavonoids promote those vasorelaxation effects and to design its molecular docking.