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Molecules 2014, 19, 21215-21225; doi:10.3390/molecules191221215
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Chemical Constituents from Licania cruegeriana and Their
Cardiovascular and Antiplatelet Effects
Omar Estrada 1, Whendy Contreras 1, Giovana Acha 1, Eva Lucena 1, Whitney Venturini 1,
Alfonso Cardozo 2 and Claudia Alvarado-Castillo 1,*
1 Centro de Biofisica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC),
Altos de Pipe 1020-A, Venezuela; E-Mails: oestrada@ivic.gob.ve (O.E.);
wcontreras@ivic.gob.ve (W.C.); gacha@ivic.gob.ve (G.A.); elucena@ivic.gob.ve (E.L.);
wventurini@ivic.gob.ve (W.V.)
2 Facultad de Agronomía, Universidad Central de Venezuela, Maracay 2101, Venezuela;
E-Mail: alfonosocardozo@gmail.com
* Author to whom correspondence should be addressed; E-Mail: calvarad@ivic.gob.ve or
cpilar.alvarado@gmail.com; Tel.: +58-212-504-1604; Fax: +58-212-504-1093.
External Editor: Nancy D. Turner
Received: 8 October 2014; in revised form: 13 November 2014 / Accepted: 14 November 2014 /
Published: 17 December 2014
Abstract: Three new lupane-type triterpenoids: 6β,30-dihydroxybetulinic acid glucopyranosyl
ester (4), 6β,30-dihydroxybetulinic acid (5) and 6β-hydroxybetulinic acid (6), were isolated
from Licania cruegeriana Urb. along with six known compounds. Their structures were
elucidated on the basis of spectroscopic methods, including IR, ESIMS, 1D- and 2D-NMR
experiments, as well as by comparison of their spectral data with those of related
compounds. All compounds were evaluated in vivo for their effects on the mean arterial
blood pressure (MABP) and heart rate (HR) of spontaneously hypertensive rats (SHR) and
also in vitro for their capacity to inhibit the human platelet aggregation. None of the
isolated flavonoids 1–3 showed cardiovascular effects on SHR and among the isolated
triterpenoids 4–9 only 5 and 6 produced a significant reduction in MABP (60.1% and 17.2%,
respectively) and an elevation in HR (11.0% and 41.2%, respectively). Compounds 3, 4, 5
and 6 were able to inhibit human platelet aggregation induced by ADP, collagen and
arachidonic acid with different selectivity profiles.
Keywords: lupane-type triterpenoids; Licania cruegeriana; platelet aggregation; SHR
OPEN ACCESS
Molecules 2014, 19 21216
1. Introduction
The genus Licania (Chrysobalanaceae) consists of more than 200 species of trees and shrubs, which
are mainly distributed in tropical regions of America and Africa [1,2]. The species of the genus
Licania have been used in South America for various medicinal purposes such as the treatment of
inflammation [3], diabetes [4], stomach ache, diarrhea, and dysentery [5]. Previous phytochemical
studies of this genus have reported the isolation of two main classes of compounds: flavonoid
glycosides based on myricetin and quercetin moieties and triterpenes of the lupane, oleane or ursane
types [4]. In the present work, we report the isolation from the leaves of Licania cruegeriana Urb. and
structure elucidation of three new lupane-type triterpenoids: 6β,30-dihydroxybetulinic acid
gluco-pyranosyl ester (4) 6β,30-dihydroxybetulinic acid (5) and 6β-hydroxybetulinic acid (6), along
with six known compounds. Additionally, all isolated compounds were evaluated for their effects on
the mean arterial blood pressure (MABP) and heart rate (HR) of spontaneously hypertensive rats
(SHR) and also for their capacity to inhibit the human platelet aggregation in vitro.
2. Results and Discussion
2.1. Extraction and Isolation
Fresh leaves (310 g) were extracted by percolation with ethanol for a week. The solvent was
evaporated in vacuo to yield 89.0 g of ethanolic extract (EE). Two fractions were obtained from EE
partition in methanol-water (1:1): A red solution that was then evaporated in vacuo to yield a red
residue (16.7 g), named methanol-water soluble fraction (MWSF), and a green residue (71.2 g) named
methanol-water insoluble fraction (MWIF). A portion of MWSF (5.0 g) was three times extracted with
acetone to obtain a brownish residue and a yellowish solution that was then concentrated to dryness
yielding a yellowish residue named AF (4.7 g). AF (1 g) was fractionated on Sephadex LH-20 column
chromatography (CC) using methanol as eluent to give three fractions named I–III. Myricetin (1,
200 mg) was separated from fraction III by CC on RP-18, eluting with a mixture methanol-water (3:2).
