Rhusflavanone and mesuaferrone B: tyrosinase and elastase inhibitory biflavonoids extracted from the stamens of Mesua ferrea L.

Chemical isolation and bioactivity studies were conducted on the stamens of Mesua ferrea L., which are being used in a traditional skincare formulation in Myanmar. Rhusflavanone and mesuaferrone B were obtained as the main biflavonoids together with lupeol, five common flavonoids, and five phenolic compounds. After being identified by NMR and other spectroscopic analyses, these compounds were evaluated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, human leukocyte elastase inhibitory, and mushroom tyrosinase inhibitory activities. The two biflavonoids exhibited strong inhibitory activities against elastase and tyrosinase, but low DPPH-radical scavenging activities. The contents of rhusflavanone and mesuaferrone B in the stamens were 0.35 ± 0.04% and 0.55 ± 0.06%, respectively. Moreover, lupeol was considered to be a cosmetically important component of the stamens because of its high content and strong elastase inhibitory activity. Rhusflavanone was reported to be isolated from M. ferrea for the first time.


Extraction and isolation
Powdered M. ferrea stamens (2.4 kg) were extracted twice with 18 L of 70% methanol at room temperature by maceration for a week, with occasional stirring. The extracts were combined and concentrated in vacuo at 40 °C, resulting in 340 g of a dry extract.
Compound 3 was obtained from the 100% methanol fraction and the dichloromethane fraction through further purification using silica gel columns. Compounds 4−8 and 9−13 were obtained from the water fraction and the combined fraction of 40% and 60% methanol fractions, respectively, through further purification with Sephadex LH-20 (eluent: methanol-water), followed by purification using silica gel (eluent: dichloromethane-methanol-water) or ODS columns (eluent: methanol-water).

Preparation of sample stock solutions
Dry extracts and the isolated compounds were dissolved in 100% DMSO at 10 g/L concentration, stored at −20 °C in the dark, and used as the sample stock solutions.
They were dissolved well at room temperature by sonication prior to use.

Determination of DPPH-radical scavenging activity
DPPH-radical scavenging activity was evaluated according to a previously reported method (Sharma and Bhat 2009), with minor modifications. Ethanol (50%) was used as the solvent for DPPH. Sample stock solutions were also diluted with 50% ethanol to obtain the final concentrations of 3, 10, 30, 100, and 300 mg/L in the assay mixtures.
Each diluted sample (100 μL) was mixed with 50 μL of 0.2 mol/L MES buffer (pH 6.0) and 50 μL of 0.8 mmol/L DPPH solution in a well of a 96-well microplate. After incubation at 25 °C for 20 min in the dark, the absorbance at 520 nm (As) was measured with a TECAN infinite F-200 PRO plate reader. A blank solution (50% ethanol) was used to obtain the control absorbance (Ac). DPPH-radical scavenging activity was calculated using the following equation:

Determination of elastase inhibitory activity
Elastase inhibitory activity was evaluated according to a previously reported method (Castrillo et al. 1979), with minor modifications. A buffer containing 0.1 mol/L HEPES and 0.5 mol/L NaCl (pH 7.5) was used as the solvent for the enzyme and the substrate To compensate the effect by DMSO carried over from the sample stock solutions, the concentration of DMSO was adjusted to 1% in all the reaction mixtures.

Determination of tyrosinase inhibitory activity
Tyrosinase inhibitory activity was evaluated according to a previously reported method ), with minor modifications. A phosphate buffer (50 mmol/L, pH 6.8) was used as the solvent for L-tyrosine and mushroom tyrosinase. Sample stock solutions were also diluted with the same buffer to obtain the final concentrations of 1, 3, 10, 30, and 100 mg/L in the assay mixtures. Each diluted sample (10 μL) was mixed with 120 μL of the phosphate buffer and 50 μL of 100 units/mL tyrosinase solution in a well of a 96-well microplate. After pre-incubation at 25 °C for 10 min, 2 mmol/L L-tyrosine (20 μL) was added to the mixture. The absorbance at 476 nm was recorded at 25 °C for 20 min at 2-min intervals. Tyrosinase inhibitory activity was calculated using the following equation: where ∆S and ∆C are the absorbance changes in the experiments with and without the test sample, respectively. The absorbance changes between 0 and 12 min were used for the calculations. To compensate the effect by DMSO carried over from the sample stock solutions, the concentration of DMSO was adjusted to 1% in all the reaction mixtures.

Calculation of EC50 and IC50
EC50 for DPPH radical scavenging and IC50 for elastase and tyrosinase inhibitions were calculated with the following equation using two adjacent data sets across the 50% scavenging or inhibition point: where EH and CH are the scavenging (%) and concentration (mg/L) data for the higher scavenging point, respectively, and EL and CL are those for the lower scavenging point. where IH and CH are the inhibition (%) and concentration (mg/L) data for the higher scavenging point, respectively, and IL and CL are those for the lower scavenging point.