Isoquercetin

Samples were analyzed on Agilent HPLC system. Separation was carried out through column 20RBAX ECLIPSE, XDB-C18, (5 μm; 4.6 × 150 mm, Agilent USA) with UV-VIS Spectra-Focus detector, injector-auto sampler. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile (v/v) with the following gradient: 0-5 min, 15% B in A, 5-10 min, 70% B in A, 10-15 min, 70% B in A are used for separation The flow rate was 1 ml/min and injection volume was 10 μl. Eleven standard compounds including rutin, myricetin, vitexin, orientin, hyperoside, isovitexin, isoquercetin, luteolin, apigenin, kaempherol, and luteolin-7-glucoside were run for comparative detection and optimized. The calibration curves were defined for each compound in the range of sample quantity 0.02-0.5 μg. All samples were assayed in triplicate.

Sirt6/ADP-ribose crystals were grown from 1.6 M (NH₄)₂SO₄, 10% PEG 400, and Bis-Tris buffer pH 5.7 with 10 mg/ml Sirt6(13–308) and 10 mM ADP-ribose by the hanging drop vapor diffusion method at 20 °C¹⁷ . The Sirt6/ADPr/quercetin and Sirt6/ADP-ribose/cyanidin complexes were obtained by soaking Sirt6/ADP-ribose crystals with 40 mM compound for one week, and the Sirt6/ADP-ribose/CG complex by soaking with 1 mM CG overnight. The structure in complex with isoquercetin was produced by transferring crystals in a new drop containing 1.6 M (NH₄)₂SO₄, 10% ethylene glycol, and Bis-Tris buffer pH 5.7 with 40 mM isoquercetin and incubation for one week. A solution of reservoir supplemented with 20% ethylene glycol, 10 mM compound and 2 mM ADP-ribose was used as cryoprotectant.
Sirt2/ADP-ribose crystals were grown in hanging drops at 20 °C with 14% PEG 10.000 and 0.1 M ammonium acetate pH 5.8 as reservoir solution^{40 (link)}. The protein solution contained 13 mg/ml Sirt2 (55–356) and 20 mM ADP-ribose. The Sirt2/ADP-ribose crystals were soaked with 40 mM quercetin for 3 days. Crystals were then transferred to a drop of reservoir supplemented with 20% glycol, 10 mM quercetin and 2 mM ADP-ribose before flash freezing in liquid nitrogen.
Diffraction data were collected at 100 K at BL14.1 operated by Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany)^{41 (link)}. Diffraction data were processed with the X-ray Detector Software (XDS) using XDSapp^{42 (link),43 (link)}. The Sirt6 crystals were detected to be twinned through L-tests in POINTLESS^{44 (link)}. Structures were solved by molecular replacement phasing with Phaser^{45 (link)} from the CCP4 software suite^{46 (link)}, using the twinned data and a Sirt6/ADP-ribose structure (PDB code 3K35)^{31 (link)} as a search model for the Sirt6 complexes and Sirt2/1,2,4-Oxadiazole/ADP-ribose structure (PDB code 5MAR)^{47 (link)} for the Sirt2 complex. The structures were manually rebuilt in COOT^{48 (link)} and refined with Refmac^{49 (link)}. The refinements were done with the amplitude-based twin refinement option and yielded twin fractions of 22–44% (Table 1). Structure figures were generated with PyMOL (Schrödinger, LLC; https://pymol.org/2/).

Powdered rose petal extract (RoseFit^™) from R. multiflora var. platyphylala, standardized to contain 2–3% isoquercetin and 63.82% of total polyphenol content, was used in the present study. The extract was formulated with maltodextrin in the form of a capsule, so that each 300 mg capsule contained 200 mg of RoseFit. The placebo capsules contained equivalent weight of maltodextrin and were identical to the extract capsules in colour and appearance.

4-methylumbelliferone, hesperidin, rutin, hesperidin methylchalcone, isoquercetin, resorcinol, phloroglucinol, methanol, ethanol, butanol, n-propanol, isopropanol, isoamyl alcohol, 2-phenylethanol, p-Nitrophenyl-β-d-glucopyranoside and p-Nitrophenyl-α-l-rhamnopyranoside were purchased in Sigma Chemical (Sigma, St. Louis, MO, USA). Narcissin, tulipanin and myrtillin were purchased in Extrasynthese company (Extrasynthese, Genay Cedex, France). HPLC-grade methanol (LiChrosolv^®) was obtained from Merck (Merck, Darmstadt, Germany). The 4-methylumbelliferyl-rutinoside reagent was synthesized as described in Mazzaferro et al. [29 (link)].