Antioxidant Capacity of Honey Enriched by Wildflowers

Abstract

The study objective was a comparative analysis of rapeseed and multifloral honey enriched by flowers of six plant species: lungwort (Pulmonaria officinalis L.), high mallow (Malva sylvestris L.), cowslip primrose (Primula veris L.), coltsfoot (Tussilago farfara L.), lawn daisy (Bellis perennis L.), and black elderberry (Sambucus nigra L.). The honey was enriched with dry flowers and plant extracts at a level of 1%, 2%, and 4% (w/w). Antioxidant capacity was measured via two different methods: DPPH and ABTS assay. Total phenolic content and total flavonoid content were determined using colorimetric methods. The highest radical scavenging capacity determined by the DPPH assay was observed in rapeseed honey with a 4% dried cowslip primrose (Primula veris L.) flower addition, which was more than 50 times higher than the activity for pure rapeseed honey. Almost 100% of the radical scavenging capacity was found for rapeseed and multifloral honeys with cowslip primrose (Primula veris L.), especially for the 4% dried flower addition, more than six times that of the control samples measured using the ABTS test. Multifloral honeys enriched with black elderberry (Sambucus nigra L.) and cowslip primrose (Primula veris L.), with a 2% and 4% plant material addition, both as an extract and as dried flowers, were characterised by the highest total phenolic content. The highest enrichment effectiveness was observed for dried flowers of lungwort (Pulmonaria officinalis L.), black elderberry (Sambucus nigra L.), and high mallow (Malva sylvestris L.), where the flavonoid content increased more than nine times compared to the honey samples without additions. The content of biologically active substances in honey enriched with flowers gives hope for new applications of the health-promoting substances contained in wild plants.

1. Introduction

Honey is a natural substance produced by honeybees (Apis mellifera) from the nectar of flowers, which is a sweet, flavourful, and viscose liquid. This unique product is highly saturated in sugars containing primarily mono- and disaccharides, such us glucose, fructose, and saccharose, the percentage of which reaches approximately 80% [1]. In addition to the ingredients, such as amino acids, minerals, vitamins, or bioactive substances, honey also contains about 14–20% water, which is an important indicator of the stability of honey [2]. Additionally, it contains a plethora of compounds with various effects, including antioxidants, which, despite occurring in small amounts in honey, have demonstrated their valuable health-promoting potential, which was already well known in antiquity. The nutritional and medicinal properties of honey are derived specifically from the presence of these antioxidants, both enzymatic and non-enzymatic [3]. The former include catalase and glutathione peroxidase [4]. Non-enzymatic antioxidants include flavonoids, non-aromatic organic acids, phenolic acids and their esters, free amino acids, mainly proline, and some vitamins and carotenoid derivatives [5,6,7]. Natural honey’s concentration of phenolic compounds that exhibit antimicrobial and antioxidant properties can exceed 250 µg·g⁻¹ [8]. The quality of honey, and especially its composition, depends on several environmental factors during production, such as the weather and the humidity inside the hive, the nectar conditions, and the treatment of the honey during extraction and storage, but primarily depends on the feeding of the bees [9].

The high health, dietary, and technological value of honey, as well as the growing demand for honey in modern times, combined with the high dependence on honey quality and its chemical composition being based on a number of factors [10,11,12], have led to the emergence of a new product—herbal honey, which is designed by beekeepers and “tailored” by the honeybees. Herbal honey is a honey-like product, produced indirectly by bees fed by the beekeeper with a syrup containing mainly saccharose enriched with herbal extracts or fruit juices [6,13]. A comparative study of three natural honey varieties and five herbal honey types demonstrated that herbal honeys were characterised by a higher antioxidative capacity than natural honeys. Furthermore, herbal honey with nettles had the highest antibacterial capacity [14,15]. The antimicrobial effect of herbal honeys against microorganisms, except E. coli, was confirmed by a study by Isidorov et al. [16]. However, herbal honeys are generally characterised by higher saccharose content than that permitted for natural honeys [14]. Despite the proven benefits of herbal honeys, from a clinical standpoint, it is definitely recommended to use only natural honeys, which are only minimally processed to aid in maintaining their natural spectrum of biological activity [17]. Therefore, in order to improve the antioxidative capabilities of beekeeping products rather than produce herbal honeys, it appears that a better solution is to enrich natural honeys with plant extracts or ground-dried medicinal plants for which honey can become a natural and tasty carrier highly acceptable to consumers, often suppressing the not very pleasant taste of health-promoting additives, such as herbs or algae [18].

Honey with various additives has become an innovative product with diversified sensory and health properties, which has expanded the range of bee products on the market and given them a new meaning, not only in terms of consumption but also in marketing, because honey with attractive additives in terms of colour and taste and that is aesthetically packaged has been sold to customers who were not even supporters of pure honey. Due to the wide variety of additives and the constantly growing customer interest in the product, it has also become the subject of research. The main goal of the experiments was to improve antioxidant properties and increase the content of bioactive substances, such as phenolic compounds and flavonoids. The results of a recent study confirmed that the antioxidant activity of honey enriched with mulberry leaves increased by even more than 50 times [19]. Increased phenol content, and consequently improved antioxidative ability, was achieved by enriching natural honey with algae extracts [18], Rubus leaf and flower extracts [20], lavender flowers (Lavandula L.), lemon balm flowers (Melissa L.), nettle (Urtica L.), peppermint flowers (Mentha L.), and ginger root (Zingiber Boehm.) [21]. Similarly successful was an attempt to enrich natural honey by macerating Melilotus officinalis and Melilotus albus flowers (coumarin content increased several-fold) at room temperature for 6 months [22]. Enriching natural honeys with medicinal plant extracts leads to increased content of pro-health ingredients in the honey. However, only the synergistic effect of honey and herbs, which has not been largely studied, can provide a product effective at curing many ailments. An example is the synergistic effect of honey and coffee, which has been scientifically verified as a highly effective means of curing persistent post-infection cough [23] and oral cavity mucous membrane inflammation [24].

In food production, flowers, primarily decorative and garden varieties, are used mainly as decorative elements in culinary products [25]. Little robust information is available on the pro-health potential of many wild-growing and medicinal plant flowers, or their potential has not been thoroughly investigated [26,27]. The literature provides information on the diversity of the bioactive compounds found in different morphological parts of medicinal plants, as well as the possibilities for selective extraction of bioactive substances from plant material [28,29]. Their potential as a pharmaceutical material has been disqualified since insufficient concentrations of medicinal substances can be a precious material for food production, providing disease prevention and health-boosting properties but not medicinal qualities, unless all of the possible contraindications have been analysed. In recent years, numerous studies have been undertaken based on traditional medicine, herbal medicine, and biomedicine [30,31]. As a result of significantly improved nutritional knowledge, consumer behaviours, such as the greening of consumption, consumer ethnocentrism, self-treatment, and subordinating consumer decisions to sustainable lifestyle principles, can all be seen. Familiarising consumers with the importance and availability for consumption of wild-growing plants and medicinal plants in other forms than the dried products found in pharmacies and health food stores is an important trend in health promotion and disease prevention activities. Enriching organic honey with dried plants and extracts from wild-growing plants with high pro-health activity can become a highly effective alternative for food supplements recommended for immunity boosting.

