Affinin (Spilanthol), Isolated from Heliopsis longipes, Induces Vasodilation via Activation of Gasotransmitters and Prostacyclin Signaling Pathways

Heliopsis longipes roots have been widely used in Mexican traditional medicine to relieve pain, mainly, toothaches. Previous studies have shown that affinin, the major alkamide of these roots, induces potent antinociceptive and anti-inflammatory activities. However, the effect of H. longipes root extracts and affinin on the cardiovascular system have not been investigated so far. In the present study, we demonstrated that the dichloromethane and ethanolic extracts of H. longipes roots, and affinin, isolated from these roots, produce a concentration-dependent vasodilation of rat aorta. Affinin-induced vasorelaxation was partly dependent on the presence of endothelium and was significantly blocked in the presence of inhibitors of NO, H2S, and CO synthesis (NG-nitro-l-arginine methyl ester (l-NAME), dl-propargylglycine (PAG), and chromium mesoporphyrin (CrMP), respectively); K+ channel blockers (glibenclamide (Gli) and tetraethyl ammonium (TEA)), and guanylate cyclase and cyclooxygenase inhibitors (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin (INDO), respectively). Our results demonstrate, for the first time, that affinin induces vasodilation by mechanisms that involve gasotransmitters, and prostacyclin signaling pathways. These findings indicate that this natural alkamide has therapeutic potential in the treatment of cardiovascular diseases.

Affinin has an adequate lipophilicity. An in vitro permeability test showed that this alkamide (10 µg/mL) permeates through CaCo-2 cell monolayer cultures via passive diffusion. Whereas in vivo assays demonstrated that it is able to permeate skin and oral mucosa, and subsequently reach blood circulation, and cross the blood-brain barrier in high amounts (~98%) [23,32]. Therefore, this compound might be considered a valuable potential drug candidate [13,18,23,33].
With respect to safety assessment studies, the acute toxicity of affinin was evaluated on ICR mice and the determined median lethal dose (LD 50 = 113 mg/kg) was significantly higher than the doses required to elicit antinociception [6,26]. No mutagenic effects were observed by using the Ames test [6] and antimutagenic effects of affinin were observed at 25 and 50 µg/mL [10]. The cytotoxic effect of affinin was determined on human HEK293 kidney cells and the calculated mean inhibitory concentration (IC 50 ) was 260 µg/mL, while the concentration used to observe biological effects was 100 µg/mL [27]. No cytotoxic effects of affinin, which elicits a stimulatory effect on nitric oxide (NO) production in RAW 264.7 murine macrophages, were observed at concentrations up to 40 µg/mL [18].
Considering involvement of the NO/cGMP/KATP pathway in the antinociceptive effect of affinin, we hypothesized that this compound might exert a vasodilator effect via activation of gasotransmitters and prostacyclin signaling pathways. Therefore, the aim of this study was to investigate whether affinin, isolated from H. longipes roots, was capable of inducing vasodilation and to explore its mechanism of action.

