Usnic acid

Identification and determination of usnic acid content in UBE dissolved in acetone and in DMSO was performed by UHPLC method [25 (link)]. The dry extract was analyzed with a Perkin-Elmer UHPLC instrument Flexar FX 20 with C18 column (150 mm/4.6 mm, 5 µm), a Binary LC Pump, PDA plus detector (PerkinElmer, Inc., Waltham, MA 02451, USA), thermostat compartment for the column, degassing system and auto-sampler. The mobile phase was an isocratic system/methanol/water/glacial acetic acid (80:15:5), and the detection was made at 282 nm. The samples were as follows: UBE solubilized in acetone and in DMSO 0.2%, diluted 1 to 10, 1 to 20, and 1 to 50. The reference substance was usnic acid (Sigma-Aldrich, St. Louis, MO 63103, USA) in acetone and in DMSO 0.2% at concentrations of 10, 20, 50, 100, 200 µg/mL, and it was injected in the chromatographic system at injection volume of 20 µL. The flow rate was 1.5 mL/min, the temperature value was 25 °C and the analysis time was 6 min; the calibration curves were drawn for acetone (y = 5.19752 × 10⁴x − 1.30654 × 10⁵; r² = 0.999808) and DMSO (y = 4.84629 × 10⁴x − 4.01679 × 10⁴; r² = 0.999877) [25 (link)].

Caprolactone (CL, Sigma Aldrich, ST. Louis, MO, USA) was distilled before use. Polyglyerol adipate (PGA) was synthesized and purified as previously reported [34 (link)]. Tetrahydrofuran (THF, Sigma Aldrich) was dehydrated on CaSO₄. Tin 2-ethylexanoate (Sigma Aldrich), methanol (Sigma Aldrich), hexane (Sigma Aldrich) and usnic acid (Sigma Aldrich) were used without further purification.

Sodium hyaluronate HA-T (MW about 1.30 MDa, glucuronic acid content 45%, protein content 0.05%) was obtained from Bloomage Freda Biopharm CO., LTD (Jinan, China). Dimethyl sulfoxide (DMSO, 99.5% ACS, MW = 78.13 g/mol) was purchased from JSC EKOS-1 (Russian Federation). Curcumin (MW = 368.38 g/mol) from Curcuma longa (Turmeric) and usnic acid (MW = 344.32 g/mol) from Usnea dasypoga were supplied by Sigma-Aldrich (St. Louis, MO, USA). All materials were used as received without additional purification. Deionized water was obtained from the laboratory distillation unit.
Sodium hyaluronate was used as biopolymer matrix for electrospun nanofibers. DMSO was utilized as a co-solvent to decrease the electrical conductivity of the polymer solution and to improve the electrospinning process. Curcumin and usnic acid were used as biologically active substances having a natural origin.

Acetone extract of lichen thalli at a concentration of 5 mg/ml were subjected to high performance liquid chromatography (HPLC) analyses (LC-20A; Shimadzu, Kyoto, Japan) on a YMC-Pack ODS-A (150 × 3.9 mm I.D.) reversed-phase column containing fully end-capped C18 material (particle size, 5 μm; pore size, 12 nm). Elution was performed at a flow rate of 1 ml/min under the following conditions before subsequent injection: column temperature, 40°C; solvent system, methanol: water: phosphoric acid (80: 20: 1, v/v/v). Analyses were monitored by a photodiode array detector (SPD-M20A; Shimadzu) with a range of 190~800 nm throughout the HPLC run. Observed peaks were scanned between 190 and 400 nm. The standard used for salazinic acid (t_R = 2.27 ± 0.2 min) was isolated from lichen Lobaria pulmonaria. Usnic acid used in our study was purchased from Sigma-Aldrich (St. Louis, USA) (329967-5G). Voucher specimens were deposited in the herbarium of the Lichen & Allied Bioresource Centre at the Korean Lichen Research Institute, Sunchon National University, South Korea.

THP-1 cells were seeded into 96-well plates at density of 6 × 10⁴ cells/well and the cells were differentiated with 160 nM of PMA. After differentiation, 10, 25, and 50 µM of schisandrin, schisandrin B, and schisandrin C was added to the wells. Usnic acid (50 µM, Sigma-Aldrich, St. Louis, MO, USA) was used as positive control and 0.25% DMSO as a vehicle control. Cells were incubated for 24–144 h. Culture medium with samples was replaced with fresh aliquots at 72 h in the 144 h experiment. After incubation, resazurin (Sigma-Aldrich, St. Louis, MO, USA) in PBS was added in a final concentration of 20 µM. The culture was incubated for further 2 h and fluorescence was recorded at 570/590 nm with Varioskan Lux plate reader. The experiments were performed with three biological replicates.

Ultrapure water (˂5 µg/L TOC) obtained by a water purification system (Mili-Q Merck Millipore, Chile) was used. For mass spectrometry analysis, HPLC-grade methanol and MS-grade formic acid were used, obtained from J.T. Baker (Phillipsburg, NJ, USA). Gallic acid, Folin Ciocalteu commercial reagent, 2,4,6-tris(2-pyridyl)-s-triazine, sodium carbonate, acetic acid, ferric chloride hexahydrate, sodium acetate, Trolox, hydrochloric acid, 2,2′-Azobis(2-amidinopropane) dihydrochloride, absolute ethanol, fluorescein solution, phosphate buffer, acetylcholinesterase enzyme (AChE), butyrylcholinesterase enzyme (BChE), galantamine, Tris-HCl buffer, Ellman’s reagent (DTNB), acetylcholine, butyrylcholine, magnesium chloride, sodium chloride, alpha-glucosidase, 4-nitrophenyl α-d-glucopyranoside, acarbose, orlistat, pancreatic lipase, 4-nitrophenyl-dodecanoate, dimethyl sulfoxide (DMSO) and HPLC standard with a purity greater than 95% (usnic acid and atranorin) were obtained from Sigma (Sigma, St. Louis, MO, USA).

Arctigenin (ARC), bergenin (BER), (+)-usnic acid (USN), and xanthohumol (XAN) were obtained from Sigma-Aldrich, USA, and their chemical structure is presented in Fig. 1.Fig. 1

The chemical structure of Arctigenin, bergenin, (+)-usnic acid and xanthohumol