Hypotensive activity of auraptene, a monoterpene coumarin from Citrus spp.

Abstract

Context: Citrus spp. (Rutaceae) are well-documented for their cardioprotective properties. Auraptene is a bioactive monoterpene coumarin ether abundantly present in the Citrus spp.

Objective: To investigate the hypotensive activity of auraptene.

Methods: Different groups of normotensive rats (n = 5 in each group) were subjected to single intravenous injections of auraptene (125, 250 and 500 µg/kg), nifedipine (as positive control; 63, 125 and 250 µg/kg) or negative control [DMSO/normal saline (1:3)]. Mean arterial blood pressure (MABP) and heart rate (HR) were evaluated following each treatment.

Results: A dose-dependent hypotensive effect was observed following auraptene injection, which was significant at 250 and 500 µg/kg (p < 0.001) but not 125 µg/kg (p > 0.05). With respect to the positive control, nifedipine reduced MABP at all tested doses, dose-dependently and significantly (p < 0.001). The MABP lowering effect of auraptene was found to be significantly lower than that of nifedipine (p < 0.001).

Conclusion: In light of the present findings, auraptene has moderate hypotensive activity. Further investigations are recommended to explore the effects of higher doses as well as oral administration of this phytochemical.

• Blood pressure
• calcium channel blockers
• cardiovascular disease
• heart rate
• hypertension
• natural product

Introduction

Cardiovascular disease (CVD) is the leading cause of mortality in the world. Arterial hypertension is among the most important and most frequent cardiovascular disorders, in particular in the developing world. Hypertension is normally silent, asymptomatic and preventable, but if remained uncontrolled, it could progress into severe cardiovascular (Davies, 1991), cerebrovascular (Ogunniyi & Talabi, 2001), peripheral vascular (Makin et al., 2001) and neurodegenerative complications (Kivipelto et al., 2002). Regulation of hypertension is the result of a delicate balance between cardiac output and peripheral vascular resistance (Vikrant & Tiwari, 2001). In many types of hypertension, an increase in peripheral vascular resistance is observed, which is associated with elevated intracellular concentrations of Ca²⁺ (Jang et al., 2004; Shehin et al., 1989; Touyz & Schiffrin, 1994). Therefore, calcium channel blockers are among the most widely used medications for the treatment of hypertension.

In recent decades, there has been an increasing interest in the use of alternative medicines and natural products for the treatment of a variety of disorders. Medicinal plants are the main category for such natural products, and there have been numerous reports on their effectiveness in controlling hypertension (Talha et al., 2011). Citrus spp. (Rutaceae) are an important class of plants with remarkable cardioprotective activity (Mulero et al., 2012). Several lines of evidence indicate the inverse association between the consumption of Citrus spp. and incidence of CVD (Mulero et al., 2012; Yamada et al., 2011). There are several mechanisms behind this cardioprotection, such as improving lipid profile by reduction of low-density lipoprotein (LDL) and boosting high-density lipoprotein (HDL) levels, reduction of inflammation, inhibition of pro-oxidant generation and LDL oxidation, and anti-ischemic, antithrombotic and vasorelaxant effects (Gaziano, 1999; Roza et al., 2007; Tribble, 1999).

Auraptene (7-geranyloxycoumarin; Figure 1) is a naturally occurring monoterpene coumarin ether found in many genus of the Rutaceae family, in particular Citrus spp. (Curini et al., 2006; Epifano et al., 2008; Ogawa et al., 2000). Being the most abundant prenyloxycoumarin in nature and the most common component of Citrus spp. (Curini et al., 2006; Epifano et al., 2008), auraptene has been reported to possess several interesting biological activities which are of therapeutic importance (Curini et al., 2006; Epifano et al., 2008; Genovese & Epifano, 2011; Sahebkar, 2011). The most documented activity of auraptene is its powerful cancer chemoprevention effect, which has been confirmed in several studies (Kohno et al., 2006; Tanaka et al., 1998). Further to this effect, auraptene has been reported to have antitumor, antioxidant, antigenotoxic, anti-inflammatory, immunomodulatory, hepatoprotective neuroprotective and antibacterial (Curini et al., 2012; Soltani et al., 2010) properties. In contrast to cancer chemoprevention, possible protective effects of auraptene on cardiovascular health have not been clarified yet. Since there have been two previous in vitro reports on the smooth muscle cell relaxant activity (Yamada et al., 1997) and inhibition of calcium-induced spontaneous heartbeat (Kakiuchi et al., 1991) by auraptene, the present study hypothesized that the plausible calcium blocking activity of this phytochemical may be of potential application in the treatment of hypertension. Hence, the present study set out to verify this hypothesis.

