BRAZILIN FROM STEM OF Caesalpinia bahamensis subsp.

orientensis. IN VITRO ANTIBACTERIAL ACTIVITY AND IN SILICO

BIOPROSPECTING OF THEIR OXIDATION PRODUCT

Luis A. Osoriaa, Alexander Dueñas Deyáb, Iraida Spenglera*, Trina H. Garcíaa,

Olga L. Hechemendiac

ABSTRACT
Brazilin was isolated as the major secondary metabolite from the ethanolic extract of
the wood of the subspecies Caesalpinia bahamensis subsp. orientensis. Its structure
was established by analyzing 1D and 2D NMR spectroscopic data and HRESIMS.
The evaluation of the in vitro antibacterial activity of the ethanolic, chloroformic
and ethyl acetate extracts against strains of Staphylococcus aureus (ATCC 6538)
and Escherichia coli (ATCC 8739), reveals that all the crude extracts are active
in a MIC range between 6.2-25 mg/mL, being S. aureus more susceptible to the
inhibitory effect of these extracts. On the other hand, due to the photooxidation
of brazilin, the evaluation of the antimicrobial potential both in vitro and in silico
was carried out for its oxidation product, brazilein, along with the bioprospecting
of its spectrum of biological activities using in silico modeling on the PASS web
platform. Brazilein only exhibited inhibitory activity against the S. aureus strain
with a MIC=12.5 mg/mL. Likewise, in silico modeling indicated 50 possible bacterial
targets on which this molecule could have antibacterial effects, which contrasts
with the set of biochemical targets identified as possible mechanisms of action
through which brazilein could exert antimicrobial effects.
Keywords: Caesalpinia bahamensis subsp. orientensis, brazilin, photooxidation,
brazilein, antibacterial, PASS

RESUMEN
La brasilina se aisló como el metabolito secundario principal del extracto etanóli-
co de la madera de la subespecie Caesalpinia bahamensis subsp. orientensis. Su
estructura se estableció mediante el análisis de datos espectroscópicos de RMN
1D y 2D y HRESIMS. La evaluación de la actividad antibacteriana in vitro de los
extractos etanólico, clorofórmico y de acetato de etilo frente a cepas de Staphylo-
coccus aureus (ATCC 6538) y Escherichia coli (ATCC 8739), revela que todos los
extractos crudos son activos en un rango de CMI entre 6,2-25 mg/mL, siendo

a
Grupo de Síntesis Química, Centro de Investigación y Desarrollo de Medicamentos, Ave.26 e/ Puentes
Grandes y Ave. Independencia, Plaza, La Habana, Cuba.
b
Facultad de Química. Universidad Nacional Autónoma de México. Ciudad Universitaria, Coyoacán 04510,
México D.F., México.
c
Instituto Finlay de Vacunas, Avenida No. 21, No. 19810, entre 198 y 200, Reparto Atabey 10400, Playa,
La Habana, Cuba.
*Correspondence: Iraida Spengler Salabarria. Centro de Estudios de Productos Naturales, Facultad de Quí-
mica, Universidad de La Habana, La Habana 10400, Cuba. Email: iraida@fq.uh.cu

7
8 Osoria, L. A., et al.

S. aureus más susceptible al efecto inhibitorio de estos extractos. Por otro lado,
debido a la fotooxidación de la brasilina, se llevó a cabo la evaluación del potencial
antimicrobiano tanto in vitro como in silico de su producto de oxidación, la bra-
zileína, y la bioprospección de su espectro de actividades biológicas mediante un
modelado in silico en la plataforma web PASS. La brazileína solo mostró actividad
inhibitoria contra la cepa S. aureus con una CMI=12,5 mg/mL. Asimismo, el mo-
delado in silico indicó 50 posibles dianas bacterianas sobre las que esta molécula
podría tener efectos antibacterianos, lo que contrasta con el conjunto de dianas
bioquímicas identificadas como posibles mecanismos de acción a través de los
cuales la brazileína podría ejercer efectos antimicrobianos.
Palabras clave: Caesalpinia bahamensis subsp. orientensis, brazilina, fotooxida-
ción, brazileína, antibacteriano, PASS

