Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro

Abstract

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids (1–3), the lignan pinoresinol (4), four hydroxylated derivatives of dehydroabietic acid (DHAA) (5 – 8), and dehydroabietic acid (9) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids–pimaric acid (10) and isopimaric acid (11)–and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

Exudates of Picea abies, Pinus nigra, and Larix decidua: Chromatographic Comparison and Pro-Migratory Effects on Keratinocytes In Vitro

“…The latter involves resin acids of the abietic- and pimaric-types sharing the same molecular weight of 302 Da and differ only in the position of one double bond and a side chain (compounds 10-1 – 10-7 , Figure 1 ). Hydroxylated resin acids ( 5 – 8 ) and dehydroabietic acid ( 9 ) are easier to separate due to their greater difference in structure and molecular weight [ 13 ]. The obtained fingerprints should facilitate comparison and differentiation of the exudates on a qualitative level.…”

“…Scientific research and literature comparing both the wound-healing properties and the phytochemical composition of the exudates of Picea abies , Pinus nigra , and Larix decidua are relatively scarce. Whereas there are studies about Norway spruce balm [ 13 , 14 , 15 ], data on European larch and black pine are still missing. Norway spruce balm consists of hydroxycinnamic acids such as ferulic acid, lignans such as pinoresinol, and hydroxylated and non-hydroxylated diterpene resin acids as main compounds [ 13 ] (see Figure 1 ).…”

“…Whereas there are studies about Norway spruce balm [ 13 , 14 , 15 ], data on European larch and black pine are still missing. Norway spruce balm consists of hydroxycinnamic acids such as ferulic acid, lignans such as pinoresinol, and hydroxylated and non-hydroxylated diterpene resin acids as main compounds [ 13 ] (see Figure 1 ). A monograph of this balm was included in the Austrian pharmacopoeia in 2016, followed by a monograph for a lard-based salve in 2019.…”

Potential Effects of Phenolic Compounds That Can Be Found in Olive Oil on Wound Healing

“…With regard to pinoresinol, the literature on its effects on wound healing is still very limited. A recent study showed that pinoresinol is able to stimulate the proliferation and migration of keratinocytes in culture, enhancing the re-epithelialisation of cell-free zones in a dose-dependent manner with better results at 10 uM concentration [ 78 ]. These results have also been observed in other cell lines, such as fibroblasts.…”

Balms of selected species of Pinaceae and their influence on keratinocyte re-epithelialization in vitro

“…Exudates of Pinaceae are used in traditional medicine to treat wounds 1 2 . The balms of Picea abies, Larix decidua and Pinus nigra were tested in a HaCaT keratinocyte based in vitro assay for their potential to boost re-epithelialization as measure for wound closure.…”