UPLC-ESI-QTOF-MS2 characterisation of Cola nitida resin fractions with inhibitory effects on NO and TNF-α released by LPS-activated J774 macrophage and on Trypanosoma cruzi and Leishmania amazonensis

Article abstract of DOI:10.1002/pca.2771

Introduction: The resin of Cola nitida is used in western Cameroon as incense for spiritual protection and during ritual ceremonies. This plant secretion has never been investigated although previous chemical and biological studies on other resins have drawn many attentions. Objective: The resin fractions which revealed inhibitory effect on nitric oxide (NO) and tumour necrosis factor alpha (TNF-α) released by lipopolysaccharide (LPS)-activated J774 macrophage as well as on intracellular forms of Leishmania amazonensis and Trypanosoma cruzi amastigote were chemically characterised. Moreover, their antiparasitic activities were compared to those of semi-synthetic triterpenes. Methodology: The anti-inflammatory activity was evaluated by measuring the nitrite production and the TNF-α concentration in the supernatants of LPS-activated macrophages by antigen capture enzyme-linked immunosorbent assay. Moreover, the antiparasitic assay was performed by infecting the host cells (THP-1) in a ratio parasite/cell 10:1 (L. amazonensis) and 2:1 (T. cruzi) and then exposed to the samples. The resin was separated in vacuo by liquid chromatography because of its sticky behaviour and the chemical profiles of the obtained fractions (F1–F4) were established by dereplication based on UPLC-ESI-MS² data while semi-synthetic triterpenes were prepared from α-amyrin by oxidation reactions. Results: Fractions F1–F4 inhibited NO and TNF-α almost similarly. However, only F1, F3 and F4 showed promising antiparasitic activities while F2 was moderately active against both parasites. Hence, F1–F4 were exclusively composed of pentacyclic triterpenes bearing oleanane and ursane skeletons. Semi-synthetic compounds revealed no to moderate antiparasitic activity compared to the fractions. Conclusion: Although it will be difficult to prove the interaction resin-spirit, interesting bioactivities were found in the resin fractions.

Full text of DOI:10.1002/pca.2771

Received: 6 November 2017
DOI: 10.1002/pca.2771
Revised: 23 January 2018
Accepted: 5 April 2018
R E S E A R C H A R T I C L E
UPLC‐ESI‐QTOF‐MS2 characterisation of Cola nitida resin
fractions with inhibitory effects on NO and TNF‐α released by
LPS‐activated J774 macrophage and on Trypanosoma cruzi and
Leishmania amazonensis
Larissa Frankenberger1 Tamara D. Mora2 Carolina D. de Siqueira2
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Fabiola B. Filippin‐Monteiro2 Milene H. de Moraes3 Maique W. Biavatti1
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Mario Steindel3 Louis P. Sandjo1
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1 Department of Pharmaceutical Sciences,
Abstract
CCS, Universidade Federal de Santa Catarina,
Florianópolis, SC, Brazil
Introduction: The resin of Cola nitida is used in western Cameroon as incense for
spiritual protection and during ritual ceremonies. This plant secretion has never been
investigated although previous chemical and biological studies on other resins have
drawn many attentions.
2 Department of Clinical Analysis,
Universidade Federal de Santa Catarina,
Florianópolis, SC, Brazil
3 Department of Microbiology, Immunology
and Parasitology, Universidade Federal de
Santa Catarina, Florianópolis, SC, Brazil
Objective: The resin fractions which revealed inhibitory effect on nitric oxide (NO)
and tumour necrosis factor alpha (TNF‐α) released by lipopolysaccharide (LPS)‐
activated J774 macrophage as well as on intracellular forms of Leishmania amazonensis
and Trypanosoma cruzi amastigote were chemically characterised. Moreover, their
antiparasitic activities were compared to those of semi‐synthetic triterpenes.
Correspondence
Louis P. Sandjo, Department of Pharmaceutical
Sciences, CCS, Universidade Federal de Santa
Catarina, 88040‐900, Florianópolis, SC, Brazil.
Email: p.l.sandjo@ufsc.br
Funding information
Methodology: The anti‐inflammatory activity was evaluated by measuring the
nitrite production and the TNF‐α concentration in the supernatants of LPS‐activated
macrophages by antigen capture enzyme‐linked immunosorbent assay. Moreover,
the antiparasitic assay was performed by infecting the host cells (THP‐1) in a ratio
parasite/cell 10:1 (L. amazonensis) and 2:1 (T. cruzi) and then exposed to the samples.
The resin was separated in vacuo by liquid chromatography because of its sticky
behaviour and the chemical profiles of the obtained fractions (F1–F4) were
established by dereplication based on UPLC‐ESI‐MS2 data while semi‐synthetic
triterpenes were prepared from α‐amyrin by oxidation reactions.
CAPES (Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior); CNPq (Conselho
Nacional de Desenvolvimento Científico e
Tecnológico)
Results: Fractions F1–F4 inhibited NO and TNF‐α almost similarly. However, only
F1, F3 and F4 showed promising antiparasitic activities while F2 was moderately
active against both parasites. Hence, F1–F4 were exclusively composed of pentacyclic
triterpenes bearing oleanane and ursane skeletons. Semi‐synthetic compounds
revealed no to moderate antiparasitic activity compared to the fractions.
Conclusion: Although it will be difficult to prove the interaction resin‐spirit,
interesting bioactivities were found in the resin fractions.
KEYWORDS
antiprotozoal activity, Cola nitida resin, NO and TNF‐α inhibition, pentacyclic triterpenes, UPLC‐
ESI‐QTOF‐MS2
Phytochemical Analysis. 2018;1–13.
wileyonlinelibrary.com/journal/pca
Copyright © 2018 John Wiley & Sons, Ltd.
1

