Phytochemistry Letters 37 (2020) 10–16

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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Phytochemical analysis of the methanolic leaves extract of Niedenzuella T

multiglandulosa (Malpighiaceae), a plant species toxic to cattle in Brazil
Helena Mannochio Russoa, Emerson Ferreira Queirozb,*, Laurence Marcourtb, Adriano Rutzb,
Pierre-Marie Allardb, Rafael Felipe de Almeidac, Nilton Marques Carvalhod, Jean-Luc Wolfenderb,
Vanderlan da Silva Bolzania,*
a
Nucleus of Bioassays, Biosynthesis and Ecophysiology of Natural Products (NuBBE), Department of Organic Chemistry, Institute of Chemistry, São Paulo State University
(UNESP), Araraquara, SP, 14800-900, Brazil
b
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1, Rue Michel-Servet, CH-1211, Geneva 4, Switzerland
c
Departament of Botany, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, 31270-901, Brazil
d
Faculty of Veterinary Medicine and Zootechnics (FAMEZ), Feredal University of Mato Grosso do Sul (UFMS), Campo Grande, MS, 79070-900, Brazil

ARTICLE INFO ABSTRACT

Keywords: Niedenzuella multiglandulosa (Malpighiaceae) is a neotropical liana responsible for cattle intoxication outbreaks
Niedenzuella multiglandulosa in Brazil that may lead to abortion and even death of the animals, causing substantial losses in Brazilian trade
Cattle intoxication balance. A phytochemical study of N. multiglandulosa leaves was performed in order to obtain chemical in-
Metabolite profile formation about secondary metabolites of this poorly studied species. The methanolic extract was purified by
UHPLC-PDA-HRMS
medium pressure liquid chromatography (MPLC) combined with semi preparative HPLC-PDA leading to the
Isolation steroids
Monofluoracetate (MFA)
isolation of 16 compounds, including a new steroid named 5-hydroxypodecdysone B (13). The structure of the
isolated compounds was determined by classical spectroscopic methods such as NMR and HRMS.
Complementary 19F NMR and UPLC-HRMS analysis did not detect the presence of monofluoracetate (MFA), a
toxic compound previously described from this species.

1. Introduction in July/October of 2004 with 7 adult cows dying and 80 % of 290

Malpighiaceae is a family of flowering plants comprising ca. 75
genera and 1300 species of trees, shrubs and lianas (Davis and
Anderson, 2010). Its species are distributed in the tropics and sub-
tropics worldwide, but are specially diverse in the neotropical region
(Davis and Anderson, 2010). Some species from the genus Mascagnia
(Bertero ex DC) Bertero and Tetrapterys Cav. have undergone molecular
and taxonomic studies and are now part of the genus Niedenzuella
(Anderson, 2006; Davis and Anderson, 2010). Species such as Nie-
denzuella multiglandulosa (A.Juss.) W. R. Anderson, Niedenzuella stannea
(Griseb.) W. R. Anderson and Niedenzuella acutifolia (Cav.) W. R. An-
derson (formerly known as Tetrapterys multiglandulosa A.Juss., Tetrap-
terys benthamiana Griseb. and Tetrapterys acutifolia Cav.) (Anderson,
2006)), are reported in the literature as toxic to cattle causing abortion
(Caldas et al., 2011) and chronic intoxication of the animals (Riet-
Correa et al., 2009) and rarely leading to sudden death syndrome
(Caldeira et al., 2017).
Two outbreaks of N. multiglandulosa intoxication by ingestion of
leaves have been described in Brazil Middle-West: the first one occurred
pregnant cows aborting or giving birth to stillborn or weak calves
(Carvalho et al., 2006). The second outbreak was reported in Sep-
tember/October of 2005, in which 9 of 285 two-year old non-pregnant
heifers died in a period of 30–40 days (Carvalho et al., 2006). The
symptoms observed in both outbreaks were: ventral subcutaneous
edema, cardiac arrhythmia, positive venous pulse, dyspnea, lethargy
and abdominal volume increase (Carvalho et al., 2006). The histolo-
gical findings consist in nutmeg liver, a globose heart increased in vo-
lume, fibrosis of myocardium and pulmonary edema (Carvalho et al.,
2006). The intoxication can be described as chronical since it took
weeks of leaves consumption for the symptoms to appear (Carvalho
et al., 2006; Peixoto et al., 2011). It was also observed that this plant is
palatable to cattle since the animals ingest it promptly when offered
(Carvalho et al., 2006), and that it becomes less toxic as the leaves
mature (Peixoto et al., 2011).
