Antileishmaniasis activity of flavonoids from Consolida oliveriana

Article abstract of DOI:10.1021/np8008122

A set of flavonoids from Consolida oliveriana, kaempferol (1), quercetin (2), trifolin (3), and acetyl hyperoside (5) and their O-acetyl derivatives (1a, 2a, 3a), and octa-O-acetylhyperoside (4) showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania spp. The cellular proliferation, metabolic, and ultrastructural studies showed that the acetylated compounds 2a, 3a, and 4 were highly active against Leishmania (V.) peruviana, while 2a as well as 4 were effective against Leishmania (V.) braziliensis. These compounds were not cytotoxic and are effective at similar concentrations up to or lower than the reference drugs (pentostam and glucantim).

Full text of DOI:10.1021/np8008122

J. Nat. Prod. 2009, 72, 1069–1074
1069
Antileishmaniasis Activity of Flavonoids from Consolida oliWeriana
Clotilde Mar´ın,† Samira Boutaleb-Charki,† Jesu´s G. D´ıaz,‡ Oscar Huertas,† Mar´ıa J. Rosales,† Gregorio Pe´rez-Cordon,†
Ramon Guitierrez-Sa´nchez,§ and Manuel Sa´nchez-Moreno*,†
Department of Parasitology, UniVersity of Granada, SeVero Ochoa s/n, E-18071 Granada, Spain, Department of Chemistry, UniVersity of La
Laguna, Instituto UniVersitario de BioOrga´nica “Antonio Gonza´lez”, E-38206 La Laguna, Tenerife, Spain, and Department of Statistics,
UniVersity of Granada, SeVero Ochoa s/n, E-18071 Granada, Spain
ReceiVed December 23, 2008
A set of flavonoids from Consolida oliVeriana, kaempferol (1), quercetin (2), trifolin (3), and acetyl hyperoside (5) and
their O-acetyl derivatives (1a, 2a, 3a), and octa-O-acetylhyperoside (4) showed leishmanicidal activity against promastigote
as well as amastigote forms of Leishmania spp. The cellular proliferation, metabolic, and ultrastructural studies showed
that the acetylated compounds 2a, 3a, and 4 were highly active against Leishmania (V.) peruViana, while 2a as well as
4 were effective against Leishmania (V.) braziliensis. These compounds were not cytotoxic and are effective at similar
concentrations up to or lower than the reference drugs (pentostam and glucantim).
In humans, Leishmania spp. cause a variety of clinical diseases
due to the ability of the organism to proliferate in deep tissue or
close to the skin surface at low temperatures. The varied manifesta-
tions of the disease have been used by the World Health Organiza-
tion as the basis for classifying leishmaniasis into four clinical
forms: (a) visceral, (b) mucocutaneous, (c) cutaneous diffuse or
disseminated, and (d) cutaneous. Certain species of the parasite have
been associated with the different clinical forms of the disease;
that is, the Leishmania donoVani complex causes visceral leish-
maniasis, while the Leishmania tropica complex is known to induce
cutaneous lesions in the Old World and Leishmania mexicana L.
(V.) peruViana does so in the New World. Some 25% of the mucous
cutaneous forms cause mucocutaneous and cutaneous diffuse
leishmaniasis in several countries of Latin America, the pathogen
being Leishmania (V.) braziliensis.1
Historically, the chemotherapy of leishmaniasis has been based
on the use of toxic heavy metals, particularly antimony compounds.
