South African Journal of Botany 146 (2022) 528–537

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Biological activity of Haematoxylum brasiletto in MCF7 and

MDA-MB-231 breast cancer cell lines
GeorginaVenecia Bello-Martínez a, Génesis García-Ramírez a, Monserrat Olea-Flores a,
Napoleón Navarro-Tito a, Alberto Hernández-Moreno a, Luz Patricia Avila-Caballero a,
Heriberto Torres-Moreno a,b, Jorge Bello-Martínez a,b,∗
a
Universidad Autónoma de Guerrero, Facultad de Ciencias Químico Biológicas, Av. Lázaro Cárdenas S/N Col. La Haciendita CU Sur C.P. 39070. Chilpancingo,
Guerrero, México
b
Universidad de Sonora – Unidad Regional Norte. Avenida K s/n Col. Eleazar Ortiz Caborca Sonora, México, CP 83600

a r t i c l e i n f o a b s t r a c t

Article history: Haematoxylum brasiletto (Fabaceae) is a wild plant from Guerrero, used in traditional medicine. How-
Received 9 March 2021 ever, there is little scientific information about anti-migratory and antioxidant activities. The aim of
Revised 19 October 2021
this study was to evaluate the anti-migratory and anti-proliferative activities of the ethanolic extract of
Accepted 15 November 2021
heartwood of H. brasiletto (HB) and their fractions of hexane (HBA) dichloromethane (HBB) and ethyl
acetate (HBC) against different breast cancer cell lines. Additionally, we analyzed their antioxidant ac-
Edited by Dr L. Rarova tivity. The antiproliferative activity of ethanolic extract and their fractions were evaluated against the
non-tumorigenic mammary epithelial cell line MCF10A and the cancer cell lines MCF7 and MDA-MB-231
Keywords:
Haematoxylum brasiletto by MTT assay, using concentrations of 3.1 to 25 μg/mL for 48 h. The anti-migratory activity was eval-
Anti-migratory activity uated by the scratch wound-healing assay. Phenolic content (TPC) was quantified by the Folin-Ciocalteu
Anti-oxidant activity assay, and flavonoid content (TFC) was investigated using the AlCl3 assay. Antioxidant activity was eval-
Breast cancer uated through the DPPH, trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant
power (FRAP) methods. Our results showed that HB (258.6mg GAE/g) presented the higher amount of
TPC. For TFC, we found that HBC (53.0 mg QE/g) presented the higher content. Antioxidant evaluation
results evidenced that HBC [DPPH IC50 : 57 μg/mL; TEAC: 61.4 μM TE/g; FRAP: 2678.2 μM Fe(II)/g] ex-
hibited the most remarkable capacity to stabilize free radicals and reduce metals. On the other hand,
HBB was the one who presented the most anti-proliferative and anti-migratory activities on MCF7. In ad-
dition, extract and fractions was less cytotoxic against the non-cancerous MCF1710 A cells, which may be
associated with their selectivity against cancer cells. To our knowledge, this is the first study that reports
the activity of the H. brasiletto plant. Besides, H. brasiletto extracts and their fractions showed an inter-
esting anti-proliferative, anti-migratory activity, and antioxidant potential, which could be considered as
a natural source of antioxidants and anti-cancer compounds.
© 2021 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Haematoxylum brasiletto species belongs to the Fabaceae fam-
ily. It grows in low deciduous forests and deciduous forests be-
tween 100 and 10 0 0 MASL (CONABIO, 2020), in open soils with
shallow and stony soils, and gullies with deep soils. The brazilian
wood stem (bark, wood, or the heart of the trunk) is extensively
used traditionally for cardiovascular system (Argueta, 2004). Addi-
tionally, it is also used in traditional medicine for depression, kid-
ney disorders, toothaches, heart, digestive problems, fevers, infec-
∗
Corresponding author:
E-mail address: belloj@uagro.mx (J. Bello-Martínez).
tions of the mucous membranes, and bleeding. The aqueous ex-
tract of the heartwood is also recognized as an effective antibiotic
(Amstrong, 1992), while the methanolic extract of the bark is an
inhibitor of bacteria and yeasts (Rivero-Cruz, 2008). The presence
of the active compounds brazilin and hematoxylin have been re-
ported in the wood of this plant (Sanchez-Marroquin, 1958). On
the other hand, wood and bark are used to obtain dyes pinks and
reds for textiles (Vigueras, 2012) and kinds of toothpaste (Conafor,
2012). The heartwood tannins of this species can be used to for-
mulate tannin-urea adhesives (Pedraza-Bucio, 2011).
H. brasiletto was investigated as a source of anti-proliferative
compounds, Bello-Martínez et al. (2017) reported that Brazilin a
compound isolated from this plant showed anti-proliferative activ-
ity on A549, LS180, H1299, HeLa, SiHa, and MDA-MB-231 cancer