In the present study, we investigated the effect of the addition of different wildflowers, such as lungwort (Pulmonaria officinalis L.), high mallow (Malva sylvestris L.), cowslip primrose (Primula veris L.), coltsfoot (Tussilago farfara L.), lawn daisy (Bellis perennis L.), and black elderberry (Sambucus nigra L.), on improving honey antioxidant activity and the level of total phenols and flavonoids. The results of this work will help verify whether honey is a good matrix for enrichment in phenolic compounds from the plants studied and also the transfer of secondary metabolites from the dried plant material or plant extract to the matrix with a high concentration of sugars, especially without extensive mechanical treatment. This research will define which flowers and the level of plant additives among those tested have the most effective influence on the increased pro-health-promoting activity of honey.

2. Materials and Methods

2.1. Chemicals and Reagents

For the purpose of determinations, analytical purity reagents designed for liquid chromatography were used: acetic acid, ethanol, and acetonitrile from Sigma-Aldrich (St. Louis, MO, USA) and methanol from J.T. Baker (Phillipsburg, NJ, USA); 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), Folin–Ciocalteu, sodium nitrite, sodium carbonate, aluminium chloride, sodium hydroxide, catechin, potassium persulfate, and gallic acid. All of the reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). Deionised water from the deioniser type HLP 5P was used (Hydrolab, Poznan, Poland).

2.2. Plant Material

The plant material in the form of flowers from six species of wild-growing plants (

Table 1. Plant species used for honey enrichment.

Latin name	Pulmonaria officinalis L.	Malva sylvestris L.	Primula veris L.	Tussilago farfara L.	Bellis perennis L.	Sambucus nigra L.
Common name	Lungwort	High mallow	Cowslip primrose	Coltsfoot	Lawn daisy	Black elderberry
Collection time	April	June	April	May	May	June

Photos source: https://wikipedia.org (accessed on 28 February 2024).

) in natural ecological habitats located in mountainous areas within the Carpathian Foothills region in southeastern Poland was collected in April–June 2022. The test material was cleaned of dust and solids and dried at 25 °C in a DanLAB (Białystok, Poland) air circulation laboratory dryer until a water content of 6–8% was achieved. After botanical identification, the material of several plants from each species was dried in the same conditions as the tested plants. The voucher specimens of the plants were deposited in the Department archive (PO/4/2022; MS/6/2022; PV/4/2022; TF/5/2022; BP/5/2022; SN/6/2022). Water content in the dried flower samples was determined using an Ohaus MB12 (Nänikon, Schweiz) moisture analyser with an infrared radiator. The dried flowers were ground in an A 11 Basic Analytical Mill (IKA, Staufen im Breisgau, Germany). The ground plant material was divided into two parts. The first part was used directly to enrich the honey, and the second part was used to prepare the ethanolic extracts.

Table 2. Ingredients used to prepare the honey samples.

	Samples Enriched by Dried Flowers			Samples Enriched by Extract
	1%	2%	4%	1%	2%	4%
Honey (g)	99.0	98.0	96.0	98.0	98.0	98.0
Dried flowers (g)	1.0	2.0	4.0	-	-	-
Extract (mL)	-	-	-	0.5	1.0	2.0
Ethanol 50% (mL)	-	-	-	1.5	1.0	0.0

shows the proportions of ingredients used to prepare the honey samples.

2.3. Preparation of the Flower Extracts

Approximately 20 grams of ground plant was extracted with 100 mL 70% ethanol using an ultrasonic bath (Sonic-6D, Polsonic, Warsaw, Poland) at 40 °C for 60 min. After ultrasonification, the samples were moved to a Biosan ES-20/60 (Riga, Latvia) rotary shaker, and the mixture was extracted at 40 °C and 180× g for 30 min. The samples were then filtered in-vacuo using Wattman 3 filter paper. Subsequently, the extracts were centrifuged at 3500× g for 20 min (Eppendorf 5702, Hamburg, Germany). The supernatant was collected, and the solution was evaporated in vacuo using a rotary evaporator at 40 °C (Hei-VAP Precision, Heidolph, Schwabach, Germany), and the dried extract was then dissolved in 10 mL of 50% ethanol and used to enrich the honey samples according to the proportions shown in Table 2.

2.4. Honey Samples and Experimental Conditions

Two varieties of honey, namely multifloral and rapeseed (Brassica napus L.), were used for the analysis. These honey samples were sourced from apiaries situated in a mountainous agro-forestry region within the Carpathian Foothills in southeastern Poland, located at precisely 49°48′09″ N 21°31′53″ E. The floral origin of the samples was specified by the beekeepers based on the hive’s location and available floral sources. The honey samples were divided into sterile glass containers equipped with lids. Plant material, either in the form of ground-dried plants or ethanol extracts, was then introduced into the containers. The mixture was carefully and thoroughly stirred to ensure proper integration. The plant addition was 1%, 2%, or 4% (w/w). For the extracts, a volume corresponding to an identical mass of plant material was used. The control samples were honey of both varieties without any plant material added. The honey samples were kept in a dark place at room temperature (21 ± 1 °C) until analysis. The sample storage time was 3 months, and the samples were stirred once every 30 days. Before further preparation procedures, the honeys were liquefied using an ultrasonic bath (Sonic-6D, Polsonic, Warsaw, Poland) at 40 °C for 1 h. For testing, 5 g portions from the honey samples were moved into sterile plastic flasks with lids and dissolved in 20 mL of water with a 1% acetic acid addition. The aqueous solutions were again placed in an ultrasonic bath for 30 min (40 °C), then purified under a vacuum on membrane filters (0.45 μm), and evaporated at 40 °C until dry in a Hei-VAP Precision (Heidolph, Schwabach, Germany) vacuum evaporator. The residue was dissolved in 15 mL of water with a 1% acetic acid addition. Honey sample solutions were applied in their entirety on conditioned cartridges with a C18 bed (Sep-Pak C18 500 mg, Waters, Milford, MA, USA). Polyphenolic compounds were eluted with 10 mL of methanol directly into a round-bottomed flask and evaporated at 40 °C until dry. The residue was dissolved in 2.5 mL MS grade methanol, filtered through nylon filters with a 0.22 µm pore diameter (Biospace, Poznań, Poland), and subsequently analysed.

2.5. Total Antioxidant Capacity

The antioxidant capacity was measured via two different methods: DPPH, as described by Brand-Williams [32], and ABTS assay, based on a procedure reported by Re et al. [33] with slight modifications. The free radical scavenging capacity (%) was calculated using the following formula (1), where A is absorbance and expressed as a percentage ± standard deviation.

