Food & Function

PAPER

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aDepartment of Chemistry, Faculty of Scien

Visegradska 33, RS-18000 Nis, Serbia. E

+381-18533014; Tel: +381-18533015bDepartment of Physiology, Faculty of Medic

Đind�ica 81, RS-18000 Nis, SerbiacFaculty of Medicine, University of Nis, Bule

SerbiadVinca Institute of Nuclear Sciences, Laborat

Matter Physics, University of Belgrade, P.O.eDepartment of Basic Sciences, Islamic Azad

Iran

† Electronic supplementary informa10.1039/c3fo60319a

Cite this: Food Funct., 2013, 4, 1751

Received 7th August 2013Accepted 9th September 2013

DOI: 10.1039/c3fo60319a

www.rsc.org/foodfunction

This journal is ª The Royal Society of Ch

Chemistry of spices: bornyl 4-methoxybenzoate fromFerula ovina (Boiss.) Boiss. (Apiaceae) induceshyperalgesia in mice†

Niko S. Radulovic,*a Dragan B. Zlatkovic,a Pavle J. Randjelovic,b Nikola M. Stojanovic,c

Slađana B. Novakovicd and Hashem Akhlaghie

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

condiments. Methods and results: GC–MS analyses of F. ovina aerial parts' essential oil revealed the

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

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

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

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

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

sensitivity to the noxious stimulus compared to that of the control group. A dynamic hot plate test was

used to demonstrate that bornyl 4-methoxybenzoate induces hyperalgesia and not allodynia.

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

potentially flavor-related properties.

1 Introduction

Ferula L. is a genus of about 170 species of umbelliferousowering plants native to the Mediterranean region and east tocentral Asia. Many species of this genus have been historicallyused for culinary andmedical purposes. Asafoetida, an oleogumresin obtained from the rhizome of F. asa-foetida and severalother Ferula species, has been commonly used in Nepal as aavoring agent in many curry recipes.1 Galbanum, a gumexudate collected from F. galbaniua and F. rubricaulis, is usedas a candy additive,2 and it has also been used for the treatmentof intestinal disorders.3 Bulgarian researchers have recentlyshown that Ferula spp. represent a new plant source of propolisin Malta.4 Ferula orientalis is used in Turkey for avoringpickles.5 Herbal products based on F. hermonis are sold as anaphrodisiac dietary supplement in the United States.6

ce and Mathematics, University of Nis,

-mail: nikoradulovic@yahoo.com; Fax:

ine, University of Nis, Bulevar dr Zorana

var dr Zorana Đind�ica 81, RS-18000 Nis,

ory of Theoretical Physics and Condensed

Box 522, RS-11001 Belgrade, Serbia

University, Sabzevar Branch, Sabzevar,

tion (ESI) available. See DOI:

emistry 2013

The Iranian ora comprises 30 species of this genus, one ofthem being F. ovina (Boiss.) Boiss. The resin obtained by cutson the stem is a traditional avoring agent and is used as aningredient of spices and condiments.7 This plant is also veryappreciated for its medicinal use. The aqueous extract ofF. ovina was found to possess antispasmodic, anticholinergicand smooth muscle relaxant activities.8 Although the essentialoil composition of F. ovina was previously reported,9,10 thepotential pharmacological activity of this oil remainsunknown up to date.

Since a meticulous knowledge of the properties, both bene-cial and adverse, of each and every food ingredient is of vitalimportance, F. ovina deserves a more detailed investigation ofits chemical and pharmacological prole. Having this and theethnopharmacological use of this plant species in mind, wedecided to perform detailed chemical composition analysis ofthe essential oil of aerial parts of F. ovina collected in Iran.

An important motivation for this study was also the fact thatplants from this genus are known to synthesize esters of substitutedbenzoic acids (vanillic, p-hydroxybenzoic and p-methoxybenzoicacids), e.g. ferutinin (ferutinol p-hydroxybenzoate), teferin(ferutinol vanillate) and jaesekanadiol p-methoxybenzoate.4,11

Previous reports on the essential oil of F. ovina surprisinglylack these esters although volatile bornyl 4-hydroxybenzoatesand vanillates, e.g. tschimganin and tschimgin, were isolatedfrom taxa belonging to this genus.12

Esters of these acids are known to activate transient receptorpotential (TRP) channels that are implicated in pain percep-tion.13 Targeting TRP channels has been an important strategy

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in the search of novel analgesics.14 Also, the TRP family channelactivity (such as TRPV1, TRPV3, TRPA1, TRPM5 and TRPM8)can contribute to aspects of taste and can modify or initiatesignals that integrate with other signals from olfactory and tastereceptors, either within the epithelium, between epithelia andspecialized cells, or at the level of neuronal input to the CNS.15

A rst compositional screening of the oil in our hands doneby GC-MS revealed the presence of several monoterpenic estersof aromatic acids. We decided to pursue with a detailed analysisof the oil in hope of nding new and pharmacologically inter-esting esters possessing antinociceptive activity.

