Vasorelaxant effect of Tribulus terrestris extract and fractions on isolated rat aortae

Abstract

Tribulus terrestris (Zygophylaceae) has been used by traditional medicine as tonic, aphrodisiac and diuretic agent, as well as to treat cardiovascular diseases. However, the vascular effect of Tribulus terrestris fruits is poorly explored. Thus, the aim of this study is to assess effect of Tribulus terrestris fruit preparations on rat aortae. Dried crude ethanol deriving from Tribulus terrestris fruit extract (CE) was partitioned to produce hexane (HEX), dichloromethane (DCM), ethyl acetate (EtOAc), butanol (BuOH) and methanol/water-soluble (MWS) fractions. These preparations had their phytochemical profile assessed though UPLC-ESI-MS. Concentration-response curves plotted for CE, or for its fractions, were built in precontracted vessels with, or without, intact endothelium. Cumulative addition of both CE and its fractions (0.1-1,000 µg/mL) to a phenylephrine-induced precontracted vessel (10-7 M) promoted vasorelaxant effect in a concentration-dependent manner, and it did not depend on the vascular endothelium. EtOAc (1,000 µg/mL) applied to endothelium-denuded rings has fully inhibited phenylephrine-induced contractions. Likewise, EtOAC (1,000 µg/mL) added to nominally Ca2+-free depolarizing solution inhibited CaCl2-induced contractions. UPLC-ESI-MS analyses have indicated that EtOAC is rich in cinnamic acid amide derivatives. Altogether, the herein analyzed data suggest that the vasorelaxant effect of EtOAc can likely be attributed to the blockage of voltage-gated calcium channels.

Key words
Zygophyllaceae; Calcium channels; UPLC-ESI-MS; Cinnamic acid amides

INTRODUCTION

Hypertension is the most common cardiovascular disease and it significantly contributes to morbidity and mortality rates, worldwide. According to estimates by the World Health Organization, 1.4 billion people in the world have hypertension, and less than 14% of them have it under control. Hypertension is a significant public health issue due to its high prevalence and underdiagnosis, as well as to patients’ poor adherence to treatment and to the high rate of patients who fail to reach adequate blood pressure targets (WHO 2022).

According to recommendations by WHO (2022), hypertensive adult individuals requiring pharmacological treatment should take drugs belonging to any of the following four pharmacological antihypertensive medication classes, as initial treatment: diuretics, angiotensin-converting enzyme inhibitors, angiotensin-receptor blockers and calcium channel blockers. Multiple antihypertensive drug classes are often indicated to reach blood pressure targets. Using several drug types leads to high medical and socioeconomic costs, side effects and drug interactions (Sear 2019).

Thus, natural products with potential antihypertensive activity and minimal side effects can be used as alternative to these drugs. In addition to contribute to blood pressure control, they can be a good substitute for synthetic drugs, mainly if they are incorporated to lifestyle changes, such as dieting and exercising (Verma et al. 2021). Moreover, multicomponent herbal products have multiple therapeutic targets. Multitarget drugs have raised considerable interest, worldwide, due to benefits brought by them to the treatment of multifactorial diseases (Koeberle & Werz 2014).

Plant species Tribulus terrestris (Zygophylaceae family) has been used in the form of infusions (leaves) or decoctions (fruits) as tonic, aphrodisiac and diuretic agent, as well as to treat cardiovascular diseases (Zhu et al. 2017). Phytochemical studies have evidenced a wide variety of phenolic compounds, flavonoids, saponins and cinnamic acid amides (Bhutani et al. 1969, Su et al. 2009, Song et al. 2016) in T. terrestris fruits. These secondary metabolites are widely known for their effects on individuals’ cardiovascular system (Semerdjieva & Zheljazkov 2019). Thus, the aim of the present study was to assess the vascular effect of crude ethanol extract and of its fractions, deriving from the T. terrestris fruits, on isolated rat aortae.

