Endothelium-Independent Vasorelaxant Effects of Sudachitin and Demethoxysudachitin, Polymethoxyflavone from the Peel of Citrus sudachi on Isolated Rat Aorta

Abstract

Although polymethoxyflavones have been reported to exhibit various pharmacological actions, the effects of polymethoxyflavones sudachitin and demethoxysudachitin from the peel of Citrus sudachi on the cardiovascular system have not been clarified. This study investigated the mechanisms of vasorelaxation induced by sudachitin and demethoxysudachitin in rat aorta. Both compounds inhibited phenylephrine-induced contractions in a concentration-dependent manner. This was also observed in the case of potassium chloride (KCl)-induced contractions although the inhibitory effect was weak. In both contraction types, no differences were found in the inhibitory effects of sudachitin and demethoxysudachitin between endothelium-intact and -denuded aorta. The relaxant effects of sudachitin in endothelium-intact aortas were not affected by the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME) or the cyclooxygenase inhibitor indomethacin. In endothelium-denuded aorta, propranolol did not affect the relaxant effect of sudachitin. Both the adenylate cyclase activator forskolin- and soluble guanylate cyclase activator sodium nitroprusside-induced relaxant effects were potentiated by preincubation of sudachitin. Furthermore, the relaxant effect of sudachitin was not affected by the adenylate and guanylate cyclase inhibitors SQ22536 and or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), respectively. Finally, we examined the effect of phosphodiesterase inhibition. Phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, cilostamide or sildenafil) alone, sudachitin alone, and a combination of phosphodiesterase inhibitors with sudachitin exhibited relaxant effects, while the lack of any interaction between each phosphodiesterase inhibitor and sudachitin indicated an additive effect between the two substance categories. These results suggest that sudachitin and demethoxysudachitin cause endothelial-independent relaxation, and that the mechanism of vasorelaxation by sudachitin is associated with the enhancement of cAMP- and guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent pathways.

INTRODUCTION

Citrus peel contains many polymethoxyflavones (PMF),¹⁾ which have been shown to have a variety of pharmacological functions including anticancer,²⁾ anti-inflammatory³⁾ and hepatoprotective⁴⁾ effects, lipolysis promotion,⁵⁾ and prevention of dementia.⁶⁾ Thus, research on citrus fruits with high PMF content for the development of health food products is increasingly attracting attention.

Citrus sudachi Hort. ex Shirai (Citrus sudachi) is a popular fruit in Tokushima Prefecture, Japan, and its peel contains high amounts of PMF⁷⁾ with the most abundant being sudachitin (SDC, Fig. 1) followed by demethoxysudachitin (DMSDC, Fig. 1). Both SDC and DMSDC are unique to Citrus sudachi and not contained in other citrus fruits.

Fig. 1. The Chemical Structure of SDC and DMSDC in the Peel of Citrus sudachi

Similar to other PMF, SDC has been reported to have anti-inflammatory effects and to improve lifestyle-related diseases such as diabetes and hyperlipidemia in cell lines and rodent models.^8–10) In a clinical study, SDC-containing sudachi peel extract powder has been shown to improve visceral fat content in human individuals at risk of developing diabetes,¹¹⁾ and the preventive effect of SDC on lifestyle-related diseases has been attracting attention. In addition, it was recently reported that long-term treatment of diet-induced obese mice with SDC modulates liver clock genes and suppresses hepatic fat accumulation.¹²⁾ However, the effects of SDC on the cardiovascular system, which may be the basis for the pathogenesis of lifestyle-related diseases, have not been sufficiently investigated.

In this light, the purpose of this study was to evaluate the effects of SDC and DMSDC from sudachi peel on vascular tonus and to investigate mechanism of action of SDC using aorta preparations isolated from rats.

MATERIALS AND METHODS

Chemicals

SDC and DMSDC were kindly provided by Ikeda Yakusou Co., Ltd., (Tokushima, Japan). Phenylephrine hydrochloride, acetylcholine hydrochloride (ACh), papaverine hydrochloride (PPV) and sodium pentacyanonitrosylferrate (III) dihydrate (SNP) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Potassium chloride (KCl) and 3-isobutyl-1-methylxanthine (IBMX) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). N-Nitro-L-arginine methyl ester hydrochloride (L-NAME), indomethacin, propranolol hydrochloride, forskolin, SQ22536, and sildenafil were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and cilostamide were purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). All other chemicals were of the highest commercially available grade.

