Effects of Datura stramonium on some key glycolytic enzymes in male albino rats

Introduction

Datura stramonium is a medicinal plant commonly referred to as Devil's trumpet and Jimson weed. It belongs in the Solanaceae family, which are known to be rich in alkaloids (Babiker et al., 2017) [4] . The plant is most commonly used as an intoxicant and hallucinogen due to the presence of anticholinergic alkaloids such as scopolamine which is known to produce hallucination and delirium (Mukhtar et al., 2019, Sharma et al., 2021) [21,28] . Datura stramonium is widely cultivated in Asia, Africa, Europe and other tropical regions (Sharma et al., 2021) [28] . Despite having hallucinogenic and poisonous potentials, D. stramonium has incredibly high medicinal property which has been deployed in ayurdevic medicine for the treatment of multiple medical conditions such as asthma, toothache, fever, infections and rheumatism. Studies and reports have indicated that the plant has bronchodilator, antiobesity, antiviral, anti-inflammatory, neuromodulatory, antioxidant and hypoglycaemic activities (Sharma et al., 2021, Alum et al., 2023) [28,3] . Owolabi et al 2023 [23] also reported significant changes in sperm analysis and testicular histomorphology following the administration of Datura stramonium extract Alkaloids in this plant include but are not limited to hyoscyamine, hyoscine, scopolamine, pseudotropine and the highly medicinal atropine (Korkmaz et al., 2019) [18] . D. stramonium also contains amino acids such as glutamate, tyrosine, alanine and phenylalanine (Melaku and Amare, 2020) [8] . Saponins, tannins, phenols, alkaloids and flavonoids have been implicated in the pharmacological effect of the plant. Some of the identified ones include pcoumaric acid, 6-hydroxyhyoscymine, anolide I, skimmianine, daturataturins A and B, quercetin, metelodine, caffeic acid, coumarins, ferulic acid, stigmasterol, campesterol, steroidal glycosides and chrysins (Singh et al., 2013, Alum et al., 2023) [29,3] . The contest between the toxic and the therapeutic effect of this plant is highly recognized. Hence, the pharmacological and therapeutic potentials are to be deployed with precise knowledge of its possible toxicological outcomes, so as to be able to evade its severe side effects which may include the most common hallucination, dry skin and other possible side effects (Mukhtar et al., 2019) [21] .

D. stramonium has been employed for a number of studies such as Alum et al. (2023) [3] reported the possible antidiabetic effect of the methanolic seed extract in diabetic rats.

Materials and methods

Chemicals and reagents: Tris-HCl, magnesium chloride, hydrazine sulfate, magnesium sulfate, glucose-6-phosphate dehydrogenase, and adenosine diphosphate were obtained from Sigma Aldrich, Missouri, USA.), reduced adenine dinucleotide (NADH), glucose, fructose-1,6-bisphosphate, phosphoenol pyruvate, and lactate dehydrogenase were obtained from Carl Roth, Germany. The kit used for the determination of lactate dehydrogenase was purchased from Cypress diagnostics, Belgium.

Plant collection and extraction

Fresh samples of Datura stramonium were collected from Ureje quarters, Ado-Ekiti, Ekiti State, Nigeria. The fresh leaves and seeds were rinsed with distilled water and airdried at room temperature until it was completely dry. The dried sample was homogenized using an electrical grinder. Extraction was carried out using a modified method of Ogunmoyele et al., (2019) [22] . Three hundred grams (300g) of the pulverized Datura stramonium was weighed and soaked in distilled water for 24 hours after which it was decanted and filtered using Whatman's filter paper, the filtrate was kept secured in a container and placed in a fridge. This process was repeated until a clear filtrate was obtained indicating that most of the active component in Datura stramonium has been extracted. The resulting filtrate was pooled and evaporated using a water bath at 40 o C. The extract was dissolved in distilled water at a concentration of 50 mg/ml, 100 mg/ml, 2000 mg/ml, and 400 mg/ml.

Experimental animals

Twenty-five (25) male albino rats weighing between 180g and 200g were used for this experiment. They were randomly assigned into five groups of five animals each and housed in different cages at the animal house of the College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti. Access to standard rat pellets and clean water were given ad libitum. They were allowed 14 days to acclimatize. Approval for this study was obtained from Afe Babalola University Health Research Ethical Committee (ABUADHREC) with the number ABUADHREC/08/03/2023/2024.