A solution of DPPH (2,2-diphenyl-1-picrylhydrazyl), 0.1 mM in MeOH, was used with an absorbance of 0.95 (±0.03) at 517 nm. Approximately 10 µL of each tested honey sample was added to 140 µL of DPPH solution in the wells of a 96-well plate. Absorption was measured after 30 min from the sample addition at 517 nm.

ABTS (2,20-azino-di-(3-ethylbenzothiazoline-sulfonic acid)) was dissolved in water for a 7.5 mM solution. ABTS radical cation (ABTS^•+) was generated by reacting 7.5 mM of ABTS with 2.5 mM of potassium persulfate. The mixture was allowed to stand in the dark at room temperature for 16 h before use. The ABTS^•+ stock solution was diluted with methanol to obtain an absorbance of 0.90 (±0.02) at 734 nm. The diluted ABTS solution (140 µL) was mixed with 10 µL of the sample prepared from a 50% stock solution of the honey sample. All measurements were performed using an Epoch microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Trolox and butylated hydroxytoluene (BHT) were used as a positive control at the concentrations of 15 µM, 45 µM, and 60 µM.

Radical scavenging (%) = [(A_(DPPH/ABTS^•+₎ − A_(sample)) × (A_(DPPH/ABTS^•+₎⁻¹] × 100

(1)

2.6. Total Phenolic Content (TPC)

Total phenolics were determined via the spectrophotometric method using a Folin–Ciocalteu reagent as described by Jańczak-Pieniążek et al. [34]. Briefly, 75 µL of the sample was mixed with 975 µL of H₂O; then, 75 µL of the Folin–Ciocalteu reagent (diluted with water 1:1) was added. After 3 min of incubation in darkness at room temperature, 125 µL of 20% Na₂CO₃ was added and mixed. The absorbance of the blue complex was measured at 725 nm. The phenolic content was read from the calibration curve for gallic acid, and the content was expressed in milligrams of gallic acid equivalent per 100 g. The solvent for gallic acid was methanol and the concentrations were in the range of 0.1–1.0 mM.

2.7. Total Flavonoid Content

Total flavonoids were determined using the colorimetric method described previously by Zhishen et al. [35]. First, 250 µL of plant extract was mixed with 637 µL of distilled water; then, 38 µL of 5% sodium nitrite was added. After 6 min, 75 µL of 10% aluminium chloride solution was added and left to stand for 5 min. Then, 250 µL of 1 M NaOH was added. The absorbance was measured at 510 nm. The flavonoid content was read from the calibration curve for catechin, and the content was expressed as mg of catechin equivalent per 100 g. The solvent for catechin was methanol and the concentrations were in the range of 0.1–1.0 mM.

2.8. Statistical Analysis

All analyses were performed in three independent replications for each honey sample. The contents of total phenols and flavonoids and antioxidant activity were expressed as the mean ± standard deviation. The obtained results were presented using hierarchical clustering analysis and heatmap visualisation. Clustering was performed using the Ward distance matrix, which was formed based on the Euclidean distance (data were standardised). Data were analysed via one-way analysis of variance (ANOVA) using Statistica, v.13.3 (StatSoft, Inc., Tulsa, OK, USA). The significances of the differences were calculated using Tukey’s multiple range test (p ≤ 0.05).

3. Results and Discussion

The two most commonly used tests, DPPH (free radical scavenging activity) and ABTS (total antioxidant capacity), were used to assess the antioxidative properties of honeys enriched with flowers and floral extracts in our study.

Table 3. Total antioxidant activity (expressed as the percentage ± standard deviation) of the tested honey products against the DPPH radical.

Sample/Plant Species	Form of Enrichment	Concentration
		1%	2%	4%
Rapeseed honey	None	1.61 ± 0.93 u
Lungwort (Pulmonaria officinalis L.)	Extract	5.83 ± 2.74 stu	11.43 ± 2.14 oprst	19.64 ± 1.79 klmn
	Dried flowers	24.84 ± 1.59 jkl	38.61 ± 5.26 gh	67.10 ± 1.79 bc
High mallow (Malva sylvestris L.)	Extract	5.16 ± 0.16 tu	8.29 ± 0.36 rstu	15.28 ± 1.31 mnopr
	Dried flowers	9.64 ± 1.31 prstu	17.09 ± 2.38 lmnop	73.61 ± 3.64 b
Cowslip primrose (Primula veris L.)	Extract	13.89 ± 0.95 noprs	18.17 ± 1.75 klmno	41.43 ± 0.08 gh
	Dried flowers	25.79 ± 5.24 jk	72.66 ± 4.96 b	83.06 ± 0.99 a
Coltsfoot (Tussilago farfara L.)	Extract	15.44 ± 0.04 mnopr	29.60 ± 1.43 ij	41.86 ± 2.34 fgh
	Dried flowers	23.13 ± 2.82 jklm	44.32 ± 0.36 efg	58.73 ± 5.15 cd
Lawn daisy (Bellis perennis L.)	Extract	7.74 ± 2.98 rstu	13.06 ± 1.39 noprs	22.88 ± 2.40 jklm
	Dried flowers	14.90 ± 2.18 mnopr	25.66 ± 4.03 jk	42.14 ± 4.05 fgh
Black elderberry (Sambucus nigra L.)	Extract	20.28 ± 0.59 klmn	35.30 ± 2.56 hi	51.31 ± 3.21 de
	Dried flowers	21.27 ± 0.24 jklmn	50.24 ± 0.87 ef	66.27 ± 0.63 bc
Multifloral honey	None	3.76 ± 0.58 s
Lungwort (Pulmonaria officinalis L.)	Extract	14.11 ± 0.87 nopr	20.48 ± 0.48 lmn	34.23 ± 2.81 gh
	Dried flowers	28.84 ± 2.39 hij	43.84 ± 2.29 de	80.32 ± 0.58 a
High mallow (Malva sylvestris L.)	Extract	8.87 ± 1.32 prs	14.64 ± 1.81 nopr	17.58 ± 0.35 lmn
	Dried flowers	14.58 ± 0.84 nopr	28.39 ± 2.58 ijk	35.97 ± 1.58 fg
Cowslip primrose (Primula veris L.)	Extract	8.35 ± 1.32 rs	14.16 ± 0.29 nopr	28.90 ± 0.58 hij
	Dried flowers	50.29 ± 2.81 c	74.64 ± 1.11 a	80.26 ± 3.16 a
Coltsfoot (Tussilago farfara L.)	Extract	13.06 ± 0.74 opr	23.23 ± 0.77 jkl	49.81 ± 4.84 cd
	Dried flowers	14.71 ± 0.90 nopr	22.35 ± 2.30 klm	48.28 ± 4.76 cd
Lawn daisy (Bellis perennis L.)	Extract	10.87 ± 1.00 opr	9.35 ± 0.34 prs	23.74 ± 0.32 jkl
	Dried flowers	16.29 ± 0.68 mno	26.93 ± 0.93 ijk	35.39 ± 1.97 fg
Black elderberry (Sambucus nigra L.)	Extract	14.90 ± 2.13 nop	29.61 ± 2.32 ghij	41.32 ± 1.58 ef
	Dried flowers	32.45 ± 1.61 ghi	47.00 ± 3.00 cde	63.42 ± 0.19 b
Positive control	Form of enrichment	Concentration
		15 µM	45 µM	60 µM
Trolox	None	12.50 ± 0.13	35.00 ± 0.50	47.50 ± 0.50
BHT	None	5.00 ± 0.05	7.50 ± 0.25	10.00 ± 0.05

Results marked by different letters are statistically different at p ≤ 0.05 according to Tukey’s test.

shows the average antiradical activity results for honeys enriched with flowers, determined using the DPPH method.

