Chemistry of spices: bornyl 4-methoxybenzoate from Ferula ovina (Boiss.) Boiss. (Apiaceae) induces...

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ISSN 2042-6496

Food & FunctionLinking the chemistry and physics of food with health and nutrition

www.rsc.org/foodfunction Volume 2 | Number 5 | May 2011 | Pages 215–280

COVER ARTICLESchini-Kerth et al.Evaluation of diff erent fruit juices and purees and optimization of a red fruit juice blend 2042-6496(2011)2:5;1-7

Food & FunctionView Article OnlineView Journal

This article can be cited before page numbers have been issued, to do this please use: N. S. Radulovi, D. Zlatkovic, P.Randjelovic, N. Stojanovic, S. Novakovic and H. Akhlaghi, Food Funct., 2013, DOI: 10.1039/C3FO60319A.

http://dx.doi.org/10.1039/c3fo60319a

http://pubs.rsc.org/en/journals/journal/FO

1

Chemistry of spices: Bornyl 4-methoxybenzoate from Ferula ovina (Boiss.) Boiss. (Apiaceae) 1

induces hyperalgesia in mice 2

3

Niko S. Radulovića , Dragan B. Zlatkovića, Pavle J. Randjelovićb, Nikola M. Stojanovićc, 4

Slađana B. Novakovićd, Hashem Akhlaghie 5

6

aDepartment of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 7

33, RS-18000 Niš, Serbia 8

bDepartment of Physiology, Faculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 9

81, RS-18000 Niš, Serbia 10

cFaculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 81, RS-18000 Niš, Serbia 11

dVinča Institute of Nuclear Sciences, Laboratory of Theoretical Physics and Condensed Matter 12

Physics, University of Belgrade, P.O. Box 522, RS-11001 Belgrade, Serbia 13

eDepartment of Basic Sciences, Islamic Azad University, Sabzevar Branch, Sabzevar, Iran 14

15

Correspondence: 16

Niko S. Radulović, Department of Chemistry, Faculty of Science and Mathematics, University of 17

Niš, Višegradska 33, RS-18000 Niš, Serbia 18

Tel.: +38118533015; fax: +38118533014. 19

E-mail address: [email protected] (N.S. Radulović). 20

21

Keywords: Ferula ovina, essential oil, bornyl p-methoxybenzoate, analgesia, hyperalgesia 22

23

Page 1 of 33 Food & Function

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View Article OnlineDOI: 10.1039/C3FO60319A


2

Table of contents 24

25

26

27

28

Ferula ovina essential oil exerted strong peripheral and moderate central analgesic activities in 29

mice. Bornyl 4-methoxybenzoate, a new natural compound induces hyperalgesia having 30

implications on its flavor. 31

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Abstract 32

Ferula ovina (Boiss.) Boiss. is a traditional Iranian flavoring agent used as an ingredient of 33

spices and condiments. 34

Methods and results: GC-MS analyses of F. ovina aerial parts’ essential oil revealed the presence 35

of a number of rare aromatic esters of monoterpenic alcohols. The structures of these esters were 36

corroborated by synthesis, and one of them, bornyl 4-methoxybenzoate, turned out to be a new 37

natural compound. The antinociceptive activities of the oil and the new ester were assessed in 38

mice using several different laboratory models. The oil exerted strong peripheral and moderate 39

central analgesic activities. Surprisingly, mice treated with bornyl 4-methoxybenzoate had an 40

increased sensitivity to the noxious stimulus compared to that of the control group. A dynamic 41

hot plate test was used to demonstrate that bornyl 4-methoxybenzoate induces hyperalgesia and 42

not allodynia. 43

Conclusion: Essential oil constituents impart this spice with both antinociceptive and 44

hyperalgesic potentially flavor-related properties. 45


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1 Introduction 46

Ferula L. is a genus of about 170 species of Umbelliferous flowering plants native to the 47

Mediterranean region and east to central Asia. Many species of this genus have been historically 48

used for culinary and medical purposes. Assafoetida, an oleogum resin obtained from the 49

rhizome of F. asa-foetida and several other Ferula species has been commonly used in Nepal as 50

a flavoring agent in many curry recipes1. Galbanum, a gum exudate collected from F. 51

galbaniflua and F. rubricaulis is used as a candy additive2, and it has also been used for the 52

treatment of intestinal disorders3. Bulgarian researchers have recently shown that Ferula spp. 53

represent a new plant source of propolis in Malta4. Ferula orientalis is used in Turkey for 54

flavoring pickles5. Herbal products based on F. hermonis are sold as an aphrodisiac dietary 55

supplement in the United States6. 56

The Iranian flora comprises 30 species of this genus, one of them being F. ovina (Boiss.) Boiss. 57

The resin obtained by cuts on the stem is a traditional flavoring agent and is used as an ingredient 58

of spices and condiments7. This plant is also very appreciated for its medicinal use. The aqueous 59

extract of F. ovina was found to possess antispasmodic, anticholinergic and smooth muscle 60

relaxant activities8. Although the essential oil composition of F. ovina was previously reported9, 61

10, the potential pharmacological activity of this oil remains unknown up to date. 62

