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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025225, enero-diciembre 2025 (Publicado Nov. 03, 2025)
Alkaloid-enriched extracts from the tree Neltuma flexuosa (Fabaceae):
chemical characterization, fractionation, and cytotoxic effects
Angelica M. Cabrera-Benítez1; https://orcid.org/0009-0009-1353-3950
Enrique N. García1; https://orcid.org/0000-0002-4224-0083
Anahi K. Alucin1, 5; https://orcid.org/0009-0000-2720-9419
Ana M. Torres2, 5; https://orcid.org/0000-0002-3793-5454
Dale Gardner3; https://orcid.org/0000-0003-3218-6893
Soledad Bustillo4, 5*; https://orcid.org/0000-0002-1875-7664
Luciana A. Cholich1, 5; https://orcid.org/0000-0002-7936-8806
1. Faculty of Veterinary Science, National University of the Northeast, Sargento Cabral 2139, Corrientes, Argentina;
angycabrerab@gmail.com, garciaenicolas@gmail.com; anahialucin@gmail.com, lucianaandreacholich@gmail.com
2. Natural Products Laboratory, IQUIBA-NEA CONICET, National University of the Northeast, Av. Libertad 5470,
Corrientes, Argentina; amtorres@exa.unne.edu.ar
3. USDA-ARS Poisonous Plant Research Laboratory, Logan, UT, 84341, USA; drglogan@gmail.com
4. Biological and Molecular Research Group (GIByM), IQUIBA-NEA CONICET, National University of the Northeast,
Av. Libertad 5470, Corrientes, Argentina; solebustillo@yahoo.es (*Correspondence)
5. The National Scientific and Technical Research Council (CONICET), Argentina.
Received 07-VII-2025. Corrected 01-VIII-2025. Accepted 22-X-2025.
ABSTRACT
Introduction: Neltuma flexuosa is a key species in arid regions, offering nutritional benefits but also containing
alkaloids that can pose toxicological risks.
Objective: To analyze the chemical composition and alkaloid content of N. flexuosa pods collected over four
years (2019-2022) in Cafayate, Argentina, considering the influence of climatic variations, and to evaluate their
potential cytotoxic effects on C6 glioma cells.
Methods: Dried and ground pods were analyzed for chemical composition using standard methods. Alkaloid-
enriched extracts (AEE1-AEE4) were obtained through acid/base extraction and analyzed by HPLC-HRMS for
juliprosine and juliprosopine quantification. AEE4 was fractionated using flash chromatography, yielding frac-
tions (F1-F5), which were also analyzed for alkaloid content. Cytotoxicity on C6 glioma cell lines was evaluated
using crystal violet staining assay, and apoptosis induction was confirmed in C6 cells treated with AEE4 using
the TUNEL assay.
Results: Chemical analysis revealed interannual variations in pod composition, particularly in fiber and pro-
tein content. HPLC-HRMS identified juliprosine and juliprosopine, with the highest concentrations in AEE4.
Fractionation of AEE4 by flash chromatography yielded an alkaloid-rich fraction (F4). Cytotoxicity assays dem-
onstrated dose-dependent reductions in cell viability, with AEE4 exhibiting the lowest 50 % cytotoxic concentra-
tion (CC50) values. Furthermore, F4 displayed the highest cytotoxicity against C6 cells. TUNEL assays confirmed
the induction of apoptosis by AEE4.
https://doi.org/10.15517/3jcswj50
OTRO
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INTRODUCTION
The genus Neltuma (Prosopis) belongs to
the Fabaceae family and is well adapted to
arid and semi-arid environments. In Argentina,
Neltuma flexuosa plays a key ecological role in
regions such as Chaco, Espinal Norte and Pam-
peana, where it is commonly known as “Algar-
robos” (Hughes et al., 2022; Palacios & Brizuela,
2005). The pods of this genus serve as a valu-
able feed source for animals due to their nutri-
tional content (Riet-Correa et al., 2012; Silva,
1981). They contain 9-17 % protein, 13-31 %
sucrose, and 17-31 % crude fiber (González-
Galán et al., 2008; Zolfaghari & Harden, 1985).
Their protein content is reported to be compa-
rable to that of major proteinaceous crops, such
as cowpea, soybean, and pigeon pea (Abbiw,
1990; Sciammaro et al., 2015). However, they
also contain antinutritional substances includ-
ing oxalates, alkaloids, saponin and tannins
(Anhwange et al., 2020).