https://doi.org/10.1016/j.sajb.2021.11.017
0254-6299/© 2021 SAAB. Published by Elsevier B.V. All rights reserved.
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al.
cell lines. Moreover, Brazilin was described as an inhibitor of NO
synthase (Kim et al., 1998), xanthine oxidase (Nguyen et al., 2005),
protein kinase C (Moon et al., 1985), and aldose reductase enzymes
(Thorn et al., 2011). However, limited phytochemical and pharma-
cological studies about H. brasiletto have been published. Further,
there are no reports about the anti-migratory activity induced by
H. brasiletto extracts in breast cancer cell lines. Considering the tra-
ditional consumption of H. brasiletto by the rural population from
Guerrero State, Mexico, as a cancer therapy, we evaluated the anti-
migratory and anti-proliferative activity of the ethanol extract of
H. brasiletto and their fractions against different breast cancer cell
lines. Additionally, we also analyzed the antioxidant properties of
this plant.
2. Materials and methods
2.1. Plant material and extracts generation
Plant specimens were collected in the Guerrero State of Mex-
ico (99°21 19.03"W; 17°29 03.27"N) during July 2018. Dra. Luz Pa-
tricia Avila Caballero carried out the botanical identification; one
specimen was deposited at the Universidad Autónoma de Guerrero
Herbarium with a voucher number UAGROHBH15. The ethanolic
extract was prepared as described by Bello-Martínez et al. (2017).
Air-dried heartwood (1 kg) was finely grounded with a Wiley mill
(200 mesh) and extracted with EtOH (1:10 w/v) by maceration at
room temperature for 10 days, with brief manual stirring twice a
day. The EtOH (HB) extract was evaporated under reduced pres-
sure obtaining a total of 43.5 g of HB; It was partitioned by a
successive liquid-liquid partition of an aqueous suspension (3:2
H2 O / MeOH) with Hx, DCM and EtOAc and evaporated under re-
duced pressure to produce the corresponding hexane (HBA, 1.7 g),
dichloromethane (HBB, 2.04 g) and ethyl acetate (HBC, 19.8 g) frac-
tions respectively. The extracts were stored at 4°C until use.
2.2. Total phenolic content (TPC)
Total phenol content (TPC) was determined according to the
method described by Velazquez et al. (2007) with a slight modi-
fication. Briefly, the extracts or fractions (10 μL) were mixed with
80 μL of distilled water, 40 μL of Folin–Ciocalteu reagent (0.25 N),
60 μL of sodium carbonate at 5%, and 80 μL of distilled water. The
mixtures were incubated in darkness for one h, and the absorbance
was read at 760 nm. Results were reported as mg of gallic acid
equivalent (mg GAE)/g d.w. as the mean of three replications.
2.3. Total flavonoid content (TFC)
Total flavonoid content (TFC) was determined according to the
method described by Saeed et al. (2012). Briefly, in a 10 mL test
tube, 0.3 mL of extracts, 3.4 mL of 30% methanol, 0.15 mL of
NaNO2 (0.5 M) and 0.15 mL of AlCl3.6.H2O (0.3 M) were mixed.
After 5 min, 1 mL of NaOH (1 M) was added. The solution was
mixed well, and the absorbance was measured against the reagent
blank at 506 nm. The standard curve for total flavonoids was made
using a quercetin standard solution (0 to 100 μg/mL), and the cal-
ibration curve was used to quantify. The results were expressed
in mg quercetin equivalents (mg QE/g d.w.) as the mean of three
replications.
2.4. Antioxidant capacity (AC) determination
2.4.1. Free-radical scavenging activity –DPPH assay
1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was carried out us-
ing a modified method described by Usia et al. (20 02). 10 0 μL of
H. brasiletto extract or their fractions were mixed with 100 μL of
529
South African Journal of Botany 146 (2022) 528–537
DPPH solution (0.3 mM). The mixtures were stored in darkness for
30 min, and the absorbance was read at 517 nm in a microplate
reader (Thermo Scientific TM Multiskan TM FC). Results were ex-
pressed as EC50, i.e., the amount of antioxidants necessary to de-
crease the initial DPPH concentration by 50%. IC50 values were ob-
tained using linear regression analysis. Measurements were carried
out in triplicate.
2.4.2. Trolox equivalent antioxidant capacity (TEAC) assay
Trolox equivalent antioxidant capacity (TEAC) assay was con-
ducted according to the method previously reported by Vidal-
Gutierrez et al. (2020. The TEAC method is based on the ability of
antioxidant compounds to quench the ABTS radical cation (ABTS. + )
and reduce the radical to the colorless neutral form. For the rad-
ical solution preparation, 19.3 mg ABTS was dissolved in 5 mL of
H2 O, then 88 μL of a solution 140 mM of K2 S2 O8 (potassium per-
sulfate) was added and the reaction was incubated for 16 h in the
dark at room temperature. After the radical absorbance was ad-
justed at an absorbance of 0.7 at a 730 nm, 245 μL of this solution
was placed in a 96 well plate, then 5 μL of H. brasiletto extract or
their fractions were added. Finally, the reaction was incubated for
5 min at room temperature and the absorbance was read using a
