Flavones and phenylpropanoids from a sedative extract of Lantana trifolia L.

Article abstract of DOI:10.1016/j.phytochem.2009.10.007

The flavone glycosides, named scutellarein-7-O-β-d-apiofuranoside and apigenin-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-apiofuranoside, and the flavone celtidifoline (5,6,4′,5′-tetrahydroxy-7,3′-dimethoxyflavone), along with other 11 known compounds, were isolated from leaves of the ethyl acetate extract of Lantana trifolia L. using step gradient High Speed Countercurrent Chromatography (HSCCC) and High Performance Liquid Chromatography (HPLC), respectively. Their structures were elucidated by spectroscopic methods, including 2D NMR and mass spectrometry (ESI-MS) techniques. The ethanolic and ethyl acetate extracts produced an intense sedative effect in mice, one hour after oral administration of 1 mg/kg. This effect was neither due to a benzodiazepine-like effect of the three flavone derivatives neither of the phenylpropanoids, betonyoside F and verbascoside, that were tested for their affinity for the [³H] flunitrazepam binding sites.

Full text of DOI:10.1016/j.phytochem.2009.10.007

Phytochemistry 71 (2010) 294–300
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Flavones and phenylpropanoids from a sedative extract of Lantana trifolia L.
c
c
c
Lisieux de Santana Julião a, Suzana Guimarães Leitão b, , Cinzia Lotti , Anna Lisa Picinelli , Luca Rastrelli ,
*
Patricia D. Fernandes d, François Noël d, Jean-Pierre Barros Thibaut d, Gilda Guimarães Leitão e
a Universidade Federal do Rio de Janeiro, Programa de Biotecnologia Vegetal, CCS, Bl. K, Rio de Janeiro, RJ, Brazil
b Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, CCS, Bl. A, Ilha do Fundão, 21.941-590 Rio de Janeiro, RJ, Brazil
c Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
d Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, CCS, Rio de Janeiro, Brazil
e Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, CCS, Bl. H, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2008
Received in revised form 10 July 2009
Available online 16 November 2009
The flavone glycosides, named scutellarein-7-O-b-D-apiofuranoside and apigenin-7-O-b-D-apiofuranosyl-
(1 ? 2)-b-D
-apiofuranoside, and the flavone celtidifoline (5,6,40,50-tetrahydroxy-7,30-dimethoxyflavone),
along with other 11 known compounds, were isolated from leaves of the ethyl acetate extract of Lantana
trifolia L. using step gradient High Speed Countercurrent Chromatography (HSCCC) and High Performance
Liquid Chromatography (HPLC), respectively. Their structures were elucidated by spectroscopic methods,
including 2D NMR and mass spectrometry (ESI-MS) techniques. The ethanolic and ethyl acetate extracts
produced an intense sedative effect in mice, one hour after oral administration of 1 mg/kg. This effect was
neither due to a benzodiazepine-like effect of the three flavone derivatives neither of the phenylpropa-
noids, betonyoside F and verbascoside, that were tested for their affinity for the [3H] flunitrazepam bind-
ing sites.
Keywords:
Lantana trifolia
Verbenaceae
HSCCC
Scutellarein-7-O-b-
Apigenin-7-O-b- -apiofuranosyl-(1 ? 2)-b-
-apiofuranoside
Celtidifoline
D-apiofuranoside
D
Ó 2009 Elsevier Ltd. All rights reserved.
D
1D and 2D NMR spectroscopy
Sedative activity
Benzodiazepine receptor
1. Introduction
(Silva et al., 2005), we reported that the crude ethanolic extract
presented anti-inflammatory, antinociceptive and sedative activity
Lantana is a neotropical genus with approximately 150 species,
many of them occurring in Brazil. Phytochemical investigations
have established the presence of phenylpropanoids, flavonoids
and terpenoids, as the main class components with relevant bio-
logical activities (Ghisalberti, 2000). Lantana trifolia L. (syn. Lantana
celtidifolia Kunth.) is a small shrub that occurs in all regions of Bra-
zil and is extensively used in folk medicine in the form of infusions
and syrups for the treatment of respiratory disorders and as seda-
tive (Lorenzi, 2000; Correa, 1926). Few studies have been carried
out on either the chemistry or pharmacology (Achola and Mun-
enge, 1996; Silva et al., 2005; Katuura et al., 2007) of this species.
Umuhengerin, a pentamethoxyflavone isolated from its leaves,
exhibited antibacterial and anti-fungal activities (Rwangabo,
in mice. The ethyl acetate extract resulting from the liquid–liquid
partition of the ethanolic extract showed a similar pharmacological
profile (data not shown) and, therefore was chosen for study. Here
we report the isolation, using a HSCCC step gradient elution ap-
proach combined with HPLC, and structural determination of two
new flavone glycosides scutellarein-7-O-b-
D
-apiofuranoside (1),
-apiofuranoside
and apigenin-7-O-b- -apiofuranosyl-(1 ? 2)-b-
D
D
(3) and one new flavone celtidifoline (5,6,40,50-tetrahydroxy-7,30-
dimethoxyflavone) (2), as well as eleven known compounds
betonyoside F (4), verbascoside (5), vanillic acid (6), protocatechuic
acid (7), 4-hydroxybenzoic acid (8), caffeic acid (9), coumaric acid
(10), martynoside (11), scutellarein-7-O-b-D-glucopyranoside (12),
sorbifolin (13) and samioside (14). We also performed experiments
in order to assess the sedative effect and affinity for the benzodiaz-
epine receptor of these three new flavones and of the phenylprop-
anoids betonyoside F and verbascoside.
1988), whereas germacrene
D and caryophyllene are described as
major components of its essential oil (Muhayimana et al., 1998).
Iridoid glycosides, which are frequently common in the Verbena-
ceae, were not detected by Rimpler and Sauerbier (1986). In the
course of a preliminary pharmacological investigation of this plant
2. Results and discussion
The ethyl acetate extract of the L. trifolia was separated by high-
speed counter-current chromatography (HSCCC) using a gradient
* Corresponding author. Tel.: +55 21 25626413; fax: +55 21 25626425.
