Polyphenol Content, Antiradical Properties, and α-Amylase Inhibition Activity of Vaccinium myrtillus L. (Bilberry) and Vaccinium vitis-idaea L. (Lingonberry) Leaf and Aerial Parts Extracts

Abstract

The utilisation of medicinal plants has gained importance due to emerging drug resistance in diseases, including type 2 diabetes mellitus (T2DM). Vaccinium myrtillus (VM) and Vaccinium vitis-idaea (VV) are particularly noteworthy as their leaves and aerial parts (no flowering leaves with stems) are rich in polyphenols and antioxidants with potential positive impacts on blood sugar levels. The aim of this study was to compare the chemical composition, antiradical, and α-amylase inhibitory activities of the leaves and aerial parts of the Latvian VM and VV. Two samples of VM and two samples of VV were collected from two different locations. Dried plants were used to prepare 70% ethanol extracts and freeze-dried samples. The total tannin and phenolic contents were determined, and the phytochemical compounds were characterised by LC-MS. The α-amylase inhibition activity and the antiradical activity in the extracts were measured. The VV ethanol extracts had the highest polyphenol content at 632.80 mg/100 g, followed by the freeze-dried extracts at 968.52 mg/100 g. The highest polyphenol content in the VV ethanol extracts was 632.80 mg/100 g and 968.52 mg/100 g in the freeze-dried extracts. The ethanol extracts of VM (1.34%) and VV (2.85%) had the highest content of tannin, whereas the freeze-dried extracts showed significantly lower tannin content in VM (0.21%) and VV (0.18%). A high correlation was observed between DPPH (2,2-diphenyl-1-picrylhydrazyl) and α-amylase inhibition activity (R = 0.86, p = 0.015). This information can be used for additional control of sugar levels in T2DM patients. Comparing both plants with each other and different types of samples (ethanol extracts, freeze-dried samples), it was concluded that the difference exists in both variants. The VM samples showed higher results (α-amylase inhibition activity, DPPH radical scavenging activity) than the VV samples, and the freeze-dried samples had higher results than the ethanol extracts.

1. Introduction

Diabetes mellitus, a common metabolic disorder characterised by hyperglycemia, is a major health problem worldwide [1]. While traditional pharmacological interventions are effective, they often come with associated side effects and long-term considerations [2]. Consequently, there has been a growing inclination towards the investigation of natural compounds from plant sources, driven by the quest for safer and more holistic alternatives [3].

Among these, bilberry (Vaccinium myrtillus) (VM) and lingonberry (Vaccinium vitis-idaea) (VV) have emerged as promising candidates, exhibiting a rich composition of bioactive compounds that can contribute to antiradical properties, which are expressed in an antioxidant and antiradical manner [4,5].

Furthermore, the research claims to explore the antiradical properties (which are considered antioxidants) of VM and VV, with a specific focus on the leaf and the aerial part extracts [6]. Understanding the impact of these natural extracts on management could offer novel insights and contribute to the development of complementary or alternative strategies for individuals affected by T2DM [7].

Understanding the α-amylase inhibition activity in VM is important for people with T2DM because α-amylase is an enzyme that helps break down complex carbohydrates into simpler sugars. In the human body, α-amylase is produced in the pancreas and salivary glands [8]. Certain natural compounds found in foods, including plant extracts, may have α-amylase inhibition activity [9].

Finding such inhibitory effects in VM and VV extracts could potentially slow down the digestion of carbohydrates, resulting in a slower release of glucose into the bloodstream [10]. This could be beneficial for individuals with T2DM by helping to manage blood sugar levels [11]. Plant-based products (from leaves, stems, roots, fruits, etc.) can serve as supplements or substitute synthetic medications in the treatment of diabetes, offering the advantage of minimally proven side effects and the potential cost-savings in the management of the condition [12].

Furthermore, a study using VM leaves in rats with T2DM demonstrated a beneficial effect on sugar levels. According to the research on VM leaf infusion, plasma glucose levels decreased by approximately 26% in two distinct stages of diabetes. The treatment also led to a 39% reduction in plasma triglyceride levels [13]. Interestingly, few articles have appeared in the past decade investigating the composition and pharmacological effects of VM leaves because previous studies have predominantly focused on the fruit aspect [11]. This study on two types of extracts from dry plant material generated new scientific data by identifying compounds in the leaves and stems of VM and VV, which had been the subject of limited prior research.

The leaves and the aerial parts of VM and VV constitute the primary by-products. Studies [14,15] have highlighted a significantly higher concentration of phenolic compounds in the leaves and stems of Vaccinium species compared to berries; however, the research findings suggest that very little is known about the tannin and derivate content of the aerial part. For example, VM leaves usually have a high number of tannins, typically between 0.8% and 6.7% [11], whereas VV leaves contain a high level of tannins [16].

Tannins are natural compounds found in different plant tissues, particularly fruits, seeds, bark, leaves, and wood [17]. Tannins are polymeric molecules [18]. They belong to a group of polyphenolic compounds and are known for their astringent taste [19]. A study looked at how adding tannin to diabetic rats on a high-fat diet affected their ability to resist oxidative damage. The findings showed that adding tannin to diabetic rats for 30 days had an antioxidant effect, lowering oxidative stress [20].

Another study describes the effects of tannins from Spondias Mombin on body and organ weight, serum amylase levels, and diabetes in streptozotocin-induced diabetic rats. Tannins from Spondias Mombin have been found to have several benefits: they improve body and organ weight, reduce serum amylase levels, and have significant antidiabetic effects. These tannins helped lower blood sugar levels and improve lipid profiles in diabetic rats. Additionally, tannin treatment provided antioxidant properties, reducing oxidative stress [21].

It is important to note that while tannins and their derivates are a component of the overall chemical composition of VM leaves, these leaves also contain other bioactive compounds, such as flavonoids and phenolic acids [20]. Some pharmacologically important compounds are galloyl glucose, chlorogenic acid, neochlorogenic acid, catechin, and epicatechin, etc. [22,23]. This study will further discuss the effects of individual phenolic compounds, especially tannin derivates.

They can lower blood sugar levels after meals and prevent or delay glucose absorption by blocking alpha-glucosidase [6].

Various analytical techniques, including chemical assays and spectroscopic methods like infrared spectroscopy, can confirm the presence of tannins in the leaves of the VM and VV aerial parts [24].

The total phenolic content in VM and VV may vary depending on the specific plant species involved, growing conditions, differences in analytical methods, and specific parts of the plant under analysis. Leaves and aerial parts exhibit a higher concentration compared to berries [25]. These aerial parts exhibit the most robust antioxidant activities, suggesting their potential as an alternative reservoir of bioactive natural substances [25]. Research in the literature indicates that although VM and VV leaves contain fewer anthocyanins than their fruits, they have a higher phenolic compound content and have promising biological activities [26,27,28].

Additionally, the choice of solvents and extraction methods can influence the results. For example, the total phenolic content can be elevated when using an 80% methanol solvent, while 70% ethanol also demonstrates effective extraction of phenolics. Interestingly, the use of acetone-water (70:30) yields even higher extraction results compared to ethanol [11,20]. In this study, 70% ethanol was used since it is less harmful compared to methanol and acetone and it extracts effectively.

Most studies compare the same type of prepared plant extracts and their effects on T2DM [29,30]. There are relatively fewer studies investigating freeze-dried samples and their successful effects on T2DM [31].

In our study, the investigation of the developing leaves and aerial parts of VM and VV in Latvia has been specifically selected. These plants are present in the human diet in the form of fruits and aerial part preparations, as well as being used historically in folk medicine [32,33,34]. VM and VV, belonging to the Vaccinium genus, are well known for their traditional uses in folk medicine, especially their leaves [7,35,36]. These plants have a history of being used to address various health concerns, including conditions related to metabolic imbalances [7,36]. Drawing on information from the scientific literature [10,17,36,37], our objectives were to characterise polyphenols and evaluate tannins, the total phenolic content, antioxidant activity, and the α-amylase inhibition activity of the leaves and the aerial parts of VM and VV in both ethanol extracts and freeze-dried samples. The plant samples were collected from two distinct locations: the Dekšāre district and the Sigulda district.