MATERIALS AND METHODS

Ethical approval

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee on the Use of Animals of the Federal University of Lavras (n° 068/2018 CEUA-UFLA).

Extract and fractions’ preparation

Dried T. terrestris L. fruits were purchased from Catedral Pharmaceutical Company, Vespasiano County, Minas Gerais State, Brazil. T. terrestris extracts and fractions were prepared according to Oliveira et al. (2015). Crude ethanol extract (CE), hexane (HEX), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH) and methanol/water soluble (MWS) fractions were herein tested.

Animals

The present study used adult male Wistar rats (mean age of 60 days, weight ranging from 250 to 300 g), who were kept at mean temperature of 22 ± 2°C, under 12/12-hour light and dark cycles, for three weeks before and during the experiments. Animals fed on standard rodent diet and had access to water ad libitum. All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, after they were approved by the Ethics Committee on the Use of Animals of Federal University of Lavras (n. 068/2018 CEUA-UFLA).

Chemicals

The following drugs were used in the current study: L-phenylephrine hydrochloride (PHE), acetylcholine chloride (ACh), 4-aminopyridine (4-AP), glibenclamide (GLIB) and tetraethylammonium (TEA) (Sigma–Aldrich, St. Louis, MO, USA). GLIB was dissolved in sodium bicarbonate, in distilled water, and the remaining drugs were only dissolved in distilled water, in order to prepare the stock solutions. All solutions were stored at 0 °C.

Solutions

The following salts were added to distilled water to prepare the Krebs Henseleit solution (KHS): sodium chloride (NaCl), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO₄.7H₂O), glucose (C₆H₁₂O₆), sodium bicarbonate (NaHCO₃), potassium phosphate dihydrate (KH₂PO₄) and calcium chloride dihydrate (CaCl₂.2H₂O). Na+ concentration was isosmotically altered to prepare Krebs Henseleit depolarizing solutions with 25 mM potassium chloride. Only CaCl₂ was omitted in calcium-free solutions. Nutritive solutions’ pH was adjusted to 7.4, whenever necessary.

Vascular reactivity

Preparing the isolated rat thoracic aortae artery rings

Rats were anesthetized with thiopental (80 mg/kg, ip). Thoracic aortae were excised, separated from connective tissue and cut into rings of approximately 4-5 mm, in length. Aortic rings were suspended in organ baths comprising Krebs Henseleit solution (KHS - NaCl 118.0 mM, KCl 4.7 mM, MgSO₄ 1.2 mM, NaHCO₃ 25.0 mM, C₆H₁₂O₆ 11.1 mM; KH₂PO₄ 1.2 mM, CaCl₂. H₂O 2.5 mM, pH 7.4), kept at 37 °C, and continuously aerated with 95% O₂ and 5% CO₂, for isometric tension recording purposes (AECAD 04F, AQCAD 2.5.0., AVS Projetos, SP). After 60-min equilibration time under resting tension of 1 g, the presence of functional endothelium in the samples was investigated based on ACh 10−6 M’s ability to induce more than 80% relaxation in PHE 10−7 M-based pre-constricted rings. Endothelium observed in some of the analyzed rings was mechanically removed. Rings were classified as not having functional endothelium whenever ACh relaxation was lower than 10%. Nutritive solution was changed every 15 minutes during stabilization to prevent metabolites’ interference (Furchgott & Zawadzki 1980, Tirapelli et al. 2004).

Experimental protocols

Effects of T. terrestris CE and fractions on aortic contraction induced by PHE or KCl

After the 60-min stabilization period was over, aortic rings’ contraction was achieved by PHE (10−7 M). CE or HEX, DCM, EtOAc, BuOH or MWS fractions of T. terrestris were cumulatively added (0.1-1,000 µg/mL) to the rings, in both the presence and absence of endothelium, when the contraction reached a given plateau. Relaxation effect was calculated as the contraction rate in response to PHE. After finding the concentration-effect curves, preparations were washed in KHS every 15 minutes and contracted again with PHE 10-7 M every 30 minutes, for 2 hours, to check whether the vasorelaxant effect was reversible (Basri et al. 2018). Only the EtOAc fraction presenting the best vasorelaxant activity was further investigated (Carullo et al. 2020, Tirapelli et al. 2004).

