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molecules
Review
Potential of Sulforaphane as a Natural Immune System
Enhancer: A Review
Andrea Mahn 1, * and Antonio Castillo 2
Citation: Mahn, A.; Castillo, A.
Potential of Sulforaphane as a
Natural Immune System Enhancer:
A Review. Molecules 2021,26, 752.
https://doi.org/10.3390/
molecules26030752
Academic Editors: Maria
JoséRodríguez-Lagunas and
Malen Massot-Cladera
Received: 30 December 2020
Accepted: 28 January 2021
Published: 1 February 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
1Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Santiago de Chile (USACH),
Santiago 8330111, Chile
2Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile (USACH),
Santiago 8330111, Chile; antonio.castillo@usach.cl
*Correspondence: andrea.mahn@usach.cl; Tel.: +56-227-181-833
Abstract:
Brassicaceae are an outstanding source of bioactive compounds such as ascorbic acid,
polyphenols, essential minerals, isothiocyanates and their precursors, glucosinolates (GSL). Recently,
GSL gained great attention because of the health promoting properties of their hydrolysis products:
isothiocyanates. Among them, sulforaphane (SFN) became the most attractive one owing to its
remarkable health-promoting properties. SFN may prevent different types of cancer and has the
ability to improve hypertensive states, to prevent type 2 diabetes–induced cardiomyopathy, and to
protect against gastric ulcer. SFN may also help in schizophrenia treatment, and recently it was
proposed that SFN has potential to help those who struggle with obesity. The mechanism underlying
the health-promoting effect of SFN relates to its indirect action at cellular level by inducing antioxidant
and Phase II detoxifying enzymes through the activation of transcription nuclear factor (erythroid-
derived 2)-like (Nrf2). The effect of SFN on immune response is generating scientific interest, because
of its bioavailability, which is much higher than other phytochemicals, and its capacity to induce
Nrf2 target genes. Clinical trials suggest that sulforaphane produces favorable results in cases
where pharmaceutical products fail. This article provides a revision about the relationship between
sulforaphane and immune response in different diseases. Special attention is given to clinical trials
related with immune system disorders.
Keywords: sulforaphane; immunological response; cellular mechanism
1. Introduction
Many of the current synthetic drugs come from natural products of plant origin. Even
some plant-derived bioactive compounds have been proposed as possible therapeutic
solutions to fight highly prevalent diseases such as cancer [1].
Sulforaphane (SFN), an isothiocyanate (ITC) widely distributed in Brassicaceae
plants, has generated great interest in the last 15 years, with an exponentially growing
number of scientific articles reaching around 250 in 2020 and a total of 2315 since 1948
(PubMed, https://pubmed.ncbi.nlm.nih.gov/, last access 21 December 2020). This is due
to the outstanding health promoting properties of SFN which are related with its high
capacity to induce Phase II detoxifying enzymes, being 14-fold higher than other potent
phytochemicals such as quercetin. Additionally, SFN exhibits the highest bioavailability
among well-known antioxidant phytochemicals, such as quercetin (20-fold higher) [
2
] and
curcumin (80-fold higher) [
3
]. This confers SFN a high potential to be used as nutraceutical
to improve health status or as pharmaceutical to treat some disease states.
SFN comes from the enzymatic hydrolysis of glucoraphanin, a glucosinolate stored
as inactive precursor in the plant cells. The hydrolysis of glucoraphanin occurs through
myrosinase (E.C. 3.2.1.147), which is compartmentalized in the vegetable inside myrosin
cells. The reaction proceeds after tissue disruption that can be produced by insects and
herbivores attack or by processing and chewing the vegetable [
4
]. The products of the
Molecules 2021,26, 752. https://doi.org/10.3390/molecules26030752 https://www.mdpi.com/journal/molecules
Molecules 2021,26, 752 2 of 14
hydrolysis reaction vary depending on the chemical conditions where the reaction occurs.
The myrosinase—glucosinolate system belongs to the defense system of the plant and there-
fore some of the products that come from glucosinolates hydrolysis are toxic [
5
].
Figure 1
depicts the myrosinase—glucosinolate (glucoraphanin) system in plants. In mammals,
SFN can be administered directly in its active form or as glucoraphanin which undergoes
the hydrolysis during digestion by the action of vegetable and gut microflora myrosinases.
After intake, SFN follows the mercapturic acid pathway until its conversion in dithiocarba-
mates and is finally excreted [
6
]. Figure 2shows the formation and metabolic degradation
of SFN.
Molecules 2021, 26, x 2 of 14
hydrolysis reaction vary depending on the chemical conditions where the reaction occurs.
The myrosinase—glucosinolate system belongs to the defense system of the plant and
therefore some of the products that come from glucosinolates hydrolysis are toxic [5]. Fig-
ure 1 depicts the myrosinase—glucosinolate (glucoraphanin) system in plants. In mam-
mals, SFN can be administered directly in its active form or as glucoraphanin which un-
dergoes the hydrolysis during digestion by the action of vegetable and gut microflora my-
rosinases. After intake, SFN follows the mercapturic acid pathway until its conversion in
dithiocarbamates and is finally excreted [6]. Figure 2 shows the formation and metabolic
degradation of SFN.
Figure 1. Myrosinase—glucoraphanin system in Brassicaceae plants. Glucosinolates are located in
specialized glucosinolate-containing cells, while myrosinase is stored in the vacuoles of the my-
rosin cells. After mechanical disruption of plant tissue, the substrate and enzyme come in contact
and the hydrolysis occurs, resulting in different products, among which it is found sulforaphane
[7].