Since a meticulous knowledge of the properties, both beneficial and adverse, of each and every 63

food ingredient is of vital importance, F. ovina deserves a more detailed investigation of its 64

chemical and pharmacological profile. Having this and the ethnopharmacological use of this 65

plant species in mind, we decided to perform detailed chemical composition analysis of the 66

essential oil of aerial parts of F. ovina collected in Iran. 67


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An important motivation for this study was also the fact that plants from this genus are known to 68

synthesize esters of substituted benzoic acids (vanillic, p-hydroxybenzoic and p-methoxybenzoic 69

acids), e.g. ferutinin (ferutinol p-hydroxybenzoate), teferin (ferutinol vanillate) and jaesekanadiol 70

p-methoxybenzoate4,11. Previous reports on the essential oil of F. ovina surprisingly lack these 71

esters although volatile bornyl 4-hydroxybenzoates and vanillates, e.g. tschimganin and 72

tschimgin, were isolated from taxa belonging to this genus12. 73

Esters of these acids are known to activate transient receptor potential channels (TRP) that are 74

implicated in pain perception13. Targeting TRP channels has been an important strategy in the 75

search of novel analgesics14. Also, the TRP family channel activity (such as TRPV1, TRPV3, 76

TRPA1, TRPM5 and TRPM8) can contribute to aspects of taste and can modify or initiate 77

signals that integrate with other signals from olfactory and taste receptors, either within the 78

epithelium, between epithelia and specialized cells, or at the level of neuronal input to the CNS 79

15. 80

A first compositional screening of the oil in our hands done by GC-MS revealed the presence of 81

several monoterpenic esters of aromatic acids. We decided to pursue with a detailed analysis of 82

the oil in hope of finding new and pharmacologically interesting esters possessing 83

antinociceptive activity. 84

85

2 Materials and methods 86

87

2.1 General experimental procedures 88

89


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Acetylsalicylic acid (ASA) (Aspirin, Bayer), morphine (Hoffmann La Roche), capsaicin (Sigma) 90

and the tested substances were freshly dissolved, in a maximum volume of 0.3 ml per animal, 91

prior to usage. All other chemicals were obtained from Aldrich (USA) or Fluka (Germany) and 92

were used as received, with the exception of the solvents that were additionally purified by 93

distillation. 1H and 13C NMR spectra were recorded on a Bruker Avance II spectrometer (Bruker, 94

Germany) operating at 400 and 100.6 MHz, respectively. 2D experiments (1H–1H COSY, 95

HSQC, HMBC and NOESY) were run on the same instrument with the usual pulse sequences. 96

All NMR spectra were measured at 25 °C in CDCl3 with TMS as internal standard. 97

Microanalysis of carbon and hydrogen were carried out with a Carlo Erba 1106 microanalyser; 98

their results agreed favorably with the calculated values. 99

Preparative medium-pressure liquid chromatography (MPLC) was performed with a pump 100

module C-601 and a pump controller C-610 Work-21 pump (Büchi, Switzerland) and was 101

carried out on pre-packed column cartridges (40 mm×75 mm), Silica-gel 60, particle size 102

distribution 40–63 μm, Büchi (Switzerland). 103

Precoated Al silica gel plates (Merck, Kieselgel 60 F254, 0.2 mm) were used for analytical TLC 104

analyses. The spots on TLC were visualized by UV light (254 nm) and by spraying with 10% 105

(w/v) solution of phosphomolybdic acid in ethanol followed by heating. 106

The GC-MS analyses were performed on a Hewlett-Packard 6890N gas chromatograph equipped 107

with a fused silica capillary column DB-5MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film 108

thickness 0.25 μm, Agilent Technologies, USA) and coupled with a 5975B mass selective 109

detector from the same company. The injector and interface were operated at 250 °C and 320 °C, 110

respectively. Oven temperature was raised from 70 to 290 °C at a heating rate of 5 °C/min and 111

then isothermally held for 10 min. As a carrier gas helium at 1.0 ml/min was used. The samples, 112


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1 μl of the solutions in diethyl ether (1 mg in 1 ml of Et2O), were injected in a pulsed split mode 113

(the flow was 1.5 ml/min for the first 0.5 min and then set to 1.0 ml/min throughout the 114

remainder of the analysis; split ratio 40:1). The mass selective detector was operated at the 115

ionization energy of 70 eV, in the 35–650 amu range and scanning speed of 0.32 s. GC (FID) 116

was carried out under the same conditions and using the same column as for GC-MS. The 117

percentage composition was computed from FID chromatogram peak areas without the use of 118

correction factors. The linear retention indices were calculated relative to the retention times of 119

C8–C24 n-alkanes on the DB-5MS column16. 120

121

2.2 Single-crystal X-ray experiment 122

123

The diffraction data were collected from a weakly diffracting colorless crystal mounted on 124

Agilent Gemini S diffractometer17. Crystal data: C18H24O3, Mr = 288.4, orthorhombic, space 125

group P212121, a = 11.0043(18), b = 11.212(3), c = 13.436(2) Å, V = 1657.8(6) Å3, Z = 4, Dc = 126

1.155g cm–3, F(000) = 624, μ = 0.614 mm–1, λ = 1.54184 Å, T = 293K. Experimental and 127

structure refinement details are given in Supplementary material (Tables S2-S3). 128

129 130

2.3 Plant material 131

132

Plant material (aerial parts of F. ovina) used in this study was collected from natural populations 133

growing at an altitude of 1650 m, on June 12th, 2011, in Sabzevar region (northeastern Iran). 134

135

2.4 Essential oil extraction 136

137


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Plant material (air dried to a constant weight, 2 batches of about 500 g) was subjected to 138

hydrodistillation with approximately 2 L of distilled water for 2.5 h using the original Clevenger-139

type apparatus. The obtained oils were separated by extraction with diethyl ether, dried over 140

anhydrous magnesium sulfate and immediately analyzed. The yield of the oil was 1.5%. 141

142

2.5 General procedure for the microsynthesis of esters 143

144

Solutions of an appropriate carboxylic acid (benzoic, 4-hydroxybenzoic, 4-methoxybenzoic or 145

vanillic acids, 0.1 mmol), alcohol (borneol, isoborneol, geraniol, menthol, nerol or endo-146

fenchol), 4-dimethylaminopyridine (DMAP, 0.5 mg), and N,N'-dicyclohexylcarbodiimide (DCC, 147

