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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

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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 (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. Keywords: terpenoids; fluorescence probe; FT-IR spectroscopy; liposomes; stratum corneum.

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Introduction

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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 GABAB 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.

Results and discussion

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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/CH3COOH medium.

CH3 C H3 CH3 CH3 C H3 CH3 CH3 C H3 CH3 O CH3 R = R = R = R = O CH3 C H2 R = CH3 C H3 C H3 R = 1 2 3 4 5 6

Synthetic pathway of compounds 1–6. Reagents and conditions: (i) DMAP, CH2Cl2, rt, 10 min; DCC, 0 °C, 30 min; rt, 10 h; (ii) HCl, CH3COOH. All esters were prepared as hydrochlorides.

R OH O H O NH RO O NH RO O NH2 + Boc Boc 1-6 i ii

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

Phospholipids Terpenoid esters

The influence of terpenoids and their esters on membrane microviscosity and polarity

CH3 C H3 CH3 O O NH3

+ Cl

1

+

*

+

*

+ +

*

+

*

+ +

*

hνM - monomer fluorescence (370-395 mn)

hνE - excimer fluorescence (470-480 nm)

Fluorescence emission spectra of pyrene incorporated into liposome membranes (control, black line) and in the presence of menthol, thymol and their GABA esters

CH3 C H3 CH3 O O NH3

+ Cl

2 350 400 450 500 550 200000 400000 600000 800000 1000000 1200000 1400000 1600000

Intensity, arb. unit Wavelength, nm

Without enhancer Menthol Compound 1

475 373 394

350 400 450 500 550 1000000 2000000 3000000 4000000 5000000

Intensity, arb. unit Wavelength, nm

Without enhancer Thymol Compound 2

475 373 394

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 carvacrol, guaiacol, borneol, eugenol and their GABA esters

CH3 C H3 CH3 O O NH3Cl 3 +

O CH3 O O NH3Cl + 4

O CH2 CH CH2 O NH3 OMe Cl

+

6

CH3 C H3 C H3 O O NH3Cl + 5

350 400 450 500 550 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 2400000

Intensity, arb. unit Wavelength, nm

Without enhancer Carvacrol Compound 3

475 373 394

350 400 450 500 550 200000 400000 600000 800000 1000000 1200000 1400000

Intensity, arb. unit Wavelength, nm

Without enhancer Guaiacol Compound 4

475 373 394

350 400 450 500 550 200000 400000 600000 800000 1000000 1200000 1400000

Intensity, arb. unit Wavelength, nm

Without enhancer Borneol Compound 5

475 373 394

350 400 450 500 550 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 2400000 2600000

Intensity, arb. unit Wavelength, nm

Without enhancer Eugenol Compound 6

475 373 394

The influence of terpenoids and their esters on membrane microviscosity and polarity Excimer to monomer ratio : IE/IM = I475/I394 Ratio of the first to third vibronic band : I373/I384

0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 Control Menthol #1 Thymol #2 Carvacrol #3 Guaiacol #4 Borneol #5 Eugenol #6

0,13 0,28 0,44 0,26 0,31 0,24 0,32 0,25 0,29 0,10 0,09 0,12 0,16

0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 Control Menthol #1 Thymol #2 Carvacrol #3 Guaiacol #4 Borneol #5 Eugenol #6

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

without enhancer menthol compound 1

Investigation of esters’ influence on phospholipid membrane using FT-IR spectroscopy

FT-IR spectra were measured with a Frontier FT-IR spectrometer (Perkin-Elmer, Hopkinton, MA, USA). The samples for FT-IR study have been prepared by dissolving the obtained lipids in carbon tetrachloride (CCl4) with subsequent addition of terpenoid esters (10% relative to lipids’ mass). FT-IR spectra were recorded for films

• btained using a method
• f slow evaporation of

solvent directly from undercover under a nitrogen atmosphere.

1800 1700 1600 Wavenumber (cm-1) 1738 1657 3600 3400 3200 Wavenumber (cm-1) 20 40 60 80 100 %Transmittance 3392 1280 1200 1120 1040 Wavenumber (cm-1) 1239 1180 1088 1062 3600 3400 3200 Wavenumber (cm-1) 20 40 60 80 100 %Transmittance 3392 1800 1700 1600 Wavenumber (cm-1) 1738 1657 1280 1200 1120 1040 Wavenumber (cm-1) 1239 1180 1088 1062

without enhancer borneol compound 5

without enhancer borneol compound 5

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

• rganic layer

was evaporated under vacuum below 40 °C under a stream

• f nitrogen.

The samples for FT-IR study have been prepared by dissolving the

• btained lipids

in carbon tetrachloride (CCl4) with subsequent addition of terpenoid esters (10% relative to lipids’ mass). FT-IR spectra were recorded for films

• btained using a

method of slow evaporation of solvent directly from undercover under a nitrogen atmosphere.

3600 3400 3200 Wavenumber (cm-1) 20 40 60 80 100 %Transmittance 3387 1775 1725 Wavenumber (cm-1) 1737 1715 3600 3400 3200 Wavenumber (cm-1) 20 40 60 80 100 %Transmittance 3387 1775 1725 Wavenumber (cm-1) 1737 1715

without enhancer menthol compound 1

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.

 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

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

Benzocaine

N H2 C OC2H5 O

Pain

O H N H O O C H3

C H2 NCS O H H

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 – PEO – 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 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

Analgesic properties of terpenoid esters investigated on AITC-induced model of pain

Anti-inflammatory activity of terpenoid esters

C H2 NCS

C H3 CH3 OH CH3 O

AITC

Ibuprofen

60 80 100 120 140 160 180 200 220 240 260 1 2 3 4 5 6 The volume of affected rat paw, % of control Time, hours Inflammation Compound 1 Compound 2 Compound 3 Ibuprofen 60 80 100 120 140 160 180 200 220 240 260 1 2 3 4 5 6 The volume of affected rat paw, % of control Time, hours Inflammation Compound 4 Compound 5 Compound 6

Conclusions

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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 IE/IM 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.