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
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
]. 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
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-α, 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
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
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 m
. 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, Differentiation, 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 differentiated 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 differentiation was induced in post-confluent cell cultures by a differentiation 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 differentiated 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 differentiation process, insulin resistance was induced in 3T3-L1 adipocytes by 10 ng/mL murine TNF-α (Sigma-Aldrich) for 5 days, with medium/TNF-α 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 × 105 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 differentiated 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-buffered 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 differentiated 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) buffer 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 buffer supplemented with 80 μM 2-NBDG (2-N
-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 × 106 cells.
2.10. Determination of IL-6, TNF-α, and PGE2 Production in RAW 264.7 Macrophages
The secretion of IL-6 and TNF-α 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 × 106 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 reagent (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 reagent (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
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 126.96.36.199) 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 188.8.131.52) 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 reagent 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 184.108.40.206) was adopted from Tan et al. [22
]. The p-nitrophenyl-α-d
-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
) 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%
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.
Excessive fat accumulation in hypertrophic adipose tissue associated with obesity is responsible for oxidative stress, chronic inflammation, and dysregulated adipokine secretion [25
]. It is believed that the therapeutic potential of natural dietary compounds against obesity and obesity-related disorders should focus on improving the fat function in pathogenic hypertrophic adipocytes by reducing oxidative stress, alleviating inflammation, and regulating underproduction or overproduction of clinically relevant adipocyte factors. However, most bioactive compounds or extracts strongly affect preadipocytes, their viability, proliferation, and differentiation into mature fat cells, without any significant effects on the pathological status of hypertrophic adipocytes. Therefore, in this work, the influence of the Sambucus nigra
fruit extract on mature fully differentiated insulin-resistant 3T3-L1 adipocytes was investigated.
In our study, we found no reduction in cell viability and lipid content in hypertrophic 3T3-L1 adipocytes after exposure to EDB extract. However, as a result of the treatment, the intracellular ROS generation was significantly down-regulated and probably, oxidative stress accompanying excessive fat accumulation was also importantly reduced. Oxidative stress induced by enhanced lipid content is reported to be involved in the pathogenesis of obesity-related comorbidities including insulin resistance and diabetes, cardiovascular complications, and cancer [26
]. It was found that ROS are intensively generated in visceral adipose tissue by adipocytes during the metabolism of excess nutrients and also by macrophages, which accumulate in adipose tissue in obesity state. The increased release of fatty acids from overproduced fat accumulated in adipose tissue, activate NADPH oxidases (NOX) and induce or aggravate ROS production. Other factors that also contribute oxidative stress to obesity include hyperleptinemia, low antioxidant defense, or chronic inflammation [27
]. Our results showed that EDB extract could reduce ROS generation by lowering the expression of NOX4, the major NOX isoform in adipocytes. Treatment of hypertrophied 3T3-L1 adipocytes with EDB extract caused a significant decreasing in NOX4
mRNA expression. Furthermore, up-regulation of mRNA expression of antioxidant enzymes, like SOD and GPx, could also contribute to enhancing adipocyte antioxidant defense efficiency. Numerous studies have shown the high antioxidant capacity of Sambucus nigra
]. However, the antioxidant effects of EDB on adipocytes have not yet been reported in the literature. In the present study, we demonstrated that introduction of EDB extract to the culture of hypertrophic adipocytes resulted in decreased ROS generation in cells. The antioxidant action of EDB extract in adipocytes may be a potential protective mechanism against obesity-associated pathological risk factors, including insulin resistance and chronic inflammation.
