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Characterization and cytotoxic activity on glial cells of alkaloid-enriched

extracts from pods of the plants Prosopis flexuosa and

Prosopis nigra (Fabaceae)

Luciana Andrea Cholich

*, María Elena Pistán

, Ana María Torres

, Hugo Héctor Ortega

Dale R. Gardner

& Soledad Bustillo

1. Clinical Department, Faculty of Veterinary Science, National University of the Northeast, Sargento Cabral 2139,

Corrientes, Argentina; lucianaandreacholich@gmail.com, maelpi007@gmail.com

2. Natural Products Laboratory, IQUIBA-NEA CONICET, National University of the Northeast, Av. Libertad 5470,

Corrientes, Argentina; amtorres39@yahoo.com.ar

3. Institute of Veterinary Sciences of Litoral (ICIVET), National University of Litoral (UNL), R.P. Kreder 2805,

Esperanza, Santa Fe, Argentina; hhortega@hotmail.com

4. USDA-ARS Poisonous Plant Research Laboratory, 1150 E 1400 N, Logan, Utah 84341, USA;

dale.gardner@usda.gov

5. Biological and Molecular Investigations Group (GIBYM), IQUIBA-NEA CONICET, National University of the

Northeast, Av. Libertad 5470, Corrientes, Argentina; solebustillo@yahoo.es

* Correspondence

Received 18-VIII-2020. Corrected 12-XI-2020. Accepted 18-XI-2020.

ABSTRACT. Introduction: Prosopis spp. pods have shown to be a potential source of protein and energy in

livestock. However, prolonged ingestion of some of these species produces neurological symptoms in ruminants.

Objective: In the present study, the alkaloid content and the in vitro neurotoxic activity of alkaloid enriched-

extracts from P. flexuosa and P. nigra pods were determined in order to elucidate the mechanism of animal

poisoning caused by these species. Methods: The main alkaloids present in both extracts were analysed by high

performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). The cytotoxic activ-

ity of Prosopis alkaloid enriched-extracts in primary mixed glial cell culture was assessed by phase contrast

microscopy and using neutral red, and lactate dehydrogenase (LDH) activity assays. Results: Juliprosine and

juliprosopine were identified in P. flexuosa pods, while the absence of these alkaloids in P. nigra was confirmed.

Both extracts (5-30 μg/mL) induced in a dose dependent manner, morphological alterations, such as swelling,

enlargement and detachment from the culture surface. Consistent with this, decrease in cell viability and release

of LDH 48 hours after exposure, revealed that P. flexuosa pods was significantly more cytotoxic than P. nigra.

Conclusions: In P. flexuosa pods, juliprosine and juliprosopine alkaloids were identified for the first time.

Moreover, the present study suggests that the cytotoxic effect displayed by both extracts is due to its alkaloid

content. However, the presence of piperidine alkaloids in P. flexuosa could explain the greater cytotoxicity on

glial cells with respect to P. nigra that was not shown to contain these alkaloids.

Key words: algarrobos; cytotoxicity; piperidine alkaloids; Prosopis species.

Cholich, L.A., Pistán, M.E., Torres, A.M., Ortega, H.H., Gardner, D.R., & Bustillo, S.

(2021). Characterization and cytotoxic activity on glial cells of alkaloid-enriched

extracts from pods of the plants Prosopis flexuosa and Prosopis nigra (Fabaceae).

Revista de Biología Tropical, 69(1), 197-206. DOI 10.15517/rbt.v69i1.43515

ISSN Printed: 0034-7744 ISSN digital: 2215-2075

DOI 10.15517/rbt.v69i1.43515

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Genus Prosopis belongs to the Fabaceae

family, subfamily Mimosoideae, and is an

important tree in arid, semi-arid, tropical and

subtropical regions of the world. Most species

are found in the Chaco biogeographical region

of South America (Schmeda-Hirschmann,

Theoduloz, Jiménez-Aspee, & Echeverría,

2020). Specifically, in Argentina, P. nigra

and P. flexuosa are geographically distributed

along with P. alba, P. hassleri and P. chilens-

sis, throughout different ecoregions, including

Chaco, Espinal Norte and Pampeana, which

are locally known as “Algarrobos” (Palacios &

Brizuela, 2005).

