Bioactivity-guided isolation of inositol as acetylcholinesterase inhibitory from endemic Campanula baskilensis Behcet: In vitro bioactivity, PCA analysis, and silico supporting studies

This study aimed to identify the bioactivity-guided molecule in the fractions of Campanula baskilensis leaf methanol: chloroform extract. The bioactivity of leaf fractions was investigated to assess and isolate bioactive molecule/molecules and structural configurations. Fractionation and isolation processes were done using advanced column chromatography techniques. In-vitro bioactivity tests were applied, including enzyme inhibition, antibacterial, and DNA protection activities. The isolated compound was characterized using the NMR technique. In silico analyses were investigated using molecular docking, molecular dynamics, and final-state free energy calculations. 14 different fractions were obtained (F1-F14) through the fractionation. F12 has the highest AChE inhibition (IC50; 6.97±2.90 μg/mL), F6 has significant inhibition against carbonic anhydrase and -amylase (IC50; 5.61±0.01 and 18.82±1.48 μg/mL). F12 and F11 have the highest antibacterial activity against E. coli (15.40±1.10 and 13.00±0.80 mm). F12 and F5 fractions have the highest protection activity in plasmid DNA, and F6 has the highest deoxyribose protection activity. Many fractions have high and varied bioactivity due to the bioactive compound components, as in F12. Principal component analysis showed that F12 positively correlated with the high inhibition activity for several bacteria and enzymes and high DNA protection. Therefore, further fractionation was applied using Sephadex LH-20 with ethyl acetate:methanol: hexane (5:5:1) to F12. Inositol was isolated according to results from the obtained fraction; the molecule characterization was clarified using the H-NMR and C13-NMR spectra. Molecular docking results showed binding between inositol and AChE. Further, molecular dynamics results showed the stability of inositol-AChE within 100 nanoseconds, and the energy calculations (gmx-MMPBSA) showed the strength of this interaction.