Comprehensive Characterisation of n-Alkylresorcinols and Other Lipid Constituents of Mercurialis tomentosa L. from Alicante, Spain

Article abstract of DOI:10.1002/cbdv.201600255

Mercurialis tomentosa L. has been used in Spanish ethnomedicine. In the present study the first phytochemical characterisation of a lipid fraction from M.?tomentosa was performed. The CHCl₃ extraction of aerial parts from M.?tomentosa and GC/MS

Full text of DOI:10.1002/cbdv.201600255

Received Date: 14-Jul-2016
Revised Date: 22-Aug-2016
Accepted Date: 31-Aug-2016
Article Type: Full Paper
Comprehensive Characterisation of n-Alkylresorcinols and other Lipid
Constituents of Mercurialis tomentosa L. from Alicate, Spain
Peter Lorenz*a), Miriam Heinricha), Jürgen Conradb), Annerose Hellerc), Florian C.
Stintzinga) and Dietmar R. Kammerera)
a)WALA Heilmittel GmbH, Department of Analytical Development & Research, Section
Phytochemical Research, Dorfstr. 1, D-73087 Bad Boll/Eckwälden, Germany.
b)University of Hohenheim, Institute of Chemistry, Bioorganic Chemistry (130b),
Garbenstraße 30, D-70599 Stuttgart, Germany.
c)University of Hohenheim, Institute of Botany (210), Garbenstraße 30, D-70599 Stuttgart,
Germany.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201600255
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*Corresponding author at: WALA Heilmittel GmbH, Department of Analytical Development
& Research, Section Phytochemical Research, Dorfstrasse 1, D-73087 Bad Boll / Eckwälden,
Germany. Tel.: +49(0)7164 930 7099.
E-mail address: peter.lorenz@wala.de
Keywords: Euphorbiaceae, Lipids, Chemotaxonomy, n-Alkylresorcinols, Antioxidants.
ABSTRACT
Mercurialis tomentosa L. has been used in Spanish ethnomedicine. In the present study the
first phytochemical characterisation of a lipid fraction from M. tomentosa was performed.
The CHCl3 extraction of aerial parts from M. tomentosa and GC-MS investigations revealed
the occurrence of cuticular lipid and wax constituents, like long chain n-alcohols and n-
aldehydes (C22-C30), besides several aromatic constituents, i.e. phenylpropanoids and n-
alkylresorcinols. The latter were further purified by CC and analysed by LC-MSn. In contrast
to other Mercurialis species, i.e. M. annua, M. perennis, which exclusively contain 5-n-
alkylresorcinols (1a-j, Cn), mainly 5-n-alkyl-2-methylresorcinols (2a-j, Cn*) with side chain
lengths of C15-C25 were found in M. tomentosa, in addition to 1a-j. Thus, the latter
compounds may be utilised for analytical characterisation and authentication of M. tomentosa
based on fingerprinting methods. For structure elucidation a novel facile total synthesis of
*
one representative 5-n-alkyl-2-methylresorcinol homologue (2d, C19 ) was developed,
starting with a Grignard reaction from a substituted benzoic acid chloride (19). The
compound obtained by synthesis was identical to the natural product 2d in terms of its
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chromatographic and spectroscopic features. Futhermore, 2d exhibited satisfactory DPPH
free radical scavenging activity (IC50 = 37.8 µM) when compared to trolox (IC50 = 21.0 µM),
corroborating the antioxidant features of these amphipathic molecules.
Introduction
Lipids are a large group of natural constituents found in higher plants and fulfill a
multitude of biological functions. Besides carbohydrates, lipids in form of lipid bodies are the
most important energy stores in all cells. Beyond, lipids are hydrophobic or amphipathic
molecules that may biosynthetically originate from carbanion-based condensations of
thioesters (acetyl-CoA to fatty acids, polyketides, etc.) and/or from carbocation-based
condensations of isoprene units (prenols, sterols, etc.) [1]. Depending on their chemical
structure, lipids may be divided into glycerol esters of fatty acids (acylglycerols), phospho-
and sphingo-lipids (ceramides), glycolipids, phenolic lipids, isoprenoids (e.g. sterols,
carotenoids) and waxes (e.g. long chain n-alkanes, ketones, fatty acids and n-aldehydes) [2].
The latter are found as cuticular waxes, constituting a natural barrier against environmental
impacts, and providing a successful strategy ensuring plant survival in the dry gaseous
environment [3][4].
In our exploration of medicinal plants as a source of novel bioactive lipid constituents we
closer investigated the genus Mercurialis (dog’s mercury), which comprises 8 to 10 species
[5]. Mercurialis species are mainly distributed in the Eurasian area, whereas M. leiocarpa is
found in Asia [6]. The plants are dioecious and belong to the Euphorbiaceae (spurge family).
Various subspecies and hybrids have been described [7]. Since ancient times, Mercurialis
species have been used in European ethnomedicine to treat several diseases such as purulent
wounds, eczema, abscesses, but also as a laxative and to cure menstrual complaints [8].
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Nowadays, particularly M. perennis is applied in medicine, especially for the treatment of
slowly healing wounds, burns, conjunctivitis (inflamed eyes) and against hemorrhoids [9].
Comprehensive phytochemical investigations of M. perennis and M. annua have revealed a
broad spectrum of nonpolar and polar secondary plant metabolites, such as low-molecular
phenolics, alkaloids, sterols, tocopherols, depsides, flavonoids, and essential oil components
[10-12]. In this course, the occurrence of 5-n-alkylresorcinol homologues in M. perennis and
M. annua, as a first proof of this compound class in the Euphorbiaceae family was reported
[13].