Whenever these kinds of drugs are no longer effective, some others
are used, including pentamidine and amphotericin B. These
chemicals have to be injected, and clinical care or hospitalization
during treatment may be necessary due to possible side effects;2,3
thus other treatments are needed. Folk medicine is very often a
valid source for researchers looking for bioactive substances
potentially useful against many diseases. Extracts from medicinal
plants or compounds derived from them are a valuable source of
new medicinal agents for treating Leishmaniasis4 and other dis-
eases.5 The range of families and species from which potentially
active leishmanicidal substances can be extracted is very broad,6
for example, Bixa orellana (Achiote), Polypodium calaguala
(Calaguala), Sida rhombifolia (Escobilla), Psidium guajaVa (Guay-
aba), Plantago major (Llante´n), Ficus dendrocida (Matapalo), Piper
angustifolium (Matico), Musa paradisiacal (Pla´tano), Lupinus tauris
(Tauri), Solanum nigra (Yerba mora), and Lepidium peruVianum
(Maca).7-12 The leishmanicidal effect may reside in its phy-
tochemical component such as flavonoids and, specifically, quer-
cetin, which is a strong candidate in the combination therapy against
the infection and the anemia associated with VL.13
diterpenoid alkaloids of other Consolida species, but reports on
the flavonoid content of members of this genus are scarce.14-19
Most of the studies directed toward the detection of secondary
plant metabolites with leishmanicidal activity have used the
promastigote form of the parasite because it is easier to maintain
under in Vitro conditions. However, since the promastigote is not
the developed form of the parasite in vertebrate hosts, evaluations
made with promastigotes have only an indicative value of the
possible leishmanicidal activity of the metabolite tested. As a result,
a preliminary evaluation using promastigotes needs to be comple-
mented with an evaluation using intracellular amastigotes in
macrophages. At the same time, an evaluation of the possible
cytotoxicity of the metabolite should be carried out using nonpara-
sitized macrophages, in order to establish whether the in Vitro
activity of the metabolite is due to its general cytotoxic activity or
whether it is selectively active against the Leishmania parasite.20
We investigated the inhibitory effects of flavonoid compounds
derived from aerial parts of C. oliVeriana (DC) Schrod, on the
extracellular promastigote and the intracellular amastigote stages
of L. (V.) peruViana and L. (V.) braziliensis, causal agents of both
cutaneous and mucocutaneous leishmaniasis. In addition, we studied
the cytotoxic effects of these compounds against a cell line of
macrophages, analyzing the mechanism by which these molecules
act.
Results and Discussion
The importance of phytotherapy as a guide in the search for new
antileishmanial drugs becomes evident in a single search of
specialized literature. Thus, the strategy for discovering new drugs
is to investigate natural products from medicina1 plants.21-23
Dozens of potential new compounds from nature have been found
in the past 5 years, especially in the rainforests of South America
and Africa, only concerning Leishmaniasis.24 Plant-derived active
principles and their semisynthetic and synthetic analogues have
served as a major path to new chemotherapeutic compounds.23,25,26
Furthermore, the leads obtained from the search for natural products
with antileshmanial activity give new impetus for developing
valuable synthetic compounds.27
Flavonoids are found in abundance in diets rich in fruits,
vegetables, and plant-derived beverages and appear to have
anticancer, antimicrobial, and antiparasitic properties.28-30 Our
Tenerife group is searching for bioactive substances potentially
useful against many diseases, and recently they have shown that
flavonoid acetates derived from flavonoids extracted from C.
oliVeriana exhibited significant impact on the growth of three human
cell lines: HL-60, U937, and SK-MEL-1.19
Part of our group (Tenerife group) has been working on
flavonoids derived from the aerial parts of Consolida oliVeriana
(DC) Schrod, a species used medicinally in parts of Anatolia
(Turkey). A large number of publications have dealt with the
* To whom correspondence should be addressed. Tel: +34 958
242369. Fax: +34 958, 243174. E-mail: msanchem@ugr.es.
† Department of Parasitology, University of Granada.
‡ University of La Laguna.
§ Department of Statistics, University of Granada.
10.1021/np8008122 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/02/2009

1070 Journal of Natural Products, 2009, Vol. 72, No. 6
Mar´ın et al.
effective against L. (L.) donoVani and T. cruzi, at doses slightly
lower than ours. These discrepancies presumably derive from the
use of different methods and different life-cycle stages of the
parasites.35
The products 2a, 3a, and 4 were selected because they had the
greatest inhibitory effect on the in Vitro growth of L. (V.) peruViana
and L. (V.) braziliensis and had less toxic effects on macrophage
cells, using the IC25 of each product as the test dosage.