Recent studies regarding the toxicity of Niedenzuella species col-
lected from the Missouri Botanical Garden herbarium (USA) have de-
tected monofluoroacetate (MFA) in N. multiglandulosa leaves by GCeMS
(Santos-Barbosa et al., 2017), which is a well-known compound present

⁎
Corresponding authors.
E-mail addresses: emerson.ferreira@unige.ch (E. Ferreira Queiroz), vanderlan.bolzani@unesp.br (V. da Silva Bolzani).

https://doi.org/10.1016/j.phytol.2020.02.005
Received 19 December 2019; Received in revised form 12 February 2020; Accepted 13 February 2020
1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
H. Mannochio Russo, et al.
in toxic plants. Plants containing MFA in its composition are known for
causing “sudden death syndrome” since it can lead to death shortly
after the initial clinical signs (Lee et al., 2014). It is estimated that these
plants are responsible for causing half of the cattle deaths in Brazil
(Tokarnia et al., 2002), leading to great economic losses. Despite the
recent findings of MFA in N. multiglandulosa herborized leaves, no
phytochemical study was ever performed in the genus to date. Ad-
ditionally, a previous study performed by our research group for Tet-
rapterys mucronata Cav. (a plant used occasionally in the preparation of
“Ayahuasca” tea) led to the isolation of several hallucinogen trypta-
mines (Queiroz et al., 2014). Those alkaloids presented high activity on
the inhibition of acetylcholinesterase enzyme (Queiroz et al., 2014),
one of the targets for the treatment of Alzheimer’s disease (Osborn and
Saunders, 2010). Therefore, it was wondered if the same class of al-
kaloids could be responsible for the chronic intoxication caused in
cattle by leaves of N. multiglandulosa, since it was once considered as
part of the genus Tetrapterys.
In the present study, the chemical composition of the methanolic
extract obtained from N. mutiglandulosa young leaves was studied in
order to explore the chemical profile of the extracts obtained, and also
to answer if alkaloids, such as those identified in Tetrapterys mucronata,
or monofluoracetate were the major compounds responsible for the
toxicity attributed to N. multiglandulosa. To answer this question, a
phytochemical study was performed on this extract, leading to the
isolation and structure characterization of sixteen compounds and both
19
F NMR and LC–MS analysis were performed attempting to detect
monofluoracetate (MFA). Our findings contribute to a better under-
standing of this poorly studied genus and on the major compounds
responsible for the toxicity attributed to the genus Niedenzuella.
2. Results and discussion
Initially, the methanolic extracts of mature and young leaves were
analyzed by HPLC-PDA in order to compare their chromatographic
profiles. It was observed that the extracts of mature and young leaves
presented the same compounds, in which only the relative intensity of
the peaks were different (Figure S1, Supporting information), however,
the mature leaves presented a higher amount of tannins, deduced by the
characteristic baseline deviation (Collins et al., 1998).
Considering the yield of the extracts and the number of peaks ob-
served in the chromatographic profile obtained for the methanolic ex-
tract of young leaves (MEYL), this extract was chosen for the phyto-
chemical study.
In order to identify the compounds present in MEYL, the extract was
initially analyzed by UHPLC-PDA-time of flight-high resolution mass
spectrometry (ToF-HRMS) for dereplication purposes (Fig. 1). UV and
HRMS data obtained were compared to compounds reported in the
“Malpighiaceae” family and the “Niedenzuella” genus (see Table S1 and
S2, Supplementary Information). A cross search of the 500 most intense
ions of the extract with a list of 82 products already reported in Mal-
pighiaceae family in the Dictionary of Natural Products led to the an-
notation of an epicatechin dimer in positive mode (m/z 579.1499 [M
+H]+, Rt =2.28 min) already reported in Byrsonima crassifolia (Geiss
et al., 1995)
Among the compounds detected, it was possible to suggest the
presence of a flavonol derivative at retention time at 5.7 min (11) by its
typical chromophore (Markham and Mabry, 1975). Based in its higher
molecular mass observed (m/z 755.1930 [M+H]+), 11 could be a
glycosylated derivative. UV and HRMS data were not sufficient for any
other early structure assignments.
