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Thymyl esters derivatives and a new natural

product modhephanone from Pulicaria
mauritanica Coss. (Asteraceae) root oil

ARTICLE in FLAVOUR AND FRAGRANCE JOURNAL · JANUARY 2015

Impact Factor: 1.97 · DOI: 10.1002/ffj.3223

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Tao xu C. Bekhechi
Université de Corse Pascal Paoli Abou Bakr Belkaid University of Tlemcen
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Retrieved on: 14 January 2016
Special Issue: Research Article
Received: 23 June 2014, Revised: 2 October 2014, Accepted: 14 October 2014 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/ffj.3223

Thymyl esters derivatives and a new natural

product modhephanone from Pulicaria
mauritanica Coss. (Asteraceae) root oil‡
Tao Xu,a† Mohammed Gherib,b† Chahrazed Bekhechi,b Fewzia Atik-Bekkara,b
Hervé Casabianca,c Félix Tomi,a* Joseph Casanovaa and Ange Bighellia
ABSTRACT: Root essential oil of Pulicaria mauritanica Coss. (Asteraceae) collected from western Algeria was analysed using a
combination of chromatographic (CC, GC/FID, GC(RI)] and spectroscopic [GC–MS, 13C-NMR] techniques. Thirty-eight com-
pounds accounting for 90.4% of the whole composition were identified. The oil composition was dominated by thymyl deriv-
atives (2,5-dimethoxy-p-cymene (37.2%), 6-methoxythymyl isobutyrate (14.2%), 10-isobutyryloxy-8,9-dehydrothymyl
isobutyrate (4.8%) and thymyl isobutyrate (3.1%)), as well as by neryl isobutyrate (11.1%). A new natural compound was
isolated and the structure was elucidated as 1S,2S,5S,8S-modhephan-3-one (relative stereochemistry). Copyright © 2014 John
Wiley & Sons, Ltd.

Keywords: Pulicaria mauritanica; root oil; thymol derivatives; structure elucidation; modhephanone

Introduction Experimental
The Pulicaria genus (Asteraceae), which consists of 100 Chemicals
species, is well distributed from Europe to North Africa, par-
ticularly around the Mediterranean basin, as well as in Asia.[1] Isobutyryl chloride and trimethylamine were obtained from Sigma-Aldrich-
Pulicaria mauritanica Coss. is an endemic species of Morocco Fluka (Saint-Quentin Fallavier, France). For measurement of response
factors (RFs), reference compounds (camphene, carvacrol, carvone, (E)-b-
and Algeria. This herbaceous plant with a strong characteris-
caryophyllene, caryophyllene oxide) were obtained in the highest available
tic odour[2] is one of the 13 Pulicaria species growing wild in purity from Sigma-Aldrich-Fluka (Saint-Quentin Fallavier, France). Carvacryl
Algeria, and its botanical characters have been described in isobutyrate was obtained by synthesis and 6-methoxythymyl isobutyrate
literature.[3] In the Nâama province (western Algeria), the was purified by preparative thin-layer chromatography from fraction
local herbalists traditionally use this wild plant to treat F3.3 (see later) obtained by column chromatography (CC).
inflammation-related diseases as well as for treatment of
intestinal disorders and headache (by fumigation). In some Plant Material
Moroccan localities, it is also used as a herbal tea and to
make various foods. Roots of P. mauritanica were collected in April 2013 (Aïn Ouarka, Nâama
province, Western Algeria). Professor A. Marouf (University of Nâama,
Recently, Liu et al.[4] reviewed the phytochemical data
Algeria) identified plant material and a voucher specimen has been de-
concerning the genus Pulicaria and a few of these studies
posited at the Laboratory of Natural Products, (Department of Biology,
concerned the chemical composition of P. mauritanica essen- University of Tlemcen, Algeria), under accession number A2778.
tial oil. Indeed, according to the recent literature[5] the occur-
rence of carvotanacetone as major component (81%) of
essential oil isolated from aerial parts was mentioned as early * Correspondence to: F. Tomi, Université de Corse-CNRS, UMR CNRS 6134,
as 1949 by Gateffossé and Igolen. A similar composition has Equipe Chimie et Biomasse, Route des Sanguinaires, 20000 Ajaccio, France.
E-mail: felix.tomi@univ-corse.fr
been reported recently for Moroccan oils (four samples,
mean content of carvotanacetone = 87.3%)[5,6] and Algerian †
T. Xu and M. Gherib contributed equally to this work.
oils (36 samples, content of carvotanacetone = 89.2–96.1%).
‡
The antimicrobial effect of the essential oil was evaluated This article is part of the virtual special issue of the Flavour and Fragrance Journal
against bacteria, yeast and filamentous fungi (lowest mini- entitled "Essential oils: chemical analysis and biological properties" edited by
Patrizia Rubiolo and Paula Dugo.
mum inhibitory concentration (MIC) value = 2 μl/ml).[7] How-
ever, to our knowledge, no phytochemical investigations on a
Université de Corse-CNRS, UMR CNRS 6134, Equipe Chimie et Biomasse,
P. mauritanica root essential oil have been undertaken to Route des Sanguinaires, 20000 Ajaccio, France
date. Thus in continuation of our work on the characteriza- b
Laboratoire des Produits Naturels, Département de Biologie, Université
tion of aromatic and medicinal plants of Africa, the present Abou Bekr Belkaid, Tlemcen, Algérie
work concerns the chemical composition of P. mauritanica
root essential oil. c
Institut des Sciences Analytiques, 5 Rue de la Doua, 69100 Villeurbanne, France
83

