Anti-Platelet Properties of Phenolic Extracts from the Leaves and Twigs of Elaeagnus rhamnoides (L.) A. Nelson

Abstract

Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a small tree or bush. It belongs to the Elaeagnaceae family, and has been used for many years in traditional medicine in both Europe and Asia. However, there is no data on the effect of sea buckthorn leaves and twigs on the properties of blood platelets. The aim of the study was to analyze the biological activity of phenolic extracts from leaves and twigs of sea buckthorn in blood platelets in vitro. Two sets of extracts were used: (1) phenolic compounds from twigs and (2) phenolic compounds from leaves. Their biological effects on human blood platelets were studied by blood platelet adhesion, platelet aggregation, arachidonic acid metabolism and the generation of superoxide anion. Cytotoxicity was also evaluated against platelets. The action of extracts from sea buckthorn twigs and leaves was compared to activities of the phenolic extract (a commercial product from the berries of Aronia melanocarpa (Aronox^®) with antioxidative and antiplatelet properties. This study is the first to demonstrate that extracts from sea buckthorn leaves and twigs are a source of bioactive compounds which may be used for the prophylaxis and treatment of cardiovascular pathologies associated with blood platelet hyperactivity. Both leaf and twig extracts were found to display anti-platelet activity in vitro. Moreover, the twig extract (rich in proanthocyanidins) displayed better anti-platelet potential than the leaf extract or aronia extract.

Keywords: E. rhamnoides; adhesion; aggregation; antiplatelet activity; blood platelets.

Figure 1

Twig (A) and leaf extract (B) (0.5–50 µg/mL; 30 min) on adhesion of resting blood platelets and thrombin-activated platelets to collagen. The inhibition of platelet adhesion by the plant extracts is expressed as the percentage of that recorded for control blood platelets (without the plant extract)–positive control. Data represent mean ± standard error (SE) of 5 (for resting platelets) and 9 (for thrombin-activated platelets) healthy volunteers (each experiment performed in triplicate). * p < 0.05, ** p < 0.02 (vs. control platelets).

Figure 2

Twig (A) and leaf extract (B) (0.5–50 µg/mL; 30 min) on adhesion of thrombin/adenosine diphosphate (ADP)-activated platelets to fibrinogen. Inhibition of platelet adhesion by the plant extract is expressed as the percentage of that recorded for control blood platelets (without the plant extract)–positive control. Data represent mean ± SE of 5 (for ADP-activated platelets) and (for thrombin-activated platelets) healthy volunteers (each experiment performed in triplicate). * p < 0.05, ** p < 0.02 (vs. control platelets).

Figure 3

Effects of twig and leaf extract (10 and 50 µg/mL; 30 min) on blood platelet aggregation stimulated by different agonists: 10 µM ADP, 2 μg/mL collagen and 1 Unit/mL thrombin. Data represent mean ± SE of 5 (for thrombin-activated platelets) and 9 (for ADP or collagen-activated platelets) healthy volunteers (each experiment performed in triplicate). Neither concentration of the tested extract (10 and 50 µg/mL) had a statistically significant effect on aggregation stimulated by ADP and collagen compared to control platelets (p > 0.05 (n.s.)). However both concentrations of the tested extract (10 and 50 µg/mL) had a statistically significant effect on aggregation stimulated by thrombin compared to controls (* p < 0.05).

Figure 4

Twig and leaf extract (1, 5 and 50 µg/mL; 30 min) on the level of thiol groups in glutathione (GSH) fraction (A) and protein fraction (B) isolated from blood platelets. Data represent mean ± SE of 3 (for GSH) and 4 (for protein fraction) healthy volunteers (each experiment done in triplicate). In these experiments, the level of GSH fraction in control sample (positive control–blood platelets not treated with plant extract) was 5.7 ± 0.8 nmol GSH/mL of platelets, and was expressed as 100% (A); the level of thiol groups in protein fraction in control sample (positive control–blood platelets not treated with plant extract) was 112.4 ± 17.4 nmol GSH/mL of platelets, and was expressed as 100% (B). None of three different concentrations of the tested extract (1, 5 and 50 µg/mL) had a statistically significant effect compared to controls (p > 0.05 (n.s.)).

