We interrupt 70%* of the signal.

The objective of the trial is to determine, through an adipogenesis screening assessment, whether the combination of berberine, catechins, and stachyose can provide statistically significant experimental data compared to the combination of berberine + catechins and berberine + stachyose. This evaluation is performed through statistical analysis aimed at demonstrating that the differences observed among samples are significant and not random.

The adipogenesis process has been widely studied to identify compounds capable of acting on fatty acid formation in adipocytes (1). Several scientific publications analyze the activity of raw materials or active molecules through in vitro studies using murine pre-adipocytes induced to differentiate, evaluating various aspects related to adipogenesis. The initial screening is based on quantifying lipids accumulating in differentiated cells through staining of lipid droplets (2,3). In a second phase, specific markers of adipogenesis and lipogenesis (C/EBPα, PPARγ) are analyzed via gene expression assessment (2,3).

Obesity is considered a chronic disease predisposing individuals to metabolic disorders such as diabetes, cardiovascular disease, and fatty liver disease (4). Differentiation from pre-adipocytes to adipocytes is a key contributor to increased fat mass during adolescence and adulthood. Adipogenesis and adipocyte apoptosis dynamically balance fat mass; approximately 10% of all adipocytes are renewed annually (5). For these reasons, adipogenesis is considered a key target for preventing or treating obesity (6).

This process occurs through multistep gene regulation primarily driven by nuclear hormone receptors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBP) α/β/δ (7). The C/EBPβ–C/EBPδ complex initiates adipogenesis by expressing PPARγ and C/EBPα (8). Later, C/EBPα dimerizes with C/EBPβ or C/EBPδ, guiding the advanced phase of adipogenesis (7,9). PPARγ, induced during the intermediate phase, leads to adipocyte maturation (10). These markers remain expressed in mature adipocytes, promoting lipid accumulation and adipokine secretion (11).

Considering their key role, evaluating their expression is essential to determine the efficacy of compounds acting on fatty acid metabolism and lipolysis.

3.1. Cell line and product solubilization

The 3T3-L1 preadipocyte cell line is one of the main cell lines used to study adipocyte differentiation in vitro, a differentiation process that requires the use of specific culture media. 3T3- L1 cells (3T3-L1 preadipocytes, SP-L1-F Zen-Bio) were grown under completely sterile conditions and maintained in an incubator at 37°C with 5% carbon dioxide (CO2). The list of culture media and reagents used is shown in Table 3.1.:

The products to be tested were dissolved in 250 ml of Dulbecco’s Modified Eagle Medium (DMEM) according to the concentrations indicated by the client.

3.2. Coloring with Oil Red O

Pre-adipocytes were seeded in 12-well plates until confluence was reached and then the differentiation protocol was performed. Cells were treated with the concentrations of product found to be non-cytotoxic by the previous cell viability test and with the positive control (Catechin). A control of undifferentiated cells and a control of differentiated and untreated cells were maintained. At the end of the treatment, the cells were fixed in formalin, washed, and stained with Oil Red O solution (Merck) for 20 min. The lipid droplets were visualized under a microscope, and lipid accumulation was quantified following extraction with isopropanol for 5 min and spectrophotometric reading at a wavelength of 492 nm

3.3. Real-Time Gene Expression Assessment

Gene expression of the PPARg marker, involved in the early stages of adipogenesis, the regulation of insulin sensitivity, and lipogenesis, was studied using qRT-PCR on 3T3-L1 cells. Real-time PCR is a technique that allows for the assessment of changes in gene expression of a specific target by extracting total RNA, reverse transcribing it into cDNA, and subsequently amplifying and quantifying it using fluorescent probes that bind to newly synthesized DNA, allowing for quantification of the amplified product at each reaction cycle. The emitted fluorescence is directly proportional to the amount of amplified product of interest. For this test, 3T3-L1 cells were seeded homogeneously in 12-well plates according to the differentiation protocol. Finally, the treatment was performed for 96 hours, changing the medium every 48 hours. Subsequently, the treatment was removed and the cells were maintained in maintenance medium for an additional 24 hours. To assess gene expression, total cellular RNA molecules were extracted from cells with the RNA isolation kit (Qiagen) according to the manufacturer's protocol. Subsequently, the concentration of extracted total RNA was quantified using MultiSkanGo (Thermo Fisher), and total RNA was reverse transcribed into cDNA using the cDNA synthesis kit (Bio-Rad). Reaction conditions consisted of denaturation of double-stranded cDNA, primer annealing, and extension of the newly synthesized strand; the last two steps were repeated for 39 consecutive cycles. RNA expression levels were normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression levels.The instrument used for the analysis is CFX (Bio-Rad) and the data were analyzed using the CFX Manager software.

The results obtained are reported in graphic form and in tables containing the values relating to cell viability and gene expression of markers involved in the phases of adipocyte differentiation, following treatment with the products to be tested compared to untreated cells. Statistical analysis was performed with the ONE WAY-ANOVA method followed by the Bonferroni test for multiple comparisons using GraphPad Prism version 10.5.0 software (GraphPad Software, Inc).

4.1. Cell viability assessment

3T3-L1 cells were treated (24h) with different concentrations of the selected products in order to identify those that did not cause a reduction in cellular respiration (intended as viability) greater than 30%.

Figure 4.1. and Table 4.1. show the graphs and the corresponding data, expressed as a percentage with respect to the control (untreated cells), of cell viability after 24 hours of treatment.

Figure 4.1. Analysis of cell viability expressed as a percentage compared to the untreated control (0) after 24 h of treatment with the tested products starting from the 100% stock and subsequent dilutions.

