molecules

Review
Recent Advances in Prodigiosin as a Bioactive Compound in
Nanocomposite Applications
Rafael G. Araújo 1 , Natalia Rodríguez Zavala 2 , Carlos Castillo-Zacarías 3 , Mario E. Barocio 4 ,
Enrique Hidalgo-Vázquez 4 , Lizeth Parra-Arroyo 4 , Jesús Alfredo Rodríguez-Hernández 4 ,
María Adriana Martínez-Prado 2 , Juan Eduardo Sosa-Hernández 1 , Manuel Martínez-Ruiz 1 ,
Wei Ning Chen 5 , Damià Barceló 6,7,8 , Hafiz M.N. Iqbal 1,4, * and Roberto Parra-Saldívar 1,4, *

1 Tecnologico de Monterrey, Institute of Advanced Materials for Sustainable Manufacturing Monterrey,

Monterrey 64849, Mexico
2 Chemical & Biochemical Engineering Department, Tecnológico Nacional de México-Instituto Tecnológico de
Durango (TecNM-ITD), Blvd. Felipe Pescador 1830 Ote. Durango, Durango 34080, Mexico
3 Universidad Autónoma de Nuevo León, Facultad de Ingeniería Civil, Departamento de Ingeniería Ambiental,
Ciudad Universitaria S/N, San Nicolás de los Garza 66455, Mexico
4 Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey 64849, Mexico
5 School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive,
Singapore 637457, Singapore
6 Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research,
IDAEA-CSIC, 08034 Barcelona, Spain
7 Catalan Institute for Water Research (ICRA-CERCA), Parc Científic i Tecnològic de la Universitat de Girona,
Edifici H2O, 17003 Girona, Spain
8 Sustainability Cluster, School of Engineering, UPES, Dehradun 248007, India
Citation: Araújo, R.G.; Zavala, N.R.; * Correspondence: [email protected] (H.M.N.I.); [email protected] (R.P.-S.)
Castillo-Zacarías, C.; Barocio, M.E.;
Hidalgo-Vázquez, E.; Parra-Arroyo,
Abstract: Bionanocomposites based on natural bioactive entities have gained importance due to
L.; Rodríguez-Hernández, J.A.;
their abundance; renewable and environmentally benign nature; and outstanding properties with
Martínez-Prado, M.A.;
applied perspective. Additionally, their formulation with biological molecules with antimicrobial,
Sosa-Hernández, J.E.; Martínez-Ruiz,
antioxidant, and anticancer activities has been produced nowadays. The present review details the
M.; et al. Recent Advances in
Prodigiosin as a Bioactive Compound
state of the art and the importance of this pyrrolic compound produced by microorganisms, with
in Nanocomposite Applications. interest towards Serratia marcescens, including production strategies at a laboratory level and scale-up
Molecules 2022, 27, 4982. https:// to bioreactors. Promising results of its biological activity have been reported to date, and the advances
doi.org/10.3390/molecules27154982 and applications in bionanocomposites are the most recent strategy to potentiate and to obtain new
carriers for the transport and controlled release of prodigiosin. Prodigiosin, a bioactive secondary
Academic Editor: Artur J. M.
metabolite, produced by Serratia marcescens, is an effective proapoptotic agent against bacterial
Valente
and fungal strains as well as cancer cell lines. Furthermore, this molecule presents antioxidant
Received: 14 July 2022 activity, which makes it ideal for treating wounds and promoting the general improvement of the
Accepted: 28 July 2022 immune system. Likewise, some of the characteristics of prodigiosin, such as hydrophobicity, limit
Published: 5 August 2022
its use for medical and biotechnological applications; however, this can be overcome by using it
Publisher’s Note: MDPI stays neutral as a component of a bionanocomposite. This review focuses on the chemistry and the structure
with regard to jurisdictional claims in of the bionanocomposites currently developed using biorenewable resources. Moreover, the work
published maps and institutional affil- illuminates recent developments in pyrrole-based bionanocomposites, with special insight to its
iations. application in the medical area.

Keywords: bionanocomposites; prodigiosin; pyrrolic compounds; Serratia marcescens; biorenew-

able resources
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
1. Introduction
conditions of the Creative Commons
Attribution (CC BY) license (https:// Bionanocomposites is a novel class of nanometric materials that has gained a lot of
creativecommons.org/licenses/by/ interest lately as it is a technology that integrates many fundamental characteristics to create
4.0/).

Molecules 2022, 27, 4982. https://doi.org/10.3390/molecules27154982 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, x FOR PEER REVIEW 2 of 23

1. Introduction
Molecules 2022, 27, 4982 2 of 20
Bionanocomposites is a novel class of nanometric materials that has gained a lot of
interest lately as it is a technology that integrates many fundamental characteristics to
create new materials with unique properties. Bionanocomposites, also known as “nano-
new materials“green
biocomposite”, with unique properties.
composite”, and/orBionanocomposites,
“bio-based plastics”, also
areknown
similarasto“nanobiocom-
nanocom-
posite”, “green composite”, and/or “bio-based plastics”, are similar to nanocomposites,
posites, but they have critical differences, such as their applications, functionalities, meth-but
they have critical differences, such as their applications, functionalities, methods
ods of preparation, compounds, properties, biodegradability, and biocompatibility, of prepa-
ration, compounds, properties, biodegradability, and biocompatibility, among
among others [1–5]. They are made up of a natural polymer (biopolymer, BP) and an in- others [1–5].
They are made up of a natural polymer (biopolymer, BP) and an inorganic
organic compound at a nano scale (nanoparticle, NP), as illustrated in Figure 1. compound at a
nano scale (nanoparticle, NP), as illustrated in Figure 1.

Figure 1. Bionanocomposites: Polymeric materials produced by bioprocesses (plants or microorgan-