Phytochemical Study of the Dichloromethane Extract Obtained from H. longipes Roots and Isolation of Affinin
Dichloromethane provided a higher yield of extract (19 g/kg roots dry weight) compared to ethanol (17 g/kg roots dry weight). Considering vasodilator potency, the dichloromethane extract was chosen to isolate the bioactive compounds. This extract (100 g) was fractionated by open column chromatography to obtain 21 fractions. Subsequent chromatography of fractions 8-17 resulted in the isolation of 28.5 g of pure affinin ( Figure 1). Affinin ( Figure 2) was identified by comparison with an authentic sample and by comparing its spectroscopic data ( 1 H-NMR and 13 C-NMR) with those previously reported in the literature (Table 1). High performance liquid chromatography/photodiode array detector (HPLC-PDA) analysis of affinin revealed a purity >94%.  Affinin ( Figure 2) was identified by comparison with an authentic sample and by comparing its spectroscopic data ( 1 H-NMR and 13 C-NMR) with those previously reported in the literature (Table 1). High performance liquid chromatography/photodiode array detector (HPLC-PDA) analysis of affinin revealed a purity >94%. The dichloromethane and ethanolic extracts of H. longipes roots, and affinin, induced a concentration-dependent relaxation of aortic rings with functional endothelium. Figure 3A shows the concentration-response curves for both extracts, affinin, and acetylcholine (ACh), which was used as a positive control. The dichloromethane extract (Emax = 100% ± 3.11% and EC50 = 76.99 ± 1.14 µg/mL) was approximately two fold more potent than the ethanolic extract (Emax = 100% ± 4.54% and EC50 = 140.5 ± 1.16 µg/mL), whereas affinin was significantly more potent than both extracts (Emax = 100% ± 3.10% and EC50 = 27.38 ± 1.20 µg/mL). Affinin turned out to be approximately twenty-five fold less potent than acetylcholine (Emax = 70.02% ± 1.43% and EC50 = 1.094 ± 1.14 µg/mL), however, this alkamide elicited a maximum vasodilator effect greater than that of the positive control ( Table 2). Carboxymethylcellulose 1% (CMC), employed as a vehicle, did not show any significant vasodilator effect.  The dichloromethane and ethanolic extracts of H. longipes roots, and affinin, induced a concentration-dependent relaxation of aortic rings with functional endothelium. Figure 3A shows the concentration-response curves for both extracts, affinin, and acetylcholine (ACh), which was used as a positive control. The dichloromethane extract (E max = 100% ± 3.11% and EC 50 = 76.99 ± 1.14 µg/mL) was approximately two fold more potent than the ethanolic extract (E max = 100% ± 4.54% and EC 50 = 140.5 ± 1.16 µg/mL), whereas affinin was significantly more potent than both extracts (E max = 100% ± 3.10% and EC 50 = 27.38 ± 1.20 µg/mL). Affinin turned out to be approximately twenty-five fold less potent than acetylcholine (E max = 70.02% ± 1.43% and EC 50 = 1.094 ± 1.14 µg/mL), however, this alkamide elicited a maximum vasodilator effect greater than that of the positive control ( Table 2). Carboxymethylcellulose 1% (CMC), employed as a vehicle, did not show any significant vasodilator effect. The dichloromethane and ethanolic extracts of H. longipes roots, and affinin, induced a concentration-dependent relaxation of aortic rings with functional endothelium. Figure 3A shows the concentration-response curves for both extracts, affinin, and acetylcholine (ACh), which was used as a positive control. The dichloromethane extract (Emax = 100% ± 3.11% and EC50 = 76.99 ± 1.14 µg/mL) was approximately two fold more potent than the ethanolic extract (Emax = 100% ± 4.54% and EC50 = 140.5 ± 1.16 µg/mL), whereas affinin was significantly more potent than both extracts (Emax = 100% ± 3.10% and EC50 = 27.38 ± 1.20 µg/mL). Affinin turned out to be approximately twenty-five fold less potent than acetylcholine (Emax = 70.02% ± 1.43% and EC50 = 1.094 ± 1.14 µg/mL), however, this alkamide elicited a maximum vasodilator effect greater than that of the positive control (Table 2). Carboxymethylcellulose 1% (CMC), employed as a vehicle, did not show any significant vasodilator effect.   Data are expressed as mean ± SEM (n = 6). Acetylcholine (ACh) is presented as positive control.

Involvement of K + Channels in Affinin-Evoked Vasodilation
To determine whether activation of K + channels participated in the vasodilatory effect of affinin, the effects of glibenclamide (Gli, a specific blocker of the K ATP channels) and tetraethyl ammonium (TEA, a non-selective K + channel inhibitor) were assessed. Both blockers significantly shifted to the right the concentration-response curve of the vasodilator effect of affinin ( Figure 4B), indicating that these channels are involved in its effect.  Data are expressed as mean ± SEM (n = 6). Acetylcholine (ACh) is presented as positive control.

Involvement of K + Channels in Affinin-Evoked Vasodilation
To determine whether activation of K + channels participated in the vasodilatory effect of affinin, the effects of glibenclamide (Gli, a specific blocker of the KATP channels) and tetraethyl ammonium (TEA, a non-selective K + channel inhibitor) were assessed. Both blockers significantly shifted to the right the concentration-response curve of the vasodilator effect of affinin ( Figure 4B), indicating that these channels are involved in its effect.

