Osthol

We studied 4 male Wistar rats groups, weighing 220–240 g, that were kept in individual acrylic cages with paper nesting and bedding material (GreenSoft, RGS, Mexico City, Mexico) and a wood chip and a black acrylic holder as enrichment materials and were randomly assigned to one of the following groups (n = 6/group): (1) control (C), healthy animals receiving a normal diet and tap water; (2) HF/HS diet [31 (link)]^, and a sweetened beverage 11% (3.8% glucose, 7.2% fructose) (HF/HS); (3) HF/HS diet and 30 mg/kg of osthol (HF/HS + OT30); and (4) HF/HS diet and 40 mg/kg of osthol (HF/HS + OT40). The content of the HF/HS diet is specified in Table 1, and the fructose percentage used in the sweetened beverage has been previously reported and is similar to some soft-drink brands [32 (link),33 (link)]. All groups were followed for 30 days, and at the end of follow-up, fasting animals were anesthetized with a low-flow vaporizer of isoflurane (Somno Suite, Kent Scientific Corpo-ration, Torrington, CT, USA) and euthanized by abdominal aorta exsanguination. Kidneys were collected and washed in cold phosphate-buffer saline (PBS) (pH 7.4) and frozen at −80 °C for further analysis.

Protein carbonylation and lipid peroxidation were evaluated using 100 mg of renal cortex by the colorimetric methods previously described [43 (link)]
Protein carbonylation: Renal cortex samples were homogenized in 20 mM PBS, incubated with 2,4-dinitrophenylhydrazine (DNPH), washed with ethanol-ethyl acetate (1:1), and resuspended in guanidine-HCL The presence of carbonyl groups in the proteins was measured using the reaction with DNPH. The protein carbonyl groups were estimated by using the molar absorption coefficient of 22,000 Molar⁻¹cm⁻¹ for DNPH derivatives, and their concentrations were expressed as nmol carbonyl groups/mg protein. Guanidine solution was used as a blank.
Lipid peroxidation: 4-Hydroxynonenal (4-HNE) was measured using a standard curve of tetramethoxypropane. A solution of 1-methyl-2-phenylindole in acetonitrile:methanol (3:1) was added to renal cortex homogenates, and the reaction was started with 37% HCl or methanesulfonic acid plus FeCl₃ to measure 4-HNE. The optical density was measured at 586 nm after 1 h of incubation at 45 °C using a Synergy HT multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Data were expressed as nmol of 4-HNE per milligram of protein (nmol HNE/mg protein).

High-performance liquid chromatography-photodiode array mass spectrometry (HPLC-PDA-MS) analysis of methanol and water extracts of fresh and steam-exploded Star Ruby, Rio Red, and Ruby Red GP and WG were completed. Water extracts were prepared by taking a 1:3 ratio of deionized water to fresh and steam-exploded Star Ruby, Rio Red, and Ruby Red GP and WG and homogenizing and or blending (Omni International homogenizer, Model GLH-01, Omni International, Marietta, GA, USA or Waring Commercial Blender, Model# 7011S, Waring Commercial, Tarrington, CT, USA). The samples were microcentrifuged, and the supernatants were used for analysis. Methanol extracts were prepared by taking 1.5 g of fresh Star Ruby, Rio Red, and Ruby Red GP or WG and homogenizing (Omni International homogenizer, Model GLH-01, Omni International, Marietta, GA, USA) in approximately 30 ml of methanol. The sample was then vacuum filtered. This was repeated two times more, and the filtered extracts were pooled and brought to 100-ml volume with methanol. A 15 ml portion was dried in a concentrator (SpeedVacConcentrator, SVC 200H, Thermo Scientific, Waltham, MA, USA) and brought to 4 ml with dimethylsulfoxide and was used for analysis. HPLC-PDA-MS analysis of methanol and water extracts of fresh and steam-exploded Star Ruby, Rio Red, and Ruby Red GP and WG was completed as described previously (18 (link)). The flavanones naringin-4′-O-glucoside, hesperidin glucoside, narirutin, naringin, naringin-6”-malonate, isosakuranetinrutinoside, and poncirin were quantified and qualified by UV-Vis at a wavelength of 285 nm. The coumarins dihydroxy-osthol and marmin were quantified and qualified by UV-Vis at a wavelength of 320 nm. Averages of triplicate flavonoid analyses and their SDs were reported. The values used to calculate the averages and SDs can be found in Supplementary Tables 9–13 of the Supporting Information section. A sample chromatogram can be seen in Supplementary Figure 7.