Based on these phytochemical analyses, we aimed to broaden our knowledge of the
pharmacognosy of this plant family. Consequently, Mercurialis tomentosa L., a species
widely spread on the Iberian Peninsula (Portugal, Spain and southern parts of France), came
into the focus of our interest. M. tomentosa (Spanish names: ‘Carra’ or ‘hierba de Santa
Quiteria’, etc.) has already been described in 1753 by Linné in the publication ‘Species
Plantarum’ [14]. The plant is white-silverish haired, has a tap root and is growing in sunny
and dry areas [15]. Compared to other Mercurialis species, which are herbaceous plants, M.
tomentosa is a subshrub with lignified stalks.
Water decoctions of the entire plant have formerly been used in Spanish ethnomedicine for
the treatment of external swellings, rheumatism, inflammations and internally as a purge or
against intestinal problems [15-17]. Interestingly, pharmacological investigations could
recently show in vitro anti-inflammatory effects by inhibition of PGE2 and TNF-α in human
monocytes, due to the application of a M. tomentosa EtOAc extract [18]. However,
information on the natural constituents of M. tomentosa, let alone the active principles, are
completely lacking in the literature. Thus, the current investigations focused on the analytical
characterisation of lipid constituents from M. tomentosa.
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Results and Discussion
For the investigation of lipid constituents, a CHCl3 extract from fresh herbal parts of
M. tomentosa was prepared and analysed by GC-MS. A complex peak profile was detected in
the GC-MS chromatograms, covering a retention time range of tR 40-60 min (Fig. 1a). By
comparing the mass spectra of these peaks with the NIST database and with those of
reference compounds, several long chain n-aldehydes and n-alcohols of carbon-chain lengths
C22-C30 were assigned, with the even-numbered homologues C24, C26 and C28 predominating.
Long chain n-aldehydes and n-alcohols are known to be typical constituents of cuticles,
covering the aerial portions of land plants and building a protective layer against dehydration,
UV irradiation, and pathogen attack [3][19]. Some cuticular wax components build
crystalline like structures as platelets, tubules or threads [3]. No conspicuous crystalline
structures were observed in the present study for M. tomentosa. SEM analysis revealed a
continuous smooth cuticular wax film on trichomes and epidermis under the thick layer of
trichomes (Fig. 2a, b, c). Thus, for M. tomentosa it becomes apparent that the trichomes have
a function in UV protection or may serve as a protection against desiccation [20] and were
developed, together with wax components, as a result of environmental adaptation. While the
occurrence of n-aldehyde and n-alcohol homologues in quince wax (Cydonia oblonga L.,
Rosaceae) has been reported recently [21], these constituents have not been analysed before
in the genus Mercurialis.
Furthermore, the CHCl3 extract of M. tomentosa was scrutinised with regard to the
occurrence of phenolic lipids, i.e. alkyl phenols. By selected ion monitoring (SIM) of base
peak fragments at m/z 124 small amounts of some 5-n-alkylresorcinol homologues were
detected in the GC-MS chromatogram of the CHCl3 extract (data not shown). Furthermore,
compounds with similar mass spectra were observed, exhibiting a 14 Da extension (CH2) of
the base peak fragment (m/z 138). However, the concentrations of these constituents were too
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low to obtain significant mass spectra. Consequently, compound enrichment was achieved by
loading a CH2Cl2 extract of M. tomentosa onto a polyamide column. After washing the
column with CH2Cl2 to remove nonpolar constituents, n-alkylresorcinols were obtained as a
highly concentrated fraction by elution with MeOH. Henceforth, GC-MS investigations of
this fraction revealed a mixture of compounds derived from two structural types of n-
alkylresorcinol homologues, as can be deduced from SIM of fragments at m/z 124 and 138,
respectively (Fig. 1b, c and Table 1). By comparison of the mass spectra with literature data
[13][22] the latter were identified as 5-n-alkylresorcinol- and 5-n-alkyl-2-methylresorcinol
homologues (1a-j and 2a-j, respectively), comprising side chain lengths of C15 – C25.
Structural assignment of the 5-n-alkylresorcinols (1, Cn) was further supported by comparison
with a reference compound (1d, C19).
While 5-n-alkylresorcinols (1, Cn) have previously been detected in M. perennis and M.
annua [13], the occurrence of 5-n-alkyl-2-methylresorcinols (2, Cn*) has so far only been
reported for two species of other plant families, i.e. Polygonum maritimum (Polygonaceae
family) and Dactylis glomerata subsp. hispanica (Poaceae family) [22][23]. Accordingly, the
newly detected homologous compounds (2, Cn*) may serve as specific chemotaxonomic
markers for M. tomentosa (Fig. 1b, c), e.g. for comparative phytochemical studies
considering further Mercurialis species.
The chain-length distribution of the n-alkylresorcinols was assessed based on the relative
peak areas of the extracted ion chromatograms (Fig. 3). The odd-numbered C19 and C21
homologues of both n-alkylresorcinol types (1d, f and 2d, f, respectively) were found to
predominate. Noteworthy, also the piperidin-2,6-dione alkaloids hermidin (5) and hermidin
quinone (8) were identified in the MeOH polyamide fraction of the CH2Cl2 extract as typical
marker components of Mercurialis [10][12], based on their mass spectra and by comparison
with synthetic reference standards (see GC-MS chromatogram, Fig. 4). In addition, several
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low-molecular aromatic compounds, like p-hydroxybenzaldehyde (3), p-hydroxybenzonitrile
(4), vanillin (9) and the phenylpropanoids coniferyl alcohol (11), o- and p-coumaric acid
methyl and ethyl esters (10, 12 and 13, respectively), and sinapyl alcohol (16), were detected
(Fig. 4). Interestingly, phenylpropanoids, like p-coumaric acid esters, have been described as
integral constituents of the cutin polyester matrix [24], while the cinnamoyl alcohols 11 and
16 are lignin monomers [25].
