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Introduction

Natural products are small molecules produced by organisms as primary and secondary metabolites and they exert various useful properties including anti-oxidant, anti-inflammatory, cancer preventive, cardiovascular, anti-Alzheimer's, platelet modulatory and pain relief effects (1-5).

These compounds are considered new therapeutic tools for the management of cardiovascular diseases, and they have received considerable attention. In this context, increasing number of reports indicated beneficial effects of foods with high levels of polyphenols (fruits, tea and cocoa) against cardiovascular diseases (6-9) in humans and similar favorable effects were noted for isolated flavonoids in animal models (10,11).

Crocin (CRO), a carotenoid from the tetraterpenes family, is responsible for the unique color of saffron, stigmas of Crocus sativus (12). Considerable amount of evidence has confirmed that CRO exhibits antioxidant, radical scavenging, cytotoxic and antitumor (12,13), renoprotective (14) and anti-hyperlipidemic (15) effects. Several studies have shown the beneficial effects of this compound against cardiovascular conditions as it could alleviate blood biomarkers associated with obesity (16), had protective effects against angiotensin II-hypertension (17) and normalized the blood pressure of rats chronically administered with desoxycorticosterone acetate (18).

In a study, CRO reduced mean arterial blood pressure (MAP) in rats and its effects were more marked in hypertensive animals compared to normotensive ones (19). It was noted that CRO inhibitory effects on the extracellular Ca2+ influx along with Ca2+ cytosolic release from intracellular Ca2+ supplies in the endoplasmic reticulum, might underlie its hypotensive effects induced by relaxing blood vessels (20,21).

Citrus fruits contain flavanone glycosides hesperidin (HES) and naringin which are members of the bigger family of flavonoids that comprises a large number of phenolic compounds. Flavanone glycosides were shown to have higher bioavailability compared to other flavonoid compounds. HES has shown various therapeutic properties such as anti-oxidant, anti-inflammatory, and anti-aging effects (22-26). Although the beneficial effects of HES on the cardiovascular system were shown by the significant reductions observed in the blood pressure and serum lipids following its administration (27-29), there is still debate regarding its value in this field since there are reports indicating that HES has no significant effect on the said cardiovascular parameters (30,31).

It was reported that 25-week administration of HES reduced blood pressure and heart rate (HR) in spontaneously hypertensive rats but in normotensive Wistar-Kyoto rats, no such effect was observed (32). Moreover, a randomized, controlled, crossover study in healthy volunteers showed that supplementation with HES during a 4-week period, led to a significant reduction in diastolic blood pressure (DBP) (29).

In another study, glucosyl HES (G-HES), a water-soluble derivative of HES, and hesperetin, aglycone HES, were given to spontaneously hypertensive rats (SHR); both compounds reduced systolic blood pressure (SBP) which was significantly inhibited by a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (33). Nevertheless, a recent meta-analysis of randomized controlled trials revealed that HES supplementation had no effect on SBP or DBP (28).

In the present study, effects of CRO and HES supplementation, alone and in combination, on MAP, SBP, DBP, and HR were assessed in rats treated with a high-fat diet (HFD) for 7 weeks.

Materials and methods

Animals.

In this study, 40 male Wistar rats (aged 8-10 weeks; 200-250 g body weight, obtained from Animal Center of Zabol University of Medical Sciences, Zabol, Iran) were kept under standard conditions (at 25˚C with 12/12 h light/dark cycles) and they had free access to food and water, ad libitum. All animal experiments were approved by the animal research Ethics Committee of Zabol University of Medical Sciences (Ethics committee approval no. IR.ZBMU.REC.1398.091) and performed in accordance with National Institute of Health Guide for the Care and Use of the Laboratory Animals.

