Caffeic acid esters and lignans from piper sanguineispicum

Article abstract of DOI:10.1021/np1005357

Three new caffeic acid esters (1-3), four new lignans (4-7), and the known compounds (7′S)-parabenzlactone (8), dihydrocubebin (9), and justiflorinol (10) have been isolated from leaves of Piper sanguineispicum. Their structures were determined by spectroscopic methods, including 1D and 2D NMR, HRCIMS, CD experiments, and chemical methods. Compounds 1-10 were assessed for their antileishmanial potential against axenic amastigote forms of Leishmania amazonensis. Caffeic acid esters 1 and 3 exhibited the best antileishmanial activity (IC₅₀ 2.0 and 1.8 μM, respectively) with moderate cytotoxicity on murine macrophages.

Full text of DOI:10.1021/np1005357

1884
J. Nat. Prod. 2010, 73, 1884–1890
Caffeic Acid Esters and Lignans from Piper sanguineispicum
Billy Joel Cabanillas,†,‡ Anne-Ce´cile Le Lamer,† Denis Castillo,§ Jorge Arevalo,§ Rosario Rojas,§ Guillaume Odonne,†
Genevie`ve Bourdy,†,‡ Be´atrice Moukarzel,† Michel Sauvain,*,†,‡ and Nicolas Fabre*,†
UniVersite´ de Toulouse, UPS, UMR 152 (Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox),
F-31062 Toulouse Cedex 9, France, IRD, UMR-152, Mission IRD Casilla 18-1209 Lima, Peru´, and Laboratorios de InVestigacio´n y
Desarrollo, Facultad de Ciencias y Filosof´ıa, UniVersidad Peruana Cayetano Heredia, AVenida Honorio Delgado 430, San Martin de Porres,
Lima, Peru´
ReceiVed July 31, 2010
Three new caffeic acid esters (1-3), four new lignans (4-7), and the known compounds (7′S)-parabenzlactone (8),
dihydrocubebin (9), and justiflorinol (10) have been isolated from leaves of Piper sanguineispicum. Their structures
were determined by spectroscopic methods, including 1D and 2D NMR, HRCIMS, CD experiments, and chemical
methods. Compounds 1-10 were assessed for their antileishmanial potential against axenic amastigote forms of
Leishmania amazonensis. Caffeic acid esters 1 and 3 exhibited the best antileishmanial activity (IC50 2.0 and 1.8 µM,
respectively) with moderate cytotoxicity on murine macrophages.
Leishmaniasis is a tropical disease caused by Trypanosomatidae
of the genus Leishmania. It has spread to 88 countries over Africa,
Asia, Europe, and America and exhibits a wide range of clinical
symptoms. The World Health Organization (WHO) has estimated
two million new cases every year, and 350 million people are
considered to be at risk.1 Chemotherapy is based largely on
antimony compounds such as Pentostam and Glucantime; however,
renal and cardiac toxicity2,3 together with clinical resistance against
these commonly used antimonial agents4 have prompted a search
for new chemicals in order to overcome these problems.5 Since
the availability of effective pharmaceuticals in remote places is also
a problem, the use of folk remedies based on medicinal plants for
the treatment of leishmaniasis is a common practice.6
kidney cells (VERO) was also determined in order to probe the
chemotherapeutic potential of most of the leishmanicidal compounds
isolated.
Results and Discussion
Compound 1 was isolated as a white, amorphous solid with a
positive optical rotation, [R]20 +43.3. The molecular formula
D
C14H18O4 was established on the basis of HRCIMS (m/z 251.1275
[M + H]+; calcd 251.1283) and from its 1H and 13C NMR spectra
(Table 1). The IR spectrum of 1 showed the presence of OH (3331
cm-1), ester (1681 cm-1), and trisubstituted aromatic (1441 cm-1
)
1
groups. The H NMR spectrum exhibited three aromatic proton
resonances [δH 6.88 (1H, d, J ) 8.2 Hz, H-5), 6.98 (1H, dd, J )
8.2, 2.0 Hz, H-6), and 7.11 (1H, d, J ) 2.0 Hz, H-2)], indicating
In this context, our ethnopharmacological surveys7,8 of the
Chayahuitas ethnic group, located in an endemic zone of Leishmania
in Peru (Loreto Department), allowed us to select various plant
species with antileishmanial potential. Among them, leaves of Piper
sanguineispicum Trel. (Piperaceae), which are used by this com-
munity as an anti-inflammatory remedy, displayed one of the most
promising in vitro leishmanicidal activity against axenic amastigote
forms of Leishmania amazonensis.
1
a 1,3,4-trisubstituted aromatic ring. The H NMR showed signals
of two trans double-bond protons [δH 7.56 (1H, d, J ) 15.9 Hz,
H-7) and 6.23 (1H, d, J ) 15.9 Hz, H-8)], suggesting a trans-
caffeoyl moiety in compound 1. The complex spin system at high
field δH 1.27-1.72 suggested the presence of an alkyl chain in the
molecule. The intense doublet at δH 1.28 (3H, d, J ) 6.27 Hz,
H-5′) and the triplet at δH 0.92 (3H, t, J ) 7.24 Hz, H-4′)
corresponding to two methyl groups are indicative of a branched
alkyl chain. The carbon signals in the 13C NMR spectrum of 1
confirmed the caffeic acid and branched alkyl side chain moieties.
The linkage of a caffeoyl moiety with the alkyl chain was solved
by analysis of the HMBC spectrum, which revealed a cross-peak
between H-1′ at δH 5.03 and the carbonyl C-9 at δC 168.4.
Compound 1 was therefore elucidated as 1-methylbutyl caffeate.
Total assignments of protons and carbons of 1 were based on the
results of the 1H-1H COSY, HSQC, and HMBC experiments (Table
1). The absolute configuration at C-1′ was determined to be S by
measurement of the optical rotation of the corresponding aliphatic
alcohol obtained after alkaline hydrolysis of 1 and by comparison
of the value with the [R]D of (S)-(+)-2-pentanol.
Phytochemical investigation of the genus Piper,9 which is widely
distributed in tropical and subtropical regions of the world, has
revealed several classes of antiprotozoal compounds including
alkaloids,10,11 lignans,12 chalcones, and dihydrochalcones.13 Com-
pounds from Piper species have also shown interesting leishmani-
cidal activity. For example, piperine, an alkaloid present in several
species, has activity similar to Pentostam against promastigotes of
L. donoVani.6 Flavokavain, isolated from leaves of P. rusbyi, is
active against L. amazonensis, L. braziliensis, and L. donoVani
promastigotes.14 In addition, chalcones and dihydrochalcones
isolated from P. aduncum and P. elongatum have been led to
moderate activity against Leishmania spp.15,16
In the present investigation, a bioassay-guided fractionation of
a 90% EtOH leaf extract led to the isolation of three new caffeic
acid esters (1-3), four new lignans (4-7), and the known lignans
(7′S)-parabenzlactone (8), dihydrocubebin (9), and justiflorinol (10).