2 Materials and methods2.1 General experimental procedures

Acetylsalicylic acid (ASA) (Aspirin, Bayer), morphine (HoffmannLa Roche), capsaicin (Sigma) and the tested substances werefreshly dissolved, in a maximum volume of 0.3 ml per animal,prior to usage. All other chemicals were obtained from Aldrich(USA) or Fluka (Germany) and were used as received, with theexception of the solvents that were additionally puried bydistillation. 1H and 13C NMR spectra were recorded on a BrukerAvance II spectrometer (Bruker, Germany) operating at 400 and100.6 MHz, respectively. 2D experiments (1H–1H COSY, HSQC,HMBC and NOESY) were run on the same instrument with theusual pulse sequences. All NMR spectra were measured at 25 �Cin CDCl3 with TMS as the internal standard. Microanalysisof carbon and hydrogen was carried out with a Carlo Erba1106 microanalyser; their results agreed favorably with thecalculated values.

Preparative medium-pressure liquid chromatography(MPLC) was performed with a pump module C-601 and apump controller C-610 Work-21 pump (Buchi, Switzerland)and was carried out on pre-packed column cartridges (40 mm �75 mm), Silica-gel 60, particle size distribution 40–63 mm, Buchi(Switzerland).

Precoated Al silica gel plates (Merck, Kieselgel 60 F254,0.2 mm) were used for analytical TLC analyses. The spots onTLC were visualized by UV light (254 nm) and by spraying with10% (w/v) solution of phosphomolybdic acid in ethanol fol-lowed by heating.

The GC-MS analyses were performed on a Hewlett–Packard6890N gas chromatograph equipped with a fused silica capillarycolumn DB-5MS (5% phenylmethylsiloxane, 30 m � 0.25 mm,lm thickness 0.25 mm, Agilent Technologies, USA) and coupledwith a 5975B mass selective detector from the same company.The injector and interface were operated at 250 �C and 320 �C,respectively. The oven temperature was raised from 70 to 290 �Cat a heating rate of 5 �C min�1 and then isothermally held for10 min. As a carrier gas helium at 1.0 ml min�1 was used. Thesamples, 1 ml of the solutions in diethyl ether (1 mg in 1 ml ofEt2O), were injected in a pulsed split mode (the ow was 1.5 mlmin�1 for the rst 0.5 min and then set to 1.0 ml min�1

throughout the remainder of the analysis; split ratio 40 : 1). Themass selective detector was operated at the ionization energy of70 eV, in the 35–650 amu range and scanning speed of 0.32 s.GC (FID) was carried out under the same conditions and using

1752 | Food Funct., 2013, 4, 1751–1758

the same column as for GC-MS. The percentage compositionwas computed from FID chromatogram peak areas without theuse of correction factors. The linear retention indices werecalculated relative to the retention times of C8–C24 n-alkanes onthe DB-5MS column.16

2.2 Single-crystal X-ray experiment

The diffraction data were collected from a weakly diffractingcolorless crystal mounted on an Agilent Gemini S diffractom-eter.17 Crystal data: C18H24O3, Mr ¼ 288.4, orthorhombic, spacegroup P212121, a ¼ 11.004(2), b ¼ 11.212(3), c ¼ 13.436(2) A, V ¼1657.8(6) A3, Z ¼ 4, Dc ¼ 1.155 g cm�3, F(000) ¼ 624, m ¼0.614 mm�1, l ¼ 1.54184 A, T ¼ 293 K. Experimental andstructure renement details are given in the ESI (Tables S2–S3†).

2.3 Plant material

Plant material (aerial parts of F. ovina) used in this study wascollected from natural populations growing at an altitude of1650 m, on June 12th, 2011, in the Sabzevar region (north-eastern Iran).

2.4 Essential oil extraction

Plant material (air dried to a constant weight, 2 batches of about500 g) was subjected to hydrodistillation with approximately 2 Lof distilled water for 2.5 h using the original Clevenger-typeapparatus. The obtained oils were separated by extraction withdiethyl ether, dried over anhydrous magnesium sulfate andimmediately analyzed. The yield of the oil was 1.5%.

2.5 General procedure for the microsynthesis of esters

Solutions of an appropriate carboxylic acid (benzoic, 4-hydroxy-benzoic, 4-methoxybenzoic or vanillic acids, 0.1 mmol), alcohol(borneol, isoborneol, geraniol, menthol, nerol or endo-fenchol),4-dimethylaminopyridine (DMAP, 0.5 mg), andN,N0-dicyclohexylcarbodiimide (DCC, 0.11 mmol) in 1.0 ml ofdry CH2Cl2 were prepared, vortexed for a few seconds and storedovernight in closed GC vials. The solutions were analyzedwithout any additional treatment.

2.5.1 Synthesis of bornyl p-methoxybenzoate (BMB).Borneol (1 mmol), 4-methoxybenzoic acid (1 mmol), DMAP(5 mg) and DCC (1.1 mmol) in dry CH2Cl2 (10 ml) were stirredovernight at room temperature protected from atmosphericmoisture by a CaCl2 guard tube. The precipitated urea wasltered off and the ltrate was concentrated in vacuo. The esterwas puried by MPLC chromatography using silica gel and a1 : 1 (v/v) mixture of hexane and CH2Cl2 as the eluent. Thepurity of the ester fractions was checked by TLC (10%, w/v,phosphomolybdic acid in ethanol) and GC-MS.

