Evaluation of Antioxidative Mechanisms In Vitro and Triterpenes Composition of Extracts from Silver Birch (Betula pendula Roth) and Black Birch (Betula obscura Kotula) Barks by FT-IR and HPLC-PDA

Abstract

Silver birch, Betula pendula Roth, is one of the most common trees in Europe. Due to its content of many biologically active substances, it has long been used in medicine and cosmetics, unlike the rare black birch, Betula obscura Kotula. The aim of the study was therefore to compare the antioxidant properties of extracts from the inner and outer bark layers of both birch trees towards the L929 line treated with acetaldehyde. Based on the lactate dehydrogenase test and the MTT test, 10 and 25% concentrations of extracts were selected for the antioxidant evaluation. All extracts at tested concentrations reduced the production of hydrogen peroxide, superoxide anion radical, and 25% extract decreased malonic aldehyde formation in acetaldehyde-treated cells. The chemical composition of bark extracts was accessed by IR and HPLC-PDA methods and surprisingly, revealed a high content of betulin and lupeol in the inner bark extract of B. obscura. Furthermore, IR analysis revealed differences in the chemical composition of the outer bark between black and silver birch extracts, indicating that black birch may be a valuable source of numerous biologically active substances. Further experiments are required to evaluate their potential against neuroinflammation, cancer, viral infections, as well as their usefulness in cosmetology.

Keywords: Betula obscura Kotula; Betula pendula Roth; black birch; pentacyclic triterpenes; silver birch.

Figure 1

Chemical structure of betulin (a) and betulinic acid (b).

Figure 2

Betula obscura Kotula (left) and Betula pendula Roth (right) outer barks.

Figure 3

Example chromatograms of birch bark extract (red line) and reference compound—lupeol (green line). Over the chromatograms the spectrum of identified and reference compound. RP18e Chromolith, acetonitrile–water (95:5 v/v), flow rate 2 mL/min, at 25 °C.

Figure 4

(A) Example chromatograms of birch bark extract (red line) and reference compounds (green line): 1 (betulinic acid), 2 (oleanolic acid), 3 (ursolic acid), 4 (betulin). Over the chromatograms the spectra of identified and reference compounds. (B,C); The spectro-chromatograms of extract and reference compounds, respectively. RP18e LiChro-spher, acetonitrile–water—phosphoric acid aqueous solution at concentration of 1% (75:25:0.5, v/v/v), flow rate 1.0 mL/min at 10 °C.

Figure 5

Evaluation of hydrogen peroxide (a) and superoxide anion radical (b) release from 24 h and 48 h incubation of L929 cell line with 10% and 25% extracts. Data presented as the means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.0001, vs. vehicle group; ^{^^} p < 0.01, ^{^^^} p < 0.001, ^{^^^^} p < 0.0001 vs. AA group; Tukey’s test. Abbreviations: i.b., inner bark; o.b., outer bark; AA, acetaldehyde (positive control).

Figure 6

Assessment of malonic dialdehyde (MDA) release from 24 h and 48 h incubation of L929 cell line with 10% and 25% extracts. Data presented as the means ± SD; *** p < 0.001, **** p < 0.0001 vs. vehicle group; ^{^} p < 0.05, ^{^^} p < 0.01, ^{^^^} p < 0.001 vs. AA group; Tukey’s test. Abbreviations: i.b., inner bark; o.b., outer bark; AA, acetaldehyde (positive control).