INTRODUCTION
Phytochemical research based on eth-
nopharmacology is considered an effective
approach in the discovery of new chemical
entities with potential as drugs for the
treatment of diseases (Brusotti et al., 2014).
According to the European Agency for Clini-
cal and Therapeutic Pharmacology, it is es-
timated that by the year 2050, deaths due
to the emergence of bacterial resistance will
begin as the greatest threat to health care
(Basu and Mukherjee, 2018). In this sense,
there is a marked increase in publications
in the field of natural products focused on
the search for new structures with potential
as possible antimicrobial agents (Liu et al.,
2022, Khan et al., 2018)
The genus Caesalpinia (Fabaceae)
constitutes a genus of plants made up of
more than 500 species distributed mainly
in the tropical and subtropical zones. Di-
fferent species of Caesalpinia are used as
part of the traditional herbal medicine of
different Latin American communities such
as: emmenagogues, for the treatment of
inflammation, digestive, to improve cir-
culation, among other purposes. These
ethnopharmacological uses are correlated
with a wide spectrum of biological activi-
ties exhibited by different species of this
genus such as: antibacterial, antifungal,
anti-inflammatory, antiproliferative, among
others (Zanin et al., 2012) .
Caesalpinia bahamensis subsp. orien-
tensis is an endemic subspecies located
mainly on the north coast of the Cuban
archipelago, in plant formations of the
type: coastal and sub-coastal xeromorphic
scrub, and microphilous semi-waste forest.
This subspecies from the point of view of its
conservation status is categorized as ‘not
threatened’ (Berazaín et al., 2005, Gonzá-
lez-Torres et al., 2016). The wood of this
shrub is used in Cuba, empirically, for the
treatment of liver and kidney diseases, as
well as diabetes and chronic ulcers (Felipe
et al., 2017, Roig, 1978). In the specialized
literature, the validation of the diuretic
activity of its aqueous extract in Wistar
rats is reported for the wood of the non-
endemic subspecies C. bahamensis subsp.
bahamensis (González et al., 2011), as well
as the antioxidant effect of three fractions
of its methanolic extract (Felipe González
et al., 2019). From the point of view of its
chemical composition for this last subspe-
cies, the absence of alkaloids is recognized
(Dominicis et al., 1995), and the presence of
fatty acids and their methyl esters, as well
as sesquiterpenes and phytosterols in an
non-polar fraction of the methanolic extract
of the wood (Felipe et al., 2017). Likewise,
a recent study refers to the isolation of
metasappanin from this same species, a
structurally novel homoisoflavonoid (Gon-
zález et al., 2020).
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis
In general, the bioguided strategies
followed in the study of natural products
from plants lead to the identification of
phytochemicals associated with their eth-
nic use. In this sense, progress has been
made with the aim of exploring more ex-
haustively the pharmacological profiles of
phytoconstituents through the application
of the PASS (Prediction of Activity Spectra
for Substances) web platform (Pramely
and Raj, 2012). This in silico approach is
capable of revealing with 96% precision
around 4000 types of biological activities
for a given chemical compound based only
on its structural formula, including phar-
macological effects, mechanisms of action,
toxic and adverse effects, interaction with
enzymes, metabolic and transporters, in-
fluence on gene expression, etc., as well
possible side effects. The estimate is based
on the analysis of the structure-activity
relationship for a broad training set that
includes drugs and candidates in various
stages of clinical and preclinical research,
chemical-pharmaceutical agents under
study, as well as compounds whose spe-
cific toxicity is known (Filimonov et al.,
2014). There are numerous investigations
that support the applicability of the PASS
platform to the study of natural products,
which include both isolated products from
terrestrial plants and marine organisms
and their use is even possible for virtual
structures that have not yet been synthe-
sized (Kurashov et al., 2016, Lagunin et al.,
2010, Dembitsky et al., 2005, Goel et al.,
2011, Anand et al., 2017, Devillers et al.,
2007, Pramely and Raj, 2012).
Taking into account that for the en-
demic subspecies C. bahamensis subsp.
orientensis there is no information about
its chemical composition, and there are no
reports in the literature on the validation
of its antibacterial activity; this work is
oriented on the qualitative phytochemical
characterization of the ethanolic extract of
the wood of C. bahamensis subsp. orien-
tensis, the evaluation of the antibacterial
Rev. Latinoamer. Quím. 50/1(2023) 9
activity of the obtained extracts and the
isolation and structural characterization
of the major metabolite of ethanolic crude
using chromatographic and spectroscopic
techniques, respectively. Likewise, it is in-
tended to estimate the profile of biological
activities and bacterial targets for the isola-
ted majority compound in order to propose
a guide for future evaluations in medicinal