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FRANKENBERGER ET AL.
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were filtered by syringe filters (13 mm, 0.22 μm, Analítica, Brazil).
1
INTRODUCTION
NMR data were recorded in an Ultrashield 300 MHz Bruker machine.
Looking back to my childhood (L.P.S) and based on souvenirs, people
from the west region of Cameroon used and are still using dried resin
from the Cola nitida tree (Sterculiaceae) as incense for spiritual
protection and during ritual ceremonies. However, resins in the
ancient centuries had many applications such as varnishes, adhesives,
incense in churches, and for mummification.1,2 This sticky solid
played a particular role in the mummification process, due to the
polymerisation of its constituents, essentially the lipophilic that
protected body tissues from microbial attacks.3 With the evolution
of sciences, investigations revealed that resins are also full of small
antimicrobial molecules.3 These findings match the chemo‐ecological
interactions observed between resins plants producer and their
environment. So, various studies reported that resins are produced
by the plant tissues as a defensive tool to fight insect's evasion or
as a defence mechanism against pathogenic infections.4,5 Moreover,
resin chemistry unveiled this secretion as a great source of secondary
metabolites where the most identified are terpenoids, aromatics,
fatty acids and terpenoic acids6 endowed with interesting biological
activities. Compounds identified from resins proved to be anti‐
inflammatory, antitumoral, antimicrobial, and larvicidal among others
bioactivities.2,6
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2.2
Material collection and preparation of the resin
fractions
The resin (2 g) was collected from the trunk of C. nitida in Bangoua,
located at the western province of Cameroon with longitude 10°29′,
and latitude 5°12′ as geographic coordinates.
The resin (1 g) was poured onto dichloromethane/methanol
(1:1, v/v), sonicated, mixed with silica gel and dried on a rotary
evaporator. The mixture was fractionated on a silica vacuum liquid
chromatography (VLC) column using hexane, dichloromethane, ethyl
acetate, and methanol as mobile phases. Fractions F1–F4 were
obtained after solvent evaporation as follows: F1 (300 mg), F2
(300 mg), F3 (150 mg) and F4 (200 mg). A portion (1 mg) of each
fraction was dissolved in acetonitrile/methanol 2:1 (5 mL) and filtered
with a syringe filter to afford 200 μg/mL for fractions F1–F4.
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2.3
Biological assays
Cell culture
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2.3.1
The murine macrophage cell line J774 was maintained in DMEM
(Dulbecco's Modified Eagle's medium) supplemented with 10%
fetal bovine serum (FBS) containing 100 U/mL of penicillin,
100 μg/mL of streptomycin and 10 mM of 4‐(2‐hydroxyethyl)‐1‐
piperazineethanesulfonic acid (HEPES). Cells were cultured at 37°C in
an atmosphere of 5% CO2 and the medium was changed every 48 h.
The cells were cultured up to confluence 80–90% and then subcultured
after dissociation by scraping using a rubber policeman.
Cola nitida (Sterculiaceae) is a well known tree because of the
caffeine high concentration in its nuts, which are largely consumed
in sub‐Saharan localities to counteract hunger, thirst, and tiredness.7
As far as we know, the resin from C. nitida has never been investigated
neither chemically nor biologically.
Since our research is dedicated to the search of new anti‐inflam-
matory and antiparasitic entities, the resin fractions were assayed for
their nitric oxide (NO) and tumour necrosis factor alpha (TNF‐α) inhi-
bition effect as well as for their anti‐protozoal activity on intracellular
forms of L. amazonensis and T. cruzi amastigote. The obtained activity
prompted us to establish the fractions UPLC‐ESI‐MS2 chemical
profile. This hyphenated technique allows simultaneous identification
of constituents in an organic mixture. Furthermore, it also permits to
avoid routine phytochemical studies that can lead to known and
well‐investigated compounds. As performing the dereplication, it
turned out that pentacyclic triterpenes were predominant in the resin
supporting the observed anti‐inflammatory activity. Previous studies
intensively reported triterpenes as inhibitors of interleukin production.
Moreover, these metabolites can also suppress in activated
macrophages, the release of NO, TNF‐α, interleukin 1 beta (IL‐1β)
and interleukin 6 (IL‐6) which by consequence lead to the decrease
of the biosynthesis and/or the release of cytokines from target cells
that results in a less aggressive inflammatory response.8-11
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2.3.2
Cytotoxicity of the resin fractions on J774
macrophage cell line
To evaluate the cytotoxicity of the fractions, resazurin‐based cytotox-
icity assay (Alamar blue®) was performed.12 For macrophages, 3 × 105
cells/well were seeded in DMEM supplemented with 10% FBS. After
24 h, the medium was replaced with medium containing the fractions
at the concentrations 1, 2.5, 5, 10, 25, 50 and 100 μg/mL and
incubated at 37°C for 24 h. Subsequently, cells were incubated with
100 μL (0.1 mg/mL) of resazurin dye for 3 h at 37°C. The fluorescence
was measured at 1 h intervals using a microplate reader (IS55, Perkin
Elmer, Waltham, MA) at 530–590 nm. Non‐linear regression of each
fraction versus cell viability in percentage was performed in order to
obtain the toxicity concentration of 10% (concentration causing 90%
cell viability) using GraphPad Prism 5.0 (GraphPad Software 5.0,
Inc., San Diego, CA).
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2.4
In vitro anti‐inflammatory activity
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2
EXPERIMENTS
The anti‐inflammatory activity was performed with modifications of
the protocol previously described by Herath et al.13 Thus, the evalua-
tion consisted by measuring the nitrite production using an indicator
of NO synthesis in the supernatants of LPS‐activated macrophages.
A quantity of cells (2 × 105 cells/well) was seeded in a 24‐well plate
and after 24 h, the medium was changed and CC10 of each fraction
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2.1
Chemicals and materials
Acetonitrile (HPLC grade), methanol, and formic acid were purchased
from Tedia (São Paulo, Brazil). Milli‐Q system (18.2 MΩ, Millipore,
Simplipak, France) was used to produce ultrapure water. All samples