Moreover, the fraction eluted with MeOH from the polyamide column was also investigated
by HPLC-APCI+-MSn to characterise the assigned n-alkylresorcinols more closely. All n-
alkylresorcinols detected by GC-MS were corroborated by LC-MS (Fig. 5, Table 1).
Expectably, differences in the fragmentation patterns of the 5-n-alkyl-2-methylresorcinols (2,
Cn*) in the EI-MS and APCI+-MS mode were observed (Scheme 1, Table 1). Thus, α-
cleavage of the aliphatic side chain and subsequent McLafferty re-arrangement of the
phenolic ring yielded a base peak fragment at m/z 138 and a minor fragment at m/z 137, due
to the formation of a dihydroxytropylium cation in the EI-MS experiments [13][26]. In
addition, fragments at m/z 151 and 180 were detected as a result of a β- and δ-cleavage,
respectively (Scheme 1, Fig. 6a). In contrast, in the APCI+ ionisation mode the preferential
fragmentation is characterised by a release of the aliphatic side chain from the aromatic
backbone, presumably by formation of a phenyloxonium cation (m/z 125) or phenoxy cation
(m/z 123; see Scheme 1, Fig. 6b and Table 1), followed by the subsequent release of water to
form a phenyl cation at m/z 107. The MSn fragmentation patterns of the individual 5-n-alkyl-
2-methylresorcinol- (2, Cn*) and 5-n-alkylresorcinol-homologues (1, Cn) in the APCI+ mode
were not consistent, which is probably due to differing concentrations of the analytes (Table
1). However, the LC-MS data supported the structure assignment of the target analytes 1 and
2.
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To further corroborate structure assignment and to obtain greater substance amounts, e.g. for
pharmacological studies, an attempt to realise a total synthesis of one representative n-
alkylresorcinol homologue, namely 5-n-nonadecyl-2-methylresorcinol (2d), was made.
Several approaches for the synthesis of n-alkylresorcinols are known from literature [27-29].
However, we here report a synthesis of 2d for the first time, starting from a Grignard reaction
(Scheme 2): The substituted benzoic acid chloride 19, obtained from 18 by chlorination with
oxalyl chloride, was treated at -78o with a THF solution of n-octadecylmagnesium chloride in
the presence of bis-[2-(N,N’-dimethylamino)ethyl]ether [30], to yield ketone 20.
Subsequently, 20 was reduced via a Wolff-Kishner reaction at 200o in the presence of
hydrazine hydrate, KOH and triethylenglycol, to obtain 21. Then, 2d was yielded from the
latter by removing the protective groups through de-methylation with BBr3 in CH2Cl2 at 0o
(Scheme 2). With respect to its chromatographic and mass spectrometric features, 2d matched
with the respective natural constituent of M. tomentosa.
Various biological activities have been reported for n-alkylresorcinols in the literature. n-
Alkylresorcinols are assumed to play an important role as phytoanticipins and
allelochemicals [22][31]. n-Alkylresorcinols also exert cytotoxic, antimicrobial [32], anti-
inflammatory (COX-1/2) [33] and antioxidant activity in vitro [34]. Exemplarily, we herein
investigated the radical-scavenging activity of 2d using the DPPH (1,1’-diphenyl-2-picryl-
hydrazyl) assay. DPPH is an artificial, stable radical, frequently used for common antioxidant
assays [35][36]. This compound dissolves in MeOH yielding a purple color. By adding
antioxidants DPPH is reduced, thus forming a decolored reaction product (yellow color). This
effect can be quantitated by spectrophotometric measurements. When 2d was applied to
DPPH a logarithmic concentration-absorption correlation was observed. From the resulting
regression equation an IC50 of 37.86 µM was calculated for 2d (Table 3). A five-fold higher
IC50 as compared to 2d has been reported in the literature for 1d [34]. The activity of 2d was
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further compared to the common antioxidant trolox, which revealed a linear concentration-
absorption correlation, from which an IC50 value of 21.03 µM was deduced, being in
agreement with literature data [34].
Conclusions
The current analytical investigations into CHCl3 extracts of M. tomentosa revealed the
occurrence of several lipid components, which were detected for the first time in this plant
species. Long-chain n-alcohols and n-aldehydes were identified as characteristic wax
compounds, which may play a significant role in the regulation of the barrier properties of
plant cuticles [3]. Furthermore, two homologous series of n-alkylresorcinols were analysed
together with further phenolic components (e.g. phenylpropanoids) of similar polarity. The
newly detected 5-n-alkyl-2-methylresorcinols (2, Cn*), which are quantitatively predominant
among the phenolic lipids in M. tomentosa, were identified as specific markers. The latter
may be utilised for chemotaxonomic differentiation of Mercurialis species and, thus, may
help to align phytochemical data with phytogenetic studies already published for Mercurialis
[6]. To support these findings a higher number of samples covering different geographic
regions should be considered in future studies.
Moreover, by investigation of one representative 5-n-alkyl-2-methylresorcinol 2d (C19*) in
the DPPH assay the antioxidant features of this compound class were assessed. Furthermore,
since n-alkylresorcinols have recently been demonstrated to exert anti-inflammatory (COX-
1/2) activities in vitro [33], it is assumed that the formerly described anti-inflammatory
effects of a M. tomentosa extract [18] could be assigned to these phenolic lipid constituents.
However, further investigations are required to ascertain this assumption.