When 1 × 105 J774.2 macrophage cells were incubated for 2
days and then infected with 1 × 106 promastigote forms of L. (V.)
peruViana and L. (V.) braziliensis (Figure 2), the parasites invaded
the cells and underwent morphological conversion to amastigotes
from the first 3 h postinfection with a steadily increasing number
of cells invaded until the last day of the assay (day 10). The values
for infected cells (control experiment) reached 95% in the case of
L. (V.) peruViana and 97% in the case of L. (V.) braziliensis.
Nevertheless, in the amastigote-cell assay with the flavonoid
compounds added simultaneously to the infection of macrophages
cells with promastigote forms, the treatment significantly reduced
the infection rate with respect to the control. That is, in the case of
L. (V.) peruViana the infection rate reached 55%, 40%, and 59%
for the compounds 4, 2a, and 3a, respectively, on day 10 (Figure
2A). Meanwhile, in the infected in Vitro cultures of L. (V.)
braziliensis the results were 46% and 67% for compounds 4 and
3a (Figure 2B). The addition of 4, 2a, and 3a had a similar effect
with a markedly lower amastigote number of L.(V.) peruViana per
infected cell with respect to the control at day 10, i.e., reaching a
value of roughly 72% (Figure 2C). When the cultured parasite was
L. (V.) braziliensis, compound 4 decreased the amastigote number
by as much as 60%, while compound 3a reached 90% (Figure 2D).
Figure 1. Chemical structure of flavonoid compounds.
Table 1. In Vitro Activity of Flavonoids on Promastigotes of L.
(V.) peruViana and L. (V.) braziliensisa
toxicity IC50 on
J774.2 macrophages
cells (µM)b
IC50 (µM)
compound L. (V.) peruViana L. (V.) braziliensis
pentostam
glucatim
1
1a
2
2a
3
3a
4
11.32
15.33
71.29
53.32
60.04
11.18
53.34
10.53
7.35
9.56
25.61
53.65
68.56
30.49
46.78
52.46
8.72
12.44
15.20
53.67
15.56
125.44
109.23
161.32
148.71
122.31
61.32
6.21
51.60
5
86.95
a IC50 ) the concentration required to give 50% inhibition, calculated
by linear regression analysis from the Kc values at the concentrations
used (0.1, 1, 10, 25, 50, and 100 µM) at 72 h culture. Note: Average of
three separate determinations. b J774.2 macrophages cells at 72 h of
culture.
Most studies on activity assays of new compounds use parasite
forms that develop in vectors (promastigote forms in the case of
Leishmania spp.),20 for the ease of working with these forms in
Vitro. However, in the present study we have included the effect
of the flavonoid compounds on the forms that are developed in the
host (amastigotes) to determine the effects in humans. For this
objective, we selected the products that had the greatest inhibitory
effect on the in Vitro growth of L. (V.) peruViana (2a, 3a, and 4)
and L. (V.) braziliensis (3a and 4) and had less toxic effects on
J774.2 macrophage cells, using the IC25 of each product at the test
dosage. The infective capacity on macrophage cells by L. (V.)
peruViana and L. (V.) braziliensis significantly decreased when
acetylated flavonoids were added. This infective capacity declined
from the early hours of culture, these compounds altering the
invasive capacity of the parasites. Until now, the mechanism by
which the parasite’s invasive capacity is lost was not known. It
was found that these compounds decrease the parasite’s multiplica-
tion rate into the macrophages. The acetylated compounds (2a, 3a,
and 4) are highly effective not only against the extracellular forms
of the parasite (promastigote forms) but also against intracellular
forms (amastigote forms).
Here, we evaluate whether the inhibitory effect on cell growth
of four flavonoids (1, 2, 3, and 5) obtained by natural means from
C. oliVeriana and their acetylated products (1a, 2a, 3a, and 4)19
(Figure 1) is active against intra- and extracellular forms of L. (V.)
peruViana and L. (V.) braziliensis. The results are displayed in Table
1, using pentostam and glucatim as the reference drugs and
including toxicity values against the J774.2 macrophage cells. After
72 h of exposure against L. (V.) peruViana, three of the acetylated
compounds tested (2a, 3a, and 4) registered IC50 values lower than
the reference drugs’ IC50 (pentostam 11.32 and glucantim 15.33
µM), i.e., an IC50 of 7.35 µM for 4, 11.18 µM for 2a, and 10.53
µM for 3a. Therefore, the toxicity values were very low when tested
on J774.2 macrophage cells. In addition to being slightly toxic,
these three compounds gave IC50 values on J774.2 macrophage cells
of about 10- to 15-fold higher than those for the reference drugs.
