Phytochemistry 72 (2011) 1848–1853

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Alkamides and a neolignan from Echinacea purpurea roots and the interaction
of alkamides with G-protein-coupled cannabinoid receptors
Judit Hohmann a,⇑, Dóra Rédei a, Peter Forgo a, Pál Szabó b, Tamás F. Freund c, József Haller c, Engin Bojnik d,
Sándor Benyhe d
a
Department of Pharmacognosy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
b
Institute of Chemistry, Chemical Research Centre, Hungarian Academy of Sciences, Pusztaszeri út 59-67, H-1525 Budapest, Hungary
c
Department of Behavioral Neurobiology, Institute of Experimental Medicine, P.O. Box 67, H-1450 Budapest, Hungary
d
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary

a r t i c l e i n f o a b s t r a c t

Article history: Multiple chromatographic separations of the CHCl3-soluble extract of the roots of Echinacea purpurea led
Received 22 April 2011 to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate,
Received in revised form 17 June 2011 were identified, and five further compounds were detected for the first time in this species. Additionally,
Available online 19 July 2011
10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry
This work is dedicated to Professor Kálmán and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in
Szendrei (Department of Pharmacognosy, [35S]GTPcS-binding experiments performed on rat brain membrane preparations. Both partial and
University of Szeged) on the occasion of his inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, char-
75th birthday. acterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-
modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with
Keywords: the CB1 receptor agonist reference compound arachidonyl-20 -chloroethylamide (ACEA). However, upon
Echinacea purpurea coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure
Asteraceae
agonist, thereby demonstrating cannabinoid receptor antagonist properties.
Alkamide
Ó 2011 Elsevier Ltd. All rights reserved.
Neolignan
Sesquiterpene
G-protein modulation
Cannabinoid receptor
GTPcS-binding assay

1. Introduction effects of five Echinacea preparations were recently reported, based

Echinacea purpurea Moench is one of the most popular medici-
nal plants; its preparations are used widely for the prevention or
treatment of common cold, coughs, bronchitis, influenza, different
wounds and inflammation of the mouth and pharynx (Barrett,
2003). Controlled clinical trials have proved that preparations of
E. purpurea may have beneficial effects on cold symptoms in adults,
especially if treatment is started early (Woelkart et al., 2008). A
series of experiments have demonstrated that Echinacea drugs ex-
ert significant immunomodulating activities, and strengthen the
immune system against pathogenic infections through the activa-
tion of neutrophils, macrophages, polymorphonuclear leukocytes
and natural killer cells (Barnes et al., 2005). Although the psycho-
tropic potential of Echinacea drugs is not widely known and this
potential was not recorded in traditional medicine, the anxiolytic
⇑ Corresponding author. Tel.: +36 62 546453; fax: +36 62 545704.
E-mail address: hohmann@pharm.u-szeged.hu (J. Hohmann).
on three animal tests of anxiety (elevated plus-maze, social inter-
action and shock-induced social avoidance tests’ (Haller et al.,
2010). Interestingly, the doses that revealed anxiolytic effects were
found to be much lower than those used in the laboratory models
of the traditional indications. Furthermore, an orally administered
Echinacea pallida root extract was reported to induce accelerated
wound healing in stressed mice, but had no apparent efficacy for
non-stressed animals (Zhai et al., 2009).
The extracts of the roots and herb of Echinacea species have a
complex chemical composition. Unsaturated lipophilic compounds
(alkamides and ketoalkenes/alkynes), glycoproteins, caffeic acid
derivatives and polysaccharides are believed to be responsible for
the observed immunostimulatory and anti-inflammatory activities
(Barnes et al., 2005). Alkamides from Echinacea have cannabinomi-
metic properties at both the cannabinoid CB1 and CB2 receptors
(Woelkart et al., 2005), reflecting their structural similarity to the
endogenous ligand anandamide. The CB1 and CB2 receptors are
heptahelical G-protein-coupled receptors with different distribu-

0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.06.008
 J. Hohmann et al. / Phytochemistry 72 (2011) 1848–1853 1849