SDC, DMSDC, L-NAME, indomethacin, SQ22536, forskolin, ODQ, IBMX, cilostamide and sildenafil were dissolved in 100% dimethyl sulfoxide (DMSO) and were added in the organ bath so that their concentration did not exceed 0.3%. All the other chemical stocks were aqueous solutions prepared and diluted with distilled water.

Animals

Male Wistar rats (8–10 weeks old; weighing 170–290 g; total number, 55; Japan SLC, Hamamatsu, Japan) were housed under controlled conditions (21–23 °C, relative air humidity 50 ± 15%) and a fixed 12/12 h light/dark cycle (8:00–20:00), with food and water available ad libitum. This study was approved by the Animal Care and Use Committee of the Mukogawa Women’s University (Approval Nos. P-17-2021-02, P-17-2022-02) and conducted according to the guidelines of the Laboratory Animal Center of the School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University.

Preparation of Rat Aorta Tissues

All rats were anesthetized with pentobarbital sodium (200 mg/kg, intraperitoneally) and euthanized by exsanguination from the carotid arteries. Subsequently, aortae were excised and placed in a well-oxygenated (95% O₂–5% CO₂) modified Krebs–Henseleit solution (KHS) (118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.9 mM NaHCO₃ and 11.1 mM glucose, pH 7.4). The aortae were then cleaned of loosely attached fat and connective tissues and cut into ring segments of 3–4 mm in length. To acquire vascular preparations without the endothelium, the intimal surface of the ring segments was gently rubbed with cotton moistened with KHS.

Recording of Tension Changes

The ring preparations were mounted using stainless hooks under the optimal resting tension of 1 g in a 10 mL organ bath containing well-oxygenated KHS at 36 °C. The changes in tension of ring preparations were isometrically recorded using a force-displacement transducer (TB-612T, Nihon Kohden, Tokyo, Japan) connected to a polygraph (RM-6100, Nihon Kohden). The ring preparations were incubated for more than 60 min to equilibrate and then contracted with high-KCl (64 mM) KHS ensuring the functional integrity of the preparations. Subsequently, the ring preparations were washed by replacing the high KCl solution with KHS to fully recover. After confirming stable contraction of aorta by phenylephrine (0.3 µM), endothelium functionality was confirmed in all preparations by determining the ability of ACh (10 µM) to induce >70% relaxation of precontracted with phenylephrine.

Evaluation of Relaxant Effects of SDC and DMSDC

In the experiments shown in Figs. 2 and 3, phenylephrine (0.3 µM) or high KCl (64 mM) was applied to produce a submaximal contraction, and after confirmation of steady-state contraction, SDC (0.3–100 µM) or DMSDC (0.3–100 µM) was added cumulatively to the bath medium. Following treatment with the highest concentration of SDC or DMSDC, PPV (100 µM) was added to determine the maximum vasorelaxation. The EC₅₀ values of SDC and DMSDC were determined for each preparation.

Fig. 2. Relaxant Effects of SDC and DMSDC on Phenylephrine-Induced Contraction of Rat Aorta

Representative examples of relaxant effects of SDC on phenylephrine (0.3 µM)-induced contraction of endothelium-intact (EC(+), A) and -denuded (EC(−), B) aorta. Concentration–response curves for the relaxant effects of SDC (C) and DMSDC (D) on phenylephrine-induced contraction of endothelium-intact and -denuded aorta. Symbol and vertical bars represent the mean ± S.E.M. of 4 or 6 preparations for each group.

Fig. 3. Relaxant Effects of SDC and DMSDC on High KCl-Induced Contraction of Rat Aorta

Representative examples of relaxant effects of SDC on KCl (64 mM)-induced contraction of endothelium-intact (EC(+), A) and -denuded (EC(−), B) aorta. Concentration–response curves for the relaxant effects of SDC (C) and DMSDC (D) on high KCl-induced contraction of endothelium-intact and -denuded aorta. Symbol and vertical bars represent the mean ± S.E.M. of 5 preparations for each group.