Another set of experiments focused on assessing the effect of cumulatively added EtOAc (0.1-1,000 µg/mL) on KCl (25 mM)-induced tonic contraction in endothelium-denuded rings. At this concentration, KCl elicits a submaximal and sustained contraction without achieving total depolarization, which facilitates the assessment of the underlying mechanisms involved in modulating depolarization-induced contraction (Carullo et al. 2020, Tirapelli et al. 2004).

Investigating the EtOAc effect on PHE-induced concentration–response curves in endothelium-denuded preparations

After the stabilization period was over, the EtOAc effect on PHE-induced contractions in endothelium-denuded rings was assessed based on the following protocol: cumulative concentration–response curves for PHE (10-9 to 10-4 M) were recorded before and after 30-min pre-incubation with EtOAc (100, 250, and 1,000 µg/mL, which corresponded to EC₂₅, EC₅₀ and EC₁₀₀, respectively). Results were expressed as maximal response rates for PHE-induced response; concentration–response curves were statistically compared to each other (Carullo et al. 2020, Tirapelli et al. 2004).

Investigating the EtOAc effect o_n CaCl2-induced contractions

After the stabilization period was over, EtOAc effect on CaCl₂-induced contractions in endothelium-denuded rings was assessed. Cumulative concentration–response curves for CaCl₂ (10-6 - 3x10-2 M) were recorded in the endothelium-denuded rings exposed to nominally Ca2+-free KCl solution (25 mM), both before and after pre-incubation with EtOAc (100, 250, and 1,000 µg/mL), for 30 min (Carullo et al. 2020, Tirapelli et al. 2004). Results were expressed as maximal response rates for CaCl₂-induced response; concentration–response curves were statistically compared to each other.

Assessing K+ channel involvement in EtOAc-induced vasorelaxant response

The involvement of K+ channels in the vasorelaxant response was assessed by incubating endothelium-denuded preparations for 30 min with TEA (3 mM), 4-AP (3 mM), or GLIB (3x10-6 M) to inhibit nonselective K+ channels, Kv-channel and K_ATP-channel, respectively. These channels subtypes represent key regulators of aortic vascular tone. Cumulative EtOAc (0.1-1,000 µg/mL) concentrations were added to the rings after the stabilization of the PHE 10-7 M-induced tonic contraction (Diniz et al. 2013, Iqbal et al. 2019).

UPLC-ESI-MS analysis

UPLC-ESI-MS analysis was performed in Waters ACQUITY UPLC system (Waters, USA) comprising binary pump, autosampler, in-line degasser and photodiode array detector (DAD) (Waters, Milford, Massachusetts, USA). Xeco™ Triple Quadrupole MS mass spectrometer (Waters Corp., Milford, Massachusetts, USA) equipped with electrospray ionization (ESI) source was used for this purpose. Data were processed in MassLynx software, version 4.1 (Waters, Milford, Massachusetts, USA). Separation was performed based on using Acquity UPLC BEH HILIC (150×2.1 mm i.d.; 1.7 μm; Waters, Milford, Massachusetts, USA), in combination to Acquity UPLC BEH HILIC guard column (5×2.1 mm; 1.7 μm; Waters, USA). The mobile phase comprised gradient water (A) and acetonitrile (B) elution, both acidified with 0.1% v/v formic acid under the following elution/re-equilibration conditions: 0-10 min, 5% → 95% B; 10-11 min, 95% B; 11-13 min, 95% →5% B, at flow rate of 0.3 mL/min, at 40 °C. Injection volume was set at 5.0 μL. The following conditions were adopted for the ESI source in negative full-scan modes (m/z = 90 to 1300): 3.50 kV capillary voltage; 30 V cone voltage; source temperature of 120 °C; desolvation temperature of 350 °C, and desolvation gas flow of 550 L h-1.