2. Materials and Methods

2.1. Plant Materials

The herbaceous material from the flowering plants was collected in the summer month of July 2023. The leaves and the aerial parts (no flowering leaves and stems together) of Vaccinium myrtillus (VM) and Vaccinium vitis-idaea (VV) were collected in two places: the Dekšāre district, 56.63311, 26.88392 and the Sigulda district, 57.173335, 24.898171.

Plant parts were air-dried at room temperature, following the standard guidelines of the World Health Organisation (WHO) [38]. They were then kept in shaded, sealed paper boxes for storage. The aerial parts were processed by being ground in a coffee mill and sifted through 2 mm sieves. The plants were identified by a PhD. Pharm. Prof. Dace Bandere, and vouchers labelled [VM leaves D 2023, VM aerial parts D 2023; VM leaves S 2023, VM aerial parts S 2023; VV leaves D 2023, VV aerial parts D 2023; VM leaves S 2023, VM aerial parts S 2023] were stored in the herbal collection of the Department of Pharmaceutical Chemistry of Riga Stradiņš University.

2.2. Reagents and Chemicals

The following were used: 2,2-diphenyl-1-picrylhydrazyl (DPPH) (from Alfa Aesar, Kandel, Germany), acarbose (from Tokyo Chemical Industry CO., Ltd., Tokyo, Japan), distilled and purified water (hereafter referred to as water), ethanol, gallic acid (from Acros Organics, Geel, Belgium), Folin–Ciocalteu reagent (from Fisher Scientific, Loughborough, UK), methanol, potassium sodium tartrate tetrahydrate, potato starch, 3,5-dinitrosalicylic acid, α-amylase from porcine pancreas (from Sigma-Aldrich, St. Louis, MO, USA), phosphomolybdic tungstic reagent R, hide powder CRS, pyrogallol, sodium chloride (from Fisher Scientific, Loughborough, UK), reagent grade formic acid (from Sigma-Aldrich, Darmstadt, Germany), sodium carbonate (from Honeywell, Charlotte, NC, USA), sodium phosphate dibasic (from Honeywell, Charlotte, NC, USA), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (from Acros Organics, Geel, Belgium), UHPLC-grade methanol (from Honeywell, CHROMASOLV, Seelze, Germany), and ultrapure water Type 1 (prepared using Stack-pure purification system; OmniaTap 6, Niederahr, Germany). We used only analytical or HPLC-grade solvents.

2.3. Sample Preparation

Sixteen samples (Figure 1) were taken from two different places, the Dekšāre district, 56.63311, 26.88392 and the Sigulda district, 57.173335, 24.898171, consisting of 4 leaf samples of Vaccinium myrtillus (VM), 4 leaf samples of VM aerial parts, 4 leaf samples of Vaccinium vitis-idaea (VV) leaf samples, and 4 leaf samples of VV aerial parts. To extract the active compounds, 5 grammes of crushed plant material were macerated in 50 mL of 70% ethanol (ratio 1:10, according to the European Pharmacopoeia, 8th edition [39]) at room temperature for 80 min. Subsequently, the solvent was removed from 8 samples (2 VM leaves, 2 VM aerial parts, 2 VV leaves, and 2 VV aerial parts) by rotary vacuum evaporation (Model: RV 3 ECO S099 IKA™, Staufen, Germany), followed by lyophilisation (Zirbus Technologies VaCo series 2.0 Standart 2018, Bad Grund, Germany), and then frozen at −70 °C for 2 days. The remaining 8 samples (2 VM leaves, 2 VM aerial parts, 2 VV leaves, and 2 VV aerial parts) were filtered and retained in the form of ethanol extracts.

In summary, 16 varieties of samples were used in the study: 8 ethanol extracts and 8 freeze-dried samples.

2.4. Determination of Total Phenolic Content

The total phenolic content (TPC) in the extracts was assessed using the Folin–Ciocalteu colorimetric method, following the protocol outlined by Dranca and Oroian [40], with slight modifications. A quantity of 0.2 g of freeze-dried sample was dissolved in 20 mL of 70% ethanol in a ratio of 1:100 or 1 mL of liquid extract had been used. Afterwards, 1 mL of the extract or the freeze-dried sample was combined with 5 mL of a 10% Folin–Ciocalteu reagent, followed by the addition of 4 mL of a 7.5% Na₂CO₃ solution.

After 30 min of incubation at room temperature in the dark with gentle shaking, the absorbance at 765 nm was recorded using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabX™, Greifensee, Switzerland). Gallic acid served as a standard for the calibration curve; 1 mL of gallic acid was combined with 5 mL of a 10% Folin–Ciocalteu reagent and 4 mL of a 7.5% Na₂CO₃ solution.

The total phenolic content (C) was quantified in mg of gallic acid equivalent (GAE) per g (mg/100 g) of freeze-dried extract, and each measurement was taken three times. According to the formula:

$$\mathrm{C}=\mathrm{a}\times \mathsf{\gamma }\times (\mathrm{V}/\mathrm{m})\times 100,$$

where a is the dilution factor, γ is the mass concentration of total phenolic compounds after the calibration line in mg/mL, V is the volume of ethanol in mL, m is the sample mass in g, and 100 is the multiplier to express the total content of phenolic compounds in the mg/100 g plant.

2.5. Determination of Tannin Content in Herbal Infusions and Ethanol Extracts

The tannin analysis was conducted using a method described in the European Pharmacopoeia, 8th edition [39]. Freeze-dried samples were prepared using 0.100 g of dry herbal material, and in the case of liquid ethanol extracts, 2 mL was treated according to the procedure described in Eur. Pharm 8 [39]. In brief, a 2 mL filtrated solution was mixed with 1 mL of phosphomolybdotungstic reagent (10% in ethanol) and 10.0 mL of water, then diluted to 25 mL with a sodium carbonate solution (29 g/100 mL). After a 30 min incubation in the dark, the absorbance was measured at 760 nm using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabX™, Greifensee, Switzerland). Pyrogallol (50 mg/100 mL) served as the standard. The results were calculated as percentages of tannins expressed as pyrogallol in dried aerial parts. All measurements were made in triplicate.

The following formula was used to calculate the percentage of tannin content:

$$\mathrm{T}\mathrm{a}\mathrm{n}\mathrm{n}\mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{g} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{d} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t},\mathrm{\%}=\frac{62.5\times \left({\mathrm{A}}_1-{\mathrm{A}}_2\right)\times {\mathrm{m}}_2}{{\mathrm{A}}_3\times {\mathrm{m}}_1},$$

where A₁ is the absorbance of the total polyphenols; A₂ is the absorbance of polyphenols not adsorbed by the hide powder; A₃ is the absorbance of standard pyrogallol; m₁ is the mass of the sample to be examined, in grammes; and m₂ is the mass of pyrogallol, in g.

2.6. Determination of Antioxidant Activity Using a DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Scavenging Assay

DPPH was used to evaluate the free radical scavenging (antioxidant) potential of the extracts. DPPH radical scavenging activity was assessed according to the methodology introduced by [41], with certain adjustments. A DPPH solution was prepared by dissolving 0.0118 g of 2,2-diphenyl-1-picrylhydrazyl in 300 mL of methanol, and it was stored in a dark place. For the assay, varying volumes (1 to 20 µL) of the ethanol extract or 16 mg of freeze-dried extract dissolved in 1 mL of water and then diluted twofold four times, diluted in 10 µL to 29 µL of methanol, were combined with 3 mL of the DPPH solution.