Flavour Fragr. J. 2015, 30, 83–90 Copyright © 2014 John Wiley & Sons, Ltd.
 T. Xu et al.

Essential Oil Isolation and Fractionation internal standard, pentafluorobromobenzene, was introduced continu-
ously into the Electron Impact source, from a reference reservoir at
Roots of P. mauritanica (3 × 350–400 g) were submitted to hydro- 50°C and through a reference inlet at 120°C. The mass m/z 181.0077
distillation using a Clevenger type apparatus for 3 h, and yielded a pale
was used as lock mass. Data were processed with MassLynx 4.1 (Waters).
yellow oil (1.932 g in total). The essential oil (1.707 g) was chro-
matographed on silica gel (200–500 μm, 40 g) and six fractions were
eluted with a gradient of solvents pentane/diethyl ether (P/DE) from
Nuclear Magnetic Resonance
100:0 to 0:100. Fraction F1 (52 mg, eluted with P) contained hydrocar-
bons; fractions F2–F5 (701, 266, 233 and 165 mg, respectively, eluted All nuclear magnetic resonance (NMR) spectra were recorded on a
1
with P:DE mixtures) contained medium polar compounds; fraction F6 Bruker AVANCE 400 (Wissembourg, France) (400.132 MHz for H and
13
(268 mg, eluted with DE) contained polar compounds. Fraction F3 was 100.623 MHz for C) equipped with a 5-mm probe, in deuterated
repeatedly fractionated with a mixture of pentane/ethyl acetate (P/EA) chloroform (CDCl3), with all shifts referred to internal tetramethylsilane
= 120:1 on silica gel (63–200 μm, 6 g) impregnated with 1.2 g of AgNO3 1
(TMS). The H-NMR spectra were recorded with the following parame-
(20% w/w). Three fractions F3.1–F3.3 (23, 178, and 67 mg respectively) ters: pulse width (PW), 4.3 μs; relaxation delay 1 s and acquisition time
were eluted. Fraction F3.2 was subjected to silica gel chromatography 2.6 s for 32 K data table with a spectral width (SW) of 6000 Hz. The
13
(63–200 μm, 4 g) using a gradient of solvents (P/DE) of increasing C-NMR spectra were recorded with the following parameters: pulse
polarity as mobile phase and yielded subfractions F3.2a (17 mg) and width, 4 μs (flip angle 45°); acquisition time, 2.7 s for 128 K data table
F3.2b (23 mg). Fraction F5 was again fractionated with P/DE = 100:1 on with a spectral width of 24 000 Hz (240 ppm); composite pulse
silica gel (63–200 μm, 4 g) and yielded F5.1 and F5.2 (12 and 125 mg decoupling (CPD) mode; digital resolution, 0.183 Hz/pt. Standard pulse
respectively). Fraction F6 was fractionated on silica gel (63–200 μm, sequences from Bruker library were used for two-dimensional spectra.
6 g) with P/DE = 90:10 to 0:100 and yielded F6.1–F6.4 (47, 69, 21 and Gradient-enhanced sequences were used for the heteronuclear two-
25 mg, respectively). dimensional experiments.

