Vasodilator effect of ethanolic extracts of Passiflora vitifolia and Passiflora edulis f. edulis seeds

Hypertension (HTA) is one of the primary risk factors in cardiovascular diseases and is considered one of the main causes of morbidity and mortality worldwide. Therapeutic alternatives focused on natural products and phytocompounds obtain more interest in the treatment of this type of disease, and species of Passiflora gender have presented scientific interest due to their chemical composition and the different bioactivities that their compounds have reported. This study aimed to evaluate the antihypertensive potential of extracts obtained from seeds of Passiflora vitifolia and Passiflora edulis f. edulis; for this, extraction was carried out by maceration with 97% ethanol of the seed flour of the two species; the extracts were characterized by qualitative and quantitative tests and UHPLC. Its cytotoxicity, in vivo antihypertensive potential, vasodilator effect, and possible mechanisms of action in vitro models were evaluated. The extracts did not show apparent toxicity, but showed a preventive effect of HTA, inhibited angiotensin-II (AT-II) contraction, and presented relaxation of contracted aortic rings with phenylephrine and KCl, and this was partially reversed in the presence of L-NAME. The results highlight the potential of these Passifloras as a source of extracts with antihypertensive capacity, having as possible mechanisms of action the synthesis of NO and inhibition or antagonism of AT-II.


INTRODUCTION
Cardiovascular diseases (CVDs) have gained a relevant role worldwide by becoming the leading cause of morbidity and mortality, generating around 17.7 million deaths in 2015, which represents 31% of all deaths recorded in the world for that year (World Health Organization, 2015). Among CVDs, arterial hypertension (HTA) stands out, which is a chronic disease that generates alterations in the organism (damage to arteries, brain, heart, eyes, kidneys, sexual dysfunction, and others) and causes 49% of related deaths with CVD. At present, HTA is controlled by the prolonged use of drugs with only one pharmacological target (angiotensin-converting enzyme inhibitors, β-blockers, diuretics, calcium blockers, ARA II, and others), which generate secondary short and long alterations, causing deterioration in the quality of life of the patients (Brown and Bussell, 2011;Piepoli et al., 2016;Rojas, 2009;Tedla and Bautista, 2016), what makes it essential to search for new alternatives such as the use of phytotherapy, diets, supplements, and functional foods that prevent condition and control symptoms without serious side effects (Mantovani et al., 2017;Shouk et al., 2014).In this regard, plants and fruits such as those belonging to the Passifloraceae family, with numerous species, of which various secondary metabolites have been reported among them, alkaloids (harman, harmine, harmalol, and harmaline), flavonoids (vitexin, isovitexin, orientin, vicenin, and isoorientin), saponins (passiflorin and quadranguloside), cyanogenic compounds (passicoriacin and epipassicoriacin), and terpenes (Alvarez et al., 2019;Alves et al., 2020;Ballesteros-Vivas et al., 2019;Ingale and Kasture, 2014;Viganó et al., 2020;Wosch et al., 2015; have presented different biological activities such as sedative, antihypertensive, antiparkinsonian, antispasmodic, antioxidant, and antiproliferative (Aguillón et al., 2013;Alves et al., 2020;Ballesteros-Vivas et al., 2019;Contreras et al., 2011;Dhawan et al., 2004;Ingale and Kasture, 2014;Ocampo et al., 2010;Pinzón et al., 2007;Rudnicki et al., 2007;Viganó et al., 2020). Colombia is recognized worldwide as the center of diversity of Passiflora and the country with the highest number of records, 172 species (58 endemic); among them, Passiflora vitifolia Kunth (gulupa de monte) and Passiflora edulis f. edulis Sims (gulupa) are studied. They are part of wild and cultivated species, of which their possible bioactivities and chemical composition are not fully known, especially their agroindustrial waste (80% of the fresh fruit).
Therefore, this work focused on characterizing chemically and biologically extracts obtained from P. vitifolia and P. edulis f. edulis, evaluating its cytotoxicity, relaxing capacity, vasodilator potential in vivo and in vitro, and possible mechanisms of antihypertensive action.

