Bauerenol acetate, the pentacyclic triterpenoid from tabernaemontana longipes, is an antitrypanosomal agent

Article abstract of DOI:10.3390/molecules23020355

The Latin American plant Tabernaemontana longipes was studied in this work as a potential source of antiparasitic agents. The chloroform extract of T. longipes leaves was separated into several fractions, and tested for antitrypanosomal activity. One of the fractions displayed significant growth inhibitory activity against Trypanosoma brucei. The active principle in the fraction was isolated, purified, and characterized by NMR and mass spectrometry. The antitrypanosomal agent in the CHCl3 extract of T. longipes leaves is the pentacyclic triterpenoid bauerenol acetate. A metabolite profiling assay suggest that the triterpenoid influences cholesterol metabolism. The molecular target(s) of bauerenol and its acetate, like many other antiparasitic pentacyclic triterpenoids is/are unknown, but they present privileged structural scaffolds that can be explored for structure-based activity optimization studies using phenotypic assays.

Full text of DOI:10.3390/molecules23020355

molecules
Communication
Bauerenol Acetate, the Pentacyclic Triterpenoid
from Tabernaemontana longipes, is an
Antitrypanosomal Agent
1
1
1
1
Simira Carothers , Rogers Nyamwihura , Jasmine Collins , Huaisheng Zhang ,
2
3
1, ID
HaJeung Park , William N. Setzer and Ifedayo Victor Ogungbe *
1
Department of Chemistry, Jackson State University, Jackson, MS 39217, USA;
Simira.carothers@gmail.com (S.C.); rogersnyamwihura@yahoo.com (R.N.); Jcollins1908@gmail.com (J.C.);
huiasheng.zhang@students.jsums.edu (H.Z.)
X-ray Crystallography Laboratory, Scripps Research Institute-FL, Jupiter, FL 33458, USA;
2
hajpark@scripps.edu
3
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA;
wsetzer@chemistry.uah.edu
*
Correspondence: ifedayo.v.ogungbe@jsums.edu; Tel.: +1-601-979-3719
Received: 21 December 2017; Accepted: 6 February 2018; Published: 8 February 2018
Abstract: The Latin American plant Tabernaemontana longipes was studied in this work as a potential
source of antiparasitic agents. The chloroform extract of T. longipes leaves was separated into several
fractions, and tested for antitrypanosomal activity. One of the fractions displayed significant growth
inhibitory activity against Trypanosoma brucei. The active principle in the fraction was isolated,
purified, and characterized by NMR and mass spectrometry. The antitrypanosomal agent in the
CHCl extract of T. longipes leaves is the pentacyclic triterpenoid bauerenol acetate. A metabolite
3
profiling assay suggest that the triterpenoid influences cholesterol metabolism. The molecular target(s)
of bauerenol and its acetate, like many other antiparasitic pentacyclic triterpenoids is/are unknown,
but they present privileged structural scaffolds that can be explored for structure-based activity
optimization studies using phenotypic assays.
Keywords: Tabernaemontana longipes; Trypanosoma brucei; trypanosomiasis; pentacyclic triterpenoid
1
. Introduction
African sleeping sickness (Human African Trypanosomiasis) caused by protozoan Trypanosoma
brucei gambiense/rhodesiense is still a public health problem in rural and remote regions of the continent,
especially in the Democratic Republic of the Congo. Despite a gradual decrease in the number of
reported cases in the past few years, the lack of effective and safe medicines, the unavailability
of adequate and rapid diagnostic tools, especially in rural and remote places, and the possibility
of continuous transmission of the parasite from animal reservoirs to humans, make the disease a
continuous threat to millions of people [1–4]. To consolidate recent gains in the fight against the
disease, continued integration of discovery and development of new drugs and new diagnostic tools
with disease control and prevention strategies is of paramount importance. Therefore, discovery of
new antitrypanosomal chemical entities using molecular target-based or phenotypic screening-based
approaches remains very attractive.
In our on-going exploration of tropical plants as potential sources of new antitrypanosomal
agents, Tabernaemontana longipes Donn. Sm. (Apocynaceae) was studied as a potential source of
antitrypanosomal phytochemicals. T. longipes is a tropical plant found in several Latin American
countries, including Nicaragua, Colombia, Ecuador, and Costa Rica. The plant’s flowers are white or
cream-colored, with cylindrical corollas and brown lobes, and the flowers smell sweet [5,6].
Molecules 2018, 23, 355; doi:10.3390/molecules23020355
www.mdpi.com/journal/molecules