The presence of different unknown metabolites and the lack of
studies in the Niedenzuella genus justify an in depth phytochemical in-
vestigation on this species. The secondary metabolites were isolated by
medium pressure liquid chromatography (MPLC-UV) and semi-pre-
parative HPLC-UV.
By applying a gradient transfer method previously developed
11
Phytochemistry Letters 37 (2020) 10–16
(Challal et al., 2015), a direct transfer of the analytical HPLC conditions
to MPLC using the same stationary phase material provided a sa-
tisfactory fractionation of 7.54 g of the crude methanolic extract. Using
this approach, it was possible to isolate compounds 2 and 16 in one
step. The MPLC fractions were further purified by semi-preparative
HPLC and 16 compounds were isolated (1–16) (Fig. 2).
The structure elucidation of the isolated compounds was performed
based on NMR spectroscopic data and HRMS data analysis. The pre-
viously known metabolites were identified as trigonelline (1)
(Marchesini et al., 2009), tryptophan (2) (Malta et al., 2009), 4-hy-
droxycinnamamide (3) (Kim et al., 2014), cis-p-coumaric acid 4-O-β-D-
glucopyranoside (4) (Wu et al., 2003), icariside F2 (5) (Bai et al., 2015),
luteoforol (6) (de Souza et al., 2008), (E)-4-hydroxycinnamic acid (7)
(Salum et al., 2010), (Z)-4-hydroxycinnamic acid (8) (Salum et al.,
2010), integristerone A (9) (Simon et al., 2009), epiecdysterone (10)
(Vokáč et al., 2002), isorhamnetin 3-O-β-D-xylopyranosyl-(1””→3”')-α-
L-rhamnopyranosyl-(1”'→6”)-β-D-galactopyranoside (11) (Ben Salah
et al., 2002), ecdysterone (12) (Vokáč et al., 1998), calonysterone (14)
(Csábi et al., 2015), podecdysone B (15) (Odinokov et al., 2003) and
cinnamic acid (17) (Wang et al., 2003). Beside these known com-
pounds, the separation afforded one new compound (13), described
below (Table 1).
Compound 13 was obtained as a yellow oil. The ESI-HRMS analysis
showed a molecular ion at m/z 479.3002 [M+H]+, consistent with the
molecular formula C27H42O7 (calcd. for C27H42O7, 478.2931, Δ ppm =
–1.5). The NMR data showed close similarity with those of podecdysone
B (15) except that the methine signal at δH 2.30 (dd, J = 12.7, 3.9 Hz,
H-5) in 15 was replaced by a singlet at δH 5.44 (OH-5) in 13. The HMBC
correlations from the hydroxyl group in C-5 to C-4, C-5, C-6 and C-10
(δC 35.3, 79.9, 208.9 and 48.3, respectively) confirmed the identifica-
tion of compound 13 as 5-hydroxypodecdysone B (Fig. 3). The ROE
correlations from OH-5 to OH-3, H-1ax, H-4eq and CH3-19 indicated
that the hydroxyl group in C-5 was on the same side than these protons
(Fig. 3). The other ROE correlations being the same than for compound
15, the relative configuration of 13 was defined as that of 15.
As previously explained, recent studies have attributed the toxicity
of N. multiglandulosa to the presence of monofluoracetate (MFA)
(Santos-Barbosa et al., 2017). Therefore, since this compound has not
been isolated in the present study, 19F NMR (Frost et al., 1989) and
LC–MS (Lee et al., 2012) analyses were performed attempting to
identify MFA in the extracts obtained (chloroform, ethyl acetate, me-
thanol and aqueous). No signal of fluorine was detected in the 19F NMR
analysis (Krebs et al., 1994). The metabolite profile obtained by the
LC–MS analysis in negative mode of all the obtained extracts could not
identify a significant abundance of an m/z 77 [M−H]− ion when
comparing to the total ion chromatogram (TIC). All of these analyses
suggested the absence of MFA of this plant.
Future in vivo toxicity studies should be performed to provide a
more complete and detailed information about the compound(s) re-
sponsible for the observed toxic effect in cattle. Another interesting
point is that the compounds isolated from the extract of young leaves of
N. multiglandulosa are structurally different from those previously iso-
lated from species of Tetrapterys. Therefore, these results corroborate
the fact that these plant species genera are indeed separated from the
chemotaxonomic point of view and that the tryptamines are not the
compounds most likely responsible for the chronic cattle intoxication.