isms)1.based
Figure on nanostructure
Bionanocomposites: of materials
Polymeric derived
materials from self-organization.
produced by bioprocesses (plants or microorgan-
isms) based on nanostructure of materials derived from self-organization.
The shape of the nanoparticle is essential in the formation of the nanocomposite, it can
beThe
divided
shapeintoofdifferent categories,
the nanoparticle is such as nanorods,
essential nanofibers,
in the formation of nanotubes, nanoparticles,
the nanocomposite, it
and nanoplates. Polymer nanocomposites (PNCs) have been of
can be divided into different categories, such as nanorods, nanofibers, nanotubes,interest due to their high
nano-
availability,
particles, cost-effectiveness,
and nanoplates. Polymer andnanocomposites
ease of production. PNCs
(PNCs) are been
have used more in thedue
of interest industry
to
due
their to their
high lightness,
availability, ease of production,
cost-effectiveness, and their
and ease malleablePNCs
of production. nature.areUnlike
used moremetals
in or
ceramics nanocomposites, polymer-based nanocomposites have low strength;
the industry due to their lightness, ease of production, and their malleable nature. Unlike however,
polymers
metals can be nanocomposites,
or ceramics filled with inorganic, synthetic, or
polymer-based natural compounds,
nanocomposites increasing
have low their
strength;
resistance, mechanical strength, and some other characteristics [1–6].
however, polymers can be filled with inorganic, synthetic, or natural compounds, increas- Polymer-based
ingnanocomposites
their resistance,have many benefits,
mechanical according
strength, and someto several
other authors [7]. For[1–6].
characteristics instance, polymer-
Polymer-
based nanocomposites are less porous than common plastic, so they are
based nanocomposites have many benefits, according to several authors [7]. For instance, perfect to use in the
protection of medical instruments, films, as a drug delivery system and
polymer-based nanocomposites are less porous than common plastic, so they are perfect so on. Additionally,
to bionanocomposites
use in the protection have great potential
of medical for various
instruments, applications
films, as a drug ranging
deliveryfrom foodand
system packing,
so
drug delivery, biosensors, gene delivery, and bioenergy production [8–14], as shown in
on. Additionally, bionanocomposites have great potential for various applications rang-
Figure 2.
ing from food packing, drug delivery, biosensors, gene delivery, and bioenergy produc-
For many biomedical applications, different bioactivities can be achieved through co-
tion [8–14], as shown in Figure 2.
polymerization with natural polysaccharides, such as starch, cellulose, and chitosan, as well
For many biomedical applications, different bioactivities can be achieved through co-
as with some proteins, such as albumin, legume and gelatin, which are widely used for the
polymerization with natural polysaccharides, such as starch, cellulose, and chitosan, as
preparation of nanostructures for delivery of different drugs derived from its non-toxicity,
well as with some proteins, such as albumin, legume and gelatin, which are widely used
stability, low size, and biodegradability. Another approach of interest is the incorporation
for the preparation of nanostructures for delivery of different drugs derived from its non-
of natural bioactive compounds in nanoencapsulated systems or in nanoemulsions that
toxicity, stability, low size, and biodegradability. Another approach of interest is the in-
can be released in a controlled manner and generate a specific bioactivity [11].
corporation of natural bioactive compounds in nanoencapsulated systems or in
Prodigiosin is a natural red pigment that is obtained through a select group of Gram-
nanoemulsions
negative bacteriathat can bePseudomonas,
of the released in a controlled
Streptomyces,manner and generate
and Serratia genera. aOnly
specific
a fewbioac-
Gram-
tivity [11].
positive bacteria, such as Streptoverticillium rubrireticuli and Streptomyces longisporus, have
been described with the ability to produce prodigiosin. It has been reported that a greater
number of Gram-negative prodigiosin producing bacteria, compared to Gram-positive
organisms, can be found in the literature, such as Pseudomonas magnesloruba, Vibrio psy-
Molecules 2022, 27, 4982 3 of 20

chroeryrhrous, Serratia rubidaea, Vibrio gazogenes, Alteromonas rubra, Rugamonas rubra, Serratia
sp, and Serratia marcescens; the latter being the characteristic microorganism in pigment
Molecules 2022, 27, x FOR PEER REVIEW
production, it has also been identified that both groups of bacteria produce prodigiosin 3 of
as23
a
secondary metabolite [14–21].

Figure 2.
Figure 2. Bionanocomposites:
Bionanocomposites: Versatility
Versatility and
and their
their applications
applications in
in different
differentindustries.
industries.

Prodigiosin is(Ca20natural
Prodigiosin H25 N3 O red pigment
) and some thatof isits
obtained
analogs, through
such as a select group of Gram-
undecylprodigiosin
negative
(C H
25 35 3 N bacteria
O ) , of the Pseudomonas,
cycloprodigiosin (C H Streptomyces,
20 23 3 N O ) , and and Serratia genera.
metacycloprodigiosin Only (Ca25few
H33Gram-
N3 O ) ,
positiveimportant
present bacteria, such as Streptoverticillium
activities of great interestrubrireticuli and Streptomyces
to the pharmacological longisporus,
industry, have
due to their
been described with the ability to produce prodigiosin. It has been
antimicrobial, antifungal, antiprotozoal, antimalarial, anticancer, immunosuppressant, and reported that a greater
number of
antiviral Gram-negative
activities. Prodigiosin prodigiosin producing
is considered as thebacteria, compared
most important to Gram-positive
candidate for cancer
organisms,due
treatment cantobeitsfound
low toxicin the literature,
effect over normalsuch as Pseudomonas
cells, and it has magnesloruba,
application asVibrio psy-
a natural
chroeryrhrous,
colorant. OtherSerratia
possiblerubidaea, Vibrio that
applications gazogenes, Alteromonas
have recently emergedrubra, is Rugamonas
the use of therubra, Serra-
molecule
tiaasp,
as pH and Serratia amarcescens;
indicator, the latteranbeing
UV ray protector, the characteristic
autofluorescence microorganism
product, in pigment
and its application as a
production,agent
controlling it hasin also
thebeen identified
formation that both
of biofilm groups ofSobacteria
[16,22–26]. far, theproduce prodigiosin
factors that have beenas
a secondary
identified thatmetabolite
affect the [14–21].
production of prodigiosin are the type of microorganism, culture
medium, incubation
Prodigiosin ( C20 H25 N
time, 3 O) and some
temperature, nitrogen,
of itsand phosphorus
analogs, such concentration, inorganic
as undecylprodigiosin
(C25 Hand
salts, 35 N3pH
O), [15,20,27–29].
cycloprodigiosin (C20 H23
Different N3 O), and metacycloprodigiosin
fermentation strategies have been applied (C25 H33toN3produce
O), pre-
prodigiosin,
sent important as well as strategies
activities of great for statistical
interest to theoptimization,
pharmacological genetic manipulation
industry, and
due to their
cloning, optimized
antimicrobial, culturesantiprotozoal,
antifungal, with gamma antimalarial,
rays, co-cultures, and even
anticancer, cultures mediated
immunosuppressant,
by
andelicitation.
antiviral activities. Prodigiosin is considered as the most important candidate for can-
In this review,
cer treatment due towe its focus on the
low toxic different
effect over normalproduction strategies
cells, and and bioactivities
it has application of
as a nat-
prodigiosin as a compound of interest for the development of
ural colorant. Other possible applications that have recently emerged is the use of the sustainable and renewable
bionanocomposites
molecule as a pH indicator, with speciala UV attention to its application
ray protector, in the medical
an autofluorescence area.and its ap-
product,
plication as a controlling agent in the formation of biofilm [16,22–26]. So far, the factors
2. Pyrrolic Compounds Produced by Microorganisms: Serratia marcescens
that have been identified that affect the production of prodigiosin are the type of micro-
Secondary
organism, culture metabolites
medium, from microorganisms
incubation are one ofnitrogen,
time, temperature, the principal sources of active
and phosphorus con-
molecules that can be used in food, health, and biotechnology
centration, inorganic salts, and pH [15,20,27–29]. Different fermentation strategies areas. Pyrrolic compoundshave
are
beenring-structured
applied to produce and electron
prodigiosin,rich asthat
wellcanasform hydrogen
strategies bonds and
for statistical coordinating
optimization, ge-
metals [30]. Pyrrolic compounds have several applications,
netic manipulation and cloning, optimized cultures with gamma rays, co-cultures, and they have been used
and
in antibiotic,
even culturesanti-inflammatory,
mediated by elicitation. antitumor, and anti-cholesterol drugs. Moreover, these
In this review, we focus on the different production strategies and bioactivities of
prodigiosin as a compound of interest for the development of sustainable and renewable
bionanocomposites with special attention to its application in the medical area.
 2. Pyrrolic Compounds Produced by Microorganisms: Serratia marcescens
Secondary metabolites from microorganisms are one of the principal sources of ac-
tive molecules that can be used in food, health, and biotechnology areas. Pyrrolic com-
Molecules 2022, 27, 4982 pounds are ring-structured and electron rich that can form hydrogen bonds and coordi- 4 of 20
nating metals [30]. Pyrrolic compounds have several applications, and they have been
used in antibiotic, anti-inflammatory, antitumor, and anti-cholesterol drugs. Moreover,
these pyrrolic
pyrrolic molecules
molecules have applications
have applications in polymerization
in polymerization and metallurgical
and metallurgical reactions
reactions and
and
as as corrosion
corrosion inhibitors
inhibitors and preservative
and preservative agentsagents [31]. Pyrrolic
[31]. Pyrrolic compounds
compounds are pro-
are produced
duced naturally
naturally by different
by different bacterial
bacterial strainsstrains
found found in nature,
in nature, Serratia
Serratia marcescens
marcescens being being the
the prin-
cipal producer
principal of these
producer compounds.
of these Serratia
compounds. marcescens
Serratia is a Gram-negative
marcescens rod-shaped
is a Gram-negative rod-
facultative bacterium,
shaped facultative from thefrom
bacterium, Enterobacteriaceae
the Enterobacteriaceaefamily. family.
According to Hardjito
According et al.,
to Hardjito
there are two types of Serratia marcescens, the unpigmented and the
et al., there are two types of Serratia marcescens, the unpigmented and the pigmentedpigmented strains, the
latter is characterized
strains, by its ability to
the latter is characterized byproduce
its abilitythetored pigment
produce thecalled prodigiosin
red pigment [32].
called Since
prodigi-
itosin
is a [32].
facultative
Since itbacterium, the pigment
is a facultative can be
bacterium, theproduced
pigmentunder
can beaerobic and anaerobic
produced con-
under aerobic
ditions [33]. There
and anaerobic are 10 species
conditions of theare
[33]. There genus Serratia,ofhowever,
10 species the genus only 3 of these
Serratia, can produce
however, only 3
prodigiosin: S. plymuthica,
of these can produce S. rubidaea,
prodigiosin: and S. marcescens.
S. plymuthica, S. rubidaea,The
andproduction
S. marcescens.of The
prodigiosin
produc-
by the bacteria Serratia marcescens depends on different conditions, such
tion of prodigiosin by the bacteria Serratia marcescens depends on different conditions, as availability of
inorganic phosphate, the composition of the medium, as well as the pH,
such as availability of inorganic phosphate, the composition of the medium, as well as the temperature, and
natural components
pH, temperature, andof natural
the growth media [34].
components of the growth media [34].
Structure of Prodigiosin
2.1. Structure of Prodigiosin
Prodigiosin is part of the family of prodiginine, secondary alkaloids with a chemical
Prodigiosin is part of the family of prodiginine, secondary alkaloids with a chemical
structure of tripyrrol, with two rings joined and the third linked through a methane bridge
structure of tripyrrol, with two rings joined and the third linked through a methane bridge
that the molecules that are part of this family are differentiated between. Due to the
that the molecules that are part of this family are differentiated between. Due to the size
size and arrangement of the branch in the last-named ring, this family is made up of
and arrangement of the branch in the last-named ring, this family is made up of Prodigi-
Prodigiosin, Undecylprodigiosin, Cycloprodigiosin, Methacycloprodigiosin, Butyl-meta-
osin, Undecylprodigiosin, Cycloprodigiosin, Methacycloprodigiosin, Butyl-meta-cyclo-
cycloprodigiosin, and Prodigiosin R1 (Figure 3). These structures are characterized by being
prodigiosin, and Prodigiosin R1 (Figure 3). These structures are characterized by being a
a part of a pigment that has aroused interest due to its antitumor, immunosuppressive, and
part of a pigment that has aroused interest due to its antitumor, immunosuppressive, and
anticancer activity. The molecular formula for prodigiosin is C20 H25 N3 O, and its molecular
anticancer activity. The molecular formula for prodigiosin is C20 H25 N3 O, and its molecu-
weight is 323.4 g/mol (232.44 Da); it can be dissolved in non-polar compounds, such as
lar weight is 323.4 g/mol (232.44 Da); it can be dissolved in non-polar compounds, such as
chloroform, methanol, acetonitrile, and dimethylsulfoxide, and it can partially be dissolved
chloroform,
in alcohol andmethanol, acetonitrile, and dimethylsulfoxide, and it can partially be dis-
ether [20,35,36].
solved in alcohol and ether [20,35,36].