chemistry.
MATERIAL AND METHODS
GENERAL EXPERIMENTAL PROCEDURES
1D and 2D NMR experiments (HSQC,
HMBC, COSY, NOESY) were recorded in
CD3OD using a Bruker DRX-400 spectro-
meter (Rheinstetten, Germany), operating
at 400 MHz for 1H and at 100 MHz for 13C.
Chemical shifts are expressed in ppm and
coupling constants in (J) in Hz. The HRES-
IMS in negative mode were obtained in an
Orbitrap Elite mass spectrometer (Ther-
mo Fisher Scientific, Bremen, Germany)
equipped with a heated ESI electrospray
ion source (spray voltage 3.5 kV, capillary
temperature 325 °C, source heater tem-
perature 45 °C, FTMS resolution 30000).
Nitrogen was used as a sheath gas. Silica
gel (200-300 mesh, Merck®) was used for
the chromatographic column. The analysis
of the chromatographic profile of the frac-
tions by TLC was carried out using 60F254
silica gel plates with glass support, the
compounds were detected by spraying the
plates with 1% vanillin/H2SO4 solution and
subsequent heating at 100°C until maxi-
mum intensity of spots.
PLANT MATERIAL
The stem of C. bahamensis subsp. orien-
tensis Borhidi, in the flowering stage,
was collected from a plant from Mayarí,
Holguín; in the National Botanical Garden
(NBG), Havana, Cuba; in June 2017. The
species was identified by Prof. Dr. Rosalina
Berazaín Iturralde, specialist at the NBG
Research Laboratory. The control material
10 Osoria, L. A., et al.
is a HAJB 89621 specimen deposited in
the Herbarium from the institution itself.
The stem was separated from the bark by
hand, the wood was dried in the shade for
15 days, and then it was reduced to small
pieces.
EXTRACTION AND ISOLATION
The chopped stem (375 g) was macerated
with 2 L of ethanol at room temperature
assisted with of ultrasound, twice. The
extract was concentrated under reduced
pressure at 45°C, obtaining 25.06 g of a
red syrup (6.68% yield). 15 g of the crude
obtained was redissolved in 600 mL of a
30% hydroalcoholic mixture in ethanol
and subsequently partitioned with chlo-
roform and ethyl acetate to give primary
fractions of 6.83 and 0.71 g, respectively. A
portion of the chloroform extract (1 g) was
subjected to a silica gel column using a
polarity gradient solvent system as mobile
phase: CH2Cl2-MeOH (100:0, 95:5, 90:10,
80:20, 300 mL of each mixture). 7 major
fractions (C1-C7) were pooled according to
their chromatographic profile. Fraction C4
(53.4 mg) shows the presence of an intense
spot, corresponding to the major metabo-
lite of ethanolic crude extract, according
to the TLC chromatographic profile of the
ethanolic extract and primary fractions. C4
was purified by preparative thin-layer chro-
matography, using CH2Cl2-MeOH (95:5) as
the solvent system. 4.8 mg of the purified
major compound was obtained.
Brazilin: amorphous solid colored oran-
ge, 1H NMR (400 MHz, Methanol-d4) δ 7.17
(d, J = 0.7 Hz, 1H) H-1, 6.70 (s, 1H) H-8,
6.59 (s, 1H) H-11, 6.46 (dd, J = 8.3, 2.5
Hz, 1H) H-2, 6.28 (d, J = 2.5 Hz, 1H) H-4,
3.96 (s, 1H) H-12, 3.91 (d, J = 11.4 Hz, 1H)
H-6, 3.68 (d, J = 11.3 Hz, 1H) H-6, 3.01 (d,
J = 15.6 Hz, 1H) H-7, 2.77 (d, J = 15.6 Hz,
1H) H-7. 13C-NMR (101 MHz, Methanol-d4)
δ 157.87 (C-4a), 155.72 (C-3), 145.66 (C-
9), 145.34 (C-10), 137.43 (C-11a), 132.21
(C-1), 131.31 (C-7a), 115.54 (C-1a), 112.83
(C-8), 112.42 (C-11), 109.93 (C-2), 104.25
(C-4), 78.07 (C-6a), 70.85 (C-6), 51.06 (C-
12), 42.91 (C-7)
The qualitative characterization of the
ethanolic extract of the wood was carried
out according to the technique described
by Rondina and Coussio (Rondina and
Coussio, 1969).
IN VITRO ANTIBACTERIAL ASSAY
The in vitro antibacterial activity of the diffe-
rent extractions and brazilein was evalua-
ted using the microdilution method (CLSI,
2000). The reference strains of Staphylococ-
cus aureus (ATCC 6538) and Escherichia
coli (ATCC 8739) were used for the test. The
microorganisms used were adjusted to 0.5
McFarland scale. Streptomycin sulfate was
used for the positive control.
From each of the extractions, double
dilutions were made from 100 mg/mL to
1.55 mg/mL in 96-well plates. The dilutions
were made using 0.9% saline solution for
the ethanolic extract and ethyl acetate ex-
tracts, while DMSO was used for the chlo-
roform extract and the isolated compound.
180 µL of each dilution of the extracts plus
20 µL of microorganism adjusted to 0.5
McFarland scale were added to each well.
The plates were incubated at 37°C for 24
hours, subsequently 5 µL of each dilution
were seeded in triplicate on plates with
Triptona Soya Agar medium, the plates
were incubated 24 hours at 37°C. The mi-
nimum inhibitory concentration (MIC) was
determined as the lowest concentration
capable of inhibiting bacterial growth.
PASS APPROACH
Keeping in mind the idea that most meta-
bolites isolated from plants are generally
conducted through a bioguided pharma-
cological evaluation and they are not fully
exploited by attending to all possible bio-
logical targets with which they could inte-
ract, this leaves out a potential number of
possible applications for a given compound.
With this objective, the spectrum of
biological activities was estimated for the
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis Rev. Latinoamer. Quím. 50/1(2023) 11