Figure 1.
Myrosinase—glucoraphanin system in Brassicaceae plants. Glucosinolates are located in
specialized glucosinolate-containing cells, while myrosinase is stored in the vacuoles of the myrosin
cells. After mechanical disruption of plant tissue, the substrate and enzyme come in contact and the
hydrolysis occurs, resulting in different products, among which it is found sulforaphane [7].
Several efforts have been conducted in order to exploit the health-promoting effects
of SFN on humans. Its direct administration has been limited because of the instability
of SFN. Some research about SFN stabilization is being conducted [
9
]. Another way to
administer SFN to humans is through broccoli sprout extracts or minimally processed
broccoli. Some processing conditions that maximize SFN content in processed broccoli
have been reported [
10
–
14
], resulting in an important amount of information about the
processing conditions that achieve this goal. Given the instability of SFN and the possibility
to maximize SFN in processed vegetable, most clinical studies about the effect of SFN use
broccoli extracts or powder, and focused on validating the efficacy of SFN-rich food, not in
SFN as a drug.
The first report about the effect of sulforaphane on health dates from 1992, when
Zhang et al. [
15
] suggested that SFN was a potent activator of cellular defense systems.
Later Bonnesen et al. [
16
] informed that SFN and other isothiocyanates showed a preventive
effect on colon tumorigenesis since these compounds stimulate apoptosis and enhance
Molecules 2021,26, 752 3 of 14
cell defense against molecules that produce gene toxicity. Since then, several
in vitro
and
in vivo
studies about the effect of SFN have been conducted, including clinical trials,
resulting in relevant information regarding prevention and treatment of diseases such as
pancreatic cancer [
17
], breast cancer [
18
], diffuse axonal injury [
19
], lymphomas [
20
], liver
cancer [
21
], leukemia [
22
], and prostate cancer [
23
]. Moreover, SFN has cardio protective
properties [
24
], the ability to prevent aging and neurodegeneration [
25
], and to protect
from gastric ulcer [
26
]. Additionally, several clinical trials are currently in progress or
already finished.
Molecules 2021, 26, x 3 of 14
Figure 2. Formation and metabolization of sulforaphane. Sulforaphane (SFN) is formed by the hydrolysis of glucoraphanin
catalyzed by either plant or bacterial myrosinase. After intake, SFN is metabolized through the mercapturic acid pathway.
Initially, isothiocyanates are conjugated with glutathione (GSH) in a glutathione transferase (GST)-catalyzed reaction.
Then, successive cleavage reactions catalyzed by γ-glutamyltranspeptidase, cysteinylglycinase, and N-acetyltransferase
occur to generate sulforaphane-N-acetylcysteine (SFR-NAC) [8].
Several efforts have been conducted in order to exploit the health-promoting effects
of SFN on humans. Its direct administration has been limited because of the instability of
SFN. Some research about SFN stabilization is being conducted [9]. Another way to ad-
minister SFN to humans is through broccoli sprout extracts or minimally processed broc-
coli. Some processing conditions that maximize SFN content in processed broccoli have
been reported [10–14], resulting in an important amount of information about the pro-
cessing conditions that achieve this goal. Given the instability of SFN and the possibility
to maximize SFN in processed vegetable, most clinical studies about the effect of SFN use
broccoli extracts or powder, and focused on validating the efficacy of SFN-rich food, not
in SFN as a drug.
The first report about the effect of sulforaphane on health dates from 1992, when
Zhang et al. [15] suggested that SFN was a potent activator of cellular defense systems.
Later Bonnesen et al. [16] informed that SFN and other isothiocyanates showed a preven-
tive effect on colon tumorigenesis since these compounds stimulate apoptosis and en-
hance cell defense against molecules that produce gene toxicity. Since then, several in vitro
and in vivo studies about the effect of SFN have been conducted, including clinical trials,
resulting in relevant information regarding prevention and treatment of diseases such as
pancreatic cancer [17], breast cancer [18], diffuse axonal injury [19], lymphomas [20], liver
cancer [21], leukemia [22], and prostate cancer [23]. Moreover, SFN has cardio protective
properties [24], the ability to prevent aging and neurodegeneration [25], and to protect
from gastric ulcer [26]. Additionally, several clinical trials are currently in progress or al-
ready finished.
This review aims at presenting the most recent advances of research about the effects
of SFN on the immune system, considering in vitro studies, which were performed using
animal or human cells in culture, and in vivo studies, which were animal or human clini-
cal intervention trials.
2. Mechanisms of Action of SFN on Immune System
Sulforaphane exerts a pleiotropic effect on immunological response. The mechanism
is based on activation of nuclear factor (erythroid-derived 2)-like (Nrf2) which triggers
cellular defense mechanisms. There is induction of Phase II detoxifying enzymes as well
as antioxidant enzymes, and down regulation of Phase I enzymes by inactivation of NFκβ.
Figure 2.
Formation and metabolization of sulforaphane. Sulforaphane (SFN) is formed by the hydrolysis of glucoraphanin
catalyzed by either plant or bacterial myrosinase. After intake, SFN is metabolized through the mercapturic acid pathway.
Initially, isothiocyanates are conjugated with glutathione (GSH) in a glutathione transferase (GST)-catalyzed reaction. Then,
successive cleavage reactions catalyzed by
γ
-glutamyltranspeptidase, cysteinylglycinase, and N-acetyltransferase occur to
generate sulforaphane-N-acetylcysteine (SFR-NAC) [8].