C. monnieri fructus was provided by CNC Korea (Seoul, Korea), and its voucher specimen (KHU-BMRI-201804) was deposited at the Bio-Medical Research Institute, Kyung Hee University, Yongin, Korea. The dried C. monnieri fructus (10 g) was extracted in 30% EtOH (200 mL × 2) at room temperature for 2 h, filtered, and evaporated under reduced pressure (CMFE). The CMFE used for in vitro experiments was diluted to a concentration of 10 mg/mL and stored in a −80 °C freezer. An ODS semi-prep LC system (Waters 600S with a Waters 2487 UV detector, Milford, MA, USA) with a semi-preparative C18 column (Supelcosil LC-18 Semi-prep, 5 μm, 250 × 10 mm, Supelco, Bellefonte, PA, USA) was used for the isolation of osthol. The obtained CMFE (300 mg) was subjected to ODS semi-preparative c.c. with the isocratic mobile phase consisting of MeOH:water (3:1) for 30 min at a flow rate of 2 mL/min under a 254 nm UV wavelength (Figure S1). A total of 35.6 mg of osthol was separated at 23.6 min and its NMR spectra (¹H- and ¹³C) were recorded on a Bruker Avance 600 (Billerica, MA, USA) (Figure S2).
Osthol: ¹H-NMR (600 MHz, CDCl₃, δ_H) 6.25 (d, 9.0, H-3), 7.63 (d, 9.0, H-4), 7.31 (d, 8.4, H-5), 6.85 (d, 8.4, H-6), 3.55 (d, 6.6, H-1′), 5.25 (d, 6.6, H-2′), 1.86 (s, H-4′), 1.69 (s, H-5′), 3.94 (s, H-7-OCH₃); ¹³C-NMR (150 MHz, CDCl₃, δ_C) 161.4 (C-2), 112.8 (C-3), 143.7 (C-4), 113.0 (C-4a), 126.2 (C-5), 107.3 (C-6), 160.2 (C-7), 118.0 (C-8), 152.8 (C-8a), 21.9 (C-1′), 121.1 (C-2′), 132.7 (C-3′), 25.8 (C-4′), 17.9 (C-5′), 56.1 (C-7-OCH₃).

Analytical-grade acetonitrile, methanol, and water were procured from J.T. Baker Inc. (Phillipsburg, NJ, USA), while trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Forty-six marker compounds, including chlorogenic acid (1), caffeic acid (2), prim-O-glucosyl-cimifugin (3), cimifugin (4), nodakenin (5), umbelliferone (6), ferulic acid (7), benzoic acid (8), senkyunolide I (9), xanthotoxol (10), senkyunolide H (11), marmesin (12), sec-O-glucosyl-hamaudol (13), oxypeucedanin hydrate (14), decursinol (15), bergaptol (16), byakangelicin (17), psoralen (18), angelol B (19), angelol H (20), angelicin (21), xanthotoxin (22), angelol A (23), angelol G (24), bergapten (25), ostenol (26), bisabolangelone (27), byakangelicol (28), oxypeucedanin (29), columbianetin acetate (30), coniferyl ferulate (31), senkyunolide A (32), 3-n-butyl-phthalide (33), imperatorin (34), ligustilide (35), phellopterin (36), osthol (37), decursin (38), decursinol angelate (39), isoimperatorin (40), suberosin (41), columbianadin (42), falcarindiol (43), praeruptorin B (44), levistilide A (45), and praeruptorin C (46), were acquired from ChemFace (Wuhan, China). The chemical structures of these marker compounds are depicted in Figure S5.

Referring to the fungicides with high efficacy and low toxicity used for the control of cobweb disease on other edible mushroom, we selected various fungicides, including Carvacrol (5% SL), Osthol (1% EW), Eugenol (0.3% SL), Propiconazole (25% EC), Triadimefon (20% EC), Trifloxystrobin and tebuconazole (75% WDG), Prochloraz-manganese chloride complex (50% WP), Pyraclostrobin (10% WDG) and Difenoconazole (10% WDG). Preliminary indoor screening of fungicides for prevention and control of cobweb disease agent on L. decastes: the test method was modified appropriately [51 ]. According to the active ingredients, nine kinds of low toxic fungicides were diluted with sterile water to make stock solutions of certain concentrations. In order to determine the concentration range of each fungicide, a pre-test was carried out with a concentration gradient of 5 times for each fungicide. According to the volume ratio, the PDA medium containing fungicide was prepared with the amount of storage fluid: PDA = 1:9 in a Petri dish with diameter of 9.0 cm. The pathogen filaments (2021062102-1) which were cultured and grown on PDA medium at 25 °C in dark for 4 days were made into cake with a 5 mm hole punch. PDA medium with equal amount of sterile water without fungicide was used as control. The fungus cakes were transferred into the prepared medium, and incubated at 24 °C in dark for 4 days. In this process, the growth of pathogen was observed to determine the initial concentration of each fungicide. Selecting the fungicide that could inhibit the pathogen and conduct further concentration screening test. According to the pretest results, each fungicide was diluted into 6 concentration gradients according to the effective components. The method of inoculation and culture for each treatment was the same as above. The diameter of colonies was measured with crossing method [18 ], when colonies in control almost covered the Petri dish. Inhibitory percentage on mycelia growth was calculated after treatment with different concentrations and fungicides. Inhibition of mycelial growth (%) = [(dimeter of mycelium in control -diameter of mycelium in treatment)/dimeter of mycelium in control]x100. Each treatment was repeated three times. The EC₅₀ value of each fungicide was evaluated by using ANOVA in three replicates. The ANOVA was performed as per Duncan’s multiple range test to determine the significant differences (* p < 0.05) [52 (link)].