wavelength of 730 nm. Results were reported as μM Trolox Equiv-
alent (TE)/g dried weight (d.w.) using a standard curve of Trolox
(0.03–2.5 mM).
2.4.3. Ferric reducing antioxidant power (FRAP) assay
Ferric reducing antioxidant power assay (FRAP) was carried out
using assay following modified protocols (Benzie and Strain 1996,
Vidal-Gutierrez et al. 2020). FRAP assay is based on the reduction
of ferric 2,4,6-tris(2-pyridyl)-1,3,5-triazine [Fe(III)-TPTZ] to the fer-
rous complex at low pH, followed by spectrophotometric analysis.
Briefly, the reagent was performed mixing 300 mM acetate buffer
(pH 3.6), TPTZ (40 mM, dissolved in 40 mM HCl), and 20 mM aque-
ous ferric chloride in a 10:1:1 proportion. 20 μL of H. brasiletto ex-
tract or their fractions were mixed with 280 μL of FRAP reagent,
and the mixture was incubated for 30 min in darkness and read at
630 nm. Results were expressed as μM Fe(II)/g d.w.
2.5. Cell culture
The non-tumorigenic mammary epithelial cell line MCF10A and
the cancer cell lines MCF7 and MDA-MB-231 (ATCC, Manassas,
VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium
DMEM/F12 culture medium (50:50, V: V; Sigma -Aldrich, St Louis,
MO, USA) supplemented with 5% fetal bovine serum (FBS) and 1%
antibiotics (penicillin G/streptomycin, Gibco, Waltham, MA, USA) in
a humidified atmosphere containing 5% CO2 at 37°C.
2.6. Cell viability assays
For the evaluation of the cell viability, the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay
with some modifications was used (Mosmann, 1983; Rascón-
Valenzuela et al., 2015). Briefly, in a 96 well plate (Costar, Corning,
N.Y. USA), 50 μL per well of a cell suspension (20 0,0 0 0 cells/mL)
was distributed and incubated for 24 h. Subsequently, extract
or fractions were dissolved in dimethyl sulfoxide (DMSO) and
re-suspended in DMEM, obtaining a final maximum concentration
of 0.25% of DMSO that showed no interference with the growth
of the cells. Later, 50 μL of the extract or fractions solutions was
added to each well for 48 h. Afterward, the plate was washed
with a phosphate buffer solution 1X (PBS 1X), and then 100 μL of
MTT solution (5 mg/mL) dissolved in DMEM (9:1 v/v) were added
per well. The plate was subsequently incubated for 4 h under
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al.
culture conditions. Once the incubation time was over, the for-
mazan crystals were dissolved with 100 μL acidified isopropanol.
The analysis was carried out in a microplate reader (Thermo
ScientificTM MultiskanTM FC) at a wavelengths of 570 and 630 nm.
The anti-proliferative activity was determined as IC50 value using
GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA)
(Torres-Moreno et al., 2015).
2.7. Morphological assessment
Cells were exposed to the HB extract and their fractions at var-
ious concentrations (0 to 400 μg/mL) at three different times (0,
24, and 48 h). Images were captured with an EVOS FL optical mi-
croscope using a 40x objective.
2.8. Scratch-wound assays
MCF10A, MCF7, and MDA-MB-231 cells were grown to conflu-
ence in 60 mm culture plates supplemented with DMEM/F12 as
described above. Cells were starved for 24 h in DMEM/F12 with-
out FBS and treated for 2 h with Cytosine β -D-Arabinofuranoside
(AraC) to inhibit cell proliferation during the experiment. After
starvation, cells were scratched using a sterile 200 μL pipette tip.
Suspended cells were removed by washing with PBS twice, and
cultures were re-fed with DMEM/F12 in the presence or absence
of H. brasiletto extract or their fractions (0 to 400 μg/mL). The
progress of cell migration into the wound was monitored at 0
and 24 h using an EVOS FL microscope with a 10x objective. The
lower part of the plate was marked as a reference. The same field
of the monolayers was photographed immediately after wounding
(time = 0 h) and 24 h after the treatments (time = 24 h); five
images were analyzed per plate. The distance between the wound
edges was measured at times 0 and 24 h, and the reported mi-
grated distance corresponds to the difference between these two.
The migration area was determined by measuring the total wound
area using ImageJ software.
2.9. Statistical analysis
Statistical analysis was performed through the GraphPad Prism
8 software. One-way analysis of variance was used, and differences
were determined using the Newman–Keuls and Tukey tests. The p
< 0.05 value was considered statistically significant. Pearson corre-
lation was also carried out.
3. Results and discussion
Plant phenolic compounds are classified into several
groups; chief flavonoids have potent antioxidant activities
(Nunes et al. 2012; Bello Martínez et al., 2021). Flavonoids
are naturally occurring in plants and are thought to have positive
effects on human health. Several studies have shown a wide
range of antibacterial, antiviral, anti-inflammatory, anti-cancer,
and anti-allergic activities (Montoro et al.,2005; Bravo,1998). These
compounds have become more critical in recent years because
they are associated with different biological activities, such as