The pods of this genus are a feed source

for animals in many regions of the world, due

to their nutritional value (Silva, 1981; Riet-

Correa et al., 2012). Recently an outbreak of

nervous disease has been reported in cattle

associated with consumption of Prosopis sp.

pods for which clinical signs include tongue

protrusion, twitches and tremors of muscles of

mastication, weight loss and lethargy (Miche-

loud, Caro, Cholich, Martinez, & Gimeno,

2019). These clinical signs are similar to those

reported in intoxicated animals by P. juliflora

from Brazil (Lima, Riet-Correa, Amorin, &

Sucupira, 2004; Tabosa, Riet-Correa, Simoes,

Medeiros, & Nobre, 2004; Silva, Riet-Correa,

Medeiros, & Oliveira, 2006; Tabosa et al.,

2006; Assis, Medeiros, Araujo, Dantas, & Riet-

Correa, 2009; Câmara et al., 2009).

Prosopis species are reported to produce a

number of different alkaloids including simple

phenolic amines such as phenethylamine, tyra-

mine and tryptamine from fruit of P. nigra and

aerial parts of P. flexuosa (Moro, Graziano,

& Coussio, 1975; Tapia et al., 2000; Pérez et

al., 2014). Besides, more complex molecules

such as the piperidine alkaloids: 2-β-methyl-3-

β-hydroxy-6-β-piperidinedodecanol, juliproso-

pine and juliprosine were isolated from leaves

of P. flexuosa (Tapia et al., 2000). Juliproso-

pine, juliprosine and juliprosinene have been

identified as the main toxins from P. juliflora

pods and leaves of Brazil (Ahmad, Sultana, &

Qazi, 1989; da Silva, da Silva, Silva, & Costa,

2018; Tabosa et al., 2000).

Although, the usage of Prosopis pods are

among the oldest foods used by the Amerindian

for animal feeding, specifically in the North

and center of Chile, Northwestern Argentina

and Southern Peru, little is known about the

composition of these Algarrobo pods (Schm-

eda-Hirschmann et al., 2020). In vitro studies

have shown that alkaloidal extract or alkaloid

fraction of the pods and leaves of P. juliflora

have a direct action in central nervous system

(Hughes et al., 2005; Silva et al., 2007; Silva

et al., 2013; Silva et al., 2017). However,

toxicological analyses of pods of species from

P. flexuosa and P. nigra, along with in vitro

cytotoxic activity of plant extracts have yet to

be reported.

The aim of this study was to characterize

the alkaloid content and in vitro neurotoxic

activity of alkaloid enriched extracts from Pro-

sospis flexuosa and Prosopis nigra pods in

order to elucidate the mechanism of animal

poisoning caused by Prosopis sp.

MATERIALS AND METHODS

Plant Material: P. flexuosa subsp. flex-

uosa pods were collected from Salta, North-

west of Argentina, while P. nigra subsp. nigra

pods were collected from Chaco, Northeastern

of Argentina. Voucher herbarium specimens

(CTES-1193, P. flexuosa and CTES-993, P.

nigra) were identified by Lic. Walter Medina

and deposited at the “Instituto de Botánica”

from Facultad de Ciencias Agrarias (UNNE-

CONICET) in Corrientes, Argentina. The pods

were dried at 37 °C to a constant weight and

then pulverized in a mill.

Preparation of alkaloid-enriched

extracts from P. flexuosa and P. nigra: Alka-

loid-enriched extracts were obtained from

500 g of P. flexuosa and P. nigra pulverized

pods by an acid/basic extraction, as previ-

ously described by Ott-Longoni, Viswanathan,

& Hesse (1980). Briefly, dry pods samples

were firstly defatted with hexane (1 L) for 10

days and then extracted with methanol (1 L)

for 7 days. The solution was filtered, and the

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methanol extract concentrated under low pres-

sure at 40 °C, stirred with 0.2 N HCl and once

more filtered. The acid extract was washed

with chloroform and basified with ammonium

hydroxide, and then was extracted with chlo-

roform. Subsequently, the chloroform solution

was evaporated to dryness and referred as the

“alkaloid-enriched extract” (AEE).