Figure 3.
Figure 3. Examples
Examples of
of natural
natural prodiginines:
prodiginines:Prodigiosin
Prodigiosin(A),
(A),Cycloprodigiosin
Cycloprodigiosin(B),
(B),Undecylprodigi-
Undecylprodi-
giosin (C), Metacycloprodigiosin (D), Butyl-meta-cycloprodigiosin (E), and ProdigiosinR1
osin (C), Metacycloprodigiosin (D), Butyl-meta-cycloprodigiosin (E), and Prodigiosin R1(F).
(F).

In total for Serratia sp. ATCC 39006 has identified a group of genes responsible for the
biosynthesis of prodigiosin ranging from PigA to PigO, with PigC being the most important.
The genes involved in MBC biosynthesis have also been found, which are PigI, PigG, PigA,
PigJ, PigH, PigM, PigF, and PigN; and, for the biosynthesis of MAP, PigD, PigE, and
PigB [37–39]. All this enzymatic complex that is activated in the production of prodigiosin
Molecules 2022, 27, 4982 5 of 20

is interconnected and stimulated by the carbon source, availability of phosphate, stationary

phase, and cyclic signaling-di-GMP. It is believed that the synthesis of prodigiosin is
accompanied by the synthesis of biosurfactants to improve its defense mechanism against
other microorganisms or when it reaches high cell density through quorum sensing [40].
Investigations through a quorum sensing modulator effect in Serratia marcescens MTCC
97 shows that the stimulation by ultrasound at a frequency of 500 and 600 Hz stimulates the
production of prodigiosin without affecting cell growth; meanwhile, frequencies of 400 and
700 Hz increase the production of prodigiosin but directly affect cell growth, which could
allow future study of the modulating effect of the enzyme complex and gene expression on
the production of prodigiosin [41]. The importance of one of the signaling molecules in the
mechanisms of quorum sensing and therefore in the production of prodigiosin, using the
hydrophobic cavity and the hydrophilic surface layer of the α-cyclodextrin immobilized
in calcium alginate, has been found to manage to capture the extracellular signaling
molecule N-acyl homoserin lactone (AHL), thereby inhibiting prodigiosin production by
S. marcescens AS-1. It has also been found that for Streptomyces coelicolor prodigiosin plays
a fundamental role in the regulation of programmed cell death, since prodigiosin has
shown hyper-accumulation in the dead mycelia of the strain and a study carried out by
suppressing the prodigiosin genes showed greater viability of the filaments, with respect to
the non-mutated strain [42].
In addition, it has been found that its synthesis is directly related to the high production
of ATP during the stage of cellular delay, just as pigmented cells reproduce at a higher
rate and accumulate at a higher ATP velocity than non-pigmented cells, showing that ATP
levels are direct regulators of prodigiosin production [43]. The use of glucose as a carbon
source also inhibits the production of pigment, but this type of inhibition is still unknown
since it would be thought that high levels of glucose would induce a high production of
ATP and the high concentration of ATP in turn would induce pigment production. It was
also thought that cAMP was a positive inducer of prodigiosin production, supported by
the hypothesis that at low glucose levels cAMP levels would be elevated, but it has been
found that both high and low glucose levels of cAMP inhibit the production of prodigiosin,
so there is still a lack of knowledge about the mechanism of repression of glucose on
pigment production [44]. On the other hand, the addition of amino acids with structures
such as pyrrole—as in the case of proline, histidine, ornithine, aspartic acid, and glutamic
acid—induce the production of prodigiosin, which suggests a direct incorporation in the
metabolic route for synthesis of MBC and MAP [32].