FRANKENBERGER ET AL.
3
was added in DMEM supplemented with 10% FBS. After 15 min, the
macrophages were stimulated with lipopolysaccharide (LPS) 10 μg/mL
and incubated for 24 h. Subsequently, the supernatants were collected
and the nitrite production was measured by adding 100 μL of Griess
reagent14 (1% sulfanilamide and 0.1% N‐(1‐naphthyl)ethylenediamine
in 0.5% phosphoric acid). The absorbance was determined with a
microplate reader (ELX800, BioTek, Winooski, VT) at 540 nm. Further-
more, the TNF‐α concentration was determined by antigen capture
enzyme‐linked immunosorbent assay (ELISA) (BD Bioscience, San
Jose, CA) according to the manufacturer's instructions.
were carefully washed with PBS and incubated for 16 h at 37°C
with 250 μL of chlorophenol red‐ß‐D‐galactopyranoside (CPRG)
(Sigma‐Aldrich Co.) at 100 μM and Nonidet P‐40 (NP‐40) (Amresco
Inc, Solon, OH) 0.1%. Optical density was read at 570/630 nm in an
Infinite M200 (Tecan, Grödig, Austria).17,18 The concentration of each
sample that reduced parasite viability by 50% when compared
to untreated control (IC50) was estimated by non‐linear regression of
concentration‐response curves. Amphotericin B (Sigma‐Aldrich) and
benznidazole (Sigma‐Aldrich) were used as positive control for
antileishmanial and antitrypanosomal activities, respectively, and
DMSO 1% as negative control. The concentration capable to inhibit
50% (IC50) of the parasites and the proliferation of THP‐1 cells
(CC50) were used to express the antiparasitic activity and cytotoxicity,
respectively. Selectivity index (SI) of each sample was determined by
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2.5
Statistical analysis
Results were shown as the mean standard deviation (SD) of three
experiments. One‐way analysis of variance followed by Dunnett's
multiple comparisons test was performed, and the level of significance
was set at p ˂ 0.05 (GraphPad Software 5.0, Inc., San Diego, CA).
the ratio of CC50/IC50
.
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2.7
Cell viability assay (MTT)
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2.6
In vitro antitrypanosomal and antileishmanial
THP‐1 cells were grown and cultivated in 96 well plates (4.0 × 104
cells/well) and treated with the compounds serially diluted in concen-
trations ranging from 2 μg/mL to 500 μg/mL and incubated for 72 h at
37°C, 5% CO2. The plates were centrifuged (3.700 ×g/7 min), the
supernatant was removed, and the cells were resuspended in 50 μL
of a solution of MTT (Amresco) at 3 mg/mL in saline buffer and
incubated for 4 h at 37°C, 5% CO2 and centrifuged (3.700 ×g/7 min)
and the formazan salt was solubilised in 100 μL DMSO. Optical
density was determined at 540 nm in aTecan® Infinite M200 spectro-
photometer. DMSO 1% (v/v) and DMSO 50% (v/v) were the negative
and positive controls, respectively. The IC50 values were calculated by
a non‐linear regression using the GraphPad Prism program.15
activities
Human macrophage cell line THP‐1 (ATCC TIB202) was grown in
RPMI‐1640 without phenol red (Sigma‐Aldrich, St Louis, MO), supple-
mented with 10% FBS (Life Technologies, Carlsbad, CA), 12.5 mM
HEPES, penicillin (100 U/mL), streptomycin (100 μg/mL) and
Glutamax (2 mM), at 37°C in a 5% CO2 incubator. Leishmania
amazonensis MHOM/BR/77/LTB0016 promastigotes, expressing β‐
galactosidase, were grown at 26°C in Schneider's insect medium
(Sigma Chemical Co., St Louis, MO) supplemented with 5% heat
inactivated FBS and 2% of human urine.
THP‐1 cells (4.0 × 104 per well) were cultivated in 96‐well plates
in RPMI‐1640 medium and incubated with 100 ng/mL of phorbol 12‐
myristate 13‐acetate (PMA) (Sigma Chemical Co.) for 72 h at 37°C in a
5% CO2, to allow THP‐1 cells differentiation into non‐dividing macro-
phages.15 Four days‐old culture promastigotes (4.0 × 106 parasites/
mL) were washed with phosphate buffered saline (PBS), pH 7.4, and
incubated in RPMI‐1640 supplemented with 10% heat‐inactivated
human B+ serum for 1 h at 34°C for parasite opsonisation. THP‐1 cells
were incubated with a parasite/cell ratio of 10:1 for 4 h at 34°C and
5% CO2. After this period non‐adherent parasite were removed by
one wash with PBS and infected cells were incubated with 180 μL
of full supplemented RPMI‐1640 medium for another 24 h to allow
the transformation of promastigotes into intracellular amastigotes.
The β‐galactosidase T. cruzi, Tulahuén strain was provided by the
Laboratory of Cellular and Molecular Parasitology, Centro de
Pesquisas René Rachou, FIOCRUZ, Belo Horizonte. Culture‐derived
typomastigotes raised from infected L929 cell line were used to infect
differentiated THP‐1 (4.0 × 104 cells/well) in 96‐well microplates in a
parasite/cell ratio of 2:1 and incubated overnight at 37°C in a 5% CO2
atmosphere. The medium containing non‐internalised parasites was
removed and replaced with 180 μL of fresh medium.16
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2.8
UPLC–MS analysis
Chromatographic separations were carried out on an Acquity UPLC
system class H (Waters, Milford, MA) equipped with a photodiode
array (PDA) detector, sample manager and a quaternary solvent
manager as well as a BEH C18 column: 50 mm, 1.0 mm, particle size
1.7 μm (Waters). Temperatures of the column and the samples tray
were 40°C and 20°C, respectively. A volume of 3 μL was injected in
a gradient condition at flow rate of 0.3 mL/min: 90% A (water/formic
acid, 99.9/0.1 (v/v)) and 10% B (acetonitrile); 1–2 min, 90% of A; 2–
10 min, 55% of A; 10–15 min, 10% of A; 15–20 min, 90% of A.
Mass data were recorded on a Xevo G2‐S QTof (Waters) with an
electrospray ionisation (ESI) probe operating in positive and negative
ionisation modes. Nebuliser gas: nitrogen; cone gas flow 60 L/h;
desolvation gas flow 900 L/h; sampling cone 40 V; source offset
80 V; collision gas: argon; Lockspray reference sample was Leucine
encephalin with reference masses at m/z 556.2771 (ESI+) and m/z
554.2615 (ESI−). Temperatures of desolvation and of the ionisation
source were 300°C and 120°C, respectively, while the capillary
voltage was 3 kV. Collision energy was 30 eV. Data were acquired in
a range of 100–1500 Da, at a scan time of 1.0 s during 20 min, and
were processed with MassLynx V4.1 (Waters).
Samples were solubilised in dimethyl sulfoxide (DMSO) and
serially diluted (500 μg/mL to 2 μg/mL). Infected cells monolayer
was treated by addition of 20 μL of each sample, in triplicate, followed
by incubation for 48 h at 34°C or 37°C, 5% CO2. After treatment, cells