In this sense, several studies have been
conducted offering insights into their nutri-
tional benefits and associated risks. In Africa,
studies have highlighted the high protein con-
tent of Neltuma africana pods and their role in
livestock diets, while also noting the presence
of toxic compounds that pose risks to animal
health (Okalebo et al., 2007). An Australian
Conclusions: These findings provide valuable insights into the chemical composition, alkaloid content, and cyto-
toxic effects of N. flexuosa extracts, contributing to the development of guidelines for their safe and sustainable
use as a feed resource.
Key words: cytotoxicity; food chemistry; piperidine alkaloids; rainfall; toxicology.
RESUMEN
Extractos enriquecidos con alcaloides del árbol Neltuma flexuosa (Fabaceae):
caracterización química, fraccionamiento y efectos citotóxicos
Introducción: Neltuma flexuosa es una especie importante en regiones áridas, ofrece beneficios nutricionales,
pero también contiene alcaloides que pueden presentar riesgos toxicológicos.
Objetivos: Analizar la composición química y el contenido de alcaloides de vainas de N. flexuosa recolectadas
durante cuatro años (2019-2022) en Cafayate, Argentina, considerando la influencia de las variaciones climáticas
y evaluar su potencial efecto citotóxico sobre células de glioma de la línea C6. Adicionalmente, evaluar los poten-
ciales efectos citotóxicos de células de glioma C6.
Métodos: Las vainas secas y molidas, fueron analizadas para su composición química mediante métodos están-
dares. Los extractos enriquecidos en alcaloides (EEA1-EEA4) se obtuvieron mediante extracción ácido/base y se
analizaron por HPLC-HRMS para cuantificar la juliprosina y la juliprosopina. El EEA4 se fraccionó mediante
cromatografía flash, obteniéndose diferentes fracciones (F1-F5), que también se analizaron para determinar su
contenido en alcaloides. Se evaluó la citotoxicidad de los extractos y fracciones a partir de la línea celular de glio-
ma C6 mediante el ensayo de tinción con violeta cristal y se confirmó la inducción de apoptosis en las células C6
tratadas con EEA4 mediante el ensayo TUNEL.
Resultados: El análisis químico reveló variaciones interanuales en la composición de las vainas, particularmente
en el contenido de fibra y proteína. El análisis por HPLC-HRMS identificó juliprosina y juliprosopina, con las
mayores concentraciones en el EEA4. El fraccionamiento de EEA4 mediante cromatografía flash produjo una
fracción rica en alcaloides (F4). Los ensayos de citotoxicidad demostraron disminución en la viabilidad celular
dependiente de la dosis, exhibiendo, EEA4, los valores más bajos de concentración citotóxica del 50 % (CC50).
Además, F4 mostró la mayor citotoxicidad contra las células C6. Los ensayos TUNEL confirmaron la inducción
de apoptosis por EEA4.
Conclusiones: Estos resultados aportan información valiosa sobre la composición química, el contenido de alca-
loides y los efectos citotóxicos de los extractos de N. flexuosa, contribuyendo al desarrollo de directrices para su
uso seguro y sostenible como recurso alimenticio.
Palabras clave: citotoxicidad; composición química del alimento; alcaloides de piperidinicos; precipitación;
toxicología.
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work has similarly assessed the impact of Nel-
tuma spp. pods on the growth and health of
grazing animals, showing that while they are a
nutritious feed source, careful management of
antinutritional factors is necessary (Kumar &
Mathur, 2014). In Brazil, Neltuma juliflora has
been extensively studied due to its widespread
use and toxicity issues with documented cases of
nervous disorders in cattle and other livestock
(Lima et al., 2004; Silva et al., 2006; Tabosa et
al., 2003). Various analytical methods, includ-
ing spectroscopic techniques and biochemical
assays, have been employed to assess toxic com-
pound levels and their effects on animal health
(Assis et al., 2009; Câmara et al., 2009). How-
ever, Riet-Correa et al. (2012) found no nervous
signs or symptoms in animals consuming N.
juliflora pods over an extended period, suggest-
ing that toxicity may vary based on factors such
as pod maturity, environmental conditions, and
regional differences in plant chemistry.
In Argentina, outbreaks of nervous dis-
ease in cattle associated with the consumption
of Neltuma nigra pods have been reported.
