**1. Introduction**

Obesity is associated with excessive adipose tissue growth, which occurs through two possible mechanisms: hypertrophy (expansion of existing adipocytes) and hyperplasia (recruitment of new adipocytes). Hypertrophic adipose tissue growth is mainly considered to be related to insulin resistance and other obesity metabolic comorbidities [1]. Abnormal expansion of adipose tissue is accompanied by local hypoxia, adipocyte death, enhanced cytokine and chemokine secretion, dysfunctional fatty acid metabolism and accumulation, and immune cell infiltration. Dysregulation of lipid metabolism in adipose tissue leads to enhanced release of free fatty acids, which initiates inflammatory signaling cascades in the infiltrating cell population. Chronic low-grade inflammation, found in abnormal fat tissue, negatively affects the insulin signal transduction pathway, and promotes insulin resistance [2,3].

Recent scientific preclinical studies have shown that bioactive dietary compounds may specifically influence hypertrophic adipose cells and mitigate the effects of extensive adipose tissue growth

by affecting various adverse phenomena, including oxidative stress, inflammation, disturbances in adipokine secretion, fatty acid release, and others. Berry fruits have been recognized as capable of counteracting obesity and obesity-related metabolic disorders, through the inhibition of adipocyte differentiation, a decrease in lipogenesis, an increase in lipolysis, or mitigation of inflammatory and insulin resistance status [4].

A promising candidate capable of attenuating obesity and complications related to excessive fat tissue growth might be *Sambucus nigra* L. (European elderberry) fruit as a valuable source of polyphenolic compounds, primarily flavonols, flavanols, phenolic acids, proanthocyanidins, and anthocyanins [5]. The unique polyphenol composition is responsible for the high biological potential of elderberry fruit (EDB), including antiviral and antimicrobial activity, as well as chemopreventive, neuroprotective, and anti-inflammatory effects that have been documented in several scientific reports [6–10]. Also, it has been suggested that EDB may be an effective remedy for diabetes, obesity, and metabolic dysfunctions [9]. Animal studies have shown the ability of *Sambucus nigra* preparations to improve glucose and lipid metabolism and diabetic osteoporosis status [11–14].

Anthocyanin-rich EDB extract has been proved to attenuate systemic inflammation and insulin resistance in high-fat diet-induced obese mice. Pro-inflammatory markers of low-grade chronic inflammation, including serum monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), were significantly reduced in EDB-fed mice. Also, the high-fat diet supplemented with EDB extract mitigated some metabolic disturbances by lowering serum triglycerides and improving insulin sensitivity [12]. Lowered insulin resistance was found in diabetic rats fed with a high-fat diet supplemented with EDB extracts rich in triterpenic acids or polyphenol compounds. The extracts modulated glucose metabolism by correcting hyperglycemia or reducing insulin secretion, respectively [13]. The anthocyanin-rich EDB extract protected against inflammation-related impairments in high-density lipoprotein (HDL) function in a mouse model of hyperlipidemia and HDL dysfunction. The decrease in total cholesterol content of the aorta in EDB-fed mice suggested limiting atherosclerosis progression [14]. Scientific reports indicate that EDB extracts possess the unique potential to modulate the immune response depending on the immune stimuli and inflammatory disorders. The EDB bioactives have evoked different immune effects by controlling pro- and anti-inflammatory cytokines and mediators (Reactive oxygen species, NO, IL-6, TNF-<sup>α</sup>, MCP-1, IL-1, IL-8, IL-10, PGE2, COX-2, iNOS, INF-γ), that play a crucial role in acute and chronic low-grade inflammatory diseases associated with obesity, diabetes, dyslipidemia, cardiovascular disturbances, and neurodegenerative diseases [7,8,10–16].

Over the last decade, significant advances in knowledge about the health-beneficial potential of EDB fruit have been achieved through extensive preclinical studies. However, the results obtained only in the few clinical trials have not enabled to express an unambiguous opinion and, so far, have not provided strong evidence of the therapeutic effects of *Sambucus nigra* fruit in obesity and metabolic disorders [9]. Recently, the scientific community has stated the need for further research on the health-promoting properties of this valuable plant as a natural constituent of food products and beneficial component of a healthy diet [6,9].