tions: the CB1 receptors are highly expressed in the central ner-
vous system (CNS), with low to moderate expressions in the
periphery, while the CB2 receptor expression is high in the im-
mune system, but with much lower and more restricted distribu-
tion in the CNS. The significant CB2 receptor activity of
alkamides, demonstrated by binding assays, is now considered to
be a possible mode of action of Echinacea alkamides as immuno-
modulatory agents (Raduner et al., 2006; Woelkart and Bauer,
2007; Chicca et al., 2009). Although the interaction between anxi-
ety and cannabinoids is complex, activation of the CB1 receptors by
endogenous ligands may play a role in the control of anxiety (Fre-
und, 2003; Haller and Bakos, 2002).
The aim of the present study was to investigate the chemical
composition of the roots of E. purpurea and to acquire new
information on the affinity of the isolated compounds for the
cannabinoid system. Multiple chromatographic separations of the
CHCl3-soluble extract of the roots were afforded 19 pure com-
pounds, including alkamides 1–3 and nitidanin diisovalerianate
(18). A further five compounds (5–8, 19) were detected for the
first time in this species, and 10 known E. purpurea metabolites
(alkamides 4, 9–17) were also identified. From among the isolated
compounds, 13 were studied for their cannabinoid receptor activ-
ities in [35S]GTPcS-binding assays.
2. Results and discussion
2.1. Structure elucidation
Repeated vacuum liquid chromatography (silica gel, RP-18) and
prep. TLC of the CHCl3-soluble extract of the roots of E. purpurea re-
sulted in the isolation of 19 compounds. The known E. purpurea
metabolites were identified as 4, 9–17 on the basis of their spectro-
scopic profiles (UV, 1H NMR and 13C NMR) through comparison
with published data (Perry et al., 1997; Chen et al., 2005; Bauer
et al., 1988; Zhang et al., 2005; Dietz and Bauer, 2001). Addition-
ally, the known alkamides 5–8 were isolated from E. purpurea for
the first time; undeca-2E,4E-diene-8,10-diynoic acid isobutyla-
mide (5) was described earlier from Echinacea atrorubens (Dietz
and Bauer, 2001), dodeca-2Z,4E,10Z-trien-8-ynoic acid isobutyla-
mide (6) (Chen et al., 2005) and dodeca-2E,4Z,10Z-trien-8-ynoic
acid isobutylamide (7) (Bauer and Remiger, 1989; Bauer et al.,
1989) from the roots of Echinacea angustifolia, and dodeca-2E,4E-
diene-8,10-diynoic acid isobutylamide (8) from Achillea species
(Bohlmann and Zdero, 1973). Our 2D NMR investigations, includ-
ing 1H–1H COSY, HSQC and HMBC experiments, allowed the unam-
biguous assignment of all 13C chemical shifts for 15 and 16.
Moreover, the 1H NMR data on 8 and the 13C NMR data on 5, 7,
8, 11, 13 and 17 were determined for the first time in this study
and are listed in Tables 1 and 2. In agreement with previous pub-
lications, 15 and 16 were obtained as an unseparable isomeric mix-
ture (Binns et al., 2002; Clifford et al., 2002). Similarly, 4 and 5
were isolated as mixtures of E and Z isomers; these could be suc-
cessfully separated by prep. RP-TLC, using MeCN–H2O (4:1), but
after separation both compounds readily formed the mixture
again. Compounds 4 and 5 in form of an inseparable mixture were
reported earlier from E. atrorubens (Dietz and Bauer, 2001).
Besides the alkamides the known sesquiterpene 1b-hydroxy-
4(15),5E,10(14)-germacratriene 19 was identified for the first time
from the Echinacea genus (Brown et al., 2003).
Compounds 1 and 2 were isolated as an isomeric mixture in ra-
tio 11:9. The HRESIMS spectrum of this mixture displayed a
[M+H]+ peak at m/z 262.2170, corresponding to the molecular for-
mula C17H27NO. The 1H and JMOD spectra of 1+2 showed signals
characteristic of alkamides (Tables 1 and 2), and it was evident
that, similarly as in the case of 15+16, isomeric dodeca-
2E,4E,8Z,10E/Z-tetraenoic acyl moieties are present in the
molecules. In contrast with isobutylamides 15+16, a 2-methylbu-
tylamide structure was identified in 1+2 via the proton resonances
at dH 3.23 m, 1.58 m, 1.17 m, 1.41 m, 0.91 t and 0.92 d, and the
carbon resonances at dC 45.7, 35.0, 26.9, 11.2 and 17.1 (Perry
et al., 1997; Bauer et al., 1988). The structures of 1 and 2 were
therefore determined to be as dodeca-2E,4E,8Z,10E-tetraenoic
acid methylbutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid
methylbutylamide, respectively.
Compound 3 was isolated as an amorphous solid. Its molecular
formula was determined from the molecular ion peak at m/z
246.1848 [M+H]+ in the HRESIMS spectrum to be C16H23NO. The
1
H NMR and JMOD spectra of 3 exhibited resonances typical of
isobutylamide (Tables 1 and 2). Three olefin bonds (dH 7.20, 6.22,
6.13, 5.91, 5.80 and 5.45; dC 140.8, 140.3, 137.5, 129.3, 124.4 and
110.1), two with E (J = 15.0 Hz) and one with Z (J = 10.6 Hz) geom-
etry, and one acetylene group (dC 93.4 and 78.0) were indicated by
the 1H and 13C chemical shifts and coupling constants. Further, two
methylene groups (dH 2.41 m and 2.49 m; dC 32.2 and 19.3), and
one secondary methyl (dH 1.83 d; dC 15.8) were observed in the
spectra besides the carbonyl carbon signal (dC 163.9). The se-
quences of correlated protons in the 1H–1H COSY spectrum
furnished information on two partial structures [(E,E) –CH = CH–
CH = CH–CH2–CH2– and (Z) –CH = CH–CH3] and suggested the

Table 1
13
C NMR data of the isolated alkamides (CDCl3, 125 MHz, d ppm).