Against L. (V.) braziliensis, two of these acetylated compounds (4
and 3a) inhibited cell growth at a IC50 of 6.21 and 8.72 µM,
respectively, this being significantly lower than the IC50 of the
reference drugs. The rest of the compounds had an IC50 higher than
the reference drugs and in some cases were toxic for the macroph-
ages. These results indicate that the acetylated compounds per-
formed better than the phenolic analogues. It appears that the
kaempferol derivatives possessing a monosubstituted B-ring are
more active than the quercetin analogues.
It has been shown that acetylation of certain flavonoids increases
the antiproliferative activities of the parent compounds against HL-
60 and other cell lines. For example, 3a induced cell death in human
leukemia cells.26 This greater efficiency may possibly be due to
acetylation, which facilitates the compound absorption, leading to
greater effectiveness.31 Little information is available on the
leishmanicidal activity of flavonoids for comparison with our results;
however, recently it has been possible to establish the activity of
some of these compounds, showing them to be effective against
promastigote development of both L. (L.) mexicana32 and L. (L.)
donoVani,33 with IC50s values similar to those found by us. In a
study by Tasdemir et al.,34 quercetin was demonstrated to be highly
Despite the good results achieved in some cases, only a few
compounds of natural origin are under clinical evaluation, as most
of them have been disapproved because of their high toxicity.36 It
should be noted that these antiprotozoal agents are basically
cytotoxic and act selectively against the parasites. Currently,
publications on antiparasitic agents include activity evaluations
against mammals and/or human cell lines.33,37 Following this
pattern, we have determined the toxicity of our compounds against
a human cell line, noting that compounds 2a, 3a, and 4 showed
little toxicity for J774.2 macrophage cells, with IC50’s of around
10 to 15 times higher than those of the reference drugs, confirming
a selective activity of these compounds against L. (V.) peruViana.
These data are consistent with findings in the case of L. (V.)
braziliensis, given that compounds 3a and 4 proved extremely
effective and with very low toxicity, opening the possibility of using

FlaVonoids from Consolida oliVeriana
Journal of Natural Products, 2009, Vol. 72, No. 6 1071
Figure 2. Effect of flavonoid compounds on the infection rate and growth of Leishmania (V.) peruViana (A) and L. (V.) braziliensis (B).
(C) Mean number of amastigotes of L. (V.) peruViana and (D) for L. (V.) braziliensis per infected J774.2 macrophage cells. -2-, control;
-b-, 2a; -+-, 3a; and -4-, 4 (at IC25 conc). The values are means of three separate experiments.
these compounds in the treatment of both cutaneous and mucocu-
taneous leishmaniasis.38
The in Vitro culture of trypanosomatids depends on the available
carbon sources (mainly glucose) present in their culture medium
for their energy metabolism.39 None of the trypanosomatids studied
are capable of completely degrading glucose to CO2 under aerobic
conditions, excreting a great part of their carbon skeleton into the
medium as fermented metabolites, this difference depending on the
species considered.39 L. (V.) peruViana and L. (V.) braziliensis
consume glucose at a high rate, thereby acidifying the culture
medium due to incomplete acid oxidation. Using 1H NMR spectra,
we determined the fermented metabolites excreted by the parasites
during their in Vitro culture, and we identified and evaluated the
inhibiting effect caused by the flavonoid compounds over final
metabolite excretion in promastigote forms of L. (V.) peruViana
and L. (V.) braziliensis cultured in Vitro in MTL medium for 96 h
(Figures 3 and 4). Figure 3B corresponds to the spectrum given by
cell-free medium 96 h after inoculation with the promastigote forms
of L. (V.) peruViana. Additional peaks, corresponding to the major
metabolites produced and excreted during growth, were detected
when the last spectrum (Figure 3B) was compared with that found
with fresh medium (Figure 3A). L. (V.) peruViana excreted acetate
and succinate as majority metabolites and L-alanine in a lower
proportion. When the trypanosomatids were treated with the
flavonoid compounds (2a, 3a, and 4; Figure 3C, D, and E), the
excretion of some of these catabolites was clearly inhibited at
the dosage tested (IC25). Acetate and succinate were decreased by
around 30 to 40%, while with compound 4 the acetate decreased
up to 67%. L-Alanine also increased, and new excreted metabolites
such as pyruvate and glycerol appeared. In the case of L. (V.)