3. Prodigiosin Producing Bacteria

The production of the red pigment as mentioned above is limited to an exclusive
group of Gram-negative bacteria and in a smaller proportion to Gram-positive bacteria,
with Serratia as the most important genus in the production of prodiginins, and Serratia
marcescens as the species with greater number of reports in the production of prodiginins,
specifically prodigiosin [45]. Other microorganisms belonging to the genus Streptomyces
have been reported for their ability to produce prodiginins (Figure 3), such as Butylcyclo-
heptylprodigiosin, Metacycloprodigiosin, Prodigiosin R1, and Undecylprodigiosin, with
a higher production report of the Streptomyces coelicolor species [26,46]. Pseudoalteromona
sp 1020R species have also been reported with the ability to produce up to four differ-
ent prodiginins (2-Methyl-3-Butylprodiginine, 2-Methyl-3-Pentylprodiginine, 2-Methyl-3-
Hexylprodiginine, and 2-Methyl-3 -Hytylprodiginine) [47]. Some microorganisms have
been reported in the last decade with the ability to produce prodiginins (Table 1). It should
be emphasized that many of the reports found in the literature to date from a red pigment
identified as prodigiosin—which were based on the spectrum of UV-VIS absorption in basic
and alkaline conditions, this spectrum can range between 470 and 537 nm in these condi-
tions, frequently reaching a maximum absorption peak at 535 nm—this information is not
sufficient for the identification of prodigiosin, since its analogs present the same spectrum
of absorption. To be able to exactly identify the type of prodiginine requires techniques
Molecules 2022, 27, 4982 6 of 20

such as mass spectrometry or nuclear magnetic resonance. Another of the confusions that
arises around prodigiosins is that they are a family of compounds of red color and that
compounds such as Uncedylprodigiosina, Cycloprodogidiosin, Cyclononylprodigiosin,
and Butylcycloheptylprodigiosina among others, are a part of this family, when the family
to which prodigiosin and its previously named analogues really belong is the family of
prodiginin, which has the characteristic skeleton pyrrolyl dipyrromethene [25,31].

Table 1. Prodigiosin producing bacteria reported in literature.

Microorganism Microorganism
Origin Prodiginine Type Origin Prodiginine Type
(Reference) (Reference)
Serratia marcescens UCP Semi-arid soil around S. coelicolor M1110
Prodigiosin N/A Undecylprodigiosin
1549 [20] banana trees [48]
Pristine soil sample
Streptomyces sp. collected from the cavy S. marcescens SMAR
Undecylprodigiosin N/A Undecylprodigiosin
JS520 [26] area of Miroc mountain [49]
in eastern Serbia
Nonylprodigiosin
From a mold and Sediment samples
S. marcescens Xd-1 [45] Prodigiosin Actinomadura sp [50] Cyclononylprodigiosin
tofu sample in Brazil
Methylcycloctilprodigiosin
From rhizospheric
soil in different sites
S. coelicolor M145 [46] N/A Undecylprodigiosin S. marcescens [51] Prodigiosin (possible)
of Salem and
Namakkal, India
Butylcycloheptylprodigiosin S. marcescens NCIM
S. parvulus [52] N/A N/A Prodigiosin
and Undecylprodigiosin 5061 [53]
From Lahul and Spiti
S. marcescens ATCC
S. nematodiphila RL2 [54] region in Himachal Prodigiosin N/A Prodigiosin
13880 [55]
Padresh, India
From clam samples
S. rubiaea RAM Alex [56] collected form Temsah Prodigiosin S. marcescens [57] From kitchen waste Prodigiosin (Possible)
Lake, Egypt
Soil sample from a
S. spectabilis BCC From soil sample
S. marcescens [58] tannery in Chennai, Prodigiosin Metacycloprodigiosin
4785 [59] collected in Thailand
Tamilnadu, India.
N/A: not applicable.

3.1. Prodigiosin Production

As the biotechnological industry expands, techniques for microbial cultivation and
growth become more sophisticated and optimal. However, at the present, large-scale
production of prodigiosin remains as an opportunity area for this technology since current
strategies and solutions for its obtention are expensive and complex [60]. As previously
mentioned, pigmented strains from the Serratia genus are often exploited as a scalable
source to produce prodigiosin and, therefore, its utilization for these purposes has been
highly reported, representing a promising solution for the current state-of-the-art of this
concern [61].
Several physical, chemical, and biological methods have been tested in order to im-
prove the synthesis of the pigment in the gram-negative bacteria. A study carried by
Gondil et al., aimed to determine the response of the wild strain S. nematodiphila RL2,
which was previously isolated and identified, to a wide number of physicochemical pa-
rameters, such as culture medium used, carbon source, nitrogen source, pH, temperature,
fermentation time, and metal ions present during fermentation, for the optimization of
biomass generation and the enhancement of prodigiosin production. For initial experi-
ments, nutrient broth, LB broth, tomato juice broth and yeast potato dextrose broth culture
mediums were tested in a 50-h fermentation, at 35 ◦ C at an initial pH 7, observing a major
production of the red pigment in nutrient broth, which was detected at up to 0.49 mg/ mL,
17.9% higher than the next best result, obtained with yeast potato dextrose, which achieved
a production of prodigiosin of 0.39 mg/mL. Nutrient medium was, hence, selected to per-
form the further experiments during this study. To select the more suitable carbon source
during fermentation, 9 carbon species were explored: sucrose, galactose, glucose, maleic
acid, ammonium acetate, citric acid, glycerol, sodium oxalate and lactose, at a concentration
Molecules 2022, 27, 4982 7 of 20

of 1% w/v in all treatments. As a result, prodigiosin was generated in higher concentrations

using lactose as a carbon source, achieving 0.52 mg/mL of pigment produced. In a similar
experiment, six different nitrogen sources were compared, determining that an optimal
production of the pigment (0.6 mg/mL) can be achieved with yeast extract at 1% w/v.
Likewise, 8 different initial pH values in the range from 3 to 10 were studied, observing the
best results at pH 6 and 7 (0.6 mg/mL). A similar result was reported during the experi-
ments to estimate the optimal temperature for prodigiosin production, in which, among
5 temperatures, the higher pigment concentration was detected at up to 0.59 mg/mL, at
35 ◦ C. In addition, during the tests with metal particles, authors demonstrated that the
presence of cobalt chloride and mercuric chloride (100 mM in each case) may severely
down-regulate the synthesis of prodigiosin in the gram-negative strain studied, reducing
its formation up to 0.16 and 0.17 mg/mL, respectively, even though fermentation is carried
under the optimal parameter previously established. On the other hand, the presence of
uranyl acetate during cultivation rocketed the pigment production up to 0.76 mg/mL [54].
In another study performed by Bhagwat and Padalia et al., authors successfully
intended to reduce the costs of bioprocesses involved in the biotechnological production
of prodigiosin by utilizing a variety of residues from the food industry as substrates for
the commercial strain S. marcescens ATCC 13880, in a circular economy model [62]. In
their research, authors tested the potential of different oil-based residues to function as
a nutrient source for fatty acids obtention for the mentioned strain in order to design
a model for waste revalorization while optimizing the cost for microbial prodigiosin
production. Powders from three different oil sources, such as peanut, sesame and mustard
were prepared for its addition to nutrient agar to run a fermentation performed under
a series of physical and chemical parameters previously optimized in this study. In all
cases the prodigiosin production increased in comparison with a treatment with non-
treated cultivations, however, the best performance was observed in treatments with oil
from peanut seed powder 4% w/v. Considering the obtained results, authors design
an optimized culture medium based on nutrient agar with the next additives: casein
hydrolysate at 1% w/v as nitrogen source, sucrose at 2% w/v as carbon source, and peanut
seed powder at 4% w/v. Likewise, the final optimal fermentation was performed with
an initial pH 7.5, at 28 ◦ C during 72 h, producing 3.5 mg/mL of prodigiosin. In a similar
series of experiments, Xia et al., utilized residues from a kitchen waste handle plant as an
innovative nutrient substrate for the optimization of prodigiosin production using a wild
strain of S. marcescens as biological model. Ideal operation parameters for fermentation,
such as initial pH, temperature, carbon source, nitrogen source and agitation speed, were
identified as well. Kitchen wastes were tested as substrates at 7 different concentrations
between 20 and 50 g/L, where authors concluded that 35 g/L was the most appropriate
substrate concentration, enhancing prodigiosin generation at >200 mg/mL [63].
Regarding chemical methods for the optimization of prodigiosin production, Chilczuk
et al., designed a culture system in which the commercial strain Serratia sp. ATCC 39006
is in interaction with four ambigols (ambigol A, ambigol B, ambigol C and ambigol D),
extracted from the cyanobacterium Fischerella ambigua. It was found out that ambigol C
at 15.6 µM may enhance prodigiosin synthesis by around 4 times in comparison with
control treatments [17]. According to transcriptomic analysis, this molecule improves
the expression of genes involved in proline consumption, which has been proved to be a
potential inductor of the synthesis of the natural red dye [63]. On the other hand, ambigols
A and D demonstrated an antimicrobial activity in the presence of the gram-negative strain.
Molecular techniques have been also reported, for instance, Sun et al. successfully
developed an efficient protocol for the rocketing of the synthesis of prodigiosin by per-
forming genetic and metabolic engineering on a wild strain of S. marcescens [64]. Their
research focused on one of the thermoregulator systems (TSs) of the strain, involved in the
silencing of the expression of pig proteins that are part of the mechanism of synthesis of
prodigiosin, particularly, on the cpx complex, a two-component regulatory system, which
operates under the activity of a sensor histidine kinase (CpxA), and a transcriptional regu-
Molecules 2022, 27, 4982 8 of 20