4
FRANKENBERGER ET AL.
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Thereafter, NO and TNF‐α were then measured in the supernatants
of macrophages. F1–F4 showed strong ability to inhibit NO and
TNF‐α release from activated macrophages (Figure 1). There was no
significant difference among fractions, neither for NO nor TNF‐α
analysis, however, all fractions were able to significantly inhibit
these inflammatory mediators in LPS‐induced macrophages (p < 0.01
and p < 0.001).
2.9
Isolation of α‐amyrin and chemical modification
Recrystallisation procedure using different solvents was performed
with F1–F4. The mixture of α‐ and β‐amyrin (60 mg) was obtained
from F4 while the others did not provide any precipitation. A chro-
matographic column on silica gel using hexane and dichloromethane
in gradient condition provided α‐amyrin (48 mg) and the mixture of
both metabolites (9 mg). Their structure was confirmed by NMR data
in conjunction with those reported in the literature. α‐Amyrin (15 mg)
was subjected to different oxidation reactions using three different
conditions: (1) pyridinium chlorochromate (PCC, 2 equivalents) and
silica gel (5 mg) in dichloromethane at room temperature for 1 h; (2).
KMnO4/KClO3 (2 equivalents each) in dichloromethane/acetone/
water (5:5:1, 11 mL), at room temperature for 20 h; (3) potassium
permanganate (KMnO4)/potassium chlorate (KClO3)/PCC (2 equiva-
lents each) in dichloromethane/acetone/water (5:5:1, 11 mL), at
room temperature for 20 h. Three triterpenes related to α‐amyrin
namely α‐amyrone, 11‐oxo‐α‐amyrin, and 11‐oxo‐α‐amyrone were
obtained after VLC purification in 70, 10, and 15% yield, respectively.
The purification was performed with hexane and ethyl acetate in a
gradient condition. Their structure was established by NMR data
(Supporting Information Figures S1–S4) in comparison to those
reported in the literature.
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3.1.2
Antiparasitic activity
Fractions F1–F4 were further assayed on intracellular amastigotes of
T. cruzi and L. amazonensis (Table 2). THP‐1 cells were used as a host
for the parasites. While F1, F3 and F4 displayed significant
antitrypanosomal activity with IC50 values of 3.94, 3.58 and
3.82 μg/mL, respectively, with good selectivity towards THP‐1 cells,
F2 showed a weak effect (24.22 μg/mL) on the same parasite with a
very low selectivity.
In addition, F1, F3 and F4 showed the most promising
antileishmanial effect with IC50 at 8.08, 7.15 and 8.23 μg/mL, respec-
tively, while F2 was weakly active (27.73 μg/mL). However, F1 and F4
displayed similar selectivity on the host cells (7.30 and 7.69, respectively)
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3
RESULTS
The sticky behaviour of C. nitida resin prompted us to perform VLC
using hexane (F1), dichloromethane (F2), ethyl acetate (F3) and
methanol (F4). Since no previous studies were found on this organic
material, the fractions were further investigated for their anti‐inflam-
matory as NO and TNF‐α inhibitors. In addition, F1–F4 were also
investigated for their antiparasitic activities against the intracellular
amastigotes of L. amazonensis and T. cruzi.
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3.1
Biological activity
|
3.1.1
Anti‐inflammatory activity
In order to evaluate the anti‐inflammatory activity of fractions F1–F4,
CC10 of each fraction was determined in order to maintain 90% of cell
viability for the anti‐inflammatory activity. As depicted in Table 1, the
macrophage J774 was less sensitive to F1 (12.63 μg/mL) compared to
F2 (8.1 μg/mL) and F4 (8.8 μg/mL), which in turn were less cytotoxic
than F3 (4.7 μg/mL).
The cells were exposed to each fraction (at a concentration
approximating the CC10 value) and then activated with LPS for 24 h.
TABLE 1 Cytotoxicity of F1–F4 on murine J774 macrophages cell line
CC10 (μg/mL)a
FIGURE 1 Cytokine release from activated macrophages. (A) •NO
determination and (B) TNF‐α in the supernatants of macrophages
J774 with or without lipopolysaccharide (LPS) (10 μg/mL) in the
presence of fractions at the concentrations 12 μg/mL (F1), 8 μg/mL
(F2), 5 μg/mL (F3), and 9 μg/mL (F4). Data expressed as
mean standard deviation of three experiments. One‐way analysis of
variance followed by Dunnett's multiple comparisons test was used
for the statistical significance. **p < 0.01, ***p < 0.001 compared with
control group LPS+
Fractions
Macrophages
F1
F2
F3
F4
12.63 6.60
8.1 3.39
4.7 1.83
8.8 1.53
aResazurin viability assay was performed in order to obtain concentrations
to maintain 90% of cell viability.

FRANKENBERGER ET AL.
5
TABLE 2 Inhibition effect of the resin fractions against Trypanosoma cruzi and Leishmania amazonensis and their selectivity index (SI) towards
THP‐1 cells
T. cruzi
L. amazonensis
THP‐1
Fractions
IC50 (μg/mL)
SI
CC50 (μg/mL)
IC50 (μg/mL)
SI
F1
3.94 0.43
24.22 1.08
3.58 0.31
3.82 0.33
NA
14.97
2.93
24.55
16.57
—
58.99 2.07
70.99 2.55
87.89 6.27
63.32 6.18
—
8.08 1.64
27.73 3.41
7.15 1.60
8.23 1.20
NA
7.30
2.56
12.29
7.69
—
F2
F3
F4
α‐Amyrin
α‐Amyrone
11‐Oxo‐α‐amyrin
11‐Oxo‐α‐amyrone
Benznidazole
Amphotericin B
NA
—
—
NA
—
8.67 0.55
16.76 0.76
2.65 0.08
—
3.52
8.40
>49.06
—
30.53 0.90
140.8 5.56
>130
11.6 2.68
19.43 2.28
—
2.63
7.24
—
25.74 0.91
0.13 0.02
198
Note: NA, not available.
while F3 and F2 showed the highest (12.29) and the lowest (2.56)
selectivity, respectively.
technique UPLC‐ESI‐MS and MS/MS. These fractions were most
sensitive to the positive ionisation mode and the base peak ionisation
parameter was employed for the acquisition of the chromatogram
(Figure 3).
An attempt to isolate single pure compounds in order to compare
their bioactivity with the fractions led to α‐amyrin. The metabolite was
further oxidised by using three different reaction conditions (Figure 2)
affording α‐amyrone, 11‐oxo‐α‐amyrone, and 11‐oxo‐α‐amyrin. These
four triterpenes were evaluated against both parasites among them,
only 11‐oxo‐α‐amyrone, and 11‐oxo‐α‐amyrin revealed, respectively,
IC50 at 16.76 and 8.67 μg/mL against T. cruzi. Moreover, both also
showed effects on L. amazonensis with IC50 at 19.42 and 11.6 μg/
mL, respectively.
LC–MS analysis of F3 showed the presence of 17 metabolites,
which were further subjected to collision‐induced dissociation (CID)
by exposing each metabolite to accelerated argon atoms.
Fraction F3 at 4.53 min exhibited the base peak ion m/z 455.3518
produced by the thermal induced loss of H3CCO2H (60 Da) from m/z
515.3730. The molecular formula [C32H50O5 + H]+ was then deduced
for this metabolite which gave in its tandem mass (Figure S5) m/z
455.3518 [M‐AcOH (60 Da) + H]+ which in turn eliminated H2O
(18 Da) and HCO2H (46 Da) to produce m/z 437.3442 and m/z
409.3488, respectively. Assuming that the ionisation occurred on
the OH group attached to ring E, the decalin ion formed from the
retro‐Diels–Alder (RDA) opening of ring C could either lead to m/z
203.1775 or to m/z 201.1653 after losing [H2O (18 Da); CO2
|
3.2
Chemistry
|
3.2.1
LC–MS analysis
Since F1–F4 revealed interesting biological activities, their chemical
constituents (Table 3) were characterised by using the hyphenated
FIGURE 2 Preparation of derivatives related
to α‐amyrin