The principles of laboratory animal care were followed during the animal research (NIH publication #85 -23, revised in 1985).

Study design: Experimental animals were randomly distributed into five groups of five animals each. Each group was exposed to graded doses of aqueous extract of Datura stramonium for twenty-eight days while the control received distilled water for the duration of the experiment. At the expiration of the experiment, animals were sacrificed under light ether anaesthesia. Brain, lungs and spleen were harvested, rinsed in normal saline to remove blood stains, blotted dry, and then kept in the freezer until further analysed. Group 1: Control group Group 2: Aqueous extract of D. stramonium 50 mg/kg Group 3: Aqueous extract of D. stramonium 100 mg/kg Group 4: Aqueous extract of D. stramonium 200 mg/kg Group 5: Aqueous extract of D. stramonium 400 mg/kg Preparation of homogenate for hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase activities Homogenates were prepared according to the method of Abdel-Hamid (2013). Ten (10) percent of the homogenate was prepared by placing 0.2g of organs in 1.8ml of 0.1 M Tris-HCl buffer (pH 7.4), this was done by 6 up and 6 down strokes at 1100rpm using a power-driven Teflon pestle in potter-Elvehjem glass homogenizing cup maintained at 4 o C. The homogenate was then centrifuged at 4000rpm at 4 o C for 10 minutes. The supernatant was carefully pipetted into an Eppendorf tube for the determination of hexokinase, aldolase, pyruvate kinase, and lactate dehydrogenase activities.

Enzyme assays

Hexokinase activity was determined by the method of Colowick (1973) [9] while aldolase was evaluated based on Boyer's modification of the hydrazine assay (Jagannathan et al., 1956) [16] . Activities of pyruvate kinase were determined according to the method of Buchner and Pfleiderer (1955) [6] , Lactate dehydrogenase activity was determined spectrophotometrically according to the method of Borgmann et al. (1974) [5] as described in Cypress Diagnostic kit manual.

Statistical analyses

Results were expressed as mean ± standard error of mean (SEM). Data was analysed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. p<0.05 was considered statistically significant.

Results

Effect of Datura stramonium on brain, spleen and lung hexokinase in male Wistar rats

Administration of Datura stramonium significantly increased hexokinase in the brain, spleen and lung of all the experimental animals when compared with control group. Effect of Datura stramonium on brain, spleen and lung lactate dehydrogenase activity in male Wistar rats Administration of Datura stramonium significantly increased LDH activity in the brain of all the experimental animals except AED100 that was significantly decreased when compared with control group. LDH activities of AED50 and AED100 but not AED200 animals were significantly increased in the spleen when compared with control. LDH activity of AED400 animals was significantly decreased when compared with control animals. However, a significant decrease was observed in the LDH activities of all the experimental animals in the lung, when compared with control group.

Discussion

Glucose is undoubtedly known to be the most preferred source of energy in the mammalian system, and as such the cells require a constant supply of glucose. Chronic and acute disturbances in glucose availability, usage and storage have been discovered to lead to diseases conditions (Szablewski, 2017) [30] . Studies involving glucose metabolism and included enzymes have generated even more interest recently as impaired glucose metabolism has been identified in diseased conditions which are often difficult to manage such as obesity, diabetes and neurological conditions such as Alzheimer's disease (Rebelos et al., 2021) [27] . Hence, the ability of D. stramonium to initiate changes in metabolic enzymes involved in glucose metabolism as reported in this study may prove to be beneficial in glucose homeostasis and management of related conditions.

Hexokinase is an isoenzyme of glucokinase that catalyzes the phosphorylation of glucose to glucose-6-phosphate and plays a pivotal role in the maintenance of glucose homeostasis. It is the first enzyme in glycogen systhesis, pentose phosphate pathway and glycolytic pathway (Prasath and Subramanian, 2011) [25] . In this study, the administration of D stramonium resulted in a dose dependent increase in the level of hexokinase in the brain, spleen and lung hexokinase levels.

A correlation has been established between insulin efficiency and hexokinase levels. An increase in hexokinase level as reported in this study is linked to an increase in insulin efficiency and enhanced glucose metabolism, which suggests that the enzyme is an insulin dependent enzyme (Gothandam et al., 2019) [15] . Hence, an increase in hexokinase levels is indicative of an increase in insulin function and efficiency and consequentially improved glucose metabolism, controlled levels of glucose availability in the blood and increased ATP production (Gothandam et al., 2019) [15] . An increase in hexokinase function as reported in this study may be indicative of the plant's ability to stimulate effective function of the spleen which has been reported to harbour stem cells that act as precussor to insulin-producing pancreatic cells (Ley et al., 2012) [20] , prevent anomalies related to impaired glucose metabolism in the lungs such as interstitial fibrosis, mucus overproduction in the airway and alveolar capillary microangiography (Khateeb et al., 2019) [17] and in the brain Alzheimer's disease (Kuehn, 2020) [19] .