In vitro activity of all the compounds was assessed against axenic
amastigote forms of L. amazonensis. Moreover, in vitro cytotoxicity
on macrophages, human breast cancer cells (MCF-7), and monkey
The molecular formula C16H22O4 (m/z 279.1586 [M + H]+; calcd
279.1596) was assigned for compound 2 by HRCIMS and cor-
responded to 28 Da more than compound 1. The 1H and 13C NMR
spectra of 2 were very similar to those observed for 1 (Table 1)
with two more methylenes in 2. Compound 2 was therefore
elucidated as 1′-methylhexyl caffeate. As for 1, the absolute
configuration at C-1′ was deduced to be S.
In the same way, the molecular formula C18H26O4 (m/z 307.1903
[M + H]+; calcd 307.1909) was assigned for compound 3 by
HRCIMS, corresponding to two more methylenes compared to 2.
The structure of 3 was therefore deduced to be 1′-methyloctyl
* To whom correspondence should be addressed. Tel: +33-5-62256848.
Fax: +33-5-61554330. E-mail: nfabre@cict.fr; michel.sauvain@ird.fr.
† Universite´ de Toulouse.
‡ Mission IRD, Peru´.
§ Universidad Peruana Cayetano Heredia.
10.1021/np1005357  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/18/2010

Caffeic Acid Esters from Piper sanguineispicum
Journal of Natural Products, 2010, Vol. 73, No. 11 1885
Chart 1
Table 1. 1H and 13C NMR Data for Compounds 1-3 in CDCl3
1a
2b
3b
position
δH (J in Hz)
δC
HMBC
3,4,6
δH (J in Hz)
δC
HMBC
3,4,6,7
δH (J in Hz)
δC
HMBC
3,4,6,7
1
2
3
4
5
6
7
8
127.3
114.4
144.1
146.8
115.5
122.4
145.3
115.6
168.4
71.7
127.5
114.5
143.8
146.4
115.5
122.3
144.7
116.1
167.7
71.6
36.0
25.1
31.7
20.1
14.0
20.1
127.5
114.5
143.8
146.4
115.5
122.3
144.8
116.1
167.7
71.6
36.0
22.6
29.4
29.2
31.8
22.6
14.1
20.1
7.11, d (2.0)
7.12, d (2.0)
7.12, d (2.0)
6.88, d (8.2)
1,3,4
2,4
1,2,6,9
1,7,9
6.88, d (8.2)
1,3,4
2,4,7
2,6,8,9
1,7,9
6.88, d (8.2)
1,3,4
2,4,7
1,2,8,9
1,7,9
6.98, dd (8.2, 2.0)
7.56, d (15.8)
6.23,d (15.8)
7.01, dd (8.2, 2.0)
7.57, d (15.9)
6.26, d (15.9)
7.01, dd (8.2, 2.0)
7.57, d (15.9)
6.26, d (15.9)
9
1′
2′
3′
4′
5′
6′
7′
8′
9′
3-OH
4-OH
5.03, m
2′,3′,9′
1′,3′,4′
4′
2′,3′
1′,2′
5.03, m
9
3′
5.03, m
9
3′
1.50-1.72, m
1.27-1.41, m
0.92, t (7.2)
1.28, d (6.2)
38.1
18.7
13.9
20.0
1.50-1.70, m
1.24-1.40, m
1.24-1.40, m
1.24-1.40, m
0.88, t (7.0)
1.28, d (6.2)
1.50-1.70, m
1.24-1.40, m
1.24-1.40, m
1.24-1.40, m
1.24-1.40, m
1.24-1.40, m
0.87, t (7.0)
1.28, d (6.2)
5.92, br
4′, 5′
1′, 2′
6′,7′
1′,2′
5.92, br
6.22, br
6.24, br
a Measured at 300 MHz. b Measured at 400 MHz.

1886 Journal of Natural Products, 2010, Vol. 73, No. 11
Cabanillas et al.
constants between H-8′/H-7′ (J ) 5.9 Hz) and H-8′/H-8 (J ) 6.1
1
Hz) and from the nonequivalence of H-9′a and H-9′b in the H
NMR spectrum. Indeed, according to Corrie et al.17 and Lopes et
al.,18 a nonequivalence of H-9′a and H-9′b indicated a trans-
dibenzylbutyrolactone, while they are almost equivalent in the cis
derivative. No significant correlations were observed in the NOESY
experiment. Thus, an all-trans relationship of H-7′, H-8′, and H-8
was deduced for compound 4. To solve the absolute configuration
at C-7′, compound 4 was treated with (S)- and (R)-MPA19 according
to Mosher’s ester method described by Latypov et al.20 Each of
the two sites showed a clear pattern of ∆δR-S values with negative
values on one side and positive values on the other (Figure 2),
following the expected pattern for Mosher’s method21 and allowing
the assignment of the S configuration at C-7′. The absolute
configuration was therefore determined as 8S,7′S,8′R. On the basis
of the above findings and according to IUPAC rules,22 compound
4 was identified as (8S,7′S,8′R)-3,3′,4,4′-bis(methylenedioxy)-7′-
hydroxy-7-oxolignano-9,9′-lactone, and it was named (-)-san-
guinolignan A.
Figure 1. Key HMBC correlations for compounds 4, 6, and 7.
caffeate. As for 1 and 2, the S configuration was deduced for C-1′
after alkaline hydrolysis.
Compound 4 was a white, amorphous solid, with a negative
specific optical rotation, [R]20 -24. The molecular formula
D
C20H16O8 was determined by HRESIMS (m/z 407.0758 [M + Na]+;
calcd 407.0743). The IR spectrum showed absorption bands at 3418,
1775, and 1660 cm-1, suggesting the presence of OH, ester, and
carbonyl functions, respectively, and the UV spectrum featured
absorption maxima at 283 and 317 nm due to the presence of
Compound 5 gave a molecular formula similar to compound 4,
C20H16O8, by HRESIMS (m/z 407.0758 [M + Na]+; calcd 407.0743).