E-mail address: sgleitao@pharma.ufrj.br (S.G. Leitão).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.10.007

Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
295
elution approach, with a biphasic solvent mixture composed of n-
hexane–ethyl acetate–n-butanol–water (0.4:1:X:1, v/v/v/v), where
X = 0.1, 0.3, 0.5 and 0.7 (solvent systems A–D). The choice of an
appropriate solvent system for a gradient elution in CCC can be a
difficult task, in part because any change in the composition of
one liquid phase of a biphasic liquid system may induce changes
in the other liquid phase (Berthod et al., 2002). In a previous work,
we used the gradient elution approach for separation of phenyl-
propanoids and iridoids by HSCCC, using a solvent system com-
posed of ethyl acetate–n-butanol–water (Leitão et al., 2005). The
same strategy of gradient elution was used here, with different ra-
tios of ethyl acetate and n-butanol being tested, but the system
proved too polar for the effective separation of compounds in Lan-
tana extract. This could be due to the complexity of the extract and
to the presence of phenylpropanoids and flavonoids instead of phe-
nylpropanoids and iridoids. Therefore, hexane was introduced as a
fourth solvent. The optimization of the number of steps of the gra-
dient composed of hexane–ethyl acetate–n-butanol–water
0.4:1:X:1 (v:v:v:v) was done with a series of test tube experiments
(see Section 4), where X varied from 0.1 to 0.7. The aim of these
experiments (monitored by simple TLC experiments) is to set
two extreme situations: one in which we have all (or at least the
major part of) compounds in the upper phase, and the other where
we have the opposite situation (all compounds in the lower phase).
The majority of the compounds on the extracts was retained in the
lower phase (aqueous) when 0.1 n-butanol was used, whereas a
gradual inversion of the partition coefficients of the target com-
pounds occurred when the n-butanol ratio was raised to 0.7. In
the step gradient HSCCC separation, seven fractions were obtained.
This fractionation afforded the isolation of the new flavone inden-
tified as scutellarein-7-O-b-apiofuranoside (1) in fraction II and of
betonyoside F (4) in fraction VII.
40-oxygenated flavonoid. Two singlets at d 6.88 and d 6.80 were as-
signed to the H-8 proton of the A ring and H-3 proton of the C ring,
respectively. The 13C NMR spectrum displayed 20 carbons (Table
1), whereas the HSQC spectrum gave a correlation between H-8
with the signal at d 94.62 (C-8). The HMBC spectrum gave correla-
tions between H-8 and C-6, C-7 and C-10. These set of experiments
led to identification of scutellarein as the aglycone (Xia et al.,
2007). The apiose moiety was confirmed by presence of an ano-
meric proton at d 5.63 (1H, J = 3.2 Hz), as well as from four other
signals between d 4.28 and d 3.42 in the 1H NMR spectrum. The
DEPT spectrum showed signals for CH2 at d 76.56 and 62.84 and
lack of any resonance at d 79.28 in agreement with values for C-
4, C-5 and C-3 of a b-apiosyl unit (Table 1). The configuration of
the b-apiofuranosyl moiety was assigned after hydrolysis of 1 with
1 N HCl. The hydrolysate was trimethylsilylated, and GC retention
times of each sugar were compared with those of authentic sam-
ples prepared in the same manner. In this way, the sugar unit of
1 was determined to be D-apiose. The apiofuranosyl ring configura-
tion was also confirmed by comparing 1H–1H scalar coupling con-
stants with those reported for methyl apiofuranosides and DL-
apioses and by NOE observations (Ishii and Yanagisawa, 1998).
The 2D ROESY spectrum of 1 showed cross-peaks between H-2
and the protons of the hydroxymethyl group, and H-2 and H-4b,
indicating that H-2, the hydroxymethyl group, and H-4b are found
on the same face of the ring for this sugar and confirming its struc-
ture as b-D-apiose. The HMBC spectrum confirmed the location of
the apiose at C-7 by showing connectivity between the anomeric
hydrogen of apiose and the carbon at d 151.89 (C-7). The HSQC
spectrum furnished all the direct correlations between protons
and carbons (Table 1). Comparison with literature data also con-
firmed the 13C values for a scutellarein-7-O-glycoside (Emam
et al., 1998). Thus, the structure of 1 could be deduced as being
Gradient elution was not effective for isolation of verbascoside
(5), the major constituent of this extract. Therefore, fraction V
was further purified by isocratic elution with the solvent system
hexane–ethyl acetate–n-butanol–water, 0.4:1:0.6:1. The concen-
tration of n-butanol chosen for the isocratic system was between
the two steps of the gradients where the fraction was collected.
This procedure allowed separation of verbascoside (5) from 1 in
this fraction. This strategy is useful since all previous isolation
and purification procedures described in the literature for these
compounds involved more than one HSCCC passage (Leitão et al.,
2005; Li et al., 2005).
Fraction I was submitted to preparative HPLC to yield vanillic
acid (6), protocatechuic acid (7), 4-hydroxybenzoic acid (8), caffeic
acid (9), coumaric acid (10) and another new flavone named celti-
difoline, 5,6,40,50-tetrahydroxy-7,30-dimethoxyflavone (2). Final
purification of the other isolated compounds was achieved by col-
umn chromatography and HPLC (see Section 4). Fraction III affor-
the new flavone glycoside scutellarein-7-O-b-D-apiofuranoside
(Fig. 1).
The HR-ESI-MS (positive-ion mode) of compound 2 exhibited a
pseudomolecular ion peak at m/z 369.0597 [M+Na]+, ascribable to
a molecular formula of C17H14O8. The ESI-MS experiment gave
(negative-ion mode) a quasi-molecular ion peak [MꢀH]ꢀ at m/z
345 indicating a molecular weight of 346 for 2. Further fragment
ion peaks in the ESI-MS-MS spectrum were observed at m/z 330
[MꢀHꢀ15]ꢀ and m/z 315 [MꢀHꢀ30]ꢀ corresponding to successive
loss of two CH3 groups. The fragment ions at m/z 182 (C8H6O5) and
at m/z 164 (C9H8O3) were the retro-Diels–Alder fragment of rings A
and B, respectively, and indicated the presence of a methoxyl
group in both rings A and B of a flavone skeleton.