Gas Chromatography with Flame Ionization Detection Identification of Individual Components

Analyses were performed on a Clarus 500 Perkin Elmer (Courtaboeuf, Identification of the individual components was based on: (i) comparison
France) chromatograph equipped with flame ionization detectors (FID) of their GC RIs on apolar and polar columns, determined relative to the
and two fused-silica capillary columns (50 m × 0.22 mm, film thickness retention times of a series of n-alkanes with linear interpolation (‘Target
0.25 μm), BP-1 (polydimethyl siloxane) and BP-20 (polyethylene glycol). Compounds’ software of Perkin-Elmer), with those of authentic com-
The oven temperature was programmed from 60 to 220°C at 2°C/min pounds, or literature data; (ii) computer search using digital libraries of
13
and then held isothermal at 220°C for 20 min: injector temperature, mass spectral data[8,9] and/or literature data[10,11]; (iii) C-NMR spectros-
250°C; detector temperature, 250°C; carrier gas, helium (0.8 mL/min); copy, following a computerized method developed in our labora-
split, 1/60. Retention indices (RI) were determined relative to the reten- tory,[12–14] using a home-made software, by comparison of the chemical
tion times of a series of n-alkanes with linear interpolation (Target Com- shift values of the signals in the essential oil spectrum with those of
pounds software from Perkin Elmer). reference compounds compiled in a laboratory-built library. This method
allows the identification of individual components of the essential oil at
content as low as 0.3–0.4%. A few compounds were identified by compar-
Gas Chromatography–Mass Spectrometry in Electron Impact ison with literature data.
Mode
Mass spectra of compounds 13, 15, 29, 31, 32, 33, 34
Fractions F1–F6 obtained by CC were analysed with a Perkin-Elmer
+
TurboMass detector (quadrupole), directly coupled to a Perkin-Elmer 2,5-dimethoxy-p-cymene 13: m/z (%), 194(45) [M ], 179(100), 164(41),
Autosystem XL (Courtaboeuf, France) equipped with a fused-silica 91(10); 8,9-dehydro-2,5-dimethoxy-p-cymene 15: m/z (%), 192(90)
+
capillary column (60 m × 0.22 mm i.d., film thickness 0.25 μm), Rtx-1 [M ], 177(100), 162(31), 91(30); modhephan-3-one 29: HRMS 220.1831
+
(polydimethylsiloxane). Carrier gas, helium at 1 mL/min; split, 1/80; (98) [M ], 149.1327(49), 121.1004(100), 93.0686(31); 6-methoxythymyl
+
injection volume, 0.2 μL; injector temperature, 250°C; oven tempera- isobutyrate 31: m/z (%), 248(10) [M ], 178(100), 180(18), 163(31), 91
ture programmed from 60 to 230°C at 2°C/min and then held isother- (10), 43(50); 6-methoxy-8,9-dehydrothymyl isobutyrate 32: m/z (%),
+
mal (45 min); ion source temperature, 150°C; energy ionization, 70 eV; 250(10) [M ], 180(100), 165(57), 91(8), 43(50); 10-isobutyryloxy thymyl
+
electron ionization mass spectra were acquired over the mass range isobutyrate 33: m/z (%), 306(3) [M ], 236(7), 218(27),175(9), 148(100),
35–350 Da. 135(35), 133(32), 105(11), 91(13), 71(40); 10-isobutyryloxy-8,9-
+
dehydrothymyl isobutyrate 34: m/z (%), 304(6) [M ], 234(10), 216(7),
146(100), 145(39), 115(9), 105(11), 91(10), 71(38).
Gas Chromatography–High-resolution Mass Spectrometry
Gas chromatography with time-of-flight mass spectrometry (GC–TOF/MS)
Quantification of Components
analyses were performed on an Agilent 6890 gas chromatograph
coupled to a TOF/MS GCT Premier from Waters equipped with a The relative amounts of volatile components, expressed as percent-
column (30 m × 0.25 mm i.d., film thickness 0.25 μm), DB-5MS UI: ages, were obtained by FID peak-area normalization by calculating
injection volume, 1 μL; mode, splitless; oven temperature programmed the response factors (RFs)[15,16] of FID for different classes of
from 60°C (1 min) to 320°C (25°C/min), held at 320°C for 10 min. volatiles (monoterpene hydrocarbons, oxygenated monoterpenes,
Transfer line temperature at 250°C and source temperature at 200°C. sesquiterpene hydrocarbons, oxygenated sesquiterpenes, oxygen-
The mass spectrometer was operated in the electron impact mode ated p-cymene derivatives) occurring in the essential oil. Owing to
(energy ionisation, 70 eV). Multichannel plate voltage was set at the large number of compounds present and the non-availability
2600 V, acquisition rate at 10 spectra/s (i.e. 5 spectra/s with ‘Dynamic of commercial standards for many of them, compounds identified
Range Enhancement’ mode on) and pusher interval at 40 μs. Acquisi- from essential oils (EOs) of each class were assumed to have the
tion was performed in the full-scan mode with a scan range of m/z same quantitative GC response factors. The RFs of reference
50–550. Calibration was undertaken using the Calibration Wizard, with compounds and the components of the EO to which they have
heptacosane as the reference. The mass resolution was around 7000 been applied are reported in Table 1. The RFs were calculated using
full-width at half maximum for m/z 218.9856. During acquisitions, an the formula:
84

wileyonlinelibrary.com/journal/ffj Copyright © 2014 John Wiley & Sons, Ltd. Flavour Fragr. J. 2015, 30, 83–90
Thymyl esters and modhephanone from Pulicaria mauritanica root oil