Its IR spectrum showed the same typical signals (3466, 1778, and
1667 cm-1), and its UV spectrum exhibited absorption maxima at
284 and 318 nm, which matched those of 4. The 1H and 13C NMR
spectra of 5 revealed the same framework showing the characteristic
signals of a dibenzylbutyrolactone lignan. The main differences
were observed for the protons and carbons of the butyrolactone
moiety and C-7′ (Tables 2 and 3). Indeed, protons H-9′ were
shielded and almost equivalent at δH 3.70 (2H, t, J ) 4.8 Hz), while
the protons H-8 at δH 5.07 (1H, d, J ) 10.4 Hz) and H-7′ at δH
5.38 (1H, d, J ) 9.4 Hz) were deshielded. The connectivity of the
carbon framework in 4 was established on the basis of combined
COSY and HMBC correlations, and the planar structure of 5 was
determined to be identical to that of compound 4. However, the
upfield shift and the equivalence of the H-9′ protons of compound
5 relative to that of compound 4 were consistent with a cis
arrangement of H-8 and H-8′. The increase in the 1H-1H coupling
constants between H-8′/H-8 (J ) 10.4 Hz) and H-8′/H-7′ (J ) 9.4
Hz) confirmed the H-8′/H-8 arrangement and also suggested a cis
relationship of H-8′ and H-7′. Mosher’s esterification with MPA
was also applied to compound 5, allowing the assignment of the S
configuration to C-7′. The absolute configuration of compound 5
was therefore determined to be 8S,7′S,8′S. Moreover, the CD
spectrum of 5 ([R]20D -34.3, CH2Cl2) showed a negative absorption
peak at 282 nm and a positive absorption peak at 334 nm (Figure
3), which is opposite that of compound 4 ([R]20D -24, (CH3)2CO),
suggesting that 4 is a diastereoisomer of 5. Thus, the structure of
1
separate aromatic rings. The H NMR spectrum displayed signals
for two sets of ABX systems of aromatic protons at δH 6.76-7.56
(6H), two methylenedioxy groups at δH 5.97 (2H, dd, J ) 5.2, 1.1
Hz, H-10′) and 6.13 (2H, s, H-10), an oxygenated methylene at δH
4.29 (1H, dd, J ) 9.0, 8.9 Hz, H-9′a) and 4.38 (1H, dd, J ) 9.0,
8.0 Hz, H-9′b), and three methine protons at δH 3.34-3.39 (1H,
m, H-8′), 4.76 (1H, d, J ) 6.1 Hz, H-8), and 4.89 (1H, d, J ) 5.9
Hz, H-7′). The 20 carbon signals observed in the DEPT and HSQC
experiments confirmed the presence of a carbonyl at δC 193.5 (C-
7), an ester group at δC 174.0 (C-9), two trisubstituted aromatic
rings, and two methylenedioxy groups at δC 102.0 (C-10′) and 103.2
(C-10), which were indicative of two 3′,4′-methylenedioxyphenyl
units, a methylene at δC 70.2 (C-9′), and three methines. The
aforementioned data strongly suggested that compound 4 was a
dibenzylbutyrolactone lignan with an additional ketone function.
Analysis of the COSY spectrum showed correlations of H-8′/H-8,
H-8′/H-9′a/H-9′b, and H-8′/H-7′. The HMBC spectrum also indi-
cated correlations of H-9′ and H-8 with the carbonyl at δC 174.0
(C-9), supporting the partial structure of a disubstituted γ-lactone
ring. In addition, long-range HMBC correlations of signals at δH
7.29 (H-2) and 7.54 (H-6) with the carbonyl C-7 at δC 193.5, and
the signals at δH 6.91 (H-2′) and 6.92 (H-6′) with the oxygenated
carbon C-7′ at δC 73.8, indicated connection of those carbons with
the corresponding aromatic unit. These data and the other COSY
and HMBC correlations (Figure 1) indicated that 4 was a diben-
zylbutyrolactone lignan with a ketone at C-7 and an OH on C-7′.
The relative configuration of 4 was elucidated from 1H-1H coupling
Figure 2. ∆δR-S (δR - δS) values for the (R)- and (S)-MPA esters of 4 and 5.

Caffeic Acid Esters from Piper sanguineispicum
Journal of Natural Products, 2010, Vol. 73, No. 11 1887
Table 2. 1H NMR Data of Lignans 4-7 (300 MHz) in (CD3)2CO
H
4
5
6
7
1
2
7.29, d (1.8)
7.55, d (1.8)
7.41, d (1.8)
7.20, d (1.8)
3
4
5
6
6.93, dd (7.5, 1.1)
7.54, dd, (8.2, 1.8)
7.02, d (8.0)
7.8, dd (8.0, 1.8)
6.97, d (8.2)
7.62, dd (8.1, 1.8)
6.91, d (8.0)
7.46, dd (8.0, 1.8)
7
8
4.76, d (6.1)
5.07, d (10.4)
4.90, d (8.0)
4.65-4.75, m
4.09-4.17, m
4.65-4.75, m
6.13, s
9-a
9-b
10
1′
2′
3′
4′
5′
6′
7′
8′
9′a
9′b
10′
6.13, s
6.16, s
6.16, s
6.91, d (1.8)
7.01, d (1.8)
6.99, d (1.8)
6.84, d (1.8)
6.77, d (8.2)
6.92, dd (8.1, 1.8)
4.89, d (5.9)
6.90, d (8.0)
6.98, d (8.0, 1.8)
5.38, d (9.4)
3.17-3.27, m
3.70, dd (4.8)
6.90, d (8.1)
6.94, dd (8.2, 1.8)
5.92, d (7.8)
3.75-3.80, m
4.17, dd (9.0, 8.0)
4.29, dd (9.0, 8.3)
6.67, d (8.0)
6.82, ddd (8.0, 1.8, 1.0)
6.20, d (3.8)
3.34-3.39, m
3.69, dd (7.4, 3.8)
4.29, dd (9.0, 5.9)
4.38, dd (9.0, 8.0)
5.97, dd (5.2, 1.1)
6.05, s
5.91, dd (12.9, 1.0)
2.10, s
Ac-CH3
3′-OCH3
4′-OCH3
1.8, s
3.79, s
3.77, s
Table 3. 13C NMR Data (δ) of Lignans 4-7 (75 MHz) in
carbonyl at δC 169.9 were assigned to an acetoxy unit attached to
C-7′. The downfield shift of H-7′ at δH 5.92 (1H, d, J ) 7.8 Hz)
relative to that of compound 4 is also consistent with the OH
substitution. Finally, the connectivity between the atoms in the
γ-lactone and the aromatic ring was identical to the previous
compounds, on the basis of COSY and HMBC correlations (Figure
1). From the H-9′ signals at δH 4.17 (1H, dd, J ) 9.0, 8.0 Hz) and
4.29 (1H, dd, J ) 9.0, 8.3 Hz) and the coupling constants between
H-8′/H-7′ (J ) 7.8 Hz) and H-8′/H-8 (J ) 8.0 Hz), an all-trans
relationship of H-7′/H-8′/H-8 was suggested for compound 6. Since
(CD3)2CO
C
4
5
6
7
1
2
3
4
5
6
7
8
131.6
108.8
149.2
153.3
108.6
126.8
193.5
50.8
132.3
108.8
149.3
153.5
109.3
127.2
192.8
51.7
131.8
108.9
149.4
153.5
112.5
126.8
192.8
51.4
131.2
108.5
149.3
153.3
108.8
125.9
195.7
44.1
the CD spectrum of 6 ([R]20 -1.2, CH2Cl2) exhibited the same
D
9
174.0
103.2
137.7
107.1
148.0
148.8
108.5
120.3
73.8
172.7
103.3
133.2
107.7
149.1
149.1
109.0
121.7
82.1
173.4
103.3
131.1
111.0
150.5
150.5
108.7
120.0
76.4
69.5
positive and negative absorption peaks as compound 4, the same
absolute configuration was proposed. Thus, compound 6 was
identified as (8S,7′S,8′R)-7′-acetoxy-3,4-methylenedioxy-3′,4′-
dimethoxy-7-oxolignano-9,9′-lactone, and it was named (-)-
sanguinolignan C.