abundant compound isolated from the is expressed as confidence in its activity,

wood of C. bahamensis subsp. orientensis.
For this, PASS online platform, free avai-
lable from http://www.way2drug.com/
passonline/index.php, was used; which
can predict around 4000 pharmacological
effects, mechanisms of action, mutageni-
city, carcinogenicity, teratogenicity and
embryotoxicity, based on the structural
formula of the compound.
IN SILICO ANTIBACTERIAL ACTIVITY
PREDICTION
There is an urgent need for new drugs
against bacterial infections because of the
increased incidence of bacterial resistance
to traditional antibiotics (Calvo and Martí-
nez-Martínez, 2009).
AntiBac-Pred free available from http://
www.way2drug.com/antibac/ allows to
predict the fact that chemical compound
can inhibit the growth of one or more of
353 bacteria in concentration below the
10000 nM. The score for each compound
which is a difference between probabili-
ties for chemical compound to inhibit (Pa)
and to do not inhibit (Pi) the growth of the
particular bacteria. The higher confidence
means the higher chance of the positive
prediction to be true. Only activities with
Pa > Pi (confidence > 0) are considered as
possible for a particular compound.
Using the web-service we could estimate
the most promising bacteria for which the
products tested would be active in order
to determine priorities during biological
assays.
RESULTS AND DISCUSSION
The results of the qualitative phyto-
chemical characterization of the ethanolic
crude from stem of C. bahamensis subsp.
bahamensis using the technique described
by Rondina and Coussio, are tabulated
in Table 1. Although this methodology is
essentially qualitative, it allows making