The samples were incubated in darkness at room temperature for 15 min, and the reduction in absorbance at 515 nm was measured using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabX™, Greifensee, Switzerland). A Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) solution (20 mg/10 mL in methanol) served as the standard, while methanol was used as a control. The samples were tested at various concentrations to determine the IC50 (the concentration that reduces the absorbance of DPPH by 50%). The radical scavenging activity was calculated using the following formula:

$$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{\%}=\left[\right({\mathrm{A}}_1-{\mathrm{A}}_0)/{\mathrm{A}}_1]\times 100,$$

where A₁ is the control absorbance and A₀ is the sample absorbance. The antioxidant activity was expressed as IC₅₀ mg/L. The antioxidant activity was quantified in terms of mg/L Trolox equivalents per gramme of dried plant. This was determined by building a calibration curve within the concentration range of 1 to 20 mg/L using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic) acid dissolved in methanol.

2.7. Determination of α-Amylase Inhibition Activity

The extracts of VV, VM leaves, aerial parts, and acarbose solutions were initially prepared in water to evaluate their inhibitory effects on porcine α-amylase inhibition activity using the dinitrosalicylic acid method [42]. Ethanol extract samples were prepared using a twofold dilution: 20 mg/mL–4.0 mL of extract and 6 mL of water. The remaining samples were prepared in the same way at 1 mg/mL. The standard acarbose was prepared the same as ethanol extracts.

The freeze-dried samples were diluted with water from 16 mg/mL to 1 mg/mL. Afterwards, they were combined with 200 μL of α-amylase, 200 μL: of 20 mg/mL sodium phosphate buffer (pH 6.9), and 200 μL of ethanol as a solvent. After pre-incubation of the samples at 25 °C for 10 min, 200 μL of a 1% starch solution in 200 μL sodium phosphate buffer (pH 6.9) was added. The reaction mixtures were then incubated at 25 °C for an additional 10 min.

The reactions were terminated by placing the mixture in a boiling water bath for 5 min after the addition of 400 μL of dinitrosalicylic acid. After the reaction mixtures reached room temperature, they were diluted in a 1:5 ratio with water, and the absorbance was measured at 540 nm using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabX™, Greifensee, Switzerland). This entire procedure was repeated for all samples. Ethanol extracts were prepared in concentrations ranging from 1 mg/mL to 20 mg/mL, while freeze-dried samples were prepared in concentrations ranging from 1 mg/mL to 16 mg/mL.

The inhibition was calculated according to the following formula:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{\%}=\left(\frac{{\mathrm{A}}_{540}^{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{540}^{\mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}}}{{\mathrm{A}}_{540}^{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\right)\times 100,$$

where ${\mathrm{A}}_{540}^{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$ is standard absorbance, ${\mathrm{A}}_{540}^{\mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}}$ is protein extract absorbance.

2.8. Qualitative Analysis of Extracts by Liquid Chromatography-Mass Spectrometry (LC-MS)

The method used in the research was based on the method described by Zolotova et al. [43], Thomas et al. [44], and Ali et al. with slight optimisation [45].

Ethanol extracts (1 mL each) were prepared, and 2 mg of the freeze-dried material was dissolved in 1 mL of methanol. UHPLC-HRMS (ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry) analyses were conducted using a Vanquish Flex UHPLC system (Thermo Fisher Scientific, Germering, Germany) equipped with a Vanquish Binary Pump F and a Vanquish Split Sampler FT. Chromatographic separation was achieved with an ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 μm; Waters, Ireland). The mobile phase consisted of 0.1% formic acid in ultrapure water (Phase A) and 0.1% formic acid in methanol (Phase B). The flow rate was set at 0.3 mL/min, and the column temperature was maintained at 30 °C. The injection volume was 4 μL, and the gradient programme was set as in

Table 1. The gradient program for UHPLC-HRMS analyses.

Time, min	Mobile Phase B, %
−3.0 (equilibration time)	5
0.0	5
1.0	5
15.0	30
20.0	50
25.0	70
26.0	95
28.0	95
29.0	5
30.0	5

.

Mass spectrometric analysis was performed using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionisation (HESI-II) probe (Thermo Fisher Scientific). The instrument operated in both negative and positive ion modes within the m/z range of 100 to 1500. For the mass spectrometer, the spray voltage was 2.5 kV (−); the sheath gas flow rate was 50; the auxiliary gas flow rate was 10; the capillary temperature was 325 °C; the probe heater temperature was 350 °C; the S-lens RF level was 70; and the scan mode was full MS (resolution 60,000) and ddMS2 (15,000), with standard collision energy and HCD normalised collision energy set to 30%. Data were processed using the software Xcalibur 4.6 (Thermo Fisher Scientific, Waltham, MA, USA) for instrument control and data handling. Compound profiling was applied to the UHPLC-HRMS raw files of the studied extracts using the TraceFinder 5.1 software (Thermo Fisher Scientific, Waltham, MA, USA). A library of 120 compounds for the identification of individual components using LC-MS was assembled from various literature sources, including mzCloud, PubChem, FoodDB, and KNApSAcK.

2.9. Statistical Analysis

Descriptive statistical analyses, Spearman correlation analysis, the post hoc Tukey test, and the Mann–Whitney U test were conducted using IBM^® SPSS^® Statistics Software (Version 27.0; IBM Corp.©, Armonk, NY, USA). Significance was established at p < 0.05 for all analyses.

3. Results

3.1. Characterisation of Phenolic Content

3.1.1. Tannin Content in Extracts of Aerial Parts and Freeze-Dried Samples

Table 2. Total tannin content in ethanol extracts and freeze-dried samples.

Plant Sample	Tannin Content %, Ethanol Extract, ±SD	Tannin Content %, Freeze-Dried Samples, ±SD
Vaccinium myrtillus leaves D	1.34 ± 0.02 e	0.14 ± 0.004 h
Vaccinium myrtillus leaves S	1.61 ± 0.05 f	0.12 ± 0.007 h
Vaccinium myrtillus aerial parts D	0.68 ± 0.07 f	0.21 ± 0.001 g
Vaccinium myrtillus aerial parts S	0.90 ± 0.09 f	0.09 ± 0.005 h
Vaccinium vitis-idaea leaves D	2.85 ± 0.07 f	0.02 ± 0.005 g
Vaccinium vitis-idaea leaves S	1.79 ± 0.09 f	0.09 ± 0.006 h
Vaccinium vitis-idaea aerial parts D	0.85 ± 0.16 e	0.18 ± 0.007 g
Vaccinium vitis-idaea aerial parts S	1.10 ± 0.03 f	0.05 ± 0.01 h

In each column, the value with the same superscript letter means the following: ^e, ^g—significant difference; ^f, ^h—insignificant difference at p < 0.05 according to the post hoc Tukey test. D—Dekšāre district, S—Sigulda district.

shows the total tannin content in ethanol extracts and freeze-dried samples. The maximum concentration of tannins was found in ethanol extracts of VV leaves from both locations (D 2.85 ± 0.07% and S 1.79 ± 0.09%). The aerial parts of VM containing 0.68 ± 0.07% and VV 0.85 ± 0.16% ethanol extracts D had the lowest concentration of tannins.

Additionally, the freeze-dried samples yielded contrasting results, revealing that the highest tannin concentrations were evident in VM aerial parts at 0.21 ± 0.001% and VV aerial parts at 0.85 ± 0.16%.

This significant variation in tannin content can be attributed to the various geographical locations where the plants are cultivated. Environmental factors, such as soil composition, climate, and altitude, likely play a crucial role in influencing the tannin composition of plant samples from different locations. Understanding these variations is essential to comprehending the potential impact of growing conditions on the bioactive components of plants.

3.1.2. Total Phenolic Content in Ethanol Extracts and Freeze-Dried Samples

The overall phenolic content in ethanol extracts and freeze-dried leaves and herbs from VM and VV, collected from two different places marked as S and D, is shown clearly in

Table 3. Total phenolic content (TPC) in ethanol extracts and freeze-dried samples, mg/100 g.