Table 1. Calculated response factors (RFs) for reference carvacryl isobutyrate (147 mg, 0.7 mmol, yield 18%), retention indices
a p 1
compounds RI , RI = 1489,1935. H-NMR (d, ppm vs. TMS; J, Hz) = 7.18 (d, J = 7.0
Hz, 1H, Ar-H), 7.04 (d, J = 7.5 Hz, Ar-H), 6.88 (s, 1H, Ar-H), 2.83–2.93
(m, 2H, CH), 2.16 (S, 3H, CH3), 1.37 (d, J = 6.5 Hz, 6H, CH3) 1.25 (d,
Compounds RFa RF applied 13
J = 7.0 Hz, 6H, CH3), C-NMR (d, ppm vs. TMS): 175.3, 149.3, 148.0,
to components 130.9, 127.2, 124.0, 119.8, 34.2, 33.6, 23.9, 19.1, 15.8): the NMR data
Camphene, p-cymene 0.93 1–4 are in agreement with those reported.[17]
(E)-β-Caryophyllene 1.07 8, 10–12, 14, 16–18,
22, 24–25
Carvacrol 1.23 7, 13, 15, Results and Discussion
Caryophyllene oxide 1.32 28–30
Carvone 1.38 5 Chemical Composition of P. mauritanica Root Oil
Geranyl acetate 1.46 6, 9, 21 When subjected to hydrodistillation, roots of P. mauritanica
Carvacryl isobutyrate 1.46 19–20, 23, 26–27, Coss. (Asteraceae) furnished a pleasant fruity pale yellow
6-Methoxythymyl 1.46 31–37 essential oil (yield = 0.17% w/w vs. dry material, mean
isobutyrate value). The essential oil has been analysed by GC/FID in
a combination with retention indices on two columns of
RFs were determined against nonane.
different polarity (Figure 1), by GC–MS with the apolar
column and by 13C-NMR following a computerized method


developed in our laboratory.[12–14] Fourteen components
RF ¼ C analyte =C is  Ais =Aanalyte
have been identified directly from the essential oil (Table 2).
where Canalyte and Aanalyte are the concentration and the absolute peak Among the components identified, compounds present in
area of the standard compound, and Cis and Ais are the concentration medium to high contents have been identified by the three
and the absolute peak area of nonane (internal standard). Component techniques. They belong to various families of compounds,
38, 8-hydroxy-9,10-diisobutyryloxy thymol, was not eluted on GC in our particularly oxygenated p-cymene derivatives (Figure 2;
13
analytical conditions. According to the C-NMR spectrum, 38 was a 2,5-dimethoxy-p-cymene 13 (37.2%), 8,9-dehydrothymyl
unique component of fraction F6.4, therefore its content has been isobutyrate 19 (1.7%), thymyl isobutyrate 20 (3.1%)) and ox-
calculated taking into account the ratio of the mass of F6.4 versus the ygenated monoterpenes (bornyl acetate 6 (1.1%) and neryl
mass of the chromatographed EO. isobutyrate 21 (11.1%)). Two sesquiterpene hydrocarbons
were also identified: modephene 11 (0.8%) and β-isocomene
Synthesis of Carvacryl Isobutyrate 14 (1.2%). Other minor components have been identified by
GC(RI) and by GC–MS: carvotanacetone 5 (0.1%), silphin-1-
Isobutyryl chloride (403 mg, 3.8 mmols) was added dropwise at 0°C to a
ene 10 (0.1%), α-isocomene 12 (0.4%), (E)-b-caryophyllene 16
mixture of carvacrol (578 mg, 3.8 mmols) and trimethylamine (535 mg,
(0.2%), β-sesquiphellandrene 25 (0.2%), caryophyllene oxide
9.1 mmols) in anhydrous CH2Cl2. The reaction mixture was stirred at 0°C
for 1 h, at 60°C for 3 h and then quenched with water and extracted with
28 (0.2%) and τ-cadinol 30 (trace).
CH2Cl2 (3 × 25 mL). Finally, the combined organic layer was washed with As several compounds remained unidentified, P. mauritanica
water and dried with anhydrous MgSO4 over night. After removal of root oil was fractionated by silica-gel CC using a gradient of
the solvent in vacuum, the residue was purified by CC using silica solvents. Non-polar components (fraction F1), medium polar
gel (63–200 μm) with P/DE = 90:10 as eluent to afford pure (98.9%) components (fractions F2–F4) and polar compounds (fractions

Figure 1. The GC/FID chromatographic profile of Pulicaria mauritanica root oil on an apolar column (BP-1)
85

Flavour Fragr. J. 2015, 30, 83–90 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj
 T. Xu et al.