The present study provides the first phytochemical information re-
garding the Niedenzuella genus. The major compound, the alkaloid tri-
gonelline (1) (Deshpande et al., 2017), occurs in many edible plants,
such as coffee (Coffea arabica) and fenugreek seeds (Trigonella foenum-
graecum). Moreover, there is also no evidence of mammalian toxicity of
this compound in the literature (Deshpande et al., 2017).
The presence of the phytoecdysteroids (compounds 9, 10, 12, 14
and 15) in N. multiglandulosa (and for the first time in Malpighiaceae
family) is interesting since these compounds are molting hormones of
arthropods insect, that have already been described in many plant
H. Mannochio Russo, et al.
Fig. 1. A) UV and HRMS data of the compounds identified. B) UHPLC-PDA-HRMS metabolite profiling (UV 280 and 254 nm) of the methanolic extract of Niedenzuella
multiglandulosa young leaves (MEYL).
species (Tarkowská and Strnad, 2016). These compounds are known to
induce “abnormal molting” in many arthropods with lethal effects, and
for this reason has the potential to be used in agricultural to control
herbivore insects (Jurenka et al., 2017). Nevertheless, the role of ec-
dysteroids in plants (also known as phytoecdysteroids) is still unknown
(Tarkowská and Strnad, 2016). This class of compounds have been
previously reported for Silene spp. (Louden et al., 2002; Simon et al.,
2004), Rhaponticum integrifolium (Sagdullaev et al., 1999), Paris poly-
phylla (Nguyen et al., 2009; Singh and Thakur, 1982; Wu et al., 2012),
among others, however, there are few information about the toxicity of
these compounds in mammals (Bajguz et al., 2015). These information,
along with the presence of ubiquitous phenolic compounds (flavonoids
and phenyl propanoid derivatives) provide interesting information of
this poorly studied species.
Additionally, efforts have been made in order to detect MFA in N.
multiglandulosa extracts; however, it was not possible to observe it in
the analyses performed. Even though MFA might be present in very low
amounts, this compound might not be the sole responsible for the
chronic toxicity observed in cattle.
3. Material and methods
3.1. General experimental procedures
Optical rotation was measured in a methanolic solution on a Perkin-
Elmer 241 polarimeter (Perkin-Elmer, Waltham, MA, USA), using 1 dm
tube. UV spectra were measured on a HPLC UV–vis DR/4000 instru-
ment (Loveland, CO, USA). NMR spectroscopic data were recorded on a
Bruker Avance III HD 600 MHz NMR spectrometer equipped with a QCI
5 mm Cryoprobe (for structure elucidation) or with a BBFO-Z plus
SmartProbe Broadband Observe (for fluorine analysis – spectral width
12
Phytochemistry Letters 37 (2020) 10–16
(SW): -150 to -250 ppm; D1: 1 s; Pulse sequence: zgfhigqn; Number of
Scans (NS): 128) and a SampleJet automated sample changer (Bruker
BioSpin, Rheinstetten, Germany). Chemical shifts are reported in parts
per million (δ) using the DMSO-d6 residual signal (δH 2.50; δC 39.5) as
internal standards for 1H and 13C NMR, and coupling constants (J) are
reported in Hz. Complete assignments were obtained based on 2D-NMR
experiments (COSY, ROESY, HSQC and HMBC). HRESIMS analysis were
performed on a Waters Acquity UPLC system coupled to a Waters
Micromass LCT Premier Time-of-Flight (ToF) mass spectrometer
(Milford, MA, USA), equipped with an electrospray interface (ESI).
LCeMS analyses were carried out at a Shimadzu chromatograph cou-
pled to an amaZon-SL ion trap Bruker Daltonics (Milford, MA, USA).
3.2. Plant material
Seedlings of Niedenzuella multiglandulosa were collected in
November 2005 in the municipality of Bataiporã, Mato Grosso do Sul
state, Brazil by Dr. Nilton Carvalho from the Federal University of Mato
Grosso do Sul (UFMS) (Figure S2, Supporting information). Those
seedlings have been cultivated since then at UFMS for toxicological
studies currently in elaboration by Dr. Carvalho. Fresh leaves from
those cultivars of Niedenzuella multiglandulosa at UFMS were collected
for this study in 2015. A voucher specimen (CGMS25848) was de-
posited at the CGMS herbarium from the UFMS representing the ori-
ginal natural population of this species. The identification of this vou-
cher specimen was based on the observation of macro-morphological
characters on a stereomicroscope following specialized taxonomic lit-
erature on Niedenzuella (Anderson, 2006).