This review aims at presenting the most recent advances of research about the effects
of SFN on the immune system, considering
in vitro
studies, which were performed using
animal or human cells in culture, and
in vivo
studies, which were animal or human clinical
intervention trials.
2. Mechanisms of Action of SFN on Immune System
Sulforaphane exerts a pleiotropic effect on immunological response. The mechanism
is based on activation of nuclear factor (erythroid-derived 2)-like (Nrf2) which triggers
cellular defense mechanisms. There is induction of Phase II detoxifying enzymes as well
as antioxidant enzymes, and down regulation of Phase I enzymes by inactivation of NF
κβ
.
The final effect of SFN varies with cell type. In T-cells, the response to SFN exposure is the
generation of a pro-oxidant environment, with an increase of intracellular reactive oxygen
species (ROS) and a decrease in intracellular glutathione (GSH) levels, that produces a block
of the T-cell-mediated immune response. SFN is able to create a pro-oxidative ROS enriched
milieu in primary human
T-cells
. It inhibits co-stimulation initiated
T-cell
activation and
proliferation by depletion of GSH and oxidation of proteins at redox active cysteine residues.
Importantly, SFN also enhances the ROS levels in lymphocytes within whole blood of RA
(rheumatoid arthritis) patients and inhibited the production of pro-inflammatory TH17
related cytokines. This immunosuppressive effect of SFN on T-cells can be desirable in
autoimmune or inflammatory diseases, but it would be detrimental in other chronic diseases
such as cancer since the T-cell-mediated immune response is important for immune surveil-
lance of tumors. Therefore, caution should be exercised, as SFN could interfere with the
successful application of immunotherapy by immune checkpoint inhibitors (e.g., CTLA-4
Molecules 2021,26, 752 4 of 14
antibodies and PD-1/PD-L1 antibodies) or CAR (chimeric antigen receptors) T-cells in
cancer patients, and a combination of both treatments could not be advisable [27].
Although there is a good amount of evidence that indicates that SFN is a potent
anticancer compound and that its main mechanism of action would occur through the
activation of Nrf2, recent publications present controversial results that indicate that the
activation of Nrf2 contributes to the whole process of pathogenesis, promotes cancer
progression and metastasis while conferring resistance to chemo- and radiotherapy, and
has a poor prognosis, a phenomenon that has been described as the “dark side” of Nrf2 [
28
].
Therefore, in accordance with the above, Nrf2 could be a promising target in cancer therapy.
However, research related to Nrf2 inhibitors is still incipient [29].
In monocytes and macrophages, SFN inhibits pro-inflammatory cytokines and acti-
vates antioxidant enzymes through Nrf2 modulation, resulting in an anti-inflammatory
effect (Figure 3) useful for the treatment of bacterial and viral related diseases. SFN is
widely recognized as among the most powerful natural anti-cancer agents, but its mecha-
nism of action is not fully understood so far. This owes to the multi-factorial nature of this
disease and to the pleiotropic effect of SFN. However there is evidence that supports SFN
to exert an antioxidant effect in tumor cells [
30
]. The mechanisms that underlay SFN effect
on immunological system in different diseases are presented below.
Molecules 2021, 26, x 7 of 14
Freeman [56] suggested that clinical trials including administration of Nrf2-activating
molecules, such as SFN, are imperative to support a possible three-party strategy to fight
the COVID-19 pandemic, which includes prevention, diagnostic, and treatment.
2.4. Bacterial Diseases
Research about the effect of SFN on immunological system during bacterial infec-
tions is incipient. Currently there are reports that consider H. pylori, S. aureus, E. coli and
M. pneumoniae. Although SFN exhibits direct bactericidal activity, it triggers an immuno-
logical response to H. pylori infection in the stomach mucosa. SFN acts by activating Nrf2
and downregulating NF-κB, whose joint action modulates antioxidant and anti-inflam-
matory response in the host [57,58]. As a consequence, SFN exerts a protective effect from
gastritis and gastric ulcer. Yanaka [59] conducted in vitro and in vivo studies about the
effect of SFN on H. pylori infection. The outcomes demonstrated that SFN significantly
reduced the bacterium viability and alleviated gastritis in animal models and in humans.
Haodang et al. [60] studied the response of monocytes stimulated with Mycoplasma
pneumoniae lipopeptide to SFN exposure. Pathological injury of M. pneumoniae in lungs
relates with inflammation that stimulates immune response of the host triggered by lipid
polysaccharide (LPS) excretion by the bacteria. The authors found that SFN inhibited the
expression of pro-inflammatory cytokines and activated the expression of HO-1 after the
induction of Nrf2. As a result, SFN reduced lung inflammation in an animal model. The
mechanism proposed by [60] is depicted in Figure 3.
Ali et al. [61] investigated the effect of four Nrf2 activators on bacteria-infected mac-
rophages, among them, SFN. Macrophages were infected either with E. coli or S. aureus
and the intracellular viability of bacteria was evaluated. SFN significantly reduced intra-
cellular bacteria survival in PBMC-derived macrophages. Even though the authors do not
present any mechanism, they propose that the intra and extra cellular bactericidal effect
of SFN relies on the anti-inflammatory and antioxidant milieu produced inside the mac-
rophages. SFN, as a potent Nrf2 activator, seems a promising therapeutic option for Gram
(+) and Gram (−) bacterial infections since it modulates antioxidant and anti-inflammatory
responses. Deramaudt et al. [62] studied the intracellular survival of S. aureus in human
and mice macrophages treated with SFN. They proposed a mechanism consisting in mod-
ulation of p38/JNK signal pathway induced by SFN in macrophages, thus reducing in-
flammatory response. Additionally, the authors reported that SFN affected S. aureus in-
tracellular survival by inducing apoptosis in the bacterium. Then, the combination of both
mechanisms supports SFN as a possible treatment for S. aureus infection.