Preparation of extract
The seeds were dried in an air circulation oven at 45°C ± 2°C and ground, obtaining a flour that was degreased. Ethanolic extracts were obtained by maceration of the flour and degreased with 97% ethanol; the mixture was vacuum filtered to obtain the crude extract, which was dried under reduced pressure and stored at −20°C (Jiménez et al., 2013); preliminary phytochemical characterization was carried out using colorimetric tests (Dominguez, 1973;Murillo and Méndez, 2012).

Determination of total phenolic compounds and tannins
Total phenolic and tannin contents of extracts were determined according to the method described by Viganó et al. (2020) with some modifications. 50 µl of each solution was mixed with 800 µl of water and 50 µl of 15% Na 2 CO 3 and was stirred and allowed to stand for 8 minutes. Then, 100 µl of Folin-Ciocalteu reagent was added. After 2 hours, absorbance was measured by a microplate lector (Multiskan™ GO Thermo scientific™) at 725 nm. The results were expressed as gallic acid equivalents (g.GAE/100 g sample). In tannins, previously, the sample was incubated with polyvinylpyrrolidone (PVPP 100 mg/ ml of sample) at 4°C/4 hours. The mixture was centrifuged at 3,000 rpm for 10 minutes and the supernatant was determined by the method described above. The results were expressed as tannic acid equivalents (g.TAE/100 g sample) (Da Silva et al., 2019).

DETERMINATION OF FLAVONOIDS
Flavonoids in the extracts were determined according to the method described by Karapetsas et al. (2019) with some modifications. 1 ml of each solution was mixed with 150 μl of 5% sodium nitrite and incubated for 5 minutes at room temperature. 150 μl of 10% AlCl 3 ethanolic solution and 2 ml of 4% sodium hydroxide were added; the solution was gauged to 5 ml with distilled water and incubated for 15 minutes at room temperature. Absorbance was measured by a microplate spectrophotometer (Multiskan™ GO; Thermo Scientific™) at 510 nm. Total flavonoid content was calculated as equivalents of g Quercetin (g.QE/100 g sample).

Extract characterization (HPLC-UV)
This was carried out using high-performance liquid chromatographic analysis on a Waters Alliance 2,695 separation module system, coupled to a dual-channel (320 and 280 nm) absorbance k detector, and a Waters Atlantis dC18 column (5 µm; 2.1 × 150 mm) using a gradient system with the conditions used by Delpino-Rius et al. (2015). The following metabolites in the extracts were quantified: chlorogenic acid, caffeic acid, coumaric acid, epicatechin, catechin, gallic acid, isoquercetin, quercetin, rutin, synaptic acid, vitexin, isovitexin, orientin, and isoorientin; by means of the elaboration of calibration curves with standards for their correct quantification.

Acute toxicity study
The acute toxicity test of extracts was carried out according to the Organization of Economic Cooperation and Development (OECD) guidelines 423 (Schlede et al., 2005). Albino mice (n = 6) were treated orally with the extracts prepared in the vehicle [polyethylene glycol:glycerol:Milli-Q water (10:10:80)] control group. The fixed-dose (2,000 mg/kg) method OECD guidelines 423 specified by CPCSEA were implemented for toxicity studies. After the administration of extracts, animals were observed for toxic effect, if any, for the first 4 hours and once daily for 15 days (Saleem et al., 2017).

Experimental animals
The Bioethics Committee of the Faculty of Sciences, National University of Colombia, Bogotá headquarters, endorsed the implementation of the project under Act 16 of September 13, 2016. Wistar rats (males and females) 9-11 weeks old, with weights between 250 and 320 g, were supplied by the Bioterium of the Pharmacy Department (Science Faculty), Universidad Nacional de Colombia, Bogotá headquarters, keeping them under controlled conditions (temperature 22°C, humidity 70%, and light/ dark 12/12 hours).

In vivo antihypertensive potential of P. vitifolia and P. edulis extracts
This was carried out using the methodology proposed by Bareño et al. (2017). The experimental animals were randomly divided into groups (n = 6), taking into account the treatments that were going to be used (Table 1). Every 48 hours for 7 weeks, the extracts and enalapril (reference drug) were administered orally and L-NAME (nitric oxide synthase inhibitor) was administered intraperitoneally. The measurement of systolic blood pressure (SBP) was made by the tail-cuff method (Tail-Cuff) in an ultrasound transducer (PANLAB-LE 5002) (Bareño et al., 2017;Vanvliet et al., 2000).

