Phenylethanoid glycosides from Lantana fucata with in vitro anti-inflammatory activity

Article abstract of DOI:10.1021/np9002383

A phytochemical analysis of Lantana fucata dried leaves led to the isolation of three new phenylethanoid glycosides, fucatosides A-C, along with parvifloroside A and six known methoxyflavones. Their structures were established by NMR and ESIMS experiments

Full text of DOI:10.1021/np9002383

1424
J. Nat. Prod. 2009, 72, 1424–1428
Phenylethanoid Glycosides from Lantana fucata with in Vitro Anti-inflammatory Activity
Lisieux de Santana Julia˜o,† Anna Lisa Piccinelli,‡ Stefania Marzocco,‡ Suzana Guimara˜es Leita˜o,† Cinzia Lotti,‡
Giuseppina Autore,‡ and Luca Rastrelli*,‡
Programa de Biotecnologia Vegetal, Faculdade de Farma´cia, UniVersidade Federal do Rio de Janeiro, CCS, Bl. A, 2 andar, Ilha do Funda˜o,
21.941-590, Rio de Janeiro, RJ, Brazil, and Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy
ReceiVed May 7, 2009
A phytochemical analysis of Lantana fucata dried leaves led to the isolation of three new phenylethanoid glycosides,
fucatosides A-C, along with parvifloroside A and six known methoxyflavones. Their structures were established by
NMR and ESIMS experiments. In Vitro assays showed that the alcoholic extract and fucatoside C have significant
anti-inflammatory effects, inhibiting NO release in the LPS-induced J774.A1 murine macrophage cell line.
Lantana (Verbenaceae) is a neotropical genus with approximately
150 species, many of them occurring in Brazil. L. fucata Lindl. (L.
lilacina Desf.) is a small shrub that produces pink or purple flowers
and is found in tropical and subtropical temperate regions in the
Americas. It is known as a weed and an ornamental plant,1 and the
leaves have been used in Brazilian traditional medicine as a
carminative and anti-inflammatory and to treat colds and bronchitis
as infusions, decoctions, and tinctures.1-3 Nowadays leaves of L.
fucata can be found in the local market as dried powder, tincture,
alcoholic, and aqueous-alcoholic preparations. However, apart from
a previous study that reported the occurrence of an acyclic
monoterpene ester glucoside and verbascoside in the leaves,3,4 there
are no literature data concerning the chemical composition and
pharmacological properties of L. fucata.
A phytochemical analysis of the EtOAc extract of L. fucata leaves
led to the isolation of phenylethanoid glycosides and methoxyfla-
vones. The structures of these compounds were elucidated by
extensive spectroscopic methods including 1D and 2D NMR
experiments as well as ESIMS analysis.
The anti-inflammatory activity of the isolated phenylethanoid
glycosides and the traditional aqueous, alcoholic, and aqueous-
alcoholic (70%) extracts of the leaves was determined in a model
of Escherichia coli lipopolysaccharide (LPS)-induced nitric oxide
(NO) release in the J774A.1 murine macrophage cell line. Alcoholic
extracts were obtained by macerating the dried leaves in alcoholic
solutions of 96% (v/v) and 70% (v/v) EtOH to obtain 1 L of tincture,
decoction, and infusion of leaves in boiling H2O, according to the
traditional preparations.
Results and Discussion
The dried leaves of L. fucata were exhaustively extracted with
EtOH to afford a brown syrup. This dried residue was partitioned
between H2O and organic solvents of increasing polarities, to afford
n-hexane, CH2Cl2, and EtOAc extracts. Extraction with low-polarity
solvents yielded more lipophilic components, while EtOAc afforded
a larger spectrum of nonpolar and polar material. Extractions with
n-hexane and CH2Cl2 were therefore used as a prepurification step
to selectively remove interfering components. The dried EtOAc
extract was fractionated by gel filtration on a Sephadex LH-20
column and by RP-HPLC, giving three new phenylethanoid gly-
cosides (1-3), together with parvifloroside A (4)5 and six meth-
oxyflavones (5-10), identified as 4′-O-methylscutellarein (5),6
nepetin (6),7 5,3′,4′-trihydroxy-6,7,5′-trimethoxyflavone (7),8 cir-
Figure 1. Phenylethanoid glycosides 1-4 isolated from L. fucata
leaves.
siliol (8),8 salvigenin (9),9 and 5,3′,5′-trihydroxy-6,7,4′-trimethoxy-
flavone (10),10 by comparison of their spectroscopic data, especially
NMR, with those in the literature. (Figures 1 and 2).
The HRESIMS of 1 gave an [M - H]- ion at m/z 609.1800,
indicating a molecular formula of C28H33O15 and in good agreement
with the observation of five methylene, 15 methine, and eight
quaternary carbon resonances in its HSQC and HMBC NMR spectra
(Table 1). The negative ESIMS spectrum showed a deprotonated
molecule [M - H]- at m/z 609, and ESIMSMS experiments showed
further fragment ions at m/z 447 [M - H - 162]-, m/z 315 [M -
H - 162 - 132]-, and m/z 297 [M - H - 162 - 132 - 18]-,
suggesting the successive loss of a caffeoyl unit, a pentosyl moiety,
* To whom correspondence should be addressed. Tel: 0039 89 969766.