Featuring attempts, based on using UPLC-ESI-MS in negative mode, were carried out by comparing the current results to the ones recorded in the literature for chemical compounds isolated from Tribulus terrestris fruits or from spectroscopic data complementary to the literature (Hong et al. 2013, Song et al. 2016, Wang et al. 2016, Wu et al. 1999).

Statistical analysis

All data were expressed as mean ± S.E.M.; n refers to the number of animals used in each protocol. EC₅₀ values (half-maximal effective concentration) were calculated through nonlinear regression applied to the concentration–response curves obtained for the CE and T. terrestris fractions used in each protocol. E_max value refers to the maximal effect induced by a given substance at a given rate, which was equal to 0%, when PHE induced maximum contraction; and to 100%, when the initial preload tension level was reached (baseline). Differences between means were statistically compared through non-paired Student’s tests or one-way ANOVA followed by Newman-Keul’s test; they were considered significant at p< 0.05. All analyses were performed in GraphPad Prism software (GraphPad Software Inc., San Diego, USA).

RESULTS

Effect of T. terrestris CE on PHE-induced pre-contractions

Based on results shown in Figure 1a, CE promoted concentration-dependent vasorelaxant effect. There was no significant difference (p>0.05) in CE’s vasorelaxant effect on PHE-induced contraction, either in the presence (EC₅₀ = 542.4 ± 154.30 µg/mL) or absence of vascular endothelium (EC₅₀ = 599.95 ± 221.09 µg/mL). This finding has suggested endothelium-independent effect.

Figure 1
a: Vasorelaxant effect induced by CE on PHE 10-7 M precontracted endothelium-intact (●) (n=6) or endothelium-denuded (○) (n=6) rat aortic rings. The values are expressed as the mean ± S.E.M.; the p>0.05. b: The maximal vasorelaxant effect (E_max) of the Tribulus terrestris extract and fractions on PHE 10-7 M-induced precontractions in isolated rat aortae. c: Log EC50 is related to the potency of the extract and fractions of Tribulus terrestris fruit on PHE-induced contraction in isolated rat aortae. Legend B and C: Values are expressed as the mean ± S.E.M.; * significant difference compared to CE and to HEX, DCM, BuOH and MWS fractions (p < 0.05 using one-way ANOVA followed by Newman-Keul’s test; n=5-7); CE: crude ethanol extract; HEX: hexane fraction; DCM: dichloromethane fraction; EtOAc: ethyl acetate fraction; BuOH: butanol fraction; MWS: methanol/water soluble fraction. d: Vasorelaxant effect induced by EtOAc on PHE 10-7 M precontracted endothelium-intact (●) (n=7) or endothelium-denuded (○) (n=4) rat aortic rings. Values are expressed as the mean ± S.E.M.; p>0.05 vs. endothelium-intact. e and f: Representative original record of the effect of the EtOAc fraction on isolated rat aortic rings with endothelium (E) or without endothelium (F) contracted by PHE 10-7 M. The arrows represent the time course of fraction administration (0.1 to 1,000 µg/mL).

Two (2) hours after CE was removed from the bath, reversion was reached, at the same amplitude as the previous one, through a new contraction promoted by PHE 10-7 M (data not shown). This finding has evidenced the viability of isolated rat aortae even after T. terrestris using, and it suggested lack of vascular toxicity.

