Journal of Ethnopharmacology 255 (2020) 112722

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Consumption of latex from Euphorbia tirucalli L. promotes a reduction of T

tumor growth and cachexia, and immunomodulation in Walker 256 tumor-
bearing rats
Carolina G. Martinsa,b,c, Marcia H. Appeld, Débora S.S. Coutinhoc, Igor P. Soaresc,
Stefani Fischerc, Bruna C. de Oliveirac, Mariana M. Fachie, Roberto Pontaroloe,
Sandro J.R. Bonattoa,b, Luiz Claudio Fernandesc, Fabíola Iagherc,∗, Lauro M. de Souzaa,b,∗∗
a
Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil
b
Faculdades Pequeno Príncipe, Curitiba, PR, Brazil
c
Department of Physiology, Federal University of Paraná, Curitiba, PR, Brazil
d
Department of Structural Biology, Molecular and Genetics, State University of Ponta Grossa, Ponta Grossa, PR, Brazil
e
Department of Pharmacy, Federal University of Paraná, Curitiba, PR, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacological relevance: Euphorbia tirucalli L. is an African plant that grows well in Brazil. Individuals
Euphorbia tirucalli L. diagnosed with cancer frequently consume latex from E. tirucalli, dissolved in drinking water. In vitro studies
Latex confirm the antitumor potential of E. tirucalli latex, but in vivo evaluations are scarce.
Oral treatment Aim of the study: To evaluate the effect of intake of an aqueous solution of E. tirucalli latex on tumor growth,
Cancer
cachexia, and immune response in Walker 256 tumor-bearing rats.
Cell proliferation
Immune response
Materials and methods: Latex from E. tirucalli was collected and analyzed by LC-MS. Sixty male Wistar rats (age,
90 days) were randomly divided into four groups: C, control group (without tumor); W, Walker 256 tumor-
bearing group; SW1, W animals but treated with 25 μL latex/mL water; and SW2, W animals but treated with
50 μL latex/mL water. Animals received 1 mL of latex solution once a day by gavage. After 15 d, animals were
euthanized, tumor mass was determined, and glucose and triacylglycerol serum levels were measured by using
commercial kits. Change in the body weight during tumor development was calculated, and proliferation ca-
pacity of tumor cells was assessed by the Alamar Blue assay. Phagocytosis and superoxide anion production by
peritoneal macrophages and circulating neutrophils were analyzed by enzymatic and colorimetric assays. Data
are analyzed by one-way ANOVA followed by Tukey's post-hoc test, with the significance level set at 5%.
Results: The analysis of the latex revealed the presence of triterpenes. The ingestion of the latex aqueous solution
promoted 40% and 60% reduction of the tumor mass in SW1 and SW2 groups, respectively (p < 0.05). The
proliferative capacity of tumor cells from SW2 group was 76% lower than that of cells from W group
(p < 0.0001). Animals treated with latex gained, on average, 20 g (SW1) and 8 g (SW2) weight. Glucose and
triacylglycerol serum levels in SW1 and SW2 animals were similar to those in C group rats. Peritoneal macro-
phages and blood neutrophils from SW1 and SW2 animals produced 30–40% less superoxide anions than those
from W group animals (p < 0.05), but neutrophils from SW2 group showed an increased phagocytic capacity
(20%, vs. W group).
Conclusions: E. tirucalli latex, administered orally for 15 d, efficiently reduced tumor growth and cachexia in
Walker 256 tumor-bearing rats. Decreased tumor cell proliferative capacity was one of the mechanisms involved
in this effect. Further, the data suggest immunomodulatory properties of E. tirucalli latex. The results agree with
folk data on the antitumor effect of latex ingestion, indicating that it may be useful as an adjunct in the treatment
of cancer patients. For this, further in vivo studies in animal and human models need to be conducted.

∗
Corresponding author. Departament of Physiology, Federal University of Paraná, Av. Cel. Francisco H. dos Santos, 100, Centro Politécnico, 81530-900, CP: 19031,
Curitiba, PR, Brazil.
∗∗
Corresponding author. Instituto de Pesquisa Pelé Pequeno Príncipe, Av. Silva Jardim, 1632, 80250-060, Curitiba, PR, Brazil.
E-mail addresses: fabiola.iagher@ufpr.br (F. Iagher), laurosouza@hotmail.com, lauro.souza@pelepequenoprincipe.org.br (L.M. de Souza).

https://doi.org/10.1016/j.jep.2020.112722
Received 23 December 2019; Received in revised form 17 February 2020; Accepted 25 February 2020
Available online 28 February 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
C.G. Martins, et al.
1. Introduction
Despite efforts of the scientific community to prevent cancer, and to
identify new substances for the treatment of cancer and the associated
cachexia, millions of people worldwide die every year from cancer.
Further, the available treatments have several side effects and re-
sistance to chemotherapy is a serious problem that limits treatment
efficacy. In addition, in many cases, access to the appropriate treatment
is not possible, especially in countries with high level of social in-
equality. In this context, the use of plants and their products for the
treatment of tumor based on empirical knowledge emerges as an easily
accessible resource.
Euphorbia tirucalli L. [syn. Euphorbia tirucalli var. rhipsaloides
(Willd.) A. Chev], is also commonly known as “Avelós”, “Aveloz” or
“Pencil Tree” (Valadares et al., 2006), originates from Africa, but also
grows in Brazil, especially in northeastern Brazil. It is a dicotyledon
from the Euphorbiaceae family (Llanes-Coronel et al., 2011; Hastilestari
et al., 2013). The medicinal use of the plant in various parts of the
world is well documented. For example, indigenous people of Africa
and Asia use latex as a purgative, and for the treatment of rheumatism,
neuralgia, and toothache (Rasool et al., 1989). In Taiwan, stems are
boiled in water and the resultant solution is used as an anticancer drink
(Lin et al., 2012). In Brazil, E. tirucalli latex, which is easily obtained by
breaking the stem, is mainly consumed orally, after dissolution in a
bottle of water by individuals diagnosed with different cancer types.