Discussion
H. longipes roots have a long tradition of culinary and medicinal use in Mexico. A number of studies have evidenced that organic extracts obtained from H. longipes roots and affinin, their major component, possess interesting biological and pharmacological activities [4][5][6]8,9,16,25,26,[28][29][30][31]58]. However, currently, no investigation has been directed toward examining the effect of H. longipes root extracts and affinin on the vascular tone.
In the present study, both the dichloromethane and ethanolic extracts from H. longipes roots were found to significantly relax the isolated rat aorta. To our knowledge, this has not been previously reported. In 2008, Wongsawatkul et al. [59] described the vasorelaxant effect of four organic extracts (hexane, chloroform, ethyl acetate, and methanol extracts) prepared from aerial parts of Spilanthes acmella (Synonym: Acmella oleracea) on rat aorta rings. In that study, the ethyl acetate extract exhibited the most potent vasorelaxant effect and, according to the authors, such an effect could be attributed to the presence of polar phenolic and triterpenoid ester compounds. Additionally, the chloroform extract showed the highest maximum vasodilator response. The authors ascribed such effect to triterpenoids and fatty alcohols or esters present in the chloroformic extract. Furthermore, vasodilation induced by the S. acmella extracts was completely abolished in the absence of endothelium and significantly reduced in the presence of L-NAME (1 µM) and indomethacin (1 µM), which strongly suggested the participation of the NO and the PGI2 pathways [59].
Other studies were carried out to test the effect of oral administration of the S. acmella ethanolic flower extract at doses ranging from 50 to 150 mg/kg on sexual performance in male rats. The extract was administered during 28 days and no toxic effects were observed. One of the main findings was the dose-dependent erectile function improvement induced by the extract and its capacity to produce long-term effects, even by day 15 after cessation of the treatment. In the same study, a good correlation was found between these results and a raise on NO levels determined in DS-1 cells (a human corpus cavernosum cell line) cultures stimulated with the S. acmella ethanolic extract (100 µg/mL). The authors suggested a possible contribution of affinin and other alkamides present in the extract on the observed effect of improved sexual function [22]. It is a well-known fact that erectile