1H NMR (CDCl3, 200 MHz) dH: 0.91 (s, 3H), 0.92 (s, 3H), 0.97(s, 3H), 1.11 (dd, J ¼ 3.4 and 13.8 Hz), 1.20–1.48 (m, 2H), 1.60–1.90 (m, 2H), 2.00–2.25 (m, 1H), 2.46 (dddd, J¼ 13.8, 9.9, 4.6, 3.4Hz, 1H), 3.86 (s, 3H), 5.09 (ddd, J ¼ 9.9, 3.4, 2.0 Hz, 1H), 6.94(dd, 8.9, 2.3 Hz, 2H), 8.20 (dd, 8.9, 2.3 Hz, 2H).

13C NMR (CDCl3, 50 MHz) dC: 13.6, 18.9, 19.7, 27.4, 28.1,36.9, 45.0, 47.8, 49.1, 55.4, 80.1, 113.6, 123.0, 131.5, 163.0, 166.7.

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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), 93 (3.8).

2.6 Pharmacological procedures

2.6.1 Animals and treatment. The essential oil (FO), BMB,acetylsalicylic acid (ASA), morphine and capsaicin were freshlydissolved, in a maximum volume of 0.3 ml per animal, prior tousage. Male BALB/c mice (20 � 5 g) 5 weeks old were obtainedfrom the Vivarium of the Institute of Biomedical Research,Medical Faculty, University of Nis, Nis, Serbia. Animals werehoused in groups of 6 males under standard laboratory condi-tions: 12 h light/dark cycle at 22 � 2 �C, with food and wateravailable ad libitum. Animals were acclimatized to the labora-tory environment for at least 12 h before testing (fasted, thoughstill allowed free access to water). Six experimental and twocontrol (positive and negative) groups, with 6 animals pergroup, for writhing, hot plate and tail ick experiments wereused. In a dynamic hot plate (DHP) test additional three groupsof animals were used and the substances were applied topicallyon the animal paws (see a detailed description in the DHP testsection). For the other tests, the essential oil of F. ovina (FO) andthe new ester (BMB) were administered intraperitoneally (i.p.)one hour prior to the experiments at doses of 50, 100 and200 mg kg�1 to six groups, whereas two more groups receivedmorphine at 5 mg kg�1 or acetylsalicylic acid at 200 mg kg�1

(ASA; used as the positive control only in the abdominalwrithing test) and vehicle (olive oil) at 10 ml kg�1. All experi-mental procedures were conducted in accordance with theprinciples of care and use of laboratory animals in research(EEC Directive of 1986; 86/609/EEC) and were approved by thelocal Ethics committee (01-10204-05).

2.6.2 Abdominal writhing test. The method for abdominalwrithing adopted here was previously described by Radulovicet al.16 Briey, aer the administration of acetic acid (1%, v/v,i.p.) the number of writhes was counted for 20 min. A writhe wasdened as a contraction of the abdominal muscles accompa-nied by an extension of the fore- and hindlimbs and elongationof the body. The % of inhibition was calculated as follows ¼100 � [number of writhes (control) � number of writhes (test)]/number of writhes (control).

2.6.3 Hot plate test. Mice were tested according to themethod described by Radulovic et al.16 Animals were placed on ahot plate apparatus set at 55 � 1 �C. The behavior of animalsconsidered as a sign of pain was licking the fore- and hind-pawsor jumping off the hot-plate. The cut-off time was xed at 20 s toavoid skin damage. Baseline (BL) was considered as the mean ofreaction time obtained at 60 and 30 min before administrationof the substances, vehicle or morphine and dened as thenormal reaction of animals to thermal stimuli. Increase inbaseline (%) was calculated using the formula: [(reaction time� 100)/BL] � 100. Antinociception was quantied as area underthe curve (AUC) of responses and was calculated as a sum ofAUC1 ¼ 15 � IB[(min15)/2 + (min30) + (min45) + (min60)/2] andAUC2 ¼ 30 � IB[(min60)/2 + (min90) + (min120)/2] where IB isthe increase in baseline (in %). % of inhibition values formorphine and each dose of FO and BMB were calculated as

This journal is ª The Royal Society of Chemistry 2013

follows: % of inhibition ¼ AUC/max, where max ¼ [(20 � 100)/BL � 100] � 105.

2.6.4 Tail immersion test. The lower 5 cm portion of the tailwas immersed in a beaker of water maintained at 50.0 �0.5 �C.18 The time in seconds for tail withdrawal from the waterwas taken as the reaction time, with a cut-off time of immersionset at 15 s. The latency of tail withdrawal was measured at 15,30, 45, 60, 90 and 120 min aer the administration of FO, BMB,morphine and vehicle. % of inhibition was calculated asdescribed in the hot plate test section.

2.6.5 Dynamic hot plate (DHP) test. This method has beendescribed recently by Yalcin et al.19 DMSO (negative control,100%), capsaicin (positive control, 10 mM dissolved in DMSO)or BMB (10 mM dissolved in DMSO) were topically applied withcotton-tipped applicators on animal paws, 15 min before theywere placed on the dynamic hot plate (DHP). The temperatureof the hot plate was 30.0� 0.1 �C when themouse was placed onit and was increased to 43 �C at a rate of 1 �C min�1. Duringeach degree interval, hind paw lickings, escape behavior(jumps) and rearing were scored, as was proposed in the orig-inal publication.19

2.6.6 Statistical analysis. Results were expressed as themean � SD. Statistically signicant differences were deter-mined by one-way analysis of variance (ANOVA) followed byTukey's post-hoc test for multiple comparisons (GraphpadPrism version 5.03, San Diego, CA, USA). Probability values (p)less than 0.05 were considered to be statistically signicant.