CE fractions were investigated after their vasorelaxant activity was checked (Figure 1a). Based on Figure 1B, HEX, DCM, BuOH and MWS fractions have shown quite low isolated rat aortae-vasorelaxation rates. Therefore, these fractions were not assessed for their action mechanisms. EtOAc has shown higher E_max (p <0.05) than the ones observed for both CE and the other fractions (Figure 1b). Thus, EtOAc recorded the highest vasorelaxation rate in PHE-induced contracted aortae; this is the reason why it was selected to be further investigated. Futhermore, the EtOAc fraction exhibited significantly lower EC₅₀ values compared to those of the CE and the other fractions (p<0.05) (Figure 1c). The lower EC₅₀ value of the EtOAc fraction reflects its higher potency and superior vasorelaxant activity compared to the other samples.

EtOAc fraction effect on PHE-induced pre-contractions

EtOAc-vasorelaxant responses took place in a concentration-dependent manner. In addition, vasorelaxation recorded similar result, either in presence (EC₅₀ = 423.7 ± 146.6 μg/mL) or absence (EC₅₀ = 227.7 ± 122.4 μg/mL) of functional endothelium (p > 0.05) (Figure 1d, e, and f). Thus, it is possible inferring that the observed vasorelaxant activity did not depend on functional endothelium.

EtOAc fraction effect on PHE-induced concentration–response curves observed for endothelium-denuded preparations

Based on Figure 2a, PHE-induced vasoconstriction was fully inhibited after EtOAc pretreatment, at concentration of 1,000 µg/mL. EC₅₀ values did not significantly differ from each other among the control, 100 µg/mL and 250 µg/mL groups.

Figure 2
a: The concentration–response curves induced by PHE (10-9 to 10-4 M) in control (●) and in pretreated vessels with of EtOAc 100 (■), 250 (▲) or 1000 (□) µg/mL (30 min). Values are expressed as the mean ± S.E.M.; n=7; ***p<0.001 vs. E_max control. b: The vasorelaxant effect induced by EtOAc on the KCl 25 mM precontracted rings (■). Values are expressed as the mean ± S.E.M; n=6. c: Concentration–response curves induced by CaCl₂ (10-6 - 3x10-2 M) in endothelium-denuded aortic rings (n=6) in control (●) and in the presence of EtOAc 100 (■), 250 (▲) or 1,000 (▼) µg/mL pretreatment for 30 min. Values are expressed as the mean ± S.E.M.; ***p<0.001 vs. E_max control. d: Effect of EtOAc on potassium channels. Vasorelaxant effect induced by EtOAc on PHE 10-7 M (●) or KCl 25 mM (■) precontracted endothelium-denuded aortic rings (n=6) in the absence or in the presence of TEA 3mM (▲), GLIB 3x10-6 M (▼) or 4AP 3mM (♦). Values are expressed as the mean ± S.E.M.

EtOAc was capable of inhibiting contractions induced by cumulative PHE addition to endothelium-denuded preparations, and it suggested that EtOAc can likely act in the contractile machinery of vascular smooth muscles.

EtOAc fraction effect on KCl 25 mM-induced pre-contracted aortic rings

Depolarizing solution-induced contraction is triggered by calcium influx. EtOAc effect on KCl 25 mM pre-contraction is shown in Figure 2b. EtOAc induced concentration-dependent relaxation in KCl pre-contracted vessels, and it suggested the involvement of voltage-gated calcium channels in this vasorelaxation process.

EtOAc fraction effect o_n CaCl2-induced contractions

CaCl₂-induced concentration–response curves were recorded both before and after EtOAc addition to the analyzed samples. EtOAc fraction of 1,000 µg/mL was capable of inhibiting cumulative CaCl₂ addition-induced contractions (E_max = 15.76 ± 4.01%***) (Figure 2c). EC₅₀ values were not significantly different among the control (E_max = 100 ± 4.21%), 100 µg/mL (E_max = 100 ± 7.01%) and 250 µg/mL (E_max = 70.68 ± 11.55%) groups. These data have indicated that the EtOAc fraction has successfully inhibited CaCl₂-induced contractions in rat thoracic aortae rings by blocking Ca_V channels. However, another mechanism may be involved in the EtOAc-induced vasorelaxation.