The highest radical scavenging capacity was observed in rapeseed honeys with a 4% dried cowslip primrose (Primula veris L.) flower addition, and it was more than 50 times higher than the activity for pure rapeseed honey. Rapeseed honeys with the addition of lungwort (Pulmonaria officinalis L.) and black elderberry (Sambucus nigra L.) flowers and multifloral honeys enriched with black elderberry (Sambucus nigra L.) flowers were characterised by antioxidative activity lower by approx. 20% compared to the highest results in this group, which was observed for the 4% plant addition. The lowest values for the 4% plant addition were noted for rapeseed honeys with added lungwort (Pulmonaria officinalis L.) and high mallow (Malva sylvestris L.), and multifloral honeys with lawn daisy (Bellis perennis L.) and high mallow (Malva sylvestris L.) extract. Enriching honeys at the 1% plant addition level confirmed the highest antioxidative activity of multifloral honeys enriched with the cowslip primrose (Primula veris L.) extract. Honeys enriched with black elderberry (Sambucus nigra L.) flowers exhibited approximately 40% lower antioxidative activity than honeys with cowslip primrose (Primula veris L.). Among the rapeseed honeys enriched with 1% flowers, honeys with dried cowslip primrose (Primula veris L.), lungwort (Pulmonaria officinalis L.), and coltsfoot (Tussilago farfara L.) exhibited the highest antiradical activity. The lowest antioxidative activity was observed for multifloral and rapeseed honeys enriched with high mallow (Malva sylvestris L.), cowslip primrose (Primula veris L.), and lawn daisy (Bellis perennis L.) (multifloral honeys), and (Pulmonaria officinalis L.) (rapeseed honeys) floral extracts. As a result of enriching the honeys with flowers and extracts from wild-growing spring flowers, the antioxidative properties were improved with dried flower additions over those with flower extracts, compared to honeys of the two varieties without the additions. Among all of the flower additions tested, dried cowslip primrose (Primula veris L.) was the most effective at improving the antioxidative properties of honey determined using the DPPH method at all enrichment ratios, with high effectiveness of the extract observed for the 2% addition, where the antioxidative activity observed was almost 3 times higher than for a flower addition half as large and only 7% lower than for a dried cowslip primrose (Primula veris L.) addition twice as large.

Higher values were observed for the antioxidative activity test using the ABTS method (

Table 4. Total antioxidant activity (expressed as percentage ± standard deviation) of the tested honey products against the ABTS radical.

Sample/Plant Species	Form of Enrichment	Concentration
		1%	2%	4%
Rapeseed honey	None	15.98 ± 0.31 s
Lungwort (Pulmonaria officinalis L.)	Extract	25.00 ± 1.82 pr	43.29 ± 1.18 klm	58.04 ± 4.78 hi
	Dried flowers	73.13 ± 3.20 ef	99.51 ± 0.11 a	99.85 ± 0.05 a
High mallow (Malva sylvestris L.)	Extract	21.35 ± 0.94 rs	40.29 ± 0.15 lmn	70.91 ± 0.69 fg
	Dried flowers	48.37 ± 2.71 jk	69.58 ± 0.34 fg	99.90 ± 0.00 a
Cowslip primrose (Primula veris L.)	Extract	38.21 ± 3.89 mno	54.59 ± 3.81 ij	92.06 ± 0.84 bc
	Dried flowers	84.86 ± 0.34 d	99.90 ± 0.00 a	100.00 ± 0.00 a
Coltsfoot (Tussilago farfara L.)	Extract	31.51 ± 0.05 op	55.32 ± 0.89 ij	86.59 ± 4.44 cd
	Dried flowers	64.30 ± 4.34 gh	98.18 ± 0.25 ab	99.70 ± 0.11 a
Lawn daisy (Bellis perennis L.)	Extract	20.81 ± 0.31 rs	34.21 ± 0.94 no	70.22 ± 1.46 fg
	Dried flowers	46.10 ± 2.42 kl	70.76 ± 1.63 fg	79.49 ± 1.48 de
Black elderberry (Sambucus nigra L.)	Extract	54.59 ± 3.01 ij	70.96 ± 4.98 fg	94.67 ± 2.17 ab
	Dried flowers	48.37 ± 2.71 gh	69.58 ± 0.34 ab	99.91 ± 0.00 a
Multifloral honey	None	13.49 ± 0.59 o
Lungwort (Pulmonaria officinalis L.)	Extract	30.73 ± 1.16 mn	55.68 ± 1.64 gh	90.08 ± 6.84 bcd
	Dried flowers	88.25 ± 0.00 cd	99.61 ± 0.00 a	99.85 ± 0.14 a
High mallow (Malva sylvestris L.)	Extract	28.52 ± 1.16 n	44.91 ± 3.08 ijkl	56.33 ± 2.31 gh
	Dried flowers	52.54 ± 2.21 ghi	80.15 ± 1.86 de	93.79 ± 1.54 abc
Cowslip primrose (Primula veris L.)	Extract	41.18 ± 3.23 kl	58.62 ± 9.78 fg	78.13 ± 3.66 e
	Dried flowers	97.41 ± 0.87 abc	99.85 ± 0.05 a	99.85 ± 0.05 a
Coltsfoot (Tussilago farfara L.)	Extract	38.44 ± 2.71 lm	59.63 ± 5.20 fg	98.84 ± 0.19 ab
	Dried flowers	44.03 ± 0.58 ijk	54.13 ± 2.61 ghi	98.84 ± 0.38 ab
Lawn daisy (Bellis perennis L.)	Extract	28.76 ± 0.43 n	41.57 ± 4.29 jkl	66.86 ± 1.06 f
	Dried flowers	50.77 ± 0.38 ghij	66.28 ± 2.50 f	83.38 ± 3.99 de
Black elderberry (Sambucus nigra L.)	Extract	48.41 ± 2.84 hijk	66.52 ± 1.20 f	98.12 ± 0.72 ab
	Dried flowers	79.38 ± 1.93 de	98.89 ± 0.05 ab	99.52 ± 0.10 a
Positive control	Form of enrichment	Concentration
		15 µM	45 µM	60 µM
Trolox	None	13.25 ± 0.75	47.50 ± 1.00	67.50 ± 1.00
BHT	None	17.75 ± 0.85	55.75 ± 1.25	76.50 ± 1.25

Results marked by different letters are statistically different at p ≤ 0.05 according to Tukey’s test.