This study aimed to evaluate the capacity of *Sambucus nigra* fruit extract to mitigate obesity-related metabolic complications through the carbohydrate and lipid metabolism regulation, glucose uptake improvement, and insulin sensitivity controlling. Also, the goal of the study was the assessment of the ability of the extract to alleviate the inflammatory response in activated macrophages, which are recruited into excessively growing fat tissue and may be a primary source of locally produced pro-inflammatory mediators.

#### **2. Materials and Methods**

#### *2.1. Preparation of Elderberry Fruit Extract*

The fruits of elderberry (*Sambucus nigra* L.) cultivar Sampo, obtained from Bio Berry Poland (Warsaw, Poland), were homogenized to fruit pulp, which was subsequently frozen at −80 ◦C and subjected to freeze-drying at a vacuum pressure of 0.1 mbar and temperature of 20 ◦C for 23 h and post-drying at 23 ◦C for 3 h using a freeze dryer (LMC-1, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). The lyophilized EDB were finely ground and packaged under nitrogen atmosphere. The EDB extract was obtained by dissolving the EDB powder in complete culture medium with the pH adjustment to 7.4. The EDB suspension was then centrifuged (3000 g, 5 min) and filtered through a 0.22 μm membrane (Merck, Germany).

#### *2.2. Determination of Individual Phenolic Compounds Using HPLC-DAD-MSn Analysis*

Analyses of phenolic compounds were performed on an Agilent 1200 series HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) that was equipped with a G1315D photodiode array detector and coupled online with an Agilent 6224 time-of-flight MS system. Phenolic compounds were identified using a mass spectrometer fitted with an electrospray ionization (ESI) source that was operated in positive-ion or negative-ion mode. Analyses were carried out using full MS scan mode, and full mass spectra were recorded in the range of 100 to 1700 *<sup>m</sup>*/*<sup>z</sup>*. Technical specification of apparatus and major HPLC/MS parameters and analysis conditions were described in detail in our previous work [17].

For quantification purposes, all anthocyanins conjugates were expressed as cyanidin-3-glucoside equivalents; all flavan-3-ols and their polymers as catechin equivalents; hydroxybenzoic acid glucoside and hydrolysable tannins as gallic acid equivalents; phenolic acids derivatives as chlorogenic acid equivalents; and flavonol glycosides as quercetin equivalents.

#### *2.3. T3-L1 Cell Culture, Di*ff*erentiation, and Treatment*

The mouse embryo 3T3-L1 cell line was purchased from the American Type Culture Collection (ATCC, CL-173). The 3T3-L1 preadipocytes were grown, passaged, and di fferentiated into adipocytes as described previously [18]. The 3T3-L1 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum supplementation (Sigma-Aldrich, Merck Group, Darmstadt, Germany). Cell di fferentiation was induced in post-confluent cell cultures by a di fferentiation mixture consisting of 1 μM insulin, 0.25 μM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) in DMEM with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Polska, Warsaw, Poland).

Fully di fferentiated 3T3-L1 cells were exposed to the EDB extract at concentrations of 5, 10, and 20 mg/mL for 24 h. The levels of intracellular ROS generation and lipid accumulation in mature adipocytes were determined. Also, the viability and metabolic activity of the mature adipocytes were analyzed after the treatment.

After completion of the di fferentiation process, insulin resistance was induced in 3T3-L1 adipocytes by 10 ng/mL murine TNFα (Sigma-Aldrich) for 5 days, with medium/TNFα replacement every 2 days. Glucose uptake measurement was performed in insulin-resistant and insulin-sensitive adipocytes subjected to the EDB treatment.

#### *2.4. Macrophage Cell Culture and Anti-Inflammatory Experiment Procedure*

RAW 264.7 murine macrophage line was obtained from the European Collection of Authenticated Cell Cultures (ECACC, 91062702) and supplied by Sigma-Aldrich. Cells were grown in DMEM supplemented with 10% heat-inactivated FBS at 37 ◦C in a humidified, 5% CO2, 95% air atmosphere. The 24-h cultures of RAW 264.7 macrophages, seeded at a density of 5 × 10<sup>5</sup> cells/cm2, were treated with EDB extract prepared in DMEM at the concentrations of 0.1, 1, and 10 μg/mL and incubated for 2 h in standard culture conditions. Controls were treated with DMEM only. Subsequently, macrophages were stimulated with 5 ng/mL of lipopolysaccharide (LPS) from *Escherichia coli* O-127 (Sigma-Aldrich). After 3-h macrophage activation, the culture media and cells were harvested to analyze the protein secretion and gene expression of pro-inflammatory mediators.

