a new antioxidant and tyrosinase inhibitor, and further active components from Trichosanthes truncata

One new amino acid derivative, (–)-β-homoarginine anhydride 1 , as well as nine known compounds were isolated from Trichosanthes truncata . The structures of the isolates were elucidated by spectroscopic methods. Among them, compounds 5 and 11 could notably dose-dependently inhibit ROS productions in HaCaT keratinocyte cells without cytotoxicity in the concentration range of 0.2 to 20 μM. In cell-free mushroom tyrosinase assay, compounds 1 – 5 , 10 and 11 had more potential anti-tyrosinase activities with IC 50 values of 106.9–255.6 μM than arbutin that were similar to predicted values of binding affinity calculated by molecule docking. The most active 2 had hydrogen bonds (Ser77, Glu309, Phe454) and electrostatic charges (Glu309, Glu248) interactions with mushroom tyrosinase, respectively. Our data manifested that T. truncata and its components are potentially to be developed as anti-aging and whitening agents for skin disorders.


Extraction and isolation
The roots of T. truncata (40.0 g) were powdered and extracted three times with MeOH, EtOH, and water (200 mL × 3) at room temperature to obtain MeOH (MTR, 1.3 g), EtOH (ETR, 0.5 g), and water (WTR, 4.0 g) extracts, respectively. Additionally, the material (40.0 g) was also extracted once with hot water to give BTR extract (4.1 g). Among above-mentioned four crude extracts, MTR showed potential bioactive properties and was selected as a candidate to clarify its active components.
The reaction mixture was concentrated in vacuo, then partitioned with CH 2 Cl 2 -H 2 O to get steroid 11 (25.4 mg) identified by NMR spectroscopy. Scavenging rate (%) = (1-A sample /A 0 ) ×100; A sample is the absorbance of the sample solution at steady state and A 0 is the absorbance of DPPH solution without sample addition.

Cytotoxicity
The cytotoxicities of HaCaT cells for test compounds were analyzed with MTT as described previously protocol with slight modifications (Cholia et al. 2017). Cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM; GIBCO Invitrogen corporation, NY, USA). The 100 μL of MTT reagent (Thermo Scientific) (0.5 mg/mL) was added to each well and then the plates were incubated for 1 h in the dark at 37 °C. The formazan product formed was dissolved in acidified DMSO and cell viabilities were measured by using a micro plate reader (SPECTROstar ® Nano, BMG LABTECH) at 570 nm.

Molecular docking
To calculate the binding affinity between compounds and mushroom tyrosinase, the molecular docking was performed by PyRx 0.8 (Dallakyan and Olson 2015)

Compound
Binding affinity