Fax: 0039 89 969602. E-mail: rastrelli@unisa.it.
† Universidade Federal do Rio de Janeiro.
‡ University of Salerno.
10.1021/np9002383 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/27/2009

Phenylethanoid Glycosides from Lantana fucata
Journal of Natural Products, 2009, Vol. 72, No. 8 1425
a
Table 1. 1H NMR and 13C NMR Data and HMBC Correlations of Compounds 1 and 4 in Methanol-d4 (600 MHz)
1
4
position
δH (JHH in Hz)
δC
HMBC
position
δH (JHH in Hz)
δC
HMBC
aglycone
1
2
3
4
5
6
R
ꢀ
aglycon
1
2
3
4
5
6
131.1
116.5
144.5
146.0
116.8
120.8
72.4
130.6
116.5
144.0
145.3
116.8
121.2
72.3
6.79, d (1.5)
3
6.72, d (1.5)
6
6.70, d (7.4)
6.61, dd (7.4, 1.5)
3.66, 4.08, m
2.83, m
3, 4
5
6.70, d (7.4)
6.59, dd (7.4, 1.5)
3.75, 4.08, m
2.81, m
1, 4
3, 5
R
ꢀ
36.1
R, 1, 2
36.3
R, 1, 2
R, 2′′
glucose
1′
glucose
1′′
2′′
3′′
4′′
5′′
6′′
4.41, d (7.4)
3.81, dd (9.2, 7.4)
3.78, t (9.2)
4.91, t (9.2)
3.50, m
104.0
81.6
75.5
70.4
76.3
62.2
4.41, d (7.9)
3.84, dd (9.5, 7.9)
3.57, t (9.5)
4.94, t (9.5)
3.40, m
104.1
81.7
75.3
70.2
76.1
62.0
2′
3′
4′
3′′, 5′′, 6′′, CO
1′′
5′
6′a
6′b
apiose
1′′
3.62, dd (12.0, 5.0)
3.75, dd (12.0, 3.5)
3.56, dd (12.0, 5.0)
3.65, dd (12.0, 3.5)
rhamnose
1′′′
5.37, d (3.5)
3.92, d (3.5)
111.2
78.3
80.2
76.0
2′, 2′′
5.22, d (1.5)
102.9
72.3
73.6
71.0
2′′, 2′′′, 5′′′
2′′
3′′
2′′′
3.95, dd (3.0, 1.5)
3.32, dd (9.5, 3.0)
3.60, t (9.5, 9.5)
3′′′
4′′a
4′′b
5′′a
5′′b
caffeoyl
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
R′′′
ꢀ′′′
CO
4.02, d (11.2)
4.15, d (11.2)
3.63, d (9.3)
3.49, d (9.3)
4′′′
65.8
1′′
5′′′ 6′′′
4.05, dq (9.5, 6.1)
1.12, d (6.1)
69.8
18.2
5′′′, 3′′′
caffeoyl
123.8
115.1
151.1
147.2
117.1
123.2
114.8
147.0
168.0
1′
127.2
115.3
146.6
146.4
116.5
123.1
117.6
148.0
167.7
7.08, d (1.5)
ꢀ′′′, 1′′′, 3′′′
2′
7.08, d (1.5)
ꢀ′, 3′, 6′
3′
4′
6.82, d (8.2)
3′′′, 4′′′, 6′′′
ꢀ′′′, 2′′′
5′
6.80, d (7.9)
4′, 1′
6.99, dd (8.2, 1.5)
6.31, d (15.4)
7.63, d (15.4)
6′
6.98, dd (7.9, 15)
6.30, d (15.8)
7.62, d (15.8)
R′, ꢀ′, 4′
1′
1′′′, CO
R′
ꢀ′
CO
R′′′, 6′′′, CO
R′, 6′, CO
a J values are in parentheses and reported in Hz; assignments by DQF- COSY, 1D TOCSY, HSQC, and HMBC experiments.
and water. The 1H NMR spectrum of 1 (see Table 1) exhibited the
characteristic signals belonging to (E)-caffeic acid and 3,4-
dihydroxyphenylethanol moieties: protons of aromatic rings (2 ×
ABX systems), two trans-olefinic protons (AB system, JAB ) 15.4
Hz), ꢀ-methylene at δ 2.83 (2H, m), and two diastereotopic protons
at δ 4.08 and 3.66 (each 1H, m) of the side-chain of the aglycon
moiety. Additionally, two anomeric proton resonances appeared at
δ 5.37 (J ) 3.5 Hz) and 4.41 (J ) 7.4 Hz). 1D-TOCSY, DQF-
COSY, and HSQC NMR experiments showed the presence of one
ꢀ-apiofuranosyl unit and one ꢀ-glucopyranosyl unit in the structures
of 1. The apiose moiety was confirmed by the observation of three
pairs of ABq signals at δ 5.37 and 3.92 (J ) 3.5 Hz), 4.15 and
the phenylethyl alcohol moiety, H-4′ (δ 4.91) of glucose and the
carbonyl carbon resonance (δ 168.00) of the acyl moiety, and H-2′
of glucose (δ 3.81) and the anomeric carbon of the apiose unit (δ
111.20, C-1′′) Therefore, the structure of 1 was established as 1-O-
3,4-(dihydroxyphenyl)ethyl-ꢀ-D-apiofuranosyl-(1f2)-4-O-caffeoyl-
ꢀ-D-glucopyranoside, named fucatoside A.