braziliensis, when the cell-free medium spectrum was compared
(Figure 4B) to that of the parasite-free medium (Figure 4A), the
excretion of acetate and succinate as major metabolites was found
together with L-alanine in a lower proportion. When compounds
Figure 3. 1H NMR study of the production of metabolites
excreted by the promastigote forms of L. (V.) peruViana treated
against flavonoid compounds (at a concentration of IC25). (Ace)
acetate; (Suc) succinate; (L-ala) L-alanine; (Pyr) pyruvate, and
(Gly) glycerol.
3a (Figure 4C) and 4 (Figure 4D) were added to the cultures, a
clear inhibition was detected in the peaks corresponding to acetate
and succinate, while the peaks corresponding to L-alanine and
D-lactate were higher, and peaks corresponding to pyruvate and
glycerol appeared.

1072 Journal of Natural Products, 2009, Vol. 72, No. 6
Mar´ın et al.
Figure 4. 1H NMR study of the production of metabolites excreted
by the promastigote forms of L. (V.) braziliensis treated against
flavonoid compounds (at a concentration of IC25). (Ace) acetate;
(Suc) succinate; (L-ala) L-alanine; (Pyr) pyruvate (Gly) glycerol,
and (D-lac) D-lactate.
Figure 5. Ultrastructural alterations by TEM in L. (V.) peruViana
and L. (V.) braziliensis treated with flavonoids compounds. (1)
Control of L. (V.) peruViana, bar ) 1.0 µm. (2 and 3) Promastigotes
of L. (V.) peruViana treated with 2a, bar ) 1.0 µm. (4) Promas-
tigotes of L. (V.) peruViana treated with 3a, bar ) 1.0 µm. (5)
Control of L. (V.) braziliensis, bar ) 1.59 µm. (6) Promastigotes
of L. (V.) braziliensis treated with 3a, bar ) 1.0 µm. (7 and 8)
Promastigotes of L. (V.) braziliensis treated with 4, bar ) 1.59 µm.
(N) nucleus; (Nu) nucleolus; (CM) cytoplasmatic membrane; (MT)
microtubules; (G) glycosomes; (R) reservosomes; (M) mitochon-
drion; (K) kinetoplast; (V) vacuole; and (LV) lipidic vacuoles.
The excretion of some of these catabolites (acetate and succinate)
was clearly inhibited at the dosages assayed (IC25). In the catabolism
of glucose in Leishmania spp., pyruvate is located at metabolic
branching points, leading to several excreted end products, such
as acetate, L-alanine, ethanol, and L-lactate.40 Acetate is a major
end product formed in the mitochondrion and excreted by simple
diffusion across the mitochondrial and cytoplasmic membranes. The
inhibition of acetate excretion may be a direct consequence of the
action of these flavonoids on the enzymes involved in their
production (pyruvate dehydrogenase complex or acetate-succinate
CoA-transferase), or else these compounds act on the mitochondrion
and even on the cytoplasmic membrane, causing a loss of
functionality. Succinate is another major end product excreted from
glucose metabolism, but the pathway leading to its production has
been the topic of a long-standing debate.41 The controversy concerns
the relevance of the NADH-dependent fumarate reductase activity
detected in most trypanosomatids, for which the contribution in
succinate production has not been clearly demonstrated. The main
role of the succinate (succinic fermentation) is probably to maintain
the glycosomal redox balance, by providing two glycosomal
oxidoreductase enzymes that allow reoxidation of NADH, produced
by glyceraldehydes-3-phosphate dehydrogenase in the glycolytic
pathway. Succinic fermentation offers the significant advantage of
requiring only half of the phosphoenol pyruvate (PEP) produced
to maintain the NAD+/NADH balance. The remaining PEP is
converted into acetate, L-lactate, L-alanine, and/or ethanol, depend-
ing on the species. The inhibition of acetate and succinate excretion
explains the observed increase in L-alanine and D-lactate production
and the appearance of two new peaks identified as glycerol and
pyruvate.