The authorization for studying this species was conceded by the
National System for Management of Genetic Heritage and Associated
Traditional Knowledge (SISGEN), n˚ A066C3E.
H. Mannochio Russo, et al.
Fig. 2. Isolated compounds from N. multiglandulosa methanolic extract of young leaves.
3.3. Plant extraction
The plant material (550 g of old and young leaves) was initially
dried in an oven at 50 °C for 72 h and shredded in a mill. 50 g of old and
young leaves were selected to an extraction by maceration with organic
solvents of increasing polarity (3 × 200 mL during 24 h): chloroform
(0.7 % NH4OH), hexane, ethyl acetate and methanol. The dry extracts
were obtained by removing the solvent by rotatory evaporation
yielding chloroform (1.85 g), hexane (0.16 g), ethyl acetate (0.17 g)
and methanol (2.78 g) of old leaves and chloroform (1.69 g), hexane
(0.12 g), ethyl acetate (0.24 g), methanol (2.13 g) of young leaves.
Large scale young leaves extracts were obtained starting from 470 g by
the same method described, yielding chloroform (15.77 g), hexane
(1.20 g), ethyl acetate (2.34 g), methanol (18.91 g) and water (43.87 g).
3.4. HPLC-PDA-ELSD analysis
Analytical HPLC-PDA-ELSD analysis were performed using an
Agilent Technologies 1260 Infinity system equipped with a photodiode
array detector (Agilent Technologies, Santa Clara, CA, USA).
Preparative medium pressure liquid chromatography (MPLC) was per-
formed using a Büchi system equipped with a C-605 module pump, C-
13
Phytochemistry Letters 37 (2020) 10–16
640 UV detector and C-684 fraction collector from Büchi (Flawil,
Switzerland) and the system was controlled by the software Sepacore
Control. A coil connected to a resistance was used to control the MPLC
column temperature. The column (460 × 49 mm i.d.) was packed with
ZEOprep® C18 as the stationary phase (ZEOprep® C18, 15–25 m,
Zeochem, Uetikon am See, Switzerland). Semi-preparative HPLC was
performed using a Shimadzu LC-8A pump (Shimadzu, Columbia, MD,
USA) equipped with a UV detector using an X-Bridge C18 column (150
× 21 mm i.d.; 5 μm) (Waters, Milford, MA, USA) and using an Armen
modular Spot prep II (Saint-Avé, France) with an Puriflash C18 column
(250 × 21.2 mm, i.d.; 10 μm) (Interchim, Montluçon, France).
3.5. UHPLC-ToF-HRMS for metabolite profiling
UHPLC-ToF-HRMS metabolite profiling of the extracts was per-
formed on a Micromass-LCT Premier time-of-flight (ToF) mass spec-
trometer (Waters) equipped with an electrospray interface and coupled
to an Acquity UPLC system (Waters). The ESI conditions were as fol-
lows: capillary voltage 2800 V, cone voltage 40 V, MCP detector voltage
2400 V, source temperature 120 °C, desolvation temperature 300 °C,
cone gas flow 20 L/h, desolvation gas flow 600 L/h. Detection was
performed in the positive and negative ion mode with a m/z range of
H. Mannochio Russo, et al. Phytochemistry Letters 37 (2020) 10–16

Table 1 The HPLC consisted in a LC-20CE solvent pump unit, a CTO-20A

1
H (600 MHz) and 13C (150 MHz) chemical shifts and HMBC correlations of column oven, a DGU-20A3R online degasser, a CBM-20A system con-
compound 13 in DMSO-d6. troller, and a SPD-M20A (190–800 nm) DAD. The injection was per-
Atom position δH, mult. (J in Hz) δC HMBC formed (2 μL) in a SIL-20A HT auto-sampler. The dry extracts were
analyzed on a Phenomenex Luna C18 column (250 × 4.6 mm i. d., 5
1 1.73, dd (14.2, 3.1), Hax 1.66, m, Heq 34.0 2, 3, 10, 9 μm particle size, 100 Å pore size) at a flow rate of 1 mL/min using a
2 3.29* 67.8
linear gradient (solvent system: A = 0.1 % formic acid − water, B =
3 3.80, dq (6.3, 3.3), Hax 68.3
4 1.98, dd (14.4, 3.3), Hax 1.62, dd 35.4 3, 5, 10 0.1 % formic acid − acetonitrile); gradient 5 − 100 % B in 40 min. The
(14.4, 3.3), Heq mass spectrum was obtained in negative ion mode considering a mass
5 79.9 range of 50–1200 m/z and target mass of m/z 77 for attempting of MFA
6 208.6 identification. The ESI conditions were set as follows: capillary voltage
7 3.42, d (21.1) 2.69, d (21.1) 39.2 6, 8, 9
8 121.3
at 4.5 kV. Nitrogen was used as the nebulizing and drying gas (50 psi,
9 136.9 10 L/min, 300 °C).