Evaluation of the SDC Mechanism of Action

As shown in Figs. 4 and 5, DMSO 0.1%, L-NAME (100 µM), indomethacin (10 µM) or propranolol (0.1 µM) were incubated for 20 or 30 min. Then, phenylephrine (0.3 µM) was added in the bath medium, and after confirming steady-state contraction, SDC (10 or 30 µM) was added to observe tension change, followed by PPV (100 µM) to ensure maximal relaxation. In the experiments shown in Figs. 6A and 7A, DMSO 0.1%, or SDC (3, 10, or 30 µM) (with propranolol 0.1 µM in Fig. 6A) was incubated for 20 min and phenylephrine (0.3 µM) was then added in the bath medium. After confirming steady-state contraction, forskolin (0.1–1000 nM) or SNP (0.1–100 nM) were added cumulatively to observe tension change. Furthermore, in the experiments shown in Figs. 6B and 7B, SQ22536 (100 µM) or ODQ (10 µM), which produced a maximum inhibition to each target, were incubated for 60 or 30 min, respectively. Then, phenylephrine (0.3 µM) was added in the bath medium, and after confirming steady-state contraction, SDC (0.3–100 µM) was added cumulatively to test tension change. Similarly, in the experiments shown in Figs. 8 and 9, phenylephrine (0.3 µM) was added to the bath medium and, after confirming steady-state contraction, DMSO 0.01% followed by DMSO 0.01%, phosphodiesterase (PDE) inhibitor (IBMX (0.5 µM), cilostamide (0.02 µM) or sildenafil (10 µM)) followed by DMSO 0.01%, DMSO 0.01% followed by SDC (5 µM) or PDE inhibitor followed by SDC (5 µM) were added to test tension change.

Fig. 4. Effects of L-NAME and Indomethacin (INM) on SDC-Induced Relaxation of Endothelium-Intact Aorta

Incubation with L-NAME (100 µM), INM (10 µM), or DMSO (0.1% control) for 30 min before adding phenylephrine indicated relaxant effects of SDC (10 or 30 µM) on phenylephrine (0.3 µM)-induced contraction. Each bar represents the mean ± S.E.M. of 4 or 5 preparations. NS: not significant vs. control.

Fig. 5. Effect of Propranolol (Pro) on SDC-Induced Relaxation of Endothelium-Denuded Aorta

Incubation with Pro (0.1 µM) or DMSO (0.1% control) for 20 min before adding phenylephrine indicated relaxant effects of SDC (10 or 30 µM) on phenylephrine (0.3 µM)-induced contraction. Each bar represents the mean ± S.E.M. of 4 preparations. NS: not significant vs. control.

Fig. 6. Effect of SDC on Forskolin-Induced Relaxation of Endothelium-Denuded Aorta (A)

Incubation of SDC (3, 10, and 30 µM) with propranolol (0.1 µM) or DMSO (0.1% control) for 20 min before adding phenylephrine indicated relaxant effects of forskolin (0.1–1000 nM) on phenylephrine (0.3 µM)-induced contraction. Symbol and vertical bars represent the mean ± S.E.M. of 5 preparations. Effect of SQ22536 (SQ) on SDC-induced relaxation of endothelium-denuded aorta (B). Incubation with SQ (100 µM) or DMSO (0.1% control) for 60 min before adding phenylephrine indicated relaxant effects of SDC (0.3–100 µM) on phenylephrine (0.3 µM)-induced contraction. Symbol and vertical bars represent the mean ± S.E.M. of 4 preparations.

Fig. 7. Effect of SDC on SNP-Induced Relaxation of Endothelium-Denuded Aorta (A)

Incubation with SDC (3, 10, and 30 µM) or DMSO (0.1% control) for 20 min before adding phenylephrine indicated relaxant effects of SNP (0.1–100 nM) on phenylephrine (0.3 µM)-induced contraction. Symbol and vertical bars represent the mean ± S.E.M. of 4 or 5 preparations. Effect of ODQ on SDC-induced relaxation of endothelium-denuded aorta (B). Incubation with ODQ (10 µM) or DMSO (0.1% control) for 30 min before adding phenylephrine indicated relaxant effects of SDC (0.3–100 µM) on phenylephrine (0.3 µM)-induced contraction. Symbol and vertical bars represent the mean ± S.E.M. of 5 preparations.

