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. 2023 Apr 20;15(4):1292.
doi: 10.3390/pharmaceutics15041292.

In Vivo Acute Toxicity and Immunomodulation Assessment of a Novel Nutraceutical in Mice

Tatiana Onisei  1 , Bianca-Maria Tihăuan  1   2   3 , Georgiana Dolete  4   5 , Mădălina Axinie Bucos  3   4 , Manuela Răscol  1 , Gheorghița Isvoranu  6
Affiliations

In Vivo Acute Toxicity and Immunomodulation Assessment of a Novel Nutraceutical in Mice

Tatiana Onisei et al. Pharmaceutics. .

Abstract

Achieving and maintaining a well-balanced immune system has righteously become an insightful task for the general population and an even more fundamental goal for those affected by immune-related diseases. Since our immune functions are indispensable in defending the body against pathogens, diseases and other external attacks, while playing a vital role in maintaining health and modulating the immune response, we require an on-point grasp of their shortcoming as a foundation for the development of functional foods and novel nutraceuticals. Seeing that immunoceuticals are considered effective in improving immune functions and reducing the incidence of immunological disorders, the main focus of this study was to assess the immunomodulatory properties and possible acute toxicity of a novel nutraceutical with active substances of natural origin on C57BL/6 mice for 21 days. We evaluated the potential hazards (microbial contamination and heavy metals) of the novel nutraceutical and addressed the acute toxicity according to OECD guidelines of a 2000 mg/kg dose on mice for 21 days. The immunomodulatory effect was assessed at three concentrations (50 mg/kg, 100 mg/kg and 200 mg/kg) by determining body and organ indexes through a leukocyte analysis; flow cytometry immunophenotyping of lymphocytes populations and their subpopulations (T lymphocytes (LyCD3+), cytotoxic suppressor T lymphocytes (CD3+CD8+), helper T lymphocytes (CD3+CD4+), B lymphocytes (CD3-CD19+) and NK cells (CD3-NK1.1.+); and the expression of the CD69 activation marker. The results obtained for the novel nutraceutical referred to as ImunoBoost indicated no acute toxicity, an increased number of lymphocytes and the stimulation of lymphocyte activation and proliferation, demonstrating its immunomodulatory effect. The safe human consumption dose was established at 30 mg/day.

Keywords: Echinacea purpurea; Vaccinium myrtillus; acute toxicity; animal studies; hydrolyzed collagen; immunomodulation; nutraceuticals; royal jelly.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
FSC/SSC dot-plot with isolated cells of interest (P1).
Figure 2
Figure 2
Unlabeled cell suspension (no specific fluorescence signals)—blue laser stimulation.
Figure 3
Figure 3
Unlabeled cell suspension (no specific fluorescence signals)—red laser stimulation.
Figure 4
Figure 4
Labeled cell suspension—blue laser stimulation.
Figure 5
Figure 5
Labeled cell suspension—red laser stimulation.
Figure 6
Figure 6
(a) Selection of singlet events. In an FSC-H/SSC-A dot-plot, the Singleti gate was constructed in which siglet events were included. The introduction of cellular aggregates into the analysis was avoided. (b) Selection of the lymphocyte population. From the gate containing the singlet events, the Ly gate (FSC-A/SSC-A) was constructed in which lymphocytes were isolated. (c) Selection of CD3ε+ lymphocytes. Using a histogram (CD3ε/Count), the population of CD3ε+ lymphocytes (total T lymphocytes) was isolated. The CD3ε- lymphocytes were virtually isolated using the ”invert gate” function. (d) Selection of Th and Ts lymphocytes. From the T lymphocytes (CD3ε+), using a CD4/CD8a dot-plot with a quadrant, we isolated the Th (helper) lymphocyte subpopulations with the phenotype CD3ɛ+CD4+CD8a− and Ts subpopulations (suppressor/cytotoxic) with the phenotype CD3ɛ+CD8a+CD4−. (e) Selection of B lymphocytes and NK cells. From CD3ε-negative lymphocytes, B lymphocytes (phenotype CD3ɛ−CD19+NK1.1−) and NK cells (phenotype CD3ɛ−CD19−NK1.1+) were isolated.
Figure 7
Figure 7
(a) Selection of singlet events. (b) Selection of the lymphocyte population. (c) Selection of lymphocytes of CD3ε+. (d) Selection of lymphocytes of CD8a+. (e) Selection of B lymphocytes. (f) Selection of NK cells. (g) Selection of lymphocytes of CD69+. (h) Selection of B lymphocytes and NK cells.
Figure 7
Figure 7
(a) Selection of singlet events. (b) Selection of the lymphocyte population. (c) Selection of lymphocytes of CD3ε+. (d) Selection of lymphocytes of CD8a+. (e) Selection of B lymphocytes. (f) Selection of NK cells. (g) Selection of lymphocytes of CD69+. (h) Selection of B lymphocytes and NK cells.
Figure 8
Figure 8
Body weight assessment—acute toxicity testing.
Figure 9
Figure 9
Assessment of leukocyte numbers in acute toxicity testing for the total numbers of leukocytes (WBC), lymphocytes (LY), neutrophils (NE) and monocytes (MO) in mice; * = p value < 0.05.
Figure 10
Figure 10
Body weight assessment of animals that received the product for 21 days.
Figure 11
Figure 11
Evaluation of the number of leukocytes in the groups that received the novel nutraceutical compared to the control group: total numbers of leukocytes (WBC), lymphocytes (LY) and neutrophils (NE) in mice.
Figure 12
Figure 12
CD69 expression on lymphocytes in the groups that received ImunoBoost compared to the control group—stimulation with LPS; * = p value < 0.05.
Figure 13
Figure 13
CD69 expression on lymphocytes in the groups that received ImunoBoost compared to the control group—stimulation with conA; * = p value < 0.05.
Figure 14
Figure 14
Lymphocyte proliferation capacity in groups that received the novel nutraceutical compared to the control group—stimulation with LPS; * = p value < 0.05.
Figure 15
Figure 15
Lymphocyte proliferation capacity levels in the groups that received the novel nutraceutical compared to the control group—stimulation with conA; * = p value < 0.05.
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