Table 2. Components of Pulicaria mauritanica root oil

Compoundsa RIb RIc RId RF g/100 g Identification

1 Camphene 950 943 1072 0.93 0.1 RI, MS, 13C-NMR
2 Myrcene 987 980 1160 0.93 tr RI, MS
3 p-Cymene 1015 1012 1273 0.93 tr RI, MS
4 (Z)-β-ocimene 1029 1023 1230 0.93 tr RI, MS
5 Carvotanacetone 1220 1221 1677 1.38 0.1 RI, MS, 13C-NMR
6 Bornyl acetate 1270 1270 1578 1.46 1.1 RI, MS, 13C-NMR
7 Carvacrol 1278 1275 2204 1.23 tr RI, MS
8 7α-H-silphiperfol-5-ene 1329 1328 1425 1.07 0.1 RI, MS, 13C-NMR
9 Neryl acetate 1342 1347 1725 1.45 0.2 RI, MS
10 Silphin-1-ene 1350 1350 1471 1.07 0.1 RI, MS, 13C-NMR
11 Modhephene 1383 1382 1522 1.07 0.8 RI, MS, 13C-NMR
12 α-Isocomene 1389 1388 1534 1.07 0.4 RI, MS, 13C-NMR
13 2,5-Dimethoxy-p-cymene 1399 1400 1866 1.23 37.2 RI, MS, 13C-NMR
14 β-Isocomene 1411 1407 1567 1.07 1.2 RI, MS, 13C-NMR
15 8,9-Dehydro-2,5-dimethoxy-p-cymene 1420 1416 1960 1.23 0.2 RI, MS, 13C-NMR
16 (E)-β-Caryophyllene 1421 1419 1471 1.07 0.2 RI, MS, 13C-NMR
17 (E)-β-Farnesene 1446 1450 1583 1.07 0.2 RI, MS, 13C-NMR
18 α-Humulene 1455 1452 1668 1.07 tr RI, MS, 13C-NMR
19 8,9-Dehydrothymyl isobutyrate 1458 1455 1933 1.46 1.7 RI, MS, 13C-NMR
20 Thymyl isobutyrate 1462 1461 1896 1.46 3.1 RI, MS, 13C-NMR
21 Neryl isobutyrate 1468 1471 1776 1.46 11.1 RI, MS, 13C-NMR
22 γ-Muurolene 1474 1477 1684 1.07 tr RI, MS, 13C-NMR
23 Carvacryl isobutyrate 1489 1487 1935 1.46 0.4 RI, MS, 13C-NMR
24 β-Bisabolene 1503 1507 1719 1.07 tr RI, MS, 13C-NMR
25 β-Sesquiphellandrene 1516 1515 1771 1.07 0.2 RI, MS, 13C-NMR
26 8,9-Dehydrothymyl 2-methylbutyrate 1548 1550 2003 1.46 tr RI, MS
27 Thymyl 2-methylbutyrate 1551 1551 1807 1.46 tr RI, MS
28 Caryophyllene oxide 1578 1565 1976 1.32 0.2 RI, MS, 13C-NMR
_
29 Modhephan-3-one 1582 2005 1.32 4.1 TOF-MS, 1D, 2D NMR
30 τ-Cadinol 1633 1624 2163 1.32 tr RI, MS, 13C-NMR
31 6-Methoxythymyl isobutyrate 1658 1659 2262 1.46 14.2 RI, MS, 13C-NMR
32 6-Methoxy-8,9-dehydrothymyl isobutyrate 1676 1666 2259 1.46 2.4 RI, MS, 13C-NMR
33 10-Isobutyryloxy thymyl isobutyrate 1891 1880 2479 1.46 2.6 RI, MS
34 10-Isobutyryloxy-8,9-dehydrothymyl isobutyrate 1891 1883 2519 1.46 4.8 RI, MS, 13C-NMR
35 10-(2-Methylbutyryloxy)-8,9-dehydrothymyl 1970 1967 2601 1.46 0.4 RI, MS, 13C-NMR
isobutyrate
_
36 10-Isobutyryloxy-8,9-epoxythymyl isobutyrate 1986 2759 1.46 0.8 RI, 13C-NMR
_
37 10-(2-Methylbutyloxy)-8,9-epoxythymyl 2056 - 1.46 1.0 RI, 13C-NMR
isobutyrate
_ 1
38 8-Hydroxy-9,10-diisobutyryloxy thymole - - - 1.5e H, 13C-NMR
Monoterpene hydrocarbons, 0.1
Oxygenated monoterpenes 12.5
Thymyl and carvacryl derivatives 70.3
Sesquiterpene hydrocarbons 3.2
Oxygenated sesquiterpenes 4.3
Total identified 90.4
RI, retention indices; RF, response factor; tr, traces less than 0.05%; 13C-NMR, components were identified by NMR in the essential
oil (EO) and obvious in at least in one fraction of chromatography; 13C-NMR, components were identified by NMR in one fraction of
chromatography.
a
Order of elution and percentages were given on an apolar column (BP1). Compound 29 was found for the first time in nature. The
RI for compounds 29 and 36–38 not found in the literature.
b
Retention indices reported in Terpenoids Library Website[29] for monoterpenes and sesquiterpenes, for thymyl and carvacryl de-
rivatives (see text).
c
Retention indices measured on BP1 capillary column.
d
Retention indices measured on BP20 capillary column.
e
According to NMR spectra, fraction F6.4 of the CC contained only compound 38, which is not eluted on the GC. Therefore, the
content of 38 (g/100 g) was calculated taking into account the mass of fraction F6.4 versus the mass of the EO.
86