Finally, Belchamber and Donnelly [63] suggested that SFN stimulates phagocytic
pathways and improves macrophage phagocytosis of S. pneumoniae, P. aeruginosa and H.
influenzae by upregulating Nrf2 in alveolar cells from COPD9 (chronic obstructive pulmo-
nary disease).
Figure 3. Mechanism of inflammatory response suppression induced by sulforaphane in bacteria-infected monocytes.
Figure 3. Mechanism of inflammatory response suppression induced by sulforaphane in bacteria-infected monocytes.
2.1. Autoimmune/Inflamatory Diseases
SFN exerts its effect on immune system through different biochemical and cellular
mechanisms, among them the downregulation of pro-inflammatory cytokines, T-cells
suppressing, and activation of adenosine monophosphate activated protein kinase (AMPK)
signaling pathway. Even though these processes have a suppressive effect, this is desirable
in cases of autoimmune/inflammatory diseases.
Townsend and Johnson [
31
] studied the effect of sulforaphane on pro-inflammatory
markers and target genes of nuclear factor erythroid 2 (NFE2)—related factor 2 (Nrf2) in
mice subjected to lipopolysaccharide (LPS) challenge. They found that SFN decreased pro-
inflammatory markers such as interleukin 1-
β
(IL-1
β
) and interleukin 6 (IL6) as response to
LPS-treatment. The authors propose that the anti-inflammatory effect of SFN was regulated
by the Nrf2 pathway.
Deng et al. [
32
] demonstrated that SFN delivered as broccoli nanoparticles to mice
is involved in prevention of colitis, an autoimmune disease that can lead to ulcers. The
mechanism consists in the induction of tolerogenic dendritic cells by adenosine monophos-
phate activated protein kinase (AMPK), thus regulating the intestinal immune homeostasis.
Accordingly, SFN could have preventive or therapeutic application on some intestinal
inflammatory diseases due to its activating effect of AMPK signaling pathway.
Liang et al. [
33
] studied the effect of sulforaphane on the redox regulation in human
T-cells, in order to uncover the mechanism that underlays the immunosuppressive effect of
Molecules 2021,26, 752 5 of 14
SFN in chronic Th17-related diseases, such as rheumatoid arthritis. They reported that SFN
exerts a redox-related immunosuppressive effect on untransformed human T-cells, downreg-
ulation of the pro-inflammatory Th17 cytokines associated with autoimmune/inflammatory
diseases (IL-17A, IL-17F and IL-22), and inhibition of cartilage-disruptive proteins. These
processes produce a significant reduction in the clinical symptoms. Since this study was
conducted ex vivo, the results cannot be extrapolated to the effect in humans.
Some authors investigated the effect of sulforaphane on immune-associated inflam-
matory diseases of the central nervous system (CNS), such as Alzheimer and Parkinson,
concluding that SFN has anti-inflammatory and anti-oxidant effect [
34
,
35
]. The mecha-
nism that underlies this kind of disease relies on promotion of leukocyte traffic across the
blood-brain barrier by the action of reactive oxygen species (ROS) [
36
]. ROS induce myelin
breakdown and neuronal injury, among other effects. Additionally, the infiltrated cells
increase the production of ROS, thus contributing to the advance of the CNS diseases [
37
].
Yoo et al. [
38
] administered SFN (orally, 50 mg/kg/day over 14 days) to an autoimmune
encephalomyelitis model (mouse). The clinical symptoms of SFN–treated animals were
diminished significantly in comparison with those observed in control animals. This was
attributed to the anti-inflammatory and anti-oxidative effects of SFN, resulting in neuro-
protection. Accordingly, SFN seems a promising alternative to traditional drugs, which are
expensive and most importantly have undesirable side-effects.
2.2. Pulmonary Diseases
Information about the effect of SFN on immune system in lung diseases is poorly
documented so far. Recently, Patel et al. [
39
] presented evidence that SFN can act as
prophylactic in hyperoxia-induced lung injury or hyperoxia-compromised macrophage
function in phagocytosis. The results presented in this study suggest that SFN can alleviate
hyperoxia-induced inflammatory acute lung injury by increasing macrophage phagocytosis
via inhibiting the accumulation of extracellular HMGB1 (high-mobility group box 1 pro-
tein). Thus, by reducing the toxic effects of extracellular HMGB1, it is possible to maintain
the functions of pulmonary macrophages and the integrity of lung tissues under oxidative
stress. This is the first report in which is shown that SFN attenuate hyperoxia-induced
macrophage dysfunction through an HMGB1-mediated pathway. The authors concluded
that the supplementation of SFN during oxygen therapy may prevent lung damage and pre-
serve lung cell functions and lung tissue integrity, thus providing a promising therapeutic
approach for patients receiving mechanical ventilation.
