Evaluation of The Immunomodulatory Activity of The Nutraceutical Formulation Aminodefence

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Volume 75, 2024 - Issue 2
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In vitro and animal studies
In vitro evaluation of the immunomodulatory

activity of the nutraceutical formulation
AminoDefence
Bianca Papotti, Mattia Dessena, Maria Pia Adorni , Davide Paleari, Laura Rinaldi &
Franco Bernini
Pages 173-184 | Received 01 Sep 2023, Accepted 10 Nov 2023, Published online: 29 Nov 2023
 Cite this article  https://doi.org/10.1080/09637486.2023.2283688
 Full Article  Figures & data  References  Citations  Metrics  Licensing
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Abstract
Immune system (IS) functionality is warranted by inter-dependent processes that

balance body defences without exceeding in inflammation. An ideal nutraceutical
approach should sustain the protective IS activity while controlling inflammation. The
potential immunomodulatory activity of the food supplement (FS) AminoDefence was
studied in resting macrophages RAW264.7 and following stimulation of bacterial- and
viral-associated inflammation trough LPS and PolyI:C treatments, respectively. In
In this article macrophages, the formulation exerted a dose-dependent
unstimulated
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https://www.tandfonline.com/doi/full/10.1080/09637486.2023.2283688?src=most-read-last-year 1/31
immunostimulant activity by up-regulating NO, IL-6, TNF-α and MCP-1 release, while it
dampened the aberrant release of these factors induced by pro-inflammatory stimuli.
Exploring the contribution of single components Echinacea purpurea (E. purpurea) extract
and quercetin, used at proportional concentrations than in whole formulation, a more
pronounced immunostimulant effect was observed for E. purpurea, and an anti-
inflammatory activity for quercetin. Hence, AminoDefence exerts an immunomodulatory
activity in macrophages by effectively stimulating a protective inflammatory response
and limiting it in cases of excessive inflammation.
 Keywords: Immune system food supplement immunomodulation inflammation quercetin
Echinacea purpurea NO cytokines
Introduction
The increasing ageing population, the greater awareness of the causal relationship
between disease and nutrition, and the importance of preventive measures to protect
individuals health, have helped make the food supplement (FS) industry one of the
fastest-growing industries (Brunelli et al. 2022). Moreover, the general public has
significantly increased the use of FSs since the start of the COVID-19 pandemic (Crawford
et al. 2022). Indeed, immunity FS sales reported by the Nutrition Business Journal (NBJ)
climbed from $3.4 billion US dollars in 2019 to almost $6 billion by the end of 2020, with
immune health representing approximately 10% of all US supplement sales (Crawford
et al. 2022). Focusing on the European context, the market size reached 13.3 billion
euros in 2021 (Perelló-Oliver 2023), with 52% of consumers that declare to use FSs
generically to maintain health status, and 45% of them that use FSs specifically intending
to support or boost the immune system (Ipsos 2022). The increased use of such FSs
specifically aimed at improving the immune system (IS) defense could be related to the
need to minimise the negative impact of seasonal infections and their symptoms on
health-related quality of life, namely the normal daily activities including swallowing,
talking, eating, sleeping, working and performing in school (Addey and Shephard 2012).
Similarly, influenza patients exhibit upper and/or lower respiratory tract signs and
symptoms, frequently accompanied by systemic illness symptoms, such as fever,
headache, myalgia, and weakness; often influenza can occur in pandemic form, leading
to serious health complications as well as social and economic disruption (Yoshino et al.
2021).
Homeostatic mechanisms regulating the IS are essential for the proper body defense
(Horwitz et al. 2019), despite understanding its composition and function is challenging
due to the high number of interdependent cell populations, their complex regulation, as
well as the variability among individuals, as a consequence of both heritable (e.g.
histocompatibility leukocyte antigen (HLA) genes) and non-heritable factors, with these
latter exerting a stronger influence on innate IS (Lakshmikanth et al. 2020). In this
regard, a systematic review of six separate studies suggests that seasonal immune
modulation might be linked to specific seasonal infections (Paynter et al. 2015), thus
supporting the hypothesis that an intervention aimed at improving immune function is
conceivable. In this respect, a growing number of reports suggest that several
nutraceutical approaches exert immunomodulatory functions by acting on the IS and
particularly by supporting the innate immunity on one side, or balancing the
exacerbated pro-inflammatory response (Alba et al. 2023) triggered by pathogens on the
other side. In particular, in a context of a varied and balanced diet and a healthy lifestyle,
FSs might sustain the immune system function by modulating inflammatory response,
both supporting the protective pro-inflammatory function and restoring immune
homeostasis (Vignesh et al. 2021), possibly representing a beneficial strategy to support
the body immune defences (Djaoudene et al. 2023). Indeed, an ideal and safe
nutraceutical approach should counteract infection initiation by stimulating the
macrophage protective pro-inflammatory function, but also control and moderate an
exaggerated inflammation during infection .
For instance, Echinacea purpurea (E. purpurea) extract, with its array of different bioactive
molecules, including alkamides, phenolic compounds and arabinogalactan proteins, is
one of the most characterised nutraceuticals with immunomodulatory properties (Kim
and Calderón 2022). Indeed, E. purpurea extract exerted an immunostimulatory activity