Despite the widespread use of E. tirucalli latex, only few studies have
focused on the effects of oral consumption of crude latex, which is the
most popular way of using the plant for cancer treatment. The majority
of published information pertains to in vitro experiments, with ob-
servations made using plant extracts or Euphol, a tetracyclic triterpene
alcohol isolated from E. tirucalli latex. Recently, Choene and Motadi
(2016) demonstrated anti-proliferative and pro-apoptotic effects of an
E. tirucalli extract on a breast cancer cell line. Euphol exerts a potent
cytotoxic effect on different tumor cell lines (Lin et al., 2012; Reis et al.,
2013; Wang et al., 2013; Palharini et al., 2017; Silva et al., 2019).
Further, in vivo studies prioritize testing compounds isolated from latex
and their effects on the inflammatory response (Passos et al., 2013;
Dutra et al., 2011, 2012bib_Dutra_et_al_2011bib_Dutra_et_al_2012;
Santana et al., 2014). To the best of our knowledge, no studies have
been published on the consumption of latex aqueous solution in an
animal model of tumor, mimicking the folkloric use of the plant ma-
terial.
Because of tumor growth, individuals with cancer develop cachexia,
a multifactorial syndrome characterized by a progressive loss of body
weight, especially of the skeletal muscle protein and fat. Weight loss is
an important prognostic indicator, and it depends on the characteristics
of both tumor and host cells (Tisdale, 2009). Peripheral hypercatabo-
lism is responsible for the death of many individuals with cancer, and
studies to identify useful biomarkers for the early diagnosis of cancer
cachexia are on-going (Loumaye and Thissen, 2017). No data on the
possible effect of latex from E. tirucalli on cancer cachexia have been
published.
In this study, we aimed to determine the effect of administration of
an aqueous solution of E. tirucalli latex on tumor growth, cachexia, and
immune response in vivo. We aimed at investigating the effect of daily
consumption of the latex as in the traditional preparation, which con-
sists of dissolve drops of latex in a water bottle, that is consumed by
individuals with cancer. Therefore, in our experimental design, we si-
mulated the traditional preparation of latex, wherein latex obtained
from E. tirucalli was dissolved in water and administered orally to
Wistar rats daily, during tumor development, using the 256 Walker
tumor model and analyzed its anti-tumor effect.
2
Journal of Ethnopharmacology 255 (2020) 112722
2. Materials and Methods
2.1. Chemicals and enzymes
Histopaque 1077 and 1119, buffer reagents, and chemicals and
enzymes used in macrophage and neutrophil assays were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Trypan blue was from GE
Healthcare BioSciences (Boston, MA, USA). Alamar Blue was from
Invitrogen (Carlsbad, CA, USA). Biochemical kits were obtained from
BioLiquid (Laborclin; Pinhais, PR, Brazil). Culture medium (RPMI
1640), antibiotics (penicillin and streptomycin), and fetal bovine serum
were obtained from Gibco (Grand Island, NY, USA). High-performance
liquid chromatography (HPLC) solvents were from Sigma-Aldrich.
2.2. Plant material
Euphorbia tirucalli L. and synonymy was verified according to “The
Plant List” (http://www.theplantlist.org) (“Plant List”, 2013). The latex
harvesting was from a species growing in São José dos Pinhais, Paraná,
Brazil (25°33′40.4″S 49°10′25.9″W). A voucher specimen (register
number UPCB-00360) was placed in the Herbarium of the Biological
Sciences Department of the Federal University of Paraná (Curitiba, PR,
Brazil). After the shaft break, the latex was collected in microtubes and
then diluted in water for aqueous solutions of 25 μL/mL and 50 μL/mL.
For the in vivo assays, the latex was collected daily, and the aqueous
solutions were prepared immediately before the treatment of animals.
2.3. Liquid chromatography-mass spectrometry (LC-MS)
The latex from E. tirucalli was collected in natura (280 mg) and kept
in an ice bath until lyophilization. The dried material (30 mg) was
dissolved in n-hexane (50 mL) and the residue was dissolved in
chloroform (50 mL). Organic solvents were removed by a rotary eva-
porator, under reduced pressure, yielding the fractions F-Hex and F-Chl,
accordingly. The samples were dissolved at 10 mg/mL in chloroform/
methanol (1:1, v/v), then diluted to 1 mg/mL in methanol, and their
chemical composition was analyzed.
The phytochemical analysis was performed using ultra-performance
liquid chromatography (UPLC™, Acquity, Waters) coupled to a high-
resolution mass spectrometer (Xevo, Waters), equipped with a BEH C18
(Waters), 100 × 2.1 mm and 1.8-μm particle size, operated at 40 °C.
Analyte separation was performed in a gradient mode, with ultra-pure
water (MilliQ, Millipore) and acetonitrile, acidified with 0.1% (v/v)
formic acid. The solvent gradient was developed at 400 μL/min, in-
itiated with 5% (v/v) acetonitrile; holding for 1 min; then increasing
acetonitrile content from 5 to 50% (v/v) in 7 min; then increasing it to
95% (v/v) at 10 min; holding to 16 min; and returning to the initial
conditions at 18 min; with 2-min column re-equilibration (total run
time of 20 min).
