2.1. Phytochemical Analysis
Bilberry leaf is one of the most popular herbal remedies for diabetes, recommended and sold by local herbalists, often without appropriate health safety and quality control. In order to investigate the safety of the plant material used in this study, total reflection X-ray fluorescence (TXRF) was applied for the simultaneous multielemental analysis of plant extract. Ca (2940 ± 4.5 mg/kg) and K (2267 ± 4.9 mg/kg) were the dominant elements in the sample. Mn (474 ± 0.95 mg/kg), P (193.5 ± 7 mg/kg), Ba (51.1 ± 0.65 mg/kg), Zn (18.05 ± 0.1 mg/kg) existed in lower, but substantial quantities. Due to their toxicity, quantity of heavy metals in the herbal sample was of special interest. As (1.05 ± 0.05 mg/kg), Cu (7.1 ± 0.1 mg/kg), Fe (14.85 ± 1.5 mg/kg), Ni (0.2 ± 0.05 mg/kg) and Pb (0.55 ± 0.01 mg/kg) were detected but their concentrations were much lower than the legal threshold limit values established for the dietary supplements and much lower than in a previous study performed in Poland [
15]. Even though there are reports that
V. myrtillus leaf contains significant amounts of chromium, an essential element involved in the action of insulin [
16], it was not detected in the analyzed sample. This result is therefore concurring with a study performed in Northern Europe [
17]. Some authors consider chrome to be the main factor responsible for potential antidiabetic effects of
V. myrtillus leaf [
16]. However, its absence in the investigated material indicates that such opinion cannot be generalized to all
V. myrtillus specimens.
Besides being sold in form of dry herbal material, bilberry leaf is often extracted, usually with water/ethanol mixtures, and sold either in form of a tincture or dry extract. However, water, ethanol and their combinations of varying ratios, have different polarity and viscosity interacting thus with different natural compounds, which is inevitably reflected in the biological properties of prepared extracts [
18]. For the preparation of
V. myrtillus leaf extracts in this study water and 80% (
v/
v) ethanol/water mixture were selected. The content of phenolic compounds in the prepared extracts is presented in
Figure 1. The amount of total phenols and flavonoids in hydroethanolic extract was more than twofold higher than their amount in aqueous extract. This is expected because of the moderate polarity and good solubility of natural phenols in 80% ethanol.
Results of HPLC analysis presented in
Figure 2 and
Table 1 revealed that the two extracts contained similar phenolic constituents but in different quantities. Quercetin-3-
O-galactoside (hyperoside) was the dominant component in both extracts, followed by chlorogenic acid. In accordance with the solvent polarity, hydroethanolic extract contained somewhat more hyperoside, while the aqueous extract was richer in chlorogenic acid. Of the other two phenolic acids,
p-coumaric acid was present in much lower amounts. A small amount of caffeic acid was detected only in the aqueous extract. In addition to that, kaempferol was detected in the hydrolyzed hydroethanolic extract, indicating the presence of its glycosides in the original extract, as reported before [
16]. The presence of hyperoside, chlorogenic and
p-coumaric acid in
V. myrtillus leaf has been confirmed on many occasions [
14,
19,
20]. Interestingly, arbutin was detected in the aqueous extract. Even though older literature allows for the possibility of arbutin presence, [
16], now it is generally considered that arbutin content is caused by admixture with
Vaccinium ×
intermedium, hybrid of
Vaccinium vitis-idaea and
V.
myrtillus. However, a detailed morphological identification according to the specialized reference [
21], excluded that possibility. Even though the amount of arbutin was relatively low, its presence in the investigated sample indicates that the excessive use of
V. myrtillus leaf aqueous extracts (e.g., infusions) should be avoided.
As expected, quercetin was the main component of the hydrolyzed extracts. Its quantity, in accordance with the previously published studies, suggests that besides hyperoside there are other quercetin derivatives in the samples. High content of protocatechuic acid, a well-known product of flavonol degradation [
22], in the aqueous extract, suggests that the degradation of quercetin occurred during the hydrolysis of aqueous extract.
2.2. Antioxidant Activity of the Extracts
Natural antioxidants may display their activity by numerous mechanisms. Antioxidants may influence oxidation reactions directly, through hydrogen atom transfer and single electron transfer, or indirectly, by their ability to bind potentially pro-oxidative metal ions [
23]. The proportion of each of those mechanisms in total antioxidant activity of an herbal extract depends on various influences. Therefore, use of more than one method is recommended to give a comprehensive analysis of antioxidant efficiency of complex mixtures such as natural extracts. Due to the growing popularity of phenolic antioxidants, many scientific methods have been developed to measure antioxidant capacity of phenolics present in medicinal plants and foods. In order to systematically evaluate the antioxidant activity of
V. myrtillus extracts in this study, multiple assay systems were used: total antioxidant activity, reducing power, ferric reducing antioxidant power, ABTS and DPPH radical scavenging and chelating activity assay. BHA, ascorbic acid, Trolox and EDTA, antioxidants and ion chelator often employed in the food and pharmaceutical industry, were used as the positive controls [
24].
Figure 3 presents the results of three assays based on the reducing abilities of the analytes. While the TAA assay is based on the reduction of Mo
6+ to Mo
5+ by the sample analyte [
25], both RP and FRAP assays measure the ability of the extracts to reduce Fe
3+ to Fe
2+ ions. Even though the FRAP assay is based on the same process as RP assay, it is more specific than the previous one. The reducing properties of the extracts were not equally pronounced toward the two ions. The aqueous extract was more active in the TAA assay, while the reducing properties of the hydroethanolic extract were more pronounced in the reaction with iron ions, especially in the reducing power assay.