The 1H NMR spectrum of 2 (Table 1) had a signal at d 7.14 (2H),
attributed to two-non equivalent protons (H-20, H-60) of the B ring
indicating oxygenation at C-30, C-40, e C-50. Two singlets at d 6.86
(1H) and at d 6.65, were assigned to the H-8 and H-3 protons,
respectively. These data as well as the intense signals at d 3.95
and 4.05 (both 3H, s), relative to two ꢀOCH3 groups, suggested
presence of a tetrahydroxyflavone with two additional methoxyl
group substitutions (Marco et al., 1988). The 13C NMR spectrum
of 2 had 17 carbons (Table 1) with typical C-5, C-6 and C-7 values
for a trioxygenated A ring (Vilegas et al., 1999). In the HMBC spec-
trum, cross-peaks disclosing the bonding site of each methoxyl
were observed: dH 3.95 correlated with dC 149.52 (C-30), and dH
4.05 correlated with dC 155.72 (C-7). Correlations were also ob-
served between H-3/C-10 and C-10, H-8/C-6, C-7 and C-10, H-20
and H-60/ C-2, C-20, C-40, C-60. Consequently, 2 was determined to
be the new 5,6,40,50-tetrahydroxy-7,30-dimethoxyflavone, named
celtidifoline (Fig. 1).
ded the new flavone apigenin-7-O-b-
apiofuranoside (3), whereas fraction IV yielded martynoside (11),
scutellarein-7-O-b- -glucopyranoside (12) and sorbifolin (13),
D-apiofuranosyl-(1 ? 2)-b-D-
D
and fraction VI afforded samioside (14) (Fig. 1).
The structures of new compounds 1, 2 and 3 were elucidated by
the use of 600 MHz NMR techniques (DQF-COSY, HMBC, and
HSQC), and ESI-mass spectrometry. The positive HR-ESI-MS of
compound 1 showed a molecular ion peak at m/z 419.0897
[M+H]+, in accordance with an empirical molecular formula of
C20H18O10. The negative ESI-MS spectrum showed a pseudomolec-
ular ion peak at 417 [MꢀH]ꢀ indicating a molecular weight of 418
for compound 1. ESI-MS-MS experiments showed further fragment
ions at m/z 285 [MꢀHꢀ132]ꢀ, suggesting the presence of a pento-
syl moiety (C5H7O4) and at m/z 168 and m/z 118 ascribable to the
retro-Diels–Alder fragments of ring A and B of a flavone skeleton.
The 1H NMR spectrum of 1 (Table 1) showed two doublets at d
7.95 (2H, J = 8.8 Hz) and d 6.90 (2H, J = 8.8 Hz), due to the B ring of a
The HR-ESI-MS (positive-ion mode) of compound 3 exhibited a
pseudomolecular ion peak at m/z 535.1462 [M+H]+, ascribable to a
molecular formula of C25H26O13. The ESI-MS experiment gave (neg-
ative-ion mode) a quasi-molecular ion peak at m/z 533 [MꢀH]ꢀ

296
Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
Fig. 1. Structures of the major compounds from Lantana trifolia.
Table 1
1H NMR and 13C NMR spectroscopic data of compounds 1, 2 and 3 in CD3OD at 600 and at 150 MHz.
1
2
3
1H
13C
1H
13C
1H
13C
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
164.5
102.8
182.7
147.2
131.1
151.9
94.6
149.4
105.9
121.7
128.9
116.4
161.6
116.4
128.9
2
3
4
5
6
7
8
9
166.5
104.0
184.1
149.7
131.4
155.7
91.89
152.1
106.5
122.4
103.1
149.5
139.1
147.1
108.8
56.9
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
167.4
103.3
184.4
163.4
101.5
164.7
96.2
159.2
107.5
122.9
129.9
118.1
164.7
118.1
129.9
6.8 (s)
6.6 (s)
6.6 (s)
6.4 (d, 1.5)
6.7 (d, 1.5)
6.8 (s)
6.8 (s)
7.1 (s)
10
10
7.9 (d, 8.8)
6.9 (d, 8.8)
20
7.8 (d, 8.3)
6.8 (d, 8.3)
30
40
6.9 (d, 8.8)
7.9 (d, 8.8)
50
6.8 (d, 8.3)
7.8 (d, 8.3)
60
7.1 (s)
3.9
4.0
OCH3 (30)
OCH3 (7)
57.1
Apiose 7
Apiose 7
100
200
300
400
500
5.6 (d, 3.2)
4.3 (d, 3.2)
108.08
76.56
79.28
75.16
62.84
100
5.8 (d, 2.6)
4.3 (d, 2.6)
107.4
84.18
83.01
75.41
64.51
200
300
3.7, 4.0 (d, 9.6)
3.4 (m)
400
4.0, 4.1 (d, 9.6)
3.6, 3.7a (m)
500
Apiose 200
1000
5.2 (d, 2.6)
4.1 (d, 2.6)
111.11
78.13
81.5
75.45
64.03
2000
3000
4000
3.8, 3.9a (d, 10.1)
3.6, 3.7a (m)
5000
Chemical shifts are in ppm from TMS and J values in Hz are presented in parentheses.
All signals were assigned by 1D-TOCSY, DQF-COSY, HSQC and HMBC experiments.
a
Interchangeable values.

Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
297
indicating a molecular weight of 534 for compound 3. Further frag-
ment ion peaks in the ESI-MS-MS spectrum were observed at m/z
401 [MꢀHꢀ132]ꢀ and m/z 269 [MꢀHꢀ132ꢀ132]ꢀ corresponding
to the successive loss of two pentosyl moieties. The 1H NMR spec-
trum of 3 (Table 1) showed signals attributable to an apigenin skel-
eton: d 6.43 (1H, d, J = 1.5 Hz, H-6), d 6.57 (1H, s, H-3), d 6.76 (1H, d,
J = 1.5 Hz, H-8), d 6.85 (2H, d, J = 8.3 Hz, H-30 and H-50), d 7.80 (2H,
d, J = 8.3 Hz, H-20and H-60). The 13C NMR shifts of the aglycone part
of 3 (Table 1) corresponded well with the shifts for apigenin, with
the only significant difference being those corresponding to C-6, C-
7 and C-8. These shifts are analogous to those reported when the 7-
hydroxy group is glycosylated in a flavone glycoside (Agrawal,
1989). Two anomeric protons, assigned to the C-1 protons of two
apiofuranosyl units, were easily identified in the spectra of 3. They
resonated at d 5.21 (d, J = 2.63 Hz) and d 5.84 (d, J = 2.63 Hz), and
they correlated to carbons at d 111.11 and d 107.4, respectively.
From the assigned aglycon and sugar values, deduced from
1H–1H COSY and HSQC experiments (Table 1), it was apparent that
a disaccharide unit was attached to C-7 of the aglycone. The chem-
ical shifts of all the individual protons of the two sugar units were
attributed on the basis of 1D-TOCSY and DQF-COSY spectroscopic
analysis, and the 13C chemical shifts of their relative attached car-
bons were clearly assigned from the HSQC spectrum (Table 1).
razepan as a classical marker of the benzodiazepine receptor.
