The effect of terpenoid esters on membrane structure investigated by fluorescence and Fourier-transform infrared spectroscopy Mariia Nesterkina 1,2, *, Sergii Smola 3 , and Iryna Kravchenko 1,2 1 Odessa National Polytechnic University, Boulevard of Shevchenko, 1, Odessa, Ukraine 2 Odessa National I.I. Mechnikov University, 2 Dvorjanskaya st., Odessa, Ukraine 3 A.V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odessa, Ukraine * Corresponding author: mashaneutron@gmail.com, kravchenko.pharm@gmail.com
The effect of terpenoid esters on membrane structure investigated by fluorescence and Fourier-transform infrared spectroscopy 2
Abstract: 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 (I E /I M ) 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 (I 1 /I 3 ) 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. Keywords: terpenoids; fluorescence probe; FT-IR spectroscopy; liposomes; stratum corneum. 3
Introduction 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. 4
Results and discussion O O O i ii R OH + NH Boc NH Boc NH 2 H O RO RO 1-6 Synthetic pathway of compounds 1 – 6 . Reagents and conditions : (i) DMAP, CH 2 Cl 2 , rt, 10 min; DCC, 0 ° C, 30 min; rt, 10 h; (ii) HCl, CH 3 COOH. All esters were prepared as hydrochlorides. CH 3 CH 3 CH 3 R = R = R = Esters based on the corresponding terpenoids ( 1 − 6 ) were synthesized H 3 C CH 3 H 3 C CH 3 H 3 C CH 3 using DCC/DMAP coupling method 1 2 3 followed by deprotection of the amino CH 3 CH 3 H 3 C CH 3 O O groups in the HCl/CH 3 COOH medium. R = R = R = H 2 C H 3 C 4 5 6 5
Investigation of esters’ influence on membrane permeability using method of fluorescence probe Steady-state Chloroform fluorescence solution of pyrene The dried mixture spectra of samples + methanol The resulting The solvents were was resuspended in containing pyrene solution of emulsion was then removed by slow 25 ml deionized were recorded on a terpenoid esters + sonicated for 10 evaporation under water and Horiba Jobin-Yvon chloroform min at 22 kHz vacuum at 40 ° C vigorously stirred Fluorog-FL 3-22 solution of lecithin frequency for 10 min spectrophotometer in a molar ratio equipped with a 1:10:100 450W Xe lamp Pyrene Phospholipids Terpenoid esters
The influence of terpenoids and their esters on membrane microviscosity and polarity * h ν M - monomer fluorescence (370-395 mn) + * * * * h ν E - excimer + + + + + + fluorescence (470-480 nm) 1600000 Without enhancer 5000000 373 373 Without enhancer 1400000 Menthol 394 394 Thymol Compound 1 CH 3 Compound 2 1200000 CH 3 4000000 Intensity, arb. unit Intensity, arb. unit O + Cl O NH 3 1000000 + Cl O NH 3 O 3000000 1 H 3 C CH 3 800000 2 H 3 C CH 3 475 600000 2000000 475 400000 1000000 200000 0 0 350 400 450 500 550 350 400 450 500 550 Wavelength, nm Wavelength, nm Fluorescence emission spectra of pyrene incorporated into liposome membranes (control, black line) and in the presence of menthol, thymol and their GABA esters
