Phytochemical, In Silico, In Vitro, and In Vivo Research on Piptadeniastrum africanum (Fabaceae) Unveiling Anti-Stereotypic, Anxiolytic, and Analgesic Effects in a Sodium Valproate-Induced Autistic Disorders Model

Abstract

Objective: Individuals with autistic spectrum disorders (ASD) primarily exhibit deficits in communication and social interaction, along with repetitive behaviors and restricted interests. This disorder is often associated with anxiety, nociceptive disorders, and pain. While medical treatment generally focuses on treating the symptoms rather than addressing the underlying causes, traditional medicine is sometimes used as an alternative. Piptadeniastrum africanum is used in Cameroonian medicinal folks to treat cognitive disorders. However, its effects and mechanisms of action regarding the inhibition of ASD-like symptoms remain unclear. The primary goal of the present study was to evaluate the anxiolytic and analgesic effects of the water extract of P. africanum on autistic triad induced in rats by sodium valproate.

Material and methods: The study investigated the secondary metabolites in P. africanum extract using UHPLC-MS. DPPH, ABTS, and FRAP tests were performed to assess the extract's ability to neutralize free radicals. Molecular docking was utilized to evaluate the extract's binding to various receptors. For the experimental study, 33 pregnant female rats were divided into two groups after pregnancy was confirmed. One group was given distilled water orally at 10 mL/kg, while the other group received sodium valproate at 800 mg/kg on gestation days 11, 12, and 13. When the male offspring reached 3 weeks old, they were evaluated for anxiety, social interaction, and pain sensitivity, with those displaying any disorders selected for further study. The remaining rats were split into six groups of five and treated with either a vehicle, bumetanide, or P. africanum extract at 190 and 760 mg/kg. Behavioral assessments focusing on sociability, anxiety, and pain sensitivity were conducted on days 28 and 37 after weaning. In the end, biochemical markers related to GABA metabolism, serotonin levels, and oxidative status were analyzed in the cerebellum, prefrontal cortex, hippocampus, and amygdala alongside histopathological analyses in the brain.

Results: UHPLC-MS allows us to identify several compounds. They bind to H3R (7F61) and HDAC2 through conventional hydrogen bonding. Findings showed that prenatal administration of sodium valproate induced in male offspring a deficit in social interaction (p < 0.001), anxiety disorders (p < 0.001), hypersensitivity to pain (p < 0.001), increased GABA and serotonin concentration (p < 0.001), disturbed oxidative status (p < 0.001), and neuronal loss (p < 0.001) as well as neuronal disorganization in the hippocampus, cerebellum and amygdala in young rats compared to neurotypical animals. P. africanum extract at doses used, like bumetanide, corrected these disorders and protected against neuronal loss. These results suggest that the extract has anxiolytic and anti-nociceptive effects. It has been found that the positive effects can be achieved by restoring GABAergic and serotonergic neurotransmission, coupled with antioxidant and neuromodulatory activity.

Conclusion: The current findings support that P. africanum induces anxiolytic and analgesic effects in a sodium valproate-induced autistic disorders model.

Keywords: Piptadeniastrum africanum; analgesic; anxiolytic; autistic disorders; sodium valproate.

FIGURE 1

Some malformations: (A) delayed eye‐opening and (B) abdominal deformity. NTC: neurotypical animals treated with distilled water (10 mL/kg). ASD: ASD animals treated with distilled water (10 mL/kg).

FIGURE 2

Identification spectrum of aqueous extract of Piptadeniastrum africanum.

FIGURE 3

Effects of Piptadeniastrum africanum on exploratory activity (A1, A2) and social interaction (B1, B2 and C1, C2). Each bar represents the mean ± MSE; n = 5. Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ¹ p < 0.05; ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg. 1 and 2 designate waves 1 and 2, respectively.