The quantitative determination of total

alkaloid content in AEEs of P. flexuosa and P.

nigra pods was performed with the bromocre-

sol green method described by Biju, Sulaiman,

Satheesh, & Reddy (2014). Total alkaloids

were calculated as mg of atropine equivalents

(mg AE) per g of extract.

HPLC Analyses: HPLC-HRMS analy-

ses were performed similar to that previously

described dos Santos et al. (2013). The analyti-

cal samples were injected onto a Synergi Hydro

column (75 x 2 mm, 4 µ). Eluting solvents

were acetonitrile (mobile phase A) and 0.05 %

TFA/0.5 % acetic acid (mobile phase B) using

a flow rate of 0.4 mL/min. A linear gradient

started at 5 % (B) from 0-2 min, increasing

to 70 % (B) from 2-15 min, returning to 5 %

(B) at 18 min. The mobile phase was deliv-

ered and samples injected using an Ultimate

3000 HPLC (Thermo Scientific, San Jose, CA,

USA) and the column eluent was connected

directly to the heated electrospray source of an

Q-Exactive high resolution mass spectrometer

(Thermo Scientific, San Jose, CA, USA), oper-

ated in the positive ion mode, and calibrated

per the manufacturer’s instructions with a

scan range 100-1 000 Da, resolution 70 000,

microscans 1, sheath gas flow 35, auxiliary gas

flow 10, spray voltage 4 kV, capillary tempera-

ture 320 °C, S lens RF field 55, and auxiliary

gas temperature 300 °C. Identification of the

alkaloids juliprosine and juliprosopine were

based on comparison of mass spectra with

that previously rerported (dos Santos et al.,

2013) and with HRMS data. Juliprosine: M+

= 626.5628 observed (626.5619 required for

C40H72N3O2; Δ 0.86 ppm). Juliprosopine:

MH+ = 630.5929 observed (630.5932 required;

Δ 0.34 ppm).

Primary Mixed Glial Cell Culture and

treatments: Primary glial cell cultures were

prepared from 1-3 days old mice (CF-1).

Animal protocols were in accordance with the

ethical principles of animal experimentation

and approved by protocol N° 0069/2016 of the

Ethics and Biosafety Committee of UNNE.

Briefly, forebrains free of meninges (N = 5)

were removed aseptically and mechanically

dissociated by repeated pipetting. Dissociated

cells were plated onto poly-D-lysine-coated

25 cm

flasks or 24-well plates for experi-

ments using a micro-full medium: Dulbecco’s

minimum essential medium (DMEM-F12)

supplemented with fetal bovine serum (FBS

10 %), (Natocor), non-essential amino acids

(MEM NEAA 100 X-1 %), L-Glutamine (1

%), Gentamicin (10 µg/mL) and Penicillin-

Streptomycin (1 %). Cells were incubated at 37

°C and 5 % CO

, medium was replaced every

5 days and confluency was achieved after 18 ±

3 days in vitro (DIV). Unless stated otherwise,

reagents were purchased from Gibco (Buenos

Aires, Argentina), or Sigma-Aldrich (St. Louis,

MO, USA).

Culture purity of mixed glial isolation was

determined by immunocytochemistry using

specific markers for microglia (Iba-1) and

astrocytes (GFAP).

After 18 ± 3 days in vitro, glial cells

were harvested from subconfluently monolay-

ers after exposure to 0.25 % trypsin/EDTA (1

X) (Gibco) at 37 °C. The resuspended cells

were seeded in 96-well microplates at an

approximate initial density of 40×103 cells per

well, in growth medium (DMEM-F12, 10 %

FBS). When monolayers reached confluence,

the media was changed to include AEE from

P. flexuosa or P. nigra, which were previously

dissolved in 0.1 % dimethylsulfoxide (DMSO,

Sigma, St. Louis, MO), at different concentra-

tions (5 to 30 μg/ml) in medium supplemented

with 10 % of FBS, and were added to cells in

a total volume of 200 µL/well. Plates were fur-

ther incubated for 48 h at 37 °C and 5 % CO

Control cells were treated with DMSO diluted

in culture medium at the higher equivalent vol-

ume used in the treated groups.