#### *2.5. Cell Viability Assay*

The viability and metabolic activity of di fferentiated 3T3-L1 adipocytes and LPS-stimulated RAW 264.7 macrophages were analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test (Sigma–Aldrich) following the protocol described previously [8].

#### *2.6. Measurement of Reactive Oxygen Species in Adipocytes*

The intracellular ROS generation was determined using nitro blue tetrazolium (NBT) according to the procedure described by Choi et al. [19]. The cells were incubated in 0.2% NBT solution for 90 min, washed with phosphate-bu ffered saline (PBS), fixed with methanol, and then air-dried. The formazan extraction was performed using KOH and DMSO for dissolving. The absorbance was measured at 620 nm using a Tecan M200 Infinite microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

#### *2.7. Measurement of Intracellular Triglyceride Content in Adipocytes*

Total concentrations of triglycerides (TG) in di fferentiated 3T3-L1 adipocytes were determined using Adipogenesis Assay Kit (Sigma-Aldrich) according to the manufacturer's protocol. Intracellular TG content was measured by a coupled enzyme assay, which resulted in a fluorometric product detected at λex = 535 nm and λem = 587 nm (Tecan M200 Infinite), which was proportional to the TG present. The TG concentration was calculated based on the curve plotted for TG standards.

#### *2.8. Glucose Uptake Measurement in Adipocytes*

Glucose uptake assay was performed according to the modified method of Alonso-Castro and Salazar-Olivo [20]. Mature 3T3-L1 adipocytes, cultured on 24-well plates for fluorescence-based assays, were starved in serum-free medium (MEM containing BSA 0.5%) overnight. Subsequently, the medium was replaced with Krebs Ringer phosphate HEPES (KRPH) bu ffer containing 0.2% BSA (KRPH/BSA) and incubated for 60 min. The cells were then exposed for 60 min to EDB extract suspended in KRPH/BSA bu ffer supplemented with 80 μM 2-NBDG (2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxy-d-glucose) (Sigma-Aldrich) used as fluorescent glucose analogue. The control cultures were treated with 100 nM insulin or 10 μM rosiglitazone (Sigma-Aldrich). After incubation, cultures were immediately washed three times with ice-cold PBS. The fluorescence intensity of 2-NBDG was measured at λex = 485 nm and λem = 535 nm (Tecan M200 Infinite).

#### *2.9. Determination of Adipokine Production in 3T3-L1 Adipocytes*

The leptin and adiponectin concentrations were measured using ELISA kits (Sigma-Aldrich, Merck Group) following the manufacturer's instructions. The adipokine concentrations were expressed in ng/mL of culture medium, which was equivalent to the amount of protein per 1 × 10<sup>6</sup> cells.

#### *2.10. Determination of IL-6, TNF-*<sup>α</sup>*, and PGE2 Production in RAW 264.7 Macrophages*

The secretion of IL-6 and TNFα cytokines as well as generation of PGE2 by LPS-stimulated RAW 264.7 macrophages were determined with ELISA kits (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer's instructions. Protein concentrations were expressed in pg/mL of culture supernatant, which was equivalent to the amount of protein per 1 × 10<sup>6</sup> cells.

#### *2.11. Determination of NO Production in RAW 264.7 Macrophages*

Griess method was applied to determine nitrite as an indicator of NO production. Equal volumes of the Griess reagen<sup>t</sup> (Sigma-Aldrich) and RAW 264.7 culture supernatant were mixed and incubated at room temperature for 15 min. The absorbance was measured at 540 nm (Tecan M200 Infinite). The standard curve plotted for sodium nitrite was used to calculate NO concentration.

#### *2.12. Quantification of Gene Expression Using Real-Time PCR*

The analysis of gene expression was carried out in accordance with the detailed protocol presented in the previous work [17]. The TRI reagen<sup>t</sup> (Sigma-Aldrich) was used to isolate total RNA, Synthesis cDNA Transcriptor First-Strand kit (Roche Diagnostics GmbH, Mannheim, Germany) for first-strand cDNA synthesis, and SYBR1 Select Master Mix (Life Technologies, Carlsbad, CA, USA) for real-time PCR. The primers used for the amplification of cDNAs are listed in Table 1.