10
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
103.3
132.8
107.1
148.8
148.2
108.7
119.9
73.4
The molecular formula of compound 7 was established as
C22H18O9 by HRESIMS. The UV and IR spectra matched those of
49.0
70.2
102.0
51.5
59.3
102.4
46.8
69.1
50.6
175.0
102.2
1
sanguinolignan C. H and 13C NMR features indicated that the
structure of 7 was also very similar to that of 6, except that a
methylenedioxy group at δH 5.91 (2H, dd, J ) 12.9, 1.0 Hz) was
present instead of the two aromatic methoxy singlets. Furthermore,
in the HMBC spectrum, H-7′ at δH 6.20 (1H, d, J ) 3.8 Hz) showed
a cross-peak with a carbonyl carbon at δC 175 (C-9′), the H-8′ at
δH 4.65-4.75 (1H, m) showed cross-peaks with both C-7′ and C-9′,
and the oxymethylene protons H-9 showed cross-peaks with both
carbonyl C-9′ and C-7 (δC 195.7). These data allowed unambiguous
assignment of a 7-oxolignano-9′,9-lactone. The downfield shift of
C-8 (δC 44.1) confirmed that the lactone moiety was reversed in
comparison to that of the other sanguinolignans. As demonstrated
above, an all-trans relationship of H-7′/H-8′/H-8 for compound 7
was deduced from the 1H NMR (Table 2). Moreover, the CD
spectrum (Figure 3) displayed a similar profile, suggesting that the
three-dimensional arrangement of substituents of the chiral centers
C-8, C-8′, and C-7′ was analogous to that of 4 and 6. Therefore,
compound 7 was identified as (8R,7′S,8′S)-7′-acetoxy-3,3′,4,4′-
bis(methylenedioxy)-7-oxolignano-9′,9-lactone, and it was named
(-)-sanguinolignan D.
3′-OCH3
4′-OCH3
Ac-CH3
Ac-CO
56.0
56.0
20.7
20.7
169.5
169.9
5 was elucidated as (8S,7′S,8′S)-3,3′,4,4′-bis(methylenedioxy)-7′-
hydroxy-7-oxolignano-9,9′-lactone and was named (-)-sanguino-
lignan B.
The HRESIMS of compound 6 (m/z 465.1158 [M + Na]+; calcd
465.1162) gave the molecular formula C23H22O9. Its UV spectrum
showed the same typical absorption maxima (282 and 320 nm),
but its IR spectrum showed the absence of hydroxyl functions and
the presence of two ester functions and a carbonyl (1777, 1757,
1
and 1677 cm-1). The H and 13C NMR spectra exhibited features
characteristic of a dibenzylbutyrolactone lignan. In comparison to
4, the most prominent changes concerned the lactone substituents.
1
The H NMR spectrum exhibited only one methylenedioxy group
at δH 6.16 (2H, s, H-10), two aromatic methoxy singlets at δH 3.77
and 3.79, and one methyl singlet at δH 1.80. Moreover, 13C NMR
confirmed the presence of an additional carbonyl at δC 169.9. From
the HMBC spectrum, it was deduced that the two methoxy groups
were connected to C-3′ and C-4′, and the methyl singlet and the
The known compounds (7′S)-parabenzlactone (8), dihydrocube-
bin (9), and justiflorinol (10) exhibited spectral data consistent with
that reported.23-25

1888 Journal of Natural Products, 2010, Vol. 73, No. 11
Cabanillas et al.
Figure 3. CD spectra for compounds 4-7.
Table 4. IC50 Values for Compounds Isolated from P. sanguineispicum
IC50
IC50
IC50
IC50
L. amazonensis
axe. amas. in µM
cytotoxicity on
on MCF7
cells in µM
on Vero
cells in µM
compound
macrophages in µMa
CARb
1
2
3
4
5
6
7
8
9
10
2.0 ( 0.11
10.0 ( 0.37
1.8 ( 0.08
36.7 ( 3.08
>130
105.4 ( 2.4
69.7 ( 2.8
79.4 ( 0.68
>140
72.0 ( 1.8
16.1 ( 1.1
12.7 ( 0.4
>260
36
1.6
7
62.0
89.8
31.0
109.4
>260
>226
82.1
113.5
83.8
14.0
10.6
3.2
>7
145.8
249.9
79.2
17.1
151.3
89.4
140.4
>550
NT
NT
NT
NT
NT
NT
>550
3.6 ( 0.3
>140
17.8 ( 0.15
0.11 ( 0.01
>281
>550
caffeic acid
amphotericin B
>31
33
a NT: not tested. b Cytotoxicity on macrophage/antileishmanial activity ratio.
specimens (GO138) are deposited at the Herbarium of the Natural
History Museum of Mayor de San Marcos University, Lima, Peru.
Extraction and Isolation. Dried leaves of P. sanguinespicum (870
g) were extracted with 90% EtOH (9 L) at room temperature. The
filtrates were combined and concentrated under vacuum to afford 100 g
of crude extract, which was then partitioned between H2O-CH2Cl2
(1:1) (2 × 2 L). After evaporation, the CH2Cl2 extract was dissolved
in MeOH and partitioned with petroleum ether (2 × 2 L). The solvent
was removed under reduced pressure to provide 17.2 and 51.5 g of
petroleum ether and MeOH fractions, respectively. The fractions were
tested on axenic amastigotes of L. amazonensis, and the best activity
was shown by the methanolic fraction (IC50 2.4 µg/mL). One part of
this extract (25 g) was chromatographed on silica gel by MPLC and
eluted with a petroleum ether-AcOEt gradient system to give five
fractions (A1-A5). The active fraction A2 (11.96 g, IC50 1.7 µg/mL)
was subjected to CC on RP-18 using a CH3CN-H2O gradient,
providing six fractions (A2A-A2F). Final purification of A2C and A2E
with CH2Cl2-AcOEt (95:5) on Sephadex LH-20 afforded compounds
2 (1.7 g) and 3 (3.0 g). Fraction A2B was further separated on a silica
gel column using a C6H12-AcOEt gradient to yield five fractions
(A2B1-A2B5). Compound 1 (60 mg) was obtained from fraction A2B2
by CC on Sephadex LH-20 using a CH2Cl2-AcOEt gradient. Fraction
Activities of the isolated compounds against axenic amastigote
forms of L. amazonensis, as well as their cytotoxicity on mouse
peritoneal macrophages and two mammalian cell lines (VERO and
MCF-7), are summarized in Table 4. The most interesting com-
pounds responsible for the leishmanicidal activity of P. san-
guineispicum were found to be the caffeic acid derivatives,
particularly 1-methylbutyl caffeate (1), exhibiting a cytotoxicity on
macrophage/activity ratio similar to that of amphotericin B (about
35). Concerning the lignans, only (-)-sanguinolignan A (4) showed
a moderate antileishmanial activity, but interestingly it had a very
low cytotoxicity on macrophage and cancer or normal cell lines.