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Morphological analysis: After 48h incu-

bation, cells were observed by phase contrast

microscopy (Axiovert 40®, Carl Zeiss Argenti-

na) to evaluate morphological changes induced

by AEEs. The images were taken with a digital

camera (Canon CCD 2272 × 1704, Argentina)

before and after treatments.

Neutral red assay: Cell viability was

quantified after 48 h by neutral red (NR)

incorporation, as described Repetto, del Peso,

& Zurita (2008), with slight modifications.

Briefly, culture medium was removed and 200

μL of DMEM without FBS and containing 4

mg/mL of NR (Sigma Aldrich) in 1:100 dilu-

tion was added to each well. After 2 h of incu-

bation, medium was removed and cells were

washed with buffered saline solution (PBS).

Subsequently, 200 μL of 1 % acetic acid in 50

% ethanol were added for dye solubilization

and gently shaken for 10 minutes for full dis-

solution. The absorbance was measured at 540

nm with a microplate spectrophotometer. The

percentage of cell viability was determined by

comparison with the mean absorbance of con-

trol wells (without AEEs), considered as 100 %.

Cytolysis assay: An additional assay was

performed to assess cytolysis determining the

release of the cytosolic enzyme lactic dehy-

drogenase (LDH), as previously described by

Lomonte, Tarkowski, & Hanson (1994). Ali-

quots of the supernatant in culture wells were

collected, and LDH activity was determined by

using a commercial kit (Wiener, LDH-P UV).

Enzymatic activity was expressed as LDH

(IU/L) activity relative to that of non-treated

cells. All assays were carried out in triplicates.

Statistical analysis: The values obtained

were expressed as the mean ± standard devia-

tion (SD). Data represents results from at

least three separate experiments, each per-

formed in triplicate. Differences among means

were tested for statistical significance using

a double-way ANOVA followed by Tukey’s

test. Statistical analysis was performed using

Infostat software. The level of significance was

set at P ≤ 0.05.

RESULTS

Analysis of alkaloids in P. flexuosa and

P. nigra enriched extracts: The total alka-

loid content in AEEs of P. flexuosa and P.

nigra were expressed in milligrams atropine

equivalent (mg AE). The results showed val-

ues of 200.3 mg AE/g AEE and 34.5 mg

AE/AEE respectively.

In order to determine the main alkaloids

from Prosopis species pods, samples of AEEs

were analysed by HPLC-HRMS. In Fig. 1, the

extract of P. flexuosa pods revealed juliprosine

m/z = 626 [M]

to be the major alkaloid detect-

ed. Juliprosopine, m/z = 630 [M + H]

, was also

detected in a relative concentration of approxi-

mately 15 % compared to juliprosine. Regard-

ing the extract of P. nigra pods, no juliprosine

(Rt = 9.34 min) or juliprosopine (Rt = 9.74)

alkaloids were detected from the HPLC-MS

chromatogram (Fig. 2) or indicated by ions at

m/z 626 or 630 (Fig. 3), confirming the absence

of these alkaloids in the extract of P. nigra. No

evidence of the previously reported alkaloids

for P. nigra (β- phenethylamine, eleagine, har-

man, tryptamine, N-acetyltryptamine and tyra-

mine) (Moro et al., 1975) were observed from

the HPLC-MS data and the alkaloids found in

low concentration by the bromocresol green

method could not be identified.

Cytotoxicity of P. flexuosa and P. nigra

alkaloid enriched extracts on mixed glial

cells: Mixed glial cultures were morphologi-

cally identified and a predominance of microg-

lia and astrocytes was demonstrated (Fig. 4A).

When P. flexuosa and P. nigra AEEs were

incubated at different concentrations with glial

cells, morphological alterations included swell-

ing, enlargement, and often detachment from

the well surface were observed. Severely dam-

aged cells, with membrane rupture and cell

debris released into the medium were detected.

These morphological alterations were more

evident at the highest doses tested and for those

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Fig. 1. HPLC-HRMS chromatogram obtained for alkaloid enriched-extract of P. flexuosa pods.