Clinical symptoms include tongue protrusion,
twitching and tremors of the masticatory mus-
cles, weight loss, and lethargy (Micheloud et al.,
2019). However, we later confirmed the pres-
ence of juliprosine and juliprosopine alkaloids
in N. flexuosa but not in N. nigra, leading us to
rule out N. nigra as the cause of spontaneous
poisoning in animals (Cholich et al., 2021).
These issues underscore the need for a
comprehensive understanding of the nutri-
tional and toxicological properties of N. flex-
uosa pods. To address this critical gap, this
study aims to analyze the chemical composition
and alkaloid content of N. flexuosa pods col-
lected over four consecutive years in Cafayate,
Argentina, considering the influence of climatic
variations, and to evaluate their potential cyto-
toxic and apoptotic effects on C6 glioma cells.
This knowledge is crucial for optimizing the
use of N. flexuosa pods as a complementary
feed resource while mitigating potential health
risks to livestock.
MATERIALS AND METHODS
Plant material: N. flexuosa subsp. flexuosa
pods were collected from Cafayate, Salta Prov-
ince, in Northwestern Argentina. A voucher
herbarium specimen (CTES-1193) was identi-
fied by Lic. Walter Medina and deposited at
the Instituto de Botánica, Facultad de Ciencias
Agrarias (UNNE, CONICET) in Corrientes,
Argentina. This region has an arid climate with
a pronounced dry season. The average annual
temperature is 17 °C, with summer highs reach-
ing 35 °C and winter lows ranging between
5 °C and -15 °C. Rainfall is confined to the
summer months, with an annual average of 207
mm occurring between November and March
(Sampietro-Vattuone & Peña-Monné, 2016).
The sampling area is located at an altitude of
1 683 m.a.s.l. (26°3’49.18” S, 65°56’52.99” W).
Pods were collected over four years (2019, 2020,
2021 and 2022), and the annual rainfall and
mean temperature for each year were recorded
from the meteorological Station in Cafayate,
National Institute for Agricultural Technology
(INTA), Argentina (Table 1). The pods were
then dried at 37 °C to a constant weight and
pulverized in a mill.
Table 1
Annual precipitation and temperature for the study region
(2019-2022). Climatic data were collected from a weather
station in Cafayate, Salta.
Year Mean temperature
(°C)
Annual precipitation
(mm)
2019 17.59 260.2
2020 17.65 264
2021 17.35 193
2022 17.7 130.4
Chemical analysis: The analytical deter-
minations were carried out on pulverized pods
of N. flexuosa, three independent samples were
collected each year from representative loca-
tions within the study area. Dry matter (DM,
method ID 934.01), ash (method ID 942.05),
ether extract (EE, method ID 920.30) and crude
protein (CP, method ID 984.13) were analyzed
following AOAC methods (Cunniff, 1999).
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Neutral detergent fiber (NDF), acid detergent
fiber (ADF) and acid detergent lignin (ADL)
were determined using an ANKOM fiber ana-
lyzer (Goering & Van-Soest, 1970). Total digest-
ible nutrients (TND) were estimated using the
equation: % TND = 92.51 – (% FDA X 0.7965)
(Chalupa et al., 1996). Based on the TND
results, digestible energy (DE) was estimated
using the equation: DE (Mcal) = % TND ×
4.409 Mcal / 100 (Ammar et al., 2004).
Alkaloid-enriched extracts (AEE) from
N. flexuosa: Alkaloid-enriched extracts were
obtained from 500 g of pulverized N. flexuosa
pods using an acid/base extraction method, as
described by Ott-Longoni et al. (1980). Briefly,
dry pods were first defatted with hexane (1.5 l/
Kg) for 10 days and then extracted with metha-
nol (1.5 l/Kg) for seven days. The solution
was filtered, and the methanolic extract was
concentrated using a BÜCHI rotary evaporator
under reduced pressure at 40 °C. The concen-
trate was then stirred with 0.2 N HCl (1:1 v/v)
and kept at room temperature for 16 h. The
resulting acidic solution was filtered, and non-
basic impurities were removed by liquid-liquid
extraction with chloroform. The aqueous phase
was then adjusted to pH 11 with ammonium
hydroxide and subsequently extracted with
chloroform. This fraction was evaporated to
dryness under reduced pressure, yielding a res-
inous, amber-colored alkaloid-enriched extract
(AEE), which was stored at -20 °C. Extracts
obtained from pods collected in 2019, 2020,
2021 and 2022 were designated as AEE1, AEE2,
AEE3, and AEE4, respectively.
