1 The protective effect of Artepillin C against lipid oxidation on model membranes 1 Wallance Moreira Pazin 1,*, Gilia Cristine Marques Ruiz 1, Marcelo José dos Santos 1, 2 Pedro Henrique Benites Aoki 2, Amando Siuiti Ito 3 and Carlos José Leopoldo 3 Constantino 1 4 5 1 São Paulo State University (UNESP), School of Technology and Applied Sciences, 6 Presidente Prudente, SP, Brazil, 19060-900; wallance.pazin@unesp.br 7 2 São Paulo State University (UNESP), School of Sciences, Humanities and Languages, 8 Assis, SP, Brazil, 19806-900; pedro.aoki@unesp.br 9 3 University of São Paulo (USP), Faculty of Philosophy, Sciences and Letters of 10 Ribeirão Preto, Ribeirão Preto, SP, Brazil, 14040-901; amandosi@ffclrp.usp.br 11 12 13 14 15 16 17 18 19 20 * Corresponding author: Wallance Moreira Pazin 21 E-mail address: wallancepazin@gmail.com 22 Rua Roberto Simonsen, 305 23 19060-900, Presidente Prudente, Brazil 24 Tel.: +55 18 3229-5461 25 2 Abstract 26 Brazilian green propolis is a well-known therapeutic product, commonly used in folk 27 medicine. Artepillin C is the major compound of Brazilian green propolis and has 28 received considerable attention owing to its lipophilic affinity and antioxidant activity, 29 enabling the use against lipid oxidation caused by free radicals, which is a first step 30 before degenerative diseases. The protective effect of Artepillin C against lipid oxidation 31 was evaluated here on models of lipid membranes based on Langmuir monolayers and 32 giant unilamellar vesicles (GUVs) formed of 1,2-dioleoyl-sn-glycero-3-phosphocholine 33 DOPC under oxidative stress induced by the photoactivated erythrosin. Our findings 34 show that the lipophilic character of Artepillin C allows the donation of a hydrogen atom 35 of the phenolic hydroxyl group to the lipid radical of both mono and bilayer, avoiding 36 the formation of truncated aldehyde lipids, interrupting oxidative reactions mediated by 37 reactive oxygen species (ROS), therefore playing a role as an antioxidant compound in 38 the lipid environment. The affinity of Artepillin C for lipid structures, together with its 39 antioxidant potential, preclude the lipid peroxidation caused by reactive species. 40 41 Keywords: Green propolis; Artepillin C; antioxidant activity; model membranes; 42 erythrosine 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3 1. Introduction 60 61 Human body is a complex system that develops its biological function with a high 62 degree of organization, going from molecular levels up to process involving tissues, 63 organs and members. However, the dynamic of metabolic process is not always 64 successful. For instance, free radicals are released in the body, and, when in excess, have 65 high reactivity with different molecules. It may trigger damages and mutation in the 66 organism by the oxidative stress [1,2]. Therefore, improper molecular combination might 67 be prejudicial to the metabolism, playing a role in the development of several deleterious 68 effects in DNA, signaling proteins and cell membranes, leading to an early aging and to 69 degenerative diseases, such as Alzheimer, Parkinson, atherosclerosis and cancer [3–5]. 70 In the case of cell membranes, free radicals attack carbon-carbon double bonds 71 present in the acyl chain, triggering lipid peroxidation [4,6]. It happens in three main 72 stages: initiation, when free radicals removes the allylic hydrogen of acyl chain, creating 73 the carbon-centered lipid radical (L•); propagation, starting from the moment that 74 molecular oxygen reacts with L• generating peroxy-radical (LOO•). This reactive species 75 captures a hydrogen from another lipid molecule in the membrane, creating a new L• that 76 will react with O2, turning this process cyclic (chain reaction) leading to the generation of 77 degradation products, as aldehydes [7]. It can be avoided by the termination step, when 78 antioxidant compounds take place donating their hydrogen atoms, ending the reaction [8]. 79 A well-known example is the α-tocopherol (vitamin E), which donates a hydrogen atom 80 to LOO• species, generating a radical compound that is able to react with another LOO•, 81 therefore forming a non-radical termination product [7,8]. 