Figure 1.  Chemical structure of auraptene.

Materials and methods

Animals and drugs

The experiments were performed under the Animals (Scientific Procedures) Act of 1986 and conform to the institutional (Mashhad University of Medical Sciences) and National Institutes of Health guidelines for the use of experimental animals. This study was carried out on male Wistar rats (200–250 g) from Razi Institute, Mashhad, Iran. Ketamine 10% and heparin (5000 IU/mL) were obtained from Merck (Rotexmeica, Germany), and xylazine 2% and nifedipine from Alfasan (Woerden, The Netherlands) and Zahravi (Tehran, Iran), respectively. Auraptene and nifedipine (as positive control) were dissolved in a solvent mixture comprising dimethylsulfoxide and normal saline (1:3).

Synthesis of auraptene

Auraptene (7-geranyloxycoumarin) was synthesized based on a previously described method (Askari et al., 2009). In brief, 7-hydroxycoumarin and trans-geranyl bromide were reacted in acetone at room temperature, in the presence of DBU (1,8-diazabicyclo [5.4.0] undec-7-ene). Auraptene was purified from the concentrated reaction mixture as white crystals using column chromatography (petroleum ether/ethyl acetate 9:1 v/v) and its structure was confirmed by ¹H- and ¹³C-NMR.

Evaluation of hypotensive activity

Rats were anaesthetized with intra-peritoneal ketamine/xylazine (60 and 6 mg/kg, respectively). Each rat’s body temperature was maintained at 36 ± 1 °C with an incandescent lamp placed over the abdomen. The trachea was cannulated and the animals were artificially ventilated (rate 40 strokes/min, stroke volume 10 mL/kg body weight). The left jugular vein was cannulated for drug administration; the right carotid artery was cannulated with a cannula containing heparinized saline (50 U/mL) and connected to a pressure transducer (MLT844 ADInstruments, Bella Vista, NSW, Australia) for continuous monitoring of arterial blood pressure. Acquisition data were performed by a computerized system Power Lab (ADInstruments, v 5.4.2). After surgery, the arterial blood pressure was allowed to stabilize for about 20 min. Mean arterial blood pressure (MABP) and heart rate (HR) were recorded before administration of graded doses of the material. MABP was calculated by software as two-third of diastolic pressure plus one-third of systolic pressure. The effect of three different doses of auraptene (125, 250 and 500 µg/kg, i.v.), positive control (nifedipine 63, 125 and 250 µg/kg, i.v.) and negative control [normal saline/DMSO (1:3); 100 μL] on MABP and HR were evaluated in different groups of normotensive animals. Each dose was injected in bolus of 100 μL.

Statistical analysis

The outcome measure included changes in MABP from baseline values. The results were expressed as mean ± SEM. Group comparisons were performed using the one-way analysis of variance (ANOVA) or Student’s t-test, where appropriate. The Tukey–Kramer test was used for post hoc multiple comparisons. A p < 0.05 was considered as statistically significant.

Results

The animals used in the present study were normetensive rats with MABP of 108 ± 0.89 mmHg and HR of 240 ± 15. The animals were injected with different doses of auraptene and nifedipine. The solvent (normal saline/DMSO) served as negative control in all experiments.