Compound 2 showed a major ion peak at m/z 741 [M - H]- in
the negative ESIMS and significant fragment ion peaks at m/z 579
[M - H - 162]-, m/z 447 [M - H - 162 - 132]-, and m/z 315
[M - H - 162 - 132 - 132]- in the ESIMS/MS experiments,
suggesting the successive loss of a caffeoyl unit and two pentosyl
moieties. In conjunction with the analysis of the HSQC and HMBC
NMR data, its molecular formula was deduced to be C33H42O19 by
HRESIMS.
1
4.02 (J ) 11.2 Hz), and 3.63 and 3.49 (J ) 9.3 Hz) in the H
NMR. The HSQC spectrum showed signals for CH2 at δ 76.03
and 65.84 and lack of a signal at δ 80.16. Analysis of HMBC
correlations and ROE measurements showed that these elements
were best arranged as a ꢀ-D-erythroapiofuranose.11 The configura-
tions of the sugar moieties were assigned after hydrolysis of 1 with
1 N HCl. The hydrolysate was dissolved in 1-(trimethylsilyl)imi-
dazole and pyridine (0.1 mL), and GC retention times of each sugar
as TMS-imidazolyl derivatives were compared with those of
authentic samples prepared in the same manner. In this way, the
sugar units of 1 were determined to be D-apiose and D-glucose.
The caffeoyl group was positioned at C-4′ of the glucose on the
basis of the strong deshielding of H-4′ of the glucose unit (δ 4.91,
t, J ) 9.2 Hz). The HSQC spectrum also showed glycosylation
shifts for C-2 (δ 81.62), suggesting that the ꢀ-apiofuranosyl was a
terminal unit. These results indicated that the structure of 1 is closely
related to that of parvifloroside A (4). An unambiguous determi-
nation of the sequence and linkage sites was obtained from the
HMBC correlations. Thus, cross-peaks were observed between the
anomeric proton of glucose (δ 4.41, H-1′) and C-R (δ 72.41) of
Compound 2 showed similar 1D and 2D NMR features to those
of compound 1 except for the presence of a third sugar unit assigned
to a ꢀ-xylose, characterized by large trans-diaxial proton coupling
constants and by 1,3- and 1,5-diaxial relationships in the ROESY
spectrum. Analysis of 2D NMR experiments (COSY and HSQC)
showed that this compound contained a ꢀ-glucose unit, as in
compound 1, whose deshielded C-3 resonance (δ 83.44) indicated
a substitution by a second pentosyl unit. Acidic hydrolysis afforded
monosaccharide components identified as D-glucose, D-xylose, and
D-apiose by GC analysis. Finally, all connectivities within 2 were
proven by an HMBC experiment, where correlations between H-1′
(δ 4.53) of the glucose unit and the R-C atom (δ 72.14) of the
phenethyl moiety, H-1′′ (δ 4.62) of the xylose and C-2′ (δ 82.50)
of the glucose unit, H-1′′′ (δ 5.33) of the apiose and C-3′ (δ 83.44)
of the glucose unit, and H-4′ (δ 4.93) of the glucose and the
carbonyl carbon (δ 169.00) of the caffeoyl moiety were observed
(Table 2). Consequently, the structure of compound 2 was estabi-
lizhed as 1-O-3,4-(dihydroxyphenyl)ethyl-ꢀ-D-apiofuranosyl-(1f2)-

1426 Journal of Natural Products, 2009, Vol. 72, No. 8
Table 2. 1H and 13C NMR Data and HMBC Correlations of Compounds 2 and 3 in Methanol-d4 (600 MHz)
Julião et al.
