Toxic essential oils. Part IV: The essential oil of

Food and Chemical Toxicology 80 (2015) 114–129

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Toxic essential oils. Part IV: The essential oil of Achillea falcata L. as a source of biologically/pharmacologically active trans-sabinyl esters Niko S. Radulovic´ a,*, Marko Z. Mladenovic´ a, Pavle J. Randjelovic b, Nikola M. Stojanovic´ c, Milan S. Dekic´ a,d, Polina D. Blagojevic´ a a

Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Department of Physiology, Faculty of Medicine, University of Niš, Zorana Ðind¯icá 81, 18000 Niš, Serbia c Faculty of Medicine, University of Niš, Zorana Ðind¯icá 81, 18000 Niš, Serbia d Department of Chemical and Technological Sciences, State University of Novi Pazar, Vuka Karadžic á bb, 36300 Novi Pazar, Serbia b

A R T I C L E

I N F O

Article history: Received 9 February 2015 Accepted 2 March 2015 Available online 9 March 2015 Keywords: Achillea falcata L. Essential oil trans-Sabinol Acute toxicity Acetylcholinesterase inhibitory activity Antinociceptive activity

A B S T R A C T

Herein we report on the comprehensive chemical analysis of the essential oils obtained from above- and underground parts of a previously unreported chemotype of Achillea falcata L. (Asteraceae) and, for the first time, on the biological/toxicological profile of its dominant/newly discovered volatile metabolites. Detailed spectral analyses, in combination with chemical synthesis and theoretical study, of selected constituents, enabled the identification of trans-sabinol and its esters – the formate, tiglate (new compounds), acetate, butanoate, isobutanoate, 2-methylbutanoate and 3-methylbutanoate – in both aerial and underground parts of A. falcata. Evaluation of acute toxicity in Artemia salina model, in vitro and in silico (molecular docking) evaluation of acetylcholinesterase inhibitory activity and in vivo (mice) evaluation of antinociceptive activity (hot plate, tail immersion and acetylcholine-induced abdominal writhing tests) of trans-sabinol and its esters suggested that they may interact with different targets in crustacean/ mammalian organisms. Alongside moderate acute toxicity (LD50 (48 h) = 0.03–0.26 mmol/L), the tested compounds exert influence on both the peripheral and central nervous systems (in the hot plate test, trans-sabinyl tiglate, at 50 mg/kg, produced a 140% baseline increase 15 min after the treatment) and to moderately inhibit acetylcholinesterase (at the concentration of 20 μg/mL, these compounds caused a reduction of acetylcholinesterase activity up to 40%). © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Achillea falcata L. (syn. A. damascena DC.) is an endemic Mediterranean species highly valued for its pharmacological properties. In a number of Middle East countries, infusion of the aerial parts of A. falcata are traditionally used to treat stomach ache, fever, hemorrhoids, and so on (Aburjai et al., 2007; Alzweiri et al., 2011). In recent years, some pharmacological properties of this species were scientifically addressed. It has been demonstrated that some A. falcata sesquiterpene lactones are cytotoxic agents (Ghantous et al., 2009; Saikali et al., 2012; Salla et al., 2013; Tohme et al., 2013); its extracts/ essential oil have antimicrobial, antioxidant and antiplatelet properties (Aburjai and Hudaib, 2006; Karaalp et al., 2009; Konyahoglu and Karamenderes, 2004; Senatore et al., 2005); A. falcata infusions have protective effects against H2O2-induced oxidative

* Corresponding author. Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia. Tel.: +381 62 80 49 210; fax: +381 18 533 014. E-mail address: [email protected] (N.S. Radulovic´). http://dx.doi.org/10.1016/j.fct.2015.03.001 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

damage in human erythrocytes and leukocytes (Konyalioglu and Karamenderes, 2005). Botanical drugs based on or including A. falcata are regarded as completely safe for human use and non-toxic (Aburjai et al., 2007; Alzweiri et al., 2011). However, such belief might be dangerous for several reasons. Firstly, there are a few examples of Achillea species that are highly toxic or can cause allergies (Burrows and Tyrl, 2012; Radulovic´ et al., 2012a). Often, minor plant constituents were reported as those responsible for adverse effects of phytopreparations (Radulovic´ et al., 2012a, 2012b). Thus, in order to claim on the safety of use of a plant species, one should have at hand an as complete as possible chemical/toxicological profile of the plant species in question. However, phytochemical and activity data regarding A. falcata are quite scarce (Bruno et al., 2003; Hofer et al., 1986). Secondly, the major issue with botanical drugs is the high variability of their composition. For example, the essential oil of A. falcata was previously investigated on three occasions (Aburjai and Hudaib, 2006; Kürkçüoglu et al., 2003; Senatore et al., 2005). According to these studies, there are at least three different chemotypes of this species. In other words, the chemical composition of the studied populations was different, while their morphological and anatomical characteristics remained unchanged. A serious problem here was