82 In a protective way, the biologic system has a defense mechanism involving redox 83 agents aiming stabilization of free radicals by means of antioxidant action. It can be, for 84 example, enzymes, vitamins (A, C and E) and natural pigments [1]. Although necessary, 85 4 this self-defense of the organism is not completely effective for the combat of oxidative 86 stress in an eventual imbalance due to the excess of free radicals, mainly for the exposition 87 of humans to the UV radiation, pollutants, tobacco smoke, heavy metals and polycyclic 88 aromatic hydrocarbons [9–11]. In this perspective, despite the action of endogenous 89 antioxidant agents, the introduction of exogenous compounds with high antioxidant 90 capacity is capable of preventing or even regenerate damages caused by undesirable redox 91 reaction. 92 Natural products rich in polyphenols, such phenolic acids, flavonoids, terpenes and 93 aromatic aldehydes, identified as secondary metabolites, are often involved in the 94 protection mechanisms to help in the adaptation of plants to the environment [12,13]. 95 They have attracted attention mainly for presenting a large spectrum of pharmacological 96 properties, including antioxidant activities against different types of free radicals [14]. 97 Taking advantage of their benefits, bees collect secondary metabolites from the plant 98 exudates for the protection of their hives against parasites and diseases, forming propolis 99 [15]. Particularly in Brazil, green propolis is constituted of several secondary metabolites 100 collected by Apis mellifera especially from Baccharis dracunculifolia [16]. To date, 101 Brazilian green propolis is extensively consumed worldwide, especially in Asiatic 102 countries, due to its several biological properties, including, besides antioxidant 103 properties, antitumor, antimicrobial, anti-inflammatory activities [17–21]. Among the 104 bioactive compounds, Artepillin C (ArtC) stands out since it is the major and the most 105 biologically relevant compound of such Brazilian propolis [22,23], with a wide range of 106 pharmacological properties, as reported above for Brazilian green propolis [23–25]. The 107 high quality of propolis correlates to the amount of ArtC in its composition [18]. It is a 108 simple phenolic acid derivative with two prenylated groups imparting hydrophobicity that 109 ensure its affinity for lipophilic environment [26–28]. This feature correlates with the 110 5 success of applying ArtC as an antioxidant agent mainly for protection of cell membranes, 111 since the hydroxyl group of aromatic ring is available for the redox process with the free 112 radical via one-step hydrogen atom transfer [29]. Szliszka et al. have reported the radical-113 scavenging activity of ArtC against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 114 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+), with 115 ED50 values 24.6 and 19.5 µM, respectively [23]. Massaeli et al. [30] reported the higher 116 potential of antioxidant agents with lipophilic affinity instead of those with hydrophilic 117 property against lipid peroxidation reaction, owed by the efficiency of transferring the 118 hydrogen atom to the lipid radical directly in the cell membrane, leading rapidly to the 119 end of the reaction by the production of a non-radical product. A recent published study 120 performed by Sadžak et al. (2020) investigated the antioxidative potential of three 121 different flavonols in both hipdrophilic and hydrophobic environments, suggesting that 122 the protective effect of lipid bilayers upon oxidative stress, preserving the supramolecular 123 and mechanical properties of the membrane, is dependent on the environment in which 124 they are located [31]. 125 As we shall demonstrate in this study, the lipophilic character of ArtC and its 126 antioxidant capacity are able to interrupt oxidative reactions mediated by the 127 photoactivated erythrosin B in a lipid environment. Langmuir monolayers and Giant 128 Unilamellar Vesicles (GUVs) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 129 were used as bioinspired systems of cell membranes. DOPC imparts one carbon-carbon 130 double-bond in each acyl chain, which is a target for reactive species. In general, ArtC 131 avoids a fast membrane permeabilization and generates an area excess of the model 132 membranes due to the production of hydroperoxides as a final step of its antioxidant 133 action. 