Plant Sample	TPC mg/100 g, Ethanol Extract, ±SD	TPC mg/100 g, Freeze-Dried Samples, ±SD
Vaccinium myrtillus leaves D	349.88 ± 0.90 m	589.25 ± 7.78 l
Vaccinium myrtillus leaves S	420.62 ± 1.05 m	706.19 ± 3.10 o
Vaccinium myrtillus aerial parts D	366.73 ± 8.26 m	645.47 ± 1.69 o
Vaccinium myrtillus aerial parts S	368.28 ± 4.66 m	714.39 ± 1.18 o
Vaccinium vitis-idaea leaves D	632.80 ± 2.71 k	825.13 ± 3.25 o
Vaccinium vitis-idaea leaves S	505.33 ± 0.75 m	861.37 ± 8.54 o
Vaccinium vitis-idaea aerial parts D	600.52 ± 2.40 k	941.78 ± 2.75 l
Vaccinium vitis-idaea aerial parts S	470.93 ± 9.00 m	968.52 ± 1.27 l

In each column, the value with the same superscript letter means the following: ^k, ^l—significant difference; ^m, ^o—insignificant difference at p < 0.05 according to the post hoc Tukey test. D—Dekšāre district, S—Sigulda district.

. Predominantly, the highest total phenolic levels are observed in leaves (632.8 mg GAE/g) and aerial parts (600.52 ± 2.40 mg/100 g) of VV D. For the leaves and herbs of VV S, the respective values are 470.8 ± 9.0 mg/100 g and 600.52 ± 2.40 mg/100 g. For VM, the phenolic content in the leaves ranges from 349.88 ± 0.9 mg/100 g to 420.62 ± 1.05 mg/100 g for D and S samples.

Freeze-dried samples exhibit significantly higher amounts compared to ethanol extract samples. In particular, the phenolic content of VV S and D aerial parts is 968.52 ± 1.27 mg/100 g and 941.78 ± 2.75 mg/100 g, respectively. It is also high in the leaves of the same species, at 825.13 ± 3.25 mg/100 g and 861.37 ± 8.54 mg/100 g. The distinct geographical locations of the plants also contribute to the disparity.

3.2. Antioxidant Activity Using a DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Scavenging Assay

Table 4. Radical scavenging activity of ethanol extracts and freeze-dried samples.

Plant Sample	IC50 Value of DPPH Radical Scavenging Activity (mg/L) of Ethanol Extract	IC50 Value of DPPH Radical Scavenging Activity (mg/L) of Freeze-Dried Sample
Vaccinium myrtillus leaves D	10.80 n	0.57 r
Vaccinium myrtillus leaves S	9.52 p	0.51s
Vaccinium myrtillus aerial parts D	8.53 n	0.54 r
Vaccinium myrtillus aerial parts S	10.37 p	0.47 s
Vaccinium vitis-idaea leaves D	5.16 n	0.43 r
Vaccinium vitis-idaea leaves S	6.54 p	0.43 s
Vaccinium vitis-idaea aerial parts D	6.64 n	0.46 r
Vaccinium vitis-idaea aerial parts S	8.05 p	0.42 s

In each column, the value with the same superscript letter means an insignificant difference, at p < 0.05 according to the post hoc Tukey test. D—Dekšāre district, S—Sigulda district. IC50 (concentration required to inhibit 50% of the free radical) of plant samples.

presents the data on the antiradical activity IC₅₀ (concentration required to inhibit 50% of the free radical) of plant samples. VM leaves from locations D and S exhibit high activity, measuring 10.8 mg/L and 9.52 mg/L, respectively. The aerial parts of these plants also show notable antiradical activity.

According to Table 4, the antiradical activity of VV leaves and aerial parts from locations S and D is approximately half that of VM, ranging between 5.16 mg/L and 8.05 mg/L. In freeze-dried samples, the antiradical activity is significantly lower compared to ethanol extracts, ranging from 0.42 mg/L to 0.57 mg/L.

The ethanol extract of VM leaves from Dekšāre exhibited an activity of 10.8 mg/L, slightly higher than that of Sigulda at 9.52 mg/L. In VV, leaves from Dekšāre demonstrated an activity of 5.16 mg/L, while Sigulda exhibited a higher value of 6.54 mg/L. Similarly, for VV aerial parts, Dekšāre displayed an activity of 6.64 mg/L, and for VV aerial parts, Sigulda showed a slightly elevated value of 8.05 mg/L.

In VM leaves, both Dekšāre and Sigulda displayed reduced activities of 0.57 mg/L and 0.51 mg/L, respectively. The Dekšāre aerial parts showed an activity of 0.54 mg/L, and Sigulda exhibited a slightly lower value of 0.47 mg/L. The leaves of VV from both locations exhibited similar activities of 0.43 mg/L, while the aerial parts of Dekšāre and Sigulda showed values of 0.46 mg/L and 0.42 mg/L, respectively.

3.3. α-Amylase Inhibition Activity of Plant Extracts

Table 5. Inhibiting effect of ethanol extracts and freeze-dried samples on α-amylase inhibition activity.

Plant Sample	IC50 Value of α-Amylase Inhibition Activity (mg/mL) of Ethanol Extract	IC50 Value of α-Amylase Inhibition Activity (mg/mL) of Freeze-Dried Sample
Vaccinium myrtillus leaves D	39.33 a	19.89 c
Vaccinium myrtillus leaves S	63.89 b	15.70 d
Vaccinium myrtillus aerial parts D	21.28 a	12.88 c
Vaccinium myrtillus aerial parts S	29.81 b	11.42 d
Vaccinium vitis-idaea leaves D	12.27 a	14.62 c
Vaccinium vitis-idaea leaves S	15.93 b	13.15 d
Vaccinium vitis-idaea aerial parts D	12.30 a	13.81 c
Vaccinium vitis-idaea aerial parts S	14.65 b	11.46 d

In each column, the value with the same superscript letter means an insignificant difference, at p < 0.05 according to the post hoc Tukey test. D—Dekšāre district, S—Sigulda district. IC50 (concentration required to inhibit 50% of the α-Amylase) of plant samples.

presents the data regarding α-amylase inhibition activity. The freeze-dried plant sample showed optimal activity, with VM aerial parts S exhibiting 11.43 mg/mL and VV ethanol extract showing 12.3 mg/mL. The freeze-dried sample exhibited the most effective activity.

The amount of phenolic components strongly correlated with the antiradical activity of the ethanol extracts from the leaves and aerial parts of VM and VV, as demonstrated by a strong negative correlation (R = −0.90, p = 0.005). A similar negative correlation was observed in freeze-dried samples (R = −0.92, p = 0.001). Additionally, in freeze-dried samples, there was a positive average correlation (R = 0.74, p = 0.03) between the number of tannins and antiradical activity.

Furthermore, a strong correlation (R = 0.95, p = 0.05) was found between leaves of VM and VV from locations S and D for DPPH and α-amylase. In ethanol extract samples, a moderate correlation (R = 0.86, p = 0.01) was identified between the DPPH and α-amylase test results. Furthermore, a strong negative correlation (R = −0.81, p = 0.028) was observed between phenolic components and the α-amylase test.

Statistical analysis using the Mann–Whitney U test revealed p-values below 0.05 (p < 0.05) for phenolic components, tannins, and antioxidant activity, indicating significant differences between the obtained results from each methodology. However, no statistically significant results were found for α-amylase inhibition activity. At p < 0.05, according to the post hoc Tukey test, there were relatively few significant differences between sample extracts.

3.4. Qualitative Analysis of Extracts by Liquid Chromatography-Mass Spectrometry (LC-MS)

The qualitative analysis of extracts by LC-MS revealed numerous chemical compounds in each type of extract for all plants.

Table 6. Tentative identification of the chemical constituents of Vaccinium myrtillus and Vaccinium vitis-idaea extracts by UHPLC-MS/MS under negative ionisation.