N.S. Radulovic´ et al.E/Food and Chemical Toxicology 80 (2015) 114–129

that one of the reported A. falcata chemotypes produced a relatively high amount (almost 40% of the oil; oil yield 0.6%) of fragranol and its ester (Radulovic´ et al., 2012a; Senatore et al., 2005). It has been recently shown (Radulovic´ et al., 2012a) that this monoterpenic alcohol was responsible for the high acute toxicity of yet another representative of the genus Achillea (A. umbellata): LD50 value for the A. umbellata oil was lower than that of well known toxic essential oils of Artemisia absinthium, Salvia officinalis or Tanacetum vulgare (Radulovic´ et al., 2012a). To the best of our knowledge, there were no previous data regarding the toxicity of A. falcata metabolites. Thus, in order to provide at least some proof regarding the alleged safety of A. falcata-based botanical drugs, we have decided to evaluate the biological activity of an A. falcata essential oil sample at hand. Surprisingly, the preliminary chemical analyses (GC-FID and GC-MS) of the essential oil hydrodistilled from the aerial parts of A. falcata (wildgrowing population from Syria) suggested that we have discovered a novel chemotype of this plant species. The dominant volatiles (more than 40% of the total oil) of the currently studied A. falcata population seems to be trans-sabinol (1) and its esters (2–9; in general, these are rare natural products) (Scheme 1). According to Casares (1964), sabinene, sabinol and sabinyl acetate are the metabolites responsible for the toxicity of Juniperus sabina, which is included in the FDA (US Food and Drug administration) Poisonous Plant Database. This toxic juniper species has an irritant effect on the mucosal lining of the intestinal tract, causes congestion of the kidneys with hematuria, congestion of other abdominal viscera, menorrhagia and abortion. Also, Judzentiene and co-workers (2012) have shown that the A. absinthium essential oil rich in trans-sabinyl acetate (45.2% of the total oil) was highly toxic. Having the previously mentioned in mind, two main goals for this study were set. Firstly, we decided to unambiguously confirm the presence of trans-sabinol and its (rare) esters in A. falcata essential oil through synthesis of the appropriate pure standards and their co-chromatography (GC-MS) with the oil sample at hand. Alongside GC-MS, the identity/correct stereochemistry of the synthetically acquired compounds would be confirmed by NMR (1D and 2D experiments) and FTIR spectroscopies. The interpretation of NMR data was envisaged to be facilitated by the prediction of NMR shifts and H-H coupling constants (DFT level of theory). Also,

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in order to assess the distribution of trans-sabinyl esters in A. falcata, the roots of this species were also screened for the presence of these compounds. Our second goal was to assess the safety and potential pharmacological usefulness of the mentioned A. falcata volatiles. Brine shrimps (Artemia salina) are often considered as the most convenient test organism for the initial toxicity studies (Pages et al., 1996; Sorgeloos et al., 1978); thus, we have decided to evaluate the acute toxicity of A. falcata constituents in this model. Since, in some cases, antinociception and analgesia might be regarded as signs of acute intoxication (Akhila et al., 2007), we have decided to screen the potential analgesic potential of trans-sabinol and its esters using several different laboratory models (ACh (acetylcholine) writhing, hot plate and tail immersion tests). Certain structural characteristics of 2–11 seem to resemble those of rivastigmine and physostigmine (Fig. 1); these are well-known inhibitors of acetylcholinesterase (AChE) and are used as drugs in the treatment of Alzheimer’s disease (Bar-On et al., 2002). Thus, one might even expect trans-sabinyl esters to have (at least some) AChE inhibitory activity. Despite the unquestionable pharmacological usefulness of some AChE inhibitors, one should not forget the high toxicity of others (for example, well-known organophosphorus nerve agents) (Millard et al., 1999). For these reasons, we decided to evaluate, by both in silico (molecular docking) and in vitro studies, the possible AChE inhibitory activity of trans-sabinol and some of its esters. 2. Materials and methods 2.1. Plant material Plant material (aerial parts and roots), originating from a wild-growing Achillea falcata L. (Asteraceae) population, was collected in July, 2010, in the vicinity of the town of Ma’loula, Syria. The plant material was air-dried at room temperature. Voucher specimens were deposited at the Herbarium of the Faculty of Science and Mathematics, University of Niš, Serbia, under the accession number MM0982. 2.2. Chemistry 2.2.1. Chemicals and drugs All solvents (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Chemicals used for syntheses, including 4-dimethylaminopyridine (DMAP), N,N′-dicyclohexylcarbodiimide (DCC), anhydrous magnesium sulfate, as well as formic, propanoic, isobutanoic (2-methylpropanoic), butanoic, 2-methylbutanoic, isovaleric (3-methylbutanoic), senecioic (3-methyl-2-butenoic), tiglic ((E)-2-methyl-2butenoic) and angelic ((Z)-2-methyl-2-butenoic) acids, were of analytical grade, commercially available and used as received (Sigma-Aldrich, USA; Merck, Germany; Carl Roth, Germany; Fluka, Germany). trans-Sabinyl acetate was available to us from an in-house collection of plant secondary metabolites. All chemicals used in the bioassays (sodium dodecyl sulfate (SDS), acetylthiocholine iodide (ATCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), acetylcholinesterase from electric eel (AChE), bovine serum albumin (BSA), Tris-HCl, physostigmine) were of the highest grade available (Sigma-Aldrich, USA; Merck, Germany; Carl Roth, Germany; Fluka, Germany; Galenika, Serbia). Morphine (Hoffmann La Roche) and acetylcholine (ACh, Sigma-Aldrich, USA) used in animal experiments were freshly dissolved and applied in a maximal dose of 0.1 mL/animal and as detailed below. 2.2.2. Essential-oil extraction The dry aerial parts (500 g) and roots (250 g) were submitted to hydrodistillation for 2.5 h, using the original Clevenger-type apparatus (Radulovic´ et al., 2012a). The obtained yellowish oils of agreeable fragrant smell were separated by extraction with diethyl ether, dried over anhydrous magnesium sulfate and immediately analyzed by GC-FID and GC-MS. The yields of the oils obtained from the aerial parts (AF1) and roots (AF2) were 0.05 and 0.004% (w/w), respectively.