134 135 6 2. Materials and Methods 136 137 ArtC (3,5-diprenyl-4-hydroxycinnamic acid), isolated and purified (98.43%) from 138 Brazilian green propolis, was purchased from Wako (Japan). 1,2-Dioleoyl-sn-glycerol-3-139 phosphocholine (DOPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, 140 USA) and used without further purification. Erythrosin, polyvinyl alcohol (PVA, M.W. 141 146.000 – 186.000 g/mol), HEPES, sodium chloride (NaCl), and the organic solvents 142 (chloroform and methanol) of analytical grade were purchased from Sigma Chemical Co. 143 (St. Louis, MO, USA). Aqueous solutions were obtained with ultrapure Milli-Q® water 144 (resistivity 18.2 MΩ.cm, surface tension 72 mN/m, 25 °C). The molecular structures of 145 ArtC, DOPC and erythrosin are displayed in Figure 1. 146 147 Figure 1. Molecular structures of Artepillin C, erythrosin and DOPC. 148 149 2.1. Surface pressure versus mean molecular area (π-A) isotherms 150 151 DOPC solution in chloroform (0.50 mg.mL-1) was spread on a 150-cm² (length = 152 30.00 cm; width = 5.00 cm) Langmuir trough (KSV Instruments Ltd., Helsinki, Finland). 153 Lipid monolayers were formed at the air/liquid interface with symmetrical compression 154 7 of the barriers at a constant rate of 10 mm/min, ten minutes after spreading DOPC solution 155 onto the subphase, required for chloroform evaporation. Surface pressure (π) vs mean 156 molecular area (Å) isotherms were obtained using a platinum plate as Wilhelmy sensor. 157 The monolayers were compressed on buffer (HEPES buffer 10 mM, pH 7.4 and NaCl 158 150 mM) and buffer containing erythrosin (10 µM) or/and ArtC (10 µM). Light-159 irradiation was performed with green LED (530 nm – 50 W, BRIWAX FFG) placed 20 160 cm above the monolayer to monitor the surface pressure stability at a constant mean 161 molecular area. A negative control was also acquired in dark, without any influence of 162 either external or LED light. All isotherms were recorded in triplicate at 23 °C (room 163 temperature). 164 2.2. Giant unilamellar vesicles (GUVs) 165 166 Giant unilamellar vesicles (GUVs) were assembled on a polyvinyl alcohol (PVA) 167 film, as previously reported by Weinberger et al [32]. This method allows GUVs to swell 168 avoiding ions interference and lipid oxidation that might happen when they are growth 169 by the traditional electroformation method [33]. Briefly, a PVA solution (5% w/w) was 170 initially prepared in milli-Q water at 90 °C and kept under stirring until complete 171 solubilisation. 500 µL of the solution was then dropped on the bottom of a well (5 cm in 172 diameter) and placed in an oven (~100 °C) for water evaporation. 20 µL of DOPC 173 chloroform solution (1 mM) was then uniformly spread on PVA-coated well and left 174 under vacuum for at least 1 hour. Afterwards, 1 mL of a sucrose solution (200 mM) 175 prepared in HEPES buffer (150 mM NaCl, 10 mM HEPES) was added to the well, 176 allowing GUVs to swell for 1h30min at room temperature. GUVs were piped from the 177 PVA-coated well and stored in Eppendorf tubes. For microscope observation, 50 µL of 178 GUVs suspensions were added to an eight-well polymer chamber (Ibidi, Munich, 179 8 Germany) containing 200 µL of iso-osmolar glucose-buffer solution with the desired 180 concentration of ArtC and/or erythrosin. A negative control was performed by adding 181 methanol (0.8% v/v) in the well, which was the necessary volume to reach the maximum 182 concentration of ArtC in the glucose-buffer solution for this study. The osmolality of both 183 sucrose and glucose solutions were checked using a freezing point Osmometer (Osmomat 184 3000 – Gonotec GmbH, Berlin, Germany). The vesicles settle at the bottom of the 185 microscope-chamber owing to the density difference between glucose and sucrose, thus 186 facilitating the observation. The experiments were performed in an inverted confocal 187 microscope (Nikon C2/C2si Eclipse Ti-E, Kyoto, Japan), using a 40x air objective, NA 188 0.9, by means of phase contrast. The samples were irradiated using an excitation band 189 filter (540/25) placed in the epi-fluorescence light path of a mercury lamp. The 190 experiments were performed at least in triplicate, at 23 °C (room temperature) and the 191 images were analyzed with ImageJ. 192 193 2.3. Statistical analysis 194 195 The experimental data were collected in triplicate, processed using OriginPro® 8.5 196 and evaluated by the analysis of standard deviation. 