The most active compound was the new scutellarein-7-O-b-
apiofuranoside (1) with an half maximal inhibitory concentration
(IC50) of 187 M (Table 3). This value is very high when com-
D
-
l
pared with classical benzodiazepines, such as clonazepam, fluni-
trazepam and diazepam, which exhibit IC50 of 1, 3 and 11 nM,
respectively, when tested in the same conditions (Noël et al.,
2007). These results indicate that compounds 1–5 have a low
affinity for the benzodiazepine site present in the GABAA recep-
tor complex, as already reported for other flavonoids (Hennebelle
et al., 2007; Fernández et al., 2006; Coleta et al., 2008). These
authors suggest that flavonoids could induce CNS effects via dif-
ferent mechanisms, such as inhibition of the NMDA (Losi et al.,
2004) or 5-HT (Coleta et al., 2008; Marder et al., 2003) receptors.
The type of sugar linkage with the aglycone should be an impor-
tant factor for the sedative activity of these flavonoids (Fernán-
dez et al., 2006). On the other hand, the glycosidic part of the
flavonoid derivatives could cause a steric hindrance responsible
for the weakness of the binding to the benzodiazepine receptor
(Fernández et al., 2006).
With respect to the methoxyflavones, anxiolytic and sedative
effects have been demonstrated by different authors (Huen et al.,
2003; Hui et al., 2002; Marder et al., 2003) who showed that, for
this class of compounds, a 20-hydroxyl group enhanced the inter-
action between the ligand and the benzodiazepine receptor
(Huen et al., 2003). Marder et al. (2001) also demonstrated that
carbons 30 and 6 are the most effective positions to place sub-
stituents that enhance the affinity for the receptor, while other
pattern of substitution do not have the same effect.
These data showed the presence of a terminal b-D-apiofuranosyl
(d 5.21) and a 2-substituted b-apiofuranosyl (d 5.84) as indicated
by the downfield shift of its C-2 (d 84.18) signal. An unambiguous
determination of the linkage site was obtained from the HMBC
spectrum, which showed key correlation peaks between the ano-
meric proton of the inner apiose (d 5.84) and the C-7 of the apige-
nin (d 164.68), and between the anomeric proton of the outer
apiose (d 5.21) and the C-2 of the inner apiofuranosyl unit (d
84.18). The b-configuration at the anomeric position for the apio-
furanosyl units was determined from their coupling constants
and by ROE observations as described for compound 1. Also in this
case, the D configuration of both apiose units was determined by
acid hydrolysis of 3 followed by GC analysis. Therefore, the struc-
ture of 3 was determined as the new flavone glycoside apigenin-
As far as phenylpropanoids usually found in Verbenaceae are
concerned, verbascoside, 5, has already been tested on benzodi-
azepine receptors (Daels-Rakotoarison et al., 2000) and its IC50
(1.9 mM) was compatible with the lack of effect at 300 lM, ob-
served in the present study (Table 3). For the other phenylprop-
anoid tested here (compound 4) there is no previous data in the
literature. Although verbascoside had no affinity for the [3H] flu-
nitrazepam binding sites, its effectiveness in the open field test
(Table 2) indicates that this compound could be one of the active
principles of the extract.
7-O-b-D-apiofuranosyl-(1 ? 2)-b-D-apiofuranoside, (Fig. 1).
The other major compounds from L. trifolia were identified as
phenolic acids vanillic acid (6), protocatechuic acid (7), 4-hydroxy-
benzoic acid (8), caffeic acid (9), coumaric acid (10), phenylpropa-
noid glycosides betonyoside F (4), verbascoside (5) (Wu et al.,
2004), martynoside (11) and samioside (14), and 6-hydroxy flav-
3. Concluding remarks
ones scutellarein-7-O-b-D-glucopyranoside (12) (Emam et al.,
1998) and sorbifolin (13) (Alam et al., 1986) by 1H and 13C NMR
spectroscopic analyses and comparison of data with those in the
literature. Betonyoside F (4) and samioside (14) were reported,
respectively, from aerial parts of Stachys officinalis (Miyase et al.,
1996) and Phlomis samia (Kyriakopoulou et al., 2001), two genus
of the family Lamiaceae (Labiatae), which is very close to the Ver-
benaceae. The occurrence of 6-hydroxy and methoxy flavones in L.
trifolia is of taxonomic interest because they are relatively uncom-
mon flavonoids in Angiosperms, and it has been reported that they
occur in some abundance in those families where they are present
(Harborne and Baxter, 1999).
The sedative properties of L. trifolia extracts were evaluated by
the open field method (Whimbey and Denenberg, 1967). One hour
after their administration, the LA and LE extracts produced an in-
tense sedative effect in all animals, reported as reduction of walked
squares, with similar pattern and intensity at 1 and 10 mg/kg
(Table 2), indicating that the maximal effect had probably been at-
tained. It was noteworthythat the effect increased with time, at least
during the two first hours after administration.
In the present study, phytochemical investigation of the ethyl
acetate extract of L. trifolia led to the isolation and identification
of flavonoids and phenylpropanoids, which are compounds re-
ported to have sedative properties (Losi et al., 2004; Marder
et al., 2003; Zétola et al., 2002; Viola et al., 1995). Flavonoids have
already been reported to be efficacious at different receptor sys-
tems in the CNS (Hui et al., 2002). They are a relatively new class
of ligands of the benzodiazepine site of the GABAA receptors, and
their glycosides are considered to form the newest group within
the growing family of flavonoids with CNS activity (Fernández
et al., 2006). Fernández et al. (2006) recently demonstrated that
a series of flavonoid glycosides were depressant of the CNS in sev-
eral in vivo assays (including locomotor activity measured in the
open field method) but had low affinity for the [3H] flunitrazepan
binding sites. The authors suggested that the flavonoid glycosides
are easily metabolized by the organism and that secondary metab-
olites could activate the GABAA receptors and mediate the sedative
effects.
Asaconclusion, weshowedthatthesedativeeffectofL. trifoliaex-
tracts demonstrated in mice cannot be attributed to the direct acti-
vation of the central benzodiazepine site by the new flavones 1–3
and the phenylpropanoids betonyoside F, 4 and verbascoside, 5.