Effect of treatments on MABP

Overall, a dose-dependent hyotensive effect was observed following auraptene injection, which was significant at 250 and 500 µg/kg (p < 0.001) but not at 125 µg/kg dose (p > 0.05). With respect to the positive control, nifedipine reduced MABP at all tested doses, 63, 125 and 250 µg/kg, dose-dependently and significantly (p < 0.001). The MABP lowering effect of auraptene was not comparable to that exerted by nifedipine, as the hypotensive effect of nifedipine at the lowest tested dose (63 µg/kg) was significantly greater than auraptene at its highest tested dose (500 µg/kg) (p < 0.001) (Figure 2).

Figure 2.  Hypotensive activity of different auraptene doses compared to negative control. ***: p < 0.001 (comparison with negative control); ††: p < 0.01, †††: p < 0.001 (comparison with the lowest dose of respective treatment); ‡‡: p < 0.01 (comparison with the medium dose of respective treatment; ♦♦♦: p < 0.001 (comparison with auraptene 500 μg/kg).

Effect of treatments on HR

Although significant increases in HR were observed following the injection of auraptene and nifedipine compared to negative control, which is indicative of a reflex tachycardia response, but these changes were not big in rats. However, no significant difference was observed in the HR between auraptene and nifedipine groups (p > 0.05) (Figure 3).

Figure 3.  Effect of auraptene versus nifedipine on HR. **: p < 0.01 (comparison with negative control).

Discussion

There have been numeorus reports on the beneficial impact of fruit and vegetable consumption in the prevention of CVD. Members of the genus Citrus are among the most promising cardioprotective plants, and are rich in antioxidant vitamins, minerals and various bioactive phytonutrients (Mulero et al., 2012; Yamada et al., 2011). Citrus spp. have also been shown to inhibit key molecules involved in the regulation of blood pressure. In an investigation by Perez et al. (2010), an aqueous extract of Citrus limetta Risso (Rutaceae, sweet lemon) leaves was found to lower both systolic and diastolic blood pressure in a significant fashion. In addition, a promising inhibition of angiotensin II action was observed from the mentioned extract. In consistence, two other investigations by Oboh and Ademosun (2011a,b) have shown that free and bound phenolic extracts from orange and grapefruit peels are capable of blocking angiotensin-I-converting enzyme, thereby posing their potential hypotensive activity. In a survey to screen natural compounds for their inhibitory activity against angiotensin-I-converting enzyme, two phenolic compounds present in Citrus spp., namely hydroxybenzoic acid and coumaric acid, were found to inhibit the enzyme by 19.3% and 2.3%, respectively (Kwon et al., 2006).

Diaz-Juarez et al. (2009) reported that Citrus paradisi Macfad. extract could decrease coronary vascular resistance and mean arterial pressure in the Langendorff isolated and perfused heart model. In addition, they showed that C. paradisi juice reduced systolic and diastolic arterial pressure both in hypertensive and normotensive subjects, and these effects were greater than those of Citrus sinensis Osbeck juice, cow milk and a vitamin C supplemented beverage. The observed coronary vasodilatory effect of C. paradisi was suggested to be NO-dependent as the effect was blocked by the inhibition of NO synthase (Diaz-Juarez et al., 2009). Although the protective effects of Citrus spp. have been mostly attributed to their high content of vitamin C, β-carotene, zinc and flavonoids, less effort has been focused to unveil the impact of other phytonutrients. Citrus bioflavonoids have also emerged as effective hypotensive agents. There have been consistent preclinical as well as clinical findings on the blood pressure (both systolic and diastolic) lowering activity of quercetin. These effects are secondary to the improvement of endothelial function through different mechanisms such as induction of NO and endothelin-1 (Guo & Bruno, 2011; Kelly, 2011; Russo et al., 2012). Hesperidin, another flavonoid present in Citrus peels, possesses hypotensive effect which has been shown to be due to the suppression of NADPH oxidase, enhancement of NO bioavailability and amelioration of endothelial dysfunction. Finally, suppression of Ca²⁺ influx, induction of NO together with vasorelaxation effect have been observed from rutin (Ari et al., 2006; Fusi et al., 2003; Xu et al., 2007). Among Citrus phytonutrients, and apart from bioflavonoids, auraptene is of special importance as it is the most common component of Citrus fruits (Curini et al., 2006; Ogawa et al., 2000).