) than with the DPPH test both for honey samples without additions and for enriched honeys at all levels.

Thirty min incubation of the extracts resulted in almost 100% of the radical scavenging capacity for rapeseed and multifloral honeys with cowslip primrose (Primula veris L.), black elderberry (Sambucus nigra L.), high mallow (Malva sylvestris L.), lungwort (Pulmonaria officinalis L.), and coltsfoot (Tussilago farfara L.) additions, especially for a 4% dried flower addition. The lowest antioxidative potential characterised rapeseed honey samples with 1% lawn daisy (Bellis perennis L.) flower extract, which was 23% higher than the antioxidative potential value of this honey variety without an addition, while for a 4-times-higher extract or dried flower addition, the antioxidative activity values observed were 70% and 80% higher, respectively, than in the samples without additions, both for rapeseed and multifloral honey. The best improved antioxidative potential was observed in rapeseed and multifloral honeys when enriched with dried cowslip primrose (Primula veris L.) at all addition ratios, with even a 1% addition in multifloral honeys providing almost complete radical scavenging capacity. The lowest antiradical activities characterised honey samples of both varieties at all enrichment ratios with extracts of lawn daisy (Bellis perennis L.), high mallow (Malva sylvestris L.), and lungwort (Pulmonaria officinalis L.) (only rapeseed honeys) on each of the three enrichment ratios.

Based on studies on the antioxidative activity of different honey varieties, Dżugan et al. [36] found that this kind of analysis can be a marker for individual honey varieties. Furthermore, they demonstrated that the antioxidative activity of rapeseed and multifloral honeys was the lowest and that they were distinguished by the brightest colour among the tested honey varieties, which supports the selection of these two varieties for our experiment. Antioxidative tests are in vitro methods designed to imitate the oxidation and reduction reactions occurring in living biological systems to assess the antioxidative potential of different chemical and biological samples. According to Gil et al. [37], the ABTS test values were, in general, significantly higher than the DPPH test values. Nevertheless, they should be considered a confirmation of the DPPH test. This was also confirmed by Aebisher et al. [38], who, in their study on determinations of the antioxidative activity of essential oils, demonstrated that the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene, which contain a phenol ring, strongly capture ABTS in comparison with the DPPH radical. The results of our total antioxidant capacity determinations in both tests are presented as percentage values to show how effectively the synthetic radical is neutralised in honeys with plant additives, because it is increasingly frequently suggested that synthetic antioxidants, such as BHA and BHT, exhibit toxic properties and are potentially harmful to human health [39,40].

The second stage of the experiment consisted of the analysis of total phenolic content (TPC) in honeys enriched with dried flowers and floral extracts. The test results are presented in

Table 5. Content of total phenols (mg of gallic acid equivalent per 100 g) in the tested honey products.

Sample/Plant Species	Form of Enrichment	Concentration
		1%	2%	4%
Rapeseed honey	None	2.24 ± 0.01 s
Lungwort (Pulmonaria officinalis L.)	Extract	4.72 ± 0.21 nop	6.80 ± 0.24 jkl	9.81 ± 0.32 fgh
	Dried flowers	8.22 ± 0.16 i	9.84 ± 0.31 gh	12.23 ± 0.61 cd
High mallow (Malva sylvestris L.)	Extract	3.55 ± 0.37 r	5.12 ± 0.30 mno	6.10 ± 0.15 klm
	Dried flowers	4.70 ± 0.44 nop	7.81 ± 0.45 ij	9.16 ± 0.44 hi
Cowslip primrose (Primula veris L.)	Extract	6.17 ± 0.43 klm	8.76 ± 0.40 hi	10.84 ± 0.32 efg
	Dried flowers	1.15 ± 0.45 de	13.44 ± 0.63 ab	13.65 ± 0.32 a
Coltsfoot (Tussilago farfara L.)	Extract	5.83 ± 0.4 lmn	6.40 ± 0.16 kl	11.00 ± 0.20 ef
	Dried flowers	4.55 ± 0.26 opr	8.78 ± 0.12 hi	12.42 ± 0.63 bc
Lawn daisy (Bellis perennis L.)	Extract	3.71 ± 0.34 pr	5.23 ± 0.27 mno	7.01 ± 0.33 jk
	Dried flowers	7.98 ± 0.22 ij	9.51 ± 0.20 h	11.44 ± 0.55 cde
Black elderberry (Sambucus nigra L.)	Extract	8.34 ± 0.20 i	10.4 ± 0.52 efg	12.18 ± 0.52 cd
	Dried flowers	8.83 ± 0.10 hi	12.2 ± 0.60 bcd	14.16 ± 0.41 a
Multifloral honey	None	3.56 ± 0.15 n
Lungwort (Pulmonaria officinalis L.)	Extract	3.76 ± 0.50 n	6.52 ± 0.10 kl	9.77 ± 0.58 gh
	Dried flowers	5.71 ± 0.18 lm	12.63 ± 0.31 cd	13.03 ± 0.54 cd
High mallow (Malva sylvestris L.)	Extract	5.32 ± 0.32 m	8.54 ± 0.00 ij	10.90 ± 0.33 efg
	Dried flowers	5.58 ± 0.34 m	8.12 ± 0.40 j	13.68 ± 0.15 ab
Cowslip primrose (Primula veris L.)	Extract	5.88 ± 0.11 lm	9.50 ± 0.33 hi	13.53 ± 0.20 abc
	Dried flowers	7.50 ± 0.39 jk	12.01 ± 0.26 de	14.21 ± 0.13 ab
Coltsfoot (Tussilago farfara L.)	Extract	5.42 ± 0.62 m	6.87 ± 0.77 kl	10.00 ± 0.35 gh
	Dried flowers	8.37 ± 0.36 ij	10.81 ± 0.42 fg	13.33 ± 0.16 bc
Lawn daisy (Bellis perennis L.)	Extract	3.69 ± 0.24 n	6.58 ± 0.39 kl	12.07 ± 0.13 de
	Dried flowers	6.10 ± 0.66 lm	10.50 ± 0.21 fgh	13.13 ± 0.34 bcd
Black elderberry (Sambucus nigra L.)	Extract	6.55 ± 0.32 kl	10.44 ± 0.36 fgh	13.40 ± 0.12 bc
	Dried flowers	11.30 ± 0.10 ef	14.30 ± 0.13 a	14.61 ± 0.26 a

Results marked by different letters are statistically different at p ≤ 0.05 according to Tukey’s test.

.

