Discovery of Dihydrophaseic Acid Glucosides from the Florets of Carthamus tinctorius

Carthamus tinctorius L. (Compositae; safflower or Hong Hua) has been used in Korean traditional medicine for maintaining the homeostasis of body circulation. Phytochemical investigation was performed on the florets of C. tinctorius by liquid chromatography–mass spectrometry (LC/MS), which afforded two dihydrophaseic acid glucosides (1 and 2). Isolated compounds were structurally confirmed using a combination of spectroscopic methods including 1D and 2D nuclear magnetic resonance and high-resolution electrospray ionization mass spectroscopy. Their absolute configurations were established by quantum chemical electronic circular dichroism calculations and enzymatic hydrolysis. The anti-adipogenesis activity of the isolated compounds was evaluated using 3T3-L1 preadipocytes. Treatment with the dihydrophaseic acid glucoside (1) during adipocyte differentiation prevented the accumulation of lipid droplets and reduced the expression of adipogenic genes, Fabp4 and Adipsin. However, compound 2 did not affect adipogenesis. Our study yielded a dihydrophaseic acid glucoside derived from C. tinctorius, which has potential advantages for treating obesity.

Merck precoated silica gel F254 plates and RP-C18 F254s plates were used for thin-layer chromatography (TLC). Spots were detected after TLC under UV light or by heating after spraying with anisaldehyde-sulfuric acid. The 3D molecular modeling was performed by using ChemBioDraw Ultra and Avogadro.

Plant material
The florets of C. tinctorius were collected in Pocheon, Gyeonggi-do, Korea and purchased from Dongyangpharm in September 2018. The

Computational analysis
To acquire the optimal conformation of 1a/1b, computational DFT calculations were performed. The first structural energy minimization of 1a/1b was performed by utilizing Avogadro 1.2.0 with a UFF force field. The ground state geometries of 1a/1b were then established by Tmolex 4.3.1 with the DFT settings of B3-LYP functional/M3 grid size, geometry optimization options of energy 10 -6 hartree, gradient norm |dE/dxyz| = 10 -3 hartree/bohr, and the basis set def-SV(P) for all atoms [23][24][25][26]. The calculated ECD spectra of the optimized structures were acquired at the B3LYP/DFT functional settings with the basis set def2-TZVPP for all atoms [23][24][25][26][27]. The obtained ECD spectra were simulated by overlying each transition, where σ is the width of the band at height 1/e; and ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. In the present study, the value of σ was 0.10 eV.

Enzymatic hydrolysis and absolute configuration determination of the sugar moiety
The absolute configuration of the sugar moiety was determined using an LC/MS-UV-based method [16]. Compound 1 (0.3 mg) was hydrolyzed with crude hesperidinase (10 mg, from Aspergillus niger; Sigma-Aldrich) at 37°C for 72 h, and EtOAc was used for the extraction.

Cell culture and differentiation
3T3-L1 preadipocytes, purchased from the American Type Culture Collection (ATCC® CL-173™), were grown in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% bovine calf serum and 1% penicillin/streptomycin (P/S). For the differentiation of 3T3-L1 cells into mature adipocytes, the cells were cultured in DMEM supplemented with 10% FBS, 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μg/mL insulin (day 0). Next, the medium was replaced every other day with DMEM containing 10% FBS, 1% P/S, and 1 μg/mL insulin. To assess the effects of compounds 1 and 2 on adipogenesis, we treated 3T3-L1 cells with compounds 1 and 2 during the entire process of adipogenesis. At day 8, the cells were harvested and subjected to further experiments, including immunoblotting or reverse transcription (RT)-quantitative PCR (qPCR).

Oil Red O staining
Oil Red O staining was conducted to visualize lipid droplets accumulated in adipocytes. Mature adipocytes were fixed with 10% formaldehyde for 1 h and washed with 60% isopropanol. Next, the cells were incubated with Oil Red O working solution for 1 h, and then washed twice with distilled water. To prepare Oil Red O stock solution, 300 mg of Oil Red O powder was dissolved in 100 mL of 99% isopropanol. The Oil Red O working solution, containing three parts of Oil Red O stock solution and two parts of distilled water, was prepared just before use.

Reverse transcription and quantitative real-time PCR
To detect RNA expression, total RNA was extracted from adipocytes utilizing Easy-Blue reagent (Intron Biotechnology). cDNA was generated by subjecting 1 μg of total RNA to reverse transcription using a Maxim RT-PreMix Kit (Intron Biotechnology). Next, qPCR was