antioxidants. H. brasiletto is a wild plant from Guerrero, México,
used in traditional medicine against various illnesses; however,
there is little information about its antioxidant potential and
anti-cancer activity.
The present investigation has been carried out to determine the
TPC and TFC present in extracts of the heartwood of H. brasiletto
(Table 1). Results of the quantitative analysis of TPC and TFC of
crude extract and organic fractions revealed that HB of heartwood
contained the highest amount of TPC (258.6 mg GAE/g), followed
by HBC (205.0 mg GAE/g) and HBB (195.2 mg GAE/g). Although,
530
South African Journal of Botany 146 (2022) 528–537
TFC was found highest in HBC (53.0 mg QE/g) followed by HBB
(49.1mg QE/g) and HB (33.5 mg QE/g) (Table 1). A high corre-
lation was found between total flavonoids and total phenol con-
tent (r = 0.7665) (Table 3). Flavonoids are highly effective scav-
engers of most oxidizing molecules, including singlet oxygen and
various free radicals (Bravo et al., 1998), implicated in several dis-
eases. In the same way as with other extracts from plant products
(Sahreen et al., 2011). Our results suggested that phenolic acids and
flavonoids may be the major contributors for the antioxidant activ-
ity of the H. brasiletto extract and fractions and their contents of
phenolics or flavonoids exhibited significant correlation.
In vitro antioxidant activity
DPPH radical was inhibited by HBB and HBC with IC50 values
of 59.2 ± 7.8 and 57.01 ± 5.4 μg/mL, respectively (Table 1). The
study revealed that HBB and HBC have prominent antioxidant ac-
tivity; the presence of phenolic compounds (flavonoids) is mainly
found in these two fractions and could be associated with their
high anti-radical properties. The electron donation ability of nat-
ural products can be measured by DPPH purple-colored solution
bleaching (Nunes et al. 2012). The method is based on the scaveng-
ing of DPPH by adding a radical species or antioxidant that decol-
orize the DPPH solution. A large decrease in the absorbance of the
reaction mixture indicates significant free radical scavenging activ-
ity of the compound under test (Krishnaiah, 2011). In the present
study, among all the fractions tested, HBB and HBC showed signifi-
cantly higher inhibition percentages and positively correlated with
TPC (r = 0.9094) and with TFC (r = 0.9615) (Table 3). Our results
suggest that the plant extract contains a phytochemical compound
capable of donating hydrogen to a free radical to scavenge the po-
tential damage.
The free radical scavenging activity of H. brasiletto extracts was
also determined through TEAC assay (Table 1). Results evidenced
that evaluated extracts exhibited scavenging activity against ABTS+
radical, presenting HBC the highest antioxidant potential (61.4
μMTE/g) followed by HBB (60 μMTE/g). Also, HB showed great ca-
pacity to stabilize the hydroxyl radical. ABTS radical scavenging as-
say involves a method that generates a blue/green ABTS+ chro-
mophore via ABTS and potassium persulfate reaction. The ABTS
radical cation is caused by the oxidation of ABTS with potassium
persulfate. Its reduction in the presence of hydrogen-donating an-
tioxidants is measured spectrophotometrically at 730 nm. All the
fractions possessed intense ABTS scavenging activity, an observa-
tion that is supported by other studies (Sahreen et al., 2011).
Previous reports suggested that the reducing properties have
been shown to exert antioxidant action by donating a hydrogen
atom to break the free radical chain (Gordon, 1990). Increasing
absorbance at 630 nm indicates an increase in reducing ability.
The antioxidants present in the fractions of H. brasiletto caused
their reduction of Fe3+ /ferricyanide complex to the ferrous form
and thus proved the reducing power. Data obtained by the FRAP
evaluation are presented in Table 1. The obtained HBC results ex-
hibited a high ferric reducing activity with 2678.2 μM Fe(II)/g
followed by HBB 2453.9 μM Fe(II)/g. Ferric reducing antioxidant
power values showed that these extracts could be classified as ex-
tracts with strong ability to reduce the ferric complex based on
the classification performed by Wong et al. (2006), according to
which natural’s extracts with FRAP values above 500 μM Fe (II)/g
are considered ferric reducing extracts. On the other hand, the re-
lationship between antioxidant activity and phenolic compounds
was determined by correlation analysis. A strong correlation was
observed in all cases because the regression coefficients (r) were
0.91, 0.90, and 0.59 for the correlation between the data obtained
from the DPPH, TEAC, and FRAP assay vs. phenolic content, re-
spectively (Table 3). The last finding suggests that the antioxidant
potential of H. brasiletto extracts is attributed to the high phe-
nolic compound content. However, higher correlations were ob-
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al. South African Journal of Botany 146 (2022) 528–537