EtOAc fraction effect on potassium channels

The role played by potassium channels in EtOAc-induced vasorelaxant response was assessed through experimental protocols applied in the presence of tetraethylammonium (TEA) 3 mM, which is a nonselective potassium channels’ blocker; of 4-AP (3 mM), which is a selective Kv-channel blocker; and of GLIB (3x10-6 M), which is a selective K_ATP-channel blocker (Figure 2d). Accordingly, as shown in Figure 2D, there was no statistically significant difference between vasorelaxant effect on KCl-induced contraction and on PHE-induced contraction in the presence of TEA (3 mM), GLIB (3x10-6 M) or 4-aminopyridine (3 mM). Therefore, it is possible saying that no potassium channel was involved in EtOAc’s vasorelaxant effect.

UPLC-DAD-ESI-MS analysis

The EtOAc fraction was qualitatively analyzed through UPLC-DAD-ESI-MS. Typical chromatographic profiles and tentatively identified chemical structures are shown in Figure 3. UPLC-DAD-ESI-MS analysis has evidenced five cinnamic acid derivative amides, namely: N-trans-feruloyloctopamine (1), N-trans-caffeoyltyramine (2), cis and trans­- terrestriamide (3,4) and terrestribisamide (5). All these amides were identified based on comparisons to data available in the literature and supported by compounds previously reported in Tribulus terrestris.

Figure 3
Typical chromatographic profiles and chemical structures of constituents tentatively identified by UPLC-ESI-MS for the ethyl acetate fraction of Tribulus terrestris. a: DAD chromatographic profile at 210 nm. b: Total ion chromatogram in ESI-MS scan negative ionization mode. Attempts to characterize the peaks: (1) N-trans-feruloyloctopamine, (2) N-trans-caffeoyltyramine, (3,4) terrestriamide (cis and trans isomers), and (5) terrestribisamide.

DISCUSSION

Tribulus terrestris has been used to treat cardiovascular diseases, such as coronary heart disease, cerebral arteriosclerosis, myocardial infarction, thrombosis and hypertension, for a long period-of-time (Verma et al. 2021). Previous studies have evidenced a wide range of medicinally important chemical constituents, mainly flavonoids, saponins and cinnamic acid amides, in Tribulus terrestris fruit and root extracts (Bhutani et al. 1969, Su et al. 2009, Song et al. 2016). Despite the well-known potential of Tribulus terrestris fruit extract to reduce systolic blood pressure and to protect vascular endothelium in animal models (Jiang et al. 2017, Semerdjieva & Zheljazkov 2019, Verma et al. 2021), its effect on vascular tone remains poorly explored.

Tribulus terrestris fruit crude extract (CE) was capable of relaxing pre-contracted aortae. This response was concentration-dependent and endothelium-independent. PHE is an α_1-adrenergic receptor agonist capable of contracting isolated rat aortae through mixed coupling, i.e., through both pharmacomechanical and electromechanical mechanisms. The pharmacomechanical mechanism involves sarcoplasmic reticulum-Ca2+ mobilization by inositol triphosphate (IP₃). The electromechanical mechanism, in its turn, involves changes in membrane potential and the activation of Ca_V channels, with subsequent Ca2+ influx from the extracellular medium and increase in intracellular Ca2+ levels.

CE likely induces rat aortae vasorelaxation through direct action on vascular smooth muscles, rather than through endothelium-derived relaxing factors, such as nitric oxide or prostacyclin, since the herein observed vasorelaxation was endothelium-independent (Figure 1a). However, according to Phillips et al. (2006), who conducted a study with the whole plant, which was collected in Kuwait, vasorelaxation promoted by T. terrestris in mesenteric rings took place in an endothelium-dependent manner and it likely involved nitric oxide release and membrane hyperpolarization. The same herb extracts may have different action mechanisms in isolated rat aortae and in the mesenteric artery due to differences in tonus control mechanisms in arteries’ conductance and resistance processes (Fisher 2010). Furthermore, the current study only used T. terrestris fruits, whose chemical composition may differ from that of other plant parts.