MS analysis was performed at source temperature of 150 °C and
desolvation at 350 °C, using cone nitrogen at a flow rate of 50 L/h, and
500 L/h for sample desolvation. The energy settings for positive ioni-
zation were: 3.5 kV for the capillary; 30 V for the cone; and 60 V for the
source offset. For negative ionization, these were 3 kV, 40 V, and 80 V,
respectively. The detected mass range was 100–1400 m/z, and the
fragmentation was induced by ramping the collision energy from 30 to
50 V. Mass accuracy was obtained by internal calibration using leu-
enkephalin, m/z 556.2771, 278.1141 [M]+, and m/z 554.2615,
236.1035 [M]–. The unsaturation degree was determined based on the
calculated double-bond equivalents (DBE) from the equation: DBE = C
− H/2 + N/2 + 1, where C, H and N mean the numbers of the ele-
ments carbon, hydrogen and nitrogen (de Souza et al., 2019).
2.4. Experimental design
Male Wistar rats (sixty, 90 d, ∼350 g) were obtained from the
C.G. Martins, et al.
bioterium of the Biological Sciences Sector of the Federal University of
Paraná (Curitiba, Brazil) after ethical procedure approval by the Ethics
Committee of the same university (certificate number 1023). Animals
were randomly divided into four groups, in three independent experi-
ments: C, control, without tumor and without latex treatment; W,
Walker 256 tumor-bearing rats, without latex treatment; SW1, 256
Walker tumor-bearing rats, treated with 25 μL/mL aqueous latex solu-
tion; and SW2, Walker 256 tumor-bearing rats, treated with 50 μL/mL
aqueous latex solution. The used concentrations of latex solutions were
determined in a pilot study (data not shown). The animals received
1 mL of water (C and W groups) or latex solution (SW1 and SW2
groups) by gavage, once a day, for 15 d. The latex treatment was in-
itiated simultaneously with the implantation of Walker tumor cells
(1 × 107) obtained from another animal intraperitoneally inoculated.
The tumor cells were subcutaneously inoculated into the right flank of
the animals from the W, SW1, and SW2 groups. The animals in all
tumor-bearing groups exhibited the same food intake and had the same
water access. The body weight was monitored every 2 d. After 15 d, all
animals were alive. The animals were then euthanized by decapitation,
the tumors were removed, weighed, and used for the cell proliferation
assay. The blood was collected via a funnel into 15-mL tubes im-
mediately after decapitation; the serum was used for glucose and tria-
cylglycerol level determinations; and neutrophils were isolated. After
blood collection, resident macrophages were obtained from the peri-
toneal cavity after inoculation with 10 mL in phosphate-buffered saline
(PBS).
2.5. Cell isolation
Tumor tissue was soaked in 10 mL of phosphate-buffered saline
(PBS), fragmented with scissors, filtered through funnel and gauze, and
centrifuged at 116×g, 21 °C, for 5 min. The obtained cell pellet was
resuspended in a hemolytic solution [17.0 mM Tris (hydroxymethyl)
aminomethane and 18.7 mM NH4Cl) and washed twice with sterile PBS,
by centrifuging at 290×g, 4 °C, for 5 min. Neutrophils were isolated
from the blood using Histopaque 1077/1119 density gradient and
washed in RPMI medium containing 100 U/mL penicillin and 100 μg/
mL streptomycin (RPMI + AB), twice, by centrifugation, as described
above. Resident macrophages were obtained by intraperitoneal lavage
with 10 mL of PBS. The peritoneal cells were collected by centrifugation
(as above), washed in RPMI + AB, twice, resuspended in 500 μL of
RPMI + AB, and counted using a Neubauer counting chamber, after
staining with a 0.5% (w/v) trypan blue solution.
2.6. Determination of cell proliferation capacity
Tumor cells were placed in 96-well plate (1 × 104 cells/well) in
180 μL of RPMI 1640 medium containing 10% (w/v) of fetal bovine
serum. Alamar Blue was added (20 μL/well) to monitor cell prolifera-
tion. The percentage reduction of resazurin to resofurin was measured
at 560 and 600 nm in an automatic microplate reader (Tecan Infinite
M200) after 24 h of culture. The obtained optical density readings were
then used in the formula recommended by the Alamar Blue manu-
facturer to calculate the proliferation of the cells. Data are presented as
percent reduction of Alamar Blue in relation to the control wells
(without the cells).
2.7. Determination of serum glucose and triacylglycerol levels
Serum glucose and triacylglycerol levels were determined using
commercial BioLiquid kits, following manufacturer's instructions. The
analyte levels were quantified by measuring sample absorbance in
Tecan Infinite M200 microplate reader at 505 nm and 540 nm, ac-
cordingly.
3
Journal of Ethnopharmacology 255 (2020) 112722
2.8. Determination of macrophage and neutrophil activity
Isolated peritoneal macrophages and peripheral blood neutrophils
were analyzed using phagocytosis capacity and superoxide anion pro-
duction assays. For the phagocytosis capacity measurements, cell sus-
pension (105 cells in 0.1 mL of RPMI + AB) was added to a 96-well flat-
bottomed tissue culture plate. The plate containing macrophages was
incubated for 1 h at 37 °C before phagocytosis measurement, to allow
cell adhesion. Then, 10 μL of neutral red-stained zymosan (108 parti-
cles/mL) was added to each well. After incubation for 30 min at 37 °C,
the cells were fixed in Baker solution [4% (v/v) formaldehyde, 2% (w/
v) sodium chloride, and 1% (w/v) calcium acetate] for 30 min. Then,
the cells were washed two times in PBS and centrifuged at 453×g,
21 °C, for 5 min. Next, 0.1 mL of acidified alcohol solution (10% acetic
acid and 40% ethanol in distilled water, v/v/v) was added to each well
to solubilize the neutral-red stain. After 30 min, sample absorbance in
each well was determined using a plate reader at 550 nm (Bonatto
et al., 2004). The data are expressed as percent values, with the C group
data as a reference.