Table 1. Main types of metabolites detected in the ethanolic crude from stem of Caesalpinia baha-
mensis subsp. orientensis
Fractions Metabolites tested Ethanolic crude
Amino groups -
A Phenolic compounds ++
Tannins +
Triterpenes/steroids ++
B.
Quinones +
C1 Alkaloids -
Alkaloids -
C2 Triterpenes/steroids ++
Cardenolides +++
Flavonoids ++
Cardenolides +
D Triterpenes/steroids ++
Proanthocyanidins/catechins ++
Alkaloids -
Proanthocyanidins/catechins ++
E Reducing sugars +++
Flavonoids +++
Saponins +
F
Amino groups -
Symbology: (-) Not detected, (+) Scarce, (++) Moderate, (+++) Abundant
12 Osoria, L. A., et al.
Fig. 1
certain inferences regarding the relative
abundance of the families of metabolites
detected according to the intensity of the
color observed in the assay or the formation
of a precipitate in some cases.
The phytochemical screening indicated
the abundant presence in the ethanolic
crude of cardenolides, flavonoids and re-
ducing sugars. The abundance of this last
group of metabolites is indicative of the
presence of glycosylated derivatives, and
taking into account the presence of flavo-
noids in the E fraction itself, it is likely that
both positive tests are associated with the
large quantity of glycosylated flavonoids. In
addition, a moderate content of triterpene/
steroidal, phenolic, and proanthocyanidin/
catechin metabolites was detected. On the
other hand, with the tests carried out it was
not possible to detect for this species the
presence of alkaloids or compounds with
amino groups.
The previous results contrast with those
presented by González et al. (2020), who
demonstrated a flavonoid content higher
than that of total phenols in the hydroalco-
holic extract for the species C. bahamensis
subsp. bahamensis, as well as the presence
of steroids, coumarins, saponins. In addi-
tion, no alkaloids were detected for said
species (González, 2020).
IN VITRO ANTIBACTERIAL ACTIVITY
The evaluation of the antibacterial activity
of the extracts of C. bahamensis subsp.
orientensis against Staphylococcus aureus
and Escherichia coli strains, Fig. 1, revealed
that both bacterial strains are sensitive to
the components of these extracts, being S.
aureus more susceptible to the effect of the-
se metabolites, by inhibiting their growth at
relatively lower concentrations compared to
E. coli. In line with these results, different
extracts and compounds of other species
of the Caesalpinia genus have exhibited a
marked antibacterial activity associated
with different types of compounds such as
homoisoflavonoids, cassane-type terpenes
or phenolic compounds (Zanin et al., 2012).
Precisely, a study with the subspecies C.
bahamensis subsp. bahamensis (Abreu et
al., 2017), demonstrates remarkable anti-
microbial activity against other strains of
these same microorganisms, despite the
fact that it uses the method of diffusion in
agar disk that does not allow a distinction
between the bacteriostatic or bactericidal
effect, and is practically qualitative (Baloui-
ri et al., 2016). Although it was not possible
to evaluate the antibacterial activity of bra-
zilin, for its oxidation product its inhibitory
effect was evaluated, showing active on
the S. aureus strain at a concentration of
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis
12.5 mg/mL, likewise this compound was
inactive against to the strain of E. coli. The-
se results are consistent with the in-silico
predictions of the biological spectrum and
antibacterial activity for brazilein, taking
into account the wide group of possible
biological targets through which brazilein
can manifest antibacterial activity, as well
as, the higher confidence value predicted
for a resistant S. aureus strain compared
to the tested strain of E. coli.
SPECTROSCOPIC CHARACTERIZATION OF
THE MAJOR METABOLITE
The major wood metabolite of C. baha-
mensis subsp. orientensis was isolated
as an amorphous solid colored orange.
From the mass spectrum, the compound
was assigned the global formula C16H14O5,
corresponding to a structure having 10
degrees of unsaturation. The 1H-NMR spec-
trum reveals the presence of two aromatic
systems in the structure, the first of them
tetrasubstituted with a 1,2,4,5 substitution
pattern and the second 1,2,4-substituted,
which is confirmed by from 5 correlation
signals in the aromatic region in the HSQC
spectrum. Likewise, in the aliphatic re-
gion there are signals corresponding to a
methylene group (δ=2.77; 3.01; J=15.6 Hz),
an oxygenated methylene (δ=3.68; 3.92;
J=11.4 Hz), both with geminal couplings;
and a methine group (δ = 3.96 ppm). From
this information, it is inferred that it is
a flavonoid, and taking into account the
Fig. 2
Rev. Latinoamer. Quím. 50/1(2023) 13
presence of 16 signals of the compound
in the 13C-NMR spectrum, the compound
is a homoisoflavonoid (Castelli and López,
2017). The assembly of the structure was
possible using coordinately the heteronu-
clear correlation information (HMBC), Fig.
2, with the rest of the spectral information.
The compound was identified as brazilin
and the spectral data coincide with those
reported for this compound, previously
isolated from the species C. sappan (Nirmal
et al., 2015).
On the other hand, prior to the eva-
luation of the antibacterial activity of the
isolated compound, its mass spectrum
was obtained in negative mode, obtaining
the global formula C16H12O5 with two mass
units lower than that reported for brazilin
as a consequence of the susceptibility of
this homoisoflavonoid to oxidation by expo-
sure to light and air as reported by Rondao
et al. (Rondao et al., 2013). This oxidation
process causes the hydroxyl group at po-
sition 9 to transform into a quinoid group
and the oxidation of C12, thus obtaining
brazilein. Fig. 3 shows the mass spectrum
obtained, and the oxidation reaction:
ESTIMATION OF BIOLOGICAL ACTIVITY
SPECTRUM OF BRAZILEIN
PASS prediction is based on the analysis
of structure-activity relationships (SAR)
of a training set containing around 1 000
000 biological active substances, including
drugs, drug-candidates, pharmaceutical
14 Osoria, L. A., et al.
Fig. 3.
leads and toxic compounds. PASS pre-
diction algorithm is described in detail in
various publications (Poroikov et al., 2003,
Filimonov et al., 2014, Filimonov and Po-
roikov, 2008). Using MOL files as input
data to PASS platform, a list of probable
biological activities for any type of drug is
generated as output.
Interpretation of the prediction results
and selection of the most prospective
compounds and biological activities are
based on flexible criteria, which depend
on the purpose of particular investigation
(Dembitsky et al., 2005). The predicted
activity spectrum of each molecule is
presented from a rank-order list of acti-
vities with probabilities ‘’to be active’’ (Pa)
and ‘’to be inactive’’ (Pi). Interpretation
of prediction results is commonly based
on consideration of Pa values. If Pa> 0.7,
the compound is very likely to reveal this
activity in experiments but the probability
that this compound is a close analogue of
known pharmaceutical agent is also high. A
very high Pa value can also mean that the
compound is included in the training set. If
0.5 <Pa <0.7, the chance of finding activity
experimentally is less and the compound
is not so similar to know chemicals. If Pa
<0.5, the chance of finding activity experi-
mentally is even less but if the presence of
this activity is confirmed by experiments,
the compound might be a new chemical
entity (Devillers et al., 2007).
Due to the rapid photooxidation that
brazilin undergoes and its transformation
into brazilein, the estimation of the profi-
le of biological activities of the latter was
carried out using the PASS web platform.
As seen in Table 2, a total of 48 biological
activities were predicted for this molecule
for a value of Pa > 0.5. The activity profile
obtained is broad, so the molecular struc-
ture of this compound does not have any
characteristics that provide high selectivity
in its biological action.
Although PASS does not explicitly
predict antibacterial activity for this com-
pound, some activities are located that
are related to the mechanisms of action
of antibacterial agents, such as inhibition
of membrane permeability, agonist action
for membrane integrity, probable kinase
inhibition, phosphatase inhibitor, gene-
ral pump inhibitor and inhibitor of NOS2
expression. From the molecular point of
view, antimicrobials for clinical use exert
their action on some of the following bac-
terial structures or functions: inhibiting
the synthesis of the bacterial wall, altering
the integrity of the cytoplasmic membrane,
preventing protein synthesis or blocking
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis Rev. Latinoamer. Quím. 50/1(2023) 15