Discussion
H. longipes roots have a long tradition of culinary and medicinal use in Mexico. A number of studies have evidenced that organic extracts obtained from H. longipes roots and affinin, their major component, possess interesting biological and pharmacological activities [4][5][6]8,9,16,25,26,[28][29][30][31]58]. However, currently, no investigation has been directed toward examining the effect of H. longipes root extracts and affinin on the vascular tone.
In the present study, both the dichloromethane and ethanolic extracts from H. longipes roots were found to significantly relax the isolated rat aorta. To our knowledge, this has not been previously reported. In 2008, Wongsawatkul et al. [59] described the vasorelaxant effect of four organic extracts (hexane, chloroform, ethyl acetate, and methanol extracts) prepared from aerial parts of Spilanthes acmella (Synonym: Acmella oleracea) on rat aorta rings. In that study, the ethyl acetate extract exhibited the most potent vasorelaxant effect and, according to the authors, such an effect could be attributed to the presence of polar phenolic and triterpenoid ester compounds. Additionally, the chloroform extract showed the highest maximum vasodilator response. The authors ascribed such effect to triterpenoids and fatty alcohols or esters present in the chloroformic extract. Furthermore, vasodilation induced by the S. acmella extracts was completely abolished in the absence of endothelium and significantly reduced in the presence of L-NAME (1 µM) and indomethacin (1 µM), which strongly suggested the participation of the NO and the PGI 2 pathways [59].
Other studies were carried out to test the effect of oral administration of the S. acmella ethanolic flower extract at doses ranging from 50 to 150 mg/kg on sexual performance in male rats. The extract was administered during 28 days and no toxic effects were observed. One of the main findings was the dose-dependent erectile function improvement induced by the extract and its capacity to produce long-term effects, even by day 15 after cessation of the treatment. In the same study, a good correlation was found between these results and a raise on NO levels determined in DS-1 cells (a human corpus cavernosum cell line) cultures stimulated with the S. acmella ethanolic extract (100 µg/mL). The authors suggested a possible contribution of affinin and other alkamides present in the extract on the observed effect of improved sexual function [22]. It is a well-known fact that erectile function is mediated by a complex integration of signals, where NO, synthesized by endothelial, inducible, and neuronal NOS, is the most important factor that contributes to vasodilation of the erectile vasculature of the penis [60][61][62].
Regarding our research, based on the vasodilator potency, we selected the dichloromethane extract of H. longipes roots to carry out a phytochemical study in order to isolate the bioactive constituents. Chromatographic analysis of the extract led to the isolation of affinin as the major component. This result is consistent with previous studies that have demonstrated that affinin is the main alkamide found in H. longipes roots [4,6,8,11,16,25,26]. Of great interest was the finding that affinin elicited a significant vasodilator effect, which was approximately three fold more potent than that of the crude extract. This finding represents the first demonstration that affinin is capable of relaxing the arterial smooth muscle. Considering that affinin was the most abundant constituent of the H. longipes dichloromethane extract, it can be inferred that this compound is responsible for the vasodilation induced by the crude extract.
Removal of endothelium significantly decreased, but did not completely block, the vasorelaxation induced by affinin, indicating that both, endothelial-dependent and independent vasodilation pathways are involved in its mechanism of action. The vasorelaxing effect was significantly reduced in the presence of NOS, CSE, and HO inhibitors, which evidenced that activation of the NO/cGMP, H 2 S/KATP, and CO/cGMP pathways contribute to affinin-induced vasodilation. The most relevant inhibition was observed when aortas were preincubated with L-NAME (p < 0.001), suggesting that activation of the NO/cGMP pathway plays a more prominent role in the effect of this alkamide than that played by the other two gasotransmitters pathways. Moreover, inhibition of sGC by ODQ (p < 0.001) significantly reduced the vasodilatory effect of affinin, revealing that it might directly activate sGC, the main receptor of NO [36]. It is important to point out that CO-sensitive sGC isoforms exist in the vascular smooth muscle [46], therefore CO is also considered to be an important activator of this class of enzymes [37]. Activation of sGC on the smooth muscle cells might underlie, at least in part, endothelium-independent vasodilation caused by affinin.
One of the key mechanisms by which NO, CO, and H 2 S, synthesized in endothelial cells, induce vasodilation is activation of potassium channels located in vascular smooth muscle cells. Regarding nitric oxide-cGMP induced vasodilation, it has been shown that cGMP-dependent protein kinase (PKG) phosphorylates calcium-activated potassium channels (K Ca ) on the smooth muscle cell membrane leading to a decrease in intracellular calcium concentration [38,39]. This same mechanism is involved in the vasorelaxation produced by CO. Moreover, this gasotransmitter is able to directly activate potassium channels, in particular K Ca [51,52]. On the other hand, H 2 S mediates vasorelaxation via direct opening of K ATP channels [47][48][49][50]. In this study, we assessed whether potassium channel blockers impaired vasodilation provoked by affinin. Glibenclamide and TEA significantly decreased affinin-evoked vasodilation, which confirmed the activation of signaling pathways for NO, CO, and H 2 S. Indomethacin (p < 0.001) caused a significant reduction in affinin-induced vasodilation, suggesting that activation of the PGI 2 /cAMP pathway is also involved in the mechanism of vasorelaxation caused by this alkamide. Along with gasotransmitters, PGI 2 is secreted by endothelial cells and elicits smooth muscle relaxation by stimulating adenylate cyclase, which subsequently increases cAMP levels. This second messenger activates calcium-activated potassium channels (K Ca ) via PKA-dependent phosphorylation [55,56]. Evidence from some previous studies suggest that cAMP also enhances K Ca activity by "cross-activation" of PKG [38,54].