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Thymyl esters and modhephanone from Pulicaria mauritanica root oil

Figure 2. Identified oxygenated p-cymene derivatives from Pulicaria mauritanica root oil

F5 and F6) were first separated. Fractions F3, F5 and F6 were matching against two spectral data libraries, the first one
then fractionated once again in order to obtain compounds with constructed with spectra recorded in our laboratory, the
a degree of purity, sufficiently high to allow for the elucidation of second one constructed in the laboratory with NMR data re-
their structures. Fractions F1–F6 of the CC were analysed using ported in the literature. However, both libraries contained
GC/FID, GC–MS and 13C-NMR. Obviously, all the components only a few data for thymol and carvacrol derivatives. There-
previously found in the essential oil were again identified in the fore, we enlarged the second library with NMR data of
fractions of the CC. Moreover, the presence of various minor oxygenated p-cymene derivatives taken from the literature.
components previously identified by MS in the EO was confirmed Computer matching against that library permitted the identi-
by 13C-NMR: silphin-1-ene 10 (10.3%, F1), α-isocomene 12 (9.3%, fication of the following components:
F1), (E)-β-caryophyllene 16 (10.2%, F1), β-sesquiphellandrene 25
(9.0%, F1) as well as carvotanacetone 5 (2.7%, F5), caryophyllene - 8,9-dehydro-2,5-dimethoxy-p-cymene 15 (1.2% in fraction F2)
oxide 28 (4.1%, F5) and τ-cadinol 30 (5.0% in F6.3). Identification was identified by comparison of its RIs, MS data and 13C-NMR
of other components was achieved by analysis of the CC and chemical shifts with those reported in the literature;[18,19]
they are reported family by family of compounds: hydrocarbons, - the structure of 6-methoxythymyl isobutyrate 31 (58.3% in
oxygenated p-cymene derivatives and oxygenated terpenes. F5.1) was easily deduced from the 1H- and 13C-NMR spectra
of that fraction when compared with those of methylcarvacrol
and thymyl isobutyrate. The 1H- and 13C-NMR data fitted
Hydrocarbons perfectly with those reported by Zee et al.,[20]
Various hydrocarbons present but not ascertained in the EO were - 10-isobutyryloxy-8,9-dehydrothymyl isobutyrate 34 (79.8% in
identified in the F1 fraction of the CC, either by GC(RI) and GC–MS F5.3) and 10-(2-methylbutyryloxy)-8,9-dehydrothymyl isobutyrate
(myrcene 2 (trace), p-cymene 3 (trace), (Z)-β-ocimene 4 (tr)) or 35 (21.0% in F6.3) were identified by comparison with the
13
by GC(RI), GC–MS and 13C-NMR (camphene 1 (0.6%), 7α-H- C-NMR data reported by Weremczuk-Jezyna et al., who iso-
silphiperfol-5-ene 8 (1.6%), α-humulene 18 (0.4%), and (E)-β- lated these compounds from the root oil of Arnica montana;[21]
farnesene 17 (3.2%), γ-muurolene 22 (0.8%), and β-bisabolene - 10-isobutyryloxy-8,9-epoxythymyl isobutyrate 36 (81.7% in F6)
24 (0.6%)). and 10-(2-methylbutyloxy) 8,9-epoxythymyl isobutyrate 37
(45.1% in F6.3) were identified by comparison of their 13C-NMR
chemical shifts with those reported by Zee et al.,[20] who isolated
Oxygenated p-Cymene Derivatives these two compounds from Carpesium divaricatum essential oil;
Concerning oxygenated p-cymene derivatives, various strate- - 8-hydroxy-9,10-diisobutyryloxy thymol 38. This highly oxy-
genated compound is not sufficiently volatile and conse-
gies have been used, depending of the content of each com-
quently was not eluted by the GC under our analytical
ponent and the published data. As previously mentioned,
2,5-dimethoxy-p-cymene 13 (37.2%), 8,9-dehydrothymyl iso- conditions. Therefore, it could not been detected and iden-
butyrate 19 (1.7%) and thymyl isobutyrate 20 (3.1%) were tified using GC–MS. However, this compound was identified
identified in the EO by GC(RI), GC–MS and 13C-NMR. Their in the fraction F6.4 eluted with diethyl oxide, by comparison
presence was confirmed in the fractions of the CC. Otherwise, of its chemical shifts with those reported by Zhang et al.,
examination of the 13C-NMR spectra of the CC fractions who isolated the compound from the ethanol extract of
demonstrated that various unidentified components probably Inula hupehensis.[22]
bore the p-cymene skeleton, with one or more oxygenated In addition:
functions (thymol or carvacrol derivatives). At this stage, we
should remember that identification of individual compo- - carvacrol 7, present at trace level in the EO, was identified by
nents by 13C-NMR spectroscopy is achieved by computer GC(RI) and GC–MS in fraction F6.3 (0.2%);
87