Preparation of the rings
By thoracotomy, the thoracic aortic artery was removed after the animal was sacrificed in a CO 2 camera. It was cleaned of connective tissue and it was divided into 4-5 mm rings that were kept in Krebs solution (NaCl 118.7; KCl 4.7; CaCl2 2.5; NaHC03 25.0; MgS04.7H20 1.2; glucose 11.0; and ascorbic acid 0.1, nM) at 37°C with carbogen mixture of 95% O 2 and 5% CO 2 . The measurement of the tension was made by fixing the rings to the base in the organ chambers containing 10 ml of Krebs solution maintained at 37°C, pH 7.4 ± 0.2, and carbogen and to the isometric transducer coupled to a signal amplification system and computerized recording (Data Trax 2, WPI) (Torres et al., 2012).

Data analysis
Data were expressed as the mean ± SEM. Statistical analysis was carried out using analysis of variance (two-way parametric), followed by Tukey's multiple difference test (p ≤ 0.05), using the GraphPad Prism 6 program.

Acute toxicity
In the acute toxicity test, there were no deaths or toxic signs after administration. The weight gain did not vary in the treated groups compared to the control group. The necropsy did not reveal any macroscopic alteration of the organs and tissues examined.
The extracts of P. vitifolia and P. edulis f. edulis are framed in the category of not classified according to the Toxicity Classes of the European Community (Commission of the European Communities, 1992), since no mortality or toxic signs are attributable to its administration. According to this result, we can affirm that the evaluated extracts did not produce significant toxicity at the limit dose in the acute toxicity test.

In vitro cytotoxic activity of the extracts (MTT Assay)
Cell viability was established with respect to the vehicle to which a 100% survival was assigned. In Figure 1, it is evident that extracts at the evaluated concentrations did not show cell viability less than 80%; therefore, these can be considered safe, complying with the parameters proposed by Rodríguez et al. (2004); on the other hand, during the time of administration of the extracts to the animals, they did not present symptoms of apparent toxicity. It is possible that the type and concentration of metabolites present are related to high levels of cell viability, since, for example, under the conditions of this study, it was not possible to detect saponins, which are reported in other plant organs (Table 1) and have been reported as toxic in cell models and if the presence of polyphenols is evident (Table 2) which are reported as antioxidants with a cellular protective effect (Orhan et al., 2003;Podolak et al., 2010).

Antihypertensive activity in vivo
The administered extracts showed an effect against the increase of the SBP of animals after the administration of the L-NAME (Fig. 2); it was evident that the basal values of SBP did not increase with the administration of L-NAME; this contrasts with the SBP of hypertensive animals which increased significantly (p < 0.0001) compared to animals treated with the extracts, enalapril, and vehicles. This favorable effect of the extracts could be attributed to different metabolites groups identified in the extracts ( Table 2), since phenolic compounds (found in high concentration) and Passiflora alkaloids have been reported to generate a protective and hypotensive effect in both in vivo and in vitro models (Carrón et al., 2010;Wang et al., 2014); it is possible that it should be noted that the rats with the lowest increase in SBP were those treated with the extract of P. edulis, which evidenced a higher content of the quantified polyphenols.

Relaxing effect of the extracts on the contraction exerted by phenylephrine (Phe) and KCl
The extracts showed relaxation in aortic rings contracted with Phe and KCl (Fig. 3), being greater in the rings treated with the extract of P. edulis. It was possible to show greater activity on the part of the two extracts in the rings contracted with Phe, finding significant differences in the maximum response between the treatments and the compounds used to contract the rings. The relaxation induced by the extracts of the two species presented concentration response behavior, suggesting that the effects may be linked to intracellular calcium movement stimulated by different mechanisms that act on voltage-dependent Ca +2 channels (Clark et al., 2001;Costa, 2013;Wang et al., 2014).