0.11 mmol) in 1.0 ml of dry CH2Cl2 were prepared, vortexed for a few seconds and stored 148

overnight in closed GC vials. The solutions were analyzed without any additional treatment. 149

150

2.5.1 Synthesis of bornyl p-methoxybenzoate (BMB) 151

Borneol (1 mmol), 4-methoxybenzoic acid (1 mmol), DMAP (5 mg) and DCC (1.1 mmol) in dry 152

CH2Cl2 (10 mL) were stirred overnight at room temperature protected from atmosphere moisture 153

by a CaCl2 guard tube. The precipitated urea was filtered off and the filtrate was concentrated in 154

vacuo. The ester was purified by MPLC chromatography using silica gel and 1:1 (v/v) mixture of 155

hexane and CH2Cl2 as the eluent. The purity of the ester fractions was checked by TLC (10%, 156

w/v, phosphomolybdic acid in ethanol) and GC-MS. 157

1H NMR (CDCl3, 200 MHz) δH: 0.91 (s, 3H), 0.92 (s, 3H), 0.97 (s, 3H), 1.11 (dd, J = 3.4 and 158

13.8 Hz), 1.20–1.48 (m, 2H), 1.60–1.90 (m, 2H), 2.00–2.25 (m, 1H), 2.46 (dddd, J = 13.8, 9.9, 159


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4.6, 3.4 Hz, 1H), 3.86 (s, 3H), 5.09 (ddd, J = 9.9, 3.4, 2.0 Hz, 1H), 6.94 (dd, 8.9, 2.3 Hz, 2H), 160

8.20 (dd, 8.9, 2.3 Hz, 2H). 161

13C NMR (CDCl3, 50 MHz) δC: 13.6, 18.9, 19.7, 27.4, 28.1, 36.9, 45.0, 47.8, 49.1, 55.4, 80.1, 162

113.6, 123.0, 131.5, 163.0, 166.7. 163

EI-MS m/z (rel. intensity, %): 135 (100.0), 136 (14.4), 77 (7.7), 92 (6.0), 288 (4.6, M+), 41 (4.1), 164

93 (3.8). 165

166

2.6 Pharmacological procedures 167 168 169 2.6.1 Animals and treatment 170

The essential oil (FO), BMB, acetylsalicylic acid (ASA), morphine and capsaicin were freshly 171

dissolved, in a maximum volume of 0.3 ml per animal, prior to usage. Male BALB/c mice (20±5 172

g) 5 weeks old were obtained from the Vivarium of the Institute of Biomedical Research, 173

Medical Faculty, University of Niš, Niš, Serbia. Animals were housed in groups of 6 males 174

under standard laboratory conditions: 12 h light/dark cycle at 22±2 °C, with food and water 175

available ad libitum. Animals were acclimatized to the laboratory environment for at least 12 h 176

before testing (fasted, though still allowed free access to water). Six experimental and two 177

control (positive and negative) groups, with 6 animals per group, for writhing, hot plate and tail 178

flick experiments were used. In a dynamic hot plate (DHP) test additional three groups of 179

animals were used and the substances were applied topically on the animal paws (see a detailed 180

description in DHP test section). For the other tests, the essential oil of F. ovina (FO) and the 181

new ester (BMB) were administered intraperitoneally (i.p.) one hour prior to the experiments at 182

doses of 50, 100 and 200 mg/kg to six groups, whereas two more groups received morphine at 5 183

mg/kg or acetylsalicylic acid at 200 mg/kg (ASA; used as the positive control only in the 184


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abdominal writhing test) and vehicle (olive oil) at 10 ml/kg. All experimental procedures were 185

conducted in accordance with the principles of care and use of laboratory animals in research 186

(EEC Directive of 1986; 86/609/EEC) and were approved by the local Ethics committee (01-187

10204-05). 188

189

2.6.2 Abdominal writhing test 190

The method for abdominal writhing adopted here was previously described by Radulović et al. 191

16. Briefly, after the administration of acetic acid (1%, v/v, i.p.) the number of writhes was 192

counted for 20 min. A writhe was defined as a contraction of the abdominal muscles 193

accompanied by an extension of the forelimbs and elongation of the body. The % of inhibition 194

was calculated as follows = 100 × [number of writhes (control) − number of writhes 195

(test)]/number of writhes (control). 196

197 2.6.3 Hot plate test 198

Mice were tested according to the method described by Radulović et al.16. Animals were placed 199

on a hot plate apparatus set at 55±1 °C. The behavior of animals considered as a sign of pain was 200

licking the fore- and hind-paws or jumping off the hot-plate. The cut-off time was fixed at 20 s to 201

avoid skin damage. Baseline (BL) was considered as the mean of reaction time obtained at 60 202

and 30 min before administration of the substances, vehicle or morphine and defined as the 203

normal reaction of animals to thermal stimuli. Increase in baseline (%) was calculated by the 204

formula: [(reaction time × 100)/BL] − 100. Antinociception was quantified as area under the 205

curve (AUC) of responses and was calculated as a sum of AUC1 = 15 × IB[(min15)/2 + (min30) 206

+ (min45) + (min60)/2] and AUC2 = 30 × IB[(min60)/2 + (min90) + (min120)/2] where IB is 207


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the increase in baseline (in %). % of inhibition for morphine and each dose of FO and BMB were 208

calculated as follows: % of inhibition=AUC/max; where max = [(20 × 100)/BL − 100] × 105. 209

210

2.6.4 Tail immersion test 211

The lower 5 cm portion of the tail was immersed in a beaker of water maintained at 50.0±0.5 °C 212

