Binding effects of Alpinia galanga oil and its nanoemulsion 1 to GABAA receptors in rat cortical membranes

21 Anesthetic activity of Alpinia galanga oil (AGO) has been reported however the mechanism of 22 action in mammals has not been clear. In the present study, the binding effects of AGO and its 23 three active components to gamma-aminobutyric acid type A (GABA A ) receptor in cortical 24 membranes of Sprague-Dawley rats were firstly investigated using a [3H]muscimol binding assay. 25 Dimethyl sulfoxide (DMSO) was used to deliver these test samples. The results showed that only 26 AGO and methyl eugenol displayed positive modulation at the highest concentration whereas 1,8- 27 cineole and 4-allylphenyl acetate were inactive. An oil-in-water nanoemulsion containing 20%w/w 28 AGO (NE-AGO) was formulated to deliver AGO instead of DMSO. This NE-AGO significantly enhanced a specific [3H]muscimol binding to 179% of the control with EC 50 of 391 µg/mL. The 30 result correlates well to the amount of methyl eugenol in AGO. This result confirms that the 31 anesthetic activity of AGO and methyl eugenol is associated with GABA A receptor modulation, 32 while that of 1,8-cineole and 4-allylphenyl acetate is not and may instead be related to other 33 mechanisms. AGO showed well-tolerated by human cells. Therefore, the formulated NE-AGO 34 might be a promising alternative anesthetic product for humans.


Introduction 36
Alpinia galanga is an edible plant of the Zingiberaceae family which is widely cultivated in Southeast Asian 37 countries 1,2 . A. galanga is well-known in Asian folk medicine and has been used for centuries as a food 38 additive, an antimicrobial agent, a local anesthetic, an analgesic, and an antipruritic 3-6 . A. galanga oil 39 (AGO) is rich in 1,8-cineole and 4-allylphenyl acetate 7 . AGO was recently shown to act as an anesthetic 40 in fish 8 . Further, 1,8-cineole has been shown to reduce locomotor activity in rodents when administered 41 orally or intraperitoneally 9,10 . To the best of our knowledge, the mechanism of anesthetic action of AGO has not yet been elucidated. Moreover, 4-allylphenyl acetate has not been reported for anesthetic effect in 43 rodents, whereas methyl eugenol, a minor compound of AGO has been reported as a surgical anesthetic in 44 rodents 11 . 45 Generally, anesthetics have been used to relieve pain and suffering during surgery and post-surgery by 46 inhibiting or depressing propagation of the signal along the nerves 12 . The mechanism of action of these 47 anesthetics are a blockade of the N-methyl-D-aspartate receptor receptors, inhibition of dopaminergic 48 receptors, or enhancement of the function of -aminobutyric acid type A (GABAA) receptors 13 . In addition, 49 GABAA receptors are the principal ionotropic receptors for fast inhibitory neurotransmission in the 50 mammalian central nervous system 14 . GABAA receptors are clinically employed targets for a range of 51 structurally diverse positive allosteric modulators such as isoflurane, etomidate, propofol, barbiturates, and 52 benzodiazepines [15][16][17] . Given the structural resemblance of the main AGO constituents with myristicin, 53 another previously reported GABAA receptor positive allosteric modulators 18 (Fig. 1), we hypothesized 54 that the anesthetic mechanism of action of AGO involves central GABAA receptors. The poor water-solubility of active compounds in essential oils remains a challenge in bioassay studies 71 employing aqueous buffers. To overcome this problem, many organic solvents have been used for 72 dissolving or delivery the active compounds before activity testing, however, potentially compromising the 73 biological evaluation 19 . AGO is also water-insoluble, therefore it needs a potential delivery system for 74 solubility enhancement. Nanoemulsion is one of the promising nano delivery system that can improve 75 aqueous solubility of many hydrophobic active compounds 20-22 . The nanoemulsions of several water-76 insoluble compounds have been developed for using in various bioassay studies 23-26 . 77 In the present study, the chemical components of AGO extracted from the fresh rhizomes of A. galanga 78 were analyzed. AGO and its three selected chemical components, 1,8-cineole, 4-allylphenyl acetate, and 79 methyl eugenol were investigated for the possible mechanism of action on anesthetic activity in mammals 80 using rat brain GABAA receptors. Moreover, a nanoemulsion containing AGO (NE-AGO) was formulated 81 to reduce the amount of organic solvent used for dissolving AGO and to investigate for its potential on 82 AGO delivery. The toxicity of AGO on normal human cells was also investigated for the further studies of 83 NE-AGO in humans. 84 Preparation and characterization of NE-AGO. As AGO is immiscible with water, DMSO is always 96 used to dissolve AGO and enhance water miscibility. However, DMSO shows several disadvantages due 97 to its toxic effects to animals and humans and binding interferences. Nanoemulsions can be prepared 98 without the use of organic solvents 29 . Incorporation of water-insoluble drugs into the o/w nanoemulsions 99