Table 2. Spectrum of biological activities predicted by PASS for brazilein (Pa > 0.5)
Pa Pi Activity
0,907 0,004 Gluconate 2-dehydrogenase (acceptor) inhibitor
0,862 0,021 CYP2C12 substrate
0,829 0,015 Chlordecone reductase inhibitor
0,767 0,014 HIF1A expression inhibitor
0,767 0,016 Membrane permeability inhibitor
0,748 0,019 Antineoplastic
0,736 0,052 Aspulvinone dimethylallyltransferase inhibitor
0,734 0,020 TP53 expression enhancer
0,726 0,004 Prostate cancer treatment
0,715 0,052 Membrane integrity agonist
0,703 0,037 Antiseborrheic
0,689 0,019 JAK2 expression inhibitor
0,681 0,006 Antineoplastic (lung cancer)
0,674 0,019 Phosphatase inhibitor
0,649 0,014 General pump inhibitor
0,649 0,031 Anaphylatoxin receptor antagonist
0,645 0,010 MAP kinase stimulant
0,635 0,009 AR expression inhibitor
0,626 0,004 NOS2 expression inhibitor
0,618 0,017 HMOX1 expression enhancer
0,614 0,014 Histidine kinase inhibitor
0,611 0,029 2-Dehydropantoate 2-reductase inhibitor
0,584 0,012 P-benzoquinone reductase (NADPH) inhibitor
0,578 0,028 Peroxidase inhibitor
0,577 0,037 Aldehyde oxidase inhibitor
0,574 0,022 UDP-glucuronosyltransferase substrate
0,572 0,016 Steroid N-acetylglucosaminyltransferase inhibitor
0,570 0,021 CYP3A4 inducer
0,570 0,111 Mucomembranous protector
0,560 0,011 CYP2E1 inducer
0,557 0,030 Ovulation inhibitor
0,557 0,035 27-Hydroxycholesterol 7alpha-monooxygenase inhibitor
0,556 0,027 Pin1 inhibitor
0,553 0,022 CYP3A inducer
0,545 0,016 UGT1A substrate
0,542 0,027 Kinase inhibitor
0,539 0,006 RELA expression inhibitor
0,539 0,022 CYP2A4 substrate
0,539 0,054 CYP3A2 substrate
0,535 0,046 Thioredoxin inhibitor
0,532 0,036 Trans-acenaphthene-1,2-diol dehydrogenase inhibitor
0,525 0,011 UGT1A1 substrate
0,524 0,004 Antineoplastic (squamous cell carcinoma)
0,522 0,025 MMP9 expression inhibitor
0,506 0,013 CYP1A inducer
0,505 0,005 Antineoplastic (carcinoma)
0,504 0,020 UGT2B12 substrate
0,500 0,017 Gonadotropin antagonist
16 Osoria, L. A., et al.
synthesis or nucleic acid functions (Dal-
hof1, 2020, Gusarov et al., 2009, Calvo
and Martínez-Martínez, 2009, Epand et al.,
2016), so the probabilistic approximation
obtained for the spectrum of biological ac-
tivities of the brazilein molecule justifies
taking it into consideration to continue
antibacterial activity tests.
One of the potential targets of novel
antibiotics that could be effective against
resistant bacteria is the bacterial cell
membrane. There is evidence that certain
antimicrobial agents can cause bacterial
toxicity by blocking the permeability of
the outer membrane. There are also cases
in which antimicrobial agents block the
permeability of solutes across the outer
membrane of Gram-negative bacteria, re-
sulting in their lethality. This membrane
barrier is also responsible for establishing
concentration and electrical gradients bet-
ween the cell and its environment. These
gradients can be dissipated by damage to
the cell membrane. One gradient that is of
particular importance is the transmembra-
ne proton gradient that is also required for
certain drug efflux mechanisms associated
with bacterial resistance. Several drugs or
drug combinations have been shown to be
potent antimicrobial agents because of in-
hibiting drug efflux as well as being effective
against resistant bacterial strains (Epand et
al., 2016, Wang et al., 2016, Ohene-Agyei
et al., 2014).
Kinases regulate bacterial metabolism,
cell division, and cell wall homeostasis and
also sense and respond to hostile environ-
mental stress factor such an immune res-
ponse or limited nutrient supply as well as
antibiotic stress. Therefore, several kinase
inhibitors of diverse structural classes used
in human medicine increases susceptibili-
ties of various bacterial species to cell wall
active antibacterial agents (Dalhof1, 2020).
Bacterial nitric oxide synthases (NOS2)
are present in many Gram-positive spe-
cies. A study carried out by Gusarov and
co-workers showed that NO generated by
NOS2 increases the resistance of bacteria
to a broad spectrum of antibiotics, enabling
the bacteria to survive and share habi-
tats with antibiotic-producing microorga-
nisms. NO-mediated resistance is achieved
through both the chemical modification of
toxic compounds and the alleviation of the
oxidative stress imposed by many antibio-
tics (Gusarov et al., 2009). These results