Fig. 2. HPLC-HRMS chromatogram obtained for alkaloid enriched-extract of P. nigra pods.

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Fig. 3. Mass spectra of the region (Retention time 8-10.5 min) for the alkaloid enriched-extracts

of P. flexuosa and P. nigra pods.

Fig. 4. Morphological analysis of primary mixed glial cells under phase contrast microscopy after 48h of incubation with

alkaloid enriched extracts. A. Control (x 200 magnification). B. P. flexuosa (30 μg/mL - x200). C. P. nigra (30 μg/mL-x

200 magnification).

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cultures incubated with AEE from P. flexuosa

(Fig. 4B, Fig. 4C).

Coincident with the observed morpho-

logical changes, neutral red assay demonstrated

that both extracts induced a dose-dependent

cytotoxicity, being AEE from P. flexuosa sig-

nificantly (P < 0.01) more toxic than control

and AEE from P. nigra. The difference in

decreased cell viability was again more evident

at the highest dose tested (30 µg/mL), being

approximately 70 % for P. flexuosa and 30

% for P. nigra (Fig. 5). The release of lactate

dehydrogenase confirmed the aforementioned

cytotoxic effects and the differences between

extracts, also indicating an alteration of the cell

membranes after 48 h of incubation (Fig. 6).

DISCUSSION

Prosopis sp. represents an important forest

resource. However, some species can become

a weed in native vegetation areas and induce

spontaneous poisoning in animals, especially

when it is used as the sole source of alimenta-

tion and during extreme drought conditions

(Riet-Correa et al., 2012; Kumar & Mathur,

2014; Ruiz-Nieto et al., 2020). Tabosa et al.

(2006), reports neurotoxic damage to the cen-

tral nervous system (CNS) in cattles fed during

6 months with a ration containing 50-75 % of

P. juliflora pods from Brasil after. Similarly,

in Argentina, Micheloud et al. (2019) have

described animal poisoning with nervous clini-

cal signs. However, it could not be specifically

confirmed that the intoxication was due to the

Prosopis species.

On the other hand, several in vitro studies

demonstrate that some Prosopis species such

as P. flexuosa, P. nigra, P. juliflora, P. ciner-

aria and P. strombulifera, exert antioxidant,

anti-inflammatory, antimicrobial, anticancer,

antidiabetic effects and enzyme inhibition asso-

ciated with metabolic syndrome (Hapon et al.,

2014; Henciya et al., 2017; Pérez et al., 2014;

Soni et al., 2018; Tapia et al., 2000). Neverthe-

less, no documented results are available about

the in vitro toxicological effects of the pods of

P. flexuosa and P. nigra.

The identification of toxic principles in

pods from Prosopis species is necessary to

limit the amount of this feed resource in live-

stock nutrition, for the prevention of animal

poisoning. Thereafter, in this study we have

identified the main alkaloids present and the

cytotoxicity on glial cells of alkaloid extracts

from pods of two species of Prosopis, widely

distributed in Argentina and in other countries

of South America (Palacios & Brizuela, 2005).

Juliprosine and juliprosopine alkaloids, had

been reported as present in P. flexuosa leaves

(Tapia et al., 2000). The analysis conducted

here by HPLC-HRMS confirmed the occur-

rence of these alkaloids in the pods of this plant

as well. According to dos Santos et al. (2013),

juliprosine and juliprosopine were the most

Fig. 5. Cell viability determined by neutral red uptake

assay. Each column represents mean ± SD of triplicates,

in three independent experiments, * P < 0.01 differences

compared to the control and

P < 0.01 P. flexuosa

vs P. nigra.

Fig. 6. LDH activity (UI/L). Each column represents mean

± SD of triplicates, in three independent experiments, * P <

0.01 differences compared to the control and

P < 0.01 P.

flexuosa vs P. nigra.

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abundant chemical compounds present in the

pods of P. juliflora. Conversely, juliprosine and

juliprospine were not found in t in the AEE of

P. nigra pods. Through these results, we would

rule out P. nigra as the cause of the spontane-

ous poisoning in animals.