6
FRANKENBERGER ET AL.
TABLE 3 Chemical profile of the Cola nitida resin fractions (F1–F4)
Fractions
Experimental
tR (Min) mass (m/z)
Theorical
mass (m/z)
Molecular
formular
Fragments
Structure proposal
F1 F2 F3 F4
4.53
455.3519/
515.3730
455.3525[M‐H3CCO2H + H]+ C30H46O3/
437.3442, 425.3455,
419.3270, 409.3488,
343.2635, 313.2548,
311.2383, 295.2458,
283.2469, 257.1937,
251.1651, 245.1920,
215.1821, 203.1775,
201.1653, 189.1643,
187.1501, 177.1674,
175.1507, 173.1347,
163.1489, 161.1333,
159.1199, 149.1355,
147.1169, 135.1182,
133.1028, 121.1030,
109.1031
Rubiprasin C or 3‐i‐acetyl
pomolic acid
p
p
/515.3736 [M + H]+
C32H50O5
5.67
6.33
425.3792
457.3659
425.3783 [M + H]+
C30H48
O
407.3693, 271.2441,
245.2273, 221.1935,
217.1968, 203.1804,
191.1808, 161.1359,
147.1194, 135.1182,
133.1028, 109.1031
Olean‐12(13),15(16)‐dien‐
3β‐ol
p
p
p
457.3683 [M + H]+
C30H48O3
439.3566, 421.3522,
411.3664, 393.3505,
343.2673, 313.2548,
295.2423, 259.2081,
247.2072, 241.1959,
229.1971, 215.1791,
187.1501, 173.1347,
159.1173, 135.1182,
121.1007, 109.1031
Oleanolic acid
6.51
6.66
425.3750
909.6996
425.3783 [M + H]+
C30H48
O
407.3651, 311.2744,
271.2441, 217.1938,
203.1804, 191.1808,
175.1480, 147.1169,
133.1028, 123.1165,
109.1031
3‐Hydroxy‐urs‐12,15‐
diene
p
p
p
p
p
p
p
909.6972 [2 M + H]+
C30H46O3
437.3442, 419.3312,
409.3447, 369.2801,
313.2548, 311.2383,
295.2458, 257.1937,
245.1920, 201.1653,
203.1833, 189.1643,
187.1501, 159.1501,
133.1028, 109.1031
3β‐Hydroxyurs‐12,18‐
dien‐28‐oic acid or
micromeric acid or 3β‐
hydroxyurs‐12,19(29)‐
dien‐28‐oic acid
6.84
6.92
457.3659
443.3867
457.3682 [M + H]+
C30H48O3
439.3609, 421.3522,
393.3505, 343.2635,
313.2584, 295.2423,
269.2303, 255.2132,
229.1971, 201.1653,
187.1501, 159.1173,
109.1031
Ursolic acid
Erythrodiol
p
p
p
p
443.3889 [M + H]+
C30H50O2
425.3792, 407.3651,
315.2705, 297.2593,
259.2048, 247.2072,
243.2106, 229.1971,
215.1821, 187.1473
175.1507, 161.1333,
149.1355, 135.1182,
123.1182, 109.1031
7.14
7.39
427.3936
441.3737
427.3940 [M + H]+
C30H50
O
409.3820, 299.2760,
271.2441, 229.1971,
217.1968, 191.1808,
189.1643, 163.1489,
149.1330, 137.1321,
135.1182, 123.1188,
121.1030, 109.1009
β‐Amyrin
p
p
p
441.3733 [M + H]+
C30H48O2
Dehydrouvaol
(Continues)

FRANKENBERGER ET AL.
7
TABLE 3 (Continued)
Fractions
Experimental
tR (Min) mass (m/z)
Theorical
mass (m/z)
Molecular
formular
Fragments
Structure proposal
F1 F2 F3 F4
423.3654, 405.3533,
315.2705, 297.2593,
287.2397, 269.2303,
259.2048, 247.2072,
241.1991, 229.1971,
215.1821, 201.1682,
189.1643, 175.1507,
161.1333, 149.1355,
135.1182, 109.1031
7.47
7.65
427.3936
441.3737
427.3940 [M + H]+
C30H50
O
409.3820, 299.2760,
271.2441, 229.1971,
217.1968, 191.1808,
189.1643, 163.1489,
149.1330, 137.1321,
123.1188, 121.1030,
109.1009
α‐Amyrin
p
441.3733 [M + H]+
C30H48O2
423.3654, 405.3533,
315.2705, 297.2593,
259.2048, 247.2072,
241.1991, 229.1971,
215.1821, 189.1671,
177.1674, 161.1359,
149.1355, 135.1182,
121.1030, 109.1031
Related to dehydrouvaol
p
p
p
7.72
443.3867
443.3889 [M + H]+
C30H50O2
425.3792, 407.3651,
315.2705, 297.2593,
259.2048, 247.2072,
243.2106, 229.1971,
215.1821, 187.1473
175.1507, 161.1333,
149.1355, 123.1182,
109.1031
Uvaol
p
7.94
8.02
485.3981
499.3794
485.3995 [M + H]+
C32H52O3
425.3750, 407.3693,
297.2628, 235.2115,
217.1968, 205.1968,
191.1808, 177.1647,
163.1489, 149.1330,
133.1028, 121.1030,
109.1031
3β‐Acetoxy‐28‐hydroxy‐
urs‐12‐ene or 3‐O‐
acetyl‐erythrodiol
p
p
499.3787 [M + H]+
C32H50O4
439.3566, 355.2642,
343,2635, 297.2593,
241.1959, 229.1971,
217.1968, 203.1804,
201.1653, 191.1808,
189.1643, 187.1501,
135.1182, 121.1007,
109.1009
Oleanolic acid 3‐acetate
p
p
p
p
p
p
8.27
467.3878
467.3889 [M + H]+
C32H50O2
407.3651, 323.2731,
311.2744, 297.2593,
283.2434, 271.2441,
269.2303 217.1968,
203.1804, 191.1808,
189.1643, 161.1333,
147.1169, 133.1028,
109.1009
Ursa‐12,20(30)‐dien‐3α‐yl
acetate or urs‐12,19(29)‐
dien‐3β‐yl acetate or
p
gigantursenyl acetate A
8.38
499.3794
499.3787 [M + H]+
C32H50O4
439.3566, 421.3480,
405.3533, 393.3505,
313.2584, 297.2593,
295.2423, 247.2072,
241.1959, 229.1971,
203.1804, 191.1808,
189.1643, 161.1359,
147.1169, 135.1182,
121.1007, 109.1009
3β‐O‐Acetylursolic acid
8.57
439.3609
439.3576 [M + H]+
C30H46O2
421.3480, 403.3424,
313.2511, 295.2423,
287.2397, 269.2303,
—
p
(Continues)