18. The time in seconds for tail withdrawal from the water was taken as the reaction time, with a 213

cut-off time of immersion set at 15 s. The latency of tail withdrawal was measured at 15, 30, 45, 214

60, 90 and 120 min after the administration of FO, BMB, morphine and vehicle. % of inhibition 215

was calculated as described in hot plate test section. 216

217

2.6.5 Dynamic hot plate (DHP) test 218

This method was described recently by Yalcin et al.19. DMSO (negative control, 100%), 219

capsaicin (positive control, 10 mM dissolved in DMSO) or BMB (10 mM dissolved in DMSO) 220

were topically applied with cotton-tipped applicators on animal paws, 15 min before they were 221

placed on the dynamic hot plate (DHP). Temperature of the hot plate was 30.0±0.1 C when the 222

mouse was placed on it and increased to 43 C with a rate of 1 C/min. During each degree 223

interval, hind paw lickings, escape behavior (jumps) and rearing were scored, as was proposed in 224

the original publication19. 225

226

2.6.6 Statistical analysis 227

Results were expressed as the mean ± SD. Statistically significant differences were determined 228

by one-way analysis of variance (ANOVA) followed by Tukey´s post hoc test for multiple 229


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comparisons (Graphpad Prism version 5.03, San Diego, CA, USA). Probability values (p) less 230

than 0.05 were considered to be statistically significant. 231

232

3 Results and discussion 233

234

3.1 Chemistry 235

236

3.1.1 Composition of the essential oil 237

GC/MS analyses indicated that the essential oil of F. ovina in our hands consisted largely of 238

monoterpene hydrocarbons (α-pinene, limonene and myrcene were the most abundant 239

constituents and represented 17.8, 14.3 and 13.0% of the oil respectively, see Table 1). Such a 240

composition is chiefly in agreement with those that were previously published for this species9,10. 241

The analyses also revealed the presence of a number of aromatic esters eluting from the GC 242

column at RI values above 1700. Carboxylic acid portion of the esters was straightforwardly 243

inferred from their mass spectra, e.g. a characteristic fragment ion of all benzoate esters was 244

located at m/z 105 (C6H5CO+). Similarly, methoxybenzoates, hydroxybenzoates and 245

hydroxymethoxybenzoates (vanillates) had prominent fragments at m/z 135, 121 and 151, 246

respectively. In the case of all esters, the molecular ions and the base peaks (ArCO+) differed in 247

153 a.m.u., indicating that the esterifying alcohols were monoterpenols of the formula C10H18O 248

(MW 154). Their MSes were not sufficiently informative to provide a means to differentiate 249

between esters of monoterpenols of the same molecular formula. The possible identity had to be 250

checked by synthesis and GC co-injection of the synthesized esters with the oil of F. ovina. We 251

assumed that the monoterpenic alcohols (borneol, endo-fenchol, geraniol and nerol, see Table 1) 252


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already detected in our sample, in the form of free alcohols and/or acetates, also esterified the 253

mentioned aromatic acids. Even though we could not deduce the right regioisomers of 254

hydroxybenzoic and methoxybenzoic acids from the MSes alone, we decided to use their para-255

isomers in the preparation of the esters since p-methoxybenzoates and p-hydroxybenzoates were 256

already known to be secondary metabolites of the taxa belonging to the genus Ferula. All 257

possible combinations of esters were prepared by Steglich coupling and GC co-injected with the 258

oil sample (Table 2). We also prepared menthyl esters of the four acids in order to probe whether 259

it is possible to recognize the carboxylic part of aromatic acid esters based on their base peaks 260

for any aliphatic monoterpene ester (no matter the total double bond equivalents). 261

Out of 24 compounds prepared, the GC co-injection confirmed the presence of 6 esters in the oil 262

(bornyl esters of all four acids, as well as the vanillate and benzoate of endo-fenchol). According 263

to a thorough literature survey, bornyl 4-methoxybenzoate represents a new natural compound. 264

An additional amount of this ester was prepared for the complete structural assignment and for 265

the pharmacological experiments. Its structure was corroborated by 1D- and 2D-NMR 266

experiments (details are contained in the Supplementary material, Figures S1-S7, Table S1) and 267

X-ray crystallographic analysis. 268

269

3.1.2. X-ray crystallographic analysis of bornyl 4-methoxybenzoate 270

271

Despite the poor crystal quality, single-crystal X-ray analysis was able to confirm the expected 272

structure of the aromatic acid ester. The established molecular structure of bornyl 4-273

methoxybenzoate is given in Figure 1. Due to severe disorder in the bornyl moiety, bond lengths 274

and angles in this part of the molecule could not be described in detail. The geometry of the 275


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methoxybenzoate moiety is within the expected ranges (see Supporting Information, Table S2). 276

The carboxylic part of the ester, C8/O2/O3 [C8–O2 = 1.210(6), C8–O3 = 1.315(6) Å], is only 277

slightly twisted with respect to the phenyl ring forming a dihedral angle of 4.0(8)º. The torsion 278

angle O2–C8–O3–C9 of 2.2(10)° describes the cis position of the bornyl moiety with respect to 279

the carboxylic O2. As evident from the torsion angle C1–O1–C2–C3 of –2.0(9)°, the methoxy 280

group only slightly deviates from the plane of the phenyl ring. 281

Regardless of the presence of three oxygen acceptors and a number of C–H donor groups, 282

the crystal structure does not contain any significant C–H…O interactions. The shortest contact 283

involving the carboxylic oxygen acceptor is C1–H1c…O2i (H…O = 2.68 Å and C–H…O = 284