Fig. 8. Combinational Effects of SDC and IBMX-Induced Relaxation of Endothelium-Denuded Aorta

Representative examples of relaxant effects of DMSO (0.01%) followed by DMSO (0.01%) (A), IBMX (0.5 µM) followed by DMSO (0.01%) (B), DMSO (0.01%) followed by SDC (5 µM) (C), and IBMX (0.5 µM) followed by SDC (5 µM) (D) on phenylephrine (0.3 µM)-induced contraction. Relaxant effects of DMSO (0.02%, Control), SDC (5 µM) alone, IBMX (0.5 µM) alone, and a combination of SDC (5 µM) and IBMX (0.5 µM) on phenylephrine-induced contraction (E). Results of a two-way repeated measures ANOVA with SDC and IBMX as two factors (F). NS: not significant. Each bar represents the mean ± S.E.M. of 5 preparations.

Fig. 9. Combinational Effects of SDC and Cilostamide (Cilo, A, B)- or SDC and Sildenafil (SDN, C, D)-Induced Relaxation of Endothelium-Denuded Aorta

Relaxant effects of DMSO (0.02%, Control), Cilo (0.02 µM) alone, SDC (5 µM) alone, and a combination of Cilo (0.02 µM) and SDC (5 µM) on phenylephrine (0.3 µM)-induced contraction (A). Relaxant effects of DMSO (0.02%, Control), SDN (10 µM) alone, SDC (5 µM) alone, and a combination of SDN (10 µM) and SDC (5 µM) on phenylephrine (0.3 µM)-induced contraction (C). Results of a two-way repeated measures ANOVA with SDC and Cilo (B) or SDC and SDN (D) as two factors. NS: not significant. Each bar represents the mean ± S.E.M. of 5 preparations.

Data Analysis

The percentage of relaxation was calculated considering 0% relaxation the maximum tension level obtained by 0.3 µM phenylephrine and 100% relaxation the tension caused by application of 100 µM PPV.

Data were plotted as a function of drug concentrations and fitted to the equation:

  

where E is the % relaxation at a given concentration, E_max is the maximum response, C is the relaxation drug concentration, nH is the Hill coefficient (slope function), and EC₅₀ is the effective drug concentration causing 50% relaxation. Logistic curve fitting was performed using the Image J software (version 1.53n, NIH, U.S.A., https://imagej.nih.gov/ij/).

Statistical Analysis

All values in the text and illustrations are expressed as mean ± standard error of the mean (S.E.M.) of the data obtained from the different numbers (n) of preparations. Statcel–the Useful Addin Forms on Excel–4th ed. (OMS Publications, Tokyo, Japan) and Microsoft^® Excel^® of Microsoft 365 were used for statistical analysis. Logarithmic transformation of EC₅₀ values was for statistical analysis. Welch’s t-test was used to evaluate the statistical significance between two groups, while for testing multiple groups, one-way ANOVA followed by Dunnett’s multiple comparison test was used considering a p value less than 0.05 statistically significant. To detect additive or synergistic effects between SDC and PDE inhibitors, a two-way repeated measures ANOVA with SDC and PDE inhibitors as two factors was performed, and a p value less than 0.05 was considered for significant interaction effects.

RESULTS

Effects of SDC and DMSDC on Phenylephrine-Induced Contraction

SDC (0.3–100 µM) caused vasorelaxation of phenylephrine (0.3 µM)-induced contraction of endothelium-intact (Fig. 2A) and -denuded (Fig. 2B) preparations, in a concentration-dependent manner. No differences in the relaxation potency of SDC between endothelium-intact (EC₅₀: 1.69 ± 0.20 × 10⁻⁵ M, n = 8) and endothelium-denuded preparations were observed (EC₅₀: 1.50 ± 0.06 × 10⁻⁵ M, n = 6, NS, Welch’s t-test, Fig. 2C). Similarly, DMSDC (0.3–100 µM) inhibited phenylephrine-induced contraction, and no differences in the relaxation potency of DMSDC between endothelium-intact (EC₅₀: 2.99 ± 1.05 × 10⁻⁵ M, n = 4) and endothelium-denuded preparations were observed (EC₅₀: 2.81 ± 1.15 × 10⁻⁵ M, n = 4, NS, Welch’s t-test, Fig. 2D).