Hemisynthesis of caffeic acid derivatives is being performed
currently in an attempt to improve leishmanicidal activity of these
compounds.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 241 polarimeter. The CD spectra were obtained
on a JASCO J-815 spectrometer. UV spectra were recorded on a
Specord 205. IR spectra were obtained on a Perkin-Elmer FT-IR
Parabon 100. 1H and 13C NMR spectra were recorded on Bru¨ker Avance
300, 400, or 500 instruments. HRCIMS and HRESI spectra were
recorded on Waters GTC Premier and Waters LCT spectrometers.
MPLC was carried out with a Bu¨chi C-605 pump and a Bu¨chi C-615
pump manager. Silica gel (Sigma-Aldrich, 230-400 mesh) and RP-18
(EM Science, LiChroprep, 40-63 µm) were used for column chroma-
tography (CC) separations, and silica gel 60 PF254 (Merck) was used
for analytical TLC (0.5 mm).
A2B4 was chromatographed on
a RP-18 column eluted with
MeOH-H2O to give three fractions (A2B4A-A2B4C). Purification
of fraction A2B4B on a silica gel column eluted with CH2Cl2-AcOEt
(98:2) afforded 8 (30 mg). Fraction A2C was fractionated by CC on
RP-18 using a MeOH-H2O gradient to give five fractions, A2C1-A2C5.
Fraction A2C2 was separated into four fractions (A2C2A-A2C2D)
on Sephadex LH-20 eluted with a CH2Cl2-AcOEt gradient system.
Fraction A2C2B gave 9 (18 mg) after purification over a silica gel
column with CH2Cl2-AcOEt (95:5). Compound 4 (60 mg) was
obtained as a precipitate after adding CH2Cl2 to fraction A2C3. After
solvent evaporation, the soluble part was subjected to CC on a silica
Plant Material. The leaves of P. sanguineispicum were collected
in May 2007 from Loreto department in Peru and identified by Ricardo
Callejas (Universidad de Antioquia, Medell´ın, Colombia). Voucher

Caffeic Acid Esters from Piper sanguineispicum
Journal of Natural Products, 2010, Vol. 73, No. 11 1889
gel column eluted with a CH2Cl2-AcOEt gradient to give four fractions
(A2C3A-A2C3D). Purification of fractions A2C3A, A2C3B, and
A2C3C on a Sephadex LH-20 column using CH2Cl2-AcOEt (95:5)
gave 7 (24 mg), 5 (14 mg), 6 (13 mg), and 10 (7 mg).
11.7 Hz, H-9′b), 4.80 (1H, d, J ) 10.6 Hz, H-8), 5.30 (1H, d, J ) 9.5
Hz, H-7′), 6.06 (2H, s, H-10′), 6.20 (2H, s, H-10), 6.90 (1H, d, J ) 8.0
Hz, H-5′), 6.96 (1H, dd, J ) 8.0, 1.6 Hz, H-6′), 7.01 (1H, d, J ) 1.6
Hz, H-2′), 7.01 (1H, d, J ) 8.2 Hz, H-5), 7.45 (1H, d, J ) 1.8 Hz,
H-2), 7.61 (1H, dd, J ) 8.2, 1.8 Hz, H-6). The R-MPA part had δ 3.28
(3H, s, CH3O), 4.73 (1H, s), 7.32-7.39 (5H, m, aromatic protons).
Preparation of (S)-(+)-r-Methoxyphenyl Acetate of 5 (5S). The
(S)-MPA ester was obtained from 4 mg (0.0104 mmol) of 5 by the
procedure described above. The reaction was stirred for 20 h.
Purification was carried out on a silica SPE cartridge using a gradient
of cyclohexane-AcOEt (from 80:20 to 70:30) to obtain 4.5 mg (83%)
(S)-1′-Methylbutyl caffeate (1): white powder; [R]20D +43.3 (c 0.53,
CH2Cl2); UV (CH2Cl2) λmax 294, 319 nm; IR (film) νmax 3331, 2916,
1681, 1601, 1275, 1181 cm-1; 1H and 13C NMR see Table 1; HRCIMS
m/z 251.1275 [M + H]+ (calcd for C14H19O4, 251.1283).
(S)-1′-Methylhexyl caffeate (2): white powder; [R]20D +43 (c 1.06,
CH2Cl2); UV (CH2Cl2) λmax 293, 319 nm; IR (film) νmax 3320, 2926,
1682, 1601, 1275, 1186 cm-1; 1H and 13C NMR see Table 1; HRCIMS
m/z 279.1586 [M + H]+ (calcd for C16H23O4, 279.1596).
1
of (S)-MPA ester: H NMR ((CD3)2CO, 300 MHz) δ 3.43-3.53 (1H,
(S)-1′-Methyloctyl caffeate (3): white powder; [R]20D +47.8 (c 1.07,
CH2Cl2); UV (CH2Cl2) λmax 294, 319 nm; IR (film) νmax 3341, 2957,
1674, 1601, 1275, 1186 cm-1; 1H and 13C NMR see Table 1; HRCIMS
m/z 307.1903 [M + H]+ (calcd for C18H27O4, 307.1909).
m, H-8′), 4.18 (1H, dd, J ) 11.7, 5.8 Hz, H-9′a), 4.31 (1H, dd, J )
11.7, 4.8 Hz, H-9′b), 4.95 (1H, d, J ) 10.7 Hz, H-8), 5.16 (1H, d, J )
9.7 Hz, H-7′), 6.06 (2H, s, H-10′), 6.20 (2H, s, H-10), 6.85 (1H, dd, J
) 8.0, 1.7 Hz, H-6′), 6.89 (1H, d, J ) 8.0 Hz, H-5′), 6.94 (1H, d, J )
1.7 Hz, H-2′), 7.03 (1H, d, J ) 8.2 Hz, H-5), 7.50 (1H, d, J ) 1.8 Hz,
H-2), 7.71 (1H, dd, J ) 8.2,1.8 Hz, H-6). The R-MPA part had δ 3.32
(3H, s, CH3O), 4.73 (1H, s), 7.30-7.34 (5H, m, aromatic protons).
