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 of such alkaloids with suspected teratogenic tendencies,however, received little attention in the literature.The study aimed to evaluate the effects of hyoscyamine fractionofD. stramoniumseeds on the neurobehavioural and histomorphology of developing hippocampus and dentate gyrus of Wistar rats. Two thousand grams of fresh and mature D. stramoniumseeds wereprocured, extracted andfractioned to detect the presence and quantify the amount of hyoscyamine using thinlayer 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 determined by vaginal cytology. Eight (8)Wistar ratscomprised of malesand females(1:1) were randomly selected and mated into four different groupsfor 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) litters were obtained after mating. Thirty (30) litters fromgroup I received200 mg/kg in uteroof normal saline and the hyoscyamine fraction of D. stramonium seeds ona gestational day (GD)15-20 respectively. Sixty (60)litters obtained from the group II and III were treated withnormal 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 fourgroups and treated withnormal saline, 200, 400 and 800 mg/kg bodyweight of the fraction on PND56-77 respectively. Litters ingroup I was physically observed for gross vii 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) was conducted on the PND4, 5 and 6 respectively to assess the effects of the fraction on motor reflexesinthe 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 inall groups when theyreachedadulthood. The animals were sacrificed at end of every phase of the treatment and in adulthood. The hippocampus was harvested for histology, stereology, Atomic Absorption Spectrophotometry of selected trace/heavy elements (Cu, Ni and Zn), and gene expressions studies. The data obtained were expressed as MeanÂSEM 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. stramoniumseeds were obtained from two thousand grams of the sample. Also,18 grams (0.01%) hyoscyamine fraction obtained from the crude extract and yielded 24.09 μg/dl hyoscyamine.No statistically significant difference (p> 0.05) in the physical growth indices observed, however, a statistically significant difference (p< 0.05) SRR, CAV and NEG were observed between the groups. There wasalso a statistically significant decrease in bodyweight, NORT, Discrimination Index (DI), Novelty Preferences, in Prenatal, Adolescence and Adulthood exposure and MWM test paradigms (p< 0.05). The neuronal numbersin region CA1, CA3 and dentate gyrus (DG) ofthe 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 when 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 neurodegenerationand lesions such as pyknosis, cytoplasmic vacuolation and nuclear hyperchromasia were observed in the treated groups. In conclusion, ingestion of hyoscyamine fraction of D. stramoniumseeds at any stage of development decreased the bodyweight, impaired motor reflexes, altered the trace elements balance Cu, Ni and Zn balance, impaired recognitionability, spatial learning and memory and downregulated the expressions of NeuroD1 and DLG3 in the hippocampus of Wistar rats and neurodegenerations.