From fraction II myricetin 3-O-α-rhamnoside (2, 100 mg) and dihydromyricetin-3-O-α-rhamnoside (3,
180 mg) were separated by low-pressure CC with a mixture of acetonitrile-water (2:3). MWIF (2 g)
was subjected to low-pressure CC with a mixture of methanol-water (7:3) as eluent to afford
6β,30-dihydroxybetulinic acid glucopyranosyl ester (4, 50 mg), 6β,30-dihydroxybetulinic acid (5,
100 mg), 6β-hydroxybetulinic acid (6, 320 mg), alphitolic acid (7, 20 mg), betulinic acid (8, 19 mg) and
lupeol (9, 15 mg). The structure of compounds 1–3 and 7–9 (Figure 1) were established by comparing
their 1H- and 13C-NMR chemical shifts and proton coupling constants with those previously reported
in the literature [6–9], whereas the structure elucidation of compounds 4, 5 and 6 is described below.
2.2. Structure Elucidation of Compounds 4, 5 and 6
The molecular formulae of compounds 5 and 6 were assigned as C30H48O5 and C30H48O4,
respectively, from their ESIMS and NMR data. The analysis of their 1H- and 13C-NMR spectra
indicated that these compounds are lupane-type triterpenoids.
Molecules 2014, 19 21217
Figure 1. Structures of isolated compounds from Licania cruegeriana.
O
O
O
H
OH
OH
OR1
OH
HO
1- R1: H
2- R1: Rham 3- R1: Rham
R2
CH3
CH2
R3
OH
R1
CH3
CH3
CH3CH3
R4
O
O
OH
OH
OH
OR1
OH
HO
4- R1:OH, R2: COOGlc, R3:OH, R4: H
5- R1:OH, R2: COOH, R3:OH, R4: H
6- R1:OH, R2: COOH, R3:H, R4: H
7- R1:H, R2: COOH, R3:H, R4: OH
8- R1:H, R2: COOH, R3:H, R4: H
9- R1:H, R2: CH3, R3:H, R4: H
The Δ20,29-functionality of the lupene skeletons were inferred from the resonances of the sp2 carbons
at C-29 (secondary carbon signal deduced by DEPT pulse sequence) at δC 106.9 and δC 110.2 ppm and
C-20 (quarternary carbon) at δC 156.3 and δC 152.0 ppm of 5 and 6 respectively [8]. From the
13C-NMR spectra is was deduced that these triterpenoids have a hydroxyl group at C-6 by the shifts of
the 13C-NMR signals at δC 19.7 ppm (C-6) of betulinic acid [8] to δC 69.3 ppm in 5 and 6. In both
compounds the axial orientation of the hydroxyl group at C-6 was inferred by the shifts of the
13C-NMR signal at δC 16.4 ppm (C-24) in 6α-hydroxybetulinic acid [9] to δC 24.8 ppm, thus compound
6 was deduced as 6β-hydroxybetulinic acid.
The signals in the 13C-NMR spectra of 5 and 6 are similar, except those at C-30 and those
corresponding to the proton coupling patterns. The shifts of the signals at δC 19.6 (C-30) in 6 to δC 65.2
in 5 suggest an additional hydroxyl substituent at this position which was confirmed in the HMBC
spectrum of 5 given the correlations between H-29a/H29b (δH 4.96, 4.85) and the carbon signal at δC
65.2 ppm and also between H-30 at δH 4.0 ppm and C-20 at δC 156.3 ppm (Figure 2), indicating
that a hydroxyl group is located at C-30 in 5. Thus, the structure of 5 was determined to be
6β,30-dihydroxybetulinic acid.
Compound 4 has a molecular formula of C36H58O10 according to ESIMS (m/z 649.4 [M−H]−).