Morphological alterations of L. (V.) peruViana treated with 2a
and 3a were examined by transmission electron microscopy (TEM).
The parasites were sensitive mainly to the treatment with these
compounds at IC25, and a considerable number of dead parasites
were observed. Several alterations could be detected when compared
with the images corresponding to control cells (Figure 5-1).
Compound 2a induced death with distortion of the parasite body
and disruption of the cytoplasm, which appeared almost empty in
some parasites but with many glycosomes throughout (Figure 5-2
and 3). Meanwhile, 3a induced intense vacuolization in the parasites
(Figure 5-4), and empty vacuoles and lipidic vacuoles were visible.
Glycosomes were abundant in the altered parasites, while other
parasites appeared to be dead.

FlaVonoids from Consolida oliVeriana
Journal of Natural Products, 2009, Vol. 72, No. 6 1073
Plant Material. Aerial parts of C. oliVeriana were collected and
identified near Pazarkik in eastern Turkey at an altitude of 980 m by
Prof. Julian Molero Briones, Department of Botany, Faculty of
Pharmacy, University of Barcelona (Spain), where a voucher specimen
(BCF-37810) has been deposited.
The treatment with 3a and 4 at IC25 for 96 h induced alterations
in L. (V.) braziliensis, which can be observed when comparing the
images made with untreated parasites (Figure 5-5). The treatment
with 3a (Figure 5-6) disrupted the cytoplasm, which in the majority
of the parasites had little electrodensity and abundant vacuoles. The
mitochondrion and kinetoplast were also affected, both organelles
appearing swollen. Some parasites had abundant lipidic vacuoles.
L. (V.) braziliensis treated with 4 (Figure 5-7 and 8) and showed
marked alterations. Figure 5-7 shows an undulating cytoplasmic
membrane and microtubules in disarray. The kinetoplasts were
intensely swollen, glycosomes were abundant, and some parasites
showed vacuolization. The rest of the compounds were highly toxic,
but it was not possible to study them by TEM.
In general, several morphological alterations were noted in L.
(V.) peruViana and L. (V.) braziliensis when additional compounds
were added to the cultures. There was an intense vacuolization as
well as disruption of the cytoplasmic, nuclear, and mitochondrial
membranes, suggesting a possible effect on tubuline, a protein
known to be affected by flavonoids.42 This disruption of the
membranes may in turn account for the strong inhibition of the
acetate that is synthesized inside the mitochondria and then diffused
to the cytoplasm and outside the cell.
In conclusion, our study demonstrates that several acetylated
flavonoids derived from C. oliVeriana (2a, 3a, and 4) were very
active in Vitro against both the extracellular as well as the
intracellular forms of L. (V.) peruViana and L. (V.) braziliensis (3a
and 4). These compounds are not toxic to the host cells and are
effective at concentrations similar to or lower than the reference
drugs used in the present study. These flavonoids have promising
leishmanicidal properties. The data from transmission electronic
microscopy and nuclear magnetic resonance raise the possibility
that the action (or part of the action) could be at the level of the
parasite membranes. The potent leishmanicidal activities described
here for flavonoids represent an exciting advance in the search for
new antiprotozoal agents.