Chemical compounds associated with vasorelaxant effect may be found at high concentration or bioavailability in the Tribulus terrestris EtOAc fraction, which recorded the best vasorelaxation outcome. This vascular effect was concentration-dependent and endothelium-independent. EtOAc fraction was capable of inhibiting contractions induced by cumulative PHE addition to endothelium-denuded preparations, and it suggested that this fraction can likely act in the contractile machinery of vascular smooth muscles. There are some mechanisms likely involved in this effect on smooth muscles, namely: decrease in Ca2+ influx caused by cell membrane calcium channel blockade, inhibited Ca2+ release from intracellular stores, increased K+ efflux due to potassium channel opening, or other mechanism inhibition in the contractile apparatus.

The Tribulus terrestris EtOAc fraction was added after depolarizing solution-based pre-contraction to assess its vascular effect; vasorelaxation was observed. The K+ ions’ concentration in the extracellular medium has increased after the Krebs Henseleit solution was exchanged for the KCl 25 mM depolarizing solution, and it led to imbalance in electrolyte homeostasis, which prevented K+ ion efflux from the intracellular medium to the extracellular one. Depolarizing solution induced both membrane depolarization and contractile response due to increased Ca2+ influx through voltage-gated calcium channels (Ca_Vs). Results suggest that EtOAc fraction-induced relaxation does not depend on potassium channels and that it likely acts through calcium channels’ blockage.

Ca2+ influx is an important mechanism involved in vascular smooth muscle contractions. Ca2+ channels are activated in the presence of high extracellular K+ or PHE concentrations (Ets et al. 2016). Tribulus terrestris EtOAc fraction was capable of inhibiting contraction induced by CaCl₂ in calcium-free solution, and it confirmed the calcium channels’ blockage hypothesis. The EtOAc fraction concentration capable of inhibiting the PHE-induced contraction was the very same concentration capable of inhibiting the CaCl₂-inducted contraction. The EtOAc fraction is likely capable of inhibiting calcium influx-induced vasoconstriction by blocking calcium channels in the cell membrane, most likely Ca_V channels. Ca_V1.2 is the main voltage-gated calcium channel type found in smooth muscles. These channels play a crucial role in the development and maintenance of smooth muscle contractions, being closely related to variations in membrane potential (Silva et al. 2015). Furthermore, Ca_V channels represent an important target for plant-derived compounds, as previously reported for the flavonoid morin and the indole-alkaloid aristoteline in the rat aortae (Carullo et al. 2021, Romero et al. 2019).

Vasorelaxant effect can be attributed to several phytochemicals previously reported in Tribulus terrestris fruits (Semerdjieva & Zheljazkov 2019). Previous studies have attributed this effect to the saponins present in the extract (Altug et al. 2009, Li et al. 2013). However, tentatively identified chemical compounds found in the EtOAc fraction were mainly detected in cinnamic acid derivatives’ amides, as shown in Figure 3. The negative ESI-MS spectra of peaks observed at 2.87 min and 3.07 min were featured by deprotonated molecular ion of m/z 328.27 Da [M-H]- and m/z 298.26 Da [M-H]-, respectively. M(H)- fragments with even molar masses suggested an odd number of N atoms in the molecule (Silverstein & Bassler 1991). Molecular masses of 329.24 and 299.62 Da were suggested for peaks observed at 2.87 min and 3.07 min, based on predicted molecular formulas, such as C₁₈H₁₉NO₅ and C₁₇H₁₇NO₄. This factor suggests the presence of N-trans-feruloyloctopamine (1) and N-trans-caffeoyltyramine (2), based on Song et al. (2016). As for constituent (2), peak at m/z 597.45 was indicative of dimeric adduct [2 M-H]-m/z (MS spectrum not shown).

