3-methylquercetin

Inhibition of platelet aggregation was evaluated by a turbidimetric method using a lumi-aggregometer (Chrono-Log, Havertown, PA, USA) [25 (link),26 (link)]. Washed platelets (3 × 10⁸ platelets/mL) were incubated for 5 min with CaCl₂ (2 mM) plus isorhamnetin (1, 10, 20, 50 and 100 µM) or vehicle (DMSO, 0.2%). Similar concentrations in vitro have been evaluated in other studies [6 (link),27 ]. Platelet aggregation was induced by TRAP-6 (5 µM), collagen (1 mg/mL), and PMA (100 nM). Platelet aggregation (transmittance) was measured for 6 min [23 (link)]. The platelet aggregation percentage was obtained with the AGGRO/LINK software (Chrono-Log, Havertown, PA, USA). Platelet inhibition was calculated as: inhibition of platelet aggregation (%) = 100 − ((platelet aggregation of isorhamnetin/platelet aggregation of negative control) × 100) [28 (link)]. The concentration necessary to reduce platelet aggregation by 50% (IC₅₀) was obtained from isorhamnetin’s concentration curves (1, 10, 20, 50, and 100 µM).

Due to the disadvantages of biological experiments as being time-consuming and of high cost, identification of ADME (absorption, distribution, metabolism, and excretion) properties by in silico tools has now become an inevitable paradigm in pharmaceutical research. In this study, three ADME-related models, namely, the evaluation of oral bioavailability (OB), Caco-2 permeability, and drug-likeness (DL), are employed to identify the potential bioactive compounds of DXP [33 (link)].
Oral Bioavailability. OB prescreening is used to determine the fraction of the oral dose of bioactive compound which reaches systemic circulation in the TCM remedy. Here, a reliable in silico model OBioavail 1.1 [34 (link)] which integrates the metabolism (P450 3A4) and transport (P-glycoprotein) information was employed to calculate the OB values of herbal ingredients.
Caco-2 Permeability. The Caco-2 cell monolayers are widely applied as standard permeability-screening assay for prediction of the compound's intestinal absorption and fraction of the oral dose absorbed in humans [35 (link)]. The Caco-2 cell permeation values of all molecules are calculated by in silico model using the VolSurf approach [36 ].
Drug-Likeness Evaluation. Drug-likeness is a qualitative profile used in drug design to evaluate whether a compound is chemically suitable for the drug, and how drug-like a molecule is with respect to parameters affecting its pharmacodynamic and pharmacokinetic profiles which ultimately impact its ADME properties [37 (link)]. In order to identify drug-like compounds, we apply a database-dependent model using the Tanimoto coefficient to calculate the DL (see (1)) of each compound in DXP. $\begin{array}{c}f\left(\mathcal{x},\mathcal{y}\right)=\frac{\mathcal{x}\mathcal{y}}{{\left|\mathcal{x}\right|}^{\mathrm{2}}+{\left|\mathcal{y}\right|}^{\mathrm{2}}-\mathcal{x}\mathcal{y}}.\end{array}$ 𝓍 represents the molecular parameters of herbal ingredients and 𝓎 represents the average molecular properties in DrugBank database (available online: http://www.drugbank.ca).
In this work, the compounds of OB ≥ 30%, Caco-2 > −0.4, and DL ≥ 0.18 are selected for subsequent research, and others are excluded.
According to these indexes, several compounds are included: ergosterol peroxide, ethyl oleate (NF), glabridin, glycyrrhetinic acid, linoleyl acetate, longikaurin A, mairin, mandenol, MOL000273, MOL001910, 508-02-1, 64997-52-0, 8β-ethoxy atractylenolide III, pachymic acid, paeonidanin, palbinone, saikosaponin C, beta-sitosterol, supraene, trametenolic acid, troxerutin, α-amyrin, MOL000285, 4-O-methylpaeoniflorin, glabrene, poricoic acid A, glycyrrhizin, sudan III, ZINC02816192, kaempferol, 7,9(11)-dehydropachymic acid, licochalcone G, paeoniflorgenone, areapillin, quercetin, stigmasterol, isoliquiritigenin, (+)-anomalin, isorhamnetin, vestitol, crocetin, 113269-36-6, α-spinasterol, licochalcone A, 113269-37-7, 3β-acetoxyatractylone, licoricone, 113269-39-9, petunidin, hederagenin, dehydroeburicoic acid, licochalcone B, ergosta-7,22E-dien-3beta-ol, MOL000280, MOL000287, mudanpioside H, NSC684433, octalupine, 18103-41-8, formononetin, 1-methoxyphaseollidin, paeoniflorin, glycyrin, ammidin, poricoic acid B, poricoic acid C, sainfuran, sitosterol, isoimperatorin, isolicoflavonol, cerevisterol, 3-methylkempferol, licoisoflavone B, cubebin, and (+)-catechin, 3′-hydroxy-4′-O-methylglabridin.