Identification and quantitation of alka-
loids in N. flexuosa alkaloid-enriched extracts
(AEE) and their fractions: The alkaloids pres-
ent in each AEE, including fractions (F1-F5)
obtained from AEE4, were identified and quan-
tified by HPLC-HRMS, following the method
described by Cholich et al. (2021). Analytical
samples (1 mg) were dissolved in 100 μl of
methanol. A 5 μl aliquot was then diluted with
995 μl of 50 % methanol containing 0.1 % for-
mic acid. Juliprosine and juliprosopine were
subsequently analyzed by HPLC-HRMS. Quan-
titation of these two alkaloids was determined
by comparing the peak area ratios with those
of a standard solution of juliprosine (m/z =
626.5619) and juliprosopine (m/z = 630.5929),
prepared at a concentration of 1 μg/ml. The
limits of detection for both alkaloids were
10 ng/ml in the final diluted solution ana-
lyzed. Juliprosine and juliprosopine (HPLC-MS
purity > 92%) were kindly provided by Dr.
Muhammad Ilias, National Center for Natural
Products Research, University of Mississippi,
USA. Identification was confirmed by matching
retention times and exact m/z values with those
of the analytical standards.
Fractionation of the alkaloid extract
(AEE4) by flash chromatography: A total
of 500 mg of AEE4, identified as the extract
with the highest concentration of piperidine
alkaloids, was dissolved in 96 % ethanol, mixed
with silica gel, and dried under reduced pres-
sure using a Büchi rotary evaporator to prepare
the sample for fractionation. The dried sample
was placed in a 24 × 400 mm column with a
stopcock and ground glass stopper to maintain
pressure. Flash chromatography was performed
using silica gel flash 60 (0.04-0.063 mm, MN)
with an elution flow rate of approximately 3.5 l/
min, provided by a CX-1000 aquarium air
pump. The extract was loaded onto the top of
the column and sequentially eluted with 50 ml
of chloroform/methanol mixtures, starting at a
48 : 2 ratio and gradually increasing the metha-
nol content as follows: 46 : 4; 40 : 10; 35 : 15; 30
: 20; 25 : 25; 20 : 30; 15 : 35; 10 : 40; 5 : 45; 2 :
48). Elution was completed with 150 ml of pure
methanol and 50 ml of methanol : ammonium
hydroxide (40 : 10). Eluted fractions were col-
lected continuously in 5 ml test tubes. Thin-
layer chromatography (TLC) was performed on
each tube using silica gel GF plates and chlo-
roform/methanol (1 : 1) as the mobile phase.
Plates were examined under UV light at 254
and 365 nm and visualized with iodine vapor.
Fractions with similar chromatographic pro-
files were pooled as follows: F1: tubes 10-23, F2:
tubes 24-31, F3: tubes 32-93, F4: tubes 94-97
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and F5: tubes 98-100. These grouped fractions
were dried under reduced pressure and reana-
lyzed by TLC followed by sequential spraying
with Dragendorff’s reagent and 10 % sodium
nitrite for alkaloid detection. Selected fractions
were subsequently analyzed by HPLC-HRMS
to determine their alkaloid composition.
Cell culture: C6 glioma cells (ATCC: CCL-
107™) were grown in 25 cm2 flasks using Dul-
becco’s minimum essential medium (DMEM)
(GIBCO-Invitrogen, Argentina) supplemented
with 10 % fetal bovine serum (FBS Sigma
Aldrich, USA), Penicillin- Streptomycin (1 %)
(GIBCO-Invitrogen) in a humidified atmo-
sphere with 5 % CO2 at 37 °C. For cytotoxic
assays, C6 cells were harvested from subconflu-
ent monolayers after exposure to 0.25 % trypsin/
EDTA (1X) (Gibco) at 37 °C. The resuspended
cells were seeded in 96-well microplates at an
approximate initial density of 3 × 104 cells per
well, in growth medium (DMEM, 10 % FBS).