Sanguinolignan C (6): white powder; [R]20D -1.2 (c 0.85, CH2Cl2);
UV (CH2Cl2) λmax 282, 319 nm; IR (film) νmax 2912, 1777, 1742, 1677,
Alkaline Hydrolysis of 1-3. To 20 mg of ester was added 2 mL of
0.1 N NaOH. The mixture was stirred in a boiling water bath for 1 h.
After this time, the reaction mixture was diluted with water (10 mL)
and extracted with CHCl3 (10 mL × 3). The organic layers were
combined and dried over anhydrous MgSO4 and evaporated to dryness
to give approximately 6-7 mg of the corresponding alcohol. The
specific optical rotations, [R]20D, were +10.3, +10.3, and +7.8 for the
corresponding alcohols obtained from 1, 2, and 3, respectively. They
were compared with [R]20D published in ref 26, giving +10.25, +10.21,
and +7.96 for (S)-(+)-2-pentanol, (S)-(+)-2-heptanol, and (S)-(+)-2-
nonanol, respectively.
1
1604, 1442, 1261, 1033 cm-1; H and 13C NMR see Tables 2 and 3;
HRESIMS m/z 465.1158 [M + Na]+ (calcd for C23H22O9Na, 465.1162).
Sanguinolignan D (7): white powder; [R]20D -47.4 (c 2.15, CH2Cl2);
UV (CH2Cl2) λmax 283, 317 nm; IR (film) νmax 2908, 1778, 1765, 1678,
1444, 1256, 1036 cm-1 1H and 13C NMR see Tables 2 and 3;
;
HRESIMS m/z 449.0855 [M + Na]+ (calcd for C22H18O9Na, 449.0849).
Sanguinolignan A (4): white powder; [R]20D -24 (c 1.0, (CH3)2CO);
UV (CH3OH) λmax 283, 317 nm; IR (film) νmax 3418, 1775, 1660, 1600,
(7′S)-Parabenzlactone (8): white powder; [R]20 -16.5 (c 2.31,
D
CH2Cl2); UV (CH2Cl2) λmax 290 nm; IR (film) νmax 3466, 1757, 1492,
1443, 1252, 1036 cm-1
;
1H and 13C NMR see Tables 2 and 3;
1244, 1036 cm-1; H NMR (CDCl3, 300 MHz) δ 2.52-2.61 (1H, m,
1
HRESIMS m/z 407.0758 [M + Na]+ (calcd for C20H17O8Na, 407.0743).
Preparation of (R)-(-)-r-Methoxyphenyl Acetate of 4 (4R). To
a solution of 20 mg of N-(3-dimethyllaminopropyl)-N′-ethylcarbodi-
imide hydrochloride (EDC ·HCl) (0.12 mmol) in acetone (1 mL) and
CH2Cl2 (0.5 mL) were added a catalytic amount of pyrrolidinopyridine
and 17 mg of (R)-MPA (0.12 mmol). After complete dissolution, 4.5
mg of the alcohol 4 (0.012 mmol) was added and the mixture was
stirred at room temperature for 2 h. The reaction mixture was diluted
with 10 mL of AcOEt and extracted with 0.1 N HCl (10 mL × 2), 0.1
N NaHCO3 (10 mL × 2), and water (10 mL × 2). The organic layer
was dried over anhydrous Na2SO4 and evaporated to dryness. The
residue was chromatographed on a silica SPE cartridge (cyclohexane-
AcOEt, 70:30) to give 5.4 mg (98%) of (R)-MPA ester: 1H NMR
((CD3)2CO, 300 MHz) δ 3.66-3.78 (1H, m, H-8′), 3.94 (1H, dd, J )
9.0, 7.5, Hz, H-9′a), 4.27 (1H, dd, J ) 9.0, 8.4 Hz, H-9′b), 4.69 (1H,
d, J ) 7.4 Hz, H-8), 5.86 (1H, d, J ) 7.0 Hz, H-7′), 5.97 (2H, dd, J
) 6.6, 1.0 Hz, H-10′), 6.18 (2H, dd, J ) 2.0, 1.0 Hz, H-10), 6.72 (1H,
d, J ) 8.5 Hz, H-5), 6.81 (1H, dd, J ) 6.4, 1.8 Hz, H-6), 6.82 (1H,
overlapped, H-2′), 6.96 (1H, d, J ) 8.2 Hz, H-5′), 7.37 (1H, overlapped,
H-2), 7.53 (1H, dd, J ) 8.3, 1.8 Hz, H-6). The R-MPA part had δ 3.25
(3H, s, CH3O), 4.84 (1H, s), 7.27-7.36 (5H, m, aromatic protons).
Preparation of the (S)-(+)-r-Methoxyphenyl Acetate of 4 (4S).
The (S)-MPA ester was obtained from 4 mg (0.0104 mmol) of 4 by
the procedure described above. The reaction was stirred for 6 h. The
residue was chromatographed on a RP-18 SPE cartridge (MeCN-H2O,
H-8′), 2.86-3.0 (3H, overlapped, H-7, H-8), 3.94 (2H, d, J ) 7.3 Hz,
H-9′), 4.61 (1H, d, J ) 6.6 Hz, H-7′), 5.92 (2H, dd, J ) 5.9, 1.4 Hz,
H-10′), 5.96 (2H, dd, J ) 3.9, 1.4 Hz, H-10), 6.58 (1H, dd, J ) 7.9,
1.6 Hz, H-6′), 6.61 (1H, d, J ) 1.6 Hz, H-2′), 6.65-6.75 (4H,
overlapped, H-5′, H-2, H-5, H-6); 13C NMR (CDCl3, 75 MHz) δ 35.3
(C-7), 43.9 (C-8′), 45.2 (C-8), 68.6 (C-9′), 75.6 (C-7′), 101.1 (C-10′),
101.4 (C-10), 106.3 (C-2′), 108.3 (C-5), 108.4 (C-5′), 110.1 (C-2), 119.5
(C-6′), 122.9 (C-6), 131.3 (C-1), 135.5 (C-1′), 146.4 (C-4′), 147.7 (C-
4), 147.8 (C-3′), 148.2 (C-3), 179.1 (C-9); HRESIMS m/z 393.0965
[M + Na]+ (calcd for C20H18O7Na, 393.0950).