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 T. Xu et al.

- the structure of carvacryl isobutyrate 23 (0.8% in F2) was Oxygenated Terpenes – Structural Elucidation of
suggested by combination of MS data and retention indices Modhephan-3-one 29
values, and was confirmed by comparison of its 13C-NMR
In addition to carvotanacetone 5, bornyl acetate 6, neryl
chemical shifts with those of the genuine compound obtained isobutyrate 21, caryophyllene oxide 28 and τ-cadinol 30, previ-
by esterification of carvacrol (see Experimental); ously mentioned, neryl acetate 9 has been identified by GC(RI)
- 8,9-dehydrothymyl-2-methylbutyrate 26 (0.2% in F3) and and GC–MS in fraction F2 (0.2%). However, compound 29
thymyl 2-methylbutyrate 27 (0.5% in F3) were identified by (4.1% of the EO, 21.2% in F3 fraction) could not be identified
computer matching against mass spectral libraries and by by GC–MS and 13C-NMR analysis, even by using all the comput-
comparing their RIs with those reported in the literature:[9,23] erized commercial MS libraries at our disposal as well as our
- 6-methoxy-8,9-dehydrothymyl isobutyrate 32 (13.8% in F4) laboratory-built 13C-NMR data library. Repeated chromato-
differed from 31 only by the presence of the C8–C9 double graphic separation of the F3 fraction produced subfraction
bond. Both compounds exhibited very close mass spectral F3.2b containing high amount of the unknown component
patterns with characteristic peaks at 248(10) [M+], 178(100), (80%) (RIs = 1580/2004 on apolar and polar columns respec-
163(31) versus 250(10) [M+], 180(100), 165(57), in agreement tively). Thus subfraction F3.2b was submitted to a full set of
with literature[21] – the structure of 32 was confirmed by com- one and two-dimensional NMR experiments (Table 3).
parison of its 13C-NMR chemical shifts, picked up on the spec- The formula C15H24O was deduced from the highresolution
tra of fraction F4, with those of 31 reported by Zee et al.,[20] mass spectrum (m/z = 220.1831, calculated = 220.1827), in agree-
- 10-isobutyryloxy thymyl isobutyrate 33 (6.5% in F5.2) differed ment with the distortionless enhancement by polarization trans-
from 34 only by the lack of the C8–C9 double bond. As above, fer spectra (4 methyls, 5 methylenes, 2 methines and 4
both compounds exhibited very close mass spectral patterns quaternary carbons). The 13C-NMR spectrum confirmed a sesqui-
with characteristic peaks at 306(3) [M+], 236(7), 218(27), 148 terpene structure bearing a ketone moiety (224.5 ppm) and
(100) versus 304(6), 234 (10), 216(7), 146(100). The MS data characterized by the lack of double bond. The occurrence of a
were in agreement with the literature.[21] tricyclic structure could be easily deduced from these data. Little

