Synergistic Inhibiting Effect of Phytochemicals in Rheum palmatum on Tyrosinase Based on Metabolomics and Isobologram Analyses

Abstract

Tyrosinase (TYR) plays a key role in the enzymatic reaction that is responsible for a range of unwanted discoloration effects, such as food browning and skin hyperpigmentation. TYR inhibitors could, therefore, be candidates for skin care products that aim to repair pigmentation problems. In this study, we used a metabolomics approach combined with the isobologram analysis to identify anti-TYR compounds within natural resources, and evaluate their possible synergism with each other. Rheum palmatum was determined to be a model plant for observing the effect, of which seven extracts with diverse phytochemicals were prepared by way of pressurized solvent extraction. Each Rheum palmatum extract (RPE) was profiled using nuclear magnetic resonance spectroscopy and its activity of tyrosinase inhibition was evaluated. According to the orthogonal partial least square analysis used to correlate phytochemicals in RPE with the corresponding activity, the goodness of fit of the model (R₂ = 0.838) and its predictive ability (Q₂ = 0.711) were high. Gallic acid and catechin were identified as the active compounds most relevant to the anti-TYR effect of RPE. Subsequently, the activity of gallic acid and catechin were evaluated individually, and when combined in various ratios by using isobologram analysis. The results showed that gallic acid and catechin in the molar ratios of 9:5 and 9:1 exhibited a synergistic inhibition on TYR, with a combination index lower than 0.77, suggesting that certain combinations of these compounds may prove effective for use in cosmetic, pharmaceutical, and food industries.

Keywords: Rheum palmatum; catechin; gallic acid; isobologram analysis; metabolomics; orthogonal partial least square-modelling; synergistic effect; tyrosinase inhibitory activity.

Figure 1

TYR-inhibition of different RPEs. RPE1 to RPE7 were extracts prepared by the mixture of n-hexane–acetone–water of different proportions. Inhibition of the positive control, KA at 0.94 μM, was 95.1 ± 0.4%. Mean value ± standard deviation was expressed (n = 3). RPE, Rheum palmatum extract; TYR, tyrosinase. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

OPLS score plot (A) and loading plot (B) of RPEs. The scores are colored according to the TYR inhibition activity. (A): The label codes consist of RPE samples prepared by different extraction solvents, as described in Materials and Methods. (B): Loading plot presents the NMR signals (green dots) which contributed for the bioactivity (blue square). Green dots in the red circle refer to the characteristic proton NMR signals at δ 6.90 (s), δ 5.87 (d, J = 2.4 Hz), and δ 5.67 (d, J = 2.4 Hz). OPLS, orthogonal partial least squares; RPE, Rheum palmatum extract; TYR, tyrosinase.

Figure 3

The chemical structures of (A) GA and (B) CH. GA, gallic acid; CH, catechin hydrate.

Figure 4

Concentration–response curves of (A) GA and (B) CH on TYR inhibition (n = 3). GA, gallic acid; CH, catechin hydrate; TYR, tyrosinase.

Figure 5

IC values of (A) GA and (B) CH in combined mixtures of different molecular ratios, and (C) CI value of GA:CH combinations at the inhibition level of 20% (IC₂₀). GA, gallic acid; CH, catechin hydrate; IC, inhibition concentration; CI: combination index.