Extraction and Isolation. Dried and powdered aerial parts of C.
oliVeriana (2.23 kg) were defatted with hexanes (6 L) for one month
and subsequently extracted repeatedly with 80% EtOH (7 L) at room
temperature for two weeks. The extract was filtered and concentrated
at reduced pressure. The remaining aqueous layer was exhaustively
extracted with n-BuOH to give, after removal of the solvent, 36 g of
a brown viscous residue. The aqueous layer was concentrated and
filtered through a column of Amberlite XAD-2 resin (8 × 40 cm) to
remove the polar compounds, while the flavonoids remaining on the
column were eluted with MeOH (see below). The viscous n-BuOH
extract (10 g) was fractionated on a 50 × 8 cm column packed with
Sephadex LH-20 and eluted with hexanes-CH2Cl2-MeOH, 1:1:2, 15
500-mL fractions (S1-S15) being collected. Fractions S1-S15 con-
tained mainly alkaloids contaminated by material exhibiting no UV
absorption, and fractions S4-S8 contained mixtures of glycosides.
Fractions S9 and S10 and fractions S11-S13, after recrystallization
from MeOH-EtAc, gave quercetin (2, 556 mg) and kaempferol (1,
472 mg), respectively. Fractions S4-S8 were chromatographed over a
40 × 4 cm Sephadex LH-20 column using hexane-CH2Cl2-MeOH
(1:1:1), the elution being monitored by TLC analysis. This resulted in
three fractions: A (430 mg), B (70 mg), and C (55 mg). Fraction B
afforded 90 mg of trifolin (3) after recrystallization from MeOH.
Rechromatography of fraction A over silica gel using 40 mL of
hexanes-EtOAc mixtures of increasing polarity rendered, from fractions
64-77 (hexanes-EtOAc, 2:8), 24 mg of 6′′-O-acetylhyperoside after
further purification over Sephadex LH-20 (hexanes-MeOH-CH2Cl2,
2:1:1). Fractions 86-90 (n-hexane-EtOHc, 1:9) yielded 21 mg of 6′′-
O-acetylhyperoside (5).
The material eluted from the Amberlite XAD-2 column with MeOH
was further purified over a 50 × 8 cm column packed with Sephadex
LH-20 and eluted with CH2Cl2-MeOH (1:1), six fractions (J1-J6) of
500 mL each being collected. Fractions J1-J3 containing mainly
alkaloids and other components exhibiting no UV absorption were
combined with fractions S1-S3. Fractions J4-J6 (2 g) were subjected
to gel filtration on Sephadex LH-20 using 42 200-mL fractions of
H2O-MeOH (1:1). Fractions 28-31 yielded 45 mg of trifolin (3) and
fractions 33-38 18 mg of octa-O-acetylhyperoside (4).
Experimental Section
General Experimental Procedures. The1H and 13C NMR spectra
were measured using a Bruker AMX-400 or a Bruker MAX-500
instrument. FAB5 and exact mass measurements were determined using
a Micromass Autospec instrument at 70 eV. ESIITMS data were
obtained by tandem electrospray ion trap mass spectrometry (LCQ Deca
XP Plus; ThermoFinnigan; San Jose, CA). Column chromatography
was performed over Sephadex LH-20 Pharmacia (ref 17-0090-01;
Upsala, Sweden), silica gel 60 (Merck 230-400 mesh; Darmstadt,
Germany, and analytical TLC, Merck Kieselgel 60 F 254. HPLC
separations were performed on a JASCO Pu-980 series pumping system
equipped with a JASCO UV-975 ultraviolet detector and with a Waters
Kromasil 5 (5 mm × 250 mm) column. A Macherey-Nagel VP 250/
10 nucleodur Sphinx RP 5 mm column (Du¨ren, Germany) was used
for HPLC-RP chromatography; chromatograms were visualized under
UV light at 255 and 366 nm and/or sprayed with oleum followed by
heating. All solvents were distilled before use; the purity of all
compounds was 99.0% as judged by HPLC. Stock solutions of 10 mM
flavonoids were made in DMSO, and aliquots were frozen at -20 °C.
The metabolite excretion 1H NMR spectra were obtained at 300 MHz
on a Bruker AM-300 spectrometer, which was operated at pulse in
Fourier transformation with quadrature detection. The temperature of
the probe was maintained at 27 °C. The acquisition parameters were
as follows: pulses of 90° in radius and a wavelength of 3287.5 Hz, 8 s
recycle time, and 160 accumulations. The chemical displacements were
expressed as parts per million (ppm) using sodium 2,2-dimethyl-2-
silapentane-5-sulfonate as the reference signal. The ultrastructural
alterations were examined with an EMCIO Zeiss transmission electron
microscope.