3 Results and discussion3.1 Chemistry

3.1.1 Composition of the essential oil. GC/MS analysesindicated that the essential oil of F. ovina in our hands consistedlargely of monoterpene hydrocarbons (a-pinene, limoneneand myrcene were the most abundant constituents and repre-sented 17.8, 14.3 and 13.0% of the oil respectively, see Table 1).Such a composition is chiey in agreement with those that werepreviously published for this species.9,10 The analyses alsorevealed the presence of a number of aromatic esters elutingfrom the GC column at RI values above 1700. The carboxylicacid portion of the esters was straightforwardly inferred fromtheir mass spectra, e.g. a characteristic fragment ion of allbenzoate esters was located at m/z 105 (C6H5CO

+). Similarly,methoxybenzoates, hydroxybenzoates and hydroxymethoxy-benzoates (vanillates) had prominent fragments at m/z 135, 121and 151, respectively. In the case of all esters, the molecularions and the base peaks (ArCO+) differed in 153 amu, indicatingthat the esterifying alcohols were monoterpenols of the formulaC10H18O (Mw 154). Their MSs were not sufficiently informativeto provide a means to differentiate between esters of mono-terpenols of the same molecular formula. The possible identityhad to be checked by synthesis and GC co-injection of thesynthesized esters with the oil of F. ovina. We assumed that themonoterpenic alcohols (borneol, endo-fenchol, geraniol andnerol, see Table 1) already detected in our sample, in the form offree alcohols and/or acetates, also esteried the mentionedaromatic acids. Even though we could not deduce the right

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Table 1 Percentage composition of Ferula ovina essential oil

RIa Compound %b IDc Classd

846 (2E)-Hexenal t RI, MS, Co-GC FAD921 Tricyclene 0.7 RI, MS M932 a-Pinene 17.8 RI, MS, Co-GC M946 Camphene 6.3 RI, MS, Co-GC M969 Sabinene 0.4 RI, MS, Co-GC M974 b-Pinene 1.9 RI, MS, Co-GC M988 Myrcene 13.0 RI, MS, Co-GC M988 Dehydro-1,8-cineole 0.1 RI, MS MO1002 a-Phellandrene 0.1 RI, MS, Co-GC M1008 d-3-Carene 0.5 RI, MS, Co-GC M1014 a-Terpinene 0.1 RI, MS, Co-GC M1022 p-Cymene 0.4 RI, MS, Co-GC M1025 Limonene 14.3 RI, MS, Co-GC M1025 b-Phellandrene 2.1 RI, MS, Co-GC M1026 1,8-Cineole 0.0 RI, MS, Co-GC MO1032 trans-b-Ocimene 0.1 RI, MS M1054 g-Terpinene 0.1 RI, MS, Co-GC M1065 cis-Sabinene hydrate 0.0 RI, MS MO1083 Fenchone 0.5 RI, MS MO1086 Terpinolene 0.4 RI, MS, Co-MS M1088 Methyl benzoate 0.0 RI, MS, Co-GC FAD1090 6,7-Epoxymyrcene 0.0 RI, MS MO1095 Linalool 1.1 RI, MS, Co-GC MO1098 trans-Sabinene hydrate 0.0 RI, MS MO1111 cis-Rose oxide 0.0 RI, MS MO1118 cis-p-Menth-2-en-1-ol t RI, MS MO1123 endo-Fenchol 0.2 RI, MS, Co-GC MO1129 a-Campholenal 0.1 RI, MS MO1132 cis-Limonene oxide t RI, MS, Co-GC MO1137 (E)-Epoxy-ocimene t RI, MS MO1137 trans-Limonene oxide t RI, MS, Co-GC MO1141 Veratrole t RI, MS, Co-GC FAD1144 cis-Verbenol 0.2 RI, MS, Co-GC MO1145 Isopulegol t RI, MS MO1148 trans-Verbenol 0.7 RI, MS, Co-GC MO1151 Citronellal 0.6 RI, MS, Co-GC MO1158 trans-Pinocamphone t RI, MS, Co-GC MO1160 Pinocarvone t RI, MS MO1172 p-Mentha-1,5-dien-8-ol 0.2 RI, MS MO1172 cis-Pinocamphone t RI, MS, Co-GC MO1175 Borneol 0.2 RI, MS, Co-GC MO1179 p-Cymen-8-ol t RI, MS MO1183 Terpinen-4-ol 0.2 RI, MS, Co-GC MO1183 Cryptone t RI, MS, Co-GC MO1196 a-Terpineol 0.9 RI, MS, Co-GC MO1204 Verbenone t RI, MS, Co-GC MO1207 trans-Piperitol t RI, MS MO1215 trans-Carveol t RI, MS MO1219 endo-Fenchyl acetate 4.1 RI, MS, Co-GC MO1225 Citronellol 0.8 RI, MS, Co-GC MO1226 cis-Carveol t RI, MS MO1229 exo-Fenchyl acetate t RI, MS MO1249 Geraniol 0.3 RI, MS, Co-GC MO1257 Methyl citronellate 0.2 RI, MS MO1283 cis-Verbenyl acetate 0.1 RI, MS MO1288 Bornyl acetate 4.6 RI, MS, Co-GC MO1324 Myrtenyl acetate t RI, MS, Co-GC MO1334 trans-Carvyl acetate 0.1 RI, MS MO1348 a-Terpinyl acetate 3.0 RI, MS, Co-GC MO1350 Citronellyl acetate t RI, MS, Co-GC MO1357 Neryl acetate 0.1 RI, MS, Co-GC MO1371 Methyl 4-methoxybenzoate t RI, MS, Co-GC FAD