In order to identify a possible mechanism of action and/or the
active substances in the extract, we tested the isolated com-
pounds 1–5 in a competition assay in rat brain, using [3H] flunit-

298
Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
Table 2
Reduction of spontaneous locomotor activity by Lantana trifolia extracts and verbascoside, 5, in the open field test. Values are expressed as mean S.D.
Extract
LE
Dose (mg/kg)
1
Hour after administration
Reduction of walked squares (%)
1st
2nd
1st
33.4 4.6*
61.3 5.8*
38.6 9.8*
62.6 6.9*
10
2nd
LA
1
1st
2nd
1st
51.1 6.1*
67.4 6.8*
59.9 6.5*
69.2 3.5*
10
2nd
Verbascoside, 5
1
3
1st
2nd
1st
38.8 5.9*
86.7 7.1*
44.7 5.2*
73.9 6.8*
2nd
LE, ethanolic extract; LA, ethyl acetate extract.
*
P < 0.05, ANOVA followed by Bonferroni0s test.
(Waters) instrument. HSCCC was performed on a P.C. Inc. (Poto-
mac, MD) apparatus, equipped with an interchangeable multi-
layer triple coil of PTFE tubing, 1.68 mm internal diameter
(15 + 80 + 240 mL). The HSCCC system was connected to a solvent
pump Rainin SD-200 Dynamax, a manual injection valve Rheo-
dyne 5020A with 5 mL loop, and a Dynamax FC-1 fraction collec-
tor. All separation were performed at 850 rpm. HPLC separations
were carried out on a Waters 590 system equipped with a Waters
R401 refractive index detector, a Phenomenex C-18 column,
Table 3
Inhibition of [3H] flunitrazepam binding in rat brain synaptosomes: inhibitory
concentrations (IC50) of some major compounds of Lantana trifolia ethyl acetate
extract.
Compound
IC50 (lM)
Scutellarein-7-O-b-
Celtidifoline, 2
Apigenin-7-O-b-D-apiofuranosyl-(1 ? 2)-b-D-apiofuranoside, 3
Betonyoside F, 4
Verbascoside, 5
D-apiofuranoside, 1
187
440
670
550
10
l
m (10 ꢁ 250 mm, flow rate 3 mL/min), U6K injector and on
»300a
an Agilent 1100 series chromatograph, equipped with a G-1312
binary pump, a G-1328A Rheodyne injector, a G-1322A degasser,
and a G-1315A photodiode array detector (Waters Corp., Milford,
MA) using the same column (flow rate 2.5 mL/min. TLC analyses
were performed with Macherey–Nagel precoated silica gel 60
F254 plates.
a
No inhibition at 300 lM indicating that the IC50 should be much higher than
this concentration.
4. Experimental
4.1. General
4.2. Plant material and extracion
Optical rotations were determined on a Jasco DIP-1000 polar-
imeter equipped with a sodium lamp (589 nm) and a 10 mm
microcell. UV spectra were obtained with a Beckman DU 670
spectrophotometer in MeOH, (c = 1). A Bruker DRX-600 NMR
spectrometer, operating at 599.19 MHz for 1H and at
150.86 MHz for 13C, using the UXNMR software package was used
for NMR experiments; chemical shifts are expressed in d (parts
per million) referring to the solvent peaks dH 3.34 and dC 49.0
for CD3OD, coupling constants, J, are in Hertz. Distortionless
enhancement by polarization transfer (DEPT) 13C, 1H–1H double
Leaves of L. trifolia were collected in Mendes, Rio de Janeiro State,
Brazil in January 2004, and identified by Prof. Dr. Fatima Regina G.
Salimena, from the Universidade Federal de Juiz de Fora, Minas Ger-
ais, Brazil, where a voucher specimen was deposited (L. trifolia L.
CESJ 30801). Dried and pulverized leaves (900 g) were exhaustively
extracted with EtOH at room temperature for one week, and the ex-
tract was concentrated under reduced pressure to afford a brown
syrup (LE). This residue was partitioned between water and organic
solvents of increasing polarities, to afford the new extracts: hexane,
CH2Cl2, EtOAc (LA), and n-BuOH, in this order.
quantum filtered correlation spectroscopy (DQF-COSY), 1H–13
C
heteronuclear single quantum coherence (HSQC), heteronuclear
multiple bond coherence (HMBC) and ROESY (Rotating-frame
Overhauser Enhancement Spectroscopy) were obtained by
employing the conventional pulse sequences. The selective excita-
tion spectra, 1D-TOCSY, were acquired using waveform genera-
tor-based GAUSS shaped pulses, mixing time ranging from 100
to 120 ms and a MLEV-17 spin-lock field of 10 kHz preceded by
a 2.5 ms trim pulse NMR experiments. Electrospray ionization
mass spectrometry (ESI-MS) was performed using a Finnigan
LC-Q Advantage instrument from Thermoquest (San Jose, CA)
equipped with Excalibur software. Samples were dissolved in
MeOH and infused in the ESI source by using a syringe pump;
4.3. Test tube experiments for the choice of the solvent system
Small amounts of the EtOAc extract of L. trifolia were dissolved
in separate test tubes containing the solvent system EtOAc–n-
BuOH–H2O in the ratios 0.8:0.2:1; 0.7:0.3:1; 0.6:0.4:1; 0.5:0.5:1;
0.3:0.7:1 and 0.2:0.8:1. The test tubes were shaken and the com-
pounds allowed to partition between the two liquid phases. Equal
aliquots of each phase were spotted beside each other, separately,
on silica gel TLC plates developed with the solvent system EtOAc–
acetone–H2O 25:8:5 (organic phase). The results were visualized
under UV light (365 nm). Another set of test tube experiments
was performed by introducing hexane into the EtOAc–n-BuOH–
H2O system. The resulting biphasic solvent mixture was hexane–
EtOAc–n-BuOH–H2O, in the ratios 0.4:1:X:1 v:v:v:v (solvent sys-
tems A–D), where X = 0.1, 0.3, 0.5 and 0.7).
the flow rate was 3 ll/min. The capillary voltage was 5 V, the
spray voltage 5 kV, and the tube lens offset 35 V. The capillary
temperature was 220 °C and the data were acquired in the MS1
and MS/MS scanning modes. The scan range was m/z 200–1000
and for the MS/MS scanning mode, the percentage of collision en-
ergy was 30%. Exact masses were measured by an ESI/Q-TOF

Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
299
4.4. HSCCC separation by gradient elution
see Table 1; (+)-HRESI-MS, m/z 419.0897 [M+H]+, calcd. for
C20H19O10, 419.0978; ESI-MS in positive mode m/z 419 [M+H]+
and in negative mode m/z 417 [MꢀH]ꢀ, 285 [MꢀHꢀ132]ꢀ, 267
[MꢀHꢀ18]ꢀ, 257 [MꢀHꢀ28]ꢀ, 239 [MꢀHꢀ46]ꢀ.