8
FRANKENBERGER ET AL.
TABLE 3 (Continued)
Fractions
Experimental
tR (Min) mass (m/z)
Theorical
mass (m/z)
Molecular
formular
Fragments
Structure proposal
F1 F2 F3 F4
241.1991, 227.1789,
217.1998, 213.1679,
203.1804, 201.1653,
187.1501, 173.1347,
161.1333, 149.1355,
135.1182, 127.1127,
123.1188, 109.1009
8.68
8.93
9.48
12.60
423.3612
441.3737
423.3612
409.3820
423.3627 [M + H]+
441.3733 [M + H]+
423.3627 [M + H]+
409.3820 [M + H]+
C30H46
O
405.3533, 297.2558,
269.2303, 255.2132,
241.1959, 229.1971,
201.1653, 189.1643,
187.1501, 149.1355,
135.1182, 121.1030,
109.1031
Olean‐9(11),12‐dien‐3‐one
Related to dehydrouvaol
Ursa‐9(11),12‐dien‐3‐one
Olean‐11,13(18)‐diene
p
p
p
p
C30H48O2
423.3612, 405.3533,
297.2558, 269.2303,
255.2132, 241.1959,
229.1971, 201.1653,
189.1643, 187.1501,
149.1355, 135.1182,
121.1030, 109.1031
p
p
C30H46O
405.3533, 297.2558,
269.2303, 255.2132,
241.1959, 229.1971,
201.1653, 189.1643,
187.1501, 149.1355,
135.1182, 121.1030,
109.1031
C30H48
299.2725, 271.2441,
257.2265, 243.2138,
231.2140, 217.1968,
205.1939, 203.1804,
191.1808, 163.1489,
149.1330, 135.1182,
123.1188, 109.1009
p
p
13.19
13.78
409.3820
409.3820
409.3820 [M + H]+
409.3820 [M + H]+
C30H48
C30H48
‐
Related to olean‐11,13(18)‐
diene
p
p
p
p
313.2910, 285.2605,
259.2443, 257.2298,
231.2109, 203.1833,
177.1647, 149.1330,
137.1345, 123.1188,
109.1031
Related to olean‐11,13(18)‐
diene
p
p
(44 Da)] or [H2O (18 Da); HCO2H (46 Da)], respectively. A careful
search in the literature of the molecular formula aforementioned led
to rubiprasin C or 3‐O‐acetyl pomolic acid structures. These com-
pounds are two metabolites differing only by the position of the
Me‐29, which could be located either at C‐19 (ursane) or C‐20
(oleanane).19 However, the daughter ions m/z 313.2548, and m/z
311.2383 (Figure 4) permitted to assign 3‐O‐acetyl pomolic acid as
structure.
The ion m/z 425.3792 [C30H48O + H]+ (5.67 min) gave a daughter
ion m/z 407.3693 after losing a molecule of H2O (18 Da). This ion fur-
ther produced the fragment m/z 191.1808 by RDA ring C opening.
Application of the charge‐driven concerted reaction (R1) concept on
m/z 407.3693 led to an intermediate with opened rings A–C. This lat-
ter underwent a [1,5]‐elimination at the position indicated in Figure S5
to afford m/z 203.1804 and m/z 271.2441; More fragments illustrated
in Figure S5 allowed the assignment of m/z 425.3792 structure as
anhydrosophoradiol. Similar fragmentation pattern was observed for
the metabolite detected at 6.51 min with the same mass value m/z
425.3750 [C30H48O + H]+. The diagnostic of its MS data suggested
this compound to be closely related to the one identified earlier and
3‐hydroxy‐urs‐12,15‐diene was proposed as the presumable structure.
The ion m/z 909.6996 detected at 6.66 min corresponded to a
cluster species with the molecular formula [2C30H46O3 + H]+. It afforded
in its MS2 spectrum, ions at m/z 437.3442 and m/z 419.3312 after losing
H2O (18 Da) and 2H2O (36 Da), respectively. This parent ion further
underwent a decarboxylation (HCO2H, 46 Da) to give m/z 409.3447;
three ion species (Figure S6) were found with the mass m/z 437.3442
among them, one with the positive charge on the carbonyl carbon. As
depicted in Figure S6, this latter underwent a ring E opening with the
elimination of a hydrocarbon portion to produce m/z 313.2526 which
in turn lost H2O (18 Da) to afford m/z 295.2558; Moreover, if the
ionisation occurred in ring E, the RDA opening of ring C gave a decalin
moiety composed of rings D and E. The ions m/z 203.1833 and m/z
201.1653 were formed out of this decalin after the decarboxylation

FRANKENBERGER ET AL.
9
FIGURE 3 LC‐ESI‐MS of F1–F4 in the positive mode ionisation
CO2 (44 Da) and HCO2H (46 Da), respectively. The aforementioned
combination of the earlier information led to the structure of
erythrodiol.21 A compound was also found in the LC–MS analysis of
F4 with almost the same MS2 behaviour. The structure of uvaol, an
ursane type was proposed and no evidence in its fragmentation
pattern permitted to distinguish its structure from erythrodiol. How-
ever, the daughter ions obtained matched those previously reported.22
The precursor m/z 427.3936 [C30H50O + H]+ (7.14 min) furnished
in its MS2 spectrum a fragment ion m/z 409.3820 resulting from the
loss of H2O (18 Da). This fragment further underwent a RDA opening
of ring C to produce m/z 191.1808. It also went through a charge‐
driven concerted reaction leading to the opening of rings A, B, and
C; the intermediate ion lost different hydrocarbon portions as
depicted in Figure S9 to afford m/z 189.1643 and m/z 271.2441; this
latter in turn gave m/z 229.1971 after eliminating propene. Another
fragment was found at m/z 217.1968 when the ionisation occurred
on the olefinic bond of m/z 427.3936 forming a tertiary carbocation.
This ion position triggered the opening of ring C and produced m/z
217.1968 after eliminating the fused rings D and E as well as H2O.
Since none of these fragments allowed to univocally identify the
metabolite at m/z 427.3936, two structures namely α‐ and β‐amyrin
were found in the literature for this metabolite. By comparing this
data with those previously reported,21 m/z 427.3936 was assigned
as β‐amyrin. Another metabolite with the same elemental composition
was detected in F4 at 7.47 min. Its tandem mass analysis revealed
fragmentation pattern similar to m/z 427.3936 [C30H50O + H]+
(7.14 min) found in F3 and the structure of α‐amyrin was proposed.
The tandem mass analysis of m/z 441.3737 [C30H48O2 + H]+
(tR 7.39 min) produced m/z 423.3654 and m/z 405.3533 from the
information conducted to
a structure related to 3β‐hydroxyurs‐
12,18‐dien‐28‐oic acid or micromeric acid or 3β‐hydroxyurs‐
12,19(29)‐dien‐28‐oic acid. These three metabolites differ from each
other by the position of the double bond which could not be well
established from the ions formed.
The metabolite at m/z 457.3659 [C30H48O3 + H]+ [retention time
(tR) 6.84 min] exhibited in its MS2 spectrum fragment ions m/z
439.3609, m/z 421.3522 and m/z 393.3505 formed after it lost H2O,
2H2O and (H2O, HCO2H), respectively. The fragmentation proposal led
to two ions with the mass value m/z 439.3609 among them, one bearing
the charge on the carbonyl carbon. Because that position of the charge,
its migration triggered the opening of rings D and E to afford m/z
313.2584 which in turn lost H2O to give m/z 295.2423 (Figure S7); from
the earlier mentioned data, the structure of oleanolic acid was assigned.
A
second compound with the same mass value m/z 457.3659
[C30H48O3 + H]+ was detected (tR 6.33 min) in F4 and the structure of
ursolic acid was attributed. However, their fragmentation pattern
proposal displayed features similar to those previously reported.20,21
The compound m/z 443.3867 [C30H50O2 + H]+ (6.92 min)
displayed in its MS2 spectrum, peaks corresponding to the loss of
H2O and 2H2O at m/z 425.3792 and m/z 407.3651, respectively. Ring
C in this latter ion opened in a RDA manner to afford an ion fragment,
which in turn produced m/z 175.1505 after losing acetylene. The ion
m/z 425.3792 containing the primary carbocation afforded m/z
315.2705 after the opening of rings D and E and the loss of a hydro-
carbon moiety (Figure S8). Furthermore, m/z 297.2593 was lighter of
18 Da from m/z 315.2705 presumably due to the loss of H2O. The

