Results and discussion Inhibitory potential of Rosa canina and Rosa sempervirens fruit extracts towards acetylcholinesterase Diandra Pintać1, Tatjana Majkić1, Jelena Nađpal1, Zorica Mrkonjić1, Kristina Bekvalac1, Marija Lesjak1, Ivana Beara1   1 Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia Introduction Results and discussion Acetylcholinesterase (AChE) is a key enzyme catalysing the hydrolysis of acetylcholine in the nervous system. The inhibitors of AChE are used in the treatment of Alzheimer’s disease. Due to numerous side effects of synthetic AChE inhibitors, there is a great need for new ones, preferably from natural sources. Objectives The aim of this study was to optimise a method for investigating the AChE inhibitory potential of water and methanol fruit extracts of Rosa canina L., traditionally used in folk medicine, and poorly investigated Rosa sempervirens L. Hence, the effects of frequently used buffers, in AChE inhibitory assay, and solvents for extract preparation, on the enzyme activity were examined. Figure 2. The effect of the pH value on the enzyme activity Figure 3. The effect of different buffers on the enzyme activity Once the optimal pH was determined, the next step was to investigate the possible effect of the frequently used buffers on the AChE activity. In order to compare the two buffers (Tris-HCl and phosphate buffer), a solution of Na-acetate, in which the pH was set to 8, was used as a reference. We decided to use Na-acetate since both components already exist in the system (Na+ is added in the buffer for preparing DTNB, while the acetate is a product of the hydrolysis of acetylthiocholine). Here, a decrease of the enzyme activity was detected in the phosphate buffer. The first step in the method optimisation was finding a suitable pH value for the activity of the enzyme. Thus, using the Tris-HCl buffer, a range of pH values from 4 to 10 was set up. The modified Ellman’s method was used for this purpose, adding only the substrate (acetylthiocholine), DTNB (5,5'-dithiobis-(2-nitrobenzoic acid) and AChE, but no inhibitor. pH 8 was chosen as optimal. Materials and methods Plant material of Rosa canina L. was collected in September 2012, in Kikinda, Vojvodina, Republic of Serbia, while plant material of Rosa sempervirens L. was collected in May 2014, in Canj, Montenegro. The specimen voucher Rosa canina L. 1753 (No. 2-1583) and Rosa sempervirens L. 1753 (2-1578) was identified and deposited in the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), Faculty of Sciences, University of Novi Sad, Republic of Serbia. Probe Correction Control 110 μl Tris-HCl buffer pH 8.0 20 μl AChE 20 μl Tris-HCl buffer pH 7.5 10 μl extract* 10 μl Tris-HCl buffer Incubation15 min at 37 ºC 40 μl DTNB 20 μl acetltiocholine 20 μl acetyltiocholine Measuring absorbance on 420nm after 4 minutes 40 g of fresh fragmented fruits (rose hips) were extracted by maceration with: •400 mL of boiling, distilled water, 1h at room temperature, for water extracts •400 mL of 80% methanol, 72h at room temperature for methanolic extracts Filtration, solvent evaporation (in vacuum at 40°C). Removal of the non-polar compounds by extraction with petrol ether (fraction 40–60°C). Complete evaporation of the solvent under vacuum and dissolution of the residue in distilled water to obtain the final concentration of 0.3 mg/mL. Figure 4. Inhibition of AChE by solvents The effect of commonly used solvents for plant extract preparation on the enzyme activity was the third parameter that was examined. The most significant finding is that dimethyl sulfoxide inhibits 64.12% of AchE activity, compared with the enzyme activity in Tris-HCl buffer pH 8. Since the activity of the AChE is dependant on the pH, and most plant extracts could exhibit a low pH, for making different extract concentrations for this assay, we would suggest using Tris-HCl buffer pH 8. Figure 1. Scheme of water and methanolic extract preparation For the development of the AChE inhibition assay, several parameters were optimized. Firstly, an evaluation of the optimal pH for the enzyme activity was conducted. Two buffers that are mentioned the most in the assays (Tris-HCl and phosphate buffer) were compared to a third system (sodium-acetate). Also a possible inhibition of AChE by commonly used solvents for plant extracts preparation (dimethyl sulfoxide, water, pure and 80% methanol and ethanol) was analysed. The inhibitory effect of the extracts was evaluated using a modified in vitro spectrophotometric Ellman´s method. 1 The extracts inhibitory potential was compared to a well-known potent inhibitor of AChE, galantamine. Figure 5. Final procedure The final step was evaluating the inhibitory potential of Rosa extracts towards AChE. All investigated extracts of R. sempervirens expressed greater activity than those of R. canina. Methanol dry fruit extract of R. sempervirens expressed the highest activity (IC50=1.2 mg/mL), while water fresh fruit extract of R. canina exhibited the lowest AChE inhibitory potential (IC50=4.52 mg/mL). In comparison with galantamine, a well-known potent inhibitor of AChE, but with adverse effects, all extracts manifested weaker activity. Acknowledgements Figure 6. Inhibitory potential of Rosa extracts Ministry of Education, Science and Technological Development of the Republic of Serbia  (Grant No. OI 172058) supported this research work. Conclusion References The optimal experimental conditions for successful determination of natural products inhibitory potential towards AChE are defined in this study. The obtained results indicate that R. sempervirens has a good potential for the application in the treatment of Alzheimer’s disease and support further investigation of this poorly investigated species. 1. Ellman, GL et al. (1961) Biochem Pharmacol. 7. 88.