Preparative fractionation of the EtOAc extract of L. trifolia was
performed in the 310 mL coil (80 + 240 mL, medium + large coils)
of the P.C. Inc. apparatus. Three grams of extract were dissolved
in 15 mL of both phases of solvent system A and injected in the
310 mL coil (previously equilibrated with stationary and mobile
phases of system A, VS = 277 mL, VM = 33 mL, SF = 89.35%). The
aqueous phase was used as the stationary phase, while the organic
phase was used as the mobile phase in the tail-to-head elution
mode, at a flow rate of 3 mL/min. Fractions of 12 mL were col-
lected, leading to final seven fractions as follows: solvent system
A: tubes 1–27 (Fractions I and II); solvent system B: tubes 28–54
(Fractions II and III); solvent system C: tubes 55–81 (Fractions
III–VI), and solvent system D: tubes 81–108 (Fractions VI and
VII). At the end of the final step of the gradient, rotor rotation
was stopped and the column content (organic and aqueous phases)
was ‘‘washed-off”. This procedure led to purified compounds 1 in
fraction II (58.7 mg, tubes 22–29) and 4 in fraction VII (118.2 mg,
tubes 96–102).
4.8. 5,6,40,50-Tetrahydroxy-7,30-dimethoxyflavone (celtidifoline, 2)
Amorphous powder; [
a
]
D = ꢀ74.0 (MeOH; c 0.9), UV kmax
(MeOH): 365, 285; for 1H NMR and 13C NMR (CD3OD, 600 MHz)
see Table 1; (+)-HRESI-MS, m/z 369.0597 [M+Na]+, calcd. for
C17H14O8 Na, 369.0586; ESI-MS in negative mode m/z 345 [MꢀH]ꢀ,
330 [MꢀHꢀ15]ꢀ, 315 [MꢀHꢀ15]ꢀ.
4.9. Apigenin-7-O–D-apiofuranosyl-(1 ? 2)–D-apiofuranoside (3)
Amorphous powder: [
a]
D = ꢀ85.2 (c 1.0; MeOH); UV kmax
(MeOH): 340, 270; for 1H NMR and 13C NMR (CD3OD, 600 MHz)
see Table 1; (+)-HRESI-MS, m/z 535.1469 [M+H]+, calcd. for
C25H27O13, 535.1452; ESI-MS in negative mode m/z 533 [MꢀH]ꢀ,
401 [MꢀHꢀ132]ꢀ, 269 [MꢀHꢀ132]ꢀ.
4.5. HSCCC separation by isocratic elution
4.10. Acid hydrolysis of compounds 1 and 3
Fraction V (400 mg, tubes 74–79) from the preparative gradient
elution (solvent C, X = 0.5 n-butanol) was further purified using an
isocratic elution mode with the same solvent system in the ratios
of 0.4:1:0.6:1. The fraction was dissolved in 5 mL of both phases
of the solvent system and injected in the 80 mL coil. The aqueous
phase was used as stationary while the organic phase was used
as mobile (tail-to-head) at a flow rate of 2 mL/min. Fractions of
4 mL were collected, rotor rotation was stopped at tube 30 and
the column content (organic and aqueous phases) was ‘‘washed-
off” leading to final 70 tubes, combined by similarity after TLC
analysis. These were obtained in total six fractions, with com-
pounds 1 (27.8 mg, fraction 1, tubes 6–8) and 5 (418.5 mg, fraction
4, tubes 28–31) purified.
A solution of compounds 1 and 3 (2.0 mg each) in 1 N HCl
(1 mL) was stirred at 80 °C in a stoppered reaction vial for 4 h.
On cooling, the solution was evaporated under a stream of N2. Each
residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine
(0.2 mL), and the solution was stirred at 60 °C for 5 min. After dry-
ing the solution with a stream of N2, the residue was separated by
H2O and CHCl3 (1 mL, 1:1 v/v). The CHCl3 layer was analyzed by GC
using an l-Chirasil-Val column (0.32 mm ꢁ 25 m). Temperatures of
the injector and detector were 200 °C for both. A temperature gra-
dient system was used for the oven, starting at 100 °C for 1 min
and increasing up to 180 °C at a rate of 5 °C/min. Peak of the hydro-
lysate of 1 and 3 was detected by comparison with retention times
of authentic sample of D-apiose (Sigma–Aldrich, St. Louis, MO) after
being treated with 1-(trimethylsilyl)imidazole in pyridine.
4.6. Isolation compounds 2–3, 6–14
4.11. Locomotor activity evaluation
Fraction I from the gradient elution HSCCC (255 mg) was sub-
mitted to HPLC using MeOH H2O (38:62) as eluent to yield com-
pounds 6 (10.7 mg, tR 5 min), 7 (2.6 mg, tR 7.5 min), 8 (2.5 mg, tR
8.5 min), 9 (2.2 mg, tR 9.75 min), 10 (5.5 mg, tR 12 min) and 2
(4.5 mg, tR 35 min). The fraction III (70 mg) from HSCCC was ap-
plied to a Sephadex LH-20 column (20 g), using MeOH as eluent
at a flow rate 1 mL/min; 29 fractions of 3 mL was obtained. Frac-
tions 17–18 afforded to pure compound 3 (3.7 mg) and fraction
19 compound 11 (5.8 mg). Fraction IV (185 mg) from HSCCC was
purified by silica gel CC leading to 97 fractions of 8 mL (EtOAc:ace-
tone:H2O, 25:8:2 from 1 to 54, 25:10:5 from 55 to 94 and MeOH
from 95 to the end). Fractions were monitored by TLC (EtOAc:ace-
tone:H2O 25:8:2) and combined by similarity. Fractions 15–31
(47 mg) containing a mixture of flavonoids and phenylpropanoids
were submitted to HPLC using MeOH–H2O (70:30) as eluent to
yield pure compounds 11 (1.7 mg, tR 6.55 min), 12 (9.4 mg, tR
5.48 min) and 13 (2.6 mg, tR 7.65 min). Fraction VI (350 mg) was
subjected to Sephadex LH-20 CC (50 ꢁ 2.5 cm) using MeOH as mo-
bile phase affording 80 fractions of 5 mL. Fractions 27–41 (110 mg)
were further purified by gradient HPLC (flow rate 2.5 mL/min) of
MeOH–H2O (from 40:60 to 48:52, stepwise) to yield compound
14 (5.9 mg, tR 36.32 min).