After 24 hours of treatment with the active ingredient blend, BOOSTER UP no green tea STACHYOSE BERBERINE shows a greater reduction in cell viability starting from a concentration of 6.25% compared to the other two products analyzed. The summary table of results shows that BOOSTER UP STACHYOSE BERBERINE GREEN TEA and EXXL FAST SLIM BERBERINE GREEN TEA are cytotoxic, leading to a reduction in cell viability of approximately 20% starting from higher concentrations, more precisely 75% for BOOSTER UP STACHYOSE BERBERINE GREEN TEA and 50% for EXXL FAST SLIM BERBERINE GREEN TEA. It can also be noted that up to a 100% concentration, the BOOSTER UP STACHYOSE BERBERINE GREEN TEA product shows stable cell viability at approximately 70%, unlike the other two products, which show a dose- dependent reduction in viability. Based on these considerations, it was decided to treat the cells for the fatty acid staining test starting from a 12.5% concentration.

4.2. Evaluation of lipid content

In order to verify the effect on the adipogenesis process, the pre-adipocytes were induced to differentiate and subsequently treated with the different products and with the positive control (Catechin), a molecule with a known activity in reducing lipid accumulation (12). The lipid content was subsequently labelled with Oil Red solution (Merck).

Figure 4.2 shows representative images of differentiated adipocytes treated with the products to be tested; a decrease in lipid content is visually noticeable in the samples treated with the positive control compared to the untreated control. The correct differentiation of pre-adipocytes into adipocytes is also evident, as demonstrated by the presence of lipid droplets in the differentiated sample compared to the pre-adipocyte sample. Quantification by spectrophotometric reading shows that no statistically significant variations in the lipid content within the cells are visible following 24 hours of treatment with the products to be tested, maintaining values comparable to the control.

Figure 4.2. Representative images of the presence of lipid droplets, indicative of adipocyte differentiation, following 24 h of treatment. Staining of the lipid content using Oil Red solution.

Table 4.3. Quantification of lipid content after 24 hours of treatment with the tested products and the positive control. Values with **** p ≤ 0.001, with *** p ≤ 0.01 are considered statistically significant compared to the CTRL

4.3. Gene expression assessment by RT-PCR

The possible ability to modulate the gene expression of one of the markers involved in the process of adipogenesis, lipogenesis and glucose metabolism was analyzed using the RT-PCR (Real-Time PCR) technique. Several transcription factors, such as those belonging to the C/EBP and PPAR families, are expressed during the process of adipocyte differentiation and lipolysis; it was therefore evaluated whether treatment with the different products to be tested could influence the expression of the PPARg marker, and therefore of fatty acid metabolism and lipolysis. Differentiated adipocytes were treated for 96 hours, changing the treatment after 48 hours. Subsequently, the samples were processed for gene expression analysis and the values reported in Figure 4.4. highlight how treatment with the products

BOOSTER UP STACHYOSE BERBERINE GREEN TEA and EXXL FAST SLIM BERBERINE GREEN TEA leads to a clear decrease in PPARg expression, compared not only with the untreated control but also with the product without green tea. In fact, comparing the same concentrations of the two products with the P1 product, it is observed that expression decreases by approximately 30% for all the conditions tested and that this decrease is statistically significant. As shown, PPARg increases in differentiated adipocytes (Ctrl) compared to the pre-adipocyte sample (C non diff.), confirming its role in the early stages of adipogenesis; furthermore, the treatment with the positive control results to be highly significant with a marker expression of about 50%, confirming the correct execution of the differentiation process.

Figure 4.4. Quantitative analysis of the PPARg marker after treatment with the products under examination. CTRL: differentiated and untreated cells; C+ Magnolol: differentiated cells treated with the positive control Magnolol; P1: differentiated cells treated with BOOSTER UP no green tea STACHYOSE BERBERINE; P2: differentiated cells treated with BOOSTER UP STACHYOSE BERBERINE GREEN TEA; P3: differentiated cells treated with EXXL FAST SLIM BERBERINE GREEN TEA. (n=3; replicates=2).

Figure 4.5. Quantitative analysis of the PPARg marker after treatment with the products under examination. CTRL: differentiated and untreated cells; P2: differentiated cells treated with BOOSTER UP STACHYOSE BERBERINE GREEN TEA; P3: differentiated cells treated with EXXL FAST SLIM BERBERINE GREEN TEA. (n=3; replicates=2).

Figure 4.5. shows the results comparing the effect of the treatment with BOOSTER UP STACHYOSE BERBERINE GREEN TEA with the sample treated with EXXL FAST SLIM BERBERINE GREEN TEA; it is clear that the first product is more effective in reducing the expression of the adipogenesis marker at the lowest concentration with an expression equal to 10%.

From the results reported here obtained through in vitro tests it can be concluded that, under the experimental conditions we tested,

THE PRODUCTS BOOSTER UP no green tea STACHYOSE BERBERINE, BOOSTER UP STACHYOSE BERBERINE GREEN TEA, EXXL FAST SLIM BERBERINE GREEN TEA (PROLIVY S.R.L.)

REDUCE THE EXPRESSION OF THE ADIPOGENESIS MARKER PPARg AFTER 96H OF TREATMENT

In particular, BOOSTER UP STACHYOSE BERBERINE GREEN TEA and EXXL FAST SLIM BERBERINE GREEN TEA show a greater efficacy in reducing the gene expression of PPARg equal to approximately 30% compared to the product without green tea.

Cura Carpignano, June 18, 2025

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