Quercetin

The reference substances used included artemisinin (Gibco, Fisher Scientific, Merelbeke, Belgium), 1, 8- cineole, α-thujone, β-thujone, rutin, chlorogenic acid, luteolin, astragalin, apigenin, caffeic acid, kaempferol, scopoletin, iso-chlorogenic acid, vitexin, fraxin, caftaric acid, 7-hydroxy coumaric acid, esculetin, quercetin, camphor, coumaric acid, and camphene (Merck, Darmstadt, Germany). The solvents applied—acetonitrile, methanol, formic acid, acetone, ethyl acetate, hexane, ethanol, and chloroform (Sigma-Aldrich, Overijse, Belgium)—were all HPLC-grade. Sulfuric acid, polyethylene glycol (PEG), vanillin powder, and diphenyl-borate diamino-ethanol (DPBAE) were obtained from Merck, Darmstadt, Germany.

Na₂SO₄, Folin–Ciocalteu reagent (FCR), sodium carbonate, gallic acid, aluminum nitrate, potassium acetate, quercetin, aluminum chloride (AlCl₃), sodium acetate (C₂H₃NaO₂), DPPH, ABTS, ferric chloride (FeCl₃), phosphate buffer, potassium ferricyanide solution (K₃[Fe(CN)₆]), trichloroacetic acid (TCA), trolox, ascorbic acid, acetylthiocholine iodide, butyrylthiocholine chloride, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and galantamine were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). All other chemicals and solvents were of analytical grade. Bacterial strains were obtained from the Pasteur Institute, Algiers, Algeria.

All surgical procedures performed on the rats were conducted under general anesthesia, ensuring the absence of pedal reflex. An intraperitoneal injection of 80 mg/kg ketamine hydrochloride (Narkamon 50 mg/mL, BIOVETA, Czechia) and 10 mg/kg xylazine (Xylazinbio 2% BIOVETA, Czechia) was administered for anesthesia induction. Furthermore, the rats were continuously monitored for pain sensation throughout the procedure, and additional anesthesia doses were administered as needed to maintain adequate pain control. Prior to incision, the surgical areas were shaved and cleansed with povidone-iodine (Batix^®, Denizpharma, Istanbul, Turkey), and all surgical procedures were carried out under sterile conditions.
A well-established tendon injury model, widely utilized in numerous studies in the literature (6 (link), 41 (link), 42 (link)), was employed in this study. Following the surgical preparation of the extremity, an incision was made along the posterior line of the ankle to expose the Achilles tendon and plantaris tendon. Tenotomy was performed approximately 0,5 cm proximal to the calcaneus insertion of the Achilles tendon using a No. 15 scalpel blade. The tendons were then repaired end-to-end using the modified Kessler method with 4/0 round polypropylene monofilament sutures (TıpKimSan Limited Company, Istanbul, Türkiye). Following irrigation of the wound area with saline, the incision was closed using 3/0 polypropylene monofilament sutures (TıpKimSan Limited Company, Istanbul, Türkiye) to achieve skin integrity. The surgical procedure is illustrated in Figure 1.
No bandages, dressings, or plasters were applied to the rats postoperatively. From the early postoperative period, all rats were allowed free joint movements and were permitted to load their lower extremities. Rats in the quercetin group received a daily dose of 100 mg/kg quercetin (Sigma-Aldrich, United States), administered as a suspension in corn oil via oral gavage at the same time each day. The control group received only corn oil via oral gavage. All rats were sacrificed on the 28th day following surgery. The quercetin dosage was adjusted based on previous studies (10 (link), 43 (link)).
Following administration of high-dose anesthesia (ketamine 75 mg/kg and xylazine 10 mg/kg), rats were euthanized by cervical dislocation. The Achilles tendons of the euthanized rats were then carefully dissected, ensuring removal distally from the bone-tendon junction of the calcaneus and proximally from the bone-tendon junctions of the femur and tibia. Subsequently, one of the Achilles tendons from each rat underwent histopathological and immunohistochemical evaluation, while the other tendon was subjected to biomechanical analysis.

Folin–Ciocalteau reagent, Bradford reagent, AlCl₃, FeCl₃, gallic acid, quercetin, albumin serum bovine, phenol, 4-aminoantipyrene, peroxidase, 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2-deoxy-D-ribose, EDTA, H₂O₂, ascorbic acid, 2-thiobarbituric acid, trichloro-acetic, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and methanol ≥99.9% were acquired at Sigma Aldrich, St. Louis, MO, USA; silica gel 60 F-254 (0.2 mm) was purchased in Merck, Darmstadt, Alemania. Mueller–Hinton broth (CAMHB) and antimicrobial agents were supplied by Laboratorios Britania S.A., Ciudad Autónoma de Buenos Aires, Argentina.

Folin and Ciocalteu’s phenol reagent, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH radical), gallic acid, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), methanol (MeOH), chloroform, acetic acid, sodium thiosulphate, protocatechuic acid, (+)-catechin, procyanidin B2, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, resveratrol, and quercetin were provided by Sigma-Aldrich (Milan, Italy). (-)-epicatechin, quercetin-3-O-glucoside, kampferol-3-O-glucoside, and petunidin-3-O-glucoside were provided by Extrasynthase (Genay, France). Cyclohexane was provided by VWR Chemicals (Milan, Italy). GP derived from the Merlot grape variety (certified by the producer) was supplied by Chiorri S.S., a local winery located in the province of Perugia (Central Italy). Its proximate composition is reported in Table S3. Commercial mayonnaise (a traditional egg-yolk mayo) and the ingredients (sunflower oil, vinegar, and lecithin) used for the formulation of the functional salad dressing were bought from a local market.