Scheme 1. Structure/synthesis of trans-sabinyl esters some of which are thought to be constituents of A. falcata oil (1 – trans-sabinol, 2 – trans-sabinyl formate, 3 – trans-sabinyl acetate, 4 – trans-sabinyl propanoate, 5 – trans-sabinyl isobutanoate, 6 – trans-sabinyl butanoate, 7 – trans-sabinyl 2-methylbutanoate, 8 – trans-sabinyl 3-methylbutanoate, 9 – trans-sabinyl tiglate, 10 – trans-sabinyl senecioate, 11 – transsabinyl angelate).

2.2.3. General experimental procedures NMR spectra (in CDCl3) were recorded at 25 °C on a Bruker Avance III 400 MHz NMR spectrometer (1H at 400 MHz, 13C at 100 MHz) using either 13CDCl3 or (CH3)4Si as reference for carbons and protons, respectively. Chemical shifts were expressed in δ (ppm) and coupling constants in hertz (Hz). The following abbreviations were used to designate multiplicities: br – broad signal, s – singlet, d – doublet, quint – quintuplet, sept – septuplet, psept – pseudoseptuplet, dd – doublet of doublets, ddd – doublet of doublets of doublets, dq – doublet of quartets, qq – quartet of quartets, pq – pseudo quartet. 2D experiments (NOESY and gradient 1H-1H COSY, HSQC and HMBC), as well as DEPT-90, DEPT-135 and selective homonuclear decoupling

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experiments, were run on the same instrument with built-in Bruker pulse sequences. IR measurements (neat, ATR-attenuated total reflectance) were carried out using a Thermo Nicolet model 6700 FT-IR instrument (Waltham, USA). Preparative medium-pressure liquid chromatography (MPLC) separations were performed on a Büchi system (C 610, Büchi, Switzerland) with pre-packed silica gel (40–63 μm) polypropylene cartridges under gradient conditions using n-hexaneEt2O mixtures as eluents at a flow rate of 2.5 mL/min. Silica gel 60 on Al plates, layer thickness 0.2 mm (Kieselgel 60 F254, Merck, Germany) was used for TLC. Spots on TLC were visualized by UV light (254 nm) and by spraying with 50% (v/v) aqueous H2SO4, followed by heating. GC-MS analyses (three repetitions) were carried out using a Hewlett-Packard 6890N gas chromatograph equipped with a fused silica capillary column DB-5MS (5% phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, USA) and coupled with a 5975B mass selective detector from the same company. The injector and interface were operated at 250 °C and 300 °C, respectively. Oven temperature was raised from 70 to 315 °C at a heating rate of 5 °C/min; the heating program ended with an isothermal period of 10 min. As a carrier gas helium at 1.0 mL/min was used. The samples (solutions of 10 mg of A. falcata essential oil, 1 mg of a pure synthesized ester or a mixture of 1 mg of an ester and 10 mg of A. falcata essential oil, in a total of 1 mL of Et2O) were injected in a pulsed split mode (injection volume was 1 μL; the flow was 1.5 mL/min for the first 0.5 min and then set to 1.0 mL/min throughout the remainder of the analysis; split ratio 40:1). MS conditions were as follows: ionization voltage of 70 eV, acquisition mass range 35–650, scan time 0.32 s. Oil constituents were identified by comparison of their GC retention indices (relative to C7-C23 n-alkanes (Van den Dool and Kratz, 1963) on the DB-5MS column) with literature values (Adams, 2007) and their mass spectra with those of authentic standards, as well as those from Wiley 6, NIST11, MassFinder 2.3, and a homemade MS library with the spectra corresponding to pure substances and components of known oils, and wherever possible, by co-injection with an authentic sample. A typical GC-MS chromatogram of sample AF1 is given in Fig. 2. The GC analyses were carried out using a Agilent 7890A GC system equipped with a single injector, one flame ionization detector (FID) and a fused silica capillary column HP-5MS (5% phenylmethylsiloxane, 30 m × 0.32 mm, film thickness 0.25 μm, Agilent Technologies, USA). The oven temperature was programmed from 150 to 300 °C at 15 °C/min and then held isothermally at 300 °C for 5 min; carrier gas was nitrogen at 3 mL/min; the injector temperature was held at 250 °C. The samples, 1 μL of the corresponding solutions, were injected in a splitless mode. The parameters of the FID detector were as follows: heater temperature – 300 °C; H2 flow – 30 mL/min; air flow – 400 mL/min; makeup flow – 23.5 mL/min; signal – 20 Hz. The amount of 9 in A. falcata essential oils was determined using GC-FID, by constructing a calibration curve (area under GC-FID peaks plotted against concentration;