Table 1
Total Phenol, Total Flavonoid, DPPH, TEAC and FRAP assay of Haematoxylum brasiletto and fractions∗ .

samples Total Phenol mg AGE/g Total Flavonoid mg QE/g DPPH IC50 μg/mL TEAC μMTE/g FRAP μM Fe(II)/g

HB 258.6 ± 13.7a 33.5± 6.5c 88.60 ± 9.7c 53.5 ± 9.6c 1283.9 ± 63.7c
HBA 56.1 ± 4.9d 2.9 ± 0.3d 350.0 ± 26.1d 11.2 ± 1.5d 447.8± 35.1d
HBB 195.2 ± 10.6c 49.1± 9.4b 59.20 ± 7.8b 60.0 ± 7.2b 2453.9 ± 86.3b
HBC 205.0 ± 16.8b 53.0 ± 9.2a 57.01± 5.4a 61.4 ± 3.4a 2678.2 ± 93.3a

Values represent a mean and standard deviation (±SD; n = 3) of three independents experiments. a-d significant difference in
statistical analysis– Independent for each experiment (column) p < 0.05. HB: Ethanolic crude extract; HBA: hexane fraction; HBB:
dichloromethane fraction; and HBC: ethyl acetate fraction.

Fig. 1. Effect of the ethanolic extract and fractions from H. brasiletto on the cell viability of MCF10A cells.
a-d
Bars with different superscripts indicate statistical differences (p < 0.05). (A) ethanolic extract from the heartwood, (B) n-hexane fraction, (C) dichloromethane fraction,
(D) ethyl acetate fraction.

served between the antioxidant capacity and the total polyphenol
(r = 0.8921, p < 0.01), antioxidant capacity and total flavonoids
(r = 0.9094, p < 0.01), and between total flavonoids and to-
tal polyphenols (r = 0.9615, p < 0.01) (Table 3).This means that
polyphenols and flavonoids are responsible for the high antioxidant
activity.
Anti-proliferative activity of ethanol extract and solvent fractions of
H. brasiletto on breast cancer cell lines and non-tumorigenic epithelial
cells
In Guerrero, Mexico, some people use H. brasiletto in everyday
practice for traditional medicine against different illnesses (Bello-
Martínez et al., 2017). Based on this widespread knowledge and
a few scientific studies demonstrating its anti-cancer property, we
decided to evaluate the anti-proliferative and anti-migratory activ-
ity of the ethanol extract of H. brasiletto and their organic fractions
in different cancer cell lines.
In order to determinate the anti-proliferative activity of the ex-
tract and different fractions obtained from H. brasiletto, the non-
invasive (MCF-7), highly invasive (MDA-MB-231) breast cancer cell
lines and the non-tumorigenic breast epithelial cell line (MCF10A)
were used. A range of extract concentrations of 0 to 25 μg/mL
was tested (Figure 1). HB inhibited the proliferation of MCF7 and
MCF10A in a dose-response manner, however, the greatest effect
was observed in MCF7 at the maximum concentration tested of 25
μg/mL (Figure 1 and 2). The IC50 values calculated for HB in MCF7
and MCF10 cells were 5.9 ± 0.7 and 3.0 ± 1.7 μg/mL, respectively.
The fractions tested exhibited potent inhibitory activity on
MCF7 and MCF10A cells. All the fractions showed a dose-response
effect on cell proliferation of both cell lines (Figure 2 and 3). HBB
showed the highest anti-proliferative activity on MCF7 cells with
IC50 values of 2.0 ± 0.1 μg/mL (Table 2), meanwhile MCF10A was