Effects of SDC and DMSDC on High KCl-Induced Contraction

SDC at concentrations equal to or higher than 30 µM induced vasorelaxation in high KCl (64 mM)-induced contraction of endothelium-intact (Fig. 3A) and -denuded (Fig. 3B) preparations, while the maximum relaxation efficacy of SDC was about 50% (Fig. 3C). DMSDC at the high concentration of 100 µM slightly inhibited KCl-induced contractions with an efficacy approximately 10% (Fig. 3D).

Effects of L-NAME and Indomethacin on SDC-Induced Relaxation

To determine any potential involvement of vascular endothelium in SDC-induced relaxation, we investigated the effects of preincubation with the nitric oxide (NO) synthase inhibitor L-NAME, or the cyclooxygenase inhibitor indomethacin on SDC-induced relaxation on endothelium-intact preparations precontracted with phenylephrine (0.3 µM). Neither L-NAME (100 µM) nor indomethacin (10 µM) affected SDC (10 or 30 µM)-induced relaxation (Fig. 4).

Effect of Propranolol on SDC-Induced Relaxation

To clarify for the potential involvement of β-adrenoceptor on SDC-induced relaxation, we evaluated the effect of preincubation with propranolol, a β-adrenoceptor antagonist on SDC-induced relaxation when precontracted with phenylephrine (0.3 µM) on endothelium-intact preparations. Results revealed that propranolol (0.1 µM) had no effects on SDC (10 or 30 µM)-induced relaxation (Fig. 5).

Effects of cAMP-Related Agents on SDC-Induced Relaxation

To examine the involvement of the cAMP-dependent pathway in SDC-induced relaxation, we investigated the effect of the adenylate cyclase activator forskolin preincubated with SDC on, the endothelium-denuded aorta precontracted with phenylephrine (0.3 µM). Forskolin (0.1–1000 nM) resulted in a concentration-dependent relaxation (Control, EC₅₀: 3.37 ± 2.03 × 10⁻⁸ M, n = 5), and the concentration–response curve of forskolin was shifted leftward in a SDC concentration-dependent manner (EC₅₀ (M) for 3, 10, and 30 µM SDC were 1.75 ± 0.74 × 10⁻⁸, 8.34 ± 4.25 × 10⁻⁹ and 4.95 ± 1.37 × 10⁻¹⁰, respectively (n = 5), Fig. 6A).

We also investigated the effect of the adenylate cyclase inhibitor SQ22536 on the SDC-induced relaxation of endothelium-denuded aortas precontracted with phenylephrine (0.3 µM). The concentration–response curve for SDC (0.3–100 µM) was not shifted upon pre-incubation with SQ22536 (100 µM) compared with the control (Fig. 6B). EC₅₀ values (M) were 1.02 ± 0.13 × 10⁻⁵ (control) vs. 1.18 ± 0.19 × 10⁻⁵ (SQ22536) (n = 4, NS).

Effects of Guanosine 3′,5′-Cyclic Monophosphate (cGMP)-Related Agents on SDC-Induced Relaxation

To examine the involvement of the cGMP-dependent pathway in SDC-induced relaxation, we evaluated the effect of preincubation with SDC on SNP-induced relaxation of the endothelium-denuded aorta precontracted with phenylephrine (0.3 µM). The soluble guanylate cyclase activator SNP (0.1–100 nM) caused a concentration-dependent relaxation (Control, EC₅₀: 1.93 ± 0.47 × 10⁻⁹ M, n = 5), and the SNP concentration–response curve was shifted leftward in a SDC concentration-dependent manner (EC₅₀ (M) for 3, 10, and 30 µM SDC were 1.30 ± 0.17 × 10⁻⁹ (n = 4), 7.87 ± 2.96 × 10⁻¹⁰ (n = 5) and 3.83 ± 0.39 × 10⁻¹⁰ (n = 4), respectively) when compared with control (Fig. 7A).

We also investigated the effect of the guanylate cyclase inhibitor ODQ on the SDC-induced relaxation of endothelium-denuded aortas precontracted with phenylephrine (0.3 µM). The concentration–response curve of SDC (0.3–100 µM) was not shifted on preincubation with ODQ (10 µM) when compared with the control (Fig. 7B). EC₅₀ values (M) were 1.19 ± 0.13 × 10⁻⁵ (control) vs. 1.18 ± 0.19 × 10⁻⁵ (ODQ) (n = 5, NS).