Position 1 2 3 5 7 8 11 13 15 16 17
1 166.4 166.4 163.9 166.2 166.1 166.2 166.5 166.0 166.4 166.4 166.7
2 122.2 122.2 124.4 123.2 124.5 123.1 122.3 122.3 122.3 122.3 122.1
3 140.9 140.9 140.8a 138.9a 135.6a 139.3 138.5 143.4 140.8 140.8 140.7
4 128.7 128.7 129.3 129.7 127.5 129.6 128.5 31.1 128.7 128.7 128.3
5 141.8 141.8 140.3a 140.3a 137.6a 140.3 140.6 26.3a 141.7 141.6 142.5
6 32.9 32.8 32.2 26.3 27.5 31.6 32.9 27.5a 32.9 32.8 32.7
7 26.9 26.7 19.3 18.6 19.7 18.8 26.4 131.9b 26.8 26.7 31.6a
8 127.8 130.0 93.4 77.3 93.4 75.2 128.3 124.1b 127.8 129.9 28.9a
9 129.3 126.7 78.1 68.1 78.0 73.5 130.0 17.3 129.2 126.6 28.7a
10 124.2 124.2 110.1 65.3 110.1 66.1 29.1 76.0 124.0 124.2 28.4a
11 129.7 126.7 137.5 65.0 137.4a 64.3 22.6 68.1 129.7 126.6 22.4a
12 18.2 13.1 15.8 - 15.8 4.1 13.6 64.4 18.2 13.1 13.8
13 - - - - - - - 65.1 - - -
10 45.7 45.7 46.9 46.9 46.9 46.9 46.8 46.7 46.9 46.9 46.9
20 35.0 35.0 28.6 28.4 28.6 28.6 28.5 28.3 28.5 28.5 28.4
30 26.9 26.9 20.1 20.0 20.1 20.1 20.0 20.0 20.1 20.1 20.0
40 11.2 11.2 20.1 20.0 20.1 20.1 20.0 20.0 20.1 20.1 20.0
50 17.1 17.1 - - - - - - - - -
a,b
Signals in each column may be interchanged.
1850 J. Hohmann et al. / Phytochemistry 72 (2011) 1848–1853

Table 2
1
H NMR data of the isolated alkamides [CDCl3, 500 MHz, d ppm (J = Hz)].

Position 1 2 3 8
2 5.81 d (14.9) 5.81 d (14.9) 5.80 d (15.0) 5.82 d (15.0)
3 7.18 dd (14.9, 10.9) 7.18 dd (14.9, 10.9) 7.20 dd (15.0, 10.6) 7.15 dd (15.0, 11.0)
4 6.13 dd (15.0, 10.9) 6.13 dd (15.0, 10.9) 6.22 dd (15.2, 10.7) 6.16 dd (15.0, 11.0)
5 6.06 dt (15.0, 6.1) 6.06 dt (15.0, 6.1) 6.13 dd (15.2, 6.6) 6.02 dt (15.0, 7.0)
6 2.28 m 2.28 m 2.41 m 2.34 m
7 2.24 m 2.24 m 2.49 t (7.0) 2.34 m
8 5.25 dt (10.7, 7.0) 5.42 dt (10.6, 7.2) - -
9 5.97 t (10.7) 6.28 dd (10.7, 7.2) - -
10 6.31 dd (14.8, 10.7) 6.24 t (10.9) 5.45 d (10.6) -
11 5.69 dq (14.8, 6.8) 5.55 dq (10.8, 7.1) 5.91 dq (10.6, 6.7) -
12 1.77 d (6.8) 1.75 d (7.2) 1.83 d (6.7) 1.88 s
10 3.23 m 3.23 m 3.16 t (6.6) 3.14 t (6.5)
20 1.58 m 1.58 m 1.80 m 1.78 m
30 1.17 m, 1.41 m 1.17 m, 1.41 m 0.93 d (6.7) 0.90 d (6.5)
40 0.91 t (7.1) 0.91 t (7.1) 0.93 d (6.7) 0.90 d (6.5)
50 0.92 d (7.2) 0.92 d (7.2) - -
NH 4.53 brs 4.35 brs 5.60 brs 5.80 brs