As shown in
Table 2, the antioxidant activity of the investigated extracts was notable, but it differed between extracts. For example, the aqueous extract was very good inhibitor of β-carotene degradation (
Figure 4), and its ANT activity was equal to the activity of BHA. Hydroethanolic extract on the other hand, displayed significant but much lower activity. Comparison of IC
50 values shows that radical scavenging ability for ABTS free radical did not differ statistically between the two extracts. Although the hydroethanolic extract showed notably better DPPH radical scavenging capabilities than the aqueous extract, its activity was still somewhat lower than the activity of the commercial antioxidant, BHA. Finally, as evident by markedly lower IC
50 values, aqueous extracts was shown to be better Fe
3+ ion chelator than hydroethanolic extract and acts thus as a better secondary antioxidant.
In the presented study both investigated
V. myrtillus leaf extracts were active in all the employed assays. They showed reducing activity (as evidenced by TAA, RP and FRAP assay), antiradical activity (in ABTS and DPPH free radical scavenging activity as well as in linoleic acid assay), as well as the potential to bind potentially pro-oxidant Fe
2+ ion. As such, they could interact with ROS and other reactive species in multiple ways reducing thus their reactivity and deleterious properties. Such notable antioxidant activity of the extracts in the performed assays can mostly be related to their high phenolic content. However, similar to other studies investigating the relationship between antioxidant activity and phenolic content, a direct relationship could not be established. Various constituents interact in specific ways making each extract most suitable for different aspects of antioxidant activity [
26].
2.3. Enzyme-Inhibitory Activity of the Extracts
The investigated
V. myrtillus extracts were tested for their inhibitory activity against two enzymes that participate in carbohydrate digestion: α-amylase and α-glucosidase. Although
V. myrtillus leaf reportedly showed a positive response in previous screening of α-amylase inhibiting properties [
16], the extracts in this study did not demonstrate any observable α-amylase-inhibiting effect. However, the α-glucosidase-inhibiting activity of the extracts was excellent (
Figure 5).
The hydroethanolic extract was a particularly good α-glucosidase inhibitor, with an IC
50 value statistically equal to the IC
50 of acarbose, an anti-diabetic drug used as standard inhibitor (
Table 2). It was previously reported that arbutin, hyperoside and chlorogenic acid, the main phenolic components of the investigated extracts, may suppress postprandial hyperglycemia by strongly inhibiting α-glucosidase [
27,
28,
29]. Additionally, current studies show that the combination of plant phenolics, such as the combination found in the investigated extracts, may have an additive effect on α-glucosidase inhibition [
30]. Furthermore, quercetin and caffeic acid, aglycones formed by hydrolysis of hyperoside and chlorogenic acid, respectively, also have notable anti-α-glucosidase activity [
27,
31]. Therefore, the observed α-glucosidase inhibitory activity may significantly contribute to the beneficial effects of V. myrtillus in diabetes.
2.4. Effect of V. myrtillus Extracts on Glucose-Induced Oxidative Stress in Hep G2 Cells
It was shown that the acute exposure to high glucose concentration produces oxidative stress in liver cells as evidenced by altered morphology, increased ROS level, lipid peroxidation, protein carbonyl and 3-nitrotyrosine adduct formation in Hep G2 cells. These changes may irreversibly damage hepatocytes leading to apoptosis. Natural metabolites and extracts may prevent the oxidative changes, normalize the concentration of intracellular antioxidants and thus prevent or even reverse cell-damage in vivo and in vitro [
32]. Besides directly reacting with ROS, natural phenols and other antioxidants may interact with molecular targets in human organism by numerous other mechanisms [
33]. One of the mechanisms by which antioxidants can protect cells and tissues from oxidative stress is regeneration of low molecular weight antioxidants, such as GSH [
33]. The influence of
V. myrtillus leaf extract on GSH levels in hepatic cells subjected to high glucose concentration has not been thoroughly investigated. Therefore, human hepatocellular carcinoma, Hep G2, cells were chosen as a model to study the potential effects of the prepared extracts on glucose-induced oxidative stress in liver.
One of the consequences of diabetic hyperglycemia is an elevated production of ROS which leads to decreased level of one of the most important antioxidant in the body, GSH [
34]. Indeed, in the performed assay the level of glutathione in glucose-treated (D) cells was significantly lower than in the non-treated cells (C) (
Figure 6).
At the higher investigated concentration, the aqueous extract showed an outstanding activity and restored the GSH concentration to the levels observed in non-stressed cells (C). Quercetin derivatives and chlorogenic acid, the main components of the extract, have well documented in vitro antioxidant properties [
35]. Furthermore, their antidiabetic activity has been confirmed in numerous in vivo studies. For example, treatment with hyperoside may prevent glomerular podocyte apoptosis in streptozotocin-induced diabetic nephropathy [
36]. Moreover, hyperoside decreased albuminuria at the early stage of diabetic nephropathy by ameliorating renal damage and podocyte injury [
37], improved the function of pancreatic islets, increased glycolysis and decreased gluconeogenesis in streptozotocin-induced diabetic rats [
38]. Besides hyperoside, the extracts contained significant amounts of chlorogenic acid. This phenolic acid has well known antidiabetic properties which have been extensively reviewed [
39] and linked to the observed diabetes protection of regular coffee consumption [
40]. Furthermore, chlorogenic acid may ameliorate oxidative stress for renal injury in streptozotocin-induced diabetic nephropathy rats [
41]. Thus we may conclude that phytochemical composition of
V. myrtillus extracts is well suited for modulation of oxidative stress in hepatic cells. Even though the observed antioxidant and hepatoprotective effects of the extracts in Hep G2 model cannot be taken as a final proof that they would display such activity in the clinical setting, they certainly indicate a positive potential which will hopefully be researched further.