2.3. Viral Diseases
Literature about the effect of SFN on immunological system during viral infection is
scarce. There are studies showing that SFN may help an organism to fight against some
types of virus, mainly HIV, influenza, hepatitis C, and most recently COVID-19. These
studies suggest that SFN acts by restoring the immune system and downregulating free
radicals production, mediated through modulation of antioxidant genes expression by the
transcription factor Nrf2.
Jin-Nyoung et al. [
40
] studied the effect of administering isothiocyanates (benzyl
isothiocyanate, indolo[3,2-b]carbazole, indole-3-carbinol, phenethyl isothiocyanate, and
sulforaphane) on the life span of leukemia retrovirus infected-mice. The authors reported
that mice treated with benzyl isothiocyanate, phenethyl isothiocyanate, or sulforaphane
significantly extended their life span in comparison with the control retrovirus-infected
group. Accordingly, those three ITC retarded the evolution of the infection with LP-BM5
retrovirus to murine AIDS. Furuya et al. [
41
] investigated the effect of SFN on human
macrophages and T-cells after infection with HIV. The authors demonstrated that, unlike
other viruses like Dengue virus (DENV) or Marburg virus (MARV) that benefit from
Nrf2, HIV infection is blocked with the activation of Nrf2 in primary macrophages. This
effect was not detected in T-cells. SFN modulates Nrf2 and results in reprogramming
Molecules 2021,26, 752 6 of 14
gene expression in macrophages. Finally, it was proposed that SFN is capable to induce
an antiviral response in human macrophages against HIV, arising as a promising therapy.
In contrast to the effect of Nrf2 on the HIV infection, the oxidative stress generation
during DENV infection stimulates the transcription factor Nrf2, which tightly regulates
ROS levels as well as innate immune and apoptotic responses to DENV infection, limiting
both antiviral and cell death responses to the virus by feedback modulation of oxidative
stress. Confirming the above, silencing of Nrf2 by RNA interference increased DENV-
associated immune and apoptotic responses [
42
]. On the other hand, MARV directly
increases Nrf2 levels through a protein called VP24. This protein, like SFN, interacts with
Keap1 (Kelch-like ECH-associated protein 1), a negative regulator of Nrf2. Binding of
VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition, promoting
persistent activation of diverse cytoprotective genes implicated in cellular responses to
oxidative stress and regulation of inflammatory responses. The authors demonstrated
that there is increased expression of Nrf2-dependent genes both during MARV infection
and upon transient expression of MARV VP24. Finally, Nrf2-deficient (Nrf2
-/-
) mice
can control MARV infection when compared to lethal infection in wild-type animals,
indicating that Nrf2 is critical for MARV infection [43].
Hepatitis C Virus (HCV) is susceptible to heme-oxigenase-1 (HO-1) which interferes
with the replication of viruses like HIV and Hepatitis B [
44
,
45
]. Since SFN is a potent
activator of phase II antioxidant enzymes, like HO-1, Yu et al. [
46
] studied the effect of
SFN on Huh-7 cells infected with HCV. The authors demonstrated that SFN suppresses
replication of HCV by inducing HO-1 expression through activation of Nrf2 pathway.
Efforts have been made to elucidate the role of SFN in immunological response to
influenza. Some phytochemicals have shown to enhance immunological response against
influenza, such as glucans [47] and sulforaphane [48], the latter associated to Nrf2 expres-
sion that blocks influenza A entry and replication in human nasal epithelial cells. Vaclav
and Jana [
49
] investigated the effect of a glucan–SFN combination on influenza in a mouse
model. They evaluated immunological response by assessing some immune reactions,
virus concentration, and animal survival. The results suggested that both phytochemicals
had a synergistic effect on stimulation of immunological system. Müller et al. [
50
] con-
ducted a clinical trial to evaluate the effect of SFN-rich broccoli sprouts homogenate on
peripheral blood mononuclear cells (PBMC) after administering a nasal vaccine dose of live
attenuated influenza virus (LAIV) to healthy subjects. They found significant differences
between the response to BSH (broccoli sprout homogenates) and placebo, observing that
LAIV significantly reduced NKT (natural killer T) and T-cell populations. The authors con-
clude that nasal influenza infection may induce complex changes in peripheral blood NK
cell activation, and that BSH (rich in SFN) effect may be important for enhanced antiviral
defense responses. Li et al. [
51
] studied the effect of SFN on influenza A virus replication in
Madin-Darby canine kidney cells. They detected an increased accumulation of Nrf2 factor
triggered by SFN, resulting in a decrease of virus replication.
During the last year, the world has been shocked by the abrupt irruption of COVID-19
and the scientific community has been devoted to find insights that help fight against this
disease. A way to reduce the severity and mortality generated by acute respiratory distress
syndrome (ARDS) produced by SARS-COV 2 is to strengthen the immune system. ARDS
produces a dysregulation of the immunological system, and in the most severe cases, the
release of pro-inflammatory cytokines and loss of T-cells in the infected organism [
52
].
There is evidence of the antiviral effect of Nrf2 on respiratory syncytial virus infection [
53
]
and on SARS-COV 1 [
54
]. Based on information about viruses that belong to the same
family, it has been proposed that compounds that activate Nrf2 could probably help to
diminish these effects. Cuadrado et al. [
55
] suggested that due to its ability to activate
Nrf2, induce antioxidant enzymes, reduce pro-inflammatory cytokines, and its efficacy and
safety, SFN is a promising candidate to counteract inflammatory reaction and protect lungs
from severe damage during SARS COV 2 infection. Finally, Horowitz and Freeman [
56
]
suggested that clinical trials including administration of Nrf2-activating molecules, such as
Molecules 2021,26, 752 7 of 14
SFN, are imperative to support a possible three-party strategy to fight the COVID-19
pandemic, which includes prevention, diagnostic, and treatment.