The influence of terpenoids and their esters on membrane microviscosity and polarity 2400000 Without enhancer 1400000 373 2200000 Without enhancer 394 373 Guaiacol Carvacrol 2000000 394 Compound 4 1200000 Compound 3 1800000 CH 3 Intensity, arb. unit Intensity, arb. unit 1600000 1000000 CH 3 O + O NH 3 Cl 1400000 O + O 800000 NH 3 Cl 1200000 3 O 4 H 3 C CH 3 1000000 600000 800000 475 400000 600000 475 400000 200000 200000 0 0 350 400 450 500 550 350 400 450 500 550 Wavelength, nm Wavelength, nm Fluorescence emission spectra of pyrene incorporated into liposome membranes (control, black line) and in the presence of carvacrol, guaiacol, borneol, eugenol and their GABA esters 2600000 Without enhancer 1400000 Without enhancer 2400000 394 394 Borneol 373 Eugenol 373 2200000 1200000 Compound 5 Compound 6 2000000 Intensity, arb. unit O 1800000 + 1000000 H 3 C CH 3 Intensity, arb. unit NH 3 Cl 1600000 O 800000 OMe 1400000 O 1200000 H 3 C + 600000 5 NH 3 Cl 1000000 O CH 2 CH CH 2 800000 400000 6 600000 475 475 400000 200000 200000 0 0 350 400 450 500 550 350 400 450 500 550 Wavelength, nm Wavelength, nm
The influence of terpenoids and their esters on membrane microviscosity and polarity Excimer to monomer ratio : I E /I M = I 475 /I 394 0,44 0,45 0,40 0,32 0,31 0,29 0,35 0,28 0,26 0,25 0,30 0,24 0,25 0,16 0,20 0,13 0,12 0,10 0,09 0,15 0,10 0,05 0,00 Control Menthol #1 Thymol #2 Carvacrol #3 Guaiacol #4 Borneol #5 Eugenol #6 Ratio of the first to third vibronic band : I 373 /I 384 1,23 1,40 1,21 1,12 1,11 1,08 1,08 1,05 1,06 1,20 1,00 0,93 0,96 0,92 0,89 1,00 0,80 0,60 0,40 0,20 0,00 Control Menthol #1 Thymol #2 Carvacrol #3 Guaiacol #4 Borneol #5 Eugenol #6
Investigation of esters’ influence on phospholipid membrane using FT -IR spectroscopy 100 FT-IR spectra were measured with a Frontier FT-IR spectrometer 80 (Perkin-Elmer, Hopkinton, %Transmittance MA, USA). 60 1657 40 1180 The samples for FT-IR study have been prepared 20 by dissolving the obtained 3392 1062 1738 1239 1088 lipids in carbon tetrachloride (CCl 4 ) with 3600 3400 3200 1800 1700 1600 1280 1200 1120 1040 Wavenumber (cm-1) Wavenumber (cm-1) subsequent addition of Wavenumber (cm-1) without enhancer menthol compound 1 terpenoid esters (10% relative to lipids’ mass). 100 80 %Transmittance 60 1657 FT-IR spectra were recorded for films obtained using a method 40 of slow evaporation of 1180 solvent directly from 20 undercover under a 3392 1062 nitrogen atmosphere. 1738 1239 1088 3600 3400 3200 1800 1700 1600 1280 1200 1120 1040 Wavenumber (cm-1) Wavenumber (cm-1) Wavenumber (cm-1) without enhancer borneol compound 5
Investigation of esters’ influence on lipids of stratum corneum using FT -IR spectroscopy The samples for FT-IR study Then the extract have been FT-IR spectra was washed prepared by were recorded twice with dissolving the for films The SC was dipped distilled water obtained lipids obtained using a Preparation of and the lower into chloroform: in carbon method of slow stratum methanol (2:1) organic layer tetrachloride evaporation of corneum solution and kept in was evaporated solvent directly (CCl 4 ) with the dark for 72 h under vacuum subsequent from undercover below 40 ° C addition of under a nitrogen under a stream atmosphere. terpenoid esters of nitrogen. (10% relative to lipids’ mass). 100 100 80 80 %Transmittance %Transmittance 60 60 1715 1715 3387 3387 1737 1737 40 40 20 20 3600 3400 3200 1775 1725 3600 3400 3200 1775 1725 Wavenumber (cm-1) Wavenumber (cm-1) Wavenumber (cm-1) Wavenumber (cm-1) without enhancer menthol compound 1 without enhancer borneol compound 5
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