Effects of SDC in Combination with PDE Inhibitor-Induced Relaxation

To examine the involvement of cAMP- and cGMP-dependent pathways in SDC-induced relaxation, we investigated the combined effects of IBMX, a non-selective PDE inhibitor, in the endothelium-denuded aorta. SDC 5 µM alone, IBMX 0.5 µM alone and the combination of SDC 5 µM with IBMX 0.5 µM inhibited phenylephrine (0.3 µM)-induced contraction compared with the control (Figs. 8A–E). A two-way ANOVA with SDC and IBMX as factors was performed and no significant interaction was observed (Fig. 8F, p = 0.94, n = 5).

We also investigated the combined effects of cilostamide, a PDE3 inhibitor, and sildenafil, a PDE5 inhibitor, on the endothelium-denuded aorta. Compared with control, SDC 5 µM alone, cilostamide 0.02 µM alone, and the combination of SDC 5 µM with cilostamide 0.02 µM inhibited phenylephrine (0.3 µM)-induced contraction (Fig. 9A). A two-way ANOVA with SDC and cilostamide as factors was performed and no significant interaction was observed (Fig. 9B, p = 0.57, n = 5). SDC 5 µM alone, sildenafil 10 µM alone, and the combination of SDC 5 µM with sildenafil 10 µM inhibited phenylephrine (0.3 µM)-induced contraction compared with the control (Fig. 9C). A two-way ANOVA with SDC and sildenafil as factors was performed and no significant interaction was observed (Fig. 9D, p = 0.77, n = 5).

DISCUSSION

Relaxant Effects of SDC and DMSDC on Phenylephrine-Induced Contraction

The mechanism responsible for phenylephrine-induced vasoconstriction involves the selective activation of α₁ adrenergic receptor by phenylephrine followed by the activation of Gq protein, and finally the increase in intracellular Ca²⁺ concentration which causes the contraction. In our study, we used rat thoracic aortic ring preparations, which exhibited α₁ adrenergic receptor-stimulated contraction via coupled Gq protein and have been used to evaluate the relaxant effects of many pharmaceuticals and natural products.^13–15) The results revealed that both SDC and DMSDC exhibited concentration-dependent relaxant effects on phenylephrine-induced contractions (Fig. 2). Comparison of the SDC- and DMSDC-induced relaxation EC₅₀ values showed that the relaxation of SDC was 1.8-fold stronger than that of DMSDC in endothelium-intact cells, and 1.9-fold stronger in endothelium-removed cells. These results indicated that the SDC relaxant effects on Gq protein signaling-dependent contractions were approximately 2-fold stronger than in DMSDC. In addition, there was no significant difference in the EC₅₀ values for the relaxant effects of SDC and DMSDC with or without the endothelium, suggesting that the site of action of SDC and DMSDC may be localized in the vascular smooth muscle but not in the endothelium.

Relaxant Effects of SDC and DMSDC on Depolarizing Contractions

The resting membrane potential of excitable cells, including those in vascular smooth muscles, is determined by the concentration ratio of intracellular and extracellular K⁺ via Na⁺-K⁺ exchange transporters. Therefore, by artificially increasing the extracellular K⁺ concentration, the membrane potential of the preparation can be depolarized, leading to the activation of voltage-dependent Ca²⁺ channels. In the present study, we induced depolarization by replacing normal KHS with high KCl KHS which resulted in extracellular Ca²⁺-dependent sustained vasoconstriction (Figs. 3A, B).

Both SDC and DMSDC caused relaxation on high KCl-induced depolarizing contractions with or without endothelium, but the relaxant effects of SDC and DMSDC at the highest concentration of 100 µM were less than 50 and 10%, respectively; thus, EC₅₀ values could not be calculated for either compound (Fig. 3). These results indicated that SDC was more potent than DMSDC in relaxing depolarizing contractions. Furthermore, the lack of any difference in the relaxant effects of SDC and DMSDC in the absence or presence of endothelium suggested that the site of action of SDC and DMSDC is the vascular smooth muscle and not the endothelium. Additionally, the vasorelaxant effects of SDC and DMSDC were more pronounced during Gq protein-coupled receptor-mediated contractions than against depolarizing contractions. The fact that SDC inhibited depolarizing contractions suggests that it may block the Ca²⁺ channels although further studies using electrophysiological techniques will be necessary to clarify the mechanism through which SDC inhibits Ca²⁺ channels.