a
2
3
position
δH (JHH in Hz)
δC
HMBC
position
δH (JHH in Hz)
δC
HMBC
aglycone
1
2
3
4
5
6
aglycon
1
2
3
4
5
6
R
ꢀ
132.8
117.3
147.4
145.6
117.1
122.0
72.1
131.3
116.6
145.7
144.3
116.6
121.0
71.5
6.73, d (1.7)
3, 6
6.70, d (1.7)
3, 6
6.70, d (7.9)
6.61, dd (7.9, 1.7)
3.73, 4.08, m
2.81, m
1, 4
4, 5
1, 1′
R, 1, 2, 3
6.67, d (8.3)
6.60, d (8.3, 1.7)
3.74, 4.06, m
2.82, m
1, 4
4, 5
R
ꢀ
36.1
36.1
R, 1, 2, 6
R, 2′
glucose
1′
glucose
1′
4.53, d (7.6)
3.66, dd (9.4, 7.6)
3.91, t (9.4)
4.93, t (9.4)
3.59, m
103.4
82.5
83.4
71.2
75.9
62.7
R, 2′
4.46, d (7.4)
3.53, dd (9.3, 7.4)
3.84, t (9.3)
4.93, t (9.3)
3.53, m
102.5
79.4
82.8
70.0
75.6
61.8
2′
1′, 3′
2′
3′
1′, 2′, 4′
3′, 5′, 6′, CO
3′
2′, 4′, 1′′
3′, 5′, CO
4′
4′
5′
5′
6′a
6′b
xylose
1′′
2′′
3′′
4′′
3.59, dd (12.0, 5.0)
3.74, dd (12.0, 3.8)
6′a
6′b
apiose
1′′
3.54, dd (12.0, 5.0)
3.64, dd (12.0, 3.5)
4.62, d (7.4)
105.7
76.0
77.8
71.3
2′
3′′
2′′, 4′′
2′′
5.22, d (1.2)
3.90, d (1.2)
111.9
78.1
79.9
75.0
3.25, dd (7.4, 8.9)
3.36, dd (8.9, 8.9)
3.56, m
2′′
3′′
4′′a
4′′b
5′′a
5′′b
apiose
1′′′
2′′′
3′′′
3′′′
4′′′
3.73, d (9.6)
3.97, d (9.6)
3.51, d (12.1),
3.66, d 12.1)
1′′, 2′′, 3′′
1′′, 4′′
5′′a
5′′b
apiose
1′′′
3.19, dd (11.0, 2.5)
3.90, dd (11.0, 5.0)
67.4
1′′, 2′′
64.7
5.33, d (3.2)
3.97, d (3.2)
112.2
78.7
81.6
81.6
75.0
3′, 2′′′, 4′′′
1′′′, 5′′′
5.33, d (1.2)
3.94, d (1.2)
111.5
77.8
80.0
80.0
74.7
2′′′
3′′′
3′′′
4′′′a
4′′′b
5′′
3.76, d (9.8)
4.11, d (9.8)
3.52, br s
1′′′, 3′′′, 5′′′
1′′′, 3′′′, 4′′′
3.78, d (9.6)
4.01, d (9.6)
3.49, d (12.1)
3.69, d (12.1)
1′′′, 2′′′, 3′′′
1′′′′, 3′′′′
65.5
5′′′a
5′′′b
caffeoyl
1′
65.3
caffeoyl
1′′′′
123.9
115.4
149.7
147.4
117.0
123.0
116.0
148.3
169.0
127.2
114.8
149.3
146.1
116.3
122.6
114.8
147.3
168.2
2′′′′
7.07, d (2.0)
1′′′′, 3′′′′, 4′′′′
2′
7.05,d (1.5)
3′′′′
3′
4′′′′
4′
5′′′′
6.80, d (8.2)
3′, 4′, 6′
ꢀ′, 2′, 4′
1′, CO
5′
6.78, d (7.5)
3′, 4′, 6′
ꢀ′, 2′
6′′′′
6.97, dd (8.2, 2.0)
6.30, d (15.9)
7.61, d (15.9)
6′
6.95, dd (7.5, 1.5)
6.30, d (15.8)
7.61, d (15.8)
R′′′′
ꢀ′′′′
CO
R′
1′, CO
1′, 2′, 6′, CO
ꢀ′
1′, 2′, CO
CO
a J values are in parentheses and reported in Hz; assignments by DQF-COSY, 1D TOCSY, HSQC, and HMBC experiments.
ꢀ-D-xylopyranosyl-(1f3)-4-O-caffeoyl-ꢀ-D-glucopyranoside, named
fucatoside B.
were best arranged as two R-threo-apiofuranoside moieties with
quaternary carbons at δ 79.95 and 79.99.11 Indeed, acid hydrolysis
of 3 afforded D-glucose and L-apiose in the ratio 1:2. All other
The ESIMS in negative mode of compound 3 exhibited a quasi-
molecular ion peak at m/z 741 [M - H]- and significant fragment
ion peaks at m/z 579 [M - H - 162]-, m/z 447 [M - H - 162 -
132]-, and m/z 315 [M - H - 162 - 132 - 132]- in ESIMSMS
experiments, suggesting, as for fucatoside B (2), the successive loss
of a caffeoyl unit and two pentosyl moieties. A high-resolution
measurement indicated the molecular formula, C33H42O19, in
accordance with HSQC and HMBC NMR analysis.
1
signals in the H NMR and 13C NMR spectra of 3 were assigned
after analysis of the 2D NMR data (Table 2). The carbon resonances
assigned to the R-apiose units showed no unusual chemical shifts,
suggesting their terminal position. HMBC experiments established
the binding site of the two apiose units at C-2 (H-1′′/C-2′) and C-3
(H-1′′′/C-3′) and the caffeoyl unit at C-4 of the glucose (H-4′/CO).
On the basis of these data, compound 3 was assigned the structure
1-O-3,4-(dihydroxyphenyl)ethyl-O-R-L-apiofuranosyl-(1f2)-O-R-
L-apiofuranosyl-(1f3)-4-O-caffeoyl-ꢀ-D-glucopyranoside, named
fucatoside C.