inhibited by HBB with an IC50 of 2.9 ± 1.3 μg/mL. For its part, HBC
showed IC50 of 5.5 ± 0.5 μg/mL on MCF7 and 2.8 μg/mL ± 0.5
in MCF10A. HBA showed lower anti-proliferative activity than HBB
and HBC against MCF7 and MCF10A cells, for this case the IC50 val-
ues of HBA in MCF7 and MCF10A cells were 18.9 ± 4.1 μg/mL and
4.7 ± 0.9 μg/mL, respectively.
On the other hand, the triple negative MDA-MB-231 (ER-/ PR-
/HER2-) was more resistant to all the treatments (extract and frac-
tions), for all the concentration tested (3.1-25 μg/mL) the % of cell
proliferation was above 75% (Figure 3), which may be associated
with their selectivity against MCF7 (ER+/EP+) cancer cells.
According to the National Cancer Institute (NCI), plant extracts
with IC50 values ≤ 30 μg/mL are considered active (Suffness &

531
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al. South African Journal of Botany 146 (2022) 528–537

Fig. 2. Effect of the ethanolic extract and fractions from H. brasiletto on the cell viability of MCF7 cells.
a-d
Bars with different superscripts indicate statistical differences (p < 0.05). (A) ethanolic extract from the heartwood, (B) n-hexane fraction, (C) dichloromethane fraction,
(D) ethyl acetate fraction.

Pezzuto, 1990). Based on the above, extract and fractions from H.

Table 2
Anti-proliferative activity of Haematoxylum brasiletto extract and or-
brasiletto can be considered cytotoxic against MCF7 cells and po-
ganic fractions on three cell lines measured by MTT assay. IC50 ± SD tential candidates to obtain antitumor agents against these types
(μg/mL)∗ . of cancer; however, its selectivity is one of the aspects that must
Sample Cell lines
also be studied.
Antimigratory activity of ethanol extract and solvent fractions of
MCF10A MCF7 MDA-MB-231
H. brasiletto on breast cancer cell lines
HB 3.02 ± 1.7 b 5.9 ± 0.7 d > 25 One of the main hallmarks of cancer is the capacity of cell
HBA 4.7 ± 0.9 c 18.9 ± 4.1 e > 25
proliferation as a consequence of genetic and epigenetic changes.
HBB 2.9 ± 1.3 a 2.0 ± 0.1 a > 25
HBC 2.8 ± 0.5 a 5.5 ± 0.5 c > 25
However, cancer cells can extend beyond their usual limits, invad-
Doxorubicin∗∗ 4.8 ± 0.7 c 4.6 ± 1.3 b > 25 ing neighboring tissues and eventually spreading to other organs
a-e
in a metastasis process. In this context, cancer cell migration is
Statistical analysis significant difference – Independent for each
cell line (column). p < 0.05 one of the early steps in the metastasis cascade. These tumor cells
∗
IC50 values represent a mean and standard deviation (± SD; use their intrinsic migratory capacity to invade adjacent tissues
n = 3) of three independents experiments.HB: Ethanolic crude ex- and intravasate blood vessels and eventually promote secondary
tract; HBA: hexane fraction; HBB: dichloromethane fraction, and tumor formation and, subsequently, the death of cancer patients
HBC: ethyl acetate fraction.
∗∗ (Yamaguchi and Condeelis, 2007).
Doxorubicin was used as a positive control.