in in vitro studies on murine and human peripheral mononuclear cells (PBMCs) and
macrophages (Rininger et al. 2000, Fu et al. 2017). Consistently, in a mouse model of
lipopolysaccharides (LPS)-induced lung injury (Zhang et al. 2020), E. purpurea
upregulated the markers of classically activated M1 macrophages, stimulated the
release of pro-inflammatory cytokines and nitric oxide (NO), thus bolstering their anti-
microbial effect. Moreover, the anti-inflammatory cytokines release induced by E.
purpurea extract has been documented in human macrophages (Zhai et al. 2009,
Dobrange et al. 2019, Vieira et al. 2022). As for human evidence, a review considering 14
studies demonstrated that the prophylactic treatment with 2400 mg/day E. purpurea
extract for 4 months appeared to be beneficial in common cold treatment and
prevention (Rondanelli et al. 2018). In addition to E. purpurea extract, a growing number
of evidence supports the involvement of quercetin in the modulation of a wide range of
biological pathways involved in the immune system, exerting an anti-inflammatory, anti-
viral and anti-oxidant properties (Li et al. 2016). Besides E. purpurea extract, this strong
evidence prompted us to analyse the immunomodulatory contribution of quercetin
among the formulation components. Quercetin, indeed, was previously shown to
effectively reduce oxidative stress (Cui et al. 2019) and to inhibit pro-inflammatory
cytokines and NO release from LPS-elicited human and murine macrophages (Cho et al.
2016, Tang et al. 2019). In addition, an inhibitory activity of quercetin on viral entry and
replication has been reported concerning Influenza A (Choi et al. 2009), and SARS-CoV (Yi
et al. 2004). Moreover, in vitro evidence reports a quercetin-derived reduction of the
inflammatory response in cultured macrophages exposed to Polyinosinic:polycytidylic
acid (PolyI:C), an experimental model of viral-related inflammation (Kim and Park 2016).
This study aimed to investigate the immunomodulatory effect of AminoDefence, a FS

consisting of a complex of amino acids, proteins, vitamins, minerals, and plant-derived
substances, including quercetin and E. purpurea extract, in cultured macrophages.
Furthermore, based on the reported activity on IS of both quercetin and E. purpurea
extract, their potential ability to stimulate macrophage pro-inflammatory activity, as well
as to suppress the macrophage inflammatory response related to bacterial LPS
stimulation was assessed. To this aim, PolyI:C was used in a limited experimental setting
to evaluate the modulating effect of the nutraceutical formulation on the viral-related
inflammation.
Materials and methods
Cell culture and treatments
Murine macrophages RAW264.7 were purchased from ATCC (VA, USA). Cells were
cultured in DMEM High Glucose (Euroclone, Italy), supplemented with 10% Fetal Calf
Serum (FCS; Euroclone, Italy) at 37 °C, 5% CO2. Twenty-four hours after plating, cells were
treated for 48 h with the nutraceutical formulation Endomune® (Laboratoires NHCO
Nutrition, Nice, France), commercialised in France with registered trademark
Endomune®, in Italy with the tradename AminoDefence and in Spain with the
tradename AminoDefense, at the concentration of 0.06 − 0.12 −0.25 − 0.5 − 1 mg/ml, or
with the single component quercetin or E. purpurea extract, at the concentrations
corresponding to those present in the formulation, to evaluate the immunostimulant
activity. A different set of cells were instead treated for 24 h with the nutraceutical
formulation, as well as with the single components quercetin or E. purpurea extract, in
combination with the inflammatory stimulus Lipopolysaccharides (LPS; Merck, Germany)
[1 µg/mL], in order to evaluate the anti-inflammatory activity of the compounds (Kim and
Park 2016, Aki et al. 2020). In these conditions, the nutraceutical formulation
AminoDefence, quercetin and E. purpurea extract were tested at the same concentration
as indicated above. As explorative test, a limited number of experiments were
performed using Polyinosinic:polycytidylic acid (PolyI:C; Merck, Germany) [50 µg/ml] as
inflammatory stimulus to specifically mimic the viral infection-related inflammation; the
experimental protocol to evaluate the anti-inflammatory effect of the compounds was
the same of that used for LPS. Cell viability of the tested compounds was preliminarily
investigated in all experimental conditions to exclude possible cytotoxic effects. The
whole formulation powder was preliminarily tested for its solubility in cell culture
medium supplemented with 0.5%v/v DMSO; in this condition ∼95% of the whole powder
was completely solubilised. The composition of the nutraceutical formulation
AminoDefence is reported in Table 1.
Table 1. Nutraceutical formulation.
Download CSV Display Table