No.	tR (min)	Tentative Compound	Class/Type	Proposed Formula	Measured (m/z)	Error (ppm)	MS/MS Ions (m/z)
1	0.59	Disaccharide	Carbohydrates	C12H22O11	341.11	−0.75	179.03; 89.02; 59.01
2	0.60	Gluconic acid	Carbohydrates and carbohydrate conjugates	C6H12O7	195.05	0.29	177.04; 129.02
3	0.61	Quinic acid	Cyclic polyols	C7H12O6	191.06	−0.34	383.12; 179.05; 85.03
4	0.69	Malic acid	Dicarboxylic acids	C4H6O5	133.01	0.13	115
5	0.71	Citric acid	Tricarboxylic acids	C6H8O7	191.02	0.32	111.01
6	1.10	Galloyl glucose	Tannins	C13H16O10	331.07	0.12	313.06; 168.01; 125.02
7	1.41	4-Hydroxybenzoic acid glucoside	Phenolic glycosides	C13H16O8	299.08	0.10	179.03; 137.02; 119.04
8	1.43	Syringoylquinic acid	Phenolic acids	C16H20O10	371.10	−1.10	743.20; 197.04; 191.06; 173.05; 135.05
9	2.04	Protocatechuic acid 4-glucoside	Phenolic glycosides	C13H16O9	315.07	0.04	315.07; 153.02; 152.01; 109.03, 108.02
10	2.75	Protocatechuic acid	Benzoic acid and derivatives	C7H6O4	153.02	0.02	153.02; 123.05; 109.03
11	3.25	(Epi)catechin O-hexoside	Polyflavonoids	C21H24O11	451.12	−0.22	289.07; 245.08; 137.02; 125.02
12	5.19	Epigallocatechin/Gallocatechin	Flavonoids	C15H14O7	305.07	−0.50	261.07; 219.07; 179.03; 167.03; 165.02; 139.04; 137.02; 125.02
13	5.27	(Epi)catechin-(4,8′)-(epi)gallocatechin	Flavonoids	C30H26O13	593.13	−0.09	423.08; 305.07;
14	5.31	Neochlorogenic acid	Phenolic acids	C16H18O9	353.09	−1.15	191.05
15	5.70	Chlorogenic acid	Phenolic acids	C16H18O9	353.09	−1.07	191.05
16	6.39	Procyanidin B-type dimer 1	Flavonoids	C30H26O12	577.13		425.09; 407.08; 289.07
17	6.85	Procyanidin B-type dimer 2	Flavonoids	C30H26O12	577.13	−0.45	425.09; 407.08; 289.07
18	7.12	Catechin	Flavonoids	C15H14O6	289.07	−0.15	245.08; 205.05; 203.07; 187.04; 179.03; 161.06
19	7.13	Pavetannin B2	Biflavonoids and polyflavonoids	C45H36O18	863.18	−0.53	711.13; 411.08; 289.07;
20	7.50	Epicatechin	Flavonoids	C15H14O6	289.07	−0.36	245.08; 205.05; 203.07; 187.04; 179.03; 161.06
21	7.74	Procyanidin A-type trimer 1	Flavonoids	C45H36O18	863.18	−0.32
22	7.95	Procyanidin B-type trimer 1	Flavonoids	C45H38O18	865.20	−0.84	739.18; 695.14; 577.14; 451.11; 407.08; 289.07
23	8.12	Procyanidin A-type trimer 2	Flavonoids	C45H36O18	863.18	0.04	711.15; 693.12; 559.08; 411.07; 289.07
24	8.29	Procyanidin B-type trimer 2	Flavonoids	C45H38O18	865.20	−0.70	739.18; 695.14; 577.14; 451.11; 407.08; 289.07
25	8.44	Procyanidin B-type tetramer 1	Flavonoids	C60H50O24	1153.26	−1.77	983.19; 865.21; 577.14; 449.09; 407.08; 287.06
26	8.74	Procyanidin B-type tetramer 2	Flavonoids	C60H50O24	1153.26	−2.72	983.19; 865.21; 577.14; 449.09; 407.08; 287.06
27	8.87	(2R,6x)-7-Methyl-3-methylene-1,2,6,7-octanetetrol 2-glucoside	Fatty acyl glycosides	C16H30O9	365.18	−1.19	347.17; 203.13; 161.05;119.03
28	9.19	3,4-Dicaffeoylquinic acid	Phenolic acids	C25H24O12	515.12	−0.22	353.09; 191.06
29	9.31	3,5-Dicaffeoylquinic acid	Phenolic acids	C25H24O12	515.12	−0.11	353.09; 191.06
30	9.58	Garcimangosone D	Phenolic glycosides	C19H20O9	391.10	−0.95	281.07; 161.05; 137.02; 109.03
31	9.59	4,5-Dicaffeoylquinic acid	Phenolic acids	C25H24O12	515.12	0.01	353.09; 191.06
32	10.14	Quercetin O-hexoside O-deoxyhexoside/Rutin	Flavonoid glycosides	C27H30O16	609.15	−0.49	463.09; 301.03; 178.998; 151.00
33	10.28	Hyperin	Flavonoid-3-o-glycosides	C21H20O12	463.09	−0.73	317.03; 178.998; 151.00
34	10.41	Quercetin 4’-glucuronide	Flavonoid glycosides	C21H18O13	477.07	−0.16	301.03
35	10.51	Quercetin 3-β-D-glucoside	Flavonoid glycosides	C21H20O12	463.09	−0.67	301.03; 300.03; 178.998; 151.00
36	10.79	Aromadendrin O-hexoside	Flavonoid glycosides	C21H22O11	449.11	−0.16	287.06; 151.00; 125.02;
37	11.73	Quercitrin	Flavonoids	C21H20O11	447.09	−0.52	300.03; 269.10; 175.04; 161.05; 151.00
38	12.32	Eriodictyol O-hexoside	Flavonoid glycosides	C21H22O11	449.11	−0.10	287.06; 151.00
39	12.66	Orientin 7,3’-dimethyl ether	Flavonoids	C23H24O11	475.12	−0.72	365.09; 179.04; 161.02
40	13.85	Quercetin derivative	Flavonoids	C30H26O13	591.14	−1.22	529.13; 489.11; 447.09; 301.03
41	15.05	Quercetin	Flavones	C15H10O7	301.04	−0.34	273.04; 178.998; 151.00; 121.03
42	15.27	Luteolin	Flavones	C15H10O6	285.04	0.77	243.03; 151.00
43	17.44	Kaempferol	Flavones	C15H10O6	285.04	0.13	257.05; 229.05; 151.00; 107.01
44	21.34	Triterpenoid	Triterpenoids	C30H48O5	487.34	−0.64	425.34
45	27.85	Oleanolic acid	Pentacyclic triterpenes	C30H48O3	455.35	−0.63	455.35

displays the detected results. The research reveals a diverse set of findings present in leaf and aerial part extracts of VM and VV.

There were different types of these compounds, such as polyflavonoids, bioflavonoids, fatty acyl glycosides, triterpenoids, flavones, and pentacyclic triterpenes. Examples of these compounds are phenolic glycosides, phenolic acids, tannins, cyclic polyols, bioflavonoids, fatty acyl glycosides, and phenolic acids.

Table 7. Presence of chemical constituents of Vaccinium myrtillus extracts by UHPLC-MS/MS under negative ionisation.