For superoxide anion production determination, the cells were
plated as described above. Macrophages and neutrophils were in-
cubated for 1 h at 37 °C in the presence of 10 μL of phorbol 12-myristate
13-acetate (5 μM final concentration). Then, 100 μL of nitroblue tetra-
zolium (0.1% final concentration) were added and the samples in-
cubated for 30 min at 37 °C in the dark. After centrifugation at 453×g,
21 °C, for 5 min, the supernatant was discarded and the cells were fixed
in 50% aqueous methanol solution. After another centrifugation step,
120 μL of 2 M KOH and 140 μL of dimethyl sulfoxide were added.
Reduction of nitroblue tetrazolium resulted in the formation of blue
formazan dye, which was detected spectrophotometrically at 550 nm.
The data are expressed as percent values, with the C group data as a
reference (Madhavi and Das, 1994).
2.9. Statistical analysis
Statistical analysis was performed by one-way ANOVA followed by
a post-hoc Tukey's test. A value for p < 0.05 indicated statistical sig-
nificance. Data were analyzed using GraphPad prism software (version
5.0; GraphPad Software; San Diego, CA, USA).
3. Results
3.1. Phytochemical analysis of latex from E. tirucalli
In addition to the steroid triterpenes Euphol and Tirucallol
(C30H50O), several other triterpenes have been recently identified in
E. tirucalli, with some structural differences concerning the presence of
hydroxyl, carbonyl, and peroxide groups (Duong et al., 2019; de Souza
et al., 2019). Further, their acylation by fatty acids has been reported in
other species of Euphorbia (Benabdelaziz et al., 2018; Ragasa and
Cornelio, 2013).
Here, dried latex from E. tirucalli was fractionated in two samples
using nonpolar solvents (n-hexane and chloroform) used to dissolve the
components of the latex extract. The compounds in these fractions were
then detected in the negative- and positive-ion mode, using the MSe
centroid acquisition mode. However, the detection was better in the
positive-ion mode [M+H]+ (Fig. 1A and B) than in the negative-ion
mode. The components were then characterized based on their exact
mass and mass fragments (m/z error < 5 ppm), assisted by the data
available in the literature.
The main peaks were the same and detected in both fractions, but
their relative abundances were somewhat different (Fig. 1). The main
components of the latex sample (compounds 1–12) were identified as
triterpenoids, structurally related to the compounds reported by Duong
et al. (2019) and de Souza et al. (2019), but differing with respect to the
presence of additional unsaturated bonds, (as determined by DBE) and
C.G. Martins, et al. Journal of Ethnopharmacology 255 (2020) 112722

Fig. 1. Phytochemical analysis of latex from E. tirucalli by LC-MS in a positive-ion mode. For a comprehensive analysis, lyophilized latex was dissolved in n-hexane
(A) and the residue was dissolved in chloroform (B).

Table 1
Components of the latex sample identified by LC-MS.
Peak tR (min) MS1→MS2 (low energy) Composition Theoretical m/z Error (ppm) DBE Compound class

+
1 8.33 475.3799 [C30H51O4] 475.3781 3.8 6 Triterpene
2 8.71 489.3961 → 457.3689, 439.3587 [C31H53O4]+ 489.3938 4.7 6 Methoxy-triterpene
3 9.23 475.3802 → 457.3685 [C30H51O4]+ 475.3781 4.4 6 Triterpene
4 9.45 489.3956 → 457.3695, 439.3590, 421.3461 [C31H53O4]+ 489.3938 3.7 6 Methoxy-triterpene
5 9.76 503.4107 → 457.3676, 439.3572 [C32H55O4]+ 503.4095 2.4 6 Ethoxy-triterpene
6 9.84 503.4102 → 457.3678, 439.3578 [C32H55O4]+ 503.4095 1.4 6 Ethoxy-triterpene
7 10.04 457.3676 → 439.3573 [C30H49O4]+ 457.3676 00 7 Triterpene
8 10.25 507.3697 → 475.3336 [C30H51O6]+ 507.3680 3.3 6 Triterpene
9 10.42 503.4104 → 457.3672, 439.3579, 421.3468 [C32H55O4]+ 503.4095 1.8 6 Ethoxy-triterpene
10 10.73 507.3681 → 475.3409, 457.3342, 439.3210 [C30H51O6]+ 507.3680 0.2 6 Triterpene
11 11.29 719.6319 [C49H83O3]+ 719.6336 −2.4 9 acyl-triterpene
12 11.56 721.6483 [C49H85O3]+ 721.6493 −1.4 8 acyl-triterpene

tR – relative retention time; DBE: double bond equivalent.

the attached hydroxyl, methoxyl, or ethoxyl groups (Table 1). At high
collision energy, all compounds, except for compounds 11 and 12,
showed a similar fragmentation pattern, consistent with that of tir-
ucallane/euphane-type triterpenes, with common main fragments ob-
served at m/z 107.085, 109.100, 121.100, 133.101, 135.115, 145.101,
159.116, 171.116, 173.131, 183.116, 185.132, 199.148, and 201.163
(Xie et al., 2014). At low energy, losses of water, methoxyl, and ethoxyl
groups were evident.
Compound 1 (m/z 475.3799) was similar to Euphorol M/N con-
taining an additional carbonyl group (or a hydroxyl group and an un-
saturated bond). Duong et al. (2019) described Euphorol L, with a de-
protonated ion at m/z 487.3416. Herein, the mass of protonated ion of
compound 2 (m/z 489.3961), although close to the expected m/z of
Euphorol L (489.3574 [M+H]+), was sufficiently different to dismiss
this possibility. Hence, compound 2 was deemed to be similar to
compound 1, but containing a methoxyl group. The latter detected in
the MS2 spectrum (m/z 457.3704) by a neutral loss (NL) of
32.0267 atomic mass units (a.m.u.) that was consistent with the
monoisotopic molecular weight of methanol (MW 32.0262). Sequential
losses of water were observed at m/z 439.3587 and 421.3464, in-
dicating the presence of free hydroxyl groups.