Aldolase converts a sixcarbon product, fructose 1,6bisphosphate (F1,6BP), into two three-carbon products: G3P and DHAP. They are in all human tissues and impaired expression and availability has been a subject of much study because it can be linked with a number of human diseases which includes but is not limited to diabetes, schizophrenia, ischemia, Alzheimer's disease, traumatic brain injury and cancer (Chang et al., 2018) [7] . The administration of D. stramonium significantly increased the level of aldolase in the brain, spleen and lung in a dose dependent manner. High level of aldolase has been implicated in a number of diseased conditions such as diabetes and traumatic brain injury (El-desoky et al., 2013, Gerst et al., 2018, Chang et al., 2018) [10,14,7] . However, high level of aldolase has yet been identified with improved glucose metabolism (Pirovich et al., 2021) [24] . These variations in results may be associated to different isoforms of aldolase, tissue of interest, diseased condition of interest and the fact that aldolase is a proficient sensor for glucose availability at low or high levels. When intracellular levels of fructose 1,6 bisphosphate is low, aldolase activates the AMP-activated protein kinase (AMPK) which stimulates alternative pathway to energy generation in the cell, especially catabolic mechanisms (Pirovich et al., 2021) [24] . The increased expression of aldolase by D. stramonium may be believed to signify an increase in glucose metabolism and usage in correlation with the activity of related enzymes which are also reported in this study. Pyruvate kinase is a ubiquitously expressed key glycolytic enzyme that catalyzes the conversion of phosphoenol pyruvate to pyruvate with the generation of ATP and the altered expression could be expected to impair the glucose metabolism and energy production (Prasath and Subramanian, 2011, Gothandam et al., 2019) [25,15] . In diabetic conditions, there is always an observable and significant decrease in the activity of pyruvate kinase in all tissues, and this is indicative of impaired glucose metabolism. Therapeutic compounds and plant molecules which have has a form of recognized or suggested protective effects against hyperglycemic identified in diabetes have always been found to significantly increase the activity of pyruvate kinase (Agius et al., 2020) [2] . Under homeostatic conditions, D. stramonium significantly increased the activity of pyruvate kinase in a dose dependent concentration in this study. This is indicative of a significant glucose regulatory effect which can be accounted for by increased glycolysis, and energy production as well as decreased gluconeogenetic production of glucose. This is also suggestive of the ability of the plant extract to significantly regulate the enzyme pyruvate kinase ensure improved energy supply to the brain (Feksa et al., 2003) [12] , possible role in the management of lung cancer which has been associated with a form of pyruvate kinase [25,13] . It is an important glucose metabolizing enzyme as increase levels of the enzyme has been implicated in the impairment of insulin secretion (Gothandam et al., 2019) [15] . In this study, the all doses of D. stramonium significantly reduced the activity of lactate dehydrogenase in the lungs, some of the doses in the spleen and brain of rats. However there was significant reduction in the lactate dehydrogenase levels in all reported organs, which may be a testament to the potentials of the plant to support insulin function, regulation of NADP/NADH ratio, improved activity of pyruvate kinase and pyruvate metabolism for mitochondrial energy production and by that improve proper metabolic processes involved in the physiological use of glucose (Gothandam et al., 2019) [15] . The regulatory effect of the plant on the considered enzyme in three different organs may also be indicative of the plants potential in the management of brain tumour, cancer, anaemia, muscle trauma, liver conditions, encephalitis and meningitis, all of which have irregular metabolism of lactate dehydrogenase in their pathogenesis (Farhana and Lappin, 2023) [11] .

Conclusion

In conclusion, the present study indicated that oral administration of D. stramonium exhibits antihyperglycemic and glucose regulatory effect as a result of the modulatory effect it exerted on enzymes responsible for glucose metabolism. Based on the result, D. stramonium has glucose regulatory and insulin enhancing effect hence has potentials in the management of diabetes and Alzheimer's disease. Further research is recommended to delineate its mechanism of action for antidiabetic and neuroprotective function

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