No.	Tentative Compound	Class/Type	Plant Samples
			FD of VM lv D	EE of VM lv D	FD of VM hb D	EE of VM hb D	FD of VM lv S	EE of VM lv S	FD of VM hb S	EE of VM hb S
1	Disaccharide	Carbohydrates	X	X	X	X	X	X	X	X
2	Gluconic acid	Carbohydrates and carbohydrate conjugates	X	X	X	X	X	X	X	X
3	Quinic acid	Cyclic polyols	X	X	X	X	X	X	X	X
4	Malic acid	Dicarboxylic acids	X	X	X	X	X	X	X	X
5	Citric acid	Tricarboxylic acids	X	X	X	X	X	X	X	X
6	Galloyl glucose	Tannins	X	X	X	X	X	X	X	X
7	4-Hydroxybenzoic acid glucoside	Phenolic glycosides	X	X	X	X	X	X	X	X
8	Syringoylquinic acid	Phenolic acids	X	n.d.	X	X	X	n.d.	X	X
9	Protocatechuic acid 4-glucoside	Phenolic glycosides	X	X	X	X	X	X	X	X
10	Protocatechuic acid	Benzoic acid and derivatives	X	X	X	X	n.d.	X	X	n.d.
11	(Epi)catechin O-hexoside	Polyflavonoids	X	X	X	X	X	X	X	X
12	Epigallocatechin/Gallocatechin	Flavonoids	X	X	X	X	X	X	X	X
13	(Epi)catechin-(4,8′)-(epi)gallocatechin	Flavonoids	X	X	X	X	X	X	X	X
14	Neochlorogenic acid	Phenolic acids	X	n.d.	X	X	n.d.	n.d.	n.d.	n.d.
15	Chlorogenic acid	Phenolic acids	X	X	X	X	X	X	X	X
16	Procyanidin B-type dimer 1	Flavonoids	n.d.	X	X	X	n.d.	n.d.	X	X
17	Procyanidin B-type dimer 2	Flavonoids	X	X	X	X	X	X	X	X
18	Catechin	Flavonoids	X	n.d.	X	n.d.	n.d.	n.d.	n.d.	n.d.
19	Pavetannin B2	Biflavonoids and polyflavonoids	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	X	n.d.
20	Epicatechin	Flavonoids	X	X	X	X	X	X	X	X
21	Procyanidin A-type trimer 1	Flavonoids	X	n.d.	X	n.d.	n.d.	n.d.	X	n.d.
22	Procyanidin B-type trimer 1	Flavonoids	X	n.d.	X	X	X	n.d.	X	n.d.
23	Procyanidin A-type trimer 2	Flavonoids	X	X	X	n.d.	X	X	X	X
24	Procyanidin B-type trimer 2	Flavonoids	X	X	X	X	X	X	X	X
25	Procyanidin B-type tetramer 1	Flavonoids	X	n.d.	X	X	X	n.d.	X	n.d.
26	Procyanidin B-type tetramer 2	Flavonoids	X	X	X	X	X	X	X	X
27	(2R,6x)-7-Methyl-3-methylene-1,2,6,7-octanetetrol 2-glucoside	Fatty acyl glycosides	X	X	X	n.d.	X	X	X	X
28	3,4-Dicaffeoylquinic acid	Phenolic acids	X	X	X	n.d.	X	X	X	X
29	3,5-Dicaffeoylquinic acid	Phenolic acids	X	X	X	n.d.	X	X	X	X
30	Garcimangosone D	Phenolic glycosides	X	X	X	n.d.	X	X	X	X
31	4,5-Dicaffeoylquinic acid	Phenolic acids	X	X	X	n.d.	X	X	X	X
32	Quercetin O-hexoside O-deoxyhexoside/Rutin	Flavonoid glycosides	X	n.d.	X	n.d.	X	n.d.	X	n.d.
33	Hyperin	Flavonoid-3-o-glycosides	X	X	X	n.d.	X	X	X	X
34	Quercetin 4′-glucuronide	Flavonoid glycosides	X	X	X	n.d.	X	X	X	X
35	Quercetin 3-β-D-glucoside	Flavonoid glycosides	X	n.d.	n.d.	n.d.	X	n.d.	X	n.d.
36	Aromadendrin O-hexoside	Flavonoid glycosides	X	X	X	n.d.	X	X	X	X
37	Quercitrin	Flavonoids	X	X	X	n.d.	X	X	X	X
38	Eriodictyol O-hexoside	Flavonoid glycosides	X	X	X	n.d.	X	X	X	X
39	Orientin 7,3′-dimethyl ether	Flavonoids	X	X	n.d.	n.d.	X	X	n.d.	n.d.
40	Quercetin derivative	Flavonoids	X	X	X	n.d.	X	X	X	X
41	Quercetin	Flavones	X	X	X	X	X	X	X	X
42	Luteolin	Flavones	X	n.d.	X	n.d.	n.d.	n.d.	n.d.	n.d.
43	Kaempferol	Flavones	X	X	X	n.d.	X	X	n.d.	n.d.
44	Triterpenoid	Triterpenoids	X	X	X	X	X	X	X	X
45	Oleanolic acid	Pentacyclic triterpenes	X	X	X	X	X	X	X	X

X—detected; n.d.—not detected; EE—ethanol extract; FD—freeze dried; VM lv D—Vaccinium myrtillus leaves D; VM hb D—Vaccinium myrtillus aerial parts D; VM lv S—Vaccinium myrtillus leaves S; VM hb S—Vaccinium myrtillus aerial parts S. D—Dekšāre district, S—Sigulda district.

and

Table 8. Presence of chemical constituents of Vaccinium vitis-idaea extracts by UHPLC-MS/MS under negative ionisation.

No.	Tentative Compound	Class/Type	Plant Samples
			FD of VV lv D	EE of VV lv D	FD of VV hb D	EE of VV hb D	FD of VV lv S	EE of VV lv S	FD of VV hb S	EE of VV hb S
1	Disaccharide	Carbohydrates	X	X	X	X	X	X	X	X
2	Gluconic acid	Carbohydrates and carbohydrate conjugates	X	X	X	X	X	X	X	X
3	Quinic acid	Cyclic polyols	X	X	X	X	X	X	X	X
4	Malic acid	Dicarboxylic acids	X	X	X	X	X	X	X	X
5	Citric acid	Tricarboxylic acids	X	X	X	X	X	X	X	X
6	Galloyl glucose	Tannins	X	n.d.	X	X	X	n.d.	X	n.d.
7	4-Hydroxybenzoic acid glucoside	Phenolic glycosides	X	X	X	X	X	X	X	X
8	Syringoylquinic acid	Phenolic acids	X	X	X	X	X	X	X	X
9	Protocatechuic acid 4-glucoside	Phenolic glycosides	X	X	X	X	X	X	X	X
10	Protocatechuic acid	Benzoic acid and derivatives	n.d.	X	n.d.	X	n.d.	X	n.d.	X
11	(Epi)catechin O-hexoside	Polyflavonoids	X	n.d.	X	X	X	X	X	X
12	Epigallocatechin/Gallocatechin	Flavonoids	n.d.	X	X	n.d.	X	X	X	n.d.
13	(Epi)catechin-(4,8′)-(epi)gallocatechin	Flavonoids	X	X	X	n.d.	n.d.	n.d.	n.d.	n.d.
14	Neochlorogenic acid	Phenolic acids	X	X	X	n.d.	n.d.	n.d.	X	n.d.
15	Chlorogenic acid	Phenolic acids	X	X	X	X	X	X	X	X
16	Procyanidin B-type dimer 1	Flavonoids	n.d.	X	n.d.	X	n.d.	X	n.d.	X
17	Procyanidin B-type dimer 2	Flavonoids	n.d.	X	X	X	X	n.d.	X	X
18	Catechin	Flavonoids	X	X	X	X	X	X	X	X
19	Pavetannin B2	Biflavonoids and polyflavonoids	n.d.	X	n.d.	X	X	n.d.	n.d.	X
20	Epicatechin	Flavonoids	X	X	X	n.d.	X	X	X	X
21	Procyanidin A-type trimer 1	Flavonoids	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	X
22	Procyanidin B-type trimer 1	Flavonoids	X	n.d.	n.d.	X	n.d.	n.d.	n.d.	n.d.
23	Procyanidin A-type trimer 2	Flavonoids	n.d.	X	X	X	X	X	X	X
24	Procyanidin B-type trimer 2	Flavonoids	X	n.d.	X	X	n.d.	n.d.	X	n.d.
25	Procyanidin B-type tetramer 1	Flavonoids	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
26	Procyanidin B-type tetramer 2	Flavonoids	n.d.	n.d.	n.d.	X	n.d.	n.d.	n.d.	n.d.
27	(2R,6x)-7-Methyl-3-methylene-1,2,6,7-octanetetrol 2-glucoside	Fatty acyl glycosides	n.d.	X	X	X	X	X	X	X
28	3,4-Dicaffeoylquinic acid	Phenolic acids	n.d.	n.d.	X	n.d.	n.d.	n.d.	n.d.	n.d.
29	3,5-Dicaffeoylquinic acid	Phenolic acids	n.d.	n.d.	X	n.d.	n.d.	n.d.	n.d.	n.d.
30	Garcimangosone D	Phenolic glycosides	n.d.	X	X	X	n.d.	X	X	X
31	4,5-Dicaffeoylquinic acid	Phenolic acids	n.d.	n.d.	X	n.d.	n.d.	X	n.d.	X
32	Quercetin O-hexoside O-deoxyhexoside/Rutin	Flavonoid glycosides	n.d.	X	X	X	X	X	n.d.	X
33	Hyperin	Flavonoid-3-o-glycosides	X	X	X	X	X	X	X	X
34	Quercetin 4′-glucuronide	Flavonoid glycosides	n.d.	X	X	X	X	X	n.d.	X
35	Quercetin 3-β-D-glucoside	Flavonoid glycosides	X	X	X	X	X	X	X	X
36	Aromadendrin O-hexoside	Flavonoid glycosides	n.d.	X	X	X	X	X	X	X
37	Quercitrin	Flavonoids	n.d.	X	X	X	X	X	X	X
38	Eriodictyol O-hexoside	Flavonoid glycosides	n.d.	X	X	X	X	X	X	X
39	Orientin 7,3′-dimethyl ether	Flavonoids	n.d.	X	X	X	X	X	X	X
40	Quercetin derivative	Flavonoids	n.d.	X	X	X	X	X	X	X
41	Quercetin	Flavones	n.d.	X	X	X	X	X	X	X
42	Luteolin	Flavones	n.d.	X	X	X	n.d.	X	n.d.	X
43	Kaempferol	Flavones	n.d.	X	X	X	X	X	X	X
44	Triterpenoid	Triterpenoids	X	X	X	X	X	X	X	X
45	Oleanolic acid	Pentacyclic triterpene	X	X	X	X	X	X	X	X