Table 3
NMR data on compound 18 500 MHz (1H), 125 MHz (13C), CDCl3, d (ppm) (J = Hz).
1 13 1 13
Position H C Position H C
0
1 - 126.6 8 6.16 dt (16.0, 6.4) 122.1
2 6.60 s 104.1 90 4.71 d (6.4) 64.7
3 - 147.3 100 - 172.9
4 - 135.6 200 2.23 d (7.0) 43.4
5 - 147.3 300 2.12 m 25.7
6 6.60 s 104.1 400 0.97 d (6.5) 22.4
7 4.86 d (8.0) 76.6 500 0.97 d (6.5) 22.4
8 4.26 m 75.6 10 00 - 172.5
9 4.35 dd (12.0, 2.5) 62.6 20 00 2.20 d (6.5) 43.0 Fig. 1. Key HMBC (H ? C) and NOESY (H M H) correlations of compound 18 (bold
4.04 dd (12.0, 5.0) 30 00 2.09 m 25.5 lines indicate partial structures elucidated from the 1H–1H COSY spectrum).
10 - 129.1 40 00 0.95 d (7.0) 22.4
20 6.60 d (1.6) 102.9 50 00 0.95 d (7.0) 22.4 carbon signals at dC 76.6 (C-7), 75.6 (C-8) and 62.6 (C-9). The long-
30 - 148.9 OCH3 3.90 s 56.3 range correlations detected in the HMBC spectrum between H-70
40 - 132.8 OCH3 3.90 s 56.3
and C-10 , C-20 and C-60 and between H-7 and C-1, C-2 and C-6 led
50 - 144.1 OCH3 3.90 s 56.2
60 6.68 d (1.6) 108.4 4-OH 5.67 s -
to the identification of two phenylpropanoid structural moieties:
70 6.53 d (16.0) 133.8 one 3,4,5-trisubstituted trans-cinnamyl alcohol unit and another
phenylpropanoid unit symmetrically substituted on the aromatic
ring. These units could be connected to a benzodioxane-type neo-
position of the acetylene group on C-8. Analysis of the HSQC and lignan with regard to the HMBC correlations between C-40 and H-9
HMBC spectra corroborated that the structure of 3 is dodeca- and the NOESY correlation between H-60 and H-7. The HMBC cor-
2E,4E,10Z-trien-8-ynoic acid isobutylamide and allowed the chem- relation of the proton signal at dH 3.90 (3  OCH3) and OH-signal at
ical shift assignments as presented in Tables 1 and 2. dH 5.67 with the carbon signals at dC 147.3, 147.3, 148.9 and 135.6
Compound 18 was isolated as an amorphous solid material. Its suggested the sites of methoxylation as C-3, C-5 and C-30 , and
HRESIMS investigation demonstrated the presence of a quasimo- hydroxylation as C-4, respectively. The positions of the isovaleroyl
lecular ion peak at m/z 595.2525 [M+Na]+ (C31H40NO10Na), which groups at C-9 and C-90 were evident from the three-bond correla-
afforded fragment ions at m/z 471.2023 [(M+H)C5H10O2]+ and tions between the ester carbonyl carbons (dC 172.5 and 172.9)
369.1323 [(M+H)2  C5H10O2]+, indicating the loss of two ali- and H2-9 and H2-90 (dH 4.04 and 4.35 and 4.71). The configuration
phatic pentanoic acid units. The 1H NMR and J-modulated 13C of the stereogenic centers of 18 was elucidated by analyzing the
NMR spectra of 18 showed typical signals of two isovaleroyl groups Overhauser effects detected in the NOESY spectrum (Fig. 1). NOE
(Table 3). A singlet signal at dH 3.90 (9H) and carbon signals at dC correlations between H-7 and H-9, and between H-8 and H-2
2  56.2 and 56.3 were indicative of the presence of three methoxy and H-6, indicated the trans stereochemistry of H-7 and H-8, which
groups. Additionally, 13 proton and 18 carbon resonances were de- was further supported by the observed coupling constant
tected in the 1H NMR and JMOD spectra, respectively, which were (J7,8 = 8.0 Hz). All of the above data are compatible with the struc-
analyzed with the aid of 1H–1H COSY, HSQC, HMBC and NOESY ture of 18 being 9,90 -diisovaleroxy nitidanin. Nitidanin was first
experiments. A –CH = CH–CH2O– structural moiety with E geome- isolated from Xanthoxylum nitidum (Rutaceae) as an antimalarial
try was readily identified on the basis of the proton resonances at agent (Ishikawa et al., 1995), and later described from other plant
dH 6.53 d (H-70 ), 6.16 dt (H-80 ) and 4.71 d (H2-90 ), showing 1H–1H families, too. Its total synthesis has also been reported (Kuboki
COSY correlations, and carbon resonances at dC 133.8 (C-70 ), et al., 2007).
122.1 (C-80 ) and 64.7 (C-90 ), exhibiting HSQC correlations with
these protons. Proton signals at dH 6.60 (2 s, 1 d) (H-2, H-6 and
H-20 ), and 6.68 d (H-60 ), and carbon signals in the interval dC 2.2. Interaction with G-protein-coupled cannabinoid receptors
102.9–148.9 (C-1-C-6 and C-10 -C-60 ), suggested two tetrasubstitut-
ed aromatic rings in the molecule. Further, a –CH(OR)–CH(OR)– The bioactivity of the compounds was studied in [35S]GTPcS-
CH2O– structural fragment was elucidated from the correlative binding experiments on rat brain membrane preparations. This
signals at dH 4.86 (H-7), 4.26 (H-8), 4.35 and 4.04 (H2-9) and the assay measures agonist-induced, receptor-mediated G-protein
 J. Hohmann et al. / Phytochemistry 72 (2011) 1848–1853 1851