Dihydrocubebin (9): white powder; UV (CH2Cl2) λmax 290 nm; IR
(film) νmax 3310, 1503, 1441, 1243, 1036 cm-1; 1H NMR (CDCl3, 300
MHz) δ 1.79-1.88 (1H, m, H-8), 2.61 (1H, dd, J ) 13.9, 6.0 Hz,
H-7a), 2.75 (1H, dd, J ) 13.9, 8.6 Hz, H-7b), 3.50 (1H, dd, J ) 11.4,
4.4 Hz, H-9a), 3.78 (1H, dd, J ) 11.4, 4.7, H-9b), 5.92 (2H, s, H-10),
6.60 (1H, dd, J ) 7.8, 1.5 Hz, H-6), 6.63 (1H, d, J ) 1.5 Hz), 6.71
(1H, d, J ) 7.8 Hz); 13C NMR (CDCl3, 75 MHz) δ 36.1 (C-7), 44.4
(C-8), 60.4 (C-9), 100.9 (C-10), 108.3 (C-5), 109.5 (C-2), 122.0 (C-
6), 134.6 (C-1), 145.9 (C-4), 147.8 (C-3); HRESIMS m/z 739.2732 [2
M + Na]+ (calcd for C40H44O12Na, 739.2730).
Justiflorinol (10): white powder; [R]20 -50.4 (c 0.55, CH2Cl2);
D
UV (CH2Cl2) λmax 279, 313 nm; IR (film) νmax 3436, 1666, 1443, 1037
1
cm-1; H NMR ((CD3)2CO, 500 MHz) δ 3.29 (1H, dd, J ) 18.0, 3.6
Hz, H-8a), 3.65 (1H, dd, J ) 18.0, 9.8 Hz, H-8b), 3.70-3.75 (1H, m,
H-9′a), 3.85-3.89 (1H, m, H-9′b), 4.19-4.29 (1H, m, H-8′), 6.12 (2H,
s, OCH2O), 6.13 (2H, s, OCH2O), 6.96 (1H, d, J ) 8.3 Hz, H-5′), 6.98
(1H, d, J ) 8.3 Hz, H-5), 7.41 (1H, d, J ) 1.7 Hz, H-2′), 7.48 (1H, d,
J ) 1.7 Hz, H-2), 7.65 (1H, dd, J ) 8.2, 1.7 Hz, H-6′), 7.69 (1H, dd,
J ) 8.2, 1.7 Hz, H-6); 13C NMR ((CD3)2CO, 125 MHz) δ 38.9 (C-8),
45.7 (C-8′), 64.3 (C-9′), 102.9 (OCH2O), 103.0 (OCH2O), 108.1 (C-
2), 108.6 (C-5′′), 108.7 (C-2′), 125.1 (C-6), 125.5 (C-6′), 132.6 (C-1)
133.1 (C-1′′), 149.0 (C-3), 149.1 (C-3′′), 152.4 (C-4), 152.7 (C-4′′),
196.9 (C-7), 199.9 (C-7′); HRESIMS m/z 379.0790 [M + Na]+ (calcd
for C19H6O7Na, 379.0794).
1
70:30) to give 5.1 mg (93%) of (S)-MPA ester: H NMR ((CD3)2CO,
300 MHz) δ 3.61-3.71 (1H, m, H-8′), 4.21 (1H, dd, J ) 9.0, 8.2 Hz,
H-9′a), 4.31 (1H, dd, J ) 9.0, 8.3 Hz, H-9′b), 4.87 (1H, d, J ) 8.2 Hz,
H-8), 5.90 (1H, d, J ) 7.5 Hz, H-7′), 5.92 (2H, dd, J ) 5.1, 1.0 Hz,
H-10′), 6.19 (1H, s, H-10), 6.43 (1H, overlapped, H-2′), 6.45 (1H, dd,
J ) 7.3, 1.8 Hz, H-6), 6.57 (1H, dd, J ) 7.2, 1.1 Hz, H-5′), 7.02 (1H,
d, J ) 8.2 Hz, H-5), 7.41 (1H, J ) 1.8 Hz, H-2), 7.63 (1H, dd, J )
8.2, 1.8 Hz, H-6). The R-MPA part had δ 3.21 (3H, s, CH3O), 4.58
(1H, s), 7.27-7.36 (5H, m, aromatic protons).
Sanguinolignan B (5): white powder; [R]20D -34.3 (c 1.18, CH2Cl2);
UV (CH2Cl2) λmax 284, 318 nm; IR (film) νmax 3466, 1778, 1667, 1602,
Antileishmanial Assay. Experiments were conducted on axenic
amastigotes of Leishmania amazonensis (strain MHOM/BR/76/LTB-
012). Axenically grown amastigotes were maintained by weekly
subpassages in MAA/20 medium at 32 ( 1 °C with 5% CO2 in 25
cm2 tissue culture flasks. Cultures were initiated with 5 × 105
amastigotes in 25 cm2 tissue culture flasks with 5 mL of medium. To
determine the activity of the extracts, the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) micromethod was used as
previously described.27 Briefly, 100 µL of axenically grown amastigotes
were seeded in 96-well flat-bottom microtiter plates. Crude extracts
were tested at 100, 50, and 1 µg/mL of DMSO. Pure compounds were
1446, 1249, 1036 cm-1 1H and 13C NMR see Tables 2 and 3;
;
HRESIMS m/z 407.0739 [M + Na]+ (calcd for C20H17O8Na, 407.0743).
Preparation of (R)-(-)-r-Methoxyphenyl Acetate of 5 (5R). The
(R)-MPA ester was obtained from 4 mg (0.0104 mmol) of 5 by the
procedure described above. The reaction was stirred for 4 h. Purification
was carried out on a silica SPE cartridge using a gradient of
cyclohexane-AcOEt (from 80:20 to 70:30) to obtain 5.4 mg (98%) of
(R)-MPA ester: 1H NMR ((CD3)2CO, 300 MHz) δ 3.40-3.50 (1H, m,
H-8′), 4.21 (1H, dd, J ) 4.7, 11.7 Hz, H-9′a), 4.32 (1H, dd, J ) 5.5,

1890 Journal of Natural Products, 2010, Vol. 73, No. 11
Cabanillas et al.
(3) Croft, S. L.; Barrett, M. P.; Urbina, J. A. Trends Parasitol. 2005, 21,
508–512.
(4) Grogl, M.; Thomason, T. N.; Franke, E. D. Am. J. Trop. Med. Hyg.
1992, 47, 117–126.
(5) Arango, V.; Robledo, S.; Se´on-Me´niel, B.; Figade`re, B.; Cardona, W.;
Sa´ez, J.; Ota´lvaro, F. J. Nat. Prod. 2010, 73, 1012–1014.
(6) Chan-Bacab, M. J.; Pena-Rodriguez, L. M. Nat. Prod. Rep. 2001, 18,
674–688.
(7) Estevez, Y.; Castillo, D.; Tangoa Pisango, M.; Arevalo, J.; Rojas, R.;
Alban, J.; Deharo, E.; Bourdy, G.; Sauvain, M. J. Ethnopharmacol.
2007, 114, 254–259.
(8) Odonne, G.; Bourdy, G.; Castillo, D.; Estevez, Y.; Lancha-Tangoa,
A.; Alban-Castillo, J.; Deharo, E.; Rojas, R.; Stien, D.; Sauvain, M.