Table 3. The NMR data of modhephan-3-one 29

C δC (ppm) DEPT δH (ppm) H Multiplicity (J, Hz) HMBC

→
COSY
1 59.7 C
2 49.7 CH 2.50 q (7.1) 1, 3, 9, 14 14
3 224.5 C
4 49.1 C
5 61.9 C
6 25.7 CH2 1.48 a m
0.95 b 4
7 39.1 CH2 1.81 a 5 6, 8
1.25 b
8 49.6 CH 1.84 m 6, 7
9 29.1 CH2 1.50 a m 2
1.36 b 1, 8
10 34.5 CH2 1.79 a m 5 9, 11
1.48 b
11 32.7 CH2 1.90 a m 4, 5 10
1.40 b
12 20.6 CH3 0.98 Broad s 3, 4, 5, 13
13 28.1 CH3 1.08 Broad s 3, 4, 5, 12
14 10.6 CH3 1.01 d (7.1) 1, 2, 3, 10 2
15 15.1 CH3 1.03 d (6.6) 1, 8, 10 8
DEPT, distortionless enhancement by polarization transfer; HMBC, heteronuclear multiple bond correlation; COSY, correlation
spectroscopy.
88

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Thymyl esters and modhephanone from Pulicaria mauritanica root oil

information was brought by the 1H-NMR spectrum, however, it species, also characterized by the pre-eminence of thymol
was noted that the signals of two methyls were doublets, and carvacrol derivatives associated with sesquiterpenes
whereas those of the two other methyls were singlets. A quartet bearing the tricyclo[3.3.3.01,5]undecane skeleton of modhe-
at 2.50 ppm (linked to the methine carbon at 49.7 ppm) belongs phane: Arnica montana,[21] Doronicum corsicum,[26] Eupatorium
to a proton probably α-positioned to the carbonyl group. In cannabinum,[23] Eupatorium fortune[27] or Telekia speciosa.[28]
addition, no cyclopropanic hydrogen was observed. From the From the analytical point of view, identification of individual com-
long-range proton–carbon correlations (heteronuclear multiple ponents of P. mauritanica root oil led us to highlight two points.
bond correlation (HMBC) spectrum) and characteristic chemical The first one consisted in the identification of polyoxygenated
shifts, it appears that three (out of four) methyls (two singlets thymol (or carvacrol) derivatives, which possessed various
and a doublet) are located in a position with respect to the methoxy and ester functions located in different positions of
carbonyl group. They are also correlated with two quaternary the p-cymene structure. The second one is related to the struc-
carbons. All these findings suggest the occurrence of a five- tural elucidation of one of the four modhephanone stereoi-
membered ring bearing a carbonyl function with three methyls somers, accounting for more than 4% of the oil composition
in α position and linked with two other rings. A survey of the ses- and reported for the first time as a natural compound. In both
quiterpenes skeletons usually found among the volatile compo- cases, NMR played a crucial role, as well as in the identification
nents of essential oils directed us towards the tricyclo[3.3.3.01,5] of 8-hydroxy-9,10-diisobutyryloxy thymol, a tetrasubstitued
undecane skeleton. Therefore, taking into account all the long- p-cymene derivative that was not eluted by the GC.
range proton–carbon correlations (Table 3), our molecule is
probably one of the four 2,4,4,8-tetramethyltricyclo[3.3.3.01,5] Acknowledgements
undecan-3-one diastereoisomers. Our hypothesis was confirmed
by comparison with literature data, this compound being This work has been done within the cooperative programme
between the Universities of Abou Bekr Belkaïd (Tlemcen) and
reported as an intermediate in the total synthesis of mod-
Pascal Paoli (Corsica). The authors gratefully acknowledge Pro-
hephene.[24] Unfortunately, 13C-NMR data are reported for the fessor M. Bensalah (Dean, Faculty of Life Sciences) for attribu-
mixture of the four diastereoisomers. tion of a travel grant, Professor A. Marouf (University of
The structure of the molecule being ensured, the next step Nâama) for his help with the plant identification, and Mr J.M.
consisted of taking advantage of the presence of a unique diaste- Desjobert (CPN laboratory, University of Corsica) who recorded
reoisomer. The precise stereochemistry of the two non- the mass spectra.
geminated methyl groups was confirmed by the observation of
spatial correlation between H2 (2.50 ppm) and H8 (1.84 ppm) in
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