Compound 3 mass (m/z 475.3802) was consistent with that of an
isomer of compound 1. Compound 4 yielded an ion at m/z 489.3956,
and fragments at m/z 457.3707 (loss of methanol), and m/z 439.3600
and 421.3461 (sequential losses of water), consistent with an isomer of
compound 2. Compounds 5 and 6 were detected at m/z 503.4107 and
503.4102, respectively. Both produced fragments at m/z 457.368, with
a NL of 46.042 a.m.u., consistent with a loss of an ethanol molecule
(MW 46.042). Other fragments were consistent with the losses of water
molecules and, therefore, compounds 5 and 6 appeared to be similar to
compounds 2 and 4, but with an attached ethoxyl group, rather than a
methoxyl group, and were isomers of compound 9 (m/z 503.4104).
Compound 7, detected at m/z 457.3676, was similar to the one
detected by de Souza et al. (2019) and differed from euphorol M/N
described by Duong et al. (2019) by 7 DBE. Zhang et al. (2017) have
identified triterpene 11-hydroxy-kansenone (tirucalla-8,24-diene-
3β,11β-diol-7-one) in Euphorbia kansui whose characteristics were si-
milar to those of compound 7 because of the presence of a ketone

4
C.G. Martins, et al.
group. The characteristics of triterpenoid kansenonol, previously
identified in E. kansui and Euphorbia resinifera (Wang et al., 2003,
2016bib_Wang_et_al_2003bib_Wang_et_al_2016), were also similar to
those of compound 7. Compounds 8 and 10 were isomers, detected at
m/z 507.3697 and 507.3681, respectively. The structure of these
compounds included six oxygen elements and DBE of 6. NL of 32.026
indicated the loss of a methoxyl group. Other fragments (cited above)
confirmed the triterpenoid structure, but the arrangement of structural
elements could not be determined.
Further, the LC-MS data were consistent with the presence of two
acylated triterpenoids, compound 11 (m/z 719.6319) and compound
12 (m/z 721.6483). The data were consistent with triterpenes with
attached fatty acids, linoleic (or equivalent) and oleic (or equivalent)
acids, as previously shown for other species in the Euphorbia genus,
E. pterococca (Benabdelaziz et al., 2018) and E. hirta (Ragasa and
Cornelio, 2013). However, additional information is needed to confirm
their detailed structures.
3.2. Effects of E. tirucalli latex on tumor development
3.2.1. Tumor mass
In the tumor-bearing animals treated with 25 μL/mL of E. tirucalli
latex (SW1), the tumor mass was reduced by approximately 40% in
comparison with the control animals (W) (p < 0.05). In animals
treated with 50 μL/mL of latex (SW2), the observed reduction was even
more pronounced, approximately 60% (p < 0.001) (Fig. 2).
3.2.2. Tumor cell proliferation
The ex vivo proliferation capacity of tumor cells cultured for 24 h in
the presence of Alamar Blue was investigated (Fig. 3). Treatment with
50 μL/mL latex (SW2) resulted in an approximately 76% reduction of
tumor cell proliferation compared with the untreated tumor (W)
(p < 0.0001). In animals from SW1 group, which received 25 μL/mL
latex, no decrease in the tumor cell proliferation compared with ani-
mals from W group was apparent (p > 0.05). To explain the observed
reduction of tumor size in SW2 group, a pathway involving the pro-
duction of hydroperoxides, which can inhibit cell proliferation, was
analyzed (data not shown). However, no alteration in the production of
hydroperoxide was observed in tumor tissue of animals treated with
E. tirucalli latex (data not shown).
In addition to the tumor cell proliferation analysis, the physical,
biochemical, and immune characteristics of animals were next ana-
lyzed. They are important parameters frequently altered in cancer
Fig. 2. Effect of latex treatment on tumor mass. Tumor mass (g) in untreated
animals with tumor (W), animals with tumor treated with 25 μL/mL aqueous
solution of latex (SW1), and animals with tumor treated with 50 μL/mL aqu-
eous solution of latex (SW2). Data are presented as the mean + SEM. W, n =
11; SW1, n = 12; SW2, n = 14. ∗p < 0.05; ∗∗p < 0.001 (one-way ANOVA
followed by a post hoc Tukey test).
5
Journal of Ethnopharmacology 255 (2020) 112722
Fig. 3. Tumor cell proliferation (% reduction of Alamar Blue staining) in un-
treated animals with tumor (W), animals with tumor treated with 25 μL/mL
aqueous solution of latex (SW1), and animals with tumor treated with 50 μL/
mL aqueous solution of latex at (SW2). Data are presented as the mean + SEM.
W, n = 11; SW1, n = 12; SW2, n = 14. ∗∗∗p < 0.0001 (one-way ANOVA
followed by a post hoc Tukey test).
development and could explain the observed relatively small tumor size
in latex-treated animals.
3.3. Effects of E. tirucalli latex on cachexia
3.3.1. Body mass
Treatment with latex did not affect the body mass of animals
(Fig. 4). The mass body gain during 15 d in rats without tumor (C
group) and tumor-bearing rats (W, SW1, and SW2 groups) was similar
(p > 0.05).
3.3.2. Glucose and triacylglycerol serum levels
In animals from W group, glucose serum levels were reduced by
approximately 30% (p < 0.001) and triacylglycerol serum levels were
increased by approximately 90% (p = 0.0262) in comparison with C
group animals (Fig. 5A and B), as anticipated for untreated Walker 256
tumor-bearing animals (Iagher et al., 2013). By contrast, treatment with
latex allowed maintenance of glucose and triacylglycerol levels similar
to those in C group (p > 0.05).