Intakes of total flavonoids and each flavonoid subclass in our study were downloaded from Flavonoid Values for US Department of Agriculture Survey Foods and Beverages (16 (link)), which are derived from (i) Database of Flavonoid Values for Food Codes; (ii) Flavonoid Intake Data Files from What We Eat in America and NHANES. The flavonoid database included data on the 6 main flavonoid subclasses: (i) flavones (apigenin and luteolin); (ii) anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin); (iii) flavanones (eriodictyol, hesperetin, and naringenin), (iv) flavonols (isorhamnetin, kaempferol, myricetin, and quercetin); (v) flavan-3-ols [catechins including: (−)-epicatechin, (−)-epicatechin 3-gallate, (−)-epigallocatechin, (−)-epigallocatechin 3-gallate, (+)-catechin, (+)-gallocatechin; theaflavin, theaflavin-3,3′-digallate, theaflavin-3′-gallate, theaflavin-3-gallate, and thearubigins]; (vi) isoflavones (daidzein, genistein, and glycitein), total flavones, total anthocyanidins, total flavanones, total flavan-3-ols, total flavonols, total isoflavones, subtotal catechins, and total flavonoids Daily flavonoid intake per participant was determined on the first and second days, and the mean of the two-day flavonoid intake was used in subsequent analyses.

Ninety participants (53 women and 37 men) were enrolled in this study between April and December 2022. The inclusion criteria were: (1) age 18–85 years, (2) written consent and (3) mental condition that enabled a one-year retrospective dietary interview. The exclusion criteria were: (1) age <18 or >85 years, (2) lack of written consent, (3) abnormal mental condition, (4) pregnancy and (5) special diet due to health reasons.
The food-frequency questionnaire dedicated to one-year specific flavonol intake assessment was administered to the participants [15 (link)]. The questionnaire gathered information about the mean consumption of 140 flavonol sources during the preceding year. The full questionnaire is available as supplementary material [15 (link)]. The selected flavonols were the four most widespread in food sources according to the USDA database [16 ]. The suggested portions of the products were based on typical servings in everyday life (e.g., one piece, a glass) and described for the participants by a suggested serving (e.g., a piece, a glass) and a weight in grams. The participants were asked to provide a frequency of selected product consumption (never or almost never, once a month, few times a month with a number of times per month given by the responder, once a week, few times a week with a number of times per week given by the responder, once a day, few times every day with a number of times per day given by the responder). The amounts of quercetin, kaempferol, isorhamnetin, and myricetin in each product were based on the data available in the USDA database [16 ]. On the basis of this information, the mean daily consumption of each product and flavonol was calculated for each participant. Total flavonol intake was calculated by adding the values of quercetin, kaempferol, isorhamnetin, and myricetin. The daily intake of each compound was expressed relative to body mass. The patient’s weight was measured with 0.05 kg accuracy by a trained professional. The patient was permitted to wear only underwear for this measurement. The information about the mean daily intake of flavonol sources was also derived from the above-described questionnaire [15 (link)].
The fasting glucose, lipid profile and creatinine level were assessed in venous blood. The patients were not allowed to eat for 12 h before the test. The blood samples were gathered by a trained nurse. The samples for glucose tests were gathered with dedicated probes (EDTA + sodium fluoride) and then measured using the enzyme (hexokinase) method with a Cobas Pro (Roche Diagnostics, Mannheim, Germany) analyzer. The lipid profile test samples were gathered with dedicated probes (heparinized) and then performed using colorimetric enzyme assays with a Cobas Pro (Roche Diagnostics, Mannheim, Germany) analyzer. Creatinine samples were gathered with dedicated probes (heparinized) and then measured using a colorimetric test based on the Jaffe method with a Cobas Pro (Roche Diagnostics, Mannheim, Germany) analyzer.