The highest total phenolic content characterised the multifloral honeys enriched with black elderberry (Sambucus nigra L.) and cowslip primrose (Primula veris L.), at a 4% plant material addition, both as an extract and as dried flowers, and honey enriched with high mallow (Malva sylvestris L.), which exceeded the total phenolic content in the multifloral honey samples by almost four times. Similar phenolic content to the samples with a 4% dried flowers addition was observed for a 2% addition of the black elderberry (Sambucus nigra L.), lungwort (Pulmonaria officinalis L.), and cowslip primrose (Primula veris L.) flowers listed previously. Therefore, this ratio can be considered an optimal enrichment for these plant species, as higher content does not significantly affect the phenolic content. On the other hand, for a 1% plant material addition, the highest total phenolic content was observed when enriched with black elderberry (Sambucus nigra L.) flowers. The dried black elderberry (Sambucus nigra L.) flower addition caused a TPC increase at each of the three levels of flower addition in multifloral honeys. The lowest total phenolic content was noted for the lungwort (Pulmonaria officinalis L.) extract addition at each enrichment ratio in multifloral honeys, with the TPC for the 1% addition not differing significantly from the phenolic content in the honey sample without plant material added. The lowest TPC in the 4% plant addition group in rapeseed honey was found for lawn daisy (Bellis perennis L.) extract and high mallow (Malva sylvestris L.) extract. In rapesesed honeys, a dried cowslip primrose (Primula veris L.) flower addition of just 2% resulted in an equally high total phenolic content as with a dried flower addition twice as high, while honey with an extract addition at the same ratio showed 35% lower total phenolic content than rapeseed honey enriched with dried cowslip primrose (Primula veris L.) flowers. Rapeseed honey enrichment with cowslip primrose (Primula veris L.) flowers significantly increased the total phenolic content in the honey samples at all addition ratios compared to honeys without additions, with the greatest increase noted at 1% and 2% additions. As with multifloral honeys, the lowest phenolic content was observed in rapeseed honeys with the lowest plant addition, in particular, samples enriched with high mallow (Malva sylvestris L.) and lawn daisy (Bellis perennis L.) extracts.

Table 6. Content of flavonoids (mg of quercetin equivalent per 100 g) in the tested honey products.

Sample/Plant Species	Form of Enrichment	Concentration
		1%	2%	4%
Rapeseed honey	None	8.42 ± 0.66 mn
Lungwort (Pulmonaria officinalis L.)	Extract	6.72 ± 0.21 no	16.12 ± 0.90 j	28.26 ± 0.63 e
	Dried flowers	16.24 ± 0.56 j	33.31 ± 0.67 d	61.00 ± 0.86 a
High mallow (Malva sylvestris L.)	Extract	5.85 ± 0.52 o	10.64 ± 0.23 lm	14.36 ± 0.68 jk
	Dried flowers	9.62 ± 0.33 lm	18.37 ± 0.75 i	24.04 ± 0.92 g
Cowslip primrose (Primula veris L.)	Extract	8.91 ± 0.14 m	14.55 ± 0.92 jk	24.71 ± 0.54 g
	Dried flowers	14.65 ± 0.83 jk	26.51 ± 0.80 ef	33.62 ± 0.72 d
Coltsfoot (Tussilago farfara L.)	Extract	11.34 ± 0.66 lm	24.82 ± 0.09 fg	37.55 ± 0.99 c
	Dried flowers	9.36 ± 0.51 lm	20.65 ± 0.63 h	40.13 ± 0.88 b
Lawn daisy (Bellis perennis L.)	Extract	10.30 ± 0.24 lm	15.26 ± 0.32 jk	23.18 ± 0.67 g
	Dried flowers	9.26 ± 0.21 m	14.92 ± 0.36 jk	23.94 ± 0.38 g
Black elderberry (Sambucus nigra L.)	Extract	13.64 ± 0.65 k	24.60 ± 0.29 fg	37.43 ± 0.43 c
	Dried flowers	27.92 ± 0.82 e	33.82 ± 0.61 d	39.36 ± 0.42 bc
Multifloral honey	None	4.33 ± 0.30 t
Lungwort (Pulmonaria officinalis L.)	Extract	9.03 ± 0.51 r	21.12 ± 0.76 gh	22.82 ± 0.77 fg
	Dried flowers	15.35 ± 0.60 lm	29.87 ± 0.58 d	40.87 ± 0.78 a
High mallow (Malva sylvestris L.)	Extract	4.32 ± 0.14 t	9.10 ± 0.62 r	16.86 ± 0.86 kl
	Dried flowers	9.27 ± 0.33 r	11.45 ± 0.56 p	35.53 ± 0.61 b
Cowslip primrose (Primula veris L.)	Extract	4.03 ± 0.45 t	9.42 ± 0.31 r	13.87 ± 0.47 mn
	Dried flowers	14.64 ± 0.56 mn	18.80 ± 0.71 ij	24.38 ± 0.52 f
Coltsfoot (Tussilago farfara L.)	Extract	5.05 ± 0.53 st	11.77 ± 0.34 op	26.84 ± 0.67 e
	Dried flowers	13.05 ± 0.34 no	20.65 ± 0.41 hi	27.26 ± 0.43 e
Lawn daisy (Bellis perennis L.)	Extract	6.32 ± 0.28 s	9.20 ± 0.30 r	22.44 ± 0.38 gh
	Dried flowers	12.51 ± 0.55 op	20.67 ± 0.68 hi	30.82 ± 0.65 d
Black elderberry (Sambucus nigra L.)	Extract	11.62 ± 0.68 op	18.92 ± 0.66 ij	36.90 ± 0.57 b
	Dried flowers	17.44 ± 0.42 jk	32.90 ± 0.39 c	40.01 ± 0.89 a

Results marked by different letters are statistically different at p ≤ 0.05 according to Tukey’s test.

shows the test results of the flavonoid content in rapeseed and multifloral honeys enriched with flowers.

The highest flavonoid content was noted in rapeseed honeys enriched with 4% dried lungwort (Pulmonaria officinalis L.), with it being more than seven times higher than for pure rapeseed honeys. In the same products enriched with a floral extract, a flavonoid content lower by half was found. On the other hand, in honeys enriched with coltsfoot (Tussilago farfara L.) and black elderberry (Sambucus nigra L.) flowers, the flavonoid content was almost five times higher compared to honey samples without additions, both for honeys enriched with floral extracts and honey enriched with dried flowers. The lowest flavonoid cotent, on the level of 4% enrichment, was noted for honeys with added cowslip primrose (Primula veris L.) and high mallow (Malva sylvestris L.) extracts in comparison with honey samples without additions. Rapeseed honey enrichment with dried lungwort (Pulmonaria officinalis L.), black elderberry (Sambucus nigra L.), and cowslip primrose (Primula veris L.) flowers at all addition ratios resulted in the greatest increases in flavonoid content within their respective groups. For honeys with coltsfoot (Tussilago farfara L.) and lawn daisy (Bellis perennis L.), a similar effectiveness of the plant material enrichment was observed for extracts and dried flowers. The highest enrichment effectiveness was observed for dried flowers of lungwort (Pulmonaria officinalis L.), black elderberry (Sambucus nigra L.), and high mallow (Malva sylvestris L.), where the flavonoid content increased more than a factor of nine compared to honey samples without additions. Statistically highly significant differences were found in the flavonoid content for all levels of enrichment and for each plant species, similar to the preceding determinations.