Additionally, EDB extract treatment modulated the leptin and adiponectin gene expression and protein secretion in hypertrophic 3T3-L1 adipocytes. Leptin and adiponectin are adipocytokines, which influence energy homeostasis, glucose and lipid metabolism, cardiovascular function, and immune response [28
]. Leptin is primarily secreted by fully differentiated adipocytes, and its crucial role is to regulate energy intake and expenditure through controlling appetite and glucose metabolism. Reflecting the increased amount of adipose tissue, obese individuals often have elevated leptin concentration and the simultaneous apparent loss of efficacy of leptin, which is a result of leptin resistance, the state that leads to uncontrolled food intake, pro-inflammatory state, diabetes mellitus, and other obesity-related complications [29
]. In contrast to leptin, adiponectin is down-regulated in obesity, and the circulating adiponectin levels are inversely correlated with body fat amount. Adiponectin enhances energy metabolism and fatty acid oxidation, promotes insulin sensitivity, improves glucose tolerance, and exerts anti-inflammatory effects [28
]. Low serum adiponectin and high serum leptin levels are considered as risk factors for developing type 2 diabetes (T2DM), obesity, dyslipidemia, hypertension, and cardiovascular diseases. In this study, a remarkable decrease in leptin expression and secretion was observed in response to EDB extract treatment of hypertrophied 3T3-L1 adipocytes, which may help counteract the leptin resistance state. Whereas, adiponectin mRNA expression and protein secretion in treated adipocytes were significantly increased. The effect of EDB extract on adiponectin production may indicate anti-inflammatory potential and insulin-sensitizing activity of Sambucus nigra
The association of visceral obesity with T2DM is a long-recognized phenomenon. The primary determinant of this correlation is the fact that central obesity is the critical factor in the emergence of insulin resistance. The insulin-resistant state results in defective insulin-stimulated glucose uptake and consequently in hyperglycemia, elevated circulating free fatty acids level, abnormal fat accumulation, and dysregulation of hepatic glucose production, that, in combination with a paucity of insulin secretion by pancreatic β-cells, leads to T2DM [30
]. These metabolic abnormalities may arise from impairment in insulin signaling pathways and subsequent defect in translocation of insulin-responsive glucose transporter protein (GLUT-4) and in adipose tissue, also from down-regulation of GLUT-4
The effects of EDB extract on glucose uptake and GLUT-4
expression were evaluated in this study. Experiments were performed both with mature 3T3-L1 adipocytes sensitive to insulin and adipocytes treated with TNF-α to induce an inflammatory status and insulin resistance. Analysis revealed that EDB extract stimulated the 2-NBDG uptake in both types of adipocytes and up-regulated mRNA expression of GLUT-4
in insulin-resistant cells, suggesting insulin-like and insulin-sensitizing activities of the extract. The signaling pathways involved in the development of these activities will be further examined in future studies. This is the first study assessing the effects of EDB extract on glucose uptake in 3T3-L1 cells. Although several recent reports have suggested the anti-diabetic and hypoglycemic properties of elderberry, it has been found that EDB methanolic extracts markedly stimulate glucose uptake in liver HepG2
cells and also exert inhibitory effect towards carbohydrate hydrolyzing enzyme [32
]. Furthermore, EDB extracts, EDB anthocyanins, mainly cyanidin-3-glucoside and cyanidin-3-sambubioside, procyanidins, and their metabolites were found to enhance glucose uptake in human skeletal muscle cells [33
]. Whereas, EDB lipophilic and polar extracts were reported to modulate glucose metabolism or lower insulin secretion contributing to the mitigation of insulin resistance in T2DM rats [13
Anti-obesity and anti-diabetic activity of EDB extract could be related to the inhibition of dietary fat and sugar absorption from the intestinal tract. There is some evidence that polyphenols from berry fruits, such as strawberry, raspberry, blueberry, bilberry, black and red currant, lingonberry, red and green gooseberry, cranberry, and chokeberry, contribute to the inhibition of digestive enzymes involved in the hydrolysis of dietary lipids and carbohydrates [34
]. Based on our research, the Sambucus nigra
fruit may be included in the class of berries considered as effective inhibitors of α-amylase, α-glucosidase, and pancreatic lipase activity.