10 48.2
11 2.24, m 22.2 8, 9, 12, 13
12 2.14, dt (12.4, 4.0) 1.46, m 36.5 9, 13, 14, 18 3.7. Purification of the methanolic extract of young leaves of N.
13 45.4
multiglandulosa
14 147.8
15 5.40, t (3.0, 2.1) 119.3 8, 13, 14, 16, 17
16 2.55, ddd (16.5, 10.7, 2.1) 2.04, ddd 30.4 13, 14, 15, 17 The conditions to the fractionation of the methanolic extract of
(16.5, 7.6, 3.0) young leaves were optimized on an HPLC column packed with Zeoprep
17 1.89, dd (10.7, 7.6) 55.8 12, 13, 16, 18 C18 as the stationary phase (15 − 25 μm, 250 × 4.6 mm i.d., Zeochem).
18 0.96, s 17.9 12, 13, 14, 17
19 0.84, s 23.6 5, 9, 10, 11
The fractionation of this extract (7.54 g) was performed by medium-
20 75.1 pressure liquid chromatography using the same stationary phase
21 1.11, s 20.0 17, 20, 22 (15−25 μm, 460 × 49 mm i.d., Zeochem) and an acidic (0.1 % formic
22 3.15, dd (11.5, 4.0) 76.5 acid) H2O (A) MeOH (B) gradient: isocratic 5 %B in 56 min, 5–38 %B in
23 1.45, m 1.12, m 26.0 24, 22
5.5 h, isocratic 38 %B in 4.7 h, 38–62 %B in 3.2 h, 62–100 %B in 9 min
24 1.65, m 1.24, m 41.4 23, 25, 26, 27
25 68.7 and isocratic 100 %B during 1.5 h. A dry load injection of the sample
26 1.07, s 29.8 24, 25, 27 was performed by mixing 7.54 g of the methanolic extract with 35 g of
27 1.04, s 29.0 24, 25, 26 of Zeoprep C18 stationary phase (40–63 μm). The dry-load cell (11.5 ×
OH-2 n.o. 2.7 cm i.d.) was connected subsequently between the pumps and the
OH-3 4.73, d (6.3) 3, 4
OH-5 5.44, s 4, 5, 6, 10
MPLC column. The flow rate was set to 20 mL min−1, and the UV ab-
OH-20 3.66, s 17, 20 sorbance was monitored at 280 nm. The MPLC fractionation yielded 79
OH-22 4.36, d (4.0) fractions of 250 mL which were analyzed by UHPLC-ToF-HRESIMS in
OH-25 4.07, s 24, 25, 26, 27 order to determine the purity of the fractions. Two compounds were
isolated in this step, in which fraction 10 yielded compound 2 (23.7
* Overlapped by another signal.
mg) and fraction 43 to 35 yielded compound 16 (53.9 mg). Besides
that, compound 1 could be detected in fraction 3 (3.3 g), even though it
100 − 1000 Da and a scan time of 0.5 s in the W-mode. The MS was
wasn’t pure and contained a very high amount of sugars. Fractions
calibrated using sodium formate. Leucine enkephalin (Sigma- Aldrich,
17–18, 20, 24, 31, 33 and 52–57 were purified by semi-preprative
Steinheim, Germany) was used as an internal reference at 2 μg/mL and
HPLC-UV using an XBridge™ C18 column (5 μm, 150 × 19 mm i.d.)