Animals were randomly assigned to the following five groups (8 animals in each group): control group [rats received standard chow diet (CD; Harlan TD.7012) for 7 weeks + normal saline on day 50], HFD control group [received HFD containing 32% kcal of fat and 0.1% cholesterol (Harlan TD.88137) for 7 weeks and normal saline on day 50(34)], and three groups of HFD-treated animals that received a single dose of either CRO (20 mg/kg), HES (20 mg/kg), or CRO + HES (20 + 20 mg/kg) on day 50.

Materials.

HES and CRO were purchased from Merck KGaA. Ketamine (Alfasan) and xylazine (Pantex Holland B.V.) were employed for induction of anesthesia. For measurement of SBP, DBP, HR and MAP, Power Lab (AD Instruments) was used (35,36).

Animal diets.

Animals were assigned into the two diet groups. The control group received purified low-fat diet (containing 10.6% kcal as fat) for 7 weeks. The rest of the animals received HFD diet containing 32% kcal as fat and 0.1% cholesterol, for 7 weeks (37). HFD was provided from Razi Institute, Tehran, Iran. The HFD animals were divided into HFD control group and CRO or HES or CRO + HES groups.

Methods.

After the 7-week period of HFD, animals were fasted overnight (minimum period of 8-10 h), and anesthetized using ketamine/xylazine. The reflexes of the animals were checked, and they were placed on a suitable rodent surgical Table with electrocardiogram (ECG) recording. The ventral side of the neck, right hind leg, and chest of the animals were carefully disinfected and shaved. A small incision (1.5-2 cm) was made in the neck of the rats for the tracheostomy procedure and carotid artery cannulation. Tracheostomy was performed using a small piece of pediatric Ryle's tube or rodent tracheal intubation tube (36). The carotid artery was identified and the cardiac end of the blood vessel was clamped by a bulldog clamp for cannulation (36). The blood vessel was cannulated employing a cannula pre-filled with heparinized normal saline (0.5 IU/ml). After cannulation, the bulldog clamp at the cardiac end of the blood vessel, was released slowly (9).

Three hours before cannulation, CRO and HES alone or in combination in the respective treatment groups or normal saline in the control and HFD-hypertensive group, were injected intraperitoneally (ip). After cannulation, the sensor was connected to the Power Lab instrument and the blood pressure, HR and ECG were recorded and analyzed using Lab Chart 8 software (36).

Statistical analysis.

Data analysis was performed using SPSS version 11.5 (SPSS, Inc.). Differences among the groups were assessed using One-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of HES and CRO on SBP in HFD-hypertensive rats.

As presented in Fig. 1, HFD-treated animals showed significant increases in SBP compared to the control group (P<0.001). In the HES and HES+CRO groups, SBP was reduced significantly in comparison to the HFD control group (for both cases P<0.001). HES treatment resulted in a significant decrease of SBP compared to the HFD control group. On the other hand, treatment of hypertensive animals with CRO alone did not reduce SBP.

Figure 1 Effects of HES, CRO and HES+CRO on systolic blood pressure. ***P<0.001 compared to the control group and ###P<0.001 compared to the HFD-Cont group. Cont, control group; HFD-Cont, HFD-treated control rats that were treated with normal saline; HES, hesperidin; CRO, crocin.

Effects of HES and CRO on DBP in HFD-hypertensive rats.

As shown in Fig. 2, DBP did not show significant variations among the different groups.

Figure 2 Effects of HES, CRO and HES+CRO on diastolic blood pressure compared to the HFD-treated control rats. Cont, control group; HFD-Cont, HFD-treated control rats that were treated with normal saline; HES, hesperidin; CRO, crocin.

Effects of HES and CRO on HR in HFD-hypertensive rats.

Fig. 3 shows that HFD rats did not present a significant difference in HR compared to the control group which did not receive HFD. Nonetheless, HES+CRO treated animals showed a significant reduction in HR in comparison to the HFD control group (P<0.001).

Figure 3 Effects of HES, CRO and HES+CRO on heart rate compared to the HFD-treated control rats. ###P<0.001 in comparison to the HFD-Cont group. Cont, control group; HFD-Cont, HFD-treated control rats that were treated with normal saline; HES, hesperidin; CRO, crocin.