Table 1 (Contd. )

RIa Compound %b IDc Classd

1374 Daucene 0.1 RI, MS S1377 Geranyl acetate 0.7 RI, MS, Co-GC MO1389 b-Elemene t RI, MS, Co-GC S1393 cis-Jasmone 0.3 RI, MS, Co-GC O1398 Methyl eugenol 0.5 RI, MS, Co-GC PP1424 trans-Caryophyllene 0.5 RI, MS, Co-GC S1424 2,5-Dimethoxy-p-cymene t RI, MS O1434 Neryl acetone t RI, MS O1452 a-Humulene t RI, MS, Co-GC S1453 (E)-b-Farnesene 0.1 RI, MS S1490 (E,E)-a-Farnesene 0.1 RI, MS S1493 b-Selinene 0.1 RI, MS S1500 a-Selinene 0.2 RI, MS S1510 b-Bisabolene 0.1 RI, MS, Co-GC S1522 d-Cadinene t RI, MS S1522 Myristicin 9.4 RI, MS, Co-GC PP1546 Elemicin 4.7 RI, MS, Co-GC PP1564 b-Calacorene t RI, MS S1587 Caryophyllene oxide 0.1 RI, MS, Co-GC SO1596 Neryl isovalerate 0.1 RI, MS, Co-GC MO1600 Hexadecane t RI, MS, Co-GC FAD1603 Geranyl isovalerate 0.1 RI, MS, Co-GC M1606 Carotol 0.2 RI, MS SO1629 1-epi-Cubenol 0.1 RI, MS SO1645 Cubenol t RI, MS SO1652 a-Cadinol t RI, MS SO1699 b-Sinensal t RI, MS SO1722 (E,E)-Farnesol t RI, MS SO1759 Benzyl benzoate t RI, MS, Co-GC FAD1773 endo-Fenchyl benzoate t RI, MS, Co-GC MO1884 Bornyl benzoate t RI, MS, Co-GC MO1959 Palmitic acid t RI, MS, Co-GC FAD1980 Camphorene t RI, MS D2185 Bornyl 4-methoxybenzoate 0.3 RI, MS, Co-GC MO2202 endo-Fenchyl Vanillate 1.1 RI, MS, Co-GC MO2251 Bornyl 4-hydroxybenzoate 0.2 RI, MS, Co-GC MO2311 Bornyl vanillate 0.8 RI, MS, Co-GC MO

Total 96.2MonoterpenoidsHydrocarbons 58.2Oxygenated 21.5SesquiterpenoidsHydrocarbons 1.2Oxygenated 0.4Diterpenes tFatty acids and fattyacid-related compounds

t

Phenylpropanoids 14.6Other 0.3

a RI: retention index relative to C8–C24 n-alkanes on a DB-5MS column.b Percentage composition computed from FID chromatogram peakareas without the use of correction factors, t represents trace amounts(<0.1%). c ID: method of identication, MS: constituent identied bymass-spectra comparison, RI: constituent identied by RI matching,Co-GC: constituent identity conrmed by GC co-injection with anauthentic sample. d O, others; FAD, fatty acids and fatty acid-relatedcompounds; M, monoterpene hydrocarbons; MO, oxygenatedmonoterpenes; S, sesquiterpene hydrocarbons; SO, oxygenatedsesquiterpenes; D, diterpenoids; PP, phenylpropanoids.

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regioisomers of hydroxybenzoic and methoxybenzoic acidsfrom the MSs alone, we decided to use their para-isomers in thepreparation of the esters since p-methoxybenzoates and

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Table 2 Retention indices of the synthesized aromatic esters on a DB-5 capillarycolumn

Benzoicacid

4-Methoxybenzoicacid

4-Hydroxybenzoicacid

Vanillicacid

Borneol 1884 2185 2251 2311Isoborneol 1893 2194 2260 2323Geraniol 1957 2245 2308 2372Menthol 1870 2150 2238 2297Nerol 1934 2220 2284 2346endo-Fenchol 1773 2086 2149 2202

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p-hydroxybenzoates were already known to be secondarymetabolites of the taxa belonging to the genus Ferula. Allpossible combinations of esters were prepared by Steglichcoupling and GC co-injected with the oil sample (Table 2). Wealso prepared menthyl esters of the four acids in order toprobe whether it is possible to recognize the carboxylic partof aromatic acid esters based on their base peaks for anyaliphatic monoterpene ester (no matter the total double bondequivalents).

