In vitro evaluation of the immunomodulatory activity of the nutraceutical formulation AminoDefence

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 unstimulated macrophages, the formulation exerted a dose-dependent 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.

COMPONENT	mg/capsule	TITRATION	% component (weight)
L-glutamine	65	100%	10.1%
Echinacea purpurea (hydroalcoholic extract, aerial parts; ratio 5:1)	65	4% polyphenol	10.1%
Whey protein isolate	60	90% proteins	9.3%
L-lysine hydrochloride	40	80% L-lysine	6.2%
L-ascorbic acid	35	100%	5.4%
Origanum vulgare leaves (aqueous extract, ratio 4:1)	30	–	4.7%
L-proline	30	100%	4.7%
Inulin (dried extract) from Cichorium intybus	30	>90% inuline	4.7%
Propolis extract from Colla apis (alcoholic extract)	20	10% polyphenols 2% flavonoids	3.1%
Eleutherococcus senticosus Maxim (hydroalcoholic extract, root; ratio 12:1)	20	0,8% eleutherosides B and E	3.1%
Panax ginseng (hydroalcoholic extract, root; ratio 7-9/1)	20	5% ginsenosides	3.1%
Lentinus edodes (hydroalcoholic extract, fruits; ratio 4–5:1)	20	15% polysaccharides	3.1%
Arthrospira platensis (Spirulina algae) (whole plant powder)	20	–	3.1%
Camellia sinensis (aqueous extract, leaf; ratio 8–10:1)	20	10–25% catechins 4–6% caffeine 30% polyphenols	3.1%
Curcuma longa (hydroalcholic extract, rhizome; ratio 6-7:1)	20	30% curcuminoids	3.1%
Quercetin di-hydrate	18	>95% quercetin	2.8%
Vitis vinifera (hydroalcoholic extract, grape skin; ratio 450:1)	15	5% resveratrol	2.3%
Euterpe oleracea (aqueous extract; fruit; ratio 4:1)	15	–	2.3%
Lycium barbarum fruit (aqueous extract, fruit; ratio 1–2:1)	15	25% vitamina C	2.3%
Eldberry Sambucus nigra fruit (aqueous extract, fruit; ratio 10–15:1)	15	6% anthocyanines	2.3%
Allium sativum (hydroalcoholic extract, bulb; ratio 100:1)	15	3% allicin	2.3%
Zinc bisglycinate	13	20% zinc	2.0%
Coenzyme Q10	10	> 98%	1.6%
D-alpha tocopherol	10	> 67.2%	1.6%
Magnesium salts of fatty acids (E470b)	8	–	1.2%
Alpha lipoic acid	5	100%	0.8%
Manganese bisglycinate	3.28	16% manganese	0.5%
Copper bisglycinate	2.65	10% copper	0.4%
L-selenomethionine (0.5% Selenium)	2.5	0.5% selenium	0.4%
Piper nigrum (hydroalcholic extract, fruit; ratio 8–12:1)	0.5	95% piperine	0.1%
Vitamin D	0.5	0.25% cholecalciferol	0.1%
Vitamin B6	0.47	99% pyridoxine HCl	0.1%
Total weight in capsule	643,90		100.0%

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.

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.

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.

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).

Figure 4. Effect of the whole formulation AminoDefence, quercetin and E. purpurea 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 Dunnett’s multiple comparison test. A value of p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ****p < 0.0001 vs basal.

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.

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).

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

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 concentrations of the formulation AminoDefence (A), quercetin (B) or E. purpurea extract (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. 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 < 0.01; ***p < 0.001; ****p < 0.0001 vs LPS-treated macrophages; °°°°p < 0.0001 vs macrophages in basal conditions.

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 concentrations of the formulation AminoDefence (A), quercetin (B) or E. purpurea extract (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. 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 < 0.01; ***p < 0.001; ****p < 0.0001 vs LPS-treated macrophages; °°°°p < 0.0001 vs macrophages in basal conditions.

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 Dunnett’s multiple comparison test. A value of p < 0.05 was considered statistically significant. ***p < 0.001; ****p < 0.0001 vs PolyI:C-treated macrophages. °°°°p < 0.0001 vs macrophages in basal conditions.

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.