Effect of Hybrid Type and Harvesting Season on Phytochemistry and Antibacterial Activity of Extracted Metabolites from Salix Bark

Abstract

Hundreds of different fast-growing Salix hybrids have been developed mainly for energy crops. In this paper, we studied water extracts from the bark of 15 willow hybrids and species as potential antimicrobial additives. Treatment of ground bark in water under mild conditions extracted 12-25% of the dry material. Preparative high-performance liquid chromatography is proven here as a fast and highly efficient tool in the small-scale recovery of raffinose from Salix bark crude extracts for structural elucidation. Less than half of the dissolved material was assigned by chromatographic (gas chromatography and liquid chromatography) and spectroscopic (mass spectrometry and nuclear magnetic resonance spectroscopy) techniques for low-molecular-weight compounds, including mono- and oligosaccharides (sucrose, raffinose, and stachyose) and aromatic phytochemicals (triandrin, catechin, salicin, and picein). The composition of the extracts varied greatly depending on the hybrid or species and the harvesting season. This information generated new scientific knowledge on the variation in the content and composition of the extracts between Salix hybrids and harvesting season depending on the desired molecule. The extracts showed high antibacterial activity on Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.6-0.8 mg/mL; however, no inhibition was observed against Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. MIC of triandrin (i.e., 1.25 mg/mL) is reported for the first time. Although antibacterial triandrin and (+)-catechin were present in extracts, clear correlation between the antibacterial effect and the chemical composition was not established, which indicates that antibacterial activity of the extracts mainly originates from some not yet elucidated substances. Aquatic toxicity and mutagenicity assessments showed the safe usage of Salix water extracts as possible antibacterial additives.

Keywords: antimicrobial activity; mutagenicity; raffinose; toxicity; triandrin; water extract; willow bark.

Figure 1

Gas chromatograms of the trimethylsilyl derivatives of the (a) bark extract of willow hybrid Tora (harvested on Dec 4) with the internal standard (Xylitol, Std) and (b) “raffinose-rich” fraction of the extract (Figure S5).

Figure 2

ESI HRMS spectrum (rt: 0.877 min) of the isolated “raffinose-rich” fraction (Figure S5).

Figure 3

Carbohydrate region of 2D HSQC NMR spectra of the (a) bark extract of hybrid Tora (harvested on Dec 4) (Table S1); (b) “raffinose-rich” fraction of the extract (Figure S5); and (c) authentic raffinose in DMSO-d₆/pyridine-d5 (4:1).

Figure 4

Chemical composition (mean = average of the content based on triplicate measurements) of water extracts quantified by GC-FID from selected Salix hybrids: (a) Tordis; (b) Schwerenee; (c) winter; and (d) Tora that were harvested in different seasons. Standard deviations (Table S7) are shown as error bars of the mean. The gravimetric extraction yields are summarized in Figure S13.

Figure 5

Chemical composition (mean = average of the content based on triplicate measurements) of water extracts from 15 Salix hybrids quantified by GC-FID. Sample abbreviations include hybrid code, harvesting month, harvesting day, and hybrid name, for example, D1_12_04_Klara refers to hybrid Klara harvested on December 4th. Standard deviations (Table S8) are shown as error bars (in red color) of the mean.