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Experimental Part
General
Source of reference compounds for GC analyses: 1-hexacosanol and 1-octacosanol
(C26/C28-Alc, respectively), 4-hydroxybenzaldehyde (3), 4-hydroxybenzonitrile (4), ethyl 4-
hydroxybenzoate (7), coniferyl alcohol (11), and n-hexadecanoic acid (14) were purchased
from Sigma-Aldrich (Steinheim, Germany). The n-aldehydes (C26/C28/C30-Ad), hermidin (5)
and hermidin quinone (8) were synthesised according to previously published procedures
[10] [21]. 5-n-Nonadecyl-resorcinol (1d, C19) was obtained from ReseaChem (Burgdorf,
Switzerland). 3,5-Dimethoxy-4-methylbenzoic acid (18) was purchased from Alfa Aesar
(Karlsruhe, Germany), and Scopoletin (15) from Phytolab (Vestenbergsgreuth, Germany).
Ethyl 4-(E)-hydroxycinnamate was obtained by synthesis from p-coumaric acid and EtOH,
based on a literature protocol [37].
NMR Spectroscopy
NMR spectra were recorded in CDCl3 using a Varian Unity Inova 500 MHz spectrometer.
The measurement temperature was 298 K. Chemical shifts were expressed in δ [ppm] and
13
referenced to residual (non-deuterated) solvent signals of CDCl3 (1H: δ(H) 7.27; C: δ(C)
13
77.00). C-NMR signal assignment of the novel compounds was based on 2D heteronuclear
NMR experiments (gHMBC and gHSQC). 13C-NMR chemical shifts of aromatic carbons
were assigned by increments [38]. For evaluation of NMR spectra the program SpinWorks
3.1.7. (Copyright® 2010, K. Marat, University of Manitoba, USA) was used.
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IR Spectroscopy
IR spectra of the synthesised compounds were recorded on a Bruker Tensor 27-FT-IR
spectrometer at the SGS Institute Fresenius (Taunusstein, Germany).
Plant material
Aerial parts of M. tomentosa were collected at Alcocer de Planes, Alicante (Spain) in
April 2015, air dried and stored at -80o until investigation. A voucher specimen of M.
tomentosa (voucher no. HOH-014615) was deposited in the herbarium of the Department of
Botany at Hohenheim University (Germany). The plant material was identified by Prof. O.
Spring.
Extraction and Chromatographic Purification of n-Alkylresorcinols
Dried herbal parts of M. tomentosa (25.0 g) were extracted with CH2Cl2 (250 ml). The
mixture was flushed with nitrogen (N2) for 10 min. and stored overnight in a refrigerator
(+ 4o). Afterwards, the plant material was filtered off over celite by vacuum suction, washed
with CH2Cl2 (250 ml), and subsequently the extraction was repeated for a second time in the
same manner. The solvent was removed from the combined extracts by vacuum rotary
evaporation to yield 0.61 g of crude material. The latter was loaded onto a polyamide column
(10.5 g, 20 cm x 2 cm, i.d.), preconditioned with CH2Cl2 (50 ml). Compound elution was
performed with CH2Cl2 (150 ml, fraction discarded) and MeOH (150 ml). The solvent was
removed in vacuo from the MeOH fraction to yield 0.04 g material, which was highly
enriched in n-alkylresorcinols and analysed by GC-MS and LC-MSn. At the same time, a
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sample of fresh plant material (11.5 g) was extracted with CHCl3 (250 ml) and stored at RT
until investigation.
GC-MS Analysis
GC-MS analyses were performed with a Clarus 500 (PerkinElmer, Inc., Shelton, CT,
USA) gas chromatograph with split injection (split ratio 30:1, injection volume 1.0 µl)
coupled to a single quadrupole mass spectrometer. The column used was a Zebron ZB-5ms
cap. column (60 m x 0.25 mm i. d. x 0.25 µm film thickness, 5% phenylpolysiloxane and
95% dimethylpolysiloxane coating; Phenomenex, Torrance, USA). Carrier gas: helium at a
flow rate of 1 ml/min. The injector used was a PSS (programmed-temperature split/splitless
injector; temperature: 250o). The temperature program for the column oven was 100 to 320o
with a linear gradient of 4o/min and a final hold time of 30 min. The mass spectrometer was
run in electron ionisation mode (70 eV).
RP-HPLC-APCI+-MSn Analyses
LC-MS analyses where carried out on an Agilent 1200 HPLC system (Agilent,
Waldbronn, Germany) equipped with a degasser, a binary pump, an autosampler, a
thermostated column compartment and a diode array detector connected to a HCTultra ion
trap MS detector equipped with an APCI ion source (Bruker Daltonik GmbH, Bremen,
Germany). For chromatographic separation, a Synergi 4u Hydro-RP 80A column (150 x 2.0
mm i.d., 4 µm particle size, Phenomenex, Torrance, USA) was used at 25°C and a flow rate
of 0.3 ml/min. The mobile phase consisted of H2O (eluent A) and MeOH (eluent B). Elution
started with 83% B, followed by a linear gradient to 100% B within 60 min, staying isocratic
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for 5 min before re-equilibration to starting conditions within 5 min. The injection volume
was 20 µl. Detection was performed by mass spectrometry with an APCI interface operating
in the positive ionisation mode, applying the following parameters: HV capillary: -4000 V;
dry gas N2: 5.00 l/min with a dry gas temperature of 300°C; nebuliser: 40 psi; vaporiser
temperature: 400°C. Full scan mass spectra (range from m/z 50 to 700) of the HPLC eluates
were recorded during chromatographic separation. MSn data were acquired in the auto
MS/MS mode. The instruments were controlled by Agilent ChemStation (Rev. B.01.03 SR1)
and Bruker Daltonik EsquireControl software (V6.1).
Synthesis of the Reference Compound 1,3-Dihydroxy-2-methyl-5-nonadecylbenzene (= 5-
Nonadecyl-2-methyl-resorcinol, 2d). 3,5-Dimethoxy-4-methyl-benzoic acid chloride (19)
A solution of 3,5-dimethoxy-4-methylbenzoic acid (18, 10.36 g, 52.80 mmol) in
toluene (244 ml) was treated with 4 drops of DMF and cooled in an ice/NaCl-mixture.