latory CpxR protein [63]. This TS activates under a variety of external stress conditions,
such as the presence of metal ions with antimicrobial activity, pH changes and high tem-
peratures (>37 ◦ C), resulting in a down-regulation of the expression of prodigiosin and
other by-products [63–66]. The molecular mechanism of action of this system relies in the
detection of such stress factors by the CpxA protein, followed by the phosphorylation of
aspartic acid residues located along the structure of the CpxR, allowing its attachment to
gene promoters that encodes pig proteins, and impeding their transcription [64,67]. To
avoid the effects of this TS, authors developed two transformed strains of S. marcescens JNB
5-1 with suppressed cpxA and cpxR genes, respectively, and evaluated their capability to
enhance the prodigiosin synthesis in comparison with a wild strain. Authors found out
that the modified strain with suppressed cpxR genes were able to synthesize up to 76%
more prodigiosin than the wild strain. Transcriptomic assays also showed a significantly
higher expression of pig genes in this strain. The fermentation was carried at 30 ◦ C, in LB
culture medium in all treatments. Further experiments in this study aimed to evaluate
the influence of temperature in the expression of cpxA and cpxR genes in the wild strain,
comparing the transcription level of these genes at 30 ◦ C and 37 ◦ C in LB medium, and
concluding that, in both cases, this was up to 6 times higher at 37 ◦ C after almost 40 h
of fermentation.

3.2. Prodigiosin Separation

Even though the most relevant factor behind the high values of prodigiosin in markets
is due to the difficulty to obtain higher production yields during large-scale fermenta-
tions [63], the development of novel downstream methods is still an important subject in
the generation of literature regarding the bioprocess of the pigment. According to Sun
et al., most of the current state-of-the-art of methodologies for extraction and purification
of prodigiosin are based on solvents, and large amounts of solvent are often required
to improve the extraction yield of the process [68]. However, using organic solvents for
large-scale extractions may be expensive and, in some cases, highly polluting [69]. At
the present, alternative technologies have emerged to avoid such problems involved with
traditional prodigiosin separation. In their research, Khanam and Chandra, determined
that it is possible to obtain higher extraction yields for prodigiosin from S. marcescens
by using inorganic solvents such as HCl, however it is possible to obtain similar yields
with organic, less contaminating solvents such as ethanol when performing extractions
at high temperatures (60 ◦ C). Authors also evidenced the contribution of using physical
cell-disruption techniques during extraction, such as ultrasonication, with which extrac-
tion yields may be doubled in comparison with using solvent-based (ethanol) extractions.
Additionally, a degree of extraction of prodigiosin almost 100% was achieved by utilizing
ultrasonication, while reducing the non-extracted fraction to almost 0% [70].
As another example of innovative separation techniques for prodigiosin produced by
microorganisms, Arivizhivendhan et al., proposed a successful methodology for a solvent-
free prodigiosin extraction by using iron oxide particles, adsorbing the pigment on their
surface, in an integrated continuous fermentation system [58]. After their synthesis, Fe3 O4
particles were functionalized using diethanolamine to work as a cross-linker between
the metal particle and the prodigiosin. Subsequently, functionalized metal particles and
pigment molecules interacted during 30 min at 150 rpm, and the resultant complex is then
recovered from both stirred batch reactors used in this study. At this point, prodigiosin can
be isolated from metal particles by using acetone, while this solvent can be easily removed
by evaporation. Authors reported a recovery yield of 98% by using the functionalized
Fe3 O4 particles, meaning a recovery of prodigiosin of around 1046 mg/L. In addition, the
synthesized iron oxide complex was proved to be highly reusable since similar adsorption
yields for prodigiosin were observed during their second extraction cycle.
Molecules 2022, 27, 4982 9 of 20

4. Biological Activity of Prodigiosin

Prodigiosin is characterized by having applications of great interest to the pharmaco-
logical industry. It has been shown in laboratory tests that prodigiosin has a good biological
activity, such as antibacterial, antifungal, and antiviral. The benefit of having a bacterium
with a secondary metabolite that has biological activity relies on the cost-effective produc-
tion and the large amount of uses that this can have, not only in the pharmacological area
but also in food preservatives, such as food coloring agents, among others.

4.1. Antimicrobial
This tripyrrole red pigment, has a notable antibacterial activity against Gram-negative
bacteria such as Escherichia coli, Aeromonas hydrophila, Klebsiella pneumoniae, Pro-
teus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, and
Salmonella Typhimurium [24,71,72], in addition to Gram-positive bacteria such as Staphy-
lococcus aureus as well as its methicillin resistant strain, MRSA, Bacillus cereus, Corynebac-
terium glutamicum, Enterococcus faecalis, Enterococcus faecium, and Listeria monocy-
togenes [71,73–75]. Table 2 shows antibacterial activity reported in different studies. The
forms of prodigiosin used in the studies varied, purified pigment was the most used, while
a few used stained textiles and others crude rhizosphere extract. Among the different
parameters for antimicrobial activity: the ATCC 100 value measures antibacterial activity in
textiles; the antibacterial rate is the percentage difference between the number of colonies
of the organism in dyed and undyed experiments; the IC50 is the concentration needed to
reduce cellular viability to 50%; the maximum zone of inhibition measures the halo left
by the compound in a petri dish seeded with a certain microorganism and the minimum
inhibitory concentration that reports the smallest amount of the compound needed in order
to affect cellular viability. Ren et al. observed the effect of pH on the antibacterial effect
of prodigiosin dyed textiles with the highest toxicity at pH 8.1, this is possibly due to
the fact that at this pH the solubility of the dye increases leading to more pigment being
loaded on the textile [75]. The general mechanisms of prodigiosin microbial action are the
cleavage of genetic material (DNA), interference in the cell cycle, affecting pH, disrupting
the plasma membrane by increasing hydrophobic stress, phototoxicity, and formation of
reactive oxygen species [74]. The effect of the pigment has been observed to vary according
to if the bacteria is Gram-positive or negative. Prodigiosin is known to lyse Gram-positive
bacterial cell walls leading to their death, while in the case of Gram-negative bacteria it
affects gene expression and protein synthesis eventually altering the cellular life cycle and
metabolism [24].

