Bio-Guided Isolation of Methanol-Soluble Metabolites of Common Spruce (Picea abies) Bark by-Products and Investigation of Their Dermo-Cosmetic Properties

Abstract

Common spruce (Picea abies L.) is a fast-growing coniferous tree, widely used in several countries for the production of sawn wood, timber and pulp. During this industrial exploitation, large quantities of barks are generated as waste materials. The aim of this study was the bio-guided investigation and the effective recovery of methanol-soluble metabolites of common spruce bark for the development of new dermo-cosmetic agents. The active methanol extract was initially fractionated by Centrifugal Partition Chromatography (CPC) using a triphasic solvent system in a step-gradient elution mode. All resulting fractions were evaluated for their antibacterial activity, antioxidant activity and their capability to inhibit tyrosinase, elastase and collagenase activity. In parallel, the chemical composition of each fraction was established by combining a ¹³C-NMR dereplication approach and 2D-NMR analyses. As a result, fourteen secondary metabolites corresponding to stilbene, flavonoid and phenolic acid derivatives were directly identified in the CPC fractions. A high amount (0.93 g) of E-astringin was recovered from 3 g of crude extract in a single 125 min run. E-Astringin significantly induced the tyrosinase activity while E-piceid, taxifolin, and taxifolin-3'-O-glucopyranoside exhibited significant anti-tyrosinase activity. The above compounds showed important anti-collagenase and antimicrobial activities, thus providing new perspectives for potential applications as cosmetic ingredients.

Keywords: 13C-NMR dereplication; Centrifugal Partition Chromatography; E-astringin; dermo-cosmetic agents; taxifolin; tyrosinase, elastase and collagenase activity.

Figure 1

Step-gradient CPC chromatogram of 3 g crude methanol extract of P. abies bark at 254 nm; Stationary phase: lower phase of the triphasic system; Mobile phase; upper and middle phases for the elution step and lower phase for the extrusion step, all in ascending mode.

Figure 2

(A) ¹³C-NMR chemical shift clusters (yellow color) obtained by applying HCA on CPC fractions of P. abies bark extract and the chemical structures of nine major compounds identified by database analysis of the marked clusters (clusters A to K). (B) Chemical structures of five minor compounds identified on CPC fractions by 2D-NMR analysis.

Figure 3

Tyrosinase inhibition activity of methanol bark extract, CPC fractions and pure compounds. (A) % inhibition activity of crude extract at 300, 100 and 60 μg/mL , (B) % inhibition activity of CPC fractions at 100 μg/mL and (C) % inhibition activity of pure taxifolin, E-piceid, E-astringin and taxifolin-3′-O-glucoside in six concentrations (0.3, 3,10, 30, 100 and 300 μM). Positive control: Kojic Acid (KA) at 2.0 μg/mL.

Figure 4

Elastase inhibitory activity of methanol bark extract, CPC fractions and pure compounds. (A) % inhibition activity of crude extract at 300, 100 and 50 μg/mL, (B) % inhibition activity of CPC fractions at 50 μg/mL and (C) % inhibition activity of pure taxifolin, catechin and taxifolin-3′-O-glucoside in six concentrations (0.3, 3,10, 30, 100 and 300 μM). Positive control: Elastatinal (ELAST) at 0.5 μg/mL.

Figure 5

Collagenase inhibitory activity of methanol bark extract, CPC fractions and pure compounds. (A) % inhibition activity of crude extract at 150, 75 and 40 μg/mL, (B) % inhibition activity of CPC fractions at 40 μg/mL and (C) % inhibition activity and IC50 value of pure taxifolin, catechin, E-piceid, E-astringin and taxifolin-3′-O-glucoside . Positive control: Phosphoramidon (PRMD) at 3.75 μg/mL.