Subsequently, oxalyl chloride (9.5 ml, resp. 14.0 g, 110.30 mmol) was added dropwise under
stirring, which was continued for 3 h 40 min, while the mixture was slowly warmed up to
room temperature. Afterwards, toluene was removed by vacuum rotary evaporation to yield a
crude product (11.3 g). For de-colorisation, the latter was dissolved in EtOAc (100 ml), 0.2 g
of activated carbon was added to the solution, stirred for 5 min, and filtered over Celite by
vacuum suction, which was washed with EtOAc (2 x 50 ml). The solvent was removed in
vacuo from the combined filtrates to yield a light beige product (19, 11.0 g, yield: 97.0 % of
the theory), which was used without further purification. GC-MS purity (70 eV): >99% (tR
min) m/z 216, 214 (6, 19, [M (35Cl/37Cl]+), 179 (100,[M-Cl]+), 151 (9), 136 (13), 121 (7), 106
(4), 91 (22), 77 (14), 65 (10), 51 (8).
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1,3-Dimethoxy-2-methyl-5-(1-oxo-nonadecyl)benzene (=5-(1-Oxononadecyl)-2-
methylresorcinol, 20)
Bis-[2-(N,N-dimethylamino)ethyl] ether (9.5 ml, resp. 8.0 g, 50 mmol) was added
dropwise under stirring to a solution of n-octadecylmagnesium chloride in THF (0.5 M, 100
ml) under N2, which was cooled with a dry ice/Me2CO mixture (-78o) [30]. Thereafter, a
solution of 3,5-dimethoxy-4-methyl-benzoic acid chloride (19, 10.73 g, 50 mmol) in dry THF
(50 ml) was added within 15 min, the reaction mixture was further stirred at -78o (20 min)
and then for 5 h at room temperature. Thereupon, the reaction was quenched by adding a sat.
ice-cooled NH4Cl solution (150 g NH4Cl/500 ml water). After extraction with Et2O (3 x 200
ml), the combined extracts were dried (Na2SO4) and the solvent was distilled off by vacuum
rotary evaporation. The crude solid product (22.7 g) obtained was treated with petroleum
ether (250 ml, b.p. 40-60o) and the mixture stirred for 15 min. Then, the sediment was filtered
off by vacuum suction, washed with petroleum ether (2 x 50 ml) and dried at room
temperature to yield the crude product 20 (11.9 g). The latter was recrystallised from ethanol
(96%, v/v, 500 ml) to give the pure 20 (10.8 g, yield: 50.0 % of the theory). White powder.
M. p. 82o (in accordance with the literature [39]). Rf (SiO2; n-hexane/EtOAc = 15:3 (v/v))
0.65. UV(ACN): 216 (4.38), 274 (4.00). GC-MS purity (70 eV): >99.9% (tR 58.0 min) m/z
432 (8, [M]+), 404 (1, [M-CO]+), 334 (0.6), 320 (0.7), 207 (6), 194 (100, [C11H14O3]+), 179
(54, [C10H11O3]+), 166 (8), 151 (7), 136 (5), 121 (3), 91 (9), 69 (6), 55 (13). APCI+-MS m/z
433* [M+H]+(MS1); 281* [M-C9H11O2+H]+, 263 [281-H2O]+, 207, 193, 179, 165*
[C10H13O2]+, 151, 137* [C8H9O2]+, 123 (MS2); 95, 81, 68 (MS3);*parent ions for the next
fragmentation step. 1H NMR (CDCl3, 500 MHz): 7.15 (s, H-C(21/21’)), 3.89 (s, H-(C25/25’),
OCH3), 2.94 (t, J = 7.4 Hz, H-C(2)), 2.14 (s, H-C(24)), 1.74 (tt, J = 7.4, 7.4 Hz, H-C(3)), 1.39
13
- 1.26 (br s, H-C(4)-C(18))**, 0.89 (t, J = 7.0 Hz, H-C(19)). C NMR (CDCl3, 125 MHz):
200.13 (C(1), C=O), 158.23 (C(22/22‘)), 135.58 (C(20)), 120.60 (C(23)), 103.32 (C(21/21‘)),
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55.86 (C(25/25‘), OCH3), 38.51 (C(2)), 31.92 (C(17)), 29.69, 29.67, 29.65, 29.63, 29.52,
29.51, 29.41, 29.35 (C(4)-C(16))**, 24.62 (C(3)), 22.68 (C(18)), 14.10 (C(19)), 8.68 (C(24)).
** 5 signals overlapped. IR (KBr, ν in cm-1): 2998 (w, aromatic CH), 2949 (m, aromatic CH),
2916 (s, alkyl-CH), 2850 (s, alkyl-CH), 1668 (m, C=O), 1584 (m), 1473 (m), 1410 (m), 1324
(w), 1309 (m), 1237 (m), 1157 (m), 1141 (s, OCH3), 861 (w), 822 (w), 715 (m).