Results and Discussion
can improve their water miscibility 30 . In the present study, NE-AGO was formulated without the use of 100 organic solvent to improve the aqueous miscibility of AGO and to avoid any binding interference of DMSO 101 in the employed binding assay. It was found that the formulated NE-AGO containing 20%w/w AGO 102 appeared as a translucent mixture with white-bluish color and showed no phase separation. It was found 103 that after adding the NE-AGO with water to make a 100-fold dilution, a fast-dispersing time of the 104 formulation with less than 5 min was observed. Moreover, the small droplet size of 49 ± 2 nm of AGO was 105 obtained with a size distribution expressed as polydispersity index (PDI) of only 0.24 ± 0.01. The small 106 PDI score indicated that this formulation presented a narrow droplet size distribution. The size distribution 107 curve showed a single peak, and the peak intensity of this formulation was 99.8% (Fig. 2). Zeta potential 108 of the diluted NE-AGO was negative with a value of -14.4 ± 0.6 mV. Adsorption of hydroxyl ions in the 109 aqueous system onto the surface of the droplets may leads to slightly negative zeta potential value, and may 110 be the explanation of the negative zeta-potential of this formulation 31,32 . 111  eugenol used (i.e. 100 µM) is about 55-folds higher than that of methyl eugenol actually found in AGO 121 (3.23%). Moreover, the concentration of 1,8-cineole (42%) and 4-allylphenyl acetate (36%) used, are also 122 3.6 and 4.9 folds higher than the actual amount found in AGO. Thus, the result showed that the modulate 123 [ 3 H]muscimol binding of these compounds was similar to that of AGO at 10 µg/mL. In this study, DMSO was used for dissolving AGO and its components, therefore, DMSO was also 139 tested separately. It was shown that DMSO significantly decreased the specific binding of [ 3 H]muscimol at 140 concentrations above 3%w/w, limiting the accuracy of the compound testing above 300 µM. Thus, an 141 accurate assessment of EC50 values could not be obtained from the oil or methyl eugenol containing DMSO. 142 Moreover, DMSO at a concentration above 1%w/w dissolved the membrane while the nanoemulsion did 143 not. To improve aqueous solubility and precluding the use of DMSO, the NE-AGO was instead used for 144 testing. The binding effect of blank NE-AGO (the nanoemulsion without AGO) as a negative control at the 145 highest tested concentration was not significantly different from 100% binding, indicated that the excipient 146 itself did not significantly modulate binding levels (Fig. 3b). Instead, the EC50 value, potent agonist value 147 (pEC50  S.E.M.), and maximum binding levels (mean ± S.E.M.) of AGO was obtained in the NE-AGO 148 formulation described and found to be 391 µg/mL (3.41  0.02) and 179  10 % of the control. 149

150
blood mononuclear cells (PBMCs) as model cells for assessing the inhibitory effects of chemicals on cell 151 proliferation of human normal cells 36,37 . Thus, PBMCs were used to evaluate cytotoxicity in this study. 152 The dose-response curves showed high viability of PBMCs (> 80%) after exposure to AGO in DMSO at 153 all contact times and concentrations (Fig. 4). Normally, PBMC viability values higher than 80% are 154 regarded as safe for human use 38 . In addition, the IC50 values of AGO in DMSO at all contact times were 155 higher than 500 µg/mL. There was no significant difference in the concentration and contact time of AGO 156 in DMSO on the viability of PBMCs among the groups exposed to AGO in DMSO for 1, 3, 6, 12, and 24 157 h (p < 0.05). However, the toxicity profile of PBMCs exposed to AGO in DMSO for 48 h showed 158 concentration-dependency (p < 0.01). These results indicated that AGO had no cytotoxic activity on 159 PBMCs. The cell viability was higher than 90% when the blank NE-AGO at a final concentration of 500 160 µg/mL was used, indicating the non-toxicity of the blank NE-AGO. However, further studies are required to determine whether the pharmacokinetic characteristics of the AGO 222 and the three main constituents; 1,8-cineole, 4-allylphenyl acetate, and methyl eugenol, will influence its 223 acceptability using this essential oil to be a new local anesthetic in human. 224 225

226
Among AGO and its three main compounds, methyl eugenol, 1,8-cineole, and 4-allylphenyl acetate, it can 227 be concluded that the possible anesthetic mechanism of action of AGO and methyl eugenol is associated 228 with GABAA receptor modulation. The activity of 1,8-cineole and 4-allylphenyl acetate is not due to 229 GABAA receptor modulation and might instead be related to other mechanisms. AGO is well-tolerated by 230 human cells. Nanoemulsion is a good delivery system for AGO. NE-AGO might be useful as an alternative 231 anesthetic nanoformulation for further studies in humans. 232 280°C, respectively. The oven temperature was 70°C. The sample was held isothermally for 3 min and the 256 temperature was then increased at 3°C/min to 188°C, and then at 20°C/min to 280°C, followed holding for 257 3 min. Helium was used as carrier gas at a flow rate of 1 mL/min. The experiments were performed in 258 triplicate with at least three independent experiments. 259