130º; i = x–1/2, y–1/2, z). The mutual orientation of the bornyl C–H fragments and of the phenyl 285

ring suggests that C–H…π interactions could have an important role in structure stabilization 286

(see Supporting Information, Figure S9). 287

288

(Figure 1 should come around here) 289

290

3.2 Pharmacological investigations 291

292

Antinociceptive properties of F. ovina essential oil and bornyl 4-methoxybenzoate were assessed 293

in three laboratory nociception models, corresponding to two different types of noxious stimuli: a 294

thermal one and a chemically-induced pain stimulus20. 295

296

3.2.1 Chemically-induced pain model 297

298


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The oil exerted, in a dose-dependent manner, a strong antinociceptive activity, being most 299

prominent in inhibition of abdominal writhings induced by acetic acid (Fig. 2). Animals treated 300

with F. ovina oil, in a dose of 200 mg/kg, showed a decreased number of writhings (~92%) 301

comparable to the effect of the standard drug in the same dosage (ASA at 200 mg/kg, ~90%) (see 302

Supporting Information, Table S4). Some of the major oil constituents (-pinene, limonene and 303

myrcene, Table 1) are known to possess certain analgesic potential21 providing at least a partial 304

explanation for the noted activity of the oil. 305

Alongside with the oil, a minor constituent – the new ester, bornyl 4-methoxybenzoate – was 306

tested. It showed almost the same effect (83%) as the oil itself in 200 mg/kg (Fig. 2, Table S4). 307

In the abdominal writhings model, a cascade of events following the injection of acetic acid – the 308

release of prostaglandins and sympathetic nervous system mediators, activation and sensitization 309

of peripheral chemosensitive nociceptive receptors and the onset of inflammation – results in 310

abdominal contortions22. A possible explanation for the observed pain threshold elevation for the 311

two tested substances might be that they act as inhibitors of prostaglandin synthesis. However, a 312

false positive result is produced by muscle relaxants, adrinergic blockers, antihistamines and 313

neuroleptics20. One might speculate that bornyl 4-methoxybenzoate possesses a myorelaxant 314

activity since the parent alcohol, borneol, is a known non-competitive inhibitor of nicotinic 315

acetylcholine receptors23. 316


318

319

3.2.2 Thermal-induced pain models 320

321


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A potential central analgesic effect of this oil was selectively evaluated in the hot plate and tail 322

immersion tests (as opposed to the abdominal writhings test where both central and peripheral 323

analgesics give positive results). The central control of pain is subject to descending modulation 324

by brainstem cell groups such as locus coeruleus/subcoeruleus and raphe complex and the hot 325

plate test is considered to be a selective test for drugs that affect supraspinally integrated 326

responses20. The oil showed a dose dependent increase of reaction time in the hot plate test (55±1 327

°C). The calculated percent of pain inhibition (%) in this test for the oil, in doses of 50, 100 and 328

200 mg/kg, and morphine (at 5 mg/kg) were: 13.2, 21.1, 28.4 and 72.7%, respectively (Table 329

S4). The first notable statistically significant effect of the highest dose of the oil on mice reaction 330

time during the hot plate test was 30 minutes after the oil administration (Fig. 3A.). The oil 331

exerted the strongest modulator effect on responses to thermal stimuli (maximal increase of 332

reaction time) 90 min after the administration (in all doses) and remained approximately the 333

same until the end of the experiment (Fig. 3A), whereas the 5 mg/kg dose of morphine 334

demonstrated its effect much more quickly - 15 min after the application of the drug (Fig. 3A). 335

A characteristic behavior of mice in the hot plate test that can be described as either hyperalgesia 336

or allodynia was observed in animals treated with bornyl 4-methoxybenzoate (BMB) (Fig. 3B). 337

While hyperalgesia is defined as an increased pain sensitivity that can include both a decrease in 338

threshold and an increase in suprathreshold response, allodynia is pain in response to a non-339

nociceptive stimulus24. A decrease in mice reaction time to thermal stimuli for all doses at some 340

point in time after the application of BMB can be seen in Fig. 3B. Only the highest dose 341

produced a temporary analgesic effect (15-45 min) after which the animals developed 342

hyperalgesia/allodynia as well. 343


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Limonene, one of the major oil constituents, failed to demonstrate any effect on supraspinal 344

nociception25, whereas our oil had an effect on supraspinal nociception. Myrcene, comprising 345

some 13% of the oil, was previously shown to stimulate the release of endogenous opioids by 346

mediation of α2-adrenoreceptors26. Hence, we can speculate that myrcene contributes to the 347

observed activity of the oil but other minor or major constituents might have an effect as well. 348


The tail immersion test is predominantly based on a spinal reflex, which could also involve 350

higher neural structures, and represents a selective test for the evaluation of central analgesic 351

activity20. The oil showed relatively weak antinociceptive activity when compared to the control 352

(Fig. 4A). Only the highest dose of the oil managed to increase the time latency (Fig. 4A) from 353

the very beginning of test (15 min after the application). For the dose of 200 mg/kg, the 354

maximum response (longest time required to react) was 60 min and the effect faded away after 355

120 min following the injection. In this time period for the animals that received the highest dose 356

of the oil, as well as during the 90-120 min period for the experimental group treated with 100 357

mg/kg of the oil, there were no statistically significant differences found between the 358

abovementioned reaction times (at 60, 90 and 120 min) and those of the morphine treated 359

animals (Fig. 4A). The determined percents of inhibition in this test were 38.5, 42.8 and 55.8 % 360

for the three doses of the oil, in an ascending dosage order, and 80.6% for morphine (Table S4). 361