10
FRANKENBERGER ET AL.
FIGURE 4 Proposal of m/z 515.3730 fragmentation pattern (4.53 min)
elimination of H2O (18 Da) and 2H2O (36 Da), respectively. Two ions
were found with the mass value m/z 423.3654 among them, the one
bearing a primary carbocation gave m/z 215.1821 through a RDA
pathway on ring C; meanwhile, the charge migration via R1 in the
same primary carbocation combined rings D and E. Furthermore, the
loss of a hydrocarbon residue as shown in Figure S10 led to m/z
315.2705 which in turn afforded lighter fragments at m/z 297.2593,
m/z 287.2397 and m/z 269.2303 after losing, respectively, H2O,
C2H4 (28 Da) and (H2O and C2H4). The second ion m/z 423.3654
bearing a secondary carbocation could also undergo R1 through a
supposed retro‐biosynthesis pathway over three isoprenyl groups.
The loss of hydrocarbon portions involved in rings A and B and MeOH
(32 Da) via charge‐remote elimination reaction (R2), gave m/z
287.2397 and m/z 241.1991; Moreover, when the ionisation occurred
on the olefinic bond of m/z 441.3737, ring C opened through R1 and
the intermediate formed lost MeOH (32 Da) to afford m/z 201.1682.

FRANKENBERGER ET AL.
11
FIGURE 5 Correlation of F1–F4 based on their chemical composition [Colour figure can be viewed at wileyonlinelibrary.com]
This latter in turn eliminated C2H2 (26 Da) yielding m/z 175.1507. The
combination of the foregoing data allowed the identification of m/z
441.3737 as dehydrouvaol. Its isomers were also detected at
7.65 min and 8.93 with the same fragmentation pattern.
405.3533 was produced after the precursor lost H2O (18 Da). As
showed in Figure S13, rings A–C in this fragment (m/z 405.3533)
further opened due to the delocalisation of the positive charge and
the loss of different hydrocarbon moieties conducted to the formation
of m/z 255.2132, m/z 297.2558, m/z 269.2303, and m/z 229.1971
(Table 3); additional fragmentation displayed in Figure S13 enabled
the identification of these metabolites as olean‐9 (11),12‐dien‐3‐one
and ursa‐9 (11),12‐dien‐3‐one.
The peak at 8.02 min flanked with the mass value m/z 499.3794
[C32H50O4 + H]+ gave ions at m/z 439.3599, m/z 421.3480, and m/z
393.3546 representing fragments formed from the elimination of
H3CCO2H (60 Da), [H3CCO2H and H2O (78 Da)], and [H3CCO2H
and HCO2H] (106 Da), respectively (Figure S11). The precursor m/z
499.3794 further lost H2O and the delocalisation of the positive
charge in the ion product followed by the loss of the hydrocarbon
moiety from rings D and E afforded m/z 343.2673; this ion, in turn,
eliminated H3CCO2H (60 Da) and the resulting ion underwent an
RDA opening of ring A to afford m/z 201.1653. The ion daughter m/
z 439.3599 showed also a charge delocalisation (R1) followed by the
opening of rings A–C in a retro‐biosynthetic manner. Moreover, the
intermediate formed, lost C6H12 (84 Da), and [C11H20 and HCO2H]
(198 Da) to furnish m/z 355.2642 and m/z 241.1959, respectively.
The foregoing data in conjunction with additional ions illustrated in
Figure S11 led to the structure of oleanolic acid 3‐acetate. Another
metabolite was detected at 8.38 min with the same mass value, which
gave almost the similar fragmentation pattern and 3β‐O‐acetylursolic
acid was assigned as its structure.
Two last metabolites were detected at 12.6 min and 13.78 min
with identical mass values m/z 409.3820, which corresponded to
[C30H48 + H]+. Based on fragmentation pattern shown in Figure S14,
the earlier detected metabolite was identified as olean‐11,13(18)‐
diene and the second on as its related derivative.
LC–MS analysis of fraction F2 displayed a unique metabolite
difference from those found in F3. So, the precursor m/z 485.3981
[C32H52O3 + H]+ furnished m/z 425.3792 and 407.3651 after losing
H3CCO2H (60 Da) and [H3CCO2H and H2O (78 Da)], respectively. A
RDA rearrangement occurred in ring C of m/z 425.3981 to afford m/z
191.1808; m/z 297.2593 was formed from m/z 407.3651 which
underwent a charge migration and a loss of a hydrocarbon moiety.
The precursor m/z 485.3981 produced through a RDA rearrangement
in ring C the ion m/z 235.2115 which in turn lost H2O (18 Da) and
[H2O and C2H2 (46 Da)] to give m/z 217.1968 and 191.1808,
respectively. Assuming that the ionisation occurred on the olefinic car-
bons, the first carbocation in C‐12 could trigger a charge delocalisation
leading to the opening of ring C. The obtained intermediate lost CH3OH
(32 Da) to afford m/z 205.1968; this latter in turn eliminated C2H4
(28 Da) to give m/z 177.1647. Moreover, the carbocation in C‐13 could
also produce m/z 217.1968 as depicted in Figure S15. From the data
outlined earlier, the structure was assigned as 3β‐acetoxy‐28‐
hydroxy‐urs‐12‐ene or 3‐O‐acetyl‐erythrodiol.
The ion m/z 467.3878 [C32H50O2 + H]+ was the mass value of the
metabolite detected at 8.31 min which provided on its tandem mass
spectrum the fragment m/z 407.3651 after losing H3CCO2H (60 Da).
This ion daughter produced in turn m/z 191.1808 after a RDA opening
of ring C. Rings A–C opened while the charge delocalisation occurred
and the intermediate formed lost two isoprenyl units (C10H16
,
136 Da) to afford m/z 271.2441; m/z 203.1804 resulted from m/z
271.2441 after another isoprenyl (68 Da) unit loss. On the basis, of
the information shown in Figure S12, the structure was established as
ursa‐12,20(30)‐dien‐3α‐yl acetate or urs‐12,19(29)‐dien‐3β‐yl acetate.
Two ions were obtained at 8.68 min and 9.48 min with similar
mass values m/z 423.3612 corresponding to [C30H46O + H]+. Both
precursors afforded similar MS2 fragments. The ion daughter m/z
LC–MS of F1 revealed metabolites already identified in F3 while
the F4 analysis showed exclusive compounds such as oleanolic acid,
α‐amyrin, uvaol and an unidentified metabolite (Table 3). Their
structures were established using the fragmentation pattern compared
to the data reported in the literature.22