Measurement of Nitric Oxide (NO) production
NO production was assessed by measuring nitrite levels in culture media through the
Griess assay (Promega, WI, USA), based on the formation of a chromophore after
reacting with Griess reagent, which was prepared fresh daily by mixing equal volumes of
stock A (1% sulphanilamide, 5% phosphoric acid) and stock B (0,1% N-[naphthyl]
ethylenediamine dihydrochloride) (Tang et al. 2019). NO production was evaluated for
immunostimulant activity, and for the anti-inflammatory activity after stimulation of cells
with LPS and PolyI:C.
Measurement of cytokine secretion
Cytokine secretion (TNF-α, IL-6 and MCP-1) in culture medium was assessed through
specific enzyme-linked immunosorbent assays (all purchased from Thermofisher, MA,
USA) following the manufacturer’s instructions. Cytokine secretion was evaluated in both
basal condition for the immunostimulant activity and for the anti-inflammatory activity
after stimulation with LPS.
Statistical analyses
Statistical analyses were performed using Prism 8 software (GraphPad Software, San
Diego, CA, USA). All data are expressed as mean ± standard deviation (SD) of
experimental conditions performed in triplicate. Statistically significant differences
among the mean values of each experimental group were investigated using the one-
way analysis of variance (ANOVA), followed by the post-hoc Dunnett’s multiple

comparison tests. A value of p < 0.05 was considered statistically significant.
Results
Immunostimulant activity
Nitric oxide (NO) release by cultured macrophages
First, the potential modulatory effect of the whole nutraceutical formulation

AminoDefence and of its compounds quercetin and E. purpurea extract was evaluated by
quantifying NO release by macrophages in resting condition. As reported in Figure 1A,
the formulation at the highest concentration tested (1 mg/ml), was able to significantly
increase NO secretion (2.3-fold increase as compared to untreated cells (vs. basal; p <
0.01). Differently, in the same experimental conditions, quercetin and E. purpurea extract
at all the concentrations tested did not influence the cellular release of NO (
Figure 1B and 1C).
Figure 1. Effect of the whole formulation AminoDefence, quercetin and E. purpurea

extract on NO release by macrophages. RAW264.7 cells were incubated for 48 h with the
indicated increasing concentrations of the formulation AminoDefence (A), quercetin (B)
or E. purpurea extract (C). NO release in the culture media was quantified through Griess
test assay. Experiments were performed in triplicate and data are expressed as mean ±
SD. Statistical analyses were performed using the one-way ANOVA coupled with
Dunnett’s multiple comparison test. A value of p < 0.05 was considered statistically
significant. **p < 0.01 vs basal.
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Cytokine and chemokine secretion by cultured macrophages
Secondly, in the same cellular model, the impact of the nutraceutical formulation
AminoDefence, as well as of quercetin and E. purpurea extract was assessed by
investigating the release of pro-inflammatory cytokines. As reported in Figure 2A,
AminoDefence significantly and dose-dependently increased the release of IL-6 in cell
culture media, reaching at the highest concentration tested a 18-fold increase as
compared to basal, p < 0.0001. Differently, quercetin did not show any significant effect
on IL-6 release (Figure 2B), except for a slight, significant, induction observed at the
concentration of 1.8 µg/ml (p < 0.01). On the other side, similarly to what observed for
the whole AminoDefence formulation, E. purpurea extract significantly induced in a dose-
dependent manner the macrophage IL-6 secretion (Figure 2C). However this effect
occurred at a significant lower extent (5.6-fold increase with E. purpurea extract [100
µg/ml] as compared to untreated macrophages, p < 0.0001, vs 18-fold with
AminoDefence [1 mg/ml], p < 0.0001).
Figure 2. Effect of AminoDefence, quercetin and E. purpurea extract on IL-6 release by

macrophages. RAW264.7 cells were incubated for 48 h with the indicated increasing
concentrations of the formulation AminoDefence (A), quercetin (B) or E. purpurea extract
(C). IL-6 release was determined through immunoenzymatic assay (ELISA). Experiments
were performed in triplicate and data are expressed as mean ± SD. Statistical analyses
were performed using the one-way ANOVA coupled with Dunnett’s multiple comparison
test. A value of p < 0.05 was considered statistically significant. **p < 0.01; ***p < 0.001;
****p < 0.0001 vs basal.
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Similarly, the formulation AminoDefence treatment significantly stimulated in a dose-