According to our results, it is evident that affinin does not only act on a specific type of cell receptor in the arteries. Since its vasodilator effect is not completely blocked in the absence of endothelium, it is clear that this compound activates both endothelium dependent and independent pathways. Our results indicated that affinin is able to activate the NO/cGMP, CO/cGMP, H 2 S/KATP, and PGI2/cAMP signaling pathways, and considering that the triggering of these four signaling pathways depends on the activation of molecular targets located on the endothelium layer, it is very likely that this alkamide might be acting on molecular targets, whose activation leads to an increase in Ca 2+ levels in the endothelial cells. The chemical structure of N-alkylamides or alkamides [63], like affinin [13,64,65] resembles that of fatty acid amides [66][67][68][69], and the endogenous cannabinoid N-arachidonylethanolamine or anandamide [70]. This molecule produces a potent vasodilator effect through several proposed mechanisms that include activation of TRPV1 channels and G-coupled receptors, such as CB 1 , CB 2 , and endothelial non-CB 1 /non-CB 2 [71][72][73]. Herradón et al. [72] showed that the vasodilator effect of anandamide in rat aorta is mainly produced by activation of the endothelial non-CB 1 /non-CB 2 cannabinoid receptor, which in turn activates the NO/cGMP pathway. Therefore, considering the similarity between the chemical structures of anandamide and affinin, it is quite possible that endothelial non-CB 1 /non-CB 2 or/and TRP channels may be the putative molecular targets for affinin in the endothelial cells.
Concerning endothelium-independent relaxation induced by affinin, our results indicate that this molecule might directly activate sGC. We can speculate that affinin might also directly activate R PGI , although this possibility needs to be confirmed. Figure 6 shows the proposed signaling pathways involved in the vasodilatory effect of affinin. in Ca 2+ levels in the endothelial cells. The chemical structure of N-alkylamides or alkamides [63], like affinin [13,64,65] resembles that of fatty acid amides [66][67][68][69], and the endogenous cannabinoid Narachidonylethanolamine or anandamide [70]. This molecule produces a potent vasodilator effect through several proposed mechanisms that include activation of TRPV1 channels and G-coupled receptors, such as CB1, CB2, and endothelial non-CB1/non-CB2 [71][72][73]. Herradón et al. [72] showed that the vasodilator effect of anandamide in rat aorta is mainly produced by activation of the endothelial non-CB1/non-CB2 cannabinoid receptor, which in turn activates the NO/cGMP pathway. Therefore, considering the similarity between the chemical structures of anandamide and affinin, it is quite possible that endothelial non-CB1/non-CB2 or/and TRP channels may be the putative molecular targets for affinin in the endothelial cells.
Concerning endothelium-independent relaxation induced by affinin, our results indicate that this molecule might directly activate sGC. We can speculate that affinin might also directly activate RPGI, although this possibility needs to be confirmed. Figure 6 shows the proposed signaling pathways involved in the vasodilatory effect of affinin. The results of the present study have clearly shown that affinin, obtained from H. longipes roots, produces vasodilation of rat aorta by activating the NO/cGMP, CO/cGMP, H2S/KATP, and PGI2/cAMP signaling pathways. This is the first report describing the vasodilator effect of this alkamide and some of the processes involved in its mechanism of action. The median effective concentration to produce vasodilation (EC50 = 27.38 µg/mL) falls within the concentration ranges at which this compound elicits other biological and pharmacological activities. Moreover, the EC50 obtained for the vasodilator effect of affinin is within the non-cytotoxic concentration range for mammalian cells, however, more cytotoxic studies must be performed in order to establish its possible adverse effects. Besides the other pharmacological properties that have been attributed to affinin, the vasodilator effect is a new interesting activity that might be ascribed to this alkamide. CSE, cystathionine-γ-lyase; sGC, soluble guanylate cyclase; PKG, protein kinase G; AC, adenylate cyclase; PKA, protein kinase A; K + Ch, K + channel; P-MLC, phosphorylated myosin light chain. Black upwards arrow, increased levels; Black downwards arrows, decreased levels. ?, pathway that remains to be confirmed.
The results of the present study have clearly shown that affinin, obtained from H. longipes roots, produces vasodilation of rat aorta by activating the NO/cGMP, CO/cGMP, H 2 S/KATP, and PGI 2 /cAMP signaling pathways. This is the first report describing the vasodilator effect of this alkamide and some of the processes involved in its mechanism of action. The median effective concentration to produce vasodilation (EC 50 = 27.38 µg/mL) falls within the concentration ranges at which this compound elicits other biological and pharmacological activities. Moreover, the EC 50 obtained for the vasodilator effect of affinin is within the non-cytotoxic concentration range for mammalian cells, however, more cytotoxic studies must be performed in order to establish its possible adverse effects. Besides the other pharmacological properties that have been attributed to affinin, the vasodilator effect is a new interesting activity that might be ascribed to this alkamide. Undoubtedly, these results contribute to support the great therapeutic potential of Heliopsis longipes roots and affinin, their main constituent.