activation. Representative dose–response curves are shown in Fig. 2. receptor-activating potency. Similar effects were previously ob-
Some of the compounds displayed either partial agonist (1+2) or served by Gertsch et al. (2006). Our results showed that on ex-
inverse agonist effects (Table 4), while the reference compound change of the isobutylamide moiety of the molecule (15+16) for
arachidonyl-20 -chloroethylamide (ACEA) is a full agonist ligand at 2-methylbutylamide (1+2), the G-protein-stimulation property
the CB1 receptor. Despite their relatively low efficacy at the CB turned from that of an inverse agonist (15+16) to one of a partial
receptors, the inverse agonist Echinacea compounds were capable agonist (1+2) and, in combination with ACEA, the antagonist effect
of inhibiting the full agonist effect of ACEA (Fig. 3). Although ACEA (1+2) changed to an additive agonist effect (15+16).
is a selective agonist of CB1 receptors, the contribution of CB2 sub-
type or other classes of GPCR to the overall effect on G-protein 3. Experimental
stimulation can not be excluded. The inhibition (antagonizing
effect) was characterized by a shift to the right and a decrease in 3.1. General experimental procedures
the plateau of the ACEA dose–response curves in the presence of
1 lM of an Echinacea ligand. The partial agonist 1+2 was also ex- Optical rotation was determined in CHCl3 with a Perkin–Elmer
pected to be a competitive antagonist when it was applied in com- 341 polarimeter. UV spectra were recorded in MeOH on a Shima-
bination with ACEA (Fig. 3). However, 1+2 significantly enhanced dzu UV-2101 PC spectrophotometer. NMR spectra were recorded
the G-protein-stimulatory effect of ACEA (F-test comparison of fits, in CDCl3 on a Bruker Avance DRX 500 spectrometer at 500 MHz
P = 0.0017). This type of additive or synergistic effect can be (1H) or 125 MHz (13C). 2D data were acquired and processed with
explained by the activation of other G-protein-coupled receptors standard Bruker software. For 1H–1H COSY, HSQC and HMBC
(probably CB2) present in the rat brain membrane preparation. experiments, gradient-enhanced versions were used. Mass spec-
trometric measurements were performed on a Shimadzu IT-TOF
2.3. Concluding remarks instrument. For vacuum liquid chromatography (VLC), silica gel
60G (15 lm, Merck) was applied. RP-VLC was performed on a
Our results demonstrate that slight differences in the structures LiChroprep RP-18 (40–63 lm, Merck) stationary phase. Separa-
of the alkamides resulted in significant differences in cannabinoid tions were monitored by TLC on 60 F254 (0.25 mm, Merck) plates.

150 150
% of GTP[ 35S] binding
% of GTP[ 35S] binding

ACEA ACEA
140 Compound 4+5 140 Compound 12
over the basal
over the basal

130 130
120 120
110 110
inverse agonist inverse agonist
100 100
90 90

-11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4

log[Ligand], M log[Ligand], M

150 150
% of GTP[ 35S] binding
% of GTP[ 35S] binding

ACEA ACEA
140 Compound 3 140 Compound 1+2
over the basal
over the basal

130 130
120 120
110 110
100 inverse agonist 100
partial agonist
90 90

-11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4

log[Ligand], M log[Ligand], M

Fig. 2. Receptor-mediated G-protein stimulation by compounds 1+2, 3, 4+5 and 12.

Table 4
Cannabinoid receptor activating properties of selected E. purpurea compounds.

Compound Propertya Efficacy (%Emax) on CB receptor(s) (%) Antagonist effectb

ACEA Reference compound, CB1 receptor agonist +47 (Full agonist effect) —
15+16 Inverse agonist 10 Yes (⁄⁄⁄)
4+5 Inverse agonist 14 Not tested
12 Inverse agonist 7 Not tested
1+2 Partial agonist +9 Additive agonist effect (⁄⁄)
3 Inverse agonist 9 Yes (⁄⁄)
6 Neutral compound 0 Not tested
13 Inverse agonist 6 Yes (NS)
8 Inverse agonist 6 Not tested
18 Weak partial agonist +4 Not tested
19 Inverse agonist 9 Not tested

Asterisks indicate the level of statistical significance of antagonism (see Fig. 3), NS = not significant.
a
Determined in [35S]GTPcS binding experiments using rat brain membranes.
b
Against ACEA in the same in vitro assay.
1852 J. Hohmann et al. / Phytochemistry 72 (2011) 1848–1853

150 *** 150 **

% of GTP[ 35S] binding

% of GTP[ 35S] binding

control control
140 + c.15+16, 1 µM 140 + c. 3, 1 µM

over the basal

over the basal

130 130

120 120

110 110

100 100

90 90
-11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4
log[ACEA], M log[ACEA], M

160
150 **
% of GTP[ 35S] binding

% of GTP[ 35S] binding

control control
150
140 + c. 13, 1 µM + c. 1+2, 1 µM
over the basal

over the basal

140
130
130
120
120
110
110
100
100
90
-11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4
log[ACEA], M log[ACEA], M

Fig. 3. Antagonist effect of compounds 1+2, 3, 13, 15+16 in [35S]GTPcS-binding experiments.