When monolayers reached confluence, medi-
um was replaced with fresh medium containing
AEE1, AEE2, AEE3, and AEE4 (10, 20, 30, 40,
50 and 100 µg/ml) or AEE4 fractions (F1, F2,
F3, F4 and F5; 2.5, 5, and 10 µg/ml) from N.
flexuosa, previously dissolved in 0.1 % ethanol.
Cells were incubated for 48 h at 37 °C, 5 % CO2
and cell viability was determined by crystal
violet staining method. Briefly, non-adherent
cells were removed by washing twice with
phosphate-buffered saline (PBS) and adherent
cells were fixed with methanol : glacial acetic
acid (3 : 1 ratio), stained with 0.5 % crystal
violet in 20 % (v/v) methanol. The dye was
released from the cells by addition of ethanol :
glacial acetic acid (3 : 1 ratio). The optical den-
sity of the released dye solution was determined
at 620 nm. The percentage of cell viability was
determined by comparing the resulting absor-
bances (620 nm) with the mean absorbance of
the control wells (without AEE, considered as
100 % viability) (Bustillo et al., 2012). The 50
% cytotoxic concentration (CC50) was defined
as the concentration of AEE required to reduce
viability by 50 %. The values of the percent-
ages of cell viability were plotted against the
logarithms of toxins concentrations, and CC50
was determined by linear regression analysis.
Determination of apoptosis by TUNEL:
Apoptosis was assessed using the in situ Cell
Death Detection Kit, AP (Roche Diagnostics).
AEE4 (10-30 µg/ml) was selected for treatment
due to its higher content of juliprosine and
juliprosopine alkaloids. After a 48 h exposure,
treated cells on coverslips were washed twice
with PBS and fixed with 4 % paraformaldehyde.
Cells were then washed with PBS, permeabi-
lized with 0.1 % Triton X-100 in a 0.1 % sodium
citrate solution, and incubated with the termi-
nal deoxynucleotidyl transferase-mediated nick
end labeling (TUNEL) reaction mixture for 60
minutes at 37 °C. Following three additional
PBS washes, Converter-POD was applied for 30
minutes at 37 °C. Immunostaining was devel-
oped using DAB (3,3’-diaminobenzidine tetra-
hydrochloride), and the reaction was stopped
by immersion in deionized water. The slides
were then counterstained with hematoxylin.
Apoptotic cells were quantified in 10 randomly
selected representative fields at 40X magnifica-
tion using a Primo Star Zeiss light microscope
equipped with an AxiocamERc 5s Zeiss digital
camera. Results were expressed as a percentage
of total nuclei per field.
Statistical analysis: All values are pre-
sented as mean ± standard deviation (SD). Data
represent the results of at least three indepen-
dent experiments, each performed in triplicate.
Statistical significance was assessed using two-
way ANOVA, followed by Tukey test for mul-
tiple comparisons. Analyses were performed
using Infostat software (Di Rienzo et al., 2019),
with significance set at p ≤ 0.05.
RESULTS
Chemical composition: The chemical
composition of N. flexuosa pod samples for
each year is presented in Table 2. All samples
exhibited relatively similar contents of dry mat-
ter, humidity, energy digestibility (ED), ash,
and ether extract contents, with no statistically
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significant differences observed across the four
years. However, crude protein content varied
from 7.60 ± 0.35 to 9.89 ± 0.89 %, with the
lowest values observed in 2020 (p < 0.05). Acid
detergent fiber (ADF) and neutral detergent
fiber (NDF) contents ranged from 15.70 ±
0.60 to 23.42 ± 0.71 % and from 28.24 ± 0.30
to 42.07 ± 0.32 %, respectively, with the lowest
values recorded in 2021 and the highest in 2022
(p < 0.05). Hemicellulose content followed the
same trend and showed significant variation,
with the highest value in 2022 (p < 0.05). In
addition, total digestible nutrients (TDN) also
varied significantly over the years, ranging from
73.85 to 80.00 % (p < 0.05).
Identification and Quantification of
Alkaloids in N. flexuosa Enriched Extracts:
Samples of each N. flexuosa extract (AEE1,
AEE2, AEE3 and AEE4) were analyzed by
HPLC-HRMS detecting juliprosine and julip-
rosopine at different concentrations (Table 3).