197 198 3. Results and discussion 199 200 3.1. π-A isotherms 201 The π-A isotherms obtained for DOPC monolayers on buffer (pH 7.4 and 150 mM 202 NaCl) and buffer containing 10 µM of ArtC, erythrosin, and erythrosin + ArtC mixture 203 are shown in Figure 2a. 204 9 (a) (b) Figure 2. a) π-A isotherms of DOPC Langmuir films at 23 °C on buffer (HEPES 205 solution at 10 mM and pH 7.4 + 150 mM of NaCl; black line) and buffer 206 containing 10 µM of ArtC (red line), 10 µM of erythrosin (blue line) and 10 µM 207 of the ArtC and erythrosin mixture (magenta line); b) Surface pressure time 208 dependence of irradiated and non-irradiated DOPC monolayer on buffer 209 (HEPES solution at 10 mM and pH 7.4 + 150 mM of NaCl) and buffer containing 210 10 µM of ArtC, 10 µM of erythrosin and 10 µM of the ArtC and erythrosin 211 mixture. 212 213 Both ArtC and erythrosin are not surface active [34,35] and cannot form Langmuir 214 monolayers when in a mixture, as shown by the subsidiary experiment in Figure S1. Such 215 compounds expand the π-A isotherms of DOPC, even at higher surface pressures, 216 suggesting their interaction on the monolayers. We have confirmed in a previous study 217 [34] a preferential adsorption of ArtC in the polar region of DPPC monolayers owing to 218 the negative charge of the carboxyl group (COO-), which form a stabilized intramolecular 219 hydrogen bonding with the water molecules at the water/lipid interface. Considering that 220 DPPC and DOPC share the same phosphatidyl head groups, it is likely that interactions 221 of the same nature governs the ArtC adsorption on DOPC monolayers. Moreover, the less 222 packed DOPC monolayers may have allowed deeper penetration of ArtC, resulting in 223 larger expansion in the π-A isotherms, relatively to DPPC. Indeed, previous studies have 224 shown ArtC embedded in deeper regions of lipid vesicles in fluid state [28], which can 225 10 be related to cohesive van der Waals interaction between the ring moiety of ArtC bearing 226 two prenyl groups with the unsaturated acyl chains. 227 The dianionic erythrosin is driven to DOPC monolayers mainly by attractive 228 electrostatic interactions with the cationic choline groups, with penetration into the 229 chains, as previously reported [35]. Compared to ArtC, erythrosin causes larger expansion 230 on DOPC monolayers, which can be notice by the higher right-shift in the π-A isotherms 231 (Figure 2a). The latter is not only related to the larger size (e.g. the presence of conjugated 232 rings) of erythrosin but also to the additional negative charge that can increase the 233 electrostatic repulsion with the anionic phosphate in the lipid heads, hindering the 234 monolayer packing. Such features can be also confirmed by the π-A isotherm formed in 235 presence of the ArtC + erythrosin mixture, which resembles the one containing only 236 erythrosin, i.e., despite ArtC interacting with DOPC monolayer, the larger effects in 237 occupied area per lipid molecule is predominantly caused by the physicochemical 238 properties of erythrosin towards lipid monolayer interaction. Although the insertion of 239 the molecules at the liquid/air interface reflects in a shift of the DOPC monolayers to 240 larger areas, it does not cause significant changes in overall monolayer elasticity, as 241 shown in Supporting Information (SI 2), preserving the shapes and profiles of the 242 isotherms. 243 Additional analysis on the collapse pressure shows a slightly decreased upon 244 erythrosin adsorption, supporting the electrostatic repulsion previously proposed. ArtC + 245 erythrosin mixture further decreased the collapse pressure, confirming the monolayer 246 instability in relation to DOPC on buffer and buffer containing ArtC or erythrosin. 