Another phenethyl alcohol glycoside, 1-O-3,4-(dihydroxyphe-
nyl)ethyl-R-L-rhamnopiranosyl-(1f2)-4-O-caffeoyl-ꢀ-D-glucopy-
ranoside (4), was also isolated from L. fucata. Its structure was
shown to be identical with parvifloroside A, reported recently from
Stachys parViflora,5 and this appears to be the second report of
this compound. Known methoxyflavones 5-10 were also isolated
from L. fucata. Their structures were elucidated by ESIMS and 1H
and 13C NMR data analysis.
Analysis of the 1H NMR spectrum of compound 3 in comparison
to that of 2 showed that the difference between the two compounds
should be confined to the sugar portion. Three doublets of anomeric
protons at δ 4.46, 5.22, and 5.33 indicated its trisaccharide nature.
Analysis of 2D NMR data (COSY, TOCSY, and HSQC) showed
that this compound contained a ꢀ-glucose unit. In the COSY
experiments, the anomeric protons at δ 5.22 and 5.33 (both J )
1.2 Hz) were observed to couple with vicinal protons at δ 3.90
and 3.94, respectively, also doublets (both J ) 1.2 Hz), and assigned
1
to H-2. These sugars were further characterized in the H NMR
spectra by two other pairs of doublets corresponding to hydroxym-
ethylene groups with attached carbons at δ 74.99 and 64.66 and at
δ 74.68 and 65.28, respectively (Table 2). Analysis of HMBC
correlations and ROE measurements showed that these elements
It is noteworthy that there are very few examples of phenyle-
thanoid glycosides with a trisaccharide moiety containing apiose.
Additionally, fucatosides A-C and parvifloroside A are closely

Phenylethanoid Glycosides from Lantana fucata
Journal of Natural Products, 2009, Vol. 72, No. 8 1427
Table 3. Effect of L. fucata Extractsa on Nitrite Release, Index
of NO Production, by J774A.1 Macrophages Activated with E.
coli Lipopolysaccharide (LPS)b
Table 5. In Vitro Antiproliferative Activity of Phenylethanoid
Glycosidesa
nitrite release inhibition (% vs LPS)
nitrite release inhibition (% vs LPS)
compound
100 µg/mL
10 µg/mL
1 µg/mL
extract
100 µg/mL
10 µg/mL
1 µg/mL
fucatoside A (1)
fucatoside B (2)
fucatoside C (3)
parvifloroside A (4)
6-mercaptopurine
a Data are expressed as cell viability % means ( SEM vs untreated
J774A.1 macrophages.
95. ( 1.9***
94.4 ( 2.7
94.3 ( 2.9
96.9 ( 3.0
31.8 ( 2.8
95.9 ( 2.9
96.1 ( 3.0
96.8 ( 2.8
95.9 ( 1.9
38.0 ( 3.0
97.3 ( 2.6
97.5 ( 3.5
96.4 ( 2.6
96.7 ( 3.2
44.1 ( 1.9
LFHA
LFA
LFD
LFI
95.6 ( 1.5***
92.4 ( 2.9***
98.9 ( 3.4***
13.26 ( 0.5***
74.4 ( 2.6***
85.9 ( 3.0***
17.5 ( 2.2
13.4 ( 0.95
35.2 ( 0.9**
0.1 ( 0.02
0.3 ( 0.3
13.4 ( 1.7
a LFA: alcoholic tincture, LFHA: 70% aqueous-alcoholic tincture,
LFD: decoction, LFI: infusion of L. fucata. b Data are expressed as
means ( SEM of percentage inhibition vs nitrite release by J774A.1
macrophage treated with LPS alone. *** and ** denote P < 0.001 and P
< 0.01, respectively, vs LPS alone.
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 δ (parts per million)
referring to the solvent peaks δH 3.34 and δC 49.0 for methanol-d4,
Table 4. Effect of Phenylethanoid Glycosides on Nitrite Re-
lease by J774A.1 Macrophages Stimulated with E. coli Lipopoly-
saccharide (LPS)a
and coupling constants, J, are in hertz. H-1H DQF-COSY, H-13C
HSQC, HMBC, and ROESY experiments were obtained by employing
the conventional pulse sequences. The selective excitation spectra, 1D
TOCSY, were acquired using waveform generator-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
experiment. ESIMS was performed using a Finnigan LC-Q Advantage
instrument from Thermoquest (San Jose, CA) equipped with Excalibur
software. Exact masses were measured by an ESI/Q-TOF (Waters)
instrument. Column chromatography was performed over Sephadex LH-
20 (Pharmacia), and HPLC separations were carried out on a Waters
590 system equipped with a Waters R401 refractive index detector, a
Phenomenex C-18 column, 10 µm (10 × 250 mm), and a U6K injector,
and on 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 a Waters C-18 column, 5 µm (3.9 × 150 mm, flow rate 1 mL/
min). TLC analyses were performed with Macherey-Nagel precoated
silica gel 60 F254 plates.