**Table 1.** The primers sequence used for real-time PCR.

The relative expression of each gene was calculated using the 2−ΔΔCT method. The mRNA levels in the control cells were designated as 1, and the relative levels of the gene transcripts in the samples were expressed as the fold change.

#### *2.13. Digestive Enzyme Inhibition Assays*

#### 2.13.1. Measurement of Pancreatic Lipase Inhibition

The EDB inhibitory activity against pancreatic lipase (EC 3.1.1.3) was evaluated according to the method of Boath et al. with minor modification [21]. The p-nitrophenyl laurate (pNP laurate) was used as a substrate. The pNP laurate was dissolved to 0.08% in 5 mM sodium acetate (pH 5.2) containing 1% Triton X-100 and 0.05% Arabic gum. The reaction mixture consisting of 350 μL of assay buffer (100 mM Tris, pH 8.2), 50 μL of EDB extract, 150 μL of pancreatic lipase type II from porcine pancreas (10 mg/mL), and 450 μL of substrate solution was incubated at 37 ◦C for 2 h. Orlistat, a known porcine pancreatic lipase inhibitor, was applied as a positive control. After incubation, the sample was centrifuged at 13,000 rpm for 3 min and read at 400 nm of wavelength (Tecan M200).

#### 2.13.2. Measurement of α-Amylase Inhibition

The inhibition of α-amylase (EC 3.2.1.1) activity was determined using the method of Tan et al. with slight modification [22]. The reaction mixture consisting of 200 μL of distilled water, 50 μL of EDB extract, 250 μL of α-amylase from porcine pancreas (30 mg/mL), and 500 μL of 0.5% starch was incubated at 37 ◦C for 10 min. Acarbose, a known pancreatic α-amylase inhibitor, was applied as a positive control. Enzymatically released reducing sugars were determined by DNS reagen<sup>t</sup> solution (96 mM 3,5-dinitrosalicylic acid, 5.31 M sodium potassium tartrate in 2 M NaOH) after heating at 95 ◦C for 10 min. Then, the mixture was diluted with distilled water and the absorbance was measured at 540 nm (Tecan M200 Infinite).

#### 2.13.3. Measurement of α-Glucosidase Inhibition

The inhibition assay of α-glucosidase (EC 3.2.1.20) was adopted from Tan et al. [22]. The p-nitrophenyl-<sup>α</sup>-<sup>d</sup>-glucuronide (pNPG) dissolved to 4 mM in 0.1 M HEPES (pH 6.8) was used as a substrate. The reaction mixture consisting of 350 μL of HEPES (pH 6.8), 50 μL of EDB extract, 150 μL of α-glucosidase (20 mg/mL), and 450 μL of substrate solution was incubated at 37 ◦C for 2 h. The release of p-nitrophenol from the pNPG substrate was measured at 410 nm (Tecan M200 Infinite). As a positive control, the glucosidase inhibitor, acarbose, was used.

All reagents used in digestive enzyme inhibition assays were provided by Sigma-Aldrich.

## 2.13.4. Data Analysis

Enzyme activity in the presence of inhibitor (EDB extract or reference inhibitor) was expressed as a percentage of the non-inhibited enzyme activity and plotted versus inhibitor concentration. Based on the dose-response curve, the inhibitor concentration required for 10% and 50% inhibition of enzyme activity (IC10 and IC50) was determined as a measure of inhibitory potency. The percentage of the non-inhibited enzyme activity was calculated by following equation:

% non-inhibited enzyme activity = [(AInhibitor − AInhibitor blank)/(AControl − AControl blank)] × 100%

where AControl is the absorbance of the sample without EDB extract/reference inhibitor; AInhibitor is the absorbance of the sample containing EDB extract/reference inhibitor; AInhibitor blank is the absorbance of the sample with EDB extract/reference inhibitor, but without enzyme addition; AControl blank is the absorbance of the sample without EDB extract/reference inhibitor and enzyme addition.

## *2.14. Statistical Analysis*

All data are expressed as the means ± SD from three independent experiments. Statistical analysis was performed using the STATISTICA version 13.3 software (Statsoft, Inc., Tulsa, OK, USA). One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine the differences between the mean values of multiple groups. The T-student's test was applied to determine the significant difference between two independent groups. The equality of variances assumption was verified with the Levene's test.
