**3. Results**

#### *3.1. Polyphenol Composition in the Elderberry Fruit Extract*

HPLC-DAD-ESI-MSn analysis of the EDB extract revealed the presence of 22 polyphenolic compounds, including anthocyanins (peaks 1–5), hydroxybenzoic acid derivative (peak 6), flavan-3-ols (peaks 7–8), polymers, tentatively identified as hydrolysable tannins (peaks 9–10), hydroxycinnamic acids (peaks 11–15), and flavonols (peaks 16–22). HPLC-DAD chromatograms and chromatographic characteristics with mass spectral data of polyphenols identified in the EDB extract are presented in Figure 1 and Table 2, respectively.


**Table 2.** HPLC-MS identification of phenolic compounds in elderberry fruit extract in positive (electrospray ionization (ESI) +) and negative (ESI −) ionization mode.

\* mg/g of lyophilized elderberry powder, values were expressed as mean ± SEM for three independent experiments.

Anthocyanins accounted for 43% of all polyphenolics; cyanidin-based anthocyanin compounds (-3,5- *O*-diglucoside, -3- *O*-sambubiosyl-5- *O*-glucoside, -3- *O*-glucoside, -3- *O*-rutinoside, and -3- *O*-sambubioside) were the main group of anthocyanins, with a significant predominance of cyanidin-3- *O*-sambubioside ([M + H]+ at *m*/*z* 287) constituting 73.2% of all anthocyanins. Peak 1 contained two compounds, identified as cyanidin-3,5- *O*-diglucoside ([M + H]+ at *m*/*z* 611) and cyanidin-3- *O*-sambubiosyl-5- *O*-glucoside ([M + H]+ at *m*/*z* 743). These anthocyanins represented 25.0% of total anthocyanin compounds, quantitatively determined in the EDB extract. In contrast, cyanidin-3- *O*-glucoside ([M + H]+ at *m*/*z* 449) and cyanidin-3- *O*-rutinoside ([M + H]+ at *m*/*z* 595) were only detected in trace amounts in the EDB extract (Table 2). The anthocyanin with a molecular ion of *m*/*z* 433, that yielded on MS<sup>2</sup> fragment at *m*/*z* 271, was identified as pelargonidin-3- *O*-glucoside and quantified in small amounts estimated at 2.3% of all anthocyanins.

Other groups of compounds: Flavan-3-ols, hydroxycinnamic acids, and flavonols amounted to 22.1%, 16.0%, and 13.7% of the total content of polyphenols, respectively. Moreover, the presence of 4-hydroxybenzoic acid glucoside (5.2%) was found in the extract. The group of non-anthocyanin compounds with the largest share in the polyphenol pool were flavan-3-ols, among which catechin and epicatechin (36%), and tannins (64%) were identified. A total of five compounds were detected within another group of hydroxycinnamic acid derivatives, including *p*-coumaric acid hexoside (57%), *p*-coumaroylquinic acid (16%), chlorogenic acid, and its isomers: Neochlorogenic and cryptochlorogenic acids (27%). Concerning flavonols, the results of the HPLC-DAD analysis revealed the presence of quercetin, kaempferol, and isorhamnetin derivatives, with quercetin-3- *O*-rutinoside (53%) and kaempferol-3- *O*-rutinoside (32%) quantified as the dominant compounds within this class.

**Figure 1.** *Cont*.

The polyphenolic compounds, described above, have been previously identified in berries of *Sambucus nigra* [7,23,24]. However, the content of individual polyphenols in EDB fruits varies, depending on the EDB genotypes as well as specific growth conditions. The total polyphenol content in the extract analyzed was determined to 31.03 mg/g of EDB lyophilized powder, including 13.34 mg of anthocyanins, 6.85 mg of flavan-3-ols, 4.98 mg of hydroxycinnamic acid derivatives, 4.26 mg of flavonols, and 1.6 mg of hydroxybenzoic acid glucoside (Table 2).

#### *3.2. Digestive Enzyme Activity Inhibition by Elderberry Fruit Extract*

The EDB extract was evaluated for the ability to inhibit digestive enzymes, including α-glucosidase, α-amylase and lipase. The extract showed similar α-glucosidase and α-amylase inhibitions (Figure 2a,b), with the same IC10 value of 1.25 mg/mL and non-significantly different IC50 values of 6.38 mg/mL and 6.70 mg/mL, respectively (Table 3).

**Figure 2.** The effect of elderberry fruit extract (EDBE) on the activity of digestive enzymes: α-glucosidase (**a**), α-amylase (**b**), and lipase (**c**). Enzyme activity is expressed in relation to the negative control (without extract addition). As positive controls, reference enzyme inhibitors, including acarbose (ACRB, 100 μg/mL) and orlistat (ORST, 5 μg/mL) were used in the experiment. The values represent the means (*n* = 3) ± SD. \* *p* < 0.05, \*\*\* *p* < 0.001 vs. control group.