The comparison of 1H- and 13C-NMR spectroscopic data of 4 with those of 5 indicated that 4 is the
glycosylated derivative of 5. The NMR spectra of 4 showed an anomeric proton at δH 5.48 ppm
(d, JH1',H2' = 8.0 Hz) with the corresponding carbon at δC 95.2 ppm. Comparison of NMR data with
those reported in the literature suggested a β-oriented glucopyranoside moiety on the basis of the large
3JH1',H2' coupling constant [10–17]. The HMBC correlations of the anomeric proton H-1' to C-28
(δC 176.2) indicated that the β-glucopyranosyl unit was attached to the carboxyl group. Thus, the
Molecules 2014, 19 21218
structure of 4 was determined to be 6β,30-dihydroxybetulinic acid glucopyranosyl ester. To the best of
our knowledge this is the first report of compounds 4, 5 and 6 from Nature.
Figure 2. Key HMBC correlations observed for compounds 4 and 5.
CH3
O
O
CH2
OH
OH
OH
CH3
CH3
CH3CH3
O
OH
OH
OH
OH
HMBC H C
4
5
CO2H
CH3
CH2
OH
OH
OH
CH3
CH3
CH3CH3
2.3. Cardiovascular Effects of the Isolated Compounds
To determine the cardiovascular effects of the isolated compounds from L. cruegeriana, all of them
were intravenously administered to pentobarbital-anaesthetized SHR over thirty seconds and the
MABP and the HR were monitored continuously during forty-five minutes as described in
Experimental Section 3.4.1. None of the isolated flavonoids showed cardiovascular effects in SHR and
among the isolated triterpenoids only compounds 5 and 6 induced changes in MABP and HR of SHR
as shown in Table 1. Compounds 5 and 6 produced significant reductions in MABP (60.1% and
17.2%, respectively) and induced elevation in HR (11.0% and 41.2%, respectively). These
cardiovascular effects exhibit a time of peak effect of three minutes and were recorded for more than
forty-five minutes without recovering baseline levels. The widely used antihypertensive drug losartan
(an AT1 receptor antagonist) served as a positive control [18–20]. In the present study, losartan
injection (0.3 mg/kg) reduced MABP by 19.3% and increased HR by 27.7% (p < 0.05). The reduction
in MABP lasted longer than forty-five minutes and showed a time of peak effect near ten minutes.
Analysis of the relationships between the molecular structures of the cardiovascular active
compounds 5 and 6 and their structurally related compounds 4, 7, 8 and 9 (Figure 1), led to conclude
that the substitution of carboxylic group in 4 with a glycoside seems to interfere with the
cardiovascular active motive of this compound and that the hydroxylation of the betulinic moieties at
C-6 and C-30 enhance its cardiovascular effects.
2.4. Antiplatelet Effects of Isolated Compounds
In Table 2 are shown the isolated compounds from L. cruegeriana which were able to inhibit
the aggregation of human platelets induced by arachidonic acid (AA), collagen and adenosine
5'-diphosphate (ADP).
Molecules 2014, 19 21219
Table 1. Effects of compounds 5 and 6 isolated from Licania cruegeriana on the mean arterial blood pressure (MABP) and heart rate (HR) of
spontaneously hypertensive rats (SHR).
Compounds MABP (mmHg)
Basal
MABP (mmHg)
After Treatment
Maximal (%)
Change
HR (bpm)
Basal
HR (bpm)
after Treatment
Maximal (%)
Change
Time
Peak (s)
Time
Recovery (min)
Vehicle 141 ± 8 137 ± 5 −3.1 422 ± 21 414 ± 12 −1.8 - -
5 138 ± 5 55 ± 10 ** −60.1 ** 290 ± 15 330 ± 12 * 11.0 180 ± 20 >45
6 145 ± 10 120 ± 12 * −17.2 * 405 ± 18 572 ± 16 ** 41.2 180 ± 10 >45
Losartan 145 ± 12 117 ± 9 * −19.3 * 350 ± 32 447 ± 21 ** 27.7 600 ± 9 >45
Compounds 5, 6 and losartan at 0.3 mg/kg were i.v. injected in 0.1 mL of 5% DMSO in physiological saline solution (vehicle) through the femoral vein of anesthetized
SHR over 30 seconds. Increases (+) and decreases (−) in MABP and HR are indicated in the maximal percent of change columns. For each MABP variation, the time of
peak effect and complete recovery to basal values are given. The time of peak effect was measured from the beginning of the injection. Values are the mean ± S.D (n = 4
of each). * p < 0.05 and ** p < 0.01 vs. basal values when unpaired Student’s test was applied. One-way ANOVA test, comparing basal values between treatment groups
of MABP and HR showed no significant differences among them (p > 0.05 for both).