Fractionation of AEE4 by flash chro-
matography and analysis of piperidinic
alkaloids: Based on the identification and
quantification of alkaloids in the AEEs, AEE4
was selected for fractionation. Analytical thin
layer chromatography (TLC) analysis of the
AEE4 fractions (F1-F5), using Dragendorff’s
reagent as developer, confirmed the presence of
alkaloids in all samples. HPLC-HRMS analysis
detected juliprosine and juliprosopine in all
fractions at different concentrations, with the
highest levels observed in F4 (Table 4).
Table 2
Chemical composition of N. flexuosa pods by year.
Year
Components 2019 2020 2021 2022
Humidity (%) 6.13 ± 0.36 5.19 ± 0.42 6.03 ± 0.28 6.09 ± 0.21
Dry matter (%) 92.59 ± 0.42 94.80 ± 1.06 96.04 ± 0.57 93.90 ± 0.60
Crude Protein (%) 9.00 ± 0.40 7.60 ± 0.35* 9.56 ± 0.62 9.89 ± 0.89
Acid detergent fibre (%) 18.42 ± 0.28 19.58 ± 0.69 15.70 ± 0.60* 23.42 ± 0.71*
Neutral detergent fibre (%) 30.00 ± 0.31* 33.09 ± 0.17* 28.24 ± 0.30* 42.07 ± 0.32*
Hemicelulosa (%) 11.58 ± 0.38* 13.51 ± 0.30 12.54 ± 0.37 18.65 ± 0.20*
TDN (%) 77.84 ± 0.42 76.91 ± 0.40 80 ± 0.40* 73.85 ± 0.32*
ED (kcal/kg) 3.43 ± 0.27 3.39 ± 0.11 3.53 ± 0.20 3.62 ± 0.21
Ash (%) 3.81 ± 0.24 3.76 ± 0.20 3.73 ± 0.15 3.60 ± 0.14
Ether Extract (%) 1.76 ± 0.07 1.66 ± 0.09 1.65 ± 0.03 1.75 ± 0.08
* Significant differences among the four years analyzed (p < 0.05).
Table 4
Alkaloid quantification by HPLC-HRMS in N. flexuosa
AEE4 Fractions.
AEE4 fractions JPS (µg/mg) JPSP (µg/mg)
F1 0.44 0.02
F2 2.05 0.58
F3 0.26 0.35
F4 33.5 104.1
F5 8.7 7.7
JPS: Juliprosine; JPSP: Juliprosopine.
Table 3
Alkaloid determination by HPLC-HRMS
in N. flexuosa extracts over 4 years.
N. flexuosa JPS (µg/mg) JPSP (µg/mg)
AEE1 (2019) 128 19
AEE2 (2020) 109 21
AEE3 (2021) 51 7
AEE4 (2022) 222 42
JPS: Juliprosine; JPSP: Juliprosopine.
Cytotoxic effects of Alkaloid-Enriched
Extracts (AEEs) and fractions of AEE4: The
results show a dose-dependent reduction in cell
viability for all extracts, indicating a cytotoxic
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effect (Fig. 1). The corresponding CC50 val-
ues are presented in the inset table (Fig. 1),
with AEE4 exhibiting the highest cytotoxic-
ity, followed by AEE1, AEE2 and AEE3, which
showed the lowest cytotoxic effects. These find-
ings indicate that AEE4 has the highest cyto-
toxic activity on C6 cells compared to the
other extracts.
On the other hand, the cytotoxicity of
AEE4 fractions on C6 cells was also assessed
using the crystal violet dye uptake assay. While
F4 exhibited dose-dependent cytotoxicity, none
of the assayed doses of F1, F2, F3 or F5 evi-
denced any significant effects on cell viability
after 48 hours of incubation (p < 0.05) (Fig. 2).
Apoptosis in AEE4 of N. flexuosa by
TUNEL assay: To determine whether the
AEE4-induced reduction in cell viability was
associated with apoptosis, C6 glioma cells were
Fig. 1. Cytotoxic activity of AEEs from Neltuma flexuosa (0-50 μg/ml) on C6 glioma cells after 48 h assessed by crystal violet
cell uptake assay in three independent experiments, *p < 0.05 differences compared to control. Inset: Cytotoxic concentration
(CC50) values.
Fig. 2. Cytotoxic activity of fractions F1-F5 from AEE4 of Neltuma flexuosa (2.5, 5 and 10 μg/ml) on C6 glioma cells.
Each column represents mean ± SD of triplicates of three independent experiments, *p < 0.05 differences compared to the
control group.