The increased sensitivity to the noxious stimulus was not observed in this test for bornyl 4-362

methoxybenzoate (Fig 4B). In this test the activity of this compound was an analgesic one. The 363

substance exerted mild antinociceptive activity with the peak activity at the doses of 100 and 200 364

mg/kg (see % of inhibition in Table S10) 45 min after the application. The decrease in pain 365

latency was observed continuously 60 min after the application of this ester (at the doses of 100 366


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and 200 mg/kg). The lowest dose of the methoxybenzoate (50 mg/kg) seems not to have any 367

significant antinociceptive effect 45 min after its application. 368


The results obtained for bornyl 4-methoxybenzoate using the hot plate and the tail immersion 370

tests indicated opposite effects. However, although both assays evaluate central control of pain 371

and thermal nociception, these finding are only seemingly conflicting. One possible explanation 372

may lay in the fact that the tail-flick response is primarily spinally mediated and that in the hot 373

plate response (paw licking or jumping) supraspinal sites play a key role27. Since bornyl 4-374

methoxybenzoate is effective only in the tail flick test, the antinociceptive activity is probably 375

mediated at the spinal level. The different activity of this compound in the two thermal-induced 376

pain tests could be due to diverse abundances of opioid receptors subtypes located supraspinally 377

(µ1, κ3, δ1, σ2) and at spinal level (µ2, κ1, δ2)28. However, further studies using specific opioid 378

receptor subtype antagonists are needed to test this hypothesis and determine the exact 379

mechanism of action of this compound. 380

381

3.2.3 Dynamic hot plat test 382

383

Prompted by the results of the hot plate test we subjected mice treated with bornyl 4-384

methoxybenzoate to another, relatively new, more specific one (a dynamic hot plate test, DHP) 385

that would allow us to discriminate allodynia from hyperalgesia19. The main reaction of 386

C57Bl/6J mice submitted to DHP observed in Yalcin’s19 work defined as the nociceptive 387

reaction was an escape behavior - jumps. In our study, no jumps were observed for the tested 388

animals (male BALB/c mice) even in the capsaicin-sensitized animals at high temperatures (data 389


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not shown). The only increased parameters noted in our experiments were rearings and paw 390

lickings (Figs S10 and 5). However, rearings can also be regarded as a nonspecific parameter 391

since even at low temperatures (30 C) the animals from the negative control group (DMSO) 392

showed a slight increase in the number of rearings. These rearings, both in the middle or up the 393

wall of the apparatus, can be classified as exploratory ones, as in the open field test, rather than 394

the reaction to the thermal stimulus (although when the animals were exposed to higher 395

temperatures (38-39 C) or were pretreated with capsaicin a dramatic increase of rearings was 396

noted (Fig S10)). Thus, we decided to use only paw lickings as the specific reaction of BALB/c 397

mice in DHP. An increase in the number of paw lickings was notable immediately after the 398

placement of the animals previously sensitized with capsaicin onto DHP (Fig. 5). On the other 399

hand, the animals treated with DMSO or bornyl 4-methoxybenzoate alone were licking their 400

paws much less frequently than the capsaicin-sensitized animals at the same temperature (Fig. 5). 401

According to the results of the previous tests and finally of the DHP test, we can conclude that 402

bornyl 4-methoxybenzoate produces hyperalgesia, and not allodynia. This activity of the new 403

ester might be important for the flavor properties of the compound and the plant species/spice as 404

well. Previous studies have shown that the omnipresent food monoterpenes, such as borneol, 405

camphor, carvacrol, eugenol and thymol, have an influence on transient receptor potential 406

vanilloid ion channels (TRPV)15. Generally, the activation of TRP channels depolarizes cells 407

from the resting membrane potential and shortens action potential duration. The mentioned 408

monoterpenes were shown to activate and sensitize TRPV3 receptors, by increasing the level of 409

cellular Ca2+ or by activation of G protein–coupled receptor that are mostly expressed in the skin 410

(keratinocytes), nasal and oral cavity (tongue and palate). The presence of these receptors in oral 411

cavity implies that they may be potential targets for flavor actions of plant derived compounds 412


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(e.g. carvacrol from oregano)29. Since TRPV3 receptors are warmth activated on temperatures 413

22-40 °C, they are predominantly activated during a DHP test. Warm to hot temperatures or the 414

penetration of lipophilic compounds (such as monoterpenes) through keratinized, non-sensory 415

layers and activation of TRPV3 on keratinocytes leads into releases of ATP to signal sensory 416

termini and thereby cause the sensation of warmth or heat30. A proposed mechanism of action of 417

camphor, whose structure is related to the bornyl moiety of the new ester, is through the 418

activation of TRPV3. Camphor also acts on TRPV1 receptors, by desensitizing them, and on 419

TRPA1 receptors by inhibiting them and thus causing antinociception31. 420


422

4 Concluding remarks 423

Several monoterpenic esters of aromatic carboxylic acids have been identified as constituents of 424

the essential oil of the aerial parts of F. ovina used traditionally in Iran as an ingredient of spice 425

and condiments. One of them, bornyl 4-methoxybenzoate turned out to be a new natural 426

compound whose structure was confirmed by synthesis and elucidated by spectral means and x-427

ray crystallographic analysis. Tests were performed to assess the antinociceptive activity of the 428

entire oil and the new compound. The results indicated that the oil of F. ovina possessed 429

analgesic properties which were mediated via central and peripheral inhibitory mechanisms. The 430

activity of the bulk oil could most certainly be connected to a variety of compounds constituting 431

the oil. However, the activity of bornyl 4-methoxybenzoate differed markedly from that of the 432

oil. Mice treated with bornyl 4-methoxybenzoate had an increased sensitivity to the noxious 433

stimulus compared to that of the control group. A dynamic hot plate test was used to demonstrate 434

that bornyl 4-methoxybenzoate induces hyperalgesia and not allodynia. Hyperalgesia might hint 435