Table 2. Effects of compounds 3, 4, 5 and 6 on human platelet aggregation.
Compounds Aggregation (%)
AA Collagen ADP
Control 100 100 100
3 1.2 ± 0 *** 18.7 ± 6 ** 40.1 ± 6 *
4 49.5 ± 6 13.6 ± 6 *** 90.6 ± 5
5 87.5 ± 2 18.5 ± 7 ** 22.9 ± 5 **
6 89.1 ± 4 32.5 ± 6 * 61.6 ± 5
Platelets in PRP were preincubated with each compound at 250 µg/mL, which correspond to 536 µM (3), 385 µM (4), 510 µM (5) and 530 µM (6), or 0.25% DMSO
(control) for 15 min, then platelet aggregation was stimulated by addition of AA (0.5 mM), collagen (1.5 µg/mL) or ADP (5 μM), at 37 °C under 1000 rpm stirring. Values
are presented as mean ± S.E. of (n 5–6) of the percent of aggregation response compared to their respective controls. One way ANOVA Kruskal-Wallis test and Dunn’s
multiple comparisons test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control) were applied.
Molecules 2014, 19 21220
Compound 3 was the only isolated flavonoid that exhibited antiplatelet effects causing total
inhibition against the action of AA, a significant decrease against collagen and near middle inhibition
against ADP. Among the isolated triterpenoids, compound 4 significantly inhibited the aggregation of
platelets induced by collagen and had an average effect against the actions of AA, and compound 5
significantly decreased the ADP and collagen-induced platelet aggregations, while compound 6
exhibited significant antiplatelet effect against collagen. The mode of action by which these
compounds exert their effects on platelet aggregation needs to be further studied.
3. Experimental Section
3.1. General Information
RediSep® Rf Reversed-phase C18 was used for low pressure CC and silica gel 60 RP18 F254
(E. Merck) for TLC on glass (Merck). Dimethylsulfoxide (DMSO), adenosine 5'-diphosphate sodium
salt (ADP) and Sephadex®LH-20 were obtained from Sigma Aldrich (St. Louis, MO, USA). 1H- and
13C-NMR spectra were obtained using a Bruker DRX 500 (500 MHz for 1H and 125 MHz for 13C) and
Bruker Avance 300 (300 MHz for 1H and 75 MHz for 13C) in CD3OD. Measurements of electrospray
ionization mass spectra were acquired in negative ion mode on an Ion Trap mass spectrometer
(Amazon SL, Bruker, Bremen, Germany). Infrared (IR) spectra (KBr discs) were recorded using a
FTIR spectrophotometer (Perkin Elmer, Shelton, CT, USA). Collagen and arachidonic acid were from
Helena Laboratories (Beaumont, TX, USA). All solvents used were of HPLC grade quality, obtained
commercially from Sigma. The purity of compounds 4, 5 and 6 was confirmed by using an HPLC-MS
Agilent 1260 series LC/MSD trap, SL model (Bruker) equipped with an electrospray interface (ESI), a
quaternary pump, degasser, autosampler and a thermostatted column compartment. A column
XBridgeTM C18 4.6 × 75 mm, 2.5 μm (Waters, Dublin, Ireland) was used and kept at 25 °C in the
column compartment. Nitrogen was used as nebulizing and drying gas at 220 °C. The ESI source was
operated in negative ion mode. Complete system control, data acquisition and processing were done
using the HyStar 3.2 for LC/MSD trap software from Bruker. The injection volume was 5 μL. The
mobile phase was delivered in isocratic mode and consisted of a mixture of methanol/water (70:30).
The chromatograms were recorded in full scan mode for compounds 4, 5 and 6 and only MS/MS mode
for compound 4 (in negative mode MW = 649). Full-scan spectra were acquired over a scan range of
m/z 70–2200 at 32.5 m/z/s.