Table 2. Recent antibacterial activity of prodigiosin reported in literature.

Prodigiosin Source Concentration Bacteria Parameter of Bactericide Action Value Reference

Serratia rubidaea Chiffon stained by AATCC 100
Escherichia coli 95 [24]
RAM_Alex prodigiosin Bacteria reduction (%)
Staphylococcus aureus 97
Satin stained by
Escherichia coli 91
prodigiosin
Staphylococcus aureus 84
Linen stained by
Escherichia coli 97
prodigiosin
Staphylococcus aureus 70
Dacron stained by
Escherichia coli 90
prodigiosin
Staphylococcus aureus 84
Gabardine stained by
Escherichia coli 19
prodigiosin
Staphylococcus aureus 15
Molecules 2022, 27, 4982 10 of 20

Table 2. Cont.

Prodigiosin Source Concentration Bacteria Parameter of Bactericide Action Value Reference

Serratia marcescens Silk pH 2.1 Staphylococcus aureus Antibacterial rate 93.17% [75]
Escherichia coli 25.12%
Silk pH 8.1 Staphylococcus aureus 87.80%
Escherichia coli 14.70%
Serratia marcescens 25–400 µg/mL Staphylococcus aureus IC50 51.17 µg/mL [71]
Listeria monocytogenes 51.54 µg/mL
Enterococcus faecium 26.18 µg/mL
Bacillus cereus 33.61 µg/mL
Salmonella enteritidis 56.56 µg/mL
Proteus vulgaris 50.81 µg/mL
Pseudomonas aeruginosa 69.71 µg/mL
klebsiella pneumoniae 48.63 µg/mL
Aeromonas hydrophila 66.98 µg/mL
Escherichia coli 44.20 µg/mL
E. coli O157:H7 20.31 µg/mL
Achromobacter
25 µg/mL Proteus mirabilis Maximum zone of inhibition (mm) 17 [72]
denitrificans SP1
Staphylococcus aureus 15
Serratia marcescens 250 µg/mL MRSA Maximum zone of inhibition (mm) 20 ± 0.33 [74]
Staphylococcus aureus 20 ± 0.0
Enterococcus faecalis 20 ± 0.88
Escherichia coli 22 ± 0.41
500 µg/mL MRSA 21 ± 0.00
Staphylococcus aureus 22 ± 0.33
Enterococcus faecalis 20 ± 0.33
Escherichia coli 27 ± 0.82
P. putida strain pig-r2 24.48 µg/mL Corynebacterium glutamicum Minimal inhibitory concentration (MIC) 2.56 µg/mL [73]

Prodigiosin has also been observed to have a toxic effect on certain fungal species, such
as Batrachochytrium dendrobatidis, B. salamandrivorans, Pythium myriotylum, Rhizoctonia solani,
Sclerotium rolfsii, Phytophthora infestans, Fusarium oxysporum, and C. nymphaeae. The pig-
ment’s antimycotic activity could be of use to control or to eradicate parasitic fungal species.
The most common parameter for comparing antifungal activity was percentage inhibition,
which is equal to the percentage difference in fungal cells with and without the pigment.
Table 3 contains different recent studies that report prodigiosin’s antimycotic effect.

Table 3. Antifungal activity of prodigiosin.

Prodigiosin Source Concentration Fungi Parameter of Antifungal Action Value Reference

Serratia plymuthica and Batrachochytrium
3.8 µM IC50 3.8 µM [76]
S. marcescens dendrobatidis (Bd)
27.3 µM B. salamandrivorans (Bsal) IC50 27.3 µM
Batrachochytrium
10 µM Minimum inhibitory concentration (MIC) 10 µM
dendrobatidis (Bd)
50 µM B. salamandrivorans (Bsal) Minimum inhibitory concentration (MIC) 50 µM
Crude extract from
Serratia sp. rhizosphere of Bacopa Pythium myriotylum Percent inhibition 71.33 [77]
monnieri (L.)
Rhizoctonia solani Percent inhibition 61.33
Sclerotium rolfsii Percent inhibition 49.33
Phytophthora infestans Percent inhibition 48.66
Fusarium oxysporum Percent inhibition 31
Serratia rubidaea
450 µg/mL C. nymphaeae Percent inhibition 29.27 [78]
Mar61-01
1000 µg/mL C. nymphaeae Percent inhibition 100
Serratia spp. isolated from
medicinal plant - Pythium myriotylum Percent inhibition 40 [79]
Plumbago indica
Molecules 2022, 27, 4982 11 of 20

4.2. Antioxidant
In the food industry, artificial additives are used to preserve food, which have shown
deterioration in health due to their consumption, such as cell damage, inflammation,
metabolic disorders, among others. Recently, an interest in natural preservative additives
has been shown, given their benefit in human health. A balanced consumption of antiox-
idant compounds or foods rich in antioxidants allows for a reduction in oxidative stress
and free radicals levels, generating protective effects in cells and balancing the immune
system to produce defenses against various diseases, such as stress, cancer, hypertension,
atherosclerosis, and gastrointestinal and hormonal disorders [80–83].
Prodigiosin has demonstrated high antioxidant potential with interest for many ap-
plications. The strong antioxidant activity may be attributed to conjugated double bond
and ring pyrrole structures of prodigiosin [84]. A recent study demonstrated the high
antioxidant activity of purified prodigiosin from Serratia marcescens, showing 92 and 99%
of scavenging at 5 mg/mL of prodigiosin, for free radicals ABTS and DPPH, respectively.
The antioxidant properties of prodigiosin also demonstrated benefits in the preservation of
foods through the decrease of rancidity and microbiological contamination, increasing shelf
life, and adding some additional properties, such as color for a pleasant appearance for
the consumer [85]. Prodigiosin purified from radio-resistant Streptomyces sp. WMA-LM31
showed antioxidant activity for DPPH with a scavenging capacity of 62%, a protein oxida-
tion inhibition of 54.8%, and a lipid peroxidation of 25.4% at 10 µg/mL, showing strong
antioxidant activity at low concentrations [84]. Prodigiosin also showed strong activity
against different free radicals, the ability to block the formation of superoxides, and the
inhibition of Fenton reactions, reducing the negative effects of protein and lipid oxidation
as well as the prevention of DNA damage [84]. Nguyen et al. produced prodigiosin from
marine chitinous wastes by a bioprocess with S. marcescens strains, reporting a moderate
antioxidant activity, with values of inhibition (IC50 ) of 115 and 235 µg/mL for ABTS and
DPPH, respectively [83].