2.4. Bacterial Diseases
Research about the effect of SFN on immunological system during bacterial infections
is incipient. Currently there are reports that consider H. pylori,S. aureus,E. coli and M. pneu-
moniae. Although SFN exhibits direct bactericidal activity, it triggers an immunological
response to H. pylori infection in the stomach mucosa. SFN acts by activating Nrf2 and
downregulating NF-
κ
B, whose joint action modulates antioxidant and anti-inflammatory
response in the host [
57
,
58
]. As a consequence, SFN exerts a protective effect from gastritis
and gastric ulcer. Yanaka [
59
] conducted
in vitro
and
in vivo
studies about the effect of
SFN on H. pylori infection. The outcomes demonstrated that SFN significantly reduced the
bacterium viability and alleviated gastritis in animal models and in humans.
Haodang et al. [
60
] studied the response of monocytes stimulated with Mycoplasma
pneumoniae lipopeptide to SFN exposure. Pathological injury of M. pneumoniae in lungs
relates with inflammation that stimulates immune response of the host triggered by lipid
polysaccharide (LPS) excretion by the bacteria. The authors found that SFN inhibited the
expression of pro-inflammatory cytokines and activated the expression of HO-1 after the
induction of Nrf2. As a result, SFN reduced lung inflammation in an animal model. The
mechanism proposed by [60] is depicted in Figure 3.
Ali et al. [
61
] investigated the effect of four Nrf2 activators on bacteria-infected
macrophages, among them, SFN. Macrophages were infected either with E. coli or S. au-
reus and the intracellular viability of bacteria was evaluated. SFN significantly reduced
intracellular bacteria survival in PBMC-derived macrophages. Even though the authors
do not present any mechanism, they propose that the intra and extra cellular bacterici-
dal effect of SFN relies on the anti-inflammatory and antioxidant milieu produced inside
the macrophages. SFN, as a potent Nrf2 activator, seems a promising therapeutic option
for Gram (+) and Gram (
−
) bacterial infections since it modulates antioxidant and anti-
inflammatory responses. Deramaudt et al. [
62
] studied the intracellular survival of S. aureus
in human and mice macrophages treated with SFN. They proposed a mechanism consisting
in modulation of p38/JNK signal pathway induced by SFN in macrophages, thus reducing
inflammatory response. Additionally, the authors reported that SFN affected S. aureus
intracellular survival by inducing apoptosis in the bacterium. Then, the combination of
both mechanisms supports SFN as a possible treatment for S. aureus infection.
Finally, Belchamber and Donnelly [
63
] suggested that SFN stimulates phagocytic
pathways and improves macrophage phagocytosis of S. pneumoniae,P. aeruginosa and
H. influenzae by upregulating Nrf2 in alveolar cells from COPD9 (chronic obstructive
pulmonary disease).
2.5. Cancer
Cancer is a multi-factorial disease responsible for around 10 million deaths worldwide
per year. The WHO estimates that 30–50% of cancer cases could be prevented. Accordingly,
several efforts are made to discover new strategies to fight and most importantly, prevent
this disease. SFN is widely recognized as the most potent natural anti-cancer compound.
This phytochemical acts at different cancer stages, from development to progression,
by exerting a pleiotropic effect. SFN can trigger apoptosis, reducing angiogenesis and
metastasis in cancerous cells. At the molecular level, it activates Nrf2, consequently
modulating cellular redox homeostasis and stimulating the immune system [
64
]. Figure 4
shows the mechanism action of SFN as chemoprotective and chemotherapeutic agent.
Singh et al. [
65
] studied the effect of administering a SFN analogue on prostate car-
cinogenesis and pulmonary metastasis in an animal model. The results showed that SFN
stimulates NK cells cytotoxicity, thus enhancing immunological function. Also, SFN increased
the infiltration of lymphocyte T-cells in prostate tumors resulting in a reduction of metastasis.
Molecules 2021,26, 752 8 of 14
Molecules 2021, 26, x 8 of 14
2.5. Cancer
Cancer is a multi-factorial disease responsible for around 10 million deaths world-
wide per year. The WHO estimates that 30–50% of cancer cases could be prevented. Ac-
cordingly, several efforts are made to discover new strategies to fight and most im-
portantly, prevent this disease. SFN is widely recognized as the most potent natural anti-
cancer compound. This phytochemical acts at different cancer stages, from development
to progression, by exerting a pleiotropic effect. SFN can trigger apoptosis, reducing angi-
ogenesis and metastasis in cancerous cells. At the molecular level, it activates Nrf2, con-
sequently modulating cellular redox homeostasis and stimulating the immune system
[64]. Figure 4 shows the mechanism action of SFN as chemoprotective and chemothera-
peutic agent.
Singh et al. [65] studied the effect of administering a SFN analogue on prostate car-
cinogenesis and pulmonary metastasis in an animal model. The results showed that SFN
stimulates NK cells cytotoxicity, thus enhancing immunological function. Also, SFN in-
creased the infiltration of lymphocyte T-cells in prostate tumors resulting in a reduction
of metastasis.