calibration equation: y = 4671.38x + 9.937; R2 = 0.999). Experimentally determined content of this compound per 100 g of dry aerial parts was 0.625 mg. 2.2.4. Hydrolysis of trans-sabinyl acetate A solution of sodium methoxide was prepared by dissolving 140 mg of metallic sodium (6.09 mmol) in anhydrous methanol (10 mL) and cooling to room temperature. Then, trans-sabinyl acetate (3) (1.02 g, 5.26 mmol) dissolved in methanol was added to this solution, the reaction mixture brought to reflux (CaCl2 drying tube) and quenched with excess ice-water. This was followed by an acidification of the reaction mixture (HCl, 1:1, v/v), and the product was taken up by Et2O (3 × 50 mL). The organic layers were combined, dried over anhydrous MgSO4 and Et2O evaporated under reduced pressure to yield 792 mg (5.21 mmol) of 1 as a colorless liquid residue (GC-MS and NMR analyses revealed this to be pure trans-sabinol). 2.2.5. Syntheses of trans-sabinyl esters Esters of trans-sabinol (1) with formic (2), propanoic (4), isobutanoic (5), butanoic (6), 2-methylbutanoic (7), 3-methylbutanoic (8), tiglic (9), senecioic (10) and angelic (11) acids were prepared according to the following general Steglich procedure (N,N′-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP)). A solution of trans-sabinol (20 mg, 0.13 mmol), the appropriate carboxylic acid (0.14 mmol), DMAP (5 mg, 0.04 mmol), and DCC (30 mg, 0.15 mmol) in 10 mL of dry CH2Cl2 was stirred overnight at room temperature protected from atmospheric moisture (CaCl 2 tube). The precipitated urea was filtered off and the filtrate was concentrated in vacuo. A further portion of urea was separated in the same way after the addition of cold n-pentane. The resulting residue was purified by MPLC chromatography on silica gel using n-hexane/Et2O mixtures of increasing polarity as the eluents. The purity of the ester fractions was checked by TLC (3%, v/v, Et2O in n-hexane) and GC-MS. According to the GC-MS analyses the yields of the synthesized esters were in the range of 49 to 78%. In the case of esters 2, 9 and 10, the synthetic procedure was repeated on a larger scale, starting with three batches of trans-sabinol (3 × 150 mg), with the aim of obtaining sufficient quantities of these compounds (139–146 mg) for biological assays. Spectral (FTIR, MS) and/or chromatographic data (RI, DB-5MS) for transsabinol and its esters are given below and Fig. 3. 1D and 2D NMR spectral data for 1–3, 9, 10 are given in Table 2, Figs. 4 and 5 and Supplementary data. (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-ol (syn. transsabinol) (1): colorless liquid; RI (DB-5MS) 1142; for 1H and 13C NMR spectral data see Table 2; FTIR (ATR) cm−1: 3361, 2956, 2929, 2871, 1656, 1464, 1364, 1313, 1200, 1090, 1068, 1049, 1013, 958, 881, 832, 785; EIMS m/z (rel. int.): 152(0.2) [M+], 134(19), 120(3), 119(23), 117(5), 110(4), 109(24), 108(3), 107(6), 105(10), 95(21), 93(14),

Fig. 1. Structural similarities of trans-sabinyl esters 2–11 and AChE (acetylcholine esterase) inhibitors, rivastigmine and physostigmine (drugs used for the treatment of Alzheimer’s disease).


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Fig. 2. Typical GC-MS total ion current (TIC) and m/z 91 partial ion current (PIC) chromatograms of AF1-oil sample revealing the presence of trans-sabinol and sabinyl esters.

Fig. 3. Mass spectra of trans-sabinol (1), trans-sabinyl acetate (3) and 7 other “trans-sabinyl acetate-like” A. falcata volatiles (peaks 2, 4–9 of the AF1 GC-MS chromatogram, Fig. 2). Diagnostic fragment ions (fragmentation routes a and b) for acyl groups with 2 to 5 carbon atoms (C = 2–5) are marked with arrows.