**Table 3.** Inhibitory concentrations IC10 and IC50 (the inhibitor concentration required for 10% and 50% inhibition of enzyme activity) of the elderberry fruit extract (EDBE), acarbose (ACRB), and orlistat (ORST) as reference pharmacological inhibitors.


Lower potency of the extract was observed in inhibiting pancreatic lipase activity. The extract concentrations reducing lipase activity by 10% and 50% were determined at 2.09 mg/mL and 10.98 mg/mL, respectively (Table 3). In inhibiting α-amylase and lipase activity, the drugs acarbose (100 μg/mL) and orlistat (5 μg/mL) were more potent than the EDB extract (Figure 2b,c). However, in α-glucosidase inhibition, the extract at concentrations of 5 mg/mL and 10 mg/mL evoked the stronger effects than acarbose at a dose of 100 μg/mL.

#### *3.3. The E*ff*ect of Elderberry Fruit Extract on Hypertrophied Adipocytes*

To determine whether EDB extract affects the condition of mature adipocytes, we examined its effect on the viability, lipid accumulation, and ROS production, as well as regulation of leptin and adiponectin expression in terminally differentiated 3T3-L1 cells. The obtained results indicated that EDB extract did not exert any significant effect on both adipocyte viability (Figure 3a) and intracellular lipid content (Figure 3b,g–i). Nevertheless, it dose-dependently inhibited the intracellular ROS generation in hypertrophied adipocytes. EDB extract caused 36%, 53%, and 58% decrease in ROS level when applied at 5, 10, and 20 mg/mL, respectively, in comparison to untreated cells (*p* < 0.001) (Figure 3c). Moreover, treatment with EDB extract led to down-regulation of *NADPH oxidase 4* (*NOX-4*) mRNA expression by approximately 49 ± 6%, independently of the extract dose. Treatment with the extract at the maximum concentration (20 mg/mL) resulted in approximately 2-fold increased mRNA expression of *superoxide dismutase* (*SOD*) and *glutathione peroxidase* (*GPx*). In contrast, the extract did not influence the mRNA expression level of *catalase* (*CAT*) (Figure 3d).

**Figure 3.** Changes in cell viability (**a**), intracellular triglyceride content (**b**), reactive oxygen species production (**c**), and mRNA expression of *NOX4*, *SOD2*, *catalase* (*CAT*), and *GPx* enzymes (**d**) as well as leptin (*LEP*) (**e**) and adiponectin (*ADIPOQ*) (**f**) expression upon the treatment of3T3-L1 mature adipocytes with the elderberry fruit extract (EDBE). The photos present 3T3-L1 preadipocytes (**g**), fully differentiated 3T3-L1 adipocytes non-treated (**h**) and treated with EDBE at the concentration of 20 mg/mL (**i**). The cells were photographed at magnification of 100 ×. The results were expressed as the means ± SD (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. control group.

Supplementation of the fully differentiated adipocyte cultures with EDB extract considerably affected the expression of key adipokines in treated adipocytes. The decrease in *leptin* (*LEP*) mRNA levels between 78% and 94% was observed in mature adipocytes exposed to the extract at concentrations ranging from 5 mg/mL to 20 mg/mL (*p* < 0.001) (Figure 3e). In contrast to leptin, the expression of adiponectin was significantly up-regulated. The highest dose of EDB extract elevated *adiponectin* (*ADIPOQ*) mRNA level by 77% compared to the control (*p* < 0.001) (Figure 3f). The exposure of mature adipocytes to EDB extract resulted in a decrease of leptin secretion. The extract at concentrations of 5 mg/mL and 10 mg/mL significantly reduced leptin synthesis (*p* < 0.05). Whereas, the highest inhibitory effect with the reduction of leptin by 86% (*p* < 0.001) was observed in the cells treated with the extract at maximum concentration. In contrast, EDB extract at the highest dose of 20 mg/mL stimulated the adiponectin secretion in treated cells. The level of adiponectin was increased by 36% respect to the control (*p* < 0.05) (Figure 3f).

#### *3.4. The E*ff*ect of Elderberry Fruit Extract on Glucose Uptake in Mature 3T3-L1 Adipocytes*

The effect of EDB extract on the glucose analogue (2-NBDG) uptake in mature 3T3-L1 adipocytes was analyzed to determine whether the extract affects the glucose uptake by adipocytes. As shown in Figure 4a, EDB extract caused a significant increase in 2-NBDG uptake at all assayed concentrations (*p* < 0.001). The extract stimulated 2-NBDG uptake by 40%, 44%, and 62% tested at 5, 10, and 20 mg/mL, respectively, in comparison to the control system. Unexpectedly, the stimulatory effect of the extract on the glucose uptake in insulin-sensitive cells was more effective than that observed with rosiglitazone (Figure 4a). Subsequently, the expression of *glucose transporter type 4* (*GLUT-4*) gene in mature 3T3-L1 cells exposed to EDB extract was investigated. The results indicated no significant effect of EDB extract on *GLUT-4* mRNA level (Figure 4d).