The efficacy of SFN as a possible therapeutic compound has been assayed in different
types of cancer cells and tissues. Bessler and Djaldetti [66] investigated the effect of SFN
on immunological interaction between PBMC and human colon cancer cell lines. The au-
thors detected a concentration-dependent effect of sulforaphane that inhibited production
of pro-inflammatory cytokines in PBMC. SFN acts against colon cancer by different mech-
anisms: (1) induction of DNA damage in cancerous cells by acetylating the DNA repair
protein; (2) activation of pro-apoptotic proteins resulting in induction of apoptosis; (3)
activation of Phase II detoxifying proteins through Nrf2; (4) cell cycle arrest by suppress-
ing histone deacetylase inhibitor and telomerase reverse transcriptase [67,68]. Suzuki et
al. [69] studied the effect of daily intake of SFN delivered as fresh broccoli sprouts on colon
cancer animal model and in humans. Their results indicate that SFN treatment suppressed
the formation of aberrant crypt foci and macroscopic tumors in mice and in colon cancer
patients.
Palliyaguru et al. [70] investigated the effect of SFN on breast cancer development in
a mouse model exposed to estradiol. The authors found that SFN enhanced cytoprotection
by mitigating DNA damage and suppressing lipogenesis. These effects were attributed to
activation of Nrf2 by SFN.
Figure 4. Chemoprotective and chemotherapeutic mechanisms of SFN in cancer cells. SFN exerts
chemoprevention by inducing HO-1 and Phase II enzymes, and increasing GSH concentration
Figure 4.
Chemoprotective and chemotherapeutic mechanisms of SFN in cancer cells. SFN exerts
chemoprevention by inducing HO-1 and Phase II enzymes, and increasing GSH concentration
expression and activating NK cells, as well as downregulating pro-inflammatory cytokines. In both
cases, the effect on immune system is mediated by transcription factors Nrf2 and Nfκβ.
The efficacy of SFN as a possible therapeutic compound has been assayed in different
types of cancer cells and tissues. Bessler and Djaldetti [
66
] investigated the effect of SFN on
immunological interaction between PBMC and human colon cancer cell lines. The authors
detected a concentration-dependent effect of sulforaphane that inhibited production of pro-
inflammatory cytokines in PBMC. SFN acts against colon cancer by different mechanisms:
(1) induction of DNA damage in cancerous cells by acetylating the DNA repair protein;
(2) activation of pro-apoptotic proteins resulting in induction of apoptosis; (3) activation
of Phase II detoxifying proteins through Nrf2; (4) cell cycle arrest by suppressing histone
deacetylase inhibitor and telomerase reverse transcriptase [
67
,
68
]. Suzuki et al. [
69
] studied
the effect of daily intake of SFN delivered as fresh broccoli sprouts on colon cancer animal
model and in humans. Their results indicate that SFN treatment suppressed the formation
of aberrant crypt foci and macroscopic tumors in mice and in colon cancer patients.
Palliyaguru et al. [
70
] investigated the effect of SFN on breast cancer development in
a mouse model exposed to estradiol. The authors found that SFN enhanced cytoprotection
by mitigating DNA damage and suppressing lipogenesis. These effects were attributed to
activation of Nrf2 by SFN.
3. Clinical Trials Regarding the Effect of Sulforaphane on Immune System
Over the last decade, there are 74 clinical studies that have aimed at evaluating the
effect of sulforaphane on different diseases; four of them focused on immune system
disorders (www.clinicaltrials.gov). Table 1shows details about the clinical trials.
Trial NCT01357070 was designed to test whether consuming a “broccoli smoothie”
containing sulforaphane could protect white blood cells from activation in the presence
of experimental stress and how long this protective effect would last. To do this, the
researchers analyzed inflammatory changes in blood samples taken at different times
during the study. The investigators suggest that inducing anti-oxidant enzymes indirectly
may be an effective means of providing vascular protection. To date, no results are available.
Molecules 2021,26, 752 9 of 14
Table 1. Clinical trials regarding the effect of sulforaphane on immune system (www.clinicaltrials.gov).
Clinical Trial
Identifier Title Study Population Duration Sulforaphane (Dose) Results Phase Status
NCT01357070
Effect of Broccoli Sprout on
Blood Levels of
Sulforaphane to Reduce
Responsiveness of
Immune System
6 healthy volunteers,
London, United
Kingdom
34 months
(May 2011–January 2014)
broccoli sprout
homogenate (70 g dry
weight, orally by three
consecutive days).
N.I. 1N.A. 2Completed
NCT01183923
Dietary Interventions in
Asthma Treatment:
Sprouts Study
1 adult, asthmatic,
male, white, United
States of America
14 months
(November 2010–
February 2012)
N.I. 1(broccoli sprouts,
one serving per day, 7
days, in a sandwich)
N.I. 1N.A. 2Halted
NCT01269723
Effects of Sulforaphane
(SFN) on Immune
Response to Live
Attenuated Influenza Virus
in Smokers and
Nonsmokers
51 adults, healthy,
smokers or
nonsmokers, United
States of America
28 months
(December 2010–
March 2013)
N.I.1(broccoli sprouts
homogenate) N.I. 1N.A. 2Completed
NCT01845493
Sulforaphane
Supplementation in Atopic
Asthmatics (brasma)
16
adults, asthmatics,
United States
of America
17 months
(April 2013–
October 2014)
N.I. 1(broccoli sprouts
homogenate orally daily,
three days)
N.I. 11 Completed
NCT01845220 Prevention of Alcohol
Intolerance
30 adults, older adults,
sensitive to alcohol on
the skin, Japanese,
United States
of America
27 months
(May 2013–July 2015)
150 nmol of
sulforaphane/cm2 of
skin in 80% acetone
SFN increased
erythema (affected skin
area) as response to
alcohol exposure
2 Completed
NCT02885025 Effects of Broccoli Sprout
Extract on Allergy Rhinitis
47 adults, older adults,
allergic rhinitis
or healthy
18 months
(October 2016–
March 2019)
N.I. 1(broccoli
sprouts extract)
SFN reduced
pro-inflammatory
cytokines with and
without combination
with fluticasone
2 Completed
1Not Informed. 2Not Applicable.