J. Ethnopharmacol. 2009, 126, 149–158.
(9) Parmar, V.; Jain, S. C.; Bisht, K. S.; Jain, R.; Taneja, P.; Jha, A.;
Tyagi, O. D.; Prasad, A. K.; Wengel, A. J.; Olsen, C. E.; Boll, P. M.
Phytochemistry 1997, 46, 591–673.
(10) Rukachaisirikul, T.; Prabpai, S.; Champung, P.; Suksamram, A. Planta
Med. 2002, 68, 853–855.
(11) Rukachaisirikul, T.; Siriwattanakit, P.; Sukcharoenphol, K.; Wongvein,
C.; Ruttanaweang, P.; Wongwattanavuch, P.; Suksamrarn, A. J.
Ethnopharmacol. 2004, 93, 173–176.
(12) Lee, A. L.; Ley, S. V. Org. Biomol. Chem. 2003, 1, 3957–3966.
(13) Jenett-Siems, K.; Mockenhaupt, F. P.; Bienzle, U.; Gupta, M. P.; Eich,
E. Trop. Med. Int. Health 1999, 4, 611–615.
(14) Flores, N.; Cabrera, G.; Jimenez, I. A.; Pinero, J.; Gimenez, A.; Bourdy,
G.; Cortes-Selva, F.; Bazzocchi, I. L. Planta Med. 2007, 73, 206–11.
(15) Torres-Santos, E. C.; Moreira, D. L.; Kaplan, M. A. C.; Meirelles,
M. N.; Rossi-Bergmann, B. Antimicrob. Agents Chemother. 1999, 43,
1234–1241.
(16) Hermoso, A.; Jimenez, I. A.; Mamani, Z. A.; Bazzocchi, I. L.; Pinero,
J. E.; Ravelo, A. G.; Valladares, B. Bioorg. Med. Chem. 2003, 11,
3975–80.
(17) Corrie, J. E. T.; Green, G. H.; Ritchie, E.; Taylor, W. C. Aust. J. Chem.
1970, 23, 133–145.
(18) Lopes, L. M. X.; Yoshida, M.; Gottlieb, O. R. Phytochemistry 1983,
22, 1515–1518.
(19) Sai Prakash, C. V.; Hoch, J. M.; Kingston, D. G. I. J. Nat. Prod. 2002,
65, 100–107.
(20) Latypov, S. K.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Org. Chem.
1996, 61, 8569–8577.
(21) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. ReV. 2004, 104, 17–118.
(22) Moss, G. P. Pure Appl. Chem. 2000, 72, 1493–1523.
(23) Raffaelli, B.; Wa¨ha¨la¨, K.; Hase, T. Org. Biomol. Chem. 2006, 4, 331–
341.
tested at four concentrations in DMSO: 50, 5, 0.5, and 0.05 µg/mL for
1 to 4 and 50, 10, 2, and 0.2 µg/mL for 5 to 10. After 72 h of incubation,
10 µL of MTT (10 mg/mL) was added to each well and plates were
further incubated for 4 h. The enzymatic reaction was then stopped
with 100 µL of 50% 2-propanol-10% sodium dodecyl sulfate and
incubated for an additional 30 min under agitation at room temperature.
Finally, the optical density (OD) was read at 570 nm with a 96-well
scanner (Bio-Rad). All experiments were performed in triplicate, and
standard deviations were calculated using Excel software.
Percent growth inhibition of the parasite was calculated by the
following formula:
(OD control - OD drugs)×100
% of inhibition )
OD control
The concentration inhibiting 50% of the parasite growth (IC50) was
calculated after evaluating percent growth inhibition at different
concentrations (Excel software). Reference compound was amphoteri-
cin B.
Cytotoxicity Assay. Vero cells (a monkey kidney cell line) and
MCF7 cells (a human breast cancer cell line) were cultured in RPMI
(Cambrex), supplemented with 10% fetal calf serum (Boehringer). Cell
growth was measured by [3H]-hypoxanthine (Perkin-Elmer, France)
incorporation after a 48 h incubation with serial drug dilutions. The
amount of [3H]-hypoxanthine incorporated in the presence of test
compounds was compared with that of control cultures without the
test compounds.28 Inhibition percentages were plotted versus concentra-
tion, and IC50 values were evaluated graphically.
Murine peritoneal macrophages were treated with appropriate
dilutions of tested compounds, and the trypan blue dye exclusion
method was used.29 Dilutions of 10, 1, and 0.1 µM in complete medium
were then added to achieve a final volume of 100 µL. The culture was
continued for another 48 h at 37 °C in 5% CO2. After this incubation,
the number of viable cells was scored by hematocytometer using 0.4%
trypan blue solution in PBS. The half-maximal cytotoxic dose for each
cell type was determined. All experiments were repeated three times.
Acknowledgment. The authors gratefully acknowledge the financial
assistance of DSF-IRD (BST) from France. We express our thanks to
members of the Chayahuitas community who were willing to share
with us their knowledge about medicinal plants. The authors are grateful
to G. Gonzalez and Y. Estevez from LID-UPCH, for technical assistance
in biological assays, and R. Duval for manuscript revision.
(24) De Carvalho, M. G.; Yoshida, M.; Gottlieb, O. R.; Gottlieb, H. E.
Phytochemistry 1987, 26, 265–267.
(25) Susplugas, S.; Nguyen Van, H.; Bignon, J.; Thoison, O.; Kruczynski,
A.; Se´venet, T.; Gue´ritte, F. J. Nat. Prod. 2005, 68, 734–738.
(26) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J. Am. Chem. Soc.
1986, 108, 162–169.
(27) Sereno, D.; Lemesre, J. L. Parasitol. Res. 1997, 83, 401–403.
(28) Roumy, V.; Garcia-Pizango, G.; Gutierrez-Choquevilca, A. L.; Ruiz,
L.; Jullian, V.; Winterton, P.; Fabre, N.; Moulis, C.; Valentin, A. J.
Ethnopharmacol. 2007, 112, 482–489.
(29) Castillo, D.; Arevalo, J.; Herrera, F.; Ruiz, C.; Rojas, R.; Rengifo, E.;
Vaisberg, A.; Lock, O.; Lemesre, J. L.; Gornitzka, H.; Sauvain, M. J.
Ethnopharmacol. 2007, 112, 410–414.
1
Supporting Information Available: H and 13C NMR spectra of
compounds 1-7. This material is available free of charge via the
Internet at http://pubs.acs.org.
References and Notes
(1) World Health Organization. Report of the Fifth ConsultatiVe Meeting
on Leishmania/HIV Coinfection, 2007.
(2) Davies, C. R.; Reithinger, R.; Campbell-Lendrum, D.; Feliciangeli,
D.; Borges, R.; Rodriguez, N. Cad. Saude Publica 2000, 16, 925–
950.
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