247 The stability of irradiated and non-irradiated DOPC monolayers on buffer and buffer 248 containing ArtC, erythrosin, and erythrosin + ArtC mixture were monitored at 30 mN/m, 249 which is believed to correspond to the lateral pressures of cell membranes [36]. Upon 250 11 reaching the target pressure, the surface area was kept constant and further modification 251 in the pressure was followed over time, as displayed in Figure 2b. In all the non-irradiated 252 monolayers, the surface pressure stabilizes at 27 ± 1 mN/m along 1 hour, after reaching 253 30mN/m. This decrease in surface pressure could be related to the monolayer stabilization 254 after a sudden stop of the barriers. Light irradiation of DOPC monolayer containing 255 erythrosin decreased the surface pressure to 20 mN/m (~ 67%), which did not occur for 256 monolayers without its presence. It is already known that erythrosin presents high 257 quantum yield of singlet oxygen (1O2) upon irradiation [37]. This reactive oxygen species 258 (ROS) is generated by the energy transfer from the excited states of erythrosin to the 259 molecular oxygen of the environment. The hydroperoxide generation has been shown to 260 be the main outcome of the 1O2 reaction with the chain unsaturations, which subsequently 261 disturb the hydrophilic-hydrophobic balance of the lipid membranes [35]. Moreover, the 262 propagation of the oxidative reaction can result in the cleavage of the chains at the 263 unsaturation site. The photoactivated erythrosin can attack the chain unsaturation, or 264 previously formed hydroperoxides, by contact-dependent reactions that result in truncated 265 lipid aldehydes [38]. The latter are solubilized by the subphase, reducing the amount of 266 material at the lipid interface and, consequently, decreasing the surface pressure of the 267 monolayer as observed in this study. On the other hand, the surface pressure of irradiated 268 DOPC monolayer on erythrosin + ArtC mixture increased to 34.5 mN/m (115%), 269 suggesting a protective action of ArtC against oxidation and, consequently, avoiding the 270 production of truncated lipid aldehydes. It is likely that a hydrogen atom of the phenolic 271 hydroxyl group is donated to the lipid radical [29], blocking the propagation of the 272 oxidative reaction. Therefore, the oxidative reaction may have terminated with the 273 hydroperoxide generation owed to the antioxidant potential of ArtC [7,8], which 274 correlates directly to the increase the mean molecular area of the phospholipid, resulting 275 12 in the overall surface pressure increase of the monolayer. An illustrative representation 276 of the involved processes is displayed in Figure 3. 277 278 Figure 3. Schematic illustration of the presence of ArtC and erythrosin towards 279 lipid monolayer (a); photoinduced erythrosin, in absence of AtC, generating 280 truncated aldehyde by the cleavage of DOPC chains (b); protective action of 281 ArtC against oxidation by the generation of hydroperoxide lipids as a termination 282 step (c); reaction representing a hydrogen atom donation from ArtC to the lipid 283 peroxyl radical (LOO•), generating lipid hydroperoxide (d). 284 285 3.2. GUVs as model membranes 286 287 Qualitative analysis was performed with GUVs in order to better understand how 288 ArtC plays antioxidant activity against photoproducts in lipophilic environment, 289 particularly in a system that represents cell size and plasma membrane curvature. GUVs 290 immersed in 10 µM of both erythrosin and ArtC were not affect (data not shown), as 291 13 expected from previous reported data [34,39]. While light-irradiation at 540 ± 25 nm did 292 not produce any visible modification on GUVs immersed in 100 µM ArtC solution (see 293 Supporting Information SI 3), lipid bilayers are significantly affected in erythrosin, as 294 shown in Figure 4. Fluctuations are observed right after the beginning of the irradiation 295 and 10 seconds later the surface area of the GUVs is increased, with no loss of contrast. 296 Membrane permeabilization is observed after 150 seconds with the pore opening and the 297 contrast fades away at ca. 300 seconds of irradiation, remaining with the same shape for 298 the whole time of observation (ca. 3600 s – data not shown). Lipid hydroperoxidation in 299 the first seconds explains the surface area increase of GUVs, resulting from 1O2 ene 300 reaction with alkenes containing allylic hydrogens [35], although the same could not be 301 observed at the beginning of surface pressure analysis in Langmuir monolayer probably 302 due to the barrier stabilization in the first 5 minutes. The pore opening and membrane 303 permeabilization is result of the cleavage of the lipid chains triggered by contact-304 dependent reactions [38], which is consistent to the data on Langmuir monolayers. 