dependent manner TNF-α release from cells, with an increase of 45.5-fold at [1 mg/ml]
as compared to untreated macrophages (p < 0.0001) (Figure 3A). No effect was instead
observed after quercetin treatment (Figure 3B), while, as reported in Figure 3C, E.
purpurea extract induced a slight, significant stimulation of TNF-α release at the two
highest concentrations tested (50 µg/ml; p < 0.05 and 100 µg/ml; p < 0.0001 as compared
to untreated macrophages). Again, the single component stimulated the cytokine release
with a significant lower magnitude with respect to the whole formulation.
Figure 3. Effect of AminoDefence, quercetin and E. purpurea extract on TNF-α release by

macrophages. RAW264.7 cells were incubated for 48 h with increasing concentrations of
the formulation AminoDefence (A), quercetin (B) or E. purpurea extract (C). TNF-α release
was determined through immunoenzymatic assay (ELISA). Experiments were performed
in triplicate and data are expressed as mean ± SD. Statistical analyses were performed
using the one-way ANOVA coupled with Dunnett’s multiple comparison test. A value of p
< 0.05 was considered statistically significant. *p < 0.05; ***p < 0.001; ****p < 0.0001 vs
basal.
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Also, the chemokine monocyte chemoattractant protein-1 (MCP-1) release from

macrophages was slightly stimulated by treatment with the AminoDefence formulation,
effect that became significant at the three highest concentrations tested (Figure 4A).
Quercetin inhibited the basal release of MCP-1 in a dose dependent manner, starting
from [7.5 µg/ml] (Figure 4B), while E. purpurea extract stimulated MCP-1 release only at
the highest concentration tested (1.3-fold increase vs untreated macrophages; p < 0.01;
Figure 4C).

extract on MCP-1 release by macrophages. RAW264.7 cells were incubated for 48 h with
the indicated increasing concentrations of the formulation AminoDefence (A), quercetin
(B) or E. purpurea extract (C). MCP-1 release was determined through immunoenzymatic
assay (ELISA). Experiments were performed in triplicate and data are expressed as mean
± SD. Statistical analyses were performed using the one-way ANOVA coupled with
significant. *p < 0.05; **p < 0.01; ****p < 0.0001 vs basal.
Display full size
Anti-inflammatory activity
LPS-treated macrophages
Nitric Oxide (NO) release
Next, the potential anti-inflammatory effect of the nutraceutical formulation

AminoDefence and of its single components quercetin and E. purpurea extract was
explored by quantifying NO release by LPS-stimulated murine macrophages, a model of
bacterial-related inflammation (Fitzgerald and Kagan 2020). As amply previously
reported (Aki et al. 2020), LPS markedly induced NO secretion by macrophages (28-fold
increase as compared to macrophages in basal condition, p < 0.0001; Figure 5A). The
whole formulation AminoDefence significantly suppressed LPS-induced NO secretion in
a dose dependent manner, with an almost complete inhibition obtained at the
concentrations of 0.5 mg/ml and 1 mg/ml (-97.4% and −94.8% vs basal, respectively; p <
0.0001 both). A similar behaviour was observed for quercetin (Figure 5B), that
significantly inhibited the LPS-induced NO release by macrophages in a dose-dependent
fashion, albeit with a lower extent as compared to the whole formulation (−81.3% and
−82.8% at [15 µg/ml] and [30 µg/ml], respectively, (p < 0.0001 both) vs LPS,
concentrations corresponding to the amount of quercetin present in [0.5 mg/ml] and [1
mg/ml] of the whole formulation). Differently, E. purpurea extract did not influence the
NO release by LPS-stimulated macrophages (Figure 5C).
Figure 5. Effect of AminoDefence, quercetin and E. purpurea extract on NO release by