3.3.3. Weight change
The weight change was calculated as the difference between the
carcass weight (body mass on day 15 without tumor mass) and body
mass on day 1. The animals from W group lost some body mass during
15 d of tumor growth (8.7 g ± 7.9), while latex-treated tumor-bearing
rats (SW1 and SW2 groups) gained some mass despite tumor growth.
SW1 and SW2 rats gained approximately 20 g and 8 g, respectively
(Fig. 5C).
3.4. Effects of E. tirucalli latex on immune function
3.4.1. Peritoneal macrophages
Table 2 presents data for the activity of peritoneal macrophages
from different animal groups. The adhesion capacity of peritoneal
macrophages was similar among animals from the tumor-bearing
groups (W, SW1, and SW2; p > 0.05), however, it was approximately
90% higher in cells from SW2 animals than in those from C group an-
imals (p = 0.0258). The phagocytic ability of macrophages from W
group animals was approximately 30% lower than that of C group
macrophages, while it was similar for SW1, SW2, and W group mac-
rophages (p > 0.05). Superoxide anion production by SW1 and SW2
macrophages was reduced by approximately 30% compared with that
of W group macrophages (p < 0.05).
C.G. Martins, et al. Journal of Ethnopharmacology 255 (2020) 112722

Fig. 4. Body mass (kg) of control animals (C),

untreated animals with tumor (W), animals with
tumor treated with 25 μL/mL aqueous solution of
latex (SW1), and animals with tumor treated with
50 μL/mL aqueous solution of latex (SW2), de-
termined every 2 d. The data are shown as
mean ± SEM. C, n = 15; W, n = 15; SW1,
n = 15; SW2, n = 15.

3.4.2. Peripheral blood neutrophils E. tirucalli. Among these latex components, the Euphol
The phagocytic capacity of neutrophils from W group animals was (3S,5R,10S,13S,14S,17S)-4,4,10,13,14-pentamethyl-17-[(2R)-6-me-
reduced by approximately 20% compared with C group neutrophils thylhept-5-en-2-yl]-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta
(Table 2). Treatment with the higher concentration of latex (SW2) [a]phenanthren-3-ol) is a well-documented triterpene that presents
promoted the phagocytosis (by approximately 20%) and reduced su- pharmacological properties. Previous investigations involving the ad-
peroxide anion production (by approximately 40%), in comparison ministration of Euphol, carried out in vitro (Lin et al., 2012; Wang et al.,
with W group cells. 2013; Chen et al., 2015) and in vivo (Passos et al., 2013), indicated that
this compound might be one of those with antitumor and anti-in-
flammatory activity observed the latex sample, since Euphol exerts anti-
4. Discussion
proliferative effects and arrests the cell cycle of cancer cells (Lin et al.,
2012; Wang et al., 2013).
In the current study, an aqueous solution of E. tirucalli latex was
Although Euphol had not been observed in our experiments, many
administered orally to Walker 256 tumor-bearing rats to mimic the
other structurally-related compounds were described. Thus, the pro-
typical preparation of the aqueous latex solution consumed by people in
nounced reduction of the proliferative capacity of cells obtained ex vivo
Brazil as a popular treatment option for cancer. The aim of the in-
from Walker 256 tumor from the SW2 group, observed in the current
vestigation was to evaluate the effects of ingestion of two different
study, may be associated with the presence of these triterpenes in latex.
doses of latex (25 and 50 μg/mL/d) on tumor growth, cachexia para-
Their chemical structures, consistent with tirucallane/euphane-type
meters, and innate immune responses in vivo, after 15 d of tumor de-
triterpenes, are related to Euphol, but with additional hydroxyl,
velopment and latex consumption. We showed that the ingestion of
methoxyl, and ethoxyl groups, as determined by the LC-MS analysis
latex/water solution during tumor development reduced tumor growth
(Table 1). On the other hand, different compounds also exhibited
and cachexia, and promoted innate immunomodulation. That was ap-
pharmacological potential, e.g., the Eutirucalin, a lectin-like protein,
parent as a 60% reduction in tumor mass (Figs. 2) and 76% reduction in
another compound described from the latex of E. tirucalli, with the
the proliferation capacity of tumor cells (Fig. 3) in the SW2 group. In
ability to reduce the proliferation of tumor cells in vitro and in vivo
addition, the plasma glucose and triacylglycerols levels remained close
(Palharini et al., 2017). In line with this, latex from different species,
to the levels in animals without tumor, and body mass gain (up to 20 g)
such as the crude latex from Ficus carica, containing different terpenoids
was apparent in both treatment groups (Fig. 5). Furthermore, 20% in-
also exhibited anti-tumor potential and successfully reduced tumor cell
crease in the phagocytic capacity of neutrophils from SW2 group ani-
proliferation, induced cell cycle arrest, inhibited tumor growth, and
mals was noted, along with a reduced superoxide anion production by
suppressed the clonogenic potential of cervical cancer cell lines
macrophages (approximately 30% reduction) and neutrophils (ap-
(Ghanbari et al., 2019).