4.11.1. Animals and extracts
Male Swiss mice (18–20 g) were obtained from our own animal
facility. Animals were maintained in a room with controlled tem-
perature (22 2 °C) for 12 h light/dark cycle with free access to
food and water. Twelve hours before each experiment animals re-
ceived only water, in order to avoid food interference with absorp-
tion of the compounds. Animal care and research protocols were in
accordance with the principles and guidelines adopted by the Bra-
zilian College of Animal Experimentation (COBEA) and approved by
the Biomedical Science Institute/UFRJ – Ethical Committee for ani-
mal research. Extracts were dissolved in DMSO in order to prepare
a stock solution at a concentration of 100 mg/ml and stored at
ꢀ70 °C. On the day of the experiment the testing solutions were di-
luted in phosphate buffered solution (PBS). In all experiments, the
final concentration of DMSO did not exceed 5% at which this sol-
vent had no effect per se.
4.11.2. Open field test
Spontaneous locomotor activity was determined according to
Whimbey and Denenberg (1967). Briefly, the open field consisted
of a box, which floor was divided into 50 squares. Animals received
oral administration of extracts or verbascoside at doses of 1, 3, 10,
or 30 mg/kg. After 1 and 2 h mice were placed individually at the
centre of the box and allowed to freely explore the new environ-
ment. The number of squares by which each animal passed during
5 min was counted.
4.7. Scutellarein-7-O– -apiofuranoside (1)
D
Amorphous powder: [
a]
D = ꢀ71.2 (c 1.1; MeOH); UV kmax
(MeOH): 335, 290; for 1H NMR and 13C NMR (CD3OD, 600 MHz),

300
Lisieux de Santana Julião et al. / Phytochemistry 71 (2010) 294–300
benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi.
4.12. [3H] flunitrazepam binding
Biochem. Pharmacol. 64, 1415–1424.
Ishii, T., Yanagisawa, M., 1998. Synthesis, separation and NMR spectral analysis of
methyl apiofuranosides. Carbohydr. Res. 313, 189–192.
Katuura, E., Waako, P., Tabuti, J.R.S., Bukenya-Ziraba, R., Ogwal-Okeng, J., 2007.
Antiplasmodial activity of extracts of selected medicinal plants used by local
communities in western Uganda for treatment of malaria. African J. Ecol. 45
(Suppl. 3), 94–98.
Brain hemispheres from albino Wistar rats were homogenized
in 15 volumes 0.32 M sucrose buffered to pH 7.4 with 5 mM
Tris–HCl. The homogenate was centrifuged at 1000gmax for
10 min. After washing of the pellet, the combined supernatants
were centrifuged at 48,000gav for 20 min to obtain crude synapto-
somes that were resuspended in physiological solution (NaCl
118 mM, KCl 4.8 mM, MgCl2 1.2 mM and CaCl2 1.2 mM) buffered
to pH 7.4 with 15 mM Tris/HCl and stored at ꢀ80 °C. Synaptosomes
Kyriakopoulou, I., Magiatis, P., Skaltsounis, A.L., Aligiannis, N., Harvala, C., 2001.
Samioside,
a new phenylethanoid glycoside with free-radical scavenging
and antimicrobial activities from Phlomis samia. J. Nat. Prod. 64, 1095–
1097.
Leitão, G.G., de Souza, P.A., Moraes, A.A., Brown, L., 2005. Step-gradient CCC
separation of phenylpropanoid and iridoid glycosides from roots of
Stachytarpheta cayennensis (Rich.) vahl. J. Liq. Chromatogr. Relat. Technol. 28,
2053–2060.
Li, L., Tsao, R., Liu, Z.Q., Liu, S.Y., Yang, R., Young, J.C., Zhu, H.H., Deng, Z.Y., Xie, M.Y.,
Fu, Z.H., 2005. Isolation and purification of acteoside and isoacteoside from
Plantago psyllium L. by high-speed counter-current chromatography. J.
Chromatogr. A 1063, 161–169.
Lorenzi, H., 2000. Plantas Daninhas do Brasil, third ed. Instituto Plantarum de
Estudos da Flora LTDA. p. 540.
Losi, G., Puia, G., Garzon, G., 2004. Apigenin modulates GABAergic and
glutamatergic transmission in cultured cortical neurons. Eur. J. Pharmacol.
502, 41–46.
Marco, J.A., Barbera, O., Rodriguez, S.C., Adell, J., 1988. Flavonoids and other
phenolics from Artemisia hispanica. Phytochemistry 27, 3155–3159.
Marder, M., Estiu, G., Blanch, L.B., Viola, H., Wasowski, C., Medina, J.H., Paladini, A.C.,
2001. Molecular modeling and QSAR analysis of the interaction of flavone
derivatives with the benzodiazepine binding site of the GABA(A) receptor
complex. Bioorg. Med. Chem. 9 (2), 323–335.
Marder, M., Viola, H., Wasowski, C., Fernandez, S., Medina, J.H., Paladini, A.C., 2003.
6-Methylapigenin and hesperidin: new valeriana flavonoids with activity on the
CNS. Pharmacol. Biochem. Behav. 75, 537–545.
(100–200 lg protein) were incubated at 4 °C in 1 mL physiological
solution containing 0.1 nM [3H] flunitrazepam (85 Ci/mmol, New
England Nuclear, USA). After 90 min, the samples were diluted
with 3 mL of ice-cold buffer and rapidly filtered under vacuum
on GF/C glass fiber filters (GMF 3, Filtrak, Germany). Filters were
further washed twice with the same washing buffer. The nonspe-
cific binding measured in the presence of 1 lM unlabeled flunitraz-
epam was less than 3% of the total binding (Noël et al., 2001).
Acknowledgements
This work was supported by CNPq (MCT – CNPq/MS-SCTIE-
DECIT. 410475/2006-8), FAPERJ and CAPES (fellowship). We are in-
debted to Dr. Fatima Regina Gonçalves Salimena, from Universid-
ade Federal de Juiz de Fora, Minas Gerais, Brazil, for plant
identification. Authors wish to thank Claudio Jose da Silva Ferreira
for plant collecting. We are also indebted to Centro Nacional de
Ressonância Magnética Nuclear Jiri Jones, UFRJ, Rio de Janeiro, for
some of the NMR experiments.