LPS-stimulated macrophages. RAW264.7 cells were incubated for 24 h at 37 °C with
Lipopolysaccharides (LPS) [1 µg/ml] and the indicated increasing concentrations of the
formulation AminoDefence (A), quercetin (B) or E. purpurea extract (C). NO release in the
culture media was quantified through Griess test assay. Experiments were performed in
triplicate and data are expressed as mean ± SD. Statistical analyses were performed
using the one-way ANOVA coupled with Dunnett’s multiple comparison test. A value of p
< 0.05 was considered statistically significant. ****p < 0.0001 vs LPS-treated
macrophages; °°°°p < 0.0001 vs macrophages in basal conditions.
Display full size
Cytokine and chemokine secretion
As also previously reported (Zhai et al. 2009, Lee et al. 2018), LPS incubation induced the
release of various inflammatory mediators, such as cytokines and chemokines.
Consistently, also under the present experimental setting, LPS treatment induced a
marked increase of the pro-inflammatory cytokine IL-6 secretion as compared to
macrophages in basal condition. AminoDefence treatment significantly inhibited LPS-
induced IL-6 release in a dose-dependent manner, starting from [0.5 mg/ml] (p < 0.001),
reaching an inhibition of 69.8% at [1 mg/ml] (vs LPS, p < 0.0001; Figure 6A). Similarly,
quercetin significantly suppressed the IL-6 secretion starting from the concentration of
15 μg/ml, corresponding to that present in [0.5 mg/ml] of formulation, reaching an

inhibition of 54.1% at [30 μg/ml] (vs LPS, p < 0.0001; Figure 6B). Differently, E. purpurea
extract showed an overall null effect on IL-6 release induced by LPS (Figure 6C).

extract on IL-6 release by LPS-stimulated macrophages. RAW264.7 cells were incubated
for 24 h at 37 °C with Lipopolysaccharides (LPS) [1 µg/ml] and the indicated increasing
concentrations of AminoDefence (A), quercetin (B) or E. purpurea extract (C). IL-6 release
in the culture media was quantified through immunoenzymatic assay (ELISA).
Experiments were performed in triplicate and data are expressed as mean ± SD.
Statistical analyses were performed using the one-way ANOVA coupled with Dunnett’s
multiple comparison test. A value of p < 0.05 was considered statistically significant. **p
< 0.01; ***p < 0.001; ****p < 0.0001 vs LPS-treated macrophages; °°°°p < 0.0001 vs
macrophages in basal conditions.
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Consistently to what observed for IL-6, the nutraceutical formulation AminoDefence

significantly reduced TNF-α secretion in a dose-dependent fashion, starting from the
concentration of 0.25 mg/ml, reaching an inhibition of 21.1% at [1 mg/ml] (vs LPS, p <
0.01; Figure 7A). Similarly, both quercetin and E. purpurea extract showed to be equally
effective in inhibiting the LPS-induced TNF-α secretion, inducing an average inhibition of
26.9% and 22.8% for quercetin and E. purpurea extract, respectively (Figure 7B and 7C).
Figure 7. Effect of the whole formulation AminoDefence, quercetin, and E. purpurea

extract on TNF-α release by LPS-stimulated macrophages. RAW264.7 cells were
incubated for 24 h at 37 °C with Lipopolysaccharides (LPS) [1 µg/ml] and increasing

(C). TNF-α release in the culture media was quantified through immunoenzymatic assay
(ELISA). Experiments were performed in triplicate and data are expressed as mean ± SD.
multiple comparison test. A value of p < 0.05 was considered statistically significant. *p <
0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs LPS-treated macrophages; °°°°p < 0.0001
vs macrophages in basal conditions.
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As reported in Figure 8A, AminoDefence inhibited also the LPS-induced MCP-1 release in
a dose-dependent fashion, starting from [0.25 mg/ml], reaching an inhibition of 87.2% at
[1 mg/ml] as compared to LPS condition (p < 0.0001). Similarly, quercetin significantly
and dose-dependently suppressed the LPS-induced MCP-1 secretion (Figure 8B),
however at a lesser extent with respect to the whole formulation (-64.3% at [30 µg/ml], p
< 0.0001). No significant changes in MCP-1 were instead observed following E. purpurea
extract treatment.
Figure 8. Effect of the whole formulation AminoDefence, quercetin, and E. purpurea

extract on MCP-1 release by LPS-stimulated macrophages. RAW264.7 cells were
incubated for 24 h at 37 °C with Lipopolysaccharides (LPS) [1 µg/ml] and increasing
(C). MCP-1 release in the culture media was quantified through immunoenzymatic assay
(ELISA). Experiments were performed in triplicate and data are expressed as mean ± SD.
multiple comparison test. A value of p < 0.05 was considered statistically significant. *p <
0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs LPS-treated macrophages; °°°°p < 0.0001
Display full size
PolyI:C-treated macrophages
Nitric Oxide (NO) release
Finally, the effect of the nutraceutical formulation AminoDefence and of its components
quercetin and E. purpurea extract was also assessed in cells treated with the pro-
inflammatory stimulus Polyinosinic:polycytidylic Acid (PolyI:C), specifically mimicking the
inflammation associated to viral infection (Kim and Park 2016).
In these experimental conditions, PolyI:C stimulated NO production by macrophages by