Figure 4

Twig and leaf extract (1, 5 and 50 µg/mL; 30 min) on the level of thiol groups in glutathione (GSH) fraction (A) and protein fraction (B) isolated from blood platelets. Data represent mean ± SE of 3 (for GSH) and 4 (for protein fraction) healthy volunteers (each experiment done in triplicate). In these experiments, the level of GSH fraction in control sample (positive control–blood platelets not treated with plant extract) was 5.7 ± 0.8 nmol GSH/mL of platelets, and was expressed as 100% (A); the level of thiol groups in protein fraction in control sample (positive control–blood platelets not treated with plant extract) was 112.4 ± 17.4 nmol GSH/mL of platelets, and was expressed as 100% (B). None of three different concentrations of the tested extract (1, 5 and 50 µg/mL) had a statistically significant effect compared to controls (p > 0.05 (n.s.)).

Figure 5

Twig and leaf extract (0.5, 5 and 50 µg/mL; 30 min) on lipid peroxidation in resting platelets (A) and in blood platelets activated by thrombin (B). In these experiments, blood platelets not treated with plant extract were used as control samples (positive control). Data represent mean ± SE of 6 healthy volunteers (each experiment done in triplicate). The three different concentrations of the twig extract (0.5, 5 and 50 µg/mL) had a statistically significant compared to controls (* p < 0.05). However, none of the three different concentrations of the leaf extract (0.5, 5 and 50 µg/mL) had any statistically significant effect compared to controls (p > 0.05 (n.s.)).

Figure 5

Twig and leaf extract (0.5, 5 and 50 µg/mL; 30 min) on lipid peroxidation in resting platelets (A) and in blood platelets activated by thrombin (B). In these experiments, blood platelets not treated with plant extract were used as control samples (positive control). Data represent mean ± SE of 6 healthy volunteers (each experiment done in triplicate). The three different concentrations of the twig extract (0.5, 5 and 50 µg/mL) had a statistically significant compared to controls (* p < 0.05). However, none of the three different concentrations of the leaf extract (0.5, 5 and 50 µg/mL) had any statistically significant effect compared to controls (p > 0.05 (n.s.)).

Figure 6

Effects of twig and leaf extract (0.5 and 5 µg/mL; 30 min) on O₂⁻ production in resting platelets (A) and in blood platelets activated by thrombin (B). Data represent mean ± SE of 5 healthy volunteers (each experiment done in triplicate). In these experiments, the O₂⁻ level in control samples (positive control – blood platelets not treated with plant extract) was 0.592 ± 0.321 nmol/10⁸ platelets (for resting platelets) and 1.222 ± 0.434 nmol/10⁸ platelets (for thrombin-activated platelets). Inhibition of O₂⁻ production was expressed as a percentage of that recorded for positive control (platelets without tested extracts). The effects of the two different concentrations of twig extract (0.5 and 5 µg/mL) were significantly different to controls (* p < 0.05; ** p < 0.02). The two different concentrations of leaf extract (0.5 and 5 µg/mL) demonstrated no statistically significant effect compared to control platelets (p > 0.05 (n.s.)).

Figure 7

The toxic effects of twig and leaf extract (0.5–50 µg/mL; 30 min) against human blood platelets. In these experiments, blood platelets not treated with plant extract were used as control samples (positive control). Data represent mean ± SE of 6 healthy volunteers (each experiment performed in triplicate). None of the five different concentrations of the tested extract (0.5, 1, 5, 10 and 50 µg/mL) had any statistically significant effect compared to controls (p > 0.05 (n.s.)).