Table 3
Pearson’s correlation coefficient for Total Phenol, Total Flavonoid, DPPH, TEAC, FRAP, and Anti-proliferative activity of H.
brasiletto

TPC TFC DPPH TEAC FRAP MCF10A MCF7 MDA-MB-231

TPC 1
TFC 0.7665∗∗ 1
DPPH 0.9094∗∗ 0.9615∗∗ 1
TEAC 0.8921∗∗ 0.9727∗∗ 0.9989∗∗ 1
FRAP 0.5861∗ 0.9695∗∗ 0.8655∗∗ 0.8865∗∗ 1
MCF10A 0.5474∗ 0.9305∗∗ 0.8103∗∗ 0.8354∗∗ 0.9697∗∗ 1
MCF7 0.8248∗∗ 0.9785∗∗ 0.9820∗∗ 0.9854∗∗ 0.9172∗∗ 0.8363∗∗ 1
MDA-MB-231 0.7031∗∗ 0.1979 0.3934 0.3687 0.0098 0.0853 0.2132 1

Pearson’s correlation between total phenols content (TPC), total flavonoids content (TFC), antioxidant capacity with
(DPPH, TEAC, and FRAP) and Anti-proliferative activity on (MCF10A, MCF7, and MDA-MB-231 cell lines).
∗
Significant at p ≤ 0.05
∗∗
significant at p ≤ 0.01