about 48-fold as compared to basal (p < 0.0001). As reported in Figure 9A, AminoDefence
was able to significantly inhibit the NO release in a dose-dependent manner starting
from [0.25 mg/ml], with an almost complete inhibition of PolyI:C-induced NO production
at [1 mg/ml]. A similar behaviour was observed for quercetin, that significantly inhibited
the NO release by PolyI:C-stimulated macrophages starting from [7.5 µg/ml], the
concentration corresponding to that present in the formulation at [0.25 mg/ml] (
Figure 9B). However, the inhibitory effect of quercetin occurred at a lower extent as
compared to the whole nutraceutical formulation (quercetin [30 µg/ml] − 55.2% and
AminoDefence [1 mg/ml] − 94.6% vs PolyI:C; p < 0.0001). Also, E. purpurea extract was
able to suppress NO release induced by PolyI:C, however starting from the
concentration of 50 µg/ml, corresponding to that present into [0.5 mg/ml] of formulation
(Figure 9C). Considering the highest concentration, E. purpurea extract was more
effective than quercetin in inhibiting PolyI:C-induced NO release from macrophage
(-84.5% E. purpurea extract at [100 µg/ml] and −55.2% quercetin at [30 µg/ml] vs PolyI:C;
p < 0.0001).
Figure 9. Effect of AminoDefence, quercetin and E. purpurea extract on NO release by

Polyinosinic:polycytidylic acid (PolyI:C)-stimulated macrophages. RAW264.7 cells were
incubated for 24 h at 37 °C with Polyinosinic:polycytidylic acid (PolyI:C) [50 µg/ml] and the
indicated increasing concentrations of the formulation AminoDefence (A), quercetin (B)
or E. purpurea extract (C). NO release in the culture media was quantified through Griess
test assay. Experiments were performed in triplicate and data are expressed as mean ±
SD. Statistical analyses were performed using the one-way ANOVA coupled with
significant. ***p < 0.001; ****p < 0.0001 vs PolyI:C-treated macrophages. °°°°p < 0.0001
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Discussion
Immune system (IS) relies on the balance between body defences and the management
of the inflammatory process (Horwitz et al. 2019). Food supplements (FSs) are not
addressed to substitute a varied and balanced diet, on the contrary their proper use
consists in combining them with a balanced and healthy diet. In this context, a FS
approach could be addressed to modulate immune activity, by supporting immunity
against infections, as well as by helping the recovery of immune homeostasis (Addey
and Shephard 2012, Vignesh et al. 2021). The aim of this in vitro study was to evaluate
the immunomodulatory potential of the FS AminoDefence, reporting its ability to
increase the activity of unstimulated macrophages, as well as to reduce the secretion of