Out of 24 compounds prepared, the GC co-injectionconrmed the presence of 6 esters in the oil (bornyl esters of allfour acids, as well as the vanillate and benzoate of endo-fen-chol). According to a thorough literature survey, bornyl4-methoxybenzoate represents a new natural compound. Anadditional amount of this ester was prepared for the completestructural assignment and for the pharmacological experi-ments. Its structure was corroborated by 1D and 2D-NMRexperiments (details are contained in the ESI, Fig. S1–S7, TableS1†) and X-ray crystallographic analysis.

3.1.2 X-ray crystallographic analysis of bornyl 4-methoxy-benzoate. Despite the poor crystal quality, single-crystal X-rayanalysis was able to conrm the expected structure of thearomatic acid ester. The established molecular structure ofbornyl 4-methoxybenzoate is given in Fig. 1. Due to severedisorder in the bornyl moiety, bond lengths and angles in thispart of the molecule could not be described in detail. Thegeometry of the methoxybenzoate moiety is within the expectedranges (see ESI, Table S2†). The carboxylic part of the ester, C8/O2/O3 [C8–O2 ¼ 1.210(6), C8–O3 ¼ 1.315(6) A], is only slightlytwisted with respect to the phenyl ring forming a dihedral angleof 4.0(8)�. The torsion angle O2–C8–O3–C9 of 2.2(10)� describesthe cis position of the bornyl moiety with respect to the

Fig. 1 Crystal structure of bornyl 4-methoxybenzoate with the atom-numberingscheme (ORTEP view). Selected bond lengths (A): O1–C2 1.352(7), C5–C81.488(7), C8–O2 1.210(6), C8–O3 1.315(6), C9–O3 1.449(7); selected bond angles(�): O2–C8–O3 123.9(5), C2–O1–C1 118.5(5).

This journal is ª The Royal Society of Chemistry 2013

carboxylic O2. As evident from the torsion angle C1–O1–C2–C3of �2.0(9)�, the methoxy group only slightly deviates from theplane of the phenyl ring.

Regardless of the presence of three oxygen acceptors and anumber of C–H donor groups, the crystal structure does notcontain any signicant C–H.O interactions. The shortestcontact involving the carboxylic oxygen acceptor is C1–H1c.O2i (H.O¼ 2.68 A and C–H.O¼ 130�; i¼ x� 1/2, y� 1/2, z). The mutual orientation of the bornyl C–H fragments andof the phenyl ring suggests that C–H.p interactions could havean important role in structure stabilization (see ESI, Fig. S9†).

3.2 Pharmacological investigations

Antinociceptive properties of F. ovina essential oil and bornyl4-methoxybenzoate were assessed in three laboratory nociceptionmodels, corresponding to two different types of noxious stimuli:a thermal one and a chemically induced pain stimulus.20

3.2.1 Chemically induced pain model. The oil exerted, in adose-dependent manner, a strong antinociceptive activity, beingmost prominent in inhibition of abdominal writhings inducedby acetic acid (Fig. 2). Animals treated with F. ovina oil, at a doseof 200 mg kg�1, showed a decreased number of writhings(�92%) comparable to the effect of the standard drug at thesame dosage (ASA at 200 mg kg�1, �90%) (see ESI, Table S4†).Some of the major oil constituents (a-pinene, limonene andmyrcene, Table 1) are known to possess certain analgesicpotential21 providing at least a partial explanation for the notedactivity of the oil.

Alongside with the oil, a minor constituent – the new ester,bornyl 4-methoxybenzoate – was tested. It showed almost thesame effect (�83%) as the oil itself in 200 mg kg�1 (Fig. 2,Table S4†). In the abdominal writhings model, a cascade ofevents following the injection of acetic acid – the release ofprostaglandins and sympathetic nervous system mediators,activation and sensitization of peripheral chemosensitive noci-ceptive receptors and the onset of inammation – results inabdominal contortions.22 A possible explanation for the observedpain threshold elevation for the two tested substances might bethat they act as inhibitors of prostaglandin synthesis. However, afalse positive result is produced by muscle relaxants, adrenergic

Fig. 2 Effect of acute administration of Ferula ovina oil (FO, 50, 100 and 200 mgkg�1), bornyl 4-methoxybenzoate (BMB, 50, 100 and 200 mg kg�1) and ASA (200mg kg�1) on the number of writhes in mice following the injection of acetic acid.Values are mean � SD, n ¼ 6. One-way ANOVA followed by Tukey's test; *p <0.001 vs. vehicle; #p < 0.001 vs. ASA.

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blockers, antihistamines and neuroleptics.20Onemight speculatethat bornyl 4-methoxybenzoate possesses a myorelaxant activitysince the parent alcohol, borneol, is a known non-competitiveinhibitor of nicotinic acetylcholine receptors.23

3.2.2 Thermally induced pain models. A potential centralanalgesic effect of this oil was selectively evaluated in the hotplate and tail immersion tests (as opposed to the abdominalwrithings test where both central and peripheral analgesics givepositive results). The central control of pain is subject todescending modulation by brainstem cell groups such as locuscoeruleus/subcoeruleus and raphe complex and the hot platetest is considered to be a selective test for drugs that affectsupraspinally integrated responses.20 The oil showed a dosedependent increase of reaction time in the hot plate test (55 �1 �C). The calculated percents of pain inhibition (%) in this testfor the oil, in doses of 50, 100 and 200 mg kg�1, and morphine(at 5 mg kg�1) were: 13.2, 21.1, 28.4 and 72.7%, respectively(Table S4†). The rst notable statistically signicant effect of thehighest dose of the oil on mice reaction time during the hotplate test was 30 minutes aer the oil administration (Fig. 3A).The oil exerted the strongest modulator effect on responses to