Reagents and Chemicals
Reagents and solvents used in the chemical study of H. longipes roots were purchased from JT Baker (Phillisburg, NJ, USA). Standards and solvents for the pharmacological assays were obtained from Sigma-Aldrich (St. Louis, MO, USA). CrMP was purchased from Porphyrin Products, Inc. (Logan, UT, USA).

Animals
All experimental protocols were performed in accordance with guidelines of the Mexican Official Standard NOM-062-ZOO-1999 [74], and approved by the Bioethics Committee of the Faculty of Natural Sciences, Autonomous University of Querétaro, México. Wistar male rats (250-300 g) were used for the pharmacological study; they were provided by the Institute of Neurobiology of the National Autonomous University of Mexico, Campus Juriquilla, Querétaro, Qro., Mexico. Animals were housed in standard cages under controlled temperature conditions with a 12:12 h light-dark cycle. Water and food were provided ad libitum.

Preparation of the Extracts Employed for the Pharmacological Evaluation
Air dried H. longipes roots were ground to a fine powder. For the preparation of H. longipes root extracts, ground plant material (10 g) was subjected to maceration with either dichloromethane or absolute ethanol for one week in a 1:10 ratio (w/v). This process was repeated three times with fresh solvent. Thereafter, the plant material was filtered and the solvents were removed by rotary evaporation. The extraction yields were: 0.019 g extract/g dried roots for the dichloromethane extract and 0.017 g extract/g dried roots for the ethanolic extract. lamp at 254 nm. Spraying TLC plates with a spray solution of anisaldehyde/sulfuric acid (0.5 mL anisaldehyde in 50 mL glacial acetic acid and 1 mL 97% sulfuric acid) developed a bright purple spot, as reported for other olefinic isobutyl-amides [75].
Pools 8-17 were combined (45 g) and further analyzed by open column chromatography (450 g, Kiesegel 60 Merck 100-230 mesh, 4.5 × 120 cm) using a step gradient of hexane and ethyl acetate 100:0 to 90:10. Based on their chromatographic similarity, determined by TLC, fractions eluted with hexane: ethyl acetate 97:3 were combined and evaporated to dryness in vacuo leaving a residue of 28.5 g of an apparently pure compound. The purity of the isolated compound was confirmed by HPLC-PDA, using an HPLC chromatograph (Waters 600 Associates, Milford, MA, USA) coupled to a photodiode array detector (Waters 2998). This analysis was carried out on a XBridge C18 (4.6 × 100 mm 3.5 µm) column. The flow rate of the mobile phase (acetonitrile/water 44:56 v/v) was 0.5 mL/min with column temperature of 30 • C and detection wavelength of 229 nm.

Determination of the Chemical Structure of Affinin
Chemical structure of the purified compound was elucidated by analysis of its proton nuclear magnetic resonance ( 1 H-NMR) and carbon-13 ( 13 C-NMR) spectra (Table 1). Nuclear magnetic resonance (NMR) spectra were taken on a Varian VNMRS 400 spectrometer with tetramethylsilane (TMS) as internal standard. Affinin was identified by comparing its spectroscopic constants with those reported in the literature [20,21].

Isolated Rat Aorta Assay
The rats were killed by decapitation. The thoracic aorta was surgically removed and placed in a Petri dish containing ice-cold (4 • C) Krebs-Henseleit solution with the following composition (mM): 126.8 NaCl; 5.9 KCl; 1.2 KH 2 PO 4 ; 1.2 MgSO 4 ; 5.0 D-glucose; 30 NaHCO 3 ; 2.5 CaCl 2 (pH 7.4), bubbled with a mixture of carbogen (95% O 2 and 5% CO 2 ). Then, the intraluminal space of aorta was rinsed with fresh solution to prevent clot formation, cleaned from surrounding connective tissue, and sliced into rings (3-4 mm in length). Aortic rings were mounted between two metallic hooks, with one being fixed and the other attached to an isometric transducer, and placed into organ baths chambers containing pre-warmed Krebs-Henseleit solution (37 • C) gassed with carbogen. The aortic segments were allowed to equilibrate for 60 min under a resting tension of 1.5 g. During the resting period, the organ bath solution was exchanged every 10 min. In order to stimulate the vascular smooth muscle, the tissues were contracted with KCl solution (100 mM). Once a stable contractile tone was reached, the bathing medium was replaced every 10 min to restore the initial resting tension of 1.5 g. Afterwards, the aortic rings were contracted with 1 µM L-phenylephrine (Phe); the contractile force induced was defined as 100%, and once the plateau was reached, the test substances were cumulatively added. Acetylcholine (ACh), dissolved in distilled water, was evaluated in a concentration range of 0.2 ng/mL-2 mg/mL; while affinin and the extracts, dissolved in vehicle (carboxymethylcellulose 1% in distilled water), were tested in a concentration range of 1 µg/mL-1 mg/mL. When used, pharmacological inhibitors were added to the organ bath chambers 20 min before the addition of Phe. The changes in tension caused by the tested concentrations were detected by Grass FT03 force transducers coupled to a Grass 7D Polygraph; they were expressed as percentages of relaxation based on the contraction generated by adding Phe [76].

