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 dessa National Polytechnic University, Boulevard of Shevchenko, 1, dessa, Ukraine 2 dessa National I.I. Mechnikov University, 2 Dvorjanskaya st., dessa, Ukraine 3 A.V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, dessa, 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=, P= 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 R H i NH Boc NH Boc H R ii R NH 2 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. All esters were prepared as hydrochlorides. R = R = R = R = C C R = C 1 2 3 R = C 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 medium. H 2 C C 4 5 6 5
Investigation of esters influence on membrane permeability using method of fluorescence probe Chloroform solution of pyrene methanol solution of terpenoid esters chloroform solution of lecithin in a molar ratio 1:10:100 The solvents were removed by slow evaporation under vacuum at 40 C The dried mixture was resuspended in 25 ml deionized water and vigorously stirred for 10 min The resulting emulsion was then sonicated for 10 min at 22 khz frequency Steady-state fluorescence spectra of samples containing pyrene were recorded on a Horiba Jobin-Yvon Fluorog-FL 3-22 spectrophotometer equipped with a 450W Xe lamp Pyrene Water Phospholipids Terpenoid esters Ultrasound
Intensity, arb. unit The influence of terpenoids and their esters on membrane microviscosity and polarity Fluorescence emission spectra of pyrene incorporated into liposome membranes (control, black line) and in the presence of menthol, thymol and their GABA esters Intensity, arb. unit * hν M - monomer fluorescence (370-395 mn) * * hν E - excimer fluorescence (470-480 nm) 1600000 1400000 1200000 1000000 800000 600000 400000 373 394 C 1 475 Without enhancer Menthol Compound 1 N Cl 5000000 4000000 3000000 2000000 373 394 C 475 2 Without enhancer Thymol Compound 2 N Cl 1000000 200000 0 350 400 450 500 550 Wavelength, nm 0 350 400 450 500 550 Wavelength, nm
Intensity, arb. unit Intensity, arb. unit Intensity, arb. unit Intensity, arb. unit The influence of terpenoids and their esters on membrane microviscosity and polarity C 3 N Cl 1000000 800000 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 1400000 1200000 1000000 800000 600000 400000 200000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 350 400 450 500 550 373 373 394 394 Wavelength, nm C C 5 475 Without enhancer Carvacrol Compound 3 N Cl 0 350 400 450 500 550 475 Wavelength, nm Without enhancer Borneol Compound 5 1400000 1200000 600000 400000 200000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 373 394 Me CH 2 CH CH 2 6 4 N Cl N Cl 0 350 400 450 500 550 373 394 475 Wavelength, nm Without enhancer Guaiacol Compound 4 0 350 400 450 500 550 475 Wavelength, nm Without enhancer Eugenol Compound 6
The influence of terpenoids and their esters on membrane microviscosity and polarity Excimer to monomer ratio : I E /I M = I 475 /I 394 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 0,44 0,31 0,32 0,28 0,29 0,26 0,24 0,25 0,16 0,13 0,10 0,09 0,12 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 1,40 1,20 1,00 0,93 0,92 1,08 1,06 1,00 1,08 1,12 1,11 1,05 1,21 1,23 0,89 0,96 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 %Transmittance %Transmittance FT-IR spectra were measured with a Frontier FT-IR spectrometer (Perkin-Elmer, Hopkinton, MA, USA). 100 80 60 1657 40 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). 20 100 3392 3600 3400 3200 1738 1800 1700 1600 1280 1200 1120 1040 without enhancer menthol compound 1 1239 1180 1062 1088 80 FT-IR spectra were recorded for films obtained using a method of slow evaporation of solvent directly from undercover under a nitrogen atmosphere. 60 40 20 3392 3600 3400 3200 1738 1657 1800 1700 1600 1239 1180 1062 1088 1280 1200 1120 1040 without enhancer borneol compound 5
%Transmittance %Transmittance Investigation of esters influence on lipids of stratum corneum using FT-IR spectroscopy Preparation of stratum corneum The SC was dipped into chloroform: methanol (2:1) solution and kept in the dark for 72 h Then the extract was washed twice with distilled water and the lower organic layer was evaporated under vacuum below 40 C under a stream of nitrogen. 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. 100 100 80 80 60 3387 1737 1715 60 3387 1737 1715 40 40 20 20 3600 3400 3200 1775 1725 without enhancer menthol compound 1 3600 3400 3200 1775 1725 without enhancer borneol compound 5
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.
Thermal methods of induction Experimental methods of pain induction Chemical methods of induction Hot plate test 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 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. 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 Compound Latency, sec Compound Latency, sec Menthol 24 ± 3,9 1 30 ± 2,6 Thymol 15 ± 0,3 2 21 ± 0,9 Carvacrol 19 ± 1,9 3 19 ± 3,8 Guaiacol 20 ± 0,5 4 17 ± 3,7 Borneol 27 ± 2,8 5 46 ± 2,2 Eugenol 21 ± 3,1 6 26 ± 2,9 Benzocaine 18 ± 0,9 Control 10 ± 0,6 C H N H C H 2 NCS H H H 2 N C C 2 H 5 Benzocaine Pain
Analgesic properties of terpenoid esters investigated on formalin-induced model of pain Compound Reaction time, sec Compound Reaction time, sec Menthol 24 ± 5,6 1 30 ± 7,4 Thymol 50 ± 5,2 2 52 ± 6,6 Carvacrol 38 ± 3,8 3 31 ± 8,3 Guaiacol 54 ± 6,8 4 47 ± 9,8 Borneol 26 ± 4,7 5 23 ± 2,0 Eugenol 40 ± 3,3 6 32 ± 4,8 Benzocaine 36 ± 2,4 Control 103 ± 8,5 Dosage form: 2% ointment Base: PEG PE 1,2-Propyleneglycol
Analgesic properties of terpenoid esters investigated on capsaicin-induced model of pain Compound Reaction time, sec Compound Reaction time, sec Menthol 11 ± 2,7 1 4 ± 0,9 Thymol 35 ± 4,1 2 20 ± 2,6 Carvacrol 23 ± 6,4 3 12 ± 1,2 Guaiacol 23 ± 2,7 4 24 ± 3,3 Borneol 12 ± 5,0 5 15 ± 3,0 Eugenol 20 ± 3,4 6 17 ± 2,2 Benzocaine 29 ± 6,6 Control 46 ± 1,8 Analgesic properties of terpenoid esters investigated on AITC-induced model of pain Compound Reaction time, sec Compound Reaction time, sec Menthol 7 ± 1,2 1 3 ± 0,3 Thymol 25 ± 3,8 2 20 ± 5,8 Carvacrol 35 ± 2,2 3 23 ± 4,3 Guaiacol 21 ± 2,8 4 25 ± 1,5 Borneol 8 ± 3,5 5 23 ± 3,0 Eugenol 30 ± 2,2 6 22 ± 3,1 Benzocaine 48 ± 2,0 Control 71 ± 1,8
The volume of affected rat paw, % of control The volume of affected rat paw, % of control Anti-inflammatory activity of terpenoid esters 260 240 220 200 180 160 140 120 100 80 60 Inflammation Compound 1 Compound 2 Compound 3 Ibuprofen 0 1 2 3 4 5 6 Time, hours C C H 2 AITC Ibuprofen NCS H 260 240 220 200 180 160 140 120 100 80 60 Inflammation Compound 4 Compound 5 Compound 6 0 1 2 3 4 5 6 Time, hours
Conclusions 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. 18