suggest that the inhibition of NOS2 activity
may increase the effectiveness of antimicro-
bial therapy, consequently brazilein could
inhibit the expression of this enzyme.
On the other hand, an analysis of the
possible pharmacological activities reveals
that brazilein could become a candidate
for possible studies as an antineoplastic
agent. The antitumor activity could be
carried out through the inhibition of the
HIF1A (Hypoxia-inducible factor 1-alpha)
gene; whose overexpression is associated
with non-papillary renal carcinoma, the
inhibition of the Pin1 gene (Peptidyl-prolyl
cis–trans isomerase NIMA-interacting 1),
the improvement in the expression of the
tumour suppressor gene TP53, as well as
the inhibition of kinases (Hanahan and
Weinberg, 2000).
The Pin1 gene has demonstrated cer-
tain functional polymorphisms associated
with cancer risk; the gene regulates con-
formation of phosphorylation sites, and it
has been involved in multiple oncogenic
signaling pathways as a critical catalyst.
TP53 is a tumor suppressor gene known
as “the cellular gatekeeper of growth and
division”, it acts controlling cellular growth
and by inducing important genes in cycle
arrest and apoptosis following DNA dama-
ge (Maruszak et al., 2009, Naccarati et al.,
2012).
Given the central role of protein kinases
in mediating diverse intracellular signaling
pathways, it is not surprising that abe-
rrant kinase activity is a common feature
of tumors and that kinase inhibitors have
attracted significant attention as targets for
cancer drugs. Some of these dysregulated
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis
kinases are bona fide oncogenes—drivers
of tumor growth (Matthews and Gerritsen,
2010).
The transcription factor NF-kB is invol-
ved in inflammatory and innate immune
responses. It is assembled as a hetero- or
homodimer from the structurally closely re-
lated subunits RelA/p65, c-Rel, RelB, p50
and p52. Deregulated NF-kB activity con-
tributes to tumorigenesis by triggering anti-
apoptotic and pro-proliferative responses.
Furthermore, activation of NF-kB may play
an important role in cancer progression
elicited by inflammatory cytokines (Wei-
chert et al., 2007). NF-kB is amplified, ove-
rexpressed, or activated in various tumor
types including: prostate cancer, melanoma
and head and neck cancer. Inappropriate
activation of NF-kB is a driver of melanoma
progression and metastasis suggesting that
its targeting may have treatment potential
(Lu and Yarbrough, 2015). PASS reveal the
possibility of brazilein to inhibit the RelA/
p65 expression and the prospect that it can
be used as modulator of NF-kB.
Taking these facts in mind and the
possible effective interaction of brazilein
Table 3. Spectrum of bacteriological targets predicted by PASS for brazilein
Name
RESISTANT Staphylococcus simulans
Staphylococcus sciuri
Nocardia transvalensis
RESISTANT Burkholderia pseudomallei
Listeria monocytogenes
RESISTANT Mycobacterium ulcerans
Staphylococcus simulans
Pseudomonas fluorescens
Yersinia pestis
RESISTANT Propionibacterium acnes
Streptococcus pneumoniae R6
Coxiella burnetii
RESISTANT Coxiella burnetii
Staphylococcus lugdunensis
Bacillus subtilis subsp. subtilis str. 168
Rev. Latinoamer. Quím. 50/1(2023) 17
with these molecular targets, a promising
candidate is presented for evaluation as a
future antineoplastic agent against various
tumor lines.
Cytochrome P450 (CYP Family) is an
important detoxification enzyme in the
body and mainly found in the liver. Cyto-
chrome P450 can oxidize foreign organic
compounds, including drugs, as well as
facilitate the excretion of these compounds.
On the other hand, enzyme inhibitors, such
as grapefruit juice, can affect the metabo-
lism of the drug, contraindicating against
the cytochrome P450. Therefore, it is im-
portant to assess the ability of compounds
that can interact with cytochrome P450
(Krihariyani et al., 2020). The metabolic
influence of brazilein on these enzymes was
shown according to the activity prediction
list obtained, acting both as substrate and
as inducer.
ESTIMATION OF THE ANTIBACTERIAL
ACTIVITY
The spectrum of plausible bacteria for
which the brazilein molecule could be ac-
tive was obtained and is shown in Table 3.
Confidence
0.4324
0.3845
0.3463
0.3111
0.2971
0.2628
0.2578
0.2522
0.2360
0.2322
0.2307
0.2305
0.2305
0.2136
0.2056
18 Osoria, L. A., et al.