1,3-Dimethoxy-2-methyl-5-nonadecylbenzene
(=5-Nonadecyl-2-methylresorcinol
dimethylether, 21), via Wolff-Kishner Reduction
A mixture of 20 (5.00 g, 11.56 mmol), triethylene glycol (63 ml), powdered KOH
(2.59 g, 46.16 mmol) and aqueous NH2OH solution (80%, v/v, 2.2 ml) was stirred under N2 at
200o for 3 h. After cooling down, the reaction mixture was extracted with Et2O (5 x 100 ml),
the unified extracts were dried (Na2SO4) and the solvent was removed in vacuo to yield a
yellow solid crude material. The latter was washed with Me2CO (5 x 100 ml) and, after
drying, the solid product recrystallised from EtOH (90 ml) to yield pure compound 21 (2.92
g, yield: 60.4% of the theory). M. p. 54.4o (in accordance with literature [39]). Rf (SiO2; n-
hexane/EtOAc = 19:1 (v/v)) 0.55. UV-VIS (ACN): 206 (4.60), sh 226. GC-MS purity (70
eV): >99.9% (tR 54.4 min) m/z 418 (27, [M]+), 376 (0.6), 334 (0.4), 250 (0.4), 208 (2), 179
(14, [C11H15O2]+), 166 (100, [C10H14O2]+), 151 (11), 135 (8), 121 (3), 105 (3), 91 (5), 79 (2),
69 (3), 57 (15). APCI+-MS m/z 419*[M+H]+ (MS1); 153* [M+C19H38+H]+ (MS2); 138*[153-
CH3] (MS3); 123 [138-CH3], 109 [138-CH2] (MS4); * parent ions for the next fragmentation
step. 1H NMR (CDCl3, 500 MHz): 6.38 (s, H-C(21/21‘)), 3.82 (s, H-C(25/C25’), OCH3), 2.58
(t, J = 7.8 Hz, H-C(1)), 2.07 (s, H-C(24)), 1.62 (tt, J = 7.4, 7.4 Hz, H-C(2)), 1.33 - 1.27 (br s,
H-C(3-C18))**, 0.89 (t, J = 6.9 Hz, H-C(19)). 13C NMR (CDCl3, 125 MHz): 158.13
(C(22/22‘)), 141.47 (C(20)), 111.60 (C(23)), 103.80 (C(21/21‘)), 55.69 (C(25/25’), OCH3),
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36.58 (C(1)), 31.92 (C(2)), 31.70 (C(3)), 29.70, 29.68, 29.65, 29.63, 29.56, 29.46, 29.36
(C(4-C17))**, 22.69 (C(18)), 14.11 (C(19)), 7.93 (C(24)).** 7 signals overlapped. IR (KBr, ν
in cm-1): 2995 (w, aromatic CH ), 2955 (m, aromatic CH), 2916 (s, alkyl-CH), 2849 (s, alkyl-
CH), 1606 (m), 1586 (s), 1471 (m), 1461 (m), 1414 (m), 1308 (w), 1239 (m), 1182 (w), 1140
(s, OCH3), 818 m), 718 (m), 663 (w), 590 (w). Noteworthy, the solvent was removed in
vacuo from the Me2CO washings (see above), and the residual yellow solid was suspended in
cooled Me2CO (20 ml), filtered off and washed with cooled Me2CO (20 ml) to yield 3-
methoxy-2-methyl-5-nonadecyl-1,4-quinone as a by-product (1.52 g, yield: 25.8% of the
theory) after drying (M. p. 77-78o, M. p.Lit. 68-74o [39]). Rf (SiO2; n-hexane/AcOEt = 19:1
(v/v)) 0.22.
1,3-Dihydroxy-2-methyl-5-nonadecylbenzene (=5-Nonadecyl-2-methylresorcinol, 2d)
A solution of 21 (1.00 g, 2.39 mmol) in CH2Cl2 (40 ml) was cooled with an ice/NaCl
mixture under a N2 atmosphere. BBr3 (1.2 ml, resp. 3.16 g, 12.64 mmol) was added under
stirring, and stirring was continued for 6 h 30 min, while the reaction mixture warmed up to
room temperature. Then the reaction was quenched by adding a sat. aqueous NaHCO3
solution (10%, w/v, 200 ml) and the mixture extracted with CH2Cl2 (3 x 100 ml). The
combined extracts were dried (Na2SO4) and the solvent was removed in vacuo. Purification
of the crude product (0.88 g) was achieved by vacuum liquid chromatography (VLC) on
silica 60 G (100 g), preconditioned with n-hexane. Elution was performed with an n-
hexane/AcOEt gradient (100/0 to 80/20, v/v). The corresponding fractions containing the
target product (analysed by TLC) were combined and the solvent was distilled off in vacuo to
afford the pure compound 2d (0.65 g, yield: 69.8% of the theory). White solid, M. p. 107.4o.
Rf (SiO2; CH2Cl2/MeOH = 19:1 (v/v)) 0.55. UV/VIS (ACN): 204 (4.58), sh 226, 270 (2.98).
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GC-MS purity (70 eV): > 98 % (tR 57.6 min) m/z 390 (9), 348 (0.4), 306 (0.5), 236 (0.2), 222
(0.6), 207 (0.4), 180 (3), 165 (0.7), 151 (10), 138 (100), 137 (39), 123 (5), 109 (1), 91 (2), 83
(2), 69 (3), 57 (9). IR (KBr, ν in cm-1): 3350 (brs, OH), 2954 (m, aromatic CH), 2917 (s,
alkyl-CH), 2847 (s, alkyl-CH), 1632 (m), 1587 (m), 1523 (w), 1466 (m), 1426 (m), 1327 (m),
1169 (m), 1095 (m), 1072 (s), 924 (w), 843 (m), 721 (m), 661 (w). The MS and NMR data
correspond to the literature (Sconamiglio et al. 2012). For APCI+-MS data see Table 1.
DPPH Radical Scavenging Assay
The DPPH assay was performed according to a literature procedure [36]. In brief,
different concentrations of the test compounds in MeOH (200 µl) were added each to 1800 µl
of 1,1’-diphenyl-2-picryl-hydrazyl (DPPH, 100 µM) and incubated for 30 min at 37o. Then,
the absorbance at λmax 516 nm was measured using a Perkin-Elmer UV/VIS-spectrometer
(Lambda 2). IC50 values were calculated from the plot of resulting absorbance/concentrations
curves by regression analysis. Scavenging activities were expressed as IC50 values, which
denote the concentrations of the test compound (µM) required to bring about a 50% reduction
in absorbance relative to that of the control. The lower the IC50 value, the higher is the
scavenging activity of the corresponding compound.