Obesity is known to be accompanied by metaflammation—low-grade chronic inflammation condition triggered by excess nutrients in metabolic cells [35
]. An attribute of obesity-related inflammation is enhanced infiltration of macrophages into expanding adipose tissue, activation of specialized immune cells, and secretion of proinflammatory cytokines such as TNF-α, IL-6, and MCP-1 leading to an unresolved inflammatory response, which affects normal metabolism and insulin action [35
]. Inhibition of obesity-induced inflammation could, thus, be a therapeutic intervention against adipose tissue dysfunction and related co-morbidities. In recent years, the use of anti-inflammatory nutrients provided through diet as a potential approach against obesity has been extensively studied [36
In the present study, we evaluated anti-inflammatory effects of EDB extract in LPS-stimulated RAW 264.7 macrophages. Activated macrophages produce cytokines such as TNF-α, IL-1β, and IL-6 as well as pro-inflammatory mediators, such as NO and PGE2
]. IL-6 and TNF-α are potent proinflammatory cytokines, which play a central role in inflammatory response and are characterized by a broad spectrum of functions with various effects in adipose tissue. TNF-α substantially influences lipid metabolism and adipocytes apoptosis. It can disrupt insulin signaling pathway promoting insulin resistance and adipocytes dysfunction [39
]. TNF-α has, thus, been believed to be the crucial mediator in the detrimental paracrine loop between adipocytes and macrophages [40
]. IL-6 has a pivotal role in acute phase reactions. It also influences hormonal balance and energy homeostasis and may affect the increase of free fatty acids level. Circulating levels of IL-6 and TNF-α are elevated in obese individuals and patients with insulin resistance [41
]. In general, the regulation of TNF-α and IL-6 secretion is considered to be a potent treatment strategy for inflammation-associated diseases [42
The research presented in this work suggests that EDB extract dose-dependently down-regulates mRNA expression and protein production of TNF-α and IL-6 in activated RAW 264.7 macrophages and therefore alleviates the cellular inflammatory response induced by LPS. In addition to TNF-α and IL-6, EDB extract significantly reduced the production of inflammatory mediators—PGE2
and NO. Increased level of PGE2
is observed in obese adipose tissue due to remarkable up-regulation of COX-2—the key enzyme in eicosanoid metabolism, of which expression is induced in inflammation state [43
]. It has been suggested that COX-2-mediated inflammation in visceral fat is responsible for insulin resistance and fatty liver development in high-fat-induced obese rats [44
]. The same study revealed that COX-2 inhibition significantly reversed adipocyte hypertrophy, macrophage infiltration, and decreased markers of adipocyte differentiation. Nitric oxide formed by iNOS is a short-lived vasodilator that acts as an important regulator of physical homeostasis, while its overproduction has been closely correlated with the pathological conditions including septic shock, osteoporosis and rheumatoid arthritis, insulin resistance, and inflammation [45
]. In the present study, EDB extract was found to suppress PGE2
and NO production via down-regulation of COX-2
expression. These findings indicate that inhibition of PGE2
and NO generation is one of the anti-inflammatory mechanisms of the extract. Several recent studies have shown the anti-inflammatory potential of EDB fruit preparations. In our previous study, we demonstrated the anti-inflammatory potential of gastrointestinally digested EDB extract following intestinal absorption in a co-culture model of intestinal epithelial Caco-2 cells and LPS-stimulated RAW 264.7 macrophages [7
]. The analyzed extract down-regulated the expression of genes (IL-1β, IL-6, TNF-α, COX-2) involving in the inflammatory pathway in a range comparable to that of budesonide. This study demonstrated adequate bioavailability and intestinal permeability of EDB compounds that are probably sufficient to evoke systemic anti-inflammatory effects [7
]. Moreover, there is increasing evidence that the EDB bioactives can penetrate the blood–brain barrier and modulate the immune response induced in different types of brain injuries, including ischemic stroke. It has been found that EDB extract and its phenolic components significantly inhibit activation of microglia, considered to be resident macrophages responsible for the initial immune response to brain injuries. Treatment of activated microglial bv-2 cells with EBD extract led to diminishing ROS and NO generation, and as a consequence, attenuating the neuroinflammatory process [16
Results of the study, as discussed above, indicate that Sambucus nigra fruit extract may offer substantial preventive and therapeutic potential for the treatment of obesity and obesity-related disorders, accompanied by oxidative stress, inflammationm and insulin resistance. Moreover, the extract can inhibit digestive enzyme activity, and consequently, significantly reduce the intestinal absorption of dietary lipids and carbohydrates, which is an effective strategy for the prevention and treatment of obesity and metabolic comorbidities.
Considering the findings of in vitro
studies, we can postulate a nutraceutical application of the Sambucus nigra
fruit extract. The scientific community focuses great attention on introducing nutraceuticals into the daily diet to prevent the occurrence of the pathological conditions, to delay or avoid the need for drug treatment and to support pharmacological therapy. Nutraceuticals as pharmafoods should be evaluated in the clinical aspects regarding safety, side effects, bioavailability, beneficial health effects, mechanisms of action and efficacy, and any possible interactions between food and drugs assumed together with them [46
]. Thus, the developing of clinical studies will be of significant importance for clinically justified promotion of the Sambucus nigra
fruit extract as a safe nutraceutical with the capacity of prevention or treatment of obesity and obesity-related immune-metabolic disorders.