Page 62 - IMO-2-1

Innovative Medicines & Omics Flavonoids against glycosidic hydrolase

transport, aldose reductase activity, and cellular glucose were purchased from Bomei Biochemical Co. (Hefei,
uptake. A practical strategy for controlling postprandial Anhui Province, China). 4-Nitrophenyl-α-D-
2-4
hyperglycemia involves using α-glucosidase and glucopyranoside (pNPG) was purchased from Beijing
α-amylase inhibitors. Implementing dietary strategies Kuerhuaxue Co (Beijing, China). Rutin, quercetin,
5
that incorporate flavonoids can help establish effective kaempferol, isorhamnetin, kaempferol-3-O-rutinoside,
dietary recommendations for healthy individuals and aid and narcissoside were purchased from Chengdu Alfa
in relieving symptoms for patients suffering from Type 2 Biotechnology Co (Chengdu, China). Acarbose tablets
Diabetes Mellitus. 6 were purchased from Bayer Healthcare Co (Leverkusen,
Germany).
The flower buds of Sophora japonica L. (FBSJ), a form
of traditional Chinese medicine (TCM), are common 2.2. Determination of flavonoids in samples
known as Flos Sophorae Immaturus, which are utilized
for treating various ailments, including hypertension and The active components of S. japonica L. were extracted using
arteriosclerosis. Flavonoids, such as quercetin, kaempferol, ethanol (5 g FBSJ: 100 mL solvent) at various concentrations
genistein, rutin, and isorhamnetin, have been isolated from of 30%, 50%, 70%, and 90%, as well as with ethyl acetate.
S. japonica L. The components found in TCM are intricate, Flavonoid standards were dissolved in methanol, while
7,8
with numerous chemicals contributing to its therapeutic the flavonoid content in the samples was extracted using
efficacy. This intricacy poses challenges in identifying ethanol. These flavonoids were subsequently separated
the specific active constituents of TCM. Nevertheless, with a Waters HPLC system (Milford, Massachusetts,
high-performance liquid chromatography (HPLC)-based USA) equipped with a C18 Agilent column. The gradient
fingerprint spectra have been employed to thoroughly elution was performed under the following conditions: (A)
analyze the diversity and concentrations of compounds in and 0.05% phosphoric acid (B), with the following time
botanical materials, thereby assessing the quality of TCM intervals: 0 – 10 min at 24 – 40% (A); 10 – 20 min at 40 –
products. Circular dichroism (CD), when paired with 50% (A); 20 – 35 min at 50 – 75% (A); 35 – 40 min at 75
9,10
fluorescence quenching methods, is frequently used to – 80% (A); 40 – 50 min at 80 – 95% (A); 50 – 55 min at 95 –
examine how flavonoids interact with proteins, offering 95% (A); and 55 – 80 min at 95 – 24% (A). Both qualitative
a crucial understanding of protein-ligand interactions, and quantitative analyses of the samples were conducted
as well as the conformational and structural changes of using standard solutions. The data were imported into
flavonoids in relation to proteins. Furthermore, molecular the “Traditional Chinese Medicine Fingerprint Similarity
11
simulation methods, including molecular docking Evaluation System,” and then HPLC fingerprint spectra
and dynamics, have become increasingly significant in were obtained.
studying interactions and have contributed to exploring 2.3. Inhibition activity assays of enzyme
the mechanisms behind inhibitor-enzyme interactions,
enhancing our comprehension from a molecular biology 2.3.1. The assessment of α-amylase inhibition activity
standpoint regarding the efficacy of these interactions. 12 The assessment of α-amylase inhibition activity was
Various screening methods have been developed and conducted as described in previous studies, with necessary
13
applied in a wide range of fields. However, these methods adjustments made. Initially, 20 of samples at various
often require specialized software and complex data concentrations were combined with 150 μL of α-amylase
processing. Therefore, there is an urgent need to develop solution (0.58 U/mL) and incubated at 37°C for 5 min.
a simple and feasible screening and comparison method. Subsequently, 150 μL of 1% starch was added to the mixture,
Chromatographic analysis combined with activity difference which was then incubated at 37°C for an additional 15 min.
analysis (CAADA) was applied. This article aimed to assess Finally, 250 μL of DNS reagent (dinitrosalicylic acid
the inhibitory potentials of the extract against α-amylase reagent, a reagent used for the determination of reducing
and α-glucosidase and to develop a new screening method sugar content, prepared by dissolving 18.2 g of potassium
using compounds found in FBSJ. The compounds that sodium tartrate in 50 mL of distilled water, heating, and
exhibited significant inhibitory effects were verified, and sequentially adding 0.63 g of 3,5-dinitrosalicylic acid, 2.1 g
the inhibitory mechanism was preliminarily studied. of NaOH, and 0.5 g of phenol to the hot solution, stirring
until dissolved, cooling, and diluting with distilled water
2. Materials and methods to 100 mL, stored in a brown bottle at room temperature)
was mixed, and the mixture was boiled for 5 min. After
2.1. Materials diluting the solution to an appropriate concentration, its
FBSJ was collected from the campus of Henan University. absorbance was recorded at 540 nm using a microplate
Porcine pancreas α-amylase and yeast α-glucosidase reader.

Volume 2 Issue 1 (2025) 56 doi: 10.36922/imo.6010