12
FRANKENBERGER ET AL.
|
Except for F2, the other fractions showed antiprotozoal activity
with moderate toxicity on the host THP‐1 cells. The predominance of
the compound m/z 467.3878 in F2 identified as ursa‐12,20(30)‐dien‐
3α‐yl acetate or urs‐12,19(29)‐dien‐3β‐yl acetate or gigantursenyl
acetate A presumably affected theTHP‐1 cells viability.
4
DISCUSSION
|
4.1
Anti‐inflammatory study
Figure
5 showed that F3 contains three components including
dehydrouvaol, olean‐9 (11),12‐dien‐3‐one and ursa‐9 (11),12‐dien‐3‐
one which were not detected in F1, F2, and F4. These metabolites might
be responsible for its strong cytotoxicity on J774. Moreover, LC–MS
analysis of F1 revealed the presence of fewer constituent than the
others and its metabolites were also detected in other fractions. Thus,
its lowest cytotoxicity suggested that a part of F2 and F4 compounds
might strongly affect the macrophage viability.
In general, the antiparasitic activity of pentacyclic triterpenes and
enriched pentacyclic triterpenes extracts is well documented. So,
oleanane and ursane derived secondary metabolites previously
revealed moderate to significant effect against both parasites.27
However, except for α‐amyrin and α‐amyrone, the dioxygenated
derivatives were active against the parasites and their potencies were
lower than those obtained for the fractions. This suggested
a
Despite their different cytotoxic effect fractions F1‐F4 almost
equally showed strong inhibition of NO and TNF‐α regardless their dif-
ferent composition. The identified constituents are mainly pentacyclic
triterpenes related to α‐ and β‐amyrin. Oleanolic acid as well as its
derivatives were previously reported to inhibit NO production by
LPS‐activated RAW 264.7 and J774A.1 cells.23 More importantly,
oleanolic acid and its 3‐oxo‐ and 3‐oxo‐28‐methyl ester derivatives
previously revealed almost the same inhibitory effect.23 This suggested
that structural differences such as oxidised functions or an increase of
the double bond equivalent observed within the constituents do not
highly change the anti‐inflammatory activity from a fraction to another.
The prepared triterpenes were not enough to be evaluated for their
anti‐inflammatory activity and compared their effects to those of the
fractions. However, previous studies on anti‐inflammatory effects of
α‐amyrin, α‐amyrone, 11‐oxo‐α‐amyrone, and 11‐oxo‐α‐amyrin
reported the ability of these metabolites to reduce the level of NO
and TNF‐α production.24-26
possible synergistic action of the constituents in the most active
fractions or eventually the presence of a principle active.
The resin collected from C. nitida was separated into four
fractions (F1–F4) because of its sticky behaviour. The obtained
fractions were evaluated for their anti‐inflammatory activity, from
which a similar potential was observed for all. It was noted that CO,
OH and olefinic functions distributed on a pentacyclic triterpene
skeleton do no create a real difference among the anti‐inflammatory
potency of the fractions. On the basis of the obtained results, the
C. nitida resin might be used to develop an anti‐inflammatory
phytomedicine. Moreover, the fractions were also evaluated for their
antiparasitic effect. Fractions F1, F3, and F4 revealed interesting anti-
parasitic effect on intracellular L. amazonensis and T. cruzi amastigotes.
The fractions antiparasitic activities were better than those of the
hemisynthetic triterpenes (prepared from three different oxidation
reactions of α‐amyrin) and α‐amyrin. This later result suggested a syn-
ergistic contribution of the fraction constituents. It also suggested that
a mixture of oleanane and ursane triterpenes can be used to prepare a
low toxic antiparasitic drug. Overall, whether this plant secretion has
an effect on spirit or not, the herein results revealed this resin as a
great source of biological active triterpenes.
|
4.2
Antiparasitic activity
Interestingly, F1, F3, and F4 also showed close antitrypanosomal
activity. Figure 5 revealed that the seven constituents of F1 are
present in other fractions. However, only four of these metabolites
including oleanolic acid 3‐acetate, olean‐11,13(18)‐diene, and two
compounds related to dehydrouvaol and to olean‐11,13(18)‐diene
were found in F3 and F4. The observation suggested that the
obtained antitrypanosomal activity and the similarity depended on
the presence of these four metabolites. Meanwhile, F2 was eight‐fold
less active than the other fractions although it contained seven
metabolites similar to those found among the F1, F3 and F4 constit-
uents. However, this fraction also contained the metabolite m/z
467.3878 (ursa‐12,20(30)‐dien‐3α‐yl acetate or urs‐12,19(29)‐dien‐
ACKNOWLEDGEMENTS
The authors would like to thank CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior) for their financial
support. This work is a collaborative production of M.S, M.W.B, and
L.P.S, members of the Research Network Natural Products against
Neglected Diseases (ResNetNPND). L.P.S would like to thank M.
Pierre Seumen for the resin collection.
ORCID
3β‐yl acetate or gigantursenyl acetate
A as the second major
Louis P. Sandjo http://orcid.org/0000-0001-5840-7733
component which could interfere in its antitrypanosomal action.
Features similar to those observed for antitrypanosomal activity,
were also found in leishmanicidal activity. F1, F3 and F4 inhibited
L. amazonensis with almost the same strength. However, the
antileishmanial effect of F3 was lightly pronounced suggesting that
the metabolites namely dehydrouvaol, olean‐9 (11),12‐dien‐3‐one,
and ursa‐9 (11),12‐dien‐3‐one only present in F3 might be responsible
for the little improvement of its activity. Fraction F2 was also weakly
active on this parasite and three‐fold less potent than the others
presumably because of its chemical composition as outlined earlier.
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