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a possible interaction with transient receptor channels (TRPV3) in the oral cavity important for 436

the flavor properties of this spice but this still needs confirmation from further studies. Also the 437

structural similarity of the new ester with monoterpenes (borneol and camphor) that are known to 438

activate these channels will guide us in future investigations. 439

440

Acknowledgments 441

This work was supported by the Ministry of Education, Science and Technological Development 442

of Serbia [Project No. 172061]. 443

444

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Table 1. Percentage composition of Ferula ovina essential oil 525

RI a) Compound % b) ID c) Class d) 846 (2E)-hexenal t RI, MS, Co-GC FAD 921 tricyclene 0.7 RI, MS M 932 α-pinene 17.8 RI, MS, Co-GC M 946 camphene 6.3 RI, MS, Co-GC M 969 sabinene 0.4 RI, MS, Co-GC M 974 β-pinene 1.9 RI, MS, Co-GC M 988 myrcene 13.0 RI, MS, Co-GC M 988 dehydro-1,8-cineole 0.1 RI, MS MO

1002 α-phellandrene 0.1 RI, MS, Co-GC M 1008 δ-3-carene 0.5 RI, MS, Co-GC M 1014 α-terpinene 0.1 RI, MS, Co-GC M 1022 p-cymene 0.4 RI, MS, Co-GC M 1025 limonene 14.3 RI, MS, Co-GC M 1025 β-phellandrene 2.1 RI, MS, Co-GC M 1026 1,8-cineole 0.0 RI, MS, Co-GC MO 1032 trans-β-ocimene 0.1 RI, MS M 1054 γ-terpinene 0.1 RI, MS, Co-GC M 1065 cis-sabinene hydrate 0.0 RI, MS MO 1083 fenchone 0.5 RI, MS MO 1086 terpinolene 0.4 RI, MS, Co-MS M 1088 methyl benzoate 0.0 RI, MS, Co-GC FAD 1090 6,7-epoxymyrcene 0.0 RI, MS MO 1095 linalool 1.1 RI, MS, Co-GC MO 1098 trans-sabinene hydrate 0.0 RI, MS MO 1111 cis-rose oxide 0.0 RI, MS MO 1118 cis-p-menth-2-en-1-ol t RI, MS MO 1123 endo-fenchol 0.2 RI, MS, Co-GC MO 1129 α-campholenal 0.1 RI, MS MO 1132 cis-limonene oxide t RI, MS, Co-GC MO 1137 (E)-epoxy-ocimene t RI, MS MO 1137 trans-limonene oxide t RI, MS, Co-GC MO 1141 veratrole t RI, MS, Co-GC FAD 1144 cis-verbenol 0.2 RI, MS, Co-GC MO 1145 isopulegol t RI, MS MO 1148 trans-verbenol 0.7 RI, MS, Co-GC MO 1151 citronellal 0.6 RI, MS, Co-GC MO


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1158 trans-pinocamphone t RI, MS, Co-GC MO 1160 pinocarvone t RI, MS MO 1172 p-mentha-1,5-dien-8-ol 0.2 RI, MS MO 1172 cis-pinocamphone t RI, MS, Co-GC MO 1175 borneol 0.2 RI, MS, Co-GC MO 1179 p-cymen-8-ol t RI, MS MO 1183 terpinen-4-ol 0.2 RI, MS, Co-GC MO 1183 cryptone t RI, MS, Co-GC MO 1196 α-terpineol 0.9 RI, MS, Co-GC MO 1204 verbenone t RI, MS, Co-GC MO 1207 trans-piperitol t RI, MS MO 1215 trans-carveol t RI, MS MO 1219 endo-fenchyl acetate 4.1 RI, MS, Co-GC MO 1225 citronellol 0.8 RI, MS, Co-GC MO 1226 cis-carveol t RI, MS MO 1229 exo-fenchyl acetate t RI, MS MO 1249 geraniol 0.3 RI, MS, Co-GC MO 1257 methyl citronellate 0.2 RI, MS MO 1283 cis-verbenyl acetate 0.1 RI, MS MO 1288 bornyl acetate 4.6 RI, MS, Co-GC MO 1324 myrtenyl acetate t RI, MS, Co-GC MO 1334 trans-carvyl acetate 0.1 RI, MS MO 1348 α-terpinyl acetate 3.0 RI, MS, Co-GC MO 1350 citronellyl acetate t RI, MS, Co-GC MO 1357 neryl acetate 0.1 RI, MS, Co-GC MO 1371 methyl 4-methoxybenzoate t RI, MS, Co-GC FAD 1374 daucene 0.1 RI, MS S 1377 geranyl acetate 0.7 RI, MS, Co-GC MO 1389 β-elemene t RI, MS, Co-GC S 1393 cis-jasmone 0.3 RI, MS, Co-GC O 1398 methyl eugenol 0.5 RI, MS, Co-GC PP 1424 trans-caryophyllene 0.5 RI, MS, Co-GC S 1424 2,5-dimethoxy-p-cymene t RI, MS O 1434 neryl acetone t RI, MS O 1452 α-humulene t RI, MS, Co-GC S 1453 (E)-β-farnesene 0.1 RI, MS S 1490 (E,E)-α-farnesene 0.1 RI, MS S 1493 β-selinene 0.1 RI, MS S 1500 α-selinene 0.2 RI, MS S