Participation of the Endothelium in the Vasodilator Response of Affinin
To determine whether the vasodilator response of affinin was dependent on the vascular endothelium, assays on aorta segments without endothelium were performed. In these experiments the endothelial layer was removed by flushing the lumen of aorta with 0.2% desoxycholate in saline solution 0.9%, as reported previously [76]. The absence of endothelium was confirmed at the start of the experiment, showing that the addition of 1 µM of acetylcholine (ACh) did not induce more than 5% relaxation. Once the cumulative concentrations of affinin were added to the bath chambers, as described above, sodium nitroprusside (100 µM) was added to the chambers to demonstrate that the artery was still capable of relaxation.  [40][41][42][43][44]49,52,77].
To determine the involvement of the prostacyclin pathway in the vasodilator effect of affinin, aortic segments were pre-incubated for 20 min in the presence of 1 µM indomethacin (INDO, inhibitor of COX) [78,79]. In addition, to assess whether activation of K + channels was involved in the vasodilation produced by affinin, the effect of pretreatment with the non-selective potassium channel blocker, 1 mM tetraethyl ammonium (TEA) and 10 µM glibenclamide (a specific blocker of the K ATP channels) was evaluated [80,81].

Statistical Analysis
Evaluations of each concentration of the tested substances were performed on aortas obtained from at least three different rats (n = 6). All values are expressed as the mean ± standard error of the mean (SEM). The resulting data obtained from each evaluation were fitted to a sigmoidal equation, plotted, and analyzed to calculate EC 50 (GraphPad Prism 7.02, San Diego, CA, USA). These results were subjected to one-way analysis of variance (ANOVA) using the statistical program GraphPad Prism 7.02, followed by the Tukey test to evaluate any significant differences between the means. Values of + p < 0.01 or * p < 0.001 were considered to be significant.

Conclusions
Our study provides a heretofore unknown evidence that affinin, isolated from H. longipes roots, is capable of inducing vasodilation via mechanisms that involve activation of gasotransmitters and prostacyclin signaling pathways. The NO/cGMP and PGI 2 /cAMP pathways appear to play a more prominent role than either the H 2 S/KATP pathway or the CO/cGMP pathway in affinin-evoked vasorelaxation. Undoubtedly, this molecule deserves further investigation in order to completely understand its mechanism of action. The results derived from this study suggest that affinin is a promising molecule for the development of drugs useful in the prevention and/or treatment of cardiovascular diseases, particularly when considering that it has an adequate lipophilicity that allows it to permeate skin and oral mucosa, and reach blood circulation.
Acknowledgments: Jesús E. Castro-Ruiz acknowledges Consejo Nacional de Ciencia y Tecnología (CONACYT) for his doctoral grant. The authors would like to thank Josué López Martínez and Yolanda Rodríguez Asa for their technical assistance.
Author Contributions: Jesús Eduardo Castro-Ruiz carried out the phytochemical study of H. longipes roots, conducted the pharmacological assays, and wrote the manuscript. Alejandra Rojas-Molina supervised the phytochemical study of H. longipes roots and contributed with the preparation of the manuscript. Francisco J. Luna-Vázquez supervised and helped in carrying out the pharmacological assays. Fausto Rivero-Cruz conducted the final identification of affinin. César Ibarra-Alvarado and Teresa García-Gasca designed this project, coordinated all the activities, and contributed with the preparation of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.