305 Indeed, the formation of truncated lipid aldehydes favors water penetration by an increase 306 in the lipid-lipid distance, causing the pore opening in the membrane and further 307 permeabilization [38,40]. 308 14 Figure 4. Phase contrast images acquired for DOPC GUVs suspended in 10 µM 309 erythrosin solution, under irradiation at 540 ± 25 nm. Scale bar: 5 µm. 310 311 The protective action of ArtC against lipid oxidation was evaluated by irradiating 312 GUVs immersed in a solution containing 10 µM of both erythrosin and ArtC. Although 313 the same photo-oxidation effects of GUVs in erythrosin solution (Figure 4) are noted in 314 Figure 5, ArtC has retarded the sequence of events. For instance, membrane fluctuations 315 and surface area increase were noticed after ca. 36 seconds and ca. 54 seconds, 316 respectively. Buds are created after ca. 88 seconds as result of the excess of area generated 317 and the phase contrast is almost completely lost after ca. 530 seconds irradiation. This 318 sequence of events is further delayed with the increase of ArtC concentration (Figure S4), 319 suggesting an increased protective effect against photosensitized species. Antioxidant 320 compounds such as ArtC plays a role in the termination step of lipid peroxidation by a 321 donation of the hydrogen atom to LOO• species, generating hydroperoxides and 322 nonradical products by the reaction of antioxidant radical with another LOO• [8], as 323 already shown in the representation of the reaction between ArtC and the lipid peroxyl 324 radical in Figure 2d. It most likely explains the reason that took the GUV to maintain for 325 15 a longer time both surface membrane fluctuation and area excess caused by the presence 326 of hydroperoxide groups. Moreover, ArtC possibly apart its antioxidant activity avoiding 327 contact-dependent mechanism, acting directly against erythrosin photoproduct, although 328 the permeabilization can be observed after GUVs recover its spherical shape, remaining 329 with the same shape even after a long time of observation. 330 Figure 5. Phase contrast images acquired for DOPC GUVs suspended in a 331 solution containing 10 µM of both erythrosin and ArtC. Scale bar: 5 µm. 332 333 4. Conclusions 334 335 The affinity of ArtC for lipophilic environment, correlated to its particular structure 336 that ensure its antioxidant activity, allow it to act as a protective compound against lipid 337 oxidation caused by the reactive species that attack the double bounds of acyl chains in 338 unsaturated lipids. It was confirmed by means of time-dependent surface-pressure 339 stability of lipid monolayers and analysis in the morphology of GUVs, formed of DOPC. 340 The first reveals that the increase in the surface pressure results from the hydroperoxides 341 16 formed at the interface, while the same effect occur for GUVs, resulting in the increase 342 in the membrane surface area. Both results suggest that ArtC donates a hydrogen atom of 343 the phenolic hydroxyl group to the lipid radical that is formed by the contact-dependent 344 reaction between photosensitized erythrosin and the double-bond of acyl chains of DOPC 345 in the monolayer. Therefore, instead the fast formation of truncated aldehyde lipids, ArtC 346 plays a role in the termination step of lipid peroxidation generating hydroperoxides, 347 which increase both lipid per area in the DOPC monolayer and surface area of DOPC 348 GUVs. It is worth to mention that, as already published recently regarding the antitumor 349 action of ArtC [24], a subject of our current investigation aims to correlate and contribute 350 the results obtained from the studies performed with model membranes with the 351 antioxidant activity of ArtC in healthy cellular models, evidencing its action in the cell 352 membrane. 353 354 Acknowledgments: We thank INEO and FAPESP (2016/09633-4 and 2018/22214-6) 355 for funding support. GCMR is grateful to CAPES for a PhD fellowship, received during 356 the development of this work. CJLP and ASI are recipients of a CNPq research grant 357 (304100/2018-8 and 305771/2016-7, respectively). PHBA thanks FAPESP (2018/16713-358 0) for his research grant. 359 Conflicts of Interest: The authors declare no conflict of interest. 360 361 References 362 363 1. Birben, E.; Sahiner, U.M.; Sackesen, C.; Erzurum, S.; Kalayci, O. Oxidative Stress 364 and Antioxidant Defense. World Allergy Organ. 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