4.3. Antitumoral
Cancer is a global health problem, representing one of the main causes of mortality
worldwide and the first or second of premature mortality in the main countries of the
world. According to the World Health Organization, in 2020 an estimated 19.3 million
new cases and 10 million deaths were estimated in which each of 5 people in the world
developed cancer during their lifetime and in which one in 11 women and one in 8 men
died from cancer [86,87]. For 2070, a strong increase in the incidence of cancer is estimated,
twice the current level, due to population growth and aging, demographic changes and an
increase in risk factors for cancer development associated with demographic increase [88].
The main types of cancer with the highest incidence in 2020 are breast, prostate, lung, and
colorectal cancers, and the cancers with the highest mortality rates were lung, colorectal,
and liver, in order of appearance [89]. The uncontrolled increase in the incidence of cancer,
high costs of treatment, and resistance to current drugs have created a great need to design,
research, and discover new compounds. The search for natural compounds produced by
different organisms has been the most important strategy, highlighting that more than half
of the approved treatments against cancer are of natural origin or derivatives [90].
Prodigiosin is the most important secondary metabolite from Serratia marcescens,
with strong biomedical applications against therapeutic diseases at low concentrations,
such as cancer, and showing a high apoptotic effect against cancer cells and low or no
toxicity on normal cells, as shown in Table 4 [91]. Prodigiosin has shown a high potential
as an antitumoral agent against colorectal cancer, inhibiting late-stage autophagy and
increasing sensitivity to 5-fluorouracil of different colorectal cancer cells (HCT116 and
SW480) through blocking autophagosome–lysosome fusion and maturation of lysosomal
cathepsin [92]. Ji et al. demonstrated that prodigiosin markedly decreases the proliferation
of K562 cells (chronic myelogenous leukemia) through increased activity of caspases-3, -8,
-9 and increased reactive oxygen species, resulting in the inhibition of autophagy and the
Molecules 2022, 27, 4982 12 of 20

induction of apoptosis [93]. Nguyen et al. demonstrated the strong anticancer effect of
prodigiosin in different cancer cell lines, MCF-7, A549, HepG2 and WiDr, with inhibition
values of 92.1%, 93.1%, 94%, and 92%, and low values of IC50 , 0.102 µg/mL, 0.182 µg/mL,
0.161 µg/mL, and 0.441 µg/mL, respectively [94]. The purified prodigiosin bioproduced
from marine chitin showed high anticancer activity against MCF-7, A549, and HepG2, and
an efficacy of 2.75, 1.67, and 3.25 times greater than Mytomycin C, a commercial anticancer
compound, respectively, proposing some mechanisms that prodigiosin affects, such as
mitogen-activated protein kinase regulators, pH modulators, DNA cleavage agents, and
cell cycle inhibitors [95]
An anticancer study demonstrated the effect of prodigiosin through tests with prostate
cancer (PC3) and human choriocarcinoma (JEG3) cell lines in vitro and PC3 and JEG3 tumor-
bearing nude mice in vivo, showing an inhibition of the proliferation and a reduction in
the size and weight of the tumors, depending on the prodigiosin concentration and the
treatment time, respectively [96]. Berning et al. demonstrated that prodigiosin increases
the sensitivity of cisplatin-resistant and sensitive urothelial carcinoma cell lines (RT-112)
to cisplatin mediated by dysregulation of lysosomal function and reduction of cathepsin
B and L activity [23]. Prodigiosin has great cytotoxic activity against many melanoma
cancers cells lines, such as NGM, 501-Mel, WM293A, HT-144, SK-Mel-19, SK-Mel-28, and
SK-Mel-147, showing mean IC50 values of 0.2 µM through kinases modulation, intracellular
acidification, DNA damage, and apoptosis induction [97]. Breast cancer is the type of
cancer with the highest incidence worldwide and the second with the highest mortality
rate. Prodigiosin has recently shown very relevant effects in the fight against this disease.
In MDA-MB-231 (hormone-independent breast cancer cell line) and MDA-MB-468 cells, the
prodigiosin at nanomolar concentrations, blocking the Wnt/β-catenin pathway, decreased
phosphorylation of GSK3β, DVL2, and LRP6 and suppressed β-catenin–stimulated Wnt
target gene expression [98].
A novel study of antitumoral activity of prodigiosin combined with PU-H71 against
MDA-MB-231 showed high levels of caspases 3, 8, and 9 and decreased the levels of
mTOR expression and HSP90α expression and transcription levels, which resulted in
a breakthrough for a new therapy against triple negative breast cancer [99]. The high
antitumor activity of prodigiosin at low concentrations and the low secondary effects
against healthy cells has strengthened and intensified research into it as a new anticancer
therapy. Obatoclax, a synthetic analog of prodigiosin, is being studied in phase I and
II clinical trials for the treatment of lymphoma, myelodysplastic syndrome, and lung
cancer [97].

Table 4. Antitumoral activity of prodigiosin against different cancer cell lines.

Cancer Type Cell Line Mechanism IC50 Units Reference

Increased activity of caspases -3, -8, -9, reactive oxygen species,
Blood/Leukemia K562 >500 µM [93]
inhibition of autophagy and apoptosis induction.
HL-60 Apoptosis induction 1.7 µg/mL [100]
Wt-p53Molt-4 Caspase-3-dependent apoptosis 1.3 µM [101]
GBM8401 ER stress/autophagy 7.36 µM
Brain [102]
U87MG ER stress/autophagy 12.29 µM
MDA-MB-231 ER stress; Wnt/β-catenin; JNK/MAPK/RAD51 62.52 nM
Breast [98]
MDA-MB-468 Inhibit Wnt/β-catenin 261.2 nM
MCF-7 Apoptosis induction 5.1 µg/mL [100]
Mitogen-activated protein kinase regulators, pH modulators,
MCF-7 0.04 µg/mL [94]
DNA cleavage agents and cell cycle inhibitors
Decreased the levels of mTOR and HSP90α expression
MDA-MB-231 2.1 nM [99]
and transcription
Dysregulation of lysosomal function and reduction of cathepsin
Urothelial RT-112 675 nM [23]
B and L activity
DLD1 c-Jun/∆Np73/p73/apoptosis; Lysosomal acidification >1.6 µM [103,104]
HCT116 c-Jun/∆Np73 p73 activation 4 µM
Colorectal SW480 c-Jun/∆Np73 p73 activation µM
SW-620 Apoptosis 0.273 µM
Blocking autophagosome–lysosome fusion and maturation of
HCT116; SW480 >0.1 µM [92]
lysosomal cathepsin
Mitogen-activated protein kinase regulators, pH modulators,
WiDr 0.05 µg/mL [97]
DNA cleavage agents and cell cycle inhibitors
Molecules 2022, 27, 4982 13 of 20

Table 4. Cont.

Cancer Type Cell Line Mechanism IC50 Units Reference

Liver HepG2 Antiproliferative effects 12.64 µg/mL [84]
Mitogen-activated protein kinase regulators, pH modulators,
HepG2 0.04 µg/mL [95]
DNA cleavage agents and cell cycle inhibitors
A549 PI3K-p85/Akt/mTOR; PKB/SKP2/p27 10 µM [105]
CNE2, NP69 PKB/SKP2/p27 4 and 0.35 mg/L [106]
Lung
NCHI-292 Apoptosis induction 3.6 µg/mL [100]
Mitogen-activated protein kinase regulators, pH modulators,
A549 0.06 µg/mL [95]
DNA cleavage agents and cell cycle inhibitors
Prostate PC3 Intrinsic apoptosis >10 µg/mL
[96]
Trophoblast JEG3 Intrinsic apoptosis >10 µg/mL
Uterus Hela Antiproliferative effects 12.75 µg/mL [84]
Gingival squamous carcinoma OECM1 Cyclin D1 inhibition, arresting cell cycle in G0/G1 phase 1.59 ± 0.77 µM
[107]
Tongue SAS cyclin D1 inhibition, arresting cell cycle in G0/G1 phase 3.25 ± 0.49 µM
Uterus Hela Intrinsic apoptosis 0.5–2.1 µg/mL [108]

4.4. Antiprotozoal
Natural compounds and their semisynthetic derivatives are the primary strategy for
obtaining and developing new drugs for the treatment of parasitic diseases [109]. Malaria
is a disease that threatens global health, as it has developed resistance to current drugs and
is causing more than a million deaths annually. Prodigiosin and derivatives, such as unde-
cylprodigiosin and metacycloprodigiosin in nanomolar concentrations and IC50 between
5–12 nM, were shown to exhibit potent in vitro antimalarial activity against Plasmodium
falciparum, the parasite that causes malaria disease in humans [110,111]. Mosquitoes are
the vectors for the transmission of deadly diseases, such as malaria and dengue. The
control of the reproduction and spread of mosquitoes is an alternative for the control and
reduction of new infections [112]. The larvicidal activity of prodigiosin has been reported,
and it is known. Prodigiosin at a concentration of 100 ppm demonstrated a larvicidal
activity against Aedes aegypti of 32% and 76% mortality with 24 and 48 h of incubation,
respectively [113]. Purified prodigiosin has shown high larvicidal activity against larval
and pupal stages of Aedes aegypti and Anopheles stephensi mosquitoes, obtaining IC50 values
between 14 to 21 µg/mL and 19 to 32 µg/mL, respectively, in the various growth stages.
The larvicidal concentration to both mosquitoes was found at 62.5 µg/mL for the three
firsts growth stages [112].