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treated with 10-30 µg/ml of AEE4 for 48 hours
and analyzed by TUNEL staining. The extract
exhibited a dose-dependent increase in the
percentage of apoptotic cells. Specifically, treat-
ment with 10, 20 and 30 µg/ml of AEE4 resulted
in apoptosis rates of 35.1 ± 3.5 %, 48.12 ± 5.5 %
and 63.6 ± 3.1 % respectively, whereas the con-
trol group displayed only 18.08 ± 2.1 % apop-
totic cells (Fig. 3).
DISCUSSION
Species of the genus Neltuma are multi-
purpose trees that thrive in arid and semi-arid
regions (Verga, 2009) and are valuable forage
resources due to their nutritional composition
(Riet-Correa et al., 2012). Their high tolerance
to drought and salinity, along with notable
growth capacity, reinforces their agronomic
importance in regions with environmental con-
straints (Pasiecznik et al., 2001). In this context,
the present study focused on evaluating the
chemical composition, alkaloid content, and
cytotoxic potential of N. flexuosa pods col-
lected over four consecutive years (2019-2022)
in Cafayate, a representative dryland region in
Northwestern Argentina.
Our results revealed substantial year-to-
year differences in pod composition, particu-
larly in crude protein, neutral detergent fiber
(NDF), and acid detergent fiber (ADF) content.
For instance, NDF ranged from 28 to 42 %,
reaching its highest level in 2022, while protein
content was lowest in 2020. These fluctuations
may be linked to environmental factors, such
as the progressive decline in rainfall observed
Fig. 3. TUNEL assay for apoptosis detection in C6 glioma cells. A. Control and AEE4-treated cells after 48 h: B. 10 µg/mL,
C. 20 µg/mL, D. 30 µg/mL. Apoptotic nuclei are indicated by black arrows; non-apoptotic (normal) nuclei are indicated by
white arrows. Scale bar = 10 μm.
9
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025225, enero-diciembre 2025 (Publicado Nov. 03, 2025)
during the study period (from 260 mm in 2019
to 130 mm in 2022), despite relatively stable
temperatures. Previous studies have shown that
drought can promote fiber accumulation and
affect protein content in legumes which sup-
ports this interpretation (Furlan et al., 2022;
Ressaissi et al., 2023).
Notably, the highest NDF values observed
in N. flexuosa pods were comparable to those
reported for soft wheat bran (42.65 %, AACC),
and exceeded those described for other Nel-
tuma species, reinforcing their potential value
as a fiber-rich feed. Protein content, although
moderately variable (7.60-9.89 %), remained
within acceptable ranges for ruminant supple-
mentation, albeit lower than values reported
for species like N. alba (up to 17 %) (Freire et
al., 2003; Zolfaghari & Harden, 1985). Total
digestible nutrients (TDN) ranged from 74 to
80 %, aligning with values for mature pods of
N. juliflora and N. laevigata (Baraza et al., 2008;
Sawal et al., 2004), confirming their potential
as an energy source. These variations in nutri-
tional composition may also be influenced by
factors such as pod maturity at harvest, soil fer-
tility, and environmental conditions during the
growing seasons (Rostagno & Degorgue, 2011).
The adverse effects of antinutritional com-
pounds, such as alkaloids, in N. flexuosa pods
have been previously reported (Cholich et al.,
2021). In this context, several studies have
described toxic effects on the nervous system
in goats (Assis et al., 2009; Lima et al. 2004;
Tabosa et al., 2000). However, one study found
that sheep and goats did not exhibit signs
of toxicity after consuming N. juliflora pods
(Riet-Correa et al., 2012). The authors pro-
posed several explanations for this discrepancy,
including variations in pod toxicity due to dif-
ferences in storage conditions and chemical
composition across harvests, which may reduce
toxicity under specific circumstances. In the
present study, N. flexuosa extracts obtained
over a four-year period exhibited marked vari-
ability in the concentrations of the piperi-
dine alkaloids juliprosine and juliprosopine,
with juliprosine consistently representing the
predominant compound.
The interannual variability observed in
both the nutritional composition and alka-
loid content of N. flexuosa pods appears to
be closely associated with climatic conditions
recorded during the study period. Notably,
the year with the lowest rainfall (2022) corre-
sponded to the highest concentrations of julip-
rosine and juliprosopine, as well as elevated
NDF values. This pattern aligns with previ-
ous findings indicating that water deficit can
induce physiological adjustments in legumes
and woody species, promoting the accumula-
tion of secondary metabolites and structural
carbohydrates (Santos et al., 2022). Similar
observations were reported by Honório et al.