1
1
nitrite release inhibition (% vs LPS)
compound
100 µg/mL
10 µg/mL
11.2 ( 0.9
1 µg/mL
fucatoside A (1)
fucatoside B (2)
fucatoside C (3)
52.5 ( 1.9***
13.3 ( 0.6
13.7 ( 0.89
4.4 ( 0.6
81.4 ( 3.9*** 16.41 ( 2.0
93.7 ( 2.5***
31.8 ( 1.2***
9.0 ( 1.6
parvifloroside A (4) 88.5 ( 2.4***
3.8 ( 0.15
a Data are expressed as inhibition percentage means ( SEM vs nitrite
production in 24 h by J774A.1 macrophages treated with LPS alone.
*** denotes P < 0.001 vs LPS-treated macrophages.
related to verbascoside previously isolated from this species.3,4 The
pharmacological activities of phenylethanoids have been extensively
reviewed.12 In particular they have been shown to possess anti-
inflammatory activity,13 which led us to investigate the in Vitro
activities of traditional aqueous extracts, obtained as decoction
(LFD) and infusion (LFI), alcoholic and aqueous-alcoholic extracts
(LFA and LFHA, respectively), and isolated phenylethanoid gly-
cosides 1-4 on inducible nitric oxide synthase (iNOS) activity,
evaluating nitrite production, index of nitric oxide biosynthesis, in
the medium of LPS-activated J744.A1 macrophage. NO release in
the cellular medium of LPS-stimulated J774.A1 macrophages,
incubated with L. fucata extracts (1, 10, and 100 µg/mL) and
phenylethanoids 1-4 (1, 10, and 100 µg/mL), was evaluated 24 h
after LPS (6 × 103 u/mL) challenge. Results were expressed as
percent inhibition evaluated versus macrophages treated with LPS
alone.
Plant Material. Leaves of L. fucata were collected in Juiz de Fora,
Minas Gerais State, Brazil, in September 2005, and identified by Prof.
Dr. F. R. G. Salimena, from Universidade Federal de Juiz de Fora,
Minas Gerais, Brazil. A specimen of the plant (voucher no. CESJ
48653) used in this study has been deposited at the Herbarium of the
same Federal University.
Extraction and Isolation. Dried and pulverized leaves (620 g) were
exhaustively extracted with EtOH, and the extract was concentrated
under reduced pressure to afford a brown syrup (90 g). This residue
was partitioned between H2O and organic solvents of increasing
polarities, to afford the new n-hexane, CH2Cl2, and EtOAc extracts
(9.4 g). Part of the EtOAc extract (6 g) was submitted to column
chromatography over Sephadex LH-20 (75 × 3.5 cm) using MeOH as
mobile phase at a flow rate of 2 mL/min. Sixty eight fractions of 8 mL
were obtained and combined into seven fractions. Fraction 3 (70 mg)
was submitted to HPLC using MeOH/H2O (40:60) as eluent in a
Phenomenex C-18 column, 10 µm (10 × 250 mm), at a flow rate of
2.5 mL/min. The major peaks (30 mg) were combined and resubjected
to HPLC using MeOH/H2O-0.05% TFA (25:75) as eluent using a
Waters C-18 column, 5 µm (3.9 × 150 mm, flow rate 1 mL/min), to
yield pure compounds 1 (12 mg, tR 18.27 min), 2 (4.2 mg, tR 22.48
min), 3 (5.0 mg, tR 31.62 min), and 4 (4.0 mg, tR 26.48 min). Fraction
6 (380 mg) was submitted to HPLC using MeOH/H2O (60:40) as eluent
on a Phenomenex C-18 column, 10 µm (10 × 250 mm), at a flow rate
of 2.5 mL/min, to yield pure compounds 5 (1.2 mg, tR 10.20 min), 6
(2 mg, tR 15.5 min), 7 and 8 (1.5 mg, tR 20.25 min), 9 (1.0 mg, tR 23.0
min), and 10 (0.5 mg, tR 29.1 min).
Among the tested extracts only the alcoholic one, added 1 h
before and simultaneously with LPS, significantly inhibited (P <
0.01 vs LPS) NO release in a concentration-related manner (92.4
( 2.9, 85.9 ( 3.0, and 35.2 ( 0.9 for 100, 10, and 1 µg/mL.
respectively, vs LPS alone; Table 3). Among L. fucata phenyle-
thanoid constituents only fucatoside C (3), added 1 h before and
simultaneously with LPS, significantly inhibited nitrite release in
a concentration-related manner (P < 0.001 vs LPS) for the
concentrations of 10 and 100 µg/mL, while fucatosides A and B
(1 and 2) and parvifloroside A (4) significantly inhibited nitrite
production release only at the highest concentration tested (100 µg/
mL; Table 4).
To establish the effects of L. fucata preparations (LFD, LFI, LFA,
and LFHA) and phenylethanoid constituents on cell viability in Vitro
(1, 10, and 100 µg/mL), extracts and compounds 1-4 were tested
on a murine macrophage cell line (J774.A1), murine fibrosarcoma
cells (WEHI-164), and human embryonic kidney cells (HEK-293)
using the MTT assay.14,15 None of the extracts (data not shown)
and compounds tested showed antiproliferative activity on J774.A1
cells (Table 5).