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92(100), 91(99), 83(12), 82(5), 81(52), 80(5), 79(38), 78(5), 77(19), 70(11), 69(9), 67(13), 66(4), 65(11), 63(4), 55(20), 53(16), 51(7), 43(13), 41(23), 39(17). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl formate (syn. trans-sabinyl formate) (2): colorless liquid; Yield: 78%; RI (DB-5MS) 1227; for 1H and 13 C NMR spectral data see Table 2; FTIR (ATR) cm−1: 2912, 2852, 1743, 1652, 1601, 1523, 1460, 1376, 1236, 1159, 1118, 1054, 1019, 950, 893, 830, 721; EIMS m/z (rel. int.): 134(3), 119(8), 117(2), 108(5), 105(4), 93(6), 92(48), 91(100), 79(8), 78(3), 77(7), 69(5), 65(4), 53(4), 43(7), 41(9), 39(5). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl acetate (syn. trans-sabinyl acetate) (3): colorless liquid; RI (DB-5MS) 1291; for 1H and 13C NMR spectral data see Table 2; FTIR (ATR) cm−1: 2958, 2873, 1735, 1653, 1605, 1523, 1450, 1368, 1233, 1188, 1115, 1054, 1018, 966, 889, 826, 746; EIMS m/z (rel. int.): 134(5), 119(11), 117(3), 109(3), 108(10), 105(5), 93(6), 92(46), 91(100), 82(5), 79(7), 78(3), 77(8), 65(5), 53(4), 43(23), 41(7), 39(5). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl propanoate (syn. trans-sabinyl propanoate) (4): colorless liquid; Yield: 75%; RI (DB-5MS) 1379; EIMS m/z (rel. int.): 134(6), 119(12), 117(6), 109(4), 108(11), 105(3), 93(6), 92(50), 91(100), 82(4), 79(3), 78(5), 77(6), 65(4), 57(3), 53(5), 43(12), 41(6), 39(3). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl 2-methylpropanoate (syn. trans-sabinyl isobutyrate) (5): colorless liquid; Yield: 72%; RI (DB-5MS) 1414; EIMS m/z (rel. int.): 134(4), 119(9), 117(4), 109(3), 108(9), 105(4), 93(7), 92(49), 91(100), 82(7), 79(5), 78(3), 77(7), 65(5), 53(4), 43(18), 41(5), 39(4). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-yl butanoate (syn. trans-sabinyl butyrate) (6): colorless liquid; Yield: 70%; RI (DB-5MS) 1465; EIMS m/z (rel. int.): 134(6), 119(11), 117(2), 109(2), 108(8), 105(4), 93(6), 92(48), 91(100), 82(5), 79(4), 78(2), 77(5), 65(3), 53(3), 43(16), 41(8), 39(3). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl 2-methylbutanoate (syn. trans-sabinyl 2-methylbutyrate) (7), a 1:1 mixture of epimers differing in the configuration of the chiral center in the acid moiety: colorless liquid; Total yield: 63%; RI (DB-5MS) 1499.6 and 1500.4; both epimers had identical mass spectra, EIMS m/z (rel. int.): 134(8), 119(12), 117(2), 109(2), 108(3), 105(3), 93(6), 92(50), 91(100), 85(6), 79(4), 78(2), 77(5), 65(2), 57(18), 53(3), 43(5), 41(8), 39(3). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl 3-methylbutanoate (syn. trans-sabinyl isovalerate) (8): colorless liquid; Yield: 65%; RI (DB-5MS) 1513; EIMS m/z (rel. int.): 134(7), 119(12), 117(2), 109(3), 108(7), 107(2), 105(3), 93(6), 92(51), 91(100), 85(10), 79(4), 78(2), 77(5), 65(2), 57(11), 53(2), 43(6), 41(8), 39(4). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl (E)-2methylbut-2-enoate (syn. trans-sabinyl tiglate) (9): colorless liquid; Yield: 59%; RI (DB-5MS) 1610; for 1H and 13C NMR spectral data see Table 2; FTIR (ATR) cm−1: 2957, 2872, 1705, 1652, 1612, 1435, 1383, 1345, 1255, 1189, 1132, 1065, 1006, 957, 890, 836, 787, 733; EIMS m/z (rel. int.): 134(11), 119(19), 117(3), 108(10), 105(4), 101(5), 93(6), 92(49), 91(100), 83(26), 79(5), 77(6), 65(4), 55(20), 53(5), 43(5), 41(6), 39(5). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl 3-methylbut2-enoate (syn. trans-sabinyl senecioate) (10): colorless liquid; Yield: 63%; RI (DB5MS) 1612; for 1H and 13C NMR spectral data see Table 2; FTIR (ATR) cm−1: 2957, 2870, 1705, 1651, 1610, 1521, 1440, 1380, 1230, 1184, 1130, 1008, 960, 891, 830, 781, 732; EIMS m/z (rel. int.): 134(10), 119(19), 117(4), 108(10), 105(4), 101(7), 93(5), 92(47), 91(100), 83(56), 79(5), 77(7), 65(5), 55(17), 53(7), 43(8), 41(9), 39(8). (1R*,3S*,5R*)-4-Methylene-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-yl (Z)-2methylbut-2-enoate (syn. trans-sabinyl angelate) (11): colorless liquid; Yield: 49%; RI (DB-5MS) 1557; EIMS m/z (rel. int.): 134(9), 119(14), 105(3), 101(3), 93(6), 92(50), 91(100), 83(22), 79(4), 77(5), 65(3), 55(19), 53(4), 43(5), 41(5), 39(4). 2.3. Theoretical study 2.3.1. Geometry optimization and calculation of NMR shifts and spin–spin coupling constants All quantum chemical calculations were performed using density functional theory (DFT), specifically Becke’s three-parameter hybrid functional combined with Lee– Yang–Parr correlation functional, B3LYP. For all atoms the 6-311G**(d,p) basis set was employed. Proton chemical shifts and H-H coupling constants were computed using GIAO-DFT method (B3LYP functional with 6-311G**(d,p) basis set. The calculations were performed using Gaussian 03 program package (Frisch et al., 2004). 2.3.2. Docking experiments trans-Sabinol (1), trans-sabinyl esters (2–11) and well known AChE inhibitors rivastigmine (RS) and physostigmine (PS) were docked into the crystal structure of acetylcholine esterase (AChE) from Torpedo californica (PDB code: 1EA5). All docking experiments were performed using AutoDock Vina 1.1.2 software (Trott and Olson, 2010), as blind dockings (Rnjan et al., 2010). Grid box (center at x, y, z = 4.91, 64.80, 56.36) covered the entire enzyme; size of search space (volume of the grid box) was set to be 60 × 66 × 60 Å. Autodock Vina docking was performed using the exhaustiveness value of 500, while the number of search modes was set to 20. All other parameters were used as defaults. The ligands were allowed to flexibly dock, but the receptor backbone and side chains remained rigid during the docking. For every docked compound, geometry optimized on DFT level of theory (6–311**(d,p) B3LYP) was used as the input conformation. Autodock Tools version 1.5.6 was used to convert the ligand and receptor molecules to proper file formats (pdbqt) for AutoDock Vina docking. The same program was used for the visualization of docking results (Sanner,