According to different authors, Polish rapeseed honey is relatively poor in phenolic compounds, containing approx. 4.5 to approx. 33.5 mg GAE·100 g⁻¹ [41]. A study conducted by Dżugan et al. [37] demonstrated that rapeseed honeys had an antioxidative activity half that of multifloral honeys, which is consistent with our DPPH test results for the same honey varieties. Wilczyńska et al. [42] reported that the antiradical activity measured using the DPPH method ranged from 23.8% in Polish nectar–honeydew honeys to 100% for Polish heather and buckwheat honeys. Jasicka-Misiak et al. [43] reported similar values (31–40%) for Polish woundwort honey, measured for a 20% w/v honey solution. In turn, Lachman et al. [44] found, after analysing multiple varieties of Czech honeys, that the antioxidant activity determined via the DPPH and ABTS methods was lowest in floral honeys. Bertoncelj et al. [45] observed low antioxidative activity in Slovenian honeys with the brightest colour, which were the acacia and linden varieties, while the lowest values were found for dark honeys: fir, spruce, and forest honey. Perna et al. [46] tested Italian honeys and discovered that free radical sweeping activity measured for honey solutions of 3–60% w/v concentration ranged from 55.06% for citrus honey to 75.37% for chestnut honey. Investigating the antioxidative activity of Serbian honeys, Srećković et al. [47] found that forest honey showed better antioxidative activity, on average 594.77 mg Trolox·kg⁻¹ in the ABTS test and 260.77 mg Trolox·kg⁻¹ in the DPPH test, than other test samples of honey. In turn, a determination of phenolic content by the same authors using the spectrophotometric method in honey samples demonstrated that the highest total phenolic content (806.10 mg GAE·kg⁻¹) and flavonoid content (146.27 mg Qu·kg⁻¹) was found for forest honey and was more than ten times higher than for acacia honey, in which the total phenolic content was determined to be 68.48 mg GAE·kg⁻¹ and the flavonoid content was determined to be 18.59 mg QU·kg⁻¹. According to Kacániová et al. [48], the radical scavenging activity in Slovakian honeydew honey samples measured for a 25% w/v honey solution ranged from 45.9 to 86.6%. Concerning the content of phenolic compounds determined as total phenolic content, especially for multifloral honeys, the authors of many studies report highly varied data. For example, Kavanagh et al. [49] found that in multifloral Irish honeys, the total phenolic content (TPC) ranged from 2.59 to 81.10 mg GAE·100 g⁻¹ of honey. In the authors’ opinion, the result of this determination was affected by the region in which the honey was collected. In Irish honeys produced in rural areas, a much lower total phenolic content was recorded (20.32 mg GAE·100 g⁻¹) than for city honeys from the same region (28.26 mg GAE·100 g⁻¹) due to the much greater diversity of floral resources in Irish urban areas. Muñoz et al. [50] reported that Peruvian wild multifloral honey showed the highest phenolic compound content, which was 207.89 ± 2.18 mg GAE·100 g⁻¹, with the results also showing the lowest variability. It also bears highlighting that there are monofloral honeys characterised by high phenolic compound content, which leads to high interest in these products, such as Manuka honey (Leptospermum scoparium), which originates from New Zealand and Australia. The latter is distinguished by the highest total phenolic content recorded to date, ranging from 217.0 to 203.0 mg GAE·100 g⁻¹, as well as high antioxidative and antibacterial activity, and is considered a medicinal honey. The Polish equivalent to this product, according to Golinski et al. [51], is buckwheat honey, whose TPC value is 211.0 ± 11.4 mg GAE·100 g⁻¹ which is almost 100 times higher than in the case of rapeseed honey without additives used in our experiments and 15 times higher than honeys of both species with the addition of dried black elderberry flowers (Sambucus nigra L.) at the level of 4% additives, which were characterised by the highest total phenolic content in our study, but without differing significantly from that of Manuka honey. Becerril-Sánchez et al. [52], based on an extensive review of previous studies on antioxidative properties and the phenolic and flavonoid contents in honey, indicated these parameters as the characteristics that distinguish individual varieties and botanical origin, their health-promoting applications, and even provided a method to detect honey falsification. The flavonoid compounds and their transformed derivatives are substances with beneficial properties for humans due to the scavenging of free radicals and increases in the stability of cell membranes; therefore, their presence in the daily diet is very important [53]. For pure raw honeys, the flavonoid content differs between varieties, ranging from 0.56–0.62 mg QE·100 g⁻¹ for multifloral and rapeseed honeys to 0.53–0.90 mg QE·100 g⁻¹ for forest honey [45].

Previously, honey enrichment with the wild-growing flowers selected by us for this study was unknown. The flowers came from plants whose therapeutic significance had not always been fully explored before, such as lawn daisy (Bellis perennis L.), but the health-promoting properties of other species belonging to the same botanical family are known, i.e., Asteraceae [54]. On the other hand, the use of cowslip primrose (Primula veris L.), both in traditional medicine and as a food additive, is well known in southeastern European countries [55]. The vast majority of the plant species used in this experiment are known, especially in pharmacy, as plants with medicinal properties and as ingredients of medicines recommended for respiratory diseases (e.g., Herbapect cough syrup) or as immunity-boosting medicines (e.g., Sinupret), although the therapeutic substances were obtained from morphological parts other than flowers [56,57,58]. Most of the plants used in our experiments are also identified in the literature as potentially effective plants with a range of beneficial qualities, such as antibacterial, anti-inflammatory, antiviral, and antioxidative properties [31]. However, they have not been used as food additives, especially as flowers. On the other hand, the choice of honey varieties for the experiment was based on literature reports on their physical (bright colour and stable, semi-liquid consistency), biochemical (relatively low antioxidative potential), and organoleptic (no distinct flavour or odour of their own) properties. The honeys enriched with floral extracts retained their semi-liquid consistency at all levels of enrichment, which for many customers is a quality that improves the value of a product. Sowa et al. [22] used multifloral honey in a study on honey enrichment with Melilotus flowers at similar levels as in our study, while Grabek-Lejko et al. [20] enriched rapeseed honey with raspberry fruits and leaves. Introducing a plant ingredient, both during the honey production stage as well as through enrichment in the form of an extract or fragmented plant material, can effectively increase the flavonoid content in the honey. High flavonoid content distinguished the herb honeys tested by Socha et al. [7], especially thyme herb honey, hawthorn herb honey, and raspberry herb honey, whose total flavonoids ranged from 20 to 28 mg QE·100 g⁻¹, as in the case of our honeys enriched with lungwort (Pulmonaria officinalis L.) extract at the level of 4%, and half lower than in rapeseed honeys with 4% addition of lungwort (Pulmonaria officinalis L.) dry flowers. The same investigators also noted very high antioxidative activity for these three products.