Preparation and characterization of NE-AGO. NE-AGO composed of 20%w/w AGO, 10%w/w 260
Tween 80, and 70% w/w water was prepared according to a method previously described as it was reported 261 that NE-AGO formulated by this method was chemically stable when stored at various temperatures (4°C, 262 20°C, and 40°C) for several weeks (0, 4, 8, 12 weeks) 7 . Briefly, the aqueous phase composed of Tween 80 263 and water was mixed using a vortex mixer for 5 min. The oil phase was composed of AGO. The water 264 phase was added to the oil phase under stirring at 50°C for 5 min before subjecting to a high-speed stirring 265 of 16,000 rpm using an Ultra-Turrax T25 (Janke and Kunkel GmbH, Germany) for 5 min and passed 266 through a high-pressure homogenizer (Avestin Inc., Canada) under a pressure of 10,000 psi for 7 cycles at 267 room temperature. 268 The droplet size, size distribution, and zeta potential of the obtained NE-AGO were determined using 269 dynamic light scattering by photon correlation spectroscopy (Zetasizer Nano ZS, Malvern Instruments Ltd., 270 UK). The PDI value indicates the width of the size distribution. The size measurements were obtained by 271 averaging at least ten measurements at a fixed angle of 173° at 25°C. in the presence of 1 mM GABA whereas 100 µM diazepam was used as a control for positive modulation. 286 After incubation for 1 h at 0-4C, the binding reaction was terminated by rapid filtration through GF/C 287 unifilters (PerkinElmer) using a 96-well Packard FilterMate cell harvester, followed by three successive 288 washes with ice-cold binding buffer, the addition of Microscint scintillation fluid (PerkinElmer) and 289 quantification of the filter-bound radioactivity in a Packard TopCount microplate scintillation counter. The 290 experiments were performed in triplicate and repeated in at least three independent experiments. Data 291 analysis was performed using GraphPad Prism 7.0b (GraphPad Software Inc, La Jolla, CA, USA). Counts 292 per min values were converted to specific binding by subtracting non-specific binding. For modulation 293 curves, data were fitted by non-linear regression analysis using the equation for sigmoidal concentration-294 response with variable slope as follows (1): 295 where Y is the response, X is the logarithm of the concentration, Top and Bottom are the plateaus in same 296 units as Y, and log IC50 is the concentration giving a response halfway between Bottom and Top. The Hill-297 Slope is the steepness of the curve. All data were determined in triplicate and repeated in at least three 298 independent experiments (as denoted in the figure legends The research presents no more than minimal risk of harm to subjects and involves no procedures. Briefly, 305 blood samples were collected by venipuncture from healthy volunteers and transferred into 15 mL of 306 heparin-coated test tubes. Blood was diluted at a 1:1 ratio (v/v) with 0.1 M PBS and layered onto 307 Lymphoprep at a volume ratio of 3:1. PBMCs were collected after centrifugation at 1000 × g for 30 min 308 and then washed 3 times with PBS. The PBMCs were resuspended in complete RPMI 1640 culture medium 309 supplemented with 10% FBS, 100 unit/mL of penicillin, 100 µg/mL of streptomycin, and 1 mM of L-310 glutamine. The PBMCs were seeded in a 96-well tissue culture plate (1×10 5 cells/well) and incubated at 311 37°C, 5% CO2 atmosphere, and 95% relative humidity for 24 h. After the incubation, various concentrations 312 of AGO or blank NE-AGO in DMSO (15-500 µg/mL) in a complete RPMI 1640 culture medium (100 µL) 313 were added into each well and incubated for another 1, 3, 6, 12, 24, and 48 h for AGO and 48 h for blank 314 NE-AGO. A mixture composed of 0.5% v/v DMSO in a complete RPMI 1640 culture medium was used as 315 a vehicle control. MTT stock dye solution (5 mg/mL MTT dye in PBS) was added to each well (15 µL) 316 after removal of 100 µL of the medium, and the plate was incubated at 37°C in a 5% CO2 atmosphere. After 317 4 h, the supernatant was removed, followed by the addition of DMSO (200 µL) to each well and mixed 318 thoroughly to dissolve the dye crystals. Absorbance was measured using an AccuReaderTM M965/965+ 319 microplate reader (Metertech Inc., Taiwan) at 570 nm with a reference wavelength of 630 nm. All 320 experiments were performed in triplicate and at least three independent experiments confirming the data. 321 The percent of cell viability was calculated using the following equation (2): 322 % Cell viability = (MAtest/MAcontrol) × 100 (2) where MAtest and MAcontrol are mean absorbance in test wells and mean absorbance in vehicle control 323 wells. 324 Statistical analysis. The data are presented as mean ± S.E.M. Statistical evaluation of cytotoxicity study 325 was performed by a one-way ANOVA followed by Tukey's posthoc or Dunnete's test where p < 0.05 was 326 considered to indicate significant differences. 327