Iron chelation as a therapeutic target in vanadium neurotoxicity and Parkinson's disease: role of medicinal plants

Abstract

Bioprospecting plant natural products has yielded significant success in the development of symptomatic treatment of neurodegenerative diseases, including the two most common, Alzheimer's and Parkinson's diseases (PD). Dysregulation of iron has been strongly implicated in the pathophysiology of these serious intractable diseases. A series of Nigerian endemic plants' methanolic extracts were explored using a Ferrozine binding iron chelation assay. This identified Spondias purpurea L. (SP) leaves as a potential therapeutic candidate and this was determined by evaluation of oxidative stress in 6-hydroxydopamine (6-OHDA)-exposed monoamine cell culture and Drosophila models of PD and vanadium neurotoxicity. SP treatment protected CAD cells against 6-OHDA toxicity and improved survival in PINK-1 mutant flies, though it had little effect on motor deficits. Furthermore, SP treatment reduced the vanadium-induced reactive oxygen species, and notably, staggered SP treatment significantly extended lifespan in vanadium-treated flies. Overall, Spondias purpurea L. leaf methanolic extract exhibited iron-chelating, antioxidant, neuroprotective, and life-extending properties, relevant to Parkinson's disease and vanadium-induced toxicity.

Keywords: Drosophila; PINK-1; Parkinson's disease; Spondias; iron homeostasis; oxidative stress.

Figure 1

Ferrozine binding assay showing the iron chelation property of the methanolic extracts using EDTA as a reference (A) Carpolobia lutea bark (B) Carpolobia lutea leaves (C) Phyllanthus muellerianus leaves (D) Spondias purpurea leaves.

Figure 2

The effect of plant extracts; Spondias purpurea leaves (SP), Carpolobia lutea bark (CB), and Carpolobia lutea leaves (CL) on 6-OHDA induced toxicity in CAD cells. (A) MTT assay (B) LDH release assay. Data presented as mean ± SEM. Group means were compared using one-way ANOVA and Tukey's post hoc test. ****P < 0.0001.

Figure 3

Effect of SP on survival and motor deficits in pink-1 mutant flies (n = 60 for all groups): (A) survival curve after 2 weeks of treatment. The proportion of control, pink-1, SP (0.05 mg/ml) and SP (0.1 mg/ml) at day 14 was 93.3%, 66.67%, 78.33%, and 81.67%, respectively. The survival curves were compared with a Log-rank (Mantel-Cox) test and found to be significantly different (P = 0.0016). Pairwise comparisons with Bonferroni adjusted p-values are as follows: Control WT vs. Control mutant, P = 0.0012; Control WT vs. Mutant + SP (0.05 mg/ml), P = 0.1008; Control WT vs. Mutant + SP (0.1 mg/ml), P = 0.2988; (B) climbing assay after 7 days of treatment. The pink-1 mutant had significantly worse climbing index compared to the control (P < 0.0001) but the SP 0.1 mg/ml supplementation of the feed worsened the performance in the climbing assay while the 0.05 mg/ml had no significant effect. Differences between group means were compared using one way ANOVA and Tukey post hoc test. ****P < 0.0001

Figure 4

Effect of SP on the survival and generation of reactive oxygen species in vanadium treated flies (n = 100 for all groups): (A) survival curve after 14 days of treatment. On day 14, the survival proportion of control, vanadium and vanadium + SP (0.06 mg/ml) treated groups were 81%, 57%, and 62%, respectively. The survival curves were compared with Log-rank (Mantel-Cox) test and vanadium treatment significantly reduced survival of the vanadium treated flies (P = 0.0007) but SP treatment was unable to mitigate this effect. Pairwise comparisons with Bonferroni adjusted p-values are as follows: Control vs. Vanadium (1 mM), P = 0.0003; Control vs. Van + SP (0.06 mg/ml), P = 0.0057; (B) DCFDA assay. After 14 days of exposure, vanadium significantly increased the generation of ROS compared with the control group as shown in this figure. SP treatment significantly reduced vanadium induced ROS generation, although the ROS level did not get as low as that of the control group. Data expressed as mean ± SEM and differences between group means compared using one-way ANOVA and Tukey's post hoc test. ****P < 0.0001.

Figure 5

Effect of intermittent SP treatment on survival of vanadium treated flies. Control group (n = 60) were raised on instant drosophila feed from day 1 – 15; Vanadium group (n = 60) received 5 mM sodium metavanadate from day 1 to day 15; S1 (n = 60) received 5 mM sodium metavanadate from day 1–15 as well as 0.05 mg/ml of Spondias purpurea extract from day 1–7; S2 (n = 42) received 5 mM sodium metavanadate from day 1–15 as well as 0.05 mg/ml of Spondias purpurea extract staggered (3 days on, 3 days off) from day 1–15. Survival proportions at day 15 are as follows: control, 88.3%; Vanadium, 51.67%; S1, 60%; S2, 71.43%. The survival curves were compared with Log-rank (Mantel-Cox) test and found to be significantly different (P < 0.0001). Pairwise comparisons with Bonferroni adjusted p-values are as follows: CTRL vs. VAN, P = 0.0006; CTRL vs. S1, P = 0.0042; CTRL vs. S2, P = 0.246.

Figure 6

Effect of intermittent SP treatment on survival and total thiol levels of vanadium treated flies (A) survival curve. The control group (n = 90) were raised on instant drosophila feed from day 1–15; vanadium group (n = 90) received 5 mM sodium metavanadate from day 1–15; S3 (n = 66) received 5 mM sodium metavanadate from day 1–15 as well as 0.05 mg/ml of Spondias purpurea extract from day 7–15; S4 (n = 90) received 5 mM sodium metavanadate from day 1–15 as well as 0.05 mg/ml of Spondias purpurea extract staggered (3 days on, 3 days off) from day 7–15. Survival proportions at day 15 are as follows: control, 75.5%; Vanadium, 50%; S3, 51.25%; S4, 63.3%. The survival curves were compared with Log-rank (Mantel-Cox) test and found to be significantly different (P = 0.0059). Pairwise comparisons with Bonferroni adjusted p-values are as follows: Control vs Vanadium (5 mM), P = 0.0144; Control vs. S3, P = 0.0204; Control vs. S4, P = 0.9906. (B) Total thiol assay. Data expressed as mean ± SEM. Group means were compared using one-way ANOVA and Tukey's multiple comparisons test. Vanadium caused a statistically significant reduction in the total thiol levels compared to the control group (****P < 0.0001). This reduction was not mitigated by SP although the S4 group showed a positive trend toward recovery (**P = 0.001).