Activity of Antioxidants from Crocus sativus L. Petals: Potential Preventive Effects towards Cardiovascular System

Abstract

The petals of the saffron crocus (Crocus sativus L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC₅₀ = 0.66 mg/mL versus EC₅₀ = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases.

Keywords: Crocus sativus L. petals; by-products; cardiovascular prevention; crocin; kaempferol; nutraceuticals.

Figure 1

Chemical structures of kaempferol and crocin.

Figure 2

(A) Negative inotropic and chronotropic effects on guinea pig left and right atrium respectively. Cumulative concentration–response curves for Saffron Petal Extract (SPE) were obtained using negative effects as percentage of the initial tension (left atria) and initial rate (right atria). (B) Spasmolytic effect of SPE on 80 mM K⁺-induced contraction on guinea pig vascular (aorta) and non-vascular (ileum) smooth muscle. Cumulative concentration–response curves for SPE were obtained using relaxant effects as percentage of the initial tension induced by 80 mM K⁺, taken as 100%. Each point is the mean ± SEM (n = 5–6). Where error bars are not shown these are covered by the point itself.

Figure 3

Negative inotropic and chronotropic effects on guinea pig left (ino SN) and right atrium (chrono DX) respectively. Cumulative concentration–response curves for kaempferol (A) and crocin (B) were obtained using negative effects as percentage of the initial tension (left atria) and initial rate (right atria). Each point is the mean ± SEM of four–six experiments (n = 4–6). Where error bars are not shown these are covered by the point itself.

Figure 4

Spasmolytic effect of crocin and kaempferol on high K⁺-induced contraction on guinea pig vascular (aorta) and non-vascular (ileum) smooth muscle. Cumulative concentration–response curves for crocin and kaempferol were obtained using relaxant effects as percentage of the initial tension induced by 40- or 80-mM K⁺, taken as 100%. Each point is the mean ± SEM (n = 5–6). Where error bars are not shown these are covered by the point itself.

Figure 5

Viability of H9c2 cells treated with crocin and kaempferol. Cells were treated for 24 h with increasing concentrations (1–100 µM) of crocin or kaempferol. Viability was measured by MTT assay, as reported in Materials and Methods. Each bar represents means ± S.E.M. of at least 4 independent experiments. (n = 4) Data were analyzed by one-way ANOVA followed by Dunnett’s test. * p < 0.05 with respect to CTRL.

Figure 6

Effect of crocin and kaempferol against H₂O₂-induced injury in H9c2 cells. Cells were pre-treated with crocin or kaempferol (0.1–10 μM) for 1 or 24 h and then exposed to 100 µM H₂O₂. Cell viability was measured by MTT assay as reported in Materials and Methods. Data are reported as % increase in respect to H₂O₂ treated cells. Each bar represents means ± SE of at least four independent experiments (n = 4). Data were analyzed by one-way ANOVA followed by Dunnett’s test. * p < 0.05 H₂O₂.

Figure 7

Effect of crocin and kaempferol on Reactive Oxygen Species (ROS) production in H9c2 cells. Cells were pre-treated with crocin or kaempferol (0.1–10 μM) for 1 or 24 h and then exposed to 100 µM H₂O₂. Intracellular ROS production was measured with the peroxide-sensitive probe DCFH-DA as reported in Materials and Methods. Data were expressed as percentage with respect to H₂O₂-treated cells. Each bar represents means ± SE. of at least four independent experiments (n = 4). Data were analyzed by one-way ANOVA followed by Dunnett’s test. * p < 0.05 with respect to H₂O₂.

Figure 8

Effect of crocin and kaempferol on ROS production at single cell level and total antioxidant activity of H9c2 cells. H9c2 cells were treated with 5 μM of the crocin or kaempferol for 1 h. (A) Intracellular ROS were evaluated at single cell level using DCFH-DA by flow cytometry as reported in Materials and Methods. (B) Total antioxidant activity was measured as reported in the Materials and Methods and expressed as percent of control cells. Each bar represents means ± SE of three independent experiments (n = 3). Data were analyzed with one-way ANOVA followed by the Bonferroni’s test. * p < 0.05 vs. CTRL.

Figure 9

Effect of kaempferol and crocin on Nrf2 and antioxidant enzyme catalase (CAT) expression. Real time-PCR was performed to detect Nrf2 and CAT mRNA levels. Data are expressed as relative abundance compared to untreated cells. Each bar represents mean ± SEM of three independent experiments. Data were analyzed with one-way ANOVA followed by the Bonferroni’s test. * p < 0.05 vs. CTRL.