The effect of terpenoid esters on membrane structure investigated by fluorescence and Fourier-transform infrared spectroscopy

The influence of esters based on gamma-aminobutyric acid (GABA) and mono-/bicyclic terpenoids on membrane structure was investigated. The mechanism of action for terpenoid esters on phospholipids of artificial membranes and lipids isolated from the rat stratum corneum was studied by fluorescence and FT-IR spectroscopy. We report here, that inclusion of monocyclic terpenoid esters in phospholipid liposomes leads to growth of excimer to monomer ratio (IE/IM) indicating a decrease of membrane microviscosity. Another mechanism of influence on biomembranes was proposed for ester of bicyclic borneol ‒ in this case a high ratio of vibronic peak intensities (I1/I3) was revealed. The addition of terpenoid esters appears in the FT-IR spectra as intensity reduction of absorption bands associated with C=O, P=O and Р‒О‒С groups of lecithin phospholipids. Similar results were obtained after esters addition to lipids isolated from stratum corneum indicating a decrease of hydrogen bonds number between polar groups of lipids.


Introduction 4
Since the discovery and detailed structure determination of transient receptor potential (TRP) channels, a significant amount of naturally occurring substances were identified as modulators of these molecular targets.Among them, terpenes and their derivatives attract great attention when applied topically due to binding to TRP channels in nerve endings or non-neuron skin cells.Despite the presence of own pharmacological activity, terpenes are widely used as penetration enhancers in transdermal delivery.
Additionally to TRP channels GABA B receptors were also found to localize in the periphery; intriguing in this case is GABA presence at the terminal endings of corneal nociceptors.Given the above, combination of terpenoid and GABA residues in one molecule is expedient for development of novel transdermal therapeutic system.Recently, the esters based on mono-/bicyclic terpenoids and GABA were synthesized and found to possess analgesic and anti-inflammatory effect after their transdermal delivery.
Despite the high efficiency of the aforementioned esters via topical application, their mechanism of interaction with membrane lipids has not been studied and described.Thus, the present paper is devoted to understanding the influence of terpenoid esters on phospholipids of artificial membranes and lipids isolated from the stratum corneum (SC).For this purpose instrumental methods such as fluorescence and Fourier transform infrared spectroscopy (FT-IR) have been used.
Esters based on the corresponding terpenoids (1−6) were synthesized using DCC/DMAP coupling method followed by deprotection of the amino groups in the HCl/CH 3 COOH medium.
Synthetic pathway of compounds 1-6.The samples for FT-IR study have been prepared by dissolving the obtained lipids in carbon tetrachloride (CCl 4 ) with subsequent addition of terpenoid esters (10% relative to lipids' mass).
FT-IR spectra were recorded for films obtained using a method of slow evaporation of solvent directly from undercover under a nitrogen atmosphere.Thus, the influence of terpenoid esters on molecular organization of the lipid matrix was confirmed by method of fluorescence probe and FT-IR spectroscopy.These data substantiate the feasibility of esters' use after their transdermal delivery in vivo.In the present study analgesic and anti-inflammatory activity of terpenoid esters has been shown after transdermal delivery.

Experimental methods of pain induction
Thermal methods of induction Chemical methods of induction "Hot plate" test The mice were placed on a hot plate maintained at 55°C one at a time.In this experiment, latency to respond to the heat stimulus was determined by the amount of time (in seconds) it takes for mouse to lick one of its paws.Cut-off time was fixed at 60 sec to minimize the tissue damage that occurs during prolonged contact with heated surface.In this study, the interaction of terpenoid esters with artificial membranes and lipids isolated from rat SC was investigated with fluorescence and FT-IR spectroscopy.
According to the obtained results, the incorporation of monocyclic terpenoid esters into membranes increased the fluidity of lecithin phospholipids.Interestingly, bicyclic terpenoid borneol and its ester when inserted into liposomes do not affect I E /I M fluorescence ratio; in turn, these compounds were shown to increase the membrane polarity.The disruption of hydrogen-bonded network formed by polar lipid groups was suggested as mechanism of terpenoid esters action confirmed by FT-IR analysis.
Thus, the influence of terpenoid esters on molecular organization of the lipid matrix substantiates the feasibility of their use after transdermal delivery in vivo.

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Capsaicin-induced licking 20 μl (6 μg/paw) of solution  Formalin-induced licking 20 μl of 2% solution  AITC-induced licking 20 μl of 0,5% solution The animal then was placed in an individual plexiglass cage.The time spent licking the injected paw was measured from 0 to 5 min after formalin/capsaicin/AITC administration and was considered as an indicator of pain response.

Analgesic properties of terpenoid esters investigated by «hot plate» test
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