Neurobehavioural and Histomorphological Effects of Hyoscyamine Fraction of Datura stramonium Seeds on the Hippocampus and Dentate Gyrus of Developing and Adult Wistar Rats

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ABSTRACT

Datura stramonium (D. stramonium) is one of the well-known traditional plants with both poisonous and therapeutic properties due to the presence of tropane alkaloids. Hyoscyamine is one such alkaloid with suspected teratogenic tendencies, however, received little attention in the literature. The study aimed to evaluate the effects of hyoscyamine fraction. Stramonium seeds on the neurobehavioural and histomorphology of developing hippocampus and dentate gyrus of Wistar rats. Two thousand grams of fresh and mature D. stramonium seeds were procured, extracted, and fractioned to detect the presence and quantify the amount of hyoscyamine using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses, respectively. Thirty-two (32) healthy Wistar rats comprised of adult males and females (1:1), weighed 150-250 g were procured. The oestrus cycles of the female rats were synchronized, and the proestrus phases were determined by vaginal cytology. Eight (8)Wistar rats comprised of males and females(1:1) were randomly selected and mated into four different groups for treatments at gestation, lactation, adolescence and adulthood, respectively. Each group was further divided equally into control and treatment subgroups. A total of one hundred and twenty (120) litres were obtained after mating. Thirty (30) litters from group I received 200 mg/kg in ute roof normal saline and the hyoscyamine fraction of D. stramonium seeds on a gestational day (GD)15-20, respectively. Sixty (60) litres obtained from groups II and III were treated with normal saline, 400, 800 mg/kg hyoscyamine fraction on a postnatal day (PND)0-21 andPND22- 42respectively. Thirty (30) litters obtained in group IV were further subdivided into four groups and treated with normal saline, 200, 400 and 800 mg/kg body weight of the fraction on PND56-77, respectively. Litters ingroup I was physically observed for gross congenital malformation of physical growth indices (eyes-opening, teeth eruption, bilateral ear detachment and hair growth). Functional battery tests for motor reflex using surface righting reflex (SRR), cliff avoidance (CAV) and negative geotaxis (NEG) were conducted on the PND4, 5 and 6, respectively, to assess the effects of the fraction on motor reflexes in the Wistar rat pups. Triple horizontal bar test (3HBT), Morris Water Maze (MWM) and Novel Objects Recognition (NORT) tests were used to assess motor coordination, spatial learning and memory and recognition ability in all groups when they reached adulthood. The animals were sacrificed at the end of every treatment phase and in adulthood. The hippocampus was harvested for histology, stereology, Atomic Absorption Spectrophotometry of selected trace/heavy elements (Cu, Ni and Zn), and gene expression studies. The data obtained were expressed as Mean±SEM using Minitab 17 statistical package. P<0.05 was considered statistically significant.

The results showed that 42 grams (2.10%) of crude ethanol extract of D. stramonium seeds were obtained from two thousand grams of the sample. Also,18 grams (0.01%) hyoscyamine fraction was obtained from the crude extract and yielded 24.09 μg/dl hyoscyamine.No statistically significant difference (p> 0.05) in the physical growth indices was observed; however, a statistically significant difference (p< 0.05) SRR, CAV and NEG were observed between the groups. There was also a statistically significant decrease in body weight, NORT, Discrimination Index (DI), Novelty Preferences in Prenatal, Adolescence and Adulthood exposure and MWM test paradigms (p< 0.05). The neuronal numbers in region CA1, CA3 and dentate gyrus (DG) of the hippocampus, surface areas and volumes of the hippocampus in the treated groups significantly differed (p<0.001) from the control groups. The expressions of the DLG3 and NeuroD1 genes were also significantly downregulated (p<0.001) in the treated groups compared to the control. Significant differences in the amounts of the Cu, Ni and Zn (p<0.005) were also observed in the treated groups during lactation, adolescence, and adulthood. A variable degree of neurodegeneration and lesions such as pyknosis, cytoplasmic vacuolation and nuclear hyperchromasia were observed in the treated groups.

In conclusion, ingestion of hyoscyamine fraction of D. stramonium seeds at any stage of development decreased the body weight, impaired motor reflexes, altered the trace elements balance Cu, Ni and Zn balance, impaired recognition ability, spatial learning and memory and downregulated the expressions of NeuroD1 and DLG3 in the hippocampus of Wistar rats and neurodegenerations.

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