Sites of Action of SDC-Induced Vasorelaxation

Vascular endothelial cells produce and release endothelium-derived relaxing factors (EDRF) such as NO and prostaglandin I₂, locally regulating vascular smooth muscle tone.¹⁶⁾

In the present study, we investigated whether EDRF was involved in the relaxant effects of SDC. The results showed that the relaxant effect of SDC in endothelium-intact preparations was not affected by the inhibitor of endothelial NO synthase L-NAME or the inhibitor of cyclooxygenase indomethacin (Fig. 4), suggesting that EDRF is not involved in the relaxant effects of SDC. This result also strongly supports that the site of action of SDC-induced relaxation is the vascular smooth muscle rather than the endothelium.

However, in this study, we used the thoracic aorta which is a large vessel and we did not evaluate small vessels. The contribution of EDRF to vascular tone has been reported to differ depending on the blood vessel diameter.¹⁷⁾ Thus, further evaluation is necessary to clarify the relaxant effects of SDC on resistant vessels, such as the mesenteric artery, to identify the specific site of action of SDC.

Mechanism of SDC-Induced Vasorelaxation

Cyclic nucleotides, such as cAMP and cGMP, which are considered key messengers that mediate vasorelaxation, are important for the physiological regulation of vascular tone.¹⁸⁾ Stimulation of β-adrenoceptors activates adenylate cyclase which catalyzes cAMP formation. An increase in cAMP activates protein kinase A (PKA), while the inactivation of myosin light chain (MLC) kinase by PKA inhibits MLC phosphorylation and induces vasorelaxation. The signaling mechanisms of the cGMP-dependent pathway are described below: Vascular endothelial cells produce NO which diffuses from the endothelial cells to smooth muscle cells, where it activates guanylyl cyclase leading to an increase in cGMP. cGMP is then activated by protein kinase G, inhibits Ca²⁺ entry into vascular smooth muscles, and activates K⁺ channels which leads to membrane hyperpolarization and relaxation. Both cAMP- and cGMP-dependent pathways induce vasorelaxation by inhibiting Ca²⁺ release from the sarcoplasmic reticulum via IP₃ receptors and thus decreasing the intracellular Ca²⁺ concentration.

In the present study, we examined the effects of both cAMP- and cGMP-dependent pathways to evaluate the mechanism of SDC-induced vasorelaxation. Results revealed that the relaxant effect of SDC in endothelium-denuded preparations was not affected by the β-adrenoceptor antagonist propranolol, suggesting that β-adrenoceptors are not involved in the observed SDC-induced relaxation (Fig. 5). Both the adenylate cyclase activator forskolin and the guanylate cyclase activator SNP in combination with SDC enhanced vasorelaxation (Figs. 6A, 7A). Moreover, the relaxant effects of SDC were not inhibited by the adenylate cyclase inhibitor SQ22536, or the guanylate cyclase inhibitor ODQ (Figs. 6B, 7B). These results suggest that SDC potentiates both cAMP- and cGMP-dependent signaling pathways without the direct activation of adenylate and guanylate cyclases.

Furthermore, the lowest concentration of SDC that potentiated forskolin- and SNP-induced relaxation was 10 µM (Figs. 6A, 7A), which is consistent with the threshold concentration of SDC that inhibits phenylephrine-induced contraction (just under 20% relaxation at 10 µM SDC, Fig. 2A). These results suggest that the relaxant effect of SDC observed at micromolar concentrations involves both cAMP- and cGMP-dependent pathways.

Next, we attempted to determine whether one of the cAMP- or cGMP-dependent pathway is predominantly involved in the relaxant effect of SDC or if the two pathways were equivalent. The concentration range of SDC that potentiates forskolin- and SNP-induced relaxation was 3–30 µM, while both activators potentiated relaxation at concentrations over 10 µM (Figs. 6A, 7A), suggesting that the cAMP- and cGMP-dependent pathways contributed almost equally to the SDC-induced relaxation.