Preparative thin-layer chromatography (PLC) was carried out on
silica gel 60 F254 (0.25 mm, Merck). In the bioassay, ACEA (Tocris
Bioscience), was used as reference CB1 receptor agonist.
3.2. Plant material
The roots were from E. purpurea cultivated and collected in
Kondoros, Hungary in August 2008. The plant was identified by
Tamás Rédei (Institute of Ecology and Botany of the Hungarian
Academy of Sciences, Vácrátót, Hungary). A voucher specimen
(No. 774) has been preserved in the Herbarium of the Department
of Pharmacognosy, University of Szeged, Szeged, Hungary. The
plant material was dried and stored at room temperature until
preparation.
3.3. Extraction and isolation
The air-dried and ground roots (17.4 kg) were percolated with
CHCl3 (90 l) at room temperature. The extract was concentrated
in vacuo, yielding 220.1 g brown, oily material, which was sub-
jected to silica gel VLC using a gradient system of n-hexane–EtOAc
(volumetric ratios of 10:0, 9:1, 17:3, 8:2, 7:3, 1:1 and 0:10). In to-
tal, seventy fractions with a volume of 500 ml were collected, and
combined in 14 main fractions (I-XIV) with regard to the results of
TLC monitoring. Alkamide-containing fractions (X, XII and XIV)
were selected for further purifications, which were carried out by
a combination of RP-VLC, using an MeCN–H2O or MeOH–H2O gra-
dient, and prep. TLC on silica gel, using the solvent system n-hex-
ane–EtOAc (3:2) (system A) or CH2Cl2–acetone (19:1) (system B).
Fraction X (6.74 g) was separated first by RP-VLC with MeCN–
H2O mixtures (1:1, 3:2, 7:3, 4:1 and 9:1). The fraction obtained
with MeCN–H2O (7:3) was purified by prep. TLC with system B,
affording 1b-hydroxy-4(15),5E,10(14)-germacratriene (19)
(26.1 mg). Fraction XII (41 g) was chromatographed by means of
RP-VLC, using mixtures of MeCN–H2O as eluents (3:2, 7:3, 4:1
and 9:1). Crystallization of the fractions eluted with MeCN–H2O
(4:1 and 9:1) afforded compounds 15+16 (3.2 g) in a ratio 11:9
as a mixture in crystalline form. The fractions eluted with
MeCN–H2O (7:3) in the RP-VLC separation were next subjected
to RP-VLC with MeOH–H2O mixtures (11:9, 12:8, 13:7, 14:6,
15:5, 16:4 and 17:3) as eluents. On standing, the subfraction ob-
tained with MeOH–H2O (15:5) afforded on standing white crystals,
which were identified as dodeca-2Z,4E-diene-8,10-diynoic acid
isobutylamide (12) (122 mg). The mother liquor of this crystalliza-
tion was separated by prep. TLC, using system B, yielding the pure
compounds 3 (4.1 mg) and dodeca-2E,4Z,10Z-trien-8-ynoic acid
isobutylamide (7) (14.9 mg). After prep. TLC on silica gel, using
developing system B, other subfractions obtained from RP-VLC
separation of fraction XII resulted in the isolation of pure unde-
ca-2E,4E-diene-8,10-diynoic acid isobutylamide (9) (1.45 g), dode-
ca-2Z,4E,10Z-trien-8-ynoic acid isobutylamide (6) (6.2 mg),
dodeca-2E,4E,8Z-trienoic acid isobutylamide (11) (64 mg), dode-
ca-2E,4E-dienoic acid isobutylamide (17) (54 mg) and an isomeric
mixture of compounds 1+2 (57 mg) in a ratio of 11:9. Alkamide-
containing fraction XIV (12 g) was separated into four subfractions
(XII/1–XII/4) by RP-VLC with an MeCN–H2O gradient (1:1, 3:2, 7:3,
4:1, and 9:1). Subfraction XII/1 was purified by RP-VLC with a
mobile phase of MeOH–H2O (1:1), to afford an isomeric mixture
of undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide (4) and
undeca-2E,4E-diene-8,10-diynoic acid isobutylamide (5) (123.7 mg)
in a ratio of 2:3. Subfraction XII/2 was chromatographed with
the same method, and the subfractions obtained were further
purified by prep. TLC, using system B (twice), yielding dodeca-
2E,4E-diene-8,10-diynoic acid isobutylamide (8) (17.6 mg), trideca-
2E,7Z-dien-10,12-diynoic acid isobutylamide (13) (93.4 mg) and
dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide (10) (96.8 mg).
Subfraction XII/3 was repeatedly fractionated by RP-VLC with
MeCN–H2O, and then purified by prep. TLC twice, using systems
A and B, to afford dodeca-2E,4Z-diene-8,10-diynoic acid 2-meth-
ylbutylamide (14) (41 mg). Repeated prep. TLC separation of sub-
fraction XII/4 resulted in the isolation of compound 18 (25 mg).
3.3.1. Dodeca-2E,4E,8Z,10E-tetraenoic acid 2-methylbutylamide (1) +
dodeca-2E,4E,8Z,10Z-tetraenoic acid 2-methylbutylamide (2)