proximately 40% reduction) in the treated groups (Table 2). These re-
Furthermore, by contrast with other investigations, in the current
sults are following the folk report on the anticancer action promoted by
study, the tumor-bearing animals received crude latex dissolved in
E. tirucalli latex.
water, as in folk medicine, which resulted in pronounced effects on
The cells of Walker 256 tumor injected subcutaneously into Wistar
tumor growth. This indicates that E. tirucalli latex may contain addi-
rat generated a pronounced tumor mass and severe cachexia within
tional active components and that purification of specific compounds is
15 d. Hence, the Walker 256 tumor-bearing rat model is a good model
not needed for a significant reduction in tumor cell proliferation and,
for the investigation of activity of antitumor and anti-cachectic com-
consequently, the tumor mass. The crude latex solution promoted the
pounds. The reduction of tumor mass observed in the treatment groups,
reduction of the tumor growth, as well as the maintenance of feed in-
especially in SW2 animals, is important, considering that it was
take and bodyweight gain, observed in both, SW1 and SW2 groups. This
achieved after ingestion of an aqueous latex solution for only 15 d.
suggested that the consumption of low concentrations of latex was not
Although the antitumor activity of E. tirucalli has been demonstrated in
toxic to the animals.
vitro (Munro et al., 2015; Choene and Motadi, 2016) and in vivo, the
Although the effect of latex solution on tumor cell death was not
latter involved plant extracts and isolated compounds (Valadares et al.,
evaluated in the current study, the effect may be responsible for the
2006; Palharini et al., 2017). Investigations involving crude latex,
reduction of tumor mass, as indicated by in vitro studies that demon-
mirroring the traditional use, are not yet reported.
strate such effect of purified compounds and crude latex. For instance,
Many types of triterpenes have been described from the latex from

6
C.G. Martins, et al.
Fig. 5. Cachexia parameters for untreated animals with tumor (W), animals
with tumor treated with 25 μL/mL aqueous solution of latex (SW1), and ani-
mals with tumor treated with 50 μL/mL aqueous solution of latex (SW2). A,
Serum glucose levels (mg/dL). C, n = 15 W, n = 5; SW1, n = 5; SW2, n = 5. B,
Serum triacylglycerol levels (mg/dL). C, n = 15 W, n = 5; SW1, n = 5; SW2, n
= 5. C, Weight change (g), calculated as a difference between the carcass
weight (body mass on day 15 without tumor mass) and body mass on day 1. W,
n = 9; SW1, n = 12; SW2, n = 13. In all panels, data are shown as mean +
SEM. ∗p < 0.05; ∗∗∗p < 0.0001 (one-way ANOVA followed by a post hoc
Tukey test).
purified Euphol upregulates BAX and downregulates BCL-2 expression
in human gastric CS12 cancer cells, promoting mitochondrial dys-
function, probably by caspase-3 activation (Lin et al., 2012). Further,
latex treatment results in an altered expression of genes involved in
7
Journal of Ethnopharmacology 255 (2020) 112722
apoptosis in Hep-2 cells (Franco-Salla et al., 2016).
The development of Walker 256 tumor promoted an increase in
serum triacylglycerol levels, with a decrease of serum glucose levels,
and a bodyweight loss in experimental animals. Together, these ob-
servations indicate the establishment of a cancer cachexia state
(Tisdale, 2009). To the best of our knowledge, the effect of intake of
E. tirucalli latex on cachexia parameters in tumor-bearing animals has
not been investigated in any other study. The reduction of cachexia
observed in SW1 and SW2 animals (i.e., reduced glycaemia and in-
creased triacylglycerolemia) was confirmed by the change in animal
body mass after 15 d of tumor development (Fig. 5). The reduction of
tumor mass observed in SW1 and SW2 animals may partially explain
the reduction of the cachectic state, since fewer tumor cells generate
fewer chemical mediators promoting hypercatabolism observed in the
cachectic state (Pinto et al., 2004). However, as reported previously,
cachexia may not always be related to the size of tumor mass (Pizato
et al., 2005; Iagher et al., 2013). In fact, serum glucose and serum
triacylglycerol levels in animals with 36% reduction of tumor mass
(SW1 group) were the same as those in animals with 60% reduction of
tumor mass (SW2 group). Therefore, the mechanisms underpinning the
effects of E. tirucalli latex in vivo require further investigation.
Hence, to elucidate the role of immune system in tumor growth and
amelioration of cachexia, we investigated the activity of the innate
immune response in the studied animals. The innate immune cells have
been shown to prevent tumor progression (Vesely et al., 2011); there-
fore, we evaluated the anti-tumor activity of macrophages and neu-
trophils from tumor-bearing animals treated with E. tirucalli latex.
E. tirucalli latex or extracts have been reported to be highly cytotoxic
(Fürstenberger and Hecker, 1977). Further, DNA damage with loss of
viability was reported in human leukocytes exposed to E. tirucalli
aqueous extract in vitro (Pansera Waczuk et al., 2015). Therefore, it
became important to evaluate the viability and functionality of immune
cells following in vivo latex-exposure experiment and we observed that
the treatment with an aqueous latex solution did not adversely affect
macrophage or neutrophil viability.
Neutrophils and macrophages have the potential to exhibit different
phenotypes in tumor-bearing individuals. Anti-tumor activities of these
cells are related to tier 1-like phenotype (N1 and M1) and correspond to
cytotoxic activity against tumor cells and recruitment and activation of
other effector cells of the immune system. Pro-tumor potential of these
cells is associated with a N2 and M2-like phenotypes that promote
upregulation of genes that increase cell proliferation capacity, increase
of the invasive and migratory potential of tumor cells, and activation of
endothelial cells and angiogenesis (Zhang et al., 2016; Hagerling et al.,
2015). A recent publication has reported that circulating neutrophils
from Walker 256 tumor-bearing animals responded to tumor growth via
expression of pro-inflammatory genes until 10 days after subcutaneous
inoculation of Walker 256 cells, and this response corresponded to an
N1-like phenotype. Further, alterations were noted in the gene ex-
pression landscape and there was a reduction in pro-inflammatory cy-
tokine expression to normal levels, indicating that neutrophils exhibit a
shift to the N2-like phenotype. Cellular factors produced by the tumors
are responsible for the shift to the pro-tumor phenotype (N2 phenotype)
of neutrophils (Kuwabara et al., 2019).