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2
. Results and Discussion
The leaves of T. longipes were obtained from several mature trees growing in Costa Rica in
2009. The leaves (1 kg) were dried, chopped, and extracted with refluxing chloroform for 6 h.
The chloroform extract (14 g) was fractionated on silica gel (700 g) and eluted with 5:1 hexane/ethyl
acetate mixture to obtain 49 fractions. The fractions were pooled together to obtain nine superfractions
(SP1–9). The fractions were screened for their ability to inhibit the growth of T. brucei in vitro.
SP2 displayed significant antitrypanosomal activity (76% growth inhibition at 10 µg/mL). SP2 (2.1 g)
was subsequently separated using 10:1 hexane/ethyl acetate mixture over 200 g of silica gel to obtain
seven fractions (SP2F1–SP2F7). The TLC profiles of the fractions showed that SP2F4 and SP2F5 have
a major and similar component while the other fractions have several minor components. SP2F4
and SP2F5 were pooled together and purified by crystallization. SP2F4-5 (227 mg), a yellowish,
oily amorphous solid, was dissolved in hexane and placed in a pentane tank for 24 h to induce
crystallization. The resulting crystals were filtered, washed four times with pentane (2 mL), and
1
13
allowed to dry, to give colorless needle-like crystals (1, 76 mg). The H- and C-NMR spectra
of 1 suggested that it is a triterpenoid. The 13C-NMR spectrum shows 31 distinguishable carbon
13
signals. The most characteristic structural features in the C spectrum include an ester carbonyl signal
1
(δ
171.1) and an alkenyl carbon signal (
δ
145.6). The H and HSQC spectra revealed that there are nine
methyl groups, nine methylene groups, one alkenyl hydrogen, one oxygenated methine group, five
non-oxygenated methine groups, and five quaternary carbons (Table 1 and Figure 1).
Table 1. H- and 13C-NMR Spectra Data for 1 and 2.
1
1
2
Position
13C
1H
13C
1H
1
2
3
4
5
6
7
8
9
36.6
24.3
81.3
37.9
48.3
24.1
116.4
145.6
50.7
35.0
32.5
31.6
37.8
37.9
28.8
16.7
41.2
55.0
35.3
31.8
29.3
37.8
15.9
27.6
13.1
23.6
22.8
37.9
25.8
22.6
21.4, 171.1
1.18, 1.66 (2H, m)
1.66 (2H, m)
4.51 (1H, dd)
37.02
27.8
79.4
39.0
48.3
24.3
116.5
145.4
50.5
35.3
32.5
31.6
37.86
37.86
29.0
16.9
41.3
55.0
35.4
31.8
29.3
37.82
14.8
27.6
13.1
23.8
22.8
38.1
25.8
22.7
1.09, 1.64 (2H, m)
1.61 (2H, m)
3.24 (1H, dd)
2.21 (1H, m)
1.97, 2.14 (2H, m)
5.40 (1H m)
2.19 (1H, m)
1.97, 2.16 (2H, m)
5.41 (1H m)
1.41 (1H, m)
1.29 (1H, m)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.48, 1.56 (2H, m)
1.14, 1.61 (2H, m)
1.48, 1.54 (2H, m)
1.14, 1.60 (2H, m)
1.40, 1.50 (2H, m)
1.48, 1.54 (2H, m)
1.41, 1.49 (2H, m)
1.48, 1.54 (2H, m)
1.29 (1H, s)
1.15 (1H, m)
1.54 (1H, m)
1.19, 1.51 (2H, m)
1.18, 1.48 (2H, m)
0.93 (3H, s)
1.29 (1H, s)
1.14 (1H, m)
1.54 (1H, m)
1.19, 1.51 (2H, m)
1.18, 1.49 (2H, m)
0.85 (3H, s)
0.85 (3H, s)
0.76 (3H, s)
0.99 (3H, s)
0.94 (3H, s)
0.96 (3H, s)
0.74 (3H, s)
0.99 (3H, s)
0.94 (3H, s)
1.02 (3H, 3)
1.04 (3H, d)
0.90 (3H, d)
1.02 (3H, s)
1.04 (3H, d)
0.90 (3H, d)
2.05 (3H, s)
OAc

