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

Figures & data

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%

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.

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.

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.

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.

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.

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.

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.

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.

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.