Ursolic acid

Leaves of S. formosum were harvested and sun-dried in September–October, 2020. Wild trees older than 5 years were selected in the Hanoi area, Vietnam. Dried C. asiatica from Indonesia was purchased online (Jung Woo-dang, Seoul, Korea). Cell culture reagent, Dulbecco Modified Eagle Medium (DMEM), was purchased from WELGENE (Daegu, Korea). Penicillin, streptomycin, and 0.05% trypsin-EDTA were purchased from Gibco (Waltham, MA, USA). Reverse transcription mix (5x master premix, Elpis-Biotech, Daejeon, Korea) and Taq PCR premix (AccuPower ProFi, Bioneer, Daejeon, Korea) were used for gene works. HPLC analysis grade asiatic acid, madecassic acid, corosolic acid, betulinic acid, oleanolic acid (4 chemicals: Sigma-Aldrich, St. Louis, MO, USA), ursolic acid (Tokyo Chemical Industry, Tokyo, Japan), and maslinic acid (Chengdu Biopurify Phytochemicals Ltd., Chengdu, China) with at least 95% purity were used.

All the chemicals used in this study were of analytical grade. Organic solvents, including 1-butanol, acetone, chloroform (also for NMR), and ethyl acetate, were purchased from KANTO CHEMICAL CO., INC., Japan. Chemicals, such as hydrochloric acid, K₂HPO₄, KH₂PO₄, L-tyrosine, silica gel, octadecyl silica gel, and tris (hydroxymethyl) aminomethane, were bought from NACALAI TESQUE, INC., Japan. Standard substances, including β-arbutin, caffeic acid, and ursolic acid, and tricine were obtained from Tokyo Chemical Industry, Japan. Enzymes, such as collagenase, elastase, and tyrosinase, including N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) and dimethyl sulfoxide (DMSO), were acquired from Sigma-Aldrich, Germany. DMSO and methanol (NMR grade) were purchased from ISOTEC^®, Sigma-Aldrich, Germany. A peptide: MOCAc-PRO-Leu-Gly-Leu-A₂pr(Dnp)-Ala-Arg-NH₂, was ordered from Peptide Institute, Inc., Japan. Instruments used included HPLC (SCL-10A sp/RID-6A/c-R3A; SHIMADZU, Kyoto, Japan), Fluorescence microplate reader (Enspire 2300 Multimode reader; PerkinElmer, Waltham, MA, USA), HR-ESI-MS (LTQ orbitrap XL; Thermo Fisher Scientific, Waltham, MA, USA), Infrared Spectrophotometer (FT-720, HORIBA), Nuclear Magnetic Resonance Spectrometer (ECA-500/600, JEOL), and UV-Vis microplate reader (Multiskan Go; Thermo Scientific, Waltham, MA, USA).

Luria broth and Luria agar were purchased from Himedia. Kanamycin and Tris were purchased MP Biomedicals, LLC (France). Ni-NTA resin was procured from Thermo Scientific (USA). BIOMOL^® Green reagent was bought from Enzo (New York, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG), quercetin, vanillin and citral were purchased from Sisco Research Laboratories. Ursolic acid, capsaicin, DL-α tocopherol acetate and limonin were bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All reagents used were of analytical grade.