Fucatoside A (1): amorphous powder; [R]25 -21.32 (c 0.34,
D
1
MeOH); UV λmax 325, 300; H NMR data (methanol-d4, 600 MHz),
see Table 1; 13C NMR data (CD3OD, 600 MHz), see Table 1; (-)-
HRESIMS, m/z 609.1800 [M - H]-, calcd for C28H33O15, 609.1819;
ESIMS (negative mode) m/z 609 [M - H]-, MS/MS m/z 447, 315,
and 297.
Fucatoside B (2): amorphous powder; [R]25D +0.03 (c 0.23, MeOH);
Experimental Section
UV λmax 330; 1H NMR data (methanol-d4, 600 MHz), see Table 2; 13
C
General Experimental Procedures. Optical rotations were deter-
mined on a Jasco DIP-1000 polarimeter equipped with a sodium lamp
(589 nm) and a 10 cm microcell. UV spectra were obtained with a
NMR data (CD3OD, 600 MHz), see Table 2; (-)-HRESIMS, m/z
741.2252 [M - H]-, calcd for C33H41O19, 741.2242; ESIMS (negative
mode) m/z 741 [M - H]-, MS/MS m/z 579, 447, and 315.

1428 Journal of Natural Products, 2009, Vol. 72, No. 8
Julião et al.
Fucatoside C (3): amorphous powder; [R]25 +5.00 (c 0.15,
line in response to treatment with tested compounds and 6-mercap-
topurine was calculated as % dead cells ) 100 - (OD treated/OD
control) × 100. To exclude the capacity of our tested compounds 1-4
to interfere with the MTT reduction assay, we performed a control
experiment testing 1-4 without cells; they did not interfere in the
reduction of MTT to formazan (data not shown).
D
1
MeOH;); UV λmax 330; H NMR data (methanol-d4, 600 MHz), see
Table 2; 13C NMR data (CD3OD, 600 MHz), see Table 2; (-)-
HRESIMS, m/z 741.2257 [M - H]-, calcd for C33H41O19, 741.2242;
ESIMS (negative mode) m/z 741 [M - H]-, MS/MS m/z 579, 447,
and 315.
Determination of the Absolute Configuration of Sugars. A solu-
tion of compounds 1-3 (1 mg) in 1 N HCl (0.25 mL) was separately
stirred at 80 °C for 4 h. On cooling, the solution was concentrated in
a stream of N2. The residue was dissolved in 1-(trimethylsilyl)imidazole
(Trisil-Z) and pyridine (0.1 mL), and the solution was stirred at 60 °C
for 5 min. After drying in a stream of N2, the residue was partitioned
between H2O and CH2Cl2 (1 mL, 1:1). The CH2Cl2 layer was analyzed
by GC (Alltech l-Chirasil-Val column, 0.32 mm × 25 m; temperatures
for injector and detector, 200 °C; temperature gradient system for the
oven, 100 °C for 1 min and then raised to 180 °C; rate 5 °C/min).
Peaks of the hydrolysate of 1-3 were detected by comparison with
retention times of authentic samples of D- and L-apiose (tR 19.25 and
17.15 min), D-glucose (tR 29.11 min), and D-xylose (tR 24.88 min)
(Sigma Aldrich, St. Louis, MO) after being treated simultaneously with
Trisil-Z.
Analysis of Nitrite. J774A.1 cells (5.0 × 104 cells/well) were plated
on 96-well microtiter plates and allowed to adhere at 37 °C in a 5%
CO2 atmosphere for 2 h. Examined extracts and compounds (1-100
mg/mL for the extracts and 1-100 µg/mL for compounds) were added
1 h before and simultaneously to LPS (6 × 103 U/mL), used to induce
-
inducible iNOS. Nitric oxide release, evaluated as nitrite (NO2
)
accumulation in the cell culture medium, was performed 24 h after
LPS stimulation by the Griess reagent.18 The amount of nitrite in the
samples was calculated from a sodium nitrite standard curve freshly
prepared in culture medium. Results are expressed as percentages of
inhibition calculated versus cells treated with LPS alone. N(G)-Nitro-
L-arginine methyl ester (L-NAME; 1 µM) has been used as reference
drug, able to inhibit nitrite production by LPS-treated macrophages,
giving rise to 46.56 ( 1.25% inhibition of nitrite release versus LPS
alone (data not shown in Table 4).
Herbal Preparations. To prepare the decoction of L. fucata leaves,
5 g of dried leaves was boiled with 100 mL of hot H2O for 5 min. The
decoctions were then lyophilized to obtain a solid residue. The infusions
were obtained by pouring 100 mL of boiling distilled water on 5 g of
dried loose leaves and steeping it for 3 min. The infusions were filtered
through filter paper, and the resulting infusion was freeze-dried. Extracts
were obtained according to the European Pharmacopoeia.16 Two-
hundred grams of dried plant material was macerated. The alcoholic
solutions used for Lantana preparations were 96% (v/v) and 70% (v/
v) to obtain 1000 mL of tincture; the extracts were concentrated under
reduced pressure to afford a brown syrup.