Scanning Electron Microscopy (SEM)
Samples of upper and lower surfaces of a fully developed leaf of a herbarium specimen
(voucher no. HOH-014615) were prepared for scanning electron microscopy. The leaf
samples, size 7-10 mm x 2-3 mm, were cut with a razor blade, and also a razor blade was
used to partly remove the trichomes from the leaves, to reveal the cuticle of the epidermis
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between the trichomes. The dry samples were mounted on adhesive carbon tabs on Al-stubs,
sputter-coated with gold-palladium (SCD 040, Balzer Union, Wallruf, Germany) and
investigated using a scanning electron microscope (DSM 940, Zeiss, Oberkochen, Germany)
at 5 and 7 kV.
The authors wish to acknowledge Prof. O. Spring (Department of Botany, Hohenheim
University, Stuttgart, Germany) for the identification of the plant specimens and F. Clamote
(Almada, Portugal) for providing a photograph of M. tomentosa. We thank Ms. C. Contreras-
Zamorano (Naturamus GmbH, Aichelberg, Germany) for procuring plant material from
Spain.
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Figures
Scan EI+
TIC
a)
100
0
Scan EI+
124
C23
C21
C19
b)
100
C17
C20
C22
C25
C24
C15
C18
0
Scan EI+
138
C17
C23
C19
C20
*
C21
*
*
*
*
c) 100
C15
C22
*
*
C18
*
C24*
C25*
0
Time [min]
40.0
45.0
50.0
55.0
60.0
65.0
70.0
75.0
Fig. 1. Sections of a GC-MS profile showing lipid constituents of M. tomentosa L. a) Crude
CHCl3 extract, displays fatty n-alcohols (Alc) and n-aldehydes (Ad). b) MeOH fraction
resulting from polyamide chromatography; Scan on m/z 124, exhibiting 5-n-alkylresorcinols
(1a-j, Cn). c) Scan on m/z 138, exhibiting 5-n-alkyl-2-methylresorcinols (2a-j, Cn*).
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a)
b)
c)
Fig. 2. Scanning electron micrographs of leaves of M. tomentosa L. a) Upper leaf surface
densely covered with trichomes. b) Upper leaf surface after removing trichomes exhibiting a
continuous smooth cuticle with only few wax particles (arrows); cut off trichome (t); cutting
edge of the cuticle (arrow head). c) Undersurface showing the cuticular ledge of a stoma.
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50
45
40
35
30
25
20
15
10
5
1a-h
2a-h
R = CnH2n+1
0
15
17
18
19
20
21
22
23
Carbon chain lengths [n]
Fig. 3. Relative distribution of 5-n-alkylresorcinols (1a-h) and 5-n-alkyl-2-methylresorcinols
(2a-h) in M. tomentosa (GC-MS area %, n = 3), based on chromatograms as shown in Fig. 1.
The homologous 1i-j (C24-C25) and 2i-j (C24*-C25*) were not covered in this calculation due
to their trace amounts.
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Scan EI+
TIC
100
80
13
3
14
8
60
11
17
40
5
12
4
16
10
15
7
9
6
20
25.0
10.0
15.0
30.0
35.0
Time [min]
20.0
Fig. 4. Section of a GC-MS chromatogram of the MeOH fraction eluted from the polyamide
column, exhibiting mostly aromatic constituents of M. tomentosa. For compound assignment
see Table 2.
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C19
*
9
x10
C21
*
C19
8
6
4
C21
C17
*
C15
*
C17
C20
C15
*
C18
C23
*
*
C23
C22
C18
*
C20
C22
C24
*
C25
*
2
0
C24
C25
30.0
40.0
45.0
55.0 Time [min]
20.0
25.0
35.0
50.0
Fig. 5. Staggered illustration of LC-MS extracted ion traces [M+H]+ of individual 5-n-alkyl-
(1a-j, Cn) and 5-n-alkyl-2-methylresorcinols (2a-j, Cn*).
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Scan EI+
138.1
100
a)
50
137.1
151.1
57.0
53.0 69.1
390.4
391.4
123.0
122
83.1
125
180.2
389.4
222.2
222
306.3
322
264.2
272
348.4
0
m/z
72
172
372
391
b)
+MS
9
3
2
1
0
x10
+MS2(391)
8
x10
2
1
137
110
107
0
1
6
+MS3(391->125)
x10
0.5
125
80
79
97
0
1
+MS4(391->125->107)
4
x10
0.5
125
0
50
m/z
100
150
200
250
300
350
400
Fig. 6. EI- and APCI+-MSn mass spectra of compound 2d (C26H46O2; MW 390.64). a) EI
spectrum (70 eV). b) APCI+-MSn data.
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Schemes
Scheme 1. Proposed mass spectrometric fragmentation of 5-n-alkyl-2-methylresorcinols (2,
Cn*) yielding their most intense daughter ions, observed in the APCI+ and EI (70 eV) mass
spectra.
APCI
EI
McLafferty
dihydroxytropylium ion
HO
HO
HO
HO
OH
OH2
OH2
OH
m/z 137
m/z 138
m/z 137
m/z 125
-cleavage
HO
-cleavage
OH2
HO
HO
HO
m/z 180
m/z 151
HO
R
HO
O
O
OH
m/z 123
-cleavage
m/z 107
OH
Scheme 2. Synthesis of 2-methyl-5-nonadecyl-resorcinol (2d).
a) (COCl)2, toluene, 0o. b) n-C18H37MgCl/THF, bis-[2-(N,N’-dimethylamino)ethyl]ether,
-78o. c) NH2OH, KOH, triethylenglycol, 200o, reflux. d) BBr3/CH2Cl2, 0o.
