Page 64 - IMO-2-1

Innovative Medicines & Omics Flavonoids against glycosidic hydrolase

2.6.2. Calculation of thermodynamic parameters the system temperature to 300 K within 10 ns. After heating,

The temperature-dependent interactions were investigated the NPT ensemble (T = 300 K and P = 1 atm) was used for 100
20
by calculating the interactions between inhibitors and ns simulation under periodic boundary conditions.
enzymes using the following equations: 2.9. Statistical analysis

1 H S
lnK - T R R (X) All data were analyzed and presented as the mean ±

a
standard deviation of the results. Three experiments were

performed for each data set. All statistical analyses were
∆G° = ∆H° – T∆S° = –RTlnK a (XI) carried out using SPSS 19.0 (IBM Corp. Armonk, NY,
Where K (Lmol ) is the binding constant, R USA) and GraphPad Prism 8 (GraphPad, San Diego, USA)
-1
a
(8.314 J mol K ) is the gas constant, ∆S° and ∆H° to evaluate the significance of differences.
-1
-1
represent the values used to evaluate the binding force, and
∆G° indicates the spontaneity of the interaction. 3. Results
2.6.3. CD spectrum analysis 3.1. Identification of flavonoids in FBSJ and
establishment of HPLC fingerprint
The α-amylase and α-glucosidase were mixed with
flavonoid samples in different proportions and scanned by Figure 1 shows the fingerprints of flavonoids in FBSJ extracted
CD spectroscopy. The combined solutions were scanned with different solvents. A total of six common peaks were
across a wavelength range of 190 – 260 nm. The results calibrated from the six samples. Compared with the peak
of the measurements were then calculated using CDNN diagram of the reference substance, the six components were
software (Aviv Biomedical Inc., Lakewood, USA). identified as rutin, kaempferol-3-O-rutinoside, narcissoside
(isorhamnetin-3-O-rutoside), quercetin, kaempferol,
2.7. Molecular docking and isorhamnetin according to the peak times, which
Molecular docking simulations were carried out using was consistent with previous reports. 21,22 The flavonoids
AutoDock 4.0. The structures of flavonoid compounds in FBSJ include three flavonols and their corresponding
were downloaded from PubChem. The protein data flavonol glycosides, which contributed to comparing the
for α-amylase (PDB ID: 1OSE) were obtained from the activities of flavonols and their corresponding glycosides.
Protein Data Bank, and α-glucosidase was obtained Combined with the fingerprints, it was evident that the main
from homology modeling. Before docking, the enzyme chromatographic peak was rutin in various ethanol solutions
structures were prepared by removing all water molecules, extract of FBSJ, while the main chromatographic peak was
adding hydrogen atoms, and distributing the Gasteiger quercetin in ethyl acetate extract of FBSJ. It was shown that the
charges to generate the PDBQT file. Finally, the docked extraction of FBSJ with different solvents resulted in varying
complexes were visualized and analyzed by PyMOL. 18,19 enzyme inhibition rates (expressed by the half inhibition rate
concentration, IC ). The order of α-amylase inhibition rates
50
2.8. Molecular dynamics (MD) was ethyl acetate >30% ethanol >90% ethanol >50% ethanol
MD simulations at 100 ns were performed using GROMACS >70% ethanol >water. The order of α-glucosidase inhibition
2020. The Amber99 SB force field was applied to the proteins in rate was ethyl acetate >70% ethanol >90% ethanol >50%
this study, and the topologies of a-amylase and α-glucosidase ethanol >30% ethanol >water (Table 1). The inhibition effect
were prepared using the pdb2gmx module of GROMACS of the ethyl acetate extract of FBSJ was significant compared
2020. The topology and coordinate parameter files for to the other extracts, although acarbose showed the most
quercetin and kaempferol were generated through simulation obvious inhibition. Based on the data in Figure 1, quercetin
on the ACPYPE server (https://www.bio2byte.be/acpype/ was the main constituent in ethyl acetate, which suggests that
status/). The physiological environment of the proteins was quercetin may be the favorite natural compound responsible
simulated by adding the TIP3P aqueous model to the protein for the inhibitory activity.
complex system, which was solvated using dodecahedral 3.2. Correlation analysis
boxes, with sodium ions added to neutralize the overall charge.
The simulations were performed using the conjugate gradient The peak areas of six common peaks, represented by P1-P6,
method combined with the most rapid descent method were established as the comparison sequence, with the IC
50
23
for 50,000 steps each to minimize energy. The V-rescale values for enzyme inhibition as the reference sequence.
thermostat was used for temperature coupling to obtain a According to references, 24-26 using the enzyme inhibition
correct trajectory distribution of the canonical ensemble. The rate as a pharmacodynamic index, the results are shown in
Berendsen pressure control method was used to slowly heat Table 2. The correlation order with α-amylase was P5, P4, P3,

Volume 2 Issue 1 (2025) 58 doi: 10.36922/imo.6010