PDE are enzymes that hydrolyze cAMP and cGMP during cell signaling, are ubiquitously distributed in mammalian tissues, and are classified into 11 families (PDE1-11).¹⁹⁾ PDE affects a wide range of biological systems including ion channel regulation, inflammatory mediator production, memory, differentiation, apoptosis, and lipogenesis.¹⁹⁾ PMF induce many pharmacological effects including PDE inhibition.²⁰⁾ Indeed, SDC has been reported to inhibit both PDE1 and PDE4 in recombinant PDE and beta-cells,^8,10) while no reports on its effects on other PDE subtypes exist. PDE3 and PDE5 are cAMP- and cGMP-specific PDEs, respectively, which are expressed in vascular smooth muscle. PDE3 promotes the degradation of cAMP, while PDE5 promotes the degradation of cGMP.²¹⁾ In this light, we hypothesized that SDC inhibits PDE3 and PDE5, both intracellular cAMP and cGMP levels will increase, finally leading to a vasorelaxation. Nobiletin, a PMF similar to SDC, has also been reported to exhibit vasodilatory effects by activating the guanylate cyclase-cGMP system¹⁵⁾ and inhibition of PDE.^8,10)

In this study, we checked the hypothesis that SDC inhibits PDE resulting in enhancement of both cAMP- and cGMP-dependent pathways by examining whether SDC in combination with various PDE inhibitors act in an additive or synergistic manner in terms of vasorelaxant activity. IBMX is a non-selective, whereas cilostamide and sildenafil are PDE inhibitors with high selectivity for PDE3 and PDE5, and are widely used in biochemistry and pharmacological research.^22–24) The concentrations of PDE inhibitors used in this study were the ones that exhibited 20–30% inhibition of phenylephrine-induced contraction, which is optimal for studying additive or synergistic pharmacological effects of drugs. Experiments using SDC in combination with PDE inhibitors demonstrated the additive effect of SDC and IBMX, suggesting a common PDE target of both compounds (Fig. 8). Moreover, additive effects were observed for both SDC in combination with cilostamide and SDC in combination with sildenafil, suggesting that SDC may inhibit both PDE3 and PDE5 subtypes (Fig. 9). However, whether SDC has a PDE inhibitory effect is not clear without examining its direct action on the PDE enzyme activity. Therefore, studies on the PDE activity using various PDE subtype are necessary to further clarify the vasorelaxant mechanism of SDC.

Human Health Promotion by SDC-Induced Vasorelaxation

The present study revealed three advantages of SDC as a supplemental agent for health promotion.

First, direct action on the vascular smooth muscle. Typical patients with hypertension and hyperlipidemia have attenuated vascular endothelial function. In cases where the endothelial function is completely impaired, drugs and functional foods that target the endothelium may not be sufficiently effective. On the contrary, since site of action of SDC is the vascular smooth muscle, SDC intake in humans may exert a stable vasorelaxant effect independent of the functional status of the endothelium.

Second, wide range of action on PDE’s. Most medical PDE inhibitors developed to date, such as cilostazol and sildenafil, act on a particular PDE subtype specifically. However, SDC is expected to produce more potent vasorelaxation because it has been shown to inhibit a wide range of PDE subtypes, including PDE1 and PDE4^8,10) as well as the PDE3 and PDE5 suggested in the present study (Fig. 9). Additionally, SDC is a natural ingredient of sudachi, a traditional Japanese fruit, and toxicity studies have demonstrated its broad safety profile.²⁵⁾

Third, clinical preventive and therapeutic effects. SDC is expected to increase the metabolism of each affected organ in a favorable manner via its vasorelaxant effect which increases blood flow and oxygen supplied. If these SDC effects can be sustained, they can lead to an overall increase in metabolism, resulting in the improvement in lipid metabolism¹¹⁾ and the prevention of lifestyle-related diseases through the intake of functional foods containing SDC. Future SDC clinical trials on metabolic syndromes, including the improvement of hypertension and peripheral circulation will validate such a claim.

Acknowledgments

We thank Ikeda Yakusou Co., Ltd. for providing us with the SDC and DMSDC samples.

Conflict of Interest

The authors declare no conflict of interest.

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