**Figure 4.** Effect of the elderberry fruit extract (EDBE) on the glucose uptake (**<sup>a</sup>**–**<sup>c</sup>**) and glucose transporter *GLUT-4* mRNA expression (**d**–**f**) in mature 3T3-L1 adipocytes non-induced (**<sup>a</sup>**,**d**) and induced by TNF-α (**b–f**) and non-treated (**b**,**<sup>e</sup>**) and treated (**<sup>c</sup>**,**f**) with insulin (INS). The cells were exposed to EDBE at concentrations of 5, 10, and 20 mg/mL, and to rosiglitazone (RGZ). Data are mean values ± SD (*n* = 3). The significance of the main effects of EDBE was determined by Tukey post hoc test; the control (INS, RGZ) significance was analyzed by T-student test; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

In the parallel experiment, the influence of EDB extract on 2-NBDG incorporation into insulin resistant adipocytes treated with TNF-α was determined. The cells were incubated with the extract in the absence or presence of insulin at 100 nM. In the insulin-resistant adipocytes cultured without insulin supplementation, the EDB extract at concentrations of 5, 10, and 20 mg/mL enhanced 2-NBDG uptake by 40%, 38%, and 39%, respectively (Figure 4b). In this case, quantitative PCR analysis revealed that treatment with EDB extract at doses from 5 mg/mL to 20 mg/mL significantly up-regulated the mRNA expression of *GLUT-4* with an increase ranging from 63% to 82% (Figure 4e).

In the experiments on insulin resistant adipocytes exposed to insulin, the EDB extract at the highest concentration showed insulin-sensitizing properties, stimulating 2-NBDG incorporation by 34% (*p* < 0.01). Its efficacy was comparable to that of rosiglitazone, which enhanced the 2-NBDG uptake by 36% (*p* < 0.01) (Figure 4c). In this study, the extract at a dose of 20 mg/mL up-regulated the expression of *GLUT-4* by 33% (*p* < 0.01), compared to control adipocytes (Figure 4f).

#### *3.5. Anti-Inflammatory E*ff*ects of Elderberry Fruit Extract*

Anti-inflammatory effects of EDB extract were evaluated in activated RAW 264.7 macrophages, considering proinflammatory cytokines and mediators determined at both molecular and cellular levels, using low non-cytotoxic extract doses of 0.1, 1, and 10 μg/mL. The obtained results demonstrated the ability of EDB extract to alleviate the cellular inflammatory response induced by LPS. The extract at concentrations of 1 and 10 μg/mL suppressed mRNA expression of *IL-6* by 34% (*p* < 0.01) and 69% (*p* < 0.001), respectively, compared to control macrophages. Moreover, a 28% decrease in *TNF-*α mRNA level (*p* < 0.05) was observed following treatment with EDB extract at a dose of 10 μg/mL (Figure 5a). The down-regulation of *IL-6* and *TNF-*α expression was consistent with the inhibited secretion of these cytokines. Namely, EDB extract assayed at 1 μg/mL reduced production of IL-6 and TNF-α by 44% and 26%, respectively (*p* < 0.05). At the extract concentration of 10 μg/mL, the 60% decrease in IL-6 secretion (*p* < 0.01) was observed while synthesis of TNF-α was reduced by 52% (*p* < 0.001) (Figure 5b).

**Figure 5.** Effect of the elderberry fruit extract (EDBE) on mRNA expression of *IL-6*, *TNF-*<sup>α</sup>, *COX-2,* and *iNOS* (**a**) and on the production of IL-6, TNF-<sup>α</sup>, PGE2 protein, and NO (**b**) in the lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Data are mean values ± SD (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 when compared with control.

The extract at a dose of 10 μg/mL also a ffected *COX-2* expression, causing a 46% reduction of *COX-2* transcripts level in activated macrophages (*p* < 0.01) (Figure 5a). As a consequence, the decrease in PGE2 production was detected after cell exposure to the extract. Namely, PGE2 level lowered by 33% and 47%, respectively, for a dose of 1 μg/mL and 10 μg/mL (Figure 5b).

Furthermore, the obtained results demonstrated the reduction in NO production as well as down-regulation of *inducible NO synthase* (*iNOS*) expression in LPS-stimulated RAW 264.7 macrophages in response to the EDB extract treatment. The extract reduced the NO synthesis by 35% (*p* < 0.05) when assayed at 1 μg/mL and by 54% (*p* < 0.01) when applied at 10 μg/mL (Figure 5b). A significant inhibitory e ffect of EDB extract on *iNOS* expression was noted only at the highest tested dose, which decreased *iNOS* mRNA level by 30% (*p* < 0.01) (Figure 5a).