The negative ESI-MS spectrum observed for peak at 3.50 min, m/z 326.4 Da [M-H]- and 653 [2 M-H]- were compatible to the quasi-molecular and dimeric adducts of terrestriamide (3,4), and it corroborated the ones described by Hong et al. (2013). The similarity between the negative ESI-MS spectra of peaks observed at 3.50 min and 3.63 min suggested cis and trans terrestriamide isomers. In addition, UV absorption at 221, 292 and 318 nm corroborates that of terrestriamide (3,4), which is typical of 3,4-dioxygenated cinnamic acid derivatives (Wu et al. 1999).

UV absorptions (UV λmax nm: 317, 287, 228 and 222) of peak with RT = 3.87 (Figure 3a) was compatible to those observed for terrestribisamide (5) and reported by Wu et al. (1999). Constituent eluted at 3.93 min (Figure 3b) led to peak with quasi-molecular ion at m/z 439.40 [M-H]-, which was also compatible to the terrestribisamide (5) proposed by Wu et al. (1999) and Hong et al. (2013). Fragmentation [M-H]- of compound 5 provided an odd mass fragment, and it suggested that this molecule may either have a pair of N atoms or no N atoms, at all (Silverstein & Bassler 1991). According to Wu et al. (1999), terrestribisamide’s molecular formula is C₂₄H₂₈N₂O₆, whose molecular ion peak has m/z of 440.31 [M]-, a fact that confirms the presence of 2 N atoms in this molecule.

Studies focused on investigating biological activities of cinnamic-related molecules have shown that they have anticancer (De et al. 2011), antidiabetic (Song et al. 2016), antituberculosis (De et al. 2012), antimalarial (Takao et al. 2017), antifungal (Tawata et al. 1996), antimicrobial (Guzman 2014), antiatherogenic (Lapeyre et al. 2005), antioxidant (Sova 2012), antispasmodic (Lima et al. 2014), antihypertensive (Ohno et al. 2008), and vasorelaxant (Othman et al. 2006) activity. Othman et al. (2006) have isolated ethyl cinnamate from crude CH₂Cl₂ extract deriving from Kaempferia galanga L. They reported that the active component had vasorelaxant effect, likely due to calcium influx inhibition in rat aortae. Later, Othman et al. (2006) have evidenced that ethyl cinnamate was capable of reducing blood pressure in anesthetized rats. Cinnamic acid derivatives found in the EtOAc fraction of Tribulus terrestris fruits can block calcium channels and contribute to the vasorelaxant effect observed in rat aortae. However, these data remain preliminary and insufficient to establish any association.

CONCLUSIONS

Based on the current results, Tribulus terrestris fruit preparations have vasorelaxant effect on isolated rat aortae. The strongest vasorelaxant effect was associated with the EtOAc fraction, which was capable of reducing calcium influx through Ca_V channels. Chemical featuring based on UPLC-DAD-ESI-MS has indicated that EtOAc fractions are rich in cinnamic acid derivatives’ amides. Overall, the current findings substantiate the use of Tribulus terrestris fruits as vasorelaxant herbal drug. However, further studies should conduct deep phytochemical analysis to unequivocally identify Tribulus terrestris cinnamic acid derivative amides and to correlate them to beneficial applications focused on treating different cardiovascular diseases.

Acknowledgements

This research is dedicated to Prof. Raimundo Vicente de Sousa, PhD. This study was financed in parts by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação de Pesquisa do Estado de Minas Gerais (FAPEMIG), and the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES – Finance Code 001).

• Data availability
  Data will be made available on request.

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Edited by

• Handling editor
  Alexander Kellner

Data availability

Data will be made available on request.

Publication Dates

• Publication in this collection
  17 July 2026
• Date of issue
  2026

History

• Received
  20 Sept 2024
• Accepted
  14 Mar 2026