Fig. 3 The effect of Ferula ovina essential oil (50, 100 and 200 mg kg�1) onnociceptive responses in the hot plate test (A) and the effect of bornyl 4-methoxybenzoate (BMB; at 50, 100 and 200 mg kg�1) in the same test (B)compared to the activity of morphine (5 mg kg�1) and vehicle (10 ml kg�1). Theresults are presented as mean� S.D. (n¼ 6), statistical significance was calculatedby one way ANOVA followed by Tukey's test. *p < 0.001, **p < 0.01, ***p < 0.05vs. vehicle; #p < 0.001 vs. morphine.

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thermal stimuli (maximal increase of reaction time) 90 minaer the administration (in all doses) and remained approxi-mately the same until the end of the experiment (Fig. 3A),whereas the 5 mg kg�1 dose of morphine demonstrated itseffect much more quickly – 15 min aer the application of thedrug (Fig. 3A).

A characteristic behavior of mice in the hot plate test that canbe described as either hyperalgesia or allodynia was observed inanimals treated with bornyl 4-methoxybenzoate (BMB) (Fig. 3B).While hyperalgesia is dened as an increased pain sensitivitythat can include both a decrease in threshold and an increase insuprathreshold response, allodynia is pain in response to a non-nociceptive stimulus.24 A decrease in mice reaction time tothermal stimuli for all doses at some point in time aer theapplication of BMB can be seen in Fig. 3B. Only the highest doseproduced a temporary analgesic effect (15–45 min) aer whichthe animals developed hyperalgesia/allodynia as well.

Limonene, one of the major oil constituents, failed todemonstrate any effect on supraspinal nociception,25 whereasour oil had an effect on supraspinal nociception. Myrcene,comprising some 13% of the oil, was previously shown tostimulate the release of endogenous opioids by mediation ofa2-adrenoreceptors.26 Hence, we can speculate that myrcenecontributes to the observed activity of the oil but other minor ormajor constituents might have an effect as well.

The tail immersion test is predominantly based on a spinalreex, which could also involve higher neural structures, andrepresents a selective test for the evaluation of central analgesicactivity.20 The oil showed relatively weak antinociceptive activitywhen compared to the control (Fig. 4A). Only the highest dose ofthe oil managed to increase the time latency (Fig. 4A) from thevery beginning of the test (15 min aer the application). For thedose of 200 mg kg�1, the maximum response (longest timerequired to react) was 60 min and the effect faded away aer120 min following the injection. In this time period for theanimals that received the highest dose of the oil, as well asduring the 90–120 min period for the experimental grouptreated with 100 mg kg�1 of the oil, there were no statisticallysignicant differences found between the abovementionedreaction times (at 60, 90 and 120 min) and those of themorphine treated animals (Fig. 4A). The determined percents ofinhibition in this test were 38.5, 42.8 and 55.8% for the threedoses of the oil, in an ascending dosage order, and 80.6% formorphine (Table S4†). The increased sensitivity to the noxiousstimulus was not observed in this test for bornyl 4-methox-ybenzoate (Fig. 4B). In this test the activity of this compoundwas an analgesic one. The substance exerted mild anti-nociceptive activity with the peak activity at the doses of 100 and200 mg kg�1 (see % of inhibition in Table S10†) 45 min aer theapplication. The decrease in pain latency was observed contin-uously 60 min aer the application of this ester (at the doses of100 and 200 mg kg�1). The lowest dose of the methoxybenzoate(50 mg kg�1) seems not to have any signicant antinociceptiveeffect 45 min aer its application.

The results obtained for bornyl 4-methoxybenzoate using thehot plate and the tail immersion tests indicated oppositeeffects. However, although both assays evaluate central control

This journal is ª The Royal Society of Chemistry 2013

Fig. 4 The effect of F. ovina essential oil (50, 100 and 200 mg kg�1) on thenociceptive responses in the tail immersion test (A) and the effect of bornyl 4-methoxybenzoate (BMB; at 50, 100 and 200 mg kg�1) in this test (B) compared tothat of morphine (5 mg kg�1) and vehicle (10 ml kg�1). The results are presentedas mean � S.D. (n ¼ 6), statistical significance was calculated by one way ANOVAfollowed by Tukey's test. *p < 0.001, **p < 0.01 vs. vehicle; #p < 0.001, ##p < 0.01,###p < 0.05 vs. morphine.

Fig. 5 Effect of the topically applied DMSO, capsaicin and bornyl 4-methoxy-benzoate (BMB) on mice performance in the DHP test. It represents the number ofpaw lickings per animal group noted. The results are presented as mean � S.D.(n ¼ 6), statistical significance was calculated by one way ANOVA followed byTukey's test. *p < 0.001, **p < 0.01, ***p < 0.05 vs. DMSO.