1999). All in silico experiments were run using Intel® Core™ i7-3930K 3.20 GHz Six core unlocked CPU Processor. 2.4. Biology and pharmacology 2.4.1. Evaluation of acute toxicity in Artemia salina Acute toxicity in Artemia salina was evaluated using the method previously described by Radulovic´ et al. (2013b). Final concentrations of the tested samples dissolved in aqueous DMSO were as follows: 0.2, 0.1, 0.05, 0.025, 0.005 and 0.0025 mg/mL, whereas the final concentration of DMSO was much less than 1% (v/v). DMSO was inactive under the stated conditions as demonstrated by a negative control. Dead nauplii were counted after 24, 48 and 72 h. LC50 (concentration lethal to 50% of nauplii) were determined after statistical analysis. Sodium dodecyl sulfate (SDS) was used as the positive control. All the tests were performed in triplicate and repeated twice. 2.4.2. AChE (acetylcholine esterase) inhibitory activity AChE inhibitory activities of the synthesized compounds 1, 2, 3, 9 and 10 were measured by a quantitative colorimetric assay based on Ellman’s method (Adhami et al., 2012; Radulovic´ et al., 2013b). A solution of 10% aqueous DMSO (v/v) was used as the negative control. The absorbance resulting from the spontaneous hydrolysis of the substrate (the absorbance measured before the addition of the enzyme (blind probe)) was subtracted from that recorded one after the addition of AChE. For validation, different concentrations of physostigmine (several concentrations in the range 0.12–15 μmol/mL) served as a positive control. Each experiment was carried out in triplicate and repeated three times. 2.4.3. Antinociceptive activity 2.4.3.1. Animals and treatment. Male BALB/c mice (20 ± 5 g), 5-weeks-old, were obtained from the Vivarium of the Institute of Biomedical Research, Medical Faculty, University of Niš, Serbia. Animals were housed in groups of 6 under standard laboratory conditions: 12 h light/dark cycle at 22 ± 2 °C, with food and water available ad libitum. Animals were acclimatized to the laboratory environment for at least 12 h before testing (fasted, though still allowed free access to water). Fifteen experimental and two control (positive and negative) groups, with 6 animals per group, for ACh writhing (AChW), hot plate (HP) and tail immersion (TI) experiments were used. For the hot plate and tail immersion tests, trans-sabinol and its five esters (the formate, acetate, tiglate, senecioate and angelate) were administered intraperitoneally (i.p.) as solutions in olive oil just prior to the experiments, at doses of 12.5, 25 and 50 mg/ kg, to fifteen groups, whereas two more groups received morphine at 5 mg/kg and olive oil (vehicle group). In the abdominal writhing test, the animals received the appropriate substances 1 hour before ACh. All experimental procedures were conducted in accordance with the principles of care and use of laboratory animals in research (EEC Directive of 1986; 86/609/EEC) and were approved by the local Ethics committee (No. 01–10204-4). 2.4.3.2. Hot plate and tail immersion tests. Mice were tested according to the method described by Radulovic´ et al. (2013d). Animals were placed on a hot plate set at 55 ± 1 °C or the lower portion of their tails were immersed in water maintained at 50 ± 0.5 °C after what the behavior was observed as previously described (Radulovic´ et al., 2013d). The latency for reaction was measured at 15, 30, 45, 60, 90 and 120 min after the administration of substances. Baseline (BL) was considered as the mean of reaction time obtained at 60 and 30 min before the administration of compounds 1, 2, 3, 9 and 10, the vehicle or morphine and defined as the normal reaction of an animal to the temperature. Increase in baseline (%) was calculated by the formula: ((reaction time × 100)/BL) − 100. Antinociception was quantified as area under the curve (AUC) of responses and was calculated as a sum of AUC1 = 15 × IB[(min15)/ 2 + (min30) + (min45) + (min60)/2] and AUC2 = 30 × IB[(min60)/2 + (min90) + (min120)/ 2] where IB is the increase in baseline (in %). Percentage of inhibition for morphine and each dose of compounds 1, 2, 3, 9 and 10 were calculated as follows: % of inhibition = AUC/max; where max = [(20 × 100)/BL−100] × 105. 2.4.3.3. Acetylcholine-induced abdominal writhing. Animals were treated as described in section 2.4.3.1.; one hour later an i.p. injection of ACh (5 mg/kg) was applied and the number of abdominal writhes was recorded during 5 min after the injection (Bittencourt et al., 1995). The percent of writhing decrease was calculated as described elsewhere (Radulovic´ et al., 2013c).

3. Results and discussion 3.1. Chemistry 3.1.1. GC-FID and GC-MS analyses of A. falcata essential oils: detection and identification of trans-sabinyl esters The two essential-oil samples isolated from the aerial and underground parts of A. falcata (samples AF1 and AF2, respectively) were analyzed in detail by GC-FID and GC-MS. These analyses enabled the identification of 180 different AF1 and AF2