Cell Lines and Reagents for Bioactivity Assays. The murine
macrophage cell line (J774A.1), the murine fibrosarcoma cells (WEHI-
164), and the human embryonic kidney cells (HEK-293) were obtained
from American Tissue Culture Collection (ATCC). E. coli lipopolysac-
charide (LPS) was obtained from Fluka (Milan, Italy). 3-(4,5-Dimeth-
ylthiazolyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) and phosphate
buffer solution (PBS) were obtained from Sigma Chemical Co. (Milan,
Italy). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/
streptomycin, HEPES, glutamine, fetal calf serum (FCS), and horse
serum were from HyClone (Euroclone-Cellbio, Pero, Milan, Italy).
J774.A1 cells were grown in adhesion on Petri dishes and maintained
at 37 °C as previously described.17 WEHI-164 and HEK-293 were
maintained in adhesion on Petri dishes with DMEM supplemented with
10% heat-inactivated FCS, 25 mM HEPES, 100 IU/mL penicillin, and
100 µg/mL streptomycin.
Antiproliferative Activity. J774A.1, WEHI-164, and HEK-293 (3.5
× 104 cells/well) were plated on 96-well microtiter plates and allowed
to adhere at 37 °C in a 5% CO2 atmosphere for 2 h. Thereafter, the
medium was replaced with fresh medium, and serial dilutions of each
test compound were added and then the cells incubated for 72 h. In
some experiments, serial dilutions of 6-mercaptopurine, as reference
drug, were added. Mitochondrial respiration, an indicator of cell
viability, was assessed by the mitochondrial-dependent reduction of
[3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bromide] (MTT)
to formazan, and cell viability was assessed according to the method
of Mosmann. Briefly, 5 µL of MTT (5 mg/mL) was added, and the
cells were incubated for an additional 3 h. Thereafter cells were lysed
and the dark blue crystals solubilized with 100 µL of a solution
containing 50% (v/v) N,N-dimethylformamide and 20% (w/v) SDS with
an adjusted pH of 4.5. The optical density (OD) of each well was
measured with a microplate spectrophotometer (Titertek Multiskan
MCC/340) equipped with a 620 nm filter. The viability of each cell
Acknowledgment. This work was supported by CNPq and FAPERJ
for financial aid and CAPES for a fellowship to L.d.S.J. We thank Dr.
F. R. Gonc¸alves-Salimena, Universidade Federal de Juiz de Fora, Minas
Gerais, Brazil, for plant identification.
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Lorenzi, H. In Plantas Daninhas do Brasil; Terrestres, Aquaticas,
Parasitas e Toxicas, 3rd ed.; Instituto Plantarum de Estudos da Flora:
LTDA Nova Odessa, 2000; p 540.
´
(2) Correa, M. P. In Diciona´rio das Plantas Uteis do Brasil e das Exo´ticas
CultiVadas; Ministe´rio da Agricultura: Rio do Jaineiro, 1984; Vol. 1,
p 747.
(3) Takeda, Y.; Takechi, A.; Masuda, T.; Otsuka, H. Planta Med. 1998,
64, 78–79.
(4) Pereira, A. C.; Carvalho, H. W. P.; Silva, G. H.; Oliveira, D. F.;
Figueiredo, H. C. P.; Cavalheiro, A. J.; Carvalho, D. A. ReV. Bras.
Pharmacogn. 2008, 18, 204–208.
(5) Ahmad, V. U.; Arshad, S.; Bader, S.; Ahmed, A.; Iqbal, S.; Tareen,
R. B. J. Asian Nat. Prod. Res. 2006, 8, 105–111.
(6) Grayer, R. J.; Veitch, N. C.; Kite, G. C.; Paton, A. J.; Garnock-Jones,
P. Phytochemistry 2002, 60, 727–731.
(7) Wollenweber, E.; Mayer, K.; Roitman, J. N. Phytochemistry 1991,
30, 2445–2446.
(8) Nagao, T.; Abe, F.; Kinjo, J.; Okabe, H. Biol. Pharm. Bull. 2002, 25,
875–879.
(9) Horie, T.; Ohtsuru, Y.; Shibatat, K.; Yamashita, K.; Tsukayama, M.;
Kawamukat, Y. Phytochemistry 1998, 4I, 865–874.
(10) Kinoshita, T.; Firman, K. Phytochemistry 1996, 42, 1207–1210.
(11) Ishii, T.; Yanagisawa, M. Carb. Res. 1998, 313, 189–192.
(12) Jimenez, C.; Riguera, R. Nat. Prod. Rep. 1994, 11, 591–606.
(13) Diaz, A. M. Life Sci. 2004, 74, 2515–2520.
(14) Setsuo, S.; Shgeya, S.; Norihiro, T.; Yiochi, N.; Keishi, N.; Madoka,
I.; Isao, I. Chem. Pharm. Bull. 1990, 38, 411–414.
(15) Kinjo, J.; Uemura, H.; Nakamura, M.; Nohara, T. L. Chem. Pharm.
Bull. 1994, 42, 1339–1341.
(16) Eudra/Q/92/021: Note for Guidance on Stability Testing of New Active
Substances and Medicinal Products.
(17) Marzocco, S.; Piacente, S.; Pizza, C.; Oleszek, W.; Stochmal, A.; Pinto,
A.; Sorrentino, R.; Autore, G. Life Sci. 2004, 75, 1491–501.
(18) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok,
J. S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131–138.
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