4.5. Antiviral
Herpes Simplex Virus (HSV) is one of the most contagious infections worldwide. Both
are lifelong, and sometimes it can cause painful blisters or ulcers at the site of the infection.
Approximately 491.5 million people had HSV type 2 infection in 2016, and approximately
3752 million people were living with HSV type 1, this is equivalent to 79.4% of the world’s
population [113,114]. This virus can also affect eye tissue and cause keratitis, eye drops are
commonly used for medical treatment of this infection. In some cases, surgical treatment
may be necessary to treat complications [115]. A recent study demonstrated the antiviral
effect of prodigiosin on HSV type 1 and HSV type 2 infections through in vitro and ex
vivo cultured mice corneas. Prodigiosin treatment significantly protects the eyes of mice,
reducing the disease’s development; it also protects against the loss of corneal sensitivity
and excessive inflammation, showing nontoxic effects [115]. At non-toxic concentrations,
prodigiosin exhibited significant in vitro antiviral activity against cells infected with Bombyx
mori nucleopolyhedrovirus (BmNPV). The result of prodigiosin action was selective death
of infected cells inhibiting viral gene transcription (ie-1) and preventing virus-mediated
membrane fusion, resulting in inhibition of virus production and replication [116].

5. Recent Advances and Applications of Prodigiosin Bionanocomposites

Prodigiosin is a microbial metabolite with numerous bioactivities and many possible
applications in biomedicine for the treatment of many diseases. However, the effectiveness
of prodigiosin in clinical treatment is limited by its poor absorption, hydrophobicity, and
low bioavailability [117]. Nanoscale drug carriers are an attractive biotechnology to over-
Molecules 2022, 27, 4982 14 of 20

come the limitations of the use of prodigiosin, which allows for obtaining formulations
with high functional yields for target delivery systems in the treatment of cancer [118]. A
study on the binding of prodigiosin to organic and inorganic matrices represents significant
importance to improving the use of prodigiosin as a bioactive compound in applied phar-
macology [119]. Natural biomolecules, such as starch, proteins, biopolymers, and chitosan,
are an attractive group of scaffold compounds compared to the synthetic, due to their low
toxicity and biodegradability [120,121]. Nanoparticle technology utilized several benefits
of biopolymer scaffolds carrying hydrophobic ligands to target sites. An essential step to
design biopolymer-based nanoparticle systems is to understand the binding sites and bind-
ing affinity of the protein-drug complex [122]. Although previous studies have shown the
interaction potential of prodigiosin (or pirrolyc) with some biopolymers or proteins, such
as bovine hemoglobin and bovine serum albumin, to the best of our knowledge, there is no
report detailing the interactions of chitosan, PLA, and other polyacids. A biodegradable
formulation of PLGA-based microparticles loaded with a bacterial prodigiosin showed
an antitumor activity against a triple negative breast tumor, reducing the cell viability
of the MDA-MB-231 cell line, being a new proposal for the controlled application of the
prodigiosin as a drug for cancer treatment [123]. A promising prodigiosin nanocarrier
as a hybrid system of nanocomposite of β-cyclodextrin and chitosan with nanoparticles
improve the activity and selectivity against cancer cells (MCF-7 and HepG2 cell lines),
compared with free prodigiosin, and showed no toxicity against healthy cells (NIH/3T3
cells) [124]. The localized and controlled administration of chemotherapeutic drugs was
demonstrated by hybrid nanofiber systems composed of PLGA, Pluronic F127, gelatin,
and prodigiosin as active compounds for the treatment of triple negative breast cancer,
decreasing cell viability and inducing apoptosis cells in MCF-7 and MDA-MB-231 cell
lines [125]. Another recent study showed nanocomposites that increase the bioavailability
of prodigiosin for use as a cytotoxic drug against Caco-2 and HCT116 carcinoma cell lines,
without negative effects in healthy cells [118].

6. Concluding Remarks and Futures Perspectives

Prodigiosin is a bioactive compound that is obtained from the secondary metabolism
of bacteria and can easily be produced at the laboratory level and scaled up to bioreactors,
as well as generating strategic genetic modifications to increase its production to use
prodigiosin in different biotechnological applications. The bioactivity of prodigiosin is
known and its positive effects have been described and accumulated over time in different
in vitro models for antioxidant, antimicrobial, anticancer, antiviral, and antiprotozoal
activity, highlighting its antimicrobial and anticancer activity due to its high efficiency at
low concentrations against different cancer cell lines and not presenting any negative effect
against healthy cells. However, the low bioavailability and low absorption of prodigiosin
are some problems that can be overcome with the prodigiosin nanocarriers strategy.
The development of prototypes of prodigiosin functionalized bionanocomposites is
the most recent strategy to potentiate and to obtain new carriers for the transport and
controlled release of prodigiosin—which are not normally achieved with free prodigiosin—
and thus achieve the desired bioactivity, however, new studies in vivo are required to
obtain new information regarding its safety and effectiveness.

Author Contributions: Conceptualization, R.G.A., N.R.Z. and M.M.-R.; methodology, C.C.-Z. and
M.E.B.; validation, R.G.A.; formal analysis, R.G.A. and E.H.-V.; investigation, R.G.A., N.R.Z., J.A.R.-H.,
E.H.-V. and J.E.S.-H.; resources, R.P.-S.; data curation, L.P.-A., J.E.S.-H. and M.M.-R.; writing—original
draft preparation, R.G.A., N.R.Z. and C.C.-Z.; writing—review and editing, R.G.A., W.N.C., D.B. and
M.A.M.-P.; visualization, W.N.C. and D.B.; supervision, R.P.-S. and M.A.M.-P.; project administration,
M.A.M.-P., R.P.-S. and H.M.N.I.; funding acquisition, R.P.-S. and H.M.N.I. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was funded by CSIC-Tecnológico de Monterrey under i-Link+program
(LINKB20030) for a project entitled “Contaminantes emergentes y prioritarios en las aguas reutilizadas
en agricultura: riesgos y efectos en suelos, producción agrícola y entorno ambiental”.
Molecules 2022, 27, 4982 15 of 20

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work was also partially supported by Consejo Nacional de Ciencia y
Tecnología (CONACyT) Mexico under Sistema Nacional de Investigadores (SNI) program awarded
to Rafael G. Araújo (CVU: 714118), Carlos Castillo-Zacarías (CVU: 359310), María Adriana Martínez
Prado (CVU: 85841), Juan Eduardo Sosa Hernández (CVU: 375202), Manuel Martínez Ruiz (CVU:
418151), Hafiz M.N. Iqbal (CVU: 735340) and Roberto Parra Saldívar (CVU: 35753). The listed
authors are highly obliged to their respective departments, institutes, and universities for providing
literature services.
Conflicts of Interest: The authors declare that they have no known competing financial interest or
personal relationship that could have appeared to influence the work reported in this paper.

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