constituents, representing 97.4–98.4% of the total GC peak areas (Table 1). Typical GC-MS total ion current (TIC) chromatogram of AF1 is given in Fig. 2. The two main constituents of sample AF1 oil were trans-sabinol (1; 19.1%) and trans-sabinyl acetate (3; 11.4%). The initial tentative GC-MS identification of these compounds, based solely on the matching of the corresponding retention indices and mass spectra with literature data (Adams, 2007; Cégiéla-Carlioz et al., 2005; Mack et al., 2013), was additionally corroborated by GC co-injection of standards with the oil. The standard of 1 was acquired by hydrolysis of 3, which was available from an in-house collection of plant secondary metabolites (the identity of both standards was confirmed by NMR; the results of spectral analyses are discussed in the following subsection). trans-Sabinol and trans-sabinyl acetate were among the major constituents of sample AF2 as well (6.5 and 4.2%, respectively). Mass spectra (MS) of trans-sabinol and trans-sabinyl acetate are given in Fig. 3. In general, MS fragmentation of both compounds was similar. For example, the most intense ions in the mass spectra of 1 and 3 were m/z 91 and 92. In addition, several low intensity (relative to the base peak) fragment ions (for example, m/z 134, 119, 55) were observable in the MS of both compounds. Fragment ion with m/z 81, abundant (rel. int. 52%) in the MS of 1, was not present in the case of 3. As expected, the diagnostic fragment ion for an acetyl group (m/z 43, CH3CO+; cleavage of C—O bond; marked with an arrow in Fig. 3), clearly observable in the MS of trans-sabinyl acetate, was of negligible intensity in the MS of 1. MS fragmentation of the GC-MS peaks designated as 4, 5, 7 and 8 (GC-MS chromatogram of AF1, Fig. 2) was almost identical with that of trans-sabinyl acetate (Fig. 3). In order to locate additional minor (trace) AF1 and AF2 constituents with the MS fragmentation analogous to that of 3, we generated a partial ion current (PIC) chromatogram of AF1/AF2 that showed changes of the m/z 91 ion current with time (Fig. 2). This allowed us to detect seven different “trans-sabinyl acetate-like” compounds (GC-MS peaks 2, 4–9, Fig. 2); we assumed that these were also esters of transsabinol. For all of these compounds, the MS base peak was at m/z 91; m/z 92 ion was the second most abundant one. The intensity of the majority of other detected fragment ions (m/z 108, 119, 134) was much lower in comparison to m/z 91 and 92 (Fig. 3). Further inspection of the mass spectra of these compounds revealed the presence of fragment ions that pointed to the identity of the acid moiety. For example, mass spectra of compounds 5 and 6 contained ions at m/z 43 and 71. These might be regarded as diagnostic fragment ions of esters of butanoic or isobutanoic acid–fragmentation routes a (C3H7CO+) and b (C3H7+) (Fig. 3). Relative to the value of RI of trans-sabinyl acetate (RI 1291), RIs of compounds 5 and 6 (DB5-MS column) were for 123, i.e. 174 units higher (RI 1414 and 1465, respectively). This also suggested that 5 and 6 might be branched/straight-chain dihomologues of 3, i.e. trans-sabinyl isobutanoate and butanoate (Table 1, Fig. 3). According to this, we have assumed that compound 4 was trans-sabinyl propanoate (fragment ion m/z 57: C2H5CO+; RI 1379 = RIacetate + 88); compounds 7 and 8 were most probably trans-sabinyl 2- and 3-methylbutanoates (fragment ions m/z 57 and 85: C4H9+ and C4H9CO+; RI 1500 or 1513 = RIbutanoate + 35 or 48); compound 1 was most probably trans-sabinyl formate (RI 1291) (Fig. 3). Presence of m/z 83 (C4H7CO+) and 55 (C4H7+) fragment ions in the MS of 9 suggested this was trans-sabinyl ester of one of the isomeric C5H8O2 unsaturated carboxylic acids (i.e. ((E)-2-methyl-2-butenoic (tiglic), (Z)-2-methyl-2-butenoic (angelic) or 3-methyl-2-butenoic (senecioic)) (Radulovic´ et al., 2013b). The value of the retention index for this compound (RI 1610) suggested this was an ester of a branched rather than a straight-chain acid. The standards of trans-sabinyl esters of formic, propanoic, isobutanoic, butanoic, 2-methylbutanoic, 3-methylbutanoic

119

Table 1 Chemical composition of the essential-oil samples obtained from above- (AF1) and underground parts (AF2) of A. falcata. RIa

Compound

731 762 765

831 844 846 865 873 904 908 928 934 944 948 955 971 975 978 978 991 992 1000 1001 1002 1008 1017 1023 1030 1032 1033 1038 1059 1061 1067 1068 1071 1073 1082 1087 1088 1096 1100 1104 1104 1107 1112 1118 1120 1122 1126 1135 1140 1142

3-Methylbutanol 1-Pentanol 3-Methyl-3-buten-1-ol (syn. Prenol) 3-Methyl-3-butenal (syn. prenal) Hexanal 3-Methylbutanoic acid (syn. isovaleric acid) Furfural Furfuryl alcohol (E)-3-Hexen-1-ol 1-Hexanol (Z)-4-Hexen-1-ol Heptanal Santolina triene α-Thujene α-Pinene 6-Methyl-2-heptanone Camphene Benzaldehyde Sabinene 1-Octen-3-ol 6-Methyl-5-hepten-2-one β-Pinene 2-Pentylfuran Dehydro-1,8-cineole Yomogi alcohol Octanal Isobutyl isovalerate (2E,4E)-2,4-Heptadienal α-Terpinene p-Cymene Santolina alcohol Benzyl alcohol 1,8-Cineole Phenylacetaldehyde Artemisia ketone γ-Terpinene (Z)-3-Hexenyl propanoate Acetophenone p-Cresol cis-Sabinene hydrate Artemisia alcohol Guaiacol Terpinolene Linalool Undecane Nonanal trans-Sabinene hydrate cis-Thujone Phenylethyl alcohol trans-Thujone Dehydrosabina ketone Isophorone Chrysanthenone cis-p-Mentha-2,8-dien-1-ol p-Menth-3-en-8-ol trans-Sabinol (1)

1144 1146 1148 1154 1157 1158 1164 1165 1168 1170 1171 1172

cis-Verbenol trans-Verbenol Camphor Eucarvone β-Pinene oxide Sabina ketone Pinocarvone 1-Nonanol Umbellulone δ-Terpineol p-Mentha-1,5-dien-8-ol Borneol

778 802 828

Percentage (%)b

Identificationc

AF1

AF2

0.1 tr tr

tr / tr

RI, MS RI, MS, Co-GC RI, MS

tr

/

RI, MS

tr 1.4

tr tr

RI, MS, Co-GC RI, MS, Co-GC

tr 1.2 tr tr tr tr tr tr 2.8 tr tr 0.2 0.4 tr tr tr tr tr 0.5 tr / tr 0.3 0.4 3.0 / 4.9 tr 1.9 0.2 0.4 tr / 0.9 0.5 tr 0.1 tr / / 1.1 0.2 0.1 1.5 tr tr 0.3 0.2 tr 19.1

tr tr / 0.4 / / / / / / / 1.6 / tr / / / / / / 0.8 / / / 0.9 1.5 tr 1.8 / / / 0.5 tr tr tr 0.4 / / tr 0.7 / / 1.3 tr / / tr tr / 6.5