FIGURE 4

Effects of Piptadeniastrum africanum aqueous extract on groomings (A1, A2) and burials (B1, B2). Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ¹ p < 0.05; ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at the dose of 190 mg/kg. 1 and 2 designate waves 1 and 2, respectively.

FIGURE 5

Effects of Piptadeniastrum africanum aqueous extract on pain sensitivity during the hot plate test. Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^c p < 0.001: significant differences versus NTC. ¹ p < 0.05; ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at the dose of 190 mg/kg. A1 and A2 designate waves 1 and 2, respectively.

FIGURE 6

Effects of Piptadeniastrum africanum aqueous extract on GABA concentration in hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^c p < 0.001: significant differences versus NTC. ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.

FIGURE 7

Effects of Piptadeniastrum africanum aqueous extract on GABA‐T activity in the hippocampus (C) and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.

FIGURE 8

Effects of Piptadeniastrum africanum aqueous extract on L‐GAD activity in the cerebellum (A), prefrontal cortex (B), hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.

FIGURE 9

Effects of Piptadeniastrum africanum aqueous extract on serotonin concentration in the cerebellum (A), prefrontal cortex (B), hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^b p < 0.01; ^c p < 0.001: significant differences from normal control. ¹ p < 0.05; ³ p < 0.001: significant differences from negative control. NTC: neurotypical animals treated with distilled water (10 mL/kg) ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.

FIGURE 10

Effects of Piptadeniastrum africanum aqueous extract on reduced glutathione concentration in the cerebellum (A), prefrontal cortex (B), hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ¹ p < 0.05; ² p < 0.01; ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.

FIGURE 11

Effects of Piptadeniastrum africanum aqueous extract on malondialdehyde concentration in the cerebellum (A), prefrontal cortex (B), hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ³ p < 0.001: significant difference versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg) ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at a dose of 190 mg/kg.

FIGURE 12

Effects of Piptadeniastrum africanum aqueous extract on superoxide dismutase activity in cerebellum (A), prefrontal cortex (B), hippocampus (C), and amygdala (D). Each bar represents the mean ± MSE; n = 5. ^b p < 0.01; ^c p < 0.001: significant differences versus NTC. ³ p < 0.001: significant differences versus ASD. NTC: neurotypical animals treated with distilled water (10 mL/kg) ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at a dose of 190 mg/kg.

FIGURE 13

Microphotographs of the microarchitecture of the cerebellum, amygdala, and hippocampus (200×, H&E). A = neurotypical animals receiving distilled water (10 mL/kg); B = ASD animals receiving distilled water (10 mL/kg); C = ASD animals receiving reference drug. D, E = ASD animals receiving the aqueous extract of P. africanum at 190 and 760 mg/kg; F = neurotypical animals receiving the aqueous extract of P. africanum at 190 mg/kg. CA1 and CA3: Ammon horn regions 1 and 2; Gc = granular cell layer; Mc = molecular layer; Pc = polymorphic cell layer; Nd = neuronal degeneration; Ce = cerebral edema; G = granular cell layer; Hn = hyperchromatic nucleus; Nn = normal neuron; On = oligodendrocyte necrosis; P = Purkinje cell layer; Nl = neuronal loss.

FIGURE 14

Effects of Piptadeniastrum africanum aqueous extract on neuron counting in the hippocampus and amygdala. Each bar represents the mean ± MSE; n = 5. ^a p < 0.05; ^c p < 0.001: significant differences versus NTC. ¹ p < 0.05; ³ p < 0.001: significant differences versus ASD. NTC: treated with distilled water (10 mL/kg); ASD: ASD animal treated with distilled water (10 mL/kg); ASD‐B: ASD animal treated at Bumetanide (4 mg/kg); ASD‐190, ASD‐760: ASD animals treated with the aqueous extract of P. africanum at doses of 190 and 760 mg/kg; NT‐190: neurotypical animal treated with the aqueous extract from P. africanum at dose of 190 mg/kg.