(2021), who demonstrated that drought stress
in Annona crassiflora increased alkaloid bio-
synthesis. In Prosopis juliflora, Al-Soqeer et al.
(2023) described significant differences in pod
composition between agro-climatic regions
with contrasting precipitation levels, highlight-
ing the impact of environmental variability
on nutritional and toxicological profiles. The
relatively stable temperatures recorded across
years suggest that precipitation was the primary
climatic factor influencing pod composition in
this study. Collectively, these results underscore
the importance of monitoring environmental
conditions when evaluating the suitability of N.
flexuosa pods as a feed resource and assessing
their potential risks.
In addition to these compositional varia-
tions, the cytotoxic effects of the extracts were
also assessed. Previous studies have reported
deleterious effects of Neltuma species, includ-
ing N. juliflora leaves and N. alpataco pods, with
low CC50 values of 31.07 μg/ml and 24.69 μg/
ml, respectively (Cholich et al., 2024; Silva et
al., 2013). Our findings align with these reports;
however, this is the first study to link cytotoxic-
ity to alkaloid content. Notably, the extract with
the highest alkaloid concentration exhibited the
lowest CC50.
In a related study, fractionation of a total
extract from N. juliflora identified one fraction
as the most cytotoxic (Silva et al., 2007), with
further analysis revealing that it was a mixture
of the alkaloids juliprosine (minor component)
10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025225, enero-diciembre 2025 (Publicado Nov. 03, 2025)
and juliprosopine (major component) (Silva et
al., 2013). Our results also showed that only
one fraction (F4) from the AEE4 extract of N.
flexuosa exhibited a dose-dependent cytotoxic
effect on the C6 cell line. In agreement with
Silva et al. (2013), HPLC-HRMS analysis iden-
tified juliprosopine as the most abundant alka-
loid in this fraction. The selective enrichment
of alkaloids in F4 suggests that its chemical
composition may facilitate the accumulation
of specific piperidine alkaloids. These find-
ings highlight the potential of fractionation to
isolate and concentrate bioactive compounds,
which can be useful for toxicological studies on
the active alkaloids of N. flexuosa.
Our previous work demonstrated that N.
alpataco extracts induced apoptosis followed
by secondary necrosis in C6 cells, as observed
using acridine orange/ethidium bromide fluo-
rescence staining. Additionally, N. juliflora has
been shown to induce apoptosis in neuron/
glial cell co-cultures (Silva et al., 2017), as
well as in various tumor cell lines, including
MDA-MB-231, MCF-7, LS-174T, and HepG2
(Abbas et al., 2022; Utage et al., 2018). In
line with these studies, the total extract of N.
juliflora triggered cell death in primary glial
cells through caspase-9 activation and nuclear
condensation (Silva et al., 2017). In the pres-
ent study, apoptotic markers increased in a
dose-dependent manner over 48 hours, further
supporting the role of apoptosis as the primary
cytotoxic mechanism of Neltuma spp. extracts.
All together, these findings suggest that
reduced precipitation may play a key role in
modulating the chemical profile of N. flexuosa
pods, particularly by promoting the accumula-
tion of fiber and piperidine alkaloids such as
juliprosine and juliprosopine. Although further
studies are needed to elucidate the underlying
mechanisms, our results highlight the impor-
tance of considering environmental variability
as a key driver of both nutritional and toxico-
logical changes in this species. Such insights
could inform the development of guidelines for
the safe use of N. flexuosa pods in animal feed,
thereby helping to prevent poisoning events.
Ethical statement: The authors declare
that they all agree with this publication and
made significant contributions; that there is no
conflict of interest of any kind; and that we fol-
lowed all pertinent ethical and legal procedures
and requirements. All financial sources are fully
and clearly stated in the acknowledgments sec-
tion. A signed document has been filed in the
journal archives.
ACKNOWLEDGMENTS
This work was supported by the Secre-
taría General de Ciencia y Técnica, Univer-
sidad Nacional del Nordeste (UNNE; grant
N° PI22B007) and by the Agencia Nacional
de Promoción Científica y Tecnológica (PICT
2020/01484).
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