salicortin and salicyl-alcohol

Salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Although their biosynthetic pathway is not known, recent work has suggested that benzyl benzoate may be a potential biosynthetic intermediate. Two candidate genes, named PtACT47 and PtACT49, encoding BAHD-type acyl transferases were identified and are predicted to produce such benzylated secondary metabolites. Herein described are the cDNA cloning, heterologous expression and in vitro functional characterization of these two BAHD acyltransferases. Recombinant PtACT47 exhibited low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM(-1) s(-1)) and lowest Km values (45.1 μM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA: salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, including benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM(-1) s(-1)) and lowest Km (55.3 μM) were observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA: benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters, such as hexenyl benzoate, at trace levels. Although the activities demonstrated are consistent with roles in salicinoid biosynthesis, direct tests of this hypothesis using transgenic poplar must still be performed.

Topics: Acyl Coenzyme A; Acyltransferases; Benzoates; Benzyl Alcohols; DNA, Complementary; Escherichia coli; Glucosides; Glycosides; Molecular Structure; Phenols; Plant Leaves; Populus

The phenolic glucoside salicortin was isolated from a Willow bark extract, and its ability to reduce the TNF- α induced ICAM-1 expression (10 ng/mL, 30 min pretreatment with salicortin) was tested IN VITRO on human microvascular endothelial cells (HMEC-1). After 24 h, 25 µM salicortin decreased the TNF- α induced ICAM-1 expression to 65.9 % compared to cells which were treated only with TNF- α. In parallel, the stability of 25 µM salicortin under assay conditions was determined by HPLC. Within 24 h, the salicortin concentration decreased to 3.1 µM whereas catechol, a known NF- κB inhibitor, rose as a metabolite. After 8 h the catechol concentration was relatively constant and varied between 8.2 and 10.9 µM. Considering this degradation in the IN VITRO test system, 10 µM catechol was added 8 h after TNF- α stimulation, and 16 h later the ICAM-1 expression was determined. In this setting, the ICAM-1 expression was reduced to 74.8 %. This is comparable to the effect obtained from 25 µM salicortin and indicates that its activity is related to the generation of catechol, as salicin, saligenin, and salicylic acid are only marginally active or inactive in this test system in a concentration up to 50 µM. These results indicate catechol as an important bioactive metabolite from salicortin.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Benzyl Alcohols; Catechols; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Stability; Endothelium, Vascular; Glucosides; Humans; Intercellular Adhesion Molecule-1; Plant Bark; Plant Extracts; Salicylic Acid; Salix; Tumor Necrosis Factor-alpha