0.5 0.9 2.9 0.3 tr 0.3 0.2 / 0.4 0.2 tr 2.0

0.1 tr tr tr / / / 0.3 / tr / 0.8

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC, NMR RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC

(continued on next page)

120


Table 1 (continued) RIa

Compound

Table 1 (continued) Percentage (%)b AF1

AF2

Identificationc

1174 1175 1179 1181 1189 1194 1195 1198 1198 1199 1200 1203 1208 1214 1219 1227

Menthol cis-Pinocamphone Terpinen-4-ol Thuj-3-en-10-al p-Cymen-8-ol Myrtenol (Z)-4-Decenal α-Terpineol Myrtenal 1-Dodecene Dodecane Decanal trans-Piperitol Fragranol trans-Carveol trans-Sabinyl formate (2)

tr tr 1.2 0.2 tr 0.2 tr 0.6 0.3 tr / 0.2 0.4 3.3 0.2 0.3

tr / 0.8 / / / tr 0.6 tr / 0.7 0.6 tr 1.8 / tr

1231 1233

cis-Carveol (Z)-3-Hexenyl 2-methylbutanoate Cumin aldehyde Carvone cis-Myrtanol Piperitone Fragranyl formate Nonanoic acid 1-Decanol Perilla aldehyde Bornyl acetate trans-Sabinyl acetate (3)

tr 0.1

/ tr

0.5 tr / 0.3 tr / tr / 0.2 11.4

tr / 1.2 0.4 / tr 0.3 tr / 4.2

0.4 tr tr 0.1 tr 0.1 / tr 0.3 / 2.0

tr 1.0 tr 1.5 tr / tr / 5.9 tr 0.4

RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC, NMR RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS RI, MS, Co-GC

tr 0.1 tr / tr / 0.3 2.7

0.6 / / tr / tr tr 0.8

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC

1241 1244 1250 1254 1259 1262 1269 1275 1287 1291 1299 1300 1305 1308 1316 1330 1341 1349 1357 1365 1379 1384 1387 1390 1391 1392 1396 1406 1414 1423 1426 1429 1433 1438 1453 1454 1465 1467 1470 1479 1482 1484 1491 1500 1512 1513 1516 1518

Thymol Tridecane Undecanal Carvacrol (2E,4E)-2,4-Decadienal p-Mentha-1,4-dien-7-ol Fragranyl acetate α-Cubebene Eugenol Decanoic acid trans-Sabinyl propanoate (4) α-Copaene β-Bourbonene β-Cubebene 1-Tetradecene (E)-Jasmone Vanillin Methyl eugenol trans-Sabinyl isobutanoate (5) α-Cedrene (E)-Caryophyllene β-Cedrene β-Copaene Fragranyl propanoate Geranyl acetone (E)-β-Farnesene trans-Sabinyl butanoate (6) 10-epi-β-Acoradiene 1-Dodecanol Fragranyl 2-methylpropanoate ar-Curcumene Germacrene D β-Selinene trans-Sabinyl 2-methylbutanoated (7) Tridecanal trans-Sabinyl 3-methylbutanoate (8) Sesquicineole Cubebol

RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS, Co-GC, NMR RI, MS RI, MS

RIa

1520 1525 1544 1563 1582 1584 1586 1587 1593 1597 1600 1610 1616 1617 1620 1638 1658 1658 1679 1679 1679 1690 1700 1719 1720 1729 1745 1751 1760 1763 1781 1801 1826 1828 1847 1888 1900 1932 1933 1966 2095 2100 2138 2220 2285 2300

Compound

δ-Cadinene β-Sesquiphellandrene α-Calacorene (E)-Nerolidol Mintoxide Fragranyl 3-methylbutanoate Spathulenol Caryophyllene oxide β-Copaen-4-α-ol 4(14)-Salvialene-1-one Hexadecane trans-Sabinyl tiglate (9) Junenol Unidentifiede Torilenol epi-α-Murrolol Acorenol B β-Eudesmol Mustakone 1-Tetradecanol Valeranone Germacra-4(15),5,10(14)trien-1α-ol Amorpha-4,9-dien-2-ol ar-Curcumen-15-al Pentadecanal Fragranyl hexanoate Isobicyclogermacrenal γ-Costol β-Acoradienol Tetradecanoic acid 14-Hydroxy-α-muurolene 14-Hydroxy-δ-cadinene Isopropyl tetradecanoate Fragranyl heptanoate Hexahydrofarnesyl acetone 1-Hexadecanol Nonadecane Methyl hexadecanoate Fragranyl octanoate Hexadecanoic acid 1-Heneicosene Heneicosane Linoleic acid Eicosanal 1-Tricosene Tricosane Total

Percentage (%)b

Identificationc

AF1

AF2

tr 1.5 tr tr 0.4 tr

tr 4.2 / 4.5 / /

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Co-GC

2.2 0.4 tr 0.5 / 1.8

6.4 2.0 / 1.7 tr 2.1

0.2 1.6 0.3 0.3 0.7 0.4 0.2 / 0.1 0.5

/ 2.7 0.8 / / 2.7 0.6 1.0 / /

RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC, NMR RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS

0.1 0.2 0.2 0.2 0.2 / 0.3 / tr 0.3 1.0 tr 1.2 tr tr tr tr tr tr tr / / / tr 98.4

tr 1.3 0.8 tr tr 8.1 1.3 tr / 0.4 tr / 0.1 0.8 tr / / 5.9 tr tr 1.2 tr tr tr 97.4

RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS, Co-GC RI, MS, Co-GC RI, MS RI, MS RI, MS, Co-GC

a

RI – experimentally determined linear retention indices on an DB-5MS column. Values are means of triplicate analyses; tr – trace amounts (