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Tables
Table 1. GC-MS and LC-MS data for 5-n-alkyl- (1a-j, Cn) and 5-n-alkyl-2-methylresorcinols
(2a-j, Cn*), identified in the polyamide fraction (MeOH) of the CHCl3 extract from M.
tomentosa.
GC-MS
(EI)
LC-
MS
tR
Intrinsic ion peaks
in the APCI+ mode
MS1(M+H)+
Compound
side chain
1a, C15
2a, C15*
1b, C17
2b, C17*
1c, C18
2c, C18*
1d, C19
2d, C19*
1e, C20
2e, C20*
1f, C21
2f, C21*
1g, C22
2g, C22*
1h, C23
2h, C23*
1i, C24
2i, C24*
1j, C25
2j, C25*
tR
m/z a)
MS2
MS3
[min]
49.4b)
50.3
53.0
53.8
54.7
55.5
56.4
57.4
58.3
59.4
60.5
61.7
63.0
64.4
65.9
67.6
69.4b)
71.4b)
73.5b)
75.9b)
[M]+ [min]
n.d.
334
348
362
362
376
376
390
390
404
404
418
418
432
432
446
n.d.
n.d.
n.d.
n.d.
20.0
19.1
28.3
27.5
32.6
31.9
36.5
35.6
41.1
40.2
44.1
43.3
47.7
47.0
50.8
50.2
53.9
53.2
57.0
56.0
321
335
349
363
363
377
377
391
391
405
405
419
419
433
433
447
447
461
461
475
111
125
111
125
111
125
111
125
70
110, 107, 79
93, 70
107, 78
93
107
70
107
66
137, 125, 111
125
114, 107, 97
95, 79
107
123
125
123
125
123
125
n.d.
n.d.
-
107, 79
-
107, 110
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a) In addition also the fragments: m/z 166*, 137**, 124 (base peak)*** and 123**** were also detected for all 5-
n-alkylresorcinols (Cn) and m/z 180*, 151**, 138 (base peak)*** and 137**** for the 5-n-alkyl-2-
*
methylresorcinols
(Cn ),
respectively
(*δ-,
**β-cleavage,
***McLafferty
rearrangement,
****dihydroxytropylium ion).b) Detected only in traces. n.d. = not detected. The corresponding ions which were
further fragmented in the MSn mode are underlined.
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Table 2. GC-MS data of constituents detected in the polyamide fraction (MeOH) of the M.
tomentosa CHCl3 extract.
No.
)
Constituent
tR [min] calc. Mr
Characteristic fragments, m/z (BPI [%])
122 (90), 121 (100), 93 (35), 65 (41)
119 (100), 91 (23), 64 (17), 63 (13)
a
[Da]
4-
Hydroxybenzaldehyde
4-
Hydroxybenzonitrile
Hermidin
15.0
16.2
16.9
17.4
19.9
122.12
119.12
171.15
136.15
166.17
3
4
5
6
7
171 (99), 156 (36), 142 (100), 128 (11), 114
(26), 69 (24)
4-
136 (34), 121 (100), 93 (31), 65 (26)
Hydroxacetophenone
Ethyl 4-
hydroxybenzoate
Hermidin quinone
166 (17), 138 (19), 121 (100), 93 (16), 65 (16)
169 (38), 112 (36), 84 (18), 69 (100)
20.6
20.8
169.13
168.15
8
9
Vanillic acid
168 (100), 153 (75), 125 (21), 97 (40), 79 (10),
69 (19)
Ethyl 2-(E)-
hydroxycinnamate
(E)-Coniferylalcohol
25.5
26.1
192.21
192 (46), 164 (11), 147 (100), 120 (38), 119
(29), 91 (28), 65 (27)
10
11
180.20 180 (75), 137 (100),131 (13), 124 (71), 119 (25),
109 (16), 103 (24), 91 (49), 77 (22), 65 (14)
Methyl 4-(E)-
hydroxycinnamate
Ethyl 4-(E)-
hydroxycinnamate
Palmitic acid
26.3
28.4
31.7
178.18
192.21
256.42
178 (66), 147 (100), 137 (22), 124 (13), 119
(37), 91 (39), 65 (27)
192 (49), 164 (12), 147 (100), 120 (36), 119
(29), 91 (29), 65 (24)
256 (31), 213 (18), 185 (10), 171 (10), 157 (9),
129 (33), 115 (13), 97 (29), 83 (37), 73 (98), 55
(100)
12
13
14
Scopoletin
31.9
32.5
192.17
210.23
192 (100), 177 (62), 164 (29), 149 (59), 121
(35), 79 (19), 69 (34)
210 (100), 182 (23), 167 (93), 154 (40), 149
(35), 135 (11), 121 (20), 106 (13), 91 (21), 77
(45)
15
16
Sinapylalcohol
35.9
278.43
278 (3), 249 (1), 222 (7), 149 (5), 135 (9), 121
(13), 108 (34), 93 (42), 79 (100), 67 (72)
17
α-Linolenic acid
a) For peak numbering see Fig. 4.
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Table 3. IC50 (µM) of 5-nonadecyl-2-methyl-resorcinol (2d, C19*) and trolox according to the
DPPH method.
Compound
2d
Regression equation
y = 18.44 ln(x)-17.011
y = 2.40 x-0.5825
R2
IC50 (µM)
37.86a)
0.9975
0.9997
trolox
21.03b)
a)Means of triplicates (CV < 5%).b)Value in accordance with the literature [3
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