X—detected; n.d.—not detected; EE—ethanol extract; FD—freeze dried; VV lv D—Vaccinium vitis-idaea leaves D; VV hb D—Vaccinium vitis-idaea aerial parts D; VV lv S—Vaccinium vitis-idaea leaves S; VV hb S—Vaccinium vitis-idaea aerial parts S. D—Dekšāre district, S—Sigulda district.

reflect the presence of biologically active substances. The slight difference in the number of certain substances may be due to the location of the collection. Furthermore, we confirm the similarity between the leaves and aerial parts of VM and VV. Furthermore, the following tannins and pseudo-tannins were found: galloyl glucose, chlorogenic acid, neochlorogenic acid, catechin, and epicatechin.

Through a literature review, it is evident that VM and VV are rich in over 20 different substances [16,46,47,48]. Some of the most important components found in VM and VV during our research are chlorogenic acid, catechin, epicatechin, quercetin, kaempferol, procyanidin a-type trimer 1, procyanidin b-type trimer 1, procyanidin a-type trimer 2, procyanidin b-type trimer 2, procyanidin b-type tetramer 1, and procyanidin b-type tetramer 2. Bujor, Pires, et al. have also described these biological substances in other research [14,47,49,50,51,52,53]. In addition, previously undescribed chemical components in VM and VV were detected: pavetannin B2, hyperin, quercitrin, oleanic acid, triterpenoid, and luteolin.

4. Discussion

All examined plants evinced the presence of phenolic acids, flavonoids, and tannins; demonstrated antiradical activity; and showed inhibitory properties towards α-amylase. The freeze-dried samples revealed a higher abundance of polyphenols. The ethanol extract samples have resulted in higher concentrations of tannins, increased antioxidant capacity, and elevated α-amylase inhibition activity.

Generally, freeze-dried extracts tend to show higher TPC values compared to ethanol extracts in both VM and VV. The type and concentration of solvent can affect TPC; the 70% ethanol/water was based on the research data of Brezoi et al. to achieve the maximum attainable concentration of polyphenols in both ethanol extracts and frozen samples [54]. Both are used in pharmaceutical preparation; the production of ethanol extract is more cost-effective and straightforward; however, freeze-dried extracts offer advantages in terms of standardisation and formulation into dosage form.

The highest TPC values were detected in freeze-dried samples of VV aerial parts from Sigulda, while the lowest values were found in Dekšāre VM leaves. The differences could be attributed to the concentration effect during the freeze-drying process. In a research study conducted by Bujor et al., the VM leaf extracts demonstrated a TPC of 142.9 ± 19.2 mg/100 g dry extract [15]. Furthermore, in a separate investigation by the same researcher, the TPC of VV leaf extracts was documented as 158.9 ± 6.0 mg/100 g dry extract [14]. The difference between the data obtained in this study and reference data lies in the location of plant germination. The increase in TPC in freeze-dried samples indicates that this method of processing may enhance the preservation of phenolic compounds compared to traditional ethanol extracts. Furthermore, geographical factors [55,56,57] contribute to differences in phenolic content, as evidenced by the variations between Dekšāre and Sigulda. The data confirmed that the amount of TPC in the VM and VV aerial parts is higher than in berries [58,59]. New sources of biological substances could be discovered by continuing the study of leaves and stems.

Variations in tannin content are observed between locations, as evidenced in both ethanol extracts and freeze-dried preparations. Ethanol extracts of VV leaves from Dekšāre have a significantly higher tannin content (2.85%) compared to Sigulda (1.79%). Tannins have previously been studied in the berries of VM and VV [60,61] and leaves [62]. The presence of tannins was indicated, but no specific results were mentioned. Therefore, the data obtained in our study can be used to compare the number of tannins in VM and VV. Freeze-dried extracts generally show lower tannin content compared to VM leaf ethanol extracts. VV consistently demonstrates a higher tannin content compared to VM in all categories analysed. According to the European Pharmacopoeia, 8th edition [39], the tannin content in VM berries must be a minimum (min) of 1%, but for the Bistortae rhizome 3%, cortex Quercus min 3%, radix Pelargonii 2%, Tormentil tincture (ethanol extract) 1.5%, and Hamamelis leaf 3%. Taking this into account, the leaves and stems of VM and VV contain the optimal amount of tannins.

Understanding the tannin content in different plant parts and processing methods is essential for applications in the food and pharmaceutical industries [63].

The DPPH radical scavenging activity is usually expressed as IC₅₀ (the concentration required to inhibit 50% of free radicals). It varied between different plants and their parts. Freeze-dried samples have lower results than ethanol extracts. Despite this, the results obtained confirm the antioxidant abilities of VM and VV leaves and aerial parts [64].

It has been proven above [65] that there is a correlation between antioxidant activity and α-amylase inhibition activity. A strong negative correlation was observed in our study as well. Additional antioxidant tests can be applied to achieve a more accurate determination of activity [65,66].

The variations observed in antioxidant activities between different parts and locations can be attributed to the diverse composition of bioactive compounds, including phenolic compounds, in these extracts [67,68]. The data obtained in Table 7 and Table 8 shows there is a difference between plants and their parts. Furthermore, the results obtained by other methods (Table 2, Table 3, Table 4 and Table 5) indicate that there is a difference in plants because the number of substances in them differs. The concentration and preservation methods used during lyophilisation may influence the higher antioxidant activities in ethanol extracts compared to freeze-dried extracts. For example, higher antioxidant results were absorbed for VM leaves D (10.8 mg/mL) and VM aerial parts S (10.37 mg/mL), but for α-amylase inhibition activity, the best results were for VM aerial parts S and VV aerial S. According to another study, VM berries show antioxidant possibilities ranging from 10.97 mg/mL to 14.87 mg/mL [69]. Furthermore, α-amylase inhibition activity in VM berries was determined to be 102.68 mg/mL and 32.71 mg/mL, respectively [70]. Plant extracts could replace synthetic food antioxidants, which could affect human health with long-term consumption [71].