Amorphous, gummy solid; UV (MeOH) kmax 235 sh, 262 nm; 1H
and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (m/z)
262.2170 [M+H]+, calcd for 262.2165 C17H28NO.
 J. Hohmann et al. / Phytochemistry 72 (2011) 1848–1853
3.3.2. Dodeca-2E,4E,10Z-trien-8-ynoic acid isobutylamide (3)
Amorphous, gummy solid; UV (MeOH) kmax 231 sh, 263 nm; 1H
and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (m/z)
246.1848 [M+H]+, calcd for 246.1858 C16H24NO.
3.3.3. Nitidanin diisovalerianate (18)
An amorphous solid; ½a25 D 0 (c 0.1, CHCl3); UV (MeOH) kmax
(log e) 208 (3.306), 226 (3.173), 275 (2.822) nm; 1H and 13C NMR
spectroscopic data, see Table 3; HRESIMS (m/z) 595.2525 [M+Na]+,
calcd for 595.2514 C31H40O10Na, 471.2023 [(M+H)C4H9COOH]+,
calcd for 471.1941 C26H30O8, 369.1323 [(M+H)2C4H9COOH]+, calcd
for 369.1260 C21H20O6.
3.4. Membrane preparations from rat brain
Inbred Wistar rats (250–300 g body weight) were housed in the
local animal house of the Biological Research Centre (Szeged, Hun-
gary). Rats were kept in groups of four, allowed free access to stan-
dard food and tap water and maintained on a 12:12-h light/dark
cycle until the time of sacrifice. Animals were handled according
to the European Communities Council Directives (86/609/ECC)
and the Hungarian Act for the Protection of Animals in Research
(XXVIII.tv. 32.§). Crude membrane fractions were prepared by dif-
ferential centrifugation of the homogenized brain tissues. Rats
were narcotized with CO2, and then decapitated, and the brains
without the cerebellum were quickly removed and washed several
times with chilled 50 mM Tris–HCl (pH 7.4) buffer. The brains were
homogenized in 5 volumes/weight (5 ml/g) of the original brain
tissue with ice-cold 50 mM Tris–HCl (pH 7.4) buffer, using an elec-
trically driven Braun Teflon-glass rota-homogenizer at 4 °C (10–15
strokes, 1000 rpm). The homogenate was diluted up to 30 ml/g of
the brain and filtered through four layers of gauze to remove any
larger aggregates. After centrifugation with a Sorvall RC5C centri-
fuge at 40,000g (18,000  rpm) for 20 min at 4 °C, the resulting
pellet was resuspended in fresh buffer (30 ml/g). The suspension
was incubated for 30 min at 37 °C to remove any endogenous li-
gands. Centrifugation was repeated under the same conditions as
described above, and the final pellet was resuspended in 5 volumes
of TEM buffer, and then rapidly frozen under liquid nitrogen. The
membranes were kept in small aliquots at 80 °C until use.
3.5. [35S]GTPcS-binding assay
Rat brain membrane fractions (10 lg of protein/sample) were
incubated at 30 °C for 60 min in TEM buffer (50 mM Tris–HCl,
1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, pH 7.4) containing
[35S]GTPcS (0.05 nM) and increasing concentrations (109–
105 M) of the compounds tested in the presence of 30 lM GDP
in a final volume of 1 ml. The [35S]GTPcS, guanosine-5-[c-35S]-tri-
phosphate (1204 Ci/mmol), was purchased from the Isotope Insti-
tute Ltd. (Budapest, Hungary). Total binding was measured in the
absence of test compound; non-specific binding was determined
in the presence of 10 lM unlabeled GTPcS and subtracted from
the total binding to calculate the specific binding. The reaction
was started by the addition of [35S]GTPcS and terminated by filter-
ing the samples through Whatman GF/B glass fiber filters. The fil-
ters were washed three times with ice-cold 50 mM Tris–HCl buffer
(pH 7.4), using a Brandel M24R Cell Harvester, and then dried, and
the bound radioactivity was detected in UltimaGold™ scintillation
cocktail (Packard). Agonist-induced receptor-mediated G-protein
stimulation is reported as a percentage of the specific [35S]GTPcS
binding observed in the absence of receptor ligands (basal activity).
The experimental data were analyzed, statistically and graphically
processed by the GraphPad Prism research software package
1853
(version 4.00 for Windows, GraphPad Software, San Diego
California USA, www.graphpad.com). G-protein stimulation data
were computed by non-linear regression, using the sigmoid
dose–response curve fit option of Prism.
Acknowledgements
This investigation was supported by the OTKA CK-78566 Grant
from the National Scientific Research Fund (Budapest, Hungary),
New Development Plan TÁMOP-4.2.2-08/1-2008-0013 and
TÁMOP-4.2.1/B-09/1/KONV-2010-0005 and by NKTH Jedlik Ányos