3.2. Plant Material
The leaves of Licania cruegeriana Urb. were collected in August 2009 at the Parque Nacional
Henri Pittier, Aragua, Venezuela. This specimen was identified by Dr. Alfonso Cardozo and a voucher
specimen (AC2706) was deposited in the herbarium of Facultad de Agronomía, UCV, Maracay.
3.3. Spectral Data
3β,6β,30-Trihydroxy-20(29)-lupen-28-O-β-glucopyranosyl ester (4). A white powder, IR (KBr) υmax
2947, 2871, 1705, 1651, 1386, 1187, 1059 cm−1; 1H-NMR (CD3OD, 500 MHz): δ 5.48 (1H, d, J = 8 Hz,
H-1'), 4.96 (1H, br s, H-29a), 4.85 (H-29b, under CD3OD signal), 4.32 (1H, br s, H-6), 4.04 (2H, br s,
Molecules 2014, 19 21221
H-30), 3.82 (1H, d, J = 12Hz, H-6'a), 3.70 (1H, d, J = 12Hz, H-6'b), 3.45 (m, H-3'), 3.37 (m, H-4'),
3.31(m, H-2'), 3.25 (1H, br s, H-3) , 2.88 (1H, m, H-19), 2.37 (2H, m, H-13 and H-16), 2.03 (3H, m,
H-12 and H-21), 1.8 (1H, t, J = 11.5 Hz, H-9), 1.6 (m, H-7), 1.25 (3H, s, Me-26), 1.20 (3H, s, Me-24),
1.18 (3H, s, Me-25), 0.99 (3H, s, Me-27), 0.97 (3H, s, Me-23); 13C-NMR (CD3OD, 125 MHz,): δ
176.2 (C-28), 156.2 (C-20), 107.1 (C-29), 95.2 (C-1'), 78.7 (C-5'), 78.5 (C-3), 78.3 (C-3'), 74.0 (C-2'),
71.1 (C-4'), 69.3 (C-6), 65.3 (C-30), 62.3 (C-6'), 58.0 (C-17), 52.2 (C-5), 51.2 (C-9), 50.1 (C-18), 43.9
(C-19), 43.7 (C-14), 42.8 (C-7), 41.3 (C-8), 39.4 (C-4), 38.4 (C-13), 38.0 (C-10), 37.3 (C-21), 36.8
(C-21), 33.2 (C-16), 32.6 (C-22), 30.9 (C-15), 28.9 (C-23), 28.1 (C-2), 26.3 (C-12), 24.8 (C-24), 22.1
(C-11), 18.0 (C-25), 17.3 (C-26), 15.4 (C-27). Negative mode ESI-MS: m/z 649.4.
3β,6β,30-Trihydroxy-20(29)-lupen-28-oic-acid (5). A white powder, IR (KBr) υmax 2946, 2870, 1704,
1645, 1452, 1397, 1202, 1058 cm−1; 1H-NMR (CD3OD, 300 MHz): δ 4.96 (1H, br s, H-29a), 4.33 (1H,
m, H-6), 4.04 (2H, br s, H2-30), 3.25 (1H, m, H-3), 2.90 (1H, m, H-19), 1.26 (3H, s, Me-26), 1.20 (3H,
s, Me-25), 1.19 (3H, s, Me-24), 1.0 (3H, s, Me-23), 0.97 (3H, s, Me-27); 13C-NMR (CD3OD,
75 MHz): δ 180.0 (C-28), 156.3 (C-20), 106.9 (C-29), 78.5 (C-3), 69.3 (C-6), 65.2 (C-30), 57.5 (C-17),
52.1 (C-5), 51.1 (C-9), 50.2 (C-18), 44.1 (C-19), 43.7 (C-14), 42.8 (C-7), 41.2 (C-8), 39.4 (C-4), 38.7
(C-13), 38.0 (C-10), 37.9 (C-1), 36.8 (C-21), 33.4 (C-16), 33.1 (C-22), 31.0 (C-15), 28.9
(C-23), 28.1 (C-2), 26.3 (C-12), 24.8 (C-24), 22.1 (C-11), 18.0 (C-25), 17.3 (C-26), 15.4 (C-27).
Negative mode ESI-MS: m/z 487.5.