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The connectivity of the carbon-carbon atoms, and their hydrogen atoms was deciphered using
a combination of HMBC, COSY, and HSQC data (Table 1 and Figure S1 in Supplementary File 1).
The HMBC data provide a clear correlation between one of the methyl groups and the carbonyl carbon
that is typical of acetylated triterpenoid, while the carbon connectivity suggested that is a pentacyclic
triterpenoid [7]. The relative stereochemistry of the compound was established by nuclear Overhauser
effect (NOE) correlations, and X-ray diffraction data from a single plate crystal.
Figure 1. Structure of compounds 1 and 2.
Its molecular formula, C H O , was established from high resolution mass spectrometry (HRMS;
3
2
52
2
+
TOFMS ESI+, [M + Li] m/z 475.4113, for C H O Li, Figure S2), and CH elemental analysis (Found:
32
52
2
C = 81.37% and H = 11.13%; Calculated: C = 81.99% and H = 11.18%). The compound (
as bauerenol acetate, a pentacyclic triterpenoid that had been previously identified in Tabernaemontana
longipes [ ]. Compound was then tested for its ability to inhibit the growth of T. brucei in vitro. The
1) was identified
8
,9
1
results showed that bauerenol acetate has an IC50 value of 3.1 µM (Table 2).
Table 2. Bioassay data of compounds 1 and 2.
Compound
T. brucei Growth Inhibition IC50 (µM)
Cytotoxicity Hep G2 IC50 (µM)
1
2
3.09 ± 0.80
2.71 ± 0.96
0.04 ± 0.01
>80
>80
n/a
Suramin
Due to the relatively poor solubility of the acetate in DMSO during the bioassay, the acetate
was subjected to hydrolysis in 5% ethanolic KOH to obtain its alcohol derivative bauerenol ( ).
The solubility of bauerenol in DMSO was somewhat better than the acetate, and its activity on T.
brucei in vitro was characterized by an IC50 value of 2.7 M. The antitrypanosomal activities of these
compounds are much lower (1–3 order of magnitude) than clinically used antitrypanosomal drugs [10].
Also, the IC50 values of and are higher than the IC50 value (<1 M) proposed by Nwaka and
2
µ
1
2
µ
Hudson as criteria for preclinical evaluation of antitrypanosomal drug candidates [11]. A number of
oleanolic and ursolic acid-based pentacyclic triterpenoids have previously reported to have growth
inhibitory activity against Leishmania spp. and T. cruzi but not T. brucei [12–17].
Several pentacyclic triterpenoids have been shown to be cytotoxic or described as potential
anticancer agents [18
hepatocarcinoma cell model (Hep G2) or if they show selective antitrypanosomal activity.
Both compounds have very little effect on cellular viability on Hep G2 cells (IC50 > 80 M). The molecular
–20]. Therefore, we decided to test if 1 and 2 are cytotoxic to human
µ
target (macromolecule or pathway) of these compounds is yet to be experimentally determined and
could be the subject of future studies, but previous docking studies have suggested T. brucei sterol
14α-demethylase to be a potential target for triterpenoids [21].

Molecules 2018, 23, 355
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Nevertheless, to understand how the pentacyclic triterpenoid (
2) affect T. brucei cells, a primary
metabolite profiling assay was carried out. The metabolites were identified and quantified using
GC-TOFMS as outlined in the methods section below. A total of 90 metabolites were unambiguously
identified and quantified (Supplementary File 2) in the assay. The metabolites with the most notable
concentration-dependent changes in the parasites are stearic acid, phosphoenolpyruvate, myristic
acid, cholesterol, arachidic acid, and adenine. The changes in the levels of cholesterol were the most
consistent and conceivably the most informative from the metabolite profiling assay. The levels of
cholesterol (Figures 2 and S3) in parasites treated with 5
compared to the control group (n = 6, p-value = 0.0023) and the group treated with 0.15
µ
M of
2
was significantly increased when
M of (n = 6,
µ
2
p-value = 0.0072). This suggests that the triterpenoid is interfering with the metabolism of endogenous
sterol(s) or a cholesterol-dependent pathway. Previous studies have shown that accumulation of
cholesterol by trypanosomes is associated with perturbation of the endogenous sterol biosynthetic
pathway [22–25]. Therefore, sterol biosynthetic pathway in T. brucei is the likely target of bauerenol (2).
Figure 2. Levels of cholesterol in parasite treated with bauerenol (2). The levels of cholesterol in the
parasite was significantly increased after 4-h exposure to 5 µM of 2.
In conclusion, bauerenol and its acetate are anti-T. brucei agents. The structure of bauerenol (2) can
be used as a starting structural motif in natural products-based diversity oriented synthesis (DOS) or
in the synthesis of derivatives that have better drug-like physicochemical properties. Further studies
are necessary to determine the specific enzyme or metabolic step directly affected by bauerenol.
3
. Materials and Methods
3
.1. Plant Material
T. longipes leaves were collected in May 2009, in the Monteverde region of northwestern Costa
◦
0
00
◦
0
00
Rica (10 18 3.4 N, 84 48 39.3 W, 1381 m elevation). The plant was identified by William A. Haber.
A voucher specimen has been deposited with the Missouri Botanical Garden herbarium (Haber
Collection No. 7104).
3
.2. Compound Characterization
All chromatographic separations were carried out on Sortech’s silica gel (Premium Rf, 60 Å,
1
13
4
0–75 µm). The 1D ( H and C) and 2D (gCOSY, gHSQC, gHMBC, NOESY) data were collected on
a 500 MHz (13C: 125 MHz) Varian INOVA spectrometer operated with Vnmrj. Chloroform-d was
used as solvent, and tetramethylsilane (TMS) as internal standard in the solvent. The mass spectra
were obtained on a Synapt G2 HDMS instrument (Waters; Milford, MA, USA) operated in positive ESI
mode. Elemental analysis was carried out on a Costech’s ECS 4010 (Valencia, CA, USA). The infrared
spectra were obtained on a Perkin Elmer Spectrum 100 FT-IR spectrometer (Waltham, MA, USA) using