Cytotoxic results here obtained, demon-

strate that AEEs from pods of P. flexuosa and P.

nigra induce a dose-dependent effect in prima-

ry mixed glial culture. Morphological changes

were compatible with the necrosis cell death

mechanism and included enlarged cells, detach-

ment and disruption of plasma membrane. This

cell membrane rupture resulted in the release

of cytoplasmic lactate dehydrogenase (LDH)

and confirmed the cytolysis triggered by these

AEEs. Decrease of cell viability, cell morpho-

logical alterations and the evidence of cytolysis

after incubation with AEEs, indicated that P.

flexuosa was more cytotoxic than P. nigra at all

doses tested. This difference could be attribut-

able to the presence of piperidine alkaloids in P.

flexuosa and the low alkaloid content detected

in P. nigra. In addition, pods of P. flexuosa

induced a cytotoxic effect similar to P. juliflora

from Brazil, which comparably releases LDH

when cells cultures were exposed at equivalent

extract concentrations (Hughes et al., 2005;

Silva et al., 2007).

In conclusion, we have described, for the

ﬁrst time, the presence of juliprosine and julip-

rosopine alkaloids in P. flexuosa pods. On the

other hand, the present study suggest that the

cytotoxic effect displayed by both AEEs from

P. flexuosa and P. nigra pods are due to its alka-

loid content. Furthermore, the presence of these

piperidine alkaloids in P. flexuosa pods could

explain the greater cytotoxicity on glial cells

with respect to P. nigra that was not shown

to contain these alkaloids. Further studies are

needed to elucidate the molecular mechanism

for these triggered toxic effects.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict 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 acknowledge-

ments section. 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° PI17B007) and by the Agencia Nacional

de Promoción Científica y Tecnológica (PICT

2016/1225). LACh, MEP and HHO are mem-

bers of CONICET (National Scientific and

Technical Research Council, Argentina).

RESUMEN

Caracterización, y actividad citotóxica en célu-

las gliales, de extractos enriquecidos con alcaloides

de vainas de las plantas Prosopis flexuosa y Prosopis

nigra (Fabaceae). Introducción: Las vainas de diversas

especies de Prosopis muestran ser una potencial fuente

de proteínas y energía para el ganado. Sin embargo, la

ingestión prolongada de algunas de estas especies produce

síntomas neurológicos en los rumiantes. Objetivo: En el

presente estudio se determinó el contenido de alcaloides y

la actividad neurotóxica in vitro de los extractos enrique-

cidos con alcaloides obtenidos en las vainas de P. flexuosa

y P. nigra, con el fin de dilucidar el mecanismo de la

intoxicación animal causada por estas especies. Métodos:

Los principales alcaloides presentes en ambos extractos

se analizaron mediante cromatografía líquida de alto

rendimiento-espectrometría de masas de alta resolución

(HPLC-HRMS). La actividad citotóxica de los extractos

enriquecidos con alcaloides de Prosopis se determinó en

cultivos primarios de células gliales mixtas y se evaluó

mediante microscopía de contraste de fase y utilizando

ensayos de actividad de rojo neutro y de deshidrogenasa

láctica (LDH). Resultados: Se identificaron la juliprosina

y la juliprosopina en las vainas de P. flexuosa, mientras que

se confirmó la ausencia de estos alcaloides piperidínicos

en P. nigra. Ambos extractos (5-30 μg/mL) indujeron, de

manera dependiente a la dosis, alteraciones morfológicas,

como hinchazón, agrandamiento y desprendimiento de la

superficie de cultivo. En consecuencia, la disminución de

la viabilidad celular y la liberación de la LDH después de

48 horas de exposición, reveló que las vainas de P. flexuosa

eran significativamente más citotóxicas que las de P. nigra.

Conclusiones: El presente estudio muestra la presencia de

los alcaloides juliprosina y juliprosopina en vainas de P.

flexuosa y sugiere que el efecto citotóxico mostrado por

ambos extractos se debe al contenido de alcaloides. Sin

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embargo, la presencia de estos alcaloides piperidínicos en

P. flexuosa podría explicar la mayor citotoxicidad en las

células gliales con respecto a P. nigra que no mostró que

tuviera estos alcaloides.

Palabras clave: alcaloides piperidínicos; algarrobos; cito-

toxicidad; Prosopis species.

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