Although the phenotype of the collected macrophages and neu-
trophils has not been evaluated in this study, we speculate that the
treatment with latex promoted immunomodulation. An increase in the
phagocytic response of neutrophils in the SW2 animals was in line with
tier 1-like phenotype cell response, and a reduction in the superoxide
anion production by macrophages and neutrophils from SW1 and SW2
rats was compatible with a loss of tier 1-like phenotype response.
Macrophages and neutrophils from untreated tumor-bearing rats (W
group) showed reduced phagocytic capacity when compared with an-
imals without tumor (C group), as reported earlier in the same tumor
model (de Lima et al., 2008; Bacurau et al., 2007). The phagocytic
activity of macrophages from SW1 and SW2 groups was similar to that
C.G. Martins, et al.
Table 2
Activity of resident macrophages and peripheral blood neutrophils from tumor-bearing untreated animals (W), animals with tumor treated with 25 μL/mL aqueous
solution of latex (SW1), and animals with tumor treated with 50 μL/mL aqueous solution of latex (SW2). The data are shown as mean ± SEM. The data were
calculated as a percentage of readings for the C group. Further, phagocytosis capacity and superoxide anion data for macrophages were normalized by the respective
adhesion capacity values.
Macrophages
Adhesion (% control) Phagocytosis (% control) Superoxide anion production (% control)
C 99.7 ± 10.5 (n = 13) 100.0 ± 6.6 (n = 15) 100.0 ± 4.6 (n = 12)
W 165.7 ± 16.2 (n = 15) 70.5 ± 3.5a (n = 20) 90.7 ± 6.2 (n = 15)
SW1 168.4 ± 18.2 (n = 18) 85.3 ± 5.0 (n = 14) 72.8 ± 4.1c (n = 16)
SW2 189.2 ± 22.5a (n = 21) 73.0 ± 5.1 (n = 15) 67.4 ± 2.9c (n = 15)
a
p < 0.05 vs. C (C vs. SW2, p = 0.0258; C vs. W p = 0.003).
b
p = 0.0349 vs. C.
c
p < 0.05 vs. W (W vs. SW1, p = 0.0312; W vs. SW2, p = 0.0034).
d
p = 0.0392 vs. W.
e
p = 0.0376 vs. W.
of W group macrophages, but the phagocytic activity of SW2 neu-
trophils showed an increase in comparison with W group neutrophils
(Table 2). An increase in the phagocytic capacity of neutrophils from
treated animals (SW2) correlates with the anti-tumor role of neu-
trophils. Therefore, the participation of these cells in tumor reduction
observed in SW1 and SW2 groups cannot be ruled out. On the contrary,
the production of superoxide anions by macrophages and neutrophils in
animals treated with the aqueous latex solution (SW1 and SW2 groups)
decreased as compared to untreated tumor-bearing animals (W). In-
creased reactive oxygen species production is an important mechanism
used by the immune system against tumor cells because it is related to
oxidative damage and tumor cell apoptosis (Zhang et al., 2016), but
does not appear to contribute to the reduction in tumor mass in SW1
and SW2 animals. In our study, latex treatment reduced the ability of
circulating neutrophils and peritoneal macrophages to produce super-
oxide anion. Since there was an important reduction in tumor mass in
the treated groups, particularly SW2, the decline in the cytotoxic ca-
pacity caused by the treatment does not seem to have compromised the
beneficial effects of latex.
5. Conclusions
Ingestion of E. tirucalli latex/water solution for 15 d, concomitant
with the development of a subcutaneous Walker 256 tumor, reduced
tumor cell proliferation, tumor mass, and cachexia in Wistar rat. The
aqueous latex solution treatment promoted a reduction of anion su-
peroxide production by macrophages and neutrophils but increased the
phagocytic capacity of neutrophils from the SW2 group in comparison
to untreated tumor-bearing rats. These data suggest that latex from
E. tirucalli has immunomodulatory effect. Further in vivo studies are
needed to delineate the mechanisms involved in the anticancer activity
of latex. The use of appropriated doses of latex may be useful as an
adjuvant in the treatment of cancer patients. For this, further in vivo
studies in animal and human models need to be conducted.
Authors' contributions
CGM was responsible for the management of the animals; data
collection, analysis and interpretation; bibliographic research; and
statistical analysis. MHA, DSSC, and IPS performed the in vitro assays.
SF and BCO prepared the botanical material. MMF, RP, and LMS were
involved in the phytochemical characterization of latex. SJRB con-
tributed to the experimental design and data interpretation. LCF con-
tributed to data interpretation and results discussion. FI conceived and
designed the experiments, and was involved in data and statistical
analysis. FI, SJRB, and LMS drafted the manuscript. All authors read
and approved the final version of the manuscript.
8
Journal of Ethnopharmacology 255 (2020) 112722
Neutrophils
Phagocytosis (% control) Superoxide anion production (% control)
100.0 ± 4.5 (n = 7) 100.0 ± 3.5 (n = 8)
82.1 ± 2.3b (n = 7) 107.0 ± 2.7 (n = 8)
95.1 ± 5.1 (n = 8) 65.1 ± 16.6 (n = 9)
99.1 ± 4.2d (n = 8) 56.6 ± 16.5e (n = 8)
Declaration of competing interest
None.
Acknowledgements
This work was supported by Instituto de Pesquisa Pelé Pequeno
Príncipe; Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq); Fundação Araucária-PR; and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Finance code
001]. The sponsors did not play any role in study design; in the col-
lection, analysis, and interpretation of data; in the writing of the report;
and in the decision to submit the article for publication.
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