proinflammatory mediators in LPS- or PolyI:C-elicited macrophages, models of bacterial-
and viral-associated inflammation, respectively. To this aim, we used cultured RAW264.7
macrophages, a widely used model to measure immunomodulatory activity of several
nutraceutical formulations (LaLone et al. 2009, Park et al. 2016), being macrophage one
of the main regulators of the IS (Gauthier and Chen 2022). The role of macrophages is
indeed to preside over immune surveillance and tissue homeostasis in basal conditions,
and, following their activation, to trigger a pro-inflammatory response to remove the
cause of tissue damage, eventually recalling other circulating immune cells (Sheu and
Hoffmann 2022). By using this in vitro model, AminoDefence activity was assessed on NO
secretion, due to its critical role in host defence and immune regulation (Swathi Krishna
et al. 2022). On one hand, NO has a protective activity by exerting an antimicrobial
effect; on the other hand, when overproduced, it may lead to tissue damage that may
generate relevant pathogenic effects (Xue et al. 2018). Interestingly, AminoDefence
treatment has shown to have a differential effect on NO release by RAW264.7 culture,
depending on the cellular inflammatory state. In unstimulated macrophages,
AminoDefence was able to promote NO secretion at the highest concentration tested,
with values far below if compared to those obtained in the inflammatory models of LPS
and PolyI:C. On the other side, in the presence of pro-inflammatory stimuli, the
activation by the TLR4 ligands LPS (Bock and Chang 2015) or the synthetic double-strand
RNA PolyI:C (Kim and Park 2016) led to a strong increase in NO release, that was
effectively and dose-dependently dampened by AminoDefence.
Moreover, the activity of AminoDefence to modulate the secretion of the two main pro-
inflammatory cytokines IL-6 and TNF-α was evaluated, due to their involvement in acute
immune response activation, as they trigger T and B cells activation and differentiation
(Arango Duque and Descoteaux 2014). The findings reported a dose-dependent increase
in both cytokines secretion following AminoDefence treatment in unstimulated
macrophages, while a strong and dose-dependent reduction in IL-6 and TNF-α release
was detected after LPS stimulation. Consistently, AminoDefence treatment was able to
promote a mild secretion of chemokine MCP-1 from unstimulated RAW264.7 cells, as
well as to reduce its excessive secretion in LPS-elicited macrophages. Notably, also MCP-
1 exerts a pivotal role in the inflammatory process, being responsible for attracting other
inflammatory cells and enhancing their activation with the release of pro-inflammatory
soluble factors. Moreover, it has been found to be a sensitive biomarker associated with
inflammatory diseases such as cardiovascular diseases, rheumatoid arthritis and viral
infections, including SARS-CoV2 (Singh et al. 2021). Based on the obtained results of this
study, AminoDefence as a whole formulation showed to increase the secretory activity of
unstimulated macrophages and to reduce the inflammatory response of macrophages
when stimulated with LPS.
Besides, this study points to an enhanced effect resulting from the combination of all the
components in the formulation, as a lower or null immunomodulatory effect was
observed when the activity of the single components E. purpurea or quercetin was
compared to that of the whole AminoDefence formulation. This observation may
suggest that the AminoDefence immunomodulatory activity might be the result of either
a synergistic interaction of the individual components of the formulation, or their
cumulative effect. With respect to the specific effects of each tested component, E.
purpurea extract exerted a better immunostimulant activity in resting macrophages,
while quercetin revealed a stronger effect in LPS- or Polyl:C-stimulated macrophages,
confirming its amply described anti-inflammatory activity (LaLone et al. 2009, Cho et al.
2016, Li et al. 2016, Cui et al. 2019, Tang et al. 2019). Moreover, E. purpurea extract
dampened NO release from PolyI:C-elicited RAW264.7 cells, thus suggesting an
important anti-viral activity that makes E. purpurea extract a potential candidate as an
immunomodulatory agent in viral infections prevention and treatment, as previously
reported (Sharma et al. 2006, Nagoor Meeran et al. 2021).
Nevertheless, this study revealed some limitations: the first one is related to the fact that
the in vitro approach does not consider the human metabolism following the intake of
most of the AminoDefence components, including E. purpurea extract and quercetin
(Shabbir et al. 2021, Burlou-Nagy et al. 2022), that accounts for different metabotypes
which can differently affect physiological functions, producing high inter-individual
differences in the biological response. Secondly, IS has an intrinsic complexity, which

hardly allows an exhaustive evaluation through an in vitro model. In order to confirm
these results, a possible evolution might be to perform an in vitro co-culture experiment,
to evaluate interactions between immune cells, and an in vivo study that would allow to
evaluate AminoDefence immunomodulatory activity taking into account digestive and
metabolic processes and bioavailability, as well as the IS complexity. We decided to focus
on macrophages since they play a pivotal role in the cross-road between innate and
adaptive immune response (Kumar 2019) and represent a solid and well recognised
experimental model, as supported by a large amount of studies (LaLone et al. 2009, Cui
et al. 2019, Chen et al. 2021). In addition, the study compared the activity of the whole
AminoDefence formulation with only 2 of its 31 total active components, i.e. quercetin
and E. purpurea extract alone, for which a robust immunomodulatory activity is reported
by the literature (Rauš et al. 2015, De Rosa et al. 2019, Hernáez et al. 2019, Vieira et al.
2022). Finally, it was only possible to evaluate the activity of the soluble fraction of whole
AminoDefence formulation (approximately 95% w/w) in which, of note, quercetin and E.
purpurea extract were completely dissolved. We hypothesize that the negligible insoluble
fraction of AminoDefence, which appeared as a waxy residue with small fibres inclusion,
may be likely related to the presence of Propolis extract from Colla apis, Coenzyme Q10,
D-alpha tocopherol and Magnesium salts of fatty acids (E470b), which solubility has been
reported to be extremely low in the medium used to dissolve the AminoDefence
formulation (Munné-Bosch 2007, Kubiliene et al. 2018, Choi et al. 2019, Zarmpi et al.
2020).
The present study could represent a ‘proof of concept’, since the analysed FS
AminoDefence is composed of a mixture of several components, all of them with a well-
described activity on IS that makes the formulation unique (Fantacone et al. 2020,
Gombart et al. 2020). Indeed, the rationale for this formulation is the complexity of the
inflammatory response, which may be sustained by different substances acting, possibly
synergistically, on the different mechanisms and factors involved in the IS.
In conclusion, the present study points out a significant in vitro immunomodulatory

activity of the FS AminoDefence on both unstimulated and LPS-elicited macrophages.
Further studies, using different models, might better clarify its potential role as a
support for IS normal function.
Authors’ contribution
All the authors contributed to the design of the study, analysis and interpretation of
data.
Experimental data were produced in the laboratories of Department of Food and Drug,
University of Parma, Parma, Italy.
All the authors drafted, revised and finally approved the version to be published.
Disclosure statement
Davide Paleari and Laura Rinaldi are employees of the Medical Department of Chiesi
Italia S.p.A., Parma, Italy.
Additional information
Funding
This publication was funded by Chiesi Italia S.p.A., Parma, Italy.
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5/21/24, 3:05 PM Full article: In vitro evaluation of the immunomodulatory activity of the nutraceutical formulation AminoDefence