Table 3. Continued
Name Confidence
RESISTANT Klebsiella oxytoca 0.1918
RESISTANT Staphylococcus aureus subsp. aureus MW2 0.1644
Burkholderia pseudomallei 0.1613
Kocuria rhizophila 0.1497
RESISTANT Bacillus subtilis 0.1382
Mycoplasma hominis 0.1192
Salmonella enterica subsp. entérica 0.1093
Nocardia otitidiscaviarum 0.1025
Porphyromonas gingivalis 0.0973
Streptococcus sp. ‘group A’ 0.0713
Mycoplasma putrefaciens 0.0633
RESISTANT Escherichia coli DH5[alpha] 0.0615
RESISTANT Helicobacter pylori 0.0585
Escherichia coli ATCC 8739 0.0570
Streptococcus sanguinis 0.0550
RESISTANT Chlamydia trachomatis 0.0547
RESISTANT Acinetobacter calcoaceticus 0.0492
Micrococcus 0.0446
Lactococcus lactis 0.0446
Legionella pneumophila 0.0437
RESISTANT Salmonella typhi 0.0358
Acinetobacter baumannii 0.0336
Staphylococcus saprophyticus 0.0326
Mycobacterium 0.0291
Streptococcus viridans 0.0279
Nocardia brasiliensis 0.0263
Listonella anguillarum 0.0231
Nocardia nova 0.0203
Corynebacterium jeikeium 0.0142
Nocardia farcinica 0.0138
Prevotella intermedia 0.0127
RESISTANT Mycoplasma pneumoniae 0.0089
RESISTANT Providencia rettgeri 0.0084
Bacillus thuringiensis 0.0042
RESISTANT Staphylococcus haemolyticus 0.0037
Brazilin from stem of Caesalpinia bahamensis subsp. Orientensis
A total of 50 bacterial strains offers
possibilities of being inhibited by brazilein.
In the previous section, we identified a set
of targets that could be the mechanisms of
action through which this molecule could
exert its antibacterial activity.
In silico tests have been conducted to
predict antibacterial activity of brazilein
by molecular docking. A study carried out
by Krihariyani and co-workers analyzed
interaction of ESBL enzyme derived from
Escherichia coli multiresistant isolates of
UTI (urinary tract infections) patients and
the molecule under study. According to the
binding energy values obtained brazilein
has potential as an antimicrobial (Krihari-
yani et al., 2020).
Propionibacterium acnes is common
bacteria that cause the inflammatory phase
of acne. In silico assays accomplished by
Arisanti and co-workers with molecular
docking showed that brazilein is potential
as anti-acne through the reducing progres-
sion acne lesion mechanism by inhibiting
the activity of endoglyceramidase; hyaluro-
nate lyase; sialidases and autolysin protein.
The molecular docking demonstrates the
high affinity interaction between brazilein
and that protein (Arisanti et al., 20-21 Octo-
ber 2018). Further studies corroborated the
in vitro antibacterial activity against P. ac-
nes (Paramita et al., 20-21 October 2018).
AntiBact-Pred results are consistent with
PASS, since one of the possible predicted
activities is anti-seborrheic.
Methicillin-resistant Staphylococcus au-
reus is a substantial contributor to morbi-
dity and mortality. In search of natural pro-
ducts capable of inhibiting this multidrug
resistant bacteria, brazilein was found to
be active against 8 different strains (ATCC
25923, ATCC 33591, KWMrI 1039, KWMrI
1040, KWMrI 1041, KWMrI 1046, KWMrI
1047, KWMrI 1048) (Lee et al., 2014).
Combination of others computer assis-
ted and in vitro tests validates the results
obtained by AntiBact-Pred platform. A
broad spectrum of new bacterial targets its
Rev. Latinoamer. Quím. 50/1(2023) 19
proposed since was no found in scientific
literature another’s important reports of
antibacterial activity of this natural meta-
bolite. This could help researchers in the
search for new antibacterial agents against
multi-resistant strains and to establish
priorities in future biological evaluations.
CONCLUSIONS
The qualitative phytochemical charac-
terization of the ethanolic extract of the
stem of the endemic species Caesalpinia
bahamensis subsp. orientensis allowed
the detection of the main families of me-
tabolites present in the extract. The most
abundant components were cardenolides,
flavonoids and glycosylated derivatives. On
the other hand, it was possible to isolate
the major metabolite of said extract, which
was identified by spectroscopic techniques
as brasiline, in accordance with previous
reports of this metabolite in other species
of the genus Caesalpinia. In addition, the
low stability of this homoisoflavonoid and
its oxidation to brasilein were verified by
mass spectrometry.
The evaluation of the in vitro antibacte-
rial activity of ethanolic extract and primary
fractions against strains of S. aureus (ATCC
6538) and E. coli (ATCC 8739), revealed
that they are capable of inhibiting bacte-
rial growth at relatively low concentrations.
While brazilein was only active against the
S. aureus strain at a concentration of 12.5
mg/mL, a result that agrees with the in
silico prediction of the biological spectrum
and the antibacterial activity for said mo-
lecule. Combination of others computer
assisted and in vitro tests validates the re-
sults obtained by AntiBact-Pred platform. A
broad spectrum of new bacterial targets its
proposed since was no found in scientific
literature another’s important reports of
antibacterial activity of this natural meta-
bolite. This could help researchers in the
search for new antibacterial agents against
multi-resistant strains and to establish
20 Osoria, L. A., et al.

priorities in future biological evaluations. Compliance with Ethical Standards

It was also possible to identify, based on Conflict of interest: The authors declare
the in silico bioprospecting of brazilein, its no conflict of interest.
potential as a promising candidate for eva-
luation as an antineoplastic agent.

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