Effect of ACh and NPS on aortic rings of hypertensive rats treated with the extracts
Chronic exposure to L-NAME generates a decreased vascular response (Bryant et al., 1995). In this work, it was not possible to maintain the vascular response of the animals by cumulative action of ACh and NPS (Figs. 4 and 5), since a decrease is observed with respect to that obtained in normotensive rats. It is evidenced that the aortic rings obtained from animals treated with the extracts showed greater relaxation than those treated with enalapril and hypertensive rats. In both cases, a shift to the right of the curve was observed with respect to the control, which could indicate a decrease in the potency and/or antagonistic action by the extracts to the relaxing action of the two compounds on the aortic rings (Aleixandre and Ortega, 2000).

Relaxation in the presence of L-NAME
The relaxing action of the two ethanolic extracts on aortic rings precontracted with Phe and incubated with the agents mentioned above did not show significant differences, except for L-NAME, which partially reversed the relaxation compared to rings without the presence of it (Fig. 6). The evaluation of the synthesis of nitric oxide by adding L-NAME to the organ bath showed that this partially reduces the relaxing capacity of the two extracts compared to that produced in its absence, which suggests that the relaxing effect of the extract could be dependent on the endothelium and would be directly related to the synthesis of nitric oxide (Pérez et al., 2010;Wang et al., 2014).

Inhibitory effect of the extracts on the contraction exerted by AT-II
Ethanolic extracts of two species inhibited contraction produced by AT-II in aortic rings. It was observed that the extract of P. edulis at the two concentrations evaluated presented a greater inhibitory effect than the extract of P. vitifolia (Fig. 7). Contraction of the rings by cumulative doses of AT-II in rings preincubated with extracts showed a concentration-dependent effect, being the P. edulis extract the one that showed the greatest inhibition of AT-II. When comparing the effect of the extracts with that of Losartan (AT-II inhibitor), higher effectiveness was obtained than that caused by the reference drug, which is a selective inhibitor of the AT1 receptor which reduces the effects of AT-II in the artery (Pérez et al., 2010). The two extracts could act as possible AT-II inhibitors; however, a greater effect (p ≤ 0.05) of the extract of P. edulis than that produced by P. vitifolia is observed; the above could be related to the concentration of phenolic compounds greater in the extract of P. edulis (Table 2).

UHPLC-DAD analysis
The results of the characterization of the two extracts by UHPLC-UV can be seen in Table 3 and Figure 8. Chromatographic analysis showed the presence and quantification of vitexin, orientin, isovitexin, isoorientin, coumaric acid, rutin, and quercetin, using curves made with the reference compounds. Other chromatographic signals were evidenced that showed characteristic UV spectra of polyphenols but that could not be identified or quantified because they did not correspond to the standards used. Similarly, quantitation of phenolic compounds (total phenols, total flavonoids, and tannins) revealed the high content of these in the two extracts (Table 2). It should be noted that some c-glycosides flavonoids such as orientin and vitexin are used in Passiflora as chemomarkers of the genus; however, these reports refer to extracts obtained from stem and leaves but not from seeds (Gomes et al., 2017;Petenatti et al., 2014;Sepúlveda et al., 2018;Wosch et al., 2017;Zeraik and Yariwake, 2010). On the other hand, some authors (Bayard et al., 2007;Occhiuto and Limardi, 1994;Occhiuto et al., 1990) indicated the positive effect of said compounds on the vascular behavior of animals with HTA and the protective potential they present on target tissues related to pathology (Orhan et al., 2003); however, it is necessary to carry out tests that allow the different compounds present in the extracts of the two Passiflora to be correlated with the different pharmacological targets related to HTA.

CONCLUSION
This study demonstrates that the two ethanolic extracts obtained from the seeds of P. edulis f. edulis and P. vitifolia prevented HTA induced by nitric oxide deficit in rats; however, P. edulis f. edulis had a greater effect, obtaining SBP similar to those of normotensive animals. The possible mechanisms of action for the extracts are primarily related to the synthesis of nitric oxide and inhibition of AT-II; these results, together with the not apparent toxicity in cell, no acute toxicity in mice and the chemical composition, that relates the activity with the presence of phenolic compounds be in agreement to studies previously carried out on seeds of the genus; the above, postulates the two species as a possible source of extracts with antihypertensive potential, reinforcing the ethnobotanical use of the genus Passiflora for the control of hypertension. In addition, this type of results contributes to the generation of added value to the agroindustrial waste of these two species through its use in obtaining extracts with biological potential.