Abstract Title: The inhibitory effects of Salvia triloba and Thymus praecox on acetylcholinesterase activity in diabetic rats

Background
Diabetes mellitus (DM) is a metabolic disease characterized by long-term complications including cerebrovascular injury and cognitive disorders. Acetylcholinesterase (AChE) which is involved in numerous pathways in the central nervous system regulates the level of acetylcholine (ACh) which plays vital functions such as memory, learning, modulation of cerebral blood flow, movement control, neurotransmission as well (1). Recent studies have shown that hypoinsulinemia and insulin resistance is the reason of the decreased level of ACh and contribute the possible biochemical link between DM and cognitive disorders including Alzheimer’s disease. Studies with plants have shed light on the correction of cognitive functions of diabetes mellitus with increased AChE activity. Some Salvia and Thymus species of the Lamiaceae family have antidiabetic effects (2). There is no study on antidiabetic activity of Salvia triloba and Thymus praecox.
Aim
To investigate the effects of Salvia triloba and Thymus praecox on brain acetylcholinesterase activities in diabetes mellitus. With the help of obtained results, further studies will be carried out for ameliorating cognitive dysfunction effectively and also supporting current treatments.
Method
The rats were divided into five groups (6 rats per group): Group-I: Normal control, Group-II: Diabetic control, Group-III: Treatment with Salvia triloba methanolic extract, (200 mg/kg/day, i.g.), Group-IV: Treatment with Thymus praecox methanolic extract, (100mg/kg/day, i.g.), Group-V: Treatment with Metformin (400 mg/kg/day, i.g.). Type 2 DM was induced experimentally in these groups of rats by the intraperitoneal administration of 55 mg/kg of STZ and 100 mg/kg nicotinamide, except control group. Treatments were carried out after induction of DM and lasted for 3 weeks except decapitation day. The AChE enzymatic assay was determined according to the method of Ellman et al. The reaction mixture (2 mL final volume) contained 100 mM K+-phosphate buffer, pH 7.5 and 1 mM 5,50-dithiobisnitrobenzoic acid (DTNB). The method is based on the formation of the yellow anion, 5, 50- dithio-bis-acid-nitrobenzoic, measured by absorbance at 412 nm during 2 min incubation at 25°C. The enzyme (40–50 mg of protein) was pre-incubated for 2 min. The reaction was initiated by adding 0.8 mM acetylthiocholine iodide (AcSCh). All samples were in triplicate readings and the enzyme activities were expressed in mmol AcSCh/h/mg of protein.
Results
Thymus praecox and Salvia triloba in methanolic extract considerably decreased (p˂0.0001) blood glucose levels compared to diabetic rats and performed this drop for 3 weeks. Thymus praecox extract reached its maximum hypoglycemic effect (141.83±7.31, p˂0.0001) in 2 weeks. At the same time, Thymus praecox and Salvia triloba extract also showed strong hypoglycemic effect (164.83±13.30, p˂0.0001). The diabetic group exhibited a significant increase (p˂0.001) in AChE activity in prefrontal cortex compared to the control and the treatment groups. However Salvia triloba extract and Thymus praecox extract reverted this increase (p˂0.001) in AChE activity induced by diabetes after 3 weeks of treatment. The metformin group decreased (%47, p˂0.001) in AChE activity compared to diabetic groups. Salvia triloba extract (%37, p˂0.001) and Thymus praecox extract group (%41, p˂0.001)  a considerably decrease in AChE activity compared to diabetic group. The metformin group did not show a significant difference in enzyme activity compared to the control group. However, Salvia triloba and Thymus praecox increased (p˂0.05) in AChE activity compared to control groups.
Discussion
Our study results showed that an increase in AChE activity in type 2 diabetic rat brains. This increase in AChE activity in the T2DM rat model can be linked to depletion in cholinergic actions leading to defective neurotransmission and cognitive dysfunction as a result of decreased levels of ACh in the synaptic cleft. We showed that the reestablishment of AChE activity by Salvia triloba extract and Thymus praecox extract which for the first time demonstrated antidiabetic activities can contribute to the maintenance of cholinergic signaling in diabetic rats. In the light of the results, these plants give a valuable direction for researchers on the future for diabetes-related cognitive disorders. 
1. Jacobson AM., et al., Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study Research Group, Long-term effect of diabetes and its treatment on cognitive function. N Engl J Med. 2007;356(18):1842–1852.
2. Davids D., et al., Ethnobotanical survey of medicinal plants used to manage High Blood Pressure and Type 2 Diabetes Mellitus in Bitterfontein, Western Cape Province, South Africa, J Ethnopharmacol. 2016 Dec 24;194:755-766.