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1510 β-bisabolene 0.1 RI, MS, Co-GC S 1522 δ-cadinene t RI, MS S 1522 myristicin 9.4 RI, MS, Co-GC PP 1546 elemicin 4.7 RI, MS, Co-GC PP 1564 β-calacorene t RI, MS S 1587 caryophyllene oxide 0.1 RI, MS, Co-GC SO 1596 neryl isovalerate 0.1 RI, MS, Co-GC MO 1600 hexadecane t RI, MS, Co-GC FAD 1603 geranyl isovalerate 0.1 RI, MS, Co-GC M 1606 carotol 0.2 RI, MS SO 1629 1-epi-cubenol 0.1 RI, MS SO 1645 cubenol t RI, MS SO 1652 α-cadinol t RI, MS SO 1699 β-sinensal t RI, MS SO 1722 (E,E)-farnesol t RI, MS SO 1759 benzyl benzoate t RI, MS, Co-GC FAD 1773 endo-fenchyl benzoate t RI, MS, Co-GC MO 1884 bornyl benzoate t RI, MS, Co-GC MO 1959 palmitic acid t RI, MS, Co-GC FAD 1980 camphorene t RI, MS D 2185 bornyl 4-methoxybenzoate 0.3 RI, MS, Co-GC MO 2202 endo-fenchyl vanillate 1.1 RI, MS, Co-GC MO 2251 bornyl 4-hydroxybenzoate 0.2 RI, MS, Co-GC MO 2311 bornyl vanillate 0.8 RI, MS, Co-GC MO

Total 96.2 Monoterpenoids Hydrocarbons 58.2 Oxygenated 21.5 Sesquiterpenoids Hydrocarbons 1.2 Oxygenated 0.4 Diterpenes t Fatty acids and fatty acid-related compounds t Phenylpropanoids 14.6

Other 0.3 a) RI: retention index relative to C8–C24 n-alkanes on a DB-5MS column b) percentage composition computed from FID chromatogram peak areas without the use of correction factors, t represents trace amounts (< 0.1%); c) ID: method of identification, MS: constituent identified by mass-spectra comparison, RI: constituent identified by RI matching, Co-GC: constituent identity confirmed by GC co-injection with an authentic sample. d) O, others; FAD, fatty acids and fatty acid-related compounds; M, monoterpene hydrocarbons; MO, oxygenated monoterpenes; S, sesquiterpene hydrocarbons; SO, oxygenated sesquiterpenes; D, diterpenoids; PP, phenylpropanoids.


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Table 2. Retention indices of the synthesized aromatic esters on a DB-5 capillary column 526

Benzoic acid 4-methoxybenzoic acid

4-hydroxybenzoic acid

vanillic acid

borneol 1884 2185 2251 2311

isoborneol 1893 2194 2260 2323

geraniol 1957 2245 2308 2372

menthol 1870 2150 2238 2297

nerol 1934 2220 2284 2346

endo-fenchol 1773 2086 2149 2202

527


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Figure captions 528 529 Figure 1. Crystal structure of bornyl 4-methoxybenzoate with the atom-numbering scheme 530

(ORTEP view). Selected bond lengths (Å): O1–C2 1.352(7), C5–C8 1.488(7), C8–O2 1.210(6), 531

C8–O3 1.315(6), C9–O3 1.449(7); Selected bond angles (°): O2–C8–O3 123.9(5), C2–O1–C1 532

118.5(5). 533

Figure 2. Effect of acute administration of Ferula ovina oil (FO, 50, 100 and 200 mg/kg), bornyl 534

4-methoxybenzoate (BMB, 50, 100 and 200 mg/kg) and ASA (200 mg/kg) on the number of 535

writhes in mice following the injection of acetic acid. Values are mean ± SD, n = 6. One-way 536

ANOVA followed by Tukey’s test; *p<0.001 vs. vehicle; #p<0.001 vs. ASA. 537

538 Figure 3. The effect of Ferula ovina essential oil (50, 100 and 200 mg/kg) on nociceptive 539

responses in the hot plate test (A) and the effect of bornyl 4-methoxybenzoate (BMB; at 50, 100 540

and 200 mg/kg) in the same test (B) compared to the activity of morphine (5 mg/kg) and vehicle 541

(10 ml/kg). The results are presented as mean ± S.D. (n = 6), statistical significance was 542

calculated by one way ANOVA followed by Tukey test. *p<0.001, **p<0.01, ***p<0.05 vs. 543

vehicle; # p<0.001 vs. morphine. 544

545 Figure 4. The effect of F. ovina essential oil (50, 100 and 200 mg/kg) on the nociceptive 546

responses in the tail immersion test (A) and the effect of bornyl 4-methoxybenzoate (BMB; at 547

50, 100 and 200 mg/kg) in this test (B) compared to that of morphine (5 mg/kg) and vehicle (10 548

ml/kg). The results are presented as mean ± S.D. (n = 6), statistical significance was calculated 549

by one way ANOVA followed by Tukey test. *p<0.001, **p<0.01 vs. vehicle; #p<0.001, 550

##p<0.01, ### p<0.05 vs. morphine. 551

552


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Figure 5. Effect of the topically applied DMSO, capsaicin and bornyl 4-methoxybenzoate (BMB) 553

on mice performance in the DHP test. It represents the number of paw likings per animal group 554

noted. The results are presented as mean ± S.D. (n = 6), statistical significance was calculated by 555

one way ANOVA followed by Tukey test. *p<0.001, **p<0.01, *** p<0.05 vs. DMSO. 556


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Figure 1 557

558

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Figure 2 560

561

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Figure 3 563

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Figure 4 565

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Figure 5 567

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Chemistry of spices: bornyl 4-methoxybenzoate from Ferula ovina (Boiss.) Boiss. (Apiaceae) induces...

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Transcript of Chemistry of spices: bornyl 4-methoxybenzoate from Ferula ovina (Boiss.) Boiss. (Apiaceae) induces...