Effects of HES and CROC on MAP in HFD-hypertensive rats.

7-week treatment of rats with HFD led to a significant increase in MAP compared to the control group (P<0.001). Furthermore, our results showed that HES+CRO administration in HFD rats significantly attenuated MAP compared to the HFD control group (P<0.001); however, treatment with HES or CRO alone did not result in significant alterations in MAP compared to the HFD control group (Fig. 4).

Figure 4 Effects of HES, CRO and HES+CRO on mean arterial blood pressure as compared to the control group, in HFD-hypertensive in rats. ***P<0.001 compared to the control group and ###P<0.001 compared to HFD-treated control rats. Cont, control group; HFD-Cont, HFD-treated control rats that were treated with normal saline; HES, hesperidin; CRO, crocin.

Discussion

In the current study, 7-week administration of HFD in rats induced systolic hypertension and increased MAP. Administration of a single dose of HES reduced high SBP. HES and the combination of HES and CRO significantly decreased SBP. HFD did not affect DBP or HR; however, it significantly increased MAP, which in turn, was significantly decreased by treatment with the combination of HES and CRO. To the best of our knowledge, this is the first experiment investigating the effect of co-administration of two natural products, HES and CRO, in a rat model of HFD-induced hypertension and reports their effects on blood pressure and HR. Co-administration of these compounds seems to have synergistic effects.

It was reported that hypotensive effects of HES are induced through nitric oxide (NO)-mediated vasodilation (33). In a study by Yamamoto et al (33), it was shown that short-term administration of HES reduces SBP by NO-induced vascular relaxation. Several studies similarly confirmed the aforementioned mechanism of action of HES (31,38,39). Another report confirmed that HES acutely stimulates phosphorylation of endothelial NO synthase to produce NO and cause vasodilation (31) and these results are in agreement with the data obtained in the present study on the short-term antihypertensive effects of HES (31). We found that HES alone reduced SBP, but HES in combination with CRO reduced simultaneously SBP, MAP and HR confirming the synergistic effects of HES and CRO in reduction of high blood pressure.

Previous studies showed that CRO can reduce hypertension in animal models (18,40). Besides, there are other reports on the antihypertensive effects of other active ingredients and extracts of Crocus sativus (41-43). Some of these studies examined possible acute antihypertensive effects of CRO and crocetin (40,42,43). In this context, Shafei et al (40) showed that CRO 50, 100 and 200 mg/kg attenuated MAP, SBP and HR induced by angiotensin II. Our results showed that CRO 20 mg/kg did not alleviate hypertension induced by HFD. In HES + CRO (20 + 20 mg/kg)-treated animals, a significant decrement of SBP and MAP was observed. In the HES (20 mg/kg)-only treated group, though SBP was decreased, no changes in DBP, HR and MAP were found. Our results point to a potentially interesting synergistic effect for these natural products that might act via similar pathways (i.e., via NO-associated mechanisms) (33,40).

In our study, the co-administration of HES and CRO exhibited favorable antihypertensive effects. The beneficial effects of these nutraceuticals could be further tested in clinical settings for the prevention and treatment of hypertension, especially in patients presenting with borderline stage 1 hypertension.

Acknowledgements

The present study was part of a Pharm. D. thesis and authors wish to thank Zabol University of Medical Sciences for support.

Funding

This work was funded by Zabol University of Medical Sciences and Students Research Committee of Zabol University of Medical Sciences.

Availability of data and materials

Not applicable.

Authors' contributions

MH and RR conceived and designed, and supervised the study. MN performed the experiments and collected the data and TKN analyzed the data. GL, KT, AT, JS and DAS interpreted the data and prepared the manuscript. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

All animal experiments were approved by the animal research Ethics Committee of Zabol University of Medical Sciences (Ethics committee approval no. IR.ZBMU.REC.1398.091).

Patient consent for publication

Not applicable.

Competing interests

DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. The other authors declare that they have no competing interests.

References