Molecules 2018, 23, 355
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an ATR accessory. Please see Figures S1, S2 and S4–S7 in Supplementary File 1 for NMR, IR, Anal, and
MS spectra as well as the crystal structure.
Crystals of
1
were obtained by slow evaporation of a chloroform solution of
1. X-ray diffraction
data were collected on a Bruker AXS Smart APEX CCD II [Mo K
α (λ
= 0.71073 Å)] (Madison, WI,
USA) at 295 K. The structure was solved by Olex2 using SHELXTL and was refined by full-matrix
least-squares procedure (Figure S6) Non-hydrogen atoms were refined anisotropically. Hydrogen
atoms were added in calculated positions and were refined. Melting points were recorded on a
MEL-TEMP 1101D apparatus (Dubuque, IA, USA). The melting point values are uncorrected.
3
.2.1. Bauerenol Acetate (1)
Colorless crystals; melting point 289–291 C; IR (CHCl ) νmax 2935, 1732, 1463, 1374, 1249,
◦
3
026 cm 1; H- and C-NMR, see Table 1; HRMS ESI+ [(M + Li) ], m/z 475.4113 [Calculated for
−
1
13
+
1
C H O Li 475.4128].
32
52
2
3
.2.2. Bauerenol (2)
Colorless solid; melting point 202–203 C; IR (CHCl ) νmax 3379, 2929, 2866, 1457, 1372, 1032
◦
3
+
cm 1; H- and C-NMR, see Table 1; HRMS ESI+ [(M + H) ], m/z 427.3922 [Calculated for C H O
−
1
13
30
51
2
4
27.3940].
3
.3. Trypanosoma brucei Assay
The growth inhibitory activity of the compounds was evaluated using the Alamar blue assay [26].
Bloodstream form of T. brucei (strain 427) cultured in HMI-9-medium supplemented with 10% FBS, 10%
Serum plus (SAFC), 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM
β
-mercaptoethanol, 0.16 mM thymidine, 1 mM pyruvate, and 0.0125% Tween 80 was used. Parasites
3
were dispensed into a sterile 96-well plate at 5
for 24 h. The compounds were prepared in DMSO containing 1.25% Tween 80. Final concentration
×
10 cells/well and treated with compound
1
and
2
of Tween 80 in the assay wells was 0.0125%. The compounds were screened in triplicate with a total
◦
assay volume of 100
µL. Next, Alamar blue (20
µ
L) was added and the plate was incubated at 37 C
for 4 h. Immediately following incubation, fluorescence signals were read (λex 530 nm, λem 590 nm).
Suramin was used as positive control.
3
.4. Cytotoxicity Assay
Human hepatocarcinoma cell line (Hep G2 CRL-11997™, ATCC) was used for cytotoxicity
studies. The cells were grown in complete medium (DMEM: F12 containing L-glutamine and sodium
◦
bicarbonate, 10% FBS, 1% penicillin/streptomycin, 0.0125% Tween 80) incubated at 37 C in a 5%
CO environment. Once 80–90% confluent, the cells were washed with phosphate-buffered saline
2
(
PBS), treated with 0.25% (w/v) of trypsin/EDTA, counted and suspended in fresh complete media.
5
Into 96-well plates, 5
×
10 cells/mL were seeded and incubated for 24 h. Cells were treated with the
compounds prepared in DMSO containing 1.25% Tween 80 for 72 h. Final concentration of Tween 80 in
the assay wells was 0.0125%. Thereafter, DMEM:F12 medium containing MTT (5 mg/mL in PBS) was
added to the cells and incubated for 1 h. The MTT-containing medium was then gently removed and
replaced with DMSO (200 µL/well), the plate was then mixed gently to allow the formazan crystals to
dissolve. Absorbance was measured at 550 nm. All compounds were tested in triplicates. SDS (10%)
was used as positive control.
3
.5. Metabolic Profiling
T. brucei cells in supplemented HMI-9 medium were treated with 5 µM and 0.15 µM of 2 for
4
−
h. After treatment, the cells were washed three times with PBS, and the cell pellets were stored at
80 C for subsequent GC-MS analysis. The treatment was carried in sextuplicate, including six no
◦

