# Evaluation of _Sambucus nigra_ L. Biopotential as an Unused Natural Resource
## 2 Materials and Methods
### Determination of Biological Activity
#### Enzyme Inhibitory Assessment  
Neuroprotective Activity (Inhibition Acetylcholinesteraseerase AChE and Butyrylcholinesteraseerase BChE)  
Cholinesterase (ChE) inhibitory activity was measured using method [24]. Sample solution (50 \(\mu\)L) was mixed with DTNB (125 \(\mu\)L) and AChE (or BChE) solution (25 \(\mu\)L) in Tris-HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C). The reaction was initiated with the addition of acetylthiocholine iodide (ATCI) or butylthiocholine chloride (BTCI) (25 \(\mu\)L). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after incubation at 25 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C) for 10 min. The absorbance of the blank was subtracted from the absorbance of samples and the cholinesterase inhibitory activity was expressed as milligrams equivalents of galantamine per gram of extract (mg GALAE/g E).  
Antityrosinase Activity (Inhibition Tyrosine)  
Tyrosinase inhibitory activity will be measure using the modified dopachrome method with L-DOPA as substrate. Sample solution (25 \(\upmu\)L) was mixed with tyrosinase solution (40 \(\upmu\)L) and phosphate buffer (100 \(\upmu\)L, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C). The reaction was initiated with the addition of L-DOPA (40 \(\upmu\)L). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after incubation at 25 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C) for 10 min. The absorbance of the blank was subtracted from the absorbance of samples and the tyrosinase inhibitory activity was expressed as milligrams equivalents of kojic acid per gram of extract (mg KAE/g E) [24].  
Antidiabetic Activity (Inhibition of \(\upalpha\)-Amylase and \(\upalpha\)-Glucosidase)  
\(\upalpha\)-amylase inhibitory activity will be perform using before described method [24]. Sample solution (25 \(\upmu\)L) was mixed with \(\upalpha\)-amylase solution (50 \(\upmu\)L) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C). After pre-incubation, the reaction was initiated with the addition of starch solution (50 \(\upmu\)L, 0.05%). Similarly, a blank sample was prepared by adding sample solution to all reaction reagents without enzyme (\(\upalpha\)-amylase) solution. The reaction mixture was incubated for 10 min at 37 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C). The reaction was stopped with the addition of HCl 25 \(\upmu\)L, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 \(\upmu\)L). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from the absorbance of samples and the \(\upalpha\)-amylase inhibitory activity was expressed as milligrams equivalents of acarbose per gram of extract (mg ACAE/g E).  
Inhibition of \(\upalpha\)-glucosidase--sample solution (50 \(\upmu\)L) was mixed with glutathione (50 \(\upmu\)L), \(\upalpha\)-glucosidase solution (50 \(\upmu\)L) in phosphate buffer (pH 6.8) and p-Nitrophenyl-\(\upbeta\)-D-Galactopyranoside (PNPG, 50 \(\upmu\)L) in a 96-well microplate and incubated for 15 min at 37 \({}^{\circ}\)C (\(\pm\)1 \({}^{\circ}\)C). Similarly, a blank sample was prepared by adding sample solution to all reaction reagents without enzyme (\(\upalpha\)-glucosidase) solution. The reaction was stopped with the addition of sodium carbonate (50 \(\upmu\)L, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from the absorbance of samples and the \(\upalpha\)-glucosidase inhibitory activity was expressed as milligrams equivalents of acarbose per gram of extract (mg ACAE/g E) [24].