Dihydrocaffeic acid (3-(3,4-dihydroxyphenyl)propanoic acid) resembles the dopamine molecule in its structure and consists of pyrocatechin and a three-carbon side chain with a carboxyl group. Its biological activity is well-known in the scientific literature, and it has antioxidant, anti-inflammatory, and cytoprotective properties. Moreover, the effect of dihydrocaffeic acid on inhibiting lipid peroxidation in human plasma, the protective effect on UV-irradiated keratinocytes, and reducing the level of lipids in the blood was confirmed. The aim of this scientific activity was to obtain dihydrocaffeic acid esters by biocatalysis and assess their antioxidant activity and other selected undesirable effects on living organisms and cell cultures in in vitro tests. Dihydrocaffeic acid is not currently used as a food additive. Its toxicity is unknown, and like many other phenolic compounds, it is poorly soluble in fats, which limits its use. One way to change the solubility of chemical compounds is their lipophilization, which can be understood as modifying the structure of the molecule by adding a hydrophobic alkyl substituent, the source of which is alcohol or carboxylic acid, in the esterification reaction. Lipophilization can be carried out using chemical and enzymatic methods.
The planned scientific activity consisted of four stages. In the first stage, the enzymatic synthesis of dihydrocaffeic acid esters was carried out. Five esters were obtained using the following primary linear alcohols: butanol, hexanol, octanol, decanol, and dodecanol. The obtained esters were purified by column chromatography, and their structure was confirmed by spectroscopic methods (1H NMR and 13C NMR). The second stage of the work involved the assessment and comparison of the antioxidant activity of the obtained compounds and their precursor (dihydrocaffeic acid) using in vitro methods with the DPPH radical (2,2-diphenyl-1-picrylhydrazyl), with the ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)), a method for determining the ability to reduce iron(III) ions, CUPRAC (a method related to the reduction of the neocuproine complex with Cu(II) ions) and CBA (a method based on the bleaching of crocin). Additionally, linoleic acid autoxidation and β-carotene bleaching tests in linoleic acid emulsion will be used to assess the antioxidant activity. Selected esters characterized by high antioxidant activity in in vitro tests and good solubility in the lipid environment were used in the third stage of the research, where the impact of the added substance on the oxidative stability of the matrix, which consisted of vegetable oils: rapeseed, sunflower and soybean, were assessed using PDSC. The oils were characterized by the fatty acid profile, acid and peroxide values, and then their oxidative stability was assessed without the addition of an antioxidant, with the addition of BHT (butylated hydroxytoluene, E321) at a concentration of 0.01% or with the addition of dihydrocaffeic acid esters. Additionally, storage tests were carried out at room and elevated temperatures, where the quality parameters of the oils, along with primary and secondary oxidation products, were assessed in selected periods of time. The last stage of the planned scientific activity involved assessing the safety of the use of the obtained compounds, i.e., considering the undesirable properties of the obtained esters, which may prevent further research on aspects of the antioxidant activity in foods with increased lipid content. Acute toxicity towards plant organisms (mono- and dicotyledonous) such as Avena sativa, Lepidium sativum, and Sinapis alba was checked, where the impact of the obtained compounds on the germination and root growth of the mentioned plants was examined. The potential cytotoxicity of dihydrocaffeic acid and selected ester derivatives obtained in this research task was assessed using the MTT test, staining with fluorescein diacetate and propidium iodide (FDA/PI), and staining with crystal violet in human HaCaT keratinocyte cultures.
(2023-07-11)