Molecules 2018, 23, 355
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treatment controls. Ten million cells per replicate were used. Metabolites were extracted from the cells by
adding 0.5 mL of 3:3:2 acetonitrile:isopropanol:water mixture to each sample followed by the addition of
3
stainless steel grinding beads and then transferred to a GenoGrinder (Metuchen, NJ, USA) for grinding.
The cells were grinded for 30 s at 1500 rpm, and centrifuged at 14,000 g for 5 min. The supernatants
×
were transferred to new tubes. The extraction was repeated twice, and the pooled supernatant was
◦
concentrated to 500
µ
L. The samples were silylated using MSTFA + 1% TMCS at 37 C, and were used
for primary metabolite profiling on a LECO Pegasus IV GC-time of flight mass spectrometer (Saint
Joseph, MI, USA) as described by Fiehn and co-workers [25]. The GC-TOFMS analysis was carried out
at the NIH West Coast Metabolomics Center at the University of California, Davis.
The samples (0.5
µL each) were analyzed using the splitless mode on a Rtx-5Sil MS capillary
column (30 m length
×
0.25 mm internal diameter with 0.25 m film made of 95% dimethyl/5%
µ
diphenylpolysiloxane, Restek Corporation, Bellefonte, PA, USA). The gas flow-rate was 1 mL/min.
Initial injection temperature was set at 50 C and ramped up to 250 C at a rate of 12 C/s. Initial
oven temperature was set at 50 C for 1 min, then ramped up to 330 C at a rate of 20 C/min,
and held constant for 5 min. The TOF mass spectrometer (LECO; Saint Joseph, MI, USA) unit
◦
◦
◦
◦
◦
◦
mass resolution was set at 17 spectra/s from 80–500 Da at
−
70 eV ionization energy and 1800 V
◦
◦
detector voltage. The transfer line and the ion source temperatures were set at 230 C and 250 C,
respectively. The primary metabolites were identified and quantified using the quantifier ions shown
in Supplementary File 2. The raw mass spectra for the metabolites were processed by BinBase (BB)
software (FiehnLab; Davis, CA, USA). The relative quantification was carried out using the peak height
of each quantifier ion. The peak heights were normalized by dividing them by the sum of all peak
heights for all identified metabolites and multiplied by the average peak heights for all identified
metabolites in each sample [27,28].
Supplementary Materials: The NMR, IR, CH elemental analysis, and MS spectra as well as the GC-TOFMS Data
are provided as supplementary material. Supplementary materials are available online.
Acknowledgments: This work was carried out in part by resources made available by the US National Institutes
of Health (SC2GM109782, SC3GM122629, and G12MD007581). SC was supported by NSF-LSAMP Bridge to the
Doctorate (HRD-1500317). We thank William Haber for plant identification. We are grateful to the Monteverde
Cloud Forest Preserve and the Tropical Science Center for permission to collect plant materials under a cooperative
rights agreement and to the Commission for the Development of Biodiversity of Costa Rica’s Ministry of the
Environment, Energy, and Telecommunications for Research Permit R-001-2006-OT-CONAGEBIO. This work
was performed as part of the activities of the Research Network Natural Products against Neglected Diseases
(ResNet-NPND), http://www.resnetnpnd.org.
Author Contributions: Simira Carothers, William N. Setzer, and Ifedayo Victor Ogungbe conceived and designed
the experiments; Simira Carothers, Jasmine Collins, Rogers Nyamwihura, Huaisheng Zhang, HaJeung Park,
William N. Setzer, and Ifedayo Victor Ogungbe performed the experiments; Simira Carothers, Jasmine Collins,
HaJeung Park, and Ifedayo Victor Ogungbe analyzed the data; Ifedayo Victor Ogungbe and William N. Setzer
contributed reagents/materials/analysis tools; Ifedayo Victor Ogungbe wrote the paper, and the paper was
reviewed by all authors.
Conflicts of Interest: The authors declare no conflict of interest. The sponsors had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of the compounds are available from the authors.
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