infused through a Lock Spray probe at a flow rate of 10 μL/min aided by
(Waters) with an acidic (0.1 % formic acid) H2O (A) ACN (B) as the
a second LC pump. The separation was performed on an Acquity BEH
mobile phase, 10 mL min−1 flow rate and UV absorbance was detected
C18 UPLC column (150 mm × 2.1 mm i.d.; 1.7 μm, Waters), using a
at 280, 210, 280, 254, 254 and 254, respectively. Fractions 17–18 (77.3
linear gradient (solvent system: A = 0.1 % formic acid − water, B =
mg) were combined and purified by an isocratic elution (7% ACN in 40
0.1 % formic acid − acetonitrile; gradient 5 − 95 % B in 30 min; flow
min) leading to the isolation of compounds 3 (1.0 mg) and 4 (6.2 mg);
rate 0.46 mL/min). The temperature was set to 40 °C. The injection
fraction 20 (48.8 mg) was purified by an isocratic elution (8% ACN in
volume was constant (2 μL).
60 min) and led to the isolation of compounds 5 (3.0 mg) and 6 (1.5
mg); fraction 24 (57.7 mg) was purified by an isocratic method (12 %
3.6. LC–MS/MS analyses ACN in 45 min) and afforded compounds 7 (2.1 mg) and 8 (1.3 mg);
fraction 31 (73.9 mg) was purified by a gradient method (5–35% ACN
The attempting to identify MFA ion was performed at a Shimadzu in 40 min) and led to the isolation of compound 9 (5.6 mg); fraction 33
chromatograph coupled to an amaZon-SL ion trap Bruker Daltonics. (87.0 mg) was purified by an gradient followed by isocratic method

Fig. 3. HMBC correlations (left) and ROESY correlations (right) of compound 13 that allowed OH to be positioned in C-5.

14
H. Mannochio Russo, et al.
(5–12% ACN in 15 min, 12 % ACN during 60 min) leading to the iso-
lation of compounds 10 (1.5 mg); fractions 52–57 were combined (63.6
mg) and submitted to isocratic separation (16 % B in 80 min) leading to
compounds 13 (2.4 mg), 14 (3.7 mg) and 15 (4.4 mg). Fraction 36
(137.0 mg) was purified by semipreprative HPLC-UV using an XBridge™
C18 with an acidic (0.1 % formic acid) H2O (A) MeOH (B) as the mobile
phase, 10 mL min−1 flow rate, UV absorbance monitored at 280 nm
and isocratic method (32 % MeOH in 60 min) leading to the isolation of
compound 14 (2.6 mg). Fraction 35 (282.6 mg) was purified by semi-
preprative HPLC-UV using an Puriflash C18 column (10 μm, 250 × 21.2
mm i.d.) (Intershim) with an acidic (0.1 % formic acid) H2O (A) MeOH
(B) as the mobile phase, 15 mL min−1 flow rate, UV absorbance
monitored at 280 nm and isocratic method (37 % MeOH in 60 min)
leading to the isolation of compounds 12 (16.8 mg) and 11 (2.5 mg).
3.8. Spectroscopic data
Compound 13: yellow oil; [α]26
D +7 (c 1.0, MeOH); UV (MeOH) λmax
(log ε) 224 nm; NMR data: see Table 1; HRESIMS m/z 479.3002 [M
+H]+ (calcd for C27H42O7, 478.2931, Δ ppm = –1.5).
UV and HRMS data of the other isolated compounds are presented
in Figure S3 (Supporting information).
There are no conflicts of interest to declare.
Supporting information
Figures S1−S8 showing HPLC-PDA analysis profile of the extracts,
peaks UV spectra, NMR spectra for the new compound 13 and pictures
of the plant. Tables S1 and S2 for the UHPLC-PDA-HRMS data used for
the dereplication.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
The authors gratefully acknowledge the financial support from the
São Paulo State Research Foundation (FAPESP) program INCT-FAPESP
(Project No. 2014/50926-0), CEPID-FAPESP (Project No. 2013/07600-
3; 2016/16970-7), and from the National Council for Scientific and
Technological Development (CNPq) for the funding INCT-CNPq
(Project No. 2014/465637-0). The School of Pharmaceutical Sciences of
the University of Geneva (Prof. J-L. Wolfender) is thankful to the Swiss
National Science Foundation for the support in the acquisition of the
NMR 600 MHz (SNF R’Equip grant 316030_164095). HMR acknowl-
edges scholarship #152341/2015-2 from National Research Council
(CNPq).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2020.02.005.
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