3β,6β-Dihydroxy-20(29)-lupen-28-oic-acid (6). A white powder, IR (KBr) υmax 2920, 2870, 1700, 1642,
1452, 1394, 1180, 1064 cm−1; 1H-NMR (CD3OD, 300 MHz): δ 4.71 and 4.59 (2H, each br s, H-29a, and
H-29b), 4.33 (1H, br s, H-6), 3.25 (1H, m, H-3), 3.05 (1H, m, H-19), 1.69 (6H, br s, Me-30 and
Me-26), 1.26 (3H, s, Me-24), 1.20 (3H, s, Me-25), 1.19 (3H, s, Me-27), 0.97 (3H, s Me-23); 13C-NMR
(CD3OD, 75 MHz): δ 180.1 (C-28), 152.0 (C-20), 110.2 (C-29), 78.5 (C-3), 69.3 (C-6), 57.5 (C-17),
52.1 (C-5), 50.5 (C-9), 50.2 (C-19), 48.4 (C-18), 43.8 (C-14), 42.8 (C-7), 41.2 (C-8), 39.3 (C-4) 38.8
(C-13), 38.1 (C-10), 38.0 (C-1), 36.8 (C-22), 33.1 (C-15), 31.7 (C-21), 30.9 (C-16), 28.9 (C-23), 27.0
(C-2), 26.3 (C-12), 24.8 (C-24), 22.0 (C-11), 19.6 (C-30), 18.1 (C-26), 17.3 (C-25), 15.4 (C-27).
Negative mode ESI-MS m/z: 471.5.
3.4. Biological Assays
3.4.1. Cardiovascular Assay
Spontaneously Hypertensive Rats (SHR), male (250–300 g) were used for all experiments and were
obtained from the animal care service of IVIC. Animals were housed under conditions of controlled
temperature (21 ± 2 °C) and lighting (lights on 06:00–18:00 h). In addition, they had free access to
food (RATARINA, Protinal, Maracay, Venezuela) and tap water. All animal procedures were
approved by the bioethical committee of IVIC (number 201417). SHR were anesthetized by an
intraperitoneal (i.p.) injection of sodium pentobarbital (40 mg/kg). The trachea was exposed and
cannulated with a polyethylene catheter to avoid ventilation disturbances. Arterial blood pressure was
recorded from the femoral artery through a catheter connected to a blood pressure transducer
(MLT844, PowerLab, Melbourne, Australia) and a bridge amplifier (ML110, PowerLab) from which
Molecules 2014, 19 21222
MABP and HR were continuously recorded using a 4/20 High Performance Data Recording System
(PowerLab). To facilitate the intravenous (i.v.) administration of isolated compound from
L. cruegeriana, an i.v. line was placed in the femoral vein using a polyethylene catheter. Once the
basal conditions remained constant for more than 45 min, the changes in MABP and HR induced by
the L. cruegeriana samples were recorded for at least 45 min after injection. Samples, at the indicated
doses, were injected as a single bolus of 0.1 mL (5% DMSO in physiological saline solution, as
vehicle) over 30 s. The doses of isolated compounds from L. cruegeriana used in this study were the
minimal doses that after inducing a significant hypotensive effect allowed the survival of rats for at
least 2 h. This protocol was evaluated and approved by the Bioethics Commission for Investigations in
Animals (COBIANIM) at the Venezuelan Institute for Scientific Research (IVIC) (Protocol 201417,
approval on November 2014), in accordance with the Code on Bioethics and Biosecurity (2008)
established by the Bioethics Commission National Fund on Science and Technology (FONACIT),
under the national legislation (LOCTI, 2005).
3.4.2. In Vitro Platelet Aggregation Assay
Human platelets were obtained from blood of healthy volunteers who did not take any drugs during
previous two weeks and gave informed consent before taking part in this study. The written informed
consent form and this protocol were evaluated and approved by the Bioethics Commission for
Investigations involving Human Subjects of the Venezuelan Institute for Scientific Research (IVIC)
(Project identification code 1316, approval on March 2009), in accordance with the Code on Bioethics
and Biosecurity (2008) established by the Bioethics Commission National Fund on Science and
Technology (FONACIT), under the national legislation (LOCTI, 2005). Platelet rich plasma (PRP)
was prepared and used in platelet aggregation assays as described earlier [21]. Inhibition experiments
were done by incubating the platelets with 250 µg/mL of each isolated compound (for 15 min) before
their stimulation by the addition of ADP (5 µM), collagen (1.5 µg/mL) and arachidonic acid (0.5 mM).