532
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al.
Fig. 3. Effect of the ethanolic extract and fractions from H. brasiletto on the cell viability of MDA-MB-231 cells.
a-d
Bars with different superscripts indicate statistical differences (p < 0.05). (A) ethanolic extract from the heartwood, (B) n-hexane fraction, (C) dichloromethane fraction,
(D) ethyl acetate fraction.
To prevent the death of cancer patients, natural products have
been used in the treatment of several cancers and have be-
come an important research area for drug discovery (Cayetano-
Salazar, 2021). Recently, Bello et al. (2017) reported that the ex-
tracts of H. brasiletto showed anti-proliferative activity in A549,
RAW 264.7, and L-929 cancer cell lines.
In our study, we used the non-invasive MCF7 breast cancer cell
line, the highly invasive MDA-MB-231 breast cancer cell line, and
the non-tumorigenic MCF10A mammary epithelial cell line as in
vitro models to evaluate the anti-migratory activity of the different
fractions obtained from H. brasiletto against cell migration.
Our results showed that the HBA fraction displayed anti-
migratory activity against the MCF7 breast cancer cell line and
the MCF10A non-tumor epithelial cell line (Figure 4, 5). Interest-
ingly, this fraction promoted apoptosis in MCF-7 and MDA-MB-
231 breast cancer cells; however, in MCF10A, non-tumorigenic ep-
ithelial cells were not observed this effect (Figure 6). Our results
are related to the findings found by Wagner & Elmadfa (2003);
they describe that terpenoids inhibit cell proliferation and tumor
growth in a wide variety of human cancers. These class of com-
pounds is beneficial in maintaining and improving health. This
compound category also exhibits multiple properties involved in
biological events such as oxidative stress, carcinogenesis, and car-
diovascular diseases.
Regarding the HBB fraction, we observed an anti-migratory ac-
tivity in the MCF10A epithelial cell line (Figure 4). Interestingly,
in MCF7 and MDA-MB-231 breast cancer cell lines, this fraction
not only showed a potent anti-migratory activity but also in-
duces apoptosis in these cells (Figure 5, 6). Furthermore, Bello-
Martínez et al. (2017) showed that the hexane fraction of H.
brasiletto contains flavonoids such as Brazilin, which can inhibit the
growth of the cancer cell lines SiHa, MDA-MB-231, A549, and NCI-
H1299. This fraction also exhibited a moderate anti-proliferative
activity in the non-cancerous human cell ARPE-19 and showed
anti-proliferative activity against human cancer cell lines.
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South African Journal of Botany 146 (2022) 528–537
Here, we also show that HBC displayed an anti-migratory ac-
tivity in the MCF10A cell line (Figure 4), while it induces apop-
tosis in MCF7 and MDA-MB-231 breast cancer cells (Figure 5, 6).
Our results match that described by Habli et al. (2017), where they
find that alkaloids can be used in cancer treatment because they
help suppress oncogenesis. Alkaloids can also modulate key signal-
ing pathways involved in proliferation, cell cycle progression, and
metastasis, making them the major components of several clinical
anti-cancer agents (Millimouno, Dong, Yang, Li, & Li, 2014).
Furthermore, Hirsutin, a significant alkaloid extracted from
plants of the genus Uncaria, selectively inhibits AKT, one of the
main proteins involved in cell proliferation in human breast cancer
cells (Lou, Takahashi, Irimura, Saiki, & Hayakawa, 2014). Most of
these medically exploited alkaloids function as therapeutic agents,
primarily causing DNA damage, inducing apoptosis, and acting as
anti-proliferative agents (Thawabteh et al., 2019).
Interestingly, such apoptosis induction was clearly visible in the
case of MDA-MB-231 and MCF7 breast cancer cell lines treated
with HBB and mainly HBC fractions. This finding could be related
to activating the intrinsic mitochondrial pathway and is triggered
by physical or chemical stimuli, such as hypoxia, growth factor de-
privation, cell shedding, or stress signals. However, this issue will
need to be evaluated in future research.
4. Conclusions
This study has demonstrated that HB heartwood extract and
their HBB and HBC fractions inhibited the proliferation of MCF-7
cells and inhibited cell migration in breast cancer cell lines. Addi-
tionally, we provide novel information about its interesting antiox-
idant activity. Our findings suggest that this activity can be asso-
ciated with phenolic compounds in extracts, especially flavonoids.
Thus, this plant could represent an excellent potential source of
phenolic extract or fractions rich in these compounds that could
serve as sources of drugs to treat cancer and other diseases re-
G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al. South African Journal of Botany 146 (2022) 528–537

Fig. 4. Confluent monolayers of MCF10A cells were treated 2 h with AraC to inhibit proliferation and then treated with different fraction concentrations. Wound-healing
assays were performed by scratching the monolayer using a sterile pipette tip. The progress of cell migration into the wound was registered at 0 (upper panel) and 48
h (lower panel). Representative light microscopy images and quantification of wounding assays were done in HB-treated MCF10A cells (A), HBA-treated MCF10A cells (B),
HBB-treated MCF10A cells (C), and HBC-treated MCF10A cells (D) were done. Data are expressed as a percentage of wound closure for each condition and represent the
mean of three independent experiments ± s.d. The Newman–Keuls test compares all experimental conditions to determine statistically significant differences; ∗∗ P < 0.01 by
one-way ANOVA.

lated to oxidative stress. However, the precise mechanisms of ac-
tion need to be explored.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.

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G. Bello-Martínez, G. García-Ramírez, M. Olea-Flores et al. South African Journal of Botany 146 (2022) 528–537

Fig. 5. Confluent monolayers of MCF7 cells were treated 2 h with AraC to inhibit proliferation and then treated with different fraction concentrations. Wound-healing assays
were performed by scratching the monolayer using a sterile pipette tip. The progress of cell migration into the wound was registered at 0 (upper panel) and 48 h (lower
panel). Representative light microscopy images and quantification of wounding assays in HB-treated MCF7 cells (A), HBA-treated MCF7 cells (B), HBB-treated MCF7 cells
(C), and HBC-treated MCF7 cells (D) were done. Data are expressed as a percentage of wound closure for each condition and represent the mean of three independent
experiments ± s.d. The Newman–Keuls test compares all experimental conditions to determine statistically significant differences; ∗∗ P < 0.01 by one-way ANOVA.

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