In vitro evaluation of the immunomodulatory

 Cite this article  https://doi.org/10.1080/09637486.2023.2283688

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Immune system (IS) functionality is warranted by inter-dependent processes that

Echinacea purpurea NO cytokines

and Calderón 2022). Indeed, E. purpurea extract exerted an immunostimulatory activity

This study aimed to investigate the immunomodulatory effect of AminoDefence, a FS

Materials and methods

Cell culture and treatments

Table 1. Nutraceutical formulation.

Download CSV Display Table

Measurement of cytokine secretion

way analysis of variance (ANOVA), followed by the post-hoc Dunnett’s multiple

Nitric oxide (NO) release by cultured macrophages

First, the potential modulatory effect of the whole nutraceutical formulation

Figure 1. Effect of the whole formulation AminoDefence, quercetin and E. purpurea

Display full size

Cytokine and chemokine secretion by cultured macrophages

Figure 2. Effect of AminoDefence, quercetin and E. purpurea extract on IL-6 release by

Display full size

Similarly, the formulation AminoDefence treatment significantly stimulated in a dose-

Figure 3. Effect of AminoDefence, quercetin and E. purpurea extract on TNF-α release by

Display full size

Also, the chemokine monocyte chemoattractant protein-1 (MCP-1) release from

Figure 4. Effect of the whole formulation AminoDefence, quercetin and E. purpurea

Display full size

Nitric Oxide (NO) release

Next, the potential anti-inflammatory effect of the nutraceutical formulation

Figure 5. Effect of AminoDefence, quercetin and E. purpurea extract on NO release by

Display full size

Cytokine and chemokine secretion

15 μg/ml, corresponding to that present in [0.5 mg/ml] of formulation, reaching an

Figure 6. Effect of the whole formulation AminoDefence, quercetin and E. purpurea

Display full size

Consistently to what observed for IL-6, the nutraceutical formulation AminoDefence

Figure 7. Effect of the whole formulation AminoDefence, quercetin, and E. purpurea

concentrations of the formulation AminoDefence (A), quercetin (B) or E. purpurea extract

Display full size

Figure 8. Effect of the whole formulation AminoDefence, quercetin, and E. purpurea

Display full size

Nitric Oxide (NO) release

In these experimental conditions, PolyI:C stimulated NO production by macrophages by

Figure 9. Effect of AminoDefence, quercetin and E. purpurea extract on NO release by

Display full size

increase the activity of unstimulated macrophages, as well as to reduce the secretion of

differences in the biological response. Secondly, IS has an intrinsic complexity, which

In conclusion, the present study points out a significant in vitro immunomodulatory

This publication was funded by Chiesi Italia S.p.A., Parma, Italy.

1. Addey D, Shephard A. 2012. Incidence, causes, severity and treatment of throat

2. Aki T, Funakoshi T, Noritake K, Unuma K, Uemura K. 2020. Extracellular glucose is

3. Alba G, Dakhaoui H, Santa-Maria C, Palomares F, Cejudo-Guillen M, Geniz I, Sobrino F,

4. Arango Duque G, Descoteaux A. 2014. Macrophage cytokines: involvement in

8. Chen C, Xie X, Li X. 2021. Immunomodulatory effects of four polysaccharides purified

immune system. JAMA Netw Open. 5(8):e2226040–e2226040. doi:

14. Cui S, Wu Q, Wang J, Li M, Qian J, Li S. 2019. Quercetin inhibits LPS-induced

15. Djaoudene O, Romano A, Bradai YD, Zebiri F, Ouchene A, Yousfi Y, Amrane-Abider M,

16. Dobrange E, Peshev D, Loedolff B, Van den Ende W. 2019. Fructans as

19. Fu A, Wang Y, Wu Y, Chen H, Zheng S, Li Y, Xu X, Li W. 2017. Echinacea purpurea

20. Gauthier T, Chen W. 2022. Modulation of macrophage immunometabolism: a new

22. Hernáez Á, Sanllorente A, Castañer O, Martínez-González MÁ, Ros E, Pintó X, Estruch R,

27. Kubiliene L, Jekabsone A, Zilius M, Trumbeckaite S, Simanaviciute D, Gerbutaviciene R,