α-amylase is an enzyme involved in carbohydrate digestion, and its inhibition can be useful for managing conditions such as T2DM [72]. α-amylase inhibition activity facilitates the enhancement of the breakdown rate of complex starch into glucose. However, excessive inhibition, as seen with acarbose, is associated with increased side effects, such as intestinal disorders [73]. Acarbose’s overactive inhibition of α-amylase leads to the release of undigested starch into the lower gastrointestinal tract, triggering abnormal fermentation by the intestinal microflora. This process results in abdominal discomfort and diarrhoea. Therefore, a moderate inhibition of α-amylase activity is preferable.

The α-amylase inhibition activity concentrations of α-amylase inhibitors in freeze-dried extracts were generally lower than those of ethanol extracts. The most effective inhibitors were found in freeze-dried VV aerial parts S and ethanol extracts of VV aerial parts D. Furthermore, according to Karcheva-Bahchevanska et al., the obtained data indicate no significant difference between the VM berries and leaves [10]. Therefore, leaves or aerial parts can serve as substitutes for berries. According to a previous study, the α-amylase inhibition activity must be at least 50% IC [74]. This is consistent with our results on the properties of VM ethanol extracts.

These findings highlight the potential of Vaccinium extracts to modulate α-amylase activity. Plant-based bioactive substances exhibit various pharmacodynamic effects, including therapeutic actions that are advantageous for people with T2DM and its associated complications [75,76,77]. Polyphenols are considered a potential means of regulating postprandial hyperglycemia through α-amylase inhibition activity. This approach aims to control the amount of glucose released during the digestive process [78]. Similar dose-dependent results have been observed in plants such as Syzygium cumini and Psidium guajava [79,80]. Many aerial parts of plants and their components can lower blood glucose levels, attributed to the presence of tannins, terpenoids, and flavonoids [81]. Tannins and flavonoids are known to possess potential inhibitory effects on α-amylase and α-glucosidase [82].

In order to influence T2DM, plants must contain biologically active substances capable of influencing hyperglycemia, insulin resistance, and oxidative stress. Research mentions substances that have this feature, such as quercetin, vitamin D, vitamin A and E, hesperidin, rutin, curcumin, polyphenols, epicatechin, kaempferol, etc. [24,25,83].

Identification of the chemical constituents was conducted in the negative ionisation mode because polyphenolic compounds were of interest in this study, and based on their structures, the negative mode is more suitable [43,84]. LC-MS analysis identified numerous valuable chemical compounds in ethanol extracts and freeze-dried leaves and aerial parts (VM and VV).

The leaf and aerial parts extracts from these plants are especially due to their bioactive polyphenolic substances (cinchonine, isoorientin, hydroquinone, epicatechin, kaempferol, quercetin, epigallocatechin, etc.) that can change the way glucose is used and have possible effects. For example, cinchonine increases the level of insulin in the plasma, while hydroquinone is a sugar-phosphatase inhibitor, and isoorientin lowers the blood glucose level and protects against cell damage; furthermore, epicatechin has several mechanisms of action, such as antioxidant (reduces oxidation with the help of free radicals), antidiabetic (stimulating insulin secretion), and cardiovascular disease (mitochondria-mediated protection), but epigallocatechin, with its antioxidant and anti-inflammatory properties, enhances glucose-stimulated insulin secretion, etc. [6,7,36,85,86].

It has also been confirmed that tannic acid helps glucose move into fat cells by activating the insulin signalling pathway [87]. It can lower blood sugar levels after meals and prevent or delay glucose absorption by blocking alpha-glucosidase [6].

VM and VV samples contained phenolic compounds known for their biological activities, such as chlorogenic acid, epicatechin, n-caffeoylquinic acid, neochlorogenic acid, kaempferol, catechin, malic acid, citric acid, and quercetin. A review article published by Yongwang Yan [88] indicated that chlorogenic acid has fewer adverse effects compared to the current hypoglycaemic drugs available. In addition, epicatechin has activity against diabetic nephropathy. Consuming epicatechin contributes to various cellular mechanisms, including improved insulin sensitivity. Studies have shown that epicatechin, present in green tea and chocolate, can reduce insulin resistance and lower blood pressure. By decreasing both insulin resistance and blood pressure, the incorporation of epicatechin-rich foods into the diet can aid in preventing the development of T2DM and numerous cardiovascular diseases [89]. The findings suggest that epicatechin could act as an anti-ageing compound. This is supported by improved survival rates in mice with T2DM and positive alterations in several age-related biomarkers [90].

In hyperlipidaemic rat models, citric acid significantly reduced blood glucose and insulin resistance while enhancing insulin sensitivity [91].

Neochlorogenic acid has an anti-inflammatory effect [92], which can be helpful in the treatment of T2DM complications. In a study involving mice with T2DM, neochlorogenic acid was found to regulate abnormalities in lipid metabolism. It also effectively reduced the accumulation of sugary substances in the kidneys’ filtering units and affected key signalling pathways linked to diabetic kidney disease [93]. The kaempferol glycosides found can help combat obesity by reducing the accumulation of fat tissue and lipid levels. Furthermore, they have positive effects on diabetes by enhancing insulin and leptin resistance in mice [94].

Additionally, catechins could offer potential benefits in preventing obesity and vascular disorders in individuals with T2DM who are not treated with insulin. Patients treated with insulinotropic agents showed an increase in insulin levels and a slight decrease in haemoglobin. These results suggest that the consumption of catechin-rich beverages could improve insulin secretion and help maintain lower haemoglobin levels in individuals treated with insulinotropic agents [95]. According to Lin Gou et al. [96], the inhibition mechanism of malic acid on alpha-glucosidase was identified through ligand binding, and the malic acid binding sites for inhibition were investigated by incorporating computational simulations. This suggests that malic acid could serve as a potent dietary supplement to treat T2DM.

According to research data from Rehman et al., using oleic acid or substituting saturated fatty acids with it may be a promising nutritional and therapeutic strategy for treating insulin resistance and T2DM. Despite its broad-spectrum antidiabetic properties, oleic acid has not received significant therapeutic attention. However, studies have demonstrated its ability to mitigate the side effects of other synthetic antidiabetic medications [97]. The quercetin derivative is effective in enhancing cardiac function, reducing lipid peroxidation, and scavenging free radicals. Furthermore, rutin improves glucose uptake both in vivo and in vitro and exhibits an antidiabetic effect by boosting insulin release and lowering blood glucose levels [92]. As these substances are found almost in all plant samples, we can conclude that VM and VV leaves and stems influence T2DM.

Our study provides an opportunity to incorporate VM and VV leaves and aerial parts into dietary considerations. Based on the data obtained, we can infer that these plants exhibit the potential to regulate blood glucose metabolism (as indicated by data on α-amylase inhibition activity) and possess antioxidant properties, which contribute to their protective attributes. Additionally, VM is rich in tannins and polyphenols, further enhancing its antioxidant properties, while VV leaves and aerial parts also display these beneficial characteristics.

5. Conclusions

It is evident that Vaccinium myrtillus and Vaccinium vitis-idaea are abundant sources of polyphenols, displaying notably high values. The presence of phenolic compounds in these plant extracts plays a substantial role in their antioxidant activity. Interestingly, the plants exhibited comparable levels of tannins, an aspect often overlooked in research despite their pharmacological significance. When comparing types of preparations, freeze-dried extracts showed a higher total phenolic content, while ethanol extracts exhibited higher levels of tannins. Furthermore, antiradical properties and α-amylase inhibition activity were more pronounced in ethanol extract samples compared to freeze-dried samples, emphasising the substantial impact of processing plant materials. The best sources of bioactive compounds and demonstrated bioactivity were the aerial parts of Vaccinium myrtillus D and Vaccinium vitis-idaea aerial parts S. The chromatographic-mass spectrometric analyses indicated the ability of Vaccinium myrtillus and Vaccinium vitis-idaea to influence type 2 diabetes mellitus. Variability in results was observed between plants collected at different locations. A potential extension of the study could involve a comparative analysis of leaves, aerial parts, and berries within plant specimens, and using other extraction methods could be additionally considered.