4. Conclusions
The present chemical investigation of the leaves of L. cruegeriana led to the isolation and
identification of myricetin, two myricetin glycosides and six triterpenoids with lupane moieties
(Figure 1). This constitutes the first phytochemical study for this species. To the best of our knowledge
the following betulinic acid derivates: 6β,30-dihydroxybetulinic acid glucopyranosyl ester (4),
6β,30-dihydroxybetulinic acid (5) and 6β-hydroxybetulinic acid (6) have never been reported before in
the literature. The myricetin derivates found in L. cruegeriana seem to follow a similar glycosylation
pattern to those reported for the Licania genus and other species of the Chrysobalanaceae family [6],
since two of them are glycosylated at C-3 having rhamnose as the common sugar. Of the six
triterpenes 4–9 with lupane skeletons identified in L. cruegeriana only betulinic acid, lupeol and
betulin have previously been reported in species of Licania [22]. 6β-Hydroxybetulinic acid was the
major chemical constituent found in L. cruegeriana leaves.
Pharmacological studies of some plants growing in Venezuela are being conducted by our research
group in order to identify secondary metabolites as potential therapeutic agents for cardiovascular
diseases. In the present study, none of the flavonoids isolated from L. cruegeriana had cardiovascular
Molecules 2014, 19 21223
effects in SHR and only two triterpenoids 5 and 6 showed significant reduction in MABP (60.1% and
17.2% respectively) inducing an elevation in HR (11.0% and 41.2% respectively) as shown in Table 1.
A simple structure–activity relationship analysis of these data would suggest that when triterpenoids
increase their oxidation state by introducing a hydroxyl group, it seems to be sufficient to significantly
increase their hypotensive properties, which is in concordance with what is reported for triterpenoids
such as pomolic acid [23] and ursolic acid [24]. On the other hand, one flavonoid (compound 3) and
three triterpenoids (compounds 4, 5 and 6) were able to inhibit the aggregation of human platelets
induced by AA, collagen and ADP as shown in Table 2. These data would suggest that the absence of
the double bound at C-2 in myricetin moiety is necessary for the antiplatelet effect of this kind of
flavonoids. In the case of lupane-type triterpenoids 4, 5 and 6 the addition of one hydroxyl group at
C-30 in compound 4 and 5 appears to increase their antiplatelet properties against collagen stimulation.
Additionally, the glycosylation at C-28 in compound 4 seems to decrease the anti-platelet effect
against ADP with respect to compound 5. None of these triterpenoids have significant effect on the
AA-induced platelet aggregation.
Taken together, the present phytochemical study of L. cruegeriana leaves is a novel contribution to
the current acknowledge of the phytochemistry of Licania genus, being remarkable that triterpenoids
with a lupane-type skeletons and flavonoids, particularly myricetin and their glycosides are the
characteristic chemotaxonomic markers for this genus [6]. Additionally, herein we report some
new triterpenoids with pharmacological activities that might be useful for the treatment of
cardiovascular diseases.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/12/21215/s1.
Acknowledgments
This work was supported by grants from IVIC (1067 and 1227) and also from the Misión Ciencia
(20071585) Fondo Nacional de Ciencia y Tecnología (FONACIT) Venezuela. We thank Lic. Daniela
Briceño and Lic. Liz Cubillan from IVIC for the helpful cooperation in the NMR and IR experiments.
Author Contributions
The contributions of the respective authors are as follows: O. Estrada. and C. Alvarado-Castillo
conceived and designed the experiments; O. Estrada, C. Alvarado-Castillo, W. Contreras, G. Acha,
W. Venturini and E. Lucena performed the experiments; O. Estrada, C. Alvarado-Castillo and
W. Contreras analyzed the data; E. Lucena performed the HPLC-MS analyses and A. Cardozo collected
and identified the leaves of Licania cruegeriana.. O. Estrada and C. Alvarado-Castillo wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
Molecules 2014, 19 21224
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Sample Availability: Not available.
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