Fig 1.
Effects of ACTPG on Aβ 25–35 fibrillogenesis.
Analysis of the deposited amyloid aggregates as assessed by thioflavin T (ThT) assays in Phase I (20, 48 h) & II (96 h & 9days). Quantitative effects of ACTPG on Aβ 25–35 fibrillogenesis by ThT assay showing the bar diagram of thioflavin-T fluorescence of Aβ 25–35 in the presence and absence of ACTPG. The emission spectrum of ACTPG and galantamine alone was subtracted, and emission data of peptide dispersions were normalized. Values are expressed as Mean ± SD (n = 3).
Fig 2.
Effects of ACTPG on antiaggregation and disaggregation property of Aβ 25–35.
Analysis of the deposited amyloid aggregates as assessed by Confocal Laser Microscope System (CLSM FV300, Olympus, Tokyo, Japan) in (A) Phase I (20, 48 h) & (B) Phase II (96 h & 9days) and processed by Adobe Photoshop (Adobe Systems, Mountain View, CA, USA). The fluorescence intensity was visualized in each of three random fields of the sample. Confocal microscopic image represents a view of deposited Aβ 25–35 amyloid aggregates, with representative fibrils from Aβ 25–35 samples (control) and Aβ 25–35 samples incubated with the presence and absence of ACTPG and galantamine.
Fig 3.
(A)Inhibitory effects of ACTPG on the transformation of secondary structure of Aβ 25–35 by FT-IR. The secondary structure of Aβ25–35 was followed by observing the amide I and amide II regions by FT-IR at different time intervals in phase I, which are the well-known vibrational IR bands particularly used to reveal the conformational changes of peptides and proteins. FTIR spectrum of Aβ25–35 (100 μM) exhibited maximum absorbance at 1600 cm-1, at 20 and 48 h in phase I, while the ACTPG treated groups showed reduction in absorbance similar to the standard galantamine, which illustrates that ACTPG prevents the formation of fibrils from oligomers. (B) Inhibitory effects of ACTPG on Aβ 25–35 peptide by FT-IR. The secondary structure of Aβ 25–35 was followed by observing the amide I and amide II regions by FT-IR at different time intervals in phase II. which are the well-known vibrational IR bands particularly used to reveal the conformational changes of peptides and proteins. In phase II (96h and 9days) FTIR spectrum exhibited increase in absorbance at 1600 cm-1 in Aβ treated groups, whereas no such shift was observed in ACTPG-treated groups, which indicates the disaggregation property of ACTPG.
Fig 4.
Bioactivity guided fractionation of ACTPG.
Bioactive fractions were eluted with linear gradient of solvent with increasing polarity from n-hexane to water and the 18 fractions (F1-F18) were collected and subjected to cholinesterase inhibition assay.
Fig 5.
AChE inhibitory activity of the column fractions (F1-F18).
The eluted fractions (F1-F18) were subjected to AChE inhibitory assay. Among the fractions, F10 (EA: MET-9:1) showed highest inhibitory activity (93%) against AChE, but similar when compared to positive control donepezil (97%). Values are expressed as Mean ± SD (n = 3).
Fig 6.
BuChE inhibitory activity of the column fractions (F1-F18).
The eluted fractions (F1-F18) were subjected to BuChE inhibitory assay. Among the fractions, F10 (EA: MET-9:1) showed highest inhibitory activity (88%) against BuChE, but similar when compared to positive control donepezil (91%). Values are expressed as Mean ± SD (n = 3).
Table 1.
LC-MS/MS profile of ACTPG showing the presence of bioactive compounds.
Fig 7.
LC-MS/MS spectrum of active fraction (F10) of ACTPG.
LC-MS/MS spectrum of active F10 fraction of ACTPG showed the presence of essential oil alpha-bisabolol.
Fig 8.
Acetylcholinesterase inhibitory activity of the active compounds (10–50 μg/ml).
The result of AChE inhibitory activity clearly shows that among the different treatment groups, alpha-bisabolol alone treatment possessed a maximum AChE inhibitory activity(87%), with the IC50 value <10 μg/ml and showed significant inhibition (*p<0.1) at 50 μg/ml. Further, standard drug donepezil (IC50 value <6 μg/ml) and the compound of our interest, alpha-bisabolol displayed a significant inhibition on AChE. Values are expressed as Mean ± SD (n = 3).
Fig 9.
Butyrylcholinesterase inhibitory activity of the active compounds (10–50 μg/ml).
The result of BuChE inhibitory activity clearly shows that among the different treatment groups, alpha-bisabolol alone treatment possessed a maximum BuChE inhibitory activity(66%), with the IC50 value <10 μg/ml and showed significant inhibition (*p<0.1) at 50 μg/ml. Further, standard drug donepezil (IC50 value <6 μg/ml) and the compound of our interest, alpha-bisabolol displayed a significant inhibition on BuChE. Values are expressed as Mean ± SD (n = 3).