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of pain and thermal nociception, these ndings are only seem-ingly conicting. One possible explanation may lay in the factthat the tail-ick response is primarily spinally mediated and thatin the hot plate response (paw licking or jumping) supraspinalsites play a key role.27 Since bornyl 4-methoxybenzoate iseffective only in the tail ick test, the antinociceptive activity isprobably mediated at the spinal level. The different activity ofthis compound in the two thermally induced pain tests couldbe due to diverse abundances of opioid receptor subtypeslocated supraspinally (m1, k3, d1, s2) and at the spinal level(m2, k1, d2).28 However, further studies using specic opioidreceptor subtype antagonists are needed to test this hypothesisand determine the exact mechanism of action of thiscompound.

3.2.3 Dynamic hot plate test. Prompted by the results of thehot plate test we subjected mice treated with bornyl 4-methox-ybenzoate to another, relatively new, more specic one (adynamic hot plate test, DHP) that would allow us to discrimi-nate allodynia from hyperalgesia.19 The main reaction of C57Bl/6J mice submitted to the DHP test observed in Yalcin's19 work

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dened as the nociceptive reaction was an escape behavior –

jumping. In our study, no jumps were observed for the testedanimals (male BALB/c mice) even in the capsaicin-sensitizedanimals at high temperatures (data not shown). The onlyincreased parameters noted in our experiments were rearingsand paw lickings (Fig. S10† and 5). However, rearings can alsobe regarded as a nonspecic parameter since even at lowtemperatures (30 �C) the animals from the negative controlgroup (DMSO) showed a slight increase in the number of rear-ings. These rearings, both in the middle or up the wall of theapparatus, can be classied as exploratory ones, as in the openeld test, rather than the reaction to the thermal stimulus(although when the animals were exposed to higher tempera-tures (38–39 �C) or when pretreated with capsaicin a dramaticincrease of rearings was noted (Fig. S10†)). Thus, we decided touse only paw lickings as the specic reaction of BALB/c mice inthe DHP test. An increase in the number of paw lickings wasnotable immediately aer the placement of the animals previ-ously sensitized with capsaicin onto the DHP (Fig. 5). On theother hand, the animals treated with DMSO or bornyl4-methoxybenzoate alone were licking their paws much lessfrequently than the capsaicin-sensitized animals at the sametemperature (Fig. 5). According to the results of the previoustests and nally of the DHP test, we can conclude that bornyl4-methoxybenzoate produces hyperalgesia, and not allodynia.This activity of the new ester might be important for the avorproperties of the compound and the plant species/spice as well.Previous studies have shown that the omnipresent foodmonoterpenes, such as borneol, camphor, carvacrol, eugenoland thymol, have an inuence on transient receptor potentialvanilloid ion channels (TRPV).15 Generally, the activation of TRPchannels depolarizes cells from the resting membrane potentialand shortens action potential duration. The mentioned mono-terpenes were shown to activate and sensitize TRPV3 receptors,by increasing the level of cellular Ca2+ or by activation of the Gprotein-coupled receptor, that are mostly expressed in the skin(keratinocytes), nasal and oral cavity (tongue and palate). Thepresence of these receptors in oral cavity implies that they may

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be potential targets for avor actions of plant derivedcompounds (e.g. carvacrol from oregano).29 Since TRPV3receptors are warmth activated on temperatures 22–40 �C, theyare predominantly activated during a DHP test. Warm to hottemperatures or the penetration of lipophilic compounds (suchas monoterpenes) through keratinized, non-sensory layers andactivation of TRPV3 on keratinocytes lead to releases of ATP tosignal sensory termini and thereby cause the sensation ofwarmth or heat.30 A proposed mechanism of action of camphor,whose structure is related to the bornyl moiety of the new ester,is through the activation of TRPV3. Camphor also acts onTRPV1 receptors by desensitizing them and on TRPA1 receptorsby inhibiting them and thus causing antinociception.31

4 Concluding remarks

Several monoterpenic esters of aromatic carboxylic acids havebeen identied as constituents of the essential oil of the aerialparts of F. ovina used traditionally in Iran as an ingredient ofspice and condiments. One of them, bornyl 4-methoxybenzoate,turned out to be a new natural compound whose structure wasconrmed by synthesis and elucidated by spectral means andX-ray crystallographic analysis. Tests were performed to assessthe antinociceptive activity of the entire oil and the newcompound. The results indicated that the oil of F. ovina possessedanalgesic properties which were mediated via central andperipheral inhibitory mechanisms. The activity of the bulk oilcould most certainly be connected to a variety of compoundsconstituting the oil. However, the activity of bornyl 4-methox-ybenzoate differed markedly from that of the oil. Mice treatedwith bornyl 4-methoxybenzoate had an increased sensitivity tothe noxious stimulus compared to that of the control group. Adynamic hot plate test was used to demonstrate that bornyl4-methoxybenzoate induces hyperalgesia and not allodynia.Hyperalgesia might hint at a possible interaction with transientreceptor channels (TRPV3) in the oral cavity important for theavor properties of this spice but this still needs conrmationfrom further studies. Also the structural similarity of the newester with monoterpenes (borneol and camphor) that are knownto activate these channels will guide us in future investigations.

Acknowledgements

This work was supported by the Ministry of Education, Scienceand Technological Development of Serbia [Project no. 172061].

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