Miyase, T., Yamamoto, R., Ueno, A., 1996. Phenylethanoid glycosides from Stachys
officinalis. Phytochemistry 43, 475–479.
Muhayimana, A., Chalchat, J.C., Garry, R.P., 1998. Chemical composition of essential
oils of Lantana trifolia L. from Rwanda. J. Essent. Oil Res. 10, 547–549.
Noël, F., Mendonça-Silva, D.L., Quintas, L.E.M., 2001. Radioligand binding assays in
the drug discovery process: high throughput screening and the risk of false
negative. Arzneim. Forsch. Drug Res. 51, 169–173.
References
Noël, F., Mendonça-Silva, D.L., Thibaut, J.P.B., Lopes, D.V.S., 2007. Characterization of
two classes of benzodiazepine binding sites in Schistosoma mansoni.
Parasitology 134 (7), 1003–1012.
Rimpler, H., Sauerbier, H., 1986. Iridoid Glucosides as taxonomic markers in the
genera Lantana, Lippia, Aloysia and Phyla. Biochem. Syst. Ecol. 14, 307–310.
Rwangabo, P.C., 1988. Umuhengerin, a new antimicrobially active flavonoid from
Lantana trifolia. J. Nat. Prod. 51, 966–968.
Alam, M.S., Qasim, M.A., Kamil, M., Ilyas, M., 1986. Sorbifolin 6-galactoside from
Garcinia andamanica. Phytochemistry 25, 2900–2901.
Silva, G.N., Martins, F.R., Matheus, M.E., Leitao, S.G., Fernandes, P.D., 2005.
Investigation of anti-inflammatory and antinociceptive activities of Lantana
trifolia. J. Ethnopharmacol. 100, 254–259.
Vilegas, W., Nehme, C.J., Dokkeddal, A.L., Piacente, S., Rastrelli, L., Pizza, C., 1999.
Quercetagetin 7-methyl ether glycosides from Paepalanthus vellozioides and
Paepalanthus latipes. Phytochemistry 51, 403–409.
Viola, H., Wasowski, C., Destein, M.L., Wolfman, C., Silveira, R., Dajas, F., Medina, J.H.,
Paladini, A.C., 1995. Apigenin, a component of Matricaria-Recutita flowers, is a
Central Benzodiazepine receptors-ligand with anxiolytic effects. Planta Med. 61,
213–216.
Whimbey, A.E., Denenberg, V.H., 1967. Two independent behavioral dimensions in
open-field performance. J. Compar. Physiol. Psychol. 63, 500–504.
Wu, J., Huang, J.S., Xiao, Q., Zhang, S., Xiao, Z.H., Li, Q.X., Long, L.J., Huang, L.M., 2004.
Spectral assignments and reference data – complete assignments of H-1 and C-
13 NMR data for 10 phenylethanoid glycosides. Magn. Reson. Chem. 42, 659–
662.
Xia, H.J., Qiu, F., Zhu, S., Zhang, T.Y., Qu, G.X., Yao, X.S., 2007. Isolation and
identification of ten metabolites of breviscapine in rat urine. Biol. Pharm. Bull.
30, 1308–1316.
Achola, K.J., Munenge, R.W., 1996. Pharmacological activities of Lantana trifolia on
isolated guinea pig trachea and rat phrenic nerve diaphragm. Int. J. Pharmacogn.
34, 273–276.
Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids, vol. 39. Elsevier Science
Publisher BV, Amsterdam, The Netherlands. p. 564.
Berthod, A., Brown, L., Leitão, G.G., Leitão, S.G., 2002. In: Barceló, D., Berthod, A.
(Eds.), Countercurrent Chromatography. The Support Free Liquid Stationary
Phase Comprehensive Analytical Chemistry, vol. XXXVIII. Elsevier, New York, p.
37.
Coleta, M., Campos, M.G., Cotrim, M.D., de Lima, T.C.M., da Cunha, A.P., 2008.
Assessment of luteolin (30,40,5,7-tetrahydroxyflavone) neuropharmacological
activity. Behav. Brain Res. 189 (1), 75–82.
Correa, M.P., 1926. Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas.
Reimpressão pelo Ministério da Agricultura (1984) 1, 747 (IBDF).
Daels-Rakotoarison, D.A., Seidel, V., Gressier, B., 2000. Neurosedative and
antioxidant activities of phenylpropanoids from Ballota nigra. Arzneim.
Forsch. 50, 16–23.
Emam, A.M., Elias, R., Moussa, A.M., Faure, R., Debrauwer, L., Balansard, G., 1998.
Two flavonoids triglycosides from Buddleja madagascariensis. Phytochemistry
48, 739–742.
Fernández, S.P., Wasowski, C., Loscalzo, L.M., 2006. Central nervous system
depressant action of flavonoid glycosides. Eur. J. Pharmacol. 539, 168–176.
Ghisalberti, E.L., 2000. Lantana camara L. (Verbenaceae). Fitoterapia 71, 467–486.
Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids, vol. I. John
Wiley and Sons Ltd., Baffins Cane, Chinchester, England. p. 889.
Hennebelle, T., Sahpaz, S., Gressier, B., Joseph, H., Bailleul, F., 2007. Antioxidant and
neurosedative properties of polyphenols and iridoids from Lippia alba.
Phytother. Res. 22 (2), 256–258.
Huen, M.S.Y., Hui, K.M., Leung, J.W.C., Sigel, E., Baur, R., Wong, J.T.F., Xue, H., 2003.
Naturally occurring 20-hydroxyl-substituted flavonoids as high-affinity
benzodiazepine site ligands. Biochem. Pharmacol. 66, 2397–2407.
Hui, K.M., Huen, M.S.Y., Leung, J.W.C., Wang, H.Y., Zheng, H., Sigel, E., Baur, R., Ren,
Zétola, M., De Lima, T.C.M., Sonaglio, D., Gonzalez-Ortega, G., Limberger, R.P.,
Petrovick, P.R., Bassani, V.L., 2002. CNS activities of liquid and spray-dried
extracts from Lippia alba
– Verbenaceae (Brazilian false melissa). J.
Ethnopharmacol. 82, 207–215.
H., Li, Z.W., Wong, J.T.F., Xue, H., 2002. Anxiolytic effect of wogonin,
a