To the best of our knowledge, there is as yet no report available on the hypotensive effects of auraptene. However, there is evidence indicating smooth muscle relaxant effects of auraptene and its analogues against different types of spasmogens such as barium ion, acetylcholine and histamine (Yamada et al., 1997). In an investigation by Kakiuchi et al. (1991) a potent inhibition of spontaneous beating of cultured mouse cardiomyocites by auraptene was reported; the IC₅₀ value for the mentioned effect was found to be 0.6 µg/mL, comparable to that of verapamil. Therefore, it may be hypothesized that auraptene exerts its bradycardic effects through a calcium antagonistic activity. There has also been a recent report indicating the positive impact of auraptene administration on improving left ventricular systolic function and repressing cardiac hypertrophy following myocardial infarction in rats. Such effects were achieved by oral auraptene supplementation at a dose of 50 mg/kg/day for 2 weeks, and accompanied by significant decreases in the mRNA expression of atrial natriuretic factor and endothelin-1 (Kawaguchi et al., 2011).

Heretofore, several studies have shown the hypotensive and vasorelaxant effects of terpenoid derivatives (Tirapelli et al., 2010). In addition, terpenoids have been reported to be active ingredients of medicinal plants with known hypotensive activity. Some examples include marrubenol from Marrubium vulgare L. (Lamiaceae) (El Bardai et al., 2003), isopimarane-type diterpenes from Orthosiphon aristatus (Blume) Miq. (Lamiaceae) (Ohashi et al., 2000), trans-dehydrocrotonin from Croton cajucara Benth. (Euphorbiaceae) (Guerrero et al., 2004), 14-deoxyandrographolide from Andrographis paniculata Nees (Acanthaceae) (Zhang et al., 1998), stevioside from Stevia rebaudiana Hemsl. (Asteraceae) (Melis & Sainati, 1991) and forskolin from Coleus forskohlii Briq. (Lamiaceae) (Lindner et al., 1978).

Apart from hypotensive properties, auraptene has been reported to possess several interesting pharmacological activities which are of potential benefit for the prevention and treatment of cardiovascular disease (Epifano et al., 2008; Genovese & Epifano, 2011). Auraptene has also anti-obesity effects and inhibits hepatic triglyceride accumulation through enhancement of lipolysis in the liver (Nagao et al., 2010). Besides, auraptene exerts antioxidant activity and suppresses oxygen radical generation (Murakami et al., 1997; Prince et al., 2009), and mitigates inflammation through a number of mechanisms including induction of peroxisome proliferator-activated receptors α (PPAR-α) and γ (PPAR-γ) (Kuroyanagi et al., 2008), down regulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) (Murakami et al., 2000) and attenuation of cyclooxygenase (Curini et al., 2006).

In summary, the present findings indicated a moderate hypotensive activity for auraptene. However, the present study was limited in several ways. First, it would be desirable to test the hypotensive effects of auraptene at higher doses. However, it must be noted that such an effect could not be evaluated using the same solvent as used in the present study since DMSO has intrinsic blood pressure lowering effects and cannot dissolve auraptene crystals at high concentrations. Second, it remains unclear whether the observed hypotensive effects of auraptene is the result of any calcium channel blocking activity and alteration of intracellular Ca²⁺ concentration. Finally, it remains to be determined whether long-term dietary supplementation with this phytochemical is of any benefit in terms of mono- or adjunctive therapy for the management of hypertension.

Declaration of interest

This study was conducted with financial support (Grant No. 87506) that was provided by the Mashhad University of Medical Sciences.

Notes

*This article was taken in part from the thesis prepared by Mohammad Eghbal to fulfill the requirements required for earning the Doctor of Pharmacy degree.