General Method for Acetylation. Dry phenolic material was
dissolved in the minimum volume of pyridine. Twice the amount of
acetic anhydride was added, and the solution was allowed to stand
overnight at ambient temperature. The mixture was diluted with H2O
and extracted three times with EtOAc. The extract was evaporated under
vacuum, and the residue containing the polyacetate was further purified
by column chromatography over silica gel using hexanes-EtOAc as
the eluent. Mass spectra of the polyacetylated compounds, all gums,
are listed below.
Tetra-O-acetylkaempferol (1a): EIMS m/z ) 412 (M+ - C2H3O2,
23), 370 (M+ - 2 C2H3O2, 57) 286 (M+ - 4 C2H3O2, 100).
Penta-O-acetylquercetin (2a): HREIMS m/z ) 513.0997, calcd for
C25H20O2 + H+ 513.1033.
Hepta-O-acetyltrifolin (3a): HRFABMS m/z ) 743.1784, calcd for
C35H34O18 + H+ 743.1823.
The compound purity of 99% was determined by HPLC.
Promastigote Assay. The compounds obtained (Figure 1) were
dissolved in DMSO (Panreac, Barcelona, Spain) at a concentration of
0.1% and were afterward assayed as nontoxic and without inhibitory
effects on the parasite growth, according to Luque et al.43 The
compounds were dissolved in the culture medium, and the dosages used
were 100, 50, 25, 10, and l µM. The effect of each compound against
promastigote forms, as well as the concentrations, was evaluated at
24, 48, and 72 h using a Neubauer hemocytometric chamber. The
leishmanicidal effect is expressed as IC50 values, i.e., the concentration
required to give 50% inhibition, calculated by linear-regression analysis
from the Kc values at the concentrations employed.
Parasite Strain, Culture. L. (V.) peruViana (MHOM/PE/1984/
LC26) and L. (V.) brazilensis (MHOM/BR /1975/M2904) were cultured
in Vitro in MTL medium plus 10% inactivated fetal bovine serum kept
in an air atmosphere at 28 °C, in Roux flasks (Corning, USA) of 75
cm2 in surface area, according to the methodology described by
Gonza´lez et al.22
Cell Culture and Cytotoxicity Tests. Macrophage line J774.2
(ECACC number 91051511) cells was obtained from a tumor in a
female BALB/c rat in 1968. Macrophages were kept in the laboratory
by cryopreservation in liquid N2 and then by successive subcultures in
RPMI medium. For the cytotoxicity test, macrophages were placed in
25 mL cone-based bottles (Sterling) and centrifuged at 1500 rpm for 5

1074 Journal of Natural Products, 2009, Vol. 72, No. 6
Mar´ın et al.
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(MEM; Gibco) supplemented with l0% inactivated fetal bovine serum
(adjusted to pH 7.2) was added to a final concentration of 105 cells/
mL. This cell suspension was distributed in a culture tray (24 wells) at
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for 96 h at 28 °C, the cells were centrifuged at 1500 rpm for 10 min.
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nuclear magnetic resonance spectroscopy (1H NMR) as previously
described by Sa´nchez-Moreno et al.44 The chemical displacements were
expressed in parts per million (ppm), using sodium 2,2 dimethyl-2-
silapentane-5-sulfonate as the reference signal. The chemical displace-
ments used to identify the respective metabolites were consistent with
those described by Fernandez-Becerra et al.45
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Acknowledgment. We wish to thank M. J. Mart´ınez-Guerrero and
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electron microscopy and NMR spectroscopy, respectively. We also
appreciate the help of L. F. Espigares-Herv´ıas for his kind assistance
in the preparation of electron microscopy photographs and D. Nesbitt
for the English revision. This investigation received financial support
from MEC (Spain), CGL2006-27889-E/BOS.
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NP8008122