Grant to H.J. and F.T.F.
References
Barnes, J., Anderson, L.A., Gibbons, S., Phillipson, J.D., 2005. Echinacea species
(Echinacea angustifolia (DC.) Hell., Echinacea pallida (Nutt.) Nutt., Echinacea
purpurea (L.) Moench): a review of their chemistry, pharmacology and clinical
properties. J. Pharm. Pharmacol. 57, 929–954.
Barrett, B., 2003. Medicinal properties of Echinacea: a critical review. Phytomedicine
10, 66–86.
Bauer, R., Remiger, P., 1989. TLC and HPLC analysis of alkamides in Echinacea drugs.
Planta Med. 55, 367–371.
Bauer, R., Remiger, P., Wagner, H., 1988. Alkamides from the roots of Echinacea
purpurea. Phytochemistry 27, 2339–2342.
Bauer, R., Remiger, P., Wagner, H., 1989. Alkamides from the roots of Echinacea
angustifolia. Phytochemistry 28, 505–508.
Binns, S.E., Livesey, J.F., Arnason, J.T., Baum, B.R., 2002. Phytochemical variation in
Echinacea from roots and flowerheads of wild and cultivated populations. Agric.
Food Chem. 50, 3673–3687.
Bohlmann, F., Zdero, C., 1973. Polyacetylenverbindungen, 215. Neue Inhaltsstoffe
aus Achillea-Arten. Chem. Ber. 106, 1328–1336.
Brown, G.D., Liang, G.Y., Sy, L.K., 2003. Terpenoids from the seeds of Artemisia annua.
Phytochemistry 64, 303–323.
Chen, Y., Fu, T., Tao, T., Yang, J., Chang, Y., Wang, M., Kim, L., Qu, L., Cassady, J., Scalzo,
R., Wang, X., 2005. Macrophage activating effects of new alkamides from the
roots of Echinacea species. J. Nat. Prod. 68, 773–776.
Chicca, A., Raduner, S., Pellati, F., Strompen, T., Altmann, K.H., Schoop, R., Gertsch, J.,
2009. Synergistic immunopharmacological effects of N-alkylamides in
Echinacea purpurea herbal extracts. Int. Immunopharmacol. 9, 850–858.
Clifford, L.J., Nair, M.G., Rana, J., Dewitt, D.L., 2002. Bioactivity of alkamides isolated
from Echinacea purpurea (L.) Moench. Phytomedicine 9, 249–253.
Dietz, B., Bauer, R., 2001. The constituents of Echinacea atrorubens roots and aerial
parts. Pharm. Biol. 39, 11–15.
Freund, T.F., 2003. Interneuron diversity series: rhythm and mood in perisomatic
inhibition. Trends Neurosci. 26, 489–495.
Gertsch, J., Raduner, S., Altmann, K.H., 2006. Review – new natural noncannabinoid
ligands for cannabinoid type-2 (CB2) receptors. J. Recept. Signal Transduct. Res.
26, 709–730.
Haller, J., Bakos, N., 2002. Stress-induced social avoidance. A new model of stress-
induced anxiety? Physiol. Behav. 77, 327–332.
Haller, J., Hohmann, J., Freund, T.F., 2010. The effect of Echinacea preparations in
three laboratory tests of anxiety: comparison with chlordiazepoxide. Phytother.
Res. 24, 1605–1613.
Ishikawa, T., Seki, M., Nishigaya, K., Miura, Y., Seki, H., Chen, I.S., Ishii, H., 1995.
Studies on the chemical constituents of Xanthoxylum nitidum (ROXB.)D. C.
(Fagara nitida ROXB.). III. The chemical constituents of the wood. Chem. Pharm.
Bull. 43, 2014–2018.
Kuboki, A., Yamamoto, T., Taira, M., Arishige, T., Ohira, S., 2007. Total synthesis of
(±)-nitidanin and novel procedures for determination of the location of the side
chains on 1,4-benzodioxane. Tetrahedron Lett. 48, 771–774.
Perry, N.B., van Klink, J.V., Burges, E.J., Parmenter, G.A., 1997. Alkamide levels in
Echinacea purpurea: a rapid analytical method revealing differences among
roots, rhizomes, stems, leaves and flowers. Planta Med. 63, 58–62.
Raduner, S., Majewska, A., Chen, J.-Z., Xie, X.Q., Hamon, J., Faller, B., Altmann, K.H.,
Gertsch, J., 2006. Alkylamides from Echinacea are a new class of
cannabinomimetics. J. Biol. Chem. 281, 14192–14206.
Woelkart, K., Bauer, R., 2007. The role of alkamides as an active principle of
Echinacea. Planta Med. 73, 615–623.
Woelkart, K., Linde, K., Bauer, R., 2008. Echinacea for preventing and treating the
common cold. Planta Med. 74, 633–637.
Woelkart, K., Xu, W., Pei, Y., Makriyannis, A., Picone, R.P., Bauer, R., 2005. The
endocannabinoid system as a target for alkamides from Echinacea angustifolia
roots. Planta Med. 71, 701–705.
Zhai, Z., Haney, D.M., Wu, L., Solco, A.K., Murphy, P.A., Wurtele, E.S., Kohut, M.L.,
Cunnick, J.E., 2009. Alcohol extract of Echinacea pallida reverses stress-delayed
wound healing in mice. Phytomedicine 16, 669–678.
Zhang, F., Chu, C.H., Xu, Q., Fu, S.P., Hu, J.H., Xiao, H.B., Liang, X.M., 2005. A new
amide from Asarum forbesii Maxim. J. Asian Nat. Prod. Res. 7, 1–5.