Conversely, in the study conducted by Grabek-Lejko et al. [20], the highest flavonoid content characterised rapeseed honeys enriched with blackberry and raspberry leaves, with the honeys also exhibiting high antioxidative activity. Tomczyk et al. [19], who enriched rapeseed honeys with mulberry leaves, found a 50-fold increase in antioxidative potential similar to the antioxidant properties of our honeys with the addition of 4% cowslip primrose (Primula veris L.) flower in the DPPH assay. Additionally, they found that the high antioxidative potential of mulberry-enriched honeys resulted primarily from the presence of phenolic acids and flavonoid glycosides. In turn, Đorđević et al. [59] demonstrated the most intense extraction of rutin and quercetin in an acacia honey sample enriched with 10% of the Sophora flower (150.24 mg/kg of rutin and 1338.93 mg/kg of quercetin), which moreover had a pleasant taste and aroma. In their study, Jović et al. [31] demonstrated the high pro-health potential of nineteen extracts from the leaves and flowers of medicinal plants and herbs, including cowslip primrose (Primula veris L.) flower, black elderberry (Sambucus nigra L.) flower, and high mallow (Malva sylvestris L.) flower. Among these, the cowslip primrose (Primula veris L.) extract was characterised by the highest antioxidative potential of 188.5 GAE mg·g⁻¹, with black elderberry (Sambucus nigra L.) extract coming second with an antioxidative activity of 170.4 GAE mg·g⁻¹, while high mallow (Malva sylvestris L.) had a potential 10 times lower than black elderberry (Sambucus nigra L.) flowers. The flavonoid contents were 52.0 RUE mg·g⁻¹ for cowslip primrose (Primula veris L.), 32.4 RUE mg·g⁻¹ for black elderberry (Sambucus nigra L.), and 35.5 RUE mg·g⁻¹ for high mallow (Malva sylvestris L.).

These results demonstrate that individual flowers and other plant parts can provide different health-promoting properties and substances, while the antioxidative activity is not always connected to high flavonoid content. A study conducted by Tarapatskyy et al. [29] demonstrated that the richest source of polyphenolic compounds was cowslip primrose (Primula veris L.) flowers and leaves, while aqueous and ethanol extracts from cowslip primrose (Primula veris L.) were characterised by a quantitatively rich profile of polyphenolic substances and high antioxidative potential. According to Wichtl [60], the total flavonoid content in cowslip primrose (Primula veris L.) flowers reaches approximately 3%, and the substances present in these flowers in the greatest quantities are rutoside, kaempferol-3-rutinoside, and isorhamnetin-3-glucoside. Tarapatskyy et al. [28] also demonstrated that as a result of enriching wine with cowslip primrose (Primula veris L.) flowers, the anthocyanin content in red wine had a four-fold increase, up to 1956.85 mg·L⁻¹ after a 10% addition of cowslip primrose (Primula veris L.) flowers, the flavonoid content had a 5-fold increase for white wines, and an almost 25-fold increase in flavonoid content was found in Carlo Rossi commercial wine samples at the lowest (2.5%) cowslip primrose (Primula veris L.) flower addition. In a study by Latypova et al. [61], the object of analysis was a solid herbal extract of cowslip primrose (Primula veris L.), which included a multi-stage purification process with standardisation of its polyphenolic composition and its cardioprotective effect in animals with experimental chronic heart failure. The authors of the study demonstrated that a solid herbal extract produced from cowslip primrose (Primula veris L.) at a dose of 30 mg·kg⁻¹ resulted in a smaller number of animal deaths and a lower level of plasma disease markers as compared to the control group.

Hierarchical clustering analysis and heatmap visualisation was performed to visualise the relationships between the analysed samples (Figure 1).

The heat map was used to depict the content of bioactive compounds (polyphenols, flavonoids) in individual samples, as well as their antioxidant activities. This facilitated the identification of samples with the highest health-promoting potential. The analysis was conducted using the Euclidean distance as the measure of distance and Ward’s method for linking objects. The variable importance was determined based on the C&RT model. The obtained predictor importance values are as follows: 1 (DPPH), 0.86 (ABTS), 0.76 (TPC), and 0.68 (TFC). The analysed samples were divided into three main clusters. In the cluster analysis, samples with similar values of analysed variables are placed close to each other. It can be observed that one cluster stands out (encompassing samples with the highest content of phenolic compounds and flavonoids, consequently exhibiting the highest antioxidant activity). Samples in the remaining clusters generally exhibited lower values, with a few exceptions, such as M_e_P.veris 4% (multifloral honey enriched with a 4% addition of extract from cowslip primrose (Primula veris L.)), showing higher values in terms of TPC.

In analysing the obtained results, it can be observed that the effectiveness of enrichment in bioactive compounds depends primarily on the plant species, where P. veris and S. nigra stand out. The form of the additive is also crucial—the dried form proved to be significantly more effective than the extract. Furthermore, as anticipated, the enrichment effectiveness increases with the applied dose (when separately analysing the dried form and the extract, respectively). Interestingly, the impact of the honey variety used as the matrix for enrichment was not as significant. A notable case is the honey enriched with dried P. veris, which exhibited very high activity in the DPPH test, a relatively high content of phenolic compounds, but a low level of flavonoids. This may indicate that, in the case of this plant species, other compounds are responsible for the antioxidant activity against DPPH, which were not detected in the analysed spectrophotometric tests. Further studies require a more in-depth analysis that considers the content of specific phenolic compounds and the identification of other bioactive compounds.

4. Conclusions

Health-promoting ingredients from wildflowers improve the antioxidant properties of honey, and their extraction is effective despite the high sugar concentration in the samples without additional mechanical intervention. This report proves that, especially in the case of dried flowers, there was an improvement in the antioxidant properties at each level of enrichment compared to honey of both varieties without additives. Cowslip primrose (Primula veris L.) improved the antioxidant properties the most effectively in honey at all levels of enrichment. The highest radical scavenging capacity was observed in rapeseed honey with a 4% dried cowslip primrose flower addition, which was more than 50 times higher than the activity for pure rapeseed honey. An almost 100% radical scavenging capacity in the DPPH and ABTS tests was found for rapeseed and multifloral honeys with cowslip primrose (Primula veris L.) additions, especially for a 4% dried flower addition. The highest total phenolic content characterised multifloral honeys enriched with black elderberry (Sambucus nigra L.) and cowslip primrose (Primula veris L.), at 2% and 4% plant material addition, both as an extract and as dried flowers. The highest flavonoid content was noted in rapeseed honey enriched with 4% dried lungwort, which was more than seven times higher than for pure rapeseed honey. Furthermore, rapeseed-type honey produces a better matrix for the transfer of natural plant metabolites into the honey. More detailed analyses of modified types of honey are needed to determine the phenolic compound profiles. On the other hand, the high sugar content in honey suggests that a comparison of the glycaemic indices of clear and enriched honey is necessary.