Molecules 2021,26, 752 10 of 14
Trial NCT01183923 hypothesized that since SFN is an inducer of Phase II antioxidant
enzymes and broccoli sprouts (BS) are rich in SFN, administration of BS would improve
lung and airways function in asthmatic subjects. As a consequence, oxidative stress and
inflammation markers would decrease after exposure to allergens. Recruited subjects
(n = 1)
ingested BS and were exposed to environmental mouse allergen challenge. After,
seven daily BS intake markers (nasal epithelial gene expression, urinary oxidative stress
biomarkers, serum inflammatory and oxidative stress biomarkers, and basophil activation)
were assessed. Unfortunately, this trial was halted because of an adverse event. While
no results are available at www.clinicaltrialls.gov, the outcomes of this trial can be found
elsewhere [
71
]. Asthmatic subjects (n = 40) ingested 100 g of BS daily for three days. Effect
of SFN was assessed by measuring antioxidant genes expression in nasal epithelial and
PBMC, inflammation, and oxidative stress biomarkers, among others. Determinations were
conducted before and after BS intake. Since no change in biomarkers and cytoprotective
genes expression could be detected, the authors concluded that despite the increase in
blood concentration of SFN, BS intake did not improve lung inflammatory response nor
antioxidant biomarkers in asthmatic subjects.
Trial NCT01269723 aimed at evaluating the short-term immunological response to live
attenuated influenza virus, and to compare the reaction between smokers and nonsmokers
treated with BS (or placebo) homogenate. As response to the treatment, they evaluated
the virus charge and inflammation biomarkers (IL6, cytokines, NK cells activation) in
nasal mucosa. There are no results available at www.clinicaltrials.gov, however the out-
comes of this trial were published in [
50
]. The main conclusions of this trial was that BS
homogenate enhanced immune response against influenza virus, demonstrated by an
increase in granzyme B production in peripheral NK cells.
Trial NCT01845493 consisted of a pilot study about the effect of SFN administration
(in the form of BS homogenate) on Nrf2 and Phase II enzymes induction. A total of
16 asthmatic subjects ingested BS homogenate for three days and the outcomes were
compared with SFN and placebo controls. Even though the trial ended on 2014, the results
are not available so far.
Trial NCT01845220 aimed at evaluating the effect of SFN (as BS extract) exposure in
alcohol-intolerant subjects. After SFN application, subjects (n = 30) were topically exposed
to alcohol, and reddened skin area was measured as indicator of SFN protection against
irritation. The outcomes indicate that topical application of SFN increased erythema after
exposure to alcohol in alcohol-sensitive subjects.
Trial NCT02885025 studied the effect of administering SFN (as BS extract) to subjects
(n = 47) suffering allergic rhinitis to grass. The randomized trial considered a three-
week treatment aiming at evaluating the effect of BS extract intake in comparison with
administration of corticosteroid (fluticasone) and the combination of both. Before treatment,
subjects were exposed (nasal way) to different varieties of grass. The results showed
that SFN alone or in combination with fluticasone reduced pro-inflammatory cytokines
expression. However SFN exhibited a more limited effect than fluticasone alone.
4. Conclusions
Despite the huge amount of information about the effect of SFN on several diseases,
especially cancer, research about the effect of SFN on immune system response at molecular
and cellular levels is scarce, as well as clinical trials focused on immune system diseases.
Sulforaphane exerts a pleiotropic effect on immunological response, and the final effect
depends on the cell type. In lymphocyte T-cells, SFN induces ROS production, GSH de-
pletion, and repression of inflammatory cytokines, resulting in suppression of immune
and inflammatory responses. This may help in treatment on autoimmune/inflammatory
diseases symptoms. In monocytes and macrophages, SFN stimulates immune response by
inducing Nrf2, thus triggering antioxidant and anti-inflammatory responses. As a conse-
quence, bacteria survival decreases in infected cells, and virus-infected cells are neutralized
by induction of antioxidant enzymes such as HO-1. Additionally, SFN improves immune
Molecules 2021,26, 752 11 of 14
system, thus helping in prevention and reducing severity of viral pulmonary diseases.
In cancer cells, SFN induces apoptosis and cell cycle arrest, as well as antioxidant enzymes
that stimulate cellular immune response. Finally, the few clinical trials about the effect of
SFN on immune system are not conclusive; this kind of study should be encouraged.
Author Contributions:
Conceptualization, A.M.; methodology, A.M. and A.C.; formal analysis, A.M.
and A.C.; investigation, A.M.; resources, A.M. and A.C.; data curation, A.M. and A.C.; writing—
original draft preparation, A.M.; writing—review and editing, A.C.; project administration, A.M.;
funding acquisition, A.M. and A.C. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was funded by Agencia Nacional de Investigación y Desarrollo (ANID),
Fondecyt program, grant number 1201418. The APC was funded by Fondecyt 1201418. Ethical review
and approval were not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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